Transcriptomic analysis of methylmercury effects on Drosophila flight muscle and ventral nerve cord development
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. Overall design: The sample types are total RNA isolated from Drosophila indirect flight muscles (IFMs) and from ventral nerve cord (VNC). Tissues were collected at two stages of development: 30 hours after pupa formation (APF) and 15 day old adults. Sexes were mixed. In addition, treatments were performed ahead of sample collection which included feeding at the larval stage on food containing 0µM (control) or 5 µM (experimental) methylmercury (MeHg).  Larval feeding results in MeHg accumulation that reaches a steady state that is maintained in the pupal stages, where there is no further uptake or excretion.  Adults were moved to MeHg-free food upon eclosion (~96 hr APF) to clear MeHg. Tissues (IFMs and VNCs) were dissected from pupae at 30h after pupa formation and from adults at 15 days post eclosion. Treatments and sample collection were carried out in triplicate. The variables being tested are MeHg exposure, developmental stage and tissue type.