EZ2414h low-fat fed 6.5 dpf zebrafish larvae (low-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.

EZ1434h high-fat fed 6.5 dpf zebrafish larvae (high-fat cohort) Reads were mapped to the zebrafish genome Zv9 by Tophat2.Refseq annotation was used as known GTF.bedgrah files for visualization were generated by custom scripts.reads falling on genes were counted by custom scripts and differentially expressed genes were called by edgeR.Genome_build: Zv9Supplementary_files_format_and_content: bedgraph files for read densities along the genome (RPKM) were generated using custom scripts.

We report the transcriptionalAresponseAof the zebrafish digestive organsAto an acute high-fat feed usingARNASeqAanalysisAand highlight the changes in geneAexpressionAinvolved in the synthesis, storage, and dispersal of lipids.AThese key physiological responses to a high-fat meal allAstem fromAthe endoplasmic reticulum (ER), where lipids are formed and assignedAtoAtheir fates.

Morpholino phosphorodiamidate antisense oligonucleotides ( MOs) and short interfering RNAs ( siRNAs) are commonly used platforms to study gene function by sequence- specific knockdown. Both technologies, however, can elicit undesirable off- target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO- injected animals with well- studied mutants. We show here indistinguishable off- targeting effects for both maternal and zygotic mRNAs and for both translational and splice- site targeting MOs. The major off- targeting effect is mediated through p53 activation, as detected through the transferase- mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off- targeting. Importantly, reversal of p53- dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse- transcriptase PCR, microarrays and whole- mount in situ hybridization assays show that MO offtargeting effects are accompanied by diagnostic transcription of an N- terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off- targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off- target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence- specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off- target effects caused by other knockdown technologies.

Pharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lipid absorption using the zebrafish system. A pilot screen of an unbiased chemical library identified novel compounds that inhibited processing of fluorescent lipid analogues in live zebrafish larvae. Secondary assays identified those compounds suitable for testing in mammals and provided insight into mechanism of action, which for several compounds could be distinguished from ezetimibe, a drug used to inhibit cholesterol absorption in humans that broadly inhibited lipid absorption in zebrafish larvae. These findings support the utility of zebrafish screening assays to identify novel compounds that target complex physiological processes.

BioEYES, a nonprofit outreach program using zebrafish to excite and educate K-12 students about science and how to think and act like scientists, has been integrated into hundreds of under-resourced schools since 2002. During the week-long experiments, students raise zebrafish embryos to learn principles of development and genetics. We have analyzed 19,463 participating students' pre-and post-tests within the program to examine their learning growth and attitude changes towards science. We found that at all grade levels, BioEYES effectively increased students' content knowledge and produced favorable shifts in students' attitudes about science. These outcomes were especially pronounced in younger students. Having served over 100,000 students, we find that our method for providing student-centered experiences and developing long-term partnerships with teachers is essential for the growth and sustainability of outreach and school collaborations.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipidrich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

The zebrafish model facilitates the study of lipid metabolism and transport during development. Here, we outline methods to introduce traceable fluorescent or radiolabeled fatty acids into zebrafish embryos and larvae at various developmental stages. Labeled fatty acids can be injected into the large yolk cell prior to the development of digestive organs when the larvae is entirely dependent on the yolk for its nutrition (lecithotrophic state). Once zebrafish are able to consume exogenous food, labeled fatty acids can be incorporated into their food. Our group and others have demonstrated that the transport and processing of these injected or ingested fatty acid analogs can be followed through microscopy and/or biochemical analysis. These techniques can be easily combined with targeted antisense approaches, transgenics, or drug treatments (see Note 1), allowing studies of lipid cell biology and metabolism that are exceedingly difficult or impossible in mammals.