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 teleostspecific 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 non-sense-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.

The developing zebrafish is a well-established model system for studies of energy metabolism, and is amenable to genetic, physiological, and biochemical approaches. For the first 5 days of life, nutrients are absorbed from its endogenous maternally deposited yolk. At 5 days post-fertilization, the yolk is exhausted and the larva has a functional digestive system including intestine, liver, gallbladder, pancreas, and intestinal microbiota. The transparency of the larval zebrafish, and the genetic and physiological similarity of its digestive system to that of mammals make it a promising system in which to address questions of energy homeostasis relevant to human health. For example, apolipoprotein expression and function is similar in zebrafish and mammals, and transgenic animals may be used to examine both the transport of lipid from yolk to body in the embryo, and the trafficking of dietary lipids in the larva. Additionally, despite the identification of many fatty acid and lipid transport proteins expressed by vertebrates, the cell biological processes that mediate the transport of dietary lipids from the intestinal lumen to the interior of enterocytes remain to be elucidated. Genetic tractability and amenability to live imaging and a range of biochemical methods make the larval zebrafish an ideal model in which to address open questions in the field of lipid transport, energy homeostasis, and nutrient metabolism.

The zebrafish larva is a powerful tool for the study of dietary triglyceride (TG) digestion and how fatty acids (FA) derived from dietary lipids are absorbed, metabolized and distributed to the body. While fluorescent FA analogues have enabled visualization of FA metabolism, methods for specifically assaying TG digestion are badly needed. Here we present a novel High Performance Liquid Chromatography (HPLC) method that quantitatively differentiates TG and phospholipid (PL) molecules with one or two fluorescent FA analogues. We show how this tool may be used to discriminate between undigested and digested TG or phosphatidylcholine (PC), and also the products of TG or PC that have been digested, absorbed and re-synthesized into new lipid molecules. Using this approach, we explored the dietary requirement of zebrafish larvae for phospholipids. Here we demonstrate that dietary TG is digested and absorbed in the intestinal epithelium, but without dietary PC, TG accumulates and is not transported out of the enterocytes. Consequently, intestinal ER stress increases and the ingested lipid is not available support the energy and metabolic needs of other tissues. In TG diets with PC, TG is readily transported from the intestine and subsequently metabolized.

EZ2111h 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.

EZ203Unfed 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.

EZ101Unfed 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.

Responding to a high-fat meal requires an interplay between multiple digestive tissues, sympathetic response pathways, and the gut microbiome. The epithelial enterocytes of the intestine are responsible for absorbing dietary nutrients and preparing them for circulation to distal tissues, which requires significant changes in cellular activity, including both morphological and transcriptional responses. Following a high-fat meal, we observe morphological changes in the enterocytes of larval zebrafish, including elongation of mitochondria, formation and expansion of lipid droplets, and the rapid and transient ruffling of the nuclear periphery. Dietary and pharmacological manipulation of zebrafish larvae demonstrated that these subcellular changes are specific to triglyceride absorption. The transcriptional changes that occur simultaneously with these morphological changes were determined using RNA sequencing, revealing a cohort of up-regulated genes associated with lipid droplet formation and lipid transport via lipoprotein particles. Using a microsomal triglyceride transfer protein (MTP) inhibitor to block -lipoprotein particle formation, we demonstrate that the transcriptional response to a high-fat meal is associated with the transfer of ER triglyceride to nascent -lipoproteins, possibly through the activation of Creb3l3/cyclic AMP-responsive element-binding protein. These data suggest that a transient increase in ER lipids is the likely mediator of the initial physiological response of intestinal enterocytes to dietary lipid.

EZ2424h 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.