Dissecting Phenotypic Variation in Pigmentation using Forward and Reverse Genetics

Sammanfattning: Coat color and patterning phenotypes have been extensively studied as a model for advancing our understanding of the relationship between genetic and phenotypic variation. In this thesis, genes of relevance for pigment cell biology were investigated. The dissertation is divided in two parts. Forward genetics was used in the first part (Paper I and II) to identify the genes controlling the Silver and Sex-linked barring loci in chicken. In the second part, reverse genetics was employed to create a mouse line in which the PMEL17 protein is inactivated (Paper III).In Paper I, we report five mutations in SLC45A2 causing plumage color variants in both chicken and Japanese quail. Normal function of the SLC45A2 gene has previously been shown to be essential for the synthesis of both red/yellow pigment (pheomelanin) and brown/black pigment (eumelanin) in numerous species, including humans. The major discovery in this paper is the specific inhibition of pheomelanin in Silver chickens, whilst null mutations at this locus cause an almost complete absence of both pheomelanin and eumelanin.In Paper II, we report that Sex-linked barring in chickens is controlled by the CDKN2A/B tumor suppressor locus. The locus encodes two proteins, INK4B and ARF. The genetic analysis indicates that missense mutations in ARF or mutations in the promoter region of the ARF transcript are causing Sex-linked barring. In previous studies, mutations inactivating the CDKN2A/B tumor suppressor locus, have been shown to be responsible for familiar forms of human melanoma. Here we propose that these mutations in chicken CDKN2A/B cause the premature cell death of melanocytes as opposed to the cell proliferation and tumor growth associated with loss-of-function alleles in humans.In Paper III, we created a mouse line in which the PMEL17 protein is inactivated. Missense mutations in the gene encoding PMEL17 have previously been shown to be associated with reduced levels of eumelanin in epidermal tissues in several vertebrate species. The knockout mice are viable, fertile, and display no obvious developmental defects. The eumelanosomes within the melanocytes of these mice are spherical in contrast to the cigar-like shaped eumelanosomes present in wild-type animals. PMEL17 protein inactivation has only a subtle diluting effect on the coat color phenotype in four different genetic backgrounds. This suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations.