The transcription factor p63 plays an essential role in epidermal morphogenesis. Animals lacking p63 fail to form many ectodermal organs, including the skin and hair follicles. Although the indispensable role of p63 in stratified epithelial skin development is well established, relatively little is known about this transcriptional regulator in directing hair follicle morphogenesis. Here, using specific antibodies, we have established the expression pattern of ΔNp63 in hair follicle development and cycling. ΔNp63 is expressed in the developing hair placode, whereas in mature hair its expression is restricted to the outer root sheath (ORS), matrix cells and to the stem cells of the hair follicle bulge. To investigate the role of ΔNp63 in hair follicle morphogenesis and cycling, we have utilized a Tet-inducible mouse model system with targeted expression of this isoform to the ORS of the hair follicle. ΔNp63 transgenic animals display dramatic defects in hair follicle development and cycling, eventually leading to severe hair loss. Strikingly, expression of ΔNp63 leads to a switch in cell fate of hair follicle keratinocytes, causing them to adopt an interfollicular epidermal (IFE) cell identity. Moreover, ΔNp63 transgenic animals exhibit a depleted hair follicle stem-cell niche, which further contributes to the overall cycling defects observed in the mutant animals. Finally, global transcriptome analysis of transgenic skin identified altered expression levels of crucial mediators of hair morphogenesis, including key members of the Wnt/β-catenin signaling pathway, which, in part, account for these effects. Our data provide evidence supporting a role for ΔNp63α in actively suppressing hair follicle differentiation and directing IFE cell lineage commitment.
INTRODUCTION
The multilayered stratified epithelium of the epidermis develops from a single layer of ectoderm progenitor cells through a tightly regulated series of events during embryogenesis. In addition to forming the epidermis, a subset of surface ectodermal cells develop into the pilosebaceous units that include the hair follicle and sebaceous gland. In mice, pelage hair follicle morphogenesis is initiated during embryonic development at E14.5 and is governed by a series of inductive cues shared between keratinocytes committed to a hair follicle cell fate and mesenchymal cells of the underlying dermis (Fuchs, 2007). These signaling events result in the formation of local thickenings, or placodes, in the overlying epithelium (Hardy, 1992). Beneath the underlying epidermal thickenings of the placodes, mesenchymal cells of the dermis localize, increase in density and form a cluster that later develops into specialized cells known as the dermal papilla (DP), which are crucial for proliferation of hair follicle matrix cells (Jahoda et al., 1984; Panteleyev et al., 2001; Paus et al., 1999; Reynolds and Jahoda, 1992).
Reciprocal mesenchymal-epithelial crosstalk continues during the early stage of development, stimulating rapid proliferation and down-growth of hair matrix cells to encase the DP. Subsequently, matrix cells differentiate into specialized structures of the hair follicle, which include the inner root sheath (IRS) and hair shaft compartments of the hair follicle (Panteleyev et al., 2001). The IRS is completely surrounded by an outer root sheath (ORS), which is continuous with the basal layer of the epidermis. After birth, by postnatal day 16 (P16), the hair follicle reaches full maturation and enters a regressive state known as catagen. At this stage, the lower two thirds of the hair follicle undergoes rapid degeneration through a process involving apoptosis, where the dermal papilla comes to rest just below the bulge region. The bulge is the permanent portion of the hair follicle and serves as a reservoir for hair follicle stem cells (Cotsarelis, 2006). It is during the resting telogen stage that the old hair shaft is shed from the hair canal. Following telogen, a new cycle of hair regeneration, anagen, is initiated. Not surprising, the complex hair development program is governed by several conserved signaling pathways including Wnt (Fuchs, 2007; Millar, 2002; Schneider et al., 2009).
Indeed, genetic studies have unequivocally demonstrated the Wnt/β-catenin signaling pathway to be crucial in regulating hair follicle morphogenesis. The importance of this pathway is illustrated by experiments where the coordinate action of β-catenin or its nuclear mediators Tcf3 or Lef1 are blocked. Thus, in mice with conditional ablation of the Ctnnb1 gene (which encodes for β-catenin) or constitutive expression of the Wnt inhibitor Dkk1, hair placode formation is severely compromised (Andl et al., 2002; Huelsken et al., 2001). In agreement with these findings, Lef1-null animals lack hair, whereas transgenic animals that express a transcriptionally inactive form of Lef1 display altered differentiation and the reprogramming of hair follicle cell fate (Merrill et al., 2001; Niemann et al., 2002; van Genderen et al., 1994; Zhou et al., 1995). These studies have established a fundamental role for the Wnt/β-catenin signaling pathway in the early epithelial-mesenchymal events that specify hair follicle cell fate, initiate hair patterning and direct hair follicle morphogenesis. As with most developmental programs, the signaling events tied to the maturation of hair follicles are intimately associated with a gene expression program dictated by transcription factors.
The transcription factor p63, a member of the p53 family, plays an important role in the development of stratified epithelium of the skin and its appendages. Mice with deletion of the Trp63 gene exhibit severe developmental abnormalities including limb truncations and defects in skin epidermal stratification and differentiation (Mills et al., 1999; Yang et al., 1999). Moreover, these animals lack ectodermal organs such as teeth, hair follicles and glandular structures. Although the p63 knockouts have provided valuable insights into understanding epidermal development, thus far it has not been an ideal model system to study hair follicle morphogenesis. This is primarily owing to the severe developmental arrest observed in these animals, including a complete block in placode formation. Another issue that has complicated studies of p63 is the existence of multiple p63 isoforms, each with potentially distinct molecular properties. The Trp63 gene encodes for multiple functionally distinct protein isoforms, including TAp63 and ΔNp63, which contain unique N-terminal segments that harbor independent activation properties (Helton et al., 2008; Yang et al., 1998). In addition, both TA and ΔN transcripts are differentially spliced at the 3′ end, generating proteins with unique C-termini designated as α, β and γ isoforms. The fact that all isoforms of p63 are absent in the conventional knockout mouse has thus far precluded studies on the biological role of individual p63 proteins (Barbieri and Pietenpol, 2006). This is particularly relevant to the ΔNp63 isoforms, which are predominantly expressed in skin epidermal keratinocytes and have recently been shown to direct keratinocyte cell fate by directly regulating the basal keratins K5 and K14 (Candi et al., 2006; Romano et al., 2007; Romano et al., 2009).
