Comparative analysis of head/tail regeneration specificity.

A) Uninjured S. mediterranea. Scale bar: 2000 µm.

B) Representative live images of scored regeneration outcomes in S.mediterranea transverse pieces. The expression of the tail marker gene fzd4-1 by fluorescent whole mount in situ hybridization (FISH) is additionally shown for the “normal” phenotype. White arrows indicate eye spots as morphological head marker. Scale bars: 500 µm.

Scoring of regeneration outcomes 14 days post-amputation (dpa), depending on C) piece length, D) A/P axis origin or E) body size. Cartoons illustrate amputation paradigms, colours regeneration outcomes as colour-coded in (B). Grey stacks in plots represent pieces with a regeneration phenotype that could not be unambiguously assigned to one of the four categories. n = 394 pieces. N = two independent experiments. Error bars represent the standard error of the mean (SEM).

F-J) As in A-E, but for G. sinensis and scored at 21 dpa. n = 540 pieces. N = two independent experiments.

Wound site-symmetric notum expression contributes to the reduced G. sinensis regeneration specificity.

A) Whole-mount notum fluorescence in situ hybridisation (FISH) time course on trunk pieces from 6 mm long S. mediterranea, as cartooned. Images represent inverted grayscale, maximum intensity projections of Z-stacks from the surface of the specimen to the dorsoventral boundary. hpa: hours post-amputation. Scale bars: 200 µm.

B) Quantification of notum fluorescence signal distribution along the A/P length of the regenerating pieces (% of total signal located in each equidistant axial bin) at the indicated time points. Error bands represent standard deviation (SD).

C) Degree of symmetry (anterior half/posterior half) derived from the data shown in (B). Each point represents one individual piece, the horizontal line the average/median. N = one representative experiment

D-F) As in (A-C), but for G. sinensis.

G) Zoom-in of boxed region in (A), with DAPI-stained nuclei and notum FISH signal shown separately and merged. Each image represents a single 3 µm optical section through subepidermal tissue. Scale bars: 50 µm.

H) As in (G), but for G. sinensis.

I) Scheme of the G. sinensis notum(RNAi) experiment.

J) Representative live images of scored regeneration outcomes. White arrows indicate eye spots as head marker. Scale bars: 500 µm.

K) Quantification of regeneration outcomes in control (egfp) versus notum(RNAi). Same colour-coding as in (J). Grey stacks represent pieces with a regeneration phenotype that could not be unambiguously assigned to one of the scored outcomes. Error bars represent SEM. n = 792 pieces. N = three independent experiments. ** p < 0.01, as assessed by Sidak multiple comparisons test.

L) The same data as in (K), but additionally segregated according to the A/P axis origin of the pieces (AP1 = anterior third; AP2= central third; AP3= posterior third).

M) Whole-mount FISH with the tail marker wnt11-2 on headless G. sinensis pieces. Representative images of pieces with respectively one and two wnt11-2 expression domains and their relative frequency are shown. n = 49 pieces. N = one representative experiment. Scale bars: 500 µm.

Conservation of the axial cWnt signalling gradient in G. sinensis.

A) Schematic illustration of the cWnt signalling pathway and number of homologues of the indicated pathway component in S. mediterranea (red) or G. sinensis (blue). B) Alignment of residues 1-238 of S. mediterranea and G. sinensis β-catenin-1 that were used as the antigen for antibody production. Black, grey and white highlighting indicate amino acid conservation, conservative replacement and non-conservative replacement.

B) Quantitative Western blot analysis of β-catenin-1 abundance as proxy for cWnt signalling activity along the A/P-axis in S. mediterranea and G. sinensis. 6 mm long specimens were cut into 6 equiproportional transverse pieces that were individually blotted; 1 = head piece; 6 = tail piece. Traces represent the average of four independent experiments (each with >12 individuals per species) and error bands the SEM. β-catenin-1 quantifications were normalised to the loading control in each lane (EF1a) and 6-point measurement sets were further normalised to their p6 sample. ns = not significant, p > 0.05; * p < 0.05, **** p < 0.0001, as assessed by Tukey multiple comparisons testing on adjacent positions (e.g. p5 vs p6).

D-E) Clustering of gene expression profiles along the A/P axis of S. mediterranea (D) and G. sinensis (E), determined by RNAseq of specimens cut into 10 (G. sinensis) or 11 (S. mediterranea) equiproportional tissue pieces after pharynx removal. Colour scales represent the maximum-normalised expression of each gene. Asterisks indicate posterior-to-anterior gradient clusters. Source data are provided in Supp. Table 3. Plotted values represent a pool of 10 individuals (D) or the average of four individuals (E). The data used to generate (D) were previously reported16,48.

F) Expression of putative cWnt target genes along the A/P axis of S. mediterranea (red) and G. sinensis (blue). S. mediterranea genes were selected from two published studies16,48.

Establishment of a pharmacological approach for time-limited cWnt gradient manipulations.

A) Quantification of C59 treatment effects on S. mediterranea regeneration outcomes 14 days post-amputation (dpa). The indicated transverse pieces (cartoon) were treated immediately after amputation with the indicated C59 concentrations for 48 h and then left to regenerate in the absence of drug. Stack colors correspond to B), Representative images of the scored regeneration outcomes plotted in (A). White arrows indicate eye spots as head marker. n = 206 pieces. N = two independent experiments. C) porcn-b(RNAi) in S. mediterranea phenocopies C59 treatment. Quantification of regeneration outcomes of the indicated transverse pieces (cartoon) at 14 dpa versus control (egfp(RNAi)). n = 181 pieces; N = two independent experiments. Stack colours correspond to the regeneration outcomes shown in (D), representative images of the scored outcomes plotted in (C). E-F) As in A-B, but in G. sinensis. n = 156 pieces. N = three independent experiments.

G) Fragmentation pattern of C59 after ms-fragmentation on an Orbitrap mass spectrometer: The singly-charged C59 precursor with m/z 380.176 Da generates two unique fragments with a mass of 183.104 Da and 210.091 Da, respectively.

H) C59 quantification by mass spectrometry with sub-micromolar sensitivity. Titration curve, obtained by adding C59 standard to untreated G. sinensis tissue extracts to the indicated final concentrations. C59 intensity was normalised to the intensity of a second compound, LGK-974, spiked into all samples at the same concentration as an internal control. Error bands represent the SD.

I) C59 clearance kinetics from 12 mm S. mediterranea. Specimens were treated for 14 days with 50 µM C59 prior to transfer into drug-free culture medium and sampling at the indicated days post wash-out. Residual C59 concentrations were normalized to the t0 tissue extract drug concentration. n = 3 individuals/time point. N = one representative experiment. J) C59 clearance kinetics from 6 mm and 18 mm G. sinensis. Specimens were treated for 7 days with 2 µM C59 prior to transfer into drug-free culture medium and sampling at the indicated days post wash-out. n = 3 individuals per size/time point combination. N = two (6 mm) or three (18 mm) independent experiments. Error bands represent the SEM. Scale bars: 500 µm.

The cWnt gradient contributes to regeneration specificity in G. sinensis.

A) Cartoon of the c59 treatment protocol used for testing cWnt gradient contributions to S. mediterranea regeneration specificity.

B) Western blot analysis of β-catenin-1 abundance/cWnt signalling activity along the A/P axis in the indicated 6 mm long S. mediterranea, after 14 days pre-treatment with 50 µM C59 or vehicle (0.75% DMSO). The β-catenin-1 signals in lysates of equiproportional transverse pieces of the indicated specimens were normalized to the loading control (Smed-EF1a) and each 6-point measurement set was further normalised to the tail piece (p6) of the DMSO-control. n = 10 individuals per group. N = three independent experiments. Error bars represent the SEM.

C) Regeneration outcome quantification of the indicated S. mediterranea transverse pieces under the same vehicle/C59 treatment protocol as in (A, B). Colour-coding of regeneration outcomes as indicated. n = 315 pieces. N = four independent experiments.

D) As in (C), but with pieces separated into three A/P axis bins as cartooned.

E) Representative live image (left) and tail marker fzd4-1 FISH image (right) of “normal” regenerated fragments, confirming wild-type regeneration in this experiment.

F-I) As in (A-D), except with G. sinensis. Note the lower drug concentrations (0.5% DMSO vehicle or 2 µM C59), shorter pre-treatment and 4 days of subsequent drug washout for these experiments. N = 195 pieces across three independent experiments (H-I).

J) Representative live images of regeneration outcomes, colour-coded as in H-I

K-N) As in (B-E), but with 15 mm long S. mediterranea. n = 3 individuals/group (K) and 373 pieces (L-M). N = 3 independent experiments (K-M).

O-R) As in (B-E) but with 18 mm long G. sinensis. n = 3 individuals/group (O) and 699 pieces (P-Q). N = five independent experiments (O-Q). ns not significant, p > 0.05; ** p < 0.01; **** p < 0.0001; as assessed by 2-way ANOVA with Bonferroni multiple comparisons test.

White arrows indicate eye spots as head marker. Scale bars: 500 µm.

Scaling of the cWnt gradient and implications for planarian tissue polarity

A) Schematic illustration of how cWnt gradient scaling and the associated slope decrease might account for the body size-dependence of regeneration specificity in G. sinensis

B-C) Western blot quantification of axial β-catenin-1 abundance in 6 mm, 12 mm and 18 mm long S. mediterranea (B) and G. sinensis (C). The β-catenin-1 signals in lysates of equiproportional transverse pieces were normalised to the loading control (Smed-EF1a) and each 6-point measurement set was further normalised to the tail piece (p6) of the DMSO-control. Traces represent the average of four independent experiments (each with 4-16 individuals per species) and are shown normalised by body length (L). Error bands represent the SEM. ns = not significant, p > 0.05; * p < 0.05, **** p < 0.0001; as assessed by Tukey multiple comparisons testing on adjacent positions (e.g. p5 vs p6). Note that the 6 mm data shown here are the same data as shown in Fig. 3C.

D) Working model of the mechanisms underlying planarian tissue polarity (a) and species-dependent regeneration specificity (b). See text for details.

A distance-based phylogenetic tree of cox1 sequences from nine different Girardia species27 and the Girardia strain used in this study, GOE00326. The placement of the GOE00326 cox1 sequence is strong support for a G. sinensis species-level classification. See Materials and Methods for a detailed explanation of how the tree was generated.

A) Quantification of regeneration outcomes in 1 mm pieces from 6 mm S. mediterranea after β-catenin-1(RNAi) or control egfp(RNAi). N = 68 pieces from one representative experiment. White arrows indicate eye spots as morphological head marker. Scale bars: 500 µm.

B) Quantitative Western blot measurements on egfp(RNAi) control and β-catenin-1(RNAi) protein extracts to verify specificity of the anti-β-catenin-1 antibody. EF1A was used as loading control. n = five individuals per RNAi condition. N = one representative experiment.

C) List of screened commercially-available cWnt signalling inhibitors and activators.

D) Graphical representation of the expression of two S. mediterranea porcupine homologues across planarian cell types. Generated with the Planaria Single Cell Atlas online resource (https://shiny.mdc-berlin.de/psca/)34.

E) Clearance of C59 from the tissue of 18 mm G. sinensis treated for 7 days with 2 µM C59 and then subjected to 4 days of drug washout. AP1, AP2 and AP3 represent different positions along the A/P axis. n = 3 individuals per time point. N = three independent experiments. Error bands represent SEM.

F) Quantitative Western blotting of cWnt signalling activity in the tail tip of 18 mm G. sinensis treated for 7 days with 2 µM C59 and then subjected to 0, 1, 2, 3 or 4 days of drug washout. Vehicle-only (VO) controls were treated for 7 days with DMSO and then subjected to 4 days of washout. Error bars represent SEM of three independent experiments. Each experiment is normalised to its respective VO measurement.

A) Representative live images of the two double-headed individuals resulting from the experiment shown in Fig. 5G-J. White arrows indicate eyes and thus head identity. Scale bars: 500 µm.

B) Overview of experimental approach. “Pre” large G. sinensis were treated with 2 µM C59 or 0.5 % DMSO as vehicle-only control for 7d, washed for 1d to allow for partial drug clearance, cut into 9ths and left to regenerate. “Post” animals were untreated prior and instead treated for 2 d with 0.5 µM C59. The shaded red rectangle represents tissue C59 levels contributed by the gradient manipulation (dark red) and persisting from 0 days post-amputation (dpa) onwards (light red). The shaded blue rectangle signifies tissue C59 levels contributed exclusively by post-amputation treatment.

C) Quantification of tissue C59 concentration in Pre vs Post regenerating pieces. Each point represents the average of three technical triplicate measurements, normalised to the respective Pre 1 dpa value. n = 14 pieces per data point. N = 4 independent experiments.

D) Representative images of live morphology and expression pattern of the anterior marker sfrp1 at 10 dpa. White arrows indicate eyes. In these experiments, regenerating pieces with biaxial expression of sfrp1 were scored as double-headed.Quantification of double-head frequency. n = 369 Pre and 326 Post pieces. N = 4 independent experiments. Statistical significance was assessed by Wilcoxon test (D) or Mann-Whitney test (E). ns = not significant (i.e. p > 0.05), * = significant (i.e. p < 0.05).

A-B) Log expression of RNA sequencing counts of S. mediterranea (left) and G. sinensis (right) shows two peaks, which are fitted by a linear combination (red) of two Gaussian functions (black dashed). We associate the Gaussian with the lower mean with noise, which allows us to define a threshold (black solid) to distinguish a meaningful signal from noise

C-D) The difference between maximum and minimum expression of each gene (blue dots) is compared to the fluctuation (Std) about the smoothed trend (moving average of 3 data points). This allows us to define a threshold (black line) to identify genes, for which the maximum expression change is not much larger (<5x) than the average fluctuations about the smoothed trend

E-F) Number of non-trivial clusters (i.e. with more than a single gene) for complete hierarchical clustering (with a correlation distance) is shown as a function of the distance cut-off. We define a threshold for our clustering (black solid) at an inflection of the curve, at which the cluster number is also stable for a range of cut-off values (i.e. gap between data points).