Fig. 2.

Derivation of organoids from the mouse MD epithelium. (A) Gross bright-field (A, a, c, e, i, and k) and GFP fluorescence (A, b, d, f, j, l, and m) images and color-inverted (A, g and n) images of the female urogenital system collected from TCF-GFP mice at E13.5 (A, a and b), E14.5 (A, c and d), E.16.5 (A, e–g), E18.5 (A, i and j), and P1 (A, k–n) (n = 5 each stage), showing real-time Wnt activity marked by GFP expression. (A, h) Quantification of FACS-sorted EpCAM+ GFP+ from MDs collected from TCF-GFP mice at E16.5; n = 6; **P = 0.005, unpaired t test with Welch’s correction. Data represent means ± SEM. Dotted lines in A, e–g outline presumptive FTs. Transverse lines in A, i and j denote FTs and the uterotubular junction, respectively. (B) In situ hybridization for Wnt7a (B, a–d), Wnt4 (B, e–h), Lgr5 (B, i–l), and Axin2 (B, m–p) messenger RNA in E14.5 (B, a, e, i, and m) and E16.5 (B, b, f, j, and n) MDs and P1 FTs (B, c, g, k, and o) and uteri (B, d, h, l, and p) (n = 5 biological replicates × 10 tissue sections per replicate). (C) Whole-well and representative images of organoids derived from E14.5 MD epithelial cells and cultured in RNEF or RNEFC and WRNEF (n = 32 MDs from 8 different dams). (D) Representative histological images of E14.5 MD epithelial cell–derived organoids cultured in RNEFC and WRNEF media. Arrows in D, a and c point to areas that are presented as higher-magnification images in D, b and d, respectively. (E) Coimmunostaining for Pax8 and Mouse AcTub (E, a–c) and Foxa2 and Ck8 (E, d–f) in E14.5 MDs (E, a and d) and P1 FTs (E, b and e) and uteri (E, c and f) (n = 3 biological replicates × 5 images per replicate). (F–I) Coimmunostaining for Pax8 and Actub (F and H) and Foxa2 and Ck8 (G and I) in organoids from D (n = 3 biological replicates × 5 images per replicate). a, adrenal glands; k, kidney; md, Müllerian duct; ut junction, uterotubular junction; wd, Wolffian duct. Data represent means ± SEM. (Scale bars, 100 μm unless indicated otherwise.)