Laure Et François

Frère Fabian

Ma Soeur Olivia

Dr Alessandra Pierrani

Diogo Le Dr

Sampaio E Castro D'

Romain Fontaine

Charles-Felix Le Dr

Calvo

Hessameh Le Dr

Hassani

Hiroko Le Dr

Marie-Stephane Nakatani

Hatem Aigrot

Corentine Hmidan

Magalie Marie

Frah

Katsuhiko Ono

Adrien Clavairoly

Tadashi Nomura

Hitoshi Gotoh

Aoi Uno

Olivier Armant

Hirohide Takebayashi

Qi Zhang

Kenji Shimamura

Shigeyoshi Itohara

Carlos M Parras

Kazuhiro Ikenaka

Dédicace A mes parents

Keywords: Dorsal thalamus, Thalamic eminence, EphA3, Microarray, In situ hybridization, Mouse

ont construit.

Table of Contents

PREFACE

The question of the embryonic development has always questioned the curious minds. Aristotle in his book -On the Generation of Animals‖ was already developing a theory of -epigenesis‖ based on the observation of developing chick eggs. As opposed to preformationist views that lived long until the 18 th century, Aristotle defined epigenesis as the progressive formation of the organism as being not fully formed at the beginning of embryonic development. In 1768 Caspar Friedrich Wolff was the first to observe that the embryo was constructed in -leaf-like layers‖ and it is Christian Heinrich Pander in 1817 that described for the first time the fate of the 3 germ layers of the embryo, the endorderm, the mesoderm and endoderm. Pionneer of the germ layer theory, he wrote in 1817 -A unique metamorphosis begins in each of these three layers, and each hurries toward its goal; although each is not yet independent enough to indicate what it truly is; it still needs its sister travellers, and therefore, although already designated for different ends, all three influence each other collectively‖ [START_REF] Pander | Induction of human neuronal cells by defined transcription factors[END_REF]. A few year later, Rudolf Virchow developed the -cellular theory‖ affirming that -Omnis cellula e cellula‖ (Virchow 1858), a statement still standing despite numerous efforts [START_REF] Miller | A production of amino acids under possible primitive earth conditions[END_REF], Gibson DG 2014;Keber F C 2014) indeed, today cells are still generated exclusively from cells by successive division. One year later, in 1859, Charles Darwin writes in "The Origin of Species by Means of Natural Selection (…)‖: -I should infer from analogy that probably all the organic beings which have ever lived on this earth have descended from some one primordial form, into which life was first breathed (Darwin C., "The Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle For Life", (1859) p. 490). Darwin's theory lead today to the -modern evolutionary synthesis‖ [START_REF] Huxley | Evolution: The Modern Synthesis[END_REF] and allowed the rise of modern genetics and evolutionary developmental biology (The Structure of Evolutionary Theory Harvard University Press, Cambridge, Gould MA 2002).

In 1908, Alexander Maksimov is the first to propose the term "stem cell" as cells able to maintain an undifferentiated state (self renewal) as well as to differentiate. The term -embryonic stem cell‖ giving rise to the three germ layers (potency) was later coined by Gail R. Martin who developped the technics to extract stem cell from the mouse blastocyst (Gail R. [START_REF] Martin | Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[END_REF]. During the process of differentiation, cells become restricted progressively from totipotency (3 germ layers ability), to pluripotency (3 germ layers but primed), multipotency (a subset of the 3 germ layers) or unipotency (only one germ layer). Carcinogenic and stem cells, able to self-renew but unable to differentiate, were later called nullipotent. In 1957, Conrad Hal Waddington published, the -epigenetic landscape‖ model depicting how a cell cell progresses during development to generate all the alternative cell fates necessary to the formation of the whole organism. Waddington depicted cell fate choices as a ball rolling down to different valleys in a three dimensional landscape defined by the X-axis as the concentration of each molecules in the cell, the Y-axis as time and the Z-axis representing the system being monostable, bistable and then multistable (Figure 1). (Waddington, C.H. 1957) where cells are depicted as a ball rolling down to a valley. New valleys represent different cell fates, and ridges maintain cells in particular epigenetic state. Landscape can be monostable, bi-stable or multistable. The solid black lines represent stable steady states, the dashed lines represent unstable steady states, and in red the bifurcations. B. Original drawing from Waddington showin correlation between the height of the ridges and combinatorial gene expression. C. Schématic representic the progressive cell fate restriction occurring during neural development. D. Mordern mathematical representation of the epigenetic landscape. Oscillatory gene expressions are generated by positive or negative feedback in the regulation network (red and black arrows) which lead to cell differentiation, a process driven by positive feedbacks stabilizing -irreversible‖ stochastic events (adapted from S Newman 2012).

After a succession of identity crises, cells can -abruptly‖ shift from one fate to another and become -canalized‖ in a particular fate -buffering‖ subsequent genetic and environmental variations to maintain their identity. Waddington also emphasizes in his drawing the direct relationship existing between the ridges of the valley and specific gene expression of individual genes that cooperate to form what he called -Creodes‖, the different pathways followed by the cell during the process of differentiation [START_REF] Waddington | The strategy of the genes; a discussion of some aspects of theoretical biology[END_REF]. Aware of the complexity of the regulations in living systems, making the cell behaviour and evolution -quasi finalistic‖ (Waddington 1962a, 88), Waddington introduced the term epigenetics as -the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being.‖ (Waddington CH. Towards a Theoretical Biology. Edinburgh, Scotland: Edinburgh University Press; 1968. The Basic Ideas of Biology).The concept of Epigenetics evolved with the emergence of modern biology and is defined today as -the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence (Wu Ct, Morris JR Science. 2001).

Waddington also defined -Attractors‖ describing the mechanism that restrict the cellular states to certain region of the three dimensional space and which allow perturbations to be bufferd. Waddington was really a pioneer in defining his model using references to thermodynamics. In 1877, Boltzman defined Entropy as a measure of -molecular disorder", Entropy of closed system being by definition always increasing toward a point of maximum entropy that defines the state of equilibrium of the system. However, cells and organisms are open systems exchanging matter, energy and information with their environnement. In 1977, Ilya Prigogine characterized the belousov-zhabotinsky reaction as a -dissipative Systems‖ which is an ensemble of nonlinear chemical reactions passing by phase of increasing and decreasing entropy and able to form oscillating pattern. Such life mimicking reactions are maintained out-ofequilibrium and can generate self-organization, complex pattern formation and increase the level of order of the system upon time thanks to continuous metabolic transformation of energy and information [START_REF] Prigogine | Self-Organization in Nonequilibrium Systems: From Dissipative Structures to Order through Fluctuations[END_REF]. Dissipative reaction does not last infinitly. As only the intial and final equilibrium states are important to measure entropy, the second law of thermodynamics is preserved. Production of entropy (from catabolic processes) is balanced by the Production of structure (from anabolic processes) or a change of the system as a whole called phase transitions, lowering the entropy of the system. In cells, while steady-state is achieved when the net change in entropy is close to zero, growth implies a net decrease in entropy for the system. In both cases, a production of entropy towards the environnement occurs to preserve the second law. Prigogine writes in -Order out of chaos‖ -We grow in direct proportion to the amount of chaos we can sustain and dissipate‖ (Ilya Prigogine, Order Out of Chaos: Man's New Dialogue with Nature).

Francisco Varela then developed the central concept of -autopoiesis‖ (selfaction) to describe caracteristics of living systems. He states that -An autopoietic machine is a network of processes that produces and degrades their own components which through their interactions continuously maintain the network of processes that produced them as a concrete unit in space. (Maturana and Varela On Machines and Living Beings, 1972). This model of self-organization highlight the fundamental role of frontiers in biology [START_REF] Mulkidjanian | Co-evolution of primordial membranes and membrane proteins[END_REF] , the notion of homeostasis (dynamic maintenance of internal environment ), metabolism (reactions that create and break down molecules), but also emphasized the fact that a system is made of a network of non-linear (positive and negative feedback) processes from which -emerge‖ the necessary functions used for its self-maintenance and organization. It is important to note that all living organism are autopoietic but not all autopoietic system are alive (for example economic systems or chemical dissipative reactions). To propose a possible definition of living systems, we could describe it as a non-linear autopoietic and dissipative system that (once) emerged from physical properties of matter and evolved since by means of natural selection.

Many studies showed evidence in support of the epigenetic landcape model. It is now recognized that most of the genes exhibit a low expression level in stem cells but are maintained in a chromatin state ready for subsequent activation upon activation (T.I. Lee, R.G. Jenner2006). Moreover, genes whose changes in expressions levels are correlated have greater probability to physically interact and increase the level of order of the cell [START_REF] Jansen | Relating whole-genome expression data with proteinprotein interactions[END_REF]. However, it is only recently that the epigenetic landscape model validity was formally demonstrated in a study covering 800 different cell types of all differentiation stages where gene expression profiles and inferred global protein network interactions were under analysis. In the -epigenetic landscape‖ paradigm, pluripotent stem cells have high entropy and low level ordering while cell differentiation lead to a decrease of entropy through progressive ordering. This study shows using a quantitative measure of -signaling entropy‖ that this parameter is sufficient to discriminate embryonic stem cell from cancer cell and differentiated cell.

However, this study failed to discriminate between more differentiated cell types (R. Banerji, D. Miranda-Saavedra 2013).

Since Waddington proposed his model, many studies have used is to describe developmental processes. However, the number of cell type and cell type transitions that exist is impossible to describe using only 3 dimensions. Moreover, a paradigm shift came with the demonstration that transdifferentiation, the conversion of cell types in different lineages, was possible. Indeed, exogenous expression of the transcription factor MyoD was shown to induce muscle-specific (mesodermal) properties in fibroblasts (ectodermal) [START_REF] Davis | Expression of a single transfected cDNA converts fibroblasts to myoblasts[END_REF]. After the pioneer work from John Gurdon who showed that cloning of a somatic nucleus into an enucleated egg was able to generate a complete organism [START_REF] Gurdon | On the origin and persistence of a cytoplasmic state inducing nuclear DNA synthesis in frogs' eggs[END_REF], cell fusion induced cell-fate reprogramming to pluripotent stem cells became possible [START_REF] Gurdon | From nuclear transfer to nuclear reprogramming: the reversal of cell differentiation[END_REF]. More recently, the revolutionary work of Yamanaka's group demonstrated that overexpression of only four transcription factors, Oct4, Sox2, Klf4, and c-Myc in adult fibroblasts, was sufficient to reprogram differentiated cells into induced pluripotent cells (iPSCs ) [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF]. -Direct‖ reprogramming of adult somatic cells into alternative cell types then became possible after the discovery that three neuronal transcription factors, Ascl1, Brn2, and Myt1l (BAM factors) were able to convert cells originating from the same (ectodermic fibroblast) or different (endodermic hepatocytes) germ layer, into functional neurons called induced neuronal (iN) cells [START_REF] Vierbuchen | Direct conversion of fibroblasts to functional neurons by defined factors[END_REF]Maro s 2011). This ground breaking discovery showed that a small number of -master regulators‖ are able to shift the expression of hundreds of genes and convert directly a cell type into another without passing by a pluripotent stage or a cell division. It is now of primary interest to understand how those cell fate change are driven at an epigenetic level and to describe precisely the

INTRODUCTION PART I: THEORETICAL BACKGROUND

Conrad Waddington had a really rich scientific carreer. He published in paleonthology, genetics, biochemistry, population genetics and embryology. In 1934, he demonstrated that the grafting of a duck -organizer‖ (the hensen's node) into a chick or a rabbit embryo at the blastoderm stage was able to induce a full sond second body axis formation [START_REF] Waddington | Experiments on embryonic induction. I. The competence of the extraembryonic ectoderm in the chick. II. Experiments on coagulated organisers in the chick. III. A note on induction by chick primitive streak transplanted to the rabbit embryo[END_REF]; Jonathan M. W. Slack 2002). As the secondary axis was containing many tissue types, Waddington understood that this -organizer effect‖ was not arising from a single molecule and more interestingly that the signals were conserved between species. He employed the word -evocation‖ as the priming of the future fate choice and the word -individuation‖ as the production of different cell responses induced by an -evocator‖. During years, Waddington tried to find the neural-inducing activity of the node and thought to find it when he purified steroids from the embryo [START_REF] Waddington | Studies on the nature of the amphibian organization centre II. Induction by synthetic polycyclic hydrocarbons[END_REF]. However in the 1930s, biochemists had no chance at all of purifying -this evocator‖and we had to wait the 1990s to finally identify the fibroblast growth factor (FGF) and transforming growth factor-β (TGFβ) families only present at picomolar concentratrion in early embryos (Slack, J. M. W.1994;[START_REF] Baedke | The epigenetic landscape in the course of time: Conrad Hal Waddington's methodological impact on the life sciences[END_REF]). Two different mechanisms were identified for creating spatially restricted patterns in living systems. The first one, proposed by Alan Turing in 1952 in -The chemical basis of morphogenesis‖ defines -morphogenes‖ as diffusing molecules able to drive morphogenetic changes (Turing A 1952). He developed a model now called -diffusion-driven instability‖ where a competition betwen a slow diffusing activator and a faster diffusing inhibitor generate periodic patterns. Even if this phenomenon was biologicaly demonstrated only recently [START_REF] Maini | Turing's model for biological pattern formation and the robustness problem[END_REF][START_REF] Marcon | Turing patterns in development: what about the horse part?[END_REF]), Turing's work introduced algorithmic and booelan logic in biology [START_REF] Davidson | Emerging properties of animal gene regulatory networks[END_REF]) and thus paved the way for modern computational model of gene regulatory network occurring during development (Corominas-Murtra, 201;Alexander Spirov2013). The second model under analysis was -the french flag gradient model‖, a system of diffusible molecule which, produced asymmetrically, creates a spatial concentration gradient. Each position along this gradient receives different concentrations of signal and gives cells access to positional information allowing them to trigger discrete response depending on their location [START_REF] Wolpert | Positional information and the spatial pattern of cellular differentiation[END_REF]. This model was later on demonstrated and refined for sonic hedgehog signaling thanks to a quantitative correlation between Shh morphogen concentration and intracellular gradual Gli signaling responses [START_REF] Dessaud | Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism[END_REF].

Life seems to be more than complex chemistry. Indeed, its unique informational management properties and modular functionality seem to be its specificity. Embryonic development is accomplished by a dynamic regulation of gene expression that lead to growth and differentiation (Bruneau, 2008;Kang et al., 2011;SiTayeb et al., 2010).

Striking differences in epigenetic landscape exist between normal and pathological embryonic, adult and cultured cells (Gifford et al., 2013;[START_REF] Heintzman | Histone modifications at human enhancers reflect global cell-type-specific gene expression[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF]Stergachis et al., 2013;Zhu et al., 2013;Ziller et al., 2013). Identifying the temporal dynamics of the genetic regulatory landscapes driving normal and pathological biological processes is thus of primary interest.

CHAPTER I: GENOME REGULATION AND NUCLEAR ORGANIZATION

General consideration on genome structure and conservation

In human, only 1.5% of the 3,38.10 9 bp of the genome serves as a template for the 20389 genes identified so far [START_REF] Lander | Initial sequencing and analysis of the human genome[END_REF]Ensembl GRCh38). The remaining proportion of the genome, mainly composed of repetitive sequences of viral origins (45%), intronic sequences (25%), intergenic sequences (~25%) and centromeric/telomeric sequences (~5%) were long thought to be -junk DNA‖ [START_REF] Ehret | Origin, development, and maturation of organelles and organelle systems of the cell surface in Paramecium[END_REF][START_REF] Ohno | So much "junk" DNA in our genome[END_REF]Venter, J.C 2001;Mandal PK, 2008). Mouse models have been used extensively to improve our understanding of mammalian development.

With their 2.7.10 9 bp and 99% of genes having human orthologs, mice played a great role in both the annotation and the study of gene function and regulation [START_REF] Buecker | Enhancers as information integration hubs in development: lessons from genomics[END_REF]. It is now well recognized that an important part of the non protein coding part of the eukaryotic genome is actually essential for the control of gene expression [START_REF] Nord | Rapid and Pervasive Changes in Genome-wide Enhancer Usage during Mammalian Development[END_REF]. Indeed, the genome is now known to undergo -pervasive transcription‖ (P Kapranov2007, MB Clark 2011) and despite a very low abundance for the majority of the RNA molecules present in a cell, around 75% of the genome is found to be transcribed in human cells in average [START_REF] Cheng | Transcriptional Maps of 10 Human Chromosomes at 5-Nucleotide Resolution[END_REF][START_REF] Manak | Biological function of unannotated transcription during the early development of Drosophila melanogaster[END_REF]The ENCODE Project Consortium 2007;[START_REF] Kapranov | RNA maps reveal new RNA classes and a possible function for pervasive transcription[END_REF][START_REF] Efroni | Global transcription in pluripotent embryonic stem cells[END_REF]Clark MB et al. 2011). Nontheless, low expression of many non-protein coding RNA have tremendous effects in many developmental process and are frequently disregulated in diseases [START_REF] Esteller | Non-coding RNAs in human disease[END_REF]. While recent comparative analysis showed that only 8.2% of the human genome is under purifying selection and thus phylogenetically conserved in mammals (Rands CM 2014), 80 % of the human genome is said to be functional, as defined by the ENCODE project by the participation of the genomic sequence under investigation in at least one biochemical RNA and/or DNA event in at least one cell type (ENCODE project consortium 2011;Dunham I 2012). However this definition of functionality is pretty loose as transcription or replication of a genomic element does not imply its functionality (D [START_REF] Graur | On the Immortality of Television Sets: -Function‖ in the Human Genome According to the Evolution-Free Gospel of ENCODE[END_REF]. A high level of sequence conservation is frequently used as evidence of functionality [START_REF] Pennacchio | Genomic strategies to identify mammalian regulatory sequences[END_REF]. However, transcription regulatory regions evolve faster than protein-coding regions and play a key role in evolution of species and specialization of organs, such as the brain (Clarke SL, 2012). Indeed, 11% and 40% of the human regulatory regions emerged since the rhesus or mouse divergence, respectively (J Cotney 2013). Moreover, conservation of regulatory regions is tissue and developmental stage dependent [START_REF] Nord | Rapid and Pervasive Changes in Genome-wide Enhancer Usage during Mammalian Development[END_REF]. Interestingly, while a rapid turnover of regulatory DNA sequences occurs during evolution [START_REF] Schmidt | Five-Vertebrate ChIP-seq Reveals the Evolutionary Dynamics of Transcription Factor Binding[END_REF][START_REF] Villar | Evolution of transcription factor binding in metazoansmechanisms and functional implications[END_REF], many studies showed a high functional conservation of these regulatory regions (Arnold CD 2014;[START_REF] Cotney | The Evolution of Lineage-Specific Regulatory Activities in the Human Embryonic Limb[END_REF]. Thus, sequence conservation and functional conservation are not strictly correlated. Nonetheless, strong regulatory functions are most of the time associated with strong purifying selection (Q. He, A.F. Bardet, B., 2011).Thus, despite usefull correlation between biological functionality and phylogenetic conservation substantial work remains to be done to understand the regulatory logic of transcription operating during development and evolution.

Basal transcription machinery and core promoter elements

Transcription is the mechanism by which genes are converted into mRNA before translation into proteins. This process needs to be tightly controlled to achieve a proper development and maintenance of the organism. Transcription of protein-coding genes and most non-coding RNA genes is driven by RNA polymerase II (pol II).

Transcription involves three steps: Initiation, the recruitment of the polymerase to the gene promoter; Elongation, the synthesis of the RNA, and Termination, the dissociation of the RNA from DNA and the transcription machinery [START_REF] Shandilya | The transcription cycle in eukaryotes: From productive initiation to RNA polymerase II recycling[END_REF]. Pol II is part of molecular complex known as the pre-initiation complex (PIC) composed of the general initiation factors, pol II and the mediator complex (Hahn, 2004;[START_REF] Kornberg | Mediator and the mechanism of transcriptional activation[END_REF]. The general initiation transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH are the minimum factors required to drive pol II binding to the -core promoter‖, the proximal cis-regulatory element (CRE) to initiate transcription (Thomas chiang, 2006). The mediator is a ubiquitous complex composed of 33 subunits required to link the core promoter with their proximal and distant cisregulatory element known as enhancers or silencers [START_REF] Takagi | Mediator as a general transcription factor[END_REF][START_REF] Poss | The Mediator complex and transcription regulation[END_REF][START_REF] Malik | The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation[END_REF][START_REF] Borggrefe | Interactions between subunits of the Mediator complex with gene-specific transcription factors[END_REF]. Core promoters are positioned between -50 and +50 relative to the +1 transcription start site (TSS) of genes and arecomposed of 4 obligatory consensus motifs: An upstream TFIIB responsive element (BREu; SSRCGCC), the TATA box consensus (TATAWAWR), the downstream BRE (BREd; RTDKKKK) and the initiator Inr (YYA+1NWYY). Additional sites were identified more recently known as the DPE (downstream promoter element), the MTE (motif ten element), the Motif 1/6 elements, the DRE (DNA replication-relatedelement), the DCE (downstream core element), the XCPE (X core promoter element), the TCE (translational control element), and the PB (the pause button) (Ohler, U.2010).

As about 160,000 PICs are detected genome-wide, the complexity of core promoter elements makes bioinformatics inference usefull but inadequate for in-vivo inference [START_REF] Müller | Chromatin and DNA sequences in defining promoters for transcription initiation[END_REF]. Accurate genome wide in-vivo identification of TSS in different cell types are now possible based on analysis of high-throughput sequencing (U. Ohler, D.A. Wassarman, 2010;B. Lenhard, A. Sandelin, 2010;[START_REF] Batut | High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression[END_REF].

Core promoters are divided in two categories: -narrow promoters‖, where Pol II transcription initiates at at a unique TSS, and -broad promoters‖ where Pol II can initiate transcription at multiple TSS (A. [START_REF] Sandelin | Mammalian RNA polymerase II core promoters: insights from genome-wide studies[END_REF]. Genes with -narrow promoters‖ have low GC-content and mainly correspond to tissue-specific genes, while genes with -broad promoters‖ have high GC content and are associated with 20 developmentally regulated or housekeeping genes (B. Lenhard, A. Sandelin, P 2010).

A former classification discriminating TATA-containing and TATA-less promoters (S.T. Smale,1997) should be abandoned. Indeed, precise analysis of TATA-less promoters demonstrated the existence of a TATA-like motif in all promoters (HS Rhee, 2012).

Transcription initiation, elongation and termination and nuclear export

After formation of the PIC, the polymerase initiate RNA synthesis in a process called elongation. Briefly, the phosphorylation of serine residues in the carboxyterminal repeat domain (CTD) of Pol II by TFIIH or P-TEFb triggers elongation [START_REF] Zhou | RNA Polymerase II Elongation Control[END_REF]. However, Pol II elongates inefficiently and often pauses within the first 100 nucleotides. Pol II pausing is caused by the association of DSIF [START_REF] Wada | DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs[END_REF], NELF [START_REF] Yamaguchi | NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation[END_REF]) and GRINL1A [START_REF] Cheng | Functional Association of Gdown1 with RNA Polymerase II Poised on Human Genes[END_REF]. Pausing of Pol II is a way to generate an accessible promoter region that can be quickly bound by activators and co-activators (TFIIH or P-TEFb) to trigger elongation. The open promoter associated with the GTFs remains after Pol II escapes and allows rapid entry of new PIC [START_REF] Yudkovsky | A transcription reinitiation intermediate that is stabilized by activator[END_REF]. Paused Pol II has been observed at genes that are rapidly induced or genes involved in early embryonic development [START_REF] Aida | Transcriptional pausing caused by NELF plays a dual role in regulating immediateearly expression of the junB gene[END_REF]Adelman, K. et al.2009). However, pausing seems to be a general mechanism as it was also observed in constitutively expressed genes [START_REF] Gilchrist | Regulating the regulators: the pervasive effects of Pol II pausing on stimulusresponsive gene networks[END_REF]. Transcription occurs in bursts, where a gene is transcribed for a short period followed by a period of inactivity (JM, O'Shea EK. 2005). Different groups of genes can be divided depending on their bursting activity [START_REF] Raj | Nature, nurture, or chance: stochastic gene expression and its consequences[END_REF]. Transcription termination, the process by which mRNA is released after synthesis, is coupled with RNA poly(A) and capping maturation [START_REF] Kuehner | Unravelling the means to an end: RNA polymerase II transcription termination[END_REF]. Most of the mRNAs have a highly conserved poly (A) signal (5′-AAUAAA-3′ followed by a G/U-rich sequence). Once the poly (A) signal is transcribed, Pol II stops. The endoribonucleolytic cleavage of the nascent transcript occurs and is followed by polyadenylation of the mRNA thanks to the recruitment of the polyadenylation specificity factor (CPSF), the cleavage stimulatory factor (CstF) and the Poly (A) polymerase on the carboxy-terminal domain (CTD) of the Pol II subunit Rpb1. (J.N. [START_REF] Kuehner | Unravelling the means to an end: RNA polymerase II transcription termination[END_REF]. Maturation of the pre-mature mRNA is a cotranscriptional process that occurs concomitantly with alternative splicing before export in the cytoplasm for translation into protein. Maturation stabilizes RNA molecules against exosomal or alternative RNA degradation pathways [START_REF] Chlebowski | RNA decay machines: the exosome[END_REF]. Disruption of the RNA degradation pathways reveals the phenomenon of pervasive transcription and highlights the importance of the degradation machinery in the control of gene expression [START_REF] Jacquier | The complex eukaryotic transcriptome: unexpected pervasive transcription and novel small RNAs[END_REF]. Recent studies estimate that in human, up to 94% of multi-exon genes undergo alternative splicing [START_REF] Pan | Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing[END_REF]Wang ET, Sandberg R et al. 2008) a phenomenon shown to underly the developmental complexity of the organism (L Chen SJ. Bush 2014). Finally, mature RNAs are exported via the nuclear pore complex thanks to receptors that directly recognize the RNA or interact with the mRNA ribonucleoprotein (mRNP) complex (Adams RL 2013). This level of RNA regulation, together with cytoplasmic P-bodies (involved in RNA degradation and strorage) as well as mechanism of riboswitch (temporary inactivation for local translation) explain the discrepancy between transcriptome and proteome. Indeed, in mice, mRNA levels explain only 40% of the variability in protein levels (Schwanhäusser 2011;[START_REF] Ghazalpour | Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages[END_REF].

The looping model of promoter-enhancer gene activation

Enhancers are proximal or distal cis-regulatory DNA sequences, commonly 20-500 bp in length, containing clustered transcription factor binding sites (TFBS) and representing 11% of the genome [START_REF] Spitz | Transcription factors: from enhancer binding to developmental control[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF]).More likely associated with developmentally regulated genes [START_REF] Levine | Transcriptional enhancers in animal development and evolution[END_REF], enhancers are responsible for the tissue specificity of gene expression (Bernstein et al., 2012;[START_REF] Ernst | Mapping and analysis of chromatin state dynamics in nine human cell types[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF][START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF]. Strikingly, enhancers are modular and work in an additive way to drive specific spatially restricted expression patterns in vivo [START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF][START_REF] Visel | A high-resolution enhancer atlas of the developing telencephalon[END_REF] independently on their location (up or downstream of the promoter) or their orientation compared to their related promoter [START_REF] Banerji | Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences[END_REF]. Intensive work allowed significant progress to identify these elements in mammalian genomes (Bernstein et al., 2012;[START_REF] Ernst | Mapping and analysis of chromatin state dynamics in nine human cell types[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF]. The major model of promoter-enhancer interaction driving gene activation is the ‗‗ looping model''where multiple enhancers (4-5 enhancer per promoter in average) activate transcription by delivering activating factors to the promoter to increase the probability of activation of the preinitiation complex as well as the probability of transition from initiation to elongation during transcription [START_REF] Bulger | Functional and mechanistic diversity of distal transcription enhancers[END_REF][START_REF] Ghavi-Helm | Analyzing transcription factor occupancy during embryo development using ChIP-seq[END_REF]Shen 2010 ). This model was higly validated by recent studies (Ong andCorces, 2011, 2012) and the emergence of technologies such as chromosome conformation capture and their derivatives (Wit and de Laat, 2012; [START_REF] Gibcus | The hierarchy of the 3D genome[END_REF], allowed the identification of the chromatin features associated with enhancers.

Interestingly, gene expression from a particular promoter is positively correlated with the number of enhancers contacting it. Promoters can be enhancers for different genes and enhancer can regulate different genes. Promoter-enhancer long range interactions display striking asymmetry with a bias for interactions with elements located ~120 Kb upstream of the TSS. Moreover, 57% of the interactions span >100 kb with a median distance of 124 kb and only ~7% occur with the nearest gene, suggesting that genomic proximity is not a good predictor for long-range interactions inference ( Sanyal A, Lajoie BR 2012;Fulai Jin, et al., & Bing Ren. Nature 13 Nov 2013). The ENCODE project which is in charge of only 1% of the genome already revealed 400,000 distinct enhancers mapped in a set of human cell lines [START_REF] Buecker | Enhancers as information integration hubs in development: lessons from genomics[END_REF]Maston1 et al., 2012). Current estimate compiling bioinformatics predictions and experimental inferences of enhancers in the human genome reach many millions (Dunham et al., 2012). High throughput in-vivo transgenic validations were recently done in mouse embryos and comprehensive enhancer databases are currently under construction (Vista Enhancer Browser http://enhancer.lbl.gov). Nuclear organization and chromosome territories. Schematic showing chromosomes occupying defined nuclear territory with little overlap with others. Despite some transregulation (inter-chromosomic), the majority of long range interaction are cis-regulatory events (intra-chromosomic). Chromatin folding generates globules of nucleosomal DNA that are organized in topologically associating domains (TADs) and SubTADs with different size and shapes. E-F. High resolution visualization of chromatin in an interphase mammalian nuclei. Chromosomes are compartmentalized and are formed of fractal successive ordering of 10 nm chromatin fibers (Mirny et al 2011). G. Subnuclear organization. Schematic representation of the nuclear bodies (adapted from YS Mao 2011). H. Chromatin regulation. Chromatin types were recently divided in five colors: Yellow (active and ubiquitously expressed genes; ~15% of the genome), Red (active and tissue specific genes; ~10%), Blue (developmentally regulated gene; ~20%), Green (heterochromatic but active genes; ~5%) and Black (heterochromatic silent genes; ~50% of the genome). Silent chromatin is characterized by lamina associated domains (LADs) which are large (0.1-10 Mb) silent domains located in periphery of the nucleus. (adapted from B van Steensel 2011).

Chromatin structure and gene regulation

Chromatin is formed of nucleosomes wrapping 147 bp of DNA around an octamer of histone (two molecules each of histone H2A, H2B, H3 and H4) in a highly stable unit that is maintained by over 120 protein-DNA interactions (Luger K, Mäder AW 1997). Chromatin is a -bead-on-a-string‖ structure with 20-60 bp long DNA linkers connecting the nucleosomes together (Grosberg andKhokhlov 1994. Routh et al. 2008) .While the volume of DNA of a human diploid genome is around 6.4 mm 3 and would occupy 5.7% of the nuclear volume, the DNA wrapped around nucleosomes occupy 20.6 mm 3 which represent 18.2% of the nuclear volume. Thus, chromatin does not compact DNA [START_REF] Fussner | Living without 30 nm chromatin fibers[END_REF]. Rather, chromatin is a highly dynamic packaging of chromosomes which allow a tight control of gene expression, DNA replication and DNA repair [START_REF] Bickmore | Genome architecture: domain organization of interphase chromosomes[END_REF]. Early electron microscopy studies showed that nucleosomes formed repetitive coiled structures of ∼30nm diameter called solenoids (JD McGhee, JM Nickol, 1983;Horowitz, R.A. et al. 1994). However, recent studies in unfixed interphase nuclei or mitotic chromosomes failed to detect solenoids in-vivo [START_REF] Maeshima | Chromatin structure: does the 30-nm fibre exist in vivo?[END_REF][START_REF] Fussner | Living without 30 nm chromatin fibers[END_REF]. Rather, most nuclei show irregular patches of local compaction consisting of globules of nucleosomes which aggregations are facilitated for example by the linker histone H1 or the Polycomb Group (PcG) protein complexes (Francis NJ, Kingston RE et al, 2004;[START_REF] Hizume | Linker histone H1 per se can induce three-dimensional folding of chromatin fiber[END_REF]. Moreover, the early distinction between -compacted and inactive heterochromatin‖ and -open and active euchromatin‖ is now recognized as an oversimplification. Indeed, accessibility of chromatin was measured in-vitro by exposing detergent-permeabilized nuclei to endonucleases DNase I and quantitative mapping of the cutting sites; the cutting frequency being thought to be proportional to the accessibility of the DNA [START_REF] Weintraub | Chromosomal subunits in active genes have an altered conformation[END_REF]. However, more recent measures of DNA accessibility by an invivo mapping of DNA methylation by Dam methyltransferase that marks any accessible GATC sequences with an adenine tag that does not occur endogenously, established that most of the genome is actually accessible (Sha K, Gu SG, 2010). Indeed, other studies showed no differences in DNA accessibility for molecules below a 10 nm (1MDa) size [START_REF] Verschure | Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules[END_REF]Gorisch et al, 2005;[START_REF] Pack | Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs[END_REF]. Thus, changes in chromatin organization do not control the binding of proteins to DNA through changes in DNA accessibility. Rather, different chromatin types may act as guides to favor targeting of specific DNA regions [START_REF] Van Steensel | Chromatin: constructing the big picture[END_REF]. Indeed, the mapping of more than 100 chromatin associated proteins divided the drosophila chromatin into five main types, each chromatin type being specifically bound by specific proteins (Filion GJ, van Bemmel JG 2010;van Bemmel JG 2013). Chromatin types were divided as follow: Yellow (active and ubiquitously expressed genes; ~15% of the genome), Red (active and tissue specific genes; ~10%), Blue (developmentally regulated gene; ~20%), Green (heterochromatic but active genes; ~5%) and Black (heterochromatic silent genes; ~50% of the genome). In human or mouse, similar chromatin types (4-5 types and 51 subtypes) were also described based on the study of post translational epigenetic marks of histone proteins [START_REF] Ernst | Discovery and characterization of chromatin states for systematic annotation of the human genome[END_REF]Oren Ram goren A 2011 ). The BLACK chromatin drives gene silencing thanks to histone H1 which stabilize nucleosome positions and decrease histone turnover [START_REF] Laybourn | Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II[END_REF]Kadonaga, 1991, Woodcock et al., 2006) as well as by lamina anchoring of the chromatin to the internal nuclear membrane [START_REF] Pickersgill | Characterization of the Drosophila melanogaster genome at the nuclear lamina[END_REF][START_REF] Towbin | The nuclear envelope--a scaffold for silencing?[END_REF]. Silent chromatin is characterized by lamina associated domains (LADs) which are large (0.1-10 Mb) silent domains located in periphery of the nucleus (L. Guelen, L. Pagie, 2008). The recent development of chromosome conformation capture techniques (3C, 4C, 5C and Hi-C) unraveled the 3D organization of chromatin at high resolution and enabled the modelization of genome-wide interactions map independently of any DNA binding factor (E. de Wit, W. de Laat 2012). Briefly, all these techniques uses formaldehyde fixation to cross-link genomic regions that interact in vivo, the sequencing of the ligation products of interacting DNA regions and reconstruction of an interaction map (E. Lieberman-Aiden 2009). ChIA-PET, a variant technique, includes an additional immuno-precipitation step that allows modelization of the interaction map for a specific TF (Jingyao Zhang, Huay Mei Poh 2012). These techniques identified in chromosomes, large discrete interacting domains called topologically associating domains (TADs) which have an average size of ~900kb in mammals [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF]. Mammalian genomes are composed of ~2,000 TADs, covering 90% of the genome. Regions located within TADs are shown to be much more likely to interact with each other than regions being in different TADs (E. [START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]. Long range interaction between promoter and cis-regulatory elements such as enhancers are particularly frequent within TADs [START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF].

Moreover, analysis of boundary regions between TADs showed enrichment for CTCF protein, although CTCF also frequently binds sites within TADs and does not block physical long-range interactions (Sanyal A, Lajoie BR 2012). Modelization of the mammalian chromatin demonstrated a -fractal globule‖ organization where nucleosomes are organized in knot-free globules that crumple together successively (as in fractal motifs) to form higher order globules (A. Y. Grosberg, S. K. Nechaev, E. I. 1998;[START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]). Hi-C analyses of mammalian genomes ( [START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF][START_REF] Kalhor | Genome architectures revealed by tethered chromosome conformation capture and population-based modeling[END_REF]Zhang R.P. McCord et al., 2012), showed that chromosomes largely occupy individual territories where the captured long-range DNA interactions are mainly in cis (intra-chromosomic) within TADs [START_REF] Simonis | Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C)[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]Hou et al., 2012;[START_REF] Kalhor | Genome architectures revealed by tethered chromosome conformation capture and population-based modeling[END_REF]Sexton et al., 2012). Despite this territorial organization, interactions in trans (inter-chromosomic) occur at the boundaries between chromosomes and through infiltration of looped-out genomic region into the territories of another chromosome [START_REF] Branco | Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations[END_REF]. This spatial organization allow the formation of -transcription factories‖ (concept first proposed to describe the clustering of transcription factors as discrete foci in the nucleus) comprising interacting chromatin regions, clustered transcription machinery and high concentration of TFs, RNA maturation and splicing machineries [START_REF] Jackson | Visualization of focal sites of transcription within human nuclei[END_REF][START_REF] Melnik | The proteomes of transcription factories containing RNA polymerases I, II or III[END_REF][START_REF] Edelman | Transcription factories: genetic programming in three dimensions[END_REF]. A well known example of transcription factory is the nucleolus which produces rRNA from 2000 clustered rRNA copies dispersed in five different chromosomes that are recruited to the nucleolus to be cotranscribed [START_REF] Hernandez-Verdun | The nucleolus: structure/function relationship in RNA metabolism[END_REF]Nemeth and Langst 2011). Many other membrane-less subnuclear nuclear structures such as Histone bodies, Cajal bodies, speckles, paraspeckles, and Polycomb bodies (Yuntao S. [START_REF] Mao | Biogenesis and function of nuclear bodies[END_REF] are ordered in the nucleus by the nucleoskeleton composed of lamins, titin, actin, myosins, and kinesin-s associated with chromatin [START_REF] Simon | The nucleoskeleton as a genome-associated dynamic 'network of networks[END_REF][START_REF] De Lanerolle | Nuclear actin and myosins: life without filaments[END_REF].

Lieberman

Transcription factors diversity and phylogenetic origin

Transcription factors (TFs) are a class of DNA-binding proteins that typically recognize small 6-12 bp-long DNA sequences (Spitz,François 2012). TFs regulate transcription through binding to defined -cis-regulatory elements‖ that are present in promoters, enhancers and silencers DNA sequences (Wingender et al 2012). In human, TFs represent 1558 genes encoding 2904 proteins grouped in 9 superclasses, 40 classes and 111 different families. Repository of every known transcription factors associated with a known transcription factor binding site (TFBS) are available on TRANSFAC (Kel-Margoulis,O.V. 2006), JASPAR (Mathelier, A. et al 2013) or Matinspector (K. [START_REF] Cartharius | MatInspector and beyond: promoter analysis based on transcription factor binding sites[END_REF] databases. Among the nine TFs superclasses, Zinc-finger domain represent 53% of all TFs, Helix-turn-helix domain represents 26% and Basic domain factors 11%. These three superclasses have in common an alpha-helix able to bind the major groove of the DNA (Wingender et al 2012).

Recent phylogenetic analysis showed that helix-turn-helix eukaryotic superclasse of transcription factors has a prokaryotic origin (D. Wilson, Charoensawan 2007).

However, most of the mammalian TFs classes are metazoan specific and originate from a diversification through duplication and divergence from their common ancestor [START_REF] Langmead | Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[END_REF][START_REF] Degnan | Early evolution of metazoan transcription factors[END_REF][START_REF] Sebé-Pedrós | Unexpected repertoire of metazoan transcription factors in the unicellular holozoan Capsaspora owczarzaki[END_REF][START_REF] Ereskovsky | Cellular and molecular processes leading to embryo formation in sponges: evidences for high conservation of processes throughout animal evolution[END_REF]. This diversification of TFs classes was a key step in the evolution from the early metazoan to a progressive complexification of organism development (Andrew H. [START_REF] Knoll | The Multiple Origins of Complex Multicellularity[END_REF].

Transcription factor functions in gene regulation

Direct target genes are genes bound by a TF in promoter or cis-regulatory element whose transcription is regulated as a consequence of that binding. Typically, direct target genes are identified combining chromatin immuno-precipitation to identify TFBS [START_REF] Gerstein | Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project[END_REF] and observation of gene regulation after an overexpression or down-regulation [START_REF] Pavlopoulos | Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis[END_REF][START_REF] Ririe | The Caenorhabditis elegans vulva: a post-embryonic gene regulatory network controlling organogenesis[END_REF]. In bacteria, early studies showed a clear correlation between binding events and transcription regulation [START_REF] Lin | The general affinity of lac repressor for E. coli DNA: implications for gene regulation in procaryotes and eucaryotes[END_REF]Riggs, A.D. 1975. Elf, J. et al. 2007). However, in eukaryotes, TFBS occupancy and gene expression are not strictly correlated (F. Gao, et al.2004, Z. Hu, et al. 2007). In prokariotes, transcription factors are expressed at low levels (typically 1 to 10 2 molecules/cell) and occupy TFBS only with the help of other transcription factors in allosteric cooperative DNA binding. The co-occurrence of appropriately spaced recognition sites in the genome (TFBS modules) is necessary to allow highly selective binding of transcription factors in vivo [START_REF] Mark | Animal Transcription Networks as Highly Connected, Quantitative Continua[END_REF][START_REF] Bolouri | Transcriptional regulatory cascades in development: initial rates, not steady state, determine network kinetics[END_REF]Johnson, 1995;Mann et al., 2009;[START_REF] Falvo | Reversal of intrinsic DNA bends in the IFN beta gene enhancer by transcription factors and the architectural protein HMG I(Y)[END_REF].

Even if clustering of TFBS and TFBS modules increase specificity in eukaryotic gene regulation (Wunderlich et al 2009), the prokaryotic -deterministic allosteric model‖ does not explain the majority of transcription events in eukaryotes. Indeed, eukaryotic TFs are expressed at really high concentration (10 3 to 10 5 molecules/nucleus) and are able to bind DNA without the help of allosteric interactions (Biggin M, 2011).

Moreover, TFBS are frequently degenerate sequences that evolve rapidly without strong evolutionary constraint (Gwenael Badis 2009;[START_REF] Villar | Evolution of transcription factor binding in metazoansmechanisms and functional implications[END_REF]. TFs binding specificities are described with a position weight matrices (PWM) which are consensus binding sequences associated with probabilistic frequencies of nucleotide distribution for each single position. Indeed, in-vitro and in-vivo experimental methods [START_REF] Geertz | Experimental strategies for studying transcription factor-DNA binding specificities[END_REF] showed that each eukaryotic TF is able to bind TFBS with different or equal affinities to primary, secondary (50% of TFs) and sometimes tertiary TFBS but also for some of them in the absence of any detectable sequence motif (Gwenael Badis 2009;Wingender et al 2012, J.A., Snyder 2012). In eukaryotes, most of the TFs are bound to thousands of genomic regions (Myers et al., 2011;[START_REF] Gerstein | Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project[END_REF]Ne` gre et al., 2011;[START_REF] Rey | Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver[END_REF], and around 10,000-60,000 regions [START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF][START_REF] Bieda | Unbiased location analysis of E2F1binding sites suggests a widespread role for E2F1 in the human genome[END_REF][START_REF] Cao | Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming[END_REF]. Importantly, the genomic regions identified in those studies typically use a 1% false discovery rate (FDR) cut-off. However, genomic regions identified at less stringent FDR cannot be explained by experimental noise.

Rather, many DNA binding are not involved in the transcriptional regulation [START_REF] Boj | Functional targets of the monogenic diabetes transcription factors HNF-1alpha and HNF-4alpha are highly conserved between mice and humans[END_REF][START_REF] Cao | Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming[END_REF][START_REF] Hoffman | Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver[END_REF][START_REF] John | Chromatin accessibility pre-determines glucocorticoid receptor binding patterns[END_REF]. Eukaryotic and more specifically mammalian transcription is most likely a continuous phenomenon where TFs bind essentially to a continuum of occupancy levels. In this -Continuous model‖ [START_REF] Carr | A comparison of in vivo and in vitro DNA-binding specificities suggests a new model for homeoprotein DNA binding in Drosophila embryos[END_REF]Biggin, 2001), genes the most highly bound by a transcription factor are the most likely to be regulated in overexpression or downregulation experiment while genes moderately bound show moderate regulation and genes bound at lower levels shows no significant regulation [START_REF] Fullwood | An oestrogen-receptor-alpha-bound human chromatin interactome[END_REF][START_REF] Macarthur | Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions[END_REF][START_REF] Rey | Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver[END_REF]. In support of this model, data indicate that functionally and structurally different TFs bind identical genomic regions [START_REF] Elf | Probing transcription factor dynamics at the single-molecule level in a living cell[END_REF][START_REF] Kao-Huang | Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: measurement of DNA-bound Escherichia coli lac repressor in vivo[END_REF][START_REF] Phair | Global nature of dynamic protein-chromatin interactions in vivo: threedimensional genome scanning and dynamic interaction networks of chromatin proteins[END_REF][START_REF] Yang | Generation of oligodendroglial cells by direct lineage conversion[END_REF]Nash, 1995 / Roy et al., 2010;[START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF][START_REF] Jenner | The transcription factors T-bet and GATA-3 control alternative pathways of Tcell differentiation through a shared set of target genes[END_REF]. Moreover, combining the fact that heterozygotic null mutation rarely leads to a phenotype [START_REF] Ashburner | Drosophila. A laboratory handbook[END_REF] and the fact that if such mutation is associated with a mutation in a second gene of the same pathway, a strong phenotype occurs [START_REF] Simon | Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase[END_REF], this -continuous model‖ seems to be more appropriate to explain the pervasive redundant cross-regulations observed between transcription factors [START_REF] Davidson | Emerging properties of animal gene regulatory networks[END_REF][START_REF] Young | Control of the embryonic stem cell state[END_REF]. However, for the ease of representation, the majority of the proposed gene regulation networks involves a short list of direct target where each transcription factor directly regulates only a defined number of genes (Georgescu et al., 2008;Oliveri et al., 2008;[START_REF] Ririe | The Caenorhabditis elegans vulva: a post-embryonic gene regulatory network controlling organogenesis[END_REF]Stathopoulos and Levine, 2005). A great effort is now made to propose networks that are far more highly connected and are more representative of the reality of eukaryotic transcription regulation [START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF][START_REF] Gerstein | Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project[END_REF]Negre et al., 2011;Sandmann et al., 2007).

Transcription factors combinatorial regulation of transcription

Different modes of TFs binding lead to different types of transcriptional outputs [START_REF] Spitz | Transcription factors: from enhancer binding to developmental control[END_REF]. The fact that enhancer activation is proportional to the concentration of the individual TFs, as recently demonstrated [START_REF] Giorgetti | Noncooperative interactions between transcription factors and clustered DNA binding sites enable graded transcriptional responses to environmental inputs[END_REF] allowed the identification of super-enhancers defined by high-density TFBS clustering and strong gene activation capacities (Hnisz D, Abraham BJ 2013;Whyte WA, Orlando DA 2013). Contrary to these graded enhancer outputs, cooperative homotypic or heterotypic bindings allow the same or different set of TFs, respectively, to demonstrate nonlinear relationship between TFs concentration, occupancy and gene regulation. In this context, cooperative binding between two TFs results in a net increase in the affinity of the two factors for their motifs, while their inherent sequence specificity remains the same [START_REF] Spitz | Transcription factors: from enhancer binding to developmental control[END_REF]. Cooperative binding can be driven by direct TFs interaction bound to adjacent sites as described above; however, an indirect cooperation mechanism termed ‗assisted loading' or ‗hit and run model' can occur when two TFs that compete for the same TFBS does not inhibit the activity of each other but rather increases the occupancy of each TFs via increased nucleosome depletion rate [START_REF] Voss | Dynamic exchange at regulatory elements during chromatin remodeling underlies assisted loading mechanism[END_REF]. TFs binding or chromatin associated protein can also trigger a change in the bending of DNA which assist the binding of neighbouring TFs keeping their DNA affinity unchanged [START_REF] Falvo | Reversal of intrinsic DNA bends in the IFN beta gene enhancer by transcription factors and the architectural protein HMG I(Y)[END_REF][START_REF] Siggers | Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex[END_REF]. Finally, TFs interactions or cofactors can increase, decrease or change the DNA sequence specificity [START_REF] Siggers | Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex[END_REF]. Together these results show how modular and context-dependent is the combinatorial action of TFs on cis-regulatory elements.

Epigenetic regulation of the chromatin: The histone code hyposthesis

In 2000, the -histone code hyposthesis‖ postulated that multiple histone posttranslational modifications acting in a combinatorial and sequential way to control chromatin remodeling (B.D. Strahl,C.D. Allis 200). Indeed, the four core histones are subject to covalent modifications, including methylation, acetylation, phosphorylation, and ubiquitination (among many others), on the N-terminal histone tails that protrude from the nucleosomal surface or present in the histone globular domains [START_REF] Vaquero | The constantly changing face of chromatin. Science of aging knowledge environment[END_REF][START_REF] Campos | Histones: annotating chromatin[END_REF]. These -epigenetic marks‖ modulate chromatin structure through effector proteins that can write, read or erase the histone code [START_REF] Taverna | How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers[END_REF][START_REF] Voigt | Histone tails: ideal motifs for probing epigenetics through chemical biology approaches[END_REF]de CA Musselman 2012). Thus, -histone marks‖ have been shown to carry epigenetic information that can be transmitted through cell division and instruct specific gene expression in daughter cells [START_REF] Probst | Epigenetic inheritance during the cell cycle[END_REF][START_REF] Margueron | Chromatin structure and the inheritance of epigenetic information[END_REF]. Proteins such as chromatin remodelers, histone modification enzymes and histone chaperones are involved in chromatin regulation [START_REF] Khorasanizadeh | The nucleosome: from genomic organization to genomic regulation[END_REF]. Chromatin remodelers act with other chromatin component to control the activity of cis-regulatory elements which can be defined by specific histone epigenetic marks (Scott B 2014). Chromatin remodelers are key proteins that use the energy of ATP hydrolysis to move, eject, or restructure the composition of nucleosomes (Becker PB, Horz W. 2002., Saha A, Wittmeyer J, Cairns BR. 2006). There are four different families of chromatin remodelers conserved from yeast to humans, the SWI/SNF, ISWI, CHD and INO80 families (for review C.

Clapier2009; [START_REF] Narlikar | Mechanisms and functions of ATPdependent chromatin-remodeling enzymes[END_REF]. All share six basic properties: They have an affinity for the nucleosome; they recognize covalent histone marks; they have a DNA-dependent ATPase domain; they have domains that regulate the ATPase domain; they interact with other chromatin and transcription factors and they have the ability to translocate on DNA [START_REF] Weake | Inducible gene expression: diverse regulatory mechanisms[END_REF]. Remodelers share the ability to translocate along DNA in an ATP dependant manner at an average speed of 25 bp/s and to bind DNA at an internal nucleosomal location to release DNA out of the nucleosome (singleton 2007 sirinakis 2011). Nucleosome repositioning is triggered without histone disassembly by the conformational change of a DNA-binding domain located on the linker DNA (between two nucleosomes) that propagates to a DNA translocation domain which generates a small DNA loop that is resolved by nucleosome sliding (C. Clapier2009). Movement of the histone is energy demanding as the hydrolysis of 2 to 4 ATP molecules is needed to move a nucleosome of one base pair (Fyodorov DV 2002).

The action of the remodelers is facilitated by the histone chaperones, which collect the histones after the histone-DNA interactions have been broken. The dynamic turnover of histones from DNA is termed histone exchange. During nucleosome formation, histones H3/H4 are deposited first onto the DNA before H2A/H2B. Conversely nucleosome disassembly involves that peripherally located histone H2A/H2B dimers are first removed, followed by removal of H3/H4 (Das, C. 2012). RNA polymerase processing facilitates histone exchange thanks to the histone chaperone FACT (Belotserkovskaya R2009). Other histone chaperone such as Sp6, Asf1, HIR, and Rtt106 promote chromatin reassembly following Pol II passage (Das C, Tyler JK. 2013). Like promoters, enhancers show a high sensitivity to the DNase I [START_REF] Gross | Nuclease hypersensitive sites in chromatin[END_REF]. However, rather than being nucleosome free, these regions are associated with nucleosomes constituted by histone variants H3.3 and H2A.Z [START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF]Jin et al., 2009). H3.3/H2A.Z-containing nucleosomes have a hyperdynamic turnover which explains the DNase I profiles (Jin et al., 2009).

However, nucleosomes flanking enhancers are far less dynamic and expose specific histone marks such as H3K4me1/2/3 (Histone 3 lysine 4 mono/di/tri methylation) and H3K27Ac (Histone 3 lysine 27 acetylation) that are now frequently used to map enhancers [START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF][START_REF] Heintzman | Histone modifications at human enhancers reflect global cell-type-specific gene expression[END_REF][START_REF] Rada-Iglesias | A unique chromatin signature uncovers early developmental enhancers in humans[END_REF][START_REF] Zentner | Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions[END_REF]. Enhancers commonly span 200-500 bp in length and containing hundreds of clustered transcription factor binding sites (TFBS) [START_REF] Spitz | Transcription factors: from enhancer binding to developmental control[END_REF]. Enhancer activation involves ‗‗ pioneer '' TFs ( aret and Carroll, 2011) Preceding occupancy of other TFs, pioneer factors help to the recruit chromatin -readers‖, writers‖, and -remodeler‖ [START_REF] Kouzarides | SnapShot: Histone-modifying enzymes[END_REF][START_REF] Ruthenburg | Multivalent engagement of chromatin modifications by linked binding modules[END_REF] and participate to the initial decompaction of the chromatin as well as the protection against inhibiting DNA methylation (ghisletti et al., 2010;serandour et al., 2011;[START_REF] Zaret | Pioneer transcription factors: establishing competence for gene expression[END_REF]. Enhancers are associated with developmentally regulated genes [START_REF] Levine | Transcriptional enhancers in animal development and evolution[END_REF] and drive tissue specificity of gene expression (Bernstein et al., 2012;[START_REF] Ernst | Mapping and analysis of chromatin state dynamics in nine human cell types[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF][START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF]. Mutations occurring in enhancers underlie evolutionary differences between species [START_REF] Jones | The genomic basis of adaptive evolution in threespine sticklebacks[END_REF] and contribute to many diseases [START_REF] Dickel | Functional anatomy of distant-acting mammalian enhancers[END_REF]Bernstein et al., 2012;[START_REF] Ernst | Mapping and analysis of chromatin state dynamics in nine human cell types[END_REF]. When enhancers are active, they bind general RNA polymerase II and mediator complex and trigger mRNA and enhancer RNAs (eRNAs ) transcription [START_REF] Natoli | Noncoding transcription at enhancers: general principles and functional models[END_REF] thanks to CTCF and cohesion long range interaction DNA stabilization [START_REF] Phillips | CTCF: master weaver of the genome[END_REF][START_REF] Nasmyth | Cohesin: its roles and mechanisms[END_REF]. eRNAs are short bidirectional and nonpolyadenylated or long unidirectional and polyadenylated which function is still under investigation [START_REF] Natoli | Noncoding transcription at enhancers: general principles and functional models[END_REF]. Intensive work allowed significant progress to identify enhancers in mammalian genomes (Bernstein et al., 2012;[START_REF] Ernst | Mapping and analysis of chromatin state dynamics in nine human cell types[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF]. H3K4me1 and H3K27Ac were the first histone modifications associated to enhancer function [START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]MP Creyghton 2010). H3K4me1 is enriched at distal enhancer sites, in contrast to H3K4me3, which is present at active promoters [START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]. However, H3K4me1 is also present at promoter regions. While a high ratio of H3K4me1 to H3K4me3 better distinguishes enhancers from promoters, an opposite ratio better define promoters [START_REF] Djebali | Landscape of transcription in human cells[END_REF][START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF][START_REF] Koch | Initiating RNA polymerase II and TIPs as hallmarks of enhancer activity and tissue-specificity[END_REF]. Interestingly, early priming of enhancers by H3K4me1 in the absence of H3K27Ac is observed before cell differentiation [START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF][START_REF] Bogdanovic | Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis[END_REF][START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF][START_REF] Mercer | Multilineage priming of enhancer repertoires precedes commitment to the B and myeloid cell lineages in hematopoietic progenitors[END_REF][START_REF] Rada-Iglesias | Epigenomic annotation of enhancers predicts transcriptional regulators of human neural crest[END_REF]Wamstad et al., 2012, Zaret and[START_REF] Zaret | Pioneer transcription factors: establishing competence for gene expression[END_REF]. Thus, H3K4me1 priming defines an enhancer state of potental activation and H3K27ac distinguishes active enhancers from inactive or poised enhancer containing H3K4me1 alone (MP Creyghton 2010). Moreover, an ‗‗ enhancer decommissing'' mechanism involving Pol II and TFs clearance, is mediated by demethylation of H3K4me1 by LSD1 [START_REF] Whyte | Enhancer decommissioning by LSD1 during embryonic stem cell differentiation[END_REF]. H3K4me2 is present at both enhancers and promoters and seems to be an intermediate state of activation [START_REF] Koch | Initiating RNA polymerase II and TIPs as hallmarks of enhancer activity and tissue-specificity[END_REF][START_REF] Pekowska | H3K4 tri-methylation provides an epigenetic signature of active enhancers[END_REF]. H3K27Ac is associated with active enhancers and is acquired exclusively in the context of the pre-existing H3K4me1 [START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF]. Many distinct Histone acetyl transferase (HATs) are involved in transcriptional activation [START_REF] Brown | The many HATs of transcription coactivators[END_REF][START_REF] Lee | Histone acetyltransferase complexes: one size doesn't fit all. Nature Reviews[END_REF]. Recently, binding of p300/CBP HATs complex, was shown to map enhancers in different cell types [START_REF] Ghazalpour | Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages[END_REF][START_REF] Heintzman | Histone modifications at human enhancers reflect global cell-type-specific gene expression[END_REF][START_REF] Kim | Widespread transcription at neuronal activity-regulated enhancers[END_REF][START_REF] Rada-Iglesias | A unique chromatin signature uncovers early developmental enhancers in humans[END_REF][START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF]. p300/CBP are ubiquitously expressed Numerous H3K4 methylation readers have been discovered in mammalian cells [START_REF] Ruthenburg | Multivalent engagement of chromatin modifications by linked binding modules[END_REF]. Unmodified H3K4 is read by BHC80 (CoREST complex), Dnmt3L (cofactor of DNA methyltransferases Dnmt3a/b), and Uhrf1 (cofactor of methyltransferase Dnmt1). Interestingly, all those proteins lead to transcription inhibition [START_REF] Lan | Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression[END_REF][START_REF] Nady | Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein[END_REF][START_REF] Ooi | DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA[END_REF]. Most of the H3K4 readers prefer trimethylated over the monomethylated form. However, chromodomains of the ATP-dependent remodelers CHD1 and CHD7 recognize all forms of methylated H3K4. Interestingly, CHD7 appears to be preferentially associated with enhancer regions [START_REF] Schnetz | Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns[END_REF]. So far, the only reader reported to prefer H3K4me1 binding is the chromodomain of the acetyltransferase TIP60 involved in the deposition and acetylation of H2A.Z (Jeong et al.,201). One of the functions of H3K4me1 could be to promote incorporation of the H2A.Z-in nucleosomes to initiate TFs accessibility [START_REF] Altaf | NuA4-dependent acetylation of nucleosomal histones H4 and H2A directly stimulates incorporation of H2A.Z by the SWR1 complex[END_REF].

Numerous histone methylation writers have also been reported. For example, genes of the Trithorax family Set1a/1b, MLL1/2/3/4 methylate histone H3K4 [START_REF] Ruthenburg | Multivalent engagement of chromatin modifications by linked binding modules[END_REF]. Studies shows that Set1a/1b/Wdr82 methylate H3K4me3 [START_REF] Ardehali | Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription[END_REF][START_REF] Hallson | dSet1 is the main H3K4 di-and tri-methyltransferase throughout Drosophila development[END_REF]Wu et al., 2008, Lee and[START_REF] Lee | Wdr82 is a C-terminal domain-binding protein that recruits the Setd1A Histone H3-Lys4 methyltransferase complex to transcription start sites of transcribed human genes[END_REF][START_REF] Wu | Molecular regulation of H3K4 trimethylation by Wdr82, a component of human Set1/COMPASS[END_REF]. In human and mouse ESCs, a large subclass of enhancers enriched for H3K4me1, but lacking H3K27Ac, is also marked by H3K27me3 and bound by the Polycomb complex [START_REF] Rada-Iglesias | A unique chromatin signature uncovers early developmental enhancers in humans[END_REF][START_REF] Zentner | Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions[END_REF]. These elements, termed ‗‗ poised enhancers,'' are located near key early developmental genes and share most of the properties of active enhancers, including similar levels of nucleosomal depletion, p300, and BRG1 binding. The loss of H3K27me3 and the gain of H3K27Ac then trigger a switch from poised enhancers to active enhancers [START_REF] Rada-Iglesias | A unique chromatin signature uncovers early developmental enhancers in humans[END_REF]. Poised enhancers are already looped to their target promoters via cohensin and CTCF looping, ready for gene activation upon proper receiving of developmental signals [START_REF] Sanyal | The long-range interaction landscape of gene promoters[END_REF]. Genes interacting with H3K27me3 enhancers commonly have bivalently marked promoters that are characterized by the simultaneous presence of the H3K4me3 and H3K27me3 and association with PRC2 [START_REF] Bernstein | A bivalent chromatin structure marks key developmental genes in embryonic stem cells[END_REF][START_REF] Buecker | Enhancers as information integration hubs in development: lessons from genomics[END_REF]Voigt T 2013).

Recently, other enhancer histone modifications have been identified such as H3K9ac and H3K18ac [START_REF] Ernst | Mapping and analysis of chromatin state dynamics in nine human cell types[END_REF][START_REF] Zentner | Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions[END_REF], or histone crotonylation [START_REF] Tan | Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification[END_REF]), H3K79me2/3 (Bonn et al., 2012;[START_REF] Djebali | Landscape of transcription in human cells[END_REF].

As many new histone and DNA epigenetic modifications are yet to be charachterized (Rothbart SB 2014) and many others will be identified in more diverse functions and cellular states, the -histone code‖ seems to be extraordinary versatile and cell type specific. Thus, the epigenetic code seems to be far from being hacked. Despite the discovery of several important epigenetic constants in differentiating cells by different leading teams [START_REF] Visel | A high-resolution enhancer atlas of the developing telencephalon[END_REF] as well as the ENCODE project (which only investigated 1% (30 Mb) of the human genome in carcinogenous cell line and cost more than 300 million dollars), unraveling the in-vivo epigenetic network in most cell types at different developmental time points remains to be done [START_REF] Graur | On the Immortality of Television Sets: -Function‖ in the Human Genome According to the Evolution-Free Gospel of ENCODE[END_REF].

Network regulation, information flow and central dogma of the molecular biology

The transcriptional program that controls gene expression is highly flexible and able to adapt to a great range of perturbations. TFs are the genes that demonstrate the most cross-regulation capacities and underwent the most genomic duplications during evolution and are thus often called the master genes of the genome [START_REF] Levine | Transcription regulation and animal diversity[END_REF]. In yeast, only ~20% of the 6000 yeast genes confer lethality when deleted (Dixon et al. 2009). In this context biological systems are defined as robust and redundant systems. In order to understand how genes regulate each other to orchestrate cell differentiation, the reconstruction of gene regulatory networks (GNR) is often done by perturbation of each component of the network via single or multiple gene knock-in and knock-out [START_REF] Kholodenko | Untangling the wires: a strategy to trace functional interactions in signaling and gene networks[END_REF]. As these perturbations propagate rapidly and cause global changes in gene expression that are stabilized to a new attractor state (if not lethal), differentiating between direct interactions and indirect interaction is one of the main challenges in genomic studies. The spatial and temporal dynamic of GNR have been extensively described with Boolean logic [START_REF] Mangan | Structure and function of the feed-forward loop network motif[END_REF]. GRNs are composed of -nodes,‖ representing the network components, joined by -edges,‖ which represent physical and/or regulatory interactions. While most of the nodes have a relatively low (in or out)-degrees, some (for example TFs) are highly connected and are called -hubs‖ (Yu and Gerstein 2006).This work allowed the description of network motifs frequently occurring in GNR such as positive or negative autoregulatory loops, activation or inhibition cascade, positive and negative feed-back loops and coherent and incoherent feedforward loops (Shoval Alon 2013). Working together as a complex logical clockwork, these motifs lead to different pattern of gene expression such as indefinite or finite oscillatons, single pulse responses or cellular state transition that are maintained and integrated by the cells [START_REF] Yosef | Impulse control: temporal dynamics in gene transcription[END_REF]. In order to generate bistable or multistable cell systems able to differentiate, GNR must include positive or double-negative feedback loops (E.H. [START_REF] Snoussi | Necessary Conditions for Multistationarity and Stable Periodicity[END_REF] or paradoxical motifs (such as the incoherent feedforward loop type 1) which are defined as motifs that simultaneously drive two opposite effects on the same target or biological process (Hart et al., 2011a[START_REF] Hart | Design principles of cell circuits with paradoxical components[END_REF]. All these motifs are pervasive in GNR [START_REF] Shoval | SnapShot: network motifs[END_REF] and cells have to process and integrate in time and space this information to allow the emergence of bistable or multistable cellular states as well as the maintenance of the final monostable state (James E. Ferrell 2012). However, biological interactions are not strictly specific and despite strict constraints, biological systems are stochastic by nature (non-linear) and degenerate (composed of elements that are structurally different but share the same function). In this context, GNR specificity is mainly driven by the subcellular and cellular spatiotemporal constraints that confer the specificity to the molecular interactions, such as cell compartmentalization and temporal separation of gene expression [START_REF] Schwartz | Principles of MAP kinase signaling specificity in Saccharomyces cerevisiae[END_REF][START_REF] Komarova | A theoretical framework for specificity in cell signaling[END_REF]. In the nucleus, while TFs and co-factors are rapidly produced and degraded upon developmental signals and mechanisms of negative titration, nuclear export or post translational regulations, chromatin epigenetic structural constraints drive the specificity of their action on specific cis-regulatory region as a continuous dialogue.

Regulation of gene expression was originally conceived as a one directional flow of information, going from one gene coded on DNA in the nucleus, to transcription of the corresponding mRNA then translated into one protein to perform its particular function. This view has been abandoned after accumulating evidence of pervasive transcription of non-coding RNA discovered between the 1950's and today (tRNA, rRNA, miRNA, LncRNA, piRNA, eRNA, PALR, PROMPT, SUTs CUTs, lincRNA) together with the discovery of alternative promoter usage, alternative splicing (>90% genes), alternative polyadenylation, RNA editing and posttranscriptional, posttranslational or epigenetic regulations. It is now well recognized that information flow goes downward, from DNA to RNA and protein as well as backward from protein to RNA and from RNA to DNA. Thus, despite local hierarchies in GNRs, a global hierarchical causality is impossible to define as both top-down and bottom-up causation are at work in cells. The gene-centric view, illustrated by Richard Dawkins in his book "The selfish gene" postulating that any DNA sequence aims at replicating itself even sometimes at the disadvantage of the organism (Dawkin 1976), restrained for decades our conception about evolution, putting the focus on genetic selection.

Besides DNA replication and genetic information transmission, the information flow generated by the interactions of actors operating in the cell as a whole (genome, epigenome, transcriptome, lipidome, metabolome) is subject to maintenance, transmission, variation and natural selection (H Nam, NE Lewis 2012). In this context, DNA sequence conservation seems to be among others a convenient hallmark (easy to conceptualize, to read, and interpret) for a more abstract multi-level -functional‖ selection [START_REF] Hogeweg | Toward a theory of multilevel evolution: long-term information integration shapes the mutational landscape and enhances evolvability[END_REF].

Methodological considerations

Transcriptomic technologies

Isolation and differential measure of RNA sequences are generally performed with different purification techniques to isolate different fractions of RNA followed by high-throughput sequencing or microarray analysis. These technics have the capacity to simultaneously measure tens of thousands of transcripts and allowed important progress in a wide range of biological questions. Despite recent progress single cell RNA experiments, generally averaged RNA content of a cell population are measured.

Microarrays remain the most used and the cheapest approach for transcriptomic analysis. However, several drawbacks, such as platform specificities (Affymetrix, Illumina, Agilent), noisy background hybridization, measurement problems with low abundance transcripts or fixed chip design. We used in this study the Affymetrix microarray (Mouse Genome 430 2.0 Array) which involves hybridization of biotinylated cDNA (obtained from reverse transcription of mRNA) against synthetic oligonucleotides (probes of 25 bases long, directed in the 3' region of each gene) directly synthesized and spotted in defined order onto a glass array. Each array contains 900,000 different oligos and each oligo is present in millions of copies. After hybridization between oligos and cDNA, the chip is stained with a fluorescent molecule (streptavidin-phycoerythrin) that binds to biotin. A series of stains AND washes with reagents binds the biotin which provides light emission that is detected by microarray scanning with a confocal laser. The distribution pattern of signal in the array is recorded and signal intensities for each gene are interpreted in relation with their locations on the array. Subsequently, replicates experiments are normalized and summarized to perform differential measure between different biological conditions. RNA-Seq, which allows sequencing of all transcripts present in biological samples is more appropriate to study refined biological differences such as novel transcripts splicing events, allele-specific expression or any non-coding RNA processes. Both techniques need powerful technical and algorithmic challenges for experimental design, sample preparation, data analysis, storage and exchange. Despite the fact that RNA-Seq better estimates absolute transcript levels, both techniques show great overlap of genes differential expression results. Details on each microarray platform and RNA-Seq technology and the challenges and benefits associated with its technology and application are reviewed (S Zhao W P F-Leung, 2014). Raw (CEL files) and processed data (tab files) are submitted to GEO Gene Expression Omnibus database (Edgar R, Domrachev M, 2013 )which store all the Chip-seq dataset published so far.

Chromatin immuno-precipitation techniques

ChIP-seq is a technique of chromatin immuno-precipitation followed by DNA sequencing (Iyer et al., 2001) which identifies specific DNA sequences bound by a specific chromatin associated protein (generally TFs or histone epigenetic marks).

Briefly, Protein-DNA interactions are fixed by formaldehyde cross-linking, chromatin is sheared in 400bp fragments by sonication, and DNA sequences binding to a specific protein are selectively enriched by immuno-precipitation with a specific antibody targeting a protein of interest. After PCR amplification and sequencing, DNA reads of the enriched fraction are aligned against the reference genome using Bowtie [START_REF] Langmead | Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[END_REF] and reads distributions are plotted to define peaks region and peaks summit that are then analysed to locate the protein bound regions. However, non-specific immuno-precipitation generate noisy signal that need to be handle by statistical methods in order to locate individual high-confidence binding sites against background sequence enrichment. MACS, a Model-based Analysis of ChIP-seq [START_REF] Feng | Identifying ChIP-seq enrichment using MACS[END_REF] is the most commonly used algorithm that identifies binding peaks (Peak calling) in ChIP-seq data. MACS consists of four steps: removing duplicate reads generated by PCR, adjusting read position by alignments to form peaks, calculating peak enrichment against background, and estimating the false discovery rate. Because TFs DNA binding is a continuous phenomenon, many weak sites are identified. Thus, the final peak list depends on the algorithm parameter settings, the FDR cut-off as well as the quality of the biological signal. An appropriate control sample is critical to estimate the background level. Two methods are used to produce control DNA samples. Either ‗‗ Input'' DNA is isolated from cells that have been crosslinked and fragmented under the same conditions but not immunoprecipitated or a ‗‗ mock'' ChIP reaction is performed using a control antibody that reacts with an irrelevant, non-nuclear antigen such as IgG. Many drawbacks are associated with this step as mock antibody rarely yield enough non-specific DNA during precipitation and the optimal input DNA level to use is hard to define a-priori. Other statistical methods have been developed to measure the intrinsic background level directly from the experimental sample and seem more appropriate to measure the background level (BA de Boer, 2014).

Similarly to microarray experiments, biological samples used in Chip-seq are cell populations rather than single cells. Thus, peaks enrichment reflects the average probability for a protein to be bound on DNA at specific regions where the most frequent binding event are the highest peaks identified. The ENCODE project defined a set of standards and guidelines to follow to increase the quality of ChIP experiments (Landt S 2014) and raw sequencing results (SRA, BAM files) and processed data (Bed files) are submitted to GEO database which store all the Chip-seq dataset published so far.

Chromosome conformation capture assays

Following the pioneering work of Job Dekker in 2002, nuclear architecture and 3D chromosome folding have been extensively studied using chromosome conformation capture techniques and derivatives [START_REF] Dekker | Capturing chromosome conformation[END_REF]J Dekker 2013).

These methods rely on cross-linking of spatially interacting chromatin, fragmentation and re-ligation of cross-linked chromatin and identification by sequencing of cisregulatory sequences (promoter, enhancers, silencers) that interact with each other via long range interactions. Hundred of thousands of significant long-range interactions between gene promoters and distal cis-regulatory are identified genome-wide thanks to complex algorithmic reconstruction of interaction probability map. As almost all of the genes engage in long range interactions, chromosome capture analysis allowed the identification of important new general concepts of gene regulation as well as a new understanding of the nuclear organization. Generalization of these techniques to the study of cell-type specific differentiation processes leads to a global overview of the chromatin remodeling mechanism at work during development. Computational inference based on correlations between gene activity and activity of distal elements across panels of cell lines are now the next frontier in our understanding of genetic and epigenetic gene regulation [START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF][START_REF] Visel | A high-resolution enhancer atlas of the developing telencephalon[END_REF]. Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) detects chromatin interactions. However, ChIA-PET uses a chromatin immunoprecipitation (ChIP) step to more specifically identify interactions with a particular bounchrod protein, such as RNA polymerase II. (adapated from S. Furey 2012 and J Dekker 2013). B. Schematic of the chromatin capture technic. C. Schematic of Hi-C data from mouse chromosome 18. Hi-C method is a genome-wide 3C. Hi-C provides a true all-by-all genome-wide interaction map, but the resolution of this map depends on the depth of sequencing. Hi-C data are represented as two-dimensional heat maps. While the probability of long range interaction is invertionaly proportional to the distance, shift from this linear model define interaction enrichement or interaction depletetion. Interacting regions detection depends on sequencing depth. Bioinformatic reconstruction allow the inference of long range regulation of gene expression (adapted from Dekker J 2013).

CHAPTER II: Nervous system development and cell type specification

The early organization of the CNS is made by the action of morphogenetic signals starting during the gastrula stage and evolving throughout the embryonic and postnatal development (echevarria D 2003). Neural development starts when ectodermal cells are specified into neurectodermal cells at the origin of the entire nervous system. Primary neural induction and antero-posterior or dorso-ventral regionalization of the early neural tube is due to the activity of the -primary organizer‖.

Indeed, after the discovery by Hans Spemann and Hilde Mangold in 1924 that grafting a salamander embryo with the dorsal blastopore lip -organizer‖ of another embryo was sufficient to induce a secondary full body axis comprising a full neural axis [START_REF] Spemann | Induction of embryonic primordia by implantation of organizers from a different species[END_REF][START_REF] De Robertis | Dorsal-ventral patterning and neural induction in Xenopus embryos[END_REF], similar -organizers‖ were identified in all vertebrates and in some nonvertebrates (Cobos et al., 2001;[START_REF] Darras | β-catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii[END_REF][START_REF] Meinhardt | Primary body axes of vertebrates: Generation of a near-cartesian coordinate system and the role of Spemann-type organizer[END_REF][START_REF] Nakamoto | Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid[END_REF]. A large number of factors expressed by -the node‖, the mammalian organizer, have now been identified. Later on, local signaling known as -secondary organizers‖, drive the antero-posterior specification of the three main domains of the brain: the forebrain, the midbrain and the hindbrain. Patterning of the central nervous system into different areas by the action of different gradients of morphogens such as Fgfs, Wnts, Shh, and BMPs lead to regional expression of homeodomain and bHLH transcription factors that specify neural precursors cells (NPC) into all specific cell types neural cell type of the central nervous system.

Neurulation: the prosomeric model

Neurulation starts at the end of gastrulation from an inductive interaction between the -node‖ and the neighboring ectoderm and endoderm. The presomptive neural plate formation is first triggered by Fgf8 signaling coming from the primary endoderm (Viera C 2010). The ectoderm surrounding the neural plate expresses BMPs while the node and the extending axial mesendoderm express Wnts and the BMP inhibitors Noggin and Chordin [START_REF] De Robertis | Dorsal-ventral patterning and neural induction in Xenopus embryos[END_REF], Munoz-Sanjuan & Brivanlou 2002;[START_REF] Stern | Evolution of the mechanisms that establish the embryonic axes[END_REF]. Two orthogonal gradients are established: a Wnt gradient along the Antero-posterior axis and a BMP gradient along the mediolateral axis and the axial mesendoderm [START_REF] Meinhardt | Primary body axes of vertebrates: Generation of a near-cartesian coordinate system and the role of Spemann-type organizer[END_REF][START_REF] Niehrs | On growth and form: a Cartesian coordinate system of Wnt and BMP signaling specifies bilaterian body axes[END_REF]. Interestingly, ectodermal cells undergo neural differentiation as their default state. Indeed, neural differentiation must be suppressed in the lateral ectoderm by TGFβ signaling in order to develop as epidermis [START_REF] Wittler | The acquisition of neural fate in the chick[END_REF]. Subsequent suppression of BMP signaling maintains neural differentiation [START_REF] Linker | Cell communication with the neural plate is required for induction of neural markers by BMP inhibition: evidence for homeogenetic induction and implications for Xenopus animal cap and chick explant assays[END_REF][START_REF] Stern | Neural induction: old problem, new findings, yet more questions[END_REF]. Neural plate formation involves pseudostratification of the neurectoderm, lengthening of the neural plate along the anteroposterior axis and bending of the neural plate triggering the formation the neural folds [START_REF] Colas | Towards a cellular and molecular understanding of neurulation[END_REF]. Elevation of the neural folds creates the neural groove becoming, after closure at the dorsal midline, the lumen of the neural tube. At this stage, the initial medio-lateral patterning is transposed into dorso-ventral polarity.

The early neural tube is formed by three primary vesicles: the forebrain (prosencephalon), the midbrain (mesencephalon) and hindbrain (rhombencephalon).

Cephalic and cervical flexures of the neural tube then start and the subdividion of the prosencephalon into telencephalon and diencephalon occurs [START_REF] Pombero | Telencephalic morphogenesis during the process of neurulation: an experimental study using quail-chick chimeras[END_REF]. Early patterning events driven by the -primary organizer‖ are then refined through the establishment of -secondary organizers‖. Secondary organizers are domains of the neural plate where localized expression of transcription factors and morphogenes gradient control morphogenesis [START_REF] Crossley | Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles[END_REF]Echevarria et al., 2003;[START_REF] Aroca | Postulated boundaries and differential fate in the developing rostral hindbrain[END_REF]. The -prosomeric segmental model‖ is used to describe the dorso-ventral and antero-posterior neural regionalization thanks to a grid-like representation of the histogenic domains by longitudinal (columnar) and transverse (segmental) boundaries based on regionalized gene expression pattern [START_REF] Puelles | Location of the rostral end of the longitudinal brain axis: review of an old topic in the light of marking experiments on the closing rostral neuropore[END_REF]Viera C 2010). Three regions in the neural tube have been identified as secondary organizers: the anterior neural ridge (ANR) at the rostral part of the neural tube, the zona limitans intrathalamica (ZLI) in the diencephalon and the isthmic organizer (IsO) at the midbrain-hindbrain boundary.

Dorso-ventral patterning

Dorso-Ventral (DV) patterning generate four longitudinal territories which are from ventral to dorsal, the floor plate, the basal plate, the alar plate and the roof plate (Kiecker, Clemens 2012). Sonic Hedgehog (Shh) signaling is necessary and sufficient to specify the ventral identity of the nervous system [START_REF] Placzek | Mesodermal control of neural cell identity: floor plate induction by the notochord[END_REF], van Straaten et al. 1985). Shh starts to be produced by the notochord, a structure underlying the midline of the neural plate. Later on, Shh is produced by the floor plate overlying the notochord [START_REF] Echelard | Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity[END_REF][START_REF] Roelink | Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord[END_REF][START_REF] Roelink | Floor plate and motor neuron induction by different concentrations of the aminoterminal cleavage product of sonic hedgehog autoproteolysis[END_REF][START_REF] Placzek | The floor plate: multiple cells, multiple signals[END_REF].

Shh is secreted and exchanged from cell to cell to form a molecular gradient that specifies different cell fates in a dose-dependent manner [START_REF] Echelard | Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity[END_REF][START_REF] Krauss | A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos[END_REF][START_REF] Roelink | Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord[END_REF][START_REF] Chiang | Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function[END_REF][START_REF] Dessaud | Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network[END_REF][START_REF] Lupo | Mechanisms of ventral patterning in the vertebrate nervous system[END_REF]. This ventral-to-dorsal gradient of shh is visible in the ventral neural tube as well as the ventral telencephalon [START_REF] Chamberlain | Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning[END_REF]. Shh downstream effectors are the activating transcription factors Gli1 and Gli2 and the inhibitor Gli3. In this context, the Shh gradient is converted into two gradients of opposite transcriptional response involving Gli1/2 and Gli3 activity along the DV axis [START_REF] Fuccillo | Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development[END_REF][START_REF] Lei | Transduction of graded Hedgehog signaling by a combination of Gli2 and Gli3 activator functions in the developing spinal cord[END_REF][START_REF] Stamataki | A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube[END_REF]. While genes such as Foxa2, Nkx2.2, Olig2, Nkx6.1, Dbx1, and Dbx2, are induced upon Gli activation, genes such as Pax6, Pax7, and Irx3 are repressed. Interestingly, repressed and activated genes cross-repress each other to define the boundaries between their expression domains [START_REF] Briscoe | A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube[END_REF].

Differential exposure duration of Shh signal and subsequent combinatorial expression of Gli participate to the cell fate diversity of diverse neural cell types in the telencephalon, the hindbrain or the spinal cord [START_REF] Dessaud | Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network[END_REF][START_REF] Pattyn | Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors[END_REF]).

In the early forebrain, the prechordal plate expresses Shh. However, Shh is not sufficient for ventralization [START_REF] Pera | Patterning of the chick forebrain anlage by the prechordal plate[END_REF]. BMP7, which is expressed in the prechordal mesoderm synergizes with Shh to induce ventral forebrain identity [START_REF] Dale | Cooperation of BMP7 and SHH in the induction of forebrain ventral midline cells by prechordal mesoderm[END_REF]. Moreover, Wnt inhibitors expressed in the prechordal plate contribute to ventralization [START_REF] Kiecker | A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus[END_REF]). In the forebrain, pallial and subpallial regions are specified by Shh secreted by the prechordal plate [START_REF] Gunhaga | Sonic hedgehog signaling at gastrula stages specifies ventral telencephalic cells in the chick embryo[END_REF]. In the subpallial region, Shh from the neural plate induce the formation of the Anterior neural ridge which will become later on, the medial ganglionic eminence [START_REF] Sousa | Sonic hedgehog functions through dynamic changes in temporal competence in the developing forebrain[END_REF].

Antero-posterior regionalization

Antero posterior (AP) regionalization generates distinct transverse domains at different axial positions which display distinct competence to respond to the same signal ( Rubenstein 1998). Transverse domains of the nervous system are called prosomeres and appear in the prosencephalon (p1-6), rhombomeres in the rhombencephalon (r1-r7) and pseudorhombomeres (r8-r11), while the mesencephalon is considered as a single unit [START_REF] Cambronero | Rostrocaudal nuclear relationships in the avian medulla oblongata: a fate map with quail chick chimeras[END_REF]. The first signal inducing AP patterning originates from the dorsal mesoderm ad specify the neuroectoderm of the forebrain and midbrain by the expression of Lim1 Otx2, Engrailed (En1) and Cerberus (Doniach, 1993). Then a other signals posteriorizes the neural plate, inducing hindbrain and spinal cord via retinoic acid that [START_REF] Durston | Retinoic acid causes an anteroposterior transformation in the developing central nervous system[END_REF] FGF2 [START_REF] Cox | Caudalization of neural fate by tissue recombination and bFGF[END_REF]) and Hox genes signaling (Pearson et al.2005). Shh is expressed along the entire AP axis and triggers Shh, HNF3 β, Nkx2.2 expressions in all regions of the ventral neural tube, but specifically induces Nkx2.1 Nkx6.1 in posterior regions [START_REF] Qiu | Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development[END_REF]. Similarly, Fgf8 applied to prosencephalon and mesencephalon induces distinct competence of receiving cells, inducing FoxG1 anteriorly and En2 posteriorly [START_REF] Shimamura | Inductive interactions direct early regionalization of the mouse forebrain[END_REF].

Secondary organizers and brain patterning

Secondary patterning mechanisms arise during central nervous system development. The anterior neural ridge (ANR), defined by the early expression of Fgf8, drive the specification of the anterior areas of the forebrain. Fgf8 cooperate with Fgf15, Bmp4, Wnt and Shh to regulates prosencephalic regionalization through inhibition of Otx2 and Emx2 [START_REF] Crossley | Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles[END_REF][START_REF] Storm | Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers[END_REF] and activation of FoxG1 [START_REF] Xuan | Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres[END_REF][START_REF] Shimamura | Inductive interactions direct early regionalization of the mouse forebrain[END_REF].

The zona limitans intrathalamica (ZLI) is the Shh expressing boundary region between prosomere 2 and prosomere 3. While prosomere 2 alar plate will develop into the thalamus, p3 alar plate will develop into the prethalamus (Martinez and Puelles, 2000). ZLI cells express the transcription factor Sim1, Nkx2.2, Wnt8b and Fgf15 (Fan et al., 1999[START_REF] Price | Regional expression of the homeobox gene Nkx-2.2 in the developing mammalian forebrain[END_REF][START_REF] Shimamura | Longitudinal organization of the anterior neural plate and neural tube[END_REF][START_REF] Gimeno | Analysis of Fgf15 expression pattern in the mouse neural tube[END_REF]. Interstingly, Shh induction in the ZLI requires Wnt8b expression (A Martinez-Ferre 2014).

The Isthmic organizer (IsO) is localized at the midbrain-hindbrain boundary which controls the development of cerebellum, the isthmic and mesencephalic regions (for review see [START_REF] Crossley | Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles[END_REF]. The isthmic organizer develops exactly at the limit between Otx2 and Gbx2 expression domains. Mutant mice lacking Gbx2, Otx2, Wnt1, Pax2, En1, or Fgf8 do not develop isthmocerebellar structures [START_REF] Mcmahon | The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain[END_REF].

Shh signaling pathway

Sonic Hedgehog is a cleaved protein that produce two secreted fragments: a 19 kDa N-terminal protein (N-Shh) that mediates signaling activities and a 25 kDa Cterminal protein (C-Shh) which possesses protease activity [START_REF] Porter | The product of hedgehog autoproteolytic cleavage active in local and long-range signalling[END_REF]. N-Shh is modified by a cholesterol moiety to the C-terminus and a palmitoyl group to the Nterminus [START_REF] Chamoun | Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal[END_REF]. Once secreted, Shh binds to patched (Ptc), a 12transmembrane protein. That prevents the constitutive inhibition of smoothened (Smo), a 7transmembrane protein [START_REF] Alcedo | The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal[END_REF][START_REF] Hynes | The seven-transmembrane receptor smoothened cell-autonomously induces multiple ventral cell types[END_REF]. Hedgehoginteracting protein (Hip) is a transmembrane protein attenuating Shh signaling by binding N-Shh with high affinity (Chuang and McMahon, 1999), whereas vitronectin, an extracellular matrix glycoprotein, enhances Shh activity (Pons and Marti, 2000).

Within the nucleus of Shh responding cells, zinc-finger transcription factors of the Gli family GL1/GL2/GLI3 are expressed [START_REF] Hynes | Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1[END_REF][START_REF] Ruiz I Altaba | Combinatorial Gli gene function in floor plate and neuronal inductions by Sonic hedgehog[END_REF]Cohen M2013). Combinatory effect of Gli1/Gli2 activation or Gli3 repression in different areas of the neural tube determines neural patterning, proliferation and cell survival [START_REF] Blaess | Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region[END_REF][START_REF] Zervas | Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1[END_REF]). Later on, Shh signaling has to decrease because repressing activity of Gli3 is necessary for neuronal differentiation [START_REF] Wang | Gli3 is required for maintenance and fate specification of cortical progenitors[END_REF]. Interestingly, decreasing Shh signaling mediates the switch from symmetric proliferative and asymmetric self-renewing divisions to symmetric neurogenic divisions through transcription of the transcription factor Hes1 [START_REF] Saade | Sonic hedgehog signaling switches the mode of division in the developing nervous system[END_REF]Dave RK, 2011). Shh mutant mice lack ventral structures in the CNS and die soon after birth (Machold R, Hayashi S 2003). However, Nestin-Cre transgenic mouse driving Shh or Smo conditional deletion in NSCs shows largely normal ganglionic eminences (Machold R, Hayashi S 2003), but disruption of postnatal neurogenic niches caused by proliferation decrease and increased apoptosis. (Balordi F, Fishell G. 2007). Interestingly, the function of Shh is dependent on the primary cilium. Ablation of the ciliary motor protein KIF3A block the formation of the primary cilia in NSC and results in decreased Shh signaling similar of Smo mutant mice animals (Han YG, Spassky 2008). Indeed, smo is needs to be translocated to the primary cilia to siplay its proper function (Breunig JJ, 2008). In the adult ventricular and subventricular zone and dentate gyrus, NSC are Shhresponsive expressing Ptc and Smo (Traiffort E, Charytoniuk D1999 1998). However in the adult, Shh expressing regions are rare and disparate with high Shh transcript found mainly in the basal forebrain and the brainstem [START_REF] Traiffort | Discrete localizations of hedgehog signalling components in the developing and adult rat nervous system[END_REF]. However, Shh protein is also detected in neurogenic niches. In the adult SVZ, the Shh-is expressed in neurons and is detected in the cerebrospinal fluid (Ihrie RA, Shah JK,2011). In the dentate gyrus, Shh protein is also detected in the subgranular zone (Ihrie RA, Shah JK,2011). Interestingly, Shh can be anterogradely transported in neurons and tranfered to neighboring cells, thus suggesting a transport mechanism to cells distant from the ventricule (Traiffort E, Moya KL, 2001). Finaly, a recent study in the limb bud showed that Shh can be transferred from one cell to another through a long extracellular filipodia, a mechanism that could also exist in the brain (Sanders TA 2013).

Shh is a well known morphogens that confer positional information to the NSC depending on the local concentration [START_REF] Rogers | Morphogen gradients: from generation to interpretation[END_REF]. The classic ‗‗ French flag model'' [START_REF] Wolpert | Positional information and the spatial pattern of cellular differentiation[END_REF] was recently updated. Indeed, a recent study using timelapse microscopy in developing zebrafish embryos tracked the relationships between Shh-GFP reporter activity and cell type patterning during the neural tube development [START_REF] Xiong | Specified neural progenitors sort to form sharp domains after noisy Shh signaling[END_REF]. Stricklingly, they found that specification of precursors does not occur in a highly ordered manner as expected. Instead, the progenitors of different lineages are mixed and specified in a salt-and-pepper pattern. Pattern precision is then recovered upon cell reorganization and migration along the dorsal-ventral axis. This reorganization is mediated by cadherin, suggesting that cellular adhesion is involved in cell sorting. In this revised -French flag model‖, precise patterning arises from early heterogeneous specification that is corrected by subsequent positional sorting. [START_REF] Xiong | Specified neural progenitors sort to form sharp domains after noisy Shh signaling[END_REF]Little SC.2013). triggers the expression of Olig2, Nkx2.2, Nkx6.1 and in restricted progenitor domains (pMN, that each generate specific neuronal and glial subtypes. Embryonic OPCs arise from the pMN domain and is Shh dependent. A dorsal OPCgenerating domain is active that is Shh-independent (Litlle SC 2013).

Notch signaling

Notch signaling pathway plays a central role in the regulation of embryonic and adult neurogenesis (Itaru Imayoshi 2008;Kageyama 2008). In the embryonic nervous system, NSC initially undergo proliferation only, then a subset of cells undergo neuronal differentiation, while others remain as progenitors for later neurogenic and gliogenic phases. During development, these processes proneural genes and Notch ligand genes are expressed at heterogenous levels in the ventricular zone also forming a ‗salt-and-pepper' gene expression pattern. This process, called lateral inhibition [START_REF] Ishibashi | Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects[END_REF]. In this process, the membrane Notch ligands Delta and Jagged activate Notch receptors on adjacent cells. The Notch intracellular domain (NICD) is then cleaved from the membrane region of on cell and is transferred to the nucleus, where NICD forms a complex with RBPj and Maml [START_REF] Jarriault | Signalling downstream of activated mammalian Notch[END_REF][START_REF] Honjo | The shortest path from the surface to the nucleus: RBP-J kappa/Su(H) transcription factor[END_REF][START_REF] Kopan | The canonical Notch signaling pathway: unfolding the activation mechanism[END_REF]. The NICD/RBPj/ Maml complex induces Hes1 and Hes5 genes. Hes genes are encode bHLH transcriptional repressors [START_REF] Sasai | Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split[END_REF][START_REF] Kageyama | The Hes gene family: repressors and oscillators that orchestrate embryogenesis[END_REF]. Through their bHLH domain, Hes factors form homodimers or heterodimers with Hes-related bHLH factors and bind to the DNA sequences called the N box (CACNAG) or the class C site (CACG(C/A)G) [START_REF] Sasai | Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split[END_REF][START_REF] Iso | HES and HERP families: multiple effectors of the Notch signaling pathway[END_REF][START_REF] Kageyama | The Hes gene family: repressors and oscillators that orchestrate embryogenesis[END_REF]. Hes1 and Hes5 directly repress proneural Ascl1 or Neurog2 expression, thereby inhibiting neural differentiation and leading to the maintenance of NSCs pool. Ciani, P.C. Salinas,2005;Faigle, Roland, 2013). In the absence of Wnt ligand, activated glycogen-synthetase-kinase-3 beta (GSK-3beta), forms a degradation complex, comprising axin, adenomatous polyposis coli (APC), and beta-transducing repeat-containing protein (beta-TrCP), which results in a phosphorylation and an ubiquitination of beta-catenin that lead to its degradation by the proteosome (R.H. 57 Chen, W.V. [START_REF] Chen | Wnt signaling to beta-catenin involves two interactive components. Glycogen synthase kinase-3beta inhibition and activation of protein kinase C[END_REF]. Continuous degradation of beta-catenin in the absence of Wnt maintains a low intracellular beta-catenin level (Faigle, Roland, 2013). However, in presence Wnt ligand, its receptor Frizzled and its co-receptor LRP5/6, activating phosphorylation of Dishevelled (Dvl) inactivates GSK-3beta by sequestering GSK-3beta from the degradation complex. (K. Tamai, M. Semenov, 2000). Beta-catenin is then stabilized and enters the nucleus to associates with TCF/LEF transcription factors which activate target genes (C.Y. [START_REF] Logan | The Wnt signaling pathway in development and disease[END_REF].

Neural stem cell diversity and cell fate competency

Embryonic and early post-natal radial glial cells

Neural stem cells (NSC) generate all the neurons and glial cells that build the nervous system [START_REF] Mcconnell | Strategies for the generation of neuronal diversity in the developing central nervous system[END_REF]Breunig et al., 2011, Kriegstein and[START_REF] Kriegstein | The glial nature of embryonic and adult neural stem cells[END_REF]. After a first neurogenic phase, NSCs undergo gliogenesis producing oligodendrocytes progenitor cells (OPC) and astrocytes [START_REF] Fujita | Transitory differentiation of matrix cells and its functional role in the morphogenesis of the developing vertebrate CNS[END_REF][START_REF] Fujita | The discovery of the matrix cell, the identification of the multipotent neural stem cell and the development of the central nervous system[END_REF][START_REF] Miller | Oligodendrocyte origins[END_REF]Temple 2001Noctor et al., 2004). The first NSCs to arise are the radial neuroepithelial cells that divide symmetrically to generate the neural plate and the neural tube. Neuroepithelial cells are oriented with a basal side attached to the pial surface and an apical side that contacts the lumen of the neural tube. After an early phase of neurogenesis, they transform into radial glial cells (RGC) increasing the size of their pial-contacting processes and through pseudostratification of the neuroepithelium by a switch from tight junctions to adherens junctions [START_REF] Aaku-Saraste | Neuroepithelial cells downregulate their plasma membrane polarity prior to neural tube closure and neurogenesis[END_REF][START_REF] Stoykova | Pax6-dependent regulation of adhesive patterning, Rcadherin expression and boundary formation in developing forebrain[END_REF].

RGC express specific markers such as Nestin ,Glast,Tenascin C, vimentin , BLBP and glycogen granules [START_REF] Morest | Precursors of neurons, neuroglia, and ependymal cells in the CNS: what are they? Where are they from? How do they get where they are going?[END_REF][START_REF] Gadisseux | Glial-neuronal relationship in the developing central nervous system. A histochemical-electron microscope study of radial glial cell particulate glycogen in normal and reeler mice and the human fetus[END_REF][START_REF] Hartfuss | Characterization of CNS precursor subtypes and radial glia[END_REF]. First observed by Camillo Golgi in 1885, it is his student Ramon y Cajal who first suggested the glial identity for these cells based on the observation of morphological similarities with astrocytes. Later confirmed, RGC were shown to share with astrocytes GFAP expression, the main astrocyte marker [START_REF] Choi | Radial glia of developing human fetal spinal cord: Golgi, immunohistochemical and electron microscopic study[END_REF][START_REF] Levitt | Immunoperoxidase localization of glial fibrillary acidic protein in radial glial cells and astrocytes of the developing rhesus monkey brain[END_REF] and the their ability to tightly contact endothelial cells (Taka1hashi et al. 1990[START_REF] Misson | The alignment of migrating neural cells in relation to the murine neopallial radial glial fiber system[END_REF]. Many RGC subtypes have been identified, including tanycytes (hypothalamus), Müller glia (retina) and Bergmann glia (cerebellum) [START_REF] Guo | Early postnatal GFAP-expressing cells produce multilineage progeny in cerebrum and astrocytes in cerebellum of adult mice[END_REF][START_REF] Surzenko | SOX2 maintains the quiescent progenitor cell state of postnatal retinal Muller glia[END_REF]. Later on, Pasko Rakic demonstrated the multifunctionality of RGC showing that the pial surface-contacting processes was used as guidance cables for neuronal migration [START_REF] Rakic | Mode of cell migration to the superficial layers of fetal monkey neocortex[END_REF][START_REF] Xu | Bergmann glia function in granule cell migration during cerebellum development[END_REF]. During development, RGC proliferate to form the ventricle zone (VZ) and subventricular zones (SVZ), a secondary germinal zone. RGC constitute the large majority of mitotic cells in ventricular zones [START_REF] Lui | Development and evolution of the human neocortex[END_REF]Malatesta and Gotz, 2013;[START_REF] Pilz | Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type[END_REF].

RGC undergo asymmetric division giving rise to one RGC and one differentiating cell or an intermediate progenitor. Intermediate progenitors then migrate into the subventricular zone (SVZ), to further proliferate [START_REF] Götz | The cell biology of neurogenesis[END_REF][START_REF] Miller | Timing is everything: making neurons versus glia in the developing cortex[END_REF]. Recently, a new type of RGC, called outer SVZ progenitors (oSVZ), has been identified [START_REF] Hansen | Neurogenic radial glia in the outer subventricular zone of human neocortex[END_REF][START_REF] Fietz | OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling[END_REF]Wang et al., 2011;[START_REF] Shitamukai | Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors[END_REF]. The expansion of the SVZ, and OSVZ progenitors, is the major cause of cortical expansion observed in mammals [START_REF] Abdel-Mannan | Evolution of cortical neurogenesis[END_REF][START_REF] Fish | Making bigger brains-the evolution of neuralprogenitor-cell division[END_REF][START_REF] Kriegstein | Patterns of neural stem and progenitor cell division may underlie evolutionary cortical expansion[END_REF].

Most of the RGC exhibit interkinetic nuclear migration (INM), a migration of their nucleus between the apical and the basal side in synchrony with their cell cycles.

During cell division (M phase), the nuclei are located at the apical surface close to the ventricule. Then, they migrate basally during the G1 phase to remain in the basal region of the VZ during the S phase. In the G2 phase, the nuclei migrate back apically to undergo a second cell division when reaching the apical surface [START_REF] Lamonica | Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex[END_REF]Tsai et al., 2010[START_REF] Taverna | The Cell Biology of Neurogenesis: Toward an Understanding of the Development and Evolution of the Neocortex[END_REF]. Observation of the INM directly by in-vivo time-lapse video imaging showed that INM has an intimate correlation with neurogenesis (Noctor SC, Flint AC 2001). However, some subtypes of neural progenitors do not exhibit INM [START_REF] Kosodo | Interkinetic nuclear migration: beyond a hallmark of neurogenesis[END_REF]). If INM is inhibited, cell cycle progression continues [START_REF] Baye | Nuclear migration during retinal development[END_REF][START_REF] Murciano | Interkinetic nuclear movement may provide spatial clues to the regulation of neurogenesis[END_REF]. However, RGC morphology seems to be involved in the long term maintenance of high proliferative capacity (Fietz SA, 2010). Notch signaling, which is known to prevent progenitor cells from differentiating, predominantly localizes to the apical side [START_REF] Del Bene | Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient[END_REF][START_REF] Murciano | Interkinetic nuclear movement may provide spatial clues to the regulation of neurogenesis[END_REF]. Interstingly, oSVZ SVZ cells undergo also some nuclear movements [START_REF] Fietz | OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling[END_REF][START_REF] Hansen | Neurogenic radial glia in the outer subventricular zone of human neocortex[END_REF].

Much work has been done to understand why the nucleuses of neuroepithelial cells migrate toward the apical surface for mitosis. First postulated by Sauer in 1935:

-mitosis are confined to the region of the lumen not because only nuclei of that region divide, but because a nucleus that is about to divide moves to the region of the lumen to do so‖ [START_REF] Sauer | Mitosis in the neural tube[END_REF]. Indeed, in neuroepithelial cells and RGC, the apical plasma membrane is the location of the primary cilium, an organelle with a key role for setting up the mitotic spindle orientation [START_REF] Chenn | Intrinsic polarity of mammalian neuroepithelial cells[END_REF][START_REF] Farkas | The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development[END_REF][START_REF] Tamai | Pax6 transcription factor is required for the interkinetic nuclear movement of neuroepithelial cells[END_REF][START_REF] Dubreuil | Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1[END_REF]. The primary cilium persists until the onset of mitosis and provides, via its basal body, the necessary anchor for the centrosomes which will drive mitotic spindle orientation [START_REF] Dubreuil | Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1[END_REF][START_REF] Santos | Building it up and taking it down: the regulation of vertebrate ciliogenesis[END_REF]Reiter, 2008, Chenn et al., 1998;[START_REF] Farkas | The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development[END_REF][START_REF] Tamai | Pax6 transcription factor is required for the interkinetic nuclear movement of neuroepithelial cells[END_REF]. Despite its small size (1-5um), primary cilia are also important sensory organelle that detects signals in the ventricular cerebrospinal fluid such as IGF and Shh and PDGFA signalings (Lehtinen MK, Walsh CA, 2011;Willaredt MA, Tasouri E, Tucker KL 2013).Thus, the main function of INM is to drive pseudostratification of the V and maximize the apical progenitors mitosis by allowing the cell nucleus to reach the centrosome [START_REF] Fish | Making bigger brains-the evolution of neuralprogenitor-cell division[END_REF]. The second main function is to control the extent to which RGC nuclei are exposed to different proliferative or differentiating signals (Taverna, Elena 2014). Interestingly, ventral RGC have a very different behavior.

Basally dividing cells quickly outnumber the apical mitosis.These subapical progenitors (SAPs) increase from 7% (LGE) and 10% (MGE) at E14 to 20% at E16 to reach 60-70% of the total divinding cell in the LGE and MGE respectively. This particular mechanism contributes to SVZ enlargement of the ventral telencephalon and the generation of all the GABAergic interneurons as well as the early generation of oligodendrocytes progenitor cells (Pilz GA 2013).

Neural stem cells in the adult subventricular zone

In adult mammals, neurogenesis is maintained in the SVZ of the lateral ventricles (Altman, J. and Das, G.D. (1965) ; Kuhn, H.G. et al. (1996[START_REF] Jessberger | H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions[END_REF]. Indeed, neurogenic niches are conserved in the adulthood by maintenance of RGC-derived NSCs and ependymal cells [START_REF] Spassky | Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis[END_REF]. The adult SVZ contains slowly dividing NSCs (B cells), which give rise to actively proliferating cells (C cells) that function as the intermediate precursor cell [START_REF] Doetsch | Subventricular zone astrocytes are neural stem cells in the adult mammalian brain[END_REF]. Type C cells give rise to immature neuroblasts (A cells) migrating in chains to the olfactory bulb in the rostral migrotary stream (RMS) where they differentiate into interneurons (Lois & AlvarezBuylla 1993, Alvarez-Buylla, A 2004). B cells display characteristics of astroglial cells, such as GFAP, GLAST, and other astroglial markers [START_REF] Platel | Control of neuroblast production and migration by converging GABA and glutamate signals in the postnatal forebrain[END_REF]). B cells maintain some important properties of RGC such as long basal processes oriented tangentially which end feet contacting blood vessels, cell bodies located in the ependymal cell layer with short apical processes extending on the ventricle or apical endings forming junctional complexes among themselves similarly to those of RGC (A Kriegstein;2009). The ventricular surface observation reveals a striking -pinwheel organization‖ containing the apical endings of B cells and in its periphery two types of Cells undergo mitosis at the ventricular surface where the primary cilia and the associated centromere was localized. Upon G1, they move toward the pial surface and receive less Shh and Notch signaling. This mechanism allow cells to differentiate and to migrate radialy toward the cortex thanks to the RGC processes (adapted from GA Pilz 2013;[START_REF] Mirzadeh | Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain[END_REF]Paridaen JTML, 2014).

Neural stem cells in the adult hippocampus

Another major adult neurogenesis region in the adult mammalian brain is the dentate gyrus of the hippocampus [START_REF] Altman | Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats[END_REF][START_REF] Kaplan | Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs[END_REF][START_REF] Gage | Mammalian neural stem cells[END_REF][START_REF] Kempermann | Milestones of neuronal development in the adult hippocampus[END_REF]. Adult neurogenesis in the dentate gyrus has been involved in memory, mood disorder and epilepsy [START_REF] Shors | Neurogenesis may relate to some but not all types of hippocampal-dependent learning[END_REF][START_REF] Zhao | Mechanisms and functional implications of adult neurogenesis[END_REF][START_REF] Dranovsky | Hippocampal neurogenesis: regulation by stress and antidepressants[END_REF][START_REF] Parent | Aberrant seizureinduced neurogenesis in experimental temporal lobe epilepsy[END_REF]. Adult neurogenesis takes place in the subgranular zone (SGZ). The SGZ contains two types of dividing cells: Radial astrocytes and darkly stained small cells [START_REF] Altman | Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats[END_REF][START_REF] Kaplan | Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs[END_REF][START_REF] Cameron | Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat[END_REF][START_REF] Palmer | Vascular niche for adult hippocampal neurogenesis[END_REF]. Radial astrocytes in the SGZ, also called type I progenitors [START_REF] Filippov | Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes[END_REF][START_REF] Fukuda | Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus[END_REF], have a long process crossing the granule cell layer as well as small horizontally oriented processes [START_REF] Kosaka | Three-dimensional structure of astrocytes in the rat dentate gyrus[END_REF][START_REF] Seri | Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus[END_REF]). Nestin [START_REF] Seri | Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus[END_REF][START_REF] Steiner | Type-2 cells as link between glial and neuronal lineage in adult hippocampal neurogenesis[END_REF]. Similarly to the SV , the Radial astrocytes express NSC markers like Nestin….and contact the vascular system [START_REF] Palmer | Vascular niche for adult hippocampal neurogenesis[END_REF]. Radial astrocytes first generate intermediate progenitor cell, the small darkly stained cells called type II progenitors [START_REF] Filippov | Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes[END_REF][START_REF] Fukuda | Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus[END_REF] and differentiate into neurons characterized by the expression of doublecortin, PSA-NCAM, Tuc4, NeuroD, Prox1, and NeuN [START_REF] Seki | Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat[END_REF][START_REF] Fukuda | Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus[END_REF][START_REF] Seri | Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus[END_REF]) to progressively acquire electrophysiological characteristics of gabaergic neurons [START_REF] Song | Contribution of T-type VDCC to TEA-induced long-term synaptic modification in hippocampal CA1 and dentate gyrus[END_REF][START_REF] Filippov | Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes[END_REF]. In addition to their role as neuronal precursors, radial astrocytes maintain their classical astrocytic functions of metabolic support Neurogenesis in the dentate gyrus is regulated by adrenal steroids [START_REF] Gould | Adrenal hormones suppress cell division in the adult rat dentate gyrus[END_REF][START_REF] Cameron | Adrenal steroids and N-methyl-D-aspartate receptor activation regulate neurogenesis in the dentate gyrus of adult rats through a common pathway[END_REF], glutamate signaling [START_REF] Gould | Blockade of NMDA receptors increases cell death and birth in the developing rat dentate gyrus[END_REF], seizures [START_REF] Parent | Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus[END_REF], enriched environment and exercise [START_REF] Kempermann | More hippocampal neurons in adult mice living in an enriched environment[END_REF]1998), inflammation [START_REF] Monje | Inflammatory blockade restores adult hippocampal neurogenesis[END_REF]antidepressants (Santarelli et al. 2003).

Oligodendrocytes progenitor cells are not adult neural stem cells

Besides self-renewal and multipotency capacities, other characteristics define stemness property. Indeed, stem cells need to have the capacity to clonally regenerate multiple different cell types in the tissue where they are, the ability to undergo asymmetric cell division and the potential to exist in as mitotically quiescent [START_REF] Hall | Stem cells: the generation and maintenance of cellular diversity[END_REF][START_REF] Morrison | Regulatory mechanisms in stem cell biology[END_REF][START_REF] Potten | Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt[END_REF]. To determine if OPCs could fall into this definition, we have to look if they fit all the required criteria. At the time of their discovery by Raff et al in 1983, OPCs were already considered as bipotential and able to generate both oligodendrocytes and type 2A astrocytes in vitro.

Later on, OPCs were shown to produce free floating balls of cells in-vitro called neurospheres whose clonal cell analysis showed a tri-potent capacity able to generate oligodendrocytes, astrocytes and neurons [START_REF] Kondo | Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells[END_REF]. However, we had to wait until recently to prove the OPCs asymmetric division capacity. A study using in vivo time lapse imaging showed that even if all OPCs visualised were generating oligodendrocytes, only 7 % differentiated directly after a cell division [START_REF] Hughes | SHH signaling acts as a chemoactrant through binding of the Patched-1 receptor (Mercha´n et al[END_REF]. However, another study bring clear evidence that the proteoglycan NG2 segregates asymmetrically during OPCs cell division and generate two distinct daughter cells, one being OPC, the other differentiating OPC [START_REF] Sugiarto | Asymmetry-defective oligodendrocyte progenitors are glioma precursors[END_REF].

Thus, these studies validate the self-renewal and multipotency capacities of OPCs.

The rate of OPCs cell division is dependent on their location in the CNS and declines with age (Young et al., 20013). Moreover, in response to stress or injury OPCs become activated and undergo proliferation and differentiation [START_REF] Zawadzka | CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination[END_REF]. Thus these studies demonstrate the quiescent capacity of OPCs as well as their tissue regenerating capacities. Finaly, Asymmetric cell division would imply an in-vivo differentiation into a different cell type i.e neuron, astrocytes or NSC. However, invivo evidence of physiological in-vivo asymmetric cell division is still lacking despite great effort to find such occurrence.Indeed, the only occurrence of asymmetric cell division was found on a verbB p53+/-mice where FACSorted NG2+ OPCs gave rise to a Nestin+/GFAP+ glioma precursor [START_REF] Sugiarto | Asymmetry-defective oligodendrocyte progenitors are glioma precursors[END_REF]. In conclusion, even if many of stem cell properties are exhibited by OPCs, they cannot be defined as true neural stem cells.

Mechanisms of Intrinsic and extrinsic regulations of neural stem cells

Stem cell niche, stemness maintance and growth factors

The NSC niche is composed of neural and vascular cells communicating by autocrine , paracrine or mechanical signallings.The embryonic NSCs niche is higly active during development but is maintained in a more quiescent stage in specif parts of the adult brain. Many factors regulate stemness maintenance and proliferation in NSCs niches (for review SJ Goldberg 2013). FGF and EGF are the main growth factors for the in vitro isolation of NSCs from the embryonic and adult brain. bFGF (FGF2) generate neurospheres in culture which differentiate into astrocytes, oligodendrocytes and neurons. Cell passaging generates secondary and tertiary neurospheres proving their self-renewal capacity. E8 to E10 NSCs are aleready responsive to bFGF rather than EGF. Similarly, EGF is a growth factor driving proliferation and NSCs survival.

Mitotic spindle orientation and asymmetric segregation of cellular components

During neurogenesis, NSCs can divide in one of three basic modes: symmetric proliferative (2NSCs) or differentiative (2 postmitotic cells) or asymmetric (1NSC and 1postmitotic cell). Recent studies have shown a link between cleavage orientation, centrosome asymmetries, ciliogenesis, asymmetric segregation of cellular components and cell fate [START_REF] Paridaen | Neurogenesis during development of the vertebrate central nervous system[END_REF]. In the vertebrate developing brain, while early RGC symmetrical divisions feature cleavage planes perpendicular to the ventricular surface, later asymmetric divisions demonstrate oblique cleavage planes (Postiglione MP2011, LaMonica BE,2013). The spindle orientation of symmetric RGC divisions, regulated by mechanisms involving centrosomes, microtubule positioning, and proteins of the internal plasma membrane, is essential to maintain symmetric RGC divisions [START_REF] Konno | Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis[END_REF]Peyre E, Jaouen F,2011). For example, mutant mice for Lis1 severely disrupts the expansion of RGC pool by inducing random cleavage planes (Xie Y, Juschke C, 2013 ). Many proteins segregate differentialy during asymmetric cell division. For example, notch signaling proteins (Notch, Dll1/3, Mindbomb, Numb) are differentially segregated between daughter cells and lead to differential Notch signaling between daughter cells, thus driving different cell fate (Bultje RS, 2009[START_REF] Kawaguchi | Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis[END_REF] In addition, the mouse Par3 protein segregates asymmetrically into the daughter cell that will remain RGC (Bultje RS 2009).

The centrosome inheritance is an other mechanism regulating asymmetric cell division.

During interphase cells possess one mother and one daughter centriole constituting the centrosome. The mother centriole is the oldest centriole and mediates nucleation of the primary cilium while older centrioles are inherited by daughter. Interestingly, cells that inherit daughter centriole maintain their stem cell identity (Wang X, Tsai jw, 2009). A recent study shows the mother centriole is able to retain ciliary membrane and reforms a new cilium before its sister cell (Paridaen JT 2013). This earlier cilium reformation is thought to allow earlier ciliary signaling involved in RGC daughter cell fate such as IGF-1. Moreover, differentiating daughter cells form their primary cilia at their basolateral instead of their apical side, thus leading to asymmetrical daughter cell behavior (Wilsch-Brauninger M 2012).

Cell cycle length of neural precursors regulation of cell fate choice

Changes in cell cycle length have been implicated in cell fate decision (Dehay C,2007;[START_REF] Salomoni | Cell cycle control of mammalian neural stem cells: putting a speed limit on G1[END_REF]. Cell cycle length in the mouse ventricular zone increases from 8 hours at the onset of neuron production at E11 to 18 hours at E16. This increase, due to a lengthening of G1 from 3 to 13 hours [START_REF] Takahashi | The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall[END_REF], is correlated with an increase in asymmetric division (Arai Y, Pulvers JN 2011;Lange C, Huttner WB,2009). To convert this correlation into causal explanation, a recent study, manipulation of overexpression cdk4/cyclinD1, two proteins thought have only a function in cell cycle control, lengthen the G1 phase, decreased neurogenesis at the expanse of RGCs. Conversely, their downregulation led to the opposite effects (Calegari, F. and Huttner, W.B. (2003), [START_REF] Lange | Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors[END_REF]).

Proneural basic helix-loop-helix factors (bHLHs) transcription factors in neural differentiation.

Basic helix-loop-helix transcription factors (bHLH) were found to play central roles in self-renewal of NSC and fate determination of neurons, oligodendrocytes, and astrocytes [START_REF] Bertrand | Proneural genes and the specification of neural cell types[END_REF][START_REF] Namihira | Mechanisms of astrocytogenesis in the mammalian brain[END_REF]. Indeed, early studies in Drosophila discovered ‗proneural' genes of the achaetescute complex [START_REF] Garcia-Bellido | Genetic analysis of maternal information in Drosophila[END_REF]. bHLH function through dimerization with ubiquitously expressed bHLH E-proteins such as E12, E47, HEB or TCF4 thanks to thei helix domain, and subsequent binding a to DNA sequence called the Ebox (CANNTG) thanks to their basic domain [START_REF] Murre | Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence[END_REF][START_REF] Bertrand | Proneural genes and the specification of neural cell types[END_REF]. The mammalian homolog of drosophilia achaetescute complex genes (Ascl1), also called Ascl1 [START_REF] Johnson | Two rat homologues of Drosophila achaete-scute specifically expressed in neuronal precursors[END_REF], is now recognized as the master regulator of neurogenesis [START_REF] Bertrand | Proneural genes and the specification of neural cell types[END_REF]. Indeed, Ascl1 is required for specific subsets of neurons or oligodendrocytes in spinal cord, telencephalon, mesencephalon, hindbrain, olfactory sensory epithelium, as well as in the autonomic nervous system [START_REF] Guillemot | Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons[END_REF][START_REF] Blaugrund | Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers and Mash-1-dependence[END_REF][START_REF] Cau | Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors[END_REF][START_REF] Hirsch | Control of noradrenergic differentiation and Phox2a expression by MASH1 in the central and peripheral nervous system[END_REF][START_REF] Casarosa | Mash1 regulates neurogenesis in the ventral telencephalon[END_REF][START_REF] Akagi | Requirement of multiple basic helix-loop-helix genes for retinal neuronal subtype specification[END_REF][START_REF] Miyoshi | Identification of a novel basic helix-loop-helix gene, Heslike, and its role in GABAergic neurogenesis[END_REF][START_REF] Pattyn | Ascl1/Mash1 is required for the development of central serotonergic neurons[END_REF][START_REF] Helms | Sequential roles for Mash1 and Ngn2 in the generation of dorsal spinal cord interneurons[END_REF][START_REF] Parras | The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes[END_REF][START_REF] Sugimori | Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord[END_REF][START_REF] Sugimori | Ascl1 is required for oligodendrocyte development in the spinal cord[END_REF]. Other bHLH proneural genes such as Atoh1 (Math1), NeuroD, NeuroM, Neurog1/2, Ptf1a and Olig1/2 were discovered later on and shown to drive different cell type specifications or differentiations [START_REF] Ma | Identification of neurogenin, a vertebrate neuronal determination gene[END_REF][START_REF] Ben-Arie | Math1 is essential for genesis of cerebellar granule neurons[END_REF][START_REF] Lu | Sonic hedgehog--regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system[END_REF]Zhou et al., 2000;[START_REF] Glasgow | Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn[END_REF][START_REF] Ross | Basic helix-loop-helix factors in cortical development[END_REF].While Atoh1 is necessary for interneurons generation in the dorsal spinal cord, granule cells in the cerebellum and glutamatergic neurons in brain stem nuclei and inner ear epithelium [START_REF] Ben-Arie | Math1 is essential for genesis of cerebellar granule neurons[END_REF][START_REF] Gowan | Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons[END_REF][START_REF] Helms | Overexpression of MATH1 disrupts the coordination of neural differentiation in cerebellum development[END_REF][START_REF] Machold | Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors[END_REF], Neurog1/2 are necessary dorsal interneurons in the spinal cord, glutamatergic neurons in the cortex, and dopaminergic neurons in the midbrain [START_REF] Ma | Identification of neurogenin, a vertebrate neuronal determination gene[END_REF][START_REF] Sommer | neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS[END_REF][START_REF] Gowan | Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons[END_REF][START_REF] Parras | Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity[END_REF]Kele et al., 2006), Ptf1a is essential for GABAergic neurons in the dorsal spinal cord, retina and cerebellum [START_REF] Glasgow | Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn[END_REF][START_REF] Hoshino | Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum[END_REF][START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF]. First, Ascl1 and Neurog1 drive differentiation through cell cycle exit thanks to the expression of p27 (Kip1), a cyclin-dependent kinase inhibitor [START_REF] Farah | Generation of neurons by transient expression of neural bHLH proteins in mammalian cells[END_REF].

Second, Ascl1 and Neurog1 trigger neuronal differentiation through direct and indirect activation neural genes such as NeuroD or NeuroM [START_REF] Fode | The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons[END_REF][START_REF] Cau | Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage[END_REF]. Ascl1 mutant mice display severe neuronal losses and proliferation defects [START_REF] Casarosa | Mash1 regulates neurogenesis in the ventral telencephalon[END_REF][START_REF] Horton | Correct coordination of neuronal differentiation events in ventral forebrain requires the bHLH factor MASH1[END_REF][START_REF] Wildner | dILA neurons in the dorsal spinal cord are the product of terminal and nonterminal asymmetric progenitor cell divisions, and require Mash1 for their development[END_REF][START_REF] Battiste | Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord[END_REF].

Conversely, overexpression of Ascl1 in neural cells drive cell cycle exit and early expression of neuronal specific markers such as Tuj1 or NeuN [START_REF] Farah | Generation of neurons by transient expression of neural bHLH proteins in mammalian cells[END_REF][START_REF] Nakada | Distinct domains within Mash1 and Math1 are required for function in neuronal differentiation versus neuronal cell-type specification[END_REF][START_REF] Helms | Sequential roles for Mash1 and Ngn2 in the generation of dorsal spinal cord interneurons[END_REF][START_REF] Kriks | Gsh2 is required for the repression of Ngn1 and specification of dorsal interneuron fate in the spinal cord[END_REF]. Knock-in experiment where Neurog2 coding sequence was replaced by Ascl1 demonstrated distinct roles in the specification of glutamatergic and GABAergic neurons in dorsal and ventral telencephalon, respectively [START_REF] Parras | Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity[END_REF]. A recent study demonstrated that Ascl1 is regulated by multisite phosphorylation on conserved serines-proline pairs by Cdk kinase activity during cell cycle and that dephosphorylation increase cell differentiation (Ali FR, Cheng K et al 2014).

Identifying transcriptional targets of bHLH factors is fundamental to provide mechanistic insights of bHLH functions in neurogenesis. However, it has been difficult to identify and validate Ascl1 direct in vivo downstream targets just based on the degenerate 6 base pair E-box DNA motif. With advanced technology using chromatin immuno-precipitation , microarray and bioinformatics, several in vivo transcriptional targets of bHLHs are beginning to be identified. Indeed, Notch ligands such as Dll1 and Dll3 were shown to be transcriptional targets of Ascl1 and Ascl1 interacts with Brn1/2 or Neurog2 on the promoter of Dll1 and Dll3 [START_REF] Castro | Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif[END_REF][START_REF] Henke | Neurog2 is a direct downstream target of the Ptf1a-Rbpj transcription complex in dorsal spinal cord[END_REF].

The Hes HLH family, the mammalian homologues of Drosophila hairy and Enhancer of split gene, and Ids, inhibitors of differentiation are transcriptional repressors. Hes1, Hes3, and Hes5 are expressed in undifferentiated neural stem cells of the VZ (Kageyama et al., 2008a). In mice mutant for Hes genes, Ascl1 and Neurog2 are upregulated and neuronal differentiation is promoted. The mechanism leads to a rapid depletion of the NSC pool [START_REF] Ishibashi | Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects[END_REF][START_REF] Chen | Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression[END_REF]. Conversely, overexpression of Hes1 or Hes5 inhibits neurogenesis [START_REF] Ohtsuka | Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation[END_REF][START_REF] Cau | Hes genes regulate sequential stages of neurogenesis in the olfactory epithelium[END_REF]. Hes proteins were shown to bind Ascl1 enhancer regions and repress their transcription [START_REF] Ishibashi | Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects[END_REF][START_REF] Chen | Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression[END_REF]. Interesingly, Hes proteins have oscillatory expression thanks to a cell autonomous negative auto-feedback loop [START_REF] Kageyama | The Hes gene family: repressors and oscillators that orchestrate embryogenesis[END_REF]. Ids proteins are HLH protein lacking the basic DNA binding domain. Ids proteins are able to inhibit neural differentiation working as a dominant negative heterodimere of bHLH proteins [START_REF] Norton | ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis[END_REF]. Ids overexpression inhibits proneural genes and increase Hes1 expression. Interestingly, Ids interact with Hes1 and suppress Hes1 negative feedback loop (Bai et al., 2007).

The Notch signaling pathway and lateral inhibition

Notch signaling pathway is the main mechanism controlling cell cycle exit and differentiation in embryonic and adult neural development [START_REF] Yoon | Notch signaling in the mammalian central nervous system: insights from mouse mutants[END_REF][START_REF] Louvi | Notch signalling in vertebrate neural development[END_REF]. Notch signaling controls neural differentiation through lateral inhibition by controlling the balance between proneural and Hes transcription factors [START_REF] Yoon | Notch signaling in the mammalian central nervous system: insights from mouse mutants[END_REF][START_REF] Louvi | Notch signalling in vertebrate neural development[END_REF].

At early stage, neural progenitors share similar levels of Notch receptor and ligand at their surface. Then, due to stochastic variation, a cell starts to express a higher level of Notch ligand Dll1. Upon activation of Notch receptor in a neighboring cell, the Notch intracellular domain (NICD) is translocated from the membrane to the nucleus. In the nucleus, NICD converts RBPj from a repressor to an activator and form an activating complex with Maml (mastermind-like) driving the expression of Hes1 or Hes5, thus suppressing the transcription Ascl1 or Neurog1/2. As Dll1 is a direct target of Ascl1 and Neurog1/2, Notch ligand expression will follow the same down regulation. Consequently, a cell having higher levels of Dll1 and Ascl1 or Neurog1/2 expression will initiate differentiation whereas a cell having higher Notch activation and Hes gene expression will remain a progenitor. Recently, this classic view of lateral inhibition was improved by the discovery of the Hes/Ascl1 oscillation mechanism (Kageyama et al., 2008b). Indeed, Hes1 expression is periodically upregulated and downregulated with a 2-3 hour frequency [START_REF] Shimojo | Oscillations in notch signaling regulate maintenance of neural progenitors[END_REF]. Dll1 and Neurog2 also oscillate but with an inverse periodicity [START_REF] Shimojo | Oscillations in notch signaling regulate maintenance of neural progenitors[END_REF]. (red), which emits mCherry fluorescence in S/G2/M phases. Time is indicated relative to the timing of cytokinesis. Two cells are generated Dll1-inheriting and non-Dll1inheriting sister cells, respectively differentiation (adapted from [START_REF] Imayoshi | Hes genes and neurogenin regulate nonneural versus neural fate specification in the dorsal telencephalic midline. Development[END_REF][START_REF] Kawaguchi | Dll1 maintains quiescence of adult neural stem cells and segregates asymmetrically during mitosis[END_REF]and EJ Kim 2009).

bHLH factors in oligodendrogenesis

During development, neurons and glia are produced from NSC in a sequential but overlapping manner. Olig1 and Olig2 bHLH transcription factors play a central role in oligodendrocyte specification and differentiation [START_REF] Meijer | Separated at birth? The functional and molecular divergence of OLIG1 and OLIG2[END_REF].

In the developing spinal cord and hindbrain, motor neurons and oligodendrocytes are sequentially produced from the pMN domain [START_REF] Goulding | Circuits controlling vertebrate locomotion: moving in a new direction[END_REF]. In the VZ and SVZ of the ventral part of the telencephalon, the ganglionic eminences, Olig1 and Olig2 are expressed where GABAergic interneurons and oligodendrocyte precursors first emerge [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF]Butt et al., 2005). Unlike Ascl1, the expression of Olig1 and Olig2 is maintained into mature oligodendrocytes. (Zhou et al., 2001;[START_REF] Lu | Oligodendrocyte lineage genes (OLIG) as molecular markers for human glial brain tumors[END_REF].

In the absence of Olig2, the formation of the pMN domain is severely diminished, and both motoneurons and oligodendrocytes are missing, [START_REF] Lu | Sonic hedgehog--regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system[END_REF][START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF][START_REF] Zhou | The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification[END_REF]Takebayashi et al., 2002). While Olig1 is requiered to Repress Dlx1/2 and inhibit GABAergic interneuron formation in the ventral telenephalon (Silbereis 2014), Olig1 knockout mice have broad defects in the maturation of oligodendrocytes [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF][START_REF] Xin | Myelinogenesis and axonal recognition by oligodendrocytes in brain are uncoupled in Olig1-null mice[END_REF] and a limited capacity to repair demyelinated lesions [START_REF] Arnett | bHLH transcription factor Olig1 is required to repair demyelinated lesions in the CNS[END_REF]. Conversely, overexpression of Olig1/2 in NPCs is sufficient to induce the specification of oligodendrocyte precursor cells (OPCs) (Zhou et al., 2001;[START_REF] Lu | Oligodendrocyte lineage genes (OLIG) as molecular markers for human glial brain tumors[END_REF]. Conditional deletions of Olig1 et Olig2 in NSC, OPCs and mature oligodendrocytes, revealed stage-specific regulatory roles. At early developmental stages, Olig2 promotes proliferation of self-renewing NPCs. Later on, Olig2 promotes motoneuron or oligodendrocytes specification [START_REF] Yue | A critical role for dorsal progenitors in cortical myelination[END_REF][START_REF] Cai | A crucial role for Olig2 in white matter astrocyte development[END_REF][START_REF] Zhu | Olig2-dependent developmental fate switch of NG2 cells[END_REF][START_REF] Mei | Stage-specific deletion of Olig2 conveys opposing functions on differentiation and maturation of oligodendrocytes[END_REF].

Interestingly, Olig2 functions mainly as a repressor [START_REF] Novitch | Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2[END_REF]Zhou et al., 2001) but also directly activates the expression of the oligodendrocyte-specific gene Sox10 [START_REF] Küspert | Olig2 regulates Sox10 expression in oligodendrocyte precursors through an evolutionary conserved distal enhancer[END_REF]. Stage-specific functions of Olig2 are regulated by posttranscriptional modifications [START_REF] Setoguchi | Nuclear export of OLIG2 in neural stem cells is essential for ciliary neurotrophic factor-induced astrocyte differentiation[END_REF][START_REF] Meijer | Separated at birth? The functional and molecular divergence of OLIG1 and OLIG2[END_REF].

Serine phosphorylation at the ST box of Olig2 by CK2 kinase activates OPCs generating function [START_REF] Huillard | Disruption of CK2beta in embryonic neural stem cells compromises proliferation and oligodendrogenesis in the mouse telencephalon[END_REF]. By contrast, while serine (S147) phosphorylation in its bHLH domain by PKA drives motor neuron formation, its dephosphorylation is involved in the transition from motor neuron to oligodendrocyte specification in the pMN domain [START_REF] Li | Phosphorylation regulates OLIG2 cofactor choice and the motor neuron-oligodendrocyte fate switch[END_REF]. This phosphorylation in the bHLH domain was shown to favor Olig2 homodimer formation, whereas dephosphorylation favor heterodimer formation with Neurog2. Olig2/ Neurog2heterodimer was shown to sequesters Neurog2 and thus prevents motoneuron formation [START_REF] Li | Phosphorylation regulates OLIG2 cofactor choice and the motor neuron-oligodendrocyte fate switch[END_REF]. In NPCs during the early stage of embryos, Olig2 is phosphorylated at 3 residues whereas in the postnatal white matter, the triple serine motif is nonphosphorylated [START_REF] Sun | Phosphorylation state of Olig2 regulates proliferation of neural progenitors[END_REF].

bHLH factors in astrogenesis

With the exception of SCL (Stem cell leukemia), a bHLH transcription factor, necessary and sufficient to drive specificiation and differentiation of astrocytes in specific regions of the developing neural tube (M Nakanishi 2007), astrocyte fate seems to be the default differentiation path in absence of proneural or oligodendroneural genes [START_REF] Ross | Basic helix-loop-helix factors in cortical development[END_REF][START_REF] Namihira | Mechanisms of astrocytogenesis in the mammalian brain[END_REF]. Indeed, Many studies described the role of Notch/ Hes signaling and Id factors in astrocyte formation, but these factors are not sufficient [START_REF] Cai | Misexpression of basic helix-loop-helix genes in the murine cerebral cortex affects cell fate choices and neuronal survival[END_REF][START_REF] Ohtsuka | Roles of the basic helix-loop-helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing brain[END_REF][START_REF] Tanigaki | Notch1 and Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells to an astroglial fate[END_REF][START_REF] Wu | Hes1 but not Hes5 regulates an astrocyte versus oligodendrocyte fate choice in glial restricted precursors[END_REF][START_REF] Namihira | Committed neuronal precursors confer astrocytic potential on residual neural precursor cells[END_REF]. For example, overexpression of Hes or Id factors in early NSCs fail to prematurely induce astrocyte formation [START_REF] Ohtsuka | Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation[END_REF][START_REF] Cai | Misexpression of basic helix-loop-helix genes in the murine cerebral cortex affects cell fate choices and neuronal survival[END_REF][START_REF] Ohtsuka | Roles of the basic helix-loop-helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing brain[END_REF][START_REF] Tanigaki | Notch1 and Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells to an astroglial fate[END_REF][START_REF] Wu | Hes1 but not Hes5 regulates an astrocyte versus oligodendrocyte fate choice in glial restricted precursors[END_REF][START_REF] Namihira | Committed neuronal precursors confer astrocytic potential on residual neural precursor cells[END_REF] and conversely, mutant mice for both Ascl1, Neurog2 or olig2 show precoce and increased astrogenesis development [START_REF] Nieto | Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors[END_REF](Nieto et al., , parras et al 2013)). Rather, accumulating evidence indicate that astrocyte fate is driven by extrinsic cues such as leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1) and BMP signaling [START_REF] Namihira | Mechanisms of astrocytogenesis in the mammalian brain[END_REF].

Dynamic Regulation of Proneural bHLH Factors

Ascl1 is dynamically regulated at the mRNA and protein levels. Ascl1 protein is unstable in human NPCs with a half-life of only 14 min but is stabilized to a half-life of 50 min when NICD is mutated [START_REF] Srinivasan | Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II[END_REF]. Moreover, Ascl1 stability is post transcriptionally regulated. Indeed, nonphosphorylated Ascl1 is very unstable, whereas CK2-mediated phosphorylation of S152 stabilizes Ascl1 (Vin˜ als et al., 2004). In the cortex, Pax6 promotes RGC proliferation and spindle orientation [START_REF] Asami | The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex[END_REF], but also triggers neuronal differentiation through the induction of Neurog2 [START_REF] Sansom | The level of the transcription factor Pax6 is essential for controlling the balance between neural stem cell self-renewal and neurogenesis[END_REF]), Tbr1/2 (Götzand Huttner, 2005), Brn1/2, Sox4 and Sox11 [START_REF] Martynoga | Molecular control of neurogenesis: a view from the mammalian cerebral cortex[END_REF].

However, even if Ascl1is predominantly expressed in the ventral telencephalon during embryonic development, dorsal Ascl1 positive cells also gives rise to cortical GABAergic interneurons and oligodendrocytes.

Ascl1 is expressed by NPCs in the ventral telencephalon, in the medial, lateral, and caudal ganglionic eminences. In Ascl1 knockout mice, the production of GABAergic neurons is largely abrogated, with a more profound phenotype in the medial ganglionic eminence [START_REF] Casarosa | Mash1 regulates neurogenesis in the ventral telencephalon[END_REF][START_REF] Horton | Correct coordination of neuronal differentiation events in ventral forebrain requires the bHLH factor MASH1[END_REF]. Conversely, overexpression of Ascl1 induces ectopic GABAergic neurons from dorsal cortical NPCs [START_REF] Berninger | Directing neurotransmitter identity of neurones derived from expanded adult neural stem cells[END_REF][START_REF] Poitras | The proneural determinant MASH1 regulates forebrain Dlx1/2 expression through the I12b intergenic enhancer[END_REF]. Moreover, transient accumulation of Ascl1 in NPCs induces the expression of GABAergic differentiation associated with Dlx1/2/5/6 and Lhx6 expression [START_REF] Yun | Modulation of the notch signaling by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon[END_REF][START_REF] Petryniak | Dlx1 and Dlx2 control neuronal versus oligodendroglial cell fate acquisition in the developing forebrain[END_REF][START_REF] Miyoshi | Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons[END_REF][START_REF] Bartolini | Integration of GABAergic interneurons into cortical cell assemblies: lessons from embryos and adults[END_REF].

Expression dynamics of bHLH factors in multipotency and cell fate choice

Oscillating expression of bHLH factors, including Hes, Ascl1, Neurog2, and Olig2, in NPCs is important to maintain their proliferative and multipotent potentials [START_REF] Imayoshi | Hes genes and neurogenin regulate nonneural versus neural fate specification in the dorsal telencephalic midline. Development[END_REF]. Single cell time-lapse imaging studies showed that in NPCs, Hes1 and Ascl1 protein expression oscillate with a period of 2-3 hr and that Olig2 protein expression oscillates with a 5-8 hr periodicity [START_REF] Shimojo | Oscillations in notch signaling regulate maintenance of neural progenitors[END_REF]. While inactivation of Hes1 abolishes Ascl1 oscillation, it does not affect Olig2 oscillations [START_REF] Imayoshi | Oscillatory control of factors determining multipotency and fate in mouse neural progenitors[END_REF]). An optogenetic approach has been employed to control the sustained Vs oscillatory Ascl1 and Olig2 mode of expressions (Wang, X., Chen, X., and Yang, Y. 2012). In this study, sustained Ascl1 expression enhances neuronal differentiation, whereas oscillatory Ascl1 expression with 3 hr periodicity activates proliferation of NPCs. These results indicate that sustained Ascl1 accumulation during 6-8h in G1 phase is necessary for neuronal fate determination. Thus, distinct expression dynamics of Ascl1 are important for the choice between proliferation and differentiation (Wang, X., Chen, X., and Yang, Y. 2012;[START_REF] Imayoshi | Oscillatory control of factors determining multipotency and fate in mouse neural progenitors[END_REF].

Interestingly, the 3 hr periodicity of Ascl1 oscillation is necessary for NPC proliferation as this effect was not observed with a 6 hr period [START_REF] Imayoshi | Oscillatory control of factors determining multipotency and fate in mouse neural progenitors[END_REF].

Whether the period of oscillatory expression correlates with that of cell cycle remains to be determined. During oligodendrocyte differentiation, oscillatory up-regulation of Olig2 occurs. When Olig2 becomes dominant the expression of Ascl1 and Hes1 is down-regulated [START_REF] Imayoshi | Oscillatory control of factors determining multipotency and fate in mouse neural progenitors[END_REF].

Before asymmetric cell division, where daughter cell remains NSC while the other differentiates, Ascl1, Hes1, and Olig2 are expressed together and oscillate. At the peak of Ascl1 or Olig2 oscillations, cells have a higher tendency to differentiate into neurons or oligodendrocytes respectively. However, this tendency is not decisive as posttranslational modifications are involved in fate choice process. Hes1 expression is first down regulated before cell division, and Ascl1 or Olig2 expression is up regulated in differentiating cells while the daughter NPC resumes Hes1 and Ascl1 oscillations.

As oscillatory Ascl1 can promote cell cycle progression, such periodic production seems to be a driving force for cell-cycle progression, although the exact mechanism remains to be determined.

While sustained Hes1 expression is able to generate quiescent NPCs [START_REF] Sang | Control of the reversibility of cellular quiescence by the transcriptional repressor HES1[END_REF], Hes1 and Ascl1 oscillations may lead to accumulation of downstream factors in NPCs and serve as a molecular clock where the number of pulses may be used to drive differentiation at the right time. Such a molecular clock mechanism is supported by many studies. Indeed, interneurons born at different times would behave differently within the same environment [START_REF] Pla | Layer acquisition by cortical GABAergic interneurons is independent of Reelin signaling[END_REF]D.G. Southwell 2012) and the differentiation timing human IPScell converted into neurons takes several months and thus mimic the human neural development timing (C.R. Nicholas 2013). It has been proposed that the microRNA-9 (miR-9), regulating negatively Hes1 mRNA, accumulate progressively every round of division to a level sufficient to abrogate Hes1 oscillation [START_REF] Bonev | MicroRNA-9 Modulates Hes1 ultradian oscillations by forming a double-negative feedback loop[END_REF]Tan et al., 2012b). Moreover, new links between Hes genes and circadian control genes (Clock/Bmal1, Per1/2) were recently discovered outside the brain (Lee YJ1, Han DH, Pak YK, Cho SH.2012). Taken together, these results show that further analyses are required to determine whether Hes and proneural genes oscillations are involved in the cellular clock mechanism.

Epigenetic mechanism of neural stem cell regulation

NSCs multipotentiality is tightly regulated by DNA methylation. In early NSCs, astrocytic gene promoters are hypermethylated, thus blocking the access of the STAT3-p300 ⁄ CBP-SMADs complex to its target sequence and thus prevent astrocytes differentiation [START_REF] Takizawa | DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain[END_REF]. Later on, demethylation occurs and allows expression of astrocytic gene expression [START_REF] Hatada | High conservation of transcription factor binding and evidence for combinatorial regulation across six Drosophila species[END_REF]). The most likely mechanism for DNA demethylation is the passive loss of CpG methylation due to successive rounds of DNA replication in the absence of DNMT1 methylase activity [START_REF] Bestor | The DNA methyltransferases of mammals[END_REF].Indeed, After conditional deletion of DNMT1 in NSCs lead a precoce astroglial differentiation [START_REF] Fan | DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling[END_REF].

The chromodomain protein family in neural stem cell

The Chromodomain Helicase DNA binding (CHD) gene family is a group of 9 chromatin reader proteins defined by the presence of two N-terminal chromodomains (chromatin modifier domain) and a sucrose non-fermenting (SNF2)-like helicase/ATPase associated with nucleosome remodeling (Feng, Weijun, 2013).

Chromodomains mediate chromatin interactions through the direct binding of DNA and methylated histone H3 [START_REF] Akhtar | Chromodomains are protein-RNA interaction modules[END_REF][START_REF] Bouazoune | The dMi-2 chromodomains are DNA binding modules important for ATPdependent nucleosome mobilization[END_REF][START_REF] Fischle | Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains[END_REF][START_REF] Min | Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27[END_REF][START_REF] Brehm | The many colours of chromodomains[END_REF][START_REF] Flanagan | Double chromodomains cooperate to recognize the methylated histone H3 tail[END_REF][START_REF] Pray-Grant | Chd1 chromodomain links histone H3 methylation with SAGA-and SLIK-dependent acetylation[END_REF][START_REF] Sims | Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains[END_REF][START_REF] Kim | Co-activation of atrial natriuretic factor promoter by Tip60 and serum response factor[END_REF]. The CHD family is further divided into 3 groups [START_REF] Hall | CHD proteins: a diverse family with strong ties[END_REF][START_REF] Marfella | The Chd family of chromatin remodelers[END_REF]. The group 1 is composed of CHD1/2 and which share 2 chromodomains, a SNF2-like helicase/ATPase domain and DNA-binding domains with a divergent N-terminus. The group 2 is composed of CHD3/4/5 and shares two N-terminal PHD zinc finger domains involved in chromatin remodeling [START_REF] Eberharter | ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts[END_REF][START_REF] Ragvin | Nucleosome binding by the bromodomain and PHD finger of the transcriptional cofactor p300[END_REF], Pena et al. 2006[START_REF] Shi | ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression[END_REF][START_REF] Sims | SnapShot: Chromatin remodeling: CHD[END_REF]. The group 3 contains CHD6/7/8/9 and share two BRK domains (Brahma and Kismet domain) with unknown function and a SANT domain (Switching-defective protein 3, Adaptor 2, Nuclear receptor corepressor, Transcription factor IIIB) which bind histone tails [START_REF] Janssen | Mutation update on the CHD7 gene involved in CHARGE syndrome[END_REF][START_REF] Boyer | The SANT domain: a unique histone-tail-binding module? Nature Reviews[END_REF][START_REF] Hall | CHD proteins: a diverse family with strong ties[END_REF].

CHD1 has been shown to assemble and slide nucleosomes [START_REF] Lusser | Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly[END_REF][START_REF] Stockdale | Analysis of nucleosome repositioning by yeast ISWI and Chd1 chromatin remodeling complexes[END_REF], and to associate with factors involved in transcription and centromeric maintenance [START_REF] Simic | Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes[END_REF][START_REF] Walfridsson | A genome-wide role for CHD remodelling factors and Nap1 in nucleosome disassembly[END_REF][START_REF] Konev | CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo[END_REF][START_REF] Okada | CENP-H-containing complex facilitates centromere deposition of CENP-A in cooperation with FACT and CHD1[END_REF]. Chd1 has a crucial role in regulating ESC pluripotency thanks to its ability to bind to H3K4me3 [START_REF] Gaspar-Maia | Chd1 regulates open chromatin and pluripotency of embryonic stem cells[END_REF][START_REF] Flanagan | Double chromodomains cooperate to recognize the methylated histone H3 tail[END_REF][START_REF] Simic | Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes[END_REF]. CHD3 and CHD4 are members of the -nucleosome remodeling and deacetylase‖ (NuRD) complex [START_REF] Mcdonel | Keeping things quiet: roles of NuRD and Sin3 co-repressor complexes during mammalian development[END_REF]. The histone deacetylation activity of NuRD complexes was shown to facilitate the recruitment of PRC2 (polycomb repressor complex 2) mediating the deposition of inhibiting H3K27me3 marks [START_REF] Reynolds | NuRD-mediated deacetylation of H3K27 facilitates recruitment of Polycomb Repressive Complex 2 to direct gene repression[END_REF]. Chd4 is necessary for the localization of Ezh2, the enzymatic subunit of PRC2 and specifically inhibit astrocyte differentiation (Sparman et al 2013). However, recent studies also showed that CHD3/4 proteins were able to activate transcription (Reynolds et al., 2013, Hu and[START_REF] Hu | NuRD and pluripotency: a complex balancing act[END_REF]. As CHD4, CHD5 has been shown to bind unmodified H3K4 [START_REF] Musselman | Binding of the CHD4 PHD2 finger to histone H3 is modulated by covalent modifications[END_REF][START_REF] Paul | Chd5 requires PHD-mediated histone 3 binding for tumor suppression[END_REF]. CHD5 is predominantly expressed in the adult CNS in neurons of the neocortex, but is absent in astrocytes and oligodendrocytes [START_REF] Gao | Defining transcriptional networks through integrative modeling of mRNA expression and transcription factor binding data[END_REF][START_REF] Potts | CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes[END_REF].

CHD5 is a tumor-suppressor gene expressed in differentiating neurons [START_REF] Brodeur | Neuroblastoma: biological insights into a clinical enigma[END_REF][START_REF] Okawa | Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor gene deleted in neuroblastomas[END_REF][START_REF] Bagchi | CHD5 is a tumor suppressor at human 1p36[END_REF][START_REF] Fujita | CHD5, a tumor suppressor gene deleted from 1p36.31 in neuroblastomas[END_REF][START_REF] Gao | Defining transcriptional networks through integrative modeling of mRNA expression and transcription factor binding data[END_REF] and is able to bind to H3K27me3 to activate neuronal differentiation and the repress Polycomb target genes (egan et al 2013). Since the discovery that CHD7 mutations lead to CHARGE syndrome in human, numerous studies have investigated its function.

Several animal models for CHARGE syndrome were created [START_REF] Siebert | Pathologic features of the CHARGE association: support for involvement of the neural crest[END_REF], Bergman et al. 2005[START_REF] Hurd | Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues[END_REF][START_REF] Bajpai | CHD7 cooperates with PBAF to control multipotent neural crest formation[END_REF], Schnetz el al. 2010[START_REF] Zentner | CHD7 functions in the nucleolus as a positive regulator of ribosomal RNA biogenesis[END_REF][START_REF] Patten | Role of Chd7 in zebrafish: a model for CHARGE syndrome[END_REF]. CHARGE syndrome is an autosomal dominant disease caused by CHD7 mutations [START_REF] Vissers | Mutations in a new member of the chromodomain gene family cause CHARGE syndrome[END_REF]). -CHARGE‖ is an acronym for Coloboma, Heart malformation, Atresia of the choanae, Retardation of Growth and development and Genital anomalies which describe the main clinical symptoms [START_REF] Pagon | Coloboma, congenital heart disease, and choanal atresia with multiple anomalies: CHARGE association[END_REF][START_REF] Blake | CHARGE association: an update and review for the primary pediatrician[END_REF][START_REF] Jongmans | CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene[END_REF][START_REF] Lalani | Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation[END_REF], Bergman et al. 2011b). Anomalies of ear (90-100% of patients with CHD7 mutation), ocular coloboma and/or microphthalmia (80-90%), cranial nerve dysfunction, swallowing and breathing difficulties (70-90%) are the major signs of CHARGE syndrome [START_REF] Blake | CHARGE association: an update and review for the primary pediatrician[END_REF]. Other symptoma could also reflect a CHARGE phenotype such as genital anomalies, growth retardation, congenital heart defects, and facial dysmorphism [START_REF] Horsch | Compendium on CHARGE syndrome multi-disciplinary and international perspectives[END_REF]. Later on, other criteria were included as hallmark for CHARGE syndrome (coloboma, choanal atresia, semicircular canals anomalies, rhombencephalic anomalies, hypothalamic-hypophyseal dysfunction, external/middle ear malformations, malformation of viscera, and mental retardation [START_REF] Verloes | Updated diagnostic criteria for CHARGE syndrome: a proposal[END_REF].

Mutant mice for Chd7 develop many of the features observed in patients with CHARGE syndrome and mice that are homozygous for such mutations die at embryonic day 10.5 [START_REF] Bosman | Multiple mutations in mouse Chd7 provide models for CHARGE syndrome[END_REF][START_REF] Hurd | Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues[END_REF]). CHD7 is composed of 38 exons spanning 188kb. CHD7 is highly conserved across evolution [START_REF] Bosman | Multiple mutations in mouse Chd7 provide models for CHARGE syndrome[END_REF][START_REF] Aramaki | Embryonic expression profile of chicken CHD7, the ortholog of the causative gene for CHARGE syndrome. Birth Defects Research[END_REF], Shrinivasan et al. 2008[START_REF] Bajpai | CHD7 cooperates with PBAF to control multipotent neural crest formation[END_REF]. Mutations in the CHD7 gene lead to different form of the disease. Nonsense and frame shift mutations (75%), missense and splice site mutations (20 %) and more rarely, chromosomal rearrangements are found in CHARGE syndrome patients [START_REF] Janssen | Mutation update on the CHD7 gene involved in CHARGE syndrome[END_REF]). CHD7 mutations are detected in 60% of patients when using the criteria of [START_REF] Blake | CHARGE association: an update and review for the primary pediatrician[END_REF] and/or [START_REF] Verloes | Updated diagnostic criteria for CHARGE syndrome: a proposal[END_REF] but mutation in other genes can trigger CHARGE syndrome [START_REF] Jongmans | CHARGE syndrome: the phenotypic spectrum of mutations in the CHD7 gene[END_REF][START_REF] Lalani | Spectrum of CHD7 mutations in 110 individuals with CHARGE syndrome and genotype-phenotype correlation[END_REF], Bergman et al. 2011a). Genetic heterogeneity in CHARGE syndrome could come from mutations in CHD7 interaction partners. The majority of CHARGE patients display mental retardation associated with olfaction deficit and anomalies of the olfactory bulb [START_REF] Blustajn | Olfactory anomalies in CHARGE syndrome: imaging findings of a potential major diagnostic criterion[END_REF][START_REF] Bergman | CHD7 mutations and CHARGE syndrome: the clinical implications of an expanding phenotype[END_REF].

Moreover, CHARGE patients also develop autistic behavior [START_REF] Hartshorne | Autistic-like behavior in CHARGE syndrome[END_REF].

Interestingly, mutation in CHD8, a protein which can physically interact with CHD7 [START_REF] Batsukh | CHD8 interacts with CHD7, a protein which is mutated in CHARGE syndrome[END_REF] was identified as one of the major genetic causes of autism spectrum disorder [START_REF] Neale | Patterns and rates of exonic de novo mutations in autism spectrum disorders[END_REF][START_REF] O'roak | Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations[END_REF][START_REF] Talkowski | Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries[END_REF].

Functional studies showed that CHD7 binds predominantly to methylated histone H3K4 in a cell type and stage specific manner [START_REF] Schnetz | Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns[END_REF][START_REF] Schnetz | CHD7 targets active gene enhancer elements to modulate ES cell-specific gene expression[END_REF][START_REF] Heintzman | Histone modifications at human enhancers reflect global cell-type-specific gene expression[END_REF][START_REF] Flanagan | Double chromodomains cooperate to recognize the methylated histone H3 tail[END_REF][START_REF] Srinivasan | Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II[END_REF]). Moreover, it has been shown that CHD7 cooperates with PBAF (polybromo and BRG1-associated factor-containing complex) to regulate neural crest differentiation and migration through activation of Sox2, Slug, and Twist [START_REF] Bajpai | CHD7 cooperates with PBAF to control multipotent neural crest formation[END_REF]. Interestingly, Kismet, the drosophila ortholog of CHD7 regulates transcription by recruiting the TRX (Trithorax) histone methyltransferases [START_REF] Srinivasan | Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II[END_REF]). In the mouse brain, CHD7 and Sox2 are able to form a complex that cooperatively activates target genes that are essential for the development of neural stem cells [START_REF] Engelen | Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes[END_REF]. A recent study showed that Chd7 is enriched in active NSCs and progenitors in the SVZ and SGZ of adult mice [START_REF] Feng | Epigenetic regulation of neuronal fate determination: the role of CHD7[END_REF]. This study showed that a NSCs specific conditional mutation of Chd7 leads triggers defects in adult neurogenesis. Indeed, loss of Chd7 in NSCs impedes the methylation of H3K4me1/3 in promoters of Sox4 and Sox11, two neuronal differentiation factors [START_REF] Bergsland | The establishment of neuronal properties is controlled by Sox4 and Sox11[END_REF][START_REF] Mu | SoxC transcription factors are required for neuronal differentiation in adult hippocampal neurogenesis[END_REF] direct effect on NSCs. Moreover, no significant difference of BrdU incorporation or Ki67 staining was observed in Chd7 mutants SVZ compared to control. Thus, loss of Chd7 impairs neurogenesis in the SVZ-OB without affecting the self-renewal of NSCs more likely indicating that Chd7 is required for the neuronal differentiation of NSCs (Feng, Weijun, 2013). Indeed, the number of transit amplifying cells marked by Ascl1 positive cells number was increased in Chd7 mutants suggesting an impairment of neuronal differentiation program. Indeed, Sox4 and Sox11, both necessary to the final differentiation of neurons were shown to be directly activated by Chd7 [START_REF] Mu | SoxC transcription factors are required for neuronal differentiation in adult hippocampal neurogenesis[END_REF].

Finally, an increase of cell death of Chd7 mutant cells was observed thus suggesting that Chd7 mutant cells undergo apoptosis when unable to properly differentiate [START_REF] Mu | SoxC transcription factors are required for neuronal differentiation in adult hippocampal neurogenesis[END_REF]. These important results bring to light the mechanism leading to the olfactory bulb and inner-ear defects and may explain some aspect of associated mental retardation and autistic behavior in CHARGE syndrome.

Direct reprogramming of fibroblasts to neurons and oligodendrocytes

Cellular reprogramming is a new emerging research field in which somatic cells can be turned into induced pluripotent stem cells (iPS cells) or other somatic cell types by the expression of specific combinations of genes [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF][START_REF] Vierbuchen | Direct conversion of fibroblasts to functional neurons by defined factors[END_REF].

In this paradigm, Ascl1 was recently shown to be sufficient to directly convert mouse fibroblasts into induced neurons (iN) in-vitro and in-vivo [START_REF] Vierbuchen | Direct conversion of fibroblasts to functional neurons by defined factors[END_REF][START_REF] Torper | Generation of induced neurons via direct conversion in vivo[END_REF]. However, the addition of Brn2 and Myt1l (BAM factors) significantly improve the conversion efficacy and results in a full maturation of neuronal characteristics [START_REF] Vierbuchen | Direct conversion of fibroblasts to functional neurons by defined factors[END_REF]. Interestingly, the BAM factors are able to converts human fibroblasts into iN but need the addition of NeuroD1 or Zic1 to achieve similar efficacy. Moreover, dephosphorylated Ascl1 significantly promotes neuronal maturity in human fibroblast converted in neurons [START_REF] Pander | Induction of human neuronal cells by defined transcription factors[END_REF]Barker RA 2014 ). Stricklingly, most of the iN cells induced by the BAM factors are glutamatergic neurons and not GABAergic as expected based on physiological condition studies [START_REF] Vierbuchen | Direct conversion of fibroblasts to functional neurons by defined factors[END_REF]. While combinatorial use of Ascl1 and Neurog2 generate a mixtures of glutamatergic and GABAergic neurons [START_REF] Ladewig | Small molecules enable highly efficient neuronal conversion of human fibroblasts[END_REF], the combination of Ascl1 and Sox2 predominantly converts cells into GABAergic neurons [START_REF] Karow | Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells[END_REF]. Other direct lineage reprogramming approaches have succefully generated dopaminergic neurons using Ascl, Nurr1 and Lmx1a [START_REF] Caiazzo | Direct generation of functional dopaminergic neurons from mouse and human fibroblasts[END_REF][START_REF] Pfisterer | Direct conversion of human fibroblasts to dopaminergic neurons[END_REF], motoneuron using BAM, Lhx3, Hb9, Isl1, and Neurog2 (Son ey 2011) or oligodendrocyte precursor cells (OPCs) using Olig2, Sox10, Nkx6.2 or Zfp536 [START_REF] Najm | Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells[END_REF]Yang N, Zuchero JB2013). For NPCs, Direct conversion was shown using only one factor, Sox2 (Ring KL 2012), but improved yield was obtained using Sox2, Fo0xG1, Brn2/4, Klf4, E47 and c-Myc (E Lujan 2012, M Their 2012, DW Han 2012).

Interestingly, BAM factors are able to directly convert cells from different embryonic layer origin into iN [START_REF] Marro | Direct lineage conversion of terminally differentiated hepatocytes to functional neurons[END_REF]. Indeed, recent genomic analysis of direct conversion by the BAM factors revealed the ‗‗pioneer factor'' activity of Ascl1which is able to bind to its direct target and activate their expression irrespective of whether genomic sites are in nucleosome depleted region or nucleosome bound. In this study, a specific histone marks (H3k4me1, H3k27ac and H3K9me3) was shown to favor Ascl1 access to its target sites [START_REF] Wapinski | Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons[END_REF].

Hoewever, protocols for direct cellular reprogramming based on overexpressing lentiviral vectors have really low efficiency (1-10%). As described above, the expression dynamic of those factors is critical for self-renewal, multipotency, and fate choice of NPCs [START_REF] Imayoshi | Oscillatory control of factors determining multipotency and fate in mouse neural progenitors[END_REF] and lentiviral vectors have only constitutively active promoters. Thus, new reprogramming protocols that control the range and mode of expression are now necessary to improve the low conversion efficacy of direct cellular reprogramming of neural cells. Moreover, the underlying mechanisms by which these reprogramming processes can be induced by just a small number of transcriptional factors remain largely unknown. To improve the efficacy and purity of direct reprogramming to neurons, it is essential to fully characterize the mechanisms by which reprogramming factors, including Ascl1 contribute to this process.

CHAPTER III: Oligodendrogenesis and myelination

Oligodendrocyte development comprises specification of oligodendrocyte precursor cells (OPCs) from NSCs and OPC differentiation into immature and mature myelinating oligodendrocytes (for review [START_REF] Rowitch | Glial specification in the vertebrate neural tube[END_REF][START_REF] Rowitch | Developmental genetics of vertebrate glial-cell specification[END_REF]Gallo V 2014;Emery, 2010b;[START_REF] Liu | Epigenetic regulation of oligodendrocyte identity[END_REF][START_REF] Meijer | Separated at birth? The functional and molecular divergence of OLIG1 and OLIG2[END_REF].

Myelination of the central nervous system

The term ‗myelin' (Greek word for ‗marrow', myelos), was first used by Rudolf Virchow in 1864 who was thinking that myelin was secreted by neurons. A century later, Pio del RioHortega revealed that myelin is actually formed as an extension of the plasma membrane of oligodendrocytes, a jawed-vertebrate specific cell type [START_REF] Zalc | The origin of the myelination program in vertebrates[END_REF]. Since, much progress has been achieved in the understanding of the myelination process (for review, see Mitew et al., 2013;[START_REF] Piaton | Axon-oligodendrocyte interactions during developmental myelination, demyelination and repair[END_REF][START_REF] Taveggia | Signals to promote myelin formation and repair[END_REF][START_REF] Simons | Axonal selection and myelin sheath generation in the central nervous system[END_REF][START_REF] Fancy | Myelin regeneration: a recapitulation of development?[END_REF]White and Kramer-Albers, 2014) . Indeed, myelin is now known to be a multi-layered lipid sheet periodically wrapped around neuronal axons and provide axonal insulation necessary for a rapid -saltatory conduction‖ of the action potential [START_REF] Lillie | FACTORS AFFECTING TRANSMISSION AND RECOVERY IN THE PASSIVE IRON NERVE MODEL[END_REF][START_REF] Tasaki | The Electro-Saltatory Transmission of the Nerve Impulse and the Effect of Narcosis Upon the Nerve Fiber[END_REF][START_REF] Huxley | Evidence for saltatory conduction in peripheral myelinated nerve fibres[END_REF], limit ephaptic conduction [START_REF] Arvanitaki | Effects Evoked in an Axon by the Activity of a Contiguous One[END_REF][START_REF] Smith | The pathophysiology of multiple sclerosis: the mechanisms underlying the production of symptoms and the natural history of the disease[END_REF]) and avoid the action potential conduction block observed in demyelinating disease (Davis FA. 1970;[START_REF] Rasminsky | Internodal conduction in undissected demyelinated nerve fibres[END_REF]. Moreover, myelin has been shown to sense axonal energy demands and provide an important trophic support to the neuron [START_REF] Lee | Circadian regulation of low density lipoprotein receptor promoter activity by CLOCK/BMAL1, Hes1 and Hes6[END_REF][START_REF] Nave | Myelination and the trophic support of long axons[END_REF]Trapp, 2008, Nave 2010) for the high energy cost of fast electrical signaling (Attwell, D. & Laughlin, S.B 2001) and for axonal long term maintenance (Griffiths I, Klugmann M,1998). Axon myelination minimizes the membrane capacitance that has to be charged to trigger action potentials, thus reducing the metabolic cost of electrical signaling [START_REF] Hartline | Rapid conduction and the evolution of giant axons and myelinated fibers[END_REF]). However, when taking into account the production of myelin and maintainance of the resting potential of oligodendrocytes, myelination has an overall positive energy cost (Harris JJ, Attwell D, 2012). Instead, myelin seems to be an evolutionary adaptation that allowed vertebrate to deal the need for a rapid information processing associated with large nervous systems and body [START_REF] Zalc | The origin of the myelination program in vertebrates[END_REF]. Indeed, myelination results in a 10 to 100-fold increase in nerve conduction velocity [START_REF] Franz | Conduction failure in myelinated and non-myelinated axons at low temperatures[END_REF][START_REF] Waxman | Determinants of conduction velocity in myelinated nerve fibers[END_REF]) reaching 100m/s. Direct observation of myelination has been possible with the development of electron and light microscopy [START_REF] Maturana | The fine anatomy of the optic nerve of anurans--an electron microscope study[END_REF]Peters, 1960a;Mobius et al., 2010). In mice, myelination starts postnatally and is almost completed at postnatal day 60 (P60) in most brain regions [START_REF] Baumann | Biology of oligodendrocyte and myelin in the mammalian central nervous system[END_REF]. We now know that myelin thickening is achieved with a single filipodia making axonal contact that spirally encircle the future internode and wrap the leading edge at the inner tongue around the axon forming microfilament-rich lamelipodia, underneath the previously deposited membrane, together with the lateral extension [START_REF] Snaidero | Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue[END_REF]. A growth zone at the inner tongue requires transport of newly synthesized membrane constituents from the soma through the oligodendroglial process. To this purpose, cytoplasmic channels transporting vesicules provides a connection from the outer and inner tongue of myelin.

Present at the peak of myelination (postnatal day 10-15, P10-P15), cytoplasmic channels are almost absent at P60 when myelination is over [START_REF] Snaidero | Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue[END_REF]. The compaction of the myelin layers is achieved by myelin basic protein (MBP) and starts after only a few wraps [START_REF] Readhead | Expression of a myelin basic protein gene in transgenic shiverer mice: correction of the dysmyelinating phenotype[END_REF]. Whereas membrane growth occurs close to the axon at the innermost region of the myelin sheath, compaction starts in the outermost layers and progresses inward. MBP is the major cytoplasmic component, and its mRNA is transported in cytoplasmic granules to myelin [START_REF] Colman | Synthesis and incorporation of myelin polypeptides into CNS myelin[END_REF] whose movements are oriented thanks to PI [START_REF] Adelman | Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling[END_REF][START_REF]Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork[END_REF]5)P3 lipid signal activating the Rho GTPases at the leading edge of the innermost layer [START_REF] Ridley | Life at the leading edge[END_REF]. MBP protein is synthesised at the innermost layer close to the axon and is regulated by axonal signals such as electrical signal, neurotransmitters, ATP, ions, or growth factors [START_REF] Colman | Synthesis and incorporation of myelin polypeptides into CNS myelin[END_REF][START_REF] Barres | Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons[END_REF][START_REF] Demerens | Induction of myelination in the central nervous system by electrical activity[END_REF][START_REF] Fields | New insights into neuron-glia communication[END_REF][START_REF] Wake | Control of local protein synthesis and initial events in myelination by action potentials[END_REF][START_REF] Laursen | Translation of myelin basic protein mRNA in oligodendrocytes is regulated by integrin activation and hnRNP-K[END_REF]. Even if MBP is synthesized in innermost layers, compaction is not fast enough to keep up with the rapid extension of the myelin sheath [START_REF] Snaidero | Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue[END_REF]. Indeed, oligodendrocyte can make new myelin sheaths in only five hours [START_REF] Czopka | Individual oligodendrocytes have only a few hours in which to generate new myelin sheaths in vivo[END_REF]. Thus, MBP is synthesized close to the axon and diffuse backward to the outermost area at which compaction is initiated.

MBP serves to bind two adjacent membranes by polymerizing into a fibrous network and establish an anchor point for myelin membrane zippering [START_REF]Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork[END_REF].

In the absence of CNP, myelin compaction occurs faster and extends to the inmost layers of the myelin sheath [START_REF] Snaidero | Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue[END_REF]. Oligodendrocytes can myelinate up to 80 small-diameter axons or fewer long-calibre axons [START_REF] Murray | The relationship between internodal length and fibre diameter in the spinal cord of the cat[END_REF][START_REF] Matthews | A quantitative study of morphological changes accompanying the initiation and progress of myelin production in the dorsal funiculus of the rat spinal cord[END_REF][START_REF] Hildebrand | Myelinated nerve fibres in the CNS[END_REF]. In-vitro, myelination occurs spontaneously when oligodendrocytes are cultured with axon-mimicking inert polystyrene tube with a diameter up to 4 mm [START_REF] Lee | Circadian regulation of low density lipoprotein receptor promoter activity by CLOCK/BMAL1, Hes1 and Hes6[END_REF]. In-vivo, oligodendrocytes are able to myelinate axons of a diameter from 0.2µm to ~1mm [START_REF] Remahl | Changing relation between onset of myelination and axon diameter range in developing feline white matter[END_REF][START_REF] Waxman | Relative conduction velocities of small myelinated and nonmyelinated fibres in the central nervous system[END_REF]. The ratio of the inner axonal diameter to the total outer diameter (gratio) is used as an index of optimal axonal myelination. Theoretical considerations have shown that axons have an optimal g-ratio of 0.6. If myelin thickness deviates from this value, conduction velocity drops [START_REF] Waxman | Relative conduction velocities of small myelinated and nonmyelinated fibres in the central nervous system[END_REF][START_REF] Chomiak | What is the optimal value of the g-ratio for myelinated fibers in the rat CNS? A theoretical approach[END_REF]. Similar considerations apply for the internodal length. A linear relationship exists between the axon diameter, the number of myelin sheaths and the internodal length [START_REF] Murray | The relationship between internodal length and fibre diameter in the spinal cord of the cat[END_REF][START_REF] Hildebrand | Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species[END_REF]. For example, the increase in the diameter of the axon from 1 to 15 µm involves a lengthening of the internode from 100 to 1500 µm. However, the maximum conduction velocity is reach at 600µm internodal distance [START_REF] Waxman | Axon-glia interactions: building a smart nerve fiber[END_REF][START_REF] Wu | Increasing internodal distance in myelinated nerves accelerates nerve conduction to a flat maximum[END_REF].

Myelin is regulated by experience both during development and in adult life (Liu J Dietz et al., 2012;Mangin JM et al., 2012). Indeed, mice socially isolated between P21 and P35 show myelin defects driven by decreased neuregulin-1/ErbB3 signaling.

Moreover, this time frame is important and constitutes a critical period, as mice isolated before or later does not develop such defects [START_REF] Makinodan | A critical period for social experience-dependent oligodendrocyte maturation and myelination[END_REF].

Interestingly, neuronal overexpression of Nrg1 increases the thickness of myelin sheaths of myelinated axons and drive myelination of small calibre axons that would not be myelinated in wildtype (Brinkmann BG, 2008).

Functional organization of myelinated axons

To fulfill those functions, myelination structures the axons in different areas: i)

The axon initial segment (AIS), an unmyelinated thin region where ion channels are clustered and action potentials are initiated; ii) the node of Ranvier, where sodium channels are clustered and action potentials are propagated (Kole MH, Stuart GJ 2012); iii) the paranode, where myelin tightly contact the axon; iv) the juxtaparanode, where potassium channels are clustered below the myelin sheath and v) the internode, the region between two node of Ranvier where the Na/K-ATPases are located (Young EA, Fowler CD 2008;Buttermore E 2013)

Lipid composition of myelinating oligodendrocytes

Myelin is characterized by an unusual high lipid to protein ratio (Chrast R2011). Indeed, compared to other cells where around 30% of the dry weight consists of lipids, myelin possesses more than 70% of lipids. Among the 700 lipid moieties identified by recent lipidomics studies (Gopalakrishnan G2013), three main classes are identified: phospholipids account for ~40%, glycosphingolipids for ~30%, and

Structural and functional organization of myelin

Protein composition of myelin

Transcriptome microarray analysis of all the neural cell types in the brain released publicly data for the oligodendrocyte lineage in which purified PDGFR+ (OPCs), GalC+ (pre-myelinating OLs) and MOG+ (myelinating OLs) are available.

However, this study focused principally on astrocyte development and did not exhaustively analysed the oligodendrocyte lineage (Cahoy JD et al 2008). Another study following the same cell purification protocol and allowed the identification of critical factors involved in myelination such as MRF (Emery B 2009) Plp1 (proteolipid protein) is the most expressed gene in myelinating oligodendrocyte (Eng et al. 1968;Norton and Poduslo 1973), but is also expressed in OPCs (Gudz et al., 2006), neurons (Bongarzone et al., 1999;Jacobs et al., 2003Jacobs et al., , 2004)), neural precursors (Spassky et al., 1998;Delaunay et al., 2009) and non-neural cells (Campagnoni et al., 1992;Skoff et al., 2004a) suggesting other functions unrelated to myelination. PLP mutant mice triggers demyelination and premyelinating OL apoptosis (Knapp et al., 1986;Boison and Stoffel, Bongarzone et al., 2001). PLP has also been involved in the regulation of ion homeostasis Gudz et al., 2006), cell migration, proliferation, differentiation (Harburger and Calderwood, 2009) and OL apoptosis. Interestingly, PLP overexpression triggers acidification and mitochondrial dysfunction and reductions in ATP levels (Hüttemann et al., 2009). DM20, its isoform generated by alternative splicing lacks an internal 35 amino acid region and is expressed in neural tube OPCs but also in the heart or thymus (Macklin et al. 1987;Nave et al. 1987;Simons et al. 1987;Campagnoni et al., 1992;Pribyl et al., 1996a,). PLP functions as an adhesive protein between two layers of the myelin sheath and it is involved in its compaction by conferring structural stability (Gow et al. 1997). PLP/DM20 is also involved in oligodendrocyte-neuron interactions (Yool et al. 2001), signaling (Gudz et al. 2003), and apoptosis regulation (Skoff et al. 2004;Heuttemann et al. 2009).

The MBP gene is very large (>100 kb) and generate mRNAs from different start site for classic oligodendrocyte-specific MBPs and peripheral golli MBPs (Campagnoni et al., 1993;Pribyl et al., 1993). The classic isoforms of MBP generate the 14 and 18.5-kDa isoforms which facilitate compaction of the myelin sheath, as well as the cytoplasmic and nuclear 17 and 21.5-kDa isoforms in pre-myelinating OLs (Allinquant et al., 1991;Hardy et al., 1996;Pedraza, 1997). MBP isoforms interact with Ca2+-CaM (calmodulin), actin, tubulin and SH3 domain (Src homology 3 domain)containing proteins (Harauz and Libich, 2009). Morevover, Golli MBP isoforms are found in pre-myelinating OLs within cell processes surrounding axons (Pribyl et al., 1996). Overexpression of golli proteins can induce processes growth and OLs maturation (Reyes and Campagnoni, 2002) and OPC Ca2+ -mediated migration (Paez et al., 2009a). MBP mutant mice (shiverer mice) shows demyelination and myelin compaction defects (Privat and al. 1979). MBP binds to negatively charged lipids on the cytosolic surface of the OL membrane through electrostatic interactions (Boggs and Moscarello, 1982;Boggs et al., 1982) and are present in compact internodal myelin (Omlin et al., 1982;Kirschner and Blaurock, 1992). MBP is involved in microtubule stabilization (Dyer et al. 1994) and form a physical barrier, preventing the diffusion of proteins in large cytoplasmic domains (such as PLP, CNPase, MAG or Tspan2) to favor their integration in compact regions of the myelin (Fitzner et al., 2006. Aggarwal et al, 2011;Zuchero and Barres, 2011;[START_REF]Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork[END_REF] .

CNPase is a cytosolic membrane-anchored enzyme present in non-compact myelin (Drummond GI, I 1962Trapp BD, Bernier L, 1988). Despite its ability to convert 2′,3′-cAMP into adenosine, the function of CNPase in myelin is still obscure. CNPase mutant mice display premature death through axonal swelling, while maintaining a normal myelin morphology. Conversely, CNPase overexpression results in oligodendrocyte process and myelin compaction defects and appear to be an autoantigen in MS (Rösener M 1997 Lovato L, Cianti R, 2008). Thus, CNPase seems to play a role in axonal maintenance after myelination, possibly through neuroprotection arising from a major contribution to the hydrolysis of 2′,3′-cAMP produced within myelinated axons (Verrier J, Jackson T 2012 Lappe-Siefke C2003)

Glial network in brain energy metabolism and axonal support

The brain is only 2% of the body's mass, but requires 20% of its energy consumption (Sokoloff, 1960). Based on the estimated energy consumption of each cell type, it is predicted that the majority ~70% should occur in neurons while the remaining ~30% should take place in glial cells [START_REF] Attwell | An energy budget for signaling in the grey matter of the brain[END_REF]. In the cortical neurons, while housekeeping energy used on non-signaling tasks (macromolecules turnover, transport and mitochondria) accounts for 25% of the total enegy consumption, signaling energy account for 75%. Signaling energy could be divided as follow: while 50% of the energy is used for postsynaptic receptors, 21% is used for action potentials, 20% on resting potentials, 5% on presynaptic events, and 4% on neurotransmitter recycling (Attwell and Laughlin, 2012). Thus, action potentials account for a significant part of the consumed energy and thus need a corresponding supply. In optic nerve for example, action potentials consume ~6.10 12 ATP/s on unmyelinated axons and ~1.5.10 11 ATP/s on myelinated axons (J Harris and D Attwell 2012) and most of the ATP is used for pumping of Na+ ions out of cells (Siesjo, 1978;[START_REF] Attwell | An energy budget for signaling in the grey matter of the brain[END_REF]. Indeed, in the neurons, action potentials cause a massive sodium entry at the nodes of Ranvier and a release of potassium below the oligodendrocyte membrane at the juxtaparanode (J.E. Rash 2010). Na+/K+-ATPases and mitochondria, located along the internodal region, are dedicated to the maintenance of membrane potential maintenance and to fit the high energetic demand of the axon, respectively (E.A. Young, C.D. Fowler,2008;N. Ohno, G.J. Kidd, D 2011). Thus, the highest energy requirements seems to occur along the internode and not at the node of Ranvier (Saab A 2013).

Astrocytes are the first metabolic support of neurons. Indeed, precise in-vitro and invivo metabolic analysis demonstrated that blood glucose is preferentially taken up by astrocytes which realease lactate to neurons by the -lactacte shuttle‖ mechanism (Pellerin andMagistretti,1998, 2003) and that neurons preferentially use lactate as their main oxidative substrate (25% of glucose and 75% of lactate) (Bouzier-Sore et al., 2006). However, in myelinated fibers, axons are almost completely covered by myelin and evidence of axonal glucose transporters (GLT1/2) at the nodes of Ranvier or direct lactate transfert by perinodal astrocytes are lacking (K.A. Nave 2010). Interestingly, perinodal astrocytes can recycle neuronal mitochondria in a process called -transmitophagy‖ (Davis CH, Kim KY 2014). Moreover, perinodal astrocytes and oligodendrocytes can form a glial (O/A) network through gap junctions in the cortex and optic nerve, (D.M. Menichella 2003). Gap junctions are channels formed of homo or heterotypic connexin hemichannels which are permeable to molecules smaller than 1.2 kDa, including ions, cyclic nucleotides, ATP, neurotransmitters and small metabolite such as glucose and lactate (Reyes EP 2014; L Hertz 2014).

Oligodendrocytes express Cx47, Cx32, andCx29 (Menichella et al., 2003, Dermietzel et al., 1989;Altevogt and Paul, 2004). Astrocytes express Cx43, Cx30 and Cx26 (Mercier and Hatton, 2001;Nagy et al., 2001). An important question remaining to answer is how myelin may be directly beneficial for maintenance of the axon integrity. Many transgenic models allowed to tackled this question. Shiverer mice (MBP mutant mice) do not show axonopathy despite absence of compact myelin (Rosenbluth, 1980a,b;[START_REF] Griffiths | Axonal swellings and degeneration in mice lacking the major proteolipid of myelin[END_REF]. In the EAE demyelinating model, early axonal defects occurs before myelin disruption (Nikic I, Merkler D, 2011). In contrary, Plp1 null mutant mice develop progressive widespread axonal degeneration, with axonal transport defects, swellings (Klugmann M, Schwab MH, 1997). Similarly, Cnp1 mutant mice develop progressive axonopathy and die prematurely despite normal myelination (Edgar JM, McLaughlin M, 2009). In another study, the specific ablation of oligodendrocytes by diphtheria toxin (DT) showed axon degeneration with axon damages occuring without complete demyelination and without involvement of inflammatory processes (Ghosh et al., 2011a;Pohl et al., 2011;Oluich et al., 2012). Thus, myelination and axonal support are not correlated but rather are two independent mechanisms, and oligodendrocytes play additional functions to just axonal myelination involved in axonal maintenance and energetic support.

Figure 20. Glial metabolic network.

A. This model of metabolic coupling between oligodendrocytes and axons shows oligodendrocytes glucose import through GLUT1, astrocytes and gap junctions. Pyruvate is metabolised in mitochondria for ATP generation. glucose serves the synthesis of fatty acid and support oligodendrocyte survival. Lactate and pyruvate can be directly transferred to axons via monocarboxylic acid transporters (MCT1, MCT2), which reside between the internodal myelin and the axonal compartment. B. Role of myelin in potassium clearance. Schematic representing myelinated axons isolated from the extracellular space by a thin periaxonal space from the oligodendroglial cytoplasm filling the inner loops of myelin (‗cytosolic channel') and paranodal loops. Electrochemical gradient of K+ allow potassium to be released in the periaxonal space and to the oligodendrocyte via the gap junction network.C. Connexin nextwork. Schematic showing specific homo and heterometic connexin coupling. Oligodendrocyte is coupled to Astrocyte, oligodendrocyte and OPCs by gap junction.

Oligodendrogenesis throughout development

Transcriptional control of oligodendrocyte progenitor cells specification

Glial progenitor cells that give rise to oligodendrocytes were first identified in cultures of developing rat optic nerve cells as O-2A, a bi-potent progenitor able to generate oligodendrocyte and type-2A astrocyte [START_REF] Raff | A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium[END_REF]. In the developing forebrain, generation of OPCs that will populate the entire brain and differentiate into myelinating oligodendrocytes occurs in ventro-dorsal and antero-posterior waves from separate domains (Kessaris et al., 2006). The anterior entopeduncular area (AEP) and the medial ganglionic eminences regions (Nkx2.1 positive) start to specify OPCs from E12.5 (Spassky et al., 2001;Tekki-Kessaris et al., 2001. Olivier, C. et al. 2001). As development progresses, the lateral and caudal ganglionic eminences (Gsh2 positive) generate OPCs from E15.5 and postnataly, the cortex (Emx1 positive precursors) start to produce OPCs (Tekki-Kessaris et al., 2006). All OPCs migrate from the VZ to populate the entire telencephalon. OPCs constitute the main proliferative population in the adult brain and around 5% of the total cell population of the brain (Levine et al., 2001;Dawson et al., 2003). Interestingly, Nkx2.1-derived OPCs are rapidly eliminated during postnatal life but if Nkx2.1, Gsh2 or Emx1 OPCs waves are ablated through conditional deletion, the remaining OPCs quickly expand to regenerate a normal number of cells in the mature sebrain (Kessaris et al., 2006). A small proportion of OPCs generated during development are maintained throughout aldulthood as an immature slowly proliferative or quiescent state (Dawson et al., 2003). OPCs density is strickingly even in the adult brain even if a higher proliferation rate is observed in the white matter (Dimou et al., 2008). Indeed, while OPCs in the white matter are proliferative and differentiate into myelinating oligodendrocytes, gray matter OPCs are quiescent or slowly proliferative (Dimou et al., 2008). Despite regional expression marker differences, adult OPCs express the same markers as embryonic OPCs (Franklin and Ffrench-Constant, 2008). Known OPCs markers are proteoglycan NG2 (Nishiyama et al., 1999) and the platelet derived growth factor receptor α (PDGFRα), the O4 antigen and the transcription factors Olig2, Olig1, Nkx2.2 and Sox10 (Fancy et al., 2004). In the embryonic spinal cord, the ventral Olig2 pMN domain of the spinal cord generate sequentialy motoneuron and OPCs ((Pringle et al., 1996 and[START_REF] Lu | Sonic hedgehog--regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system[END_REF]. However, it is now recognized that spinal oligodendrocyte also have a dorsal origin from domains dP3-6 starting at E15.5. Indeed, Nkx6.1/Nkx6.2 double mutant mice, in which OPCs are lacking due to impaired Olig2 expression, are still producing OPCs in the dorsal spinal cord (Cai et al., 2005;Vallstedt et al., 2005). Embryonic dorsal OPCs arise from different domains and represent around 15% of dorsal OLs and are also generated later in development (Fogarty et al., 2005, Cai et al., 2005;Vallstedt et al., 2005). OPCs represent heterogeneous populations with distinct functions. Indeed, gray and white matter OPCs have different resting membrane potentials and ion channel expression profiles. Moreover, some OPCs generate action potentials and are able to sense neuronal activity by forming synapses with axons (Yuan et al., 2002;Karadottir et al., 2008). In support to this heterogeneity, dorsally or ventrally generated oligodendrocytes respectively myelinate specific axonal tracts (Tripathi et al., 2011) and have different proliferative potency depending on their origin (Vigano et al., 2013).

While Shh gradient produced by the notochord and the floor plate triggers Olig2 expression in the pMN domain, Shh gradient produced by the AEP triggers Olig2 expression in the ventral telencephalon [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF]. While, dorsal BMPs and Wnts signal support generation of astrocytes and prevent OL generation at earlier embryonic stages (Mekki-Dauriac et al., 2002;Shimizu et al., 2005;Vallstedt et al., 2005), dorsal

Oligodendrogenesis during postnatal development and adulthood

In the mice postnatal and adult brain, transit amplifying precursors (also called C-type cells) are generate OPCs that migrate radially out of the SVZ into the overlying white matter and cortex where they stop dividing to differentiate into myelinating OLs (Aguirre et al., 2004;Aguirre et al., 2007;Menn et al., 2006;Nait Oumesmar et al., 1999). NSC in the SVZ generates OPCs or neuroblast but never both (Ortega et al., 2013). Interestingly, a ten fold higher number of OPCs are produced in the posterior part of the SVZ compared to rostral SVZ (Menn et al., 2006). Contrary to neuroblasts in the RMS, OPCs production from the adult SVZ is maintained with age (Capilla-Gonzalez et al., 2013). Another major source of OPCs comes from proliferation of OPCs present in the white matter and the brain parenchyma (Scafidi et al., 2014;Jablonska et al., 2012). OPCs are the major proliferative population of the adult brain and virtually all oligodendrocyte precursor cells continue to divide in adult mice with a cell-cycle time of 20 to 40 days [START_REF] Young | Oligodendrocyte dynamics in the healthy adult CNS: evidence for myelin remodeling[END_REF][START_REF] Simon | The nucleoskeleton as a genome-associated dynamic 'network of networks[END_REF]Kang et al., 2010). Neonatal OPCs have been shown to divide either symmetrically, generating pairs of OPCs or mature oligodendrocytes, or asymmetrically to generate one OPC and one oligodendrocyte [START_REF] Sugiarto | Asymmetry-defective oligodendrocyte progenitors are glioma precursors[END_REF]Zhu et al., 2011). In the adult brain, while half of the OPCs are dividing, the remaining OPCs are quiescent or divide even more slowly (Rivers et al., 2008). Different studies showed that adult OPCs have an increased cell cycle and migrate more slowly (Wolswijk and Noble, 1989;[START_REF] Simon | The nucleoskeleton as a genome-associated dynamic 'network of networks[END_REF][START_REF] Clark | Human developmental enhancers conserved between deuterostomes and protostomes[END_REF][START_REF] Hughes | SHH signaling acts as a chemoactrant through binding of the Patched-1 receptor (Mercha´n et al[END_REF][START_REF] Young | Oligodendrocyte dynamics in the healthy adult CNS: evidence for myelin remodeling[END_REF] but also differentiate more quickly than embryonic OPCs (Lin et al., 2009;Windrem et al., 2004). However, embryonic OPCs have better remyelinating capacity than adult OPCs thanks to their increased proliferative capacities (Ruckh et al., 2012;van Wijngaarden and Franklin, 2013).

Proliferation and survival of oligodendrocyte progenitor cells

While proliferation of OPCs depends in Cdk4 activation in the SVZ after demyelinating insult (Jablonska et al., 2012), OPC proliferation in WM depends on Cdk2/CyclinE activation in both developing and adult brains (Jablonska et al., 2012;[START_REF] Belachew | Migration of oligodendrocyte progenitor cells OPCs achieve an homogenous distribution in the brain. it appears OPCs are a mediator of their own migration[END_REF].

PDGFRα is a specific marker of OPCs among neural cells (Heldin. C-H Westermark, B, 1981). PDGF-A, its ligand, is perhaps the most important mitogen for OPCs signaling (Noble et al., 1988;Richardson et al., 1988;Pringle et al., 1992).

Indeed, OPC proliferation is tightly coupled to PDGF-A level (Calver et al., 1998;Fruttiger et al., 1999). Proliferative and pro-survival signaling through PDGFR is linked to the α6β1 and αvβ3 integrin receptor interacting together in lipid rafts and driving phosphoinositide 3-kinase (PI3K)/PKC signaling (Baron et al., 2002(Baron et al., , 2003;;Decker and ffrench-Constant, 2004).

FGF-2 and FGF-18 act on FGFR1/2 or FGFR2/3, respectively, to stimulate OPC proliferation and inhibit differentiation (Chandran et al., 2003;Fortin et al., 2005).

FGF-8 and FGF-17 primarily inhibit OPC differentiation through FGFR3 alone (Furusho et al., 2011). FGF-2 has also the capacity to promote PDGFR expression and thus induces OPC proliferation and inhibits differentiation (McKinnon et al., 1990;Wolswijk and Noble, 1992;Baron et al., 2000).

IGF-1/ IGF-1R promote OPC proliferation and survival through the PI3K/Akt pathway phosphorylating GSK3 that prevent the phosphorylation and degradation of Cyclin-D1 (Pang et al., 2007;Zeger et al., 2007;Bibollet-Bahena and Almazan, 2009;Romanelli et al., 2009;Frederick et al., 2007). Moreover, FGF-2 and IGF-1 work synergistically to activate the ERK1/2 pathway that activate proliferating function of Cyclin-D1 in OPC (Frederick and Wood, 2004;Frederick et al., 2007).

Neuregulin-1 (NRG-1), an axonally-bound or secreted protein, has been shown to bind the OPC expressed ErbB tyrosine kinase receptors and promote OPC survival though the PI3K/ Akt pathway (Canoll et al., 1999;Fernandez et al., 2000;Flores et al., 2000). (PDGF) and fibroblast growth factor 2 (FGF2)-that act either individually or cooperatively through PDGFR and FGFR1 to generate mature OLs (Calver et al., 1998;Fortin et al., 2005;McKinnon et al., 1990;Miller, 2002). Expression of the ligand PDGF-A is upregulated in reactive astrocytes of demyelinated lesions (Armstrong et al., 2002: Messersmith et al., 2000), and both FGF2 and FGFRs are upregulated in areas of acute or chronic cuprizone-induced demyelination (Messersmith et al., 2000). PDGF and FGF2 control differential responses in the OL lineage after demyelination (Murtie et al., 2005). Consistent with a role of PDGF in proliferation, OPC amplification and subsequent repopulation of demyelinated lesions are impaired in a heterozygote PDGFR +/-or in a GFAP-driven PDGF-A null mouse (Murtie et al., 2005). Conversely, FGF2 acts to inhibit remyelination through FGFR1 (see below). Stimulation of epidermal growth factor (EGF) exerts multiple effects on the OL lineage, both OPCs and mature OLs (Aguirre et al., 2007;Scafidi et al., 2014).

Selective EGFR overexpression in OL lineage cells promotes OPC proliferation and migration, enhancing the extent of functional remyelination of the corpus callosum after LPC-induced demyelination (Aguirre et al., 2007). Consistent with these findings, EGF infusion into the lateral ventricle (Gonzalez-Perez et al., 2009) or intranasal HB-EGF administration (Cantarella et al., 2008) promoted OPC recruitment from SVZ to demyelinated lesions. Furthermore, viral-mediated EGFR overexpression in WM OPCs increased proliferation and maintained an undifferentiated phenotype (Ivkovic et al., 2008). brain-derived neurotrophic factor (BDNF) is required to regulate the number of OPCs during normal development and during remyelination (Vondran et al., 2010;VonDran et al., 2011).

Ciliary neurotrophic factor (CNTF) promote OL maturation during development and after demyelination (Stankoff et al., 2002;Albrecht et al., 2003).

The neurotrophin NT-3 promotes OPC proliferation and survival of mature OLs through TrkC receptors and MAPK phosphorylation (Cohen et al., 1996;Jean et al., 2003).

Insulin-like growth factor 1 (IGF-1) is a necessary factor for OL differentiation and myelination in vitro and in vivo (Mozell and McMorris, 1991;Werther et al., 1998;Beck et al., 1995;Ye et al., 2002). The IGF type 1 receptor (IGF-1R) is continuously expressed throughout the entire OL lineage, and IGF-1 appears to act at multiple developmental stages, promoting OPC proliferation, OL survival, and myelin synthesis [START_REF] Barres | Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons[END_REF]Ye and D'Ercole, 1999;e ger et al., 2007).

Hepatocyte growth factor (HGF) has been identified as a positive regulator of oligodendrocyte proliferation and migration and an inhibitor of differentiation (Yan andRivkees, 2002: Ohya et al., 2007;Mela and Goldman, 2013).

MS occurs more frequently in females and undergoes remission during pregnancy (Confavreux et al., 1998), although the hormonal causes are poorly understood.

Interestingly, pregnant mice demonstrate an increase in newly generated OLs and in the number of myelinated axons in corpus callosum (Gregg et al., 2007), prolactin has been identified as a major regulator of OL plasticity and WM repair during pregnancy, inducing OPC proliferation and enhancing OL regeneration in vivo (Gregg et al., 2007). Sexual dimorphism exists in rodent subcortical WM, where females display a higher proportion of unmyelinated axons (Kim and Juraska, 1997) and higher OL turnover (Cerghet et al., 2006).

Tumor necrosis factor  (TNF) is an inflammatory cytokine which effects are mediated by the activation of TNFR1 (p55) and TNFR2 (p75) (Raine et al., 1998). In the brain, TNF is synthesized and released by astrocytes and microglia (Liu et al., 1998). TNF promote OPC proliferation (Arnett et al., 2001). More recent analysis demonstrated that TNFR2 also regulates expression of the chemokine CXCL12, which acts on the CXCR4 receptor to modulate remyelination. (Patel et al., 2012). dispensable for postnatal OPC migration (Ortega et al., 2012). Netrin-1/Unc5A receptor act as chemorepellent in the second wave of telencephalic and spinal OPCs Jarjour et al., 2003;Tsai et al., 2003Tsai et al., , 2006)). CXCL1/CXCR2 act as a stop signal and is central for proper OPC positioning (Tsai et al., 2002;Padovani-Claudio et al., 2006).

Secreted Semaphorin-3A and membrane-bound Semaphorin-4D/ F [START_REF] Spassky | NRG-1 has been shown to be chemoattractant during development but completely References[END_REF][START_REF] Vue | Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice[END_REF].

Molecular mechanisms involved in myelination and remyelination

The mechanisms underlying the failure of remyelination in demyelinating diseases include depletion of the OPC pool, failure of correct migration of OPCs to the lesion, irreversible axonal loss, and presence of inhibitors of differentiation produced by reactive astrocytes, microglia or lymphocytes (Chong and Chan, 2010;Mason et al., 2004;Miron et al., 2013). In order to tackle each of those aspects, many studies tried to uncover the transcriptional network and the signaling pathways promoting or inhibiting myelination.

Transcriptional regulation of myelination

During development, Olig1, Olig2, and Nkx2.2 are central in OPCs specification and differentiation [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF]Soula et al., 2001, Gallo, Vittorio 2014). Olig2 and Nkx2.2 are upregulated during acute phases of myelination and remyelination (Fancy et al., 2004). The role of Olig2 was recently extensively studied by Chip-seq and RNAseq analysis (Yu Chen Kim 2013). This groundbreaking study focuses on the collaborative role of Olig2 and Brg1 (Smarca4) in the transition between OPCs and myelinating oligodendrocyte. Olig2 was shown to activate different target genes depending on the differentiation stage and to recruit the chromatin-remodeling enzyme Brg1 (ATPase subunit of the SWI/SNF chromatin-remodeling complex) at the time of myelination (Yu Chen Kim 2013;Marathe HG 2013).This study made available important epigenetic data from H3K4me3, H3K27ac (active enhancers), RNA PolII, Olig2 and Brg1 Chip-seq in OPCs, pre-myelinating OLs and myelinating OLs (GSE42447). Olig2 was shown to be involved in almost all aspect of oligodendrocyte physiology such as specification, proliferation, survival, migration and signalings of OPCs together with myelination, survival and signalings of mature oligodendrocyte. These processes are mainly controlled by the Olig2 phosphorylation status (Maire et al., 2010). While mutation of Olig2 in OPCs (CNPase-promoter) significantly inhibits differentiation, deletion in immature OLs (Plp1 promoter) significantly enhances myelination. In contrast, ablation in the mature cells had no detrimental effect on myelination because of Olig1 compensatory role [START_REF] Mei | Stage-specific deletion of Olig2 conveys opposing functions on differentiation and maturation of oligodendrocytes[END_REF]). As Olig2, Olig1 is expressed all along the oligodendrocyte lineage but is mainly involved in remyelination [START_REF] Arnett | bHLH transcription factor Olig1 is required to repair demyelinated lesions in the CNS[END_REF]. Indeed, while Olig1 function in OPCs is largely overlapping and compensated by Olig2, Olig1 mutant oligodendrocytes hardy differentiate into remyelinating oligodendrocytes [START_REF] Arnett | bHLH transcription factor Olig1 is required to repair demyelinated lesions in the CNS[END_REF]. Interestingly, Olig1 expression is nuclear in OPCs, immature oligodendrocytes and early remyelinating lesions, but become cytosolic in normal appearing white matter (Ligon et al., 2006). While the cytoplasmic function of Olig1 was recently shown to drive membrane extension and depend on the phosphorylation status of Olig1 (Niu et al., 2012), the nuclear function was shown to involve synergistic action with Olig2 and Sox10 on different promoters such as Mbp promoter [START_REF] Xin | Myelinogenesis and axonal recognition by oligodendrocytes in brain are uncoupled in Olig1-null mice[END_REF]Li et al., 2007). This kind of localization switch is also observed for Myt1, a TFs involved in late maturation of oligodendrocytes (Nielsen et al., 2004;Vana et al., 2007).

Sox10 is also expressed all along the oligodendrocyte lineage. Sox10 mutant mice have relatively normal numbers of OPCs thanks to transcriptional redundancy with Sox9 and Sox8 (Stolt C, 2002;Finzsch M 2008;[START_REF] Emery | Regulation of oligodendrocyte differentiation and myelination[END_REF]. However, Sox10 mutant mice display a complete loss of myelinating oligodendrocytes (Stolt et al., 2002;Finzsch M 2008;Takada et al., 2010;Hornig et al., 2013). Sox10 mutant oligodendrocytes are able to ensheath axons but are unable to fully myelinate and finaly die by apoptosis (Takada N, 2010). Sox10 is necessary for Mbp promoter activation (Li et al., 2007) and it binds the mediator subunit 12 (Med12) and thus stabilize the general transcription machinery via the mediator complex (Vogl et al., 2013). Myrf and Sox10 share many common direct targets and work in a synergistic manner on many myelin specific genes (Bujalka et al., 2013;Hornig et al., 2013). Myrf is specifically induced by Sox10 during oligodendrocyte differentiation (Emery et al., 2009. Hornig et al., 2013). Conditional mutation of Myrf in oligodendrocytes results in a loss of myelin genes and progressive demyelination (Koenning et al., 2012). Sox17, which expression is maximal at the peak of myelination (P15) is involved in cell-cycle exit and the initiation of differentiation (Sohn et al., 2006). Sox17 promotes myelination by inhibiting the Wnt/-catenin pathway that maintain OPC proliferation (Chew et al., 2011;Ming et al., 2013).

Interestingly, Sox17 is highly expressed in Multiple Sclerosis chronic active lesions (Moll et al., 2013). Different other TFs were recently shown to be involved in myelination such as ZFP488 and ZFP191 which drive MBP or CNP expressions (Wang et al., 2006;Howng et al., 2010) but most of their genome-wide targets are yet to be discovered.

Specific role of Ascl1 in myelination

In addition to its function in OPCs generation in the neonatal olfactory bulb (Parras et al., 2004), Ascl1 is also involved in OLs specification in the embryonic wave of OPCs from the ventral telencephalon [START_REF] Parras | The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes[END_REF] and in the postnatal wave from the dorsal SVZ [START_REF] Nakatani | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination[END_REF]. Indeed, Ascl1 conditional knock-out mice in neonatal SVZ progenitors, show defects in the generation of OPC/oligodendrocytes at the profit of astrocyte generation [START_REF] Nakatani | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination[END_REF]. Moreover, conditional deletion of Ascl1 in OPCs results in an increase OPC proliferation at the expense of a reduce differentiation/maturation into myelinating oligodendrocytes. This could be explained by an increase of self-renewing symmetrical divisions of OPCs generating OPCs pairs at the expense of differentiating asymmetrical divisions generating OLs pairs [START_REF] Nakatani | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination[END_REF]. Interestingly, Ascl1 knockout results in a decrease expression of Nkx2.2 in OLs [START_REF] Sugimori | Ascl1 is required for oligodendrocyte development in the spinal cord[END_REF] and could explain part of the myelination defects observed in the Ascl1 knock-out [START_REF] Sugimori | Ascl1 is required for oligodendrocyte development in the spinal cord[END_REF][START_REF] Nakatani | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination[END_REF]. In focal demyelination experiments, Ascl1 levels are upregulated both in the adult SVZ and OPCs close to the demyelinated region in cells co-expressing Olig2 and Nkx2.2 sugesting that this transcription factors could be part of a common transcriptional code promoting remyelination [START_REF] Nakatani | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination[END_REF]. Indeed, during OLs regeneration, Ascl1 function is also important for the normal differentiation of OPCs during the remyelination as shown by the conditional deletion of Ascl1 in adult OPCs before inducing a brain demyelinating lesion [START_REF] Nakatani | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination[END_REF].

Importantly, ASCL1 expression is also up-regulated around Multiple Sclerosis chronic active lesions suggesting that the function of Ascl1 in remyelination is conserved between rodents and humans [START_REF] Nakatani | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination[END_REF]. Thus these results show that Ascl1 is involved in a cell autonomous manner in the neural stem cell (NSC) cell-fate choice following cell division and regulate genes involved in the specification and differentiation of OPCs. However, most of the direct targets of Ascl1 are unknown. In this study, we aimed at uncovering the specific transcriptional role of Ascl1 in the process of OPCs to OLs differentiation.

Epigenetic Regulation of oligodendrogenesis

The oligodendrocyte epigenetics only begins to be unraveled compared to epigenetic in other lineages [START_REF] Liu | Epigenetic regulation of oligodendrocyte identity[END_REF]JL Huynh, 2013, Yu Chen Kim 2013). Contrary to other neural lineages, OPC differentiation is initiated by histone deacetylation (Marin-Husstege et al., 2002). HDACs, which catalyses the removal of acetyl groups on histone lysine residues favoring transcription inhibition, repress proliferative genes such as Sox2 or Sox9, whose downregulation is necessary for myelination (Lyssiotis et al., 2007;Shen and Casaccia-Bonnefil, 2008). HDAC inhibitors decrease the number of OPCs at the expanse of astrocytes and neurons (Siebzehnrubl et al., 2007). Oligodendrocyte differentiation is initiated by HDAC1/2 recruitement to the promoters of myelination inhibitors (Shen and Casaccia-Bonnefil, 2008) such as Hes5 [START_REF] Kondo | Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells[END_REF], Sox11 andTcf4 (He al., 2007), Id2 andId4 (Samanta andKessler, 2004). YY1, Yin Yang 1 facilitates the recruitment of HDAC1 to the promoters of some of these transcriptional inhibitors (He et al., 2007). SIRT2 another HDAC, is upregulated during differentiation. TCF7L2/TCF4 as described above mediates a cross talk between HDAC1/2 and Wnt signaling (Ye et al., 2009).

Histone methyltransferases catalyze the addition of methyl groups to histones.

Methylation at H3K27 by

Promoting signals of myelination

A multitude of growth and trophic factors regulate myelination (Casaccia-Bonnefil, 2000;[START_REF] Nave | Axon-glial signaling and the glial support of axon function[END_REF][START_REF] Mitew | Mechanisms regulating the development of oligodendrocytes and central nervous system myelin[END_REF]. The thyroid hormone signaling is maybe the most important pathway driving myelination. While, hypothyroidism is associated with myelin defects (Schoonover et al., 2004), hyperthyroidism results in early myelination (Pombo et al., 1998).

Triiodothyronine/thyroid hormone 3 (T3) triggers OPCs cell cycle exit and myelination through Thra and P53 mediated signalings (Barres et al., 1994;Tokumoto et al., 2001).

IGF-1 signaling causes an increase in overall brain growth and an increased number of oligodendrocytes (Goddard et al., 1999). Fyn mutant mice display fewer myelinated axons with fewer mature oligodendrocytes and thinner myelin sheaths (Umemori et al., 1994;Osterhout et al., 1999;Goto et al., 2008). The fibronectin pathway blocks the myelin process outgrowth, while the laminin-2 and laminin-1 integrin receptor pathway triggers process expansion [START_REF][END_REF]Relucio et al., 2009). Axons produce laminin-2 before myelination (Colognato et al., 2002) which then binds to 1-integrin receptors on oligodendrocytes to promote the initial process extension through dephosphorylation of the tyrosine kinase Fyn [START_REF] Hu | Relucio et al[END_REF]Liang et al., 2004;Bauer et al., 2009). Fyn induces actin and microtubule polymerization via FAKmediated activation of the RhoGTPases Cdc42 and Rac1 [START_REF][END_REF]Bacon et al., 2007;Kim et al., 2008;O'Meara et al., 2013) .Brain-derived neurotrophic factor (BDNF) and FGF-2 activate the Raf-MEK-ERK pathway which favor myelination (Furusho et al., 2012;Xiao et al., 2012), and act through laminins, dystroglycan and integrin 1 receptors [START_REF] Lee | Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells[END_REF]Galvin et al., 2010).

The PI3K/Akt/mTOR (mammalian target of rapamycin) pathway is necessary to increase protein and lipid synthesis ability at the myelinating stage (Guardiola-Diaz et al., 2012). Interestingly, stimulation of this signaling triggers hypermyelination (Flores et al., 2008;Narayanan et al., 2009). Finally, LINGO-1, involved in cell-cell interactions, is specifically expressed in axons and OPCs being a key regulator of myelination timing because downregulation of LINGO-1 promotes OL differentiation and myelination during development (Mi et al., 2005).

Inhibiting molecular mechanisms of myelination

BMP, FGF2, Notch, and Wnt signaling prevent oligodendrogenesis in favor of neurogenesis (Colak et al., 2008;Hsieh et al., 2004) and astrogliogenesis (Gomes et al., 2003;Samanta et al., 2007;See et al., 2007;Grinspan, 2009). These effects involve Id genes expression and inhibition of Olig2 (Samanta and Kessler, 2004;Chen and Panchision, 2007). The BMP antagonist Noggin increases the density of mature OLs during remyelination (Sabo et al., 2011) and the BMP antagonist chordin promotes oligodendrogenesis from GAD65 expressing neuroblasts of the adult SVZ (Jablonska et al., 2010). Blockade of Wnt/-catenin signaling inhibit oligodendrogenesis (Langseth et al., 2010), being the effects of Wnt mediated by BMPs (Feigenson et al., 2011).

Notch1 activation in OPCs inhibits myelination (Wang et al., 1998;[START_REF] Kondo | Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells[END_REF]Genoud et al., 2002). Indeed Jag1 and Dll1 ligand are expressed on axons and binds to Notch1 in OPCs. Subsequent NICD nuclear transclocation increases the expression of the Hes1/5 that inhibit OPC differentiation (Wang et al., 1998;[START_REF] Kondo | Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells[END_REF]. In turn, Hes5 is inhibits Ascl1 and Sox10 (Liu et al., 2006;Wang et al., 1998). However, the presence of axonally expressed F3/contactin revert Notch1 signaling to a pro differentiation effect (Hu et al., 2003). Suppressors of Notch signaling enhance OPC maturation during normal development (Aguirre et al., 2010).

Moreover, Notch signaling in adult brain is reactivated after demyelination (Hammond et al., 2014) and Jagged 1 is upregulated in MS chronic lesions (John et al., 2002).

Eventually, a recent study showed that Endothelin-1 (ET-1) is a major regulator of Notch after demyelination (Hammond et al., 2014).

BMP and GPR17 signalings (Cheng et al., 2007(Cheng et al., , 2009) ) are inhibiting differentiation by inducing the bHLH molecules ID2/4, which directly inhibit Olig1 and Olig2 activity (Samanta and Kessler, 2004;Fumagalli et al., 2011). Wnt3A / Frizzled signaling, leads to the nuclear localization of -catenin and strongly inhibits differentiation (Shimizu et al., 2005). β-catening recruits TCF7L2(TCF4) to Id2 and Id4 promoters but Tcf7l2 may act as a molecular switch between proliferation and differentiation depending on binding partners. (Fu et al., 2012;Memezawa et al., 2007). Finally, in Sox5 and Sox6 mutant mice present premature expression of PLP and MBP indicating that the also form part of an inhibitory mechanism of oligodendrocyte differentiation and myelination (Stolt 11C 2006)

Therapeutic Aspect of oligodendroglial pathologies

Multiple sclerosis is an inflammatory degenerative disease of the central nervous system, in which an autoimmune attack driven by infiltration of the brain by immune cells causes a progressive or relapsing-remitting destruction of myelin sheaths together with the underlying axons and lead to action potential conduction block and associated disabilities (Noseworthy et al., 2000;Compston and Coles, 2002). MS is a disease found in every region of the world, with increased prevalence in northern countries, affecting 2.3 million persons worldwide with a female to male sex ratio female/males has increased from 2:1 to 3:1 from data accumulated between 1941 to 1980. The development of therapies are mainly based on immunosuppressants, immunomodulatory and anti-inflammatory drugs (Compston and Coles, 2002;Kieseier et al., 2007), but none is able to directly promote myelin repair and therefore, improve patients disabilities. Demyelinated plaques can be normally repaired by endogenous OPCs proliferation and differentiation, especially in acute and early stages of the disease (Chang et al., 2002;Wolswijk, 2002). However, remyelination with time and desease progression becomes more and more ineffective, leading to chronically demyelinated lesions with substantial axonal loss (Trapp et al., 1998). Although endogenous myelin repair could be complete in some MS patients (Patrikios et al., 2006), this process is, in most cases, insufficient and abnormalities in myelin ultrastructure subsist even if the overall myelination appears normal (Jablonska et al., 2012) As demonstrated in the experimental autoimmune-encephalomyelitis (EAE) and the cuprizone-induced demyelination models that are respectively used to tackle preferentialy the immune or the oligodendroglial aspect of the disease, axonal damage and neuronal loss play a central role in determining the extent of the disability of demyelinating diseases (Scafidi et al., 2014). Therefore, one of the major therapeutic challenges is to design new multi-steps treatments enhancing sequentially effective neuro-protection, OPCs proliferation, OL differentiation and axonal support.

Conclusions and Future Directions

Much progress have been made in the understanding of oligodendrocyte physiology since the discovery of Olig2 as the oligodendrocyte master gene (Zhou et al., 2001;[START_REF] Lu | Oligodendrocyte lineage genes (OLIG) as molecular markers for human glial brain tumors[END_REF]. However, really few therapeutic approaches have succeeded in driving potent remyelination. In order to find specific oligodendroglial therapeutic targets, it is now necessary to integrate the exhaustive genomic (Cahoy J 2008, Fu et al., 2009;Fancy et al., 2009)

Animals and genotyping

All animal procedures were performed according to the guidelines and regulations of the Inserm ethical committees (authorization #A75-13-19) and animal experimentation license A75-17-72 (C.P.). Both males and females were included in the study. Mice were maintained in standard conditions with food and water ad-libitum in the ICM animal facilities. Ascl1 KO and Olig2 KO mouse strains were used in this study. Because of the lethality in the first postnatal week in both knock-out mice, these strains were maintained by backcrossing to OF1 mice. For staging of embryos, midday of the vaginal plug was calculated as E0.5. Polymerase chain reaction was performed with wild type and knock-out primers to genotype the embryos. Wild-type, heterozygous and homozygous mutant embryos were obtained from intercrosses of Ascl1 +/or Olig2+/mice. Genotyping of the different Ascl1 alleles was performed by PCR using the following primers and conditions: for the Ascl1 knock-out allele, forward primer 5'-AGATGTAATTGTGGGCGAAG-3' and reverse primer 5'-ACAGTTTGGCCCGGCATGGA-3' with 35 cycles of 95°C/1 min, 60°C/1 min, 72°C/1 min were used. For the wild type allele, forward primer 5'-CTCCGGGAGCATGTCCCCAA-3' and reverse primer 5'-CCAGGACTCAATACGCAGGG-3' with 35 cycles of 95°C/1 min, 58°C/1 min, 72°C/1 min were used. For the Olig2 wild type allele, forward primer 5'-CTCGTAGCTCGGAGCTCAGCTCTGGCGGGC-3' and reverse primer 5'-TAGAAGGCCAGACTGCCGCGGCGTAGATGC-3', and for the Olig2 knock-out allele forward primer 5'-TGGCTACCCGTGATATTGCTGAAGAGCTTG-3' and reverse primer 5'-GCATGCCGCCCCAGTGCTGGAAGCCACCGC-3', both with 35 cycles of 95°C/4 min, 62°C/1 min, 72°C/1 min were used. Because no differences in the phenotypes between Wild-type and heterozygous brains have been detected, they were both used as controls.

Immunofluorescence

Adult mice were perfused with freshly made chilled 2% paraformaldehyde (Sigma) with 25 ml, brain was dissected out, cryoprotected in PBS with 20% sucrose overnight, and included in OCT (BDH) before freezing and sectioning (14µ mthick) with a cryostated microtome (Leica). Sections were then either processed for immunohistochemistry or stored at -80°C. The following primary antibodies were used: mouse monoclonal antibodies to APC (Calbiochem, 1:200), Ascl1 (BD Biosciences, 1:200), Olig2 (Ligon et al., 2006), GFP (Invitrogen, 1:2000), Gpr17

(Cayman Chemical, 1:400), myelin basic protein (MBP, Millipore, 1:1000), Olig2

(Millipore, 1:1000), Ascl1 (gift from J. Johnson, 1:1000); PDGFRa (BD Biosciences, 1:800), and chicken polyclonal antibodies to GFP (Aves Laboratories, 1:2000) and MBP (Millipore, 1:50). Fluorescent secondary antibodies included the following:

AlexaFluor-488, AlexaFluor-594, and AlexaFluor-647 Ig (1:1000, Invitrogen). Antigen retrieval for Ki67 antibody staining was performed using antigen retrieval solution

Demyelinating lesions

Before surgery, adult (2-3months) WT mice were weighted and anesthetized by intraperitoneal injection of mixture of ketamine (0.1 mg/g) and xylacine (0.01 mg/g).

An analgesic (buprenorphine, 30 mg/g) was administered intraperitoneally to prevent postsurgical pain. Focal demyelinating lesions were induced by stereotaxic injection of 1.5 llysolecithinsolution(LPC,Sigma, 1% in 0.9%NaCl) into the corpus callosum (CC; at coordinates: 1 mm lateral, 1.3 mm rostral to bregma, 1.7 mm deep to brain surface) using a glass-capillary connected to a 10µl Hamilton syringe. Animals were left to recover in a warm chamber before being returned into their housing cages.

Microarray experiments

Global gene expression analysis provides quantitative data about the population of RNA species in cells and tissues. Thus, microarray analysis is among the most commonly used methods in modern biology and there are more than a million expression datasets in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) public database [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF]. DNA microarrays are the most frequently used technique for RNA expression analysis. In this study, we performed microarray experiment using Ascl1 KO [START_REF] Guillemot | Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons[END_REF] and Olig2 KO [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF] mice. Different genotypes (Ascl1 KO/KO , Olig2 KO/KO and wild-type, i.e.

Ascl1 +/+ , Olig2 +/+ ) were obtained from intercrosses of Ascl1 KO/+ ; Olig2 KO/+ doble heterozygous mice. RNA was purified from disected E13.5 ventral forebrain (Fig. 23B,C). RNA samples were prepared by pooling RNAs extracted from 3 brains to constitute one replicate and each sample type was prepared in five replicates. cRNA hybridization probes were hybridized to Affymetrix Mouse Whole Genome MOE430

2.0 arrays. The R-CRAN statistical language and Bioconductor software package [START_REF] Gentleman | Bioconductor: open software development for computational biology and bioinformatics[END_REF] were used throughout the expression analyses. Raw image files were processed using Affymetrix GCOS. The probe signal levels were quantilenormalized, summarized and then transformed to the log 2 expression using GCRMA (gcrma R-package, [START_REF] Irizarry | Use of mixture models in a microarray-based screening procedure for detecting differentially represented yeast mutants[END_REF] and Frozen RMA (fRMA package; McCall and Irizarry., 2011). Gene filtering was performed using the Wilcoxon signed-rank test to select probe sets that were -consistently expressed‖ (p <0.04) in at least one group (mas5calls R-package; Bioconductor) (Fig. 24). All samples showed good quality control according to MAS5.0 guidelines and the AffyQC report (R-package;

Bioconductor). Principle component analysis (PCA) was used to reduce the dimensionality of the whole dataset and measure the degree of similarity between samples (dudi.pca R-package; [START_REF] Thioulouse | Interactive multivariate data analysis in R with the ade4 and ade4TkGUI packages[END_REF]. We decided to withdraw two replicates from each sample type as they were outliers. The subsequent threedimensional PCA plot confirmed a high degree of similarity among samples within the same genotype and much less similarity among samples of different genotypes. We used LIMMA statistical analysis (Smyt et al., 2004) to determine the differentially expressed genes. The resulting p-values were adjusted to FDR <5% with the Benjamini-Hochberg method to control the mean proportion of false positives (q-value R-package, [START_REF] Storey | Statistical significance for genomewide studies[END_REF]. Our microarray processing choices are explained below.

Technical aspects of microarray processing

A normalization step is necessary to remove non-biological variation among samples. Normalization comprises background correction, a within and between chip normalization and a probe summarization process to generate the final expression values for each probesets, a group of probes representing one particular mRNA.

Several normalization protocols have been proposed based on different biological and statistical assumption. Thus, it is necessary to choose the method that most effectively normalizes the microarray data. In order to measure the background, Affymetrix microarrays includes for each gene 11 to 20 probesets that are composed by a perfect match (PM) and a mismatch (MM) probe. The PM is a 25-mer oligonucleotide designed to be complementary to its target cDNA and the MM probe contains a mutation at the 13th position of the probe. MAS 5.0 (Affymetrix algorithm) corrects for cross-hybridization by subtracting the MM signal from the PM to determine specific binding. A major problem of this approach is that the MM measures also cross-hybridization. Indeed, hybridization still occurs with the MM probe and leads to a MM intensity levels greater than expected. Thus, MAS5.0 normalization lead to false negatives and a great variability of the fold change estimate at low intensity [START_REF] Irizarry | Use of mixture models in a microarray-based screening procedure for detecting differentially represented yeast mutants[END_REF]. In our study, we preferred the GCRMA algorithm (GC-Robust Multichip Average) which uses the nearest-neighbor model (Santa Lucia, 1998) this study, we had to deal with dataset of multiple sources and had to find a proper normalization methods that allow good fold change estimate. We choose the frozen RMA (fRMA) method, which uses precomputed and -frozen‖ information from the large publicly available microarray databases to estimates each probe-specific effects and variances (McCall and Irizarry, 2010). Even if GCRMA is best to compare a unique batch of experiment, fRMA greatly outperforms any other method to compare different datasets and properly estimate fold changes and statistical significance when testing for differential expression. To assess statistical significance when comparing between different conditions, each gene could be tested independently from one another using a classical t-test or ANOVA. However, the incidence of false positives is proportional to the number of tests performed and the significance level (p-value cutoff). For example, a p-value of 0.05 signifies a 5% probability that a gene expression level is different between the two groups by chance. If 40,000 genes are tested, 2000 genes could be called significant by chance. Thus, it is important to correct the p-value to assess the false discovery rate of each gene when performing such statistical test. In this study, we used a recent approach to correct the p-value using LIMMA (Smyt, 2004). LIMMA is designed to easily analyze complex experiments involving comparisons between many arrays and correct for multiple testing using the bonferroni Hochberg FDR method thanks to a linear model of the whole dataset matrix that increase to normalization power of the pre-processing GCRMA or fRMA methods.

In order to assess the quality of each microarray replicates, we used principal component analysis (PCA) and clustering methods (hierarchical clustering) to measure the degree of similarity between samples (Fig. 24C). These methods are used to determine the relationships among a group of variables when it is not appropriate to make a priori grouping decisions. They determine the effect of each variable on the total variance of the data set, and extract the maximum variance possible from the data to group similar replicates. All along our microarray analysis, we confirmed a high degree of similarity between samples within the same sample type and much less similarity between samples from different sample type and withdrowed of the analysis the necessary outlier replicates.

Cell culture

Neurospheres cultures

Neurospheres were isolated from CD1 mouse fetuses at embryonic day 13.5.

Briefly, pregnant mice were euthanized by cervical dislocation and the fetuses were rapidly decapitated. After removal of meninges, the cortices were collected in Hanks' balanced salt solution without Ca2+ and Mg2+ (HBSS, Invitrogen, Carlsbad, USA)

and dissociated in trypsin-EDTA (5 g/L trypsin-2 g/L EDTA, Sigma, St. Louis, USA) plus 1 U/mL DNAse I (Sigma) solution, for 30 min at 37 °C. Following trypsinization, cells were washed three times with HBSS, resuspended in a final volume of stem cell culture medium (xx) and mechanically dissociated using a pipette. Approximately 1 × 10 6 cells/mL were cultured in the presence of 10 ng/mL murine epidermal growth factor (EGF, PeproTech), 10 ng/mL murine basic fibroblast growth factor (bFGF, PeproTech) and 1% antibiotic peniciline /streptavidine (Sigma), and maintained at 37

°C in a humidified atmosphere of 5% CO2. Under these proliferating conditions, the cells grow as free-floating neurospheres. After mechanical dissociation of neurospheres, proliferating medium was changed after 2 DIV. For differentiation analysis, cells were plated on coated coverslips pre-coated with poly-d-lysine (PDL, Sigma) at 1 × 10

Cell transfection

Dissociated neurospheres were transfected with a NEON electroporation system (Invitrogen) using 100 μl cells at a concentration of 5 × 10 6 cells/ml in electroporation buffer and 400 nM pCAG-Ascl1-ires-GFP or pCAG-Olig2-ires-GFP plasmids.

Electroporation was carried out using the following settings: 100 μl tip volume, 1300 V pulse voltage, 20 ms pulse width, one pulse. Electroporated cells were transferred to antibiotic-free EGF/FGF neurosphere medium, and experiments were carried out 24 h after transfection. Successful overexpression in 70% of living cells was confirmed by counting of the GFP positive cells 24h post-electroporation. Successful overexpression was then confirmed by real-time RT-PCR.

Drug screening

Drug repositioning is the process of finding new uses of existing drugs. The availability of several established clinical drug libraries and rapid advances in nextwork biology, genomics and bioinformatics has accelerated the development in silico drug screening. Here we used the Comparative Toxicogenomics Database (Davis AP, Murphy CG 2013) to searchevery drugs that were shown in a any system to modify the expression of one of the selected genes in enriched and specific gene lists generated for the oligodendrglial lineage cell types (OPCs, Premature OLs and myelinating OLs). Aiming at finding potential drug that could affect OPCs proliferation, survival or myelination. We selected drugs identified in at least two different studies with minimal toxic effect that were shown to significantly modify the expression of our gene of interest. We identified 184 drugs involved in OPC gene regulation and 519 drugs involved in myelin genes. As positive control, we identified many drugs already tested in the demyelinating/remyelinating context such as Cuprizone (demyelinating drug), Fingolimod, dimethyl fumarate (remyelinating drug in MS). Focusing on drugs that were untested in remyelinating studies, we selected Creatine as genes involved in creatine metabolism Gatm and Gtam were found to be specific or enriched in myelinating oligodendrocytes.

Chapter II: Data analysis and interpretation

ChIP-Sequencing Peak-Calling and Data Analysis

All the Chip-sequencing analysis were done using Cisgenome2.0. Sequencing data were mapped to rat genome assembly (Baylor 3.4/rn4) using bowtie (version 0.12.7). Quality control and peak calling was performed using MACS (Model-based Analysis of ChIP-seq version 1.4.2) with default parameters. Filter was apply using a p value cutoff < 10 -9 ; an 5-fold enrichment compared to control; tag number > 20. The genome-wide distribution of protein binding regions was determined by genomatix in reference to Ensembl RGSC3.4.61 release and converted into mouse mm9 assembly for comparison with mouse data using the liftover tool of Cisgenome2.0. Phylogenetic conservation analysis was performed using Cisgenome2.0. Chip-sequencing peaks (BED file) were uploaded into genomtix to generate overview statistics of overlapping regions such as promoters, exons, introns, repeats, microRNAs etc. We then performed a next-neighbor analysis selecting all hit within a genes and genes associated with peaks in less than 5kb regions. We then associated integenic peaks with epigenetic and Hi-C data obtained from embryonic and adult brain tissue to detect long range promoter-enhancer interactions (Visel et al, 2010). Finally, we performed diverse gene set enrichment analysis with the identified direct targets either with pathway studio or genomatix and finally reconstructed the transcriptional network of Ascl1 and Olig2.

RT-PCR Analysis of ver-expression of Ascl1 and Olig2 in neurosphere

Total RNA was isolated from dissociated neurosphere cultures using the RNeasy Mini Lipid Kit (Qiagen) with DNase treatment. Tissues were homogenized in 1 mL QIAzol Lysis Reagent (Qiagen) using electric homogenizer. cDNA was generated from 500 ng of total RNA using SuperScript-III (Invitrogen

Transcription factor binding site enrichement analysis

Methods that detect occurrences of TFBSs and clusters of TFBSs from DNA sequence data are a usefull tool to understand the structure of cis-regulatory regions (Hardison and Taylor 2012). Methods used in this analysis detect clusters of motif matches appearing more frequently than expected given a promoter or whole-genome background model (Berman et al., 2002;Frith et al., 2002;Halfon et al., 2002;Markstein et al., 2002;Rebeiz et al., 2002). This Analysis searches transcription factor binding sites (TFBS) within the input sequences (BED files); and generates statistics on single TFBSs and TFBS pairs (modules) together with overrepresentation values and Z-scores. TFBS pairs are defined as two TFBS in a distance of 10-50 bp and allow to assess preferred distance ranges between TFBS. All occurrences of TFBS matches are calculated and the overrepresentation values are given based on the background of occurrences of the TFBS within the whole genomic sequence of the selected species or within all promoters as already annotated (500/100bp up/downstream of the TSS).

Analysis output can be either to TFs family matrices or to individual TFs matrices. We also used de novo methods for the discovery of TFBS, such as MEME-CHIP (Timothy L. Bailey, 2009);

OBJECTIVES & HYPOTHESES

Our project aims to provide a new molecular understanding of the transcription program involved in neural stem cells differentiation into oligodendrocytes. The rational of this work relies on previous studies demonstrating that the bHLH transcription factors Olig2 and Ascl1 work in synergy to specify OPCs, the oligodendrocyte progenitor cells [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF][START_REF] Zhou | The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification[END_REF]Parras et al., 2004;[START_REF] Xin | Myelinogenesis and axonal recognition by oligodendrocytes in brain are uncoupled in Olig1-null mice[END_REF]Wang et al., 2006;[START_REF] Parras | The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes[END_REF][START_REF] Sugimori | Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord[END_REF][START_REF] Sugimori | Ascl1 is required for oligodendrocyte development in the spinal cord[END_REF].

One central goal of this work was to understand at a genomic and transcriptomic level, how Ascl1 and Olig2 work together to specify OPCs. To do so, we followed a strategy using genome-wide transcriptome analysis and chromatin immuno-precipitation. We characterized Ascl1 and Olig2 regulated genes using genome-wide microarray analysis of embryonic ventral telencephalon from Ascl1 KO , Olig2 KO and wild-type (WT). Next, we identified functional high-confidence whole-genome transcription factor binding sites profiles for Ascl1 and Olig2 using chromatin-immuno-precipitation techniques in embryonic ventral telencephalon and purified OPCs. In order to perform an integrative analysis of the neural stem cell (NSCs) and oligodendroglial lineage differentiation processes, we coupled our Ascl1/Olig2 analysis with epigenetic information for active transcriptional enhancers in neural stem cells and OPCs that we combined with a celltype specific transcriptomic analysis of the major cell types in the brain. We identified new specific markers of different neural lineages and new important genes driving OPCs differentiation. We focused on Chd7 and Tns3, two genes whose expressions are driven by Ascl1 and Olig2 and enriched in the oligodendroglial lineage at two interesting stage, the early specification stage and the transition between migrating and differentiating oligodendrocytes, respectively.

Another major challenge of this work was to find therapeutical molecules promoting myelin repair. As currently, most of the available treatments for demyelinating diseases are based on immuno-modulatory and anti-inflammatory drugs and only a few are able to promote myelin repair, we used an approach of toxicogenomic and drug repositioning to identify new molecules known to modify OPCs and myelin genes but untested in the context of demyelinating diseases. We identified creatine as a potential metabolite important in the oligodendrocyte and axonal metabolism.

RESULTS

Role of Ascl1 and Olig2 in neural stem cell and oligodendrocyte differentiation Ascl1 and Olig2 regulated genes in embryonic ventral telencephalon identification by microarray analysis

In order to characterize the role of Ascl1 in NSCs and OPCs, we identified the Ascl1 regulated genes using genome-wide microarray analysis of E13.5 ventral telencephalon from Ascl1 KO and WT (Fig. 24A). The embryonic ventral telencephalon is formed by the ganglionic eminences and is mainly composed of NSCs and migrating neuroblasts but is also the first place of OPCs specification in the forebrain. We dissected part of ventral telencephalon containing the medial, lateral and caudal ganglionic eminences (Fig 24A -D), including also the following regions: subpallium (Spall), rostral secondary prosencephalon (RSP) and part of the prethalamus (PTh)

according to Allen Brain E13.5 reference atlas (http://developingmouse.brainmap.org/static/atlas). We identified 2337 unique genes significantly regulated by Ascl1 (P-value < 0.05 and fold-change >1.2 fold) in this region. We subsequently analyzed the functional meaning of the genes identified using a -gene-set enrichment analysis‖ to detect statistically significant signalling pathways regulated by Ascl1 (Fig. 24). As expected, we found Notch/Hes pathway as significantly regulated. Interestingly, we found that Ascl1 regulates Notch signalling at different level: Ascl1 regulates Notch at the receptor level (Notch1), at the ligand level (Jag1, Dll1, Dll3), at the downstream target genes level (Hes1, Hes5, Hes6, Hey1) but also at the regulatory level (Mfng, Fbxw7, Itch). Similarly, Ascl1 regulates the Wnt pathway at different levels. Indeed, Ascl1 regulates the Wnt5a ligand gene, Fzd8 receptor gene, but also the Gsk3b, Tcf7l2, Axin1 and Ctnnb1 effectors genes. We also found important regulators of the cell cycle and mitosis such as Ccnd1, Ccnd2, Cdca8, Aurkb, Cdk7, Cdk6, Csnk2a1 Cdc25b, as being significantly regulated, thus providing evidence of a functional control of Ascl1 in the G1-S and G2-M transitions. Moreover, Ascl1 is involved in other important signaling in NSCs such as, Integrin signaling (Itgb3bp, Itgb1, Itgb5), Semaphorin signaling (Sema7a, Sema5a, Sema3a, Sema3e, Sema3c, Sema4c) and Ephrin signaling (Ephb2,Ephb3,Epha3,Epha4,Epha5).

Then, we performed a genome-wide microarray analysis of E13.5 ventral telencephalon from Olig2 KO and WT and identified 1318 unique genes regulated by Olig2 (P-value < 0.05 and fold-change >1.2 fold, Fig. 26A). We identified 674 unique genes regulated by both Ascl1 and Olig2 and 644 unique genes regulated only by

Olig2. Interestingly, we found that a large proportion of genes regulated by both Ascl1 and Olig2 follow the same pattern of regulation (and really few having opposite regulation) thus demonstrating the synergistic action of Ascl1 and Olig2 in NSCs physiology. Moreover, the gene-set enrichment analysis of the shared and unique regulated genes showed that Olig2 and Ascl1 mostly regulate the same signalling pathways despite some specificity (Fig. 26B). Indeed, Olig2 also regulate genes involved in cell cycle (Ccnl2,Cdc73,Mcm4,Ccnd2,Csnk2a2,Cenpl,Cdk5rap1,Atm,Ccnc,Ccnd1,Cdc27,Ccnt2), Notch signalling (Notch1, Notch2, Hes1, Itch, Jag1),

Wnt signalling (Gsk3b,Tcf7l2,Fzd5 Fzd1,Dvl1,Axin1,Fbxw11) showing the most down-regulated and statistically significant Ascl1 regulated genes (maximal fold change and FDR 1%). As expected, Ascl1 transcript is highly down-regulated, together with members of the Notch pathway such as Dll1, Dll3, Mnfg or Hes5. Interestingly, another Ascl1 probeset, measure an increase in gene expression in the knock-out. Partial transcription of the Ascl1 exon 1 region may be up-regulated larger selection of Ascl1 regulated genes can be found in annex.

Ascl1 direct target in embryonic ventral telencephalon identification by chromatinimmuno-precipitation

We used promoter-specific ChIP-on-chip and whole genome ChIP-sequencing techniques generated with Ascl1 antibody on whole E12.

Ascl1 and Olig2 direct target Identification in oligodendrocyte progenitor cells.

In order to find Ascl1 direct targets in OPCs, we generated Ascl1 ChIPsequencing data from purified OPCs (see methods for details, in collaboration with Richard Q. Lu) and identified 3252 regions (Fig. 30A) significantly bound by Ascl1 from which 1507 region were located inside a gene and 1745 in intergenic regions.

Following the same method previously mentioned, we inferred functional cisregulatory regions from epigenetic information for H3K4me3 and H3K27Ac in OPCs We then analysed Olig2 ChIP-sequencing data from purified OPCs (GSM1040156)

and identified 21297 regions bound with high-confidence by Olig2. We compared the Ascl1 and Olig2 bound regions identified in OPCs and found 1514/3252 regions that are Ascl1 specific, 19588/21297 regions that are Olig2 specific and 1738 shared regions. repartition of the OPCs Olig2 binding sites. Among the 21297 identified peaks in Olig2 Chip-seq in OPCs, 45% are intronic, 35% are intergenic and 14.8% of peaks are located in promoters. This reflects the enrichment of TFBS in promoters, enhancers and exon-intron junction where they play a role in the splicing regulation.

Interestingly, Ascl1 binding sites specific for OPCs are located in or near genes known to have key functions in the oligodendroglial lineage such as the early marker Olig2, Pdgfra, Cspg4 (NG2) or Gpr17 but interestingly also in genes involved in later stages and myelination such as Mbp, Qk, Plp1, Brg1, Myt1, Thra or Fa2h (see Fig. 31). We then analysed Olig2 ChIP-sequencing data from purified OPCs (GSM1040156)

and identified 21297 regions bound with high-confidence by Olig2. We compared the Ascl1 and Olig2 bound regions identified in OPCs and found 1514/3252 regions that are Ascl1 specific, 19588/21297 regions that are Olig2 specific and 1738 shared regions.

In order to construct a genomic profiles, we intrgrated our datasets to the Olig2 Chip- Together, these results demonstrate the synergistic role played by Ascl and Olig2 in OPCs physiology in driving important signaling pathways such as Notch and Wnt but also their role in transcriptional activation of myelin genes whose protein expression will only be effective later on the development. Interestingly, the number of high confidence binding sites is much higher for Olig2. As similar binding site number differences were observed in different replicated Chip-seq experiment for Ascl1 and Olig2 in different contexts, this discrepancy is unlikely to be caused by technical or processing issues. In order to understand this difference, we identified all the binding site matrices of both Ascl1 and Olig2 searching for matrix specifity.

Ascl1 and Olig2 consensus DNA binding sequences analysis

bHLH proteins form heterodimers with E-proteins to bind DNA sequences called E-boxes (CANNTG). Thanks to ChIP sequencing data, it has been possible to define specificities in E-boxes for different bHLH proteins. In order to determine if Ascl1 and Olig2 have such binding preference for an E-box subtype, we searched in all the regions identified in Ascl1 and Olig2 ChIP-seq, all occurrences of E-boxes matches to perform a de-novo motif discovery and an E-box subtype overrepresentation analysis. We found that the E-boxes recognized in Ascl1 ChIP-seq correspond to degenerate TFBS matrices defined by binding frequency order as Ascl2, Ascl1, MyoD matrices (Fig. 35) and E-boxes recognized in Olig2 ChIP-seq correspond to Atoh1 (Fig. 35). As identified ChIP-seq regions can be bound by both Ascl1 and Olig2 and can display many E-boxes, we confirmed this sub-type specificity by selecting Ascl1 sepcific or Olig2 specific regions containing only one E-box. We obtained the same preferential binding order and confirmed the larger scale results (Fig. 35). Interestingly, Atoh1 and NeuroD1 matrices contain a CA/TC/TG E-box, while Ascl12 and Ascl1 contain a CA/CG/TG matrix. This difference could be in part explain the difference in binding frequence observed for each TFs in the Chip-seq datasets.

Bioinformatic identification of Ascl1 and Olig2 cofactors

Models of combinatorial regulation by several TFs can be inferred from in-silico analysis of ChIP-seq binding regions (Hnisz D, Abraham BJ 2013). To identify potential transcription co-factors and identified the precise chromosomic position of all the E-boxes present in those regions. We extended those chromosomic positions of 50bp up and downstream to screen those regions in search of other known transcription factors TFBS matrices or transcription factor modules (one E-box flanking another TFBS matrix located a less than 50bp). This in-silico analysis showed that Ascl1 and Olig2 E-boxes are most frequently associated in pairs of E-boxes (tandem of TFBS) separated by a stable distance of 15bp (Fig. 37). We found that Ascl1 preferentially make TFBS clusters with E47, TCF15, MAZ, SP1, LHX8 (Fig 36), while Olig2 preferentially associate with Nf1, Evx1/2, Gsh2, Emx2, Zic1/2/3, Sox2/9, Gli2/3 (Fig. 40). Interestingly, the potential co-factors identified by this bioinformatics screen all share the same expression pattern than Ascl1 and Olig2 (as cross checked on Allen brain atlas), thus reinforcing the inference about their potential action as co-factors.

Gene expression cell-type specificity of the oligodendroglial lineage

In order to prioritize the genes regulated by Ascl1 and Olig2 in OPCs and the ventral telencephalon, we reprocessed microarray data obtained from purified OPCs, pre-myelinating and myelinating oligodendrocytes (GSE15303; GSE9566) that we compared using different clustering methods in order to identify the genes involved at each stage of the oligodendrocyte lineage (Emery et al., 2008, Cahoy et al., 2006). We identified the genes involved in the transition from NSCs to OPCs comparing microarray data obtained from purified OPCs with different microarray data obtained from embryonic and postanatal CD133/Prominin NSCs (GSE8034; GSE18326; GSE17760). We refined our screen using microarray data from all available brain celltypes such as different subtypes of postmitotic neurons (GABAergic, glutamatergic, dopaminergic, serotoninergic), astrocytes, microglia and endothelial cells to assess for strict cell-specificity (GSE29949; GSE9566; GSE11870). We validated our approach by the identification of known and unknown specific markers of each cell lineage, selected based on gene clustering analysis (see material and methods and annexes) and RNA expression pattern at E14.5 (Eurexpress) and P56 (Allen brain atlas). We selected different gene lists whose expressions are enriched in different stages of the oligodendrocyte lineage or from other cell types for subsequent analysis and paid particular attention to transcriptional factors (Fig. 38). presenting Olig2 bound by Olig2 (Chip-seq) in H3k27ac region(Chip-seq) whose expression is enriched genes in myelinating OLs compared to other cell types. Expression data for NSCs, OPCs, PremyelinatingOL, myelinating OLs, neurons and astrocytes. One regulatory chromosomic position is indicated for each gene. This kind of cis-regulatory sequence is higly enriched in TFBS cluster specific to the defined cell-type and can be use in the context of genetic therapies to build synthetic promoters able to drive specific gene expression or repression.

Identification of Ascl1 and Olig2 regulated genes involved in oligodendrocyte lineage

Identification of Cdh7 as putative regulator of oligodendrogenesis and myelination.

As mentioned in the introduction, Chd7 is a member of the Chromodomain Helicase DNA binding domain (CHD) family that plays a role in controlling gene expression by ATP-dependent chromatin remodeling. Chd7 is known to interact with components of the BAF (Brahma associated factor complex) and PBAF complexes (Polybromo containing complex) and thus help recruiting transcriptional activators or repressors depending on the cellular context, such as embryonic stem cells [START_REF] Schnetz | Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns[END_REF] or neural crest cells where it interact with Brg1 (Li et al., 2013). We found that intronic regions of Chd7 locus are bound by Ascl1 and Olig2 in OPCs, at regulatory elements presenting epigenetic marks associated with transcription activation such as H3K27Ac in OPCs, immature or myelinating oligodendrocytes (Fig. 39A, red rectangles). Moreover, Brg1 remodeling complex factor that interacts with Olig2 also binds in these elements in OPCs, suggesting that this gene is part of the regulatory mechanism of Olig2 function in oligodendrogenesis. Both Chd7 expression pattern at embryonic (Fig. 39B) and postnatal/adult stages (Fig. 39C) indicate that Chd7 is expressed like Ascl1 and Olig2 in VZ/SVZ progenitors and in oligodendroglial containing regions (such as the corpus callosum or the fimbria, arrowheads). Indeed, the transcriptome comparison of neural subpopulations clearly indicates that Chd7 mRNA is expressed mainly in OPCs and immature oligodendrocytes (Fig. 39D). We investigated if Chd7 was the only member of the chromodomain family expressed in OPCs and found that Chd8, the paralog of Chd7 to the common Drosophila ortholog, Kismet, was also express in OPCs (Fig. 39E). We then searched for evidences that Chd7 could be involved in de/remyelination and found that CHD7 is significantly downregulated in human active MS plaque [START_REF] Han | Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors[END_REF]Fig. 39E) as well as in a FACSorted OPCs purified after a cuprizone induced demyelination (S. Moyon and C. Lubetzki,unpublished;.007; Fig. 39F). Transcriptional regulation of Chd7 by Ascl1 and Olig2 was obtained not only by our microarrays analysis of Ascl1 KO and Olig2 KO (Fig. 39F) but also by overexpressing Ascl1 or Olig2 in neurosphere cultures, each of which was sufficient to upregulate Cdh7 levels compared to controls (Fig. 39I,H). Finally, Chd7 has been shown to bind directly to different sequences located near the Olig2 locus in the SH-SY5Y neural cell line known to express Olig2 [START_REF] Schnetz | Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns[END_REF]. Base in data integration, we show here that those sites defined as active enhancers (H3k27ac, P300 marks) are able to interact with the Olig2 transcription start site (Hi-C data) most likely due to the binding of Olig2, Ascl1, and Gli1 (Fig. 39K). Therefore, those sites may constitute important regulatory sequences for Olig2 expression in oligodendrogenesis suggesting that Chd7 together with Ascl1 and Olig2 play a role in this process. All these data strongly suggest that Chd7 is an important Ascl1/Olig2 target controlling oligodendrogenesis.

Chd7 protein expression during myelination

Given the Chd7 mRNA expression in oligodendroglia and white matter regions, we characterized Chd7 protein expression by immunostaining at postnatal stages. Chd7 protein was coexpressed during neonatal stages (P3) with Ascl1 (but not Olig2) in the SVZ progenitors (expressing Mcm2 proliferation marker, Fig. 40C) and in all OPCs (Fig. 40B) as expected from our transcriptome analysis. During the peak of myelination, at 3 weeks postnatal (P24), we found Chd7 protein in all OPCs (PDGFRα + cells, Fig. 40D-F) while its expression levels were highest in maturing oligodendrocytes (APC/CC1 + cells; Fig. 40E,F) and was reduced in mature oligodendrocytes (not shown). Therefore, Chd7 protein expression correlates with OPC differentiation suggesting a role in chromatin remodeling involved in this process during myelination. Given the expression pattern of Chd7, we investigated if its paralog gene, Chd8, was also expressed in oligodendrocyte lineage cells as expected from its expression in postnatal (P7) OPCs expression arrays (Fig. 39E). Indeed, Allen Brain Atlas in situ hybridization database (www.allen-brain-atlas) shows expression of Chd8 transcript in some myelinating cells in adult brain (P56, Fig. 41A) and microarray expression data from neonatal (P7) rat brain neural subtypes shows Chd8 expression in both OPCs and oligodendrocytes (Cajoy et al., 2007;Fig. 41B). We therefore investigated Chd8 protein expression in postnatal brain and found that it was strongly expressed in maturing oligodendrocytes (CC1/APC + cells, Fig. 41C,D), maintained at lower levels in mature oligodendrocytes (Fig. 41D) but differently to the mRNA, rarely detected in OPCs (PDGFR+ cells, Fig. 41C,D). Therefore, during brain myelination while Chd7 is expressed both in OPCs and differentiating oligodendrocytes, Chd8 is only expressed in differentiating oligodendrocytes (Fig. 41D) suggesting a role in the process of differentiation and myelination.

CHD8 protein expression in multiple sclerosis brain lesions

The data of Chd8 expression in demyelinating mouse models led us to investigate if CHD8 could also be expressed in oligodendroglia during remyelination in MS patients. We therefore have started to characterize CHD8 expression in different types of MS lesions, either chronic or actively remyelinating. Our preliminary data show that CHD8 can be found in oligodendroglial cells in the white matter close to demyelinated lesions (Fig. 43A,B) presenting typical alignments of white matter OLs (Fig. 43A). Moreover, we could find Chd8 co-expression with Sox10 oligodendroglial marker in cells at the border of the demyelinated lesion (Fig. 43C). We are currently characterizing CHD8 expression in different areas and lesions of MS patients to assess if CHD8 expression correlates with remyelination and it could be used as a hallmark of remyelination.

Identification of TNS3 expression in premyelinating oligodendrocyte

Tensin 3 (Tns3) was one of the genes downregulated in Ascl1 KO (Fig. 44F) and Olig2 KO presenting binding by these two factor in an intragenic enhancer region showing active epigenetic marks in OPCs and differentiating oligodendrocytes (Fig. 44A). The expression pattern by in situ hybridization presented a strong specificity in white matter cells (such as the corpus callosum or the fimbria, Fig. 44B arrowheads) in the adult brain, and it was expressed like Ascl1 and Olig2 in the VZ zone of the embryonic (E14.5) ventral telencephalon (Fig. 44C). Moreover, the enrichment in the oligodendroglial cells was found also in the expression arrays of oligodendroglial cells in the postnatal brain (Fig. 44D), being Tns3 the only member of the family expressed in the brain (Fig. 44E).

TNS3 EXPRESSION DURING MYELINATION

Tensin family is a group of focal adhesion proteins that interact with actin (by an actin binding motif), integrins and phosphotyrosine-containing proteins (through a SH-PBT motif). Tns3 has been found to be a negative regulator of cell migration and to be downregulated in human kidney cancer (Martuszewska et al., 2009). We therefore, investigated the expression of Tns3 in the postnatal brain. Indeed an anti-Tns3 polyclonal antibody showed specific expression in white matter regions correlating with the start of OPC differentiation in the brain (starting in the brainstem at P4 and appearing in the corpus callosum in the second postnatal week (Fig. 45A). Differently from the mRNA expression seen at P7 brain cell-types, we rarely detect Tns3 protein expression in PDGFR + OPCs (Fig. 45A,B). As expected from the mRNA data mentioned before, its expression was strong in differentiating oligodendrocytes (strong APC-expressing cells). Surprisingly, Tns3 was localized mainly in the nucleus of the differentiating oligodendrocytes and some neuronal population (Fig. 45A). The specificity of our anti-Tns3 antibody was confirm with a Tns3-LacZ gene-trap line, which show expression in Olig2 + / PDGFR -cells (oligodendrocytes) in the postnatal brain (Fig. 45C,D) and its by its cytoplasmic expression in the kidney and other regions

TNS3 EXPRESSION DURING REMYELINATION

Given the expression of Tns3 during myelination we investigated if it could also play a role during the process of remyelination. Using the LPC-induced focal demyelination model mentioned before, our preliminary studies has found that Tns3 is strongly expressed soon after the demyelinating insult (2, 4 or 8 days post-injection, dpi), in and around the LPC demyelinating lesion (compare Fig. 46A-A showing strong Tns3 expression in APC + /Olig1 citoplasmic cells (immature OLs) around the lesion. Note the absence of APC + /Olig1 + cells (oligodendrocytes) in the lesion. A 1 and A 2 are higher magnification of corresponding insets (red squares). Demyelinated lesion is shown by the dotted circle area. Note the absence of Tns3 expression and presence of APC + /Olig1 + oligodendrocytes in the lesion. Scale bar, 20 m.

Together, all this data indicates that Tns3 expression clearly correlates with oligodendrocyte differentiation during myelination and remyelination and therefore it will be important to characterize its function in these processes and its possible implication in Multiple Sclerosis pathology.

Myelinating oligodendrocyte control the creatine metabolism in the brain

Creatine is a small metabolite that serves as an energy shuttle between the mitochondrial sites of production and the cytosol, the site of ATP utilization. As ATP diffusion is very limited, phosphocreatine and creatine, much smaller molecules, are able to diffuse very easily and they can accumulate to high concentrations without affecting metabolism (Wyss and Kaddurah-Daouk, 2000). showing drugs shown to affect oligodendrocyte genes and never tested in the context of demyelinating diseases. As creatine was among the identified molecules, we focused on its role in myelination and remyelination.

Introduction

During development, the basic-helix-loop-helix factor (TF) Olig2 is both and, in some contexts, suffi-166 cient for oligodendrocyte precursor (OPC) generation [START_REF] Lu | Oligodendrocyte lineage genes (OLIG) as molecular markers for human glial brain tumors[END_REF]Zhou et al., 2001;[START_REF] Zhou | The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification[END_REF]Yue et al., Zhu et Despite this role, not a master and it acts in concert with additional cofactors to regulate oligodendrocyte For must act with controlling its phosphorylation state acquire an oligodendrogenic [START_REF] Li | Phosphorylation regulates OLIG2 cofactor choice and the motor neuron-oligodendrocyte fate switch[END_REF]. The pattern in the embryonic brain sugthat interacts with multiple providing positional cues and regulating its action in progenitor et al., et et 2012). is another factor a role at different and potent ral and reprogramming (Bertrand et al., chen et al., 2010;Vierbuchen and Wernig, 2012).

is also expressed by embryonic and neonatal OPCs [START_REF] Kondo | Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells[END_REF]Wang et al., 2001;Parras et al., 2004;[START_REF] Parras | The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes[END_REF].

operates in interaction with during OPC specification in the embryonic telencephalon, and the loss of Ascl1 function reduces embryonic oligodendrogenesis (Parras et al., Sugimori et 2007). Therefore, beyond its role in in the regulation oligodendrogenesis.

167

tissue

The importance of action in postnatal oligodendrogenesis and myelination as well as its requirement for proper myelin repair have however not yet been

To ascertain its involvement in oligodendrogenesis myelination and remyelination, we have examined expression and function in oligodendroglial during the period of development and during myelin repair in the brain. At all ages examined, Ascl1 expression was restricted immature (i.e., cortical subventricular zone genitor and OPCs of gray matter and white matter). Using different genetic models to delete Ascl1 in progenitor cells OPCs, and to trace the we have characterized Ascl1 requirement in oligodendrogenesis both under physiological conditions and after demyelinating lesions. We found that was required in progenitors for oligodendroglial cell fate decision as well as in OPCs for their differentiation into oligodendrocytes. After demyelinating lesions, was expressed by intralesional progenitors OPCs, which regenerated myelin-forming and thus pated in myelin repair of demyelinated lesions. Although Ascl1 dispensable for recruitment and maturation of intralesional progenitors and OPCs, we showed that it was required for a proper balance between the number of OPCs and cytes. Finally, examination of human periventricular (MS) lesions confirmed that expression is a hallmark of OPCs involved in myelin repair, both in mice and mans. Together, our findings support a promoting role for Ascl1 during postnatal and in the of elination, both in progenitors for OPC as as OPCs for their Postnatal brain electroporation (Boutin al., 2008) was adapted to target the dorsal postnatal day 2 (P2) pups were cryoanesthetized for 3 min on ice, and 2 l of plasmid solution (3 g pCX-Cre) was injected into the ventricle using a glass capillary. pCX-Cre plasmid drive Cre expression under the CMVmodified strong and ubiquitous promoter (kindly gift from Alain dotal, Institut de la Vision, Paris). Electrodes (Nepagene CUY650P10) were positioned in the dorsoventral axis with the positive pole dorsal. Five electric pulses of 100 V, 50 ms pulse ON, 850 ms pulse OFF were applied using a Nepagene electroporator. Pups were warmed up in a heating chamber and brought to their at end of the Tamoxifen administration. Tamoxifen (Sigma) was dissolved in oil (Sigma) at 20 mg/ml. Postnatal mice received one subcutaneous jection 400 (P5) or one intraperitoneal injection 800 Adult animals received one intraperitoneal injection per day of 200 g for 5 days starting 7 d before lesion.

MS and

Autopsy brain tissue samples from 4 patients with confirmed secondary MS and two tients without neurological diseases (Table 1) were obtained from United Kingdom MS tissue bank (Richards Imperial College, London). MS and control brain samples were excised from periventricular regions or white matter. Four MS tissue blocks ing active (n 3) and chronic active lesions (n 3), lesions, and periplaque white matter were selected for further analysis.

were as reported (Lassmann, 1998). Ascl1 Cre/LacZ mice (P120) were perfused with and 0.5% glutaraldehyde in buffer. Brains were sectioned (300 m) with a Tissue chopper (McIlwain) and subjected overnight to X-gal reaction. Tissue was postfixed in 2.5% glutaraldehyde for 2 Regions containing the CC were dissected out under a binocular scope (MZ16, Leica). Tissue samples were osmicated, dehydrated in and embedded in Epon. Ultrathin sections were made using a Leica electronic microscopy (EM) UC7 ultramicrotome, and were taken with a Philips electronic microscope. We optimized immunostaining for which is very sensitive to fixation conditions. Mice were perfused with freshly made 2% paraformaldehyde (Sigma) with the (P0-P10, 15 ml; P10-P16, 20 ml; adult, 25 ml), brain was dissected cryoprotected in with 20% sucrose overnight, and included in OCT (BDH) before freezing and sectioning (14 m thick) with a cryostated microtome were then either processed for tochemistry or stored at 80°C. P0-P16 brain sections were postfixed with 4% for 10 min. (gift from K. Campbell, Ki67 (Novocastra, 1:1000), myelin basic protein (MBP, Millipore, 1:1000), NG2 (Millipore, 1:1000), Olig1 [START_REF] Mizuguchi | Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtypespecific properties of motoneurons[END_REF], Olig2 (Millipore, 1:1000), Sox9 (R&D Systems, 1:500); guinea pig antibodies Sox10 (gift from M. Wegner, 1:1000), Ascl1 (gift from J.

rat monoclonal antibodies to (Dako, 1:50), PDGFR (BD and antibodies to Laboratories, and (Millipore, secondary tibodies included the following: AlexaFluor-488, AlexaFluor-594, Ig Invitrogen). Antigen retrieval for body staining was performed using antigen retrieval solution (Vector, H-330), heating in citrate-based solution up to boiling and allowing cooling down at room temperature for 20 min. Sox9 (80%; Fig. 1C,G), two TFs expressed by immature progenitor/stem (Hack et al., 2004;Scott et al., 2010). The majority of Ascl1 /Olig2 /Sox9 cells expressed PDGFR , the most marker for OPCs (Hall et al., 1996;et 2006), nor other OPC markers, such as NG2 not shown), or in such as Nkx2.2 TFs (Fig.

G). In the CC, however, Ascl1 cells included equal populations ( 50%) of Olig2 /Sox9 PDGFR progenitor cells (Fig. 1A-E,G) and PDGFR cells (Fig.

)

. It is noteworthy that all Ascl1 /PDGFR cells ( 10% of CC and 2% of cells) also expressed Olig2 (Fig.

) and both nuclear and cytoplasmic Olig1 (Fig.

), ), and Sox9 (Fig.

)

. This phenotype suggests that all Ascl1 /PDGFR cells are OPCs and a fraction of Ascl1 PDGFR correspond to immature neural in the process of generating OPCs (Fig.

)

. Supporting possibility, we found that 48% of Ascl1 /PDGFR not express either Dlx or Gsx2, two neurogenic cofactors ryniak et al., et al., 2009) that are not expressed OPCs (Fig.

)

. Therefore, Ascl1 /Olig2 /PDGFR Dlx /Gsx2 (corresponding to 5-7% of or CC cells) are immature oligodendroglial cells.

/PDGFR in is upon oligodendrocyte differentiation We then investigated Ascl1 expression during OPC into myelinating

In the CC, when many OPCs start to differentiate during the second postnatal week, is still expressed in all PDGFR OPCs (Fig. 2A), but APC/CC1expressing maturing oligodendrocytes (Bhat et al., 1996) were Ascl1 B ) that maturing 168 Ascl1 B ) that maturing regulate expression. Indeed, using the Ascl1 GFP transgenic line, in which GFP remains expressed longer than Ascl1, we found that most APC at this were GFP (Fig. 2B). ). In Cko OPC , a two fold increase in OPC/OPC pairs (48.4 4A ,A ), a twofold in OPC/OL pairs (14.6 3.1%), and no change in numbers of OL/OL pairs (37.0 5.9%; Fig. 4A ,A ), suggesting that, in the absence of Ascl1, OPCs that would divide (OPC/OL pairs) divide instead generate two OPCs

). results obtained when tamoxifen was injected at P5 or P8-P9 (Fig. ).

thus regulates and promotes asymmetric divisions of OPCs. quently, the ratio of OPC/oligodendrocytes within the YFP population was increased in Cko OPC cells (1.4-fold) with 1.5% PDGFR n Ctr OPC : 47.6 1.8% APC vs 52.4 1.5% PDGFR n 3, indicating that loss of in OPCs favors proliferation over differentiation. The differentiation defect of Ascl1-mutant appeared more nounced regarding their expression of myelin proteins, such as CNP (Fig. 4C-C 2 ,E). To whether these differences were maintained at later stages, we studied the proportion of OPCs and oligodendrocytes present at 28 dpi. A similar ratio of 4: In control experiments using a GFP-expressing plasmid, many GFP cells radial thus demonstrating that dorsal neural progenitor/stem cells were targeted by electroporation technique (Fig. 3B). In brains with plasmids, protein detected only but not in cells (Fig. 3C,C 1 ,C ,C 1 ), that could be deleted by Cre recombination. the fate of electroporated cells, we analyzed the brains 30 d postelectroporation (dpe). The majority of (62.5 n 5, high ), some were astrocytes (32.1 5.9%, n 5, GFAP low ), and few were OPCs (9.3 0.8%, n 3, PDGFR ) (Fig.

D ,E). were OPCs (9.3 0.8%, n 3, PDGFR ) (Fig. D ,E). E). E 4.1%, 2.7-fold), only 15.3 1.8% were oligodendrocytes (3.9fold reduction), and 1.6 0.5% OPCs (5.5-fold (Fig. 3D ,D 1 ,E), indicating that loss of inhibits oligoden drogenesis for the benefit of astrogenesis. We did not observe a death of at 3 dpe (as shown by the absence of activated caspase3 expression; data not There was also a 1.7-fold increase in the number of Ascl1deficient GFAP cells in the SVZ compared with (88.9 11.1% vs 48. is therefore required for the proper generation of oligodendrocytes from the cortical and, in the absence of the progenitor's fate is predominantly astroglial.

171 Demyelination levels in adult OPCs expression is lost with oligodendrocyte differentiation, we then extended our study to adulthood to whether Ascl1 was still expressed in OPCs under normal demyelinating conditions.

an optimized protocol for Ascl1 immunofluorescence (see Materials and Methods), we detected intermediate to low levels of Ascl1 in young (P70) and aged (P200) brain, in (99%) in the high were observed in the neurogenic regions (i.e., SVZ/rostral migratory stream also detected at in majority ( 90%) in brain regions, including in the matter (in the thalamus, olfactory bulb; Fig. 5C; and data not shown) and white matter (in the fimbria, striatum, cerebellum, data not shown). In the a large fraction (67%) of OPCs (Fig. 5D). These results are accordance with the expression of the GFP reporter in Ascl1 GFP mice in the adult brain (Fig. 5B) [START_REF] Parras | The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes[END_REF].

In the adult demyelination in the upregulation of several TF involved in oligodendrogenesis, including tion of Nkx2.2, and Sox10 transcripts (Fancy et al., 2004, Watanabe et To determine whether expression is also altered in response to demyelination, we used Ascl1 GFP animals to perform LPC-induced focal demyelinating in the CC, as reported (Nait-Oumesmar et al. expression in OPCs (relative to its expression in SVZ upregulated to similar postnatal OPCs 5G). Moreover, Ascl1 GFP in the lesion (Fig. ) and in the SVZ (data not shown) coexpressed high of Olig2 and Nkx2.2, that all three TFs are upregulated in progenitors nearby OPCs involved in expression was not induced in oligodendrocytes after demyelination given that protein not detected Olig2 /APC cells (Fig. 5H, arrowheads). However, the GFP transgene was expressed in maturing oligodendrocytes of animals high of and Sox10, I 1 ), as we observed at postnatal These data indicate that, in both the postnatal brain and in sponse to demyelination, a combination Ascl1 is induced in OPCs/progenitors and likely involved mice, and the presence of intralesional APC /YFP oligoden drocytes at 5, 14, and 28 dpl. At all examined, the majority (67%) of intralesional PDGFR OPCs APC oligodendrocytes expressed and belong to the Ascl1 cell lineage (Fig.

D), with a similar proportion of YFP godendrocytes present in nonlesion territories (Fig. 7D). At 14 dpl, intralesional APC /YFP cells expressed myelin such as MOG (Fig.

C,C 1 ,C 2 ), that these were maturing into used identify intralesional -galactosidase /X-gal myelinating godendrocytes using Ascl1 Cre/LacZ mice. X-galreaction deposits were detected within the lesion area, in with characteristics of newly formed oligodendrocytes multiple axons (Fig.

E 1 ,E 2
). These observations that Ascl1 progenitors/OPCs generate myelin-forming cells that participate in myelin cells Ascl1 progenitor/OPCs are localized in and around focal LPC that they may differentiate into myelinating oligodendrocytes and participate to myelin repair. Ascl1 GFP mice, GFP expression persists long after that Ascl1 is downregulated, which fate mapping of progeny. We have examined the contribution of oligodendrocyte regeneration and lesion repair in mice. The demyelinated can be by a dense cellularity, the absence of myelin proteins (such as Fig. 6A), and oligodendrocyte depletion (APC Fig. 6C,E). At 2 dpl, were many GFP in the lesion, most of them being OPCs (79%; PDGFR /Olig2 6D) and the remaining cells being immature progenitors. At 5 dpl, the number of GFP cells in the lesion was increased sevenfold, 60% being OPCs (Fig.

D). both time points, OPCs GFP , indicating OPCs present within the lesion derive from

In addition, at 5 dpl, 25% of GFP in the lesion were APC oligodendrocytes (Fig. 6C,E), suggesting that these cells had differentiated from Ascl1 OPCs contributing to myelin lesion repair in the CC may derive from progenitors located in the (Nait-Oumesmar al., 1999, 2008;Aguirre et al., 2007;Franklin and ffrench-Constant, 2008;Staugaitis and Trapp, 2009). LPC-induced Ascl1 GFP mice showed OPCs associated with GFP /PDGFR progenitors in the displaying a bipolar shape and with the main cell body axis oriented toward the CC where the lesion is located (Fig. for either oligoden droglial (PDGFR or APC) or astroglial (GFAP) markers at stage (Fig. B ). Some of them had typical oligodendrocyte morphology despite not expressing APC (Fig. 8B ), most likely corresponding to with abnormal differentiation. In at 15 dpl, YFP -Cko OPC had a ratio of cyte to OPCs than Ctr OPC (Fig. 8C,C ,E), whereas the glial marker-negative population was very much reduced (Fig. To test this hypothesis, we examined Ascl1 expression in four MS and two non-neurological control mortem brain samples. Lesions were first characterized using Luxol fast blue/cresyl violet (Fig. 9B), MOG, and CD68

The and glial markers (Olig1 and Sox10) was examined in actively remyelinating borders (patchy MOG staining/CD68 ), in chronic core of the lesions (MOG /CD68 ; Fig. 9C), and normal-appearing white matter (Fig. C). Ascl1 cells were more numerous in active borders of MS lesions compared with chronic core or normal-appearing matter. Moreover, the number of Ascl1 cells was reduced in the core of lesions compared with the other regions studied. Interestingly, Ascl1 cells were more concentrated within the CD68 active oligodendrocytes To the contribution of Ascl1 progenitors to the remyelination process, we used the Cre/loxP recombination system trace the Ascl1-cell lineage in LPC-demyelinated mice. LPCinduced lesions were performed in the CC of adult borders of periventricular MS lesions (Fig. 9D) compared with other regions studied. Indeed, in other brain regions, cortex and the normal-appearing white matter, expression most resulting from poor detection due to the fixation constraints of human brain samples.

the majority of in MS periventricular lesions were Olig1 and Sox10 (Fig.

), that upregulation in the endogenous may play a role in oligodendrocyte regeneration under pathological 176 tions.

our findings on tissue are in agreement with data on animal models demyelination and demonstrate that Ascl1 is at higher in in plaques. may therefore be involved in oligodendrocyte ifferentiation and myelin repair in demyelinating pathology.

In this paper, we show that is expressed during brain elination and remyelination in progenitors and OPCs, play- promotes specification Ascl1-conditional deletion in dorsal SVZ cells strongly reduces the generation of oligodendroglial in the dorsal telencephalon and the majority of become instead astrocytes. This effect could be the result of the following: cell death of oligodendroglial reduced proliferation of oligodendroglial in the absence of ( 3) blocking of at immature genitor or ( 4) from oligodendrocyte in the of did not find preferential cell death of or of their progeny. Also, OPCs proliferate more than controls. In we found a 1.7-fold increase in the number of Ascl1-deficient GFAP in the compared with controls. Ascl1 is most likely required for OPC specification in the and in the absence of the majority of cells come astrocytes or remain undifferentiated progenitors. In vivo clonal studies would be required to discriminate between these two Indeed, a role in oligodendrocyte specification is supported by previous reports on in experiments of Ascl1 loss-and gain-of-function in progenitors. Clonal cultures of progenitors a reduced capacity to generate (and neurons) and differentiate stead into astrocytes (Parras et al., 2004), whereas Ascl1progenitors oligodendrocyte at the expense of an astrocytic fate [START_REF] Sugimori | Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord[END_REF]. elination (postnatal) or remyelination (adult). Indeed, one or 2 weeks after Ascl1 deletion, more Ascl1-deficient cells are still OPCs and fewer have started differentiation. Our data suggest that cooperates with other factors to activate OPC differentiation and myelin protein expression. quirement is compensated at later stages, and no phenotype is found in Ascl1-mutant Although known to regulate transcription in (Gokhan al., 2005), a genome-wide analysis of targets in oligodendrogenesis will be important to identify the Ascl1-target genes involved in the different of oligodendrocyte (specification, proliferation, division mode, differentiation, of during and remyelination Demyelination has been shown to upregulate

Nkx2.2, transcript in and around lesion and many factors (Fancy et al., 2004(Fancy et al., , 2009)), raising the question of similarities and differences between oligodendrogenesis ing myelination and remyelination. Our detailed characterization of the expression of Ascl1 and other TFs involved oligodendrogenesis (Figs. 2 and5) indicates that the same types of progenitors and OPCs, characterized by the same combined of markers, are in during myelination and remyelination.

demyelination, we found that upregulated in OPCs at levels found during myelination. Moreover, the levels of Ascl1, Olig2, Nkx2.2, and Sox9/10 expression at different stages of oligodendrogenesis were similar during remyelination myelination (Fig. 5; and data not shown). Therefore, the regulation of these factors during active remyelination likely results from the rejuvenation of a developmental genetic gram enabling OPCs to proliferate, migrate, and differentiate into new remyelinating oligodendrocytes.

Demyelination induces axonal functional and structural loss, leading to major neurological and motor dysfunctions. In MS, despite the recent therapeutic advances aimed at reducing inflammatory component and stopping demyelination, ments to improve myelin repair are lacking because of the understanding of mechanisms to In this context, our finding on the role of during myelin formation and in particular after demyelination, and future work characterizing its target may pave the toward a understanding of the in oligodendrogenesis after demyelination and therefore help tic targets aimed at favoring myelin function in oligodendrogenesis It has been suggested that plays an indirect role in oligodendrogenesis by Notch-mediated repression of the determinants Dlx1/2 [START_REF] Petryniak | Dlx1 and Dlx2 control neuronal versus oligodendroglial cell fate acquisition in the developing forebrain[END_REF]. However, in trast with the embryonic expression of in a subset of OPCs (Parras et al., Sugimori et al., 2007), we demonstrate that protein is present in the majority of postnatal OPCs, that a cell-autonomous function oligodendrogenesis. Some immature progenitors coexpress and Dlx in oligodendrocyte-generating regions of onic (Petryniak et and postnatal telencephalon study), leaving space for Dlx inhibition of OPC specification. Nevertheless, Ascl1 conditional deletion in either SVZ cells OPCs not only alters oligodendrocyte specification, but OPC proliferation differentiation demonstrating cellautonomous role of at different of esis. It is worth noting that and Olig2 are coexpressed immature that OPC requires the downregulation of Dlx1/2 in their progenitor cells.

function in versus balance

Even though is expressed in proliferating cells, its tional deletion in OPCs increased proliferation as measured by Ki67 expression. This could result from the fact that vates target genes involved either in proliferation or in cycle depending on the context, reported for (Castro et In the inhibitors, such as Id2/4 (Wang et al., Yun et al., 2004) Thalamocortical axons (TCAs) send sensory information and feedback of motor programming from the caudal brain areas, and these connections are organized in a topographical manner [START_REF] Vanderhaeghen | Developmental mechanisms patterning thalamocortical projections: intrinsic, extrinsic and in between[END_REF]. Formation of the topographic connections is regulated by several axon guidance molecules [START_REF] Braisted | Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection[END_REF][START_REF] Braisted | Slits are chemorepellents endogenous to hypothalamus and steer thalamocortical axons into ventral telencephalon[END_REF][START_REF] Dufour | Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes[END_REF][START_REF] Vanderhaeghen | Developmental mechanisms patterning thalamocortical projections: intrinsic, extrinsic and in between[END_REF][START_REF] Torii | Dissociation of corticothalamic and thalamocortical axon targeting by an EphA7-mediated mechanism[END_REF][START_REF] Uemura | OL-protocadherin is essential for growth of striatal axons and thalamocortical projections[END_REF]. Developing thalamic neurons send axons towards the ventral telencephalon through the prethalamus (or ventral thalamus). Special guidance cells named corridor cells in the ventral telencephalon guide TCAs to the pallium (López-Bendito et al., 2006). Thus, the ventral telencephalon is regarded as an important intermediate target for the formation of reciprocal connections. TCAs need to pass through the prethalamus on exiting from the dorsal thalamus to the ventral telencephalon as the prethalamus occupies exiting points of TCAs. The prethalamus has been supposed to be an intermediate target of TCAs [START_REF] Deng | Corticothalamic and thalamocortical pathfinding in the mouse: dependence on intermediate targets and guidance axis[END_REF]Molnár et al., 2012); however, evidence is scarce that identifies the molecular mechanisms underlying the axon guidance role of the prethalamus, and functions of the prethalamus in thalamocortical projection are not fully understood (Leyva-Diaz and Lopez-Bendito, 2013). Mouse lines showing defects in prethalamus formation would be a useful model for analyzing the functional role of the prethalamus in thalamocortical projection formation.

Olig2 is a bHLH transcription factor that is essential for oligodendrocyte and somatic motoneuron development [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF]Takebayashi et al., 2002b;[START_REF] Zhou | The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification[END_REF], and is also involved in dorsoventral patterning of the spinal cord, which is required for pMN domain specification. In the diencephalon, Olig2 is expressed in the VZ of the prethalamus at early fetal stages, such as E9.5 in mice [START_REF] Ono | Regional-and Temporal-dependent changes in the differentiation of Olig2 progenitors in the forebrain, and the impact on astrocyte development in the dorsal pallium[END_REF]. These Olig2+ cells differentiate into GABAergic neurons in the thalamic reticular nucleus (TRN) as well as into macroglial cells in the diencephalon, whereas loss of Olig2 does not affect GABAergic neuron differentiation [START_REF] Takebayashi | Tamoxifen modulates apoptosis in multiple modes of action in CreER mice[END_REF]. The function of Olig2 in this area has not been elucidated. Here, we report that loss of Olig2 results in hypoplasia of the prethalamus, which leads to defects of TCA extension. The prethalamus is devoid of Epha3 and Epha5 expression whereas ventrally adjacent thalamic eminence (TE) expresses Epha3 and Epha5 (referred to here as Ephas positive) and, in the E13.5 Olig2-KO diencephalon, Ephas-positive TE expanded dorsally. Furthermore, the substrate-bound Epha3 suppresses neurite extension in cultured thalamic neurons. These results together indicate that Olig2 regulates proper formation of the prethalamus, which leads to exclusion of the EphA3-expressing non-permissive region for TCA and is crucial for proper TCA formation.

RESULTS

Reduced size of the prethalamus in Olig2-KO mice

We first explored early development of the prethalamus in the Olig2-KO mouse to examine whether Olig2-KO mice can be used to analyze functions in the prethalamus for thalamocortical projection formation. The prethalamus is demarcated by Dlx2, as well as by Islet1/2, expression [START_REF] Bulfone | Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries[END_REF].

As Olig2 expression in the diencephalon is observed as early as E9.5 [START_REF] Ono | Regional-and Temporal-dependent changes in the differentiation of Olig2 progenitors in the forebrain, and the impact on astrocyte development in the dorsal pallium[END_REF]; supplementary material Fig. S1), wholemount Dlx2 in situ hybridization was performed in the E10.5 forebrain. Dlx2+ prethalamus was much smaller in Olig2-KO mice (n=3) than in wild-type animals (n=4) (Fig. 1A,B, arrows). To observe prethalamus formation more precisely, coronal sections of the E11.5 forebrain were double-stained with Dlx2 in situ hybridization and Islet1/2 immunohistochemistry. In wild-type or heterozygous mice, Dlx2+ cells were observed in the middle part of the diencephalon (Fig. 1C,E,G,I). Islet1/2+ cells were located laterally to Dlx2+ cells (Fig. 1I). Because no significant difference was observed between wild-type and Olig2 heterozygous animals, they are both referred to as normal control animals. Dlx2+ cells in the Olig2-KO diencephalon were also observed at a similar level; however, as shown in the whole-mount in situ hybridization, the Dlx2+ area was much smaller than that in normal control animals (Fig. 1D,F,H,J). The mean area of the Dlx2+ region in each section of Olig2-KO was ∼60% smaller than that of the wild type (Fig. 1K). In addition, the Islet1/2+ region was also greatly decreased in the Olig2-KO prethalamus (Fig. 1I,J). The prethalamus in E13.5 Olig2-KO mice was also smaller (not shown); thus, loss of Olig2 results in hypoplasia of the prethalamus in early development, as early as E10.5.

We then examined whether reduced proliferation or elevated apoptosis contributes to the hypoplasia of the Olig2-KO prethalamus. Sections of control and Olig2-KO mice at E10.5 and E11.5 were double labeled with Dlx2 in situ hybridization and cleaved caspase 3 immunohistochemistry (a marker of apoptotic cells) or pH3 immunohistochemistry (a marker of mitotic cells). At E10.5, cleaved caspase 3+ spots were more abundantly observed in the prethalamus of Olig2-KO than in control mice (supplementary material Fig. S2A-E), whereas pH3+ cell density was similar between the control and Olig2-KO (supplementary material Fig. S2G). At E11.5, the density of cleaved caspase 3+ cells was similar between the control and Olig2-KO prethalamus, whereas that of pH3+ cells was slightly higher in the Olig2-KO prethalamus than in the normal control (supplementary material Fig. S2F,H). These results indicate that transiently elevated apoptosis at E10.5 may be, at least in part, involved in reduction of the size of the prethalamus in Olig2-KO mice.

Dorsal shift of the border between the prethalamus and thalamic eminence in the Olig2-KO diencephalon To better understand the defects of prethalamus formation in Olig2-KO mice, areas adjacent to the prethalamus were examined by the expression of region marker molecules. The thalamic eminence (TE) is a dorsal part of prosomere 3, although, in coronal sections of the fetal diencephalon, TE is observed ventral to the prethalamus (Fig. 2; López-Bendito and Molnár, 2003;[START_REF] Puelles | Brain segmentation and forebrain development in amniotes[END_REF]. The TE is demarcated by the expression of calretinin [START_REF] Abbott | Developmental expression of calretinin-immunoreactivity in the thalamic eminence of the fetal mouse[END_REF], Tbr1 and Tbr2 (Eomes -Mouse Genome Informatics) [START_REF] Bulfone | T-Brain-1: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex[END_REF][START_REF] Puelles | Brain segmentation and forebrain development in amniotes[END_REF]. Tbr2 is expressed in basal progenitors of the TE, and calretinin and Tbr1 are expressed in the mantle layer. In normal control animals, Tbr2 expression was observed ventrally to the Olig2+ domain (Fig. 2A). In the Olig2-KO diencephalon, Tbr2 was also expressed ventral to the CreER expression that recapitulates intrinsic Olig2 expression (Fig. 2B), and the Tbr2+ area was much wider than that in normal control animals (Fig. 2A,B, flanked by arrows), whereas the CreER+ prethalamus was narrower (Fig. 2B, flanked by arrowheads). Although Lhx5 expression was reported to demarcate the prethalamus, in our observation, Lhx5 was expressed in the dorsal border of the prethalamus and the main body of the TE whereas the main part of the prethalamus was devoid of Lhx5 expression (supplementary material Fig. S3A,F). In the E12.5 Olig2-KO diencephalon, the Lhx5negative area was much reduced in size and was compatible with the reduction of the Dlx2+ area (supplementary material Fig. S3B,G). We then measured the relative positions of Tbr2-and Olig2-or Creexpressing domains within the dorsoventral axis of the E13.5 diencephalon (Fig. 2C). Total height of the diencephalon in a coronal section was regarded as 100% height. As shown in Fig. 2C, the dorsal border of the prethalamus and the ventral border of the TE were unchanged in the Olig2-KO diencephalon. However, the border between the prethalamus and TE was significantly shifted dorsally in knockout mice. In addition, the Tbr2-expressing thalamic eminence occupied 10±1.44% width within the total height of the diencephalon in control animals, whereas that in Olig2-KO mice was 20.7±2.38%, which was also statistically significant (P<0.01). These observations clearly demonstrate hypoplasia of the prethalamus accompanied by expansion of the TE in Olig2-KO mice.

To examine whether the reduction of the prethalamus with dorsal expansion of the TE involves a fate change of Olig2 lineage cells, we performed genetic fate mapping. We generated Olig2 KICreER/+ ; Rosa26-GAP-EGFP and Olig2 KICreER/KICreER ;Rosa26-GAP-EGFP (hereafter named Olig2-hetero;Rosa26 GFP and Olig2-KO;Rosa26 GFP , respectively) mice, and tamoxifen was administered to dams at E10.5 to induce GFP expression in Olig2+ cells. Animals were analyzed at E13.5, and sections were double-stained with calretinin, Tbr1 or Islet1/2 and with GFP immunohistochemistry. In the Olig2hetero;Rosa26 GFP diencephalon, GFP+ cells and calretinin+ cells were mostly mutually exclusive and thus only a few cells were double immunoreactive (Fig. 3A,C). By contrast, calretinin+ cells, as well as Tbr1+ cells, expanded dorsally and showed overlapping distribution with GFP+ cells in the Olig2-KO;Rosa26 GFP diencephalon (Fig. 3B,D; supplementary material Fig. S4A,B), and many calretinin+ cells were also GFP+, indicating a fate change of Olig2 lineage cells (Fig. 3D, inset). Islet1/2+ cells in the Olig2-KO prethalamus were also GFP+ (supplementary material Fig. S4C,D) and therefore Olig2 lineage cells in the Olig2-KO diencephalon showed dual phenotypes, both prethalamus and TE.

Although the border between the prethalamus and the TE was dorsally shifted in Olig2-KO mice, other areas of the forebrain seemed to be properly formed. For example, the Olig3+ dorsal thalamus was positioned dorsal to the prethalamus [START_REF] Vue | Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice[END_REF]; supplementary material Fig. S3C,H). Whereas the ventral and dorsal borders of the Olig3+ area were unchanged in the Olig2-KO mouse (supplementary material Fig. S3C,H), the relative width of the Olig3 domain within the dorsoventral axis was slightly reduced in the Olig2-KO diencephalon (supplementary material Fig. S3K). Shh and Fgf8 expression in ZLI was unchanged in the absence of Olig2 (supplementary material Fig. S3D,E,I,J). In addition, Islet1/2+ cells, including corridor cells (López-Bendito et al., 2006), were positioned normally in the ventral telencephalon at E13.5 in both the normal control and Olig2-KO mice (supplementary material Fig. S5). Overall, the prethalamus and TE are specifically malformed in the early forebrain development of the Olig2-KO mouse, although a slight defect was noticed in the dorsal thalamus.

Defect of TCA extension in the Olig2-KO diencephalon

We next examined whether the prethalamus has an impact on the formation of the thalamocortical projection. Axons in the fetal forebrain were stained with anti-neurofilament M (NF-M) antibody. At E13.5, when TCAs start to extend into the prethalamus and the ventral telencephalon (López-Bendito et al., 2006), NF-M+ axons extended dorsoventrally and in parallel in the mantle layer, and they gradually converged in the ventral part (Fig. 4A,B, arrowheads). In age-matched Olig2-KO mice, NF-M+ axons were disorganized and followed a tortuous course in the dorsal thalamus; they sometimes formed small aggregates (Fig. 4C,D, arrowheads). To elucidate whether these NF-M+ axons are TCAs, they were co-immunostained with anti-netrin G1 antibody [START_REF] Niimi | Monoclonal antibodies discriminating netrin-G1 and netrin-G2 neuronal pathways[END_REF] because netrin G1 is expressed by developing dorsal thalamic neurons [START_REF] Nakashiba | Complementary expression and neurite outgrowth activity of netrin-G subfamily members[END_REF]. Indeed, NF-M+ axons co-expressed netrin G1 in both the wild-type and Olig2-KO dorsal thalamus (Fig. 4B,D, arrowheads), indicating that the disorganized axons in Olig2-KO mice were TCAs (Fig. 4D). By E17.5, wild-type TCAs had formed a thin fasciculus in the dorsal thalamus and prethalamus including TRN, which extended in parallel, orienting ventrolaterally (Fig. 4E). By contrast, Olig2-KO netrin G1+ fibers formed an abnormally thick fasciculus and extended randomly in the dorsal thalamus (Fig. 4F,arrowheads). These observations indicate that, in the Olig2-KO diencephalon, the initial changes in prethalamus/TE regionalization are followed by later disorganized axonal fasciculation and extension (Fig. 4G).

Retarded and disorganized thalamocortical projection in the Olig2-KO forebrain The Olig2-KO mouse prethalamus shows defects as early as E10.5, before the onset of TCA formation, suggesting that the initial stage of projection formation would be disorganized in Olig2-KO mice. We thus examined TCA at E13.5 using DiI axonal tracing. DiI-labeled TCAs were observed, orienting dorsolaterally in the ventral telencephalon of wild-type animals (Fig. 5A). By contrast, in Olig2-KO mice, DiI-labeled TCAs had just crossed the telencephalon-diencephalon boundary (TDB), and had not yet reached the lateral ganglionic eminence (Fig. 5B). Measuring the length of the TCAs in the ventral telencephalon between the growing tip and the TDB, Olig2-KO TCAs were much shorter (around 60% reduction) than those of wild-type animals (Fig. 5C). The result clearly shows a reduction of TCA extension at early stages in the absence of Olig2 (Fig. 5A,B, insets).

We further examined whether TCAs reach the cerebral cortex and whether topographic thalamocortical projection is formed in the Olig2-KO mouse. DiI was injected into the middle part of the E18.5 cortex, and DiA was injected into the frontal and occipital poles of the same brain (Fig. 6A,B, insets). The frontal cortex had a connection with the medial part of the thalamus, the middle part with the central part, and occipital cortex with the lateral part in both normal control and Olig2-KO mice (Fig. 6C,D). Therefore, the topography of the reciprocal projection was roughly preserved in the absence of Olig2. However, axon arrangement was markedly disorganized in the Olig2-KO thalamus: whereas axons were arranged in parallel in the wild-type thalamus, those in Olig2-KO formed a thick fasciculus and crossed each other with abnormal overlapping course of axons (Fig. 6C-F). On closer examination, retrogradely labeled cell bodies were observed in the Olig2-KO dorsal thalamus (Fig. 6F, arrows), indicating that TCAs in Olig2-KO reached the cerebral cortex in spite of the initial delay of their extension.

Disorganized TCAs extend from non-Olig2 lineage cells Olig2 is strongly expressed in the VZ of the prethalamus and weakly in the caudoventral part of the dorsal thalamus, whereas TCAs have defects of axon extension; therefore, we examined whether the axonal extension defect in the Olig2-KO dorsal thalamus is induced in a cellautonomous or a non-cell-autonomous manner. If TCA defects in Olig2-KO mice are induced in a cell-autonomous manner, they should be shown by Olig2 lineage neurons. To analyze this, we again used Olig2-KO;Rosa26 GFP mice, and tamoxifen was administered to pregnant mice at E11.5 and/or E12.5 to label Olig2+ cells. At E13.5, when NF-M+ axons were disorganized in the lateral part of the dorsal thalamus of Olig2-KO;Rosa26 GFP mouse, axon aggregates were found in the nucleus-free zones (supplementary material Fig. S6A,B,arrows). Nevertheless, processes of GFP+ Olig2-lineage cells were present in the prethalamus showing radial extension, but were not present in cell-free zones (supplementary material Fig. S6C,D, asterisks). At E17.5, GFP+ axons of Olig2-KO;Rosa26 GFP mice were observed in the TRN, whereas no GFP+ axons were present in the dorsal thalamus (supplementary material Fig. S6E). Abnormally oriented axons extending randomly were GFP negative (supplementary material Fig. S6F, arrowheads). These results indicated that axons showing disorganized elongation do not belong to Olig2 lineage neurons, suggesting non-cell autonomous defect of axonal extension in the Olig2-KO dorsal thalamus.

Altered expression of axon guidance molecules in the Olig2-KO prethalamus

As axon arrangement was impaired in the Olig2-KO mouse dorsal thalamus, and axonal disorganization occurred in a noncell-autonomous manner, it is highly probable that the expressions of axon guidance molecules are altered in the terrain through which TCAs pass, including the prethalamus and the TE. We therefore conducted a microarray analysis for changes in the gene expression of axon guidance molecules in the diencephalon or basal forebrain comparing the transcriptomes of wild-type and Olig2-KO mice at E13.5 (Table 1). Efna, Epha and Sema genes, and Unc5c were shown to change their expression in the absence of Olig2. Using Allen Institute for Brain Science Web in situ hybridization data (http://developingmouse.brain-map.org/), we confirmed that Epha3 and Epha5 were expressed in the E13.5 diencephalon. We then examined the expression pattern of these molecules in the Olig2-KO diencephalon by in situ hybridization. In the E13.5 wild-type forebrain, the prethalamus is devoid of mRNA for Epha3 and Epha5, and the Ephas-negative region continues to the TDB (Fig. 7A,C, asterisks), and both Epha3 and Epha5 are expressed in the TE (Fig. 7A,C; supplementary material Fig. S7A,C,E,G,I,K,M,O; [START_REF] Kudo | Expression profiles of EphA3 at both the RNA and protein level in the developing mammalian forebrain[END_REF]. In the age-matched Olig2-KO forebrain, the Epha3-and Epha5-expressing TE was dorsally expanded and, conversely, the Ephas-negative prethalamus were reduced in size (Fig. 7B,D; supplementary material Fig. S7B,D,F,H,J,L,N,P). Thus, malformed formation of the prethalamus and TE was accompanied with the altered expression of axon guidance molecules, likely leading to the altered extension of TCAs. EphrinA5 (Efna5), which encodes one of the ligands for EphA3, was expressed in the dorsal thalamus, which seemed to be unchanged in the absence of Olig2 (Fig. 7E,F).

Sema5a and Unc5c also showed changed expression in microarray analysis (Table 1); however, in situ hybridization analysis demonstrated that the expression patterns of Sema5a and Unc5c were not altered in the E13.5 diencephalon of Olig2-KO mice (not shown).

EphA3 suppresses axonal extension from cultured thalamic neurons

The above results strongly suggest that EphA3 and EphA5 are candidate molecules affecting the initial extension of TCAs. We next examined the roles of EphA3 in the neurite extension of dorsal thalamic neurons in vitro. Dissociated dorsal thalamic progenitor cells were cultured on the substrate double coated with PLL and either EphA3-Fc chimeric protein or human Fc for 40 h. Neurons were identified with class-III β tubulin immunohistochemistry (Fig. 8A,B). Neurite length on Fc-coated substrate was slightly but definitely different from that on EphA3 (33.3±2.38 µm and 24.4±2.90 µm, respectively, mean±s.e.m.) (Fig. 8C); thus, substrate-bound EphA3 apparently inhibited neurite extension from dorsal thalamic neurons.

Ectopic Olig2 does not affect EphA3 expression

We then examined whether Olig2 affects or suppresses EphA3 expression. In this experiment, we used a chick embryo neural tube, because gene expression hierarchy is well known in this region of the chick embryo [START_REF] Liu | Induction of oligodendrocyte differentiation by Olig2 and Sox10: evidence for reciprocal interactions and dosage-dependent mechanisms[END_REF][START_REF] Mizuguchi | Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtypespecific properties of motoneurons[END_REF] when compared with the fetal mouse diencephalon. Epha3 expression in the

DISCUSSION

In the present study, the Olig2-KO mouse diencephalon was examined morphologically, and we found malformation of the prethalamus and TE, followed by disorganized extension of TCAs.

These results indicate that Olig2 controls proper formation of the prethalamus and also that proper formation of the prethalamus controls the initial extension of TCAs. Together, our results provide possible molecular mechanisms underlying initial TCA extension in the prethalamus.

Olig2 regulates proper formation of the prethalamus Olig2 is expressed in VZ cells of the prethalamus, which develops into TRN and the zona incerta [START_REF] Inamura | Olig2 lineage cells generate GABAergic neurons in the prethalamic nuclei, including the zona incerta, ventral lateral geniculate nucleus and reticular thalamic nucleus[END_REF], which are mostly composed of GABAergic neurons [START_REF] Ottersen | GABA-containing neurons in the thalamus and pretectum of the rodent. An immunohistochemical study[END_REF]. We previously demonstrated that distribution of GABAergic neurons is nearly normal in the Olig2-KO forebrain compared with normal control animals [START_REF] Ono | Regional-and Temporal-dependent changes in the differentiation of Olig2 progenitors in the forebrain, and the impact on astrocyte development in the dorsal pallium[END_REF], and a fatemapping study demonstrated that Olig2 lineage cells differentiate into GABAergic neurons in the absence of Olig2 [START_REF] Takebayashi | Tamoxifen modulates apoptosis in multiple modes of action in CreER mice[END_REF]. In the present study, we examined the formation of the prethalamus at much younger stages: E10.5-E13.5. The Dlx2positive, as well as Lhx5-negative, prethalamus in Olig2-KO mice was markedly reduced in size (Figs 1,2). Accompanying the size reduction of the prethalamus, the TE expanded dorsally. The reduced size of the prethalamus is probably caused both by elevated apoptosis at E10.5 (supplementary material Fig. S2) and by the fate change of Olig2 lineage cells from prethalamus cells to TE cells (Fig. 3; supplementary material Fig. S4). It is noteworthy that this is the first demonstration of Olig2 function in early forebrain patterning beyond its known role in glial development [START_REF] Cai | A crucial role for Olig2 in white matter astrocyte development[END_REF][START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF][START_REF] Ono | Regional-and Temporal-dependent changes in the differentiation of Olig2 progenitors in the forebrain, and the impact on astrocyte development in the dorsal pallium[END_REF][START_REF] Ono | Olig2 transcription factor in the developing and injured forebrain; Cell lineage and glial development[END_REF]. We previously reported that loss of Olig2 does not affect apoptosis in the pMN domain [START_REF] Takebayashi | Tamoxifen modulates apoptosis in multiple modes of action in CreER mice[END_REF]. Other parts of the E10.5 forebrain regions, such as the Dlx2-positive ganglionic eminence or (Dlx2-negative) cerebral cortex, revealed a similar level of apoptosis (not shown). Therefore, involvement of Olig2 in the apoptotic pathway might be dependent on the stage or region of the central nervous system. In addition, it is probable that Olig2 interacts with other transcription factors, such as Tbr2, for proper boundary formation in the TE, as has been reported in the developing neural tube [START_REF] Helms | Specification of dorsal spinal cord interneurons[END_REF]. Further studies are necessary to clarify the mechanisms of Olig2 function in forebrain patterning.

Proper formation of the prethalamus and TE is required for early thalamocortical projection

Another important new finding is that loss of Olig2 results in retardation of initial TCA extension, which leads to abnormal fasciculation and random orientation of TCA at the late fetal stage . It is highly probable that the delay of TCA extension is caused by reduction of prethalamus formation and expansion of the TE, both of which are induced by loss of Olig2: (1) the prethalamus occupies an exit region for TCAs; (2) a defect in the thalamocortical projection was probably induced in a non-cell-autonomous manner (see supplementary material Fig. S6); (3) EphA3 and EphA5 expression is observed in the TE but not in the prethalamus; (4) EphA3 and EphA5 expression occupies the route of TCA extension in the Olig2-KO diencephalon (Fig. 7; supplementary material Fig. S7);

(5) ephrinA5, a possible counterpart molecule of EphA3, is expressed in the dorsal thalamus; (6) substrate-bound EphA3 suppresses neurite extension from thalamic neurons in vitro (Fig. 8). Eph receptors and ephrin ligands act as a repellant molecule system in neural circuit formation. In addition, ephrin A proteins also recognize and transduce the reverse signaling of EphA proteins, which also includes axon repulsion [START_REF] Rashid | Opposing gradients of ephrin-As and EphA7 in the superior colliculus are essential for topographic mapping in the mammalian visual system[END_REF][START_REF] Xu | Ephrin reverse signaling in axon guidance and synapstogenesis[END_REF]. Together, these observations suggest that the Epha3-and Epha5-negative prethalamus may provide a permissive or less inhibitory substrate for TCAs, and that occupation of Epha3 and Epha5 in the exit route impairs initial extension of TCAs in the Olig2-KO diencephalon. It is probable that EphA3 negatively regulates TCA elongation, and, therefore, expansion of Ephas-positive TE may impair initial TCA extension in the Olig2-KO forebrain (Figs 4,5).

Another possible functional role of the prethalamus in TCA extension is contact guidance of prethalamic axons to TCAs. [START_REF] Mitrofanis | Development of the thalamic reticular and perireticular nuclei in rats and their relationship to the course of growing corticofugal and corticopetal axons[END_REF] proposed that pioneer fibers from the prethalamus extending into the thalamus guide TCAs to the prethalamus. Our observation might support the possible association of thalamic and prethalamic axons as these axons do not express a combination of repellant molecules; while thalamic axons may express ephrinA5, its counterpart molecules EphA3 and EphA5 are not expressed in prethalamic axons (Fig. 7). It remains unknown whether these axons express molecules that mediate contact attraction.

The Olig2-KO forebrain includes other minor defects. For example, the prethalamus region at E11.5 and E12.5 was constricted in Olig2-KO mice (Fig. 1, Fig. 7E,F). This constriction was, however, partially recovered by E13.5 (Figs 2, 3, Fig. 7A-D) [ probably owing to elevated proliferation in the Olig2-KO prethalamus (supplementary material Fig. S2H)], and cytoarchitecture, such as radial arrangement of the prethalamic cells and radial fiber extension, was preserved normally in the absence of Olig2 (Fig. 3D; supplementary material Fig. S6). Therefore, the constriction of the prethalamus at early stages may not be the cause of the retardation of TCAs in the Olig2-KO forebrain, although this possibility cannot be ruled out completely. In addition, the dorsal thalamus of the Olig2-KO mouse shows slight hypoplasia (supplementary material Fig. S3K), and the EphA3-positive region is also slightly reduced in the Olig2-KO thalamus (Fig. 7). We could not clarify whether the slight reduction of EphA3 expression in the dorsal thalamus affects the initial delay of TCA extension. However, as EphA3 negatively regulates TCA extension, reduction of EphA3 expression might not have a strong effect on the initial delay of axon elongation. As hypoplasia of the prethalamus is the severest defect within the Olig2-KO diencephalon, it is the most likely cause of the initial retardation and impairment of TCA guidance.

The prethalamus and TE have been supposed to be intermediate targets for formation of the thalamocortical projection, as loss of transcription factors expressed in these regions induces defects of thalamocortical projection, which includes Pax6, Mash1 (Ascl1 -Mouse Genome Informatics), Tbr1 (López-Bendito and Molnár, 2003), Dlx1/2 and Ebf1 [START_REF] Garel | The early topography of thalamocortical projections is shifted in Ebf1 and Dlx1/2 mutant mice[END_REF] and Olig2 ( present study). Transcription factors regulate region identity in the rostralcaudal axis [START_REF] Kiecker | Hedgehog signaling from the ZLI regulates diencephalic regional identity[END_REF][START_REF] Puelles | Forebrain gene expression domains and the evolving prosomeric model[END_REF][START_REF] Rubenstein | Regionalization of the prosencephalic neural plate[END_REF] and cell-fate determination in the dorsoventral axis [START_REF] Helms | Specification of dorsal spinal cord interneurons[END_REF][START_REF] Tanabe | Diversity and pattern in the developing spinal cord[END_REF]. Recently, evidence has supported that transcription factors regulating cell-type specification also regulate cell migration and neural circuit formation (Chédotal and Rijli, 2009;[START_REF] Shirasaki | FGF as a targetderived chemoattractant for developing motor axons genetically programmed by the LIM code[END_REF]. Cell-fate determination may include the cell-type-specific behavior of committed cells through regulation of the expression of cytoskeletal molecules and receptors for axon guidance molecules.

In our observations, prethalamus formation is controlled by Olig2, which leads to EphA3-and EphA5-negative terrain for the initial extension of TCAs; therefore, transcription factors also control the milieu or environment for axon elongation, regulating neural circuit formation indirectly. As Olig2 may not suppress or regulate EphA3 expression directly (supplementary material Fig. S8) and as the expression of morphogen molecules that may affect EphA3 expression was not altered in the Olig2-KO diencephalon (supplementary material Fig. S3D,E,I,J), it is probable that putative programs for TE formation regulate EphA3 expression. Olig2 is involved in multistep processes of diencephalon formation and may indirectly repress EphA3 expression through interacting with transcription factors involved in TE development.

In conclusion, the present study elucidates that Olig2 is crucial for proper formation of the prethalamus, especially the boundary between the prethalamus and TE, and that proper formation of the prethalamus is crucial for correct extension of TCAs. Therefore, appropriate regionalization of the prethalamus and TE provides a pathway from the dorsal thalamus to the ventral telencephalon.

MATERIALS AND METHODS

Animals and tissue preparation

The animals used in this study were Olig2 KICreER (Takebayashi et al., 2002b), Olig2 -/- [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF], ROSA26-GAP43-EGFP [START_REF] Nakahira | Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes[END_REF] and wild-type ICR mice (Slc, Shizuoka, Japan). Genotyping was performed as described previously (Takebayashi et al., 2002b;[START_REF] Tatsumi | Genetic fate mapping of Olig2 progenitors in the injured adult cerebral cortex reveals preferential differentiation into astrocytes[END_REF]. Fertilized chick eggs were purchased from Yamagishi Corporation (Mie, Japan) and were incubated at 37°C. Embryonic stages of chicks were determined according to [START_REF] Hamburger | A series of normal stages in the development of the chick embryo[END_REF]. At least three independent experiments were carried out in all histological and culture analyses, including DiI tracing. All animal procedures were approved by the Animal Research Committee of Kyoto Prefectural University of Medicine and of the National Institute for Physiological Sciences.

To obtain Olig2-KO mice, Olig2 KICreER heterozygous mice were mated and the day when the vaginal plug was found was regarded as embryonic day 0.5 (E0.5). To label Olig2 lineage cells, Olig2 KICreER ;ROSA26-GAP43-EGFP double heterozygous mice were mated, and tamoxifen (TM; 3 mg/animal) was injected intraperitoneally into pregnant mothers with fetuses at E10.5, E11.5 or E11.5 and E12.5, as described previously [START_REF] Masahira | Olig2-positive progenitors in the embryonic spinal cord give rise not only to motoneurons and oligodendrocytes, but also to a subset of astrocytes and ependymal cells[END_REF]. Pregnant mice were deeply anesthetized with pentobarbital (100 mg/kg body weight), and fetal mice were removed from the uterus. Fetal mouse brains were fixed with 4% paraformaldehyde (PFA) in PBS overnight, and subsequently with 20% sucrose in PBS. Fixed brains were cut coronally with a cryostat at 20 µm, and sections were thaw-mounted onto MAS-coated glass slides (Matsunami Glass, Tokyo, Japan).

In situ hybridization

In situ hybridization was performed using digoxygenin-labeled antisense riboprobes, as described previously [START_REF] Ding | Short-term lineage analysis of dorsally derived Olig3 cells in the developing spinal cord[END_REF][START_REF] Wilkinson | In situ Hybridization, A Practical Approach[END_REF]. cDNAs used in this study were as follows: mDlx2 (NM_010054, nt_746-1355), mephrinA5 (NM_207654, nt204-831), mUuc5c [START_REF] Watanabe | Dorsally derived netrin 1 provides an inhibitory cue and elaborates the ‗waiting period' for primary sensory axons in the developing spinal cord[END_REF], mEpha3 (NM_010140, nt_2760-3340), mEpha5 (NM_007937, nt_1135-2100), mFgf8 [START_REF] Ohuchi | Involvement of androgen-induced growth factor (FGF-8) gene in mouse embryogenesis and morphogenesis[END_REF], mOlig3 (Takebayashi et al., 2002a), mLhx5 [START_REF] Hirata | Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions[END_REF], mShh [START_REF] Iseki | Sonic hedgehog is expressed in epithelial cells during development of whisker, hair, and tooth[END_REF], mSema5a (NM_009154, nt_634-1162) and chick EphA3 [START_REF] Iwamasa | Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles[END_REF]. Sense probes were used as a negative control, which did not show specific signals.

Immunohistochemistry

Immunohistochemistry was carried out as previously described [START_REF] Ono | Lower rhombic lip-derived cells undergo transmedian tangential migration followed by radial migration in the chick embryo brainstem[END_REF][START_REF] Ono | Regional-and Temporal-dependent changes in the differentiation of Olig2 progenitors in the forebrain, and the impact on astrocyte development in the dorsal pallium[END_REF]. The GFP signal in the Rosa26-GAP43-EGFP reporter mouse brain was enhanced by incubation with fluorescein-conjugated tyramide (Tyramide Signal Amplification-Fluorescein Systems; PerkinElmer Life Science, Boston, MA, USA). Primary antibodies used were: rabbit anti-Olig2 (1:1000, AB9610; Millipore, Temecula, CA, USA; 1:500, 0B-905; IBL, Takasaki, Japan), rabbit anti-GFP (1:1000, AB3080; Millipore), rat anti-GFP (1:1000, 04404-84; Nacalai Tesque, Kyoto, Japan), rabbit anti-calretinin (1:1000, 7697; Swant, Marly, Switzerland), rabbit anti-Tbr1 (1:500, ab31940; Abcam, Cambridge, UK), rabbit anti-Tbr2 (1:500, ab23345; Abcam), mouse anti-Islet1/2 (4D5, 1:50; DSHB), mouse anti-neurofilament M [START_REF] Watanabe | Dorsally derived netrin 1 provides an inhibitory cue and elaborates the ‗waiting period' for primary sensory axons in the developing spinal cord[END_REF], mouse anti-netrin G1 (1:500;[START_REF] Niimi | Monoclonal antibodies discriminating netrin-G1 and netrin-G2 neuronal pathways[END_REF], rabbit anti-netrin G1 (1:10;[START_REF] Niimi | Monoclonal antibodies discriminating netrin-G1 and netrin-G2 neuronal pathways[END_REF], mouse anti-HB9 (1:50, 5C10; DSHB), rabbit anti-cleaved caspase 3 (1:500, 9661; Cell Signaling, Danvers, MA), rabbit anti-Cre (1:1000, 69050-3; Novagen, San Diego, CA) and rabbit antiphospho-histone H3 ( pH3; 1:500, 06-570; Millipore). Stained sections were observed under a bright field and epifluorescent microscope (BX-51; Olympus, Tokyo, Japan), and images were captured via a CCD camera (DP-71; Olympus).

Axonal tracing with lipophilic carbocyanine dye

In order to label early TCAs in the E13.5 brain, crystals of lipophilic dye DiI (1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyanine perchlorate; Invitrogen, Eugene, OR, USA) were applied to the dorsal thalamus of wildtype and Olig2-KO animals after fixation. Tissues were kept in 4% PFA at 37°C for 4 days. To label reciprocal connections between the dorsal thalamus and cortex in the E17.5 and E18.5 cortex, 2% solutions of DiI and DiA [4-(4-(dihexadecylamino)styryl )-N-methylpyridinium iodide; Invitrogen] were used as tracers. DiA was injected into the frontal and occipital poles, and DiI was injected into the middle part of the cortex (Fig. 6A,B, insets). They were kept at 37°C in the fixative for 2 weeks and then cut coronally with a vibratome at 100 µm. Sections were stained with Hoechst33342, and observed under an epifluorescent microscope and a confocal laser scanning microscope (FV-1000; Olympus).

Microarray preparation, processing and statistical analysis

The samples were grouped into two sample types: Olig2-KO [START_REF] Lu | Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection[END_REF] and the wild type. RNA was purified from the E13.5 ventral forebrain from Olig2-KO (Olig2 -/-) and the wild type (Olig2 +/+ ). RNA samples were prepared by pooling RNAs extracted from three brains to constitute one replicate and each sample type was prepared in five replicates. cRNA hybridization probes were hybridized to Affymetrix Mouse Whole Genome MOE430 2.0 arrays. The R-CRAN statistical language and Bioconductor software package [START_REF] Gentleman | Bioconductor: open software development for computational biology and bioinformatics[END_REF] were used throughout the expression analyses. Raw image files were processed using Affymetrix GCOS and the Microarray Suite (MAS) 5.0 algorithm. The probe signal levels were quantile normalized, summarized and then transformed to the log 2 expression using GCRMA (gcrma R-package; [START_REF] Irizarry | Use of mixture models in a microarray-based screening procedure for detecting differentially represented yeast mutants[END_REF] and Frozen RMA ( fRMA package; [START_REF] Mccall | Thawing frozen robust multi-array analysis ( fRMA)[END_REF]. Gene filtering was performed using the Wilcoxon signed-rank test to select probe sets that were ‗consistently expressed' (P<0.04) in at least one group (mas5calls R-package; Bioconductor). All samples showed good quality control according to MAS5.0 guidelines and the AffyQC report (R-package; Bioconductor). Principal component analysis (PCA) was used to reduce the dimensionality of the whole dataset and measure the degree of similarity between samples (dudi.pca R-package; [START_REF] Thioulouse | Interactive multivariate data analysis in R with the ade4 and ade4TkGUI packages[END_REF]. We had to withdraw two replicates from each sample type as they were aberrant. The subsequent three-dimensional PCA plot confirmed a high degree of similarity among samples of the same sample type and much less similarity among samples of different sample types. We used LIMMA statistical analysis (Smyt, 2004) to determine the differentially expressed genes in Olig2-KO. The resulting P-values were adjusted to FDR <5% with the Benjamini-Hochberg method to control the mean proportion of false positives (q-value R-package; [START_REF] Storey | Statistical significance for genomewide studies[END_REF] and a kernelbased FDR method to assess the probability of being false positive for a specific gene (kerfdr R-package; [START_REF] Guedj | Kerfdr: a semi-parametric kernel-based approach to local false discovery rate estimation[END_REF]. Microarray data have been deposited in Gene Expression Omnibus with accession number GSE56389.

Dissociation culture of dorsal thalamus neurons

The dorsal thalamus at E12.5 was dissected free from the fetal brain, minced into small pieces, treated with 0.1% trypsin for 15 min at 37°C, and then triturated with a fire-polished pasture pipette. Dissociated cell suspension (1×10 5 cells/ml) was prepared in culture medium (MEM containing 5% fetal bovine serum and 5% horse serum). For analyses of the substratebound form of EphA3, plastic dishes (35 mm in diameter) were sequentially coated with poly-L lysine (PLL, 80 µg/ml; Sigma, St Louis, MO, USA) overnight at 4°C and then with mouse EphA3-human Fc or human Fc (100 nM; R&D Systems, Minneapolis, MN, USA) for 5 h at 37°C. After rinsing the culture substrates with PBS, 2 ml thalamic cell suspension was plated onto the culture dishes. Cultures were maintained for 40 h. They were fixed with 4% PFA and stained with anti-class III β tubulin antibody (1:1000, G7121; Promega) to examine neurite extension. Three independent culture experiments were performed. In each experiment, epifluorescent photomicrographs were taken of 9-15 randomly chosen fields and the length of neurites with more than 50 labeled cells was measured with ImageJ software (NIH).

Electroporation

In ovo electroporation was performed as previously described [START_REF] Ono | Lower rhombic lip-derived cells undergo transmedian tangential migration followed by radial migration in the chick embryo brainstem[END_REF]. E3 chick embryos (HH stage 17-20;[START_REF] Hamburger | A series of normal stages in the development of the chick embryo[END_REF] were used. DNA of pCAGGS-EGFP [START_REF] Gotoh | Genetically-defined lineage tracing of Nkx2.2-expressing cells in chick spinal cord[END_REF] solution with or without pCAGGS-mOlig2 [START_REF] Mizuguchi | Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtypespecific properties of motoneurons[END_REF] was injected into the central canal, and square pulses (30 V,50 ms,twice;Nihon Koden, were delivered to the embryos. The embryos were fixed at E6 (HH stage [START_REF] Bai | NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells[END_REF][START_REF] Bajpai | CHD7 cooperates with PBAF to control multipotent neural crest formation[END_REF][START_REF] Balordi | Hedgehog signaling in the subventricular zone is required for both the maintenance of stem cells and the migration of newborn neurons[END_REF] and analyzed histologically. Gene expression was analyzed as the ratio of signal intensity between the electroporated side and non-electroporated side, and the ratio of Olig2-transfected samples was compared with that of EGFP-transfected samples.

Image analysis

The area of the Dlx2+ region and neurite length both in vivo and in vitro was measured with ImageJ software after images were captured with a CCD camera as above. In all cases, quantitative analysis was carried out using at least three Olig2-KO or wild-type/heterozygous mice.

PART III: DISCUSSION

Since 2010, the year of publication of the groundbreaking discovery of the direct conversion mechanism of fibroblast into neuron by the BAM factors, Brn2, Ascll1 and Myt1l [START_REF] Vierbuchen | Direct conversion of fibroblasts to functional neurons by defined factors[END_REF], more than 500 article about Ascl1 were published. Most of them followed the same approach of viral transduction of combination of TFs. Starting from a constructs combining more than 20 different TFs, Verbruchen et al, identified the core BAM factors sufficient to induce neurogenesis. Similar experiments were done to generate OPCs and oligodendrocytes through direct conversion [START_REF] Najm | Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells[END_REF]Yang N, Zuchero JB 2013). Despite successful, this approach display really low yield and do not explain the chromosomic remodeling mechanisms which triggers direct cell lineage conversion. Recent studies tackled this question for Ascl1 (D. Castro 2011;[START_REF] Wapinski | Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons[END_REF]. MD Borromeo, 2014) [START_REF] Imayoshi | Hes genes and neurogenin regulate nonneural versus neural fate specification in the dorsal telencephalic midline. Development[END_REF]. This study shed a light on a regulatory process involving an oscillatory or stable mode of expression to regulate cell proliferation or cell fate choice respectively. This kind of regulation involving the mode of expression rather than specific expression of a new TFs opens the way for wider comprehension of the genetic regulatory logic.

One particular goal of this study was to untangle the specific and shared role of Ascl1 and Olig2 in the specification of oligodendrocyte progenitors. We demonstrated that Ascl1 directly binds and regulates key genes involved in the specification, proliferation and differentiation of OPCs. Using comparison of transcriptomic and binding profile of Ascl1 and Olig2, we showed that the downstream direct targets of both TFs were highly overlapping despite clear specificities in regulated genes, in binding sequence preferences and on average coverage of the genome. Four time more peaks were identified in OPCs in Olig2 Chip-seq compared to Ascl1 Chip-seq.

Our work confronted us with the multiple testing problem. In microarray and chip-seq experiments, several thousand events are simultaneously measured across different conditions. When testing for differential expression, each gene is tested independently from one another using a t-test or ANOVA. The incidence of false positives is proportional to the number of tests performed and the significance level (p-value cut-off). A p-value of 0.05 signifies a 5% probability that a gene expression level is different between the two groups by chance. If 40,000 genes are tested, 2,000 genes could be called significant by chance. Thus, it is important to correct the p-value to assess the false discovery rate (FDR) for each gene when performing such large statistical test. In this study, we used a combination of algorithm (GCRMA, fRMA, LIMMA) to normalize perform the multiple comparisons and estimate the best FDR cut-off. A FDR of 5% signifies a 5% probability that a particular test result is a false positive. Using this strategy is really of helped when we compared multiple conditions.

Stringent FDR coupled to clustering allowed us to identify previously unknown genes that are both co-regulated and specific of the process we studied during this work, the differentiation of oligodendrocytes. In our Ascl1/Olig2 microarray experiment, the different replicates displayed rather high variability (see PCA in material and methods Fig. 24C). Dissection variability is the most likely explanation; however, this could also be caused by biological or other technical variability. Mice of the same genetic background and the same parents were used to generate the samples. Despite this variability, comparison with Ascl1 KO yield robust results. Control genes such as Ascl1 Dll1, Hes5 (Dlx genes being stable) were highly regulated and statistically significant and were used to define the appropriate FDR cut-off.

Ascl1 had two different probe-sets which undergo differential up and down regulation. The detection of an up-regulated transcript in the Ascl1 -/-mice is in accordance with the upregulation of the in-situ-hybridization signal observed with RNA probe targeting the most 5' region of the gene [START_REF] Cau | Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors[END_REF]. To avoid to miss information, being too stringent, FDR of 20% and Fold-change >1.2 were used to compare microarray and ChIP-seq experiments further down the workflow. Olig2 KO results were more puzzling because of the detection of only a small down-regulation for Olig2 (Fc 0.63 FDR 3%; Fig 24B) while a complete absence was expected. However, Olig2 targets were identified as control, such as Olig1 or Brevican.

Corrected p-value were set also at 20% (p-value <0.01) for subsequent analysis. Both datasets behave similarly, with only few genes strongly regulated and many genes regulated (2300 by Ascl1 and 1200 by Olig2) with low amplitude. This result could be attributed to technical biases described above, however, it also fit with the -continuous model‖ described in the introduction and perhaps reflect some biological causality (Biggin 2011). The small number of replicates used was initially 5 per condition but only 3 were available for subsequent analysis, as 2/5 replicates were discarded for each condition after being identified by principal component analysis as outliers (see material and methods and annex for R-script). As demonstrated by the reprocessing of published data, increasing the replicate number rapidly overcome the biological and technical variability (see annex 12). In humans, hundreds of microarrays are necessary. Using statistical tests as a proxy to prioritize the genes identified, we always used internal controls as well as external control (Allen brain atlas, Eurexpress In Combined with other cell type specific microarrays such as different neural stem cells, microglia, endothelial cells and neural subtypes (see material and methods for complete references), the global cell type comparison presented in this work allowed us to identify for each cell type, genes involved in the shaping of the epigenetic landscape such as transcription factor network and epigenetic regulators. The information contained in those gene lists was used to generate functional hypothesis about the oligodendrocyte physiology. For example, the gene up/down-regulated between the NSCs to OPC are involved in OPCs activation or inhibition. The gene highly expressed in NSCs and OPCs but not in post-mitotic cells must be involved in cell cycle control and mitosis. However, differential expression is not sufficient to detect important functions. Olig2 being stable but highly expressed from the NSCs to the myelinating stage, would not have been detected only based on fold-change consideration. As mentioned above, the mode of regulation is a key determinant of the gene signaling. Gene regulation involves that both the expression level and the mode of expression are important to keep in mind to understand the complete regulatory logic. In this regards, assessing the oscillatory expression of Ascl1 and Hes genes in OPCs will bring a new understanding of OPCs specificity. Indeed, stable expression of Ascl1 and down regulation of Hes1 is necessary for the NSCs to become post-mitotic and start to differentiate. However, OPCs maintain their Ascl1 expression and continue to divide after the embryonic and postnatal gliogenic phase, constituting the main proliferative population of the brain. Thus, an interesting question is to know is if Ascl1 maintain its oscillatory activity in order to maintain its proliferative state. This parameter may explain why conversion yield are so low in OPCs generation experiment. Generation of new kind of synthetic promoters able to drive different mode of gene activation will be of great help in controlling cell reprogramming more precisely. On other central question is the possible link between Ascl1 / Hes1 / Hes5 and the circadian gene such as clock or Bma1 which are also bHLH proteins. A recent study showed an intrinsic timing of differentiation for the gabaergic neuroblast migrating tangentially from the ganglionc eminence to the cortex. When grafting cell from the MGE, directly in the cortex, cells took the same time to differentiate or undergo apoptosis than the one that were travelling in-vivo DG (DG Southwell 2012).

Recent technological progresses in biology, such as high throughput sequencing favored the emergence of high dimensional data. Our ability to benefit from this large knowledge was only made possible since the organization of a publicly accessible data repository (GEO) and the emergence of the computational tool necessary to process the high-throughput sequencing data. The emergence of internet profoundly changed the scientific potentialities. Having access to raw data of studies performed by other laboratory is particularly important to assess their validity and compare with ours. Indeed, we realized during this work how most of the experiments were done when dealing with sequencing and microarray analysis. Most of the time, the minimal amount (3) of replicates is used, while many studies (even on human) does not reach this requirement. In this line, only a few microarray analysis of demyelinated and remyelinating lesions in mice were performed. Having robust data about the process of remyelination is indeed necessary to tackle the complexity of the phenomenon at work in Human diseases. The most careful experimental approach of demyelination and remyelination to date was performed in rat where demyelination was induced by 0.1 % ethidium bromide injection and lesions where micro-dissected at 5, 14 and 28 days post lesion and analysed by microarray experiment (Huang JK, Jarjour A et al ., 2010). Microdissected lesions are enriched in inflammatory, and cell death associated genes, comparing day 14 with d5 captured genes regulated upon remyelination. Interrogating this dataset with our oligodendrocyte screen discriminated the inflammatory part from the oligodendroglial part. However, this brilliant study suffered from the lack of statistical robustness with only 3 replicates. A far more exhaustive view of the remyelination process would have been gained with only few more replicates.

In contrast, we have been fortunate to benefit from replicated Chip-seq experiments analysis done in different cellular contexts for Ascl1, Chip-seq from purified OPCs, whole ventral telencephalon, SH-SY5Y neuronal cultures (B martynoga) cell line and in different species (rat OPCs, mouse ventral telencephalon, mouse cell line). Established algorithm pipeline to process chip-seq data from sequencing to peak calling (BOWTIE for reads alignement, MACS for peak calling) are now available online and make the procedure rather standard (see material and methods).

The recent discovery allowing to question the role of the genome structure in gene regulation (Dekker 2013) made us reconsidering carefully the classical next neighbor analysis method to infer regulatory region to the closest gene. Indeed, only 7% of the cis-regulatory regions interact with their closest gene. We used epigenetic information about active enhancers and active promoters to assess the functionality of the Ascl1 and Olig2 binding event and infer long-distance regulation from available Hi-C data capturing interaction profile from embryonic and postnatal brain. Chip-seq peak detection and comparisons of the Chip-seq experiments were done using cistrome genome and Genomatix. Those powerful bioinformatics tools allowed us to convert Ascl1 bound chromosomic position in rat into mouse chromosomic position (liftover tool) and update from previous genome assembly (mm9 to mm10). Mouse genome is much better annotated than the rat genome. Results were loaded on a genome browser for subsequent analysis and observation of ChIP-seq binding profile (Genomatix, UCSC). This general overview of the chromatin state associated to particular genomic loci is a powerful tool. We identification high confidence Ascl1 and Olig2 binding site as well as the enriched regulated signaling pathway they compose. Ascl1 and Olig2 regulate overlapping function by clusters of modules of TFBS. We generated new hypotheses about gene regulatory networks and validate them by different approaches paying particular attention to the chromatin remodelers. A precise analysis of promoter-enhancer interactions involved in the oligodendrocyte lineage allowed the construction of a catalogue of regulatory sequence greatly enriched in oligodendrocyte specific regulatory sequence(Annex). This information was used to define short synthetic promoter for viral construction that could efficiently drive gene expression to the oligodendrocytes. We also analyzed OPCs specific cis-regulatory element such as the Pdgfrα or Cspg4. Transfection analysis of miniTOL2-Tomato integrative plasmids containing 6 putative enhancers of Pdgfra or MOG were done in differentiating neurospheres (data not shown). We obtained preliminary successful expression in Pdgfra positive OPCs for the Pdgfra construct. However, NSCs were also expressing the construct, likely because of one of the enhancer being involved in another process. The MOG miniTOL2-Tomato construct was expressed in myelinating oligodendrocyte but replication of the experiment has to be performed.

Creatine

The creatine metabolism seems to be a function of the oligodendrocytes. Creatine was thought to come principally from periphery through the blood brain barrier (Wyss and Kaddurah-Daouk, 2000) In situ hybridization shows stronger GATM expression in white matter region compared to grey matter (Fig. 47G). It will be of great interest to explore further the metabolic coupling between myelin, axons and astrocytes. As phosphocreatine acts as a metabolic intermediate transported from mitochondria to areas with high energy demands. This gradient of GATM expression could indeed be correlated to the specific metabolic needs of the axonal fibers passing by the white matter structures such as the corpus callosum or the fimbria. If so, specific transporter such as CT1 or specific channels such as gap junction could be involved. The role of creatine in myelination has already been explored and shown to be beneficial in different neurological diseases. A clinical trial is currently in phase III in the context of Huntington's disease (M. Flint Beal 2011). Moreover, increased creatine is found in MS lesion (M Inglese 2003; M. V. Davydovskaya 2013). These results suggest that the increased creatine content in MS lesion is due to the release of its content by the dying oligodendrocyte and associated metabolic changes. A recent study demonstrated the expression of CT1, the creatin transporter in neurons and oligodendrocytes. This finding is in accordance with the microarray data. Moreover, Gamt and Gatm are clearly expressed in young adult white matter. Gatm knock-out mice was recently generated and they provide a good model to test the role of creatine in myelin formation (Choe et al. 2012;C. I. Nabuurs 2013). Gatm mutant mice are viable, despite growth retardation and weight loss. Overall size is reduced despite normal gross morphology appearance. However, precise characterization of its role in oligodendrogenesis and myelin methabolism has not yet been assessed. We are currently exploring this function in the lab using 2 and 3 weeks old brains from Gatm mutant mice.

Chd7

Chd7 was our first selected candidate, Bcas being already understudy in another lab. Indeed, all the criteria made from it an interesting factor to study in the role of oligodendrocyte differentiation and myelination: i) it was downregulated in both Ascl1and Olig2 mutant arrays; ii) it presented binding of Ascl1 and Olig2 in intragenic regions by ChIP-seq made in purified OPCs; iii) it was expressed in the ventricular zone of the ventral telencephalon like Ascl1 and Olig2; iv) its expression was enriched in oligodendroglia in expression microarrays; v) its expression in white matter was confirmed by in situ hybridization datasets; vi) it was downrelated both in demyelinating mouse models (cuprizone and lysolecytine) and in MS microarrays; vii) tools for its expression and functional characterization were available (antibody working in immunofluorescence) and both regular and conditional mutant mice (Chd7 Gt and Chd7 Flox mice); viii) Chd7

We therefore, started a detailed characterization of Chd7 protein expression demonstrating that like the Chd7 mRNA, Chd7 protein is found both in OPCs and in immature OLs being downregulated to very low levels in mature OLs (Fig. 40). We also demonstrated that Chd7 was co-expressed with Ascl1 in SVZ progenitors (confirming the data that was published at that time by the lab of HK Liu [START_REF] Feng | Epigenetic regulation of neuronal fate determination: the role of CHD7[END_REF] suggesting that both factors likely regulate each other and/or function in the same transcriptional complex. Our recent data also indicates that Chd7 is strongly upregulated in maturing oligodendrocytes in and around remyelinating lesions in mice (not shown), suggesting an important role in both myelination and remyelination. We obtained Chd7 Gt and Chd7 Flox mice from Dr. Donna Martin (Michigan, USA) but due to health status of the mice and cold weather during last winter, plus the 6 months needed to combine the three alleles to trace mutant cells (Cre, Chd7 fl , tracer), all our functional studies have been delayed until now. We are nevertheless, starting the first experiments using two Chd7 conditional mutations in oligodendroglia. One using Olig1 Cre mice, that will remove Chd7 in around 50% of oligodendroglia from embryonic stages (experience from RQ. Lu's lab). Another, T , that will permit us to specifically delete Chd7 in some OPCs the postnatal brain during myelination (Tamoxifen injection in the first week postnatal) or after a focal demyelinating lesion (lysolecytin, LPC) in the adult brain (Tamoxifen injection before demyelination). In both cases, we are going to trace the Chd7 mutant cells using a Cre-induceable reporter background (Rosa26 stop-Tomato ). We hope from this studies to demonstrate the function/requirement of Chd7 in oligodendrocyte differentiation and myelination.

Moreover, in collaboration with RQ Lu's lab we have started to improve ChIP protocols to make Chd7 ChIP-seq in purified OPCs and immature oligodendrocytes. From this data we expect to characterize the Chd7 transcriptional regulation mechanisms in oligodendrogenesis during (re)myelination.

Chd8

The choice of Chd7 as a important candidate to regulate OPC/OL differentiation pushed us to investigate the role of its paralog gene, Chd8, given its expression in oligodendroglia from microarray neural subtypes. Indeed, this data was confirmed by in situ hybridization expression data from postnatal and adult stages showing enrichment in white matter cells. Our immunofluorescence characterization of Chd8 protein expression in the postnatal brain has confirmed that Chd8 is present during the phase of OL differentiation and being downregulated to very low levels in mature/myelinating OL. Moreover, Chd8 is induced in OPCs close to demyelinating lesions and its expression timing correlates with factors inducing remyelination such as Olig2, Ascl1 or Nkx2.2. This function in remyelination could be conserved up to humans given that our preliminary data shows that Chd8 is expressed in active remyelinating MS lesions. Given that Chd7 conditional mutations (Olig1-Cre, on-going work in collaboration with Richard Q. Lu's lab) shows strong impairment in oligodendrocyte differentiation, we could assume and the upregulation of Chd8 in OPCs during remyelination we could expect that Chd7 and Chd8 regulate different steps/genes in OL differentiation and myelination. Therefore, the characterization of Chd8 function in (re)myelination and its target genes in chromatin remodeling could constitute an important step forward in our understanding of the (re)myelination process, and future therapeutic strategies could be derived from this knowledge.

Tns3

From our microarray and ChIP integrative strategy we found that Tns3 was an interesting common target of Ascl1 and Olig2 given the expression pattern similar to these TFs in the ventral thelencephalon and from the expression in oligodendroglial cell-types in the postnatal brain. These data was confirmed by the expression found in Allen brain and Euroexpress In situ hybridization databases, which shown an enriched expression in white matter regions at postnatal and adult stages, in sparse cells that could constitute a precise stage of oligendroglial lineage different to OPCs given the it was less dense than Pdgfra in situ expression pattern. We could confirm the oligodendroglial expression using an antibody for Tns3, and moreover, characterize that its expression perfectly match the start of OPC/OL differentiation, being downregulation to very low levels in the mature OL. This specific expression of Tns3 to the moment of OL differentiation and myelination suggests that it plays a relevant role in these processes and that is therefore the kind of genes that we set off to find in our analysis.

Another criteria for which Tns3 was selected was its implication human tumors. Contrary to the expression data in tumors and mouse kidney, Tns3 antibody signal was localized to the nucleus. Given that Tns3 has been shown to mediate integrin signaling from the cell surface to the actin skeleton, we were surprise to find Tns3 nuclear localization. Nevertheless, search for actin functions led us to discover more than ten years of documenting the nuclear function of actin in transcriptional regulation, that place Tns3 in the center of the research focus of the lab. We have started to analyze brains of Tns3 mutant mice (from a gene-trap line) but our preliminary data (from two and three postnatal mice) have not yet unravel a phenotype in the process of oligodendrogenesis or myelination. This contrary to our expectation rises the problem of gene functional redundancy and reliability of expression databases. We have explored all the data published for other Tensin family members without finding a clear signal in oligodendroglial cells. Tns2 is expressed in vessels of the brain and maybe there is a weak signal in the white matter that needs to be confirm. We recently got an antibody working in immunofluorescence showing weak signal of Tns2 in white matter regions and we are testing the possibility of an upregulation of Tns2 levels in Tns3 mutant white matter and therefore a possible functional compensation of Tns3 function by Tns2.

CONCLUSION AND PERSPECTIVES

In the present work we used integrated analyses of expression microarrays and chromatin immuno-precipitation analysis to characterize the genes that are regulated by Ascl1 and Olig2 in NSCs and the oligodendroglial lineage. We identified Ascl1 and Olig2 target genes in Notch, Wnt and Sonic Hedgehog signaling pathways. We identified Chd7 and Tns3 as new important players regulated by both Ascl1 and Olig2 during the oligodendrocyte differentiation and compared the transcriptome of OPC and differentiating oligodendrocytes with other neural cell types to identify genes with specific function at different stages of the oligodendroglial lineage. We also brought attention to the creatine metabolism function in oligodendrocyte. We found a highly enriched expression of Gatm, Gamt and Slc68a, two genes that code for the enzymes necessary for creatine synthesis. Finally, we generated a list of potential beneficial drugs able to change myelin gene expression and which were never tested in the context of remyelination. we hope that these identified genes and regulatory sequence involved in oligodendrogenesis and whose expression are regulated in demyelinated lesions will allow the development of new therapeutic strategies promoting an efficient remyelination in demyelinating diseases such as Multiple sclerosis or leukodystrophies.

ANNEX

Annex1 Sagittal section of Ascl1 and Olig2 in-situ hybridization of E13.5 and P56 mouse brain .Similar expression pattern in the major part of the ventricular zone at E13.5. At P56, Ascl1 expression is scatterd in the brain and highly enriched in the neurogenic zone in the adult SVZ and SGZ in the hippocampus. Olig2 expression is strong all over the brain and more visible in white matter regions such as the corpus callosum and the fimbria. Olig2 work together to specify OPCs. We followed a strategy using genome-wide transcriptome analysis and chromatin immuno-precipitation to characterize Ascl1 and Olig2 directly regulated genes in OPCs and during oligodendrocyte differentiation. We identified new specific markers of different stage of the neural lineages and new important genes correlated to OPCs differentiation. We focused on Chd7 and Tns3, two genes which expressions are driven by Ascl1 and Olig2 and enriched in the oligodendroglial lineage at two interesting stage, the early specification stage and the transition between migrating and differentiating oligodendrocytes, respectively. Moreover, we identified the myelinating oligodendrocyte as the cell in charge of the creatine synthesis in the brain and potentially driving axonal metabolic support. We also used an approach a toxicogenomic and drug repositioning approach to identify new molecules known to modify OPCs and myelin genes but untested in the context of demyelinating diseases. As currently, most of the available treatments for demyelinating diseases are based on immuno-modulatory and antiinflammatory drugs but none are able to directly promote myelin repair, we expect that these identified genes involved in oligodendrogenesis and whose expression are regulated in demyelinated lesions will allow the development of new therapeutic strategies promoting an efficient remyelination in demyelinating diseases such as Multiple sclerosis or leukodystrophies. tester dans le cadre de maladie demyélinisantesLa plupart des traitements disponibles pour traiter les maladies démyélinisantes sont basées sur une approche immune modulatrice et antiinflammatoire. A ce jour, aucun n'est en mesure de promouvoir directement la réparation de la myéline de manière efficace. Nous espérons que les gènes dont l'expression est régulée dans les lésions de démyélinisation identifiés lors de cette étude permettront de mieux comprendre le mécanisme de remyelinisation et le développement de nouvelles stratégies dans les maladies démyélinisantes telles que la sclérose en plaques ou dans les leucodystrophies.

RESUME EN FRANÇAIS

Ce

  Remerciements ............................................................................................................................ PREFACE ....................................................................................................................................... INTRODUCTION ......................................................................................................................16 PART I: THEORETICAL BACKGROUND .............................................................................16

Figure 1 .

 1 Figure 1. Evolution of Epigenetic landscape. A. Original drawing of Waddington (Waddington, C.H. 1957) where cells are depicted as a ball rolling down to a valley. New valleys represent different cell fates, and ridges maintain cells in particular epigenetic state. Landscape can be monostable, bi-stable or multistable. The solid black lines represent stable steady states, the dashed lines represent unstable steady states, and in red the bifurcations. B. Original drawing from Waddington showin correlation between the height of the ridges and combinatorial gene expression. C. Schématic representic the progressive cell fate restriction occurring during neural development. D. Mordern mathematical representation of the epigenetic landscape. Oscillatory gene expressions are generated by positive or negative feedback in the regulation network (red and black arrows) which lead to cell differentiation, a process driven by positive feedbacks stabilizing -irreversible‖ stochastic events (adapted from S Newman 2012).

Figure (A) Figure 1 .Figure 2 .

 12 Figure (A) Figure 1. Epigenetic change during development. Schematic showing the mouse developmental change in epigenetic profile between E11.5 and P56. The Chipseq profiles of the active enhancer mark H3K27Ac are shown for 3 different tissues, the brain, the heart and the liver. (B) Parrallel analysis of Chip-seq peaks. The absence of overlap between peaks of each tissue indicated cell type specificity of the identified enhancers. (adapted from B van Steensel 2011.(B) Stuctural organization of the chromatin and gene regulation. Schematin showinh the DNA packed around histone and forming globules of nucleosomes. DNA accessibility is driver by Histone turn over and competition with TFs and chromatin remodelers. P300 is shown to bind on active enhancer and acetylate H3k27. Pararallele view of different epigenetic marks or DNAse accessibility regions.(C) Model of chromatin organization in an interphase mammalian nuclei.(D) RNA PolII core promoter element motifs. Sequence elements that can contribute to basal transcription from the core promoter are indicated.The location given for the different motis are indicated relative to the start site (adapted from AS Nord 2013; B van Steensel 2011; Mirny et al 2011)

  which can directly associate with DNA in packed nucleosomes (J Xu, SD Pope 2007; AA Sérandour, S Avner, 2011).

Figure 3 .

 3 Figure 3. Histone code of functional elements in mammalian genomes. Table2. Numerous other epigenetic marks define the epigenetic code (adapted from ENCODE project consortium).

Figure 3 .

 3 Figure 3. Histone code of functional elements in mammalian genomes. Schematic showing the epigenetic marks associated with promoters, enhancer and transcription elongation. Active promoters are marked by histone H3 lysine 4 dimethylation (H3K4me2), H3K4me3, acetylation (ac), and H2A.Z. Transcribed regions are enriched for H3K36me3 and H3K79me2. Repressed genes may be located in large domains of H3K9me2 and/or H3K9me3 or H3K27me3. Enhancers are relatively enriched for H3K4me1, H3K4me2, H3K27ac and the histone acetyltransferase p300.

Figure 4 .

 4 Figure 4. Affymetrix arrays, one population is used as starting material. Total RNA is extracted and cDNA is prepared. The cDNA is used in an IVT reaction to generate biotinylated cRNA. After fragmentation, this cRNA is hybridized to microarrays, washed and stained with PE-conjugated streptavidin, and subsequently scanned on a laser scanner (adapted from F J T Staal, 2003).

Figure 5

 5 Figure 5 Chromatin immunoprecipitation techniques. Simplified schematics of the main steps are shown. Left: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for DNA-binding proteins such as transcription factors. Right: ChIP-seq for histone modifications (adapated from S. Furey 2012).

Figure 6 .

 6 Figure 6. Detecting chromatin interactions with chromosome capture. A. Distant genomic regions interaction are mediated by DNA-binding proteins. Chromatin conformation capture technics (3C and derivatives) use a cromatin cross-linking folled by a fragmentation and a ligation step to join distant fragments that are interacting in 3D space, thus providing information on possible promoter-enhancer interactions. Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) detects chromatin interactions. However, ChIA-PET uses a chromatin immunoprecipitation (ChIP) step to more specifically identify interactions with a particular bounchrod protein, such as RNA polymerase II. (adapated from S. Furey 2012 and J Dekker 2013). B. Schematic of the chromatin capture technic. C. Schematic of Hi-C data from mouse chromosome 18. Hi-C method is a genome-wide 3C. Hi-C provides a true all-by-all genome-wide interaction map, but the resolution of this map depends on the depth of sequencing. Hi-C data are represented as two-dimensional heat maps. While the probability of long range interaction is invertionaly proportional to the distance, shift from this linear model define interaction enrichement or interaction depletetion. Interacting regions detection depends on sequencing depth. Bioinformatic reconstruction allow the inference of long range regulation of gene expression (adapted from Dekker J 2013).

Figure 7 .

 7 Figure 7. Neural and brain development. (A).Schematic representing the mouse embryonic development from E0.5 to E5.5. Embryonic Pluripotency factors, Nanog, Gata6, Oct4 are indicated. Epiblast cells are the embryonic stem cells. (B) From E6.5, neurulation begins and lead to the formation of the neural system. (C) Neurulation. At the neural plate stage, vertical induction from the mesendoderm (notochord and prechordal plate), and planar induction from Hensen's node and ectoderm drive dorsoventral and antero-posterior patterning of the nervous system. (D) Schematic representations of TFs expression domains at the neural plate stage. Gene symbol and color codes are identified. Medio-lateral (dorso-ventral) and antero-posterior (rostro-

Figure 8 .

 8 Figure 8. Sonic Hedgehog signaling pathway (adapted from Cell Signaling technology).

Figure 8 .

 8 Figure 8. Cell sorting occurring after stochastic salt-and-pepper specification of neural progenitors. A. Patterning of the neural tube by signals (Shh, Wnt, BMP)

Figure 9 .

 9 Figure 9. Notch signaling pathway. (adapted from Cell Signaling technology)

Figure 10 .

 10 Figure 10. Wnt signaling pathway. (adapted from Cell Signaling technology)

Figure 11 .

 11 Figure 11. Overview of neurogenesis during development. The principal cell types of NPCs with the progeny they produce are indicated by different colors. Additional NPC types that are typically found in mammalian neocortex are indicated in the box..

Figure 12 .

 12 Figure 12. Ventricular zone organization. (A). Dividing NSCs are clustered in the cortical ventricular zone (VZ) but are scattered in the ganglionic eminence where subapical progenitors divide actively in the subventricular zone (SVZ). (B). Pinwheel architecture of the ventricular surface. Multiciliatted cells are the ependimal cells which circle proper NSCs which extend their primary cilia outside the ventricule. (C). Time laps imaging of interkinetic migration in mouse cortical slice. (D). Schematic of the nterkinetic migration. Nucleus move up and down depending on the cell cycle phase.Cells undergo mitosis at the ventricular surface where the primary cilia and the associated centromere was localized. Upon G1, they move toward the pial surface and receive less Shh and Notch signaling. This mechanism allow cells to differentiate and to migrate radialy toward the cortex thanks to the RGC processes (adapted fromGA Pilz 2013;[START_REF] Mirzadeh | Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain[END_REF] Paridaen JTML, 2014).

Figure 13 .

 13 Figure 13. Comparison between SVZ and SGZ adult neurogenesis. None of the indicated NSCs marker is specific on its own. Combination of markers define the quiescent NSCs stage. (adapted from Ming Song 2011).

  Cultures with bFGF alone divide slowly while those cultured with EGF alone divide at a faster rate. FGF-2 and EGF are mainly tranfered by endothelial cells contacting directly the NSCs. Other important factors regulate NSCs maintenance and survival such as VEGF, Wnt, Shh, Notch or CTNF (Fig.15).

Figure 14 .

 14 Figure 14. A. Sources of vascular -derived adult NSC niche effectors. Growth factors originate from the endothelial cell, blood circulation, extracellular matrix deposition, and perivascular cell types (adapted from SJ Goldberg 2013).

Figure 15 .

 15 Figure 15. Table show in-vivo and in vitro -specific‖ markers together with survival factors involved in NSCs maintenance. Currently, a combination of marker is necessary to fully define the NSCs stage. (adapted from Ramasamy 2014).

Figure 16 .

 16 Figure 16. Asymetric segregation of cellular position and spindle orientation. Cell fate determinants may be equally (symmetric division, A) or unequally (Asymemetric B) distributed between daughter cells. (C, D) Examples of asymmetries between daughter cells that were introduced by asymmetric inheritance of differently aged centrioles, ciliary membrane, Par 3 and Notch signaling components (adapted from JTML Paridaen 2014).

Figure 17 .

 17 Figure 17. Ascl1 and Olig2 are Proneural bHLH transcription factors. (A)Schematic of a DNA bound by a bHLH:E47 dimere (B) Phylogenetic tree of the proneural bHLH. Ash1 is the more ancient proneural HLH, gene evolved by

Figure 18 Dynamic

 18 Figure 18 Dynamic Regulation of cell fate by Proneural bHLH Factors. (A).Schematic representation of proneural gene oscillations NSCs. Time-lapse clearly showed that Hes1 and Ascl1 protein expression oscillate with a period of 2-3 hr, while Olig2 protein expression oscillates with a period of about 5 -8 hr in NSCs.(B).Notch lateral inhibition. Ascl1, Dll1 and Hes1 forming salt-and-pepper patterns. Substantial accumulation of Ascl1 and Delta or Hes1 is stabilized by their oscillatory dynamics. In the cell having higher level of Ascl1, Delta expression is increased and activates Notch signaling in the neighboring cell. In the cell having Notch signaling activation, RBPj upregulates Hes1 and Hes5. Then, the transcription Ascl1 is inhibited by Hes1 and Hes5 . (C) Roles of bHLH Factors in NPC Self-Renewal and Cell Fate Determination. Schematic representing Ascl1 up regulation upon neuron and oligodendrocyte differentiation, Hes1 cycling up-regulation upon Astrocyte differentiation and Olig2 up-regulation upon oligodendrocyte (D) Dll1 asymmetric inheritance between sister cells. Time-lapse of dividing NSCs. Cultured NSCs infected with retroviruses expressing Dll1-EGFP (green) and mCherry-Geminin

Fig 19 .

 19 Fig 19. Ascl1 and Olig2 requirement for the generation of OPCs. (A-H) sections of E13.5 brain In-situ hybridization showing expression of Ascl1 (A, coronal and B, sagittal) and Olig2 (E,F). (C,D) Ascl1 knock-out morphological changes observed in E13.5 sagital section of WT (G) and Ascl1-/-(H) mice. (G,H) Olig2 knock-out morphological changes observed in E13.5 sagital section of WT (G) and Olig2-/-(H) mice. (I) Ascl1 requirement for OPCs specification in embryonic telencephalon: Telencephalic coronal sections of wild-type (A-A1) and Ascl1-/-(A'-A1') embryos at E14.5 (Parras et al 2007). (J) Olig2 requirement for OPCs specification in spinal cord coronal sections of wild-type (A) and Olig2-/-(D) embryos (Zhu et al 2001). (K) Genetic interaction between Ascl1 and Olig2. Quantification of OPCS in the forebrain of E13.5embryos. Percentages represent the ratio of OPCs observed in embryos of indicated genotypes over wild-type embryos (Parras et al 2007).

  , thus inducing transcriptional repression by inhibitory chromatin remodeling of their promoters. Insitu hybridization and immunostaining data show that Chd7 is highly expressed in adult SVZ, RMS and SGZ regions of the mouse brain and in cells co-expressing GFAP, Mcm2, Nestin and Sox2, indicating that Chd7 is expressed in a subpopulation of dividing NSC. Moreover, most of Ascl1 and DCX positive cells express Chd7 thus showing Chd7 persistent expression in neural progenitors and neuroblasts. Using a Chd7 conditional knockout mouse line (Chd7 fl/fl ) injected with BrdU and traced for 4 weeks showed a dramatic decrease of BrdU-positive cells and newborn DCX positive neurons in the olfactory bulb of Chd7 mutants. Interestingly, no changes were observed in Nestin and Sox2 positive cells in the SVZ of Chd7 knock-out mice, showing no

  . The wrapping of oligodendrocyte stabilize the clustering of axonal Nav1.1 and Nav1.6 sodium channels at nodes of Ranvier and the Kv1, Kv3 and Kv 7 potassium channels at the paranode (Buttermore E 2013). Interestingly, clustering of Nav channels at the node of Ranvier occurs before myelination (feinberg et al. 2010)

  cholesterol for ~30%. (Gopalakrishnan G2013; Chrast R2011). More specifically, galactocerebrosides are the most abundant component of myelin, sulfatide (sGalCer) representing almost 20% alone. Phosphatidylethanolamine (plasmalogens) and Phosphatidylcholine (lecithin) are also major constituent, representing 16% and 12% respectively. Sphingomyelin, a minor component, associates with cholesterol in -lipid rafts‖ and are involved in many processes, such as protein and lipid sorting or cell signaling (Dupree JL, 2010; K. Simons, D. Toomre 2001) Interstingly, despite their abundance, glycosphingolipids are not essential for myelination. Indeed, mutant mice for CGT (UDP-galactose:ceramide galactosyltransferase) and CST (galactosylceramide 30 -sulfotransferase), two enzymes essential their synthesis, does not prevent formation of compact myelin sheaths despite paranodal and axonal defects (Coetzee T, Fujita 1996). Paranodal disruption was shown to be partly due to the lack of SGalCer, since mice lacking galactosylceramide-3-Osulfotransferase (Gal3st1), the enzyme converting GalC into SGalC (Honke K, Hirahara Y, 2002-Hirahara Y, Bansal R2004), also showed nodes defects. Finaly, mice lacking Fa2h (fatty acid 2-hydroxylase), the enzyme hydroxylating GalC and SGalC showed important delay in myelination (Zoller I, Meixner M 2008). Together, these results highlight the importance of lipid synthesis and lipid signaling in oligodendrocytes metabolism and demyelinating diseases, a question that only begin to be tackled (Aggarwal 2011 ; Podbielska M 2011).

  . As those datasets are made available publicly on GEO database, they are now used by many others to analyse oligodendrocyte lineage and allow identification new critical factors (Monasterio-Schrader 2012). To date, mass-spectrometry is the most sensitive method to systematically identify the protein content of myelin. Up to now, eight proteomic studies of CNS myelin have been published, providing 11 datasets for myelin from mouse (Jahn O, Tenzer S 2009 Werner HB et al 2007; Vanrobaeys F et al 2005; Taylor CM et al 2004), Rat (Roth AD, Ivanova A, Colman DR 2006; Baer AS et al 2009), and human (Ishii A et al 2009; Dhaunchak AS et al 2010). The integration of all myelin proteomic datasets identified more than 1,280 proteins (Monasterio-Schrader 2012). Among them, the main myelin proteins identified are PLP/ DM20 (proteolipid protein) representing 17% of the protein content, MBP (myelin basic protein) representing 8% and CNP (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) representing 4% ( Monasterio-Schrader 2012). Other major compact myelin proteins are MOBP (myelinassociated oligodendrocytic basic protein), MAL (myelin and lymphocyte protein), MOG (myelin oligodendrocyte protein), MAG (myelin associated glycoprotein) representing each around 1% of the total protein content (Monasterio-Schrader 2012).

  Oligodendrocytes are bidirectionaly coupled to astrocytes thanks to Cx47/Cx43 and Cx32/Cx30 heterotypic channels in the cortex (D.M. Menichella 2003, Wasseff 2011). However, in the corpus callosum, where only a few astrocytes reside, only oligodendrocyte to oligodendrocyte couplings occurs through Cx47/ Cx32 (Wasseff 2011, M. Maglione, 2011). Interestingly, Cx32/Cx47 mutant mice have no O/A or O/O coupling and exhibit a severe demyelination phenotype with myelin vacuolization phenotype, seizures, conduction block and increased oligodendrocytic input resistance typical of potassium homeostasis problems (D.M. Menichella 2003; M. Maglione, 2011). Similarly, in Cx43/Cx30 mutant mice, O/A coupling is disrupted and lead to vacuolization and myelination defect (S.E. Lutz, Y. Zhao 2009; A. Wallraff, R. Kohlin 2006). Those studies demonstrated that such O/A or O/O networks are involved in the buffering of the potassium generated at the periaxonal space (below the myelin membrane) through a mechanism called potassium syphonning (D.M. Menichella 2006).

FGF

  signaling was shown to overcome inhibiting signal and specify OPCs in the dorsal spinal cord(Vallstedt et al., 2005).

Figure 21 .

 21 Figure 21. Schematic of the transcriptional control of oligodendrogensis. The diagram focuses on the NSCs to OPCs specification and maturation. Because of their undifferentiated state, stem cells defined by only few marker. Combination of marker is necessary to discriminate between active and quiescent NSCs.While the combination of GFAP, CD133 and EGFR define active NSCs, Spot14 or S1A and PGD were recently shown to define quiescent NSCs. OPCs are express only two specific markers, PDGFRα or NG2(CSPG4), whereas oligodendrocytes express APC (adenomatous polyposis coli) and proteolipid protein (PLP). Myelinating oligodendrocytes express

  Neurotransmitters released to OPCs by axonal synapses are involved in cell proliferation through IP3/Ca2+ signaling cascade (QK Yang Xiong 2013). Calcium enter in OPCs through GluR3/GluR4 receptors (P. P. Maldonado 2011; T. Itoh, J. Beesley, 2002, C. Y. Brazel, 2005), ATP /P2X and P2Y receptors (C. Agresti 2004-64), α-adrenergic receptors (R. Papay 2005), VDCC of the L-, N-, and R-type oligodendrocyte lineages in vivo (A. M. Butt, 2006). During normal development, OPC proliferation and cell-cycle progression are positively regulated by two potent mitogenic factors-platelet-derived growth factor

Nkx2. 2

 2 mutant mice have a normal OPC numbers (Qi Y,Cai J, Wu Y 2001; Soula C, Danesin C2001) but display a severe defect in differentiation(Qi et al., 2001). Despite the fact that Nkx2.2 is required for myelination, it is transiently downregulated upon cytoplasmic translocation of Olig1, a process associated with Nkx6.2 upregulation(Cai et al., 2010). As Nkx2.2 inhibit Mbp(Wei et al., 2005) thus, its down-regulation may be necessary to initiate myelination. Nkx6.2 mutant mice display myelin defect and aberrant expression of the paranodal proteins stathmin1, neurofascin and contactin(Southwood et al., 2004). Nkx6.2 and Nkx2.2 may participate to paranodal region and axo-glial interactions (refXX).Retinoid X receptor gamma (RXR) positively regulates OL differentiation during myelination and remyelination(Huang et al., 2011). RXR nuclear localization was demonstrated during OPC differentiation and OL regeneration in demyelinating lesions multiple sclerosis tissue. Moreover, treatment with 9-cis retinoic acid after demyelination promoted axonal remyelination(Huang et al., 2011).

  Ezh2 plays a role in the selection of the oligodendrocyte lineage. Overexpression of Ezh2 in NSCs triggers oligodendrocyte fate choice, while Ezh2 silencing causes the opposite (Sher et al., 2008b). The dynamic changes in activation and repression of promoter and enhancers occurring during the process of OPCs differentiation and myelination was recently studied by Chip-seq analysis of H3K4me3 and H3K27ac marks at the stages of oligodendrogenesis: OPCs, premature OLs and myelinating OLs. (Yu Chen Kim 2013). This is nevertheless, just the begging of a deeper understanding of the logic undelaying the regulation of key regulatory genes promoting and inhibiting these stage transitions in the process of oligodendrogenesis.

Figure 22 .

 22 Figure 22. Table showing the main signaling controlling different aspect of the OPCs physiology (Adapted from S. Mitew et al.2013).

  and epigenetic (Yu Chen Kim 2013) analysis of the oligodendroglial lineage together with the characterization of the intra and extracellular regulatory signaling pathways to bring a new understanding about the specificity of oligodendrocyte development and remyelination comparable to other cell types. Unravelling the particular transcriptional program of OPCs specification and differentiation will allow to progress in cell based therapies for example by increasing the yield of OPC production in direct lineage conversion experiments or to find new oligodendrocyte specific druggable molecules that avoid side effects.

Figure 23 .

