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First published online 8 March 2006
doi: 10.1242/dev.02325
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1 Institute of Biotechnology, University of Helsinki, 00014 Helsinki,
Finland.
2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Author for correspondence (e-mail:
irma.thesleff{at}helsinki.fi)
Accepted 8 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Np63) was expressed at high levels in embryonic ectoderm at all stages
of tooth and hair development, and it was already dominant over the
transactivating TAp63 isoform prior to epidermal stratification. Bmp7,
Fgfr2b, Jag1 and Notch1 transcripts were co-expressed with
Np63 in wild-type embryos, but were not detectable in the ectoderm of
p63 mutants. In addition, ß-catenin and Edar
transcripts were significantly reduced in skin ectoderm. We also demonstrate
that BMP2, BMP7 and FGF10 are potent inducers of p63 in cultured
tissue explants. Hence, we suggest that p63 regulates the morphogenesis of
surface ectoderm and its derivatives via multiple signalling pathways.
Key words: BMP7, FGF10, FGFR2b, Notch1, ß-Catenin
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). p63 possesses
at least two promoters that direct the expression of two fundamentally
different classes of protein, namely one which contains an N-terminal
transactivation (TA) domain and one which lacks this domain, the N-terminally
truncated (N) isoform (Yang et al.,
1998). Extensive alternative splicing is seen at the 3' end
of p63 transcripts resulting in three different C termini: 
, ß and
. Exogenously expressed p63 can bind to consensus p53 target sequences,
and can activate and repress the promoters of several p53 responsive genes. It
is generally assumed that Np63 isoforms are repressor molecules against
TAp63 and p53. However, the physiological targets, either induced or repressed
by p63, are largely unknown (Levrero et
al., 2000; Westfall and
Pietenpol, 2004).
Despite the high similarity in their transcriptional activities, members of
the p53 family seem to play mostly distinct functions in tumour suppression
and development (Melino et al.,
2003). Heterozygous germline mutations in p63 result in a
plethora of human syndromes involving defective development of the limbs,
and/or ectodermal dysplasia characterised by defects in skin and its
associated structures (van Bokhoven and
McKeon, 2002). Mice lacking all p63 isoforms die at birth and show
severe developmental abnormalities, including limb truncations, and defects in
the epidermis and its appendages (Mills et
al., 1999; Yang et al.,
1999). The surface epithelium is thin, lacks stratification, and
does not express markers of epithelial differentiation. The epithelial
phenotype has been interpreted to result from either a lack of commitment of
the immature ectoderm to epidermal lineages
(Mills et al., 1999), or a
lack of proliferative potential of the p63-deficient epidermal stem cells
(Yang et al., 1999).
Ectodermal organs such as hairs, whiskers, teeth and several glands, including
mammary, salivary and lacrimal glands, are lacking in p63-deficient
mice (Mills et al., 1999;
Yang et al., 1999).
A common theme in the development of ectodermal organs is that their
morphogenesis is regulated by a complex series of reciprocal interactions
between epithelial and mesenchymal tissues. Tooth morphogenesis is governed by
interactions between the oral ectoderm and neural crest-derived mesenchyme.
Development starts from the dental lamina, which forms as a stripe of
thickened epithelium at the site of the future dental arch. The initiation of
both molars and incisors becomes morphologically visible in the mouse at
embryonic day 11 (E11) when the dental lamina epithelium thickens locally. The
dental placodes form from this epithelium at E12. The placodal epithelium buds
into the mesenchyme during E13, and subsequent epithelial growth and folding
determine the shape of the tooth crown
(Jernvall and Thesleff, 2000).
The program of hair follicle morphogenesis is similar to that of tooth
morphogenesis; the onset of both of these morphogenetic processes is heralded
by the formation of the ectodermal placode
(Pispa and Thesleff, 2003).
The primary pelage hair placodes are visible at E14 in the dorsal back skin;
these will form the guard hairs. The placodes bud into the mesenchyme and
undergo morphogenesis in co-operation with the mesenchymal dermal papilla
(Millar, 2002).
The molecular mechanisms regulating the development of distinct epithelial
organs are shared to a great extent (Pispa
and Thesleff, 2003). Many genes required for placode initiation
have been identified in studies of genetically modified mice. Inhibition of
the WNT pathway by overexpression of the WNT inhibitor DKK1 caused
developmental arrest prior to the placode stage in all ectodermal organs
analysed (Andl et al., 2002).
The ectodermal organ phenotype is similar also in Msx1/Msx2
double-mutant mice and indicates a role of BMP signalling in placode formation
(Bei and Maas, 1998). The
formation of tooth placodes is inhibited in double mutants of
Dlx1/Dlx2 and Gli2/Gli3
(Thomas et al., 1997;
Hardcastle et al., 1998),
highlighting the importance of FGF and SHH signalling for placode formation.
Furthermore, signalling by EDA via the EDAR receptor is required for the
placodes of guard hairs to be initiated
(Thesleff and Mikkola, 2002a),
and stimulation of this specific TNF signal pathway stimulates placode
formation in several ectodermal organs
(Mustonen et al., 2004).
Placode development also requires an intricate control of cell adhesion
(Jamora et al., 2003). Thus,
formation of the ectodermal placode requires the coordination of multiple
signalling pathways. However, precisely how these diverse pathways are
integrated is currently unknown.
In this study, we have examined the function of p63 in tooth and
hair development. We show that it is required for the formation of individual
dental and hair placodes, but not for the specification of the dental field.
Intriguingly, the truncated Np63 isoform was the main isoform
expressed at all stages of development. Our results indicate that Bmp7,
Fgfr2b, jagged 1 (Jag1) and Notch1 lie downstream of
p63, whereas BMP2, BMP7 and FGF10 were potent inducers of p63 expression in
cultured tissue explants. We conclude that Np63 integrates multiple
signalling pathways required for the formation of tooth and hair placodes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
De Moerlooze et al., 2000).
Mice were mated overnight, and the day of formation of a vaginal plug was
taken as embryonic day 0.
Histology
The embryonic and postnatal tissues for histology, radioactive in situ
hybridisation and immunostaining were fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS; 4°C; overnight), dehydrated, embedded in
paraffin wax, and serially sectioned at 7 µm. Sections for normal histology
were stained with Haematoxylin and Eosin.
Organ cultures
First mandibular molar tooth germs were dissected from E11-E14 mouse
embryos and cultured with protein-releasing beads as previously described
(Laurikkala et al., 2001;
Laurikkala et al., 2002). The
recombinant proteins were: activin A, FGF4, FGF8, FGF10 and SHH (50 ng/µl;
all from R&D Systems, Abingdon, UK); BMP2 (75 ng/µl, a kind gift from
J. Wozney, Genetics Institute); BMP7 (100 ng/µl, kind gift from Creative
Biomolecules, USA); EGF (25ng/µl; Boehringer Mannheim, Germany); TGFß1
(10 ng/µl; R&D Systems) and bovine serum albumin (BSA; 1 µg/µl;
Sigma). The NIH3T3 cell line expressing WNT6
(Kettunen et al., 2000) was a
kind gift from Seppo Vainio.
In situ hybridisation and immunohistochemistry
Radioactive in situ hybridisation was carried out as described earlier
(Wilkinson and Green 1990).
Probes were labelled with 35S-UTP (Amersham) and exposure time was
10-14 days. Whole-mount in situ hybridisation was performed as described
earlier (Raatikainen-Ahokas et al.,
2000), by using the InSituPro Robot (Intavis AG, Germany). The
digoxigenin-labelled probes were detected with BM Purple AP Substrate
Precipitating Solution (Boehringer Mannheim Gmbh, Germany). The p63
probe (pan-p63) has been described (Mills
et al., 1999); the Fgf20 probe was a kind gift from Dr N.
Itoh (Kyoto University). The probe detecting a 324 bp fragment of murine
Pvrl1 (nucleotides 58-382, GenBank Accession Number AF 297665) was
made by cloning the PCR fragment into the pCRII-TOPO vector (Invitrogen).
Probes specific for the transactivating (TA) and N-terminally truncated
(N) isoforms of p63 were made by cloning a 269 bp PCR fragment of
murine TAp63 (nucleotides 35-303, GenBank Accession Number AF 075434)
and a 154 bp PCR fragment from Np63 cDNA (nucleotides 20-173,
EST BB 649754) into pCRII-TOPO (Invitrogen). Details for other probes, all
previously described, are available upon request. Immunostaining was performed
as described earlier (Laurikkala et al.,
2002). The primary antibodies used were anti-p63 (4A4, 1:500;
NeoMarkers) and anti-Np63 (sc-8609, 1:100, Santa Cruz
Biotechnology).
