Extended Data Figure 5. Oln-mTomato⁺ osteoblasts and osteocytes in the metaphysis do not express Scf-GFP and Oln-mTomato⁺ cells in the diaphysis expand during fracture healing.

The differences among treatments in cell frequencies were also evident in absolute numbers. (a-c) Epiphysis (a) and metaphysis (b, c) of Oln^mT/+; Scf^GFP/+ femur bone marrow showing that hypertrophic chondrocytes (arrowhead in a; scale bars = 30μm) as well as osteoblasts and osteocytes associated with trabecular bone (arrowhead in c) were negative for Scf-GFP (images are representative of 3 independent experiments; scale bars = 400μm (b) and 100μm (c)). Most of the Oln-Tomato staining in the metaphysis reflects Col1a1*2.3-GFP⁺ osteoblasts and osteocytes associated with trabecular bone, as shown in Fig. 1d and Extended Data Fig. 2b. The boxed area in panel b is magnified in panel c. (d) Image of the metaphysis of Scf^GFP/+ femur bone marrow showing limited non-specific staining by anti-tdTomato antibody (scale bar = 100mm). (e-g) Oln^iCreER/+; Scf^fl/fl and Scf^fl/fl littermate control mice were fed tamoxifen chow from 2–4 months of age. They exhibited no significant differences in the frequencies of HSCs, MPPs, GMPs, MEPs, or CMPs in the bone marrow (e), or B220⁺ B cells (f), or CD3⁺ T cells (g) in the bone marrow or spleen. The differences between Oln^iCreER/+; Scf^fl/fl and Scf^fl/fl mice were not statistically significant in e-g (5 mice per genotype analyzed in 3 independent experiments). (h, i) Three days after tamoxifen, femurs were fractured in 2-month-old Oln^mT/iCreER; Scf^fl/fl mice and Oln^mT/+; Scf^fl/fl littermate controls then the bone marrow was analyzed two weeks later. HSC and MPP frequencies did not significantly change during fracture healing. In contrast, CLP frequencies significantly increased in Oln^mT/+; Scf^fl/fl control but not in Oln^mT/iCreER; Scf^fl/fl mice (h). The frequency of Osteolectin⁺ cells significantly increased in both Oln^mT/iCreER; Scf^fl/fl mice and Oln^mT/+; Scf^fl/fl controls (I; 5 mice per genotype analyzed in 3 independent experiments). (j) Localization of IL7Rα⁺Lineage⁻ lymphoid progenitors in the marrow of the fractured as compared to control (CON) femur (4 mice per treatment analyzed in 4 independent experiments). All data represent mean ± SD. Statistical significance was assessed using matched samples two-way ANOVAs followed by Sidak’s (e-i) or Tukey’s multiple comparisons tests (h), or Cochran-Mantel-Haenszel test (j).