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First published online March 7, 2008
doi: 10.1242/10.1242/dev.011759
1 Dulbecco Telethon Institute, Molecular Biotechnology Center, University of
Torino, Via Nizza 52, Torino, 10126, Italy.
2 Department of Biomolecular Science and Biotechnology, University of Milan, Via
Celoria 26, 20133 Milan, Italy.
3 CNR Istituto Tecnologie Biomediche, Segrate Milano, Italy.
4 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
5 Developmental Skin Biology Unit, NIAMS, NIH, Bethesda, MD, USA.
6 Department of Dermatology, University of Rome, TorVergata, Italy.
7 Evolution des Régulations Endocriniennes CNRS, UMR5166, Muséum
National d'Histoire Naturelle, Paris, France.
Authors for correspondence (e-mails:
luisa.guerrini{at}unimi.it;
gmerlo{at}dti.telethon.it)
Accepted 31 January 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Np63
induces transcription from the Dlx5 and
Dlx6 promoters, an activity abolished by EEC and SHFM-IV mutations,
but not by Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC)
mutations. ChIP analysis shows that p63 is directly associated with the
Dlx5 and Dlx6 promoters. Thus, our data strongly implicate
p63 and the Dlx5-Dlx6 locus in a pathway relevant in the
aetio-pathogenesis of SHFM.
Key words: Dlx, p63, Ectrodactyly, Limb development, Transcription regulation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
malformation Split-Hand/Foot (SHFM, MIM 183600), also known as ectrodactyly,
affects the distal portion of the upper and lower limbs, and is characterized
by a deep medial cleft and missing central fingers
(Sifakis et al., 2001).
Genetically, SHFM comprises both isolated and hereditary forms, linked to five
distinct loci (types I-V).
For SHFM-II (MIM 313350) and SHFM-V (MIM 606708), the disease genes have
not been identified. SHFM-III (MIM 600095) is associated with genomic
alterations on chromosome 10q24-q25
(deMollerat et al., 2003),
which results in a complex rearrangement around the Dactylyn locus, possibly
associated with gene inactivation. Dactylyn is also involved in a complex
rearrangement/duplication in dactylaplasia (dac) mutant
mice, which exhibit ectrodactyly-like limb defects
(Chai, 1981;
Johnson et al., 1995;
Crackower et al., 1998). In
spite of these findings, no demonstration that Dactylyn is the disease gene
for SHFM-III, or in the dac mice, has been provided.
SHFM-I, the most common form, is associated with deletions of variable
extent on chromosome 7q21. The minimal common deletion includes DSS1
and the distalless-related homeogenes DLX5 and DLX6
(Simeone et al., 1994;
Scherer et al., 1994a;
Scherer et al., 1994b;
Crackower et al., 1996). The
double knock-out (DKO) of Dlx5 and Dlx6 in the mouse leads
to ectrodactyly (Robledo et al.,
2002; Merlo et al.,
2002; Merlo et al.,
2003), implicating the human orthologs DLX5 and
DLX6 in this pathology. Mutations in the DLX5-DLX6 locus
have not been found, therefore the molecular alteration remains unknown;
however, a `position effect' mutagenic mechanism for SHFM-I has been proposed
(Scherer et al., 2003). Dlx
genes code for six distalless-related homeodomain transcription
factors (Dlx1-Dlx6) that play key roles in the development and morphogenesis
of the head and limb skeleton (Merlo et
al., 2000; Merlo et al.,
2003; Panganiban and
Rubenstein, 2002). Expression of Dlx5 and Dlx6
has been detected in the apical ectodermal ridge (AER) of the embryonic limb
buds, in the pharyngeal arches, in the osteoblasts of developing bones and in
interneurons of the basal forebrain
(Simeone et al., 1994;
Acampora et al., 1999;
Levi et al., 2003). In spite
of known functions of distalless for the development of insect
appendages, little is known about the molecular regulation of Dlx genes in
mammalian limbs. Defining the upstream regulation of the Dlx5-Dlx6
locus during limb development might help to clarify the molecular basis of
SHFM.
SHFM-IV (MIM 605289) is caused by mutations in p63, a gene coding
for a transcription factor homologous to p53 and p73
(Ianakiev et al., 2000;
vanBokhoven and Brunner, 2002;
Berdon-Zapata et al., 2004). In
50 unrelated SHFM patients, five mutations in p63 were found,
suggesting that these may account for about 10% of sporadic SHFM
(vanBokhoven et al., 2001;
vanBokhoven et al., 2002).
