Fig. 5: Osteoblast-derived RSPO3 increases osteoblast proliferation and differentiation.

a Rspo3 mRNA expression in primary calvarial osteoblasts (cOBL) isolated from CAGGCre-ER-Rspo3^flox/flox mice cultured for 2 and 6 days (d) with or without prior Rspo3 inactivation using tamoxifen (TAM). Veh=vehicle. b Representative photos of alkaline phosphatase staining of CAGGCre-ER-Rspo3^flox/flox and Rspo3^flox/flox cOBL cultured for 6 days after veh treatment or Rspo3 inactivation. c, d Representative photos (c) and quantification (d) of mineralized noduli in cultures of CAGGCre-ER-Rspo3^flox/flox and Rspo3^flox/flox cOBL cultured for 14 days after veh treatment or Rspo3 inactivation. e, f mRNA expression analysis of Alpl (e) and Col1a1 (f) in CAGGCre-ER-Rspo3^flox/flox cOBL cultured for 2 and 6 days with or without prior Rspo3 inactivation. g Haematoxylin (Htx) and Dapi staining of CAGGCre-ER-Rspo3^flox/flox cOBL cultured for 3 days with or without prior Rspo3 inactivation. Scale bar 100 μm. h Amount of DNA per well in CAGGCre-ER-Rspo3^flox/flox cOBL at the time of TAM addition (day −1), directly after removal of TAM (day 0) and after culture for 1, 3, 6, and 10 days. i Mki67 mRNA expression in CAGGCre-ER-Rspo3^flox/flox cOBL cultured for 2 days after Rspo3 inactivation. j, k mRNA expression of Tnfsf11 (j) and Tnfrsf11b (k) in CAGGCre-ER-Rspo3^flox/flox cOBL cultured with or without PTH and PGE2 for 5 days with or without prior Rspo3 inactivation. n = 3–4 wells. l–n TRAP staining (l), counting (m), and Acp5 mRNA expression (n) of bone marrow macrophages cultured in M-CSF (M) or in M-CSF and RANKL (RL) to induce osteoclastogenesis in the absence or presence of recombinant RSPO3 (rRSPO3). Scale bar 200 μm. Individual values are presented in all graphs with the mean presented as horizontal lines and ±SEM as vertical lines. If not otherwise stated, n = 4 wells per group. Experiments were repeated two (g, j–n) or at least three (a–f, h, i) times. For a, e, f, h, two-way ANOVA was used to determine the overall effect of Rspo3 inactivation by treatment of TAM or veh, time, as well as their interaction. For j, k, two-way ANOVA was used to determine the overall effect of PTH/PGE2 treatment, Rspo3 inactivation by treatment of TAM or veh, as well as their interaction. When only the effect of Rspo3 inactivation was evaluated, two-sided Student’s t was used. Source data are provided as a Source Data file.