Fig 2: Analysis of the female reproductive tract of adultFgfr2f/f andFgfr2d/d mice.A) Histology of the ovary, oviduct, cervix, and vagina of nulliparousFgfr2f/f andFgfr2d/d mice. Reproductive tract tissue sections were stained with hematoxylin and eosin. Bar = 100 µm.B) Histological examination of the uteri collected on Gestational Day (GD) 3.5 from nulliparousFgfr2f/f (on the left) andFgfr2d/d mice (middle and right images). Sections of uteri were stained with hematoxylin and eosin. Note the presence of stratified LE in the uterus ofFgfr2d/d mutant mice. LE, luminal epithelium; GE, glandular epithelium. Bar = 100 µm.C)Krt14 andSprr2b mRNA in the uterus ofFgfr2f/f andFgfr2d/d mice on GD 3.5. The relative mRNA levels ofKrt14 andSprr2b were measured in the GD 3.5 uterus by real-time RT-PCR. Data are presented as fold change relative to the mRNA level in uteri fromFgfr2f/f mice (n = 4 mice/genotype).

Fig 3: Epithelial Esr1 deletion results in a stratified uterine epithelial layer(A) Top panel: uteri of PND 60 Esr1f/f (WT), Esr1-/-, PgrCre/+Esr1f/f, and Foxa2Cre/+Esr1f/f mice (scale bars, 1 cm). Middle panel: ESR1 immunolocalization in uterine cross-sections (scale bars, 100 µm). Bottom panel: gross morphology of passage 3 endometrial epithelial organoids (EEO) (scale bars = 500 µm).(B–D) Immunolocalization of the basal markers KRT5 (B), KRT14 (C), and p63 (D) (scale bars, 250 µm). For (B), (C), and (D), panels on the left show uterine cross-sections and dashed panels on the right are insets of luminal and glandular epithelium. White and black arrows indicate the presence of basal cells in the uterine glandular epithelium of Foxa2Cre/+Esr1f/f mice (n = 3 mice/genotype). LE, luminal epithelium; GE, glandular epithelium; S, stroma; M, mesometrial; AM, antimesometrial.

Fig 4: Basal cell differentiation in the uterine epithelium is mediated by E2 actions on the stroma(A and B) Immunolocalization of p63 (A) and KRT5 (B) in uterine cross-sections of Esr1f/f (WT) and Foxa2Cre/+Esr1f/f mice. Histological analysis was performed on PND 30 (top panels) and PND 60 (bottom panels) females (scale bars, 100 µm). White and black arrows indicate the presence of basal cells in the uterine glandular epithelium. LE, luminal epithelium; GE, glandular epithelium; S, stroma.(C) Experimental design. Esr1f/f (WT) and Foxa2Cre/+Esr1f/f females were ovariectomized (OVX) at PND 30 and supplemented with E2 (20 µg/pellet) for 10 days.(D–F) Immunolocalization of the basal markers KRT5 (D), KRT14 (E), and p63 (F). White and black arrows indicate the presence of basal cells in the uterine glandular epithelium (scale bars, 100 µm).(G) Experimental design for the co-culture experiment. Stromal cells isolated WT females were co-cultured with organoids established from individual WT or Esr1-/- mice (n = 3 females per genotype). Co-cultures were treated with vehicle (100% Ethanol) or 100 nM E2 for 20 days.(H) RT-qPCR for the basal cell markers Trp63, Krt5, and Krt14. Gene expression data were normalized to Actb and Ppia mRNA and are presented as mean relative expression in comparison to the value of an appropriate control sample using the 2-?Ct method ±S.E.M. *, p < 0.05 (Student’s t test) (n = 3 mice per genotype).

Fig 5: Stromal-epithelial co-culture impacts Esr1-/- organoid development(A) Experimental design. Epithelial cells were seeded in Cultrex on transwell inserts over a bed of stromal fibroblasts adhered to the bottom of 6 well plates and cultured for 20 days (n = 5 mice per genotype).(B) Representative brightfield images at day 20 of culture (scale bars, 500 µm).(C) Quantification of the number of organoids at day 20 of culture. Solid dots represent average organoid number per biological replicate.(D) RT-qPCR analysis for the basal cell markers Trp63, Krt5, and Krt14 (n = 3 mice per genotype/treatment). Data are presented as fold change compared to WT EEO cultured in the absence of stromal fibroblasts. *p < 0.05 and ***p < 0.001 (Student’s t test).