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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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Altered MeHg tolerance in kirre mutant flies.(A) Schematic representation of mutant lines targeting kirre. kirreM107148 is located at X: 2921499 targeting kirre transcripts E, F, B and G. kirreMI00678 is located at X: 2952881 targeting E, F, B and G transcripts. kirreG1566 is located at X: 3025507 in Exon 10 targeting all 7 splice variants of kirre. Gray arrows depict the location of the forward and reverse primers used for qRT-PCR analysis. (B) Eclosion assays of kirre mutant lines (MiMIC and EP) compared to y1w67c23 control line. Statistical analyses done by z- test. N = 300 flies/bar. * p<0.01, **p<0.001 and *** p<0.0001 relative to y1w67c23. (C) Expression of kirre mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.
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pone-0110375-g007: Altered MeHg tolerance in kirre mutant flies.(A) Schematic representation of mutant lines targeting kirre. kirreM107148 is located at X: 2921499 targeting kirre transcripts E, F, B and G. kirreMI00678 is located at X: 2952881 targeting E, F, B and G transcripts. kirreG1566 is located at X: 3025507 in Exon 10 targeting all 7 splice variants of kirre. Gray arrows depict the location of the forward and reverse primers used for qRT-PCR analysis. (B) Eclosion assays of kirre mutant lines (MiMIC and EP) compared to y1w67c23 control line. Statistical analyses done by z- test. N = 300 flies/bar. * p<0.01, **p<0.001 and *** p<0.0001 relative to y1w67c23. (C) Expression of kirre mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.

Mentions: We further examined flies carrying mutations in kirre, one of the core myogenic candidate genes identified in our GWA study. The kirreG1566 mutation is a viable mutant of kirre carrying a P-element insert in exon 10 (Fig. 7A). The kirreMI07148 and kirreMI00678 mutations carry a Minos element insert in an intronic region of four of the seven transcripts (Fig. 7A). Analysis by qRT-PCR demonstrates that transcript levels are substantially reduced in the kirreMI07148 mutant and essentially absent in the kirreG1566 mutant (Fig. 7C), consistent with the predicted effect on transcripts encompassing the respective insertions. A moderate but significant reduction in MeHg tolerance is seen with both the kirreMI07148 and kirreG1566 mutants, specifically with exposure to 10 µM MeHg (Fig. 7B). Consistent with the corresponding expression levels, the kirreG1566 mutant showed the lowest MeHg tolerance with the kirreMI07148mutant giving an intermediate tolerance relative to the yw control line at 10 µM exposures (Fig. 7B). Unexpectedly, the kirreMI00678 mutant shows a significant increase in MeHg tolerance at the 5, 10 and 15 µM exposures (Fig. 7B), and concomitantly, kirre mRNA levels are seen to be elevated relative to the y1w67c23 control line (Fig. 7C). These findings are consistent with the notion that modulation in the levels of kirre expression affects sensitivity of muscle development to MeHg.


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)

Altered MeHg tolerance in kirre mutant flies.(A) Schematic representation of mutant lines targeting kirre. kirreM107148 is located at X: 2921499 targeting kirre transcripts E, F, B and G. kirreMI00678 is located at X: 2952881 targeting E, F, B and G transcripts. kirreG1566 is located at X: 3025507 in Exon 10 targeting all 7 splice variants of kirre. Gray arrows depict the location of the forward and reverse primers used for qRT-PCR analysis. (B) Eclosion assays of kirre mutant lines (MiMIC and EP) compared to y1w67c23 control line. Statistical analyses done by z- test. N = 300 flies/bar. * p<0.01, **p<0.001 and *** p<0.0001 relative to y1w67c23. (C) Expression of kirre mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.
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getmorefigures.php?uid=PMC4215868&req=5

pone-0110375-g007: Altered MeHg tolerance in kirre mutant flies.(A) Schematic representation of mutant lines targeting kirre. kirreM107148 is located at X: 2921499 targeting kirre transcripts E, F, B and G. kirreMI00678 is located at X: 2952881 targeting E, F, B and G transcripts. kirreG1566 is located at X: 3025507 in Exon 10 targeting all 7 splice variants of kirre. Gray arrows depict the location of the forward and reverse primers used for qRT-PCR analysis. (B) Eclosion assays of kirre mutant lines (MiMIC and EP) compared to y1w67c23 control line. Statistical analyses done by z- test. N = 300 flies/bar. * p<0.01, **p<0.001 and *** p<0.0001 relative to y1w67c23. (C) Expression of kirre mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.
Mentions: We further examined flies carrying mutations in kirre, one of the core myogenic candidate genes identified in our GWA study. The kirreG1566 mutation is a viable mutant of kirre carrying a P-element insert in exon 10 (Fig. 7A). The kirreMI07148 and kirreMI00678 mutations carry a Minos element insert in an intronic region of four of the seven transcripts (Fig. 7A). Analysis by qRT-PCR demonstrates that transcript levels are substantially reduced in the kirreMI07148 mutant and essentially absent in the kirreG1566 mutant (Fig. 7C), consistent with the predicted effect on transcripts encompassing the respective insertions. A moderate but significant reduction in MeHg tolerance is seen with both the kirreMI07148 and kirreG1566 mutants, specifically with exposure to 10 µM MeHg (Fig. 7B). Consistent with the corresponding expression levels, the kirreG1566 mutant showed the lowest MeHg tolerance with the kirreMI07148mutant giving an intermediate tolerance relative to the yw control line at 10 µM exposures (Fig. 7B). Unexpectedly, the kirreMI00678 mutant shows a significant increase in MeHg tolerance at the 5, 10 and 15 µM exposures (Fig. 7B), and concomitantly, kirre mRNA levels are seen to be elevated relative to the y1w67c23 control line (Fig. 7C). These findings are consistent with the notion that modulation in the levels of kirre expression affects sensitivity of muscle development to MeHg.

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