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.

Specific small molecule inhibitors of the de novo cholesterol synthesis pathway (statins) and the protein prenylation pathway were used to study their effect on germ cell migration. Hydroxymethylglutaryl-Coenzyme A reductase (HMGCoAR) catalyzes the rate-limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. Pharmacological HMGCoAR inhibition by statins alters zebrafish development and germ cell migration. These effects were completely blocked by prior injection of mevalonate, the product of HMGCoAR activity, or the prenylation precursors farnesol and geranylgeraniol. Finally, pharmacological inhibition of geranylgeranyl transferase I activity, an enzyme downstream from mevalonate synthesis and responsible for the transfer of a lipid to target proteins, resulted in abnormal germ cell migration. These data together with new data from Drosophila demonstrate that protein prenylation is an evolutionarily conserved pathway mediating germ cell migration. Further, pharmacological block-and-rescue approach provided detailed information about the elements of isoprenoid biosynthesis that contribute to germ cell migration. A key question raised by this work is the identity of the prenylated protein which facilitates proper germ cell migration. Work from other laboratories suggests that germ cell migration might be a general model for the long-range migration of other cell types including cancer metastasis.

Lipids serve essential functions in cells as signaling molecules, membrane components, and sources of energy. Defects in lipid metabolism are implicated in a number of pandemic human diseases, including diabetes, obesity, and hypercholesterolemia. Many aspects of how fatty acids and cholesterol are absorbed and processed by intestinal cells remain unclear and present a hurdle to developing approaches for disease prevention and treatment. Numerous studies have shown that the zebrafish is an excellent model for vertebrate lipid metabolism. In this chapter, we review studies that employ zebrafish to better understand lipid signaling and metabolism.

Heterotrimeric G protein signaling is involved in many pathways essential to development including those controlling cell migration, proliferation, differentiation and apoptosis. One key developmental event known to rely on proper heterotrimeric G protein signaling is primordial germ cell (PGC) migration. We previously developed an in vivo PGC migration assay that identified differences in the signaling capacity of G protein gamma subunits. In this study we developed G gamma subunit chimeras to determine the regions of G gamma isoforms that are responsible for these differences. The central section of the G gamma subunit was found to be necessary for the ability of a G gamma subunit to mediate signaling involved in PGC migration. Residues found in the carboxyterminal segment of G gamma transducin (gngt1) were found to be responsible for the ability of this subunit to disrupt PGC migration. The type of prenylation did not affect the ability of a G gamma subunit to reverse prenylation-deficient-G gamma-induced PGC migration defects. However, a version of gng2. engineered to be farnesylated instead of geranylgeranylated. still lacks the ability to reverse PGC migration defects known to result from treatment of zebrafish with geranylgeranyl transferase inhibitors (GGTI), supporting the notion that G gamma subunits are one of several protein targets that need to be geranylgeranylated to orchestrate the proper long-range migration of PGCs. (C) 2011 Elsevier Inc. All rights reserved.

Lipids are essential for cellular function as sources of fuel, critical signaling molecules and membrane components. Deficiencies in lipid processing and transport underlie many metabolic diseases. To better understand metabolic function as it relates to disease etiology, a whole animal approach is advantageous, one in which multiple organs and cell types can be assessed simultaneously in vivo. Towards this end, we have developed an assay to visualize fatty acid (FA) metabolism in larval zebrafish (Danio rerio). The method utilizes egg yolk liposomes to deliver different chain length FA analogs (BODIPY-FL) to six day-old larvae. Following liposome incubation, larvae accumulate the analogs throughout their digestive organs, providing a comprehensive readout of organ structure and physiology. Using this assay we have observed that different chain length FAs are differentially transported and metabolized by the larval digestive system. We show that this assay can also reveal structural and metabolic defects in digestive mutants. Because this labeling technique can be used to investigate digestive organ morphology and function, we foresee its application in diverse studies of organ development and physiology. (C) 2011 Elsevier Inc. All rights reserved.

The challenge of studying complex protein networks in whole animals has driven the development of new methods for manipulating protein function with spatial and temporal precision. A novel combination of chemical and genetic protein regulation (Rodriguez and Wolfgang, in this issue of Chemistry & Biology) achieves levels of control that will revolutionize the study of protein function.