Dr Anne Schmidt

Dr Estelle Oberlin

Pr Joelle Sobczak

Pierre Laurence Petit

Yves Canto

Isabelle Cacciapuoti

progress and to learn a lot in

You know you are never alone when you have in your life someone like Viviane PELTIER, it is never easy to be far from home, but Viviane made it easy for me. Vivi you were like my mother during these 5 years, always here, taking care of me, discussing with me, every problem seems easy to solve when discussed with you.

Thank you for literally everything, thank you for each lunch, each coffee, each discussion we shared.

Every person has a guardian angel somewhere, I am more than confident that Cecile DREVON were mine starting my very first day in the lab. Cecile, you believed in me and always pushed me forward, always told me to pursue my dream and to have faith in myself. It was hard to see you leaving 2 years ago but I knew that will not change the fact that you are always here for me. I promise you even if my journey in this lab has finished, we will always maintain this special relationship and you will always be my guardian angel.

To Gregoire STIK with whom I shared the office for almost 2 years and above all I shared great memories and great scientific exchanges, thank you Greg. I also owe a lot to all the people who were part of my daily life at one time or another and without whom these years wouldn't be the same, all the people of the Developmental Biology Laboratory especially Sylvie SCHNEIDER MANNOURY who welcomed me in the unit and was always ready to answer my needs. I am thinking also about all the master students I worked with especially Lea, Marietta and Shako and Olivera with whom I shared the office for 6 months. As well as our neighbours of the 6 th floor: Delphine, Marie Ange, Cedrine, Claire, Emmanuelle, Estelle, Berbang and the two amazing friends who always gave me strength Joanna and Sonya thank you. My special thanks also go to Sophie GOURNET who initiated me to Photoshop and was always ready to help me presenting my results using her great graphic talent. I have no choice but to mention a special person who followed my evolution starting my Master 1, Sophie LOUVET, thank you for every word you said to recomfort me, for every advice and every "Bonjour souriant", you are the kind of teacher and colleague that always stay in mind.

There is two persons who enlighten my stay in the lab and who truly became more than friends and whom I can call without hesitation "My Lebanese", Keirththana and Ludovic. To all the selfies we took, to the Christmas trees we set up to all the coffee breaks and the laugh we had, to each time we mutually supported each other, to the different culture we exchanged: I am more than grateful! Thank you Kiki et Ludo.

To all the PhD students I represented in the UMR 7622 laboratory council, I hope I was able to meet your expectations and demands. It was a pleasure for me to organize the «Club des Docs» of the UMR7622 and to meet each and every one of you.

If I could finish this thesis, it was also thanks to my friends and the outside activities that were there to change my ideas. I am thinking about all the great meetings and friendships I have made during these six years in Paris as well as my childhood and university friends back home. I will not list everyone but be sure that each one of you my friends helped me somehow to achieve my goal.

There are two peoples who left a special print during this journey, Lama meeting you was one of the best thing that happened to me, you were a source of inspiration and of strength thank you ! and Sandy you were always here when I needed you, listening to me, advising me, helping me every time you could, thank you! Wassim, thank you for your creativity and your help in defining each chapter with a wonderful illustration.

Well now the most important part has arrived, I don't know if I will be able to express what I am feeling for my two families. Bassam and Rebecca without you I would never ever think about moving to France. Bassam you made everything easier, you believed in me and helped me achieve my dream. Rebecca my travel partner being around you always gave me kind of positive energy. Thank you so much.

What can I say to you? Rita, Remie and Nadine my three sweet sisters and my brothers in law (Ziad, Rawad and Georges). Sharing with you the happiest events in your life these past years (engagement, marriage, parenthood) made me the luckiest sister ever. Thank you for everything, but foremost thank you for giving me the best niece and nephews in the world, to Chris, Rhea and Tony, I am the most delighted aunt ever, sometimes it was enough looking to some of your pictures or videos to cheer me up.

Maroun, my caring brother, the best thing that happened to me was you moving to Paris and living together. You have filled my life, we shared moments of joy, Hematopoietic Stem Cells (HSCs) are multipotent stem cells, sitting at the top of a complex hierarchy and are capable, through complex subtlety regulated pathways, of giving birth to the multi-lineage progenitors. Those progenitors have enhanced proliferative capacities and give rise to committed progenitors, which will differentiate into specific blood cell types. Normal hematopoiesis is required to constantly generate new mature blood cells, allowing, for example, the turnover of erythrocytes, and to preserve the HSC pool.

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A small disruption of this subtle equilibrium leads to hematological disorders, like leukemia, multiple myeloma, or non-Hodgkin's lymphoma. In these diseases, the HSC pool homeostasis is severely affected, and the consequences can be lethal.

Bone marrow and peripheral blood stem cell transplants are the gold standard when it comes to treating blood disorders. The injection of allogenic HSCs allows the patients to restore their hematopoietic system after radiation therapy or chemotherapy.

As many as 50,000 transplants are performed each year, therefore, their improvement is of crucial significance to contemporary medicine. It is undeniable that this strategy has improved over the years, with the increase of donors, development of alternative sources to the bone marrow, such as the cord blood, or improvement of the conditioning methods. Nevertheless, HSCs transplant still presents flaws. HSCs are difficult to harvest (HSCs represent only 0,01 to 0,1% of the bone marrow cell population), and complications can occur, such as engraftment failure, graft-versushost disease, and delayed reconstitution. Therefore, theoretical and applied researches need to be conducted to solve this public health matter problem.

Throughout an adult lifetime, HSCs reside in the bone marrow. Although several waves of hematopoietic emergence occur early and transiently in multiple sites during embryonic development, only one site produces bona fide HSCs. The first two waves of blood cells emergence are initiated in the extra-embryonic yolk sac and generate mainly primitive erythrocytes and then erythroid-myeloid progenitors. Adult blood, including HSCs, originates from a next wave that occurs in the aorta-gonadmesonephros (AGM) region. It was shown that HSCs emerge from clusters in the ventral floor of the dorsal aorta, from a specific type of endothelium named the hemogenic endothelium HE. They lose their endothelial characteristics and acquire hematopoietic traits, in a process called the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms controlling EHT and the specification and emergence of HSCs are still poorly understood. Different systems are utilized to disclose those factors. Embryonic (ESCs) and induced (IPSCs) pluripotent stem cells are widely used to study how to orient non-committed cells towards the hematopoietic lineage. One of the advantages of these cells is that they can be genetically modified to study the precise targets involved in hematopoietic development. On the other hand, a different approach consists of culturing immature cells in strictly defined culture media and trying to produce hematopoietic cells and eventually HSCs.

Characterization of hematopoietic cells is achieved with surface markers, analysis of specific expression markers and functional tests. However, this identification is always retrospective.

Those approaches led to a better understanding of the EHT (e.g. role of Runx1, GFI1-GFI1B, GATA2…), but these models do not recapitulate all the steps of development, and the yield of HEC is very low. In spite of those studies, it is still impossible to produce HSCs de novo or amplify them ex vivo. Even though some notable advances were made in which cocktails of transcription factors are used to generate hemogenic ECs directly from differentiated cells through direct reprogramming [START_REF] Sugimura | Haematopoietic stem and progenitor cells from human pluripotent stem cells[END_REF], Lis et al., 2017). This brake needs to be overcome, and thus a better understanding of cellular and molecular mechanisms controlling HSCs emergence and expansion is required to develop future therapeutic approaches.

In the lab, we developed an ex vivo model recapitulating the different steps of mesoderm commitment towards hematopoietic production, including EHT. This model uses uncommitted pre-somitic quail mesoderm (PSM) that, following culture in a specific media, will orient the cells towards the endothelial and hemogenic endothelial commitment. Hemogenic endothelial cells (HECs) commitment is testified by the expression of the transcription factor Runx1. HECs undergo an endothelial-tohematopoietic transition, losing their endothelial features and acquiring a hematopoietic phenotype, producing HSCs [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. The progression through the different phenotypes follows a precise calendar, characterized by the expression of specific genes and markers.

In this study, we investigated the molecular mechanisms controlling EHT in this culture system. To do so we generated transcriptomic data for each population subsets (mesodermal cells, endothelial cells, hemogenic endothelial cells, and hematopoietic cells), and bioinformatics analyses were carried out to identify key genes and pathways regulating the transition from endothelial cells to hematopoietic production. For each population, differentially expressed genes were selected (up or down-regulated genes). WGCNA (data mining method) was performed using the matrix of specific up-regulated genes, it revealed the existence of 5 modules, 1 for EC, 1 for M, 1 for HC and 2 for HEC. In the present work, I focused on studying the two networks of HEC. Based on the list of genes positively correlated to the HEC state obtained following WGCNA analysis and the measure of gene connectivity, we selected several candidate genes shown as hubs in our networks and which we chose to functionally validate in our culture system. We could demonstrate a prominent role of two novel genes POFUT2 and CREB3L1 (on going) implicated in this transition.

Furthermore, we validated our culture system by testing two pathways known to play a crucial role in the EHT Wnt and Notch. We showed that they are implicated in the regulation of the EHT in our system.

1

CHAPTER 1: SCIENTIFIC CONTEXT

Hematopoietic stem cells: intriguing yet remarkable cells 1. Generalities about hematopoiesis

Blood is a mesenchymal tissue composed of cells and an aqueous fluid known as plasma which allows cells to circulate continuously in all the vessels of the body.

Two major functions of the blood include transporting substances to and from our cells and providing immunity and protection against infectious agents such as bacteria and viruses.

Blood consists of several elements and represents approximately 8% of total body weight. The major components include plasma (55%) and cellular elements (red blood cells, white blood cells, and platelets: 45%) Figure 1. Those cells named hematopoietic cells (HCs) are generated during a process called hematopoiesis and possess, depending on their type, special functions. The red blood cells (erythrocytes) ensure the transport of oxygen. The white blood cells (leukocytes) play an important role in the immune system and lymphatic system by defending the body against infection. The platelets (thrombocytes) circulate through the bloodstream and play a major role in clotting.

Mature blood cells have varying life spans. Red blood cells circulate for about 4 months, platelets for about 9 days, and white blood cells range from about a few hours to several days. When oxygen in tissues is low, the body responds by stimulating the bone marrow (BM) to produce more red blood cells. When the body is infected, more white blood cells are produced.

Figure 1: Schematic representation of blood cell constituents

The major blood components include plasma (55%) and cellular elements (red blood cells, white blood cells, and platelets: 45%)

The turnover of cells of the hematopoietic system may be estimated to be close to 1 trillion cells per day [START_REF] Ogawa | Differentiation and proliferation of hematopoietic stem cells[END_REF]. This remarkable cell renewal process is supported by a small population of BM cells termed hematopoietic stem cells HSCs.

Stem Cells SCs and HSCs

A. Self-renewal and multipotency

There are four types of SCs: totipotent, pluripotent, multipotent and unipotent.

They appear during development and some are also present in the adult. Totipotent SCs are perhaps the most versatile of the SC types. A totipotent fertilized egg has the potential to give rise to virtually all human cells, such as nerve, heart, skin... including gametes and all the embryonic appendages. So far totipotency comprises the zygote and extends to some early cleavage-stage blastomeres; the precise early-stage depends on the species. Shortly after the early cleavage stage, totipotency is lost and is replaced by a second SC state i.e., pluripotency. In the mouse embryo, pluripotency occurs during the first fourth days of development. Pluripotent SCs can do everything the totipotent ones except that they are not able to give rise to the placenta.

Trophectoderm and inner cell mass fates separate early due to the expression of the transcription factor Cdx2. During development, the pluripotent SC state is limited to the inner cell mass stage. Multipotent SCs reside in most, if not all the tissues. They are limited in numbers and their role is to replenish the organs or tissues when cells are dying. Examples of multipotent SCs are neural SCs that give rise to different types of neural cells and glia, intestinal SCs that renew the intestinal crypts or hematopoietic SCs which can give rise to different blood cell types. Finally, a unipotent SC refers to a cell that can differentiate along only one lineage. The word 'uni' itself is derived from the Latin word 'unus,' meaning one. Found in adult tissues, a unipotent SC, in comparison with other types of SCs, has the lowest differentiation potential. A good example is the epidermis SC that gives rise only to keratinocytes. Two modes of SC divisions are reported in the literature. The asymmetric division, in which a SC produces one differentiated cell and one SC. This suggests that the homeostatic control of the SC pool is maintained at the level of single cells.

The symmetric division model also starts from a SC and produces two differentiated cells or two SCs. In this context, homeostasis is controlled at the population level, rather than at the individual cell level [START_REF] Shahriyari | Symmetric vs. Asymmetric Stem Cell Divisions: An Adaptation against Cancer?[END_REF]. Overall, the number of adult SCs within a tissue is constant, because of the balance between symmetric and asymmetric divisions Figure 2.

In most tissues, SCs are rare and reside in a specific microenvironment named niche. Niche cells regulate the signals sent to SCs by the surrounding tissues and maintain their two specific SC properties: self-renewal and multipotency. Like all SCs, HSCs are able to differentiate into a series of more and more committed progenitors that will proliferate to produce precursors, engaged into specific lineages to finally give rise to all mature blood cells such as lymphoid, myeloid, and erythroid cells. The hematopoietic system is a complex system formed of a progression of different cell types each defined by specific self-renewal, multilineage capacities, and maturity degrees. HCs are organized into different compartments according to a pyramidal hierarchy, starting from HSCs and ending in all differentiated mature blood cells, through progenitors and hematopoietic precursors. Progenitors are more specific than a SC and are pushed to differentiate into their "target" cell. The most important difference between SCs and progenitor cells is that SCs can replicate indefinitely, whereas progenitor cells can divide only a limited number of times. While precursors are a partially differentiated cell, usually referred to as a unipotent cell that has lost most of its SC properties. HSCs sit at the apex of the tree with their capacity of long-term self-renewal and multilineage differentiation. They are very rare and represent 0.05% of the total mononucleated cells of the BM. Those cells are divided based on their capacity to selfrenew into Long-Term HSCs (LT-HSCs) and Short-Term HSCs (ST-HSCs). The use of the classical technologies (in vitro colony-forming cell assays, other clonal assays and transient in vivo repopulation) led to the identification of multipotent progenitor MPP, which is still able to produce all mature blood cells but lost the capacity to selfrenew [START_REF] Adolfsson | Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c kit(+) stem cell compartment is accompanied by loss of self-renewal capacity[END_REF]. MPP produces two types of progenitors, the Common Myeloid Progenitor CMP [START_REF] Akashi | A clonogenic common myeloid progenitor that gives rise to all myeloid lineages[END_REF] and the Common Lymphoid Progenitor CLP [START_REF] Hao | Identification of a novel, human multilymphoid progenitor in cord blood[END_REF][START_REF] Kondo | Identification of Clonogenic Common Lymphoid Progenitors in Mouse Bone Marrow[END_REF]. CMPs and CLPs lose the multipotency and generate successively the myeloid and lymphoid lineages. CMPs produce 2 types of progenitors: GMPs (granulocyte and monocyte progenitors) and MEPs (megakaryocyte and erythrocyte progenitors) whereas CLPs differentiate into B and T Lymphocytes and Natural Killer (NK) cells. Regarding the dendritic cells, it has been shown according to their phenotype, that they can derive from CMP or CLP as well [START_REF] Manz | Dendritic cell potentials of early lymphoid and myeloid progenitors[END_REF][START_REF] Manz | Dendritic Cell Development from Common Myeloid Progenitors[END_REF][START_REF] Traver | Development of CD8 -Positive Dendritic Cells from a Common Myeloid Progenitor[END_REF]. In the embryo as in the adult, HSCs are the only HCs able to self-renew. However, unlike the embryonic HSCs, adult HSCs have low proliferative potential [START_REF] Abkowitz | Evidence that the number of hematopoietic stem cells per animal is conserved in mammals[END_REF][START_REF] Gordon | Of mice and men ... and elephants[END_REF] and the maintenance of mature blood cells is insured by the progenitors and hematopoietic precursors.

The study of HSC differentiation which allowed the generation of this hierarchical pyramid Figure 3, has relied mainly on ex vivo colony assays and the transplantation of hematopoietic stem and progenitor cells (HSPCs) into irradiated animals.

Figure 3: Schematic representation of the hierarchy of hematopoietic cells

A-Multipotency degree B-Self-renewal and amplification degree HSCs sit at the apex of the tree with their capacity of long-term self-renewal and multilineage differentiation. They give rise to multipotent progenitor MPP, which is still able to produce all mature blood cells but lost the capacity to self-renew. MPPs produce two types of progenitor CMP and CLP which lose the multipotency and generate successively the myeloid and lymphoid lineages.

Abbreviations: HSC: Hematopoietic Stem Cell, LT-HSC: Long Term HSC, ST-HSC: Short-Term HSC, MPP: Multipotent Progenitor, CMP: Common Myeloid Progenitor, CLP: Common Lymphoid Progenitor, GMP: granulocyte and monocyte progenitors, MEP: megakaryocyte and erythrocyte progenitors, LB: Lymphocyte B, LT: Lymphocyte T, NK: Natural Killer In all proposed hierarchies, hematopoiesis originates in multipotent repopulating HSCs, and lineage development occurs via discrete and increasingly lineage-restricted progenitor populations. However, as predominantly purified cell populations were analyzed as opposed to individual cells, functional heterogeneity within the identified populations might have been underestimated. In addition, although transplantation of purified prospective HSC populations into irradiated recipient mice is a powerful assay to evaluate repopulating HSCs at the single-cell level, it may not be able to de-convolute HSC heterogeneity and function in steadystate hematopoiesis. Recently, several studies have explored emerging single-cell technologies that have the potential to overcome many of these limitations. Single-cell whole transcriptome analysis has allowed molecular heterogeneity to be systematically explored across the hematopoietic system. In addition, in vivo barcoding has emerged as a means to study the fate and contribution of individual stem and progenitor cells within larger populations, including their roles in unperturbed hematopoiesis. Together, these innovative single-cell technologies promise to considerably advance our understanding of hematopoiesis. However, as for the more classical single-cell technologies, recent single-cell approaches have technical and conceptual limits that need to be considered when interpreting the results, they generate [START_REF] Jacobsen | Haematopoiesis in the era of advanced single-cell technologies[END_REF].

Concerning new genetic fate mapping, clonal marking techniques and singlecell analysis, they shed light on hematopoiesis under physiological conditions. The inducible lineage tracing of cells emerging from HSCs is assessed by using mice expressing a dependent version of Cre recombinase from different loci: Scl, Runx1, Hoxb4, Fgd5… or by using Knock-In (KI) mouse expressing Tie2 locus Cre recombinase flanked on both ends by a modified estrogen receptor domain. This system combined with limiting dilution and mathematical modeling revealed quantitative properties of hematopoiesis from SCs in the BM [START_REF] Busch | Unperturbed vs. post-transplantation hematopoiesis: both in vivo but different[END_REF][START_REF] Busch | Fundamental properties of unperturbed haematopoiesis from stem cells in vivo[END_REF][START_REF] Henninger | Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development[END_REF][START_REF] Maetzig | A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking[END_REF][START_REF] Upadhaya | New genetic tools for the in vivo study of hematopoietic stem cell function[END_REF].

Together the ex vivo and in vivo analysis of hematopoiesis allowed a new global and more precise overview of the implications of each component, HSCs progenitors and precursors, in producing the mature blood cells.

B. The balance of the number of HSCs

The number of HSCs in the BM is roughly constant (1-4 x 10 5 mononuclear cells, about 150 are solicited each day) however how the balance between selfrenewal and quiescence is controlled is not totally understood. The number of HSCs is controlled by specific genetic programs defined by three major axes: the specification of HSCs, their self-renewal (expansion and maintenance) and finally their commitment, proliferation, and differentiation [START_REF] Lessard | Genetic programs regulating HSC specification, maintenance and expansion[END_REF].

The specification of HSCs occurs earlier during development and generates a pool present in the BM at birth. HSC amplification occurs in 2 defined embryonic sites: the placenta and the fetal liver. It is assumed that expansion of the HSC pool is mainly driven by symmetric division. The self-renewal capacity allows the conservation of a sufficient number of HSCs throughout the lifetime of a person. Those cells undergo asymmetric divisions in order to produce at the same time HSCs and MPPs. Thus, we talk about maintenance Figure 4. Despite strong efforts over the past ten years, the directed generation of clinical-grade, transplantable human HSCs has not yet been achieved although the efficient generation of several more specialized blood cell types useful in human therapies was reported including erythroid cells [START_REF] Lu | Directed differentiation of red blood cells from human embryonic stem cells[END_REF]) B and T lymphoid cells and megakaryocytes. There is, therefore, an urgent need to establish novel procedures to produce HSCs de novo since our lack of knowledge has a profound impact on therapies, weighs heavily on health budgets and is responsible for the poor quality of life for patients suffering from hematological diseases. to mimic radiation effects on mice, showed that spleen and BM cells of the adult mouse were able to reconstitute all hematopoietic cells of a lethally irradiated animal, proving the existence of cells responsible for the replenishment of hematopoietic cells [START_REF] Jacobson | Recovery from radiation injury[END_REF][START_REF] Lorenz | Modification of irradiation injury in mice and guinea pigs by bone marrow injections[END_REF].

Functional and phenotypic identification of HSPCs

After those discoveries, [START_REF] Mcculloch | The Radiation Sensitivity of Normal Mouse Bone Marrow Cells, Determined by Quantitative Marrow Transplantation into Irradiated Mice[END_REF], revealed that the injection of BM cells into lethally irradiated mice led to the formation of hematopoietic nodules within the spleen. They proposed the term: Colony Forming Unit in the Spleen CFU-S [START_REF] Mcculloch | The Radiation Sensitivity of Normal Mouse Bone Marrow Cells, Determined by Quantitative Marrow Transplantation into Irradiated Mice[END_REF] for these nodules and showed that day 8 post-injection nodules were essentially formed of unipotent HCs engaged in the myeloid, erythroid or megakaryocytic lineages, whereas day 12 post-injection nodules contained less differentiated HCs Figure 6. This difference between nodules suggests that the CFU-S giving rise to these 2 nodule types does not have the same degree of immaturity hence demonstrating a CFU-S hierarchy. Only day 12 CFU-S were able to reconstitute a secondary irradiated mice recipient and to form nodules within the spleen. This suggests that day 12 CFU-S possess a self-renewal potential and are more immature than day 8 CFU-S [START_REF] Charbord | Hemopoietic stem cells: Analysis of some parameters critical for engraftment[END_REF].

Several experiments showed that CFU-S are not able to maintain long-term hematopoiesis and are more mature than HSCs [START_REF] Hodgson | Properties of haematopoietic stem cells surviving 5fluorouracil treatment: evidence for a pre-CFU-S cell?[END_REF][START_REF] Jones | Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells[END_REF]. In order to better characterize CFU-S, Ploemacher and Brons introduced for the first time the notion of pre-CFU-S [START_REF] Ploemacher | Isolation of hemopoietic stem cell subsets from murine bone marrow: II. Evidence for an early precursor of day-12 CFU-S and cells associated with radioprotective ability[END_REF]. Thirteen days after the primary injection, BM cells of an irradiated mouse were retrieved and injected in an another lethally irradiated recipient. Twelve days after the secondary injection, CFU-S were described in the spleen. Cells at the origin of this secondary day 12 CFU-S were named pre-CFU-S. They were shown to be able to migrate in the BM of the first mouse recipient and to be more immature than the primary day 12 CFU-S. Those pre-CFU-S were compared to the LT-HSCs and shown as cells able to reconstitute the hematopoietic system of the lethally irradiated mouse in the long-term. Nowadays the CFU-S test remains a classical way to assess the hematopoietic potential of certain embryonic and adult cell populations in vivo. In the embryo, as well as in the adult, HSCs are rare and difficult to characterize, which limits considerably their fundamental study and their use in clinical therapies.

The paradigm of HSCs is that it is still impossible to isolate HSCs at purity since no specific marker of these cells is highlighted. Due to the combination of membrane and intracellular markers, some populations of cells more or less enriched in HSCs can be isolated, and their respective hematopoietic potentials can be evaluated retrospectively through in vivo and ex vivo tests.

B. Ex vivo tests a. Colony Forming Cell assay: CFC

The Colony Forming Cell assay is used in human and mouse to identify progenitors and hematopoietic precursors in a given population. It uses the property of committed progenitors to form colonies in a semi-solid medium in the presence of defined growth factors. Each colony is derived from a single progenitor, therefore counting the number of colonies provides the frequency and the total number of progenitors in the cell suspension. This assay also defines the quality of the committed progenitor which is assessed by looking at the size of the colony and its cell composition. MPPs will give rise to mixed colonies containing various types of differentiated HCs whereas, at the opposite, unipotent progenitors will give rise to colonies containing only one type of blood cells i.e., erythrocytes, granulocytes, macrophages… After 7 days of culture, colonies formed contained different lineages:

Erythrocytes, granulocytes, macrophages, megakaryocytes [START_REF] Johnson | Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin[END_REF] However, the CFU test does not allow the identification of T lymphocyte progenitors since these cells need interaction with a permissive microenvironment.

Starting Day 10 we can find MPPs belonging to more than one lineage: CFU-GEMM colony-forming unit granulocyte-erythroid-macrophage-megakaryocyte.

In addition to these above-cited types of colonies, the CFC assay allows the formation of HPP-CFCs (High Proliferative Potential Colony Forming Cells) [START_REF] Hodgson | Properties of haematopoietic stem cells surviving 5fluorouracil treatment: evidence for a pre-CFU-S cell?[END_REF][START_REF] Srour | Long-Term Generation and Expansion of Human Primitive Hematopoietic Progenitor Cells In Vitro[END_REF]. They are characterized by their ability to generate large colonies which, after 10-12 days of culture, contain more than 50,000 cells of the type granulocyte, erythrocyte, megakaryocyte and macrophage. presence of CA-FC allows to say that the initial population contains hematopoietic progenitors, while CA-FC at day 28 shows that the initial population contain more immature cells able to persist longer. This test was also used to determine the susceptibility of HCs to radiation [START_REF] Ploemacher | Murine haemopoietic stem cells with long-term engraftment and marrow repopulating ability are more resistant to gammaradiation than are spleen colony forming cells[END_REF] and cytotoxic drugs [START_REF] Down | Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: differential effects of host conditioning with gamma radiation and cytotoxic drugs[END_REF].

d. Long-Term Culture-Initiating Cell LTC-IC

In the direct continuity of the previously described co-culture, it is possible to go further in determining the degree of the immaturity of HCs contained in the population seeded on the stromal cell layer. Thus, after 28 to 35 days of culture, the cells of this layer, as well as the supernatant, can be recovered and grown in methylcellulose medium. If 7 to 14 days later, CFCs representative of all myeloid lineage are observable, it is possible to conclude retrospectively that the starting population initially seeded contained LTC-IC, HCs more immature than CA-FC D28. Originally, the growing conditions for the identification of LTC-IC in a population had been identified

to study the myeloid differentiation of very immature human CHs ex vivo [START_REF] Sutherland | Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro[END_REF], but they turn out to be also relevant to the mouse. Some disadvantages to keep in mind is the absence of lymphoid differentiation and the loss of erythroid potential and heterogeneity of the progenitors Figure 9.

In order to estimate the lymphocyte potential, a variation of the LTC-IC test is set up. After 28 days of culture, the supernatant is totally replaced by a medium promoting the B lymphocyte production [START_REF] Whitlock | Long-term culture of B lymphocytes and their precursors from murine bone marrow[END_REF]. The culture is maintained for 7 to 14 days. Cells of the supernatant are analyzed by flow cytometry. Thus, the current co-culture systems allow at different times of the culture, to determine the frequency of HCs and their degree of immaturity in a given population.

Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay

The population to test is added to a stromal cell layer and co-cultured for 28 days. Then all the cells are taken and cultivated on methylcellulose medium for 7 days and the number of colonies is assessed.

Abbreviation: CA-FC Cobblestone Area-Forming Cell, HCs: Hematopoietic Cells

C. In vivo tests

The worldwide recognized test to assess the potential of HSCs is their transplantation into lethally irradiated mice recipients. These experiments are based on Till&McChulloch assay in the 1960s. When irradiated, the mouse loses all its cycling HCs including progenitors and its survival depends on the existence of HSCs within the fraction during transplantation. The short-term survival depends on progenitor cells and injected cells are defined as ST-HSCs, while long-term survival depends on the presence of HSCs able to produce all the hematopoietic lineages of the mouse and defined as LT-HSCs.

The analysis is based on the expression of different markers especially the CD45 marker having different alleles in human and mouse and allowing the differentiation between donor and receiver cells Figure 10.

The first reconstitution is named the primary reconstitution and assesses the multipotency of the injected cells. This step is followed by secondary transplantation in order to assess the long-term self-renewal potential Figure 11.

D. Phenotypic identification of HSPCs

Depending on the type, lineage and maturation degree, hematopoietic cells express specific cell-surface proteins. These proteins can mediate several cellular functions such as receptors, ligands, adhesion or migration… Together with the discovery of Fluorescent Activating Cell Sorting FACS and the production of monoclonal antibodies, those markers allowed the identification and the follow up of the hematopoietic system.

While many aspects of HSC biology are shared between species, the cell surface markers between mice and humans appear to show a lack of congruence.

In the mouse BM, two cell populations can be distinguished: cells expressing markers of mature lineages (CD3, CD4, CD8, CD19, NK1.1, Gr1, CD11b and Ter119, Lin + ), and cells negative for these markers. This population called lineage minus (Lin -

) was shown to contain hematopoietic stem and progenitor cells. HSCs and MPPs can be fractionated according to a high level of Stem Cell Antigen (Sca)-1 and c-Kit tyrosine kinase receptor (Lin -Sca-1 hi c-Kit hi , LSK) (Okada et al.). Using CD34 and FLT3 markers, LT-HSCs can be distinguished from ST-HSCs and MPPs. Within the LSK CD34 -FLT3 -subset, around 35% are HSCs with LT repopulation activity. In contrast, LSK CD34 + FLT3 -cells are multipotent but have a limited repopulation activity in lethally irradiated mice [START_REF] Yang | Identification of Lin-Sca1+kit+CD34+Flt3-short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients[END_REF]. The most commonly used method nowadays to purify mouse HSCs is the use of cell surface receptors of the SLAM (Signaling Lymphocyte Activation Molecule) family. The SLAM family is a group of 10-11 cell surface receptors that are tandemly arrayed at a single locus on chromosome 1 [START_REF] Engel | The SAP and SLAM families in immune responses and Xlinked lymphoproliferative disease[END_REF]. Using the SLAM code, HSCs were highly purified as CD150 + CD244 -CD48 -cells while MPPs were CD244 + CD150 -CD48 -and most restricted progenitors were CD48 + CD244 + CD150 - (Kiel et al., 2005). Among the LSK CD150 + CD48 -cell population, 30% of cells do not express CD34 and this population might be highly enriched in HSCs.

In human, HSCs express several surface markers, but their number is not enough to specify their determination/self-renewal potential. The most used surface marker is the CD34 adhesion mediated glycoprotein. CD34 + cells account about 1% of the BM cells and are enriched in progenitors and HSCs. CD34 expression is not exclusive to HSCs as some endothelial cells (ECs), some stromal BM cells and many others also carry the CD34 marker [START_REF] Sidney | Concise Review: Evidence for CD34 as a Common Marker for Diverse Progenitors[END_REF] Figure 12.

Several works showed that HSCs can also be found in CD34 -Lin -cells. Other studies conclude that CD34 -cells could be the ancestors of CD34 + cells [START_REF] Anjos-Afonso | CD34-Cells at the Apex of the Human Hematopoietic Stem Cell Hierarchy Have Distinctive Cellular and Molecular Signatures[END_REF]. Since CD34 also marks committed progenitors, other markers are to be combined to enrich in more immature cells. The most used are CD38, Thy1/CD90, and CD133/prominim1.

These markers can be combined with the ability of HSCs to efflux the Rhodamine or Hoechst dye to assess the ability of HSCs at the clonal level.

In grey cell types In yellow mouse markers In orange human markers

On the origin of blood

The origins of HSCs have been the subject of intense debate. Development of the hematopoietic system is characterized by the existence of multiple waves, in multiple origins and changing locations. There are also specificities related to the species. The chick embryo was instrumental in decoding many rules of developmental hematopoiesis. In order to investigate how the blood system forms during development, Moore and Owen chose the chicken embryo, already the subject of many descriptive studies since the end of the XIXth century, because it is directly visible after shell opening and develops independently from a maternal circulation.

They applied an array of experimental approaches: irradiation/restoration, parabiosis, grafting of rudiments (thymus, spleen, bursa of Fabricius) on the chorioallantoic membrane [START_REF] Moore | Ontogeny of the Haemopoietic System: Yolk Sac Origin of In Vivo and In Vitro Colony Forming Cells in the Developing Mouse Embryo[END_REF]For review) To follow cell origins in these different designs, Moore and Owen used the pair of sexual chromosomes as a cell marker. Despite several drawbacks inherent to this labelling system (the chromosomes can be observed only in 5-10% of cells arrested at the metaphase stage of mitosis after a colcemid treatment which elicits high mortality of the embryos; furthermore the adequate opposite sex combination is left to chance), these investigators discovered an important trait which characterizes the development of the blood system: HSCs colonize all rudiments to the exception of the yolk sac (YS). They also grafted mouse embryo rudiments on the chicken chorioallantois and observed that, in contrast with cell type-specific recognition capacities displayed by most other embryonic cell types across different classes of vertebrates, attraction or recognition did not work between mouse stromal rudiments and chicken HSCs. Retrieved at an early stage and grafted on the chorioallantois, the mouse thymic rudiment remained lymphoid, allowing Moore and Owen to conclude in 1970 that, in mammalian developmental hematopoiesis also, HSC emerge in one location and differentiate in another [START_REF] Moore | Ontogeny of the Haemopoietic System: Yolk Sac Origin of In Vivo and In Vitro Colony Forming Cells in the Developing Mouse Embryo[END_REF].

Benefiting from accurate functional testing, the mouse embryo took the lead and was instrumental in building a precise, comprehensive picture of hematopoietic development in mammals. Developmental hematopoiesis is organized into 3 separated waves. The first one takes place in the YS at embryonic day (E)7.5 in the mouse embryo [START_REF] Palis | Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse[END_REF]) and at 19-22 hours in the chick embryo [START_REF] Minko | From mesoderm to blood islands: patterns of key molecules during yolk sac erythropoiesis[END_REF]. This wave gives rise to primitive erythroid cells, macrophages, and megakaryocytes [START_REF] Palis | Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse[END_REF]. Shortly after the onset of this primitive hematopoiesis, the first definitive erythro-myeloid progenitors and immune-restricted progenitors emerge from the YS at E8.5 in the mouse and E3 in the chick embryo. The third wave occurs in the embryo proper in a specific region named aorta gonad mesonephros (AGM) and is characterized by the production of HSCs and adult type lympho-myeloid progenitors [START_REF] Drevon | Cell interactions and cell signaling during hematopoietic development[END_REF][START_REF] Gritz | Specification and function of hemogenic endothelium during embryogenesis[END_REF].

HSCs detectable by direct transplantation in adult recipients are not present until E10.5 in the AGM but hematopoietic emergence is detected from E9.5 closely associated with vitelline and umbilical arteries [START_REF] De Bruijn | Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo[END_REF][START_REF] Tavian | Myeloid Stem Cells: Mapping Multipotent Hematopoietic Cell Fate in Intraembryonic Mesoderm[END_REF].

Subsequently, transplantable HSCs are found in the YS, the placenta and the head [START_REF] Li | VCAM-1(+) macrophages guide the homing of HSPCs to a vascular niche[END_REF], Alvarez-Silva et al., 2003) but it is not clear whether HSCs emerge de novo in these sites. Shortly after this period, HSCs migrate and either colonize the fetal liver in mammals or reach the sub-aortic mesenchyme in birds. In both situations, they undergo multiplication and amplification and eventually migrate to the definitive hematopoietic organs (BM, Thymus, spleen…) where they will reside during the lifetime of the organism Figure 13.

. Yolk sac blood islands form between the single-cell layer of endoderm cells (E) and mesothelial cells (open arrow).

Blood islands develop from undifferentiated mesoderm cells (E7.5, closed arrow) that give rise to inner blood cells surrounded by an outer endothelial lining (E9. 5, closed arrow). (Palis and Yoder, 2001) a. The first primitive hematopoietic wave.

The first morphological evidence of hematopoietic activity in multiple mammalian and non-mammalian embryos is the appearance of cohorts of immature erythroid cells within the YS mesoderm beginning at E7.5 in the mouse embryo. These "blood islands" consist of immature erythroid cells wrapped by a monolayer of ECs emerging from seemingly identical mesoderm cells. This close spatial and temporal relationship between the two cell types suggested to [START_REF] Maximow | Untersuchungen uber Blut und Bindegewebe. I. Die Fruhesten Entwicklongsstadien des Blut-und Bindewegebezellen beim Saugetierembryo, bis zum Anfang des Blutbildung in der Leber[END_REF], Vera Dantschakoff (1909) and Florence [START_REF] Sabin | Studies on the Origin of Bllod-Vessels and of Rde Blood-Corpuscules as seen in the Living Blastoderm of Chicks during the Second Day of incubation[END_REF] that these cells arise from a common mesoderm precursor designated as the hemangioblast [START_REF] Murray | The development in vitro of the blood of the early chick embryo[END_REF]. See part III page 55 for a description of the hemangioblast.

The fact that primitive erythroid cells emerge from the YS blood islands and constitute the first circulating cells of the embryo was first recognized by Maximov (1909). The large, nucleated erythroid cells contrasted with small, enucleated, red blood cells that circulate during the life span. These primitive erythrocytes have some specific features that distinguish them from definitive erythrocytes: 1° they are several folds larger and contain six-fold more hemoglobin when mature. Palis and co-workers showed that, contrary to expectations, primitive erythroblasts progressively enucleate between embryonic days 12.5 and 16.5, generating mature primitive erythrocytes that are similar in size to their nucleated counterparts. These enucleated primitive erythrocytes circulate as late as 5 days after birth (Kingsley, 2004). 2° primitive and definitive red blood cells express different combinations of globin chains. Primitive erythroblasts express both embryonic (Zeta, ßH1, and ey) and adult (alpha1, 2, ß1, and ß2) globins during cell maturation. z-and alpha-globin mRNAs have been detected in YS blood islands in pre-somite stage embryos. In functional ex vivo tests, primitive erythrocytes arise from a unique progenitor termed EryP-CFC that generates within 5 days compact erythroid colonies comprised of large erythroid cells that express both embryonic and adult globin chains (Palis, 1999a;[START_REF] Wong | Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac[END_REF].

In addition to primitive erythroid cells, the early YS also generates colonies of macrophages that emerge temporarily and spatially with the primitive erythroid cells at E7.5. These colonies are generated from a unipotent progenitor and are not a component of a bi-or multipotent progenitors until E8.5. Most of these macrophages give rise to resident macrophages involved in cell debris removal during tissue remodeling but it was shown recently that a cohort enters the central nervous system and differentiate into microglia [START_REF] Ginhoux | Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages[END_REF]. The first experimental evidence was obtained on the avian model by constructing YS chimeras where a chick embryo is grafted onto a quail YS or vice versa and immunostain them with an antibody recognizing the hemangioblastic lineage QH1 or MB1. This experimental design showed that a phagocytic lineage originating from the YS seeds the embryo body and invade the tissues including the central nervous system (Cuadros et al., 1992). Slightly later on, the existence of a microglial progenitor was shown from E8 onward in the mouse embryo. Later on, using inducible lineage or gene expression mapping experiments using Runx1, c-Kit, CSFR1, Tie2 or Flt3 reporter mice, tracing of microglial progenitors was achieved, and their YS origin formerly demonstrated [START_REF] Mcgrath | Early hematopoiesis and macrophage development[END_REF] Megakaryocyte progenitors have also been isolated from the E7.5 YS of the mouse embryo. Mk-CFCs increase with time from E7.5 to E9.5 indicating that megakaryocyte formation overlaps between primitive and definitive YS hematopoiesis.

However, megakaryocyte precursors revealed with anti Gp1bß are not detected until E9.5 in the YS. Interestingly, if in the BM, a bipotent erythro-megakaryocyte progenitor has been revealed, most of the erythroid and megakaryocyte colonies were composed of a single cell type indicating a unipotent progenitor. However, some bipotential progenitors were detected that are biased towards the erythroid lineage [START_REF] Tober | The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis[END_REF].

b. The second definitive hematopoietic wave.

Interestingly, the first definitive erythroid progenitors Burst Forming Unit-Erythroid (BFU-E) were detected from E8.5 in the mouse YS. They will massively expand over the next 48 hours and will be found in increasing numbers in the bloodstream between E9.5 and 10.5. As the liver emerges as a hematopoietic organ, they will be massively amplified from E10. Small numbers of BFU-E are detected within the YS after the appearance of CFU-E. Large numbers of CFU-E are detected in the liver, prior to the colonization by adult-repopulating HSCs, indicating a major role in definitive erythropoiesis. Definitive erythrocytes enter the bloodstream between E11.5 and E12.5.

In addition to definitive erythrocytes, several additional hematopoietic progenitors are detected in the YS. These are unipotent mast cell progenitor and a bipotential granulocyte-macrophage progenitor and multilineage, high proliferative potential colony-forming cells (HPP-CFC) which give rise ex vivo to macrophages, mastocytes, and granulocytes. This myeloid potential originates from erythro-myeloid progenitors that express high levels of c-kit and CD41 at their surface [START_REF] Ferkowicz | CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo[END_REF][START_REF] Mikkola | Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo[END_REF]. Single E9.5 Erythro-Myeloid Progenitors (EMPs) have the capacity to generate multiple myeloid lineages ex vivo as well as definitive erythroid potential [START_REF] Mcgrath | Early hematopoiesis and macrophage development[END_REF]. However, EMP does not have B-lymphoid potential.

Of note, EMP arises via an endothelial-to-hematopoietic transition (EHT) in a Runx1dependent manner (Chen et al., 2011;[START_REF] Frame | Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity: EMPs Arise from Arteriovenous Hemogenic Endothelia[END_REF][START_REF] Miller | The core-binding factor β subunit is required for bone formation and hematopoietic maturation[END_REF][START_REF] North | Cbfa2 is required for the formation of intra-aortic hematopoietic clusters[END_REF]. Macrophage and megakaryocyte progenitors persist after primitive erythropoiesis indicating that these progenitors are component of the two waves.

Finally, small numbers of lymphoid progenitors have been detected around E9

in the YS and the embryo proper. However, it is not clear whether these lymphoid progenitors arise from the YS or seed the YS from the embryo. Thorough examination of the YS and para-aortic splanchnopleura lymphoid potential indicates that this potential is not present in the YS until the onset of circulation but is detected in the future aortic region as early as E7.5 days [START_REF] Delassus | Circulation of Hematopoietic Progenitors in the Mouse Embryo[END_REF]. However, YS chimeras indicate that the generation of B-lymphoid cells from the YS is possible but at very low frequency [START_REF] Sugiyama | B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency[END_REF]. Another study indicates that B1 progenitor cells, that preferentially differentiate into innate type-B1 and marginal zone B cells, but not into B2 cells upon transplantation, arise directly from the YS through an Endothelial to Hematopoietic Transition (EHT) independently of HSCs [START_REF] Yoshimoto | Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential[END_REF]. A similar YS-derived immune-restricted and lymphoid-primed progenitor was also described prior to the detection of definitive HSCs and contribute to giving rise to lymphoid as well as myeloid components of the immune system. Dissection of the YS and the Para-aortic-Splanchnopleura (P-Sp) prior to circulation followed by a short culture period show that these cells emerge autonomously from the YS and are detected based on Rag1 expression as early as E9.5 [START_REF] Böiers | Lymphomyeloid Contribution of an Immune-Restricted Progenitor Emerging Prior to Definitive Hematopoietic Stem Cells[END_REF].

B. The allantois/placenta

A hematopoietic function for the placenta was considered a long time ago.

While the presence of reconstituting HSC [START_REF] Mcculloch | The Radiation Sensitivity of Normal Mouse Bone Marrow Cells, Determined by Quantitative Marrow Transplantation into Irradiated Mice[END_REF]; [START_REF] Dancis | Hematopoietic cells in mouse placenta[END_REF]) and of B lymphoid progenitors [START_REF] Melchers | Murine embryonic B lymphocyte development in the placenta[END_REF] in the placenta has been reported many years ago, a contribution of the placenta to fetal hematopoiesis was not seriously considered then.

It started with the quail chicken system that revealed an additional hematopoietic organ starting at E3.5, the allantois, an embryonic appendage situated at the caudal part of the embryo and developing from the intestine. The allantois is basically formed of endoderm associated with mesoderm, allowing it to produce ECs and HCs. Quail allantois grafted in the chicken host could produce ECs and HCs that colonize the BM. Moreover, HCs produced by the allantois express different globin isoforms including the adult ß globin [START_REF] Caprioli | Blood-borne seeding by hematopoietic and endothelial precursors from the allantois[END_REF]. Detailed examination of the earliest phases of allantois development showed that this structure develops blood islands harboring red blood cells and CD45+ cells. Ex vivo culture of the allantois indicated that the red blood cells were of definitive lineage in keeping with [START_REF] Caprioli | Blood-borne seeding by hematopoietic and endothelial precursors from the allantois[END_REF][START_REF] Caprioli | Hemangioblast commitment in the avian allantois: cellular and molecular aspects[END_REF] Figure 15.

Like the avian chorioallantoic membrane, the placenta is an extra-embryonic appendage richly vascularized. It is formed in the mouse, from E8,5 and provides gas and nutrient exchange necessary for the survival of the embryo. It is formed by the fusion of the allantois with the ecto-placental cone at E8.5 and is composed of these two structures, the first of fetal origin and the second of maternal origin. The hematopoietic potential of the placenta was detected in both allantois and pre-fusion chorion (Corbel et al., 2007) Figure 16. The presence of clonogenic hematopoietic progenitors was investigated in the E8/E9 placenta and compared to the fetal liver of YS at the same stage. HPP-CFC were 2-4 times more frequent in the placenta [START_REF] Alvarez-Silva | Mouse placenta is a major hematopoietic organ[END_REF]. The placenta was also shown to be an amplification niche for HSCs between E12.5 to E13.5 [START_REF] Gekas | The Placenta Is a Niche for Hematopoietic Stem Cells[END_REF] in the labyrinth region [START_REF] Ottersbach | The Murine Placenta Contains Hematopoietic Stem Cells within the Vascular Labyrinth Region[END_REF]. However, it was not clear whether the allantois/placenta was a site of maturation or a source of hematopoietic cells. The allantois isolated before the establishment of the circulation is capable of giving rise to myeloid and definitive erythroid cells [START_REF] Ziegler | The allantois and chorion, when isolated before circulation or chorioallantoic fusion, have hematopoietic potential[END_REF]. In contrast to the fetal liver, the placenta is enriched in immature progenitors and few progenitors involved in specific lineages are observed. In addition to being the emergence site of HSCs, this extraembryonic structure plays a temporary niche role in the maturation and expansion of HSCs [START_REF] Alvarez-Silva | Mouse placenta is a major hematopoietic organ[END_REF][START_REF] Gekas | The Placenta Is a Niche for Hematopoietic Stem Cells[END_REF] Using mouse embryos deficient for NCX1

and lacking heartbeat suggest that placenta is able to generate B, T and erythroid cells suggesting the in-situ emergence of hematopoietic progenitors [START_REF] Rhodes | The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation[END_REF]. The approach is controversial as the blood flow itself plays a role in maintaining the endothelial structure and the appearance of HSCs. Indeed, recent experiments in Zebrafish and Mouse show that the absence of blood circulation causes a severe decrease in the number of HSCs in the embryo [START_REF] North | Hematopoietic Stem Cell Development Is Dependent on Blood Flow[END_REF]. Additional in vivo and in vitro experiments were performed, showing that blood flow has a significant influence on the level of hematopoietic gene expression, such as runx1 [START_REF] Adamo | Biomechanical forces promote embryonic haematopoiesis[END_REF]. Abbreviations: E, ectoderm; En,allantoic endoderm. Adapted from [START_REF] Caprioli | Hemangioblast commitment in the avian allantois: cellular and molecular aspects[END_REF] Similar investigations were undertaken in the human embryo. Multiple types of progenitors i.e., myeloid, erythroid, NK and B cells were detected in the labyrinth of the human placenta [START_REF] Barcena | A New Role for the Human Placenta as a Hematopoietic Site Throughout Gestation[END_REF]. In addition, progenitors and HSC were detected in the placenta all along with development. However, no amplification of HSC or progenitor could be detected indicating a slightly different role than the mouse placenta [START_REF] Robin | Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development[END_REF]

C. The aortic region

At the dawn of embryology, scientists described the seemingly coincident emergence of endothelial and hematopoietic cells in the YS giving rise to the concept of hemangioblast [START_REF] Murray | The development in vitro of the blood of the early chick embryo[END_REF]. It is not difficult to imagine why the YS was the first hematopoietic site to be investigated. Indeed, the YS shows the first signs of hematopoiesis testified by the presence of the red blood islands and was naturally proposed to be the site of HSC production. In the mid and late 60s, Moore and Owen reported that the YS was the site where hematopoietic stem cells emerge in a unique, early, developmental event and subsequently colonize the hematopoietic organs [START_REF] Moore | Experimental studies on the development of the thymus[END_REF]. The experimental design used a series of graft and parabiosis experiments relying on the identification of sex chromosomes Figure 17. In addition, they also showed that HSPCs were of extrinsic origin (see above). These two pillars made the core of the hematogenous theory. During the 70s the relevance of this hypothesis was investigated in mammalian species by culturing either E7 whole mouse embryo or separated YS and embryo. The conclusion was that the YS was the only site where HSC were formed reinforcing the central role of this site in HSC generation [START_REF] Moore | Ontogeny of the Haemopoietic System: Yolk Sac Origin of In Vivo and In Vitro Colony Forming Cells in the Developing Mouse Embryo[END_REF]. Based on this and on other reports [START_REF] Weissman | Normal and Neoplastic Maturation of T-lineage Lymphocytes[END_REF], the hematogenous theory became a dogma and was widely accepted among the scientific community. In 1972, a surgical technique to construct a unique type of quail/chick chimera was devised by [START_REF] Martin | Technique d'explantation in ovo de blastodermes d'embryons d'oiseaux[END_REF]. The flatness of the avian embryo as it lies on the surface of the spherical YS facilitates its dissection, allowing it to be replaced with the embryo of another species in ovo. When such manipulations were carried out, it was noted that there were a rapid reconstitution and joining of blood vessels between the grafted embryo body and the host YS and that the free circulation of blood cells was established between the YS and the body, without any disruption to normal development in the resulting chimeras. The quail/chick chimera consisted in grafting a quail embryo on a chick YS at a very early stage of development, before the onset of heart beating Figure 18 The embryos resulting from this operation possessed hemopoietic organs in which the cells belonging to blood lineages in the thymus, spleen, bursa, and BM were all quail while stromal cells were chicken (Dieterlen-Lievre, 1975); [START_REF] Martin | Developmental relationships between vitelline and intra-embryonic haemopoiesis studied in avian 'yolk sac chimaeras[END_REF]. Circulating blood was chicken (derived from YS progenitors) until day 5 of incubation, then became mixed, and then richer and richer in quail erythrocytes [START_REF] Beaupain | Related to the Origin of Stem Cells and Site of Erythropoiesis?[END_REF]. The replacement of erythrocytes derived from YS progenitors by these derived from intraembryonic HSC was strikingly confirmed when the chimeras were built between congenic strains of chickens, differing in their immunoglobulin allotypes or in their major histocompatibility antigens [START_REF] Lassila | The origin of lymphoid stem cells studied in chick yolk sac-embryo chimaeras[END_REF]. These results led to the indisputable conclusion that YS progenitors give rise to a short-lived progeny, hence cannot be considered as long-life hematopoietic stem cells, since this term specifies the property of long-term selfrenewal. The extinction of YS progenitors may result from the differentiation pressure resulting from the exponential need for red cells in the rapidly developing embryo.

