Fig. 3. RANK–RANKL controls intestinal villus expansion in pregnancy and lactation.

a, Representative 3D reconstruction of the upper small intestine from nulliparous and lactating (5 days after delivery, L5) Rank^WT and Rank^Δvil female mice (top). Grid spacing is 200 μm. Bottom, the small intestinal villus length, volume and surface areas in nulliparous Rank^WT (n = 5 mice, n = 88 villi), nulliparous Rank^Δvil (n = 5 mice, n = 97 villi), P18.5 Rank^WT (n = 6 mice, n = 89 villi), P18.5 Rank^Δvil (n = 6 mice, n = 93 villi), L5 Rank^WT (n = 5 mice, n = 91 villi) and L5 Rank^Δvil (n = 6 mice, n = 141 villi) mice. b, Representative OLFM4 immunostaining in the small intestines of the indicated mice (top). Scale bars, 20 μm. Bottom, quantification of OLFM4⁺ cells per crypt in nulliparous Rank^WT (n = 3 mice, n = 87 crypts), nulliparous Rank^Δvil (n = 4 mice, n = 130 crypts), L5 Rank^WT (n = 6 mice, n = 160 crypts) and L5 Rank^Δvil (n = 7 mice, n = 181 crypts) mice, and Rank^WT (n = 4 mice, n = 59 crypts) and Rank^Δvil (n = 5 mice, n = 44 crypts) mice 6 weeks after weaning of the offspring. c, Representative 3D images (left) and villi lengths of small intestines from age-matched nulliparous (n = 3 mice, n = 70 villi analysed) and lactating (n = 5 mice, n = 94 villi) germ-free C57BL6 mice (right). Grid spacing is 200 μm. d, Left, representative small intestinal sections (haematoxylin and eosin (H&E) staining) of L5 wild-type mice and wild-type mice whose offspring were removed on 1 day after delivery (no lactation). Scale bars, 100 μm. Right, quantification of villus length in L5 dams (with offspring) (n = 7 mice, n = 170 villi) and female mice with their offspring removed (without offspring) (n = 5 mice, n = 158 villi). e, Representative small intestinal sections (H&E staining) (left) and quantification of villi lengths (right) of L5 wild-type mice treated with DMSO or cabergoline (5 mg per kg) for 5 consecutive days starting on the day of delivery. Villi were assessed on day 5 after delivery. Scale bars, 100 μm. Right, quantification of villus length in mice treated with DMSO (n = 4 mice, n = 148 villi) and mice treated with cabergoline (n = 4 mice, n = 179 villi). f, Representative H&E-stained intestinal sections from nulliparous Traf6^WT and Traf6^Δvil mice, and L5 lactating Traf6^WT and Traf6^Δvil mice (left). Scale bars, 100 μm. Right, quantification of villous length in nulliparous Traf6^WT (n = 4 mice, n = 76 villi) and Traf6^Δvil (n = 5 mice, n = 73 villi) mice, and L5 Traf6^WT (n = 6 mice, n = 198 villi) and Traf6^Δvil (n = 5 mice, n = 226 villi) mice. Data are mean ± s.e.m. Each point represents the measurement of length, volume and surface area (a (bottom) and c–f (right)), and the number of OLFM4⁺ cells per crypt (b, bottom). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (a, b and f) and two-tailed Student’s t-tests (c–e); **P < 0.01, ***P < 0.001.

Source Data