Ana Martin-Villalba

Nadine Mestre-Frances

Keywords: Cellules souches neurales, Progéniteurs, Zone sous-ventriculaire, Séquençage Neural stem cells, Progenitors, Subventricular zone, Single-cell RNA sequencing, Cortical lesion, Transcriptional profiling

Here, I would like to share my thanks to all the people who have participated one way or another to this PhD work. Basic Research is so psychologically demanding because of Up and Downs that team work, supports, encouraging

Résumé

Objectif 2 : J'ai mis au point un modèle de lésion corticale qui permet l'ablation ciblée de neurones de couches corticales définies pour étudier la capacité régénérative et la spécification appropriée des progéniteurs corticaux postnataux. Une analyse quantitative des régions adjacentes, incluant la partie dorsale de la SVZ, a révélé une réponse transitoire de populations de progéniteurs définis.

Objectif 3 : Nous avons développé une lignée de souris transgénique nommée Neurog2 CreERT2Ai14 , qui permet le marquage de façon conditionnelle de cohortes de progéniteurs glutamatergiques et de leurs descendants. Nous avons utilisé des approches de cartographie et montré qu'une large fraction de ces progéniteurs persiste dans le cerveau antérieur postnatal après la fermeture de la période de neurogénèse corticale. Ils ne s'accumulent pas pendant le développement embryonnaire mais sont produits par des RGs qui persistent après la naissance dans la SVZ et qui continuent de générer des neurones corticaux, bien que l'efficacité soit faible. Le séquençage d'ARN sur cellule unique a révélé une dérégulation transcriptionnelle qui corrèle avec le déclin progressif observé in vivo de la neurogénèse corticale.

Ensemble, ces résultats soulignent le potentiel des études transcriptomiques à résoudre mais aussi à soulever des questions fondamentales comme les changements trancriptionnels intervenant dans une population de progéniteurs au cours du temps et participant aux changements de leur destinée. Cette connaissance sera la clé du développement d'approches novatrices pour recruter et promouvoir la génération de types cellulaires spécifiques, incluant les sous-types neuronaux dans un contexte pathologique.

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Abstract

During development, a remarkable coordination of molecular and cellular events leads to the generation of the cortex, which orchestrates most sensorimotor and cognitive functions. Cortex development occurs in a stepwise manner: radial glia cells (RGs) -the neural stem cells (NSCs) of the developing brain -and progenitor cells from the ventricular zone (VZ) and the subventricular zone (SVZ) sequentially give rise to distinct waves of nascent neurons that form cortical layers in an inside-out manner. Around birth, RGs switch fate to produce glial cells. A fraction of neurogenic RGs that lose their radial morphology however persists throughout postnatal life in the subventricular zone that lines the lateral ventricles. These NSCs give rise to different subtypes of olfactory bulb interneurons and glial cells, according to their spatial origin and location within the postnatal SVZ.

These observations raise important unresolved questions on 1) the transcriptional coding of postnatal SVZ regionalization, 2) the potential of postnatal NSCs for cellular regeneration and forebrain repair, and 3) the lineage relationship and transcriptional specificities of postnatal NSCs and of their progenies.

My PhD work built upon a previously published comparative transcriptional study of defined microdomains of the postnatal SVZ. This study highlighted a high degree of transcriptional heterogeneity within NSCs and progenitors and revealed transcriptional regulators as major hallmarks sustaining postnatal SVZ regionalization. I developed bioinformatics approaches to explore these datasets further and relate expression of defined transcription factors (TFs) to the regional generation of distinct neural lineages. I then developed a model of targeted ablation that can be used to investigate the regenerative potential of postnatal progenitors in various contexts. Finally, I participated to the development of a pipeline for exploring and comparing select populations of pre-and postnatal progenitors at the single cell level.

Objective 1: Transcriptomic as well as fate mapping were used to investigate the relationship between regional expression of TFs by NSCs and their acquisition of distinct neural lineage fates. Our results supported an early priming of NSCs to produce defined cell types depending of their spatial location in the SVZ and identified HOPX as a marker of a subpopulation biased to generate astrocytes.

Objective 2: I established a cortical lesion model, which allowed the targeted ablation of neurons of defined cortical layers to investigate the regenerative capacity and appropriate VI specification of postnatal cortical progenitors. Quantitative assessment of surrounding brain regions, including the dorsal SVZ, revealed a transient response of defined progenitor populations.

Objective 3: We developed a transgenic mouse line, i.e. Neurog2 CreERT2Ai14 , which allowed the conditional labeling of birth-dated cohorts of glutamatergic progenitors and their progeny. We used fate-mapping approaches to show that a large fraction of Glu progenitors persist in the postnatal forebrain after closure of the cortical neurogenesis period. Postnatal Glu progenitors do not accumulate during embryonal development but are produced by embryonal RGs that persist after birth in the dorsal SVZ and continue to give rise to cortical neurons, although with low efficiency. Single-cell RNA sequencing revealed a dysregulation of transcriptional programs, which correlates with the gradual decline in cortical neurogenesis observed in vivo.

Altogether, these data highlight the potential of transcriptomic studies to unravel but also to approach fundamental questions such as transcriptional changes occurring in a population of progenitors over time and participating to changes in their fate potential. This knowledge will be key in developing innovative approaches to recruit and promote the generation of selected cell types, including neuronal subtypes in pathologies.

My gratitude goes to all the past and current lab members. Thank you for the great atmosphere you created, your friendship and the funny moments we've shared. I still think that you all were a bit crazy (me being normal) but it is good to be crazy. Wish you all, all the best! It was good to leave "abroad in France" (Switzerland, Holland, Vendée and France origins! What a good mix and good image for Europe!) I have a special thanks to Stefan Zweifel and Vanessa Donega, because we have shared projects and all the up and downs that go with it. Finally we made it through, congrats! Thank you Dr. Stefan for your friendship and your refreshing personality. I was impressed by your creativity, your meticulous way of work and the way you managed all the things during your last months.

I wish I could be as good as you regarding that. Didn't honor my drinking evening with you. Hope that in the near future I can honor my promise. Geil! (I think I understood it means awesome…). Dr. Vanessa, well, I really appreciated working with you. Thanks for all the help, you were always available and enthusiastic. I could always count on you.

Thanks for the discussions we had on everything, it was really nice to share things of life in general with you. I may apologize for the 2 fingers you lost while I was cutting the mouse tails. But the vision declines when you get older you know… And also for the StarWars spoilers. May the force be with you. Thank you Dr. Elodie Gaborieau for you refreshing and enthusiastic personality which brought fun in the office. Thanks for the easy way it was to work with you. I must say you impressed me the way you finished up the thesis and the papers. It was really an amazing job so wish you to find the right direction that make you happy now. I can't forget Quentin Lo Giudice who basically saved my life with bioinformatics (ha the geeks…). I don't really have words to express my gratitude for the contribution you made. And I don't forget that you saved my computer!! You showed me so many things and it was so pleasant to work with such a great person. Can't wait to assist to your thesis defense in Geneva! I also would like to thank Charlotte Verrier, who was a very great technician student, for the quality work you made, Pierre Marijon for establishing bioinformatics tools and Diane Angonin. My last words will go to Louis Foucault, our new PhD student. I wish you all the best and am really glad to continue working with you. Easy going, nice and always showing the desire to learn. It's impressive! Good luck! I may teach you how to ifly in the future because it's definitely your weakness… I also have thoughts to Kaz, a previous postdoc in Olivier's former lab who teached me and provided me with a lot informations and advices at the beginning of the different projects.

I would like to thank all the different people that I knew before or those I learnt to know over the last years at the SBRI who helped me and encouraged me. I have worked here during 6 years from 2003 to 2009 so it's with a particular emotion that I am turning the page of this part of my life here. It is great to work with you and can't cite and thank all of you here. Still, my thankfulness to Colette Dehay and Henry Kennedy for their support, Chandara Nay and Jennifer Beneyton for their administrative support and to all the animal facility technicians for their help. I would like to thank some people outside SBRI for their technical assistance, expertise and contribution: people from ProfilExpert and Isabelle Durand.

The last but not the least, I have no doubt when I am saying that you helped me to hold when things are not working and that it would have been so much more difficult without you. You are always here to give me more strength and self-confidence and to push me forward. Thank you for being in my life and thank you for the sacrifice you made, professionally and personally, to allow me to concentrate to finish this up. My forever thank to you Irène. And of course, how can I not say a word on my two little monsters. I wasn't sure if I would thank you kids because you didn't really help me to rest and relax!! but only you can transform the worse day ever into peanuts by a simple look or smile which make me realizing what's really important in life. Love you and hope you will be proud of me because it's a bit for you that I was doing it.

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Long Résumé

Une activité germinale persiste dans le cerveau postnatal des mammifères dans des niches spécialisées, à savoir le gyrus denté (DG) de l'hippocampe et la zone sousventriculaire (SVZ) qui entoure le ventricule latéral (LV). Les cellules souches neuronales (NSCs) de la SVZ postnatale se divisent et donnent naissance à des progéniteurs transitoires (TAPs) qui génèrent des neuroblastes migrant via la voie de migration rostrale (RMS) vers le bulbe olfactif (OB), où ils se différencient en neurones. La SVZ génère en outre des progéniteurs gliaux qui envahissent le parenchyme local. Récemment, de plus en plus de preuves ont mis en évidence la nature hétérogène de la SVZ postnatale en ce qui concerne ses différents microdomaines générant des lignages neuraux distincts. Par exemple, les progéniteurs des neurones GABAergiques sont principalement dérivés de la SVZ latérale (lSVZ), tandis que la production des progéniteurs des neurones glutamatergiques est limitée à la SVZ dorsale (dSVZ). De plus, des oligodendrocytes postnataux sont générés à partir de la dSVZ.

Cette hétérogénéité a pour origine le développement embryonnaire précoce et est intrinsèquement codée par l'expression de facteurs de transcription (TFs) spécifiques.

Ainsi, l'expression régionalisée de TFs enrichis dans des régions du cerveau antérieur embryonnaire persiste dans les microdomaines correspondants de la SVZ postnatale.

Nous avons récemment décrit les hétérogénéités transcriptionnelles des différentes populations de cellules contenues dans la SVZ postnatale. Ainsi, un nombre élevé et inattendu de transcrits (1900) étaient exprimés de manière différentielle dans des NSCs et TAPs isolés à partir de microdomaines définis de la SVZ. Nous avions observé que l'hétérogénéité transcriptionnelle observée entre les NSCs latérales et dorsales (dNSCs et lNSCs) était due à l'expression de facteurs de transcription. Notamment, la protéine HOPX a été identifiée avec une expression abondante spécifiquement dans les dNSCs.

HOPX est une petite protéine atypique (73 acides aminés) de la famille homeobox dépourvue de sites de liaison à l'ADN. L'expression d'Hopx est minimale au jour embryonnaire 14,5 (E14,5) et atteint un pic autour d'E16,5 avec un gradient rostromédial à caudolatéral. La protéine HOPX est exprimée dans les cellules souches du gyrus denté de l'hippocampe chez l'adulte, alors qu'elle est décrite comme étant systématiquement absente de la SVZ adulte. De plus, HOPX a récemment fait l'objet d'une attention croissante en raison de son expression dans les NSCs quiescentes, dans les astrocytes matures du DG de la souris adulte [START_REF] Li | Hopx distinguishes hippocampal from lateral ventricle neural stem cells[END_REF], ainsi que dans les cellules de la glie radiaire externe (oRG) du cerveau humain en développement [START_REF] Pollen | Molecular Identity of Human Outer Radial Glia during Cortical Development[END_REF]Thomsen et al., 2015a). Dans cette étude, les TFs qui régulent des lignages neuronaux distincts ont été caractérisées pour leur enrichissement dans des microdomaines spécifiques de la SVZ.

En sélectionnant l'un de ces transcrits, nous montrons que les NSCs se distinguent spatialement et qu'elles sont amorcées dans la différenciation vers des destins neuronaux spécifiques. Nos résultats identifient Hopx comme un gène révélant une hétérogénéité accrue de la SVZ dorsale (dSVZ) régulant certains aspects de l'astrogenèse.

La diversité des sous-types de neurones générés par les NSCs de la SVZ après la naissance est beaucoup plus grande qu'on ne le pensait. Le concept de régionalisation de la SVZ, dans lequel la genèse de lignées neuronales distinctes est régulée spatialement et temporellement, est de plus en plus étudié. Les NSCs situées dans les microdomaines SVZ proviennent de régions spécifiques du cerveau antérieur en développement (Fuentealba et al., 2015a) et génèrent une grande diversité de cellules neurales, y compris des sous-types neuronaux, en fonction de l'expression de programmes de transcription spécifiques. En conséquence, l'expression des TFs est directement corrélée à l'acquisition de destins neuronaux définis, un concept qui a été exploré dans cette première étude.

Nous avons tiré parti du transcriptome du génome entier des NSCs postnatales spécifiques aux différentes régions de la SVZ, récemment décrit [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. Une méta-analyse des TFs exprimés dans les NSCs latérales et dorsales avec des jeux de données de sous-populations neuronales et gliales isolées à partir du cerveau antérieur [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF] a mis en évidence des réseaux transcriptionels correspondant aux XIII lignages dérivés des NSCs de chaque micro-domaines. Nous démontrons ainsi que l'expression précoce de TFs spécifiques amorcent les NSCs dans un destin spécifique.

Une tel amorçage précoce est corroboré par un séquençage de l'ARN en cellule unique des NSCs de la SVZ adulte [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF]. L'identification de marqueurs de NSCs régionalisées, tels que HOPX, facilitera grandement l'exploration de l'hétérogénéité spatiale et de la nature restreinte des NSCs lors de la génération de lignées neuronales spécifiques. En utilisant deux approches distinctes, nous démontrons que l'expression d'HOPX est limitée à une sous-population de NScs dorsales (dNSCs) alors que son expression est minimale dans les NSCs latérales (lNSCs). De plus, nos résultats impliquent une association d'HOPX dans le lignage astroglial. De même, au stade embryonnaire 17. 5 (E17.5), l'ARNm d'Hopx est confiné (20%) à une sous-population de cellules gliales radiaires (RGCs)les cellules souches du cerveau en développement -(20%) caractérisée par un enrichissement en marqueurs astrocytaires issus des astrocytes de la SVZ adulte. Ceci est en corrélation avec la cartographie récente réalisée de manière clonale, du destin des RGCs suggérant une transition neurogénique à gliogénique en grande partie incomplète, dans la mesure où seulement une fraction des RGCs (estimée à environ 1/6) produit des cellules gliales. Il est important de noter, bien que l'expression d'HOPX soit observée dans une sous-population de NSCs produisant principalement des astrocytes, HOPX ne peut pas être considérée comme un marqueur astrocytaire général. En effet, l'expression d'HOPX est limitée dans l'espace et est donc susceptible d'être associée à la génération d'une sous-population d'astrocytes. Des études de cartographie de lignage ont révélé que les astrocytes sont localement produits selon le site embryonnaire d'origine dans la zone ventriculaire [START_REF] Tsai | Regional astrocyte allocation regulates CNS synaptogenesis and repair[END_REF]. En outre, l'analyse transcriptomique d'astrocytes isolés de différentes régions du cerveau révèle une expression hétérogène de plusieurs marqueurs astrocytaires. Par exemple, HOPX s'est avéré être enrichi dans les astrocytes du cerveau antérieur dorsal (cortex et hippocampe) et faiblement exprimé dans les astrocytes des régions sous-corticales (thalamus et hypothalamus; [START_REF] Morel | Molecular and Functional Properties of Regional Astrocytes in the Adult Brain[END_REF]. L'hétérogénéité des astrocytes dans le CNS a récemment été décrite comme influençant la synaptogenèse et la maturation neuronale via la sécrétion de plusieurs protéines de la matrice extracellulaire [START_REF] Eroglu | Regulation of synaptic connectivity by glia[END_REF]. De plus, la densité d'astrocytes varie considérablement entre les régions du cerveau [START_REF] Azevedo | Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain[END_REF]. Le rôle d'HOPX dans l'acquisition des propriétés et / ou la régulation des densités des astrocytes régionaux reste à explorer.

Les mécanismes par lesquels HOPX assure ses fonctions restent largement inconnus.

HOPX est un TF atypique qui ne se lie pas directement à l'ADN, mais module d'autres TFs et / ou effecteurs de voies de signalisation au niveau post-transcriptionnel. Une interaction XIV d'HOPX avec SRF a été démontrée au cours du développement cardiaque [START_REF] Shin | Modulation of cardiac growth and development by HOP, an unusual homeodomain protein[END_REF], mais il est peu probable qu'elle se produise dans la SVZ où l'expression de SRF reste faible (données non présentées). Une fonction plus probable d'HOPX est la modulation des voies de signalisation dorsalement actives, telles que les voies BMP et WNT (voir aussi Azim et al. 2014;Azim et al. 2017), qui ont été démontrées comme raffinant l'astrogenèse avec la neurogenèse au cours de corticogenèse [START_REF] Gross | Bone morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells[END_REF][START_REF] Takizawa | DNA Methylation Is a Critical Cell-Intrinsic Determinant of Astrocyte Differentiation in the Fetal Brain[END_REF][START_REF] Tiberi | Cortical neurogenesis and morphogens: Diversity of cues, sources and functions[END_REF]. Une action réciproque entre BMP et WNT a été rapportée dans plusieurs populations de progéniteurs [START_REF] He | BMP signaling inhibits intestinal stem cell selfrenewal through suppression of Wnt-β-catenin signaling[END_REF][START_REF] Plikus | Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration[END_REF][START_REF] Kandyba | Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation[END_REF][START_REF] Genander | BMP signaling and its pSMADS1/5 target genes differentially regulate hair follicle stem cell lineages[END_REF][START_REF] Song | Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells[END_REF]. et peut être intégrée par l'expression dHOPX, comme récemment démontré dans les cardiomyoblastes [START_REF] Jain | Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts[END_REF]. Des études futures visant à manipuler l'activité de ces deux voies de signalisation chez les animaux HOPX KO pourraient nous permettre d'aborder ces questions et d'étudier le rôle de l'intégration du signal extrinsèque dans la spécification du lignage des populations de NSCs voisines.

Il est intéressant de penser que d'autres voies de signalisation peuvent influencer le profil d'expression d'HOPX et pourraient être impliquées dans son évolution chez les primates.

Curieusement, l'expression d'HOPX dans la SVZ de souris suit la maturation spatiotemporelle des cellules épendymaires, ce qui peut limiter progressivement le contact des

RGCs avec le liquide céphalorachidien [START_REF] Mery | Resectión de lesiones cerebrales con asistencia de mapeo cortical intraoperatorio[END_REF] [START_REF] Li | GSK3 Temporally Regulates Neurogenin 2 Proneural Activity in the Neocortex[END_REF]. Cependant, une fraction importante des progéniteurs neuraux ne change pas de destin. Par exemple, une population de progéniteurs subsistant dans la SVZ postnatale contribue à la neurogenèse du bulbe olfactif et à la gliogenèse parenchymateuse tout au long de la vie (Doetsch et al., 1999b). Certains au moins de ces progéniteurs proviennent de cellules de la glie radiaire (RGCs) embryonnaires, cyclant lentement ou en quiescence, qui se divisent entre E13.5 et E15.5 (Fuentealba et al., 2015b;[START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. L'analyse de la cartographie de leur destinée a montré qu'elles donnaient lieu à des lignages neuronaux et / ou gliaux distincts, en fonction de leur localisation dans la SVZ [START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF]. Étonnamment, plusieurs études suggèrent la persistance des progéniteurs Glu dans la SVZ dorsale (dSVZ) jusqu'au début de l'âge adulte [START_REF] Brill | Adult generation of glutamatergic olfactory bulb interneurons[END_REF][START_REF] Winpenny | Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells[END_REF] [START_REF] Frazer | Transcriptomic and anatomic parcellation of 5-HT3AR expressing cortical interneuron subtypes revealed by singlecell RNA sequencing[END_REF]. De plus, il a été démontré que la migration des interneurones dans le cortex frontal persistait tôt après la naissance chez les rongeurs [START_REF] Inta | Neurogenesis and widespread forebrain migration of distinct GABAergic neurons from the postnatal subventricular zone[END_REF][START_REF] Magueresse | Subventricular zone-derived neuroblasts use vasculature as a scaffold to migrate radially to the cortex in neonatal mice[END_REF], ainsi que chez les bébés humains (Nogueira et al., 2003). 2017).

Nos résultats révèlent que cette neurogenèse persistante ne se limite pas à la lignée

GABAergique mais inclut également les neurones de la lignée glutamatergique. En effet, notre travail identifie une petite population de neurones corticaux Satb2/Cux1 + générés à la naissance. Les neurones survivants développent des épines et des projections axonales intracorticales, favorisant ainsi leur intégration dans les réseaux corticaux. Nos résultats indiquent en outre que ces neurones proviennent d'une large population palliale de RGCs qui ne basculent pas vers l'astrogenèse et persistent dans la dSVZ. Ces résultats sont en accord avec des expériences MADM suggérant que seulement 1 cellule neurogénique radiaire sur 6 produit des cellules gliales (Gao et al., 2014a). Nos résultats soulignent un déclin rapide de la capacité des progéniteurs Glu à se différencier et à migrer, contribuant ainsi à la fermeture de la période de neurogenèse corticale.

Nos données de scRNAseq ont mis en lumière les mécanismes à l'origine de cette perte progressive du pouvoir neurogène. Les changements épitranscriptomiques apparaissent comme des mécanismes clés dans la médiation du contrôle temporel sur la progression du lignage. La modification de l'ARNm m6A est la modification la plus répandue dans les cellules eucaryotes [START_REF] Desrosiers | Identification of Methylated Nucleosides in Messenger RNA from Novikoff Hepatoma Cells[END_REF] et a récemment été suggérée dans la régulation de la corticogenèse [START_REF] Yoon | Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation[END_REF]. Nos résultats identifient cette méthylation comme un mécanisme possible conduisant à la dérégulation de la transcription que nous observons chez les progéniteurs Glu postnataux. Outre ces modifications épitranscriptomiques, l'analyse KEGG met en évidence les cahngements de plusieurs voies de signalisation clés, telles que celles impliquées dans l'astrogenèse (comme les voies de signalisation Jak-Stat et Notch;[START_REF] Rowitch | Developmental genetics of vertebrate glial-cell specification[END_REF], XVII suggérant qu'elles peuvent affecter simultanément le potentiel de différenciation des progéniteurs de Glu. Une autre voie de signalisation dérégulée est la voie de signalisation Wnt. Ceci est en accord avec une étude précédente décrivant une augmentation progressive de l'activité de GSK3β à partir de E15.5 et se traduisant par la phosphorylation de Neurog2, effecteur du lignage neuronal glutamatergique, affectant ainsi son activité [START_REF] Li | GSK3 Temporally Regulates Neurogenin 2 Proneural Activity in the Neocortex[END_REF]. Parallèlement à une activité transcriptionelle réduite de Neurog2, 64% de ses gènes cibles [START_REF] Gohlke | Characterization of the proneural gene regulatory network during mouse telencephalon development[END_REF] sont régulés négativement à P2, alors que 2% seulement sont régulés à la hausse, malgré la persistance de l'expression de Neurog2.

Il est important de noter que les progéniteurs postnataux Glu semblent toujours être permissifs à la manipulation intrinsèque/extrinsèque. Nous montrons que leur prolifération et migration Glu peuvent être favorisées par des manipulations génétiques ou pharmacologiques. Nos expériences révèlent toutefois que ces manipulations ne sont pas suffisantes pour favoriser la survie à long terme des neurones, ce qui suggère que le cortex n'est pas permissif à l'intégration de ces neurones nouveau-nés dans des conditions physiologiques. Cependant, des observations récentes suggèrent que la permissivité de l'environnement pourrait être accrue après une lésion, comme après une hypoxie chronique néonatale, où une neurogenèse corticale de novo a été observée [START_REF] Fagel | Fgfr1 Is Required for Cortical Regeneration and Repair after Perinatal Hypoxia[END_REF][START_REF] Bi | Cortical Glial Fibrillary Acidic Protein-Positive Cells Generate Neurons after Perinatal Hypoxic Injury[END_REF]Falkner et al., 2011[START_REF] Falkner | Transplanted embryonic neurons integrate into adult neocortical circuits[END_REF]Azim et al., 2017). Il est probable que nos résultats fourniront des informations importantes pour orienter les recherches futures dans ce contexte.

Les régions germinales ne sont pas homogènes, mais très hétérogènes, avec des différences de transcription conduisant à la production de lignées cellulaires divergentes.

Récemment, notre laboratoire a contribué à résoudre cette hétérogénéité en démontrant un niveau inattendu d'hétérogénéité transcriptionnelle entre la zone sous-ventriculaire dorsale et latérale du cerveau antérieur de la souris postnatale, ainsi que dans leurs cellules souches neurales et leurs progéniteurs [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. Dans le premier chapitre de ma thèse, mes résultats ont contribué à décrire et à discuter un nouveau niveau d'hétérogénéité régionale et spécifique à la lignée dans la SVZ dorsale basée sur l'expression de Hopx (chapitre 1). Ces travaux soutiennent la coexistence de NSCs biaisés vers un lignage, les NSCs voisines exprimant des facteurs de transcription distincts qui influencent leurs comportements respectifs et les guident tout au long de l'acquisition de différents destins.

Dans le deuxième chapitre de ma thèse, j'ai poursuivi l'exploration de l'hétérogénéité régionale et spécifique à la lignée de la SVZ dorsale en caractérisant les progéniteurs des XVIII neurones glutamatergiques corticaux (c.-à-d. Les progéniteurs Glu) dont il a déjà été montré qu'ils persistaient après la naissance (Donega et al., 2018a Development and Postnatal Life ...................................................................................... 1.1. The Developing Forebrain Generates Neuronal Diversity ..................................... 1.1.1 Diversity of the Progenitors during Cortical Neurogenesis ............................... 1.1.2 Regionalization of the Developing Brain .......................................................... 1.1.3. Basic Neural Stem Cells ......................................................................................................... 2.1. Introduction ....................................................................................................... Transcriptional Featrues with adult Astrocytes ....................................................... 46 2.4. Discussion .......................................................................................................... 48 2.5. Supplementary Figures ....................................................................................... 51 3. Experimental Chapter 2: Transcriptional .............................................................................................................................. 88 4.3. Implication for Brain Repair................................................................................. 92 4.3 Here I describe its gradual development and complexification over time, with a special emphasis on the forebrain and of the cortex, the regions of interest of this PhD work.

Germinal activity does not sharply stop at birth, but persists within the subventricular zone of the lateral ventricle. In a second part of the introduction, I describe the transformation of this germinal region at postnatal stages. I finally describe its specificities and similarities compared to its embryonic counterpart, both in term of cellular and regional organization, which were both explored further during my PhD.

The Developing Forebrain Generates Neuronal Diversity

The neocortex is by far one of the most complex regions of the mammalian brain, characterized by a remarkably diversity of neuronal and non-neuronal cell types, whose spatial and temporal coordinated development and function guarantee the execution of high-order cognitive, sensory, and motor behaviors. Decoding its heterogeneity have been at the core of neuroscientists' work for decades. In this first part of the introduction I would like to introduce some of the key aspects of its organization and development.

Diversity of the Progenitors during Cortical Neurogenesis

Once neurulation is completed, the neural tube forms three primary vesicles, namely the forebrain, the midbrain and the hindbrain (Figure 1A). The forebrain rapidly evolves in further subdivisions to form secondary vesicles including the telencephalon. Its dorsal part becomes the pallium where the cerebral cortex derives. RGCs maintain a bipolar morphology with apical and basal processes (Figure 2A) and produce most of the neurons in the brain, either directly or indirectly [START_REF] Haubensak | From The Cover: Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: A major site of neurogenesis[END_REF][START_REF] Miyata | Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells[END_REF][START_REF] Noctor | Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases[END_REF]. Cortical neurogenesis which extends from E10.5 to E18.5 in mice is followed by the gliogenesis period, which terminates after birth. At early stage of corticogenesis, RGCs in the pallium divide asymmetrically to self-renew and generate a projecting neuron. This process is defined as direct neurogenesis. As corticogenesis proceeds, RGCs give rise to intermediate progenitors (IPs) that delaminate from the apical surface to invade the subventricular zone (SVZ) (Figure 2B). Contrary to RGCs, IPs divide symmetrically to give birth to two identical neurons following further round of cell division. This process is defined as indirect neurogenesis and is critical to increase the number of neurons that are generated from a given number of RGCs [START_REF] Haubensak | From The Cover: Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: A major site of neurogenesis[END_REF]. IPs differ from RGCs in terms of genes they express. For example, they specifically express the genes that encode the transcription factor Tbr2 [START_REF] Englund | Pax6, Tbr2, and Tbr1 Are Expressed Sequentially by Radial Glia, Intermediate Progenitor Cells, and Postmitotic Neurons in Developing Neocortex[END_REF]. Newborn neurons generated directly or indirectly from RGCs migrate to reach the cortical plate (CP). Early born neurons move basally through somal translocation and integrate the deep layers of the cortex [START_REF] Tabata | Differences of migratory behavior between direct progeny of apical progenitors and basal progenitors in the developing cerebral cortex[END_REF]. In contrast, later born neurons transit through multiple morphologies and use the processes of RGCs as a scaffold for their locomotion towards the upper cortical layers [START_REF] Evsyukova | Integrative Mechanisms of Oriented Neuronal Migration in the Developing Brain[END_REF][START_REF] Hippenmeyer | Molecular pathways controlling the sequential steps of cortical projection neuron migration[END_REF]. Cortical layering occurs in an 'inside-out' fashion whereby earlier born neurons populate deep layers and later born neurons progressively occupy upper layers [START_REF] Angevine | Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse[END_REF]. These successive waves produce six layers in the neocortex.

Figure

Regionalization of the Developing Brain

Within the telencephalon, a dorso-ventral polarity rapidly develops leading to the emergence of the pallium at its dorsal part. This is due to the reciprocal action of transcription factors and morphogens secreted by three signaling centers, the cortical hem (CH) in the mediodorsal region, the anti-hem (AH) in the lateral aspect, and the anterior neural ridge (ANR) in the anteromedial region (Figure 1B and1B). These patterning molecules diffuse in complementary gradients and induce expression of regionally defined transcription factors (TFs). Some of the main TFs involved in the regionalization of the embryonic pallium are presented here. Some of these morphogens and their role in neural stem cell activity are covered in more details in part 1.3.5. of this introduction.

The early forebrain patterning in mouse embryo is established around E8.5. Otx2 first establishes the forebrain and midbrain territories [START_REF] Inoue | Gbx2 Directly Restricts Otx2 Expression to Forebrain and Midbrain, Competing with Class III POU Factors[END_REF]. Then, Emx2 and Pax6 functions redundantly to establish the caudal forebrain, which contributes to the medial pallium, ventral pallium and diencephalon while Six3 establishes the rostral forebrain domain [START_REF] Lagutin | Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development[END_REF] (Figure 3). The expression of Fgf8 in the anterior neural ridge induces Foxg1 in the Six3-expressing domain and establishes the dorsal pallium and subpallium [START_REF] Kobayashi | Early subdivisions in the neural plate define distinct competence for inductive signals[END_REF][START_REF] Lagutin | Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development[END_REF]. The boundary between the pallium and subpallium is regulated by cross-repression between Gli3 and Shh, and Fgf8 expression [START_REF] Gutin | FGF signalling generates ventral telencephalic cells independently of SHH[END_REF]. Through these interactions, the border between the Pax6 + pallium and the Nkx2.1 + subpallium is first established around E9.5 [START_REF] Shimamura | Inductive interactions direct early regionalization of the mouse forebrain[END_REF], which is subsequently replaced by a Pax6/Gsx2 boundary by E12.5 [START_REF] Yun | Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon[END_REF]. After this, the entire pallium is defined by Pax6 expression in the progenitors and Tbr1 expression in the postmitotic neurons [START_REF] Puelles | Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1[END_REF], although

Tbr1 is downregulated in many of the layers II-V neurons [START_REF] Han | TBR1 directly represses Fezf2 to control the laminar origin and development of the corticospinal tract[END_REF]. Emx1 further delineates an expression boundary between the dorsal and ventral pallium [START_REF] Puelles | Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1[END_REF]. Thus, excitatory glutamatergic projection neurons arise from progenitor cells of the Emx1-positive dorsal pallial area [START_REF] Louvi | The derivatives of the Wnt3a lineage in the central nervous system[END_REF], whereas the subpallium which includes the ganglionic eminences (GE) produces inhibitory interneurons [START_REF] Hébert | The genetics of early telencephalon patterning: Some assembly required[END_REF][START_REF] Moreno | Development and evolution of the subpallium[END_REF].

Following E9.5 several transcription factors have a clear regionalized expression pattern. This is particularly clear for markers of the pallium and the subpallium. Members of the Dlx family (Distal-less), namely homeobox genes Dlx1, Dlx2 [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], Dlx5 and Dlx6 [START_REF] Simeone | Cloning and characterization of two members of the vertebrate Dlx gene family[END_REF] are enriched in the ganglionic eminences of the subpallium and consistently absent from the pallial domain. Other ventrally enriched genes are Nkx2, Nkx6.2, Gsx1 and Gsx2 [START_REF] Zhong | HomeoDB: A database of homeobox gene diversity[END_REF][START_REF] Zhong | HomeoDB2: Functional expansion of a comparative homeobox gene database for evolutionary developmental biology[END_REF].

On the other hand, homeobox genes Emx1, Emx2 and Pax6 show restricted expression to the pallium as well as the T-box transcription factors Tbr1 and Tbr2 (also referred as Eomes) [START_REF] Bulfone | T-Brain-1: A homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex[END_REF][START_REF] Bulfone | Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development[END_REF], as well as the bHLH transcription factor Neurog2 (reviewed in [START_REF] Lee | Basic helix-loop-helix genes in neural development[END_REF]. Expression of these TFs antagonize a subpallial expression of the bHLH transcription factor Mash1 [START_REF] Casarosa | Mash1 regulates neurogenesis in the ventral telencephalon[END_REF]. In sagittal view, the medial and lateral/ventral pallium connects at caudal levels. DP, dorsal pallium; LP, lateral pallium; me, mesencephalon; MP, medial pallium; pr, prosencephalon; SP, subpallium; VP, ventral pallium. Adapted from [START_REF] Kumamoto | Neuronal subtype specification in establishing mammalian neocortical circuits[END_REF].

Basic Principles of Cortical Organization

Following embryonic development, the mouse neocortex consists of four primary areas:

the somatosensory cortex (which processes sensory modalities, such as input from the vibrissae), the auditory cortex (which processes sound), the visual cortex (which processes the sense of sight), and the motor cortex (which outputs information to control fine motor behavior) [START_REF] Rash | Area and layer patterning in the developing cerebral cortex[END_REF] (Figure 5A). These primary cortical areas occupy most of the cortical surface, unlike in humans where secondary or associative cortical areas are more developed. The cortex displays unique cytoarchitectural characteristics, which are the result of the organization and composition of the different cell types and circuits and vary in an area-specific manner. The neocortex is organized into six horizontal layers, historically defined as supragranular (layer I/II-III), granular (layer IV), and infragranular (layers V and VI). Layers contain different classes of neurons and vary in thickness and tissue architecture depending on their areal identity [START_REF] Greig | Molecular logic of neocortical projection neuron specification, development and diversity[END_REF]. Neurons can be classified in excitatory glutamatergic projection neurons (PNs) (also referred as pyramidal neurons) and GABAergic inhibitory interneurons (INs).

GABAergic inhibitory interneurons

Inhibitory INs are approximately 20-30% of the total number of cortical neurons and are often referred to as 'short axons neurons', although several long projecting INs have been recently traced [START_REF] Tremblay | GABAergic Interneurons in the Neocortex: From Cellular Properties to Circuits[END_REF]. Aspiny INs are the main inhibitory component of neocortical circuits, finely modulating projection neuron activity by regulating both synaptic function and the timing of action potential generation [START_REF] Kepecs | Interneuron cell types are fit to function[END_REF].

Generated from the ventral forebrain progenitors located in medial and caudal ganglionic eminences (MGE and CGE), as well as in the preoptic area (POA), inhibitory INs reach their final destinations in the appropriate cortical layer through a highly coordinated process of tangential migration and radial dispersion in the cortex. The MGE has long been regarded as a primary source of cortical interneuron population in mice (~ 60%) [START_REF] Butt | The temporal and spatial origins of cortical interneurons predict their physiological subtype[END_REF] while the CGE has been shown to be the second-greatest contributor of interneuron progenitors, producing approximately 30% of all cortical interneurons [START_REF] Anderson | Distinct cortical migrations from the medial and lateral ganglionic eminences[END_REF]. Cortical GABAergic INs are very diverse; they contain subtypes that differ in morphology, molecular identity, firing properties, and patterns of local connectivity [START_REF] Wamsley | Genetic and activity-dependent mechanisms underlying interneuron diversity[END_REF]. Although it is far from being an accurate representation, neocortical interneurons can be largely classified into several cardinal groups based on the expression of key molecular markers [START_REF] Tremblay | GABAergic Interneurons in the Neocortex: From Cellular Properties to Circuits[END_REF] [START_REF] Rudy | Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons[END_REF] (Figure 4). However, compared with PNs, the laminar distribution of the molecularly distinct IN groups is much less precise.

Glutamatergic projection neurons

On the other hand, PNs are excitatory glutamatergic neurons and make up the vast majority of neurons of the cortex population (approx. 80%). They connect the cerebral cortex to its distal intracortical, subcortical, and subcerebral targets through their long axonal projections. PNs are generated from dorsal forebrain progenitors and can be broadly classified into intracortical and corticofugal neurons. Intracortical PNs are present in all six layers, but they are predominantly represented in the upper layer II/III and can be further divided into associative and commissural PNs (CoPNs) (Figure 5BC). PNs that project their axons either to targets in the same hemisphere or to different layers of the same area or column are called associative PNs (Molyneaux et al., 2007). By (Molyneaux et al., 2007).

Molecular Diversity of Anatomically Defined PNs

Nevertheless, the full picture of PNs types is incomplete as some of them do not fit into these classical classes [START_REF] Cederquist | Lmo4 Establishes Rostral Motor Cortex Projection Neuron Subtype Diversity[END_REF]. Several studies have however recently tackled this problem by using single cell resolution approaches [START_REF] Zeisel | Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq[END_REF][START_REF] Han | The logic of single-cell projections from visual cortex[END_REF].The authors revealed an additional level of diversity of intracortical PNs, which collectively with previous studies [START_REF] Molyneaux | DeCoN: Genome-wide analysis of invivo transcriptional dynamics during pyramidal neuron fate selection in neocortex[END_REF], provide PN class-specific signature genes. Examples are Fezf2,Cntn6,Cad13,Ctip2,Igfbp4 and Ldb2 for layer V and CSMNs,and Cux2,Svet1,Lhx2,Mef2c,Inhba,Btg1,Lpl,Cited2,and PlexinD1 for CPNs [START_REF] Arlotta | Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo[END_REF][START_REF] Molyneaux | Novel Subtype-Specific Genes Identify Distinct Subpopulations of Callosal Projection Neurons[END_REF][START_REF] Molyneaux | DeCoN: Genome-wide analysis of invivo transcriptional dynamics during pyramidal neuron fate selection in neocortex[END_REF][START_REF] Lodato | Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons[END_REF]. Sox5, Tle4, FoxP2 are specifically expressed in corticothalamic PNs of layer VI. Molecular differences also allow distinguishing between more closely related classes. Several important conclusions can be drawn from those profiling studies. First, the identity of a defined neuronal population relies on the expression of multiple class-specific genes rather than the expression of specific single genes. Second, it implies also that laminar coordinates of a neuron do not fully define its class-specific identity. Third, transcriptional profiling suggests a higher degree of heterogeneity within PN subtypes than what it looks like from their connectivity pattern. For example the expression of Cux2, Ptn, EphA3 and Nnmt parcellate CPNs [START_REF] Molyneaux | Novel Subtype-Specific Genes Identify Distinct Subpopulations of Callosal Projection Neurons[END_REF][START_REF] Molyneaux | DeCoN: Genome-wide analysis of invivo transcriptional dynamics during pyramidal neuron fate selection in neocortex[END_REF]. Finally, class-specific profiles of gene expression are temporally dynamic, changing dramatically during neuronal maturation. With the recent advance of single-cell transcriptomics, it is now possible to tackle the fundamental problem of neuronal diversity at the necessary resolution of individual cells.

Single-cell RNA sequencing technology (scRNA-Seq) had opened new avenue for cell type classification and unbiased discovery of cell types [START_REF] Macosko | Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets[END_REF][START_REF] Zeisel | Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq[END_REF][START_REF] Tasic | Adult mouse cortical cell taxonomy revealed by single cell transcriptomics[END_REF]. The first unbiased sampling of mouse primary somatosensory cortex from Zeisel and colleagues allowed the identification of 7 excitatory and 16 inhibitory neuronal cell types that correctly fitted the already identified molecularly-defined classes.

However, they used an unbiased approach, in which a heterogeneous mixture of cells from all cortical layers was subjected to scRNA-Seq without a priori selection, whereas Tasic and colleagues combined scRNA-Seq with reporter mice to generate a most complete description of cortical neurons although in a more restricted area of the adult mouse cortex (i.e. the primary visual cortex). The limit of the former study is the under sampling of rare and fragile neurons due to unbiased tissue isolation and single cell dissociation. As a result, it favors the most common types of neurons at the expense of the most fragile ones.

In the later study, the authors purified and sequenced specific subsets of cortical neurons using transgenic mouse lines. They identified 19 excitatory and 23 inhibitory neuronal cell types. More and more elegant and pioneered studies are recently emerging such as integrative approaches of multidimensional profiling to draw a more complete cell-type atlas in the mouse cerebral cortex. Activity-induced transcriptional states and electrophysiological features followed by scRNA-Seq (P. Hu et al. 2017;[START_REF] Chevée | Variation in Activity State, Axonal Projection, and Position Define the Transcriptional Identity of Individual Neocortical Projection Neurons[END_REF][START_REF] Fuzik | Integration of electrophysiological recordings with single-cell RNAseq data identifies neuronal subtypes[END_REF] are novel strategies that will deepened our understanding of the cortical cellular composition.

Germinal Activity Persists in the Postnatal SVZ

Germinal activity does not stop sharply at birth, but persists in the vicinity of the lateral ventricles. In this region, RGCs change their morphology to gradually transform into postnatal, then adult NSCs (also named Type B1 cells). They continue proliferating to give rise to both glial cells and neurons. Interestingly, although major differences exist in the cellular composition and organization of the SVZ when compared to its embryonic counterpart, significant similarities are also evidenced. In particular the regionalization of postnatal/adult NSCs is inherited from early development, leading to the local production of defined progenitor populations. Unraveling the signaling pathway and transcriptional correlates of this regionalization has become an active area of research.

The Postnatal Niche

The postnatal and adult mouse brain contains several hundreds (estimated to ~1200 in young adult mice) of NSCs distributed along the lateral ventricle walls [START_REF] Ponti | Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice[END_REF].

These SVZ NSCs are named type B1 cells in adult animals and share characteristics with brain astrocytes such as the expression of several glial markers including the glial-fibrillary acidic protein (GFAP), glutamate aspartate transporter (GLAST), and brain lipid-binding protein (BLBP); morphology, and ultrastructure [START_REF] Paterson | Investigation of glial cells in semithin sections. III. Transformation of subependymal cells into glial cells, as shown by radioautography after3H-thymidine injection into the lateral ventricle of the brain of young rats[END_REF]Doetsch et al., 1997aDoetsch et al., , 1999a)). Type B1 cells are heterogeneous and exists in a quiescent (qNSCs) or activated state (aNSCs) [START_REF] Codega | Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche[END_REF][START_REF] Mich | Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain[END_REF]. These cardinal features underline molecular and cellular differences, which have implications on disease and repair ability. For instance, it is important to understand the molecular specificities and mechanisms controlling the induction of these two ground states to be able to use adult

NSCs in therapy without depleting them. Activated type B1 cells give rise to transit amplifying progenitors (type C cells), which generate neuroblasts (type A cells) that migrate to the OB. Type A cells move along as chains, which are ensheathed by GFAP

positive cells (Lois 1996;Lois et al. 1996;Wichterle et al. 1997) (Figure 6). This neuroblast chain migration defines the rostral migratory stream (RMS) that originates at the anterior tip of the SVZ [START_REF] Doetsch | Network of tangential pathways for neuronal migration in adult mammalian brain[END_REF]. Inside the OB, neuroblasts then migrate radially from the RMS to invade the distinct OB layers where they differentiate into distinct interneuron subtypes. They become integrated into functional circuits replacing throughout life the older cells. This has been suggested to be important for neuronal plasticity and the processing of olfactory information [START_REF] Cecchi | Unsupervised learning and adaptation in a model of adult neurogenesis[END_REF][START_REF] Petreanu | Maturation and Death of Adult-Born Olfactory Bulb Granule Neurons: Role of Olfaction[END_REF][START_REF] Wachowiak | Coding and synaptic processing of sensory information in the glomerular layer of the olfactory bulb[END_REF][START_REF] Imayoshi | Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain[END_REF][START_REF] Lledo | Origin and function of olfactory bulb interneuron diversity[END_REF][START_REF] Sakamoto | The functional significance of newly born neurons integrated into olfactory bulb circuits[END_REF]. The transcription factors Ascl1 and Dlx2 are used as markers of type C cells whereas doublecortin (DCX) and polysialylated neural-cell-adhesion molecule (PSA-NCAM) are expressed by type A cells (Doetsch et al., 1997b). Type B1 cells also give rise to oligodendrocytes for the corpus callosum but in a much lower magnitude as compare to OB neurogenesis [START_REF] Menn | Origin of Oligodendrocytes in the Subventricular Zone of the Adult Brain[END_REF]. Brain injury has also been suggested to induce the SVZ to produce astrocytes that migrate to the injury site [START_REF] Benner | Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4[END_REF].

Nonetheless, it is still unclear whether type B1 cells are multipotent and, thus, able to generate both neurons and glial cells. To address this question, Ortega and colleagues isolated single activated NSC in vitro and performed live imaging to continuously track them. Their results reveal that while aNSCs can give rise to both astrocytes and neurons, none were capable of giving rise to both oligodendroglia and neurons [START_REF] Ortega | Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling[END_REF].

While single cell linages tracing studies were later performed in vivo [START_REF] Calzolari | Fast clonal expansion and limited neural stem cell self-renewal in the adult subependymal zone[END_REF], the use of a Glast promoter to induce recombination in a small number of NSCs prevented the authors to conclude on the multipotency of adult NSCs. Indeed, Glast is active in NSCs, but also in glia cells surrounding the lateral ventricle.

The Radial Glia Origin of Type B1 Cells:

Type B1 cells and embryonic radial glia share both spatial and morphological similarities.

The ventricular contact of type B1 cells, their long-term neurogenic potential, and their unique apical process containing a primary cilium suggest the direct lineage relationship of the two cell types. The original and prevailing model suggested that radial glia transform into B1 cells during the first postnatal weeks, after they have produced the neurons and glial cells of the forebrain [START_REF] Kriegstein | The Glial Nature of Embryonic and Adult Neural Stem Cells[END_REF]. This simple view has recently been challenged by lineage tracing studies (Fuentealba et al., 2015a;[START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. The authors of these studies show that postnatal B1 cells are indeed derived from embryonic NSCs that produce also neurons and glia for other forebrain regions (including cortex, striatum, and septum), but also reveal that the lineage progression of NSCs is not linear. Indeed, their results demonstrate that a subpopulation of radial glial cells, bifurcates from the main population during embryogenesis (i.e. between E13 and E15), and enter quiescence. At postnatal stages, these cells appear to reactivate gradually. These new findings suggest that the lineage for postnatal NSCs is not linear, but bifurcates in the embryo. I will come back on their origin in more details in the general discussion of this manuscript.

Type B1 Cells: A Specialized Astrocyte

Type B1 cells are located within the ventricular epithelium which form a tight barrier of multiciliated ependymal cells to separate the brain parenchyma from the ventricle lumen.

Type B1 cells make direct contact with the ventricle by extending a thin cellular process in-between ependymal cells (Doetsch et al., 1999b) (Figure 7). These apical processes contain each a primary cilium and tend to cluster together at the center of a rosette of ependymal cells in a repeating "pinwheel" pattern. The integrity of the ependymal cell layer as well as the primary cilium and apical contact with the CSF of type B1 cells, are primordial for the maintenance of SVZ germinal activity and neurogenesis. Similarly to embryonic NECs and RGCs this cellular structure likely transduces signaling molecules present within the CSF. For instance, Shh, Wnts, retinoic acid (RA), and BMPs are all found in CSF [START_REF] Huang | Smad4-Shh-Nfic signaling cascade-mediated epithelial-mesenchymal interaction is crucial in regulating tooth root development[END_REF]Lehtinen and Walsh, 2011;Lehtinen et al., 2011), and these factors regulate SVZ neurogenesis. Prominin-1 cell-surface marker has been used to enrich for SVZ NSCs, but expression of CD133 is variable in type B1 cells and is also prominently expressed on ependymal cells [START_REF] Mirzadeh | Neural Stem Cells Confer Unique Pinwheel Architecture to the Ventricular Surface in Neurogenic Regions of the Adult Brain[END_REF][START_REF] Beckervordersandforth | In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells[END_REF]. LewisX (LeX) or CD15, is a cell-surface carbohydrate antigen that has also been found on SVZ NSCs, but this marker is also present on many non-GFAP-expressing cells as well as in some type A cells [START_REF] Capela | LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal[END_REF][START_REF] Imura | Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: Differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes[END_REF][START_REF] Shen | Adult SVZ Stem Cells Lie in a Vascular Niche: A Quantitative Analysis of Niche Cell-Cell Interactions[END_REF][START_REF] Obermair | A novel classification of quiescent and transit amplifying adult neural stem cells by surface and metabolic markers permits a defined simultaneous isolation[END_REF].

The epidermal growth factor receptor (EGFR) is found on "activated" type B1 cells, but remains highly expressed in type C cells [START_REF] Pastrana | Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny[END_REF]. Similarly, GLAST is found in both type B1 cells and some type C cells, as well as in some mature non-neurogenic astrocytes. Although high levels of Id1 have been found in type B1 cells, this transcription factor is present at lower levels in type C cells [START_REF] Nam H Song | High Levels of Id1 Expression Define B1 Type Adult Neural Stem Cells[END_REF]. Finally, VCAM1

has been found to be expressed on the apical process of type B1 cells and shown to play a critical role in NSC maintenance [START_REF] Kokovay | VCAM1 is essential to maintain the structure of the SVZ niche and acts as an environmental sensor to regulate SVZ lineage progression[END_REF].

These patterns of protein expression demonstrate the difficulty of identifying unique marker of adult NSCs. As a consequence, identification of type B1 cells relies on multiple markers which combined and/or differential expression, allows identifying and FAC-sorting adult NSCs. While many of these protocols rely on the use of transgenic animals, Daynac and colleagues established a FACS protocol based on a combination of cell surface markers to isolate type B1 cells from their progenies and to distinguish the quiescent and activated state. They used LeX combined with an anti-CD24 antibody to label neuroblasts and a fluorescent EGF to label EGFR on actively proliferating cells [START_REF] Daynac | TGFβ lengthens the G1 phase of stem cells in aged mouse brain[END_REF].

They described 6 cell populations: the type C neuroblast cells as CD24 + LeX -EGFR -, the immature neuroblasts as CD24 + LeX -EGFR + , the TAPS as The CD24-LeX-EGFR + , the activated NSCs as CD24 -LeX + EGFR + and finally the Lex bright (CD24-LeX + EGFR -) as the quiescent NSCs. Isolation of the two cell populations allowed the transcriptional identification of distinct molecular signatures in quiescent and activated NSCs [START_REF] Morizur | Distinct Molecular Signatures of Quiescent and Activated Adult Neural Stem Cells Reveal Specific Interactions with Their Microenvironment[END_REF]. This, together with a previous study based on the use of alternative markers [START_REF] Codega | Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche[END_REF], reveal specific interactions of NSCs with their microenvironment during quiescence.

Thanks to advances of sequencing technologies, a recent single cell transcriptomic study revealed additional insights in the molecular profiles of adult NSCs [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF] by describing in exquisite details their sequential pattern of activation. It demonstrated that qNSCs can be divided into 2 further subpopulations: a dormant state (qNSC1) preceding a primed-quiescent state (qNSC2) which can be identified based on Glast expression level. Indeed, Prom1 + EGFR -Glast +low or Glast +high can distinguish qNSC2 from qNSC1, respectively. Moreover, the authors described CD9 as new marker of NSCs versus mature parenchymal astrocytes.

Regionalization and heterogeneity of Postnatal SVZ NSCs

Three regions of the developing forebrain contribute to the formation of the lateral ventricular walls, and therefore to the formation of the SVZ: i.e. the septum, the pallium and the ganglionic eminences, that contribute to the medial, dorsal and lateral SVZ, respectively. This result in NSCs of different embryonic origin being distributed within all the 3 walls of the postnatal lateral ventricles. Accumulating evidences have proven that their origin (and therefore their location) drives their specification in defined interneuron subtypes in the OB (Merkle et al., 2007a;[START_REF] Alvarez-Buylla | The heterogeneity of adult neural stem cells and the emerging complexity of their niche[END_REF][START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF].

Thanks to viral infection, genetic fate mapping or targeted electroporation, several landmark experiments revealed that CR + [expressing calretinin] PGCs are preferentially generated by the medial SVZ NSCs (Merkle et al., 2007a;[START_REF] Young | Subventricular Zone Stem Cells Are Heterogeneous with Respect to Their Embryonic Origins and Neurogenic Fates in the Adult Olfactory Bulb[END_REF][START_REF] Fernández | Targeted electroporation of defined lateral ventricular walls: A novel and rapid method to study fate specification during postnatal forebrain neurogenesis[END_REF][START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF]. Superficial GCs, tyrosine hydroxylase (TH)-expressing PGCs and a small number of glutamatergic juxtaglomerular cells are generated by NSCs in the dorsal SVZ (Merkle et al., 2007a;[START_REF] Young | Subventricular Zone Stem Cells Are Heterogeneous with Respect to Their Embryonic Origins and Neurogenic Fates in the Adult Olfactory Bulb[END_REF][START_REF] Brill | Adult generation of glutamatergic olfactory bulb interneurons[END_REF][START_REF] Fernández | Targeted electroporation of defined lateral ventricular walls: A novel and rapid method to study fate specification during postnatal forebrain neurogenesis[END_REF][START_REF] De Chevigny | Dynamic expression of the pro-dopaminergic transcription factors Pax6 and Dlx2 during postnatal olfactory bulb neurogenesis[END_REF]. In contrast, deep GCs as well as CB + [expressing calbindin] PGCs preferentially originate from NSCs of the lateral wall (Merkle et al., 2007a;[START_REF] Young | Subventricular Zone Stem Cells Are Heterogeneous with Respect to Their Embryonic Origins and Neurogenic Fates in the Adult Olfactory Bulb[END_REF][START_REF] Fernández | Targeted electroporation of defined lateral ventricular walls: A novel and rapid method to study fate specification during postnatal forebrain neurogenesis[END_REF]. A subsequent studies has more recently revealed an extra level of heterogeneity by identifying the site of origin of additional, rare, subtypes of interneurons [START_REF] Merkle | Adult neural stem cells in distinct microdomains generate previously unknown interneuron types[END_REF]. Moreover, it is worth mentioning that rostral and medial/dorsal localization primes NSCs to generate neurons that populate more superficial layers of the OB, whereas a caudal and lateral origin correlates with deeper fates [START_REF] Fernández | Targeted electroporation of defined lateral ventricular walls: A novel and rapid method to study fate specification during postnatal forebrain neurogenesis[END_REF][START_REF] De Chevigny | Dynamic expression of the pro-dopaminergic transcription factors Pax6 and Dlx2 during postnatal olfactory bulb neurogenesis[END_REF][START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF].

Taken together, these observations underline that NSCs are not a homogenous population and that their regional specification in defined interneuron subtypes originates from early embryonic development [START_REF] Willaime-Morawek | Embryonic cortical neural stem cells migrate ventrally and persist as postnatal striatal stem cells[END_REF][START_REF] Kohwi | A Subpopulation of Olfactory Bulb GABAergic Interneurons Is Derived from Emx1-and Dlx5/6-Expressing Progenitors[END_REF][START_REF] Ventura | Dorsal Radial Glia Generate Olfactory Bulb Interneurons in the Postnatal Murine Brain[END_REF][START_REF] Young | Subventricular Zone Stem Cells Are Heterogeneous with Respect to Their Embryonic Origins and Neurogenic Fates in the Adult Olfactory Bulb[END_REF][START_REF] Willaime-Morawek | Cortex-and striatum-derived neural stem cells produce distinct progeny in the olfactory bulb and striatum[END_REF]Fuentealba et al., 2015b). Indeed, the embryonic NSCs of the septum, the MGE, the LGE and the neocortex generate NSCs that respectively populate the medial, ventral, lateral and dorsal areas of the adult SVZ. A small level of mixing appears to exist between specific walls. Indeed a degree of NSCs migration has been reported between defined SVZ, such as dorsal/pallial NSCs which have been described to migrate into the lateral SVZ at perinatal timepoints to ventral markers [START_REF] Willaime-Morawek | Embryonic cortical neural stem cells migrate ventrally and persist as postnatal striatal stem cells[END_REF]. Therefore, OB neuronal diversity emerges from a broad postnatal germinal zone, organized in microdomains that relate to the major subdivisions of the telencephalon. It is likely that molecular patterning of each subregion contribute to unique combinations of transcription factors. In agreement with their developmental origin, pallial markers (Emx1, Pax6, Tbr2

(Eomes), Tbr1, Neurog2) are expressed in the dorsal most regions of the neonatal and adult LV, whereas subpallial (Dlx1/2/5, Gsx1/2, Ascl1, Nkx2.1, Nkx6.2) and septal (Zic1/3) markers are expressed in the lateral and medial walls of the LV, respectively [START_REF] Kohwi | A Subpopulation of Olfactory Bulb GABAergic Interneurons Is Derived from Emx1-and Dlx5/6-Expressing Progenitors[END_REF][START_REF] Winpenny | Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells[END_REF]Azim et al., 2012a;[START_REF] López-Juárez | Gsx2 controls region-specific activation of neural stem cells and injury-induced neurogenesis in the adult subventricular zone[END_REF][START_REF] Merkle | Adult neural stem cells in distinct microdomains generate previously unknown interneuron types[END_REF]. The expression pattern of these TFs can also vary significantly along the rostrocaudal extent of the SVZ, a characteristic recently described for Zic1/3, Nkx6.2 and Nkx2.1 in the most antero-ventral SVZ producing additional subtypes of OB neurons recently revealed [START_REF] Merkle | Adult neural stem cells in distinct microdomains generate previously unknown interneuron types[END_REF]. A large number of region-specific TFs can be observed earlier during development [START_REF] Flames | Delineation of Multiple Subpallial Progenitor Domains by the Combinatorial Expression of Transcriptional Codes[END_REF], suggesting that a greater number of differentially expressed TFs might be observed in the SVZ at postnatal ages, which remain to be fully explored. Such intrinsic coding programs contribute to the OB neuronal diversity. For instance, different combinations of the expression of Pax6, Dlx2

and Meis2 drive the differentiation of NSCs toward TH + , CB + or CR + PGCs [START_REF] Brill | A Dlx2-and Pax6-Dependent Transcriptional Code for Periglomerular Neuron Specification in the Adult Olfactory Bulb[END_REF][START_REF] Agoston | Meis2 is a Pax6 co-factor in neurogenesis and dopaminergic periglomerular fate specification in the adult olfactory bulb[END_REF]. Other regionally expressed TFs, including Etv1, Nurr1 (Nr4a2), Sall3, Zic1/3 might be associated with the acquisition of defined neuronal fates [START_REF] Díaz-Guerra | Transcriptional regulation of olfactory bulb neurogenesis[END_REF].

