Activation And Function Of TGF Signals During Early Development Of Zebrafish Embryos

Nodal and Activin, members of TGF-βsuperfamily, are mediated by the TGF-βreceptors ALK4 or ALK7, and they play important roles in mesendoderm induction and anteroposterior patterning of the neuroectoderm. It remains unclear when maternal TGF-βsignals are activated and how these signals regulate embryonic development.SB-431542 is a small compound. Studies in cultured mammalian cells have revealed that this compound is able to specifically inhibit ALK4/ALK5/ALK7 mediated TGF-βsignal transduction. This study demonstrates that it can also specifically inhibit ALK4/ALK7-mediated TGF-βsignaling in zebrafish embryos. First, embryos incubated in SB-431542 exhibit defects similar to those seen in Nodal-defective mutants. Second, the expression of Activin/TGF-βresponsive luciferase reporter, but not that of BMP-responsive luciferase, is inhibited in embryos in the presence of SB-431542. Third, induction of mesoderm gene expression by activin or sqt is blocked in embryos treated by SB-431542 while induction of the ventral ectoderm marker bmp2 is not inhibited by SB-431542. Fourth, the presence of SB-431542 inhibits gsc expression induced by overexpression of smad3a or smad3b. Fifth, injection of the constitutively active Smad2 protein rescues defects caused by SB-431542 treatment.To determine the activation time of maternal TGF-βsignals in embryos, embryos were treated with 50μM SB-431542, which started at different time points prior to the midblastula transition (before expression of zygotic genes), and were subsequently examined for morphological and molecular changes. Results shows that the earlier the treatment starts, the more the decrease of gsc expression in late blastula, the fewer the number of somites at 24 h, and the more severe the defects in axial mesoderm. This suggests that maternal TGF-βsignals are activated immediately after fertilization and such activation continues before the onset of zygotic gene expression. When embryos were incubated in SB-431542 for a short time during cleavage period, they also showed defects in axial tissues at 24 h, implying that activation of maternal TGF-βs?ignals before the midblastula transition is essential for normal development of zebrafish embryos. When embryos were treated with SB-431542 at the midblastula transition, they developed with defects at 24 h, suggesting that activated maternal TGF-β s?ignals before the midblastula transition are not sufficient for normal development and later supply of activated TGF-βsignals is also required for normal development.In addition, this study performed gene chip screening using mRNAs isolated from Nodal-defective MZoep mutants, sqt mRNA-injected embryos and wildtype embryos. A number of genes that may be regulated by Nodal signals have been identified. These genes will be useful for further studying mechanisms regulating embryonic development by Nodal signals.