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Fig. 3. Neural crest progenitors with glial potential persist in Wnt1-Cre⁺ Rbpsuh^fl/fl sensory ganglia despite the lack of gliogenesis in vivo, but require stimulation by a gliogenic factor to undergo gliogenesis in culture. (A-N) We dissociated and cultured dorsal root ganglion cells from E13.5 control and Wnt1-Cre⁺ Rbpsuh^fl/fl mouse embryos at clonal density (500 cells per 35 mm dish) under adherent conditions. (A,B) Multilineage colonies (N+G+M) contained neurons (N, peripherin⁺), glia (G, Gfap⁺) and myofibroblasts [M, Sma⁺ (Acta2/Actg1 - Mouse Genome Informatics)]. Other colonies were also counted. The data are expressed as the percentage of cells added to culture that formed each type of colony. Wnt1-Cre⁺ Rbpsuh^fl/fl cells formed significantly fewer multilineage and glia-containing (G-containing) colonies (which included all multilineage, glia-only, and other colonies that contained glia) as compared with control cells (A; ^*, P<0.05). Wnt1-Cre⁺ Rbpsuh^fl/fl cells did not differ from control cells in their ability to form neuron-containing or myofibroblast-containing colonies (A). Addition of the gliogenic factor neuregulin (Nrg) to sister cultures allowed Wnt1-Cre⁺ Rbpsuh^fl/fl cells to form normal numbers of multilineage, glia-containing and other types of colonies as compared with control cells (B). (C-N) Representative photos of single fields of view from within multilineage colonies cultured from control cells in standard medium (C-E), Wnt1-Cre⁺ Rbpsuh^fl/fl cells in standard medium (F-H), control cells in the presence of Nrg (I-K) and Wnt1-Cre⁺ Rbpsuh^fl/fl cells in Nrg (L-N) show that Rbpsuh-deficient NCSCs rarely formed glia (H, Gfap, red), except in the presence of Nrg (N) (three to six independent experiments).

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