Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias.
ABSTRACT Ectodermal dysplasias (EDs) are a group of human pathological conditions characterized by anomalies in organs derived from epithelial-mesenchymal interactions during development. Dlx3 and p63 act as part of the transcriptional regulatory pathways relevant in ectoderm derivatives, and autosomal mutations in either of these genes are associated with human EDs. However, the functional relationship between both proteins is unknown. Here, we demonstrate that Dlx3 is a downstream target of p63. Moreover, we show that transcription of Dlx3 is abrogated by mutations in the sterile alpha-motif (SAM) domain of p63 that are associated with ankyloblepharon-ectodermal dysplasia-clefting (AEC) dysplasias, but not by mutations found in ectrodactylyectodermal dysplasia-cleft lip/palate (EEC), Limb-mammary syndrome (LMS) and split hand-foot malformation (SHFM) dysplasias. Our results unravel aspects of the transcriptional cascade of events that contribute to ectoderm development and pathogenesis associated with p63 mutations.
- SourceAvailable from: Guobin Yang[Show abstract] [Hide abstract]
ABSTRACT: Dlx3 is essential for osteoblast differentiation and bone formation, and its expression is regulated by bone morphogenetic protein-2 (BMP-2). However, the intimate mechanism of BMP-2 regulation of Dlx3 transcription in osteoblasts is still unknown. Considering the important roles of Smad5 and p38 in osteoblast differentiation, we hypothesized that Smad5 and p38 mediated BMP-2-induced Dlx3 transcription in osteoblasts. We found activation of Smad5 and p38 increased the expression of Dlx3, whereas knocking down Smad5 or inactivation of p38 inhibited BMP-2-induced Dlx3 expression. Both Smad5 and p38 were able to activate Dlx3 promoter activity and p38/Smad5 response elements were located from -698 to -368 in Dlx3 promoter. Two Smad5 binding sites (SBEI and SBEII, TGTCT box) were identified in this region by EMSA and ChIP assay. Deletions and mutagenesis study of the Dlx3 promoter region indicated that the TGTCT boxes are crucial for p38/Smad5-induced Dlx3 promoter activity. At last, we found a cross-talk between p38 and Smad5, and that activation of p38 is necessary for BMP-2-induced Smad5 phosphorylation and nuclear translocation. Overall, we provide a novel insight that BMP-2-induced Dlx3 expression is regulated by p38/Smad5 signaling pathway in osteoblasts. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.Journal of Cellular Physiology 12/2013; 229(7). · 3.87 Impact Factor
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ABSTRACT: Mouse models have greatly helped in elucidating the molecular mechanisms involved in hair formation and regeneration. Recent publications have reviewed the genes involved in mouse hair development based on the phenotype of transgenic, knockout and mutant animal models. While much of this information has been instrumental in determining molecular aspects of human hair development and cycling, mice exhibit a specific pattern of hair morphogenesis and hair distribution throughout the body that cannot be directly correlated to human hair. In this mini-review, we discuss specific aspects of human hair follicle development and present an up-to-date summary of human genetic disorders associated with abnormalities in hair follicle morphogenesis, structure or regeneration.Seminars in Cell and Developmental Biology 12/2013; · 6.20 Impact Factor
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ABSTRACT: Ectrodactyly, or Split Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the EEC (Ectodermal dysplasia-Ectrodactyly-Cleft lip/palate) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the Apical Ectodermal Ridge (AER) of the developing limb buds. FGF8 is the key signalling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1- ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated to SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations.Human Molecular Genetics 02/2014; · 6.68 Impact Factor
During embryonic development and organ formation, a series of
signals between epithelial cells and the underlying mesenchymal
cells are the basis for the formation of a variety of appendages and/or
organs (Pispa and Thesleff, 2003). Anomalies in epithelial-
mesenchymal-derived organs are characteristics of human
pathological conditions defined as ectodermal dysplasias (EDs)
(Priolo and Lagana, 2001).
Mutations in DLX3 and p63, among other genes, have been
directly linked with EDs. The Dlx and p63 families of transcriptional
effectors are essential for the development of the epidermis and/or
embryonic appendages (Panganiban and Rubenstein, 2002; Merlo
et al., 2003; Morasso and Radoja, 2005; Koster and Roop, 2004).
Dlx3 expression has been detected in the hair follicle, tooth, limb
bud, branchial arches, labyrinthe layer of the placenta, osteoblasts
and epidermis (Morasso et al., 1995; Morasso et al., 1999; Hassan
et al., 2004). Here, we present evidence that Dlx3is regulated by p63
as part of a transcriptional regulatory pathway relevant to specific
p63 regulates multiple signaling pathways, such as the bone
morphogenetic protein (BMP) and fibroblast growth factor (FGF)
pathways (Laurikkala et al., 2006; Barbieri and Pietenpol, 2006).
Transgenic and knockout (KO) mouse models indicate that p63 has
essential roles in the development and maintenance of the stratified
epidermis (Yang et al., 1999; Mills et al., 1999; Koster et al., 2004;
Koster and Roop, 2004). The p63 gene is transcribed from two
distinct promoters, giving rise to proteins that either contain
(TAp63) or lack (?Np63) the amino terminal transactivating
domain. The TA and ?N isoforms both possess the DNA-binding
and oligomerization domains, and, by alternative splicing at the 3?
end, produce isoforms with different C-termini, termed alpha (?),
beta (?) and gamma (?) (Yang et al., 1998). The ? isoforms
contain a sterile ? motif (SAM) – a domain with reputed
importance in protein-protein interactions (Qiao and Bowie, 2005).
p63 isoforms act as transcriptional activators and/or repressors
(Ghioni et al., 2002; King et al., 2003; Wu et al., 2005), and bind
to two or more tandem repeats of RRRCWWGYYY, but
preferentially activate the RRRCGTGYYY sequence (Osada et al.,
Mutations in the p63 gene have been associated with EDs
that include ectrodactyly-ectodermal dysplasia-cleft lip/palate
(EEC), limb-mammary syndrome (LMS), split hand-foot
malformation (SHFM) and
dysplasia-clefting (AEC) syndrome. There is a correlation
between the position of the mutation and the observed abnormal
phenotype (van Bokhoven et al., 2001; McGrath et al., 2001; van
Bokhoven and Brunner, 2002). Mutations in the DLX3 gene are
linked to tricho-dento-osseus (TDO) syndrome, which, like AEC,
is characterized by defects in the development of hair, teeth and
bone, and by absence of overt limb malformations (Price et al.,
Partial-overlapping mRNA expression and phenotypes of specific
human malformations caused by molecular lesions in either p63 or
DLX3suggest that these genes are components of common signaling
pathways during embryonic development. Here, we show that p63
is able to bind and transactivate Dlx3 both in vitro and in vivo.
Mutant p63 proteins derived from AEC patients exhibit an impaired
ability to transactivate Dlx3, indicating that the misregulation of the
DLX3 gene is involved in the pathogenesis of human syndromes
associated to AEC.
Homeobox gene Dlx3 is regulated by p63 during ectoderm
development: relevance in the pathogenesis of ectodermal
Nadezda Radoja1, Luisa Guerrini2, Nadia Lo Iacono2,3, Giorgio R. Merlo3, Antonio Costanzo4,
Wendy C. Weinberg5, Girolama La Mantia6, Viola Calabrò6,* and Maria I. Morasso1,*,†
Ectodermal dysplasias (EDs) are a group of human pathological conditions characterized by anomalies in organs derived from
epithelial-mesenchymal interactions during development. Dlx3 and p63 act as part of the transcriptional regulatory pathways
relevant in ectoderm derivatives, and autosomal mutations in either of these genes are associated with human EDs. However, the
functional relationship between both proteins is unknown. Here, we demonstrate that Dlx3 is a downstream target of p63.
Moreover, we show that transcription of Dlx3 is abrogated by mutations in the sterile ?-motif (SAM) domain of p63 that are
associated with ankyloblepharon-ectodermal dysplasia-clefting (AEC) dysplasias, but not by mutations found in ectrodactyly-
ectodermal dysplasia-cleft lip/palate (EEC), Limb-mammary syndrome (LMS) and split hand-foot malformation (SHFM) dysplasias.
Our results unravel aspects of the transcriptional cascade of events that contribute to ectoderm development and pathogenesis
associated with p63 mutations.
KEY WORDS: Dlx3, p63, Transcription, Ectodermal dysplasias, Mouse development
Development 134, 13-18 (2007) doi:10.1242/dev.02703
1Developmental Skin Biology Unit, NIAMS, NIH, Bethesda, MD 20892, USA.
2Department of Biomolecular and Biotechnological Sciences, University of Milan, Via
Celoria 26. 20133, Milan, Italy. 3Dulbecco Telethon Institute c/o Istituto Tecnologie
Biomediche CNR, 20100 Milan, Italy. 4Department of Dermatology, University of
Rome ‘Tor Vergata’, Viale Oxford 81. 00133 Rome, Italy. 5Division of Monoclonal
Antibodies, CDER/FDA, Bethesda, MD 20892, USA. 6Department of Structural and
Functional Biology, University of Naples, Via Cinzia 26. 80126 Naples, Italy.
†Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 18 October 2006
MATERIALS AND METHODS
The –117 to +60 DNA fragment of the Dlx3 promoter (Park and Morasso,
1999) was inserted into the pGL3-Basic and pCAT-Basic vectors (Promega).
Mutations in the p63-binding sites of the Dlx3promoter were obtained using
the ExSite Mutagenesis kit (Stratagene). The coding sequences for ?Np63?,
?Np63?, and ?Np63? were cloned into pBK-CMV (Stratagene). The
TAp63?, TAp63? and TAp63? constructs were a gift from E. Candi
(University of Rome ‘Tor Vergata’, Rome, Italy) and G. Melino (University
of Rome ‘Tor Vergata’, Rome, Italy). The p63 mutants L518F, L518V and
Q540L(AEC); E639X(SHFM); FS525 (EEC); and G76Wand ?AA(LMS)
were kindly provided by H. van Bokhoven (Radboud University, Nijmegan,
Primary mouse keratinocytes were grown according to Park and Morasso
(Park and Morasso, 1999). Normal human epidermal keratinocytes [NHEK;
a gift of M. Simon (SUNY, Stony Brook, NY, USA)] were derived from
newborn foreskin and cultured in K-SFM (Invitrogen). The human
osteosarcoma Saos-2 cell line was maintained in RPMI 1640 and 10% FCS.
The immortalized human keratinocyte HaCaT and H1299 non-small-lung-
carcinoma cell lines were grown in DMEM and 10% FBS.
Transient transfections of keratinocytes were performed with FuGENE6
(Roche) in a 1:3 ratio. PRL-SV40 vector was used as an internal control.
Luciferase activity was measured 24-36 hours after transfection using the
Dual-Luciferase Reporter Assay System (Promega). Transient transfections
of Saos-2 and H1299 cells were performed according to Calabrò et al.
(Calabrò et al., 2002). CAT reaction was performed 48 hours after
transfection using 90 ?g of cell extract. ?-Gal was used to normalize for
Nuclear extracts and EMSA analysis were carried out according to Park and
Morasso (Park and Morasso, 1999) using the Dlx3 p63site1+2 and non-
specific competitor AP-2 binding site.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) was performed with chromatin
from mouse keratinocytes and H1299 cells transfected with TAp63?
according to Caretti et al. (Caretti et al., 2004) using no antibody, IgG or p63
antibody (Santa Cruz, H137 and 4A4). Real-time PCR was performed using
the Mx3000P System (Stratagene) and SyberGreen MasterMix (Applied
Biosystems) with independent DNA samples and the following
oligonucleotides: for mouse, Dlx3(F) 5?-GAGAAAGCGCG AGCGT -
GTTTTGCC-3? and Dlx3(R) 5?-CCGGCTGTCGGTCAGTCG CTGCGT-
3?; for human, DLX3(F) 5?-AGAGAGGCGGAAGAGACGAG-3? and
DLX3(R) 5?-GAGGAGGGAGGAGAGAAGGA-3?; and for JAG2(F)
5?-CAAGTGGTGAACAAGGGAGACT-3? and JAG2(R) 5?-ACTG -
CTGCCTTCTGGAAACTC-3?. Data are presented as fold differences
relative to input and values are obtained by IgG with the formula
2[(CtIgG–CtInput)–(CtAb–CtInput)], where Ct is threshold cycles, IgG is normal
rabbit IgG, Ab is specific antibody and Input is input genomic DNA. ACHR
amplification was performed as a control for H1299 transfected with
TAp63? using ACHR(F) 5?-TGCCTCGGGTGAACTAAGATG-3? and
For analysis of the expression of p63in mouse keratinocytes and embryonic
tissues, the following oligonucleotides were used: TAp63(F) 5?-AGA -
CAAGCGAGTTCCTCAGC-3?, TAp63(R) 5?-TGCGGATACAATCC -
?Np63(R) 5?-GATGGAGAGAGGGCATCAAA-3?. These oligonucleotides
are designed in regions of the mRNA common to all isoforms (?, ? and ?),
and do not distinguish between these variants.
