Fig. 6.

ROS kills unfit cells downstream of cadherin and Smad signalling. a Representative confocal images showing whole-mount immunostaining of Sephs1 (magenta) in mosaic embryos expressing membrane GFP ± β-catCA or GSK3β (green). Scale bar, 50 μm. b, c Cells with unfit Wnt/β-catenin activity activate ROS production through E-cadherin and Smad signaling. b Fluorescence images showing ROS probe (CellRox Green)-stained mosaic embryos expressing membrane mKO2 ± indicated genes. Scale bars, 200 μm. c Confocal images showing whole-mount immunostaining of 8-OHdG (magenta) in mosaic embryos expressing membrane GFP ± indicated genes (green). Arrowheads indicate 8-OHdG-positive oxidized nuclei. Scale bar, 50 μm. Box plots of fluorescence intensity of 8-OHdG staining show 75th, 50th (median) and 25th percentiles (right). Whiskers indicate minimum and maximum. Each dot represents one embryo. (n = 40 or more cells, two independent experiments) **p < 0.01, *p < 0.05 (one-way ANOVA). d ROS downregulation blocks Wnt-activated or -inhibited cell apoptosis. Confocal images showing whole-mount immunostaining of active caspase-3 (magenta) in mosaic embryos expressing membrane GFP ± β-catCA- or Axin1 (green) and injected with SOD1 or sephs1 mRNA. Arrowheads indicate caspase-3-active cells. Scale bar, 50 μm (left). Graphs show the means ± SEM (n = 5 or 6 embryos, two independent experiments) of GFP⁺ caspase-3-active cell frequencies (right). **p < 0.01, *p < 0.05 (one-way ANOVA). e Cells with unfit Wnt/β-catenin activity reduce Bcl-2 protein levels. Confocal images of mosaic embryos expressing membrane mKO2 ± β-catCA- or Axin1 (magenta) and injected with GFP-Bcl2 mRNA. Scale bar, 50 μm (left). Graphs show the means ± SEM (n = 16 or more cells, two independent experiments) of fluorescence intensity of GFP-Bcl-2 (right). **p < 0.01, *p < 0.05 (one-way ANOVA). See also Supplementary Fig. 6