Studies On The Role Of CXXC5Gene In Zebrafish Heart Develpment

In this study, we used zebrafish and C2C12cell line as models to explore the biological functions of the gene CXXC5in heart and skeletal muscle development. The zebrafish CXXC5gene is located in chromosome21, containing4exons and3introns and being up to73.5kb whole-length on genome. Its mRNA is2622bp nucleotides long and contains an ORF of855bp nucleotides encoding a protein of284amino acids with a molecular weight of32.24kDa. Bioinformatics analysis shows that CXXC5contains one zinc finger domain of CXXC type which is highly conserved during evolution among a variety of species.We cloned the cDNA and generated the polyclonal antibody against zebrafish CXXC5protein. Western blot showed that CXXC5protein is highly expressed in various zebrafish tissues such as heart. zCXXC5gene was also detected to be highly expressed in heart by whole amount in situ hybridization of zebrafish embryos. Fluorescent sub-cellular localizaion analysis suggested that zCXXC5protein is mainly located in the nucleus.Using morpholino oligo-nucleotide knock-down techniques, we found that knocking down the expression of CXXC5led to severe heart malformations in zebrafish embryos, including unable to loop normly, varying degrees of enlarged pericardial cavity and relatively smaller heart ventricle, which resulting in smaller cardiac output and therefore affecting its function seriously. We used a heart-specific GFP-positive zebrafish line Tg (nppa:gfp) which was established by this lab as a model to analyze heart phenotype, the number of cardiac muscle cells and morphology. Based on the results of the number of the myocardial cells analysed by biometrics software analysis, we found that the number of the ventricle myocardial cells in those embryos which unable to normly loop were remarkably decreased, which demonstrated that CXXC5was involved in the proliferation regulation of cardiac ventricle myocytes during embryo early development.Detecting the human Smad protein family by coimmunoprecipitation analysis, we obtained the Smad2and Smad3protiens that interacts with zCXXC5. We showed that hSmad2and hSmad3protiens did interact with zCXXC5by GST-Pull down and sub-cellular colocalizaion analysis. By sub-cloning and coimmunoprecipitation analysis, we identified that the CXXC domain of zCXXC5protien interacted with the DWA domain of Smad2and Smad3protiens.RT-PCR results showed that an obvious down-regulation of cardiac marker proteins Nkx2.5、Hand2、Has2、Myh6and Vmhc was observed in the embryos when knocking down the expression of CXXC5by the morpholino, which suggested that CXXC5gene is located upstream of these marker genes. We used the capped mRNA of Hand2gene to conduct the rescue experiment and the result showed that Hand2mRNA could well rescue the malformations in the zebrafish embryos with CXXC5protein expression knocked-down by the specific morpholino of this gene.It have been reported that CXXC5is a responsive factor of retinoic acid signing pathway and additionally, involved in WNT signing pathway by interacting with DVL, which suggests that CXXC5play regulatory roles through various signing pathways. We detected role of the gene CXXC5on various signing pathways by using luciferase report system. The result indicated that CXXC5plays an important role in skeletal myogenesis. And we used C2C12cell line to further verify the role of CXXC5in skeletal myogenesis. The expression level of CXXC5increased as C2C12elongated and fused to form myotubes and was well matched with that of the marker genes of myocyte differentiation. Overexpression of CXXC5in C2C12myoblasts promoted myocyte differentiation and myotube formation and expression level of the marker genes also increased accordingly, which were whereas remarkably inhibited by interfering the entogenous expression of CXXC5. The results indicated that CXXC5also plays an important role in the regulation of skeletal muscle development and differentiation.