© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 282-290 doi:10.1242/dev.118307
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
The carboxy-terminus of p63 links cell cycle control and the
proliferative potential of epidermal progenitor cells
ABSTRACT
The transcription factor p63 (Trp63) plays a key role in homeostasis
and regeneration of the skin. The p63 gene is transcribed from dual
promoters, generating TAp63 isoforms with growth suppressive
functions and dominant-negative ΔNp63 isoforms with opposing
properties. p63 also encodes multiple carboxy (C)-terminal variants.
Although mutations of C-terminal variants have been linked to
the pathogenesis of p63-associated ectodermal disorders, the
physiological role of the p63 C-terminus is poorly understood. We
report here that deletion of the p63 C-terminus in mice leads to
ectodermal malformation and hypoplasia, accompanied by a reduced
proliferative capacity of epidermal progenitor cells. Notably, unlike the
p63-null condition, we find that p63 C-terminus deficiency promotes
expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1
(Cdkn1a), a factor associated with reduced proliferative capacity of
both hematopoietic and neuronal stem cells. These data suggest that
the p63 C-terminus plays a key role in the cell cycle progression
required to maintain the proliferative potential of stem cells of many
different lineages. Mechanistically, we show that loss of Cα, the
predominant C-terminal p63 variant in epithelia, promotes the
transcriptional activity of TAp63 and also impairs the dominantnegative activity of ΔNp63, thereby controlling p21Waf1/Cip1
expression. We propose that the p63 C-terminus links cell cycle
control and the proliferative potential of epidermal progenitor cells via
mechanisms that equilibrate TAp63 and ΔNp63 isoform function.
KEY WORDS: Cell cycle, Epithelia, Mouse, p21, p63, Proliferation,
Stem cells
INTRODUCTION
Adult stem cells are capable of long-term self-renewal and
differentiation, enabling their use as an important therapeutic
approach in regenerative medicine (Green, 2008; Pellegrini et al.,
2009; Rama et al., 2010). Epidermal stem cells are essential for
maintaining homeostasis and regeneration of the skin, but the
specific mechanisms that control their fate are not well understood
(Bickenbach et al., 2006; Blanpain and Fuchs, 2009; Ghadially,
2012; Kaur, 2006; Watt and Jensen, 2009). We previously identified
p63 (Trp63), a homolog of the tumor suppressor p53 (Trp53)
(Osada et al., 1998; Schmale and Bamberger, 1997; Senoo et al.,
1998; Trink et al., 1998; Yang et al., 1998), and showed that p63
deficiency leads to a loss of proliferative potential of epidermal stem
1
Department of Animal Biology, University of Pennsylvania School of Veterinary
2
Medicine, Philadelphia, PA 19104, USA. Institute for Regenerative Medicine,
University of Pennsylvania, Philadelphia, PA 19104, USA.
*Present address: Department of Zoology, APC College, West Bengal State
University, New Barrackpore, Kolkata, West Bengal 700131, India.
‡
Author for correspondence ([email protected])
Received 30 September 2014; Accepted 14 November 2014
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cells (Senoo et al., 2007). Indeed, p63-null mice display severe
defects in epithelial development (Mills et al., 1999; Yang et al.,
1999). Genetic studies suggest that mutations in p63 contribute to
the pathogenesis of a wide range of ectodermal dysplasias (EDs),
characterized by ectodermal malformations and hypoplasias (Celli
et al., 1999; Koster, 2010; Rinne et al., 2007; van Bokhoven and
McKeon, 2002), and we have recently noted decreased p63
expression in chronic equine laminitis in which the proliferative
epidermal layers appear dysplastic (Carter et al., 2011). Thus, it is
clear that p63 plays a key role in both the normal physiology and
pathophysiology of the epidermis.
The p63 gene is transcribed from dual promoters, generating
TAp63 isoforms that contain a transactivation domain with growth
suppressive functions (Guo et al., 2009) and dominant-negative
ΔNp63 isoforms that lack this domain and exhibit opposing
oncogenic properties (Keyes et al., 2011). Studies on isoformspecific knockout (KO) mice revealed that loss of ΔNp63 leads to
the identical epidermal hypoplasia observed in p63-null mice
(Romano et al., 2012). As ΔNp63 is the predominant isoform
expressed in epidermal progenitor cells, including stem cells and
transit-amplifying cells, ΔNp63 appears to play the major role in
maintaining the proliferative potential of epidermal progenitors
(Senoo, 2013). However, ΔNp63-specific KO mice also show an
early onset of terminal differentiation (Romano et al., 2012) that is
not observed in p63-null mice, suggesting that ΔNp63 normally
counterbalances TAp63-driven differentiation and the consequent
reduction in proliferative potential of epidermal stem cells. Notably,
although TAp63-specific KO mice show grossly normal epidermal
development, their isolated epidermal progenitor cells show
reduced proliferative capacity in vitro (Su et al., 2009). These
results suggest that TAp63 opposes ΔNp63 function, thereby
preventing a premature reduction in proliferative potential. Thus, it
is likely that p63 function reflects a cooperative effect between
TAp63 and ΔNp63 isoforms (Candi et al., 2006; Truong et al., 2006;
Zhang et al., 2014).
Whereas the amino (N)-terminal functions of p63 are relatively
well studied, carboxy (C)-terminal functions in vivo are poorly
understood. By alternative splicing, the p63 gene generates at least
three C-terminus variants, termed Cα, Cβ and Cγ, for both the
TAp63 and ΔNp63 isoforms (Yang et al., 1998). Notably, Cα
uniquely harbors the sterile α-motif (SAM) domain ( p63SAM),
which is a protein-protein interaction domain (Qiao and Bowie,
2005; Thanos and Bowie, 1999), and the transcription inhibitory
(TI) domain ( p63TI) (Serber et al., 2002). The significance of Cα is
evident from genetic studies of p63-associated EDs, showing that
mutations in either the p63SAM or p63TI domain or a complete
absence of Cα/β but not Cγ cause ankyloblepharon-ectodermal
defects-cleft lip/palate (AEC) syndrome, ectrodactyly-ectodermal
dysplasia-clefting (EEC) syndrome and limb-mammary syndrome
(LMS) (Barrow et al., 2002; Celli et al., 1999; Rinne et al., 2009;
van Bokhoven et al., 2001). A recent study has shown that a point
DEVELOPMENT
Daisuke Suzuki1,2, Raju Sahu1,2, *, N. Adrian Leu1 and Makoto Senoo1,2,‡
mutation in the p63SAM domain reduces the number of epidermal
progenitor cells (Ferone et al., 2012), highlighting the significance
of Cα in regulating stem cell properties. However, how Cα exerts its
function and the relative contribution of Cα in TAp63 and ΔNp63
isoform functions remain largely unknown.
A precise coupling between cell proliferation and differentiation
is required during embryonic development and self-renewal of adult
tissues. The cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1
(Cdkn1a) belongs to the Cip/Kip family of CDK inhibitors and was
originally identified as a mediator of p53-induced growth arrest
(El-Deiry et al., 1993; Harper et al., 1993). However, induction of
p21Waf1/Cip1 in epidermal progenitor cells is independent of p53
function (Missero et al., 1995), indicating that alternative regulatory
mechanisms are involved in this cell type. Indeed, accumulating
evidence suggests that p63 can control p21Waf1/Cip1 expression (Guo
et al., 2009; Su et al., 2013; Truong et al., 2006; Westfall et al.,
2003) and, notably, p21Waf1/Cip1 itself acts as a negative regulator
of the proliferative potential of epidermal progenitor cells (Topley
et al., 1999). However, whether p63 regulates p21Waf1/Cip1
expression in vivo to influence the proliferative potential of
epidermal progenitor cells remains unknown.
To further investigate the global function of the p63SAM and p63TI
domains, we have generated mutant mice lacking Cα/β by gene
targeting and found that homozygous mutant (referred to here as
p63C−/−) mice show multiple phenotypes including ectodermal
hypoplasia, limb malformation and orofacial clefting. We further
demonstrate that mice with p63 C-terminus deficiency show reduced
cell cycle progression and enhanced p21Waf1/Cip1 expression in
epidermal progenitor cells, leading to their decreased proliferative
capacity. Although the function of p63 is complex owing to the
existence of multiple isoforms as well as inter- and intramolecular
interactions, our present study shows that loss of Cα both promotes
transcriptional activity of TAp63 and reduces the dominant-negative
activity of ΔNp63 in the control of p21Waf1/Cip1 expression. Based
on these data, we propose that p63 links cell cycle control and
proliferative potential of epidermal progenitor cells through
C-terminus-dependent mechanisms that balance TAp63 and
ΔNp63 isoform functions.
RESULTS
Generation of mice lacking the C-terminus of p63
The SAM and TI domains of p63 are encoded by exons 12-14 of the
p63 gene (Fig. 1A). To generate mice lacking these two domains, we
deleted exon 12 of p63 by gene targeting (supplementary material
Fig. S1). This strategy allowed us to delete both p63SAM and p63TI
from Cα while leaving the Cγ isoform intact, as it is encoded by
alternative exon 10′ (Fig. 1A). As Cα and Cβ share exon 12, these
mice also lack full-length p63β isoforms. We confirmed that
expression of both full-length Cα and Cβ was absent in homozygous
mutant ( p63C−/−) mice, whereas expression of Cγ was similar
between p63C−/− mice and the wild-type (WT) control (Fig. 1B).
To analyze alternative splicing at the C-terminus resulting from
the deletion of exon 12, we sequenced the fragments amplified from
p63C−/− epidermal cell cDNA (Fig. 1B,C). Our data show that the
major transcript was encoded by exon 11 spliced to exon 13 (termed
Cα′), while a minor transcript resulted from splicing of exon 11 to
exon 14 (termed Cβ′). In both transcripts, stop codons appear
shortly after the splicing sites by frameshift, resulting in the addition
of only one and eight amino acids after exon 11, respectively
(Fig. 1C; supplementary material Fig. S2). These Cα′ and Cβ′
isoforms in p63C−/− mice were collectively referred to as the ΔC
isoform (Fig. 1D).
Development (2015) 142, 282-290 doi:10.1242/dev.118307
Fig. 1. Alterative splicing at the p63 C-terminus in p63C−/− mice.
(A) Structure and splicing of the p63 C-terminus in WT and ΔC p63 alleles.
Arrowheads indicate stop codons in each isoform. The p63SAM and p63TI
domains are illustrated. Exons are numbered. (B) Expression of p63 Cterminus variants in WT and p63C−/− epidermis as assessed by PCR. p63C−/−
epidermal cells predominantly express Cα′, which lacks both the p63SAM and
p63TI domains, whereas WT cells express full-length Cα. (C) Sequencing of
Cα′ and Cβ′ expressed in p63C−/− mice. Stop codons are underlined. Arrows in
A indicate the primer pair used to amplify the Cα′ and Cβ′ splicing variants.
(D) Structure of p63 isoforms expressed in WT and p63C−/− mice. p63Cα′ and
p63Cβ′ isoforms are collectively referred to as p63ΔC. DBD, DNA-binding
domain; OD, oligomerization domain. (E) qPCR analysis of the N-terminus of
the p63 gene in WT and p63C−/− epidermal cells. Shown is expression of
TAp63 relative to ΔNp63, with the ΔNp63 level in WT being set to 1.0. Error bars
indicate s.d. (F) Western blot with anti-p63 antibody of E14.5 whole epidermal
cell extracts from WT, p63C−/− and p63C+/− mice. Asterisk indicates nonspecific signal. Full, full-length ΔNp63α; ΔC, ΔNp63ΔC.
To determine whether targeting of exon 12 influences p63 gene
promoter activities, we performed qPCR using TAp63- and
ΔNp63-specific primers (Fig. 1E). Our data show that relative
expression of TAp63 and ΔNp63 was unchanged in p63C−/− mice
compared with WT littermates, indicating that the manipulation of
the p63 locus at the distal region does not affect p63 gene promoter
activities.
We next investigated p63 protein expression in p63C−/− mice by
western blot (Fig. 1F). As ΔNp63α is the predominant p63 isoform
expressed in WT epidermal cells, the major p63 isoform detected in
p63C−/− keratinocytes likely represents the ΔNp63 isoform lacking
Cα (hereafter refered to as ΔNp63ΔC). Together, these data
demonstrate that p63C−/− mice lack both p63SAM and p63TI
domains from Cα without significant changes to either p63 gene
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RESEARCH ARTICLE
promoter activity or p63γ isoform expression. Thus, p63C−/− mice
are useful for elucidating the function of Cα/β in the context of both
the TAp63 and ΔNp63 isoforms.
Craniofacial and skeletal phenotypes in p63C−/− mice
Heterozygous mutant ( p63C+/−) mice were grossly normal and
fertile. Although homozygotes were born at near Mendelian ratios
( p63C+/+:p63C+/−:p63C−/−=27.9%:43.7%:28.4%, n=197) they
died shortly after birth due to a significant loss of the skin,
resulting in dehydration and neglect by mothers. Although this
neonatal lethality is similar to that observed in p63-null mice (Mills
et al., 1999; Yang et al., 1999), we noted that craniofacial defects in
p63C−/− embryos were less significant than those in p63-null mice
(Fig. 2A). Whereas all p63-null mice show both cleft lip and cleft
palate, some p63C−/− embryos (35%, n=23) lack cleft lip although
cleft palate is still evident (Fig. 2B). As p63-null mice used in this
Development (2015) 142, 282-290 doi:10.1242/dev.118307
study were generated in parallel with p63C−/− mice in the same
genetic background (supplementary material Fig. S3), these data
indicate that p63C−/− and p63-null mice are phenotypically
different and highlight the functional significance of the p63
C-terminus during embryonic development.
Skeletal analysis of whole embryos revealed further differences
between p63-null and p63C−/− mice. Consistent with previous
studies (Mills et al., 1999; Yang et al., 1999), p63-null embryos
exhibit severely impaired forelimb development and a complete
absence of hindlimbs (Fig. 2C). By contrast, both fore- and
hindlimbs in p63C−/− embryos were more developed (Fig. 2C,D).
For example, the majority of forelimbs in p63C−/− mice (81%,
n=42) show grossly normal development of the humerus and ulna,
although the radius is lacking, with either syndactyly or ectrodactyly
(Fig. 2D). Strikingly, in 75% of p63C−/− mice (n=52), hindlimbs
show normal femur development (Fig. 2D). The frequency of
mice showing development of further hindlimb structure was
lower; however, 23% and 8% of p63C−/− embryos showed
relatively normal development of tibia and fibula, respectively
(Fig. 2D). Approximately 14% of p63C−/− embryos developed
digits with either syndactyly or ectrodactyly (Fig. 2D). Together,
these data indicate that, in contrast to the p63-null condition, p63ΔC
proteins support the initial development of limbs but that alterations
in p63 C-terminus function restrict further progression of limb
development.
Fig. 2. Craniofacial and skeletal abnormalities in p63C−/− mice. (A) Gross
appearance of the head of E17.5 WT, p63C−/− and p63-null mice. Whereas
p63-null mice show cleft lip, p63C−/− mice frequently lack this characteristic as
indicated by asterisks. (B) H&E staining of coronal sections of E17.5 WT and
p63C−/− mouse heads showing the absence and presence, respectively, of
cleft of the secondary palate. nc, nasal cavity; p, palatal shelf; t, tongue. Scale
bar: 250 µm. (C) Skeletal staining of E17.5 WT, p63C−/− and p63-null mice
showing relatively well developed forelimbs and hindlimbs in p63C−/− mice
compared with those in p63-null mice. (D) Enlarged views of forelimbs and
hindlimbs of WT and p63C−/− mice. s, scapula; h, humerus; r, radius; u, ulna;
d, digits; fe, femur; fi, fibula; t, tibia.
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Normal development of limbs requires a close interplay between the
ectoderm and underlying mesenchyme (Johnson and Tabin, 1997).
As described above, p63C−/− embryos show more advanced limb
development than p63-null mice, suggesting that epidermal cells
in p63C−/− embryos are still capable of supporting epithelialmesenchymal interactions. Indeed, macroscopic examination
revealed that, whereas p63-null embryos are translucent and
devoid of epidermis by embryonic day (E) 17.5 (supplementary
material Fig. S3C,D), skin covered 60-80% of the body of p63C−/−
embryos at the same developmental stage, although it was severely
eroded (Fig. 3A). Microscopic analysis using Hematoxylin and
Eosin (H&E) staining showed that p63C−/− mouse epidermis is
hypoplastic and significantly thinner than that of WT littermates and
lacks hair follicle development (Fig. 3B,C).
