ERRATUM

Development 137, 1775 (2010) doi:10.1242/dev.053116

Abnormal hair follicle development and altered cell fate of follicular keratinocytes in transgenic mice expressing Np63 Rose-Anne Romano, Kirsten Smalley, Song Liu and Satrajit Sinha

There was a technical problem with the ePress version of Development 137, 1431-1439 published on 24 March 2010.

A number of Greek symbols were missing from the text and figure headings in the Results section.

A replacement ePress article was published on 31 March 2010. The online issue and print copy are correct.

We apologise to authors and readers for this error. DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 1431

Development 137, 1431-1439 (2010) doi:10.1242/dev.045427 © 2010. Published by The Company of Biologists Ltd Abnormal hair follicle development and altered cell fate of follicular keratinocytes in transgenic mice expressing DNp63a Rose-Anne Romano1, Kirsten Smalley1, Song Liu2,3 and Satrajit Sinha1,*

SUMMARY 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 DNp63 in hair follicle development and cycling. DNp63 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 DNp63 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. DNp63 transgenic animals display dramatic defects in hair follicle development and cycling, eventually leading to severe hair loss. Strikingly, expression of DNp63 leads to a switch in cell fate of hair follicle keratinocytes, causing them to adopt an interfollicular epidermal (IFE) cell identity. Moreover, DNp63 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/b-catenin signaling pathway, which, in part, account for these effects. Our data provide evidence supporting a role for DNp63a in actively suppressing hair follicle differentiation and directing IFE cell lineage commitment.

KEY WORDS: Differentiation, Hair follicle, Transcription, p63, Mouse

INTRODUCTION continuous with the basal layer of the epidermis. After birth, by The multilayered stratified epithelium of the epidermis develops from postnatal day 16 (P16), the hair follicle reaches full maturation and a single layer of ectoderm progenitor cells through a tightly regulated enters a regressive state known as catagen. At this stage, the lower series of events during embryogenesis. In addition to forming the two thirds of the hair follicle undergoes rapid degeneration through epidermis, a subset of surface ectodermal cells develop into the a process involving apoptosis, where the dermal papilla comes to pilosebaceous units that include the hair follicle and sebaceous gland. rest just below the bulge region. The bulge is the permanent portion In mice, pelage hair follicle morphogenesis is initiated during of the hair follicle and serves as a reservoir for hair follicle stem cells embryonic development at E14.5 and is governed by a series of (Cotsarelis, 2006). It is during the resting telogen stage that the old inductive cues shared between keratinocytes committed to a hair hair shaft is shed from the hair canal. Following telogen, a new cycle follicle cell fate and mesenchymal cells of the underlying dermis of hair regeneration, anagen, is initiated. Not surprising, the complex (Fuchs, 2007). These signaling events result in the formation of local hair development program is governed by several conserved thickenings, or placodes, in the overlying epithelium (Hardy, 1992). signaling pathways including Wnt (Fuchs, 2007; Millar, 2002; Beneath the underlying epidermal thickenings of the placodes, Schneider et al., 2009). mesenchymal cells of the dermis localize, increase in density and Indeed, genetic studies have unequivocally demonstrated the form a cluster that later develops into specialized cells known as the Wnt/b-catenin signaling pathway to be crucial in regulating hair dermal papilla (DP), which are crucial for proliferation of hair follicle follicle morphogenesis. The importance of this pathway is illustrated matrix cells (Jahoda et al., 1984; Panteleyev et al., 2001; Paus et al., by experiments where the coordinate action of b-catenin or its 1999; Reynolds and Jahoda, 1992). nuclear mediators Tcf3 or Lef1 are blocked. Thus, in mice with Reciprocal mesenchymal-epithelial crosstalk continues during the conditional ablation of the Ctnnb1 gene (which encodes for b- early stage of development, stimulating rapid proliferation and catenin) or constitutive expression of the Wnt inhibitor Dkk1, hair down-growth of hair matrix cells to encase the DP. Subsequently, placode formation is severely compromised (Andl et al., 2002; matrix cells differentiate into specialized structures of the hair Huelsken et al., 2001). In agreement with these findings, Lef1-null follicle, which include the inner root sheath (IRS) and hair shaft animals lack hair, whereas transgenic animals that express a compartments of the hair follicle (Panteleyev et al., 2001). The IRS transcriptionally inactive form of Lef1 display altered differentiation is completely surrounded by an outer root sheath (ORS), which is 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/b- 1 2 Department of Biochemistry, and Department of Biostatistics, Center for Excellence catenin signaling pathway in the early epithelial-mesenchymal in Bioinformatics and Life Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA. 3Department of Biostatistics, Roswell Park Cancer Institute, Buffalo, events that specify hair follicle cell fate, initiate hair patterning and NY 14263, USA. direct hair follicle morphogenesis. As with most developmental programs, the signaling events tied to the maturation of hair follicles *Author for correspondence ([email protected]) are intimately associated with a gene expression program dictated

Accepted 25 February 2010 by transcription factors. DEVELOPMENT 1432 RESEARCH ARTICLE Development 137 (9)

The transcription factor p63, a member of the p53 family, plays by Dox chow withdrawal from the lactating mother. For triple transgenic an important role in the development of stratified epithelium of the experiments, DNp63aBG mice were mated to TOPGAL.lacZ mice, in the skin and its appendages. Mice with deletion of the Trp63 gene absence of Dox, to generate TOPGALDNp63aBG animals. exhibit severe developmental abnormalities including limb Western blot truncations and defects in skin epidermal stratification and Western blot analysis was performed as previously described (Romano et differentiation (Mills et al., 1999; Yang et al., 1999). Moreover, these al., 2009). Primary antibodies were used at the following dilutions: HA animals lack ectodermal organs such as teeth, hair follicles and (Roche, 1:5000), b-tubulin (Chemicon, 1:10000), Lef1 (Upstate, 1:2000), glandular structures. Although the p63 knockouts have provided b-catenin (Sigma 1:2000), K15 (Thermo Scientific, 1:2000), Gata3 (Santa valuable insights into understanding epidermal development, thus Cruz, 1:2000), RR-14 (1:5000) and Sox9 (Santa Cruz, 1:2000). far it has not been an ideal model system to study hair follicle morphogenesis. This is primarily owing to the severe developmental Immunostaining Stainings using paraffin embedded sections were performed as previously arrest observed in these animals, including a complete block in described (Romano et al., 2009). Primary antibodies used at the indicated placode formation. Another issue that has complicated studies of dilutions were DNp63 (RR-14; 1:50), p63 (1:50; Santa Cruz, 4A4), K5, K10, p63 is the existence of multiple p63 isoforms, each with potentially K6, loricrin and filaggrin (1:200; gift from Dr Julie Segre, NHGRI, distinct molecular properties. The Trp63 gene encodes for multiple Bethesda, USA), Sox9 (1:50; Santa Cruz), K15 (1:50; Thermo Scientific), functionally distinct protein isoforms, including TAp63 and DNp63, K17 (1:200; gift from Dr Pierre Coulombe, John Hopkins University, New which contain unique N-terminal segments that harbor independent York, USA), AE13/AE15 (1:10; gift from Dr T. T. Sun, New York activation properties (Helton et al., 2008; Yang et al., 1998). In University, New York, USA), Elf5 (1:50, Santa Cruz), Gata3 (1:50, Santa addition, both TA and DN transcripts are differentially spliced at the Cruz), b-catenin (1:100, Santa Cruz), Lef1 (1:100, Upstate), Ki67 (1:100, 3Ј end, generating proteins with unique C-termini designated as a, Novacastra), PCNA (1:50, Dako Cytomation) and S100A6 (1:100, Thermo b and g isoforms. The fact that all isoforms of p63 are absent in the Scientific). When staining with mouse monoclonal antibodies, we used the reagents and protocol from the MOM Basic Kit (Vector Labs). Slides were conventional knockout mouse has thus far precluded studies on the mounted using Vectashield Mounting Medium with DAPI (Vector Labs) and biological role of individual p63 proteins (Barbieri and Pietenpol, viewed with a Nikon FXA fluorescence microscope. Images were captured 2006). This is particularly relevant to the DNp63 isoforms, which using a Nikon digital camera and analyzed using ImageJ, Adobe Photoshop are predominantly expressed in skin epidermal keratinocytes and and Adobe Illustrator software. have recently been shown to direct keratinocyte cell fate by directly regulating the basal keratins K5 and K14 (Candi et al., 2006; b-galactosidase staining Romano et al., 2007; Romano et al., 2009). 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 To investigate the role of DNp63a in hair follicle development, then incubated in 1 mg/ml X-Gal solution [100 mM sodium phosphate buffer we have engineered tetracycline-inducible transgenic animals with (pH 7.3), 0.01% (w/v) sodium deoxycholate, 0.02% (v/v) Nonidet P-40, targeted expression of DNp63a to the ORS of the hair follicle. 2 mM magnesium chloride, 5 mM potassium ferricyanide and 5 mM Interestingly, DNp63 transgenic animals develop severe hair growth potassium ferrocyanide] at 37°C overnight. Samples were then washed in and cycling defects leading to eventual hair loss. Transgenic animals PBS for 20 minutes and post-fixed in 10% NBF for 6 hours and then display a progressive increase in hair follicle size with an expanded immediately dehydrated, paraffin embedded and sectioned to 4 mm thickness. ORS and dramatic defects in differentiation of the matrix cells. Furthermore, mutant hair follicle keratinocytes undergo a switch in Semi-quantitative RT-PCR cell lineage and adopt an interfollicular cell fate. Our results provide Total RNA from dorsal skin of wild-type and BG animals was isolated and purified using TRIzol (Invitrogen) according to established protocols. Two novel insight into the function of DNp63a in regulating various micrograms of total RNA was reverse transcribed using the ThermoScript facets of the hair differentiation program and reveal a key role for RT-PCR System (Invitrogen). PCR amplifications were carried out using several members of the Wnt/b-catenin signaling pathway in the Fermentas Taq DNA Polymerase LC (Fermentas, MD, USA). All primers observed hair phenotype. were designed to span at least one intron. Primer sequences are available upon request. MATERIALS AND METHODS Generation of transgenic animals and animal procedures Microarray analysis The HA-DNp63a construct was generated by cloning the full-length mouse Total RNA was extracted from wild-type and BG skin using TRIzol DNp63a containing a 5Ј HA epitope tag into the pTRE-Tight plasmid (BD (Invitrogen, Carlsbad, CA, USA) and then purified using the RNeasy Bioscience). Transgenic mouse lines were generated by microinjecting the Mini Kit (Qiagen, Valencia, CA, USA). Purified total RNA was analyzed purified DNA construct into fertilized mouse oocytes derived from a mixed on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, genetic background (C3Hf/HeRos ϫ C57BL/10 Rospd). Seven HA- USA) and labeled to obtain biotinylated cRNA for hybridization to DNp63a transgenic founder lines were identified by PCR analysis of tail Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Two independent DNA. The following primers were used to genotype the HA-DNp63 sets of biological replicates (BG and control from P16 dorsal skin) were founders: forward, 5Ј-GGAGAATTCGAGCTCGGTACCCG-3Ј and used. Scanned microarray images were imported into GeneChip reverse, 5Ј-CGCTATTCTGTGCGTGGTCTG-3Ј. The founders were then Operating Software (GCOS, Affymetrix) to generate raw signal values crossed to K5-tTA mice (Diamond et al., 2000) in the absence of Dox to for each probe. The MAS5.0 algorithm in the ‘Affy’ package of determine which of the founders express the transgene. Four founding lines Bioconductor in the R statistical computing environment was used to were identified to express the transgene by western blot analysis. generate expression summary values, followed by trimmed mean global Protocols for mouse experimentation were performed according to SUNY normalization to bring the median expression values of all four at Buffalo and RPCI IACUC protocols. The K5-tTA and TOPGAL.lacZ GeneChips to the same scale (Gentleman et al., 2004). For data quality mice have been previously described (DasGupta and Fuchs, 1999; Diamond control, MAS5.0 present/absent calls were used to filter out probe sets et al., 2000). Mice of appropriate genotype were mated and noon of the day whose expression intensities were close to background noise across the the vaginal plug was observed was considered E0.5. In experiments when majority of samples. Specifically, filtering of two ‘present calls’ was transgene expression was repressed in BG (bi-transgenic) pups, pregnant applied to either the wild-type or BG group, with greater than 13,000 females were administered Dox through rodent chow at a concentration of unique genes passing the quality control. Genes that were altered at a P-

