Cells of the vertebrate neural crest (crest cells) differentiate in vitro to melanocytes and sympathoadrenal (SA) progenitor cells. We have shown previously, using primary J. quail neural crest cultures, the combinatorial effect of bone morphogenetic protein-2 (BMP-2) and cAMP signaling on SA cell development. Herein, we report that in primary J. quail neural crest cultures, BMP-2 and cAMP signaling similarly exert a combinatorial effect on melanocyte development. We demonstrate that BMP-2 treatment of neural crest cells increases melanogenesis by promoting the synthesis of melanin. This increased melanin synthesis by BMP-2 is effected by the selective increase in the transcription of the tyrosinase gene, encoding the rate-limiting enzyme of the melanin biosynthetic pathway. By contrast, BMP-2 exerts no effect on the expression of the tyrosine-related proteins 1 and 2 (Tyrpl and Dct), also involved in the melanin biosynthetic process, or on the expression of microphalmia (Mitf) gene, supporting the fact that BMP-2 does not affect melanocyte differentiation. Employing transient transfection analysis of tyrosinase-reporter constructs in B16 melanoma cells, we demonstrate that the BMP-2 response-element is localized between 900 and 1,100 bp upstream from the tyrosinase transcriptional start site. These studies support a role for BMP-2 in melanogenesis by selectively targeting the expression of the tyrosinase gene involved in melanin biosynthesis.

Bone morphogenetic protein-2 (BMP-2) promotes the development of primary neural crest cells grown in tissue culture to the sympathoadrenal (SA) lineage. Independent studies have characterized the expression patterns of SA-lineage genes in developing chicken embryo; however, studies using cultured primary neural crest cells have characterized only the expression patterns of the catecholaminergic markers, tyrosine hydroxylase (TH) and catecholamines (CAs). To further explore the molecular mechanisms that control SA-cell development using the in vitro model system, it is crucial to define the expression patterns of both the catecholaminergic markers and the genes regulating SA-lineage determination. Accordingly, we defined, in the absence and presence of BMP-2, the temporal expression patterns of TH and CA, the SA lineage-determining genes ASH-1, Phox2a, and Phox2b, the GATA-2 gene, and the pan-neuronal SCG10 gene. Comparison of these data with the reported temporal and spatial patterns of expression in vivo demonstrate that the inductive steps of SA-lineage determination, including the specification of neurotransmitter identity and neuronal fate, are recapitulated in the neural-crest culture system.