This dissertation is divided into three chapters, each discussing the study of different regulatory molecules involved in sensory neurogenesis occurring in the trigeminal placode. Chapter one is a spatiotemporal description of Notch pathway genes in chick opV placode by stage-specific expression analysis, showing expression of many Notch pathway genes and effectors in the opV placode. Notch pathway gene expression is primarily confined to the ectoderm with highest expression of these genes at the beginning stages of peak neuronal differentiation. This information preceded studies of the functional roles that Notch signaling has in the opV placode and how it may affect the transcription factor, Pax3. Chapter two is a study of the transcription factor Pax3 and its role in opV placode development and sensory neuron differentiation. Pax3 is known to activate or repress gene transcription, and its activity may be dependent on the splice variant or isoform present. We show through RT-PCR that alternative splice forms of Pax3 are present at stages of chick development corresponding to cellular competence, cellular differentiation and ingression, and cellular aggregation. We have named these splice forms, Pax3V1 and Pax3V2. Using quantitative RT-PCR we show that Pax3V2 is consistently expressed at lower levels compared to Pax3 during cellular competence and differentiation. In order to determine the function of the three splice forms, we misexpressed them in the opV placode and analyzed the effect on neurogenesis. We looked at markers for neuronal differentiation of targeted cells after in ovo electroporation of Pax3, Pax3V1, and Pax3V2, which showed a significant difference between the control and each construct, but not between the groups of constructs. To enhance the process of neurogenesis we exposed the electroporated embryos to DAPT, a Notch signaling inhibitor that enhances sensory neurogenesis. Using this method we found that misexpression of Pax3 and Pax3V1 resulted in cells failing to differentiate, while Pax3V2 misexpression more closely resembles the neuronal differentiation seen in controls. These results show that the Pax3V2 isoform allows for neuronal differentiation of opV placodal cells after misexpression, while the Pax3 isoform and the Pax3V1 isoform block neuronal differentiation. Chapter three is a study of the necessity of Wnt signaling originating from the neural tube to induce Pax3 expression in the opV placode. A double knockout of Wnt1 and Wnt3a was produced to determine the necessity of these genes in opV placode development. Pax3 expression in the opV placode at E8.5 and E9.5 was markedly reduced in the double mutants when compared to wild type mice. This study shows that Wnt1 and Wnt3a genes are necessary for normal Pax3 expression, but that other signals may contribute to its induction.

No description available.

No description available.

La destruction des cellules ciliées de la cochlée entraine des surdités sensorielles. Chez les mammifères ces cellules ne se régénèrent pas et les déficits auditifs occasionnés sont définitifs. Aucune thérapie visant à remplacer les cellules ciliées détruites n'est actuellement proposée.L'objectif de cette thèse est de contribuer au développement d'une thérapie cellulaire basée sur la greffe de cellules souches / progénitrices cochléaires et destinée à promouvoir la régénération des cellules ciliées.Au cours de nos travaux, nous avons isolé une population de cellules souches cochléaires chez des souris néonatales appartenant à la « side population » (Savary et al. 2007). Nous avons également montré, par des expériences de perte et de gain de fonction in vitro, que la voie de signalisation Notch est nécessaire pour l'auto-renouvellement et la différenciation de ces cellules (Savary et al., 2008). Des lignées de souris transgéniques exprimant la GFP sous le promoteur de la GFAP et de la Nestine nous ont permis de suivre l'expression de ces marqueurs de cellules souches dans des cochlées de souris P3 et adultes. En étudiant l'expression combinée d'autres marqueurs comme Sox2 et Abcg2, nous avons montré que les cellules progénitrices cochléaires sont réparties différemment chez les souris néonatales et les souris adultes (Smeti, Savary et al 2010).Nos expériences préliminaires de transplantation in vitro dans un modèle murin de surdité génétique humaine de type DFNA15 démontrent que les cellules souches / progénitrices greffées sont capables d'intégrer l'épithélium sensoriel lésé et de se différencier en cellules exprimant un marqueur de cellules ciliées. / The destruction of cochlear hair cells causes sensory deafness. In Mammals these cells do not regenerate and damages are irreversible. Currently, there is no proposed therapy to replace the destroyed hair cells.The focus of this thesis is to develop a novel cell therapy based on transplantation of cochlear progenitor cells in order to promote regeneration of hair cells.We first isolated a population of cochlear stem cells from neonatal mice by using the side population analysis technique (Savary et al. 2007). Then, we showed, by in vitro loss and gain of function experiments, that the Notch signaling pathway is necessary for cellular self-renewal and differentiation (Savary et al., 2008).Transgenic mice strains expressing GFP under the control of GFAP and Nestin promotors allowed us to monitor the expression of these markers of stem cells in the P3 and adult mice cochleae. By studying the combined expression of other stem cells markers such as Sox2 and ABCG2, we showed that the niches of cochlear progenitor cells are differently distributed in neonatal and adult mice (Smati, Savary et al 2010).Our preliminary in vitro transplantation experiments in a mouse model that mimics human genetic deafness DFNA15 show that the transplanted stem / progenitor cells are able to migrate to the lesion site, to integrate the damaged sensory epithelium and to differentiate into cells expressing a marker of hair cells.