expression in mouse keratinocytes, the following
oligonucleotides were used: Dlx3(F) 5?-ATTACAGCGCTCCTCAGCAT-
3? and Dlx3(R) 5?-GCCTATAGGATCCCCCGTAG-3?. In embryonic
Development 134 (1)
Fig. 1.Transcriptional regulation of the Dlx3 promoter by p63. (A)
Fold-induction increase, compared with wild type, of Dlx3–117/+60co-
transfected with vectors expressing the different p63 isoforms. (B)
Sequence of the mouse Dlx3-promoter region containing the two
overlapping p63-binding sites (p63 site1 and p63 site2; underlined) and
CCAAT box (italics). The mutated Dlx3 sequences are shown in gray. (C-
D) Dlx3–117/+60, Dlx3p63M1 and Dlx3p63M2 constructs were co-
transfected with vectors expressing the TAp63 (C) and ?Np63 (D)
isoforms, with activity shown relative to the basic activity of
Dlx3–117/+60. Transient transfection experiments were performed using
mouse keratinocytes (C-D) and Saos-2 cells (E). Basal activity of the
reporter was set to 1. Each histogram bar represents the mean of three
independent transfection duplicates. Standard deviations are indicated.
Dlx3 p63M1, mutated at p63-binding-site 1; Dlx3p63M2, mutated at
tissues: Dlx3(F) 5?-CGTTTCCAGAAAGCCCGTA-3? and Dlx3(R) 5?-
CGTGGAATGGGAAGATGTGT-3?. For normalization: GAPDH(F)
5?-TGTCAGCAATGCATCCTGCA-3? and GADPH(R) 5?-TGTATGC -
For the analysis of p63 and Dlx3 mRNA levels in embryonic tissues,
E10.5, E11.5 and E12.5 anterior or posterior limb buds from wild-type
embryos were dissected, pooled in TRIZOL (Roche) and extracted, as
indicated by the manufacturer. Real-time PCR was performed with a
LightCycler (Roche) using FastStart DNA MasterPLUS SYBR-Green I
(Roche). Standard curves were performed using wild-type cDNA with
four calibration points: TQ; 1:3; 1:9 and 1:27. All samples were done in
duplicates and the analysis was repeated twice. GAPDH was used
for normalization, calculated using LightCycler Software 3.5.3. The
results are expressed with the value relative to E10.5 (set at 1) for each
Immunohistochemistry and whole-mount in situ hybridization
Immunohistochemistry was performed on 11 ?m cryostatic sections of
E10.5 embryonic forelimbs. Sections were blocked with 10% goat serum in
PBS for 40 minutes at room temperature. Antibodies used were: mouse
monoclonal anti-p63 (4A4, 1:100, Santa Cruz) and rabbit anti-distal-less
[pan-anti-Dlx, 1:100; kindly provided by G. Boekhoff-Falk (University of
Wisconsin Medical School, WI, USA)]. As secondary antibody, anti-mouse-
Cy2, anti-rabbit-Cy3 (1:100; Jackson Immuno-Research) and Envision anti-
rabbit HRP (Dako) were used. Fluorescence micrographs were taken by
Whole-mount in situ hybridization was performed according to
Acampora et al. (Acampora et al., 1999) on E10.5 p63KO embryos [Brdm2
line of p63 KO kindly provided by D. Roop (Baylor College of Medicine,
Houston, USA)] using a Dlx3 probe (Morasso et al., 1995).
