Notch target gene E(spl)mδ is a mediator of methylmercury-induced myotoxicity in Drosophila

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Abstract

Methylmercury (MeHg) is a ubiquitous environmental contaminant and neurotoxicant that has long been known to cause a variety of motor deficits. These motor deficits have primarily been attributed to MeHg targeting of developing neurons and induction of oxidative stress and calcium dysregulation. Few studies have looked at how MeHg may be affecting fundamental signaling mechanisms in development, particularly in developing muscle. Studies in Drosophila recently revealed that MeHg perturbs embryonic muscle formation and upregulates Notch target genes, reflected predominantly by expression of the downstream transcriptional repressor Enhancer of Split mdelta [E(spl)mδ]. An E(spl)mδ reporter gene shows expression primarily in the myogenic domain, and both MeHg exposure and genetic upregulation of E(spl)mδ can disrupt embryonic muscle development. Here, we tested the hypothesis that developing muscle is targeted by MeHg via upregulation of E(spl)mδ using genetic modulation of E(spl)mδ expression in combination with MeHg exposure in developing flies. Developmental MeHg exposure causes a decreased rate of eclosion that parallels gross disruption of indirect flight muscle (IFM) development. An increase in E(spl) expression across the pupal stages, with preferential E(spl)mδ upregulation occurring at early (p5) stages, is also observed. E(spl)mδ overexpression in myogenic lineages under the Mef2 promoter was seen to phenocopy eclosion and IFM effects of developmental MeHg exposure; whereas reduced expression of E(spl)mδ shows rescue of eclosion and IFM morphology effects of MeHg exposure. No effects were seen on eclosion with E(spl)mδ overexpression in neural and gut tissues. Our data indicate that muscle development is a target for MeHg and that E(spl)mδ is a muscle-specific mediator of this myotoxicity. This research advances our knowledge of the target pathways that mediate susceptibility to MeHg toxicity, as well as a potential muscle development-specific role for E(spl)mδ.

