Distinct stages of melanocyte differentiation revealed by anlaysis of nonuniform pigmentation patterns.

The injection of an antagonistic anti-murine c-kit monoclonal antibody ACK2 during mouse embryonic development produced three distinctive pigmentation patterns on the coat of the offspring. Pattern 1 consisted of pigmentation in craniofacial and caudal regions and was induced by an ACK2 injection between 9.5 and 11.5 days post coitum (dpc). In pattern 2, the entire coat was unpigmented and was induced by the injection at around 13.0 dpc. Pattern 3 consisted of pigmented patches spreading ventrolaterally from the dorsoanterior trunk regions towards the anterior and posterior directions and it was induced by ACK2 administered at 14.5-15.0 dpc. We investigated the embryological basis of these nonuniform pigmentation patterns to elucidate the process of melanoblast differentiation between lineage commitment and colonization into developing hair follicles. The results showed the following. (1) Melanocyte differentiation at the embryonic stage from 10.5 to 12.5 dpc progresses in a spatially nonuniform fashion, being faster in the craniofacial and caudal regions than in the trunk; pattern 1 reflects this. (2) Melanoblasts are activated to proliferate synchronously upon entering into the epidermis; pattern 2 correlates with this process. (3) c-kit functions as a survival signal for proliferating melanoblasts in the epidermis. (4) The melanoblasts that enter developing hair follicles can survive without a c-kit signal; pattern 3 essentially represents the hair follicles colonized by these cells. Analysis of the melanoblast distribution of ls/ls embryos that bear a loss-of-function mutation in the endothelin 3 gene suggested that endothelin 3 is required for early melanoblast differentiation before entering into the epidermis, whereas proliferation in the epidermis takes place without this molecule. Based on these data, we propose 4 distinct steps of embryonic melanocyte differentiation: (1) migration in the dermis, which requires both c-kit and endothelin 3; (2) a state before epidermal entry that is resistant to anti-c-kit mAb; (3) cell proliferation after entering the epidermal layer, which requires c-kit and endothelin receptor B but not endothelin 3 and (4) integration into developing hair follicles, which renders melanoblasts resistant to anti-c-kit mAb. Thus, melanoblast differentiation proceeds by alternately repeating c-kit -dependent and c-kit-independent stages and c-kit functions as a survival factor for the proliferating melanoblasts.