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed as described previously
(Alberts et al., 1998). Back
skin was dissected from E13 NMRI mouse embryos in Dulbecco's PBS. For the
separation of skin epithelium from mesenchyme, the explants were incubated in
0.75% pancreatin (Gibco), 2.25% trypsin (Difco) for 25 minutes at room
temperature. The epithelium was further dissociated by trypsin-EDTA solution
for 30 minutes at room temperature. The cells were allowed to recover in organ
culture medium for 5 minutes at room temperature, fixed with 1% formaldehyde
for 15 minutes at room temperature, lysed (10 mM EDTA, 50 mM Tris, 1% SDS),
sonicated five times for 10 seconds, and diluted 10-fold (1.2 mM EDTA, 16.7 mM
Tris, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS) with a cocktail of proteinase
inhibitors (Complete mini, Roche). After preclearing treatment, cell extracts
were incubated with p63 antibody (4A4; NeoMarkers) overnight at 4°C
followed by precipitation with ProteinA sepharose. Washing and elution of the
immune complexes, as well as precipitation of DNA, were performed as described
previously (Braunstein et al.,
1993). The presence of the putative p63 target sites of the
Bmp7, Notch1 and p21 genes in the immune complexes were
detected by PCR using primers amplifying the following genomic regions (a
detailed description of primers is available upon request): Bmp7 gene, -1797
to -1586, as a control -209 to -90; Notch1, -4949 to -4765, -3402 to -3232,
+13,151 to +13,324, as a control -194 and -25; p21, -2875 to -2715. Positions
of PCR fragments correspond to NCBI Mouse genome Build 33.1.
Quantitative RT-PCR
For real-time RT-PCR, back skin from wild-type E13 embryos was dissected.
In order to get corresponding samples from E9 embryos, head as well as the
most posterior part of the embryo were cut out, all internal organs were
excised, and the remaining trunk skin (including somites) was used for the
analysis. E7 (n=5) and E8 (n=5) whole embryos, or tissues
from individual E9 (n=4) or E13 embryos (n=6), were placed
straight into 350 µl lysis buffer of the RNeasy mini kit (Qiagen)
containing 1% ß-mercaptoethanol. Total RNA was isolated as specified by
the manufacturer and quantified using UV spectroscopy. RNA (100 ng) was
reverse transcribed using random hexamers (Promega) and Superscript II
(Invitrogen), according to the manufacturer's instructions. Quantitative PCR
was carried out using the 2xSYBR-green PCR master mix (Applied
Biosystems) and Applied Biosystems' default PCR conditions for the ABI 7000.
Amplification was performed with the following primers: TAp63 forward,
5'-GTGGATGAACCTTCCGAAAA-3'; Np63 forward,
5'-CAAAACCCTGGAAGCAGAAA-3'; and reverse for both isoforms,
5'-GAGGAGCCGTTCTGAATCTG-3'. This resulted in 158 and 159 bp
products specific for the TAp63 and Np63 isoforms, respectively. PCR
products were run on a 2% agarose gel to verify the absence of non-specific
reaction products and primer dimers. Gene expression was quantified by
comparing the sample data against a dilution series of plasmids containing the
corresponding cDNA fragments of the TAp63 and Np63 isoforms. Data were
analysed using Applied Biosystems' Prism SDS software and normalised against
Hprt.
Western blotting
The open reading frames of the , ß and isoforms of
Np63 were cloned into the pcDNA3 vector (Invitrogen). An optimised
Kozak sequence (ACCACCATG) was tailored at the 5' end of each
construct. NIH3T3 cells were transfected 24 hours prior use with Np63
constructs or with empty vector using Lipofectamine 2000 (Invitrogen), and
directly lysed in Laemmli sample buffer. Dissected skin from E13 and E14
p63-/- mouse embryos or control littermates (combined +/-
and +/+) was homogenised in 2% SDS in PBS by boiling, and by using a syringe
and needle. Protein (20 µg) was separated in 10% SDS-PAGE, transferred onto
a Hybond-C-extra membrane (Amersham) and probed with a p63 antibody (4A4,
1:1000, NeoMarkers), detecting all p63 isoforms; finally, blots were developed
by enhanced chemiluminesence (ECL, Amersham).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the back skin, pan-p63 transcripts were detected throughout the simple ectoderm in E11-E13 embryos (Fig. 1G). At E15, expression was intense in the ectoderm, except for in the most superficial cell layers (Fig. 1H). At E17, pan-p63 staining was seen in the basal epithelial cells and in the maturing stage 3-4 hair follicles of guard hairs, as well as in the initiated epithelial placodes of the awl hairs (Fig. 1I). Expression was also detected in the eye, in tongue mesenchyme, and in some nerves (data not shown).
