- Email: [email protected]
Immunolocalization of β-catenin and Lef-1 during postnatal hair follicle development in mice
Acta Histochemica 114 (2012) 773–778
Contents lists available at SciVerse ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Immunolocalization of -catenin and Lef-1 during postnatal hair follicle development in mice Hai-Dong Wang a , Lei Yang a , Xiu-Ju Yu a , Jun-Ping He a , Lin-Hua Fan b , Yan-Jun Dong a , Chang-Sheng Dong a,∗ , Tian-Fu Liu b a b
College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, China Laboratory Animal Centre, Shanxi Medical University, Taiyuan, China
a r t i c l e
i n f o
Article history: Received 20 November 2011 Received in revised form 6 January 2012 Accepted 9 January 2012
Keywords: -Catenin Lef-1 Hair follicle Hair follicle cycle Mice
a b s t r a c t It is well recognized that the Wnt pathway, in which -catenin and Lef-1 are important factors, is associated with many physiological processes, including embryogenesis and postnatal development. The Wnt pathway also plays a critical role in the development of skin. It regulates the formation of the dorsal dermis and epidermal appendages in the skin and the activity of epithelial stem cells. In this study, we investigated the presence and localization of -catenin and Lef-1 in murine hair follicles through the first postnatal month, which encompasses the first hair cycle in mice, using Western blotting and immunohistochemistry. Our results show that -catenin and Lef-1 are expressed during all stages in a hair cycle, most strongly in the anagen and weakly in the catagen and telogen phases. The results also suggest that the -catenin-Lef-1 complex may regulate hair follicle cycling. This process will be of considerable interest to future studies. © 2012 Elsevier GmbH. All rights reserved.
Introduction The hair follicle is an accessory organ of the skin that produces hair. The formation of hair follicles depends on the interactions between the epidermal and the mesenchymal cells during embryogenesis. The first wave of signal is derived from the mesenchyme to induce the formation of the placode in the epidermis. The second wave of signal comes from the epidermal placode and induces the condensation of dermal cells underneath the placodes. Further interactions between the placode and dermal condensation lead to the formation of hair follicles (Duverger and Morasso, 2009). The hair follicle renews itself cyclically through the hair cycle, which can be divided into three phases: anagen, catagen, and telogen (Schmidt-Ullrich and Paus, 2005; Alonso and Fuchs, 2006). Hair follicles at each phase have distinct morphological characteristics (Müller-Röver et al., 2001). During anagen, the hair bulb is enlarged and encloses the dermal papilla, which has a loose consistency. The hair matrix cells proliferate and differentiate into daughter cells, which move upwards to form the inner root sheath and hair shaft. The length of the hair follicle increases continuously and the hair bulb enters the hypodermis. The skin becomes thicker at this stage. As hair follicles enter catagen, the proliferation of matrix cells and growth of the hair stops and the bottom of the hair shaft forms a
∗ Corresponding author. E-mail address: [email protected] (C.-S. Dong). 0065-1281/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2012.01.004
club-shaped structure. With the degeneration of the lower follicle, it forms the epithelial strand and retracts. The dermal papilla condenses into a ball-shaped structure and retracts upwards together with the epithelial strand and the skin becomes thinner. The hair follicles enter telogen after this transient catagen phase. The dermal papilla remains a dense ball-shaped structure and resides in the dermis. When stem cells in the hair follicle are activated, the follicle enters a new anagen and a new hair shaft is made (MüllerRöver et al., 2001; Alonso and Fuchs, 2006). Eventually the club hair is shed and the new hair occupies the hair canal. Hair follicles undergo these regenerative cycles continuously throughout life. Though large differences exist between cycle times for hair from different body sites, and for hair of different species, the development of all hair follicles follows this general pattern. Each hair follicle in human and guinea pig skin, for example, has its own rhythms and time scales. However, the first hair cycle in mouse skin is synchronized (Stenn and Paus, 2001). Thus mouse skin presents a useful tool to study the molecular control of hair follicle cycling. It is well recognized that the Wnt pathway, in which -catenin and Lef-1 play major roles, is associated with many physiological processes, such as embryogenesis and postnatal development (Goding, 2000; Kühl et al., 2000; Boras-Granic et al., 2006; Driskell et al., 2007). In response to a canonic Wnt signal, -catenin accumulates in the nucleus and binds Lef-1 or other Lef/Tcf family transcription factors, and regulates the transcription of downstream genes. Several studies have suggested that -catenin and Lef-1 control keratinocyte cell fate decision and hair follicle
774
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
morphogenesis and cycling. Niemann et al. (2002) reported that in K14NLef1 transgenic mice in which the binding of -catenin/Lef1 was disrupted, there was a progressive hair loss and development of dermal cysts after six weeks indicating that -catenin/Lef-1 signaling determines whether keratinocytes differentiate into hair or interfollicular epidermis. Moreover, the onset of the first postnatal hair cycle, which occurs around 4 weeks of age in normal mice, was delayed (Niemann et al., 2002). Using ˇ-catenin conditional knockout mice, Huelsken et al. (2001) showed that when ˇ-catenin was deleted during embryogenesis, hair follicle formation was blocked due to the lack of placode formation. However, when ˇ-catenin was deleted after hair follicle formation, hair is lost after the first hair cycle, as a new hair growth phase failed to initiate (Huelsken et al., 2001). Lef-1 null mice contained no whisker follicles and only a reduced number of pelage hair follicles, which failed to grow hair (van Genderen et al., 1994). These data indicate that -catenin and Lef-1 are necessary for the growth of hair (van Genderen et al., 1994; Huelsken et al., 2001; Niemann et al., 2002). The roles catenin and Lef-1 play in the hair cycle and whether their protein levels and localization are changed through the cyclical changes of the hair follicles, however, have not yet been reported. We hypothesized that -catenin and Lef-1 function during the murine hair follicle cycle. To determine the dynamics of ˇ-catenin and Lef-1 expression during the murine hair cycle, we examined the levels and localizations of -catenin and Lef-1 proteins throughout the first hair cycle. This provides a basis for further investigation of the mechanisms controlling hair follicle cycling. Materials and methods Experimental animals and sample collection The housing and care of Kunming (KM) mice and collection of skin samples for use in the experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals (http://www.cioms.ch/publications/guidelines/1985 texts of guidelines.htm). Skin biopsies were obtained from ninety mice, with three mice for each postnatal day from day 1 until day 30. Every effort was made to minimize both the suffering and the number of animals used. Two biopsies were taken from each mouse, one for protein extraction, and the other was fixed in 10% formalin overnight, processed and embedded in paraffin, and 5-m thick sections were cut from the embedded tissue. Protein extraction and Western blotting Total protein was extracted from mouse skins using a protein extraction kit (RIPA Lysis Buffer, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein concentrations were measured by the BCA method using bovine serum albumin as the standard. Extracts were heat denatured at 95 ◦ C for 5 min and equal amounts (100 g/lane) of protein from each sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Dong et al., 2010; Wang et al., 2011). Western blotting was carried out using primary antibodies at the following dilutions: anti--catenin (C-18, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Lef-1 (C-19, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti--actin (1:1000, CWBIO, Beijing, China). Dilutions were all performed in fresh 5% skim milk blocking buffer and incubations took place overnight at 4 ◦ C. After washing in TBS with 0.1% Tween-20, the blots were incubated with the appropriate horseradish-peroxidase-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA, USA)
for 1 h at room temperature (1:5000 in 3% blocking buffer). Following washes, the blots were developed with the Supersignal West Pico substrate (Tiangen, Beijing, China) and exposed to film. The intensity of the signals for each protein was quantified using ImagePro Plus Software, version 6.0 (Media Cybernetics, Bethesda, MD, USA) and normalized to values obtained for -actin. All experiments were performed in triplicates.
Immunohistochemistry The optimal conditions for the immunohistological application were previously described (Tibary and Vaughan, 2006; Yan et al., 2008). We used the Streptavidin–Biotin-enzyme Complex, SABC System, Peroxidase (Biomeda Staining Kit, Dako, Glostrup, Denmark), which is an indirect immunohistochemistry method using streptavidin–biotin complex that allows signal amplification. Free-floating sections of mouse skin were washed three times for 3 min each, in 0.1 M PBS and were then incubated at room temperature in 3% hydrogen peroxide for 15 min to block the activity of endogenous peroxidase. After washing three times with 0.1 M PBS for 15 min each, the sections were boiled in 0.01 M citric acid for 10 min. This was followed by a 20-min immersion in PBS containing 5% BSA (bovine serum albumin) at 37 ◦ C. Afterwards, sections were incubated for 15–24 h at 4 ◦ C with one of the primary antibodies (polyclonal antisera for -catenin or Lef-1, both diluted at 1:75), and for 30 min at room temperature. After three washes in 0.1 M PBS for 5 min each, sections were incubated with secondary antibodies (biotinylated anti-rabbit IgG, 1:100, Boster) for 20–30 min at 37 ◦ C. After washing three times with 0.1 PBS for 15 min each, sections were incubated with an avidin–biotin–peroxidase reagent (1:200, SABC Elite, Boster), and the immunoreaction products were visualized by placing the sections in a staining solution containing 0.04% DAB (3,3 -diaminobenzidine), 0.06% nickel sulfate, and 0.06% hydrogen peroxide for 5–10 min. Nuclei were counterstained with hematoxylin. Then they were mounted, dehydrated, coverslipped and observed under a light microscope (Nikon). For negative controls, PBS was substituted for the primary antibody (Ayer-LeLievre et al., 1988).
Statistical analysis All experiments were performed in triplicates. Values were represented as mean ± SD in the figures. Differences in the protein abundance of -catenin and Lef-1 in mouse skin samples at distinct stages of the hair cycle were determined by variance analysis. P-values under 0.05 were considered statistically significant, and under 0.01 extremely significant.
Results The histological characteristics of hair follicles at different stages of the hair cycle To determine how the first hair cycle progresses in the Kunming mouse strain used in our study, we collected skin samples from mice of different ages, from postnatal day 1 (P1) to postnatal day 30 (P30) on a daily basis, and examined hair follicle morphology with hematoxylin–eosin (H&E) staining of skin sections. Classification of hair follicles in hair cycle stages was performed according to Müller-Röver et al. (2001). We found that in Kunming mice, hair follicles are in anagen from P1 to P19, catagen from P20 to P22, and telogen from P23 to P28, after which a new anagen starts. Representative pictures of each hair cycle stage are shown in Fig. 1.
