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Genome-wide association analysis of tolerance to methylmercury toxicity in Drosophila implicates myogenic and neuromuscular developmental pathways.

Montgomery SL, Vorojeikina D, Huang W, Mackay TF, Anholt RR, Rand MD - PLoS ONE (2014)

Bottom Line: We found significant genetic variation in the effects of MeHg on development, measured by eclosion rate, giving a broad sense heritability of 0.86.We found that caffeine counteracts the deleterious effects of MeHg in the majority of lines, and there is significant genetic variance in the magnitude of this effect, with a broad sense heritability of 0.80.Furthermore, our observations that caffeine can ameliorate the toxic effects of MeHg show that nutritional factors and dietary manipulations may offer protection against the deleterious effects of MeHg exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

ABSTRACT
Methylmercury (MeHg) is a persistent environmental toxin present in seafood that can compromise the developing nervous system in humans. The effects of MeHg toxicity varies among individuals, despite similar levels of exposure, indicating that genetic differences contribute to MeHg susceptibility. To examine how genetic variation impacts MeHg tolerance, we assessed developmental tolerance to MeHg using the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP). We found significant genetic variation in the effects of MeHg on development, measured by eclosion rate, giving a broad sense heritability of 0.86. To investigate the influence of dietary factors, we measured MeHg toxicity with caffeine supplementation in the DGRP lines. We found that caffeine counteracts the deleterious effects of MeHg in the majority of lines, and there is significant genetic variance in the magnitude of this effect, with a broad sense heritability of 0.80. We performed genome-wide association (GWA) analysis for both traits, and identified candidate genes that fall into several gene ontology categories, with enrichment for genes involved in muscle and neuromuscular development. Overexpression of glutamate-cysteine ligase, a MeHg protective enzyme, in a muscle-specific manner leads to a robust rescue of eclosion of flies reared on MeHg food. Conversely, mutations in kirre, a pivotal myogenic gene identified in our GWA analyses, modulate tolerance to MeHg during development in accordance with kirre expression levels. Finally, we observe disruptions of indirect flight muscle morphogenesis in MeHg-exposed pupae. Since the pathways for muscle development are evolutionarily conserved, it is likely that the effects of MeHg observed in Drosophila can be generalized across phyla, implicating muscle as an additional hitherto unrecognized target for MeHg toxicity. Furthermore, our observations that caffeine can ameliorate the toxic effects of MeHg show that nutritional factors and dietary manipulations may offer protection against the deleterious effects of MeHg exposure.

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Muscle-specific expression of glutamate-cysteine ligase (GCL) rescues eclosion on MeHg.(A) Eclosion assays comparing flies expressing the catalytic subunit of GCL in two independent UAS responder lines (GCLc5 and GCLc6) using the muscle-specific Mef2-GAL4 driver (Mef2) with control flies (Mef2>w1118). Statistical analyses by z- test, n = 150 flies/bar. * p <0.001 and **p<0.0001 relative to Mef2>w1118. (B) Expression of GCLc mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.
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pone-0110375-g006: Muscle-specific expression of glutamate-cysteine ligase (GCL) rescues eclosion on MeHg.(A) Eclosion assays comparing flies expressing the catalytic subunit of GCL in two independent UAS responder lines (GCLc5 and GCLc6) using the muscle-specific Mef2-GAL4 driver (Mef2) with control flies (Mef2>w1118). Statistical analyses by z- test, n = 150 flies/bar. * p <0.001 and **p<0.0001 relative to Mef2>w1118. (B) Expression of GCLc mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.

Mentions: To functionally assay developing muscle tissue as a sensitive MeHg target, we examined flies that carry a gene known to affect MeHg tolerance expressed under the muscle specific enhancer myocyte enhancing factor 2 (Mef2) [40]. We induced expression of glutamate-cysteine ligase (GCL), the highly conserved, rate-limiting enzyme for the synthesis of glutathione (GSH). GSH is a small molecule thiol compound present in all cells and is a first line of defense to MeHg toxicity, forming a conjugate that enhances MeHg excretion [41]. Elevated expression of GCL gives resistance to MeHg toxicity [42]. Using the Gal4>UAS system, we over-expressed the catalytic subunit of GCL (GCLc) using two independent lines carrying the UAS-GCLc construct (GCLc5, GCLc6) with the Mef2-Gal4 driver (Fig. 6B). GCLc expression in muscle shows a robust enhancement of tolerance to MeHg in the eclosion assay (Fig. 6A), consistent with the notion that protection of muscle development is critical to overall development of the fly and completion of eclosion under MeHg toxicity stress.


