RESEARCH ARTICLE
743
Development 135, 743-753 (2008) doi:10.1242/dev.006718
P-cadherin is a p63 target gene with a crucial role in the
developing human limb bud and hair follicle
Yutaka Shimomura1, Muhammad Wajid1, Lawrence Shapiro2 and Angela M. Christiano1,3,*
P-cadherin is a member of the classical cadherin family that forms the transmembrane core of adherens junctions. Recently,
mutations in the P-cadherin gene (CDH3) have been shown to cause two inherited diseases in humans: hypotrichosis with juvenile
macular dystrophy (HJMD) and ectodermal dysplasia, ectrodactyly, macular dystrophy (EEM syndrome). The common features of
both diseases are sparse hair and macular dystrophy of the retina, while only EEM syndrome shows the additional finding of split
hand/foot malformation (SHFM). We identified five consanguineous Pakistani families with either HJMD or EEM syndrome, and
detected pathogenic mutations in the CDH3 gene of all five families. In order to define the role of P-cadherin in hair follicle and
limb development, we performed expression studies on P-cadherin in the mouse embryo, and demonstrated the predominant
expression of P-cadherin not only in the hair follicle placode, but also at the apical ectodermal ridge (AER) of the limb bud. Based
on the evidence that mutations in the p63 gene also result in hypotrichosis and SHFM, and that the expression patterns of p63 and
P-cadherin overlap in the hair follicle placode and AER, we postulated that CDH3 could be a direct transcriptional target gene of
p63. We performed promoter assays and ChIP, which revealed that p63 directly interacts with two distinct regions of the CDH3
promoter. We conclude that P-cadherin is a newly defined transcriptional target gene of p63, with a crucial role in hair follicle
morphogenesis as well as the AER during limb bud outgrowth in humans, whereas it is not required for either in mice.
INTRODUCTION
The classical cadherins are a major structural component of
adherens junctions in many tissues. Previous studies have shown the
involvement of the classical cadherins in many biological processes,
such as cell recognition, cell signaling, morphogenesis and tumor
development (Goodwin and Yap, 2004). Recently, two members of
the classical cadherin family, E-cadherin and P-cadherin, encoded
by the genes CDH1 and CDH3, have been shown to be involved in
hair follicle (HF) morphogenesis (Jamora et al., 2003). HF
development begins with the formation of a placode, which is
composed of groups of rearranged epidermal cells, and subsequent
formation of mesenchymal condensate just beneath the placode. The
interaction between these specific structures further induces the
downgrowth of the placode (Schmidt-Ullrich and Paus, 2005). In the
epidermis, E-cadherin is abundantly expressed in the basal and
suprabasal layers, while P-cadherin is expressed only weakly in the
basal layer (Hirai et al., 1989). As the HF placode begins to form,
the expression level of E-cadherin markedly decreases, whereas that
of P-cadherin becomes much stronger in the placode than in the
interfollicular epidermis (Jamora et al., 2003). The downregulation
of E-cadherin, as well as the predominant expression of P-cadherin,
continues throughout HF development (Muller-Rover et al., 1999).
This ‘cadherin switch’ has been considered to be essential to change
cell-cell contacts and potentially facilitate cell movement (Jamora
et al., 2003).
1
Department of Dermatology, Columbia University, New York, NY 10032, USA.
Departments of Biochemistry and Molecular Biophysics, and Ophthalmology,
Columbia University, New York, NY 10032, USA. 3Department of Genetics and
Development, Columbia University, New York, NY 10032, USA.
2
*Author for correspondence (e-mail: [email protected])
Accepted 15 November 2007
The importance of P-cadherin in HF morphogenesis has been
further highlighted by recent evidence that human P-cadherin
mutations are associated with two congenital diseases affecting the
HF. Recessively inherited mutations in the human CDH3 gene were
identified in affected individuals with hypotrichosis with juvenile
macular dystrophy (HJMD) characterized by congenital sparse hair
and early blindness due to macular dystrophy of the retina (Sprecher
et al., 2001; Indelman et al., 2002; Indelman et al., 2003; Indelman
et al., 2005; Indelman et al., 2007). More recently, mutations in the
CDH3 were also reported to cause another autosomal recessive
human disease in two families: ectodermal dysplasia, ectrodactyly
and macular dystrophy (EEM syndrome) (Kjaer et al., 2005).
Patients with EEM syndrome exhibit hair and eye symptoms similar
to those with HJMD; however, they show the additional phenotype
of a severe split hand/foot malformation (SHFM) or ectrodactyly.
Previous studies have clearly demonstrated the expression of Pcadherin in the HF placode (Jamora et al., 2003) and retinal
pigmented epithelium (RPE) of the eye (Xu et al., 2002) during
mouse embryogenesis, which corresponds to the sites of the human
phenotype caused by dysfunction of P-cadherin. To date, however,
the precise expression of P-cadherin during limb formation has not
been determined. The development of the HF and limb bud share
some characteristic features. Several common molecules such as
Wnt, sonic hedgehog and bone morphogenic proteins contribute to
generate both structures (Duijf et al., 2003). Morphologically, the
apical ectodermal ridge (AER), which is the thickened rim of
ectoderm at the tip of limb bud, may correspond in some ways to the
HF placode. The AER mediates limb bud outgrowth via the
interaction with underlying mesenchyme (Sanz-Ezquerro and
Tickle, 2001).
Interestingly, the only other gene known to cause SHFM in
humans is p63. Mice that lack p63 have striking developmental
defects and die shortly after birth from dehydration. They lack
stratified squamous epithelia and associated appendages (Mills et
al., 1999; Yang et al., 1999). They also display severe defects in limb
DEVELOPMENT
KEY WORDS: P-cadherin, p63, Apical ectodermal ridge, Hair follicle placode
RESEARCH ARTICLE
and craniofacial development, including cleft lip. Humans that
possess dominant mutations in p63 similarly have dramatic
developmental defects, depending on the location of the mutation
within the p63 gene. One example is ectrodactyly-ectodermal
dysplasia-cleft lip/palate (EEC) syndrome. Most patients who are
affected with EEC have mutations that abolish the ability of p63 to
bind to DNA (Celli et al., 1999). Other syndromes that result from
mutations in p63 include ankyloblepharon-ectodermal dysplasiaclefting (AEC) syndrome, acro-dermato-ungual-lacrimal-tooth
syndrome, non-syndromic SHFM and limb-mammary syndrome
(Rinne et al., 2006). All of these syndromes are autosomal dominant
and can exhibit SHFM, with the exception of AEC.
The transcription factor p63 is primarily expressed in epithelia. It
was first identified as a member of the p53 family (Augustin et al.,
1998; Yang et al., 1998; Osada et al., 1998). p53 is a tumor
suppressor that is mutated in many human cancers. Although there
is a high degree of similarity in sequence and structure between p53
and p63, they are functionally divergent. p63 can be expressed from
two different promoters and transcriptional start sites, generating the
TA and ⌬N isoforms of p63. Each isoform has three additional splice
variants, designated ␣, ␤ and ␥ (Yang et al., 1998). All p63 proteins
possess a DNA-binding domain, which is 60% identical to that of
p53. The TAp63 isoforms additionally have an N-terminal
transactivation region, which is only weakly homologous (22%) to
that of p53. The ⌬Np63 isoforms are truncated in this region. The
diversity of isoforms, splice variants and expression suggests
complexity in the function of p63.
The role of p63 during development is incompletely understood.
The two p63 isoforms are differentially expressed in embryonic
mouse epidermis. One model is that the TAp63 isoform, which is
expressed earlier in development, has a role in the commitment to
epithelial stratification, whereas the ⌬Np63 is involved in terminal
differentiation (Koster et al., 2004; Koster et al., 2005). Although
the expression patterns of p63 in normal epithelia as well as in
epithelial tumors have been determined in part (Nylander et al.,
2000; Nylander et al., 2002; Di Como et al., 2002), the targets of p63
regulation have remained largely elusive. Recently, however, several
studies using microarrays, chromatin immunoprecipitation and/or
RNA interference approaches have identified several candidate p63target genes, such as EGFR, ICAM3 and ␤4 integrin (Carroll et al.,
2006; Barbieri et al., 2006; Vigano et al., 2006; Yang et al., 2006;
Testoni et al., 2006). Interestingly, these studies have gradually
disclosed that p63 regulates gene expression programs involved in
cell adhesion (Carroll et al., 2006).
p63 and CDH3 are the only genes that have been known to be
responsible for SHFM in humans. At present, the CDH3 mutations
reported so far are limited, and the genes that control the expression
of P-cadherin remain unknown. The phenotypic overlap led us to
hypothesize that p63 might be a transcriptional regulator of CDH3.
In this study, we identified pathogenic mutations in the CDH3 of five
consanguineous Pakistani families affected with either HJMD or
EEM syndrome. In addition, we determined the expression pattern
of P-cadherin during hair and limb development in the mouse
embryo. Finally, we show that P-cadherin is a direct transcriptional
target gene of p63.
MATERIALS AND METHODS
Mutation analysis of the CDH3 gene
After obtaining informed consent, we collected peripheral blood samples from
members of five Pakistani families (Families 1-5) (under institutional approval
and in adherence to the Declaration of Helsinki Principles). Genomic DNA
was isolated from these samples according to standard techniques. Using
Development 135 (4)
genomic DNA and primers described previously (Kjaer et al., 2005), all exons
of CDH3 with adjacent sequences of exon-intron borders were amplified by
PCR. The PCR products were directly sequenced on an ABI Prism 310
Automated Sequencer, using the ABI Prism BigDye Terminator Cycle
Sequencing Ready Reaction Kit (PE Applied Biosystems).
Indirect immunofluorescence and whole-mount
immunohistochemistry
Cryosections were prepared from C57BL/6J embryos at E9.5 (9.5 days post
coitus, day of plug considered 0.5 days), E10.5, E15.5 and E17.5. Sections
were fixed in cold methanol:acetone (1:1) for 7 minutes, followed by
blocking with 10% normal goat serum in PBS for 1 hour at room
temperature. Primary antibodies used were anti-P-cadherin rat monoclonal
PCD-1 (1:100; ZYMED Laboratories), anti-p63 rabbit polyclonal H-129
(1:200; Santa Cruz Biotechnology), anti-N-cadherin rabbit polyclonal H-63
(1:200; Santa Cruz Biotechnology) and mouse monoclonal anti-E-cadherin
(1:200; BD Biosciences). After the incubation with primary antibodies,
FITC and/or Alexa-Fluor-labeled secondary antibodies (Jackson
ImmunoResearch) were applied to the sections. Nuclei were counterstained
with DAPI. The images were processed with Adobe Photoshop (version 8.0)
software. Whole-mount immunohistochemistry was performed as
previously described (Yoshida et al., 1996) with some modifications.
C57BL/6J embryos were incubated with anti-P-cadherin antibody PCD-1
(1:100), and subsequently with 1 ␮g/ml HRP-conjugated anti-rat-IgG
antibody. Antibody binding was visualized with diaminobenzidine substrate.
p63-expression vectors
Expression constructs encoding c-myc-tagged mouse TAp63␣, TAp63␥,
⌬Np63␣ and ⌬Np63␥ were kindly provided by Drs Dennis Roop and
Maranke Koster (University of Colorado, Aurora, Colorado, USA). To
generate the mutant (R280C) TAp63␥, the upstream coding region of
TAp63␥ was PCR-amplified using the forward primer (5⬘AAAAGGATCCCTCGCAGAGCACCCAGACAAG-3⬘) and the reverse
primer (5⬘-ATGATTAAAATTGGACATCTGTTCA-3⬘) and wild-type
TAp63␥ expression construct as a template. Note that the mutation of
interest was introduced into the reverse primer as underlined. The remaining
coding region of TAp63␥ was also amplified by PCR using the forward
primer (5⬘-AATTTTAATCATCGTTACTCTGGAAAC-3⬘) and the reverse
primer (5⬘-AAAACTCGAGCTATGGGTACACGGAGTGGT-3⬘). The two
amplified fragments were then ligated at the MseI restriction enzyme site,
which was subcloned in frame into the BamHI and XhoI sites of pCMV-Tag3
vector (Stratagene).
Transient transfections and quantitative PCR analysis
HEK293 (human embryonic kidney epithelial) cells were plated in 12-well
plates the day before transfection. Expression plasmids of TAp63␣, TAp63␥,
⌬Np63␣ and ⌬Np63␥ (1.6 ␮g each) were transfected with Lipofectamine
2000 (Invitrogen) according to the manufacturer’s recommendations. Empty
vector was also transfected as a control. Twenty-four hours after transfection,
total RNA was isolated from the cells using the RNeasy Minikit (Qiagen).
Total RNA (2 ␮g) was reverse-transcribed using random primers and
SuperScript III (Invitrogen). Real-time PCR was performed on an ABI 7300
(Applied Biosystems). PCR reactions were performed using ABI SYBR
Green PCR Master Mix, 300 nM primers, 200 ng cDNA at the following
consecutive steps: (1) 50°C for 2 minutes; (2) 95°C for 10 minutes; (3) 40
cycles of 95°C for 15 seconds and 60°C for 1 minute. The samples were run
in triplicate and normalized to an internal control (GAPDH) using the
accompanying software. The following primers were used: CDH3 (forward
5⬘-CGAAGAGGACCAGGACTATGA-3⬘, reverse 5⬘-GTCTGTGTTAGCCGCCTTCA-3⬘), GAPDH (forward 5⬘-TCACCAGGGCTGCTTTTAACTC-3⬘, reverse 5⬘-GGGTGGAATCATATTGGAACATG-3⬘). The
transfection and real-time PCR experiments were performed in triplicate.
Statistical analysis was performed using the ANOVA procedure, followed
by the Dunnett’s t-test. P<0.05 was considered significant.
Promoter analyses and reporter gene assays
The whole genome rVISTA tool (http://genome.lbl.gov/vista/index.shtml)
evaluates conservation of genomes between pairs of species (specified as
mouse and humans) and pinpoints transcription-factor-binding sites that are
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744
over-represented in upstream regions of genes of interest. PCR-amplified
CDH3 promoter regions were cloned into the NheI and XhoI sites of the pGL3
basic luciferase reporter vector (Promega). To generate the mutated constructs,
mutations of interest were introduced into the forward primers. HeLa (human
cervical cell carcinoma) cells were seeded in a 12-well plate and the next day
transfected with the appropriate plasmids using Lipofectamine 2000
(Invitrogen). A constitutive ␤-galactosidase reporter was used for
normalization of transfection efficiency. The cells were lysed 24 hours after
transfection and the signals were assayed using the appropriate substrates for
luciferase (Steady-Glo Luciferase Assay System) and ␤-galactosidase
(Promega) on a 20/20n luminometer (Turner Biosystems) for luciferase and a
Model 680 microplate reader (BioRad) for ␤-galactosidase.
Chromatin immunoprecipitation (ChIP)
HEK293 cells were seeded at 1⫻106 per 60 mm dish and transfected 24
hours later with 8 ␮g TAp63␣ or TAp63␥ expression vector, or empty vector
as described above. After 24 hours, the cells were fixed in 1% formaldehyde
for 10 minutes and the Chromatin Immunoprecipitation Kit (Upstate) was
used per the manufacturer’s recommendations. In addition, HaCaT (human
epidermal keratinocytes) cells were cultured in a 100 mm dish and used for
in vivo ChIP. An anti-p63 antibody mouse monoclonal 4A4 (Santa Cruz
Biotechnology) was used for immunoprecipitation. Cell lysates were also
precipitated with normal mouse IgG as a negative control. PCR was
performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen).
The amplification conditions for each PCR were 94°C for 2 minutes,
followed by 33 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 68°C
for 30 seconds, with a final extension at 68°C for 7 minutes. p21 is a bona
fide target of p63 and was used as a positive control (Shimada et al., 1999).
The following primers were used: p21 promoter (–1519 to –1245; forward
5⬘-GCAGTGGGGCTTAGAGTGGGG-3⬘, reverse 5⬘-CAGGCTTGGAGCAGCTACAATTAC-3⬘) (Osada et al., 2005), CDH3 promoter-region 1
(–2246 to –2000; forward 5⬘-TGCCCGAGAAGGAGGCCGGAAATG-3⬘,
reverse 5⬘-GCTTTTTATGCCCGAGGGGAGGT-3⬘), CDH3 promoterregion 2 (+6 to +227; forward 5⬘-TGTAGCCGCGTGTGGGAGGA, reverse
5⬘-TCTCGCAGTTCTGGGTTAGAGGAG). A 248 bp fragment spanning
exon 3 of the CDH3 gene was also amplified as a negative control using the
primers described previously (Kjaer et al., 2005).
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745
homozygous mutation 490insA in exon 5 of CDH3 in affected
individuals of Family 1 (Fig. 1B). Mismatch allele-specific PCR
showed that 50 unrelated healthy control individuals did not possess
the mutation (data not shown). Next, affected individuals in both
Families 2 and 3 were homozygous for a novel mutation Ivs10-1G>T
in CDH3 (Fig. 1C). This mutation abolished a BpmI restriction
enzyme site, which was used to exclude the mutation from 50 control
individuals (data not shown). Affected individuals in Family 4 were
found to carry a novel homozygous substitution 353A>G in CDH3,
which results in a substitution of glutamic acid by glycine at amino
acid 118 (Fig. 1D). Multiple amino-acid sequence alignments showed
that the glutamic acid at position 118 is completely conserved among
P-cadherin of other species, as well as other human classical cadherins
(Fig. 1D). In addition, the mutation generated a BstNI restriction
enzyme site, which was used to exclude the possibility that it
represents a common polymorphism (data not shown). Finally,
patients in Family 5 possessed a homozygous transition 965A>T
(N322I) (Fig. 1E), which was previously detected in a Danish family
with EEM syndrome (Kjaer et al., 2005).
