Myosin 18B is an unconventional myosin that has been implicated in tumor progression in humans. In addition, loss-offunction mutations of the MYO18B gene have recently been identified in several patients exhibiting symptoms of nemaline myopathy. In mouse, mutation of Myo18B results in early developmental arrest associated with cardiomyopathy, precluding analysis of its effects on skeletal muscle development. The zebrafish, frozen (fro) mutant was identified as one of a group of immotile mutants in the 1996 Tübingen genetic screen. Mutant embryos display a loss of birefringency in their skeletal muscle, indicative of disrupted sarcomeric organization. Using meiotic mapping, we localized the fro locus to the previously unannotated zebrafish myo18b gene, the product of which shares close to 50% identity with its human ortholog. Transcription of myo18b is restricted to fast-twitch myocytes in the zebrafish embryo; consistent with this, fro mutant embryos exhibit defects specifically in their fast-twitch skeletal muscles. We show that sarcomeric assembly is blocked at an early stage in fro mutants, leading to the disorganized accumulation of actin, myosin, and a-actinin and a complete loss of myofibrillar organization in fast-twitch muscles.
Differentiation of skeletal muscle myoblasts involves activation of muscle-specific markers such as MyoD, Myf5, MRF4, and myogenin, followed by exit from the cell cycle, expression of structural proteins, and fusion into multinucleated myotubes. Cyclin D3 is upregulated during muscle differentiation, and expression of cyclin D3 in proliferating myoblasts causes early activation of myogenesis. In this study, we have identified the genes activated by cyclin D3 expression in C2C12 myoblasts and differentiated cells by real-time PCR analysis. Cyclin D3 expression induced faster differentiation kinetics and increase in levels of myogenic genes such as MyoD, Myf5, and myogenin at an early stage during the differentiation process, although long-term myogenic differentiation was not affected. Transcript levels of the transcription factor Pax7 that is expressed in muscle progenitors were enhanced by cyclin D3 expression in myoblasts. Components of a histone methyltransferase complex recruited by Pax7 to myogenic gene promoters were also regulated by cyclin D3. Further, the Pax7 promoter was upregulated in myoblasts expressing cyclin D3. Myoblasts that expressed cyclin D3 showed moderately higher levels of the cyclin-dependent kinase inhibitor p21 and were stalled in G2/M phase of the cell cycle. Our findings suggest that cyclin D3 primes myoblasts for differentiation by enhancing muscle specific gene expression and cell cycle exit.
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