The liver performs several essential functions including the storage and filtration of blood, the secretion of bile, the synthesis and secretion of amino acids, and the conversions of sugars into glycogen. Despite these essential functions, little is known about the cellular and molecular mechanisms leading to the formation of the liver. We propose to use a forward genetic approach in zebrafish to investigate liver development. The gut tube ad its associated organs are not readily visible in the developing zebrafish embryo and thus have not been very accessible to morphological screens. Using a transgenic line which expresses GFP in the developing gut tube and its associated organs, the liver and pancreas, we plan to conduct a large-scale diploid screen for mutations that affect the formation of these organs. We will focus our future studies on mutations that affect the development of the liver. However, mutations that affect the formation of the pancreas and/or intestine will also be identified in our screen. We will set up a system to publish the phenotype of these mutations on the Web and make them available as soon as they are identified. In order to screen for mutations affecting the development of the liver, pancreas and intestine using this GFP transgenic line, we propose the following specific aims: 1) Characterize zebrafish liver development in detail. These studies will include further analysis of the GFP transgenic line as well as detailed examination of liver gene expression in wild-type and specific mutant backgrounds. 2) Screen for mutations that affect the formation of the liver, pancreas, and intestine. We will conduct a large- scale diploid screen using the GFP transgenic line. 3) Classify the liver mutations and begin to analyze several of them in detail. Complementation analysis, genetic mapping and molecular characterization will allow us to classify these mutations and select a few for further work. These molecular genetic studies of liver development will help provide the necessary context for further investigating the pathophysiology of malformations and malfunctions of the human liver, and may also facilitate differentiation-based strategies for treatment of a variety of diseased states.
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