Characterization and isolation of melanocyte progenitors from mouse embryos

Whole mount immunohistochemistry and flow cytometry have been used to determine the morphological and molecular features that distinguish melanoblasts from surrounding cells. Whole mount immunohistochemistry of mouse embryos using anti-c-Kit monoclonal antibody revealed two distinct types of c-Kit(+) cells; one dendritic and the other round in shape. The distribution of c-Kit(+) dendritic cells in 12.5 days postcoitem embryos correlated well with that of tyrosinase-related protein-2 expression, while the distribution of c-Kit(+) round cells overlaps that of CD45(+) cells. This observation suggests that melanoblasts are distinguishable from other c-Kit(+) cells by their dendritic shape. Mice homozygous for the steel-Dickie mutation (S/(d)/S/(d)) were analyzed to further distinguish melanoblasts from hematopoietic progenitor cells. S/(d)/S/(d) mice are unpigmented but contain hematopoietic cells, although reduced in number. Although no c-Kit(+) dendritic cells were detectable in the S/(d)/S/(d) embryos, a significant number of c-Kit(+) round cells were present in the same embryos. To further analyze characteristic features of melanoblasts, c-Kit(+)CD45- and c-Kit(+)CD45(+) cells were isolated from dissociated embryonic skin by fluorescent activated cell sorter and the expression of TRP2 melanogenic enzyme was analyzed. Consistent with histological analysis, most c-Kit(+)CD45- cells were TRP2(+). c-Kit(+)CD45(+) cells failed to express TRP2. These results show that most of the melanoblasts are c-Kit(+)TRP2(+)CD45- dendritic cells and can be discriminated from other cells by flow cytometry or by their morphology.