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. 2019 Oct 17;10:4710. doi: 10.1038/s41467-019-12609-4

Fig. 2.

Fig. 2

Substantial difference in Wnt/β-catenin activity between unfit and neighbouring cells triggers unfit cell apoptosis. a Schematic illustration of experimental introduction of fluorescent Wnt/β-catenin-abnormal cells in zebrafish early embryo through heat shock induction. b, c Artificially introduced Wnt/β-catenin-abnormal cells undergo apoptosis. Confocal microscopy images showing whole-mount immunostaining of active caspase-3 (magenta) in mosaic embryos expressing membrane GFP ± Wnt activators or inhibitors. Scale bar, 50 μm. Arrowheads indicate caspase-3-active cells. c Box plots of GFP+ caspase-3-active cell frequencies show 75th, 50th (median), and 25th percentiles (right). Whiskers indicate minimum and maximum. Each dot represents one embryo (n = 23, 21, 16, 17 and 16 embryos, two or more independent experiments). **p < 0.01 (one-way ANOVA). d Embryos artificially introduced with cells expressing membrane GFP alone (GFP) or with β-catCA or Axin1 with or without caspase inhibitor p35. Fluorescence and bright-field images after heat-shock. p35 expression (magenta). Scale bar, 200 μm. (See also Supplementary Movies 3 and 4). e Surrounding normal cells are required for apoptosis induction of β-catCA- or GSK-3β-overexpressing cells. [Tg(hsp70l:GFP-T2A-β-catCA)] or [Tg(hsp70l:GFP-T2A-GSK-3β)] transgenic lines were exposed to heat shock. Percentages of embryos showing similar phenotype and number of embryos are shown. Scale bar, 200 μm. f Alleviation of Wnt/β-catenin activity difference between β-catCA-overexpressing cells and surrounding normal cells by injecting APC MO blocks β-catCA-overexpressing cell elimination. Cell membrane was visualized by injecting membrane-tagged mKO2 mRNA (red). Scale bar, 200 μm. g Cells causing excess noise in Wnt/β-catenin-gradients efficiently undergo apoptosis. Top panels show the maps of artificially introduced cells in zebrafish embryos. Bottom graphs show the means ± SEM of caspase-3-active cell frequencies within the divided range along the AP axis (GSK-3βDN, n = 8 embryos, 1131 cells; Axin1, n = 14 embryos, 1308 cells). See also Supplementary Fig. 3