PhD Pauline Spéder
email: pauline.speder@pasteur.fr

V-SVZ Ventricular-Subventricular Zone SGZ Subgranular zone

Keywords: Neural stem cell, glia, niche interactions, immunoglobulin superfamily of cell adhesion molecules, proliferation, actomyosin contractility, Drosophila melanogaster

ACKNOWLEDGMENTS

Many people helped me during these research years, and I want to express my sincere appreciation to all of them, and each person reading this thesis, I hope you enjoy it! First I would like to thank the PCRE lab. Thank you to my PhD supervisor, Dr. Pauline Spéder, for the opportunity of joining the lab and carrying on this PhD project. Dear Pauline, thank you for sharing all your knowledge, your positivism and so much of your time with me. I learned a lot during these years in the lab and I am truly grateful for all the guidance and help you gave me throughout my research as well as during the writing of this thesis. Thank you, Agata, Billel, David, Marco, and Maria for teaching me all the secrets of Drosophila, for continuously answering my (many times naive) questions, for all the favors I asked, and for your precious advice. Thank you, Sabine and Vesna, for your kindness constantly proposing your help somehow.

A big thanks to the members of my thesis committee for agreeing to take part in the jury of my PhD defense. Thank you to the reviewers of this thesis, Dr. Ruth Beckervordersandforth and Dr. Roland Le Borgne, for dedicating some of your time to review my project and for your valuable feedback. Thank you also to the president of the jury and other examiners, Dr. Fiona Francis, Dr. Isabel Fariñas, and Dr. Véronique Marthiens, for taking the time to read this thesis and allowing me to share and discuss my work with you.

I want to thank my thesis advisor committee, Dr. Laure Bally-Cuif, Dr. Yohanns Bellaïce and Dr. Antoine Bovin, for your enlightening recommendations pertaining to this study. It was a pleasure for me to share the progress of my work with you, and I am thankful that this led to a collaboration. Thank you, Yohanns, for welcoming me to your lab to learn the CRISPR technique and to Amina for teaching it. I enjoyed the cloning a lot and it even worked! Multiple times the imaging hub at Pasteur helped me to solve many issues analyzing my data and I want to thank its members for that, especially, Stéphane, for all the hours dedicated to troubleshooting with the neural stem cell segmentation of my dissected brains. I am extremely thankful to my Spanish team at Pasteur for being there from the beginning with invaluable support and great moments around food and drinks. Feeling home in your workplace when you live abroad, away from your family, is the best I could ever ask for. Infinitas gracias, Cristina, Eva, Lorea, Maria, Rebe, Alicia, Alejandro y Rober, por todo el tiempo, el afecto y los consejos que habéis compartido conmigo.

Our little PhDs team from the very first days at Pasteur, Anna, Erminia, Ferdinand, Melania and Serena, I thank you for all the cheerful moments we shared, and for your hundreds of wonderful words of advice. It has been a pleasure to meet you and go through this PhD challenge together.

My life at the Institut Pasteur would have not been the same without many other pasteurians with whom I shared countless moments along this adventure. I want to thank you for your scientific feedback, but also for the endless conversations around a beer or tea, or even the short ones, just around the corner of the lab. Thank you to those that have shared their contagious smiles throughout the campus, as well as to the ones that have carried on with me some of the associative experiences. With you, I managed to couple a PhD project with the organization of a StaPa retreat and a BIOTechno forum, the participation to an iGEM competition, and the creation of an innovation team at Pasteur (merci Marc pour cela et toutes les heures partagées à me conseiller sur mon avenir, c'était un plaisir de croiser ton chemin à Pasteur). All these activities have taught me a lot and have contributed to making this journey worth it. I am also thankful to the best flatmates I could have ever wished to have. Biel and Cristina, thank you for accepting my non-stop life and always being there when I needed someone to talk to. Gracias, Cristina, por compartir tanto conmigo a lo largo de esta tesis, he aprendido muchísimo a tu lado y me quedo con tu buen humor contagioso, tus aventuras divertidas y todo tu cariño. No podría imaginarme este PhD sin ti, te quiero.

It is another must to thank my human towers team, always there to make my time outside the lab a total blast. Gràcies Castellers de Paris per acollir-me des de la meva primera setmana a París, per tota la confiança i per haver-me permès viure tantes emocions a flor de pell amb els castells compartits. M'enduc records molt bonics d'aquesta experiència, entre riures, mirades de complicitat, abraçades de celebració, i el desig de divendres setmana rere setmana. Gràcies a les meves mosquis estimades per alegrar-me els dies de laboratori que acabaven amb reunions adorables en els restaurants més cuquis de París. Gràcies al meu pom de dalt imaginari preferit, Anna i Jesús, per compartir tant d'amor amb mi, em sento molt afortunada de tenir-vos en la meva vida. Thank you also to my climbing (and lately brunching) partners, responsible of my fascination for this sport and my meticulously organized week schedule to try to fit some climbing time on it. Merci pour votre encouragement continu tout au long de cette thèse.

Before my PhD I had the possibility to learn from many other scientists to which I owe many things I know and the motivation I had to pursue a PhD. Thank you to the members of my previous internships for showing me everything, from how to use a pipette to what is a plasmid and how cool are place cells. I keep very precious moments of these experiences and I am truly thankful to many of you. Claire, JK, Michaël, Paul, and Valentina, thank you for accepting me in the team when I had very little or no experience at all in the model of the lab and sometimes the field. You gave me the opportunity to learn from very different models and this curious person that I am, enjoyed it very much. Céline, je ne pourrais jamais exprimer à quelle point je suis reconnaissante de tout le bonheur et l'aide que tu m'as donné depuis le premier jour, you and Rali could not have welcomed me in a better way in the lab, I truly felt like home and coming to work was a total pleasure. Thank you for all the moments we shared and for making my first long experience working abroad a remarkable one. Thank you Jeff and Dimitrii for all your patience, the project was challenging but I can only remember how your everyday good mood helped me throughout the internship and taught me so much of the determination I have today to achieve my goals. Thank you Fabio for your kindness and all the precious advice. A huge thanks to many of my teachers during my studies that shared their passion for biology and undoubtedly contributed to my will to pursue a professional career in biomedical science.

Furthermore, I would like to acknowledge my friends from my master's degree for their support and for the multiple times we shared ups and downs during both master and PhD years. It was very nice to have you there.

I also want to thank my three lovely friends, Anna, Eva and Rut, from my bachelor's degree back in Barcelona. Tot i la distància, sempre he pogut comptar amb vosaltres i no sabeu quant us ho agraeixo, gràcies per fer la vida més fàcil i divertida. Us estimo i us trobo a faltar.

In addition, I am grateful to have grown next to many sweethearts in or near my little sunny town of Cambrils. M'agradaria donar-vos les gràcies a molts ja que no estic segura que sigueu conscients de fins a quin punt poder veure-us i saber de vosaltres de tant en tant m'ha ajudat a carregar piles i seguir endavant. I believe it is almost impossible to achieve anything alone in live. Here I want to thank my amazing unconventional family without which I would not have been able to complete this work, from my four inspiring parents, to my five awesome sisters and brothers, my marvelous grandparents here and up there, my kind aunties, cousins and uncles, my cool sisters-in-law, and my beloved life partner. Gràcies família per creure en mi i haver-me deixat escollir el camí que volia tot i els meus dubtes infinits. És gràcies al vostre suport incondicional que avui sóc aquí. Moltíssimes gràcies per l'amor amb el que m'heu educat i que continueu compartint dia a dia. Gràcies per créixer al meu costat, ensenyar-me a gaudir de la vida i viure en el present, on tot i la distància, hi heu sigut en cada moment. Gràcies per trobar sempre les paraules per calmar les meves inquietuds al llarg de la tesis, teníeu raó totes i cadascuna de les vegades. Us prometo que he gaudit de l'aventura fins al final. Us estimo infinit a tots i cadascun de vosaltres.

Et finalement, mon p'tit pote d'amour, quoi te dire à toi ? Tu as vécu le quotidien de ces années de thèse avec moi, tu as écouté plein de fois mes peurs et tu as toujours su comment gérer la situation quand je ne pouvais pas le faire. On a beaucoup privilégié cette thèse même si ça n'a pas toujours été évident, j'ai énormément apprécié et je te remercie encore. Merci pour avoir facilité tellement les choses dans les moments compliqués, pour m'avoir encouragé autant de fois sans vraiment comprendre ce monde de la science et pour tout l'amour que tu me partages. Je t'aime Lucas.

Last, I would like to take some courage here and thank myself for all the work done, for having tried my best to combine learning, experiments, and personal life along these years of research.

I learned a lot about multiple scientific fields, conducting experiments, divulgating science, overcoming conflicts or failures, collaborating with others, favorite hypotheses and perseverance, innovation, living abroad, meeting new people, and living in the present as much as possible. Still, there is a long path to walk ahead to improve some things. However, I am happy to say that writing this thesis was an unforgettable experience that magically allowed me to learn how to listen a bit more to myself and I am grateful for that.

Importance of stem cells

The development from a single cell to a multicellular adult organism relies on the proliferative potential of stem cells. These cells were defined as undifferentiated cells capable of proliferation, self-maintenance, production of differentiated functional progeny, and tissue regeneration after injury [START_REF] Potten | Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt[END_REF]. The ability to self-renew and generate copies of itself is an essential property of stem cells to perpetuate themselves while maintaining the stem cell pool or expanding it. The ability to give rise to progeny with a more restricted potential is the other crucial property.

The remarkable capacity that stem cells have to self-renew while producing different cell types makes them unique. To accomplish that, they undergo a balance of symmetric and asymmetric divisions to generate two daughter cells that will acquire identical or different developmental fates, respectively [START_REF] Lu | Control of cell divisions in the nervous system: Symmetry and asymmetry[END_REF]. Stem cells symmetric divisions generate either a)

two copies of the same mother cell to increase the stem cell population; or b) two daughter cells identical to each other but more committed to differentiation than the mother cell. Asymmetric divisions generate either a) one copy of the mother cell to maintain the stem cell pool, and a daughter cell with a more restricted potential of differentiation; or b) two daughter cells both destined to differentiation but along distinct paths. As development proceeds, cells committed to differentiate will progressively lose the capability to proliferate and differentiate into tissuespecialized cell types. The choice between symmetric and asymmetric divisions must be tightly coordinated to ensure the generation of the appropriate number of cells in each tissue. restricted, it is committed to undergo terminal differentiation into a single cell type [START_REF] Fortier | Stem Cells: Classifications, Controversies, and Clinical Applications[END_REF][START_REF] Jaenisch | Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear Reprogramming[END_REF].

Stem cells can be

In adults, stem cells are involved in maintaining homeostasis and contributing to tissue repair after damage. Therefore, the proliferative potential of stem cells is crucial for tissue growth, development, homeostasis, and repair. Accordingly, stem cell division has to be tightly regulated to avoid any imbalance leading to either tissue undergrowth, affecting its ability to adapt and repair lesions; or overgrowth, potentially resulting in cancer-like development or exhaustion of the stem cell population [START_REF] Rumman | Concise Review: Quiescence in Adult Stem Cells: Biological Significance and Relevance to Tissue Regeneration[END_REF]. Understanding stem cell behavior is hence fundamental in the field of developmental biology.

Mammal neural stem cells

Neural stem cells (NSCs) are multipotent stem cells that produce both neuronal and glial lineages, the cellular components of the nervous system [START_REF] Mckay | Stem cells in the central nervous system[END_REF][START_REF] Alvarez-Buylla | Stem cells in the developing and adult nervous system[END_REF][START_REF] Gage | Mammalian neural stem cells[END_REF]. Proper brain function requires a sufficient production number of neurons and glia, their maintenance during the animal's lifetime, and a suitable interaction between these cells both temporally and spatially. Neurons process and communicate information making specific contacts with other cells, whereas glia orchestrate essential roles in the development, maintenance, and function of the nervous system [START_REF] Somjen | Nervenkitt: Notes on the history of the concept of neuroglia[END_REF][START_REF] Allen | Glia as architects of central nervous system formation and function[END_REF].

Origin and development

In the early development of the vertebrate embryo, the neural fate is induced in the ectoderm, from which NSCs are derived. They constitute the neural plate that first folds into the neural tube and then builds the central nervous system (CNS). These NSCs form the pseudostratified neuroepithelium that constitutes the wall of the developing neural tube and lines the ventricular system that ultimately shapes the cerebral ventricles. They undergo symmetric proliferative divisions to generate new copies of neuroepithelial cells and expand. Later on, neuroepithelial cells transition to radial glia cells, which now take the role of NSCs, and neurogenesis begins (Figure 1). These NSCs divide asymmetrically to self-renew and generate terminally defaced a long-held dogma that the adult central CNS was unable to generate new neurons, a concept previously challenged with radioactive thymidine [START_REF] Altman | Are new neurons formed in the brains of adult mammals?[END_REF]; radioactive thymidine and electron microscopy [START_REF] Kaplan | Neurogenesis in the adult rat: Electron microscopic analysis of light radioautographs[END_REF]; BrdU, neural markers and confocal microscopy [START_REF] Paton | Neurons generated in the adult brain are recruited into functional circuits[END_REF] experiments but not widely accepted in the field. Adult neurogenesis depends on NSCs that share similar properties with radial glia cells, for this reason, adult NSCs are considered radial glia-like cells. These cells appear mainly in a quiescent state and when activated, they produce new neurons that integrate into the preexisting neuronal network as well as astrocytes and oligodendrocytes. Adult neurogenesis is more tissue-restricted than embryonic neurogenesis and, in function of their location, NSCs may only generate one or two specific types of neurons. The adult mammalian brain harbors populations of NSCs in at least two neurogenic regions: the ventricular-subventricular zone (V-SVZ) of the lateral ventricles, and the subgranular zone (SGZ) of the hippocampus [START_REF] Song | Adult Neurogenesis in the Mammalian Brain: Significant Answers and Significant Questions[END_REF].

Drosophila neural stem cells

In the mammalian nervous system, the diversity of structures, functions, and cell types entangles the study of stem cells more than in simpler organisms like Drosophila melanogaster.

The powerful genetics, short life cycle, ease to grow and maintain, low ethics regulation, and high conservation of basic mechanisms makes the Drosophila melanogaster a formidable system for research purposes. Specifically, NSCs in Drosophila share many features with those in mammals, such as their ability to self-renew, their multipotency to produce several types of neurons and glia, their neuroectodermal origin, the quiescent state to preserve stemness, and their direct or indirect modes of division [START_REF] Doe | Neural stem cells: From fly to vertebrates[END_REF]Egger, Chell and Brand, 2008;[START_REF] Brand | Neural Stem Cell Biology in Vertebrates and Invertebrates: More Alike than Different?[END_REF]Homem and Knoblich, 2012).

The Drosophila CNS includes different types of NSCs also known in the field as neuroblasts.

At larval stages, there exist four types of NSCs: type I neuroblasts, type II neuroblasts, optic lobe neuroblasts, and mushroom body neuroblasts. These NSC subtypes are present in different regions of the developing CNS: the central brain, which contains mainly type I neuroblasts, 16

type II neuroblasts, and 8 mushroom body neuroblasts; the ventral nerve cord (VNC), which contains only type I neuroblasts; and the two optic lobes, which contain optic lobe neuroblasts (Sousa-Nunes, [START_REF] Sousa-Nunes | Regulating neural proliferation in the Drosophila CNS[END_REF]Homem and Knoblich, 2012).

the neuroectoderm are selected to acquire a neuroblast fate from a proneural equivalence group of five to six cells. This is achieved by the process of lateral inhibition and is based on a molecular regulatory loop between the adjacent cells. (Egger, Chell and Brand, 2008;Homem and Knoblich, 2012).

At early larval stages, the intake of dietary amino acids initiates a signaling cascade that results

in NSCs synchronously re-entering the cell cycle [START_REF] Britton | Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms[END_REF][START_REF] Chell | Nutrition-responsive glia control exit of neural stem cells from quiescence[END_REF][START_REF] Sousa-Nunes | Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila[END_REF]. Upon reactivation, NSCs resume proliferation in a characteristic spatiotemporal pattern generating 90% of the secondary neuronal lineages that form the adult CNS [START_REF] White | Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster[END_REF][START_REF] Truman | Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster[END_REF][START_REF] Prokop | The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster[END_REF][START_REF] Ito | Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster[END_REF]. This corresponds to the second wave of neurogenesis and gliogenesis (Figure 3A,D) (Homem and Knoblich, 2012;[START_REF] Kang | Control of neural stem cell self-renewal and differentiation in Drosophila[END_REF]. This larval stage is considered a post-embryonic, juvenile growth phase. Later during development, NSCs proliferation becomes largely independent of dietary amino acids (Cheng et al., 2011).

In the future optic lobe region of the embryonic brain, neuroepithelial cells first increase their population through symmetric division and remain mitotically dormant throughout the rest of embryogenesis [START_REF] White | Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster[END_REF][START_REF] Hofbauer | Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster[END_REF]. At mid-larval stages, a proneural wave across the neuroepithelium gradually transforms those cells into optic lobe neuroblasts. These NSCs undergo asymmetric divisions to self-renew and ultimately produce neurons and glia [START_REF] Hofbauer | Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster[END_REF][START_REF] Egger | Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe[END_REF][START_REF] Sousa-Nunes | Regulating neural proliferation in the Drosophila CNS[END_REF]. Recent studies have shifted the timeline of NSC activity in the optic lobe, showing that neuroepithelial cells divide throughout embryogenesis not to expand their pool but to already generate embryonic optic lobe neuroblasts [START_REF] Hakes | A newly discovered neural stem cell population is generated by the optic lobe neuroepithelium during embryogenesis in drosophila melanogaster[END_REF].

Mushroom body neuroblasts also delaminate from the neuroepithelium. Nevertheless, these NSCs continuously proliferate from late embryonic stages until the end of pupation. They divide asymmetrically to self-maintain themselves while generating a GMC, which will produce two identical neurons. A total of 2000 neurons belonging to three different sequentially produced neuronal types constitute the mushroom bodies [START_REF] Ito | Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster[END_REF][START_REF] Prokop | Normal function of the mushroom body defect gene of Drosophila is required for the regulation of the number and proliferation of neuroblasts[END_REF][START_REF] Lee | Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast[END_REF][START_REF] Kunz | Origin of Drosophila mushroom body neuroblasts and generation of divergent embryonic lineages[END_REF].

By pupal stages, where the animal sexual maturation occurs, NSC division ceases and most NSCs withdraw the cell cycle (Figure 3A) while few undergo apoptosis [START_REF] Ito | Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster[END_REF][START_REF] Maurange | Temporal Transcription Factors and Their Targets Schedule the End of Neural Proliferation in Drosophila[END_REF]. Nowadays, there is some ongoing debate about the existence of adult neurogenesis in Drosophila, although it is widely accepted that the adult dividing cells are not proper NSCs [START_REF] Li | Adult neurogenesis in the drosophila brain: The evidence and the void[END_REF].

The stem cell niche

The idea of the stem cell niche was first proposed as an association of a stem cell with its surrounding cells, which is required for stemness [START_REF] Schofield | The relationship between the spleen colony-forming cell and the haemopoietic stem cell. A hypothesis[END_REF]. Since then, the concept of stem cell niche has evolved to describe a dynamic cellular microenvironment that is tailored to the stem cell needs and ensures the maintenance of the stem cell fate. The stem cell niche varies between tissues and can regulate several features of the stem cell behavior, including maintenance, differentiation, proliferation, and quiescence [START_REF] Lander | What does the concept of the stem cell niche really mean today?[END_REF]. It comprises cellular structures, membrane bound molecules, extracellular matrix proteins, and soluble factors [START_REF] Alvarez-Buylla | For the long run: Maintaining germinal niches in the adult brain[END_REF].

Mammalian neural stem cell niches

Mammalian NSCs reside in specific microenvironments that support their behavior. The composition and structure of these niches evolve throughout life and behave differently depending on their location in the CNS. NSC niches have been mostly investigated during postnatal stages and in rodent models. The core cellular components of the adult mammalian NSC niche are the NSCs themselves, neurons, glial astrocytes, microglia, ependymal cells (in those niches lining the cerebral ventricles) and the blood-brain barrier cells [START_REF] Doetsch | Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain[END_REF][START_REF] Seri | Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus[END_REF].

The compactness in the tissue brings the different cell populations in close proximity and makes the niche an important signaling information hub for NSCs [START_REF] Bjornsson | It takes a village: Constructing the neurogenic niche[END_REF][START_REF] Bond | Adult Mammalian Neural Stem Cells and Neurogenesis: Five Decades Later[END_REF]. Morphogens, growth factors, neurotransmitters, and membrane-associated ligands are examples of diffusible signals that influence the behavior of NSCs. Most of these signaling molecules derive from the niche populations and act in a paracrine, non-cell autonomous, way [START_REF] Andreotti | Neural stem cell niche heterogeneity[END_REF]. Accordingly, the various cellular components surrounding NSCs coordinate the NSC integration of all these factors, acting like obstacles or transmitters of the signals they are exposed to. Their position also makes them likely mediators of the NSC response to external stimuli.

Niche components

Neural stem cells

NSCs, the key components of the niche whose roles have already been presented before, can also modulate the proliferation and differentiation of other NSCs through calcium signaling mechanisms. Both in the developing embryonic neocortex and in the postnatal SGZ and V-SVZ, NSCs have been shown to be connected to each other by gap junctions, allowing for communication between the cell's cytoplasm and extracellular space, thus potentially coordinating their actions [START_REF] Weissman | Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex[END_REF][START_REF] Kunze | Connexin expression by radial glia-like cells is required for neurogenesis in the adult dentate gyrus[END_REF][START_REF] Lacar | Gap junction-mediated calcium waves define communication networks among murine postnatal neural progenitor cells[END_REF].

Neurons

Neurons, a big part of the NSC progeny, are able to release neurotransmitters to signal to other neurons, and also other cells present in the microenvironment. Recent studies have shown that

NSCs are able to respond to the alteration of several neurotransmitters [START_REF] Berg | Neurotransmitter-mediated control of neurogenesis in the adult vertebrate brain[END_REF][START_REF] Tong | Axonal control of the adult neural stem cell niche[END_REF][START_REF] Paul | Hypothalamic regulation of regionally distinct adult neural stem cells and neurogenesis[END_REF][START_REF] Perez-Villalba | Synaptic regulator α-synuclein in dopaminergic fibers is essentially required for the maintenance of subependymal neural stem cells[END_REF], emphasizing that neurons can impact NSC behavior. The cerebral blood flow can similarly be regulated through neurotransmitter-mediated signaling [START_REF] Mishra | Binaural blood flow control by astrocytes: listening to synapses and the vasculature[END_REF].

Glia: astrocytes

Glial cells were traditionally regarded as supporting cells. However, emerging evidence has shown they do have crucial roles in the NSCs niches [START_REF] Ma | Glial influences on neural stem cell development: Cellular niches for adult neurogenesis[END_REF]. Astrocytes are star-shaped glial cells very abundant in the mammalian NSC niche. Through their many processes, they directly connect to the CNS vasculature, NSCs and neurons, even enwrapping their synapses [START_REF] Moss | Fine processes of Nestin-GFP-positive radial glia-like stem cells in the adult dentate gyrus ensheathe local synapses and vasculature[END_REF]. On one side, astrocytes express and secrete molecules through which they can influence NSC activity mediating the balance between quiescence and active states, and promoting their proliferation when needed [START_REF] Lim | Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis[END_REF]H. Song, Stevens and Gage, 2002;[START_REF] Ma | Glial influences on neural stem cell development: Cellular niches for adult neurogenesis[END_REF][START_REF] Morrens | Glial cells in adult neurogenesis[END_REF]. On the other side, they can also control neuronal activity participating in numerous steps: neuronal fate specification (H. Song, Stevens and Gage, 2002;[START_REF] Barkho | Identification of astrocyte-expressed factors that modulate neural stem/progenitor cell differentiation[END_REF], neuronal maturation (H. J. Song, Stevens and Gage, 2002), migration and axonal guidance [START_REF] Lois | Chain migration of neuronal precursors[END_REF][START_REF] Peretto | Glial tubes in the rostral migratory stream of the adult rat[END_REF][START_REF] Bolteus | GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone[END_REF], synapse formation, modulation and maintenance [START_REF] Pfrieger | Synaptic efficacy enhanced by glial cells in vitro[END_REF][START_REF] Ullian | Control of synapse number by glia[END_REF][START_REF] Christopherson | Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis[END_REF][START_REF] Perea | Neuron-glia networks: Integral gear of brain function[END_REF], and integration of the newborn neurons into pre-existing neuronal circuits in the adult brain [START_REF] Krzisch | Pre-existing astrocytes form functional perisynaptic processes on neurons generated in the adult hippocampus[END_REF]. Gap junctions can also be present along the astrocytes' processes permitting a syncytial network that facilitates communication to distal brain regions through calcium waves, although it remains to be determined if it is the case for astrocytes in close contact with NSCs [START_REF] Buskila | Generating Brain Waves, the Power of Astrocytes[END_REF]. Besides, astrocytes forming the migration path of new neurons express Robo receptors and respond to the repulsive activity of the neuron-secreted Slit changing the organization of their processes to facilitate neuronal migration [START_REF] Kaneko | New neurons clear the path of astrocytic processes for their rapid migration in the adult brain[END_REF].

Neurovascularization: endothelial cells, pericytes and astrocytes (mammalian BBB)

Blood vessels deliver oxygen and nutrients, as well as remove carbon dioxide and metabolic waste from all the tissues. Those vascularizing the CNS have unique properties controlling their permeability. This "blood-brain barrier" (BBB) is a neuroprotective diffusion barrier that selectively limits the movement of ions and molecules between the blood and the brain.

Endothelial cells form the core element of the BBB and are connected to the astrocytes and pericytes. They form tight junctions' seals that block the entrance of pathogens and harmful compounds to the brain while ensuring the delivery of nutrients and other essential molecules via specific transporters [START_REF] Daneman | The blood-brain barrier[END_REF]. Pericytes are in direct contact with endothelial cells and participate in the formation of the BBB, as well as maintain the structural and functional integrity of the vessels they contact [START_REF] Hill | Emerging Roles of Pericytes in the Regulation of the Neurovascular Unit in Health and Disease[END_REF][START_REF] Langen | Development and cell biology of the blood-brain barrier[END_REF]. Astrocytes project cellular processes to enwrap the vascular network of the brain [START_REF] Abbott | Astrocyte-endothelial interactions at the blood-brain barrier[END_REF]. Though they do not directly interact with blood vessels but pericytes, astrocytes significantly contribute to the regulation of the cerebral blood flow [START_REF] Mishra | Binaural blood flow control by astrocytes: listening to synapses and the vasculature[END_REF]. Besides, their secreted molecules may be necessary to maintain the BBB properties [START_REF] Langen | Development and cell biology of the blood-brain barrier[END_REF]. Both pericytes and astrocytic processes are completely embedded in a structural matrix, the basement membrane, produced by secreted molecules of endothelial cells, pericytes, and astrocytes. This extracellular matrix supports BBB integrity by anchoring cells in place and is an important hub for their intercellular communication [START_REF] Obermeier | The blood-brain barrier[END_REF].

A direct impact on NSC behavior by the vasculature has also been elucidated: endothelial cells stimulate NSCs self-renewal and differentiation in vitro [START_REF] Shen | Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells[END_REF], NSCs reside in close association with blood vessels [START_REF] Palmer | Vascular niche for adult hippocampal neurogenesis[END_REF] or extend processes to contact them if they are further away [START_REF] Tavazoie | A Specialized Vascular Niche for Adult Neural Stem Cells[END_REF]. Direct contact to the neurovasculature has been shown to maintain NSC quiescence (Ottone et al., 2014). In addition, circulating blood carries systemic factors that can impact neurogenesis.

The development and maintenance of the BBB properties are complex processes that involve the interaction with a variety of cell types, as well as different environmental cues, some of which are released by neurons. Hence, this interplay of cellular and acellular components tightly controls the brain homeostasis and protects it from toxins, pathogens, and inflammation in order to ensure the optimal environment for the proper functioning of the neuronal circuits and neurogenesis.

Microglia

Microglia, the CNS resident immune cells, are also part of the NSC niche. Adult microglia exhibit ramified extensions that extend and retract to constantly scan their surroundings [START_REF] Nimmerjahn | Neuroscience: Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo[END_REF]. In pathological conditions, resulting from inflammation, injury, infection, or disease, microglial cells can release inflammatory mediators and acquire an activated ameboid shape that allows them to phagocytize. Their highly efficient phagocytic activity enables them to clear pathogens, dead or dying cells, and other cellular debris. Microglia are also able to interact with other cells in the CNS impacting on their functions. They contribute to the correct formation, maintenance, and remodeling of neural circuits by mediating the removal of supranumerary newborn neurons, the survival of neuroblasts, the maturation of synapses, and the elimination of immature ones [START_REF] Barres | The Mystery and Magic of Glia: A Perspective on Their Roles in Health and Disease[END_REF][START_REF] Sierra | Cien Años de Microglía: Milestones in a Century of Microglial Research[END_REF]. In addition to axonal or synaptic modulation, these macrophages interact with NSCs in the developing SVZ and both adults SVZ and SGZ.

Through the release of soluble factors, microglia influence neurogenesis in both life stages, and in front of an injury [START_REF] Matarredona | Interactions between neural progenitor cells and microglia in the subventricular zone: Physiological implications in the neurogenic niche and after implantation in the injured brain[END_REF][START_REF] Sierra | Cien Años de Microglía: Milestones in a Century of Microglial Research[END_REF]. Microglia also promote the survival of oligodendrocyte precursors for normal myelinogenesis and phagocyte myelin waste [START_REF] Li | Microglia and macrophages in brain homeostasis and disease[END_REF]. Through the binding to astrocytes of the BBB, they support its function to maintain CNS homeostasis. Besides, microglia participate in the formation of vascular networks [START_REF] Sierra | Cien Años de Microglía: Milestones in a Century of Microglial Research[END_REF]. Microglial cells have been involved in the progression of neurogenerative disorders, traumatic brain injury, and psychiatric diseases, during which can have beneficial or detrimental roles depending on the context (reviewed in [START_REF] Wolf | Microglia in Physiology and Disease[END_REF].

Ependymal cells

Ciliated ependymal cells, another type of glial cell, line the ventricular wall of the adult SVZ forming a barrier. Specialized ependymal cells in some regions are known as choroid plexus and secrete the cerebrospinal fluid filling the cerebral ventricles. This fluid is a reservoir of trophic molecules and other systemic signals essentials for the maintenance and proliferation of NSCs [START_REF] Kaiser | Choroid Plexus: The Orchestrator of Long-Range Signalling Within the CNS[END_REF]. Ependymal cells enter in close contact with NSCs and their progeny actively regulating adult NSCs neuronal fate commitment [START_REF] Lim | Noggin antagonizes BMP signaling to create a niche for adult neurogenesis[END_REF] and contributing to the formation of a gradient concentration that guides neuroblasts migration [START_REF] Sawamoto | New neurons follow the flow of cerebrospinal fluid in the adult brain[END_REF]. Ependymal/cerebrospinal fluid-derived factors activate NSCs proliferation and neurogenesis [START_REF] Kazanis | Quiescence and activation of stem and precursor cell populations in the subependymal zone of the mammalian brain are associated with distinct cellular and extracellular matrix signals[END_REF], while endothelial cells-derived factors maintained them in a quiescent state (Ottone et al., 2014).

Progression to the adult NSC niche

During brain development, the structure of NSC niches is transient. At early stages, embryonic NSCs (radial glia) form a homogeneous population lining the wall of the cerebral ventricles (Figure 4A). They start to rapidly generate intermediate progenitors that amplify the progenitor pool and consequently the neural progeny [START_REF] Temple | The development of neural stem cells[END_REF]. Newborn neurons use their parental cells as scaffolds to migrate along their radially extended cellular fibers to their corresponding position in the nascent cortex (Figure 4A). These immature cells become postmitotic cells very early during development yet they may remain alive and functional for decades [START_REF] Aranda-Anzaldo | The post-mitotic state in neurons correlates with a stable nuclear higher-order structure[END_REF]. In parallel, a second group of newborn neurons migrate tangentially, thus in parallel to the ventricles, to occupy the entire cerebral cortex [START_REF] Marín | Cellular and molecular mechanisms controlling the migration of neocortical interneurons[END_REF]. At later stages, the developing NSC niche also includes some astrocytes and microglia, directly interacting with

In the adult mammalian brain, there are two major neurogenic niches where NSCs persist in a mitotically inactive state: the V-SVZ, also known as subependymal zone (SEZ), and the SGZ (Figure 5A) [START_REF] Song | Adult Neurogenesis in the Mammalian Brain: Significant Answers and Significant Questions[END_REF]. Microenvironmental cues and cell-cell interactions are crucial to balance each of the NSC behaviors: proliferation, self-renewal, differentiation into neuronal or glial lineages and quiescence. Adult NSCs remain quiescent for long time periods and can enter the cell cycle and mature into active NSC that slowly proliferate.

In the V-SVZ, adult NSCs (also known as B1 cells) line the walls of the lateral ventricles (Figure 5B). Their apical side contacts the cerebrospinal fluid with a single cilia and their basal side blood vessels with their radial processes [START_REF] Doetsch | Subventricular zone astrocytes are neural stem cells in the adult mammalian brain[END_REF][START_REF] Shen | Adult SVZ Stem Cells Lie in a Vascular Niche: A Quantitative Analysis of Niche Cell-Cell Interactions[END_REF]. NSCs receive cues from both, allowing them to sense external changes. Once activated, they produce transit-amplifying intermediate progenitors (C cells) that also proliferate few times to generate migrating neurons called neuroblasts (A cells) [START_REF] Doetsch | Subventricular zone astrocytes are neural stem cells in the adult mammalian brain[END_REF][START_REF] Bond | Adult Mammalian Neural Stem Cells and Neurogenesis: Five Decades Later[END_REF]. In most mammals, neuroblasts migrate inside glial tubes structures formed by surrounding astrocytes that guide them through the rostral migratory stream to the olfactory bulb [START_REF] Luskin | Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone[END_REF]. Once in they reach their corresponding location, they differentiate into local interneurons that integrate the existing neuronal circuitry [START_REF] Lois | Long-distance neuronal migration in the adult mammalian brain[END_REF]. Whereas neurogenesis precedes gliogenesis during embryonic development, and as such neurons are produced in environments devoid of mature astrocytes, in adults, astrocytes are distributed through the neurogenic niches in which they play fundamental roles. Adult NSC niches are also composed by microglia and ependymal cells whose roles have been previously

explained [START_REF] Doetsch | Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain[END_REF]. While robust adult V-SVZ neurogenesis has been characterized in rodents and non-human primates, in humans, little to no neurogenesis has been characterized (Conover and Todd, 2017).

In the dentate gyrus, adult NSCs (also known as type 1 cells) are located in the SGZ layer (Figure 5C). Therefore, unlike adult NSCs from the V-SVZ they do not contact the ventricle, but they are also in close contact with blood vessels [START_REF] Palmer | Vascular niche for adult hippocampal neurogenesis[END_REF]. Once activated, they give rise to intermediate progenitor cells (type 2 cells) which produce neuroblasts (type 3 cells). These very immature neurons do not move very far and integrate the granule cell layer, where they differentiate into granule neurons [START_REF] Song | Adult Neurogenesis in the Mammalian Brain: Significant Answers and Significant Questions[END_REF]. They form synaptic integration into hippocampal neuronal networks and play key roles in the formation of short-term and spatial memory learning, memory and pattern separation [START_REF] Tuncdemir | Contributions of adult neurogenesis to dentate gyrus network activity and computations[END_REF]. Therefore, adult NSCs in the SGZ are in close proximity to their differentiated daughter cells, to astrocytes and to microglia, all of which influence their neurons and the Drosophila NSC or neuroblast (NB), the Drosophila NSCs. Ensheathing glia (EG) and astrocyte-like glia (AG) cover the neuropil and send projections within the neuropil to enwrap axons and dendrites. Adapted from [START_REF] Skeath | The extracellular Metalloprotease AdamTS-A anchors neural lineages in place within and preserves the architecture of the central nervous system[END_REF]

Niche components

Neural stem cells

During embryonic and larval development, NSCs are distributed along the CNS and not far from its surface (except the dorsal VNC which is unoccupied by NSC). Strikingly, in Drosophila, each embryonic NSC can be identified by its unique gene expression profile, its progeny, and its birth time and place. This is explained because NSCs have an intrinsic program of cell proliferation where different patterning genes act along the anteroposterior and dorsoventral axes to generate unique pairs of NSCs along the CNS. The expression of specific combinations of regulatory genes determines precise NSC identities and restricts the fate of their progeny [START_REF] Truman | Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster[END_REF][START_REF] Doe | Identification and cell lineage of individual neural precursors in the Drosophila CNS[END_REF][START_REF] Schmidt | The embryonic central nervous system lineages of Drosophila melanogaster: II. Neuroblast lineages derived from the dorsal part of the nueroectoderm[END_REF][START_REF] Skeath | Genetic control of Drosophila nerve cord development[END_REF][START_REF] Beckervordersandforth | Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression[END_REF][START_REF] Sousa-Nunes | Regulating neural proliferation in the Drosophila CNS[END_REF]. A certain continuity in the identity of embryonic and larval NSCs has been proposed [START_REF] Birkholz | Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster[END_REF][START_REF] Lacin | Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system[END_REF], although precise tracing and characterization remain to be done.

Neurons

Once born, neurons are located near their mother cells and all the lineage remains kind-of attached together. Along NSC proliferation, neurons are pushed to deeper regions of the CNS and with maturation, they tend to detach from the lineage. Neurons project their axons into the neuropil where they form synapses and exchange neurotransmitters to communicate with other neurons coming from the peripheral nervous system [START_REF] Shin | Drosophila as a Model System for Neurotransmitter Measurements[END_REF]. Due to the close proximity between newborn neurons and NSCs, it is likely that neurons interact with NSCs though their secreted signaling molecules but no specific mechanisms have been characterized so far in Drosophila.

Glia

Gliogenesis starts during embryonic stages and the variety of glial cells present in postembryonic NSC niches play important functions influencing NSC behavior and match those in mammals (

Surface glia: perineurial and subperineurial glia (Drosophila BBB)

There are two cell types, perineurial glia and subperineurial glia. They ensheath the CNS and separate the nervous system from the open circulatory system establishing the Drosophila BBB from late embryonic stages (Figure 6B) [START_REF] Yildirim | Drosophila glia: Few cell types and many conserved functions[END_REF]. They also appear to produce signals preventing the premature re-entry of NSCs into the cell cycle [START_REF] Ebens | The Drosophila anachronism locus: A glycoprotein secreted by glia inhibits neuroblast proliferation[END_REF].

The most outer layer is formed by many perineurial glial cells, which exhibit star-like shapes forming extensive thin cell protrusions [START_REF] Stork | Organization and function of the blood-brain barrier in Drosophila[END_REF]. They contribute to the secretion and regulation of a layer of extracellular matrix that covers the CNS and peripheral nerves called neural lamella [START_REF] Meyer | Glia ECM interactions are required to shape the Drosophila nervous system[END_REF][START_REF] Petley-Ragan | Accumulation of Laminin monomers in Drosophila Glia leads to Glial endoplasmic reticulum stress and disrupted larval locomotion[END_REF]. Perineurial glia express many transporter proteins responsible for the uptake of nutrients [START_REF] Volkenhoff | Glial glycolysis is essential for neuronal survival in drosophila[END_REF][START_REF] Weiler | Metabolite transport across the mammalian and insect brain diffusion barriers[END_REF]. In addition, these cells express an extracellular factor involved in the modulation of NSCs proliferation [START_REF] Kanai | Regulation of neuroblast proliferation by surface glia in the Drosophila larval brain[END_REF], raising the question of how NSCs and perineurial glia communicate.

While the perineurial layer forms a rather cellular mesh, the subperineurial glia layer below is a very effective shield of few cells, attached to each other by septate junctions blocking all paracellular diffusion [START_REF] Carlson | Blood Barriers of the Insect[END_REF]. Their function is similar to the tight junctions of the endothelial cells present in the mammalian BBB, offering chemoprotection and selective transcellular transport of nutrients [START_REF] Limmer | The drosophila blood-brain barrier: Development and function of a glial endothelium[END_REF][START_REF] Schirmeier | The Drosophila blood-brain barrier as interface between neurons and hemolymph[END_REF].

Subperineurial glial cells are large polyploid cells that undergo endoreplication to increase their DNA content and nuclei number. Thanks to this strategy, they keep the cells number constant and the septate junctions unperturbed by cytokinesis to sustain cell growth [START_REF] Unhavaithaya | Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity[END_REF]. Subperineurial glia contact NSCs during embryogenesis and also the most superficial neuronal cell bodies in the cortex (Pereanu, Shy and Hartenstein, 2005;Spéder and Brand, 2018). In addition to its BBB role, this glia fulfills a true niche function for NSCs by mediating the impact of nutrition on NSC reactivation from their quiescent state. Newly hatched larvae start to feed and the fat body, the functional equivalent of the mammalian liver and adipose tissue, is able to sense the intake of dietary amino acids [START_REF] Colombani | A nutrient sensor mechanism controls Drosophila growth[END_REF]. The fat body provides an unknown signal which induces calcium oscillations in the subperineurial glia, triggering the release of Drosophila insulin-like peptides. These peptides act locally, bind to the insulin receptor on quiescent NSCs and activate the phosphatidylinositol 3-kinase (PI3K)/ anaplastic lymphoma kinase (Akt)/ target of rapamycin (TOR) pathway (Figure 7B) [START_REF] Chell | Nutrition-responsive glia control exit of neural stem cells from quiescence[END_REF][START_REF] Sousa-Nunes | Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila[END_REF]. NSC enlarge re-entering the cell cycle in a synchronous way thanks to gap junctions present in the subperineurial glia, and which coordinate the nutrition-induced calcium oscillations [START_REF] Spéder | Gap junction proteins in the blood-brain barrier control nutrient-dependent reactivation of Drosophila neural stem cells[END_REF].

Cortex glia

Beneath the surface glia, the cortex glia (CG) ensheath the cell bodies of the NSCs and their progeny (Figure 6B). They form a huge network while individually encasing cycling NSCs and their lineages with their membranes (Figure 7A). CG are differentiated during late embryonic stages from specific NSC lineages [START_REF] Schmidt | The embryonic central nervous system lineages of Drosophila melanogaster: II. Neuroblast lineages derived from the dorsal part of the nueroectoderm[END_REF]. At the beginning CG rapidly enwrap newborn neurons, by the time of larval hatching, CG membranes enclose multiple primary neurons, and later, they end up enclosing them all (Pereanu, Shy and Hartenstein, 2005;[START_REF] Coutinho-Budd | The secreted neurotrophin spätzle 3 promotes glial morphogenesis and supports neuronal survival and function[END_REF]. These membranes continuously expand and by 48h after larval hatching (ALH) they closely encase entire NSCs lineages in "chambers" (Spéder and Brand, 2018). Inside, NSCs remain closer to the CNS surface and neurons are pushed to the deeper regions as they mature [START_REF] Dumstrei | Role of DE-cadherin in neuroblast proliferation, neural morphogenesis, and axon tract formation in Drosophila larval brain development[END_REF]. CG morphology and promote continued enwrapping of mature neuronal cell body and proximal axons through a cell-autonomous control (Coutinho-Budd, Sheehan and Freeman, 2017). In addition, CG also help with the clearance, through phagocytosis, of young dead neurons during larval development [START_REF] Nakano | Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe[END_REF].

Besides, CG also protect NSCs against environmental pressures. Under nutritional restriction tissue growth is generally shutdown, however, CG are able to sustain NSC proliferation, therefore sparing the brain from undergrowth. Particularly, CG are able to secrete a growth factor, Jelly Belly, to stimulate PI3K/Akt signaling pathway in NSCs even if nutrients are not available in late larval stages (Cheng et al., 2011). Moreover, under hypoxia and oxidative stress, CG (as well as subperineurial glia) synthesize lipid droplets that protect NSCs from oxidative damage (Bailey et al., 2015). These examples show the important function of glialderived signaling within the NSC niche.

Neuropil-associated glia: astrocyte-like and ensheathing glia

At the heart of the CNS lie the neuropil-associated glia ensheating axons, dendrites and synapses: namely the astrocyte-like glia and the ensheathing glia. However, their cell bodies lie in the interphase between the CNS cortex and the neuropil. They both originate during embryonic stages and persist until adult after proliferation during late larval stages [START_REF] Omoto | Origin and development of neuropil glia of the Drosophila larval and adult brain: Two distinct glial populations derived from separate progenitors[END_REF][START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF].

Whereas mammalian astrocytes are the most frequent glia cell type in postembryonic stages, flies only harbor few numbers of astrocyte-like glial cells. Nevertheless, they share many resemblances in morphological, molecular and functional aspects. They are highly ramified and extend their processes into the neuropil where they influence the function of axons, dendrites and synapses [START_REF] Awasaki | Organization and postembryonic development of glial cells in the adult central brain of Drosophila[END_REF][START_REF] Freeman | Drosophila central nervous system glia[END_REF]. Astrocyte-like glia development into the neuropil is tightly coupled with synaptogenesis during the formation of the adult CNS, where they are involved in the modulation of neuronal circuit function regulating excitatory/inhibitory balance in the nervous system [START_REF] Muthukumar | Activity-dependent regulation of astrocyte GAT levels during synaptogenesis[END_REF]. Moreover, they can also take over microglia-like functions, phagocytizing neuronal debris and participating in the clearing of neurotransmitters in the synapses [START_REF] Tasdemir-Yilmaz | Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons[END_REF].

Compared to astrocyte-like glia, ensheathing glia do not infiltrate the neuropil but rather provide a compartmentalization by enwrapping the neuropil and the axon bundles leaving the CNS [START_REF] Yildirim | Drosophila glia: Few cell types and many conserved functions[END_REF]. The role of this compartmentalization is poorly known. Besides, a recent role of ensheathing glia in neuromodulation has been depicted [START_REF] Otto | The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia[END_REF]. They also contribute to the phagocytic clearance of neuronal debris [START_REF] Doherty | Ensheathing glia function as phagocytes in the adult Drosophila brain[END_REF].

Regulation of neural stem cell behavior

Understanding stem cell behavior includes understanding how and when stem cells proliferate and differentiate. NSC are able to proliferate to increase or replenish their pool, to self-renew while producing a differentiated cell, and to differentiate into more committed cells. They do it by regulating their modes (symmetric versus asymmetric) and their rate of divisions [START_REF] Hardwick | Nervous decision-making: To divide or differentiate[END_REF]. In addition, they can switch between proliferative and non-proliferative states by entering and exiting the cell cycle, respectively.

The cell cycle

The cell cycle is a sequence of highly coordinated stages through which a cell duplicates its content and divides. It comprises a long interphase, which consists of the G1, S and G2 phases;

and a short mitotic (M) phase, which includes mitosis and cytokinesis (Figure 8). During interphase the cell grows, doubles its amount of proteins and organelles, and duplicates its DNA content to prepare for the start of mitosis. This mitotic phase involves a complex series of events and is conventionally divided into five phases: prophase, prometaphase, metaphase, anaphase and telophase. Prophase starts with the condensation of the duplicated DNA strands into compact structures. Each condensed and replicated chromosome consists now of a pair of sister chromatids held together by a centromere [START_REF] Alberts | Molecular Biology of the Cell, Sixth Edition[END_REF]. A pair of cytoplasmic nonmembranous cell organelles, called the centrosomes, go to opposite poles of the cell and organize the nucleation of microtubules that will ultimately form the mitotic spindle [START_REF] Jaiswal | Centrosome: A Microtubule Nucleating Cellular Machinery[END_REF]. In many cells, the transition for the next phase is the breakdown of the nuclear membrane, allowing the entry of the spindle microtubules (De Magistris and Antonin, 2018).

During prometaphase, the spindle extends from the poles and binds to the chromosomes through some protein formations called kinetochores that are associated with the centromeres.

This bipolar attachment of the sister chromatids to microtubules of the spindle leads to chromosomes' migration to the equatorial plane at the center of the cell. Metaphase is the alignment of the chromosomes along the equatorial plane. The kinetochores pull the chromosomes back and forth until they totally align them waiting for segregation. Anaphase remains more or less intact. In any case, at the end of mitosis, the nuclear envelope will be formed around the segregated chromatin. Between these theoretical extreme possibilities, a plethora of forms of semi-open/close mitosis exist in eukaryotes to allow nuclear envelope remodeling where some nuclei undergo only partial nuclear breakdown, and others lose the permeability barrier of the nuclear pores connecting cytoplasm and nucleoplasm (Figure 9). For example, Drosophila NSCs undergo a semi-closed mitosis where the nuclear envelope persists from prophase to anaphase with an underlying lamina (a meshwork of lamin proteins) enveloping the spindle. This remodeling is characterized by the disassembly of nuclear pores and the loss of the barrier function, which upon mitotic entry becomes discontinuous but still keeps large structures out [START_REF] Katsani | In vivo dynamics of Drosophila nuclear envelope components[END_REF][START_REF] Roubinet | Asymmetric nuclear division of neural stem cells contributes to the formation of sibling nuclei with different identities[END_REF].

Figure 9. Plethora of possible mitotic nuclear remodeling strategies.

The concentrations of the three major cyclin types oscillate during the cell cycle, while the concentrations of Cdks (not shown) do not change and exceed cyclin amounts. In late G1, rising G1/S-cyclin levels lead to the formation of G1/S-Cdk complexes that trigger progression through the start transition, resulting in a commitment to cell-cycle entry. S-Cdk complexes form at the start of S phase and trigger DNA replication, as well as some early mitotic events. M-Cdk complexes form during G2 but are held in an inactive state; they are activated at the end of G2 and trigger entry into mitosis at the G2/M transition. A separate regulatory protein complex, the APC/C, initiates the metaphase-to-anaphase transition. From [START_REF] Alberts | Molecular Biology of the Cell, Sixth Edition[END_REF].

The three major checkpoints in the cell cycle are: the G1 checkpoint before the G1/S transition, the G2 checkpoint before the G2/M transition and the spindle assembly checkpoint at the transition from metaphase to anaphase (Figure 10) [START_REF] Alberts | Molecular Biology of the Cell, Sixth Edition[END_REF]. G1 checkpoint is influenced primarily by cell size, growth factors, cell nutrition, DNA damage, and other molecular and mechanical cues. Extended periods of G1 arrest may lead the cell to exit the cell cycle and enter to G0, a state of quiescence. Normal quiescent cells are still capable of reentering the cell cycle in response to certain signals. G2 checkpoint checks if the DNA is completely replicated and its whole integrity maintained. Any evidence of DNA damage would pause the cell in this phase to allow for DNA reparation. The entry into mitosis is triggered by the M-Cdk complex (Figure 10). The spindle assembly checkpoint assesses whether all sister chromatids are correctly attached to the mitotic spindle. Because the separation of the sister chromatids during anaphase is an irreversible step, the cycle will not proceed until each chromosome kinetochore is firmly attached to at least two spindle microtubules from opposite poles of the cell. If there is any chromosome misplacement, the cell will wait until the spindle captures any lagging chromosome before continuing.

Spindle assembly checkpoint

A central event of cell division is the equal distribution of the genetic material between the daughter cells. Correct chromosome segregation requires functional microtubule organizing centers that promote the formation of the mitotic spindle. This microtubule-based structure binds to the chromosomal kinetochores and pulls sister chromatids apart in anaphase, thus segregating the two sets of chromosomes to opposite sides of the cell. Three distinct sets of microtubules emanate from the microtubule organizing centers of each pole (Figure 11).

Kinetochore microtubules attach directly to chromosomes at the kinetochores; interpolar microtubules connect at the spindle equator with other interpolar microtubules emanating from chromosomes are properly attached, at which point the SAC becomes "satisfied" (it subsides), and stops signaling. From [START_REF] Krenn | The Aurora B kinase in chromosome bi-orientation and spindle checkpoint signaling[END_REF].

Several checkpoint proteins have been identified as required for proper mitotic progression.

Thus, alteration of the pathways for the correct microtubule-kinetochore attachment and the SAC surveillance mechanism can delay the onset of anaphase and arrest the cells at metaphase for more or less longer periods of time [START_REF] Gardner | The spindle checkpoint: Two transitions, two pathways[END_REF][START_REF] Donaldson | Metaphase arrest with centromere separation in polo mutants of Drosophila[END_REF][START_REF] Wainman | Roles of the Drosophila NudE protein in kinetochore function and centrosome migration[END_REF][START_REF] Rubin | Cenp-meta is required for sustained spindle checkpoint[END_REF]. In parallel, DNA damage could be another cause of a delay in the metaphase-to-anaphase transition [START_REF] Royou | The Drosophila Grp/Chk1 DNA damage checkpoint controls entry into anaphase[END_REF]. Following a prolonged mitotic arrest, cells usually either die in mitosis via apoptosis or they undergo a process called 'slippage' where they exit mitosis without chromosome segregation or cytokinesis [START_REF] Topham | Mitosis and apoptosis: How is the balance set?[END_REF]. In slippage, cells return to the G1 phase of the cell cycle but with the double of genetic material. It occurs because, despite the persistence of an active checkpoint signal, the M-Cdk complex ultimately falls below the level needed to maintain the mitotic state [START_REF] Brito | Mitotic Checkpoint Slippage in Humans Occurs via Cyclin B Destruction in the Presence of an Active Checkpoint[END_REF]. The outcomes that can follow are cell cycle progression, G1 growth arrest and apoptosis.

Balance between proliferation and quiescence

Following cell division, a daughter cell may choose to enter immediately into another cycle of growth and division, leading repeated rounds of cell division and resulting in an increase of the cell population. Otherwise, a daughter cell may choose to cease active growth and exit the cell cycle to enter a state of quiescence termed G0 [START_REF] Alberts | Molecular Biology of the Cell, Sixth Edition[END_REF].

In mammals, NSCs are widespread during embryonic development and differentiate in most regions of the CNS at late embryonic or early postnatal stages. Yet, in most mammalian brains some NSC persist in few specific regions. These adult NSCs reside mainly in a non-proliferative state, the previously mentioned quiescent state [START_REF] Morshead | Neural stem cells in the adult mammalian forebrain: A relatively quiescent subpopulation of subependymal cells[END_REF]. This mitotically dormant state is suggested to be key in preserving stemness, by preventing precocious differentiation and premature exhaustion of the NSC pool. It is a reversible state in which the cell cycle is arrested and divisions cease. In the presence of the appropriate signal, NSCs reactivate and resume proliferation [START_REF] Rumman | Concise Review: Quiescence in Adult Stem Cells: Biological Significance and Relevance to Tissue Regeneration[END_REF]. In this way, NSCs transition between quiescence and proliferation by exiting and entering the cell cycle, respectively. With this balance, these amazing cells ideally guarantee lifelong neurogenesis by avoiding accumulation of damage to DNA or to other cellular components as the animal ages.

NSC choice between remaining quiescent or entering an active state is one of the most important points of regulation of NSC behavior.

Despite being associated with low metabolic activity, quiescence is an active state where the cell performs normal cellular functions except division and ensures reversibility of the cell cycle-arrest. Quiescent NSCs are suggested to be highly sensitive to external stimuli due to their upregulation of genes that code for membrane proteins involved in intercellular communication, cell adhesion and transport [START_REF] Shin | Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis[END_REF]. Accordingly, transition from quiescence to active states entails changes in the cell physiology such as an increase in the expression of genes related to cell cycle and protein synthesis, as well as a gradual shutdown of extrinsic signaling pathways [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF][START_REF] Urbán | Quiescence of Adult Mammalian Neural Stem Cells: A Highly Regulated Rest[END_REF]). An heterogeneity in the stem cell population seems to coexist in the adult stem cell niches to support rapid response and regeneration after tissue stress [START_REF] Li | Coexistence of quiescent and active adult stem cells in mammals[END_REF].

In response to environmental inputs, quiescent NSC reactivate and are able to renew the stem cell population with the possibility of generating also more committed cells, and return to quiescence or differentiate without division [START_REF] Bonaguidi | In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics[END_REF][START_REF] Urbán | Return to Quiescence of mouse neural stem cells by degradation of a proactivation protein[END_REF]. Selfrenewing divisions may lead to intrinsic changes like aging of the stem cell lineage. This selfrenewal capacity has been shown to also decrease with the number of NSCs that occupy the same niche-corroborating the important role that the stem cell's microenvironment has on them [START_REF] Basak | Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy[END_REF]. The vasculature of the NSC niche can also act as a regulator of quiescence, direct contact between quiescent NSCs and endothelial cells impairs NSC response to extrinsic proliferative and differentiative cues in order to maintain them quiescent (Ottone et al., 2014).

Interestingly, the NSC microenvironment shows an essential role in the regulation of NSC fate and number by controlling the reversible transition between quiescent and active NSC states.

In Drosophila, NSCs divide throughout embryogenesis, then cease proliferation before larval hatching and some enter quiescence while others undergo apoptosis [START_REF] Prokop | The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster[END_REF]. Their entry into quiescence is intrinsically determined by a temporal cascade of transcription factors [START_REF] Tsuji | Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors[END_REF]. They remain quiescent for around 24h and then reactivate in a coordinated fashion [START_REF] Spéder | Gap junction proteins in the blood-brain barrier control nutrient-dependent reactivation of Drosophila neural stem cells[END_REF] in response to extrinsic nutritional stimulus [START_REF] Britton | Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms[END_REF]. Feeding is detected by the fat body which promotes the release of an insulin-like peptide by surface glia. In turn, the insulin receptor in quiescent NSC is then activated and initiates a downstream signaling cascade via the conserved PI3K/Akt/TOR pathway (Figure 7B). This stimulates NSC re-entry to the cell cycle, first growth and then proliferation [START_REF] Chell | Nutrition-responsive glia control exit of neural stem cells from quiescence[END_REF][START_REF] Sousa-Nunes | Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila[END_REF]. Either dietary amino acids or the genetic activation of the PI3K/Akt pathway, are sufficient for the reactivation of quiescent NSCs and are not essential afterwards in activated NSC to continue division [START_REF] Britton | Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms[END_REF]Cheng et al., 2011).

Regulators of neural stem cell proliferation

NSCs proliferation is finely regulated by changes in the surrounding microenvironment. These changes are controlled by both extrinsic and intrinsic mechanisms. Many molecular players and signaling pathways have been identified including niche (like Wnt, Notch, Sonic hedgehog and the receptor tyrosine kinase family), cytoplasmic (like small non-coding RNA modulators), transcription (like TLX and the Sox family) and epigenetic regulators (like histone modifications and DNA methylations) [START_REF] Shi | Neural stem cell self-renewal[END_REF][START_REF] Song | Adult Neurogenesis in the Mammalian Brain: Significant Answers and Significant Questions[END_REF]. A wide range of systemic cues can also alter NSCs behavior. Changes in the metabolic state, such as high fat diets, malnutrition, obesity, diabetes and exercise, play important roles influencing NSCs behavior and their progeny [START_REF] Spéder | Nutrient control of neural stem cells[END_REF].

Cellular interactions within the NSCs niche

Cells express a multitude of cell adhesion molecules at the surface of their plasma membranes.

They function as receptors enabling cells to bind to adjacent ones or to the extracellular matrix, hence to adhere and communicate with their environment. Most of these surface receptors serve also as signal transductors initiating intracellular signaling pathways that modulate important cellular events including proliferation, survival, and differentiation. Some cell adhesion

Immunoglobulins

The immunoglobulin superfamily is defined by the presence of one or more immunoglobulin domains and, in many cases, they also contain copies of fibronectin type III domains. Their interactions can be either homophilic or heterophilic. Immunoglobulins can be secreted or localized to the cell surface. They perform both cis-(on the same side) and trans-(on the other side) interactions at the level of the plasma membrane. One of the most important contexts for immunoglobulins is the developing nervous system, where many different members are involved in axon guidance and neural circuit formation. Neural immunoglobulins serve also as guidance cues not only for axonal pathfinding but also for cell migration. Moreover, they participate in the maintenance and plasticity of synapses. The human genome encodes about 500 non-antibody, non-T cell receptor immunoglobulins, whereas Drosophila melanogaster has 130 (Zinn and Özkan, 2017).

In the nervous system, the immunoglobulin superfamily represents a large group which includes: i) the L1 subfamily [START_REF] Rathjen | Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion[END_REF], involved mainly in the formation and maintenance of the nervous system; ii) the NCAM (Neural Cell Adhesion Molecule) [START_REF] Thiery | Adhesion among neural cells of the chick embryo. II. Purification and characterization of a cell adhesion molecule from neural retina[END_REF], important for cell migration, synaptic plasticity, axonal growth and fasciculation; iii) VCAM1 (Vascular Cell Adhesion Molecule), expressed in NSCs and which maintains their correct niche positioning and quiescent state [START_REF] Kokovay | VCAM1 is essential to maintain the structure of the SVZ niche and acts as an environmental sensor to regulate SVZ lineage progression[END_REF]; iv) the Dscam (Down Syndrome Cell Adhesion Molecule) [START_REF] Yamakawa | DSCAM: A novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system[END_REF], named after its identification in the human chromosome 21 serves as an axon guidance receptor; v) the Robo (Roundabout) subfamily [START_REF] Kidd | Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors[END_REF], receptors for Slit ligands to mediate axonal repulsion; vi) nectins, crucial components of cadherin-based adherens junctions in many different tissues contribute to cell-cell junctions, cell polarization, movement, proliferation, differentiation, and survival [START_REF] Takahashi | Nectin/PRR: An immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with afadin, a PDZ domaincontaining protein[END_REF]; and vii) IgLONs [START_REF] Pimenta | The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting[END_REF], with roles in dendritic arborization or synapse formation.

In Drosophila, a recent in vitro extracellular interactome assay, elucidated many previously unknown interactions among the immunoglobulin superfamily interactome and identified 4 main subfamilies (Figure 15) (Özkan et al., 2013): the Dpr (Defective in Proboscis Extension Response), the DIP (Dpr Interacting Proteins), the Beat (Beaten Path), and the Side (Side-step).

They all mainly function as heterophilic neuronal wiring molecules, the 21-members Dpr subfamily interacting with the 11-members DIP one and the 14-members Beat subfamily with the 8-members Side one.

homolog, and IgLONs share homology with Drosophila Lachesin and DIPs, although the latter resemble to nectins, their closer vertebrate counterpart are IgLONs [START_REF] Silies | Adhesion and signaling between neurons and glial cells in Drosophila[END_REF]Carrillo et al., 2015).

Integrins

The integrin superfamily primarily acts as receptors for extracellular matrix proteins such as laminin, fibronectin and collagen. They form heterodimers of different α and β subunits, each having a large extracellular domain, a single membrane spanning domain and, in most cases, a short cytoplasmic one. Combinations of these subunits give distinct properties to the integrin molecules. For instance, the integrin α6/β1 interaction with the extracellular matrix has been discovered to be an essential attribute of stemness [START_REF] Ramalho-Santos | Stemness": Transcriptional profiling of embryonic and adult stem cells[END_REF]. Inside the In the CNS, integrins are expressed in a variety of cell types including neurons, glia, endothelial cells and NSCs [START_REF] Del Zoppo | Integrin-matrix interactions in the cerebral microvasculature[END_REF]. Integrin-mediated adhesion provides physical support for cells, guides cell positioning and enables microenvironmental sensing [START_REF] Morgan | Synergistic control of cell adhesion by integrins and syndecans[END_REF]. Another relevant feature of integrin function in the CNS is the regulation of cell migration. In particular, integrins are involved in the migration of neuroblasts and the proper formation of the rostral migratory chain [START_REF] Emsley | α6β1 integrin directs migration of neuronal precursors in adult mouse forebrain[END_REF][START_REF] Belvindrah | β1 integrins control the formation of cell chains in the adult rostral migratory stream[END_REF].

During CNS development, embryonic NSCs display a bipolar radial morphology that is supported by two adhesion sites (Figure 16A). At the apical end, NSCs lining the ventricular surface are attached to their neighboring cells through adherens junctions; whereas at the basal end, they adhere to extracellular matrix under the pia surface via integrin interactions [START_REF] Campos | β1 integrins and neural stem cells: Making sense of the extracellular environment[END_REF][START_REF] Miyamoto | N-cadherin-based adherens junction regulates the maintenance, proliferation, And differentiation of neural progenitor cells during development[END_REF]. Adult NSCs of the V-SVZ retain similar type of adhesions and the apico-basal polarity with short apical processes penetrating the ependymal layer to contact the cerebrospinal fluid, and long basal processes extending to blood vessels to adhere to the molecules present in their dense surrounding extracellular matrix (Figure 16B) [START_REF] Shen | Adult SVZ Stem Cells Lie in a Vascular Niche: A Quantitative Analysis of Niche Cell-Cell Interactions[END_REF]Morante-Redolat and Porlan, 2019). In some mammals, such as humans, NSCs can also reside away from the ventricular zone and lack apical processes [START_REF] Hansen | Neurogenic radial glia in the outer subventricular zone of human neocortex[END_REF]. In the SGZ, NSC basal processes also contact blood vessels and can form adhesion points with astrocytic processes when they meet on the endothelial surface [START_REF] Moss | Fine processes of Nestin-GFP-positive radial glia-like stem cells in the adult dentate gyrus ensheathe local synapses and vasculature[END_REF].

Adherens junctions show critical roles during the development of the mammalian nervous system (Figure 14) and N-cadherin mediates many of them. N-cadherin is expressed in embryonic NSCs and it is highly concentrated in their apical side, maintaining the polarized architecture of these NSCs and interconnecting them to each other to preserve tissue organization [START_REF] Kadowaki | N-cadherin mediates cortical organization in the mouse brain[END_REF]. In addition, N-cadherin-based adhesions are also important to control the balance between NSCs self-renewal and differentiation. Fine-tuned levels of N-cadherin are required in NSCs since its downregulation increases NSCs proliferation and neuronal differentiation [START_REF] Zhang | Cortical Neural Precursors Inhibit Their Own Differentiation via N-Cadherin Maintenance of β-Catenin Signaling[END_REF][START_REF] Rago | miR379-410 cluster miRNAs regulate neurogenesis and neuronal migration by fine-tuning N-cadherin[END_REF]. Together with other studies in different vertebrate models, this suggests that N-cadherin-mediated adhesions are crucial for NSC niche formation and CNS development [START_REF] Miyamoto | N-cadherin-based adherens junction regulates the maintenance, proliferation, And differentiation of neural progenitor cells during development[END_REF]. In the adult V-SVZ, N-cadherin is also important for the integrity of the niche architecture and the maintenance of quiescent NSCs. N-cadherin-mediated anchorage of NSCs to ependymal cells dynamically modulates NSCs' proliferative status by acting as quiescent signals [START_REF] Porlan | MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin[END_REF]). E-Cadherin, another adhesion molecule expressed in the embryonic and adult ventricles, where NSCs reside, also influences NSCs behavior promoting self-renewal [START_REF] Karpowicz | E-Cadherin regulates neural stem cell self-renewal[END_REF].

Like in most tissues, NSC adhesion to the extracellular matrix is paramount to integrate NSCs with their microenvironment. These attachments provide cells with physical support and can trigger cellular signaling. Mammalian NSCs actually participate in the secretion of an extracellular matrix that form the basement membrane, a layer rich in laminins, tenascins and other glycoproteins. These extracellular matrix molecules adhere to their receptors located in

Adhesion within the Drosophila NSCs niche

In Drosophila, cell-cell adhesion directs the delamination of embryonic NSCs from the neuroepithelium. These cells constrict their apical membrane while sequentially disassembling their adherens junctions with their neighboring cells. New cell-cell junctions between the remaining cells of the epithelium will be built (Figure 19) [START_REF] Simões | Myosin II promotes the anisotropic loss of the apical domain during Drosophila neuroblast ingression[END_REF]. Another function of the adherens junctions presents in the epithelium is to prevent neuroepithelial cells from dividing asymmetrically [START_REF] Lu | Adherens junctions inhibit asymmetric division in the Drosophila epithelium[END_REF]. They also play other major roles including the establishment and maintenance of the apico-basal polarity in epithelia, which then is inherited in NSCs; the formation of adhesive junctions at the synapse levels; the control of neuron-glia interactions; and the regulation of spindle orientation in NSC division [START_REF] Mandai | Afadin/AF-6 and canoe: Roles in cell adhesion and beyond[END_REF].

After delamination, NSCs start dividing asymmetrically to give rise to GMCs. These Drosophila embryonic NSCs, called neuroblasts, undergo apical constriction and disassemble their adherens junctions while being extruded from the epithelium. While these cells detach from their neighbors new adherens junctions are formed between the remaining epithelial cells.

From [START_REF] Simões | Myosin II promotes the anisotropic loss of the apical domain during Drosophila neuroblast ingression[END_REF] Integrins mediate cell contacts to the extracellular matrix proteins also in flies. During larval stages, the Drosophila nervous system grows while surface glia have to adapt to maintain the glial sheath throughout development. These glial cells bind to the neural lamella via integrins to provide and modulate the adhesive force that shapes the nervous system [START_REF] Xie | Integrins are necessary for the development and maintenance of the glial layers in the Drosophila peripheral nerve[END_REF][START_REF] Meyer | Glia ECM interactions are required to shape the Drosophila nervous system[END_REF]. A role for integrins in Slit-mediated axonal guidance has been described too, whereby integrin-mediated adhesion of neurons impacts axonal responsiveness to Slit-mediated chemo-repulsion to control their direction [START_REF] Stevens | Integrins Regulate Responsiveness to Slit Repellent Signals[END_REF].

Mechanics of cell shapes

All cells have a nonuniform cytoplasm and a continuously active plasma membrane that interacts with the underlying cytoskeleton, which consist of actin filaments, microtubules, several types of intermediate filaments and septins [START_REF] Alberts | Molecular Biology of the Cell, Sixth Edition[END_REF]. Together, this diverse and dynamic composition provides structural, biochemical and mechanical properties to each cell and, further, to the tissue. Every cell is exposed to mechanical forces and responds to them with different degrees. The tension that cells experience in different locations can arise from both outside and inside the cell. Cell adhesion molecules such as cadherins, immunoglobulins and integrins bind through their extracellular domains to other cells or matrix proteins, while their intracellular domain links them to the cytoskeleton. These adhesions act as dynamic tension sensors bidirectionally linking mechanical forces outside the cells with those inside.

Ultimately, these mechanical forces can be transduced into molecular signaling activities playing critical roles on cellular morphology, function, and fate determination. The overall cellular response to mechanical stimuli is called mechanotransduction [START_REF] Mammoto | Mechanobiology and Developmental Control[END_REF].

Mechanotransduction

Cells sense externally applied mechanical forces. These ones can result from the compression generated by neighboring cells as well as from the stiffness of the surrounding extracellular matrix [START_REF] Gattazzo | Extracellular matrix: A dynamic microenvironment for stem cell niche[END_REF]. These mechanical forces are exerted at the plasma membrane and can be altered via changes in cell-matrix or cell-cell adhesions. Cell adhesion molecules at the cell surface, such as integrins, end up triggering a variety of signaling pathways in the cell (Figure 20). Cells are not purely passive observers of mechanical forces.

Inside the cell, the cortex, a submembranous network of actin, myosin and actin-binding the subsequent inactivation the actin-depolymerizing protein cofilin (CLF), and directly promotes actomyosin contraction through the phosphorylation of myosin II (MyoII). mDia promotes actin polymerization. FAK, TLN, VCL, P130 Cas and VASP illustrate a range of adapter and signaling proteins associated with integrin(IT)-mediated focal adhesions. INM, inner nuclear membrane; NPC, nuclear pore complex; ONM, outer nuclear membrane; PS, perinuclear space. Adapted from [START_REF] Rubin | Cenp-meta is required for sustained spindle checkpoint[END_REF] Actin monomers polymerize into filaments that can be highly crosslinked and impart rigidity to the cell cortex, providing integral support to the cell. In conjunction with non-muscle myosin II, these actin networks generate actomyosin contractility. Myosins are molecular motors that use ATP hydrolysis to produce force on actin filaments (Munjal and Lecuit, 2014). The actomyosin interaction is responsible for the response of cells to external forces and for the internal generation of forces. Actin networks are regulated by proteins that control i) the rate of nucleation, elongation or depolymerization of actin filaments, ii) the architecture of the filaments by crosslinking them together, branching them into cables or unbinding the networks, iii) and their stability by severing the long filaments or capping their ends to block further addition of actin monomers [START_REF] Pollard | Actin and actin-binding proteins[END_REF]. Myosins are composed of one or two heavy chains and several light chains, and their activity is primarily modulated through the phosphorylation of their regulatory light chain. This phosphorylation promotes myosin assembly into bipolar filaments and enhances its ATPase activity [START_REF] Heissler | Various Themes of Myosin Regulation[END_REF].

Whether the forces are exogenously applied on cells or are endogenously generated, through the reorganization of cytoskeletal proteins, they trigger a variety of signaling pathways, ranging from GTPases, activation of kinase cascades to tension-activated ion channels [START_REF] Mammoto | Mechanobiology and Developmental Control[END_REF]. In some cases, the response is transmitted to the surrounding matrix or to neighboring cells via cell adhesion molecules to contribute to major changes in cell morphology [START_REF] Lessey | From mechanical force to RhoA activation[END_REF]. In other cases, the response is at the level of the nucleus where nuclear components physically connected to the cytoskeleton, such as nuclear envelope proteins, nuclear lamina, and nuclear pore complexes, regulate the nuclear morphology and chromatin organization (Figure 20) [START_REF] Janota | The role of the cell nucleus in mechanotransduction[END_REF].

The molecular composition and structure of the actomyosin network are important determinants of its function. High concentrations of myosin can lead to the disassembly of complex cytoskeletal structures composed of actin, myosin and other crosslinking proteins [START_REF] Backouche | Active gels: Dynamics of patterning and self-organization[END_REF]. Different proteins can alter the conformation of the actomyosin bundles, hence regulating the actomyosin activity. Through their binding, these proteins influence the interaction between actin filaments and myosin and can even increase the efficiency of generating contractile forces [START_REF] Lee | Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics[END_REF]. Actomyosin structures are connected to the plasma membrane or the nucleus and can also be linked to anchor points of cell-matrix or cellcell adhesion. These subcellular anchoring sites control the outcome of actomyosin contraction.

Besides its function generating contractile forces, myosin II activity is also itself regulated by mechanical forces. Experiments applying external forces to the Drosophila embryo show myosin recruitment to the cortex, furthermore, laser ablations cause the rapid loss of myosin from the cortex [START_REF] Fernandez-Gonzalez | Myosin II Dynamics Are Regulated by Tension in Intercalating Cells[END_REF]. This demonstrates that tension can regulate cortical myosin localization, implying the existence of feedback loops.

Nucleus and mechanotransduction

One may think that since the nucleus is located inside the cell, it could be protected from external mechanical stress. However, the nuclear envelope, like the plasma membrane, can also be exposed to mechanical forces emanating from the cytoskeleton or the cellular microenvironment. For proper cellular function, the cytoskeleton is connected to the cell nucleus allowing the transfer of various molecules and signals to and from the nucleus. In many cases, this connection is mediated by the linker of nucleoskeleton and cytoskeleton (LINC)

complex which also provides spatial and structural integrity to the nucleus [START_REF] Crisp | Coupling of the nucleus and cytoplasm: Role of the LINC complex[END_REF].

This adhesion complex, similar to integrin based-adhesions, interacts with the cytoskeleton on one side and with a matrix of proteins on the other side and enables the bidirectional transmission of information. In terms of mechanotransduction, the LINC complex participates in the mechanism by which extracellular forces generated by changes in matrix stiffness can end up regulating the nucleus and gene expression through cytoskeleton modulation [START_REF] Aureille | Mechanotransduction via the nuclear envelope: a distant reflection of the cell surface[END_REF].

The nucleus is delimited by two membranes forming the nuclear envelope. The outer nuclear membrane facing the cytoplasm is connected to the inner nuclear membrane facing the nucleoplasm through the LINC complex, which is highly conserve among species. Two transmembrane classes of proteins constitute this complex: KASH-domain proteins (also known as nesprins in mammals) span the outer nuclear membrane and SUN-domain proteins the inner one. Both types of proteins interact with each other in the perinuclear space laying inbetween the nuclear envelope membranes. Besides, nesprins interact with cytoskeletal molecules, and SUN proteins interact with lamins and few other molecules (Figure 22) [START_REF] Crisp | Coupling of the nucleus and cytoplasm: Role of the LINC complex[END_REF][START_REF] Starr | Interactions between nuclei and the cytoskeleton are mediated by SUN-KASH nuclear-envelope bridges[END_REF]. Nuclear pore complexes are channels formed by the fusion of both outer and inner nuclear envelope membranes that allow selective transport between the nucleoplasm and the cytoplasm. The nuclear lamina lays in contact with the chromatin and regulates different nuclear properties including size, stiffness and reinforcement of the nuclear envelope, as well as chromatin and gene expression [START_REF] Gruenbaum | Intermediate filaments: A dynamic network that controls cell mechanics[END_REF]. The nuclear lamina mainly includes four isoforms of lamin: two A-type lamins (A and C isoforms) encoded by the LMNA gene, and two B-type lamins (B1 and B2 isoforms) are encoded by two different genes LMNB1 and LMNB2.

Recent work showed that acute perturbations to the extracellular matrix elasticity or the actomyosin contractility can cause nuclear rupture, which leads to the leakage of DNA repair factors to the cytoplasm, resulting in increased DNA damage and cell cycle arrest [START_REF] Cho | Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest[END_REF]. Surprisingly, A-type lamin levels changed in tight coordination with tissue stiffness and cytoskeletal stress indicating a mechanosensitive role for the nuclear lamina. These results also suggested that the nuclear lamina serves as a DNA mechanoprotector increasing its structural meshwork to oppose stress-driven nuclear rupture and to retain DNA repair factors in the nucleus. Together with the nuclear-to-cytoskeletal adhesion complexes, the nuclear lamina mediates the transmission of mechanical forces between the nucleus and the cytoskeleton, where perinuclear actin builds the main network. The physical arrangement of the nucleus, the structure of the nuclear lamina, the molecular traffic across the nuclear envelope and the organization of the chromatin can be modulated through nuclear mechanotransduction [START_REF] Hamouda | Nuclear mechanotransduction in stem cells[END_REF]. A defect in the connection of cytoskeleton and nucleus could lead to dysregulation of the signaling activity and have serious impacts on cellular functions, including on stem cells differentiation [START_REF] Heo | Mechano-adaptation of the stem cell nucleus[END_REF]. How these cytoskeleton-nucleus connectors affect stem cell function remains to be studied. Accordingly, the second goal was to investigate the impact of these interacting molecules on NSCs. Strikingly, their downregulation disrupted NSC nuclear shape and affected their proliferation.

OBJECTIVES

Then, the third and final goal was to determine the cellular and molecular mechanisms through which these immunoglobulins act. I hypothesized that they could serve as adhesion complexes bringing NSCs and CG membranes together, and that this contact was required for NSC proliferation. I found that the triad of immunoglobulins is required for proper contact between NSC and CG membranes, and determined that they regulate actomyosin cytoskeleton in the CG. In turn, modulation of CG actomyosin contractility is important for proper NSC proliferation.

MATERIALS AND METHODS

Fly stocks

In this study we used several lines of the fruit fly Drosophila melanogaster. They were maintained in plastic vials with porous bungs to allow breathing. Each vial contained 10 mL of culture medium consisting of osmotic water, glucose, dry yeast, corn flour, nipagin, agar agar, and propionic acid. Each 4 weeks flies were transferred to new freshly poured vials to renew their food source. Flies were conserved at 18ºC and transferred to 25ºC for crosses and experimental procedures, unless stated otherwise. The specific fly strains used are listed below (Table 2).

Strains

Generation of fluorescently tagged lines

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system was used to insert DNA fragments encoding GFP or mKate2 at the C-or N-terminal of Dpr10 and DIPα generating the following alleles: DIPα∷GFP, Dpr10∷GFP and Dpr10∷mKate2. To direct the Cas9 endonuclease to specific DNA sites, two different and concatenated guide RNAs were cloned into a single CFD5 plasmid vector under the strong ubiquitous U6:3 promoter and were preceded by tRNAs [START_REF] Port | Augmenting CRISPR applications in Drosophila with tRNAflanked sgRNAs[END_REF].

This strategy uses the endogenous tRNA-processing system to boost the CRISPR/Cas9 system.

The oligonucleotides used for CRISPR/Cas9 target sites are shown below (Table 3).

Table 4. List of primers used for the cloning of the two homologous regions around the CRISPR/Cas9 target sites inside the plasmid vector containing the fluorescent labels.

All the cloning was done following the sequence and ligation-independent cloning (SLIC) method [START_REF] Li | Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC[END_REF]. Both reporter and guide RNA plasmid vectors were injected into vasa-Cas9 flies (BDSC reference 55821 for DIPα, 51324 for Dpr10) by BestGene®.

Larval culture and dissection

Flies were kept in cages for breeding at 25ºC and their embryos were collected on plates previously filled with 12 mL of a mix of osmotic water, grape juice, sugar, agar agar and nipagin, boiled and left to solidify. Plates were changed each 2 hours to stimulate mating. At the time of larval hatching, larvae were transferred each hour to plates filled with culture medium to stage them. Plates were kept at 25ºC except if the cross involved a GAL4-UAS system driving an RNAi line, in which case they were at 29ºC to boost the system; or a thermosensitive tubulin-GAL80 transgene, and then were at 18ºC to repress the activity for 72 hours and then transferred to 29ºC to activate the system. Larvae from each staged plate were dissected at the required timepoint. The dissection was performed in phosphate-buffered saline (PBS) with the use of a pair of forceps. Larvae were cut at the middle and an inside-out technique was used to expose the CNS while manually removing some extra tissue.

Fixation, immunostaining and imaging

CNS were fixed for 30 minutes in 4% methanol-free formaldehyde (ThermoFisher 28908) while rocking at room temperature. They were washed in PBS once and permeabilized in PBS containing 0.3% Triton (Sigma T9284) three times for 10 minutes each. CNS were incubated with blocking solution (0.3% PBS-Triton, 5% Bovine Serum Albumin (Sigma A3608), 2%

Normal Goat Serum (Abcam ab7481) for 1 hour at room temperature while rocking. Primary antibody (Table 5) dilution was prepared in blocking solution and CNS were incubated at 4°C for 16-40 hours, then washed three times with 0.3% PBS-Triton while rocking. Secondary antibodies were diluted in blocking solution and CNS were incubated at 4 °C overnight (around 18h). CNS were washed three times with 0.3% PBS-Triton while rocking and kept in PBS at 4ºC until mounted.

Mowiol mounting medium (Sigma 81381) was used to mount the CNS. All images were acquired with a confocal microscope (Zeiss LSM880) on a ZEN software, with an optimal distance between each slice of 0.38 µm and a 40x/1.3 oil immersion objective. with live imaging medium. Dissected CNS were loaded in the wells and oriented with the ventral side facing down, a lid was placed on the top of the dish. After a couple of minutes, the CNS were settled down and the sample ready to image. Live imaging was performed with a temperature-controlled environmental chamber set at 25ºC and a 40x/1.3 oil immersion objective. Images were collected at 1024x1024 pixels following time series of 60s interval.

Antigens/Dyes

Transcriptional profiling

We used Targeted DamID (Southall et al., 2013;Marshall and Brand, 2015;[START_REF] Marshall | Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing[END_REF] to profile transcription in cortex glia at different times along niche formation (T1, T2 and T3). UAS-Dam and UAS-Dam-Pol II flies were crossed to tubulin-GAL80 ts ; cyp4g15-GAL4 flies. Different tests were performed to center the expression of Dam-Pol II at the right time windows. Each time, progeny was raised at 18˚C then shifted to 29˚C at the following time: T1: 32 hours after egg laying; T2: 32 hours after larval hatching; T3: 76 hours after larval hatching.

In all cases, they were dissected 18 hours after the 29°C shift. Processing of genomic DNA and data analysis were performed as previously described (Southall et al., 2013;Marshall and Brand, 2015;[START_REF] Marshall | Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing[END_REF]. Three biological replicates were performed. 3436 genes were called with an FDR < 0.01 and categorized following a HK-means clustering into 8 groups.

Image analysis

Confocal data, both fixed and live, was processed using Icy, ImageJ and Imaris software.

GraphPad Prism software was used for all analyses and figures were assembled using Adobe Photoshop and Illustrator.

NSCs nuclear shape, mitotic index, mitotic phase distribution, NSC-CG membranes proximity and modulation of the actomyosin contractility of CG were assessed on fixed Drosophila VNCs.

The length of mitosis in NSCs and the deformation level of CG chamber while NSCs divide were calculated on live imaging data.

For the mitotic index and phase distribution, quantifications were manually done in the ventral VNC, mitotic NSCs were identified as the colocalization of Dpn (NSC transcription factor) and PH3 markers and they were classified into prophase, prometaphase, metaphase, anaphase or telophase.

To determine the NSC-CG membranes proximity, the nuclei area was segmented using surface segmentation from Imaris on the Dpn signal. The resulted nuclei mask Mnuclei was first dilated with an element of size 11x11x3 pixels generating the mask Mdilate, in order to extend the considered area to include the area corresponding to the NSCs membrane. Mnuclei was also eroded with an element of size 3x3x1 pixels to generate the mask Merode. The membrane mask Mmembrane was computed using the difference between Mdilate and Merode. Any region not containing the LexAop-myr-tomato signal was removed from the quantification. We then computed an intensity ratio of the split-GFP signal by comparing the number of pixel higher than an Otsu threshold Mdilate ROI, with the number of pixels contained in the Mmembrane ROI.

The python quantification script was based on scikit-image library [START_REF] Van Der Walt | Scikit-image: Image processing in python[END_REF].

Live imaging data was manually analyzed to count the length of NSCs mitosis (considering time 0 the first observation of clear chromatin condensation with a H2Av∷mRFP reporter) and the deformation level of the CG chamber on the plane of NSCs division.

To compare the mitotic length of our control and dpr10 knockdown conditions, we first plotted on a histogram the distribution of the NSCs division times in both groups. Overall, distributions in both groups looked highly similar except for a few outliers. Then, we tested for the existence of outliers in both groups by assuming that variation in mitotic length could be suitably captured by an exponentially modified Gaussian distribution (henceforth, exGaussian distribution). This distribution was considered flexible enough to model intermitotic times in a wide range of cells and conditions [START_REF] Tyson | Fractional proliferation: A method to deconvolve cell population dynamics from single-cell data[END_REF] with only three parameters (namely, the mean, the standard deviation as well as the exponential rate).

We sampled a particular value for each of the parameters where the probability to sample a particular combination of three was proportional to the likelihood of the corresponding distribution fitting the observed values of the control group (Metropolis-Hastings algorithm).

Based on the sampled parameter values, the number of values corresponding to the size of each group (98 for control and 162 for the dpr10 knockdown) was randomly obtained from the theoretical exGaussian distribution. The highest value was recorded in each set of simulated values (control and dpr10 knockdown group). This step was repeated 100 times (and in each group) so that 100 "highest values" are recorded for a particular set of parameter values (mean, standard deviation, rate). After repeating the procedure 20 000 times, p-values were estimated for each observed value as: the number of times the simulated "highest values" exceed the given observed value, divided by the total number of repetitions (i.e., 100 x 20,000). Each single value could be considered a significant outlier whenever a p-value < 0.05.

Statistics

All statistical analyses were performed in GraphPad Prism. On fixed data, in order to perform statistical tests on several experimental and biological replicates, each value (corresponding to one VNC) was normalized to the mean of the control condition of the corresponding replicate.

RESULTS

This section will be presented in the form of a manuscript in preparation, including abstract, introduction, results and figures. The experiments presented comprise the main work of my PhD.

Immunoglobulin-mediated adhesions regulate individual neural stem cell proliferation through control of cortical tension in the glial niche Authors

Anna Segú Cristina 1,2 , Agata Banach-Latapy 1 , Yohanns Bellaïche 3 and Pauline Spéder

Introduction

Development of a functional central nervous system (CNS) relies on the precise generation of a diversity of neurons, that will later connect to form organized networks. This neurogenic task is devolved to neural stem cells (NSCs), the multipotent progenitors able to self-renew while producing neuronal and glial progeny 1 . A tight regulation of their proliferative capacity is thus crucial to ensure the timely production of the correct number and types of neurons. Such control is first fueled by intrinsic regulators, including asymmetric division machinery, fate determinants and epigenetic marking 2-4 . In addition, NSCs integrate external cues, ranging from systemic information to local signals. A key player in both generating and modulating extrinsic regulations is the specific cellular microenvironment in which NSCs reside, the niche 5 .

The neurogenic niche is made of a diversity of cell types, including NSCs themselves, glial cells (astrocyte), resident immune cells (microglia) and blood vessels, as well as of acellular components, such as the extracellular matrix. Perhaps surprisingly, niche roles have been mostly defined in the adult, taking advantage of a more restricted perimeter 6,7 . There, the niche has been shown to provide a wealth and breadth of paracrine, soluble signals able to modulate NSC behavior, including balance between quiescence, proliferation, and differentiation. In addition to being a signaling hub, the niche is a concentrate of tight, direct interactions between cell types, arranged in an exquisite architecture. For example, NSCs can directly contacts the endothelial wall of the blood vessels, which are also covered by a meshwork of astrocytic end feet 5,7 . NSCs can also be targeted by neuronal axons, and microglia squeeze in between these cell types. Several types of classical cell-cell recognition and adhesion molecules support these interactions 8,9 . While integrins rather mediate adhesions of NSCs and niche cells to the extracellular matrix, N-cadherin-mediated adherens junctions can support interactions between NSCs or between NSCs and ependymal cells. While removing these molecules does alter NSC behavior and neurogenesis, it is unclear whether it is a result of their role as transducer of upstream signaling, or of their architectural functions, or of both 8-11 . Despite being such an eyecatching attribute of the NSC niche, how its architecture influences neurogenesis, either at single NSC level or at the population level, has been scarcely explored. In particular, whether and how the topological and mechanical inputs resulting from adhesions and tight interactions within the niche directly regulate NSC proliferation remain mysterious.

Understanding the structural contribution of cell-cell interactions and adhesion to NSC behavior in a complex and heterogeneous niche requires the ability to track and manipulate precisely, independently and in parallel both the different cell populations and the supporting molecules themselves, while keeping the three-dimensional, in vivo organization.

The Drosophila CNS has risen to be a powerful model to decipher the cellular and molecular mechanisms controlling neurogenesis, combining conservation of core cellular properties with compelling genetics and ease of manipulation 12,13 . Drosophila NSCs are born from the neuroectoderm during embryonic development, during which they proliferate actively to generate primary neurons 14,15 . They then enter a period of quiescence at the end of embryogenesis, from which they will awake at larval hatching upon nutrition. They will first grow and then re-enter the cell cycle, leading to a second neurogenic wave generating secondary neurons, which themselves will form the majority of the adult CNS.

These actively proliferating NSCs are found in a multilayered cellular niche, comprising in particular a glial subtype called the cortex glia (CG). The CG have been previously shown to be crucial for normal neurogenesis at several levels, either in physiological conditions, by enabling NSC division 16-18 and survival of newborn neurons 19 , or protecting NSC from external stresses 20,21 . Interestingly, CG form a striking architecture around the NSC population, encasing individual NSC lineage in a tailored membrane chamber while spanning the whole CNS as a network 19,22,23 and as such creating a grid-type niche pattern in the CNS (Fig. 1A). Formation of the CG structure is progressive and parallels NSC behavior. They first grow and send membranes, then enclose NSCs at the time the first NSC division takes place, and finally keep adapting to the growth of the NSC lineages 19,24 (Fig. 1B). Thus, dividing NSCs are found tightly enclosed in a close-fitting chamber made of CG membranes (Fig. 1A), implying that regulating such structure and association must be necessary to adapt to NSC deformation during division and ultimately allow proper neurogenesis.

Leveraging this model of NSC niche, we uncovered a mechanism by which adhesion between NSC and their glial niche regulates NSC division by controlling actomyosin contractility in glia. First, we identified a triad of immunoglobulins, namely DIPa, Dpr10 and Dpr6 as expressed in the NSC niche and important for its functions. We reveal that interactions between these immunoglobulins regulate membrane contacts between NSC and CG as well as cortical tension in CG. Finally, we demonstrate that immunoglobulin-mediated adhesions are required for correct NSC cycling and nuclear shape through modulation of actomyosin contractility in the CG.

Results

Transcriptional analysis of cortex glia during niche morphogenesis identifies the immunoglobulin superfamily as a regulator of NSC and CG niche features.

In order to identify potential adhesions established between NSC and CG during chamber formation, we decided to assess the dynamics of gene expression in CG along this process (Fig. 1B). To do so, we made use of the Targeted DamID (TaDa) technique 25,26 , which allows the profiling of gene expression in a specific cell type and at chosen timepoints without the need for cell isolation. Briefly, it relies on the use of a DNA adenine methyltransferase (Dam) fused to a core subunit of the RNA polymerase II (Pol II). Expression of this fusion results in adenine methylation of GATC sequences at the sites of Pol II binding, a readout of both paused and active transcription (Supp. Fig. S1A) Placing this expression under the control of a bipartite system, such as the GAL4/UAS, allows the extraction of the methylation signal for specific cells (Fig. 1C). The processed DNA is then analyzed through Next Generation Sequencing, and genes enriched for Poll II binding are called through a stereotyped pipeline 27 (see Materials and Methods for details). We thus expressed Dam-Pol II under the control of a GAL4 driver strongly expressed in the CG (cyp4g15-GAL4 19 ), choosing time windows covering each of the three main steps of chamber formation: T1, before visible nutrition-induced membrane growth;

T2, during membrane growth; and T3, at the time of NSC encasing (Supp. Fig. 1B). A first functional annotation of the genes unique to T1, T2 and T3 (Fig. 1D) revealed enrichments matching the nature of the biological events, with functions pertaining to cellular growth at T2, and guidance as well as adhesion pathways at T3. Further, statistical analysis and HK-means clustering of these data identified genes that increase, decrease or remain the same over the time period (Fig. E). Focusing on genes whose expression increases over time (classes 1 and 2) illuminated a striking enrichment in members of the immunoglobulin (Ig) superfamily (Fig. E).

Igs are cell recognition molecules that can bind to themselves or other members of the superfamily, forming homophilic and heterophilic complexes respectively 28,29 . As such they provide a frame for a diversity of cell-cell interactions, both in strength and in nature. They actually represent the most frequent extracellular domain in humans (around 3% of proteincoding genes) and have been involved in many biological processes requiring cell interaction and communication, including migration, adhesion, guidance and self-recognition. Importantly, their expression and roles have been highlighted previously in the CNS, where they are important mediators of neuronal connections and neurogenesis 30,31 .

The Drosophila Ig superfamily comprises 130 non-antibody, non-T cell receptor members and can be divided in four main subfamilies 32,33 (Fig. 1F). The DIP subfamily interacts with the Dpr subfamily, and the Beat subfamily with the Side subfamily. Similarly to mammals, several studies have pinpointed the role of Igs in the assembly of neural circuitry, by mediating the targeting and anchoring of opposing synaptic partners 34,35 . Differential expression of Ig members between neuronal cell types will generate unique combinations of cell interactions, resulting in the generation of specific synapses. We found that out of 35 Igs members expressed in CG (Fig. 1F), 26 were increasing overtime in our transcriptional dataset, pinpointing a strong and complex role of the Ig superfamily in the NSC niche. Interestingly, while several Dpr are expressed in the CG, we did not find any member of the DIP family in the CG dataset, suggesting that these partners must be mediating interactions with other cell types of the NSC niche.

We next performed a functional screen on these Ig candidates, by knocking them down specifically in the CG and scoring for any defects in either CG morphology or NSC features (Fig. 1G). We uncovered a range of phenotypes spanning all candidates tested, indicating that Ig enrichment in CG transcriptome over time was indeed revealing of a crucial and multifacetted involvement of this superfamily in niche morphogenesis and function. Amongst the candidates tested, knocking down the Dpr member dpr10 in CG resulted in a striking, nonautonomous phenotype at the level of the NSC. We decided to focus on this candidate.

Interactions between DIPa, Dpr10 and Dpr6 regulate the shape of NSC nuclei

Interestingly we observed that dpr10 knockdown in the CG did not lead to any detectable phenotype on CG morphology, while resulting in a striking alteration of nuclear shapes of NSCs (Fig. 2A). We relied on the NSC-specific transcription factor Deadpan (Dpn) as a proxy for NSC shape during interphase. Indeed, Dpn stays within the boundary of the nuclear envelope during interphase and prophase, only getting out and becoming cytoplasmic during prometaphase and throughout the mitotic steps (Supp. Fig. S2A). Using this readout, we noticed that, instead of the rounder, oblong typical form of control nuclei in interphase, NSC nuclei under dpr10 knockdown in CG displayed a range of contorted shapes, appearing multilobulated, constricted or stretched in places (Fig. 2B). Knocking dpr10 down in NSC confirmed the specificity of the non-autonomous phenotype when downregulating it in CG (Fig. 2D). An alteration in NSCs nuclear shape was confirmed in null dpr10 mutants (Supp.

Fig. S2B).

Previous biochemical as well as biological approaches 32,34,36 have shown that Dpr10 can establish heterophilic interactions with DIP-alpha (DIP-a), which itself can interact with Dpr6 as well as set up homophilic interactions with itself (Fig. 2C). We thus wondered whether the role in CG of dpr10 for the regulation of NSC nuclear shape depends on the heterophilic interactions between DIP-a and Dpr10 or Dpr6, or is independent of Dpr10 interactions with potential partners. To do so, we knocked down DIP-a and dpr6 in either CG or NSC. We found that the loss of DIP-a in NSC and the loss of dpr6 in CG mimicked dpr10 knockdown in CG, with altered nuclear shapes of NSCs in interphase while CG grid-type organization was conserved (Fig. 2A,D). Conversely, knocking down DIP-a in CG did not result in any obvious phenotypes (Fig. 2A). However, dpr6 knockdown in NSCs led to a mild alteration in NSC nuclear shape (Fig. 2D), an intriguing observation that could be explained by cis-binding in NSC, indirect regulation or trans-binding to other DIP partners (see Discussion). This potential role for Dpr6 in NSC will not be further investigated here.

All together these data show that DIP-a is required autonomously in NSCs and its partners Dpr10 or Dpr6 are required non-autonomously in CG for controlling NSC nuclear shape. This suggests that DIP-a establishes heterophilic interactions with its partners Dpr10 and Dpr6 across NSC-CG membranes, that are involved in regulating NSC features.

Loss of Ig function in the NSC niche alters NSC division

NSCs are highly proliferative cells, undergoing mitotic division almost every hour at their peak speed, in mid-L3 stage 37,38 . Considering the alterations of nuclear shapes, we wondered whether NSC division was affected, either as a consequence or a cause. We thus assayed NSC proliferation under Ig loss of function compared to control at 66 h after larval hatching (ALH66) (29°C), using the mitotic marker Phospho-Histone 3 (PH3), which stains condensed chromatin.

DIP-a knockdown in NSC and dpr10 or dpr6 knockdowns in CG resulted in a significant decrease of NSC mitotic index (Fig. 3A-D).

Interestingly, we also found a mild change in the proportion of the different mitotic steps, with a general increase in prometaphase and metaphase, often at the expense of prophase (Fig. 3E).

Notably, arrests in prometaphase and metaphase are linked to a safety mechanism, the spindle assembly checkpoint (SAC), which ensures that all chromosomes are attached and aligned, ready to separate 39 . Such arrests thus are revealing of defects in mitotic mechanisms happening prior to or at this stage. We hypothesized that loss of Ig interactions within the NSC niche affects normal mitotic machinery leading to prometaphase/metaphase arrest and delayed mitosis. Then, ultimately NSCs fail to enter division either as a result of repetitive damages or of stochastic stronger Ig downregulation. This interpretation predicted that we should be able to observe live prometaphase/metaphase arrest and mitotic delays under Ig knockdown. We recorded NSC division using a fluorescently tagged Histone2Av in a live imaging set-up (see Materials and Methods). While all NSCs analyzed in a control condition were able to complete mitosis in an average of 17 min, we noticed several examples of NSC experiencing very long metaphase ranging from 1h to, at least, 5 when dpr10 was knocked down in the CG (Fig. 3F-G and Movie 1). We wondered whether mechanisms of asymmetric division were also affected in these conditions. We assessed the localization of the basal determinant Miranda, an adapter protein required for proper asymmetric segregation of fate determinants during mitotis 3,40 , and did not observe differences under dpr10 knockdown in CG compared to a control condition (Supp. Fig. 3A).

Taken together, these results demonstrate that mitotic progression is affected under Ig knockdown in the NSC niche, leading to long arrests in prometaphase/metaphase and to a decreased ability to re-enter the cell cycle.

DIP-a, Dpr10 and Dpr6 are expressed in the NSC niche

To understand how Ig-related proteins could affect NSC cycling, we first assessed their localization in the NSC niche. Interestingly, the DIP-a and Dpr10 or Dpr6 interacting triad has been previously implicated in CNS development in the Drosophila larva. In particular, heterophilic interactions between DIP-a and Dpr10/Dpr6 are crucial for the development of the visual system, by allowing neuronal targeting and synaptic pairing in specific layers 36 . DIP-a and Dpr10 also interact at the neuro-muscular junction, where they are expressed in the motor neuron and muscle sides respectively, and are required for proper muscle innervation 41,42 . While expressions and localizations of Dpr10, Dpr6 and DIP-a were thus observed in neuronal bodies and processes 35,36 , there was no report of their presence in CG or NSCs.

First, we took advantage of knock-in GAL4 driver lines under the control of Dpr10, Dpr6 and DIP-a enhancers 36 to drive nuclear and membrane reporters. Using this approach, we first were able to detect a clear and expected expression in neurons for all three members (Fig. 4A and Supp. Fig. S4A). We also observed, in a much fainter fashion, the characteristic pattern of CG network around NSCs with both Dpr10 and Dpr6-GAL4 lines (Fig. 4A). Conversely, we could not detect any signals in the NSC nor CG with the DIP-a driver line (Supp. Fig. S4A).

Nevertheless, driving under specific enhancers might not recapitulate the whole or exact expression pattern. We then took advantage of an antibody generated against DIP-α and we were able to detect a faint staining in the whole CNS, including along a marker for the NSC membrane, which was lost in a null DIP-α mutant condition 36 (Fig. 4B).

To be able to directly visualize the proteins themselves as well as bypass potential limitations of enhancer or antibodies approaches, we then decided to create knock-in fluorescent fusions through CRISPR engineering (see Materials and Methods). Dpr10 and DIP-a were tagged with mKate2 in N-terminal or GFP in C-terminal. Using a live imaging approach, we were first able to visualize Dpr10∷GFP and DIP-a∷GFP and in neurons as reported before 36 (Fig. 4C,E). In addition, we detected a punctuated dynamic mKate2∷Dpr10 signal along the CG membrane and in the CG cytoplasm (Fig. 4D and Movie 2). Similarly, we observed DIP-α dots along the NSC membrane (Fig. 4E and Movie 3). While these expressions matched the expected cell types, such speckled subcellular localizations were unexpected compared to what was reported and what we saw in neurons and suggested a differential localization depending on their specific function in these cell types. Next, we wondered whether these two interacting partners could be found localized next to each other. Combining mKate2∷Dpr10 with DIP-a∷GFP revealed some colocalizing dots (Supp. Fig. S4B) at the confocal resolution, suggesting that they might be acting in close-by localizations in CG and NSC, respectively.

To conclude, these different approaches show that Dpr10 and Dpr6 are expressed in the CG while DIP-a is expressed in NSCs, in accordance with the RNAi phenotype (Fig. 2).

Immunoglobulin-mediated adhesions are required for membrane contact between the

NSC and CG

With interacting and binding Ig partners expressed in the membrane of different cell types, we postulated that they could serve as adhesion complexes between NSCs and CG, bringing their membranes close together. The GFP Reconstitution Across Synaptic Partners (GRASP) system 43 relies on the expression in supposed cellular partners of two complementary fragments of GFP (Split-GFP 44 ) bound to the extracellular domain of transmembrane proteins (Fig. 5A).

When these Split GFP fragments come close enough, GFP fluorescence will be restored. As such, the GRASP system is a spatial readout of membrane proximity, and likely contact, between two cells. We took advantage of the existence of parallel genetic binary systems in Drosophila (GAL4/UAS and LexO/LexAOp) to express one GFP fragment in the CG and the complementary other in NSCs (Fig. 5A, see Materials and Methods). In control condition, we were able to detect a continuous and mostly uniform GFP signal overlapping with a reporter for NSC membrane (Fig. 5B). Conversely, the GFP signal under dpr10 knockdown in CG appeared discontinuous and heterogenous compared to the marker for NSC membrane. We quantified NSC-CG proximity by measuring the ratios between the GFP signal and the NSC membrane across the entire NSC population in both conditions, and found that it was indeed decreased when dpr10 was knockdown in the CG (Fig. 5C). These results show that Igmediated interactions within the niche are required for proper contact between NSC and CG membranes.

To address the relationship between loss of cell contact and impairment in NSC division, we tested whether Ig interactions were important for physically allowing mitosis. Indeed, when NSC division takes place, the CG membrane deforms to accommodate for NSC elongation along the spindle axis, as visualized through live imaging of NSC and CG membranes using population-specific transmembrane proteins fused to fluorophores (Supp.

Regulation of CG cortical tension by Ig interactions is required for proper NSC proliferation

Adhesion molecules mechanically link the cells they bring together across the extracellular space. In addition, they are classically connected on the intracellular side to each cell's architecture through interactions with cytoskeletal components. As such they behave as mechanotransducers that can propagate changes in cell structural and mechanical properties between the two connected cells 45-47 . Moreover, cell-cell adhesion not only transmits but can also generate and modulate mechanical cues themselves 48 . This reciprocal function is supported by several molecular mechanisms, including recruitment and nucleation of cytoskeletal complexes, and leads to the formation of localized, junctional tension at the site of interaction.

A major scaffolding component associated with cell adhesions is the cortical actomyosin network. The actomyosin network is composed by actin, polymerized into filaments, and by the non-muscular myosin II, a molecular motor that produces contractile force 49 . Classically, myosin II converts chemical energy, by ATP hydrolysis, into directed movement and generation of pulling forces on anti-parallel actin filaments 50 . This ultimately results in contraction of the cortical actin network, generating tension. Myosin II function can be regulated through activating phosphorylation of its regulatory light chain by a number of kinases, including RhoA and ROCK. Activated forms of such kinases will thus promote Myosin II activity, hence cortical tension, whereas conversely their inhibition will disrupt Myosin II function and decrease cortical tension. In Drosophila, a similar role and regulation of myosin II has been described

depending on Rho1 and Rok, the fly equivalents of RhoA and ROCK respectively (Fig 6A).

We wondered what the level of interplay between Ig-mediated interaction and cortical tension was, and whether a change in Ig interaction could directly impinge on cortical tension. We first assessed whether loss of Ig interactions in the NSC niche altered myosin II expression pattern and localization. Using a ubiquitous reporter, we could not detect any difference (Supp. Fig. S6A). However, as discriminating between different cellular pools is difficult in this context, it might mask a mild effect. We thus decided to test whether modulating cortical tension in the CG, either by increasing or decreasing myosin II activity would mimic the phenotype seen under loss of Ig interactions in the NSC niche (Fig. 2 and3). To do so, we expressed a dominant-negative form of Rho1 (Rho1 DN ) or a constitutively active form of Rok (Rok CA ) in CG to decrease or increase cortical tension in these cells, respectively. We did not detect any changes in nuclear shape, our first visual readout for Ig loss-of-function phenotype, when

Rho1 DN was expressed in the CG (Fig. 6B). However, to our surprise, we found that expressing Rok CA in CG resulted in alterations in the NSCs nuclear shape (Fig 6C), and in an increased mitotic index with a prominent shift to prometaphase/metaphase (Fig. 6D-E). These changes in NSC parameters are reminiscent of dpr10 non-autonomous phenotype, albeit at an earlier stage with NSCs lingering in prometaphase/metaphase rather than having exited the cell cycle. These results suggest that loss of Ig interaction within the NSC niche might result in increased cortical tension in the CG. To confirm this finding, we next tested whether decreasing cortical tension in CG would rescue the NSC phenotype associated with loss of Ig interactions within the NSC niche. To do so, we expressed Rho1 DN specifically in the CG in a dpr10 mutant background. In these conditions, we observed a striking rescue of both NSC mitotic index and nuclear shape (Fig. 6F andG). All together, these data demonstrate that Ig-mediated adhesion/interaction 305 between NSC and CG regulate cortical tension in the latter. 306 morphology (See Results Figure 1G). Amongst the candidates tested, the knockdown of the dpr10 immunoglobulin in CG produced an intriguing non-autonomous phenotype in NSCs.

Earlier findings stated that Dpr10 exhibited heterophilic interactions with DIP-α and that Dpr6 was the direct competitor of Dpr10 for the binding to DIP-α (Özkan et al., 2013). Hence, I concentrated my research project on this interacting triad of immunoglobulins. demonstrate that, in addition to NSC nuclear shape, immunoglobulin knockdown in the niche affects mitotic progression, transiently arresting cells in prometaphase-metaphase stages and reducing their ability to cycle again. The downregulation of dpr10 in CG creates a rare evidence in the NSC niche of a cell non-autonomous phenotype affecting NSC function.

Immunoglobulin interactions control NSC features

The interacting triad of immunoglobulins is expressed in the niche

To understand where the interactions between these immunoglobulins are taking place, I checked for their expression in the NSC niche. While neuronal expression of the same interacting triad had been previously described (Tan et al., 2015), no expression of these immunoglobulins had been reported in NSCs nor in CG. With transcription (GAL4) trap lines of Dpr10, DIP-α and Dpr6 I also detected a clear expression in neurons for the three. In addition, I observed a weak expression of Dpr10 and Dpr6 around NSCs resembling the CG network pattern (See Results Figure 4A). Nevertheless, DIP-α signal outside neurons was not detectable.

To some extent, these observations could be due to the enhancer trap driver not reproducing the whole expression pattern of the protein. Then, with an available antibody for DIP-α we noticed a weak dotty staining along NSC membrane, which was lost in DIP-α null larvae (See Results

Immunoglobulins mediate adhesion between NSC and CG membranes

Considering the roles of these immunoglobulins in cell-cell interactions, I proposed that defects in NSC parameters could result from a defective adhesion between NSC and CG membranes.

Therefore, I investigated whether the loss of immunoglobulins affected the contact between NSC and CG membranes. Using a Split-GFP approach I observed that whilst the control condition displayed a rather uniform GFP signal along the interphase of NSC and CG membranes, dpr10 downregulation in CG showed a discontinuous GFP contour. This data indicated that NSC and CG membranes relay on DIP-α binding to Dpr10 to maintain their adhesion. I quantified NSC and CG membrane proximity and observed a decrease in the GFP signal (See Results Figure 5B-C). The addition of another replicate is required to increase the N and potentially obtain a significative decrease comparing dpr10 knockdown to control conditions. Improving of the methodology used to calculate the signal could also be considered.

Then, I hypothesized that the lack of such physical interaction could potentially prevent CG membranes to adequately deform in response to NSC division, in return impacting on NSC cell cycle progression. Using a live imaging approach, I measured the length of CG membrane deformation along the NSC spindle axis under dpr10 knockdown in CG and control conditions, but we found no difference between them. Taken together, these data indicate that immunoglobulin-mediated interactions are necessary for proper contact between NSC and CG membranes, though dispensable for CG membrane deformation around cycling NSCs.

Subcellular location and structure of the interacting triad

Tracking the specific subcellular location of these immunoglobulins has been a real challenge. I tried to validate the expression pattern of these immunoglobulins in either CG or NSCs with a variety of tools. I started using available GFP trap lines made with the insertion of the transposon Minos-Mediated Integration Cassette to assess their expression in the niche. As previously reported, I was able to observe a well-defined expression in neurons and I also noticed a very faint reminiscent pattern of CG network. Then, I combined those lines with specific cell-type driven RNAi of the proteins to see whether I could see a disappearance of the GFP expression at the level of NSC or CG in fixed samples. Unfortunately, I was unable to appreciate a clear difference between the RNAi and the control conditions. I also used specific

Supplemental Movies S2, S3).

Live-imaging show labile dots displayed not only along the membrane (although mostly) but also inside the cells. Could the cytoplasmic expression correspond to cargo proteins in their way to the membrane? Protein translation begins in the cytosol. There, different molecular tags guide the protein shipment to their final destination. Proteins targeted to the plasma membrane or to the extracellular space are sorted into vesicles along their transport. Assessing potential co-localization between these immunoglobulins and staining for endosomal vesicles could allow us to understand their cytoplasmic expression.

A situation could be imagined in which these immunoglobulins would be present in membrane organelles like the nucleus. Full-length transmembrane cell surface proteins, as well as proteolytic fragments of L1 immunoglobulins, have already been detected in the nucleus after translocation from the plasma membrane in both mammals and Drosophila [START_REF] Planque | Nuclear trafficking of secreted factors and cell-surface receptors: New pathways to regulate cell proliferation and differentiation, and involvement in cancers[END_REF][START_REF] Lutz | Generation and nuclear translocation of sumoylated transmembrane fragment of cell adhesion molecule L1[END_REF][START_REF] Kakad | An ankyrin-binding motif regulates nuclear levels of L1-type neuroglian and expression of the oncogene Myc in Drosophila neurons[END_REF]. This could be an alternative explanation of the internal presence of Dpr10 and DIP-α in case they were specifically in the nucleus, something that should be verified before.

Interestingly, the molecular structure of these proteins has been modelized before although without reaching a full consensus. Dpr10 and Dpr6 have been predicted as either membraneattached (transmembrane or glycosyl phosphatidylinositol(gpi)-anchored) or secreted proteins and DIP-α as a transmembrane one (Özkan et al., 2013;[START_REF] Cheng | Molecular basis of synaptic specificity by immunoglobulin superfamily receptors in Drosophila[END_REF]. Further characterization of their specific domains is required. A co-localization experiment between these immunoglobulins and a marker for gpi-anchoring sites could also give us an insight of their structure [START_REF] Brankatschk | Lipoprotein particles cross the blood-brain barrier in Drosophila[END_REF].

Relaying signaling through the plasma membrane

Previous research reported that both the Dpr and DIP subfamilies lack intracellular regions [START_REF] Cheng | Family of neural wiring receptors in bilaterians defined by phylogenetic, biochemical, and structural evidence[END_REF]. Therefore, these immunoglobulins may require the binding to coreceptors or the presence of secondary messengers to relay signals to the cytoplasm. Since many immunoglobulins bind to integrins, we could theorize integrins as cellular co-receptors for Dpr10, DIP-α, or Dpr6, but it has never been proven. Nevertheless, interactions with the cytoskeleton have been shown for immunoglobulins without intracellular domains linked to the plasma membrane, through GPI-anchoring (Leshchyns'ka et al., 2003;[START_REF] Chen | The transmembrane protein CBP plays a role in transiently anchoring small clusters of Thy-1, a GPI-anchored protein, to the cytoskeleton[END_REF].

Further experiments proving that Dpr10 and Dpr6 are GPI-anchored proteins would strengthen the hypothesis that these immunoglobulins relay on lipids to transduce signaling to the cytoskeleton and vice versa.

Since these molecules seem to be dynamically expressed in the membrane, I suggest that their role is to mediate adhesion between CG and NSC plasma membranes bringing them together at specific times and regions. Accordingly, changes of the adhesion in the intercellular contacts can also be sensed by mechanosensors at the plasma membrane, such integrins or ion channels [START_REF] Wu | Touch, Tension, and Transduction -The Function and Regulation of Piezo Ion Channels[END_REF], which then transmit the signal internally; or by other mechanosensors inside the cells, like the nucleus, able to translate spatial constrains into actomyosin contractility [START_REF] Lomakin | The nucleus acts as a ruler tailoring cell responses to spatial constraints[END_REF][START_REF] Venturini | The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior[END_REF]. With the idea that contractile forces created in CG could be responsible of the NSC phenotype, we started looking at the expression of myosin II (See Results Figure S6), a major player in the contractility of the cell cortex. However, due to the close proximity of these membranes we were unable to differentiate the pool of myosin II coming from the CG from the one of NSCs. To overcome this issue, we could try to make GFP/mRFP clones of myosin II [START_REF] Herszterg | Interplay between the Dividing Cell and Its Neighbors Regulates Adherens Junction Formation during Cytokinesis in Epithelial Tissue[END_REF] and focus on the interplay of those clones to distinguish the myosin II contribution of NSC from the one of CG.

Another interesting aspect that could be determined is whether these immunoglobulins provide a stable adhesion or rather transient. I began developing genetic combinations to perform live imaging experiments with markers of both NSC and CG membranes, with the goal of shedding light on the coordination of these plasma membranes (See Results Supplemental Movie S4). In particular, my aim it to understand how NSC and CG membranes behave under the loss of Dpr10. In addition, I wonder whether the NSC nuclear envelope and its plasma membrane follow the same axis of deformation during mitosis. Hence, in the future I plan to track both at the same time when dpr10 is downregulated.

The dynamic expression of CRISPR constructs is also an interesting observation that could be taken as a hint to understand how these molecules act. One could speculate that they exhibit lateral mobility and polarize to specific regions of the membrane around cycling NSCs, thus being necessary to reinforce adhesion at concrete points and moments of the mitotic process.

In a way, we could imagine that the perturbation of the binding triad could cause a relocation of these immunoglobulins elsewhere than where they are required, maybe even binding to other partners. Then, when NSCs try to divide, the new adhesion created or the lack of it impairs their proper mitosis.

The immunoglobulin subfamily of DIPs and Dpr interact with each other through common binding domains. Various Dprs proteins can bind to the same DIP, albeit with different affinities. These diverse affinities may be exploited by developing neurons to specifically shape neural circuitry [START_REF] Cheng | Molecular basis of synaptic specificity by immunoglobulin superfamily receptors in Drosophila[END_REF]. Some DIPs form homodimers adding another layer of complexity to the regulation of these immunoglobulin interactions. For instance, DIP-α homodimerization is weaker than its heterophilic binding to its Dpr partners, Dpr10 and Dpr6. Indeed, genetic rescue experiments showed that, in some contexts, homophilic bindings could substitute for heterophilic interactions (Xu et al., 2018). This binding promiscuity may be a source of functional redundancy. In our model, redundancy between Dpr10 and Dpr6 should be considered, as well as with other Dpr and DIP partners (see Results

Signal transduction from the cytoskeleton to the nucleus

I have discovered that these immunoglobulins mediate adhesion between the membrane of CG and NSC, on one side influencing cortical tension in the glial niche cells, and, on the other side, regulating NSC nuclear shape and proliferation, respectively. Both mechanisms seem correlated through the binding of these immunoglobulins. Now, further studies need to be conducted to determine how the cortical tension in CG, and its associated cytoskeletal remodeling, impacts on NSC nuclei.

It is tempting to imagine that the LINC complex may be implicated in the process. This conserved complex forms a bridge allowing direct transmission of mechanical cues from the cytoskeleton to the nucleus and vice versa. As discussed before, mechanotransduction of external forces to the cytoplasm depends on mechanosensors like integrins or ion channels.

Once the signal is in the cytoplasm, LINC complex and other nuclear envelope associated proteins play an important role to transduce to the nucleus, impacting on chromatin conformation, histone modifications and gene expression, among others. In Drosophila, the LINC complex is formed of Klarsicht, the equivalent KASH-domain commonly known as nesprins in mammals, and Klaroid, the vertebrate counterpart of SUN-domain proteins. It would be interesting to assess whether a change in the adhesion between CG and NSC membranes could be relayed to the nucleus through the LINC complex. For that, a first experiment could be directed to investigate whether this complex can control NSCs features (nuclear shape and proliferation) per se using RNAi lines or mutants of the components of the LINC to check for defects in NSCs parameters. Another idea would be to combine these RNAi or mutant lines with the knockdown of DIP-α or dpr10 checking for a potential rescue of the phenotype associated with the loss of function of these immunoglobulins. The reasoning behind the last suggestion is that when cells experience tension, the nucleus undergoes significant morphological changes due to its connection with the cytoskeleton that transmits mechanical stress to the nuclear envelope [START_REF] Maniotis | Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure[END_REF][START_REF] Khatau | A perinuclear actin cap regulates nuclear shape[END_REF]. Thus, if the change of mechanical forces, generated with the loss of interaction between the immunoglobulins, is transmitted through the LINC complex into the nucleus, disrupting one component of this complex could uncouple, to some extent, the cytoskeleton-nucleoskeleton link and help avoiding NSC nuclear disruption under a mechanical change.

Another protein that could have a role in relaying the information to the nucleus is Moesin (the unique Drosophila protein of the Ezrin, Radixin and Moesin family). This protein can be at the interplay between microtubules and the cytoskeleton in mitosis [START_REF] Solinet | The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex[END_REF]. Moesin regulates cell shape changes during cell division [START_REF] Carreno | Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells[END_REF][START_REF] Kunda | Moesin Controls Cortical Rigidity, Cell Rounding, and Spindle Morphogenesis during Mitosis[END_REF]. Activated Moesin spreads around the cortical cytoskeleton during prometaphase and helps to increase rigidity, which contributes to metaphase cell rounding. Moesin-microtubule interaction regulate spindle organization in metaphase and mediates the coordination between mitotic spindle and actin cortex [START_REF] Solinet | The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex[END_REF]. Redistribution of active Moesin at the equatorial plane of the cell controls cell elongation and subsequent cytokinesis [START_REF] Roubinet | Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis[END_REF]. A change in the organization of the NSC cytoskeleton could be relayed to the mitotic spindle and disrupt the proper process of mitosis. Like the experiment proposed with the LINC components, moesin downregulation could provide insight on its contribution.

Another possibility to link changes in cortical and cytoskeletal properties to nuclear features would be that biochemical molecules shuttling through the nuclear pore complex between the cytoplasm and the nucleus may contribute to regulate NSC nuclei in response to a mechanical signal [START_REF] Elosegui-Artola | Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores[END_REF].

Reasoning behind nuclear shape abnormalities and mitotic defects

The observation of abnormal NSC nuclear shapes was intriguing. Despite having so far only assessed the phenotype qualitatively, we have established a collaboration with the Imaging Hub of Institut Pasteur to get a quantitative analysis of it. Nuclear segmentation accurate enough to allow precise quantification of geometric properties has been a challenge, though we expect that ongoing research may overcome it.

One explanation under this strange nuclear morphology could be a problem in the nuclear lamina. This meshwork with mechanical properties has been shown to protect the nucleus from external physical disturbances mediating the assembly of perinuclear actin fibers in other systems [START_REF] Kim | Nuclear lamin A/C harnesses the perinuclear apical actin cables to protect nuclear morphology[END_REF]. The mediation is through the LINC complex, which bridges many cytoskeletal structures to the nuclear lamina. Similar to mammalian species where there are two types of lamin, A-types and B-types, flies also have two types, lamin C (equivalent to A-type lamins) and lamin Dm0 (equivalent to B-type lamins) [START_REF] Melcer | Invertebrate lamins[END_REF].

Besides, these intermediate filaments also play important roles in the morphology of the nucleus in Drosophila [START_REF] Petrovsky | Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes[END_REF]. As a future perspective, I propose to study the properties of the overall nuclear envelope, including the lining meshwork of lamins.

Using nuclear lamin markers, we could try to document the behavior of this protein layer throughout the cell cycle. Since Drosophila NSC undergo a semi-closed mitosis, the nuclear envelop remains more or less intact. B-type lamins become more permeable in mitotic stages [START_REF] Roubinet | Asymmetric nuclear division of neural stem cells contributes to the formation of sibling nuclei with different identities[END_REF]. Any alteration of behavior of the nuclear envelop under loss of immunoglobulin-mediated adhesion would open a new line of research where we could broaden the characterization of its properties. Are lamins not dissociating as much as they should, leading to an increase in the rigidity of the nucleus? Is the whole meshwork in coordination with the movements of the mitotic spindle?

In addition, an increase of DNA damage could also suggest a problem in the nuclear lamina. Indeed, A-type lamins act as mechanosensors to protect against DNA damage [START_REF] Cho | Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest[END_REF]. Assessing DNA damage with a double-strand break marker would be interesting to assess a potential hint on a lamina issue. Furthermore, a DNA fluorescence in situ hybridization approach in NSCs could be used to estimate polyploidy. One possibility is to speculate that NSCs fail to finish mitosis after a prolongated metaphase, hence exiting mitosis without chromosome segregation nor cytokinesis and becoming polyploid. In addition, the observation of an increased DNA damage may help to support this theory, as it has been recently reported that DNA damage occurs in nuclei that are not ready to enter mitosis but are force to do so [START_REF] Nano | Cell-Cycle Asynchrony Generates DNA Damage at Mitotic Entry in Polyploid Cells[END_REF]. Interestingly, I was occasionally able to observe abnormal mitoses with lagging chromosomes or multiple poles of them, reminiscent of multipolar mitosis. Additional research staining for the mitotic spindle should be considered. A possible explanation concerning transient mitotic arrests could be a defective kinetochore attachment to the spindle microtubules. These defects are responsible of arrested metaphases through the activation of the spindle assembly checkpoint (SAC). This mitotic machinery is a surveillance mechanism that coordinates cell cycle progression with the state of chromosomal attachment to the mitotic spindle. It prevents cells from entering anaphase when chromosomes are not properly attached (Musacchio, 2015). Based on this fact, I could hypothesize that the loss of immunoglobulin interaction within the NSC niche leads to improper attachment of spindle microtubules to chromosomal kinetochores, resulting in activation of the SAC. The underlying mechanism at the level of microtubule attachment remain unclear. In this context, it would be interesting to try to rescue the transient metaphase arrest with mutants or RNAi lines of the SAC pathway, such as bub3 [START_REF] Lopes | The Drosophila Bub3 protein is required for the mitotic checkpoint and for normal accumulation of cyclins during G2 and early stages of mitosis[END_REF]. Another experiment could be to check whether cyclin B is properly being degraded in NSCs under dpr10 knockdown since its accumulation would also indicate a SAC problem.

Despite the strong resemblance of the NSC nuclear shape phenotype obtained through the myosin II upregulation to the non-autonomous phenotype of dpr10 knockdown, the mitotic index was unexpectedly augmented instead of reduced. I propose a hypothesis in which the mitotic index is dependent on the timeline of the phenotype. Earlier timepoints may show a high number of NSCs proliferating due to multiple arrests in prometaphase-metaphase. Then, NSCs might accumulate damages during these abnormal cycles, what may compromise their ability to undergo another mitosis. This would eventually result in a lower mitotic index at later timepoints. Future experiments with replicates of dpr10 knockdown and myosin II upregulation at earlier or later timepoints, respectively, would allow us to evaluate the validity of this explanation.

An intriguing question remains regarding the causal relationship between the nuclear disruption and the altered cell cycle progression observed in NSCs. While we have shown that the downregulation of this triad of immunoglobulins in the niche leads to both phenotypes, it will be interesting to determine which one precedes the other one. Either aberrant nuclear shapes lead to problems in mitosis, either the other way around. Assessing the causality of these two phenotypes may not be very straightforward. Still, we could try to mimic mitotic arrests with the help of mutants or RNAi lines of the SAC effectors and observe whether similar nuclear aberrations are present.

Global picture

A tight control of NSC proliferative potential is essential for proper CNS development. The niche is an essential player in such control by locally concentrating specific cells and secreted signaling molecules. These extracellular stimuli act together with intrinsic programs to influence NSC development and behavior [START_REF] Bjornsson | It takes a village: Constructing the neurogenic niche[END_REF]. The specificities of NSC interactions with their surrounding environment remain unclear. Drosophila is a powerful and practical model to address these questions. First, the NSC niche comprise similar players to the mammalian microenvironment, including glial cells. In particular, the CG has been shown to perform genuine niche function and is very tightly associated with NSCs. In addition, specific markers exist to track the different populations in the niche. Moreover, the existence of several binary genetic systems allows the independent genetic targeting of the different cell populations.

Although the crucial role of niche cells on NSCs is now unquestionable, their implications have mostly been related to the secretion of factors that regulate NSC proliferation, self-renewal and differentiation. Here we present a novel mechanism by which physical interactions between niche glial cells and NSCs modulates the proliferation of the latter. A triad of binding immunoglobulins serve as bridges between extrinsic signals from neighboring CG cells and intracellular signaling in the NSC. In our Drosophila system, individual NSCs lineages are in direct contact with CG membrane whereas in mammals NSCs are mainly surrounded by their progeny. However, direct contact between mammalian astrocytes and NSCs has been suggested to be important to promote neurogenesis [START_REF] Lim | Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis[END_REF][START_REF] Ashton | Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling[END_REF].

Fascinating 3D reconstructions from electron microscopy images of the adult NSC SGZ showed that astrocytic processes form adhesion points with NSC processes at the surface of blood vessels [START_REF] Moss | Fine processes of Nestin-GFP-positive radial glia-like stem cells in the adult dentate gyrus ensheathe local synapses and vasculature[END_REF]. Unraveling intercellular interactions in the Drosophila melanogaster NSC niche model will provide new concepts on the diversity of roles performed by niche adhesions, that could help addressing what happens in the niche of more complex organisms such as mammals.

Whilst the focus of this thesis was to assess the dynamics of interaction between niche glial cells and NSCs, our data also offer insights into the impact of niche cortical tension in NSCs.

They also provide a novel framework to understand how (immunoglobulin-mediated) adhesion modulates NSC division. Finally, we may have uncovered new exploitable models to study delayed metaphase-to-anaphase transitions in Drosophila NSCs and mechanotransduction between niche cells.

LIST OF FIGURES

LIST OF TABLES

1. 2 .

 2 Immunoglobulin interactions control NSC features ...............................................108 1.3. The interacting triad of immunoglobulins is expressed in the niche .......................108 1.4. Immunoglobulins mediate adhesion between NSC and CG membranes ................109 1.5. Cortical cytoskeleton modulation through immunoglobulin interaction regulates NSC proliferation ............................................................................................................110 2. Subcellular location and structure of the interacting triad......................................111 3. Relaying signaling through the plasma membrane ..................................................112 4. Signal transduction from the cytoskeleton to the nucleus ........................................114 5. Reasoning behind nuclear shape abnormalities and mitotic defects .......................115 6. Global picture ............................................................................................................117 LIST OF FIGURES ..........................................................................................................119 LIST OF MOVIES ...........................................................................................................121 LIST OF TABLES ............................................................................................................122 REFERENCES .................................................................................................................123 ABSTRACT ....................................................................................................................

  classified according to their potential to proliferate and differentiate. First, there are totipotent stem cells able to generate all cell types of an organism, including germ cells and extra-embryonic tissues. The only totipotent stem cells in the development of an organism are the fertilized egg and the cells produced in the first few divisions of it. The descendants of those cells are pluripotent stem cells able to generate a variety of cell types within different lineages. Embryonic stem cells derived from the pre-implanted embryo are pluripotent stem cells and give rise to all the derivatives of the three embryonic germ layers: ectoderm, mesoderm, and endoderm. Within each germ layer, multipotent stem cells are reservoirs of undifferentiated cells that divide to generate cell types belonging to a common specific lineage. These cells can be found among differentiated cells in specialized niches even in adulthood. With successive divisions, cells lose their potential and go through the last pathways of differentiation to become specialized. When a cell's potential has been completely

  Continuous maintenance and adaptation of the brain physiology is also crucial to face neuroinflammation triggered by an acute infection or a chronic diseases, which ultimately alter neurogenesis (Pérez-Domínguez, Tovar-Y-Romo and Zepeda, 2017). Many of those systemic signals are relayed to NSCs through the local interactions with the niche populations. These different mechanisms are coordinated to regulate the development, maintenance, self-renewal and differentiation of stem cells. Hence, to achieve CNS homeostasis, NSCs react to both systemic, local and internal changes, many of which are mediated by the cells constituting the microenvironment in which NSCs reside.

  cell, integrins indirectly associate to actin filaments forming focal contacts, or to intermediate filaments forming hemidesmosomes. These bindings depend on a mixture of proteins, which include the adaptor Talin in many cases. Integrins undergo structural conformational changes, which reflect their ligand binding affinity, hence, their states of activity. This binding affinity is regulated by a range of external (e.g. divalent cations) or internal (e.g. GTPases) stimuli coordinating in function of the cell needs (Van der Flier and Sonnenberg, 2001). Integrins convey signals in both directions across the plasma membrane. For instance, they can sense mechanical cues arising from the extracellular matrix and transmit the stimuli to downstream signaling modulating the cell[START_REF] Kechagia | Integrins as biomechanical sensors of the microenvironment[END_REF]. Similar to the other cell adhesion molecules, integrins operate in clusters to increase the affinity of their binding to the extracellular matrix proteins. In few cases, integrins can bind to specific immunoglobulins and mediate cell to cell interactions. Humans contain 24 types of integrins whereas Drosophila 10 types[START_REF] Bulgakova | Cell adhesion in Drosophila: versatility of cadherin and integrin complexes during development[END_REF].

  intermediate progenitors will produce neurons and glial cells. At mid larval stages, CG will end up enwrapping NSCs lineages. Hence, larval NSCs reside in direct contact with CG on their apical and lateral sides. Drosophila E-cadherin-mediated adherens junctions are present in this NSC-CG interphase and also at a variable and transient level within the NSC lineage. This adhesion to glial cells has been proposed to promote NSC proliferation[START_REF] Dumstrei | Role of DE-cadherin in neuroblast proliferation, neural morphogenesis, and axon tract formation in Drosophila larval brain development[END_REF], though we think further experiments are required to characterize it. During neuronal differentiation, cell adhesion mediated by the Drosophila N-cadherin is necessary for axonal guidance and synaptogenesis[START_REF] Kurusu | Developmental changes in expression, subcellular distribution, and function of Drosophila N-cadherin, guided by a cell-intrinsic program during neuronal differentiation[END_REF].

Figure 19 .

 19 Figure 19. Drosophila NSCs delaminate via a progressive loss of apical cell-cell junctions.

For

  proper CNS development and function, neural stem cell (NSC) proliferation must be strictly regulated. The microcellular environment that surrounds them play an important role in this process. NSCs reside in tailored niches with which they necessarily communicate. The diversity of contacts and interactions between NSCs and their neighboring cells are key to maintain their stemness and to control the mode and timing of their divisions. Despite the great interest of understanding the extent of this exchange of signals in the niche, the molecular mechanisms mediating the different interactions and their impact on neurogenesis are still poorly explored. This PhD project is part of a relevant research line in Dr. Pauline Spéder's lab that aims to decipher the dynamics of interaction between NSCs and niche cells during CNS development.To address this matter, we take advantage of the Drosophila melanogaster post-embryonic, late larval CNS, where each NSC and their progeny are individually enwrapped by cortex glia (CG). This precise encasing of NSC lineages in CG chambers pinpoints tightly controlled contacts between NSCs and CG. The purpose of this PhD research was to use this morphogenetic event as a model to elucidate cellular processes that support the interactions between NSCs and niche cells, as well as and their importance for NSCs.My first goal was to identify novel regulators of the NSCs niche. Previous work from the Spéder lab yielded a transcriptomic dataset of genes expressed in CG chamber morphogenesis. An enrichment of the members of the immunoglobulin superfamily appeared remarkable. I started this project screening multiple immunoglobulin genes potentially involved in the NSC niche formation. From these results, I retained an interacting triad of immunoglobulins and used it as a proxy to study how this precise architecture that CG build around NSCs could influence their function.

Table 3 .

 3 List of oligonucleotides used as guide RNAs to target Cas9 endonuclease.CRISPR/Cas9-mediated homologous recombination was used to insert the fluorescent labels at the specific nucleated sites. Homologous sequences of 1 kb flanking the CRISPR/Cas9 target sites were stacked into plasmid vectors harboring the C-or N-terminal GFP or mKate2 sequence and a hsp70∷miniwhite gene flanked by two loxP sites allowing the removal of the miniwhite gene in the final strain by Cre recombinase. The primers used to clone the homologous regions inside the plasmid vector containing the fluorescent tags are

  Statistical tests were then run on all the normalized values from all replicates. For most experiments, at least two biologicals replicated were performed and analyzed qualitatively for phenotype reproducibility. One biological replicate was quantified for the mitotic index, phase distribution, NSC-CG membrane proximity and modulation of the actomyosin contractility of CG. All fixed data were represented with box and whiskers plots, except for the phase distribution experiments that were displayed in stacked bars. All box and whiskers plots display minimal value (bottom whisker), first quartile (25th percentile, lower limit of the box), a median of the interquartile range (middle horizontal line), third quartile (75th percentile, the upper limit of the box) and maximal value (top whisker). All individual points are plotted. Stacked bars were generated using the mean for each phase distribution. On live imaging data, each value corresponds to one cell. At least three biological replicated were quantified for the mitotic length and CG membrane deformation. The data were represented with scatter plots.Statistical significance was determined using unpaired t-test when comparing two different conditions that passed a normality test, Mann-Whitney test when comparing two different conditions that did not pass the normality test and 2way ANOVA when comparing multiple grouped conditions. In each figure the following convention is used: n.s. (p > 0.05), *(p ≤ 0.05), **(p ≤0.01), ***(P ≤ 0.001) and ****(P ≤ 0.0001).