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
775
Fig. 1. The histological characteristics of the hair cycle (H&E stain). (A, D) Anagen-phase mouse skin; (B, E) catagen-phase mouse skin; (C, F) telogen-phase mouse skin. 1: hair shaft; 2: inner root sheath; 3: dermal papilla; 4: hair matrix; 5: club hair; 6: epithelial strand; 7: sebaceous glands; 8: old hair; Ep: epidermis; De: dermis; Su: subcutis.
Western blot assay of ˇ-catenin and Lef-1 during different hair cycle stages
Immunolocalization of ˇ-catenin and Lef-1 during different hair cycle stages
We analyzed the protein level of -catenin and Lef-1 by immunoblot. As shown in Fig. 2, -catenin and Lef-1 exist in the total protein extracts taken from the skin of mice during each stage (Fig. 2A). -Catenin appears as an 85 kDa band and Lef-1 as a 45 kDa band, both at the expected size. -Catenin and Lef-1 protein levels were both the highest during the anagen phase, 2.9 and 3.9 times higher than levels of the catagen phase, and 1.8 and 3.4 times higher than those of the telogen phase for -catenin and Lef-1, respectively. -Catenin level was found to be higher in telogen than in catagen, while Lef-1 level showed no change from catagen to telogen (Fig. 2B). The levels of both proteins seem to rise and fall with the cyclical growth of the mouse hair follicle, indicating that they may be important regulators of natural hair follicle growth.
Immunohistochemical assays were performed to determine the localization of -catenin and Lef-1during different hair cycle stages. -Catenin was found in the epidermis, sebaceous glands, inner root sheath, and dermal papilla in the skin. Different levels of -catenin are found during different stages. In anagen, -catenin staining is strongly positive, especially in the dermal papilla and inner root sheath (Fig. 3A). -Catenin level decreases in catagen, and the retracting epithelial strand and dermal papilla are only weakly positive (Fig. 3B). In telogen, the positive staining of -catenin increases slightly in the epidermis, sebaceous glands, root sheath, and dermal papilla (Fig. 3C). Lef-1 was found in the sebaceous glands, inner root sheath and dermal papilla in the skin. It was also present at different levels
Fig. 2. Protein immunoblot results for -catenin and lef-1. 1: Anagen; 2: catagen; 3: telogen. Note: ** refers to extremely significant differences (P < 0.01).
776
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
Fig. 3. Expression levels of -catenin protein during different stages of the mouse hair follicle cycle shown by immunohistochemical methods. (A–C) Experimental groups; (D–F) control groups. (A, D) Expression of -catenin in anagen-phase mouse skin. (B, E) Expression of -catenin in catagen-phase mouse skin. (C, F) Expression of -catenin in telogen-phase mouse skin. 1: Epidermis; 2: sebaceous glands; 3: inner root sheath; 4: dermal papilla; 5: hair shaft; 6: club hair.
during different periods. Notably, it was not present in the epidermis. At anagen, catagen, and telogen, the changes of Lef-1 level showed a similar pattern as that of -catenin (Fig. 4A–C). The specificity of the assay was confirmed by a lack of positive staining in the negative control group (Figs. 3D–F and 4D–F).
Discussion The Wnt pathway plays a critical role in the development of skin. It regulates the formation of the dermis, epidermal appendages in the skin such as hair and feathers, and the activity of hair
follicle stem cells (Widelitz, 2008). In an experiment involving dermal papilla cell cultures, Kishimoto et al. (2000) found that Wnt maintains the expression of specific hair-inducing genes in dermal papilla cells. Recent research shows that -catenin, as a main signaling molecule of the Wnt pathway, is expressed more strongly in hair follicles (Goding, 2000). Huelsken et al. (2001) found that -catenin mRNA is expressed at a higher level during hair follicle development, and Widelitz et al. (2000) observed similar phenomena during the process of feather formation in chicken (Barker, 2008). Lef-1, another important signaling molecule in the Wnt pathway, also shows important functions in skin development (Liu et al., 2004). Most studies on Lef-1 have focused on the embryonic
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
777
Fig. 4. Expression levels of lef-1 protein in different stages of the mouse hair follicle cycle shown by immunohistochemical methods. (A–C) Experimental groups; (D–F) control groups. (A, D) Expression of lef-1 in anagen-phase mouse skin. (B, E) Expression of lef-1 in catagen-phase mouse skin. (C, F) Expression of lef-1 in telogen-phase mouse skin. 1: Root sheath; 2: dermal papilla; 3: sebaceous glands; 4: new hair follicle.
development period and organ formation. According to a report by Quinn and Rudolf (1999), knocking out Lef-1 impacts the development of many organs during the embryonic period, including hair follicles (van Genderen et al., 1994; Quinn and Rudolf, 1999). However, there are few references on the function of Lef-1 during postnatal development. It was pointed out by Oosterwegel et al. (1993) that the lymphoid system is the only place in which Lef-1 is expressed more strongly postnatally than in embryos (Oosterwegel et al., 1993). In this study, we showed that Lef-1 was present in postnatal murine skin, and its level was high in the hair follicles during anagen, but low during catagen and telogen. -Catenin protein level showed a similar dynamic pattern through the hair cycle.
Huelsken et al. (2001) reported that the presence and distribution of stem cells was disrupted when hair follicles of -catenin-deficient skin disappeared. Similar phenomena were observed in other studies in which -catenin and Lef-1 were mutated (Botchkarev et al., 2002). Gat et al. (1998) applied transgenic techniques to delete the terminal amino-phosphorylated sites of -catenin, thus resulting in unusually high levels of stable -catenin. Gat et al. (1998) found that in transgenic mice, interfollicular epidermis grew new hair follicles without disturbing the differentiation of the epidermis. However, adult mice did not emerge with new hair follicles until the new hair cycle began. Zhou et al. (1995) reported that the differentiation and maturation
778
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
of hair follicles were enhanced, and hair follicles and tooth cells grew ectopically when the level of Lef-1 in epithelial cells was raised. These studies indicate that -catenin and Lef-1 have similar functions in the activation and directional differentiation of hair follicle stem cells. This study suggests that -catenin and Lef-1 are both necessary for the development and differentiation of hair follicles. Both proteins may have a function in maintaining the hair cycle. In addition, they appeared in the same areas at the same times and showed the same patterns of changes in protein levels. This implies that -catenin and Lef-1 function through the formation of a catenin-Lef-1 complex during the cyclical growth of hair follicles. This study provides a theoretical and experimental foundation for further research into the regularity of the hair follicle growth cycle. Acknowledgements This research was sponsored by the China National Natural Science Foundation (Grant No. 30671512) and by the Science and Technology Innovation Foundation of SXAU (Grant No. 2009001). References Alonso L, Fuchs E. The hair cycle. J Cell Sci 2006;119:391–3. Ayer-LeLievre C, Olson L, Ebendal T, Hallböök F, Persson H. Nerve growth factor mRNA and protein in the testis and epididymis of mouse and rat. Proc Natl Acad Sci U S A 1988;85:2628–32. Barker N. The canonical Wnt/-catenin signalling pathway. Methods Mol Biol 2008;468:5–15. Boras-Granic K, Chang H, Grosschedl R, Hamel PA. Lef-1 is required for the transition of Wnt signaling from mesenchymal to epithelial cells in the mouse embryonic mammary gland. Dev Biol 2006;295:219–31. Botchkarev VA, Botchkareva NV, Sharov AA, Funa K, Huber O, Gilchrest BA. Modulation of BMP signaling by noggin is required for induction of the secondary (nontylotrich) hair follicles. J Invest Dermatol 2002;118:3–10. Dong Y, Cao J, Wang H, Zhang J, Zhu Z, Bai R, et al. Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to a-melanocyte-stimulating hormone by upregulating melanocortin-1 receptor. Biochem Biophys Res Commun 2010;396:849–53. Driskell RR, Goodheart M, Neff T, Liu X, Luo M, Moothart C, et al. Wnt3a regulates Lef-1 expression during airway submucosal gland morphogenesis. Dev Biol 2007;305:90–102. Duverger O, Morasso MI. Epidermal patterning and induction of different hair types during mouse embryonic development. Birth Defects Res C Embryo Today 2009;87:263–72. Gat U, DasGupta R, Degenstein L, Fuchs E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated -catenin in skin. Cell 1998;95:605–14. Goding CR. Mitf from neural crest to melanoma: signal transduction and transcriptionin the melanocyte lineage. Genes Dev 2000;14:1712–28.
Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 2001;105:533–45. Kishimoto J, Burgeson RE, Morgan BA. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev 2000;14:1181–5. Kühl M, Sheldahl LC, Park M, Miller JR, Moon RT. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 2000;16:279–83. Liu X, Driskell RR, Luo M, Abbott D, Filali M, Cheng N, et al. Characterization of LEF-1 promoter segments that facilitate inductive developmental expression in skin. J Invest Dermatol 2004;123:264–74. Müller-Röver S, Handjiski B, van der Veen C, Eichmüller S, Foitzik K, McKay IA, et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 2001;117:3–15. Niemann C, Owens DM, Hülsken J, Birchmeier W, Watt FM. Expression of Lef-1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours. Development 2002;129:95–109. Oosterwegel M, van de Wetering M, Timmerman J, Kruisbeek A, Destree O, Meijlink F, et al. Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis. Development 1993;118:439–48. Quinn E, Rudolf G. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biol 1999;11:233–40. Schmidt-Ullrich R, Paus R. Molecular principles of hair follicle induction and morphogenesis. Bioessays 2005;27:247–61. Stenn KS, Paus R. Controls of hair follicle cycling. Physiol Rev 2001;81:449–94. Tibary A, Vaughan JL. Reproductive physiology and infertility in male South America camelids: a review and clinical observations. Small Ruminant Res 2006;61:283–98. ˜ van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, et al. Development of several organs that require inductive epithelial–mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev 1994;8:2691–703. Wang H, Dong Y, Chen W, Hei J, Dong C. Expression and localization of nerve growth factor (NGF) in the testis of alpaca (llama pacos). Folia Histochem Cytobiol 2011:55–61. Widelitz RB. Wnt signaling in skin organogenesis. Organogenesis 2008;4:123–33. Widelitz RB, Jiang TX, Lu J, Chuong CM. -Catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin. Dev Biol 2000;219: 98–114. Yan Y, Dong C, He J, Ren Y, He X, Bai R, et al. Expression and localization of transforming growth factor-b1of alpaca testis. Acta Vet Zootech Sin 2008;39:97–102. Zhou P, Byrne C, Jacobs J, Fuchs E. Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev 1995;9:700–13.
Contents lists available at SciVerse ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Immunolocalization of -catenin and Lef-1 during postnatal hair follicle development in mice Hai-Dong Wang a , Lei Yang a , Xiu-Ju Yu a , Jun-Ping He a , Lin-Hua Fan b , Yan-Jun Dong a , Chang-Sheng Dong a,∗ , Tian-Fu Liu b a b
College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, China Laboratory Animal Centre, Shanxi Medical University, Taiyuan, China
a r t i c l e
i n f o
Article history: Received 20 November 2011 Received in revised form 6 January 2012 Accepted 9 January 2012
Keywords: -Catenin Lef-1 Hair follicle Hair follicle cycle Mice
a b s t r a c t It is well recognized that the Wnt pathway, in which -catenin and Lef-1 are important factors, is associated with many physiological processes, including embryogenesis and postnatal development. The Wnt pathway also plays a critical role in the development of skin. It regulates the formation of the dorsal dermis and epidermal appendages in the skin and the activity of epithelial stem cells. In this study, we investigated the presence and localization of -catenin and Lef-1 in murine hair follicles through the first postnatal month, which encompasses the first hair cycle in mice, using Western blotting and immunohistochemistry. Our results show that -catenin and Lef-1 are expressed during all stages in a hair cycle, most strongly in the anagen and weakly in the catagen and telogen phases. The results also suggest that the -catenin-Lef-1 complex may regulate hair follicle cycling. This process will be of considerable interest to future studies. © 2012 Elsevier GmbH. All rights reserved.
Introduction The hair follicle is an accessory organ of the skin that produces hair. The formation of hair follicles depends on the interactions between the epidermal and the mesenchymal cells during embryogenesis. The first wave of signal is derived from the mesenchyme to induce the formation of the placode in the epidermis. The second wave of signal comes from the epidermal placode and induces the condensation of dermal cells underneath the placodes. Further interactions between the placode and dermal condensation lead to the formation of hair follicles (Duverger and Morasso, 2009). The hair follicle renews itself cyclically through the hair cycle, which can be divided into three phases: anagen, catagen, and telogen (Schmidt-Ullrich and Paus, 2005; Alonso and Fuchs, 2006). Hair follicles at each phase have distinct morphological characteristics (Müller-Röver et al., 2001). During anagen, the hair bulb is enlarged and encloses the dermal papilla, which has a loose consistency. The hair matrix cells proliferate and differentiate into daughter cells, which move upwards to form the inner root sheath and hair shaft. The length of the hair follicle increases continuously and the hair bulb enters the hypodermis. The skin becomes thicker at this stage. As hair follicles enter catagen, the proliferation of matrix cells and growth of the hair stops and the bottom of the hair shaft forms a
∗ Corresponding author. E-mail address: [email protected] (C.-S. Dong). 0065-1281/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2012.01.004
club-shaped structure. With the degeneration of the lower follicle, it forms the epithelial strand and retracts. The dermal papilla condenses into a ball-shaped structure and retracts upwards together with the epithelial strand and the skin becomes thinner. The hair follicles enter telogen after this transient catagen phase. The dermal papilla remains a dense ball-shaped structure and resides in the dermis. When stem cells in the hair follicle are activated, the follicle enters a new anagen and a new hair shaft is made (MüllerRöver et al., 2001; Alonso and Fuchs, 2006). Eventually the club hair is shed and the new hair occupies the hair canal. Hair follicles undergo these regenerative cycles continuously throughout life. Though large differences exist between cycle times for hair from different body sites, and for hair of different species, the development of all hair follicles follows this general pattern. Each hair follicle in human and guinea pig skin, for example, has its own rhythms and time scales. However, the first hair cycle in mouse skin is synchronized (Stenn and Paus, 2001). Thus mouse skin presents a useful tool to study the molecular control of hair follicle cycling. It is well recognized that the Wnt pathway, in which -catenin and Lef-1 play major roles, is associated with many physiological processes, such as embryogenesis and postnatal development (Goding, 2000; Kühl et al., 2000; Boras-Granic et al., 2006; Driskell et al., 2007). In response to a canonic Wnt signal, -catenin accumulates in the nucleus and binds Lef-1 or other Lef/Tcf family transcription factors, and regulates the transcription of downstream genes. Several studies have suggested that -catenin and Lef-1 control keratinocyte cell fate decision and hair follicle
774
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
morphogenesis and cycling. Niemann et al. (2002) reported that in K14NLef1 transgenic mice in which the binding of -catenin/Lef1 was disrupted, there was a progressive hair loss and development of dermal cysts after six weeks indicating that -catenin/Lef-1 signaling determines whether keratinocytes differentiate into hair or interfollicular epidermis. Moreover, the onset of the first postnatal hair cycle, which occurs around 4 weeks of age in normal mice, was delayed (Niemann et al., 2002). Using ˇ-catenin conditional knockout mice, Huelsken et al. (2001) showed that when ˇ-catenin was deleted during embryogenesis, hair follicle formation was blocked due to the lack of placode formation. However, when ˇ-catenin was deleted after hair follicle formation, hair is lost after the first hair cycle, as a new hair growth phase failed to initiate (Huelsken et al., 2001). Lef-1 null mice contained no whisker follicles and only a reduced number of pelage hair follicles, which failed to grow hair (van Genderen et al., 1994). These data indicate that -catenin and Lef-1 are necessary for the growth of hair (van Genderen et al., 1994; Huelsken et al., 2001; Niemann et al., 2002). The roles catenin and Lef-1 play in the hair cycle and whether their protein levels and localization are changed through the cyclical changes of the hair follicles, however, have not yet been reported. We hypothesized that -catenin and Lef-1 function during the murine hair follicle cycle. To determine the dynamics of ˇ-catenin and Lef-1 expression during the murine hair cycle, we examined the levels and localizations of -catenin and Lef-1 proteins throughout the first hair cycle. This provides a basis for further investigation of the mechanisms controlling hair follicle cycling. Materials and methods Experimental animals and sample collection The housing and care of Kunming (KM) mice and collection of skin samples for use in the experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals (http://www.cioms.ch/publications/guidelines/1985 texts of guidelines.htm). Skin biopsies were obtained from ninety mice, with three mice for each postnatal day from day 1 until day 30. Every effort was made to minimize both the suffering and the number of animals used. Two biopsies were taken from each mouse, one for protein extraction, and the other was fixed in 10% formalin overnight, processed and embedded in paraffin, and 5-m thick sections were cut from the embedded tissue. Protein extraction and Western blotting Total protein was extracted from mouse skins using a protein extraction kit (RIPA Lysis Buffer, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein concentrations were measured by the BCA method using bovine serum albumin as the standard. Extracts were heat denatured at 95 ◦ C for 5 min and equal amounts (100 g/lane) of protein from each sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Dong et al., 2010; Wang et al., 2011). Western blotting was carried out using primary antibodies at the following dilutions: anti--catenin (C-18, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Lef-1 (C-19, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti--actin (1:1000, CWBIO, Beijing, China). Dilutions were all performed in fresh 5% skim milk blocking buffer and incubations took place overnight at 4 ◦ C. After washing in TBS with 0.1% Tween-20, the blots were incubated with the appropriate horseradish-peroxidase-conjugated secondary antibody (Zymed Laboratories, San Francisco, CA, USA)
for 1 h at room temperature (1:5000 in 3% blocking buffer). Following washes, the blots were developed with the Supersignal West Pico substrate (Tiangen, Beijing, China) and exposed to film. The intensity of the signals for each protein was quantified using ImagePro Plus Software, version 6.0 (Media Cybernetics, Bethesda, MD, USA) and normalized to values obtained for -actin. All experiments were performed in triplicates.
Immunohistochemistry The optimal conditions for the immunohistological application were previously described (Tibary and Vaughan, 2006; Yan et al., 2008). We used the Streptavidin–Biotin-enzyme Complex, SABC System, Peroxidase (Biomeda Staining Kit, Dako, Glostrup, Denmark), which is an indirect immunohistochemistry method using streptavidin–biotin complex that allows signal amplification. Free-floating sections of mouse skin were washed three times for 3 min each, in 0.1 M PBS and were then incubated at room temperature in 3% hydrogen peroxide for 15 min to block the activity of endogenous peroxidase. After washing three times with 0.1 M PBS for 15 min each, the sections were boiled in 0.01 M citric acid for 10 min. This was followed by a 20-min immersion in PBS containing 5% BSA (bovine serum albumin) at 37 ◦ C. Afterwards, sections were incubated for 15–24 h at 4 ◦ C with one of the primary antibodies (polyclonal antisera for -catenin or Lef-1, both diluted at 1:75), and for 30 min at room temperature. After three washes in 0.1 M PBS for 5 min each, sections were incubated with secondary antibodies (biotinylated anti-rabbit IgG, 1:100, Boster) for 20–30 min at 37 ◦ C. After washing three times with 0.1 PBS for 15 min each, sections were incubated with an avidin–biotin–peroxidase reagent (1:200, SABC Elite, Boster), and the immunoreaction products were visualized by placing the sections in a staining solution containing 0.04% DAB (3,3 -diaminobenzidine), 0.06% nickel sulfate, and 0.06% hydrogen peroxide for 5–10 min. Nuclei were counterstained with hematoxylin. Then they were mounted, dehydrated, coverslipped and observed under a light microscope (Nikon). For negative controls, PBS was substituted for the primary antibody (Ayer-LeLievre et al., 1988).
Statistical analysis All experiments were performed in triplicates. Values were represented as mean ± SD in the figures. Differences in the protein abundance of -catenin and Lef-1 in mouse skin samples at distinct stages of the hair cycle were determined by variance analysis. P-values under 0.05 were considered statistically significant, and under 0.01 extremely significant.
Results The histological characteristics of hair follicles at different stages of the hair cycle To determine how the first hair cycle progresses in the Kunming mouse strain used in our study, we collected skin samples from mice of different ages, from postnatal day 1 (P1) to postnatal day 30 (P30) on a daily basis, and examined hair follicle morphology with hematoxylin–eosin (H&E) staining of skin sections. Classification of hair follicles in hair cycle stages was performed according to Müller-Röver et al. (2001). We found that in Kunming mice, hair follicles are in anagen from P1 to P19, catagen from P20 to P22, and telogen from P23 to P28, after which a new anagen starts. Representative pictures of each hair cycle stage are shown in Fig. 1.