Because little is known about the spatiotemporal expression of the
different p63 isoforms during embryogenesis, we next analysed the
expression of Np63 and TAp63 isoforms using probes
specific to the different 5' ends of these transcripts. A similar
distribution of p63 in the surface epithelium, hair follicles and teeth was
revealed by the Np63 probe as by the pan-p63 probe (Figs
1,
2). Np63
transcripts were seen throughout the simple oral epithelium and in the dental
lamina at E10 (data not shown), and in all dental epithelia from E11 onwards
(Fig. 2D-G); they were lost
from the inner enamel epithelium when it differentiated into ameloblasts
(Fig. 2I). No expression was
seen in developing teeth and hairs with the TAp63-specific probe
(Fig. 2A-C,H). TAp63
transcripts were detected in some mesenchymal structures of the tongue at E14
and E15, as well as in the eye and in some nerves, in patterns similar to
those seen with the pan-p63 probe (data not shown).
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Np63 isoforms (see Fig. S1 in the supplementary material) confirmed
that Np63 is the major p63 splice variant expressed during embryonic
tooth and hair formation. Protein and mRNA expression patterns corresponded
well with each other. Western blot analysis preformed on wild-type E13 and E14
skin samples revealed that the alpha isoform of p63 is the predominant one
expressed during the initiation of hair development and epidermal
stratification; although a faint band corresponding to the gamma isoform was
also discernible (Fig. 2M).
To further verify our in situ hybridisation and immunohistochemical
results, we analysed E13 skin samples by quantitative RT-PCR
(Fig. 2N). Only one percent of
all p63 represented the TA isoform, confirming our in situ hybridisation
results. We also analysed E7 and E8 whole embryos, as well as E9 skin samples
by RT-PCR. Intriguingly, p63 was not expressed at E7, whereas at E8 and E9
only Np63 was detected (Fig.
2N).
p63 is required for the formation of both tooth and hair placodes
The original phenotypic analyses of p63-/- mice
revealed a lack of tooth and hair development, but the pathogenesis and exact
stage of arrest were not studied (Mills et
al., 1999; Yang et al.,
1999). In order to characterise this defect more thoroughly, we
examined back skin sections and serial frontal sections of the heads and jaws
of p63 mutants and their wild-type littermates between E10 and E17.
No morphological difference was noted in surface ectoderm at E10 (not shown).
At E11, the dental lamina, which forms a horse shoeshaped stripe of
multilayered epithelium in the lower jaw, was morphologically normal in
p63 mutant embryos, and was increased in thickness in the molar and
incisor regions (Fig. 3A,B). At
E12, dental placodes had formed in the wild-type dental lamina at the sites of
incisors and molars, and at E13 they had progressed into tooth buds surrounded
by condensed mesenchyme (Fig.
3C,E). In p63-deficient embryos, development did not
advance from the dental lamina stage. Dental placodes were absent in E12 and
E13 embryos (Fig. 3D,F), and
the thickened dental lamina epithelium appeared to regress in
p63-/- embryos during later stages of development
(Fig. 3G,H; and data not
shown). Only occasionally (2/15), was a rudimentary bud-like structure seen in
a mutant embryo (data not shown).
|
;
Yang et al., 1999). The
placodes of guard and awl hairs, which are seen in the wild-type mouse embryo,
were not detected in p63-deficient embryos
(Fig. 3I,J).
To confirm the presence of the dental lamina in the mutants, we analysed
the expression of two dental lamina markers Pitx2 and Shh in
E11-E12 embryonic lower jaws by whole-mount in situ hybridisation
(Mucchielli et al., 1997;
Keränen et al., 1999).
Pitx2 and Shh transcripts were co-expressed in the dental
lamina both in wild-type and p63 mutant jaws at E11 (data not shown).
At E12, Pitx2 and Shh expression became restricted to the
incisor and molar placodes, and their expression was downregulated in the
diastema region between the incisors and molars in wild-type mandibles;
interestingly, however, their expression remained continuous in the mutant
dental lamina (Fig. 4A-D).
The placodes of guard hairs become morphologically evident in the back skin
at E14 and they can be visualised by the punctuate expression of several
marker genes, such as ß-catenin and Edar
(Huelsken et al., 2001;
Laurikkala et al., 2002).
Whole-mount in situ hybridisation analysis of p63 mutant embryos at
E14 showed that both ß-catenin and Edar were absent
(Fig. 4E-H). In conclusion,
these results show that, in the absence of p63, tooth development arrests at
the dental lamina stage and hair follicle development is not initiated,
suggesting that p63 is required in the ectoderm for the formation of both the
tooth and hair placodes.
