Fig. 2. Dual origin of trabecular osteoblasts in the spongiosa of Col10CreYFP⁺ specimens.

(A) Co-immunostaining for YFP and Col1 of a P1 Col10CreYFP⁺ tibia shows that YFP⁺ cells in the spongiosa are to a large part associated with Col1⁺ trabeculae. Panels a and b show different magnifications. (B) Triple staining of a Col10CreYFP⁺ tibia (P1) for YFP (green), osteocalcin (Ocn, blue and CD31 (red), confirms the presence of YFP⁺Ocn⁺ osteoblasts in the spongiosa, whereas endothelial cells are YFP negative. (b–e) Enlarged images of the boxed area in panel a showing single channels (b,c), two channels (d) and all three channels (e). (Ca–f) Immunofluorescence staining for Osx (red) and YFP (green) shows that a substantial number of Osx⁺ cells in the spongiosa and endosteum ‘E’ of P1 (a–c) and E18.5 (d–i) tibiae are YFP⁺ (yellow cells in c–e; white cells in f). In the periosteum ‘P’ in, all Osx⁺ cells are negative for YFP (c,d). pro = proliferating zone, ph = prehypertrophic zone; h = hypertrophic zone. (g–i) Triple immunostaining for Osx, Col1 and YFP: most YFP⁺Osx⁺ cells (yellow) and YFP⁻Osx⁺ cells (red) are associated with trabeculae, stained for Col1 (blue). (D) Immunostaining of paraffin sections shows Osx⁺ cells in the groove of Ranvier ‘R’, periosteum ‘P’, pre-hypertrophic chondrocytes ‘ph’ and trabecular bone ‘T’. E18.5 Col10CreYFP⁺ tibia. (E) Pie chart showing the relative amounts of chondrocyte-derived (YFP⁺) cells of all DAPI⁺ cells and other cells in the spongiosa of P2 tibiae. 15% of all DAPI⁺ cells are YFP⁺Osx⁺; 19% are YFP⁺Osx⁻ cells and thus not osteogenic; 31% are Osx⁺YFP⁻. Cells were counted in the trabecular zone in five humeri or tibia sections (100–300 cells per field). (F) Bar graph showing ratios of YFP⁺Osx⁺ double positive (yellow) cells to total Osx⁺ cells (red and yellow) in the spongiosa at various stages of development. Error bars: SD.