Mutations of p63 are also responsible for other autosomal, dominantly
inherited human syndromes, including Ectrodactyly-Ectodermal dysplasia-Cleft
lip (EEC), Limb-Mammary Syndrome (LMS) and Ankyloblepharon-Ectodermal
defects-Cleft lip/palate (AEC) (Celli et
al., 1999; vanBokhoven et al.,
2001; Rinne et al.,
2006; Rinne et al.,
2007). p63-null mice show severe defects affecting their
skin, limbs, craniofacial skeleton, and they lack teeth, hair and mammary
glands. In p63-/- newborn animals, the hindlimbs (HLs) are
absent, whereas the forelimbs (FLs) are severely truncated in the distal
segments (Mills et al., 1999;
Yang et al., 1999).
p63 is transcribed as two classes (TA and
N) of mRNAs. The use of alternative promoters drives the
transcription of either TAp63 proteins, comprising a p53-related N-terminal
transactivation (TA) domain, a DNA-binding (DB) and an oligomerization (OD)
domain, or Np63 proteins, lacking the TA domain. Additional TA domains
have been identified that account for the transcriptional activities of the
N isoforms (Dohn et al.,
2001; Ghioni et al.,
2002; Laurikkala et al.,
2006). Three alternative splicing routes at the 3'-end
generate TAp63 and Np63 proteins with different C-termini, denoted
, β and 
(vanBokhoven
and Brunner, 2002). A Sterile-Alpha-Motif (SAM) and a
Transcription-Inhibitory-Domain (TID) are present only in the -isoforms
(Serber et al., 2002;
Qiao and Bowie, 2005).
The limb defects of p63-/- mice have been associated
with a failure of AER formation and the loss of expression of key morphogens.
p63 is expressed in several ectoderm-derived tissues: it is essential
for the initiation of the epithelial stratification program during embryonic
development (Koster et al.,
2004; Barbieri and Pietenpol,
2006; Laurikkala et al.,
2006) and to maintain the proliferation potential of epithelial
stem cells (Senoo et al.,
2007). The AER is a stratified embryonic epithelium, therefore it
seems logical that p63 might control its function and maintenance via
regulation of AER-restricted target genes
(Koster and Roop, 2004;
Koster et al., 2007), such as
the Dlx genes.
Here, we provide evidence that p63 is genetically upstream of Dlx
genes in the AER, a region critical for normal limb development. We identify
Np63 as the main regulatory isoform, able to induce
transcription of the Dlx5 and Dlx6 promoters, whereas EEC
and SHFM-IV mutations impair this activity. In vivo, combining the
p63+/EEC mutation with the incomplete loss of
Dlx5-Dlx6 results in aggravated limb defects. These data indicate
that alteration of the p63-Dlx pathway is the most likely molecular
basis of SHFM-I and SHFM-IV.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Merlo et al., 2002). The
Brdm2 mice carry a null allele, also denominated
p63- (Mills et al.,
1999), and were genotyped by RT-PCR on embryonic RNA, with primers
annealing to the C terminus of the isoform mRNA and amplifying a 450
bp fragment. Primers were:
Mice carrying the p63R279H (p63EEC) mutation (an EEC mutation in humans) were generated by A. Mills (Cold Spring Harbor Laboratory, USA; E.G. and A.A.M., unpublished). The mutation abrogates a BsaHI restriction site. The genotype was determined by PCR amplification with primers flanking codon 279, followed by digestion with BsaHI to distinguish the two alleles. Primers were:
Preparation of tissue samples
All experiments involving the use of animals were approved by the
Institutional Animal Care Committee and by the Ministry of Health. Pregnant
mice were sacrificed between 10.5 and 12.5 days of pregnancy, embryos were
collected by caesarean section. For histochemistry and section in situ
hybridization, limbs were dissected, fixed in 4% paraformaldehyde (PFA)
overnight, rinsed in PBS at 4°C, cryoprotected with 20% sucrose, embedded
in OCT and sectioned at 11 µm. For whole-mount hybridization, PFA-fixed
embryos were stored in methanol at -20°C. Skeletal staining was done
according to published procedures (Wallin
et al., 1994). For RT-PCR or RealTime analyses, limbs were
dissected in PBS, transferred in RNAlater (Ambion) and stored at -20°C
until extraction.
Immunohistochemistry and in situ hybridization
Antibodies used were: mouse monoclonal anti-p63 (4A4, Santa Cruz, 1:100),
rabbit anti-distalless (from Dr G. Boekhoff-Falk, 1:100) and rabbit
anti-E-cadherin (C20820, Translucent Laboratory, 1:200). Secondary antibodies
were: anti-mouse-Cy2 and anti-rabbit-Cy3 (Jackson ImmunoResearch, 1:200).
Confocal micrographs were obtained using the sequential frame-scanning mode,
followed by stacking and digital merging.
In situ hybridization was carried out with digoxigenin (DIG)-labeled
antisense murine RNA probes corresponding to the complete coding sequence of
Dlx5 and Dlx2, and to a fragment of Dlx6 comprising
exons III and IV. For each probe, two normal and two mutant specimens were
examined. Section and whole-mount hybridization was done according to
published procedures (Levi et al.,
2006).
