Muriel Perron

Elena Sena
email: elena.sena@curie.fr

Nathalie Rocques

Caroline Borday

Harem Sabr

Muhamad Amin

Hugo Juraver-Geslin

Karine Parain

Albert Chesneau

Béatrice C Durand
email: beatrice.durand@curie.fr

Kerstin Feistel
email: k.feistel@uni-hohenheim.de

Henk Roelink

Simon J Conway

A nuclear brake on T-cell

Keywords: Groucho/Tle, Organizer, Barhl2, Stem Cells, Hdac, Relative Normalized Expression barhl2 Relative Normalized Expression siamois1 chordin Relative Normalized Expression mcl1, barhl2, neural plate, xenopus, apoptosis developmental biology, embryogenesis, neural, segregation, compartment, sonic hedgehog, forebrain, thalamus, holoprosencephaly, patterning

Ces années passées à l'Institut ont été enrichissantes et un grand apprentissage professionnel et personnel. Je suis très heureuse de finir l'écriture de ce manuscrit en remerciant tous les gens qui m'ont aidée et soutenue pendant cette période.

Je tiens en tout premier lieu à remercier l'ensemble des membres de mon jury de thèse en commençant par mes rapporteurs, le Dr. Laurent Sachs et le Pr Muriel Umbhauer pour le temps consacré à lire et examiner mon manuscrit. Je remercie également le Dr.

Madeleine, Juliette, Valerie, Ha-Yeon et Zackie de la team Larue avec qui j'ai partagé autant de repas. Merci également pour les soirées ! Cha, Mag, MoMo (sans les départs tardifs rien n'aurait été si efficace…), Audrey, Bennedetta, Claire et Chloé. Pour les soirées filles. Aux membres de l'équipe Eychene, Ayrault et Dutreix et tous les autres membres de l'Institut avec qui j'ai partagé des soirées, des happy Fridays et des bonjours dans le couloir (je vous nommerais bien tous mais on ne finirait jamais).

A tous les chercheurs et ingénieurs que j'ai rencontrés pendant ces années et qui m'ont autant aidée, spécialement à Karine, Albert et Clémence.

Merci à Julie, qui a toujours été une constante dans l'Institut. Mon point de support quand j'avais juste envie de déconnecter et une amie sur laquelle je pourrais toujours compter. Moi en train de faire de randonnées, qui allait le dire ! A Malika et Ana, pour être un soutien très important. Je suis très heureuse de vous avoir rencontrées et d'avoir vécu autant des choses ensemble. Vous me manquez tous les jours et je suis très heureuse de l'avenir qui vous arrive.

A mes amis Giuliana, Fra et Nico. Pour être ma famille italienne et pour ne pas me laisser tomber. Pour tous les pique-niques et apéros, le repas de famille le dimanche, le cinéma, les promenades, les châteaux… et pour être là.

A tous mes amis à Barcelone. A mes meilleures copines, qui me manquent. A Irene, parce que les années passent et la plage elle reste. Rosari et Claudia, vous êtes tout et avoir dû nous séparer pendant ces années ça a été dur, triste et compliqué mais notre amitié est plus forte chaque année et il n'y a rien qui me rend plus fière. On va fêter tout ça au Tickets.

A ma famille, spécialement à mon père pour m'avoir permis de réaliser mes aspirations sans poser trop de questions.

A Francine, évidemment, pour avoir été un soutien constant pendants ces trois dernières années. A Sonia et Ruben, car ils rendent heureuses les personnes les plus importantes de ma vie.

A mon frère, que j'aime et sur lequel je pourrais toujours compter.

Et enfin, à ma soeur. La personne la plus importante de ma vie. Sans toi je suis perdue, et sans toi rien ne serait pareil.

LIST OF FIGURES

EARLY EMBRYONIC DEVELOPMENT IN VERTEBRATES

Developmental biology focuses in the study of all developmental processes that occur during and after embryogenesis. Embryonic development is a finely regulated process that will give rise to a whole organism. Embryogenesis comprises the stages of development between fertilization and birth and consists of six fundamental steps:

-Fertilization: it is the fusion of the gametes, the sperm and the egg. This fusion stimulates the egg to begin development. The fusion of the male and female pronuclei gives the embryo its genome. In humans, the zygote nucleus is diploid but in other species, such as Xenopus laevis (X. laevis) the resulting oocyte is tetraploid, this means the genome contains four copies of each gene.

-Cleavage: it is a series of rapid mitotic divisions that follow fertilization. The zygote is divided into smaller cells called blastomeres. By the end of cleavage in amphibians, the structure is called blastula and contains a cavity on its interior called blastocoel.

-Gastrulation: it consists in a series of cell movements were blastomeres rearrange themselves to form three germ layers: ectoderm, endoderm and mesoderm. At this stage, the embryo is called gastrula. These layers will give rise to all tissues and organs in the body: the ectoderm especially is the precursor of the epidermis, brain and nerves and stays in the outside of the embryo; the endoderm is at the origin of the gut and respiratory systems and stays in the inside; and the mesoderm gives rise to the connective tissue, blood, heart, skeleton, gonads and kidneys and is located between the endoderm and the ectoderm.

-Organogenesis: it is the process in which the cells in the embryo divide and associate to generate a whole individual by producing tissues and organs.

-Metamorphosis: in some species, at the end of the organogenesis, the organism called larva needs to undergo this process to make a sexually mature adult.

-Gametogenesis: is the process in which a group of cells is set aside to produce the next generation. These are the germ cells and are the precursors of gametes. Germ cells migrate to the gonads, where they differentiate into gametes.

These processes are shared in vertebrates although there are some differences between them. In humans there is no metamorphosis and, in the amphibian, fertilization occurs outside the organism when the sperm fertilizes the eggs just laid by the female on the water. In amphibians the point of sperm entry initiates the formation of the dorsal-ventral (DV) axis of the embryo. This axis is established after the cortical rotation the cytoplasm effectuates after the fertilization [START_REF] Vincent | Subcortical rotation in Xenopus eggs: An early step in embryonic axis specification[END_REF]. After the fusion of the female and male eggs cell divisions occur without gap phases until the embryo has around 4000 cells and it is called blastula. The blastula has three distinct regions: the animal cap, the equatorial or marginal zone and the vegetal hemisphere. The animal cap cells are considered naïve/pluripotent cells because they can be induced to differentiate in different cell types depending on the signals provided.

Before gastrulation occurs, there is a crucial step called the mid-blastula transition (MBT). This process begins at stage 8,5 and marks the beginning of zygotic gene transcription and the presence of gap phases in the cell cycle (Saka and Smith, 2001). Until this moment, the transcription machinery in the cells was not fully activated and the embryo only expressed maternal messenger RNA (mRNA). Some of these maternal mRNA are genome-wide transcriptional repressors such as t-cell factor 3 (tcf3 a.k.a. tcf7l1) (Houston et al., 2002) and transcriptional activators such as Forkhead box H1 (FoxH1) [START_REF] Kofron | New roles for FoxH1 in patterning the early embryo[END_REF]), the T box protein

VegT [START_REF] Zhang | The role of maternal VegT in establishing the primary germ layers in Xenopus embryos[END_REF] and the cyclic AMP response element-binding protein (CREB) [START_REF] Sundaram | The role of maternal CREB in early embryogenesis of Xenopus laevis[END_REF]. These mRNAs are strategically localized in the egg so that specific cells inherit them. The events that trigger the MBT involve chromatin modifications. The genes that begin to be transcribed after the MBT are hypo-acetylated and have H3-methylated chromatin [START_REF] Meehan | Epigenetic silencing in embryogenesis[END_REF]. Histones are responsible for allowing or repressing gene expression. This is achieved by modifying the tails of histones H2 and H4 with methyl and acetyl residues. Histone acetylation loosens the histones allowing the chromatin to be accessible to RNA polymerase (RNA pol) and to activate transcription. This reaction is made by histone acetyltransferases (Hat). The inverse role is made by the histone deacetylases (Hdac). Chromatin modification can also be achieved by methylation of DNA by the enzyme DNA methyltransferase (Dnmt). When the MBT occurs, certain promoters are demethylated allowing gene transcription. This demethylation is specific of genes that are activated in the MBT [START_REF] Akkers | A Hierarchy of H3K4me3 and H3K27me3 Acquisition in Spatial Gene Regulation in Xenopus Embryos[END_REF][START_REF] Hontelez | Embryonic transcription is controlled by maternally defined chromatin state[END_REF][START_REF] Stancheva | DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos[END_REF]. When chromatin at the promoters becomes accessible, transcription factors bind to them and initiate new transcription. Even if this has been an established opinion through the years recent results claim that before the MBT there is already gene transcription [START_REF] Collart | High-resolution analysis of gene activity during the Xenopus mid-blastula transition[END_REF]Owens et al., 2016;[START_REF] Paranjpe | A genome-wide survey of maternal and embryonic transcripts during Xenopus tropicalis development[END_REF]Session et al., 2016;[START_REF] Yang | beta-Catenin/Tcf-regulated transcription prior to the midblastula transition[END_REF].

Gastrulation in amphibians begins at 180º opposite of the point of sperm entry with the formation of the blastopore, the most dorsal part of the embryo. This structure is the point where cells invaginate inside the embryo during gastrulation to form the three germ layers.

During this process, there is formation of a cavity in the animal hemisphere that allows the cell movements during gastrulation called the blastocoel. This cavity also prevents the cells under it to interact with the cells of the animal cap.

The major signaling pathways that are active during the first steps of embryonic patterning are summarized in the next section.

SIGNALING PATHWAYS INVOLVED IN EARLY EMBRYONIC DEVELOPMENT

The signaling pathways described here are well conserved between species and are essential for the mechanisms of early development. They are active after the MBT and achieve different roles through gastrulation and organogenesis. They are classified depending on the paracrine factors involved. Paracrine factors bind receptors in the cell inducing a conformational change on the receptor and initiate enzymatic reactions. These reactions can affect either regulation of transcription factors and/or the cytoskeleton.

THE TRANSFORMING GROWTH FACTOR-b SUPERFAMILY

The transforming growth factor (Tgf)-b superfamily members are classified according to their protein sequence and include among others Tgf-b, bone morphogenetic proteins (Bmp), Activin and Nodal. They bind to two types of receptors that have tyrosine kinase activity in the cytoplasm of the cell, receptor type I, and receptor type II. Each Tgf-b family member binds to a combination of these two receptors forming a complex. Receptor II phosphorylates and activates receptor I (Figure 1). In the cytoplasm, the phosphorylated receptor I phosphorylates Smad2/3 proteins in the case of Tgf-b, Nodal and Activin or Smad 1/5/8 in the case of Bmp and this activates them. When Smads are phosphorylated they dissociate from the receptor and bind to Smad4 in the cytoplasm. The Smad complex translocates into the nucleus, where it binds other proteins related with gene expression to induce transcription of Tgf-b target genes.

BONE MORPHOGENETIC PROTEINS

The members of the Bmp family were first described as responsible for induction of bone formation but they are also important in embryonic development. They have been found to regulate cell division, apoptosis, cell migration and differentiation. The Bmp family members can be divided into different subfamilies depending on their amino acid sequence: Bmp2 and 4; Bmp3, Bmp5, 6, 7, 8a and 8b; growth differentiation factor (Gdf) 5, 6, 7 (Carreira et al., 2014). They all contain seven conserved cysteine residues in their active site. There are many antagonists of Bmp ligands that bind to their receptors inhibiting their activity. Two of these antagonists are Chordin and Noggin, which are important in nervous system development. Bmp has a role in DV axial patterning. Mechanisms on neural induction will be discussed later, but Bmp proteins are involved in the influence on the ectoderm to become neuroectoderm.

During many years it was believed that no specific signal activated neural fate and that this was the "default state" for the ectoderm [START_REF] Godsave | Neural induction and patterning in embryos deficient in FGF signaling[END_REF][START_REF] Muñoz-Sanjuán | Neural induction, the default model and Embryonic Stem Cells[END_REF]. In Xenopus animal caps, absence of signaling resulted in development of neural tissue. When the explants were cultured in the presence of Bmp, they became epidermis. These experiments suggested that the fate of the ectoderm was to become neural tissue in the absence of instructive signals, Bmp. However, this was not true for chick embryos, which added controversial to the default state model [START_REF] Streit | Neural induction[END_REF][START_REF] Streit | Chordin regulates primitive streak development and the stability of induced neural cells, but is not sufficient for neural induction in the chick embryo[END_REF]. It is now understood that Bmp ligands and its antagonists, Chordin and Noggin, are expressed in opposed regions in the DV axis. Thus, epidermal fate is determined by Bmp signaling and neural specification requires Bmp antagonists. Moreover, fibroblast growth factor (Fgf) signaling is too required for neural specification [START_REF] Dorey | FGF signalling: diverse roles during early vertebrate embryogenesis[END_REF][START_REF] Reversade | Regulation of ADMP and BMP2/4/7 at Opposite Embryonic Poles Generates a Self-Regulating Morphogenetic Field[END_REF]Stern, 2006).

NODAL RELATED PROTEINS

These proteins are important for specifying the different regions of the mesoderm and for establishing the left-right asymmetry. Vertebrates have morphological and functional left-right asymmetries in the brain, heart, lungs and gut. This process is controlled by genes including members of the Nodal cell-signaling family.

In Xenopus there are four nodal genes expressed during gastrulation: nodal homolog 1 (nodal1, a.k.a. xnr1), nodal homolog 2 (nodal2, a.k.a. xnr2) [START_REF] Jones | Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation[END_REF], nodal homolog 3 gene 1 (nodal3.1, a.k.a. xnr3) [START_REF] Smith | A nodal-related gene defines a physical and functional domain within the Spemann organizer[END_REF] and nodal growth differentiation factor (nodal, a.k.a. xnr4) [START_REF] Joseph | Xnr4: A Xenopus nodal-related gene expressed in the Spemann organizer[END_REF]. xnr1 and xnr2 are first expressed in the vegetal hemisphere and their expression is triggered by the accumulation of nuclear b-catenin in the dorsal region [START_REF] Kofron | Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFbeta growth factors[END_REF]. xnr3 is expressed in the Spemann organizer only at the gastrula stage whereas xnr4 is expressed in the Spemann organizer at gastrula stages but at later stages it can also be detected in the notochord and neural tube. Loss of nodal genes leads to defects in gastrulation as well as a disturbed anterior-posterior (AP) axis. The other nodal genes nodal homolog 5 (nodal5, a.k.a. xnr5) and nodal homolog 6 (nodal6, a.k.a. xnr6) are expressed at the MBT under the control of maternal b-catenin and VegT [START_REF] Takahashi | Two novel nodal-related genes initiate early inductive events in Xenopus Nieuwkoop center[END_REF].

VegT is a protein transcribed from maternal mRNA in the vegetal hemisphere of the embryo.

It activates expression of genes that determine the endodermal fate and it also has roles in mesoderm induction and gastrulation [START_REF] Kofron | Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFbeta growth factors[END_REF].

Activin is essential for normal development. It induces the mesoderm and regulates dorsal zygotic genes like brachyury (bra) [START_REF] Smith | A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2-and msh-class homeoproteins, mediates active transcriptional repression in vivo[END_REF], goosecoid (gsc) (Cho et al., 1991) and chordin (chd). It also regulates the transcription of Nodal proteins.

SONIC HEDGEHOG

Sonic hedgehog (Shh) secreted factor acts as local morphogen. It is secreted from the cell and induces differential gene expression depending on a concentration gradient. During neurulation, Shh is induced in the notochord, the floor plate and anteriorly in the ventral neuroepithelium (Sena et al., 2016). During development it has many roles, for example it specifies the AP axis in the diencephalon (Epstein, 2012;Sena et al., 2016), it participates in the DV patterning of the anterior brain [START_REF] Dessaud | Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network[END_REF] and it is important for limb formation in vertebrates [START_REF] Tickle | How the embryo makes a limb: Determination, polarity and identity[END_REF]. Its active form is attached to a fatty acid chain and cholesterol [START_REF] Gallet | Hedgehog morphogen: From secretion to reception[END_REF]. The transducing mechanism was first described in Drosophila melanogaster (D. melanogaster). In the fly, in the absence of Shh ligand, the twelve-domain transmembrane receptor Patched (Ptc) is kidnapping the seven-domain receptor Smoothened (Smo) in intracellular vesicles (Figure 2Aa). When Shh is present, it binds to the co-receptors Interference hedgehog (Ihog) and

Brother of Ihog (Boi). This allows the binding and inactivation of Ptc by Shh. Smo is phosphorylated and translocated into the plasma membrane, where it can bind Shh (Figure 2Ab). When the pathway is inactive, the protein Cubitus interruptus (Ci) is being sequestered in the cytoplasm by a multiprotein complex containing protein kinase A (PkA), Glycogen Synthase 3 (Gsk3) and Casein kinase I (Ck1). This complex phosphorylates Ci so that is cleaved into smaller fragments. One of these fragments can translocate into the nucleus, where it can act as transcriptional repressor of Shh target genes. When the Shh ligand is present, the multiprotein complex is phosphorylated at the cell membrane and Ci can accumulate and translocate into the nucleus, where it activates transcription of Shh target genes.

Whereas the Shh mode of action has been extensively studied in Drosophila, there are pending questions on vertebrates' mechanisms of Shh signal transduction. In vertebrates Shh signaling takes place in the cell's primary cilium (Figure 2B). The primary cilium is a specialized sensory organelle. Signal transduction in the primary cilium requires intraflagellar transport mediated by microtubules. This allows the transport of the receptors and effectors of the pathway. The vertebrates homologues for Ihog and Boi are Cdo and Boc [START_REF] Briscoe | The mechanisms of Hedgehog signalling and its roles in development and disease[END_REF]. They bind to Shh in the primary cilium and allow signaling by Smo (Figure 2b). The homologous of Ci in vertebrates are the Gli family zinc finger proteins (Gli). In vertebrates there are at least three Gli. It has been demonstrated that Gli2 and Gli3 act as transcriptional repressors and activators but Gli1 acts only as transcriptional activators. A balance between the production of activating and repressor forms of Gli allows modulation of Shh response. When Smo is inhibited, Gli2 and Gli3 are phosphorylated and targeted to the proteasome, then cleaved. The cleaved forms correspond to the repressive Gli. When Shh binds Ptc, Smo translocates into the cytoplasm and inhibits the degradation of Gli2, that in turns activates the secretion of the activated form of Gli1 [START_REF] Varjosalo | Hedgehog: functions and mechanisms[END_REF].

FIBROBLAST GROWTH FACTORS

The Fgf family is comprised by at least 24 secreted factors. They contain 140 conserved aminoacids and bind to Fgf Receptors (FgfRs), a type of tyrosine kinase receptor. In vertebrates we can find 4 FgfRs that can generate different variants depending on their splicing. They are expressed in almost all tissues and have different roles during development [START_REF] Lea | Temporal and spatial expression of FGF ligands and receptors during Xenopus development[END_REF][START_REF] Ornitz | The fibroblast growth factor signaling pathway[END_REF]. Fgf bind to their receptors together with co-factors such as heparin sulfate to form a dimer. The binding of the ligand to its receptor induces the phosphorylation of tyrosine by the kinase on the plasma membrane triggering intracellular signaling. Some of the activated pathways are the Ras-MAPK, Pi2K-Akt, Phospholipase C (PLC)g and STAT.

Fgf are implicated in different processes during development such as cell proliferation and differentiation, cell survival or cell morphology and migration [START_REF] Dorey | FGF signalling: diverse roles during early vertebrate embryogenesis[END_REF]. As mentioned above, during embryogenesis, Fgf has a role in the induction of neuroectoderm. In Xenopus, absence of Fgf signaling even in the presence of Bmp antagonists results in lack of neural markers in the ectoderm. Fgf induces the territory that will become neuroectoderm and helps to maintain the anti-Bmp signaling in the organizer [START_REF] Branney | Characterisation of the fibroblast growth factor dependent transcriptome in early development[END_REF][START_REF] Delaune | Neural induction in Xenopus requires early FGF signalling in addition to BMP inhibition[END_REF][START_REF] Lee | Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos[END_REF]. This mechanism is also true in chick embryos [START_REF] Linker | Neural induction requires BMP inhibition only as a late step, and involves signals other than FGF and Wnt antagonists[END_REF].

Fgf signaling is also implicated in the coordination of cell movements during gastrulation [START_REF] Amaya | Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos[END_REF] and it is also necessary for the induction of paraxial mesoderm and the maintenance of axial mesoderm signaling [START_REF] Fletcher | The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus[END_REF].

WNT SIGNALING

Wingless-Int (Wnt) proteins are secreted signaling molecules that act as mediators and morphogens, controlling diverse aspects of development such as embryonic axis formation and segmentation, organogenesis and stem cell proliferation. In the absence of Wnt signaling activation, the responsive Wnt genes remain silent through an inhibiting complex of gene regulatory proteins. If this pathway is altered it can be at the origin of disease (Clevers and Nusse, 2012;Sokol, 2011[START_REF] Sokol | Wnt signaling axis specification in vertebrates[END_REF]. Wnt1 was the first discovered member of the family and at first was studied as a proto-oncogene [START_REF] Nusse | Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome[END_REF]. Currently we know that vertebrates contain a family of at least 19 Wnt-related genes.

Overall, it has been defined that the different Wnt family members are specific of the canonical or non-canonical pathways. For example, Wnt3a is meant to act through canonical signaling whereas Wnt5 is non-canonical. However, some results argue that this classification should not been done on the activation of b-catenin but on their ability to activate receptors and their co-factors (van Amerongen and Nusse, 2009). In fact, many Wnt proteins can act in both signaling pathways when used in specific models.

NON-CANONICAL PATHWAYS

In the non-canonical pathways -"planar cell polarity" pathway and "Wnt/calcium" pathway -Wnt influences cell function shape and behavior.

The planar cell polarity pathway (PCP), regulates the actin and microtubule cytoskeleton influencing cell shape. Wnt activates Disheveled (Dsh) by binding to its receptor Frizzled (Frz) paired with Ror. The Ror receptor complex phosphorylates Dsh allowing it to interact with the small GTPases Rho and Rac. These induce cytoskeletal rearrangements that lead to the development of lateral asymmetry in epithelial sheets and other structures. In vertebrates, regulation of cell division and migration is important for establishing germ layers, and for anterior-posterior axis extension during gastrulation and neurulation.

The Wnt/calcium pathway leads to the release of calcium stored within cells, and this calcium acts as a secondary messenger to modulate many downstream targets. Binding of Wnt to its receptors activates the PLC, which causes the release of calcium ions from the smooth endoplasmic reticulum. Release of calcium can activate enzymes, transcription factors and translation factors.

CANONICAL PATHWAY

Discovery of Wnt1 opened a field of investigation that linked cancer and embryogenesis. The first studied pathway was the so-called "Wnt canonical pathway" also known as the "Wnt/bcatenin pathway", which is characterized by the activation of b-catenin and modulation of gene expression. In the Wnt canonical pathway the transcriptional co-factor b-catenin is constantly being degraded in the proteasome [START_REF] Aberle | β-Catenin is a target for the ubiquitin-proteasome pathway[END_REF][START_REF] Yost | The axisinducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3[END_REF] by a multiprotein complex formed by the scaffold protein Axin, the adenomatous polyposis coli suppressor protein (Apc) [START_REF] Rubinfeld | Association of the APC gene product with β-catenin[END_REF], Ck1 and Gsk3 [START_REF] Dominguez | Role of glycogen synthase kinase 3β as a negative regulator of dorsoventral axis formation in Xenopus embryos[END_REF][START_REF] He | Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos[END_REF][START_REF] Pierce | Overexpression of Xgsk-3 disrupts anterior ectodermal patterning in Xenopus[END_REF]. In the absence of Wnt ligands, Gsk3 and Ck1 phosphorylate bcatenin and target it for ubiquitination and degradation in the proteasome (Figure 3Aa).

When the Wnt ligand is present, it binds to Frz [START_REF] Vinson | A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains[END_REF] that interacts with the Low Density Lipoprotein Receptor-related protein (LRP) family of receptors 5 and 6 [START_REF] Pinson | An LDL-receptor-related protein mediates Wnt signalling in mice[END_REF] forming a multimeric complex. The cytoplasmic tail of LRP5/6 is phosphorylated and interacts with Gsk3 and Axin dissociating the multiprotein complex that was degrading bcatenin [START_REF] Mao | Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway[END_REF]. Frz binds to Dsh [START_REF] Perrimon | Multiple functions of segment polarity genes in Drosophila[END_REF], which keeps Axin and Gsk3 inactive. Gsk3 is inactivated by its kidnapping in mutivesicular endosomes in the cytoplasm [START_REF] Taelman | Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes[END_REF]. b-catenin then accumulates and translocates into the nucleus, where it binds the transcription factor T-cell factor(Tcf)/Lymphoid enhancer-binding factor (Lef) promoting transcription of Wnt target genes (Behrens et al., 1996;[START_REF] Huber | Nuclear localization of β-catenin by interaction with transcription factor LEF-1[END_REF]Molenaar et al., 1996) (Figure 3Ab). In the nucleus, Tcf/Lef alone cannot activate gene transcription and needs binding of co-factors (van de [START_REF] Van De Wetering | Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes[END_REF]. b-catenin allows gene transcription whereas the co-factor Groucho (Gro)/Transducer like-enhancer (Tle) plays a role on gene repression (Daniels and Weis, 2005;[START_REF] Levanon | Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors[END_REF]. In addition to bcatenin and Gro, Tcfs can interact with other molecules that regulate their stability, localization and ability to bind DNA (Cadigan, 2012;[START_REF] Hurlstone | T-cell factors: turn-ons and turn-offs[END_REF].

The Tcf/Lef family of transcription factors contain a DNA-binding domain, the high mobility group (HMG) domain, a small peptide motif of basic residues called the basic tail and the Cclamp domain, located in the carboxyl (C)-terminal region. The HMG domain induces a sharp bend in the DNA helix allowing binding of target genes DNA [START_REF] Castrop | A gene family of HMG box factors with homology to TCF-1[END_REF][START_REF] Giese | The HMG Domain of Lymphoid Enhancer Factor 1 Bends DNA and Facilitates Assembly of Functional Nucleoprotein Structures[END_REF][START_REF] Love | Structural basis for DNA bending by the architectural transcription factor LEF-1[END_REF]. The basic tail binds to DNA together with the HMG domain, and the C-clamp domain is a second DNA-binding domain which is not present in all vertebrates' Tcfs (Cadigan and Waterman, 2012). Tcfs are highly conserved between species and during evolution [START_REF] Hurlstone | T-cell factors: turn-ons and turn-offs[END_REF] (Table 1 and Figure 4A). Tcfs are redundant in their activity, except for Tcf7l1, whose single knockout induces a lethal phenotype in Xenopus embryos (Liu et al., 2005). In Xenopus embryos, Tcf7l1 is the most predominant expressed maternal Tcf and acts as a transcription repressor.

Name (human) Homologue Mouse Homologue Xenopus

Transcription factor 7 (TCF7, a.k.a. TCF-1) [START_REF] Van De Wetering | Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequencespecific HMG box[END_REF] Tcf-7 (Oosterwegel et al., 1991) tcf7 (Molenaar et al., 1996) Lymphoid enhancer binding factor 1 (LEF1) [START_REF] Travis | LEF-1, a gene encoding a lymphoid-specific with protein, an HMG domain, regulates T-cell receptor α enhancer function[END_REF] Lef-1 [START_REF] Travis | LEF-1, a gene encoding a lymphoid-specific with protein, an HMG domain, regulates T-cell receptor α enhancer function[END_REF] lef1 (a.k.a. xlef-1) [START_REF] Molenaar | Differential expression of the HMG box transcription factors XTcf-3 and XLef-1 during early Xenopus development[END_REF] Transcription factor 7 like 1 (TCF7L1, a.k.a. TCF-3) [START_REF] Castrop | A gene family of HMG box factors with homology to TCF-1[END_REF] Tcf7l1 [START_REF] Castrop | A gene family of HMG box factors with homology to TCF-1[END_REF] tcf7l1 (a.k.a. xtcf-3) (Molenaar et al., 1996) Transcription factor 7 like 2 (TCF7L2, a.k.a. TCF-4) [START_REF] Castrop | A gene family of HMG box factors with homology to TCF-1[END_REF] Tcf7l2 [START_REF] Castrop | A gene family of HMG box factors with homology to TCF-1[END_REF] tcf7l2 (a.k.a. xtcf-4) (Ah Cho and Dressler, 1998) containing the two short domains LVPQ and SXXSS act as repressor Tcfs (Liu et al., 2005).

However, Tcf7 and Tcf7l2 have both activator and repressor roles depending on the tissue and developmental stage (Houston et al., 2002;[START_REF] Standley | Maternal XTcf1 and XTcf4 have distinct roles in regulating Wnt target genes[END_REF]. Thus, the classical switch of Tcf from repressor to activator is reconsidered into a switch of the Tcf involved in gene regulation (Figure 3B). Hikasa and colleagues demonstrated that the homeodomaininteracting protein kinase 2 (HIPK2) phosphorylates Lef1 and Tcf7l1 inhibiting their role as activator and repressors of gene transcription respectively. Moreover, upon phosphorylation of Tcf7l1, the binding to its target genes is lost on behalf of non-phosphorylated Tcf7, that can interact with b-catenin to activate Wnt target genes transcription (Hikasa et al., 2010;[START_REF] Hikasa | Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2[END_REF] (Figure 3Bb).

The following sections describe in detail the interaction and mode of action of Tcf and its cofactors b-catenin and Gro in the nucleus.

b-CATENIN

b-catenin is the main effector of Wnt canonical signaling. When the pathway is active it stabilizes and translocates to the nucleus to promote gene transcription. The role of b-catenin as the main effector of Wnt signaling was at first surprising, since its role had been defined as an adherent junction linking cadherin-E with the cortical axin cytoskeleton. Its role as Wnt signaling effector is conserved between species, the homologue Armadillo in Drosophila is also effector of the Wingless signaling pathway and the sequence is highly conserved between invertebrates and vertebrates [START_REF] Peifer | The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation[END_REF]. Together with Gro, many observations and interactions described on the Wnt pathway were first discovered in Drosophila.

The amino (N)-terminal domain of b-catenin contains four conserved serine/threonine residues that can be phosphorylated by Gsk3, targeting b-catenin for degradation in the proteasome.

The central domain comprises 12 amino-acid repeats called armadillo repeats. These mediate most of the interactions between b-catenin and its partners such as E-cadherin, Apc, Axin and Tcf. b-catenin binds to the first 60 amino-acids of Tcf via its repeats 3 to 10 (Behrens et al., 1996;[START_REF] Graham | Crystal Structure of a β-Catenin/Tcf Complex[END_REF][START_REF] Huber | Nuclear localization of β-catenin by interaction with transcription factor LEF-1[END_REF]Molenaar et al., 1996;[START_REF] Poy | Structure of a human Tcf4-??catenin complex[END_REF]. The Cterminal domain contains transcriptional transactivation elements that recruit the transcription machinery such as the TATA binding protein (TBP) [START_REF] Hecht | Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA-binding protein in vitro[END_REF] (Figure 4B).

Stabilization of b-catenin is associated with its nuclear localization despite the lack of a nuclear localization sequence (NLS) [START_REF] Hsu | Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin[END_REF].

In Drosophila, it has been observed that Tcf and Armadillo bind to Wnt responsive elements (WRE) in target loci promote the recruitment of CREB-binding protein (CBP) and p300. They are Hats that bind to b-catenin and increase transcription [START_REF] Parker | Wingless Signaling Induces Widespread Chromatin Remodeling of Target Loci[END_REF]. This modification on chromatin is made long distance and when transcription is still inactive. In

Xenopus there are also studies on chromatin structure that describe the recruitment of bcatenin on target genes loci (Nakamura and Hoppler, 2017). p300 binds to closed chromatin to induce the recruitment of chromatin modifiers that results in a more accessible chromatin state [START_REF] Hontelez | Embryonic transcription is controlled by maternally defined chromatin state[END_REF]. There is recruitment of b-catenin and Tcf7l2 into regions where histones are acetylated, this means open to transcription (Blythe et al., 2010). This recruitment is independent of zygotic gene transcription, meaning that this process' objective is to induce chromatin reorganization and not direct transcription.

GROUCHO

The Gro family members are evolutionary conserved nuclear proteins and they share five protein domains (Jennings and Ish-Horowicz, 2008) (Figure 4C). At the N-terminal region there is a glutamine (Q)-rich domain that interacts with Tcf [START_REF] Brantjes | All Tcf HMG box transcription factors interact with Groucho-related co-repressors[END_REF]Daniels and Weis, 2005). Tcf interacts with Gro through a region located between the b-catenin binding domain and the HMG box (Chen et al., 1999;Roose et al., 1998). The next domain on Gro proteins is a glycine (G) and proline (P)-rich domain that interacts with Hdacs (Chen et al., 1999); a domain containing casein kinase phosphorylation sites and a nuclear localization signal (CcN); a serine (S) and P rich domain and finally four WD40 repeats that allow interaction of Groucho with other proteins. Some examples of the transcription factors that interact with Groucho through the WD40 repeats to act as transcriptional repressors are Hairy, Runt, Engrailed (Eng)

and Dorsal [START_REF] Brantjes | TCF: Lady Justice casting the final verdict on the outcome of Wnt signalling[END_REF]Fisher and Caudy, 1998). Groucho-5 (Gro5) only contains the Q and GP domains and is a dominant negative, thus impeding its binding to Hdac, which explains its lack of repression activity [START_REF] Brantjes | All Tcf HMG box transcription factors interact with Groucho-related co-repressors[END_REF]Roose et al., 1998). In the absence of Wnt signaling Tcf binds to Groucho [START_REF] Cavallo | Drosophila Tcf and Groucho interact to repress Wingless signalling activity[END_REF]Roose et al., 1998), which either increases RNA pol II pausing time and/or recruits histone deacetylase 1 (Hdac1)

to mediate transcriptional repression [START_REF] Brantjes | All Tcf HMG box transcription factors interact with Groucho-related co-repressors[END_REF]Chen et al., 1999[START_REF] Chen | Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus[END_REF][START_REF] Kaul | ainsi que les effets répressifs transcriptionnels de la modification post-traductionnelle des histones par cette enzyme modifiant la chromatine sont documentés[END_REF][START_REF] Palaparti | The Groucho/transducin-like enhancer of split transcriptional repressors interact with the genetically defined amino-terminal silencing domain of histone H3[END_REF], although there may be other mechanisms involved (Jennings and Ish-Horowicz, 2008). An established model is that Gro spreads along chromatin via its Q domain oligomerization to act as a long-range repressor [START_REF] Chen | A role for Groucho tetramerization in transcriptional repression[END_REF][START_REF] Palaparti | The Groucho/transducin-like enhancer of split transcriptional repressors interact with the genetically defined amino-terminal silencing domain of histone H3[END_REF], but recent studies contradict this model [START_REF] Kaul | The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription[END_REF]. The interaction between Gro and Hdac1 together with the transcriptional repressive effects of the post-translational modification of histones by this chromatin-modifying enzyme have been well studied. However very little is known on what controls the specificity of Gro-Hdac1 activity and/or the role(s) of these epigenetic regulations driven by this protein complex during early development.

PATTERNING THE EMBRYO: THE ORGANIZING CENTERS

Neural organization is set up during gastrulation, when cells receive signals that will determine their fate and commitment to one tissue or another. An organizer center is composed of a group of specialized cells that secrete signals involved in patterning its neighboring structures.

The main embryonic organizer center is the Spemann organizer, which is capable to induce a secondary axis when grafted on the ventral side of the blastula embryo. Throughout development secondary organizing centers emerge that have crucial activities in patterning of the nervous system. One of them is the zona limitans intrathalamica (ZLI). I participated to the writing of a review describing the developmental events supporting development of the ZLI (Sena et al., 2016) (Section IV, Annex 1). Therefore, in this section I will not present the ZLI and I will focus on the molecular events involved in DV and AP axis specification in Xenopus embryos.

THE SPEMANN ORGANIZER

In 1924 Hilde Mangold, doctoral student of Hans Spemann, discovered that one region of the dorsal side of the newt embryo was capable of inducing gastrulation and formation of a new organism in the opposite side of the embryo when transplanted [START_REF] Mangold | Über induktion von embryoanlagen durch implantation artfremder organisatoren[END_REF]. The transplanted region gives rise to a new head and dorsal region with its own notochord and surrounding somites. This region is called the Mangold-Spemann Organizer (the Spemann Organizer, SO) and corresponds to the area where the dorsal lip of the blastopore forms. The SO has different roles: it induces the neural plate in the dorsal ectoderm, it is involved in endodermal development and it controls morphogenetic movements of cells during gastrulation that define the AP axis in the embryo. It has equivalent structures in other vertebrates such as Hensen's node in amniotes (Anderson and Stern, 2016).

In the following years after this discovery researchers focused on elucidating the signals that emanated from this group of cells and how the SO was induced.

INDUCTION OF THE SPEMANN ORGANIZER

The cells of the Nieuwkoop center located opposite to the point of sperm entry in blastula (presumptive dorsal part of the embryo) are responsible for inducing the SO before the beginning of zygotic transcription. Signals derived from the Nieuwkoop center induce a population of cells near the equatorial zone to initiate dorsal mesodermal differentiation; therefore, the SO is formed by newly induced dorsal mesodermal cells. These cells emit signals that dorsalize the fate of adjacent ventral mesodermal cells, and that induce overlying ectodermal cells to adopt a neural fate. The Nieuwkoop center and the SO share some molecular components and are both induced by maternal Wnt signaling [START_REF] Vonica | The Xenopus Nieuwkoop center and Spemann-Mangold organizer share molecular components and a requirement for maternal Wnt activity[END_REF].

Experiments done with b-catenin proved that its overexpression could induce a double axis in embryos [START_REF] Funayama | Embryonic Axis Induction by the Armadillo Repeat Domain of β-catenin: Evidence for Intracellular Signaling[END_REF][START_REF] Guger | β-Catenin has Wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning[END_REF][START_REF] Heasman | Overexpression of cadherins and underexpression of β-catenin inhibit dorsal mesoderm induction in early Xenopus embryos[END_REF][START_REF] Mccrea | Induction of a Secondary Body Axis in Xenopus by Antibodies to β-catenin[END_REF]. These suggested that b-catenin was responsible for the SO's induction. b-catenin is synthetized from maternal mRNA at early cleavage stages and accumulates in the dorsal side of the embryo [START_REF] Larabell | Establishment of the Dorso-ventral Axis in Xenopus Embryos is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway[END_REF]Schneider et al., 1996). As mentioned before, maternal mRNAs are localized in specific regions of the egg cytoplasm so when cleavage occurs some blastomeres inherit certain mRNA and not others. Dsh and Gsk3-binding protein (Gbp) locate in the dorsal side of the embryo due to cortical rotation movements (Figure 5C).

These two proteins inhibit Gsk3, allowing the accumulation of b-catenin in the nuclei of dorsal cells (Figure 5D,E). Finally Wnt-11 through stabilization of Dsh and Gbp participate to increase b-catenin nuclear levels [START_REF] Heasman | Patterning the early Xenopus embryo[END_REF].

WNT ACTIVITY

The [START_REF] Mccrea | Induction of a Secondary Body Axis in Xenopus by Antibodies to β-catenin[END_REF], and inversely dorsal suppression of b-catenin caused ventralized embryos (Heasman et al., 2000[START_REF] Heasman | Overexpression of cadherins and underexpression of β-catenin inhibit dorsal mesoderm induction in early Xenopus embryos[END_REF]. These experiments demonstrated the link between Wnt and b-catenin in patterning the embryo. Before the MBT, maternal Wnt signaling regulates specific genes transcription that function to establish dorsal development [START_REF] Larabell | Establishment of the Dorso-ventral Axis in Xenopus Embryos is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway[END_REF]Schneider et al., 1996;[START_REF] Schohl | β-catenin, MAPK and Smad signaling during early Xenopus development[END_REF][START_REF] Yang | beta-Catenin/Tcf-regulated transcription prior to the midblastula transition[END_REF] but shortly after Wnt signaling assures the development of ventrolateral mesoderm (Christian and Moon, 1993).

Among

F MBT and organizer genes transcription

Gene transcription

Gro

β-cat

Tcf sia, twin is wnt-11, that is present in the oocyte and due to the cortical rotation gets located in the dorsal equatorial zone [START_REF] Ku | Xwnt-11: a maternally expressed Xenopus wnt gene[END_REF]. After the MBT it is expressed in the dorsal marginal zone and later ventrally. Its ectopic injection on the ventral blastomeres cannot induce a complete dorsal axis with anterior structures. Opposite to wnt-11 there are other wnt genes whose ectopic expression can induce formation of a complete double axis such as wnt-1 [START_REF] Mcmahon | Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis[END_REF] and xwnt-8 (Sokol et al., 1991). However, wnt-1 and wnt-8 start to be expressed at late blastula or early gastrula, long after mesoderm induction and display other roles. For example, wnt8 is repressed in the dorsal mesoderm after the SO induction to ensure the correct development of neural structures and plays a ventralizing role at this stage.

Therefore, Wnt ligands expression varies during the first stages of development and achieve different signaling roles (Christian and Moon, 1993).

ZYGOTIC GENE TRANSCRIPTION AFTER THE MID-BLASTULA TRANSITION

The events that trigger the formation of the SO also trigger zygotic transcription in the embryo.

