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.
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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.
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
1
Department of Biochemistry, and 2Department of Biostatistics, Center for Excellence
in Bioinformatics and Life Sciences, State University of New York at Buffalo, Buffalo,
NY 14203, USA. 3Department of Biostatistics, Roswell Park Cancer Institute, Buffalo,
NY 14263, USA.
*Author for correspondence ([email protected])
Accepted 25 February 2010
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/b-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 b-catenin or its
nuclear mediators Tcf3 or Lef1 are blocked. Thus, in mice with
conditional ablation of the Ctnnb1 gene (which encodes for bcatenin) 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/bcatenin 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.
DEVELOPMENT
KEY WORDS: Differentiation, Hair follicle, Transcription, p63, Mouse
1432 RESEARCH ARTICLE
MATERIALS AND METHODS
Generation of transgenic animals and animal procedures
The HA-DNp63a construct was generated by cloning the full-length mouse
DNp63a 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 HADNp63a transgenic founder lines were identified by PCR analysis of tail
DNA. The following primers were used to genotype the HA-DNp63
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, DNp63aBG mice were mated to TOPGAL.lacZ mice, in the
absence of Dox, to generate TOPGALDNp63aBG 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), b-tubulin (Chemicon, 1:10000), Lef1 (Upstate, 1:2000),
b-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 DNp63 (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), b-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.
b-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 mm 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 Pvalue less than 0.05 between BG and wild-type populations were
DEVELOPMENT
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 DNp63,
which contain unique N-terminal segments that harbor independent
activation properties (Helton et al., 2008; Yang et al., 1998). In
addition, both TA and DN transcripts are differentially spliced at the
3⬘ end, generating proteins with unique C-termini designated as a,
b and g 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 DNp63 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 DNp63a in hair follicle development,
we have engineered tetracycline-inducible transgenic animals with
targeted expression of DNp63a to the ORS of the hair follicle.
Interestingly, DNp63 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 DNp63a in regulating various
facets of the hair differentiation program and reveal a key role for
several members of the Wnt/b-catenin signaling pathway in the
observed hair phenotype.
Development 137 (9)
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.
Generation of DNp63a transgenic mice
To investigate the role of DNp63a in hair follicle morphogenesis,
we generated tetracycline-inducible DNp63a 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-DNp63a bi-transgenic animals
(here after referred to as DNp63aBG) express HA-DNp63a 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. DNp63aBG 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 DNp63aBGs 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, DNp63aBG
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 DNp63aBGs, 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
Fig. 1. DNp63 expression analysis during hair follicle development
and cycling. (A,B) At E14.5, expression of DNp63 protein can be detected
in the developing placodes and continues to be expressed in the downgrowing hair germ at E16.5 (B). Dotted lines indicate the epidermaldermal junction. (C) By postnatal day 1 (P1), DNp63 is expressed along the
entire length of the outer root sheath (ORS) as well as the matrix cells.
(D) At P21 (telogen), DNp63 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 DNp63 to hair follicle stem cells. Scale bars: 50mm.
western blot analysis using skin samples from the DNp63aBG
animals and controls. The transgene was expressed in the
DNp63aBG animals, as demonstrated by anti-HA antibodies as well
as increased DNp63a levels, as judged by anti-p63 antibodies (Fig.
2B). Owing to the moderate levels of HA-DNp63a 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.
Histological analysis of hair development and
cycling in DNp63aBG animals
To analyze the effects of overexpression of DNp63a 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 DNp63aBG 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 DNp63aBG 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 DNp63aBG-expressing lines (see Fig. S1 in the
supplementary material).
DEVELOPMENT
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
developing hair placodes (Fig. 1A) (Romano et al., 2009).
Expression continues during placode down-growth and, by P1,
DNp63 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, DNp63 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).
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 timepoints 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: 75 mm in upper and middle panels;
100 mm in lower panels.