Au cours de l’ovogenèse, les stades vitellogéniques nécessitent une énergie considérable, et leur formation doit être ajustée en fonction d’autres besoins physiologiques. En utilisant la drosophile comme modèle, j’ai montré que la signalisation Insuline régule une transition du cycle cellulaire, mitose/ endocyle (M/E), une étape critique qui contrôle l’entrée des follicules en vitellogenèse. Mes travaux montrent que la transition M/E porte le rôle d’un point de contrôle nutritionnel. La carence protéique induit un blocage de cette transition au travers d’une interaction entre FoxO, Cut et Notch, empêchant une perte d’énergie. Ce blocage reste réversible, autorisant la reprise de l’ovogenèse sous retour à une alimentation normale. Ce travail montre qu’un point de contrôle nutritionnel au cours de l’ovogenèse permet de coupler des signaux métaboliques et développementaux pour protéger les tissus des dommages liés à la carence. D’autre part, j’ai montré que la signalisation Insuline contrôle la migration d’une cohorte de cellules d’origine épithéliale pour assurer la fertilité de l’ovocyte. L’insuline participe à la formation d’extensions cytoplasmiques riches en actine. Lors de ce processus, la signalisation Insuline contrôle notamment l’expression de chickadee, qui code pour la Profiline, une protéine nécessaire pour la polymérisation de l’actine qui permet la motilité des cellules. L’ensemble de ce travail montre que des tissus somatiques assurent l’homéostasie de l’ovogenèse malgré des conditions de nutritions fluctuantes. Ces travaux posent les bases de l’étude de nouveaux aspects de l’ovogenèse, potentiellement conservés chez les mammifères. / How oogenesis is controlled upon nutrient challenge is a key biological question to understand the balance between reproduction and adult fitness. During Drosophila oogenesis, vitellogenic stages are highly energy consuming so their formation has to be balanced with other physiological needs. We reveal the role of the Insulin pathway and FoxO in regulating the transition from Mitotic-to-Endocycle, a critical step controlling the entry of egg chambers into vitellogenesis. We show that the M/E switch functions as a nutrient checkpoint, blocking the entry into vitellogenesis upon starvation and therefore protecting adults from energy loss. Pausing of the M/E switch involves a previously unknown crosstalk between FoxO, Cut and Notch, a fully reversible process ensuring rapid resuming of oogenesis upon re-feeding. This work reveals a FoxO-dependent nutrient checkpoint integrating metabolic cues with reproduction and protecting tissues from starvation-induced damages. In addition, we show that the Insulin pathway regulates the migration of a subset of epithelial cells to ensure oocyte fertilization. We demonstrate that Insulin signaling regulates the formation of actin-rich cellular extensions in invasive cells. During this process, FoxO represses chickadee expression, which encodes Profilin. Insulin signaling activity leads to the inhibition of FoxO and subsequent Profilin accumulation, which further allows actin polymerization, necessary for cell motility. Altogether, data reveal a crucial role for the conserved Insulin signaling pathway in regulating ovarian follicles through somatic tissues, a process which is likely to share much in common with oogenesis in mammals.

Les canaux potassiques à deux domaines pore (K2P) sont des régulateurs principaux de l’excitabilité cellulaire car ils jouent un rôle central dans l’établissement et le maintien du potentiel de repos des cellules animales. Malgré leur rôle fondamental, peu d’informations sont connues sur les processus cellulaires qui contrôlent la fonction des canaux K2P in vivo. En particulier, nous ne connaissons que quelques facteurs qui contrôlent directement le nombre, l’activité et la localisation des K2P à la surface des cellules.Durant ma thèse, j’ai utilisé des stratégies d’ingénierie du génome que j’ai associé à des approches génétiques afin de caractériser le canal potassique EGL-23. Pour cela, j’ai réalisé un crible suppresseur du phénotype de défaut de ponte du mutant egl-23(n601) et un crible visuel sur le rapporteur fluorescent traductionnel egl-23::TagRFP-T. Grâce au reséquençagecomplet du génome, j’ai pu cloner 4 gènes impliqués dans la régulation du canal EGL-23. / Two-pore domain potassium channels (K2P) are major regulators of cell excitability, playing a central role in the establishment and maintenance of the resting potential of animal cells. Despite their fundamental role, little is known about the cellular processes that control K2P channels function in vivo. In particular, we know only few factors that directly control thenumber, activity, and localization of K2P on the cell surface.During my thesis, I used state-of-the art genome engineering technologies combined with genetic approaches to characterize the C. elegans potassium channel EGL-23. For this, I realized a phenotypic suppressor screen of the egg-laying defective mutant egl-23(n601) and a visual screen on an egl-23 translational fluorescent reporter. Using whole genome sequencing, I was able to clone for new genes involved in EGL-23 regulation

Notch receptors and ligands are type I transmembrane proteins that regulate development and differentiation during cell-cell contact. There are four Notch receptor homologues and five notch ligands, identified in humans till date. Upon ligand activation, Notch1 intracellular domain (NIC-1) is released into the cytoplasm, which binds to several proteins as well as translocates into the nucleus to effect the Notch signaling. In the absence of the activated Notch signaling, the Notch target genes are kept repressed by the transcriptional repressor C protein binding factor 1 (CBF1) also known as RBPjk or CSL for CBF1/Su(H)/Lag1. RBPjk binds to the sequence “CGTGGGAA” and acts as a constitutive repressor. Upon ligand dependent activation, NIC-1 enters into the nucler and converts RBPjk from transcriptional repressor to an activator. Notch binding to CSL replaces the SMRT corepressor complex with a coactivator complex including SKIP, Mastermind like 1 (MAML1) (Mastermind in Drosophila), and histone acetyl transferases PCAF, GCN5 and p300 activating the transcription of target genes. Mastermind-like (MAML), a family of transcriptional activator proteins comprising of 3 members 1 to 3, has been shown to be required for Notch signaling. MAML forms a ternary complex with RBPjk-NIC by directly interacting with NIC. In turn, MAML recruits the histone acetyl transferase p300/CBP, which acetylates the histones, thereby altering the structure of chromatin amenable for transcription. Activation of Notch pathway induces oncogenesis, which can be divided into two categories including 1) Inhibition of Apoptosis and 2) Induction of proliferation. In T cells, activation of Notch1 protects cells from T cell receptor, dexamethasone and etoposide-mediated apoptosis, Fas receptor-mediated signaling by up regulating IAP (Inhibitor of Apoptosis) and Bcl-2 families, as well as FLIP (FLICE-like inhibitor protein). Notch signaling also promotes the survival of T cells through maintenance of cell size as well as through the promotion of glucose uptake and metabolism. Notch-1 has been shown to protect against anoikis (apoptosis induced by matrix withdrawal) or p53-mediated apoptosis in immortalized epithelial cells, T cell receptor-induced apoptosis in mature cells and dexamethasone-mediated apoptosis in thymocytes. This study was carried out to functionally characterize NIC-1 (human Notch1-intracellular domain) as an inhibitor of apoptosis and to evaluate the therapeutic potential of reversal of this apoptosis inhibition. The main objectives of this study are 1. Construction of recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and characterization of NIC-1 as an inhibitor of chemotherapy and p53-induced cytotoxicity and apoptosis. 2. Role of PI3 kinase -Akt/PKB -mTOR pathway in NIC-1-mediated inhibition of p53-induced apoptosis. 3. Essential role of association between mTOR and NIC-1 and the dependent NIC-1 phosphorylation in Notch1-mediated transcription and survival signaling. 4. Identification of NIC-1 as an inhibitor of E1A-induced apoptosis and the role of mTOR in NIC-1-mediated inhibition of E1A-induced apoptosis. Activated Notch1 was first linked to tumorigenesis through identification of a recurrent t(7;9)(q34;q34.3) chromosomal translocation involving the human Notch1 gene that is found in a subset of human pre-T-cell acute lymphoblastic leukemia’s (T-ALL). Deregulated Notch signaling is oncogenic, inhibits apoptosis and promotes survival. In order to understand survival signaling induced by Notch1 and its possible role in chemoresistance, we have generated a replication deficient recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and shown that it produces functional NIC-1 protein. Using this overexpression system, we characterized that activated Notch1-inhibits chemotherapy and in particular p53 induced apoptosis. Notch1-mediated inhibition of p53-induced apoptosis does not include coactivator squelching. p53 was inefficient in binding to its DNA in NIC-1 overexpressing cells. The levels of phosphorylation at Ser15, Ser20, and Ser392 of p53 expressed from Ad-p53 significantly reduced in NIC-1 preinfected cells. These results suggest that NIC-1-mediated inhibition of p53-mediated apoptosis involves reduced DNA binding, reduced nuclear localization and reduced post translational modifications and thus reduced transactivation of its target genes. Notch1-mediated inhibition of p53 was found to occur mainly through mammalian target of rapamycin (mTOR) using PI3 kinase-Akt/PKB pathway, as the mTOR inhibitor; rapamycin treatment was able to reverse Notch-1 mediated inhibition of p53 and chemoresistance. Consistent with this, rapamycin failed to reverse NIC-1 induced chemoresistance in cells expressing rapamycin resistant mTOR. Our results also suggest that the N-terminal HEAT repeat and the kinase function of mTOR are essential for Notch mediated inhibition of p53. Further, ectopic expression of eIF4E, a translational regulator that acts downstream of mTOR, inhibited p53-induced apoptosis and conferred protection against p53-mediated cytotoxicity to similar extent as that of NIC-1 overexpression, but was not reversed by rapamycin, which indicates that eIF4E is the major target of mTOR in Notch1-mediated survival signaling. Notch1-intracellular domain (NIC-1), following proteolytic cleavage, binds to RBPjk and regulates transcription. Active NIC-1 located in the nucleus is phosphorylated, which makes it more stable and bind better to RBPjk. NIC-1 was also shown to bind to Deltex1 in the cytoplasm. Next, we studied the requirement of components of Notch1 signaling pathway for this function. By using variety of approaches, we found that both RBPjk and Maml1 and hence transcription activation is required for NIC-1-mediated survival signaling and inhibition of p53 functions. Interestingly, while we found the other Notch1 effector, Deltex1 is also required for above functions, Notch1 failed to activate PI3 kinase -Akt/PKB -mTOR pathway in Deltex1, but not in RBPjk silenced cells. Our results suggest that Notch-Deltex1 pathway activates PI3 kinase. Previous studies show that NIC-1 interacts with Deltex1 and Grb2 interacts with PI3 kinase. Our data shows that Deltex1 interacts with SH3 domain of Grb2. Since Notch1-Deltex1 and PI3 kinase-Grb2 interactions are known, we conclude that Notch1 activation of PI3 kinase involves Deltex1 and Grb2. We found activated mTOR was able to binds to NIC-1 and regulates its phosphorylation. Inhibition of mTOR either by PI3 kinase inhibitors or mTOR inhibitor treatment or silencing of Akt/PKB or mTOR reduced the phosphorylation of NIC-1 with the concomitant reduction in NIC-1-mediated transcription. Further, endogenous Notch1 receptor activated by the DSL ligand failed to activate transcription efficiently in rapamycin treated cells, implying a positive role for mTOR in mammalian Notch signaling. These studies reveal that Notch1 activates PI3 kinase -Akt/PKB -mTOR signaling through Deltex1 and subsequently activated mTOR modulates Notch1 signaling by direct binding and possibly thorough phosphorylation of the intracellular domain of Notch. Adenoviral E1A, in the absence of cooperating oncogene, suppresses primary tumor growth and reverses the transformed phenotype of human tumor cells by inducing apoptosis. E1A requires p53 for efficient induction of apoptosis and was shown to induce apoptosis by down regulating Akt and the activation of pro apoptotic factor p38 MAP kinase. Since our results suggest Notch1 inhibits chemotherapy and p53-induced apoptosis, we analyzed the ability of Notch1 to protect cells from E1A-induced apoptosis. Here we show that NIC-1 suppresses the ability of E1A to induce apoptosis. NIC-1 requires mTOR-dependent signal to inhibit E1A-mediated apoptosis, as the rapamycin, an mTOR inhibitor was able to completely reverse the ability of Notch1 to protect cells against E1A-induced apoptosis. The role of mTOR in NIC-1-mediated survival signaling was further confirmed by using the cells stably expressing rapamycin resistant mTOR. Rapamycin was able to reverse Notch1-mediated protection in cells expressing wild type mTOR but not in rapamycin resistant mTOR expressing cells. We also found that E1A was able to induce apoptosis in cells silenced for the pro apoptotic factor p38 and NIC-1 continued to inhibit E1A-induced apoptosis in these cells. These results confirm that Notch1 requires the activation of mTOR signaling but not p38 MAP kinase for inhibition of E1A-induced apoptosis. These results also suggest that the combination therapy utilizing E1A-mediated gene delivery in combination with inhibition of mTOR pathway may prove successful in treating Notch overexpressing cancers. Chemotherapy remains a major treatment modality for human cancers. Chemoresistance is a clinical problem that severely limits treatment success. It can be divided into two forms: intrinsic and acquired. Intrinsic resistance is the essence of oncogenic transformation, resulting from activation of oncogenes and the loss of tumor suppressors, and manifests itself as alterations in cell cycle checkpoints and apoptotic pathways. It is now widely accepted that the apoptotic capacity of the cancer cell is crucial in determining the response to chemotherapeutic agents. Indeed, several gene products that regulate apoptosis, i.e., p53, Akt and PI3K are frequently altered in cancer cells. In this study, we identified that cells with aberrant Notch1 signaling are chemoresistant. Activated Notch1 overexpression makes cells resistant to chemotherapy in a wild type p53 dependent manner. Notch protected p53 wild type cells but not p53 mutated or p53 deleted cells against chemotherapy induced cytotoxicity. Further, inactivation of p53 by specific silencing abrogated the ability of NIC-1 to protect H460 cells against adriamycin induced cytotoxicity. Most importantly, NIC-1 mediated chemoresistance can be reversed by blocking PI3 kinase -Akt/PKB -mTOR pathway. Collectively, these results suggest that cancers with activated Notch1 signaling are chemoresistant and provide basis for the reversal of chemoresistance.

Les Glioblastomes (GBM, grade IV selon l’OMS) sont les tumeurs cérébrales primaires les plus agressives et sont caractérisées par une néovascularisation importante associée à l’hypoxie et à la nécrose. L’origine cellulaire des GBM est controversée, mais des sous-populations de cellules multipotentes ont été identifiées au sein des tumeurs, et seraient responsables de la radio/chimiorésistance des GBM. Ces cellules souches de glioblastome (GSC) contrôlent activement la vascularisation tumorale par leur interaction étroite avec les cellules vasculaires composant les niches tumorales. La voie Notch est une signalisation canonique essentielle au développement et à l’homéostasie du système nerveux central et son réseau vasculaire associé. Dans le contexte des GBM, cette cascade serait nécessaire à la gliomagénèse, par le maintien du réservoir de GSC au sein de la niche périvasculaire. Cependant, le mode d’action moléculaire de Notch dans les GBM reste encore à démontrer, du fait de résultats divergents observés dans plusieurs études. Dans la première partie de mon travail de thèse, j’ai contribué à l’exploration de la signalisation Notch1 dans des cultures de GSC établies et caractérisées au sein du laboratoire. Le niveau basal d’activation de Notch1 étant faible dans nos GSC, l’approche a été d’activer constitutivement cet axe par transduction lentivirale. Suite à cette activation forcée, les GSC subissent un changement phénotypique majeur et se différencient en cellules périvasculaires ou cellules « pericyte-like ». Cette transition neurovasculaire des GSC promeut la vascularisation active des tumeurs par la normalisation du réseau vasculaire in vivo. Par la suite, j’ai posé la question des mécanismes moléculaires en aval de Notch1 ; par l’étude des facteurs de transcription TAL1 et SLUG, deux candidats potentiels au contrôle de cette plasticité neurovasculaire. Dans ce but, j’ai examiné leur contribution au phénotype vasculaire des GSC dans un modèle in vitro de la niche périvasculaire ; et in vivo par l’analyse d’échantillons humains de GBM. Enfin, j’ai également observé que l’activation de Notch1 module l’activité de la machinerie du protéasome, ce qui pourrait contribuer activement à la transition moléculaire observée dans les GSC. Ces travaux mettent en avant la plasticité phénotypique des GSC: une meilleure compréhension de ces processus pourrait mener à la conception de thérapies ciblant efficacement les GSC et leur vascularisation associée. / Glioblastomas (GBM, WHO grade IV) are highly aggressive brain tumors in which extensive vascularization is associated with hypoxia and necrosis. GBM cell of origin is controversial; however multipotent stem-like subpopulations have been identified within tumors, and could account for GBM radio/chemoresistance. These glioblastoma stem-like cells (GSC) actively promote tumoral vascularization processes by closely interacting with vascular cells composing tumoral niches. The Notch cascade is a canonical signaling pathway required during developmental stages and adult homeostasis of the central nervous system and the associated vascular network. In the context of GBM, this molecular axis could induce gliomagenesis by promoting GSC maintenance in the perivascular niche. However, Notch-induced molecular mechanisms controlling GBM progression still remain elusive, due to divergent results observed in numerous reports. During the first part of my thesis work, I contributed to the assessment of Notch1 functions in GSC cultures isolated and characterized in our lab. Given a low Notch1 basal activation status in our GSCs, our approach was to constitutively activate this axis via lentiviral transduction. Following this forced activation, GSCs undergo drastic phenotypic changes and differenciate into perivascular-like or “pericyte-like” cells. This neurovascular transition of GSCs induces active tumoral vascularization by promoting normalization of the vascular network in vivo. Consequently, I questioned the molecular mechanisms downstream of Notch1 by focusing on TAL1 and SLUG transcription factors, two potential candidates controlling this neurovascular plasticity. For this purpose, I examined their contribution to the GSC vascular-like phenotype in an in vitro model of the perivascular niche; and in vivo by analyzing human GBM samples. Finally, I also observed that Notch1 activation modulates the activity of the proteasomal machinery, which could actively contribute to the molecular transition occurring in GSCs. This work highlights GSC phenotypic plasticity: a better understanding of these processes could lead to the design of therapies efficiently targeting GSCs and their associated vasculature.

