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.

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.

The small intestine is the primary site of dietary lipid absorption in mammals. The balance of nutrients, microorganisms, bile, and mucus that determine intestinal luminal environment cannot be recapitulated ex vivo, thus complicating studies of lipid absorption. We show that fluorescently labeled lipids can be used to visualize and study lipid absorption in live zebrafish larvae. We demonstrate that the addition of a BODIPY-fatty acid to a diet high in atherogenic lipids enables imaging of enterocyte lipid droplet dynamics in real time. We find that a lipid-rich meal promotes BODIPY-cholesterol absorption into an endosomal compartment distinguishable from lipid droplets. We also show that dietary fatty acids promote intestinal cholesterol absorption by rapid re-localization of NPC1L1 to the intestinal brush border. These data illustrate the power of the zebrafish system to address longstanding questions in vertebrate digestive physiology.

Challenges in imaging lipid-processing events in live, intact vertebrate models have historically led to reliance on cultured cell studies, thus hampering our understanding of lipid metabolism and gastrointestinal physiology. Fluorescently-labeled molecules, such as BODIPY-labeled lipids, can reveal lipid-processing events in live zebrafish (Danio rerio) and has expanded our understanding of digestive physiology. This review will cover recent advances from the past two to three years in the use of fluorescence-based imaging techniques in live zebrafish to characterize gastrointestinal physiology in health and disease and to conduct small molecule screens to discover therapeutic compounds.