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First published online 1 November 2006
doi: 10.1242/dev.02621
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Np63 plays an anti-apoptotic role in ventral bladder development
1 Department of Surgery, Hospital for Sick Children, Toronto, M5G 1X8,
Canada.
2 Program for Infection, Immunity, Injury and Repair, Hospital for Sick
Children, Toronto, M5G 1X8, Canada.
3 Program of Developmental Biology, Hospital for Sick Children, Toronto, M5G
1X8, Canada.
4 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
* Author for correspondence (e-mail: peter.kim{at}sickkids.ca)
Accepted 8 September 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Np63, is known to have
anti-apoptotic characteristics. We have established that Np63
is not only the predominant isoform expressed throughout the bladder, but is
also preferentially expressed in the ventral bladder urothelium during early
development. We observed a host of ventral defects in
p63-/- embryos, including the absence of the abdominal and
ventral bladder walls. This number of ventral defects is identical to bladder
exstrophy, a congenital anomaly exhibited in human neonates. In the absence of
p63, the ventral urothelium was neither committed nor differentiated,
whereas the dorsal urothelium was both committed and differentiated.
Furthermore, in p63-/- bladders, apoptosis in the ventral
urothelium was significantly increased. This was accompanied by the
upregulation of mitochondrial apoptotic mediators Bax and
Apaf1, and concurrent upregulation of p53. Overexpression of
Np63
and Np63ß in
p63-/- bladder primary cell cultures resulted in a rescue,
evidenced by significantly reduced expressions of Bax and
Apaf1. We conclude that Np63 plays a crucial
anti-apoptotic role in normal bladder development.
Key words: p63, Bladder exstrophy, Apoptosis, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In cases of BE, the ventral abdominal and bladder walls are
either absent, leaving the bladder cavity exposed, or covered only by an
amniotic sac; the pubic bones, external genitalia and rectus abdominis muscles
are separated along the midline, whereas the anus is displaced ventrally,
often with an associated narrowing or atresia. Treatment of BE is complicated
and involves major reconstructive surgical procedures. Although advanced
parental age (Boyadjiev et al.,
2004), familial links (Shapiro
et al., 1984) and racial predilection
(Roberts et al., 1995) imply a
genetic cause, the molecular mechanisms underlying the formation of BE remain
unknown. As such, our understanding of the pathogenesis of BE remains limited
to that provided by a previous descriptive study
(Muecke, 1964).
During embryogenesis, the cloacal cavity at the posterior end of the embryo
is partitioned by the uro-rectal septum into the ventral urogenital sinus
(UGS) and the dorsal hindgut. The UGS subsequently develops into the urethra,
bladder and urachus. The UGS epithelium differentiates into a stratified
transitional epithelium, known as the urothelium, whereas the mesenchyme of
the UGS differentiates into the lamina propria and the smooth muscle of the
bladder, known as the detrusor muscle. Interaction between the UGS epithelium
and its mesenchyme is crucial for proper development of the detrusor muscle,
as previous studies have shown that the UGS epithelium provides key signaling
input that promotes differentiation of the UGS mesenchyme into smooth muscle
(Baskin et al., 1996b).
Homologs p53, p63 and p73 comprise the p53 gene family
(Levine, 1997;
Murray-Zmijewski et al.,
2006). p63 is highly expressed in all stratified
epithelia and its expression can be detected in the urothelium as early as
E11.5 and persisting thereafter (Kurita
and Cunha, 2001; Kurita et
al., 2004a; Kurita et al.,
2004b). We therefore hypothesize that p63 plays a role in
bladder urothelium development that, in turn, affects bladder development.
p63-/- mice exhibit severe developmental anomalies,
including failure of skin morphogenesis, truncation of limbs and craniofacial
abnormalities (Mills et al.,
1999; Yang et al.,
1999). The specific mechanism underlying the regulation of
epithelial stratification and development by p63 is not fully
delineated and remains controversial. Some investigators suggest that failure
of epithelial stratification in the absence of p63 is related to a
lack of commitment (Koster et al.,
2004; Mills et al.,
1999), whereas others suggest that it results from a defect in
epithelial cell proliferation (McKeon,
2004; Yang et al.,
1999).
p63 expresses multiple N-terminal isoforms, known as
TAp63 and Np63, because of the presence of an
alternative promoter located in intron 3. The full-length isoform,
TAp63, contains a transactivation (TA) domain similar to the TA
domain of p53. TAp63 is capable of activating numerous p53
target genes, promoting cell-cycle arrest
(Yang et al., 1999) and
inducing apoptosis (Jacobs et al.,
2005). Conversely, the truncated isoform, Np63,
acts in a dominant-negative manner towards the TA isoforms of p63 and p53
(Yang et al., 1998).
