Molecular analysis of mammalian muscle differentiation, fusion and regeneration

Molecular study of mammalian skeletal muscle has become an archetype for the mechanisms that determine cell fates during embryonic development and adult tissue maintenance by closely linking cell proliferation and differentiation. Early developmental studies have led to the identification of the origins of skeletal muscle, as well as roles played by inductive signals and transcriptional regulatory factors that initiate and aid during muscle specification and differentiation. However after the initiation of the myogenic program, and the progression of myogenic differentiation by means of expression of transcriptional activators, the molecular and morphological events ultimately concluding in the formation of multi-nucleated myotubes are unknown. Here, I have studied the molecular basis of muscle cell fusion, which leads to the formation and maturation of muscle cells, and of muscle regeneration, in their potential to re-enter the cell cycle and de-differentiate. To initiate these studies in mammals, I have taken advantage of the evolutionary conservation of these processes. For fusion, I selected, Br˙ag2 and Dock180 since their roles as guanine nucleotide exchange factors, of Arf6 and Rac respectively have been shown to be necessary in Drosophila development. Herein the roles of these two molecules in mammalian myotube formation have been investigated and shown to be dependent on dissimilar molecular mechanisms. Their effects on myotube formation are distinct due to the modes of action of their respective GTPases. Furthermore, I show that the essential role of Dock180 in myotube formation is conserved in other syncytial structures, such as macrophages, thus providing evidence that fusogenic machinery in cells of different origins may be shared.;In the context of regeneration, the fused and multi-nucleated myotube structure of mammalian muscle provides an inherent physical barrier. However in urodele amphibians, regeneration occurs frequently in muscle, and other tissues, following tail and limb amputation. Unlike the de-differentiation and regeneration observed in newts and axolotls, analysis of mammalian muscle de-differentiation has been hindered by the post-mitotic and non-responsive state of mammalian myonuclei to serum stimulation. Here I have investigated the molecular basis of cell cycle re-entry in fused and differentiated mammalian myotubes, and concluded that at least two regulatory barriers, presented by Rb and Ink4a genes, must be removed in order for mammalian myonuclei to initiate DNA synthesis in mature myotubes. This initiation of cell cycle machinery is followed by upregulation of mitotic and cytokinetic components and culminates in direct evidence of de-differentiation as seen by loss of structural myogenic components and collapse of myotube morphology.;By employing this strategy of myotube de-differentiation on single-nucleated, post-mitotic myocytes, which normally never re-enter the cell cycle, I have been able to isolate differentiated muscle cells capable of multiple rounds of division and expansion. Myoblast colonies obtained from myocytes transiently deficient in Rb and Ink4a are capable proliferation and upon serum starvation can recapitulate the myogenic program and producing mature myotubes in culture. These de-differentiated cells are also capable of fusing to muscle fibers in vivo, thus completing for the first time a cycle of regeneration in a mammalian muscle.