200 mg/kg (Bio-Serv). Transgene expression in BG pups was then induced value less than 0.05 between BG and wild-type populations were DEVELOPMENT p63 and hair development RESEARCH ARTICLE 1433 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 DNp63 expression during hair follicle development and cycling To investigate the role DNp63 plays in hair follicle morphogenesis, we initially sought to establish its expression pattern during normal murine hair follicle development and cycling. DNp63 expression was analyzed using the RR-14 antibody that specifically recognizes the DNp63 isoform of p63 (Romano et al., 2006). We have previously shown that DNp63 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 Fig. 1. DNp63 expression analysis during hair follicle development developing hair placodes (Fig. 1A) (Romano et al., 2009). and cycling. (A,B) At E14.5, expression of DNp63 protein can be detected Expression continues during placode down-growth and, by P1, in the developing placodes and continues to be expressed in the down- DNp63 is restricted to the basal layer of the skin, the ORS and the growing hair germ at E16.5 (B). Dotted lines indicate the epidermal- undifferentiated cells of the matrix region (Fig. 1C) (Rendl et al., dermal junction. (C) By postnatal day 1 (P1), DNp63 is expressed along the 2005). At the telogen stage (P21), in addition to the basal layer, ORS entire length of the outer root sheath (ORS) as well as the matrix cells. and sebaceous gland, DNp63 expression is detected in the bulge (D) At P21 (telogen), DNp63 expression is localized to the basal layer, ORS region of the hair follicle as well as the secondary hair germ, and the bulge region (brackets, insets) of the hair follicle and secondary structures that serve as reservoirs for hair follicle stem cells and are hair germ (Hg). (E,F) Co-staining with K15 and Sox9 reveals co-localization important for the initiation of anagen (Fig. 1D) (Schneider et al., of DNp63 to hair follicle stem cells. Scale bars: 50mm. 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, western blot analysis using skin samples from the DNp63aBG establish that p63 is expressed in the stem cell compartment of the animals and controls. The transgene was expressed in the hair follicle (Fig. 1E,F). DNp63aBG animals, as demonstrated by anti-HA antibodies as well as increased DNp63a levels, as judged by anti-p63 antibodies (Fig. Generation of DNp63a transgenic mice 2B). Owing to the moderate levels of HA-DNp63a in line D and the To investigate the role of DNp63a in hair follicle morphogenesis, fact that animals from this line survived well into the second phase we generated tetracycline-inducible DNp63a transgenic animals. of hair follicle cycling, we focused on this line for the studies These animals were crossed to K5-tTA tet-OFF transgenic mice, described here. which express the tetracycline transactivator (tTA) under the control of the bovine K5 promoter (Diamond et al., 2000). In the absence of Histological analysis of hair development and doxycycline (Dox), K5-tTA/pTRE-DNp63a bi-transgenic animals cycling in DNp63aBG animals (here after referred to as DNp63aBG) express HA-DNp63a in the To analyze the effects of overexpression of DNp63a in hair follicle basal layer of the epidermis and ORS of the hair follicle. Of the cycling, we examined the histology of mid-dorsal skin sections seven transgenic founders, four expressed the transgene with stained with Hematoxylin and Eosin (H&E) from BG and wild-type varying phenotypic severities. DNp63aBG animals corresponding littermates. Although no obvious histological differences were to line D, expressing the lowest levels of the transgene, were observed at the newborn (NB) stage, by P16 DNp63aBG animals indistinguishable at birth compared with wild-type littermates. In displayed a thickened, hyperproliferative epidermis with a dramatic contrast to control animals, line D DNp63aBGs failed to grow a reduction of the granular layer (Fig. 2C, middle panel inset). Hair normal coat, whereas the whiskers appeared to develop normally. follicles from the BG animals appeared hypertrophic and By P16, there were signs of sparse hair growth and the skin of the considerably larger than wild-type control hair follicles (Fig. 2C, BG animals appeared dry and scaly (Fig. 2A). Typically, these middle panel, yellow arrow). Strikingly, the hair shafts of the BG animals survived until approximately P35, after which they were animals were replaced by thick keratinized tissues (middle and euthanized for morbidity and overall poor health. For the second line lower panels, black arrows). These histological data suggested a E, which expressed the transgene at moderate levels, DNp63aBG possible switch of keratinocytes from a hair follicle to the animals were indistinguishable from control littermates at birth. By interfollicular epidermal (IFE) cell fate. In mice, the normal hair P2, however, the skin appeared wrinkled and, by P4, the skin began cycle lasts approximately 28 days, with the onset of catagen to peel and the animals had to be euthanized (see Fig. S1 in the beginning around P16 and telogen starting at P21. The second phase supplementary material). Conversely, the highest expressing lines, of hair growth (anagen) commences at P28 (Muller-Rover et al., B and F DNp63aBGs, displayed an eye open at birth phenotype and 2001). Hair follicles in the DNp63aBG animals failed to enter died shortly after birth. This neonatal lethality could be overcome catagen and, by day P28, remained in anagen, as demonstrated by by Dox administration to the pregnant dams, thereby preventing their continued down-growth into the underlying dermis (Fig. 2C, transgene expression. When Dox was withdrawn during late lower panel). These dramatic changes in the overall appearance of pregnancy, the BG offspring exhibited severe skin and hair the hair follicles and the IFE were a common feature observed in phenotypes within 2-3 weeks (see Fig. S1 in the supplementary all of the DNp63aBG-expressing lines (see Fig. S1 in the

material). To confirm expression of the transgene, we performed supplementary material). DEVELOPMENT 1434 RESEARCH ARTICLE Development 137 (9)

Fig. 2. Gross morphology and histological analysis of DNp63a 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 (a-HA) as well as increased levels of DNp63 (a-DNp63a) as compared with wild-type control littermates at P16. b-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: 75mm in upper and middle panels; 100 mm in lower panels.

Alterations in the keratinocyte differentiation therefore examined the expression of a range of markers involved in program in the IFE of DNp63aBG animals hair follicle differentiation. In mutant skin, K5 and K17 staining The histology shown in Fig. 2 suggested that the normal revealed an expanded ORS as compared with wild-type control differentiation program of the epidermal keratinocytes was disrupted littermates, whereas K6 staining demonstrated an expanded by overexpression of DNp63a in the basal layer of the epidermis. To companion layer in the BG animals (Fig. 4A-F). Differentiation confirm these observations, we analyzed the distribution of a variety of the hair cortex, medulla and cuticle, as assayed by of epidermal-specific markers by staining dorsal skin sections of BG immunofluorescence for the hair keratin proteins (AE13), and control mice at P16. As shown in Fig. 3, keratinocytes from the trichohyalins (AE15) and the transcriptional regulator Elf5 (IRS), skin of BG animals demonstrated significant alterations in the revealed a dramatic reduction in the expression of these markers in expression of these markers. For example, expression of K5, which is the hair follicles of BG animals as compared with control animals normally localized to the proliferating basal layer of the epidermis, (Fig. 4G-L) (Choi et al., 2008; Lynch et al., 1986). These results was expanded in the BG animals, with most of the cellular layers of suggest a defect in the differentiation of these cellular compartments the epidermis staining positive for this keratin marker (Fig. 3A,B). in the mutant follicles. The alterations in the differentiation of the Similar results were obtained for K14 (data not shown). In addition, IRS compartment was further confirmed by a loss of Gata3 when we examined the expression of K1 and K10, markers normally expression (Fig. 4M-N) (Kaufman et al., 2003). 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 DNp63a 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 DNp63a 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 DNp63a 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 DNp63a 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 Fig. 3. Analysis of the interfollicular epidermis. differentiation program. (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. Expansion of the ORS and severely reduced IRS and (G,H) Conversely, there is a dramatic reduction in loricrin expression in the hair shaft compartments in DNp63aBG animals BG animals. (I-L) Evaluation of involucrin and filaggrin expression In light of the histological appearance of the epidermis, the hair demonstrates increased expression levels in the BG IFE as compared with follicles and the apparent paucity of hair in the DNp63aBG animals, wild-type animals. Dotted lines indicate the epidermal-dermal junction.

we next wanted to probe the extent of follicular defects. We Scale bar: 75mm. DEVELOPMENT p63 and hair development RESEARCH ARTICLE 1435

Fig. 4. Alterations in the hair follicle differentiation program of DNp63aBG animals. (A-R) Immunofluorescence staining used to detect changes in the hair follicle differentiation program of DNp63aBG animals at P13. Antibodies used for each staining are shown in lower left panels. Scale bars: 100mm in C-H; 75 mm in A,B,I-L,O,P; 60mm in Q,R; 50mm in M,N.

The canonical Wnt/wingless signaling pathway has been shown to expression of K5 and K14 is severely attenuated in p63-null animals be necessary for the formation of many ectodermal organs, including (Mills et al., 1999; Yang et al., 1999). Given this correlation and the the hair follicle (Andl et al., 2002). Given the importance of b-catenin histological appearance of the skin, we investigated the possibility and Lef1 in hair follicle differentiation and hair cycling, we that overexpression of DNp63 in the ORS could alter the inherent investigated the expression of these proteins in the mutant hair cellular identity and initiate an epidermal differentiation program in follicles of the transgenic animals. In control anagen hair follicles at hair follicle keratinocytes. Surprisingly, cytokeratin markers K1 and P13, b-catenin is expressed in most cellular layers of the hair follicle, K10, which in wild-type skin are restricted to the spinous layer of while Lef1 expression is restricted to the precortex region (Fig. 4O,Q) the epidermis and the infundibulum, were expressed in the (Merrill et al., 2001). However, in the DNp63aBG hair follicles, there keratinocytes of the hair follicles of transgenic animals as is a dramatic reduction in the expression of both b-catenin and Lef1 demonstrated by double staining with K14 (Fig. 6, top panel; data (Fig. 4P,R). The loss of Lef1 expression, especially in the precortex not shown). Similarly, filaggrin, a marker normally expressed in the region of the BG hair follicles, might be causally associated with the terminally differentiated layer of the epidermis, was expressed in the blocked differentiation of both the IRS and hair shaft compartments. hair follicle keratinocytes of the BG animals (Fig. 6, lower panel). The reduced expression levels of some of these crucial markers of the The misplaced expression of these IFE differentiation markers in the hair follicles, such as Gata3 and Lef1, was also evident by western blot DNp63aBG animals was widespread, extending into the lower analysis (see Fig. S2 in the supplementary material). portion of the hair follicle near the bulb. These data suggest the possibility that elevated levels of DNp63a in the ORS of the hair Decrease in matrix cell proliferation results in a follicle results in a switch of hair follicle keratinocytes to adopt an block in IRS and hair shaft development IFE cell fate, thus affecting hair follicle development and cycling in Given the observed defects in the development of the IRS, cortex and the mutant animals. medulla in the DNp63aBG 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 DNp63a 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 Fig. 5. Reduced matrix cell proliferation in the hair follicles of follicles expressing DNp63a DNp63aBG animals. Dorsal skin sections from P13 were stained with DNp63 can directly regulate the expression of K5 and K14, two Ki67 (upper panel) and PCNA (lower panel). Matrix cells of BG hair follicles markers coinciding with the initiation of the stratification program show reduced numbers of proliferating cells as compared with control

in the skin epidermis (Romano et al., 2009). Furthermore, littermates (arrows). Scale bar: 75mm. DEVELOPMENT 1436 RESEARCH ARTICLE Development 137 (9)

Fig. 7. Forced expression of DNp63a results in hair follicle stem cell Fig. 6. Follicular keratinocyte transformation in BG animals. depletion. Dorsal skin sections from P21 were stained with various stem Overexpression of DNp63a results in the transition of hair follicle cell markers of the hair follicle bulge. As compared with wild-type control keratinocytes to adopt an IFE cell fate. Dorsal skin sections from P28 were hair follicles, follicles of the BG animal show a complete loss of K15 stained with K1 and filaggrin, markers of the IFE. Compared with wild- (green), Sox9 (red) and S100A6 (red) expression, suggesting a depletion of type hair follicles, BG hair follicles express K1 and filaggrin. A higher hair follicle stem cells of the bulge. A higher magnification is shown in the magnification is shown in the insets. Arrows indicate cells expressing both insets. Scale bar: 100mm. K14/K1 or K14/Fil respectively. All sections are stained with K14 (red), K1 or Fil (green), and DAPI (blue). Scale bar: 75mm.

Sustained activation of DNp63a stimulates a downregulation as compared with wild-type (see Table S1 in the depletion of hair follicle stem cells supplementary material). Indeed, there were major alterations in the In mouse skin, the bulge serves as an important reservoir of stem expression of many structural genes and crucial components of cells necessary for the cyclic bouts of degeneration (catagen) and several signaling pathways demonstrated to be important regulators regeneration (anagen) occurring in mammalian hair follicles of epidermal differentiation and hair follicle morphogenesis and (Cotsarelis, 2006). Given the apparent failure of hair follicles to cycling (a heat map of selected genes is shown in Fig. 8). undergo proper hair follicle cycling in the BG animals, we In support of the defects observed in the hair shaft of the BG investigated the status of the stem cell compartment in these animals. animals and the results of the immunostaining experiments for hair To determine the effects of sustained expression of DNp63a on hair keratin proteins and trichohyalins, transcript levels of various hair follicle stem cells, we analyzed the expression of the stem cell shaft-specific keratins and keratin-associated proteins were markers K15, Sox9 and S100-A6. In contrast to wild-type hair dramatically downregulated in the mutant skin (Fig. 8). These data follicles, which express robust levels of K15, there is a loss of K15 were confirmed for selected hair keratin genes by semi-quantitative expression in the hair follicles of the transgenic animals (Fig. 7, left RT-PCR (Krt25, Krt28 and Krt31; see Fig. S3A in the panel). Similarly, we observed a loss of both Sox9 and S100-A6 supplementary material). Also significantly downregulated were expression (Fig. 7, middle and right panel) in transgenic follicles at numerous genes expressed in hair shaft precursor cells and genes P21, suggesting a loss of follicular stem cells. Western blot analysis required for hair shaft differentiation. Several hair follicle stem- further confirmed a dramatic reduction in the protein expression cell-specific genes such as Krt15, Sox9 and Lhx2 were also levels of both K15 and Sox9 (see Fig. S2 in the supplementary downregulated in the mutant skin as compared with the control, material). Taken together, these results suggest that forced consistent with our immunofluorescence and western blot analysis. expression of DNp63a in the ORS causes a depletion of the hair In agreement with immunofluorescence experiments, the follicle stem cell niche, indicating an important role for this isoform microarray analysis revealed a dramatic reduction in some of the in bulge stem-cell maintenance. genes involved in the Wnt/b-catenin signaling pathway (Fig. 8). Semi-quantitative RT-PCR confirmed reduced levels of Lef1, Global changes in gene expression patterns in Wnt5a, Wnt11 and Tcf3 transcripts, suggesting an alteration in the DNp63a mutant skin Wnt/b-catenin signaling pathway (Fig. 9A). As the microarray To understand the molecular mechanisms underlying the dramatic analysis was performed using skin samples from P16, it is possible defects observed in mutant hair follicle development and cycling, that the downregulation of Wnt/b-catenin signaling was not an we next evaluated global changes in gene expression by performing immediate and/or direct effect of DNp63a overexpression. To test transcriptional profiling of transgenic and control dorsal skin at P16. this, we performed semi-quantitative RT-PCR using RNA isolated In agreement with the dramatic phenotype observed in the BG from BG animals after a short induction of DNp63a. Skin samples animals, the microarray analysis revealed major changes in the analyzed from E18.5 BG animals that were induced during transcriptional profile of BG skin, with 705 genes displaying at least embryogenesis revealed reduced Lef1, Tcf3 and Wnt5 levels, but not

a 2-fold upregulation and 1352 genes showing at least a 2-fold Wnt11 (Fig. 9A). The fact that the expression of at least some of the DEVELOPMENT p63 and hair development RESEARCH ARTICLE 1437

Fig. 8. Altered hair-follicle-specific genes and signaling pathways in DNp63a 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 DNp63a 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.

crucial genes belonging to the Wnt pathway were altered in the Closer examination using specific markers reveals a failure of the DNp63a embryonic skin suggests that downregulation of this mutant follicles to undergo proper differentiation of the IRS and hair pathway occurs prior to the development of the skin phenotype, shaft compartments as evidenced by the loss of expression of several rather than merely as a consequence of botched hair follicle hair-specific keratins and IRS markers. Interestingly, we also differentiation. Conversely, we did not observe any significant observed a reduction in the proliferation of matrix cells, which give changes in b-catenin transcript levels at any of the time points that rise to both the IRS and hair shaft compartments. Unexpectedly, the were examined (data not shown). Microarray analysis also keratinocytes within the hair follicle adopted a new cell fate, demonstrated a downregulation of some of the members of the Shh expressing markers of the IFE rather than hair. The propensity of pathway (Fig. 8). This is not surprising, given the intimate epithelial cells that express higher levels of DNp63a to undergo connection between the Shh pathway and hair follicle development squamous metaplasia is in agreement with our previous studies (Callahan and Oro, 2001). Altered expression of members of the Shh where DNp63a overexpression in simple lung epithelium converted pathway in the BG animals was confirmed by semi-quantitative RT- PCR and results are shown in Fig. S3B in the supplementary material. 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 DNp63aBG animals to the TOPGAL animals to generate triple transgenic animals (TOPGALDNp63aBG). 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/b-catenin signaling, possibly resulting from reduced Lef1 expression levels (Fig. 9B). Fig. 9. Loss of Wnt/b-catenin signaling in DNp63a animals. (A) R T- DISCUSSION PCR analysis of mRNA transcripts from wild-type and BG animals. Semi- Given the dynamic expression pattern of DNp63 in the hair follicle, quantitative RT-PCR reveals a dramatic downregulation in the levels of we wanted to investigate the role of this isoform in hair follicle several genes belonging to the Wnt/b-catenin signaling pathway at P16 and E18.5. (B) TOPGAL and TOPGALDNp63aBG dorsal skin sections at morphogenesis, an area that has received little attention thus far. P13 were stained for lacZ expression and counter-stained with Eosin. With that goal in mind, we have generated a Tet-inducible system Staining reveals Wnt/b-catenin activity in the lower portion of the hair that allows expression of DNp63a in a temporal-spatial fashion. We shaft and matrix region of the TOPGAL animals (left panel). lacZ expression find that targeted expression of DNp63a to the ORS of the hair in TOPGALDNp63aBG hair follicles is dramatically reduced (right panel).

