The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this thesis I have shown that in the absence of pRb, differentiating primary myoblasts fused to form short myotubes that never twitched and degenerated via a non-apoptotic mechanism. The shortened myotubes exhibited an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation and reduced ATP production. Bcl-2 and autophagy inhibitors restored mitochondrial function and rescued muscle degeneration, leading to twitching myotubes that expressed normal levels of muscle-specific proteins and eventually exited the cell-cycle. A hypoxia-induced glycolytic switch also rescued the myogenic defect after chronic or acute inactivation of Rb in a HIF-1-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program. I next tested the effect of retinoblastoma protein family members – p107 and p130 – on skeletal myogenesis in the absence of Rb. Chronic or acute inactivation of Rb plus p130 or Rb plus p107 increased myoblast cell death and reduced myotube formation, yet expression of Bcl-2, treatment with autophagy antagonist or exposure to hypoxia extended myotube survival, leading to long, contracting myotubes that appeared indistinguishable from control myotubes. Triple mutations in Rb family genes further accelerated cell death and led to elongated myocytes or myotubes containing two nuclei, some of which survived and twitched under hypoxia. Whereas nuclei in Rb-/- myotubes were unable to stably exit the cell-cycle, myotubes lacking both p107/p130 became permanently post-mitotic, suggesting that pRb, but not p107 or p130 may be lost in cancer because of the unique requirement for cell-cycle exit during terminal differentiation. This thesis demonstrates that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and reveal a novel link between pRb and cell metabolism.

The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this thesis I have shown that in the absence of pRb, differentiating primary myoblasts fused to form short myotubes that never twitched and degenerated via a non-apoptotic mechanism. The shortened myotubes exhibited an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation and reduced ATP production. Bcl-2 and autophagy inhibitors restored mitochondrial function and rescued muscle degeneration, leading to twitching myotubes that expressed normal levels of muscle-specific proteins and eventually exited the cell-cycle. A hypoxia-induced glycolytic switch also rescued the myogenic defect after chronic or acute inactivation of Rb in a HIF-1-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program. I next tested the effect of retinoblastoma protein family members – p107 and p130 – on skeletal myogenesis in the absence of Rb. Chronic or acute inactivation of Rb plus p130 or Rb plus p107 increased myoblast cell death and reduced myotube formation, yet expression of Bcl-2, treatment with autophagy antagonist or exposure to hypoxia extended myotube survival, leading to long, contracting myotubes that appeared indistinguishable from control myotubes. Triple mutations in Rb family genes further accelerated cell death and led to elongated myocytes or myotubes containing two nuclei, some of which survived and twitched under hypoxia. Whereas nuclei in Rb-/- myotubes were unable to stably exit the cell-cycle, myotubes lacking both p107/p130 became permanently post-mitotic, suggesting that pRb, but not p107 or p130 may be lost in cancer because of the unique requirement for cell-cycle exit during terminal differentiation. This thesis demonstrates that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and reveal a novel link between pRb and cell metabolism.

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.

Thesis (Ph.D.)--Baylor University, 2008. / Includes bibliographical references (p. 121-133)

AN ABSTRACT OF THE DISSERTATION OF Priya Londhe, for the Doctor of Philosophy degree in Biochemistry and Molecular Biology, presented on 30th July, 2013 at Southern Illinois University Carbondale. TITLE: UNRAVELING THE MOLECULAR MECHANISMS OF THE CLASS II TRANSACTIVATOR IN SKELETAL MUSCLE MAJOR PROFESSOR: Dr. Judy Davie The inflammatory cytokine, interferon gamma, IFN-gamma orchestrates a diverse array of fundamental physiological processes and exhibits complex effects on myogenesis. IFN-gamma also induces the class II transactivator, CIITA, which is a critical mediator of IFN-gamma mediated repression and activation. The aims in my dissertation are directed towards understanding the role of IFN-gamma and CIITA in muscle. Stimulation by IFN-gamma in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. We show that the IFN-gamma induced inhibition of myogenesis is mediated by CIITA, which specifically interacts with myogenin. CIITA acts by both, repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. The IFN-gamma mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-gamma. We also show that CIITA is indispensible for the inhibition of myogenesis. To gain a mechanistic insight into the IFN-gamma induced repression of myogenesis, we have discovered that IFN-gamma and CIITA inhibit myogenesis by modifying gene regulation in a muscle cell subject to inflammation. We show that CIITA first interacts with JARID2, a non catalytic subunit of PRC2 complex, which induces a paused RNAPII, phosphorylated at serine 5 and then interacts with the catalytic subunit EZH2, in a JARID2 dependent manner. Our data show that both CIITA and IFN-gamma block myogenesis by the induction and recruitment of the PRC2 complex, which is normally silenced in a differentiating muscle cell. One of my dissertation aims sheds light on the silencing of CIITA in Rhabdomyosarcoma. Silencing of CIITA prevents the expression of MHC Class I and II genes. We have found that IFN-gamma signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells, but shows a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have also observed that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-gamma treatment, is blocked in both types of RMS cells. Further, our studies also impart a novel role for IFN-gamma and CIITA in inhibiting the IGF induced activation of muscle specific genes. Our data show that IFN-gamma does not block the signaling cascade of IGF. However, blocking exogenous IFN-gamma restores IGF activation of muscle specific genes. My dissertation also reveals an important role for the FACT complex in the early steps of gene activation through its histone chaperone activities that serve to open chromatin structure and facilitate transcription promoting muscle differentiation. We show that myogenin interacts with the FACT complex and the recruitment of FACT complex to muscle specific genes is dependent on myogenin. The final aim in my dissertation highlights the distinct binding profiles of the MRFs and E proteins during proliferation and differentiation. Our sequential ChIP assays show that MYOD, MYOG, and MYF5 co-occupy promoters. Taken together, my dissertation provides a comprehensive understanding of the molecular mechanisms during myogenesis and reveals the deleterious effects of chronic inflammation in skeletal muscle.

This study aimed to determine the modulation of skeletal muscle fibers of Longissimus dorsi, Semitendinosus and Supraspinatus, due to the variation in body weight and diet, evaluating its relationship with meat quality of lambs of Texel. For assessment of body mass were used thirty lambs distributed in five treatments with six replicates each: six lambs were slaughtered at 20 kg (slaughter control), 24 animals were slaughtered at 25, 30, 35 and 40 kg. For the evaluation of the influence of diet twenty-four lambs were randomly divided into four treatments, where six lambs were soon slaughtered after adjusting the experimental conditions (slaughter control), 12 animals were submitted to two food restriction levels (55 and 70% of ad libitum intake, six per group) and 6 animals were submitted to ad libitum consumption and slaughtered with a body mass of 40 kg. The different body mass slaughter, provided modulation of muscle fibers type IIB and IIC for type IIA fibers to the Longissimus dorsi, Semitendinosus and Supraspinatus, and provide changes in diameter, area and relative frequency of muscle fibers. Regarding the quality of meat, observed effect (P <0.05) of different body masses, the moisture, lipids, cholesterol, pH, hardness, color and lipid profile. Already, for different levels of food restriction it was observed that animals submitted the restrictions 55 and 70% had lower values of the characteristics of muscle fibers, whereas the opposite for animals submitted to ad libitum intake. Food restriction also influenced the meat quality characteristics, such as moisture, ash, protein, lipids, cholesterol, loss of cooking and color attributes. Thus, we conclude that different body mass slaughter and different feeding