Indeed, associated in vitro with an attractive thymic rudiment, the extraembryonic area (i.e. future YS) was capable of providing lymphoid progenitors. It was clear that another source was responsible for the production of hematopoietic progenitors that seeded the hematopoietic organs as well as for HSCs.

Intra-embryonic clusters had been described from the beginning of the 20th century in both avian and mammalian species as groups of hematopoietic cells, located on the aortic floor, protruding into the aortic lumen [START_REF] Dieterlen-Lièvre | Are intra-aortic hemopoietic cells derived from endothelial cells during ontogeny?[END_REF] Figure 19.

This region thus appeared as an interesting candidate to produce the cells that seeded the definitive hematopoietic organs. In quail/chick chimeras, these intra-aortic clusters are quail, i.e. they have an in situ intra-embryonic origin (Dieterlen-Livre and [START_REF] Dieterlen-Livre | Diffuse lntraembryonic Hemopoiesis in Normal and Chimeric Avian Development[END_REF]. Moreover, their affinity for anti-ITAG2b integrin (also known as CD41) antibody

authenticates their hematopoietic progenitor nature (Corbel, 2002). Indeed the periaortic region of the chicken embryo, dissociated into single cells and seeded in a semisolid medium, gave rise to 3-4 times more colonies (erythroid or macrophages) than the BM from a newly hatched animal (Cormier and Dieterlen-Lievre, 1988). In 1993, two groups, based on bird results, identified the aorta as a major site of HSC emergence in mice [START_REF] Medvinsky | An early pre-liver intraembryonic source of CFU-S in the developing mouse[END_REF]; [START_REF] Godin | Para aortic splanchnopleura from early mouse embryos contains B1a cell progenitors[END_REF]. Prior observations had made it possible to highlight the embryological origin of the aorta: between E7 and E8.5, the presumptive territory of the aorta corresponds to the splanchnopleural mesoderm located in the caudal region of the embryo [START_REF] Delassus | Circulation of Hematopoietic Progenitors in the Mouse Embryo[END_REF]. From E8.5, the splanchnopleure (Sp), located in the splanchnopleural mesenchyme and associated endoderm, evolves into a trunk and abdominal structure including the paired aortas, the subaortic mesenchyme, and the endoderm of the digestive tract, this structure is called Para-aortic Splanchnopleura (P-Sp) [START_REF] Godin | Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus[END_REF].

This P-Sp develops at E10.5-E11.5 to form the Aorte-Gonad-Mesonephros (AGM) region comprising the dorsal aorta (resulting of the merge of the paired aortas), its underlying mesenchyme, and the urogenital system [START_REF] Medvinsky | An early pre-liver intraembryonic source of CFU-S in the developing mouse[END_REF]. It is within this AGM that we can observe between E10 and E11.5 the budding of the clusters of round cells in the light of the aorta from the endothelium Figure 20.

These structures are similar to those observed in Chickens and are mainly observed at the ventral wall of the aorta. On rare occasions, however, hematopoietic buds are seen in the vitelline arteries or in the dorsal position of the aorta, the latter marking here a difference from the situation observed in the bird [START_REF] De Bruijn | Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo[END_REF].

Evidence of the aortic origin of the final HSCs was provided through experiments of P-Sp or YS transplants taken prior to the establishment of circulation between the YS and the embryo under the SCID mouse renal capsule [START_REF] Godin | Para aortic splanchnopleura from early mouse embryos contains B1a cell progenitors[END_REF].

The authors observed that only the aortic region allowed a reconstruction of the lymphoid lineage of the host. At the same time, another group showed that the AGM carries a CFU-S activity initiated by E9 and which peaks at E10 and then declines.

Concurrently, CFU-S activity increases in the fetal liver, suggesting that hematopoietic progenitors emerging at the aorta subsequently colonize the fetal liver [START_REF] Medvinsky | An early pre-liver intraembryonic source of CFU-S in the developing mouse[END_REF].

The first progenitors are detected in the Sp while the circulation between the YS and the embryo is already established, which does not allow us to conclude on the true site of emergence of the aortic HSC. The experiments conducted by [START_REF] Delassus | Circulation of Hematopoietic Progenitors in the Mouse Embryo[END_REF] were able to respond to this question: the authors took before the establishment of the blood circulation, the YS, and the Sp and separately placed them in organotypic culture for 2 to 4 days before testing their hematopoietic potential [START_REF] Delassus | Circulation of Hematopoietic Progenitors in the Mouse Embryo[END_REF]. The authors show that before establishing blood flow between the YS and the embryo, Sp contains progenitors capable of producing B and T lymphocytes. Experiments conducted by E. Dzierzak and A. Medvinsky in the same year complement these results [START_REF] Medvinsky | Definitive Hematopoiesis Is Autonomously Initiated by the AGM Region[END_REF]. The authors show that cells from the AGM of mice at E11 are able to reconstitute the different hematopoietic blood lineages in irradiated adult mice, and are therefore capable of self-renewal. Similar experiments conducted prior to the establishment of blood flow between the embryo and the YS were conducted in 2001 and confirmed that HSCs of the AGM are produced in this tissue [START_REF] Cumano | Intraembryonic, but Not Yolk Sac Hematopoietic Precursors, Isolated before Circulation, Provide Long-Term Multilineage Reconstitution[END_REF]. Whereas the culture of YS alone gives rise only to erythromyeloid progenitors that have reduced self-renew potential.

The embryo grown in the absence of YS and Sp has no hematopoietic activity:

the intra-embryonic hematopoietic potential is therefore specifically restricted to the aortic region. Recent experiments with genetically marked YS transplants in mice showed that this tissue is capable of generating lymphocyte B progenitors, in very limited quantities: less than 1% [START_REF] Sugiyama | B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency[END_REF]. However, no contribution of the YS to the T lymphoid population has been demonstrated: the contribution of the YS to adult hematopoiesis in the mouse, if it exists, is very limited.

The aortic hematopoiesis was subsequently slightly better characterized: 500 to 1000 progenitors of which only a dozen HSCs are generated between E8.5 and E13 in the AGM. The peak production is detected at E10.5-E11, and the quantity in the aortic region then decreases to E12.5-E13 [START_REF] Godin | The hare and the tortoise: an embryonic haematopoietic race[END_REF][START_REF] Godin | Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus[END_REF][START_REF] Godin | Stem Cell Emergence and Hemopoietic Activity Are Incompatible in Mouse Intraembryonic Sites[END_REF]. The aortic region is mainly dedicated to the production of HSCs, multipotent progenitors capable of long-term self-renewal, the expansion and differentiation of these progenitors taking place in other hematopoietic sites, including the fetal liver that becomes a major organ of hematopoietic expansion and differentiation [START_REF] Ema | Expansion of hematopoietic stem cells in the developing liver of a mouse embryo[END_REF][START_REF] Godin | Stem Cell Emergence and Hemopoietic Activity Are Incompatible in Mouse Intraembryonic Sites[END_REF].

b. Amphibians aorta

In amphibians, removal of the ventral blood island resulted in the absence of circulating red blood cells. The origin of adult progenitors was hence first exclusively attributed to ventral blood island (VBI) [START_REF] Federici | Recherches experimentales sur les potentialités de l'îlot sanguin chez l'embryon Rana fusca[END_REF]. This hypothesis was then invalidated in the amphibian Rana pipiens by transplanting various territories subsequently identified according to their ploidy difference (Hollyfeild, 1966). These experiments first showed the existence of an alternative source of progenitors. When the cytogenetically labelled VBI was transplanted to the neurula stage, no larval erythrocyte came from the graft. This approach then made it possible to locate an alternative source of progenitors: if the dorsal mesodermal territory containing the pronephros is transplanted, erythrocytes from the graft are detected in the larva and in adults indicating that this region could be at the origin of the adult hematopoietic system. In agreement with this hypothesis, the lymphoid population of the larva is also derived from the dorsal region [START_REF] Turpen | The early ontogeny of hematopoietic cells studied by grafting cytogenetically labeled tissue anlagen: Localization of a prospective stem cell compartment[END_REF][START_REF] Turpen | PERITONEAL EXUDATE IN LARVAL RANA PIPIENS CONTAINS CELLS THAT ARE EMBRYOLOGICALLY DERIVED FROM DORSAL LATERAL[END_REF]. The emergence of HSCs from the dorsal region was then demonstrated in Xenopus [START_REF] Tompkins | Origin of hemopoietic stem cells in amphibian ontogeny[END_REF]. The activity of the hematopoietic region is characterized by the presence of HCs in the pronephric tubules, the aorta and the cardinal veins [START_REF] Turpen | The early ontogeny of hematopoietic cells studied by grafting cytogenetically labeled tissue anlagen: Localization of a prospective stem cell compartment[END_REF]. The origin of these cells was precisely located in the Dorsal Lateral Plate (DLP) [START_REF] Turpen | Ontogeny of hematopoietic cells in Rana pipiens: precursor cell migration during embryogenesis[END_REF][START_REF] Kau | Dual contribution of embryonic ventral blood island and dorsal lateral plate mesoderm during ontogeny of hemopoietic cells in Xenopus laevis[END_REF]. Data indicate that the DLP produces SCs contributing to larval and adult hematopoiesis. In particular, this territory participates to definitive erythropoiesis, thymic and hepatic hematopoietic activities [START_REF] Bechtold | Differential stem cell contributions to thymocyte succession during development of Xenopus laevis[END_REF]; [START_REF] Kau | Dual contribution of embryonic ventral blood island and dorsal lateral plate mesoderm during ontogeny of hemopoietic cells in Xenopus laevis[END_REF]Maneo et al., 1985a, b;Chen & Turpen, 1995). These results were later confirmed by two groups using dye injection (rhodamine dextran, GFP or βgalactosidase) into blastomeres and the follow up of the blastomere progeny (Ciau-Uitz et al., 2000;[START_REF] Lane | Primitive blood origins[END_REF].

In particular, R. Patient's group was able to map the origin of different hematopoietic progenitors in stage 32 blastula cells. Regarding the ISV, the authors showed that the anterior part of the structure comes from blastomeres C1 and D1 while the posterior part comes from blastomere D4 (Ciau-Uitz et al., 2000). This study also highlighted that the dorsolateral plate (DLP) and the VBI lineages are already separated at a very early stage: only the C3 dorsal blastomere contributes to the formation of the DLP and the emergence of HCs in the aortic region of the embryo Figure 21. Nevertheless, a more recent study suggests that embryonic and adult lineages could have a common origin in Xenopus [START_REF] Lane | Primitive and Definitive Blood Share a Common Origin in Xenopus: A Comparison of Lineage Techniques Used to Construct Fate Maps[END_REF].

c. Zebrafish aorta

In developmental biology, and for the past two decades, the zebrafish model (Danio rerio) has been increasingly used for the study of HSCs due to its many advantages. In the same way as the Xenopus embryo, the fertilization of the zebrafish is external and almost 200 eggs are obtained each spawning. The body of these embryos is translucent, which facilitates phenotypic observations, especially when they are coupled with fluorescent markers. Finally, this model is particularly powerful for conducting genetic analyses, notably through the realization of large-scale screens and obtaining mutating lines.

The establishment of the hematopoietic system in the zebrafish is accomplished as in the avian model, via two waves, one primitive and the other definitive emerging from two distinct sites. The primitive wave occurs in the Inner Cell Mass (ICM) equivalent to the YS of birds. When the heart begins to beat (26 to 28 hours post-fertilization) erythrocytes and endothelial precursors within the ICM depart in circulation. At the same time, macrophages and granulocytes differentiate in an anterior area named Rostral Blood Island RBI [START_REF] Herbomel | Ontogeny and behaviour of early macrophages in zebrafish[END_REF][START_REF] Lieschke | Zebrafish SPI-1 (PU.1) Marks a Site of Myeloid Development Independent of Primitive Erythropoiesis: Implications for Axial Patterning[END_REF].

Starting 26 hours post-fertilization, definitive hematopoiesis is initiated in the aortic region. Hematopoietic genes (runx1, c-myb) start to be expressed in the aortic endothelium reminding the situation observed in the bird [START_REF] Thompson | TheclocheandspadetailGenes Differentially Affect Hematopoiesis and Vasculogenesis[END_REF] In 2002, runx1, the key gene in HSCs specification in vertebrates was described in zebrafish [START_REF] Kalev-Zylinska | Runx1 in zebrafish hematopoiesis[END_REF]. The hematopoietic potential of the aortic region was revealed in 2006. The group of P. Herbomel was able to establish that progenitors from the aortic region migrated via blood flow to an area called Caudal Hematopoietic Tissue (CHT), a probable equivalent of the para-aortic foci of birds, or the fetal liver of the mouse before colonizing the final hematopoietic organs [START_REF] Murayama | Tracing Hematopoietic Precursor Migration to Successive Hematopoietic Organs during Zebrafish Development[END_REF].

The first lymphoid progenitors were detected at E3 in the thymus. At E5, they are found in the pronephros, equivalent of the BM [START_REF] Zapata | Ultrastructural study of the teleost fish kidney[END_REF] During its formation, the coelom divides the lateral mesoderm into two layers: a dorsal one designated as the somatopleure comprises the somatopleural mesoderm associated with the ectoderm; a ventral one designated as the splanchnopleure comprises the splanchnopleural mesoderm associated with the endoderm.

Hematopoietic cells and a cohort of endothelial progenitors arise from the splanchnopleure. The blood islands are formed from the extra-embryonic splanchnopleural mesoderm and associate endothelial and hematopoietic progenitors. In the embryo proper, the splanchnopleural mesoderm gives rise to angioblasts which form capillaries that will merge to form a vascular network closely associated with the endoderm. The vascular system of the viscera and the aorta are formed this way. This origin of blood and endothelium in the different embryonic tissues was demonstrated by quail/chicken grafts experiments followed by QH1 (a marker of endothelial and hematopoietic quail cells) staining Figure 24. Results differed according to the germ layer constitution of the grafted rudiments. In the case of the limb buds and body wall, ECs from the host invaded the graft through an angiogenic process. Hematopoietic progenitors from the host also colonized the grafted BM. In contrast, rudiments of internal organs provided their own contingent of endothelial precursors, a process termed vasculogenesis. Nevertheless, HCs in these organs were all derived from the host [START_REF] Pardanaud | Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny[END_REF].

Seeking the origins of ECs Pardanaud and co-workers traced the distribution of QH1 + cells after replacement of the last somite or heterotopic grafts of lateral plate mesoderm. When the authors grafted quail somitic tissue on a chicken host, they found QH1 + cells in the endothelium of somatopleural derivatives, i.e the body of the embryo, limbs and renal endothelium. When the splanchnopleural mesoderm was grafted, quail QH1 + cells contributed to the formation of the vascular system of the viscera as well as to the ventral endothelium of the aorta [START_REF] Pardanaud | Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis[END_REF].

The results lead to the conclusion that the embryo becomes vascularized by endothelial precursors from two distinct regions, splanchnopleural mesoderm and paraxial mesoderm i.e., somites. The territories respectively vascularized are complementary, precursors from the paraxial mesoderm occupy the body wall and kidney, i.e., they settle along with the other paraxial mesoderm derivatives and colonize the somatopleure. Interestingly, ECs originating from the somites are barred to contribute to vascularization of the internal organs and from initially integrating the aortic floor thus never giving rise to hematopoiesis. ECs originating from the splanchnopleural mesoderm colonize internal organs and contribute to hematopoiesis [START_REF] Pardanaud | Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis[END_REF]. Finally, the endothelial and hematopoietic potential of these two mesodermic tissues can be flip-flopped by prior culturing them with endoderm or ectoderm or by treatment with several growth factors [START_REF] Pardanaud | Manipulation of angiopoietc/hematopoietic potentials in the avian embryo[END_REF]. The endoderm induced a hemangiopoietic potential in the associated mesoderm. Indeed, the association of somatopleural mesoderm with endoderm promoted the 'ventral homing' and the production of hemopoietic cells from mesoderm not normally endowed with this potential. The hemangiopoietic induction by endoderm could be mimicked by VEGF, bFGF, and TGFβ1. In contrast, contact with ectoderm or EGF/TGFα treatments totally abrogated the hemangiopoietic capacity of the splanchnopleural mesoderm, which produced pure angioblasts with no 'ventral homing' behavior.

Given the difficulties encountered during in vitro mouse embryo cultures, an interspecific grafting approach has been used to demonstrate the endothelial potential of different mesodermal regions in mice (Fontaine-Perus and Jarno, 1995). Mouse somites transplanted orthotopically in a chicken embryo produce ECs that colonize the neural tube, limbs, mesonephros, and roof of the aorta (Ambler et al., 2001).

The splanchnopleural origin of hematopoietic precursors may extend to other sites of embryonic hematopoiesis. In fact, the yolk sac progenitors emerge from the splanchnopleural layer of the lateral plate, and the mesoderm involved in the formation of the allantois also corresponds to mesoderm associated with endoderm, therefore having a splanchnopleural identity [START_REF] Caprioli | Blood-borne seeding by hematopoietic and endothelial precursors from the allantois[END_REF][START_REF] Caprioli | Hemangioblast commitment in the avian allantois: cellular and molecular aspects[END_REF].

Interspecific grafting experiments have shown that, as soon as the coelom is formed, somatopleural and splanchnopleural mesoderms have different potentials [START_REF] Pardanaud | Emergence of endothelial and hemopoietic cells in the avian embryo[END_REF]. The hematopoietic potential would, therefore, be restricted to ECs of splanchnopleural origin. origin for the ECs that form the dorsal embryonic aorta [START_REF] Pardanaud | Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis[END_REF].

Ventral cells with a hemogenic potential are of splanchnopleural origin whereas those in the dorsal position have a somitic origin. This demonstration was confirmed and completed in the team (Pouget et al., 2006;[START_REF] Pouget | Sclerotomal origin of vascular smooth muscle cells and pericytes in the embryo[END_REF] somites) and analyzed the contribution of the graft to the formation of the aortic endothelium at different stages (Pouget et al., 2006). In sum, during their formation, the paired aortas are uniquely composed of ECs of splanchnopleural origin. From E2 in the bird embryo, while the aortas are still paired, somitic ECs migrate into the roof and sides of the aorta and replace the initial roof and sides of splanchnopleural origin and FOXC2 are shortly expressed by the forming smooth muscle cells and are downregulated as the cell layer forms [START_REF] Pouget | Sclerotomal origin of vascular smooth muscle cells and pericytes in the embryo[END_REF][START_REF] Wiegreffe | Sclerotomal origin of smooth muscle cells in the wall of the avian dorsal aorta[END_REF]. In the mouse, the study of FLK1 expression shows that in the dorsolateral position, a cell population exists expressing this characteristic marker of ECs [START_REF] Ema | Deletion of the selection cassette, but not cis -acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors[END_REF].

More recently, during orthotopic grafts of mouse somites in a chicken embryo followed by tracing the fate of ECs, there is also a gradual and complete replacement of the aortic endothelium starting with the roof of the vessel (Yvernogeau et al., 2012).

Smooth muscle cells forming the tunica of the aorta are also established in two successive stages: cells originating from the lateral plate and placed under the aorta differentiate under the vessel, then a second generation of smooth muscle cells of sclerotomous origin forms the final tunica around the aorta concomitantly with the replacement of ECs [START_REF] Wasteson | Developmental origin of smooth muscle cells in the descending aorta in mice[END_REF][START_REF] Wiegreffe | Remodeling of aortic smooth muscle during avian embryonic development[END_REF].

This set of experiments shows that it exists at the time of hematopoiesis a complete replacement of the aortic endothelium of splanchnopleural origin by ECs of somitic origin. This total replacement suggests that all the aortic ECs of splanchnopleural origin have a hematopoietic destiny: the tissue origin seems to play a fundamental role in the hemogenic potential of the ECs. with several other studies in the mouse and human embryos, which could not find any HSC activity in the embryonic head (Muller et al., 1994;[START_REF] Kumaravelu | Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver[END_REF][START_REF] Ivanovs | Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonadmesonephros region[END_REF].

Ultimately, a study uncovered a hemogenic activity in the early cardiac endothelium in the mouse. The authors used Ncx1 -/-embryos that lack heartbeat and found a hematopoietic-forming potential in a subset of ECs expressing the CD41antigen. These CD41+ cells arise from Nkx2.5+ cells i.e., cardiac cells. This hematopoietic activity was detected in the E8.5-E9.5 embryo anticipating the AGM activity. The hematopoietic production resembles EMP produced in the yolk sac [START_REF] Nakano | Haemogenic endocardium contributes to transient definitive haematopoiesis[END_REF].

E. The Fetal liver

The fetal liver FL, of mesodermal and endodermal origin, is the major organ of amplification and differentiation of embryonic HSCs. For the moment, researchers showed that the FL is not capable of producing HSCs de novo and is simply colonized by circulating HCs of extrinsic origin starting E10-E10.5. [START_REF] Johnson | Role of stem cell migration in initiation of mouse foetal liver haemopoiesis[END_REF][START_REF] Houssaint | Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line[END_REF].

The FL is colonized by two successive waves of HCs: the first wave establish a connection between the YS and the embryo via the vitelline vessels and are mainly enriched with primitive and definitive hematopoietic precursors. Since the BM is not yet formed at the time the FL begins to be colonized, the primary hematopoietic role of the FL is to form mature erythrocytes from primitive and definitive YS-derived precursors to ensure the needs of the embryo. The FL contains "erythroblast islands" that also exist in the BM. Those islands consist of a central macrophage that extends cytoplasmic protrusions to a ring of surrounding erythroblast and are the site of production of all the erythrocytes (Chasis, 2006;[START_REF] Palis | No red cell is an island[END_REF] Figure 26.

The second wave of hematopoiesis in the FL begins at E11.5 and corresponds to populations enriched in hematopoietic progenitors/immature HSCs mainly from the AGM and the Placenta via the umbilical vessels (the second vascular network pathway of HCs). At this stage, FL HCs are capable of forming in vitro BFU-E and CFU-E colonies and are able to proliferate for several days and to generate definitive erythroid cells.

At E12.5, the CFU-S activity of the FL begins to be detected, when the AGM has practically no more activity of this kind. Between E12.5 and E15.5, the total number of definitive hematopoietic precursors/HSCs increases exponentially in the FL, doubling practically every day. The total number of myeloid and lymphoid B precursors sequentially increases till near-birth stage [START_REF] Barker | Development of the mouse hematopoietic system. II. Estimation of spleen and liver ?stem? cell number[END_REF][START_REF] Gunji | Support of Early B-Cell Differentiation in Mouse Fetal Liver by Stromal Cells and Interleukin-7[END_REF][START_REF] Paige | Differentiation of murine B cell precursors in agar culture. Frequency, surface marker analysis and requirements for growth of clonable pre-B cells[END_REF]. Concerning the number of T-lymphoid precursors, they reached a peak at E13 and then decline [START_REF] Ema | Development of T cell precursor activity in the murine fetal liver[END_REF]. Finally, the number of HSCs increases sharply to E16 (the stage in which the FL contains approximately 1,600) and then decreases gradually [START_REF] Ema | Expansion of hematopoietic stem cells in the developing liver of a mouse embryo[END_REF][START_REF] Gekas | The Placenta Is a Niche for Hematopoietic Stem Cells[END_REF][START_REF] Morrison | The purification and characterization of fetal liver hematopoietic stem cells[END_REF]. The results of Kurata et al. show that a small number of hematopoietic precursors subsist for up to five weeks after birth [START_REF] Kurata | Stem cell factor induces proliferation and differentiation of fetal progenitor cells in the mouse[END_REF], these results are correlated to Taniguchi et al. work which showed that the adult liver contains immature HCs capable of restoring the hematopoietic system of a lethally irradiated mouse [START_REF] Taniguchi | Presence of hematopoietic stem cells in the adult liver[END_REF]. Several studies have shown that the HSCs present in the FL possess a very strong proliferative capacity compared to the HSCs of the adult BM [START_REF] Micklem | Competitivein vivo proliferation of foetal and adult haematopoietic cells in lethally irradiated mice[END_REF]Rebel et al., 1996a a). However, the frequency of HSCs of FL at E14.5 is comparable to that of the adult BM (Rebel et al., 1996b). Assuming that the AGM is the only real site for the emergence of definitive HSCs and since the AGM at E12.5 is intended to contain only 3 definitive HSCs [START_REF] Kumaravelu | Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver[END_REF], it was however difficult to explain the large number of HSCs found in the FL at E14.5 only by the speed of cell division. Thus just as the study of the emergence of HSCs in the YS, researchers are looking for the emergence and amplification capacity of the Placenta.

F. Definitive hematopoietic organs

Starting E15.5, the frequency of the HSCs present in the FL begins to decrease [START_REF] Morrison | The purification and characterization of fetal liver hematopoietic stem cells[END_REF]. This decrease is due to the gradual migration of HCs/HSCs from FL to the definitive hematopoietic organs: the thymus, the spleen and finally the BM, where HSCs are maintained during a lifetime. These three organs possess specific microenvironments in which the lymphocyte T differentiation, lymphocyte B differentiation, and the maintenance/differentiation of the HSCs are supported respectively.

a. The thymus Since HCs capable of colonizing the thymus are present in the blood several days before they enter the thymic rudiment, the onset of stem cell inflow into the thymus is dependent upon maturation of the thymic epithelium, which acquires a specific "receptivity" for lymphocyte precursor cells at a precise stage of development.

In early thymic ontogeny, the "receptivity" for hematopoietic cells falls sharply and probably stops completely for a transient period.

Two teams showed that the thymic rudiments are colonized by lymphoid precursors from E10.5 [START_REF] Fontaine-Perus | Seeding of the 10-day mouse embryo thymic rudiment by lymphocyte precursors in vitro[END_REF]; [START_REF] Owen | Tissue interaction in the development of thymus lymphocytes[END_REF]. During Red blood cells are produced in the FL hence its red color. The maturation of erythroblasts takes place in erythroblastic islands. Each of them has a macrophage at its centre, in contact with a ring of erythroblasts, immature nucleated red blood cells.

Abbreviations: FL: Fetal Liver. Adapted from [START_REF] Palis | No red cell is an island[END_REF]Chasis et al 2006 the following days, the colonization of the thymus accelerated and the first mature lymphocytes appeared at E15. The E12-E16 thymus transplants show the existence of a second wave of thymic colonization. The resulting mature HCs are the only ones that will subsist in the thymus thereafter [START_REF] Mathieson | Cell Surface Antigen Expression on Thymocytes: Development and Phenotypic Differentiation of Intrathymic Subsets[END_REF].

b. The spleen

The spleen doesn't have the capacity to generate HSCs. It is colonized by

HSCs coming from the FL. The microenvironment of the spleen supports hematopoietic differentiation, but unlike the FL, it is not capable of amplifying HCs.

The first HCs to colonize the spleen are multipotent (lymphomyeloid progenitors) and are detectable starting E12.5-E13 [START_REF] Godin | Stem Cell Emergence and Hemopoietic Activity Are Incompatible in Mouse Intraembryonic Sites[END_REF]. The work of

Christensen et al. demonstrates, through hematopoietic reconstitution experiments,

that LT-HSCs are detectable starting E14.5 [START_REF] Christensen | Circulation and chemotaxis of fetal hematopoietic stem cells[END_REF]. The hematopoietic activity of the spleen then increases daily until E17.5 and remains detectable up to 2 weeks after birth. In cooperation with FL and BM, the spleen plays an important role in maintaining the homeostasis of the hematopoietic system [START_REF] Plett | Treatment of circulating CD34(+) cells with SDF-1alpha or anti-CXCR4 antibody enhances migration and NOD/SCID repopulating potential[END_REF].

c. The Bone Marrow

The first colonization of the BM occurs starting E15.5 by the progenitors followed by the colonization starting E17.5 by the HSCs [START_REF] Christensen | Circulation and chemotaxis of fetal hematopoietic stem cells[END_REF][START_REF] Delassus | Circulation of Hematopoietic Progenitors in the Mouse Embryo[END_REF]. As we discussed earlier for the thymus, the HSCs able to colonize the BM are present in the blood several days before the colonization, suggesting that the BM microenvironment needs certain maturation before receiving the HSCs. A cascade of chemotactic interactions governs the migration of progenitors / HSCs to the BM. The Stromal Cell-Derived Factor-1 (SDF-1) chemokine, also known as CXCL12, seems to play a particularly important role. Mice deficient for SDF-1 or its receptor CXCR4 (Chemokine Receptor type 4) fail to establish hematopoietic activity in BM although the FL develops normally [START_REF] Ara | Long-Term Hematopoietic Stem Cells Require Stromal Cell-Derived Factor-1 for Colonizing Bone Marrow during Ontogeny[END_REF][START_REF] Nagasawa | Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1[END_REF][START_REF] Zou | Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development[END_REF]. In addition, it has been shown that HSCs of the BM migrate selectively in vitro in response to SDF-1 [START_REF] Wright | Hematopoietic Stem Cells Are Uniquely Selective in Their Migratory Response to Chemokines[END_REF]. In humans, a study has shown that in response to SDF-1, CD34 + BM cells injected into lethally irradiated mice have an increased hematopoietic reconstitution potential [START_REF] Plett | Treatment of circulating CD34(+) cells with SDF-1alpha or anti-CXCR4 antibody enhances migration and NOD/SCID repopulating potential[END_REF]. Thus, SDF-1 plays an important role both as a chemokine [START_REF] Imai | Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow[END_REF], as an activator of HSC adhesion molecules [START_REF] Peled | The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow[END_REF][START_REF] Peled | The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34ϩ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice[END_REF] and as a molecule involved in the maintenance of embryonic HSCs when they arrived in a hematopoietic niche [START_REF] Kawabata | A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution[END_REF][START_REF] Ma | The Chemokine Receptor CXCR4 Is Required for the Retention of B Lineage and Granulocytic Precursors within the Bone Marrow Microenvironment[END_REF].

The establishment of human hematopoiesis

Several aspects of hematopoietic development of mice are conserved in humans Figure 13.Human HSCs have been extensively studied in newborns and adults through transplantation into immunodeficient mice (Conneally et al., 1997;[START_REF] Wang | Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay[END_REF], but much less in the embryo. Despite the current difficulties in conducting research on the human embryo, several teams have studied the AGM's HSCs, the FL, and the placenta both in vitro by LTC-IC tests, and in vivo through hematopoietic reconstructions in the NOD-SCID mouse. The study of several animal models is therefore essential to have an overview of the embryonic hematopoietic process during evolution, as well as to better understand these processes in human.

In Human as well as in mouse, hematopoiesis begins with a first wave of primitive HCs appearing prior to the introduction of blood circulation at the level of the blood islands of the YS, visible between E17-E18 and E24 [START_REF] Luckett | Origin and differentiation of the yolk sac and extraembryonic mesoderm in presomite human and rhesus monkey embryos[END_REF][START_REF] Tavian | Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo[END_REF]. This hematopoietic population is transient since it contains only progenitors with erythro-myeloid potential. However, several types of HCs were detectable in the YS later during development, and between E25 and E50, YS contained immature HCs capable of forming myeloid and erythroid colonies ex vivo [START_REF] Huyhn | Characterization of Hematopoietic Progenitors From Human Yolk Sacs and Embryos[END_REF]; [START_REF] Dommergues | Hematopoiesis in the human yolk sac: quantitation of erythroid and granulopoietic progenitors between 3.5 and 8 weeks of development[END_REF], suggesting that it still possesses hematopoietic activity in vivo at this time of embryonic development. The frequency of these precursors remains constant between E35 and E50, but no hematopoietic activity is detectable after E60.

Unlike the YS, the aortic region contains multilineage HCs (lymphoid and myeloid), and these cells were initially characterized ex vivo. Starting E28 of development, the embryo contains GMPs capable of forming hematopoietic colonies in clonogenic tests [START_REF] Huyhn | Characterization of Hematopoietic Progenitors From Human Yolk Sacs and Embryos[END_REF]) and these progenitors are CD34 + [START_REF] Huyhn | Characterization of Hematopoietic Progenitors From Human Yolk Sacs and Embryos[END_REF]. The work of Tavian et al. shows that these intra-embryonic hematopoietic progenitors are adjacent to the ventral wall of the aorta [START_REF] Tavian | Aorta-Associated CD34+ Hematopoietic Cells in the Early Human Embryo[END_REF] and can produce HCs for 4 to 5 weeks when cocultured with Sys-1 murine stromal cells [START_REF] Tavian | Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo[END_REF]. This population has a specific phenotypic profile (Lin - CD45 + CD34 + CD31 + ckit + CD38 -) and molecular profile (GATA-2 + GATA-3 + cmyb + Scl + flk1 + ) characteristic of HSCs [START_REF] Labastie | Molecular Identity of Hematopoietic Precursor Cells Emerging in the Human Embryo[END_REF][START_REF] Tavian | Aorta-Associated CD34+ Hematopoietic Cells in the Early Human Embryo[END_REF].

In 2001 the Péault team showed using culture explant followed by coculture on MS5 stromal cells, that hematopoietic progenitors are detected in the aorta region starting E19. Unlike the immature HCs of the YS, which can produce only myeloid cells, the aorta also contains cells capable of producing lymphoid HCs. Starting 2011, Ivanos et al tested the hematopoietic potential of these cells in vivo. Using the reconstitution assays, they showed that the AGM is the only site of definitive HSC emergence between E35 and E44. A single HSCs of the AGM is able to produce at least 300 HSCs on a period of 8 months [START_REF] Ivanovs | Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonadmesonephros region[END_REF].

The hematopoietic potential of the human placenta has been assessed using the flow cytometry analysis and the confocal microscopy [START_REF] Barcena | A New Role for the Human Placenta as a Hematopoietic Site Throughout Gestation[END_REF]. Researchers were able to demonstrate the existence, in the placenta, of a population CD34 + CD45 + of fetal origin expressing a combination of markers specific of hematopoietic progenitors and able to produce erythroid, myeloid and lymphoid B populations.

Therefore, they suggested that in human as well as in mouse, the placenta is a major hematopoietic site.

The colonization of the definitive hematopoietic organs starts with the colonization of the thymus, between wk 7 and 8.5 of fetal gestation, by the first lymphocyte T precursors [START_REF] Haynes | Early events in human T cell ontogeny. Phenotypic characterization and immunohistologic localization of T cell precursors in early human fetal tissues[END_REF]. Regarding the spleen, the first studies showed that it receives the first HCs between wk 18 and 19 [START_REF] Vellguth | The development of the human spleen. Ultrastructural studies in fetuses from the 14th to 24th week of gestation[END_REF]; [START_REF] Hann | Development of pluripotent hematopoietic progenitor cells in the human fetus[END_REF]. While new studies showed that the first erythrocytes are present in spleen starting Wk 11 or 12 [START_REF] Steiniger | Fetal and early post-natal development of the human spleen: from primordial arterial B cell lobules to a non-segmented organ[END_REF].

The BM, the principal hematopoietic organ of the adult mammals, starts to be colonized when the hematopoiesis in the YS is finished and the FL one is still present.

Using immunohistochemistry, Charbord et al, studied the establishment of hematopoiesis in the medullary cavities which begins during the 11 th SG [START_REF] Charbord | Early Ontogeny of the Human Marrow From Long Bones: An Immunohistochemical Study of Hematopoiesis and Its Microenvironment[END_REF]. The authors indicate that the CD15 + myeloid HCs are the first to differentiate, followed closely by erythrocytes. According to the ex vivo and in vivo results of the Péault team, the HSCs of the BM between 18 and 24 wk possess the phenotype thy-1 + Lin-CD34 + [START_REF] Baum | Isolation of a candidate human hematopoietic stem-cell population[END_REF].

55

The Endothelial to Hematopoietic Transition EHT

As described in the previous chapter, the AGM region is the primordial site of HSCs emergence. The hematopoietic region of the aorta is characterized systematically by the presence of clusters of round cells attached to the endothelial wall budding in the light of the vessel and this is the case of the different studied animal models Figure 20. This process is stereotyped in space and time and researchers aim in understanding the mechanisms governing the onset of the intra-aortic budding.

The Hemangioblast concept

Mapping studies have shown that the extraembryonic mesoderm comes from the posterior lateral plate (LP) [START_REF] Schoenwolf | Mesoderm movement and fate during avian gastrulation and neurulation[END_REF]; [START_REF] Psychoyos | Fates and migratory routes of primitive streak cells in the chick embryo[END_REF][START_REF] Kinder | The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo[END_REF]. It is within this extraembryonic mesoderm that the earliest signs of vertebrate hematopoiesis occur. Based on the pioneering work of Sabin, Maximov and others in birds, Murray coined the term hemangioblast to indicate the thickening of the mesoderm in the chick yolk sac, the mesodermal "masses" located at the sites where later the blood islands emerge [START_REF] Murray | The development in vitro of the blood of the early chick embryo[END_REF]. It is worth noting that Murray defines the hemangioblast as a population of cells, which, as a whole, gives rise to the blood and endothelium of the blood islands. At the time, he could not say whether extrinsic cues determine the fate of individual hemangioblast cells or whether inherently different precursors for the 2 lineages coexist within his hemangioblast. In birds, the mesoderm from this region colonized the area opaqua by extending laterally and anteriorly. The expression profile of the Fetal Liver Kinase FLK-1 (VEGFR2) has been described in detail during this process. FLK-1 is strongly expressed in cells exiting the posterior LP and marks forming blood islands in the yolk sac. When the islands mature, receptor expression becomes restricted to EC [START_REF] Dieterlen-Lièvre | Are intra-aortic hemopoietic cells derived from endothelial cells during ontogeny?[END_REF][START_REF] Eichmann | Two molecules related to the VEGF receptor are expressed in early endothelial cells during avian embryonic development[END_REF]. The earliest experimental proof of the existence of the hemangioblast was brought by the deletion of VEGFR2 by gene targeting. In the absence of VEGFR2, both endothelial and HCs were absent in homozygous null mice [START_REF] Shalaby | Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice[END_REF]. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages.

Moreover, clonal cultures of VEGFR2 + cells isolated from the posterior primitive streak of the avian embryo give rise to either a hematopoietic or an endothelial cell colony.

The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hematopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2 [START_REF] Eichmann | Liganddependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2[END_REF]. At the same time, a clonal mesodermal precursor for blood and endothelium was identified in Embryonic Stem (ES) cell differentiation cultures (Choi et al., 1998;[START_REF] Nishikawa | In Vitro Generation of Lymphohematopoietic Cells from Endothelial Cells Purified from Murine Embryos[END_REF]. Termed blast colony-forming cell (BL-CFC) by G. Keller and co-workers, this cell was thought to be the ex-vivo equivalent of the in vivo hemangioblast, implying a conceptual change, where "hemangioblast" no longer represents a population of cells, but a clonal bipotent progenitor for blood and endothelium. However, the work by Ema et al. suggests that this precursor may also undertake smooth muscle differentiation [START_REF] Ema | Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse[END_REF]. The BL-CFC expresses FLK1 [START_REF] Faloon | Basic fibroblast growth factor positively regulates hematopoietic development[END_REF] and the mesodermal marker Brachyury [START_REF] Fehling | Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation[END_REF] but no marker of blood or ECs (with the exception of FLK-1) [START_REF] Lancrin | Blood cell generation from the hemangioblast[END_REF]. In addition to these two markers, Runx1 [START_REF] North | Cbfa2 is required for the formation of intra-aortic hematopoietic clusters[END_REF], LIM Only domain 2 or LMO2 [START_REF] Manaia | Lmo2 and GATA-3 hemopoietic expression[END_REF] and Scl (Chung et al., 2002) have also been described as expressed by the hemangioblast.

FLK1 and Brachyury allowed Keller's group to finally track the hemangioblast from the primitive streak of the gastrulating E7-E7.5 mouse embryo [START_REF] Huber | Haemangioblast commitment is initiated in the primitive streak of the mouse embryo[END_REF] and showed that this cell is present in a very small number (33 per mouse) and for a very short period of time Figure 27. Using different approaches, the presence of hemangioblasts was also shown in vivo in the zebrafish [START_REF] Vogeli | A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula[END_REF], Drosophila [START_REF] Mandal | Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm[END_REF], mouse [START_REF] Huber | Haemangioblast commitment is initiated in the primitive streak of the mouse embryo[END_REF] and avian [START_REF] Weng | On Hemangioblasts in Chicken[END_REF] embryos. The percentage of hemangioblasts varies from one species to another and, probably, with the different tracking techniques used. Thus 12.5% of mesoderm cells in zebrafish gives rise to hemangioblasts, while only 7% have this fate in birds.

Despite these publications, the in vivo existence of the hemangioblast has been challenged by many studies. Fate mapping studies suggested that the endothelial and hematopoietic precursors of the yolk sac are specified at the pregastrula stage [START_REF] Kinder | The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo[END_REF]. ES cells injections into blastocysts followed by thorough clonal analysis failed to formally demonstrate the existence of the hemangioblast [START_REF] Ueno | Clonal Analysis of Mouse Development Reveals a Polyclonal Origin for Yolk Sac Blood Islands[END_REF].

More recently, an elegant in vivo clonal analysis conducted in the early developing mouse embryo found that early YS blood and endothelial lineages mostly derive from independent epiblast populations, specified before gastrulation (Padrón- [START_REF] Padrón-Barthe | Clonal analysis identifies hemogenic endothelium as the source of the blood-endothelial common lineage in the mouse embryo[END_REF] in keeping with [START_REF] Kinder | The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo[END_REF]. It is worth noting here that the definition of the hemangioblast raised mostly by Keller's group and some others is based on the potential of the cells ex vivo and not on their fate. It should also be noted that the last published experimental analysis (Padrón-Barthe et al., 2014) suffers drawbacks. One good example is the use of Tie2-Cre to track the hemangioblast. Tie2

is not expressed by the gastrulating mesoderm but is switched on at the onset of endothelial differentiation. Another one is the fact that Ueno and Weissmann claimed that HCs were derived from FLK1 -cells whereas ECs were generated from FLK1 + cells. This has been refuted by another study using a simpler and more efficient lineage tracing system demonstrating that indeed most, if not all, blood island HCs and adult bone marrow resident HCs are the progeny of FLK-1-expressing cells [START_REF] Lugus | Both primitive and definitive blood cells are derived from Flk-1+ mesoderm[END_REF]. Taken together the demonstration that hemangioblasts exist in vivo is still to be done and needs additional, more appropriate approaches.

The existence of the hemangioblast has also been investigated using human ES cell (hESC) cultures. hES cells form Embryonic Bodies (EBs) and produce hematopoietic progenitors [START_REF] Thomson | Embryonic Stem Cell Lines Derived from Human Blastocysts[END_REF][START_REF] Vodyanik | Human embryonic stem cellderived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential[END_REF][START_REF] Zambidis | Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development[END_REF]. The existence of a hemangioblast equivalent to that described in the mouse model has been demonstrated later. Placed on proper culture conditions, human EBs indeed produce a progenitor equivalent to murine BL-CFC, expressing FLK1 and which gives rise to cell colonies mixing erythrocytes and ECs. A human hemangioblast dependent of BMP4 and able to clonally generate both endothelial and HCs was first reported in 2007 [START_REF] Kennedy | Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures[END_REF] Figure 28. Another study using a slightly different approach revealed hemangioblasts that, when injected into an animal with diabetes or vascular injury, were able to contribute to tissue repair [START_REF] Lu | Generation of functional hemangioblasts from human embryonic stem cells[END_REF].

Two studies used the expression of the angiotensin-converting enzyme (CD133) to enrich in human hemangioblasts. Cultures of CD133+ cells in the presence of VEGF, stem cell growth factor and Flt3 ligand was shown to give rise to cells giving rise to dual endo-hematopoietic colonies in one-quarter of the cells [START_REF] Loges | Identification of the Adult Human Hemangioblast[END_REF].

CD133, in addition with the lack of CD45 expression and the presence of CD34, was demonstrated as a strong combination to isolate cells capable to give rise to endothelial and lymphohematopoietic cells from hES cells [START_REF] Zambidis | Expression of angiotensin-converting enzyme (CD143) identifies and regulates primitive hemangioblasts derived from human pluripotent stem cells[END_REF].

Blast cells giving rise to endo-hematopoietic colonies were shown to express low levels of class 1 antigen compared to their non-hemangioblast counterparts.

If the hemangioblast exists in vivo and is functional in giving rise to blood and endothelial progenies, it certainly contributes marginally to the formation of the blood system hence asking the question of the mechanisms of blood cell formation.

Figure 27: A model of hemangioblast development in the mouse embryo

The hemangioblast is a brachyury+ and FLK-1+ cell that arises in the primitive streak and migrates onto the yolk sac where it differentiates into hematopoietic (H), endothelial (E) and vascular smooth muscle (VSM) progenitor cells.

Hemogenic endothelium: a bunch of convincing arguments

The existence of a close link between endothelial and HCs has long been recognized. The formal demonstration that ECs were giving rise to blood was achieved using lineage tracing (Jaffredo et al., 1998;[START_REF] Zovein | Fate Tracing Reveals the Endothelial Origin of Hematopoietic Stem Cells[END_REF] and time-lapse imaging [START_REF] Bertrand | Haematopoietic stem cells derive directly from aortic endothelium during development[END_REF][START_REF] Boisset | In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium[END_REF][START_REF] Eilken | Continuous single-cell imaging of blood generation from haemogenic endothelium[END_REF]Kissa and Herbomel, 2010;Lam et al., 2010;[START_REF] Lancrin | The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage[END_REF]. These ECs endowed with the potential to give rise to blood were coined "hemogenic endothelium" [START_REF] Jordan | Evidence of hemogenic capacity of endothelium[END_REF]. This hemogenic endothelium gives rise to blood through an endothelial-to-hematopoietic transition (EHT) not preceded by asymmetric divisions (Kissa and Herbomel, 2010).

Hemogenic ECs display an endothelial-specific signature and morphology and is integrated into a vascular tree hence ensuring bona fide endothelial-specific functions.

These cells are also capable to give rise to blood cells by turning on a blood-specific program and turning off the endothelial-specific program. Hemogenic ECs have been reported in all the vertebrate species described to date [START_REF] Dieterlen-Lièvre | Are intra-aortic hemopoietic cells derived from endothelial cells during ontogeny?[END_REF].

Most of the data regarding the existence and the phenotypic and functional characterization of the hemogenic endothelium in vivo were collected on the mouse and zebrafish embryos. The hemogenic endothelium has long been associated with the production of HSCs in the aorta however, it also extends to non-HSC production and has been shown in the yolk sac and probably in the placenta.

Since primitive hematopoiesis occurs before the formation of the vasculature, it is unlikely that HCs derived from this primitive wave were generated from hemogenic endothelium. However, experimental evidence indicates that this primitive wave emerges from precursors expressing endothelial cell markers such as VE-Cadherin [START_REF] Fraser | Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherinϩ population[END_REF] CD31 and CD34 [START_REF] Ema | Deletion of the selection cassette, but not cis -acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors[END_REF]. These precursor cells are not organized into endothelial layers but rather grouped into cell masses. Lacaud and coworkers have proposed the term "angiogenic angioblast" for this immature, likely hemogenic ECs. EMPs emerging in the yolk sac at E8.5 arise at the time of yolk sac vascular plexus remodeling [START_REF] Marcelo | Regulation of Endothelial Cell Differentiation and Specification[END_REF][START_REF] Palis | Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse[END_REF]. There are also reports indicating the presence of c-Kit + or CD41 + cells clusters within the yolk sac early during development [START_REF] Ferkowicz | CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo[END_REF][START_REF] Li | Endothelial cells in the early murine yolk sac give rise to CD41-expressing hematopoietic cells[END_REF]Yokomizo et al., 2001).

In 1998, Nishikawa and colleagues demonstrated that E9.5 VE-Cadherin + CD45 -yolk sac cells were able to generate HCs in culture [START_REF] Nishikawa | In Vitro Generation of Lymphohematopoietic Cells from Endothelial Cells Purified from Murine Embryos[END_REF] hence bringing the first evidence of hemogenic endothelium in the mouse embryo. A recent precise morphological study indicated that EMPs arise from a rare population of c-Kit lo Runx1 + cells expressing endothelial markers detected from E8.5 until E10.

Interestingly these flat cells were shown to become cuboid and then to round, forming clusters. Although circulation initiates at E8.25, all EMPs remained attached as clusters to the vasculature until E9.5. The study also shows that both arterial identity and blood flow are dispensable for EMP emergence, a major difference regarding the aorta [START_REF] Frame | Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity: EMPs Arise from Arteriovenous Hemogenic Endothelia[END_REF]. In addition, it was shown that yolk sac endothelium gives rise to AA4.1 + CD19 + B220 lo-neg B progenitor cells that preferentially differentiate into innate type B-1 and marginal zone B cells but not into B-2 cells upon transplantation and can also give rise to T cell progenitors that colonize the thymus [START_REF] Yoshimoto | Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential[END_REF]. Interestingly in the aorta pan endothelial expression of the core binding factor ß (the non-DNA-binding partner of Runx1) in Tek (Tie2) expressing cells was shown sufficient to produce EMPs but not HSCs whereas HSCs are produced from Ly6Aexpressing ECs (Chen et al., 2011).

The first hematopoietic site described to date as harboring hemogenic ECs was the aorta and later on its associated arteries i.e., vitelline and umbilical arteries. The existence of the hemogenic endothelium in the aorta was first demonstrated in the avian embryo. Its easy accessibility allowed the use of intracardiac inoculation to tag vascular ECs and trace their progeny. Starting at E.2.5, Intra-aortic Hematopoietic cell Clusters (IAHCs) extended from the branchial arches to the pre-umbilical levels [START_REF] Yvernogeau | Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken[END_REF]. They developed as two longitudinal symmetric ridges according to a rostrocaudal gradient located on the floor of the aorta. By injecting 1,10

-dioctadecyl-3,3,30,30-tetramethyl indocarbocyanine perchlorate-labeled human acetylated low-density lipo-protein (AcLDL-DiI) 1 day before the emergence of IAHCs, the whole endothelium of the embryo was readily labeled. Human low-density lipoprotein (LDL) is a large protein complex (~500,000 Da) that binds to a specific receptor on the surface of vertebrate cells and delivers cholesterol via receptormediated endocytosis. ECs and macrophages express this specific receptor i.e., the scavenger receptor, and can hence endocytose human acetylated LDLs.

The following day, not only the vasculature but also the newly formed IAHCs identified by expression of CD45 were DiI + , confirming that IAHCs derived from the endothelium (Jaffredo et al., 1998) Figure 29. Similar results were obtained with nonreplicative retroviral vectors (Jaffredo et al., 2000) or liposome-delivered eukaryote expression vectors [START_REF] Bollerot | Core binding factor in the early avian embryo: cloning of Cbfbeta and combinatorial expression patterns with Runx1[END_REF]. The use of a long-term tracing method using non-replicative viruses, allowed to follow the fate of the infected ECs (Jaffredo et al., 2000). In addition, the authors showed that the clones obtained are either endothelial or hematopoietic, suggesting that the production of aortic hematopoietic progenitors would pass through a phenotypical switch but not by an asymmetric division of the ECs. Cells of the intra-aortic clusters no longer express FLK1 + or VE-Cadherin mRNA but acquired the expression of the pan leukocyte antigen CD45 but also of the transcription factors PU1 and c-MYB [START_REF] Richard | Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis[END_REF]. FLK1/VE-Cadherin and CD45 are mutually exclusive at the level of the aortic floor. No ECs are visible under the budding cells, suggesting that HCs are formed from ECs that have changed their identity to acquire a hematopoietic fate.

The cell tracing approach developed in the bird embryo was adapted to the mouse. Injections of AcLDL-DiI were performed at E10 in the circulation allowing to mark ECs. On sections, DiI staining was observed in the endothelial layer a few hours after AcLDL-DiI inoculation. Flow cytometry analysis showed that the DiI + cells expressed CD31 and CD34 but not CD45. The embryo was cultured in vitro for an additional period of 12 hours. Ac-LDL-DiI + , c-Kit + multipotent progenitors as well as erythroid progenitors were revealed using clonogenic tests suggesting that they derived from pre-existing ECs [START_REF] Sugiyama | Erythropoiesis from acetyl LDL incorporating endothelial cells at the preliver stage[END_REF]. The experimental design does not allow the authors to tell which EC i.e., yolk sac versus embryo, contributed to this production. The endothelial origin of HSCs from the aorta in mice has formally been

shown by using a temporally restricted endothelial cell tracing to selectively label the endothelium. Lineage tracing endothelium, via an inducible VE-cadherin Cre line, reveals that the aortic endothelium is capable of HSC emergence. The endothelial progeny migrates to the FL, and later to the BM and are capable of expansion, selfrenewal, and multilineage hematopoietic differentiation [START_REF] Zovein | Fate Tracing Reveals the Endothelial Origin of Hematopoietic Stem Cells[END_REF]. In addition, this study shows that the sub-aortic tissues are incapable of giving rise to hematopoiesis. Finally, the ultimate proof of hemogenic endothelium has been carried out with the use of time lapse-confocal imaging on living tissues or embryos. Thick slices made on living Ly-6A-GFP mouse embryos were imaged over a period of 15h and showed flat ly-6A-GFP + cells becoming round and bud into the lumen. Tissue staining with an anti-CD31 antibody showed that the budding cells are hematopoietic.

Identical conclusions were drawn using a CD41-YFP mouse strain [START_REF] Boisset | In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium[END_REF] Figure 30. These conclusions were recently confirmed by [START_REF] Eich | In vivo single cell analysis reveals Gata2 dynamics in cells transitioning to hematopoietic fate[END_REF] and extended to the molecular control of EHT by the transcription factor Gata-2.

The zebrafish model also conveyed undisputable evidence for the existence of hemogenic endothelium in the aorta. The use of high-resolution live imaging of ECs of the dorsal aorta in the zebrafish allowed to observe the precise steps leading to the formation of the first HSCs. Using the KDR promoter driving a fluorescent protein, [START_REF] Bertrand | Haematopoietic stem cells derive directly from aortic endothelium during development[END_REF] showed that a portion of HCs in the adult was stained indicating that their ancestors had undergone an endothelial step at an earlier stage of their development. Three independent groups used confocal microscopy to look at the aortic region of Kdr-GFP zebrafish embryos during different time points of embryonic development: 24.5-30 hours post fertilization (h.p.f.) (Lam et al., 2010), 30-38 h.p.f. [START_REF] Bertrand | Haematopoietic stem cells derive directly from aortic endothelium during development[END_REF] and 36-52 h.p.f. (Kissa and Herbomel, 2010). In all cases, the emergence of HCs from ventral ECs of the dorsal aorta was observed. This hematopoietic commitment coincided with CD41, Lmo2, c-myb or Runx1 expression (all being HSC markers). The cells bud in the sub-aortic mesenchyme and later access the circulation through the neighboring axial vein. It should be noted that IAHCs do not form in the lumen of the embryonic aorta of zebrafish due to the small size of the aorta.

The transition from ECs to HCs does not require cell division but rather cell shape transformations. Indeed, hemogenic ECs bend toward the mesenchyme and round up, leading to detachment from the neighboring ECs. Interestingly, the knock-down of Runx1 leads to the death of the emerging cells (Kissa and Herbomel, 2010). The zebrafish model therefore clearly shows that hematopoietic stem/ progenitor cells emerge from a polarized hemogenic endothelium located in the ventral part of the dorsal aorta.

Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo (A-B) 3 hours after inoculation. AcLDL-DiI (red) has been endocytosed by endothelial cells) No CD45+ cells in the aorta (B). (D-E) 20 hours after inoculation. AcLDLDiI has been taken up by CD45-endothelial cells of the aorta and CD45+ budding cells (arrows) as well as CD45+ cells in the dorsal mesentery (arrowheads).

Abbreviations: Ao, Aorta; E, endoderm; NT, Neural tube, W, Wolffian duct Photos from jaffredo et al 1998 Abbreviations: s, somites; nt, neural tube; u, urogenital ridge; ao, aorta; bw, body wall; hg,hind gut. Adapted from Boisset et al 2010, De Bruijn andDzierzak, 2017 Not all the ECs are endowed with a hemogenic potential. How could we identify them and to what extent do they differ from their non-hemogenic counterparts?

As there are no unique surface marker that allow for the phenotypic identification and isolation of the hemogenic endothelium, people have sought for different cell surface markers. CD31 (PECAM), CD34 and FLK1 are shared by both endothelial and HCs. Positive and negative markers with a more pronounced lineage specificity have been used to enrich in hemogenic ECs. CD144 (VE-Cadherin) was used as a positive marker with CD45 and Ter119 being excluded [START_REF] Fraser | Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherinϩ population[END_REF][START_REF] Nishikawa | In Vitro Generation of Lymphohematopoietic Cells from Endothelial Cells Purified from Murine Embryos[END_REF]. The markers used to isolate the hemogenic ECs have varied in stringency. For instance, in several papers, CD41 + progenitor cells were not excluded. Further dissection has included the use of integrin alpha4 (CD49d) [START_REF] Ogawa | Expression of alpha4-integrin defines the earliest precursor of hematopoietic cell lineage diverged from endothelial cells[END_REF]. Based on the expression of the transcription factor Runx1 (see chapter IV, 1-D page 76), only a subset of ECs in hematopoietic sites is thought to be hemogenic. However, when considering the aorta, Runx1 is not expressed until the formation of the sub-aortic mesenchyme that acts as a switch to induce Runx1 and to trigger the hematopoietic program [START_REF] Richard | Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis[END_REF]. In this respect, a silencing of Runx1 expression mediated by BMI was recently uncovered elucidating the blockade of Runx1 expression in the angioblasts [START_REF] Eliades | The Hemogenic Competence of Endothelial Progenitors Is Restricted by Runx1 Silencing during Embryonic Development[END_REF]. At the functional level, the hemogenic endothelium is characterized by displaying an endothelial phenotype and morphology and having the capacity to form HCs and endothelial tubules in culture (Nishikawa et al., 1998a b;[START_REF] Nishikawa | In Vitro Generation of Lymphohematopoietic Cells from Endothelial Cells Purified from Murine Embryos[END_REF]. In an effort to identify hemogenic endothelium in the mouse embryo, De Bruijn and co-workers used the Runx1+23 hematopoietic enhancer they identified (Bee et al., 2010;Nottingham et al., 2007) to generate reporter mice expressing either LacZ or GFP. These mice were used to sort +23-GFP+ hemogenic ECs that were probed for EHT potential and identified for their transcriptional program. They showed that initiation of the hematopoietic program is initiated earlier than expected and is associated with a decreasing endothelial program. Hemogenic ECs were detected from E8.5 until E9.5 the time point at which the hematopoietic program is initiated. At E10.5, no hemogenic EC was detected and all the Runx1+23 cells are hematopoietic (Swiers et al., 2013). Trying to tackle the problem from the hematopoietic side, Medvinsky and co-workers identified two distinct hematopoietic cell types within the embryonic dorsal aorta that they designated type I and type II pre-HSC. They showed that type II pre-HSCs give rise to HSCs. Type I and II pre-HSCs differ in their expression of several surface markers. Type I and II pre-HSCs have a VE-Cadh + CD41 lo CD45and VE-Cadh + CD41 lo CD45 + phenotype, respectively, and lack the capacity to form endothelial tubules and sheets in culture [START_REF] Rybtsov | Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region[END_REF][START_REF] Taoudi | Functional identification of the hematopoietic stem cell niche in the ventral domain of the embryonic dorsal aorta[END_REF]. In addition to the in vivo studies, the hemogenic potential of ECs was also probed ex vivo using different culture conditions.

The conditional extinction of the runx1 hematopoietic gene in the cells expressing Ve-Cadherin causes a phenotype similar to that observed in embryos Runx1-/-represented by a lack of hematopoietic and definitive hematopoietic progenitors budding [START_REF] Chen | Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter[END_REF]. The authors thus prove that the cells of the clusters are produced from the aortic endothelium that expresses runx1, and that runx1 is necessary for the transition between the ECs state and HCs state.

In vitro approaches in the human embryo.

Since no manipulation is ethically possible on the human embryo, all the approaches were made using either observation on sections or in vitro/ex vivo culture systems. Cortes et al., (1999) reported the presence of FLK1+CD34-cells likely to develop into endothelial precursors in the mesoderm of the early human embryo at the beginning of the 4th week of gestation. CD34 which marks human ECs at 27 days of gestation has been shown to label IAHCs [START_REF] Tavian | Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo[END_REF]. CD34+CD45-cells purified from the 28-44 gestational days were shown to give rise to myeloid, lymphoid and NK cell populations following co-culture on stromal cells (Oberlin et al., 2002).

However, based on the assumption above it is unlikely that these populations were composed of pure ECs.

Since much of the past work has considered the hemangioblast as a source for hematopoietic progenitors and stem cells, several studies have reconciled this view and proposed that the hemogenic endothelium was the progeny of the hemangioblast.

The pioneering work by Bhatia and co-workers identified a population of primitive ECs that express CD31, FLK1, and Ve-Cadh that was able to give rise to both ECs and HCs. Despite the fact that the authors designated these cells as hemangioblast, they have all the characteristics of ECs endowed with a hemogenic potential [START_REF] Wang | Endothelial and Hematopoietic Cell Fate of Human Embryonic Stem Cells Originates from Primitive Endothelium with Hemangioblastic Properties[END_REF]. Furthermore, using the ES murine cells, several groups demonstrated that BL-CFC generated blood progenitors through a transient and specialized population of hemogenic ECs (Dieterlen-Lièvre and Jaffredo, 2009;[START_REF] Eilken | Continuous single-cell imaging of blood generation from haemogenic endothelium[END_REF]Lancrin et al., 2009;[START_REF] Pearson | The Sequential Expression of CD40 and Icam2 Defines Progressive Steps in the Formation of Blood Precursors from the Mesoderm Germ Layer[END_REF] for review [START_REF] Dieterlen-Lièvre | Decoding the hemogenic endothelium in mammals[END_REF]. These ECs form clusters from which blood cells emerge [START_REF] Eilken | Continuous single-cell imaging of blood generation from haemogenic endothelium[END_REF][START_REF] Lancrin | The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage[END_REF] Figure 31. showed that FLK1 + cells give rise to VE-Cadh + cells and differentiate into CD41 + and finally CD45 + HCs. Several studies using human ES cells now confirmed this mouse model (Choi et al., 2012;[START_REF] Kennedy | Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures[END_REF][START_REF] Kennedy | T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures[END_REF][START_REF] Wang | Endothelial and Hematopoietic Cell Fate of Human Embryonic Stem Cells Originates from Primitive Endothelium with Hemangioblastic Properties[END_REF]. Examining the hematopoietic commitment using VE-Cadh and CD41 reporters, it was shown that there are several subsets of hemogenic ES cells in keeping with in vivo studies and that the quality of the hematopoiesis produced varies with time un culture [START_REF] Rafii | Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition[END_REF]. In an effort to produce engraftable HSCs, several groups have refined their differentiation protocol of either murine or human EC or iPS cells and in particular have avoided the use of serum in culture. Lacaud and Kousskof's groups produced hemogenic endothelium from mouse ES cells that were capable of giving rise to myeloid, erythroid and lymphoid lineages upon engraftment into immunocompromised recipients. Primary transplantation was successful up to 22

weeks, but secondary transplantation failed. In addition, they showed that the hemogenic ECs were extremely transient in culture [START_REF] Pearson | In Vivo Repopulating Activity Emerges at the Onset of Hematopoietic Specification during Embryonic Stem Cell Differentiation[END_REF]. Another strategy to generate hemogenic endothelium is to reprogram ES cell-derived cell types of differentiated cells. Overexpression of SOX17, a transcription factor involved in EC differentiation, in CD34 + CD43 -cells generated hemogenic ECs with hematopoietic potential. Sox17 was shown to play a key role in priming hemogenic potential in ECs [START_REF] Nakajima-Takagi | Role of SOX17 in hematopoietic development from human embryonic stem cells[END_REF]. Another strategy consisted of screening an ensemble of 27 transcription factors for their capacity to induce hematopoiesis from human pluripotent stem cells. The authors identified two optimal combinations of TFs capable of inducing distinct, robust hematopoietic programs from pluripotent stem cells: panmyeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1).

Interestingly, both transcription factor combinations directly induced hemogenic ECs, which subsequently transformed into blood progenitors with a distinct spectrum of hematopoietic differentiation [START_REF] Elcheva | Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators[END_REF].

Another aspect consists in generating hemogenic ECs directly from differentiated cells through direct reprogramming. The team of K. Moore showed that the overexpression of four transcription factors, Gata2, Gfi1b, c-Fos, and Etv6 within fibroblasts induced a hemogenic endothelial cell fate in murine fibroblasts that subsequently produced hematopoietic precursor cells that express CD34, SCA1 and PROMININ1 within a global endothelial program [START_REF] Pereira | Induction of a Hemogenic Program in Mouse Fibroblasts[END_REF]. However, these cells displayed poor multilineage potential in vitro. This hemogenic phenotype completed by the absence of CD45 expression was later shown to be present in ECs of the placenta. When grafted into irradiated mice following co-culture with stromal cells, these hemogenic ECs engraft into recipient mice and give rise to primary and secondary recipients [START_REF] Pereira | Hematopoietic Reprogramming In Vitro Informs In Vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells[END_REF]. Lacaud and co-workers showed that the ectopic expression of five transcription factors (Erg, Gata2, Lmo2, Runx1c, and Scl) reprogrammed fibroblasts to hematopoietic progenitors with erythrocyte, granulocyte, macrophage, and megakaryocyte potential. This reprogramming passes through a hemogenic endothelium state, that in contrast to Peirera et al. gives rise to robust clonogenic potential [START_REF] Batta | Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells[END_REF]. In recent reports, Daley and co-workers showed that seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) that are sufficient to convert hemogenic endothelium into hematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. These factors are transferred into so-called CD34 + hemogenic ECs and are likely to mimic molecular events occurring within the dorsal embryonic aorta [START_REF] Sugimura | Haematopoietic stem and progenitor cells from human pluripotent stem cells[END_REF]. In another study published in the same issue of Nature, Rafii and colleagues reported constitutive expression of transcription factors FOSB, GFI1, RUNX1, and SPI1 within adult ECs and cocultivation of these cells with vascular niche induce a hemogenic state characterized by the expression of RUNX1

and the subsequent expression of CD45. When transplanted into immunocompromised recipients, these cells were capable of primary and secondary engraftment [START_REF] Lis | Conversion of adult endothelium to immunocompetent haematopoietic stem cells[END_REF]. Interestingly, this protocol is a refinement of a previous study published in 2014 identifying the transcription factors and the requirement for a vascular niche [START_REF] Sandler | Reprogramming human endothelial cells to haematopoietic cells requires vascular induction[END_REF].

SubAortic Patches

At E4 in the bird embryo, IAHCs disappeared from the aortic floor. Instead of being produced into the lumen, some cells accumulate in the inner side of the aorta being also the progeny of the aortic endothelium (Jaffredo et al., 2000). Those cells emitted toward the inner side of the aorta would multiply in the mesentery giving rise to the para-aortic foci (Dieterlen-Livre and [START_REF] Dieterlen-Livre | Diffuse lntraembryonic Hemopoiesis in Normal and Chimeric Avian Development[END_REF]Jaffredo et al., 2000) Figure 32. These para-aortic foci contain cells expressing the surface molecules α 2 β 1 integrin, c-kit, thrombomucin/MEP21, HEMCAM, and chL12. Importantly, the presence of these antigens was found to correlate with the recolonization of the recipient thymus following intrathymic cell transfers. These intra-embryonic cells were also found to express the Ikaros transcription factor, the molecular function of which is considered to be prerequisite for embryonic lymphoid development [START_REF] Lampisuo | Characterization of prethymic progenitors within the chicken embryo[END_REF]. In the mouse embryo, Godin and co-workers described the presence of cells localized in the sub-aortic space that express c-kit, AA4.1 [START_REF] Petrenko | The Molecular Characterization of the Fetal Stem Cell Marker AA4[END_REF], CD41 and Gata3 [START_REF] Manaia | Lmo2 and GATA-3 hemopoietic expression[END_REF]Figure 33 but lack the expression of FLK1 and CD45.

They designated these cells as sub-aortic patches and proposed they were at the origin of IAHCs [START_REF] Bertrand | Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin[END_REF]. However, in accordance with the results of VeCad + CD45 + present in aortic clusters at E11.5 originates from a VeCad + CD45 - CD41 + E10.5 population localized not only in the aortic endothelium but also in the subaortic mesenchyme, relaying again the debate on a potential mesenchymal origin of HSCs [START_REF] Rybtsov | Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region[END_REF]. Then, one study has shown that the ubiquitin ligase mind bomb-1, a regulator of the Notch pathway, is implicated in the hematopoiesis of SAPs [START_REF] Yoon | Mind Bomb 1-Expressing Intermediate Progenitors Generate Notch Signaling to Maintain Radial Glial Cells[END_REF]. Although not totally solved these Gata3-positive patches were shown to be part of the AGM microenvironment and belonging to the sympathetic nervous system and catecholamine-producing cells [START_REF] Fitch | Signaling from the Sympathetic Nervous System Regulates Hematopoietic Stem Cell Emergence during Embryogenesis[END_REF]. [START_REF] Millauer | High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis[END_REF]. Flk1 is expressed by cells dedicated to giving the cardiac tissue at the level of the primitive streak and is described as a marker of endothelial precursors [START_REF] Yamaguchi | flk-1, an fltrelated receptor tyrosine kinase is an early marker for endothelial cell precursors[END_REF]. Flk1 knock out shows that this receptor is essential for both the development of HCs and vasculogenesis. Embryos invalidated for the Flk1 gene present a total absence of ECs and HCs and die in utero between E 8.5 and E 9.5. In this respect, the Flk1 knockout phenotype has been considered as the first functional proof of the existence of the hemangioblast [START_REF] Eichmann | Liganddependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2[END_REF][START_REF] Shalaby | Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice[END_REF][START_REF] Shalaby | A Requirement for Flk1 in Primitive and Definitive Hematopoiesis and Vasculogenesis[END_REF]. Later in development, Flk1 is expressed at the base of IAHCs and in the adjacent ECs at the protein level for the chicken (Jaffredo et al., 1998) and mouse [START_REF] Yokomizo | Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos[END_REF], and at the mRNA level for the human embryo [START_REF] Labastie | Molecular Identity of Hematopoietic Precursor Cells Emerging in the Human Embryo[END_REF]. The expression profiles of Flk1 and CD45 are therefore mutually exclusive in the aorta as previously shown by Nishikawa et al. with ES cells (Nishikawa et al., 1998b a).

Interestingly, Runx1 represses the expression of Flk1 contributing to orchestrate EHT.

In view of all these results, the Flk1 expression was attached to the hemangioblast.

The work of Fehling et al. on ES cells confirm this hypothesis [START_REF] Fehling | Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation[END_REF].

B. Stem Cell Leukemia Scl, Lmo2 and Lyl1

The SCL gene encoding for a bHLH domain transcription factor was initially identified by its involvement in a number of chromosomal translocations observed in leukemic T cells [START_REF] Begley | The SCL Gene: From Case Report to Critical Hematopoietic Regulator[END_REF][START_REF] Begley | The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNAbinding motif[END_REF]Chen et al., 1990).

This gene is expressed early in the blood islands and when differentiated, remains expressed in the hematopoietic cells and is detected very weakly in ECs [START_REF] Drake | Vasculogenesis in the day 6.5 to 9.5 mouse embryo[END_REF][START_REF] Elefanty | SCL Expression in the Mouse Embryo Detected With a Targeted lacZ Reporter Gene Demonstrates Its Localization to Hematopoietic[END_REF][START_REF] Minko | From mesoderm to blood islands: patterns of key molecules during yolk sac erythropoiesis[END_REF]. In the aortic region, SCL is expressed in the aortic endothelium prior to the onset of intra-aortic budding, and its expression appears to be strengthened at the level of the ventral endothelium. When the hematopoietic budding appears, SCL is strongly expressed and its expression in endothelial cells is low [START_REF] Jaffredo | Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells[END_REF]. Functional experiments in mice and zebrafish have helped to highlight the role of SCL during hematopoiesis. When the gene is invalidated in mice, the embryos die at E9,5 with a complete absence of hematopoiesis in the yolk sac [START_REF] Robb | Absence of yolk sac hematopoiesis from mice with a targeted disruption of the sci gene[END_REF]. Later on, the authors showed that SCL is indispensable for the development of all the hematopoietic lineages, primitive and definitive [START_REF] Porcher | The T Cell Leukemia Oncoprotein SCL/tal-1 Is Essential for Development of All Hematopoietic Lineages[END_REF][START_REF] Robb | The sci gene product is required for the generation of all hematopoietic lineages in the adult mouse[END_REF]. Although it was proposed that SCL would be secondarily required for remodeling of the primitive vascular network (D 'Souza et al., 2005;[START_REF] Elefanty | SCL Expression in the Mouse Embryo Detected With a Targeted lacZ Reporter Gene Demonstrates Its Localization to Hematopoietic[END_REF][START_REF] Visvader | Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development[END_REF], several reports indicate a crucial role for the specification of the hemogenic endothelium. In the absence of SCL in BL-CFC, all the hematopoietic lineages are, as expected, compromised but all ECs differentiation including hemogenic ECs is abrogated [START_REF] Lancrin | Blood cell generation from the hemangioblast[END_REF]. This is consistent with a report showing that SCL was required for the formation of VE-Cadherin+ cells from Flk1 + cells [START_REF] Endoh | SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation[END_REF]. Several years after, Mikkola and co-workers showed that SCL activated a network of transcription factors necessary for the specification of the hemogenic endothelium and showed a repressive role of SCL to limit the commitment of ectopic cardiomyocytes [START_REF] Van Handel | Scl represses cardiomyogenesis in prospective hemogenic endothelium and endocardium[END_REF]. On the other hand, it has been shown that the ectopic expression of SCL can restore the differentiation of all hematopoietic lineages in SCL - /-mouse embryos, suggesting a role in the differentiation of hemogenic progenitors [START_REF] Visvader | Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development[END_REF]. In addition, in zebrafish, morpholinos targeting of SCL blocks hematopoiesis and GATA1 expression without altering the expression of other hematopoietic markers such as GATA2 or Lmo2. The absence of hematopoiesis would not be due to the absence of progenitors but SCL would play a key role in hematopoietic differentiation [START_REF] Dooley | Zebrafish scl functions independently in hematopoietic and endothelial development[END_REF][START_REF] Patterson | Scl is required for dorsal aorta as well as blood formation in zebrafish embryos[END_REF]. Interestingly, SCL becomes dispensable at the end of hematopoietic development due to redundancy with another bHLH protein, Lyl1.

Lmo2 is also a transcription factor involved in lymphoid leukemia induced by chromosomal translocations [START_REF] Royer-Pokora | TTG-2, a new gene encoding a cysteine-rich protein with the LIM motif, is overexpressed in acute T-cell leukaemia with the t(11;14)(p13;q11)[END_REF]. Its expression in hematopoietic sites is similar to that of SCL although a little more extensive.

Invalidation of the gene in mice leads to the same phenotype as the one described for SCL [START_REF] Yamada | The oncogenic LIM-only transcription factor Lmo2 regulates angiogenesis but not vasculogenesis in mice[END_REF]. In vitro functional interaction experiments have shown that SCL and Lmo2 interact within of multimeric transcriptional complexes: through its LIM domain, Lmo2 plays a role in between SCL and GATA1 [START_REF] Wadman | The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/ NLI proteins[END_REF].

Studies to determine whether there was a synergy of action between the two transcription factors were performed in zebrafish: the combined overexpression of SCL and lmo2 have indeed shown that the presence of both factors is necessary to respecify a mesodermal territory of origin inducing the expression of endothelial and hematopoietic genes.

The lyl-1 gene also codes for a bHLH domain transcription factor and has been identified, along with SCL, as being implicated in some lymphoid leukemia in humans.

Although gene deletion in mice is not lethal, it results in a decrease in the number of B lymphocytes as well as immature progenitors, reducing their potential for reconstitution [START_REF] Capron | The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell function and B-cell differentiation[END_REF]. Analysis of the expression patterns at different stages shows that the SCL and lyl-1 expression overlap widely in hematopoietic sites in mice, particularly in the aortic endothelium and the fetal liver, suggesting possible cooperation with SCL in the regulation of hematopoiesis [START_REF] Giroux | lyl-1 and tal-1/scl, two genes encoding closely related bHLH transcription factors, display highly overlapping expression patterns during cardiovascular and hematopoietic ontogeny[END_REF]. Analysis of the regulation of the expression of lyl-1 as well as functional studies through the obtaining of SCL -/-and Lyl-1 -/-embryos yielded a number of responses to the respective roles of Lyl-1 and SCL in hematopoiesis: If both genes are regulated by the same molecular combinatorial, the SCL expression is initiated before that of lyl-1. In addition, Lyl-1 is unable to save the hematopoietic phenotype of ES SCL-/-cells, suggesting that these two transcription factors play a different role during hematopoiesis.

C. Gata transcription factors

The Gata protein family includes six highly conserved zinc finger transcription factors, of which three members (GATA-1, 2 and 3) are involved in hematopoiesis at different levels. GATA-1 and 2 are required very early for primitive hematopoiesis (Fujiwara et al., 2004). The expression patterns established in different species have the same characteristics: the expression of GATA-1 and 2 is detected very early in the mesodermal masses in the blood islands. During their formation, the expression of GATA-1 persists while that of GATA-2 gradually disappears from the most mature islands [START_REF] Minko | From mesoderm to blood islands: patterns of key molecules during yolk sac erythropoiesis[END_REF]; [START_REF] Silver | Initiation of murine embryonic erythropoiesis: a spatial analysis[END_REF][START_REF] Walmsley | Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands[END_REF][START_REF] Zon | Expression of GATA-binding proteins during embryonic development in Xenopus laevis[END_REF]. These observations suggest an early role for both genes in definitive erythropoiesis, which could be subsequently confirmed through GATA-1 invalidation experiments: erythroid cells are present in the blood islands, but their maturation is blocked, and they enter into apoptosis [START_REF] Fujiwara | Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1[END_REF]Fujiwara et al., 2004).

The in vivo deletion of GATA-2 leads to a reduced number of hematopoietic progenitors that normally develop [START_REF] Tsai | An early haematopoietic defect in mice lacking the transcription factor GATA-2[END_REF]. Thus, the Gata-2 factor would be required for the commitment to the erythropoietic pathway while GATA-1 would allow the maturation of erythroid progenitors. However, double mutant embryos have a lack of primitive erythrocytes in blood islands, suggesting a partially redundant role of two transcription factors (Fujiwara et al., 2004;[START_REF] Takahashi | GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies[END_REF]. In the aortic endothelium, GATA-2 appears in the ventral endothelium of the aorta at the same time as SCL and lmo2, at the time of fusion of the two pair aorta. The expression of GATA-2 is then strengthened in the budding cells the expression of Gata-3 is also initiated but in a lower manner [START_REF] Jaffredo | Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells[END_REF]. In 2004, a double role of Gata2 dosedependent could be shown in mice: it would be involved in the production and expansion of HSCs in the AGM as well as the proliferation stage of HSCs in the adult bone marrow [START_REF] Ling | GATA-2 Plays Two Functionally Distinct Roles during the Ontogeny of Hematopoietic Stem Cells[END_REF]. Notch1 specifically associated with the GATA2

promoter suggesting a role in the formation of HSPCs by the hemogenic ECs [START_REF] Robert-Moreno | RBPj{kappa}-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells[END_REF] but multiple roles for GATA2 at different levels has been demonstrated in culture using conditional GATA2 induction in BL-CFC. Conditional GATA2 deletion where CRE is under the control of the vascular-specific GATA2 enhancer leads to defect of HSCs in the fetal liver and in the bone marrow.

Interestingly, enforced expression of GATA2 in fibroblasts together with Gfi1b, Fos and ETV6 induces a hemogenic endothelium program in fibroblasts with the subsequent appearance of blood cells [START_REF] Pereira | Induction of a Hemogenic Program in Mouse Fibroblasts[END_REF]. Another study using Erg, Lmo2, Runx1c SCl and GATA2 reached to the same conclusions [START_REF] Batta | Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells[END_REF] Similar to Runx1 (see the Runx1 chapter), the requirement for GATA2 has been studied both in the context of ECs or HSPCs. Deletion of GATA2 in an endothelial i.e., VE-Cadherin-Cre, context leads to a complete loss of HSCs in the AGM whereas deletion in hematopoietic cells i.e., Vav-Cre impacts HSC survival [START_REF] De Pater | Gata2 is required for HSC generation and survival[END_REF]. In this respect, abrogation of the +9.5 GATA2 regulatory element completely abrogates IAHCs formation and HSC emergence from the AGM. Transcriptome analysis indicates that the +9.5 element regulates an ensemble of genes involved in hemogenic endothelium and HSCs [START_REF] Gao | Gata2 cis-element is required for hematopoietic stem cell generation in the mammalian embryo[END_REF]. Interestingly, the existence of two GATA2 orthologues in the zebrafish reveals a tight control of GATA2b, under the control of Notch, over the hemogenic endothelium and Runx1 expression and a broader role of GATA2a on the vascular endothelium [START_REF] Butko | Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo[END_REF]. Conditional expression of GATA2 in GATA2 knockout human embryonic stem cells under hematopoietic differentiation reveals that GATA2 is not required for the generation of EC or hemogenic ECs but is required for hemogenic ECs to proceed EHT [START_REF] Kang | GATA2 Is Dispensable for Specification of Hemogenic Endothelium but Promotes Endothelial-to-Hematopoietic Transition[END_REF]. Recently, time-lapse imaging of GATA2 expression within the aorta has uncovered the existence of a pulsatile expression in cells undergoing EHT. GATA2 haploinsufficiency alters the pulsatile pattern, reduces the number of EHT and reduces hematopoiesis [START_REF] Eich | In vivo single cell analysis reveals Gata2 dynamics in cells transitioning to hematopoietic fate[END_REF]. Concerning Gata3, it is expressed in adult and embryonic HSCs [START_REF] Bertrand | Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin[END_REF][START_REF] Frelin | GATA-3 regulates the self-renewal of longterm hematopoietic stem cells[END_REF] It is also involved in the development of T lymphocytes [START_REF] Ting | Transcription factor GATA-3 is required for development of the T-cell lineage[END_REF] and NK cells [START_REF] Vosshenrich | A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127[END_REF]. KO embryos for Gata3 die around E11.5 and exhibit abnormal fetal hematopoiesis [START_REF] Pandolfi | Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis[END_REF]. Nevertheless, the deletion of Gata3 under the control of pan-hematopoietic marker Vav1 does not affect the activity of HSC [START_REF] Buza-Vidas | GATA3 is redundant for maintenance and self-renewal of hematopoietic stem cells[END_REF]. However, another analysis showed that Gata3 was required for HSC maintenance by regulating their entry into the cell cycle.

D. The Runx1 transcription factor

Runt-related transcription factor 1 (RUNX1) also known as acute myeloid leukemia 1 protein (AML1) or core-binding factor subunit alpha- Runx1 needs a heterodimeric partner to bind DNA. CBFß has no DNA binding activity by itself but potentiates Runx1 binding to DNA and protects it from degradation by the ubiquitin-proteasome [START_REF] Wang | Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis[END_REF].

Runx1 knockout mice show a drastic decrease in the number of erythrocytes in fetal liver, and premature death at E12.5-E13.5 accompanied by large brain hemorrhages [START_REF] Wang | Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis[END_REF]. If primitive erythropoiesis is marginally affected (Yokomizo et al., 20010), no definitive hematopoietic progenitor and HSCs is detected in the Runx1-/-embryo and are severely reduced in CBFß-/-embryos [START_REF] Wang | Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis[END_REF]. Of the primitive lineages affected, the yolk sac -derived macrophages are absent from Runx1-/-embryos and from ES cell cultures [START_REF] Lacaud | Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro[END_REF]. These observations therefore suggest that Runx1 would play a key role in the induction of definitive hematopoietic progenitors at the level of the aortic endothelium.

In the embryo, Runx1 expression is found in all sites of hematopoiesis.

Strikingly, Runx1 expression is found in a subset of ECs in the vitelline and umbilical arteries, in the yolk sac, the placenta and the ventral aspect of the dorsal aorta in the mouse embryo. Runx1 is also found expressed in some peri-aortic tissue underneath the aorta. A polarized expression of Runx1 is also found conserved in all the vertebrate species described [START_REF] Bollerot | Core binding factor in the early avian embryo: cloning of Cbfbeta and combinatorial expression patterns with Runx1[END_REF]Ciau-Uitz et al., 2000;[START_REF] Kalev-Zylinska | Runx1 in zebrafish hematopoiesis[END_REF][START_REF] North | Cbfa2 is required for the formation of intra-aortic hematopoietic clusters[END_REF] giving a molecular credit to the existence of hemogenic endothelium. In the aorta, Runx1 knock-down abrogates the formation of the IAHCs [START_REF] North | Cbfa2 is required for the formation of intra-aortic hematopoietic clusters[END_REF]Yokomizo et al., 2001). In the Zebrafish, morpholino knock-down impedes IAHCs formation without affecting the aortic endothelium (Kalev-Zylinska, 2002); [START_REF] Burns | Hematopoietic stem cell fate is established by the Notch-Runx pathway[END_REF][START_REF] Gering | Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos[END_REF]. In contrast, in the adult, Runx1 deletion causes rather an expansion of HSCs and progenitors in the bone marrow [START_REF] Chen | Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter[END_REF]; [START_REF] Growney | Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype[END_REF] Vav gene regulatory sequences demonstrates that Runx1 is required for the EHT but is no longer required after hematopoietic cells were formed [START_REF] Chen | Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter[END_REF]. In line, retroviral transduction of Runx1 into cells from E11.5 AGM did not rescue hematopoiesis wile the same approach made at E9.5 did so [START_REF] Goyama | The transcriptionally active form of AML1 is required for hematopoietic rescue of the AML1-deficient embryonic para-aortic splanchnopleural (P-Sp) region[END_REF][START_REF] Mukouyama | The AML1 Transcription Factor Functions to Develop and Maintain Hematogenic Precursor Cells in the Embryonic Aorta-Gonad-Mesonephros Region[END_REF]. Restauration of Runx1 expression in ECs, using a Tekdriven construct showed that Runx1 expression is sufficient to rescue lymphoid, myeloid and HSCs and to prolong the life of the embryos until birth [START_REF] Liakhovitskaia | Restoration of Runx1 expression in the Tie2 cell compartment rescues definitive hematopoietic stem cells and extends life of Runx1 knockout animals until birth[END_REF], post-natal lethality resulting from the loss of Runx1 expression in nonhematopoietic tissues. Conditional, timed deletion shows that Runx1 deletion between E7.5 and E10.5 impairs EMP formation but not HSCs which are eliminated at E11.5.

hence revealing a temporal requirement for Runx1 function [START_REF] Tober | Distinct temporal requirements for Runx1 in hematopoietic progenitors and stem cells[END_REF].

Conditional expression of Runx1 in endothelial cells at different time points during development indicates a temporal window for Runx1 to generate hemogenic endothelium. Runx1 expression between E7.5 and 8.5 in non-hemogenic ECs promotes the formation of erythro-myeloid progenitors in the yolk sac, the AGM and the heart. This formation is abrogated when Runx1 is expressed at E8.5-9.5 [START_REF] Yzaguirre | Runx1 is sufficient for blood cell formation from non-hemogenic endothelial cells in vivo only during early embryogenesis[END_REF].

Runx1 thus appears as an orchestrator of EHT rather than a master gene to promote the hemogenic endothelial fate. Runx1 was first shown to regulate the expression of a number of hematopoietic-specific genes such as Spi1, IL3, GM-CSF but also to interact with either activation cofactors, such as p300 and CBP (Creb Binding Protein), histone acetyltransferases, or repression cofactors such as SUV39H1, a histone methyltransferase. Regarding the EHT, two direct targets of Runx1, Gfi1 and Gfi1b, that encode nuclear zinc finger transcriptional repressors have been identified and shown to inhibit the expression of endothelial-specific genes [START_REF] Lancrin | GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment[END_REF]. Embryos deficient for Gfi1/Gfi1B display a block in EHT wherein the cells remain attached to the basal layer in the yolk sac. However, when cells are set free, they are able to form clonogenic colonies [START_REF] Lancrin | GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment[END_REF]. However, expression of Gfi1/Gfi1b in Runx1 deficient mouse embryos shows that cells can undergo EHT but don't form hematopoietic colonies [START_REF] Lancrin | GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment[END_REF]. A more recent study indicates that IAHCS did not form in Gfi1/Gfi1b-deficient embryos but cells remain attached to the endothelial layer. Contrary to the yolk sac, cells are not able to give rise to hematopoietic colonies when dissociated and placed in clonogenic conditions [START_REF] Thambyrajah | GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1[END_REF]. Despite advances, more significant information is needed to understand how EHT is controlled. In order to broaden the search, Lacaud repressor or activator in the same cell type [START_REF] Canon | In vivo analysis of a developmental circuit for direct transcriptional activation and repression in the same cell by a Runx protein[END_REF]. Interestingly, target genes that were positively correlated with Runx1 expression were associated with cell adhesion, integrin signaling, cellular movement and interaction with the extracellular matrix but few hematopoietic-specific genes were identified (Lie-A- [START_REF] Lie-A-Ling | Regulation of RUNX1 dosage is crucial for efficient blood formation from hemogenic endothelium[END_REF].

In addition to the activation or repression of target genes, Runx1 was also shown to have an epigenetic role in chromatin remodeling. It was shown that the transient binding of Runx1 to PU1 cis-regulatory elements is required for further DNA binding-dependent transactivation of PU1 [START_REF] Hoogenkamp | Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program[END_REF]. More widely, Runx1 was shown to be able to associate with numerous de novo DNA sites upon Runx1 expression hence initiating a global reorganization of transcription factors assemblies [START_REF] Lichtinger | RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis: RUNX1 shifts transcription factor binding patterns[END_REF]. A genome-wide analysis of Runx1-dependent binding of factors involved in transcription elongation shows that Runx1 induction moves several transcription factors from their previous sites and relocates them to other ones and recruits the basal transcription factors CDK9, BRD4, the Mediator complex, and the looping factor LDB1 thus being able to shortly activates genes essential for EHT [START_REF] Gilmour | The Co-operation of RUNX1 with LDB1, CDK9 and BRD4 Drives Transcription Factor Complex Relocation During Haematopoietic Specification[END_REF]. Runx1 also contributes to site-specific demethylation via the recruitment of Tet2, a demethylation enzyme [START_REF] Suzuki | RUNX1 regulates site specificity of DNA demethylation by recruitment of DNA demethylation machineries in hematopoietic cells[END_REF].

E. Gfi1 and Gfi1b

Gfi1 and Gfi1b genes encode 2 highly homologous nuclear zinc finger proteins that function as transcriptional repressors [START_REF] Grimes | The Gfi-1 protooncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal[END_REF][START_REF] Tong | The Gfi-1B Proto-Oncoprotein Repressesp21WAF1 and Inhibits Myeloid Cell Differentiation[END_REF].

They both harbor a DNA binding domain localized in the C-terminal domain of the molecules and embedded within 6 zinc finger motifs while the repressor activity is localized within the N-terminal domain [START_REF] Grimes | The Gfi-1 protooncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal[END_REF][START_REF] Zweidler-Mckay | Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor[END_REF]. Gfi1 is expressed in many hematopoietic cells including HSCs, lymphoid and myeloid progenitors and mature cells and Gfi1b is mostly expressed in erythroid and megakaryocytic lineages. Invalidation of Gfi1 leads to neutropenia and reduction of HSCs whereas invalidation of Gfi1b leads to embryonic lethality at E14.5 due to an impairment in erythropoiesis and megakaryocytopoiesis.

Both were identified as direct targets of Runx1 and critical regulators of EHT based on pluripotent-based cell culture approaches. In the absence of Runx1, Gfi1

and Gfi1b are able to induce the loss of EC identity required for EHT and induce the formation of round cells that are not endowed with the full hematopoietic competence.

Conversely, blood cells generated in the absence of Gfi1 and Gfi1b maintain the expression of EC markers and cannot be released into the circulation [START_REF] Lancrin | GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment[END_REF]. Gfi1 and Gfi1b binding sites obtained using Dam-ID reveals genes associated with cell adhesion, integrin signaling, cellular movement and interaction with the extracellular matrix [START_REF] Thambyrajah | GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1[END_REF]. In addition, Gfi1 or Gfi1b also belong to the small set of genes used to reprogram cells into HSCs [START_REF] Pereira | Induction of a Hemogenic Program in Mouse Fibroblasts[END_REF][START_REF] Sandler | Reprogramming human endothelial cells to haematopoietic cells requires vascular induction[END_REF][START_REF] Tsukada | In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells by Gfi1b, c-Fos , and Gata2 Overexpression within Teratoma[END_REF]. et al., 2013). Sox17 was shown to actively repress hematopoietic-specific genes and thus contribute to maintain and modulate the endothelial identity and the EHT [START_REF] Lizama | Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition[END_REF] through an interplay with Runx1 [START_REF] Bos | Single-cell resolution of morphological changes in hemogenic endothelium[END_REF].

G. HoxA3

Another gene which is shown to control Runx1 expression and EHT is HoxA3.

HoxA3 is a member of the paralogue group 3 that was shown to play a role in the specification of pharyngeal tissues. One of these paralogue HoxA3 is also shown to play a role in vascular development. Mice lacking HoxA3 displays cardiovascular anomalies (Chisaka and Capecchi, 1991) and vascular expression of group 3 paralogues is associated with revascularization and wound repair [START_REF] Boudreau | Induction of the Angiogenic Phenotype by Hox D3[END_REF][START_REF] Myers | Homeobox B3 Promotes Capillary Morphogenesis and Angiogenesis[END_REF]. In the embryo, HoxA3 is expressed in vascular ECs of the embryo as early as 8. to maintain an endothelial phenotype (Iacovino et al., 2011, 3). A recent report indicates that this repressive mechanism is also passing through the Notch pathway in upregulating Jag1 in ECs thus reinforcing the endothelial phenotype [START_REF] Sanghez | Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation[END_REF].

H. NOTCH pathway

The involvement of the Notch pathway in developmental processes has been described in multicellular organisms, from nematodes to humans. It plays an essential role in many cell lineage selections and can regulate processes such as proliferation, survival or cell differentiation, both during development and in adults. The Notch gene was characterized for the first time in Drosophila in 1917: its mutation causes in animals the appearance of a notch on the wings. It was not until 1985 that this gene was cloned in Drosophila [START_REF] Kidd | Sequence of the notch locus of Drosophila melanogaster: relationship of the encoded protein to mammalian clotting and growth factors[END_REF] and then in 1990 in Vertebrates (Coffman et al., 1990). In mammals, four Notch genes were identified and cloned, in the Bird, several sequences with homologies to the mouse genes were identified in the avian genome, but only two genes, notch1 and notch2, were cloned. Notch is a transmembrane protein and has a stereotyped structure, whatever the isoform. The extracellular part of Notch includes repetitions of a particular motif, the EGF-like motif, which allows it to link with other proteins: in fact, Notch acts as a receptor on the surface of the target cell and its extracellular domain allows him to interact with the ligands of the signaling pathway. Notch ligands are numerous; they are also transmembrane proteins which, in addition to EGF-like repeats, have a DSL (Delta Serrate Lag2) domain in their extracellular domain. Notch ligands include Delta proteins as well as Serrate / Jagged proteins.

Notch signaling is pleiotropic but has been shown to play a major role in both embryonic and adult hematopoiesis. Indeed, Notch 1, 2 and 3 are expressed in mature and immature progenitors, as well as in blood cells and lymphocytes. Notch ligands are predominantly expressed in the stroma of adult hematopoietic sites as well as on antigen-presenting cells. The Notch pathway is implicated, for example, in the induction of the T lymphoid lineage [START_REF] Radtke | Deficient T Cell Fate Specification in Mice with an Induced Inactivation of Notch1[END_REF] at the expense of lymphocyte B differentiation [START_REF] Pui | Notch1 Expression in Early Lymphopoiesis Influences B versus T Lineage Determination[END_REF]. In addition, in mice a GFP reporter study of Notch activity showed that in the cKit+ HSCs in the bone marrow, Notch pathway is activated.

The authors suggest that it plays a role in maintaining the undifferentiated state of HSCs. Bone marrow stromal cells express Notch ligands such as Jagged-1, Delta-1

or Delta-4. Activation of the Notch pathway in HSCs is probably involved in the differentiation of hematopoietic cells such as dendritic cells (Cheng and Gabrilovich, 2008). In vitro, HSC culture experiments in which a constitutively active form of Notch is transfected showed that the Notch pathway inhibits the differentiation of HSCs and allows expansion of the number of progenitors.

During development, NOTCH1 a member of the notch pathway has been defined as a key gene in EHT. Evidence for the involvement of the notch pathway in embryonic aortic hematopoiesis was provided by the study of mutant mice for Notch1

or Notch2 [START_REF] Kumano | Notch1 but Not Notch2 Is Essential for Generating Hematopoietic Stem Cells from Endothelial Cells[END_REF]. The authors showed that the hematopoietic activity of the endothelium in Notch1-/-embryos was abolished, whereas Notch2-/-embryos exhibited normal hematopoietic activity, showing that, unlike Notch2, Notch1 was essential for normal aortic hematopoiesis. These results have been confirmed by the analysis of RBP-JK-/-mutants, which are therefore unable to transduce Notch signaling via the classical pathway. These mutants exhibit an absence of hematopoietic progenitors, accompanied by a lack of expression of the transcription factors Runx1, SCL and GATA-2 [START_REF] Robert-Moreno | RBPj{kappa}-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells[END_REF]. At the aortic endothelium level, Notch1 and Notch4 were conserved in the mutant but Jagged-1, and Jagged-2, expressions were lost in the mutant RBP-JK-/-. In addition, Notch 1 and Jagged-1 expressions are regionalized to the ventral endothelium of the aorta, suggesting a specific role in aortic hematopoiesis. Finally, careful examination of the Notch1 and Gata-2 patterns shows that these two genes are co-expressed at E9, 5 in the ventral endothelium of the aorta. A direct interaction between Notch1 and the Gata-2 promoter has also been highlighted, reinforcing the idea that the Notch pathway plays a fundamental role in the molecular network controlling the hematopoietic process in the aortic region [START_REF] Robert-Moreno | RBPj{kappa}-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells[END_REF]. A relationship between the Notch pathway and the regulation of hematopoiesis by Runx1 was also shown in the zebrafish. The transient activation of the notch pathway in mutant embryos deficient for Notch induces expression of Runx1 in the aortic region as well as the production of HSC [START_REF] Burns | Hematopoietic stem cell fate is established by the Notch-Runx pathway[END_REF]. In addition, the inhibition of the notch pathway in zebrafish inhibits definitive hematopoiesis, while primitive hematopoiesis occurs normally [START_REF] Gering | Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos[END_REF]. Finally, it has been shown that the rescue of Notch1-/-by in vitro transfection of Runx1 was capable of restoring the hematopoietic potential, suggesting an effect of Runx1 downstream from the notch pathway [START_REF] Nakagawa | AML1/Runx1 rescues Notch1-null mutation-induced deficiency of para-aortic splanchnopleural hematopoiesis[END_REF]. Even though Notch1 signaling is required to initiate expression of key hematopoietic regulators such as Gata2, its activity also needs to be restricted to allow the completion of the EHT, as high Notch activity promotes an arterial program in endothelial cells, while suppressing the hematopoietic program [START_REF] Lizama | Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition[END_REF].

In fact, a reporter system that differentially labels cells with high or low Notch activity has identified two populations in the dorsal aorta, an arterial-restricted population with high Notch activity and a hematopoietic competent population with low Notch activity [START_REF] Gama-Norton | Notch signal strength controls cell fate in the haemogenic endothelium[END_REF]. That same study further revealed the involvement of two different Notch ligands, with Dll4 inducing high Notch activity and thus implementing the arterial program, while Jag1 facilitated the hematopoietic program by restricting Notch activity. A down-regulation of Notch signaling was also observed during the maturation of pre-HSCs into fully competent HSCs.

Furthermore, the notch ligands regulator mind bomb-1 was shown to be crucial for intra-embryonic hematopoietic progenitor formation, mainly when expressed in the non-endothelial compartment. This suggests a paracrine action of the notch ligands on the endothelial cells [START_REF] Yoon | Mind Bomb 1-Expressing Intermediate Progenitors Generate Notch Signaling to Maintain Radial Glial Cells[END_REF]. c-Mpl, the receptor for thrombopoietin has been shown to be expressed by IAHC in mice, and its absence led to diminished HSC activity in E11 AGMs [START_REF] Petit-Cocault | Dual role of Mpl receptor during the establishment of definitive hematopoiesis[END_REF].

Mechanisms that allow a cell to leave a tissue during development ubiquitin ligase, inflammation [START_REF] Zeisberg | Biomarkers for epithelial-mesenchymal transitions[END_REF].

EMT is defined as a metastable state which describes a transiently stable state that is different from the more stable end state and often expresses both epithelial and mesenchymal markers. This definition allowed the description of three separable elements in the EMT: two end stable states with epithelial or mesenchymal identity and a third transitional one which corresponds to the overlapping of transitional states named intermediate or metastable states. It is likely that all three subtypes of EMT will share part of a signature, but some aspects of the signature may be unique to a particular subtype. The most visible protein signatures include at least one changing cytoskeletal protein, transcription factor, adherence protein, and perhaps the changing levels of discrete microRNAs.

The parallel between EHT and EMT

During development, Endothelial to Hematopoietic Transition EHT is described as a partial EMT process in which the hemogenic endothelium, like most of the endothelial structures, contains stereotypic epithelial characteristics such as apicobasal polarity, tight junctions, adherens junctions, and basement membrane. It also involves similar cellular processes found in canonical EMTs.

A parallel can be drawn between EHT and EMT. As described in the previous chapter (genes and pathways) it has been shown that NOTCH1/Jagged1 as a main EHT driver [START_REF] Robert-Moreno | Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1[END_REF]. When this pathway is activated in nonhemogenic ECs, it results in an EMT like process called endothelial to mesenchymal transition EndMT and ECs lose their endothelial markers (PECAM1, Ve-Cad) but this time instead of acquiring hematopoietic fate they acquire mesenchymal features and expresses FN1 and a-SMA. In addition to this pathway Runx1 as described earlier is a critical factor for the EHT, it has been shown to be implicated in the initiation of the EMT process through TGFB dependent pathway, resulting in decreasing epithelial markers or through TGFB independent process, resulting in increasing ZEB2 expression which is expressed in the AGM of E10.5 mouse. HSPCS need ZEB2 mediated transition towards a more mesenchymal intermediate EMT state in order to acquire migratory properties necessary for the invasion and colonization of primary and secondary hematopoietic organs Figure 35.

In the bone marrow, several lines of evidence suggested that the interaction of HSCs with the different components of the niche is essential for their maintenance, quiescence and self-renewal balance. At the molecular level, in addition to E-cadherin and Notch pathway members, several groups identified other cell-cell interaction proteins i.e Tie2, JamC, Esam and EPCR as playing a role in HSC niche interactions. [START_REF] Roch | Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells[END_REF]. Together all these findings suggest that LT-HSCs are strongly associated with an appropriate metastable state of EMT regulated by the niche architecture. The majority of POFUT2 targets are constitutive component of ECM or secreted matrix-associated proteins able of influencing several processes as cell adhesion/migration, ECM synthesis, and remodeling and finally modulating growth factor synthesis. Consequently, the loss of POFUT2 disrupts embryonic processes.

To investigate the biological role of POFUT2 in mice, they characterized two POFUT2 gene trap insertions: Pofut2 Gt(RST434)Byg and Pofut2 Gt(neo)699Lex inactivating one copy of POFUT2 at a time. They could demonstrate that POFUT2 was a key modulator of gastrulation (cell differentiation, tissue morphology, and tissue boundaries) and that the disruption of POFUT2 gene resulted in embryonic lethality by E10.5. POFUT2 mutant showed from one hand an abundance of mesenchymal cells, markedly expanded the expression of SNAIL and a reduction in the expression of pluripotency markers in the epiblast suggesting that O-fucosylation of TSR protein by POFUT2 is essential to restrict EMT. From the other hand, it shows an abundance of FLK1 and PECAM1 positive cells and absence of differentiated primitive erythrocytes, demonstrating that O-fucosylation of TSR by POFUT2 was important for the distinction between the hematopoietic and vascular endothelial lineage.

Together these results showed that the loss of POFUT2 in mice resulted in unrestricted EMT and biased differentiation of vascular endothelial cells.

Table 2: Mouse Proteins with putative O-fucosylation site within TSRs

The Extracellular Matrix and cytoskeleton and their implication in hematopoiesis

The extracellular matrix (ECM) is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support of surrounding cells. It is well described that ECM provides critical support for vascular endothelium through adhesive interaction with integrin on the ECs surface. Therefore, ECM provides a scaffold, important for maintaining the organization of vascular ECs into blood cells. As angiogenesis proceeds, the basement membrane matrix is degraded by proteinases among which membrane-type matrix metalloproteinases (MT-MMPs) are particularly significant. In this case ECM serves essential function in supporting key signaling events involved in regulating EC migration, invasion, proliferation and survival. Moreover, through specific integrin-dependent signal transduction pathways, ECM controls the EC cytoskeleton (a complex, dynamic network of interlinking protein filaments that extends from the cell nucleus to the cell membrane) to orchestrate the complex process of vascular morphogenesis [START_REF] Davis | Endothelial Extracellular Matrix: Biosynthesis, Remodeling, and Functions During Vascular Morphogenesis and Neovessel Stabilization[END_REF].

Concerning hematopoiesis, ECM was described as an important component of the BM hematopoietic microenvironment regulating some cytokines and their downstream signaling. Earlier during development, in the pre-umbilical AGM region, directly underlying the hematopoietic cluster, this region of mesenchyme expresses tenascin-C, an extracellular matrix glycoprotein known to facilitate cell-cell interactions and migration. This region of cells may, therefore, provide the microenvironmental support for the intraembryonic development of definitive hematopoietic stem cells, a process in which tenascin-C may play a pivotal role [START_REF] Marshall | Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer[END_REF].

However, the cellular mechanism(s) allowing HSPC egress and migration to secondary niches are incompletely understood. Using zebrafish, [START_REF] Theodore | Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization[END_REF] showed that Mmp2 and Mmp9 are induced in response to inflammatory signals Further understanding of the intersections between inflammatory signaling, ECM remodeling, cytoskeleton, and hematopoiesis could be exploited to improve in vitro and in vivo HSPC expansion efforts, as well as HSC mobilization and transplantation.

EMILIN 1, an extracellular matrix could potentially play a role in hematopoiesis.

It is an ECM glycoprotein associated with elastic fibers and composed of an N-terminal cysteine-rich domain and the EMI domain, followed by a coiled-coil structure, a short collagenous stalk, and a C-terminal gC1q domain. EMILIN1 is particularly abundant in the walls of large blood vessels, such as the aorta, and has been implicated in multiple functions. EMILIN1 is involved in elastogenesis and in the maintenance of blood vascular cell morphology. It interacts with the α4β1 integrin through the gC1q1 domain and has strong adhesive and migratory properties for different cell types [START_REF] Zanetti | EMILIN-1 Deficiency Induces Elastogenesis and Vascular Cell Defects[END_REF]. Concerning EMILIN 2 (multimerin) a closely related molecule to EMILIN1, it is a protein shown to be secreted by endothelial cells and platelets [START_REF] Hayward | The cDNA Sequence of Human Endothelial Cell Multimerin A UNIQUE PROTEIN WITH RGDS, COILED-COIL, AND EPIDERMAL GROWTH FACTOR-LIKE DOMAINS AND A CARBOXYL TERMINUS SIMILAR TO THE GLOBULAR DOMAIN OF COMPLEMENT C1q AND COLLAGENS TYPE VIII AND X[END_REF]. Therefore, EMILIN1 and 2 could play a role in the endothelial to hematopoietic transition.

Pre-somitic mesoderm culture system

Cell culture

PSM were cultured under the following conditions Opti-MEM® with GlutaMAX™I supplemented with 5% FCS, 1% Chicken Serum, 100 Units per ml penicillin/streptomycin and the following growth factors (PromoCell/PromoKine): hVEGF (≠C64410); 2ng/ml), hFGF (≠C60240) ; 4ng/ml), hIGF (≠C60840) ; 3ng/ml), hEGF (≠C60170) 10ng/ml), hydrocortisone (200ng/ml SIGMA ≠H6909), ascorbic acid (75ug/ml SIGMA ≠A4544). PSM were cultured in 35mm collagen Ior IV -coated dishes from Corning BioCoat (Corning -Dutscher France). Medium was changed every two days unless specified (Figure M1A).

Functional tests with small molecules

We selected 3 small molecules to test. PKF 118-310 (K4394 Sigma-Aldrich) an inhibitor of the Wnt pathway, SB216763 (S3442 Sigma-Aldrich) an activator of the Wnt pathway and DAPT (D5942 Sigma-Aldrich) an inhibitor of the Notch pathway. For the initial experiments, we started from reported concentrations found on the literature.

PKF118-310: 0,66μM/mL, 1μM/mL, 1,5μM/mL DAPT: 25μM/mL and 50μM/mL SB216763: 10μM/mL, 20μM/mL and 30μM/mL DAPT and SB216763 were diluted in DMSO, and PKF118-310 was diluted in H20. To ensure the diluent (H20 or DMSO) did not influence the culture, we also cultured cells with media supplemented with either DMSO or water (the same volume as for the solutions of small molecules) as positive controls.

siRNA design and lipofection-mediated transfer

We used a knock-down strategy utilizing siRNA to inhibit our candidate genes.

We designed three different siRNAs against POFUT2 and three others against TESTIN (Table M1). Lipofectamine RNAiMax was used to transfect cells at day 1 or 2 with specific siRNA or Scramble si control following the manufacturer's protocols. The transfection efficiency was evaluated by analysing the mRNA expression of the targeted gene by qPCR.

Transcriptomic analysis 1. Next Generation Sequencing

RNA libraries were prepared by Fasteris Life Science (Switzerland). In brief, total RNA was submitted to a poly-A mRNA purification using oligodT magnetic beads.

Supernatants were kept for small RNA library preparation, and poly-A-RNAs after elution from the beads were prepared according to the manufacturer's protocol (TruSeq RNA Sample Prep-Kit V2; Illumina). Transcripts were broken at 95°C in presence of zinc, and first-strand cDNA syntheses were performed using random primers. A second-strand cDNA synthesis was performed in the presence of deoxyuridine triphosphate, and after a 3′ A addition step, adapters were ligated, and an amplification by PCR was per-formed to generate the DNA colony template libraries. Small RNA libraries were performed according to the manufacturer's protocol (TruSeq Small RNA Library Prep kit; Illumina). After acrylamide gel purification of small RNA between 18-30 nt, single-stranded ligation of 3′ adapter and the 5′ adapter were performed before reverse transcription and PCR amplification to generate the DNA colonies template. All the samples were sequenced using 1 × 125-bp single reads high-throughput sequencing (RNA-Seq) in single lane on a HiSeq 2000 sequencing system (Illumina).

2. Single-cell RNA-seq from D0, D4 and D6 cell culture using 10x Genomics Chromium Samples were prepared as outlined by the 10x Genomics Single Cell 3′ v2

Reagent Kit user guide. Briefly, the samples were washed twice in PBS (Life Technologies) + 0.04% BSA (Sigma) and re-suspended in PBS + 0.04% BSA. Sample viability was assessed via Trypan Blue (Thermo Fisher). Following counting, the appropriate volume for each sample was calculated for a target capture of 6000 cells.

Samples below the required cell concentration as defined by the user guide (i.e., <400 cells/µl) were pelleted and re-suspended in a reduced volume and counted again prior to loading onto the 10x Genomics single-cell-A chip. After the reverse transcription, cDNA was recovered using Recovery Agent provided by 10x followed by a Silane DynaBead clean-up (Thermo Fisher) as outlined in the user guide. Purified cDNA was amplified for 12 cycles before being cleaned up using SPRIselect beads (Beckman).

Samples were diluted 4:1 (elution buffer (Qiagen): cDNA) and run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell 3′ Reagent Kits v2 user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration (as recommended by 10X Genomics). Analysis were performed using partek flow single cell workflow.

Bioinformatic analysis

A. RNA-Seq data analysis RNA-Seq analysis was performed on Partek Flow™. For mRNA libraries, sequence reads in fastq format were aligned to the quail reference genome assembly RefSeq GCF_001577835. using the STAR aligner. The number of reads for all the features were then counted using the quantification to a model E/M and normalized for each library, and then ANOVA two ways were performed to compare read values between different libraries (Figure M2).

Figure M2: Workflow of RNAseq analysis

The unaligned reads of the 20 samples were analyzed for the quality of the nucleotide base composition and then aligned to the quail genome. The quality of the alignment was assessed by the QA/QC post-alignment quality. We quantified 20332 genes and then normalized to be able to perform the statistical analysis.

The lists of differentially expressed genes were generated using ANOVA and Venn Diagram. GO was performed using DAVID (http://david.abcc.ncifcrf.gov). To find out the network structure of the gene set HEC, we used WGCNA (Langfelder and Horvath, 2008). Adjacencies were given according to signed Pearson correlation. The soft threshold power b that resulted in approximate scale-free topology was 36.

Modules were constructed with average linkage hierarchical clustering.

B. Single cell data analysis 10x data were processed using Cell Ranger 2.1.0 with default parameters.

Using Partek Flow reads were aligned to the coturnix japonica reference GCF_001577835. Partek Flow single cell workflow was used to perform graph-based clustering of the cells. Briefly, cells with <200 detected genes and with a cell counts > 9000 were filtered out. In addition, cells that showed lower number of genes than expected from the number of UMIs detected were filtered out as low quality cells. We then excluded features where value equal to zero. The data were log normalized (Counts per million, log2+1). Principle Component analysis PCA was performed using variable genes. Eigenvalues was used to determine the statistically Significant PCs to be used for graph-based clustering. t-SNE was used to visualize the clusters. Cell types were classified according to the expression of previously reported marker gene and GO categories.

Statistical analysis

Data were processed using GraphPad Prism v.

CHAPTER 3: RESULTS

LDL int cells represent an EC population upregulating RUNX1 and down-regulating CD31 and CD144.

One advantage of the culture system [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF] is the stepwise progression of the mesoderm towards the hematopoietic fate that allows isolating discrete cellular steps over the culture period of 12 days. In addition, by removing serum or VEGF from the culture, it is possible to prevent hemogenic EC commitment and to orient the cells towards a more advanced vascular endothelium state. We previously showed that ECs emerging in culture uptake LDL in keeping with their endothelial phenotype including those expressing RUNX1. We reasoned to use the levels of LDL uptake to separate ECs from hemogenic ECs. This is based on the observation that RUNX1 initiated expression is initiated at D3 in only a few ECs but is found expressed in almost 100% of the cells at D5. RUNX1 expression is shortly followed by that of the earliest hematopoietic-specific transcription factors, in particular, PU1, a direct target of RUNX1. In this respect, hemogenic ECs i.e., RUNX1expressing cells, should undergo a decrease of LDL uptake in keeping with the progressive loss of the endothelial program. Based on this assumption, D2 and D4 adherent cells were incubated with Ac-LDL coupled to Alexa 488 (hereafter referred as LDL) over a period of two hours, trypsinized and Fluorescence-Activated Cell Sorted (FACS) based on the LDL level of expression. At D2, FACS analysis showed cells harboring a wide, albeit continuous, range of fluorescence from nil to high (Figure R1A). At D4, we separated two populations: one with a high level of expression of LDL (LDL hi ) and another with an intermediate level of expression (LDL int ) (Figure R1B). In the absence of serum, most of the population is within the LDL hi (Figure R1C).

The different LDL populations displayed on Figures R1A-C were sorted and analyzed for the expression of two endothelial markers i.e., CD31 (PECAM) and CD144 (VE-Cadherin) and the canonical hemogenic endothelial marker RUNX1.

Gene expression on the D0 pre-somitic mesoderm served as a baseline. Both CD31 mRNA was highly expressed in LDL hi vs LDL int cells with a mean fold change of two (Figure R1D) CD144 mRNA expression was high at D2 and D4 with no clear change on the level of expression between D2 and D4 and between D4 and D4 without SVF but was at least 8-fold decreased in D4 LDL int cells (Figure R1E). However, RUNX1 mRNA expression was strongly increased in LDL int compared with the other LDLpositive populations (Figure R1F).

Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium

A-C: Flow cytometry analysis showing the expression of LDL uptake in the culture. A: analysis at D2. The cells display a continuum of LDL uptake from nil to high. Frame shows the sorted population. B: analysis at D4 in normal culture conditions. Two distinct populations with contrasted LDL uptake are seen. The left frame isolates the LDL int population and the right frame, the LDL hi population. C: analysis at D4 in the absence of fetal calf serum known to orientate the cells towards the vascular endothelium lineage [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. A large percentage of the population is contained within the LDL hi population in keeping with their strong endothelial commitment. D-F: quantitative PCR on the different cell fractions for CD31 (D), CD144 (E) and RUNX1 (F). CD31 and CD144 are decreased in the LDL int population compared with the other endothelial populations. RUNX1 is strongly increased in the LDL int population compared with the other endothelial fractions (n=2). Significance is given compared with the D4 LDL hi fraction. Data are shown as means ± SEM.

Sample isolation strategy

Based on this result, we isolated the different cell fractions to perform NGS. We used the level of LDL uptake to isolate ECs (LDL high or +) from hemogenic ECs (LDL int ).

Presomitic mesoderm was directly isolated from the embryo whereas hematopoietic cells were collected as the floating fraction, in keeping with [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF] (Figure R2A). We used high-throughput sequencing to compare the molecular signatures of the different cell fractions. We isolated 20 samples corresponding to the different cell fractions. mRNAs were isolated and the libraries were sequenced using next-generation sequencing Illumina technology. According to the quality control, we detected 20 332 genes. The review of the following analysis is shown in Figure R2B. showed some discrepancies in the ordering with the M fraction found between the HEC and HC fractions (Figure R3B).

Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes)

A-3D PCA: PC1 (30.2% of the variance) and PC3 (9.85% of the variance) allowed to discriminate ECs from pre-somitic mesoderm (M) and hemogenic ECs (HEC) from hematopoietic cells (HC), respectively. B-Hierarchical clustering: showed some discrepancies in the ordering with the M fraction found between the HEC and HC fractions.

To identify the genes that were specifically up-or down-regulated in the different cell populations we followed a two-step procedure. First, we compared pairwise using ANOVA (with p-values <0.06 and fold changes ≥1.45 or ≤-1.45) the M, EC, HC and HEC cell populations. Second, we considered using Venn diagrams the gene sets that were up-regulated (or down-regulated) in a given population according to the 4 comparisons (Figure R4A). This procedure led to the identification of 2462 upregulated and 2087 down-regulated genes (upregulated DEGs in the 4 populations Table S1; Table S2). We then performed a novel PCA that provided a clear-cut discrimination between the 4 populations, according to PC1 (43% of the variance) corresponding to the contrast between M and EC and to PC3 (17% of the variance) corresponding to that contrast between HC and HEC (Figure R4B). And this time hierarchical clustering showed a perfect ordering going from M through EC and HEC fractions until HC population (Figure R4C).

Figure R4: Identification of the specifically up-regulated genes in the different cell populations

A-we used Venn diagrams to isolate the gene sets that were upregulated in a given population according to the 3 comparisons. This led to 4 Venn diagrams one for each population. B-3D PCA provided a clear-cut discrimination between the 4 populations, according to PC1 (43% of the variance) corresponding to the contrast between M and EC and to PC3 (17% of the variance) corresponding to that contrast between HC and HEC. C-Hierarchical clustering showed a perfect ordering going from M through EC and HEC fractions until HC population.

To find the structure of the gene network indicating how genes from our dataset are interconnected, we used weighted gene correlation network analysis (WGCNA).

WGCNA makes use of correlation between genes to identify coordinately expressed genes (Langfelder and Horvath, 2008). Moreover, this method indicates how genes are correlated to an external genetic trait, corresponding in this work to the factor support quantified for each line by PCA. This analysis allowed network construction corresponding to the set of 2452 up regulated genes without relying upon literature mining. WGCNA revealed the existence of 5 modules, one for EC, one for M, one for HC and two for HEC (Figure R6A). Each module corresponds to a network, in order

A-Dendrogram of modules identified by WGCNA: Green module was attributed to Meso population, Salmon module to HC population, Blue module to EC population, Black module to HEC as well as Red module. B-Cytoscape visualization of the Green M network C-Cytoscape visualization of the Blue EC network D-Cytoscape visualization of the Salmon HC network

B A

Analysis of the GO categories revealed shared and distinctive patterns between the two HSCs networks. Both networks showed an enrichment in molecules implicated in the ECM, with Pleckstrin homology domain, SH3 domain, components of the Golgi apparatus, cell adherent junction, membrane and cell signaling. On the contrary, genes implicated in Focal adhesion were enriched in the red module only, while the black module showed enrichment in genes implicated in microtubule, migration and phosphatase activity (Figure R8).

In order to confirm the predictive value of our culture system and in order to validate candidate genes that potentially play a crucial role in the EHT, we selected two genes i.e., POFUT2 and TESTIN corresponding to two of the hubs of the HEC networks. To fully validate the system, we also selected the Wnt and Notch pathways already demonstrated as playing a role in the EHT. In order to do so, we followed a lipotransfection strategy with siRNA for POFUT2 and TESTIN and small molecules compounds for Notch and Wnt.

Figure R8: Analysis of the gene ontology categories of HEC networks

Both networks showed an enrichment in molecules implicated in the extracellular matrix ECM, with Pleckstrin homology domain PH, SH3 domain, components of the Golgi apparatus Golg, cell adherent junction Junc, membrane Memb and cell signaling sig. On the contrary, genes implicated in Focal adhesion FA were enriched in the red module only, while the black module showed enrichment in genes implicated in microtubule MT, migration Mig and phosphatase activity Phase.

Modulation of the Wnt pathway contributes to the endo-hematopoietic balance

To investigate whether ß-catenin activity influences the generation of HEC and the EHT, we tested the effect of inhibitors or activators of the pathway on culture. As an inhibitor, we used the small molecule PKF118-310 (hereafter referred as PKF118),

an inhibitor of the TCF4 ß-CATENIN protein interaction (Hallet et al., 2012). We tested 3 different concentrations of PKF118 (0.66µM, 1µM and 1.5µM). The last concentration provoked toxic effects whereas the first one did not produce significant results (not shown). The concentration of 1µM caused drastic changes to the culture.

Treated cells were flatter and had fewer round cells than the non-treated samples (Figure R9A; Figure R9B; Figure R9D; Figure R9E). Flow cytometry analysis confirmed the increase of LDL hi cells at the expense of LDL int cells (Figure R9C; Figure R9F; Figure R9G; Figure R9H) and the strong decrease of the floating cells (Figure R9I) (n=5). QRT-PCR on adherent cells revealed the reinforcement of CD144 mRNA expression (Figure R9J) in keeping with the increase of the endothelial phenotype and the decrease of RUNX1 expression (Figure R9K) consistent with the lower number of LDL int cells.

We also tested the Wnt activator SB216763 hereafter referred as SB, that inhibits GSK3 alpha and GSK3 beta. We used two different concentrations i.e., 10µM and 20µM with 20µM displaying the strongest effect. The result is an increase in the number of round hematopoietic cells produced compared with the mock-(DMSO) treated culture (Compare Figure R10A to Figure R10D and Figure R10B to Figure R10E). Flow cytometry analysis reveals a 1.6-fold change in the number of LDL int cells at the expense of LDL hi cells (Figure R10C; Figure R10F The Gamma secretase inhibitor DAPT stimulates the EHT We used gamma secretase as an inhibitor of Notch to inquire into the role of this signaling pathway in hemogenic endothelium and EHT. DAPT 25µM was applied to the culture at D2. Analysis was performed at D4. DAPT readily produced a dramatic change in culture. Within the two days of DAPT contact, many cells rounded up, detached from the adherent layer and were found in the supernatant (Compare Figure R11A; Figure R11B). Cells in the supernatant were round and very bright suggestive of a hematopoietic production (Figure R11B) and [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. FACS analysis showed a sharp increase in the number of LDL int cells at the expense of LDL hi cells (Figure R11C; Figure R11D and Figure R11G; Figure R11H) accompanied by a parallel increase of hematopoietic cells (Figure R11E; Figure R11 F and Figure R11I).

QRT-PCR analysis showed a highly significant increase of CD45 expression characteristic of hematopoietic differentiation (Figure R11J). We designed three different siRNAs against POFUT2 (Table M1), that were transfected at the culture incipience at Day 1 or 2. The analysis was performed at day 4 (Figure R9A) and was compared to Lipofectamine alone or a scrambled POFUT2 siRNA.

We tested 3 concentrations of siRNA against POFUT2 i.e. 30, 60 and 120nM (n=5) and compared their knock-down efficiency by measuring POFUT2 mRNA expression by q-PCR on transfected cells. The lowest concentration decreased POFUT2 mRNA expression by 80% whereas the highest displayed signs of toxicity and was not retained (not shown). The 60nM siRNA concentration decreased POFUT2 by 90% on transfected cells and was selected for further use (Figure R12B, R14B). POFUT2 mRNA knock-down at D1 resulted in the appearance of more round hematopoietic cells in the culture compared with Lipofectamine or scramble si controls (Figure R12C; Figure R12E; Figure R12G). Flow cytometry analysis revealed an increase in the LDL int population at the expense of the LDL hi population (Figure R12 D, F, H). Taken together POFUT2 knock-down resulted in a decrease of the number of ECs (p<0.001) and an increase in the number of HEC (p<0.05) (Figure R12I; Figure R12J).

To further validate the gene network and its predictive value, we selected the first neighbors of POFUT2 (Figure R13) and examined if a decrease of POFUT2 mRNA expression was also associated with a modulation of several of its first neighbors. Based on gene connectivity, we selected 3 genes among the most connected to POFUT2. COL5A2 encodes an alpha chain for one of the low abundances fibrillar collagens; DNAJB4, is a molecular chaperone, tumor suppressor, and member of the heat shock protein-40 family that binds and regulates E-Cadherin and EMILIN1A (Elastin Microfibril Interfacer 1) is thought to regulate smooth muscle cell adhesion and vessel assembly. COL5A2, the most connected gene to POFUT2

showed a significant decrease upon POFUT2 siRNA mediated knock-down when compared to the Lipofectamine control but not to the scramble (p<0.001and 0.3 respectively) (Figure R12K). DNAJB4 and EMILIN1A both showed a significant decrease upon POFUT2 siRNA knock-down whatever the control used (Figure R12L;

Figure R12M). Taken together, this indicates that POFUT2 and, at least, 3 of their first neighbors are acting coordinately to regulate the HEC fate and the hematopoietic production.

We also tested POFUT2 mRNA knock-down at D2. The 60nM siRNA concentration decreased POFUT2 by almost 90% on transfected cells (Figure R14B).

Figure R13: POFUT2 first neighbors

The color code is the same as Figure R7 125

Figure R14: POFUT2 knock down at day 2 doesn't affect EHT.

A We designed three different siRNAs against TESTIN (Figure R15A) that were transfected at day 1 of culture incipience. The analysis was performed at day 4 (Figure R15B) and was compared to Lipofectamine alone or a scrambled TESTIN siRNA. We tested 3 concentrations of siRNA against TESTIN i.e. 30, 60 and 120nM (n=3) and compared their knockdown efficiency by measuring TESTIN mRNA expression by q-PCR on transfected cells. The lowest concentration decreased TESTIN mRNA expression by 70% whereas the highest displayed signs of toxicity and was not retained (not shown). The 60nM concentration decreased TESTIN by 80% on transfected cells and was selected for further use (Figure R15C). TESTIN mRNA knockdown at D1 did not show any significant difference in the culture compared with Lipofectamine or scramble si controls (Figure R15D-E'). Flow cytometry analysis revealed no changes in LDL int population nor the LDL hi population (Figure R15F-H).

Taken together TESTIN knock-down resulted in no changes in the number of ECs and HEC (Figure R15I-J).

To further validate if the inhibition of TESTIN even affects the gene network, we selected the TESTIN's first neighbors and examined if a decrease of TESTIN mRNA expression was also associated with a modulation of several of its first neighbors.

Based on gene connectivity (Figure R16) we selected 3 genes among the most connected to TESTIN. BICC1 is a putative RNA-binding protein that acts as a negative regulator of Wnt signaling, CREB3L1 a protein with a DNA-binding transcription factor activity and chromatin binding required for the assembly of collagen extracellular

Figure R16: TESTIN first neighbors

The color code is the same as Figure R7 The next step was to attribute each of these clusters to a well-defined population according to the expression of previously reported marker genes. Clusters 3,4,5,8 and 12 showed enrichment in mesodermal markers: FGF8, MEOX1, TBXT, HOXA1, ADD3 (Figure R18). Clusters 1, 2, 7, 13 showed enrichment in endothelial markers: CDH5, ESAM, KDR, CD34 (Figure R19). Whereas, cluster 6 showed enrichment in hemogenic endothelial markers: RUNX1, CD44 (Figure R20); The cluster 9 and 10 did not show a specific enrichment with defined markers apart from some cells expressing PTPRC and RUNX1. No evidence of endothelial or mesodermal expression were detected in these two populations suggesting that these clusters might be of hematopietic nature. In summary, the single cell approach recapitulates the presence of 4 defined populations: mesodermal, endothelial cells, hemogenic endothelial cells and hematopoietic cells. showed an enrichment in carbon metabolism, apoptosis and hemopoiesis, which could correspond to hematopoietic cluster (Figure R21).

This analysis allowed us to confirm the presence of the 4 specific populations in our culture system going from Mesoderm to Endothelium to Hemogenic endothelium and finally the Hematopoietic population. This part of the work is still preliminary, and this data set will be further investigated in more refined way. Complementary results

LOC assignment workflow

One of the issues with our gene list (after alignment to the quail genome) is that it included around 8000 non-assigned sequences, named LOC. Those LOC correspond to sequences not yet linked with any orthologs. It was vital for our future investigation that these sequences were matched with known genes, so we had to identify most of them manually. We used the databases of NCBI Genes, NBCI Nucleotides and Uniprot, as well as the alignment search program BLAST (blastn algorithm) to assign our ARN sequences to known genes (Figure CR1). We could almost identify 4000 of these LOCs (Annex table 1). In this study, I aimed at defining molecularly the hemogenic endothelium undergoing EHT. For this, I took advantage of the precise culture system I contributed to set up [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF] to isolate discrete cell populations from the noncommitted mesoderm to hematopoietic cells including hemogenic endothelium as an intermediate. I used the level of LDL uptake to discriminate the endothelial cells from the hemogenic ECs that display a lower LDL uptake. I confirmed this point by validating the expression of several lineage-specific genes in particular RUNX1, CD144, and CD31. I obtained the transcriptomes of the different cell types and initiated a bioinformatic approach aiming at revealing the molecular identity of HECs. The first step of the analysis was to control the expression of the known marker in our population, we found that mesodermal population expresses specific mesoderm genes i.e TBOX, ZIC3, MYC, the endothelial population expresses VWF, KDR, FLI1, CDH5, GATA2, Tal1 … Whereas HEC population in addition to expressing endothelial-specific genes, it expresses RUNX1, GFI1, KIT, and CD44, finally the hematopoietic population expresses CD45, SPI1 … Together the expression of these key genes validates the specificity of the isolated and sequenced populations.

Figure CR1: LOC Assignment workflow

LOC107311523 RASSF2 ADCY2 ICA1 MOB1B UEVLD ALOX5 LOC107311560 RASSF5 ADGRA1 IDS MPPE1 UNC13C ANK1 LOC107311590 RBPJ ADGRF4 IFNGR1 MPZL1 UPP1 ANKRD12 LOC107311827 RGR ADGRF5 IGDCC4 MTMR10 UPP2 ANKRD13A LOC107312292 RHCE ADGRG1 IGFBP3 MTRF1 USP12 AP1AR LOC107312344 RHOBTB3 ADGRL4 IGFBP7 MYCT1 USP38 AQP2 LOC107312365 RHOF ADSSL1 IMPA1 MYLK USP6NL ARFGEF2 LOC107312428 RIMS4 AGA ING4 MYO6 UTRN ARL8B LOC107312433 RNF145 AGT INPP4B MYO7A VANGL1 ARSB LOC107312463 RNF212 AHR INTS10 MYRIP VAT1 ASB6 LOC107312591 RRP36 AHRR ISCA2 NAAA VPS33B ASB8 LOC107312649 S1PR4 AK4 ISPD NAGA VWC2 ASTL LOC107312725 SAMSN1 AKT3 ISYNA1 NCKAP1 VWF ASXL2 LOC107313061 SASH3 AKTIP ITM2A NDFIP2 WDFY2 ATP6 LOC107313680 SBNO1 ALS2CL ITM2B NECTIN3 WSCD1 ATP6V0B LOC107313817 SCIN ANGEL1 ITSN2 NEMP2 WWTR1 ATP6V1A LOC107313856 SCNN1D ANKRD50 IVNS1ABP NFIA XYLB ATP6V1B2 LOC107313879 SESN1 ANPEP JAG2 NHLRC1 ZC3H7B ATP6V1C1 LOC107314004 SGCZ ANXA7 KCNB2 NOS1AP ZCCHC24 ATP6V1C2 LOC107314262 SLC15A5 AP5M1 KCNE3 NOVA1 ZDHHC14 ATP8 LOC107314505 SLC1A1 APOLD1 KCNMB2 NPL ZDHHC23 B4GALNT1 LOC107314560 SLC25A37 APP KCTD18 NR0B1 ZFAND2B BCL11A LOC107314689 SLC35D1 AQP1 KDR NR3C2 ZFPM2 BHLHA15 LOC107314794 SLC40A1 AQP9 KIAA1210 NR5A2 ZMYND11 BPI LOC107314877 SLC43A3 ARFGEF3 KIAA1462 NRBP2 ZNF521 BRI3 LOC107315482 SLC4A1 ARHGAP31 KIF13A NRXN3 BRIP1 LOC107315614 SLC9A3R1 ARHGEF10 KIF13B NTNG1 BST1 LOC107316070 SMIM5 ARHGEF26 KLF3 NTRK3 BTK LOC107316219 SPATA2L ARHGEF9 KLHL24 NUDT14 C17H9orf91 LOC107316229 SPG21 ARID4B KLHL3 OSBPL10 C18H17orf62 LOC107316493 SPNS3 ARL11 KLHL4 OSBPL8 C18H17orf89 LOC107316612 STARD5 ARL15 KLHL5 OSBPL9 138 C21H1orf158 LOC107316955 STK10 ARPP19 L3HYPDH PAOX C21H1orf167 LOC107317373 STK4 ARSA LAMA4 PAQR5 C26H6orf106 LOC107317685 STX11 AS3MT LAMB1 PAQR9 C6H10orf54 LOC107318005 SULT1B1 ASAP1 LAMP2 PBXIP1 C8H1orf228 LOC107318300 SUOX ASB5 LDB2 PCNX4 CADM3 LOC107318468 SUSD3 ATG13 LEMD3 PCSK2 CARD9 LOC107318673 SYNGR3 ATG4A LGMN PDE8A CC2D1B LOC107318831 TALDO1 ATOH8 LGR5 PDGFB CCDC171 LOC107318879 TAS1R1 ATP8B2 LIN7C PEX26 CCND3 LOC107319031 TDP2 ATP9B LNX2 PEX5 CCNDBP1 LOC107319050 TECTA AXDND1 LOC107306001 PHACTR2 CD59 LOC107319126 TFRC B3GLCT LOC107306678 PHKA1 CEBPA LOC107319205 TGM4 BACH2 LOC107306761 PIK3C2G CEL LOC107319240 TMBIM4 BAMBI LOC107306762 PIK3CA CHST12 LOC107319258 TMED3 BBX LOC107306776 PKP4 CLEC3A LOC107319261 TMEM156 BMF LOC107306817 PLA2G12A CLK4 LOC107319266 TMEM173 BMP6 LOC107306852 PLA2G7 CLPB LOC107320068 TMEM40 BMPR2 LOC107307576 PLCB1 CNIH3 LOC107320098 TMEM52 BTD LOC107308050 PLCD1 COX1 LOC107320103 TMEM56 BVES LOC107308084 PLD1 COX2 LOC107320131 TMEM63A C1HXorf36 LOC107308152 PLEKHB2 COX3 LOC107320199 TMEM71 C2CD2 LOC107308164 PPFIBP2 CPN2 LOC107320215 TMOD4 C2H18orf8 LOC107308181 PPM1K CPOX LOC107320470 TNFAIP8 C4H4orf33 LOC107308460 PPP3CA CREG1 LOC107320472 TNFRSF18 C4H8orf48 LOC107308462 PQLC3 CRLF2 LOC107320548 TRADD C5H11orf74 LOC107308654 PRDM11 CSF1R LOC107320703 TRPV2 C8H1orf210 LOC107308722 PRDM4 CTAGE5 LOC107320986 TTC39C C8H1orf226 LOC107308787 PREX2 CTSC LOC107321029 TTC7A CABLES1 LOC107308858 PRKD1 CTSD LOC107321102 TTPAL CADPS2 LOC107308953 PROCR CYTB LOC107321358 TUBB1 CALCRL LOC107309030 PROX1 CZH5orf51 LOC107321408 TUBD1 CAPN5 LOC107309087 PRR5L DECR1 LOC107321519 TUSC1 CAT LOC107309092 PRRG3 DNAAF1 LOC107321546 TYR CCDC85A LOC107309179 PRSS23 DNAH12 LOC107321550 TYRP1 CCNI LOC107309559 PTCHD1 DNAH14 LOC107321620 UIMC1 CCNY LOC107309563 PTP4A1 DNAH17 LOC107321658 UROD CCNYL1 LOC107309600 PTPN14 DNAH3 LOC107321681 UROS CD151 LOC107309698 PTPRB DOCK11 LOC107321684 USP45 CD2AP LOC107309880 PTPRM DOCK5 LOC107322046 VPS11 CD81 LOC107309932 PXDC1 DOCK8 LOC107322132 VRK3 CD9 LOC107310100 PXDN DOPEY2 LOC107322293 WAS CD93 LOC107310202 RAB12 DSCR3 LOC107322314 WDR41 CDC42EP1 LOC107310334 RAI2 EFHD2 LOC107322386 WDTC1 CDK17 LOC107310341 RANBP9 EMB LOC107322421 WIPF1 CDKL2 LOC107310343 RAP2B ENO3 LOC107322440 ZBTB8OS CEP85L LOC107310620 RAPGEF5 139 ENTPD5 LOC107322461 ZNF831 CGNL1 LOC107310625 RARRES2 EPN3 LOC107322623 ZNFX1 CHMP4C LOC107310736 RASAL2 ERAL1 LOC107322695 rna47256 CHRM3 LOC107310737 RASGEF1A EVI2A LOC107322790 rna47258 CHST7 LOC107310746 RASGRP3 EVI2B LOC107322824 CIB1 LOC107310815 RASSF8 F13A1 LOC107322904 CLCN3 LOC107310833 RECK FABP3 LOC107322910 CLN5 LOC107310894 RGL1 FAHD1 LOC107322928 CNDP2 LOC107310896 RGS2 FAM117A LOC107323028 CNTNAP2 LOC107310913 RHOB FAM129A LOC107323038 COBLL1 LOC107311894 RHOJ FAM134C LOC107323081 COLGALT2 LOC107312011 RIPK2 FAM13B LOC107323147 CORO2B LOC107312057 RMDN2 FAM210B LOC107323592 CPE LOC107312129 RNF11 FBXO18 LOC107323637 CPEB2 LOC107312133 RNF144A FBXO9 LOC107323914 CPN1 LOC107312528 RNF150 FCHO1 LOC107323924 CRIM1 LOC107312575 RNF19A FECH LOC107324314 CRY1 LOC107312640 RSU1 FETUB LOC107324323 CSGALNACT1 LOC107312798 RUFY3 FGFR1OP2 LOC107324364 CTDSP1 LOC107312799 RUNX1T1 FLVCR2 LOC107324389 CTDSPL LOC107312816 S1PR1 FMNL1 LOC107324445 CTNNB1 LOC107312822 SAMD12 FNIP1 LOC107324452 CUL4A LOC107312840 SASH1 FRRS1 LOC107324453 CYYR1 LOC107312977 SCARB2 FUCA2 LOC107324456 DCAF6 LOC107313022 SCP2 FUT7 LOC107324465 DEPTOR LOC107313134 SDCBP FUT8 LOC107324551 DESI1 LOC107313185 SDPR FYB LOC107324681 DGKZ LOC107313228 SECISBP2L GCNT4 LOC107324776 DHRS12 LOC107313257 SERF2 GDA LOC107324840 DHRS7 LOC107313495 SERHL2 GDPGP1 LOC107324841 DIRC2 LOC107313546 SETD7 GHITM LOC107324873 DISC1 LOC107313549 SFRP4 GMIP LOC107324930 DLEC1 LOC107313570 SFT2D1 GNA13 LOC107324939 DNM3 LOC107313670 SGCB GNAT2 LOC107325029 DNMBP LOC107313674 SGK1 GOLGA7B LOC107325190 DOCK1 LOC107313678 SH3GLB1 GPNMB LOC107325339 DOCK9 LOC107314087 SH3RF3 GPR157 LOC107325340 DPYD LOC107314285 SH3TC1 GPR174 LOC107325522 DYNC1I1 LOC107314378 SHANK3 GPRC6A LOC107325555 DYSF LOC107314473 SHROOM4 GPT2 LOC107325677 DZIP1 LOC107314525 SLC12A7 GPX1 LOC107325871 EDN1 LOC107314526 SLC12A9 GRB2 LRCH3 EDNRB LOC107314670 SLC17A5 GUCY2F LRRC25 EEPD1 LOC107314707 SLC22A23 HAX1 LY86 EGLN1 LOC107314714 SLC2A12 HCK LYRM9 EHD4 LOC107314972 SLC2A13 HDHD3 M6PR ELF2 LOC107315000 SLC30A1 HFM1 MADD ELK3 LOC107315001 SLC30A4 HIPK1 MAP3K6 ELMO1 LOC107315340 SLC35C1 HK3 MAP3K8 EMCN LOC107315364 SLC7A6 HMBS MAPT ENPP2 LOC107315680 SLCO2A1 HMGCL MAT1A EPAS1 LOC107315726 SOAT1 HPCAL1 MATN4 ERBIN LOC107315820 SOCS6 HSPB9 MBNL1 ERG LOC107315823 SORBS2 HYAL3 MGST1 ETFBKMT LOC107315983 SOS2 HYLS1 MITF EVC LOC107316143 SOX7 IFITM10 MKNK1 EVI5 LOC107316352 SPG20 IFNAR2 MOB1A EXOC3L1 LOC107316486 SPPL2A IGF2R MPHOSPH9 F10 LOC107316541 SPTBN1 IGLL1 MPP1 F8 LOC107316772 SRI IL22 MS4A15 FAM120B LOC107317020 SRPK2 IL2RG MSL1 FAM124B LOC107317216 SSFA2 IL5RA MTMR3 FAM13A LOC107317314 ST3GAL2 ING5 MXD1 FAM160A1 LOC107317332 ST3GAL6 IRAK4 MXD3 FAM160B1 LOC107317338 STAB2 IRF5 MYD88 FAM171A1 LOC107317376 STARD3NL IRF8 MYO1F FAM185A LOC107317519 STARD9 IRG1 MYO7B FAM214A LOC107317595 STK32C KATNA1 NADK FAM21C LOC107317596 STON1 KCNG2 NARF FAM234B LOC107317652 STS KCTD12 NCEH1 FAM43A LOC107318030 STXBP3 KIF1BP NCF2 FAM89A LOC107318190 STYK1 KIT ND1 FAT4 LOC107318199 SYNJ2BP KMT5A ND2 FBXL2 LOC107318369 SYT16 LARP1B ND4 FBXO8 LOC107318370 SYTL4 LDB1 ND4L FCHSD2 LOC107318671 SYTL5 LECT2 ND5 FGD6 LOC107318756 TANC1 LOC107305709 NEU2 FHOD1 LOC107318851 TBC1D1 LOC107305764 NFAM1 FJX1 LOC107319006 TBC1D22A LOC107305832 NFE2L2 FLRT2 LOC107319130 TBC1D30 LOC107305842 NHLRC4 FNDC3A LOC107320404 TBC1D8 LOC107305889 NIT1 FOXO1 LOC107321499 TBCK LOC107305946 NKX3-1 FOXO4 LOC107322457 TDRD5 LOC107306192 NKX6-3 FPGT LOC107322477 TGFBR2 LOC107306243 NLRC5 FRYL LOC107324276 TGFBR3 LOC107306265 NMBR FYCO1 LPAR4 THBD LOC107306618 NOS2 FYN LPAR6 THPO LOC107306830 NRROS FZD4 LRCH1 TIE1 LOC107306906 NT5DC4 FZD6 LRP1B TJP1 LOC107306909 NUDT7 GAB1 LRP6 TM2D1 LOC107307041 NUP210 GALK2 LRRC57 TM6SF1 LOC107307042 OAZ1 GALNT4 LRRC58 TMCC3 LOC107307139 OMG GDF7 LRRK2 TMCO3 141 LOC107307169 OXGR1 GLB1 LSAMP TMEM106B LOC107307292 PAPD4 GLB1L LTBP2 TMEM108 LOC107307293 PAX5 GLTSCR1L LYRM2 TMEM117 LOC107307355 PDE3B GNAL LYVE1 TMEM135 LOC107307435 PGD GNB4 MAB21L3 TMEM140 LOC107307508 PGP GNB5 MAN2A2 TMEM181 LOC107307602 PI16 GPHN MANSC1 TMEM241 LOC107307701 PI4KA GPR158 MAP3K9 TMEM26 LOC107307710 PIGV GPR50 MAP4K4 TMEM30A LOC107307799 PIK3AP1 GPR75 MAST4 TMOD3 LOC107307838 PIK3CD GPR89B MBTPS1 TMPPE LOC107307845 PIK3CG GPRIN3 MC5R TNFRSF11A LOC107308175 PLA2G15 GYPC MCEE TNFRSF1A LOC107308400 PLCB2 HACE1 MCTP2 TRAF3IP2 LOC107308694 PLCL1 HAPLN3 MEF2A TRIM45 LOC107308713 PLEKHA2 HDLBP MEI1 TRIM67 LOC107309064 PNPLA2 HEBP1 MERTK TSC22D1

Table S2: upregulated DEGs in M and HEC

M UP HEC UP

AAGAB GTF3C3 NAT10 TFAM ABCB5 GALNT16 NTF3 TUBB6 AASDH GUK1 NBAS TFDP2 ACOT11 GALNT5 OLFM3 TULP3 ABCA4 HACD2 NCOA1 TGFBR1 ACOT9 GALNT7 OPHN1 TWSG1 ABCE1 HACL1 NDC1 TGS1 ACTA1 GCK OPN4 UCHL1 ABCG5 HAS2 NDP THOC2 ACTA2 GIPC2 OST4 UCK2 ABI2 HDAC2 NEBL THUMPD2 ACTC1 GJC2 OTUD7B UHRF1BP1L ACBD7 HDAC4 NEDD1 TIAL1 ACTR1A GLRB OTULIN UNC45A ACTL6A HDX NEO1 TM7SF3 ACTR3 GLUD1 P3H2 UNC5C ACTR3B HEATR1 NFXL1 TMEFF1 ACVR2A GNA11 P3H3 UPRT ACVR2B HEATR3 NGB TMEM151B ADAM33 GNAS P4HA1 USP46 ADAMTS20 HMGB3 NKAIN3 TMEM163 ADAM8 GNG12 P4HA3 USP53 ADCK3 HMGN1 NKD1 TMEM168 ADAMTS14 GNG5 PAFAH1B2 USP8 ADGRA3 HNRNPDL NKRF TMEM169 ADGRG5 GORASP1 PALLD USP9X ADNP2 HNRNPH3 NKTR TMEM192 ADM2 GPR162 PAMR1 VCL ADSL HOMER2 NOC3L TMEM237 AFAP1 GPR37 PAPD5 VIM AGPAT3 HOXA1 NONO TMEM266 AHDC1 GPR68 PAPSS1 VOPP1 AKAP12 HOXA2 NRDE2 TMEM33 AKAP6 GRAMD4 PAPSS2 VTCN1 AKT1 HOXA3 NRG4 TMEM55A ALG5 GRIA3 PARVA WDR1 ALMS1 HOXA4 NSUN2 TMEM64 AMFR GSTK1 PCDH11X WLS ALS2 HS6ST2 NSUN3 TMPRSS3 AMIGO2 GYLTL1B PCDH18 WNT5B AMER1 HSF2 NUDCD1 TMTC4 AMOTL1 HGF PCDH19 WNT9A ANK3 HSPA14 NUFIP1 TNFRSF19 ANGPT1 HHAT PCGF2 YBX3 ANOS1 HSPD1 NUP153 TOMM70 ANKMY2 HIBADH PCOLCE2 YWHAQ AP3B2 HUNK NUP93 TP53BP1 ANKRD22 HPDL PDCD6 ZBED1 API5 ICE1 NUP98 TPH1 ANKRD29 HPS6 PDCL3 ZBTB1 AQR ICE2 NVL TPI1 ANKRD34C HSPA13 PDE10A ZC2HC1A ARGLU1 IFT27 ODC1 TPP2 ANKRD52 HSPA2 PDE5A ZC3H12C ARHGAP28 IGF2BP3 OGFOD1 TPR ANO1 HTR2B PDE6D ZDBF2 ARHGAP8 ILDR2 ORC2 TRA2A ANO10 HTRA3 PDGFC ZDHHC5 ARID1B ING3 ORC3 TRA2B ANO3 IFFO1 PGAM1 ZDHHC9 ARID3B INTS7 OTUD4 TRABD ANO5 IFNGR2 PGBD5 ZFHX4 ARL13B INTS8 OXSR1 TRHR ANTXR2 IFT43 PGM3 ZNF362 ARL3 INTS9 PAK3 TRMT1L ANXA2 IGF2BP2 PGRMC1 ZNF425 ARL5A IPO13 PANK1 TSHZ1 ANXA5 IGHMBP2 PHLDA1 ZNF622 ARL5B IPO5 PANX1 TSKU APBB2 IGSF3 PHLDB2 ZNF804B ASB7 ITPR2 PAQR3 TSN ARFIP1 IKBIP PICK1 ZNHIT6 ASNSD1 JARID2 PARD3 TSSC1 ARFIP2 IL34 PITPNM1 ZSWIM5 ATG4C JMJD4 PARD3B TTC14 ARHGAP42 ILK PKDCC ZYX ATRX JMJD7 PARG TTC21B ARHGEF11 IMPACT PKIA ATXN10 KCNAB1 PARP1 TTC26 ARHGEF17 INHBA PLCE1 B3GALNT1 KCNMB4 PAX2 TTLL12 ARHGEF4 INPP5A PLEKHG3 B3GNT5 KCNS2 PCCA TUBGCP2 ARID5B INPP5F PLEKHH2 BAZ2B KCNS3 PCID2 TUBGCP3 ARL1 IQGAP1 PLOD2 BBS5 KDM4A PCM1 TULP4 ARNTL IRX2 PLPP6 143 BCHE KDM5A PCMTD1 TXLNG ARPC2 ITFG1 PLXDC2 BCLAF1 KIAA0586 PCNP U2AF1 ARSJ ITGA11 PODXL BEND3 KIAA1549 PDCD11 U2SURP ASB2 ITGA8 POFUT2 BEND4 KIAA1549L PDGFRA UBE2D1 ATG9B ITGAV PPM1L BIVM KIAA2022 PELI1 UBE2E1 ATP10A JKAMP PPP1R3C BLOC1S4 KIF16B PEX13 UBE2E3 ATXN1 KANK4 PQLC2L BMPR1B KIF21A PGK1 UBE2K B3GALT2 KANSL1L PRDM5 BRD1 KIZ PHC1 UBE2N B3GNT9 KATNAL1 PRDX6 BRD7 KLHL13 PHF14 UBE2QL1 B4GALT2 KDELC2 PRKAG2 BRWD1 KLHL23 PHF5A UBE3A B4GALT4 KDELR3 PRRG4 BTBD9 KRAS PHF6 UBN2 BAG2 KIF26B PRSS12 BTF3L4 KRIT1 PHIP UBR5 BAG3 KIFC3 PSMD13 BTRC KRR1 PHKB UBXN7 BAK1 KLC4 PTCHD3 C10H15orf61 LAS1L PHOSPHO2 UCN3 BASP1 KLF10 PTK2 C1H12orf4 LDHB PIBF1 ULK4 BBS9 KLHL36 PTPN9 C1H7orf60 LEF1 PIH1D3 URB1 BDNF KTN1 PTPRN C2H8orf88 LEO1 PKNOX1 URB2 BICC1 LACTB PTPRN2 C8H1orf27 LGI2 PLEKHA5 USP16 BIN1 LBH PTTG1IP CACHD1 LIN28B PLEKHH1 UST BMPER LCMT2 PTX3 CACNB4 LIN52 PLRG1 UTP20 BORCS5 LEPROTL1 PUDP CAPRIN1 LNX1 PLXNA4 VBP1 BTBD10 LETM1 RAB10 CASP10 LOC107306757 PNISR VEZT C1GALT1C1 LIMA1 RAB15 CASP3 LOC107306862 PNPT1 VTA1 C1H2orf40 LIMD1 RAB23 CCDC173 LOC107306866 POLR2B WASF1 C1QTNF4 LIMK2 RAP1GDS1 CCDC59 LOC107308079 POMT2 WASF3 C8H1orf21 LIN7A RAPH1 CCDC61 LOC107308218 POU2F1 WDR11 CACNB2 LMF2 RARG CCDC88C LOC107308222 PPHLN1 WDR19 CALD1 LMO7 RASGRP1 CCNG2 LOC107308341 PPP1R3G WDR48 CAMK2D LOC107305692 RASL11B CCNJ LOC107308547 PPP2R3B WDR61 CAPN11 LOC107306754 RBPMS CCNT2 LOC107308730 PPP4R3B WDR75 CAPN2 LOC107306800 RCN1 CCT2 LOC107308740 PREP WRB CAPRIN2 LOC107306825 REST CCT5 LOC107308746 PRMT7 WRN CASC4 LOC107306834 RFTN1 CD24 LOC107308812 PROSER1 XPNPEP1 CBLB LOC107306841 RGS4 CDC40 LOC107308919 PRPF4B XPO5 CCDC102A LOC107306871 RGS9BP CDC5L LOC107308983 PRTG XRCC3 CCDC113 LOC107307951 RIC3 CDK8 LOC107309187 PSMA4 XRCC5 CCDC80 LOC107308888 RINT1 CDKN2AIP LOC107309251 PSMD14 XRN2 CCM2 LOC107308912 RIOK3 CECR2 LOC107309267 PSPC1 YAE1D1 CCSER2 LOC107308948 RNF149 CELF1 LOC107309281 PTBP2 YAP1 CD99 LOC107309052 ROBO1 CEP162 LOC107309305 PTCH2 YEATS2 CDC42BPB LOC107309196 ROR1 CEP295 LOC107309667 PTER YME1L1 CDKL1 LOC107309558 RPS6KA5 CEP97 LOC107309750 PTGDR ZBTB18 CDV3 LOC107309564 RPS6KL1 CFAP36 LOC107309809 PTGR2 ZBTB42 CELSR1 LOC107309567 RRAS2 CFAP61 LOC107310641 PTMA ZC3H14 CEP126 LOC107309618 RRBP1 CGGBP1 LOC107310649 PTN ZFAND4 CEP170 LOC107309619 RTN4 CHD7 LOC107310657 PTPN4 ZHX2 CEP170B LOC107309896 RYK CHGB LOC107310798 PWP2 ZIC3 CEP68 LOC107309997 SAMD4A CHIC2 LOC107310951 PYGO1 ZMAT3 CERS2 LOC107310048 SCAP CHRFAM7A LOC107311283 QRFPR ZMYM2 CFL2 LOC107310176 SCCPDH CKAP5 LOC107311432 RAB28 ZNF248 CHADL LOC107310367 SDC1 CLASP1 LOC107311492 RAB30 ZNF277 CHPF LOC107310519 SDC2 CLSTN3 LOC107311494 RBFA ZNF280D CHPF2 LOC107310722 SEC23A CMTR1 LOC107311554 RBM33 ZNF292 CHRDL2 LOC107310842 SEC23IP CNIH4 LOC107311589 RBM45 ZNF318 CHRM4 LOC107310852 SEC24D CNKSR3 LOC107312091 RBMX ZNF326 CHRNA2 LOC107310998 SEC31B COQ3 LOC107312144 RBP5 ZNF648 CHST10 LOC107311125 SEC61A2 CPSF2 LOC107312240 RCAN2 ZNF654 CHST14 LOC107311256 SEMA3C CPSF6 LOC107312272 RCOR3 ZRANB1 CHST2 LOC107311290 SEMA3E CREBZF LOC107312446 RFWD2 ZRANB2 CHST3 LOC107311795 SEMA4B CRIPT LOC107312514 RGMA CKAP4 LOC107312120 SERPINH1 CRNKL1 LOC107312724 RHOU CLCC1 LOC107312420 SERPINI1 CRYZL1 LOC107312753 RLIM CNIH1 LOC107312697 SERTAD2 CSDC2 LOC107312790 RNF2 CNN3 LOC107312843 SGCE CSNK1G1 LOC107312794 RNF4 COL11A1 LOC107313248 SH3BP4 CTDSPL2 LOC107312844 RNF8 COL12A1 LOC107313342 SH3D19 CTH LOC107312851 RNGTT COL1A2 LOC107313525 SH3PXD2B CTNND2 LOC107313038 RNMT COL3A1 LOC107313631 SHC4 CTR9 LOC107313438 RPAP3 COL5A2 LOC107313922 SHF CTTNBP2 LOC107313528 RPGR COL6A1 LOC107314317 SIPA1L1 CUL1 LOC107313656 RPP40 COL6A2 LOC107314438 SLC12A2 CUL4B LOC107313717 RPS6KA6 COL6A3 LOC107314540 SLC12A4 CXADR LOC107313962 RPUSD2 COPG1 LOC107314557 SLC16A2 DACT2 LOC107314132 RRP15 CREB3L1 LOC107314568 SLC20A2 DCLK2 LOC107314371 RRP1B CSNK2A2 LOC107314574 SLC25A4 DCX LOC107314487 RSPO3 CSPG4 LOC107314696 SLC27A4 DDX1 LOC107314585 RTCA CSRP2 LOC107314709 SLC2A3 DDX24 LOC107314723 RTF1 CTBP1 LOC107314914 SLC30A7 DDX3X LOC107314750 RTN4IP1 CTHRC1 LOC107314917 SLC35A5 DENND5B LOC107314806 RWDD1 DAGLA LOC107314954 SLC37A3 DESI2 LOC107314815 SACM1L DAP LOC107315268 SLC38A7 DET1 LOC107314847 SALL1 DCAF5 LOC107315292 SLC41A2 DHTKD1 LOC107315283 SALL3 DCN LOC107316274 SLC4A2 DHX15 LOC107315722 SBF2 DCTN1 LOC107316275 SLC4A3 DIEXF LOC107315759 SCAF11 DDAH1 LOC107316423 SLC5A6 DIS3 LOC107315955 SCAF8 DES LOC107316440 SLC9A6 DIS3L2 LOC107316090 SEC24B DFNA5 LOC107316593 SLIT2 DLG1 LOC107316524 SEMA7A DHRSX LOC107316691 SLX4IP DLG3 LOC107316637 SENP6 DHX36 LOC107316692 SMIM18 DLG5 LOC107316774 SEPSECS DLC1 LOC107316866 SNAI2 DLL1 LOC107316860 SETD2 DLGAP4 LOC107316896 SNX12 DNAJC17 LOC107317002 SF3B1 DLK2 LOC107316904 SNX33 DNMT3A LOC107317006 SF3B3 DNAH7 LOC107316980 SNX7 145 DOCK7 LOC107317009 SF3B6 DNAJB4 LOC107317105 SOGA3 DPYSL4 LOC107317050 SH3BGR DNAJC10 LOC107317449 SORCS2 DROSHA LOC107317055 SH3RF1 DNAJC25 LOC107317468 SOX4 DTNA LOC107317315 SHOC2 DNER LOC107317657 SPATA4 DYNC2H1 LOC107317372 SIPA1L2 DNPEP LOC107317794 SPEG DYRK1A LOC107317472 SIX4 DOK4 LOC107318056 SPRED1 EAF1 LOC107317506 SKAP2 DOK7 LOC107318415 SPTLC3 EDA LOC107317605 SLAIN1 DPYSL5 LOC107318625 SREBF2 EFCAB1 LOC107317619 SLC16A14 DRD5 LOC107318809 SRF EIF4A3 LOC107318221 SLC24A5 DSEL LOC107319543 SRP72 EIF4E LOC107318346 SLC35F3 DSG2 LOC107320701 SSR1 ELAVL4 LOC107318438 SLC38A8 DSTN LOC107321229 SSR3 ENY2 LOC107318449 SLC39A6 DUSP10 LOC107321238 ST3GAL1 EPCAM LOC107318520 SLC8A3 DUSP5 LOC107322296 ST5 EPHA4 LOC107318577 SLITRK3 DYNC1H1 LOC107323964 STARD13 EPHA5 LOC107318655 SLK DYNC1LI1 LOC107324203 STIM1 ERI3 LOC107318989 SMARCAD1 DYNC1LI2 LOXL3 STXBP6 ESF1 LOC107320437 SMARCC1 EDA2R LPP STYX EVX1 LOC107321247 SMARCD3 EDEM3 LRCH2 SULF1 EXOSC9 LOC107321951 SMC3 EDNRA LRFN5 SYDE2 EXTL3 LOC107323918 SMCHD1 EFHC1 LRP12 SYNJ2 EYA4 LPCAT1 SMOC1 EHHADH LRP4 SYNPO2 FAM103A1 LRIG3 SNCG EMC2 LRP8 TBC1D2B FAM110B LRPPRC SNRPB2 EMILIN1 LRRC20 TBCE FAM155B LRRC1 SNTG1 EMILIN2 LRRC6 TES FAM171B LRRC40 SNW1 EML6 LRRFIP2 TG FARP2 LYPD6B SNX6 ENAH LRRN4 TGDS FARSB LYPLA1 SOCS2 EPM2A LTBP1 TGFB2 FASTK MAP1A SOX11 ERBB4 MAP2K1 TGFB3 FASTKD2 MAP3K4 SP4 ERC1 MAP3K5 THBS1 FAXC MAP4K3 SP5 ERP44 MAP4 THBS2 FBXO3 MAP4K5 SPAG6 ETV1 MAP7D1 TIMP3 FBXW7 MB21D2 SPATA13 EVA1A MAPK8IP1 TMCO1 FERD3L MBD5 SPDYA EXOSC8 MAPRE3 TMEM138 FGF13 MBIP SPICE1 EXT1 MED28 TMEM150A FGF4 MDN1 SPOPL EXT2 MESDC2 TMEM150C FGF8 MEIS1 SPRTN FAM114A1 MFHAS1 TMEM209 FKBP4 METTL16 SPSB4 FAM13C MICU3 TMEM255A FMNL2 METTL5 SRSF11 FAM150B MINPP1 TMEM263 FMR1 METTL6 SS18 FAM155A MMP15 TMEM43 FRMD1 MGEA5 SSB FAM177A1 MMS19 TMEM61 FUBP1 MIPOL1 SSBP3 FAM49A MOB4 TMEM63B FXR1 MORF4L1 ST6GALNAC3 FAM81A MORN4 TMSB15B FYTTD1 MPP4 ST8SIA2 FAT1 MREG TMSB4X FZD3 MPP6 STAG1 FBN1 MSMO1 TMTC2 FZD7 MRAP STK3 FBXL15 MUC4 TNK2 146 G3BP2 MRE11A SUGT1 FERMT2 MYH15 TNKS1BP1 GAB2 MRPL1 SUMO1 FEZ2 MYH9 TNS1 GABPA MRPL53 SUPV3L1 FGF2 MYO1B TOMM7 GALNT13 MRPS14 SUV39H2 FHL2 NACAD TPM1 GAS2 MRPS18A SYNE2 FHL5 NALCN TPM3 GFRA4 MSANTD3 T FKBP14 NAV1 TPM4 GINS3 MSGN1 TAF1 FKBP7 NAV3 TRAM1 GLI3 MSH6 TAF2 FKBP9 NBEA TRAPPC10 GLRX2 MTA3 TBC1D19 FN1 NEK9 TRAPPC2 GOLT1B MTF2 TBX15 FNDC1 NEURL1 TRIB2 GPATCH2 MTMR9 TBX18 FOSL2 NFKB2 TRIM35 GPC3 MTURN TCP1 FRMPD4 NID1 TRIM54 GPLD1 MYEF2 TDG FURIN NOTCH2 TROVE2 GPR85 NAALADL2 TDRD9 FUT11 NPM3 TRPC4 GREB1L NAF1 TENM4 FZD8 NPNT TSC22D2 GRIP1 NAP1L4 TERT GADD45A NRBP1
My approach is driven by the fact that, despite efforts made by several groups worldwide, the precise identity of the hemogenic endothelium remains largely inaccessible. Indeed, several essential regulators such as Runx1, Gfi1, Gfi1b, CD44 Gpr56, cdca7… have been identified but the complete molecular identity is still escaping our scrutiny.

To reach this aim, I asked several questions:

-How could I define molecularly a hemogenic endothelium?

-What are the specific differences between endothelium and hemogenic endothelium?

-Could I reveal specific features that have not been previously described?

To this end, I undertook isolating a specific gene signature for each of the specific cell types compared to all the other ones. This strategy allows isolating gene sets that are specifically upregulated by a given cell type but excludes genes which expression is shared by several cell types. Indeed, several essential factors present in both the endothelium and hemogenic endothelium are erased as well as factors shared between the hemogenic endothelium and hematopoietic cells. Runx1, Gfi1, Flk1… disappear because they are not specific. Then I used this specific gene sets to create a unique expression matrix and reconstruct gene interaction networks using WGCNA. Interestingly, WGCNA reconstructed two HEC networks of up-regulated genes and one HEC network of down-regulated genes. Cytoscape representation of these gene networks reveals gene interactions within the network and genes with multiple connections. These genes are called hubs and carry essential functions in the network [START_REF] Charbord | A systems biology approach for defining the molecular framework of the hematopoietic stem cell niche[END_REF]Langfelder and Horvath, 2008).

In an attempt to decode the hidden identity of the hemogenic endothelium and the EHT, several bulk or single-cell transcriptomes have been produced over the past few years (For a review see [START_REF] Ottersbach | Endothelial-to-haematopoietic transition: an update on the process of making blood[END_REF]). All the papers report isolation of EHT based on a combination of endothelial-specific markers genes such as VE-Cadherin or ESAM, the putative hemogenic endothelium-specific marker Ly6A, Runx1 expression driven by the +23 enhancer, the combination of the transcription factors Gfi1 and Gfi1b, the expression of the endo-hematopoietic marker CD31 and the presence or absence of several hematopoietic-specific markers such as CD41, c-Kit or CD45. In brief, despite common markers used between the different teams, it is not clear whether the same, or even close, cell types or cell fractions are isolated.

Whatever the samples, the bioinformatics strategies are diverse for isolating specific differentially expressed gene sets.

Starting a few years ago, Swiers et al, 2013 were the first to establish the fact that HECs gradually lose their endothelial identity between E8.5-E10.5 as their hematopoietic potential increase and the endothelial and hematopoietic potential increase as well as that the endothelial and hematopoietic capacities are mutually exclusive. They took advantage of the RUNX1 +23 enhancer which is active in definitive HSPCs and HECs and performed the first functional and molecular analysis of embryonic HECs undergoing EHT. Therefore, they showed that the initiation of the ECs to become HECs is coordinated by RUNX1 (Swiers et al., 2013).

In 2005, Solaimani Kartalaei et al, took advantage of the Ly6aGFP (Sca1) mouse model, in which all HSCs throughout development are GFP + (de Bruijn et al., 2002; Joosten et al., 2002), to study the EHT. They present a set of RNA sequencing data obtained from highly enriched small numbers of EHT cells from Ly6aGFP embryos, aortic ECs, HECs, and emerging HSCs. Among the few (530) DEGs during EHT, they found Gpr56 as the highest up-regulated gene encoding a cell surface receptor and then showed the functional involvement of Gpr56 in HSC emergence during EHT. However, on the three experiments performed, one was clearly an outlier (Figure 2B) explained by lower sequencing depth. Nevertheless, the experiment was maintained for further analysis certainly inducing a bias in the final picture.

Using the same mouse model, this time li et al 2014, used another combination of marker (CD31, Cdh5, Esam and cKit) in order to compare GFP + and GFP - endothelial cells and intra-aortic cluster cells. They focused on a specific trait of their microarray analysis which is the inflammatory pathway in HECs. They found that interferon signaling increased HSPC production [START_REF] Li | Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production[END_REF]. The role of proinflammatory signals has also been shown to promote HSPC emergence from HECs in zebrafish [START_REF] Espín-Palazón | Proinflammatory signaling regulates hematopoietic stem cell emergence[END_REF][START_REF] He | Inflammatory signaling regulates hematopoietic stem and progenitor cell emergence in vertebrates[END_REF].

In 2012, and this time using the ES culture system, Lancrin et al aimed to look to downstream RUNX1 effectors that drive the EHT. To do so, they compared the transcriptomes of RUNX1 +/-and RUNX1 -/-hemogenic endothelium in ES culture (transcriptome generated by cDNA microarray). They found that the genes encoding Gfi1 and Gfi1b transcriptional repressors displayed significantly lower expression levels in the absence of RUNX1. Therefore, after chromatin immunoprecipitation, they demonstrated that RUNX1 directly regulates the expression of Gfi1 and Gfi1b at the onset of hematopoietic development [START_REF] Lancrin | GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment[END_REF]. These findings were further investigated by Thambryajah et al in 2015, who by combining mouse reporter lines and functional assays, they established that Gfi1 expression is initiated in a rare population of HE whereas GFI1B expression is upregulated in emerging IAHC. And for the first time, they described that the repressive CoREST complex is implicated in the transition through the recruitment by GFI1 and GFI1b, of LSD1 a member of this complex [START_REF] Thambyrajah | GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1[END_REF].

In 2014, Guiu et al focused on the role of NOTCH effectors in EHT this time and used genome-wide analysis of RBPj-targeted genes during embryonic hematopoiesis, CHIP assay and ChIP on chip experiments, coupled with the analysis in the jag 1 deficient mice. They could demonstrate cdca7 gene as a new Notch target gene involved in early hematopoietic development. And confirmed the requirement of cdca7 for the HSC and hematopoietic progenitor emergence in zebrafish [START_REF] Guiu | Identification of Cdca7 as a novel Notch transcriptional target involved in hematopoietic stem cell emergence[END_REF].

Back to RUNX1, Gao et al in 2018 point to the differences between the transcriptomic profiles of HE depending on the hematopoietic site comparing YS and AGM at E9.5 and E10.5. They supposed that the drivers of the different lineage potential are likely to be the distinct molecular properties of the HE cells from which they are derived. Using the expression of GFP from the endogenous RUNX1 locus, they isolated and compared HE from different anatomic sites. They get a global view of the TFs expressed in the different compartment validating some already discovered genes implicated in EHT [START_REF] Gao | RUNX1 and the endothelial origin of blood[END_REF].

The same year Baron et al, used single-cell transcriptomics to reveal the dynamic of HSC production in the aorta. This elegant study in vivo focused on the analysis of the composition of the intra-aortic clusters and found these to contain type I and type II pre-HSCs as well as more committed HSPCs. They were the first to isolate single whole IAHCs in the ventral and dorsal side of the aorta of mice embryos and demonstrated that they have similar transcriptomes. Concerning the silencing of the endothelial program during EHT, they identified a set of genes and transcription factors activated in the initiation of the hematopoietic program during EHT. They showed that very few genes were differentially expressed between HE and non-HE cells at E10 and E11 [START_REF] Baron | Single-cell transcriptomics reveal the dynamic of haematopoietic stem cell production in the aorta[END_REF].

In the most recent single-cell RNA-Seq study, CD44 was identified as a marker that becomes detectable specifically in the transitioning population in which endothelial and hematopoietic programs are co-expressed. In fact, together with ckit and Cdh5, CD44 allowed an accurate isolation of cell stages from non-HEC to mature HSPCs.

Furthermore, CD44 appears to have a functional role, as a disruption of the interaction with its ligand hyaluronan interfered with the EHT [START_REF] Oatley | Single-cell transcriptomics identifies CD44 as a new marker and regulator of haematopoietic stem cells development[END_REF]. In the same lab and using single cell transcriptomic techniques, Bergiers et al, studied the gene regulatory networks of the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium at the single cell levels. They found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically coexpressed in the hemogenic population. They surprisingly suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1 [START_REF] Bergiers | Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis[END_REF].

A close look at all these studies allows spotting flagrantly the heterogeneity in defining the HE population with different markers (review [START_REF] Ottersbach | Endothelial-to-haematopoietic transition: an update on the process of making blood[END_REF])) as well FA, phosphatase and Golgi apparatus. Hence in our culture system, we were able to define 2 distinct categories of HE and non-HE cells.

The difference in the number of DEGs in the previous analysis is due to the use of different bioinformatic methodology going from the isolation and comparison of populations, the exclusion of out layers or not, the definition of p-values and fold changes. In their analysis, many of the groups aimed to look for gene candidates, surface markers and transcription factors. However, in our study, the use of WGCNA allowed us to have a global view of each step of the transition considering networks and not specific genes (5 specific networks going from M to EC to HEC and HC). It allowed us to define the implication of ECM, cytoskeleton and morphological changes in HE going through EHT. In this study, we focused on studying the HEC networks, but indeed in the future, it is interesting to look for the specification of the endothelium from the mesoderm and the switch endothelium to hemogenic endothelium.

Since the EHT is transient and since not all the HE cells go through the transition together, we decided to perform single-cell transcriptomics on our culture system. To do so we isolated 4 samples (1 M, 2 HEC, and 1 HC) and we performed single-cell transcriptomics using 10X genomics. The analysis I showed in the results are preliminary and will be more refined, but we believe that this analysis will help us to capture a specific signature of cells undergoing EHT.

The parallel between EMT and EHT: what is the common side?

At first glance, EHT seems to share similarities with another major cell fate change: the epithelial-to-mesenchymal transition (EMT). It has been demonstrated

that TGFB signaling is shared between these two transitions. Activation of the TGFB pathway was shown to promote the EMT, while its role in the EHT has been seemingly controversial. While some reports suggested that activation of TGFB signaling promotes the EHT [START_REF] Monteiro | Transforming Growth Factor β Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells[END_REF], others suggested that TGFB activity needs to be down-regulated for the EHT to proceed [START_REF] Lempereur | The TGFβ pathway is a key player for the endothelial-to-hematopoietic transition in the embryonic aorta[END_REF]. Interestingly, in our HEC networks, we found some TGFB signaling members: TGFB1, TGFB3, TGFBR1, RBPMS… confirming that TGFB plays a role in the EHT.

More globally in our HEC networks we found some up-regulated genes known to play a role in the EMT as TESTIN, POFUT2, ITGAV, BMPER as well as downregulated genes like CTBP2 and TEAD4. This finding suggests that effectively EHT and EMT share some molecular signature.

While looking to the connectivity in our HEC network, we found between the most connected genes POFUT2 a GDP-fucose protein O-fucosyltransferase 2. It is an enzyme responsible for adding fucose sugars in O linkage to serine or threonine residues in Thrombospondin Repeats TSRs. The majority of POFUT2 targets are constitutive component of ECM or secreted matrix associated proteins able of influencing several processes as cell adhesion/migration, ECM synthesis, remodeling and finally modulating growth factor synthesis. In 2010, DU et al showed that the loss of POFUT2 in mice resulted in unrestricted EMT [START_REF] Du | O-Fucosylation of Thrombospondin Type 1 Repeats Restricts Epithelial to Mesenchymal Transition (EMT) and Maintains Epiblast Pluripotency During Mouse Gastrulation[END_REF].

Since POFUT2 is a hub in one of our HEC networks we thought that it could play an important role in the transition, so we decided to functionally validate it in our culture system. To do so we designed three siRNA against POFUT2 and performed transfection in our culture system. The knock-down of POFUT2 induces a decrease in the endothelial fraction and an increase in the hemogenic endothelial one suggesting that the loss of POFUT2 enhances the EHT. In addition, to further validate the gene network and its predictive value, we selected the POFUT2's first neighbors and examined if a decrease of POFUT2 mRNA expression was also associated with a modulation of several of its first neighbors. Based on gene connectivity, we selected 3 genes among the most connected to POFUT2. COL5A2 DNAJB4 and EMILIN1A.

The three chosen genes showed a significant decrease upon POFUT2 siRNA knockdown. Taken together, this indicates that POFUT2 and, at least, 3 of their first neighbors are acting coordinately to regulate the HEC fate and the hematopoietic production.

The knock-down of TESTIN another hub of our HEC network didn't show any effect on the EHT this time. The loss of TESTIN altered one of the tested first neighbors but not the others. It was interesting to look for the half-life of this protein and indeed we found that proteins of the extracellular matrix and the cytoskeleton have a half-life of the order of a few days and even a few months whereas the majority of metabolic enzymes last only a few hours which can explain that even if we inhibit the mRNA of TESTIN we still have the protein which can compensate this loss. This experiment point to the importance of looking to the half-life of the protein before performing the knock-down experiments.

NOTCH and WNT pathway: Do our culture system recapitulate the known role of Notch and wnt during embryonic hematopoiesis?

The involvement of the Notch pathway in hematopoietic development has been described in different system and species. Notch expression in aortic ECs and HCs displays a conserved signature between species. HC cluster production is accompanied by a decrease of Notch ligands. This pattern takes place as early as runx1 expression initiates in the hemogenic endothelium. This pattern is also prominent as HCs mature from CD41+ to CD45+ cells. Our transcriptomic data indicates that notch1 signaling (NOTCH1, JAG1, JAG2 …) is downregulated in our HC population. A requirement for Notch signaling and intraembryonic HSC production has been shown for mouse and zebrafish embryos. Loss-of-function experiments demonstrate that ablating Notch signaling suppresses definitive (embryo-derived), but not primitive (yolk sac-derived), hematopoiesis [START_REF] Burns | Hematopoietic stem cell fate is established by the Notch-Runx pathway[END_REF][START_REF] Kumano | Notch1 but Not Notch2 Is Essential for Generating Hematopoietic Stem Cells from Endothelial Cells[END_REF][START_REF] Robert-Moreno | Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1[END_REF]. Here, we show that the suppression of Notch signaling using DAPT enhances the hemogenic endothelial phenotype and the production of CD45 + HCs in our culture system. However, this phenotype was already shown in our lab by the use of DAPT directly in the chicken embryo, and mouse aorta organotypic culture causing an increase in the HC production [START_REF] Richard | Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis[END_REF]. Together these results suggest that the downregulation of Notch signaling in the hemogenic endothelium is required for aortic hematopoiesis to occur.

Concerning the Wnt pathway, when looking to the HEC networks, we find several actors of the Wnt pathway i.e CTNNB1, CTNNBIP1, MESD2, WLS, NPHP3, PORCN… Since it has been demonstrated that the generation of HSCs depends on β-catenin activity and becomes gradually β-catenin independent, we thought about testing an activator and an inhibitor of the β-catenin. We demonstrated that when using β-catenin inhibitor (PKF) we induced an increase of endothelial cells and a decrease of the hemogenic population. Whereas the use of β-catenin activator (SB) induces a decrease in the endothelial population and an increase in the hemogenic population.

These results are in keeping with the results published by [START_REF] Ruiz-Herguido | Hematopoietic stem cell development requires transient Wnt/β-catenin activity[END_REF] on the mouse AGM. Taken together we confirmed that the Wnt pathway plays a role in EHT.

Conclusion and Perspectives

Finally, I could demonstrate that our culture system does recapitulate all the steps of the EHT occurring in the dorsal aorta of the embryo. It goes from a nonengaged mesoderm which will acquire endothelial phenotype and then hemogenic endothelial fate, those HEC will then go through EHT and will produce HCs.

Bioinformatics and statistical tests allowed me to identify specific DEGs in each population compared to the others, therefore, to determine a single and specific signature of each population. Those DEGs were used to perform WGCNA a powerful tool to generate gene networks, it generates 5 modules (1M, 1EC, 1HC, and 2 HEC) represented as networks.

We focused on studying the HEC population and looked at the GO of the HEC networks. It shows specifically an enrichment with ECM, cytoskeleton and EMT actors, categories that have not been fully studied in the EHT yet. We demonstrated that in keeping with the literature, NOTCH and wnt pathways play a role in the EHT. And identified a new actor of this transition POFUT2 known to play a role in the EMT and could demonstrate that the loss of POFUT2 enhances the EHT. Together these results confirmed the predictive value of our gene networks.

Nevertheless, we are aware of some potential drawbacks of our culture system recapitulated as:

-Is the quail model relevant for the study of hematopoiesis?

-Is there any way to synchronize our culture system? -Does the system recapitulate the EHT of the embryo?

-Does the EHT occurring in our culture system recapitulate YS or AGM hematopoiesis and what type of hematopoietic cells are we generating? -Does our data set concur with the already published EHT in vivo and in vitro data sets?

As already described in the introduction, hematopoiesis is well conserved between species, the quail has been demonstrated as a key model for the study of this multistep process (quail-chicken chimeras…). We are convinced that hematopoiesis in quail is in keeping with the chicken and mammal ones. We faced different problems with this model and tried to find solutions. The first one is the absence of antibodies that can be used to identify the hematopoietic population. We managed to perform qPCR to validate the presence of the different hematopoietic cell categories using key lineage gene markers. The second one is that the culture system is not synchronized. Even if as described in Yvernogeau et al. 2016, almost 75% of the cells at day 2 are ECs, the EHT process is not synchronized and not all the HEC go through EHT and produce

HCs at the same time. In Yvernogeau et al., we identified a culture condition where in when the fetal serum is removed from the medium, 90% of the cells became endothelial. However, the culture is blocked at this stage and no EHT nor HC production occurs. This specific condition could be the key to synchronize the culture by removing the serum in the beginning of the culture and reintroducing it at day 4 when cells are ready to undergo EHT. The other one is to ensure that the hubs we have identified are actually expressed in the hematopoietic aorta. In order to assess that the culture system recapitulates cellular events occurring in the embryo, we are performing in situ hybridization in toto on chicken embryos to validate POFUT2 expression in the hematopoietic sites in particular the aorta.

To further test whether the culture system produces EMP or HSCs, I would perform grafts of hematopoietic cells produced by the culture into chicken, agematched, recipients.

The 2 data sets we generated will be further studied in the lab. Many perspectives are established. We will start by a comparison of our transcriptome with transcriptomes of cells isolated from embryos and ES cell cultures by others. This can be done in silico by the use of different transcriptomes generated by colleagues in the field. (cite les articles) and can contribute to a global view of the EHT leading to a better understanding of this process. It is true that we focused in our study on the HEC population and identified new actors in the EHT. Since our culture system recapitulates all the EHT steps, we will look in details to the first step of the commitment between EC toward HEC, a key question in the EHT field that have implications in regenerative medicine. Finally, since our cells can be efficiently transfected, our culture system can be further used to the validation of the expression of novel genes playing role in the EHT.

In vivo haematopoietic cell production by bone marrow-derived hemogenic endothelium

Yvernogeau L 1,¥ , Gautier R 1# , Petit, L 1# , Khoury, H 1 , Relaix F 2 , Ribes V 3 , Sang H 4 , Charbord, P 1 , Souyri M 1,7 , Robin C 5,6 and Jaffredo T *Correspondence to : thierry.jaffredo@upmc.fr

One of the major challenges in regenerative medicine is to reproduce tailor-made tissue-specific stem cells or early committed progenitors for cell replacement therapies. Despite recent progress 1,2 , controlled production of bona fide Haematopoietic Stem/Progenitor Cells (HSPCs) from pluripotent precursors still requires a better understanding of where, when and how these cells are physiologically produced during development. It is now well established that HSPCs derive from a specialized subset of Endothelial Cells (ECs) designated as haemogenic through an Endothelial-to-Haematopoietic Transition (EHT) 3-8 . EHT occurs early during embryogenesis in the main arteries of the embryo such as the aorta 9,10 . Whether EHT occurs at later stages and in other organs (e.g. bone marrow (BM)) is unknown. Here, we discovered the existence of a haemogenic endothelium capable of de novo generating HSPCs in the forming BM of chicken and mouse late foetuses and newborns. We identified the somite as the source of BM vascularization by tracing the BM-forming ECs through experimental embryology, genetic labelling and live imaging approaches and subsequently by analysing their haematopoietic progeny in adults. We demonstrated that the late foetal/newborn BM haemogenic ECs produce a small cohort of HSPCs capable of circulating and colonizing the secondary haematopoietic organs. Phenotypic and functional analyses disclosed that BM endothelium-derived HSPCs are mainly Multipotent Progenitors (MPPs) and a few Haematopoietic Stem Cells (HSCs). These cells harbour a very specific molecular signature close to the one of embryonic ECs with a prominent Notch pathway, endothelial-specific genes and transcription factors involved in EHT. Our results demonstrate that HSPCs are de novo generated past embryonic stages. We therefore disclose a new haematopoietic wave occurring in late foetal/newborn stage that fills the gap between the completion of embryonic haematopoiesis and the beginning of adult BM production. Identifying all steps of haematopoietic production and the molecular events controlling this process is of fundamental interest and might help to devise innovative stem cell therapies in the future.

To determine whether haemogenic ECs exist in the BM vascular network and their potential origin, we used a combination of experimental embryology, genetic approaches, transcriptomic and functional analyses on chicken and mouse models. Based on previous research 11-14 , the somite appeared as a promising site of BM endothelial origin. The somite is a mesoderm-derived tissue that produces axial skeleton, muscles of the body wall and limbs, dermis and endothelium 15 . Somite-derived ECs form the vascular network of the body wall, kidney and limbs 11-13 . To trace the endothelial derivatives of the somite in the chicken we performed isotopic and isochronic grafts of GFP + chicken 16 presomitic mesoderm (PSM), the somite anlage, into wild-type (WT) chicken embryos at embryonic day (E)2 (15 somite pairs) (Fig. 1a). GFP + somite-derived cells were followed in individual grafted chickens (n=34) from the time of grafting (E2) to the adult stage by performing immunostaining and flow cytometry analyses on circulating blood, embryo sections or whole mount organs (Extended Data Table 1). A collection of endothelial/haematopoietic antibodies (MEP21) 17 or haematopoietic-specific antibodies against CD45

(or PTPRC, pan-haematopoietic marker), CD41 (or ITGA2B, HSPC marker) 18 , c-Kit (or CD117, HSPC marker), KUL1 (macrophage/monocyte marker) and CD3 (T cell marker) were used. EHT was examined by live confocal imaging of BM thick slices (Extended Data Table 1). In agreement with our previous work 12 , PSM removal did not affect the aortic integrity (Extended Data Fig. 1a).

Importantly, PSM did not contain neither HCs nor ECs, as demonstrated here by immunostainings and flow cytometry analyses on dissected PSMs (Extended Data Fig. 1b-d) or previously by qRT-PCR 19 . It thereby rules out graft contamination by these two cell types later on during development.

Two days after grafting (at E4, n=5), PSM-derived GFP + cells (including emigrating ECs and myoblasts)

were visible in the limb bud (Extended Data Fig. 2a). GFP + cells were found around the neural tube, in limbs and the aorta. Of note, no GFP + cells were found within the intra-aortic clusters that harbour the first HSPCs (Extended Data Fig. 2b,c,d), as expected from previous reports 11,12 . By E8, the grafted tissue has contributed to the body wall and limb muscle masses and to vascular ECs (Extended Data Fig. 2e, f, g). No GFP + cells were found in the para-aortic foci (n=2) known to contain CD41 + HSPCs 20

(Extended Data Fig. 2h,i). These data therefore proved that GFP + cells do not contribute to the formation of the haematopoietic system at that stage, which is in keeping with the absence of GFP + circulating cells and with the established relationship between intra-aortic clusters and para-aortic foci 21 . The limb BM remained avascular until E12 (n=2), a stage when the first blood vessels associated with circulating CD41 + and CD45 + cells penetrated the cartilage tissue (Extended Data Fig. 2j-m). No GFP + circulating cell was detected from E4 to E12 (not shown). The contribution of GFP + cells to the BM was extensively analysed at E16/E17, a developmental stage when sufficient BM cells could be observed or collected for flow cytometry analysis (n=16). Whole BM mounts showed the colonization of the grafted limb by GFP + cells (Fig. 1b) and the presence of a dense GFP + vascular network in the forming BM (Fig. 1c,d and Supplementary Movie 1). Cross section of the BM confirmed the presence of GFP + sinusoids hence demonstrating the somite origin of the BM vascular network (Fig. 1e,f). Numerous GFP -CD45 + blood cells outside the GFP + vascular network were observed (Extended Data Fig. 3a-c). However, a subpopulation of GFP + CD41 + and to a less extent of GFP + CD45 + cells was tightly associated with the GFP + sinusoids (Fig. 1g-j, Extended Data Fig. 3). The co-expression of blood cell markers and GFP (as shown on confocal sections) clearly demonstrated that a fraction of CD45 + cells (Extended Data Fig. 3dl and Supplementary Movie 2) and CD41 + cells (Extended Data Fig. 4a-f and Supplementary Movie 3)

arose from GFP + somite-derived ECs in the BM. To ascertain the haematopoietic production in situ, we adapted the previously described time-lapse live confocal imaging 8 to BM thick slices of E16/E17 grafted embryos that were stained with anti-CD41 antibodies. We were able to witness flat GFP + CD41 -ECs becoming round while expressing CD41 + (as shown by CD41 staining after overnight imaging), therefore demonstrating the EHT (Supplementary Movie 4 and Extended Data Fig. 5). At E16/E17, a small definite fraction (0.1 to 1%) of GFP + blood cells was found circulating in line with the presence of round GFP + CD41 + or GFP + CD45 + cells in the contralateral left wing and hind limb BMs. In the grafted limbs, GFP + cells represented an average of 1.10±0.53% of the total BM mononucleated cells (Fig. 1k) that were found unevenly distributed among the endothelial and haematopoietic lineages. Strikingly, the GFP + cells significantly contributed to the BM haematopoietic production, representing 13.7±7.3% of the CD41 + cells, 3.0±1.3% of c-Kit + cells, 7.4±2.9% of CD3 + cells and also to 14.2±5.1% of the MEP21 + population and 0.97±0.52% of CD45 + cells (Fig. 1l). On the other hand, 42.9±5.4% of the GFP + cells were MEP21 + endothelial/haematopoietic cells whereas 32.4±10.4%, 28.4±8.7%, 17.2±4.7% and 5.0±2.8% of the GFP + cells were CD45 + , CD41 + , c-Kit + or CD3 + , respectively (Fig. 1m and Extended

To further specifically trace the progeny of ECs in the long term after birth, we used an indelible marking system to target the endothelium. For this purpose, floxed reporter YFP transgene mice 23 were crossed to mice with a tamoxifen-inducible Cre driven via the complete endothelial-specific VE-cadherin (CDH5) upstream regulatory sequences 24 . Day (D)1 pups received one injection of 50µg tamoxifen every day during 3 days (Fig. 3A). BMs were then analysed at different time points up to 15 weeks (105 days)

for the presence of YFP + endothelial and haematopoietic cells (n=2-6). Importantly, no CD45 + cells displayed YFP expression immediately following the last tamoxifen injection ruling out contamination from haematopoietic progenitors expressing CD144 (Extended Data Fig. 10a). D27 BM analysis uncovered the presence of 0.096±0.011% YFP + cells in total mononuclear cells (Fig. 3b).

Haematopoietic cells (4.1±0.4% of CD144 + CD45 + and 0.0054±0.0013% of CD144 -CD45 + ) and ECs (42.4±6.8 % of CD144 + CD45 -) were YFP + (Fig. 3c). This staining is accurate regarding the absence of YFP staining observed in the non-induced animals (Extended Data Fig. 10b). Following-up the total number of YFP + cells, we observed that CD144 + CD45 -ECs and the rare lineage-negative Sca1 + c-Kit + (LSK) HSPCs decreased over time to reach almost zero 105 days after birth, indicating a progressive disappearance of the haemogenic endothelium and haematopoietic production with age (Fig. 3d). Of note, inductions at later stages after birth (n=3-7) resulted in a reduced number of YFP + CD144+CD45cells and LSK cells, confirming the progressive loss of haemogenic endothelium with age (Extended Data Fig. 11). Aiming to better identify the immature YFP + cell population, we analysed LSK cells using the SLAM family markers known to distinguish HSCs from MPPs 25 (Fig. 3e). We disclosed the presence of 7.8±1.6% of YFP + cells in the LSK population (Fig. 3e). YFP + cells were highly enriched in the CD48 - CD150 -MPP fraction 26 (23.1±4.7%) and also present in the CD150 + CD48 -HSC fraction (5.0±1.6%) (Fig. 3e,f). Further analysis using CD229 and CD244 markers 25 to hierarchically discriminate the different haematopoietic progenitor populations revealed the presence of 65.3±8.9% of YFP + cells in the CD150 -CD48 -CD229 + CD244 + MMP3 fraction that represents transiently reconstituting haematopoietic progenitors and to a less extent in the MMP1 population that represents intermediate and transiently reconstituting progenitors (5.9±4.2%, Fig. 3g). Again, this staining was accurate when compared to the non-induced animals (Extended Data Fig. 10c).

To identify the HCs originating from a VE-cadherin ancestor at the molecular level, we performed transcriptomic analysis on YFP -and YFP + LSK cells isolated from D27 BM. Principal component analysis (PCA) with the entire set of 30,922 genes separated YFP + from YFP -LSK cells (Extended Data Fig. 12a and Table S3). Gene Ontology (GO) using database for annotation, visualization, and integrated discovery (DAVID, http://david.abcc.ncifcrf.gov) performed on differentially expressed genes (DEG) upregulated in the two populations (i.e. 1,417 genes in the YFP + and 1,552 genes in the YFP -LSK fraction, p-value= 0.03), disclosed differentially represented GO categories (Extended Data Fig. 12b). 12c

) now identified 4 groups that are circled in Fig. 4a (lower panel). To find out how DEGs are interrelated within the two populations, we resorted to weighted gene correlation network analysis (WGCNA) 27 , which disclosed two major gene modules (sub networks). The magenta and black modules corresponded to genes up-regulated in YFP -and YFP + LSK cells, respectively (Fig. 4b). GO analysis of the two gene sets revealed strong differences in their biological functions. The magenta network contained genes implicated in cell proliferation (cell cycle, DNA replication, protein translation, mRNA splicing). The black module was enriched in molecules implicated in ECM, actin cytoskeleton and ECMto-actin cytoskeleton interaction (Fig. 4c). Close-up view of the genes within the cyan network confirmed the presence of a core of transcription factors including key regulators of haematopoiesis, such as Hoxa9, Meis1 and Etv6 (Fig. 4d). The presence of several Notch genes, from canonical (Notch4, Dll4)

and non-canonical (Mamld1), pathways and many endothelial genes such as Pecam1, Kdr, Flt1, Flt4

and Cdh5, Aplnr (Fig. 4d) is a compelling argument for the endothelial origin of YFP + LSK cells. We finally tested the HSC potential of the YFP + and YFP -LSK cells using transplantation experiments into irradiated recipients. YFP -LSK cells contributed to a robust, multilineage haematopoiesis (Fig. 4e).

Remarkably, YFP + LSK cells also contributed, albeit to a less extend, to multilineage haematopoiesis with a preferential differentiation towards the lymphoid and erythroid lineages (Fig. 4e, f and Extended Data Fig. 13).

In conclusion, our cell tracing studies in both chicken and mouse species have identified a new wave of haematopoiesis (MMP3 and HSCs) occurring de novo in the BM of late embryonic/newborn and deriving from resident haemogenic ECs of somite origin. It is well known that the haematopoietic cells originating from the yolk sac will colonize the foetal liver with definitive erythroid, megakaryocyte, macrophage, neutrophil, and mast cell progenitors 28 . These progenitors will be sufficient for the mouse embryo to survive until birth, when the HSC-dependent wave will take over. One may speculate that the new transient haematopoietic production discovered in our study fills the gap between the end of the yolk sac haematopoiesis and the bone marrow HSC-dependent production. Interestingly, a transient HSC population harbouring robust adult haematopoietic reconstitution potential was recently uncovered during mouse embryonic life. This precise HSC population was shown to be lymphoid biased 29 . This, together with our finding reveals an unappreciated complexity of the ontogeny of the haematopoietic system. Indeed, the pool of HSCs that expended in the foetal liver starts to colonize the bone marrow at E17 30 . Since at birth bone marrow colonization is low and HSCs are present in very few numbers, some time might be required before HSCs find their final adult-type niches and start to differentiate and proliferate into more committed progenitors and mature blood cells. Alternatively, this transient haematopoietic wave might prepare the bone marrow niches to mature to an appropriate state to accommodate the HSCs generated in the foetal liver. Such hypotheses will need to be verified in the future. A recent paper 31 indicates that yolk sac-derived erythromyeloid progenitors may contribute to intraembryonic vascular endothelium where they persist until adulthood, therefore unraveling an unexpected source of ECs. In our approaches, PSM does not contain erythromyeloid progenitors hence ruling out a contribution of this cell type to HSPC formation by the BM. A model of cell origins and lineage relationships in both chicken and mouse species is displayed in Fig. 5. Overall, our study proved that HSPCs could be produced through a haemogenic endothelium intermediate past the embryonic aortic and yolk sac stages, an important knowledge that should help designing novel therapies for haematopoietic disorders.

Methods

Antibodies for immunohistochemistry and flow cytometry.

See Extended Data Table 2.

Grafts of avian presomitic mesoderm (PSM).

Wild-type and transgenic GFP chicken (Gallus gallus) eggs were incubated at 38°C ± 1 °C in humidified atmosphere until embryos reached the appropriate stage established using Hamburger and Hamilton development tables 32 . GFP chicken were generated and kindly provided by the Roslin Institute 16 . An Chicken cell staining and analysis.

Chicken BM cells were flushed with PBS containing 5% FCS (Gibco) (PBS/FCS). The suspension was washed and passed through successive gauge needles [START_REF] Makarova | Replication and transmission of influenza viruses in Japanese quail[END_REF]23 and 26G,Terumo) to obtain a single cell suspension. Thymus, spleen and bursa of Fabricius were crushed; cell suspensions were filtered through a 40µm Nylon Cell Strainer (Falcon), and then washed twice in PBS/FCS.

Cell suspensions were then incubated with antibodies for 20 minutes on ice. Cells were washed and suspended in PBS supplemented with DAPI or 7-AAD (to exclude dead cells). Stained cells were analysed on a MACSquant (Miltenyi Biotec) or FACScalibur (Becton Dickinson).

Immunostainings on whole bone marrow, thymus, thick embryo slices and PSM.

Bone marrow and thymus were prepared as previously described in 31 . Tissues were stained with anti-GFP, anti-CD45 and anti-MEP21 antibodies (described above). A goat anti-rabbit IgG (H+L) coupled to Alexa Fluor 488, a goat anti-mouse IgG2a coupled to Alexa Fluor 555 and a goat anti-mouse IgG1 coupled to Alexa Fluor 647 (all from ThermoFisher Scientific) were used to reveal the anti -GFP, anti-CD45 and anti-MEP21 antibodies, respectively. Thick slices and dissected PSM were stained with anti-CD45 and anti-MEP21 antibodies. PSM were counterstained with DAPI. Transparent bone marrow, thymus, thick slice and PSM were imaged with a Zeiss LSM700 confocal microscope with a 10xPlanApo dry objective. Three-dimensional reconstructions were generated from z-stacks with Imaris software and converted to QuickTime files (.Mov).

Time-lapse confocal imaging of the chicken grafted bone marrow.

Transversal slices of E16 PSM grafted bone marrows were processed for time-lapse confocal imaging as described in 8,33 . Briefly, E16 grafted bone marrows were gently flushed out of the bones. Transversal Mice.

Experiments were carried out in accordance with the guidelines of the French Veterinary Department.

Approval was obtained from the French Ministry of Agriculture institutional review board for these studies. ROSA26 mT/mG reporter mice contain a double-fluorescent Cre reporter that expresses membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision 23 . ROSA26-stop-EYFP mutant mice have a loxP-flanked STOP sequence followed by the Enhanced Yellow Fluorescent Protein gene (EYFP) inserted into the Gt(ROSA)26Sor locus 34 . Pax3-Cre mice were generated by replacing the first Pax3-coding exon with a gene encoding Cre recombinase. Mice were grown as heterozygous 35 . Tg(Cdh5Cre/ERT2) transgenic mouse line was generated by insertion of a tamoxifen-inducible Cre (CreERT2) into a large PAC clone of the Cadh5 (VE-Cadherin) gene resulting in endothelium-specific expression 24 .

Pax3-mTmG mice generation

Homozygous mT/mG females were mated with heterozygous Pax3-Cre males. Foetuses were collected between E19 and E21, phenotyped based on the GFP expression in the central nervous system and analysed for their BM content.

Cdh5-YFP mice generation and induction

Homozygous ROSA-YFP females were mated with homozygous Tg(Cdh5-Cre/ERT2) males. After birth, inductions were made by intra-peritoneal injections of tamoxifen (T5648, Sigma) diluted in corn oil (C8267, Sigma).

Mouse bone marrow analysis and cell sorting.

Femurs and tibias were flushed with DMEM (Gibco) supplemented with 10% FCS (Eurobio). Cells were dissociated with Collagenase type 1 (C0130, Sigma) 1.25mg/ml in PBS+10% FCS for 30 min at 37°C, and washed twice with DMEM+10% FCS. 1 to 5.10 6 cells were stained with fluorescent antibodies (Extended Data Table 2) in PBS+0.5% BSA for at least 30 min at 4°C. Cells were washed, and suspended in PBS+0.5% BSA supplemented with 1.25 µg/ml DAPI (D9542, Sigma) for cell viability and analysed on a MACSQuant VYB (Miltenyi Biotec) or a LSR II (Becton-Dickinson) flow cytometer. Data were reanalysed with FlowJo X software.

For cell sorting, after collagenase treatment and washing, red blood cells were lysed using Gey's solution for 5 min at 4°C. After two washes with DMEM+10% FCS, Lin depletion was performed by incubation of cells with purified Gr-1, Ter119, B220, CD11b, CD4 and CD8 antibodies in PBS+0.5% BSA for 30 min at 4°C. After a wash, cells were incubated with Goat anti-rat microbeads (Miltenyi Biotec) for 30 min at 4°C. After washing, cells were applied on a LD column (Miltenyi Biotec) according to the manufacturer's guidelines and eluted Lin-cells were collected. Lin-cells were then stained with Sca1-PE and CD117(c-Kit)-PE-Cy7. LSK YFP + and YFP-cells were sorted using a FACS Aria III (Becton-Dickinson) cell sorter.

Analysis of transplantation capabilities.

Transcriptomic analysis

Sorted LSK YFP + and YFP -cells were lysed with 350µL RLT+ buffer (RNeasy Plus Micro kit, Qiagen) and conserved at -80°C. All RNA isolations were performed at the same time, according to the manufacturer's guidelines.

Affymetrix Mouse Gene 2.0 ST arrays were used. RNA concentration and integrity were evaluated with the Agilent bioanalysor 2100 (Genomi'C Facility platform, Cochin Institute, Paris). To correct from probe set definition inaccuracy, we used the version 17 of the custom Chip Definition File (CDF).

Design of the screening strategy and microarray analysis

Non-supervised analyses were performed on the global transcriptome of 6 samples (3 YFP + and 3

YFP -replicates) and data were represented with Principal Component Analysis (PCA). Gene upregulated in YFP + vs YFP -cells were selected using ANOVA. Selected gene sets were then processed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov) 36,37 to identify representative biological activities of YFP + vs YFP -LSKs.

For PCA, hierarchical clustering and other statistical analyses, we used Partek and JMP softwares. To find how our neonate bone marrow samples were positioned in relation to E11.5 AGM and 3 week-old bone marrow samples, we merged the present data with previously generated AGM and adult bone a, Scheme depicting the isotopic and isochronic graft of a PSM, the somite anlage, from a ubiquitous GFP transgenic chicken embryo into a wild-type chicken embryo host. PSM grafts were performed at E2, at the level of somites 14-21, which corresponds to the wing bud development 38,39 . b, The E16/E17 limb was colonized by GFP + somite-derived cells that formed visible muscle masses (arrows). c, d,

Isolated

Legend of the Table box

Not done

Not applicable

Extended Data Table 1. Number of grafted chickens, time points of analyses and type of analyses performed.

This table summarizes the number of chicken samples according to the age at analysis; the organs or tissues analysed, i.e., thymus, bone marrow, spleen; the kind of analysis applied i.e., FACS, immunostaining, confocal imaging or videos; and the number of cases analysed for each time point.

INTRODUCTION

Japanese quail (Coturnix japonica) is a popular domestic poultry species raised for meat and eggs in Asia and Europe. Quail have been used in genetics research since 1940 1 , and are an increasingly important model in developmental biology, behaviour and biomedical studies 2 . Quail belong to the same family as chickens (Phasianidae) but have several advantages over chickens as a research model. They are small and easy to raise, have a rapid growth rate and a short life cycle, becoming sexually mature only seven to eight weeks after hatching 3 .

Quail have become a key model in several research fields. The avian embryo has long been a popular model for studying developmental biology due to the accessibility of the embryo. The quail embryo survives manipulation and culture better than chicken embryos making them ideal for this type of research 3 . Quail have been used as model for stem cell differentiation, for example a culture system that mimics the development of hematopoietic stem cells has been recently developed, as quail show greater cell multiplication in these cultures than chickens 4 . Quail are also used to study the genetics underlying social behaviours 5 , sexual behaviour 6,7 , pre-and postnatal stress programming 8 , and emotional reactivity 9-12 . Japanese quail have a fast and reliable reproductive response to increased photoperiod, making them an important model species for investigation into seasonal behaviour and reproduction in birds 13-15 . The molecular mechanisms behind seasonality including metabolism and growth, immunity, reproduction, behaviour and feather moult is poorly understood despite its importance in the management of avian species.

Quail are also important in disease research. Strains of quail have been developed as models of human disease such as albinism 16 or necrotizing enterocolitis in neonates 17 , and quail lines have been selected on their immunological response 18 . There are key differences in the immunogenetics of quail and chicken -particularly in the major histocompatibility complex (MHC) 19,20 . Investigating the immunology quail is important for understanding infectious disease spread and control in poultry. For example they are a key species for influenza transmission, with previous research showing that quail may play a key role as an intermediate host in evolution of avian influenza 21-23 . Zoonotic H5N1 influenza strains have crossed from quail to human causing mortality in the past 24,25 , making them a potential pandemic source.

We have produced a high-quality annotated genome of the Japanese quail (Coturnix japonica). Here we describe the assembly and annotation of the quail genome and demonstrate key uses of the quail genome in immunogenetics, disease, seasonality and behavioural research demonstrating its utility as an avian model species.

RESULTS

Genome assembly and annotation

We sequenced a male Coturnix japonica individual from an inbred quail line using an Illumina HiSeq 2500 instrument. Total sequence genome input coverage of Illumina reads was ~73x, using a genome size estimate of 1.1 Gb. Additionally, 20x coverage of long PacBio reads were sequenced and used to close gaps. The assembled male genome Coturnix japonica 2.0 is made up of a total of 2,531 scaffolds (including single contigs with no scaffold association) with an N50 scaffold length of 2.9 Mb (N50 contig length is 511 kb). The assembly sequence size is 0.927 Gb with only 1.7 % (16 Mb) not assigned to 33 total chromosomes. Coturnix japonica 2.0 assembly metrics were comparable to previous assemblies of Galliformes, and superior to other quail genomes 26,27 in ungapped (contigs) sequence length metrics (Table 1). Specifically, in comparison to recently published genomic data from the Japanese quail 27 , our genome is substantially less fragmented (contig N50 of 0.511 Mb vs 0.027 Mb), has been assigned to more chromosomes, and has more complete annotation with ncRNA, mRNA and pseudogenes predicted. Our estimate of total interspersed repetitive elements was 19% genome-wide based on masking with Windowmasker 28 . In the genomes of other quail species the estimated repeat content was much lower, ~10% less in both species 26 .

To improve the quantity and quality of data used for the annotation of the genome, we sequenced RNA extracted from seven tissues sampled from the same animal used for the genome assembly. Using the same inbred animal increases the alignment rate and accuracy. The amount of data produced for annotation from the 7 tissues is respectively in giga bases: 18.9 in brain, 35.6 in heart, 19.3 in intestine, 27.8 in kidney, 39.0 in liver, 18.8 in lung and 34.0 in muscle. High sequencing depth was aimed at, to help detect low expression genes, including tissue-specific ones, in these various tissues. In total we predicted 16,057 protein-coding genes and 39,075 transcripts in the Coturnix japonica genome (Table 2). In comparison to other assembled and annotated Galliformes, transcript and protein alignments of known chicken RefSeq proteins to Coturnix japonica suggest the gene representation is sufficient for all analyses described herein (Table 3). However, we find ~1000 fewer protein coding genes in the Japanese quail than the northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) genomes 26 . We attribute this to the use of different gene prediction algorithms, and the slightly lower assembled size of Japanese quail, 927 Mb compared to 1 Gb in other quail genomes 26 (Table 1).

For further annotation, a set of genes unnamed by the automated pipeline were manually annotated. As part of an ongoing project to investigate hemogenic endothelium commitment and HSC production 4 , transcriptomes were produced for two cultured cell fractions. Study of these cells is critical for developmental biology and regenerative medicine, and quail are an excellent model for studying these as they produce much more hematopoietic cells than similar chicken cultures. Approximately 8000 genes were expressed in these cells lines which lacked genes names or annotation from the automated annotation pipeline. Using BLAST 29 searches to identify homology to other genes, approximately 3000 of these were manually annotated (Supplementary Data 1).

Genome completeness was also quantitatively assessed by analyzing 4,915 single copy, orthologous genes derived from OrthoDB v7 and v9 30 . Presence and contiguity of these conserved, avian specific genes were tested with BUSCO v3.0.2 31 . A comparison with the chicken assembly (Gallus gallus 5.0 32 ) indicates that 95% of these genes are present and full length in all three assemblies. The percentage of duplicated, fragmented and missing genes are also very similar between the assemblies (Supplementary Fig. 1). The quail genome has 10 more missing and 23 more fragmented genes than the Gallus gallus 5.0 assembly. However, relative to the total number of genes in the benchmarking set, these increases amount to just 0.2% and 0.5%, respectively. This indicates that the quail genome, like the chicken genome, is highly contiguous and in terms of its expected gene content, it is close to complete.

Galliforme genome synteny

Immune gene repertoire

We investigated the immune genes in the quail genome in detail due to the importance of quail in disease research. The MHC-B complex of the quail has been previously sequenced and found to be generally conserved compared to chicken in terms of gene content and order 19,20 . However the quail MHC contains a higher copy number of several gene families within the MHC-B 19 and shows increased structural flexibility 20 , as well as an inversion in the TAP region 19 . The MHC-B sequence in the quail genome extends from the previously sequenced scaffold, and this additional region also contains similar gene content and order to chicken, but with gene copy number variations. As in the chicken, the CD1A and B genes are found downstream of the MHC I region, while many TRIM family genes and IL4I1 are encoded upstream. The BG region, which encodes a family of butrophylin genes known as BG genes in the chicken, was also present in the quail. Within this region, six BG genes were identified in the quail, compared to thirteen in the chicken 40 . At least five of these BG genes are transcribed in the quail lung and ileum. The chicken and turkey have an additional MHC locus known as the Rfp-Y or MHC-Y locus, which contains several copies of non-classical MHCI-Y and MHCIIB-Y genes. However, no MHC-Y genes have been previously identified in quail. BLAST searches of both the quail genome and quail transcriptomes, as well as the bobwhite and scaled quail genomes, failed to identify any MHC-Y genes, indicating this locus probably does not exist in the quail.

Cathelicidins and defensins are two families of antimicrobial peptides that have activities against a broad range of pathogens and exhibit immune-modulatory effects. Orthologs of all four chicken cathelicidins and of thirteen chicken defensins 41 were identified in the quail genome (Supplementary Data 5). Due to their high divergence, of the thirteen defensins only four were annotated through the annotation pipeline, with the remainder identified through BLAST and HMMER searches with chicken defensins. The only poultry defensin missing from the quail genome was AvBD7. The defensins were encoded in a 42 kb cluster on quail chromosome 3, as in chickens. A 4 kb gap in the scaffold in this region may explain the missing AvBD7 sequence.

Several genes are thought to be key for influenza resistance in both humans and birds, including RIG-I, TLR and IFITM genes. RIG-I has not previously been identified in chicken, despite being present in ducks and many other bird orders, and is considered highly likely to be deleted from the chicken genome 42 . In addition, an important RIG-I binding protein RNF135 has also not been identified in chicken 43 . Likewise, an ortholog of RIG-I or RNF135 could not be identified in the quail genome or transcriptomes through BLAST searches and therefore is likely missing in the quail also. Orthologs of all five chicken IFITM genes (IFTIM1, 2, 3, 5 and 10) were identified in the quail genome and transcriptomes. In addition, orthologs of each chicken TLR, including key TLRs for viral recognition, TLR4 and TLR7, were identified in the quail genome, except for TLR1A.

Selection for social motivation

Quail has been used as a model to study the genetic determinism of behaviour traits such as social behaviours and emotional reactivity 11,12,44 , these being major factors in animal adaptation. Moreover quail selected with a low social motivation behave in a way that can be related to autistic-like traits, so the genes and causal variants are of wider interest to the biomedical community. Here we use the new quail genome assembly to improve previous results on the detection of selection signatures in lines selected for sociability. Due to the non-availability of a useable quail reference genome at the start of these studies, genomic sequence data produced from two DNA pools of 10 individuals each from two quail lines diverging for social motivation had been aligned to the chicken reference genome, GallusWU2.58 45 . As a result, only 55% of the reads had mapped in proper pairs, whereas by using our quail genome as a reference, this number increased to 92%. This corresponds to an improvement of the averaged coverage from 9x to 20x and of the number of analysed SNPs from 12,364,867 to 13,506,139. The FLK 46 and local 45 score analysis led to the detection of 32 significant selection signature regions (p < 0.05), (Supplementary Data 6). Supplementary Fig. 2 shows an example of such a region on Chr20. This represents a substantial improvement in the number of detected regions, compared with the 10 regions obtained when using the chicken genome as a reference 45 . Of the 32 detected regions, six may be merged in pairs due to their physical proximity, four regions map to new linkage groups absent in the previous analysis, and eight correspond with results obtained in the previous study (Supplementary Data 6). Altogether, 17 new regions were detected. Of these, eight could be seen in the previous analysis, but had not been considered as they did not reach the significance threshold, and nine are solely due to the availability of our quail assembly. Two very short selection signatures previously detected using the chicken assembly as reference are not recovered here and were most probably false positives.

These results confirm the selection signature regions harbouring genes involved in human autistic disorders or being related to social behaviour 45 (PTPRE, ARL13B, IMPK, CTNNA2). Among the genes localised in the newly detected genomic regions, several have also been shown to be implicated in autism spectrum disorders or synaptogenic activity (Supplementary Data 6): mutations in the EEF1A2 gene (Eukaryotic elongation factor 1, alpha-2 ) have been discovered in patients with autistic behaviours 47 ; EHMT1 (Euchromatin Histone Methyltransferase 1) is involved in autistic syndrome and social behaviour disorders in human and mouse 47-50 ; LRRTM4 (Leucine Rich Repeat Transmembrane Neuronal 4) is a synapse organizing protein, member of the LRRTM family, involved in mechanisms underlying experience-dependent synaptic plasticity 51 . Autistic spectrum disorders are observed in several disorders that have very different aetiology, including fragile X Syndrome, Rett Syndrome or Foetal Anticonvulsant Syndrome. While these disorders have very different underlying etiologies, they share common qualitative behavioural abnormalities in domains particularly relevant for social behaviours such as language, communication and social interaction 52,53 . In line with this, several experiments conducted on high social (HSR) and low social (LSR) reinstatement behaviour quail indicate that the selection program carried out with these lines is not limited to selection on a single response, social reinstatement, but affect more generally the ability of the quail to process social information 8 . Differences in social motivation, but also individual recognition have been described between LSR and HSR quail 49,50 . Inter-individual distances are longer in LSR quail 49 and LSR young quail have decreased interest in unfamiliar birds 51 and lower isolation distress than HSR ones 12 .

Further experiments will be required to examine the possible functional link between the selected genes and the divergent phenotype observed in these lines. Also, by analyses of genes known to be differentially expressed in the zebra finch during song learning we hope to comparatively understand molecular systems linked to behaviour in the avian brain.

A model for avian seasonal biology

Quail is an important model for studying seasonal biology. Seminal work in quail established that pineal melatonin 57,58 is regulated by the circadian clock 59 . In mammals, photosensing is dependent on a single retinal photoreceptor melanopsin (OPN4) that regulates pineal melatonin release. Nocturnal melatonin is critical for mammalian neuroendocrine response to photoperiod and is likely to target melatonin receptors in the pars tubularis (PT) 60 . Birds have a distinct non-retinal mechanism for photoreception through deep-brain photoreceptors 61 and melatonin does not appear to be critical for most avian seasonal cycles 62 . The medial basal hypothalamus (MBH) seems to be a critical region for avian perception of photoperiod 63 . There are currently three main candidates for avian deep-brain photoreceptors that communicate the photoperiod signal to seasonal cycles: OPN4 64 , neuropsin 65 (OPN5) and vertebrate ancient 66 (VA).

While melatonin may not be a critical component to avian photoperiod signal transduction it may play a role. Photoperiodic regulation of Gonadotropin-inhibitory hormone (GnIH), first identified in quail, has been shown to be regulated by melatonin 67 . Melatonin receptors are also located in the quail PT 68 and like the mammalian PT 69 the expression of core clock genes in the quail PT 70 are phase-shifted with photoperiod. Previously, two studies 63,71 have examined temperature dependent effects of photoperiod on core clock genes, TSHβ in the PT and DIO2 and DIO3 in the MBH. Here, we leverage the new quail genome for genome-wide analysis to determine how photoperiod and temperature interact to determine the MBH transcriptome (Fig. 2A).

We examined the effect of short-(SD) and long-day (LD) photoperiod (SD, 6L18D & LD, 20L4D) and temperature (9°C and 23°C) (Fig. 2A; Supplementary Fig. 3) on genome-wide transcription and identified 269 significantly differentially expressed genes (DEGs; FDR<0.05, log 2 FC>1; Supplementary Data 7). 127 DEGs were regulated irrespective of temperature, 60 and 82 DEGs were specific to the contrast with SD 9°C and 23°C, respectively.

We identified 16 temperature dependent DEGs with a large modulating effect of temperature (log 2 FC>1) (Fig. 2E). With the exception of aldehyde dehydrogenase (ALDH1A1), the temperature-dependent photoperiod effected DEGs were down-regulated in LD. There was an equal division of genes between temperature dependent amplification and suppression of LD down-regulated genes.

The MBH shows strong TSHβ induction in LD (Fig. 2C-D, log 2 FC=7.96 at 9°C, 8.36 at 23°C), indicating the stamp contains the adjacent PT as well as the MBH. Ikegami et al. 71 in-situ data support the localisation of TSHβ in the quail PT. Consistent with the MBH findings of Ikegami et al. 71 , we observed significant up-regulation of DIO2 and down-regulation of DIO3, in LD. We also observed a significant effect of cold (9°C) in short days as an amplifier of DIO3 LP downregulation (Fig. 2E, log 2 FC=-3.86 at 9°C, -2.51 at 23°C). We were unable to confirm any significant effect of cold on DIO2. We note significant photoperiod-dependent down-regulation of the thyroid hormone specific transporter SLC16A2 in LP that was amplified at 9°C (log 2 FC=-1.19 at 9°C, -1.63 at 23°C).

Differential regulation of G-protein coupled receptor (GPCR) signalling was the most enriched pathway regulated by photoperiod (Fig. 2F; Supplementary Data 8). It also emerged as the largest connecting component within the String interaction network of DEG genes (Fig. 2G).

TSHβ itself binds to the GPCR THR 72 . G-protein signalling is also critical for opsin signalling 73 . We also observed transcriptional regulation in other GPCR hormone receptors, including Relaxin, Vasopressin, LH, Prolactin, and GH. GnRH is associated with VA opsins in AVT neurones and has been suggested as a photoperiod sensor 66 . We also noted down-regulation of the neuronally important GPCR GPR20 (Fig. 2G). In mice, deficiency of GPR20 is associated with hyperactivity and may play a role in cAMP-dependent mitogenesis 74 . There was a strong enrichment of collagen biosynthetic processes and extracellular matrix organisation processes (Fig. 2F) and a large body of genes associated with cell differentiation and development (Fig. 2H).

We observed photoperiod-dependent regulation of a single clock gene, CRY4. CRY4 is upregulated in LP (log 2 FC=0.85 at 23°C, 1.37 at 9°C). This is consistent with the finding of Yasuo et al. 63 , that the expression of PER2-3, CLOCK, BMAL1, CRY1-2 and E4BP4 remain stable across photoperiods. Intriguingly, CRY4 has recently been proposed as a component of light-dependent magnetoreception in the avian retina 75 .

We detected photoperiod effects on OPN4 transcripts, which were up-regulated in LD. Photoperiod-dependent expression in OPN4 may well play a role in the photoperiod-refractory response. Encephalopsin (OPN3) was found to be highly expressed in the MBH (2.31..2.42 log 2 CPM) but without significant changes in expression. OPN3 has recently been identified in the hypothalamus of chick hatchlings 76 but not as yet to the MBH of adult birds. OPN5 (-0.46..-0.89 log 2 CPM) and VA (-0.11..0.31 log 2 CPM) were also unchanging and expressed at a low level in the MBH sample.

In conclusion, we confirm the importance of temperature and photoperiod-dependent regulation of thyroid hormone metabolism in the avian MBH (Fig. 3). Temperature-dependent amplification and suppression of the photoperiod response may indicate qualitative differences in the MBH pathways or simply reflect different stages of progression through seasonally phased processes. This could be further investigates by contrasting across time series at different temperatures. We also observed concurrent regulation of multiple hormonal signalling pathways, this may reflects a diversity of pathways and cell types in the MBH or reflect a corrective mechanism to account for cross-talk with other GPCR pathways. We observed LH, PRL and GH receptor transcripts changes which may indicate modulation of a GnRH-anterior pituitary feedback mechanism. Intriguingly, we also note LD induction of CRY4 which raises the question, is there a role for seasonally active magneto-sensing in the MBH? In addition to observing high OPN3 expression in the MBH, we also noted LD overexpression of OPN4, which could provide a potential component for an avian photoperiod-refractory mechanism. This study has demonstrated the utility of genome-wide transcriptome analysis in quail to provide valuable insights and novel hypothesis for avian seasonal biology.

Quail response to H5N1 influenza

Highly pathogenic influenza A viruses (HPAI), such as H5N1, are responsible for enormous economic losses in the poultry industry and pose a serious threat to public health. While quail can survive infection with low pathogenic influenza viruses (LPAI), they experience high mortality when infected with strains of HPAI 77 . Quail are more susceptible than chickens to infection by some strains of H5N1 including one that caused human mortality 25 . Previous research has shown that quail may play a key role as an intermediate host in the evolution of avian influenza, allowing viral strains to spread from wild birds to chickens and mammals 21,22,25,78 . Unlike quail and chicken, aquatic reservoir species such as duck are tolerant of most HPAI strains 79 . The generation of a high-quality quail genome has enabled us to perform an RNA-seq differential analysis of gene expression in quail infected with LPAI and HPAI, to better understand the response of quail to influenza infection. Lung and ileum samples were collected at 1 day post infection (1dpi) and 3 days post infection (3dpi). We also reanalysed previous data collected from duck and chickens 80 and compare this to the quail response.

To provide an overview of the response to LPAI and HPAI in quail we examined pathway and GO term enrichment of DEGs (see Supplementary Data 9, 10 and Supplementary Figs. 4567). In response to LPAI infection, pathways enriched in the ileum included metabolism, JAK/STAT signalling, IL6 signalling and regulation of T-cells (Supplementary Fig. 4). In the lung, pathways upregulated included complement, IL8 signalling, and leukocyte activation (Supplementary Fig. 5). In the lung at 3dpi highly enriched GO terms included 'response to interferon-gamma', 'regulation of NF-kappaB', 'granulocyte chemotaxis' and 'response to virus' (Supplementary Data 9), which are key influenza responses. This indicates an active immune response occurs to LPAI infection in quail, involving both ileum and lung, but with the strongest immune response occurring in the lung.

Genes upregulated in response to HPAI in the ileum were related to metabolism and transport, while inflammatory response was downregulated at 1dpi (Supplementary Fig. 6). Downregulated pathways at 1dpi included IL-6, IL-9 and neuroinflammation signalling pathways (Supplementary Fig. 6). In the quail lung many genes were downregulated after HPAI infection (Supplementary Data 9). At 3dpi, most downregulated pathways and terms were linked to immune system processes. GO terms with the highest fold enrichment in downregulated genes at this time included T and B cell proliferation, TNF signalling pathway, TLR pathway and IFN-G production (Supplementary Data 10). Pathways downregulated included both Th1 and Th2 pathways, T cell, B cell and macrophage signalling pathways (Supplementary Fig. 7). This indicates that key immune responses in quail, are downregulated in ileum, and particularly in the lung at day 3, following HPAI infection.

To compare the response of quail, duck and chicken, clustering of gene counts was examined using BioLayout 3D 81 . This revealed a cluster of 189 genes that were strongly upregulated at 1dpi in the duck, which showed no response in chicken and quail (Supplementary Table 3). This cluster was dominated by RIG-I pathway and IFN response genes including IFNG, DDX60, DHX58, IRF1, IRF2, and MX1. Pathways associated with this cluster includes MHCI processing and death receptor signalling (Fig. 4). Thus, the lack of this early anti-viral response may be key to the susceptibility of Galliformes to HPAI.

To further compare the responses between the three species, enrichment of pathways in each species was examined (Fig. 5). This revealed very few commonly regulated pathways between the three species. However, at 1dpi in the ileum and 3dpi in the lung there were many pathways that were downregulated in the quail, not altered in chicken, and upregulated in the duck. In the ileum at 1dpi, this included pattern recognition and death receptor signalling. In the lung at 3dpi this involved host of immune related pathways including production of NOS by macrophages, pattern recognition, B and T cell signalling and NK-KB, IL8 and IL2 signalling.

The proportion of genes commonly regulated between quail, chicken and duck to HPAI infection was also examined (Fig. 6). Consistent with the heatmap comparison (Fig. 5), the response of chicken, quail and duck were largely unique, with few genes commonly differentially expressed. There was a large set of genes that were upregulated in duck, while being downregulated in quail at 3dpi, in both ileum and lung. In lung these genes were related primarily to innate immune system pathways, including pattern recognition pathways, cytokine production, leukocyte adhesion, TNF production, interferon production, B cell signalling and response to virus (Supplementary Data 10). Genes with the greatest differential expression included RSAD2 which inhibits viruses including influenza, IFIT5 which senses viral RNA and OASL which has antiviral activity. These differences further highlight that the anti-viral immune response is dysregulated in quail. Additionally in both ileum and lung the apoptosis pathway was enriched in duck, but not quail (Supplementary Data 10). Apoptosis is known to be a key difference in the response of chickens and ducks to HPAI infection 82 .

Lastly, we examined the response of key families involved in influenza and immune response, focussing on the lung (Supplementary Table 5, Supplementary Data 11). IFTIM genes have previously been found to have a key role in HPAI resistance 80 and may block AIV from entering cells 83 . Consistent with previous findings in the chicken 80 , quail showed no significant upregulation of IFTIM genes, while these genes in duck were strongly upregulated, (Supplementary Table 5). TLRs and MHC receptors are involved in recognition of foreign molecules and triggering either an innate (TLR) or adaptive (MHC) immune response. TLR3, 4 and 7, which bind viral RNAs, were upregulated in response to LPAI in quail. A reversal was seen in response to HPAI, with TLR4, and 7 substantially downregulated. Likewise, genes of both MHC class I and II were upregulated in response to LPAI and downregulated in response to HPAI. By comparison there was no perturbation of TLR and MHC genes in chicken and upregulation of class I genes in duck. The quail seems to have a highly dysfunctional response to HPAI infection with key innate and adaptive immune markers downregulated at 3dpi, which contrasts with the strong immune response mounted by the duck and minimal immune response in the chicken.

In conclusion, we found that quail have a robust immune response to infection with LPAI, allowing them to survive the infection. However, they show dysregulation of the immune response after infection with HPAI, and this may explain their susceptibility to HPAI strains. IFITM response was not seen to HPAI while genes associated with apoptosis were downregulated, and this may allow the virus to easily enter cells and spread early in infection. Antiviral and innate immune genes, including those involved in antigen recognition, immune system activation, and anti-viral responses were downregulated at 3dpi, which would prevent an effective immune response and viral clearance once infection is established. This study provides crucial data that can be used to understand the differing response of bird species to AIV, which will be critical for managing and mitigating these diseases in the future.

DISCUSSION

Here we describe the assembly, annotation and use of a high-quality quail genome, an important avian model in biological and biomedical research. We have demonstrated the utility of this genome in both infectious disease and behavioural research providing further confirmation of the importance of quail as a research model, and for its role in agricultural and animal health studies. Specifically, the availability of this genome has allowed us to make significant discoveries in the unique response of quail to highly pathogenic avian influenza infection, helping elucidate the basis for extreme susceptibility seen in this species. It has also allowed us to identify and confirm genes and genomic regions associated with social behaviour, showing many similarities to genes associated with autism in humans and thus represents a possible biomedical model for autism. Furthermore, we have shown that genome-wide transcriptomics using this genomes facilitated further insights and hypothesis into the mechanism of photoperiodism in avian seasonal biology. Moving forward, the availability of a high-quality quail genome will facilitate the study of diverse topics in both avian and human biology, including disease, behaviour, comparative genomics, seasonality and developmental biology.

METHODS

Whole Genome Sequencing and Assembly

To facilitate genome assembly by avoiding polymorphism, we produced an individual as inbred as possible. We started with a quail line previously selected for early egg production and having a high inbreeding coefficient 84 and four generations of brother-sister matings produced a dedicated line "ConsDD" (PEAT, INRA Tours, France). A 15 week-old male Coturnix japonica (id. 7356) was then selected from this line for the sequencing project. Genomic DNA was extracted from a blood sample using a high-salt extraction method 85 . Our sequencing plan followed the recommendations provided in the ALLPATHS2 assembler 86 . This model requires 45x sequence coverage of each fragment (overlapping paired reads ~180 bp length) from 3 kb paired end (PE) reads as well as 5x coverage of 8 kb PE reads. These sequences were generated on the HiSeq2500 Illumina instrument. Long-reads used for gap filling were generated at 20x coverage on the same DNA source using a RSII instrument (Pacific Biosciences). The Illumina sequence reads were assembled using ALLPATHS2 software 86 using default parameter settings and where possible, and scaffold gaps were closed by mapping and local assembly of long-reads using PBJelly 87 . The Illumina long insert paired-end reads (3 kb and 8kb PE) were used to further extend assembled scaffolds using SSPACE 83 . The draft assembly scaffolds were then aligned to the genetic linkage map 44 and the Galgal4.0 chicken reference (Genbank accession: GCA_000002315.2) to construct chromosome files following previously established methods 32 . Finally, all contaminating contigs identified by NCBI filters (alignments to non-avian species at the highest BLAST score obtained), and all contigs < 200 bp were removed prior to final assembly submission.

Gene Annotation

Specific RNA-seq data for the genome annotation was produced from the same animal used for the genome assembly. RNA was extracted from heart, kidney, lung, brain, liver, intestine, and muscle using Trizol and the Nucleospin® RNA II kit (MACHEREY-NAGEL), following the manufacturer's protocol.

The Coturnix japonica assembly was annotated using the NCBI pipeline, including masking of repeats prior to ab initio gene predictions, for evidence-supported gene model building. We utilized an extensive variety of RNA-Seq data to further improve gene model accuracy by alignment to nascent gene models that are necessary to delineate boundaries of untranslated regions as well as to identify genes not found through interspecific similarity evidence from other species. A full description of the NCBI gene annotation pipeline was previously described 89 . Around 8,000 lacked gene symbols from this pipeline, and these were further annotated manually by using BLAST searches using the corresponding sequences and extracting protein names from Uniprot.

Comparative analyses

A set of single copy, orthologous, avian-specific genes were selected from OrthoDB v. 9 30 and their status (present, duplicated, fragment or missing) were tested with BUSCO v.3.0.2 31 in the Gallus gallus 5.0 and Coturnix japonica 2.0 genomes. Ab initio gene predictions were done within the BUSCO framework using tBLASTn matches 90 followed by avian specific gene predictions with Augustus v. 3.3 91 . Gene status was assessed by running HMMER 92 with the BUSCO HMM profiles of the orthologous sequences. Comparative maps and breakpoint data were generated using AutoGRAPH 93 using chicken and quail gff annotation files, using default settings.

Endogenous retrovirus identification

Endogenous retroviruses (ERVs) were identified in the Coturnix japonica 2.0 and Turkey 5.0 genome assemblies using the LocaTR identification pipeline 35 and compared to a previous analysis of ERVs in the Gallus gallus 5.0 genome assembly 32 . LocaTR is an iterative pipeline which incorporates LTR_STRUC 94 , LTRharvest 95 , MGEScan_LTR 96 and RepeatMasker 97 search algorithms.

Sociability selection study

The data and methods used have been described previously 45 . Briefly, two quail lines were used, divergently selected on their sociability 9 : high social (HSR) and low social (LSR) reinstatement behaviour. A total of 10 individuals from generation 50 of each quail line were sequenced after equimolar DNA pooling. Sequencing was performed (paired-ends, 100 bp) on a HiSeq 2000 sequencer (Illumina), using one lane per line (TruSeq sbs kit version 3). The reads (190,159,084 and 230,805,732 reads, respectively, for the HSR and LSR lines) were mapped to the CoJa2.2 genome assembly using BWA 98 , with the mem algorithm. Data are publicly available under SRA accession number SRP047364. Within each line, the frequency of the reference allele was estimated for all SNPs covered by at least 5 reads, using Pool-HMM 99 . This analysis provided 13,506,139 SNPs with allele frequency estimates in the two lines. FLK values 46 were computed for all these SNPs, and the local score method 45 was applied to the p-value on single-marker tests.

Photoperiod study

MBH tissue was collected as previously 71 . Male 4-week old quail were obtained from a local dealer in Japan and kept under SD conditions (6L18D) for 4 weeks. At 8 weeks of age, quail were transferred to LD conditions (20L4D) and kept under LD conditions for 4 weeks to develop their testes. And then, 12 week-old LD quail were transferred to short day and low temperature (SL: 6L18D 9C) conditions for another 4 weeks to fully regress their testes. All samples were collected at ZT18. (Light onset is same for LD and SD and light offset was extended in LD group). RNA-Seq was performed using a TruSeq stranded mRNA prep (Revision E 15031047) with 125bp paired-end reads on a HiSeq Illumina 2500 with four replicates in each of the three conditions.

Reads were quality (Phred>25) and adapter trimmed with trim galore (version 0.4.5). Tophat (version 2.1.0) 100 with bowtie2 (version 2.2.6) was used to map reads to the quail genome (GCA_001577835.1 Coturnix japonica 2.0), using the NCBI annotation. We determined feature counts for gene loci using the featureCounts program 101 in the subread (version 1.5.0) package 102 . Statistical analysis was performed using the limma package 103 (version 3.36.1) in the R programming environment (version 3.5.0). The trimmed mean of M-values normalization method (TMM) was used for normalisation with Voom for error estimation (Sup. Table 2). We retained gene loci with more than 10x coverage in three replicates in at least two conditions. A categorical least squared regression model was fitted using LD 23°C, SD 23°C, and SD 9°C conditions. Statistics for pairwise comparisons were then recalculated by refitting contrasts to the model for LD 23°C vs SD 23°C, LD 23°C vs SD 9°C and SD 23°C vs SD. The Benjamini Hochberg 104 approach was used to estimate the false discovery rate. For reporting numbers of photoperiod significant genes, we applied thresholds of FDR <0.05, log2 CPM > 0, and absolute log2 fold change > 1. Temperature-dependent genes are reported as those with a photoperiod significant effect at either 23°C or 9°C and a significant effect when contrasting SD 9°C and SD 23°C at the same thresholds defined across photoperiods.

Influenza response study

All experiments involving animals were approved by the Animal Care and Use Committee of St. Jude Children's Research Hospital and performed in compliance with relevant policies of the National Institutes of Health and the Animal Welfare Act. All animal challenge experiments were performed in animal biosafety level 2 containment facilities for the LPAI challenges and in biosafety level 3 enhanced containment laboratories for the HPAI challenges. Viral challenges of quail, tissue collection, RNA extractions and sequencing were carried out as previously described for chicken 80 . Briefly, fifteen mixed-sex quail were challenged with 106 EID50 intranasally, intratracheally, and intraocularly of LPAI A/Mallard/British Columbia/500/2005 (H5N2) in phosphate buffered saline (PBS). Fifteen quail were challenged with 101.5 EID50 intranasally, intratracheally, and intraocularly of HPAI A/Vietnam/1203/2004 (H5N1) in PBS. Mock infection control groups (n=12) were also inoculated, receiving an equivalent volume and route of administration with PBS. Animals were monitored daily for clinical signs. Lung and ileum samples were collected from all birds on 1dpi and 3 dpi. RNA extractions were performed using Trizol and QIAGEN's RNeasy kit. For sequencing thirty-six cycle single-ended sequencing was carried out on the Genome Analyser IIx using Illumina v3 Sequencing by Synthesis kits.

All quail as well as duck and chicken RNA-seq reads from the previous study 80 were analysed as follows. Ileum and lung RNAs were analysed from PBS infected control (3 samples from each of 1dpi and 3dpi), H5N1-infected (3 samples from each of 1dpi and 3dpi, except quail ileum 1dpi which had 2 samples) and H5N2-infected (3 samples from each of 1dpi and 3dpi). 251 million reads of 36 nucleotides in length were generated in total for quail. Reads were quality checked using FastQC and trimmed for quality using Trim-galore. Mapping was performed to the quail genome (GCA_001577835.1 Coturnix japonica 2.0), chicken genome (GCA_000002315.3 Gallus_gallus-5.0) and duck (GCA_000355885.1 BGI_duck_1.0) using Tophat2 100 using default options. For quantification and differential analysis, the following pipeline was used. First transcripts were assembled and quantified using cufflinks 105 , guided with the NCBI annotation for the relevant genome, and the multi-read correct option was used. The transcriptomes were merged using stringtie merge 106 and cuffdiff 105 was used for differential analysis using default settings. To determine orthology between quail, duck and chicken genes, reciprocal BLAST searches were performed. For analysis of GO term enrichment the PANTHER overrepresentation test 107 was used and for pathway analysis Ingenuity Pathway Analysis software (QIAGEN) was used. For clustering analysis BioLayout 3D 76 was used using default settings except 1.4 inflation for Markov clustering.

committee approved the rearing protocol (authorization number 00915.02). The use of quail in photoperiod experiments were approved by the Animal Experiment Committee of Nagoya University. All experiments involving animals in the infection studies were approved by the Animal Care and Use Committee of St. Jude Children's Research Hospital and performed in compliance with relevant policies of the National Institutes of Health and the Animal Welfare Act.

Introduction

Hematopoiesis is an essential biological process leading to the daily formation of blood cells able to maintain the homeostasis of the body. This production relies on rare cells designated as hematopoietic stem cells (HSCs) able to produce both all the hematopoietic lineages and to maintain their number roughly constant through selfrenewal mechanisms. In all the vertebrate species described, HSCs are formed early during development at the level of the embryonic dorsal aorta and associated arteries through an endothelial-to-hematopoietic commitment (EHT) proposed more than a century ago [START_REF] Jordan | Evidence of hemogenic capacity of endothelium[END_REF]. In addition to the aorta, EHT also occurs earlier on in development at the level of the yolk sac where it was shown to produce a wave of Erythro-Myeloid Progenitors (EMPs) as well as lympho-myeloid progenitors. This unique type of cell emergence is characterized by the progressive loss of the endothelial transcriptional program, the switching on of the hematopoietic program accompanied by a morphological change that conduct to the rounding of the flat endothelial cells, the breaking of the tight junctions and the release into the vessel lumen. Thus, EHT appears as a unique method to produce most of the hematopoietic cells including HSCs. The use of mouse or human pluripotent stem cells has allowed to first demonstrate EHT [START_REF] Lancrin | Blood cell generation from the hemangioblast[END_REF]Schröder et al., 2010) and to further permit significant advances in the cellular and molecular control of EHT. Recent advances in live imaging using the zebrafish or murine embryos have allowed EHT to be observed in real time and hematopoietic cells to be produced unambiguously demonstrating EHT within the aorta.

Due to the use of pluripotent stem cells and on transcriptomic approaches, progress have been made in the past few years on the molecular control of EHT. In addition to the RUNX1 transcription factor (TF), a master regulator of EHT (Okuda et al., 1996;[START_REF] Wang | Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis[END_REF][START_REF] Chen | Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter[END_REF], the GATA2 TF was also shown to be a master regulator of EHT [START_REF] Ottersbach | Endothelial-to-haematopoietic transition: an update on the process of making blood[END_REF] in addition with other TFs called heptad (Wilson et al., Cell Stem Cell 2010). In addition, two downstream targets of Runx1, Gfi1, and Gfi1B were shown to be essential for hematopoietic production from the hemogenic endothelium (Tambyrajah et al., 2016;[START_REF] Lancrin | GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment[END_REF]. A recent study also indicates that Bmi1, a member of the polycomb complex contributes to Runx1 silencing to prevent premature EHT [START_REF] Eliades | The Hemogenic Competence of Endothelial Progenitors Is Restricted by Runx1 Silencing during Embryonic Development[END_REF]. In addition to these TFs and their modulators, several signaling pathways were shown to control EHT either by acting directly on endothelial cells such as Notch or Wnt (Bigas and Espinoza, 2012) or by acting on the immediate microenvironment such as BMP or Hedgehog (Dzierzak and Bigas, 2018).

Results

LDL int cells represent an EC population upregulating RUNX1 and down-regulating CD31 and CD144.

One advantage of the culture system [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF] is the stepwise progression of the mesoderm towards the hematopoietic fate that allows isolating discrete cellular steps over the culture period of 12 days. In addition, by removing serum or VEGF from the culture, it is possible to prevent hemogenic EC commitment and to orient the cells towards a more advanced vascular endothelium state. We previously showed that ECs emerging in culture uptake AcLDL in keeping with their endothelial phenotype including those expressing RUNX1. We reasoned to use the levels of AcLDL uptake to separate ECs from hemogenic ECs. This is based on the observation that RUNX1 initiated expression is found at D3 in only a few ECs, but in almost 100% of the cells at D5. RUNX1 expression is shortly followed by that of the earliest hematopoieticspecific transcription factors, in particular, PU1, a direct target of RUNX1. In this respect, hemogenic ECs i.e., RUNX1-expressing cells, should undergo a decrease of AcLDL uptake in keeping with the progressive loss of the endothelial program. Based on this assumption, D2 and D4 adherent cells were incubated with AcLDL over a period of two hours, trypsinized and Fluorescence-Activated Cell Sorted (FACS) based on the AcLDL level of expression. At D2, FACS analysis showed cells harboring a wide, albeit continuous, range of fluorescence from nil to high (Figure A1A). At D4, we separated two populations: one with a high level of expression of AcLDL (LDL hi ) and another with an intermediate level of expression (LDL int ) (Figure A1B). In the absence of serum, the majority of the population is within the LDL hi (Figure A1C).

The different LDL populations displayed on Figures 1A-C were sorted and analyzed for the expression of two endothelial markers i.e., CD31 (PECAM) and CD144 (VE-Cadherin) and the canonical hemogenic endothelial marker RUNX1. Gene expression on the D0 pre-somitic mesoderm served as a baseline. Both CD31 mRNA was highly expressed in LDL hi vs LDL int cells with a mean fold change of two (Figure A1D) CD144 mRNA expression was high at D2 and D4 with no clear change on the level of expression between D2 and D4 and between D4 and D4 without SVF but was at least 8-fold decreased in D4 LDL int cells (Figure A1E). However, RUNX1 mRNA expression was strongly increased in LDL int compared with the other LDL-positive populations (Figure A1F). Identification of the gene network characteristic of hemogenic ECs enrichment in lysosome, FoxO, Wnt and Rap1 signaling pathways and glycosaminoglycan degradation. The HEC population was enriched in genes implicated in focal adhesion, ECM, regulation of actin cytoskeleton, MAPK and PI3K-Akt pathways, glycosaminoglycan biosynthesis and axon guidance. Finally, the HC population was enriched in genes implicated in metabolic pathways, oxidative phosphorylation, Jak-STAT and B and T cell receptor signaling pathways (Figure A2E). WGCNA then performed using the matrix of up-regulated genes revealed the existence of 5 modules, one for EC, one for M, one for HC and two for HEC (Figure A2F).

From this point, we decided to focus on the two HEC modules of up-regulated genes.

The designated green (Figure A2G) and red (Figure S2A) modules corresponding to 192 and 253 genes respectively significantly up-regulated in HEC had a very particular extended topology consisting in relatively dense cores connected by a few genes; hence there were very prominent hubs: the ECM components EMILIN1, EMILIN2, COL5A1, FBN1 and COL1A2, COL6A2, COL6A3, CSPG4 in the red and green modules, respectively, the ECM enzyme PLOD2, the migration cue SLIT2, the transcription factor CREB3L1 in the red module, and the transcription factor NFKB2 and the cytokines TGFB3 and INHBA in the green module. The gene connectivity of the most connected genes in each module is displayed in (Figure A2H and Figure S2B). Analysis of the GO categories revealed shared and distinctive patterns between the two HSCs networks.

Both networks showed an enrichment in molecules implicated in the ECM, with Pleckstrin homology domain, SH3 domain, components of the Golgi apparatus, cell adherent junction, membrane and cell signaling. On the contrary, genes implicated in Focal adhesion were enriched in the green module only, while the red module showed enrichment in genes implicated in microtubule, migration and phosphatase activity (Figure A2I).

Modulation of the Wnt pathway contributes to the endo-hematopoietic balance

To investigate whether ß-catenin activity influences the generation of HEC and the EHT, we tested the effect of inhibitors or activators of the pathway on culture. As an inhibitor, we used the small molecule PKF118-310 (hereafter referred as PKF118), an inhibitor of the TCF4 ß-CATENIN protein interaction (Hallet et al., 2012). We tested 3 different concentrations of PKF118 (0.66µM, 1µM and 1.5µM). The last concentration provoked toxic effects whereas the first one did not produce significant results (not shown). The concentration of 1µM caused drastic changes to the culture. Treated cells were flatter and had fewer round cells than the non-treated samples (Figure A3A; Figure A3B; Figure A3D; Figure A3E). Flow cytometry analysis confirmed the increase of LDL hi cells at the expense of LDL int cells (Figure A3C; Figure A3F; Figure A3G; Figure A3H) and the strong decrease of the floating cells (Figure A3I) (n=5). QRT-PCR on adherent cells revealed the reinforcement of CD144 mRNA expression (Figure A3J) in keeping with the increase of the endothelial phenotype and the decrease of RUNX1 expression (Figure A3K) consistent with the lower number of LDL int cells.

We also tested the Wnt activator SB216763 hereafter referred as SB, that inhibits GSK3 alpha and GSK3 beta. We used two different concentrations i. Cells in the supernatant were round and very bright suggestive of a hematopoietic production (Figure S4B) and [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. FACS analysis showed a sharp increase in the number of LDL int cells at the expense of LDL hi cells (Figure S4C; Figure S4D and Figure S4G; Figure S4H) accompanied by a parallel increase of hematopoietic cells (Figure S4E; Figure S4F and Figure S4I). QRT-PCR analysis showed a highly significant increase of CD45 expression characteristic of hematopoietic differentiation (Figure S4J). (ref). We used a knock-down strategy utilizing siRNA. We designed three different siRNAs against POFUT2 (Table A1) that were transfected at day 1 of culture incipience.

The analysis was performed at day 4 (Figure A4A) and was compared to Lipofectamine alone or a scrambled POFUT2 siRNA. We tested 3 concentrations of siRNA against POFUT2 i.e. 30, 60 and 120nM (n=5) and compared their knock-down efficiency by measuring POFUT2 mRNA expression by q-PCR on transfected cells. The lowest concentration decreased POFUT2 mRNA expression by 80% whereas the highest displayed signs of toxicity and was not retained (not shown). The 60nM concentration decreased POFUT2 by 90% on transfected cells and was selected for further use (Figure A4B). POFUT2 mRNA knock-down at D1 resulted in the appearance of more round hematopoietic cells in the culture compared with Lipofectamine or scramble si controls (Figure A4C; Figure A4E; Figure A4G). Flow cytometry analysis revealed an increase in the AcLDL int population at the expense of the AcLDL hi population (Figure 4D, F, H). Taken together POFUT2 knock-down resulted in a decrease of the number of ECs (p<0.001) and an increase in the number of HEC (p<0.08) (Figure A4I; Figure A4J).

To further validate the gene network and its predictive value, we selected the POFUT2's first neighbors (Figure S2C) and examined if a decrease of POFUT2 mRNA expression was also associated with a modulation of several of its first neighbors. Based on gene connectivity (Figure S2C), we selected 3 genes among the most connected to POFUT2. COL5A2 encodes an alpha chain for one of the low abundance fibrillar collagens; DNAJB4, is a molecular chaperone, tumor suppressor, and member of the heat shock protein-40 family that binds and regulates E-Cadherin and EMILIN1A (Elastin Microfibril Interfacer 1) is thought to regulate smooth muscle cell adhesion and vessel assembly. COL5A2, the most connected gene to POFUT2 showed a significant decrease upon POFUT2 siRNA mediated knock-down when compared to the Lipofectamine control but not to the scramble (p<0.001and 0.3 respectively) (Figure A4K). DNAJB4 and EMILIN1A both showed a significant decrease upon POFUT2 siRNA knock-down whatever the control used (Figure A4L; Figure A4M). Taken together, this indicates that and 1696210) to determine the endothelial phenotype of PSM-derived cells. AcLDL-A488

(1/100 in PBS) was incubated for 2 hours at 37°C in the culture dish. Cultures were then washed 3 times in PBS before flow cytometry and cell sorting.

FACS analysis

Cells were stained with AcLDL coupled to Alexa-488. When adherent cells were used, cells were tripsinized and rinsed thrice in PBS. When floating cells were used, supernatant cells were gently removed from the culture dish by pipetting. For LDL analysis, cells were cells were suspended in PBS containing 7AAD to exclude dead cells and analyzed with a MacsQuant analyzer 10. Analyses were made with FlowJo™ 10.

Statistics were performed with GraphPad Prism.

Lipofection-mediated siRNA transfer

Lipofectamine RNAiMax was used to transfect cells at day 1 or 2 with specific siRNA or Scramble si control following the manufacturer's protocols. The transfection efficiency was evaluated by analysing the mRNA expression of the targeted gene by qPCR.

RNA sequencing and bioinformatic analyses

RNA libraries were prepared by Fasteris Life Science (Switzerland). In brief, total RNA was submitted to a poly-A mRNA purification using oligodT magnetic beads.

Supernatants were kept for small RNA library preparation, and poly-A-RNAs after elution from the beads were prepared according to the manufacturer's protocol (TruSeq RNA Sample Prep-Kit V2; Illumina). Transcripts were broken at 95°C in presence of zinc, and first-strand cDNA syntheses were performed using random primers. A second-strand cDNA synthesis was performed in the presence of deoxyuridine triphosphate, and after a 3′ A addition step, adapters were ligated, and an amplification by PCR was performed to generate the DNA colony template libraries. Small RNA libraries were performed according to the manufacturer's protocol (TruSeq Small RNA Library Prep kit; Illumina).

After acrylamide gel purification of small RNA between 18-30 nt, single-stranded ligation of 3′ adapter and the 5′ adapter were performed before reverse transcription and PCR amplification to generate the DNA colonies template. All the samples were sequenced using 1 × 125-bp single reads high-throughput sequencing (RNA-Seq) in single lane on a HiSeq 2000 sequencing system (Illumina). RNA-Seq analysis was performed on Partek Flow™. For mRNA libraries, sequence reads in fastq format were aligned to the quail reference genome assembly RefSeq GCF_001577835.1 (Figure S1B workflow) using the STAR aligner. The number of reads for all the features were then counted using the quantification to a model E/M and normalized for each library, and then ANOVA were performed to compare read values between different libraries.

Statistical analysis

Statistical analyses (t tests) were performed with GraphPad and Excel software.

Data were considered statistically significant for P < 0.05.

Data analysis

The lists of differentially expressed genes were generated by ANOVA and Venn

Diagram . GO was performed using DAVID (http://david.abcc.ncifcrf.gov). To find out the network structure of the gene set HEC, we used WGCNA (Langfelder and Horvath, 2008). Adjacencies were given according to signed Pearson correlation. The soft threshold power b that resulted in approximate scale-free topology was 36. Modules were constructed with average linkage hierarchical clustering. Minimum module size was 30. A: experimental design. PSM cells were put in culture at D0, transfected at D1 and analyzed at D4 for AcLDL uptake by flow cytometry and q-PCR for the candidate gene and some of its immediate neighbors in the gene network. B: Effect of Lipofectamine (Lipo), scramble (Scr) and POFUT2 siRNA transduction on the expression of endogenous POFUT2. At 60nM, POFUT2 mRNA expression was decreased by more than 90% compared with the liposome and scramble siRNA controls.

C, E, G: culture at D4 that received Lipofectamine (C), scramble si (E) and POFUT2 si (F) at D1. Note that the number of round cells is more important in the siPOFUT2 condition than with the two other ones. D, F, H: representative flow cytometry experiment showing the balance between AcLDLint and AcLDLhi cells when cells received Lipofectamine (D), scramble si (F) and POFUT2 si (H). Note that the number of AcLDLint cells has increased at the expense of AcLDLhi cells in the POFUT2 si condition. I: percentage of AcLDLhi cells in the three conditions. A significant (p<0,001) decrease of LDLhi cells is visible in the POFUT2 si conditions (n=5).

J: percentage of AcLDLint cells in the three conditions. A low but significant (p<0.08) increase of AcLDLint cells is observed in the POFUT2 si condition compared with the two others. K: mRNA expression of COL5A2 is significantly decreased when POFUT2 is knock down by siRNA. L: DNAJB4 mRNA expression is decreased in POFUT2 knock down condition. M: EMILIN1A mRNA expression is significantly decreased in POFUT2 knock down condition. C, E, G, bar= 400µm.

Figure 1 :

 1 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 2 :

 2 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 3 :

 3 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 4 :

 4 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 5 :

 5 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 6 :

 6 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 7 :

 7 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 8 :Figure 10 :

 810 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 11 :Figure 12 :

 1112 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 13 :

 13 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 14 :

 14 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 15 :

 15 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 17 :

 17 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 18 :

 18 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 19 :

 19 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 20 :

 20 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 21 :

 21 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 22 :

 22 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 23 :

 23 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 24 :

 24 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 25 :

 25 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 26 :

 26 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 27 :

 27 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 29 :

 29 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 30 :

 30 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 31 :

 31 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 32 :

 32 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 33 :

 33 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 34 :

 34 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure 35 :

 35 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure M1 :

 M1 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure M2 :

 M2 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure R1 :

 R1 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure R2 :

 R2 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure R3 :

 R3 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure R5 :

 R5 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure R6 :

 R6 Figure 1: Schematic representation of blood cell constituents .........................................................Figure 2: Symmetric and asymmetric divisions schematic representation ........................................Figure 3: Schematic representation of the hierarchy of hematopoietic cells ....................................Figure 4: Genetic programs in hematopoiesis ...................................................................................Figure 5: Ernst Neumann illustrating his "stem cell" 1914 ..............................................................Figure 6: Colony Forming Unit in the Spleen: CFU-S ........................................................................Figure 7: Colony-Forming Cell (CFC) assay .......................................................................................Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Figure 9: Test LTC-IC: Long-term Culture Initiating Cell assay.......................................................... Figure 10: Primary and secondary hematopoietic reconstitution ...................................................Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy18 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. .............................................................................................Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. .....Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 ........................Figure 15: The Allantois a hematopoietic emergence site............................................................... Figure 16: The extraembryonic membranes in human, mouse and chick embryos. ........................Figure 17: Parabiosis procedures between the YS of two chicken embryos ....................................Figure 18: Quail-chicken yolk sac chimera .....................................................................................Figure 19: Hematopoietic clusters in the aorta of different species ................................................Figure 20: Location of intra-embryonic HSCs. .................................................................................Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus .................Figure 22: Hematopoietic sites in zebrafish ....................................................................................Figure 23: The aorta a source of hematopoietic progenitors ..........................................................Figure 24: Mapping of the two endothelial lineages .......................................................................Figure 25: Developmental history of the aorta in relationship with endothelial remodeling .........Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5 .........................Figure 27: A model of hemangioblast development in the mouse embryo..................................... Figure 28: Developmental potential of human blast colonies .........................................................Figure 29: Tracing of aortic endothelium derivatives labelled by Ac-LDL-DIL injection in HH13 chick embryo ...........................................................................................................................................Figure 30: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 31: The endothelial origin of HSCs from the aorta in mice ...................................................Figure 32: Labeling of hematopoietic cells in the aortic region .......................................................Figure 33: Existence of para aortic foci in mice ...............................................................................Figure 34: EMT entails profound morphological and phenotypic changes to a cell .........................Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. ..Figure M1: PSM harvesting in the quail embryo .............................................................................Figure M2: Workflow of RNAseq analysis .....................................................................................Figure R1: The level of LDL uptake separates endothelium from hemogenic endothelium ..........Figure R2: Sample isolation strategy .............................................................................................Figure R3: Analysis of the entire set of mRNA and samples (20 samples, 20332 genes) ............... Figure R4: Identification of the specifically up-regulated genes in the different cell populations .Figure R5: Analysis of the gene ontology categories .....................................................................Figure R6: Identification of five cell specific modules using WGCNA and their attributed networks. ......................................................................................................................................................

Figure R7 :

 R7 Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

Figure R10 :

 R10 Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

Figure R13 :

 R13 Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

Figure R14 :

 R14 Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

Figure R15 :

 R15 Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

Figure R16 :

 R16 Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

  Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

Figure R20 :

 R20 Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

  Figure R7: HEC modules and gene connectivity ............................................................................ Figure R8: Analysis of the gene ontology categories of HEC networks .......................................... Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype .Figure R10: The Wnt agonist SB enhances EHT ............................................................................. Figure R11: The gamma secretase inhibitor DAPT enhances EHT .................................................. Figure R12: POFUT2 knock down enhances EHT. ..........................................................................Figure R13: POFUT2 first neighbors ..............................................................................................Figure R14: POFUT2 knock down at day 2 doesn't affect EHT. ......................................................Figure R15: TESTIN knock down doesn't affect EHT. .....................................................................Figure R16: TESTIN first neighbors ................................................................................................ Figure R17: t-SNE analysis revealed 12 well defined clusters ........................................................ Figure R18: Mesodermal markers ................................................................................................. Figure R19: Endothelial markers ...................................................................................................Figure R20: Hemogenic endothelial and hematopoietic markers ................................................. Figure R21: Analysis of the KEGG pathways categories by PCA .....................................................Figure CR1: LOC Assignment workflow .........................................................................................

Table 1 :

 1 The different types of colonies in the CFC assay ................................................................ Table 2: Mouse Proteins with putative O-fucosylation site within TSRs ......................................... Table M1: Sequences of the siRNA POFUT2, TESTIN and Scramble ................................................. Table S1: upregulated DEGs in HC and EC ..................................................................................... Table S2: upregulated DEGs in M and HEC .................................................................................... EHT: Endothelial-to-Hematopoietic Transition ELTC-IC: Extended Long-Term Culture-

Figure 2 :

 2 Figure 2: Symmetric and asymmetric divisions schematic representation Symmetric divisions start from a SC and produces two differentiated cells or two SCs Asymmetric division starts from a SC and produces one differentiated cell and one SC Pink cells: Stem cells Green cells: Differentiated cells

Figure 4 :

 4 Figure 4: Genetic programs in hematopoiesis At least three genetic programs are required for the development of the blood system. (a) The specification of HSCs (b) The self-renewal, including expansion and maintenance of HSCs (c) The commitment, proliferation, and differentiation of HSCs

A.

  History of the concept of HSCsIn 1908, a Russian histologist named Alexander Maksimov, first put forward the existence of the SC (and coming up with the term himself) as part of his theory of hematopoiesis but, the first studies of the hematopoietic system started in 1914 when Franz Ernst Christian Neumann described the BM as the organ of blood cell formation Figure5.In 1945, the United States detonated two nuclear bombs over the cities of Hiroshima and Nagasaki. Many people who did not die immediately from the blast and heat died afterward because of a hematopoietic failure due to a lethal dose of irradiation. This was the starting point for several seminal discoveries concerning the field of hematopoietic cell transplantation.Jacobson and Lorenz in early 1951, trying

Figure 5 :

 5 Figure 5: Ernst Neumann illustrating his "stem cell" 1914Draft byErnst Neumann himself, showing the "great-lymphozyt-stem-cell" (1912) or "greatlymphocyt" as stem cell for the postembryonic and embryonic development of erythropoiesis and leukopoiesis in the bone marrow and, as shown here, in the embryonic liver; GrLK: nucleus of greatlymphocyt-stem-cell; Erblk: Erythroblast;(Neumann 1914)

Figure 6 :

 6 Figure 6: Colony Forming Unit in the Spleen: CFU-S After injecting bone marrow cells into an irradiated animal, isolated colonies of hematopoietic cells develop on the surface of the spleen (Till 1981, from "Developmental Biology 6th edition" Scott F. Gilbert).

Populations resistant to 5 -

 5 fluorouracil (5-FU-used to destroy cells in cycle) are enriched with HPP-CFC, suggesting the quiescent state of these cells in vivo. HPP-CFCs are capable of producing new colonies once they are replated, demonstrating their ability to self-renew, a characteristic common to CFC and HSCs.

Figure 7 : 8 .

 78 Figure 7: Colony-Forming Cell (CFC) assayThe suspension of hematopoietic progenitors and precursors is cultured in a semi-solid medium with defined growth factors for 7 days and then the number and the type of colonies formed are assessed. Abbreviations: CFU Colony-Forming Unit, BFU Burst-Forming Unit, CFU -E Erythrocyte, CFU -E Erythrocyte, CFU -G Granulocyte, CFU -M Monocyte/Macrophage, CFU -GM Granulocyte/Macrophage, CFU -GEMM Granulocyte/Erythrocyte/Macrophage/Megakaryocyte

Figure 8 :

 8 Figure 8: Illustration of a coculture of murine hematopoietic cells on a layer of MS5 stromal cells Two types of cells arise: in the supernatant, round and refractive HCs and CA-FC cells that make paved colonies in close contact with the stromal supporting cellsAbbreviations: CA-FC Cobblestone Area-Forming Cell, HCs Hematopoietic Cells.

Figure 10 :

 10 Figure 10: Primary and secondary hematopoietic reconstitution Abbreviations: LTRC: Long-Term Repopulating Cell FACS: Fluorescence Activated Cell Sorting, BM: Bone Marrow

Figure 11 :

 11 Figure 11: Recapitulative of Ex vivo and In vivo tests in parallel with the hematopoietic hierarchy Abbreviations: HSC: Hematopoietic Stem Cell; CFC: Colony-Forming Cell; LTC-IC: Long-term Culture Initiating Cell assay

Figure 12 :

 12 Figure 12: Parallel between human and mouse most definitive markers used to identify the various types of hematopoietic cells. In grey cell types In yellow mouse markers In orange human markers

Figure 13 :

 13 Figure 13: The Journey of HSC from in different species: Human, Mouse, Chick and Zebrafish. Starting with fertilization, circulation, birth and larvae and finishing with the Adult life. The four species share two major waves of hematopoiesis: primitive and definitive waves. Human, Mouse and Chick have the same hematopoietic organs, however in Chick the Fetal Liver is replaced by the para-aortic foci. Concerning the Zebrafish, it shares the AGM and the Thymus with the other species. It got the ICM as an equivalent of the YS, the CHT as equivalent of the Fetal Liver and the Kidney marrow as equivalent of the bone marrow. Abbreviations: YS: Yolk Sac, ICM: Inner Cell Mass, CHT: Caudal Hematopoietic Tissue. d: day, h: hour, W: Week, hpf: hours post fertilization, Mo: month Adapted from Rowe et al 2016

Figure 14 :

 14 Figure 14: Maturation of the blood island in mice embryo between E7,5 and E9,5 Development of yolk sac blood islands in the mouse embryo between E7.5 and E9.5. Yolk sac blood islands form between the single-cell layer of endoderm cells (E) and mesothelial cells (open arrow). Blood islands develop from undifferentiated mesoderm cells (E7.5, closed arrow) that give rise to inner blood cells surrounded by an outer endothelial lining (E9.5, closed arrow).(Palis and Yoder, 2001)

Figure 15 :

 15 Figure 15: The Allantois a hematopoietic emergence site A-HH20 chick embryo. India ink inoculation followed by methyl salicylate clearing. The whole vascular network including plexuses in the brain, neural tube, and posterior intestinal portal are filled with ink. Only the base of the allantois is vascularized, while the large aggregates of red blood cells at the apex remain free of ink (arrows). B-Benzidine staining 1 phase contrast, cross section. Bar 5 20 mm C-Benzidine staining 1 Nomarski's interference contrast of yolk sac blood islands from an E2 chicken embryo. Allantoic and yolk sac blood islands are very similar.

Figure 16 :

 16 Figure 16: The extraembryonic membranes in human, mouse and chick embryos. In the middle: embryo In yellow: Yolk Sac In green: The allantois In blue: The Amnion In pink: Chorion The placenta of human and mouse embryos is shown on the upper part in pink.

Figure 17 :

 17 Figure 17: Parabiosis procedures between the YS of two chicken embryos Embryos joined by a yolk sac anastomosis (arrowed) Image from Moore&Owen 1967

Figure 18 :

 18 Figure 18: Quail-chicken yolk sac chimera A-Immediately after surgical construction B-E13 chimera developed in ovo. The quail embryo (with typical pigmentation) is associated to a chicken yolk sac. C-Erythropoiesis in the chick embryo spleen, 13 days. Early erythroblast nuclei show fine lumps of chromatin, regularly distributed, as schematized in the frame D-Early erythroblasts have Feulgen-positive material in a heavy central mass and smaller peripheral patches in their nuclei (schematized in the frame) (Images fromDieterlen-Lièvre,1975 and Dieterlen-Lièvre & Le Douarin 2004)

Figure 19 :

 19 Figure 19: Hematopoietic clusters in the aorta of different speciesIllustrations respectively from : Emmel et al 1916,[START_REF] Tavian | Aorta-Associated CD34+ Hematopoietic Cells in the Early Human Embryo[END_REF], Ciau-Uitz et al 2010, Dieterlen-Lièvre et al 1981 and Wood et al 1997

Figure 20 :

 20 Figure 20: Location of intra-embryonic HSCs. A-schematic of a transverse section through the embryo, showing the internal structure of the embryo at the level of the truncal AGM. B-An enlargement of the aortic region, schematically showing the intra-aortic clusters, which are restricted to the ventral part (floor) of the vessel, and the sub-aortic patches. The area that has hemogenic activity is shown in a red box.Abbreviations: AGM, aorta-gonad-mesonephros; NT: Neural Tube, S: Somite, M: Mesonephros, G: Gonad Adapted from[START_REF] Godin | The hare and the tortoise: an embryonic haematopoietic race[END_REF]

Figure 21 :

 21 Figure 21: Ontogeny and hematopoietic potential of the different regions of Xenopus A-The aVBI and its precursor, the embryonic hemangioblasts, derive from dorsal blastomeres of the 32-cell stage embryo B-Ventral blastomere D4 gives rise to the pVBI. C-The C3 blastomere of the 32-cell stage embryo gives rise to the DLP which later generates HSCs in the ventral wall of the dorsal aorta. Abbreviations: VBI: Ventral Blood Island, DLP: Dorsal Lateral Plate, aVBI: anterior VBI, pVBI: posterior VBI. Adapted from Ciau-Uitz et al 2010.

Figure 22 ,

 22 Figure 23.

Figure 22 :

 22 Figure 22: Hematopoietic sites in zebrafish A,B-.GFP expression driven by the gata 1 promoter in a 12 hour and a 22 hour transgenic zebrafish embryo, respectively reporting the hematopoietic activity of the ICM. C-c-myb Expression Reveals the Sites of Larval and Definitive Hematopoiesis. Blue arrows, thymus; red arrows, pronephros Adapted from Long et al 1997 and Murayama et al 2006.

Figure 23 :

 23 Figure 23: The aorta a source of hematopoietic progenitors A-arrows point at the DA-PCV joint targeted with the laser. B-Fluorescence of fluorescein just laser-uncaged all along the DA-PCV joint C-Cells marked by laser-uncaged migrate to the thymus (blue arrow) and to the pronephros (red arrow) and form cell aggregates in the CHT D-E-F: Magnifying of C showing the progenitors in the CHT Abbreviations: CHT: caudal hematopoietic tissue, PCV: posterior cardinal vein, DA: Dorsal aorta, cv: caudal vein, ca: caudal artery, Y: yolk sac, c: cloaca, ntc: notochord. Adapted from Murayama et al 2006

Figure 25 .

 25 Figure 25. Thus, when the aortas fuse into a single vessel, the ECs derived from the splanchnopleura are restricted to the aortic floor hence restricting the blood-forming ability to the ventral side of the aorta. The second phase of aortic remodeling occurs during and after aortic hematopoiesis. At the time of hematopoietic cluster formation, ECs from the somite begin to migrate and locate immediately underneath the clusters. Since hematopoietic cells are produced at the expense of splanchnopleural ECs, cells of somitic origin progressively replace the ECs of the floor ensuring the integrity of the vessel during the phase of HSC emergence. At the completion of hematopoiesis, the aortic floor of splachnopleural origin has been replaced by ECs originating from the somite. Hence within a short period of time, the origin of the aortic endothelium has changed from splanchnopleural to somatopleural (somite). In addition to replacing the aortic endothelium, the somite was also shown to give rise to the aorta smooth muscle cells. Interestingly, this aortic cell layer derives from cells of the ventral side of the sclerotome that turn their sclerotome fate and acquire a smooth muscle cell fate. In particular two sclerotome-specific markers Pax1

Figure 25 :

 25 Figure 25: Developmental history of the aorta in relationship with endothelial remodeling A-E6.5 chick host, 5 days after the graft. Cross-section at the trunk level. Triple labeling with QH1 (green)/MEP21 (red)/DAPI (blue). The vascular networks of the neural tube, dorsal root ganglia, dermis, mesonephros, body wall and limb bud derive entirely from the graft. Inset: closer view of the right side of the aorta. The aortic endothelium on the grafted side mostly derives from the graft B-Recap of the dynamics of the aorta Abbreviations: NT: Neural Tube, N: Notochord, Ao: Aorta, L: Lung, WB: Wing Bud, H: Heart, E: Ectoderm, En: Endoderm, D: Dermomyotom, Sc: Sclerotom. Illustartions from Pouget et al 2006

Figure 26 :

 26 Figure 26: Schematic representation of an erythroblast islands of the FL at E13.5Red blood cells are produced in the FL hence its red color. The maturation of erythroblasts takes place in erythroblastic islands. Each of them has a macrophage at its centre, in contact with a ring of erythroblasts, immature nucleated red blood cells.

Figure from Huber et al 2005 Figure 28 :

 200528 Figure from Huber et al 2005

Figure 30 :

 30 Figure 30: The endothelial origin of HSCs from the aorta in mice A-Protocol scheme. Slices were stained with antibodies before or after embryo slicing and imaged by confocal microscopy. Schematic of an embryo slice. B-Emergence of a GFP + cell (arrow). Green, Ly-6A-GFP; red, CD31-PE.

Figure 31 :

 31 Figure 31: The endothelial origin of HSCs from the aorta in mice Link between HSCs, hemangioblast and hemogenic endothelium Genes in red are cellular markers of the different cell types, genes in blue are differentiation factors.Adapted from[START_REF] Eilken | Continuous single-cell imaging of blood generation from haemogenic endothelium[END_REF], Lancrin et al 2009

  Bertrand et al. (2005) Rybtsov et al. showed that the population enriched with HSCs

Figure 33 :

 33 Figure 33: Existence of para aortic foci in miceThe analysis of Gata3 showed the existence of cells underneath the aorta at E11. the authors called this group of cells "para-aortic foci".A, B. In blue: Gata3 B. Gata3 positive cells do not overlap with Smooth muscle actin positive cells in red C. AA4-positive cells in blue encircle GATA-3-positive patches in yellow;Photos from[START_REF] Manaia | Lmo2 and GATA-3 hemopoietic expression[END_REF]

Figure 32 :

 32 Figure 32: Labeling of hematopoietic cells in the aortic region Cross-sections of the aortic region of E3, E4, E7 and E8 chicken embryos, first showing the hematopoietic cells inserted into the aortic endothelium and then the para-aortic foci that form in the mesenchyme underlying the aorta. A. In green: Endothelial cells FLK1, in pink the hematopoietic cells CD45. B, C. CD45 cells that have ingressed in mesenchyme. Arrowheads: macrophages. D. Para-aortic foci (pink and green) are basophile cells. Photos from jaffredo et al 2000

  indicating a change in HSPC requirements regarding Runx1. Interestingly, conditional deletion of Runx1 made either in an endothelial context where the Cre is driven by the VE-Cadherin regulatory sequences or in a hematopoietic context where the Cre is driven by the hematopoietic-specific

  and co-workers implemented a highly sensitive DNA adenine methyltransferase (Dam) identification-based methodology, to map early RUNX1 transcriptional targets in hemogenic ECs differentiated from mouse ES cells deficient for Runx1 and containing doxycycline inducible Runx1-Dam. Comparison of the Runx1-DamID methylation and RNA-Seq datasets led to the identification of 235 genes that were both bound by Runx1 and differential expressed in hemogenic ECs generated from wild type and Runx1 -/-ES cells. The expression of 80 of the genes was negatively correlated with Runx1 occupancy and 155 genes were positively correlated (Lie-A-Ling et al., 2018), consistent with Runx1's ability to function as a transcriptional

  Examination of Gfi1 and Gfi1b expression patterns indicates that during the period of hematopoietic production Gfi1 expression is associated with ECs and IAHCs whereas Gfi1b is more associated with IAHCs. Transplantation of Gfi1/1b + cells from the AGM into irradiated mice indicates that the reconstituting fraction is associated with the Gfi fraction. Double Gfi1/Gfi1b inactivation results in earlier lethality than either single invalidation. IAHCs formation is totally impaired in the AGM but cells expressing c-Kit or CD31 markers were found embedded within the endothelial layer. When isolated and dissociated, these cells were able to form clonogenic colonies. To gain insights into the molecular regulation of EHT by Gfi1 molecules, Lacaud and coworkers examined the role of LSD1, belonging to the CoREST complex, known to interact with Gfi1 proteins. Pharmacological or genetic inactivation of LSD1 leads to an impairment of EHT in culture. The analysis of global gene expression profiles upon LSD1 inhibition reveals an upregulation of cardiac-specific genes at the expense of hematopoietic-specific genes. Comparison of transcriptome in LSD1 inactivation and

F.

  Sox 7, 17 and 18 Sox genes encompasses a family of SRY (Sex Determining Region Y)-related HMG box of DNA binding proteins, referred to as SOX transcription factors, which consists of more than 20 Sox genes characterized by the evolutionarily conserved high mobility group (HMG) box, a 79-amino-acid DNA-binding motif that binds to a common consensus site with variable efficiency[START_REF] She | SOX family transcription factors involved in diverse cellular events during development[END_REF]. The Sox transcription factors associated with the vasculature are Sox7, Sox17 and Sox18. They are expressed at the onset of endothelial differentiation as early as E7.5 in a subset of Etv2 + Flk1 + CD41 -cells. Sox7 and Sox 18 continue to be expressed in the aorta, cardinal vein and intersomitic vessels at later stages whereas Sox17 becomes restricted to arterial ECs from E10.5. This pattern continues after birth. Besides roles in cardiovascular development (reviewed in[START_REF] Lilly | SOX7 expression is critically required in FLK1-expressing cells for vasculogenesis and angiogenesis during mouse embryonic development[END_REF]), Sox7, 17 and 18 were shown to play a growing role in the hemogenic endothelium and EHT. Sox 17 was shown to be expressed in hemogenic ECs during ES cell differentiation and to bind and activate the expression ofVE-Cadherin (Costa et al., 2012). Another study demonstrated that Sox17 is expressed by hemogenic ECs in vivo and that it is required for the expansion of hemogenic endothelium through the Notch signalling pathway (Clarke

  25 and is found expressed in aortic ECs but is absent from ECs of the yolk sac. As EHT takes place, HoxA3 and Runx1 expression are mutually exclusive. Forced expression of HoxA3 in ES cells under endo-hematopoietic differentiation results in the repression of CD41 and CD45 hematopoietic-specific genes and the enforced expression of VE-Cadherin and Flk1. This correlates with the loss of hematopoietic-forming potential in a clonogenic assay. A similar effect has been observed when HoxA3 is transduced in AGM cells. Transcriptome analysis and chromatin immunoprecipitation indicates that HoxA3 targets the key hematopoietic transcription factors Runx1, Gata1, Gfi1b, Ikaros and Pu1 and down regulates them

1 .

 1 Epithelial-mesenchymal transition (EMT)Epithelial-mesenchymal transition (EMT) is a morphogenetic process of cells that adopt an epithelial organization in their developmental ontogeny or homeostatic maintenance. During EMT, epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types. EMT is essential for numerous developmental processes of many tissues and organs in the developing embryo, and numerous embryonic events such as gastrulation, neural crest formation[START_REF] Shook | Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development[END_REF], heart valve formation, secondary palate development, and myogenesis. In multicellular organisms, epithelial and mesenchymal cells differ in phenotype as well as function, though both share inherent plasticity. Epithelial cells organize themselves with a uniform stable apicobasal polarity and are often associated with barrier junctions and a specialized layer of the extracellular matrix, whereas mesenchymal cells lack this polarization, have a spindleshaped morphology and interact with each other only through focal points[START_REF] Hamidi | Epithelial-mesenchymal transition in haematopoietic stem cell development and homeostasis[END_REF]. It is well known that epithelial cells express high levels of E-cadherin, whereas mesenchymal cells express those of N-cadherin, fibronectin, and vimentin.Hence, EMT entails profound morphological and phenotypic changes to a cell Figure34.

Figure 34 :

 34 Figure 34: EMT entails profound morphological and phenotypic changes to a cell EMT involves progressive loss of epithelial markers and acquisition of mesenchymal ones Co-expression of both epithelial and mesenchymal markers defines intermediate states of EMT. The relative ratio between epithelial and mesenchymal markers is used to define either an epithelial-or mesenchymal-like intermediate state.

Figure

  Figure from hamidi and Sheng 2018

Figure 35 :

 35 Figure 35: Comparative view of molecular determinants involved in EndMT and EHT processes. Upon TGF-b and shear stress stimulation, some endothelial cells undergo an EMT-like process characterized by downregulation of epithelial markers and upregulation of mesenchymal ones.EndMT and EHT processes share a set of EMT regulators, but fundamental differences can also be observed (e.g. Runx3 for the EndMT and Runx1 for the EHT).

Figure from hamidi and Sheng 2018 90 3.

 90 Figure from hamidi and Sheng 2018

  to perform necessary, but spatially and temporally distinct, functions during definitive hematopoiesis. The inhibition of MMP2 function caused accumulation of fibronectinrich ECM, retention of runx1/ cmyb+ HSPCs in the VDA, and delayed caudal hematopoietic tissue (CHT) colonization; these defects were absent in fibronectin mutants, indicating that Mmp2 facilitates endothelial-to-hematopoietic transition via ECM remodeling. Whereas, Mmp9 was dispensable for HSPC budding, being instead required for proper colonization of secondary niches. They demonstrated that MMP2 and MMP9, which represent the gelatinase family, have been implicated in angiogenic ECM remodeling downstream of Notch and VEGF signaling.

1 .

 1 PSM harvesting in the quail embryoWe used quail (Coturnix coturnix japonica) embryo pre-somitic mesoderm (PSM) according to[START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. Eggs were incubated for 36-45 hours at 37±1°C in a humidified atmosphere to reach 10-18 somite pairs. Microsurgery was performed as previously described[START_REF] Pardanaud | Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis[END_REF]Pardanaud, #2110]]. The ectoderm layer was gently cut following a line between the neural tube and the PSM. Dispase (1U/ml) was used to facilitate tissue separation. The PSM was harvested over a length of approximately 10 somites (from the most posterior formed somite to the rhomboidal sinus) on both sides of the embryo (FigureM1 B, C). Then they were sectioned into 5-6 pieces each to increase the surface exposed to the culture media and allow a better spreading during the first phases of cell attachment. Five embryos (i.e. 10 PSM) were used per culture dish. Each PSM was cut into 4 to 5 equal pieces, rinsed in Opti-MEM plus GlutaMAX™ I (Gibco Life Technologies) +5% Fetal Calf Serum (FCS, Gibco Life Technologies) +100 Units per ml penicillin/streptomycin (Gibco Life Technologies) + 1% Chicken Serum (Gibco Life Technologies) before culture.

Figure M1 :

 M1 Figure M1: PSM harvesting in the quail embryo A. Culture system (PSM in red) (Yvernogeau et al 2016) B-C. Photos of the quail embryo dissection, Leica Binocular Loupe, magnification 40X. The ectoderm is cut following the red line (B). PSM (white arrows) are detached from the embryo on both sides of the neural tube (black arrow) (C).

Figure R2 :

 R2 Figure R2: Sample isolation strategyA-In total we isolated 20 samples. 4 samples for each of the populations: PSM, EC D2, EC D4, HEC D4 and 2 samples for each of HC and EC woSVF populations. PSM were directly isolated from embryos, HC were collected as floating cells whereas EC and HEC were cell sorted based on the level of LDL uptake. B-Roadmap of the used strategy of the analysis. Starting from the sequenced data going through the bioinformatics and statistical analysis and finishing with the functional characterization of HEC specific genes.

  Analysis of the GO categories clearly confirmed the phenotypic differences between the 4 distinct populations. The M cell population was enriched in genes implicated in spliceosome, Hippo and Hedgehog signaling pathways and shared the category adherens junctions with the EC population. The EC population showed enrichment in lysosome, FoxO, Wnt and Rap1 signaling pathways and glycosaminoglycan degradation. The HEC population was enriched in genes implicated in focal adhesion, ECM, regulation of actin cytoskeleton, MAPK and PI3K-Akt pathways, glycosaminoglycan biosynthesis and axon guidance. Finally, the HC population was enriched in genes implicated in metabolic pathways, oxidative phosphorylation, Jak-STAT and B and T cell receptor signaling pathways (FigureR5).

Figure R5 :

 R5 Figure R5: Analysis of the gene ontology categoriesA-Score plot (PC1 v/s PC3) on 28 GO categories and 4 populations allowed the discrimination between HC and HEC (PC1 46%), EC and M (PC3 16.9%). B-Loading plot (variables as red arrows) defined the specific categories of GO for each population.

  Figure R7 C,D.

Figure R7 :

 R7 Figure R7: HEC modules and gene connectivityA-Cytoscape visualization of the Black HEC network B-Cytoscape visualization of the Red HEC network C-The gene connectivity chart of the most connected genes in red module D-The gene connectivity chart of the most connected genes in black module

  and Figure R10G; Figure R10H). Taken together the Wnt pathway plays a role in EHT. These results are completely in keeping with results published by Ruiz-Herguido et al., 2012 on the mouse AGM.

Figure R9 :

 R9 Figure R9: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype A: PSM culture mock treated at D1 and observed at D4. B: enlargement of the frame in A showing the flat endothelial layer and the few round cells resulting from the initiation of EHT at this stage. C: Representative FACS analysis showing the percentages of LDL hi et LDL int in the mock-(water) treated culture. D: PSM culture treated with PKF118 1µM at D1 and observed at D4. Note the flattening of the cell layer treated with PKF118 compared with the mock-treated culture. E: Enlargement of the frame in D showing the flattening of the cells and the presence of fewer round cells. F: representative FACS analysis showing the percentages of LDL hi et LDL int in the PKF118-treated culture (G). G: Percentage of the LDL hi cells in mock-vs PKF118-treated cells (n=5, p<0.02). H: Percentage of the LDL int cells in mockvs PKF118-treated cells (n=5, p<0.05). I: Percentage of floating cells in mock-vs PKF118-treated cells (n=5, p<0.05). J: qRT-PCR for CD144 on mock-and PKF118-treated cells. A significant increase of CD144 mRNA expression is detected in the PKF118-treated cultures. K: qRT-PCR for RUNX1 on mock-and PKF118-treated cells. A significant decrease of RUNX1 mRNA expression is detected in the PKF118treated cultures. Means are ± SEM. A, D, bar= 400µm; B, E, bar= 20µm.

Figure R10 :

 R10 Figure R10: The Wnt agonist SB enhances EHT A: PSM culture mock treated at D1 and observed at D4. B: enlargement of the frame in A showing the flat endothelial layer and the few round cells resulting from the initiation of EHT at this stage. C: Representative FACS analysis showing the percentages of LDLhi and LDLint in the mock-(DMSO) treated culture. D: PSM culture treated with SB216763 20µM at D1 and observed at D4. Note the presence of more round hematopoietic cells in the sample treated with SB216763 compared with the mock-treated culture. E: Enlargement of the frame in D showing the flattening of the cells and the presence of fewer round cells. F: representative FACS analysis showing the percentages of LDLhi et LDLint in the SB216763-treated culture. G: Percentage of the LDLhi cells in mock-vs SB216763-treated cells (n=5, p<0.04). H: Percentage of the LDLint cells in mock-vs SB216763-treated cells (n=5, p<0.02). Means are ± SEM. A, D bar= 400µm; B, E, bar= 20µm.

Figure R11 :

 R11 Figure R11: The gamma secretase inhibitor DAPT enhances EHT A: Aspect of a control culture at D4. B: aspect of the DAPT-treated culture (25µM) at D4. Note the presence of numerous round cells. B': Enlargement of a significant area of a DAPT-treated culture. Numerous rounds, bright, floating cells are visible. C: Representative FACS analysis showing the percentages of LDLhi and LDLint in the mock-(DMSO) treated culture. D: Representative FACS analysis showing the percentages of LDLhi and LDLint in the DAPT-treated culture. E: Representative FACS analysis showing the percentages of floating hematopoietic cells in the mock-(DMSO) treated culture. F: Representative FACS analysis showing the percentages of floating hematopoietic cells in the DAPT-treated culture. A 3.2-fold increase is obtained compared to the DMSO-treated culture. G: LDLhi cells in DMSO-vs DAPT-treated cultures. H: LDLint cells in DMSOvs DAPT-treated cultures. I: Hematopoietic cells in DMSO-vs DAPT-treated cultures (p<0.001). J: q-RT-PCR of CD45 expression in DMSO-vs DAPT-treated cultures showing a more than two-fold increase in CD45 expression. A, B bar= 400µm; B' bar= 20µm.

Figure R12 :

 R12 Figure R12: POFUT2 knock down enhances EHT.A: experimental design. PSM cells were put in culture at D0, transfected at D1 and analyzed at D4 for LDL uptake by flow cytometry and q-PCR for the candidate gene and some of its immediate neighbors in the gene network. B: Effect of Lipofectamine (Lipo), scramble (Scr) and POFUT2 siRNA transduction on the expression of endogenous POFUT2. At 60nM, POFUT2 mRNA expression was decreased by more than 90% compared with the liposome and scramble siRNA controls. C, E, G: culture at D4 that received Lipofectamine (C), scramble si (E) and POFUT2 si (F) at D1. Note that the number of round cells is more important in the siPOFUT2 condition than with the two other ones. D, F, H: representative flow cytometry experiment showing the balance between LDL int and LDL hi cells when cells received Lipofectamine (D), scramble si (F) and POFUT2 si (H). Note that the number of LDLint cells has increased at the expense of LDL hi cells in the POFUT2 si condition. I: percentage of LDL hi cells in the three conditions. A significant (p<0,001) decrease of LDL hi cells is visible in the POFUT2 si conditions (n=5). J: percentage of LDL int cells in the three conditions. A low but significant (p<0.05) increase of LDL int cells is observed in the POFUT2 si condition compared with the two others. K: mRNA expression of COL5A2 is significantly decreased when POFUT2 is knock down by siRNA. L: DNAJB4 mRNA expression is decreased in POFUT2 knock down condition. M: EMILIN1A mRNA expression is significantly decreased in POFUT2 knock down condition. C, E, G, bar= 400µm.

  TESTIN a hub of the HEC network Based on the list of genes positively correlated to the HEC state obtained following WGCNA analysis and the measure of gene connectivity, we selected TESTIN for potentially playing a role in HEC. TESTIN is upregulated in HEC compared to EC or HC. It is a protein expressed in almost all normal human tissues. It locates in the cytoplasm along stress fibers being recruited to focal adhesions. it forms complexes with various cytoskeleton proteins such as Actin, Talin and Paxilin playing a significant role in cell motility and adhesion. Decreased Testin expression associate with loss of epithelial morphology and gain of migratory and invasive properties of mesenchymal cells. Testin plays a significant role in cell adhesion, cell spreading and the reorganization of actin cytoskeleton.

129 Figure R15 :

 129R15 Figure R15: TESTIN knock down doesn't affect EHT.A: Sequences of the siRNA and scramble designed for Testin knock down. B: experimental design. PSM cells were put in culture at D0, transfected at D1 and analyzed at D4 for LDL uptake by flow cytometry. C: Effect of Lipofectamine (Lipo), scramble (Scr) and POFUT2 siRNA transduction on the expression of endogenous TESTIN. At 60nM, TESTIN mRNA expression was decreased by more than 90% compared with the liposome and scramble siRNA controls. D, E: culture at D4 that received Lipofectamine (D,D'), TESTIN si (E,E') at D1. Note that no changes in the number of round cells in the siTESTIN condition compared to the two other ones. F, G,H: representative flow cytometry experiment showing the balance between LDL int and LDL hi cells when cells received Lipofectamine (F), scramble si (G) and TESTIN si (H). I, J: percentage of LDL hi and LDL int cells in the three conditions. Note that no significant changes in the % of LDL hi nor the LDL int fraction with TESTIN si compared to Scr and Lipo. K: The 11 first highly connected first neighbors of TESTIN. L: mRNA expression of BICC1 is decreased when TESTIN is knock down by siRNA. M, N: CREB3L1 mRNA expression and CSPG4 showed no differences in TESTIN knock down condition.

FigureFigure R20 :

 R20 Figure R18: Mesodermal markers

Figure R21 :

 R21 Figure R21: Analysis of the KEGG pathways categories by PCA -Left: the score plot (PC1 v/s PC2) on 82 KEGG pathways categories and 13 clusters allowed the discrimination between 4 major populations HEC, HC, M and EC -Right: Loading plot (variables as red arrows) defined the specific categories of KEGG pathways for each population.

  EHT: what we know and what we are still missing! Endothelial to hematopoietic transition is driven by an incredibly complex yet to be fully elucidated interplay between developmental timing and tissue microenvironment factors. Despite having identified some of the key transcriptional and pathways playing a role in this transition, still there remain several unanswered questions surrounding the progression from embryonic mesoderm to endothelium to HSPCs and adult HCs, especially the morphological changes and restructuring occurrence during this transition. Efficient generation and differentiation of hematopoietic cells in vitro to date has been accompanied with technical difficulties, and these difficulties are almost certainly worsened by a need for a more complete understanding of in vivo endothelial and hematopoietic development.

  as the time point chosen for the analyses. i.e Swiers et al looked to HE at E8.5 and E10.5 whereas Guiu et al looked at E11.5, Baron et al looked at E9 and E10. This points to a lack in the definition of a well-defined timeframe of the HE identification. Baron et al and Gao et al discussed a close relationship between HE and EC in the major arteries with a shortlist of deferentially expressed genes in each population. Or in our analysis using WGCNA, we could spot 2 unique signatures of EC and HEC populations represented by different gene networks. The network of ECs composed of 600 genes and the two networks of HECs composed respectively of 260 and 200 genes showed distinct GO categories. EC module was enriched with Pleckstrin homology domain, membrane genes, lysosome, SH3 domain, cell adherens junctions and wnt pathway. Whereas HEC were enriched with cell migration, ECM,

  Whil e GO characteristics of YFP -LSK cells focused on transcription, translation and DNA repair, GO of the YFP + LSK cells were more diverse and included membrane, extra-cellular matrix (ECM), focal adhesion and actin-binding molecules. To more accurately determine the identity of the YFP + LSK cells, we included transcriptomes of several BM and AGM-derived endothelial and HC types (i.e. BM lin + HCs, BM CD150 + CD48 -LSK HSCs, AGM CD144 + CD45 -ECs, AGM CD144 -CD45 + HCs and AGM CD144 + CD45 + HSPCs to the YFP + and YFP -LSK cell transcriptomes, n=23 samples). PCA analysis on the whole gene set (18,920 genes) revealed three main groups. The first encompassed BM and AGM HCs, the second comprised adult and embryonic HSPCs including YFP -LSK cells while the third group encompassed AGM ECs and YFP + LSK cells (Fig.4a, upper panel). Further analysis of DEGs identified by ANOVA (p-value= 0.001) on the 23 samples as observations and the gene set of 2,056 DEGs (986 up; 1,070 down) confirmed that the first principal component highlighted the difference between YFP + and YFP -LSK cells (Fig.4a, lower panel). Hierarchical clustering (Extended Data Fig.

  artificial dark field was obtained by injecting Indian ink, diluted 1:1 in phosphate -buffered saline (PBS), beneath the host embryos. Microsurgery was performed on the right side of the host. The PSM attached to the last formed somite was removed over a length corresponding to 5-10 somites. PSMs were rinsed in DMEM (PAA Laboratories)/10% foetal calf serum (FCS, Eurobio) and transplanted into a host submitted to the same ablation. Grafts were performed according to the original dorso-ventral and antero-posterior orientations. The chimeric embryos were incubated and analysed at several time points as indicated in Extended Data Table1.After hatching, PSM grafted chickens were housed at the Gemeenschappelijk Dierenlaboratorium facility (Utrecht, Netherlands) until they reached adulthood. Animals were housed according to institutional guidelines, and procedures were performed in compliance with Standards for Care and Use of Laboratory Animals, with approval from the Dutch Animal Experiment Committee.

  slices (200µm thickness) of the marrows were obtained using a tissue chopper and st ained with CD41-PE (clone 11C3, Bio-Rad) antibody for 20 minutes on ice. After 3 washes in PBS, slices were placed in a culture chamber, embedded in a 1% low melting point gel agarose. Five to 6 slices per experiments were over-night sequentially time-lapse imaged (every 20 minutes) on a Zeiss LSM700 confocal microscope. The following day, slices were stained again with CD41-PE (clone 11C3, Bio-Rad) antibody, washed and imaged to assess the emergence of new HSPCs during the overnight imaging.

FiguresFigure 1 .

 1 Figures

  radius showing conspicuous signs of BM haematopoiesis observed under visible (c) or UV illumination (d). GFP tubule-like structures were observed in the BM. The arrow indicates the level of section seen in (e). e, f, Representative cross section (arrow in (d)) through the bone stained for DAPI (cell nuclei) and GFP showing GFP + cells forming sinusoids. g-j, Representative fluorescent images showing the co-distribution of GFP + sinusoids (h) and CD41 + HCs (i) (arrows), counterstained with DAPI (j). Merged picture is shown in (g). k, Percentage of GFP + cells in E16 grafted limbs. Each dot represents a grafted recipient. l, Distribution of GFP + cells in the endothelial and haematopoietic lineages. MEP21, endothelial/haematopoietic cell marker; CD45, panhaematopoietic cell marker; CD41 and c-Kit, HSPC markers; CD3, lymphoid cell marker. m, Distribution of the endothelial and the different haematopoietic lineages within the GFP + population. Numbers in brackets under the x axis (l and m) are the numbers of grafted recipients. All data represent mean ± SEM from at least n=8 independent experimental animals. n, Representative flow cytometry analysis identifying the MEP21 + CD45 + population known to be enriched in multipotent progenitors 17 and the contribution of the GFP + PSM-derived cells to the endothelial and/or haematopoietic (multipotent) progenitors. Scale bars in b, 1cm; in c, d, 300µm; in e, 200µm; in f, 80µm; in g, 15µm.

Figure 2 .

 2 Figure 2. HSPCs derived from the late foetal BM endothelium persists after hatching. a, b, PSM grafted chimeras at 8 days (a) and 5 months (b) post-grafting, respectively. c, Percentage of GFP + cells in grafted limbs at 5 months. Each dot represents a grafted recipient. d, Distribution of GFP + cells in the different BM endothelial and haematopoietic lineages of a 5.5 month-old recipient. MEP21, endothelial/haematopoietic marker; CD45, pan-haematopoietic marker; CD41, c-Kit, HSPC markers; KUL1, macrophage/monocyte marker; CD3, lymphoid marker. e, Distribution of the BM endothelial and haematopoietic lineages contained in the GFP + population. Numbers in brackets under the x axis (d and e) indicate the numbers of analysed transplanted recipients. All data represent mean ± SEM (n=4-7 independent experimental animals). f, Combined flow cytometry analysis of the adult grafted BM for MEP21 and CD45 populations showing the percentages of MEP21 -CD45 + , MEP21 + CD45 + and MEP21 + CD45 -populations and the contribution of GFP + cells within each

Figure 3 .

 3 Figure 3. Conditional expression of YFP in VE-Cadherin cells in BM at birth discloses a contribution of vascular ECs to MPPs and Lin -Sca1 + c-kit + CD150 + CD48 -HSCs. a, Scheme showing the activation of VE-Cadherin (CD144, CDH5) by tamoxifen injection and the different time points of analysis. Bottom arrows, time of tamoxifen injections. Top arrows, time points of analyses. b, YFP expression in mononucleated BM cells at 27 days. c, Representative flow cytometry analysis of CD144 -CD45 + , CD144 + CD45 + and CD144 + CD45 -populations and YFP expression in each population. d, Time course analysis of total YFP + cells, and YFP + cells in CD144 + CD45 -and LSK populations. n=2-5 individuals per time point. Error bars are mean ± SEM. e, f, g, Representative flow cytometry analyses of the SLAM/LSK cells (e), HSCs (f) and MPPs (f, g) revealed an enrichment of YFP + cells in the MPP3 fraction and HSCs.

Fig. 4 .

 4 Fig. 4. BM vascular EC-derived HSPCs display an endo-haematopoietic transcriptomic profile and show long-term multilineage reconstitution. a, PCA (upper panel): PC1 vs. PC2 score plot corresponding to the PCA of 23 samples as observations, and the whole gene set (18920 genes) as variables. PCA (lower panel): PC1 vs. PC2 score plot corresponding to the 23 samples as observations and the gene set of 2056 DEGs (986 up, 1070 down) as variables; the gene set was obtained by ANOVA (YFP + vs. YFP -, p < 0,001, f > 1 or f < -1); PC1 corresponds to the contrast between YFP+ and YFP-cells. b, Module detection by clustering tree cut. Same data as for PCA (lower panel). The parameters were as follows: power: 20; adjacency: signed; correlation: Pearson's; linkage: average; minimum module size: 30. Two major highlighted modules, black and magenta, are positively and negatively correlated to the YFP + to YFP -cells contrast, respectively. c, Major GO categories corresponding to genes belonging to the black and magenta modules. Enrichment scores as given by DAVID database. Abbreviations: ECM-Receptor: ECMreceptor interaction; Actin: actin binding; ECM: extracellular matrix; Notch: notch binding/pathway; Replication: DNA replication; Repair: DNA repair; Splicing: mRNA splicing. d, Cytoscape connectivity plot of the black (up-regulated in YFP + cells) and magenta (up-regulated in YFP -cells) modules. e, Comparison of levels of circulating blood cells analysed at different time points following engraftment between recipients transplanted with YFP + or YFP -LSK cells. Empty triangles, YFP -LSK cells grafted in six animals. Black circles, YFP + LSK cells grafted in 4 animals. Bars represent means. f, Flow cytometry analysis of representative C57Bl6-CD45.1 recipients injected with 2,000 YFP -(upper plots) or YFP + (lower plots) LSK cells (C57Bl6-CD45.2), respectively (left plots). The right plots show YFP expression in the engrafted CD45.2 + populations.

Fig. 5 .

 5 Fig. 5. Summary of presomitic and lateral plate mesoderm contribution to the endothelial and haematopoietic compartments of the bone marrow. a-c, Scheme summarizing the location and fate of presomitic mesoderm (PSM, green) and lateral plate mesoderm (LPM, blue) at three representative and equivalent time points of chicken and mouse development. a, Location of PSM and LPM in chicken (at E1.5, time point of graft isolation) and mouse (E9.5) embryos. PSM: unsegmented plate located at the caudal part of the developing embryo, on each side of the neural tube. LPM: mesoderm located laterally and ventrally to the PSM. b, The PSM progressively segments to form somites, which eventually differentiate into dermomyotome and sclerotome. They later give rise to muscles, vasculature and bones (i.e. from trunk, limbs). The haemogenic endothelial cells of the aorta and intra-aortic haematopoietic cluster (IAHC) cells derive exclusively from the LPM, which also gives rise to the limb mesenchyme. c,In young adults, bones and marrows are derived from the LPM while the vasculature is entirely derived from the PSM. Most haematopoietic stem and progenitor cells (HSPCs) come from LPM -derived HSCs (initially formed in the embryonic aorta). HSPCs are also de novo generated from the PSM-derived endothelium of the bone marrow (this study).

  and haematopoietic cells expressing GFP in adult BM. Distribution of CD45, MEP21, CD41, c-KIT, CD3 and KUL1 populations in grafted (a, b) and wild-type (c, d) adult BM mononucleated cells. Numbers within the parenthesis indicate the number of samples analysed for each marker. b, Representative multilineage flow cytometry analysis of a grafted adult BM showing that GFP + cells derived from the PSM graft contributed to all lineages. Numbers in green indicate the percentages of GFP + cells in each population (i.e. in CD45 + , MEP21 + , CD41 + , c-KIT + , CD3 + and KUL1 + populations). d, Representative multilineage flow cytometry analysis of a wild-type adult BM showing the absence of GFP staining. Error bars represent mean ± SEM. Extended Data Figure 9. Pax3 + somite-derived cells do not contribute to the aorta haematopoieisis but contribute to the endothelial and haematopoietic lineages in the late foetal BM. a, Transverse section through the aorta of an E10.5 Pax3 GFP mouse embryo at the mid -trunk level. CD31 (red, left), Pax3-GFP (green, middle) double staining counterstained with DAPI (blue). Right panel represents the merge. b, Flow cytometry analysis of the endothelial and haematopoietic lineages in the BM of Pax3-Cre/mTmG foetuses. c, Flow cytometry analysis showing GFP + cell (i.e. somite-derived cell) contribution to the different endothelial and haematopoietic populations. Mouse somite -derived cells mostly contributed to new-born BM CD3 + , Sca1 + c-Kit + and CD144 + CD45 + cell populations. Data in b, c represent mean ± SEM from 3-5 pooled foetuses in 3 independent experiments. d, Representative GFP expression in mononucleated BM cells. e, Representative flow cytometry analysis of the mononucleated BM cells revealed GFP + cells mainly in the CD144 + CD45 + population. f, Flow cytometry analysis showing the presence of GFP + cells in the Sca1 + c-KIT + population (i.e. HSPC population). g, Negative control, i.e. non-recombined foetuses. Extended Data Figure 11. YFP induction in VE-cadherin + cells at different time points after birth reveals a decrease in the number of YFP + endothelial and haematopoietic cells. a, Scheme showing the activation of YFP in VE-Cadherin + (Cdh5) cells by tamoxifen injection at different time points after birth (coloured arrows). b, Analysis of the percentages of total YFP + cells, and YFP + cells in CD144 + CD45 -and LSK populations at 21 days post tamoxifen injection. Of note, the x axis values represent the age of the mice from the time of tamoxifen injection. Error bars are mean ± SEM. Extended Data Figure 12. PCA, Gene Ontology and hierarchical clustering analysis show major differences between YFP + and YFP -LSK cells. a, PCA with the entire set of mRNAs (30,922 genes) as variables and the basic set of YFP + (green) and YFP -(red) LSK cells as observations. The two types of transcriptomes were strongly separated. b, Major GO categories given by DAVID for the gene sets up-regulated in YFP + (green) and YFP -LSK (red) cells. c, Hierarchical clustering obtained with the 23 samples as observations and the gene set of 2056 DEGs (986 up, 1070 down) as variables was generated from the PCA displayed in Fig. 4a, bottom panel.Branch organisation reflects the association between the different samples displayed on the PCA. for each type of analysis.

Figure 2 :

 2 Figure 2: Genome-wide analysis of temperature-dependent transcriptome responses to photoperiod in quail. Experimental design showing the 3 time-points each sampled after 4 weeks of the target photoperiod (circled) with RNASeq at n=4 A.Intersection of DEGs between LD 23°C vs SD 23°C and LD 23°C vs SD 9°C B. Volcano plots comparing LD 23°C vs SD 23°C showing 71 up (yellow) and 42 down (blue) DEGs C and LD 23°C vs SD 23°C D. Grey labels do not pass fold change threshold at 23°C. Temperaturedependent effects on fold change in DEGs when comparing SD at 23°C and SD 9°C. Arrows point from 23°C to 9°C and indicate a significant amplifying (green) or dampening (orange) effect of 9°C on

Figure 3 :Figure 4 :

 34 Figure 3: Photoperiod signalling in the MBH incorporating observations from RNASeq

Figure 5 :

 5 Figure 5: Heatmap comparison between pathways upregulated (orange) and downregulated (orange) in quail, chicken and duck, in ileum day 1 (A), ileum day 3 (B), lung day 1 (C) and lung day 3 (D).

Figure 6 :

 6 Figure 6: Proportion of genes commonly regulated between quail and chicken or duck to H5N1 infection on day 3 in ileum (A) and lung (B)

  e., 10µM and 20µM with 20µM displaying the strongest effect. The result is an increase in the number of round hematopoietic cells produced compared with the mock-(DMSO) treated culture (Compare Figure S3A to Figure S3D and Figure S3B to Figure S3E). Flow cytometry analysis reveals a 1.6-fold change in the number of LDL int cells at the expense of LDL hi cells (Figure S3C; Figure S3F and Figure S3G; Figure S3H). Taken together the Wnt pathway plays a role in EHT. These results are completely in keeping with results published by Ruiz-Herguido et al., 2012 on the mouse AGM.The Gamma secretase inhibitor DAPT stimulates the EHT We used gamma secretase as an inhibitor of Notch to inquire into the role of this signaling pathway in hemogenic endothelium and EHT. DAPT 25µM was applied to the culture at D2. Analysis was performed at D4. DAPT readily produced a dramatic change in culture. Within the two days of DAPT contact, many cells rounded up, detached from the adherent layer and were found in the supernatant (Compare FigureS4A; FigureS4B).

  POFUT2 loss of function enhances the EHTBased on the list of genes positively correlated to the HEC state obtained following WGCNA analysis and the measure of gene connectivity, we selected POFUT2 for potentially playing a strong role in HEC. POFUT2 is upregulated in HEC compared to EC or HC. POFUT2 (Protein O-Fucosyltransferase 2) catalyzes the reaction that attaches fucose through an O-glycosidic linkage to a conserved serine or threonine residue in the consensus sequence C1-X(2,3)-S/T-C2-X(2)-G of thrombospondin type 1 repeats where C1 and C2 are the first and second cysteines, respectively. It fucosylates members of the ADAMTS family, the Trombosporin and Spondin families. This fucosylation is required to regulate EMT during formation of the mesoderm and definitive endoderm

Figure A1 :

 A1 Figure A1: The level of AcLDL uptake separates endothelium from hemogenic endotheliumA-C: Flow cytometry analysis showing the expression of AcLDL uptake in the culture. A: analysis at D2. The cells display a continuum of AcLDL uptake from nil to high. Frame shows the sorted population. B: analysis at D4 in normal culture conditions. Two distinct populations with contrasted AcLDL uptake are seen. The left frame isolates the LDL int population and the right frame, the LDL hi population. C: analysis at D4 in the absence of fetal calf serum known to orientate the cells towards the vascular endothelium lineage[START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. A large percentage of the population is contained within the LDL hi population in keeping with their strong endothelial commitment. D-F: quantitative PCR on the different cell fractions for CD31 (D), CD144 (E) and RUNX1 (F). CD31 and CD144 are decreased in the LDL int population compared with the other endothelial populations. RUNX1 is strongly increased in the LDL int population compared with the other endothelial fractions (n=2). Significance is given compared with the D4 LDL hi fraction. Data are shown as means ± SEM.

Figure A2 :

 A2 Figure A2: General outline of the study and transcriptome analysis A: flow chart outlining the analyses on the different cell fraction transcriptomes. B: PCA on the 20322 genes expressed by the 4 cell fractions. Mesoderm (M) are in purple. Endothelial cells (ECs) are in red.Hemogenic ECs (HEC) are in green and Hematopoietic Cells (HC) are in blue. PC1(30.2% of the variance) separates M from ECs whereas PC3 (9.85% of the variance) separates HEC from HC. C: Venn diagram of genes differentially upregulated in HEC vs. the other cell types. D: Major GO categories given by DAVID using the genes specifically up-regulated in the 4 cell populations. E: PCA using the genes specifically up-regulated in the 4 cell populations. PC1 (43% of the variance discriminates M from ECs whereas PC3 (17% of the variance) discriminates HEC from EC. F: Dendrogram and correlation map of the genes up-regulated revealing the cell-specific modules. G: Organization of the HEC-specific Green module of genes upregulated. H: Connectivity plot of the 19 most connected genes in the Green module. I: Major GO categories given by DAVID for genes belonging to the two HEC-specific modules i.e.,Green and Red. Golg, golgi; PH, pleckstrin homology domain; FA, focal adhesion; ECM, extracellular matrix; SH3, SH3 domain; MT, microtubule; Mig, migration; Phase, phosphatase; Junc, cell adherens junctions; Memb, membrane; Sig, cell signaling.

Figure A3 :

 A3 Figure A3: The Wnt antagonist, PKF118, inhibits EHT by reinforcing the endothelial phenotype A: PSM culture mock treated at D1 and observed at D4. B: enlargement of the frame in A showing the flat endothelial layer and the few round cells resulting from the initiation of EHT at this stage. C: Representative FACS analysis showing the percentages of LDL hi et LDL int in the mock-(water) treated culture. D: PSM culture treated with PKF118 1µM at D1 and observed at D4. Note the flattening of the cell layer treated with PKF118 compared with the mock-treated culture. E: Enlargement of the frame in D showing the flattening of the cells and the presence of fewer round cells. F: representative FACS analysis showing the percentages of LDL hi et LDL int in the PKF118-treated culture (G). G: Percentage of the LDL hi cells in mock-vs PKF118-treated cells (n=5, p<0.02). H: Percentage of the LDL int cells in mockvs PKF118-treated cells (n=5, p<0.1). I: Percentage of floating cells in mock-vs PKF118-treated cells (n=5, p<0.1). J: qRT-PCR for CD144 on mock-and PKF118-treated cells. A significant increase of CD144 mRNA expression is detected in the PKF118-treated cultures. K: qRT-PCR for RUNX1 on mock-and PKF118-treated cells. A significant decrease of RUNX1 mRNA expression is detected in the PKF118treated cultures. Means are ± SEM. A, D, bar= 400µm; B, E, bar= 20µm.

Figure A4 :

 A4 Figure A4: POFUT2 knock down enhances EHT.

Figure S1 :

 S1 Figure S1: Outlines of transcriptome and statistical analysis A: Chart of the different cell fractions isolated, their LDL characteristics and their numbers. B: Partek flow pipeline of transcriptome analysis. C: Hierarchical clustering of the 20 samples based on the expression of the 20332 genes. Note that M is found in between HEC and HC D: Venn diagrams of the hematopoietic-(HC), mesoderm-(M) and endothelial-(EC) specific up-regulated genes. HC= 492, M= 701, EC= 635. E: Hierarchical clustering on the sets of differentially expressed genes. The series now starts from M and ends at HC.

Figure S2 :

 S2 Figure S2: HEC-specific red gene network and POFUT2 first neighbors Cytoscape representation of the HEC-specific red network (up-regulated in HEC). The color code of the different molecule categories is indicated. B: Gene connectivity chart of the HEC-specific red network. The first 19 genes are represented. C: POFUT2 first neighbors. The color code is the same than in A.

Figure S3 :

 S3 Figure S3: The Wnt agonist SB enhances EHT A: PSM culture mock treated at D1 and observed at D4. B: enlargement of the frame in A showing the flat endothelial layer and the few round cells resulting from the initiation of EHT at this stage. C: Representative FACS analysis showing the percentages of LDLhi and LDLint in the mock-(DMSO) treated culture. D: PSM culture treated with SB216763 20µM at D1 and observed at D4. Note the presence of more round hematopoietic cells in the sample treated with SB216763 compared with the mock-treated culture. E: Enlargement of the frame in D showing the flattening of the cells and the presence of fewer round cells. F: representative FACS analysis showing the percentages of LDLhi et LDLint in the SB216763-treated culture. G: Percentage of the LDLhi cells in mock-vs SB216763-treated cells (n=5, p<0.04). H: Percentage of the LDLint cells in mock-vs SB216763-treated cells (n=5, p<0.02). Means are ± SEM. A, D bar= 400µm; B, E, bar= 20µm.

Figure S4 :

 S4 Figure S4: The gamma secretase inhibitor DAPT enhances EHT A: Aspect of a control culture at D4. B: aspect of the DAPT-treated culture (25µM) at D4. Note the presence of numerous round cells. B': Enlargement of a significant area of a DAPT-treated culture. Numerous round, bright, floating cells are visible. C: Representative FACS analysis showing the percentages of LDL hi and LDLint in the mock-(DMSO) treated culture. D: Representative FACS analysis showing the percentages of LDL hi and LDL int in the DAPT-treated culture. E: Representative FACS analysis showing the percentages of floating hematopoietic cells in the mock-(DMSO) treated culture. F: Representative FACS analysis showing the percentages of floating hematopoietic cells in the DAPT-treated culture. A 3.2-fold increase is obtained compared to the DMSO-treated culture. G: LDL hi cells in DMSO-vs DAPT-treated cultures. H: LDL int cells in DMSOvs DAPT-treated cultures. I: Hematopoietic cells in DMSO-vs DAPT-treated cultures (p<0.001). J: q-RT-PCR of CD45 expression in DMSO-vs DAPT-treated cultures showing a more than two-fold increase in CD45 expression. A, B bar= 400µm; B' bar= 20µm.

Table

Table 1 : The different types of colonies in the CFC assay

 1 , Figure7.

	CFU-E	Colony Forming Unit-Erythroid	Erythroid progenitors
	CFU-Mk	Colony Forming Unit-Megakaryocyte	Megakaryocyte progenitors
	CFU-G	Colony Forming Unit-Granulocyte	Granulocyte progenitors
	CFU-M	Colony Forming Unit-Monocyte/Macrophage	Monocyte/macrophage progenitors
	CFU-B	Colony Forming Unit-Lymphocyte B	Lymphocyte B progenitors
	CFU-GM Colony Forming Unit-granulocyte/macrophage	GMP progenitors
	CFU-E/Mk Colony Forming Unit-Erythroid/Megakaryocyte	MEP progenitors

Figure 24: Mapping of the two endothelial lineages A-3-day chick host, 2 days after quail splanchnopleural mesoderm grafting. QH1+EC are integrated in the floor of the aorta B-3-day chick host, 2 days after the graft of quail rostral segmental plate. QH1+EC have colonized the ipsilateral body wall of the chick host; they participate in the vascularization of the neural tube, somitic derivatives, wing bud , mesonephros (arrow) and roof of the aorta C-The aorta appears to have a mosaic origin, roof and sides from the somite, floor from splanchnopleural mesoderm.

	ii. The aortic endothelium remodeling by cells of
	somitic origin
	Experimental approaches by Pardanaud and collaborators indicate a double
	Abbreviations: CV, cardinal vein; G, gut; L, limb bud, NT: Neural Tube, W: Wing bud, S: Somite, So:
	somatopleural mesoderm, Sp: splanchnopleural mesoderm.
	Illustartions from Pardonaud et al 1996

Table 2 .

 2 ).POFUT2 seems to have 3 isoforms produced by alternative splicing and it is widely expressed in the embryo. In normal development, isoform A is expressed in fetal liver and peripheral blood lymphocytes. Isoform B is expressed in spleen, lung, testis, bone marrow, thymus, pancreas, prostate, fetal brain, fetal liver, and fetal kidney. Isoform C is expressed in brain, heart, spleen, liver, lung, stomach, testis, placenta, skin, thymus, pancreas, mammary gland, prostate, fetal brain, fetal liver and fetal heart[START_REF] Menzel | The Caenorhabditis elegans ortholog of C21orf80, a potential new protein Ofucosyltransferase, is required for normal development[END_REF]. Using SMART database, Du et al looked for the TSR proteins containing the POFUT2 consensus recognition sequence, they identified 51 putative targets in mice. 10 of these targets are transmembrane proteins and 41 are secreted proteins

Table M1 : Sequences of the siRNA POFUT2, TESTIN and Scramble

 M1

	siRNA	Sequence
	Scr sense	GGUUUCGUCUAUAGAUUGUTT
	Scr antisense	ACAAUCUAUAGACGAAACCTT
	POFUT2 si1 sense	GCUUCACUUAUGAAGACUUTT
	POFUT2 si1 antisense	AAGUCUUCAUAAGUGAAGCTT
	POFUT2 si2 sense	GCAGAUUUAUGUCCUGCAATT
	POFUT2 si2 antisense	UUGCAGGACAUAAAUCUGCTT
	POFUT2 si3 sense	AAAUACCAUACUACGACGGTT
	POFUT2 si3 antisense	CCGUCGUAGUAUGGUAUUUTT
	TESTIN si1 sense	GGAUAAGUGUGAAGGAUUUTT
	TESTIN si1 antisense	AAAUCCUUCACACUUAUCCTT
	TESTIN si2 sense	CCAGAAGGCAUCUCAGUAUTT
	TESTIN si2 antisense	AUACUGAGAUGCCUUCUGGTT
	TESTIN si3 sense	GCAUGCUGCGAUUUGCCAATT
	TESTIN si3 antisense	UUGGCAAAUCGCAGCAUGCTT
	6. Staining of in vitro culture, FACS analysis and cell
	sorting
	Endothelial cells were incubated with Acetylated LDL (10μL/mL) (acetylated low-
	density lipoprotein from human plasma coupled to Alexa Fluor 488-AcLDL-A488; 1
	mg/ml; (Life Technologies) diluted in culture medium for two hours at 37°C, 5% CO2,
	the dishes were rinsed in PBS/SVF (PBS 1X, 10% SVF) solution. Cells were

5. Cell preparation and isolation for RNA extraction

Non-adherent cells were collected by thoroughly pipetting and rinsing the culture dishes. Adherent cells were first trypsinized, washed in PBS/FCS (10%), centrifuged and collected. All RNA extractions were performed using the RNeasy™ kit from Qiagen SAS France. Freshly isolated PSM

[START_REF] Jones | Divergent selection for social reinstatement behaviour in Japanese quail: Effects on sociality and social discrimination[END_REF]

were re-suspended into the RNeasy™ buffer solution (RLT). For RNA extraction on cultured cells, the supernatant cells are first centrifuged to remove the medium and re-suspended into the RNeasy™ buffer solution. Adherent cells after trypsination are re-suspended into the RNeasy™ buffer solution. Quality/quantity of the extracted RNA was evaluated using a Nanodrop. dissociated using 0,25% Trypsin-EDTA 1X (Gibco Life Technologies) for 1min. After 2 washes with PBS/SVF to inhibit the trypsin, the supernatant was removed, the bottom was resuspended in remaining solution and the obtained samples were plated in round bottom, 96 wells cytometry plate. Plates were centrifuged, and a DAPI solution added (200μL of DAPI/BSA solution at 0,2% DAPI). Flow cytometry was performed with MACSQuant VYB, and data were analysed with Flowjo. Cell sorting was performed with BD FACSAria Cell sorter.

  : experimental design. PSM cells were put in culture at D0, transfected at D2 and analyzed at D4 for LDL uptake by flow cytometry. B: Effect of Lipofectamine (Lipo), scramble (Scr) and POFUT2 siRNA transduction on the expression of endogenous POFUT2. At 60nM, POFUT2 mRNA expression was decreased by more than 90% compared with the liposome and scramble siRNA controls.

	C, E, G: culture at D4 that received Lipofectamine (C,C'), scramble si (E,E') and POFUT2 si (G,G') at D2.
	Note that no changes in the number of round cells in the siPOFUT2 condition compared to the two
	other ones. D, F, H: representative flow cytometry experiment showing the balance between LDL int
	and LDLhi cells when cells received Lipofectamine (D), scramble si (F) and POFUT2 si (H). I,J:
	percentage of LDLhi and LDLint cells in the three conditions. Note that no significant changes in the %
	of LDL hi nor the LDL int fraction with POFUT2 compared to Scr and Lipo. K: RPKM value of the expression
	of POFUT2 in our transcriptome.

Table S1 : upregulated DEGs in HC and EC

 S1

		HC UP			EC UP
	ABCB10	LOC107309112	PPM1H	A4GNT	HEG1	METTL21A	TSPAN12
	ABCB7	LOC107309121	PPOX	AASS	HEXA	MFGE8	TSPAN13
	ACOT13	LOC107309332	PRAM1	ABCA13	HGSNAT	MFSD1	TSPAN6
	ACSS2	LOC107309555	PRKCB	ABCD4	HIPK2	MFSD10	TSPAN9
	ADA	LOC107309690	PSTPIP1	ABLIM1	HIPK3	MFSD4B	TTC38
	ADAP2	LOC107309869	PTAFR	ABLIM2	HIVEP1	MFSD6	TTC9
	ADIPOR2	LOC107310095	PTPN6	ACAP2	HMGCLL1	MGAT5	TTLL2
	ADRBK2	LOC107310166	PTPRC	ACBD5	HRASLS	MICAL3	TUSC3
	AFTPH	LOC107310573	RAB11FIP5	ACER3	HS1BP3	MID2	TXNDC16
	AGL	LOC107311215	RAB27A	ADAM17	HS3ST1	MKL2	TXNDC5
	AGTPBP1	LOC107311272	RAB32	ADAMTS7	HTATIP2	MMRN1	UACA
	AK2	LOC107311500	RAB44	ADCK2	HYI	MMRN2	UBE2H
	ALDOB

publications[START_REF] Beaumont | A genome scan with AFLP markers to detect fearfulness-related QTLs in Japanese quail[END_REF][START_REF] Recoquillay | Evidence of phenotypic and genetic relationships between sociality, emotional reactivity and production traits in Japanese quail[END_REF] . c, d, Immediate adjacent section of b. MEP21 and GFP immunohistochemistry and

analysis of the mononucleated BM cells showing the absence of YFP expression in the Sca1 + c-KIT + population (i.e. HSPC population) and in the different HSC, HPC and MPP populations.

Predicted transcripts per species aligned to Aves known RefSeq transcripts (n=8,776); turkey aligned to same species Genebank (n=380).

Predicted proteins per species aligned to Aves known RefSeq (n=7,733).

Acknowledgements:

The authors apologize to those investigators whose important work we were unable to cite or describe due to space constraints. We thank D. Traver for critical reading of the manuscript, and S. Gournet for help in image preparation. We are indebted to R. Adams for his sharing of the Tg(Cdh5Cre/ERT2) transgenic mouse line and to S. Germain for providing us with the founders. We thank the Cell Imaging and Flow Cytometry facility of the IBPS (Paris, France) for access and technical support in confocal image acquisition. This work was supported by CNRS, UPMC, Fondation Les Treilles (LY), EMBO short term fellowship (LY) and Grants from FRM (DEQ20100318258) and ANR/CIRM joint grant (ANR/CIRM 0001-02) in Jaffredo lab. The production of the GFP + chicken embryos was supported by grants from the BBSRC and the Wellcome Trust. Part of the work and LY were supported by an European Research Council grant (ERC, project number 220 -H75001EU/HSCOrigin-309361) and a TOP-subsidy from NWO/ZonMw (912.15.017) in Robin lab.

The authors declare no financial competing interests.

Correspondence and request for materials should be addressed to thierry.jaffredo@upmc.fr.

Acknowledgements

The authors would like to thank the Edinburgh Genomics sequencing facility (Edinburgh, UK) for carrying out the transcriptomic sequencing, RNA sequencing of the reference individual was performed on the GeT-Plage platform (http://get.genotoul.fr/en/) and funded by the INRA Genetics Division (QuailAnnot program). HSR and LSR lines sequencing was supported by the French Agence Nationale de la Recherche (SNP-BB project, ANR-009-GENM-008). We are grateful to the genotoul bioinformatics platform Toulouse Midi-Pyrenees (Bioinfo Genotoul) for providing help, computing and storage for these resources and to the ITAVI SeqVol program for financing gallo-anseriformes genome sequencing. KMM was supported by a National Health and Medical Research Council Overseas Postdoctoral Fellowship. This work was funded in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under contract numbers HHSN266200700005C and HHSN272201400006C, by ALSAC and by BB/N015347/1 and an HFSP 2015 award (RGP0030/2015).

Data availability

YFP + and YFP -cells sequence data used in this study have been deposited in the NCBI Gene Expression Omnibus database (accession number GSE122857). Source data for c, 8a, 9b, c and 11 are provided in Supplementary Table 4. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Availability of supporting data

Data has been submitted to the public databses under the following accession numbers: GCA_001577835.1 [genome sequence data]; SRA SRR2968870 -SRR2968911 [transcription annotation data]; Array Express accession numbers E-MTAB-3311(quail), E-MTAB-2909 (duck), E-MTAB-2908 (chicken) [RNAseq data for infection studies]; SRA accession number SRP047364 [HSR/LSR sequencing]; SRA accession number PRJNA490454 [RNA-seq data for photoperiod study].

CHAPTER 2 : MATERIALS & METHODES B-Green Mesoderm network C-Blue EC network

Single-cell profiling identifies several welldefined clusters

In order to reveal specific signatures of cells undergoing EHT and to decipher the changes that occur between endothelial and hemogenic endothelial fates, we performed single-cell RNA-seq (scRNAseq) using 8326 cells from 4 independent samples (1 D0, 2 D4, 1 D6). Single-cell t-Distributed Stochastic Neighbor Embedding (tSNE) analysis revealed 12 well defined clusters (Figure R17). An in vitro model of hemogenic endothelium commitment and hematopoietic production In this article, I performed and analysed the experiments in order to answer to the reviewers demand. I did the PSM dissection, the flow cytometry experiments as well as the analysis on different culture conditions. Article 2 In vivo haematopoietic cell production by bone marrowderived hemogenic endothelium Accepted manuscript in Nature Cell Biology.

In this article I analyzed the YFP + vs YFP -transcriptome and suggested the use of our culture conditions to validate the endothelial and hematopoietic populations in this study.

Data Fig. 6a,b). Moreover, the MEP21 + CD45 + population, known to be enriched in multipotent progenitors 17 and representing 2.4% of the mononucleated cells, harboured 15.2% of GFP + cells (Fig. 1n). Of note, no obvious difference was found in the haematopoietic composition of the BM between grafted (Extended Data Fig. 6a,b) and WT embryos (Extended Data Fig. 6c,d).

Secondary haematopoietic organs were colonized by GFP + cells. For example, GFP + cells were visible in the thymic lobules of E16/E17 grafted recipients. GFP + cells were CD4 + (1.4%), indicative of T cell differentiation (Extended Data Fig. 7a-e). Contrary to BM, the vascular network of the thymus was not made by GFP + cells (Supplementary Movie 5). A rather small, but clear GFP + population was also present in the spleen (Extended Data Fig. 7f-h) and the bursa of Fabricius (Fig. 7i-k) therefore supporting the multilineage contribution by the BM endothelium-derived haematopoietic cell population.

GFP chimeras were also examined at different time points post-hatching (up to 41 weeks, Fig. 2a,b and Supplementary Table 1) and analysed for the presence of GFP + cells associated with endothelial and haematopoietic lineages in the BM (n=7). The continued presence of GFP + cells in the BM (Fig. 2c) and a rather similar distribution of GFP among the endothelial and haematopoietic fractions (Fig. 2d,e and Extended Data Fig. 8a-d) compared to E16/E17 embryos (Fig. 1l, m and Extended Data Fig. 6) was found in the growing chicken. The MEP21 + CD45 + population harboured 6.1% of GFP + cells (Fig. 2f). FACS sorting followed by cytospin and May-Grünwald Giemsa staining of the GFP + MEP21 -CD45 - cell fraction disclosed an erythroblast/erythrocyte population (Fig. 2g) whereas the GFP + MEP21 -CD45 + fraction displayed eosinophils, heterophils, monocytes and thrombocytes (Fig. 2h).

To assess whether the process of haematopoietic production by somite-derived BM haemogenic ECs disclosed in Birds were conserved in Mammals, two mouse models were used. The contribution of the somite to the perinatal BM was traced by using Pax3 CRE/+ mice crossed to ROSA mT/mG mice. We first assessed that no Pax3 + cells were present in the embryonic aorta at E10.5, when the first HSPCs emerge (Extended Data Fig. 9a). Similar to the chicken, endothelial and haematopoietic cells associated with GFP expression were found in E19 to E21 foetal BM. The presence of mature cells from different haematopoietic lineages (CD45 + , CD3 + , B220 + , Gr1 + , CD11b + , Ter119 + ), HSPCs (Sca1 + c-Kit + , CD144 + CD45 + ) and endothelial cells (CD144 + CD45 -) were analysed in the perinatal mouse BM (Extended Data Fig. 9b). Moreover, the distribution of GFP + cells in each population was measured (Extended Data Fig. 9c) (n=3). GFP + cells contributed to all lineages. However, the highest contribution was found in the CD3 + T cell population, the CD144 + CD45 + population which is similar to the first HSPCs emerging from the aorta 22 , and the Sca1 + c-Kit + population (Extended Data Fig. 9c-f). These results were significant compared to control samples (Extended Data Fig. 9g). BM mononucleated cells contained an average of 0.30±0.02% GFP + cells (Extended Data Fig. 9d) in line with the numbers found in birds (Fig. 2c and1k), with 4.2±1.2% of CD144 + CD45 + cells expressing GFP (Extended Data Fig. 9c ande). The rare Sca1 + c-Kit + population (known to harbour HSPCs) displayed a conspicuous fraction of GFP + cells (10.0±0.3%) (Extended Data Fig. 9f), indicating the production of HSPCs by somite -derived BM endothelium. marrow transcriptomes Dr. M. Souyri, unpublished; available upon request after publication). The AGM samples consisted in cells sorted according to the expression of CD144 (CDH5), CD34, CD45 (PTPRC) and c-Kit (CD117), enabling to discriminate immature HCs, broadly equivalent to HSC (CD144 med CD45 + or CD34 + c-Kit + ) from mature HCs (CD144 med CD45 + or CD34 med c-Kit med ) and endothelial cells (CD144 + CD45 -). The bone marrow samples consisted in cells sorted according to the expression of CD150 (SLAMF1), c-Kit, Sca-1 and markers of the different haematopoietic lineages (Lin), enabling to discriminate immature HCs, broadly equivalent to HSC (CD150 + c-Kit + Sca-1 + Lin -) from mature HCs (Lin + ) (Available upon request. A new matrix was therefore generated corresponding to 28 samples and including 18,920 genes. After removal of the batch effect (3 different experiments were merged), PCA was carried out. ANOVA was then performed contrasting YFP + vs. YFP -cells with p < 0.001, resulting in 2,479 differentially expressed genes (DEGs), 1,123 up-regulated and 1,356 down-regulated. Since on the PC1 vs. PC2 score plot of the PCA YFP + cells were clearly discriminated from YFP -cells, we resorted to Weighted Gene Correlation Network Analysis (WGCNA) 27 to unravel the different gene networks. The parameters were as follows: power: 18, adjacency: signed, correlation: Pearson's, linkage: average, minimum module size: 30. The chosen weight threshold for network representation using Netscape was 0.1.

Reporting Summary

Further information on experimental design is available in the Reporting Summary.

Supplementary Information is linked to the online version of the paper at www.nature.com/nature Author Contributions L.Y and T.J. conceived the project. L.Y. and T.J. designed experiments on chicken embryos, performed chicken somite grafts and interpreted data. L.Y. performed chicken grafts, made immunohistological analysis, performed FACS analysis. R.G. performed chicken graft analysis.

Author information

Reprints and permissions information is available at www.nature.com/reprints

Extended Data Figures

Extended Data Figure The YFP population was more prominent in B220, CD19, CD71, Gr1 and CD8 fractions. * not available anymore Extended Data Table 2. List of anti-chicken and anti-mouse antibodies.

List of anti-mouse (first) and anti-chicken (second) antibodies used in the study, including the conjugated fluorochrome when applicable, clone of origin, cell specificity, supplier and catalogue number. Movie S5. Whole-mount staining of an E16 chicken thymus after GFP + PSM graft.

Extended Data

The thymus was stained with anti-MEP21 (endothelial marker, red), anti-CD45 (haematopoietic marker, blue) and anti-GFP (green, which revealed grafted PSM-derived cells) antibodies. The thymus was colonized by GFP + PSM-derived cells, some of the lobes being more colonized than others. GFP + cells could colonize secondary haematopoietic organs that are located far from the grafting site. Extended data movie 5 is visible at https://figshare.com/s/d8e15dcd3ab9e509ce13. 88, 180-191 (1981).

Article 3

The quail as an avian model system: its genome provides insights into social behaviour, seasonal biology and infectious disease response

Article in revision in BMCB and available on biorxiv https://www.biorxiv.org/content/10.1101/575332v1

In this article, I contributed to the manual annotation of around 8000 unannotated genes of the quail genome.

You can find the LOC assignment list I annotated in the appendix 4.

The quail as an avian model system: its genome provides insights into social behaviour, seasonal biology and infectious disease response

ABSTRACT

The Japanese quail (Coturnix japonica) is a popular domestic poultry species and an increasingly significant model species in avian developmental, behavioural and disease research. We have produced a high-quality quail genome sequence, spanning 0.93 Gb assigned to 33 chromosomes. In terms of contiguity, assembly statistics, gene content and chromosomal organization, the quail genome shows high similarity to the chicken genome. We demonstrate the utility of this genome through three diverse applications. First, we identify selection signatures and candidate genes associated with social behaviour in the quail genome, an important agricultural and domestication trait. Second, we investigate the effects and interaction of photoperiod and temperature on the transcriptome of the quail medial basal hypothalamus, revealing mechanisms of photoperiodism. Finally, we investigate the response of quail to H5N1 influenza infection. In quail lung, many key immune genes and pathways were downregulated, and this may be key to the susceptibility of quail to H5N1. This genome will facilitate further research into diverse research questions using the quail as a model avian species.

Comparative mapping of the quail and chicken genomes revealed a high conservation of the chromosomal arrangement (Fig. 1; Supplementary Data 2), with no major rearrangements since the divergence of the two species approximately 23 MYA 33 . All identified quail chromosomes showed synteny conservation to their chicken chromosomal counterparts. By comparison, the turkey (Meleagris gallopavo) genome is more highly rearranged with two chromosomes having synteny conservation to each of chicken chromosomes 2 and 4 34 . No large intrachromosomal translocations were seen between chicken and quail chromosomes, compared to the two seen in the turkey 34 . Inversions and inter-chromosomal translocations were common, with 33 large (>1Mb) inversions or translocations occurring between chicken and quail chromosomes (Fig. 2; Supplementary Data 2). The quail chromosomes are more compact than their chicken counterparts (14% smaller on average).

Orthologous genes between quail and closely related species were identified through reciprocal BLAST searches. One-to-one orthologs in chicken were identified for 78.2% of all quail genes and 91.8% of protein-coding quail genes (Supplementary Table 1), indicating a high degree of genic conservation in the quail genome. Fewer orthologs were seen between turkey and quail genes (69.3%), although the number of orthologs of protein-coding genes was similar (91.7%), so the discrepancy is likely due to missing non-coding gene predictions in the turkey genome. As expected, conservation of one-to-one orthologs was lower with the mallard duck (Anas platyrhynchos), with duck orthologs identified for 64.5% of quail genes (78.9% proteincoding genes).

Endogenous retroviruses (ERVs)

ERVs represent retroviral integrations into the germline over millions of years and are the only Long Terminal Repeat (LTR) retrotransposons which remain in avian genomes 35,36 . Whilst the majority of ERVs have been degraded or epigenetically silenced, more recent integrations retain the ability to produce retroviral proteins, impacting the host immune response to novel exogneous infections 37,38 . A total of 19.4 Mb of the Coturnix japonica 2.0 assembly was identified as ERV sequence using the LocaTR pipeline 35 (Supplementary Data 3 and 4). ERVs therefore account for 2.1% of the quail genome sequence, levels similar to those in the chicken and turkey 32 (Supplementary Table 2), and similarly analysed passerine birds 35 .

The majority of ERV sequences in all three genomes were short and fragmented, but 393 intact ERVs were identified in the quail, most of which were identified as alpha-, beta-or gammaretroviral sequences by reverse transcriptase homology. It is possible that the smaller genome size of the quail compared to other birds reflects a more limited expansion of ERVs and other repeats (such as the LINE CR1 element; Supplementary Table 2) within the genome, following the basal avian lineage genome contraction 36,39 . However, ERV content is highly species-specific 35 .

Despite variation in total and intact ERV content, the overall genomic ERV distribution in these three gallinaceous birds was highly similar. ERV sequence density was strongly correlated with chromosome length on the macrochromosomes and Z (r > 0.97; P < 0.001), but there was no significant correlation across the other smaller chromosomes. Furthermore, ERV density on each Z chromosome was at least 50% greater than would be expected on an autosome of equal length. These results support the depletion of repetitive elements in gene dense areas of the genome, and the persistence of insertions in poorly recombining regions, as was seen in the chicken 35 . This is further supported by the presence of clusters of intact ERVs (where density was five times the genome-wide level) on the macrochromosomes and sex chromosomes (Supplementary Table 2).

Competing interests

The authors declare there are no competing interests However, few is known about how these TFs and signaling pathways orchestrate the EHT and few essential regulators have been isolated. A recent study pointed out on the role of the blood flow and shear stress to modulate EHT and the existence of pulsatile cycles involving members of the cytoskeleton to trigger EHT (Lancino et al., 2018).

Recently we developed an ex vivo culture system that allows stepwise commitment of the mesoderm into hemogenic endothelial cells that undergo EHT to give rise to multilineage hematopoiesis [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. Here we have utilized this system to purify cell types belonging to key steps on the road to hematopoietic production and to isolate specific mRNA signatures using transcriptome analysis. Through comprehensive meta-analysis and isolation of specific gene expression signatures, 798 genes arranged in three networks representing the hemogenic endothelium state. These networks do not contain the standard hemogenic endothelium regulators since present in upstream or downstream cell types but reveal unique gene sets with previously unrecognized regulators of hemogenic endothelium. Two gene sets are positively regulated in hemogenic endothelium and one is negatively regulated. Within gene sets, genes are interconnected and contain members that are highly interconnected defining hubs as well as several genes belonging to specific signaling pathways. Experimental knock-down of one signaling pathway and two positively regulated hubs strongly modifies the culture and modulates EHT giving networks a strong predictive value. In addition to hubs, genes connected to the hubs also appeared deregulated following hub knock-down strengthening the existence and the robustness of the networks. These gene sets uncovered new, unique regulators of the hemogenic endothelium state and encompasses genes with strong predictive value.

Sample isolation strategy

Based on this result, we isolated the different cell fractions to perform NGS (Figure S1A). We used the level of LDL uptake to isolate ECs (LDL high or + ) from hemogenic ECs (LDL int ). Presomitic mesoderm was directly isolated from the embryo whereas hematopoietic cells were collected as the floating fraction, in keeping with [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF].

Bioinformatic analysis

We used high-throughput sequencing to compare the molecular signatures of the different cell fractions. We isolated 20 samples corresponding to the different cell fractions (Figure A2A; Figure S1A). mRNAs were isolated and the libraries were sequenced using next-generation sequencing Illumina technology. According to the quality control, we detected 20 332 genes. PCA on the entire set of mRNAs and samples (20 samples; 20 332 genes) indicated differences between the different cell fractions. PC1 (30.2% of the variance) and PC3 (9.85% of the variance) allowed to discriminate ECs from pre-somitic mesoderm (M) and hemogenic ECs (HEC) from hematopoietic cells (HC), respectively (Figure A2B). A first hierarchical clustering on the entire set of mRNAs and samples showed some discrepancies in the ordering with the M fraction found between the EHC and HC fractions (Figure S1C). To identify the genes that were specifically up-or down-regulated in the different cell populations we followed a two-step procedure. First, we compared pairwise using ANOVA (with p-values <0.06 and fold changes ≥1.45 or ≤-1.45) the M, EC, HC and HEC cell populations. Second, we considered using Venn diagrams the gene sets that were up-regulated (or downregulated) in a given population according to the 4 comparisons (Figure A2C; Figure S1D). This procedure led to the identification of 2462 up-regulated and 2087 downregulated genes (upregulated DEGs in the 4 populations Table S1; Table S2). We then performed a novel PCA that provided a clearcut discrimination between the 4 populations, according to PC1 (43% of the variance) corresponding to the contrast between M and EC and to PC3 (17% of the variance) corresponding to that contrast between HC and HEC (Figure A2D). And this time hierarchical clustering showed a perfect ordering going from M through EC and HEC fractions until HC population (Figure S1E). Analysis of the GO categories clearly confirmed the phenotypic differences between the 4 distinct populations. The M cell population was enriched in genes implicated in spliceosome, Hippo and hedgehog signaling pathways and shared the category adherent junction with the EC population. The EC population showed POFUT2 and, at least, 3 of their first neighbors are acting coordinately to regulate the EHC fate and the hematopoietic production.

Material and Methods

Presomitic mesoderm isolation

We used quail (Coturnix coturnix japonica) embryo pre-somitic mesoderm according to [START_REF] Yvernogeau | An in vitro model of hemogenic endothelium commitment and hematopoietic production[END_REF]. Eggs were incubated for 36-45 hours at 37±1°C in a humidified atmosphere to reach 10-18 somite pairs. Microsurgery was performed as previously described [START_REF] Pardanaud | Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis[END_REF]Pardanaud, #2110]]. The PSM was removed over a length corresponding to 10 somites from both sides of the embryo. Five embryos (i.e. 10 PSM)

were used per culture dish. Each PSM was cut into 4 to 5 equal pieces, rinsed in Opti-MEM plus GlutaMAX™ I (Gibco Life Technologies) +5% Fetal Calf Serum (FCS, Gibco Life Technologies) +100 Units per ml penicillin/streptomycin (Gibco Life Technologies) + 1% Chicken Serum (Gibco Life Technologies) before culture.

Medium, culture dishes PSM were cultured under the following conditions Opti-MEM® with GlutaMAX™ I supplemented with 5% FCS, 1% Chicken Serum, 100 Units per ml penicillin/streptomycin and the following growth factors (PromoCell/PromoKine): hVEGF (≠C64410); 2ng/ml), hFGF (≠C60240) ; 4ng/ml), hIGF (≠C60840) ; 3ng/ml), hEGF (≠C60170) 10ng/ml), hydrocortisone (200ng/ml SIGMA ≠H6909), ascorbic acid (75ug/ml SIGMA ≠A4544).

PSM were cultured in 35mm collagen I-or IV -coated dishes from Corning BioCoat (Corning -Dutscher France). Medium was changed every two days unless specified.

RNA extraction, qRT-PCR, primers

All RNA extractions were performed using the RNeasy™ kit from Qiagen SAS France. Freshly isolated PSM [START_REF] Jones | Divergent selection for social reinstatement behaviour in Japanese quail: Effects on sociality and social discrimination[END_REF] were re-suspended into the RNeasy™ buffer solution (RLT). For RNA extraction on cultured cells, the supernatant cells were first centrifuged to remove the medium and re-suspended into the RNeasy™ buffer solution. Adherent cells were first trypsinized, washed in PBS/FCS (10%), centrifuged and then resuspended into the RNeasy™ buffer solution. Quality/quantity of the extracted RNA was evaluated using a Nanodrop.

Staining of in vitro culture

We used Low Density Lipoprotein from Human Plasma, Acetylated, coupled to Alexa-488 (AcLDL-A488 1mg/ml Life Technologies, Ref. L23380, batches N° 1291485