Expression of Calcium-Binding S100 Proteins A4 and A6 in Regions of the Epithelial Sac Associated with the Onset of Hair Follicle Regeneration  Mayumi Ito, Kenji Kizawa  Journal of Investigative Dermatology  Volume 116, Issue 6, Pages 956-963 (June 2001) DOI: 10.1046/j.0022-202x.2001.01369.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic illustrations of two proposed scenarios for hair follicle regeneration in a telogen follicle. (a) Onset by bulge activation. Signals from the dermal papilla induce the bulge cells residing in the outer layer of the epithelial sac to give rise to transit-amplifying cells. Bulge daughter cells undergo mitosis as they migrate down the hair germ prior to entering the growing phase. (b) Onset by hair germ activation. Inductive signals from the dermal papilla are transmitted directly to the resting hair germ. Consequently, transit-amplifying cells are conceived in the hair germ. DP, dermal papilla; SG, sebaceous gland. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Transcripts and translation products of S100 genes A4 and A6 extracted from skin tissue. (a) Northern blot analyses. Total RNA from mouse skin (2 μg) was size-separated on 1.2% agarose gel containing 0.4 M formaldehyde, and transferred to a nylon membrane. Blots were hybridized with cRNA probes to either S100A4 (left lane) or S100A6 (right lane). (b) Western blot analyses. Protein extract of mouse whole skin (100 μg in left lane; 20 μg in right lane) was separated on 16.5% Tricine-SDS-PAGE. S100 proteins A4 and A6 transferred onto a polyvinylidene difluoride membrane were detected with specific antibodies raised against each peptide antigen. d, dimer; m, monomer. Relative positions of molecular weight makers are shown on the left. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Distribution of S100A4 and S100A6 proteins during hair cycle transition and follicle morphogenesis. Immunohistochemistry was performed with antibodies for S100 proteins A4 and A6 on mouse dorsal skin tissues at various stages of postnatal hair cycles and embryonic morphogenesis. At catagen phase (18 dpp), S100A4 staining is seen in the bulge region at the insertion site of the arrector pili (a), and continuous S100A6 staining is observed from the innermost cell layer of the ORS to the bulge area (b). In an early anagen follicle (20 dpp), S100A4 staining is seen in the ventral region of the sac (c). S100A6 staining is extended over the epithelial sac regions, but is attenuated in the proliferating hair germ (d). Most follicular cells in the mid-anagen phase (24 dpp) lose S100A4 staining (e), whereas S100A6 staining persists in both layers of the sac supporting the club and extending to the innermost cell layer of the ORS through the next follicle generation (f). Neither S100A4 (g) nor S100A6 (h) staining is seen in the epithelial part of the embryonic hair germ (16.5 dpc). Both proteins are present in dermal condensation. APM, arrector pili muscle; Bu, bulge; DP, dermal papilla; I, inner cellular layer of epithelial sac; O, outer cellular layer of epithelial sac. Scale bar: (a–e, g, h) 100 μm; (f) 200 μm. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression of S100A4 and S100A6 in the epithelial sac at the onset of follicle regeneration. Prolonged telogen follicle sections (56 dpp) were subjected to immunohistochemistry and in situ hybridization. Anti-S100A4 antibody staining is restricted to the outer layer of the epithelial sac (a), whereas anti-S100A6 antibody staining is preferentially seen in the inner layer (b). Insets show cross-sections. In situ hybridization with antisense probe to S100A4 showed very faint staining (c). Staining with antisense probe to S100A6 was confined to the inner layer of the epithelial sac (d). In situ hybridization of clipped follicle sections at 6 h after shaving showed the S100A4 mRNA signal in the outer layer of the sac (e), whereas S100A6 signal is found in both layers and the upper region of the hair germ (f). Note the apparent S100A4 mRNA induction in the bulge region at the beginning of the anagen phase. In situ hybridization combined with BrdU immunohistochemistry was performed. BrdU-labeled nuclei are observed in the hair germ, and S100A4 mRNA expression has almost ceased in the outer layer of the sac 24 h after shaving (g). S100A6 mRNA is lost from BrdU-labeled cells in the early second anagen follicle (20 dpc, h). Bu, bulge; DP, dermal papilla; HG, hair germ. Scale bar: 50 μm. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Summarized expression profile of S100A4 and S100A6 mRNAs during spontaneous follicle regeneration. A diagram of the S100A4 and S100A6 mRNA expression profiles. As cell division rarely occurs in the bulge region and dermal papilla, translation products of S100A4 and S100A6 mRNAs, which previously transcribed during the catagen phase, persist throughout the following telogen phase. Thus, this mRNA expression profile is compatible with the protein distribution shown in Figure 3 and Figure 4. Note that clipped follicles show a similar expression pattern. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Plucking-induced follicle regeneration commenced from S100A6 expression in the hair germ. Six hours after plucking, mouse dorsal skin tissues were subjected to hematoxylin-eosin staining (a), and TUNEL detection of apoptotic cell death (b). Most cells in the outer layer of the epithelial sac are TUNEL-positive. Skin tissues excised at 6 h (c), 24 h (d), and 48 h (e) after plucking were subjected to combined S100A6 in situ hybridization/BrdU immunohistochemistry. S100A6 mRNA staining is seen in the upper region of the hair germ at 6 h. During 24–48 h, the BrdU-labeled cells are observed at the proximal location to the S100A6 mRNA signals in the reconstructing bulge region. Bu, bulge; DP, dermal papilla; HG, hair germ. Scale bar: 100 μm. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Optional follicle induction mechanism revealed by plucking. The expression profile shown is based on the distribution of mRNAs for S100A4 and S100A6 following induction by plucking. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Follicle renewal caused by skin incision is correlated with S100A4 and S100A6 expressions in the epithelial sac.In situ hybridization shows the appearance of mRNA signals for S100A4 (a) and S100A6 (b) in the regions of the epithelial sac at 6 h after skin incisions. Injured skin tissues after 24 h (c) and 48 h (d) were subjected to combined in situ hybridization for S100A6/immunohistochemistry for BrdU. Numerous BrdU-labeled nuclei are present throughout the ORS and the basal layer of the epidermis, which were preceded by intense staining for S100A6 mRNA. (e) Hematoxylin-eosin staining of skin tissue contains regenerating hair follicles 7 d later. Note that anagen follicles in advanced stages are observed in regions closer to the skin incision. Arrows indicate the incisions. Arrowheads indicate newly generated hair bulbs. Bu, bulge; HG, hair germ; SG, sebaceous gland. Scale bar: (a–d) 100 μm; (e) 200 μm. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Follicle induction upon wounding stimuli. The expression profile shown is based on the distribution of mRNAs for S100A4 and S100A6. Journal of Investigative Dermatology 2001 116, 956-963DOI: (10.1046/j.0022-202x.2001.01369.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions