Expression And Possible Function Of Myostatin During Early Embryonic Skeletal Muscle Development

Myostatin, a potent inhibitor of myogenesis, is a member of transforming growth factor-?superfamily because the skeletal muscle mass of Myostatin null mice was significantly larger than that of wild-type animals which was resulted from a combination of muscle cell hyperplasia and hypertrophy. Quantification of the number of muscle fibers showed that at the widest portion of the tibialis cranialis muscle, the total cell number was 86% higher in mutant animals compared to wild-type littermates. It also been reported that mutation of Myostatin gene was responsible for the increased skeletal muscle mass in the double-muscled cattle. In vertebrates, with the exception of fish, the number of muscle fibers in adult animal is largely determined by muscle fibers formed prenatally. The satellite cells located between the plasma membrane of a muscle fiber and the basal membrane that wraps up the muscle fiber along its whole length contribute to the growth of skeletal muscle in postnatal muscle development. They undergo proliferation and differentiation prior to fusion with fibers developed prenatally. As a result, the fibers display hypertrophy. Based on the phenotypes of muscle cell hyperplasia and hypertrophy observed in the Myostatin null mice and double-muscled cattle, it implies that Myostatin plays a critical role in early myogenesis. However, little is known about the functional roles of Myostatin during early muscle development.To explore the functional roles of Myostatin in early myogenesis during embryonic development, in this study we systematically examined the expression patterns of Myostatin in chicken embryos by using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and whole mount in situ hybridization analysis, respectively. Our results showed that Myostatin mRNAs were detected in chicken embryos at HH stage 15 by RT-PCR and in the three most anteriorly situated occipital somites at HH stage 16 by whole mount in situ hybridization. With the embryo development, Myostatin expression extended behind above somites. Myostatin positive signals were noticed in the interlimb somites at HH stage 19. The majority of somites throughout the embryo expressed Myostatin at HH stages 23?24. Interestingly, we found that Myostatin initiated its expression at the centre of dermomyotome in individual somites and as development processed, Myostatin expression extended more dorsomedially and ventrolaterally. Myostatin expression in hypoglossal cord which formed prospective tongue muscle was observed at HH stage 17. The abundance of Myostatin mRNA increased in the following stages, and decreased from HH stage 23. First expression in a proximocentral domain of limb buds was observed at HH stage 21 and its expression was extended along proximal-distal axis in the following stages. Additionally, Myostatin expression didn't confined to the dorsal and ventral domains in which the prospective muscle was developed, whereas it expressed at the central part of limb buds in which the mesenchymal cells were resided. At HH stage 21, faint expression of Myostatin was detected in branchial arch 2, by HH stage 22 Myostatin expression in both branchial arch 2 and 1 was evident at the proximal aspect. Taking together, Myostatin was expressed at high abundance in which the prospective skeletal muscle development was proceeded.Most intriguingly, our data revealed that the expression pattern of Myostatin in the dermomyotome was correlated well with an important event of epithelial mesenchymal transition (EMT) which is essential for muscle progenitor cell specification. To further investigate the relationship between Myostatin expression and EMT, the progress of EMT at interlimb somites from HH stage 18 to 22 was examined. EMT took place in the central part of dermomyotome at HH stage 19, with the development progress, EMT extended more dorsomedially and ventrolaterally. By HH stage 22, the majority of dermomyotome underwent EMT, with the exception of dorsomedial and ventrolateral lips. By comparing the sites of EMT in dermomyotome and patterns of Myostatin expression at the same HH stages, we demonstrated that Myostatin expression was highly correlated to EMT. They shared the similar developmental pattern and both events took place firstly at the central parts of dermomyotome, and extended more dorsomedially and ventrolaterally in the following development. Most significantly, our results indicated that EMT was happened where Myostatin was expressed. The preliminary data from knocking down Myostatin experiments with its corresponding siRNA showed that EMT was delayed during development. In summary, the specific patterns of Myostatin expression observed in this study imply its functional roles in regulating early muscle development.