Le gène H19, soumis à l’empreinte parentale, est fortement exprimé durant le développement embryonnaire, cependant, son expression est réprimée après la naissance dans l’ensemble des tissus à l’exception du muscle squelettique, et plus particulièrement des cellules souches musculaires : les cellules satellites. L’objectif de ma thèse a été de déterminer le rôle du gène H19 dans la mise en place et dans la fonction de ces cellules souches durant la myogénèse adulte. En utilisant un modèle murin présentant une délétion du gène H19, les souris H19∆3, notre laboratoire avait montré que le gène H19 est capable de moduler, dans le muscle embryonnaire, l’expression de neuf gènes appartenant à un réseau de gènes soumis à l’empreinte parentale (IGN) impliqué dans la croissance. Au cours de ma thèse, j’ai étudié le phénotype des muscles de ces souris mutantes qui présentent une hyperplasie et une hypertrophie des fibres musculaires. Ce phénotype est accompagné d’une diminution du nombre de cellules satellites qui apparait lors de l’entrée en quiescence de ces cellules. De façon étonnante, nous avons observé une meilleure capacité de régénération, malgré le nombre réduit de cellules satellites, dans les muscles H19∆3 comparée à celle des muscles wt. Cela indique que la capacité d’auto-renouvellement des cellules satellites n’est pas influencée par l’absence du gène H19. De même, nous avons observé une surexpression de plusieurs gènes appartenant à l’IGN lors de la régénération musculaire des muscles mutants comparés aux muscles wt. Ces résultats indiquent que le gène H19 module l’expression des gènes de l’IGN durant l’embryogénèse et par la suite, durant les étapes de régénération de la myogénèse adulte. / The imprinted H19 gene is highly expressed during embryonic development. H19 is fully repressed after birth in all tissues, with the exception of skeletal muscle, and especially of the muscle stem cells: the satellite cells. The aim of my thesis was to define the function of the H19 gene in the satellite cells establishment and function during adult myogenesis. Using loss-of-function H19∆3 mice, the laboratory had shown that the H19 gene was able to modulate the expression of several genes belonging to an imprinted gene network (IGN) in the embryonic muscle. During my thesis, I studied the muscle phenotype of these adult mice, which present both fiber hyperplasia and hypertrophy. This phenotype is accompanied by an important reduction of the satellite cell number, probably due to a delay in their entry into quiescence. Unexpectedly, despite the reduction in the number of satellite cells in mutant mice, the self-renewal capacity of the satellite cells is fully retained. In addition, we observe a better regeneration potential of the mutant muscles compared with wt muscles. This is accompanied by the enhanced expression of several genes from the IGN. These results indicate that H19 gene can modulate IGN gene expression both during embryogenesis and after birth, in adult myogenesis.

