Fig 2: WNT4 acts downstream of CTNNB1 to cause germ cell loss in vivo.(A–D) Photomicrographs of testes from 8 week-old animals of the indicated genotypes. Insets show sections of epididymides from the corresponding animals. (E) Photomicrograph of a testis of the indicated genotype showing the severe testicular degeneration, coagulation necrosis and intratubular hemorrhage phenotypes described in the text. (F) Testis weights from 8 week-old animals of the indicated genotypes, n = 4 animals/genotype. C;Wf/-: Ctnnb1 tm1Mmt/+;Wnt4 flox/- (control), C;A: Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+, C;W+/-;A: Ctnnb1 tm1Mmt/+;Wnt4 +/-;Amhr2 tm3(cre)Bhr/+, C;Wf/-;A: Ctnnb1 tm1Mmt/+;Wnt4 flox/-;Amhr2 tm3(cre)Bhr/+. (G) Wnt4 mRNA levels in the mice described in panel F. Note the logarithmic scale on the Y axis. (H) CTNNB1 immunoblot analyses of testes from 8 week-old animals of the indicate genotypes. The lower band corresponds to the dominant-stable CTNNB1 mutant protein produced by the recombined Ctnnb1 tm1Mmt/+ allele. ACTB was used as a loading control. Animals showing the severe degenerative phenotype described in the text and shown in panel E were excluded from the data analyses shown in panels F–H. All data are expressed as mean (columns) ± SEM (error bars). Groups labeled with different letters (a, b, c) were significantly different (P<0.05).

Fig 3: Ectopic expression of ?-cateninS28A in mESCs activates the Wnt pathway without altering cytosolic ß-catenin levels.(A) mESCs were stably transfected with a Myc-tagged S28A variant of ?-catenin. Clones were isolated, expanded and screened for ?-catS28A expression by western blotting of hypotonic lysates using a tag-specific antibody. (B) Alkaline phosphatase staining of WT, ?-catS28A-expressing and ß-catS33A-expressing mESC colonies, visualized using bright-field microscopy. Bar = 400 µm. (C) Western blot analyses of nuclear and cytosolic extracts prepared from WT and ?-catS28A cell lines, as indicated. Elevated levels of ?-cat protein were detected in both cytoplasmic and nuclear lysates of ?-catS28A-overexpressing cell lines. (D) TCF reporter activity of mESCs stably expressing ?-catS28A. (E) Induction of the ß-catenin/TCF target genes, Axin2, Brachyury and Cdx1, but not c-Myc, in mESCs stably expressing ?-catS28A or ß-catS33A. (F) Expression of stabilized ?-catenin in WT mESCs does not result in detectable levels of cytosolic ß-catenin as determined by western blot analysis of hypotonic lysates. Cytosolic levels of ß-catenin in GSK-3 DKO mESCs or WT mESCs treated with CHIR99021 (24 h, 15 µM) are provided as positive controls. For (D) and (E), Bars represent means and error bars indicate s.e.m. (n = 3).

Fig 4: LV-delivered mitogens drive hiPSC-CM proliferation in monolayers.(A) Schematic of experimental design in hiPSC-CM monolayers. (B–F) Flow cytometry analysis of mCherry+ hiPSC-CMs showing (B) fold-change (FC) in EdU incorporation relative to control LV-transduced hiPSC-CMs, (C) percentage of H3P+ CMs, (D, E) percentage of 2N and >2N cells in all CMs (D) and EdU+ CMs (E), and (F) percentage of apoptotic Cc3+ CMs. (G, H) Analysis of relative (G) gene expression of Yap and its targets Ctgf and Cyr61, and (H) total and active Yap protein abundance in caYap8SA-transduced vs. control hiPSC-CMs. (I,J) Representative immunostaining images (I) and quantified nuclear mean fluorescence intensity (MFI; J) of YAP in caYap8SA-transduced versus control hiPSC-CMs. (K, L) Analysis of relative (K) expression of Ctnnb1 and Wnt-signaling genes Lef1 and Axin2 and (L) Ctnnb1 protein abundance in caCtnnb1-transduced versus control hiPSC-CMs. (M, N) Analysis of relative Ccnd2 (M) gene and (N) protein expression and (O, P) representative immunostaining images (O) and quantified nuclear MFI (P) of Ccnd2 in Ccnd2-transduced versus control hiPSC-CMs. Data: box and whiskers showing distribution and min to max. Column graphs showing mean+ SD (*p<0.05, **p<0.01, ***p<0.001 vs. Ctrl LV). See Supplementary file 1 for sample numbers and complete statistical information for all figures. Cc3, cleaved caspase-3; hiPSC-CM, human-induced pluripotent stem cell-derived cardiomyocyte; LV, lentiviral vector.

Fig 5: Stem cell dynamics are accelerated within Ctnnb1-driven tumours but inhibited adjacently to them. (A) Schematic diagram and real example of measurements used to characterise clones inside and outside of tumours. (B) Graph displaying average clone size across binned median of tumour sizes. Each bin contains 50 clones. Points and bars show median and interquartile range. (C) Graph comparing the proportion of surviving clones that have monoclonally converged within tumours and in surrounding tissue. (D) As in C but with the intra-tumour data split into those clones that are further from (tumour edge) or closer to the Tc (falling without or within 75% of the average tumour radius, respectively). (E) The proportion of surviving non-fixed clones (in crypt quartiles) inside and outside tumours evolve differently in time when the monoclonal proportion inside tumours has been rescaled to be identical to the monoclonal proportion outside tumours. (F) Divergence from theoretical wild type of the clonal dynamics in crypts occupying adjacent intestinal epithelia surrounding ß-catenin tumours. Clones are grouped into overlapping bins containing equal numbers of crypts (100 clones per bin) increasing in distance from the edge of the tumour. Line and ribbon shading show the 80% and 95% credible intervals around the median of 1000 simulated datasets of 100 wild-type crypts. Point sizes show the average clone size in each bin. Distances are measured in crypt diameters. (G) The daily replacement rate ? of stem cells undergoing neutral drift is inferred for clones in tumour-adjacent epithelia. Close to tumour (Re<7 crypt diameters), ?=0.07 [80% confidence interval (c.i.): 0.061-0.083]; far from tumour (Re=7 crypt diameters), ?=0.09 (80% c.i.: 0.079-0.103).