To investigate the role of ΔNp63α in hair follicle development, we have engineered tetracycline-inducible transgenic animals with targeted expression of ΔNp63α to the ORS of the hair follicle. Interestingly, ΔNp63 transgenic animals develop severe hair growth and cycling defects leading to eventual hair loss. Transgenic animals display a progressive increase in hair follicle size with an expanded ORS and dramatic defects in differentiation of the matrix cells. Furthermore, mutant hair follicle keratinocytes undergo a switch in cell lineage and adopt an interfollicular cell fate. Our results provide novel insight into the function of ΔNp63α in regulating various facets of the hair differentiation program and reveal a key role for several members of the Wnt/β-catenin signaling pathway in the observed hair phenotype.
MATERIALS AND METHODS
Generation of transgenic animals and animal procedures
The HA-ΔNp63α construct was generated by cloning the full-length mouse ΔNp63α containing a 5′ HA epitope tag into the pTRE-Tight plasmid (BD Bioscience). Transgenic mouse lines were generated by microinjecting the purified DNA construct into fertilized mouse oocytes derived from a mixed genetic background (C3Hf/HeRos × C57BL/10 Rospd). Seven HA-ΔNp63α transgenic founder lines were identified by PCR analysis of tail DNA. The following primers were used to genotype the HA-ΔNp63 founders: forward, 5′-GGAGAATTCGAGCTCGGTACCCG-3′ and reverse, 5′-CGCTATTCTGTGCGTGGTCTG-3′. The founders were then crossed to K5-tTA mice (Diamond et al., 2000) in the absence of Dox to determine which of the founders express the transgene. Four founding lines were identified to express the transgene by western blot analysis.
Protocols for mouse experimentation were performed according to SUNY at Buffalo and RPCI IACUC protocols. The K5-tTA and TOPGAL.lacZ mice have been previously described (DasGupta and Fuchs, 1999; Diamond et al., 2000). Mice of appropriate genotype were mated and noon of the day the vaginal plug was observed was considered E0.5. In experiments when transgene expression was repressed in BG (bi-transgenic) pups, pregnant females were administered Dox through rodent chow at a concentration of 200 mg/kg (Bio-Serv). Transgene expression in BG pups was then induced by Dox chow withdrawal from the lactating mother. For triple transgenic experiments, ΔNp63αBG mice were mated to TOPGAL.lacZ mice, in the absence of Dox, to generate TOPGALΔNp63αBG animals.
Western blot
Western blot analysis was performed as previously described (Romano et al., 2009). Primary antibodies were used at the following dilutions: HA (Roche, 1:5000), β-tubulin (Chemicon, 1:10000), Lef1 (Upstate, 1:2000), β-catenin (Sigma 1:2000), K15 (Thermo Scientific, 1:2000), Gata3 (Santa Cruz, 1:2000), RR-14 (1:5000) and Sox9 (Santa Cruz, 1:2000).
Immunostaining
Stainings using paraffin embedded sections were performed as previously described (Romano et al., 2009). Primary antibodies used at the indicated dilutions were ΔNp63 (RR-14; 1:50), p63 (1:50; Santa Cruz, 4A4), K5, K10, K6, loricrin and filaggrin (1:200; gift from Dr Julie Segre, NHGRI, Bethesda, USA), Sox9 (1:50; Santa Cruz), K15 (1:50; Thermo Scientific), K17 (1:200; gift from Dr Pierre Coulombe, John Hopkins University, New York, USA), AE13/AE15 (1:10; gift from Dr T. T. Sun, New York University, New York, USA), Elf5 (1:50, Santa Cruz), Gata3 (1:50, Santa Cruz), β-catenin (1:100, Santa Cruz), Lef1 (1:100, Upstate), Ki67 (1:100, Novacastra), PCNA (1:50, Dako Cytomation) and S100A6 (1:100, Thermo Scientific). When staining with mouse monoclonal antibodies, we used the reagents and protocol from the MOM Basic Kit (Vector Labs). Slides were mounted using Vectashield Mounting Medium with DAPI (Vector Labs) and viewed with a Nikon FXA fluorescence microscope. Images were captured using a Nikon digital camera and analyzed using ImageJ, Adobe Photoshop and Adobe Illustrator software.
β-galactosidase staining
Dorsal skin samples were fixed in 1% formaldehyde and 2% gluteraldehyde (in PBS) for 2 hours. Samples were then washed in PBS for 20 minutes and then incubated in 1 mg/ml X-Gal solution [100 mM sodium phosphate buffer (pH 7.3), 0.01% (w/v) sodium deoxycholate, 0.02% (v/v) Nonidet P-40, 2 mM magnesium chloride, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide] at 37°C overnight. Samples were then washed in PBS for 20 minutes and post-fixed in 10% NBF for 6 hours and then immediately dehydrated, paraffin embedded and sectioned to 4 μm thickness.
Semi-quantitative RT-PCR
Total RNA from dorsal skin of wild-type and BG animals was isolated and purified using TRIzol (Invitrogen) according to established protocols. Two micrograms of total RNA was reverse transcribed using the ThermoScript RT-PCR System (Invitrogen). PCR amplifications were carried out using Fermentas Taq DNA Polymerase LC (Fermentas, MD, USA). All primers were designed to span at least one intron. Primer sequences are available upon request.
