Muscle Development In Fish And Evolution Of Myogenic Factors Directed By Pei-jun Zhang And Sho Jun Du

In this thesis, the production of partial protein of Gli2 which is one of transcription factors in Hedgehog signaling that involve in muscle development; its purification and the production of its antibody; the characters of transcription factors in muscle development in fish- differentiation step and the analysis of its evolution through comparing MyoD of amphioxus with that of other species was studied.A gene that have critical function in muscle development-differentiation and maturation of muscle cells was got through yeast two-hybrid system. The specific character of these promoters of these genes was also studied. During the process of production of partial Gli2 protein, a soluble protein - -Gli2N2protien was got. A antibody that can recognize ectopic Gli2 was produced from rabbit injected with Gli2N2 protein. The temporal expression of ectopic Gli2 protein was analysed by anti-Gli2 antibody.At 12hpf,ectopic Gli2 protein was full length ,but from 15hpf most Gli2 protein was C-terminal truncated form.This is the first anti-Gli2 antibody and the first time ectopic expression of Gli2 was detected by anti-Gli2 antibody. From Striped bass , Myf-5 and myogenin wasisolated. Analysis of its character show these two gene share the same gene structure(three exon and two intron) with other myogenic factors-MyoD family.Two MyoD was isolated from Seabream.They have different expression pattern in Seabream embryos, the expression of MyoD1was affected by Hedgehog.The studied of evolution history of evolution of MyoD was done by comparing them with other MyoD of other species.The results show these two MyoD was produced through independent gene duplication or duplication of entire genomes but in some species one of myogenic gene was lost . All of the promoters of these genes of MyoD family can drive report gene –GFP muscle specific expression in zebrafish embryos.This suggested that there is muscle specific regulatory elements in the promoters and the elements are conserved between species and have function cross species. In evolution, Amphioxus, a cephalochordate, has been regarded as the living fossil closest to the vertebrates. In this study, we have isolated and characterized a new member of the MyoD family (AmphiMyoD) in amphioxus. Sequence analysis revealed that AmphiMyoD shares more identity with vertebrate and invertebrate myogenic genes compared with other three amphioxus MyoD-like genes. These data suggest that AmphiMyoD may be closely related to the ancestral myogenic gene that gave rise to the four vertebrate myogenic genes during evolution. Moreover, because the four myogenic genes in amphioxus are grouped together in a phylogenetic tree analysis rather than segregated with the four members of the MyoD family in vertebrates, it suggests that gene duplications that generated the multiple myogenic genes in amphioxus were independent from duplications that produced the four vertebrate myogenic genes. Its promoter can drive reporter gene-GFP in skeletal and cardiac muscles in zebrafish embryos. These data indicate that the activity of AmphiMyoD promoter might not be exclusively restricted to skeletal muscles as in vertebrates. The AmphiMyoD promoter may contain regulatory elements for cardiac muscle expression This data suggests that some of the regulatory elements for skeletal muscle-specific expression might be conserved during evolution. The cardiac muscle-specificity of the AmphiMyoD promoter is intriguing. Bop which came from yeast two-hybrid system produce at least two isoforms of Bop in zebrafish by alternative splicing. Expression analysis revealed that Bop is specifically expressed in developing somite and heart in zebrafish embryos. Functional studies by antisense knockdown approach demonstrated that Bop is involved in skeletal muscle maturation and heart formation. Blocking Bop expression in zebrafish embryos resulted in malfunction of motivation of zerbrafish embryos, and moreover the blood circulation. Together, these data suggest that Bop plays a critical role in the differentiation of skel...