Copy number variants (CNVs) represent a substantial source of genomic variation in vertebrates and have been associated with numerous human diseases. Despite this, the extent of CNVs in the zebrafish, an important model for human disease, remains unknown. Using 80 zebrafish genomes, representing three commonly used laboratory strains and one native population, we constructed a genome-wide, high-resolution CNV map for the zebrafish comprising 6,080 CNV elements and encompassing 14.6% of the zebrafish reference genome. This amount of copy number variation is four times that previously observed in other vertebrates, including humans. Moreover, 69% of the CNV elements exhibited strain specificity, with the highest number observed for Tubingen. This variation likely arose, in part, from Tubingen's large founding size and composite population origin. Additional population genetic studies also provided important insight into the origins and substructure of these commonly used laboratory strains. This extensive variation among and within zebrafish strains may have functional effects that impact phenotype and, if not properly addressed, such extensive levels of germ-line variation and population substructure in this commonly used model organism can potentially confound studies intended for translation to human diseases.

Atrazine, a herbicide commonly applied to agricultural areas and a common contaminant of potable water supplies, is implicated as an endocrine-disrupting chemical (EDC) and potential carcinogen. Studies show that EDCs can cause irreversible changes in tissue formation, decreased reproductive potential, obesity, and cancer. The U.S. Environmental Protection Agency considers an atrazine concentration of ≤ 3 ppb in drinking water safe for consumption. The specific adverse human health effects associated with a developmental atrazine exposure and the underlying genetic mechanisms of these effects are not well defined. In this study, zebrafish embryos were exposed to a range of atrazine concentrations to establish toxicity. Morphological, transcriptomic, and protein alterations were then assessed at 72h postfertilization following developmental atrazine exposure at 0, 0.3, 3, or 30 ppb. A significant increase in head length was observed in all three atrazine treatments. Transcriptomic profiles revealed 21, 62, and 64 genes with altered expression in the 0.3, 3, and 30 ppb atrazine treatments, respectively. Altered genes were associated with neuroendocrine and reproductive system development, function, and disease; cell cycle control; and carcinogenesis. There was a significant overlap (42 genes) between the 3 and 30 ppb differentially expressed gene lists, with two of these genes (CYP17A1 and SAMHD1) present in all three atrazine treatments. Increased transcript levels were translated to significant upregulation in protein expression. Overall, this study identifies genetic and molecular targets altered in response to a developmental atrazine exposure to further define the biological pathways and mechanisms of toxicity.

Many fields of biology – including vertebrate Evo-Devo research – are facing an explosion of genomic and transcriptomic sequence information and a multitude of fish species are now swimming in this ‘genomic tsunami’. Here, we first give an overview of recent developments in sequencing fish genomes and transcriptomes that identify properties of fish genomes requiring particular attention and propose strategies to overcome common challenges in fish genomics. We suggest that the generation of chromosome-level genome assemblies - for which we introduce the term ‘chromonome’ – should be a key component of genomic investigations in fish because they enable large-scale conserved synteny analyses that inform orthology detection, a process critical for connectivity of genomes. Orthology calls in vertebrates, especially in teleost fish, are complicated by divergent evolution of gene repertoires and functions following two rounds of genome duplication in the ancestor of vertebrates and a third round at the base of teleost fish. Second, using examples of spotted gar, basal teleosts, zebrafish-related cyprinids, cavefish, livebearers, icefish, and lobefin fish, we illustrate how next generation sequencing technologies liberate emerging fish systems from genomic ignorance and transform them into a new model army to answer longstanding questions on the genomic and developmental basis of their biodiversity. Finally, we discuss recent progress in the genetic toolbox for the major fish models for functional analysis, zebrafish and medaka, that can be transferred to many other fish species to study in vivo the functional effect of evolutionary genomic change as Evo-Devo research enters the postgenomic era.

BackgroundThe underlying genetic mechanisms specific to subtle neurological alterations associated with environmental lead (Pb) exposures have not been clearly elucidated.ObjectivesThe goal of this study was to identify novel gene targets and the underlying genetic mechanisms associated with developmental Pb neurotoxicity.MethodsWe first exposed zebrafish embryos to a range of Pb concentrations throughout early development to establish relative toxicity. Using the data from that experiment, we exposed another group of zebrafish embryos to a sublethal dose of Pb (100 ppb) immediately after fertilization through 72 or 120 hr postfertilization (hpf). Global gene expression was then analyzed for molecular pathways and gene ontology enrichment, and Western blot analysis was performed to investigate the translation of gene expression changes to protein levels.ResultsAfter 72 hpf, we identified 231 probes representing 90 nonredundant genes with well-established function or orthology to human genes as being altered by Pb exposure. This gene set was both confirmatory and novel in nature and was highly enriched for neurological development, function, and disease. Moreover, gene changes at this time point were correlated to altered protein levels. Alternatively, the gene set at 120 hpf did not share association with neurological development.ConclusionsGlobal gene expression alterations associated with developmental Pb exposure were dynamic and dependent on developmental stage. Gene expression alterations at the 72-hpf time point were highly enriched with genes and molecular pathways associated with neurological development and disease. Moreover, we identified a number of novel targets for future exploration into their role in the genetic mechanisms underlying Pb-induced neurological alterations.

Many important and complex laboratory procedures require an input of high quality, intact RNA. A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications. It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample. Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated. The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage. Protocol Part 1: Extraction of Total RNA using TRIzol ReagentWhen working with RNA it is very important to work in an environment that is free of RNAses. Simple precautions such as having reserved pipettes for use only with RNA procedures and spraying the work area with a decontaminant reagent (e.g., RNAse Away) before beginning the procedure are very helpful. Part 2: RNA Cleanup using the Qiagen RNEasy Mini KitThe Qiagen RNEasy Mini Kit 2 is useful in removing contaminants and impurities from an RNA sample isolated from other methods. For this procedure, β-mercaptoethanol, ethanol, and nuclease-free water are needed in addition to the components included in the kit. An optional DNAse treatment is recommended during this procedure.