systems influence meat quality characteristics and the modulation of skeletal muscle fibers. / Objetivou-se determinar a modulação das fibras musculares esqueléticas dos músculos Longissimus dorsi, Semitendinosus e Supraspinatus, em função da variação da massa corporal e do regime alimentar, avaliando sua relação com a qualidade da carne de cordeiros da raça Texel. Para avaliação da massa corporal foram utilizados trinta cordeiros, distribuídos em cinco tratamentos com seis repetições cada: seis cordeiros foram abatidos com 20 kg (abate controle), 24 animais foram abatidos aos 25, 30, 35 e 40 kg. Para a avaliação da influência do regime alimentar, foram utilizados vinte e quatro cordeiros, distribuídos em quatro tratamentos, onde seis cordeiros foram abatidos logo após adaptação as condições experimentais (abate controle), 12 animais foram submetidos a dois níveis de restrição alimentar (55 e 70% do consumo ad libitum, seis por grupo) e 6 animais foram submetidos ao consumo ad libitum e abatidos com massa corporal de 40 kg. As diferentes massas corporais ao abate, proporcionaram modulação das fibras musculares do tipo IIB e IIC para fibras do tipo IIA para os músculos Longissimus dorsi, Semitendinoso e Supraspinatus, além de proporcionar alterações no diâmetro, área e frequência relativa das fibras musculares. Em relação a qualidade da carne, observou efeito (P<0,05) das diferentes massas corporais, nos teores de umidade, lipídios, colesterol, pH, dureza, cor e perfil lipídico. Já, para os diferentes níveis de restrição alimentar observou-se que os animais submetidos as restrições de 55 e 70% apresentaram menores valores das características das fibras musculares, sendo observado o contrário para os animais submetidos ao consumo ad libitum. A restrição alimentar, influenciou também nas características da qualidade da carne, como a umidade, cinzas, proteína, lipídios, colesterol, perdas de cocção e atributos de cor. Sendo assim, é possível concluir que diferentes massas corporais ao abate e diferentes sistemas de alimentação influenciam as características da qualidade da carne e a modulação das fibras musculares esqueléticas.

Orientador: Kleber Gomes Franchini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T13:44:01Z (GMT). No. of bitstreams: 1 Oliveira_MichelVazde_M.pdf: 1365391 bytes, checksum: a8c1dd22f85ed3c7df93982f678f95f7 (MD5) Previous issue date: 2008 / Resumo: Os mioblastos que formam o músculo esquelético são derivados de regiões do miótomo dos somitos. No processo de miogênese ocorre transição de estado proliferativo para um estado de diferenciação que se caracteriza por interrupção do ciclo celular, expressão de genes músculo-específico, reconhecimento célula-célula e formação de miotubos multinucleados. Para o estudo de diferenciação miogênica in vitro a linhagem celular C2C12 é um modelo bem estabelecido. Quando os mitógenos são retirados do meio de cultura (privação de soro fetal bovino), fatores de transcrição específicos são ativados, levando à diferenciação em miotubos. Na progressão do estado proliferativo para o de diferenciação participam diversas proteínas sinalizadoras. Em estudos anteriores demonstramos que a modulação da atividade da FAK tem papel crítico no processo de miogênese de células C2C12. Níveis relativamente elevados de atividade da FAK determinam a manutenção do estado proliferativo (indiferenciado) através de ativação de ciclinas. Também demonstramos que redução transitória da atividade da FAK é essencial para que as mioblastos C2C12 iniciem o processo de diferenciação terminal em miotubos. No presente estudo foi avaliada a hipótese de que a ação da tirosino-fosfatase SHP-2 determina a redução transitória da quinase de adesão focal (FAK) na transição do estado proliferativo para o estado de diferenciação no modelo de miogênese em células C2C12. A privação do soro fetal bovino induziu uma redução transiente (cerca de 80 % por ~2 horas) da fosforilação da FAK no resíduo de Tyr-397, detectada através de anticorpo fosfoespecífico anti-FAK-pY397. Não foi observada mudança na expressão da proteína FAK durante o período experimental. Experimentos de co-imunoprecipitação foram realizados para avaliar se a redução do nível de fosforilação da FAK é acompanhada de sua associação com SHP-2. Observou-se aumento de cerca de 2 vezes na associação entre FAK e SHP-2 no período coincidente com os nível baixos de fosforilação da FAK. Para avaliar o efeito da SHP-2 na redução transitória da pFAK padronizou-se o silenciamento de SHP-2 em células C2C12 nos estados de proliferação e diferenciação. Após a transfecção com siRNASHP2 nas células C2C12 foi feita a extração total de proteínas em diferentes tempos para avaliar o nível de expressão da SHP-2 no estado de proliferação. Foi observada menor expressão desta proteína após 12 horas ao período de transfecção. Depois da transfecção em meio de cultivo com o silenciamento de SHP-2 padronizados em 12 horas as células foram induzidas a se diferenciarem através da privação de soro fetal bovino. A depleção de SHP-2 aboliu a redução transiente da fosforilação da FAK. Observou-se que a depleção de SHP-2 também impediu a diferenciação