The zebrafish fat-free (ffr) mutation was identified in a physiological screen for genes that regulate lipid metabolism. ffr mutant larvae are morphologically indistinguishable from wild-type sibling larvae, but their absorption of fluorescent lipids is severely impaired. Through positional cloning, we have identified a causative mutation in a highly conserved and ubiquitously expressed gene within the ffr locus. The Ffr protein contains a Dor-1 like domain typical of oligomeric Golgi complex (COG) gene, cog8. Golgi complex ultrastructure is disrupted in the ffr digestive tract. Consistent with a possible role in COG-mediated Golgi function, wild-type Ffr-GFP and COG8-mRFIP fusion proteins partially colocalize in zebrafish blastomeres. Enterocyte retention of an endosomal lipid marker in ffr larvae support the idea that altered vesicle trafficking contributes to the ffr mutant defect. These data indicate that ffr is required for both Golgi structure and vesicular trafficking, and ultimately lipid transport.

Background. Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown. Results. We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies. Conclusion(s). This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.

Using a spotted 65-mer oligonucleotide microarray, we have characterized the developmental expression profile from mid-gastrulation (75% epiboly) to 5 days post-fertilization (dpf) for >16,000 unique transcripts in the zebrafish genome. Microarray profiling data sets are often immense, and one challenge is validating the results and prioritizing genes for further study. The purpose of the current study was to address such issues, as well as to generate a publicly available resource for investigators to examine the developmental expression profile of any of the over 16,000 zebrafish genes on the array. On the chips, there are 16,459 printed spots corresponding to 16,288 unique transcripts and 172 beta-actin (AF025305) spots spatially distributed throughout the chip as a positive control. We have collected 55 microarray gene expression profiling results from various zebrafish laboratories and created a Per1/CGI-based software tool (http:Hserine.umdnj.edu/similar to ouyangmi/cgi-bin/zebrafish/profile.htm) for researchers to look for the expression patterns of their gene of interest. Users can search for their genes of interest by entering the accession numbers or the nucleotide sequences and the expression profiling will be reported in the form of expression intensities versus time-course graphical displays. In order to validate this web tool, we compared 74 genes' expression results between our web tool and the in situ hybridization results from Thisse et al. [Thisse, B., Heyer, V., Lux, A., Alunni, A., Degrave, A., Seiliez, L, Kirchner, J., Parkhill, J.-P., Thisse, C., 2004. Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Meth. Cell. Biol. 77, 505-519] as well as those reported by Mathavan et al. [Mathavan, S., Lee, S.G., mark, A., Miller, L.D., Murthy, K.R., Tong, Y., Wu, Y.L., Lam, S.H., Yang, H., Ruan, Y., Korzh, V., Gong, Z., Liu, E.T., Lufkin, T., 2005. Transcriptome analysis of zebrafish embryogenesis using microarrays. PLoS Genet. 1, 260-276]. The comparison indicates that our microarray-derived expression patterns are 80% and 75% in agreement with the in situ database (Thisse et al., 2004) and previously published microarray data (Mathavan et al., 2005), respectively. Those genes that conflict between our web tool and the in situ database either have high sequence similarity with other genes or the in situ probes are not reliable. Among those genes that disagree between our web tool and those reported by Mathavan et al. (2005), 93% of the genes are in agreement between our web tool and the in situ database, indicating our web tool results are quite reliable. Thus, this resource provides a user-friendly web based platform for researchers to determine the developmental profile of their gene of interest and to prioritize genes identified in microarray analyses by their developmental expression profile. (c) 2007 Elsevier B.V. All rights reserved.

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.

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.

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.