follicle results in enlarged hair follicles with an expanded ORS. Scale bar: 50mm. DEVELOPMENT 1438 RESEARCH ARTICLE Development 137 (9) these cells to a stratified state with ectopic expression of basal and reduced levels of b-catenin in the hair follicles of our mutant mice. suprabasal keratinocyte markers that are normally associated with Therefore, it is possible that the reduced b-catenin and Lef1 levels the IFE (Romano et al., 2009). in the mutant hair follicles reprogram the hair follicle keratinocytes Using global transcriptional analysis, we identified significant to an IFE cell fate, similar to what has been reported in b-catenin alterations to genes involved in Wnt/b-catenin signaling in the BG ablated mice (Huelsken et al., 2001). animals as compared with wild-type control littermates. In The capacity of hair follicles to maintain and activate a program addition to Wnt5a, we detected reduced levels of Tcf3, a member of self-renewal is primarily dependent on stem cells located within of the Lef/Tcf family of HMG domain-containing DNA-binding the bulge region of the hair follicle. Bulge stem cells have been proteins. This is of particular interest considering the recently shown to express several markers including Krt15, Sox9, S100A6 described roles for Tcf3 as well as another family member, Tcf4, and Lhx2 (Fuchs, 2007). Interestingly, our transgenic animals in hair follicle stem cell maintenance (Nguyen et al., 2009). demonstrate a depletion of the stem cell compartment, with reduced Indeed, Tcf3–Tcf4 compound homozygous animals initiate hair expression levels of all of the aforementioned genes. Recently, Sox9 follicle development, but display defects in hair follicle stem cell has been implicated in hair follicle morphogenesis as well as in the maintenance and follicle down-growth. Given the attenuated levels formation of the hair follicle stem cell compartment (Nowak et al., of Tcf3 in our BG animals, it is plausible that Tcf3 could be one 2008; Vidal et al., 2005). In addition, in Sox9-null animals, the ORS contributing factor in the loss of the hair follicle stem cell niche of the hair follicles acquire epidermal characteristics, a feature observed in these animals. In addition to Wnt5a and Tcf3, we similar to the phenotype observed in our transgenic animals. found Lef1 levels to be considerably downregulated in the hair Although there is no experimental evidence supporting a role for p63 follicles of the BG animals at both the mRNA transcript and in directly regulating the expression of Sox9, it is interesting to note protein levels. This was surprising given the existing data showing that siRNA-mediated knockdown of DNp63 isoforms in human a putative role for p63 as a direct and positive transcriptional keratinocytes leads to a strong upregulation of Sox9, suggesting that activator of Lef1 (Pozzi et al., 2009). However, this observation is DNp63 might be a negative regulator of Sox9 (Truong et al., 2006). based solely on knockdown studies in a transformed human Intriguingly, studies with p63 knockout animals have clearly shown keratinocyte cell line and thus could reflect context-dependent that p63 is required for the high proliferative potential and self- variability or isoform-specific effects. Alternatively, it is possible renewal of epithelial stem cells (Blanpain and Fuchs, 2007; Senoo that the downregulation of Lef1 in the DNp63aBG animals et al., 2007). It is possible that the seemingly opposite effects of represents an indirect effect. In the future, a detailed examination overexpression of DNp63a on stem cell behavior in the transgenic of the potential interaction of p63 with the regulatory elements of animals described in this study might reflect an imbalance of p63 Lef1 in the in vivo context is warranted to resolve this discrepancy isoforms or secondary effects due to global signaling changes. and address the underlying mechanism. In summary, we have shown that targeted overexpression of The hair follicle phenotype of the transgenic animals is in good DNp63a to the ORS of the hair follicle leads to a loss of both the IRS agreement with our microarray data, which revealed reduced levels and hair shaft compartments, resulting in progressive hair loss in of many hair-specific keratin genes and hair shaft- and IRS-specific mutant animals. We posit the hair follicle defects to be primarily genes in the mutant animals. Moreover, crossing our BG animals to attributed to a loss of Wnt/b-catenin and other signaling molecules the well-characterized TOPGAL transgenic mice confirmed a loss as supported by global transcriptome analysis. These data suggest of Wnt signaling in the mutant hair follicles, suggesting that the an important role for DNp63a in hair follicle morphogenesis and observed defects in the IRS and hair shaft compartments are a direct hair shaft differentiation. Given the distinctive expression pattern of result of reduced Wnt signaling. We posit that the hair abnormalities DNp63 in the developing placodes, it is very probable that this might be further augmented by other key regulators of hair shaft transcription factor is crucial in the early stages of hair follicle differentiation including Dlx3 and Runx1, both of which are development. In mature hair follicles, DNp63 expression remains established p63 target genes and show dramatic downregulation in restricted to the bulge and matrix regions where it might play an the transgenic animals based on microarray data (Hwang et al., important role in stem cell maintenance and self-renewal and in 2008; Ortt et al., 2008; Osorio et al., 2008; Raveh et al., 2006). balancing the proliferation and differentiation of matrix cells, Indeed, a careful evaluation of the microarray data shows significant respectively. The definitive analysis of the roles for DNp63 in hair alterations in a number of genes associated with additional signaling follicle biology awaits the development of new tools and strategies, pathways [insulin growth factor-binding protein 5 (Igfbp5) and including an isoform-specific knockout. fibroblast growth factor 5 (Fgf5) for example], which might, in part, contribute to some aspects of the observed BG hair phenotype such Acknowledgements We thank Dr Adam Glick for generously providing the K5-tTA animals. We are as the cycling defects (Hebert et al., 1994; Schlake, 2005; Schneider especially grateful to Irene Kulik for technical assistance and past and present et al., 2009). members of our laboratory for useful comments on this study. This work was It is plausible that the apparent transformation of hair follicle supported by a grant from NIH R01AR049238 to S.S. Deposited in PMC for keratinocytes to adopt an IFE cell fate is most probably the result of release after 12 months. reduced Wnt/b-catenin signaling as this phenotype recapitulates Competing interests statement some of the changes described in mouse model studies where b- The authors declare no competing financial interests. catenin levels have been augmented using either gain-of-function or loss-of-function approaches (Zhang et al., 2008). Knockout and Supplementary material transgenic mouse model studies have shown that mice with targeted Supplementary material for this article is available at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.045427/-/DC1 deletion of Ctnnb1 fail to develop hair follicles, whereas mice expressing an activated form of b-catenin result in the programming References of embryonic epidermis to a hair follicle cell fate. Overall, these data Andl, T., Reddy, S. T., Gaddapara, T. and Millar, S. E. (2002). 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