Prior to this phase maternal genes are the only ones expressed in the embryo while the others are repressed by histone modifications. Maternal genes are important and induce the first signals that trigger the developmental signaling cascade [START_REF] Paranjpe | A genome-wide survey of maternal and embryonic transcripts during Xenopus tropicalis development[END_REF][START_REF] White | Maternal Control of Pattern Formation in Xenopus laevis[END_REF]. To facilitate the transcription of zygotic genes after the MBT, some of them are poised for activation. This means they are in a transcriptional state that can be easily reverted and allows for a rapid response to transducing signals. The genes poised for activation are characterized by the presence of activating (H3K4me3) and repressive (H3K27me4) histone marks. When is necessary, the repressive or activating histone modifications disappear to facilitate the transcription or repression of the gene.

The first genes to be induced after the MBT at the dorsal region of the embryo are the transcription factor siamois (sia) and siamois homologue 2 (twin) [START_REF] Carnac | The homeobox gene Siamois is a target of the Wnt dorsalisation pathway and triggers organiser activity in the absence of mesoderm[END_REF][START_REF] Lemaire | Expression cloning of Siamois, a xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis[END_REF] (Figure 5F). They are directly regulated by binding of Tcfs to their promoters [START_REF] Brannon | A βcatenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus[END_REF][START_REF] Fan | Wnt signaling and transcriptional control of Siamois in Xenopus embryos[END_REF]. sia and twin are redundant and induce the dorsal genes gsc, chd, xnr3 and noggin and inhibit Bmp4 [START_REF] Bae | Siamois and Twin are redundant and essential in formation of the Spemann organizer[END_REF][START_REF] Carnac | The homeobox gene Siamois is a target of the Wnt dorsalisation pathway and triggers organiser activity in the absence of mesoderm[END_REF]Cho et al., 1991;[START_REF] Laurent | The Xenopus homeobox gene twin mediates Wnt induction of goosecoid in establishment of Spemann's organizer[END_REF][START_REF] Sasai | Xenopus chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes[END_REF]. The gsc promoter contains a distal element responsible to Tgf-b and Wnt signals [START_REF] Watabe | Molecular mechanisms of Spemann's organizer formation : conserved growth factor synergy between Xenopus and mouse[END_REF]. Indeed the gsc promoter also binds to VegT, Lim1, Sia and Orthodenticle Homebox 2 (Otx2) in vivo during the Spemann organizer formation (Sudou et al., 2012). xnr3 can also be activated by direct binding of Tcf [START_REF] Mckendry | LEF-1/TCF proteins mediate Wnt-inducible transcription from the xenopus nodal-related 3 promoter[END_REF]. In the absence of β-catenin, tcf7l1 and Gro inhibit sia and twin transcription (Roose et al., 1998).

The Smad signaling pathway is also responsible for the induction of the SO genes [START_REF] Hurlstone | T-cell factors: turn-ons and turn-offs[END_REF][START_REF] Nishita | Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer[END_REF]. vg1 and vegT are maternally encoded and located in the vegetal cytoplasm before the MBT. b-catenin interacts with Vg1 and VegT to induce xnr genes transcription. This creates a gradient across the endoderm that is higher in the dorsal region and that will induce the marginal zone cells to become mesoderm. Mesodermal regions with lower levels of xnr have high levels of Bmp4 and Wnt8 and become ventral mesoderm.

Those having intermediate concentration become lateral mesoderm and finally where there is high concentration of nodal genes there is activation of gsc and other dorsal mesoderm genes and the mesodermal tissue becomes the organizer [START_REF] Agius | Endodermal Nodal-related signals and mesoderm induction in Xenopus[END_REF][START_REF] Chea | Nodal signaling and the evolution of deuterostome gastrulation[END_REF].

Embryos with inhibition of Tgf-b signaling cannot express organizer-specific genes [START_REF] Hemmati-Brivanlou | A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos[END_REF]. sia requires the Tgf-b signals present in the vegetal cytoplasm to induce formation of the organizer [START_REF] Engleka | Siamois cooperates with TGFβ signals to induce the complete function of the Spemann-Mangold Organizer[END_REF]. Moreover, VegT can activate sia expression by binding to the promoter independently of Wnt signaling [START_REF] Li | Direct regulation of siamois by VegT is required for axis formation in Xenopus embryo[END_REF].

Different approaches contributed to the identification of genes specific for the organizer [START_REF] Hufton | Genomic analysis of Xenopus organizer function[END_REF][START_REF] Wessely | Analysis of Spemann organizer formation in Xenopus embryos by cDNA macroarrays[END_REF]. Moreover, the publishing of the complete genome for X. laevis has increased the interest in the study of embryological processes in this amphibian model and promoted the development of more sophisticated techniques (Ding et al., 2016;Y. Ding et al., 2017;Session et al., 2016). However, most of these studies have analyzed the effect of b-catenin in gene activation and less is known on the role of Groucho and Tcf in the in vivo repression of the SO organizer genes after the first phase of SO induction (Yasuoka et al., 2014). Even if the genes regulated by maternal Wnt signaling such as siamois and xnr3 have been extensively studied, the genes regulated by post-MBT Wnt signaling are poorly known. We currently do not understand completely how Tcf activity can mediate expression of genes after the MBT. Which are the signals that activate or repress Tcf transcriptional activity in the promoters of zygotic genes? Is it dependent on the cellular context and time? The identification, in vivo, of the mechanisms regulating Tcf transcriptional activity is still pending.

THE AXIAL ORGANIZER

During gastrulation, the signals secreted by the organizer establish the three germ layers of the embryo: ectoderm, mesoderm and endoderm (Figure 6). The first cells that invaginate into the embryo through the dorsal lip of the blastopore will give rise to the prechordal plate, which is the precursor of the head mesoderm. These cells express gsc. The following cells to involute are the ones that will become the chordamesoderm, which that will generate the notochord.

These cells express bra. The prechordal plate and the notochord together with the floor plate constitute a secondary organizer center called the axial organizer that secretes Shh and anti-Bmp factors that initiate neural induction (Juraver-Geslin and Durand, 2015).

Figure 6 Gastrulation events leading to neural plate formation. A: at stage 10 the events leading to gastrulation have begun with the appearance of the dorsal lip of the blastopore at the dorsal side of the embryo. Cells will invaginate through this. There are three distinct regions: the animal cap (blue), the equatorial zone (magenta) and the vegetal hemisphere (yellow). The image represents a sagittal section from the miniature embryo in the upper right corner. Arrows indicate cell movements. B: at stage 13, gastrulation is coming to an end. The first cells from the equatorial zone to invaginate become the prechordal plate, followed by the paraxial mesoderm. A new cavity, the archenteron, develops and the blastopore lip has a ventral and a dorsal region. The image represents a parasagittal section from the miniature embryo in the upper right corner. Arrows indicate cell movements. C: at stage 14, gastrulation has finished and neurulation begins. The blastocoel has disappeared. The three germ layers are established and the mesoderm can be divided in the chordal mesoderm (notochord) and lateral mesoderm (somites). The notochord secretes signals that influence the ectoderm to become neuroectoderm.

NEURAL INDUCTION

Neural induction is the process by which ectodermal cells become specified as neural precursor cells. The "default state" of the animal cap cells establishes that, if isolated, the fate of this population is to become epidermis, and this is achieved by the binding of Bmp in those cells [START_REF] Piccolo | Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of chordin to BMP-4[END_REF][START_REF] Wilson | Induction of epidermis and inhibition of neural fate by Bmp-4[END_REF][START_REF] Zimmerman | The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4[END_REF].

Actually, because of the expression of the Bmp antagonists chd and noggin, Bmp signaling pathway, initially activated at MBT downstream of Bmp2 and Bmp7 activity, is excluded from the dorsal animal region of the embryo [START_REF] Kuroda | Neural induction in Xenopus: Requirement for ectodermal and endomesodermal signals via Chordin, Noggin, β-Catenin, and Cerberus[END_REF]. The SO secretes anti-Bmp factors that, along with Fgf, induce a change in the ectoderm cells so that they become neural tissue [START_REF] Delaune | Neural induction in Xenopus requires early FGF signalling in addition to BMP inhibition[END_REF][START_REF] Khokha | Depletion of three BMP antagonists from Spemann's organizer leads to a catastrophic loss of dorsal structures[END_REF][START_REF] Reversade | Regulation of ADMP and BMP2/4/7 at Opposite Embryonic Poles Generates a Self-Regulating Morphogenetic Field[END_REF]) (Figure 7). The classical anti Bmp factors are Noggin, Chd and Follistatin and a fourth BMP inhibitor, Norrin, appears to be stored in the animal pole of the oocyte and functions to block Bmp signaling in the dorsal ectoderm [START_REF] Khokha | Depletion of three BMP antagonists from Spemann's organizer leads to a catastrophic loss of dorsal structures[END_REF][START_REF] Xu | Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus[END_REF]. Noggin is first expressed in the dorsal lip region and then in the notochord [START_REF] Smith | Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos[END_REF]. It induces the dorsal ectoderm to become neural tissue and it dorsalizes mesodermal cells that would otherwise become ventral mesoderm. chd is expressed in the region that becomes the blastopore lip prior gastrulation, and after it remains in the dorsal blastopore lip [START_REF] Sasai | Xenopus chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes[END_REF]. Noggin

and Chd bind to Bmp4 and Bmp2 preventing their binding to their receptors [START_REF] Piccolo | Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of chordin to BMP-4[END_REF][START_REF] Zimmerman | The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4[END_REF]. Follistatin is an inhibitor of both BMP and activin.

Gsc, among other transcription factors, prevents the transcription of bmp4 in the dorsal region of the embryo restricting its expression to the ventral side [START_REF] Blitz | Anterior neurectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle[END_REF][START_REF] Yao | Goosecoid promotes head organizer activity by direct repression of Xwnt8 in Spemann's organizer[END_REF]. Some genes downstream BMP signaling are Xvent2 and msh homebox 1 (msx1), which activate the epidermal pathway and also suppress pro-neural genes when ectopically overexpressed.

NEURULATION

Neurulation corresponds to the formation of the neural tube. At the end of gastrulation and as a consequence of neural induction, a portion of the dorsal ectoderm is specified to form the neural ectoderm, also called the neural plate [START_REF] Wilson | Neural induction: Toward a unifying mechanism[END_REF]. The cells of the neural plate express the genes SRY-box (sox) 1, 2 and 3 [START_REF] Archer | Interaction of Sox1, Sox2, Sox3 and Oct4 during primary neurogenesis[END_REF]. Later, the neural plate bends to form the neural folds. When the folds get closer they fuse forming the neural tube.

During this process the embryo is called a neurula. The neural tube will give rise to the brain, the neural pituitary, the spinal cord, and the retina. The neuroepithelium is composed of a single layer of rapidly dividing Neural Stem Cells (NSCs) and progenitors. Early in mammalian development, the majority of cells of the neural tube proliferate, as seen by incorporation of thymidine analogues into DNA [START_REF] Fujita | Analysis of neuron differentiation in the central nervous system by tritiated thymidine autoradiography[END_REF]. As the cells adjacent to the lumen continue to proliferate, the neural progenitors migrate and form a second layer around the neural tube. This layer becomes progressively thicker and is called the mantle zone (MZ), whereas the germinal neuroepithelium adopts the name of ventricular zone (VZ), which will be later called the ependyma. Cells in the MZ differentiate into both neurons and glia [START_REF] Cayuso | Morphogens in motion: growth control of the neural tube[END_REF].

Many signaling pathways are responsible for the proliferation and patterning of the neuroepithelium. These molecules are responsible for the transcription of genes related to progenitors' proliferation and/or differentiation. Regarding the Wnt canonical signaling pathway, b-catenin mediated signaling is essential for the maintenance of proliferation of neuronal progenitors [START_REF] Zechner | β-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system[END_REF] and the establishment of a DV axis in the neural tube (De Robertis and Kuroda, 2004;[START_REF] Tsuji | Choice of either β -catenin or Groucho / TLE as a co-factor for Xtcf-3 determines dorsal-ventral cell fate of diencephalon during Xenopus development[END_REF][START_REF] Ulloa | Morphogens and the control of cell proliferation and patterning in the spinal cord[END_REF][START_REF] Wylie | Maternal beta-catenin establishes a "dorsal signal" in early Xenopus embryos[END_REF].

ROLE OF APOPTOSIS DURING DEVELOPMENT

The growth of the neural tube is a process controlled by the coordinate equilibrium between proliferation, differentiation and apoptosis. Thus, these three processes act together to regulate progenitors' proliferation or elimination of supernumerary cells. In the following sections I discuss the importance of apoptosis during development and I also argue about the non-apoptotic functions of some components involved in this mechanism.

The apoptosis term was first used by Kerr and colleagues describing a specific cell morphology characterized by cell condensation, and cytoskeleton collapse. The nuclear cell membrane disaggregates and the nuclear chromatin is condensed and fragmented (Kerr, 2002;[START_REF] Kerr | La cellule apoptotique sécrète des facteurs apoptotiques et est finalement absorbée par les macrophages ou une cellule adjacente. Au cours de ce processus, une endonucléase nucléaire clive l'ADN chromosomique[END_REF]. The apoptotic cell secretes apoptotic factors and is finally absorbed by macrophages or an adjacent cell. During this process a nuclear endonuclease cleaves chromosomic DNA and these double-strand DNA breaks generate DNA fragments. Their 3′hydroxyl termini can be detected by a technique called Terminal deoxynucleotidyl transferase mediated dUTP-biotin Nick End Labeling (TUNEL) that marks apoptotic cells [START_REF] Gavrieli | Idenitification of Programmed Cell Death In Situ via Specific Labeling of Nuclear DNA Fragmentation[END_REF]. Apoptosis has extensively been studied in Caenorhabditis elegans (C. elegans) (Ellis and Horvitz, 1986), as well as in Drosophila and mammals (Arya and White, 2015). However, apoptosis roles and modes of regulation in X. laevis embryonic development are less described.

APOPTOSIS REGULATION

The balance between pro-apoptotic and anti-apoptotic proteins is crucial in cell homeostasis and survival. The apoptotic cascade is ultimately controlled by activation of its downstream effectors: Caspases. Caspases are proteolytic enzymes that contain a cysteine in their active site and cleave their targets on specific residues of aspartic acid. They are synthetized in the cell as inactive precursors and need to be cleaved to be active. We can distinguish two types of Caspases: the initiator Caspases and the effector Caspases. The initiator Caspases cleave each other and generate two subunits that form a tetramer with the other two subunits of another initiator Caspase. This tetramer is responsible for the cleavage and activation of the effector Caspases. The effector Caspases cleave specific proteins such as nuclear laminas, the inhibitor of the specific DNA endonuclease or cell-cell adhesion proteins and this chain reaction induces the morphological changes for apoptosis. Other than these specific activities involved in mediating apoptosis, Caspases have been described to have non-apoptotic roles (Juraver-Geslin and Durand, 2015;[START_REF] White | Controlling caspase activity in life and death[END_REF]. In the intrinsic pathway, upon death stimuli the BH3-only proteins are activated and can inhibit the anti-apoptotic family proteins Bcl2. These proteins are responsible for inhibition of pro-apoptotic proteins Bax and Bak. Once this inhibition is released, Bax and Bak can promote cytochrome C release from the mitochondria. Cytochrome C associates with Apaf1 and this induces an oligomerization of the complex. This complex, the apoptosome, activates the initiator pro-caspase-9. Once activated Caspase-9 can cleave and activate effector pro-caspases-3 and -7. Activated effector caspases can cleave specific apoptotic proteins to induce cell death. In the extrinsic pathway, a TNF receptor is activated by a death ligand on the cell surface. This activates the initiator Caspase-8, that in turn activates the effector Caspase-7. -2, -4, -5, -9, -11 and -12), or a Death Effector Domain (DED) . Both domains allow the binding to the scaffold protein allowing initiator pro-Caspases to bind and cleave one another and further recruitment of the apoptotic machinery. Initiation of the apoptotic cascade can be triggered in two ways: the extrinsic pathway and the intrinsic pathway.

THE EXTRINSIC PATHWAY

In the extrinsic pathway, apoptosis is triggered by the binding of a ligand to a specific death receptor in the cell membrane. This pathway is mostly active in immune system in cells such as lymphocytes that contain a death receptor that belongs to the Tumor Necrosis Factor (TNF) family of receptors. This transmembrane protein contains one external domain for the binding of ligand, one transmembrane domain and one intracellular death domain.

In the cell that needs to be eliminated there is a Fas receptor on the cell surface. The cytotoxic lymphocytes responsible for the cell elimination secrete a Fas ligand. When the receptor is activated, the death domains in the cytoplasm of the cell recruit the scaffold protein Fasassociating death domain containing-protein (FADD) that activates pro-caspases-8 and -10

(Figure 8). The binding of the receptor, the scaffold proteins and the pro-Caspases forms the Death Inducing Signaling Complex (DISC). This complex activates the effector Caspases.

THE INTRINSIC PATHWAY

The intrinsic pathway is activated in response to injury or other stress factors such as DNA damage and oxygen, nutrient or absence of extracellular survival signals (Green et al., 2014).

This pathway depends on the release of mitochondrial proteins in the cytosol that are normally located in the intermembrane space of the mitochondria and is regulated by the B-cell lymphoma 2 (Bcl2) protein family (Figure 8).

The most important protein that is released from the mitochondria into the cytoplasm is the cytochrome C. When this protein is released it binds to the scaffold protein Apoptotic Protease

Activating Factor 1 (Apaf1). Apaf1 oligomerizes in a heptametrical structure called the apoptosome. In the apoptosome, Apaf1 recruits the initiator pro-caspase-9 and activates it.

Caspase-9 in turn activates the effector caspases.

BCL2 PROTEIN FAMILY

The Bcl2 protein family is the main regulator of the apoptosis intrinsic pathway. These proteins contain the evolutionary conserved Bcl2 homology (BH) domain. It can be divided into two major sub-groups depending on its pro-or anti-apoptotic function (Table 2).

In mammals, the main anti-apoptotic proteins are Bcl2, Myeloid cell leukemia level can be regulated at the RNA and/or at the protein levels.

The human mcl1 promoter has been characterized [START_REF] Akgul | Functional analysis of the human MCL-1 gene[END_REF] and it contains some putative sites for transcription factors fixation. The down-regulation of mcl1 RNA levels can be achieved by inhibition of the transcription factors responsible for mcl1 expression including the E2F transcription factor 1 that directly repressed mcl1 transcription [START_REF] Croxton | Direct repression of the Mcl-1 promoter by E2F1[END_REF].

However, Mcl1 levels are mostly control at the protein level. Mcl1 contains three BH domains and two proline-glutamic acid-serine-threonine motifs on its N-terminal region. Mcl1 can be phosphorylated at these N-terminals regions by different kinases such as for example Gsk3 (Ding et al., 2007). Gsk3 phosphorylates Mcl1 on its S155, S159 and S163 promoting its degradation (Thomas et al., 2010). Moreover different E3 ubiquitin ligase can polyubiquitinate

Mcl1 to target it for degradation in the proteasome: Mcl1 ubiquitin ligase E3 (MULE) and btransducing containing protein (b-TrCP) (Ding et al., 2007;Zhong et al., 2005). Finally, Mcl1

can also be eliminated in a non-proteasomal pathway by Caspase cleavage on its N-terminal region (Clohessy et al., 2004).

INHIBITORS OF APOPTOSIS PROTEINS

Apart from the Bcl-2 proteins, apoptosis can also be regulated by a family of proteins that are the Inhibitors of Apoptosis Proteins (IAPs) [START_REF] Kenneth | IAP proteins: regulators of cell migration and development[END_REF][START_REF] Liston | The inhibitors of apoptosis: there is more to life than Bcl2[END_REF][START_REF] Wei | Inhibitor of apoptosis proteins and apoptosis[END_REF]. IAPs were first discovered in virus where they regulate endogenous Caspase's activity. IAPs are a large family of proteins with evolutionary conserved domains whose members can vary between species. The N-terminal region contains a 70-80 aminoacid baculoviral IAP repeat (BIR). The BIR domain is a cysteine-and histidine-rich folding domain that recognizes and binds to specific substrates. On its C-terminal region they contain a Really Interesting New Gene (RING) zinc-finger that has E3-ubiquitin ligase activity and polyubiquitinates Caspases marking them for degradation in the proteasome. The number of BIR domains varies depending on the IAP (Figure 9).

Apoptosis can be induced through inhibition of IAPs by specific antagonists: Reaper, Hid, Grim and Sickle in Drosophila, Smac/Diablo in mammals. Importantly IAPs can also inhibit their own antagonists. Through this mechanism IAPs with low affinity for Caspases can initiate apoptosis [START_REF] Riedl | Structural basis for the inhibition of caspase-3 by XIAP[END_REF][START_REF] Wilkinson | Neutralization of Smac/Diablo by inhibitors of apoptosis (IAPs): A caspaseindependent mechanism for apoptotic inhibition[END_REF].

XIAP

The best-characterized IAP in mammals is the X-linked inhibitor of apoptosis protein (Xiap).

Xiap contains three BIR domains and one RING finger (Figure 9). It inhibits Caspases-7, -9 and -3 [START_REF] Chai | Structural basis of caspase-7 inhibition by XIAP[END_REF][START_REF] Riedl | Structural basis for the inhibition of caspase-3 by XIAP[END_REF][START_REF] Shiozaki | Mechanism of XIAP-mediated inhibition of caspase-9[END_REF].Caspase-9 [START_REF] Shiozaki | Mechanism of XIAP-mediated inhibition of caspase-9[END_REF]. Xiap BIR1 displays no Caspase inhibiting activity. In Drosophila, the Xiap homologue Death associated inhibitor of apoptosis protein 1 (Diap1) inhibit Caspase activity. The human paralogues of XIAP, cellular IAP (cIAP) 1 and 2, bind to Caspases but cannot repress their activity in vivo [START_REF] Eckelman | The human anti-apoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases[END_REF].

In a cell, upon exposure to apoptotic stimuli, XIAPs can be inhibited by pro-apoptotic proteins such as XIAP-associated factor 1, Smac and Omi. In Drosophila, the XIAP homologous DIAP1 is regulated by a Drosophila IKK-related kinase (DmIKKe) [START_REF] Kuranaga | Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs[END_REF]. This kinase phosphorylates DIAP1 and promotes its degradation trough the proteasome. As a consequence, the Caspases orthologues in Drosophila Death related ICE-like caspase (DrICE) and Death regulator Nedd2-like Caspase (DrONC) become active.

APOPTOSIS IS REQUIRED FOR NEURAL DEVELOPMENT

Apoptosis takes place at different developmental steps during embryogenesis (Baehrecke, 2002;Miura, 2011). It is active in four principal processes:

-Formation of new structures: neural tube formation [START_REF] Copp | The genetic basis of mammalian neurulation[END_REF][START_REF] Weil | Is programmed cell death required for neural tube closure?[END_REF][START_REF] Yamaguchi | Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure[END_REF].

-Removal of unneeded tissue: interdigital membranes on vertebrate limb buds [START_REF] Zuzarte-Luís | Programmed cell death in the developing limb[END_REF].

-Removal of supernumerary cells: removal of neurons at the final steps of neuronal development [START_REF] Buss | Adaptive Roles of Programmed Cell Death During Nervous System Development[END_REF][START_REF] Yamaguchi | Programmed Cell Death in Neurodevelopment[END_REF].

-Removal of defective cells: cells containing DNA mutations.

The requirement of apoptosis in nervous system development is observed in mice models deficient for apoptotic members. Mice deficient for pro-apoptotic proteins Caspase-3, -9 and/or Apaf1 exhibit brain malformations as a consequence of neural precursors and patterning defects. In these mice there is brain ventricle compression and exencephaly. Conversely, Mice deficient for the anti-apoptotic proteins Bcl-X L or Mcl1 prematurely die due to massive apoptosis of immature neurons and hematopoietic cells [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]Motoyama et al., 1995;Opferman and Kothari, 2018). In the sub-ventricular zone (SVZ) of the adult brain there neural progenitor cells (NPCs) and is the only anti-apoptotic Bcl-2 protein known to regulate their survival [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]Malone et al., 2012).

In Xenopus, the earliest stage where apoptosis is detected is stage 10.5 at the beginning of the gastrulation (Hensey and Gautier, 1998). This may be due to the presence of maternal IAPs in the egg [START_REF] Tsuchiya | Apoptosis-inhibiting activities of BIR family proteins in Xenopus egg extracts[END_REF]. During gastrulation apoptosis is observed in the prospective neural plate and at neurula stage it remains in the dorsal region of the neural plate (Hensey and Gautier, 1998;Offner et al., 2005). At stage 14 and 15 apoptosis is detected at the anterior edge of the neural plate. Apoptotic cells are confined in the anterior regions where the brain and placodes will form. At later stages apoptosis is located in specific brain regions, developing sensory organs and the spinal cord (Hensey and Gautier, 1998). Inhibition of apoptosis by overexpression of Bcl2 anti-apoptotic proteins leads to an expansion of neural cells and there is a decrease in apoptosis when cell differentiation is blocked (Yeo and Gautier, 2003). Beside apoptosis plays an important role in limiting the quantity of morphogens secreted by the organizing centers. In Xenopus, apoptosis is observed at the axial organizer (Offner et al., 2005), where it limits the quantity of Shh and Bmp antagonists-secreting cells. This is also true for other vertebrates, in the chick it is observed in the floor plate (Homma et al., 1994), and in mice apoptosis regulates morphogen secretion in the anterior neural ridge (ANR) (Nonomura et al., 2013). Interestingly in fly, one of the consequences of apoptosis is the release of growth factors such as Bmp, Wnt and Shh from the apoptotic cell that induce proliferation on the adjacent cells. This mechanism is called compensatory proliferation and it is necessary to maintain homeostasis and cell number [START_REF] Mollereau | Compensatory proliferation and apoptosis-induced proliferation: A need for clarification[END_REF][START_REF] Yamaguchi | Programmed Cell Death in Neurodevelopment[END_REF].

During the early phase of embryonic development apoptosis is mostly regulated by the signaling pathways that are essential for early patterning in the embryo (Arya and White, 2015;Juraver-Geslin and Durand, 2015). The involved pathways are, for example, Notch, phosphatidylinositol 3-kinase (PI3K) and Ras/Mapk signaling. XIAP RING zinc-finger can bind to the cytoplasmic tail of Bmp receptors [START_REF] Yamaguchi | XIAP , a cellular member of the inhibitor of apoptosis protein family , links the receptors to TAB1 -TAK1 in the BMP signaling pathway[END_REF]. In mice, XIAP can interact with the C-terminal region of Ptc to coordinate craniofacial development and promote cell survival [START_REF] Aoto | Co-ordinated brain and craniofacial development depend upon Patched1/XIAP regulation of cell survival[END_REF]. XIAP is also involved in cerebellar granule neurons survival in mice postnatal cerebellums [START_REF] Blancas | Endogenous XIAP, but not other members of the inhibitory apoptosis protein family modulates cerebellar granule neurons survival[END_REF]. In Drosophila, Caspases interact with Wnt signaling components to mediate NPCs proliferation [START_REF] Kanuka | Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development[END_REF][START_REF] Kuranaga | Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs[END_REF].

NON-APOPTOTIC ROLES OF THE APOPTOTIC CASCADE MEMBERS

In the recent years, many investigations have highlighted the role of some apoptotic components in non-apoptotic functions that are also important for development [START_REF] Gross | Non-apoptotic functions of BCL-2 family proteins[END_REF][START_REF] Kutscher | Non-apoptotic cell death in animal development[END_REF].

CASPASES

The non-apoptotic role of Caspases is important in many processes such as cell differentiation, embryonic development and compensatory proliferation [START_REF] Connolly | New roles for old enzymes: killer caspases as the engine of cell behavior changes[END_REF][START_REF] Miura | Apoptotic and nonapoptotic Caspase function in animal development[END_REF][START_REF] Nakajima | Caspase-dependent non-apoptotic processes in development[END_REF][START_REF] Shalini | Old, new and emerging functions of caspases[END_REF][START_REF] Yi | The Jekyll and Hyde functions of caspases[END_REF]) (Table 3). However, the mechanisms involved in these processes are poorly understood, especially in vertebrates.

There are two possible modes of action for these functions:

-Cell autonomous or direct model: Caspases activity directly modulates regulatory networks within the cell.

-Non-autonomous or indirect model: Caspases activity occurs in one cell that signals to another cell.

It has also been proposed that the non-apoptotic function of Caspases depends on their localization in the cell and the duration and timing of the cascade to avoid cell death.

Noteworthy the low activity of Caspases has been proposed as a mechanism for Caspase function in the emergence of NPCs in combination with Wingless signaling in Drosophila [START_REF] Kanuka | Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development[END_REF][START_REF] Kuranaga | Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs[END_REF] and to be controlled by ubiquitination [START_REF] Kamber Kaya | An inhibitory monoubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways[END_REF][START_REF] White | Controlling caspase activity in life and death[END_REF]. (Juraver-Geslin and Durand, 2015). [START_REF] Kuida | Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice[END_REF] described an hyperplasia in the brain of Caspase-3 deficient mice, and proposed that this was due to a decrease in apoptosis. Later, Juraver-Geslin et al. (Juraver-Geslin et al., 2011) showed that depletion of Caspase-3 in X. laevis caused an increase in cell number in the neural tube, and they also observed a disruption of the neuroepithelium architecture and an increase in Wnt activity. In contrast to the explanations of Kuida et al., Juraver-Geslin and colleagues described that the role of Caspase-3 in the proliferation of neuroepithelial cells was due to a non-apoptotic function of this protease. This was assessed by the phenotypic differences observed between embryos depleted of Caspase-3 that exhibited an hyperplasia, and embryos overexpressing the anti-apoptotic proteins BclXL and Bcl2 that did not exhibit an hyperplasia (Juraver-Geslin et al., 2011).

A non-apoptotic role for Caspase-3 has also been described in mouse Embryonic Stem Cells (ESCs). There is an increase in Caspase-3 activity when ESCs begin to differentiate [START_REF] Fujita | Caspase activity mediates the differentiation of embryonic stem cells[END_REF]. Cells lacking Caspase-3 are unable to differentiate even in the presence of retinoic acid arguing that Caspase-3 activity is necessary to their differentiation. Caspase-3 is also important for the homeostasis of primitive hematopoietic cells [START_REF] Janzen | Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3[END_REF]. Caspase-3 deficient hematopoietic stem cells (HSCs) display accelerated proliferation and retarded differentiation. NSCs differentiation is also dependent on Caspase-3 non-apoptotic activities [START_REF] Fernando | Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates[END_REF]. Inhibition of Caspase-3 impairs the differentiation of neurospheres in vitro. Thus, Caspase-3 is necessary for differentiation of different kind of stem cells.

Apart from being necessary to stem cells differentiation, Caspase-3 has proved to be necessary for the induction of induced pluripotent stem cells (iPSCs) from human fibroblasts.

Caspase-3 and -8 are activated upon induction of iPSCs by the transcription factor Oct4 (Li et al., 2010).

XIAP

IAPs have also non-apoptotic roles important for development. It has been demonstrated that in the development of the sensory organ in the fly, DIAP1 has different roles depending on the cell differentiation state [START_REF] Koto | Temporal regulation of Drosophila IAP1 determines caspase functions in sensory organ development[END_REF]. Thus, DIAP1 can act both in cell proliferation and morphogenesis and this reinforces the previously mentioned hypothesis that apoptotic components can mediate non-apoptotic functions depending on their location and time of expression in the cell. Moreover, DIAP1 inhibition by the DmIKKe activates Caspases DrONC and DrICE promoting NPCs [START_REF] Kuranaga | Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs[END_REF]. The role of DmIKKe seems to be conserved in mammals. The DmIKKe homologous Tank-binding kinase 1 (Tbk1) has also been reported to be a direct activator of the PI3K/Akt pathway [START_REF] Xie | IκB kinase ε and TANK-binding kinase 1 activate AKT by direct phosphorylation[END_REF]. This activation in turn inhibits Gsk3 resulting in a b-catenin activation that will eventually control the survival of NSCs and progenitors in the neuroepithelium.

XIAP can also interact with the Wnt signaling pathway in a non-apoptotic way. In response to Wnt activation XIAP is recruited to Tcf/Lef transcriptional complexes where it ubiquitinates Gro in mammalian cells and in Xenopus. As a consequence, the affinity of Gro for Tcf is decreased [START_REF] Hanson | XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling[END_REF].

THE TRANSCRIPTION FACTOR BARHL2

The BarH-like (Barhl) are transcription factors homologous to the transcription factor Hox Barclass HD (BarH) in Drosophila. They were first described as genes important for the eye development in the fly (Higashijima et al., 1992b(Higashijima et al., , 1992a;;[START_REF] Kojima | Identification of a different-type homeobox gene, BarH1, possibly causing Bar (B) and Om(1D) mutations in Drosophila[END_REF]. Barhl are evolutionary conserved and its homologues can be found in different species (Figure 11). In vertebrates there are two barhl genes, barhl1 and barhl2. Vertebrate Barhl are expressed exclusively in the nervous system, where they control cell identity, migration and survival.

Barhl genes contain a homeodomain required for its fixation to DNA responsive elements, a NLS and an N-terminal region that contains one or two highly conserved Phenylalanine/Isoleucine/Leucine (FIL) rich domains, FIL1 and FIL2 (Figure 10). These domains, also called engrailed homology (Eh), are present in other classes of homeoproteins such as Eng. In Drosophila, Eng has been shown to interact with Groucho to repress gene transcription via its Eh domains [START_REF] Jennings | Molecular Recognition of Transcriptional Repressor Motifs by the WD Domain of the Groucho/TLE Corepressor[END_REF][START_REF] Jiménez | Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed[END_REF]Smith and Jaynes, 1996). Because Barhl2 contains two Ehs domains, it could be possible that Barhl2 mediates transcriptional repression by binding to Groucho.

BARHL EXPRESSION DURING EMBRYONIC NERVOUS SYSTEM DEVELOPMENT

The homeobox gene barhl2 and its paralogue barhl1 differ in their expression pattern in the developing nervous system specifically in the developing caudal forebrain. Their expression has been well characterized in mice [START_REF] Bulfone | Barhl1, a gene belonging to a new subfamily of mammalian homeobox genes, is expressed in migrating neurons of the CNS[END_REF]Mo et al., 2004;[START_REF] Rachidi | Differential transcription of Barhl1 homeobox gene in restricted functional domains of the central nervous system suggests a role in brain patterning[END_REF] and rat [START_REF] Saito | Mammalian BarH homologue is a potential regulator of neural bHLH genes[END_REF] and in less extent, Zebrafish (Colombo et al., 2006) and Xenopus (Juraver-Geslin et al., 2011;Patterson et al., 2000).

BARHL1 EXPRESSION

The Zebrafish genome contains two copies of barhl1. Its expression is restricted to the diencephalon. Later, barhl1 is found on the prospective cerebellum. It is also expressed in the tectum and retina (Colombo et al., 2006).

In mice, barhl1 is first detected at embryonic day (E)9.5 in the caudal diencephalon, specifically in the ventral plate of prosomer (p)1 and p2. At E10.5 barhl1 expression extends from the isthmic boundary through the rhombencephalon until the dorsal spinal cord. By E11.5 it appears in the mesencephalic basal plate and its expression expands laterally in the spinal cord following the ventral migrating cells from the dorsal alar plate to the boundary between the basal and alar plate. At E12.5 its expression is restricted to the mammillary region in the diencephalon and in the mesencephalon its expression is located on the presumptive area of the superior colliculus. At post-natal stage, barhl1 is expressed in the colliculus, in the external cerebellar granule layer, in the pontine nucleus and in a restricted hypothalamic area [START_REF] Bulfone | Barhl1, a gene belonging to a new subfamily of mammalian homeobox genes, is expressed in migrating neurons of the CNS[END_REF][START_REF] Rachidi | Differential transcription of Barhl1 homeobox gene in restricted functional domains of the central nervous system suggests a role in brain patterning[END_REF].

In X. laevis embryo barhl1 is expressed in the neural plate starting at stage 14. At later stages barhl1 is expressed only in the basal forebrain (Patterson et al., 2000).

BARHL2 EXPRESSION

The Zebrafish genome contains one copy of barhl2. It is expressed in the diencephalon and later in the prospective epithalamus and thalamus. Later, it is also located in the dorsal telencephalon. Like barhl1, it is also be expressed in the tectum and retina but at higher levels than its homologue (Colombo et al., 2006).

In mice at E9.5, barhl2 is expressed in the alar diencephalon. At E10.5 it is located in the pretectum and throughout the thalamic ventricular zone. Its expression is excluded from the prethalamus [START_REF] Parish | Expression of Barhl2 and its relationship with Pax6 expression in the forebrain of the mouse embryo[END_REF]. At E11.5 its expression extends to the mesencephalon along the dorsal neural tube. From E14.5 to post-natal stage 1, barhl2 is found on neurons of the pyriform cortex and diagonal band of the telencephalon. In the post-natal diencephalon barhl2 is expressed in the epithalamic and intermediate thalamic neuroepithelium, ventral thalamus, preoptic area, paraventricular nucleus and mammillary body. It is also expressed in the upper rhombic lip and the external granule cell layer. In the retina, barhl2 is expressed by amacrine, horizontal and ganglion cells (Mo et al., 2004).

In X. laevis embryos barhl2 is expressed in the neural plate starting at stage 10,5 and specifically in the presumptive caudal forebrain from stage 14 onwards (Figure 12). At stage 24 barhl2 marks the p2 and at stage 31 barhl2 is expressed in the cortical hem, p2 and the midbrain (Juraver-Geslin et al., 2011;Offner et al., 2005;Patterson et al., 2000).

ROLES OF BARHL GENES IN VERTEBRATE DEVELOPMENT

barH genes were first described in Drosophila for having a role in retina neurogenesis [START_REF] Kojima | Identification of a different-type homeobox gene, BarH1, possibly causing Bar (B) and Om(1D) mutations in Drosophila[END_REF]. In this region, BarH interacts with the basic helix loop helix (bHLH) proneural gene atonal (ato) and restricts neurogenesis [START_REF] Lim | Bar homeodomain proteins are anti-proneural in the Drosophila eye: transcriptional repression of atonal by Bar prevents ectopic retinal neurogenesis[END_REF]. In mice, Barhl1 is important for the maintenance of cochlear hair cells. Its deletion results in hearing loss (Li et al., 2002).

Here I will focus on the main functions of barhl2 throughout vertebrate development.

ROLE OF BARHL2 IN ZLI FORMATION

In X. laevis, embryos depleted from Barhl2 have an abnormal zli formation and Shh secretion in this area is decreased. Juraver-Geslin and colleagues demonstrated that Barhl2 is important for the establishment of the zli (Juraver-Geslin et al., 2014). Its expression overlaps with that of the zli markers otx2 and irx3 and depletion of Barhl2 impairs the establishment of the p2 alar plate territories (Juraver-Geslin et al., 2014;Sena et al., 2016). In Zebrafish, loss of Otx2 induces a loss of Barhl2. Pax6 might also have a repressive role on barhl2. They are expressed in complementary areas during diencephalon development, and there are thirteen potential Pax6 binding sites in the transcription start site of barhl2 [START_REF] Parish | Expression of Barhl2 and its relationship with Pax6 expression in the forebrain of the mouse embryo[END_REF].

It has been described that Barhl2 and Otx2 bind to conserved cis-regulatory motifs in the shh promoter to induce zli formation in mice (Yao et al., 2016). Barhl2 acts upstream of Shh and plays a crucial role in establishing the positional identities of progenitor cells in the diencephalon (Q. Ding et al., 2017). The role of Barhl2 in the establishment of the zli and its interaction with the gene regulatory network in this region is described in (Sena et al., 2016) (Section IV, Annex 1).

A B

ROLE OF BARHL2 IN DIFFERENTIATION OF RETINAL CELLS

In X. laevis, barhl2 acts as a transcriptional repressor downstream of neuronal differentiation 4 (neurod4, a.k.a. ath3) and atonal bHLH transcription factor 7 (atoh7, a.k.a. ath5) to promote radial glial cells (RGC) markers expression [START_REF] Poggi | The homeobox gene Xbh1 cooperates with proneural genes to specify ganglion cell fate within the Xenopus neural retina[END_REF]Reig et al., 2007). In mice, barhl2 promotes differentiation of glycinergic amacrine cells [START_REF] Ding | BARHL2 Differentially Regulates the Development of Retinal Amacrine and Ganglion Neurons[END_REF]Mo et al., 2004).

This opposed role in both maintenance of pluripotency and differentiation of cells suggests that barhl2 function can vary depending on the cellular context.

ROLE OF BARHL2 IN APOPTOSIS

Barhl2 is expressed in the posterior dorsal mesoderm at neurula stages (Offner et al., 2005).

Barhl2 gain-of-function (GOF) leads to an increase in apoptosis in this area, especially in the midline, affecting the gradient of secreted Shh and Bmp.

A role of Barhl2 in controlling non-apoptotic functions of Caspase-3 has also been described

in Xenopus (Juraver-Geslin et al., 2011). Here, Barhl2 LOF induces a hyperplasia of the neural tube and increased proliferation via Caspase-3 in a non-apoptotic way. Importantly, Barhl2 Wingless pathway [START_REF] Kanuka | Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development[END_REF]. Genetic analysis in C. elegans demonstrate that ceh-30, the orthologue of barhl2, modifies Caspase-3 activity (Peden et al., 2007;Schwartz and Horvitz, 2007) indicating that Barhl2 function as a regulator of an unconventional Caspase-3 function may be conserved in metazoans.

