Human superficial basal cell carcinoma (BCC) expresses hair bud lineage markers. (a, b) H&E-stained sections of early human superficial BCC and embryonic hair buds. Dashed line in b indicates epithelial component of embryonic hair follicle, with adjacent mesenchymal condensate marked with an arrowhead. Note absence of morphologically recognizable mesenchymal condensate near superficial BCC. (c, d) Hair placode marker K17 is expressed in superficial BCC and hair bud. At this stage of embryonic development, surrounding epidermis (d) also expresses this marker. (e, f) The suprabasal cell marker K1 is not expressed in superficial BCC or in hair bud. (g–j) Outer root sheath marker Sox9 and hair matrix/inner root sheath marker CDP are expressed in superficial BCC and hair bud. Note largely nonoverlapping expression patterns of Sox9 and CDP in hair bud (brackets in h and j), as compared with overlapping expression in superficial BCC. (k, l) Increased proliferation in superficial BCC and hair bud relative to adjacent epidermis, assessed by Ki67 staining.

Ectopic Hh signaling in M2SMO-expressing hairless mouse skin drives superficial BCC-like downgrowths resembling hair buds. Histological appearance, marker expression and increased proliferation rate of ectopic epithelial buds in volar skin from M2SMO-expressing mice and hair bud from embryonic day 16.5 mouse embryo. (a) Whole-mount view of control skin from ventral aspect of mouse hind limb, with H&E-stained sections from indicated regions. Triangle outlines a region completely devoid of hair follicles or other skin appendages (b′), compared to eccrine glands in the footpad region (a′) and hair follicles at the periphery (c′). (b, c) Whole-mount view of hairless control (WT) and M2SMO-transgenic volar skin. Activation of Hh signaling using M2SMO results in the appearance of scattered buds and hyperpigmentation in hairless skin (c). (d, e) H&E-stained sections showing distribution of buds in M2SMO volar skin (e). (f, g) Endogenous alkaline phosphatase (Alk Phos), a marker for follicle-associated mesenchymal condensates, is not detected in control or M2SMO volar skin. Inset in g shows alkaline phosphatase–positive (red) dermal papillae in hair follicles. (h, i) H&E-stained sections of de novo epithelial bud in M2SMO volar skin and embryonic mouse hair bud. The mesenchymal condensate neighboring normal hair buds (arrowhead in i) is not detected in association with the M2SMO-induced bud (h). Immunostaining revealed expression of hair follicle outer root sheath markers K17 and Sox9 (j–m), and the hair matrix/inner root sheath marker CDP (n, o) in ectopic buds and embryonic hair buds. Note nonoverlapping expression of Sox9 and CDP in hair buds (brackets in m and o), as compared with M2SMO-induced bud. (p, q) The suprabasal marker K1 is not expressed in M2SMO-induced buds or embryonic hair buds, whereas proliferation (r, s) is increased in both. (t, u) The hair bud marker P-cadherin (P-cad) is upregulated in both M2SMO and embryonic hair buds, whereas downregulation of E-cadherin (E-cad) is detected only in hair buds (v, w). White dotted line shows epidermal-dermal junction.

Canonical Wnt signaling is activated in human superficial BCC and epithelial buds in M2SMO-expressing hairless mouse skin. (a, b) β-catenin immunostaining reveals nuclear-cytoplasmic β-catenin localization in human superficial BCC and hair bud, consistent with activation of canonical Wnt signaling. Note intense nuclear staining in mesenchymal cells adjacent to hair bud (arrowhead in b) but not BCC (a). Adjacent epidermis (*) exhibits β-catenin localized to the cell membrane. (c, d) Similarly, β-catenin immunostaining reveals nuclear-cytoplasmic localization in mouse M2SMO bud cells arising from hairless volar skin and embryonic day 16.5 mouse embryonic hair bud, with stained mesenchymal cells also noted adjacent to the hair bud (arrowhead). (e) Immunoblotting using mouse ABC antibody showing presence of nonphosphorylated (active) β-catenin in lysates from M2SMO volar skin, but not hairless control volar skin. Skin lysate from postnatal day 8 mouse with actively growing hair follicles (hf) was used as a positive control. (f) Semiquantitative RT-PCR was performed on RNA isolated from control volar skin (Ctrl), early bud-stage M2SMO-transgenic volar skin (M2SMO Bud) and later, basaloid follicular hamartoma-containing (Supplementary Fig. 1) M2SMO volar skin (M2SMO BFH), with postnatal day 8 skin as a positive control (hf). Upregulation of Gli1 and Ptch1 expression in M2SMO skin confirms activation of Hh signaling. M2SMO-expressing skin expresses multiple Wnt genes, Tcf4 and endogenous Wnt target genes Axin2, Sp5 and cyclin D1. In control volar skin there is negligible expression of Gli1, Ptch1 and Wnt pathway-associated genes.

Inhibition of canonical Wnt signaling with Dkk1 blocks M2SMO-induced development of epithelial buds and follicular hamartomas. (a) Experimental timeline for induction of Dkk1 expression in M2SMO + Dkk1 mice during embryogenesis (E16.5) with analysis at postnatal day 7 (P7). (b) Expression of Dkk1 blocks development of de novo buds seen in whole-mount or H&E-stained sections. (c) Experimental timeline for Dkk1 induction at P1 with analysis at P35 to assess effects on hamartoma development. (d) Gross view and H&E-stained sections of volar skin from control, M2SMO and M2SMO + Dkk1 mice. Note increased pigmentation and hamartoma development in M2SMO skin, as compared with control. The M2SMO-induced phenotype is markedly inhibited by coexpression of Dkk1. Foci of hyperpigmentation were sometimes detected in M2SMO + Dkk1 volar skin near footpads (arrowhead in d), and histological examination of these regions revealed buds and/or hamartomas with nuclear β-catenin (not shown), indicating incomplete blockade of canonical Wnt signaling in these regions. (e) Gross view and H&E-stained sections of tail skin from control, M2SMO and M2SMO + Dkk1 mice. Note disrupted hair formation, increased pigmentation and hamartomas in M2SMO skin, as compared with control. The M2SMO-induced phenotype is again markedly inhibited by coexpression of Dkk1. (f) Immunofluorescence for β-catenin in volar skin. Cells with nuclear β-catenin in M2SMO skin, indicating activation of canonical Wnt signaling, are marked with arrowheads. Inhibition of nuclear β-catenin localization in M2SMO + Dkk1 mice, indicating effective blockade of canonical Wnt signaling. White dotted lines represent epidermal-dermal junction. (g) Semiquantitative RT-PCR using RNA from control, M2SMO and M2SMO + Dkk1 tail skin reveals downregulation of Axin2 and Sp5 in M2SMO + Dkk1 volar skin, confirming blockade of canonical Wnt signaling. In contrast, expression of Hh target genes Gli1 and Ptch1, as well as Wnt genes, is essentially unaltered in M2SMO + Dkk1 skin as compared with M2SMO skin. (h) The Hh-responsive keratin, K17, is not detected in control volar skin, but ectopically expressed in both M2SMO and M2SMO + Dkk1 volar skin, in keeping with sustained activation of Hh signaling in these mice. M2SMO-associated expression of Sox9, CDP and Ki67 are blocked or inhibited in mice also expressing Dkk1.