Fig. 5

TGF-β-Smad signalling mediates unfit cell killing. a Schematic representation of FACS (top) and mRNA expression changes by RNA-Seq between β-catCA-mosaically introduced (Mosaic), ubiquitously β-catCA-expressing (Ubiquitous), or uninjected embryos (bottom). (See also Supplementary Fig. 6e). b GFP-Smad2 translocates into unfit Wnt/β-catenin-activity cell nuclei. Confocal fluorescence images of cells overexpressing indicated genes in GFP-Smad2 mRNA (200 pg)-injected embryos. Scale bar, 50 μm. Box plots of nuclear/cytoplasmic GFP-Smad2 ratio show 75th, 50th (median) and 25th percentiles (right). Whiskers indicate minimum and maximum. Each dot represents one embryo. (n = 34, 43, 44 and 80 cells, two independent experiments) **p < 0.01 (one-way ANOVA). c Wnt/β-catenin-abnormal cells activate a Smad2/3/4-dependent reporter gene (SBE-luc). Confocal images showing whole-mount fluorescent in situ hybridization of luciferase mRNA in mosaic embryos expressing membrane GFP ± β-catCA or Axin1 (green) and injected with SBE-luc. Arrowheads indicate SBE-luc-active cells. Scale bar, 50 μm. SBE-luc active cell frequencies are graphed. (n = 5 or more embryos, two independent experiments) *p < 0.05 (one-way ANOVA). d, e Smad4 knockdown (d) or forced expression of smad2/3 dominant negative mutants (smad2/3DN) (e) blocks apoptosis of βcatCA- or Axin1-expressing cells. 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 control MO, smad4 MO, or smad2/3DN mRNA. Arrowheads indicate caspase-3-active cells. Scale bar, 50 μm. Graphs show the means ± SEM (n = 6 or more embryos, two independent experiments) of GFP⁺ (β-catCA- or Axin1-expressing) caspase-3-active cell frequencies (right). **p < 0.01, *p < 0.05 (t test). f Mosaic Smad3 activation is sufficient to induce apoptosis. Confocal images showing whole-mount immunostaining of active caspase-3 (magenta) in mosaic embryos expressing membrane GFP ± smad3CA. Arrowheads indicate caspase-3-active cells. Scale bar, 50 μm. Graph shows the means ± SEM (n = 4 or more embryos, two independent experiments) of GFP⁺ caspase-3-active cell frequencies (right). **p < 0.01 (t test). g E-cadherin αC mutant-expressing cell apoptosis is inhibited in smad2/3DN- or skilb-overexpressing embryos. Confocal images showing whole-mount immunostaining of active caspase-3 (magenta) in mosaic embryos expressing membrane GFP and E-cadherin mutant and injected without (−) or with smad2/3DN or skilb mRNA. Arrowheads indicate caspase-3-active cells. Scale bar, 50 μm. Graph shows the means ± SEM (n = 5 or more embryos, two independent experiments) of GFP⁺ caspase-3-active cell frequencies (right). *p < 0.05 ⁽one-way ANOVA). See also Supplementary Fig. 5