Fate of melanocytes during development of the hair follicle pigmentary unit

During hair follicle morphogenesis, melanocyte precursors migrate into developing hair follicles and give rise to differentiated melanocytes that actively produce and transport pigment into the keratinocytes that form the hair shaft; however, patterns of melanocyte proliferation and differentiation during formation of the hair pigmentation unit remain to be elucidated. Using multicolor confocal microscopy and double immunofluorescence of melanogenic proteins (tyrosinase-related proteins 1 and 2, tyrosinase) and the proliferative marker Ki67, we have studied melanocyte development in C57BL/6 mouse embryonic hair follicles. Proliferating melanocyte precursors (tyrosinase-related protein-2/Ki67(+) cells) are seen in the hair follicles at stages 1-2 of morphogenesis, as follicular invagination begins. In stage 3-4 hair follicles, the majority of intrafollicular melanocytes remain tyrosinase-related protein-2(+) and Ki67(+), whereas some located adjacent to the forming dermal papilla begin to express tyrosinase-related protein-1, an early marker of differentiation. Melanin granules appear in stage 5 hair follicles coincident with tyrosinase expression in nonproliferating tyrosinase-related protein-2(+)/tyrosinase-related protein-1(+) melanocytes. Stage 6-8 hair follicles, those actively producing hair, show nonproliferating tyrosinase-related protein-2(+) melanocytes in the bulge area, tyrosinase-related protein-2(+)/tyrosinase-related protein-1(+) melanocytes in the outer root sheath, and tyrosinase-related protein-2(+)/tyrosinase-related protein-1(+)/tyrosinase(+) melanocytes above the dermal papilla. These data suggest that melanocyte precursor cells proliferate extensively at the onset of follicle development. Progeny of these cells migrate down the developing follicle, differentiating further until reaching the area immediately above the dermal papilla, where fully differentiated nonproliferative melanin-producing melanocytes persist, contributing pigment to the growing hair shaft.