One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

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 HIFalpha 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.

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 HIF alpha 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.

Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real time from the subcellular to the whole organism level. Fluorescently tagging the lipid droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models.

Background: The ability of animals and their microbiomes to adapt to starvation and then restore homeostasis after refeeding is fundamental to their continued survival and symbiosis. The intestine is the primary site of nutrient absorption and microbiome interaction, however our understanding of intestinal adaptations to starvation and refeeding remains limited. Here we used RNA sequencing and 16S rRNA gene sequencing to uncover changes in the intestinal transcriptome and microbiome of zebrafish subjected to long-term starvation and refeeding compared to continuously fed controls.

This study examined the feasibility, acceptability, and efficacy of a video game-based digital therapeutic combining applied behavior analysis techniques and gaze-contingent eye tracking to target emotion recognition in youth with autism spectrum disorder (ASD). Children aged 4-14 years with ASD were randomized to complete Lookware (TM) (n = 25) or a control video game (n = 29). Results from a 2 x 2 mixed ANOVA revealed that children in the intervention condition demonstrated significant improvements in emotion recognition from pre- to post-intervention compared to children in the control condition, F(1,52) = 17.48, p < 0.001. Children and staff perceived high feasibility and acceptability of Lookware (TM). Study results demonstrated the feasibility, acceptability, and preliminary efficacy of Lookware (TM).

Interactions between the dinoflagellate endosymbiont Symbiodinium and its cnidarian hosts (e.g. corals, sea anemones) are the foundation of coral-reef ecosystems. Carbon flow between the partners is a hallmark of this mutualism, but the mechanisms governing this flow and its impact on symbiosis remain poorly understood. We showed previously that although Symbiodinium strain SSB01 can grow photoautotrophically, it can grow mixotrophically or heterotrophically when supplied with Glc, a metabolite normally transferred from the alga to its host. Here we show that Glc supplementation of SSB01 cultures causes a loss of pigmentation and photosynthetic activity, disorganization of thylakoid membranes, accumulation of lipid bodies, and alterations of cell-surface morphology. We used global transcriptome analyses to determine if these physiological changes were correlated with changes in gene expression. Glc-supplemented cells exhibited a marked reduction in levels of plastid transcripts encoding photosynthetic proteins, although most nuclear-encoded transcripts (including those for proteins involved in lipid synthesis and formation of the extracellular matrix) exhibited little change in their abundances. However, the altered carbon metabolism in Glc-supplemented cells was correlated with modest alterations (approximately 2x) in the levels of some nuclear-encoded transcripts for sugar transporters. Finally, Glc-bleached SSB01 cells appeared unable to efficiently populate anemone larvae. Together, these results suggest links between energy metabolism and cellular physiology, morphology, and symbiotic interactions. However, the results also show that in contrast to many other organisms, Symbiodinium can undergo dramatic physiological changes that are not reflected by major changes in the abundances of nuclear-encoded transcripts and thus presumably reflect posttranscriptional regulatory processes.

Reef-building corals are keystone species that are threatened by anthropogenic stresses including climate change. To investigate corals' responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating many hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals or closely related cnidarians. CRISPR technology seems likely to alleviate this problem. Indeed, we show here that microinjection of single-guide RNA/Cas9 ribonucleoprotein complexes into fertilized eggs of the coral Acropora millepora can produce a sufficiently high frequency of mutations to detect a clear phenotype in the injected generation. Based in part on experiments in a sea-anemone model system, we targeted the gene encoding Heat Shock Transcription Factor 1 (HSF1) and obtained larvae in which >90% of the gene copies were mutant. The mutant larvae survived well at 27 degrees C but died rapidly at 34 degrees C, a temperature that did not produce detectable mortality over the duration of the experiment in wild-type (WT) larvae or larvae injected with Cas9 alone. We conclude that HSF1 function (presumably its induction of genes in response to heat stress) plays an important protective role in corals. More broadly, we conclude that CRISPR mutagenesis in corals should allow wide-ranging and rigorous tests of gene function in both larval and adult corals.

Loss of endosymbiotic algae ("bleaching") under heat stress has become a major problem for reef-building corals worldwide. To identify genes that might be involved in triggering or executing bleaching, or in protecting corals from it, we used RNAseq to an-alyze gene-expression changes during heat stress in a coral relative, the sea anemone Aiptasia. We identified >500 genes that showed rapid and extensive up-regulation upon temperature increase. These genes fell into two clusters. In both clusters, most genes showed similar expression patterns in symbiotic and aposymbiotic anemones, suggesting that this early stress response is largely independent of the symbiosis. Cluster I was highly enriched for genes involved in innate immunity and apoptosis, and most transcript levels returned to baseline many hours before bleaching was first detected, raising doubts about their possible roles in this process. Cluster II was highly enriched for genes involved in protein folding, and most transcript levels returned more slowly to baseline, so that roles in either promoting or preventing bleaching seem plausible. Many of the genes in clusters I and II appear to be targets of the transcription factors NF kappa B and HSF1, respectively. We also examined the behavior of 337 genes whose much higher levels of expression in symbiotic than aposymbiotic anemones in the absence of stress suggest that they are important for the symbiosis. Unexpectedly, in many cases, these expression levels declined precipitously long before bleaching itself was evident, suggesting that loss of expression of symbiosis-supporting genes may be involved in triggering bleaching.

Understanding the response of the coral holobiont to environmental change is crucial to inform conservation efforts. The most pressing problem is "coral bleaching," usually precipitated by prolonged thermal stress. We used untargeted, polar metabolite profiling to investigate the physiological response of the coral species Montipora capitata and Pocillopora acuta to heat stress. Our goal was to identify diagnostic markers present early in the bleaching response. From the untargeted UHPLC-MS data, a variety of co-regulated dipeptides were found that have the highest differential accumulation in both species. The structures of four dipeptides were determined and showed differential accumulation in symbiotic and aposymbiotic (alga-free) populations of the sea anemone Aiptasia (Exaiptasia pallida), suggesting the deep evolutionary origins of these dipeptides and their involvement in symbiosis. These and other metabolites may be used as diagnostic markers for thermal stress in wild coral.