Fig. 2: Active Wnt/β-catenin signalling blocks the establishment of epithelial polarity.

a E14 ES cells grown in 3D culture conditions and stained for Par6, Oct4 and DAPI. b Percentage of ES cell clumps that formed Par6-positive polarised rosettes at 48 h of culture. Data represent mean ± SD, three independent experiments. c Stabilisation of β-catenin via Cre-mediated exon-3 deletion. β-catenin exon-3 fl/fl Cre-ERT2 ES cells were exposed to 4OHT for 2 days before 3D culture. After that, the cells were culture for 48 h and stained for Par6 and DAPI. d Stabilisation of β-catenin via ectopic expression of tetracycline-inducible Gsk3-mutated-β-catenin-IRES-Venus transgene in E14 ES cells. The transgene expression was induced via treatment with Dox for 2 days before 3D culture. The Venus-positive cells were collected by FACS and cultured in Matrigel for 48 h in Dox containing medium supplemented with DMSO, 2i, CH or PD. e Percentage of β-catenin exon-3 Δ/Δ and exon-3 fl/fl ES cells that formed Par6-positive rosettes at 48 h of culture. Data represent mean ± SD, three independent experiments. f Percentage of control (−Dox) and Dox-treated ES cells harbouring Gsk3-mutated-β-catenin transgene that formed Par6-positive rosettes at 48 h of culture. Data represent mean ± SD, four independent experiments. g Schematic representation of the dual role of β-catenin and the experimental approach compensating for the β-catenin function on the cell membrane using E-cadherin-α-catenin fusion. h Deletion of β-catenin via Cre-mediated recombination. β-catenin fl/fl Cre-ERT2 ES cells were exposed to 4OHT for 2 days before 3D culture. After that, the cells were cultured for 48 h and stained for E-cadherin, podocalyxin and DAPI. i β-catenin-deficient (+4OHT) and control (−4OHT) β-catenin fl/fl Cre-ERT2 ES cells were cultured for 48 h and stained for β-catenin, Par6 and DAPI. j Conditional ablation of β-catenin in β-catenin fl/fl Cre-ERT2 ES cells expressing E-cadherin–α-catenin fusion (Eα-fusion). The cells were exposed to 4OHT for 2 days and after that cultured for 48 h and stained for β-catenin, Par6 and DAPI. Scale bars, 10 µm.