Fig 2: Osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and is a major regulator of trabecular bone mass.a Schematic figure of the conditional osteoblast-lineage specific Rspo3-inactivated mouse model. b mRNA expression analyses of Rspo3 in vertebral trabecular bone, cortical diaphyseal bone, liver, and brain cortex in male Runx2-creRspo3flox/flox mice, compared to Rspo3flox/flox mice. c Body weight in Runx2-creRspo3flox/flox mice compared to Rspo3flox/flox mice at 5, 9, and 13 weeks-of-age. d Weight of liver, gonadal fat, kidneys, and spleen, per body weight (BW) in Runx2-creRspo3flox/flox mice, and Rspo3flox/flox mice. e Representative 3D µCT images of vertebra L5 in Runx2-creRspo3flox/flox mouse (left) and Rspo3flox/flox mouse (right). f–i Trabecular bone volume over total volume (BV/TV; f), trabecular thickness (Tb.Th; g), trabecular number (Tb.N; h), and trabecular separation (Tb.Sp; i) in vertebra L5 from Runx2-creRspo3flox/flox mice, compared to Rspo3flox/flox mice, as measured by µCT. j Correlation between Rspo3 mRNA levels in vertebral body (relative quantity, x-axis) and trabecular BV/TV (%, y-axis) in male Runx2-creRspo3flox/flox and Rspo3flox/flox mice. Variance explained (r2) is given in the figure. k Maximal load at failure (N) of vertebra L5 as measured by compression test in 14-week-old male Runx2-creRspo3flox/flox (n = 10) mice and Rspo3flox/flox (n = 9) mice. l, m Cortical volumetric bone mineral density (Ct.vBMD; l) and cortical thickness (Ct.Th; m) in femur from Runx2-creRspo3flox/flox mice, compared to Rspo3flox/flox mice. n mRNA expression of sclerostin (Sost), Dickkopf-1 (Dkk1), and R-spondins 1, -2, and -4 (Rspo1, Rspo2, and Rspo4) in vertebral body in male Runx2-creRspo3flox/flox mice, compared to Rspo3flox/flox mice. o Levels of procollagen type I N-terminal propeptide (P1NP, left) and C-terminal type I collagen (CTX, right) in male Runx2-creRspo3flox/flox mice, compared to Rspo3flox/flox mice. Unless otherwise stated, the results refer to 13-week-old Runx2-creRspo3flox/flox mice, males n = 14; females n = 12, and Rspo3flox/flox mice, males n = 15; females n = 12. Individual values are presented in all graphs with the mean presented as horizontal lines and ±SEM as vertical lines. Statistical analyses were performed using two-sided Student’s t test. Source data are provided as a Source Data file.

Fig 3: Stromal RSPO3 and Wnts are critical for adult intestinal homeostasis. (A) Intestinal stromal cells contact Lgr5-expressing cells in vitro. mTomato-expressing stromal cells from RosamTmG mice were combined with purified epithelial crypts from Lgr5-GFP mice. The mixed cells were cultured without added RSPO for 5 d and then were imaged using an inverted Zeiss LSM 710 microscope. Intestinal stroma, Lgr5+ cells, and nuclei are labeled red, green, and blue, respectively. (Scale bar: 20 μm.) (B) Stromal RSPO3 is required for organoid growth. PORCN-deficient intestinal epithelial cells requiring Wnts from stroma were cultured either alone or in combination with intestinal stromal cells. Rspo3 expression was targeted using siRNA (Upper) or adenoviral-expressed Cre (Lower). Mock targeting was performed with scrambled siRNA or GFP-expressing adenovirus, respectively. In the rescue, RSPO3 (50 ng/mL) was added. Combined data from four siRNA and three viral-targeting experiments are presented. **P < 0.001, Wilcoxon rank sum test.

Fig 4: RSPO3 is expressed in osteoblasts but not in osteocytes or osteoclasts.aRspo3 mRNA expression pattern in various tissues of male, wildtype mice (n = 6). Mean of individual values are presented as horizontal lines and ±SEM as vertical lines. Data are presented as % of expression in cortical diaphyseal bone. b Rspo3 is expressed in primary calvarial osteoblasts (OBL), but not in bone marrow-derived macrophages (BMM) or RANKL-differentiated osteoclasts (OCL). Rspo3 gene expression increase from day 2 (d2) to day 7 (d7) in primary calvarial osteoblasts cultured in osteogenic media. Presented as % of expression in osteoblasts day 2. ND=not detectable. Mean of individual values are presented as horizontal lines and ±SEM as vertical lines. Difference in expression between day 2 and 7 was analyzed by two-sided Student’s t test, n = 4 wells per group. c–f Representative in situ hybridization images in mice. Transverse sections across lumbar vertebra 5 in mouse showing the mRNA expression pattern (red) of Rspo3 (c–f, upper) and Runx2 (c, lower), Col1a1 (d, lower), Dmp1 (e, lower), and Acp5/Trap (f, lower). The lower images are consecutive to the upper images. Rspo3 mRNA could be observed in Runx2-expressing (c), Col1a1-expressing (d), and Dmp1-expressing (e) osteoblast lineage cells (OBL) on the bone surface. In contrast, Rspo3 was not detectable in osteocytes (Ocy; e), or osteoclasts (OCL; f). Scale bar 50 µm. g–i Representative in situ hybridization images in humans. Sections from human proximal femur (g) and iliac crest (h, i) confirmed moderate RSPO3 mRNA expression. Staining for RSPO3 mRNA (red) were observed in immature osteoblast lineage cells (OBL) on eroded lining surfaces (reversal cells), and on quiescent bone surfaces (bone lining cells), as well as in a subset of cells close to blood vessels (bv; g, h). No RSPO3 mRNA expression was observed in osteocytes (Ocy) or TRAP-immunostained osteoclasts (OCL, black; i). Scale bar 20 µm. Experiments were repeated one (a), two (g–i) or at least three (b–f) times. Source data are provided as a Source Data file.

Fig 5: RSPO3 is more potent than RSPO1 in activating a β-catenin reporter and supporting intestinal organoid cultures. (A) STF3A cells were transfected with the indicated amounts of RSPO1 and RSPO3 expression plasmids, and the resultant Wnt/β-catenin–dependent luciferase signal was measured as described. (B) Comparative RSPO1-Myc and RSPO3-Myc expression levels in transfected STF3A cells assessed by anti-MYC immunoblot. Mw, molecular weight. (C) Recombinant RSPO3 and RSPO1 were added at the indicated concentrations to HEK293 STF3A cells with robust endogenous Wnt signaling as described. Data from two independent experiments with similar results were normalized and merged, with the activity induced by 200 ng/mL RSPO1 set as 100%. (D) RSPO3 is more potent than RSPO1 in promoting organoid growth. Organoids were cultured with the indicated concentration of RSPO and counted in three wells per group on day 5. Data from two independent experiments were equalized using the organoid count in the RSPO1 500 ng/mL group as 100% response. (E) RSPO1- and RSPO3-stimulated organoids have similar morphologic features. Organoids were cultured in the presence of the indicated R-spondin concentrations and photographed on day 5. (Scale bar: 100 μm.) (F) RSPO3 is more potent than RSPO1 in regulating differentiation in intestinal epithelial organoids. The expression of the indicated differentiation markers from the organoids in D was assessed at day 5, normalized to β-actin expression levels. The figures combine two independent experiments, equalized by setting the expression in the RSPO3 100 ng/mL group as 100% response. *P < 0.05, Wilcoxon rank sum test.