terminal dos mioblastos privados de soro fetal bovino em miotubos. Esses dados sugerem que a SHP-2 modula o nível de fosforilação da FAK exercendo papel inibitório na ativação da FAK na transição das células C2C12 no estado de proliferação para diferenciação, influenciando a entrada destas células na diferenciação / Abstract: The myoblasts that form the skeletal muscle are derived from regions of the myotome of somites. In the process of myogenesis occurs transition of proliferative status to a state of differentiation which is characterised by disruption of the cell cycle, expression of muscle-specific genes, cell-cell recognition and training of myotubes multinucleate. For the study of the in vitro differentiation myogenic cell line C2C12 is a model well established. Where mitogens are moved from the culture medium (deprivation of fetal bovine serum), specific transcription factors are activated, leading to the differentiation in myotubes. The progression to the proliferative status of differentiation are involves several signaling proteins. In previous studies we showed that the modulation of the activity of FAK plays a critical role in the process of myogenesis of C2C12 cells. Relatively high levels of activity of FAK determine the maintenance of the state proliferative (indistinct) through activation of cyclin. It also demonstrated that transient reduction of the activity of FAK is essential for the myoblasts C2C12 begin the process of terminal differentiation in myotubes. In the present study was evaluated the hypothesis that the action of tirosino-phosphatase SHP-2 determines the reduction of transient focal membership of kinase (FAK) in the transition of proliferative state to the state of differentiation in the model of miogênese in C2C12 cells. The deprivation of fetal bovine serum produces a transient reduction (about 80% by ~ 2 hours) of the phosphorylation of FAK in the residue of Tyr-397, detected by antibody phsphoespecific anti-FAK-pY397. No change was observed in the expression of FAK protein during the trial period. Coimmunoprecitation experiments were performed to evaluate whether the reduction in the level of phosphorylation of FAK is accompanied by its association with SHP-2. There was an increase of about 2 times in the association between FAK and SHP-2 in the period coinciding with the low level of phosphorylation of FAK. To evaluate the effect of SHP-2 on the reduction of transient pFAK standardized up the silencing of SHP-2 in C2C12 cells in the states of proliferation and differentiation. It was observed lower expression of this protein after 12 hours the period of transfection. After Transfection in the midst of cultivation with the silencing of SHP-2 standard in 12 hours the cells were induced to differentiate by deprivation of fetal bovine serum. The depletion of SHP-2 abolished the reduction of transient phosphorylation of FAK. It was observed that the depletion of SHP-2 also prevented the terminal differentiation of myoblasts deprived of fetal bovine serum in myotubes. These data suggest that SHP-2 modulates the level of phosphorylation of FAK acting inhibitory role in the activation of FAK in the transition of C2C12 cells in the state of proliferation to differentiation, influencing the entry of these cells in the differentiation / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Le développement du muscle squelettique est un processus complexe très finement régulé, qui inclus des étapes de prolifération de cellules progénitrices appelées myoblastes et des étapes de différenciation pour former des myotubes multi nucléés. La glycosylation est la principale modification post-traductionnelle des protéines. Son rôle dans divers processus biologiques et pathologiques est largement documenté, mais les mécanismes intimes de son implication lors du processus myogénique restent mal élucidés. Nous avons pris comme modèle cellulaire la lignée myoblastique C2C12 car elle est capable de mimer in vitro les étapes de prolifération et de différenciation de la cellule musculaire. En utilisant différentes lectines, nous montrons un changement de la sialylation périphérique en α2-6 des glycoconjugués de surface de la cellule C2C12 durant la différenciation myoblastique. En complément, nous avons analysé les N-glycannes des glycoprotéines par spectrométrie de masse et mesuré les niveaux d’expression des gènes des α2-6 sialyl-transférases et neuraminidases. Tous les résultats obtenus confirment bien que la différenciation des cellules C2C12 est accompagnée d’une diminution du taux de sialylation des glycoconjugués. Pour mieux comprendre l’implication de la sialylation en α2-6 dans la myogenèse, nous avons réalisé une étude fonctionnelle sur des cellules C2C12 qui sous-expriment St6gal1 du fait de l’introduction d’un shRNA spécifique. Les clones obtenus présentent de plus forts index de fusion et génèrent un plus grand nombre de myotubes qui, de surcroit, sont de grande taille. Ce phénotype est probablement dû à un engagement accru des cellules de réserve en différenciation. En effet, les clones sous-exprimant St6gal1 contiennent une plus petite proportion de cellules Pax7+, c’est-à-dire