Araf Regulates Early Embryonic Germ Layer Formation Via Different Signalling Pathways

Vertebrate embryos form three germ layers of different cell fates during embryogenesis. The germ layer formation involves the precise modulation of multiple regulatory factors and signalling pathways. Nodal and FGF signalling pathways play very important roles in regulating the mesendoderm induction and dorsoventral axis formation. Besides, these two signals cross-talk and regulate each other to provide a proper signal strength for embryonic development. This project aims to study the function of araf gene in regulating the germ layer formation during zebrafish early embryogenesis, and to reveal the molecular mechanism of araf regulating Nodal and FGF signals via its diffenent isoforms during this process.First, the function of full-length Araf during zebrafish embryonic germ layer formation is investigated. Knocking-down of araf promotes the mesendoderm induction and dorsal tissue formation. Genetic interaction assays show that overexpression of full-length araf mRNA could counteract the effects of ectopic Nodal signal on the marker gene expression. Besides, the inhibitory role of Araf in regulating mesendoderm induction is independent of the function of its downstream effector Erk. At the same time, a series of biochemical experiments in cultured cell lines uncover the molecular mechanism that full-length Araf regulates Nodal/Smad2 signal through directly phosphorylating the linker region of Smad2. Thus, the project identifies a new path for Raf regulation of Nodal/Smad2 signalling pathway.Next, this project deeply investigates the function of the araf ’s alternative polyadenylation isoform, araf tv2, which lacks the kinase activity domain at the C-terminus. The results indicate that the expression level of araf tv2 increases during early embryonic development. Overexpression of araf tv2 leads to developmental defects of embryos with decreased expression levels of mesodermal, endodermal and neuroectodermal makers and increased expression levels of epidermal ectoderm markers. Further studies show that Araf tv2 could directly interacts with full-length Araf without impairing its inhibitory role in regulating TGF-β/Smad2 signalling. Araf tv2 could inhibit the FGF/MAPK signalling efficiently, and counteract the activation of Erk and marker genes expression change induced by overexpression of FGF ligands in embryos.Mechanistically, Araf tv2 physically associates with Ras protein, and then competitively inhibits the interaction of Ras with Raf, ultimately interferring with the signal transduction of the Ras-Raf-Mek-Erk cascade. Overexpression of araf tv2 in embryos could counteract the activation of Erk and marker genes expression change induced by overexpression of constitutively active Ras. Finally, the inhibitory role of Araf tv2 in regulating FGF/MAPK is dependent on its binding with Ras.In summary, this project identifies a novel key gene—araf in cross-talking of Ras-Raf-Mek-Erk cascade and Nodal/Smad2 signal. araf regulates Nodal/Smad2 and FGF/MAPK signalling pathways through its different isoforms, and participates in the regulation of germ layer formation during zebrafish early embryogenesis.