Characterization of FGFR1/2⁺ skeletal cells in vivo. (A) Immunofluorescence of trabecular BM tissue section from anterior iliac visualized with confocal microscopy. A 3D reconstruction of a Z-stack of the whole 10-μm section taken at 0.3-μm intervals is shown. Hematopoietic lineage markers (Lin) and CD31 were used to exclude hematopoietic cells and endothelial cells, respectively. The dotted line shows a trabecular bone surface. (B) Whole-mount immunofluorescence staining of perichondrium (acquired as in Figure 1). Collagen 1 (Col.1) and PTHrP showed a similar expression pattern to FGFR1/2⁺ cells in the perichondrium and were sometimes irrigated by CD31⁺ microvasculature. (C) Cells surrounded by perichondrium-specific, FGF-18–expressing cells. The proliferation marker Ki67 was only detected in perichondrial cells and colocalized with FGFR1/2⁺ cells (D). (E) Three D reconstruction and surface rendering demonstrating that most periosteal FGFR1⁺ cells were pericytes wrapped around CD31⁺ blood vessels. In the mostly avascular perichondrium, the pericyte marker NG2 did not colocalize with FGFR1⁺ cells, but appeared to be expressed by chondrocytic progenitors (F). In vascularized periosteum, FGFR1⁺/NG2⁺ pericytes could be observed; however, not all FGFR1⁺ cells coexpressed NG2, which was also detected in nonpericytic cells (G). Expression of the pericytic MSC marker CD146 in periosteum also showed only limited colocalization with NG2 (H).