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Transcriptomic analysis of methylmercury effects on Drosophila flight muscle and ventral nerve cord development

ABSTRACT: The greatest concern for exposure to methylmercury (MeHg) lies with the fetus and young children, as exemplified in historic cases of Congenital Minamata Disease (CMD) resulting from prenatal exposure. Children that experience CMD characteristically present with various degrees of cognitive and motor symptoms and signs, much like cerebral palsy. MeHg has thus been characterized as a neurotoxicant, where motor deficits are ascribed to central nervous system targets. Skeletal muscle as a post-synaptic MeHg target and contributor to the etiology of CMD has garnered far less attention. Prior studies using Drosophila to model CMD revealed that developmental exposure of MeHg in the larval/pupal stages can elicit graded and latent dose responses affecting adult flight behavior at lower doses (0.4-2.5 ppm in food) and eclosion (emergence from the pupa case) at higher doses (>2.5 ppm in food). The latter phenotype is accompanied by dysmorphogenesis of skeletal muscles. Here, we investigate respective roles for muscle and neural targets in MeHg toxicity. Using RNA-seq analysis, we find that developmental MeHg exposure produces 10-times as many differentially expressed transcripts in indirect flight muscle (IFM) compared to the VNC. Among known MeHg response genes, Nrf2 antioxidant response pathway genes showed muscle-specific MeHg-induced expression changes. Within the muscle transcriptome, the most enriched and significant Gene Ontology terms identified genes required for mitochondrial ribosomal translation at the pupa stage and mitochondrial function (respiratory chain complex I) and vesicle trafficking (ESCRT III) pathways in adults, all showing decreased expression with MeHg exposure. By using an intact, whole-animal developmental model, we identify preferential candidates to evaluate a novel role for muscle-specific mitochondria and intercellular vesicular communication mechanisms as targets in MeHg toxicity and the etiology of CMD.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE282865 | GEO | 2024/11/26

REPOSITORIES: GEO

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Project description:The developing human brain is uniquely vulnerable to methylmercury (MeHg) intoxication. Given the limited experimental access to MeHg-intoxicated human fetal brain, neither the threshold levels for neurotoxic effects nor the mechanisms underlying neurodevelopmental MeHg toxicity are clear. The observed persistent and latent effects in human and animal exposures and the relatively nonspecific neurodevelopmental changes suggest that MeHg impacts fundamental neurodevelopmental processes. Human cortical development is characterized by the sequential emergence of different neural stem cells, precursors, intermediate progenitors and postmitotic neurons, a sequence that can be replicated in human-induced pluripotent stem cell (hiPSC)-derived differentiating neuronal cultures. As the developing human cortex has been shown to be particularly vulnerable to fetal MeHg exposure, we assessed here by means of single cell RNA sequencing (scRNAseq) MeHg’s effects on different stages of differentiation and cell types present in early cortical cultures differentiated from hiPSC cells. Cortical cultures differentiating from hiPSC were exposed to toxicologically relevant MeHg (0.1 and 1.0 µM) continuously for 6 days at two different time points (days 4-10; and/or days 14-20) of early cortical differentiation under conditions below the cytotoxic threshold. Previous analysis at days 21-23 revealed subtle changes in cortical differentiation markers and cellular energetics, but no effect on glutathione levels, a biomarker of acute MeHg toxicity. Therefore, here we assessed the possibility of persistent effects on neurodevelopment at day 38 of differentiation (18 or 28 days after cessation of MeHg exposure). At this time, we observed subtle but significant changes in the population size of different cell types and the fractions of cells present in the different phases of the cell cycle. The observed MeHg-induced changes were developmental-stage and MeHg paradigm-specific. We further assessed overall differential gene expression, quantifying the number of genes showing significantly up- or down-regulated expression and identifying the genes affected at least partially identified biological processes affected. Importantly, the MeHg-induced changes in gene expression were also developmental-stage and MeHg-exposure paradigm specific. Thus, our studies demonstrate for the first time in a human model that MeHg exposure induces persistent changes in gene expression well after cessation of exposure, these changes are cell type- and developmental stage-specific, and this outcome is highly dependent on the concentration and timing of MeHg exposure. We conclude hiPSC-derived developing neuronal cultures provide an excellent human model to study persistent neurodevelopmental toxicity of MeHg in the developing human cortex.

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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Transcriptomic analysis of methylmercury effects on Drosophila flight muscle and ventral nerve cord development

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