RT-PCR, RealTime PCR and western blot analyses
FL and HL buds (eight for each genotype) were dissected, genotyped and
pooled in TriPure (Roche) for RNA extraction, as indicated by the supplier.
One µg of RNA was reverse-transcribed using SuperScript II (Invitrogen).
RealTime quantitative PCR (qPCR) was performed with LightCycler and FastStart
DNA MasterPLUS SYBR-Green I (Roche). Five µl of diluted cDNA were used in
each reaction, standard curves were determined using wild-type cDNA with four
calibration points. Samples were tested in duplicates. Specificity and absence
of primer dimers was controlled by denaturation curves. GAPDH mRNA
was used for normalization, results were calculated using LightCycler Software
3.5.3. For primers for RT-PCR and RealTime qPCR, see Tables
1 and
2.
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). Total proteins were extracted in 2% SDS, 30% glycerol,
300 µM β-mercaptoethanol, 100 mM Tris-HCl (pH 6.8) from pools of (at
least eight) HLs and FLs of E10.5 and E12.5 normal embryos.
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed on sheared genomic DNA from cultured mouse keratinocytes
and from H1299-tet-on Np63, using 2 µg of the 4A4 antibody,
as described (Ceribelli et al.,
2006; LoIacono et al.,
2006). IP material was analysed by PCR using primers designed to
amplify three fragments of approximately 200 bp of the mouse Dlx5
promoter, two fragments of the Dlx6 promoter, and one region of the
I
K and Np63 promoters, as
positive control (see Table
3).
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), except
for the R279H mutation, which was obtained by site-directed mutagenesis and
sequence verified.
Cell culture and transfection
Primary mouse keratinocytes were isolated from newborn mice and cultured in
Keratinocyte Basal Medium (Clonetics, San Diego CA) with EGF (10 ng/ml). The
human U2OS osteosarcoma and H1299 lung carcinoma cell lines were grown in DMEM
with 10% FBS. The procedure and expression vectors for luciferase assay have
been previously described (Beretta et al.,
2005). H1299 cells stably transfected with a tet-on
Np63 expression plasmid were induced with Doxycyclin, as
described (LoIacono et al.,
2006).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
observed colocalization in nearly all nuclei of the AER of both the FLs and
the HLs (Fig. 1E-H).
Colocalization was also observed in distal regions of the dorsal limb
ectoderm, where Dlx3 is present
(Radoja et al., 2007). p63 and
Dlx proteins do not always colocalize: in the olfactory placodes, Dlx and p63
are coexpressed in nuclei of the non-neural region but not in the neural
region, where only Dlx proteins are present
(Fig. 1J,K).
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Np63 mRNAs in the developing ectoderm
(Nylander et al., 2002;
Koster et al., 2004;
Laurikkala et al., 2006).
Furthermore, the dynamic expression of p63 and Dlx genes during limb
development has not been addressed. We decided to quantify, by RealTime qPCR,
the levels of TAp63 and Np63 mRNAs, and to compare
them with those of Dlx1, Dlx2, Dlx5 and Dlx6 mRNAs in
embryonic FLs and HLs at different ages (E10.5-E12.5). Primers were designed
to amplify either all TA or all N mRNA isoforms. The
abundance of TAp63 and Np63 mRNAs always increased
from E10.5 to E12.5 (4- and 2.5-fold, respectively;
Fig. 2A). However, the
TA cDNAs were detected at a higher cycle number (CP=29) than the
N cDNAs (CP=21), indicating that the N mRNAs
are more abundant than the TA. A 4- and a 7-fold increase in
Dlx5 expression was observed, respectively, in FLs and HLs; in the
same samples, a 4- and 3-fold increase in Dlx6 expression was
observed, whereas Dlx1 and Dlx2 expression increased
modestly (Fig. 2A).
To clarify which p63 isoforms are predominantly expressed, we applied
semi-quantitative RT-PCR using primers that can distinguish each isoform, to
examine p63 expression in embryonic FLs and HLs at different ages
(E10.5-E12.5). The TAp63 mRNA was detected at 38 cycles, but
not at 29 cycles. On the contrary, the Np63 mRNAs was
easily detected at 29 cycles and the reaction had reached a plateau before 38
cycles. The TAp63β and Np63β mRNAs were
both detected only after 38 cycles (Fig.
2B). The TAp63 and Np63
mRNAs were both detected at 29 cycles. In general, the expression of p63
isoforms increased from E10.5 to E12.5, and expression in the HL was generally
lower than in the FL.
Finally, we carried out western blot analysis on extracts from wild-type
embryonic limbs collected at E10.5-E12.5. Np63 expression was
detected in the HaCaT cell extracts and in the limb samples
(Fig. 2C). The abundance of
Np63 protein increased sharply from E10.5 to E12.5 in both FLs
(Fig. 2C) and HLs (data not
shown), consistent with the mRNA expression data. By contrast, TAp63
isoforms could not be detected.
These data indicate that the Np63 isoforms are the predominant
isoforms expressed in the embryonic limbs at the time of AER function and
maintenance (E10.5-E12.5), and that the increase of Dlx gene expression
parallels that of p63 at these developmental stages.
Np63 activates the Dlx5 and Dlx6 promoters in vitro
To test the possibility that p63 may regulate Dlx5 and
Dlx6 expression, we isolated genomic fragments comprising the
proximal promoters of murine Dlx5 and Dlx6, of 1150 and 1740
bp, respectively, to generate the mDlx5-luc and
mDlx6-luc reporter vectors. These vectors were
co-transfected with plasmids expressing TA- and Np63
cDNA isoforms into U2OS cells; U2OS cells do not normally express p63. The
TAp63 isoforms showed little (Dlx6) or no (Dlx5) activity.
Conversely, a 7- and 10-fold activation of Dlx5- and
Dlx6-dependent transcription, respectively, was observed with
Np63 co-transfection (Fig.
3A). Np63β showed modest activity (4- and 3-fold,
respectively), and Np63 was slightly more active on the
Dlx6 promoter (7-fold) than the Dlx5 promoter (4-fold;
Fig. 3A). We repeated these
experiments in the HaCaT keratinocyte cell line, which expresses
Np63 endogenously: similar results were obtained,
although the overall fold-activation was lower than in U2OS cells (data not
shown).
We then examined whether p63 mutations associated with SHFM-IV
(K193E and K194E), EEC (R279H and C306R) or AEC (L518F) affect the capacity of
p63 to activate the Dlx5 and Dlx6 promoters. Except for
L518F, these mutations fall within the DNA-binding domain of p63 and should
result in the loss of DNA binding (Celli et
al., 1999). The SHFM and EEC mutant p63 proteins showed a strongly
reduced transactivation potential on both Dlx promoters
(Fig. 3A), whereas AEC mutant
p63 behaved as wild type. Importantly, AEC patients do not exhibit
limb abnormalities.
|
Np63 expression could activate
transcription from the endogenous DLX genes, we used H1299 cells stably
transfected with a Doxycyclin-inducible Np63 expression
vector. Induction of Np63 resulted in a 1.8- and 2-fold
increased expression of DLX5 and DLX6, respectively. By
contrast, the expression of DLX1 and DLX2 was minimally
increased (Fig. 3B).
These data suggest that Np63 positively regulates the transcription
of both isolated Dlx5 and Dlx6 promoters, and of the
endogenous DLX5 and DLX6 genes, and that EEC and SHFM-IV
mutations abolish this capacity.
p63 binds to the Dlx5 and Dlx6 promoter in vivo
We searched the murine and human Dlx5 and Dlx6 promoters
and conserved intergenic sequences
(Zerucha et al., 2000) for p53
consensus binding sites, using the PatSearch prediction algorithm
(Grillo et al., 2003;
Osada et al., 2005;
Sbisà et al., 2007). No
p53 sites were found in the intergenic enhancer sequences. p53 binding sites
were predicted in the promoter regions of mDlx5 (-967, -858, -611,
-344 and -89 from the start site, designated A, B, C, D and E, respectively)
and mDlx6 (-1670 and -1190 from the start site, designated F and G,
respectively; see Fig. S1 in the supplementary material).
To determine whether p63 binds to the Dlx5 and Dlx6
promoters, we carried out ChIP analysis for Dlx5 on genomic DNA from
mouse keratinocytes, in which both Np63 and Dlx5 are
expressed (Morasso et al.,
1999), and for DLX6 on H1299 cells induced to express
Np63 (LoIacono et
al., 2006). PCR primers were designed to amplify genomic fragments
from the Dlx5 and DLX6 promoters
(Fig. 3C). p63-specific
enrichment was observed in two regions of the Dlx5 promoter, roughly
corresponding to -1200/-800 and -500/-100
(Fig. 3C,D), named
X5-R1 and X5-R2, respectively. Importantly, the X5-R1 and
X5-R2 regions comprise, respectively, the A and B and the D and E
predicted p53-binding sites. As a control, PCR amplification of an upstream
region (X5-2000) yielded no enrichment
(Fig. 3C). p63-specific
enrichment was observed in one region of the DLX6 promoter, named
X6-R2 and corresponding to -500/-100
(Fig. 3C,E), but not in the
region named X6-R1, comprising the F and G predicted p53-binding
sites. As a positive control, ChIP assays were performed on fragments of the
IK and Np63 promoters
(Candi et al., 2006;
Lanza et al., 2006)
(Fig. 3C). In summary, p63 is
physically associated to both Dlx5 and Dlx6 promoters in
vivo.
|
Np63
showed that Dlx5 promoter activation was progressively reduced in the
deleted versions, compared with mDlx5-luc, but it was not abolished
(Fig. 3D). This result suggests
that both of the regions of the Dlx5 promoter (X5-R1 and
X5-R2) to which p63 binds play a role in mediating the observed
p63-dependent transcriptional activation. We also generated two deleted
versions of the Dlx6 promoter: one removing the F+G p53-binding sites
and the second removing an additional 600-bp of sequences. Surprisingly,
transactivation of these promoter fragments by Np63 was
similar to the full-length fragment (Fig.
3E). These results suggest that the Dlx6 promoter is not
activated by p63 through the predicted p53-binding sites, but is possibly
activated through interaction with other transcription factors (Koutsondis et
al., 2005; Testoni and Mantovani,
2006).
Dlx expression is reduced in p63 mutant limbs
To determine whether the inactivation or mutation of p63 result in
altered Dlx gene expression in vivo, we used the p63EEC
mouse strain, in which the EEC mutation R279H
(Celli et al., 1999) was
introduced into the mouse germline by homologous recombination.
p63EEC/EEC mice show severe truncations of the FLs,
absence of the HLs, ectodermal dysplasia and craniofacial anomalies, similar
to those of p63-/- mice (see
Fig. 6 and next paragraph). The
limb buds of p63EEC/EEC E10.5 embryos are reduced in size
and have a pointed shape (Fig.
4A,A'), and the AER appears unstratified;
p63+/EEC mice have normal limbs and a stratified AER
(Fig. 4B-E). A mild
ectrodactyly is rarely observed (1/40) in p63+/EEC limbs
(see Fig. 6C,N). Other
phenotypes of p63+/EEC mice (skin, palate, genitalia) will
be reported separately (E.G. and A.A.M., unpublished).
First, we carried out whole-mount in situ hybridization for Dlx5
on E10 embryos. In p63EEC/EEC FLs and HLs, the signal was
strongly reduced when compared with wild-type or heterozygous limbs
(Fig. 4H-K), whereas the signal
in the pharyngeal arches, olfactory and otic placodes was only slightly
reduced (Fig. 4F,G). To
determine whether this signal reduction might be due to failure of AER
induction in the first place, we carried out in situ hybridization for
Dlx5 on E9.5 embryos, using Dlx5 expression as a marker for
pre-AER cells (Kimmel et al.,
2000; Robledo et al.,
2002). Dlx5 signal was present in both the FLs and the
HLs of normal and p63-null embryos, although it was reduced in the
later (Fig. 4L-O), indicating
that the pre-AER is properly induced.
Next, we hybridized sections of the HLs from wild-type, p63+/EEC, p63EEC/EEC and p63-/- E10.5 embryos for Dlx2 and Dlx5. The expression of both genes was strongly reduced in the AER of p63 homozygous mutant limbs (Fig. 4P-W). Immunostaining of sections of E10.5 HLs with anti-Dll antibody revealed a marked reduction of Dlx immunoreactivity in the AER of p63 homozygous mutant limbs (Fig. 4X-Z).
To quantify Dlx gene expression in p63 mutant limbs, we applied RealTime qPCR to detect Dlx1, Dlx2, Dlx5 and Dlx6 mRNAs in samples from E10.5 wild-type and p63EEC/EEC limbs. The abundance of Dlx1, Dlx2, Dlx5 and Dlx6 mRNAs was significantly reduced in p63EEC/EEC FLs (-50, -60, -70 and -70%, respectively) and HLs (-75, -75, -80 and -80%, respectively), compared with wild type (Table 4A). A similar reduction in Dlx gene expression was observed in p63 null embryos at the same age (Table 4A).
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;
Yang et al., 1999). We
examined Dlx gene expression in wild-type and p63+/- and
p63+/EEC limbs. In p63+/- and
p63+/EEC HLs, Dlx5 and Dlx6 mRNAs were
reduced (-40% to -50%), relative to in wild type. Dlx1 and
Dlx2 mRNAs were reduced (-40%) in the p63+/- HL,
but were minimally changed (-10%) in the p63+/EEC HL. In
the FLs of heterozygous embryos, the expression of Dlx genes was not reduced,
in fact it was slightly increased (see
Table 4B). Thus, the loss of
Dlx gene expression in homozygous p63 mutant HL is unlikely to be the
mere consequence of AER failure. Furthermore, these data indicate that FLs and
HLs do not share the same regulation of Dlx gene expression.
To complete the analysis, we compared the expression of Dlx genes with that
of Perp, a know p63 target
(Ihrie et al., 2005), in
normal and p63 mutant limbs. In p63EEC/EEC FLs
and HLs, Perp mRNA was reduced, respectively, by -45% and -60%,
whereas an intermediate reduction (-30%) was observed in
p63+/EEC HLs (Table
4B). In p63-/- FLs and HLs, Perp mRNA
was reduced, respectively, by -80% and -70%.
To exclude that the entire Dlx5-Dlx6 genomic region might be
silenced in p63 mutant limbs, we determined the abundance of two RNAs
transcribed within the locus, Dlx6 antisense (as) and Evf2
ncRNA (Liu et al., 1997;
Feng et al., 2006), in
wild-type and p63 mutant limbs. Although Evf2 was
practically undetectable, the expression of Dlx6as was decreased in
p63-/- FLs and increased in p63EEC/EEC
FLs and HLs (2- and 3-fold, respectively,
Table 4A). These data suggest
that p63 regulates Dlx5 and Dlx6 independently of the
high-order epigenetic regulations of this locus
(Horike et al., 2005).
Finally, we examined the possibility that p63 expression might be
controlled by Dlx. Very similar p63 immunostaining was observed in the AER and
ectoderm of E11 Dlx5;Dlx6 double knock-out (DKO) FLs and HLs compared
with wild type (Fig. 5C-F). We
then isolated RNA from the central wedge of wild-type and Dlx DKO limbs and
determined the abundance of TA and Np63 mRNAs by
qPCR; we did not observe significant differences compared with wild type
(Fig. 5G,H). Thus, Dlx5 and
Dlx6 are unlikely to control p63 expression. We also determined the
abundance of Dactylyn mRNA in samples from E11 wild-type, Dlx5;Dlx6
DKO and p63EEC/EEC limbs by qPCR. We observed a modest
increase in Dactylyn expression in p63 and Dlx mutant limbs, when
compared with wild type (Fig.
5H; Table 4A).
These data tend to exclude the participation of Dactylyn in Dlx- or
p63-dependent regulation.
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Dlx5;Dlx6 DKO mice show ectrodactyly of the HL
(Fig. 6T), but neither the
Dlx5-/- nor the
Dlx5+/-;Dlx6+/- mice display limb defects
(Fig. 6F,R). In mice with the
genotype Dlx5-/-;Dlx6+/- the HLs are normal
(Fig. 6G,S), although a
deviation of central phalanges has rarely (1:30) been observed. We crossbred
p63+/EEC mice with Dlx5+/- and with
Dlx5+/-;Dlx6+/- mice, to obtain animals
doublely (p63EEC;Dlx5) or triplely
(p63EEC;Dlx5;Dlx6) heterozygous. None of these mice showed
limb abnormalities (0/8 and 0/9, respectively). We then crossbred
p63+/EEC;Dlx5+/- mice with
Dlx5+/-;Dlx6+/- mice, to obtain mice with the
genotype p63+/EEC;Dlx5-/-;Dlx6+/-,
in which three out of four alleles of the Dlx5-Dlx6 cluster are
deleted. At birth, these mice (3/3) showed severe ectrodactyly (in one case
monodactyly) of their HLs, with two to four missing fingers, syndactyly, and
dysmorphologies of the most proximal segments of the central phalanges
(Fig. 6U,V). These defects were
never observed with any of the two mutations (p63+/EEC or
Dlx5-/-;Dlx6+/-) individually.
p63+/EEC;Dlx5-/-;Dlx6+/- mice also
showed anomalies of their FLs (two out of three), comprising missing posterior
fingers and syndactyly (Fig.
6K). Importantly, FL defects were never observed in our strain of
Dlx5;Dlx6 DKO mice (Merlo et al.,
2002) (Fig. 6) or
in p63+/- ones, although a mild deviation of the central
phalanges was rarely (1:40) observed in p63+/EEC mice. In
summary, the HL defects in
p63+/EEC;Dlx5-/-;Dlx6+/- animals
represent a significant aggravation and point to a developmental function for
p63-Dlx regulation, in vivo.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
corresponding murine models p63-, p63EEC
(SHFM-IV), Dlx5;Dlx6 DKO (SHFM-I) and dactylaplasia
(SHFM-III) show defects in limb development of varying severity. The existence
of these phenotypic similarities suggested the possibility that the
corresponding disease genes might participate in a regulatory cascade. Here we
show that: (1) p63 colocalizes with Dlx proteins in the embryonic AER and is
associated with the Dlx5 and Dlx6 promoters in vivo; (2)
Np63, the predominant p63 isoform expressed in the developing
limbs, can activate Dlx5 and Dlx6 transcription; (3) EEC and
SHFM, but not AEC, mutations nearly abrogate the transcriptional activity of
Dlx5 and Dlx6 promoters; (4) the expression of Dlx5
and Dlx6 is reduced in p63- and
p63EEC heterozygous and homozygous limbs; and (5)
heterozygous p63EEC mutations combined with the incomplete
loss of Dlx5 and Dlx6 alleles results in aggravated limb
phenotypes. These observations indicate that p63 lies genetically upstream of
the Dlx genes during limb development.
