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

Fig. 3.

Fig. 3

Membrane β-catenin and cadherin proteins form their concentration gradient along the AP-axis in a Wnt/β-catenin gradient-dependent manner. a Direct β-catenin-E-cadherin binding is required for unfit cell apoptosis induction. Schematic illustration of nuclear and membrane β-catenin functions (right). Graph show means ± SEM of GFP+ caspase-3-active cell frequencies in Lef1 or β-catCA mutant mosaic embryos (left) (n = 9, 11, 13, 13, 31, 14 and 6 embryos, two or more independent experiments). **p < 0.01 (one-way ANOVA). b Representative confocal fluorescence images show GFP (green), active caspase-3 (magenta), and DNA (blue) in embryos introduced with cells expressing Lef1 mutants or β-catCA mutants. Scale bar, 50 μm. c Both β-catCA and NES-β-catCA localize to the plasma membrane in zebrafish embryos. Scale bar, 20 μm. d Membrane β-catenin and E-cadherin protein levels correlate with Wnt/β-catenin signalling activity. Optical sagittal cross-section (dorsal side) in 8.3 hpf embryos. Panels show fluorescence of OTM:d2EGFP (green) and DNA (blue), fluorescence intensity of β-catenin and E-cadherin staining, and their magnified views. Scale bar, 50 μm. Bottom graph shows fluorescence intensity (means ± SEM, n = 12 cells per region) of β-catenin and E-cadherin staining at intercellular boundaries within three evenly divided regions across the AP axis. (Also see Methods). **p < 0.01 (t test). e Inhibition of Wnt signalling (Dkk1 overexpression) reduces nuclear as well as membrane β-catenin. Dorsal side of whole-mount β-catenin immunostaining of Tg(HS:dkk1b-GFP) zebrafish embryos and sibling embryos at 9 hpf exposed to heat shock at 37 °C from 4.3 to 5.3 hpf. +/− and −/− indicate the heterozygous transgenic sibling and non-transgenic wild-type sibling, respectively. Scale bar, 50 μm. Bottom graph shows fluorescent intensity (means ± SEM, n = 10 cells). **p < 0.01 (t test). f, g E-cadherin protein level correlates with Wnt/β-catenin signalling activity. 9 hpf embryos injected with dkk1b mRNA or Tg(HS:hsp70l:GFP-T2A-β-catCA) embryos exposed heat shock at 37 °C from 4.3 to 5.3 hpf were extracted and then subjected into immunoblotting with anti-E-cadherin and anti-α-tubulin antibodies (f) or qPCR (g)