RESULTS
Clinical features
Families 1-3 are large consanguineous Pakistani pedigrees with four,
twelve and six affected individuals, respectively, who were born
with sparse hair that fails to grow (Fig. 1A). Retinal degeneration
manifested in all affected individuals with decreased visual acuity
during the first decade of life, whereas none of them had limb or
finger anomalies. Family 4 has five affected individuals whose
parents are first-degree cousins (Fig. 1A). All affected individuals
have sparse hair, weak eyesight, and the additional phenotype of
severe SHFM (Fig. 1A). Family 5 contains two affected individuals
whose parents are first-degree cousins (Fig. 1A). The younger
patient (II-6) exhibits severe hypotrichosis with a trichorrhexis
nodosa-like hair shaft, weak eyesight and severe syndactyly.
Trichorrhexis nodosa is a particular type of hair shaft anomaly
characterized by nodular swelling of the hair due to its fragility. The
elder patient (II-5) shows mild hypotrichosis with pili torti-like hair
shaft twisting, weak eyesight and a less severe syndactyly (Fig. 1A).
Based on these clinical features, we diagnosed the affected
individuals in Families 1-3 with HJMD, and those in Families 4 and
5 with EEM syndrome.
P-cadherin is expressed in the hair follicle placode
and the AER of limb bud, overlapping with p63
In order to determine the precise expression pattern of P-cadherin in
the developing HF, eye and limb bud, we performed immunostaining
using cryosections of whole mouse embryos. At E15.5, P-cadherin
was strongly expressed in the HF placode, wheareas it was only
weakly expressed at basal layer of the interfolliclular epidermis (Fig.
2A). In addition, P-cadherin expression was detected in the RPE of
the developing eye at E17.5 (Fig. 2B). These results are consistent
with previous studies by others (Jamora et al., 2003; Xu et al., 2002).
In forelimb bud at E9.5 (Fig. 2C) and E10.5 (Fig. 2D), P-cadherin was
strongly expressed throughout the AER. The positive signal was also
detected in dorsal and ventral surface ectoderm around the AER, but
the expression was more prominent in the AER (Fig. 2C,D). At E15.5,
P-cadherin was ubiquitously expressed at the epithelial cell junctions
of the forelimb digits (Fig. 2E,F). We also performed whole-mount
immunohistochemistry using E9.5 embryos, which precisely
confirmed the expression of P-cadherin at the AER of forelimb buds
(Fig. 2G,H).
Double immunostaining with antibodies for P-cadherin and p63
clearly demonstrated overlapping expression of the two proteins in
the HF placode (Fig. 3A,B). In addition, p63 was strongly expressed
throughout the nuclei of the forelimb bud epithelium, especially in
the AER (Fig. 3C,D), which overlaps with P-cadherin expression
(Fig. 3E-H).
In addition to P-cadherin, we analyzed the expression of two
other classical cadherin members in the developing mouse limb
bud. At E9.5, E-cadherin was markedly expressed in the AER of
forelimb bud (Fig. 4A), and its expression almost completely
overlapped with that of P-cadherin (Fig. 4B,C). The strong
expression of E-cadherin was similarly detected throughout the
AER at E10.5 (Fig. 4D,E). N-cadherin expression was widely
detected in the mesenchyme, but not in the overlying epithelium
(Fig. 4F). In particular, the expression of N-cadherin was
prominent just beneath the AER and never overlapped with Ecadherin expression (Fig. 4G,H).
Identification of three novel and one recurrent
mutation in the CDH3 gene
Using genomic DNA from members of the five Pakistani families, we
performed mutation analysis and identified pathogenic mutations in
the CDH3 gene in all five families. First, we found a novel
p63 induces the expression of the P-cadherin
(CDH3) gene
In order to investigate the role of p63 isoforms in regulating the
expression of CDH3, we tested the ability of p63 to induce CDH3
expression. HEK293 cells were transfected with TAp63␣,
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P-cadherin in the developing limb and HF
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RESEARCH ARTICLE
Development 135 (4)
TAp63␥, ⌬Np63␣, ⌬Np63␥ or empty vector, and the relative
CDH3-transcript endogenous levels were examined by real-time
PCR. Cells transfected with TAp63 isoforms showed significant
increase in the expression of CDH3 (Fig. 5). In addition, cells
transfected with ⌬Np63 isoforms also exhibited a moderate, albeit
statistically significant, increase (P<0.05) in CDH3 expression
(Fig. 5).
Identification of two regions in the CDH3
promoter responsive to p63
Comparison of the CDH3 promoter sequences among different
species using the whole genome rVISTA tool resulted in the
identification of two distinct regions that were highly conserved
between mouse and human (Fig. 6A). These are located at –2400/
–2000 bp and +1/+500 bp of the CDH3 promoter, which we termed
DEVELOPMENT
Fig. 1. Identification of mutations in the CDH3 gene of five consanguineous Pakistani families. (A) Pedigrees and clinical appearance of
affected individuals of five Pakistani families (Families 1-5) with either HJMD or EEM syndrome. In Families 1-3, arrows indicate affected individuals
whose clinical photographs are shown. (B) A novel mutation 490insA (arrow) in Family 1. (C) A novel mutation Ivs10-1G>T (arrows) in Families 2
and 3. (D) A novel mutation E118G in Family 4 (top) and amino-acid sequence alignment of human classical cadherins and P-cadherin of other
species (bottom). The glutamic acid residue at position 118 is colored light blue. (E) The recurrent mutation N322I (arrows) in Family 5.
Fig. 2. Expression of P-cadherin in the developing mouse hair
follicle, eye and limb bud. (A) P-cadherin expression is upregulated in
the HF placode at E15.5. (B) P-cadherin is expressed in the pigmented
epithelium of the retina (white arrow) at E17.5. (C,D) Transverse
sections of forelimb bud at E9.5 (C) and E10.5 (D) show prominent
expression of P-cadherin along the AER (white arrows). Note that, in
the transverse section, the tip of forelimb bud corresponds to the AER.
For a better view, counterstaining with DAPI is shown in red instead of
blue (B-D). (E,F) Longitudinal section of forelimb bud at E15.5 shows
that P-cadherin is widely expressed throughout the epithelial cells of the
forelimb bud. F is a higher magnification view of E. (G,H) Whole-mount
immunohistochemistry also localizes the P-cadherin in the AER of the
forelimb bud at E9.5 (white arrows). Scale bars: 100 ␮m.
Region 1 and Region 2, respectively (Fig. 6A). Region 2
corresponds to exon 1 of the CDH3 that contains the start
codon. In addition, analysis with the NCBI database
(http://www.ncbi.nlm.nih.gov/) showed that both regions are
included within CpG islands (Fig. 6A). Based on this information,
we first cloned –2472 to +500 bp of the CDH3 promoter, which
contained both regions, into the pGL3 basic plasmid, and tested
whether the luciferase reporter gene activity is induced by TAp63␥
and ⌬Np63␥. As shown in Fig. 6B, both TAp63␥ and ⌬Np63␥
markedly upregulated luciferase activity (more than 25-fold relative
to control).
Subsequently, we cloned four shorter fragments of the CDH3promoter, –3366 to –2452 (low homology region upstream of
Region 1), –2472 to –1833 (Region 1), –1944 to –1 (low homology
region between Regions 1 and 2) and +1 to +500 (Region 2) into the
pGL3 vector (Fig. 6A), and analyzed the promoter activity in the
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Fig. 3. P-cadherin overlaps with p63 in the hair follicle placode
and the AER in mouse. (A,B) Double-immunostaining shows
overlapping expression of P-cadherin (red) and p63 (green) at the HF
placode at E15.5. (C,D) Longitudinal section of forelimb bud at E9.5 (C)
and transverse section of forelimb bud at E10.5 (D) demonstrate that
p63 is predominantly expressed at nuclei of the AER. (E-H) Doubleimmunostaining using a transverse section of forelimb bud at E10.5
shows that both P-cadherin (green) and p63 (red) are expressed
throughout the AER. H is the merged image of F and G. Scale bars:
100 ␮m in A,C,D,E; 20 ␮m in B,F-H.
presence of p63. We observed that both Region 1 and Region 2
showed significant levels of transcription, as measured by relative
luciferase activity (Fig. 6B). It has been well established that the
binding sequence of p63 is similar to the canonical responsive
element of p53 (RRRCWWGYYY), with minor differences (Osada
et al., 2005). We analyzed the sequence of Regions 1 and 2 and
found five and two sites, respectively, that show high homology
(7/10 to 9/10) with the sequence ‘RRRCWWGYYY’, which could
be regarded as potential p63-binding sites (Fig. 6C,D). Indeed, both
regions were sufficient to show a significant increase in transcription
by p63 when they were truncated to ~230 bp fragments containing
these potential p63-binding sites (Fig. 6E). By contrast, we noted
that p53 did not induce transactivation of the CDH3 in any of the
constructs analyzed (data not shown). Of the five potential p63binding sites in Region 1, the downstream two sites partially
overlapped with each other at –2088/–2070 (Fig. 6C). We focused
on these successive binding sites and further analyzed them in detail.
In order to test each binding site, we compared the activities of the
CDH3-2106/-2004-Wild, CDH3-2106/-2004-M1 (both sites were mutated),
CDH3-2106/-2004-M2 (only the upstream site was mutated), and
CDH3-2106/-2004-M3 (only the downstream site was mutated)
constructs in the presence of TAp63␥ (Fig. 6F). All three mutated
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Development 135 (4)
Fig. 5. CDH3 expression is upregulated by p63 in 293 cells.
HEK293 cells were transfected with TAp63␣, TAp63␥, ⌬Np63␣,
⌬Np63␥ or empty vector. At 24 hours post-transfection, the CDH3
expression levels were measured by real-time PCR. All four p63
isoforms upregulated CDH3 expression. Asterisks indicate a statistically
significant difference (P<0.05) compared with the empty vector.
the reporter activity in either Region 1 (Fig. 7A) or Region 2 (Fig.
7B). Furthermore, in both regions, wild-type TAp63␥-induced
transactivation of the CDH3 reporter was markedly inhibited by the
mutant TAp63␥ in a dose-dependent manner (Fig. 7A,B).
constructs showed markedly decreased luciferase activity compared
with CDH3-2106/-2004-Wild construct, indicating that both sites are
required for the transactivation by TA-p63␥ (Fig. 6F). In addition,
we also introduced mutations into the putative binding site at +31/40
of Region 2 and compared the induction of transcription by TAp63␥
between CDH3+1/+124-Wild and CDH3+1/+124-Mutant constructs
(Fig. 6G). Significant reduction in luciferase activity was detected
using the CDH3+1/+124-Mutant construct (Fig. 6G).
TAp63␥ (R280C) mutant fails to transactivate
CDH3 expression
Previous studies have shown that mutations in the DNA-binding
domain of p63 result in SHFM (Celli et al., 1999; Ianakiev et al.,
2000). In particular, missense mutations at codon 279 or 280 have
been shown to cause SHFM at a high frequency (Rinne et al., 2006).
Therefore, we generated a construct expressing TAp63␥ with the
common SHFM mutation R280C, and tested whether this mutation
affects the CDH3 expression. The mutant TAp63␥ did not activate
DISCUSSION
P-cadherin mutations cause HJMD or EEM
syndrome in humans
Recent advances in molecular genetics have revealed that two
recessively inherited human diseases, HJMD and EEM syndrome,
are caused by mutations in the CDH3 gene, encoding P-cadherin
(Sprecher et al., 2001; Kjaer et al., 2005). In this study, we identified
five consanguineous Pakistani families (Families 1-5) affected with
either HJMD or EEM syndrome and found mutations in the CDH3
in all five families. The mutation 490insA identified in Family 1 with
HJMD is a novel insertion mutation that results in a frameshift at
codon 164 and premature termination codon (PTC) at codon 171
(Fig. 1B). The mutation Ivs10-1G>T found in Families 2 and 3 with
HJMD is a novel splice site mutation that is predicted to cause out
of frame skipping of exon 11, 146 bp in size, and generate a PTC
within exon 12 (Fig. 1C). It is possible that truncated proteins
lacking most of the essential domains could be generated from both
the 490insA and the Ivs10-1G>T mutant alleles. Alternatively, the
DEVELOPMENT
Fig. 4. Expression of classical cadherins in the forelimb bud of
mouse. (A-C) Double immunostaining of a longitudinal section of the
forelimb bud at E9.5 shows that the expression of E-cadherin (green, A)
and P-cadherin (red, B) overlaps along the AER (yellow in merged
image, C). (D,E) Transverse section of the forelimb bud at E10.5 shows
the strong expression of E-cadherin throughout the AER. (F-H) Ncadherin is strongly expressed at mesenchyme just beneath the AER (F),
whereas E-cadherin is prominently expressed in the AER (G). The
merged image of F and G clearly demonstrates their distinct expression
patterns (H). Counterstaining with DAPI is shown in blue in C and in
red in D,E. Scale bars: 100 ␮m in A-D,F-H; 20 ␮m in E.
p63 binds directly to two regions in the CDH3
promoter
We investigated the ability of p63 to bind to the CDH3 promoter
using ChIP assays. First, we overexpressed both TAp63␣ and
TAp63␥ isoforms in HEK293 cells, and precipitated the DNAprotein complexes with anti-p63 4A4 antibody. The precipitated
DNA fragments were PCR-amplified. In the cells in which
TAp63␣ and TAp63␥ were induced, anti-p63 antibody clearly
immunoprecipitated both Region 1 and Region 2 of the CDH3
promoter, which contain putative p63-binding sites, whereas it did
not immunoprecipitate the CDH3 coding region (Fig. 8A). Finally,
we performed in vivo ChIP using HaCaT cells that predominantly
express ⌬Np63␣ isoform (Vigano et al., 2006). Similarly, both
Region 1 and Region 2 were PCR-amplified from the sample
precipitated with anti-p63 antibody (Fig. 8B). These results
demonstrate that both TAp63 and ⌬Np63 isoforms directly bind to
two distinct regions within the CDH3 promoter.
RESEARCH ARTICLE
749
Fig. 6. Identification of two distinct regions in the CDH3 promoter that are responsive to p63. (A) Schematic of the CDH3 promoter.
Regions 1 and 2 that show high homology with mouse sequences are boxed. CpG islands are indicated by shaded boxes. Five templates that were
cloned into the pGL3 basic vector are shown at the bottom. (B) Reporter gene assay in HeLa cells. The longest construct (–2472/+500), which
contains both Regions 1 and 2, showed more than 25-fold transactivation of the reporter gene by TAp63␥ and ⌬Np63␥. Furthermore, shorter
constructs that contain either Region 1 (–2472/-1833) or Region 2 (+1/+500) resulted in a much greater activation of luciferase expression by p63.
(C,D) Potential p63-binding sites in Region 1 (C) and Region 2 (D) are shaded with the canonical p53-binding sequence shown either above or
below. Asterisks show nucleotide residues that correspond to the ‘RRRCWWGYYY’ sequence. The positions where primers for ChIP assay were
designated are underlined. (E) Both Regions 1 and 2 showed further increase of the reporter gene activity by p63, when they were truncated to
about 230 bp fragments with potential p63-binding sites. (F) Sequence of the two partially overlapping p63-binding sites in Region 1 (–2088 to
–2070). Sequences of the three mutated constructs (M1-M3) are also shown. All three mutated constructs led to a marked reduction of luciferase
activity by TAp63␥. (G) Sequence of a p63-binding site in Region 2 (+31 to +40). Sequence of the mutated construct is also shown. TAp63␥
showed only weak transactivation of the reporter gene in the mutated construct.
DEVELOPMENT
P-cadherin in the developing limb and HF
RESEARCH ARTICLE
Fig. 7. A dominant-negative effect of TAp63␥ (R280C) mutant.
(A,B) HeLa cells were transfected with the indicated amount of the
expression constructs, and reporter gene activity was examined in both
Region 1 (–2233/–2004, A) and Region 2 (+1/+223, B). Mutant TAp63␥
did not activate luciferase expression in either region. In addition, it
inhibited the wild-type TAp63␥-induced transactivation in a dosedependent manner in both regions.
aberrant CDH3-transcripts containing the PTC might be largely
degraded due to nonsense-mediated messenger RNA decay
(Maquat, 1996; Frischmeyer and Dietz, 1999). E118G is a novel
missense mutation detected in Family 4 with EEM syndrome
showing severe SHFM (Fig. 1D). The glutamic acid at position 118
(E118), which corresponds to E11 in the mature protein, is a highly
conserved amino acid residue within the EC1 domain of P-cadherin
(Fig. 1D, Fig. 9). The side chain of E11 coordinates calcium in the
EC1-EC2 calcium-binding site (Fig. 9), and without this side chain,
the structure of the calcium-binding site will be compromised.