Total RNA from human Saos-2 and HaCaT cells was prepared with TRIZOL
(Roche). For reverse transcription (RT)-PCR, 3-4 ?g of total RNA were
reverse-transcribed using SuperScript II (Invitrogen). The following
oligonucleotides were used: Dlx3(F) 5?-ACCTACGGAGCCTCCTACCG-
3?, Dlx3(R) 5?-ACTCAGGTTCTGTGCGTGAT-3?, p63?(F) 5?-GTCT -
CCATCTTCATATGGTAAC-3?, p63?(R) 5?-CACACTGACTGTAGAG -
GCA-3?, p63?(F) 5?-GTCTCCATCTTCATATGGTAAC-3?, p63?(R)
5?-CTTGCCAAATCCTGACAATGCTGC-3?, p63?(F) 5?-GAGGATAG -
CATCAGAAAACAGCAAG-3? and p63?(R) 5?-CTCCACAAGCTCAT -
TCCTGAAGC-3?. For normalization: Cyclophillin(F) 5?-ATCACCA -
TTGCTGACTGTGG-3?, Cyclophillin(R) 5?-ACTCTGCAATCCAGC -
TAGGC-3?, GAPDH(F) 5?-GTCTCCATCTTCATATGGTAA-3? and
GAPDH(R) 5?-CCACAGTCCATGCCATCACT-3? were used.
RESULTS AND DISCUSSION
The existence of malformations caused by either p63 or DLX3 gene
mutations that translate to partially overlapping phenotypes suggests
that these genes are transcriptional effectors in common signaling
cascades regulating epidermal development. The severity of the
phenotype in p63-null mice suggests that it is a crucial upstream
regulator of these signaling pathways (Mills et al., 1999; Yang et al.,
1999). Detailed analysis of the Dlx3 proximal promoter region
revealed a sequence with two p63-like overlapping binding sites
immediately upstream of the CCAAT box, located from –89 to –80
bp (site 1) and from –84 to –75 bp (site 2) of the transcriptional start
site (Park and Morasso, 1999). Because the expression patterns of
p63and Dlx3overlap throughout embryonic development (Morasso
and Radoja, 2005), we proceeded to test the ability of different p63
isoforms to transactivate the Dlx3 promoter. The Dlx3–117/+60
construct, which contains the two overlapping sites, was transiently
transfected into primary mouse keratinocytes in either the absence or
presence of expression vectors encoding TAp63?, TAp63?, TAp63?,
?Np63?, ?Np63? or ?Np63?. The TA isoforms activated the
Dlx3–117/+60promoter at a magnitude of twelve-, three- and seven-
Dlx3 and p63 in ectoderm development
Fig. 2. p63 and Dlx3 expression in primary mouse keratinocytes
cultured in vitro. Dlx3 (A) and TAp63 (B) mRNAs were induced, and
?Np63 (C) mRNA was downregulated after 12- and 24-hours of high-
[Ca+2] treatment. (D-F) p63 binds to the Dlx3 promoter region in vitro
and in vivo. (D) EMSA assay performed with a DNA fragment that
included the Dlx3 p63-binding site 1 and 2 using nuclear extract from
primary keratinocytes (NE), and in the presence of 100 M excess of
specific (SC) and nonspecific (NC) competitors. (E-F) ChIP analysis on
mouse keratinocytes with either control IgG or p63 antibody (p63 Ab)
on the region of the Dlx3 promoter containing the p63-binding sites by
regular (E) and real-time (F) PCR. (G) ChIP analysis on TAp63?-
transfected H1299 cells with no antibody (no Ab) or p63 antibody on
DLX3 and JAG2 promoters. ACHR was used as a control.
fold, respectively, compared to normal activation, whereas the
exogenous expression of the ?N isoforms resulted in a two- to four-
fold greater transactivation compared with wild type (Fig. 1A). These
effects are specific for p63 isoforms, because p53 did not transactivate
the Dlx3 promoter (data not shown).
In order to test each overlapping site (p63-binding-site 1 and
p63-binding-site 2, Fig. 1B), we compared the activities of
the Dlx3–117/+60, Dlx3–117/+60p63M1 (mutated p63 site 1) and
Dlx3–117/+60p63M2 (mutated p63 site 2) constructs (Fig. 1B) by co-
transfection performed in the presence of the TA and ?Np63
isoforms (Fig. 1C,D). Our results indicate that TAp63?induction of
Dlx3 is mediated through either of the two p63 sites, whereas
TAp63? required an intact regulatory region. These results are not
cell-type specific, because a similar profile was obtained using Saos-
2 cells (Fig. 1E).
Since Dlx3 is induced in keratinocytes cultured in 0.12 mM Ca2+
(Park and Morasso, 1999), we compared the endogenous expression
of the p63 isoforms with Dlx3 after 12- and 24-hours of 0.12 mM
Ca2+treatment (Fig. 2A-C). The real-time PCR results showed a
correlation between the upregulation of Dlx3 and TAp63 and the
downregulation of ?Np63 mRNAs associated by Ca2+-induced
differentiation, and are consistent with the recent report by King et
al. (King et al., 2006). The specificity of the PCR products was
corroborated by sequencing (data not shown). TAp63 proteins are
found in normal adult tissues (Nylander et al., 2002) and during
mouse embryonic development (Koster et al., 2004) (also our own
data). Findings of Koster et al. (Koster et al., 2004) support a role for
TAp63 as a molecular switch for the initiation of epithelial
stratification. Our findings support a working model in which, once
transactivated by TAp63?, Dlx3 will in turn regulate the expression
of terminal differentiation markers (Morasso et al., 1996).
In order to demonstrate direct binding to the p63 region in the Dlx3
promoter, we performed EMSA with a fragment comprising –89 to
–75 bp (Dlx3 p63 sites 1 and 2) using nuclear extracts from primary
keratinocytes (NE). A shift was detected (Fig. 2D, lane NE), and the
complexes were competed with a specific competitor (Fig. 2D, lane
SC), but not with a nonspecific DNA competitor (Fig. 2D, lane NC).
We next evaluated whether p63 bound this region of the Dlx3
promoter in vivo. In mouse keratinocytes, ChIP experiments were
performed with a p63-specific antibody andanalyzed by regular PCR
(Fig. 2E) and real-time PCR (Fig. 2F). The data shows that p63
specifically binds to the Dlx3promoter in vivo (eightfold higher than
with IgG control). Moreover, ChIP experiments on TAp63?-
transfected H1299 cells, which are devoid of p63, demonstrated the
direct binding of TAp63? to the Dlx3 promoter (Fig. 2G).
To further explore the relationship of Dlx3 and p63 in vivo, we
analyzed their colocalization by immunofluorescence on E10.5
embryonic forelimb sections with anti-p63 and anti-distal-less
antibodies. The latter reagent recognizes Dlx3 in the limb ectoderm.
p63 and Dlx3 immunoreactivity were found to colocalize in the same
nuclei (Fig. 3A). Comparison of the expression of Dlx3, TAp63 and
?Np63 in the limbs at embryonic stages E10.5, E11.5 and E12.5 was
performed by real-time PCR. Between E11.5 and E12.5, the relative
abundance of both TAp63 and Dlx3 mRNA increased from three- to
eight-fold relative to their expression at E10.5 in the anterior (AL)
and posterior limbs (PL) (Fig. 3B), whereas expression of ?Np63 was
only moderately increased. These results for p63 in the limb ectoderm
of embryos at E10.5-E12.5 are in agreement with reported data
(Koster et al., 2004) and show for the first time that a good correlation
is observed between the expressions of Dlx3 and TAp63.
To provide further evidence that p63 is an upstream regulator of
Dlx3, we studied the effect of p63 ablation on Dlx3 mRNA and
protein expression in the Brdm2 p63 KO mice (Mills et al., 1999)
(Fig. 3C-D). As assessed by immunohistochemistry, the abundance
of Dlx3 protein was significantly reduced in p63-KO limb ectoderm
(Fig. 3C). Analysis by whole-mount in situ hybridization with a
Dlx3 antisense probe demonstrated that the absence of p63 led to a
downregulation of Dlx3 (Fig. 3D).
