Figure 3. The non-chondrocytic reporter⁺ cells in the primary spongiosa of tamoxifen-treated Agc1-CreERT2 embryos and mice are derived from mature chondrocytes.

A: The LacZ stained femur section of E16.5 Agc1-CreERT2; ROSA26R embryo treated with tamoxifen at E11.5. The black arrows indicate the non-chondrocytic LacZ⁺ cells in the primary spongiosa. Black brackets: hypertrophic zone. No LacZ⁺ cells were detected within the perichondrium (red arrows) and periosteum (red arrowheads). B: LacZ staining of Agc1-CreERT2; ROSA26R femurs treated with tamoxifen at E14.5 and euthanized 1 and 2 days after injections or at 1 month postnatally. At E15.5, LacZ⁺ non-chondrocytic cells (black arrows) were only present right beneath the growth plates, while at E16.5, the number of LacZ⁺ non-chondrocytic cells was increased. At 1 month, LacZ⁺ cells were still present on the trabecular surfaces (black arrows), endosteum (green arrows) and within bone matrix (red arrows). Black brackets: hypertrophic zone. C: Anti-EGFP Immunohistochemical analysis (IHC) of Agc1-CreERT2; Osx ^flox/+ mice treated with tamoxifen at E13.5 or E14.5 and euthanized 1, 2 and 3 days after injections or 2 weeks postnatally. The data show that there were abundant non-chondrocyte EGFP⁺ (Osx^−/+) cells in the primary spongiosa of the femur of E15.5 Agc1-CreERT2; Osx ^flox/+ embryos treated with tamoxifen at E13.5. However, there were almost no non-chondrocyte EGFP⁺ cells in the primary spongiosa of E15.5 Agc1-CreERT2; Osx ^flox/+ embryos treated with tamoxifen at E14.5. Red arrows: EGFP⁺ (Osx^−/+) hypertrophic chondrocytes. Green arrows: preosteoblast-like EGFP⁺ (Osx^−/+) cells. Purple arrows: mature osteoblast-like EGFP⁺ (Osx^−/+) cells. Blue arrows: EGFP⁺ (Osx^−/+) osteocytes. Black brackets: hypertrophic zone; ps: primary spongiosa.