Fig 2: Yap-induced Epiregulin (Ereg) expression directs ductal progenitor specification.(A) qPCR analysis of Ereg expression in E15.5 WT vs. Yap-cnull SMGs. The average of three SMGs from different litters is shown +S.E.M. [one sample t-test: ***p<0.0001]. (B) In situ hybridization of Ereg mRNA in E13.5 WT (duct and bud) and Yap-cnull (bud) SMGs. (C) Combined in situ hybridization for Ereg mRNA (red) and IF for Yap (green) in E13.5 WT SMGs. (D,E) E13.5 WT and Yap-cnull SMGs were dissected and cultured for 24 hr in the presence or absence of 0.5 µg/mL of exogenous Ereg protein and analyzed by phase-contrast and IF for Krt14 (K14, magenta), Krt5 (K5, green), Krt19 (K19, red), TuJ1 (yellow), and F-actin (white, Phalloidin). DAPI was used to mark the nuclei (blue). Scale = 100 µm. (F) qPCR analysis of Yap, Krt5, and Krt14 expression in the conditions of (D) and (E). The average of three SMGs from different litters is shown +S.E.M. [one sample t-test: ***p<0.0001]. All images represent observations made from a minimum of three biological repeats.DOI: http://dx.doi.org/10.7554/eLife.23499.010

Fig 3: Overexpression of epithelial wild-type FGFR1 synergizes with paracrine FGF10 to induce EMT. (A) Diagram of the bi-cistronic lentiviral vector for the aberrant expression of FGFR1, FGFR2, FGF10, or Src. While the oncogenic gene was controlled by the ubiquitin promoter, RFP or GFP was regulated by the CMV promoter. (B) 293 T cells were transduced with control vector, FGFR1, or FGFR2 by lentiviral infection. The protein lysates were harvested for immunoblotting. The expression levels of FGFR1 or FGFR2, and ?-tubulin were examined by Western blot. Aberrant expression levels of FGFR1 and FGFR2 were confirmed. (C) UGSM cells were transduced with control vector or FGF10 by lentiviral infection. Total mRNA was extracted for the analysis of FGF10 expression by RT-PCR. FGF10 was highly expressed in FGF10-UGSM cells. (D) Diagram for evaluation of FGFR1/2 overexpression in epithelium and aberrant paracrine FGF10-induced tumorigenesis by the prostate tissue regeneration assay in vivo. Freshly isolated prostate epithelial cells were transduced with control vector (FUCRW), FGFR1, or FGFR2 by lentiviral infection. UGSM cells were isolated from 16.5-day-old mouse embryos. UGSM cells were transduced with GFP (control) or FGF10 by lentiviral infection. The FGFR1/2-transduced prostate epithelial cells were combined with GFP- or FGF10-UGSM. The combined cells were mixed with collagen and implanted under SCID mouse kidney capsule. The regenerated prostate tissues were harvested after 8-week incubation. (E) The regenerated prostate tissues derived from the experimental groups including PrECs-control + GFP-UGSM, PrECs-FGFR1 + GFP-UGSM, PrECs-control + FGF10-UGSM, and PrECs-FGFR1 + FGF10-UGSM were analyzed for H&E, RFP signal, and IHC staining of CK5 (red)/CK8 (green)/DAPI (blue), and E-Cadherin (red)/vimentin (green)/DAPI (blue). Scale bar, 100 µm. The coexpression of E-Cadherin (red), vimentin (green), and DAPI staining (blue) in tissues from PrECs-FGFR1 + FGF10-UGSM group are presented in Figure S1.Overexpression of epithelial wild-type FGFR1 synergizes with paracrine FGF10 to induce EMT. (A) Diagram of the bi-cistronic lentiviral vector for the aberrant expression of FGFR1, FGFR2, FGF10, or Src. While the oncogenic gene was controlled by the ubiquitin promoter, RFP or GFP was regulated by the CMV promoter. (B) 293 T cells were transduced with control vector, FGFR1, or FGFR2 by lentiviral infection. The protein lysates were harvested for immunoblotting. The expression levels of FGFR1 or FGFR2, and ?-tubulin were examined by Western blot. Aberrant expression levels of FGFR1 and FGFR2 were confirmed. (C) UGSM cells were transduced with control vector or FGF10 by lentiviral infection. Total mRNA was extracted for the analysis of FGF10 expression by RT-PCR. FGF10 was highly expressed in FGF10-UGSM cells. (D) Diagram for evaluation of FGFR1/2 overexpression in epithelium and aberrant paracrine FGF10-induced tumorigenesis by the prostate tissue regeneration assay in vivo. Freshly isolated prostate epithelial cells were transduced with control vector (FUCRW), FGFR1, or FGFR2 by lentiviral infection. UGSM cells were isolated from 16.5-day-old mouse embryos. UGSM cells were transduced with GFP (control) or FGF10 by lentiviral infection. The FGFR1/2-transduced prostate epithelial cells were combined with GFP- or FGF10-UGSM. The combined cells were mixed with collagen and implanted under SCID mouse kidney capsule. The regenerated prostate tissues were harvested after 8-week incubation. (E) The regenerated prostate tissues derived from the experimental groups including PrECs-control + GFP-UGSM, PrECs-FGFR1 + GFP-UGSM, PrECs-control + FGF10-UGSM, and PrECs-FGFR1 + FGF10-UGSM were analyzed for H&E, RFP signal, and IHC staining of CK5 (red)/CK8 (green)/DAPI (blue), and E-Cadherin (red)/vimentin (green)/DAPI (blue). Scale bar, 100 µm. The coexpression of E-Cadherin (red), vimentin (green), and DAPI staining (blue) in tissues from PrECs-FGFR1 + FGF10-UGSM group are presented in Figure S1.

