Fig. 3

Inhibition of ectopic tentacle phenotype by knockdown of β-catenin and identification of Wnt/β-catenin target genes. To identify the β-catenin- mediated gene regulation by Wnt signalling, transcriptome analysis was performed following ectopic activation of Wnt signalling and knockdown of β-catenin. a, b Inhibition of the ectopic tentacle phenotype caused by systemic activation of Wnt signalling at 62%, 72% and 25% upon knockdown of β-catenin, Margin and Gorget, respectively compared to siGFP controls. Scale bar in a, 500 µm; N = 3; n = x in b, number of polyps; ***p < 0.001. c Heatmap of significantly differentially regulated TFs by Wnt signalling in β-catenin-dependent manner. siGFP.1 and siGFP.2, are the biological replicates of the controls where siGFP was used; siBCAT.1 and siBCAT.2 are the biological replicates of β-catenin knockdown samples; intensity of red and blue colours indicates respectively the higher and lower expression of genes compared to the controls; significantly dysregulated genes were selected based on log₂ fold change cut-off of ±0.58 with a p value of 0.05; TFs highlighted in red colour are investigated in this study; UNC, uncharacterized genes with no clear homology to known genes. d Venn diagram depicts the overlap of TFs upregulated by activation of Wnt signalling and downregulated by β-catenin knockdown. ALP-UP, upregulated TFs upon Alsterpaullone treatment; siβ-catenin-DOWN, downregulated TFs after β-catenin knockdown. e TCF7L2-binding motif used to scan the Margin promoter (2 kb upstream to TSS). Binding motifs are highlighted in purple coloured bars where the numbers adjacent to these bars denote the motif containing region selected for ChIP qRT-PCR; TCF7L2-binding motif sequences are given below the bars and genomic locations relative to TSS are given above the bars. f ChIP qRT-PCR performed with different sets of primers designed to amplify the TCF7L2-binding motifs in Margin promoter after Alsterpaullone (5 µm) treatment and in DMSO solvent control. Plot displays log₂ fold enrichment of β-catenin occupancy over IgG control; Desert denotes the region with no variable occupancy under control and treatment conditions; N = 3; Students two-tailed t-test was performed; *p value < 0.1.