Cloning, Expression And Functional Analysis Of Genes Regulating Musle Development From Flounder (Paralichthys Olivaceus)

This study focused on the cloning and expression of flounder muscle regulatory genes, MyoD and Myf5, and Forkhead domain genes, FoxD1, FoxD3 and FoxD5, and the analysis of their functions during flounder muscle development.The genomic sequences of both flounder MyoD and Myf5 contain three exons and two introns, and a highly conserved bHLH domain. The flounder FoxD1, FoxD3 and FoxD5 contain only one exon and a conserved winged-helix DNA binding domain. In situ hybridization indicated that, Myf5 transcripts were first detected in the paraxial mesoderm, during somitogenesis, Myf5 expression was found in developing somites; MyoD expression was first detected as two rows of presomitic cells in the segmental plate, and then was present in the adaxial cells and the lateral somitic cells. With the development of embryo, Myf5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites; After 30 somites formed, MyoD expression decreased in the somites except the caudal somites. At the hatching stage, MyoD and Myf5 were expressed in head muscle cells and fin muscle cells. In the growing fish, Myf5 was expressed in the skeletal muscle and intestine, and in adult flounder, Myf5 was only expressed in muscle. In the growing fish and adult fish, MyoD was only expressed in muscle.The MyoD promoter and Myf5 promoter could drive GFP specifically expressed in zebrafish muscle fibers, indicating that the two promoters contained the core sequences required for the normal expression of the two genes, also, the two promoters were conserved because they could function across species.During the early stage of embryogenesis, FoxD1 was mainly expressed in the somites, kidney progenitor cells, brain progenitor cells and intestine. FoxD3 was expressed in premigratory neural crest cells, somites, post otic placodes, cranial and trunk neural crest cells, and pineal gland. FoxD5 was mainly expressed in the somites, tail bud, forebrain and otic vesicle etc.Flounder FoxD3 mRNA was over expressed in one cell of the two-cell stage zebrafish by microinjection, zebrafish homologue FoxD3 was used to confirm the effect of flounder FoxD3. After over expression, the two sides separated by the anterior-posterior axis of zebrafish embryo showed an asymmetrical development, the expression of MyoD and Myf5 in the paraxial mesoderm was inhibited. So the flounder FoxD3 might function in the muscle development regulatory network by interacting with MyoD and Myf5.Flounder FoxD1 mRNA was over expressed in one cell of the two-cell stage zebrafish, MyoD expression in the somites on one side was reduced, but the expression in the adaxial cells was not affected; Myf5 expression in the presomitic mesoderm, adaxial cells and somites on one side was reduced. It is indicated that FoxD1 might play a role in the muscle development by regulating MyoD and Myf5.Flounder FoxD5 mRNA was over expressed in one cell of the two-cell stage zebrafish, MyoD expression in the somites on one side was enhanced, but the expression in the adaxial cells was not affected; Myf5 expression in the somites and presomitic mesoderm on one side was also enhanced. FoxD5 might regulate the muscle development by regulating the expression of MyoD and Myf5.