Fig 2: Effects of S1P on endothelial cells and macrophages in radiated salivary glands. (A–D) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. (E,F) Levels of p-H2ax in Cd31+ endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. (G–I) Expression of F4/80 and levels of p-H2ax in F4/80+ macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. (J) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, #: p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

Fig 3: TGF-ß neutralizing antibody attenuates HO induced by BMP-2/gelatin implantation and FOP mice. a–c Mice were treated with 5 mg/kg body weight of the TGF-ß neutralizing antibody 1D11 three times a week for 2 weeks from 1 day (D1), 2 weeks (W2), and 4 weeks (W4) after BGI and analyzed 8 weeks after BGI or sham surgery. a micro CT images and b quantitative analysis of bone volume of heterotopic bone in hamstring muscles of mice. Scar bar, 4 mm. c H&E staining of ectopic bone in hamstring muscles. Scale bar, 100 µm. d–k Mice were treated with 5 mg/kg body weight of 1D11 three times a week for 2 weeks from 1 day (D1) and 2 weeks (W2) after BGI and analyzed 4 weeks after BGI or sham surgery. d Immunostaining and e quantification of pSmad2/3+ cells in ectopic bone marrow. Scale bar, 50 µm. f Nestin+ (red) cells in the ectopic bone marrow and g quantification. Scale bar, 50 µm. Blue indicates DAPI staining of nuclei. h CD31+ (red) and Emcn+ (green) cells in the ectopic bone marrow and i quantification of the fold change of type H vessels normalized to that of Sham mice. Scale bar, 100 µm. Yellow indicates type H vessels. j Immunostaining and k quantification of Ocn+ cells of in ectopic bone marrow. Scale bar, 50 µm. l, m Ad.Cre and cobra venom factor-injected caALK2 transgenic mice were treated with vehicle or 1D11 three times a week for 3 weeks. l Micro CT images of hamstring muscles after vehicle or 1D11 treatment and m quantitative analysis of bone volume. Scale bar, 4 mm. All data are shown as the mean ± s.d. n = 8 per group. b, e, g, i, k *p < 0.05 as determined by one-way ANOVA. m *p < 0.05 as determined by unpaired, two-tailed Student’s t-test

Fig 4: Nestin+ cells at the injured site are predominantly MSPCs. a The percentage of the CD45-GFP+ cells that express LepR. n = 16. b The percentages of the GFP+LepR+ cells or c GFP+LepR- cells that express Sca-1, CD105, or CD31, respectively. d Alizarin red staining of GFP+ cells cultured in osteogenic differentiation medium for 14 days and e tube formation of GFP+ cells by Matrigel assay. f Immunostaining of COLII+ cells (red) and YFP+ cells (green) and g quantitation of Nestin-creERT2::EYFPflox/flox mice 3 weeks post ATP. Scale bar, 50 µm, n = 8 per group. h, left: CD31+ cells (red), YFP+ cells (green), and right: Emcn+ cells (red), YFP+ cells (green), and i quantitation in ectopic bone marrow of Nestin-creERT2::EYFPflox/flox mice 6 weeks post ATP. Scale bar, 100 µm, n = 8 per group. All data are shown as the mean ± s.d

Fig 5: Osteolectin contains conserved integrin binding motifs and binds with high affinity to integrin a11ß1.(A, B) The human (A) and mouse (B) Osteolectin proteins contain RGD and LDT sequences. (C) Alignment of Osteolectin amino acid sequences shows that the RGD and LDT domains are evolutionarily conserved among bony vertebrates. (D, E) RNA-seq analysis of integrin a (D) and ß (E) subunits in PDGFRa+CD45-Ter119-CD31- bone marrow stromal cells from enzymatically dissociated adult bone marrow (n = 2 independent samples). These cells are uniformly positive for LepR expression (Zhou et al., 2014). (F) RNA-seq analysis of Itga1, Itga6, Itga11, and Itgav in PDGFRa+CD45-Ter119-CD31- bone marrow stromal cells, VE-Cadherin+ bone marrow endothelial cells, and whole bone marrow cells (n = 2 independent samples per cell population). (G) Itga11 expression in cell populations from mouse bone marrow by qRT-PCR (n = 3 independent samples per cell population). The markers used for the isolation of each cell population are shown in Supplementary file 1. (H) In MC3T3-E1 preosteoblast cells expressing Flag-tagged Osteolectin, anti-Flag antibody co-immunoprecipitated endogenous integrin ß1 and integrin a11 with Flag-tagged Osteolectin (results are representative of two independent experiments). (I) Recombinant human Osteolectin (rhOln) selectively bound to recombinant human integrin a11ß1 and a10ß1, but not to other integrins (n = 3 independent experiments). (J) Integrin a11ß1 bound Osteolectin and recombinant human Pro-Collagen 1a (rhCol1A) with similar affinities, but not bovine serum albumin (BSA) (n = 3 independent experiments). (K) Osteolectin, but not Pro-Collagen 1a, promoted osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells (n = 3 independent experiments). (L) 200 nM RGDS peptide inhibited the binding of integrin a11ß1 to recombinant human Osteolectin. (M) 100 µM RGDS peptide inhibited osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells in response to 30 ng/ml of recombinant human Osteolectin. All numerical data reflect mean ±standard deviation. Statistical significance was determined with one-way (G) or two-way ANOVAs with Dunnett’s multiple comparisons tests (K) or Tukey’s multiple comparisons tests (M).10.7554/eLife.42274.004Figure 1—source data 1.Data for Figure 1.