de cellules de réserve maintenues dans un état de quiescence.Ainsi, nos résultats montrent l’importante implication de la sialylation périphérique en α2-6 au cours de la différenciation myogénique. / Skeletal muscle development is a complex process highly regulated and which includes proliferation then differentiation of progenitor cells or myoblasts into multi-nucleated myotubes. Glycosylation is the main post-translational modification of proteins. Its role in various biological and pathological processes is well documented, but the precise mechanisms of its involvement during myogenesis are still poorly understood.We have used the C2C12 myoblast as a model cell line since it is able to mimic in vitro the steps of muscle cell proliferation and differentiation. Using different lectins we showed a change in the peripheral α2-6 sialylation of the cell surface glycoconjugates, during C2C12 differentiation. Besides, we also analyzed by mass spectrometry the N-glycans carried by glycoproteins and measured the expression levels of α2-6 sialyl-transferases and neuraminidases genes. All the results confirm that C2C12 differentiation is accompanied by a decrease of glycoconjugates sialylation. To highlight the involvement of α2-6 sialylation in myogenesis, we performed a functional study of C2C12 cells knockdown for St6gal1 by a specific shRNA. The generated clones exhibit a higher fusion index and generate more elongated myotubes. This phenotype probably results from an increased commitment of reserve cell in differentiation. Indeed, the clones knockdown for St6gal1 contain a lower proportion of Pax7+ cells, i.e. of reserve cells maintained in a quiescent state. Thus, our results show the significant involvement of the peripheral α2-6 sialylation during myogenic differentiation.

Ce travail de thèse a porté sur l'étude de l'équilibre entre la prolifération et la différenciation dans le cadre de la myogenèse embryonaire et postnatale. Chez l'embryon, le sortie du cycle cellulaire est contrôlé par p57 et p21 pendant la myogenèse. Nous avons montré que la voie de signalisation Notch ainsi que les facteurs de régulation myogéniques (MRFs) régulent l'expression de p57 dans les cellules progénitrices et les myoblastes en différentiation. Chez l'adulte, p21 et p57 ne sont pas exriés dans la population quiescente de cellules souches du muscle (cellules satellites - SCs). p21 et p57 sont rapidement induits après activation et durant la différentiation des SCs. Ex vivo, les myoblastes déficients pour le gène p21 présentent des défauts de prolifération et de différenciation. In vivo, l'étude de la régénération musculaire chez les mutants p21 a montré une réduction précoce des SCs, avec un retard de reconstitution du tissu musculaire. Afin de pouvoir étudier le rôle de p57 après la naissance (les mutants p57 meurent à la naissance) nous avons généré un modèle murin permettant de muter le gène p57 de manière spatio-temporelle avec le système de recombinaison Cre/LoxP. Nous avons combiner notre allèle p57 conditionnel avec une Cre exprimée de manière ubiquitaire, et observé des phénotypes identiques aux phénotypes décrits précédemment chez les souris présentant une perte du p57. L'ablation conditionnelle de p57 dans les SCs adultes, a conduit à une diminution de la différenciation myogénique in vitro. Notre travail suggère que p21 et p57 jouent un rôle important dans la régulation de la différenciation et le cycle cellulaire dans le muscle adulte. / This thesis focuses on the coordination of proliferation and differentiation in embryonic and adult myogenesis. During development, we demonstrated that skeletal muscle progenitors interact with the differentiating myoblasts via the Notch pathway to maintain their pool. It has previously been established that p57 and p21 redundantly promote cell cycle exit in developing muscle and we showed that Notch and Myogenic Regulatory Factors act through muscle-specific regulation of p57. We then examined p21 and p57 in adult skeletal muscle stem cells, called satellite cells (SCs). Although absent from quiescent SCs, p21 and p57 are expressed upon activation (including proliferating myoblasts) and differentiation. p21-null myoblasts exhibited proliferation and differentiation defects in myofiber cultures, implicating p21 at the early activation phase. In vivo muscle regeneration studies with p21 mutants showed an early impact on the SC pool, while SCs and muscle structure were re-established by the end of regeneration. Since p57-deficient mice die at birth, we generated a conditional knock-out (KO) allele for postnatal studies using the loxP/Cre recombination system. With a ubiquitous Cre we observed developmental and perinatal phenotypes similar to previously described KO embryos. The new p57 allele also includes a β-galactosidase reporter and we showed that it recapitulates p57 expression profile in embryonic and adult tissues. Conditional ablation of p57 in adult SCs reduced myogenic differentiation in primary myoblast culture. Our work suggests that p21 and p57 are involved in adult myogenesis and cell cycle exit, working at the early steps of satellite cell activation.

