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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. 3 — a Diagram of protocols to isolate Fgfr3-creER⁺ cells from bone marrow (top) or periosteum (bottom). b CFU-F assay. Fgfr3^CE-tdTomato⁺ periosteal cells (leftmost), Fgfr3^CE-tdTomato⁺ (left center), Gas1^CE-tdTomato⁺ (center), and Gli1^CE-tdTomato⁺ (right center) bone marrow cells at P23 (pulsed at P21), and Lepr^cre-tdTomato⁺ (rightmost) bone marrow cells at P21. Upper: tdTomato⁺ colonies, Lower: total colonies stained by methylene blue. Scale bar: 5 mm. c Percentage of tdTomato⁺ colonies among total CFU-Fs in young bone (left). Fgfr3^CE-periosteum, Fgfr3^CE-bone marrow, Gas1^CE, Gli1^CE and Lepr^cre. n = 8 (Fgfr3^CE-periosteum), n = 4 (Fgfr3^CE-bone marrow), n = 3 (Gas1^CE), n = 7 (Gli1^CE), n = 8 (Lepr^cre) mice. Two-tailed, one-way ANOVA followed by Dunnett’s multiple comparison test. Survival curve of individual tdTomato⁺ clones over serial passages (right). n = 46 (Fgfr3^CE-periosteum), n = 25 (Fgfr3^CE-bone marrow), n = 41 (Gas1^CE) clones. Log-rank (Mantel–Cox) test. d CFU-F assay of Fgfr3^CE-tdTomato⁺ bone marrow cells at 1 week after the pulse at 8W or 9M, and Lepr^cre-tdTomato⁺ bone marrow cells at 8W and 9M. Scale bar: 5 mm. e Percentage of tdTomato⁺ colonies among total CFU-Fs across young and adult stages. Fgfr3^CE (pulsed at P21, 8W, or 9M) or Lepr^cre bone marrow cells. Fgfr3^CE: n = 4 (P21), n = 4 (8W), n = 6 (9M). Lepr^cre: n = 8 (P21), n = 5 (8W), n = 4 (9M). Two-tailed, Mann–Whitney’s U-test. f In vitro trilineage differentiation assay of Fgfr3^CE-tdTomato⁺ clones (Passage 2–7). Left: Chondrogenic condition, Alcian Blue staining. Center: Osteogenic conditions, Alizarin Red staining. Right two panels: Adipogenic conditions, Oil red O (right center) and LipidTOX staining (rightmost), Green: LipidTOX-Alexa488, red: tdTomato. Scale bar: 200 µm. n = 17 clones. g qPCR analyses of Fgfr3^CE-tdTomato⁺ clones, expression of chondrocyte markers (Acan, Col2a1), osteoblast markers (Sp7, Dmp1), adipocyte markers (Pparg, Adipoq) under chondrogenic, osteogenic and adipogenic conditions, respectively. n = 4 clones (#5, 6, 7, 17). Two-tailed, Mann–Whitney’s U-test. Data were presented as mean ± s.d. Exact P value is indicated in the figures. Source data are provided as a Source Data file.