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
775
Fig. 1. The histological characteristics of the hair cycle (H&E stain). (A, D) Anagen-phase mouse skin; (B, E) catagen-phase mouse skin; (C, F) telogen-phase mouse skin. 1: hair shaft; 2: inner root sheath; 3: dermal papilla; 4: hair matrix; 5: club hair; 6: epithelial strand; 7: sebaceous glands; 8: old hair; Ep: epidermis; De: dermis; Su: subcutis.
Western blot assay of ˇ-catenin and Lef-1 during different hair cycle stages
Immunolocalization of ˇ-catenin and Lef-1 during different hair cycle stages
We analyzed the protein level of -catenin and Lef-1 by immunoblot. As shown in Fig. 2, -catenin and Lef-1 exist in the total protein extracts taken from the skin of mice during each stage (Fig. 2A). -Catenin appears as an 85 kDa band and Lef-1 as a 45 kDa band, both at the expected size. -Catenin and Lef-1 protein levels were both the highest during the anagen phase, 2.9 and 3.9 times higher than levels of the catagen phase, and 1.8 and 3.4 times higher than those of the telogen phase for -catenin and Lef-1, respectively. -Catenin level was found to be higher in telogen than in catagen, while Lef-1 level showed no change from catagen to telogen (Fig. 2B). The levels of both proteins seem to rise and fall with the cyclical growth of the mouse hair follicle, indicating that they may be important regulators of natural hair follicle growth.
Immunohistochemical assays were performed to determine the localization of -catenin and Lef-1during different hair cycle stages. -Catenin was found in the epidermis, sebaceous glands, inner root sheath, and dermal papilla in the skin. Different levels of -catenin are found during different stages. In anagen, -catenin staining is strongly positive, especially in the dermal papilla and inner root sheath (Fig. 3A). -Catenin level decreases in catagen, and the retracting epithelial strand and dermal papilla are only weakly positive (Fig. 3B). In telogen, the positive staining of -catenin increases slightly in the epidermis, sebaceous glands, root sheath, and dermal papilla (Fig. 3C). Lef-1 was found in the sebaceous glands, inner root sheath and dermal papilla in the skin. It was also present at different levels
Fig. 2. Protein immunoblot results for -catenin and lef-1. 1: Anagen; 2: catagen; 3: telogen. Note: ** refers to extremely significant differences (P < 0.01).
776
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
Fig. 3. Expression levels of -catenin protein during different stages of the mouse hair follicle cycle shown by immunohistochemical methods. (A–C) Experimental groups; (D–F) control groups. (A, D) Expression of -catenin in anagen-phase mouse skin. (B, E) Expression of -catenin in catagen-phase mouse skin. (C, F) Expression of -catenin in telogen-phase mouse skin. 1: Epidermis; 2: sebaceous glands; 3: inner root sheath; 4: dermal papilla; 5: hair shaft; 6: club hair.
during different periods. Notably, it was not present in the epidermis. At anagen, catagen, and telogen, the changes of Lef-1 level showed a similar pattern as that of -catenin (Fig. 4A–C). The specificity of the assay was confirmed by a lack of positive staining in the negative control group (Figs. 3D–F and 4D–F).
Discussion The Wnt pathway plays a critical role in the development of skin. It regulates the formation of the dermis, epidermal appendages in the skin such as hair and feathers, and the activity of hair
follicle stem cells (Widelitz, 2008). In an experiment involving dermal papilla cell cultures, Kishimoto et al. (2000) found that Wnt maintains the expression of specific hair-inducing genes in dermal papilla cells. Recent research shows that -catenin, as a main signaling molecule of the Wnt pathway, is expressed more strongly in hair follicles (Goding, 2000). Huelsken et al. (2001) found that -catenin mRNA is expressed at a higher level during hair follicle development, and Widelitz et al. (2000) observed similar phenomena during the process of feather formation in chicken (Barker, 2008). Lef-1, another important signaling molecule in the Wnt pathway, also shows important functions in skin development (Liu et al., 2004). Most studies on Lef-1 have focused on the embryonic
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
777
Fig. 4. Expression levels of lef-1 protein in different stages of the mouse hair follicle cycle shown by immunohistochemical methods. (A–C) Experimental groups; (D–F) control groups. (A, D) Expression of lef-1 in anagen-phase mouse skin. (B, E) Expression of lef-1 in catagen-phase mouse skin. (C, F) Expression of lef-1 in telogen-phase mouse skin. 1: Root sheath; 2: dermal papilla; 3: sebaceous glands; 4: new hair follicle.
development period and organ formation. According to a report by Quinn and Rudolf (1999), knocking out Lef-1 impacts the development of many organs during the embryonic period, including hair follicles (van Genderen et al., 1994; Quinn and Rudolf, 1999). However, there are few references on the function of Lef-1 during postnatal development. It was pointed out by Oosterwegel et al. (1993) that the lymphoid system is the only place in which Lef-1 is expressed more strongly postnatally than in embryos (Oosterwegel et al., 1993). In this study, we showed that Lef-1 was present in postnatal murine skin, and its level was high in the hair follicles during anagen, but low during catagen and telogen. -Catenin protein level showed a similar dynamic pattern through the hair cycle.