Search for downstream targets of p63 by in situ hybridisation
We studied the expression of 29 potential downstream target genes of p63 by
in situ hybridisation analysis. The expression patterns of the following genes
are shown: Shh, Pitx2, Fgf8, Fgf9, Fgfr2b, Bmp4, Bmp7, Msx1, activin
ßA, Notch1, Notch2, Notch3, Jag1, Jag2, Wnt3a, Wnt6,
Wnt10b, ß-catenin, Lef1, Edar, Eda, Tnfrsf19, Pvrl1 and
Ptc1 (Figs 5,
6; see also Figs S2, S3 in the
supplementary material). Expression patterns are not shown for Egfr,
Hes1, lunatic fringe, Msx2 and Pax9. We compared serial
tissue sections from the heads of E10-E12 embryos. The sections included the
molar area, as well as surface ectoderm of the head and neck. In addition,
sections from E12-E14 back skin were examined. We focused on genes that have
been associated with the initiation and morphogenesis of teeth and hairs.
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).
Fgfr2b is expressed in the epithelial cells of developing teeth
(E11-P4) (Kettunen et al.,
1998), and we saw expression in wild-type embryos throughout the
oral epithelium, including the tooth at E11-E12
(Fig. 5A,C). Interestingly,
p63 mutant epithelium completely lacked Fgfr2b expression
(Fig. 5B,D). Similarly,
Fgfr2b transcripts were completely missing from
p63-deficient epidermis at E12
(Fig. 5E,F). FGF3, FGF7 and
FGF10 use exclusively FGFR2b, and are expressed in developing teeth and hair
follicles (Chuong, 1998;
Millar, 2002). Hence, our
results suggest that signalling by these FGFs is impaired in p63 null
embryos.
|
). It was
reported previously that Fgf8 transcripts were severely
downregulated/absent in the p63 mutant limb buds, which fail to
develop further (Mills et al.,
1999; Yang et al.,
1999). However, we detected similar expression domains of
Fgf8 (Fig. 5G,H), as
well as Fgf9, in the branchial arch ectoderm (E10) and in the dental
lamina (E11-E12) in wild-type embryos and p63 mutants (see Fig. S2 in
the supplementary material).
Several BMPs show developmentally regulated expression patterns during
murine tooth morphogenesis (Åberg et
al., 1997). Bmp7 is co-expressed with p63 in the
oral epithelium during the formation of dental lamina and early tooth buds
(Fig. 5I; data not shown).
Interestingly, Bmp7 transcripts were not detected in the oral
epithelium of p63-deficient embryos
(Fig. 5J). Accordingly,
Bmp7 transcripts were also completely absent from the back skin
epithelium of p63 mutants at E14
(Fig. 5K,L). No apparent
changes were detected in the expression of two mesenchymal TGFß
superfamily members, Bmp4 and activin ßA (see Fig. S2 in the
supplementary material).
ß-Catenin, the key mediator of the WNT signals, was expressed similarly in the oral epithelium of wild type and p63 mutants at E12 (Fig. 5M,N). Interestingly, it was significantly downregulated in the surface epithelium of p63 mutants, although some transcripts were occasionally observed (Fig. 5O,P). We also analysed the expression of WNT ligands WNT3, WNT6 and WNT10b in developing teeth. Wnt3 and Wnt6 transcripts were similarly expressed throughout oral and dental epithelia in wild-type and p63-/- embryos (see Fig. S3 in the supplementary material). Wnt10b was expressed throughout the epithelium at E11 and was localised to the tooth placode at E12 in wild-type embryo. By contrast, in p63 null embryos, the Wnt10b hybridisation signal remained continuous throughout the dental and oral epithelia (see Fig. S3 in the supplementary material).
|
;
Mitsiadis et al., 1995;
Fuchs and Segre, 2000). During
the early stages of tooth development (E11-E12), Notch1, Notch2 and
Notch3 were co-expressed in wild-type embryos in the suprabasal cells
of oral and dental epithelium, but the basal cells were devoid of transcripts
(Fig. 6A,C,E,G).
Notch1 expression was not detected in the mutant epithelium at
E11-E12, whereas Notch2 and Notch3 transcripts were
expressed normally (Fig.
6B,D,F,H). The Notch ligands Jag1 and Jag2 were
co-expressed with Notch1 and Notch2 in wild-type dental
epithelium at E11-E12 (Fig.
6I,K). Mutant embryos lacked epithelial Jag1, and the
mesenchymal expression of Jag1 was significantly downregulated
(Fig. 6J). Notch1 and
Jag1 were intensely expressed in dorsal back skin ectoderm in E12 and
E14 wild-type embryos (Fig.
6M,O; data not shown), but their transcripts were generally absent
in the mutants (Fig. 6N,P),
although we occasionally detected Notch1 and Jag1 expression
in some epithelial cells (data not shown). Recently, Jag1 and
Jag2 were shown to be upregulated by ectopic expression of p63 and
p73 in several human cancer cell lines
(Sasaki et al., 2002).
