Promotion of chromatophore differentiation in isolated premigratory neural crest cells by extracellular matrix material explanted on microcarriers.

This study was undertaken to determine whether premigratory neural crest cells of the axolotl embryo differentiate autonomously into chromatophores, or whether stimuli from the environment, particularly from the extracellular matrix, are required for this process. Neural crest cells were excised from the dorsal part of the premigratory crest cord and cultured alone, either in a serum-free salt solution or in the presence of fetal calf serum (FCS), and together with explants of the neural tube or dorsal epidermis. A "microcarrier" technique was developed to assay the possible effects of subepidermal extracellular matrix (ECM) on chromatophore differentiation. ECM was adsorbed in vivo onto microcarriers prepared from Nuclepore filters, by inserting such carriers under the dorsolateral epidermis in the embryonic trunk. Neural crest cells were then cultured on the substrate of ECM deposited on the carriers. Melanophores were detected by DOPA incubation, revealing phenol oxidase activity, or by externally visible accumulation of melanin. Prospective xanthophores were visualized before they became overtly differentiated by alkali-induced pteridine fluorescence. Isolated premigratory neural crest cells did not transform autonomously into any of these phenotypes. Conversely, coculture with the neural tube or the dorsal epidermis, and also the initial presence or later addition of FCS during incubation, resulted in differentiation of neural crest cells into chromatophores. Both chromatophore phenotypes were also expressed on the ECM substrate deposited on the microcarriers. The results indicate that neural crest cells do not differentiate autonomously into melanophores and xanthophores, but that interactions with components of, or factors associated with the extra cellular matrix surrounding the premigratory neural crest and present along the dorsolateral migratory pathway are crucial for the expression of these chromatophore phenotypes in the embryo.