To characterize epidermal cell differentiation in p63C−/− embryos,
we stained sections of the epidermis with markers of basal cells
[cytokeratin 14 (K14; also known as keratin 14, Krt14) and K5], a
marker of the spinous layer (K10) and markers of terminally
differentiated epithelium (loricrin and involucrin) (Eckert and Green,
1986; Freedberg et al., 2001; Hohl et al., 1991; Ivanyi et al., 1989).
Our data show that marker expression was comparable in p63C−/−
and WT epidermis (Fig. 3D), suggesting that the hypoplastic
epidermis in p63C−/− mice is not due to a block in differentiation.
Notably, however, staining of E15.5 epidermis with anti-p63
antibody showed that, whereas a large number of p63-positive
cells were found in the suprabasal layer of WT epidermis, the
majority of p63-positive cells in p63C−/− mice remained in the basal
layer (Fig. 3D, top). This hypoplastic characteristic of progenitor
cells was also seen in other epithelial tissues in p63C−/− mice
(supplementary material Fig. S4). For instance, developing eyelids
remained thinner and unfused in p63C−/− mice, whereas the eyelids
of WT littermates are densely populated by p63-positive epithelial
progenitor cells and are completely fused at the midline. Likewise,
dental epithelial progenitors expand and develop into a large
number of outer and inner enamel epithelia of the molar in WT
DEVELOPMENT
Epidermal phenotypes in p63C−/− mice
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p63C−/− mice were low to negative for this marker (Fig. 3E,F),
suggesting that epidermal progenitor cells in p63C−/− mice are less
proliferative than those in the WT control. We extended our analysis
of S66/68 phosphorylation to tongue epithelium, another epithelial
tissue showing hypoplasia in p63C−/− mice, and obtained similar
results (supplementary material Fig. S5A).
To determine whether increased apoptosis promotes the
hypoplastic epidermis observed in p63C−/− mice, we stained
sections prepared from p63C−/− and WT embryos with anti-cleaved
caspase 3 antibody. However, no evidence of enhanced apoptosis in
p63C−/− mouse epidermis was detected at E15.5 (supplementary
material Fig. S6), indicating that increased apoptosis does not account
for the hypoplastic epidermis of p63C−/− mice. Collectively, these
data demonstrate that the C-terminus of p63 plays a crucial role in
regulating the expansion of epidermal progenitor cells but is
dispensable for their differentiation and survival.
Decreased proliferative capacity of epidermal progenitor
cells in p63C−/− mice
embryos, but they remain in only a few cell layers in p63C−/− mice.
Together, these data indicate that epithelia remain hypoplastic in
many tissues of p63C−/− mice due to reduced expansion of
epithelial progenitor cells.
We have shown previously that phosphorylation of serine residues
at position 66/68 of ΔNp63 is associated with expansion of epidermal
progenitor cells (Suzuki and Senoo, 2012, 2013). To determine
whether S66/68 phosphorylation of ΔNp63 is reduced in p63C−/−
epidermal cells, we co-stained E15.5 epidermis with anti-pan-p63 and
anti-S66/68 antibodies (Fig. 3E). Our data show that, whereas
virtually all basal cells in WT epidermis exhibit relatively high levels
of S66/68 phosphorylation, the majority of p63-positive cells in
Increased expression of p21Waf1/Cip1 in epidermal progenitor
cells of p63C−/− mice
As epidermal progenitor cells in p63C−/− mice showed reduced
cell cycle progression (Fig. 4), we next investigated expression of
the CDK inhibitor p21Waf1/Cip1, a well characterized downstream
target of p63 (Su et al., 2013) with growth suppressive function in
keratinocytes (Di Cunto et al., 1998; Missero et al., 1996). We first
stained sections of E15.5 WT and p63C−/− epidermis with antip21Waf1/Cip1 antibody and found that, unlike the WT control,
p63C−/− epidermis had a high frequency of epidermal cells with
high p21Waf1/Cip1 expression (Fig. 5A). We confirmed that
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DEVELOPMENT
Fig. 3. Epidermal hypoplasia in p63C−/− mice. (A) Representative E17.5 WT
and p63C−/− mouse embryos (top) and an enlarged view of the p63C−/− mouse
abdomen showing severe skin erosion (bottom). (B) H&E staining of E17.5 WT
and p63C−/− mouse epidermis. Note that p63C−/− mouse epidermis is
significantly thinner than that of WT and lacks hair follicle development.
(C) Changes in epidermal thickness during development (mean±s.d.).
*P<0.01; n.s., not significant. (D) Immunofluorescence of WT and p63C−/−
mouse epidermis with the indicated antibodies, counterstained with Hoechst
33342 (DNA, blue). Arrows indicate p63-positive cells expanding into
the suprabasal layers in WT epidermis. Lori, loricrin; Inv, involucrin.
(E) Immunofluorescence of E15.5 WT and p63C−/− mouse epidermis with antip63 and anti-phosphorylated p63 ( pp63) antibodies, counterstained with
Hoechst 33342. Arrows indicate high p63-positive cells with low
phosphorylation in the basal layer of the p63C−/− mouse epidermis.
(F) Western blot of whole epidermal cell extracts from E15.5 WT and p63C−/−
mice. The membrane was probed first with anti-pp63 antibody, stripped and
then reprobed with anti-pan-p63 antibody. Full, full-length ΔNp63α; ΔC,
ΔNp63ΔC. Scale bars: 50 µm.
Having excluded obvious alterations in differentiation and
apoptosis, we next investigated whether hypoplastic epidermis in
p63C−/− embryos is accompanied by decreased proliferation of
epidermal progenitor cells. We first investigated the proliferation of
p63C−/− epidermal progenitor cells in vivo by injecting BrdU into
pregnant mice of p63C+/− intercrosses 2 h prior to sacrifice,
followed by sectioning and staining of WT and p63C−/− mouse
epidermis with anti-p63 and anti-BrdU antibodies (Fig. 4A).
Notably, our data show that the relative frequency of p63+ BrdU+
epidermal progenitor cells was significantly lower in p63C−/− mice
than in WT littermates at E15.5 (Fig. 4B), indicating that the
C-terminus of p63 is crucial for the proliferation of epidermal
progenitors.
Next, to determine the long-term consequence of loss of the p63
C-terminus for the proliferative capacity of epidermal progenitor
cells, we isolated epidermal cells from E14.0 WT and p63C−/−
embryos and cultured them at clonal density in a defined
keratinocyte medium. Our data show that, in contrast to the
densely packed and highly proliferative WT keratinocytes, p63C−/−
keratinocytes became large and flattened within the first few days,
with reduced growth as determined by Ki67 staining and high
expression of involucrin (Fig. 4C,D). These data are consistent with
our in vivo observation that p63C−/− epidermal cells undergo
premature differentiation with a reduced ability to propagate into
suprabasal cell populations (Fig. 3D,E). Accordingly, extended
cultivation of these keratinocytes showed that, whereas WT cells
grew into large clones, p63C−/− cells did not yield any
macroscopically visible clones at 3 weeks of culture as determined
by Rhodamine B staining (Fig. 4E). Together, these data indicate
that p63C−/− epidermal progenitor cells have a reduced proliferative
capacity compared with their WT counterparts.
Fig. 4. Reduced proliferative capacity of p63C−/− epidermal cells.
(A) Immunofluorescence of E15.5 WT and p63C−/− mouse epidermis with antiBrdU and anti-p63 antibodies, counterstained with Hoechst 33342. Arrows
indicate representative p63+ BrdU+ epidermal progenitor cells. Dotted lines
indicate the epidermal-dermal border. (B) Quantification of A (mean±s.e.m.).
*P<0.01. (C) Epidermal cells were isolated from E14.0 WT and p63C−/− mouse
embryos, seeded at clonal density in Matrigel-coated dishes and cultivated for
7 days. Inset shows high expression of involucrin (red) in p63C−/− epidermal
cells, which is absent in the WT counterpart. Nuclei were counterstained with
Hoechst 33342. (D) Proliferation of WT and p63C−/− mouse epidermal cells at
day 5 of cultivation. Shown are percentages of CK+ Ki67+ epidermal cells
(mean±s.e.m.). *P<0.01. CK refers to pan-cytokeratin. (E) Staining of WT and
p63C−/− mouse epidermal cells with Rhodamine B at 3 weeks of culture. No
macroscopically visible clones were detected in p63C−/− cell cultures. Scale
bars: 25 µm in A,C; 1 cm in E.
expression of p21Waf1/Cip1 was significantly higher in p63C−/−
mice than in WT littermates by western blot using whole epidermal
cell extracts (Fig. 5B). Expression of p53, a strong activator of
p21Waf1/Cip1 expression, was undetectable in epidermal cells from
either p63C−/− or WT mice (supplementary material Fig. S7),
indicating that increased expression of p21Waf1/Cip1 in p63C−/−
mice is not due to dysregulated p53 expression. We also showed
that expression of two other closely related CDK inhibitors,
p27Kip1 (Cdkn1b) and p57Kip2 (Cdkn1c), was similar in p63C−/−
and WT mice (Fig. 5B). As p57Kip2 is a known p63 target gene
(Beretta et al., 2005; Su et al., 2009), these data indicate that
alterations to p63 C-terminus function selectively influence a
subset of p63 target genes.
To determine whether the increase in p21Waf1/Cip1 expression in
p63C−/− mice is specific to the skin epidermis or is common to other
epithelia, we performed additional staining of sections of WT and
p63C−/− mouse esophagus, a tissue that expresses high levels of
p63. Like epidermis, the esophageal epithelium in p63C−/− mice
exhibited high p21Waf1/Cip1 expression, whereas cells with
detectable p21Waf1/Cip1 levels in WT mice were rare (Fig. 5C).
These data suggest that an increase in p21Waf1/Cip1 expression in
p63C−/− mice is common among different epithelial cell types that
normally express p63.
Finally, our data further show that esophageal epithelial cells in
p63-null mice had no detectable p21Waf1/Cip1 expression (Fig. 5C),
indicating that increased expression of p21Waf1/Cip1 in p63C−/− mice
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Fig. 5. Upregulation of p21Waf1/Cip1 expression in p63C−/− mouse
epithelia. (A) Immunofluorescence of E15.5 WT and p63C−/− mouse
epidermis with anti-p21Waf1/Cip1 and anti-pan-cytokeratin (CK) antibodies,
counterstained with Hoechst 33342. Dotted lines indicate epidermal-dermal
border. (B) Western blot of whole epidermal cell extracts from E15.5 WT and
p63C−/− mice with the antibodies to p21Waf1/Cip1, p27Kip1 and p57Kip2. AntiTubulin α antibody was used as a loading control. (C) Immunohistochemistry of
mouse esophagus with anti-p63 (left) and anti-p21Waf1/Cip1 (right) antibodies.
Data shown are representative of WT and p63C−/− mice at E15.5 and p63-null
mice at E17.5. Arrows indicate epidermal cells with high p21Waf1/Cip1
expression. Scale bars: 25 µm in A; 50 µm in C.
does not result from the loss of function of p63 but is instead likely
to reflect altered functions of p63ΔC isoforms. Collectively, these
data suggest that p63ΔC proteins, in the context of either the TAp63
or ΔNp63 isoform or a combination of both, promote p21Waf1/Cip1
expression in p63C−/− epithelia.
Cα balances TAp63 and ΔNp63 functions in the control of
p21Waf1/Cip1 expression
To gain insight into how deletion of the p63 C-terminus promotes
p21Waf1/Cip1 expression, we first compared the transcriptional
activity of TAp63ΔC with that of other TAp63 isoforms in the
non-small cell lung carcinoma cell line H1299 using a minimal
p21Waf1/Cip-luciferase reporter construct that harbors the p63/p53
binding site (Suzuki et al., 2012). H1299 cells are ideal for our study
as they have a homozygous deletion of the p53 gene with no
detectable endogenous p63 expression and thus induce p21Waf1/Cip1
gene expression in response to ectopic p63 (Zeng et al., 2002).
Unlike TAp63α, our data show that TAp63ΔC upregulates
p21Waf1/Cip1 reporter activity to a similar extent as transcriptionally
active TAp63β and TAp63γ isoforms (Fig. 6A). We also show that
the stimulation of the p21Waf1/Cip1 reporter reflects endogenous
p21Waf1/Cip1 expression at both mRNA and protein levels (Fig. 6B,C).
This raises the possibility that the loss of the p63 C-terminus enhances
the total activity of TAp63 in stimulating p21Waf1/Cip1 gene expression
in p63C−/− mice as compared with the WT control. However, as
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Fig. 6. Altered p63ΔC functions in control of p21Waf1/Cip1 expression.
(A-C) Stimulation of p21Waf1/Cip1 expression by TAp63ΔC. H1299 cells were
transfected with 1.0 µg plasmid expressing each p63 isoform. Samples
were prepared 24 h after transfection. (A) Reporter assays using the minimal
p21Waf1/Cip1 reporter (mean±s.d.). RLU, relative light unit. (B) qPCR analysis
of endogenous p21Waf1/Cip1 gene expression. Values are mean±s.d. with
the basal level set to 1.0. *P<0.05. (C) Western blot with anti-p63 and
anti-p21Waf1/Cip1 antibodies. (D-F) Loss of dominant-negative activity of
ΔNp63ΔC against TAp63-dependent transactivation of p21Waf1/Cip1.
(D) Reporter assays using the minimal p21Waf1/Cip1 reporter. H1299 cells were
transfected with 0.3 µg TAp63 plasmid alone or together with an increasing
amount of ΔNp63 plasmid at ratios of 100:1 or 30:1 and cells were lysed 24 h
after transfection. Data shown are mean±s.d. Transactivation by TAp63 alone
was set to 1.0. (E) qPCR analysis of endogenous p21Waf1/Cip1 gene
expression. H1299 cells were transfected with 1.0 µg TAp63 plasmid alone or
together with ΔNp63 plasmid at a 50:1 ratio and RNA was extracted 24 h
after transfection. Values are mean±s.d. with the basal level set to 1.0.
*P<0.05. (F) Western blot analysis of p21Waf1/Cip1 expression. H1299 cells
were transfected with 1.0 µg TAp63 plasmid alone or together with ΔNp63
plasmid at a 20:1 ratio and cells were lysed 36 h after transfection. The same
cell extracts were used to verify expression of each p63 isoform (top).
(C,F) Anti-tubulin α antibody was used as a loading control.
ΔNp63α normally counterbalances TAp63-dependent transcriptional
activity in a WT setting (Yang et al., 1998), we next determined
whether ΔNp63ΔC has an impaired dominant-negative activity
against TAp63γ and TAp63ΔC, two transcriptionally active TAp63
isoforms expressed in p63C−/− mice. Indeed, our data show that,
whereas ΔNp63α inhibited both TAp63β- and TAp63γ-dependent
transcriptional activation of the p21Waf1/Cip1 reporter in a dosedependent manner, ΔNp63ΔC lacked such ability against both the
TAp63γ and TAp63ΔC isoforms (Fig. 6D). We confirmed that
changes in p21Waf1/Cip1 reporter activity correlated with those of
endogenous p21Waf1/Cip1 expression at both the mRNA and protein
levels (Fig. 6E,F). We conclude that Cα balances TAp63 and ΔNp63
isoform functions in the control of p21Waf1/Cip1 expression.
DISCUSSION
Studies of mice deficient for p63 have shown that it is a key
regulator of ectodermal development (Mills et al., 1999; Yang et al.,
1999). In humans, mutations in the p63 gene (TP63) cause at least
seven syndromic and non-syndromic disorders, each with different
combinations of ectodermal dysplasia, split hand/foot malformation
Development (2015) 142, 282-290 doi:10.1242/dev.118307
and orofacial clefting (Rinne et al., 2007). Our data show that
p63C−/− mice exhibit all of these characteristics (Figs 2 and 3),
indicating that the C-terminus is central to p63 function during
embryonic development. Cα is frequently mutated or deleted in
AEC patients and indeed p63C−/− mice show AEC-like severe skin
erosion and fragility (Fig. 3A). However, the presence of
abnormalities in orofacial and limb development in p63C−/− mice
indicates that they will also be useful for assessment of additional
p63-associated disorders. Specifically, relatively rare mutations that
lead to the generation of p63ΔC-like proteins are found in patients
with EEC syndrome and LMS, both of which exhibit the three p63associated characteristics mentioned above (Celli et al., 1999; van
Bokhoven et al., 2001). Our studies reveal that loss of Cα leads to
the dysregulation of both TAp63 and ΔNp63 isoform functions
(Fig. 6). Although these changes can be explained by the loss of
function of the TI domain, the SAM domain also plays a key role in
regulating p63 function in vivo (Ferone et al., 2012). Thus, it will be
important to determine the specific roles of the p63SAM and p63TI
domains and whether a mutation in one domain affects the function
(s) of the other during development. Answering these questions will
contribute to our understanding of the molecular mechanisms
underlying p63-associated EDs.
As a member of the p53 family of transcription factors, p63 is
likely to carry out its function through controlling the expression of
genes that are crucial for stem cell properties (Yang et al., 2006).
p21Waf1/Cip1 is one of the best-characterized downstream targets of
p63, and the growth-suppressing function of this CDK inhibitor is
well established. However, how p21Waf1/Cip1 expression is regulated
in epidermal progenitor cells in vivo remains an open question
(Ohtani et al., 2007). In the present study, we found that p63 can
regulate p21Waf1/Cip1 gene expression through a mechanism that is
dependent on the function of the p63 C-terminus. Our data suggest
that the p63 C-terminus regulates the functional equilibrium
between TAp63 and ΔNp63 isoforms in controlling target gene
expression (Fig. 6). However, as p63C−/− epidermal cells show
significantly elevated levels of p21Waf1/Cip1 but not of the closely
related p63 target p57Kip2 (Beretta et al., 2005; Su et al., 2009), our
studies indicate that the p63 C-terminus influences only a subset of
downstream target genes. Further investigation of the mechanistic
basis for this effect will provide significant insight into stem cell
regulatory pathways governed by p63.
Notably, p21Waf1/Cip1 KO mice have an increased number of
epidermal progenitor cells with high proliferative potential compared
with WT mice (Topley et al., 1999), indicating that p21Waf1/Cip1 plays
a key role in restricting the proliferative capacity of epidermal
progenitors. The function of p21Waf1/Cip1 in stem cell maintenance is
operative in other cell types, including hematopoietic and neuronal
progenitor cells (Cheng et al., 2000; Kippin et al., 2005). However, as
these cells do not express p63, it would be interesting to investigate
whether cell type-specific regulators of stem cell maintenance
(Lessard and Sauvageau, 2003; Molofsky et al., 2003; Park et al.,
2003) are involved in the regulation of p21Waf1/Cip1 expression. We
show in the present study that epidermal progenitor cells derived from
p63C−/− mice have reduced proliferative capacity with enhanced
p21Waf1/Cip1 expression (Figs 4 and 5). Thus, a connection has been
established between p63 C-terminus functions and cell cycle
progression in control of the proliferative potential of epidermal
progenitor cells. However, as we have reported previously, a complete
absence of all p63 isoforms also reduces the proliferative capacity of
epidermal progenitor cells (Senoo et al., 2007). Thus, it is likely that
alternative pathways exist by which p63 regulates the proliferative
potential of epidermal progenitor cells independently of p21Waf1/Cip1
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MATERIALS AND METHODS
Animals
All procedures were performed in accordance with a protocol approved by
the Institutional Animal Care and Use Committee at the University of
Pennsylvania. For staged embryos, the day of the vaginal plug was
designated E0.5.
Generation of p63C−/− mice
Genomic p63 DNA was isolated from BAC clones (Children’s Hospital
Oakland Research Institute, Oakland, CA, USA). The targeting vector
consists of a 3.2 kb HindIII/HincII homologous fragment containing exon
11 followed by a PGK-NeoR cassette flanked by loxP sites and a 2.3 kb
homologous fragment containing exon 13, and a PGK-TK cassette at the
3′-end. Gene targeting was performed in V6.5 (129×B6 F1 hybrid)
embryonic stem cells (ESCs) (Eggan et al., 2001). Two correctly targeted
ESC clones were microinjected into C57BL/6 blastocysts to obtain chimeric
mice. Male chimeras were mated to female C57BL/6 mice to achieve
germline transmission of the targeted (Neo) allele and heterozygous mutant
mice were crossed with Actβ-Cre transgenic mice in a FVB/N background
(Jackson Labs) to remove the Neo cassette (ΔNeo allele). Genotyping of
progeny was performed by PCR using tail DNA and the Neo and ΔNeo
allele and Cre transgene primers listed in supplementary material Table S1.
Mutant mouse colony was maintained by intercrossing heterozygous mice
in the absence of the Actβ-Cre transgene
with [32P]dCTP (GE Healthcare) using the Rediprime II DNA Labeling
System (GE Healthcare) and used as probes. To confirm homologous
recombination at the p63 DNA-binding domain, 600 bp and 350 bp
genomic DNA fragments outside the targeting vector were radiolabeled and
used as probes.
Skeletal staining
Skeletal staining was performed as previously described (Wallin et al.,
1994) with some modifications. Briefly, embryos were eviscerated and the
skin removed, then fixed in 95% ethanol for 5 days and then in 100%
acetone for an additional 2 days at room temperature. Specimens were
incubated in staining solution (0.015% Alcian Blue and 0.005% Alizarin
Red S in 90% ethanol and 5% acetic acid) for 3 days at 37°C. Subsequently,
specimens were rinsed in water and kept in 1% KOH for 2 days at room
temperature, followed by clearing in a series of 0.8% KOH/20% glycerol,
0.5% KOH/50% glycerol and 0.2% KOH/80% glycerol for 3-5 days each.
After complete clearing, specimens were stored in 100% glycerol.
Antibodies
The primary antibodies used in this study were mouse anti-p63 (4A4, Santa
Cruz Biotechnology; 1:250), rabbit anti-phosphorylated p63 (#4981,
corresponding to S66/68 of ΔNp63α, Cell Signaling Technology; 1:100),
mouse anti-p21Waf1/Cip1 (F-5, Santa Cruz Biotechnology; 1:50 for
immunohistochemistry and immunofluorescence, and 1:300 for western
blotting), mouse anti-p27Kip1 (p27/Kip1, BD Biosciences; 1:2000), rabbit
anti-p57Kip2 (NBP1-61640, Novus Biologicals; 1:2000), mouse anti-p53
(PAb421, EMD Millipore; 1:2000), rabbit anti-mouse Ki67 (NB600-1209,
Novus Biologicals; 1:50), rat anti-BrdU (ICR1, Abcam; 1:100), rabbit anticleaved caspase 3 (5A1E, Cell Signaling Technology; 1:200), rabbit anticytokeratin 5 (EP1601Y, Abcam; 1:4000), mouse anti-cytokeratin 14 (LL001,
Santa Cruz Biotechnology; 1:300), mouse anti-cytokeratin 10 (RSKE60,
Abcam; 1:400), rabbit anti-loricrin (PRB-145, Covance; 1:1000), mouse antiinvolucrin (SY5, NeoMarkers; 1:1000), mouse anti-pan-cytokeratin (AE1/
AE3, Thermo Fisher Scientific; 1:200), rabbit anti-pan-cytokeratin
(PA521985, Thermo Fisher Scientific; 1:100) and mouse anti-tubulin α
(12G10, Developmental Studies Hybridoma Bank, University of Iowa, Iowa
City, IA, USA; 1:500). Secondary antibodies used for immunofluorescence
were Alexa 488-goat anti-mouse IgG, Alexa 488-goat anti-rabbit IgG, Alexa
594-goat anti-mouse IgG and Alexa 594-goat anti-rabbit IgG (Molecular
Probes). When a combination of mouse and rat primary antibodies was used,
pre-adsorbed DyLight 594-goat anti-mouse IgG and pre-adsorbed DyLight
488-goat anti-rat IgG antibodies (Abcam) were used to avoid cross-reactivity.
For DAB staining and western blot, horseradish peroxidase (HRP)-conjugated
goat anti-mouse IgG (KPL) and HRP-conjugated goat anti-rabbit IgG (Cell
Signaling Technology) were used.
Immunohistochemistry
Mice deficient for all p63 isoforms were generated and maintained in the
same genetic background as described above. The 5′ homologous arm
included a 5.0 kb fragment extending to the end of the coding sequences of
p63 exon 5, ligated in frame to a start codon (ATG)-depleted EGFP
expression cassette derived from pEGFP-N1 (BD Biosciences). A 4.2 kb
SphI fragment harboring p63 exons 8 and 9 was used as the 3′ homologous
arm. Mutant p63 alleles were identified by PCR using tail DNA and the
primer pairs listed in supplementary material Table S1.
Tissues were fixed in 10% buffered formalin overnight at 4°C. Paraffinembedded sections were cut into 6 µm sections and antigen retrieval was
performed by incubating the slides in 0.01 M citric acid buffer ( pH 6.0) at
95°C for 20 min. Sections were then blocked with 10% fetal bovine serum
(FBS) (Atlanta Biologicals) at room temperature for 30 min. Cultured
epidermal cells were fixed in 10% buffered formalin for 10 min and
permeabilized in 0.1% Triton X-100 in phosphate buffered saline (PBS) for
5 min, followed by blocking at room temperature for 30 min. Subsequently,
samples were incubated with primary antibodies overnight at 4°C. After
three washes in PBS containing 0.1% Tween 20, samples were incubated
with either fluorescently labeled secondary antibodies for 1 h at room
temperature followed by counterstaining with Hoechst 33342 (Molecular
Probes), or HRP-conjugated secondary antibodies followed by detection of
the antigens using a DAB reagent kit (KPL).
Southern blot
BrdU labeling and detection
Genomic DNA was digested with restriction enzymes, separated in 0.8%
agarose gels and transferred to nylon membranes (Sigma-Aldrich). To
confirm homologous recombination at the C-terminus of p63, 1.4 kb and a
560 bp genomic fragments outside the targeting vector were radiolabeled
Pregnant mice received an intraperitoneal injection of 100 mg/kg body
weight 5-bromo-2′-deoxyuridine (BrdU) (BD Biosciences). Two hours after
injection, embryos were collected, fixed, dehydrated and embedded in
paraffin blocks. Sections were treated with 2 N HCl for 10 min at 37°C and
Generation of p63-null mice
288
DEVELOPMENT
function. Deciphering gene programs altered by p63 C-terminus
deficiency versus total loss of p63 function will provide further
insight into the molecular mechanisms that regulate proliferative
potential.
Finally, cell proliferation and differentiation are coupled during
embryonic development and in self-renewing tissues of the adult,
including the skin. Skewing this balance toward proliferation can
cause tumorigenesis (Owens and Watt, 2003; Perez-Losada and
Balmain, 2003). In this regard, p63 and p21Waf1/Cip1 might share
common tumor-suppressive machineries. For instance, loss of
p21Waf1/Cip1 cooperates with Ras oncogene (Hras) in the induction
of aggressive and relatively undifferentiated tumors in vivo (Missero
et al., 1996). Conversely, Ras-driven transformation of p53deficient cells is counteracted by ectopic expression of TAp63 in
a p21Waf1/Cip1-dependent manner (Guo et al., 2009), whereas the
ΔNp63 isoform cooperates with Ras to promote tumor-initiating
stem-like proliferation (Keyes et al., 2011). It will be important to
determine the molecular mechanisms by which p63 and loss of
p21Waf1/Cip1 cooperate in the regulation of genes required for the
proliferative potential of epidermal progenitor cells versus those
favoring malignant transformation. Thus, an exciting area of future
study is the close interplay between the classical cell cycle and stem
cell pathways in the control of self-renewal and tumorigenesis of
epithelia.
Development (2015) 142, 282-290 doi:10.1242/dev.118307
RESEARCH ARTICLE
washed three times in PBS, followed by treatment with 0.025% trypsin for
15 min at 37°C. Staining was then performed as described (Suzuki and
Senoo, 2013).
Western blot
Cells were lysed in a protein lysis buffer containing 50 mM Tris-HCl ( pH
6.8), 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate, 10% glycerol,
1 mM phenylmethanesulfonyl fluoride and 1 mM sodium orthovanadate.
After boiling for 5 min, whole cell extracts were subjected to 8-12%
SDS-PAGE followed by western blot.
Development (2015) 142, 282-290 doi:10.1242/dev.118307
minimal p21Waf1/Cip1 reporter and 0.3 µg Renilla luciferase vector pRL-TK
(Promega). The minimal p21Waf1/Cip1 reporter was constructed by inserting
the 72 bp HindIII-SacI fragment harboring the p63/p53 binding site (Suzuki
et al., 2012) of WWP-luc (El-Deiry et al., 1993) into the pGL3-Basic vector
(Promega). Luciferase activities were measured using the Dual-Luciferase
Reporter Assay System (Promega). Firefly luciferase activities were
normalized to Renilla luciferase control activities. In co-transfection
experiments, pcDNA3 was used as carrier so that all wells received the
same total amount of DNA.
Statistical analysis
Cell culture
Human embryonic kidney 293T (HEK293T) cells and human lung
carcinoma H1299 cells were grown in DMEM containing 10% FBS,
10 U/ml penicillin and 100 μg/ml streptomycin in a humidified chamber
at 37°C with 5% CO2. Primary epidermal cells were isolated from
E14.0 mouse embryos. Briefly, embryos were collected into 1.5 ml
microcentrifuge tubes and incubated in 0.25% trypsin at 37°C for 20 min
with gentle agitation every 5 min without disrupting embryo structures. Cell
suspensions were transferred into new tubes, neutralized and centrifuged at
1000 rpm (180 g) for 5 min. Cells were then suspended in CnT-57 basal
keratinocyte medium supplemented with growth supplements (Cellntec)
and 1×105 cells were seeded onto 35 mm dishes coated with Matrigel (BD
Biosciences) and incubated at 37°C with 5% CO2. After 3 weeks of culture,
epidermal cell clones were fixed in 10% buffered formalin and visualized by
staining with 1% Rhodamine B (Sigma-Aldrich).
Student’s t-tests were performed and P<0.05 was considered statistically
significant.
Acknowledgements
We thank Leslie King for critical reading of the manuscript, and Hirokuni Kobayashi
and Malavika Bhattacharya for help in the initial development of this study.
Competing interests
The authors declare no competing financial interests.
Author contributions
D.S. designed and performed the majority of the experiments and analyzed data.
N.A.L., R.S., D.S. and M.S. generated mutant mice. M.S. conceived the project,
performed experiments, analyzed data and wrote the manuscript with input from
coauthors.
Funding
Total RNA was purified using Trizol reagent (Life Technologies) and 1 µg
was reverse transcribed using the ProtoScript M-MuLV First Strand cDNA
Synthesis Kit (New England BioLabs). Real-time PCR was performed
using SYBR Green Master Mix (Life Technologies) in an ABI 7900 HT
machine. Relative expression of each gene to the housekeeping gene Rps18
or GAPDH was determined by the ΔΔCt method. Primer sequences used for
qPCR are listed in supplementary material Table S1.
To analyze alternative splicing at the p63 C-terminus in p63C−/− mice,
epidermal cell cDNAs were amplified with a primer pair Exon 11-forward and
Exon 14-reverse. PCR products were separated in 1.2% agarose gels and DNA
fragments corresponding to Cα′ (494 bp) and Cβ′ (400 bp) were purified and
directly sequenced with primer 5′-GTGAGCCAGCTTATCAACCC-3′. Note
that although the Exon 11-forward/Exon 14-reverse primer pair is also able to
detect Cβ transcripts, they were below detection level in WT mice, probably
owing to low expression and competition with Cα transcripts. Therefore, a
second primer pair was used to detect Cβ transcripts: Exon 6-forward and
Exon 12/14-reverse (852 bp). Cγ transcripts were amplified using Exon
6-forward and Exon 10′-reverse (648 bp). Primers are listed in supplementary
material Table S1.
Construction of p63 plasmids
All p63 cDNAs used in this study were of human origin. ΔNp63 isoforms
(α, β and γ) were myc-tagged at the N-terminus and subcloned into the
pcDNA3 vector (Life Technologies), while TAp63 isoforms (α, β and γ)
were 2×HA-tagged at the N-terminus and subcloned into the pcDNA3.1
vector (Life Technologies). To generate p63ΔC isoforms, site-directed
mutagenesis was performed using ΔNp63α in pUK21 as template with
primer pair 5′-TTTCTTAGCGAGGTTGGGCTGTTC-3′ and 5′-TGTTGGCTCCCATGCCATCAGGAA-3′, followed by self-ligation and direct
sequencing to verify correct amplification. Mutant C-terminus was excised
as a BsrGI/XhoI fragment and replaced with that in TAp63α and ΔNp63α.
Transient transfection and reporter assay
H1299 and HEK293T cells were seeded into 12-well plates at 1×105 cells/
well the day before transfection. Transfections were performed using
SuperFect transfection reagent (Qiagen). Twenty-four to 36 h later, cells
were harvested and whole cell extracts and total RNA were prepared for
western blot and qPCR, respectively. For reporter assays, H1299 cells were
transiently transfected with p63-expressing plasmids along with 0.5 µg
This study was supported by grants from the Skin Disease Research Center
[P30AR057217] and National Institutes of Health [R01AR066755] to M.S. Deposited
in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.118307/-/DC1
References
Ali-Khan, S. E. and Hales, B. F. (2003). Caspase-3 mediates retinoid-induced
apoptosis in the organogenesis-stage mouse limb. Birth Defects Res. A Clin. Mol.
Teratol. 67, 848-860.
Barrow, L. L., van Bokhoven, H., Daack-Hirsch, S., Andersen, T., van Beersum,
S. E. C., Gorlin, R. and Murray, J. C. (2002). Analysis of the p63 gene in classical
EEC syndrome, related syndromes, and non-syndromic orofacial clefts. J. Med.
Genet. 39, 559-566.
Beretta, C., Chiarelli, A., Testoni, B., Mantovani, R. and Guerrini, L. (2005).
Regulation of the cyclin-dependent kinase inhibitor p57Kip2 expression by p63.
Cell Cycle 4, 1625-1631.
Bickenbach, J. R., Stern, M. M., Grinnell, K. L., Manuel, A. and Chinnathambi, S.
(2006). Epidermal stem cells have the potential to assist in healing damaged
tissues. J. Investig. Dermatol. Symp. Proc. 11, 118-123.
Blanpain, C. and Fuchs, E. (2009). Epidermal homeostasis: a balancing act of stem
cells in the skin. Nat. Rev. Mol. Cell Biol. 10, 207-217.
Candi, E., Rufini, A., Terrinoni, A., Dinsdale, D., Ranalli, M., Paradisi, A., De
Laurenzi, V., Spagnoli, L. G., Catani, M. V., Ramadan, S. et al. (2006).
Differential roles of p63 isoforms in epidermal development: selective genetic
complementation in p63 null mice. Cell Death Differ. 13, 1037-1047.
Carter, R. A., Engiles, J. B., Megee, S. O., Senoo, M. and Galantino-Homer, H. L.
(2011). Decreased expression of p63, a regulator of epidermal stem cells, in the
chronic laminitic equine hoof. Equine Vet. J. 43, 543-551.
Celli, J., Duijf, P., Hamel, B. C. J., Bamshad, M., Kramer, B., Smits, A. P. T.,
Newbury-Ecob, R., Hennekam, R. C. M., Van Buggenhout, G., van Haeringen, A.
et al. (1999). Heterozygous germline mutations in the p53 homolog p63 are the
cause of EEC syndrome. Cell 99, 143-153.
Cheng, T., Rodrigues, N., Shen, H., Yang, Y.-g., Dombkowski, D., Sykes, M. and
Scadden, D. T. (2000). Hematopoietic stem cell quiescence maintained by
p21cip1/waf1. Science 287, 1804-1808.
Di Cunto, F., Topley, G., Calautti, E., Hsiao, J., Ong, L., Seth, P. K. and Dotto,
G. P. (1998). Inhibitory function of p21Cip1/WAF1 in differentiation of primary
mouse keratinocytes independent of cell cycle control. Science 280, 1069-1072.
Eckert, R. L. and Green, H. (1986). Structure and evolution of the human involucrin
gene. Cell 46, 583-589.
Eggan, K., Akutsu, H., Loring, J., Jackson-Grusby, L., Klemm, M., Rideout,
W. M., III, Yanagimachi, R. and Jaenisch, R. (2001). Hybrid vigor, fetal
overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo
complementation. Proc. Natl. Acad. Sci. USA 98, 6209-6214.
289
DEVELOPMENT
qPCR and RT-PCR
El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent,
J. M., Lin, D., Mercer, W. E., Kinzler, K. W. and Vogelstein, B. (1993). WAF1, a
potential mediator of p53 tumor suppression. Cell 75, 817-825.
Ferone, G., Thomason, H. A., Antonini, D., De Rosa, L., Hu, B., Gemei, M., Zhou,
H., Ambrosio, R., Rice, D. P., Acampora, D. et al. (2012). Mutant p63 causes
defective expansion of ectodermal progenitor cells and impaired FGF signalling in
AEC syndrome. EMBO Mol. Med. 4, 192-205.
Freedberg, I. M., Tomic-Canic, M., Komine, M. and Blumenberg, M. (2001).
Keratins and the keratinocyte activation cycle. J. Invest. Dermatol. 116, 633-640.
Ghadially, R. (2012). 25 years of epidermal stem cell research. J. Invest. Dermatol.
132, 797-810.
Green, H. (2008). The birth of therapy with cultured cells. Bioessays 30, 897-903.
Guo, X., Keyes, W. M., Papazoglu, C., Zuber, J., Li, W., Lowe, S. W., Vogel, H.
and Mills, A. A. (2009). TAp63 induces senescence and suppresses
tumorigenesis in vivo. Nat. Cell Biol. 11, 1451-1457.
Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. and Elledge, S. J. (1993). The
p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent
kinases. Cell 75, 805-816.
Hohl, D., Mehrel, T., Lichti, U., Turner, M. L., Roop, D. R. and Steinert, P. M.
(1991). Characterization of human loricrin. Structure and function of a new class of
epidermal cell envelope proteins. J. Biol. Chem. 266, 6626-6636.
Ivanyi, D., Ansink, A., Groeneveld, E., Hageman, P. C., Mooi, W. J. and Heintz,
A. P. M. (1989). New monoclonal antibodies recognizing epidermal differentiationassociated keratins in formalin-fixed, paraffin-embedded tissue. Keratin 10
expression in carcinoma of the vulva. J. Pathol. 159, 7-12.
Johnson, R. L. and Tabin, C. J. (1997). Molecular models for vertebrate limb
development. Cell 90, 979-990.
Kaur, P. (2006). Interfollicular epidermal stem cells: identification, challenges,
potential. J. Invest. Dermatol. 126, 1450-1458.
Keyes, W. M., Pecoraro, M., Aranda, V., Vernersson-Lindahl, E., Li, W., Vogel,
H., Guo, X., Garcia, E. L., Michurina, T. V., Enikolopov, G. et al. (2011). ΔNp63α
is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell
proliferation and tumorigenesis. Cell Stem Cell 8, 164-176.
Kippin, T. E., Martens, D. J. and van der Kooy, D. (2005). p21 loss compromises
the relative quiescence of forebrain stem cell proliferation leading to exhaustion of
their proliferation capacity. Genes Dev. 19, 756-767.
Koster, M. I. (2010). p63 in skin development and ectodermal dysplasias. J. Invest.
Dermatol. 130, 2352-2358.
Lessard, J. and Sauvageau, G. (2003). Bmi-1 determines the proliferative capacity
of normal and leukaemic stem cells. Nature 423, 255-260.
Mills, A. A., Zheng, B., Wang, X.-J., Vogel, H., Roop, D. R. and Bradley, A. (1999).
p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature
398, 708-713.
Missero, C., Calautti, E., Eckner, R., Chin, J., Tsai, L. H., Livingston, D. M. and
Dotto, G. P. (1995). Involvement of the cell-cycle inhibitor Cip1/WAF1 and the
E1A-associated p300 protein in terminal differentiation. Proc. Natl. Acad. Sci. USA
92, 5451-5455.
Missero, C., Di Cunto, F., Kiyokawa, H., Koff, A. and Dotto, G. P. (1996). The
absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and
promotes ras-tumor progression. Genes Dev. 10, 3065-3075.
Molofsky, A. V., Pardal, R., Iwashita, T., Park, I.-K., Clarke, M. F. and Morrison,
S. J. (2003). Bmi-1 dependence distinguishes neural stem cell self-renewal from
progenitor proliferation. Nature 425, 962-967.
Ohtani, N., Imamura, Y., Yamakoshi, K., Hirota, F., Nakayama, R., Kubo, Y.,
Ishimaru, N., Takahashi, A., Hirao, A., Shimizu, T. et al. (2007). Visualizing the
dynamics of p21(Waf1/Cip1) cyclin-dependent kinase inhibitor expression in living
animals. Proc. Natl. Acad. Sci. USA 104, 15034-15039.
Osada, M., Ohba, M., Kawahara, C., Ishioka, C., Kanamaru, R., Katoh, I., Ikawa,
Y., Nimura, Y., Nakagawara, A., Obinata, M. et al. (1998). Cloning and functional
analysis of human p51, which structurally and functionally resembles p53. Nat.
Med. 4, 839-843.
Owens, D. M. and Watt, F. M. (2003). Contribution of stem cells and differentiated
cells to epidermal tumours. Nat. Rev. Cancer 3, 444-451.
Park, I.-K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L.,
Morrison, S. J. and Clarke, M. F. (2003). Bmi-1 is required for maintenance of
adult self-renewing haematopoietic stem cells. Nature 423, 302-305.
Pellegrini, G., Rama, P., Mavilio, F. and De Luca, M. (2009). Epithelial stem cells in
corneal regeneration and epidermal gene therapy. J. Pathol. 217, 217-228.
Perez-Losada, J. and Balmain, A. (2003). Stem-cell hierarchy in skin cancer. Nat.
Rev. Cancer 3, 434-443.
Qiao, F. and Bowie, J. U. (2005). The many faces of SAM. Sci. STKE. 286, pre7.
Rama, P., Matuska, S., Paganoni, G., Spenelli, A., De Luca, M. and Pellegrini, G.
(2010). Limbal stem-cell therapy and long-term corneal regeneration.
N. Engl. J. Med. 363, 147-155.
Rinne, T., Brunner, H. G. and van Bokhoven, H. (2007). p63-associated disorders.
Cell Cycle 6, 262-268.
290
Development (2015) 142, 282-290 doi:10.1242/dev.118307
Rinne, T., Bolat, E., Meijer, R., Scheffer, H. and van Bokhoven, H. (2009).
Spectrum of p63 mutations in a selected patient cohort affected with
ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC).
Am. J. Med. Genet. A 149A, 1948-1951.
Romano, R.-A., Smalley, K., Magraw, C., Serna, V. A., Kurita, T., Raghavan, S.
and Sinha, S. (2012). ΔNp63 knockout mice reveal its indispensable role as a
master regulator of epithelial development and differentiation. Development 139,
772-782.
Schmale, H. and Bamberger, C. (1997). A novel protein with strong homology to
the tumor suppressor p53. Oncogene 15, 1363-1367.
Senoo, M. (2013). Epidermal stem cells in homeostasis and wound repair of the
skin. Adv. Wound Care 2, 273-282.
Senoo, M., Seki, N., Ohira, M., Sugano, S., Watanabe, M., Tachibana, M.,
Tanaka, T., Shinkai, Y. and Kato, H. (1998). A second p53-related protein, p73L,
with high homology to p73. Biochem. Biophys. Res. Commun. 248, 603-607.
Senoo, M., Pinto, F., Crum, C. P. and McKeon, F. (2007). p63 is essential for the
proliferative potential of stem cells in stratified epithelia. Cell 129, 523-536.
Serber, Z., Lai, H. C., Yang, A., Ou, H. D., Sigal, M. S., Kelly, A. E., Darimont,
B. D., Duijf, P. H. G., van Bokhoven, H., McKeon, F. et al. (2002). A C-terminal
inhibitory domain controls the activity of p63 by an intramolecular mechanism.
Mol. Cell. Biol. 22, 8601-8611.
Su, X., Paris, M., Gi, Y. J., Tsai, K. Y., Cho, M. S., Lin, Y.-L., Biernaskie, J. A.,
Sinha, S., Prives, C., Pevny, L. H. et al. (2009). TAp63 prevents premature aging
by promoting adult stem cell maintenance. Cell Stem Cell 5, 64-75.
Su, X., Chakravarti, D. and Flores, E. R. (2013). p63 steps into the limelight: crucial
roles in the suppression of tumorigenesis and metastasis. Nat. Rev. Cancer 13,
136-143.
Suzuki, D. and Senoo, M. (2012). Increased p63 phosphorylation marks early
transition of epidermal stem cells to progenitors. J. Invest. Dermatol. 132,
2461-2464.
Suzuki, D. and Senoo, M. (2013). Expansion of epidermal progenitors with high p63
phosphorylation during wound healing of mouse epidermis. Exp. Dermatol. 22,
374-376.
Suzuki, H., Ito, R., Ikeda, K. and Tamura, T.-a. (2012). TATA-binding protein (TBP)like protein is required for p53-dependent transcriptional activation of upstream
promoter of p21Waf1/Cip1 gene. J. Biol. Chem. 287, 19792-19803.
Thanos, C. D. and Bowie, J. U. (1999). p53 Family members p63 and p73 are SAM
domain-containing proteins. Protein Sci. 8, 1708-1710.
Topley, G. I., Okuyama, R., Gonzales, J. G., Conti, C. and Dotto, G. P. (1999). p21
(WAF1/Cip1) functions as a suppressor of malignant skin tumor formation and a
determinant of keratinocyte stem-cell potential. Proc. Natl. Acad. Sci. USA 96,
9089-9094.
Trink, B., Okami, K., Wu, L., Sriuranpong, V., Jen, J. and Sidransky, D. (1998).
A new human p53 homologue. Nat. Med. 4, 747-748.
Truong, A. B., Kretz, M., Ridky, T. W., Kimmel, R. and Khavari, P. A. (2006). p63
regulates proliferation and differentiation of developmentally mature
keratinocytes. Genes Dev. 20, 3185-3197.
van Bokhoven, H. and McKeon, F. (2002). Mutations in the p53 homolog p63:
allele-specific developmental syndromes in humans. Trends Mol. Med. 8,
133-139.
van Bokhoven, H., Hamel, B. C. J., Bamshad, M., Sangiorgi, E., Gurrieri, F.,
Duijf, P. H. G., Vanmolkot, K. R. J., van Beusekom, E., van Beersum, S. E. C.,
Celli, J. et al. (2001). p63 Gene mutations in EEC syndrome, limb-mammary
syndrome, and isolated split hand-split foot malformation suggest a genotypephenotype correlation. Am. J. Hum. Genet. 69, 481-492.
Wallin, J., Wilting, J., Koseki, H., Fritsch, R., Christ, B. and Balling, R. (1994).
The role of Pax-1 in axial skeleton development. Development 120, 1109-1121.
Watt, F. M. and Jensen, K. B. (2009). Epidermal stem cell diversity and quiescence.
EMBO Mol. Med. 1, 260-267.
Westfall, M. D., Mays, D. J., Sniezek, J. C. and Pietenpol, J. A. (2003). The Delta
Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and
has transcriptional repressor activity that is reduced by Hay-Wells syndromederived mutations. Mol. Cell. Biol. 23, 2264-2276.
Yang, A., Kaghad, M., Wang, Y., Gillett, E., Fleming, M. D., Dö tsch, V., Andrews,
N. C., Caput, D. and McKeon, F. (1998). p63, a p53 homolog at 3q27-29,
encodes multiple products with transactivating, death-inducing, and dominantnegative activities. Mol. Cell 2, 305-316.
Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R. T., Tabin,
C., Sharpe, A., Caput, D., Crum, C. et al. (1999). p63 is essential for regenerative
proliferation in limb, craniofacial and epithelial development. Nature 398, 714-718.
Yang, A., Zhu, Z., Kapranov, P., McKeon, F., Church, G. M., Gingeras, T. R. and
Struhl, K. (2006). Relationships between p63 binding, DNA sequence,
transcription activity, and biological function in human cells. Mol. Cell 24, 593-602.
Zeng, S. X., Dai, M.-S., Keller, D. M. and Lu, H. (2002). SSRP1 functions as a coactivator of the transcriptional activator p63. EMBO J. 21, 5487-5497.
Zhang, Y., Yan, W. and Chen, X. (2014). P63 regulates tubular formation via
epithelial-to-mesenchymal transition. Oncogene 33, 1548-1557.
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