wnt Signaling in Liver Development and Disease

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DESCRIPTION (provided by applicant): Liver disease is a common cause for morbidity and mortality in the U.S., and understanding the critical pathways during liver development is essential for our knowledge of normal liver function and disease. The zebrafish is an excellent model to study early vertebrate development because of its translucency during development and the ability to produce large numbers of offspring. Our laboratory has made significant contributions by identifying genes important in zebrafish development using the advantage of forward genetics, and recently characterized several cancer-prone mutants in a genetic screen. Recent reports have demonstrated the feasibility of the zebrafish system for the study of liver development. Liver morphogenesis has been described by functional genomics approaches and use of fluorescent transgenic fish lines. Several studies suggest that the wnt pathway, especially beta-catenin, is of great importance for liver growth, hepatocyte and biliary differentiation and neoplasia. To test this hypothesis, in Specific Aim 1 transgenic zebrafish will be created overexpressing stabilized beta-catenin fused with green fluorescent protein under the control of the liver specific promoter of liver fatty acid binding protein. These fish will be examined using histology, immunohistochemistry and in situ hybridization to analyze the expression patterns of liver-specific genes. The fluorescent labeling allows for hepatocyte isolation by FACS and subsequent microarray analysis in comparison to wild-type fish. Other transgenic strains, expressing inhibitors of the wnt pathway under a heatshock promoter, and small molecule inhibitors of the wnt pathway will be studied for the effects of decreased wnt signaling for liver development. Specific Aim 2 will determine the importance of wnt signaling in liver neoplasia by using the transgenic beta-catenin fish and other transgenic fish expressing the effectors of the wnt pathway, cyclin D1 and myc. Resulting tumors will be detected using advanced multispectral fluorescence imaging technology and ultrasound, and analyzed by histology, cytogenetics, and microarray analysis. Fish carrying mutant genes of the tumor suppressors p53 and APC will be mated to transgenic fish to study synergistic effects with the wnt pathway. These studies will provide new insight into the role of the wnt signaling pathway during liver development and neoplasia, and may identify new therapeutic targets for the treatment of liver disease.