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. 2023 Apr 25;14:2383. doi: 10.1038/s41467-023-38034-2

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Diagram of lineage-tracing analyses of Fgfr3-creER⁺ and Gas1-creER⁺ cells. Right: a proposed model of bone marrow stromal cell differentiation. b, c Fgfr3-creER; R26R^tdTomato (b) and Gas1-creER; R26R^tdTomato (c) femur endosteal space at P23 (pulsed at P21) with Col1a1-GFP (left) and Cxcl12-GFP (right). Scale bar: 20 µm. n = 4 mice per each group. BM: bone marrow. d Flow cytometry analysis of CD45/Ter119/CD31^neg bone marrow cells isolated from Fgfr3-creER; R26R^tdTomato (left) and Gas1-creER; R26R^tdTomato femurs (center) with Cxcl12-GFP (upper) or Col1a1-GFP (lower) at P23 (pulsed at P21). Cxcl12^GFP/+; Lepr-cre; R26R^tdTomato femurs at P21 (upper right) Lower right panel: Percentage of tdTomato⁺ cells among Cxcl12-GFP⁺ cells (tdTomato expression induced by Fgfr3-creER, Gas1-creER, or Lepr-cre). Two-tailed, one-way ANOVA followed by Tukey’s multiple comparison test. n = 5 (Fgfr3^CE), n = 4 (Gas1^CE), n = 5 (Lepr^cre) mice. e, f Col1a1-GFP; Fgfr3-creER; R26R^tdTomato (e) and Col1a1-GFP; Gas1-creER; R26R^tdTomato (f) distal femurs at P49 and 6M (pulsed at P21). Scale bar: 500 µm (left and center right), 20 µm (center left and right). n = 4 mice per each time point. BM: bone marrow. g Quantification of tdTomato⁺ bone marrow stromal cells (upper) and osteoblasts and osteocytes (lower) in marrow space at P49 and 6M, aligned based on distance from the growth plate. Fgfr3^CE-tdTomato⁺ cells (red), Gas1^CE-tdTomato⁺ cells (blue). n = 4 mice per each group. Two-tailed, Mann–Whitney’s U-test. h Flow cytometry analysis of CD45/Ter119/CD31^neg bone marrow cells after 2 days, 1 week, 4 weeks, and 8 weeks of chase (pulsed at P21), isolated from Fgfr3-creER; R26R^tdTomato (red line) or Gas1-creER; R26R^tdTomato femurs (blue line). Percentage of lineage-marked tdTomato⁺ cells among Cxcl12-GFP⁺ cells (upper) and Col1a1-GFP⁺ cells (lower). n = 3 mice for Col1a1-GFP/Fgfr3-creER at P49 and P77, Col1a1-GFP/Gas1-creER at P23, Cxcl12-GFP/Fgfr3-creER at P77, Cxcl12-GFP/Gas1-creER at P49), n = 4 mice for Col1a1-GFP/Fgfr3-creER at P23 and P28, Col1a1-GFP/Gas1-creER at P28 and P49, Cxcl12-GFP/Fgfr3-creER at P28, Cxcl12-GFP/Gas1-creER at P23, P28, and P77, n = 5 mice for Col1a1-GFP/Gas1-creER at P77, Cxcl12-GFP/Fgfr3-creER at P23 and P49). Data were presented as mean ± s.d. Exact P value is indicated in the figures. Source data are provided as a Source Data file.