J Mol Hist DOI 10.1007/s10735-015-9616-5


Expression of Wnt/b-catenin signaling, stem-cell markers and proliferating cell markers in rat whisker hair follicles Chang-min Lin1 • Yan-ping Yuan1 • Xian-cai Chen1 • Hai-hong Li2 Bo-zhi Cai3 • Yang Liu1 • Huan Zhang1 • Yu Li3 • Keng Huang2

Received: 5 January 2015 / Accepted: 17 March 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract The rat whisker hair follicle (HF) is a model for studying the reconstruction of the HF or dermal papilla (DP), and involves the Wnt/b-catenin signaling pathway, which is a key pathway in HF development and HF cycling after birth. It has been reported that Wnt/catenin signaling plays an indispensable role in human or rat pelages development and postnatal growth. However, the distribution of some Wnt/bcatenin signaling pathway factors and their relationship with the epithelial stem cell markers in whisker follicles has not been characterized. In this study, we investigated the immunolocalization of Wnt/catenin signaling pathway members, including Wnt10b, Wnt10a, Wnt5a, b-catenin, and downstream lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 3 (TCF3), as well as, HF stem-cell markers CD34, CK15 and proliferating cell nuclear antigen (PCNA) protein, in rat anagen phase whisker follicles. bcatenin, Wnt5a, Wnt10b, Wnt10a, LEF1, and TCF3 were expressed in the outer root sheath (ORS), inner root sheath, Chang-min Lin and Yan-ping Yuan have contributed equally to this work. & Keng Huang [email protected] 1

Department of Histology and Embryology, Shantou University Medical College, No. 22 XinLing Road, Shantou 515041, Guangdong Province, People’s Republic of China


Emergency Department, The Second Affiliated Hospital, Shantou University Medical College, North DongXia Road, Shantou 515041, Guangdong Province, People’s Republic of China


Tissue Engineering Laboratory, First Affiliated Hospital, Shantou University Medical College, No. 57 ChangPing Road, Shantou 515041, Guangdong Province, People’s Republic of China

matrix and hair shaft of anagen follicles. b-catenin, Wnt10b, LEF1, and TCF3 were highly expressed and Wnt5a and Wnt10a weakly expressed in DP and dermal sheath (DS) regions. The expression of a-smooth muscle actin was strong in the lower DS and it was also detected in some DP cells. CD34, CK15 and PCNA were all expressed in the ORS; and CD34 and PCNA were also detected in the matrix, however CD34 was extensively expressed in DP and DS regions. Our studies located the position of Wnts, downstream LEF1 and TCF3 and stem cell marker proteins, which provide new information in understanding the role of the Wnt singaling pathway in whisker follicles’ growth. Keywords Wnt/b-catenin signaling  Hair follicle  Anagen  Immunofluorescence staining  Stem cells Abbreviations Bu Bulge C Capsule DP Dermal papilla DS Dermal sheath GM Glassy membrane HF Hair follicle HS Hair shaft IRS Inner root sheath Mx Matrix ORS Outer root sheath P Precortex

Introduction Whisker follicles of rats and mice, and human hair follicles are the most specialized mini-organs in mammals because of their remarkable regenerative abilities (Kim and Choi


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1995; Kobayashi and Nishimura 1989; Oliver 1966a). HF undergo cycles of growth (anagen), regression (catagen), and quiescence (telogen) throughout the lifetime of the animal. The mature hair follicles are composed primarily of keratinocytes arranged in concentric layers of differentiated cell types that comprise the hair shaft (HS), inner root sheath (IRS) and outer root sheath (ORS). Keratinocytes in direct contact with the dermal papilla, a specialized mesenchymal component embedded in the hair bulb at the base of the follicle, undergo asymmetric division to renew this ‘‘matrix stem cell’’ compartment. Daughter cells move upwards, adopting one of six lineages of the IRS and HS. From outermost to innermost, the layers include Henley, Huxley and cuticle layers of the IRS, and the cuticle, cortex and medulla layers of the HS (Alonso and Fuchs 2006; Krause and Foitzik 2006). Proliferating matrix cells have a cell cycle length of approximately 18 h (Lavker et al. 2003). The anagen follicle represents a remarkable model for studies of hair growth and organ regeneration and an attractive experimental system to study epithelial–mesenchymal interactions (Cohen 1961; Kim and Choi 1995; Oliver 1966b; Waters et al. 2011). HF development and regeneration in vitro are strictly regulated by various growth factors, hormones and signaling molecules, with the Wnt signaling pathway being one of the most important factors (Andl et al. 2002; Li et al. 2013; Millar et al. 1999; Myung et al. 2013; Reddy et al. 2001). It has also been reported that the Wnt signaling pathway plays critical role in human and mouse tooth development (Wang et al. 2014). Wnt10b, Wnt10a, Wnt5a, b-catenin, and downstream lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 3 (TCF3) all play key roles in HF development (Andl et al. 2002; DasGupta and Fuchs 1999; Reddy et al. 2001). The expression and function of Wnt5a in vibrissae follicles’ growth have been reported, while the distribution of Wnt10b, Wnt10a, LEF1 and TCF3 in rat whisker follicles is still unclear (Li et al. 2011; Xing et al. 2011, 2013). The expression of CD34 and CK15 in vibrissae follicles has been reported: CK15 is expressed in the ORS, and CD34, an epidermal stem cell marker, is always expressed in the dermal region (Larouche et al. 2008; Tucker et al. 2013). CK15 may be more suitable as a marker of follicle stem cells. Wnt proteins and stem-cell marker proteins are both important factors in follicles but their connection is unclear. In the present study, we investigated the expression of the Wnt/b-catenin signaling pathway in anagen vibrissae follicle of the rat. We examined Wnt10a, Wnt10b, Wnt5a, b-catenin and their downstream molecules LEF1 and TCF3, and HF stem-cell markers, including CD34 and CK15, as well as proliferating cell nuclear antigen (PCNA) and a-smooth muscle actin (a-SMA), in relation to the differentiation of dermal


papilla and dermal sheath. We investigated the immunolocalization of Wnt/b-catenin signaling molecules, follicular stem cell markers and DS markers during the anagen of vibrissae follicles, and our results mainly demonstrate that Wnt10a plays a role in regulation of hair shaft growth.

Materials and methods Tissue samples Anagen vibrissae follicles were exposed and isolated from Sprague-Dawley rats purchased from the Experimental Animal Center of Shantou University Medical College. All animal-related procedures were in accordance with maintenance protocols and principles of laboratory animal care established by the Ethics Committee of Shantou University Medical College (Shantou, China). As previously described (Iida et al. 2007; Kobayashi and Nishimura 1989; Young and Oliver 1976), 2–3-month-old rats were killed after anaesthetization, and their upper lip skins, containing the vibrissae follicles, were dissected. Connective tissue surrounding the vibrissae follicles was carefully removed from the skin and follicles were removed using forceps. According to the ratio of hair length of the growing hair shaft to the fully grown club hair formed in the previous hair cycle, i.e., less than 1/3, 1/3–2/3, and over 2/3, anagen follicles were categorized into early anagen, mid-anagen, and late anagen phase, respectively. About 40 hair follicles in the anagen stage of the hair cycle were examined as frozen samples. Only 32 hair follicles cut along the long axis of the follicles were submitted for immunofluorescence analysis. Serial sections were cut at 5 lm thickness, and eleven consecutive single sections were retrieved and mounted on separate slides for different stains. Hematoxylin and eosin (H&E) staining Freshly dissected follicles were cut and retrieved on slides, then stained with H&E after fixing with 4 % paraformaldehyde for 10 min at 4 °C and washed with PBS for 15 min. Immunofluorescence staining Frozen tissue sections were air dried for 10 min and fixed with 4 % paraformaldehyde for 10 min at 4 °C. After being washed with PBS for 15 min, sections were incubated with 10 % donkey serum in phosphate buffer solution (PBS) for 30 min at 37 °C to block nonspecific binding, then incubated at 4 °C overnight with mouse anti-CK15 (1:25, sc-47679, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-PCNA (1:50, BA0104, Boster, Wuhan, China), rabbit

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anti-b-catenin (ab32572), anti-Wnt10b (ab70816), antiWnt10a (ab106522), anti-a-SMA (ab66133),anti-LEF1 (ab85052), anti-TCF3 (ab69999, all Abcam, Cambridge, UK), rabbit anti-CD34 (BA0532, Boster, Wuhan, China), or mouse anti-Wnt5a (sc-30224, Santa Cruz Biotechnology, Santa Cruz, CA, USA) all at 1:100 dilution. Following washing with PBS, sections were incubated with secondary AlexaFluor488 or 594-conjugated antibodies (1:150, Jackson) for 1 h in the dark at room temperature. Finally, sections were counter stained with 4,6-diamidino-2-phenylindole (DAPI) (1 lg/ml, C1005, Beyotime, Jiangsu, China) for 10 min at room temperature in the dark and mounted with antifade mounting medium (P0126, Beyotime, Jiangsu, China). Sections were observed by fluorescence microscopy (Nikon H600L, Japan).

low levels in the DP (Figs. 2b, 3b1, b2). Wnt5a was strongly expressed in some matrix cells adjacent to the basal stalk (Fig. 3b1). Wnt10b was expressed in all areas of the anagen whisker, including the HS, IRS, ORS, matrix, DS and DP (Fig. 2c). The highest levels were in the IRS, ORS and HS, and high levels were detected in a cluster of DP cells (Fig. 3c1, c2). Wnt10a was strongly expressed in the matrix, IRS, ORS and capsule, which packages the vibrissa follicle; but weakly expressed in the DP and DS (Figs. 2d, 3d1–d3). Wnt10a was highly expressed in the capsule (Fig. 3d1). Except for the distribution of Wnt10a in the capsule, other expression patterns were consistent with the b-catenin staining.

Expression of LEF1 and TCF3

Results H&E and DAPI staining of the rat anagen vibrissae follicle The structural features of anagen follicles are well shown by HE and DAPI staining (Fig. 1a) (Young and Oliver 1976). The follicles were composed of epidermal tissues, dermal tissues, HS and capsule (Fig. 1b1, c1). The epidermis contained the outer root sheath (ORS) and inner root sheath (IRS) (Fig. 1b2, c2), and the bulge was located at the upper end of the ORS (Fig. 1b1, c1). The dermis contained the dermal papilla (DP), the command center of the HFs, and the dermal sheath (DS), considered a source of cells for the DP (Fig. 1b3, c3). The findings suggested that the vibrissae follicles were in growing stage (anagen phase) and the bulge area was well observed. Expression of b-catenin b-catenin is the central factor in Wnt/b-catenin signaling pathway and associated with many physiological processes, such as embryogenesis and postnatal development (Huelsken et al. 2001; Enshell-Seijffers et al. 2010) b-catenin was strongly expressed in the ORS, IRS, matrix and HS (Figs. 2a, 3a1). The highest levels were in the matrix and ORS cells and some DP cells (Fig. 3a1–a3); b-catenin was expressed both in the cytoplasm and the nucleus (Fig. 3a2, a3). However, it was weakly expressed in the DS cells that surrounded the ORS (Fig. 3a2). b-catenin is active in the epithelial region of follicles, but not in the dermal areas. Expression of Wnt5a, Wnt10b and Wnt10a Wnt5a protein was moderately expressed and distributed extensively, including the HS, IRS, ORS and matrix, with

LEF1and TCF3 expression patterns during the postnatal vibrissae follicle cycle have not yet been characterized. We found LEF1 protein to be strongly expressed throughout the entire vibrissae follicle, including the IRS, ORS, DS, DP and matrix (Figs. 2e, 3e1, e2). LEF1 accumulated in the nucleus of the DP and matrix cells (Fig. 3e3). We found TCF3 expression to be similar to that of LEF1. TCF3 was extensively expressed in the IRS, ORS, DS, DP and matrix (Figs. 2f, 3f1, f2). However, contrary to LEF1, TCF3 was strongly expressed in the IRS, but weakly in the matrix (Fig. 3f1). The different expression patterns suggest they may exert functions in multiple ways.

Expression of a-SMA a-SMA is considered a marker of the dermis of HFs, and its expression has been described in vibrissae, pelage and human follicles (Jahoda et al. 1991; Morioka et al. 2011). We found strong expression of a-SMA in the lower half of the dermal sheath of rat vibrissae follicles (Figs. 2g, 4a1). Some cells located at the bottom of the DP showed a-SMA signals (Fig. 4a3). The gap, which is the basement membrane between the DS and ORS, was unstained (Fig. 4a2).

Expression of PCNA PCNA was expressed in distal matrix cells and the ORS of the anagen vibrissa follicle (Fig. 4b1–b4). However, the highest levels were in the basal layer of the ORS adjacent to the glassy membrane (Figs. 2h, 4b3). This basal layer of the ORS that belongs to the bulge is considered to contain the stem cells of the HF epithelium (Lavker et al. 1991; Lavker and Sun 2000).


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Fig. 1 Anagen vibrissae follicle of Sprague-Dawley rats. a A single follicle containing two fibers [club and hair shaft (HS)] stripped of surrounding tissue. b1–b3 Frozen section of an anagen follicle stained with hematoxylin and eosin. b1 Follicle contains both club and HS. b2–b3 Magnified follicle structures, which containing bulb, DS, GM,

HS, IRS, ORS. c1–c3 Section of an anagen follicle stained with DAPI. Bu bulge, C capsule, DP dermal papilla, DS dermal sheath, GM glassy membrane, HS hair shaft, IRS inner root sheath, Mx matrix, ORS outer root sheath, P precortex. Scale bars 100 lm

Expression of CK15 and CD34

2003; Trempus et al. 2003), are both expressed strongly at the bulge areas, which indicates a relationship between Wnt signaling and the proliferation of epithelial stem cells. Previous studies (Hendaoui et al. 2014; Tucker et al. 2013) showed that CD34-positive stem cells bind Wnt ligands, which leads to localized increased concentration of Wnt factors. As a result, Wnt/b-catenin signaling is activated and the stem cell reservoir is maintained. Although not as widely expressed as CD34, CK15 is detected only in the ORS, especially the bulge area, the site of HF stem cells (Larouche et al. 2008; Tucker et al. 2013). CD34 expression is indicative of proliferating cells rather than stem cells. Compared with CD34, CK15 is an ideal marker for HF stem cells. Wnt5a is only moderately expressed in the precortex region of the ORS, IRS and matrix. Wnt5a could not be detected during the other phases of the HF cycle or other sites. The relationship between Wnt5a and the proliferation of matrix cells needs further study. DP regulates the development of the epidermal follicle and depends on signals from the epidermis for development and maintenance (Myung et al. 2013). However, directly demonstrating the inductive activity of the DP in mature HF is difficult. In our study, Wnt10b, b-catenin, LEF1, TCF3 and CD34 were expressed in the DP region, which indicates that Wnt signaling is also active in the anagen phase of the dermal section of the HF. Because

CK15 and CD34 are markers of HF stem cells. We detected the expressions of both in the bulge area of the ORS (Fig. 4e4). CK15 was slightly expressed in the outer most layer cells of the ORS. However, its expression gradually attenuated down to the matrix, and it was not expressed in the DP and DS, as expected (Fig. 4c1–c3, e2). CD34 was detected in the ORS, HS, DP and dermal region immediately adjacent to the bulge area (Fig. 4d1–d3, e1). The results confirm that CD34, expressed at the bulge area and dermal region immediately adjacent to it, acts as the niche for follicular epithelial stem cells.

Discussion Here, we found that in anagen follicles of rat whiskers, the IRS and ORS cells, as well as their precursor cells, show strong expression of Wnt10b and its downstream genes, including b-catenin. LEF1 and TCF3 are expressed consistently at the matrix and lower part of the HS. As well, bcatenin staining shows strong expression in HS and matrix cell nuclei, indicative of active Wnt/b-catenin signaling at the anagen stage (Millar et al. 1999). CD34 and CK15, HF stem cell markers (Larouche et al. 2008; Lavker et al.


