Regeneration of Hair Follicles Is Modulated by Flightless I (Flii) in a Rodent Vibrissa Model  James M. Waters, Jessica E. Lindo, Ruth M. Arkell, Allison J. Cowin  Journal of Investigative Dermatology  Volume 131, Issue 4, Pages 838-847 (April 2011) DOI: 10.1038/jid.2010.393 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunolabeling of flightless I (Flii) in rodent hair follicles. (a) Flii is expressed in anagen vibrissae from rat. (b) Flii staining is localized to the dermal sheath (ds), outer root sheath (ors), and inner root sheath (irs) to the point of hair fiber (hf) detachment, but is absent from the companion layer (cp) and hair fiber (hf). Highest levels of Flii are detected in the outermost layer of the ors, directly adjacent to the Flii-negative companion layer. (c) Flii is strongly expressed in patches of the ors basal layer at the site of club fiber anchorage (arrowhead) but absent from the ors basal layer immediately below the club fiber (arrow) (an=anagen fiber). (d) Strong expression of Flii is also detected in some cells in the dermal papillae of anagen vibrissae. Dorsal mouse skin containing anagen follicles (e) shows immunolabeling of Flii (f) in the irs, from the base of the follicles to the point of hair fiber detachment (arrow), but low levels in the dermal papilla and other cell layers of hair follicle. 4,6-Diamidino-2-phenylindole (DAPI) was used as nuclear counterstain in a–d. Scale bars=100μm. The image in a is a mosaic of images taken using a × 10 objective lens. Journal of Investigative Dermatology 2011 131, 838-847DOI: (10.1038/jid.2010.393) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Flightless I (Flii) is dynamically expressed during the progression of hair follicle regeneration in rat vibrissae. Expression of Flii changes throughout the process of hair follicle regeneration. Flii is strongly expressed throughout the epithelium and fibroblasts of the lower follicle during early stages of regeneration (day 4). Flii levels are reduced by day 8, with strongest expression found in fibroblasts closest to the follicle and basal epithelial cells. At day 14, Flii levels are highest in the differentiating epithelium and closely associated fibroblasts. No Flii is detected in the newly formed dermal papilla at day 14. By day 25, Flii staining resembles that of unwounded follicles with strong expression in the differentiating epithelium and some cells of the dermal papilla. Scale bars=50μm. Journal of Investigative Dermatology 2011 131, 838-847DOI: (10.1038/jid.2010.393) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Comparison of hair follicle regeneration (HFR) success rates, between mice expressing varying levels of flightless I (Flii). (a) HFR outcomes for Flii+/- (+/-), wild-type (+/+), and FLIITg/Tg (Tg/Tg) mice 25 days after amputation. Follicles were classified into one of four categories: successful hair shaft production, visible externally (black), no externally visible hair shaft, but large encapsulated dermal papilla with extensive epithelial differentiation (gray), small dermal condensation with no epithelial differentiation (chequered), or complete failure to regenerate any endbulb structures (white). (b) Rates of successful HFR in a subset of standardized follicles in mid-anagen at the time of amputation show similar results. Successful regeneration by day 25 was significantly reduced in Flii+/- follicles (*P<0.05). (c) Terminal hair fiber lengths of regenerated follicles relative to unwounded controls measured >10 weeks after amputation (mean±SEM). Length of regenerated hair fibers in FLIITg/Tg follicles were significantly longer than wild-type and Flii+/- follicles (P<0.05). The number of hair follicles (HF) and mice, in parentheses, analyzed are below each column. NS, not significant. Journal of Investigative Dermatology 2011 131, 838-847DOI: (10.1038/jid.2010.393) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Hair cycle is unaffected among mice expressing varying levels of flightless I (Flii). Pelage follicles from (a–e) Flii+/-, (f–j) Flii+/+, and (k–o) FLIITg/Tg were depilated and allowed to enter a new, synchronous hair cycle. Histological analysis of samples at day 3 (anagen II—a, f, k), day 6 (anagen IV—b, g, l), day 12 (anagen VI—c, h, m), day 19 (catagen—d, i, n), and day 25 (telogen—e, j, o) show no difference in the progression of the hair cycle among all three strains of mice. Scale bars=50μm (day 3) and 100μm (days 6–25). Journal of Investigative Dermatology 2011 131, 838-847DOI: (10.1038/jid.2010.393) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunofluorescent analysis of hair follicle regeneration. (a, d) α-Smooth muscle actin (αSMA) was detected in the dermal cells surrounding the early epithelial outgrowths at day 4 and (b, e) in the newly forming dermal papilla at day 8 but (c, f) was absent from the fully regenerated dermal papilla in (a–c) Flii+/- and (d–f) FLIITg/Tg follicles. Proliferating cell nuclear antigen (PCNA) was detected at day 4 throughout the upper epithelium of all follicles and in the epithelial outgrowth of (g, arrow) Flii+/- follicles, but not (h, arrowhead) Flii+/+ or (i, arrowhead) FLIITg/Tg follicles. (j, k) Syndecan-1 (CD138) and P-cadherin (P-cad) were detected in regenerating Flii+/- follicles at days 8 and 14, respectively. Pattern and intensity of β-catenin staining were consistent between (m, n) Flii+/- and (p, q) Flii+/+ follicles on (m, p) day 4 and (n, q) day 8. (l) Flii+/- follicles that had failed to regenerate any endbulb structures by day 25 showed marked reduction in syndecan-1 and fibroblast growth factor receptor 2 (FGFR2) expression, (o, r) compared with fully regenerated follicles from Flii+/- and Flii+/+ mice. 4,6-Diamidino-2-phenylindole (DAPI) was used as nuclear counterstain in a–f and j–l. col IV, collagen IV. Scale bars=50μm. Journal of Investigative Dermatology 2011 131, 838-847DOI: (10.1038/jid.2010.393) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of cytokeratins and hair cortex keratins during hair follicle regeneration. (a) K17 was upregulated on day 4 in the distal tip of regenerating follicles, and (b) throughout the lower follicle by day 8. (c, d) K17 expression was lost around the dermal papilla and the innermost layers of the newly regenerated hair follicle as expression of new hair cortex keratins (AE13) were upregulated by days 14 and 25. (e) Both K14 and K15 were also upregulated on day 4 throughout the distal tip of the regenerating follicle with strong basal expression of K15 and suprabasal expression of K14 continuing up the follicle. (f) By day 8, K14 and K15 expression was diminished around the newly forming dermal papilla. (g, h) K14 and K15 expression was further restricted to the basal layers of the regenerated follicle. Scale bars=50μm. Journal of Investigative Dermatology 2011 131, 838-847DOI: (10.1038/jid.2010.393) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions