Figure 1.

The fate and localization of bone-forming cells: Col2-, Osx- and Col1-positive cells during bone development (see also Supplementary Figure S1). (a) Experimental design. R26R-tdTomato reporter mice were crossed with transgenic mice expressing creER under the control of Col1, Osx and Col2 promoters. A single dose of tamoxifen (0.1 mg/g) was intraperitoneally (i.p.) injected into pregnant mothers at E18.5. (b-d) Hematoxylin/eosin-stained paraffin sections of the proximal metaphysis of the tibia at indicated ages (P=postnatal day, TB=trabecular bone, BM=bone marrow, HZ=hypertrophic chondrocyte zone). (e-g) Fluorescent images of the tibia of B-D (× 10). Blue:Dapi; Red:Tomato red fluorescent protein. (h) A cryosection (× 20 magnification) of the secondary ossification center in the Col2-creER;R26R^tomato mouse tibia at P28. Tomato-labeled cells are observed on the bone surface (osteoblasts; thick arrow), inside of the bone matrix (osteocytes; thin arrow), and in the bone marrow (dotted arrow). (i-j) Cryosections (× 10 magnification) of the Osx-creER;R26R^tomato mouse tibiae at indicated ages. Blue, DAPI; red, Tomato red fluorescent protein. (k) Cryosection (× 10 magnification) of the Osx-creER;R26R^tomato mouse tibia bone marrow at P28. Blue: DAPI; red: Tomato red fluorescent protein; thick arrow: osteoblasts; thin arrow: osteocytes; dotted arrow: stromal cells. (l-m) Cryosections (× 10 magnification) of the Col1-creER;R26R^tomato mouse tibia pulsed with tamoxifen at age E18.5. Tomato (red fluorescent protein) labeled the Col1-positive cells at age E18.5 and cell fate was chased at ages P1.5 and P28. (n) Cryosection of Col1-creER;R26R^tomato mouse tibia showing the secondary ossification center (× 20 magnification) at P28. Blue, DAPI; red, Tomato red fluorescent protein. thin arrow: osteocytes; thick arrow: osteoblasts. No tomato-positive cells were found in the bone marrow. n=5 mice per group