Preliminary Study In The Negative Regulation Of Wnt Pathway By Rspo3 In Zebrafish

Wnt signaling pathway is one of the key signaling pathways which control cell proliferation, differentiation and morphogenesis in organism. The R-spondins (Rspos) are a recently described family of 4 secreted proteins (Rspo1-4). Reported activities for Rspo proteins include essential roles in vertebrate development. Their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both Rspo and canonical Wnt ligands could induce the activation ofβ-catenin. However, their structures and functions in teleost fish are poorly understood. Therefore, this study aims at zebrafish Rspo3, and make a preliminary study of its influence to the Wnt pathway and the function in the development of zebrafish.Luciferase reporter gene is used to find that Rspo3 in zebrafish can inhibit the Wnt signaling pathway not only when activated by the exogenous Wnt3a ligands but in the endogenous of it. And it also can restrain the expression of boz and sp5l actived by exogenous Wnt3a ligand, which is reported as the downstream target gene of Wnt pathway. And then, morpholinos are used to knock down the zebrafish Rspo3 and find that the Wnt pathway is obviously activated, resulting in the phenomenon of decreased head and eyes. Furthermore, Rspo3 gene mutants are constructed which miss the Fu domain, TB domain, TSP domain and NLS domain respectively, finding that the Fu domain of Rspo3 has a strong inhibition to Wnt pathway using the micro-injection. And most interestingly, different from the other species reported in previous studies, Rspo3 in zebrafish does not interact the structure of LRP6 at the cell surface to achieve the regulation of Wnt pathway, but by acting on theβ-catenin protein or some signaling factors upstreaming theβ-catenin or the other membrane receptors to inhibit the Wnt pathway.At this point, a different regulation mode of Wnt pathway by Rspo3 protein in zebrafish is found. As a new model organism, the zebrafish has shown a high conservation in structure and function of genes and proteins compared to other vertebrates. And has a great significance for the study of vertebrates in the growth and development, even in the construction of human disease model. So, confirming the differences between zebrafish and the higher organisms in signal transduction and other aspects will help us combine it to the growth and development of higher organisms well, and use it as an accurate and effective genetic tool more quickly, more accurately and more thoroughly.