Genes required for lipid processing and digestive organ function in larval zebraf

Carnegie Institution Of Washington, D.C., Washington DC

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Abstract

A number of widespread diseases that impact both quality of life and longevity are influenced by dietary fat. One major impediment to improving our understanding of lipid metabolism and its related disorders is that so few metabolic studies have been carried out in live organisms. As a result, the dynamic regulatory signals that coordinate the absorption and transport of fatty acids (FA) in vivo, such as their uptake from the intestinal lumen and secretion to the rest of the body, are not fully understood. To this end, we have developed an animal model of dietary fat (triacylglycerol) metabolism that can track FAs from their site of absorption at the intestinal brush border of 6-day old zebrafish larvae, through the circulatory system, and ultimately to lipid drops within a variety of organs. While it is well known that intestinal enterocytes are the main absorptive cell that can take in large amounts of dietary lipid and export it in the form of lipoproteins to the rest of the organism, many questions remain regarding the precise cell biological processes involved. The optically clear 6-day old zebrafish larva is ideally suited to address these gaps in our understanding of vertebrate intestinal lipid absorption and processing. We have developed methods to feed larvae a high-fat diet and subsequently characterize intestinal enterocytes by electron microscopy (EM). This [unreadable]supersized[unreadable] diet produced prodigious subcellular lipid accumulations that had all the morphological features of lipid drops. While EM analysis was informative, it precluded real-time live imaging of fed larvae. To address this limitation, we included fluorescent lipids in our feeding protocol that enabled visualization of intestinal lipids in live animals. We developed microscopy techniques to visualize lipid accumulations at the whole organ level (1x objective) and at the subcellular level (63x objective). Varying the type of fluorescent lipid enabled us to elucidate two specific lipid-dependent transport processes (cholesterol vs triacylglycerol). We also found that very short chain fluorescent FAs are processed profoundly differently than medium and long chain FA in that they accumulate exclusively in the ductile networks of the liver and pancreas. The recent availability of zebrafish lines with known mutations provides a unique opportunity to functionally annotate the vertebrate genome. A recent community-wide meeting set out broad guidelines for a strategy to phenotype large numbers of zebrafish lines generated from the existing TILLING consortiums and stressed the importance of novel screening tools. We propose to screen existing mutant lines using our high-fat feeding paradigm with a variety of fluorescent reagents. The work described focuses on assays of digestive organ function that go significantly beyond previous approaches that described mutations from only the perspective of organ development and patterning. It is expected that we will identify genes that influence FA and lipoprotein metabolism, making them potential targets for future therapeutics.