Gene Regulation Of Skeletal Muscle Growth And Development By Sonic Hedgehog In Siniperca Chuatsi

Sonic hedgehog(Shh)was one of the important signaling molecules in the Hedgehog gene family,which played an important role in regulating cell proliferation and differentiation in early embryos of individuals.In this study,we investigated the role of the Shh gene in the regulation of skeletal muscle growth and development of Siniperca chuatsi.We cloned the sequence of the ORF region of the Shh gene of Siniperca chuatsi and used bioinformatic techniques to analyze the protein encoded by the Shh gene and its homologous evolutionary features.In addition,the gene suppression and overexpression treatments were used to in vestigate its regulatory effects of the Shh gene on skeletal muscle development and differentiation in Siniperca chuatsi by HE sectioning,immunofluorescence and in situ hybridization.The main results obtained were as followings:1.A bioinformatic approach was used to analyse the proteins encoded by the Shh gene of Siniperca chuatsi and the homologous evolutionary features.The protein encoded by this gene was found to have two functional structural domains,namely Hh-N and Hh-C.The Hh-N structural domain had the signaling activity of the Hh protein,while Hh-C functions as a cholesterol transferase-like enzyme,influencing the distribution of the signaling molecule.The results of the sequence homology evolutionary analysis showed that the Shh protein of Siniperca chuatsi had a high homology with the Shh protein of the Perciformes to which it belongs,as well as a conserved amino acid sequence of the Hh-N structural domain of the protein between different species.2.The spatial-temporal expression characteristics and patterns of the Shh gene of Siniperca chuatsi were assayed using RT-q PCR and whole in situ hybridization.The RT-q PCR results showed that the expression of Shh was relatively low in the early stages of embryonic development,significantly up-regulated in the neuroembryonic stage,and expressed at a higher level in the middle and later stages of embryonic development,with peak expression at the emergence stage.In juvenile fish at 1-7 d of development,expression decreased from 1 d to 4 d significant difference(P<0.05),while Shh expression remained constant in the later stages of juvenile fish.The spatial expression of Shh in juvenile C.chuatsi tissues was also examined and showed some differences in expression in different tissues,with higher expression in the brain and intestine and differentially expressed the red muscle,white muscle,spleen,liver,kidney and heart.The receptor Ptch gene was expressed in the somites,neural tube and brain.Therefore,it is hypothesized that Shh signalling molecules may have a regulatory role in muscle development during the development of Siniperca chuatsi.3.Preliminary functional regulatory role of Shh on myoblast differentiation and development were assayed by using Shh signalling inhibitor-cyclopamine in the embryos and juveniles of Siniperca chuatsi and by Shh overexpression in the embryos.The obtained results showed that the development of the embryos of Siniperca chuatsi was hindered and the tail end of the trunk was bent by use of cyclopamine soaked embryos and intramuscular injection of juvenile fish,which resulting in a 100%teratogenicity rate;In situ hybridization of embryos showed that Ptch,Myod and Mrf5 gene expression was inhibited to varying degrees;HE sections of skeletal muscle from juvenile fish showed increased gaps between muscle fibres and the fiber volumn reduced,and then with the transmission electron microscopy observed that the muscle fibres were uneven in thickness and some of them were broken,discontinuous and exhibited degenerative changes.Using immunofluorescence analysis,the mean number of Pax7-positive cells and the number of Brd U-positive cells were found to be significantly decreased.The expression of myocyte differentiation and development-related transcription factors,myogenic regulators and muscle structure genes was significantly reduced by the inhibition of Shh signalling molecules,and the expression of all genes tested increased significantly after Shh overexpression,except for the Mylk and Myh2 genes.The above results suggest that Shh is presumably involved in regulating the early developmental formation of myoblasts in Siniperca chuatsi.