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The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A

View Article: PubMed Central - PubMed

ABSTRACT

Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner.

No MeSH data available.


Proposed mechanism for development of the Sex-linked barring phenotype.(A) The non-coding mutation(s) present in the B0, B1 and B2 allele cause a tissue specific up-regulation of CDKN2A encoding the ARF protein. ARF inhibits MDM2-mediated degradation of p53. p53 will activate downstream targets possibly initiating premature melanocyte differentiation and thereby loss of mature pigment cells. (B) The missense mutations present in the B1 and B2 alleles impair the interaction between ARF and MDM2, which counteract the consequences of up-regulated ARF expression. (C) In solid colored feathers, melanocyte progenitor cells migrate up from the feather base and start expressing CDKN2A in the barb region leading to differentiation of melanocytes and pigment production without exhausting the pool of undifferentiated melanocytes. In sex-linked barred feathers, up-regulated ARF expression may lead to premature differentiation of pigment cells and a lack of undifferentiated melanocytes that can replenish the ones producing pigment. As the feather keeps on growing, no more melanocytes are available to produce pigment resulting in the white bar. A plausible explanation for the cyclic appearance of white and black bars is that new recruitment of melanocyte progenitor cells takes place after the undifferentiated melanocytes have been depleted.
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pgen.1006665.g005: Proposed mechanism for development of the Sex-linked barring phenotype.(A) The non-coding mutation(s) present in the B0, B1 and B2 allele cause a tissue specific up-regulation of CDKN2A encoding the ARF protein. ARF inhibits MDM2-mediated degradation of p53. p53 will activate downstream targets possibly initiating premature melanocyte differentiation and thereby loss of mature pigment cells. (B) The missense mutations present in the B1 and B2 alleles impair the interaction between ARF and MDM2, which counteract the consequences of up-regulated ARF expression. (C) In solid colored feathers, melanocyte progenitor cells migrate up from the feather base and start expressing CDKN2A in the barb region leading to differentiation of melanocytes and pigment production without exhausting the pool of undifferentiated melanocytes. In sex-linked barred feathers, up-regulated ARF expression may lead to premature differentiation of pigment cells and a lack of undifferentiated melanocytes that can replenish the ones producing pigment. As the feather keeps on growing, no more melanocytes are available to produce pigment resulting in the white bar. A plausible explanation for the cyclic appearance of white and black bars is that new recruitment of melanocyte progenitor cells takes place after the undifferentiated melanocytes have been depleted.

Mentions: Available phenotypic data indicate a ranking of the three variant alleles regarding pigment reduction, as follows: Sex-linked extreme dilution > Sex-linked dilution > Sex-linked barring. Our functional data are fully consistent with the proposed ranking. Firstly, expression analysis shows that one or both of the non-coding changes cause an up-regulation of CDKN2A expression in feather follicles during feather growth (Fig 5A). A higher expression of ARF, encoded by CDKN2A, is expected to lead to a reduction of pigment cells due to apoptosis, cell cycle arrest or premature differentiation of melanocytes. The Sex-linked extreme dilution (B0) allele carries only these non-coding changes and is associated with a drastic reduction in pigmentation. In contrast, our three functional assays (CD, ITC, and luciferase reporter assay) all indicate that the two missense mutations (V9D and R10C) result in hypomorphic ARF alleles. Thus, these mutations are expected to counteract the effect of up-regulated ARF expression, most likely by impairing the ARF-MDM2 interaction and thereby lead to a less severe reduction in pigmentation (Fig 5B). Furthermore, the three functional assays all indicate that the V9D substitution (B1) is expected to limit the effect of the non-coding mutations (B0) on pigment dilution to a larger extent than does R10C (B2), which is consistent with the observation that Sex-linked dilution (B2) shows a stronger reduction in pigmentation than does Sex-linked barring (B1), at least in the homozygous condition (Fig 1A and 1C).


The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A
Proposed mechanism for development of the Sex-linked barring phenotype.(A) The non-coding mutation(s) present in the B0, B1 and B2 allele cause a tissue specific up-regulation of CDKN2A encoding the ARF protein. ARF inhibits MDM2-mediated degradation of p53. p53 will activate downstream targets possibly initiating premature melanocyte differentiation and thereby loss of mature pigment cells. (B) The missense mutations present in the B1 and B2 alleles impair the interaction between ARF and MDM2, which counteract the consequences of up-regulated ARF expression. (C) In solid colored feathers, melanocyte progenitor cells migrate up from the feather base and start expressing CDKN2A in the barb region leading to differentiation of melanocytes and pigment production without exhausting the pool of undifferentiated melanocytes. In sex-linked barred feathers, up-regulated ARF expression may lead to premature differentiation of pigment cells and a lack of undifferentiated melanocytes that can replenish the ones producing pigment. As the feather keeps on growing, no more melanocytes are available to produce pigment resulting in the white bar. A plausible explanation for the cyclic appearance of white and black bars is that new recruitment of melanocyte progenitor cells takes place after the undifferentiated melanocytes have been depleted.
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Related In: Results  -  Collection

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pgen.1006665.g005: Proposed mechanism for development of the Sex-linked barring phenotype.(A) The non-coding mutation(s) present in the B0, B1 and B2 allele cause a tissue specific up-regulation of CDKN2A encoding the ARF protein. ARF inhibits MDM2-mediated degradation of p53. p53 will activate downstream targets possibly initiating premature melanocyte differentiation and thereby loss of mature pigment cells. (B) The missense mutations present in the B1 and B2 alleles impair the interaction between ARF and MDM2, which counteract the consequences of up-regulated ARF expression. (C) In solid colored feathers, melanocyte progenitor cells migrate up from the feather base and start expressing CDKN2A in the barb region leading to differentiation of melanocytes and pigment production without exhausting the pool of undifferentiated melanocytes. In sex-linked barred feathers, up-regulated ARF expression may lead to premature differentiation of pigment cells and a lack of undifferentiated melanocytes that can replenish the ones producing pigment. As the feather keeps on growing, no more melanocytes are available to produce pigment resulting in the white bar. A plausible explanation for the cyclic appearance of white and black bars is that new recruitment of melanocyte progenitor cells takes place after the undifferentiated melanocytes have been depleted.
Mentions: Available phenotypic data indicate a ranking of the three variant alleles regarding pigment reduction, as follows: Sex-linked extreme dilution > Sex-linked dilution > Sex-linked barring. Our functional data are fully consistent with the proposed ranking. Firstly, expression analysis shows that one or both of the non-coding changes cause an up-regulation of CDKN2A expression in feather follicles during feather growth (Fig 5A). A higher expression of ARF, encoded by CDKN2A, is expected to lead to a reduction of pigment cells due to apoptosis, cell cycle arrest or premature differentiation of melanocytes. The Sex-linked extreme dilution (B0) allele carries only these non-coding changes and is associated with a drastic reduction in pigmentation. In contrast, our three functional assays (CD, ITC, and luciferase reporter assay) all indicate that the two missense mutations (V9D and R10C) result in hypomorphic ARF alleles. Thus, these mutations are expected to counteract the effect of up-regulated ARF expression, most likely by impairing the ARF-MDM2 interaction and thereby lead to a less severe reduction in pigmentation (Fig 5B). Furthermore, the three functional assays all indicate that the V9D substitution (B1) is expected to limit the effect of the non-coding mutations (B0) on pigment dilution to a larger extent than does R10C (B2), which is consistent with the observation that Sex-linked dilution (B2) shows a stronger reduction in pigmentation than does Sex-linked barring (B1), at least in the homozygous condition (Fig 1A and 1C).

View Article: PubMed Central - PubMed

ABSTRACT

Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner.

No MeSH data available.