Regionalization also applied to gliogenesis

Regionalization is not restricted to the production of distinct neuronal subtypes. Indeed, similar principle apply for macroglial cells, namely astrocytes and oligodendrocytes.

Astrocytes are generated towards the end of the embryonic development, following the period of neurogenesis. The classical model, is that RGCs switch fate and start to produce astrocytes precursors that migrate to the cortex and further amplify locally to associate with previously generated neurons in so called cortical columns [START_REF] Ge | Local generation of glia is a major astrocyte source in postnatal cortex[END_REF][START_REF] Magavi | Coincident Generation of Pyramidal Neurons and Protoplasmic Astrocytes in Neocortical Columns[END_REF][START_REF] Tabata | Diverse subtypes of astrocytes and their development during corticogenesis[END_REF]. Recent studies indicate that astrocytes production is highly regionalized, with newborn astrocyte precursors migrating radially away from their region of origin [START_REF] Tsai | Regional astrocyte allocation regulates CNS synaptogenesis and repair[END_REF]. Thus, cortical astrocytes derive from the Emx1-expressing pallial VZ, while striatal astrocytes derive from the Dbx1-expressing sub-pallial VZ. It is likely that these different origins reflect some phenotypic and functional heterogeneity of astrocytes present in distinct brain regions. Indeed, the densities and morphologies of astrocytes vary greatly between brain regions [START_REF] Azevedo | Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain[END_REF]. Furthermore, astrocyte heterogeneity in the CNS has recently been described to influence neuronal synaptogenesis and maturation through secretion of several extracellular matrix proteins [START_REF] Eroglu | Regulation of synaptic connectivity by glia[END_REF].

The other macroglia lineage, oligodendrocytes, were long believed to be generated by all RGCs, because of their presence in all regions of the forebrain [START_REF] Richardson | Oligodendrocyte wars[END_REF].

Later studies have revealed that they are produced in 3 different temporal and spatial waves. The first wave originates from the MGE around E12.5 and the medial part of the ventral forebrain, while the second is more laterally generated by the LGE around E15,5.

The final third wave appears at perinatal ages from Emx1 + NSCs that reside in the dorsal SVZ microdomain [START_REF] Kessaris | Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage[END_REF]. Interestingly, while oligodendrocytes formed by all 3 waves are rather homogeneously distributed throughout the forebrain at birth, their differential survival results in their heterogeneous distribution at later timepoint. As a result, ventral forebrain regions are mainly populated by oligodendrocytes generated from the first and second "ventral" waves, while dorsal forebrain regions including the corpus callosum are mainly populated by oligodendrocytes generated from the third "dorsal" [START_REF] Kessaris | Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage[END_REF].

Altogether, these observations indicate that regionalization is not restricted to neurogenesis but also embraces gliogenesis. The exact transcriptional mechanisms involved in the regional production and functional importance of glial cell heterogeneity remains however to be fully explored.

Signals from the CSF and the niche act in concert to regulate NSCs activity and regionalization throughout pre-and postnatal life

In the developing brain, morphogen signaling have a global influence on the organization and development of the cerebral cortex. RGCs harbor at their apical surface a primary cilium contacting the cerebrospinal fluid (CSF) within the ventricles [START_REF] Cohen | Ultrastructural analysis of primary cilium in the embryonic nervous tissue of mouse[END_REF]. This primary cilium acts like a sensor to probe extracellular signals secreted by the choroid plexus and initiate intracellular transduction of specific molecular pathways [START_REF] Tasouri | Primary cilia and brain development[END_REF][START_REF] Lepanto | Neuron's little helper: The role of primary cilia in neurogenesis[END_REF]. The CSF composition changes during the corticogenesis period, as well as during postnatal life, and contains diffusible morphogens that act as key drivers of cortical development (Lehtinen et al., 2011;[START_REF] Silva-Vargas | Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells[END_REF] including FGFs, Shh, transforming growth factor beta (TGF-β)/BMPs and Wnts.

Studies have shown that CSF from developing lateral ventricles (E17) is sufficient to stimulate progenitor proliferation and maintenance in neocortical explants and in neurosphere cultures (Lehtinen et al., 2011). Moreover, secreted factors from the lateral ventricle choroid plexus, a primary producer of CSF, directly and dynamically regulate multiple aspects of the behavior of adult NSCs, such as quiescence, and their progeny throughout the life of the animal [START_REF] Silva-Vargas | Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells[END_REF]. In addition, the basal processes of RGCs contact meninges, which secrete regulatory factors for progenitor maintenance. β1-integrins anchor RGC basal processes to the extracellular matrix, allowing exposure to meningeal-derived trophic signals that maintain progenitor survival and proliferation [START_REF] Radakovits | Regulation of Radial Glial Survival by Signals from the Meninges[END_REF]. Lastly, on the striatal end of the SVZ, the basal processes of type B1 cells contact blood vessels at sites that lack unsheathing astrocytes or pericytes, facilitating direct access to circulating trophic factors [START_REF] Tavazoie | A Specialized Vascular Niche for Adult Neural Stem Cells[END_REF].

This intricate constant cross talk between the vasculature and the SVZ has been further

highlighted by coupling whole-mount preparation with automated 3D analysis of confocal images. Immediately underneath the ependymal cell layer the blood vessels form a dense plexus that is spatially associated with proliferating NSCs and type C progenitors [START_REF] Shen | Adult SVZ Stem Cells Lie in a Vascular Niche: A Quantitative Analysis of Niche Cell-Cell Interactions[END_REF][START_REF] Tavazoie | A Specialized Vascular Niche for Adult Neural Stem Cells[END_REF]. An important component of blood vessels is the associated extracellular matrix, which forms extravascular structures named fractones.

These fractones engulf astrocytic processes, as well as ependymal, microglial and progenitor cells, underlying their intimate association with the neurogenic niche and adding to the complexity of the NSC environment [START_REF] Mercier | Anatomy of the brain neurogenic zones revisited: Fractones and the fibroblast/macrophage network[END_REF][START_REF] Shen | Adult SVZ Stem Cells Lie in a Vascular Niche: A Quantitative Analysis of Niche Cell-Cell Interactions[END_REF]. This specialized cytoarchitecture results in a unique microenvironment in which multiple signals act to maintain germinal activity in the adult brain [START_REF] Ihrie | Lake-Front Property: A Unique Germinal Niche by the Lateral Ventricles of the Adult Brain[END_REF].

In addition to the CSF, the specialized cellular SVZ microenvironment, or "niche," in which postnatal/adult NSCs reside play a key role in maintaining their activity and regionalization at postnatal stages. This is best illustrated by transplantation experiments. Homotopic grafts of SVZ cells give rise to large numbers of OB interneurons in the host mouse [START_REF] Lois | Long-distance neuronal migration in the adult mammalian brain[END_REF]. In contrast, SVZ cells transplanted to non-neurogenic brain regions produce few, if any neurons [START_REF] Herrera | Adult-derived neural precursors transplanted into multiple regions in the adult brain[END_REF] [START_REF] Zheng | Fibroblast growth factor 2 is required for maintaining the neural stem cell pool in the mouse brain subventricular zone[END_REF] or the EGFR ligand-transforming growth factor a (TGF-a) [START_REF] Tropepe | Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon[END_REF].

Interestingly, in vitro experiments suggested that local astrocytes can provide these proliferative signals for the SVZ niche [START_REF] Morita | Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex[END_REF]. Other factors have been implicated in the regulation of the SVZ such as ciliary neurotrophic factor (CNTF) [START_REF] Scott | Loan officer turnover and credit availability for small firms[END_REF], Heparin-binding EGF (HB-EGF). Although most of these factors act on NSCs activation, those involved in maintaining NSCs quiescence remain to be fully explored.

I describe in more details below current knowledge on key diffusible and cell contact signaling pathways and on their role in NSCs activity and regionalization.

Sonic hedgehog

Shh is both a classical morphogen during development and a mitogenic factor for a variety of neural precursors (Ruiz i Altaba et al. 2003). Sonic hedgehog (Shh) is a diffusible protein that belongs to the hedgehog family members. In the developing forebrain, Shh is mostly secreted from the ventral telencephalon into the CSF [START_REF] Echelard | Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity[END_REF] (Figure 1C).

Shh binding to its receptor Patched1 (Ptch1) at the primary cilium site induces a signaling cascade which leads to the activation of the downstream Gli2 and Gli3 transcription factors. They, in turn, activate numerous effectors including Gli1 and Nkx2.1 that participate to various aspects of ventral telencephalon specification including the generation of oligodendrocytes, astrocytes and GABAergic interneurons. The cortical interneurons invade the CP by tangential migration [START_REF] Fuccillo | Temporal requirement for hedgehog signaling in ventral telencephalic patterning[END_REF]. Their intracortical dispersion from the tangential migratory streams is controlled by a short primary cilium probing Shh signaling [START_REF] Graef | Zahnkariesfrequenz Bei Jugendlichen in Gemeinden Mit Naturlich Erhohtem Und Geringem Trinkwasserfluoridgehalt[END_REF]. In contrast to the ventral telencephalon, the developing cortex shows limited sensitivity to Shh signaling whose physiological role remained unclear. However, changes (increase or decrease) affecting Shh signaling impair the generation of IPs and result in size modification of the dorsal telencephalon [START_REF] Yabut | Suppressor of Fused Is Critical for Maintenance of Neuronal Progenitor Identity during Corticogenesis[END_REF][START_REF] Wang | Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex[END_REF] and prevent generation of the postnatal progenitors in the dentate gyrus [START_REF] Han | Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells[END_REF].

In the postnatal/adult SVZ, it appears that Shh has multiple roles and cells responding to Shh signaling express Gli1 transcription factor [START_REF] Bai | Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway[END_REF]. Gli1 + cells produce OB interneurons and are found in ventral SVZ NSCs (Machold et al. 2003;Palma et al. 2005;Ihrie et al. 2011) but also in the dorsal domain producing cells of the oligodendrogilal lineage [START_REF] Tong | A dorsal SHH-dependent domain in the V-SVZ produces large numbers of oligodendroglial lineage cells in the postnatal brain[END_REF]. Shh mRNA is primarily detected in ventral forebrain neurons, suggesting that local expression of Shh activates Gli1 in the ventral SVZ (Ihrie et al., 2011).

Conditional deletion of Shh in adult mice reduces the production of ventrally derived OB

interneurons, suggesting that Shh activity underlies type B1 cell regional heterogeneity (Ihrie et al., 2011). Dorsal SVZ cells however appear to be resistant to Shh-pathway activation, suggesting that the regional identities of type B1 cells are epigenetically restricted. This may explain the absence of re-specification observed in heterochronic transplantation experiments, as previously discussed (Merkle et al., 2007c).

The Wnt/β-catenin signaling pathway

In neural development, the Wnt family of secreted signaling molecules plays multiple roles in stem cell maintenance, cellular proliferation, differentiation, migration, and axon guidance [START_REF] Ille | Wnt signaling: Multiple functions in neural development[END_REF][START_REF] Sj | Wnt signaling and forebrain development[END_REF]. Wnt ligands are secreted glycoproteins that bind to the receptors Frizzled and LRP 5/6 localized at the plasma membrane of APs inducing stabilization of the cytoplasmic β-catenin. Without signaling, activation β-catenin is degraded following its phosphorylation by GSK3β. Once stabilized, β-catenin translocates into the nucleus and associates to TCF/LEF transcription factors family, which become activators and promote the transcription of downstream targets [START_REF] Cadigan | TCFs and Wnt/β-catenin Signaling. More than One Way to Throw the Switch[END_REF]. The cortical hem of dorsomedial telencephalon secretes BMP and Wnt (2b,3a,5a,7b,8b) (Figure 1C)and regulates the patterning of the dorsoventral cerebral cortex [START_REF] Caronia-Brown | The cortical hem regulates the size and patterning of neocortex[END_REF]. Moreover, Wnt signaling plays a major role in the regulation of cortical development. During early corticogenesis, the increase of βcatenin induces proliferation of NECs by decreasing their cell cycle exit and by delaying the neurogenic [START_REF] Hirabayashi | The Wnt/ -catenin pathway directs neuronal differentiation of cortical neural precursor cells[END_REF][START_REF] Wrobel | Persistent expression of stabilized β-catenin delays maturation of radial glial cells into intermediate progenitors[END_REF]. In contrast, later, it induces the expansion of the SVZ, the IPs pool and the neuronal output [START_REF] Munji | Wnt Signaling Regulates Neuronal Differentiation of Cortical Intermediate Progenitors[END_REF]. The forced repression of the Wnt signaling pathway through genetic ablation of β-catenin during midgestation decreases Pax6 expression and leads to the depletion of APs, IPs and projection neurons, impairs oligodendrogenesis while increasing astrogliogenesis [START_REF] Gan | Pax6 mediates β-catenin signaling for self-renewal and neurogenesis by neocortical radial glial stem cells[END_REF][START_REF] Draganova | Wnt/β-catenin signaling regulates sequential fate decisions of murine cortical precursor cells[END_REF]. Indeed, Wnt signaling promotes the generation of oligodendrocyte progenitors by RGCs during late embryogenesis, while Shh supports the specification of ventrally derived oligodendrocyte progenitors [START_REF] Langseth | Wnts Influence the Timing and Efficiency of Oligodendrocyte Precursor Cell Generation in the Telencephalon[END_REF].

Interestingly, these observations highlight the stage dependent activity of the canonical Wnt pathway. First it promotes self-renewal of NECs and RGCs and then promotes differentiation to IPs and finally induces oligodendrocyte differentiation from RGCs at the end of corticogenesis.

In the postnatal SVZ, Wnt signaling is restricted to the dorsal-most microdomain, in NSCs and progenitors that generate oligodendrocytes and OB glutamatergic neurons [START_REF] Azim | Persistent Wnt/β-catenin signaling determines dorsalization of the postnatal subventricular zone and neural stem cell specification into oligodendrocytes and glutamatergic neurons[END_REF]. Genetic and pharmacological modulation approaches demonstrated that Wnt signaling regulates both proliferation and fate specification in the dSVZ [START_REF] Azim | Persistent Wnt/β-catenin signaling determines dorsalization of the postnatal subventricular zone and neural stem cell specification into oligodendrocytes and glutamatergic neurons[END_REF].

and identify canonical Wnts as the key dorsalizing factor in the postnatal SVZ, opposing ventrally localized Shh activity to determine dorso-ventral NSC fate [START_REF] Backman | Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon[END_REF].

The highest functional Wnt activity defines the dSVZ-NSCs and their progeny, Tbr2 + NPs and Olig2 + OPs. Furthermore, Gli1 expression is not affected in dSVZ-NSCs providing further evidence that Wnt signaling exerts a direct dorsalizing effect on the postnatal SVZ.

In the adult, increased β-catenin by expression of a stabilized form of this protein or inhibition of the GSK3β kinase, which is required for targeting β-catenin for degradation, increases the proliferation of Ascl1-expressing SVZ cells. This proliferation is also observed in the lSVZ of early postnatal brain but involves other signaling pathways as no induction of downstream Wnt target genes have been detected [START_REF] Azim | Persistent Wnt/β-catenin signaling determines dorsalization of the postnatal subventricular zone and neural stem cell specification into oligodendrocytes and glutamatergic neurons[END_REF] [START_REF] Backman | Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon[END_REF]. Recently, Non canonical Wnt signaling regulates neural stem cell quiescence during homeostasis and after demyelination [START_REF] Chavali | Non-canonical Wnt signaling regulates neural stem cell quiescence during homeostasis and after demyelination[END_REF].

Bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) are members of the TGF-b superfamily. BMPs (2,[START_REF]Targeted Neuronal Ablation as a Model to Study Competence of Postnatal NSCs for Cortical Repair[END_REF][START_REF] Annexes | [END_REF][START_REF] References Agirman | Cerebral cortex development: an outside-in perspective[END_REF][START_REF] Cv | [END_REF] are secreted by the cortical hem and cooperate with Wnts to promote the dorsomedial patterning of the telencephalon [START_REF] Furuta | Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development[END_REF] (Figure 1C) and control midline development [START_REF] Hébert | BMP signaling is required locally to pattern the dorsal telencephalic midline[END_REF]. Without BMPs receptors, cortical progenitor specification is preserved and neurogenesis is maintained [START_REF] Fernandes | Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly[END_REF]. In contrast, increase of BMP2/4 signaling during cortical development induces premature differentiation of RGCs into neurons [START_REF] Shakèd | Histone deacetylases control neurogenesis in embryonic brain by inhibition of BMP2/4 signaling Chan-Ling T[END_REF]. During gliogenesis stage, overexpression of BMP4 impairs the production of oligodendrocytes by RGCs at the expense of astrocytes [START_REF] Gomes | Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment[END_REF].

BMPs also regulate adult brain germinal niches such as the SVZ. BMP signaling promotes astrocyte differentiation of cultured embryonic neural precursors at the expense of oligodendrogliogenesis and neurogenesis [START_REF] Gross | Bone morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells[END_REF]. Adult SVZ cells produce BMPs and their receptors [START_REF] Peretto | BMP mRNA and protein expression in the developing mouse olfactory system[END_REF][START_REF] Lim | The adult ventricular-subventricular zone (V-SVZ) and olfactory bulb (OB) neurogenesis[END_REF]. NOGGIN, a secreted BMP antagonist, is also locally expressed, most strongly in the ependymal cells. This locally derived BMP antagonist may contribute to the neurogenic niche.

Downregulation of BMP signaling in NSCs impairs neurogenesis [START_REF] Colak | Adult Neurogenesis Requires Smad4-Mediated Bone Morphogenic Protein Signaling in Stem Cells[END_REF].

Overexpression of BMP7 in the SVZ suppresses neurogenesis [START_REF] Lim | Postnatal mouse subventricular zone neuronal precursors can migrate and differentiate within multiple levels of the developing neuraxis[END_REF], and overexpression of Noggin from the ependymal suppresses local gliogenesis [START_REF] Chmielnicki | Adenovirally Expressed Noggin and Brain-Derived Neurotrophic Factor Cooperate to Induce New Medium Spiny Neurons from Resident Progenitor Cells in the Adult Striatal Ventricular Zone[END_REF]. Thus, a "balance" between BMP and their antagonists may control the levels of neurogenesis and gliogenesis from NSCs in adult brain niches. In embryonic stem cells, BMPs act in concert with leukemia inhibitory factor (LIF) to sustain self-renewal and suppress differentiation [START_REF] Ying | BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3[END_REF]. In the adult, it is possible that the level of their expression regulates the fate of type B1 cells into niche astrocytes or self-renewing stem cells. For example, overexpression of LIF promotes SVZ NSC self-renewal, expanding the population of NSCs in vivo while concomitantly decreasing OB neurogenesis [START_REF] Bauer | Leukemia Inhibitory Factor Promotes Neural Stem Cell Self-Renewal in the Adult Brain[END_REF].

Fibroblast growth factors

The fibroblast growth factors (FGFs) family is composed by 22 FGFs arranged into seven families [START_REF] Mason | Initiation to end point: The multiple roles of fibroblast growth factors in neural development[END_REF] [START_REF] Sahara | Fgf10 Regulates Transition Period of Cortical Stem Cell Differentiation to Radial Glia Controlling Generation of Neurons and Basal Progenitors[END_REF]. FGF receptors are expressed with specific spatial patterns along the progenitor region. However, absence of all FGFRs in the dorsal telencephalon leads to precocious neurogenesis, resulting in a reduced cortical surface area in both rostral and caudal parts [START_REF] Kang | The Transition from Radial Glial to Intermediate Progenitor Cell Is Inhibited by FGF Signaling during Corticogenesis[END_REF]. In addition, Notch related genes are downregulated in FGFR mutants and gain-of-function of Notch signaling can rescue the phenotype [START_REF] Rash | Cortical Gyrification Induced by Fibroblast Growth Factor 2 in the Mouse Brain[END_REF], suggesting that Notch lies downstream of FGFR signaling in the same pathway regulating cortical neurogenesis. Different FGF ligands can either promote progenitor differentiation or self-renewal [START_REF] Raballo | Basic Fibroblast Growth Factor (Fgf2) Is Necessary for Cell Proliferation and Neurogenesis in the Developing Cerebral Cortex[END_REF][START_REF] Borello | FGF15 promotes neurogenesis and opposes FGF8 function during neocortical development[END_REF] therefore controlling in fine the normal growth of the brain. FGF signaling also participate to some extent to the differentiation of ventral progenitors independently of Shh signaling [START_REF] Gutin | FGF signalling generates ventral telencephalic cells independently of SHH[END_REF]. FGF signals, are required for the specification, survival, proliferation, and patterning of progenitor cells in the ventral telencephalon.

Pharmacological inhibition of FGF signaling in embryos results in an increase in ventral apoptosis, loss of ventral gene expression, and ventral expansion of dorsal gene

expression [START_REF] Shinya | Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos[END_REF]. Thus, FGF signaling participates in patterning the telencephalon into ventral and dorsal domains, potentially via regulation of anti-hem signals/functions.

In the postnatal SVZ, FGF2 promotes NSC self-renewal, amplification of TAPs and subsequently OB neurons generation [START_REF] Zheng | Fibroblast growth factor 2 is required for maintaining the neural stem cell pool in the mouse brain subventricular zone[END_REF]Azim et al., 2012b;[START_REF] Li | Organocatalytic Biomimetic Decarboxylative Aldol Reaction of Fluorinated β-Keto Acids with Unprotected Isatins[END_REF]. Further, direct administration of FGF2 into the lateral ventricle increased the generation of oligodendrocyte progenitors (OPCs) throughout the SVZ, both within the dSVZ and ectopically in the lSVZ and ependymal wall of the SVZ (Azim et al., 2012b).

Besides diffusible factors, other external factors arising from the direct cellular environment

of NSCs influence their activity in a regional manner.

Notch signaling

Notch signaling is activated by interactions between Notch receptors and their ligands.

The mammalian Notch receptors (Notch1 -Notch4) are transmembrane proteins expressed on the cell surface as non-covalently linked heterodimers. The proteins comprise of an extracellular domain, which functions as the signal receiver and a transmembraneintracellular domain that functions as the signal transducer. The extracellular domain contains epidermal growth factor (EGF)-like repeats, which are required for ligand binding [START_REF] Zhang | Notch: an interactive player in neurogenesis and disease[END_REF] [START_REF] Lutolf | Notch1 is required for neuronal and glial dif-ferentiation in the cerebellum[END_REF]Nyfeler et al., 2005a;[START_REF] Mizutani | Differential Notch signalling distinguishes neural stem cells from intermediate progenitors[END_REF][START_REF] Ehm | RBPJ -Dependent Signaling Is Essential for Long-Term Maintenance of Neural Stem Cells in the Adult Hippocampus[END_REF][START_REF] Imayoshi | Essential Roles of Notch Signaling in Maintenance of Neural Stem Cells in Developing and Adult Brains[END_REF][START_REF] Basak | Neurogenic Subventricular Zone Stem/Progenitor Cells Are Notch1-Dependent in Their Active But Not Quiescent State[END_REF], inhibition of neuronal differentiation and even terminal differentiation into an astrocyte lineage [START_REF] Gaiano | The Role of Notch in Promoting Glial and Neural Stem Cell Fates[END_REF]. Once the ligand binds Notch receptor, the transcription of several genes, including the Hairy enhancer of split (Hes) genes, are initiated. Hes genes are basic-helix-loop-helix (bHLH) transcription factors that repress the expression of proneural genes, further ensuring RGCs stemness maintenance [START_REF] Kageyama | Roles of Hes genes in neural development[END_REF]. Hes genes expression oscillate due to the negative feedback loop on their own promoter [START_REF] Imayoshi | Oscillatory control of factors determining multipotency and fate in mouse neural progenitors[END_REF]. This characteristic pattern is critical for RGCs cell-cycle progression and a sustained Hes expression impairs neuronal generation [START_REF] Baek | Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system[END_REF]. In contrast absence of Hes1, the key effector of the Notch signaling, leads to premature neurogenesis [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]. et al., 1997;[START_REF] Stump | Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain[END_REF][START_REF] Gratton | Hes6 Promotes Cortical Neurogenesis and Inhibits Hes1 Transcription Repression Activity by Multiple Mechanisms[END_REF]. The RING finger E3 ubiquitin ligase mind bomb 1 (MIB1) is essential to activate Notch signaling [START_REF] Itoh | Mind bomb is a ubiquitin ligase that is essential for efficient activation of notch signaling by delta[END_REF]. Interestingly, MIB1 is expressed in neurons and particularly in IPs, indicating that different progenitor populations can interact and mutually regulate each other to control the balance between self-renewal and neuronal differentiation in the mouse cortex [START_REF] Yoon | Mind Bomb 1-Expressing Intermediate Progenitors Generate Notch Signaling to Maintain Radial Glial Cells[END_REF]. A recent work provided evidence that during neurogenic divisions, MIB1 is asymmetrically localized at the daughter centriole and then inherited by the future neuron, identifying the Notch regulator MIB1 as an intrinsic fate determinant [START_REF] Tozer | Differential Routing of Mindbomb1 via Centriolar Satellites Regulates Asymmetric Divisions of Neural Progenitors[END_REF]. Preventing MIB1 centrosomal association induces reciprocal Notch activation between sister cells and promotes symmetric divisions.

In postnatal/adult NSCs, Notch signaling suppresses neuronal differentiation and maintains precursor cell properties [START_REF] Gaiano | The Role of Notch in Promoting Glial and Neural Stem Cell Fates[END_REF]. Notch1, Jagged1 and Delta1, are expressed in the adult SVZ [START_REF] Stump | Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain[END_REF]Nyfeler et al., 2005b;[START_REF] Givogri | Notch Signaling in Astrocytes and Neuroblasts of the Adult Subventricular Zone in Health and after Cortical Injury[END_REF]. In postnatal and adult SVZ cells, retrovirally transduced activated Notch prevents cell migration to the OB, suppresses neuronal differentiation, and decreases proliferation, creating an apparently more "quiescent" cell type [START_REF] Chambers | Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors[END_REF].

Conversely, inactivation of canonical Notch signaling causes essentially all NSCs in the SVZ to differentiate into type C and A cells, leading to the eventual depletion of NSCs from this adult region [START_REF] Imayoshi | Essential Roles of Notch Signaling in Maintenance of Neural Stem Cells in Developing and Adult Brains[END_REF]. Thus, Notch signaling is a critical component of type B1 cell maintenance. Interestingly, the ligands Jagged1 and Delta1 are expressed in type C and A cells [START_REF] Givogri | Notch Signaling in Astrocytes and Neuroblasts of the Adult Subventricular Zone in Health and after Cortical Injury[END_REF][START_REF] Aguirre | Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal[END_REF] [START_REF] Givogri | Notch Signaling in Astrocytes and Neuroblasts of the Adult Subventricular Zone in Health and after Cortical Injury[END_REF][START_REF] Aguirre | Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal[END_REF]), which suggest a potential feedback regulation of the SVZ niche. Accumulation of newly born type A cells expressing Jagged1 or Delta1 may activate Notch signaling in type B1 cells, suppressing differentiation and potentiating self-renewal.

In the postnatal SVZ, regional differences in the composition of the extracellular matrix such as collagens, laminins or fibronectins and differential distribution of their receptors will likely contribute to the diffusion and exposition of these diffused molecules. Their role in NSCs activity and regionalization remains to be fully explored.

Transcriptional correlates of SVZ NSCs regionalization

As described above, a multitude of external factors (diffusible and cell-contact factors) act in concert to orchestrate NSCs activity and regionalization throughout pre-and postnatal development. Cross-talks exist at multiple levels in between these pathways resulting in the regional expression of define gene networks that regulate NSCs activity, as well as their production of distinct cell progenies (see for exemple [START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF][START_REF] Azim | Mosaic subventricular origins of forebrain oligodendrogenesis[END_REF]. Understanding the transcriptional specificities of postnatal NSCs have proved to provide a wealth of information on their regional diversity. and SVZ NSCs identified IGF2 to be highly enriched in the DG. In this region, IGF2 was shown to control the NSCs proliferation through AKT-signaling [START_REF] Bracko | Gene Expression Profiling of Neural Stem Cells and Their Neuronal Progeny Reveals IGF2 as a Regulator of Adult Hippocampal Neurogenesis[END_REF]. In the SVZ the early expression of neuronal markers in NSCs has been demonstrated, in agreement with an early priming of NSCs to differentiate into specific lineage. Further, the importance of cilia-and Ca-dependent pathways were emphasized [START_REF] Beckervordersandforth | In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells[END_REF]. In another study, EGF receptor expression was used to isolate activated and quiescent NSCs from the SVZ. This allowed discovering multiple markers for these two stages and uncovered signaling pathways that may be targeted by small bioactive molecules to regulate NSCs activity [START_REF] Codega | Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche[END_REF].

We recently performed a similar transcriptional analysis to gain insight into the transcriptional correlates of SVZ NSCs. In order to probe heterogeneity of the postnatal SVZ, we microdissected its dorsal and lateral walls at different postnatal ages and isolated

NSCs and their immediate progeny based on their expression of Hes5EGFP/Prominin1

and Ascl1-EGFP, respectively. Whole genome comparative transcriptome analysis revealed transcriptional regulators as major hallmarks that sustain postnatal SVZ regionalization. This comparative analysis, which was also applied to the NSCs and TAPs they contain, revealed the existence of an unsuspected heterogeneity in the postnatal SVZ, at both the regional and temporal levels [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. This was evidenced by the surprisingly high number of genes differentially expressed in the microdissected dorsal and lateral SVZ. Roughly, 30% of the genes were age-specific, whereas 30%-40% were shared by all three ages analyzed. Classification based on GO terms revealed the importance of transcription-related cues that were further abundant in NSCs. We identified several other transcriptional regulators aside from TFs. Those include chromatin modifiers, epigenetic modulators, downstream signaling mechanisms (nuclear), all of which could act in concert to dynamically regulate the diversity of neuronal and glial lineages generated by SVZ-NSCs during early postnatal life.

Objectives of the PhD thesis

Objectives of my PhD were twice.

First, to explore further the transcriptional datasets of regionalized NSCs populations we have previously produced [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF], by combining them with transcriptional datasets of specific neural lineages produced by others [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF]. To use such meta-analysis to explore differences in the lineage priming of regionalized NSC populations (Azim et al., 2017) and to identify new TFs associated with SVZ NSCs biased to acquire specific fate (see experimental chapter 1).

Second, to explore the origin, fate and transcriptional specificities of cortical progenitors that are still present in the dorsal-most domain of the postnatal SVZ, despite of the closure of the cortical neurogenic period. In particular, to develop a single-cell RNA-Sequencing approach to directly compare the transcriptome of this peculiar progenitor population at pre-and postnatal stages.

To achieve these objectives, I acquired expertise in bioinformatics and developed all the scripts necessary to perform those studies. I also remained directly involved in the histology inherent to the completion of this work. In this chapter, transcriptomics and fate-mapping approaches were employed to explore the relationship between regional expression of transcription factors by neural stem cells (NSCs) and their specification in defined neural lineages. I developed in collaboration with Q. Lo Giudice a bioinformatic tool called Heatmap Generator (see annexes for the complete script), which aims at combining and comparing published transcriptional datasets in order to perform a meta-analysis. I also performed histology experiments as well as a meta-analysis of a recently published single-cell RNA sequencing experiment.

EXPERIMENTAL CHAPTERS

The result supports the previous observation regarding the role of HOPX in the astrocytic lineage. Our results support an early priming of NSCs for the genesis of defined cell types depending on their spatial location in the SVZ and identify HOPX as a marker of a subpopulation primed toward astrocytic fates.

N.B. experiments aimed at investigating the role of HOPX in SVZ NSCs by gain-and loss-

of-function were all performed by S. Zweifel, and were therefore not included in this chapter (although they can be found in the published manuscript). [START_REF] Lois | Long-distance neuronal migration in the adult mammalian brain[END_REF]. The SVZ additionally generates glial progenitors that invade the local parenchyma (reviewed in [START_REF] Azim | Mosaic subventricular origins of forebrain oligodendrogenesis[END_REF]. Recently, accumulating evidence has highlighted the heterogeneous nature of postnatal SVZ in respect to different microdomains generating distinct neural lineages. For example, progenitors of GABAergic neurons are predominantly derived from the lateral SVZ (lSVZ), while the genesis of glutamatergic neuron progenitors is restricted to the dorsal SVZ (dSVZ; Azim et al. 2012;reviewed in Fiorelli et al. 2015). Furthermore, postnatally derived oligodendrocytes are generated from the dSVZ [START_REF] Kessaris | Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage[END_REF].

Introduction

This heterogeneity originates from early embryonic development (Fuentealba et al., 2015b) and is intrinsically encoded by expression of selected transcription factors (TFs).

Therefore, TFs enriched in specific embryonic forebrain regions are persistent in their expression in corresponding microdomains of the postnatal SVZ. Examples of such TFs include EMX1 (pallium; dSVZ), GSX1/2 (lateral and medial ganglionic eminence; lSVZ), and ZIC1/3 (septum; medial SVZ; reviewed in [START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF]. We recently resolved the transcriptional heterogeneities of different cell populations of the postnatal SVZ, in which an unexpected large number of transcripts (i.e. 1900) were differentially expressed in NSCs and TAPs sorted from defined SVZ microdomains. Intriguingly, most of the transcriptional heterogeneity observed between the dorsal and lateral NSCs (dNSCs and lNSCs) was due to the expression of transcriptional cues. Notably, HOPX was identified with specific abundant expression in dNSCs [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. HOPX is a small (73 amino acids) atypical homeodomain protein that lacks DNA binding sites [START_REF] Chen | Hop is an unusual homeobox gene that modulates cardiac development[END_REF][START_REF] Shin | Modulation of cardiac growth and development by HOP, an unusual homeodomain protein[END_REF]. Hopx expression is minimal at embryonic day 14.5 (E14.5) and peaks around E16.5 with a rostromedial to caudolateral gradient [START_REF] Mühlfriedel | A roof platedependent enhancer controls the expression of Homeodomain only protein in the developing cerebral cortex[END_REF]. HOPX expression has been found in radial astrocytes of the adult DG, while it is described to be consistently absent from the adult SVZ [START_REF] Toni | Regulation of survival in adult hippocampal and glioblastoma stem cell lineages by the homeodomain-only protein HOP[END_REF]. Moreover, the expression of HOPX has recently received increasing attention due to its expression in quiescent NSCs, in mature astrocytes in the adult mouse DG [START_REF] Li | Hopx distinguishes hippocampal from lateral ventricle neural stem cells[END_REF], as well as in outer radial glia (oRG) cells of the developing human brain [START_REF] Pollen | Molecular Identity of Human Outer Radial Glia during Cortical Development[END_REF]Thomsen et al., 2015a).

Here, we used various approaches to further investigate the regionalization of the postnatal SVZ and of resident subpopulations of NSCs. In particular, we characterized the spatiotemporal and lineage-specific patterns of HOPX expression in the postnatal SVZ Mice used in this study were: OF1 wild-type (Charles Rivers; France), HES5:EGFP [START_REF] Basak | Stem cells of the adult mammalian brain and their niche[END_REF], HOPX Creert2 [START_REF] Takeda | Interconversion between intestinal stem cell populations in distinct niches[END_REF] and Rosa-EYFP mice [START_REF] Srinivas | Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus[END_REF].

Experimental procedures

Animals and Ethics

Plasmid Preparation and Electroporation

Plasmids used for electroporation are listed in the Supplemental Experimental Procedures.

Plasmids were prepared and electroporated as previously described [START_REF] Fernández | Targeted electroporation of defined lateral ventricular walls: A novel and rapid method to study fate specification during postnatal forebrain neurogenesis[END_REF] P1 to P2 (postnatal day 1 to 2) pups as previously described [START_REF] Fernández | Targeted electroporation of defined lateral ventricular walls: A novel and rapid method to study fate specification during postnatal forebrain neurogenesis[END_REF]. In Hopx Creert2 animals subcutaneous tamoxifen (Tam; SIGMAAldrich; T5648) administration

(1 mg per pup) was performed 2 to 3 hrs after electroporation.

Primary Antibodies for Immunohistochemistry

The following primary antibodies were used for immunohistochemical procedures: Rabbit

Fluorescence-Activated Cell Sorting and qPCR

Hes5::EGFP (with C57BL/6 background) of the age P2-P4 were used for sorting for NSCs as previously described and using the exact same parameters [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. 4 to 5 animals of one litter was used for 1 "n" number. Microdissection of SVZ domains (dorsomedial; dorso-lateral and lateral) was performed in RNase free, sterile conditions.

Microdissection of SVZ domains (dorso-medial; dorso-lateral and lateral) was performed in RNase free, sterile conditions. Tissues were dissociated using a trypsin-based Neural Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). For additional purification of the Hes5-EGFP population, an APC conjugated NSC antibody against the transmembrane-protein prominin-1 (1:100; ebiosciences) was applied for 15 mins at RT, before suspension was subjected to Fluorescence Activated Cell Sorting (FACS Aria III;

BD Bioscience, Franklin Lakes, New Jersey, USA). Dead cells were excluded by forward and sideward scatter. Gating settings were gained using an EGFP-wildtype animal and a prominin-1 isotype control conjugated to APC (rat anti-IgG; 1:100, ebiosciences).

Brightest 30% of EGFP + cells, which where prominin-1 + were collected directly into RNA lysis buffer and snap-frozen for further gene expression analysis. RNA extraction was performed using the RNeasy microkit (Qiagen; 74004) following manufacturer's guidelines. RNA amplification of 3 ng input material was done using the Nugene Pico Ovation WT kit (NuGen Technologies, Inc., San Carlos, CA) as described previously [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. qPCR was performed according to the procedures described elsewhere (Azim et al., 2012b[START_REF] Azim | Persistent Wnt/β-catenin signaling determines dorsalization of the postnatal subventricular zone and neural stem cell specification into oligodendrocytes and glutamatergic neurons[END_REF] (Azim et al., 2012b, Azim et al., 2014b)

Meta-Analysis of Transcriptional Profiles

To generate the lists of TFs that are enriched in dNSCs and lNSCs, we made use of previously published datasets [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF][START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF], accessible on the Gene Expression Omnibus database (GEO: GSE60905 and GSE9566). We analyzed them on the "Gene Expression Omnibus" (https://www.ncbi.nlm.nih.gov/geo/) for transcripts that are differentially expressed between dNSCs and lNSCs (≥1.8 fold enrichment and p-values <5%). Finally, we selected transcripts for transcription factor activity and regulation of transcription using "DAVID Analysis Wizard"

(https://david.ncifcrf.gov/). Lists of transcripts were analyzed for enrichments in the neuronal, astrocytic or oligodendrocytic lineage using the transcriptional dataset of the Barres group [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF]GSE9566). Heatmaps were produced using a self-made R script "Heatmap Generator" which is described below.

Heatmap Generator

We developed a tool to combine different transcriptomic datasets available via the "Gene Expression Omnibus" (GEO) and generate heatmaps named HeatMap Generator. This tool is a self-made R script that can be run as a local application. The procedure involves to compile on the one hand the datasets of interest with their GSE references, and on the other hand the genes of interest. Both files should be saved as csv files with the same name and placed in a DataSet and Genes folders accordingly. Then the application GSEtoHeatmaps.sh can be launch. The software will interrogate the GEO database and generate heatmaps of pre-selected genes. The R script is provided as Annex 1.

Meta-Analysis of Single-Cell RNA Sequencing datasets

We analyzed the transcriptomes of embryonic cortical cells (E15.5 and E17.5) produced by Yuzwa et al. (Yuzwa et al. 2017, GEO: GSE107122.). The Seurat bioinformatic pipeline was used for the analysis as follow. We first created a "Seurat object" including cells expressing more than 100 genes and genes expressed in more than 2 cells. We next performed a Principal Components Analysis (PCA), using the prcomp function of R, after scaling and centering of the data across these genes. This ensured robust identification of the primary structures in the data. We identified statistically significant principal components and used the most significant genes for each of these PCs as input for t-Distributed Stochastic Neighbour Embedding (t-SNE, R package, "perplexity parameter" = 30). To identify clusters, a graph-based clustering approach was used. For the subclustering analysis of E17.5 RGCs only we used the astrocyte marker genes described in [START_REF] Zywitza | Single-cell transcriptomics characterizes cell types in the subventricular zone and uncovers molecular defects underlying impaired adult neurogenesis[END_REF]

Results

Hopx Is Enriched in NSCs of the dSVZ and in Cells of the Astrocytic Lineage.

In a previous study, we examined the transcriptome of spatially distinct domains of the postnatal SVZ and revealed differential transcriptional networks in region-specific NSCs [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. This heterogeneity was explored further by analysis of TFs and transcriptional regulators (termed hereafter as TFs) as well as their association with defined neural lineages. 7D). Among transcripts enriched in dNSCs (Figure 8B), 5 out of the top 10 (NeuroD6, Eomes, NeuroD1, Tbr1, Neurog2) are known major determinants of the glutamatergic neuronal lineage (Schuurmans et al., 2004;Hevner et al., 2006;[START_REF] Brill | Adult generation of glutamatergic olfactory bulb interneurons[END_REF][START_REF] Winpenny | Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells[END_REF]. In addition, a meta-analysis was performed on publicly available datasets of isolated glial cells and neurons for the characterization of TFs into defined neural lineages [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF]. Interestingly, this analysis revealed that TFs enriched in dNSCs formed at least three clusters corresponding to astroglia (18/61), neurons (15/61), and oligodendroglia (11/61; Figure 8E). In contrast, lNSCs enriched TFs identified a single large neuronal cluster (42/51), while those of oligodendroglia were relatively fewer (3/51) and the astroglial cluster was absent (Figure S1E). These observations underline the greater diversity of lineages arising from the dSVZ, whereas the lSVZ generates almost exclusively interneurons (reviewed in [START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF][START_REF] Azim | Mosaic subventricular origins of forebrain oligodendrogenesis[END_REF].

We then focused our analysis onto HOPX, an atypical homeodomain protein, which was notably enriched in both dNSCs (rank 7; 7-fold enriched in dNSCs) and the astrocytic lineage (Figures 8A, 8B, 8D, and8E). Immunodetection of HOPX confirmed that it was not expressed in migrating neuroblasts (DCX + ) of the RMS nor in OLIG2 + oligodendrocytes of the corpus callosum (CC; Figures 8F and8G). In contrast, HOPX was expressed by astrocytes in the CC (glial fibrillary acidic protein [GFAP] + ; Figure 8H). In the dSVZ, HOPX expression was evident in astrocyte-like lineages while absent in the other lineages (Figures S1F-S1H), in agreement with the transcriptional meta-analysis (Figure 8E).

Such an expression pattern supports an early expression of HOPX and its association with

the astroglial lineage.

HOPX Expression Reveals Intraregional Heterogeneity within the dSVZ

We next focused our analysis on HOPX expression within the dSVZ. Using two different antibodies, HOPX protein expression was found to be restricted to the dSVZ, while it was consistently absent from its lateral counterpart (Figure 9A; see also Figure S2).

A high HOPX expression was already detectable throughout the dorsal region of the VZ/SVZ at E16. At early postnatal time points (postnatal day 1 [P1] and P4), its expression remained high but declined sharply thereafter in the young adult SVZ. Throughout its period of expression, a clear mediolateral gradient persisted, with the highest expression observed in the medial aspects of the dorsal wall and declining in its lateral aspects (i.e., high medial-to-lateral expression), which has not yet been described for any other gene (Figure 9A).

To further investigate HOPX expression within dNSCs we used the HES5:EGFP mouse line, which efficiently labels NSCs, as previously reported [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]Giachino et al., 2014). Quantification of HOPX expression in the dSVZ in this mouse line revealed that a large proportion (70.1% ± 5.0%) of EGFP + NSCs expressed HOPX. Due to the notable mediolateral gradient of HOPX expression in the dSVZ, quantifications of EGFP + /HOPX + cells were performed in the medial and lateral subdomains. There was a significantly higher overlap in the medial subdomain of the dSVZ (90.9% ± 6.6%; dmSVZ) compared with the lateral subdomain (53.7% ± 6.2%; dlSVZ; Figure 9B). These results were confirmed by performing electroporations (EPOs) of a GFP-encoding plasmid into the dSVZ of pups, which were sacrificed 8 hr later. This enables labeling of cells in direct contact with the lumen of the ventricle, i.e., those with radial glia (RG) morphology, as previously described [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]Tiveron et al., 2017). Similar results were obtained with 76.0% ± 2.4% of the electroporated (GFP + ) cells expressing HOPX, which were significantly larger in the dorsomedial subdomain compared with its dorsolateral counterpart (93.0% ± 2.4% versus 69.3% ± 3.4%; Figure 9C).

Taken together, the gradient of HOPX expression in dNSCs demonstrates intraregional

NSC heterogeneity within the dSVZ wall and proposes its regulation of NSC identity and fate.

The dSVZ Is Defined by Microdomains with Distinct Lineage Outputs

Both the astrocytic expression of HOPX and its regional enrichment within a subpopulation of dNSCs suggest further transcriptional heterogeneity within the dSVZ microdomains. To test this hypothesis, we took advantage of the HES5:EGFP mice and microdissected the medial and lateral parts of the dSVZ (dmSVZ and dlSVZ, respectively) and isolated the NSCs (dmNSCs and dlNSCs, respectively) based on their EGFP expression, as previously described [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. qRT-PCR was performed to compare the expression of lineage-specific markers in regional NSC populations (Figures 10A and10B).

We selected known transcripts enriched in NSCs as well as in defined neural lineages [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF][START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF] and compared their expression levels by qPCR.

Measured transcripts were all enriched in NSCs compared with the dSVZ, thereby validating our fluorescence-activated cell sorting strategy. In addition, there was no overall regional enrichment of stem cell markers (Hes5, Egfp; [START_REF] Basak | Identification of self-replicating multipotent progenitors in the embryonic nervous system by high Notch activity and Hes5 expression[END_REF]) and a proliferation marker (Pcna) within the different NSC populations, although the neuroblast marker Dcx was enriched in lNSCs, consistent with a greater number of neuroblasts generated by this SVZ microdomain [START_REF] Yang | Distribution of Doublecortin Expressing Cells Near the Lateral Ventricles in the Adult Mouse Brain[END_REF]; Figure 10A, top panel). Markers for the astroglial, oligodendroglial, and neuronal lineages were confirmed using the transcriptional datasets from the Barres group [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF]; Figure S3). Markers In contrast, the population of GFP + astrocytes in the vicinity of the LV (assessed by morphology and GFAP expression; Figures 3H and3I), was substantially larger following dmEPO than dlEPO (34. 6.5 ± 6.3 versus 16.5 ± 4.1; Figure 10J). Taken together, our data reveal that dmNSCs are primed to generate astrocytes (74.9% ± 3.0% astrocytes versus 25.1% ± 3.0% neurons), whilst dlNSCs generate largely olfactory neurons (15.2% ± 3.8% astrocytes versus 84.8% ± 3.8% neurons; Figure 10K).

Altogether, these findings demonstrate a high degree of heterogeneity within the dSVZ in containing specialized NSC populations that generate either astrocytes or neurons according to their location.

HOPX-Expressing dNSCs Are Biased to Acquire an Astroglial Fate

To confirm a direct relationship between HOPX expression and the generation of distinct neural lineages by dNSCs, we fate-mapped HOPX-expressing NSCs. To this end, we coelectroporated an inducible fluorescent plasmid (pFloxpA-DsRed) with an EGFP plasmid (pCX-GFP) in the dSVZ of P1 HOPX Creert2 mice and analyzed brain sections at 7 and 21 dpe (Figures 11A and11B). Tamoxifen-mediated activation of the CRE-recombinase in HOPX + NSCs led to DsRed/GFP co-expression (hereafter termed dsRed) in electroporated cells and their progenies, whilst the HOPX -lineage expressed EGFP only (Figure 11C). Electroporated cell distribution and fate were assessed at both time points on serial sections encompassing the LV and the OB (Figures 11D and11E). These results revealed the presence of DsRed + and GFP + cells at both 7 and 21 dpe (Figure 11F).

Remarkably, while the majority of GFP + cells were found in the OB (7 dpe: 66.2% ± 2.1%; 21 dpe: 77.7% ± 2.5%) and acquired the typical morphology of granule neurons, the majority of DsRed + cells remained in close proximity to the dSVZ, i.e., in the CC and the cortex (7 dpe: 64.6% ± 5.6%; 21 dpe: 63.8% ± 6.8%) at both time points (Figure 11G).

The distinct neural fates adopted by HOPX + or HOPX -NSCs were examined by immunolabeling of GFAP in DsRed + and GFP + -expressing progenies in the periventricular regions at 21 dpe. This analysis confirmed that the generation of GFAP + astrocytes produced by HOPX-expressing NSCs was approximately twice as large compared witth dNSCs that do not express HOPX (59.2% ± 7.1% versus 32.8% ± 4.3%, Figure 11H).

These findings confirm HOPX as a marker for a subpopulation of dNSCs primed for astrogenesis.

Hopx-expressing RGCs are Present in the Late Developing pallium and Share Transcriptional Featrues with adult Astrocytes

We next wondered if a subpopulation of dNSCs primed for astrogenesis is already present during the embryonic period of cortical neurogenesis. To address this question, we took advantage of two recently published single-cell RNA sequencing datasets ( [START_REF] Yuzwa | Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling[END_REF][START_REF] Zywitza | Single-cell transcriptomics characterizes cell types in the subventricular zone and uncovers molecular defects underlying impaired adult neurogenesis[END_REF]. We used the dataset of Yuzwa and colleagues, consisting of a single cell RNA sequencing analysis of cortical cells at distinct embryonic timepoints, to isolate E15.5 and E17.5 radial glia cells. We next used the dataset of Zywitza and colleagues, consisting of a large scale single-cell RNA sequencing of the entire SVZ, to isolate the list of markers associated to niche astrocytes.

We first focused our analysis on E15. [START_REF] Annexes | [END_REF]

and E17.5 isolated cortical cells. Genes with high variance were used to compute principal components as input to visualize cells in two dimensions using t-distributed stochastic neighbor embedding (t-SNE). To identify RGCs at both time points we identified cells with Sox2 expression, representing Sox2 relative expression as a color gradient, with grey indicating no detectable mRNA and red indicating

the highest mRNA level detected (Figure 12A). This resulted in the identification of 334 radial glial cells at E15.5 and 76 at E15.5. We combined the 2 populations together (Figure 12B) and identified differentially expressed genes (386 and 639 enriched genes at E15.5 and E17.5 respectively, bimod test and threshold 0.25). A functional clustering analysis was performed using the DAVID software, which revealed enrichment of cell cycle related genes (Figure 12C) and neuronal related genes (Figure 12D) in E15.5 RGCs, whereas astroglial-related genes such as Sparcl1 were enriched in E17.5 RGCs (Figure 12E, Annex 3), in agreement with the neurogenic to gliogenic switch occurring at this late embryonic timepoint. These results are consistent with the known developmental trajectories described during development. We next asked whether subpopulations of RGCs coexist at E17.5. We first attempted at identifying RGCs clusters using the graph-based clustering method from the Seurat R package. However, no significant cluster emerged through this unbiased strategy (Not shown). We next reasoned that if a subpopulation of RGCs was biased to produce astrocytes, they should share some of the genes associated with adult astrocytes. We used a list of 254 astrocytes specific genes (Annex 4), described in a recent single-cell RNA sequencing study of the entire SVZ [START_REF] Zywitza | Single-cell transcriptomics characterizes cell types in the subventricular zone and uncovers molecular defects underlying impaired adult neurogenesis[END_REF] (Figure S4A), to visualize cells in a new t-SNE. The graph-based clustering method identified 2 clusters (Figure 12F). Strikingly, 44% of the Zywitza astrocyte markers were present in cluster 1 enriched genes, such as the classical astrocyte marker Aldh1l1 (Figure 12GH), whereas only 7% were present in cluster 2 enriched genes. This result suggest that cluster 1 contains RGCs primed toward the astroglial lineage. Remarkably, enriched genes in cluster 1 which contains Aqp4, ApoE, Aldoc, Id3, Clu (Figure S4B and Annex 5) also include Hopx. This indicates that Hopx defines a subpopulation of pallial RGCs primed towards astrogenesis, as early as E17.5 a timepoint corresponding to the neurogenic to gliogenic switch.

Discussion

In this study, the expression hallmarks of TFs that regulate distinct neural lineages were further characterized for their enrichment in specific microdomains of the SVZ. By selecting one of these transcripts, we show that NSCs are spatially segregated and primed to differentiate toward specific neural fates. Our results identify Hopx as a gene that unravels further heterogeneity of the dSVZ in mediating aspects of astrogenesis and suggest its association with the emergence of germinal traits observed in higher-order mammals.

The diversity of neural subtypes generated by SVZ-NSCs after birth is much larger than first believed. The concept of SVZ regionalization whereby the genesis of distinct neural lineages are spatially and temporally regulated is being increasingly investigated (reviewed in [START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF][START_REF] Azim | Mosaic subventricular origins of forebrain oligodendrogenesis[END_REF]. NSCs located in SVZ microdomains originate from specific regions of the developing forebrain (Fuentealba et al., 2015a), and generate a large diversity of neural cells, including neuronal subtypes, depending on the expression of particular transcriptional programs. Consequently, the expression of TFs directly correlates with the acquisition of defined neural fates, a concept that was explored in the present study.

We took advantage of the whole-genome transcriptome of region-specific postnatal NSCs that were recently resolved [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. A meta-analysis of TFs expressed in dorsal and lateral NSCs with datasets of isolated forebrain neuronal and glial subpopulations [START_REF] Cahoy | A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function[END_REF] expression is observed in a subpopulation of NSCs that mainly produces astrocytes, it cannot be considered a pan-astrocytic marker. Indeed, HOPX expression is spatially restricted and is therefore likely to be associated with the generation of a subpopulation of astrocytes. Fate-mapping studies revealed that astrocytes are allocated to spatial domains in accordance with their embryonic sites of origin in the ventricular zone [START_REF] Tsai | Regional astrocyte allocation regulates CNS synaptogenesis and repair[END_REF].

Furthermore, transcriptomic analysis of astrocytes isolated from various brain regions reveals heterogeneous expression of several astrocytic markers. For instance, HOPX was shown to be enriched in astrocytes of the dorsal forebrain (cortex and hippocampus) and lowly expressed in astrocytes of subcortical regions (thalamus and hypothalamus;[START_REF] Morel | Molecular and Functional Properties of Regional Astrocytes in the Adult Brain[END_REF]. Astrocyte heterogeneity in the CNS has recently been described to influence neuronal synaptogenesis and maturation through secretion of several extracellular matrix proteins [START_REF] Eroglu | Regulation of synaptic connectivity by glia[END_REF]. In addition, the densities of astrocytes vary greatly between brain regions [START_REF] Azevedo | Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain[END_REF]. The role of HOPX in influencing regional astrocytes properties and/or densities remains to be explored.

Recently, a number of key studies have reported HOPX expression in human oRG [START_REF] Pollen | Molecular Identity of Human Outer Radial Glia during Cortical Development[END_REF]Thomsen et al., 2015b). Furthermore, ectopic overexpression of the hominoidspecific gene Tbc1d3 into RG in rodents induces HOPX-expressing oRG that contribute to cortical folding [START_REF] Ju | The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice[END_REF]. In line with these observations, our overexpression results (not presented here, see Zweifel et al. 2018) suggest an instructive role for HOPX in oRG cell formation.

The mechanisms by which HOPX mediates its functions remain largely unknown. HOPX is an atypical TF that does not bind to DNA directly, but modulates other TFs and/or effectors of signaling pathways at the posttranscriptional level. An interaction of HOPX with SRF has been demonstrated during cardiac development [START_REF] Shin | Modulation of cardiac growth and development by HOP, an unusual homeodomain protein[END_REF], but is unlikely to occur in the SVZ where SRF expression remains low (data not shown). A more likely function of HOPX is the modulation of dorsally active signaling pathways, such as the BMP and WNT pathways (see also [START_REF] Azim | Persistent Wnt/β-catenin signaling determines dorsalization of the postnatal subventricular zone and neural stem cell specification into oligodendrocytes and glutamatergic neurons[END_REF]Azim et al. 2017), which have been demonstrated to fine-tune astrogenesis with neurogenesis during corticogenesis [START_REF] Gross | Bone morphogenetic proteins promote astroglial lineage commitment by mammalian subventricular zone progenitor cells[END_REF][START_REF] Takizawa | DNA Methylation Is a Critical Cell-Intrinsic Determinant of Astrocyte Differentiation in the Fetal Brain[END_REF][START_REF] Tiberi | Cortical neurogenesis and morphogens: Diversity of cues, sources and functions[END_REF]. Reciprocal signaling between BMP and WNT has been reported in multiple progenitor populations [START_REF] He | BMP signaling inhibits intestinal stem cell selfrenewal through suppression of Wnt-β-catenin signaling[END_REF][START_REF] Plikus | Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration[END_REF][START_REF] Kandyba | Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation[END_REF][START_REF] Genander | BMP signaling and its pSMADS1/5 target genes differentially regulate hair follicle stem cell lineages[END_REF][START_REF] Song | Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells[END_REF],

and may be integrated by HOPX expression, as recently demonstrated in cardiomyoblasts [START_REF] Jain | Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts[END_REF]. Future studies aimed at manipulating the activity of these two signaling pathways in HOPX KO animals may allow us to address these questions and investigate the role of extrinsic signal integration in lineage fate specification of neighboring populations of NSCs.