Genome-wide association analysis of tolerance to methylmercury toxicity in Drosophila implicates myogenic and neuromuscular developmental pathways.

Montgomery SL, Vorojeikina D, Huang W, Mackay TF, Anholt RR, Rand MD - PLoS ONE (2014)

Muscle-specific expression of glutamate-cysteine ligase (GCL) rescues eclosion on MeHg.(A) Eclosion assays comparing flies expressing the catalytic subunit of GCL in two independent UAS responder lines (GCLc5 and GCLc6) using the muscle-specific Mef2-GAL4 driver (Mef2) with control flies (Mef2>w1118). Statistical analyses by z- test, n = 150 flies/bar. * p <0.001 and **p<0.0001 relative to Mef2>w1118. (B) Expression of GCLc mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215868&req=5

pone-0110375-g006: Muscle-specific expression of glutamate-cysteine ligase (GCL) rescues eclosion on MeHg.(A) Eclosion assays comparing flies expressing the catalytic subunit of GCL in two independent UAS responder lines (GCLc5 and GCLc6) using the muscle-specific Mef2-GAL4 driver (Mef2) with control flies (Mef2>w1118). Statistical analyses by z- test, n = 150 flies/bar. * p <0.001 and **p<0.0001 relative to Mef2>w1118. (B) Expression of GCLc mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.
Mentions: To functionally assay developing muscle tissue as a sensitive MeHg target, we examined flies that carry a gene known to affect MeHg tolerance expressed under the muscle specific enhancer myocyte enhancing factor 2 (Mef2) [40]. We induced expression of glutamate-cysteine ligase (GCL), the highly conserved, rate-limiting enzyme for the synthesis of glutathione (GSH). GSH is a small molecule thiol compound present in all cells and is a first line of defense to MeHg toxicity, forming a conjugate that enhances MeHg excretion [41]. Elevated expression of GCL gives resistance to MeHg toxicity [42]. Using the Gal4>UAS system, we over-expressed the catalytic subunit of GCL (GCLc) using two independent lines carrying the UAS-GCLc construct (GCLc5, GCLc6) with the Mef2-Gal4 driver (Fig. 6B). GCLc expression in muscle shows a robust enhancement of tolerance to MeHg in the eclosion assay (Fig. 6A), consistent with the notion that protection of muscle development is critical to overall development of the fly and completion of eclosion under MeHg toxicity stress.

Bottom Line: We found significant genetic variation in the effects of MeHg on development, measured by eclosion rate, giving a broad sense heritability of 0.86.We found that caffeine counteracts the deleterious effects of MeHg in the majority of lines, and there is significant genetic variance in the magnitude of this effect, with a broad sense heritability of 0.80.Furthermore, our observations that caffeine can ameliorate the toxic effects of MeHg show that nutritional factors and dietary manipulations may offer protection against the deleterious effects of MeHg exposure.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

ABSTRACT
Methylmercury (MeHg) is a persistent environmental toxin present in seafood that can compromise the developing nervous system in humans. The effects of MeHg toxicity varies among individuals, despite similar levels of exposure, indicating that genetic differences contribute to MeHg susceptibility. To examine how genetic variation impacts MeHg tolerance, we assessed developmental tolerance to MeHg using the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP). We found significant genetic variation in the effects of MeHg on development, measured by eclosion rate, giving a broad sense heritability of 0.86. To investigate the influence of dietary factors, we measured MeHg toxicity with caffeine supplementation in the DGRP lines. We found that caffeine counteracts the deleterious effects of MeHg in the majority of lines, and there is significant genetic variance in the magnitude of this effect, with a broad sense heritability of 0.80. We performed genome-wide association (GWA) analysis for both traits, and identified candidate genes that fall into several gene ontology categories, with enrichment for genes involved in muscle and neuromuscular development. Overexpression of glutamate-cysteine ligase, a MeHg protective enzyme, in a muscle-specific manner leads to a robust rescue of eclosion of flies reared on MeHg food. Conversely, mutations in kirre, a pivotal myogenic gene identified in our GWA analyses, modulate tolerance to MeHg during development in accordance with kirre expression levels. Finally, we observe disruptions of indirect flight muscle morphogenesis in MeHg-exposed pupae. Since the pathways for muscle development are evolutionarily conserved, it is likely that the effects of MeHg observed in Drosophila can be generalized across phyla, implicating muscle as an additional hitherto unrecognized target for MeHg toxicity. Furthermore, our observations that caffeine can ameliorate the toxic effects of MeHg show that nutritional factors and dietary manipulations may offer protection against the deleterious effects of MeHg exposure.

Show MeSH
Related in: MedlinePlus