The long-term goals of the proposed research are to identify the genes involved in the establishment of the dorsal-ventral axis and determine the precise molecular mechanisms by which they function to specify the different cell types generated along the dorsal-ventral axis of the vertebrate embryo. An extensive screen for mutations affecting morphogenesis in the zebrafish resulted in the identification of 6 genes with key functions in the establishment of the dorsal/ventral axis. These are among the first mutations affecting dorsal-ventral patterning in a vertebrate model system, and they provide a unique opportunity to study the mechanism of this important developmental process. Cell transplantation experiments between wild-type and mutant embryos will determine: (1) which of the 6 genes function as a signaling molecule or in the generation of a signal; (2) whether the cellular function of the gene is in the establishment within a cell of its identity; and (3) the functional domains of each gene within the embryo. Ectopic expression of Xenopus ventralizing genes in the zebrafish dorsalized mutant embryos will determine (4) the order in which the genes function in the establishment of the dorsal-ventral axis and provide a molecular framework for the pathway. Three different approaches will be utilized to identify the molecular nature of the dorsalized genes: a candidate gene approach, expression cloning methodologies, and establishment of the foundation for a positional cloning approach. With the cloned genes in hand, numerous direct tests of function will be performed depending on the biochemical properties expected of the particular dorsalized gene and the functional properties established in the studies proposed here. Mutations in these genes in humans are likely to result in miscarriage and also birth defects. The understanding of the mechanisms by which these dorsal-ventral genes function in normal development is likely to have direct implications in the understanding of the progression of cancer and molecular mechanisms of human inherited disorders.
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