O músculo estriado esquelético apresenta em sua constituição células satélites (CS) que se encontram em estado quiescente localizadas entre o sarcolema e a lâmina basal das fibras musculares. As CS podem ser ativadas, diferenciando em mioblastos, contribuindo para regeneração e/ou crescimento do tecido muscular. Os Fusos neuromusculares são mecanorreceptores localizados no interior dos músculos esqueléticos considerados a unidade contrátil reguladora, monitorando a velocidade e duração do alongamento do músculo. Está composto de fibras intrafusais (FIF), circundadas por uma bainha de tecido conjuntivo e encontra-se paralelo às fibras extrafusais. A desnervação promove alterações no músculo esquelético, tanto em CS, quanto nos fusos neuromusculares. Este trabalho analisou quantitativamente as FIF e a proliferação de CS em músculos esquelético de ratos desnervados por longo período. Foram utilizados ratos Wistar. Os animais foram divididos em grupos desnervados e controle. Os músculos Sóleo e Extensor longo dos dedos (EDL) foram desnervados experimentalmente. Após os períodos de 0, 12, 16, 19, 30 e 38 semanas, os músculos foram dissecados, removidos e preparados histológicamente. A porcentagem de CS em músculos imediatamente após desnervação aumenta em relação ao músculo normal e depois decresce em ambos os músculos. Durante o progresso do tempo de desnervação ocorreu um aumento no número de FIF, se comparado com o grupo normal. O número de CS diminui significantemente entre os períodos de desnervação, em ambos os grupos. Nos músculos estudados quanto menor a porcentagem de CS maior é o número de FIF e, aumentando o tempo de desnervação, diminui o número de CS. Em relação às FIF, no grupo controle com o aumento do tempo, o número de fibras não se altera. Já para o grupo experimental, com o aumento do tempo de desnervação, diminui o número de CS e aumenta o número de FIF significantemente. Concluimos então que nos músculos desnervados por longo período ocorre diminuição na porcentagem de células satélites e aumento no número de FIF. Finalmente nossos resultados sugerem que entre 16ª e 19ª semana pós-desnervação encontra-se o melhor período para reinervação de um músculo desnervados. / The skeletal muscle consists of satellite cells (SC) which are in a quiescent state located between the sarcolemma and basal lamina of the muscle fibers. The SC can get activated, differentiating into myoblasts, contributing to regeneration and/or growth of muscle tissue. The neuromuscular spindles are mechanoreceptors located within the skeletal muscle and are considered as contractile regulatory unit, monitoring the speed and duration of muscle stretching. It is composed of Intrafusal muscle fibers (FIF), surrounded by a sheath and is parallel to extrafusal fibers. Denervation cause changes in skeletal muscles both in the CS and neuromuscular spindles. This study analyzed quantitatively the FIF and the proliferation of CS in rat skeletal muscle, denervated for long period. We used Wistar rats to perform this study. The animals were divided into control and denervated groups. The soleus and extensor digitorum longus (EDL) were denervated experimentally. After periods of 0, 12, 16, 19, 30 and 38 weeks, the muscles were dissected, removed and were prepared for histological analysis. The percentage of SC in muscles immediately after denervation, increases in relation to normal muscle and later decreases in both the groups. During the process of denervation, there was an increase in FIF when compared with normal group. The number of SC reduces significantly between the periods of denervation in both the groups. In the muscles studied, the smaller the percentage of SC, higher is the number of FIF and increase in the duration of denervation, reduces the number of SC. As for FIF, with the increase in time in control group, the number of fibres was unaltered. However, in the experimental group, with increase in the time of denervation, the number of SC decreases while there is increase in the number of FIF significantly. We thus conclude that in denervated mucles for long period, there is decrease in the percentage of satellite cells and increase in FIF. Finally our results suggest that the period between 16th and 19th week of post denervation is the best time for reinnervation of denervated muscle.

No músculo reside um conjunto de células miogênicas indiferenciadas denominadas células satélites (CS), que são essenciais na homeostasia e na regeneração muscular. No entanto, as fases de ativação, proliferação e diferenciação das CS podem ser influenciadas por vários fatores extracelulares, como, a adiponectina. Essa adipocina tem sido amplamente estudada em relação a seus efeitos anti-inflamatórios e antidiabéticos, e foi proposta como reguladora da miogênese in vitro, porém sua atuação na regeneração in vivo, ainda não é bem elucidada. O objetivo deste trabalho foi avaliar a regeneração muscular em camundongos deficientes de adiponectina. A lesão foi induzida pela injeção de 50 ul de cloreto de bário no músculo tibial anterior esquerdo de animais machos selvagens (WT) e deficientes em adiponectina (AdKO). Após 3, 7 e 14 dias os animais foram eutanasiados e as amostras coletadas para análises morfológicas, gênica e proteica. O grupo AdKO apresentou maior quantidade de núcleos centralizados comparado com o grupo WT após 7 dias da lesão. Foram encontrados redução da expressão gênica de Pax7, MyoD e Miogenina nos AdKO após 3 dias. Houve aumento dos níveis das citocinas anti-inflamatórias IL-10 e IL-4 após 3 e 7 dias, respectivamente, e aumento das citocinas pró-inflamatórias IL-1 &#946, IL-17, TNF- &#945 e IFN após 7 dias nos animais AdKO. Apesar de não haver diferença entre os genótipos na expressão gênica de F4/80, os animais AdKO apresentaram aumento de CD206. Os animais WT tiveram aumento de mRNA de adiponectina com 7 dias da lesão. Não foram encontradas diferenças significativas entre os genótipos quanto ao infiltrado inflamatório, a deposição de colágeno total, a área de secção transversal e a recuperação da massa muscular. Concluímos que a adiponectina é importante no processo de remodelamento tecidual durante a regeneração e que sua deficiência não compromete a maturação das fibras musculares, devido aumento da resposta anti-inflamatória, apesar de haver um possível comprometimento na resposta pró-inflamatória. / In the muscle resides a set of undifferentiated myogenic cells, called satellite cells (SC), which are essential in the maintenance of homeostasis and muscle regeneration. However, the control and activation of SC can be influenced by several extracellular myogenic factors, such as adiponectin. Adiponectin, an adipokine, mainly produced by subcutaneous adipose tissue, has been widely studied in relation to its anti-inflammatory and anti-diabetic effects, but its role as regulator of in vivo regeneration is not yet well elucidated. Here we evaluate muscle regeneration in adiponectin deficient mice. Barium chloride was administered to the left anterior tibial muscle of wild type (WT) and adiponectin deficient (AdKO) animals. The right anterior tibial muscle was not injured and was used as control. The animals were euthanized after 3, 7 and 14 days after injury. We measured the area of the fibers by laminin, in addition to qualitatively evaluating the response to injury by HE and picrosirius. The inflammatory response was verified by the concentration of several cytokines in the muscle, by ELISA, and by the gene expression of some inflammation pathways (RT-qPCR). The regeneration response was obtained by the analysis of regulatory genes of this process by RT-pPCR. The data were compared statistically and the significant differences considered presented p <0.05. The lesion was effective in causing tissue damage, and the activation of regulatory genes of the regeneration process besides inducing inflammation in WT. However, we observed that the animals with deletion for the adiponectin gene presented a myogenic response potentially similar to the control animals, however, the cellular response presents differences. We conclude that adiponectin deficiency does not compromise muscle regeneration, although there is a possible compromise of the pro-inflammatory response.

