Members of the Wnt gene family are related to the proto-oncogene Wnt-1 and the Drosophila segment polarity gene wingless. Wnts are thought to be secreted proteins acting during vertebrate embryonic development in a paracrine or autocrine fashion to activate receptors on target cells. While the functional significance, receptors, and mechanisms of Wnt-signal transduction are largely unknown, it is thought that Wnts are transiently expressed in localized tissues of embryos, and that they play an important role in intercellular communication involved in pattern formation. Supporting this, deregulation of the timing and spatial expression of the neural-specific Wnt-1 causes pattern defects in Xenopus embryos, and deletion of these genes in mouse results in midbrain and hindbrain defects. The current project will directly test the hypothesis that the Wnt gene family plays an essential role in embryonic pattern formation in vertebrates and will investigate the signal transduction pathway involved in intercellular signalling by Wnts. The zebrafish (Brachydanio rerio) will be used for these studies, as it offers genetic analysis as well as accessible early embryonic development. The specific goals follow: 1. The Wnt gene family will be cloned by amplification of Wnt sequences with degenerate oligonucleotide primers, followed by cDNA library screening to isolate the full coding sequences. 2. cDNAs from Aim 1 will be employed for RNA blot analysis and for in situ hybridization while, in parallel, antisera will be developed and employed in immunocytochemistry at the light and EM level. These studies will establish the temporal and spatial pattern of expression of each Wnt. 3. Genetic approaches will be employed to alter Wnt expression, or interfere with Wnt function. Analysis of the mutant phenotypes may reveal insights into the roles of Wnts in development. 4. The pathways by which Wnts signal spatial information between cells will be studied by examining whether deregulation of Wnt patterns of expression affects gap junctional communication and known second messenger systems and by pursuing zebrafish homologues of genes which operate in the Wnt pathway in Drosophila. Collectively, these studies will directly test the functions and mechanisms of actions of newly discovered signalling polypeptides which may, when aberrantly expressed, be involved in birth defects and transformation of cells.
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