Analysis of lineage segregation during mammalian neural crest development has 
not been sufficiently performed due to technical difficulties. In the present 
study, therefore, we established a clonal culture system of mouse neural crest 
cells in order to analyze developmental potentials of individual neural crest 
cells and their patterns of lineage segregation. 
12-O-Tetradecanoylphorbol-13-acetate (TPA) and cholera toxin (CT) were applied 
to culture medium to trigger melanogenic differentiation of mouse neural crest 
cells. Three morphologically distinct types of clones were observed. (1) 
"Pigmented clones" consisted of melanocytes only, suggesting that the 
clone-forming cells were committed to the melanogenic lineage. These clones were 
observed only in the presence of TPA and CT. The proportion of this type of 
clone (8%) was much lower than that of the equivalent type of clone in quail 
trunk neural crest (40-60%; Sieber-Blum and Cohen, 1980, Dev. Biol. 80, 96-106). 
It therefore appears that the segregation pattern to the melanogenic lineage 
during mouse neural crest development in vitro differs quantitatively from that 
in the quail. (2) "Mixed clones" consisted of pigmented and unpigmented cells. 
Like pigmented clones, they were observed only in the presence of TPA and CT. 
The clones contained up to four types of cells: melanocytes, S100-positive cells 
(Schwann cells or melanogenic precursor cells), serotonin (5-HT)-positive 
autonomic neuron-like cells, and substance P (SP)-immunoreactive sensory 
neuron-like cells. Thus, at least some mixed clone-forming cells are 
pluripotent. (3) Two classes of "unpigmented clones" were observed that 
consisted of unpigmented cells only. These clones developed in the presence and 
absence of TPA and CT. Unpigmented clones in one class contained up to three 
types of cells as well as other, as yet unidentified cells: S100-, 5-HT-, and 
SP-positive cells. This observation suggests that at least some of these clones 
originate from cells with a partially restricted developmental potential. Clones 
in another class consisted of S100- or SP-positive cells only. These clones 
might be derived from cells restricted to the SP-positive neuronal cell or 
melanocyte/Schwann cell lineage. The present data indicate that at initiation of 
migration, the mouse neural crest of the trunk region is a heterogeneous 
population of cells containing pluripotent cells, cells with a restricted 
developmental potential, and cells apparently committed to the melanogenic cell 
lineage.