Fig. 2
Epidermal Shh is capable of regenerating HF in wounds without alteration of collagen. a, b K14-CreER; LSL-Shh; Gli1-LacZ (K14-Shh-Gli1-LacZ) and littermate controls with Gli1-LacZ were subjected to SW and treated with TAM from PW1d until tissue harvest at indicated time (n = 12 W (4 M) per condition). X-gal staining was analyzed at PW16d (a), and PW35d (b). Arrowheads show regenerated HFs (b). c–e Distribution of regenerated DP was analyzed from three representative LWs of K14-CreER; LSL-Shh (K14-Shh) and littermate controls treated with TAM from PW3d until tissue harvesting at PW35d (n = 9 W (9 M) per condition). AP+ DP in each picture were converted into dots with three different colors (red, green, and cyan) and merged into one picture (c, right). The original images and the corresponding colors represented in three columns on the left (c). Quantifications of AP+ DP per wound (d) and area occupied by DP (e). “Area occupied by DP” was defined by drawing a line that connects the outermost regenerated DP in wound. f–k K14-CreER; LSL-Shh (K14-Shh) and littermate controls were subjected to SW and treated with TAM from PW1d until tissue harvest at indicated time. RNA-seq analyses at PW11d showing heatmap of differentially expressed genes for K14-Shh and control mice (f, g). Red and green correspond to high and low expression levels, respectively (n = 12 W (4 M) per condition). Hydroxyproline assay to measure collagen contents at PW11d (n = 12 W (4 M) per condition) (h). Diameter of collagen fiber at PW30d (TEM images on the left, n = 2 W (2 M) per condition) (i). Detection of collagen fiber by Masson’s trichrome staining (j) and Picrosirius red staining (k) at PW11d and PW35d (insets). n: number of wounds (W) or mice (M), Data are represented as mean ± s.d., **p < 0.01; ***p < 0.001; ns: non-significant; Student’s t-test, Zigzag line and dashed white circle: wound boundary, Dashed line: epidermis–dermis border, SW small wound, LW large wound, PW post-wound, DP dermal papilla, AP alkaline phosphatase, FE follicular epithelium. Scale bars represent 500 μm (b (whole mount), c), 50 μm (b (section), j, k), 10 μm (a), 100 nm (i)