Microarray analysis
Total RNA was extracted from wild-type and BG skin using TRIzol (Invitrogen, Carlsbad, CA, USA) and then purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Purified total RNA was analyzed on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and labeled to obtain biotinylated cRNA for hybridization to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Two independent sets of biological replicates (BG and control from P16 dorsal skin) were used. Scanned microarray images were imported into GeneChip Operating Software (GCOS, Affymetrix) to generate raw signal values for each probe. The MAS5.0 algorithm in the ‘Affy’ package of Bioconductor in the R statistical computing environment was used to generate expression summary values, followed by trimmed mean global normalization to bring the median expression values of all four GeneChips to the same scale (Gentleman et al., 2004). For data quality control, MAS5.0 present/absent calls were used to filter out probe sets whose expression intensities were close to background noise across the majority of samples. Specifically, filtering of two ‘present calls’ was applied to either the wild-type or BG group, with greater than 13,000 unique genes passing the quality control. Genes that were altered at a P-value less than 0.05 between BG and wild-type populations were considered significant for further analysis. The mRNA expression profiling datasets have been deposited in the NCBI Gene Expression Omnibus (GEO) data repository (http://www.ncbi.nlm.nih.gov/geo/) under Accession number GSE20514.
RESULTS
Analysis of ΔNp63 expression during hair follicle development and cycling
To investigate the role ΔNp63 plays in hair follicle morphogenesis, we initially sought to establish its expression pattern during normal murine hair follicle development and cycling. ΔNp63 expression was analyzed using the RR-14 antibody that specifically recognizes the ΔNp63 isoform of p63 (Romano et al., 2006). We have previously shown that ΔNp63 expression is first detected in skin as early as E10.5. By E14.5, it is expressed in the basal layer of the epidermis and the focal epidermal thickenings corresponding to the developing hair placodes (Fig. 1A) (Romano et al., 2009). Expression continues during placode down-growth and, by P1, ΔNp63 is restricted to the basal layer of the skin, the ORS and the undifferentiated cells of the matrix region (Fig. 1C) (Rendl et al., 2005). At the telogen stage (P21), in addition to the basal layer, ORS and sebaceous gland, ΔNp63 expression is detected in the bulge region of the hair follicle as well as the secondary hair germ, structures that serve as reservoirs for hair follicle stem cells and are important for the initiation of anagen (Fig. 1D) (Schneider et al., 2009). Stem cells in the bulge compartment have been demonstrated to express high levels of K15 and Sox9 (Liu et al., 2003; Vidal et al., 2005). Double staining with p63 and K15, as well as p63 and Sox9, establish that p63 is expressed in the stem cell compartment of the hair follicle (Fig. 1E,F).
Generation of ΔNp63α transgenic mice
To investigate the role of ΔNp63α in hair follicle morphogenesis, we generated tetracycline-inducible ΔNp63α transgenic animals. These animals were crossed to K5-tTA tet-OFF transgenic mice, which express the tetracycline transactivator (tTA) under the control of the bovine K5 promoter (Diamond et al., 2000). In the absence of doxycycline (Dox), K5-tTA/pTRE-ΔNp63α bi-transgenic animals (here after referred to as ΔNp63αBG) express HA-ΔNp63α in the basal layer of the epidermis and ORS of the hair follicle. Of the seven transgenic founders, four expressed the transgene with varying phenotypic severities. ΔNp63αBG animals corresponding to line D, expressing the lowest levels of the transgene, were indistinguishable at birth compared with wild-type littermates. In contrast to control animals, line D ΔNp63αBGs failed to grow a normal coat, whereas the whiskers appeared to develop normally. By P16, there were signs of sparse hair growth and the skin of the BG animals appeared dry and scaly (Fig. 2A). Typically, these animals survived until approximately P35, after which they were euthanized for morbidity and overall poor health. For the second line E, which expressed the transgene at moderate levels, ΔNp63αBG animals were indistinguishable from control littermates at birth. By P2, however, the skin appeared wrinkled and, by P4, the skin began to peel and the animals had to be euthanized (see Fig. S1 in the supplementary material). Conversely, the highest expressing lines, B and F ΔNp63αBGs, displayed an eye open at birth phenotype and died shortly after birth. This neonatal lethality could be overcome by Dox administration to the pregnant dams, thereby preventing transgene expression. When Dox was withdrawn during late pregnancy, the BG offspring exhibited severe skin and hair phenotypes within 2-3 weeks (see Fig. S1 in the supplementary material). To confirm expression of the transgene, we performed western blot analysis using skin samples from the ΔNp63αBG animals and controls. The transgene was expressed in the ΔNp63αBG animals, as demonstrated by anti-HA antibodies as well as increased ΔNp63α levels, as judged by anti-p63 antibodies (Fig. 2B). Owing to the moderate levels of HA-ΔNp63α in line D and the fact that animals from this line survived well into the second phase of hair follicle cycling, we focused on this line for the studies described here.
ΔNp63 expression analysis during hair follicle development and cycling. (A,B) At E14.5, expression of ΔNp63 protein can be detected in the developing placodes and continues to be expressed in the down-growing hair germ at E16.5 (B). Dotted lines indicate the epidermal-dermal junction. (C) By postnatal day 1 (P1), ΔNp63 is expressed along the entire length of the outer root sheath (ORS) as well as the matrix cells. (D) At P21 (telogen), ΔNp63 expression is localized to the basal layer, ORS and the bulge region (brackets, insets) of the hair follicle and secondary hair germ (Hg). (E,F) Co-staining with K15 and Sox9 reveals co-localization of ΔNp63 to hair follicle stem cells. Scale bars: 50μm.
ΔNp63 expression analysis during hair follicle development and cycling. (A,B) At E14.5, expression of ΔNp63 protein can be detected in the developing placodes and continues to be expressed in the down-growing hair germ at E16.5 (B). Dotted lines indicate the epidermal-dermal junction. (C) By postnatal day 1 (P1), ΔNp63 is expressed along the entire length of the outer root sheath (ORS) as well as the matrix cells. (D) At P21 (telogen), ΔNp63 expression is localized to the basal layer, ORS and the bulge region (brackets, insets) of the hair follicle and secondary hair germ (Hg). (E,F) Co-staining with K15 and Sox9 reveals co-localization of ΔNp63 to hair follicle stem cells. Scale bars: 50μm.
