A high-cholesterol zebrafish diet promotes hypercholesterolemia and fasting-associated liver steatosis.

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. Unlike murine models, to which various standardized high lipid diets such as a high-cholesterol diet (HCD) are available, there has yet to be a uniformly adopted zebrafish HCD protocol. In this study, we have developed an improved HCD protocol and thoroughly tested its impact on zebrafish lipid deposition and lipoprotein regulation in a dose- and time-dependent manner. The diet stability, reproducibility, and fish palatability were also validated. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LPs) and increased plasma levels of cholesterol and cholesterol esters. Feeding of the HCD to larvae for 8 days produced hepatic steatosis that became more stable and sever after 1 day of fasting and was associated with an opaque liver phenotype (dark under transmitted light). Unlike larvae, adult fish fed HCD for 14 days followed by a 3-day fast did not develop a stable fatty liver phenotype, though the fish had higher ApoB-LP levels in plasma and an upregulated lipogenesis gene fasn in adipose tissue. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease.

Serum lipoprotein-derived fatty acids regulate hypoxia-inducible factor.

Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. Whereas much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is un-known. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an independent regulator of HIF. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of the HIFα subunit of these heterodimeric transcription factors comparable with hypoxia with activation of downstream target genes. The addition of fatty acids to culture medium suppressed this signal, which required an intact mitochondrial respiratory chain. Mechanistically, fatty acids and oxygen are distinct signals integrated to control HIF activity. Finally, we observed lipid signaling to HIF and changes in target gene expression in developing zebrafish and adult mice, and this pathway operates in cancer cells from a range of tissues. This study identifies fatty acids as a physiological modulator of HIF, defining a mechanism for lipoprotein regulation that functions in parallel to oxygen.

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

Dietary cholesterol and apolipoprotein A-I are trafficked in endosomes and lysosomes in the live zebrafish intestine.

Difficulty in imaging the vertebrate intestine in vivo has hindered our ability to model nutrient and protein trafficking from both the lumenal and basolateral aspects of enterocytes. Our goal was to use live confocal imaging to increase understanding of intestinal trafficking of dietary cholesterol and apolipoprotein A-I (APOA-I), the main structural component of high-density lipoproteins. We developed a novel assay to visualize live dietary cholesterol trafficking in the zebrafish intestine by feeding TopFluor-cholesterol (TF-cholesterol), a fluorescent cholesterol analog, in a lipid-rich, chicken egg yolk feed. Quantitative microscopy of transgenic zebrafish expressing fluorescently tagged protein markers of early, recycling, and late endosomes/lysosomes provided the first evidence, to our knowledge, of cholesterol transport in the intestinal endosomal-lysosomal trafficking system. To study APOA-I dynamics, transgenic zebrafish expressing an APOA-I fluorescent fusion protein (APOA-I-mCherry) from tissue-specific promoters were created. These zebrafish demonstrated that APOA-I-mCherry derived from the intestine accumulated in the liver and vice versa. Additionally, intracellular APOA-I-mCherry localized to endosomes and lysosomes in the intestine and liver. Moreover, live imaging demonstrated that APOA-I-mCherry colocalized with dietary TF-cholesterol in enterocytes, and this colocalization increased with feeding time. This study provides a new set of tools for the study of cellular lipid biology and elucidates a key role for endosomal-lysosomal trafficking of intestinal cholesterol and APOA-I. NEW & NOTEWORTHY A fluorescent cholesterol analog was fed to live, translucent larval zebrafish to visualize intracellular cholesterol and apolipoprotein A-I (APOA-I) trafficking. With this model intestinal endosomal-lysosomal cholesterol trafficking was observed for the first time. A new APOA-I fusion protein (APOA-I-mCherry) expressed from tissue-specific promoters was secreted into the circulation and revealed that liver-derived APOA-I-mCherry accumulates in the intestine and vice versa. Intestinal, intracellular APOA-I-mCherry was observed in endosomes and lysosomes and colocalized with dietary cholesterol.

In vivo monitoring of cardiomyocyte proliferation to identify chemical modifiers of heart regeneration.

Adult mammalian cardiomyocytes have little capacity to proliferate in response to injury, a deficiency that underlies the poor regenerative ability of human hearts after myocardial infarction. By contrast, zebrafish regenerate heart muscle after trauma by inducing proliferation of spared cardiomyocytes, providing a model for identifying manipulations that block or enhance these events. Although direct genetic or chemical screens of heart regeneration in adult zebrafish present several challenges, zebrafish embryos are ideal for high-throughput screening. Here, to visualize cardiomyocyte proliferation events in live zebrafish embryos, we generated transgenic zebrafish lines that employ fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. We then performed a chemical screen and identified several small molecules that increase or reduce cardiomyocyte proliferation during heart development. These compounds act via Hedgehog, Insulin-like growth factor or Transforming growth factor ß signaling pathways. Direct examination of heart regeneration after mechanical or genetic ablation injuries indicated that these pathways are activated in regenerating cardiomyocytes and that they can be pharmacologically manipulated to inhibit or enhance cardiomyocyte proliferation during adult heart regeneration. Our findings describe a new screening system that identifies molecules and pathways with the potential to modify heart regeneration.

Heat-shock-mediated conditional regulation of hedgehog/gli signaling in zebrafish.

BACKGROUND: Hedgehog (Hh) signaling is required for embryogenesis and continues to play key roles postembryonically in many tissues, influencing growth, stem cell proliferation, and tumorigenesis. Systems for conditional regulation of Hh signaling facilitate the study of these postembryonic Hh functions. RESULTS: We used the hsp70l promoter to generated three heat-shock-inducible transgenic lines that activate Hh signaling and one line that represses Hh signaling. Heat-shock activation of these transgenes appropriately recapitulates early embryonic loss or gain of Hh function phenotypes. Hh signaling remains activated 24 hr after heat shock in the Tg(hsp70l:shha-EGFP) and Tg(hsp70l:dnPKA-BGFP) lines, while a single heat shock of the Tg(hsp70l:gli1-EGFP) or Tg(hsp70l:gli2aDR-EGFP) lines results in a 6- to 12-hr pulse of Hh signal activation or inactivation, respectively. Using both in situ hybridization and quantitative polymerase chain reaction, we show that these lines can be used to manipulate Hh signaling through larval and juvenile stages. A ptch2 promoter element was used to generate new reporter lines that allow clear visualization of Hh responding cells throughout the life cycle, including graded Hh responses in the embryonic central nervous system. CONCLUSIONS: These zebrafish transgenic lines provide important new experimental tools to study the embryonic and postembryonic roles of Hh signaling.

Pla2g12b drives expansion of triglyceride-rich lipoproteins.

Vertebrates transport hydrophobic triglycerides through the circulatory system by packaging them within amphipathic particles called Triglyceride-Rich Lipoproteins. Yet, it remains largely unknown how triglycerides are loaded onto these particles. Mutations in Phospholipase A2 group 12B (PLA2G12B) are known to disrupt lipoprotein homeostasis, but its mechanistic role in this process remains unclear. Here we report that PLA2G12B channels lipids within the lumen of the endoplasmic reticulum into nascent lipoproteins. This activity promotes efficient lipid secretion while preventing excess accumulation of intracellular lipids. We characterize the functional domains, subcellular localization, and interacting partners of PLA2G12B, demonstrating that PLA2G12B is calcium-dependent and tightly associated with the membrane of the endoplasmic reticulum. We also detect profound resistance to atherosclerosis in PLA2G12B mutant mice, suggesting an evolutionary tradeoff between triglyceride transport and cardiovascular disease risk. Here we identify PLA2G12B as a key driver of triglyceride incorporation into vertebrate lipoproteins.

A high-cholesterol zebrafish diet promotes hypercholesterolemia and fasting-associated liver triglycerides accumulation.

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. In this study, we provide an improved protocol to assay the impact of a high-cholesterol diet (HCD) on zebrafish lipid deposition and lipoprotein regulation. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LP) and increased plasma levels of cholesterol and cholesterol esters. Feeding of the HCD to larvae (8 days followed by a 1 day fast) and adult female fish (2 weeks, followed by 3 days of fasting) was also associated with a fatty liver phenotype that presented as severe hepatic steatosis. The HCD feeding paradigm doubled the levels of liver triacylglycerol (TG), which was striking because our HCD was only supplemented with cholesterol. The accumulated liver TG was unlikely due to increased de novo lipogenesis or inhibited ß-oxidation since no differentially expressed genes in these pathways were found between the livers of fish fed the HCD versus control diets. However, fasted HCD fish had significantly increased lipogenesis gene fasn in adipose tissue and higher free fatty acids (FFA) in plasma. This suggested that elevated dietary cholesterol resulted in lipid accumulation in adipocytes, which supplied more FFA during fasting, promoting hepatic steatosis. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease.

A dynamic Gli code interprets Hh signals to regulate induction, patterning, and endocrine cell specification in the zebrafish pituitary.

Hedgehog (Hh) signaling is necessary for the induction and functional patterning of the pituitary placode, however the mechanisms by which Hh signals are interpreted by placodal cells are unknown. Here we show distinct temporal requirements for Hh signaling in endocrine cell differentiation and describe a dynamic Gli transcriptional response code that interprets these Hh signals within the developing adenohypophysis. Gli1 is required for the differentiation of selected endocrine cell types and acts as the major activator of Hh-mediated pituitary induction, while Gli2a and Gli2b contribute more minor activator functions. Intriguingly, this Gli response code changes as development proceeds. Gli1 continues to be required for the activation of the Hh response anteriorly in the pars distalis. In contrast, Gli2b is required to repress Hh target gene expression posteriorly in the pars intermedia. Consistent with these changing roles, gli1, gli2a, and gli2b, but not gli3, are expressed in pituitary precursor cells at the anterior neural ridge. Later in development, gli1 expression is maintained throughout the adenohypophysis while gli2a and gli2b expression are restricted to the pars intermedia. Given the link between Hh signaling and pituitary adenomas in humans, our data suggest misregulation of Gli function may contribute to these common pituitary tumors.

Hedgehog Signaling Regulates Neurogenesis in the Larval and Adult Zebrafish Hypothalamus.

Neurogenesis is now known to play a role in adult hypothalamic function, yet the cell-cell mechanisms regulating this neurogenesis remain poorly understood. Here, we show that Hedgehog (Hh)/Gli signaling positively regulates hypothalamic neurogenesis in both larval and adult zebrafish and is necessary and sufficient for normal hypothalamic proliferation rates. Hh-responsive radial glia represent a relatively highly proliferative precursor population that gives rise to dopaminergic, serotonergic, and GABAergic neurons. In situ and transgenic reporter analyses revealed substantial heterogeneity in cell-cell signaling within the hypothalamic niche, with slow cycling Nestin-expressing cells residing among distinct and overlapping populations of Sonic Hh (Shh)-expressing, Hh-responsive, Notch-responsive, and Wnt-responsive radial glia. This work shows for the first time that Hh/Gli signaling is a key component of the complex cell-cell signaling environment that regulates hypothalamic neurogenesis throughout life.

A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish.

BACKGROUND: Tissue heating has been employed to study a variety of biological processes, including the study of genes that control embryonic development. Conditional regulation of gene expression is a particularly powerful approach for understanding gene function. One popular method for mis-expressing a gene of interest employs heat-inducible heat shock protein (hsp) promoters. Global heat shock of hsp-promoter-containing transgenic animals induces gene expression throughout all tissues, but does not allow for spatial control. Local heating allows for spatial control of hsp-promoter-driven transgenes, but methods for local heating are cumbersome and variably effective. RESULTS: We describe a simple, highly controllable, and versatile apparatus for heating biological tissue and other materials on the micron-scale. This microheater employs micron-scale fiber optics and uses an inexpensive laser-pointer as a power source. Optical fibers can be pulled on a standard electrode puller to produce tips of varying sizes that can then be used to reliably heat 20-100 mum targets. We demonstrate precise spatiotemporal control of hsp70l:GFP transgene expression in a variety of tissue types in zebrafish embryos and larvae. We also show how this system can be employed as part of a new method for lineage tracing that would greatly facilitate the study of organogenesis and tissue regulation at any time in the life cycle. CONCLUSION: This versatile and simple local heater has broad utility for the study of gene function and for lineage tracing. This system could be used to control hsp-driven gene expression in any organism simply by bringing the fiber optic tip in contact with the tissue of interest. Beyond these uses for the study of gene function, this device has wide-ranging utility in materials science and could easily be adapted for therapeutic purposes in humans.

Expression profiling identifies novel Hh/Gli-regulated genes in developing zebrafish embryos.

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Misregulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants that affect different components of the Hh/Gli signaling system have been identified. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified known (e.g., ptc1 and nkx2.2a) and novel Hh-regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue-specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli-regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of misregulation during tumorigenesis.

Intestinal epithelial cell caveolin 1 regulates fatty acid and lipoprotein cholesterol plasma levels.

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 diet-induced 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.

Embryonic exposure to excess thyroid hormone causes thyrotrope cell death.

Central congenital hypothyroidism (CCH) is more prevalent in children born to women with hyperthyroidism during pregnancy, suggesting a role for thyroid hormone (TH) in the development of central thyroid regulation. Using the zebrafish embryo as a model for thyroid axis development, we have characterized the ontogeny of negative feedback regulation of thyrotrope function and examined the effect of excess TH on thyrotrope development. We found that thyroid-stimulating hormone ß subunit (tshb) and type 2 deiodinase (dio2) are coexpressed in zebrafish thyrotropes by 48 hours after fertilization and that TH-driven negative feedback regulation of tshb transcription appears in the thyroid axis by 96 hours after fertilization. Negative feedback regulation correlated with increased systemic TH levels from the developing thyroid follicles. We used a transgenic zebrafish that expresses GFP under the control of the tshb promoter to follow thyrotrope fates in vivo. Time-lapse imaging revealed that early exposure to elevated TH leads to thyrotrope cell death. Thyrotrope numbers slowly recovered following the removal of excess TH. These data demonstrate that transient TH exposure profoundly impacts the thyrotrope population during a critical period of pituitary development and may have long-term implications for the functional reserve of thyroid-stimulating hormone (TSH) production and the TSH set point later in life.

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells.

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by (59)Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.