BIOLOGICAL QUESTION AND PHD PROJECT

Embryonic development is a complex process that requires precise signaling between cells. In my first manuscript (Sena et al., in preparation), I present evidence that Barhl2 and Groucho interact in a complex containing the transcription factor Tcf7l1. Both in vitro and in vivo Barhl2 reinforces the binding between Groucho and Tcf7l1 and increases Wnt signaling repression. I provide a demonstration that Barhl2 normally limits formation of the Spemann organizer in X.

Many transduction pathways act

laevis embryos. I also demonstrate that Hdac1 is part of a protein complex containing Tcf7l1 and Gro4, and that Hdac1 activity mediates transcriptional silencing in this developmental context.

The laboratory had previously demonstrated that in X. laevis embryos Barhl2 increased the apoptosis naturally occurring in the axial organizer at gastrula stages (Offner et al., 2005).

However, the mechanism underlying this effect was unknown. In mice, it has been described PART II: RESULTS

BARHL2 REGULATES WNT SIGNALING 1.1 SCIENTIFIC CONTEXT

Wnt signaling is present in many developmental stages and is also important in adult organisms. It is a transducing pathway that when deregulated can lead to developmental malformations as well as pathologies such as cancer. Among its different roles, Wnt can regulate cell proliferation and differentiation, and in the embryo, it is responsible for the formation of the Spemann organizer. The Spemann organizer is the first embryonic organizer to appear and determines the corporal axis in the embryos.

The Wnt canonical pathway relies on the effector b-catenin to regulate gene transcription in the nucleus. This protein is regulated at its protein levels in the cytoplasm by a multiprotein During embryonic development, Wnt ligands have different roles depending on their location and time of expression. In the case of the Spemann organizer, Wnt has at first a dorsalizing role inducing formation of this structure, but when zygotic transcription begins Wnt is responsible for ventralizing cells. This is also translated into a switch between active to repressive Tcf in the dorsal cells where the organizer develops. The interaction of the complex formed by Groucho and Tcf with Wnt target genes to silence transcription seems to be implicated in this switch (Yasuoka et al., 2014).

In the following paper, we describe the interaction between the transcription factor Barhl2, Groucho and Tcf in vitro and in vivo. We show that this complex has an active role during the formation of the Spemann organizer as it controls the switch of Tcf from its active to its repressive form. Our results confirmed the importance of Groucho mediated repression on Spemann Organizer. Moreover, we provide evidence that the association between Barhl2, Groucho and Tcf is maintained in later developmental stages to mediate the confinement of a stem cell niche in the rhombic lip.

Introduction

Wnt/b-catenin signaling plays major roles in embryonic development, stem cell biology, regeneration, and tumorigenesis (Reviewed in (Clevers & Nusse, 2012;[START_REF] Holland | Wnt signaling in stem and cancer stem cells[END_REF][START_REF] Nusse | Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities[END_REF]). The key step for transcriptional activation of Wnt canonical activity is the increase of β-catenin levels within the nucleus where it engages T-Cell Factor (Tcf)/Lymphoid enhancer binding factor 1 (Lef1) bound on Wntresponsive enhancers [START_REF] Schuijers | Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF[END_REF]. b-catenin transiently converts Tcf into a transcriptional activator that induces its target genes ( [START_REF] Schuijers | Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF[END_REF]; Reviewed in (Behrens et al., 1996;Cadigan, 2012;Clevers & Nusse, 2012;Nakamura & Hoppler, 2017)). In the Wnt ''off'' state, Tcf interacts with Groucho (Gro)/Transducin-like Enhancer-of-split (Tle) proteins, and acts as a transcriptional repressor eventually through the recruitment of histone deacetylases (Hdac), which establish regional repressive chromatin structures (Chen, Fernandez, Mische, & Courey, 1999) (Reviewed in [START_REF] Laugesen | Chromatin repressive complexes in stem cells, development, and cancer[END_REF][START_REF] Seto | Erasers of histone acetylation: the histone deacetylase enzymes[END_REF]). During early vertebrate development Tcf, specifically Tcf7-like 1 (Tcf7l1 also known as Tcf3), is abundantly expressed [START_REF] Korinek | Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse[END_REF][START_REF] Roel | Lef-1 and Tcf-3 transcription factors mediate tissue-specific Wnt signaling during Xenopus development[END_REF]. The current model holds that inactivation of Tcf7l1-mediated repression is a core event in vertebrate organizer formation (Hikasa et al., 2010;[START_REF] Merrill | Tcf3: a transcriptional regulator of axis induction in the early embryo[END_REF]Molenaar et al., 1996;Roose et al., 1998;S. Schneider, Steinbeisser, Warga, & Hausen, 1996), early lineage specification (reviewed in [START_REF] Arnold | Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo[END_REF]), and regulation of pluripotency in mouse embryonic stem cells (Cole, Johnstone, Newman, Kagey, & Young, 2008;Tam et al., 2008;Yi et al., 2011;[START_REF] Yi | Tcf3 functions as a steady-state limiter of transcriptional programs of mouse embryonic stem cell self-renewal[END_REF]. Whereas the role of bcatenin in transcriptional activation of Wnt/Tcf target genes is largely documented (Reviewed in (Behrens et al., 1996;Cadigan, 2012;Clevers & Nusse, 2012;[START_REF] Nusse | Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities[END_REF])), the nature and function of other developmental controls on Tcf transcriptional activity remain elusive (reviewed in (S. Y. Sokol, 2011)). Understanding Tcf7l1 regulatory modes is therefore a priority.

In vertebrate embryos, important early developmental events are driven by signals from the organizer named the "Spemann organizer" in amphibians, and the "node" in birds and mammals. In amphibian and mice maternally encoded Tcf7l1 represses organizer genes transcription throughout the embryo (Houston et al., 2002;[START_REF] Kim | Repressor activity of Headless/Tcf3 is essential for vertebrate head formation[END_REF]. The dorsal development program is initiated by b-catenin that locally de-represses Tcf7l1 target genes in the presumptive organizer territory, a process counter-acted by overexpression of Gro (Blythe, Cha, Tadjuidje, Heasman, & Klein, 2010;Houston et al., 2002;Molenaar et al., 1996;Roose et al., 1998;S. Schneider et al., 1996;[START_REF] Tao | Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos[END_REF][START_REF] Xanthos | The roles of three signaling pathways in the formation and function of the Spemann Organizer[END_REF]Yasuoka et al., 2014). Whereas b-catenin driven transcriptional activation of organizer genes has recently been explored in depth (Ding et al., 2017;[START_REF] Kjolby | Genome-wide identification of Wnt/beta-catenin transcriptional targets during Xenopus gastrulation[END_REF][START_REF] Nakamura | Tissue-and stagespecific Wnt target gene expression is controlled subsequent to beta-catenin recruitment to cis-regulatory modules[END_REF], less is known on Gro co-repressor activities in organizer development.

Gro proteins do not have a DNA binding domain. They rely on their interaction with transcription factors such as Tcf for their specific recognition of promoter and/or enhancer sequences. Gro proteins bind Tcfs via their glutamine-rich (Q) domain (Cadigan & Waterman, 2012). They also contain a WD-repeat known to interact with the Engrailed Homology-1 (Eh1) motif present in numerous homeodomain-containing transcription factors (Supplementary Figure 1) (Jennings & Ish-Horowicz, 2008;[START_REF] Jimenez | Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed[END_REF][START_REF] Pickles | Crystal structure of the C-terminal WD40 repeat domain of the human Groucho/TLE1 transcriptional corepressor[END_REF]Smith & Jaynes, 1996;Yasuoka et al., 2014). Barhlike homebox 2 (Barhl2) is an evolutionarily conserved member of the BarH family of transcription factors (Schuhmacher, Albadri, Ramialison, & Poggi, 2011). Whereas most homeodomain-containing proteins possess one Eh1 repression domain vertebrates Barhl proteins contain two Eh1 motifs (Offner, Duval, Jamrich, & Durand, 2005). In amphibian Barhl2 limits Wnt/b-catenin activity in the neural tube (Juraver-Geslin, Ausseil, Wassef, & Durand, 2011), and participates to formation of the axial organizing center, which derives from the Spemann organizer (Offner et al., 2005). Little is known about its molecular mode of action.

In the present study we investigated how Barhl2 limits Wnt transcriptional activity using bcatenin-dependent induction of the Xenopus organizer as a model system. Using embryological and real time (RT)-qPCR approaches we demonstrate that Barhl2 is expressed in the Xenopus blastula, and normally limits the size and the signaling activity of the Spemann organizer. Using co-immunoprecipitation (co-IP) assays we show that Barhl2 directly binds Gro via its Eh1 domains, interacts with Tcf7l1 independently of Gro, and promotes the recruitment of Gro on Tcf7l1. Using Wnt/Tcf reporter activity assays in mammalian cells and a

Xenopus tropicalis (X. tropicalis) Wnt/Tcf reporter line, we demonstrate that Barhl2 limits Tcf transcriptional activation in vitro and in vivo, and consequently switches off Spemann organizer formation. We also show that Barhl2 belongs with Gro to a multipartite protein complex containing Tcf7l1 and Hdac1 and promotes Hdac1-dependent repression of Spemann organizer formation. Finally our data in a X. tropicalis Wnt/Tcf reporter line demonstrate that Barhl2 normally enhances Tcf7l1 repressor activity in the cerebellar rhombic lip, and the cortical hem, two germinative zones that produce neuronal progenitors at postembryonic stages [START_REF] Ben-Arie | Math1 is essential for genesis of cerebellar granule neurons[END_REF][START_REF] Takiguchi-Hayashi | Generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles[END_REF].

Results

Barhl2 limits the size of the organizer

We first assessed the effect of Barhl2 gain (GOF) and loss-of-function (LOF) in stage 25Xenopus laevis (X. laevis) embryos (Figure 1A). We injected two previously characterized morpholino (MO) against Barhl2 (MObarhl2-1 andMObarhl2-2 previously Xbarhl2ASI andXbarhl2ASII), or a control MO (MObarhl2-ct previously Xbarhl2ASIII) (Offner et al., 2005), or mRNA coding for either X. laevis or mouse barhl2 in two dorsal blastomeres at the 2-cell stage together with b-gal or gfp mRNA as tracer. We observed similar effects for Barhl2 depletion with the two specific MOs that is an anteriorization of embryos that exhibited an enlarged cement gland and an expansion of dorsal structures. Barhl2 GOF produces the reverse phenotype that is ventralized embryos lacking anterior structures. Injection of the MObarhl2-ct was phenotypically silent. For each condition the penetrance of the phenotype was assessed (Figure 1A). As the effects of MObarhl2-1 and MObarhl2-2 were similar, we refer to it as

MObarhl2. These observations indicate that Barhl2 influences establishment of the dorsoventral (DV) axis in Xenopus.

We next investigated barhl2 expression in X. laevis embryos between blastula and gastrula stages. Using RT-qPCR, we detected a peak of barhl2 transcripts at stage 8.5 (Figure 1B). An average of 4000 barhl2 transcripts are reported to be present in blastula stage embryos whereas about 10 5 barhl2 transcripts are detected at stage 14 (Owens et al., 2016;Session et al., 2016). Using whole-mount in situ hybridization (ISH) we detected barhl2 in the presumptive organizer region, a territory known to participate in formation of the dorsal blastopore lip (Figure 1B) (Sudou, Yamamoto, Ogino, & Taira, 2012). At stage 9 we also detected transcripts coding for barhl1 the closest homologue of barhl2 (Figure 1B; (Session et al., 2016)). Therefore, barhl2 transcripts are present at the right place and time for this transcription factor to participate in formation of the organizer.

We next investigated the impact of Barhl2 GOF and LOF on Spemann organizer formation.

MObarhl2 together with a tracer were injected either into one blastomere (Figure 1C), or into two blastomeres (Figure 1D) at the 2-cell stage in X. laevis embryos. The expression of dorsal mesodermal genes as that of ventro-lateral markers was analyzed using either ISH and/or RT-qPCR. When analyzed at stage 10, barhl2 overexpression decreased both chordin and goosecoid (gsc) expression whereas Barhl2 depletion generated an increase in expression of both markers (Figure 1C).

For chordin ISH we further quantified differences between the injected and control sides using an ImageJ macro (see Material and Methods and Figure 1C). In both GOF and LOF embryos we measured significant differences between the injected and control sides for the surface of chordin stained areas and their integrated densities (Figure D; Supplementary Figure 1)).

Using RT-qPCR on embryos depleted for Barhl2 activity, we quantified the expression changes in organizer genes: siamois1 (sia1), barhl2, chordin, gsc, orthodenticle homebox 2 (otx2), notochord homebox (not), and ventro/lateral mesoderm markers: vent homebox 2 (vent2), wnt family member 8a (wnt8a), brachyury, between stage 8 and stage 10 (Figure 1E). We measured an increase of 2-to 5-fold in the expression of all dorsal mesodermal genes analyzed. The increase in RNA levels started at stage 9 for gsc and chordin, at stage 10 for otx2 and not. In contrast we measured a decrease in all ventro/lateral mesoderm markers starting at stage 9 for vent2, and stage 10 for wnt8a and brachyury. Finally, we did not detect any change in the early expression of sia1 at stage 8 and 9 (Figure 1D). Representative embryos depleted for Barhl2 using probes against otx2, not and vent2 are shown (Figure 1F).

In conclusion, in Xenopus embryos barhl2 participates to establishment of the DV axis. The mRNA coding for barhl2 is expressed in the presumptive organizer territory at midblastula stage. Depletion of Barhl2 activity promotes the formation of dorsal mesodermal tissues at the expense of the ventro/lateral mesodermal tissues. These results indicate that Barhl2 normally limits extension of the Spemann organizer between stage 8 and stage 10.

Barhl2 limits organizer signaling and indirectly affects neural plate specification

During gastrulation the Spemann organizer, and the structures that derive from it, the prechordal plate and the notochord, send planar and vertical signals -Bone Morphogenetic Protein (Bmp) inhibitors, Sonic Hedgehog (Shh), Fibroblast Growth Factors (Fgfs) and Wnt pathway factors. These signals induce and pattern dorsal structures including the mesoderm and the emerging nervous system (reviewed in (De Robertis & Kuroda, 2004;[START_REF] Niehrs | Regionally specific induction by the Spemann-Mangold organizer[END_REF]Stern, 2006)). We asked whether changes in Barhl2 activity affect organizer function, and consequently specification of the neuroepithelium.

Our previous analysis of Barhl2 GOF and LOF early neurula phenotypes demonstrate that Barhl2 influences antero-posterior (AP) and DV neuroepithelium specification, and changes the Shh and BMP signaling activity gradients within the neural plate (Juraver-Geslin et al., 2011;Offner et al., 2005). Using sry-box 3 (sox3), an early marker of the neural plate, cyclinD1 (ccnd1) normally only expressed in the anterior neural plate and regulated by both Shh and Wnt signaling [START_REF] Borday | Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation[END_REF], and forkhead box B1 (foxb1), a marker of the hindbrain and the diencephalic territories [START_REF] Gamse | Early anteroposterior division of the presumptive neurectoderm in Xenopus[END_REF], we further observed that in neurula embryos Barhl2 depletion generates a widening of the neuroepithelium (Figure 2A, B), associated to an ectopic expression of ccnd1 in the posterior neural plate (Figure 2C).

All these phenotypic changes in neural plate specification could be indirect consequences of Barhl2 requirement for organizer formation. To exclude the possibility that Barhl2, present in undetectable amount in the neuroepithelium, acted by changing the response of neuroepithelial cells to extracellular signals we targeted Barhl2 depletion in different parts of the dorsal tissues. Xenopus embryos were injected into one dorsal blastomere at eight-or sixteen-cell stage with MObarhl2 and β-gal RNA as a tracer. Based on the expression of β-Gal we assessed retrospectively whether MObarhl2 was injected in the entire neural plate, or in the axial midline, or in medio-lateral neuroectodermal cells (Figure 2F). We followed the increase in neural plate size using sox3 as a neural plate marker. We quantified the neural plate enlargement phenotype for each type of injections (Figure 2D, F). We observed that Barhl2 depletion either in the entire neural plate, or in the midline (axial cells, derived from the organizer) generated the characteristic increase in sox3 territory (Figure 2D-F). In contrast injection of MObarhl2 in the medio-lateral neuroectodermal cells had no effect on the size of the neuroepithelium (Figure 2E,F).

Therefore, Barhl2 acts on Spemann organizer signaling activity. Barhl2 LOF and GOF phenotypes on the neural plate size are at least partly an indirect consequence of its activity on the Spemann organizer.

Barhl2 ability to limit the organizer size requires its binding to DNA and a Eh1-mediated protein-protein interaction

In Xenopus embryos Gro suppresses Tcf7l1 dependent axis formation and represses transcription of Tcf/b-catenin target genes (Roose et al., 1998). Barhl2 protein contains a homeodomain, a nuclear localization sequence (NLS) and two Eh1 domains known to interact with Gro (Figure 3A).

We asked whether Barhl2 activity on organizer formation requires its DNA binding domain, its Eh1 domains, or both. In the barhl2 full-length (barhl2fl) cDNA we either removed the Eh1 domains to generate barhl2∆EHs or mutated 3 aminoacids (AAs) in each Eh1 motif to generate barhl2flEHsM. We also mutated 4 AAs within the helix 3 of the homeodomain, known to be instrumental for the correct interaction between the homeodomain and the target DNA, to generate barhl2flHoxM [START_REF] Burglin | Homeodomain proteins: an update[END_REF] (Figure 3A). The barhl-derived constructs were injected into one dorsal blastomere of Xenopus embryos at the 4-cell stage, together with β-gal as tracer. We assessed the size of the neural plate using sox3 as a marker. At stage 14, barhl2 GOF causes a decrease in the size of the neural plate whereas Barhl2 depletion generates its enlargement (Figure 3Ba, e) (Offner et al., 2005). Overexpression of Barhl2 with a mutated homeodomain (barhl2flHoxM) or depleted of its N-terminal part (barhl2∆EHs), or with two mutated Eh1 domains (barhl2flEHsM), did not affect the size of the neural plate (Figure 3Bb-d; C). These results suggest that this activity of Barhl2 requires both its DNAbinding domain and its Eh1 domains.

To further investigate the function of the Eh1 motif of Barhl2 we generated barhl2EHs and barhl1EHs that contain the N-terminal part of barhl2 or barhl1 cDNA respectively. In the barhl2EHs construct we mutated 3 AAs in each Eh1 motif either separately or together to generate respectively barhl2EH1M, barhl2EH2M, and barhl2EHsM (Figure 3A). We overexpressed these barhl-derived constructs and analyzed their effect on neural plate size.

Overexpression of the N-terminal part of Barhl2 containing at least one functional Eh1 domain (barhl2EH1M and barhl2EH2M), or overexpression of Barhl1 N-terminal part (barhl1EHs), mimics the Barhl2 depletion phenotype (Figure 3Bf-i). This activity is dependent on the presence of functional Eh1 domains: overexpression of barhl2EHsM, whose Eh1 domains have been mutated, did not cause any change in neural plate size (Figure 3Bj). A quantification of the phenotypes is shown on Figure 3C.

These results suggest that overexpression of the N-terminal part of Barhl2 (Barhl2EHs) interferes with Barhl2 normal activity. To test whether Barhl2EHs is indeed a dominant negative form of Barhl2, we first assessed its activity on Spemann organizer, and also its ability to rescue the Barhl2 overexpression phenotype. Using chordin and not as markers we followed development of the notochord -a Spemann organizer derivative. In agreement with its normal role in limiting Spemann organizer size, Barhl2 depletion increases the size of the notochord whereas Barhl2 overexpression decreases it (Figure 3Da,b; Ea,b) (Offner et al., 2005). We also observed that Barhl2 N-terminal part (Barhl2EHs) increased notochord size whereas Barhl2EHsM, whose EH1 domains have been mutated, did not (Figure 3Dc,d;Ec,d).

Moreover, at stage 10 barhl2EHs overexpression expands the expression of other organizer markers, chordin, gsc and otx2, decreases the expression of the ventro-lateral mesodermal marker vent homebox 1 (vent1) (Figure 3F). Therefore, similarly to Barhl2 depletion, overexpression of its N-terminal part (Barhl2EHs) increases Spemann organizer size. We also assessed and quantified the neural plate enlargement phenotype in embryos injected with RNAs coding for Barhl2 and/or Barhl2EHs separately or together. Indeed, Barhl2EHs overexpression can rescue the Barhl2 GOF phenotype (Figure 3G).

We propose that the ability of Barhl2 to limit the size of the Spemann organizer requires its binding to DNA and an Eh1-mediated protein-protein interaction, and that this is efficiently antagonized by overexpression of the N-terminal part of Barhl2, which contains two Eh1 motifs.

Barhl2EHs competes with Barhl2fl for Gro binding

Transcripts coding for Gro1, 2 and 4 proteins are present in blastula embryos (Owens et al., 2016). We hypothesized that Barhl2 N-terminal part (Barhl2EHs) acts as a dominant negative of Barhl2 by binding and buffering Gro proteins present in the blastula embryos. We first investigated interactions between Barhl2EHs, Barhl2fl, and Gro performing co-IP experiments on protein extracts from HEK293T cells transfected with vectors encoding Myc, Flag, HA, AM or GFP tagged version of the proteins (Supplementary Figure 1). Gro4 (Gro) has been demonstrated to be required for Xenopus organizer formation [START_REF] Roose | qui augmente le temps de pause de l'ARN pol II sur les séquences promotrices -processus d'atténuation-et/ou recrute l'histone désacétylase 1 (Hdac1) pour favoriser la répression transcriptionnelle longue distance ou silencing[END_REF]. However Gro1 and Gro2 also act as co-repressors and interact with both Tcf and Eh1 motifs (Houston et al., 2002;Roose et al., 1998). We investigated Barhl2EHs ability to interact with Gro4 and Gro1. We observed that Barhl2EHs co-immunoprecipitates both Gro1

and Gro4 but Gro1 less efficiently that Gro4 (Supplementary Figure 2). We therefore performed all our experiments using vectors coding for gro4.

We first observed that Barhl2fl co-immunoprecipitates Gro (Figure 4A). We next asked whether Barhl2EHs binds Gro through its Eh1 domains, and whether it competes with Barhl2fl for Gro binding. We observed that Gro co-immunoprecipitates with Barhl2EHs but not with Barhl2EHsM whose Eh1 domains have been mutated (Figure 4B). In co-IP experiments using a limiting amount of Gro as bait in the presence of Barhl2fl, we observed that increasing the amount of Barhl2EHs diminishes the interaction of Gro with Barhl2fl. These results suggest that there is direct competition between Barhl2 N-terminal part and Barhl2fl for binding to Gro via the Eh1 motifs (Figure 4C).

We next investigated the subcellular localization of Gro in HeLa cells in the presence or absence of Barhl2fl, Barhl2EHs and Barhl2EHsM. In agreement with their activities on transcription, both Barhl2fl and Gro were detected inside the nucleus when transfected separately or together (Supplementary Figure 3A, B; Figure 3Da-c). Barhl2EHs and Barhl2EHsM were detected in the nucleus and the cytoplasm (Supplementary Figure 3C,D).

However, when Gro was co-transfected with Barhl2 N-terminal part (Barhl2EHs) we observed a significant presence of Gro protein in the cytoplasm (Figure 4Dd-f). This shift in sub-cellular localization was not observed when Gro was co-transfected with Barhl2EHsM (Figure 4Dg-i).

Then, we investigated the functional consequences of Barhl2EHs interaction with Gro. Gro inhibits activation of synthetic Tcf reporter gene transcription in transient transfection assays (Roose et al., 1998). We asked whether Barhl2EHs inhibits Gro counter-activity on β-catenin transactivation of pTop-Flash, a Wnt-reporter construct that carries 3 Tcf-binding sites upstream of the luciferase reporter gene [START_REF] Korinek | Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/-colon carcinoma[END_REF]. Mean luciferase activities were measured in HEK293T cells transfected with pTop-Flash, a constitutively active form of ß-cat (ß-cat*) [START_REF] Wei | Identification of the LIM protein FHL2 as a coactivator of beta-catenin[END_REF], Gro alone or together with Barhl2EHs or with Barhl2EHsM used as a control (Figure 4E). pTop-Flash activity was measured relative to the pFOP-flash reporter, an altered version of pTOP-flash carrying point mutations in the Tcf-binding sites to render it unresponsive to ß-cat activity [START_REF] Korinek | Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/-colon carcinoma[END_REF]. We observed a strong decrease of pTop-Flash activity when ß-cat* was co-transfected with Gro. This Gro-induced decrease of pTop-Flash activity was significantly alleviated in the presence of Barhl2EHs, but not in the presence of Barhl2EHsM (Figure 4E).

Therefore, Barhl2EHs competes with Barhl2fl for binding to Gro independently of the presence of DNA and interferes with Barhl2 normal function. Barhl2EHs induces a nuclear to cytoplasmic translocation in Gro and alleviates Gro inhibition of Tcf transcriptional activity. Our data argue that Barhl2 N-terminal part (Barhl2EHs) captures Gro and prevents its co-repressor activity, at least on Tcf.

Barhl2 binds Gro and Tcf7l1 independently and in cells enhances the Tcf7l1/Gro interaction

These data indicate that Barhl2 binding to Gro is crucial for its activity in regulating the size of the Spemann organizer. We next investigated interactions between Barhl2, Gro and Tcf by performing co-IP experiments in HEK293T cells using tagged versions of Xenopus Tcf7l1, which together with Gro4 mediates Tcf repressor activity in blastula stage embryos (Supplementary Figure 1) (Houston et al., 2002;Roose et al., 1998).

Using Tcf7l1 as bait, we asked whether Barhl2 binds Tcf, and whether it acts on formation of the Tcf-Gro complex. We first observed that Tcf7l1 co-immunoprecipitates with Barhl2fl both in the presence and in the absence of Gro (Figure 5A). Tcf7l1 co-immunoprecipitates with Barhl2fl, and in a similar way with Barhl2flEHsM whose Eh1 motifs have been mutated (Figure 5A). Therefore, Barhl2 binds to Tcf independently of Gro.

We observed that in the absence of Barhl2fl, the interaction between Tcf7l1 and Gro could only be detected when the co-IP experiment was performed overnight (Figure 5B), but not when it was performed for 2h (Figure 5A). Moreover, in the presence of Barhl2fl the ability of Tcf7l1 to co-immunoprecipitate with Gro was dramatically increased: after 2h Tcf7l1 immunoprecipitated Gro very efficiently (Figure 5A). The effect is dependent on the Eh1 motifs because Barhl2flEHsM, whose Eh1 motifs have been mutated, does not promote the ability of Tcf7l1 to precipitate Gro (Figure 5A).

We investigated whether the ability of Barhl2 to stabilize the Tcf7l1-Gro interaction limits the switch from the repressive to the active form of Tcf7l1 normally induced by b-catenin. Mean luciferase activities of pTop-Flash relative to pFOP-flash reporter were measured in HEK293T cells transfected with increasing amounts of ß-cat* together with two different doses of a vector coding for Barhl2fl. Increasing doses of ß-cat* generated increasing levels of pTop-Flash activity. When pTop-Flash and ß-cat* were co-transfected with Barhl2fl, luciferase activity decreases at all doses of β-cat* tested, and the decrease was Barhl2 dose-dependent (Figure 5C).

We next asked whether Barhl2EHs limits Barhl2 ability to stabilize the Tcf7l1-Gro interaction.

We transfected HEK293T cells with pTop-Flash together with b-cat* alone, together with Barhl2fl, or in combination with Barhl2fl and Barhl2EHs. As previously described, we observed a decrease of pTop-Flash activity when b-cat* was co-transfected with Barhl2fl compared with b-cat* alone (Figure 5D). In contrast, cotransfection of Barhl2EHs reverted the inhibitory activity of Barhl2fl on b-cat* activation of pTop-Flash (Figure 5D).

Therefore, in cells Barhl2fl interacts with Gro through its Eh1 motifs, binds Tcf7l1 independently of Gro, stabilizes the interaction between Tcf7l1 and Gro and prevents β-catenin driven activation of Tcf target genes. Barhl2EHs prevents Barhl2fl inhibitory activity on β-catenin mediated induction of pTop-Flash.

Barhl2 promotes transcriptional repression of Tcf target genes in embryos

Results in the previous sections suggest that Barhl2 limits Spemann organizer development by blocking β-catenin induced de-repression of Tcf7l1 target genes.

To determine whether Barhl2 interacted with Gro and Tcf7l1, and whether Barhl2EHs interacts with Gro in Xenopus embryos, we performed co-IP experiments using protein extracts from stage 10 Xenopus embryos injected in two blastomeres at the two-cell stage with RNA coding for Gro4-HA, Tcf7l1-Myc, Barhl2-HA and Barhl2EHs-Gfp as indicated (Figure 6A). Similarly to our observations in HEK293T cells, we observed that in Xenopus embryos Gro coimmunoprecipitated Barhl2EHs (Figure 6A), and Barhl2fl (Figure 6B). We observed that Tcf7l1 co-immunoprecipitated Gro4, Barhl2fl, and Barhl2fl together with Gro4 (Figure 6C).

Interestingly we did not detect the enhancement of Tcf7l1, Gro4 interaction previously observed in HEK293T cells.

To test whether our observations are relevant in vivo, we investigated the impact of Barhl2 on the Tcf repressor to Tcf activator switch normally occurring in Xenopus embryos. We first asked whether Barhl2 prevents the axis duplication normally induced by a ventral injection of wnt8b RNA, which increases β-catenin activity (S. Sokol, Christian, Moon, & Melton, 1991).

RNA coding for wnt8b was injected into one ventral blastomere at the 4-cell stage either alone, or together with Xbarhl2fl or with mbarhl2fl RNA, using gfp as tracer. Injection of wnt8b induces formation of double axis in all embryos, whereas a ventral injection of Xbarhl2fl or of mbarhl2fl was phenotypically silent (Figure 6D). We observed that injection of Xbarhl2fl or of mbarhl2fl together with wnt8b RNA abolishes the number of embryos developing secondary axis (Figure 6D). Co-injection of MObarhl2 with wnt8b RNA had no effect. However, the presence of MObarhl2 abolishes Xbarhl2fl inhibitory effect on wnt8b double axis induction, and in similar conditions had no influence on mbarhl2 RNA ability to block Wnt8b inducing effect (Figure 6D).

Therefore, Barhl2 inhibits wnt8b ability to induce secondary axis in Xenopus embryos.

Quantification of the experiments and its statistical significance is shown on Figure 6E.

We then used a X. tropicalis transgenic Wnt reporter line called pbin7LefdGFP (Tran, Sekkali, Van Imschoot, Janssens, & Vleminckx, 2010). This transgenic line contains one copy of a Wnt reporter gene consisting of a synthetic promoter carrying seven copies of an optimal binding sequence for Tcf7l1 upstream of an eGFP coding sequence (Supplementary Figure 4). This line allows read-out of Wnt/Tcf activity in vivo, through ISH using a gfp antisense probe or RT-qPCR for gfp [START_REF] Borday | Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation[END_REF]Tran et al., 2010). We injected MObarhl2 with a tracer, in dorsal blastomeres of X. tropicalis transgenic embryos at the 2-cell or 4-cell stage either unilaterally for ISH, or on both sides for RT-qPCR. At stage 8.5 we observed a localized Tcf activity in an area corresponding to the presumptive organizer territory (Figure 6Fa). Barhl2

depletion increased the number of Wnt-responsive cells in this territory at both stages 8 and 8.5 (Figure 6Fb,c). A RT-qPCR quantification of Barhl2 depletion effect at stage 8.5 demonstrated a 2-fold increase in gfp mRNA levels in Barhl2 depleted embryos compared to control (Figure 6G). Therefore, Barhl2 depletion increases Tcf activity in Xenopus blastula.

Whereas at stage 8.5 Tcf activity marks the presumptive organizer territory (Figure 6Fa), at stage 10 the activity of Tcf is null within the dorsal blastopore lip (Figure 6Ha). We injected either RNA coding for barhl2, or MObarhl2 in one dorsal blastomere of X. tropicalis transgenic embryos at the 2-cell or 4-cell stage. We assessed Tcf activity in these embryos. At stage 10.5 overexpression of Barhl2 diminishes the size of the dorsal blastopore lip area exhibiting a null activity for Tcf (Figure 6Hb,c). On the contrary depletion of Barhl2 enlarges the dorsal mesoderm territory (Figure 6Hd, e). The Spemann organizer expresses a number of genes encoding growth factor antagonists including Wnt antagonists such as Frzb-1 (Frizzled), Crescent, sFRP-2, Dickkopf and Cerberus (Reviewed in (De Robertis & Kuroda, 2004;[START_REF] Niehrs | Regionally specific induction by the Spemann-Mangold organizer[END_REF]). We observed that a decrease in the organizer size upon Barhl2 overexpression results in an increase in Wnt/Tcf activity on the injected side probably due to a diminution of Wnt antagonists' secretion (Figure 6Hb,c). Depletion of Barhl2 activity leads to the reverse phenotype (Figure 6Hd, e).

Our data argue that in blastula embryos Barhl2 promotes Tcf7l1 repressor activity. Within the dorsal animal territory Barhl2 limits β-catenin induced de-repression of Tcf7l1 target genes and contributes to repress transcription of organizer genes.

Hdac1 activity participates in Barhl2 mediated transcriptional repression of organizer genes

The Tcf-Gro complex normally acts as a transcriptional repressor. Both Tcf and Gro interact with Hdac1, a chromatin compacting enzyme known to have a global repressive effect on transcription (Billin, Thirlwell, & Ayer, 2000;Chen et al., 1999). Little is known about the function of these interactions during early development. We asked whether Hdac1 activity participates in Barhl2-mediated repression of Tcf target genes during organizer formation.

We first investigated whether Barhl2fl influences the ability of Tcf7l1 to recruit Hdac1, and/or influences the ability of Tcf7l1 to recruit Gro in the presence of Hdac1. As previously described, Tcf7l1 co-immunoprecipitates with Hdac1 (Billin et al., 2000), irrespective of the presence or absence of Gro (Figure 7A). In agreement with our previous observations in the presence of Hdac1 the ability of Tcf7l1 to recruit Gro is dramatically increased by Barhl2fl and Tcf7l1

immunoprecipitates a complex containing Barhl2fl, Gro4 and Hdac1. The interaction of Tcf7l1 and Barhl2fl with Hdac1 appears independent of Gro and of the EH1 motifs as it is also observed with Barhl2flEHsM (Figure 7A).

We next tested whether Barhl2fl interacted with Hdac1 independently of Tcf7l1 or of Gro4. We performed co-IP experiments using Barhl2 as bait in the presence of Gro4 and Hdac1. Barhl2 precipitated Hdac1, as expected Gro4, and both proteins together (Figure 7B). These results indicate that Barhl2, Tcf7l1, Gro4 and Hdac1 are part of the same protein complex.

To investigate the importance of Hdac1 activity in Spemann organizer formation we asked whether Barhl2EHs inhibits Gro interaction with Hdac1. We performed co-IP experiments using Gro in limiting amount as bait in the presence of Hdac1 and increasing amounts of Barhl2EHs (Figure 7C). We observed that increasing the amount of Barhl2EHs diminishes the interaction of Gro with Hdac1 arguing that Barhl2EHs prevents Gro interaction with Hdac1.

We next investigated whether Hdac1 activity contributes to Barhl2 driven transcriptional repression of Tcf target genes during organizer formation. We first asked whether Hdac1 activity participates in the repression of Tcf7l1 target genes in Xenopus blastula embryos. We depleted Hdac1 activity using previously characterized MOhdac1 (Y. Tao, Ruan, Guo, [START_REF] Tao | HDAC1 regulates the proliferation of radial glial cells in the developing Xenopus tectum[END_REF] injected unilaterally or on two sides of the pbin7LefdGFP X. tropicalis transgenic embryos at the 2-cell and 4-cell stages. We measured Wnt/β-catenin activity at stage 8.5 using either ISH or RT-qPCR for gfp. We observed an increase in the number of Wnt-responsive cells in the Hdac1 depleted territory (Figure 7D), and an increase in the levels of gfp mRNA in embryos depleted for Hdac activity (Figure 7E). Using RT-qPCR on stage 10 we observed an increase in both chordin and gsc mRNA levels in embryos depleted for Hdac1 activity (Figure 7F). Therefore, Hdac1 participates in transcriptional repression in blastula embryos.

We further asked whether Hdac1 participates in Barhl2 repression of organizer genes. We investigated whether depletion of Hdac1 activity prevents Barhl2 inhibition of organizer formation. barhl2 RNAs and/or MOhdac1 were injected separately or together in one dorsal blastomere in X. laevis embryos at the 2-cell stage. The organizer size was assessed by following the expression of the dorsal blastopore lip marker, chordin. As previously described (Figure 1C, D) we observed a decrease in chordin expression within the barhl2-injected side of stage 10.5 embryos (Figure 7Ga). In contrast, depletion of Hdac1 activity generated an enlargement of the chordin expression territory (Figure 7Gb). When both MOhdac1 and barhl2

RNA were injected together, we observed a rescue of the Barhl2 overexpression phenotype (Figure 7Gc,H). We quantified changes in chordin expression in barhl2-, MOhdac1-and barhl2

together with MOhdac1-injected embryos. Indeed coinjection of barhl2 together with MOhdac1 significantly rescues the changes in organizer size (Figure 7H).

In conclusion, Barhl2 does not affect the affinity of Gro for Hdac1, but rather promotes the recruitment of Gro to a protein complex containing Tcf7l1 and Hdac1. Hdac1 activity participates in Barhl2 driven transcriptional repression of Tcf7l1 target genes during Spemann organizer formation.

Barhl2 limits the Tcf repressor to activator switch in other biological processes

Our data suggest that Barhl2 plays an important part in an Hdac1 dependent repression process that extinguishes Tcf7l1 target genes transcription during Spemann organizer formation. Genetic and functional evidence argue that Tcf7l1 mostly acts as a transcriptional repressor during embryonic development (Houston et al., 2002;[START_REF] Kim | Repressor activity of Headless/Tcf3 is essential for vertebrate head formation[END_REF]Liu, van den Broek, Destree, & Hoppler, 2005). barhl2 is expressed from late neurulation onwards in the developing neural tube (Juraver-Geslin et al., 2011;Juraver-Geslin, Gomez-Skarmeta, & Durand, 2014;Offner et al., 2005). We asked whether Barhl2 activity as a repressor of Tcf transcriptional activation is operative in neural progenitors at later developmental stages. We chose developmental stage 41, when embryonic development is considered complete. We focused on two neural germinative zones known in mice to generate pools of neural progenitors at postembryonic stages: the cerebellar rhombic lip and the cortical hem (Figure 8A). The mouse cerebellar rhombic lip exhibits Tcf transcriptional activity at late developmental stages [START_REF] Selvadurai | Wnt/beta-catenin signalling is active in a highly dynamic pattern during development of the mouse cerebellum[END_REF]. It is a source of migratory neuron populations including granular neurons, a subpopulation of neurons of the cerebellar nuclei, and neurons of several pre-cerebellar nuclei [START_REF] Ben-Arie | Math1 is essential for genesis of cerebellar granule neurons[END_REF]. The cortical hem is a telencephalic organizing center, which expresses Wnt ligands including wnt2b and wnt3a [START_REF] Grove | The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice[END_REF]. The mouse cortical hem contains the progenitors of the Cajal-Retzius cells that migrate to the neocortex [START_REF] Takiguchi-Hayashi | Generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles[END_REF]. We first investigated barhl2 expression relative to Wnt/Tcf transcriptional activity. We observed that at stage 38 barhl2

expression borders that of Tcf activity in the cerebellar rhombic lip and in the cortical hem (Figure 8A).

We performed barhl2 GOF and LOF in the X. tropicalis pbin7LefdGFP transgenic line and followed Tcf transcriptional activity during embryogenesis. MObarhl2, or mRNA coding barhl2, was injected with a tracer at the 4-cell or 8-stage in one dorsal blastomere of X. tropicalis transgenic embryos. Stage 41 embryos injected unilaterally in the anterior neural tube were selected. Expression of gfp was followed using ISH (Figure 8B). We quantified relative differences in areas and pixel intensities between the injected and control sides in transverse sections using the ImageJ macro for normalization (Figure 8C, D; Supplementary Figure 5A-D) and using fluorescence red staining as a reference for the injected area. We observed that Barhl2 depletion induces a significant increase of Tcf activity in both the cerebellar rhombic lip and the cortical hem. In contrast, Barhl2 GOF decreases Tcf activity in both structures (Figure 8B-D).

We conclude that the ability of Barhl2 to enhance Tcf repressor activity is not a phenomenon specific to early development but plays a role at later developmental stages.

Discussion

The most important finding of this study is that the transcription factor Barhl2 normally enhances Tcf7l1 transcriptional repressive activity. This function of Barhl2 contributes to limit Spemann organizer size and activity. It contributes to driving Hdac1 activity on Tcf target genes (Figure 9). It participates in the biology of germinative zones at later developmental stages.

Whereas it had been previously proposed that β-catenin displacement of Gro is responsible for activation of the Wnt/β-catenin pathway (Cadigan, 2012;Daniels & Weis, 2005;[START_REF] Roose | qui augmente le temps de pause de l'ARN pol II sur les séquences promotrices -processus d'atténuation-et/ou recrute l'histone désacétylase 1 (Hdac1) pour favoriser la répression transcriptionnelle longue distance ou silencing[END_REF], recent studies revealed that Gro is not simply displaced by β-catenin, but the whole Tcf7l1-Gro repressor complex is replaced by an activator complex containing another Tcf (Tcf7) associated to β-catenin that promotes gene activation [START_REF] Chodaparambil | Molecular functions of the TLE tetramerization domain in Wnt target gene repression[END_REF]Hikasa et al., 2010). In all cases the switch from repression to activation of transcription of Wnt/β-catenin target genes is controlled by the stability of a functional Tcf7l1-Gro repressor complex, which can promote compaction of chromatin when the pathway is switched off [START_REF] Shy | Regulation of Tcf7l1 DNA binding and protein stability as principal mechanisms of Wnt/beta-catenin signaling[END_REF]. Tcf7l1 gene disruption in Xenopus (Houston et al., 2002;[START_REF] Kim | Repressor activity of Headless/Tcf3 is essential for vertebrate head formation[END_REF]Liu et al., 2005), zebrafish [START_REF] Kim | Repressor activity of Headless/Tcf3 is essential for vertebrate head formation[END_REF], and mice [START_REF] Shy | Regulation of Tcf7l1 DNA binding and protein stability as principal mechanisms of Wnt/beta-catenin signaling[END_REF] indicate that Tcf7l1 mostly acts as a transcriptional repressor during embryonic development. Our study establishes that Barhl2 is part of a tripartite complex with Tcf7l1 and Gro that increases Tcf target genes repression in vitro and in vivo.

Barhl2 activity is timely and locally controlled: barhl2 RNA is detected after stage 8 in the animal pole and in the presumptive organizer. Before the mid-blastula transition (MBT) βcatenin establishes transcriptionally poised chromatin architecture at promoters of key organizer genes, via the recruitment of the arginine methyltransferase Prmt2 (Blythe et al., 2010). For example, the promoter of sia1 is poised for activation before the MBT and barhl2

has no influence on sia1 early expression. Our data indicate that Barhl2 limits the size of the organizer. We suggest that Barhl2 participates in the progression of the gastrulation developmental program and in turning-off β-catenin transcriptional activity after the initial transcription of key organizer genes. Due to the very small number of barhl2 transcripts in a blastula embryo we do not know whether barhl2 normally turns off the organizer program within the entire organizer territory, or whether it normally acts to define the organizer boundaries.

Mouse embryos that have lost the tcf7l1 gene exhibit a de-repression of Wnt target genes, and expanded axial mesoderm structures [START_REF] Merrill | Tcf3: a transcriptional regulator of axis induction in the early embryo[END_REF]. This phenotype is reminiscent to that of Barhl2 depleted Xenopus embryos in which, similarly, Wnt signaling is de-repressed (this study and (Offner et al., 2005)). Wnt/β-catenin signaling role in formation of a blastoporeassociated axis organizer is conserved in evolution, and appears to predate the cnidarianbilaterian split over 600 million years ago [START_REF] Kraus | Pre-bilaterian origin of the blastoporal axial organizer[END_REF].

barhl2 is highly evolutionarily conserved (Schuhmacher et al., 2011;Sena, Feistel, & Durand, 2016). The orthologue of barH, in the hemichordate Saccoglossus kowalevskii displays some transcriptional activity conservation (Yao et al., 2016). Taken together these observations suggest that the Barhl2 function as an enhancer of Tcf7l1 repression could be evolutionarily conserved.

Gro proteins do not contain a DNA binding domain and are dependent on transcription factors for their recognition of DNA binding motifs. The ectopic expression of N-terminal parts of Barhl2 or of Barhl1 prevent Barhl2 normal function by binding and buffering Gro proteins present in the Xenopus blastula, indicating that the interaction of Barhl2 with Gro, most probably Gro4, is at the core of its normal activity. The differences in homeodomain-containing transcription factors' affinity for Gro is instrumental for their biological activity, either by repressing transcription (Muhr, Andersson, Persson, Jessell, & Ericson, 2001;Yasuoka et al., 2014) or by decreasing Tcf7l1-Gro interaction and consequently Tcf7l1 activity [START_REF] Lu | Tissue-specific derepression of TCF/LEF controls the activity of the Wnt/beta-catenin pathway[END_REF].

In cells Barhl2 induces important changes in Gro stoichiometry in a protein complex containing Tcf7l1 alone or with Hdac1 [START_REF] Arce | Groucho binds two conserved regions of LEF-1 for HDAC-dependent repression[END_REF]Chen et al., 1999;[START_REF] Chodaparambil | Molecular functions of the TLE tetramerization domain in Wnt target gene repression[END_REF]. We provide evidence that Hdac1 activity participates in reducing expression of early Tcf7l1 target gene (our data and [START_REF] Gao | A novel role for Ascl1 in the regulation of mesendoderm formation via HDAC-dependent antagonism of VegT[END_REF]), and contributes to Barhl2-mediated limitation of Spemann organizer size and activity. Gro co-repressors can attenuate gene transcription [START_REF] Kaul | Recent insights into Groucho corepressor recruitment and function[END_REF], and/or silence target genes expression through their ability to tetramerize, associate with histones [START_REF] Chodaparambil | Molecular functions of the TLE tetramerization domain in Wnt target gene repression[END_REF], and recruit histone deacetylases, which establish regional repressive chromatin structures (Chen et al., 1999) (Reviewed in [START_REF] Laugesen | Chromatin repressive complexes in stem cells, development, and cancer[END_REF][START_REF] Seto | Erasers of histone acetylation: the histone deacetylase enzymes[END_REF]). Gro tetramerization on a Tcf-Gro complex is the key step that promotes structural transitions of chromatin to mediate repression [START_REF] Chodaparambil | Molecular functions of the TLE tetramerization domain in Wnt target gene repression[END_REF] (reviewed in [START_REF] Kwong | The Central Region of the Drosophila Co-repressor Groucho as a Regulatory Hub[END_REF]). Therefore, the influence of Barhl2 on Gro stoichiometry may promote direct binding of a Barhl2-Tcf7l1-Gro-Hdac1 complex to histone, influence the repression potential of Tcfs, and allow change in chromatin structure.

A form of Barhl2fl mutated for DNA binding is inactive in embryos. In transfected cells and in embryos, Barhl2 limits β-catenin-induced Tcf7l1 activation from two synthetic promoters;

neither of them contains Barhl2 DNA binding motifs. In transfected cells, Barhl2 limits β-catenin activation of pTop-Flash but does not extinguish it. We suggest that by stabilizing the Tcf7l1-Gro interaction, Barhl2 increases the promoter proximal pausing time of RNA Pol II, a mechanism described for Gro-mediated attenuation of transcription [START_REF] Kaul | Recent insights into Groucho corepressor recruitment and function[END_REF]. In the X. tropicalis pbin7LefdGFP Wnt reporter line, Barhl2 could similarly act locally, without DNA binding. However, it could also interact with Tcf7l1 while bound on distant enhancers and participate to gene silencing via the recruitment of Gro together with chromatin modifiers.

Further studies are necessary to determine the contribution of DNA in the transcriptional repression process controlled by Barhl2 and Tcf7l1. However our observations are in agreement with a long-range activity of Barhl2 via its specific binding on DNA, maybe on superenhancers as previously described (Lin et al., 2016).

In conclusion, we describe a novel mechanism by which the transcription factor Barhl2 enhances the repression strength of Tcf7l1-Gro, and perhaps contributes to Gro-mediated regulation of the chromatin opening state allowing for the silencing of the transcriptional response to a given amount of Wnt ligand. Barhl2 decreases Tcf target gene transcription in mammalian cells, and in the Xenopus organizer. At later stages of development, Barhl2 changes the cellular response to Wnt stimulation in the cerebellar rhombic lip and the cortical hem, two post-embryonic germinative zones for neural progenitors. Genetic and functional evidence accumulates demonstrating that Tcf7l1 negatively regulates the pluripotency network in mouse embryonic stem (ES) cells, which express barhl2 (Cole et al., 2008;[START_REF] Lee | Control of developmental regulators by Polycomb in human embryonic stem cells[END_REF]Tam et al., 2008;Yamamizu et al., 2016;Yi et al., 2011;[START_REF] Yi | Tcf3 functions as a steady-state limiter of transcriptional programs of mouse embryonic stem cell self-renewal[END_REF]. In these cells most genes repressed by Tcf7l1 are different from those activated by Wnt factors, suggesting that Tcf7l1 might have Wnt/β-catenin independent activities (Cole et al., 2008;Yi et al., 2011).

Further studies are necessary to establish the contribution of Barhl2 enhancing Tcf7l1 repression strength to neural stem /progenitor cells formation and function, ES cell differentiation (Yamamizu et al., 2016), and cerebellar carcinogenesis (Lin et al., 2016).

Materials and Methods

Xenopus embryos care and husbandry X. laevis embryos were obtained and staged using standard procedures (Nieuwkoop & Faber, 1994;Sive, Grainger, & Harland, 2010). X. tropicalis transgenic embryos were obtained by conventional methods of hormone-induced egg laying and in vitro fertilization between a wild type female and a transgenic male carrying the Wnt reporter pbin7LefdGFP previously described (Tran et al., 2010;[START_REF] Tran | Design and use of transgenic reporter strains for detecting activity of signaling pathways in Xenopus[END_REF]. The male was beforehand selected as having a single insertion site of the transgene (as inferred by mendelian ratios in its progeny) in order to insure homogeneous levels of gfp expression in the offspring. Experimental (YPYDVPDYATSTIV) at the C-terminal part of Tcf7l1 was a gift from S. Sokol (Hikasa et al., 2010). pCS2+ Myc-Tcf7l1 (Xenopus) was a gift from Marc Kirschner (Addgene plasmid # 13439). p181 pK7-Hdac1 (GFP) was a gift from Ramesh Shivdasani (Addgene plasmid # 11054). pCS2+ Flag-Groucho4 and pCS2+ Myc-Groucho4 (X. laevis) were gifts from Phil

Jones. pCS2-XGroucho1-Flag was a gift from Nancy Papalopulu. The sequence of all plasmids is available upon request.

mRNA synthesis, Antisense Morpholinos and Injections

Synthetic capped RNAs were prepared from pCS2+ derivatives with mMessage mMachine kit (Ambion). Antisense oligonucleotides either with no 5' capping, either coupled to fluorescein were made by Gene-Tools as previously published: MObarhl2 (Offner et al., 2005) (30ng), MOhdac1 (Y. [START_REF] Tao | HDAC1 regulates the proliferation of radial glial cells in the developing Xenopus tectum[END_REF](0,3ng) and control MO MOct (3ng) (Gene-Tools). The specificity of MObarhl2 effect has been previously demonstrated (Offner et al., 2005), and confirmed by the similarity of X. laevis Barhl2-depleted, and mouse barhl2-/-phenotypes in the anterior brain (Juraver-Geslin et al., 2014;Yao et al., 2016). The overall barhl2 mRNA sequence is conserved at 100% between X. laevis and X. tropicalis in the first 25 nucleotides on which the MObarhl2 hybridizes. We used MObarhl2 to deplete Barhl2 activity in X. tropicalis embryos.

We used a previously validated translation-blocking MO against the X. laevis Hdac1.L (Y. [START_REF] Tao | HDAC1 regulates the proliferation of radial glial cells in the developing Xenopus tectum[END_REF]. The hybridization site for the X. laevis Hdac1.L MO has only two mismatches with that of Hdac1.S and hybridizes perfectly on its last 23 nucleotides with X. tropicalis Hdac1 translation initiation site sequence. Therefore, this MO binds all Xenopus Hdac1 transcripts with good affinity. MO were resuspended in RNAse free water at 1nmol/µl, kept at -80°C and heated 10min at 65°C before use. Except when otherwise specified, MOs or mRNAs were coinjected with β-gal mRNA (100 pg) or gfp mRNA (100 pg) or MOct coupled to fluorescein for lineage tracing. ß-Galactosidase activity was revealed in Red-Gal (Research Organics) staining solution (Offner et al., 2005). Embryos were injected unilaterally for ISH/morphology, on both sides for RT-qPCR experiments. Three independent experiments were performed and the results were pooled. A phenotype exhibited by at least 60% of the embryos was considered significant. For all rescue experiments, we tested a range of cRNA (50-500pg) and selected the minimal cRNA quantity that both induced the specific phenotype and displayed no toxicity.

For rescue experiments the means between different data sets were compared using the Wilcoxon signed-rank test and significant differences are indicated with asterisks.

Real Time quantitative Polymerase Chain Reaction (RT-qPCR)

For each time point RNA extract was obtained twice from 5 individual embryos obtained from the same fertilization. Embryos were dissociated by pipetting up-and-down with a 26G needle in Trizol (Invitrogen) and extracted according to manufacturer instructions. The RNA pellet was suspended in RNAse free water and kept at -80ºC. RNA samples were reverse-transcripted twice, using MMLV-RT enzyme (Promega). RT-qPCR was carried out using IQ SYBR green Supermix (BioRad) on a CFX96 touch Real-Time PCR Detection System (BioRad). RT-qPCRs were performed on embryos using standard procedures (39 cycles) using technical triplicates for each time point. Primers were designed using Primer 3 (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi/). Primers for barhl2, barhl1, goosecoid and elongation factor-1 (ef1) were selected based on their amplification efficiency and specificity. Primers oligomeres used in qPCR are resumed in Table S1. EF1 and Ornithine decarboxylase (ODC) primers were used as controls.

Whole mount In Situ Hybridization (ISH)

Control or injected embryos were collected at indicated stages. Double and single ISH were performed using digoxigenin or fluorescein labeled probes. Antisense probes were generated for Xbarhl2, Xsox3, Xvent2, chordin, Xccdn1, Xpax6, Xfoxb1, Xnot2, Xotx2, Xvent1 according to the manufacturer's instructions (RNA Labeling Mix, Roche). pCS2-Gfp was a gift from David Turner. ISH was processed following protocol described in (Juraver-Geslin et al., 2014).

Briefly, following overnight incubation with the probes and then with alkaline Phosphataseconjugated anti-DIG and anti-Fluorescein antibody, enzymatic activity was revealed using NBT/BCIP or BCIP or INT/BCIP substrates (Roche). Then, bleaching treatment was performed after the post-fixation of the embryos. Whole mount images were captured using stereomicroscope Lumar V12. Sections (40μm thickness) were cut using a Leica VT1000 vibratome after gelatin-albumin embedding for stages up to stage 26 and in 3% agarose for later stages. Pictures were captured using a digital Axiocam 506 color camera on a Zeiss microscope on PICT-IBiSA@Orsay Histology facility of Institut Curie and processed with Zen program (version2.3), ImageJ and Adobe Photoshop CS6 software.

Firefly luciferase activity

To perform luciferase assays, HEK293T cells grown at about 60% confluence in 24-well plates and transfected using lipofectamine (GIBCOBRL, #11668-027) with 50ng of pTopFlash or pFopFlash (firefly luciferase) reporter plasmid and pCS2-β-galactosidase as a transfection control. Proteins were extracted and a luciferase assay was performed according to the manufacturer instruction (Luciferase Assay system Promega) using a bioluminometer TriStar LB 941(Berthold). Firefly luciferase activity was normalized to ß-gal (beta-Glo Promega) activity. The assay was performed in triplicate.

Immunocytochemistry

HeLa cells were spread on cover-slips in 24-well plates with a concentration of 5x10 4 cells/well and incubated overnight in DMEM (GIBCO, 31966) medium containing 10% FCS and 1% pen/strep in a humidified 5% CO 2 incubator at 37C. In each well, the 60-70% confluent cells were transfected with 1μl lipofectamine (GIBCOBRL, #11668-027) using 6.25-50ng of plasmid DNA as indicated. pSK+ plasmid was used as a carrier. Transfected cells were incubated for 24 hours in complete-DMEM in a humidified 5% CO 2 incubator at 37⁰C then fixed in 4% PFA for 20 minutes at RT and washed 8 times with PBT, then permeabilized with PGT (PBS 1X, 0.2% Gelatin & 0.5% Triton) for 45 minutes. The cells were co-incubated with 1/10000 DAPI and 1/1000 (diluted in PGT primary rabbit anti-Flag and/or chicken anti-GFP antibodies for 1 hour at room temperature (Tables S2 andS3). After successive washing with PBT, cells were incubated with 1/5000 Alexa Fluor 488 or Cy3 conjugated secondary goat anti-rabbit (Jackson)

and/or donkey anti-chicken (Jackson) antibodies for 1 hour at room temperature and obscurity.

Finally, coverslips were mounted with fluoromount and kept at 4⁰C . Cells were analyzed using a 100x oil immersion objective of a wide-field microscope equipped with a Nikon DS-5Mc camera.

Immunoprecipitation in transfected cells and in embryos

Using Phosphate Calcium method, cells were transfected with 0.5µg to 2µg of expression vectors coding for tagged proteins as indicated using Phosphate Calcium method. Plasmids coding for pCS2+ or pSK+ were used as a supplement to ensure that in the same experiment, cells in different wells were transfected with the same quantity of expression vectors and plasmids. Thirty-six hours after transfection, cells were harvested and lysed on ice in lysis buffer containing 20mM Tris pH7.6, 150mM NaCl, 1% Triton, 1mM EDTA and c0mplete

proteases inhibitor (Roche 11697498001). Whole cell lysates were cleared via centrifugation at 14.000x g for 15min. Alternatively forty stage 10 embryos injected on both sides with 0,7ng of synthetic RNA were homogenized in lysis buffer containing 0,5% NP40, 5mM EDTA, 10mM

Tris pH 7.5, 100mM NaCl, c0mplete proteases inhibitor and PhosSTOP (Sigma-Aldrich).

Extracts were cleared by centrifugation (30 min at 14000rpm, 4ºC).

In both cases protein complexes were precipitated from the whole-cell lysates with anti-c-Myc

Tag antibody (clone 9E10). Protein complexes were then precipitated with protein Asepharose beads (Sigma P9424). Beads were pelleted by centrifugation and washed four times with lysis buffer. Boiling the beads in loading buffer eluted immunoprecipitates. Western blotting with cell lysates or immunoprecipitated protein elutes was performed using conventional methods. Blots were blocked with 5% milk and were detected with an ECL Western Blotting Detection Reagent (Amersham RPN2109). Antibodies used can be found in Tables S2 andS3.

Antibodies

The primary antibodies and folds of dilutions of each antibody used for the detection of tagged proteins were as follows are described in Tables S2 andS3.

Image processing

X. tropicalis stage 41 specimens were embedded in 3% agarose, and sections (40 µm) were cut on a Leica VT1000E vibratome. Each embryo was individually analyzed. In order to perform an objective comparison of the different expression levels of gfp RNA revealed by ISH in X.

tropicalis stage 41 sections RGB images were analyzed by using an ImageJ macro (http://rsb.info.nih.gov/ij/) [START_REF] Abramoff | Image Processing with ImageJ[END_REF][START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. Such the expression level is proportional to the signal recorded on the blue channel, RGB images were split and only pixel values corresponding to the blue channel were analyzed. With the goal of removing unspecific signal, background value, estimated as the average intensity of pixels outside the region of interest (ROI), were subtracted to each image pixel. The ROI was determined by automatic segmentation using IsoData auto-thresholding based determination [START_REF] Ridler | Picture thresholding using an iterative selection method[END_REF]. This prevents for any subjectivity in ROI determination. Surface and integrated densities on ROIs were computed for each background corrected image. Macro and pictures corresponding to original data after histogram equalization using ImageJ with percentage of saturated pixel 0.4. are available upon request.

Statistical analysis

Statistical analyses were performed using Real statistics plugin from Microsoft Excel. For

Firefly luciferase activity and images analysis the Gaussian distribution of the data between embryos for the data sets and sections of one embryo respectively was verified using the Shapiro Wilk statistic test. The results are shown as mean ± standard error (s.e.). The means between two different data sets were compared using the paired sample T-test using Real statistics plugin from Microsoft Excel considering that samples were independent and variances unequal. Significant differences are indicated with asterisks.

MObarhl2-ct (20ng) HEK293T cells were co-transfected with vectors (0.5µg to 2µg) encoding indicated proteins.

Xbarhl2 (100ng) mbarhl2 (100ng) MObarhl2-2 (20ng) MObarhl2-1 (10ng) A
Co-IP was performed on cell protein extracts using an anti-Myc antibody. Total cell lysates and the immunoprecipitated complexes were analyzed with WB analysis using anti-Myc, anti-Flag, anti-HA, anti-GFP, and anti-AM antibodies as indicated.

(A) Gro interacts with Barhl2EHs but not with Barhl2EHsM that carries mutations in its two The means between different data sets were compared using the Wilcoxon signed-rank test and differences were significant as indicated with asterisks).

(F, G) Barhl2 depletion increases β-catenin transcriptional activation in stage 8.5 embryos. A scheme of the transgene Wnt reporter construct is given in Supplementary Fig. Co-IP experiments were performed on HEK293T cells protein extracts using an anti-Myc antibody. Total cell lysates and the immunoprecipitated complex were analyzed with WB analysis using anti-Myc, anti-Flag, anti-HA and anti-GFP antibodies as indicated.

A) Barhl2 enhances Gro stoichiometry in a protein complex containing Tcf7l1 and Hdac1.

Tcf7l1 ability to recruit Gro in a complex containing Hdac1 was dramatically increased in the presence of Barhl2fl. The IP membrane was first hybridized with an Anti-Flag antibody (Gro)

and secondly with an anti-GFP antibody (Hdac1). The band marked with a star corresponds to the left over Gro signal. In the presence of Hdac1 Barhl2fl potentiates Tcf7l1 ability to immunoprecipitate Gro4 and Hdac1. In contrast Tcf7l1 co-immunoprecipitated Barhl2flEHsM and Hdac1 but not Gro4. The black arrow indicates the denatured heavy chains of the anti-Myc antibody at 55kDa.

(B) Barhl2 recruits Gro and Hdac1 independently of the presence of one or another. HEK293T cells were transfected with Barhl2-Myc (2µg), Hdac1-GFP (2µg), and Gro4-Flag (2µg). Barhl2

immunoprecipitates both Hdac1 and Gro4 similarly when the proteins are transfected alone or together.

(C) Barhl2EHs prevents Gro interaction with Hdac1. HEK293T cells were transfected with Gro4-Myc (0,5µg), Hdac1-GFP (1µg), and increasing amount of Barhl2EHs-GFP (0,5µg to 1,5µg). In the absence of Barhl2EHs Gro4 binds Hdac1. Increasing the amount of Barhl2fl decreases the interaction between Gro4 and Hdac1.

(D, E) Hdac1 depletion increases Tcf transcriptional activation in midblastula embryos.

MOhdac1 was injected into one or two dorsal blastomeres at the 2-cell or 4-cell stage, together with MOct (red) as tracer in X. tropicalis transgenic embryos. We followed gfp RNA levels on Table S1. RT-qPCR Primers used in this study.

MCL-1 REGULATES APOPTOSIS IN THE NEURAL PLATE

SCIENTIFIC CONTEXT

Apoptosis is a finely regulated process that ensures cell homeostasis by regulating the number of cells in the organism. Analysis of apoptosis in zebrafish, mouse and Xenopus embryos demonstrated the presence of TUNEL-positive nuclei at specific developmental space and time indicating that programmed cell death (PCD) is part of normal development. Little is known on the identity of factors involved in early apoptosis, or on pathways initiating PCD at these early developmental stages (Hensey and Gautier, 1998;Yeo and Gautier, 2003).

One of the main regulators of apoptosis are the Bcl2 protein family members, which comprise pro-and anti-apoptotic proteins. When an apoptotic stimulus in the cell is present, the Bh3 only pro-apoptotic proteins will inhibit the activities of the anti-apoptotic ones (Figure 9). The two main anti-apoptotic proteins involved in nervous system development in vertebrates are Bcl-XL and Mcl1. Mice lacking one of these proteins show smaller brains with decreased number of cells due to increase of apoptosis. In Xenopus, apoptosis appears from stage 10.5 onwards in the neural plate and the dorsal midline (Hensey and Gautier, 1998;Malikova et al., 2007;Offner et al., 2005). TUNEL staining in these embryos showed the presence of apoptotic nuclei in the forming axial organizer formed by the notochord and the floor plate and at the border between the neural plate and the ectoderm. Cells from the axial organizer, secrete signals such as Shh and Chordin that influence development of the neural plate. An increase or decrease in the number of cells secreting these morphogens leads to defects in neural plate patterning.

Our team previously described that the transcription factor Barhl2 promotes apoptosis in the axial organizer of Xenopus embryos. Barhl2 LOF leads to a decrease in apoptotic cells and a disruption in the Shh and anti-Bmp gradients emanating from organizer. How Barhl2 regulates apoptosis in this area is currently unknown. barhl2 is expressed from the onset of gastrulation in two longitudinal stripes along the Xenopus axial organizer (Offner et al., 2005).

In the following paper we present the expression pattern of Mcl1 and Bcl-X L in X. laevis embryos at early developmental stages. In agreement with its expression pattern we demonstrate that Mcl1 controls the survival of axial organizer cells in late gastrula-early neurula. We present evidence that Barhl2, and the anti-apoptotic protein Mcl1, participate to the same developmental process. We show that Barhl2 does not affect transcription of most apoptotic factors including mcl1. We also provide data supporting a role for Barhl2 in regulating directly or indirectly Mcl1 levels post-transcriptionally.

Introduction

Apoptosis is a type of programmed cell death (PCD). It induces a series of morphological changes in the apoptotic cell such as fragmentation and condensation of the chromatin as well as secretion of apoptotic factors that induce the absorption of the apoptotic cell my macrophages (Kerr, 2002;Kerr et al., 1972). Apoptosis takes place during development to ensure cell homeostasis and removal of unwanted cells (Baehrecke, 2002;Miura, 2011). It has extensively been studied in Caenorhabditis elegans (C. elegans) (Ellis and Horvitz, 1986), as well as in Drosophila and mammals (Arya and White, 2015). However, its regulation in Xenopus laevis (X. laevis) is less studied. In Xenopus embryos, apoptosis is not detected before stage 10.5 (Hensey and Gautier, 1998). During gastrulation and neurulation apoptotic nuclei are detected in the prospective neural plate (Hensey and Gautier, 1998;Offner et al., 2005). Vertebrate brain patterning relies on localized organizing centers that secrete proteins controlling neural tube development. Apoptosis plays an important role in delimiting the quantity of morphogens secreted by the organizing centers (Homma et al., 1994;Nonomura et al., 2013;Offner et al., 2005). The axial organizer derives from the Spemann organizer and comprises the notochord and the floor plate. In Xenopus, apoptosis is observed within the axial organizer and limits the quantity of Shh and Bmp antagonists-secreting cells (Offner et al., 2005). At later developmental stages stage 23 and 30, apoptosis is involved in notochord formation (Malikova et al., 2007). Little is known on the identity of factors involved in early apoptosis, or on pathways initiating PCD at these early developmental stages (Juraver-Geslin and Durand, 2015).

The cell death machinery is evolutionary conserved and its main components are the B-cell lymphoma 2 (Bcl2) family proteins and the downstream effectors Caspases. The apoptotic pathway can be triggered in response to stress or DNA damage (Green et al., 2014) [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]Motoyama et al., 1995;Opferman and Kothari, 2018).

Mcl1 is required for the survival of the stem cell niche in the sub-ventricular zone (SVZ) and at date, is the only anti-apoptotic Bcl-2 protein that regulates neural progenitor cells (NPCs) survival [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]Malone et al., 2012). Analysis of RNA expression levels of members of the Bcl2 family revealed that the anti-apoptotic bcl2, bcl2-like 10 (bcl2l10, a.k.a.

bcl-b), bcl2-like 2 (bcl2l2, a.k.a. bcl-w), bcl-xl and mcl1 transcripts are detected in Xenopus, and Bcl-w, Bcl-XL and Mcl1 efficiently inhibit apoptosis (Tsuchiya and Yamashita, 2011).

Overexpression of Bcl-XL, Bcl2 or Mcl1 decreases cell death in Xenopus embryos (Coen et al., 2000;Johnston et al., 2005;Offner et al., 2005;Yeo and Gautier, 2003). However, a role of Bcl2-protein family members in the formation of the axial organizer has not been investigated.

The homeodomain-containing transcription factors Bar-class HD (Barh) contain two members, BarH1 and BarH2 that are well conserved in metazoans during evolution. In both Xenopus and mice, the vertebrate homologue BarH-like 2 (barhl2) is expressed in the developing neuroepithelium from early gastrulation onwards, at a time period overlapping with the initial steps of neural plate patterning (Juraver-Geslin et al., 2014, 2011;Offner et al., 2005;Patterson et al., 2000). Investigation of BarH proteins activities in nematode, Xenopus and mice revealed roles for these transcription factors in the developmental control of cell death (Juraver-Geslin et al., 2011;Li et al., 2004Li et al., , 2002;;Nehme et al., 2010;Offner et al., 2005;Peden et al., 2007;Schwartz and Horvitz, 2007). These activities are strictly dependent on the cellular context and their regulatory modes in vertebrates have not yet been elucidated. How Barhl2 regulates PCD during development is unknown. We previously demonstrated that barhl2 is normally expressed from the onset of gastrulation in two longitudinal stripes along the Xenopus axial organizer (Offner et al., 2005). We showed that specific inhibition of barhl2 activity or over-expression of human anti-apoptotic gene bcl2 indirectly affects neural plate size by increasing the survival of Chordin-and Sonic Hedgehog (Shh)-expressing midline cells (Offner et al., 2005). On the basis of the patterns of apoptosis (Hensey and Gautier, 1998), we previously proposed that Barhl2 normally acts to control cell survival in the neural plate and dorsal mesoderm and that it is necessary for the normal formation of the axial organizing center of the Xenopus neural plate (Offner et al., 2005). However, whether Barhl2 directly or indirectly regulates the apoptotic cascade in neuroepithelial cells is unknown.

In the present study we investigated the role of Bcl2 family members and Caspases proteins in the formation of the axial organizer in late gastrula-early neurula Xenopus embryos. We describe that Mcl1 and Bcl-X L are expressed in the neural plate of neurula embryos. Barhl2

does not transcriptionally regulate any of the anti-or pro-apoptotic proteins studied but we have evidence that Barhl2 regulates post-transcriptionally the half-life of Mcl1. Our work provides an explanation for the regulation of apoptosis in the midline cells of the neurula embryos and the mode of action of Barhl2 in those cells.

Results

Apoptosis is detected in the axial organizer and it limits neuroepithelium's size

A previous study demonstrated that in X. laevis embryos cells do not undergo apoptosis before stage 10.5 (Hensey and Gautier, 1998). They also show that between stage 10 and stage 14

TUNEL positive nuclei cells are detected around the blastopore and in the midline of the dorsal ectoderm (Hensey and Gautier, 1998). We confirmed these observations performing TUNEL staining on stage 12.5 Xenopus embryos (Figure 1A a, b). In contrast, using immunostaining we did not detect any cells expressing an activated form of Caspase3 between stage 9 and stage 14 (Figure 1B). We asked whether interference with normal apoptosis, either by overexpression of the anti-apoptotic protein Bcl-XL or depletion of the pro-apoptotic enzyme was injected in the entire neural plate (Figure 1Dab), in the axial midline (Figure 1Ca) or in medio-lateral neuroectodermal cells (Figure 1Dcd). We quantified the increase in neural plate size using sox3 for each type of injections (Figure 1E). We observed that Bcl-XL overexpression either in the entire neural plate or in the midline cells derived from the organizer generated an increase in sox3 staining (Figure 1Ca, Dab, E). In contrast, injection of bcl-xL in the medio-lateral neuroectodermal cells had no effect on the size of the neuroepithelium (Figure 1Dcd,E). These results demonstrate that the increase of the neural plate size observed by the overexpression of the anti-apoptotic protein Bcl-XL is due to an effect on cells of the notochord. We observed that in all conditions depletion of Casp3 had no effect on the size of the neural plate (Figure 1Cb,E).

These results suggest that apoptosis is normally part of axial organizer formation. Inhibition of apoptosis through Bcl-XL overexpression generates an increase in the neural plate size that is at least partly an indirect consequence of Bcl-XL activity on axial organizer formation. Our observations demonstrate that at these developmental stages Casp3 activity is not required for the endogenous apoptotic program.

mcl1 expression pattern indicates a possible role in axial organizer development

We next aimed to identify which apoptotic cascade members were important for endogenous apoptosis at these stages. We dissected the neural plate of stage 12.5 embryos and investigated the presence of RNA encoding apoptotic genes using Reverse Transcription PCR (RT-PCR). The genes studied were members of the Bcl2 family, which include the antiapoptotic genes bcl2, bcl2l1, mcl1, bcl-w, bcl-b and the pro-apoptotic genes NADPH oxidase activator 1 (noxa1), bak, bh3 interacting domain death agonist (bid) and bcl2 like 11 (bcl2l11, aka bim). We observed that most transcripts encoded by bcl2 family genes are present in the neural plate at stage 13, specifically those exhibiting strong anti-apoptotic activities during nervous system development: mcl1 and bcl-x l (Figure 2A). Given their role in murine neural development we decided to further investigate the expression of mcl1 and bcl-xl in X. laevis embryos.

Using ISH we observed that mcl1 mRNA is weakly detected at stage 11 but between stage 13 and stage 20 mcl1 transcripts are present within the neuroepithelium (Figure 2B). Between stage 13 and stage 16 mcl1 transcripts are present in the anterior neuroectoderm, in two stripes along the forming axial organizer, which appear on transverse section to be in the mesoderm and the neuroectoderm (Figure 2Bc-e), and at the border between the neuroectoderm and the ectoderm. At stage 20 mcl1 expression is detected within the future neural tube specifically in the eye primordium. According to databases (Yanai et al., 2011) mcl1 transcripts are present during neurulation in X. laevis. Taken together, these results are coherent with an early activity for mcl1 in neural plate development and is consistent with a putative activity for this protein in the survival of notochord cells.

In contrast, bcl-x l RNA is weakly expressed before stage 20 where it is detected within the developing neuroectoderm (Supplementary Figure 1). Therefore, we focused our study on mcl1 activity in axial organizer development.

Depletion of Mcl1 activity limits neuroepithelium size and shape

We first asked whether depletion of Mcl1 activity affected development of the neuroepithelium.

To inhibit Mcl1 activity we designed in its 5' untranslated region two antisense morpholinos (MOmcl1-1 and MOmcl1-2) together with a control morpholino (MOmcl1-Ct) (Figure 3Aa). We We injected MOs into one dorsal blastomere of four-cell stage embryos. At stage 14, we observed a dose-dependent decrease in the width of the sox3 expression domain on embryos injected with either MOmcl1-1, or MOmcl1-2, but not on embryos injected with MOmcl1-Ct (Figure 3B). The effect was observed along the entire anterior-posterior (AP) axis and its extend was dose-dependent.

To establish the specificity of the MOs effect, we tested the ability of MOmcl1 to inhibit translation of the mcl1-myc mRNA. mcl1-myc mRNA was injected alone or together with We next evaluated whether the decrease in the width of the sox3 expression observed in Mcl1depleted neural plate was compensated by a mirror increase in the ectoderm territory.

MOmcl1-1 was co-injected with b-gal mRNA in one dorsal blastomere of four-cell stage embryos. At stage 13.5 we followed formation of the epidermal territory using epidermal keratin (EpKer) as a marker. We observed an increase in EpKer expression on the MOmcl1 injected side compared to the non-injected side (Figure 4B).

Taken together these observations suggest that a decrease in mcl1 specifically affects survival of notochord cells and that depletion of Mcl1 indirectly affects neural plate specification.

Mcl1 and Barhl2 are part of the same pathway

We previously demonstrated that Barhl2 overexpression promotes apoptosis in the Xenopus dorsal region between stage 10 and stage 14 and affects survival of axial organizer cells (Offner et al., 2005). Moreover, we also demonstrated that Barhl2 controls the size of the Spemann organizer and that Barhl2 depletion increases the number of chordin expressing cells at stage 10.5 (Sena et al, in preparation). We asked whether Barhl2 LOF compensated for Mcl1 LOF during neuroepithelium establishment.

To inhibit Barhl2 activity we used either MO against Barhl2 (MObarhl2) or mRNA coding for the N-terminal region of Barhl2 (Barhl2EHs) that acts as a Barhl2 dominant negative (Sena et al, in preparation) We conclude that at these developmental stages Barhl2 LOF compensates for the loss of Mcl1.

Taken together our observations argue that Mcl1 controls the survival of axial organizer cells and suggest that Barhl2 directly or indirectly participate to their apoptosis.

Depletion of Barhl2 activity does not affect apoptotic cascade components gene expression

Studies in nematode suggest that the temporal and spatial regulation of PCD during development is primarily regulated by transcriptional control of the expression of cell-death execution machinery. We asked whether Barhl2 directly or indirectly compensates Mcl1 ability to inhibit axial organizer cells decision to die by transcriptional up-regulation of pro-apoptotic genes or downregulation of anti-apoptotic genes. MObarhl2 was injected into two dorsal blastomeres at the two-cell stage in embryos. The expression of pro-apoptotic or anti-apoptotic genes was analyzed using RT-qPCR. When analyzed at stage 13, barhl2 LOF does not significantly modify the RNA levels of neither anti-nor pro-apoptotic proteins (Figure 6A).

A peak of barhl2 transcripts in the animal hemisphere and in the presumptive organizer region is detected at stage 8.5 in X. laevis embryos (Session et al., 2016, Sena et al. in preparation).

We further quantified the expression changes in the three main anti-apoptotic genes implicated in nervous system development between stage 8 and stage 10. We did not measure any significant change in the expression levels of mcl1, bcl-Xl or xiap neither at the blastula nor at the gastrula stages (Figure 6B).

We conclude that Barhl2 does not normally affect apoptosis through transcriptional regulation of mcl1, bcl-xl or xiap, and other apoptosis-related genes between stage 8 and stage 13.

Therefore, the mechanism by which Barhl2 downregulation rescues Mcl1 LOF is not related to transcriptional regulation.

Overexpression of a stabilized form of Mcl1 increases the size of the neural plate

Mcl1 has the strongest activity as an apoptosis inhibitor during early neural development [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]. We asked whether overexpression of Mcl1 was as efficient as Bcl-XL in preventing the loss of axial organizer cells (Figure 1B,C). xmcl1-myc or xmcl1 mRNA were injected into one dorsal blastomere of four-cell stage embryos together with b-gal as a tracer.

At stage 14 we observed for both xmcl1-myc and xmcl1 an increase in the width of the sox3 expression domain along the entire AP axis (Figure 7Aa,b). However, only one third of the embryos exhibited the phenotype. To determine whether the low penetrance was due to an effect on Mcl1 protein levels we investigated the phenotype penetrance of a mutated form of human mcl1 (hmcl1) carrying an Alanine in position 159 instead of a Serine (hMcl1S159A) (Maurer et al., 2006). This mutation prevents Gsk3-mediated phosphorylation of Mcl1 and its subsequent degradation via the proteasome. hMcl1 and hMcl1S159A mRNA were injected into one dorsal blastomere of four-cell stage embryos. In both cases we observed the typical enlargement of the neural plate, however the penetrance of the phenotype was significantly higher in hMcl1S159A-injected embryos compared with hMcl1-injected ones (Figure 7A,C).

These results demonstrate that in comparison with Bcl-XL overexpression (Figure 1B

Discussion

In this paper we investigated an early role for Mcl1 in neural plate development. We describe the expression pattern of two anti-apoptotic proteins: bcl-xl and mcl1. The expression pattern of mcl1 expression is consistent with a role for this protein in regulating programmed cell death within the neural plate. We demonstrate that Mcl1 is involved in controlling survival of axial organizer cells between stage 10 and stage 14 and therefore indirectly affects neural plate size and patterning. Following observations on a role for the transcription factor Barhl2 in the neural plate of X. laevis at stage 14 (Offner et al., 2005), we show that barhl2 does not affect transcription of apoptotic cascade factors, and specifically the expression of mcl1, bcl-xl, xiap.

Our data are coherent with a role for Barhl2 in directly or indirectly affecting Mcl1 stability.

Mcl1 apoptotic activities in neural plate development

In Xenopus the pattern of developmental apoptosis has been described in gastrula and neurula embryos (Hensey and Gautier, 1998). Cells undergoing developmental apoptosis are detected only after stage 10 in the axial organizer and at the neural border (Hensey and Gautier, 1998;Johnston et al., 2005;Juraver-Geslin and Durand, 2015;Malikova et al., 2007;Offner et al., 2005;Van Stry et al., 2004;Yeo and Gautier, 2003). Overexpression of Bcl-X L inhibits cell death in Xenopus embryos and generates an increase in the neural plate size (Offner et al., 2005;Yeo and Gautier, 2003). We observed that mcl1 transcripts are detected in neuroepithelium areas exhibiting EPCD including the axial organizer and the border between the neuroepithelium and the ectoderm (Hensey and Gautier, 1998;Juraver-Geslin and Durand, 2015;Yeo and Gautier, 2003). Mcl1 is, along with Bcl-X L, one of the main anti-apoptotic proteins involved in neural development. Mice deficient for mcl1 exhibit reduced size of the telencephalon and atrophied ventricles, with increased apoptosis in neural cells [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]. Moreover, Mcl1 is involved in the survival of stem cells from the SVZ and it regulates NPCs survival (Arbour et al., 2008;Malone et al., 2012). Our results confirm an important role for Mcl1 as the main regulator of neural progenitor and stem cell survival.

