Roles Of Smad2/3 During Early Development Of Zebrafish Embryos

Embryogenesis of vertebrates is one of the hottest fields in life science. Development of embryos is a very complicated process, in which many signaling pathways, including TGF-β, Fgf and Wnt signaling pathways, play important roles together. This study focuses on the roles of TGF-β/Nodal signals and their transducers/effectors Smad2/3 during early development of zebrafish embryos.Functional studies of smad2/3 genes in zebrafish embryos have been impeded because of the presence of three smad genes, that is, smad2, smad3a, smad3b and their maternal expression. In this study, I generated potent and specific dominant-negative forms of zebrafish Smad2, Smad3a, and Smad3b by mutating multiple amino acids. These Smad2/3 mutants are useful for investigating involvements of smad2/3 genes in development of zebrafish embryos.Although Activin, Nodal and Vg1, members of the TGF-beta superfamily, have been found to play important roles in mesendoderm induction and patterning during vertebrate embryogenesis, involvements of their downstream effectors Smad2 and Smad3 in these processes are largely remain unclear. In this study, it is demonstrated that Smad2/3 activities are absolutely required in mesendoderm induction by Nodal signals, and that interference with Smad2/3 activities inhibits development of mesendodermal tissues. Smad2/3-abrogated cells in the blastodermal margin differentiate predominantly into neural tissues instead of mesendodermal tissues. Studies in MZoep indicate that Oep-independent non-Nodal signals mediated by Smad2/3 may also play a role in mesoderm induction.Wnt, Fgf, BMP and Retinoic Acid signals have been shown to be critical for neural induction and neuroectodermal posteriorization. Roles of Nodal and Smad2/3 in neural induction and neuroectodermal patterning were studied in this thesis. It is found that overexpression of sqt, a zebrafish Nodal ligand, and smad2/3 both can promote neural induction and neuroectodermal posteriorization, while overexpression of dnsmad3b inhibits neural induction. Additionally, inhibition of Smad2/3 activities rescues the neuroectodermal posteriorization caused by enhancing Nodal, Fgf or Wnt signals. Experiments in MZoep show that Smad2/3 may mediate the functions of other Oep-independent non-Nodal signals in neural induction and neuroectodermal posteriorization.In view of the important roles of Smad2/3 in early embryonic development, it is valuable to identify their interacting factors. Through yeast two hybrid screen, this study has identified several maternal proteins that interact with Smad2/3. Studies about these factors would help to further understand functions and mechanisms of Smad2/3 in embryonic development.