Activating Hair Follicle Stem Cells via R-spondin2 to Stimulate Hair Growth  Andrew A. Smith, Jingtao Li, Bo Liu, Daniel Hunter, Malcolm Pyles, Martin Gillette, Girija R. Dhamdhere, Arie Abo, Anthony Oro, Jill A. Helms  Journal of Investigative Dermatology  Volume 136, Issue 8, Pages 1549-1558 (August 2016) DOI: 10.1016/j.jid.2016.01.041 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Wnt signaling flutuates with the hair cycle. Gomori trichrome staining of tissue sections through HFs (a) in anagen and (b) in telogen (dotted yellow lines). Whole-mount Xgal staining of dorsal skin from an Axin2LacZ/+ mouse showing (c) HFs in anagen and (d) telogen. (e) qRT-PCR for Axin2 expression in the skin of 5-week-old, 9-week-old, and waxed 12-week-old mice. GFP and KRT72 immunostaining in HFs from Axin2Cre/+R26RmTmG/+ mice in (f) anagen. (g) GFP immunostaining in HFs from Axin2Cre/+R26RmTmG/+ mice in anagen and (h, i) telogen. Scale bars = 50 μm; *P < 0.05. Axin2, axis inhibition protein 2; DAIP, 4',6-diamidino-2-phenylindole; dp, dermal papilla; GFP, green fluorescent protein; HF, hair folicle; irs, inner root sheath; m, matrix; ors, outer root sheath; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; Xgal, x-galactosidase. Journal of Investigative Dermatology 2016 136, 1549-1558DOI: (10.1016/j.jid.2016.01.041) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Wnt signaling is elevated during anagen and lowered at the onset catagen. (a) Waxing synchronizes the hair cycle, which has a duration of 21 days. (b) Localization of BrdU+ve cells (asterisk indicates autofluorescent hair shaft), (c) TUNEL activity, and (d) Lef1 immunostaining in tissue sections of waxed skin, collected on the days indicated. (e) qRT-PCR analyses of Axin2 and DKK1 expression in punch biopsies from waxed skin, collected on the days indicated. (f) qRT-PCR analysis of proliferating cell nuclear antigen (PCNA) expression in 4-mm punch biopsies from waxed skin collected on the days indicated. Scale bar = 50 μm. Axin2, axis inhibition protein 2; BrdU+ve, Bromodeoxyuridine+ve; DKK1, dickkopf1; dp, dermal papilla; m, matrix; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; rs, root sheath; se, sebaceous gland; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; um, upper matrix. Journal of Investigative Dermatology 2016 136, 1549-1558DOI: (10.1016/j.jid.2016.01.041) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Rspo2 amplifies endogenous and exogenous Wnt signals. (a) qRT-PCR analysis of endogenous Rspo2 expression in the intestine (positive control, purple bar), and in skin whose HFs were in anagen (solid red bar), catagen (striped bar), and telogen (dotted bar). For primer sequences see Supplementary Table S1 online. (b) Endogenous Rspo expression domains during early and (c) late anagen; (d) during late catagen and (e) during telogen. (f) Rspo2+WNT3A, but not Rspo2 alone, amplifies Wnt signaling approximately 5-fold in mouse LSL cells. A minimum Wnt3a concentration of 0.05 ng/μl was required for Wnt signal amplification by Rspo2 in LSL cells. (g) Rspo2+Wnt3a amplifies Wnt signaling 2.5-fold in MEFs. (h) Rspo2 (100 ng/μl) delivered subcutaneously to 6-week-old mice whose synchronized hair cycle was in anagen, or to 8-week-old mice whose synchronized hair cycle was in telogen; after 24 hours, qRT-PCR was used to analyze Axin2 and (i) Lef1 expression. *P < 0.05; **P < 0.01. Scale bar = 50 μm. Axin2, axis inhibition protein 2; HF, hair folicle; MEF, mouse embryonic fibroblast; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; Rspo2, R-spondin2. Journal of Investigative Dermatology 2016 136, 1549-1558DOI: (10.1016/j.jid.2016.01.041) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Rspo2 prolongs anagen. (a) Schematic of experimental design. (b) Brackets indicate Rspo2 injection sites. (c, d) Whole-mount Xgal staining of an Axin2LacZ/+ pelt; brackets indicate Rspo2 injection sites. (e) Length and thickness of individual guard hairs after 10 days of Rspo2 measured. (f) Gomori staining of a representative tissue section at (f) Rspo2 and (g) PBS injection sites. (h) Lef1, (i) TUNEL, and (j) Ki67 expression in anagen HFs. (k) Lef1, (l) TUNEL, and (m) Ki67 expression in telogen HFs. Scale bars = 50 μm. *P < 0.05; **P < 0.01. Axin2, axis inhibition protein 2; HF, hair folicle; lm, lower matrix; PBS, phosphate buffered saline; Rspo2, R-spondin2; s, shaft; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; um, upper matrix; Xgal, x-galactosidase. Journal of Investigative Dermatology 2016 136, 1549-1558DOI: (10.1016/j.jid.2016.01.041) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Rspo2 stimulates hair growth via activation of Lgr5+ve stem cells in the HF. Xgal staining of HFs from Lgr5LacZ/+ mice on (a) PWD1, (b) 7, (c) 10, and (d) 14. (e) Quantitative RT-PCR for endogenous Lgr5 expression in the skin after waxing at time points indicated. (f) Lgr5 expression following intradermal PBS (black line) or Rspo2 (red line); skin was harvested and interrogated at the time points indicated. (g) On PWD22, representative whole-mount Xgal staining of an Lgr5LacZ/+ pelt from the ventral surface and (h) on its side; dotted lines indicate the Rspo2 injection site. (i) Whole-mount Xgal staining of an Lgr5LacZ/+ pelt showing HFs in telogen at a noninjected region and (j) in anagen at the site of Rspo2 injection. Scale bars = 50 μm. *P < 0.05. HF, hair folicle; Lgr5, leucine-rich repeat-containing G-protein coupled receptor 5; ors, outer root sheath; PBS, phosphate buffered saline; PWD, post-wax day; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; Rspo2, R-spondin2; Xgal, x-galactosidase. Journal of Investigative Dermatology 2016 136, 1549-1558DOI: (10.1016/j.jid.2016.01.041) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Human hair grows in response to a Wnt stimulus. (a) Schematic showing human HFs transplanted into SCID mice or cultured in vitro. The number of HFs is indicated. (b) Survival curve of transplanted HFs after treatment with PBS or Rspo2. (c) Relative growth rate of HFs incubated in media (dotted line), WNT3A (gray), Rspo2 (red), or WNT3A+Rspo2 (purple) over a 14-day time period. (d) Representative human HFs after 7 days in culture. Scale bar = 1 mm. *P < 0.05. HF, hair folicle; PBS, phosphate buffered saline; Rspo2, R-spondin2. Journal of Investigative Dermatology 2016 136, 1549-1558DOI: (10.1016/j.jid.2016.01.041) Copyright © 2016 The Authors Terms and Conditions