However, we found normal Jag2 expression in p63 mutants
(Fig. 6L).
Mutations in the genes encoding the TNF receptor EDAR and its ligand EDA
cause hypohidrotic ectodermal dysplasia
(Thesleff and Mikkola, 2002a).
Edar is expressed throughout the branchial arch ectoderm in E10
wild-type embryos, and becomes upregulated in the thickened dental epithelium
at E11 and in the dental placode at E12
(Fig. 6Q; data not shown)
(Tucker et al., 2000;
Laurikkala et al., 2001). In
p63 mutants, Edar was expressed as in wild-type mice at E10
(data not shown), but we did not notice localised upregulation at E11 that
occurs in wild-type embryos (Fig.
6R). Edar was also expressed at reduced levels in mutant
epidermis at E12 (Fig. 6S,T).
Expression of Tnfrsf19 (also called Taj or Troy),
which encodes a TNF receptor homologous to EDAR, was unaffected in the
p63 mutant dental epithelium (see Fig. S3 in the supplementary
material). Likewise, expression of Eda, which colocalises with
p63 in the early ectoderm
(Laurikkala et al., 2001;
Laurikkala et al., 2002), was
normal in p63-/- embryos at E10-E12 (Fig. S3 in the
supplementary material).
|
Our in situ hybridisation analysis indicated that Bmp7 and
Notch1 lie downstream of p63. To determine whether p63 directly
regulates their transcription, we performed chromatin immunoprecipitation
(ChIP) assays using freshly isolated E13 epidermal cells. p63 is known to bind
to p53-responsive elements (El-Deiry et
al., 1992; Sasaki et al.,
2002; Westfall et al.,
2003), and therefore we tested several putative p53-responsive
elements found in the promoter/intron regions of Bmp7 and
Notch1 genes in ChIP (see Fig. S4 in the supplementary material). The
p53 binding site (site 1) in the p21 gene, which is known to be
regulated by p63, was used as a positive control
(Westfall et al., 2003). ChIP
analysis revealed that one candidate sequence on the Bmp7 promoter,
as well as one out of three candidate sites in the Notch1 gene, could
be amplified from the p63 immune complexes (Fig. S4 in the supplementary
material).
p63 expression is induced by BMPs and FGF10
To analyse the genes upstream of p63, we studied several
signalling molecules for their ability to regulate p63 expression in
cultured whole tooth explants or isolated dental epithelia (E11-E14). The
expression of p63 was maintained in the epithelium even in the
absence of the mesenchyme (data not shown). Beads releasing BMP2 and BMP7
induced the expression of p63 in the dental epithelium in whole tooth
explants at all stages analysed (E11-E14;
Fig. 7A,B), as well as in the
isolated dental epithelium (data not shown). In addition to BMPs, FGF10
induced the expression of p63, whereas FGF4 and FGF8 did not
(Fig. 7C-E). Similar induction
with BMPs and FGF10 was seen with the Np63-specific probe as
with the pan-p63 probe, whereas no induction of the TAp63
isoform was observed (data not shown). FGF10 signals exclusively via FGFR2b,
yet we found intact expression of p63 in Fgfr2b mutant
epidermis (Fig. 7K,L),
indicating that FGF10 is not necessary for p63 expression. None of
the other signal molecules analysed, including activin A, EGF, SHH, TGFß1
and WNT6, stimulated p63 expression (Fig.
7F-J; data not shown), and we found intact expression of p63 in
Lef1-/- embryonic ectoderm (data not shown).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Np63 isoform, but not the TAp63 isoform, of p63 is expressed in epithelial cells during morphogenesis of the epidermis, hair follicles and teethNp63 isoform is
expressed in the simple ectoderm prior to the morphological onset of tooth and
hair formation. During subsequent stages, Np63 expression continued in
epithelial cells, but was downregulated during the differentiation of
keratinocytes and ameloblasts. Transactivating TAp63 isoforms were not
detected during early development (E8-E9), and only trace amounts were
observed during ectodermal appendage development. These results contradict
with those recently reported by Koster et al., who proposed that TAp63
isoforms dominate over Np63 isoforms in the epidermis until E18.5,
based on RT-PCR analysis (Koster et al.,
2004). The reason for this discrepancy is currently unclear.
However, the degree of normalisation of these PCR reactions was not described
(Koster et al., 2004). On the
basis of our data, we conclude that Np63 is the main isoform expressed
in the developing tooth and hair, as well as in the embryonic epidermis.
Accordingly, Np63 was the only isoform detected in the developing
epidermis in zebrafish, where it is required for proper epidermal and limb
development (Lee and Kimelman,
2002; Bakkers et al.,
2002).