Np63 has been shown to inhibit apoptosis
(Jacobs et al., 2005) and to
promote stem-cell proliferation in vitro
(Moll and Slade, 2004). In
addition to these N-terminal isoforms, alternative splicing at the C-terminus
of p63 generates three isoforms: 
, ß and . In
combination with the N-terminal isoforms, six p63 isoforms can be
generated (Yang et al.,
1998).
In the current study, we find that the Np63 isoform is the
predominant isoform in the ventral bladder throughout development. In the
absence of p63, the abdominal and ventral bladder walls are absent;
these defects epitomize the BE complex in humans. In addition, the ventral
epithelium of the p63-/- bladder is neither committed to
stratification nor differentiated, and exhibits significantly increased
apoptotic activity. Pro-apoptotic mediators Bax and Apaf1
are upregulated in the p63-/- bladder. Restoration of
Np63ß or Np63 protein levels in
p63-/- bladders partially rescues expression of
Bax and Apaf1. Furthermore, absence of p63 in the
bladder epithelium leads to failure of induction of the adjacent UGS
mesenchyme, resulting in a significant reduction of mesenchymal proliferation.
Taken together, these observations lead us to conclude that
Np63 plays a crucial anti-apoptotic role in the development of
the ventral bladder epithelium.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Homozygous embryos were identified by phenotype. Heterozygous embryos were
genotyped by PCR (primers 5'-GTGTTGGCAAGGATTCTGAGACC-3' and
5'-GGAAGACAATAGCAGGCATGCTG-3').
Histochemistry and immunohistochemistry
Specimen sections (7'm) were stained with 50% hematoxylin and 0.5%
Eosin in 70% ethanol. Carbohydrates were stained with periodic acid and Schiff
reaction (PAS, Surgipath). Alkaline phosphatase (AP) reaction was studied by
treating slides with 0.1 M Tris-buffered solution (pH 9.5) followed by the
addition of BM purple AP substrate (Roche).
Immunochemistry was performed as follows: after quenching the endogenous
peroxidases with 3% H2O2 in 10% methanol, the antigens
were retrieved by boiling the slides in an antigen-unmasking solution (H-3300,
Vector Laboratories). The sections were blocked with blocking reagent (Roche).
Primary antibodies at the following dilutions were applied: cytokeratin 18
(CK18) (1:100, Santa Cruz Biotechnology), p63 (4A4, 1:100, Santa Cruz
Biotechnology), TAp63 (1:20, Santa Cruz Biotechnology),
Np63 (1:100, gift from Dr K Nylander)
(Nylander et al., 2002),
p53 (1:250, Abcam, ab26), p73 (1:200, Abcam, ab17230),
villin (1:100, Santa Cruz Biotechnology), uroplakin 3 (undiluted, Santa Cruz
Biotechnology), smooth-muscle -actin (undiluted, Sigma Chemicals),
cleaved caspase-3 (1:100, Sigma Chemicals), Msx1 (1:500, Covance Research
Products) and smooth-muscle heavy-chain myosin (1: 2000, Santa Cruz
Biotechnology). Appropriate secondary antibodies were applied at 1:200
dilutions. Avidin-biotin-peroxidase complex (ABC)-buffer washing was followed
by substrate diaminobenzidine (DAB) staining. Cell proliferation was assayed
by 5-bromo-2'deoxyuridine (BrdU) incorporation (animals were injected
with 100 µm BrdU per gram of bodyweight). Apoptosis was studied using the
terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL)
assay and FragEL DNA Fragmentation Detection Kit (Calbiochem).