A Role for Barhl2 in controlling Mcl1 levels

We previously showed that the transcription factor Barhl2 affects the anti-Bmp and Shh gradient generated by secreted signals from the axial organizer (Offner et al., 2005). We demonstrated that Barhl2 promotes apoptosis in the Xenopus neuroectoderm and mesoderm at late gastrula/early neurula stages (Offner et al., 2005). In contrast Barhl2 LOF decreases the number of apoptotic nuclei within the axial organizer (Offner et al., 2005). Here we demonstrate that Mcl1 LOF compensates for Barhl2 LOF arguing that both proteins act on the size of the organizer.

Using RT-qPCR we followed the expression of pro-and anti-apoptotic genes from stage 8 to stage 13. We observed no differences in the levels of most apoptotic proteins mRNA in the presence or in the absence of Barhl2. Therefore, at these developmental stages, Barhl2 does (Ding et al., 2007;Zhong et al., 2005). Mcl1 can also be cleaved on its N-terminal region and degraded (Clohessy et al., 2004).

Mcl1 can be phosphorylated at its N-terminals region by different kinases such as for example Gsk-3 (Ding et al., 2007). Gsk-3 phosphorylates Mcl1 on its Serine155, Serine 159 and Serine163 promoting its phosphorylation and degradation (Thomas et al., 2010). In the nematode C. elegans, the Bh3-only protein Egl1, a pro-apoptotic member of the Bcl2 family, is the key effector of developmental cell death and is transcriptionally controlled. The transcriptional activation of egl1 is sufficient for the activation of the cell death machinery.

Overexpression of Xenopus or

Three C. elegans genetic screens performed independently highlighted the regulation of the apoptotic pathway by the orthologue of Barh proteins ceh30 (Nehme et al., 2010;Peden et al., 2007;Schwartz and Horvitz, 2007). In C. elegans, the cephalic male-specific (CEM) sensory neurons in hermaphrodites undergo PCD by inducing the expression of egl1, which activates the caspase-3 orthologue, Ced-3. ceh30, blocks apoptosis and allows the survival of four specific CEM neurons. In males, ceh30 acts as a transcriptional repressor of ced-3 and egl-1 genes independently of the bcl2 homologue ced9. Importantly ectopic expression of vertebrate barhl1 or barhl2 transgenes in C. elegans rescue the CEM survival defect of male Ceh30 mutants. Therefore, barhl genes encode proteins that retain the functions and target specificity of Ceh30 (Schwartz and Horvitz, 2007). Together with our results it appears that Barhl2 ability to regulate the cells death machinery is conserved between Xenopus and nematode. However, a role for Barhl2 in transcriptional regulation of the apoptotic machinery does not appear to be conserved (Juraver-Geslin et al., 2011).

bleaching treatment was performed after the post-fixation of the embryos. Whole mount images were captured using stereomicroscope Lumar V12. Sections (40μm thickness) were cut using a Leica VT1000 vibratome after gelatin-albumin embedding. Pictures were captured using a digital Axiocam 506 color camera on a Zeiss microscope on PICT-IBiSA@Orsay Histology facility of Institut Curie and processed with Zen program (version2.3) and ImageJ and Adobe Photoshop CS6 software.

Protein extraction and Western Blot

Embryos were lysed on ice in lysis buffer containing 1% Triton x-100, 5mM EDTA, 5mM EGTA, 50mM Tris pH7.5, 0.3M NaCl, 1x c0mplete proteases inhibitor (Roche 11697498001) and 1x

PhosSTOP phosphatase inhibitor (Roche 04906837001). Whole cell lysates were cleared via centrifugation at 14.000x g for 30min. Protein quantification was performed using the BioRad Pc Protein Assay (BioRad). Samples were denatured with ½ volume of 2x Laemli Buffer (BioRad) and b-mercaptoethanol and boiled for 5min. 20µg of protein extracts were separated by a 10% or 12% SDS-polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P, Millipore). Blots were blocked with 5% milk and were detected with an ECL Western Blotting Detection Reagent (Amersham RPN2109). Densitometry analysis, standardized to actin as a control for protein loading, was performed using ImageJ software (National Institutes of Health, USA). The primary antibodies and folds of dilutions of each antibody used for the detection of tagged or endogenous proteins are described in Tables S3 and Table S4.

Statistical analysis

Statistical analyses were performed using Real statistics plugin from Microsoft Excel.

Significant differences are indicated with asterisks.

Equal

Supplementary data

1.REGULATION OF WNT SIGNALING BY BARHL2 1.1 ROLE OF BARHL2 DURING THE SPEMANN ORGANIZER DEVELOPMENT

In the manuscript "A nuclear brake on T-cell factors/Lef1 transcriptional activation switches off Wnt/b-catenin signaling" (Paper 1), we describe inhibition of Wnt canonical pathway by the transcription factor Barhl2. Barhl2 interacts with the co-repressor Gro4 via its engrailed domains and they form together a tripartite complex with Tcf7l1 preventing its activity as a transcriptional activator. We provide in vitro and in vivo evidence of Barhl2 inhibitory effect on b-catenin activity as an activator of transcription. In embryos we showed that Barhl2 specifically inhibits Wnt ability to induce a double axis when injected ventrally. We made use of a transgenic X. tropicalis line that expresses gfp when Tcf fixation sites are bound by b-catenin.

This transgenic line (Tran et al., 2010) allowed us to follow Wnt activity throughout Xenopus development. Using this reporter line, we demonstrated that Barhl2 depletion generates an increase in Tcf transcriptional activity.

Wnt signaling is important in many developmental steps at both embryonic and adult stages.

During my PhD I focused on the regulation of Wnt signaling during the formation of the Spemann organizer, a process known to be strictly dependent on b-catenin activity. Before Spemann organizer formation b-catenin accumulates in the dorsal region of the embryo. There it binds to Tcf and activates the transcription of siamois and twin, which in turn activates transcription of goosecoid and chordin. We establish that overexpression and downregulation of Barhl2 respectively decreased or increased the expression of head and trunk organizer genes. Our experiments demonstrate that Barhl2 inhibitory effect takes place after the induction of siamois mRNA and that Barhl2 repressive effect on goosecoid and chordin expression is downstream of the master gene siamois. These data demonstrate that Barhl2 is part of a developmental program that shuts down organizer induction. Due to the very small number of cells expressing barhl2 it is difficult to be absolutely certain of which cells express or not this transcription factor. We therefore do not know whether barhl2 normally turns off the organizer program within the entire organizer territory, or whether it normally acts to define the organizer boundaries. In any case we observed expansion of chordin and goosecoid expression within the ventro-lateral mesoderm territory in Barhl2 depleted embryos.

We do not have a good antibody for Xenopus Barhl2. We therefore rely on indirect evidence that Barhl2 activity starts when its mRNA is detected i.e. at the MBT. We demonstrated that Barhl2EHs acts as a dominant negative (DN) (detailed in the next paragraph). We observe that dorsal overexpression of Barhl2EHs induces the same phenotype as barhl2 depletion meaning that it starts acting at the MBT, when barhl2 mRNA are detected. We also observe that neither barhl2 mRNA nor MObarhl2 injection in the ventral part of the embryos generate a phenotype. Taken together these observations argue that there is neither a presence, nor an effect of maternal Barhl2.

INTERACTION BETWEEN BARHL2, GROUCHO AND TCF

To analyze how Barhl2 regulates the formation of the Spemann organizer I studied the interaction between Barhl2, Gro4 and Tcf7l1. Gro is a known repressor of Tcf. I focused on Barhl2 regulation of Gro and Tcf binding, and its effects on the Tcf-activator to repressor switch in the nucleus.

Barhl2 contains two engrailed domains known to directly bind the WD-repeat motif of Gro [START_REF] Jiménez | A conserved motif in goosecoid mediates groucho-dependent repression in Drosophila embryos[END_REF][START_REF] Jiménez | Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed[END_REF] to inhibit gene transcription [START_REF] Jaynes | Active repression of transcription by the Engrailed homeodomain protein[END_REF]Smith and Jaynes, 1996). Our data in cells and embryos demonstrate that Barhl2 interacts with Gro via its engrailed domains. We provide biochemical evidence of this direct interaction in cells and in embryos.

The importance of Barhl2 interaction with Gro for its activity on Spemann Organizer formation was confirmed using Barhl2EHs that only contains the engrailed domains, and acts as a dominant negative. We demonstrated that Barhl2EHs inhibits Barhl2 activity by binding to Gro.

Indeed, Barhl2EHS impedes the interaction between Barhl2 full length and Gro in the dorsal part of embryos. Importantly, because Barhl2EHs captures Groucho its DN effect is not specific of Barhl2 but reveals that Barhl2 normally acts on Spemann Organizer formation via its interaction with Groucho.

As had been first described using the two-yeast hybrid assay (Roose et al., 1998), Gro interact with Tcf via its Q domain. We first confirm the Tcf7l1, Gro4 interaction using immunoprecipitation assays in cells. Interestingly, in HEK293T cells Barhl2 dramatically increases the binding of Gro4 to Tcf7l1. Moreover, luciferase assays using TopFlash, a reporter for Tcf activity, confirmed that Barhl2 limits the ability of b-catenin to activate Tcf in cultured cells. Also, Barhl2EHs acts as a DN in this system and prevents Barhl2 inhibitory effect on b-catenin TopFlash activation.

To validate these observations in vivo we performed immunoprecipitations on extracts from Xenopus embryos injected with tagged-mRNAs encoding Groucho, Tcf and Barhl2. In embryos the interactions between Gro and/or Tcf7l1 and/or Barhl2 were confirmed. However, in striking contrast to what we observe in cells we did not observed that Barhl2 enhanced the interaction between Gro and Tcf. Whereas we do not know why there is such a difference between cells and embryos we suggest two main hypotheses. First whereas HEK293T cells do not express barhl2 mRNA, in embryos Barhl2 protein is present. Endogenous Barhl2 could contribute to stabilize the interaction between Tcf and Gro in the absence of mRNA coding for tagged-Barhl2. To investigate this hypothesis, we shall perform the same immunoprecipitations on extracts from Xenopus embryos injected with tagged-mRNAs encoding Gro, Tcf and mouse Barhl2 but in the presence of MObarhl2 that will inhibit endogenous XBarhl2 activity. The second and most probable hypothesis is that the interactions between Tcf, Gro and Barhl2 are finely regulated, eventually by posttranslational modifications including phosphorylation, sumoylation or ubiquitination. We hypothesize that whereas in HEK293T cells -a homogenous system -such modifications occur, in embryos composed of a lot of different cell types, the necessary machinery is not present/active in all cells and the same modifications do not take place. In the future it will be extremely interesting to assess further the nature and functions of such modifications on Gro, Tcf or Barhl2.

Finally, and importantly, we validated in vivo that Barhl2 prevents the activation of Tcf by bcatenin during the formation of the Spemann organizer. First using the double axis induction assay by ventral injection of Wnt8b mRNA, secondly by using the transgenic X. tropicalis line that expressed gfp upon Tcf activation.

LONG-DISTANCE TRANSCRIPTIONAL REGULATION BY BINDING TO SPECIFIC

ENHANCERS

Enhancers, also called cis-regulatory elements, are important to control gene transcription.

The promoter of a determined gene can be active at a distal region of expression of its mRNA.

The way the genome can express the same gene at different places and times depends on the activity of the cis-regulatory elements. They recruit co-factors and transcription factors to transcribe genes at the right time and place via a long-distance mechanism (Visel et al., 2009a(Visel et al., , 2009c)). The enhancer sequences are the same in all cells but each cell differs on the combination of transcription factors that bind to each enhancer.

Barhl2 is important for the development of the ZLI (Juraver-Geslin et al., 2014;Sena et al., 2016). It forms part of an evolutionary conserved group of transcription factors that bind enhancers to promote shh expression (Yao et al., 2016). More experiments should be done to prove it, but it is possible that similarly Barhl2 ability to repress Wnt signaling relies on its binding to enhancers and long-distance inhibition of Spemann organizer genes. There are no evident putative fixation sites for Barhl2 on the promoters of head and trunk Spemann organizer genes. This suggests that there could be no binding of Barhl2 on the promoter of these genes. Some transcription factors bind enhancers and at the same time recruit enzymes that modify the structure of chromatin, and consequently affect the transcription of genes through a long-distance mechanism. Gro can mediate its repressive activities either via increasing the RNA pol II pausing time or by recruiting Hdac1 to mediate gene silencing (Billin et al., 2000;[START_REF] Brantjes | TCF: Lady Justice casting the final verdict on the outcome of Wnt signalling[END_REF]. We showed that Barhl2, Gro, Tcf and Hdac1 are part of the same multiprotein complex. We tested the effect of Hdac1 downregulation on the Wnt reporter transgenic line and demonstrated that Hdac1 loss-of-function induces Tcf activation. This observation argues that Hdac1 is part of the Tcf repressive machinery at these developmental stages.

We investigated the interaction between Barhl2 and Hdac1 during the formation of the Spemann organizer. We demonstrated that Barhl2 gain-of-function is compensated by Hdac1 depletion, indicating that Hdac1 activity is involved in Barhl2 inhibition of Spemann Organizer program. To validate the role of Hdac1 in Barhl2 mediated inhibition of Spemann organizer genes we are currently performing a chromatin immunoprecipitation assay using an antibody against Histone H3 Lysine 9 Acetylated, followed by a quantitative PCR for promoters of Spemann organizer genes. Our aim is to compare the acetylation of histones on the promoters of siamois, chordin, goosecoid, otx2, vent2, brachyury and wnt8a in the presence, or absence of Barhl2. This experiment should provide information on Barhl2 mediated Hdac1 activity during Spemann Organizer formation. It should help us determine whether Hdac1 is part of the Barhl2, Gro, Tcf7l1 complex mediating long distance silencing of Spemann organizer genes during normal development.

It has been demonstrated that Gro in association with transcription factors like Otx2 and Gsc bind to cis-regulatory elements to control gene transcription [START_REF] Kaul | The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription[END_REF]Yasuoka et al., 2014). The binding of the co-activator p300 has classically been an indicator of tissue-specific cis-regulatory modules (Visel et al., 2009b). However, Yasuoka and colleagues describe that the binding of Gro to some promoter's regions is a better indicator of the presence of enhancers because they mark all the regions were the genes are not being transcribed. It is unknown whether the binding of Groucho to these enhancers is dependent or not of Tcf.

We observe that dorsal overexpression of Barhl2EHs that captures Gro, reproduces Barhl2 depletion effect. This DN activity reveals that Barhl2 normally acts on Spemann Organizer formation via its interaction with Gro. These results raise the possibility that Barhl2 acts long distance at least partly via the recruitment of Gro eventually independently of Tcf. More experiments are necessary to accurately address the respective roles of Barhl2, Tcf7l1 and Gro on long distance transcription inhibition of Spemann Organizer genes.

A CONSERVED ROLE FOR BARHL2 ACTIVITY ON TCF AT POSTEMBRYONIC

STAGES

We investigated whether the molecular interaction we described between Barhl2, Gro and Tcf, and the ability of Barhl2 to limit Tcf activation was specific or early development or also occurs at later developmental stages. To answer this question, we first studied barhl2 expression pattern relative to Tcf activity at postembryonic stages i.e. when organogenesis is finished. At post-embryonic stages we observed that Barhl2 is expressed in areas flanking regions where Tcf is active. This is specifically true at the level of the cerebellar rhombic lip and cortical hems, two regions that continue to generate neural progenitors after embryonic development is terminated. Barhl2 transcripts being present in a domain bordering an area producing progenitors' cells is also true in the ciliary marginal zone (CMZ) of the retina, a bona fide stem cell niche (Figure 13). Taken together these observations suggest that Barhl2 plays a role in the switch Tcf-active to Tcf-repressive that controls stem cells pluripotency (Cole et al., 2008;Sokol, 2011;Tam et al., 2008;Yi et al., 2011). We analyzed the effect of Barhl2 LOF and GOF on Tcf activity using the X. tropicalis transgenic line. We indeed observe that Barhl2 controls Tcf activity at post-embryonic stages. At stage 41 in the cerebellar rhombic Lip and the cortical hem Barhl2 GOF limits Tcf activity whereas Barhl2 LOF increases it. Barhl2 activity in controlling Tcf activity could be conserved in other vertebrates. Particularly in mice, Barhl2 is expressed in the rhombic lip in a pattern complementary of that of Tcf activity ( (Mo et al., 2004) and unpublished data).

Whereas further work is necessary to address the role of Barhl2 in stem cells some papers have already highlighted the importance of Barhl2 in the differentiation of ESCs (Yamamizu et al., 2016). Indeed, Yamamizu and colleagues analyzed the global expression profile of some ESCs lines after overexpression of Barhl2. Among other transcription factors Barhl2 was the only one that induced a significant fold-change in more than three thousand genes analyzed.

A role for the regulation of progenitors pluripotency has also been described for Groucho in the neural tube (Muhr et al., 2001) and on ESCs [START_REF] Laing | Gro/TLE enables embryonic stem cell differentiation by repressing pluripotent gene expression[END_REF]. Moreover, it has been shown that Wnt stimulates self-renewal of ESCs by inhibiting Tcf7l1 (Yi et al., 2011). Overall, these results suggest that Barhl2 could regulate a Wnt signaling pathway implicated in ESCs differentiation. We have developed a lentivirus tool expressing a shRNA that specifically silence mouse Barhl2. These molecular tools could be used to investigate Barhl2 activity in stem cell biology in mice. Specifically, we could silence Barhl2 in ESCs and assess their differentiation rates compared to non-transfected cells.

The implication of Barhl2 in the maintenance or differentiation of this pool of stem cells could give us insights on its putative role in development of medulloblastoma, a malignant pediatric tumor (Lin et al., 2016). Analysis on tumor derived from patients highlighted that both barhl genes are deregulated in these pediatric tumors: Barhl2 being associated to the group Shh and Barhl1 to group 3 and 4. Understanding the exact function(s) of Barhl2 and/or Barhl1 in granular cell progenitors and the origin of their deregulation in medulloblastoma should provide important information of molecular mechanisms involved in the origin of this disease.

BARHL2 AND TCF IN PROLIFERATION OF THE NEURAL TUBE

The neural tube emerges from the neural plate, when the neuroectoderm folds and closes in a tubular shape. In the neural tube there are proliferating cells, the neural progenitors that either produce other progenitors or differentiate. The balance between proliferating and differentiating cells is crucial during development and long term allows maintenance of a progenitor pool in this region.

Multiple factors intrinsic and extrinsic including signaling pathways contribute to maintain the pool of progenitors in the neural tube. One of these signaling pathways is the canonical Wnt signaling pathway. When the pathway is active cells proliferate and maintain an undifferentiated state. There are different transcription factors that contribute to the inhibition of this pathway. Tcf is one of them. When active, Tcf maintains progression of the cell cycle.

When repressed, Tcf induces cell cycle exit and differentiation. Indeed among the genes activated by b-catenin when the Wnt pathway is active are c-myc, which is a powerful stimulator of cell growth and proliferation; and cyclinD1, which is involved in the regulation of cell cycle. Thus, Wnt signaling controls cell cycle exit and the proliferation of NSCs and progenitors in the neural tube.

We previously demonstrated that the transcription factor Barhl2 limits Wnt-dependent proliferation in the prospective diencephalon of the Xenopus embryo. Indeed Barhl2 depletion via morpholino (MO) injection induces a hyperplasia of the neural tube associated to an increased proliferation of progenitor cells. Barhl2 LOF phenotype can be compensated by depletion of b-catenin, or expression of GSK3b (Juraver-Geslin et al., 2011).

We investigated whether Barhl2 was limiting the switch Tcf-Repressor to Tcf-Activator in the developing forebrain. Using the X. tropicalis Tcf reporter line we observed that at stage 30 Tcf activity is repressed in Barhl2 GOF embryos, and Tcf activity is increase in Barhl2 LOF embryos (Figure 14). These observations on stage 30 embryos demonstrate that Barhl2

participates to the Tcf-active to Tcf-repressive switch in the diencephalon. It is probable that this effect is mediated by the ability of Barhl2 to stabilize Groucho, Tcf interaction, and consequently inhibits b-catenin activity. Therefore, a signaling pathway involving Barhl2, Groucho and Tcf is active in the diencephalon. It has also been demonstrated that Xiap can monoubiquitinilate the co-repressor Groucho to induce Wnt signaling [START_REF] Hanson | XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling[END_REF]. Indeed Xiap monoubiquitination of Groucho interferes with Groucho ability to bind Tcf, consequently inhibits its repressor activity and further promote Wnt signaling [START_REF] Hanson | XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling[END_REF]. During formation of the Spemann organizer Barhl2 and Groucho are part of the same protein complex that inhibits b-catenin induction of Tcf activity. A question arises, does XIAP participate in the Barhl2, Groucho mediated inhibition of Wnt signaling on the diencephalon? Indeed, these observations suggest that crosstalk between Barhl2, Gro, Tcf and Xiap could be at play in the control of caudal forebrain NSCs proliferation.

Further experiments should be done to accurately describe the signaling mechanism controlling Tcf activity in the proliferating neural tube. Specifically, we should investigate XIAP expression pattern at neurula and tadpole stages, XIAP LOF and GOF phenotypes in the caudal forebrain, and the interactions between Barhl2 and XIAP signaling pathways in neural tube development. It should also be tested whether Xiap ubiquitination of Groucho affects Barhl2 affinity for Groucho, as it does for Tcf [START_REF] Hanson | XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling[END_REF]. Performing immunoprecipitation and luciferase assays in cells and embryos shall allow us to evaluate whether and how Xiap ubiquitination of Groucho could modify Barhl2 activity as a repressor of Tcf activity.

In any case these observations indicate that Barhl2 ability to inhibit Wnt signaling could be involved in numerous aspects of Xenopus development.

BARHL2 IN APOPTOSIS AND A NON-APOPTOTIC FUNCTION OF CASPASE3 2.1 INVOLVEMENT OF SIGNALING PATHWAYS IN BARHL2 ACTIVITIES

Groucho is at the interplay between different signaling pathways. It is a target in Wnt pathway, Notch and Receptor Tyrosine Kinase (RTK) signaling as it is a target of Xiap, an inhibitor of apoptosis (Cinnamon and Paroush, 2008;[START_REF] Hanson | XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling[END_REF].

The results presented in this manuscript describe how Barhl2 regulates Wnt signaling via the recruitment of Groucho during the Spemann organizer development. As suggested above, this mechanism could be conserved at different stages of development with different types of regulatory interactions involved, and ultimately different functions. In the Spemann Organizer Barhl2 limits the size of the organizing center. In the early neural tube, Barhl2 controls the rate of proliferation of neural progenitors. Juraver-Geslin described that Barhl2 acts via Caspase3 non-apoptotic activity to limit proliferation in the neural tube (Juraver-Geslin et al., 2011). As described previously XIAP activity as an ubiquitin ligase plays an important role in regulating Gro affinity for Tcf and could participate in regulating Barhl2-Gro interaction. One of the main inhibitors of Caspase-3 proteolytic activity is Xiap. Xiap can bind Caspase3 catalytic sites and prevents Caspase-3 proteolytic activity, Xiap can also ubiquitinate Caspase3 and target it for degradation [START_REF] Suzuki | Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death[END_REF]. A recent study demonstrated that monoubiquitination of the Drosophila Caspase DrONC by Diap1, a homologue for Xiap, inhibits its apoptotic function and promotes its non-lethal activities [START_REF] Kamber Kaya | An inhibitory monoubiquitylation of the Drosophila initiator caspase Dronc functions in both apoptotic and non-apoptotic pathways[END_REF]. It is therefore possible that in the diencephalic territory besides Groucho, Xiap monoubiquitinates Caspase3 and potentiates other activities independent of its pro-apoptotic role.

Importantly observations in different species argue that a Barhl2 -Caspase3 pathway could be conserved during neural development. In Drosophila cleavage dependent activation of a Gsk-3β isoform, Sgg-46 protein, by the fly Caspases DrONC or DrICE, leads to the downstream phosphorylation and subsequent inhibition of the Wingless pathway [START_REF] Kanuka | Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development[END_REF]. The fly Caspases are regulated in turn by Diap1, the homologue for Xiap. Genetic analysis in C. elegans demonstrate that ceh-30, the orthologue of barhl2, modifies Caspase-3 activity (Peden et al., 2007;Schwartz and Horvitz, 2007). Noteworthy non-apoptotic roles of Caspase3 in the control of stem cell pluripotency have been described [START_REF] Fujita | Caspase activity mediates the differentiation of embryonic stem cells[END_REF][START_REF] Janzen | Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3[END_REF][START_REF] Yi | The Jekyll and Hyde functions of caspases[END_REF]. In conclusion It will be interesting to further investigate Barhl2 functions as a regulator of an unconventional Caspase-3 function, eventually via Xiap activity, at it appears conserved in metazoans.

In the postembryonic neural progenitors Barhl2 could control the rate of differentiation of putative "stem cells" in the cerebellar rhombic lip, in the cortical hem and in the CMZ of the retina. It is well documented that beside Wnt, the Notch and the RTK pathways regulates

Groucho repressor activity and participate to the biology of stem cells (Cinnamon et al., 2008;Cinnamon and Paroush, 2008;[START_REF] Turki-Judeh | Groucho : A Corepressor with Instructive Roles in Development[END_REF]. It will be interesting to evaluate whether Barhl2 role on Tcf is affected by Notch, and or RTK signaling at late developmental stages.

BARHL2 AND THE APOPTOTIC MACHINERY

The second axis of my PhD focused on the regulation of apoptosis during neural development on Xenopus. The laboratory previously described that apoptosis on the neural plate was affected by the overexpression or downregulation of Barhl2 (Offner et al., 2005). It was showed that Barhl2 affected the anti-Bmp and Shh gradient from the axial organizer and this disruption affected in turn the neural plate size. One of my aim was to elucidate the molecular intermediates that played a role on this regulation.

I focused on the two main anti-apoptotic genes that are implicated in nervous system development in vertebrates, bcl-xl and mcl1 [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]Motoyama et al., 1995). We provide the expression pattern of these two genes in Xenopus. The results show that mcl1 is expressed in the neural plate at stage 14 and its expression remains in the neural tube at later stages. We focused on the study of mcl1 regulation because its pattern of expression mcl1 can be degraded by different methods. It can be directly inactivated by cleavage (Clohessy et al., 2004) and it can also be phosphorylated at its N-terminal region by Gsk3 and be posteriorly ubiquitinated (Ding et al., 2007;Thomas et al., 2010). Gsk3 can phosphorylate

Mcl1 to promote its degradation. This is clear when we overexpress a mutant for Mcl1 that cannot be phosphorylated at the S159 by this kinase. We have previously demonstrated that in the neural tube of Xenopus Gsk3 can partially compensate Barhl2 loss-of-function phenotype (Juraver-Geslin et al., 2011). We hypothesize that Barhl2 could be regulating Gsk3 activities in the cytoplasm to mediate Mcl1 degradation. This however, need to be studied and further experiments regarding the effect of Barhl2 on Gsk3 kinase activities should be done to confirm this hypothesis.

SUMMARY AND CONCLUSION

The results presented in this manuscript describe a regulation for Tcf activity during Xenopus embryonic development. We describe the molecular mechanism by which the transcription factor Barhl2 interacts with Groucho and, together with Hdac1, inhibit Wnt activity by binding to Tcf. We demonstrate that this interaction disrupts the formation of the Spemann organizer and that it is maintained throughout development. This mechanism could have implications in regulation of proliferation, and in the maintenance of progenitors' cells niches.

Overall, our results reveal a new and important mechanism for the control of Wnt transcriptional activity.

IV: ANNEX DOCUMENTS

Introduction

In the last decades, functional experiments in numerous model systems provided crucial insights into the early molecular, cellular, and morphological events underlying forebrain development. They revealed the importance of brain signaling centers: small groups of specialized cells acting as local sources of secreted factors. These secreted factors regulate survival, proliferation, and early patterning of neuroepithelial cells and facilitate compartmentalization of the neuroepithelium into functional histological units [START_REF] Jessell | The decade of the developing brain[END_REF][START_REF] Hebert | The genetics of early telencephalon patterning: Some assembly required[END_REF][3][START_REF] Cavodeassi | Brain regionalization: Of signaling centers and boundaries[END_REF][START_REF] Kiecker | The role of organizers in patterning the nervous system[END_REF][6][START_REF] Anderson | Organizers in development[END_REF].

The forebrain (also called prosencephalon) is derived from the most anterior part of the neuroepithelium, the prosencephalic neural plate. Its rostral part, the telencephalon, develops into the cerebrum, which gets divided into the cerebral hemispheres. The caudal forebrain or diencephalon is situated between the cerebral cortex and the midbrain. It gives rise to the thalamic complex, a bilateral structure that contains the pre-thalamus (ventral thalamus) rostrally and the thalamus (dorsal thalamus) caudally.

Development of the thalamus is orchestrated by a brain signaling center called the zona limitans intrathalamica or interthalamica (zli), also referred to as the mid-diencephalic organizer (MDO) [3]. The zli emerges after the completion of neural tube closure and is characterized by a dorso-ventrally (DV) extending gene expression domain of the morphogen Sonic hedgehog (Shh). Shh secreted from the zli and the basal plate participates in the survival, growth, and patterning of neuronal progenitor subpopulations within the thalamic complex [START_REF] Scholpp | Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon[END_REF][START_REF] Vieira | Thalamic development induced by shh in the chick embryo[END_REF][START_REF] Kiecker | Hedgehog signaling from the zli regulates diencephalic regional identity[END_REF][START_REF] Hashimoto-Torii | Differential activities of sonic hedgehog mediated by gli transcription factors define distinct neuronal subtypes in the dorsal thalamus[END_REF][START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Epstein | Regulation of thalamic development by sonic hedgehog[END_REF][START_REF] Jeong | Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity[END_REF][START_REF] Szabo | The role of sonic hedgehog of neural origin in thalamic differentiation in the mouse[END_REF].

In-depth reviews detail the role of Shh in diencephalic patterning [3,[START_REF] Epstein | Regulation of thalamic development by sonic hedgehog[END_REF][START_REF] Chatterjee | Patterning and compartment formation in the diencephalon[END_REF][START_REF] Martinez-Ferre | Molecular regionalization of the diencephalon[END_REF]. Here we focus on the principles underlying emergence of the zli. Specifically, we describe the network of transcription factors and cis-regulatory sequences that confer competence for zli establishment. We review work analyzing their evolutionary conservation. We also highlight the contribution of signaling pathways to zli formation, discuss implications for the fact that Shh relies on primary cilia for signal transduction and speculate about zli development for the etiology of brain malformations.

The Zli: A Unique Shh-Expressing Compartment in the Caudal Forebrain

Forebrain Morphology and Zli Positioning

On the basis of morphology [START_REF] Bergquist | Notes on the early histogenesis and morphogenesis of the central nervous system in vertebrates[END_REF][START_REF] Coggeshall | A study of diencephalic development in the albino rat[END_REF][20][START_REF] Vaage | The segmentation of the primitive neural tube in chick embryos (gallus domesticus): A morphological, histochemical and autoradiographical investigation[END_REF] and gene expression [22,[START_REF] Rubenstein | The embryonic vertebrate forebrain: The prosomeric model[END_REF], the diencephalic primordium is divided into three transverse segments called prosomeres (p) that generate three distinct histogenic fields: p3, or anterior parencephalon, corresponds to the presumptive prethalamus and the eminentia thalami; p2, or posterior parencephalon, gives rise to the epithalamus and the thalamus; and p1, or synencephalon, generates the presumptive pretectum (reviewed in [START_REF] Martinez-Ferre | Molecular regionalization of the diencephalon[END_REF][START_REF] Figdor | Segmental organization of embryonic diencephalon[END_REF][START_REF] Puelles | Forebrain gene expression domains and the evolving prosomeric model[END_REF][START_REF] Thompson | A high-resolution spatiotemporal atlas of gene expression of the developing mouse brain[END_REF][START_REF] Ferran | Molecular codes defining rostrocaudal domains in the embryonic mouse hypothalamus[END_REF]). Each prosomere is divided into a ventral (basal) and a dorsal (alar) part (Figure 1A).

The zli demarcates the boundary between pre-thalamus and thalamus, separating the posterior diencephalon (p1 and p2) from the anterior diencephalon (p3) (Figure 1; [START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF][START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF][START_REF] Shimamura | Longitudinal organization of the anterior neural plate and neural tube[END_REF]). This position corresponds approximately to the junction between the prechordal neuraxis overlying the prechordal mesendoderm, and the epichordal neuraxis overlying the notochord [START_REF] Vieira | Thalamic development induced by shh in the chick embryo[END_REF]. The zli is defined by a dorsalward continuation of shh expression into the alar plate of the diencephalon (Figure 1A,Bb-d).

The zli is the last brain signaling center to emerge: its dorsal progression within the alar plate starts at stage 24 in frog [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]), HH12 to HH26 in chicken [START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF][START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF][START_REF] Zeltser | A new developmental compartment in the forebrain regulated by lunatic fringe[END_REF], E9 in mouse, and between the 12-and 15-somite stage in zebrafish [START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF]. It is unique in brain regionalization because it represents the only neural area in which Shh, normally a DV patterning signal, regulates anterior-posterior (AP) regionalization [START_REF] Ericson | Two critical periods of sonic hedgehog signaling required for the specification of motor neuron identity[END_REF][START_REF] Watanabe | Control of chick tectum territory along dorsoventral axis by sonic hedgehog[END_REF].

Shh Expression Initiates and Demarcates Zli Development

During gastrulation and early neurulation, shh is expressed in the prechordal plate and notochord. Notochord-derived Shh activates the Hedgehog signaling pathway and its own neural midline expression in the overlying neuroepithelial cells ( [START_REF] Placzek | Mesodermal control of neural cell identity: Floor plate induction by the notochord[END_REF]36]; reviewed in [START_REF] Placzek | The floor plate: Multiple cells, multiple signals[END_REF]). In the posterior part of the neural tube, shh gene expression remains restricted to the medial floor plate, whereas in the mesencephalon and prosencephalon shh expression spreads from the floor plate to the basal plate. It is only at the p2 to p3 boundary that a triangle-shaped expression of shh extends dorsalward (i.e., more lateral) on either side of the diencephalic walls (Figure 1; reviewed in [START_REF] Placzek | The floor plate: Multiple cells, multiple signals[END_REF]). Studies in chicken revealed that a sequential induction process, initiated by Shh secreted by floor plate cells, underlies the appearance of the shh-expressing line of cells corresponding to the zli [START_REF] Zeltser | Shh-dependent formation of the zli is opposed by signals from the dorsal diencephalon[END_REF]. This homeotic induction of shh occuring from one cell to its neighbor is not understood. It could necessitate specific Shh maturation and secretion processes, together with modification at the level of the primary cilium that mediates Shh read-out. Because the zli is not formed through cell migration from the basal to the alar plate, the zli is considered an alar structure [START_REF] Zeltser | Shh-dependent formation of the zli is opposed by signals from the dorsal diencephalon[END_REF]. Observations in frog, chicken, and mouse demonstrate that a continuous source of Shh signals, provided in vivo by secretion of Shh initially from floor plate cells, and then from the basal plate, is strictly necessary both for induction of shh expression within the zli and for the correct segregation of zli cells from the thalamus [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF][START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF][START_REF] Zeltser | Shh-dependent formation of the zli is opposed by signals from the dorsal diencephalon[END_REF][START_REF] Vieira | Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure[END_REF].

In zebrafish, however, a small domain of shh expression at the position of the zli appears in smoothened (smo) mutants, in which signaling downstream of all Hedgehog family ligands is defective [START_REF] Mathieu | Distinct and cooperative roles for nodal and hedgehog signals during hypothalamic development[END_REF][START_REF] Holzschuh | Genetic analysis of the roles of hh, fgf8, and nodal signaling during catecholaminergic system development in the zebrafish brain[END_REF]. Similarly, a population of "zli cells" develops in a quarter of zebrafish cyclops mutants, which lack a floor plate in the diencephalon [START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF][START_REF] Zeltser | Shh-dependent formation of the zli is opposed by signals from the dorsal diencephalon[END_REF][START_REF] Vieira | Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure[END_REF][START_REF] Sampath | Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling[END_REF]. Taken together, these observations suggest that, contrary to observations in other model organisms, the continuous presence of a neural midline-derived Shh signal is not required for emergence of the zli in zebrafish.

Taken together, these observations suggest that, contrary to observations in other model organisms, the continuous presence of a neural midline-derived Shh signal is not required for emergence of the zli in zebrafish.

Physical Separation: The Zli as a Tissue Compartment

To fulfill their localized patterning function, cells of a signaling center need to be arranged in a coherent compartment with clear boundaries and lineage restriction. This enables the signaling

Physical Separation: The Zli as a Tissue Compartment

To fulfill their localized patterning function, cells of a signaling center need to be arranged in a coherent compartment with clear boundaries and lineage restriction. This enables the signaling center to maintain its position relative to the surrounding tissue [START_REF] Figdor | Segmental organization of embryonic diencephalon[END_REF][START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF][START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF][START_REF] Martinez | Neurogenetic compartments of the mouse diencephalon and some characteristic gene expression patterns[END_REF] (reviewed in [START_REF] Martinez-Ferre | Molecular regionalization of the diencephalon[END_REF][START_REF] Kiecker | Compartments and their boundaries in vertebrate brain development[END_REF]). An analysis of chick diencephalon development revealed that early on, the caudal forebrain territory is not overtly segmented [START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF]. Development of the zli, however, correlates with acquisition of cell lineage-specific properties. Cell movements become restricted at both the anterior and posterior limits of the zli and zli formation is associated with acquisition of properties observed at tissue boundaries: 1) interkinetic nuclear migration movements are disrupted and 2) markers of immiscible interfaces such as chondroitin sulfate proteoglycans, laminin, weakly polysialylated neural cell adhesion molecules, and vimentin are detected within the zli territory [START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF][START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF]. These studies established that the zli is a narrow compartment with cell lineage-restricted boundaries, introducing physical separation of the diencephalon into the prethalamus anteriorly and the thalamus posteriorly [START_REF] Figdor | Segmental organization of embryonic diencephalon[END_REF][START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF][START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF][START_REF] Zeltser | A new developmental compartment in the forebrain regulated by lunatic fringe[END_REF].

This physical separation brought about by lineage restriction within the zli may in addition be instructive for AP segmentation on a forebrain-wide scale. According to the prosomeric model the forebrain becomes subdivided into an anterior and a posterior part. Anteriorly, the so-called secondary prosencephalon contains the telencephalon, optic vesicles, and hypothalamus. More posteriorly, the alar plate of the diencephalon is divided into the anterior p3, p2 and p1 ( [START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF]; reviewed in [START_REF] Martinez-Ferre | Molecular regionalization of the diencephalon[END_REF][START_REF] Puelles | Forebrain gene expression domains and the evolving prosomeric model[END_REF]). Note that whereas the prosomeric model differentiates p2 and p3 basal plates from the anterior basal hypothalamus, in zebrafish the hypothalamus is thought to develop within the basal diencephalon, a difference that may generate misinterpretations. Partitioning of the anterior forebrain from the remainder of the neural tube partly relies on the distinct processes inducing specification of the prechordal versus epichordal neural plate. It is still under debate whether the alar plate of p3, which generates the prethalamus, is under the influence of the chordal, or of the prechordal mesendoderm [START_REF] Puelles | Forebrain gene expression domains and the evolving prosomeric model[END_REF][START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF]. However, cell lineage analysis indicates that cells do not segregate between the prethalamus and the secondary prosencephalon [START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF]. Moreover, the competence of the prethalamus (p3) to respond to instructive factors such as fibroblast growth factor (Fgf) 8 is different from that of the other diencephalic prosomeres (p2 and p1) [START_REF] Crossley | Midbrain development induced by fgf8 in the chick embryo[END_REF][START_REF] Robertshaw | Irx3 and pax6 establish differential competence for shh-mediated induction of gabaergic and glutamatergic neurons of the thalamus[END_REF]. This raises the question whether the zli could represent a pivotal structure along the neuraxis segregating a "large prosencephalon" from the remainder of the neural tube. At minimum, the zli separates territories with differential competence to respond to morphogens [START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF][START_REF] Rubenstein | Regionalization of the prosencephalic neural plate[END_REF].

In conclusion, the zli is a narrow compartment, located in the alar plate of the caudal forebrain that expresses shh. It is the last signaling center to form. It introduces segmentation within the diencephalon and has an important function in diencephalic patterning. The zli is characterized by the same positioning as similar gene expression patterns in all model organisms. However, the mode of its formation may vary between species. From studies in zebrafish, frog, chicken and mouse, it is now possible to propose a model for the GRN and the inductive cues allowing formation of this unique signaling center.

A Role for Wnt Ligands in Generating a Zli-Permissive Compartment

Observations in zebrafish, amphibian, and chicken have highlighted early and late roles for Wnt ligands in zli positioning, induction, and development [START_REF] Mattes | Wnt3 and wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain[END_REF][START_REF] Martinez-Ferre | Wnt signal specifies the intrathalamic limit and its organizer properties by regulating shh induction in the alar plate[END_REF] (Figure 2). Canonical Wnt signaling is involved in AP patterning of the forebrain [START_REF] Houart | A small population of anterior cells patterns the forebrain during zebrafish gastrulation[END_REF] (reviewed in [START_REF] Wilson | Review: Early steps in the development of the forebrain[END_REF]). Receptors, ligands and modifiers of the Wnt pathway are expressed during caudal forebrain regionalization. Specifically, expression of the Wnt ligands Wnt3, Wnt3a, and Wnt8b mark the alar plate of p2 and the zli primordium [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Zeltser | A new developmental compartment in the forebrain regulated by lunatic fringe[END_REF][START_REF] Martinez-Ferre | Wnt signal specifies the intrathalamic limit and its organizer properties by regulating shh induction in the alar plate[END_REF]. In zebrafish, lack of canonical Wnt signaling brought about by a Wnt signaling antagonist induces loss of the pre-zli territory. Conversely, enhancement of Wnt signaling using the Glycogen Synthase Kinase 3β (GSK3β) inhibitor BIO leads to a broader expression domain of shh at the zli [START_REF] Mattes | Wnt3 and wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain[END_REF]. The depletion of both Wnt3 and Wnt3a leads to an increase of apoptosis and a loss of the diencephalic organizer primordium indicating that Wnt3 and Wnt3a are normally required for survival of zli anlage cells. Interestingly, the effect of canonical Wnt signaling on the survival of zli anlage cells is restricted to a time window of 4 hours during somitogenesis. In embryos depleted for Wnt3 and Wnt3a the size of the prethalamic and thalamic markers fezf2, irx1b and otx2 expression domains are unaltered (Figures 1A and2). In Wnt3/3a depleted embryos the concomitant depletion of fezf2, or of Irx1b, activity rescues zli formation, indicating that both Fezf2 and Irx1b normally restrict the zli territory [START_REF] Mattes | Wnt3 and wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain[END_REF]. Therefore, Wnt3/3a function is required for maintenance of the zli anlage, but not for the maintenance of the prethalamus and thalamus territories (Figure 2). In zebrafish, lack of canonical Wnt signaling brought about by a Wnt signaling antagonist induces loss of the pre-zli territory. Conversely, enhancement of Wnt signaling using the Glycogen Synthase Kinase 3 (GSK3 ) inhibitor BIO leads to a broader expression domain of shh at the zli [START_REF] Mattes | Wnt3 and wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain[END_REF]. The depletion of both Wnt3 and Wnt3a leads to an increase of apoptosis and a loss of the diencephalic organizer primordium indicating that Wnt3 and Wnt3a are normally required for survival of zli anlage cells. Interestingly, the effect of canonical Wnt signaling on the survival of zli anlage cells is restricted to a time window of 4 hours during somitogenesis. In embryos depleted for Wnt3 and Wnt3a the size of the prethalamic and thalamic markers fezf2, irx1b and otx2 expression domains are unaltered (Figures 1A and2). In Wnt3/3a depleted embryos the concomitant depletion of fezf2, or of Irx1b, activity rescues zli formation, indicating that both Fezf2 and Irx1b normally restrict the zli territory [START_REF] Mattes | Wnt3 and wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain[END_REF].

Therefore, Wnt3/3a function is required for maintenance of the zli anlage, but not for the maintenance of the prethalamus and thalamus territories (Figure 2).

A study by Martinez-Ferre et al. reveals a requirement for a Wnt8b-mediated signal as a permissive step for the subsequent induction of shh expression and for emergence of the zli in the diencephalic primordium [START_REF] Martinez-Ferre | Wnt signal specifies the intrathalamic limit and its organizer properties by regulating shh induction in the alar plate[END_REF]. Gli3 is a Shh-regulated transcriptional repressor [START_REF] Ruiz I Altaba | The emergent design of the neural tube: Prepattern, shh morphogen and gli code[END_REF][START_REF] Jacob | Gli proteins and the control of spinal-cord patterning[END_REF][START_REF] Jessell | Neuronal specification in the spinal cord: Inductive signals and transcriptional codes[END_REF]. During early patterning stages of the neural plate gli3 transcripts are detected within the alar neural plate and specifically in the alar diencephalon. In chick, this wide expression domain gets restricted to a narrow band of cells at the center of the wnt8b expression domain. The transverse alar stripe of wnt8b which is now devoid of gli3 expression is the prospective zli anlage [START_REF] Hashimoto-Torii | Differential activities of sonic hedgehog mediated by gli transcription factors define distinct neuronal subtypes in the dorsal thalamus[END_REF]. Over-expression of gli3 in the zli inhibits shh induction, indicating that the local repression of gli3 is necessary to allow shh homeotic induction during zli formation. Martinez-Ferre et al. demonstrated that the local downregulation of gli3 at the future zli requires a Wnt-signal, which in chicken is mediated by the presence of Wnt8b. Wnt8b signal appears necessary to locally downregulate gli3 at the center of the wnt8b expression domain. It is, however, not sufficient, as the entire domain that expresses wnt8b does not lose gli3 expression. Finally, once Shh is expressed in the zli, inhibition of the Wnt pathway does not have any effect on its maintenance [START_REF] Martinez-Ferre | Wnt signal specifies the intrathalamic limit and its organizer properties by regulating shh induction in the alar plate[END_REF].

In conclusion, while Wnt-mediated signals are important for maintenance of the zli anlage, and to generate the permissive conditions for the activation of Shh in the zli, they must act in collaboration with a combination of factors at the prethalamic (Fezf/Fez) and thalamic (Barhl2, Irx3, and Otx2) forebrain borders.

Laying the Ground for the zli: Otx2 and Barhl2 Binding to cis-Regulatory Sequences Confers Competence for zli Formation

During neural induction, an underlying pre-pattern, partly encoded by TFs, emerges in the neural plate. These early patterning cues contribute to the specification of forebrain territories and influence the way in which neighboring cell populations respond differentially to similar morphogens [START_REF] Robertshaw | Irx3 and pax6 establish differential competence for shh-mediated induction of gabaergic and glutamatergic neurons of the thalamus[END_REF][START_REF] Kobayashi | Early subdivisions in the neural plate define distinct competence for inductive signals[END_REF][START_REF] Gomez-Skarmeta | Half a century of neural prepatterning: The story of a few bristles and many genes[END_REF]. Analysis of TF expression dynamics in the anterior neural plate provides crucial information about the cues involved in emergence of the zli and reveals that zli induction starts during neurulation.

Two TFs are especially involved in conferring competence to the future zli tissue to express shh. Orthodenticle homeobox (Otx) 1 and 2 are homeodomain-containing proteins involved in specification and regionalization of the forebrain (reviewed in [START_REF] Acampora | The role of otx and otp genes in brain development[END_REF][START_REF] Beby | The homeobox gene otx2 in development and disease[END_REF]). The otx2 expression territory marks the anterior neural plate from gastrulation onwards [START_REF] Pannese | The xenopus homologue of otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions[END_REF]. In both zebrafish and frog otx expression decreases in the telencephalic territory during neurulation. At the onset of zli development otx expression is restricted to the p2 and midbrain territories ( [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF]; Figure 1Ba). The Bar-class homeodomain-containing (BarH) Barh1 and Barh2 are also homeodomain-containing TFs (reviewed in [START_REF] Schuhmacher | Evolutionary relationships and diversification of barhl genes within retinal cell lineages[END_REF][START_REF] Reig | Functions of barh transcription factors during embryonic development[END_REF][START_REF] Juraver-Geslin | Early development of the neural plate: New roles for apoptosis and for one of its main effectors caspase-3[END_REF]). Transcripts encoding BarH-like (barhl) 2 are detected in the diencephalic primordium of amphibian [START_REF] Offner | The pro-apoptotic activity of a vertebrate bar-like homeobox gene plays a key role in patterning the xenopus neural plate by limiting the number of chordin-and shh-expressing cells[END_REF][START_REF] Juraver-Geslin | Barhl2 limits growth of the diencephalic primordium through caspase3 inhibition of beta-catenin activation[END_REF], zebrafish [START_REF] Staudt | The prethalamus is established during gastrulation and influences diencephalic regionalization[END_REF], and mouse [START_REF] Mo | Role of the barhl2 homeobox gene in the specification of glycinergic amacrine cells[END_REF][START_REF] Yao | cis-Regulatory architecture of a brain signaling center predates the origin of chordates[END_REF]. Similar to otx genes, at the onset of zli development barhl1 and barhl2 expression are restricted to prosomere p2. While barhl2 is expressed in the entire p2, barhl1 expression is restricted to basal p2 ( [START_REF] Patterson | Distinct expression patterns for two xenopus bar homeobox genes[END_REF][START_REF] Colombo | Zebrafish barh-like genes define discrete neural domains in the early embryo[END_REF]; Figure 1A,Bb).

In zebrafish, the lack of Otx1l/2 (the zebrafish ortholog of Otx2) function leads to absence of the zli and subsequently of zli-dependent target genes. With a lack of Otx function, the thalamus is mis-specified prior to, and independently from, zli formation. The adjacent territories of the prethalamus and pretectum expand into the mis-specified territory and form a new interface. Mouse embryos with reduced otx1/2 transcripts similarly show a lack of shh zli expression [START_REF] Yao | cis-Regulatory architecture of a brain signaling center predates the origin of chordates[END_REF]. Therefore, the presence of Otx1/2 is required to establish a competence area, allowing induction of shh in the zli [START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF][START_REF] Yao | cis-Regulatory architecture of a brain signaling center predates the origin of chordates[END_REF][START_REF] Hirata | Zinc-finger genes fez and fez-like function in the establishment of diencephalon subdivisions[END_REF]. In Otx-depleted zebrafish embryos, ectopic expression of Otx rescues formation of the zli solely within the diencephalic territory, anterior to the presumptive thalamus [START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF]. In X. laevis embryos depleted for Barhl2, development of the zli is abolished [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]. Similarly, in barhl2 / mice, shh expression within the zli is significantly reduced [START_REF] Yao | cis-Regulatory architecture of a brain signaling center predates the origin of chordates[END_REF][START_REF] Ding | Barhl2 determines the early patterning of the diencephalon by regulating shh[END_REF]. Barhl2-depleted X. laevis embryos resemble zebrafish Otx1l/2-depleted embryos: although most forebrain markers are unaltered, the embryos exhibit defects in shh expression in the zli and in the formation of the mid-diencephalic furrow [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Ding | Barhl2 determines the early patterning of the diencephalon by regulating shh[END_REF].

Noteworthy, in zebrafish loss of Otx2 induces a loss of barhl2 expression while otx2 expression is maintained in Barhl2-depleted X. laevis. Therefore, Otx1l/2 proteins are necessary for maintenance of barhl2 in the future zli territory, and the loss of barhl2 contributes to the zli defects observed in Otx-deficient zebrafish [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF].

Synergistic activities of Barhl2 and Otx2 in zli formation are confirmed by analysis of the cis-regulatory-motifs controlling shh expression within the mouse zli. Yao et al. identified two enhancers, SBE1 and SBE5, driving shh expression within the zli. Both enhancers function in a partially redundant manner and their activity relies on six position-independent motifs directly regulated by a combination of Otx2, Barhl2 and the TEA-domain family member 2 (Tead2), a key mediator of Hippo signaling, and its co-transcriptional activation partner Yap. Using large-scale genomic approaches associated with bioinformatic analytical tools, Yao et al. characterized a 116-bp homology block, referred to as SBE1-like-enhancer, present in enhancers scattered throughout the mouse and human genomes. The SBE1-like-enhancer sequence is conserved from human to zebrafish. Using luciferase reporter assays, chromatin immunoprecipitation (ChIP), and transgenic mouse reporter assays, Yao et al. showed that the six motifs are necessary and sufficient for full enhancer activity and paired three motifs with cognate transcription factors. Motifs 1 and 6 correspond to recognition sequences for Otx1/Otx2 and Barhl2, respectively. Indeed, the combined action of Barhl2 and Otx2 resulted in a synergistic induction of reporters whose activity is under the control of SBE1-like enhancers containing motifs 1 and 6. Similarly, Tead2 and Yap recognize and contribute to shh expression through binding on motif 2. Embryos with conditional loss of Yap1 showed a selective reduction in zli shh expression. Tead2 and Yap activities on zli formation have not been assessed in species others than mouse. The second enhancer SBE5 contains a cluster of permuted motifs similar to those identified in the SBE1 enhancer in the absence of any other overt sequence homology. This second enhancer, located in the vicinity of the shh locus, performs equivalently to SBE1 in cell-based reporter and ChIP assays. Deletion of both enhancers in mouse entirely abolishes expression of shh within the zli [START_REF] Yao | cis-Regulatory architecture of a brain signaling center predates the origin of chordates[END_REF].

Taken together, these studies reveal the presence of a "zli developmental cassette" that uses two main TFs, Otx2 and Barhl2, in combination, at least in mice, with the Tead2-Yap1 activation complex and conserved cis-regulatory motifs to induce shh expression in a narrow band of cells within the anterior brain.

Hedgehog Sequential Induction Process in zli Formation: Shh Is a Secreted Signal Read out through a Primary Cilium

During zli formation the N-terminal part of Shh secreted by basal plate cells is released into the ventricular lumen and N-Shh activates its own expression only in its neighboring cell [START_REF] Zeltser | Shh-dependent formation of the zli is opposed by signals from the dorsal diencephalon[END_REF]. Pre-zli cells respond differently to Shh compared to neighboring prethalamus and thalamus cells. The cues controlling sequential induction and the differential response to Shh are not fully deciphered. Modifications in Shh maturation and secretion, and/or in the primary cilium that mediates Shh read-out, could participate in this unique process.

Shh Maturation and Secretion

The secretion of Hedgehog (HH) proteins into the ventricular lumen necessitates that HH proteins undergo an autocatalytic internal cleavage associated with the addition of lipid molecules, specifically cholesterol and palmitic acid moieties (reviewed in [START_REF] Ramsbottom | Regulation of hedgehog signalling inside and outside the cell[END_REF]). Cleavage confers a hydrophobic character to HH that is required for its association with the cell membrane. HH proteins truncated at the site of internal cleavage diffused more largely [START_REF] Porter | Hedgehog patterning activity: Role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain[END_REF]. The internal cleavage occurs in the endoplasmic reticulum and produces a 20 kD amino-terminal domain (N-HH) and a 25 kD carboxy-terminal domain (C-HH) [START_REF] Porter | The product of hedgehog autoproteolytic cleavage active in local and long-range signalling[END_REF][START_REF] Bumcrot | Proteolytic processing yields two secreted forms of sonic hedgehog[END_REF][START_REF] Chen | Processing and turnover of the hedgehog protein in the endoplasmic reticulum[END_REF]. The C-HH part recruits a cholesterol molecule that binds to the C-terminus of N-HH [START_REF] Chen | Processing and turnover of the hedgehog protein in the endoplasmic reticulum[END_REF][START_REF] Porter | Cholesterol modification of hedgehog signaling proteins in animal development[END_REF]. The binding of cholesterol to N-HH is essential in limiting the range of HH signaling. In mouse, N-HH lacking cholesterol has an extended signaling range in the limb bud [START_REF] Li | Cholesterol modification restricts the spread of shh gradient in the limb bud[END_REF].

Besides cholesterol, the N-HH protein is also modified by the attachment of a palmitic acid group on its N-terminal part that has proved important for HH activity [START_REF] Pepinsky | Identification of a palmitic acid-modified form of human sonic hedgehog[END_REF]. The transfer is mediated by an acyltransferase named Skinny Hedgehog (SKI) in Drosophila and mouse and Hedgehog acyltransferase (HHAT) in humans [START_REF] Buglino | Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of sonic hedgehog[END_REF][START_REF] Chamoun | Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal[END_REF][82]. Mouse embryos lacking N-HH palmitoylation exhibit decreased Shh signaling in limb buds [START_REF] Chen | Palmitoylation is required for the production of a soluble multimeric hedgehog protein complex and long-range signaling in vertebrates[END_REF] as well as in ventral forebrain formation [START_REF] Kohtz | N-Terminal fatty-acylation of sonic hedgehog enhances the induction of rodent ventral forebrain neurons[END_REF].

Different mechanisms have been described for the release of processed N-HH (pN-HH) into the extracellular space. pN-HH is strongly hydrophobic. It can be secreted as a monomer but needs to bind to other secreted proteins. In Drosophila, the transmembrane protein Dispatched-1 (Disp1) [START_REF] Burke | Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells[END_REF] binds to pN-HH along with the secreted protein Scube2. Disp1 and Scube2 bind to different parts of the cholesterol attached to N-HH and promote the release of pN-HH from the cell surface [START_REF] Creanga | Scube/you activity mediates release of dually lipid-modified hedgehog signal in soluble form[END_REF][START_REF] Tukachinsky | Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand[END_REF]. There is also evidence that pN-HH secretion is mediated through a lipoprotein complex [START_REF] Eugster | Lipoprotein-heparan sulfate interactions in the hh pathway[END_REF][START_REF] Panakova | Lipoprotein particles are required for hedgehog and wingless signalling[END_REF]. Lipoproteins consist of a phospholipid monolayer that embraces the lipids present in the pN-HH leaving the hydrophilic areas outside, decreasing the strong hydrophobicity of the pN-HH molecule. In Caenorhabditis elegans (C. elegans) [START_REF] Liegeois | The v0-atpase mediates apical secretion of exosomes containing hedgehog-related proteins in caenorhabditis elegans[END_REF] and Drosophila [START_REF] Callejo | Dispatched mediates hedgehog basolateral release to form the long-range morphogenetic gradient in the drosophila wing disk epithelium[END_REF], an exosome-mediated release has been described. In vertebrates pN-HH can also be released in a vesicular form [START_REF] Vyas | Vertebrate hedgehog is secreted on two types of extracellular vesicles with different signaling properties[END_REF].

The Shh Signal Is Read through a Primary Cilium

Shh-related patterning defects in mice carrying mutations in genes essential for cilia function revealed that Shh signaling in vertebrates requires the presence of primary cilia [START_REF] Huangfu | Hedgehog signalling in the mouse requires intraflagellar transport proteins[END_REF]. Cilia are small, membrane-sheathed cell protrusions that occur on almost all cells during development and adulthood. Motile cilia can be bent by dynein motors, which results in rotational or beating movement, whereas primary cilia are immotile. Owing to the fact that their membranes are studded with a range of different mechano-and chemo-receptors, primary cilia are considered highly specialized sensory organelles [START_REF] Hilgendorf | The primary cilium as a cellular receiver: Organizing ciliary gpcr signaling[END_REF]. Signal transduction from the sensory primary cilium requires motor protein-driven transport along axonemal microtubules, termed intraflagellar transport (IFT). IFT is required for Hedgehog signal transduction since Patched (Ptc), a twelve-transmembrane domain receptor, Smo, a G-protein coupled receptor, and the zinc finger containing Gli family of TFs are moving into, within, and out of the cilium, depending on pathway activation status ( [START_REF] Bai | All mouse ventral spinal cord patterning by hedgehog is gli dependent and involves an activator function of gli3[END_REF]; reviewed in [START_REF] Ramsbottom | Regulation of hedgehog signalling inside and outside the cell[END_REF][START_REF] Briscoe | Making a grade: Sonic hedgehog signalling and the control of neural cell fate[END_REF][97][START_REF] Mukhopadhyay | G-Protein-coupled receptors, hedgehog signaling and primary cilia[END_REF]).

During embryogenesis, neural tube cells in mouse [START_REF] Huangfu | Hedgehog signalling in the mouse requires intraflagellar transport proteins[END_REF][START_REF] Willaredt | Foxj1 regulates floor plate cilia architecture and modifies the response of cells to sonic hedgehog signalling[END_REF], chicken [100], X. laevis [101], and zebrafish [102] carry primary cilia on their apical surface. These are crucial for Shh signal transduction governing DV patterning of the neural tube [START_REF] Huangfu | Hedgehog signalling in the mouse requires intraflagellar transport proteins[END_REF]. Cilia have also been identified on ventricle-contacting cells specifically in the forebrain region in mouse [103] and on diencephalic cells corresponding to the region of the zli in X. laevis (Figure 3;[104]). Interestingly, cilia on cells within shh-expressing brain signaling centers including the floor plate, rhombomere boundaries, and zli are specifically elongated compared to surrounding primary cilia on non-shh-expressing cells ([100,101,104] and Figure 3). This could indicate a way of differentially adapting signaling pathway activation, as longer cilia seem to correlate with reduced pathway activation [100].

In conclusion, the mechanisms controlling Shh secretion and diffusion rate in the context of zli development are still being investigated. The strong hydrophobic character of N-Shh suggests that it diffuses very slowly and tends to stay close to the membrane surface. This could at least partly explain the sequential induction process observed during zli development. Moreover, a primary cilium is present in diencephalic progenitor cells. The primary cilium is structurally different on pre-zli cells versus prethalamic and thalamic cells and little is known on the biological consequences of such differences.

ventricle-contacting cells specifically in the forebrain region in mouse [103] and on diencephalic cells corresponding to the region of the zli in X. laevis (Figure 3;[104]). Interestingly, cilia on cells within shh-expressing brain signaling centers including the floor plate, rhombomere boundaries, and zli are specifically elongated compared to surrounding primary cilia on non-shh-expressing cells ([100,101,104] and Figure 3). This could indicate a way of differentially adapting signaling pathway activation, as longer cilia seem to correlate with reduced pathway activation [100].

Building Compartments: The Iroquois Genes Refine the Zli Borders

The Iroquois (irx) genes encode for homeodomain-containing TFs, highly conserved from Drosophila to mammals (reviewed in [105,106]). Most vertebrates contain six irx genes grouped in two paralog clusters of three genes each [107,108]. The irxA cluster contains irx1, irx2 and irx4, while the irxB cluster corresponds to irx3, irx5 and irx6, and irx7 in zebrafish. The Irx proteins participate in defining territories and in specifying cell identity. irx gene activities in boundary formation have been previously described (reviewed in [105]).

Observations in zebrafish and amphibian promote the idea that Irx factors participate in acquisition of zli compartment identity [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF]. In all species studied, irx1, irx2, and irx3 are co-expressed in the anterior neural plate during the early stages of neural patterning and mark the future p2 (Figures 1 and3; [START_REF] Offner | The pro-apoptotic activity of a vertebrate bar-like homeobox gene plays a key role in patterning the xenopus neural plate by limiting the number of chordin-and shh-expressing cells[END_REF]109,110]). In zebrafish, irx3b is strongly expressed in the developing zli together with irx5a, irx5b and irx7. The irx3b/5/7 expression pattern subdivides the zli into distinct DV domains. In contrast, irx1a, irx1b, and irx2a expression domains abut the zli posteriorly [110]. In zebrafish, irx1b function is dispensable for zli development; however, depletion of the irx1 orthologs irx1b and irx7 induces a posterior shift of the zli caudal border and an expansion of the shh expression domain at the expense of the thalamic field [START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF][START_REF] Hirata | Zinc-finger genes fez and fez-like function in the establishment of diencephalon subdivisions[END_REF].

Expression analysis of irx genes during X. laevis zli development reveals a temporally dynamic process [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]. At neural plate stages, irx1, 2 and 3 are co-expressed in the future p2 domain expressing barhl2 and otx2. At the onset of zli formation the rostral p2, i.e., the future zli, expresses solely irx3, whereas the caudal p2, i.e., future thalamus, expresses irx1, 2 and 3. After the zli has fully developed, the rostral p2 expresses irx3 and shh, and the caudal p2 expresses irx1 and irx2 (Figure 1A,Bc,d and Figure 2 and [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]). Furthermore, the mis-expression of the Irx factors within the barhl2/otx2-expressing field in frog indicates that the ratio of Irx3 to Irx1/2 is essential for zli specification and for establishment of the zli posterior boundary. Both overexpression of irx3 as well as irx1/2 depletion promotes the acquisition of a zli fate at the expense of a thalamic fate. It is unknown whether Irx1, Irx2, and Irx3 proteins regulate one another's expression.

Observations in chick embryos are divergent from those described in other vertebrates. Chicken irx3 is described as being expressed posterior to the zli and appears to have a repressive function on zli formation [START_REF] Kiecker | Hedgehog signaling from the zli regulates diencephalic regional identity[END_REF]. In chicken, Irx3 does not appear to participate in zli development but in establishment of the differential cellular competence to respond to Shh signaling from the zli [START_REF] Robertshaw | Irx3 and pax6 establish differential competence for shh-mediated induction of gabaergic and glutamatergic neurons of the thalamus[END_REF]. Misexpression experiments in the caudal forebrain through in ovo electroporation approaches indicate that Irx3 together with Pax6 alters the competence of caudal forebrain cells to respond to both Fgf8 and Shh. Irx3 ectopic expression in the prethalamic anlage induces expression of thalamic markers and represses prethalamic markers [START_REF] Robertshaw | Irx3 and pax6 establish differential competence for shh-mediated induction of gabaergic and glutamatergic neurons of the thalamus[END_REF].

In conclusion, numerous observations indicate that Irx activities are important to establish the anterior and posterior borders of the zli, and to acquire cell segregation properties. Irx activities may vary depending on species. Whether the Irx proteins are strictly required for the expression of shh within the zli territory, and what is their exact function in zli formation, remain open questions.

Sticking Together: GRNs and Signaling Pathways Modulate Zli Cell Differential Adhesive Properties and Proliferation Rate

Compartments exhibit specific features: they are characterized by a reduced rate of cell proliferation and cells of neighboring compartments separate along boundaries. Cells segregate from one another based on differences in the adhesive properties (affinity) of their cell surfaces. At the boundary, cells deposit an extracellular matrix that acts as a mechanical barrier between different cell populations. Establishment of adhesive differences constitutes the first step of lineage restriction, whereas fence-type mechanisms might stabilize compartments at later stages (reviewed in [START_REF] Kiecker | Compartments and their boundaries in vertebrate brain development[END_REF]111]).

Canonical Wnt Signaling Regulates Thalamic Cell Adhesiveness and Segregation

Various canonical Wnt ligands are markers of the alar plate of p2. Analysis of caudal forebrain proliferation kinetics in chicken and mouse reveal that zli cells divide slowly relative to cells in their flanking territories [START_REF] Martinez | Neurogenetic compartments of the mouse diencephalon and some characteristic gene expression patterns[END_REF]112]. -catenin, the main effector of the canonical Wnt pathway, controls neuroepithelial cell proliferation (reviewed in [113][114][115]). -catenin mediates the interactions between the intracellular cytoskeleton and the cadherins, a group of cell-cell adhesion proteins important in the formation of neural boundaries. Members of the cadherin superfamily mediate differential adhesive properties and are expressed differentially in the forebrain subdivisions. Therefore, it is thought that cadherins participate in formation of compartment boundaries (reviewed in [116][117][118]). In the zebrafish thalamus, -catenin regulates the expression of protocadherin 10b (Pcdh10b, formerly known as [118]). Alteration of pcdh10b expression in the thalamus territory leads to an intermingling of thalamic cells with the neighboring brain areas, predominantly with the pretectum, indicating an important role for pcdh10b in thalamus separation. Stabilization and nuclear translocation of -catenin leads to a broadening of the expression domain of pcdh10b, whereas inhibition of Wnt signaling decreases pcdh10b expression. Therefore, the Wnt canonical pathway participates in acquisition of the adhesive differences supporting differential cell segregation behaviors within p2.

The Notch/Delta Pathway Contributes to Correct Separation of the Zli and Thalamic Fields

Notch signaling mediates lateral inhibition in embryonic tissues and during neural development (reviewed in [119]). delta as well as radical fringe (rfng) and lunatic fringe (lfng), two glycosyltransferases that regulate Notch signaling, are expressed and have been involved in the establishment of boundaries. In zebrafish, in vivo activation of the Notch pathway directs cells to the rhombomere boundaries, whereas inhibition of Notch activity excludes cells from boundaries [120]. In avian embryos, a forebrain wedge-shaped domain referred to as the "pre-zli" is characterized by a gap in the expression of lfng. Ectopic expression of lfng in this "pre-zli" compartment results in sorting of the electroporated cells into the lfng-expressing border regions, indicating that lfng contributes to specification of the zli borders and acquisition of its compartment properties [START_REF] Zeltser | A new developmental compartment in the forebrain regulated by lunatic fringe[END_REF]121]. Of note, cell lineage analysis suggests that this "pre-zli" domain has compartment properties and is thought to collapse during development to form the final zli [START_REF] Zeltser | A new developmental compartment in the forebrain regulated by lunatic fringe[END_REF]. However, the mechanism allowing this allosteric growth are not yet explained, and are not attributable to either cellular movements in the epithelium [START_REF] Garcia-Lopez | Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos[END_REF] or to cell death [START_REF] Zeltser | A new developmental compartment in the forebrain regulated by lunatic fringe[END_REF].

The TFs Barhl2 and Irx Facilitate Acquisition of Zli Compartment Properties

As described above, Barhl2 participates in shh induction within the zli territory. Moreover, Barhl2 acts as a brake on p2 neuroepithelial cell proliferation and plays a key role in the maintenance of diencephalic neuroepithelial architecture by limiting activation of the Wnt canonical pathway [START_REF] Juraver-Geslin | Barhl2 limits growth of the diencephalic primordium through caspase3 inhibition of beta-catenin activation[END_REF]. In fly imaginal discs, the reduced activity of irx genes promotes cell proliferation by accelerating the G1 to S transition whereas their increased expression causes cell-cycle arrest contributing to the size determination of the imaginal disc [122]. Irx proteins act on establishment of distinct cell affinities in dorsal versus ventral cells in the Drosophila eye [123]. In zebrafish, iro7 is required for the proper positioning of the prospective r4/r5 hindbrain rhombomeric boundary [110,124]. Taken together, these observations indicate that Barhl2 along with the Irx factors could participate in limiting zli cell proliferation as well as enabling adhesion and facilitating acquisition of zli compartment properties (reviewed in [START_REF] Juraver-Geslin | Early development of the neural plate: New roles for apoptosis and for one of its main effectors caspase-3[END_REF]).

In conclusion, the Wnt, Shh, and Notch signaling pathways act in a coordinate way to drive segregation of zli cells. The identification of genetic and epigenetic modifications initiated by the co-expression of otx2, barhl2, and irx genes in the presence or absence of Shh should identify the molecular cues driving segregation of zli and thalamic cells.

The Programming of A "Zli-Like" Structure in X. laevis Animal Cap Explants Confirms Otx2, Barhl2 and Iroquois Cell Autonomous and Non-Autonomous Activities in Zli Formation

In amphibian, cells from the roof of the blastocoel are pluripotent. These cells can be isolated and programmed to generate tissues through manipulation of gene expression-injection of synthetic mRNA or morpholino (MO)-or induction by secreted factors. The investigation of the GRN underlying zli formation was performed using such explants [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]125].

In the presence of Shh, explants co-expressing barhl2, otx2 and irx3 acquire a zli-like identity. The shape of the shh-expressing domain observed in the programmed explants-wide where in contact with the source of Shh signal and pointed at its other extremity-is consistent with an induction process occurring sequentially from one cell expressing shh to the next. Concomitant in time with acquisition of a Shh-expression program, zli-like cells acquire cell segregation properties that depend on the presence or absence of a Shh signal: explant cells mimic their in vivo segregation behaviors. Moreover, when grafted into a developing neural plate and continuously exposed to a Shh signal, neuroepithelial cells co-expressing barhl2, otx2, and irx3 form an ectopic zli in vivo [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]125].

These approaches confirmed that the GRN which governs competence and refinement of the zli can be reconstructed in an ex vivo system, and a cell-autonomous role for Otx2 and Barhl2 [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]125]. Moreover, these data indicate that there could be an ongoing recruitment of irx3 expressing cells from the thalamus into the zli during zli formation. It is not known whether Shh induces the segregation of zli and thalamus cells in vivo, however, such recruitment would participate in giving the zli its wedged shape, wide at the bottom and pointed at its extremity, a morphology observed in most vertebrate species (Figures 1Bb and2D). Finally, these data also reveal that the efficiency of shh induction-i.e., the average size of the shh-expressing area-is increased in the presence of a thalamus-like explant [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF]125]. Further experiments are necessary to determine whether the thalamus territory through a cell non-autonomous mechanism facilitates the induction of shh and contributes to the dorsal progression of shh expression.

Inductive Cues and Zli Positioning: A Role for the p2/p3 Border and for Fezf/Fez TFs

Using grafting experiments in chicken embryos, Vieira et al. showed that an ectopic border between neural tissue from a prechordal (defined as being six3-positive) and epichordal (defined as being irx3-positive) origin is sufficient to induce an ectopic zli [START_REF] Vieira | Thalamic development induced by shh in the chick embryo[END_REF]. Indeed, comparison of barhl2 and shh expression patterns during neurulation support the idea that the zli forms in immediate proximity to the interface between the prechordal neuraxis, induced by prechordal plate mesoderm, and the epichordal neuraxis, induced by the chordamesoderm ( [START_REF] Larsen | Boundary formation and compartition in the avian diencephalon[END_REF][START_REF] Shimamura | Longitudinal organization of the anterior neural plate and neural tube[END_REF]; Figure 1Bg). In any case, the zli anterior border develops at the interface between the expression domains of the FEZ family zinc finger 2 (fezf2), which marks the alar plate of p3 rostral to the zli, and irx3 that marks p2 Figure 1Be; [START_REF] Shimamura | Longitudinal organization of the anterior neural plate and neural tube[END_REF][START_REF] Zeltser | Shh-dependent formation of the zli is opposed by signals from the dorsal diencephalon[END_REF][START_REF] Vieira | Sonic hedgehog from the basal plate and the zona limitans intrathalamica exhibits differential activity on diencephalic molecular regionalization and nuclear structure[END_REF][START_REF] Martinez-Ferre | Wnt signal specifies the intrathalamic limit and its organizer properties by regulating shh induction in the alar plate[END_REF]).

fez-like (fezl) genes are highly conserved during evolution from flies to men. They are expressed in the presumptive pre-thalamus during early segmentation stages but are not detected within the zli territory (Figures 1A,Be and 2). Functional analysis of Fez and Fezl in zebrafish and mouse indicate an evolutionarily conserved role for these TFs in zli formation. Analysis of E12.5 mouse embryos in which both Fez and Fezl genes have been disrupted reveals defects in both prethalamus and diencephalic development, including loss of shh expression in the zli, ectopic expression of pax6 in the mid-diencephalic furrow, and extension of wnt3a expression within the p2 alar plate. In contrast, in zebrafish the knock down of fezl results in anterior expansion of the zli, associated with concomitant expansion of irx3a. This difference in loss of function phenotypes remains unexplained. It could be species-dependent or it could be due to a difference in levels of expression. The zebrafish fezl morphant represents a weaker loss of function of fezl with fez being intact, whereas fez/fezl double-mutant mice represent true null conditions. Conversely, in both zebrafish and mouse the over-expression of fezl, or fez abolishes expression of shh in the zli. fezl over-expression in late gastrula zebrafish embryos expands the prethalamus and hypothalamus territories at the expense of the zli and posterior forebrain and/or mid-brain regions. Therefore fezl/fez gene activity, and specifically fezf2 activity, plays a key role in formation of the p2/p3 boundary and co-localizes with the zli anterior boundary [START_REF] Hirata | Zinc-finger genes fez and fez-like function in the establishment of diencephalon subdivisions[END_REF]126].

By analogy with induction of the midbrain-hindbrain boundary, which develops at the interface of otx2 and gbx2 expression, it has been suggested, but not tested, that the interface between the fezf2 and irx3 expression domains is the inductive cue at the origin of zli formation (reviewed in [3,[START_REF] Epstein | Regulation of thalamic development by sonic hedgehog[END_REF][START_REF] Martinez-Ferre | Molecular regionalization of the diencephalon[END_REF][START_REF] Kiecker | Compartments and their boundaries in vertebrate brain development[END_REF]127]). Indeed, the p2/p3 boundary localizes at the interface between the expression domains of fezf2 and irx3, but also fezf2 and otx2, and fezf2 and barhl2 (Figures 1Be and3). In amphibian, neither over-expression of irx3, nor down-regulation of barhl2, affects formation of the p2/p3 border [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF]. Similarly, in zebrafish, the loss of otx does not affect zli anterior border location [START_REF] Scholpp | Otx1l, otx2 and irx1b establish and position the zli in the diencephalon[END_REF]. It is possible, however not tested, that Otx2, Barhl2 and Irx TFs have redundant activities in establishment of the interface with the Fezf/Fez TFs that set up the p2/p3 border. Results in frog explants suggest that whereas the p2/p3 limit is important in setting up the zli anterior border, it is the co-expression of barhl2, otx2 and irx3 that initiates zli development. This observation is important, as it indicates that the zli developmental mode may be different from that of the midbrain-hindbrain boundary.

Finally, grafts of dorsal diencephalic tissue in chicken inhibit zli propagation, arguing that the progression of shh expression is limited dorsally by inhibitory factors [START_REF] Zeltser | Shh-dependent formation of the zli is opposed by signals from the dorsal diencephalon[END_REF]. The identity of such signals is unknown. However, since shh signaling from the zli represses pax6 within the mid-diencephalic furrow [128], Pax6 together with unidentified dorsal signals could in turn prevent shh from being expressed beyond the zli (Figure 1A,Bf).

In conclusion, the molecular cues initiating zli positioning remain partially unknown. In this regard, the determination of signals controlling otx, barhl2 and irx neural plate expression will provide important information.

The GRN Involved in Zli Formation is Evolutionarily Conserved

Yao et al. analyzed whether the cis-and trans-regulatory landscape underlying zli development was conserved in the chordate phylum. They investigated conservation of the "zli developmental cassette" in the hemichordate Saccoglossus kowalevskii (S. kowalevskii) that is closest to the central basic reference animal at the root of the chordate phylogenetic tree [129,130]. In S. kowalevskii, the narrow band of cells at the proboscis-collar boundary is considered zli-like as it expresses hh. A study of expression patterns for barhl2 (barH), otx, and irx orthologs was performed in this hemichordate. Indeed, the proboscis-collar boundary expresses the orthologs of barhl2, otx and irx at the right time and place to perform their patterning function [129,130]. Yao et al. identified in S. kowalevskii a 1.1-kb region containing a cis-regulatory element containing the six motifs of the mouse SBE1: skSBE1. Functional experiments in mouse and in S. kowalevskii demonstrate that skSBE1 is a functional ortholog of the mmSBE1 enhancer [START_REF] Yao | cis-Regulatory architecture of a brain signaling center predates the origin of chordates[END_REF]. Similar SBE1 cis-regulatory elements, intact or with a shuffled motif arrangement, were discovered in lamprey and all jawed vertebrates that display "zli-like" structures. In contrast, similar SBE1-like motifs were not found in amphioxus (cephalochordate) or in ascidian (tunicate) that both lack an hh-expressing domain in the anterior brain.

In conclusion, these studies support the hypothesis that early chordates inherited an hh cis-regulatory-motif from a deuterostome ancestor that was subsequently lost in the invertebrate chordate lineages. A conserved hh cis-regulatory-motif (SBE1-like) was maintained in the vertebrate shh gene and used to activate its transcription in the zli, paving the way for the establishment of this brain-signaling center more than 500 millions years ago [START_REF] Yao | cis-Regulatory architecture of a brain signaling center predates the origin of chordates[END_REF].

Forebrain Malformations Associated with Defects in the GRN and Signaling Pathways Supporting Zli Formation

The GRN controlling zli development is also involved in thalamic primordium induction and in thalamic growth, patterning, and organogenesis. Indeed, depletion of otx genes impairs forebrain development (reviewed in [START_REF] Acampora | The role of otx and otp genes in brain development[END_REF]). In both frog and mouse, targeted-depletion of barhl2 generates thalamic developmental defects [START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Ding | Barhl2 determines the early patterning of the diencephalon by regulating shh[END_REF]. In barhl2 / mice, p2 thalamic progenitors acquire a pretectal fate and there is an absence of thalamo-cortical axon projections [START_REF] Ding | Barhl2 determines the early patterning of the diencephalon by regulating shh[END_REF]. Finally, irx gene depletion in frog, chicken, and zebrafish disrupts thalamus development [3,[START_REF] Juraver-Geslin | The conserved barh-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by sonic hedgehog[END_REF][START_REF] Chatterjee | Patterning and compartment formation in the diencephalon[END_REF][START_REF] Martinez-Ferre | Molecular regionalization of the diencephalon[END_REF][START_REF] Robertshaw | Irx3 and pax6 establish differential competence for shh-mediated induction of gabaergic and glutamatergic neurons of the thalamus[END_REF]109,131]. Some genetic disorders such as holoprosencephaly (HPE) or the ciliopathies, which are characterized or accompanied by brain malformations in humans and model organisms, are associated with thalamic and thalamo-cortical developmental defects (reviewed in [132,133]). Neuropsychiatric disorders such as obsessive-compulsive disorder and attention deficit hyperactivity disorder have been associated with abnormal neuronal activity within the thalamus (reviewed in [131,[134][135][136][137]).

