Fig. 7

Fig. 7 Cell-autonomous induction of ngn1 by Sox10. (A) In situ hybridisation reveals robust ngn1 induction by 4 hours after injection of heatshock construct driving wild-type sox10 (hs>sox10) into one-cell stage wild-type embryos followed by 2 hour heat shock at 37°C from 2 hpf (A). Heat-shocked uninjected embryos and embryos injected with an empty heat-shock construct, never contained ngn1⁺ cells at this stage. Injection of a heat-shock vector driving expression of the Sox10^m618 mutant protein (hs>sox10^m618(L142Q)) gave only very weak induction of ngn1. (B) Relative proportions of embryos showing very strong, strong, weak or no induction of ngn1 after each treatment. (C) Double in situ hybridisation of injected embryos with sox10 (red/fluorescence) and ngn1 (blue) showed that ngn1⁺ cells had detectable sox10 message (99/103 cells scored in ten embryos). Nuclear localisation of ngn1 message suggests that nuclear export of these transcripts was limited within the timeframe of this experiment. (D,E) Double immunofluorescence confocal images (single focal plane) of 48 hpf wild-type sox10:eGFP embryos showed perduring GFP protein (green, left panel) in Hu⁺ (red, middle panel) nascent DRG neurons (merged, right panel). At this stage, 54/71 (76%) DRG neurons in five wild-type embryos were detectably GFP⁺; colocalisation has been confirmed in an independent sox10:eGFP line (data not shown). Hu^-/GFP⁺ glial precursors (green) surrounding the Hu⁺/GFP⁺ neurons (yellow) were revealed using a larger confocal pinhole size (E). (F) Double immunofluorescence confocal images (single focal plane) of 40 hpf wild-type ngn1:egfp embryo showing overlap of weak (arrowed) GFP (green, left panel) with Sox10⁺ (red, middle) nascent DRG neurons (merged, right panel). Cells expressing higher levels of GFP, likely to be more mature neurons, lack Sox10 expression (asterisks). Scale bars: 100 μm in A; 10 μm in C; 50 μm in D; 20 μm in E,F.