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Abstract
Hama K, Provost E, Baranowski TC, Rubinstein AL, Anderson JL, Leach SD, Farber SA. In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters. Am J Physiol Gastrointest Liver Physiol 296: G445-G453, 2009. First published December 4, 2008; doi:10.1152/ajpgi.90513.2008.-Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.
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Abstract
Many fundamental questions remain regarding the cellular and molecular mechanisms of digestive lipid metabolism. One major impediment to answering important questions in the field has been the lack of a tractable and sufficiently complex model system. Until recently, most studies of lipid metabolism have been performed in vitro or in mice, yet each approach possesses certain limitations. The zebrafish (Danio rerio) offers an excellent model system in which to study lipid metabolism in vivo, owing to its small size, genetic tractability and optical clarity. Fluorescent lipid dyes and optical reporters of lipid-modifying enzymes are now being used in live zebrafish to generate visible readouts of digestive physiology. Here we review recent advances in visualizing intestinal lipid metabolism in live larval zebrafish.
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Abstract
Prenylation of G protein gamma (gamma) subunits is necessary for the membrane localization of heterotrimeric G proteins and for functional heterotrimeric G protein coupled receptor (GPCR) signaling. To evaluate GPCR signaling pathways during development, we injected zebrafish embryos with mRNAs encoding G gamma subunits mutated so that they can no longer be prenylated. Low-level expression of these prenylation-deficient G gamma subunits driven either ubiquitously or specifically in the primordial germ cells (PGCs) disrupts GPCR signaling and manifests as a PGC migration defect. This disruption results in a reduction of calcium accumulation in the protrusions of migrating PGCs and a failure of PGCs to directionally migrate. When co-expressed with a prenylation-deficient G gamma, 8 of the 17 wildtype G gamma isoforms individually confer the ability to restore calcium accumulation and directional migration. These results suggest that while the G gamma subunits possess the ability to interact with G Beta (beta) proteins, only a subset of wildtype G gamma proteins are stable within PGCs and can interact with key signaling components necessary for PGC migration. This in vivo study highlights the functional redundancy of these signaling components and demonstrates that prenylation-deficient G gamma subunits are an effective tool to investigate the roles of GPCR signaling events during vertebrate development. (C) 2009 Elsevier Inc. All rights reserved.
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Abstract
Research involving model organisms necessitates recording and archiving many types of animal maintenance and use data. We developed a comprehensive inventory system using FileMaker Pro (R) to incorporate, record, and archive data on zebrafish stocks, tank organization, husbandry, and fish usage. Our relational database is constructed of tables containing detailed information on fish identity, parents of origin, tank location, mutant phenotypes, caretakers, natural mating and in vitro fertilization experiments, and fish mortality. In addition to its basic annotation and reporting capabilities, the database allows barcode scan entry of several actions, for example, moving a tank of fish, mating or performing in vitro fertilization with specific fish, and recording dead fish. All data are input in real time using either barcode scanning or manual entry. The database provides several types of preformatted reports, as well as printed labels for tank location and stock identification. In summary, we have created a versatile, multipurpose inventory system that can be personalized and enhanced for any zebrafish facility and can be further adapted to organize data and archival information for other model systems or applications.
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