Fig. 4. Smo is dispensable in callus cells after 4 dpi in a large-scale injury model.

a To broadly knock out Smo in callus cells at the time of injury (“Early Smo cKO”), Sox9:CreER;R26:tdTomato;Smo^flox/flox animals received tamoxifen injections on −1, 1, and 2 dpi. All rib resections were performed on day 0 and tissue was harvested at 14 dpi to assay regeneration outcomes. b To broadly knock out Smo in callus cells at 4 dpi (“Late Smo cKO”), Sox9:CreER;R26^tdT;Smo^flox/flox animals received tamoxifen injections at 4, 5, and 6 dpi. All rib resections were performed on day 0 and tissue was harvested at 14 dpi to assay regeneration outcomes. c tdTomato (tdT) reporter expression shows that Sox9:CreER targets the majority of the callus when tamoxifen is administered according to the scheme outlined in A and B. Scale bar = 200 microns. d At 14 dpi, H&E staining reveals that Early Smo cKO mice generate some bone and cartilage adjacent to the cut ends, but they largely fail to differentiate in the central region of the callus. Late Smo cKO mice generate a substantial callus that is largely composed of bone. N = at least three for each regimen. Surgical cut sites are indicated with yellow dotted lines. Scale bar = 200 microns. e Quantification analysis of H&E sections at 14 dpi reveals impaired skeletal regeneration in early Smo cKO animals compared to late Smo cKO animals (−29.9%, 95% CI = [−57.2–−2.61]). See Methods for quantification details.