Epidermal Wnt Controls Hair Follicle Induction by Orchestrating Dynamic Signaling Crosstalk between the Epidermis and Dermis Jiang Fu, Wei Hsu Journal.

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Epidermal Wnt Controls Hair Follicle Induction by Orchestrating Dynamic Signaling Crosstalk between the Epidermis and Dermis Jiang Fu, Wei Hsu Journal of Investigative Dermatology Volume 133, Issue 4, Pages 890-898 (April 2013) DOI: 10.1038/jid.2012.407 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Gpr177 is expressed in hair follicle development. Immunostaining of the (a) embryonic day 11.5 (E11.5) and (b) E13.5 skin shows the expression of Gpr177 in the epithelium and underlying mesenchyme. A restricted elevation is found in the epidermal basal cells and hair follicular cells at (c) E15.5 and (d) E17.5, respectively. (d) The inset shows nonuniform expression of Gpr177 in the hair follicle. (e–l) Double labeling of Gpr177 with (e, g, i, k) AE3, a marker for the entire epidermis, or (f, h, j, l) keratin 14 (K14), a marker for the epidermal basal layer, identifies the Gpr177-expressing cells at (e, f, i, j) E14.5 and (g, h, k, l) E17.5. Bars=50μm (a–l). Journal of Investigative Dermatology 2013 133, 890-898DOI: (10.1038/jid.2012.407) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Epidermal deletion of Gpr177 abrogates the induction of hair follicles. β-Galactosidase (β-gal) staining of the (a) embryonic day 13.5 (E13.5) and (b) E14.5 K5-Cre; R26R embryos analyzes the effectiveness of Cre recombination in the epidermis and hair placode. Sections of the control and Gpr177K5 embryos were analyzed by coimmunostaining of (c, d) Gpr177 and keratin 14 (K14) and (e–h) hematoxylin/eosin staining at (c–f) E14.5 and (g, h) E17.5. Whole-mount in situ hybridization of the (i, k, m, o) control and (j, l, n, p) Gpr177K5 embryos reveals the expression of (i, j) Edar, (k, l) Bmp2, (m, n) Bmp4, and (o, p) Shh at E14.5. Control genotype: Gpr177Fx/Fx or K5-Cre; Gpr177Fx/+. Bars=50μm (a–h); 200μm (i–p). Journal of Investigative Dermatology 2013 133, 890-898DOI: (10.1038/jid.2012.407) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Wnt expression and signaling are affected by the epidermal deletion of Gpr177. In situ hybridization in sections shows the expression of Wnt genes in the (a–k) embryonic day 14.5 (E14.5) control and (a′–k′) Gpr177K5 skin. Sections of (l–o) control and (l′–o′) Gpr177K5 skin examine the signaling activity of Wnt by immunostaining of an activated form of β-catenin (ABC) and β-galactosidase (β-gal) staining of the Axin2lacZ allele at (n, n′) E13.5, (l, l′, o, o′) E14.5, and (m, m′) E17.5. In situ hybridization analyzes the expression of Wnt downstream targets, (p, p′) Lef1 and (q, q′) Dkk4 in the E14.5 (p, q) control and (p′, q′) Gpr177K5 skin. Control genotype: Gpr177Fx/Fx or K5-Cre; Gpr177Fx/+. Bars=50μm (a–q, a′–q′). Journal of Investigative Dermatology 2013 133, 890-898DOI: (10.1038/jid.2012.407) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Epidermal stimulation of β-catenin alleviates the hair follicle defects of Gpr177K5. Sections of the control, Gpr177K5, and Gpr177K5; sβcatK5 were analyzed by (a–c) hematoxylin and eosin (H&E) staining, (d–f) immunostaining of Gpr177, (g–i) ABC, and (p–r) keratin 17 (K17), in situ hybridization of (j–l) Edar and (m–o) Shh, and double labeling of (s–u) CD133 and AE3 or (v–x) Sox2 and AE3 at embryonic day 14.5 (E14.5) and E15.5. Arrowhead indicates the dermal activation of β-catenin in the (g) control, but not in the (h, i) Gpr177K5 and Gpr177K5; sβcatK5 mutants. (s, v) Asterisks indicating the dermal papilla markers, CD133 and Sox2, are detected in the control, (q, r, t, u) but absent in the Gpr177K5; sβcatK5 mutants. Control genotype: Gpr177Fx/Fx or K5-Cre. Bars=50μm (a–x). Journal of Investigative Dermatology 2013 133, 890-898DOI: (10.1038/jid.2012.407) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The Gpr177-mediated regulation of Wnt in the dermis is dispensable for hair follicle initiation. (a, b) β-Galactosidase (β-gal) staining of the embryonic day 14.5 (E14.5) control and Gpr177Dermo1 skin carrying R26R shows Cre effectiveness. Sections are analyzed by colabeling of (c–f) Gpr177 and keratin 14 (K14) or (q, r) CD133 and AE3, (g, h) hematoxylin and eosin (H&E), immunostaining of (i–l) Cadherin and (s, t) ABC and in situ hybridization of (m, n) Edar and (o, p) Shh, and (u–x) β-gal staining of the Axin2lacZ allele at E13.5, E14.5, and E15.5. (y) Graph indicates quantitative analysis for the placode region and the expression domain for ABC, Edar, and Shh. Control genotype: Gpr177Fx/Fx or Dermo1-Cre; Gpr177Fx/+. Bars=50μm (a–x). (z) Model for epidermal Wnt in orchestrating signaling interaction between the epidermis and dermis during hair follicle development. Journal of Investigative Dermatology 2013 133, 890-898DOI: (10.1038/jid.2012.407) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions