Fig 2: fscn1a-depletion inhibits Nodal signalling.(a) Reduction of ARE-luciferase reporter expression in fscn1a morphants. Embryos were co-injected with the reporter plasmids and mis-MO1 or MO1 at the one-cell stage and collected at the 75% epiboly stage for luciferase activity analysis. Data presented as mean with s.d. Student's t test, n=3, ***P<0.001. (b,c) Wild-type and MZfscn1a mutant embryos (b) and embryos injected with mis-MO1 or MO1 (c) were collected at indicated stages for immunoblotting. (d) The effectiveness of mouse fscn1 shRNAs. NMuMG cells were transfected with 8 µg indicated shRNA plasmids per 100 mm dish and collected 48 h after transfection for western blot analyses. (e–g) NIH3T3 and NMuMG cells transfected with indicated shRNA plasmids (2 µg shRNA plasmids together with or without 0.5 µg reporter plasmids in one well of six-well plate) were treated with TGF-ß1 (5 ng ml-l) for 12 h (e,f) or 3 h (g), and collected for luciferase measurements (e,f) or immunoblotting (g). The relative luciferase activity was the mean with s.d. from three independent biological repeats. Student's t test, *P<0.05, ***P<0.001. (h) Overexpression of sqt or casmad2 rescues endoderm induction in fscn1a morphants. Embryos were injected with indicated MOs or mRNAs at the one-cell stage and collected at the 75% epiboly stage for in situ hybridization with sox32 and sox17 probes. Injection doses: mis-MO1, 4 ng; MO1, 4 ng; sqt mRNA, 1 pg; casmad2 mRNA, 100 pg. Scale bar, 200 µm. (i) EB5 cells transfected with ARE-luciferase reporter together shRNA plasmids and treated with or without 25 ng ml-1 Activin A for 12 h before collected for luciferase assays. Data presented as mean with s.d. Student's t test, n=3, *P<0.05. (j) Western blots for the expression of Nanog, FoxA2, Sox17 and Fscn1 in samples taken at specified time points during embryoid body formation. The expression of Tubulin was examined as loading control. (k) Fscn1 knockdown disturbs the formation of endodermal cell lineage during embryoid body differentiation. Embryoid bodies on day 8, derived from EB5 cells infected with the indicated lentiviral shRNAs, were collected for western blots with indicated antibodies.

Fig 3: Pre-pubertal Lgr5high uterus cells behave as Wnt-regulated stem/progenitor cells in ex vivo organoid culture assays. a Representative image of a developing uterine organoid generated from a single EPCAM+GFPhigh cell isolated from a P14 Lgr5-2A-EGFP uterus. b H&E and IHC for K8 on a Lgr5high cell-derived organoid. c Endogenous EGFP fluorescence on a Lgr5high cell-derived organoid. Yellow arrowheads highlight the heterogenous expression of Lgr5-EGFP within individual organoids. Scale bars, 50 µm. d Organoids generated from single EPCAM+GFPhigh cells and EPCAM+GFPneg cells (image representative of five replicates). Scale bars, 1000 µm. e Quantification of organoid formation efficacy from single EPCAM+GFPhigh and EPCAM+GFPneg cells. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test. *** P < 0.001. f Passage frequency of organoids derived from single EPCAM+GFPhigh and EPCAM+GFPneg cells. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (P = 7E-07). g Experimental strategy to ablate Lgr5+ cells in vitro. h Organoid cultures derived from Lgr5-DTR-EGFP and wild-type (control) EPCAM+ uterus cells following Lgr5+ cell ablation by DT treatment in vitro (image representative of five replicates). Scale bars, 200 µm. i Outgrowth efficiency of organoids derived from Lgr5-DTR-EGFP and wild-type mouse EPCAM+ uterus cells following DT treatment in vitro. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (P = 4E-06). j Organoids derived from wild-type EPCAM+ uterus cells cultured under low Wnt3a and low Wnt3a + CHIR conditions (image representative of five replicates). Scale bars, 200 µm. k The average proportions of organoid types under low Wnt3a, low Wnt3a + CHIR and high Wnt3a conditions. Data from five independent experiments are presented. Data were tested for significance by a chi-square test (Low Wnt3a vs. Low Wnt3a + CHIR: P = 2E-17, Low Wnt3a vs. High Wnt3a: P = 2E-08). l QPCR analysis of Lgr5 and Fzd10 expression on organoids generated under each culture condition. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (Lgr5: Low Wnt3a vs. Low Wnt3a + CHIR: P = 0.002, Low Wnt3a vs. High Wnt3a: P = 0.012, Fzd10: Low Wnt3a vs. Low Wnt3a + CHIR: P = 0.018, Low Wnt3a vs. High Wnt3a: P = 0.027). m QPCR analysis of GE markers (Foxa2, Lif, Prss28) and LE markers (Wnt7a, Scnn1a, Irg1) expression on organoids generated under each culture condition Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (Foxa2: P = 9E-04, Lif: P = 0.006, Prss28: P = 0.54, Wnt7a: P = 0.46, Scnn1a: P = 0.49, Irg1: P = 5E-06). n, o Immunostaining for Foxa2, Lif, and Ki67 on round and vacuolated-type organoids. Scale bars, 50 µm. Red arrows indicate Foxa2-positive cells (image representative of three independent mice). ***P < 0.001, **P < 0.01, *P < 0.05, N.S., not significant.