Huelsken et al. (2001) reported that the presence and distribution of stem cells was disrupted when hair follicles of -catenin-deficient skin disappeared. Similar phenomena were observed in other studies in which -catenin and Lef-1 were mutated (Botchkarev et al., 2002). Gat et al. (1998) applied transgenic techniques to delete the terminal amino-phosphorylated sites of -catenin, thus resulting in unusually high levels of stable -catenin. Gat et al. (1998) found that in transgenic mice, interfollicular epidermis grew new hair follicles without disturbing the differentiation of the epidermis. However, adult mice did not emerge with new hair follicles until the new hair cycle began. Zhou et al. (1995) reported that the differentiation and maturation
778
H.-D. Wang et al. / Acta Histochemica 114 (2012) 773–778
of hair follicles were enhanced, and hair follicles and tooth cells grew ectopically when the level of Lef-1 in epithelial cells was raised. These studies indicate that -catenin and Lef-1 have similar functions in the activation and directional differentiation of hair follicle stem cells. This study suggests that -catenin and Lef-1 are both necessary for the development and differentiation of hair follicles. Both proteins may have a function in maintaining the hair cycle. In addition, they appeared in the same areas at the same times and showed the same patterns of changes in protein levels. This implies that -catenin and Lef-1 function through the formation of a catenin-Lef-1 complex during the cyclical growth of hair follicles. This study provides a theoretical and experimental foundation for further research into the regularity of the hair follicle growth cycle. Acknowledgements This research was sponsored by the China National Natural Science Foundation (Grant No. 30671512) and by the Science and Technology Innovation Foundation of SXAU (Grant No. 2009001). References Alonso L, Fuchs E. The hair cycle. J Cell Sci 2006;119:391–3. Ayer-LeLievre C, Olson L, Ebendal T, Hallböök F, Persson H. Nerve growth factor mRNA and protein in the testis and epididymis of mouse and rat. Proc Natl Acad Sci U S A 1988;85:2628–32. Barker N. The canonical Wnt/-catenin signalling pathway. Methods Mol Biol 2008;468:5–15. Boras-Granic K, Chang H, Grosschedl R, Hamel PA. Lef-1 is required for the transition of Wnt signaling from mesenchymal to epithelial cells in the mouse embryonic mammary gland. Dev Biol 2006;295:219–31. Botchkarev VA, Botchkareva NV, Sharov AA, Funa K, Huber O, Gilchrest BA. Modulation of BMP signaling by noggin is required for induction of the secondary (nontylotrich) hair follicles. J Invest Dermatol 2002;118:3–10. Dong Y, Cao J, Wang H, Zhang J, Zhu Z, Bai R, et al. Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to a-melanocyte-stimulating hormone by upregulating melanocortin-1 receptor. Biochem Biophys Res Commun 2010;396:849–53. Driskell RR, Goodheart M, Neff T, Liu X, Luo M, Moothart C, et al. Wnt3a regulates Lef-1 expression during airway submucosal gland morphogenesis. Dev Biol 2007;305:90–102. Duverger O, Morasso MI. Epidermal patterning and induction of different hair types during mouse embryonic development. Birth Defects Res C Embryo Today 2009;87:263–72. Gat U, DasGupta R, Degenstein L, Fuchs E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated -catenin in skin. Cell 1998;95:605–14. Goding CR. Mitf from neural crest to melanoma: signal transduction and transcriptionin the melanocyte lineage. Genes Dev 2000;14:1712–28.
Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 2001;105:533–45. Kishimoto J, Burgeson RE, Morgan BA. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev 2000;14:1181–5. Kühl M, Sheldahl LC, Park M, Miller JR, Moon RT. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 2000;16:279–83. Liu X, Driskell RR, Luo M, Abbott D, Filali M, Cheng N, et al. Characterization of LEF-1 promoter segments that facilitate inductive developmental expression in skin. J Invest Dermatol 2004;123:264–74. Müller-Röver S, Handjiski B, van der Veen C, Eichmüller S, Foitzik K, McKay IA, et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 2001;117:3–15. Niemann C, Owens DM, Hülsken J, Birchmeier W, Watt FM. Expression of Lef-1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours. Development 2002;129:95–109. Oosterwegel M, van de Wetering M, Timmerman J, Kruisbeek A, Destree O, Meijlink F, et al. Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis. Development 1993;118:439–48. Quinn E, Rudolf G. Regulation of LEF-1/TCF transcription factors by Wnt and other signals. Curr Opin Cell Biol 1999;11:233–40. Schmidt-Ullrich R, Paus R. Molecular principles of hair follicle induction and morphogenesis. Bioessays 2005;27:247–61. Stenn KS, Paus R. Controls of hair follicle cycling. Physiol Rev 2001;81:449–94. Tibary A, Vaughan JL. Reproductive physiology and infertility in male South America camelids: a review and clinical observations. Small Ruminant Res 2006;61:283–98. ˜ van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, et al. Development of several organs that require inductive epithelial–mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev 1994;8:2691–703. Wang H, Dong Y, Chen W, Hei J, Dong C. Expression and localization of nerve growth factor (NGF) in the testis of alpaca (llama pacos). Folia Histochem Cytobiol 2011:55–61. Widelitz RB. Wnt signaling in skin organogenesis. Organogenesis 2008;4:123–33. Widelitz RB, Jiang TX, Lu J, Chuong CM. -Catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin. Dev Biol 2000;219: 98–114. Yan Y, Dong C, He J, Ren Y, He X, Bai R, et al. Expression and localization of transforming growth factor-b1of alpaca testis. Acta Vet Zootech Sin 2008;39:97–102. Zhou P, Byrne C, Jacobs J, Fuchs E. Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev 1995;9:700–13.