A Novel Deletion Mutation of Mouse ruby-eye 2 Named ru2d/Hps5ru2-d Inhibits Melanocyte Differentiation and Its Impaired Differentiation is Rescued by L-tyrosine

In our laboratory, a single autosomal recessive mutation in a phenotype similar to ruby-eye (ru/Hps6ru) or ruby-eye 2 (ru2/Hps5ru2) spontaneously occurred in siblings of C57BL/10JHir (+/+, black) mice in 2006. RT-PCR analysis revealed that this novel mutation, named ru2d/Hps5ru2-d, exhibited frameshift by 997G deletion in the Hps5 gene. To clarify the mechanism of the hypopigmentation, the characteristics of proliferation and differentiation of ru2d/ru2d epidermal melanoblasts and melanocytes cultured in a serum-free medium were investigated. The proliferation of ru2d/ru2d melanoblasts and melanocytes did not differ from that of +/+ melanoblasts and melanocytes. However, the differentiation of ru2d/ru2d melanocytes was greatly inhibited. Tyrosinase (TYR) activity, expression of TYR, TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT), eumelanin synthesis, and the number of stage IV melanosomes markedly decreased in ru2d/ru2d melanocytes. However, excess L-tyrosine (Tyr) added to culture media from initiation of the primary culture rescued the reduced differentiation through increase in TYR activity, expression of TYR, TRP1, TRP2 and Kit, eumelanin synthesis, and stage IV melanosomes. L-Tyr injected into ru2d/ru2d mice also stimulated melanocyte differentiation. These results suggest that the ru2d allele inhibits melanocyte differentiation, and that its impaired differentiation is rescued by excess Tyr.