Extended Data Fig. 7. Cellular characterization of intestinal villi expansion in pregnancy and lactation.

a, Mating strategy to generate intestinal epithelial cell specific Rank deleted mice (left) and anti-RANK immunostaining on intestinal cryo-sections from nulliparous control Rank^WT and Rank^ΔVil mice (right). Scale bars, 50 μm. b, Quantitative RT–PCR analysis of Rank mRNA expression in isolated small intestinal epithelial cells from nulliparous control Rank^WT (n = 3) and Rank^ΔVil (n = 5) littermates. c, Representative immunostaining to detect CRE expression (blue) in the small intestine (left panel) and mammary gland (right panel) of Villin1-Cre mice. Paraffin sections were counterstained with hematoxylin. Scale bars, 50 μm. d, Representative whole mount images (hematoxylin staining) of mammary glands from nulliparous Rank^WT, nulliparous Rank^ΔVil, pregnant Rank^WT (day 18.5 of pregnancy, P18.5), pregnant Rank^ΔVil females, lactating Rank^WT (5 days after delivery, L5) and lactating L5 Rank^ΔVil females. Scale bars, 500 μm. e, Small intestinal villi length, volume and surface areas in indicated mice. Each data point represents average villi length, volume and surface area per mouse in the indicated mice described in Fig. 3a. f, Quantification of the average number of OLFM4⁺ cells in each crypt per mouse in the indicated mice described in Fig.  3b. g,h, Cell death of villous epithelial cells from nulliparous Rank^WT and Rank^ΔVil females and age-matched lactating (L5) Rank^WT and Rank^ΔVil dams, as determined by immunostaining of cleaved caspase 3 (CLC3). Representative immunostaining. Scale bars, 100 μm (left in g). Quantification of CLC3 positive cells in villi per 2 mm of the intestine in nulliparous Rank^WT (n = 4 mice, n = 49 regions analysed) and Rank^ΔVil females (n = 5/59) and age-matched lactating (L5) Rank^WT (n = 8/162) and Rank^ΔVil dams (n = 8/135) (right in g and h). Each data point represents the numbers of CLC3 positive cells in villi per each 2 mm of the intestine (g) and average number per mouse (h). i,j, Quantification of the EdU-labelled intestinal epithelial cell migration along the crypt-villus axis in nulliparous Rank^WT (n = 3 mice, n = 29 crypt-villus axis analysed) and Rank^ΔVil females (n = 3/37) and age-matched lactating (L5) Rank^WT (n = 4/52) and Rank^ΔVil dams (n = 4/62). Tissues were harvested 24 h after EdU administration. Each data point represents the measurements of the migration distance in individual crypt-villus axis (i) and the average distance per mouse (j). k, Representative images of jejunal organoids generated from Rank^WT and Rank^ΔVil mice. Scale bars, 100 μm. l, Ratios of the number of jejunal organoids derived from Rank^WT and Rank^ΔVil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). The number of organoids was counted at each passage. The ratio of organoid numbers in the Rank^WT + RANKL group was normalized to control Rank^WT organoids, whereas the ratio of organoid numbers in the Rank^ΔVil + RANKL group was normalized to untreated Rank^ΔVil organoids. Data are from two independent experiments. n = 6 (Rank^WT), n = 6 (Rank^WT + RANKL), n = 6 (Rank^ΔVil), n = 6 (Rank^ΔVil + RANKL). m,n, Representative hematoxylin and eosin (H&E) stained images of the upper small intestine (left in m) and quantification of upper small intestinal villus lengths (right in m, n) in age matched wild type nulliparous females (n = 4 mice, n = 310 villi analysed), pregnant females (P18.5) (n = 4/272), lactating dams (L5, 5 days after delivery) (n = 4/212) and females 6 weeks after weaning of the offspring (n = 4/49). Scale bars, 100 μm. Each data point represents the length of individual villi (m) and average length per mouse (n). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Two-tailed Mann–Whitney U-test (e,f,h,j,n); One-way analysis of variance (ANOVA) with Tukey’s post hoc test (g,i,l,m).

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