Histological analysis of hair development and cycling in ΔNp63αBG animals
To analyze the effects of overexpression of ΔNp63α in hair follicle cycling, we examined the histology of mid-dorsal skin sections stained with Hematoxylin and Eosin (H&E) from BG and wild-type littermates. Although no obvious histological differences were observed at the newborn (NB) stage, by P16 ΔNp63αBG animals displayed a thickened, hyperproliferative epidermis with a dramatic reduction of the granular layer (Fig. 2C, middle panel inset). Hair follicles from the BG animals appeared hypertrophic and considerably larger than wild-type control hair follicles (Fig. 2C, middle panel, yellow arrow). Strikingly, the hair shafts of the BG animals were replaced by thick keratinized tissues (middle and lower panels, black arrows). These histological data suggested a possible switch of keratinocytes from a hair follicle to the interfollicular epidermal (IFE) cell fate. In mice, the normal hair cycle lasts approximately 28 days, with the onset of catagen beginning around P16 and telogen starting at P21. The second phase of hair growth (anagen) commences at P28 (Muller-Rover et al., 2001). Hair follicles in the ΔNp63αBG animals failed to enter catagen and, by day P28, remained in anagen, as demonstrated by their continued down-growth into the underlying dermis (Fig. 2C, lower panel). These dramatic changes in the overall appearance of the hair follicles and the IFE were a common feature observed in all of the ΔNp63αBG-expressing lines (see Fig. S1 in the supplementary material).
Gross morphology and histological analysis of ΔNp63α transgenic mice. (A) Gross morphology reveals that the BG animals have thickened scaly skin and fail to grow hair as compared with control littermates at P16. (B) Western blot analysis using skin whole-cell extracts reveals that BG animals express the transgene (α-HA) as well as increased levels of ΔNp63 (α-ΔNp63α) as compared with wild-type control littermates at P16. β-tubulin serves as a loading control. (C) Dorsal skin sections from various developmental time-points stained with Hematoxylin and Eosin (H&E). At newborn (NB), BG animals are indistinguishable from their control littermates (top panel). At P16, the BG interfollicular epidermis (IFE) is hyperplastic and displays a reduced granular layer (middle panel inset). Hair follicles from the BG animals (middle panel) are enlarged (yellow arrow) and have increased amounts of keratinized tissue localized to the hair shaft region (black arrows). By P28, when wild-type hair follicles are in telogen, the BG follicles continue to grow into the dermis (arrow, lower panel), suggesting a failure of these follicles to cycle properly. Scale bars: 75μm in upper and middle panels; 100 μm in lower panels.
Gross morphology and histological analysis of ΔNp63α transgenic mice. (A) Gross morphology reveals that the BG animals have thickened scaly skin and fail to grow hair as compared with control littermates at P16. (B) Western blot analysis using skin whole-cell extracts reveals that BG animals express the transgene (α-HA) as well as increased levels of ΔNp63 (α-ΔNp63α) as compared with wild-type control littermates at P16. β-tubulin serves as a loading control. (C) Dorsal skin sections from various developmental time-points stained with Hematoxylin and Eosin (H&E). At newborn (NB), BG animals are indistinguishable from their control littermates (top panel). At P16, the BG interfollicular epidermis (IFE) is hyperplastic and displays a reduced granular layer (middle panel inset). Hair follicles from the BG animals (middle panel) are enlarged (yellow arrow) and have increased amounts of keratinized tissue localized to the hair shaft region (black arrows). By P28, when wild-type hair follicles are in telogen, the BG follicles continue to grow into the dermis (arrow, lower panel), suggesting a failure of these follicles to cycle properly. Scale bars: 75μm in upper and middle panels; 100 μm in lower panels.
Alterations in the keratinocyte differentiation program in the IFE of ΔNp63αBG animals
The histology shown in Fig. 2 suggested that the normal differentiation program of the epidermal keratinocytes was disrupted by overexpression of ΔNp63α in the basal layer of the epidermis. To confirm these observations, we analyzed the distribution of a variety of epidermal-specific markers by staining dorsal skin sections of BG and control mice at P16. As shown in Fig. 3, keratinocytes from the skin of BG animals demonstrated significant alterations in the expression of these markers. For example, expression of K5, which is normally localized to the proliferating basal layer of the epidermis, was expanded in the BG animals, with most of the cellular layers of the epidermis staining positive for this keratin marker (Fig. 3A,B). Similar results were obtained for K14 (data not shown). In addition, when we examined the expression of K1 and K10, markers normally confined to the spinous layer of the epidermis, we observed an expansion of this layer in the IFE of the BG animals as compared with control mice (Fig. 3C-F). This is in agreement with recent studies demonstrating that ΔNp63α can induce K1 in cell culture experiments (Ogawa et al., 2008; Truong et al., 2006). To determine whether there was a delay in the later stages of terminal differentiation, we evaluated the expression pattern of involucrin, loricrin and filaggrin. Whereas sustained expression of ΔNp63α resulted in a dramatic reduction in loricrin expression, expression of involucrin and filaggrin were not reduced but, instead surprisingly, appeared to be increased (Fig. 3G-L). It is plausible that the elevated levels of involucrin and filaggrin might be due to direct transcriptional effects of overexpressed ΔNp63α or secondary to compensatory mechanisms. The specific changes in expression levels of structural genes, as observed by immunostaining, were also reproducible by western blot analysis (see Fig. S2 in the supplementary material). These results indicate that expression of ΔNp63α in the basal layer of the epidermis results in an expansion of both the basal and spinous layers of the epidermis, while only partially interrupting the normal keratinocyte terminal differentiation program.
