Adenovirus-Mediated Wnt10b Overexpression Induces Hair Follicle Regeneration Yu-Hong Li, Kun Zhang, Ke Yang, Ji-Xing Ye, Yi-Zhan Xing, Hai-Ying Guo, Fang.

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Adenovirus-Mediated Wnt10b Overexpression Induces Hair Follicle Regeneration Yu-Hong Li, Kun Zhang, Ke Yang, Ji-Xing Ye, Yi-Zhan Xing, Hai-Ying Guo, Fang Deng, Xiao-Hua Lian, Tian Yang Journal of Investigative Dermatology Volume 133, Issue 1, Pages 42-48 (January 2013) DOI: 10.1038/jid.2012.235 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The expression pattern of Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b) throughout the hair follicle cycle. The expression pattern of Wnt10b in the dorsal skin of C57 mice was determined by in situ hybridization (a–c) and immunofluorescence (d–f). Wnt10b was expressed in anagen (a, d) but not in catagen (b, e) or telogen (c, f). The upper right corner shows a magnification of the framed labeled area. The dashed line delineates the hair follicle structure. Arrowheads in a and d show the expression of Wnt10b in hair matrix cells. DAPI, 4’,6-diamidino-2-phenylindole; DP, dermal papilla. Scale bar=10μm. Journal of Investigative Dermatology 2013 133, 42-48DOI: (10.1038/jid.2012.235) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The expression pattern of Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b) after the overexpression of Wnt10b. Telogen C57 mice were intradermally injected with AdWnt10b, and the expression of Wnt10b was detected by in situ hybridization (a–c) and immunofluorescence (d–f) at 24 (a, d), 48 (b, e), and 72hours (c, f) after injection. The upper right corner shows a magnification of the framed labeled area. The dashed line delineates the hair follicle structure. Arrowheads show the expression of Wnt10b. DAPI, 4’,6-diamidino-2-phenylindole; DP, dermal papilla; SG, sebaceous gland. Scale bar=10μm. Journal of Investigative Dermatology 2013 133, 42-48DOI: (10.1038/jid.2012.235) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Hair follicles after the overexpression of Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b) in telogen C57 mice. Telogen C57 mice were intradermally injected with adenovirus and observed under gross observation (a–d) or with hematoxylin and eosin staining (e–i). The observation time points were 3 days (a, e), 5 days (f), 9 days (g, h), 11 days (b), 14 days (c), 28 eight days (i), and 56 days (d) after adenovirus injection. (a, b, d–i) Mice injected with AdWnt10b. (c) From left to right, mice injected with phosphate-buffered saline, AdGFP (adenovirus with green fluorescent protein), AdWnt10b, AdWnt10b, and AdWnt10b. (j) Mice injected with AdGFP. Arrowheads show the injection areas. (e–g, i, j) The right panel shows a magnification of the framed labeled area in the left panel. Scale bar=100μm (left panel and h) and 10μm (right panel). Journal of Investigative Dermatology 2013 133, 42-48DOI: (10.1038/jid.2012.235) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression patterns of structure markers in Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b)-overexpressed skin. Telogen C57 mice were intradermally injected with AdWnt10b, and expressions of AE15, β-catenin, K6, K10, Wnt10b, and Sox9 were detected by immunohistochemistry 9 days after injection (left panel) and compared with those in hair follicles at the corresponding stage (right panel). The upper right corner shows a magnification of the framed labeled area. The dashed line delineates the hair follicle structure. Arrowheads show the expressions of AE15 (a), β-catenin (b), K10 (c), K6 (d), Wnt10b (e), and Sox9 (f). DAPI, 4’,6-diamidino-2-phenylindole; DP, dermal papilla. Scale bar=10μm. Journal of Investigative Dermatology 2013 133, 42-48DOI: (10.1038/jid.2012.235) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Hair follicles after the knockdown of Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b). C57 mice at the period of transition from telogen to anagen (postnatal day 30) were intradermally injected with AdGFP (adenovirus with green fluorescent protein; a) or AdSim10b (b–d). (a, b) Five days after adenovirus injection. (c, d) Nine days after AdSim10b injection. (c) The dorsal skins of treated mice were spread out on paper and photographed. (d) The skin in c was cut and stained with hematoxylin and eosin. Hair follicles in the injection area were still in telogen, whereas follicles in the surrounding area around the skin were in anagen. Arrowheads show the injection area (wheal around the injection site). AdSim10b, siRNA of Wnt10b mediated by adenovirus. Scale bar=500μm. Journal of Investigative Dermatology 2013 133, 42-48DOI: (10.1038/jid.2012.235) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 AdSimBC abrogates the hair follicle regeneration initiated by the overexpression of Wnt10b (wingless-type mouse mammary tumor virus integration site family member 10b). (a–c) Telogen C57 mice were depilated and intradermally injected with AdSimBC. (a) Depilated mice. (b) Seven days after AdSimBC injection. Arrowheads show the injection area (wheal around the injection site). The triangle shows the delay of anagen entry resulting from the wound, which is different from the delay of anagen entry resulting from AdSimBC treatment. (c) Nine days after AdSimBC injection. Arrowheads show the skin of the injection area, which turned gray. (d) A telogen C57 mouse was first injected with AdSimBC and then injected with AdWnt10b 24hours later at the same injection area. Arrowheads show that there were no visible changes in the skin adjacent to the injection areas 7 days after AdWnt10b injection. AdSimBC, siRNA of β-catenin mediated by adenovirus. Journal of Investigative Dermatology 2013 133, 42-48DOI: (10.1038/jid.2012.235) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions