Au cours de la mitose, la ségrégation des chromatides, la partition du matériel cytoplasmique entre cellules filles et leur position relative se fait selon un plan qui est préfiguré par la plaque métaphasique. Ainsi, l'orientation de ce plan est un processus crucial pour le contrôle du destin des cellules, pour la morphogenèse durant l'embryogenèse et pour l'homéostasie tissulaire. Jusqu'à aujourd'hui, les mécanismes intrinsèques impliqués dans le positionnement du plan de division ont reçu beaucoup d'attention. En revanche, peu d'études ont exploré l'implication de signaux extracellulaires dans l'orientation du plan de division. Pourtant, l'axe des divisions cellulaires dont la position est souvent stéréotypée est largement associé aux axes de polarités du tissu. Au cours de ma thèse, je me suis demandé si des signaux extracellulaires capables de délivrer des informations de position spatiale aux cellules dans le cadre de leur migration, de leur différenciation morphologique, ou de leur polarisation, pouvaient influencer l'orientation des divisions cellulaires. En particulier, je me suis intéressée aux facteurs impliqués dans le guidage axonal à travers l'étude des mitoses des progéniteurs neuraux chez l'embryon de souris. Dans la moelle épinière en développement, les progéniteurs neuraux effectuent leur division au contact du canal central, lequel renferme le liquide céphalo-rachidien (LCR), une source de nombreux facteurs extracellulaires comme les morphogènes. Nous avons montré que la présence de molécules du LCR était nécessaire pour une orientation appropriée du plan de divisions des progéniteurs neuraux localisés au contact du canal central. Priver les progéniteurs neuraux de LCR par l'ouverture du tube neural ou provoquer génétiquement l'obstruction du canal central affecte les proportions de divisions planaires et obliques. Nous avons identifié la protéine Sémaphorine 3B, secrétée par les cellules de la plaque du plancher et les plexus choroïdes, comme un signal extrinsèque contrôlant l'orientation des divisions des progéniteurs neuraux dans la moelle épinière. L'invalidation génétique de Sema3B chez la souris phénocopie la perte d'accès au LCR des progéniteurs. Une application exogène de Sema3B sur des embryons dont le tube neural a été ouvert compense la déficience de LCR. Nous avons pu montrer que Sema3B se lie à ses récepteurs Neuropilines à la surface apicale des progéniteurs mitotiques et agit sur l'architecture des microtubules via l'activation de la voie GSK3/CRMP2, voie initialement mise en évidence dans le contexte du guidage axonal. Afin d'identifier de nouveaux facteurs influençant le positionnement du fuseau mitotique en réponse à ce facteur de guidage, une analyse transcriptomique des progéniteurs neuraux des mutants Sema3B-/- a été réalisée et des gènes candidats dérégulés en contexte d'invalidation de Sema3B ont été considérés. Durant la seconde partie de ma thèse, j'ai exploré l'implication du gène Norbin/Neurochondrin. De manière intéressante, le knock- down de Norbin dans les cellules HeLa altère l'orientation du fuseau mitotique. L'ensemble de ces travaux révèle donc la contribution d'une large famille de signaux topographiques jusqu'à présent inexplorée, dans l'orientation des divisions cellulaires et ouvre un large champ d'investigation passionnant concernant leur action moléculaire et cellulaire dans la neurogenèse et la morphogenèse / During development, the orientation of cell division is crucial to correctly organize andshape tissues and organs and also to generate cellular diversity. As cell mitosis proceeds, thesegregation of chromatids and cytoplasmic material occurs along a division axis. Itsorientation largely determines the relative position of daughter cells and the partition ofmother cell subcellular domain between them. The orientation of the cell division isprefigured by the position of a complex microtubule-based scaffold, the mitotic spindle.Until now, the intrinsic molecular machinery positioning the mitotic spindle and its couplingto cell polarities have been study in details. In contrast, the contribution of extracellularsignals to cell division orientation is less characterised. My research shows that these signalsin the CSF contribute to the orientation of cell division in neural progenitors. Removal theCSF cues by opening the neural tube or by genetic engineering affects the proportion ofplanar and oblique divisions. We identified Semaphorin 3B (Sema3B), released from thefloor plate