(A) To target a sub-population of callus cells that arise from the periosteum, Sox9-CreERT2;tdTomato mice were injected for 3 consecutive days starting 7 days before analysis or before surgery. This tamoxifen induction schedule is referred to as ‘Pre’. The pie chart is a visual representation of number of cells affected in the callus at 10 dpr (red) and will be used throughout the figures as a reminder. (B) In uninjured Sox9-CreERT2;tdTomato mice, Sox9+ cells can be observed within the periosteum of the diaphysis by immunofluorescence (IF) for the tdTomato protein (red) (n = 3). They make up 6 ± 0.3% of the periosteal cells. (C) To determine if these cells participate in repair, rib resections were performed. At 10 dpr, lineage tracing of the Sox9+ cells show that the tdTomato+ expressing cells, 22 ± 1.3%, contribute to both cartilage and bone (n = 3) (IF for tdTomato). The enlarged images show chondrocytes, some of which are positive for tdTomato along with a near-adjacent section showing the histology of the area. At 14 dpr, tdTomato-expressing cells can be seen contributing to the trabecular bone. The enlarged image shows cells lining the trabeculae that are positive for tdTomato. (D) To activate Cre in a larger percentage of cells that build the repair callus, Sox9-CreERT2;tdTomato mice were injected with tamoxifen for 4 consecutive days, starting the day before surgery took place, the day of surgery, and for 2 more days. This tamoxifen induction schedule is referred to as ‘Post’. Another pie chart is used to visualize that a much greater portion of the callus is affected using this tamoxifen induction schedule at 10 dpr (85.5 ± 2.8%). (E) At 10 dpr, tdTomato+ cells are present in developing cartilage and bone, as well as in the periosteum surrounding the callus (native tdTomato fluoresence from a cryosection). The enlarged panel shows cells with chondrocyte morphology that are tdTomato+. At 14 dpr, tdTomato+ cells can be seen building new trabecular bone. In comparison to the Pre induced mice, these mice have significantly more tdTomato+ cells within the repair callus (n > 2 for all time points). Scale bar B = 100 microns, enlarged box = 50 microns; C, E = 200 microns.