Calcium binding rigidifies cadherin ectodomains, which is thought
to geometrically constrain cadherins so as to interact preferentially
with cadherins from apposing cells rather than the same cell
(Boggon et al., 2002; Patel et al., 2006). The mutation E118G may
thus enable the interaction of mutant proteins on the same, rather
than opposing, cell surfaces, thereby negating adhesive function.
The mutation N322I found in Family 5 with EEM syndrome is a
non-conservative amino acid change in the EC2-EC3 calciumbinding site (Fig. 9), which was previously identified in a Danish
family with EEM syndrome (Kjaer et al., 2005). It is noteworthy that
the affected individuals in the Danish family exhibited more severe
hand involvement (Kjaer et al., 2005) than those in Family 5 (Fig.
1A). In addition, the severity is significantly different between two
affected individuals in Family 5 (Fig. 1A). These data indicate that
the N322I shows variation in severity between different families and
even between members in a family, even though the same organs
(hair, retina and hand/foot) are involved in both families.
Paradoxically, previous reports have demonstrated that the
identical mutation 829delG resulted in HJMD in one family
(Indelman et al., 2003), while it led to EEM syndrome in another
Development 135 (4)
Fig. 8. Both TAp63 and ⌬Np63 isoforms directly bind to two
distinct regions in the CDH3 promoter. (A) ChIP assay in HEK293
cells in which either TAp63␣ or TAp63␥ was overexpressed. (B) In vivo
ChIP assay in HaCaT cells. An antibody against p63 immunoprecipitated
both Region 1 and Region 2 of the CDH3 promoter (black arrows)
similar to the positive control p21 (gray arrow). The CDH3 coding
region was not immunoprecipitated (black arrowhead).
family (Kjaer et al., 2005), which suggests that an identical mutation
can give rise to different phenotypes between families (Fig. 9).
Nevertheless, in HJMD families, all affected individuals have hair
and eye involvement only (Sprecher et al., 2001; Indelman et al.,
2002; Indelman et al., 2003; Indelman et al., 2005; Indelman et al.,
2007). Similarly, in EEM families, all affected individuals show not
only hair and eye phenotype but also hand/foot involvement (Kjaer
et al., 2005). An obvious genotype-phenotype correlation between
CDH3 mutations has yet to emerge.
Balanced expression of classical cadherin
members might be crucial for limb bud
development
To our knowledge, the expression of P-cadherin in the limb bud has
not been previously reported. Here, we clearly demonstrated that Pcadherin expression is upregulated in the AER of the limb bud in the
developing mouse embryo (Fig. 2C-H). Our data suggest that Pcadherin plays an important role in outgrowth of limb bud, perhaps
akin to its role in the downgrowth of the HF placode. In addition to
P-cadherin, we also examined the expression of N- and E-cadherins
in the limb bud. Like previous reports (Yajima et al., 2002), we
showed that N-cadherin expression is detected in mesenchyme
underlying the AER (Fig. 4F-H). Surprisingly, E-cadherin was
strongly expressed in the AER, overlapping with P-cadherin (Fig.
4A-E), suggesting that the E-cadherin to P-cadherin switch does not
occur in the AER of the developing mouse embryo as it does in the
HF placode.
The identification of mutations in the human CDH3 gene
underscores the involvement of P-cadherin in the human HF, eye
and limb development. Despite these findings in humans, it is
DEVELOPMENT
750
P-cadherin in the developing limb and HF
RESEARCH ARTICLE
751
somewhat surprising that P-cadherin-knockout mice, which are on
a C57BL/6 background, show only precocious mammary gland
development, whereas hair, eye and limb anomalies are not observed
in these animals (Radice et al., 1997). These differences may be due
to the influence of some modifier genes and/or genetic background
in the animal models. The strong expression of E-cadherin in the
AER raises the possibility that E-cadherin could play a role as a
modifier and compensate for the P-cadherin deficiency in the mouse
model. In addition, as it is known that the sensitivity to teratogeninduced limb malformations is variable among different mouse
strains (Shimizu et al., 2007), there is the possibility that P-cadherin
deficiency might cause more severe anomalies, including hair and
limb phenotypes on different mouse strains.
P-cadherin is a direct transcriptional target gene
of p63
Double immunostainings with anti-P-cadherin and anti-p63
antibodies clearly showed that the expression patterns of p63 and Pcadherin overlap in the HF placode (Fig. 3A,B) and in the AER of
the limb bud (Fig. 3E-H). The transcription factor p63 has been
known to be involved in the HF and limb development because p63
mutations result in developmental defects of hair and limb in both
human (Celli et al., 1999) and mouse (Mills et al., 1999; Yang et al.,
1999). In particular, SHFM caused by human p63 mutations is
phenotypically similar to that in affected individuals with CDH3
mutations. Our expression studies, as well as the phenotypic
similarity, prompted us to postulate that P-cadherin could be a direct
transcriptional target of p63. In order to test this hypothesis, we first
examined whether P-cadherin expression is affected when p63 is
overexpressed in 293 cells. We found that both TAp63 and ⌬Np63
isoforms upregulated the expression of the CDH3 mRNA (Fig. 5).
Osada et al. have recently shown that a total of 129 genes were
activated more than four-fold when TAp63␥ was ectopically
expressed in 293 cells, and CDH3 was found among these genes
(8.1-fold upregulation) (Osada et al., 2005).
Subsequently, we focused on p63␥ isoforms and performed
promoter assays using the luciferase reporter gene. We identified
two distinct regions (Regions 1 and 2), which were significantly
responsive in reporter gene expression analysis by both TAp63␥ and
⌬Np63␥ (Fig. 6A,B,E). The reporter gene activity in the longest
construct containing both regions was lower than that of a shorter
construct with either region (Fig. 6B). A suppressor element might
be present in the region between Regions 1 and 2, thereby repressing
the expression of P-cadherin. Furthermore, a mutant (R280C)
TAp63␥ inhibited the transactivation by the wild-type TAp63␥ in a
dose-dependent manner in both regions (Fig. 7A,B), indicating that
the mutant TAp63␥ would cause a dominant-negative effect to the
wild-type TAp63␥ and thereby reduce P-cadherin expression.