and the nascent choroid plexus, as an important actor in this extrinsic control ofprogenitor division. Knockout of Sema3B phenocopies the loss of progenitor access to CSF.Delivery of exogenous Sema3B to progenitors in living embryos compensates this deficiency.We showed that Sema3B binds to Neuropilin receptors at the apical surface of mitoticprogenitors and exerts its effect through GSK3b activation and subsequent inhibition of themicrotubule stabilizer CRMP2. Thus extrinsic signaling mediated by Semaphorins directs theorientation of progenitor division in neurogenic zones.In order to identify new factors implicated in Sema3B-dependant mitotic spindleposition, we performed a transcriptomic analysis of Sema3B -/- neural progenitors. Severalderegulated candidate genes were considered. In the second part of my thesis, I focus onone of this, Norbin/Neurochondrin. Interestingly, the invalidation of Norbin/Neurochondrinalters the orientation of the mitotic spindle in HeLa cells.My PhD work reveals the contribution of a large family of topographic cues known tofunction in axon guidance has a novel role in the orientation of cell division

La libération d'hormones glucocorticoïdes (GC), en réponse au stress, est un mécanisme important du contrôle de la neurogenèse chez l'adulte. Une question non résolue est de savoir si ces hormones agissent directement sur les précurseurs neuronaux (NPCs) ou indirectement, en agissant sur d'autres types cellulaires, modifiant la libération de facteurs de croissance ou l'activité de réseaux neuronaux. Afin de répondre à cette question, nous avons développé un modèle murin dans lequel le gène du récepteur des GC (GR) est invalidé, de façon inductible, dans les précurseurs neuraux adultes. Nous avons montré qu'en présence ou en absence du GR, un traitement chronique avec des GC affecte de façon similaire la différentiation et la survie des neurones nés chez l'adulte. L'effet connu des GC sur la suppression de la neurogenèse adulte n'est donc pas du à une action directe de ces hormones sur les NPCs. L'absence du GR n'affecte pas non plus le comportement des souris mutantes lorsque les GC circulent à un niveau de base. En revanche, un traitement chronique avec des GC induit chez les animaux contrôles un phénotype anxieux (observé dans les tests de novelty-suppressed feeding, light/dark box, and elevated O-maze) alors que les animaux mutés sont préservé de ce changement comportemental. De façon similaire, un traitement chronique avec des GC facilite l'apprentissage des souris contrôles lors d'un test d'apprentissage par la peur. L'invalidation du gène GR dans les NPC bloque cet effet. L'apprentissage des souris. Ces résultats précisent le rôle du GR dans le contrôle de la neurogenèse dans l'hippocampe adulte et dans la modulation des comportements de type anxieux. / Stress hormones are known as one of the strongest and most ecologically relevant mediators of adult neurogenesis. A lingering question in adult neurogenesis is whether these hormones, known as glucocorticoids (CG), act directly on neural progenitor cells (NPCs), or indirectly through secreted factors or changes in network activity. To address these unknowns, we generated a transgenic mouse model whose GC receptors (GRs) could be inducibly inactivated specifically in NPCs. We investigated the effect of this cell-specific GR knockout model on hippocampal survival and differentiation and found them to be similarly affected by chronic GC treatment compared to controls. This implies that GC-suppressed neurogenesis and its impact on morphology is indirect, and GR in other cells may be mediating the effects. Furthermore, mice with GR inactivation in newborn neurons behaved similarly to controls in all tasks observed under basal levels of GC. When mice were chronically treated with GC, however, controls exhibited an anxious phenotype, whereas transgenic mice behaved like untreated control groups in all anxiety measures except latency to feed in NSF. Neither GC nor inactivation of GR in adult-born neurons altered depression-like behaviors in the forced swim test, nor percent freezing in contextual fear discrimination. Lastly, we found that GC increased the rate of learning in 1-trial contextual fear conditioning, an effect not mediated by reducing GR signaling in the neurogenic pool. These results highlight the functional contributions of adult neurogenesis as well as how their GRs mediate anxiety-relevant behaviors irrespective of suppressed neurogenesis.

