Endoderm forms the epithelial component of the vertebrate digestive tract, and embryological studies in amphibians suggest that maternal determinants regulate endodermal fate. The long-term objective of the proposed research is to define the molecular mechanisms that govern the establishment and patterning of embryonic endoderm, processes that underlie the development of the functional digestive tract. The identification and analysis of maternal inducers regulating endodermal fate is our primary goal. Results indicate that Vg1, a maternal TGFbeta-related factor, can specify endodermal fate, consistent with its localization to prospective endoderm. The following hypothesis will be tested: Vg1 regulates formation of embryonic endoderm and initiates a series of inductions resulting in the precise spatial organization of the vertebrate digestive tract. To test this hypothesis, dominant inhibitory mutants of the Vg1signaling pathway will be used to perturb endogenous Vg1 signaling. Two approaches will be used: 1) Mutagenesis of conserved residues in the Vg1 mature domain will generate dominant inhibitory ligands; and 2) A Vg1 specific receptor will be isolated using a defined Vg1 responsive transcriptional element in a functional cloning strategy, and dominant inhibitory and activated mutants will be prepared. Expression of mutant signaling molecules in embryos and explants of prospective endoderm will permit a direct analysis of the role of Vg1 in the development of embryonic endoderm. A functional screen will identify additional maternal regulatory genes that specify and pattern endoderm, including those acting in response to Vg1. Concurrently, cloned members of the TGFbeta, FGF, wnt and hedgehog family will be examined for regulation of endodermal development. In all, the experiments will elucidate the molecular pathways that specify endoderm and establish regional pattern in the primitive gut, processes crucial for the future organization of the functional digestive tract. The development of vertebrate mesodermal and neural tissues has been described in great detail; a similar description of early endodermal development is now possible. The experiments will provide an embryological and molecular foundation for a more complete understanding of digestive tract biology and may elucidate the basis of gut malformation and dysfuntion.