It is interesting to speculate that other signaling pathways may influence the pattern of HOPX expression and may be implicated in its evolution in primates. Intriguingly, HOPX expression in the mouse SVZ follows the spatiotemporal maturation of ependymal cells, which may gradually restrict RG cell contact with the cerebrospinal fluid [START_REF] Mery | Resectión de lesiones cerebrales con asistencia de mapeo cortical intraoperatorio[END_REF].

This, combined with the expression of HOPX in oRG, which lack apical processes in primates, suggests that an unknown cerebrospinal fluid-derived signal may regulate HOPX expression. Such signals might modulate SHH signaling that has been recently identified in regulating oRG cell formation [START_REF] Wang | Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex[END_REF] Rescuing experiments show that postnatal progenitors are partially permissive to genetic and pharmacological manipulations.

Introduction

During neocortical development, glutamatergic neurons are born from progenitors (Glu progenitors) located in the ventricular zone (VZ) and subventricular zone (SVZ) and assemble to form the circuits that underlie cognitive functions. It is classically accepted that the period of cortical neurogenesis closes around embryonic day (E)17.5 in the mouse, with neuronal progenitors switching fate to produce astrocytes [START_REF] Li | GSK3 Temporally Regulates Neurogenin 2 Proneural Activity in the Neocortex[END_REF].

However, a significant fraction of neural progenitors do not switch fate. For instance, a population of progenitors remain in the postnatal SVZ, contributing to olfactory bulb neurogenesis and parenchymal gliogenesis throughout life (Doetsch et al., 1999b). At least some of these progenitors arise from slow cycling/ quiescent embryonal radial glial cells that divide between E13.5 and E15.5 (Fuentealba et al., 2015b;[START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF].

Fate-mapping analysis demonstrated that they give rise to distinct neuronal and/or glial lineages, depending on their location in the SVZ [START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF]. Surprisingly, several reports suggest the persistence of Glu progenitors in the dorsal SVZ (dSVZ) until early adulthood [START_REF] Brill | Adult generation of glutamatergic olfactory bulb interneurons[END_REF][START_REF] Winpenny | Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells[END_REF]. We used Neurog2 Creert2 /tdTom mice to permanently and specifically label synchronous cohorts of prenatal and postnatal Glu progenitors to study their lineage relationship and transcriptional specificities. Our results

show that Glu progenitors continue to be produced after closure of the period of cortical neurogenesis. Single-cell RNA sequencing (scRNA-seq) reveals that postnatal Glu progenitors show dysregulation in genes involved in metabolism, differentiation, and migration, which parallels a rapid decline in their capacity to migrate and differentiate. Our data suggest that this transcriptional dysregulation in postnatal Glu progenitors may result from decreased N6-methyladenosine (m6A) methylation of certain proneural genes.

Nevertheless, postnatal Glu progenitors remain partially amenable to pharmacological and genetic manipulations.

Experimental Procedures

Ethical Statement

All animal experiments were performed in accordance with international guidelines from the EU directive 2012/63/EU and approved by the Animal Care and Use Committee CELYNE (APAFIS#187 & #188).

Animals

The Neurog2 Creert2 transgenic mouse line was crossed with the reporter line RosatdTomato (tdTom) (Madisen et al., 2010), allowing the specific labeling of Glu progenitors and their immediate progeny. For fate mapping of birthdated cohorts of Glu progenitors, tamoxifen was administered to Neurog2 Creert2 /tdTom transgenic mice at various embryonal and postnatal time points. The morning when a plug was observed was considered as E0.5, and the day of birth was defined as P0.

Tamoxifen injections

Tamoxifen St. Louis Missouri,USA) and progesterone (Sigma-Aldrich, P8783-1G) were dissolved in Corn oil (Sigma-Aldrich, C8267) at final concentration of 20mg/ml and 10mg/ml, respectively. Pregnant Neurog2 Creert2 / tdTom females were administrated 0.5mg tamoxifen and 0.25mg progesterone by gavage at E13.5, E15.5 or E17.5. The morning when a plug was observed was considered as E0.5

and the day of birth is defined as P0. Mouse pups were injected subcutaneously with 1mg tamoxifen at P1 and were terminated at P2, P3, P5, P7, P11, P21 or P45.

In Utero Electroporation

In utero electroporation was performed at E13.5 or E15. [START_REF] Annexes | [END_REF]

Immunohistochemistry

Animals were sacrificed with an overdose of pentobarbital and fixed by transcardial perfusion with PBS followed by 4% paraformaldehyde (PFA; w/v). Brains were dissected and post-fixed in 4% PFA at 4°C. Free-floating vibratome serial sections were cut at a thickness of 50 mm. Immunostainings were performed as described previously.

For immunostainings, sections were blocked in TNB solution for 2 hrs at room temperature and incubated overnight at 4°C with primary antibodies diluted in TNB with 0.4% Triton-X.

The following primary antibodies were used: FoxP2 (rabbit, 1:1000, Millipore), Cux1

Quality control, identification of outliers and analysis

The obtained RNA-seq data were prepared and analyzed as previously described [START_REF] Telley | Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex[END_REF]. Briefly, sequenced reads were aligned on the latest mouse reference genome assembly (GRCm38) using STAR (Spliced Transcripts Alignment to a Reference) [START_REF] Dobin | STAR: Ultrafast universal RNA-seq aligner[END_REF] (See summary report in Table 1) and the number of reads per transcript calculated with FeatureCounts [START_REF] Liao | FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features[END_REF]. For each cell, Reads per Million (RPM) are divided by the length of the gene to obtain normalized Reads Per Kilobase Million (RPKM) and transformed in log2 when necessary. Identification of progenitors, nascent neurons, and transcripts was performed using the Seurat bioinformatic pipeline as follows:

We first created a "Seurat object" including cells expressing more than 500 genes and genes expressed in more than 2 cells, identifying 11.718 genes across 228 cells. We next performed a Principal Components Analysis (PCA), using the prcomp function of R, after scaling and centering of the data across these genes. This ensured robust identification of the primary structures in the data. We identified statistically significant principal components and used the most significant genes for each of these PCs as input for t-Distributed Stochastic Neighbour Embedding (t-SNE, R package, "perplexity parameter" = 30). To identify clusters, a density clustering approach was used. Single-Cell Differential Expression (SCDE; [START_REF] Kharchenko | Bayesian approach to single-cell differential expression analysis[END_REF]) was used to identify differentially expressed genes across progenitors and nascent neurons between developmental time-points (cut off: absolute Z score ≥ 2, corresponding to a classical P value ≤ 0.05).

Table 1. Sequencing Report. Sequencing depth (Reads/cell) and % of assigned reads are consistent in P2 and E15.5 progenitors.

Gene ontology enrichment analyses

GO analysis were performed in identified dysregulated genes with the GO biological process complete annotation set from PANTHER (v12) (http://pantherdb.org/). Kegg pathway analysis was performed with a homemade R code querying the Kegg pathway database (http://www.kegg.jp/kegg/pathway.html). The proteinprotein interaction (PPI) analysis was performed in differentially regulated transcripts using Enrichr web-based tools [START_REF] Chen | Enrichr: Interactive and collaborative HTML5 gene list enrichment analysis tool[END_REF][START_REF] Wang | Real-time quantitative description of gas invasion in deepwater drilling[END_REF] (http://amp.pharm.mssm.edu/Enrichr/).

Data and Software Accessibility

The accession numbers for the data reported in this paper are GEO: GSE109556 and have been posted to Mendeley at https://doi.org/10.17632/k659jr9gvv.1.

m6A dot blot assay

The pallium and dSVZ were isolated (n=4) at E15. 5 and P2,respectively. mRNA

Bcl11a gain of function

Briefly, P2 mouse pups were anesthetized on ice and were fixed on a plate placed in a stereotaxic rig. A mixture of Bcl11a or control plasmid (LacZ) (final concentration 3μg/μL) and the dye Fast Green (0.2μL) was prepared. Injection (2μL) was done at the level of the right lateral ventricle at a depth of 2mm from the surface of the skull using a Hamilton syringe with a 34G needle (Nevada, USA). Successfully injected mice were subjected to five electrical pulses (95V, 50ms, separated by 950ms intervals) using the ECM CUY21 type 2 electroporator (NepaGene, Chiba, Japan).

Results

Fate Mapping of Birth-Dated Cohorts of Glutamatergic Neurons

The recombination efficiency and specificity of the Neurog2 Creert2 /tdTom mice was verified by injecting tamoxifen (Tam) at different embryonal time points (i.e., E13.5 and E15.5) and examining brains after 12 and 24 hr (Figures 13A and13B).

The recombined cells initially expressed the Glu progenitor marker, Tbr2, and the proliferation marker Ki67, and rapidly translocated from the VZ to the cortical plate. Fate mapping of recombined cells was assessed at postnatal day (P)21. Tam injections at either E13.5, E15.5, or E17.5 labeled neurons in subcortical and cortical brain regions in accordance with their date of birth (Figure S5).

To confirm the precise labeling of birth-dated cohorts of glutamatergic neurons, we performed a more detailed analysis of recombined cells in the cortex. Tam injection at E13.5 labeled neurons in the deep cortical layers (i.e., L5-L6), which expressed the deep cortical layer marker FoxP2, (Figures 13C-13E).

In contrast, recombination at E15.5 labeled neurons in L3-L4, which expressed the upper layer marker Cux1 (Figures 13C-13E). Importantly, only a few glial-like cells were labeled (<1% of total tdTom + cells) from E15.5 on. These glial cells were found in clusters, suggesting that only a very limited number of progenitors switched from a glutamatergic to an astrocytic fate (Figure S5). Altogether, these observations confirm that Neurog2 Creert2 /tdTom mice allow the specific labeling of birth-dated cohorts of glutamatergic neurons.

Recombined cells were confirmed to be Glu progenitors by their expression of Tbr2

(Figure 13G) but exclusion of other lineage markers (e.g., Olig2). Twenty-four hours following recombination, 20% of Glu progenitors were still actively cycling, but most had exited the cell cycle by P3 (Figure 13H).

To follow the long-term fate of these progenitors, we injected Tam in P0.5 pups and sacrificed them at various time points. At P2, most recombined cells were observed in the dSVZ, but their number gradually decreased with age, disappearing by P21 (Figure 13I).

In parallel, Glu progenitors gave rise to a cohort of migrating neurons that transiently increased in the corpus callosum and cingulum, as they left the dSVZ and migrated toward the cortex (Figures 13I and13J). At P21, tdTom + neurons were observed in the cortex, in the dentate gyrus, and in some subcortical nuclei (Figure 13J; Figure S6B).

The number of recombined cortical neurons increased from frontal to caudal brain sections (Figure S6C), with most neurons located in sensory rather than motor regions (Figure S2D). A marked decrease in the number of recombined neurons was observed over time.

Indeed, comparison of the number of tdTom + cells at short and long survival times (i.e., 12 hr post-Tam injection or at P21, respectively), revealed a large decrease (81.3%) following a P0.5 Tam injection. In comparison, a marginal drop was observed in the number of cells recombined at E15. 5 (11.8%), indicating that while most recombined embryonal progenitors produce neurons that efficiently survive until P21, only a fraction do so at postnatal time points.

We next looked in more detail at the location and phenotype of recombined cortical neurons. Most recombined glutamatergic neurons were located in upper cortical layers L2-L3, and a few were located in L5-L6 (Figures S7A-S7D). By P45, most recombined neurons expressed the marker Satb2. While recombined neurons located in L2-L3 also expressed the upper layer marker Cux1, those in L5-L6 did not express the markers of deep cortical layers, Ctip2 or FoxP2 (Figures 13K and13L; Figure S7E).

By P45, the decrease in number of recombined neurons was more pronounced in deeper cortical layers than in superficial layers (Figure 13M), as around 1% of the total number of tdTom + neurons were located in L5-L6 (i.e., 30-48 cells per brain). This decreased survival correlated with a slower maturation of surviving neurons in deep cortical layers (Figures 13N and13O). Nevertheless, surviving glutamatergic neurons of both upper and deep layers developed spines (Figure S8A), thereby suggesting proper integration of newborn glutamatergic neurons. In contrast to neurons produced at E13.5, which formed corticofugal (i.e., internal capsule or corticospinal) projections, postnatally born neurons only formed intracortical projections, as shown by the presence of axons in the corpus callosum (Figure S8B).

Taken together, these results reveal that closure of the cortical neurogenic period is not due to the disappearance of Glu progenitors but rather to a gradual decline in their capacity to differentiate and survive.

ScRNA-Seq Reveals Transcriptional Dysregulation in Postnatal Glu Progenitors

We next investigated the origin and transcriptional specificities of postnatal Glu progenitors. Injection of Tam at E13.5 or E15.5 failed to label Glu progenitors at P2 (Figures 14A-14F). In contrast, in utero electroporation of a transposon-GFP plasmid [START_REF] Siddiqi | Fate mapping by piggybac transposase reveals that neocortical glast+ progenitors generate more astrocytes than nestin+ progenitors in rat neocortex[END_REF] at either E13.5 or E15.5 revealed a large pool of GFP + cells that persisted in the postnatal dSVZ and continued generating proliferating Tbr2 + cells at P2

(Figures 14G and14H) as well as at P21 (Figures 14I and14J).

Together, these results indicate that postnatal Glu progenitors do not accumulate during embryonic development but are produced by embryonic radial glial cells (RGCs) that transcripts, such as Neurog2 and Tbr2, confirmed their Glu identity (Figure 15C).

Transcriptional differences increased during differentiation, with 597 (8%) and 1. 425 (20%) genes being differentially regulated between E15.5 and P2 progenitors and nascent neurons, respectively (Figures 15D and15E; see Table S1 in online supporting material of the article and see https:// genebrowser. lyon.inserm.fr/ for an interactive dataset). Thus, while the vast majority (90%) of E15.5 top 1,000 expressed genes were also expressed in P2 progenitors, only 40% of the E15.5 top 1,000 genes were similarly expressed in nascent neurons at P2 (Figure 15F).

Interestingly, transcripts enriched in Glu progenitors (i.e.,Sox2,Pax6,Neurog2,and Tbr2) were upregulated at P2, suggesting a transcriptional dysregulation leading to the persistence of progenitor traits in nascent neurons (Figure 15G).

Knowing that m6A methylation plays a role in cortical neurogenesis by regulating the expression of proneural transcripts [START_REF] Yoon | Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation[END_REF], we assessed whether this mRNA modification was decreased in postnatal Glu progenitors. Transcriptional analysis confirmed a downregulation of transcripts coding for proteins of the methyltransferase complex, Mettl3 and Mettl14 (Figure 15G), while expression of YTHDF2, which has been shown to localize m6A-tagged transcripts to decay sites [START_REF] Jain | Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts[END_REF], was not changed (data not shown). To assess m6A methylation level in Glu progenitors, we performed an mRNA dot blot assay to detect m6A modifications (Figures 15H and15I).

Our results reveal a 65% decrease in m6A level in the postnatal dSVZ, compared to the E15.5 pallium.

All together, these data suggest that epitranscriptomic modifications participate in the transcriptional dysregulation observed in postnatal Glu progenitors and their immediate progeny.

Postnatal Glu Progenitor Differentiation Can Be Partially Rescued

We next focused on the transcriptional specificities of P2 Glu progenitors and nascent neurons. Classification of transcripts differentially expressed in Glu progenitors highlighted depressed transcriptional and metabolic activities at P2 that correlates with reduced potential of Glu progenitors to differentiate postnatally (Figure 16J). Classification of differentially expressed transcripts in nascent neurons highlighted downregulation of generic programs of differentiation and migration, and an upregulation of genes involved in neuronal death (Figure 16K), in line with our histological observations. A KEGG analysis further highlighted paralleled perturbation of several key signaling pathways, such as Wnt canonical signaling (Figure 16L), as previously suggested [START_REF] Li | GSK3 Temporally Regulates Neurogenin 2 Proneural Activity in the Neocortex[END_REF].

A more detailed analysis into downregulated GO terms, revealed a decrease in several genes, including Ctnnb1 (i.e., β-catenin) and other Wnt signaling transcripts such as, Fzd1

and Lef1 (Figures 16A and16B). Protein-protein interaction (PPI) analysis further emphasized the central role of β-catenin, as most of the downregulated genes at P2 encode for proteins that interact with it (Figure 16C).

To assess if migration and differentiation of postnatal Glu progenitors could be reactivated,

we performed experiments to rescue downregulated signaling and transcriptional pathways. We first used a glycogen synthase kinase 3β (GSK3β) inhibitor, AR-A014418, to activate the canonical Wnt signaling pathway. Our results showed increased tdTom + cells in the dSVZ, which was paralleled by increased proliferation (Figures 16D-16F).

Next, we selected Bcl11a as a candidate gene for overexpression, as this transcription factor is among the top 10 differentially expressed (i.e., downregulated) transcription factors at P2 (Figure 16G). Bcl11a has been shown to regulate both migration and fate specification during embryonic development [START_REF] Wiegreffe | Bcl11a (Ctip1) Controls Migration of Cortical Projection Neurons through Regulation of Sema3c[END_REF][START_REF] Greig | Ctip1 Controls Acquisition of Sensory Area Identity and Establishment of Sensory Input Fields in the Developing Neocortex[END_REF].

Overexpression of Bcl11a increased proliferation in the dSVZ (Figure 16H). In addition, our results show a significant increase in migration towards the cortex and increased number of cells that adopted bipolar morphologies (Figures 16H-16J).

Together, these results demonstrate that Glu progenitors remain permissive to both pharmacological and genetic manipulation.

Discussion

Our work highlights the persistence of a large population of Glu progenitors in the postnatal forebrain, after the closure of the cortical neurogenic period. Fate mapping and scRNAseq reveal a dysregulation of transcriptional and signaling pathways that contribute to restricting the neurogenic potential of postnatal Glu progenitors early after birth. Rescuing experiments, however, show that postnatal progenitors remain partially permissive to genetic and pharmacological manipulations, suggesting that they could be recruited for cortical repair. The perinatal period has long been considered as a period of exclusive cortical gliogenesis associated with the maturation of embryonically born neurons into functional circuits. This view is currently challenged by the demonstration of neurogenesis in specific cortical regions. Thus, GABAergic progenitors accumulate in the early postnatal white matter and give rise to a subpopulation of cortical interstitial interneurons [START_REF] Frazer | Transcriptomic and anatomic parcellation of 5-HT3AR expressing cortical interneuron subtypes revealed by singlecell RNA sequencing[END_REF]. In addition, the migration of interneurons into the frontal cortex has been shown to persist early after birth in rodents [START_REF] Inta | Neurogenesis and widespread forebrain migration of distinct GABAergic neurons from the postnatal subventricular zone[END_REF][START_REF] Magueresse | Subventricular zone-derived neuroblasts use vasculature as a scaffold to migrate radially to the cortex in neonatal mice[END_REF], as well as in human babies [START_REF] Nogueira | Letter: Extensive Migration of Young Neurons into the Infant Human Frontal Lobe[END_REF].

Our results reveal that this persisting neurogenesis is not restricted to the GABAergic lineage but also includes neurons of the glutamatergic lineage. Indeed, our work identifies a small population of cortical Satb2/Cux1 + neurons that are generated at birth. Surviving neurons develop spines and intracortical axonal projections, supporting their integration into cortical networks. Our results further indicate that these neurons arise from a large population of pallial RGCs that do not switch fate toward astrogenesis and persist in the dSVZ. These results are in line with recent mosaic analysis with double markers (MADM) experiments suggesting that only 1 out of 6 neurogenic RGCs produce glia (Gao et al., 2014a). Our results underline a rapid decline in the capacity of Glu progenitors to differentiate and migrate, thereby contributing to the closure of the period of cortical neurogenesis.

Our scRNAseq data shed light on the mechanisms mediating this gradual loss of neurogenic potency. Epitranscriptomic changes are emerging as key mechanisms in mediating temporal control over lineage progression. m6A is the most prevalent mRNA modification in eukaryotic cells [START_REF] Desrosiers | Identification of Methylated Nucleosides in Messenger RNA from Novikoff Hepatoma Cells[END_REF] and has recently been suggested to regulate transcriptional prepatterning during corticogenesis [START_REF] Yoon | Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation[END_REF]. Our results identify m6A methylation as a possible mechanism leading to the transcriptional dysregulation that we observe in postnatal Glu progenitors. In addition to these epitranscriptomic modifications, KEGG pathway analysis highlights changes in several key signaling pathways, such as those involved in astrogenesis (i.e., Jak-Stat and Notch signaling pathways; [START_REF] Rowitch | Developmental genetics of vertebrate glial-cell specification[END_REF], suggesting that they may concomitantly affect the differentiation potential of Glu progenitors. Another signaling pathway that is dysregulated is the Wnt signaling pathway. This is in agreement with a previous study describing a gradual increase in GSK3β activity from E15.5 on, which results in the phosphorylation of Neurog2, thereby affecting its activity [START_REF] Li | GSK3 Temporally Regulates Neurogenin 2 Proneural Activity in the Neocortex[END_REF]. In line with a decreased transcriptional activity of Neurog2, 64% of its target genes [START_REF] Gohlke | Characterization of the proneural gene regulatory network during mouse telencephalon development[END_REF] are downregulated at P2, while only 2% are upregulated, despite the persistence of Neurog2 expression.

Importantly, postnatal Glu progenitors appear to be still permissive to intrinsic/extrinsic manipulation. We show that proliferation and migration of postnatal Glu progenitors can be promoted by genetic or pharmacological manipulations. Our experiments, however, reveal that these manipulations are not sufficient for promoting long-term neuron survival, suggesting that the cortex is not permissive to the integration of these newborn neurons under physiological conditions. However, recent observations suggest that permissiveness of the environment might be increased following injury, such as after neonatal chronic hypoxia, where cortical de novo neurogenesis has been observed [START_REF] Fagel | Fgfr1 Is Required for Cortical Regeneration and Repair after Perinatal Hypoxia[END_REF][START_REF] Bi | Cortical Glial Fibrillary Acidic Protein-Positive Cells Generate Neurons after Perinatal Hypoxic Injury[END_REF][START_REF] Falkner | Transplanted embryonic neurons integrate into adult neocortical circuits[END_REF]Azim et al., 2017). It is likely that our results will provide important information to guide future research in this context.

GENERAL DISCUSSION

General Discussion

Summary and Opened Questions

Germinal regions are not homogeneous, but rather highly heterogeneous with transcriptional differences driving the production of divergent cell lineages. Recently our lab contributed to resolve this heterogeneity by demonstrating an unexpected level of transcriptional heterogeneity between the dorsal and lateral subventricular zone of the postnatal mouse forebrain, as well as in their neural stem cells and transient amplifying progenitors [START_REF] Azim | Transcriptional hallmarks of heterogeneous neural stem cell niches of the subventricular zone[END_REF]. In the first chapter of my thesis, my results contributed to describe and discuss a new level of regional and lineage-specific heterogeneity in the dorsal SVZ based on Hopx expression (Chapter 1). This work support the co-existence of lineage-biased NSC, with neighboring NSCs expressing distinct transcription factors that influence their respective behaviors and guide them through the acquisition of different fates.

In the second chapter of my thesis, I continued exploring regional and lineage-specific heterogeneity in the dorsal SVZ, by characterizing progenitors of cortical glutamatergic neurons (i.e. Glu progenitors) that we have previously shown to persist after birth (Donega et al., 2018a). Progenitors of cortical glutamatergic neurons are usually thought to switch fate before birth to produce astrocytes. My results show that this switch is largely incomplete and that a large fraction of Glu progenitors continues to be produced in the postnatal forebrain after closure of the cortical neurogenesis period. Single cell RNAsequencing however reveals a profound transcriptional dysregulation at postnatal stages, which correlates with the gradual decline in cortical neurogenesis observed in vivo (Chapter 2).

Taken together, my work illustrates the existence of a profound NSCs heterogeneity in the postnatal SVZ, which can be explored from the regional to the single cell levels.

Clonal Techniques in Histology and Transcriptomic

The recent single cell studies involve different set of tools which I describe in this paragraph as a technical box.

Barcoding [START_REF] Golden | Construction and characterization of a highly complex retroviral library for lineage analysis[END_REF] Barcoded GFP-expressing retroviral library, in which infection of progenitor cells of a given region is achieved by local expression of an avian virus receptor (Fuentealba et al., 2015b;[START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF]. This approach is superior to lineage tracing methods based on the use of fluorescent reporters of different colors in that it provides the complexity in tag diversity to unequivocally demonstrate that two cells in different regions are derived from a single progenitor. It may, however, underestimate clone size and/or complexity, because of viral inactivation and partial recovery of barcode sequence in laser microdissected cells.

MADM (Gao et al., 2014b;Mayer et al., 2015a;[START_REF] Beattie | Mosaic Analysis with Double Markers Reveals Distinct Sequential Functions of Lgl1 in Neural Stem Cells[END_REF] A key feature of this approach is the ability to induce clones of distinctly labeled neurons originating from a single dividing progenitor cell in a temporally defined fashion using tamoxifen. This approach also allows introduction of gene mutations allowing clonal twocolor labeling with concomitant genetic manipulation. It however relies on transgenic animals and requires very low number of clones for conclusive analysis, which makes it both expensive and time consuming.

Multicolor Approaches [START_REF] García-Marqués | Clonal identity determines astrocyte cortical heterogeneity[END_REF][START_REF] Roy | Concise review: Understanding clonal dynamics in homeostasis and injury through multicolor lineage tracing[END_REF][START_REF] Figueres-Onãte | UbC-StarTrack, a clonal method to target the entire progeny of individual progenitors[END_REF] Spectral fluorescent protein variants are stochastically expressed in cells of interest (by transgenesis, plasmid transfection or viral transduction), which both highlight cells of a tissue and differentiate them from one another when expressed in a mosaic manner. The main limitation of this approach is that juxtaposed clones labeled with a same color (due to limited number of possible colors or of their detection) cannot be distinguished and must be taken into account by modeling approaches Single-cell RNA sequencing [START_REF] Prakadan | Scaling by shrinking: Empowering singlecell "omics" with microfluidic devices[END_REF] Single-cell RNA sequencing (scRNA-seq) allows the analysis of the transcriptome from individual cells, and is ideal to address the complexity and dynamics of transcriptional changes occurring during neural stem cell differentiation. There are multiple experimental approaches to capture and sequence single cells, which lead to different levels of cellular and tissue coverage. Currently, the two most popular technologies are the Drop-Seq [START_REF] Yuzwa | Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling[END_REF][START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF] and the integrated fluidic circuits (IFC) [START_REF] Nowakowski | Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex[END_REF]. An advantage of droplet-based approaches is their scale and speed at the expense of sequencing depth and RNA capture efficiency [START_REF] Macosko | Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets[END_REF] whereas valve-based systems (IFCs) still provide the greatest molecular efficiency and are useful for applications in which the highest-quality transcriptomes are needed at moderate scale. Original attempts at clonal RGs fate mapping made use of retroviral vectors. In the pallium, analysis of clones 1 to 3 days following viral transduction revealed that neurons migrate along clonally-related RG to form radial units. While some degree of dispersion occurs, the majority of clones populate columns thereby contributing to the functional radial organization of the neocortex [START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF]. Similar conclusions were reached in more recent studies relying on near clonal recombination [START_REF] Magavi | Coincident Generation of Pyramidal Neurons and Protoplasmic Astrocytes in Neocortical Columns[END_REF], as well as more recently by MADM clonal analysis (Gao et al., 2014a).

Visualizing and clustering tools

Similar principles are likely to apply in the subpallium. Thus, radial clusters composed of a radial cell, IPCs and newborn neurons are observed < 4 days post viral transduction at E12.5 in the MGE [START_REF] Harwell | Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons[END_REF]. Subpallial RGs appear to be the primary neural stem cells, dividing asymmetrically to produce IPCs that subsequently divide symmetrically to expand their numbers, or more frequently, to produce pairs of neurons [START_REF] Magavi | Coincident Generation of Pyramidal Neurons and Protoplasmic Astrocytes in Neocortical Columns[END_REF]Mayer et al., 2015b). Similarities however stop there, as interneurons produced by RG from distinct ganglionic eminences then migrate tangentially rather than radially to invade distinct telencephalic regions (review Hu et al., 2017a, see also [START_REF] Wichterle | In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain[END_REF]. Clonal analysis in the MGE, which contributes to 60% of cortical interneuron [START_REF] Miyoshi | Physiologically Distinct Temporal Cohorts of Cortical Interneurons Arise from Telencephalic Olig2-Expressing Precursors[END_REF], reveal a dramatic dispersion of sibling interneurons. Thus, interneurons migrate to widespread regions within the telencephalon, although they do not cross the segmental boundary of the diencephalon nor the midline [START_REF] Magavi | Coincident Generation of Pyramidal Neurons and Protoplasmic Astrocytes in Neocortical Columns[END_REF]Mayer et al., 2015b).

scRNA-Seq studies have investigated the transcriptional identity of both pallial and subpallial RGs and the lineages they are producing. A direct comparison of results obtained in various studies is made difficult by differences in the methods of cell capture, sequencing depth, and methods of analysis (Table 2). For instance, while the VZ and SVZ are separated in some studies, this is not always the case. Important and convergent conclusions however emerge from this work.