We next studied the clinical relevance of p63-mediated regulation
of Dlx3 expression. We examined the transcriptional activity of the
Dlx3 promoter in the presence of p63 mutants causative of human
AEC, EEC, SHFM or LMS (Fig. 4A) in the H1299 cell line. The p63
AEC mutants – L518V, L518F and Q540L – are all point
Development 134 (1)
Fig. 3. p63 and Dlx3 colocalize in the embryonic ectoderm, and
Dlx3 expression is downregulated in p63-KO embryos. (A)
Histochemistry with anti-distal-less (pan-anti-Dlx; red) and p63 (green)
antibodies on the dorsal forelimb ectoderm of E10.5 wild-type embryos
(merge of Dlx and p63 expression is yellow). DAPI staining is also
shown (blue). Arrows indicate the dlx-p63 double-positive nucleus. (B)
Relative mRNA abundance, determined by real-time PCR, for Dlx3,
TAp63 and ?Np63 in the anterior limb (AL) and posterior limb (PL) of
E10.5, E11.5 and E12.5 wild-type embryos. The relative abundance is
expressed as fold-induction relative to the value at E10.5 (set at 1). (C)
Histochemistry with anti-p63 and anti-Dlx on E10.5 wild-type and p63-
KO limb-bud ectoderm. (D) Whole-mount in situ hybridization on wild-
type and p63-KO E10.5 embryos with a Dlx3 antisense probe. Arrows
indicate limb buds. M, mesoderm; E, ectoderm. Scale bars: 10 ?M in A
and 40 ?M in C. wt, wild type.
substitutions within the SAM domain, present only in TAp63? and
?Np63?. The p63?FS mutant contains a mutation found in EEC
that generates a frameshift at amino acid 525, which leads to a
premature stop. The E639X mutation, in exon 14, was isolated in a
SHFM patient, whereas the 2-bp deletion, in exon 14 (?AA), was
present in one family with LMS. The G76W mutation, in exon 3,
was isolated from a LMS patient and affects all p63 isoforms. Co-
transfection experiments were performed with expression vectors
encoding TAp63 and ?Np63 mutants. As shown in Fig. 4B and 4C,
the AEC mutants failed to yield a significant level of reporter-gene
expression despite having intact amino terminal and DNA-binding
domains. These results point to a crucial role of the C-terminus of
p63? on the regulation of Dlx3 transcription. The LMS-derived
mutants G76W and ?AA; as well as the FSEEC mutant, SHFM-
derived E639X TA and ?N proteins; showed a similar mode of
regulation of Dlx3-promoter activity compared with the
corresponding wild-type proteins (Fig. 4B,C). A similar profile,
albeit with a lower amount of induction, was obtained upon
transfection in HaCaT cells (data not shown). The level of
expression of the mutant proteins was corroborated by immunoblot
analysis with anti-p63 antibody (Fig. 4D).
The hampered ability of the AEC mutants, as well as the partial
overlapping phenotypes of specific malformations caused by to p63
(i.e. AEC) or Dlx3 (i.e. TDO) gene mutations, suggest that Dlx3
misregulation is involved in aspects of the pathogenesis of AEC. AEC
is characterized by ectodermal dysplasia, ankyloblepharon and cleft
lip with cleft palate, and by the lack of limb involvement. The absence
of limb defects in AEC may reflect the possibility that a putative role
of Dlx3 in the limb is compensated for by other Dlx proteins
(Panganiban and Rubenstein, 2002; Morasso and Radoja, 2005).
To determine the modulation of endogenous Dlx3 by p63, we
used Saos-2 cells to express wild-type and mutant p63 (Fig. 4E).
Interestingly, TAp63? increased Dlx3 expression (Fig. 4E, lane 2),
and this effect was shared, although to different extents, with the
EEC, SHFM and LMS mutants (Fig. 4E; lanes 6, 7 and 8). TAp63?
did not alter Dlx3 levels (Fig. 4E, lane 3). Remarkably, an AEC
mutant (Fig. 4E, lane 5) and the TAp63? isoform abolished Dlx3
expression. Transfections of ?Np63 isoforms, both wild-type and
mutant, had no significant effect on Dlx3 transcription (Fig. 4E,
bottom panel). TAp63? and TAp63? are potent transactivators
(Barbieri and Pietenpol, 2006) and, in our in vitro studies, both
isoforms were able to transactivate Dlx3 at similar levels. However,
Dlx3 and p63 in ectoderm development
Fig. 4. Differential ability of p63-mutant
proteins to transactivate Dlx3. (A) Schematic
representation of p63 transcripts and mutations.
(B-C) Transcriptional regulation of the Dlx3
promoter by TAp63 (B) and ?Np63 (C) wild-type
and mutant proteins. H1299 cells were co-
transfected with the Dlx3 reporter plasmid and
expression vectors for TAp63- and ?Np63-mutant
isoforms. The basal activity of the reporter was set
to 1. Each histogram bar represents the mean of
three independent transfections. Standard
deviations are indicated. (D) Proteins were
corroborated by western blot analysis with anti-
p63 4A4 antibody (Santa Cruz). (E) The level of
endogenous Dlx3 mRNA upon transfection with
p63 mutants in Saos-2 cells. Top panel, lanes: 1,
mock; 2, TAp63?; 3, TAp63?; 4, TAp63?; 5,
TAp63F518V-AEC; 6, TAp63FS-EEC; 7,
TAp63E639X-SHFM; and 8, TAp63?AA-LMS.
Bottom panel, lanes: 1, mock; 2, ?Np63?; 3,
?Np63?; 4, ?Np63?; 5, ?Np63F518V-AEC; 6,
?Np63FS-EEC; 7, ?Np63E639X-SHFM; and 8,
?Np63?AA-LMS. GAPDH was used for
normalization. (F) The level of endogenous Dlx3
mRNA upon transfection with TAp63? and
TAp63? in HaCaT cells. Lanes: 1, mock; 2, TAp63?
0.5 ?g; 3, TAp63? 2 ?g; 4, TAp63? 4 ?g; 5,
TAp63? 0.5 ?g; 6, TAp63? 2 ?g; 7, TAp63? 4 ?g.
Cyclophillin was used for normalization.
whereas TAp63? induction was mediated through either of the two
p63 sites in the Dlx3promoter, TAp63?required an intact regulatory
region. Surprisingly, a slightly different outcome was obtained for
the endogenous Dlx3 regulation, where upregulation of Dlx3 was
detected with TAp63? and a complete downregulation was found
when expressing TAp63? in Soas-2 cells. To determine if these
results could be attributed to cell context, the experiments were also
performed in HaCaT cells. In these cells, overexpression of TAp63?
showed upregulation of endogenous Dlx3, whereas TAp63? once
again caused a complete downregulation of Dlx3 expression (Fig.
4F). These differences might be attributed to the specific p63RE-
CCAAT-box chromatin architecture. An important feature of the
Dlx3 promoter is that the overlapping p63-binding sites are in close
proximity to CCAAT box that binds NF-Y in keratinocytes (Park
and Morasso, 1999). NF-Y is a general promoter organizer that pre-
sets chromatin structure locally. A recent report shows that p63?
regulates the transcription of the hsp70 gene through interactions
with NF-Y (Wu et al., 2005). Although we have not determined the
significance of NF-Y and p63 interactions on Dlx3 transcriptional
regulation, it might be proposed that there is a dual role for the
overlapping p63-binding sites, and that Dlx3will be transcriptionally
active or repressed depending on the specific p63 isoform bound to
the promoter, on which of the p63 sites is occupied and on
interactions with NF-Y CCAAT binding factor.
Dlx3 and p63 both function as part of a complex series of
cascades that ultimately lead to the formation of ectoderm-derived
organs. Unraveling the function of each protein at specific times of
embryonic development will prove to be complex because of the
differential expression of the p63 isoforms in distinct tissues
(Nylander et al., 2002) and the cross-regulation with other
developmentally relevant signaling pathways [i.e. FGF, BMP, and
Notch (Laurikkala et al., 2006; Nguyen et al., 2006)]. The
characterization of p63 target genes promises to improve our
knowledge of the signaling cascades that are directly involved in
normal ectodermal development. In summary, our study proves a
functional relationship between p63 and Dlx3, with Dlx3
demonstrated to be a direct target of p63. The findings also provide
evidence that the misregulation of Dlx3 is involved in the
pathogenesis of p63 molecular lesions in AEC.
We thank S. J. Stimpson, Y. Rivera, S. Bertuzzi, T. Lozito, A. Pollice, K. King and
G. Caretti for helpful comments; H. van Bokhoven, E. Candi and G. Melino for
their gifts of plasmids; M. Simon for NHEK; G. Boekhoff-Falk for the anti-distal-
less antibody; and D. Roop for providing the Bmdr2 p63-KO mouse line. The
research was supported by the Intramural Research Program of the NIAMS of
the National Institutes of Health; Telethon (GGP05056) to L.G.; Telethon
(GGP030326), AIRC and MIUR to G.L.M.; CIB to V.C.; and G.R.M. is supported
by a career award from Fondazione Telethon (TCP99003) and Fondazione San
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