Figures

  • FIGURE 1 | Multidrug Resistance Protein (MRP) upregulation in myogenic lineages conveys MeHg tolerance during development. (A) Expression of MRP, a xenobiotic and MeHg transporter, was assessed by crossing UAS-MRP (MRPEY11919) with Actin-GAL4 and conducting RT-qPCR on RNA extracted from pupal offspring (****p < 0.0001, t-test). Tolerance to MeHg during development was determined using an eclosion assay, with offspring of control (YW) or UAS-MRP flies crossed to various driver lines: (B) NP1-G4 (gut driver), (C) ELAV-G4 (neural driver), and (D) Mef2-G4 (muscle driver). Asterisks mark statistical significance in comparison to control at each treatment (*p ≤ 0.05, **p ≤ 0.01, ****p < 0.0001, z-test).
  • FIGURE 2 | MeHg effects on indirect flight muscle (IFM) development are rescued with MRP upregulation. Epifluorescence images of IFMs of pupae at stage p10. (A–C) Mef2-RFP > YW (control) and (D–F) Mef2-RFP > MRP. Pupae were imaged after treatment with the indicated concentration of MeHg from the 1st instar larval stage. Asterisks mark the dorsal ventral muscles (DVM) and closed arrows mark the dorsal longitudinal muscles (DLM). Open arrows indicate failure of muscle fiber development (see Figure S1 for additional images).
  • FIGURE 3 | E(spl) expression in pupae developmentally exposed to MeHg. RT-qPCR on RNA extracted from pupae was performed to assess expression levels of E(spl)mδ and other E(spl) transcription factors. (A) E(spl)mδ expression over pupal development in Canton S. (B–D) Canton S. 1st instar larvae were exposed to either 0 or 10µM MeHg and allowed to develop to (B) p5, (C) p6, and (D) p10 stages of pupal development. Asterisks mark statistical significance from respective 0µM treatment (*p ≤ 0.05, t-test).
  • FIGURE 4 | E(spl)mδ is expressed in the developing IFM. Live epifluorescent imaging of developing IFMs in pupae carrying both Mef2-RFP and E(spl)mδ-GFP at the indicated stages (A–C) p5, (D–F) p6, and (G–I) p10. (C,F,I) Merged images of RFP and GFP represent overlapping regions of expression of Mef2 in developing IFMs and E(spl)mδ. Pupae were dissected from their pupal cases and imaged directly. Asterisks mark the dorsal ventral muscles (DVM) and closed arrows mark the dorsal longitudinal muscles (DLM). Brackets mark the regions of DLM development that encompass myoblast fusing to the larval templates. Green arrow points to E(spl)mδ expression in the developing eye.
  • FIGURE 5 | Effects on eclosion rate upon upregulation of various E(spl)s in neurons, gut, and muscle. Developmental effects of genetic upregulation of E(spl)mδ ORF, E(spl)mγ ORF, and E(spl)m3 ORF were assessed by eclosion assay in the absence of MeHg exposure. UAS-E(spl) ORF responders were crossed with various drivers: (A) Mef2-G4 (muscle driver), (B) ELAV-G4 (neural driver), and (C) NP1-G4 (gut driver). The number of flies successfully eclosed were scored (****p < 0.0001, in comparison to Mef2-Gal4; #p < 0.05, ###p < 0.001, in comparison to Mef2 > mδ, z-test).
  • FIGURE 6 | E(spl)mδ overexpression in myogenic lineage perturbs IFM development. (A) Expression levels of E(spl)mδ using two independent constructs, UAS-mδ h8 and UAS-mδ ORF, was assessed by RT-qPCR with RNA from p5 pupae (**p ≤ 0.01, ***p < 0.001 in comparison to Mef2-G4; #p ≤ 0.05, in comparison to Mef2 > mδ h8, one-way ANOVA). (B) Eclosion of Mef2 > mδ h8 and Mef2 > mδ ORF (***p < 0.001 in comparison to Mef2-G4; ###p < 0.001 in comparison to Mef2 > mδ h8, z-test). (C–N) Developing IFMs in pupae at indicated stages were imaged by epifluorescence. Overexpression of E(spl)mδ was compared to knockdown of the myoblast fusion protein Sticks and Stones (SNS). (C–E) Mef2-RFP (control), (F–H) Mef2-RFP > mδ ORF, (I–K) Mef2-RFP > mδ h8, and (L–N) Mef2-RFP > SNS RNAi pupae were dissected from their pupal cases and imaged directly. Open arrows point to IFMs undergoing extension. White arrows point to partially formed DLM fibers. Asterisk mark absence of DVM formation.
  • FIGURE 7 | Knockdown of E(spl)mδ conveys MeHg tolerance during development. Expression of E(spl)mδ was assessed by RT-qPCR with RNA extracted from p5 pupae of (A) E(spl)mδ deficiency (P3) and its background control strain (K33) and (C) Mef2 > E(spl)mδ RNAi and Mef2 > Attp2 (control) (***p < 0.001, t-test). Tolerance to MeHg was determined through an eclosion assay of (B) E(spl)mδ deficiency (P3) and its background control strain (K33) and (D) Mef2 > E(spl)mδ RNAi and Mef2 > Attp2 (control) strain (*p ≤ 0.05, ***p < 0.001, ****p < 0.0001, z-test).
  • FIGURE 8 | Rescue of MeHg effects on IFM development with downregulation of E(spl)mδ in myogenic lineages. Developing IFMs in pupae, at the p10 stage after treatment with the indicated concentrations of MeHg were imaged by epifluorescence of (A–C) Mef2-RFP > Attp2 (control) and (D–F) Mef2-RFP > mδ RNAi. Pupae were dissected from their pupal cases and imaged directly. Asterisks and arrows mark failure of IFM development (see Figure S2 for additional images).

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Prince, L. M., & Rand, M. D. (2018). Notch target gene E(spl)mδ is a mediator of methylmercury-induced myotoxicity in Drosophila. Frontiers in Genetics, 8(JAN). https://doi.org/10.3389/fgene.2017.00233

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