Extended Data Figure 2. Bone marrow Oln-mTomato⁺ cells localized mainly around arterioles in the diaphysis.

(a) A low magnification view of the femur diaphysis showed Oln-Tomato⁺ stromal cells associated with arterioles enriched in the center of the marrow. In this image, arteriolar and sinusoidal blood vessels were distinguished based on size, morphology, and continuity of the basal lamina, visualized using anti-laminin antibody staining as previously described²¹ (images are representative of 3 independent experiments; scale bar = 200μm). (b) Oln^mT/+ femur bone marrow showing that, in contrast to the diaphysis (Fig. 1h), most Sca-1⁺ arterioles were not surrounded by Oln-Tomato⁺ stromal cells in the metaphysis. The Oln-Tomato⁺ cells in this panel were osteoblasts and osteocytes associated with trabecular bone (scale bar = 500μm). (c, d) Gene set enrichment analysis showing significant enrichment of genes associated with osteogenesis in CD45⁻Ter119⁻CD31⁻Scf-GFP⁺Oln-mTomato⁺ stromal cells and adipogenesis in CD45⁻Ter119⁻CD31⁻Scf-GFP⁺Oln-mTomato⁻ stromal cells (FDR, false discovery rate; NES, normalized enrichment score; 4 mice analyzed in 4 independent experiments). (e-f) The mouse Osteolectin gene was modified by inserting an iCreER-WPRE-pA cassette between the 5’ untranslated region and exon 3 to generate the targeting vector. These sites were selected to avoid disrupting conserved intronic sequences. Open boxes indicate untranslated regions and black boxes indicate translated regions of Osteolectin. The targeted founder mouse (F0) was identified by Southern blotting (f) using 5’ and WPRE probes (black bars). (g) PCR genotyping of genomic DNA confirmed germline transmission of the Oln^iCreER allele. Mice were backcrossed at least three times onto a C57BL/Ka background before analysis. (h) Deep imaging of Oln^iCreER/+; Rosa26^{loxp-tdTomato/+} femur bone marrow 3 days after tamoxifen administration at 2 months of age, showing that Oln-Tomato⁺ cells were exclusively peri-arteriolar in the diaphysis (images are representative of 3 independent experiments; scale bar = 200μm). (i and j) Deep imaging of the femur epiphysis (i) and diaphysis (j) one month after tamoxifen administration at 2 months of age (images are representative of 3 independent experiments). Panel i shows Oln-Tomato⁺ hypertrophic chondrocytes in the growth plate (arrow) and Oln-Tomato⁺Col1a1*2.3-EGFP⁺ osteoblasts in trabecular bone (arrowhead; scale bar = 60mm). Panel j shows Oln-Tomato⁺Col1a1*2.3-EGFP⁺ osteoblasts at the endosteum (arrow), an Oln-Tomato⁺ osteocyte (arrowhead), and Oln-Tomato⁺ periosteal cells (asterisk; scale bars = 30μm). (k) Col1a1-CreER; Rosa26^{loxp-tdTomato/+}; Col1a1*2.3-EGFP mice were treated with tamoxifen at 2 months of age and the percentage of Col1a1*2.3-EGFP⁺ osteoblasts that were Tomato⁺ was assessed 3 days to 1 month later (3 mice per time point analyzed in 3 independent experiments). All data represent mean ± SD.