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

Proper central nervous system development is critical for survival and depends on complex intracellular and extracellular signaling to regulate neural progenitor cell growth and differentiation; however, the mechanisms that mediate molecular crosstalk between pathways during neurogenesis are not fully understood. Here, we explored the integration of the Hedgehog (Hh) signaling pathway with the two critical developmental pathways, Receptor Tyrosine Kinase (RTK) and Notch signaling, in the growth and maintenance of neural progenitors in the developing neuroretina. We found combined and sustained RTK and Hh signaling was sufficient to establish long-term retinal progenitor cell (RPC) cultures and these cells maintained neurogenic and gliogenic, but not retinogenic, competence in vitro and in vivo. In addition, we identified crosstalk between Notch and Hh signaling, where Notch is required for Hh-mediated proliferation and Gli protein accumulation, and gain-of-function of Notch is sufficient to extend the window of Hh responsiveness in a subset of Müller glia. Both Hh-RPC monolayer establishment and Notch mediated Hh-responsiveness required Gli2. Taken together, we identified molecular cross-communication between the Hh pathway and two major pathways, Notch and RTK, during retinogenesis, advancing our understanding of mechanisms that influence Hh to control neural progenitor growth.

One mechanism of plasticity within the adult mammalian brain is the dynamic process of adult neurogenesis that is functionally important in physiological and pathological conditions. During this process, neurons develop from adult neural stem cells (NSCs) via intermediate neural progenitors (NPCs) through several processes including proliferation, survival, differentiation, migration and integration. Despite neurogenesis during development sharing these same processes, there is growing evidence highlighting unique mechanisms that regulate adult versus embryonic neurogenesis. The studies in this thesis test the cell-intrinsic function of genes that have defined roles in embryonic neurogenesis and undefined roles in adult hippocampal neurogenesis using a combination of transgenic inducible mice and in vivo retroviral techniques. The first study examines the microtubule associated protein Doublecortin (DCX), which is transiently expressed by NPCs and is critical for neuronal migration. Our results show that, in the context of adult hippocampal neurogenesis, DCX is not required for the survival or differentiation of the NPCs within the subgranular zone (SGZ). The second study examines the functional role of the autophagy-associated gene 5 (Atg5) which is critical for embryonic neurogenesis and survival. Our findings demonstrate that the intracellular recycling process of autophagy is active throughout maturation of adult hippocampal NPCs and that ablation of Atg5 produces a drastic reduction in NPC survival, without altering the neuronal fate of these cells. The third study examines the requirement of the familial-Alzheimer’s disease associated genes, presenilin 1 and presenilin 2 (PS1 & PS2), which are critical for embryonic NSC maintenance and differentiation. Similar to the findings with DCX, our results demonstrate that presenilins are dispensable for adult neurogenesis. Altogether, these studies add to the growing evidence suggesting differences in the regulation of adult versus embryonic neurogenesis, and highlight autophagy as a novel regulator of survival for adult generated granule neurons in the hippocampus.

