Structure and function of the zebrafish embryonic patterning gene one-eyed pinhead: Implication in nodal signaling

Components in signaling pathways playing roles in germ layer formation during vertebrate embryogenesis are being identified continuously. One resource for identifying these genes is zebrafish mutants generated by mutagenesis screens. One-eyed pinhead (oep ) is a zebrafish embryonic patterning mutant with deficits in endoderm, anterior mesoderm and ventral neuroectoderm. The recently cloned oep gene belongs to the EGF-CFC family. To fully elucidate the role of oep in germ layer patterning, homozygous oep- fish were generated by microinjection of oep mRNA into oep- embryos. From the phenotypic and molecular analyses of homozygous maternal-zygotic oep - embryos (MZoep) and homozygous zygotic oep - embryos (Zoep), an essential early function of oep in mesoderm determination was revealed, which was masked in Zoep by the presence of maternal oep mRNA. The maternal oep mRNA itself was shown not to be essential for embryogenesis. The phenotype of MZoep is strikingly similar to that of the double mutants for the zebrafish nodal related genes, cyc and sqt, suggesting that oep functions in the nodal signaling pathway. To characterize the molecular role of Oep, the subcellular localization of the Oep protein was analysed by western blot analysis and immunocytochernistry. The results show that Oep is a cell surface protein. Cell surface anchoring mediated by the C-terminus is required for concentrating Oep at its functional site. EGF-CFC proteins have divergent functions in embryonic patterning. To address the structure-function relationship and specificity of Oep and other EGF-CFC proteins, mRNAs coding for Oep derivatives and heterologous EGF-CFC proteins were microinjected into oep mutants and assayed for phenotypic rescue. In contrast to previous results that suggested the EGF domain is sufficient for the activity of EGF-CFC proteins in cell culture, both the EGF and CFC domains are necessary and the EGF-CFC region is sufficient for Oep function in vivo. The structure and function analysis confirms a previous computer modeling study suggesting that the EGF domains in EGF-CFC proteins take on an EGF fold. The EGF-CFC proteins Cripto, Cryptic and Frl-1 have the similar in vivo rescuing activity as Oep, establishing that EGF-CFC proteins have a common molecular specificity mediated by the EGF-CFC region.