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Gene expression alteration associated with early embryonic MeHg exposure in zebrafish (Danio rerio)

ABSTRACT: Methylmercury (MeHg) is an environmental neurotoxicant known to cause adverse effects in fish, such as locomotor abnormalities, visual deficits or teratogenesis. However, very few studies have investigated the effects of environmentally realistic MeHg exposures on the gene expression of fish embryos. Since the primary source of MeHg exposure in wild fish is through the diet, this study analyzed differential gene expression in zebrafish embryos from parents that had been subjected to environmentally relevant dietary MeHg exposures (0, 1, 3, and 10ppm) throughout their whole life cycle.

ORGANISM(S): Danio rerio

PROVIDER: GSE74416 | GEO | 2018/01/01

SECONDARY ACCESSION(S): PRJNA300316

REPOSITORIES: GEO

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Project description:Methylmercury (MeHg) toxicity in humans manifests deficits in neurological function. Cases of prenatal exposure to mercury have established that the developing nervous system is most highly susceptible to perturbation by MeHg. At a cellular level, MeHg-induced defects result from altered neuronal proliferation, migration and pathfinding. However, the molecular targets of MeHg that give rise to these outcomes are not fully understood. In an overall effort to identify the fundamental molecular targets of MeHg in neural development, we are investigating the effects of MeHg on gene expression and protein function in the Drosophila model. Since the fundamental signaling pathways in development of multicellular animals have been extensively characterized in Drosophila, and demonstrate high degree of conservation in vertebrates, we believe these data will lead us to the most pertinent pathways affected by MeHg and begin to elucidate the mechanism of MeHg neural toxicity relevant to cases of human exposure to this prevalent environmental toxin. Our aim is to identify fundamental signaling pathways in neural development that are targets for MeHg poisoning. Our hypothesis is that MeHg, by direct interaction with cysteine thiol groups, alters the function signaling pathway proteins and subsequently alters transcription of target genes in the respective pathways. Our hypothesis is supported by our recent data demonstrating a direct action of MeHg in activating the Notch receptor pathway and upregulating target gene expression. We anticipate a microarray analysis will elucidate additional fundamental signaling pathways where transcription is affected by this toxin. Fertilized female flies are fed on food containing methylmercury (1-20 micromolar) or solvent control (DMSO) for a period of five days to allow for MeHg penetration in to eggs. Flies are then transfered to a cage for embryo collection for a discrete window of time (e.g. 1hr). Embryos are aged 16-24 hours and collected for total RNA extraction using the Trizol reagent. Total RNA is quantitated by spectrophotometry with a Nanodrop reader. While concentration dependent effects of MeHg are of interest, initial experiments will be conducted on samples exposed to a single concetration known to illicit effect in other bioassays (e.g. 10 micromolar). The goal is to see which genes are up- or down-regulated with MeHg exposure, as compared to control embryos that are not exposed. Emphasis in the analysis stage will be in identifying target genes of known signaling pathways.

Project description:Three-month old zebrafish were fed Biodiet starter (4% body weight per day) with MeHg added at 0, 0.5, 5 and 50 ppm for six weeks. Atomic absorption spectrometry was applied to measure the level of MeHg in the whole fish body. Zebrafish at six weeks were sampled from each group for gene expression analysis by NimbleGen Gene Expression 12X135K zebrafish microarrays. MeHg-exposed trout and zebrafish did not show overt signs of toxicity, nor were significant differences seen in mortality, length, mass, or condition factor. The chronic accumulation of total Hg in zebrafish exhibited dose- and time-dependent patterns. The dysregulated genes in MeHg-treated fish have multiple functional annotations, such as involving metabolism, cellular development, ion binding, stress response, transcriptional regulation, and apoptotic pathways. These results show that numerous molecular pathways involved in the growth and development of multiple organ systems are disrupted by exposure to moderate levels of dietary MeHg. The dysregulated genes will be selected by further analysis and used as biomarkers for MeHg exposure in aquatic environments. This study will allow us to assess the potential impacts of low-level exposure to environmental MeHg in the food chain and on the health of humans and animals. Three-month old zebrafish (Danio rerio, average body 0.149 g) were fed Biodiet Starter (Bio-Oregon) 4% of body weight per day with MeHg added at 0ppm, 0.5ppm, 5ppm and 50ppm (with ethanol as vehicle). Fish at six weeks were sampled from each group for gene expression analysis. Three fish from each treatment group were used for microarray experiments. Total RNA from individual fish was isolated using Trizol reagent (Invitrogen) and further purified using the RNeasy MiniElute cleanup kit (Qiagen). Double-strand cDNA was synthesized from 10ug total RNA of individual fish by using Superscript Double-Stranded cDNA synthesis Kit (Invitrogen). ). One ug double strand DNA was labeled with Cy3 using One-Color DNA labeling Kit (NimbleGen), then hybridized to NimbleGen 12X135K array. The arrays were scanned at 2um on a NimbleGen MS200 scanner with auto-gain adjust. The TIFF images were gridded and extracted using NimbleScan v. 2.6. Expression data were normalized using the Robust Multichip Average (RMA) algorithm.

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Gene expression alteration associated with early embryonic MeHg exposure in zebrafish (Danio rerio)

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