Fig 4: Defining an epithelial Wnt source for neonatal Lgr5high stem/progenitor cells in the developing uterus. a Experimental design of co-culturing EPCAM+GFPhigh and EPCAM+GFPneg cells sorted from P14 Lgr5-2A-EGFP mouse. b Organoid cultures obtained using the indicated conditions (image representative of five replicates). Scale bars, 200 µm. c The efficiency of organoid formation from single EPCAM+GFPhigh cells using the indicated culture conditions. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (Low Wnt3a vs .+GFP neg: P = 0.025, +GFP neg vs. +GFP neg + ETC159: P = 0.002). d Quantitation of organoid size under each culture condition. Three independent fields from each condition were analyzed. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t-test (Low Wnt3a vs. +GFP neg: P = 0.036,+GFP neg vs. +GFP neg + ETC159: P = 0.006). e The average proportions of organoid types generated using the indicated culture conditions from five independent experiments. Data were tested for significance by a chi-square test (Low Wnt3a vs.+GFP neg: P = 9E-13, +GFP neg vs. +GFP neg + ETC159: P = 8E-10). f Experimental strategy of in vivo ETC159 treatment to suppress Wnt secretion in neonatal uterus. g Immunostaining for Foxa2 on ETC159 treated (n = 5) and vehicle-treated control mouse (n = 5) at P14. The insets with solid lines indicate developing GE. Scale bars, 200 µm. h Quantification of the number of Foxa2+ glands in ETC159- treated and vehicle-treated control mouse uterus at P14. Three independent fields from each mouse were analyzed. Data from five independent mice are presented. Red bars represent median. Data were tested for significance using unpaired two-tailed t-test (P = 2E-11). i 3D images of P14 vehicle and ETC159-treated WT mouse, stained for K8. The insets show glandular areas. Scale bars, 100 µm. All images are representative of five independent mice per treatment. ***P < 0.001, **P < 0.01, *P < 0.05.

Fig 5: PD0325901 prevented formation of the primitive streak and inhibits EpiSC self-renewal. a Western blot analysis showed PD0325901 (PD, 1 µM) prevented expression of PS marker T in the presence of CHIR99021 (CHIR, 3 µM) in EpiSCs cultured in N2B27 for 24 hours. b PD0325901 prevented expression of PS marker T and endoderm marker FOXA2 in the presence of Activin A (10 ng/ml) in EpiSCs cultured in N2B27 for 24 hours. c Real-time PCR showed that PD0325901 inhibited differentiation of PS even in the presence of Activin A or CHIR99021. d Immunostaining showed that PD0325901 inhibited both OCT4 and NANOG expression, whereas SB431542 (2 µM) only inhibited NANOG expression in EpiSCs differentiated in N2B27 for 24 hours. Bar, 100 µm. e PD0325901 inhibited both OCT4 and NANOG expression detected by western blot analysis. f Oct4 knockdown promoted PS differentiation in EpiSCs cultured in N2B27 medium for 24 hours. Pluripotent markers, Oct4 and Nanog; primitive streak markers, T and Mixl1; endoderm marker, Foxa2; neuroectoderm markers, Sox1, Sox2 and Pax6. AA Activin A, NC negative control, DMSO dimethyl sulfoxide, EpiSC epiblast stem cell, FGF fibroblast growth factor, T brachyury, ERK extracellular signal-regulated protein kinase, OCT4 octamer-binding transcription factor 4, FOXA2 forkhead box protein A2, SOX2 sex determining region Y-box 2, Mixl1 mix paired-like homeobox, Pax6 paired box 6