La transplantation de cellules souches de muscle possède un grand potentiel pour la réparation à long terme du muscle dystrophique. Cependant, la croissance ex vivo des cellules souches musculaires réduit de manière significative l'efficacité de leur greffe puisque le potentiel myogénique est considérablement réduit lors de la mise en culture. La voie de signalisation Notch a émergé comme un régulateur majeur des cellules souches musculaires (MuSCs) et il a également été décrit que la sur-activation de Notch est crucial pour le maintien du caractère souche des MuSC. Cette découverte pourrait être traduite comme un bénéfice thérapeutique potentiel. Des MuSCs murines ont été fraîchement isolées et ensemencées sur des boîtes de culture recouverte de Dll1-Fc, le domaine extracellulaire de Delta-like-1 est fusionné au fragment Fc humain, afin d'activer la voie de signalisation Notch et avec un IgG hu-main comme contrôle. Nous avons utilisé le rAAV afin d’exprimer le Dll1 spécifique-ment dans les muscles de souris. Les souris P3 ont été traitées avec de l’AAV pendant 3 semaines et 6 semaines afin d’étudier l'effet de Dll1 au cours du développement postnatal. Afin d’étudier le processus de régénération, l'AAV a également été injecté dans les muscles de souris mdx alors que les souris de type sauvage ont été utilisées comme contrôle. Un potentiel caractère souche supérieur (marquée avec le Pax7) est observé dans les cultures des MuSCs qui sont recouverte de Dll1-Fc par rapport à leurs homologues contrôles, par contre le taux de proliférer est réduit. Au cours du développement postnatal, la sur-activation de la voie de signalisation Notch par Dll1 sur les fibres musculaires a été en mesure d'élargir le pool des cellules Pax7+, cependant elle entraîne une diminution de la masse musculaire avec réduction de la taille des fibres et ceci sans affecter l'accumulation des myonuclei. Dans les MuSCs quiescentes (de type sauvage), la sur-activation de la voie de signalisation Notch ne présente pas de réel effet. La surexpression de Dll1 dans le muscle mdx a diminué la masse musculaire et agrandit le pool de cellules souches musculaires, ce-pendant le taux de régénération n'a pas été affecté. L’augmentation des MuSCs est attribuée à une différenciation entravée des cellules souches musculaires. En étudiant la stimulation de la voie de signalisation Notch dans les MuSCs à la fois in vitro et in vivo, nous démontrons que sur-activation de Notch préserve le caractère souche des cellules via l’inhibition de la prolifération et de la différenciation myogénique des MuSCs. / Muscle stem cell transplantation possesses great potential for long-term repair of dys-trophic muscle. However expansion of muscle stem cells ex vivo significantly reduces their engraftment efficiency since the myogenic potential is dramatically lost in culture. The Notch signaling pathway has emerged as a major regulator of muscle stem cells (MuSCs) and it has recently been discovered that high Notch activity is crucial for maintaining stemness in MuSCs. This feature might be exploited and developed into a novel therapeutic approach.Murine MuSCs were freshly isolated and seeded on culture vessels coated with Dll1-Fc, which fused Delta-like-1 extracellular domain with human Fc, to activate Notch sig-naling and with human IgG as a control. The rAAV gene delivery system was em-ployed to express Dll1 in murine muscles. P3 mice were treated with AAV for 3 weeks and 6 weeks to investigate the effect of Dll1 during postnatal development. To investi-gate the regeneration process, AAV were injected into mdx muscles whereas wild-type mice were used as control.Higher potential stemness (marked by Pax7 positivity) was observed in MuSCs grow-ing on a Dll1-Fc surface as compared to their counterparts on the control surface, while their proliferation rate was reduced. During postnatal development, overstimulation of Notch signaling by Dll1 on the mus-cle fibers was able to enlarge the Pax7+ cell pool, while also resulting in decreased muscle mass and smaller muscle fibers without affecting the accretion of myonuclei into the fiber. In quiescent (wild-type) MuSCs, overstimulation of Notch signaling did not have any discernible effect. Overexpression of Dll1 in mdx muscle decreased the muscle mass and enlarged the muscle stem cell pool, while muscle regeneration re-mained unaffected. By investigating Notch stimulation in MuSCs both in vitro and in vivo, we demonstrate that high Notch activity preserves stemness via inhibition of MuSCs proliferation and myogenic differentiation. Our findings point out that the Dll1 molecule, as a canonical Notch ligand, might have a therapeutic potential in cell-based therapies against muscu-lar dystrophies.

