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.

The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. Conclusions: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins.

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach, we found that lipids labeled with the fluorescent tags, 4,4-difluoro5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene (BODIPY FL), 4,4-difluoro-5-(2-thienyl)-4-bora-3a, 4a-diaza-s-indacene [BODIPY(558/568)], and dipyrrometheneboron difluoride undecanoic acid (TopFluor) are all metabolized into varying arrays of polar and nonpolar fluorescent lipid products when they are fed to larval zebrafish. Quantitative metabolic labeling experiments performed in this system revealed significant effects of total dietary lipid composition on fluorescent lipid partitioning. We provide evidence that cholesterol metabolism in the intestine is important in determining the metabolic fates of dietary FAs. Using this method, we found that inhibitors of dietary cholesterol absorption and esterification both decreased incorporation of dietary fluorescent FAs into cholesterol esters (CEs), suggesting that CE synthesis in enterocytes is primarily responsive to the availability of dietary cholesterol.(jlr) These results are the first to comprehensively characterize fluorescent FA metabolism and to demonstrate their utility as metabolic labeling reagents, effectively coupling quantitative biochemistry with live imaging studies.

Caveolae and their structural protein caveolin 1 (CAV1) have roles in cellular lipid processing and systemic lipid metabolism. Global deletion of CAV1 in mice results in insulin resistance and increases in atherogenic plasma lipids and cholesterol, but protects from dietinduced obesity and atherosclerosis. Despite the fundamental role of the intestinal epithelia in the regulation of dietary lipid processing and metabolism, the contributions of CAV1 to lipid metabolism in this tissue have never been directly investigated. In this study the cellular dynamics of intestinal Cav1 were visualized in zebrafish and the metabolic contributions of CAV1 were determined with mice lacking CAV1 in intestinal epithelial cells (CAV1IEC-KO). Live imaging of Cav1-GFP and fluorescently labeled caveolae cargos shows localization to the basolateral and lateral enterocyte plasma membrane (PM), suggesting Cav1 mediates transport between enterocytes and the submucosa. CAV1IEC-KO mice are protected from the elevation in circulating fasted low-density lipoprotein (LDL) cholesterol associated with a high-fat diet (HFD), but have increased postprandial LDL cholesterol, total free fatty acids (FFAs), palmitoleic acid, and palmitic acid. The increase in circulating FAs in HFD CAV1IEC-KO mice is mirrored by decreased hepatic FAs, suggesting a non-cell-autonomous role for intestinal epithelial cell CAV1 in promoting hepatic FA storage. In conclusion, CAV1 regulates circulating LDL cholesterol and several FA species via the basolateral PM of enterocytes. These results point to intestinal epithelial cell CAV1 as a potential therapeutic target to lower circulating FFAs and LDL cholesterol, as high levels are associated with development of type II diabetes and cardiovascular disease.