Fig. 2: Smad1/5 cKO mice develop abnormal uterine glands that appear enlarged and cystic.

a–h Histological analysis of control (a, c, e, g) and Smad1/5 cKO (b, d, f, h) uteri stained with FOXA2 (a–d) or H&E (e–h). Uteri were analyzed at 3 weeks (a–b), 6 weeks (c–d), 12 weeks (e–f), and 24 weeks of age (g–h). i Expression of the WNT-pathway inhibitors (Sfrp1-5) was analyzed using qPCR of 12-week-old uterine tissues of control (n = 3) and Smad1/5 cKO (n = 3) mice. Histograms represent mean ± standard error of the mean (SEM), paired, two-tailed, t test, *p < 0.05, **p < 0.01, ***P < 0.001. Sfrp1, p = 0.017; Sfrp2, p = 0.105; Sfrp3, p = 0.007; Sfrp4, p = 0.0107; Sfrp5, p = 0.042. j–r Whole-mount immunostaining with FOXA2 antibody followed by multiphoton microscopy (j–o) in the uteri of non-pregnant control (j), or Smad1/5 cKO mice (k). l–r show analyses performed on individual glands from control (l, l’, l”) or Smad1/5 cKO mice (m, m’, m”) and the corresponding quantification of the width, length, and density of the glands (p–r). Total fields counted for gland density analysis: n = 8 in three control mice and n = 18 in three Smad1/5 cKO mice. Histograms represent mean ± standard error of the mean (SEM). Unpaired, two-tailed t test, *p < 0.033, **p < 0.002, ***P < 0.001. Size bar: j–k is 500 µm; l–m” is 30 µm. Arrow in o indicates enlarged cystic endometrial gland from Smad1/5 cKO. s–x Uterine lumen and endometrial glands stained with E-cadherin antibody and scanned by Optical Projection Tomography (OPT). Control (s, s’, s”, v) and Smad1/5 cKO (t–u”, w–x) whole-mount (s–u”) and optical cross-sections (v–x) are displayed. Arrows in v–x point to the uterine glands and arrowheads indicate the uterine lumen. s’–u”) Surface rendering of the mouse uterus emphasizes uterine glands (arrows in s’–u’) and folds in uterine lumen (arrowheads in s”–u”). Scale bar s–u’ (500 µm), and v–x, s”–u” (300 µm). OPT scans and multiphoton imaging were performed in the tissues of at least three control and three Smad1/5 cKO mice.