VISUALIZATION OF LIPID METABOLISM AND SIGNALING IN THE ZEBRAFISH

Work in my laboratory utilizes the zebrafish, to visualize biochemical processes in living embryos by exploiting their accessibility and optical clarity. Specifically, the laboratory has focused on studying lipid modifying and transport processes in the developing embryo. Lipids are known to play a role in a host of physiological processes, most notably inflammation, and in cancer.  Despite these findings, little is known about their role in cell signaling events important during embryonic development. A novel aspect of this work is that it examines the regulation of lipid metabolism in vivo by feeding embryos fluorescent lipids and visualizing their uptake, and processing using time-lapse microscopy. We couple these tools with mutagenesis screening to identify genes that regulate lipid metabolism, germ cell migration, intestinal and liver transport, and development of the biliary tract.  Identification of these genes has important implications for cancer research and research related to diseases of the liver, intestine and cardiovascular system.

Major Research Efforts:

1. Modulators of lipid metabolism in vivo: Real-time imaging of intestinal absorption

The uptake and efflux of sterol is tightly regulated at the cellular and organismal level by multiple mechanisms. While it is well established that the intestine is the primary site of dietary cholesterol absorption, the mechanisms involved in the vectorial transport of cholesterol across the intestinal epithelium are poorly understood. A better understanding of these mechanisms of sterol transport may have direct translational impact on developing treatment strategies for addressing diet-induced obesity, diabetes and cardiovascular disease. A major effort in the lab is the visualization of fluorescent fusion proteins together with lipid dyes in single cells of the zebrafish intestine.

A live zebrafish intestine after swallowing of an endosomal dye revealing the brush boarder, goblet cells, and enterocytes.

2. Forward genetic screening

Dietary fat consumption is thought to be one of the risk factors to develop cardiovascular diseases, diabetes and obesity.  Significant effort has focused on how dietary lipids are assimilated in the gastrointestinal tract, however, many of basic mechanisms remain unclear.  Recently, numerous studies have shown that bile metabolism is regulated by dietary lipids through nuclear receptors  however, bile homeostasis is not yet fully understood. Genetic analysis in zebrafish is a powerful tool for identifying genes that direct vertebrate development.  Screening methods that employ fluorescent lipids as optical biosensors of specific biochemical processes can identify mutations that perturb lipid metabolism.  By applying these reagents to ethylnitrosurea(ENU) -mutagenized zebrafish larvae, we have identified a number of mutants.  One mutant, fat-free, whose digestive system appears morphologically normal yet has impaired digestive lipid processing is the focus of our most intensive efforts (Science, 2001, 292:1385-1388).We eventually succeeded in the positional cloning of the fat-free locus to find that it encodes a hypothetical protein that has no known function. New data indicates that the fat-free protein is involved in the endoplasmic reticulum - Golgi trafficking.

3. A reverse genetic screen with guts

The vertebrate genome contains a predicted 30,000 genes many of which with unknown function. The recent development of morpholino-based gene knockdown technology in zebrafish has opened the door to the genome-wide assignment of function based on sequence in a model vertebrate. This effort explores the molecular mechanisms that control fundamental vertebrate embryonic processes of patterning and organ formation, biological problems in which cell-cell communication is critical during development. My lab is part of a multicenter consortium  to systematically assign the biological function to a set of 100-200 putative secreted proteins by injecting morpholinos targeted to these genes.

4. The role of HMG-CoA reductase activity in regulating germ cell migration

Hydroxymethylglutaryl-Coenzyme A (HMG-CoA) reductase is the rate-limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. In Drosophila melanogaster, reduced HMG-CoA reductase activity results in germ cell migration defects as exemplified by the columbus mutation. Work in my lab has demonstrated that pharmacological HMG-CoA reductase inhibition by statins alters zebrafish development and germ cell migration. Our data demonstrates that protein lipidation by geranylgeranyl transferase (GGT) is an evolutionarily conserved pathway mediating vertebrate germ cell migration and might also be a model for long range cell migration as seen in many cancers.  We are now focused on identifying the target of GGT that mediates this migratory behavior.

The migration of germ cells are imaged in live animals

5. Science Outreach Program

Together with Ms Jamie Shuda, I have created a Science Outreach Program, Project BioEYES, that incorporates life science and laboratory education using zebrafish. The outreach program has two main components: educating students and community members through hands-on tours of a Zebrafish Facility, and bringing the zebrafish to 4-12th grade classrooms for hands-on experiments. The program teaches students about science literacy, genetics, the experimental process and the cardiovascular system through the use of live zebrafish.

The mission of the Science Outreach Program is to foster an enthusiasm for science education, promote interest for future participation in a biology-related field, and allow all students the opportunity to learn life science through a hands-on, student-centered approach to instruction. The goals of the program encompass educating the community about life science and incorporating live research as the primary teaching tool. We provide our services to all students and teachers regardless of community, poverty or race. Project BioEYES is currently serving children in Philadelphia, Baltimore, Lehigh and Notre Dame public and private school districts.

This program has garnered significant excitement among primary school faculty in the region and the scientific press. Since we began, over 10,000 students have participated in our in-class zebrafish unit. (Schaefer, J., and Farber, S. A. (2004) PLoS Biol.)