Fig S5A and Movie 4 ).

 4 Fig S5B, see Materials and Methods). We did not find any difference in CG deformation between the two conditions (Supp. Fig S5C), suggesting that Ig interactions within the niche are not required for allowing proper CG deformation during NSC division.

Figure 1 .

 1 Figure 1. Transcriptional analysis of CG morphogenesis identifies niche regulators.

Figure 2 .

 2 Figure 2. The interacting triad, Dpr10, DIP-α and Dpr6, control NSC nuclear shape.

Figure 3 .

 3 Figure 3. Dpr10, DIP-α and Dpr6 are required for proper cycle progression in NSCs.

Figure 4 .

 4 Figure 4. The interacting triad of immunoglobulins is expressed in the NSC niche.

  dpr10::mKate shows a punctuated expression that colocalizes partly with CG membrane. Scale bar is 15µm. E) Confocal images of Drosophila VNCs at ALH72 at 25ºC of DIP-α::GFP (green) and myr-mScarlet (magenta) under the grh-GAL4 driver. DIP-α::GFP is expressed in neurons and colocalizes also with NSC membrane marker. Scale bar is 15µm. Close ups of a NSC highlights the expression of DIP-α over the NSC membrane. Scale bar is 5 µm.

Figure 5 .

 5 Figure 5. Loss of the interacting partner Dpr10 in CG decreases NSC-CG membrane proximity.

Figure 6 .

 6 Figure 6. The role of immunoglobulin-mediated adhesion on NSC involves actomyosin modulation.

  Interestingly, precise dpr10 knockdown in CG consistently showed abnormalities in the nuclear morphology of NSCs (See Results Figure 2A-B). The downregulation of DIP-α in NSCs and Dpr6 in CG led to similar phenotypes (See Results Figure 2A,D). These data indicate that DIP-α is required in NSC while its interacting partners, Dpr10 or Dpr6, are required in CG for controlling NSC nuclear shape. The different disruptions of NSC nuclei pinpointed a potential problem in the cell cycle. An analysis of the rate of NSC proliferation under the loss of function of these immunoglobulins in the corresponding cell type revealed a significant decrease of the NSC mitotic index (See Results Figure 3A-D). Furthermore, from their mitotic phase distribution, a mild increase of NSCs in prometaphase and metaphase was perceived (See Results Figure 3E). Live imaging experiments corroborated this finding with several examples of NSCs undergoing delays in anaphase onset (See Results Figure 3F-G and Supplemental Movie S1). Overall, these results

Figure

  Figure 4B). Using the CRISPR/Cas9 homologous recombination technique I generated fluorescent Dpr10 and DIP-α knock-in lines. Live imaging of dpr10∷mKate2 and DIP-α∷GFP revealed a mobile, punctuated expression of these immunoglobulins in the CG and NSC membranes, respectively (See Results Figure 4C-D). This technique confirmed the weak and dotty expression of these proteins in the NSC niche and added an unexpected dynamic characteristic to them.

  antibodies and GAL4 trap lines, as previously shown. Thanks to the dpr10∷GFP and DIP-α∷GFP CIRSPR/Cas9 lines I engineered, I was able to look at their expression pattern using the live imaging technique. With these tools, I observed a striking punctuated expression along the NSC (DIP-α∷GFP, See Results Figure 4E) or CG membranes (Dpr10∷GFP or mKate2::Dpr10, See Results Figure 4C-D). I combined dpr10∷GFP and DIP-α∷GFP with specific cell-type driven RNAi. The experiment was done in fixed tissue, and I was no longer able to see the clear dots nor a difference between control and RNAi conditions. The several issues I encountered in detecting a clear expression pattern for these proteins in fix samples could be due to an alteration of the proteins in the fixation process. Live imaging of dpr10∷mKate2 and DIP-α∷GFP turned out to be the most promising approach (See Results

  Figure. 1F). Indeed, Dpr10 and Dpr6 can contact other DIPs (DIP-λ for both and DIP-β for Dpr6) and we cannot exclude than in DIP-α absence these other DIPs could be expressed and take their role. In the future, investigating what happens to these immunoglobulins under overexpression or downregulation of their binding partner(s) would help to shed light on this intriguing redundancy between Dpr6 and Dpr10.

Figure 1 .

 1 Figure 1. Mammalian embryonic neurogenesis. ...................................................................

Figure 2 .

 2 Figure 2. Comparison of mammalian and Drosophila NSC origin. .......................................

Figure 3 .

 3 Figure 3. Neurogenesis in Drosophila embryos and larvae ...................................................

Figure 4 .

 4 Figure 4. Development of the NSC niches. ..........................................................................

Figure 5 .

 5 Figure 5. Major adult NSC niches of the mammalian brain. .................................................

Figure 6 .

 6 Figure 6. The Drosophila CNS and the NSC niche. ..............................................................

Figure 7 .

 7 Figure 7. Cortex glia chambers and model of its morphogenesis. .........................................

Figure 8 .

 8 Figure 8. The cell cycle phases. ...........................................................................................

Figure 9 .

 9 Figure 9. Plethora of possible mitotic nuclear remodeling strategies.....................................

Figure 10 .

 10 Figure 10. Cyclin-Cdk complexes of the cell-cycle control. .................................................

Figure 11 .

 11 Figure 11. The metaphase mitotic spindle in an animal cell. .................................................

Figure 12 .

 12 Figure 12. The spindle assembly checkpoint. .......................................................................

Figure 13 .

 13 Figure 13. Main cell adhesion molecules. ............................................................................

Figure 14 .

 14 Figure 14. Cadherin-mediated adhesions in the nervous system development. .....................

Figure 15 .

 15 Figure 15. Extracellular interactome of the four immunoglobulin subfamilies in Drosophila.

Figure 16 .

 16 Figure 16. NSC niche adhesion in mammals. .......................................................................

Figure 17 .

 17 Figure 17. Model through which NSCs modulate the expression of extracellular matrix receptors to regulate their activity. .......................................................................................

Figure 18 .

 18 Figure 18. Summary of the roles previously described of different cell adhesion molecules in NSCs proliferation, quiescence, self-renewal and differentiation. .........................................

Figure 19 .

 19 Figure 19. Drosophila NSCs delaminate via a progressive loss of apical cell-cell junctions. .

Figure 20 .Figure 21 .Figure 22 .

 202122 Figure 20. Cellular mechanotransduction. ............................................................................ 53

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Transcriptional analysis of CG morphogenesis identifies niche regulators. ............ 81 Figure 2. The interacting triad, Dpr10, DIP-α and Dpr6, control NSC nuclear shape............. 83 Figure 3. Dpr10, DIP-α and Dpr6 are required for proper cycle progression in NSCs. .......... 85

Figure 4 .Figure 5 .Figure 6 .Figure 23 .

 45623 Figure 4. The interacting triad of immunoglobulins is expressed in the NSC niche. .............. 87 Figure 5. Loss of the interacting partner Dpr10 in CG decreases NSC-CG membrane proximity. ............................................................................................................................................. 89 Figure 6. The role of immunoglobulin-mediated adhesion on NSC involves actomyosin modulation............................................................................................................................ 91 Figure S1. The TaDa technique. ........................................................................................... 93 Figure S2. Visualization of NSC nuclear shape in control and mutant phenotype. ................. 94 Figure S3. Dpr10 is dispensable for proper asymmetric division. .......................................... 95 Figure S4. The interacting triad of immunoglobulins is expressed in the NSC niche. ............ 96 Figure S5. Dpr10 is not required for CG deformation along NSC mitosis ............................. 97 Figure S6. Expression of myosin II in the NSC niche ........................................................... 98 Figure 23. Schematic view of the working model with an outline of the proposed timeline of the events............................................................................................................................110

  The selected neuroblast delaminates basally into the embryo. The remaining cells of each proneural cluster adopt an alternative epidermal fate. (C) In the Drosophila embryo NBs delaminate from neuroectodermal cells. The polarity of the first embryonic NB division is inherited from the original epithelial cells. NBs divide to generate a ganglion mother cell (GMC) that divides once more, giving rise to two neurons and later also glia. Some NBs will divide through an intermediate neural progenitor (INP) ultimately producing neurons and glia. (D) The larval brain can be subdivided into: ventral nerve cord (VNC) with its thoracic and abdominal NBs; brain lobes with optic lobes (OL); central brain (CB) mushroom body NBs (MB). (E) Type I NBs divide asymmetrically to self-renew and generate a GMC, which divide once to generate neurons or glia. (F) Type II NBs divide asymmetrically to self-renew and generate an immature INP. After a period of maturation, INPs start dividing asymmetrically to self-renew and to generate a GMC. The GMCs divide once into two differentiating neurons or glial cells. Through INPs, type II lineages give rise to more neurons than do type I NBs. Adapted from

Table 1

 1

	) (Losada-Perez, 2018). Glial cells are classified based on their
	positions, morphology and gene expression profiles into surface glia, cortex glia and neuropil-
	associated glia.
	Glia functions in CNS	Mammals	Drosophila
	NSC regulation	Astrocytes Ependymal cells	Cortex glia Subperineurial glia Perineurial glia
	BBB	Astrocytes	Subperineurial glia Perineurial glia
			Astrocyte-like glia
	Phagocytic activity	Microglia	Ensheating glia
			Cortex glia
	Neuronal support	Astrocytes	Cortex glia Astrocyte-like glia
	Axonal guidance	Astrocytes	Astrocyte-like glia Ensheating glia
	Synaptic control	Astrocytes	Astrocytes-like glia

Table 1 . Postembryonic glial functions comparison between mammals and Drosophila.

 1

Table 2 . List of Drosophila melanogaster lines used in this project.

 2

		Chromosome Source	Reference
	w 1118	I	BDSC	5905
	cyp4g15(cyp)-GAL4	III	BDSC	39103
	NP577-GAL4	I	T Lee lab	Awasaki et al., 2008
	worniu(wor)-GAL4	II	C Doe lab	Albertson et al., 2004
	dpr10-GAL4	III	L Zipursky lab	Xu et al., 2018
	dpr6-GAL4	III	L Zipursky lab	Xu et al., 2018
	UAS-Histone(H)2B::YFP	II	F Schweisguth lab Bellaïche et al., 2001
	UAS-H2B::RFP	III	Y Bellaïche lab	Langevin et al., 2005
	UAS-mCD::GFP	III	BDSC	5130
	UAS-Dam	III	A Brand lab	Southall et al., 2013
	UAS-Dam-Pol II	III	A Brand lab	Southall et al., 2013
	tubulin-GAL80 thermosensitive(ts)	II	BDSC	65406
	UAS-Rho1 Dominant Negative(DN)	I	BDSC	7327
	UAS-Rho1 Dominant Negative(DN)	II	BDSC	58818
	UAS-Rok Constitutively Active(CA)	II	BDSC	6668
	UAS-CD4::splitGFP 1-10 , LexAop-CD4::splitGFP 11	II	K Scott lab	Gordon and Scott, 2009
	UAS-DIPα RNAi	II	VDRC	104044 KK
	UAS-Dpr10 RNAi	II	VDRC	103511 KK
	UAS-Dpr6 RNAi	II	VDRC	41161 GD
	grh-FRT-STOP-FRT-LexA	III	P Spéder lab	Unpublished

Table 5 . List of primary antibodies and dyes used in this project. 5. Live imaging

 5

		Specie	Concentration	Source
	DAPI	-	1/5000	ThermoFisher, 62247
	Deadpan (Dpn)	Guinea pig 1/5000	Pauline Spéder lab
	DIPa	Mouse	1/10	Lawrance Zipursky lab
	ElaV	Rat	1/100	DSHB, 7E8A10
	GFP	Chicken	1/2000	Abcam, ab13970
	Lamin Dm0 (LamDm0)	Mouse	1/10	DSHB, ADL101
	Miranda (Mira)	rabbit	1/500	Cayetano González lab
	Phospho-histone 3 (PH3)	rabbit	1/200	Millipore, 06-570

CNS were dissected at 60 hours after larval hatching in freshly prepared live imaging medium composed of Schneider's medium (ThermoFisher 21720024), 5% Fetal Bovine Serum (Sigma F4135), 1/100 larval lysate and 1/100 Penicillin/Streptomycin (ThermoFisher 15140). Larval lysate was prepared with 20 larvae mixed in 200 uL of Schneider's until obtaining a whole homogenate that was spun down at 6000 rpm for 5 minutes at 4°C and the supernatant was recovered. 15 mm glass bottom dishes were previously coated with 1% low melting point agarose (ThermoFisher 16520100) in Schneider's with 1/100 Penicillin/Streptomycin. Wells of approximately 2 mm diameter were made in the solidified agarose and the surface was filled

Table 1 .

 1 Postembryonic glial functions comparison between mammals and Drosophila. ......

Table 2 .

 2 List of Drosophila melanogaster lines used in this project. .....................................

Table 3 .

 3 List of oligonucleotides used as guide RNAs to target Cas9 endonuclease. .............

Table 4 .

 4 List of primers used for the cloning of the two homologous regions around the CRISPR/Cas9 target sites inside the plasmid vector containing the fluorescent labels. .........

Table 4 .

 4 List of primary antibodies and dyes used in this project. .........................................

Acknowledgments

We thank members of the Spéder lab for helpful discussion and suggestions; S. Rigaud for general input on the analyses and, in particular, for the split-GFP ones; P. Campagne for the analysis of the NSC mitotic length; N. Owens for performing the HK-means clustering in the processed TaDa datasets; A. Joudat for sharing her expertise on the generation of the fluorescently tagged CRISPR/Cas9-engineered lines; R. Levayer for input on the modulation of cortical tension. We thank L. Zipursky, C. González, K. Scott, the VDRC and BDSC stock centers for reagents. This work has been funded by a starting package from Institut Pasteur/LabEx Revive and a JCJC grant from Agence Nationale de la Recherche (NeuraSteNic, ANR-17-CE13-0010-01) to PS.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions

ASC performed all experiments, except the Targeted DamID who was performed by ABL and PS. ASC and PS analyzed the data. PS and ASC wrote the article. YB provided expertise on tension manipulation and the generation of the CRISPR/Cas9-based lines as well as unpublished transgenic lines.

Declaration of Interests

The authors declare no competing interest.

DISCUSSION

The neural stem cell (NSC) niche is a highly specialized environment composed of NSCs and other niche populations surrounded by extracellular matrix. In there, localized and specific intercellular and extracellular matrix interactions, as well as soluble factors, influence NSC proliferation, self-renewal and differentiation. How the precise cytoarchitecture of the niche provides spatial constraints to modulate NSCs remains uncertain. The PhD research presented here emphasizes the role of intercellular adhesion within the juvenile, larval NSC niche in Drosophila melanogaster in which cortex glia (CG) tightly enwrap NSCs lineages. By investigating the dynamics of interaction between NSCs and CG, we uncovered a mechanism by which adhesion between NSC and their glial niche regulates NSC division through the control of the cortical cytoskeleton in glia.

Key findings of the PhD project will be discussed in this section exploring their implications in the context of the NSC niche. Improvements of this work and future perspectives to address the questions that this research raises will follow.

Summary of results

Identification of a novel niche regulator: the immunoglobulin superfamily

An initial objective of the project was to identify NSC niche regulators. A transcriptional analysis of CG morphogenesis highlighted an enrichment of genes from the immunoglobulin superfamily (See Results Figure 1E). Investigating this category of genes was particularly promising because i) such molecular function (specific cell-to-cell recognition and adhesion) seemed highly relevant with respect to the interaction between or within the niche; ii) this class contains distinct types of pathways and several very closely related members, suggesting a fine regulation of their function [START_REF] Quarles | Immunoglobulin Superfamily and the Nervous System[END_REF]Zinn and Özkan, 2017). Therefore, we expected that members of this immunoglobulin superfamily could have a role in the niche development.

A functional screening of multiple members of the extracellular interactome of these immunoglobulins resulted in a variety of phenotypes in the CG network, as well as in NSC

LIST OF MOVIES

ABSTRACT English

Proper brain development depends on the activity of neural stem cells (NSCs). These multipotent progenitors are found in the niche, a complex and dynamic cellular microenvironment. The niche must detect and respond to NSC needs while also mediating the impact of both local and systemic inputs to support successful neurogenesis. In particular, the intricate structure of the niche, rich in direct cell-cell interactions and adhesions, is poised to be a critical regulator of NSC behavior. However how interactions between the NSCs and their niche control neurogenesis remains poorly explored. This study takes advantage of the Drosophila juvenile, larval brain, which contains a specific subpopulation of glial cells, the cortex glia (CG), that enclose each NSC and its neuronal progeny within membrane chambers. The precise encasing of single NSC lineages pinpoints the existence of tightly controlled contacts between CG and NSCs lineages.

Transcriptional analyses of CG morphogenesis revealed a binding triad of immunoglobulins as novel regulators of the NSCs niche. The independent knockdown of these genes in CG or NSC resulted in a remarkable phenotype in NSCs, affecting their nuclear shape and cell cycle progression. Furthermore, we discovered that these immunoglobulins could act as adhesion complexes bringing together NSCs and CG membranes. Finally, we found that immunoglobulin-mediated adhesion regulates NSC function by modulating the cortical tension of CG. By investigating the dynamics of interaction between NSCs and CG, this work demonstrates that the niche exerts mechanical constraints impacting on NSC behavior, a concept poorly documented.

French

Le développement du cerveau dépend de l'activité des cellules souches neurales (CSN). Ces progéniteurs multipotents existent dans la niche, un microenvironnement cellulaire complexe et dynamique. La niche doit détecter et répondre aux besoins des CSN tout en relayant l'impact des signaux locaux et systémiques afin de favoriser la neurogenèse. En particulier, la structure de la niche occupe une position idéale pour réguler le comportement des CSN. Toutefois, l'impact des interactions directes entre CSN et leur niche reste mal connu. Cette étude profite du cerveau juvénile, larvaire de la drosophile, qui contient une sous-population spécifique de cellules gliales, la glie du cortex (CG), qui enferme chaque CSN et sa progéniture neuronale dans des chambres membranaires. Ce partitionnement précis des lignages individuels des CSN suggère l'existence d'interactions étroitement contrôlés entre les CSN et la CG.

L'analyse transcriptionnelle de la morphogenèse de la CG a révélé une triade d'immunoglobulines (Igs) comme nouveaux régulateurs de la niche. La régulation négative de ces gènes dans la CG ou les CSN entraine un phénotype remarquable dans les CSN, affectant la forme de leur noyau et la progression de leur cycle cellulaire. De plus, nous avons découvert que ces Igs pouvaient agir comme des complexes d'adhésion en rapprochant les membranes des CSN et de la CG. Enfin, cette adhésion médiée par des Igs régule la fonction des CSN en modulant la tension corticale de la CG. En étudiant la dynamique d'interaction entre les CSN et la CG, ce travail démontre que la niche exerce des contraintes mécaniques ayant un impact sur le comportement des CSN, un concept peu documenté.