The most prominent pathological condition in which development of the thalamus is affected is HPE. HPE is a rare developmental disorder with an occurrence of 1 case in about 16,000 live births. However, approximately 1/250 conceptuses are thought to be affected by HPE which makes it the most common forebrain defect in humans. HPE presents itself as variable degrees of fusion between the left and right halves of the cerebral hemispheres, basal ganglia and, interestingly, also the thalamus [132,138]. However, rather than representing a fusion event, HPE arises from a failure to separate the two halves of the forebrain along the midline. Most studies focus on correct separation between the cerebral hemispheres (reviewed in [139]). However, little is known on the thalamic contribution to the etiology of HPE.

Mutations in genes linked to HPE have been identified in animal models of HPE and human patients. All of them play important roles in major brain developmental signaling pathways [140][141][142][143][144][145]. Whereas defects in the Shh pathway are the most frequent cause of HPE, genetic screening of HPE patients and studies in animal models also involved the Fgf, Nodal and Notch pathways as major contributors to HPE etiology [140][141][142][143][144][145]. Most mutations found in patients with HPE are in genes that participate in midline formation and early neural plate patterning, strengthening the hypothesis that developmental defects at the origin of these malformations take place early during neural development.

As described above, the Shh pathway relies on primary cilia for signal transduction. It is thus not surprising that mutations in certain cilia-related genes affect diencephalon morphology and induce HPE or HPE-like phenotypes. In mouse, mutants carrying deletions in the anterograde intraflagellar transport (IFT) 172 gene, Shh signaling in the forebrain is down-regulated. Consequently, the mutant diencephalon is severely reduced and embryos exhibit lobar or semilobar HPE [146]. The hypomorphic cobblestone allele of another anterograde IFT gene, IFT88, also affects diencephalon development. In this mouse mutant, the border between tel-and diencephalon is dissolved and cells mix freely between the two parts [START_REF] Willaredt | Foxj1 regulates floor plate cilia architecture and modifies the response of cells to sonic hedgehog signalling[END_REF], a feature reminiscent of a failure to establish zli-mediated compartmentalization and therefore correct establishment of segregation properties. Loss of function of forkhead box J1 (foxj1), a TF regulating the biogenesis of motile cilia, caused striking forebrain defects in X. laevis [104]. While cilia on zli cells in Xenopus are longer than other primary cilia in the wildtype ([104]; Figure 2), zli cilia are shortened in foxj1 morphants and diencephalon size is massively reduced. Taken together, cilia dysfunction in the forebrain leads to variable molecular and morphological defects, pinpointing the dependence of diencephalon development on cilia-based Shh signaling.

In conclusion, even though thalamic fusion in human patients has not yet been connected to diencephalic primordium and zli development, it appears possible that malformation of p2 and concurrent mis-patterning may prevent separation of the thalamic complex and plays an as yet unrecognized role in the etiology of HPE. Specifically, failure to establish the anterior medial source of Shh, a lack of competence in the presumptive zli region, defective spreading and/or signaling of Shh can cause problems with setting up the p2/p3 border, the zli, and correct Shh, Notch and Fgf signaling in the thalamic field. All these events can lead to brain malformations.

Conclusions

The morphogenetic events supporting brain development are controlled and coordinated by a handful of extracellular signaling networks. Shh is one of the most prominent and collaborates with the Wnt, Bone Morphogenetic Protein, Fgf, Nodal, and retinoid acid (RA) pathways in neurogenesis. The patterning and growth of the diencephalon is, in that sense, particularly meaningful as Wnt, Shh, Fgf, and RA signaling act together in a strictly regulated chronology and topology to orchestrate the development and neurogenesis of the diencephalon and specifically of the thalamus (reviewed in [3,[START_REF] Epstein | Regulation of thalamic development by sonic hedgehog[END_REF][START_REF] Martinez-Ferre | Molecular regionalization of the diencephalon[END_REF]127]). Whereas recent efforts have allowed partial identification of the zli developmental cis-and trans-determinants, important questions remain unresolved. Specifically, it remains to identify: (i) the molecular cues initiating zli emergence and positioning; (ii) the molecular cues driving segregation of zli and thalamic cells; and (iii) the contribution of otx, barhl, and irx genes to thalamic development. It will also be interesting to investigate: (i) how the Shh signal is distributed and read in the developing zli and the thalamus; and (ii) which signals generate the splitting of the midline in the forebrain region, knowing that this process is defective in HPE. In the years to come, an important focus should be to better understand the gene-environment interactions involved in thalamic organogenesis and to develop animal models (chicken, zebrafish, mouse, Xenopus) mimicking human thalamic developmental disorders, a strategy that shall provide critical help into possible clinical interventions.

ANNEX 2 : RESUMEE EN FRANÇAIS 1. INTRODUCTION

La biologie du développement est focalisée sur l'étude de tous les processus développementaux qui ont lieu durant et après l'embryogenèse. Le développement embryonnaire est un processus finement régulé qui est à l'origine d'un nouvel organisme.

Différents mécanismes développementaux se produisent pendant le l'embryogénèse. Dans ce travail nous nous concentrons sur la prolifération cellulaire et la mort cellulaire programmée.

LA VOIE DE SIGNALISATION WNT CANONIQUE

Une de voie de signalisation qui contrôle la prolifération cellulaire est la voie canonique Wnt.

Ce mécanisme de transduction cellulaire est impliqué dans plusieurs processus développementaux, comme la formation de l'organisateur de Spemann ou la maintenance de la prolifération des cellules souches. La dérégulation de cette voie de signalisation est à l'origine de pathologies comme des malformations développementales ou des cancers (Clevers and Nusse, 2012). L'effecteur principal de la signalisation Wnt est la b-catenin. En l'absence de signalisation Wnt, la b-catenine est dégradée dans le cytoplasme par un complexe multiprotéique qui la phosphoryle pour la cibler vers le protéasome [START_REF] Aberle | β-Catenin is a target for the ubiquitin-proteasome pathway[END_REF][START_REF] Yost | The axisinducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3[END_REF]. Lorsque le ligand Wnt se lie à son récepteur Frizzled sur la surface cellulaire, le complexe multiprotéique est inactivé par différents mécanismes et la b-caténine s'accumule et se transloque vers le noyau [START_REF] Mao | Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway[END_REF][START_REF] Perrimon | Multiple functions of segment polarity genes in Drosophila[END_REF][START_REF] Pinson | An LDL-receptor-related protein mediates Wnt signalling in mice[END_REF][START_REF] Taelman | Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes[END_REF]. Dans le noyau, la b-caténine se lie au facteur de transcription Tcf pour activer la transcription des gènes cibles de la voie Wnt (Behrens et al., 1996;[START_REF] Huber | Nuclear localization of β-catenin by interaction with transcription factor LEF-1[END_REF][START_REF] Molenaar | Differential expression of the HMG box transcription factors XTcf-3 and XLef-1 during early Xenopus development[END_REF] (Figure 15). Au cours de ma thèse, je me suis concentré sur les mécanismes qui ont lieu dans le noyau et qui ont un impact sur la régulation de la voie. Lorsque la signalisation Wnt est inactive, Tcf est lié au corépresseur Groucho.

Groucho interagit avec Tcf pour favoriser l'inhibition de la transcription génique (Cadigan and Waterman, 2012;Daniels and Weis, 2005;[START_REF] Levanon | Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors[END_REF].

Les Tcfs sont hautement conservés entre les espèces et durant l'évolution [START_REF] Hurlstone | T-cell factors: turn-ons and turn-offs[END_REF]. Les Tcfs sont redondants dans leur activité, à l'exception de Tcf71l, dont l'inactivation induit un phénotype létal dans les embryons de Xénope (Liu et al., 2005). Dans les embryons de Xénope, Tcf71 est le Tcf maternel exprimé le plus prédominant et agit comme un répresseur de la transcription. par la terminaison dexonucléotidyl transférase (TUNEL) qui marque les cellules apoptotiques [START_REF] Gavrieli | Idenitification of Programmed Cell Death In Situ via Specific Labeling of Nuclear DNA Fragmentation[END_REF]. L'apoptose a été largement étudiée chez Caernorhabditis elegans (C. elegans) (Ellis and Horvitz, 1986), ainsi que chez la drosophile et les mammifères (Arya and White, 2015). Cependant, sa régulation chez X. laevis est moins décrite.

Les souris déficientes pour les protéines anti-apoptotiques Bcl-XL ou Mcl1 meurent prématurément en raison de l'apoptose massive des neurones immatures et des cellules hématopoïétiques [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]Motoyama et al., 1995;Opferman and Kothari, 2018). pour réguler leur survie [START_REF] Arbour | Mcl-1 Is a Key Regulator of Apoptosis during CNS Development and after DNA damage[END_REF]Malone et al., 2012). Chez le Xenope, l'apoptose est observée au niveau de l'organisateur axial (Offner et al., 2005), où elle limite la quantité de cellules sécrétrices d'antagonistes de Shh et de Bmp.

LE FACTEUR DE TRANSCRIPTION BARHL2

Des travaux antérieurs effectués par l'équipe ont identifié Barhl2 comme un facteur de transcription impliqué dans le développement de l'organisateur axial (Offner et al., 2005). Chez les embryons de X. laevis, barhl2 est exprimé dans la plaque neurale, dans le futur diencéphale, à partir de l'étape 14. Le gène barhl2 contient un homéodomaine (HD) nécessaire pour sa fixation à l'ADN, un signal de localisation nucléaire et un région N terminal qui contient deux domaines EHs hautement conservés, EH1 et EH2 (Figure 16). Il a été montré que les domaines EHs interagissent avec Groucho in vitro et in vivo et sont déterminant pour la répression transcriptionnelle. Le gain-de-fonction ou la perte de fonction de Barhl2 provoque une perturbation du gradient des facteurs Shh et anti-BMP dans la plaque neurale, et Barhl2 contrôle la survie des cellules dans cette zone en favorisant l'apoptose (Offner et al., 2005). Dans le premier manuscrit de cette thèse nous décrivons l'interaction entre le facteur de transcription Barhl2, Groucho et Tcf in vitro et in vivo (Figure 18). Nous montrons que ce complexe joue un rôle actif lors de la formation de l'organisateur de Spemann puisqu'il contrôle le passage de Tcf de sa forme active à sa forme répressive (Figure 17). Nos résultats ont confirmé l'importance de la répression médiée par Groucho sur l'organisateur de Spemann (Figure 19). De plus, nous apportons la preuve que l'association entre Barhl2, Groucho et Tcf est maintenue à des stades de développement ultérieurs et joue un rôle dans l'émergence d'une niche de cellules souches dans la lèvre rhombique.

CONCLUSIONS ET PERSPECTIVES

Au cours de ma thèse, j'ai étudié les effets du facteur de transcription Barhl2 sur la régulation V: BIBLIOGRAPHY

Figure 1 28 Figure 2 30 Figure 3 35 Figure 4 38 Figure 5 41 Figure 6 44 Figure 7 46 Figure 8 48 Figure 9 52 Figure 10 59 Figure 11 60 Figure 12

 1282303354385416447468489521059116012 Figure 1 Tgf-b signaling overview. ........................................................................................28 Figure 2 Shh signaling mechanism in Drosophila and vertebrates ........................................30 Figure 3 The Wnt signaling pathway in invertebrates and vertebrates. .................................35 Figure 4 Tcf, Groucho and b-catenin homologues structure .................................................38 Figure 5 Events leading to b-catenin dorsal accumulation and mid-blastula transition in the Xenopus embryo ..........................................................................................................41 Figure 6 Gastrulation events leading to neural plate formation .............................................44 Figure 7 Neural induction .....................................................................................................46 Figure 8 Apoptosis pathway in vertebrates. ..........................................................................48 Figure 9 Schematic representation of IAP proteins in different species ................................52 Figure 10 xBarhl1 and xBarhl2 protein structure. ..................................................................59 Figure 11 Sequence comparison of mouse, human and X. laevis barhl genes .....................60 Figure 12 Whole Mount in situ Hybridization (ISH) using barhl2 as probe on X. laevis embryo at stage 14 or dissected neural tube at stage 26...........................................................62

Figure 13 :

 13 Figure 13: Barhl2 ability to promote Tcf7l1 repressor activity is present at the retina CMZ . 183

Figure 14 :Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 :

 1415161718192021 Figure 14: Barhl2 ability to promote Tcf7l1 repressor activity is present at stage 30 ........... 185 Figure 15 La voie de signalisation Wnt chez les invertébrés et les vertébrés...................... 216 Figure 16 Structure des protéines xBarhl1 et xBarhl2 ......................................................... 218 Figure 17 Barhl2 limite la taille des organisateurs de la tête et corps ................................. 220 Figure 18 Barhl2 interagis avec Gro et Tcf71l indépendamment et améliore l'interaction Tcf71-Gro ............................................................................................................................. 221 Figure 19 Barhl2 augmente l'activité du répresseur Tcf7l1 et limite la dé-répression β-caténine dépendante des gènes cibles Tcf7l1 dans le territoire dorsal des embryons de blastula ................................................................................................................................... 222 Figure 20 Expression de mcl1 pendant la neurulation chez X. laevis ................................. 223 Figure 21: Barhl2 et Mcl1 font partie de la même voie de signalisation............................... 224

Figure 1

 1 Figure 1 Tgf-b signaling overview. When the Tgf-b ligand binds to the type I and II Tgf-b receptors, type II receptor phosphorylates type I. The protein Smad in the cytoplasm is recruited to the type I receptor and is phosphorylated and activated. Activated Smad recruit Smad4 and translocate in to the nucleus. This complex binds to gene promoters to induce gene transcription.

Figure 2

 2 Figure 2 Shh signaling mechanism in Drosophila and vertebrates. A: Shh signaling overview in the fly model. (a) In the absence of Shh signaling the receptor Patched (Ptc) inhibits Smoothened (Smo) activity and maintains it in intravesicular bodies in the cytoplasm. The downstream effector Cubitus interruptus (Ci) is cleaved in the cytoplasm to be degraded in the proteasome. A small fragment translocates into the nucleus and acts as

Figure 3 The

 3 Figure 3 The Wnt signaling pathway in invertebrates and vertebrates. A: (a) In the absence of Wnt signaling b-catenin is sequestrated in the cytoplasm by a protein complex composed of Axin, Apc and Gsk3 that phosphorylates b-catenin and marks it for degradation. In the nucleus the co-repressive factor Groucho binds to the transcription factor Tcf and inhibits Wnt targets gene transcription. (b) Wnt binds to its receptor in the cell membrane, Frizzled, and thanks to LRP protein it recruits and phosphorylates the protein complex responsible for b-catenin degradation. b-catenin is therefore stabilized and can enter to the nucleus where it displaces Groucho and binds to Tcf allowing gene transcription. B: in vertebrates there are different kinds of Tcf that act as activators or repressors. (a) In the absence of Wnt signaling Tcf7l1 and Gro-4 bind to promoters in Wnt target genes to inhibit transcription. (b) When the Wnt ligand binds its receptor, Hipk2 phosphorylates Tcf7l1 and Tcf7 binds to b-catenin to activate gene transcription in the nucleus.

Figure 4

 4 Figure 4 Tcf, Groucho and b-catenin homologues structure. A: Domain structure of Lef/Tcf proteins in mouse (m), human (h) and Xenopus (x). B: Domain structure of Xenopus b-catenin. C: Domain structure of Gro proteins in Xenopus (x) and mouse.

  importance of Wnt signaling in embryonic development became clear in the experiments by McMahon and Moon that showed that Wnt1 was implicated in embryonic axis formation in X. laevis (McMahon and Moon, 1989). They ventrally injected mouse Wnt1 mRNA in the Xenopus embryo and this resulted in a duplication of the embryonic axis. This was the first experiment that demonstrated the implication of Wnt in vertebrate development and suggested a role of Wnt in the SO formation. Following McMahon and Moon experiments, other experiments showed that ventral b-catenin overexpression produced a secondary axis

Figure 5

 5 Figure 5Events leading to b-catenin dorsal accumulation and mid-blastula transition in the Xenopus embryo. A: the sperm fertilizes the egg and the sperm entry determines the future dorsal lip of the blastopore, at 180º. B: as a consequence of sperm entry there is a cortical rotation and maternal stored mRNA change position. C: Gbp, Wnt and Dsh accumulate in the presumptive dorsal side of the embryo. D: Dsh and Gbp along with Wnt inhibit Gsk3 activity in this region. In the opposite of the embryo there is Gsk3 activity. E: as a consequence of Gsk3 activity inhibition there is b-catenin accumulation in the cell nucleus of the dorsal cells. F: at the nuclei of dorsal cells b-catenin binds the transcription factor Tcf and transcription of organizer genes siamois and twin begins. An: animal hemisphere, Vg: vegetal hemisphere, DL; dorsal lip of the blastopore, Dsh: disheveled, V: ventral, D: dorsal, Bl: blastocoel, Gbp: Gsk3 binding protein, b-cat: b-catenin, Tcf: T-cell factor, Gro: Groucho, Sia: siamois.

  the different forms of Wnt ligands, not all of them have the same role during the SO induction and ventrolateral mesoderm formation. The only maternal mRNA of the Wnt family

Figure 7

 7 Figure 7 Neural induction. After the MBT siamois and twin induce the organizer genes chordin, noggin, goosecoid and xnr3. These genes encode for anti Bmp proteins that inhibit the Bmp signal coming from the ventral equatorial cells. This Bmp gradient patterns the mesoderm, high Bmp induces ventral mesoderm and low amounts of Bmp result in dorsal mesoderm, the Spemann organizer. Anti Bmp signals, along with Fgf, induce neural ectoderm from the ectoderm.

Figure 8

 8 Figure 8 Apoptosis pathway in vertebrates.In the intrinsic pathway, upon death stimuli the BH3-only proteins are activated and can inhibit the anti-apoptotic family proteins Bcl2. These proteins are responsible for inhibition of pro-apoptotic proteins Bax and Bak. Once this inhibition is released, Bax and Bak can promote cytochrome C release from the mitochondria. Cytochrome C associates with Apaf1 and this induces an oligomerization of the complex. This complex, the apoptosome, activates the initiator pro-caspase-9. Once activated Caspase-9 can cleave and activate effector pro-caspases-3 and -7. Activated effector caspases can cleave specific apoptotic proteins to induce cell death. In the extrinsic pathway, a TNF receptor is activated by a death ligand on the cell surface. This activates the initiator Caspase-8, that in turn activates the effector Caspase-7.

  Throughout development, cells are constantly synthetizing pro-Caspases and other components of the apoptotic machinery. The initiator pro-Caspases are characterized by the presence of a Caspase Recruiting Domain (CARD) (Caspase-1,

Figure 9

 9 Figure 9 Schematic representation of IAP proteins in different species. DIAP1 and DIAP2 in Drosophila, cIAP1. cIAP2 and XIAP in Xenopus.

  is a niche of stem cells. Mcl1 loss-of-function (LOF) increases apoptosis in this niche. Mcl1 is thus required for the survival of these cells. Therefore, Mcl1 regulates the survival of adult

Figure 10 xBarhl1

 10 Figure 10 xBarhl1 and xBarhl2 protein structure. Schematic representation of Xenopus Barhl1 and Barhl2 proteins. The FIL domain, NLS and Homeodomain are represented.

Figure 11

 11 Figure 11 Sequence comparison of mouse, human and X. laevis barhl genes. The most conserved regions are the homeodomain and FIL domains. Human, mouse and X. laevis barhl2 genes contain two FIL domains, as the nematode and Drosophila (data not shown). Amino acids identities within the homeodomain and FIL domains are highlighted in bold. H: human; m: mouse; x: X. laevis.

Figure 12 Whole

 12 Figure 12 Whole Mount in situ Hybridization (ISH) using barhl2 as probe on X. laevis embryo at stage 14 or dissected neural tube at stage 26. A: At stage 14 (dorsal view) barhl2 is expressed in the anterior dorsal side of the embryo. B: At stage 26 (dissected neural tube) barhl2 expression can be detected at the level of the prosomere P2, in the caudal forebrain. A scheme of Xenopus laevis developmental stage is shown at the same stage as the WISH. Ant: anterior; post: posterior; st: stage. Adapted from (Juraver-Geslin et al., 2011)

  LOF is partially compensated by Gsk3b overexpression. Barhl2 acts in negatively regulating Wnt signaling in these cells. The mechanisms by which Barhl2 controls Wnt signaling or Caspase-3 non-apoptotic activities have not been elucidated. Observations in different species argue that a Barhl2 -non-apoptotic Caspase-3 activity -Gsk3 pathway could be conserved during neural development. In Drosophila cleavage dependent activation of a Gsk3-β isoform, Shaggy (Sgg)-46 protein, by the fly Caspases DrONC or DrICE, leads to the downstream phosphorylation and subsequent inhibition of the

  through development and different processes assure cell homeostasis. During my PhD I focused on the study of two mechanisms important for embryonic development that are regulated by the same transcription factor: Barhl2. My first work focused on the regulation of Wnt signaling pathway by Barhl2. Wnt signaling pathway has been extensively studied and the main focus has been the elucidation of the cytoplasmic mechanisms controlling b-catenin degradation. Tcf-driven inhibition of transcription has not been as well deciphered. In particular, elucidating the roles of the corepressor Groucho in developmental regulation of Wnt signaling is still pending. It has been suggested that Groucho acts long distance to repress gene transcription. However, the evidence is mostly biochemical and there are few studies trying to comprehend Gro transcriptional silencing activities during development. During my PhD I focused on the study of Wnt regulation in the context of the Spemann organizer formation using the X. laevis embryo as a model system. Indeed, formation of the Spemann organizer requires Wnt signaling as well described, and we discover a new role for Barhl2 in organizer formation.

  that bcl-xl and mcl1 are the main anti-apoptotic members of the bcl2 family responsible for development of the central nervous system. The second aim of my PhD was to elucidate the molecular mechanisms by which Barhl2 was increasing apoptosis at these early developmental stages. In parallel(Sena et al., in preparation) we describe the spatio-temporal expression of mcl1 and bcl-xl and demonstrate that Barhl2 and Mcl1 are part of the same signaling pathway.

  complex comprised, among others, by Gsk3. The cytoplasmic degradation of b-catenin has extensively been studied. When b-catenin is stabilized it translocates to the cell nucleus where it binds to the transcription factor Tcf to mediate Wnt target genes transcription. The developmental constraints modulating the downstream events driven by b-catenin fixation to the promoters of Wnt responsive target genes are not fully understood. In a mechanism that slightly differs from invertebrates and vertebrates, the co-repressor Groucho antagonizes bcatenin transcriptional functions on its binding to Tcf. When the Wnt pathway is inactive, Groucho binds to Tcf to form a repressive complex that maintains genes non-transcribed. The transcriptional consequences and the mechanisms involving the repressive role of Groucho in the nucleus are still a matter of study.

  procedures were specifically approved by the ethics committee of the Institut Curie (Authorization APAFIS#10934-2017081019149808v2 given by National Authority) and are in compliance with the international guidelines (2010/63/UE). B Durand carries the Authorization for vertebrates' experimental use N°75-1548.PlasmidsAll mutated and/or truncated forms of Barhl2 were generated from mouse barhl2 full-length cDNA in pCS2+(Offner et al., 2005). Constructs were generated by sequential PCR amplification followed by subcloning and sequencing in pTOPO (TOPO TA cloning kit Thermofisher) and subcloned in pCS2+. The full-length and mutated cDNAs tagged constructs were generated by subcloning in either pCS2-Myc (C-terminal Myc tag for Barhl2EHs-Myc and Barhl2fl-Myc), in pEGFP-N1 (N-terminal GFP tag for Barhl2fl-GFP) or in pCS2-GFP (Cterminal GFP tag for Barhl2EHs-GFP and Barhl2EHsM-GFP). HA Tag (YPYDVPDYA) and AM-Tag (CQDPQRKGNVILSQAYGCQDPQRKGNVILSQAY) encoding oligonucleotides were subcloned in C-terminal of pCS2-barhl2fl and pCS2-barhl2flEHsM.HA Tag (YPYDVPDYATSTIV) encoding oligonucleotides was subcloned in C-terminal of pCS2-XGroucho4 (gift Phil Jones). A vector carrying a Flag tag at the N-terminal end and HA

Figure 1 Figure 1 :

 11 Figure 1

Figure 2 :

 2 Figure 2: Depletion of Barhl2 affects organizer signaling. (A-C) Barhl2 depletion affects neural plate patterning. ISH on embryos depleted for Barhl2 (MObarhl2) using sox3 (marker of neural induction), foxb1 (marker of hindbrain and diencephalon), ccnd1 (cyclin D1, marker of anterior neural plate), as probes. MObarhl2 together with gfp mRNA were injected into one dorsal blastomere at the 4-cell stage in X. laevis embryos. The markers and stages are indicated. Representative embryos are shown dorsal view, anterior up. Inj stands for injected side. (D-F) Barhl2 effect neural plate patterning via its activity on Spemann organizer formation. ISH on embryos depleted for Barhl2 (MObarhl2) using sox3 as a probe. Embryos were injected into one dorsal blastomere of eight-or sixteen-cell stage using β-gal RNA (red) as a tracer. Representative embryos in which Barhl2 activity was depleted either in the axial midline (D), or in medio-lateral neuroectodermal cells (E) are shown dorsal view, anterior up, together with representative transverse sections (50µm) at the antero-posterior axis positions indicated with a white-dashed arrow. (F) Quantification of the neural plate enlargement phenotype. The scheme shows a dorsal view of eight-or sixteen-cell stage embryos with the site of injection (red dot) and the corresponding targeted territory (in red) in stage 14 embryos shown dorsal view anterior up. The graph indicates the % of embryos exhibiting the neural plate enlargement phenotype (blue), and of embryos with no phenotype (light green). N is the number of embryos analyzed.

Figure 3 :

 3 Figure 3: Barhl2 activity on the organizer requires both its DNA binding domain and its Eh1 domains and is efficiently antagonized by Barhl2 N-terminal part. (A) Scheme of barhl2-derived constructs. All mutated and/or truncated forms of Barhl2 were generated from mouse barhl2 full-length cDNA (barhl2fl). Barhl2 AAs sequence is partly indicated, specifically the homeodomain (HD-60AAs) with its three helices (AAs in blue), a nuclear localization sequence (NLS, AAs in black) and its two Eh1 domains composed of phenylalanine (F), isoleucine (I) and leucine (L). In Barhl2∆EHs the first 182 AAs of barhl2fl were deleted. In Barhl2flHoxM we mutated 4 AAs in the third helix of the homeodomain (HD-60AAs) known to interact with DNA -AA 44 Q to E, AA47 T to G, AA51 Q to E and AA52 N to R as indicated (AAs in red). Barhl2EHs contains the first 177AAs of Barhl2fl. Barhl1EHs contains the first 119AAs of Barhl1fl. The homology at the AAs level between barhl1 and barhl2 outside the two Eh1 domains is 61%. In Barhl2EH1M, 2M or EHsM and Barhl2flEHsM the F, I and L residues in position 1, 3 and 6 of each Eh1 motif were mutated in C, K, N respectively as indicated (AAs in red). Mutated Eh1 domains are shown in black. B) Analysis of barhl2-derived constructs effects on the organizer ISH using sox3 as a probe on embryos injected into one dorsal blastomere at the 4-cell stage, together with β-gal (red) as tracer. Stage 14 representative embryos injected with the barhl-derived constructs or MObarhl2 as indicated, are shown dorsal view, anterior up. A dashed line indicates the neural plate midline. inj stands for injected side. C) Graph of B quantification. The % of embryos exhibiting the neural plate phenotype either an increase (The ratio of neural plate size inj versus control side is over 1.2) in blue or decrease (The ratio of neural plate size inj versus control side is below 0,8) in red, or no phenotype in light green, as the number of embryo analyzed (N), are indicated. (D, E) Barhl2EHs induces an increase in the organizer size at stage 14. ISH using chordin (D) or not (E) as probes on embryos injected into one dorsal blastomere at the 4-cell stage, together with β-gal (red) as tracer. Stage 14 (st.14) representative embryos injected with (a) MObarhl2, or (b) barhlfl, (c) barhl2EHs or (d) barhl2EHsM encoding RNA are shown dorsal view, anterior up. A dashed line indicates the neural plate midline. F) Barhl2EHs induces an increase in the organizer size at stage 10. ISH using chordin, goosecoid, otx2 or vent1 as probes on embryos injected into one dorsal blastomere at the 4cell stage, together with β-gal (red) as tracer. Stage 10 (st. 10) representative embryos injected with barhl2EHs are shown dorsal view, anterior up. G) Barhl2EHs rescues Barhl2 GOF phenotype Using sox3 as a neural plate marker the ability of Barhl2EHs to rescue Barhl2fl neural plate enlargement phenotype was assessed in embryos injected with RNAs coding for Barhl2 and/or Barhl2EHs separately or together. The % of

Figure 4 :

 4 Figure 4: Barhl2EHs directly competes with Barhl2fl for Gro binding.

  Figure 5

Figure 5 :

 5 Figure 5: Barhl2 binds Gro and Tcf7l1 independently and enhances the Tcf7l1-Gro interaction.(A, B) Tcf7l1 and Gro interact with one another but the Tcf7l1-Gro interaction is stabilized by the presence of Barhl2fl. Both Barhl2fl and Barhl2EHsM interact with Tcf7l1 with the same efficiency in the presence or absence of Gro. Gro interaction with Tcf7l1 is detected either (A) in the presence of Barhl2fl when the co-IP experiment is performed for 2h or (B) when the co-IP experiment is performed for 12h. HEK293T cells were co-transfected with vectors (2µg) encoding indicated proteins. Co-IP was performed on cell protein extracts using an anti-Myc antibody. Total cell lysates and the immunoprecipitated complexes were analyzed with WB analysis using anti-Myc, anti-Flag, anti-HA, antibodies as indicated. The black arrow indicates the denatured heavy chains of the anti-Myc antibody at 55kDa. (C) Barhl2 attenuates activated β-catenin (β-cat*) activation of pTop-Flash. A graph of pTopFlash relative to pFopFlash luciferase activities upon transfection of increasing amount of a vector coding for β-cat* (5ng to 50ng) (blue curve) together with vector coding for Barhl2 (200ng, red curve or 400ng, green curve) are shown as mean ± s.e. A paired sample T-test (unequal variances, two tails) was used to generate the P value (β-cat* versus β-cat* + Barhl2-200ng P value 0,039737206 ; β-cat* versus β-cat* + Barhl2-400ng P value 0,010236345). (D) Barhl2EHs limits Barhl2fl attenuation on b-cat. A graph of pTopFlash relative to pFopFLash luciferase activites upon transfection of b-cat*, Barhl2fl and Barhl2EHs. Results are shown as mean ± s.e. A paired sample T-test (unequal variances, two tails) was used to generate the P value.

Figure 6 :

 6 Figure 6: Barhl2 enhances Tcf7l1 repressor activity and limits β-catenin dependent derepression of Tcf7l1 target genes within the dorsal territory of blastula embryos. (A, B, C) Barhl2, Groucho and Tcf interact in Xenopus embryos. Both Barhl2fl and Barhl2EHs interact with Gro. Tcf7l1 interacts both with Barhl2fl and Gro. Embryos were injected with tagged mRNA coding for tcf7l1-Myc, gro-HA or barhl2fl-HA. Co-IP was performed on embryos protein extracts using an anti-Myc antibody. Total cell lysates and the immunoprecipitated complexes were analyzed with WB analysis using anti-Myc and anti-HA, antibodies as indicated. (D) Barhl2 prevents wnt8b-induced formation of a double axis. Xenopus embryos were coinjected into one ventral blastomere at the 4-cell stage, with wnt8b (4pg) alone or together with Xbarhl2fl (200pg), mbarhl2fl (200pg), or MObarhl2-2 (20ng) together with gfp as tracer. (D) Ventral (a), or dorsal (b, c, d) view of representative stage 25 embryos exhibiting (a) a double axis, (b) a partial double axis, (c) an aborted double axis, or (d) a normal phenotype. (E) Graph of D quantification. The percentage of embryos with each phenotype is graphed.

4 .

 4 scheme of the transgene Wnt reporter construct is given in Supplementary Fig. 4. MObarhl2 were injected into one or two dorsal blastomeres at the 2-cell or 4-cell stage, together with a tracer in X. tropicalis transgenic embryos. gfp RNA levels were analyzed by (F) ISH on wt (a), stage 8 (b) and stage 8.5 (c) embryos or by (G) RT-qPCR analysis on wt (blue) or MObarhl2 (orange) injected embryos. The results are normalized relative to control expression. Results are shown as mean ± s.e and evaluated to be significant by paired sample T-test (P=0,013, unequal variances, two tailed). (H) Barhl2 GOF and LOF change Tcf transcriptional activity in stage 10.5 X. tropicalis Wnt reporter embryos. ISH using gfp probe was performed on stage 10.5 pbin7LefdGFP transgenic embryos injected with RNA encoding barhl2 (barhl2) (b, c) or with MObarhl2 (d, e) together with a tracer. (H) Stage 10.5 representative embryos are shown either posterior (a, b, d) or dorsal views (c, e). The area devoid of Tcf activity and corresponding to the dorsal blastopore lip at stage 10.5 is delimitated by a white line.

Figure 7 :

 7 Figure 7: Barhl2fl promotes the recruitment of Gro within a protein complex containing Tcf7l1 and Hdac1.

stage 8 .

 8 Figure 8

Figure 8 :Figure 9 125Figure 9 :

 899 Figure 8: Barhl2 ability to promote Tcf7l1 repressor activity persists at postembryonic stages. ISH using gfp or barhl2 as probes was performed on stage 38 pbin7LefdGFP transgenic embryos either wt or injected with RNA encoding barhl2 or MObarhl2 together with MOct (red) as tracer. Representative embryos are shown dorsal view. (A) barhl2 expression pattern is complementary to that of Wnt transcriptional activity. ISH using gfp (a) or barhl2 (b) as probes. ch: cortical hem; cRhL: cerebellar rhombic lip. (B) Barhl2 depletion and overexpression modifies the cellular response to Wnt activation at post-embryonic stages. ISH using gfp as probe. Dorsal view of representative stage 41 transgenic embryos either injected with barhl2 RNA (a) or MObarhl2 (b) together with MOct (red) as tracer. (C, D) In the cortical hems and the cerebellar rhombic lip territories Barhl2 GOF decreases, and Barhl2 LOF increases, Tcf transcriptional activity. Representative transversal sections are shown (a, b) together with (c) a graph representing the ratio of injected versus control measurement of 1-the area of the region of interest (ROI), 2-the mean pixel intensity of the ROI and 3-the integrated pixel density measured within the ROI. To prevent for any subjectivity in ROI determination the ROI was determine for each section of each embryo with an ImageJ macro. N is the number of embryos. Results are shown as mean ± s.e and evaluated to be significant by paired sample T-test (P=0,022316088 for cortical hems, P=0,016402055 for the cerebellar rhombic lip, unequal variances, two tailed).

  . The BH3 only proteins are translated in response to cell damage and induce the production of Bcl2 associated protein X (Bax) and Bcl2 antagonist killer (Bak), responsible for the release of cytochrome C from the mitochondria. The cytochrome C release will lead to the activation of the initiator pro-Caspase-9 and the effector Caspases-3 and -7. Bax and Bak are inhibited by the Bcl2 anti-apoptotic proteins Bcl2 like 1 (Bcl2l1) and myeloid cell leukemia 1 (Mcl1). Bcl-X L is the long isoform encoded by the bcl2l1 gene. Mcl1 and Bcl-XL act as the main anti-apoptotic Bcl2 family member in the developing central nervous system (CNS). Mice deficient for Bcl-XL or Mcl1 prematurely die due to massive apoptosis of progenitors, immature neurons and hematopoietic cells

Caspase3(

  Casp3), affected development of the neural plate. Messenger RNA (mRNA) encoding Xbcl-xl or previously validated morpholino (MO) specific of Casp-3 (MOcasp3) (Juraver-Geslin et al., 2011) were injected into one dorsal blastomere at the 4-cell stage, together with b-gal mRNA as a tracer. Using the early marker of neural plate sry-box 3 (sox3) on whole in situ hybridization (ISH), we observed that Bcl-XL overexpression generated a widening of the neuroepithelium whereas MOcasp3 did not (Figure 1C a, b, D). In agreement with previous results (Offner et al., 2005) we suggest that the change in neuroepithelium size generated by Bcl-XL overexpression could be an indirect consequence of apoptosis requirement in axial organizer formation. To determine if cells normally undergoing apoptosis between stage 10 and stage 14 are part of the axial organizer we targeted bcl-xl mRNA injection in different regions of the dorsal tissue. Xenopus embryos were injected into one dorsal blastomere at eight-or sixteen-cell stage with bcl-xl and β-gal. Based on the expression of β-gal we assessed retrospectively whether bcl-xl

  generated constructs coding for two tagged versions of Mcl1: a Flag-Mcl1 and a Mcl1-Myc (Figure 3Ab). Whereas MOmcl1-1 and MOmcl1-2 bind myc-mcl1 mRNA, they do not bind flag-mcl1 mRNA. For all constructs we injected either the MO, or the specified mRNA, together with b-gal mRNA used as a tracer.

MOmcl1- 1 ,

 1 MOmcl1-2 or MOmcl1-Ct in two dorsal blastomeres of two-cell stage Xenopus embryos. Western blot (WB) analysis of protein extracts from injected embryos revealed that both MOmcl1-1 and MOmcl1-2 inhibited the translation of Mcl1-Myc while MOmcl1-Ct did not (Figure 3C). Results were similar with MOmcl1-1 and MOmcl1-2, however MOmcl1-2 generated stronger developmental disruptions. We therefore used MOmcl1-1 for further experiments. We tested MOmcl1-1 as a specific inhibitor of endogenous Mcl1 activity by coinjecting MOmcl1-1 with flag-mcl1 mRNA, which is not targeted by the designed morpholino (Figure 3D). We observed that Flag-Mcl1 significantly rescued the decrease in neuroepithelium width induced by endogenous mcl1 depletion (Figure 3Db). Taken together these results provide strong evidence that MOmcl1-1 acts by specifically inhibiting endogenous Mcl1 activity and that Mcl1 loss-of-function (LOF) affects the size and shape of the neural plate suggesting a role of Mcl1 in the neural plate development.Depletion of Mcl1 activity induces a specific loss of notochord cellsConsidering the expression pattern of mcl1 within the neural plate we investigated whether the changes in neural plate size induced by depletion of endogenous Mcl1 could be a consequence of alterations in notochord formation. MOmcl1-1 was co-injected with b-gal mRNA into one dorsal blastomere of four-cell stage embryos. At stage 14 the notochord was observed by ISH with probes against the dorsal mesoderm gene chordin. We observed a decrease of chordin-expressing cells on the injected size (Figure4Ab). Transverse section of MOmcl1-1 injected embryos confirmed a diminution of the notochord size in the injected embryo (Figure4Ad). Mcl1 is an anti-apoptotic protein. Consequently, a decrease of Mcl1 levels could increase apoptosis in notochord but also in neural plate cells. Embryos were injected in one dorsal blastomere at the 4-cell stage with different doses -0.5ng to 5ng -of MOmcl1. TUNEL staining in these embryos indicate that apoptotic nuclei appear preferentially detected in the axial organizer and not randomly within the neuroepithelium (ongoing experiment). This experiment argues that depletion of Mcl1 preferentially induce apoptosis in axial organizer cells.

  . MObarhl2 or barhl2ehs mRNA and MOmcl1-1 were injected individually or together in one dorsal blastomere at the two-or four-cell stage. The effect of the injections on the axial organizer was assessed with analysis of sox3 expression. As expected, we observed an increase in sox3 expression territory within either the MObarhl2-, or the barhl2ehs-injected side of stage 14 embryos (Figure 5Aa, Ba). Depletion of Mcl1 activity generated a diminution of the sox3 expression territory (Figure 5Ac, Bc). Injection of both MOmcl1 and MObarhl2 significantly reduced both the number of embryos exhibiting neuroepithelium size defects, and the severity of the phenotypes (Figure 5Ab, C). Injection of MOmcl1 with the dominant negative barhl2EHs mRNA produced the same effect (Figure 5Bb, C).

  ,C), Mcl1 inhibits apoptosis with a low efficiency. The higher penetrance of the non-phosphorylable Mcl1 on sox3 expression suggests that Mcl1 protein expression levels could be important during axial organizer development. Depletion of Barhl2 activity increases Mcl1 protein half-life We next asked whether Barhl2 directly or indirectly modified Mcl1 protein levels. xmcl1-myc mRNA was injected alone or together with MObarhl2 into two blastomeres of two-cell stage embryos. From stage 10 onwards we either let the embryos develop normally or treated them with cycloheximide (CHX), a pharmacological inhibitor that prevented mcl1 mRNA to be translated. Protein extracts were prepared 30minutes (min), 2hours (h) and 4h after CHX treatment. The presence of both Mcl1-Myc and the loading control Actin were assessed by Western Blot analysis (Fig 8A) and quantified using ImageJ software (Fig 8B). Whereas in control conditions Mcl1-Myc protein levels remain steady (Figure 8A, B), in the presence of CHX we observed a sharp decrease of Mcl1-Myc protein levels that reached undetectable levels after 4h. Mcl1-Myc protein half-life was measured as the length of time necessary to decrease the total level of Mcl1-Myc protein measured at t0 by half. The quantification of Mcl1-Myc protein levels in the wild type conditions or in absence of Barhl2 reveals that Mcl1-Myc protein half-life is increased by 1h when Barhl2 is downregulated. These ongoing experiments suggest that Barhl2 contributes to limit survival of notochord cells by decreasing Mcl1 antiapoptotic protein half-life.

  At the same developmental stages bcl-xl transcripts are not detected. Mcl1 LOF decreases the number of notochord cells and the neural plate size indicating that Mcl1 plays an important role in neural plate development partly through its ability to control the survival or organizer cells. We followed cleaved-Caspase3 activity in stage 10 to stage 14 embryos. We did not observe activated-Caspase3 positive cells in the neuroepithelium of WT neurula embryos. Moreover Mo-mediated inhibition of Caspase-3 activity in midline cells is phenotypically silent. These observations suggest that at these developmental stages EPCD occurs independently of Caspase3. In conclusion, the apoptosis observed in the neural plate in late gastrula early neurula embryos is Caspase3 independent and regulated by changes in levels of the Bcl2 anti-apoptotic protein Mcl1.

  not regulate the apoptotic cascade at the transcriptional level. Different E3 ubiquitin ligases can polyubiqutinate Mcl1 to target it for degradation in the proteasome: Mcl1 ubiquitin ligase E3 (MULE) and b-transducing containing protein (b-TrCP)

  Human mcl1 poorly protects notochord cells from apoptosis. However, overexpression of a form of Mcl1 that cannot be phosphorylated, and subsequently degraded by the proteasome, increases Mcl1 protective effect on notochord cells. Therefore, Mcl1 stability is an important parameter in the control of developmental apoptosis in the neural plate. We are currently assessing Barhl2 ability to regulate Mcl1 protein stability by measuring Mcl1 half-life time in the presence or absence of Barhl2.

  Figure 1

Figure 1 :Figure 2 :

 12 Figure 1: Apoptosis is detected in the dorsal region of Xenopus embryos. A: TUNEL-staining in wild type Xenopus embryos at stage 12.5. B: Immunostaining against cleaved Caspase3 on WT embryos at different stages during neurulation. Blue staining is background noise. No positive nucleus for cleaved Caspase3 staining are observed. C: ISH with probes against sox3 to evaluate neuroepithelium size in (a) overexpression of the antiapoptotic protein Bcl-xl or (b) down-regulation of the pro-apoptotic protein Caspase-3 in Xenopus embryos at stage 14. b-gal (in red) is used as injection tracer. D: ISH with probes against sox3 to evaluate neuroepithelium size in stage 14 embryos overexpressing the proapoptotic protein Bcl-xl. Embryos are injected in (a,b) one dorsal blastomere at 4-cell stage and (c,d) one dorsal blastomere at 8-cell stage. (b,d) are histological sections corresponding to the white arrow level in (a,c). b-gal (in red) is used as injection tracer. Black arrow indicates b-gal staining. E: counting of embryos showing increased sox3 expression phenotype upon injection of bcl-xl. Schematic embryos indicate in red the area of bcl-xl injection corresponding to neural plate cells, mediolateral cells and lateral cells respectively. Bars indicate the percentage of embryos exhibiting the phenotype upon injection of bcl-xl in the whole neural plate, MOcasp-3 in the whole neural plate, bcl-xl in mediolateral cells and bcl-xl in lateral cells. N indicates number of embryos. White vertical dashed line indicated midline. All embryos are shown dorsal view up and posterior to the bottom.

Figure 3 : 4 BFigure 4 :

 344 Figure 3: Effect of Mcl1 loss-of-function on neuroepithelium size. A: (a) morpholino sequences for mcl-1 downregulation (b) Mcl1-tagged constructs for the study of Mcl1 overexpression. Mcl1-Myc contains the target sequences for the two morpholino B: (a) ISH at stage 14 embryos using probes against sox3 on embryos depleted of Mcl1 or injected with the control MO (b) percentage of embryos exhibiting a decrease on sox3 expression on the injected side. C: Western Blot showing the inhibition of overexpressed Mcl1-Myc by morpholino against Mcl1. Actin is used as a loading control. D: (a) ISH at stage 14 embryos using probes against sox3 on embryos injected with (a) MOmcl1, (b) MOmcl1 and flag-mcl1 or (c) flag-mcl1 alone. (e) percentage of embryos exhibiting an increase or decrease of sox3 upon injection. (d) Western blot showing Flag detection of the overexpressed construction flag-mcl1. N indicates number of embryos. b-gal (in red) is used as injection tracer. White vertical dashed line indicated midline. All embryos are showed dorsal side up and posterior to the bottom.

Figure 5 :Figure 6 :

 56 Figure 5: Barhl2 and Mcl1 are part of the same signaling pathway. A: ISH at stage 14 embryos using probes against sox3 on embryos injected with (a) MObarhl2 (b) MObarhl2 and MOmcl1 (c) MOmcl1. B: ISH at stage 14 embryos using probes against sox3 on embryos injected with (a) barhl2ehs (b) barhl2ehs and MOmcl1 (c) MOmcl1. C: percentage of embryos exhibiting a change on sox3 expression on the injected side compared to the noninjected side. White vertical dashed line indicated midline. N indicates number of embryos. bgal (in red) is used as injection tracer.

Figure 7 :

 7 Figure 7: Effect of Mcl1 overexpression on neuroepithelium size. A: ISH at stage 14 embryos using probes against sox3 on embryos injected with (a) xmcl1 (b) xmcl1-myc (c) hmcl1 (d) hmcl1s159a. B: Western blot showing Myc detection of the overexpressed construction xmcl1-myc. C: percentage of embryos exhibiting an increase on sox3 expression on the injected side compared to the non-injected side. White vertical dashed line indicated midline. N indicates number of embryos. b-gal (in red) is used as injection tracer.

Figure 8 :

 8 Figure 8: Barhl2 regulates Mcl1 half-life. A: Western Blot gels of injected embryos protein extracts treated or not with the pharmacological inhibitor cycloheximide (CHX) at different times after treatment. Actine is used as a loading reference. T: time in hours. B: graph showing half-life of Mcl1 in the presence or absence of Barhl2. In CHX treated embryos, Mcl1 half-life is about 2,5h and in the absence of Barhl2 is increased by an hour.

DISCUSSION

  During my PhD I studied the effects of the transcription factor Barhl2 on the regulation of two important processes during embryonic development: cell survival and Wnt signaling. The results presented in this thesis describe that Barhl2 limits Wnt signaling by interacting with the co-repressor Gro and Tcf in a signaling mechanism that impedes Tcf activation by b-catenin. This role appears to be conserved at different stages during development and its effects on development are dependent on the location and time the interaction takes place. I also investigated Barhl2 function on regulation of the anti-apoptotic factor Mcl1 during neural plate formation. The implications of our findings are discussed below.

Figure 13 :

 13 Figure 13: Barhl2 ability to promote Tcf7l1 repressor activity is present at the retina CMZ. ISH using gfp and barhl2 as probes was performed on stage 26 pbin7LefdGFP wild-type transgenic embryos. Histological sections of representative embryos are shown. The red arrow indicates the CMZ. Barhl2 and Wnt activity are present in excluding regions.

Figure 14 :

 14 Figure 14: Barhl2 ability to promote Tcf7l1 repressor activity is present at stage 30. ISH using gfp as probe was performed on stage 30 pbin7LefdGFP transgenic embryos either injected with RNA encoding barhl2 or MObarhl2 together with MOct (red) as tracer. Representative embryos are shown dorsal view. Barhl2 depletion and overexpression modifies the cellular response to Wnt activation at post-embryonic stages. In the CMZ of the retina Barhl2 GOF decreases and Barhl2 LOF increases Tcf transcriptional activity. A graph representing the ratio of injected barhl2 versus MObarhl2 is shown of the area of the region of interest (ROI), the mean pixel intensity of the ROI and the integrated pixel density measured within the ROI. To prevent for any subjectivity in ROI determination the ROI was determined for each section of each embryo with an ImageJ macro. Results are shown as mean ± s.e and evaluated to be significant by paired sample T-test.

  Indeed, similarly to Barhl2 Caspase3 depletion generates a hyperplasia not associated to a decrease in apoptosis. Caspase3 LOF phenotypes can also be compensated by depletion of b-Catenin, or expression of GSK3b (Juraver-Geslin et al., 2011). Whereas Juraver-Geslin et al. demonstrated that Caspase3 acts downstream of Barhl2 we do not know the mechanisms by which Barhl2 controls Caspase-3 activity.

  suggested a role for this gene in neural plate organizer formation. The results presented in my second publication demonstrate that Mcl1 LOF increases neural plate size and this is due to an effect on the notochord size. I also provide evidence that Barhl2 and Mcl1 are part of the same signaling pathway. Our results suggest that Barhl2 regulates Mcl1 protein half-life during neurulation.

Figure 1 .

 1 Figure 1. Schematic of Xenopus (X.) laevis forebrain markers and flat-mounted neural tubes from X. laevis and Mus musculus embryo after whole-mount double in situ-hybridization (WM-dISH). (A) Schematic of X. laevis forebrain markers at st. 30. (a) Areas of expression are shown and indicated for fezf2 (pink), pax6 (checkerboard pattern), irx3 (yellow), wnt3a (light green), shh (orange), barhl2 (hatched lines), and irx1 (red). (b) Enlargement of the diencephalic part indicated by a rectangle in a. The pineal gland located on top of p2 is marked by an asterisk. The future zli is indicated in b. p: prosomere; Tel: telencephalon; Mes: mesencephalon; Pre-Th: pre-thalamus; zli: zona limitans intrathalamica; Th: thalamus. (B) WM-dISH is performed using otx2, irx1, irx3, fezf2, pax6, and shh or barhl2 as probes on (a-g). X. laevis and mouse embryos at different developmental stages as indicated. Neural tubes (a-f) or heads (h,i) of representative embryos, dissected and flat-mounted, are shown from a side view, dorsal up, anterior left except for (g), shown in anterior view, dorsal side up. The markers and stages are indicated. Black arrows indicate the rostral and caudal boundaries of p2. White arrows indicate the bilateral zli. Scale bar: 0.5 mm.

Figure 1 .

 1 Figure 1. Schematic of Xenopus (X.) laevis forebrain markers and flat-mounted neural tubes from X. laevis and Mus musculus embryo after whole-mount double in situ-hybridization (WM-dISH). (A) Schematic of X. laevis forebrain markers at st. 30. (a) Areas of expression are shown and indicated for fezf2 (pink), pax6 (checkerboard pattern), irx3 (yellow), wnt3a (light green), shh (orange), barhl2 (hatched lines), and irx1 (red). (b) Enlargement of the diencephalic part indicated by a rectangle in (a). The pineal gland located on top of p2 is marked by an asterisk. The future zli is indicated in (b). p: prosomere; Tel: telencephalon; Mes: mesencephalon; Pre-Th: pre-thalamus; zli: zona limitans intrathalamica; Th: thalamus. (B) WM-dISH is performed using otx2, irx1, irx3, fezf2, pax6, and shh or barhl2 as probes on (a-g). X. laevis and mouse embryos at different developmental stages as indicated. Neural tubes (a-f) or heads (h,i) of representative embryos, dissected and flat-mounted, are shown from a side view, dorsal up, anterior left except for (g), shown in anterior view, dorsal side up. The markers and stages are indicated. Black arrows indicate the rostral and caudal boundaries of p2. White arrows indicate the bilateral zli. Scale bar: 0.5 mm.

Figure 2 .

 2 Figure 2. A model for zli formation. (A) During neural plate patterning the diencephalic primordium expresses otx2, barhl2 and irx1, irx2, irx3 (orange). Cross-repressive interactions between Fezf/Fez and Irx homeoproteins contribute to formation of the p2/p3 alar border. shh (light blue) is expressed in the p2 basal plate. (B) Concomitantly, a Wnt-mediated signal promotes survival of zli anlage cells, and gli3 expression is repressed through a Wnt-mediated signal in a narrow band of cells that correspond to the zli primordium. (C) At the onset of zli formation a permissive environment is established in the future zli, and zli cells acquire the competence to express shh. The zli anlage is characterized by the expression of barhl2, otx2, irx3 (orange), and the future thalamus by the expression of barhl2, otx2, irx1/2/3 (green). At the onset of zli development, irx3 is expressed in the future thalamus territory. (D) Using a sequential induction mechanism, Shh secreted by the basal plate initiates the spreading of shh expression into the pre-zli domain. Transcription factors (TFs) including Otx1, Otx2, Barhl2, Yap-Tead, and possibly unidentified TFs (TF-X) are recruited to the enhancers SBE1 and SBE5 to initiate shh transcription within the zli. Under the inductive influence of secreted N-Shh, some irx3-expressing cells located in caudal p2 sort out from thalamic cells. Shh signaling from the zli induces the repression of pax6 within the mid-diencephalic furrow. Pax6 together with unidentified dorsal signals could in turn prevent Shh from being expressed beyond the zli. The thalamus (p2) and the prethalamus (p3) receive a combination of Shh signals coming from two orthogonally oriented signaling centers, the zli and the basal plate. Together, they establish a morphogenetic gradient of Shh protein that patterns the thalamus posterior to the zli and the prethalamus anterior to the zli. p: prosomere; zli: zona limitans intrathalamica.

Figure 2 .

 2 Figure 2. A model for zli formation. (A) During neural plate patterning the diencephalic primordium expresses otx2, barhl2 and irx1, irx2, irx3 (orange). Cross-repressive interactions between Fezf/Fez and Irx homeoproteins contribute to formation of the p2/p3 alar border. shh (light blue) is expressed in the p2 basal plate. (B) Concomitantly, a Wnt-mediated signal promotes survival of zli anlage cells, and gli3 expression is repressed through a Wnt-mediated signal in a narrow band of cells that correspond to the zli primordium. (C) At the onset of zli formation a permissive environment is established in the future zli, and zli cells acquire the competence to express shh. The zli anlage is characterized by the expression of barhl2, otx2, irx3 (orange), and the future thalamus by the expression of barhl2, otx2, irx1/2/3 (green). At the onset of zli development, irx3 is expressed in the future thalamus territory. (D) Using a sequential induction mechanism, Shh secreted by the basal plate initiates the spreading of shh expression into the pre-zli domain. Transcription factors (TFs) including Otx1, Otx2, Barhl2, Yap-Tead, and possibly unidentified TFs (TF-X) are recruited to the enhancers SBE1 and SBE5 to initiate shh transcription within the zli. Under the inductive influence of secreted N-Shh, some irx3-expressing cells located in caudal p2 sort out from thalamic cells. Shh signaling from the zli induces the repression of pax6 within the mid-diencephalic furrow. Pax6 together with unidentified dorsal signals could in turn prevent Shh from being expressed beyond the zli. The thalamus (p2) and the prethalamus (p3) receive a combination of Shh signals coming from two orthogonally oriented signaling centers, the zli and the basal plate.Together, they establish a morphogenetic gradient of Shh protein that patterns the thalamus posterior to the zli and the prethalamus anterior to the zli. p: prosomere; zli: zona limitans intrathalamica.

Figure 3 .

 3 Figure 3. Elongated cilia on the zli. (A) Schematic overview of the Xenopus tadpole brain comprised of (from anterior to posterior) telencephalon (tel), diencephalon (di), mesencephalon (mes) and rhombencephalon (rhomb). The position of the zli is indicated by orange dashed line. (B) Magnification of area indicated by white dashed line in (A). Whole mount immunofluorescence staining on the ventricular surface of a tadpole brain. Antibodies detecting acetylated alpha-tubulin (ac. ↵-tub.) and gamma-tubulin ( -tub.) mark cilia axonemes and basal bodies, respectively. Note that zli cilia are markedly elongated compared to cilia on neighboring cells (scale bars: 5 µm).

Figure 15

 15 Figure 15 La voie de signalisation Wnt chez les invertébrés et les vertébrés. A : (a) En absence de signalisation Wnt, la b-catenin est séquestré dans le cytoplasme par un complexe protéique composé d'Axin, Apc et Gsk3 qui phosphoryle la b-caténine et le marque pour la dégradation. Dans le noyau, le facteur corépresseur Groucho se lie au facteur de transcription Tcf et inhibe la transcription génique des cibles Wnt. (b) Wnt se lie à son récepteur dans la membrane cellulaire, Frizzled, et grâce à la protéine LRP il recrute et phosphoryle le complexe protéique responsable de la dégradation de la b-caténine. La b-caténine est donc stabilisée et peut entrer dans le noyau où elle déplace Groucho et se lie à Tcf permettant la transcription des gènes. B : chez les vertébrés, il existe différents types de Tcf qui agissent comme activateurs ou répresseurs. (a) En l'absence de signalisation Wnt, Tcf71I et Gro-4 se lient aux promoteurs dans les gènes cibles Wnt pour inhiber la transcription. (b) Lorsque le ligand Wnt se lie à son récepteur, Hipk2 phosphoryle Tcf7l1 et Tcf7 se lie à la b-caténine pour activer la transcription des gènes dans le noyau.

  Dans la zone sous-ventriculaire (SVZ) du cerveau adulte, il existe une niche de cellules souches. La perte de fonction de Mcl1 augmente l'apoptose dans cette niche. Mcl1 est donc nécessaire à la survie de ces cellules. Par conséquent, Mcl1 régule la survie des cellules progénitrices neurales adultes (NPC) et est la seule protéine Bcl-2 anti-apoptotique connue

Figure 16

 16 Figure 16 Structure des protéines xBarhl1 et xBarhl2. Représentation schématique des protéines Xenopus Barhl1 et Barhl2. Le domaine FIL, NLS et Homeodomain sont représentés.

Figure 17

 17 Figure 17 Barhl2 limite la taille des organisateurs de la tête et corps. (A) barhl2 est exprimé dans le territoire de l'organisateur présomptif au stade mi-blastula. Analyse RT-qPCR de l'expression de barhl2 et barhl1 sur des embryons de stade 7 à 10. L'expression relative a été normalisée au stade 10. Les résultats sont présentés en

Figure 18

 18 Figure 18Barhl2 interagis avec Gro et Tcf71l indépendamment et améliore l'interaction Tcf71-Gro. Tcf7l1 et Gro interagissent entre eux mais l'interaction Tcf7l1-Gro est stabilisée par la présence de Barhl2fl. Les deux Barhl2fl et Barhl2EHsM interagissent avec Tcf7l1 avec la même efficacité en présence ou en l'absence de Gro. L'interaction Gro avec Tcf7l1 est détectée en présence de Barhl2fl lorsque l'expérience co-IP est réalisée pendant 2h Les cellules HEK293T ont été co-transfectées avec des vecteurs (2 ug) codant pour des protéines indiquées. La Co-IP a été réalisée sur des extraits de protéines cellulaires en utilisant un anticorps anti-Myc. Les lysats cellulaires totaux et les complexes immunoprécipités ont été analysés par analyse WB en utilisant des anticorps anti-Myc, anti-Flag, anti-HA, comme indiqué. La flèche noire indique les chaînes lourdes dénaturées de l'anticorps anti-Myc à 55 kDa.

Figure 19

 19 Figure 19 Barhl2 augmente l'activité du répresseur Tcf7l1 et limite la dé-répression β-caténine dépendante des gènes cibles Tcf7l1 dans le territoire dorsal des embryons de blastula. (A, B, C) Barhl2, Groucho et Tcf interagissent chez les embryons Xenopus. Barhl2fl et Barhl2EH interagissent avec Gro. Tcf7l1 interagit à la fois avec Barhl2fl et Gro. Des embryons ont été injectés avec de l'ARNm marqué codant pour tcf71-Myc, gro-HA ou barhl2fl-HA. La co-IP a été réalisée sur des extraits protéiques d'embryons en utilisant un anticorps anti-Myc. Les lysats cellulaires totaux et les complexes immunoprécipités ont été analysés par analyse WB en utilisant des anticorps anti-Myc et anti-HA, comme indiqué. (D) Barhl2 empêche la formation d'un double axe induite par wnt8b. Des embryons de Xenopus ont été co-injectés dans un blastomère ventral au stade 4 cellules, avec wnt8b (4pg) seul ou avec Xbarhl2fl (200pg), mbarhl2fl (200pg), ou MObarhl2-2 (20ng) ensemble avec gfp comme traceur. (D) vue ventrale (a), ou dorsale (b, c, d) des embryons représentatifs de stade 25 présentant (a) un double axe, (b) un double axe partiel, (c) un double axe avorté, ou (d) un phénotype normal. (E) Graphique de la quantification D. Le pourcentage d'embryons avec chaque phénotype est représenté graphiquement. Les moyennes entre les différents ensembles de données ont été comparées en utilisant le test de Wilcoxon signé-rank et les différences étaient significatives comme indiqué avec des astérisques). (F, G) La déplétion de Barhl2 augmente l'activation transcriptionnelle de la β-caténine chez les embryons de stade 8.5. MObarhl2 a été injecté dans un ou deux blastomères dorsaux au stade à 2 cellules ou à 4 cellules, avec un traceur dans des embryons transgéniques de

3. 2

 2 MCL1 REGULE L'APOPTOSE A LA PLAQUE NEURALE Notre équipe a précédemment décrit que le facteur de transcription Barhl2 favorise l'apoptose dans l'organisateur axial des embryons Xenopus. La perte de fonction de Barhl2 conduit à une diminution des cellules apoptotiques et une perturbation dans les gradients Shh et anti-Bmp émanant de l'organisateur. Comment Barhl2 régule l'apoptose dans ce domaine est actuellement inconnu (Offner et al., 2005). Dans la deuxième partie de résultats nous présentons le profil d'expression de mcl1 et bcl-XL chez des embryons de X. laevis au début du développement (Figure 20). En accord avec son profil d'expression, nous démontrons que Mcl1 contrôle la survie des cellules organisatrices axiales dans la neurula gastrula-précoce tardive. Nous présentons des preuves que Barhl2, et la protéine anti-apoptotique Mcl1, participent au même processus de développement (Figure 21). Nous montrons que Barhl2 n'affecte pas la transcription de la plupart des facteurs apoptotiques, y compris mcl1.

Figure 20

 20 Figure 20 Expression de mcl1 pendant la neurulation chez X. laevis. ISH montrant le profil d'expression du gène anti-apoptotique mcl1 chez les embryons de la fin de la gastrula aux stades tardifs de la neurula. Les embryons sont montrés face dorsale vers le haut, sauf à partir de (g) montrant la face antérieure vers le haut. (e) correspond à une section sagittale à l'étape 14, la coloration bleue correspond à la coloration de la sonde ISH.

Figure 21 :

 21 Figure 21: Barhl2 et Mcl1 font partie de la même voie de signalisation. A: ISH au stade 14 embryons en utilisant des sondes contre sox3 sur des embryons injectés avec (a) MObarhl2 (b) MObarhl2 et MOmcl1 (c) MOmcl1. B: ISH au stade 14 utilisant des sondes contre sox3 sur des embryons injectés avec (a) barhl2Ehs (b) barhl2Ehs et MOmcl1 (c) MOmcl1. C: pourcentage d'embryons présentant un changement d'expression de sox3 du côté injecté par rapport au côté non injecté. La ligne pointillée verticale blanche indiquée sur la ligne médiane. N indique le nombre d'embryons. b-gal (en rouge) est utilisé comme traceur d'injection.

  de deux processus importants au cours du développement embryonnaire : la survie cellulaire et la signalisation Wnt. Les résultats présentés dans cette thèse décrivent que Barhl2 limite la signalisation Wnt en interagissant avec le corépresseur Gro et Tcf dans un mécanisme de signalisation qui empêche l'activation de Tcf par la b-caténine. Ce rôle semble être conservé à différentes étapes du développement et ses effets sur le développement dépendent du lieu et du moment où l'interaction a lieu. J'ai également étudié la fonction de Barhl2 sur la régulation du facteur anti-apoptotique Mcl1 lors de la formation de la plaque neurale.

4. 1

 1 LA REGULATION DE LA SIGNALISATION WNT PAR BARHL2Nous avons étudié si l'interaction moléculaire que nous avons décrite entre Barhl2, Gro et Tcf, et la capacité de Barhl2 à limiter l'activation de Tcf était spécifique ou développement précoce ou se produit également à des stades de développement plus tardives. Pour répondre à cette question, nous avons d'abord étudié le modèle d'expression barhl2 par rapport à l'activité Tcf aux stades post-embryonnaires, c'est-à-dire lorsque l'organogenèse est terminée. Aux stades post-embryonnaires, nous avons observé que Barhl2 est exprimé dans les régions proches où Tcf est active. Ceci est particulièrement vrai au niveau de la lèvre rhombique cérébelleuse et des ourlets corticaux, deux régions qui continuent à générer des progéniteurs neuraux après la fin du développement embryonnaire. Les transcrits barhl2 étant présents dans un domaine bordant une zone produisant des cellules progénitrices est également vrai dans la zone marginale ciliaire (CMZ) de la rétine, une niche de cellules souches de bonne foi. Prises ensemble, ces observations suggèrent que Barhl2 joue un rôle dans le passage Tcf-actif à Tcf-répressif qui contrôle la pluripotence des cellules souches.L'implication de Barhl2 dans le maintien ou la différenciation de ce pool de cellules souches pourrait nous éclairer sur son rôle supposé dans le développement du médulloblastome, une tumeur pédiatrique maligne(Lin et al., 2016). L'analyse des tumeurs dérivées des patients a mis en évidence que les deux gènes barhl sont dérégulés dans ces tumeurs pédiatriques: Barhl2 étant associé au groupe Shh et Barhl1 aux groupes 3 et 4. Comprendre les fonctions exactes de Barhl2 et/ou Barhl1 dans les progéniteurs granulaires et l'origine de leur dérégulation dans le médulloblastome devrait fournir des informations importantes sur les mécanismes moléculaires impliqués dans l'origine de cette maladie.

4. 2

 2 LE ROLE DE BARHL2 DANS L'APOPTOSE Nos résultats montrent que mcl1 est exprimée dans la plaque neurale à l'étape 14 et son expression reste dans le tube neural à des stades plus avancés. Nous nous sommes concentrés sur l'étude de la régulation mcl1 parce que son patron d'expression suggérait un rôle pour ce gène dans la formation des organisateurs de la plaque neurale. Les résultats présentés dans ma deuxième publication démontrent que la perte de fonction de Mcl1 augmente la taille de la plaque neurale et cela est dû à un effet sur la taille de la notocorde. Nos résultats suggèrent que Barhl2 régule la demi-vie de la protéine Mcl1 pendant la neurulation. La protéine Mcl1 peut être dégradée par différentes méthodes. Elle peut être directement inactivée par clivage (Clohessy et al., 2004) et peut également être phosphorylée dans sa région N-terminale par Gsk3 et être postérieurement ubiquitinée (Ding et al., 2007; Thomas et al., 2010). Gsk3 peut phosphoryler Mcl1 pour favoriser sa dégradation. L'analyse de la surexpression d'un mutant Mcl1 qui ne peut pas être phosphorylé au S159 par GSK3 confirme nos observations. Nous avons déjà démontré que dans le tube neural de Xenopus, Gsk3 peut partiellement compenser le phénotype de perte de fonction de Barhl2 (Juraver-Geslin et al., 2011). Nous posons l'hypothèse que Barhl2 pourrait réguler les activités de Gsk3 dans le cytoplasme et induire la dégradation de Mcl1. Ceci, cependant, doit être étudié et d'autres expériences concernant l'effet de Barhl2 sur les activités kinases Gsk3 devraient être effectuées pour confirmer cette hypothèse. Les résultats présentés dans ce manuscrit décrivent une régulation de l'activité Tcf au cours du développement embryonnaire de Xenopus. Nous décrivons le mécanisme moléculaire par lequel le facteur de transcription Barhl2 interagit avec Groucho et, avec Hdac1, inhibe l'activité Wnt en se liant à Tcf. Nous démontrons que cette interaction perturbe la formation de l'organisateur Spemann et qu'elle est maintenue tout au long du développement. Ce mécanisme pourrait avoir des implications dans la régulation de la prolifération, et dans le maintien des niches des cellules progénitrices. Dans l'ensemble, nos résultats révèlent un mécanisme nouveau et important pour le contrôle de l'activité transcriptionnelle Wnt.

Table

Abbreviation Full name Anti-or pro-apoptotic BH domains

				1 (Mcl1) and
	Bcl2-like 1 (Bcl2l1, a.k.a. Bcl-xL), and they share three or four BH domains. The pro-apoptotic
	Bcl2 proteins can be divided into the BH123 and the BH3 only proteins. Bcl2 antagonist killer
	(Bak) and Bcl2 associated protein X (Bax) are BH123 proteins and Bcl2 like 11 (Bcl2l11, a.k.a.
	Bim), Bh3 interacting domain death agonist (Bid), p53 upregulated modulator of apoptosis
	(Puma) and NADPH oxidase activator 1 (Noxa1) are BH3 only proteins. The BH3 only proteins
	are secreted upon apoptotic stimuli and are often transcriptionally controlled (Kumar and
	Cakouros, 2004; Nehme et al., 2010).
	Bak	Bcl2 antagonist killer	Pro-apoptotic	BH1, 2, 3
	Bax	Bcl2 associated protein X	Pro-apoptotic	BH1, 2, 3
	Bcl2	B-cell lymphoma 2	Anti-apoptotic	BH1, 2, 3, 4
	Bcl2l1 (Bcl-XL)	B-cell lymphoma 2 like 1	Anti-apoptotic	BH1, 2, 3, 4
		Bh3 interacting
	Bid	domain death	Pro-apoptotic	BH-3 only
		antagonist
	Bcl2l11 (Bim)	B-cell lymphoma 2 like 11	Pro-apoptotic	BH-3 only
	Mcl1	Myeloid cell leukemia	Anti-apoptotic	BH1, 2, 3
	Noxa1	NADPH oxidase activator	Pro-apoptotic	BH-3 only
		p53 upregulated
	PUMA	modulator of	Pro-apoptotic	BH-3 only
		apoptosis

Table 2 Bcl2 family protein members. In

 2 the table there is indication of the apoptotic activity and number of domains of each member.

	MCL1
	Mcl1 is one of the main anti-apoptotic proteins of the Bcl2 family. Mcl1 blocks apoptosis by
	binding and kidnapping the pro-apoptotic proteins Bak and Bax, which are responsible for
	Cytochrome C release. Mcl1 can also bind some of the BH3 only pro-apoptotic proteins.
	Consequently, Mcl1 protein level is an important parameter in the control of cell survival. Mcl1

Table 3 Non-apoptotic role of caspases. In

 3 the table are noted some non-apoptotic roles for Caspases in different species. Adapted from[START_REF] Yi | The Jekyll and Hyde functions of caspases[END_REF].

	CASPASE-3
	Caspase-3 non-apoptotic activities have been described repeatedly in different developmental
	contexts, but the mechanisms involved are poorly understood. Numerous observations
	indicate that Caspase-3 has roles in controlling proliferation and/or differentiation of NPCs

Table S1 .

 S1 Primers used for RT-PCR in this study.

	Gene	Forward	Reverse	Reference
	bak	TGTGGAGCCCCAGCAGTCAGA	TGCCGCCCAACCTCCTCTGT	This study
	bcl-b	GCGGCACAGACCCTGCTGAG	CCGTCCCAGCCTCCGCTACT	This study
	bcl-xl	CACGGCCCAGCAGAGCTTCC	TGCTCTGGGCTGCGGCATTC	This study
	bcl-w	GAGCAAGGCTGGATGGCGCA	TGAGACAGCAGCAAGTCCAGA	This study
	bcl-2	GCAGCCAGTCCCTTCGGCAG	CCACGCACATGACCCCACCA	This study
	bim	GAGGGGCCCCCACCTCTCTT	TTCTCGGCCCGTCTCGGAGT	This study
	histone 4	ATGTCCGGCCGCGGCAAAGG	AGCCGTAGAGAGTGCGGCCC	This study
	mcl1	GGTTCCACGTCTCCCCCGGA	CGCCAAACCGGCCCCAAGAA	This study
	noxa	ATGAAGCCCCGCACTGCATC	TATCAAGTTCCGGAAGAGAG	This study
	Gene	Forward	Reverse	Reference
	apaf-1	TGCTTCACGAGGGGTACAAA	CTCCCTCACAGAGTACGGTT	This study
	bak	TAATTGGAAGAATGCCGCCC	GAATGGGCAGCGAGGATTTT	This study
	barhl2	TTAGGGATGTCAGGGCTACG	ATGGACGCTGTCCACTAACC	This study
	bax	GAATGGGCAGCGAGGATTTT	TAATTGGAAGAATGCCGCCC	This study
	bcl-b	GGACTGGCTGCAGAGTAGC	TGCCCGCTGAGTCCTCT	This study
	bcl-xl	TGTGGAGAGTGCAAACAAGG	ACAGGCCAACAAAAGCTTCC	This study
	bcl-w	TGAGAGGCTGGATTCAGAGC	GCCTCCTGGCTTCTTCTATGG	This study
	bcl-2	GACAGATCTCAGGGCTCCTC	CCTTCCCCAGTTTACCCCAT	This study
	bim	GGAGGAGCAGCGATGAAGAA	TTGTAACTGGCCACCTTCAC	This study
	caspase-3	AGAGGAGACCGATGCAAGAC	CTCCACTGGAATACGCTGGA	This study
	caspase-7	ACTGACCACTCTCTTCCGTG	ATCTGCCTCCAACCCATCAT	This study
	caspase-9	ATGCCACCCCTGTTTTCTCT	ATCCCTCCATGACACGTAGC	This study
	ef1	ACACTGCTCACATTGCTTGC	AGAAGCTCTCCACGCACATT	This study
	mcl1	CCTTCACGGGCATGCTACA	AGCGGGAACCTCAGAAAGTTT	This study
	odc	CATGGCATTCTCCCTGAAGT	TGGTCCCAAGGCTAAAGTTG	(Milet et al., 2013)
	xiap	TCACCTGCATTACCCAAGGA	ATCCGCACATACAGCACATG	This study

Table S2 .

 S2 Primers used for RT-qPCR in this study.

J. Dev. Biol. 2016, 4, 31; doi:10.3390/jdb4040031 www.mdpi.com/journal/jdb

Remerciements

Acknowledgements We thank Muriel Umbhauer, Phil Jones, Nancy Papalopulu, Muriel Perron, David Turner for gifts of materials; Sergio Marco for his advice on image processing and ImageJ macro writing; Simon Saule and Muriel Perron for help and advice; The authors greatly acknowledge the PICT-IBiSA@Orsay, Histology and Imaging Facilities, of the Institut Curie supported by the French National Research Agency through the "Investments for the Future" program (Fran-BioImaging, ANR-10-INSB-04); The "plateau technique de biologie moléculaire" of I2BC (Véronique Henriot) for construction of plasmids; Elodie Belloir, Virginie Dangles-Marie and Isabelle Grandjean for animal care. Marion Wassef and Claudio Stern for their comments on the manuscript. Paul Johnson for his editing work on the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS -UMR3347), the Institut National de la santé et de la recherche médicale (INSERM -U1021) l'Institut Curie (ETIC) and donors to B Durand project, l'Université Paris-Sud and the Fondation Pierre Gilles de Gennes (FPGG0039). The Centre National de la Recherche Scientifique supports B. Durand. E. Sena is supported by a fellowship from the "Ministère de la Recherche" and "Association pour la Recherche sur le Cancer" (DOC20170505923). Acknowledgements The authors greatly acknowledge the PICT-IBiSA@Orsay, Histology and Imaging Facilities, of the Institut Curie supported by the French National Research Agency through the "Investments for the Future" program (Fran-BioImaging, ANR-10-INSB-04), the "plateau technique de biologie moléculaire" of I2BC (Véronique Henriot) for construction of plasmids and Elodie Belloir, Virginie Dangles and Isabelle Grandjean for animal care. This work was supported by the Centre National de la Recherche Scientifique (CNRS -UMR3347), the Institut National de la santé et de la recherche médicale (INSERM -U1021) l'Institut Curie (ETIC) l'Université Paris-Sud and the Fondation Pierre Gilles de Gennes (FPGG0039). The Centre National de la Recherche Scientifique funds B.Durand. E. Sena is supported by a fellowship from the Acknowledgments: We thank Paul Johnson for his editing work on the manuscript and Elisabeth Schuster and Nathalie Rocques for technical assistance. B. Durand is funded by the Centre National de la Recherche Scientifique (Institut Curie-CNRS UMR3387, INSERM U1021), and the Fondation Pierre Gilles de Gennes (FPGG0039). E. Sena is supported by a fellowship from the "Ministère de la Recherche".

Supplementary Fig. 2: Co-IP experiments were performed on HEK293T cells protein extracts using an anti-Myc antibody. Total cell lysates and the immunoprecipitated complex were analyzed with WB analysis using anti-Myc, anti-Flag antibodies as indicated. Barhl2EHs coimmunoprecipitates with both Gro4 and Gro1 but exhibits a stronger affinity for Gro4. Supplementary Fig. 4: A scheme of the transgene Wnt reporter construct containing chicken b-globin insulators and seven Tcf consensus binding sites driving expression of a destabilized eGFP. Table S3. Secondary antibodies used in this study.

DAPI

Gro4-Flag Merge

Supplementary information

PAPER 2

The

Material and Methods

Xenopus embryos care and husbandry

X. laevis embryos were obtained by in vitro fertilization and staged using standard procedures (Nieuwkoop and Faber, 1994;Sive et al., 2010). Experimental procedures were specifically approved by the ethics committee of the Institut Curie (Authorization 2016-013 given by CEEA-IC #118) and are in compliance with the international guidelines (2010/63/UE). B Durand carries the Authorization for vertebrates' experimental use N°75-1548.

Plasmids

Pharmacological assays

Protein translation was inhibited using cycloheximide (Sigma-Aldrich C4859) at 100µg/ml.

mRNA synthesis, Antisense Morpholinos and Injections

Synthetic capped RNAs were prepared from pCS2+ derivatives with mMessage mMachine kit (Ambion). Antisense morpholinos either with no 5' capping, either coupled to fluorescein were made by Gene-Tools as previously published: MObarhl2 (Offner et al., 2005) independent experiments were performed and the results were pooled. A phenotype exhibited by at least 60% of the embryos was considered significant. For all rescue experiments, we tested a range of cRNA (50-500pg) and selected the minimal cRNA quantity that both induced the specific phenotype and displayed no toxicity. For rescue experiments the means between different data sets were compared using the Wilcoxon signed-rank test and significant differences are indicated with asterisks.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Total RNA from embryos was extracted using the Qiagen RNeasy Kit. RT-PCR analysis was carried out with the Superscript One-step Kit (Invitrogen) using specific primers resumed in Table S1. Histone 4 was used as control and water was used as a negative control.

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

For each time point RNA extract was obtained twice from 5 individual embryos obtained from the same fertilization. Embryos were dissociated by pipetting up-and-down with a 26G needle in Trizol (Invitrogen) and extracted according to manufacturer instructions. The RNA pellet was suspended in RNAse free water and kept at -80ºC. RNA samples were reverse-transcripted twice, using MMLV-RT enzyme (Promega). RT-qPCR was carried out using IQ SYBR green Supermix (BioRad) on a CFX96 touch Real-Time PCR Detection System (BioRad). RT-qPCRs were performed on embryos using standard procedures (39 cycles) using technical triplicates for each time point. Primers were designed using Primer 3 (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi/). Primers for were selected based on their amplification efficiency and specificity. Primers oligomeres used in RT-qPCR are resumed in Table S2.

Elongation Factor 1 (EF1) and Ornithine decarboxylase (ODC) primers were used as controls.

TUNEL staining

Whole-mount TUNEL staining was performed as previously described (Hensey and Gautier, 1998;Yeo and Gautier, 2003).

Immunostaining

Immunostaining was performed using either a rabbit polyclonal anti-Cleaved Caspase3 antibody, as described previously (Saka and Smith, 2001). The anti-Cleaved Caspase3 antibody was detected using anti-rabbit (Ig) antibodies conjugated to alkaline phosphatase (Roche), followed by a staining using NBT (4-Nitro blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3-indoyl-phosphate) substrates (Roche).

Whole mount In Situ Hybridization (ISH)

Control or injected embryos were collected at indicated stages. ISH were performed using digoxigenin labeled probes. Antisense probes were generated for XEpKer, Xmcl1, Xbcl-Xl and Xsox3 according to the manufacturer's instructions (RNA Labeling Mix, Roche). ISH was processed following protocol described elsewhere (Juraver-Geslin et al., 2014). Briefly, following overnight incubation with the probes and then with alkaline Phosphatase-conjugated anti-DIG enzymatic activity was revealed using NBT/BCIP substrates (Roche). Then, "Ministère de la Recherche" and "Association pour la Recherche sur le Cancer" (DOC20170505923). The results presented in this manuscript provide evidence that the evolutionarily conserved transcription factor Barhl2 limits the development of the Spemann organizer. In this territory Barhl2 inhibits Wnt activity via its interaction with the co-repressor Groucho and the transcription factor Tcf. It participates to the recruitment of the chromatin remodeling enzyme, Hdac1 that represses the expression of Spemann organizer genes. Using a Xenopus tropicalis Tcf reporter line we demonstrate that Barhl2 inhibitory effect on Groucho-Tcf activities is maintained during embryogenesis and plays a role in the confinement of neural progenitors in the brain. Together, our results provide a new and important mechanism for the control of Wnt transcriptional activity.

Supplementary Figures