Expansion of the ORS and severely reduced IRS and hair shaft compartments in ΔNp63αBG animals
In light of the histological appearance of the epidermis, the hair follicles and the apparent paucity of hair in the ΔNp63αBG animals, we next wanted to probe the extent of follicular defects. We therefore examined the expression of a range of markers involved in hair follicle differentiation. In mutant skin, K5 and K17 staining revealed an expanded ORS as compared with wild-type control littermates, whereas K6 staining demonstrated an expanded companion layer in the BG animals (Fig. 4A-F). Differentiation of the hair cortex, medulla and cuticle, as assayed by immunofluorescence for the hair keratin proteins (AE13), trichohyalins (AE15) and the transcriptional regulator Elf5 (IRS), revealed a dramatic reduction in the expression of these markers in the hair follicles of BG animals as compared with control animals (Fig. 4G-L) (Choi et al., 2008; Lynch et al., 1986). These results suggest a defect in the differentiation of these cellular compartments in the mutant follicles. The alterations in the differentiation of the IRS compartment was further confirmed by a loss of Gata3 expression (Fig. 4M-N) (Kaufman et al., 2003).
Analysis of the interfollicular epidermis. (A-F) Immunofluorescence staining of dorsal skin sections using antibodies against K5, K1 and K10 shows an expansion of the proliferating basal layer (K5) and spinous layer (K1/K10) in the IFE of the BG animals. (G,H) Conversely, there is a dramatic reduction in loricrin expression in the BG animals. (I-L) Evaluation of involucrin and filaggrin expression demonstrates increased expression levels in the BG IFE as compared with wild-type animals. Dotted lines indicate the epidermal-dermal junction. Scale bar: 75μm.
Analysis of the interfollicular epidermis. (A-F) Immunofluorescence staining of dorsal skin sections using antibodies against K5, K1 and K10 shows an expansion of the proliferating basal layer (K5) and spinous layer (K1/K10) in the IFE of the BG animals. (G,H) Conversely, there is a dramatic reduction in loricrin expression in the BG animals. (I-L) Evaluation of involucrin and filaggrin expression demonstrates increased expression levels in the BG IFE as compared with wild-type animals. Dotted lines indicate the epidermal-dermal junction. Scale bar: 75μm.
Alterations in the hair follicle differentiation program of ΔNp63αBG animals. (A-R) Immunofluorescence staining used to detect changes in the hair follicle differentiation program of ΔNp63αBG animals at P13. Antibodies used for each staining are shown in lower left panels. Scale bars: 100μm in C-H; 75μm in A,B,I-L,O,P; 60 μm in Q,R; 50 μm in M,N.
Alterations in the hair follicle differentiation program of ΔNp63αBG animals. (A-R) Immunofluorescence staining used to detect changes in the hair follicle differentiation program of ΔNp63αBG animals at P13. Antibodies used for each staining are shown in lower left panels. Scale bars: 100μm in C-H; 75μm in A,B,I-L,O,P; 60 μm in Q,R; 50 μm in M,N.
The canonical Wnt/wingless signaling pathway has been shown to be necessary for the formation of many ectodermal organs, including the hair follicle (Andl et al., 2002). Given the importance of β-catenin and Lef1 in hair follicle differentiation and hair cycling, we investigated the expression of these proteins in the mutant hair follicles of the transgenic animals. In control anagen hair follicles at P13, β-catenin is expressed in most cellular layers of the hair follicle, while Lef1 expression is restricted to the precortex region (Fig. 4O,Q) (Merrill et al., 2001). However, in the ΔNp63αBG hair follicles, there is a dramatic reduction in the expression of both β-catenin and Lef1 (Fig. 4P,R). The loss of Lef1 expression, especially in the precortex region of the BG hair follicles, might be causally associated with the blocked differentiation of both the IRS and hair shaft compartments. The reduced expression levels of some of these crucial markers of the hair follicles, such as Gata3 and Lef1, was also evident by western blot analysis (see Fig. S2 in the supplementary material).
Decrease in matrix cell proliferation results in a block in IRS and hair shaft development
Given the observed defects in the development of the IRS, cortex and medulla in the ΔNp63αBG hair follicles, we next examined the status of matrix cells of the hair follicle bulb region. Matrix cells are transiently amplifying cells that are thought to terminally differentiate, giving rise to both the IRS and the hair shaft (Millar, 2002). We therefore reasoned that the failure of mutant hair follicles to properly generate the IRS and hair shaft was probably due to a loss of proliferation potential of the matrix cells of the bulb region. To investigate this possibility, we stained wild-type and mutant hair follicle bulbs from P13 animals using antibodies against cell cycle markers Ki67 and proliferating cell nuclear antigen (PCNA). As seen in Fig. 5, transgenic hair follicle bulbs demonstrated reduced numbers of Ki67+ cells as compared with control hair follicles (upper panel, arrows). Similar results were observed with PCNA (Fig. 5, lower panel, arrows). This data suggests that overexpression of ΔNp63α in the ORS of the hair follicle induces a loss of proliferation potential in matrix cells of the hair follicle, which might be responsible for the IRS and hair shaft defects seen in the BG hair follicles.
Changes in follicular keratinocyte identity in hair follicles expressing ΔNp63α
ΔNp63 can directly regulate the expression of K5 and K14, two markers coinciding with the initiation of the stratification program in the skin epidermis (Romano et al., 2009). Furthermore, expression of K5 and K14 is severely attenuated in p63-null animals (Mills et al., 1999; Yang et al., 1999). Given this correlation and the histological appearance of the skin, we investigated the possibility that overexpression of ΔNp63 in the ORS could alter the inherent cellular identity and initiate an epidermal differentiation program in hair follicle keratinocytes. Surprisingly, cytokeratin markers K1 and K10, which in wild-type skin are restricted to the spinous layer of the epidermis and the infundibulum, were expressed in the keratinocytes of the hair follicles of transgenic animals as demonstrated by double staining with K14 (Fig. 6, top panel; data not shown). Similarly, filaggrin, a marker normally expressed in the terminally differentiated layer of the epidermis, was expressed in the hair follicle keratinocytes of the BG animals (Fig. 6, lower panel). The misplaced expression of these IFE differentiation markers in the ΔNp63αBG animals was widespread, extending into the lower portion of the hair follicle near the bulb. These data suggest the possibility that elevated levels of ΔNp63α in the ORS of the hair follicle results in a switch of hair follicle keratinocytes to adopt an IFE cell fate, thus affecting hair follicle development and cycling in the mutant animals.
Reduced matrix cell proliferation in the hair follicles of ΔNp63αBG animals. Dorsal skin sections from P13 were stained with Ki67 (upper panel) and PCNA (lower panel). Matrix cells of BG hair follicles show reduced numbers of proliferating cells as compared with control littermates (arrows). Scale bar: 75 μm.
Reduced matrix cell proliferation in the hair follicles of ΔNp63αBG animals. Dorsal skin sections from P13 were stained with Ki67 (upper panel) and PCNA (lower panel). Matrix cells of BG hair follicles show reduced numbers of proliferating cells as compared with control littermates (arrows). Scale bar: 75 μm.
Follicular keratinocyte transformation in BG animals. Overexpression of ΔNp63α results in the transition of hair follicle keratinocytes to adopt an IFE cell fate. Dorsal skin sections from P28 were stained with K1 and filaggrin, markers of the IFE. Compared with wild-type hair follicles, BG hair follicles express K1 and filaggrin. A higher magnification is shown in the insets. Arrows indicate cells expressing both K14/K1 or K14/Fil respectively. All sections are stained with K14 (red), K1 or Fil (green), and DAPI (blue). Scale bar: 75μm.
Follicular keratinocyte transformation in BG animals. Overexpression of ΔNp63α results in the transition of hair follicle keratinocytes to adopt an IFE cell fate. Dorsal skin sections from P28 were stained with K1 and filaggrin, markers of the IFE. Compared with wild-type hair follicles, BG hair follicles express K1 and filaggrin. A higher magnification is shown in the insets. Arrows indicate cells expressing both K14/K1 or K14/Fil respectively. All sections are stained with K14 (red), K1 or Fil (green), and DAPI (blue). Scale bar: 75μm.
Sustained activation of ΔNp63α stimulates a depletion of hair follicle stem cells
In mouse skin, the bulge serves as an important reservoir of stem cells necessary for the cyclic bouts of degeneration (catagen) and regeneration (anagen) occurring in mammalian hair follicles (Cotsarelis, 2006). Given the apparent failure of hair follicles to undergo proper hair follicle cycling in the BG animals, we investigated the status of the stem cell compartment in these animals. To determine the effects of sustained expression of ΔNp63α on hair follicle stem cells, we analyzed the expression of the stem cell markers K15, Sox9 and S100-A6. In contrast to wild-type hair follicles, which express robust levels of K15, there is a loss of K15 expression in the hair follicles of the transgenic animals (Fig. 7, left panel). Similarly, we observed a loss of both Sox9 and S100-A6 expression (Fig. 7, middle and right panel) in transgenic follicles at P21, suggesting a loss of follicular stem cells. Western blot analysis further confirmed a dramatic reduction in the protein expression levels of both K15 and Sox9 (see Fig. S2 in the supplementary material). Taken together, these results suggest that forced expression of ΔNp63α in the ORS causes a depletion of the hair follicle stem cell niche, indicating an important role for this isoform in bulge stem-cell maintenance.
Global changes in gene expression patterns in ΔNp63α mutant skin
To understand the molecular mechanisms underlying the dramatic defects observed in mutant hair follicle development and cycling, we next evaluated global changes in gene expression by performing transcriptional profiling of transgenic and control dorsal skin at P16. In agreement with the dramatic phenotype observed in the BG animals, the microarray analysis revealed major changes in the transcriptional profile of BG skin, with 705 genes displaying at least a 2-fold upregulation and 1352 genes showing at least a 2-fold downregulation as compared with wild-type (see Table S1 in the supplementary material). Indeed, there were major alterations in the expression of many structural genes and crucial components of several signaling pathways demonstrated to be important regulators of epidermal differentiation and hair follicle morphogenesis and cycling (a heat map of selected genes is shown in Fig. 8).
Forced expression of ΔNp63α results in hair follicle stem cell depletion. Dorsal skin sections from P21 were stained with various stem cell markers of the hair follicle bulge. As compared with wild-type control hair follicles, follicles of the BG animal show a complete loss of K15 (green), Sox9 (red) and S100A6 (red) expression, suggesting a depletion of hair follicle stem cells of the bulge. A higher magnification is shown in the insets. Scale bar: 100 μm.
Forced expression of ΔNp63α results in hair follicle stem cell depletion. Dorsal skin sections from P21 were stained with various stem cell markers of the hair follicle bulge. As compared with wild-type control hair follicles, follicles of the BG animal show a complete loss of K15 (green), Sox9 (red) and S100A6 (red) expression, suggesting a depletion of hair follicle stem cells of the bulge. A higher magnification is shown in the insets. Scale bar: 100 μm.
In support of the defects observed in the hair shaft of the BG animals and the results of the immunostaining experiments for hair keratin proteins and trichohyalins, transcript levels of various hair shaft-specific keratins and keratin-associated proteins were dramatically downregulated in the mutant skin (Fig. 8). These data were confirmed for selected hair keratin genes by semi-quantitative RT-PCR (Krt25, Krt28 and Krt31; see Fig. S3A in the supplementary material). Also significantly downregulated were numerous genes expressed in hair shaft precursor cells and genes required for hair shaft differentiation. Several hair follicle stem-cell-specific genes such as Krt15, Sox9 and Lhx2 were also downregulated in the mutant skin as compared with the control, consistent with our immunofluorescence and western blot analysis.
In agreement with immunofluorescence experiments, the microarray analysis revealed a dramatic reduction in some of the genes involved in the Wnt/β-catenin signaling pathway (Fig. 8). Semi-quantitative RT-PCR confirmed reduced levels of Lef1, Wnt5a, Wnt11 and Tcf3 transcripts, suggesting an alteration in the Wnt/β-catenin signaling pathway (Fig. 9A). As the microarray analysis was performed using skin samples from P16, it is possible that the downregulation of Wnt/β-catenin signaling was not an immediate and/or direct effect of ΔNp63α overexpression. To test this, we performed semi-quantitative RT-PCR using RNA isolated from BG animals after a short induction of ΔNp63α. Skin samples analyzed from E18.5 BG animals that were induced during embryogenesis revealed reduced Lef1, Tcf3 and Wnt5 levels, but not Wnt11 (Fig. 9A). The fact that the expression of at least some of the crucial genes belonging to the Wnt pathway were altered in the ΔNp63α embryonic skin suggests that downregulation of this pathway occurs prior to the development of the skin phenotype, rather than merely as a consequence of botched hair follicle differentiation. Conversely, we did not observe any significant changes in β-catenin transcript levels at any of the time points that were examined (data not shown). Microarray analysis also demonstrated a downregulation of some of the members of the Shh pathway (Fig. 8). This is not surprising, given the intimate connection between the Shh pathway and hair follicle development (Callahan and Oro, 2001). Altered expression of members of the Shh pathway in the BG animals was confirmed by semi-quantitative RT-PCR and results are shown in Fig. S3B in the supplementary material.
Altered hair-follicle-specific genes and signaling pathways in ΔNp63α mutant animals. Heat map representation of microarray data demonstrating a downregulation of genes involved hair shaft and inner root sheath (IRS) development, genes involved in hair follicle stem cell maintenance and various signaling pathways in ΔNp63α animals. The colour scale represents the expression level of a gene above (red), below (green), or at the mean expression level (black) across all samples.
Altered hair-follicle-specific genes and signaling pathways in ΔNp63α mutant animals. Heat map representation of microarray data demonstrating a downregulation of genes involved hair shaft and inner root sheath (IRS) development, genes involved in hair follicle stem cell maintenance and various signaling pathways in ΔNp63α animals. The colour scale represents the expression level of a gene above (red), below (green), or at the mean expression level (black) across all samples.
Finally, given the results of the gene expression study, we wondered whether a severe attenuation of Wnt activity in the hair follicles of our BG animals might explain the defects in hair follicle cycling and hair shaft development. We therefore sought to characterize any changes in the levels of Wnt activity in our BG animals by using the well-characterized transgenic mouse reporter line TOPGAL.lacZ (TOPGAL), which allows direct assessment of the temporal and spatial activity of the Wnt pathway (DasGupta and Fuchs, 1999). We therefore mated our ΔNp63αBG animals to the TOPGAL animals to generate triple transgenic animals (TOPGALΔNp63αBG). Dorsal skin sections of triple transgenic animals stained with X-Gal reveal a dramatic reduction of Wnt activity in the hair follicle matrix cells as compared with control animals, suggesting that the alterations in hair shaft morphogenesis in the BG animals are due to a loss of Wnt/β-catenin signaling, possibly resulting from reduced Lef1 expression levels (Fig. 9B).
DISCUSSION
Given the dynamic expression pattern of ΔNp63 in the hair follicle, we wanted to investigate the role of this isoform in hair follicle morphogenesis, an area that has received little attention thus far. With that goal in mind, we have generated a Tet-inducible system that allows expression of ΔNp63α in a temporal-spatial fashion. We find that targeted expression of ΔNp63α to the ORS of the hair follicle results in enlarged hair follicles with an expanded ORS. Closer examination using specific markers reveals a failure of the mutant follicles to undergo proper differentiation of the IRS and hair shaft compartments as evidenced by the loss of expression of several hair-specific keratins and IRS markers. Interestingly, we also observed a reduction in the proliferation of matrix cells, which give rise to both the IRS and hair shaft compartments. Unexpectedly, the keratinocytes within the hair follicle adopted a new cell fate, expressing markers of the IFE rather than hair. The propensity of epithelial cells that express higher levels of ΔNp63α to undergo squamous metaplasia is in agreement with our previous studies where ΔNp63α overexpression in simple lung epithelium converted these cells to a stratified state with ectopic expression of basal and suprabasal keratinocyte markers that are normally associated with the IFE (Romano et al., 2009).
Loss of Wnt/β-catenin signaling in ΔNp63α animals. (A)RT-PCR analysis of mRNA transcripts from wild-type and BG animals. Semi-quantitative RT-PCR reveals a dramatic downregulation in the levels of several genes belonging to the Wnt/β-catenin signaling pathway at P16 and E18.5. (B) TOPGAL and TOPGALΔNp63αBG dorsal skin sections at P13 were stained for lacZ expression and counter-stained with Eosin. Staining reveals Wnt/β-catenin activity in the lower portion of the hair shaft and matrix region of the TOPGAL animals (left panel). lacZ expression in TOPGALΔNp63αBG hair follicles is dramatically reduced (right panel). Scale bar: 50μm.
Loss of Wnt/β-catenin signaling in ΔNp63α animals. (A)RT-PCR analysis of mRNA transcripts from wild-type and BG animals. Semi-quantitative RT-PCR reveals a dramatic downregulation in the levels of several genes belonging to the Wnt/β-catenin signaling pathway at P16 and E18.5. (B) TOPGAL and TOPGALΔNp63αBG dorsal skin sections at P13 were stained for lacZ expression and counter-stained with Eosin. Staining reveals Wnt/β-catenin activity in the lower portion of the hair shaft and matrix region of the TOPGAL animals (left panel). lacZ expression in TOPGALΔNp63αBG hair follicles is dramatically reduced (right panel). Scale bar: 50μm.
