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. 2018 Apr 6;6:12. doi: 10.1038/s41413-018-0013-z

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© The Author(s) 2018

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 — SHP2 deficiency in PRRX1-expressing OCPs delays endochondral ossification and leads to ectopic cartilage formation. a Representative images of H&E-stained longitudinal sections of femurs demonstrate impaired ossification of appendicular bones, enhanced chondrogenesis and ectopic cartilage formation in 7-day-old SHP2_Prrx1KO mice, compared with SHP2_Prrx1CTR Mice. Bottom panels are enlarged views (×10) of corresponding boxed areas in the top panel showing ectopic chondrocytes in the bone cortex and islands of chondrocytes in the bone marrow (BM, arrow) of SHP2_Prrx1KO mice (n = 4). b Images of sagittal sections of proximal tibiae (top), stained with Safranin O/fast green, showing the broad and elongated growth plate cartilage in SHP2_Prrx1KO mice. Bar graphs (bottom) show 2- and 5-fold increases in height of the epiphyseal cartilage (blue+yellow lines) and the pre-hypertrophic and hypertrophic layers, (yellow line) respectively, in SHP2_Prrx1KO mice, compared with SHP2_Prrx1CTR mice (n = 4). *P < 0.05 (Student’s t test). c Fluorescence microscopy of frozen tibia sections demonstrates that PRRX1-expressing cells (GFP⁺) primarily exist as a thin layer (periosteum, arrow) on the surface of the mineralized bone cortex and also appear in the epiphyseal cartilage of 2-day-old SHP2_Prrx1CTR;R26^mTmG reporter mice. By contrast, in age-matched SHP2_Prrx1KO;R26^mTmG mice, there was no mineralized cortical bone, and the GFP+ cells were not restricted to the thin periosteal soft tissue layer along what would have been the cortex (arrows). Instead, GFP⁺ OCPs and their derivatives were scattered in non-mineralized areas where cortical bone should have formed and in cartilage islands within the bone marrow (arrow head) (n = 4). Bottom panels are enlarged views (×10) of the corresponding boxed areas in top panels. Dashed lines denote the boundary between muscle and bone. Bone marrow, BM. d Images of anti-BrdU staining of proximal tibia sections demonstrate the increase of BrdU⁺ cells in the proliferative zone of SHP2_Prrx1KO mice, compared to that of SHP2_Prrx1CTR. Bar graphs at the bottom show quantification of these data from replicates (n = 3, *P < 0.01, Student’s t test).