Fig. 4: Canonical Wnt pathway inhibition rescues head-regeneration defects across planarian phylogeny.

a, Fluorometric Western blot demonstration of anti-ß-CATENIN-1 antibody G78 crossreactivity with tail tip lysates of different planarian species. Species and amount of lysate/lane as indicated. b, RNAi-mediated Wnt pathway activity modulation in the indicated species from regeneration groups A, B or C (colour coding). Animal cohorts were treated with enhanced green fluorescent protein (eGFP) (RNAi) (control), APC(RNAi) (gain of Wnt signalling) or ß-catenin-1(RNAi) (loss of Wnt signalling; Methods). Top, representative quantitative Western blots for ß-CATENIN-1 and histone H3 as loading control. Bottom, bar graph representation of G78 signal intensity relative to the control in each species (eGFP(RNAi)). Data are presented as mean ± s.d. of the mean of four technical replicates (dots). c, Head-regeneration rescue assay upon Wnt inhibition/ß-catenin-1(RNAi) in the indicated category B or C species. Live images at the indicated day postamputation (dpa) and RNAi conditions; number pairs represent the observed frequency of the shown phenotype; head or eye regeneration. Red triangles, regenerated eyes. Scale bar, 500 µm. ph, pharynx. d, Phylogenetic representation of documented head-regeneration rescued by ß-catenin-1(RNAi) (green check-mark) (phylogeny extracted from Fig. 3d). This study (red): C. pinguis, P. torva, C. robusta and B. candida. Previous studies (blue): D. lacteum²⁵ and P. fluviatilis²⁶. Phagocata kawakatsui, for which head-regeneration rescue was also reported²⁷, was excluded due to the lack of a publicly available transcriptome. ß-Cat-1, ß-catenin-1.