Fig 4: BCL11A overexpression leads to thickening of the airways and abnormal organoid formation. a Schematic representing strategy to explore the role of BCL11A in vivo and ex vivo. Left Panel: Adenovirus-Cre was nasally administered to BCL11Aovx mice and the lungs were analysed after eight months. Right panel: for the tracheosphere organoid model, basal cells from the trachea of either BCL11Aovx or BCL11Acko mice were FACS sorted, embedded in matrigel and analysed after 15 days. Three independent mice were used for each experiment. b Images of airways from control and BCL11Aovx. Arrows indicate small hyperchromatic cells with irregular nuclei. c Quantificaiton of airway epithelial layer hyperplasia from two control and BCL11Aovx mice. d Bright field images of organoids from Bcl11acko and BCL11Aovx mice treated with vehicle or tamoxifen. e Sectioned organoids from BCL11Aovx mice stained with haematoxylin and eosin, BCL11A, Sox2, Krt5, Trp63 and Krt8. Arrows indicate positive staining. Scale bar indicates 50 µm

Fig 5: Klf5K358R knockin in basal progenitors increases hyper proliferative luminal cells.Tamoxifen was applied to mice at postnatal day 18 to induce Cre expression via p63 promoter to knock in the Klf5KR mutant and activate YFP expression in basal cells, and prostates were collected at postnatal week 8 for analysis. a, b Knockin of the Klf5KR mutant decreased lumen areas (a) and reduced prostate weights in mice (b). In b, Klf5+/-, n = 12 mouse prostates; Klf5-/KR, n = 14 mouse prostates. c, d Knockin of the Klf5KR mutant did not significantly affect the number of basal cells, as indicated by costaining YFP and the p63 basal cell marker. e–i Knockin of Klf5KR significantly increased luminal cells, as indicated by costaining YFP and the luminal cell markers CK18 (e) or CK8 (f) and the percentages of YFP+ luminal cells (g), YFP+ luminal clusters (a cluster of 2 or more adjacent YFP+/CK18+ cells, h), and YFP+/CK18+ units (either a YFP+/CK18+ single cell or a cluster of YFP+/CK18+ cells, i). In d–i, the number of mice are as follows: Klf5+/-, n = 6; Klf5-/KR, n = 6. Arrowheads indicate YFP expressing p63+ cells (basal), arrows indicate YFP expressing CK18+ cells (luminal), and asterisks indicate YFP+ luminal clusters. j, k Knockin of Klf5KR did not affect the percentage of YFP+ cells in hybrid CK5+/CK8+ cells, as indicated by costaining YFP, CK5 and CK8 (k) and statistical analysis (j). Three mice were used for each genotype in j. l, m Knockin of the Klf5KR mutant increased the proliferation index of luminal cells, as revealed by costaining Ki67, YFP and the CK18 luminal marker. Arrows indicate Ki67+ and YFP+ luminal cells. In m, the number of mice are as follows: Klf5+/-, n = 5; Klf5-/KR, n = 4. Representative images are selected based on the statistical analysis. Scale bar in a, 100 µm; scale bars in others, 50 µm. Data are shown in mean ± S.E.M. NS not significant; *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student’s t-test). Source data are provided as a Source Data file.