Fig. 2. Experimental design for clonal analysis of molecularly identified cells. (Top) Because we were able to molecularly identify distinct neural crest populations in live cultures (see Fig. 1), we were able to perform clonal analysis of these cells by microinjection of lineage dye (photomicrograph). The lineage dye is inherited by all clonal descendants of the labeled precursor, which permits them to be distinguished in differentiated cultures and phenotypically analyzed using cell type-specific markers. (Bottom) The time periods at which we immunolabeled and injected individual receptor-expressing cells are expressed relative to the initiation of segregation of neural crest cells from neural tube explants (see Results, and Materials and Methods). We labeled individual TrkC-expressing cells in 1-6 and 13-16 hour populations. We labeled C-Kit-expressing cells in 13-16 and 30-36 hour populations. The initial overt differentiation of melanocytes (melanization) does not begin until 54 hours and initial overt neuronal differentiation begins at 72 hours. `Complete' differentiation (>95% of all cells) of the cultures occurs by 108 hours.