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.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 18, 2010). Includes bibliographical references.

Skeletal muscle has a remarkable capacity for regeneration, mediated by muscle stem cells that can self-renew or differentiate to form the mature myofibers of the tissue. Several human diseases are characterized by a loss of function and strength in skeletal muscle, with impairments in the ability to regenerate and consequent decreases in quality of life and increases in mortality. The work in this dissertation has focused on developing methods for combating muscle disease. This goal has been approached through attempts at cell replacement therapy - by generating muscle cells that can be engrafted in vivo. I also investigated the influence on regeneration of the skeletal muscle microenvironment (skeletal muscle-resident fibroblasts), and systemic environment (inflammation in myogenic and non-myogenic tissues), both of which were found to affect skeletal muscle stem cell behavior and the efficiency of myogenic regeneration. Ultimately, these studies identified novel factors that impair or improve skeletal muscle differentiation, and that offer the potential to modulate the process of muscle regeneration. In the process of investigating if induced pluripotent stem cells from skeletal muscle retain an epigenetic memory conducive to myogenic differentiation, I discovered that precursor cells in skeletal muscle reprogram to a pluripotent state more efficiently. However, these induced pluripotent stem cells, like embryonic stem cells, retain strong barriers to skeletal muscle differentiation. Together, these findings offer insights into the process of muscle regeneration, and suggest new potential pathways towards treatment of muscle disease.

Skeletal satellite cells are adult stem cells located among muscle fibers. Proliferation, migration and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates skeletal satellite cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22phox, p47phox and p67 phox were detected in primary human and murine skeletal muscle satellite cells. In human satellite cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47 phox, which was detected in the nucleus. In proliferating subconfluent satellite cells, both Nox2 and Nox4 contributed to O2- production. However, Nox4 expression was significantly attenuated in confluent cells and in differentiated myotubes. Proliferation of satellite cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22phox expression using siRNA oligonucleotides, and reduction of Nox4 and p47phox activities with dominant-negative vectors resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal satellite cells and that its activity plays an important role in promoting proliferation of these cells.

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.

The sexual differentiation of the spinal nucleus of bulbocavernosus (SNB) and the bulbocavernosus (BC) and levator ani (LA) muscles that the SNB innervates, are masculinized by androgens acting on the BC/LA. The site of androgen receptors (AR) responsible for the masculinization of the neuromuscular system is not known. A potential site of action is satellite cells: proliferation of these cells is androgen-dependent and satellite cells seem to contribute to the size of the LA. Fluorescent immunohistochemistry and confocal microscopy were used to co-localize satellite cells and AR within the LA of postnatal day one and three male and female rats. Results indicate that satellite cells express AR and reveal a difference in proportion of satellite cells expressing AR between the LA and control muscle. Interpretations of these findings, including whether the relatively small proportion of AR accounted for by satellite cells is enough to masculinize the SNB system, are discussed.

The sexual differentiation of the spinal nucleus of bulbocavernosus (SNB) and the bulbocavernosus (BC) and levator ani (LA) muscles that the SNB innervates, are masculinized by androgens acting on the BC/LA. The site of androgen receptors (AR) responsible for the masculinization of the neuromuscular system is not known. A potential site of action is satellite cells: proliferation of these cells is androgen-dependent and satellite cells seem to contribute to the size of the LA. Fluorescent immunohistochemistry and confocal microscopy were used to co-localize satellite cells and AR within the LA of postnatal day one and three male and female rats. Results indicate that satellite cells express AR and reveal a difference in proportion of satellite cells expressing AR between the LA and control muscle. Interpretations of these findings, including whether the relatively small proportion of AR accounted for by satellite cells is enough to masculinize the SNB system, are discussed.