Table 2. ScRNA-Seq experiments and their parameters

First, it appears that transcriptional differences between RGs that reside in distinct pallial and subpallial regions are small and rely on a rather restricted number of genes (Novotortsev et al., 2010;[START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF][START_REF] Mi | Early emergence of cortical interneuron diversity in the mouse embryo[END_REF]. For instance, only 16 TFs are differentially expressed within mitotic progenitors of MGE vs. CGE, while the majority of dynamically expressed genes follow robust and highly reproducible sequential waves of gene expression in both eminences [START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF]. Similarly, in the pallium, RGs from the PFC and V1 cortical areas have only 68 differentially expressed genes and share a common molecular differentiation program [START_REF] Nowakowski | Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex[END_REF]. The subtle transcriptional differences which distinguish progenitors get likely superimposed onto a large pan-developmental program and therefore render the threshold of detection of progenitor subtypes rather tedious and variable between studies [START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF][START_REF] Mi | Early emergence of cortical interneuron diversity in the mouse embryo[END_REF]. A second observation is that clearly distinctive transcriptional programs are only consistently emerging at the postmitotic level when newborn neurons from distinct germinal regions initiate migration and maturation [START_REF] Nowakowski | Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex[END_REF][START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF][START_REF] Mi | Early emergence of cortical interneuron diversity in the mouse embryo[END_REF].

Altogether, these observations support a model for emergence of neuronal diversity in which small differences across cycling progenitors are sufficient to drive neuronal diversity, which amplifies over time in postmitotic neurons (Figure 17BC). The small differences across cycling progenitors might appear disconcerting, however previous studies have shown that swapping of individual TFs from subpallial to pallial progenitors is sufficient to re-specify them [START_REF] Parras | Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity[END_REF], illustrating the critical impact of a small number of genes on cell fate.

The introduction of an approach (i.e. flashtag) allowing labeling of isochronic cohorts of RGs and of their progeny, offers an extra level of refinement to these transcriptomic studies [START_REF] Telley | Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex[END_REF][START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF]. Labeled cells can be isolated at distinct time points following injection and tracked during their differentiation, thereby giving a precise overview of transcriptional waves regulating the acquisition of neuronal identity [START_REF] Telley | Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex[END_REF]. This approach demonstrates the importance of the proper sequence of transcriptional waves for normal progression through neuronal differentiation and the relative high degree of their overlap. In the first hours following their labeling, RGs rapidly repress proliferation-associated transcripts while upregulating transcripts associated with protein translation. Later transcriptional waves include transcripts associated with DNA repair, revealing a critical period following mitosis during which neocortical neurons are susceptible to somatic mutations. As early as twelve hours post-labeling, differentiation programs unravel with transcriptional waves initiating late-occurring processes such as synaptogenesis. The early occurrence and overlapping nature of these transcriptional waves suggest an early priming of terminal differentiation events and provide discrete time windows during which specific transcriptional complexes are present simultaneously and can interact [START_REF] Nowakowski | Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex[END_REF].The same approach was applied by Mayer and colleagues, which conclude of common dynamics in all three eminences (Gao et al., 2014b). While some transcriptional programs are generic to all cells (generic genes), others are expressed at different stages of their differentiation but remain generic to all lineages (stage specific genes). Line age specification is coded by a small number of genes which progressively increase with the acquisition of defined morphology, connectivity and functions. Clustering sensitivity following single cell RNA-Sequencing rely on differentially expressed genes wh ich only become clearly distinctive between distinct neuronal lineages at the post -mitotic level. It is therefore highly dependent on sequencing depth and number of cells analyzed.

Contributions of Single cell Approaches to Probe Temporal Identity Specification

Neural progenitors change over time to contribute to the generation of cell diversity, a process termed as temporal identity specification. Thus, while RGs initially undergo proliferative divisions to amplify their pool, they later switch into a differentiating mode of division to produce specialized cells.

In the pallium, RGs progressively change their fates to sequentially produce deep-layer, then upper-layer neurons before generating glial cells at late embryonic times [START_REF] Okano | Cell types to order: temporal specification of CNS stem cells[END_REF]. While it was originally proposed that subtype of fate-biased RG cells coexist, recent clonal studies have revealed that pallial RGs follow a deterministic behavior and unitary production of projection neurons. Thus, RGs from the neocortex appear to invariably produce combinations of layer specific subtypes of cortical neurons (Gao et al., 2014a), implying that the behavior of individual RG is remarkable predictable across developmental stages and cortical areas. RGs first undergo symmetric divisions for several rounds before transiting to asymmetrical division and producing 8 to 9 neurons in different cortical layers. Towards the end of the embryonic period, RGs then change fate and produce astrocytes that migrate to the cortex, where they amplify locally [START_REF] Ge | Local generation of glia is a major astrocyte source in postnatal cortex[END_REF], to form clones [START_REF] García-Marqués | Clonal identity determines astrocyte cortical heterogeneity[END_REF] and associate to defined cortical columns [START_REF] Magavi | Coincident Generation of Pyramidal Neurons and Protoplasmic Astrocytes in Neocortical Columns[END_REF]. Interestingly, recent clonal fate mapping suggest that this neurogenic to gliogenic transition is largely incomplete, as only a fraction of RG cells (estimated to roughly 1/6), switch to produce glial cells. Other pallial RGs appear to continue producing IPCs (Tbr2/Neurog2 positive), whose contribution to cortical neurogenesis rapidly declines postnatally (Donega et al., 2018b), but continue producing a small population of glutamatergic interneurons in the periglomerular cell layer of the olfactory bulb for several weeks after birth [START_REF] Brill | Adult generation of glutamatergic olfactory bulb interneurons[END_REF]. Finally a population of pallial RGs, identified by Emx1 expression, change fate to contribute to the generation of calretinin-expressing GABAergic, as well as dopaminergic interneurons in the olfactory bulb [START_REF] Kohwi | A Subpopulation of Olfactory Bulb GABAergic Interneurons Is Derived from Emx1-and Dlx5/6-Expressing Progenitors[END_REF]Fuentealba et al., 2015b).

The advantage but also inherent limitation of clonal fate-mapping studies is the low number of cells that can be analyzed at once. On the other hand, scRNA-Seq experiments analyze genes expression of a large number of cells across the embryonic development allowing the investigation of temporal transcriptional heterogeneity. Is transcriptional profiling using scRNA-Seq supporting clonal analysis observations? In line with clonal histology experiments, the analysis of RGs transcriptional profile at different developmental stages, indicates a single linear trajectory of RGs from E13,5 to E17,5 [START_REF] Yuzwa | Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling[END_REF], reflecting a gradual shifting of the population of RGs, rather the co-existence of divergent fated subpopulations. In line with fate mapping studies, emergence of gliogenic related genes are observed later in the development (Novotortsev et al., 2010). Although this has not been systematically addressed in the subpallium, current evidences however support the existence of a subtle heterogeneous RGs population in the MGE from E12.5 to E14.5 whereas in the CGE RGs appear more homogeneous [START_REF] Mi | Early emergence of cortical interneuron diversity in the mouse embryo[END_REF]. This likely reflects the larger GABAergic neuron diversity, and sequential generation of PV and SST interneurons by the MGE. Future studies making use of the "flashmap" to label isochronic cohorts of RGs and of their progeny throughout embryonic development will be necessary to investigate in detail the transcriptional changes occurring in a population of RGs while it gives rise sequentially to distinct subtypes of neurons.

How can we explain then that relatively transcriptionally close RGs sequentially give rise to distinct neuronal subtypes? Noticeably, layer specific genes, encoding for layer specific proteins (such as Cux1 or Ctip2), are highly co-expressed in RGs throughout the neurogenic period (>70% coexpression) [START_REF] Zahr | A Translational Repression Complex in Developing Mammalian Neural Stem Cells that Regulates Neuronal Specification[END_REF]. This situation contributes to the relative homogeneity of RGs and sheds light on a strong post-transcriptional regulation of RGs fate [START_REF] Zahr | A Translational Repression Complex in Developing Mammalian Neural Stem Cells that Regulates Neuronal Specification[END_REF]. The authors identified 4E-T/Pum2 complex as a major player regulating fate specification of neurogenesis through post-transcriptional repression of appropriate mRNAs. In absence of this complex, aberrant specification of neurons is observed [START_REF] Zahr | A Translational Repression Complex in Developing Mammalian Neural Stem Cells that Regulates Neuronal Specification[END_REF]. Other epitranscriptomic changes mediating temporal control over lineage progression, include m6A methylation, recently suggested to regulate transcriptional pre-patterning during corticogenesis [START_REF] Yoon | Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation[END_REF]. The observation of a large decrease in m6A level in cortical progenitors persisting at birth suggest that epitranscriptomic modifications participate in the closure of the period of cortical neurogenesis (Donega et al., 2018a). Taken together, these studies highlight that the temporal production of defined neuronal subtypes is the result of numerous regulating mechanisms, not restricted to the mRNA expression revealed by scRNA-Seq, but including post-translational modifications of histones, DNA modifications and chromatin remodeling [START_REF] Albert | Epigenetic and transcriptional pre-patterning-An emerging theme in cortical neurogenesis[END_REF][START_REF] Sokpor | ATPdependent chromatin remodeling during cortical neurogenesis[END_REF][START_REF] Stricker | DNA-methylation: Master or slave of neural fate decisions?[END_REF].

Contributions of Single cell Approaches to Probe Adult NSCs Origin and Biology

The persistence of a germinal activity in the ventricular zone of the postnatal and adult forebrain prompted the question of the origin of these NSCs. While original studies had suggested a continuum in the differentiation of RGs in adult NSCs at perinatal times [START_REF] Tramontin | Postnatal development of radial glia and the ventricular zone (VZ): A continuum of the neural stem cell compartment[END_REF], recent clonal fate mapping studies revealed that adult NSCs derive from embryonic RGCs, which are distinct from those contributing to the neuronal and glial lineage of the developing forebrain (Figure 18).

Two distinct approaches were used to demonstrate that a subpopulation of RG slow their proliferation and segregate from the rest of RG that continue proliferating. Label retaining protocols demonstrate the prevalent emergence of a slowly dividing NPC population between E14.5 and E16.5 (Fuentealba et al., 2015b;[START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. This time points coincides with cell cycle exit of a subpopulation of RG biased to generate ependymal cells [START_REF] Spassky | Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis[END_REF]. While this timing coincides, it is still unclear if they are generated by a common pool of RGs. Interestingly, the level of labels retained in young adult NSCs were higher than in ependymal cells, suggesting that if NSCs and ependymal cells are produced sequentially by the same RG population, then NSCs may be produced first [START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. Bar coding tagging of RG at distinct embryonic time points resulted in similar conclusions (Fuentealba et al., 2015b).

Interestingly, the induction of RG quiescence is not homogeneous along the lateral ventricle and is likely to explain the persistence of germinal activity to restricted regions of the lateral ventricle at adult ages. Indeed, while label-retaining cells are frequently observed in the ganglionic eminences, in particular the LGE, they are rare in the pallium [START_REF] Tramontin | Postnatal development of radial glia and the ventricular zone (VZ): A continuum of the neural stem cell compartment[END_REF]. This goes in line with the different dynamics observed in the generation of pallial (i.e. Tbr2 + ) and subpallial (i.e. DLX2 + ) progenitors observed at postnatal stages. Thus, while Tbr2 progenitors continue to be produced by pallial RGs at early postnatal timepoints (Donega et al., 2018a), their production rapidly decreases over time to become almost extinguished by P90 [START_REF] Brill | Adult generation of glutamatergic olfactory bulb interneurons[END_REF]. On the contrary, DLX2 + progenitors continue being produced throughout adulthood, in agreement with the preferential accumulation of quiescent NSCs in the lateral SVZ [START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF].

In the adult, quiescent NSCs reactivate asynchronously to divide symmetrically. While 1/5 th self-renew in P21 mice, the remaining population generate type C cells that cycle 3 times before generating neuroblasts that undergo one or two additional cycles [START_REF] Ponti | Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice[END_REF][START_REF] Obernier | Adult Neurogenesis Is Sustained by Symmetric Self-Renewal and Differentiation[END_REF]. This results in a fast clonal expansion but limited self-renewal of adult NSCs as demonstrated by clonal analysis [START_REF] Calzolari | Fast clonal expansion and limited neural stem cell self-renewal in the adult subependymal zone[END_REF]. The rapid consequent depletion of activated NSCs explain the sparsity of clones containing both B1 cells and OB interneurons following bar coding tagging of RGs during embryonic development [START_REF] Fuentealba | Adult neural stem cells bridge their niche[END_REF]. Thus, a population of NSCs self-renew to dampen their early depletion, while a second population reactivates to produce a progeny before rapidly extinguishing [START_REF] Calzolari | Fast clonal expansion and limited neural stem cell self-renewal in the adult subependymal zone[END_REF][START_REF] Obernier | Adult Neurogenesis Is Sustained by Symmetric Self-Renewal and Differentiation[END_REF]. The asynchronous activation of adult NSCs allows SVZ germinal activity to persist throughout life, despite of a gradual decline. This asynchronous, gradual activation is further supported by the observation that E15.5 label retaining NSCs produce a neuronal progeny with undisguisable BrdU levels at different postnatal ages (Fuentealba et al., 2015b). It is still unclear if NSCs then produce glial cells following their final division, as the transgenic mice used in these studies did not allow discriminating between NSCs and surrounding glial cells.

Single cell transcriptomic studies revealed that RGs from the subpallium (regardless of the eminence of origin), as well as from the pallium, have different maturation statuses but no sign of fate bifurcation [START_REF] Nowakowski | Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex[END_REF][START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF]. A transcriptional variance is however observed across RGs, which primary origin is developmental and cell cycle progression. This likely reflects multiple modes of divisions (symmetric vs. asymmetric) at distinct embryonic stages [START_REF] Mayer | Developmental diversification of cortical inhibitory interneurons[END_REF], as well as the cell cycle exit of some RGs. Analysis of a larger number of cells, indeed reveal the co-existence of mitotic vs. non-mitotic RGs within the pallium as early as E13 [START_REF] Yuzwa | Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling[END_REF] [START_REF] Tramontin | Postnatal development of radial glia and the ventricular zone (VZ): A continuum of the neural stem cell compartment[END_REF]. Detection of transcriptional similarities between slowly/non mitotic embryonic RGs from E13.15 to quiescent adult SVZ support the existence of the subpopulation of quiescent cells accumulating over development, to be reactivated in adulthood. Further investigations will reinforce the extent of transcriptional overlap of these RGs with quiescent NSCs isolated from the adult brain [START_REF] Codega | Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche[END_REF][START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF].

Single cell transcriptomic approaches were recently applied to adult NSCs [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF] to reveal the co-existence of NSCs in multiple states of activation.

Similar to the sequential transcriptional waves observed newborn neurons in the mouse neocortex [START_REF] Telley | Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex[END_REF], it reveal that NSC activation and early lineage progression is organized as a continuum of molecular events, along which cells express sets of coregulated genes. This work highlighted an association of NSCs quiescence with high glycolytic and lipid metabolism, as well as with the previously reported high expression of transcripts encoding membrane receptors [START_REF] Codega | Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche[END_REF]. Further, NSCs activation and differentiation correlated with a progressive up-regulation of the protein synthesis machinery. Interestingly, this single cell transcriptomic analysis provides important information on the priming of individual adult NSCs towards specific lineages. Indeed, the postnatal and adult SVZ is regionalized in respect to different microdomains generating distinct neural lineages. This regionalization appears to originate from early embryonic development (Fuentealba et al., 2015b) and is intrinsically encoded by expression of selected TFs (reviewed in [START_REF] Fiorelli | Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis[END_REF]. Single cell analysis identified single cells exhibiting expression of oligodendroglial or neuronal subtype-specific TF combinations, thereby revealing the early priming of adult NSCs towards specific fates [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF]. This observation is in line with ex vivo lineage tracing of individual cells

showing that neurogenic and oligodendrogenic NSCs constitute distinct lineages, while both can produce astrocytes. Recent lineage tracing of a subpopulation of dorsal NSCs expressing the TF HOPX at early postnatal timepoints, however suggests the existence of a NSCs population biased to produce astrocytes (Zweifel et al., 2018). Thus, the production of distinct cell types by adult NSCs relies on the co-existence of fate-biased NSCs, which are heterogeneously distributed along the wall of the lateral ventricle.

In conclusion, single cell approaches contribute to our understanding of NSC biology and of their specification into distinct cell types at defined developmental and postnatal stages. [START_REF] Qian | Timing of CNS cell generation: A programmed sequence of neuron and glial cell production from isolated murine cortical stem cells[END_REF][START_REF] Shen | The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells[END_REF] . Further, the competence of cortical progenitors appears to become increasingly restricted. Noticeably, mid-gestational progenitors can still be manipulated to generate early neuronal fates, while late-gestational progenitors have lost this competence [START_REF] Shen | The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells[END_REF]. This progressive loss of competence has also been illustrated in vivo, by transplantation experiments. Early heterochronic transplantation approaches have found that the potential of different progenitor populations to generate distinct neuronal subtypes becomes gradually restricted over time (for review [START_REF] Gaiano | Transplantation as a tool to study progenitors within the vertebrate nervous system[END_REF]. For instance, cycling progenitors from late stages of the ferret corticogenesis, keep their intrinsic restriction, even if transplanted into an early stage environment. Indeed they keep generating upper layer neurons and fail to produce deep cortical layers, which are normally generated at this age [START_REF] Frantz | Restriction of late cerebral cortical progenitors to an upper-layer fate[END_REF]. In contrast, early progenitors are able to adapt to the host environment to generate upper layer neurons, when transplanted into a late stage environment [START_REF] Gümüssoy | Changes in IGF-I, IGFBP-3 and ghrelin levels after adenotonsillectomy in children with sleep disordered breathing[END_REF] [START_REF] Ortega | Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling[END_REF]. Further, heterochronic transplantation experiments have shown that postnatal SVZ progenitors can migrate and differentiate within multiple levels of the developing neuraxis, when transplantated into embryonic mouse brain ventricles at day 15 of gestation [START_REF] Lim | Postnatal mouse subventricular zone neuronal precursors can migrate and differentiate within multiple levels of the developing neuraxis[END_REF]. Although expression of subtype specific markers was not assessed, their acquisition of morphologies typical of resident neurons support their capacity to generate other neurons subtypes than those they normally produce within the olfactory bulb. Finally, ex vivo experiments showed that postnatal and adult SVZ explants have the capacity to generate pyramidal neurons. In these experiments, adult SVZ explants were exposed to an embryonic environment by juxtaposing them to organotypic slices of the pallium. Neurons were produced by the SVZ explants, which invaded the pallium. Their expression of Tbr1

suggested their acquisition of a glutamatergic phenotype, although their maturation could not be investigated at long term [START_REF] Sequerra | Generation of glutamatergic neurons from postnatal and adult subventricular zone with pyramidal-like morphology[END_REF].

Taken together, these observations challenge the model of gradual fate restriction observed during embryogenesis. They rather suggest that embryonic and postnatal/adult NSCs represent distinct populations in term of diversity and competence to generate various cell types. The recent demonstration that postnatal/adult NSCs derive from embryonic RGCs that are distinct from those contributing to the neuronal and glial lineage of the developing forebrain, support this hypothesis (Fuentealba et al., 2015b;[START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. Further, single cell studies discussed above provide a roadmap to guide postnatal/adult NSCs to differentiate into defined cell types, and give insight in the mechanisms at play that restrict their recruitment and successful differentiation.

Unravelling the diversity and competence of postnatal/adult NSCs to produce distinct neural cell types is of major importance for further considering them as a potential source for cellular repair. Unravelling their diversity relies on the identification of markers expressed in subpopulation of NSCs biased to acquired defined fate. To this end, the identification of markers expressed in SVZ microdomains giving rise to distinct cell lineage represent a powerful approach, as illustrated in Chapter 1. This work resulted in the identification of HOPX as a marker of NSCs within the dorsal SVZ, biased to acquire an astrocytic fate. Unravelling their competence to generate diverse neural cell types relies on understanding the transcriptional changes that occur in neural progenitors over time, as illustrated in Chapter 2. It finally relies on testing their capacity to participate to forebrain development, or to participate to its regeneration. While the former can be achieved by heterochronic transplantation, as previously [START_REF] Lim | Postnatal mouse subventricular zone neuronal precursors can migrate and differentiate within multiple levels of the developing neuraxis[END_REF] the later relies on establishment of new models in which define cell types are ablated at early postnatal time points, when its regenerative potential is maximal. Please find below a description of such model.

Targeted Neuronal Ablation as a Model to Study Competence of Postnatal NSCs for Cortical Repair

Early brain injury following perinatal insults such as hypoxic-ischemic episodes, often result in severe neurological disabilities. The common feature of these lesions is the massive death of various cell types including neurons and glial cells [START_REF] Ferriero | Neonatal brain injury[END_REF][START_REF] Kaindl | Molecular mechanisms involved in injury to the preterm brain[END_REF][START_REF] Deng | Neurobiology of injury to the developing brain[END_REF]. The capacity of the adult mammalian cerebral cortex to compensate adequately for neuronal and glial loss appears limited. Indeed, there are only few evidences of a significant contribution of germinal regions to brain repair in the adult.

Nonetheless, this situation appears to be strikingly different during the early postnatal period [START_REF] Plane | Neonatal hypoxicischemic injury increases forebrain subventricular zone neurogenesis in the mouse[END_REF][START_REF] Ong | Hypoxic-ischemic injury stimulates subventricular zone proliferation and neurogenesis in the neonatal rat[END_REF]. Convergent observations indicate that neonatal trauma can lead to regenerative cortical neurogenesis. Newly generated neurons appear to arise from the rostral subventricular zone (SVZ), to be recruited to the damaged cortex [START_REF] Jin | Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum[END_REF][START_REF] Saha | Potentials of endogenous neural stem cells in cortical repair[END_REF][START_REF] Thomsen | Traumatic brain injury reveals novel cell lineage relationships within the subventricular zone[END_REF]. However, although de novo neurogenesis is well documented in newborn, fundamental questions remain to be addressed to fully appreciate the inner capacity of the neonatal forebrain to repair. In particular, the capacity of newborn neurons to specify in appropriate neuronal subtypes have not been studied in details because only generic markers have been used in most of the studies [START_REF] Saha | Cortical lesion stimulates adult subventricular zone neural progenitor cell proliferation and migration to the site of injury[END_REF]. Thus, do newborn neurons correctly specify in neuronal subtypes and functionally integrate? This open question is of particular interest, as an incorrectly specification and integration of these neurons may result adverse effects.

Different cortical lesion models have been developed over the years, in order to test the capacity of the neural tissue to regenerate in newborn rodents. In 2008, Kadam and colleagues, used a hypoxic-ischemia model to induce a diffuse cortical lesion [START_REF] Kadam | Neurogenesis and neuronal commitment following ischemia in a new mouse model for neonatal stroke[END_REF]. In 2011, Bi and colleagues described a chronic hypoxia model in 3-days-old mice [START_REF] Bi | Cortical Glial Fibrillary Acidic Protein-Positive Cells Generate Neurons after Perinatal Hypoxic Injury[END_REF] Other models include ischemic insults [START_REF] Biran | Cerebellar abnormalities following hypoxia alone compared to hypoxic-ischemic forebrain injury in the developing rat brain[END_REF]. Importantly, all of these models result in diffuse injuries, characterized by the loss of various cell types often accompanied by a severe disorganization of the tissue. Thus, these models are not appropriate to study the recruitment of specific progenitor populations, nor their specification and integration.

I aimed at developing a versatile injury model that circumvent most of these limitations by

developing a model of targeted ablation of specific neuron populations. This approach is based on the targeting of select cohorts of cortical progenitor during embryonic development, and the control of the onset of ablation through diphtheria toxin (DT) and diphtheria toxin receptor (DTR) system [START_REF] Petrenko | Apoptotic neurons induce proliferative responses of progenitor cells in the postnatal neocortex[END_REF] 19G) which demonstrates that number of dead neurons is sufficient to induce a clear and robust phenotype. Despite an important apoptosis, our results showed a mild inflammation at the lesion site, in agreement with previous studies [START_REF] Petrenko | Apoptotic neurons induce proliferative responses of progenitor cells in the postnatal neocortex[END_REF], as revealed by an increase of microglia (Iba1 + ) and astrocytes (GFAP + ) in the cortex several days following the induction of the lesion (Figure 19I-J).

Nevertheless, those cells kept a clear organization and did not form a scar as observed in more invasive injury models. The timecourse and possible persistence of this inflammation remains however to be fully assessed.

I next focused on the dorsal SVZ to investigate the consequence of the cortical ablation on cellular proliferation 5 and 12 days later. An increased proportion of Ki67 + cells was observed in the dorsal SVZ, five days following DT injection, when compared to controls (Figure 20AB). This demonstrated that the severity of the lesion was sufficient to induce a rapid proliferative response, which attenuated over time. The number of Glu progenitors, identified by Tbr2 immunolabeling, did not vary following DT injection (Figure 20AB). In contrast, the number of oligodendrocyte progenitors identified by Olig2 immunolabeling, increased rapidly and persistently following ablation (Figure 20AB). This could be due to an important axonal degeneration following targeted cortical ablation, resulting in a reactive oligodendrogenesis. [START_REF] Azim | Persistent Wnt/β-catenin signaling determines dorsalization of the postnatal subventricular zone and neural stem cell specification into oligodendrocytes and glutamatergic neurons[END_REF](Azim et al., , 2017;;Donega et al., 2018a) (Figure 20C). Similarly, DT-injected animals were treated for 3 days following the start of ablation with AR-A014418. This resulted in a significant increase of Tbr2 + cells (Figure 20D). In parallel, a significant reduction of GFAP expression was observed around the site of neuronal ablation (Figure 20E). These observations support a regenerative potential for Wnt/β-catenin stimulation following neonatal brain injuries, which remains to be fully explored. For instance, it will be interesting to study if this activation of Tbr2 + progenitors translate into cortical neurogenesis. If new neurons are observed, their appropriate specification in layer IV cortical neurons can then be assessed.

Altogether this work demonstrates that DT-induced cortical ablation may prove to be an interesting model to study the competence of postnatal progenitors to produce select neuronal subtypes following injury. paste0(str_split_fixed(list.files()[which(nchar(list.files())>14)][s],"_",2)[1],".CEL.gz")) } } # gunzip(paste0(i,".CEL.gz")) system(paste0("gunzip ",paste0(i,".CEL.gz")))

Annexes

if (!(paste0(i,".CEL") paste0(str_split_fixed(list.files()[which(nchar(list.

files())>14)][s],"_",2)[1],".CEL.gz"))

} } # gunzip(paste0(i,".CEL.gz")) system(paste0 ("gunzip ",paste0(i,".CEL.gz"))) } if(!(paste0(i,".CEL") %in% list.files())){ # If download still fails, error message cat("ERROR GSM not Downloaded : ") cat(i) cat(" \n") } # downl = paste0("ftp://ftp.ncbi.nlm.nih.gov/geo/samples/",str_sub (i,1,6),"nnn/",i,"/suppl/",i,"%2ECE L%2Egz") # download.file(downl,destfile = paste0("./",i,".CEL.gz")) #download files matching GSM }} system(paste0("gunzip ",paste0(i,".CEL.gz"))) # gunzip(paste0(i,".CEL.gz")) file.copy(paste0(i,".CEL"),"../temp/") } setwd("../temp/") # Read in the CEL files in the directory, then normalize the data data=ReadAffy() eset <-affy::rma(data,verbose = T) data2 = eset@assayData$exprs if(data@cdfName!="Mouse430_2"){ cat("ERROR WITH GENE NAMES") print("ERROR WITH GENE NAMES") } # library("annotate") # library ("mouse4302.db", lib.loc="~/R/x86_64-pc-linux-gnu-library/3.3") # biocLite("mouse4302.db") # Affymetrix Mouse Genome 430 2.0 Array annotation data (chip mouse4302) # Verify that experiments match chip setwd("../tables/") if (!(paste0(colnames(inputf)[o],".csv") %in% list. files(paste0("./",str_split_fixed(w,".csv",2) str_c(paste0(colnames(inputf) [o],"_"),as.character(1:dim(data2) [2]))

# columns(mouse4302.db) write.csv(data2,paste0("./",colnames(inputf)[o],".csv")) } }#End of the loop on columns # load the affy library ## Normalize experiments setwd(paste0 ("./",str_split_fixed(w,".csv",2)[1])) dir.create("./TablesPurif/") tablestab = list.files(paste0("./",str_split_fixed(w,".csv",2)[1],"/"),pattern = ".csv",include.dirs = T) for(i in tablestab){ #lecture des tableaux créés # i = tablestab [1] if(!(i %in% list.files("./TablesPurif/"))){ # Test if file already exists a = read.csv(paste0("./",i)) a = na.omit (a) for (z in unique(as.character(read.csv(paste0("../../genes/",w),header = F)$V1))){ #loop on gene names # z = "Tcf" if(z %in% a[,1]){ # check if gene already exist experiences = str_c("./",colnames(inputf),".csv") # ListGenes = c ("Neurod6","Eomes","Neurod1","Zic1","Zic5","Tbr1","Hopx","Dmrta2","Tfap2c","Neurog2 ") results = c(1:length(ListGenes)) # initializing result namescol = NULL for(j in experiences){ # loop on table and combine results fic = read.csv(j,row.names = c( 1)) results = cbind (resultats,fic[ListGenes,) namescol=c (namescol,colnames(fic)[-1]) } results = results [,-1] # Remove unwanted columns colnames(results)<-namescol # cell identification of the table results = na.omit(results) jpeg(paste0("../../../",str_split_fixed(w,".csv",2)[1],"_heatmap.jpeg"),width = 1000,height = 1000) pheatmap (as.matrix(results),Rowv=NULL,Colv=NULL,col=redgreen(256), labRow="" ,border_color = "black",scale = "row",clustering_distance_cols = 'correlation') dev.off() } length(yuzwaE17_seurat@var.genes) yuzwaE17_seurat pc.genes = yuzwaE17_seurat@var.genes,do.print = FALSE) PCAPlot(object = yuzwaE17_seurat, dim.1 = 1, dim.2 = 2) yuzwaE17_seurat <-JackStraw(object = yuzwaE17_seurat, num.replicate = 100, display.progress = FALSE) JackStrawPlot(yuzwaE17_seurat, PCs = 1:12) yuzwaE17_seurat dims.use = 1:12,do.fast = TRUE,perplexity = 30) TSNEPlot(yuzwaE17_seurat,do.label = FALSE,pt.size = 4) FeaturePlot(yuzwaE17_seurat,"Sox2",pt.size = 5,cols.use = c("grey","red")) t-SNE Generation on RGCs Related to Figure 12B. yuzwaE15RPcellsRename.data = yuzwaE15.data[,RPE15.cells] yuzwaE17RPcellsRename.data = yuzwaE17.data[,RPE17.cells] yuzwaAll.data = cbind(yuzwaE15RPcellsRename.data[row.names(GenesAll),],yuzwaE17RPcellsRename.data[row.names(GenesAll),]) yuzwaAll_seurat = CreateSeuratObject(raw.data = yuzwaAll.data,min.cells = 0,min.genes = 100,project = "YuzwaEAll",names.field = 1, names.delim = "_") yuzwaAll_seurat <-NormalizeData(object = yuzwaAll_seurat, normalization.method = "LogNormalize", scale.factor = 10000) yuzwaAll_seurat mean.function = ExpMean,dispersion.function = LogVMR,x.low.cutoff = 0.0125,x.high.cutoff = 5,y.cutoff = 1.5) length(yuzwaAll_seurat@var.genes) yuzwaAll_seurat <-ScaleData(object = yuzwaAll_seurat, vars.to.regress = NULL) yuzwaAll_seurat <-RunPCA(yuzwaAll_seurat, pc.genes = yuzwaAll_seurat@var.genes, do.print = FALSE) PCAPlot(object = yuzwaAll_seurat, dim.1 = 1, dim.2 = 2) yuzwaAll_seurat <-JackStraw(object = yuzwaAll_seurat, num.replicate = 100, display.progress = FALSE) JackStrawPlot(yuzwaAll_seurat, PCs = 1:12) yuzwaAll_seurat dims.use = 1:12,do.fast = TRUE,perplexity = 30) TSNEPlot (yuzwaAll_seurat,do.label = FALSE,ident.1 = "E17",thresh.use = 0.25,min.pct = 0.20,test.use = "bimod",only.pos = F) write.table(RPE17E15.markers,"RPE17E15.markers.csv",sep = ",") t-SNE Generation on E17.5 RGCs with Zywitza Markers Related to Figure 12. ##Download first astrocytes markers from Zywitza et al. AstroGenes.data = read.table("AstroGenes.csv",sep=",",header = TRUE,row.names = 1) yuzwaZywitza_seurat = CreateSeuratObject(raw.data = yuzwaRPE17.data,min.cells = 2,min.genes = 100,project = "yuzwaZywitza.data",names.field = 1, names.delim = "_") yuzwaZywitza_seurat <-NormalizeData(object = yuzwaZywitza_seurat, normalization.method = "LogNormalize", scale.factor = 10000) yuzwaZywitza_seurat@var.genes = AstroGenes yuzwaZywitza_seurat <-ScaleData(object = yuzwaZywitza_seurat, vars.to.regress = NULL) yuzwaZywitza_seurat <-RunPCA(yuzwaZywitza_seurat, pc.genes = yuzwaZywitza_seurat@var.genes, do.print = FALSE) PCAPlot(object = yuzwaZywitza_seurat, dim.1 = 1, dim.2 = 2) yuzwaZywitza_seurat <-JackStraw(object = yuzwaZywitza_seurat, num.replicate = 100, display.progress = FALSE) JackStrawPlot(yuzwaZywitza_seurat, PCs = 1:12) yuzwaZywitza_seurat dims.use = 1:4,do.fast = TRUE,perplexity = 25) TSNEPlot(yuzwaZywitza_seurat,do.label = FALSE,pt.size = 3) FeaturePlot(yuzwaZywitza_seurat,c("Sulf1"),pt.size = 6,cols.use = c("grey","red")) ##Clustering yuzwaZywitza_seurat <-FindClusters(object = yuzwaZywitza_seurat, reduction.type = "pca", dims.use = 1:3, resolution = 1, print.output = FALSE, save.SNN = TRUE, force.recalc = TRUE) #Result: cluster 1 = "0" TSNEPlot (yuzwaZywitza_seurat,do.label = FALSE,pt.size = 6) ##Markers yuzwaZywitzaE17.markers ident.1 = 0,thresh.use = 0.25,min.pct = 0.20,test.use = "bimod",only.pos = F) write.table(yuzwaZywitzaE17.markers,"yuzwaZywitzaE17.markers.csv",sep = ",") FeaturePlot(yuzwaZywitza_seurat, c("Aldh1l1"), pt.size = 6, cols.use = c("grey", "red"))

Abstract

During development, remarkable coordination of molecular and cellular events leads to the generation of the cortex, which orchestrates most sensorimotor and cognitive functions. Its development occurs in a stepwise manner: radial glia cells (RGs) -the neural stem cells (NSCs) of the developing brain -sequentially give rise to distinct waves of nascent neurons that form cortical layers. Around birth, RGs switch fate to produce glial cells. A fraction of neurogenic RGs however persists throughout postnatal life in the subventricular zone (SVZ) that lines the lateral ventricles. These NSCs give rise to different subtypes of olfactory bulb interneurons and glial cells, according to their spatial origin within the postnatal SVZ. These observations raise important unresolved questions on 1) the transcriptional coding of postnatal SVZ regionalization, 2) the potential of postnatal NSCs for cellular regeneration and brain repair, and 3) the lineage relationship and transcriptional specificities of postnatal NSCs and of their progenies. I developed bioinformatics approaches to relate expression of defined transcription factors to the regional generation of distinct neural lineages and to explore and compare select populations of pre-and postnatal progenitors at the single cell level. These data highlight the potential of transcriptomic studies to unravel fundamental questions such as transcriptional changes occurring in a population of progenitors over time and participating to changes in their fate potential. This knowledge will be key in developing innovative approaches to recruit and promote the generation of specific cell types, including neuronal subtypes in pathologies.

  Pendant le développement, la coordination remarquable d'évènements moléculaires et cellulaires mène à la production du cortex cérébral quiorchestre les fonctions sensorimotrices et cognitives. Son développement s'effectue par étapes : les cellules gliales radiaires (RGs)les cellules souches neurales (NSCs) du cerveau en développementet les cellules progénitrices de la zone ventriculaire (VZ) et de la zone sous ventriculaire (SVZ) génèrent séquentiellement des vagues distinctes de nouveaux neurones qui formeront les différentes couches corticales. Autour de la naissance, les RGs changent de devenir et produisent des cellules gliales. Cependant, une fraction d'entre elles persiste tout au long de la vie dans la SVZ qui borde le ventricule, perdant au passage leur morphologie radiale. Ces NSCs produisent ensuite les différents sous types d'interneurones du bulbe olfactif ainsi que des cellules gliales en fonction de leur emplacement d'origine et de leur localisation dans la SVZ périnatale. Ces observations soulèvent d'importantes questions non résolues sur 1) le codage transcriptionnel régulant la régionalisation de la SVZ, 2) le potentiel des NSCs postnatales à la régénération cellulaire et à la réparation cérébrale, et 3) la relation de lignage et les spécificités transcriptionnelles entre les NSCs et leur descendants. Mon travail de doctorat s'est construit à partir d'une étude transcriptionnelle des différents microdomaines de la SVZ postnatale. Cette étude surlignait le haut degré d'hétérogénéité transcriptionnelle entre les NSCs et les progéniteurs et identifiait des régulateurs transcriptionnels clés soutenant la régionalisation de la SVZ. J'ai développé des approches bio-informatiques pour explorer ces banques de données et connecté l'expression de facteurs de transcription avec la génération régionale de lignages neuraux distincts. J'ai ensuite développé un modèle d'ablation ciblée qui peut être utilisé pour étudier le potentiel régénératif des progéniteurs postnataux dans divers contextes. Finalement, j'ai participé au développement d'une procédure pour explorer et comparer des populations sélectionnées de progéniteurs pré et postnataux à l'échelle de la cellule unique. Objectif 1 : Des expériences de transcriptomique ainsi que de cartographie ont été réalisées pour étudier la relation entre l'expression régionale de facteurs de transcription par les NSCs et l'acquisition de leur devenir dans des lignages neuraux distincts. Nos résultats suggèrent un engagement précoce des NSCs à produire des types cellulaires définis selon leur localisation spatiale dans la SVZ et identifient la protéine HOPX comme un marqueur d'une sous population biaisé à générer des astrocytes.

XII

  Dans mon premier chapitre expérimental, des approches transcriptomiques et de cartographie du devenir cellulaire ont été utilisées pour explorer la relation entre l'expression régionale de facteurs de transcription par des cellules souches neurales (NSCs) et leur spécification dans des lignages neuraux définis. En particulier, nous avons caractérisé le lignage et le profil d'expression spatio-temporelle d'HOPX dans la SVZ postnatale. J'ai développé en collaboration avec Q. Lo Giudice un outil bio-informatique appelé Heatmap Generator qui vise à combiner et à comparer des ensembles de données de transcription publiés afin de réaliser une méta-analyse. J'ai également effectué des expériences histologiques ainsi qu'une méta-analyse d'une expérience de séquençage d'ARN sur cellule unique récemment publiée. Nos résultats démontrent un amorçage précoce des NSCs pour la genèse de types cellualires définis en fonction de leurs localisations spatiales dans la SVZ et identifient HOPX comme marqueur d'une souspopulation amorcée vers un destin astrocytaire.

  . Nous avons utilisé des souris Neurog2 Creert2 / tdTom pour marquer de manière permanente et spécifique des cohortes synchrones de progéniteurs prénatux et postnataux Glutamatergiques afin d'étudier leurs relations de lignage et leurs spécificités transcriptionnelles. Nos résultats montrent que les progéniteurs Glu continuent à être produits après la fermeture de la période de neurogenèse corticale. Le séquençage d'ARN sur cellule unique (scRNA-seq) révèle que les progéniteurs postnataux Glu présentent une dérégulation des gènes impliqués dans le métabolisme, la différenciation et la migration, parallèlement à un déclin rapide de leur capacité à migrer et à se différencier. Nos données suggèrent que cette dérégulation de la transcription chez les progéniteurs de Glu postnataux pourrait résulter d'une diminution de la méthylation de la N6-méthyladénosine (m6A) de certains gènes proneuronaux. Néanmoins, les progéniteurs postnataux Glu XVI restent partiellement permissifs aux manipulations pharmacologiques et génétiques ce qui suggère qu'ils pourraient être recrutés pour une réparation corticale. La période périnatale a longtemps été considérée comme une période de gliogenèse corticale exclusive associée à la maturation des neurones nés pendant le développement embryonnaire et là leur intégration dans des circuits fonctionnels. Cette vision est actuellement contestée par la démonstration de la neurogenèse dans des régions corticales spécifiques. Ainsi, les progéniteurs GABAergiques s'accumulent dans la substance blanche postnatale précoce et donnent naissance à une sous-population d'interneurones interstitiels corticaux

Figure 3 :

 3 Figure 3: The genetics of early pallial patterning. Coronal view indicates spatial subdivisions of the pallium, where dorsal pallium lies between the medial and lateral/ventral pallium.In sagittal view, the medial and lateral/ventral pallium connects at caudal levels. DP, dorsal pallium; LP, lateral pallium; me, mesencephalon; MP, medial pallium; pr, prosencephalon; SP, subpallium; VP, ventral pallium. Adapted from[START_REF] Kumamoto | Neuronal subtype specification in establishing mammalian neocortical circuits[END_REF].

Figure 4 :

 4 Figure 4: Schematic representation of the major neocortical interneuron subtypes. Schematic representation of the distinct interneuron classes within the six cortical layers. Excitatory spiny interneurons display mainly stellate and pyramidal morphology and are primarily located in the intragranular layer IV of the somatosensory cortex (barrel cortex, shown in the yellow boxes). In contrast, each cortical layer contains different types of inhibitory interneurons with different morphologies and molecular identities. (molecular markers are listed on right). Illustration from Lodato and Arlotta, 2015.

Figure 6 :

 6 Figure 6: The SVZ replenishes olfactory interneurons throughout life . Schematic depiction of the neurogenic process along the entire extent of the ventricular system of the neonatal/adult rodent forebrain. (A) 3D representation of SVZ neurogenesis. Neural stem cells (NSCs) are located in the walls of the lateral ventricle (LV) and generate an irregular network of migratory neuroblasts (orange) that converge into the rostral migratory stream

Figure 7 :

 7 Figure 7: Cellular composition of the ventricular -subventricular zone (SVZ). (A+B): Representative micrographs of the SVZ (A) and the higher magnification of the lateral microdomain (B) show the zone of interest to illustrate the SVZ cytoarchitecture.(C+D): Ciliated ependymal cells (E; yellow) align the LV and isolate the lumen from the proliferative SVZ. NSC (type B1; blue) give rise to type C cells (green) which generate migrating neuroblasts (type A; red) (illustrations were modified from Doetsch et al., 1997 and Tong and Alvarez-Buylla, 2014). Scale bars: A = 500 µm; B = 100 µm; C = 20 µm. Abbreviations: BV, blood vessel; LV, lateral ventricle; SVZ, subventricular zone.

Figure 8 .

 8 Figure 8. A Meta-Analysis of TFs Enrichment in dNSCs Highlights Their Association with Distinct Neural Lineages. (A) Heatmap showing enrichment of 61 TFs in dNSCs compared with lNSCs (R1.8-fold and p < 0.05).(B) Top ten TFs enriched in dNSCs. (C and D): Dorsal enrichment of select transcripts was confirmed using the Allen Brain Atlas for Eomes (C) and by immunohistochemistry for HOPX (D). (E) Heatmap of dNSC enriched TFs reveals three clusters corresponding to defined neural lineages: oligodendrocytes (purple, 11/61); astrocytes (yellow, 18/61); neurons (turquoise, 15/61). Hopx (highlighted in bold) associates with the astrocytic lineage. (F-H) Confirmation of astroglial lineage-specific enrichment of HOPX by immunohistochemistry. HOPX is largely absent in neuroblasts of the RMS (DCX; F) and oligodendrocytes in the CC (OLIG2; G), but is observed in astrocytes of the CC (GFAP; H, arrows indicate double positive cells). CC, corpus callosum; dNSC, dorsal NSCs; lNSC, lateral NSCs; RMS, rostral migratory stream; OPC, oligodendrocyte precursor cell; OL, oligodendrocyte. Scale bars, 500 mm (C and D) and 25 mm (H).

Figure 9 .

 9 Figure 9. HOPX Exhibits a Complex Spatial and Temporal Expression Pattern within the SVZ Where it Labels a Subpopulation of dNSCs . (A) Representative micrographs of HOPX expression in coronal sections at E16, P1, P4, P21, and P60 (top panels). Higher magnifications (lower pa nels) show high expression in the embryo (E16) and at early postnatal stages (P1, P4) and a decline thereafter (P21, P60). A clear medial -tolateral HOPX expression gradient is apparent in the dSVZ (see also Figure S2 for a more complet e rostrocaudal overview at P4). (B and C) Analysis of HOPX expression in dNSCs was performed in HES5:EGFP mice (B; n = 3 animals) and after short -term (8 hr) targeted EPO of a pCX-GFP plasmid (C; n = 6 animals). Crops show overlap of HOPX with HES5:EGFPand GFP-positive cells, respectively, which was more pronounced in the medial than lateral subdomain of the dSVZ (crops and graphs). EPO, electroporation; dSVZ, dorsal SVZ; lSVZ, lateral SVZ; dNSC, dorsal NSC. Error bars represent the SEM. Scale bars in (A) represent 1 mm (overview) and 500 mm (crops). Scale bars in (B) and (C) represent 200 mm (DAPI overview), 100 mm (higher magnification), and 25 mm (crops). *p < 0.05;***p < 0.001.

  that were highly specific to astrocytes(Aqp4, Aldh1l1, Fgfr3, Id3), including Hopx, were enriched in dmNSCs (Figure10B, top panel), and those for oligodendroglial lineage(Pdgfra, Plp1, Olig1, Olig2, Dct) were partly enriched in dmNSCs but overall more homogeneously distributed (Figure10B, middle panel). Finally, transcripts of the neuronal lineage(Dlx2, Sp8, Eomes, Vax1, Ebf1) were generally enriched in dlNSCs or in lNSCs (Figure10B, bottom panel), implying spatial segregation of lineages in the dSVZ. Taken together, Hopx and astroglial markers exhibit expression patterns in the dSVZ that are opposite to neuronal markers (Figure10C). Interestingly, target genes for morphogens, i.e., WNT and SHH, showed that WNT signaling is homogenously distributed throughout the dorsal wall (Lef1, Tcf7), whereas Gli1 is highly expressed in lNSCs (Figure10A, bottom panel). Another signaling pathway target gene, Id3, used as a readout for bone morphogenetic protein (BMP) signaling, showed very marked enrichment in dmNSCs (Figure10B, top panel). Next, to test whether NSCs harbored by those two subdomains are biased to generate specific neural progenies, we performed targeted EPO of the dmSVZ and dlSVZ (Figure10D). At a short time point(i.e., 12 hr), EPO of both microdomains with a GFP-encoding plasmid resulted in efficient GFP expression in numerous RG cells. Accurate spatial targeting was assessed by measuring the GFP fluorescence intensity within 50-mm probes distributed along the mediolateral aspects of the dSVZ at 14 days post-electroporation (dpe)(Not shown). This confirmed the precise and reliable targeting of NSCs of the dmSVZ and dlSVZ. Both regions had given rise to large cohorts of progenies whose distribution and fate appeared to be strikingly different. Indeed, in agreement with the transcriptional profile of dlNSCs, a large population of GFP + neurons in the OB (93.3 ± 11.1, average number of cells/section) were found following dorsolateral EPO (dlEPO), while those derived from the dorsomedialEPO (dmEPO) remained fewer (11.1 ± 1.8; Figures9E-9G).

Figure 10 .

 10 Figure 10. Lineage-Specific Markers Highlight Acquisition of Divergent Cell Fates by NSCs Located in Different dSVZ Subdomains . (A and B) Heatmaps show enrichment of transcripts in NSCs of distinct SVZ regions (dmNSCs, dlNSCs, lNSCs) compared with the dorsal environment (dSVZ). Transcripts of stem cell, proliferation, and neurogenesis markers (A; top panel) and selected signaling pathways . (A; bottom panel) were analyzed. Selected markers of the astroglial (B; top panel), oligodendroglial (B; middle panel), and neuronal lineage (B; bottom panel) were analyzed. Astrocyte and neuronal markers show regional enrichment in dmNSCs and dlNSCs, respectively, while oligode ndroglial markers show partial preference for enrichment in dmNSC. Note that HOPX transcripts were enriched in dmNSCs. (C) Scheme representing the counter gradients of the expression of Hopx (red) and astroglial

Figure 11 .

 11 Figure 11. Conditional Fate Mapping Reveals HOPX -Expressing NSCs Are Biased to Generate Astrocytes. (A-C) Co-electroporation of a pCX-GFP and an inducible pFloxpA-DsRed plasmid (1:2; A) in HOPX Cr e er t 2 mice, allows lineage tracing of HOPX + and HOPXpopulations at short term (7 dpe) and long term (21 dpe; B). This approach results in GFPonly progenies from HOPX + NSCs, whilst HOPX + NSCs give rise to dsRED/GFP (termed as DsRed) progenies (C). (D and E) Representative micrographs of an LV (D) and OB (E) section

Figure 12 .

 12 Figure 12. Single-cell RNA Sequencing Meta -analysis Reveals Hopx-expressing RGCs Share Transcriptional Features with Adult Astrocytes. (A) t-SNE of E15.5 cortical cells (left panel) and E17.5 cortical cells (right panel) generated from Yuzwa et al. raw datasets and Feature Plots showing Sox2 expression (in red) identifying the RGCs cluster at both timepoints. (B) t-SNE of E15.5 and E17.5 RGCs indicating transcriptional changes occur over embryonic development. (C-E) E15.5 RGCs show higher cell cycle activity as indi cated by enrichment of cell cycle gene ontologies (C), as well as higher neurogenic potential, as illustrated by NeuroD6 expression. (D) In contrast, E17.5 RGCs show astroglial potential as indicated by Sparcl1 expression (E), correlating with the neurogenic to astrogenic switch occurring at this late embryonic time point. (F-G) t-SNE of E17.5 RGCs using adult astrocytes marker from Zywitza et al. as input genes, reveals 2 identified clusters (F). Cluster 1 contains 44% of the astrocyte markers list whereas Cluster 2 contains only 7% (G). (H) Feature Plots showing Aldh1l1 expression, a classical astrocytic marker, as illustration.

Figure 13 .Figure

 13 Figure 13. Neurog2 Cr e er t 2 /tdTom Mice Allow Fate Mapping of Birth-Dated Cohorts of Glu Progenitors and Reveal Their Persistence at Postnatal Stages. (A) In situ hybridization from the Allen Brain Atlas showing Neurog2 expression in the VZ and SVZ at E15.5. (B)

Figure 15 .

 15 Figure 15. Dysregulation of Neurogenic Transcriptional Coding in Postnatal Glu Progenitors. (A and B) Hierarchical clustering (A) and t-SNE of individual cells recombined at E15.5 and P2 allows distinguishing 2 clusters of cells, actively cycling cells (i.e., progenitors), and their immediate progeny (i.e., nascent neurons) (B). (C) Feature plot showing the expression of transcripts of the Glu lineage. (D) Venn diagrams showing the number of differentially expressed transcripts in progenitors or nascent neurons . (E and F) Transcriptional differences increase as differentiation progresses, as reflected by the percentage of transcripts differentially expressed between E15.5 and P2 (E), as well as the gradual loss of transcripts expressed at E15.5 in P2 -sorted cells (F). (G) Feature plots showing downregulation of methyltransferases Mettl3 and Mettl14 in P2 progenitors and upregulation of m6A-tagged transcripts Sox2, Pax6, Neurog2, and Tbr2. (H) Dot blot showing decreased m6A levels at P2 and methylene blue staining confirming equal mRNA loading on membrane. (I) Quantification of m6A levels. (J) GO showing downregulation in P2 progenitors of transcripts involved in transcription and metabolism. (K) GO analysis showing downregulation of migration, differentiation, and cell death in P2 nascent neurons. (L) KEGG pathway analysis highlighting dysfunctional signaling pathways. Data are presented as mean ± SEM.

Figure 16 .

 16 Figure 16. Partial Rescue of Impaired Migration and Differentiation of Postna tal Glu Progenitors by Pharmacological and Genetic Manipulations . (A) Feature plot showing decreased expression Ctnnb1 (β-catenin) in P2 nascent neurons. (B) Feature plots showing altered expression of two Wnt-signaling transcripts. (C) PPI analysis showing the top 10 proteins to interact with proteins coded by genes downregulated at P2. (D-F') Pharmacological activation of the Wnt canonical pathway increases the (D) number and (E) proliferation (Ki67) of tdTom + cells in the dSVZ at 72 hr. (F and F') . Representative images showing tdTom + progenitors migrating away from the dSVZ. (G) Feature plot showing decreased expression of Bcl11a in P2 nascent neurons. (H) Overexpression of Bcl11a increases proliferation in the dSVZ while promoting cell migration and bipolar cell morphology outside the dSVZ. (I-J') Representative images after electroporation of control plasmid (LacZ) (I-I') or Bcl11a (J-J') overexpression. Scale bars: 100 mm in (F); 50 mm in (J); and 25 mm in (F') and (J'). *p < 0.05; **p < 0.005. Data are presented as mean ± SEM.

Figure S7 .

 S7 Figure S7. Related to Figure 13. Neurog2 Cr e er t 2 /tdTom mice reveal a late wave of corticogenesis early postnatally. (A-E) tdTom + cells in both upper and deeper cortical layers express markers of glutamatergic projection neurons. (A) Overview showing the expression of Satb2 throughout cortical layers and Ctip2, which is strongly expressed in L5 -6. (B-C) Examples of tdTom + Satb2 + cells. (D) The majority of tdTom + cells express Satb2, but not the deep cortical layer marker Ctip2 at P7. (E) Quantification of the percentage of tdTom + cells that express Cux1, Satb2, Ctip2 and FoxP2 at P45. Scale bars 75μm (A); 25μm (B-C). Data are represented as mean ± SEM.

Figure 17 :

 17 Figure 17: (A) Schematic representation of the organization of the periventricular germinal niche at pre-and postnatal timepoints. The terminology used to define neural progenitors at pre-and postnatal timepoints is summarized. The multiple layers of the embryonic periventricular germinal region merge following birth and become separated from the ventricular lumen by ependymal cells. Radial Glia (also termed basal progenitors) are the bona fide neural stem cells. They generate neurons direct ly or through intermediate progenitors, as well as glial cells, ependymal cells and adult neural stem cells. Different colors and shades of blue are used to illustrate their heterogeneity in term of fate and transcriptional profiles, as discussed in this r eview. Adapted with permission of Development.(B) Schematic representation of the germinal regions of the embryonic forebrain. Distinct neuronal subtypes emerge from neural progenitors located in defined pallial and subpallial regions. Gradients represent the transcriptional differences observed between these neural progenitor populations. Note that transcriptional differences are minimal in neural progenitors and exacerbated during differentiation. (C) Schematic representation of transcriptional programs superimposition during cellular differentiation. While some transcriptional programs are generic to all cells (generic genes), others are expressed at different stages of their differentiation but remain generic to all lineages (stage specific genes). Line age specification is coded by a small number of genes which progressively increase with the acquisition of defined morphology, connectivity and functions. Clustering sensitivity following single cell RNA-Sequencing rely on differentially expressed genes wh ich only become clearly distinctive between distinct neuronal lineages at the post -mitotic level. It is therefore highly dependent on sequencing depth and number of cells analyzed.

Future

  developments are likely to allow integrating emerging approaches to investigate the three-dimensional genomic organization (e.g. Chi-C mapping) or chromatin accessibility (e.g. ATAC-Seq) at the single cell level. This will provide a complete picture of transcriptional and epigenetic changes occurring in neural progenitors. Extension of these methods to pathological situations will inform on mechanisms involved in NSC activation and will guide research in their priming towards specific fate for the development of regenerative therapies.

Figure 18 :

 18 Figure 18: A unifying scheme of pre-and postnatal germinal activity, integrating single cell findings discussed in this review . (A) Schematic representation of the germinal region of the embryonic forebrain and of th eir evolution during postnatal life. (B) During development, neural progenitors from the pallium and subpallium generate distinct neuronal subtypes.Whereas the coding of this diversity is primarily spatial in the subpallium, it is temporal in the pallium with the sequential generation of deep, then upper layer neurons. Ependymal cells are produced from a subpopulation of neural progenitors around E14.5, in a time course concomitant to the generation of adult NSCs in the subpallium. Around birth, a fraction of neural progenitors change fate to produce astrocytes in both regions, while others continue generating a small population of glutamatergic neurons in the cortex. Following birth, germinal activity remains sustained in SVZ regions originating from the subpallium (i.e. mSVZ and to a larger extent lSVZ), while it gradually disappears from those derived from the pallium (dSVZ). Abbreviations: LGE: lateral ganglionic eminence; MGE: medial ganglionic eminence; dSVZ, lSVZ, mSVZ: dorsal, lateral and medial subv entricular zone.

Figure 19 :

 19 Figure 19: Specific Targeting of Layer IV Cortical Neurons induces fast and efficient neuronal ablation. (A) 3D reconstruction of DTR-GFP electroporated brains representing the average surface of the targeted area based on GFP expression. (B-C) Representative images of coronal brain sections showing localization of GFP electroporated cells at low and high magnification using epifluorescent ( B) and confocal microscopy (C), respectively. In panel C, co-immunolabeling of GFP with Cux1, a upper layers marker, and Ctip2, a lower layer marker, were used to define borders of cortical layers. (D) High magnification of GFP/Cux1 immunolabeling image shows that all GFP + cells are Cux1 + upper cortical neurons. In average, GFP + cells represent 30% of the total number of layer IV Cux1 + neurons (Not shown). (E) Representative image of GFP targeted neurons co -expressing the Diphtheria toxin (DT) receptor dete cted with a HB-EGF antibody (Heparin Binding EGF-like Growth Factor). Note that all electroporated neurons express the DT receptor. (F-G) Representative image of pyknotic Dapi + cells in targeted area (F) resulting in a 20% decrease of Cux1 + thickness 5 days following ablation induction (G) and demonstrating efficiency of the ablation approach. (I-J) Representative images of brain response 5 days following DT injection. GFAP expression, a marker of astroglial reactivity, is increased at the lesion site (I), such as Iba1 + cells number, a marker of responsive microglia (J). Arrow indicating GFP + cells engulfed by a microglia. UL, upper layers; DL, deeper layers. Scale bars: 500 µm in (B); 50 µm in (B); 10 µm in (C), 20 µm in (E/J) and 100 µm in (I). ***p < 0.00 1, data are presented as mean ± SEM from n=3 animals.