La myogenèse fœtale repose sur les cellules progénitrices musculaires PAX7+ qui assurent la croissance musculaire au cours du développement et qui sont à l’origine des cellules satellites. Nous avons cherché à interpréter les signaux régulant la myogenèse fœtale et leur lien avec le cycle cellulaire. Nous avons effectué une analyse exhaustive du cycle cellulaire des cellules myogéniques au cours de la myogenèse fœtale. Nous avons aussi identifié que les cellules PAX7+ progressant dans le cycle cellulaire (phases S, G2, et M) sont régionalisées aux extrémités des muscles. Les voies de signalisation BMP et NOTCH régulent positivement le nombre de cellules PAX7+ pendant le développement fœtal mais ont un effet différent sur la différenciation musculaire. Nous avons montré que les voies de signalisation BMP ou NOTCH augmentent le nombre de cellules PAX7+ de manière indépendante. Nous avons aussi identifié des interactions antagonistes entre ces deux voies lors de la différenciation musculaire. Nous avons testé l'importance de la contraction musculaire pendant la myogenèse fœtale chez l’embryon de poulet. Le blocage des contractions musculaires mime un phénotype de perte de fonction NOTCH, à savoir une diminution du nombre de cellules progénitrices musculaires avec une tendance à la différenciation musculaire. Nous avons aussi montré que les forces mécaniques produites par les contractions musculaires sont détectées par le co-activateur transcriptionnel YAP1 qui régule l'expression d’un ligand de NOTCH au sein des fibres musculaires, qui à son tour va maintenir le pool de cellules progénitrices musculaires fœtaux. / Foetal myogenesis relies on PAX7+ muscle progenitors that provide the source of cells for muscle growth during development and for the generation of the satellite cell pool. We aimed to decipher the signals that regulate the balance between myogenic differentiation and proliferation. We performed an exhaustive analysis of the cell cycle phases of myogenic cells during foetal myogenesis. I defined that PAX7+ cells in the S/G2/M phases were enriched at the contact points to the tendons. BMP and NOTCH signals increase the number of PAX7+ cells during foetal development, but affect differentiation in a positive and negative manner, respectively. I revealed that BMP and NOTCH increase the number of PAX7+ cells independently of each other. However, they act antagonistically during differentiation. Thus, the interplay between NOTCH and BMP signalling differs in proliferation and differentiation. Because muscle is a mechanical tissue, we tested the importance of muscle contraction for foetal myogenesis in chick embryos. I found that the block of muscle contraction during foetal myogenesis mimicked a NOTCH loss-of-function, i.e. decreased the number of foetal muscle progenitors and shifted the balance between proliferation and differentiation towards a differentiation fate. Mechanical forces provided by muscle contractions are sensed in myonuclei by the transcriptional co-activator YAP1 that regulates expression of the NOTCH ligand JAGGED2 in muscle fibres. This JAGGED2 signal keeps the muscle progenitors in an undifferentiated state and suppresses differentiation.