Previous findings indicate that the protein c-KIT and its ligand, stem cell factor (SCF) play a crucial role in the development of melanocytes from their precursors in the embryonic neural crest cells. Using a monoclonal anti-c-KIT antibody, ACK2, which is an antagonistic blocker of c-KIT function, we and colleagues demonstrated that mouse melanocytes disappeared with the injection of ACK2 during certain periods of embryonic and postnatal life. The precise mechanisms of this disappearance, however, remain unclear. Because melanocytes disappeared without any inflammation in these in vivo studies, we suspect that apoptosis was a main cause of their disappearance. In this study, to clarify the underlying mechanism, we studied whether ACK2 induces apoptosis in c-KIT-positive melanoblasts, which appear in mouse neural crest cells cultured with SCF from 9.5 d old mouse embryos. With an in situ apoptosis detection kit, a significant increase in apoptosis was detected after the removal of SCF, which further increased with the addition of ACK2 during SCF-dependent periods. The occurrence of apoptosis in the cultured cells was also demonstrated by a DNA analysis and electron microscopy. Immunohistochemical double staining confirmed that the apoptotic cells were c-KIT positive, and the electron microscopy showed that these apoptotic cells were melanocyte precursors. It was therefore demonstrated that apoptosis was induced in the SCF-dependent c-KIT-positive melanocytes in vitro when the SCF/c-KIT interaction was obstructed. These findings elucidate the mechanism of the regulation of melanocyte development, and the survival and proliferation of these precursor cells, by SCF/c-KIT interaction.

Previous studies indicate that c-Kit is required for postnatal melanocyte development. To understand the precise mechanisms of c-Kit dependence, we studied melanocyte development in newborn C57BL/6 mice by means of peritoneal injection of a monoclonal anti-c-Kit antibody (ACK2), which blocks c-Kit functions. The mice were injected once or more with ACK2 at various intervals after birth. In experiment 1, skin samples were examined on day 10 post-partum and in experiment 2 they were examined daily until day 10 post-partum. We studied melanocytes in the hair follicles, epidermis, and dermis by light and electron microscopy with dopa reactions and immunohistochemistry. Epidermal melanocytes in untreated mice were dopa negative and c-Kit positive on day 0 post-partum but became dopa positive soon thereafter. In ACK2-treated mice, the earlier the mice received ACK2 injections after birth, the fewer melanocytes they had, not only in the epidermis, but also in follicles. In these mice, melanocytes that had undergone apoptosis in the dermis and the follicles were detected ultrastructurally. Some appeared to have produced tyrosinase, because they had dopa-positive melanosomes. These results suggest that melanocytes in newborn mice are c-Kit dependent and undergo apoptosis when c-Kit receptors are blocked by ACK2 in the early days after birth. During this c-Kit-dependent period, melanocytes differentiate from dopa negative to positive and migrate from the epidermis to hair follicles.

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