Our in vitro analysis showed that mesenchymal signals are not needed for
the maintenance of p63 expression. Our results indicate that BMPs may
be epithelial signals regulating Np63 expression in embryonic
ectoderm. This is in line with earlier findings showing that the expression of
zebrafish Np63 is activated by Smad4/Smad5-mediated Bmp
signalling, and that Np63 is downregulated in bmp7
mutant zebrafish embryos (Bakkers et al.,
2002). Of the other signalling molecules tested, only FGF10
stimulated Np63 expression in dental epithelium.
Search for downstream targets: Fgfr2b, Jag1, Notch1 and Bmp7 are downstream of Np63
In our search for downstream targets of p63, we found several genes that
were co-expressed with p63 in wild-type embryos but that were
downregulated in p63-deficient embryos. We show, for the first time,
the absence of Bmp7, Jag1, Notch1 and Fgfr2b, and the
downregulation of Edar and ß-catenin, in embryos deficient for
p63. The finding that Np63, which was initially
classified as a non-transactivating molecule
(Yang et al., 2002), is the
main isoform during the crucial early stages of tooth and hair development is
intriguing. However, there are several possibilities as to how these genes
could be regulated by Np63. First, there is increasing evidence that
Np63 isoforms can act as transcriptional activators and regulate the
same genes as the TA isoforms (Ghioni et
al., 2002; Wu et al.,
2003) in a cell-specific manner
(King et al., 2003;
Ihrie et al., 2005), possibly
via a second transactivating domain present within the first 26 N-terminal
amino acids (Dohn et al.,
2001). Indeed, our ChIP results suggest that both Bmp7
and Notch1 could be direct transcriptional targets of
Np63. However, additional experiments will be required to
validate these suggestive data. Previous studies using cells overexpressing
TAp63 have also implicated Jag1 as a direct transcriptional target of
p63 (Sasaki et al., 2002;
Ross and Kadesch, 2004).
Second, Np63 isoforms could regulate the expression of specific
repressors (Wu et al., 2003).
Third, they could exert their effects via protein-protein interactions
(Patturajan et al., 2002).
Recently, Fomenkov et al. showed that wild-type Np63 binds to
ABBP1, a member of the RNA-processing machinery, promoting the formation of
the IIIb splice variant of FGFR2, whereas Np63 harbouring an
ectodermal dysplasia-associated mutation failed to do so
(Fomenkov et al., 2003). In
line with our results, they also noted downregulation of the FGFR2b splice
variant in the skin samples of p63 knockout mice by semi-quantitative
RT-PCR. Apparently, a lack of Np63 expression leads to the absence of
several, direct or indirect, downstream targets via multiple mechanisms.
|
). Also,
the dental lamina, a stripe of thickened epithelium prefiguring the dental
arches in the upper and lower jaws, formed in its normal location in p63
mutants, but dental placodes did not form. Hence, although the epithelial
stratification failed in the p63-deficient surface epidermis, the multilayered
epithelial thickenings were apparently normal in the oral ectoderm. This
contrasts to the limb phenotype of p63-/- embryos, where no
morphologically distinct apical ectodermal ridge can be seen
(Mills et al., 1999;
Yang et al., 1999). In
embryonic skin, we did not detect localised expression of any of the marker
genes of the hair placode, indicating that hair development had already failed
at the initiation stage.
The severity of the dental and hair phenotype of p63 mutants most
likely results from the abrogation of multiple signalling pathways. The
importance of ß-catenin for hair development was highlighted by its
conditional ablation in the epidermis, which resulted in an early block of
hair placode formation that apparently was caused by the inability of the
epithelium to respond to WNT signals
(Huelsken et al., 2001).
Mutations in both p63 and EDAR cause ectodermal dysplasia
syndromes in humans, with quite similar hair and tooth phenotypes
(Kere and Elomaa, 2002;
van Bokhoven and McKeon,
2002), and primary hair placodes do not form in mice lacking the
requisite components of the EDAR signalling pathway
(Thesleff and Mikkola, 2002a).
A role for the Notch pathway in tooth morphogenesis is suggested by the
dynamic expression of several Notch receptors and ligands during tooth
development (Mitsiadis et al.,
1995; Mustonen et al.,
2002). However, the phenotype of mice with conditional ablation of
-secretase, an obligate activator of all Notch receptors, suggests that
the Notch pathway is dispensable for the initiation of hair development
(Pan et al., 2004). The
expression of Bmp7 was also completely downregulated in oral and skin
ectoderm of p63 mutants. Because both teeth and hair form in
Bmp7 mutant embryos (Dudley et
al., 1995; Luo et al.,
1995), BMP7 is conceivably redundant with other BMPs, perhaps
BMP2, in the regulation of ectodermal organogenesis.
