The identification and characterization of an enhancer essential for melanogenesis

The neural crest is a transient embryonic cell population that gives rise to many vertebrate organs. Defects in neural crest cell development can lead to defects throughout the body and diseases such as Hirschsprung Disease and piebaldism. cKit is a receptor tyrosine kinase that marks several cell lineages, including neural crest, hematopoietic, and germ-line stem cells. Signaling from cKit is crucial for normal hematopoiesis, pigmentation, fertility, gut movement, and neural development. However, the mechanisms underlying cKit's role in melanogenesis remain poorly understood. We focused on characterizing a mouse harboring a spontaneous cKit mutation, cKitW44, which leads to defects in melanogenesis and gametogenesis in mice homozygous for cKitW44. While the cKitW44 mutation was reported over 60 years ago, it remains uncharacterized at the molecular level. To define the cKitW44 mutation, we performed an RT-PCR-based tiling strategy to clone and sequence the cKit genomic locus in wild-type and mutant mice. We demonstrated that the cKitW44 mutation results from an insertion of a retrotransposon into an intron within the cKit locus. Based on this data, we hypothesized that the insertion mutation disrupted a potential enhancer region essential for melanogenesis. We discovered an enhancer necessary for melanocyte development within the cKit locus by cloning this region and making a transgenic mouse line. These findings elucidate the impact of the cKitW44 mutation on melanogenesis, shedding light on the mechanisms regulating neural crest development and related disease states.