Regions 1 and 2 have a total of seven potential p63-binding sites
(Fig. 6C,D). Even though they show high homology with the
canonical p53-binding sequence (RRRCWWGYYY), p53 did not
activate the luciferase expression in either region (data not shown),
indicating that these binding sites are specific to p63. Four of seven
binding sites possess a CCTG core sequence (Fig. 6C,D), which has
been found in the promoter regions of recently identified p63-target
genes as well (Yan and Chen, 2006; Romano et al., 2006). We
generated mutated constructs in which mutations were introduced
to abolish the core sequence, and showed significant reduction of
transactivation compared with wild-type constructs (Fig. 6F,G).
These data indicate that the CCTG core sequence represents a p63specific element in addition to the recently identified CGTG core
sequence (Osada et al., 2005). Finally, we performed a ChIP assay
using HEK293 cells in which either TAp63␣ or TAp63␥ was
induced, and demonstrated that TAp63 isoforms bind to both
Regions 1 and 2 of the CDH3 promoter containing potential p63binding sites (Fig. 8A). Furthermore, we also performed in vivo
ChIP using HaCaT cells in which endogenous ⌬Np63␣ is strongly
expressed, and clearly showed that p63, especially ⌬Np63␣, binds
to the CDH3 promoter in vivo (Fig. 8B). Collectively, our data
indicate that P-cadherin is a direct transcriptional target of p63.
The interaction between p63 and P-cadherin gene
is crucial for HF and limb bud development in
humans
Using large-scale genomic approaches, a number of potential p63target genes have been predicted by several groups (Carroll et al.,
2006; Barbieri et al., 2006; Vigano et al., 2006; Yang et al., 2006;
Testoni et al., 2006). Of these, Carroll et al. showed the upregulation
DEVELOPMENT
Fig. 9. Schematic representation of
P-cadherin protein and CDH3 mutations,
and diagram of the EC1-EC2 calciumbinding site of P-cadherin. Mutations that
were identified in this study are indicated in
bold and underlined. Note that the mutation
E118G corresponds to E11 in the mature
protein. In the diagram, the side chain of E11
is colored in red. EC1-EC5, extracellular
cadherin repeat domains; IC, intracellular
domain; TM, transmembrane domain.
RESEARCH ARTICLE
of CDH3 expression when TAp63␥ was overexpressed in MCF-10A
(normal human breast epithelial) cells (Carroll et al., 2006). In
addition, Yang et al. performed ChIP using an anti-p63 antibody and
ME180 (human cervical carcinoma) cells, which was followed by
microarray studies (Yang et al., 2006). As a result, approximately
5800 p63-target sites were identified in the whole genome. One of
these sites corresponded to intron 2 of the CDH3, which is not
conserved between human and mouse, and is distant from the
transcription start site. These data, together with Osada et al. (Osada
et al., 2005), suggested a potential relationship between p63 and the
CDH3. For the first time, definitive evidence to prove that the CDH3
is a direct target of p63 is provided in this study, and our results are
consistent with the recent report that implicates p63 as a key
regulator of broader cell adhesion gene expression programs
(Carroll et al., 2006). Recently, Senoo et al. showed that one role of
p63 is to control programmed cell death and maintain the
proliferative potential of embryonic and adult stem cells (Senoo et
al., 2007). p63 might prevent cell death through regulating the
expression of some adhesion molecules that are essential for
epithelial integrity and maintenance of proliferation (Blanpain and
Fuchs, 2007). P-cadherin might be involved in this mechanism.
During mouse embryogenesis, TAp63 isoforms have been shown
to be expressed much more strongly than ⌬Np63 isoforms (Koster
et al., 2004; Radoja et al., 2007), whereas ⌬Np63 isoforms are
predominantly expressed in the basal layer of the epidermis and
govern basal-epidermal gene expression (Laurikkaka et al., 2006;
Candi et al., 2006). Recently, a TAp63-specific knockout mouse, in
which ⌬Np63 isoforms are expressed normally, has been reported
to show neither hair nor limb anomalies (Suh et al., 2006). In our
study, we showed that not only TAp63, but also ⌬Np63 isoforms,
bind to the CDH3 promoter and activate CDH3 expression. We
conclude that TAp63 isoforms, together with ⌬Np63, play an
essential role in maintaining the P-cadherin expression in the AER
during limb bud outgrowth, as well as in the HF placode. As p63 is
more broadly expressed than P-cadherin, not only p63 but also other
factors are likely to play a role in regulating P-cadherin expression
in the HF placode and the AER. Nevertheless, the phenotypic
similarities caused by p63 and P-cadherin mutations, as well as our
results, strongly suggest that the interaction between p63 and Pcadherin gene is crucial for HF and limb bud development in
humans, whereas it does not appear to be essential for either in mice.
We have defined a functional relationship between p63 and Pcadherin, and also determined their role in failure of the HF placode
and the AER that can lead to sparse hair and SHFM when either
gene is mutated in humans.
We gratefully acknowledge the family members for having participated in this
study. We thank Drs Dennis Roop and Maranke Koster for generously sharing
p63 constructs. We appreciate insightful comments from Dr Gail Martin (UCSF)
and Dr Victor Luria (Columbia University) regarding limb development, and Dr
Richard Hynes (Howard Hughes Medical Institute) for helpful discussion about
P-cadherin knockout mice. We thank Mr Ming Zhang for excellent technical
assistance, and Hisham Bazzi, Yoshiyuki Ishii, Katherine Fantauzzo and Eiichi
Hinoi of Columbia University for helpful assistance and discussions. We
appreciate the help of Lynn Petukhova for the statistical analysis. This work
was in part supported by a grant from USPHS NIH RO1AR44924 (to A.M.C.).
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DEVELOPMENT
P-cadherin in the developing limb and HF