 23 Figure 23. Isolation of the ventral forebrain region containing the ventral source of OPCs during development. (A) Picture of a mouse embryo at 13 days of gestation. (B) Dissected brain in a horizontal perspective with the dorsal telencephalon removed showing the dissection steps to follow to get remove the dorsal forebrain. (C) Steps to follow to dissect the ventral region containing the source of embryonic OPCs in the forebrain. Cutting steps, lines represent the cutting plane perpendicular to the plane of view, are represented by scissors and step numbers. (D) Brain sagittal section with the different regions represented schematically, according to the reference brain atlas of E13.5 embryo (Allen brain atlas: http://atlas.brain-map.org) that are contained in our dissected tissue (grey square). (E) Morphological difference between a wild-type and a Ascl1 mutant ventral telencephalon. Note that the MGE is absent in the mutant (asterisk). MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; CGE, caudal ganglionic eminence.

  to estimate probe affinity to non specific binding. Taking into account the thermodynamics of binding (DNA duplex stability arises from hydrogen bonding between Crick's pairs and base stacking interactions between adjacent base pairs (Van der Waals interactions, electrostatic and hydrophobic interactions) as well as the biotin labeling interferences, GCRMA gives a more robust background correction and fold change estimate as well as less variability at low intensity (Mieczkowski et al., 2010). However, GCRMA is cannot normalize properly batch of experiments done in different condition or in different labs because variability in such case overcome the normalization capacity. In

Figure 24 .

 24 Figure 24. Generation, normalization and analysis of Ascl1 KO and Olig2 KO microarrays. (A) Scheme of arrays production. (B) Arrays samples before and after normalization by GCRMA showing a good normalization of all arrays after the treatment. (C) 2D projection of a 3D plot global representation of arrays showing the similarity of gene expression in each array, represented by the principal component analysis. One Ascl1 and one Olig2 arrays were not included in further analysis given their difference with respect of the others of the same genotype. Note the proximity of 4 Ascl1 KO arrays making the data of Ascl1 arrays very robust. (D) Clustering heat map of the 300 top genes more significantly regulated in the arrays, showing that selected arrays cluster together by genotype, presenting 6 different regulation pattern clusters. M1 1-5 , Ascl1KO arrays; O2 1-5 , Olig2KO arrays; WT 1-5 , wild-type arrays. Dissection picture A in shows a forebrain where the dorsal region has been removed to show the ventral part; red are indicates the region generating OPC at embryonic stages (MGE/AEP). MGE, medial ganglionic eminence; AEP, anterior entopenduncular area; OB, olfactory bulb; LGE, lateral ganglionic eminence; Cx, cortex.

Figure 25 .

 25 Figure 25. Project experimental design for identification of Ascl1/Olig2 common direct targets. Scheme representation of the different sets of data generated or processed to identify both the direct targets common to Aslc1 and Olig2, and those that are enriched in the oligodendroglial cells. Each Venn circle represents a gene dataset. Time flows from let to right. In silico studies are depictures with grey arrows and biological experiments with red arrows. Final focus in few selected genes is shown with relative size corresponding to the functional data cumulated for each selected gene (Chd7, Tns3, Chd8, GAMT).

Figure 26 .

 26 Figure 26. Ascl1 and Olig2 commonly regulated genes. (A) Venn diagrams representing the gene subsets differently regaulated between Ascl1 mutant versus wildtype and Olig2 mutant versus wild-type. (B) Main pathways regulated in both Ascl1 and Olig2 mutant microarrays. Table showing significant signaling pathways regulated by Ascl1 (Yellow), Olig2 (Blue) or Ascl1 and Olig2 (Red). Most of the regulated pathways are regulted by both Ascl1 and Olig2. Important signaling, such as Notch,

Figure 27 .

 27 Figure 27. Regulation of Ascl1 and Olig2 target genes in E13.5 vental telencephalon. (A-B) Tableshowingthe most regulated and statistically significant genes (A) down and (B) up-regulated by Olig2 (maximal fold change and FDR 1%). A larger selection of Ascl1 regulated genes can be found in annex. Olig2 is not detected as significantly down regulated. However, Olig1 is higly up regulated and

  5 ventral telencephalon material to investigate which genes were regulated through direct binding of Ascl1 on gene promoter or associated cis-regulatory sequences (collaboration with D.Castro & F. Guillemot). First, we analyzed Ascl1 ChIP-on-chip experimental data (D. Castro 2011) and identified 1265 promoters significantly bound by Ascl1 (Fig.28). Then, we analyzed Ascl1 in ChIP-sequencing experiment and identified 21902 regions significantly bound all along the genome by Ascl1 from which 8020 region were located inside a gene (promoter, intron, exon) and 13882 region were located in intergenic regions. We found a good overlap between the two experiments as 22013 unique regions bound by Ascl1 were found combining the two datasets. In order to infer regulatory capacities of those regions, we combined these datasets with NSCs H3K4me1, H3K4me3 and H3K27Ac epigenetic information to identify promoters and cis-regulatory regions that are actively regulated (GEO GSE12241). We then used a -next neighbor's analysis‖ and we associated regions to the closest gene with a maximum distance of 5kb. Finally, we took advantage of recent Hi-C data obtained from embryonic and postnatal brain tissues to infer long range promoter-enhancer interactions from our ChIP-seq dataset (Y Shen 2012). This integrative method (Fig.28and Fig.29) allowed us to map the position of Ascl1 binding in the regulatory genetic landscape of NSCs and subsequent comparison with Ascl1/Olig2 knock-out microarray data allowed us to detect numerous high confidence functional Ascl1 binding sites.

Figure 28 .

 28 Figure 28. Analysis of Ascl1 and Olig2 function in oligodendrocyte differentiation. (A) Scheme of array production from Ascl1 KO , Olig2 KO and WT. Dissection picture shows the MGE/AEP region of mouse ventral forebrain (in red) where embryonic OPC are generated. MGE, medial ganglionic eminence; AEP, anterior entopenduncular area. (B) Identification of oligodendrocyte genes reprocessing published microarray data from all the main neural cell types. (C,D) Identification of Ascl1 and Olig2 binding sites using ChIP-Chip and Chip-seq analysis on mouse ventral forebrain (C), purified rat postnatal OPCs and NS5 cell line (D). (E) Identification of Ascl1/Olig2 regulated active enhancers using H3K27ac in the oligodendrocyte lineage. (F) Identification of genome-wide long range interactions in embryonic and adult mouse brain using Hi-C technics. Picture shows 3D organisation of chromosomes, each one occupying its own

Figure 29 .

 29 Figure 29. Ascl1 direct targets in E12.5 ventral telencephalon. Table presenting a selection of Ascl1 direct tragets in E12.5 ventral telencephalon. Symbols of genes are indicated together with chromosomic position of the bindind peaks and associated Fold-change enrichement value compared to control and -log10(p-value). The list is sorted in decreasing order Fold-change enrichement. As expected, Notch genes are the main target of Ascl1 (Fbxw7, Dll3, Hes6. However, important epigenetic chromatin remodelers are aslo directly regulated by Ascl1 such as Chd7 or Smarcc1 (BRG1-Associated Factor 155). A list showing other directly regulated genes is available in the Annex 4.

(

  GSM1040159; GSM1040162), and from brain Hi-C data (Y Shen 2012). First, we compared the Ascl1 bound regions identified in OPCs with Ascl1 bound regions identified in ventral telencephalon. We found only 734 shared regions, while 2495/3252 regions were OPCs specific and 21213/22013 regions were ventral telencephalon specific. We show that the shared regions are involved in the same signalling pathways than in the ventral telencephalon but with some peculiarities. For example, genes involved in Notch signalling such as Notch1, Notch2, Dll1, Fbxw7 are bound in OPCs; however, downstream targets like Hes1, Hes5 or Hey1 are not bound anymore. Similarly some of the genes of the Wnt signalling are significantly bound by Ascl1 such as Gsk3b, Fzd1, Dvl1, Axin1, Fbxw11, Tcf7l2 (for other signalling pathway, seeFig 31).

Figure 30 .

 30 Figure 30. Ascl1 and Olig2 ChIP binding sites analyses. (A) Table showing repartition of the Ascl1 peaks on the genome. Peaks originate from pooling datasets from the ventral telencephalon Chip-seq and Chip-on-chip and Chip-seq in OPCs. 38.4% of peaks are located in promoters. However, Chip-on-chip experiment are designed specifically for promoters, and thus bias proportions. (B) Tableshowingrepartition of the OPCs Olig2 binding sites. Among the 21297 identified peaks in Olig2 Chip-seq in OPCs, 45% are intronic, 35% are intergenic and 14.8% of peaks are located in promoters. This reflects the enrichment of TFBS in promoters, enhancers and exon-intron junction where they play a role in the splicing regulation.

Figure 31 .

 31 Figure 31. (A). Colaborative binding of Ascl1 and Olig2 on the Plp1 gene. Schematic representing the Plp1 locus. Plp1 gene is transcribed from left to right and indicated by different black horizontal lines representing different transcript existing for the Plp1 gene. Ascl1(red) and Olig2 (blue) binding region are indicated in parallel with H3K4me1/2 and HK27ac in neural stem cells (absent) as well as H3K27ac in the oligodendrogial lineage (light blue). This cobinding event of Ascl1 and Olig2 on active regulatory sites of genes involved in myelination demonstrate overlapping functions. Importantly, Chip-seq experiment are based on cell population pooling and thus reflect average chromatin state. Different mode of co-binding are possible and can occur at the same time. Ascl1 and Olig2 likely synergize via the mechanism of ‗assisted loading' or ‗hit and run model' where two TFs that compete for the same TFBS does not inhibit the activity of each other but rather increases the occupancy of each TFs via increased nucleosome depletion rate (Voss, T. C. 2011). Increased H3K27 acetylation of the chromatin promoter region is visible by the enlargement of the region between the OPC and the myelinating OLS stage. Ascl1 and Olig2 bind at secondary promoter site localized at the second exon of the gene and likely regulate the alternative promoter usage. Hi-C data (purple) indicate a long range interaction between the TSS of Plp1 and the region located at the last exon.

  seq dataset from the whole oligodendrocyte lineage, (OPCs (21297regions), premyelinating OLs (25106regions),and myelinating OLs(2662 regions) , together with epigenetic marks from H3K27Ac, H3K4me1, and Brg1 (Yu et al 2013) as well as from Olig2-ChIPseq in SH-SY5Y (21523 regions) which is a neural proliferative cell line (Obtained from Ben Martinoga). This allowed us to map the genome with transcriptional and epigenetic information at different stages of the differentiation process and compare the overlapping and unique regions of botch Ascl1 and Olig2 in different contexts. This data integration is invaluable to generate hypothesis about transcriptional regulations, even in highly complex chromosomic regions such as the Olig2 locus or a simpler one such as THE Ascl1 locus (Fig32).

Figure 32 .Figure 33 .

 3233 Figure 32. Integration of ChIP datasets in Ascl1 and Olig2 locus: Schematic representation of the Ascl1 (A) and Olig2 (B) loci integrating ChIP data depictured as vertical bars. Red rectangles indicate regulatory elements bound by Ascl1 and Olig2 in neural cells. Ascl1 binding are shown in red, Olig2 in blue, Epigenetic marks in NSCs in light green, Epigenetic marks in oligodendroglial lineage in light blue. In purple are indicated long range interacting enhancers identified by Hi-C analysis (data references in Fig. 31)

Figure 35 :

 35 Figure 35: E-box subtype enrichment analysis in Ascl1 and Olig2 bound region (A) Table presenting E-box subtype analysis of all Ascl1 and Olig2 chipseq peaks containing E-boxes (containing at least one E-box. peaks without Eboxes are withdrawn). Over representation of E-boxes subtypes is given for Ascl1 specific binding regions (Ascl1 only), Olig2 specific regions (olig2 only) and Ascl1/Olig2 shared regions. Enrichment is given compared to E-box average frequency in promoters. Associated statistical test is indicated (Z-score >2 are equivalent to a corrected p-value of 0.5). Ascl1 is more likely to bind Ascl2 or

Figure 36 .Figure 37 .

 3637 Figure 36. Enrichment analysis of TFBS-module in Ascl1/H3K27Ac or Olig2/H3K27Ac bound regions. Table presenting most enriched Ascl1 (left) or Olig2 (right) associated modules of TFBS. Modules are defined as two TFBS located at less than 50bp to each other. As demonstrated by single TFBS analysis, Ascl2 matrix is more frequent than Ascl1 matrix. Interestingly, Ascl1 binds more frequently genomic region containing tandem repeat or clusters of E-boxes. Other binding sites enriched together with Ascl1/2 E-boxes binding site are indicated. Those TFs are likely to work in synergy or inhibit the action of Ascl1 on identified bound regions. We found Tcf15, Dlx1/2 or Gli to be significantly associated with Ascl1 TFBS. B.Table presenting most enriched Olig2 associated TFBS. Similarly, Olig2 binding region are screened for

Figure 38 .

 38 Figure 38. Identification of oligodendroglial enriched genes. (A) Clustering of expression data from different brain cell-type expression microarrays sorted for increasing expression in oligodendrocytes. Red indicates higher expression values and blue lower ones (data from (data from Cahoy et al 2009 and others cited in material and methods). (B-G) In situ hybridization of myelin oligodendrocyte glicoprotein (MOG) gene and selected candidates in sagittal sections of P56 mouse brains showing

Figure 38bis .

 38bis Figure 38bis. Identification of cis-regulatory oligodendroglial specific genes. (A) Table presenting Olig2 regulated genes (microarray) and bound by Olig2 (Chip-seq) in H3k27ac region(Chip-seq) whose expression is enriched genes in OPCs compare to other cell types. Expression data for NSCs, OPCs, PremyelinatingOL, myelinating OLs, neurons and astrocytes. One regulatory chromosomic position is indicated for each gene. (A) Table presenting Olig2 bound by Olig2 (Chip-seq) in H3k27ac region(Chip-seq) whose expression is enriched genes in myelinating OLs compared to other cell types. Expression data for NSCs, OPCs, PremyelinatingOL, myelinating OLs, neurons and astrocytes. One regulatory chromosomic position is indicated for each gene. This kind of cis-regulatory sequence is higly enriched in TFBS cluster

Figure 39 .

 39 Figure 39. Chd7 putative Ascl1-Olig2 target driving oligodendrogenesis. (A) Schematic representation from Genomatix genome browser of Chd7 locus integrating ChIP data depictured as rectangles/bars in each line. Ascl1 and Olig2 ChIP binding peaks are shown in red and blue respectively, Brg1 ChIP sites in pink and epigenetic marks in cyan. Red vertical rectangles indicate regulatory elements bound by Ascl1 and Olig2 in OPCs and immature oligodendrocytes, presenting also Brg1 binding sites and active epigenetic marks in oligodendroglial cells. (B) Sagittal section of E14.5 embryo (www.euroexpress.org) showing Chd7 expression pattern in the forebrain ventricular zone (VZ, arrows) by in situ hybridization; red square indicates the higher magnification area. (C) Brain sagittal section of P56 brain showing Chd7 expression in telencephalic SVZ (arrow), and corpus callosum and fimbria (some cells highlighted by arrowheads); red square indicates the higher magnification area (D) Histograms representing raw expression levels of Chd7 in different brain subtypes expression microarrays. Note the highest expression in OPCs, iOLs and microglia. (E) Histograms representing raw expression levels of different Chd genes in OPC expression microarrays. (F) Histograms representing raw expression levels of Chd7 in human MS plaques compared to controls processed from GEO GSE38010 microarrays. (Fc=0.42, BH p-value=0.05). (G) Histograms representing raw expression levels of Chd7 in FACSorted OPCs from corpus callosum tissue of cuprizone-treated demyelinated mice and controls (obtained from S. Moyon et al., C. Lubetzki J.Neurosci. in press. (H) Histograms representing raw expression levels of Chd7 in ventral telencephalon expression microarrays of wild-type and Ascl1 mutant mice. (I,J) Histograms representing qPCR obtain fold change gene expression levels in neurosphere cells transfected with Ascl1 (I) or Olig2 (J) overexpressing vectors after 48 hours. (K) See figure Schematic representation of the Olig2 locus integrating ChIP data depictured as vertical bars. Red rectangles indicate regulatory elements bound by Ascl1 and Olig2 and Chd7 in neural cells. Chd7 Chip binding peaks are

Figure 40 .

 40 Figure 40. Chd7 protein expression during in postnatal brain. Sagittal sections of P3 (A-C) and P24 (D-F) brains immunostained for Chd7 antibody. Arrows and arrowheads indicate some examples of labelled cells. (A) Coexpression of Chd7 and Ascl1 in SVZ and CC cells either expressing Olig2 (arrowheads) or not (most likely transit amplifying precursors, arrows). (B) Chd7 and Ascl1 co-expression in PDGFR+ (arrows), PDGFR-(transit amplifying cells, arrowheads). Note that Chd7 is also expressed in Ascl1-negative cells in SVZ, most likely neuroblasts. (C) Chd7 and Aslc1 co-expression with Mcm2, proliferation marker in SVZ cells. (D) Chd7 and Ascl1 co-expression in SVZ cells (arrows) or PDGFR+ OPCs in the CC (arrowheads). (E, F) Chd7-expressing cells in the CC (E) or in the fimbria (F) are either OPCs (PDGFR + cells, arrowheads) or maturing oligodendrocytes (APC + cells, arrows). Note that the highest levels of Chd7 protein expression are found in APC + cells. V, ventricle; Fi, fimbria; CC, corpus callosum, Stri, striatum. Scale bar, 20 m.

Figure 41 .

 41 Figure 41. Chd8 is expressed during myelination in differentiating OL. (A) Expression of Chd8 mRNA in P56 brain showing expression in some cells of the white matter (arrows) and many neurons in the grey matter. (B) Chd8 RNA expression levels in P7 rat brain neural subtypes showing enriched expression in immature and myelinating OL. (C) Chd8 immunoflurorescence in P24 brain sections showing expression with in all APC + cells but rarely in PDGFR+ OPCs. (D) Summary schematic showing Chd7 and Chd8 expression at different stages of the oligodendroglial lineage together with transcription factors involved in oligodendrogenesis.

Figure 39 .

 39 Figure 39. CHD8 is expressed oligodendroglial cells in MS brain. (A) Sagittal section of a periventricular brain tissue showing a demyelinated lesion by the absence of MBP expression. (B) CHD8 immunostaining in white matter region close to the demyelinating lesion showing CHD8 + cells presenting typical alignments of OLs. (C) Coexpression of Sox10 and CHD8 in some cells close to lesion areas indicating that they are oligodendroglial cells.

Figure 44 .

 44 Figure 44. Tns3 putative Ascl1-Olig2 target in oligodendrogenesis. (A) Schematic representation from Genomatix genome browser of Tns3 locus integrating ChIP data depictured as rectangles/bars in each line. Ascl1 and Olig2 ChIP binding peaks are shown in red and blue respectively, Brg1 ChIP sites in pink and epigenetic marks in cyan. Red vertical rectangles indicate regulatory elements bound by Ascl1 and Olig2 in OPCs and immature oligodendrocytes, presenting also Brg1 binding sites and active epigenetic marks in oligodendroglial cells. (B) Sagittal section of E14.5 embryo showing Tns3 expression pattern in the forebrain ventricular zone (VZ, arrows) by in situ hybridization; red square indicates the higher magnification area. (C) Brain sagittal section of P56 brain showing Chd7 expression in corpus callosum (CC), cortex (Ctx) and fimbria (Fi) (some cells highlighted by arrowheads). (D) Histograms representing raw expression levels of Tns3 in different brain subtypes expression microarrays. Note the higher expression in differentiating oligodendrocytes (OLs). (E) Histograms representing raw expression levels of different Chd genes in OPC expression microarrays. (F) Histograms representing raw expression levels of Tns3 in ventral telencephalon expression microarrays of wild-type and Ascl1 mutant mice.

  outside the brain (data not shown). In line with the nuclear localization of Tns3, a role of nuclear actin in transcriptional regulation has been clearly demonstrated in the last decade (Miyamoto and Gurdon. Cell Mol Sci. 2013), suggesting that Tns3 nuclear expression in maturing oligodendrocytes could also be involved in the transcriptional regulation of myelination.

Figure 45 :

 45 Figure 45: Tns3 protein expression during brain myelination. Sagittal sections of P12 (A,B), P21 (C,D) brains immunostained for Tns3 antibody. Arrows and arrowheads indicate some examples of labelled cells. (A,B) Expression of Tns3 in APC + oligodendrocytes but not in PDGFR+ OPCs in the CC (A) or the brainstem (B) cells either expressing Olig2 (arrowheads) or not (most likely transit amplifying precursors, arrows). (C,D) gal expression in the Tns3-LacZ mice showing expression with Olig2 + /PDGFR -cells (oligodendrocytes) but not in Olig2 + /PDGFR + cells (OPCs) and Ascl1 co-expression in PDGFR+ (arrows), (transit amplifying cells, arrowheads). Note that Chd7 supporting specificity anti-Tns3 antibody. (C) Chd7 and Aslc1 co-expression with Mcm2, proliferation marker in SVZ cells. (D) Chd7 and Ascl1 co-expression in SVZ cells (arrows) or PDGFR+ OPCs in the CC (arrowheads). HP, hippocampus; CC, corpus callosum, GM, grey matter, WM, white matter. Scale bar, 20 m.

  2 ) specifically in maturing oligodendrocytes (cells expressing high levels of APC and Olig1 in the cytoplasm) suggesting that Tns3 could be involved in the mechanisms leading to remyelination.

Figure 46 .

 46 Figure 46. Tns3 expression in newly generated remyelinating oligodendrocytes. (A-A 2 ) Tns3-expressing cells in the CC of adult (P90) mice, 4 days after demyelination

Figure 47 .

 47 Figure 47. Creatine producing enzymes mRNA expression in the brain. (A-E).Cell-type specific microarray expression study of Gamt (A), Gatm (B), Slc68a (C),

Figure 48 .

 48 Figure 48. Toxicogenomic selection of drug affecting oligodendrocyte genes. A. Table showing the drug identified in this study as modifying oligodendrocyte gene expression and already testes in the context of demyelinating diseases. B. Table

  Rosa26 stop-YFP (Srinivas et al., 2001), Rosa26 stop-LacZ 1999), and PDGFR ::CreER T2 (Rivers et al., 2008). For simplicity, we renamed the different mice as follows: Ascl1 GFP (for Ascl1 Cre/YFP (for Rosa26 stop-YFP ), Ascl1 Cre/LacZ (for gical pain. Focal demyelinating lesions were induced by stereotaxic 167 injection of 1.5 l solution (LPC, 1% in 0.9% NaCl) into the corpus callosum (CC; at coordinates: 1 mm lateral, 1.3 rostral to bregma, 1.7 mm deep to brain surface) using a glass-capillary connected to a 10 l Hamilton were to recover in a warm chamber before being returned into their housing cages.

  and then with primary antibodies overnight at 4°C. The following primary antibodies were used: mouse monoclonal bodies to (Calbiochem, CNP (Millipore, 1:500), MOG (8-18C5, 1:5, Dr. C. Linington, Max-Planck Rosa26 stop-LacZ ), (for conditional knock-out in OPCs: Cre-electroporated Ascl1 fl/ ; Rosa26 stop-YFP ), and (for dorsal Cre-electroporated Ascl1 fl/ ; Rosa26 stop-YFP ). Both males and females were included in the study. All experiments were carried out in accordance to Inserm ethical committees (authorization #A75-13-19) personal animal experimentation Demyelinating mouse lesions. Before surgery, adult -6 Demyelinating Ascl1 GFP , Demyelinating surgery, surgery, or Cko/Ctr OPC mice were weighted and anesthetized by intraperitoneal injection of mixture of ketamine (0.1 mg/g) and (0.01 mg/g). An phine, 30 mg/g) was administered intraperitoneally to prevent tissue Institute, Tuebingen, Germany), Olig2 (Ligon et al., 2006), Nkx2.2 (clone 74.5A5, Developmental Studies Hybridoma Bank, 1

  Human brain tissues were immunostained as previously described (Nait-Oumesmar et al., 2007) with mouse monoclonal antibodies human fied for expression of TFs by manual or automated counting using ageJ software. To quantify Ascl1 staining intensity at different stages studied (P2, P14, adult control, and adult 5 d after lesion), confocal pictures of brain sections, immunolabeled for and PDGFR from images (n 3), such as those shown in Figure 2. Data are presented as a line chart in Figure Values range from 0 to 255, with values 50 considered as background levels. All values are shown as mean performed using t test Prism 4, GraphPad p values in graphic are represented as follows: *p 0.05, **p 0.01, ***p 0.001. Black bars in histograms correspond to SEM.

1 .

 1 tion in the CC, Ascl1 is coexpressed with Sox9, Sox10, Olig2, Olig1, and Nkx2.2 in PDGFR (OPCs; labeled #1 in Fig. ). According to the expression of these TFs markers, could divide premyelinating oligodendrocytes in two ): (1) premyelinating oligodendrocyte stage I (mOLI) characterized by downregulation of Olig1, and PDGFR and upregulation of Nkx2.2/Gpr17/ in ); and (2) ing oligodendrocyte stage II (pre-mOLII), implicating downregulation of Nkx2.2/Gpr17 and upregulation of Olig1cytoplasmic expression while presenting the highest levels of (labeled #3 in ). Ascl1 GFP transgene at premyelinating characterized by coexpression of high of Nkx2.2/Olig2 or dicating that OPCs do into ing oligodendrocytes (Fig. ). APC to express myelin proteins, such as MOG, present a larger cytoplasm and intermediate levels of expression with persistent Olig1 cytoplasmic expression (labeled #4 in Fig. OPC generation in the forebrain and spinal cord (Parras et al., 2007; Sugimori et al., its during neonatal and myelination are currently unknown. Postnatal OPCs derive from cortical wall of lateral ventricles both in neonates (Gorski et al.expression in newborn mice (P2) by immunofluorescence. Ascl1 was tected in subsets of located in the cortical ventricle in the CC ( 15% and 25% of total The notype characterized ies against TFs expressed by oligodendroglial progenitors precursors, including and et al., Sun et al., Zhou et al., Zhou and son, Stolt et al., Arnett et al., Kitada Rowitch, 2006; Liu et al., 2007; Cai et al., 2010). In the dorsal Ascl1 G) ation is marked by a transient upregulation of Nkx2.2/Gpr17 correlating with a downregulation of (Fig. whereas further steps of transition toward a myelin-forming cell correlate with Nkx2.2/Gpr17 downregulation together with expression of in SVZ-progenitors and in OPCs during the postnatal wave of oligodendrogenesis described above, we first investigated its role in cortical SVZ and neonatal oligodendrogenesis. For this purpose, we deleted Ascl1 in dorsal SVZ cells in a temporally controlled by targeting a Cre-expressing plasmid to the dorsal SVZ at P2 sence of could result from a change in OPC mode of division, we quantified the different types of YFP cell pairs in CC at 7 dpi. Ctr OPC (tamoxifen at P5) showed 23.1 4.0% of OPC/OPC pairs, 29.0 of OPC/OL pairs, and 47.8 3.7% of pairs

  3 4.7%; p 0.0082, t test, n 4; Fig. D 1 ).

  , 5 d postlesion (dpl), high to ate of and Ascl1 GFP high /Ascl1 GFP cells) were detected both in the and within and around the which was characterized by a very dense cellularity (Fig. 5E-E 2 ). of Cko OPC and Ctr OPC genotypes using PDGFR mice (Rivers et al., 2008; see Materials and Methods). We deletion at postnatal when OPC numbers peak in the brain (tamoxifen injection at P5 or P8-P9) and assessed their and differentiation OPCs used marker of tamoxifen injection (dpi) Cko OPC YFP OPCs showed a 1.7-fold increase in the proportion of Ki67 cells (36.4 5.2%, Fig. 4A ,D) compared with Ctr OPC (21.3 D), ing an increase of OPC proliferation in the absence of Ascl1. Similar results were obtained when tamoxifen injected at P5 or P8-P9 4D). Therefore, OPC proliferation in absence of Ascl1. Neonatal OPCs have been shown to divide either generating pairs of OPCs (OPC/OPC pairs) or oligodendrocytes (APC named OL/OL pairs), or asymmetrically to generate one OPC and one oligodendrocyte (OPC/OL pairs) (Sugiarto et Zhu et To assess whether the difference in proliferation rate observed in the

  F 1

  -autonomous role both in OPC specification and in fine-tuning of OPC proliferation versus differentiation (Fig.demonstrate that expressed in neonatal cortical tors and that its conditional strongly suppress OPC generation, demonstrating that Ascl1 is required postnatal oligodendrocyte specification.in OPCs most brain regions and its ablation in OPCs favors proliferative over OPC leading to a reduced number of cytes. Third, Ascl1 is not expressed differentiating oligodendrocytes, but lineagetracing studies show that Ascl1-expressing differentiate into myelinating and myelinating oligodendrocytes. cells (OPCs and ture progenitors) are found in and active lesions both in mouse and MS brain, Ascl1 being required for OPC differentiation during in mice and most implicated in man 10.

  and other cofactors in proliferation and differentiation, could balance Ascl1 activation of proliferation versus cell exit. Supporting hypothesis, Ascl1 genome-wide chromatin tion data (ChIP-seq) in purified OPCs suggest that regulatory regions of both positive and factors Parras unpublished observations). deletion in OPCs also differentiation during 180 References Aguirre A, Dupree JL, Mangin JM, Gallo V (2007) A functional role in myelination and remyelination. Nat Neurosci -Medline Arnett HA, Fancy SP, Alberta JA, Zhao C, Plant SR, Kaing S, Raine CS, Rowitch DH, Franklin RJ, CD (2004) bHLH transcription factor is required to repair demyelinated in the 306: Medline Bertrand N, Castro DS, Guillemot F (2002) Proneural genes and the specification of neural cell types. Nat Rev Neurosci 3:517-530. CrossRef Medline vivo cell maturation and participation in acute and chronic damage. 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Fig. 1 .

 1 Fig. 1. Defective prethalamus development in the Olig2-KO mouse. (A,B) Whole-mount in situ hybridization of E10.5 forebrain with Dlx2. Arrows indicate prethalamus. The prethalamus of the Olig2-KO mouse shows hypoplasia. (C-J) Double staining with Dlx2 in situ hybridization (purple) and Islet1/2 immunohistochemistry (brown). (C,E,G,I) E11.5 wild-type mouse. (D,F,H,J) E11.5 Olig2-KO mouse. The Dlx2+ and Islet1/2+ area is much smaller in Olig2-KO mouse than in normal control animals. Cb, cerebellum; DTh, dorsal thalamus; GE, ganglionic eminence; Pth, prethalamus; TE, thalamic eminence. (K) Quantitative analysis of E11.5 Dlx2+ prethalamus region, showing 60% reduction of the area in the Olig2-KO mouse. Data are mean±s.e.m. (wild type, n=3; Olig2-KO, n=3; Student's t-test). Scale bars: 1 mm in B; 500 µm in H; 100 µm in J.

Fig. 2 .

 2 Fig. 2. Altered formation of the prethalamus and TE in Olig2-KO mice. (A,B) Comparison of formation of the prethalamus and thalamic eminence in normal control and Olig2-KO mouse at E13.5. Both are composite pictures, in which left and right halves are adjacent sections immunostained with Islet1/2 (green in left half ), Olig2 or Cre (red in left half ), and Tbr2 (red in right half ). The prethalamus is shown by Islet1/2 and the TE by Tbr2. The VZ of the prethalamus is indicated by arrowheads and that of the TE by arrows. The mutant embryo brain was taken at a slightly caudal level compared with the wild-type embryo brain. Scale bar: 500 µm in B. (C) Relative position of borders of the dorsal thalamus (yellow), prethalamus (red) and TE (blue) in coronal sections of the diencephalon. Total height of the diencephalon in the coronal section is regarded as 100% height, and relative position is expressed as percentage from the bottom (mean±s.d.; wild type, n=3; Olig2-KO, n=3; Student's t-test).

Fig. 3 .

 3 Fig. 3. Fate change of Olig2 lineage cells in the absence of Olig2. (A,B) E13.5 Olig2-hetero;Rosa26 GFP and Olig2-KO;Rosa26 GFP mouse diencephalon, stained with calretinin (red) and GFP (green). Boxed areas in A and B are magnified in C and D, respectively. (C,D) Higher magnification pictures. In the heterozygous mouse, GFP+ and calretinin+ cells are mutually exclusive (C), whereas overlapping distribution is apparent in Olig2-KO mouse (D). Inset shows representative double-labeled cells (arrows). Scale bars: 500 µm in B; 200 µm in D; 20 µm in inset.

Fig. 4 .

 4 Fig. 4. Disorganization of TCAs in the Olig2-KO dorsal thalamus. (A-D) E13.5 diencephalon immunostained with anti-NF-M (red) and anti-netrin G1 (green) antibodies. NF-M+ axons in the dorsal thalamus (DTh) are also netrin-G1+, indicating that they are TCAs. Wild-type TCAs extend in parallel and converge ventrally in the prethalamus (A,B; arrowheads in B). TCAs in the Olig2-KO mouse show disorganized extension and sometimes form axon aggregates (C,D; arrowheads in D). (E,F) Netrin G1+ TCAs in the E17.5 DTh and prethalamus (thalamic reticular nucleus, TRN). In the wild type, netrin G1+ axons are extended in parallel in the DTh and TRN (E), whereas those in the Olig2-KO mouse form abnormal thick axon bundles orienting randomly (arrowheads in F). (G) Summary of the observed defects in Olig2-KO diencephalon. Scale bars: 200 μm in C; 100 μm in D; 500 μm in F.

Fig. 5 .

 5 Fig. 5. Arrest of TCA extension at the initial stage of projection formation. (A,B) E13.5 forebrain with DiI crystal injection into the dorsal thalamus. Broken lines indicate the telencephalon-diencephalon boundary (TDB). Labeled TCAs had reached the lateral ganglionic eminence of the ventral telencephalon in the wild-type animal (arrow in A) whereas those in the Olig2-KO mouse had just crossed the TDB in the ventral telencephalon (arrow in B). DTh, dorsal thalamus; GE, ganglionic eminence; Pth, prethalamu. Insets show schematics of diencephalon regionalization and TCA extension. Scale bar: 500 µm. (C) Mean distance between the distal tip of DiI-labeled fibers and the TDB. Data are mean±s.e.m. (wild type, n=3; Olig2-KO, n=3; Student's t-test)

Fig. 6 .

 6 Fig. 6. Axonal tracing of reciprocal connection between the thalamus and cortex at E18.5. (A,B) Low-magnification pictures. Insets show injection sites of DiI (i, intermediate) and DiA (r, rostral; c, caudal). (C,D) Higher magnification pictures of the dorsal thalamus. c, i, r of the thalamus represent connections from c, i, r, respectively, of the cortical injection site. Arrows in C and D indicate positions of the external medullary laminae. Arrowheads in D indicate abnormal overlapping course of axons showing yellow color. (E,F) Laser scanning confocal micrographs. Arrows indicate retrogradely labeled cell bodies. Axons in the wild type are arranged in parallel (E), whereas those in Olig2-KO cross each other (F). Scale bars: 1 mm in B and inset; 200 µm in D; 100 µm in F.

Fig. 7 .

 7 Fig. 7. Altered expression of Epha3 and Epha5 in the E13.5 Olig2-KO mouse diencephalon. (A,B) Epha3 in situ hybridization. (C,D) Epha5 in situ hybridization. Note that an Ephas-negative area is observed in the control prethalamus, being intercalated between the dorsal thalamus and the ventral telencephalon (asterisks in A and C). By contrast, the Ephas-negative area is very narrow in the Olig2-KO diencephalon (asterisks in B and D). (E,F) EphrinA5 (Efna5) in situ hybridization. EphrinA5 is expressed in both the wild-type and Olig2-KO dorsal thalamus (arrows). Scale bars: 1 mm.

Fig. 8 .

 8 Fig. 8. Suppression of neurite extension from cultured thalamic neurons by EphA3. (A,B) Dissociated thalamus progenitor cells were cultured on substrate double-coated with poly-L-lysine and with human Fc (A) or mouse EphA3-human Fc (B) chimeric protein. Cultured cells were stained with class-III β tubulin immunohistochemistry (red). (C) Neurite extension was apparently impaired on substrate coated with EphA3. n=3 independent cultures. Scale bar: 100 µm in B.

  and for Olig2 (Yu et al 2013) and unraveled some of the mechanisms of action of Ascl1 and Olig2 at a genome-wide level. While Castro et al, focused on the role of Ascl1 in the ventral telencephalon using promoter-specific Chip-on-chip, Borromero et al, focused on the role of Ascl1 in the spinal cord using genome-wide Chip-sequencing analysis. Wapinsky et al demonstrated the preferential binding of Ascl1 on H3k27Ac/H3K4me1 active enhancers and characterized its role of pioneer TFs. Yu et al characterized the whole oligodendrocyte lineage and provide a great insight in the epigenetic of differentiating oligodendrocytes. Another great progress was made by the characterization of oscillatory expression of Ascl1 and Olig2 in proliferating neural stem cells

  situ hybridization databases) to define the necessary stringency. Inspired by the work of Cahoy et al 2009 and Emery et al 2009, we took advantage of their published microarrays done in the whole oligodendrocyte lineage as well as post-mitotic neurons and astrocytes.

Annex 2 :

 2 Sagital view of in-situ hybridization of Chd7 in E14.5 mice (eurexpress). early appearance of OPCs is visible in the rhombencephalon. Expression of Chd7 is mainly in the SVZ and VZ. Annex Annex 3 : Table showing the gene the most significantly upregulated in Olig2 knock-out compared to wild type. Fold change are indicated Fc and associated p-value. Annex 4: Table showing the gene the most significantly downregulated in Olig2 knock-out compared to wild type. Fold change are indicated Fc and associated p-value. Annex 5: Table showing the gene the most significantly downregulated in Ascl1 knock-out compared to wild type. Fold change are indicated Fc and associated p-value. Annex : Table of Ascl1/Olig2 down regulated genes in ventral telencephalon ABSTRACT Our project aims to provide a new molecular understanding of the transcription program involved in neural stem cells differentiation into oligodendrocytes. The rational of this work relies on previous studies demonstrating that the bHLH transcription factors Olig2 and Ascl1 work in synergy to specify OPCs, the oligodendrocyte progenitor cells. One central goal of this work was to understand at a genomic and transcriptomic level, how Ascl1 and

  projet vise à fournir une nouvelle compréhension moléculaire du programme de transcription impliqué dans la différenciation des cellules souches neurales en oligodendrocytes myélinisant. La logique de ce travail repose sur des études antérieures ayant montré le rôle des facteurs de transcription bHLH Olig2 et Ascl1, opérant en synergie dans la spécification des OPCs, les cellules progénitrices d'oligodendrocytes . L'objectif central de ce travail était de comprendre au niveau génomique et transcriptomique les mécanismes par lesquels Ascl1 et Olig2 agissent pour spécifier les OPCs. Nous avons suivi une stratégie utilisant l'analyse du transcriptome et des profils de fixation des facteurs de transcription par immuno-précipitation de la chromatine . Nous avons pu identifier les cibles directes de Ascl1 et Olig2 dans les OPC et lors de la différenciation des oligodendrocytes. Nous avons également identifié de nouveaux marqueurs spécifiques des différents stades des lignées oligodendrocyte et nous sommes concentrés sur Chd7 et Tns3, deux gènes régulé par Ascl1 et Olig2 et enrichis dans la lignée oligodendrogliale à deux stades intéressants, la phase de spécification précoce et la transition entre la migration et la différenciation des oligodendrocytes, respectivement. De plus, nous avons porté notre attention sur le rôle spécifique des oligodendrocyte dans la synthèse de la créatine et son rôle possible de support métabolique dans la synthèse de myéline et de support axonal. Nous avons également initié une approche de repositionnement toxicogénomique pour identifier de nouvelles molécules à

  ). This observation Ascl1 progenitors and OPCs generated in the genic zone can migrate away from the toward CC lesions, recruited with delivered from the injured tissue.

	is	for	timing of	differentiation
	during remyelination
	To determine Ascl1 requirement for proper remyelination, we
	deleted		in OPCs before inducing CC
	myelin	We	whether YFP -Cko OPC	able
	to migrate into lesions, proliferate, and differentiate in the
	sence of	At 8 and 15 dpl, remyelinating lesions in
	Cko OPC and Ctr OPC	YFP	being PDGFR OPCs
	and APC		A	B ). This result
	cates that	is dispensable for OPC recruitment and subse-
	quent oligodendrocyte maturation in demyelinated	At 8
	dpi, the proportion of YFP OPCs and YFP oligodendrocytes
	similar in Cko OPC and Ctr OPC .	a	large
	number of YFP -Cko OPC

  Moreover, we found fewer YFP cells expressing CNP myelin protein in Cko OPC than in Ctr OPC

					D	these
	data strongly	that	is also required for normal OPC
	differentiation during remyelination and, as during myelination,
	other	most	compensate its	of function at later
	points of the differentiation process.
	is	in
	In the mouse brain,		expression is a hallmark of OPCs
	precursor		in remyelination. We hypothesized
	in humans, immature oligodendroglial cells in MS lesions may
	also express
					174

Table 1 .

 1 Microarray expression analysis of control versus Olig2-KO brains at E13.5

		Gene			FcOlig2/wild
	Gene name	symbol	RefSeq transcript ID	Probe set ID	type	P value
	Ephrin A1	Efna1	NM_001162425	1416895_at	1.37	0.01
			/NM_010107
	Ephrin B1	Efnb1	NM_010110	1418286_a_at	1.46	0.00
	Ephrin B2	Efnb2	NM_010111	1449549_at	1.75	0.02
	Ephrin B2	Efnb2		1419639_at	1.40	0.03
	Eph receptor A3	Epha3	NM_010140	1426057_a_at	1.95	0.00
	Eph receptor A3	Epha3		1425574_at	1.59	0.00
	Eph receptor A3	Epha3		1455426_at	1.35	0.00
	Eph receptor A3	Epha3		1425575_at	1.29	0.01
	Eph receptor A5	Epha5	NM_007937	1420557_at	1.67	0.01
	Eph receptor A5	Epha5		1435286_at	1.12	0.04
	Semaphorin 3E	Sema3e	NM_011348	1442226_at	1.56	0.00
	Semaphorin 4B	Sema4b	NM_013659	1455678_at	1.41	0.03
	Semaphorin 5A	Sema5a	NM_009154	1437422_at	0.61	0.01
	UNC-5 homolog C	Unc5c	NM_009472	1449522_at	0.79	0.01
	Significant changes were observed in genes encoding these axon guidance molecules.

normal E6 chick spinal cord was observed in the ventral VZ and ventral horn (VH). In the VZ, Epha3 expression was partially overlapped with Olig2, whereas motoneurons in the ventral horn, which are of Olig2 lineage, do not express Olig2. Olig2 was transfected to the E3 chick neural tube and E6 spinal cord was examined with GFP, Olig2, Islet1/2 and HB9 immunohistochemistry,

170 that

4.

5.

7.

Remerciements

Acknowledgements Some monoclonal antibodies were obtained from DSHB. We thank Dr Martyn Goulding (The Salk Institute) for providing the reporter mouse, and Drs Hideaki Tanaka (Kumamoto University), Hideyo Ohuchi (Okayama University), Masahiko Hibi (Nagoya University), Jun-ichi Miyazaki (Osaka University) and Kazunori Nakajima (Keio University) for cDNAs and helpful comments.

Dr Boris Zalc de m'avoir accueilli au

Funding This work was supported by Grants-in-Aid for Scientific Research (C) and an Innovated Area (Neural Diversity and Neocortical Organization) provided by JSPS and MEXT, Japan (to K.O.); by the Cooperative Study Program of NIPS (to K.O., H.T., H.G. and K.I.); by Kumamoto University (K.O., H.T., H.G. and K.S.); by FIRST program (to S.I.); by ARSEP scholarships (A.C.); and by Institut National de la Sante et de la Recherche Medicale and the program ‗Investissements d'Avenir' [ANR-1-IAIHU-06] (C.M.P).

CHD7/8 protein expression during remyelination

We therefore, investigated if Chd7 and Chd8 could play a role during the process of remyelination. Using the LPC-induced focal demyelination model in the mouse corpus callosum, our preliminary analysis has found that soon after the demyelinating insult [START_REF] Abdel-Mannan | Evolution of cortical neurogenesis[END_REF][START_REF]Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork[END_REF] or 8 days post-injection, dpi), Chd7 (not shown) and Chd8 are highly expressed in and around the LPC demyelinating lesion (compare Fig. 42A and A') not only in the OLs close to the lesion region but also in the OPCs in/around the lesion (Fig. 42B,B 1 ). This upregulation in OPCs due to demyelination strongly suggests that Chd8 is mostlikely involved in promoting the remyelinating process.

ARTICLE I: Ascl1/Mash1 Promotes Brain Oligodendrogenesis during Myelination and Remyelination during Remyelination

Maire, 1,2,3 Weber, 4 Nait-Oumesmar, 1,2,3,6 Parras 1,2,3

Competing interests

The authors declare no competing financial interests.

Author contributions

The project was designed by K.O., H.

Bioconductor Packages to install from www.bioconductor.org or R-cran repository:

Affy; gcrma; oligo; frma; rgl ; mass; Ade4;limma;kerfdr

Load packages: library(affy) library(gcrma) library(oligo) library(frma) library(ade4) library(rgl) library(MASS) library(limma) library(kerfdr)

Fonctions to source in R-CRAN:

pxt.h1Proportion = function(pv, method = "storey", lambda = 0.5){ method = match.arg(method) pi1 = 1 -mean(pv > lambda, na.rm = TRUE)/(1 -lambda) if (pi1 < 0) { warning(paste("estimated pi1 =", round(pi1, digit = 4), "set to 0.0")) pi1 = 0 } if (pi1 > 1) { warning(paste("estimated pi1 =", round(pi1, digit = 4), "set to 1.0")) pi1 = 1 } return(pi1) } pxt.rainbow = function(n, default2Col = c("darkgrey","orange"), default6Col = c("red", "green", "blue", "orange", "purple", "pink" ), mode = c("rainbow", "gray", "grey")){ mode = match.arg(mode) if (mode == "grey") mode = "gray" if (n == 1) return

return(gray (seq (0.8, 0.2, length = n))) } pxt.plotGroup3d = function(xyz, group = NULL, group.display = TRUE, group.font = 4, group.family = c("serif", "sans", "mono", "symbol"), group.cex = 1.5, group.xyz = NULL, labels = NULL, type = c("s", "p", "l", "h"), alpha = 0.6, box = FALSE, size = 0.4, col = NULL, fcol = pxt.rainbow, star = FALSE, ellipse = TRUE, ellipse.alpha = 0.1,ellipse.back = c("fill", "line"), labels.col = "black", labels.cex = 0.6, labels.adj = c(0,-1), labels.family = c("serif", "sans", "mono", "symbol"),labels.font = 2, . text3d(xyz, text=labels,adj = labels.adj, color = labels.col, family = labels.family, cex = labels.cex, font = labels.font) } pxt.plotGroup2d = function(xy, group = NULL, group.display = FALSE, group.font = 4, group.family = c("serif", "sans", "mono", "symbol"), group.cex = 1.5, group.xy = NULL, pch = 16, labels = NULL, col = NULL, fcol = pxt.rainbow, star = FALSE, star.lty = "dotted", ellipse = TRUE, ellipse.axes = TRUE, labels.cex = 0.8, labels.adj = c(0,-1), labels.family = c("serif", "sans", "mono", "symbol"),labels.font = 2, legend = TRUE, legend.location = "bottomright", legend.title = NULL, legend.cex = 1, .

Read and load Affymetrix MOE430 2.0 arrays CEL files:

Normalize affymetrix MOE430 2.0 arrays data: eset <-gcrma(Mydata) (choose rma, gcrma or frma)

write.exprs(eset, file="file_name.txt", sep=",")

Load data for statistical analysis: exp.norm = read.table("name_file.txt", sep = "\t", h = TRUE, stringsAsFactors = FALSE) rownames(exp.norm) = exp.norm[, 1] exp.norm = exp.norm [, 2:ncol

Expression data distribution : pdf("distributionExpression.pdf") hist(as.numeric(as.matrix(exp.norm)), main = "distribution expression") dev.off()

Probeset selection for Principal component analysis: d90 <-apply(exp.norm,1,function(x)quantile(x,probs=.9)) cv = apply(exp.norm, MARGIN = 1, FUN = pxt.cv) pbsok = rownames(exp.norm) [which(d90>log(10,2)

Principle component analysis: grp = rep(c("KO", "WT"), each = 5) pca = dudi.pca(t(exp.norm[pbsok, ]), nf = 3, scannf = FALSE) pxt.plotGroup3d(pca$li, group = grp) pdf("pca.pdf") pxt.plotGroup2d(pca$li[, 1:2], , group = grp, labels = names(exp.norm)) dev.off()

pxt.h1Proportion(pv) pdf("distributionPv.pdf") hist(pv, main = "percent of differentialy expressed genes") dev.off() (pbsok, pv, bonf, FDR_bh, kerfdr) write.table(resultats, file = "resultats_pvalues.txt", sep = "\t", quote = FALSE, row.names = FALSE) -model.matrix(~ -1+factor(c(1,1,1,2,2,2,3,3,3))) colnames(design) <-c("group1", "group2", "group3") fit <-lmFit(exp.norm, design) contrast.matrix <-makeContrasts(group2-group1, group3-group2, group3-group1, levels=design) fit2 <-contrasts.fit(fit, contrast.matrix) fit2 <-eBayes(fit2) write.table(topTable(fit2, coef=1, adjust="fdr", sort.by="B", number=50000), file="name_comparison1.xls", row.names=F, sep="\t") write.table(topTable(fit2, coef=2, adjust="fdr", sort.by="B", number=50000), file=" name_comparison2.xls", row.names=F, sep="\t") Increase coefficient for more comparisons: write.table(topTable(fit2, coef=3…, adjust="fdr", sort.by="B", number=50000), file=" name_comparison3….xls", row.names=F, sep="\t")

Affymetrix mogene_1_0_st_v1 analysis: library(oligo) geneCELs <-list.celfiles("C:/Users/Desktop/geneExperiment", full.names = TRUE) affyGeneFS <-read.celfiles(geneCELs) genePS <-rma(affyGeneFS, target = "probeset") theIDs <-getBM(attributes = "affy_mogene_1_0_st_v1", filters = "entrezgene", mart = ensembl) geneCore <-rma(affyGeneFS, target = "core") featureData(genePS) <-getNetAffx(genePS, "probeset") featureData(geneCore) <-getNetAffx(geneCore, "transcript") featureData(exonCore) varLabels(featureData(geneCore)) write.exprs(genePS, file="Myproject_oligo_rma.xls", sep=",")