Impaired growth and differentiation of diploid but not immortal melanoblasts from endothelin receptor B mutant (piebald) mice.

Endothelin 3 (Edn3) and its preferred receptor, endothelin receptor B (Ednrb), are implicated in development, especially that of two neural-crest-derived cell lineages: melanocytes and enteric ganglion cells. Mice and humans with a null mutation at either locus can show major deficiencies in both cell types: congenital white spotting and aganglionic megacolon (Hirschsprung disease in human). Numbers of early (migrating) embryonic melanoblasts are low in Ednrb(ls) mutant mice, while added Edn3 appears to promote the growth of melanocyte precursors in neural crest cultures. However, it is hard to assess cell differentiation in these mixed cultures, and it is not known whether Ednrb has any role in the postnatal melanocytic lineage. We have therefore studied primary cultures of neonatal melanoblasts homozygous for the piebald (Ednrb(s)) mutation. These mutant melanoblasts showed severe impairment of both net cell growth and differentiation compared to wild-type melanoblasts. They were also unresponsive to stimulation of growth by cholera toxin. We have established three immortal lines of melanoblasts and one of melanocytes homozygous for Ednrb(s). These immortal lines, however, had no detectable deficiency of growth or differentiation as judged by cell counts, induced pigmentation and immunocytochemistry for melanocytic markers. Consistent with this, neither Ednrb nor Edn3 mRNA was detected in 3/3 tested immortal lines of mouse melanoblasts and 5/5 lines of melanocytes, of various genotypes. We also report for the first time a method to grow immortal melanoblasts in pure culture, without feeder cells.