Finally, the absence of Fgfr2b, and therefore of FGF3, FGF7 and
FGF10 signalling, in p63-deficient ectoderm may play a prominent role in the
pathogenesis of p63 mutant phenotype. A link between FGFR2b and p63 is
supported by the similar mouse knockout phenotypes, although the phenotype in
ectodermal organs is less severe in the Fgfr2b mutants
(Mills et al., 1999;
Yang et al., 1999;
De Moerlooze et al., 2000;
Petiot et al., 2003). Tooth
development of Fgfr2b-/- mice is arrested at the bud stage
and hair development shows a reduced number of hair placodes. Taken together,
our findings that p63 regulates many genes in different signalling pathways
involved in placode initiation conceivably explain the more universal
inhibition of placode formation in the p63 mouse mutants, as compared
with most mutants where placodes are affected by the inactivation of genes in
one pathway only (Hardcastle et al.,
1998; Satokata and Maas,
1994; Laurikkala et al.,
2002; van Genderen et al.,
1994).
P63 and regulation of epidermal development
The surface epithelium of p63 null embryos is thin, lacks
stratification (Yang et al.,
1999; Mills et al.,
1999), and cultured keratinocytes from these embryos express K18,
a marker of all simple epithelia, including the epidermis prior to
stratification (Koster et al.,
2004). However, our results indicate that molecular
differentiation of the p63-deficient epidermis partially took place, as
Notch2, Notch3 and Jag2, as well as Pvrl1, which
encodes the cell adhesion molecule, nectin 1, that is essential for proper
development of the ectoderm (see Fig. S3 in the supplementary material),
showed normal expression in the suprabasal cells, with concomitant
downregulation in the basal cells.
To date, few p63 target genes involved in epithelial maturation have been
identified, and the cause of the failed ectodermal development of p63
null mice has been a matter of debate
(McKeon, 2004). Absence of a
cohort of genes found in this report may, at least partially, explain the
epidermal phenotype of p63 mutants. The epidermis of Fgfr2b
null embryos is reminiscent of that of p63 mutants in that it is
abnormally thin (De Moerlooze et al.,
2000; Petiot et al.,
2003). It appears that p63 regulates epidermal proliferation (at
least) via FGFR2b, as a reduction in keratinocyte proliferation was reported
to cause the hypoplastic epidermis in Fgfr2b null embryos. In
addition, the proliferative capacity of p63-deficient embryonic
epidermis may further be reduced by p21, which is a target of direct
transcriptional repression by Np63
(Westfall et al., 2003).
However, all of the usual epidermal cell lineages are found in
Fgfr2b-deficient epidermis, indicating that other targets genes of
p63 are involved in triggering differentiation.
Notch signalling has been implicated in epidermal stratification and
keratinocyte differentiation (Rangarajan
et al., 2001; Nickoloff et
al., 2002; Okuyama et al.,
2004). Application of JAG1 to an epidermal equivalent model system
induces epidermal maturation (Nickoloff et
al., 2002), and ectopic expression of activated NOTCH1 in cultured
keratinocytes causes a substantial induction of early differentiation markers
(Rangarajan et al., 2001). Our
observations suggest that NOTCH1 and JAG1 may be important downstream
mediators of p63 during epidermal maturation in vivo, although, apparently,
other target genes such as Perp are also involved
(Ihrie et al., 2005).
The relative contribution of individual p63 isoforms during ectodermal
differentiation and organogenesis is still far from understood. Recently, a
model was put forth that suggests that TAp63 isoforms are the first p63
isoforms to be expressed during embryonic development, and that they are
necessary for the commitment to stratification while simultaneously blocking
the differentiation programme. Therefore, a shift towards Np63 isoforms
during later stages would be required to counterbalance the activity of TAp63,
thereby allowing cells to respond to terminal differentiation cues
(Koster et al., 2004;
Koster and Roop, 2004). Our
results, however, appear to contradict this model, as they indicate that
Np63 isoforms dominate throughout the development of the epidermis and
its appendages. We propose that, during embryonic development, Np63
isoforms have an independent role as transcriptional regulators and do not
merely act as inhibitors towards transactivating molecules of the p53 family.
The clarification of this issue must wait for the generation of
isoform-specific knockouts. In conclusion, we suggest that Np63
isoforms, most notably Np63, are crucial for the development of
the ectoderm and its appendages.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
* These authors contributed equally to this work 
|
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