Expansion of the ORS and severely reduced IRS and
hair shaft compartments in DNp63aBG animals
In light of the histological appearance of the epidermis, the hair
follicles and the apparent paucity of hair in the DNp63aBG 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).
Fig. 3. 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: 75 mm.
DEVELOPMENT
Alterations in the keratinocyte differentiation
program in the IFE of DNp63aBG animals
The histology shown in Fig. 2 suggested that the normal
differentiation program of the epidermal keratinocytes was disrupted
by overexpression of DNp63a 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 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. 3GL). 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
differentiation program.
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: 100 mm in C-H;
75 mm in A,B,I-L,O,P; 60 mm in Q,R; 50 mm in M,N.
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 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
follicles expressing DNp63a
DNp63 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 DNp63 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
DNp63aBG animals was widespread, extending into the lower
portion of the hair follicle near the bulb. These data suggest the
possibility that elevated levels of DNp63a 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.
Fig. 5. Reduced matrix cell proliferation in the hair follicles of
DNp63aBG 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 mm.
DEVELOPMENT
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 b-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, b-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 DNp63aBG hair follicles, there
is a dramatic reduction in the expression of both b-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).
Fig. 6. Follicular keratinocyte transformation in BG animals.
Overexpression of DNp63a 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 wildtype 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: 75mm.
Sustained activation of DNp63a 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 DNp63a 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 DNp63a 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
DNp63a 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
Development 137 (9)
Fig. 7. Forced expression of DNp63a 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 mm.
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).
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 stemcell-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/b-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/b-catenin signaling pathway (Fig. 9A). As the microarray
analysis was performed using skin samples from P16, it is possible
that the downregulation of Wnt/b-catenin signaling was not an
immediate and/or direct effect of DNp63a overexpression. To test
this, we performed semi-quantitative RT-PCR using RNA isolated
from BG animals after a short induction of DNp63a. 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
DEVELOPMENT
1436 RESEARCH ARTICLE
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.
DISCUSSION
Given the dynamic expression pattern of DNp63 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 DNp63a in a temporal-spatial fashion. We
find that targeted expression of DNp63a 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 DNp63a to undergo
squamous metaplasia is in agreement with our previous studies
where DNp63a overexpression in simple lung epithelium converted
Fig. 9. Loss of Wnt/b-catenin signaling in DNp63a animals. (A) RTPCR analysis of mRNA transcripts from wild-type and BG animals. Semiquantitative RT-PCR reveals a dramatic downregulation in the levels of
several genes belonging to the Wnt/b-catenin signaling pathway at P16
and E18.5. (B) TOPGAL and TOPGALDNp63aBG dorsal skin sections at
P13 were stained for lacZ expression and counter-stained with Eosin.
Staining reveals Wnt/b-catenin activity in the lower portion of the hair
shaft and matrix region of the TOPGAL animals (left panel). lacZ expression
in TOPGALDNp63aBG hair follicles is dramatically reduced (right panel).
Scale bar: 50 mm.
DEVELOPMENT
crucial genes belonging to the Wnt pathway were altered in the
DNp63a 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 b-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 RTPCR 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).
1438 RESEARCH ARTICLE
reduced levels of b-catenin in the hair follicles of our mutant mice.
Therefore, it is possible that the reduced b-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 b-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 DNp63 isoforms in human
keratinocytes leads to a strong upregulation of Sox9, suggesting that
DNp63 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 selfrenewal of epithelial stem cells (Blanpain and Fuchs, 2007; Senoo
et al., 2007). It is possible that the seemingly opposite effects of
overexpression of DNp63a 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
DNp63a 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/b-catenin and other signaling molecules
as supported by global transcriptome analysis. These data suggest
an important role for DNp63a in hair follicle morphogenesis and
hair shaft differentiation. Given the distinctive expression pattern of
DNp63 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, DNp63 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 DNp63 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.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.045427/-/DC1
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p63 and hair development