Figure 20 :

 20 Figure 20: Targeted cortical ablation induces progenitor responses within the dSVZ. (A) Representative pictures of Tbr2 (red, top panel), Ki67 (red, bottom panel) and Olig2 (green, bottom panel) immunostaining in the dSVZ, 5 days post -ablation. (B) Histograms showing the proportion of Ki67 + , Tbr2 + and Olig2 + cells 5 days and 12 days following ablation induction, relative to control animals. The relative numbers of proliferative progenitors (Ki67 + ) and oligodendrocyte progenitors (Olig2 + ) increase at 5 days, whereas only the Olig2 + increase persists at 12 days. The relative number of glutamatergic progenitor (Tbr2 + ) do not vary at any time points. (C) Administration of AR-A014418 in control animal efficiently increases the relative proportion of Ki67 + and Tbr2 + cells 5 days post-treatment, as previously described (see experimental chapter 2; Azim et al. 2014), thereby validating the pharmacological Wnt-signaling pathway activation. (D) Histogram showing the efficient increase of Tbr2 + progenitor following DT injection and AR -A014418 administration. (E) Histograms of GFAP optical density within the cortical lesion site reveal a significant decrease in GFAP + reactive astrogliosis following AR-A014418 treatment, indicating a potential effect on tissue inflammation.

  yuzwaE15_seurat reduction.type = "pca", dims.use = 1:12, resolution = 1.2, print.output = FALSE, save.SNN = TRUE, force.recalc = TRUE) TSNEPlot(yuzwaE15_seurat, do.label = FALSE, pt.size = 3) #RP cells = cluster 4 yuzwaE17_seurat <-FindClusters(object = yuzwaE17_seurat, reduction.type = "pca", dims.use = 1:12, resolution = 0.5, print.output = FALSE, save.SNN = TRUE, force.recalc = TRUE) TSNEPlot(yuzwaE17_seurat, do.label = FALSE, pt.size = 3) #RP cells = cluster 3 #Analysis only in RGCs RPE15.cells=WhichCells(yuzwaE15_seurat, 4) RPE17.cells=WhichCells(yuzwaE17_seurat, 3) #RPE15 vs RPE17

  le cerveau antérieur postnatal après la fermeture de la période de neurogenèse corticale. Les progéniteurs postnataux Glu ne s'accumulent pas au cours du développement embryonnaire mais sont produits par des cellules gliales radiales embryonnaires qui persistent après la naissance dans la zone sous-ventriculaire dorsale

	(dSVZ) et continuent à donner naissance à des neurones corticaux, bien que l'efficacité
	soit faible. Le séquençage d'ARN sur cellule unique révèle une dérégulation des
	programmes transcriptionels corrélée au déclin progressif de la neurogenèse corticale
	observé in vivo. Des manipulations génétiques et pharmacologiques montrent que les
	progéniteurs postnataux sont partiellement permissifs.
	Au cours du développement cortical, les neurones glutamatergiques naissent de
	progéniteurs Glu situés dans la zone ventriculaire (VZ) et la zone sous-ventriculaire (SVZ)
	et s'assemblent pour former les circuits sous-jacents aux fonctions cognitives. Comme
	décrit précédemment, il est généralement admis que la période de neurogenèse corticale
	s'achève autour du jour embryonnaire (E) 17,5 chez la souris, les progéniteurs neuronaux
	changeant alors de destin pour produire des astrocytes
	. Ceci, combiné à l'expression
	d'HOPX dans les oRGCs, qui manquent de processus apicaux chez les primates, suggère
	qu'un signal inconnu sécrété par dans le liquide céphalo-rachidien pourrait réguler
	l'expression d'HOPX. En résumé, nos travaux démontrent que la dSVZ est beaucoup plus
	hétérogène qu'on ne le pensait auparavant en termes de ségrégation spatiale et
	d'amorçage précoce des NSCs lors de la génération de lignages neuronaux spécifiques.
	L'expression abondante du TF HOPX contribue à l'hétérogénéité intrarégionale de la
	dSVZ chez les rongeurs.
	Dans mon deuxième chapitre expérimental, des approches transcriptomiques et de
	cartographie du devenir cellulaire ont été utilisées pour étudier l'origine, les spécificités
	transcriptionnelles et la compétence des progéniteurs glutamatergiques (Glu) postnataux.
	J'ai établi le pipeline complet de l'isolement des progéniteurs Glu des souris Ng2 Creert2 Ai14
	-après optimisation des injections de tamoxifène -à l'analyse bioinformatique du
	séquençage d'ARN en cellule unique à l'aide du logiciel Seurat sous R. J'ai également

effectué des manipulations génétiques in vivo sur des embryons ainsi que sur des animaux nouveau-nés pour aborder la question de l'origine et de la compétence des progéniteurs Glu. Nos résultats ont montré qu'une grande partie des progéniteurs Glu XV persistaient dans

  ). On pense généralement que les progéniteurs de neurones glutamatergiques corticaux changent de destin avant la naissance pour produire des astrocytes. Mes résultats montrent que ce changement est en grande partie incomplet et qu'une grande partie des progéniteurs Glu Pris ensemble, mon travail de thèse illustre l'existence d'une profonde hétérogénéité des NSCs dans la SVZ postnatale, qui peut être explorée du niveau régional au niveau cellulaire. Cela met également en évidence l'intérêt des études transcriptionnelles pour explorer la diversité de l'hétérogénéité des NSCs à ces différentes échelles. Il démontre

	List of Abbreviations IPC intermediate progenitor cell
	continue à être produite dans le cerveau antérieur postnatal après la fermeture de la période de neurogenèse corticale. Le séquençage d'ARN sur cellule unique révèle toutefois une dérégulation transcriptionnelle profonde aux stades postnataux, ce qui est en corrélation avec le déclin progressif de la neurogenèse corticale observé in vivo (chapitre 2). IUE in utero electroporation AC anterior commissure LGE lateral ganglionic eminence AH anti Hem LIF leukemia inhibitory factor Aldh1l1 aldehyd dehydrogenase lNSC lateral neural stem cell ANR anterior neural ridge lSVZ lateral subventricular zone microdomain BLBP brain lipid-binding protein LV lateral ventricle BMP bone morphogenetic protein MADM mosaic analysis with double markers BrdU 5-bromo-2'-deoxyuridin MAP2 microtubule-associated protein-2 CB calbindin MGE medial ganglionic eminence CC corpus callosum mSVZ medial subventricular zone microdomain CGE caudal ganglionic eminence NECs neuroepithelial stem cells CH cortical hem NeuN neuronal nuclei CNS central nervous system NP neural progenitor CoPNs commissural PNs NSC neural stem cell CP cortical plate OB olfactory bulb CPNs callosal PNs Olig2 oligodendrocyte transcription factor 2 CR calretinin OPC oligodendrocyte precursor cell CSF cerebrospinal fluid oRG outre radial glia CTPNs corticotectal PNs PCA principal Component Analysis Ctrl. control PN projection Neuron DCX doublecortin POA preoptic area dEPO dorsal electroporation PPI proteinprotein interaction DG dentate gyrus Prog progenitor dlEPO dorso-lateral electroporation PSA-NCAM polysialylated-neural cell adhesion molecule dlNSC dorso-lateral neural stem cell PV parvalbumin dlSVZ dorso-lateral subventricular zone microdomain qNSCs quiescent NSCs dmEPO dorso-medial electroporation qPCR quantitative polymerase chain reaction dmNSC dorso-medial neural stem cell RG radial glia dmSVZ dorso-medial subventricular zone microdomain RMS rostral migratory stream dNSC dorsal neural stem cell S100β s100 calcium-binding protein B
	dpe SCDE dSVZ scRNA-seq DT SCZ DTR SGZ EGFR Shh EPO SST FACS St FGF SVZ GFAP Tam GFP TAP	days post electroporation single-cell differential expression dorsal subventricular zone microdomain Single cell RNA sequencing diphtheria toxin subcallosal zone diphtheria toxin receptor subgranular zone epidermal growth factor receptor sonic hedgog electroporation somatostatin fluorescence activated cell sorting striatum fibroblast growth factors subventricular zone glial fibrillary acidic protein tamoxifen green fluoresent protein transient amplifying progenitor
	GL Tbr2 GLAST TF Glu TH IFC t-SNE IN VZ	glomerular layer T-box brain protein 2 glutamate aspartate transporter transcription factor glutamatergic tyrosine hydroxylase integrated fluidic circuits distributed stochastic neighbour embedding inhibitory neuron ventricular zone

enfin la nécessité de l'analyse clonale en histologie et de la transcriptomique unicellulaire pour éclairer de manière plus approfondie notre compréhension de l'activité germinative du cerveau antérieur aux moments pré et postnataux. Dans la discussion générale de ce manuscrit de thèse, je passe en revue la contribution récente des approches unicellulaires, y compris les travaux décrits dans cette thèse, sur la progression du lignage et des compétences des progéniteurs neuraux tout au long du développement pré et postnatal (revue concise soumise et en cours de révision). Enfin, je conclus en examinant l'implication de ces résultats dans la réparation du CNS et en proposant un nouveau modèle expérimental pour étudier la compétence des progéniteurs neuronaux postnataux dans la régénération de sous-types neuronaux définis à la suite d'une lésion cérébrale.

Table of Contents

 of

1. Regional Heterogeneity and Competence of Neural Stem Cells Throughout

  Principles of Cortical Organization ........................................................ 1.1.4. Molecular Diversity of Anatomically Defined PNs ........................................... 1.2. Germinal Activity Persists in the Postnatal SVZ ..................................................

1.2.1. The Postnatal Niche ..................................................................................... 1.2.2. The Radial Glia Origin of Type B1 Cells: ...................................................... 1.2.3. Type B1 Cells: A Specialized Astrocyte ........................................................ 1.2.4

. Regionalization and heterogeneity of Postnatal SVZ NSCs .......................... 1.2.5. Regionalization also applied to gliogenesis .................................................. 1.2.6. Signals from the CSF and the niche act in concert to regulate NSCs activity and regionalization throughout

2. Experimental Chapter 1: HOPX Defines Heterogeneity of Postnatal Subventricular Zone

  Dysregulation in Postnatal Glutamatergic Progenitors Contributes to Closure of the Cortical Neurogenic Period .......................... 55 3.1. Introduction ......................................................................................................... 56 3.2. Experimental Procedures ................................................................................... 56 3.3. Results ............................................................................................................... 61

	..................................................................................................... 80
	4.2.1. Clonal Techniques in Histology and Transcriptomic ..................................... 80
	4.2.2. Contributions of Single Cell Approaches to Probe Spatial Identity Specification
	.............................................................................................................................. 82
	4.2.3. Contributions of Single cell Approaches to Probe Temporal Identity
	Specification .......................................................................................................... 86
	4.2.4. Contributions of Single cell Approaches to Probe Adult NSCs Origin and Biology

Fate Mapping of

92 INTRODUCTION XXVIII 1. Regional Heterogeneity and Competence of Neural Stem Cells Throughout Development and Postnatal Life

	The central nervous is a tissue of exquisite complexity. It forms in a stepwise manner
	during embryonic development from a population of specialized cells within the ectoderm.

.1 Are embryonic and Postnatal/adult NSCs Representing Distinct Populations in term of

Diversity and Competence? .......................................................................

Figure 1: Cerebral cortex development and morphogens expression. (A) The

	rostral part
	of the neural tube evolves rostrally into the primary forebrain (FB), midbrain (MB) and
	hindbrain (HB) vesicles while the caudal part gives the spinal cord (SC). The secondary
	vesicles telencephalon (Tel), diencephalon (Di), metencephalon (Met) and myelencephalon
	(Myel) develop from the primary vesicles. (B) The anterior neural ridge (ANR), cortical hem
	(CH) and anti-hem (AH) are secreting centers. ANR secretes FGFs, CH secretes Wnts/BMPs
	and AH secretes FGF15 morphogens in the telencephalon. Shh is mainly present in the
	ventral part of the telencephalon, where ganglionic eminences are specified and generate
	interneurons that follow dorsal tangential path to invade the developing cortex. All
	morphogens are also found in the CSF secreted by choroid plexus (CP) and filling t he lateral
	ventricles (LV). (C) Morphogens gradients in the dorsal telencephalon. Illustration from
	Agirman et al., 2017.
	At the end of the neural tube closure, the neuroepithelium is composed of neuroepithelial
	stem cells (NECs), the earliest progenitors of the cortex. They are organized in a
	pseudostratified neuroepithelium because their nuclei migrate up and down the apical-
	basal axis during the cell cycle. NECs show typical epithelial features and are highly
	polarized along their apical-basal axis. These cells initially expand their pool by successive
	symmetric proliferative divisions and later divide asymmetrically to generate radial glial
	cells (RGCs) that sit in the ventricular zone (VZ) of the dorsal part of the telencephalon,
	the most apical cell layer that lines the ventricles. During this process, NECs lose some of
	their epithelial traits to acquire astroglial hallmarks. For example, in contrast to NECs,
	RGCs show several astroglial properties such as the presence of glycogen granules and
	the expression of astrocytic proteins like GLAST, S100β, GFAP, Vimentin and BLBP. This
	transition marks the onset of neurogenesis in mice and occurs throughout most of the brain
	between embryonic days 10 (E10.5) and E12.5.

2: Neural progenitor subtypes and neurogenic phases during cerebral cortex development in mice. (A) RGCs

	maintain a bipolar morphology with apical and basal
	processes. (B) Sequential steps of neurogenesis in the mouse: neuroepithelial cells (NECs,
	grey) self-renew by symmetric division, then turn into apical radial glial cells (RGCs, pink)
	that divide either symmetrically to self-renew, or asymmetrically to give rise first to primary
	neurons including Cajal-Retzius cells (dark green) (direct neurogenesis) which migrate to
	the cortical surface to form the marginal zone (MZ), and to intermediate progenitor cells (I PC)
	at E12.5 and onwards; IPCs populate the subventricular zone (SVZ) and generate cortical
	layer neurons (dark to light blue), which migr ate along the basal process of RGCs through
	the intermediate zone or subplate (IZ/SP) towards their destined layer. At later stages, some
	RGCs can undergo final symmetric divisions generating two neurons or switch to gliogenesis.
	Illustration adapted from Jiang and Nardelli, 2015.

  PV-and SST-positive INs are primarily found in the deep layers of the cortex, and 5HT3aR-positive INs preferentially populate the upper layers

	and others.
	, which
	include: parvalbumin (PV), somatostatin (SST) produced mainly by MGE, vasoactive
	intestinal peptide (VIP), serotonin receptor 3a (5HT3aR), calretinin (CR) produced by CGE

Figure 5: Schematic illustration of the six horizontal layers characteristic of the cerebral neocortex. (A) Schematic

	representation of primary neocortical areas . (B) Cortical
	columns contain horizontally arranged layers with very diverse neuronal compositions. (C)
	Layer II/III contains different classes of commissural neurons, primarily of distinct CPN
	identities. Layer V contains CPNs, often maintaining distinctive collaterals to the striatum
	and different classes of subcerebral PNs that connect to the brainstem, spinal cord, and
	superior colliculus. Layer VI has different classes of CThPNs, connecting to separate
	thalamic nuclei, and CPNs that connect through the CC. Cortical PN subtypes express unique
	gene signatures that in specific combinations identify each class (listed on right).
	Abbreviations: A1, auditory cortex; CC, corpus callosum; CPN, callosal projection neuron;
	CTPN, corticotectal projection neuron; CThPN, corticothalamic; F/M, frontal/moto r cortex; IN,
	interneurons; IT, intratelencephalic; PN, projection neuron; S1, somatosensory cortex; Th,
	thalamus; V1, visual cortex. Illustration from Lodato and Arlotta, 2015.

  . The ligands of Notch signaling are transmembrane proteins that are present on the surface of neighboring cells. Such as IPs

or neurons in the developing brain. There are five mammalian Notch ligands; Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3 and DLL4, belonging to the Jagged and Delta-like families, respectively. As transmembrane ligands, they provide short-range signals. Notch in neural stem cells (NSCs) has been implicated in the maintenance of the undifferentiated and multipotent state

  Germinal activity persists in the postnatal mammalian brain in specialized niches, namely the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) that

	surrounds the lateral ventricle (LV). Neural stem cells (NSCs) of the postnatal SVZ divide
	and give rise to transient amplifying progenitors (TAPs) that generate neuroblasts, which
	migrate via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they
	differentiate into neurons

  All animal experiments in Zurich were performed according to the Ethics Committee of the Veterinary Department of the Canton of Zurich (Approval ID 182/2011). Experiments in France were performed in accordance with European requirements 2010/63/ UE and have been approved by the Animal Care and Use Committee CELYNE (APAFIS #187 & 188).

Contributions of Single cell Approaches to Probe Neural Progenitor's Heterogeneity and Dynamics

	under review). Finally, I conclude by discussing implication of these findings for CNS repair
	and propose a new experimental model to investigate the competence of postnatal neural
	progenitors in regenerating defined neuronal subtypes following brain injury.
	4.2. The forebrain is the result of a remarkable coordination of molecular and cellular
	embryonic events, which transform the simple neuroepithelium (E10) into complex neural
	networks orchestrating sensorimotor and cognitive functions of the body. Development of
	the forebrain occurs in a stepwise manner. Neural progenitors (NPs) first form the
	pseudostratified epithelium of the ventricular zone. They initially divide symmetrically,
	undergoing interkinetic nuclear migration as they progress through the cell cycle, to
	expand their pool. Around E11.5 in the mouse, NPs acquire a radial morphology to form
	the Radial Glia (RG) that function as the primary neural stem cells of the developing CNS
	(Figure 15A). RGs have a bipolar morphology with apical and basal contacts with the
	surface of the ventricle and of the brain, respectively. They sequentially produce distinct
	subtypes of neurons before generating glial cells around birth. Later, germinal activity,
	including neurogenesis, persists throughout life in restricted forebrain regions, in particular
	the subventricular zone that lines the lateral ventricles.
	Although this stepwise progression through competence states is well accepted at the
	population level, it is still debated if it applies to all NPs or if subpopulations of fate-biased
	NPs coexist. Clonal analyses in histology or transcriptomics have recently emerged as
	approaches to explore NPs lineage progression and competence. Here I discuss recent
	single cell studies made in the developing forebrain to probe the spatial and temporal
	neural progenitor's identity specification. I also discuss how single cell studies are
	It further contributing to our understanding of pre-and postnatal neural progenitor's changes in their
	highlights the benefit of transcriptional studies in exploring the diversity of NSCs competence and distribution over time.
	heterogeneity at these different scales. It finally demonstrates the necessity of clonal
	analysis in histology and single cell transcriptomic in shedding new lights on our
	understanding of forebrain germinal activity at pre-and postnatal time points. In this
	general discussion, I review recent contribution of single cell approaches, including the
	work described in this thesis, in probing neural progenitors lineage progression and
	competence throughout pre-and postnatal development (concise review submitted and

  Although, generation of new cortical neurons was not spontaneously observed, this model might be used to test strategies to recruit Glu progenitors, as discussed in Chapter 2.

	Results
	Diphtheria toxin is a well-characterized bacterial toxin composed of an A and a B chain.
	The toxin can only enter into the cell by endocytosis if a specific receptor (DTR), bound by
	the B chain, is expressed on the cell surface. Inside the cell the A (DTA) chain inactivates
	elongation factor 2 via ADP ribosylation, leading to cessation of protein synthesis and
	subsequent cell death. Rodent cells are naturally resistant to DT, due to the absence of
	DTR on their membrane (Buch et al., 2005). Sensitivity of select neuronal populations to
	DT can therefore be induced by selective DTR expression. We focused on layer IV cortical
	neurons, which integrates thalamic inputs into cortical networks. Due to the sequential
	generation of cortical neurons, neurons of select cortical layers can be efficiently
	electroporated by in utero electroporation at defined embryonic timepoints. Electroporation
	at E14.5 of a DTR-GFP plasmid (at 1.5µM) resulted in an efficient transfection of a large
	number of layer IV neurons, i.e. 30% of Cux1
	. Although I did not manage
	to complete this project due to time constrains, I obtained encouraging data showing that
	DT-mediated apoptotic death of select neuronal population is efficient and sufficient to

induce a proliferative response of progenitors in the dSVZ. This model present several advantages that will prove to be of interest to test repair capacity under various contexts, the spatio-temporal control of ablation and the restrained inflammation response. + layer IV neurons in our average (Figure

19A-D

). Electroporated neurons were distributed over a large area, covering in average 8.5 mm² of cortex. Five days following a single intraperitoneally DT injection, at the concentration of 25ng/g in P2 animals, we observed a significant 20% decrease of CUX1 + upper layer thickness (Figure

  I next assessed if de novo cortical neurogenesis was observed following targeted cortical ablation. The mitotic marker BrdU was administered at the time of DT injection to label the cohort of Glu progenitors and the presence of BrdU + /NeuN + newborn neurons assessed within cortical layer 12 days post ablation. Strikingly, no BrdU + /NeuN + newborn neurons were observed indicating that spontaneous neurogenesis does not occur in this model, although this still need to be confirmed with the use of additional neuronal markers (e.g. finally assessed if a pharmacological activation of canonical Wnt signaling could promote recruitment of Tbr2 + progenitors. The GSK3β inhibitor AR-A014418 was then administered for 3 days in P2 control animals to confirm that the relative proportion of Ki67 + and Tbr2 + cells 5 days post-treatment efficiently increases, as previously reported

	Dcx, Cux1…).

I

Annex 1: R Script To Run HeatMaps Generator Software Related to Figure 8 and S1

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	str_split_fixed(list.files(),"_",2)[,1]))){ ## If file already here, no download
	# getGEOSuppFiles(i,makeDirectory = F)
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	# Check file names with too many letters, select them and rename them as
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	# # source("http://bioconductor.org/biocLite.R") if(length(list.files()[which(nchar(list.files())>14)])>0){
	# # biocLite("affy") for(s in 1:length(list.files()[which(nchar(list.files())>14)])){
	# # biocLite("oligo") file.rename(list.files()[which(nchar(list.files())>14)][s]		,
	# # biocLite("limma")
	# library(oligo)
	library(affy)
	library(stringr)
	library(pheatmap)
	library(stringr)
	library(gplots)
	library(GEOquery)
	library(mouse4302.db)
	library(R.utils)
	# library(affyio)
	# setwd("~/Desktop/Quentin/heatmaps sur GEO gsm/")
	setwd("./")
	pathex = getwd()
	for(w in list.files("./DataSet/")){ # Loop on file name
	# w = list.files("./DataSet/")[1]
	setwd(pathex)
	inputf = read.csv(paste0("./DataSet/",w)) # Open a dataset file containing GSM
	# ## checker :
	# colnames(inputf)
	# list.dirs()
	# dir.create(paste0("./",str_split_fixed(w,".csv",2)[1]))
	# setwd(paste0("./",str_split_fixed(w,".csv",2)[1]))
	for(o in 1:dim(inputf)[2]){ ## Loop on column number
	# o = 1
	setwd(pathex)
	setwd("./temp/")
	file.remove(list.files())
	setwd("../GSM/")
	# setwd(paste0("./",str_split_fixed(w,".csv",2)[1]))
	# dir.create(paste0("./",colnames(inputf)[o]))
	# setwd(paste0("./",colnames(inputf)[o]))
	for(i in as.character(inputf[,o])){
	# i = as.character(inputf[,o])[1]
	if(nchar(i)!=0){ # If there is no empty GSM

# install.packages("/home/affxparser_1.48.0.tar.gz", repos = NULL, type="source") # install.packages("/home/xps_1.36.1.tar.gz", repos = NULL, type="source")

########################### Define Genes :

  %in% list.files())){ # If download fails, do it again try(getGEOSuppFiles(i,makeDirectory = F))# Check file names with too many letters, select them and rename them as GSMxxxxxxx, looping if several

if(length(list.files()[which(nchar(list.

files())>14)])>0){ for(s in 1:length(list.files()[which(nchar(list.files())>14)])){ file.rename(list.files()[which(nchar(list.files())>14)][s]

,

IdentityMarkers (1)

IdentityMarkers (2)

Acknowledgments

List of Figures

Supplementary Figure S2

persist after birth in the dSVZ, in accordance with recent studies (Fuentealba et al., 2015b;[START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. To explore the mechanisms of the decreased neurogenic capacity of postnatal Glu progenitors, we performed scRNA-seq of tdTom + cells isolated by flow cytometry from microdissected pallium at E15. [START_REF] Annexes | [END_REF] 15A and15B), corresponding to actively cycling recombined cells (i.e., progenitors) and their immediate progeny (i.e., nascent neurons) (Figure 15B and Annex 9). Expression of lineage-specific [-c(as.integer(names(temp)

or P2. Unbiased clustering combined with interrogation of cell-cycle and post-mitotic markers revealed several cell clusters (Figures

Supplementary Figures

ANNEXES if(length(which(a$X == z))!=1){ ## test if genes already exist if(dim(a)[2]>2){ temp = rowMeans(a[which(a$X == z),-1]) # Mean of gene expression names(temp) = which(a$X == z) indice = which(temp==max(temp)) # indice to keep, max value of the gene

)[-indice]),] ## remove doublet lines excluding the indice to keep }else{ # if table has one cell : temp = (a[which(a$X == z),-1]) # mean of gene expression names(temp) = which(a$X == z) indice = which(temp==max(temp)) # indice to keep, max value for this gene # temp = a[which(a$X == z),] if(length(indice)>1){# If two have same mean indice=indice[1] } a = a[-c(as.integer(names(temp))[-indice]),] ## remove doublet lines excluding the indice to keep

a[which(a$X==unique(as.character(read.csv(paste0("../../genes/",w),header = F)$V1))),] akeep=NULL for (b in unique(as.character(read.csv(paste0("..

/../genes/",w),header = F)$V1))){ # loop on gene name of current table akeep = c(akeep,which(a$X == b)) } a<-a[akeep,]

rownames(a)<-a$X write.csv(a,paste0("./TablesPurif/",i)) } } ListGenes= as.character(read.csv(paste0("../../genes/",str_split_fixed(w,".csv",2)[1],".csv"),header = F)[,1]) # ListGenes = c("Hdac2","Emx1","Hopx","Gfap","Slc39a12","Aqp4","Slc14a1","2900052N01Rik","Chrdl1 ","Aldh1a1","S100b","2900052N01Rik-203","Ttr","Ddn","Hpcal4","Scn4b","Syndig1l","Rasd2","Rasgef1a","Camk2a","Ermn","Mal ","Mog","Mobp","") ## Experiments list conssitent with inputf table setwd("./TablesPurif/") Annex 2: R Scripts Used for Meta-Analysis of E17.5 Hopx Expressing RGCs. Related to Figure 12 Raw data : 1. Read counts are available in the supplementary information of [START_REF] Yuzwa | Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling[END_REF] 2. Astrocyte markers are available in supplementary information of Zywitza et al. I used the R Seurat package (V2.3) for unbiased clustering and gene expression analysis t-SNE Generation Related to Figure 12A. library(Seurat) library(dplyr) setwd("D:/Yuzwa") E15.5 yuzwaE15.data = read.table("GSE107122_E155_Combined_Only_Cortical_Cells_DGE.csv", sep=",", header = TRUE, row.names = 1) yuzwaE15_seurat = CreateSeuratObject(raw.data = yuzwaE15.data, min.cells = 2, min.genes = 100, project = "YuzwaE15.5", names.field = 1) yuzwaE15_seurat <-NormalizeData(object = yuzwaE15_seurat, normalization.method = "LogNormalize", scale.factor = 10000) yuzwaE15_seurat mean.function = ExpMean,dispersion.function = LogVMR,x.low.cutoff = 0.0125,x.high.cutoff = 5,y.cutoff = 1.5) length(yuzwaE15_seurat@var.genes) yuzwaE15_seurat vars.to.regress = NULL) yuzwaE15_seurat <-RunPCA(yuzwaE15_seurat, pc.genes = yuzwaE15_seurat@var.genes, do.print = FALSE) PCAPlot(object = yuzwaE15_seurat, dim.1 = 1, dim.2 = 2) yuzwaE15_seurat <-JackStraw(object = yuzwaE15_seurat, num.replicate = 100, display.progress = FALSE) JackStrawPlot(yuzwaE15_seurat, PCs = 1:12) yuzwaE15_seurat dims.use = 1:12,do.fast = TRUE,perplexity = 30) TSNEPlot(yuzwaE15_seurat, do.label = FALSE, pt.size = 4) yuzwaE15_seurat = SetAllIdent(yuzwaE15_seurat, "orig.ident") pt.size = 3, reduction.use = "tsne") FeaturePlot(yuzwaE15_seurat, "Sox2", pt.size = 5, cols.use = c("grey", "red")) E17.5 yuzwaE17.data = read.table("GSM2861514_E175_Only_Cortical_Cells_DGE.csv", sep=",", header = TRUE, row.names = 1) yuzwaE17_seurat = CreateSeuratObject(raw.data = yuzwaE17.data, min.cells = 2, min.genes = 100, project = "YuzwaE17.5", names.field = 1) yuzwaE17_seurat <-NormalizeData(object = yuzwaE17_seurat, normalization.method = "LogNormalize", scale.factor = 10000) yuzwaE17_seurat mean.function = ExpMean,dispersion.function = LogVMR,x.low.cutoff = 0.0125,x.high.cutoff = 5,y.cutoff