Patched 1 and Patched 2 Redundancy Has a Key Role in Regulating Epidermal Differentiation Christelle Adolphe, Erica Nieuwenhuis, Rehan Villani, Zhu Juan.

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Patched 1 and Patched 2 Redundancy Has a Key Role in Regulating Epidermal Differentiation Christelle Adolphe, Erica Nieuwenhuis, Rehan Villani, Zhu Juan Li, Pritinder Kaur, Chi-chung Hui, Brandon J. Wainwright Journal of Investigative Dermatology Volume 134, Issue 7, Pages 1981-1990 (July 2014) DOI: 10.1038/jid.2014.63 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 K14Cre:Patched-deficient skin is maintained in a hyperproliferative state. (a–d) Histology of E18.5 transgenic epidermis. Note loss of typical elongated follicular structures in K14Cre:Ptch1lox/lox; Ptch2tm1/tm1 invaginations (d: arrows). (e–h) Basal (K14), (i–l) suprabasal (K10), and (m, n) cornified (loricrin) cell layers present in E18.5 transgenic skin. Expanded PCNA (proliferating cell nuclear antigen; n) and maintained expression of (p: arrows) cycling D1 in K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 invaginations compared with (m: arrows and o) K14Cre:Ptch1lox/lox control skin. bcl: basal cell layer, cl: cornified layer; sbl: suprabasal cell layer. Bar=50 μm. Journal of Investigative Dermatology 2014 134, 1981-1990DOI: (10.1038/jid.2014.63) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 K14Cre:Patched-deficient follicular structures exhibit a primordial hair follicle (HF)–like state. Hair germ stages of follicular development in control epidermis express (a) p-cadherin, (c) CDP, and (e) Sox9. K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 invaginations also express (b) p-cadherin, (d) CDP, and (f) Sox9. A proportion of K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 invaginations did not express CDP (d: asterisks). (g: yellow arrows) Lef1 expression in the HF downgrowths of E18.5 control epidermis. (h: asterisks) Mutant follicular-like structures invading the dermis fail to express Lef1. (i) HF structures present in control skin grafts (9 weeks). (j) Presence of basal cell carcinoma (BCC) lesions and lack of HF structures in K14Cre:Ptch1lox/lox;Ptch2tm1/tm1 skin grafts. Dermal papillae structures associated with control HFs (white arrows: a, c, and e) are absent in mutant epidermis. Bar=50 μm. Journal of Investigative Dermatology 2014 134, 1981-1990DOI: (10.1038/jid.2014.63) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Loss of glandular, follicular, and interfollicular epidermal lineage specification and differentiation in K5Cre:Patched-deficient skin. Gross morphology indicating epidermal transparency of (a: LHS and a’) control and (a: RHS and a’’) K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 E18.5 embryos. Epidermal barrier assay revealing penetration of dye in (b) K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis. (d) Histology of E18.5 transgenic epidermis revealing perturbed hair follicles (hf) in K5Cre:Ptch1lox/lox epidermis and (e: arrows) loss of all epidermal appendages and hyperplastic epithelial invaginations in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis compared with (c) controls. Immunohistochemical staining of interfollicular differentiation markers (f–h) K10 and (i–k) loricrin revealing absence of suprabasal (sbl) (h: arrows) and (k: arrows) cornified cell layers (cl) in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis. Bar=50 μm. Journal of Investigative Dermatology 2014 134, 1981-1990DOI: (10.1038/jid.2014.63) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Proliferation and progenitor cell state of K5Cre:Patched-deficient epidermis. Protein expression of the proliferation markers (a–c) PCNA (proliferating cell nuclear antigen), (d–f) Ki67, and transcriptional expression of (g–i) cyclin D1 mRNA. Protein expression of epidermal stem/progenitor cell markers (m–o) p63 and transcriptional expression of (p–r) K17 and (s–u) K15 mRNA. Immunohistochemical staining of the basal cell markers (k–l) K5 and (v–x) K14. Note decrease in the expression of basal cell markers in K5Cre:Ptch1lox/lox;Ptch2tm1/tm1 epidermis (l: arrows, x: arrows). Bar=50 μm. Journal of Investigative Dermatology 2014 134, 1981-1990DOI: (10.1038/jid.2014.63) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions