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Melanism in peromyscus is caused by independent mutations in agouti.

Kingsley EP, Manceau M, Wiley CD, Hoekstra HE - PLoS ONE (2009)

Bottom Line: Identifying the molecular basis of phenotypes that have evolved independently can provide insight into the ways genetic and developmental constraints influence the maintenance of phenotypic diversity.While variation in the Mc1r coding region does not correlate with melanism in any population, in a New Hampshire population, we find that a 125-kb deletion, which includes the upstream regulatory region and exons 1 and 2 of Agouti, results in a loss of Agouti expression and is perfectly associated with melanic color.In a second population from Alaska, we find that a premature stop codon in exon 3 of Agouti is associated with a similar melanic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Organismic and Evolutionary Biology and the Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, United States of America. ekingsley@oeb.harvard.edu

ABSTRACT
Identifying the molecular basis of phenotypes that have evolved independently can provide insight into the ways genetic and developmental constraints influence the maintenance of phenotypic diversity. Melanic (darkly pigmented) phenotypes in mammals provide a potent system in which to study the genetic basis of naturally occurring mutant phenotypes because melanism occurs in many mammals, and the mammalian pigmentation pathway is well understood. Spontaneous alleles of a few key pigmentation loci are known to cause melanism in domestic or laboratory populations of mammals, but in natural populations, mutations at one gene, the melanocortin-1 receptor (Mc1r), have been implicated in the vast majority of cases, possibly due to its minimal pleiotropic effects. To investigate whether mutations in this or other genes cause melanism in the wild, we investigated the genetic basis of melanism in the rodent genus Peromyscus, in which melanic mice have been reported in several populations. We focused on two genes known to cause melanism in other taxa, Mc1r and its antagonist, the agouti signaling protein (Agouti). While variation in the Mc1r coding region does not correlate with melanism in any population, in a New Hampshire population, we find that a 125-kb deletion, which includes the upstream regulatory region and exons 1 and 2 of Agouti, results in a loss of Agouti expression and is perfectly associated with melanic color. In a second population from Alaska, we find that a premature stop codon in exon 3 of Agouti is associated with a similar melanic phenotype. These results show that melanism has evolved independently in these populations through mutations in the same gene, and suggest that melanism produced by mutations in genes other than Mc1r may be more common than previously thought.

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Agouti and Mc1r expression in wild type and melanic mice.(A, B) Relative expression of Agouti and Mc1r transcripts in dorsal skin of P4 P. maniculatus was measured by quantitative RT-PCR. Expression level of the target gene is standardized with that of β-actin. We compared relative expression levels of each gene with Student's t-test (two-tailed, unequal variance). For each phenotype class, N = 5. (A) Agouti expression is significantly higher in the dorsal skin of wild type mice than in melanic mice; expression level in melanic mice is not significantly different from zero. (B) Mc1r expression in wild type and melanic mice does not significantly differ. Bars indicate standard error. (C,D) Lateral views of whole-mount in situ hybridizations for Agouti in E12.5 embryos. (C) Wild type embryos express Agouti in the whisker plate and the limbs (arrows). (D) Agouti expression is not detected in aΔ125kb homozygote embryos.
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pone-0006435-g003: Agouti and Mc1r expression in wild type and melanic mice.(A, B) Relative expression of Agouti and Mc1r transcripts in dorsal skin of P4 P. maniculatus was measured by quantitative RT-PCR. Expression level of the target gene is standardized with that of β-actin. We compared relative expression levels of each gene with Student's t-test (two-tailed, unequal variance). For each phenotype class, N = 5. (A) Agouti expression is significantly higher in the dorsal skin of wild type mice than in melanic mice; expression level in melanic mice is not significantly different from zero. (B) Mc1r expression in wild type and melanic mice does not significantly differ. Bars indicate standard error. (C,D) Lateral views of whole-mount in situ hybridizations for Agouti in E12.5 embryos. (C) Wild type embryos express Agouti in the whisker plate and the limbs (arrows). (D) Agouti expression is not detected in aΔ125kb homozygote embryos.

Mentions: To test whether this 125 kb deletion affects the abundance of Agouti transcript, we measured Agouti mRNA in the skin of P4 pups. In animals heterozygous for the wild type and the aΔ125kb alleles, levels of Agouti expression were significantly higher than those of animals homozygous for aΔ125kb (Figure 3A). These data show that the aΔ125kb allele produces significantly less Agouti mRNA transcript and is thus likely the cause of melanism. Mc1r transcript levels, on the other hand, were not significantly different between melanic and wild type individuals (Figure 3B). In addition, we performed in situ hybridizations on 12.5 day-old embryos to determine whether Agouti is expressed in melanic embryos. At this stage, wild type embryos express Agouti in the whisker plate and in parts of the limbs (Figure 3C), an expression pattern similar to that seen in Mus [20]. We did not detect any Agouti expression in melanic embryos (Figure 3D).


Melanism in peromyscus is caused by independent mutations in agouti.

Kingsley EP, Manceau M, Wiley CD, Hoekstra HE - PLoS ONE (2009)

Agouti and Mc1r expression in wild type and melanic mice.(A, B) Relative expression of Agouti and Mc1r transcripts in dorsal skin of P4 P. maniculatus was measured by quantitative RT-PCR. Expression level of the target gene is standardized with that of β-actin. We compared relative expression levels of each gene with Student's t-test (two-tailed, unequal variance). For each phenotype class, N = 5. (A) Agouti expression is significantly higher in the dorsal skin of wild type mice than in melanic mice; expression level in melanic mice is not significantly different from zero. (B) Mc1r expression in wild type and melanic mice does not significantly differ. Bars indicate standard error. (C,D) Lateral views of whole-mount in situ hybridizations for Agouti in E12.5 embryos. (C) Wild type embryos express Agouti in the whisker plate and the limbs (arrows). (D) Agouti expression is not detected in aΔ125kb homozygote embryos.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713407&req=5

pone-0006435-g003: Agouti and Mc1r expression in wild type and melanic mice.(A, B) Relative expression of Agouti and Mc1r transcripts in dorsal skin of P4 P. maniculatus was measured by quantitative RT-PCR. Expression level of the target gene is standardized with that of β-actin. We compared relative expression levels of each gene with Student's t-test (two-tailed, unequal variance). For each phenotype class, N = 5. (A) Agouti expression is significantly higher in the dorsal skin of wild type mice than in melanic mice; expression level in melanic mice is not significantly different from zero. (B) Mc1r expression in wild type and melanic mice does not significantly differ. Bars indicate standard error. (C,D) Lateral views of whole-mount in situ hybridizations for Agouti in E12.5 embryos. (C) Wild type embryos express Agouti in the whisker plate and the limbs (arrows). (D) Agouti expression is not detected in aΔ125kb homozygote embryos.
Mentions: To test whether this 125 kb deletion affects the abundance of Agouti transcript, we measured Agouti mRNA in the skin of P4 pups. In animals heterozygous for the wild type and the aΔ125kb alleles, levels of Agouti expression were significantly higher than those of animals homozygous for aΔ125kb (Figure 3A). These data show that the aΔ125kb allele produces significantly less Agouti mRNA transcript and is thus likely the cause of melanism. Mc1r transcript levels, on the other hand, were not significantly different between melanic and wild type individuals (Figure 3B). In addition, we performed in situ hybridizations on 12.5 day-old embryos to determine whether Agouti is expressed in melanic embryos. At this stage, wild type embryos express Agouti in the whisker plate and in parts of the limbs (Figure 3C), an expression pattern similar to that seen in Mus [20]. We did not detect any Agouti expression in melanic embryos (Figure 3D).

Bottom Line: Identifying the molecular basis of phenotypes that have evolved independently can provide insight into the ways genetic and developmental constraints influence the maintenance of phenotypic diversity.While variation in the Mc1r coding region does not correlate with melanism in any population, in a New Hampshire population, we find that a 125-kb deletion, which includes the upstream regulatory region and exons 1 and 2 of Agouti, results in a loss of Agouti expression and is perfectly associated with melanic color.In a second population from Alaska, we find that a premature stop codon in exon 3 of Agouti is associated with a similar melanic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Organismic and Evolutionary Biology and the Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, United States of America. ekingsley@oeb.harvard.edu

ABSTRACT
Identifying the molecular basis of phenotypes that have evolved independently can provide insight into the ways genetic and developmental constraints influence the maintenance of phenotypic diversity. Melanic (darkly pigmented) phenotypes in mammals provide a potent system in which to study the genetic basis of naturally occurring mutant phenotypes because melanism occurs in many mammals, and the mammalian pigmentation pathway is well understood. Spontaneous alleles of a few key pigmentation loci are known to cause melanism in domestic or laboratory populations of mammals, but in natural populations, mutations at one gene, the melanocortin-1 receptor (Mc1r), have been implicated in the vast majority of cases, possibly due to its minimal pleiotropic effects. To investigate whether mutations in this or other genes cause melanism in the wild, we investigated the genetic basis of melanism in the rodent genus Peromyscus, in which melanic mice have been reported in several populations. We focused on two genes known to cause melanism in other taxa, Mc1r and its antagonist, the agouti signaling protein (Agouti). While variation in the Mc1r coding region does not correlate with melanism in any population, in a New Hampshire population, we find that a 125-kb deletion, which includes the upstream regulatory region and exons 1 and 2 of Agouti, results in a loss of Agouti expression and is perfectly associated with melanic color. In a second population from Alaska, we find that a premature stop codon in exon 3 of Agouti is associated with a similar melanic phenotype. These results show that melanism has evolved independently in these populations through mutations in the same gene, and suggest that melanism produced by mutations in genes other than Mc1r may be more common than previously thought.

Show MeSH
Related in: MedlinePlus