Fig. 2. Notch signaling dominates the proliferative phase while canonical Wnt signaling governs the differentiation phase.
a Temporospatial expression profile of Notch downstream target Hey1 by immunostaning and co-localization with osterix-positive cells. To label osterix-positive cells, iOsx/tdTom mice were pulsed by tamoxifen injection (1 mg/day/mouse) twice at 1 day before surgery and 1 day after surgery. b, c Temporal Notch downstream target gene expression during the early injury response (uninjured, POD 1–10) in SSCs and BCSPs (n = 6–9). d, e Notch activation using Jagged1-coated tissue culture plates resulted in upregulation of the Notch target genes Hey1 and Hes1. f In response to Notch activation, proliferation increased, shown by BrdU quantification. g The number of tdTomato-positive (osx-positive OPCs) cells decreased in the POD 3 injury site after Notch inhibition in iOSX/tdTom/Rbpjfl/fl mice. Tamoxifen (1 mg/day/mouse) was injected from day −1 until day 2. h Bone volume, i tissue volume, and j bone volume/total volume, at postoperative day 10 using microCT histomorphometry demonstrates a smaller bone volume in the regenerate in the iOSX/tdTom/Rbpjfl/fl mice (tamoxifen (1 mg/day/mouse daily) from day −1 to day 6). k Spatial expression of Wnt responsiveness within osx-positive (IF) OPCs using iAxin2/GFP reporter mice (Tamoxifen administration: 1 day before euthanasia). Scale bar = 50 μm. l, m Axin2 gene expression SSCs and BCSPs in uninjured and POD 7–21 days (n = 4). BCSP bone-cartilage-stromal progenitor cells, Jag1 Jagged1, POD postoperative day, SSC skeletal stem cell. *p < 0.05, **p < 0.01, ***p < 0.001. Data were represented as mean ± s.e.m.