Fig. 7.

Foxd3 controls multipotency of individual NC cells. (A) NC cells were cultured at clonal density in self-renewal medium for 6 days and then differentiation was promoted for 8 days by changing culture medium. Immunocytochemical analysis of multilineage differentiation into neurons (peripherin), glia (GFAP, red) and myofibroblasts (SMA, green). Images of a single field from a control (N+G+M) multilineage containing colony (left), a rare Foxd3 mutant (N+G) colony (center) and a more typical (M-only) mutant colony (right panels) are shown. Insets show higher magnification of Peripherin-positive neurons. (B,C) Quantification of lineage composition of single-cell derived colonies. Both vagal and trunk Foxd3 mutant NC formed bi-potent colonies with neural potential (N+G and N+M) at reduced frequency and rarely formed multipotent colonies compared with control cultures. Mutant clonal cultures contained an increased percentage of myofibroblast-only (M-only) colonies and were unchanged from control NC in establishing restricted progenitor colonies (N-only or G-only). Data represent six independent experiments with at least three technical replicates of clonal cultures from ten mutant and six wild-type littermate control embryos. All statistics are mean ± s.e.m.; ^*, P<0.05 comparing control with mutant. (D) A model of Foxd3 action in multipotent NCSCs. Foxd3 is required for self-renewal of NCSCs (curved arrows) and for maintenance of the neural lineage choice (N). Without Foxd3 (right side of diagram) NCSCs have reduced self-renewal capacity (dashed X) and preferentially and precociously differentiate towards mesenchymal fates.