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Fig. 2 Immunofluorescence analysis of protein expression in the entire anagen vibrissa follicle: b-catenin, Wnt5a, Wnt10b, Wnt10a, lymphoid enhancer-binding factor 1 (LEF1), transcription factor 3

(TCF3) a-smooth muscle actin (a-SMA), proliferating cell nuclear antigen (PCNA), CK15 and CD34. Scale bars 100 lm

expression of Wnt10b is synchronous with the epidermis region of the HF, Wnt10b cannot act as the initiating signal for the anagen phase. Determining which signal activates the tologen-to-anagen transition is still a challenge (Kim et al. 2014; Li et al. 2013; Stenn and Paus 2001; Xing et al. 2013). a-SMA is strongly expressed in the DS region, a site of DP precursor cells, as previously shown (Jahoda et al. 1991; Morioka et al. 2011). However, we find that a-SMA is also expressed in a portion of cells at the base of the DP, indicating that DP cells originate from the surrounding DS cells. In addition, the expression of PCNA, a marker of

proliferating cells, is strongly expressed in the outer layer cells of the ORS, but is not found in the DS region. Therefore, the DS may be situated in a dormant state during anagen. In conclusion, hair follicles are small, but complicated organs as compared with other organs. Epithelial and dermal tissues interact with each other and show different marker expressions during the HF cycle. Here we demonstrated the expression of HF stem cell markers and Wnt signaling pathway components in the anagen phase of rat whisker follicles and elucidate their underlying connection by immunofluorescence analysis. The relationship among Wnt


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Fig. 3 Factors of Wnt/b-catenin signaling pathway expression patterns in the anagen vibrissa follicles. b-catenin was expressed in the ORS, IRS, matrix and HS (a1, a2), with strong expression occasionally detected in DP cells (a3). Wnt5a expression was extensive in the lower part of the vibrissae follicle, including the HS, IRS, ORS and matrix (b1, b2), with low expression in the DP as compared with the matrix (b3). Wnt10b was detected in all areas of the follicle, including the HS, IRS, ORS, matrix, DS and DP (c1); Wnt10bwas highest in the IRS and in a cluster of DP cells (c2, c3). Wnt10a was expressed in the


IRS, ORS, matrix and capsule (d1), but weakly expressed in the DP (d3) and DS (d2); Wnt10a was highest in the IRS and capsule (d1, d2). LEF1 expression was strong in the entire vibrissae follicle, with strongest expression in the matrix (e1, e2); LEF1 was strong in the nucleus of the DP and matrix cells (e3). The expression pattern of TCF3 was similar to that of LEF1 (f1, f2); however, TCF3 was weakly expressed in the matrix (f3). C capsule, DP dermal papilla, DS dermal sheath, HS hair shaft, IRS inner root sheath, Mx matrix, ORS outer root sheath, P precortex. Scale bars 100 lm

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Fig. 4 Protein of stem cell and the special markers of DP expression patterns in the anagen vibrissae follicles. a-SMA expression was strong in lower dermal sheath cells adjacent to the ORS (a1, a2) and also detected in some ORS cells (a2) and DP cells (a3) located at the bottom of the DP. PCNA was strongly expressed in distal matrix cells (b2, b4) and the ORS (b1, b3), and weakly expressed in the DP region (b2, b4). CK15 and CD34 were both detected in the bulge area of the

ORS (e4) and HS (c1, d1), and CK15 expression was highest in the outermost layer cells (c2, e2) of the ORS. CD34, a stem-cell marker, was detected in the ORS, HS (e1), DP (d3) and dermal region (d2) adjacent to the bulge area. Bu bulge, DP dermal papilla, DS dermal sheath, HS hair shaft, IRS inner root sheath, Mx matrix, ORS outer root sheath. Scale bars 100 lm

proteins, LEF1/TCF3 and stem cells is indispensable in the anagen HF. Further study should focus on the very beginning of the Wnt/b-catenin signaling pathway in the HF cycle, so as

to unlock the mysteries of follicle stem cell activation and hence, contribute to the understanding of development and regeneration of HF.


J Mol Hist Acknowledgments This work was supported by grants from the National Science Foundation of China (Nos. 81372084, 81171832) and the Guangdong Province Outstanding Young Teacher Training Program (No. Yq2013078).

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β-catenin signaling, stem-cell markers and proliferating cell markers in rat whisker hair follicles.

The rat whisker hair follicle (HF) is a model for studying the reconstruction of the HF or dermal papilla (DP), and involves the Wnt/β-catenin signali...
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