Project description:Methylmercury (MeHg) is a ubiquitous environmental toxicant that is often detected in the tissues of fish-eating species. It has been well established that prenatal exposure to MeHg can lead to widespread brain damage and impaired neurological development resulting in defects ranging from severe cerebral palsy and cognitive deficits to impaired motor and sensory function. A wide range of environmental toxicants have been shown to induce transgenerational inheritance of diseases via changes in DNA methylation—a well-known epigenetic modification. Our previous research has demonstrated that developmental MeHg exposure may yield transgenerational inheritance of neurological dysfunction in adult F3-lineage zebrafish via quantitative neurobehavioral assays that evaluated the visual startle response, retinal electrophysiology, and locomotor function. The objective of the current study was to examine the correlation between neurobehavioral phenotypes and the transcriptome activity in the brain and retina of F3 zebrafish by RNA sequencing (RNAseq). Transcriptomic analyses of F3 generation MeHg-treated zebrafish (compared to control) revealed significant gene dysregulation in both the brain and retina. There were 1648 and 138 differentially expressed genes in the retina and brain, respectively (FDR <0.05). Thirty-five genes were commonly dysregulated in both organs. Gene set enrichment analysis revealed significantly enriched pathways including: neurodevelopment, visual functions, phototransduction, and motor movement. Moreover, commonly dysregulated genes were associated with circadian rhythm and metabolic pathways, as well as arginine and proline metabolism. To our knowledge, this is the first evidence of a transgenerational transcriptome induced by ancestral developmental exposure to MeHg in any species. If the transgenerational phenotypes, transcriptome, homologous biomarkers, or similar molecular pathways hold true for human populations, our findings have significant impact on global public health in terms of identifying the susceptible populations using biomarkers and preventing transgenerational inheritance of MeHg-induced neurobehavioral deficits.

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.

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.

Dataset Information

Methylmercury (MeHg) induced Transgenerational Inheritance of Brain Transcriptome in Zebrafish (Danio rerio)

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