(A) Gli1creERT2;R26Tom;Ptch1fl/fl mice were treated with tamoxifen (TAM) at 8 weeks and dorsal skin was analyzed 5 weeks post TAM treatment (n = 3 mice). The TD area shows a de novo anagen HF (turquois frame). Additionally, traced pre-existing (old) telogen HFs with basal cell carcinoma (BCC)-like growth are present. Inset (white frame): anagen hair bulb of de novo HF showing K71-positive Henle’s layer, K6-positive companion layer, hair pigment and continuous Tomato-tracing from the hair bulb into the TD. (B) Immunofluorescent co-staining of K71 (Henle’s layer) and K6 (companion layer) in a wild type HF of a similar hair cycle stage (Anagen IIIa, P27) (n = 2 mice). (C) Quantification of hair size in Gli1creERT2;R26Tom;Ptch1fl/fl and Gli1creERT2;R26Tom;Ptch1fl/wt mice that were treated with TAM at 8 weeks and dorsal skin was analyzed 9 weeks post TAM treatment. Right panel: De novo telogen HFs with a thin hair shaft formed in the TDs of Gli1creERT2;R26Tom;Ptch1fl/fl skin (white arrow). For the quantification, we analyzed hair shafts of de novo HFs from Gli1creERT2;R26Tom;Ptch1fl/fl mice (blue bracket), old/pre-existing Zig-zag HFs from the same mice (white bracket), and Zig-zag HFs from wild-type-phenotype control mice (Gli1creERT2;R26Tom;Ptch1fl/wt) (n = 3 mice for each genotype; 34 de novo, 314 old/pre-existing, and 437 control HFs; Figure 2—source data 1). Hair shaft length was measured in telogen stage hair shafts from the hair club to the HF opening (as indicated by the blue and white brackets). Left panel: Each dot represents a hair shaft and the dots are colored according to the HF type (blue for de novo HFs, gray for pre-existing Zig-zag HFs, orange for control Zig-zag HFs). De novo hair shafts were significantly smaller (p-value<1.10−6) and thinner (p-value<1.10−6) compared to old/pre-existing and control Zig-zag hairs (Mann-Whitney U test). Arrowheads on the x- and y-axis indicate the mean values of hair shaft length and width of de novo, old/pre-existing or control Zig-zag HFs, respectively. TD: touch dome. HF: hair follcile. TAM: tamoxifen. BF: bright field. Scale bars: 100 μm (A), 50 μm (B, C).