Using global transcriptional analysis, we identified significant alterations to genes involved in Wnt/β-catenin signaling in the BG animals as compared with wild-type control littermates. In addition to Wnt5a, we detected reduced levels of Tcf3, a member of the Lef/Tcf family of HMG domain-containing DNA-binding proteins. This is of particular interest considering the recently described roles for Tcf3 as well as another family member, Tcf4, in hair follicle stem cell maintenance (Nguyen et al., 2009). Indeed, Tcf3–Tcf4 compound homozygous animals initiate hair follicle development, but display defects in hair follicle stem cell maintenance and follicle down-growth. Given the attenuated levels of Tcf3 in our BG animals, it is plausible that Tcf3 could be one contributing factor in the loss of the hair follicle stem cell niche observed in these animals. In addition to Wnt5a and Tcf3, we found Lef1 levels to be considerably downregulated in the hair follicles of the BG animals at both the mRNA transcript and protein levels. This was surprising given the existing data showing a putative role for p63 as a direct and positive transcriptional activator of Lef1 (Pozzi et al., 2009). However, this observation is based solely on knockdown studies in a transformed human keratinocyte cell line and thus could reflect context-dependent variability or isoform-specific effects. Alternatively, it is possible that the downregulation of Lef1 in the ΔNp63αBG animals represents an indirect effect. In the future, a detailed examination of the potential interaction of p63 with the regulatory elements of Lef1 in the in vivo context is warranted to resolve this discrepancy and address the underlying mechanism.
The hair follicle phenotype of the transgenic animals is in good agreement with our microarray data, which revealed reduced levels of many hair-specific keratin genes and hair shaft- and IRS-specific genes in the mutant animals. Moreover, crossing our BG animals to the well-characterized TOPGAL transgenic mice confirmed a loss of Wnt signaling in the mutant hair follicles, suggesting that the observed defects in the IRS and hair shaft compartments are a direct result of reduced Wnt signaling. We posit that the hair abnormalities might be further augmented by other key regulators of hair shaft differentiation including Dlx3 and Runx1, both of which are established p63 target genes and show dramatic downregulation in the transgenic animals based on microarray data (Hwang et al., 2008; Ortt et al., 2008; Osorio et al., 2008; Raveh et al., 2006). Indeed, a careful evaluation of the microarray data shows significant alterations in a number of genes associated with additional signaling pathways [insulin growth factor-binding protein 5 (Igfbp5) and fibroblast growth factor 5 (Fgf5) for example], which might, in part, contribute to some aspects of the observed BG hair phenotype such as the cycling defects (Hebert et al., 1994; Schlake, 2005; Schneider et al., 2009).
It is plausible that the apparent transformation of hair follicle keratinocytes to adopt an IFE cell fate is most probably the result of reduced Wnt/β-catenin signaling as this phenotype recapitulates some of the changes described in mouse model studies where β-catenin levels have been augmented using either gain-of-function or loss-of-function approaches (Zhang et al., 2008). Knockout and transgenic mouse model studies have shown that mice with targeted deletion of Ctnnb1 fail to develop hair follicles, whereas mice expressing an activated form of β-catenin result in the programming of embryonic epidermis to a hair follicle cell fate. Overall, these data implicate a crucial role for β-catenin in regulating hair shaft and IRS cell fate. Both immunostaining and western blot analysis reveal reduced levels of β-catenin in the hair follicles of our mutant mice. Therefore, it is possible that the reduced β-catenin and Lef1 levels in the mutant hair follicles reprogram the hair follicle keratinocytes to an IFE cell fate, similar to what has been reported in β-catenin ablated mice (Huelsken et al., 2001).
The capacity of hair follicles to maintain and activate a program of self-renewal is primarily dependent on stem cells located within the bulge region of the hair follicle. Bulge stem cells have been shown to express several markers including Krt15, Sox9, S100A6 and Lhx2 (Fuchs, 2007). Interestingly, our transgenic animals demonstrate a depletion of the stem cell compartment, with reduced expression levels of all of the aforementioned genes. Recently, Sox9 has been implicated in hair follicle morphogenesis as well as in the formation of the hair follicle stem cell compartment (Nowak et al., 2008; Vidal et al., 2005). In addition, in Sox9-null animals, the ORS of the hair follicles acquire epidermal characteristics, a feature similar to the phenotype observed in our transgenic animals. Although there is no experimental evidence supporting a role for p63 in directly regulating the expression of Sox9, it is interesting to note that siRNA-mediated knockdown of ΔNp63 isoforms in human keratinocytes leads to a strong upregulation of Sox9, suggesting that ΔNp63 might be a negative regulator of Sox9 (Truong et al., 2006). Intriguingly, studies with p63 knockout animals have clearly shown that p63 is required for the high proliferative potential and self-renewal of epithelial stem cells (Blanpain and Fuchs, 2007; Senoo et al., 2007). It is possible that the seemingly opposite effects of overexpression of ΔNp63α on stem cell behavior in the transgenic animals described in this study might reflect an imbalance of p63 isoforms or secondary effects due to global signaling changes.
In summary, we have shown that targeted overexpression of ΔNp63α to the ORS of the hair follicle leads to a loss of both the IRS and hair shaft compartments, resulting in progressive hair loss in mutant animals. We posit the hair follicle defects to be primarily attributed to a loss of Wnt/β-catenin and other signaling molecules as supported by global transcriptome analysis. These data suggest an important role for ΔNp63α in hair follicle morphogenesis and hair shaft differentiation. Given the distinctive expression pattern of ΔNp63 in the developing placodes, it is very probable that this transcription factor is crucial in the early stages of hair follicle development. In mature hair follicles, ΔNp63 expression remains restricted to the bulge and matrix regions where it might play an important role in stem cell maintenance and self-renewal and in balancing the proliferation and differentiation of matrix cells, respectively. The definitive analysis of the roles for ΔNp63 in hair follicle biology awaits the development of new tools and strategies, including an isoform-specific knockout.
Acknowledgements
We thank Dr Adam Glick for generously providing the K5-tTA animals. We are especially grateful to Irene Kulik for technical assistance and past and present members of our laboratory for useful comments on this study. This work was supported by a grant from NIH R01AR049238 to S.S. Deposited in PMC for release after 12 months.
References
Competing interests statement
The authors declare no competing financial interests.