The trunk neural crest of vertebrate embryos is a transient collection of precursor cells present along the dorsal aspect of the neural tube. These cells migrate on two distinct pathways and give rise to specific derivatives in precise embryonic locations. One group of crest cells migrates early on a ventral pathway and generates neurons and glial cells. A later-dispersing group migrates laterally and gives rise to melanocytes in the skin. These observations raise the possibility that the appearance of distinct derivatives in different embryonic locations is a consequence of lineage restrictions specified before or soon after the onset of neural crest cell migration.

To test this notion, we have assessed when and in what order distinct cell fates are specified during neural crest development. We determined the proportions of different types of precursor cells in cultured neural crest populations immediately after emergence from the neural tube and at intervals as development proceeds. We found that the initial neural crest population was a heterogeneous mixture of precursors almost half of which generated singlephenotype clones. Distinct neurogenic and melanogenic sublineages were also present in the outgrowth population almost immediately, but melanogenic precursors dispersed from the neural tube only after many neurogenic precursors had already done so. A discrete fate-restricted neuronal precursor population was distinguished before entirely separate fate-restricted melanocyte and glial precursor populations were present, and well before initial neuronal differentiation. Taken together, our results demonstrate that lineage-restricted subpopulations constitute a major portion of the initial neural crest population and that neural crest diversification occurs well before overt differentiation by the asynchronous restriction of distinct cell fates. Thus, the different morphogenetic and differentiative behavior of neural crest subsets in vivo may result from earlier cell fate-specification events that generate developmentally distinct subpopulations that respond differentially to environmental cues.