Although morphology-based genetic screens have identified mutations that affect specification and patterning of zebrafish embryonic structures, few reagents have been applied to assess physiological functions in vivo. We produced fluorescent lipids that are substrates for phospholipase A2 (PLA2), an important signaling and digestive enzyme present in the cytoplasm and brush border of the intestinal epithelium. Cleavage of these lipids by PLA2 increases or shifts their wavelength of fluorescent emission, thereby revealing localized enzymatic activity in live animals. When administered to optically transparent zebrafish larvae, these lipids provide a real-time visual assay of PLA2 activity. Fluorescent lipids ingested by day 5 post-fertilization zebrafish larvae produce intense gallbladder fluorescence, a finding we have shown reflects lipid processing by intestinal PLA2 and subsequent transport through the hepatobiliary system. Since the rate and degree of gallbladder fluorescence are easily observed in zebrafish larvae, these reagents provide a sensitive readout of digestive physiology that in the context of a mutagenesis screen can identify genes that influence lipid processing. We administered fluorescent lipids to mutagenized 5-day post-fertilization zebrafish larvae and have identified one mutation that perturbs intestinal phospholipase activity. The work described in this proposal outlines our plan for a large scale mutagenesis screen using these reagents. By identifying mutations that perturb lipid metabolism, we hope to recover genes that regulate intestinal and hepatobiliary development as well as PLA2, a gene with an imprint role in inflammation, hemostasis and cell proliferation. Genetic characterization of these developmental and physiological processes has important implications for research related to congenital diseases of the intestine and liver, cancer, and cardiovascular and inflammatory diseases.
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