The classical cell cycle regulatory pathway is well appreciated as a key regulator of cell fate determination during neurogenesis; however, the extent of pRB/E2F function in neural stem and progenitor cells is not fully understood, and insight into the mechanisms underlying its connection with cell fate regulation are lacking. The E2F3 transcription factor has emerged as an important regulator of neural precursor cell (NPC) proliferation in the embryonic and adult forebrain, and we demonstrate here that it also influences the self-renewal potential of NPCs. Using knockout mouse models of individual E2F3 isoforms, we demonstrate the surprising result that the classical transcriptional activator E2F3a represses NPC self-renewal and promotes neuronal differentiation, while E2F3b promotes the expansion of the NPC pool and inhibits differentiation. We attribute these opposing activities to a unique mechanism of transcriptional regulation at the Sox2 locus, a key regulator of stem cell pluripotency, whereby E2F3a recruits transcriptional repressors to this site, and E2F3b promotes Sox2 activation. Importantly, E2F3a-mediated Sox2 regulation is necessary for cognitive function in the adult. Additionally, through the determination of genome-wide promoter binding sites for E2f3 isoforms as well as E2F4, another key regulator of NPC self-renewal, we determined that E2Fs are poised to regulate an extensive set of target genes with key roles in regulating diverse cell fate choices in NPCs, including self-renewal, cell death, progenitor expansion, maintenance of the precursor state, and differentiation. Together, these results reveal a diversity of function for E2Fs in the control of neural precursor cell fate, and identify E2F3 isoforms as important regulators of the pluripotency and stem cell maintenance gene Sox2.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to characterize the ependymal cells and neural population in the Tropidurus hispidus medial cortex. It s well known nowadays that the nervous system isn t so constant the way was believed and detains morphological and functional properties that was influenced by environmental, sexual, age alterations and origin of the lesion like others. There s a vast literature about studies of amphibians, birds, reptiles even mammals brains, but one of the major difficulties found in the nervous system s plasticity research area is the appropriated experimental model. The medial cortex of Tropidurus hispidus specimens was studied by several techniques as TIMM, Nissl, immunostainings to BrDU, GABA, EC40, by Lectin and mainly the Golgi method. The test results showed the existence of 17 neuronal types in the T. hispidus medial cortex, with the major prevalence of the unipolar granular neuron, showed 12 ependymal cells, showed the positive reactivity to the GABA, BrDU and EC40 immunostainings too, the two lasts shows a post-natal neurogenic activity in the cortical region of this animal. Also was positive reactivity to the Lectin utilization in the microglia visualization. With the analysis of all this aspects, the study in the Tropidurus hispidus medial cortex showed a structural complexity as other characteristics that turn this animal a viable experimental model to the neurogenic processes studies. / O objetivo deste trabalho foi caracterizar a população neural e de células ependimárias no córtex medial do lagarto Tropidurus hispidus. Sabe-se atualmente que o sistema nervoso não é tão constante como se pensava e que detêm propriedades morfológicas e funcionais que são influenciadas por alterações ambientais, sexuais, etárias, da natureza da lesão dentre outras. Toda essa problemática vem sendo analisada tendo como base a regeneração do sistema nervoso. Há vasta literatura sobre estudo dos cérebros desde anfíbios, aves répteis até mamíferos e mesmo humanos, mas uma das maiores dificuldades para a pesquisa na área de plasticidade do sistema nervoso é um modelo experimental adequado. Para isto, no presente estudo o córtex medial dos espécimes de Tropidurus hispidus foi estudado por diversas técnicas como TIMM, Nissl, imunohistoquímicas para BrDU, GABA, EC40, pela Lectina e principalmente pelo método de Golgi. Os resultados dos testes demonstraram a existência de 17 tipos de neurônios no córtex medial do cérebro do T. hispidus, sendo que o tipo de maior prevalência foi o do neurônio granular unipolar, o teste de Golgi também mostrou 12 tipos de ependimócitos, verificando-se reatividade positiva à imunohistoquímica para GABA, para BrDU e para EC40, esses dois últimos demonstraram uma atividade neurogênica pós-natal na região cortical desse animal. Também houve uma reatividade positiva à utilização da Lectina na visualização da micróglia. Haja vista aos aspectos analisados, o estudo no córtex medial do T. hispidus denotou uma complexidade estrutural bem como características que o tornam um modelo experimental viável para os estudos dos processos neurogênicos.