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.
Stem cell factor (SCF) and endothelin-3 (ET3) are both necessary for melanocyte development. In order to obtain immortal cell populations of melanoblasts that can survive without feeder cells, we first obtained an immortal cell population of neural crest cells (NCCs) from Sl/+ and +/+ mice of strain WB by incubating with a culture medium supplemented with SCF and ET3, and then we designated them as NCC-SE3 cells. NCC-SE3 cells were bipolar, polygonal, or round in shape and possessed melanosomes of stages I-III (mainly stage I). They were positive to dihydroxyphenylalanine (DOPA) reaction and expressed KIT (a receptor tyrosine kinase), tyrosinase, tyrosinase-related protein-1 (TRP1), tyrosinase-related protein-2 (TRP2), and endothelin-B receptor (ETRB) as determined by immunostaining. We next cultured NCC-SE3 cells by changing culture medium from the one supplemented with SCF + ET3 to the one supplemented with SCF or ET3. NCC-SE3 cells cultured with ET3 alone, designated as NCC-E3 cells, were bipolar in shape and had mainly stage II melanosomes and expressed the same proteins as did NCC-SE3 cells. However, NCC-SE3 cells cultured with SCF alone, designated as NCC-S4.1 cells, were polygonal in shape and had mainly stage I melanosomes. They are thought to be more immature because they were positive to KIT, TRP1, and TRP2, but not to ETR(B), tyrosinase, and DOPA reaction. When 12-O-tetradecanoylphorbol 13-acetate and cholera toxin were added to the culture medium, NCC-S4.1 cells changed shape from polygonal to bipolar and became DOPA-positive. This suggests that NCC-S4.1 cells are melanoblasts that have the potential to differentiate into melanocytes. These cell populations will be extremely useful to study factors that affect melanocyte development and melanogenesis.
Stem cell factor (SCF) has been suggested to be indispensable for the development of neural crest cells into melanocytes because Steel mutant mice (i.e., Sl/Sl(d)) have no pigmented hairs. On the other hand, it has been demonstrated that the addition of endothelin 3 (ET-3) or TPA to neural crest cell cultures can induce melanocyte differentiation without addition of extrinsic SCF. In this study, we excluded the influence of intrinsic SCF by using Sl/Sl mouse embryos to study more precisely the effects of natural cytokines, such as extrinsic soluble SCF or ET-3, or chemical reagents, such as TPA or cholera toxin. We found that SCF is supplied within the wild-type neural crest explants and that ET-3 cannot induce melanocyte differentiation or proliferation without SCF. These results indicate that SCF plays a critical role in survival or G1/S entry of melanocyte progenitors and that SCF initially stimulates their proliferation and then ET-3 accelerates their proliferation and differentiation. TPA has the ability to elicit neural crest cell differentiation into melanocytes without exogenously added SCF but it is not as effective as SCF because many more melanocytes developed in the wild-type neural crest explants cultured with TPA.
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