Dans le système nerveux central adulte, la glycine est principalement connue pour son rôle de transmission d’un signal inhibiteur à l'intérieur des neurones matures, régulant ainsi l'activité du réseau neuronal. Paradoxalement, durant l'embryogenèse, ce même neurotransmetteur génère une transmission excitatrice produisant ainsi le premier signal électrique dans les neurones immatures. Le rôle et la signification fonctionnelle de ce changement d’activité durant le développement neurologique restent toujours inconnus. En utilisant l’embryon du poisson-zèbre comme modèle, nous avons exploré les mécanismes moléculaires et cellulaires dépendants de la signalisation de glycine dans les cellules souches neuronales (CSNs). En premier lieu, nous avons développé un outil d’analyse basé sur une combinaison de deux éléments: une lignée transgénique qui exprime du GFP dans les CSNs et la technique de séquençage de l’ARN total. Nous avons utilisé cette technique pour isoler et déterminer les mécanismes moléculaires régulés par la glycine dans les CSNs. Ceci a permis d’identifier plusieurs gènes candidats dont l’expression est modulée par l’activité glycinergique. Ces gènes appartiennent principalement à cinq différentes voies de signalisation canoniques incluant la voie de signalisation du calcium, TGF-bêta, Shh, Wnt et p53. Pour en apprendre davantage sur ces mécanismes moléculaires, nous avons exploré l’un d’entre eux soit la régulation de la signalisation p53 par l’activité glycinergique. En effet, nous avons démontré que l’activité glycinergique favorise la survie des CSNs par la régulation de la signalisation de p53 et agit spécifiquement sur la sous-population CSN-nestin+ durant la neurogenèse. Dans un autre projet, nous avons examiné la régulation de l’expression de lnx1 par l’activité glycinergique. Nous avons démontré que la signalisation de glycine/lnx1 régule la prolifération des CSNs via la modulation de l’activité de Notch durant la neurogenèse. En conclusion, dans ce projet de thèse, j’ai mis en lumière plusieurs mécanismes moléculaires et cellulaires modulés par l’activité glycinergique dans les CSNs. Ceci peut contribuer dans le futur à la compréhension de la physiopathologie liée au dysfonctionnement de cette dernière ainsi qu’à l’identification de nouvelles cibles thérapeutiques. / In the adult central nervous system, glycine is mainly known as an inhibitory neurotransmitter in mature neurons, thereby regulating the neural network activity. Paradoxically, during embryogenesis, the same neurotransmitter generates excitatory transmission and induces the first electrical signal in immature neurons. The role and functional significance of this change in glycinergic activity during neurogenesis are still unknown. In this study, we used zebrafish embryos as a model to explore the glycine-dependent molecular and cellular mechanisms in neural stem cells (NSCs). First, we developed an in vivo analysis method based on two main elements: a transgenic line that expresses GFP within NSCs and the RNA sequencing technique. This method of analysis was used to determine glycine-dependent molecular mechanisms in NSCs. We identified several candidate genes whose expression is modulated by the glycinergic activity. These genes participate in five different canonical signaling pathways including the calcium signaling pathway, TGF-beta, Shh, Wnt and p53. To further understand these molecular mechanisms, we focused our investigation on the regulation of p53 signaling by the glycinergic activity. Indeed, we have demonstrated that glycinergic activity promotes the survival of NSCs by regulating p53 signaling and more specifically acting on NSC-nestin + subpopulation during neurogenesis. Finally, we explored the regulation of lnx1 expression by glycinergic activity. We have demonstrated that glycine/lnx1 signaling regulates the proliferation of NSCs via the modulation of Notch activity during neurogenesis. In conclusion, during this thesis project, I highlighted several molecular and cellular mechanisms modulated by the glycinergic activity in NSCs. These relevant results may contribute in the future to the understanding of the physiopathology related to glycinergic activity dysfunctions and the identification of new therapeutic targets.