Fig. 4. Normal SOX10 activity and EDN3 signalling are required for formation of NLBs and EPCs. Guts from E11.5 wild-type (A), Sox10^+/LacZ (B) or Ret^51/51 (C) embryos were dissociated and cultured under conditions that promote the formation of NLBs. After fixation, cultures were immunostained for TuJ1 (red) and GFAP (green), and counterstained with DAPI. NLBs form readily in gut cultures from wild-type and Ret^51/51 embryos but fail to form in cultures from embryos hemizygous for Sox10. In wild-type gut cultures maintained in standard medium (CM), NLBs form characteristic colonies composed of undifferentiated cells, neurons and glia (Bondurand et al., 2003). Such a colony immunostained for TuJ1 (red) and GFAP (green) is shown (D). These characteristic colonies do not appear in the presence of BQ788 (E). In the absence of CEE, embryo gut cultures failed to form NLBs (F). However, addition of EDN3 was sufficient to rescue the formation of NLBs (G) and EPCs capable of generating multilineage colonies. Part of such an EPC colony is shown in the inset of G.