The involvement of p63, Dlx5 and Dlx6 in SHFM-related
limb phenotype is well established, although their molecular functions in the
maintenance of the AER are incompletely known. On the contrary, the disease
genes responsible for SHFM-III and SHFM-V are still unknown. SHFM-V has been
associated with deletions on chromosome 2q24-q31, a large cytogenetic region
proximal to, and perhaps including, DLX1 and DLX2
(Boles et al., 1995;
DelCampo et al., 1999;
Maas et al., 2000). More
recently, Goodman and co-authors (Goodman
et al., 2002) proposed that the SHFM-V locus is located in the
interval between EVX2 (2q31-q32) and marker D2S294, 5 Mb centromeric
to EVX2. This raises the possibility that SHFM-V and other digit
anomalies may be caused by haploinsufficiency of the 5' HOXD,
EVX2 or DLX1 and DLX2 genes. In p63
heterozygous and homozygous limbs, expression of Dlx1 and
Dlx2 is reduced, but overall to a lower extent than Dlx5 and
Dlx6. Thus, our findings are compatible with a role for the
DLX1-DLX2 locus in SHFM-V, although they do not actually demonstrate
this. The single inactivation of Dlx1 or Dlx2 in mice does
not result in limb phenotypes; however, a possible role of these two genes
will be clarified by examining the Dlx1;Dlx2 DKO model.
Dactylyn is the proposed disease gene for SHFM-III (human) and
dac+/- (mouse)
(Johnson et al., 1995;
Sidow et al., 1999;
deMollerat et al., 2003).
However, reduced Dactylyn expression has been documented only in lymphocytes
of SHFM-III patients (Basel et al.,
2003), and not in dac+/- limbs. Of note, in
the SHFM-III and dac+/- rearranged locus, one normal copy
of Dactylyn is retained; therefore doubts can be raised as to whether Dactylyn
is the SHFM-III disease gene (deMollerat
et al., 2003; Lyle et al.,
2006). Our data indicate that the expression of Dactylyn is
unchanged in Dlx mutant limbs and is minimally increased in p63
mutant limbs; thus, it is unlikely that Dactylyn lies genetically downstream
of p63 or the Dlx5-Dlx6 locus. SHFM-III has an alternative
explanation: three AER-expressed genes Fgf8, NFB2 and
Lbx1 are located in the Dactylyn chromosomal neighbourhood. Of these
Fgf8, a morphogen essential for limb development
(Lewandoski et al., 2000;
Sun et al., 2002;
Tickle, 2003), is
downregulated in both p63 and Dlx mutant limbs
(Mills et al., 1999;
Robledo et al., 2002). A
downregulation of Fgf8 (and/or NFB2 and
Lbx1) could explain the limb defects in SHFM-III patients and in
dac+/- mice.
|
). This suggests that Dlx5 and Dlx6 may play
partially redundant roles in AER function, and that a threshold level of Dlx
expression needs to be reached. If this were true, it would be expected that
the DKO of other Dlx genes would result in the appearance of SHFM-type limb
defects. Indeed Dlx2;Dlx5 DKO mice show ectrodactyly
(Panganiban and Rubenstein,
2002). Limb phenotypes in combined Dlx DKO mice have not been
reported as yet, but these data would help to clarify this issue.
The regulation of Dlx5 and Dlx6 by p63
Transcriptional regulation within the Dlx5-Dlx6 bigenic cluster
involves at least four mechanisms: (1) tissue-specific enhancers, shared by
the two genes in the cluster, and operating at distance, such as the
intergenic elements I56i and I56ii and the Mef2c-response element; (2)
Mecp2-dependent chromatin looping, possibly linked to partial imprinting of
these genes; (3) interaction between Evf2 ncRNA and Dlx proteins, and
(4) cis-acting regulation on the proximal promoter region, which is expected
to be specific for each gene (Zerucha et
al., 2000; Horike et al.,
2005; Feng et al.,
2006; Verzi et al.,
2007) (this manuscript).
Enhancer-type regulation and chromatin folding
(Zerucha et al., 2000;
Ghanem et al., 2003;
Horike et al., 2005;
Feng et al., 2006) are likely
to be shared by Dlx5 and Dlx6, and accounts for their nearly
identical expression pattern. The highly conserved I56i and I56ii regulatory
elements, located in the intergenic region, comprise homeodomain-binding sites
to which Dlx1 and Dlx2 bind and drive reporter expression in
the embryonic forebrain, pharyngeal arches, and limbs
(Zerucha et al., 2000;
Ghanem et al., 2003). We could
not identify any p53-binding element in the I56i and I56ii sequences, and
consistently could not detect binding of p63 (L.G., unpublished). Thus, we
exclude that p63 might regulate Dlx5-Dlx6 expression via these
intergenic enhancers. Evf2-dependent regulation can also be excluded,
as Evf2 ncRNA could not be detected in embryonic limbs.
Our data indicate that p63 regulates Dlx5 and Dlx6
transcription, at least in part, by cis-acting regulation at the promoter
level. We show that the Np63 isoforms are able to activate
transcription of Dlx5 and Dlx6, whereas the TA isoforms are
largely inactive. Due to the presence of the TA domain, the TAp63 and
- isoforms have been considered to be the transcriptionally active
forms (Barbieri and Pietenpol,
2006); however, the Np63 isoforms can activate
transcription via alternative activation domains
(Ghioni et al., 2002;
King et al., 2003;
Wu et al., 2005). Considering
that Np63 is the predominantly expressed isoform in the AER, the
results are consistent with p63 being genetically upstream of the Dlx
genes. By contrast, expression of Dlx3 in the limb ectoderm is
regulated by TAp63 (Radoja et al.,
2007), which suggests that individual Dlx genes might be regulated
differently by p63 isoforms. It is difficult to determine the exact function
of individual p63 isoforms on Dlx gene expression in vivo, as both the
deletion and the point mutation introduced into the mouse genome will equally
affect both classes. The development of isoform-specific mutant animal models
will be essential to resolve this issue.
p63, Dlx and limb development
Np63 is essential to endow stem cells of the stratified
epithelia with their proliferation potential and to maintain the stratified
organization of some epithelia (Signoretti
et al., 2000; Nylander et al.,
2002; Koster et al.,
2004; Laurikkala et al.,
2006; Senoo et al.,
2007). The AER is a transitory multi-layered ectoderm acting as a
signalling centre essential for distal limb development, digit patterning and
morphogenesis (Niswander,
2002; Tickle,
2003). Consistently, the stratified organization of the AER and
the expression of morphogenetic molecules are dramatically compromised in
p63 mutant limbs (Mills et al.,
1999; Yang et al.,
1999). AER stratification is also lost in Dlx5;Dlx6 DKO
limbs (Robledo et al.,
2002; Merlo et al.,
2002), although restricted to the central AER wedge, and in
dac+/- limbs (Seto et
al., 1997; Crackower et al.,
1998). Thus, it appears that the activity of these three genes is
required for maintaining the AER organization. However, the time of onset of
this defect in p63 mutant limbs is earlier, compared with the other
models (Mills et al., 1999;
Yang et al., 1999). We have
considered the possibility that changes in Dlx gene expression are the mere
consequence of loss of AER stratification; here, we report that Dlx5
and Dlx6, and to a lesser extent Dlx1 and Dlx2, are
downregulated in heterozygous p63- and
p63EEC HLs, which display a normally stratified AER.
Because Dlx5 expression is detectable (although reduced) in the
pre-AER of early p63-/- embryos, it appears unlikely that
Dlx gene expression is altered solely as a consequence of AER failure.
Conversely, Dlx gene expression is not reduced, but is rather increased, in
the heterozygous p63 FLs. This suggests that the
p63EEC mutation behaves differently in FLs and HLs. In the
Dlx- and the p63-mutant mice, as well as in the combined
p63EEC;Dlx mutant ones, HL defects are more prominent and
more severe that those of the FLs. FL defects might therefore result from
different molecular mis-regulations. In the compound
p63+/EEC;Dlx5-/-;Dlx6+/- mice, FL
defects have been observed that affect the distal region but that do not
resemble ectrodactyly. The meaning of this is still unclear: Dlx and p63 may
converge on the regulation of a common target or may engage in an
autoregulatory loop.
Np63 might regulate the expression of a large number of (direct and
indirect) genes (Yang et al.,
2006), only some of which might be essential for AER function. The
expression of Fgf8, the key AER-secreted morphogen essential for
proximal-distal limb growth, is severely downregulated in p63 null
limbs (Mills et al., 1999).
Perp, another p63 target gene, which codes for a desmosome-associated
protein required for the integrity of stratified epithelia
(Ihrie et al., 2005), is also
downregulated in p63EEC mutant limbs. However, no limb
defects have been reported in Perp-null mice, and therefore its role
remains uncertain. One possibility is that Perp downregulation might
contribute to the increased severity of the p63-null phenotype when
combined with reduced Dlx gene expression. Ik,
a p63 transcriptional target required for epithelia stratification and
differentiation, is transactivated directly by TA and Np63, and
indirectly by Np63 via Gata3 (Candi
et al., 2006; Koster et al.,
2007). Ik is also required for
keratinocyte differentiation through its kinase-independent (nuclear)
activity. During embryonic development, Ik
acts cell-autonomously in the ectoderm to maintain normal
epithelial-mesenchyme interactions, acting via repression of Fgf8 and
other Fgfs. These, in turn, are essential for normal craniofacial and limb
development; in fact, Ik-null mice show distal
limb defects (Sil et al.,
2004). The genetic and functional relationship between
p63, Dlx genes, Perp and Ik
in maintaining the AER stratification and function is currently unclear, and
will need to be addressed by crossbreeding the corresponding mutant mice and
examining their limb development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1377/DC1
|
ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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