Global molecular changes that result in a drastic phenotypic alteration with expanded hypostome-like features upon systemic activation of Wnt signalling. Wnt-regulated genes were identified by differential gene expression (DE) profiling after inhibiting GSK-3β with Alsterpaullone (5 μM). a Cartoon depicting the experimental design for activation of Wnt signalling and collection of samples at different time points for transcriptome and phenotype analysis. b Morphological changes after 24 h and ectopic tentacle formation after 72 h of Alsterpaullone treatment (5 µM). Scale bar represents 1 mm. c Expression pattern of HvWnt3a in DMSO control and ALP-treated polyps 24 h post treatment. Scale bar represents 200 µm. d Smear plot showing logarithmic fold change of transcript expression compared to control on y-axis and log of read counts per million on x-axis. Red and Green colour dots denote significantly up- and downregulated genes with log₂ fold change cut-off of ±0.58 with a p value of 0.01 respectively; two biological replicates for each condition were used for DE analysis. e Bar plot showing the percentage of genes differentially regulated in selected GO term classes, upon activation of Wnt signalling. Numbers in the bars represent genes that exhibit altered expression over total genes of respective GO categories.

Homeobox family transcription factors (TFs) upregulated upon activation of Wnt signalling are expressed in head (oral side) of the polyp. Hierarchical clustering of differentially expressed TFs was carried out and domain organization of the upregulated TFs was studied. Expression analysis of selected TFs was carried out by in situ hybridization. a Heatmap of differentially expressed TFs after activation of Wnt signalling. VC.1, VC.2, biological replicates of vehicle (DMSO) controls; ALP.1, ALP.2, biological replicates of Alsterpaullone (5 μM)-treated samples; intensity of red and blue colours indicates respectively the higher and lower expression levels of genes compared to the controls; red and blue colours denote significantly up- and downregulated genes with log₂ fold change cut-off of ±0.58 with a p value of 0.01, respectively; genes that have been previously reported and characterized in Hydra are labelled in black and genes investigated in the current study are labelled in green. b Domain organization of TFs upregulated by Wnt signalling. c Expression pattern of HvOtp1, HvOtx1, Margin and Gorget and magnified images of selected regions of the polyps displaying expression. Scale bar represents 100 µm. d Expression pattern of HvOtp1, HvOtx1, Margin and Gorget in DMSO controls and Alsterpaullone (5 µM)-treated polyps. Scale bar represents 200 µm.

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.

Hydra Wnt signalling targets regulate genes that play a crucial role in dorsoventral patterning. Upregulation of Wnt signalling results in negative regulation of bilaterian-specific ventral markers. To test the role of Wnt/β-catenin targets in dorsoventral patterning we have used a heterologous model system, zebrafish. Hydra Margin and HvOtp1 genes were selected and in vitro synthesized capped mRNAs were injected into one-cell stage zebrafish embryos. a Heatmap displays the significantly differential regulated TGF-β family members upon activation of Wnt signalling. Red and blue colours denote significantly up- and down- regulated genes with log₂ fold change cut-off of ±0.58 with a p value of 0.01 respectively. b Morphological changes upon injections of Margin and HvOtp1 mRNA. Embryos were imaged at 30 hpf (lateral view, anterior to the left) and further classified based on the extent of phenotypic changes into three sub-classes. c Percentage of total embryos observed with different classes of phenotypic changes (N = 3). d Expression levels of axis determining genes upon injections of Hydra Margin and HvOtp1 mRNAs are represented as fold change with respect to antisense mRNA-injected control embryos at 75% epiboly stage. N = 3; Students two-tailed t-test was performed; *p value < 0.03; **p value < 0.002. e Whole-mount in situ hybridization analysis of dorsal organizer gene chordin (dorsal view).

Conserved and novel homologues of bilaterian genes involved in axis patterning and regulated by the Wnt/β-catenin signalling pathway in Hydra. a Stacked bar plot depicts shared homologues of planaria and Xenopus that are differentially regulated by the Wnt/β-catenin signalling pathway in Hydra. b Predicted gene regulatory network of dorsoventral patterning genes based on differentially expressed gene under wnt activation condition. Genes highlighted in red colour are upregulated and genes highlighted in blue are downregulated; dotted line, β-catenin-dependent or -independent regulation. c Transcription factor targets of Wnt/β-catenin signalling identified in Hydra. TFs highlighted in red are also observed as part of the Spemann’s organizer signature⁴⁶.