Fig. 5. The primary source of SHH is not Sox9-derived SSPCs.

a Experimental overview. To lineage-trace Sox9+ lineage cells, Sox9:CreER;R26:tdTomato animals received tamoxifen injections on days −7, −6, and −5. All rib resections were performed on day 0 and tissue was harvested at 7 dpi to assay Shh expression in b. b RNA-ISH for Shh (green) on histological sections at 7 dpi. Sox9+ lineage cells express tdTomato (tdT, red). Shh expression is strongly detected within cells located in the central resection gap and in the surrounding periosteal tissues. Although a few tdT+ lineage-traced cells express Shh (white arrowheads), the majority of Shh-expressing cells are tdT-negative. Surgical cut sites are indicated with yellow dotted lines. N = 3. Scale bar = 200 microns. c Experimental overview. To knock out Shh in Sox9+ lineage cells, Sox9:CreER;Shh^flox/flox animals received tamoxifen injections on days −7, −6, and −5. All rib resections were performed on day 0 and tissue was harvested at 10 or 14 dpi to assay regeneration outcomes. d Blinded scoring of regeneration outcomes. Histological sections were used to score regeneration outcomes as Good, Moderate, or Poor (see Methods for details regarding scoring criteria). Control animals largely healed well and scored as Good (4/9 animals) or Moderate (3/9 animals). Similarly, Sox9:CreER;Shh^flox/flox animals largely healed well (3/8 animals) or Moderate (2/8). Animals from 10 and 14 dpi were pooled for graphical representation. e At 10 dpi, Sox9:CreER;Shh^flox/+ controls and Sox9:CreER;Shh^flox/flox mice both generate a large Safranin-O stained callus composed largely of cells with chondrocyte morphology and with the central-most portion beginning osteogenic differentiation. At 14 dpi, Sox9:CreER;Shh^flox/+ controls and Sox9:CreER;Shh^flox/flox mice have large Safranin-O-stained calluses with a central region undergoing bone conversion. Surgical cut sites are indicated with yellow dotted lines. Scale bar = 200 microns.