Fig. 3: Wnt signalling counters epithelialization in the context of naïve pluripotency via Esrrb.

a TCF/Lef:H2B-GFP ES cells and TCF/Lef:H2B-GFP EpiLC stained for GFP, Par6 and DAPI. b ES cells and EpiSC cultured in the presence of DMSO or CH and stained for Par6 and DAPI. c Percentage of ES cells and EpiSC that formed Par6-positive polarised rosettes. Data represent mean ± SD, three independent experiments. d Principal component analysis (PCA) plot of RNA-seq datasets. The cells were grown in 3D culture for 48 h, each culture condition is represented by three replicates. e Scatter plot of gene expression of CH and DMSO-treated cells with differentially expressed genes that are significantly up- or downregulated shown in red, adjusted P value < 0.01, three replicates per culture condition. f Gene tracks representing the binding of Tcf3 at the indicated loci. The x axis represents the linear sequence of genomic DNA, and the y axis represents the total number of mapped reads. g Expression of Wnt target genes with respect to the mean expression across DMSO, 2i, CH or Fgf2/Activin culture conditions. h E14 ES cells expressing ectopically Nanog or Esrrb transgenes, cultured in the presence of DMSO or CH and stained for Par6 and DAPI. i Percentage of ES cells ectopically expressing Tfcp2l1, Klf2, Nanog, Nr0b1 or Esrrb that formed Par6-positive polarised rosettes. Data represent mean ± SD, three independent experiments, two-tailed unpaired Student’s t test, the exact P value is noted in the figure. j EpiLC expressing inducible Esrrb transgene were cultured without Dox (control), in the presence of Dox or in medium supplemented with both of Dox and Lif. After 24 and 48 h, the cells were stained for Par6, Esrrb and DAPI. k Endogenous Nanog expression during EpiLC reprogramming. l Percentage of Par6-positive polarised rosettes at 24 and 48 h of EpiLC reprogramming. Data represent mean ± SD, three independent experiments, two-tailed unpaired Student’s t test, the exact P value is noted in the figure. Scale bars, 10 µm.