Studies On The Roles Of Mpr5 And Tcap Genes In Zebrafish Cardiac Development

In the process of embryonic development and growth,the heart is the earliest internal organ,and also an important organ for maintaining individual development and growth.It is known that the cardiac development and formation are regulated by the synergistic effect of multiple gene signaling networks,and the mutation or abnormal expression of any one of these factors may lead to cardiac development defects.Abnormal human cardiac development is the cause of congenital heart disease,which has the highest fatality rate in babies born.Therefore,the studies on the function of cardiac development genes and their regulatory mechanisms will lay a theoretical foundation for the development of effective treatment for congenital heart disease.This project mainly discussed the role of the heart development candidate genes Mpr5 and Tcap in the zebrafish heart development.In situ hybridization method was used to study the spatiotemporal expression of the Mpr5 and Tcap genes during zebrafish embryonic development.The results showed that both the Mpr5 and Tcap genes were highly expressed in the heart.In the early studies in our laboratory,it has shown that Mpr5is a maternal gene,and its unmodified maternal protein has an obvious compensatory effect on the phenotype of early embryonic heart development caused by loss of Mpr5 zygotic gene expression in Mpr5knockout homozygous embryos.Therefore,in this study,a new method called Trim-Away technology for degrading target protein was used to degrade Mpr5 maternal protein in zebrafish embryos for studying its effect on embryo development.First explored the optimal injection concentration for this technique is 1.5 ng per embryo.And found that Trim-Away technology shows a clear advantage in operability.Then compared Trim-Away technology,Morpholino knockdown and CRISPR/Cas9 three schemes,the results showed that Trim-Away technology is more effective than morpholino knockdown and CRISPR/Cas9 technologies for studying the function of maternal regulatory factors in early embryo development.Next,Trim-Away technology was used to study the effect of zebrafish Mpr5 maternal protein on cardiac development.The results showed that the degradation of the Mpr5 maternal protein caused obvious developmental defects in the embryonic heart.In order to explore whether Mpr5 protein is involved in the regulation mechanism of ubiquitination as a substrate for ubiquitination,the ubiquitinated E₃ ligase acting on Mpr5protein was predicted and found to be closely related to TP53.Subsequently,Co-IP was used to discover that Mpr5 interacted with Skp1,which is the ubiquitin ligase complex.These results suggested that Mpr5protein may participate in the ubiquitin-protease system as a ubiquitination substrate to regulate zebrafish heart development.In addition,the early laboratory research indicated that Foxp4 plays a role in regulating cardiac development,and Mpr5 regulates the expression of Foxp4 and Foxp4regulates the expression of Tcap.Therefore,the hypothesis that the Mpr5-Foxp4-Tcap signaling pathway regulates cardiac development is proposed.In this study,the Chip experiment found that Mpr5 bound to the-266～-626 region upstream of the ATG of the Foxp4 promoter,indicating that Mpr5 participates in the regulation of zebrafish cardiac development by regulating the expression of Foxp4 gene.Then,the role of Tcap gene in cardiac development was explored.Firstly,p Tol2-cmlc2-IRES-EGFP plasmid was used to construct a Tcap overexpression zebrafish strain.In the F₀ generation,individuals with heart-specific expression of green fluorescent protein were screened,and it was found that the embryonic heart showed an abnormal phenotype with enlarged pericardium,suggesting that the Tcap gene might be involved in the cardiac development of zebrafish.Subsequently,the Tcap protein was degraded by the Trim-Away technology.The results found that the degradation of Tcap protein caused severe malformation of the zebrafish heart.Thus indicating that the Tcap gene controls embryonic cardiac development.At the same time,Co-IP was used to explore whether Mpr5 interacts with Tcap,and the results showed that there is no interaction between the two in the cells,which is consistent with the early prediction of the experiment.These findings indicated that Mpr5 may be located upstream of Foxp4 in the regulatory signaling pathway for zebrafish cardiac development,while Tcap may be located downstream of the signaling pathway.In summary,this project explored the role of Mpr5 and Tcap in the development of zebrafish heart by using a new technology to degrade target proteins.It also showd that Trim-Away technology is an effective new method for studying the function of proteins in early embryo development.At the same time,the molecular biology technologies were used to study the relationship amoung Mpr5,Foxp4 and Tcap.The results supported the hypothrsis that the Mpr5,Foxp4 and Tcap genes may form a signaling pathway to regulate the differentiation and development of the heart.The results of this study laid the foundation for further exploring the roles and their mechanisms of these genes in heart development.