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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. 2 — a UMAP plots colored by expression of representative OCT Stem (Fgfr3), ORT 1 (Gas1), chondrocyte (Col2a1), osteoblast (Col1a1), and reticular cell (Lepr) markers. Violet: high expression, yellow: low expression. b Fgfr3-GFP femur at P21. Left panel: Scale bar: 500 µm. n = 4 mice. Center panels: Growth plate (upper) and cortical bone (lower) in high magnification. Immunohistostaining for FGFR3 (left) and RNAscope analyses of Fgfr3 (right). Scale bar: 20 µm. n = 4 mice. Right panels: Magnified images of endosteal space with Osx-mCherry (left), ALPL staining (center), and ACAN staining (right). GP: growth plate, PO: periosteum, BM: bone marrow. Scale bar: 20 µm. n = 4 mice. c Fgfr3-GFP; Fgfr3-creER; R26R^tdTomato femur at P23 (pulsed at P21). Histology and flow cytometry analysis of CD45/Ter119/CD31^neg bone marrow cells isolated from distal femurs. Fgfr3-GFP expression in Fgfr3^CE-tdTomato⁺ cells. Blue lines: GFP^neg control cells. GP: growth plate, BM: bone marrow. Scale bar: 20 µm. n = 3 mice. d UMAP visualization of three datasets (Prrx1^cre-tdTomato⁺ cells at P21, Fgfr3^CE-tdTomato⁺ cells at P23, and Gas1^CE-tdTomato⁺ cells at P23) merged by LIGER. Prrx1^cre-tdTomato⁺: 10,062 cells, pooled from n = 3 mice, Fgfr3^CE-tdTomato⁺: 5615 cells, pooled from n = 8 mice (pulsed at P21), Gas1^CE-tdTomato⁺: 2050 cells, pooled from n = 7 mice (pulsed at P21). Leftmost: UMAP visualization of major sub-clusters. Center to the right: Density plots of Prrx1-cre (left center), Fgfr3-creER (right center), and Gas1-creER (rightmost). Red dotted contour: OCT Stem cluster (enriched in Fgfr3-creER dataset). Blue dotted contour: ORT 1 cluster (enriched in Gas1-creER dataset). Colored scale, violet: high expression, yellow: low expression. e Flow cytometry analyses of CD45/Ter119/CD31^neg bone marrow cells isolated from Fgfr3-creER; R26R^tdTomato and Gas1-creER; R26R^tdTomato femurs at P23 (pulsed at P21), or Lepr-cre; R26R^tdTomato, Col1a1-GFP, and Cxcl12-GFP femurs at P21. Lower right: Percentage of tdTomato⁺ cells within CD45/Ter119/CD31^neg fraction. n = 7 (Fgfr3^CE), n = 6 (Gas1^CE), n = 5 (Lepr^cre), n = 8 (Col1a1-GFP), and n = 6 (Cxcl12-GFP) mice. Two-tailed, one-way ANOVA followed by Dunnett’s multiple comparison test. f Percentage of mSSCs among Fgfr3^CE+/neg or Gas1^CE+/neg cells (upper). Percentage of Fgfr3^CE+ and Gas1^CE+ cells among mSSCs (lower). n = 7 (Fgfr3^CE), n = 4 (Gas1^CE) mice. Data were presented as mean ± s.d. Exact P value is indicated in the figures. Source data are provided as a Source Data file.