RNA extraction, qPCR and RT-PCR
Total RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA was
synthesized using the SuperScript II First-Strand Synthesis Kit (Invitrogen),
then purified using a QIAquick PCR Purification Kit (Qiagen). Quantitative
polymerase chain reactions (qPCRs) were performed using commercially available
p53 primers (SuperArray Bioscience, PPM02931A-24) and selfdesigned
primers: p53 (5'-CACCTCACTGCATGGACGATC-3',
5'-GTCTGCCTGTCTTCCAGATACTCG-3', T: 59.1°C); p73
(5'-CAAGAAGGCAGAGCATGTGA-3',
5'-TCATACGGCACAACCACACT-3', T: 50.1°C);'-actin
(internal control, 5'-CCTTTTCCAGCCTTCCTTC-3',
5'-TACTCCTGCTTGCTGATCC-3', T: 55.0°C); Bax
(5'-CGAGCTGATCAGAACCATCA-3',
5'-CTCAGCCCATCTTCTTCCAG-3', T: 50.1°C); and Apaf1
(5'-GAGAAAACCCTGAGGCACAA-3',
5'-TAATTAAAGCGGCTGCTCGT-3', T: 50.4°C). The relative
expressions were analyzed according to Pfaffl's methods
(Pfaffl, 2001).
Reverse transcriptase-polymerase chain reactions (RT-PCRs) were performed
using the following primers: Np63
(5'-CAATGCCCAGACTCAATTTAGTGA-3',
5'-GGCCCGGGTAATCTGTGTTGG-3', 221bp, T: 51.4°C); TAp63
(5'-AACCCCAGCTCATTTCTCTG-3',
5'-GGCCCGGGTAATCTGTGTTGG-3', 449 bp, T: 57.0°C);
p63' (5'-ACGGGGTGGAAAAGAGATGGTC-3',
5'-AAGAGACCGGAAGGCAGATGAAG-3', 919 bp, T: 59.5°C);
p63 (5'-GACTTGCCAAATCCTGACA-3',
5'-AAGAGACCGGAAGGCAGATGAAG-3', 619 bp, 55.1°C); and
p63ß (5'-CTCCCCGGGGCTCCACAAG-3',
5'-AAGAGACCGGAAGGCAGATGAAG-3', 338 bp, T: 56.2°C).
Immunoblot
Immunoblot was performed as previously described
(Qiu et al., 2004). Briefly,
the cultured cells were washed twice with 1xPBS and lysed using a
solubilizing buffer (1xPBS containing 1% Nonidet P-40, 1% deoxycholate,
5 mM EDTA, 1 mM EGTA, 2 mM PMSF, 0.1 mM leupeptin, 100 KIU/mL Trasylol and 0.5
µm ALLN), and an equal amount of cell lysates were resolved on 8% SDS-PAGE
mini gels. Following SDS-PAGE, the protein was transferred electrophoretically
for 18 hours at 4°C onto PVDF. The membranes were blocked with a 4%
solution of fat-free dry milk powder, incubated with the primary antibodies
(anti-Bax antibody, Upstate Cell Signaling, cat. no. 06-499, 1:500;
anti-Apaf1, Chemicon, cat. no. MAB3504, 1:500; and anti-ß-actin
antibody, Sigma Chemicals, cat. no. A 5441, 1:1000), washed, and incubated
with a secondary antibody conjugated to horseradish peroxidase. Membranes were
then incubated in an enhanced chemiluminescence-detection reagent (Amersham
Pharmacia Biotech) and exposed to Kodak Hyperfilm (Eastman Kodak). Films were
developed and quantitative analysis was performed using an imaging
densitometer.
Organ culture, primary cell culture and transfection
The dissected bladders were cultured in 50% BGJb medium (Invitrogen), plus
50% A10 (Wisent) culture medium with supplements of transferrin (20 ng/ml),
insulin (10 ng/ml) and epithelial growth factor (10 ng/ml). Primary cells were
cultured with Eagle's minimum essential medium (EMEM; Wisent) and 20% fresh
bovine serum (FBS). Green fluorescent protein (GFP)-tagged recombinant
bicistronic adenoviruses with Np63,
Np63ß, and TAp63 constructs were
generated, purified and titered as previously reported
(Jacobs et al., 2005). The
adenoviruses were added to the culture media at the ratio of 5-10 pfu per
cell.
In situ hybridization
In situ hybridization of paraffin sections with a DIG-labeled RNA probe was
performed as previously described (Hui and
Joyner, 1993). Briefly, the dewaxed slides were pre-fixed with 4%
paraformaldehyde, permeabilized with proteinase K (0.02 mg/ml), and treated
with 0.2 M HCl solution and 0.1 M triethanoloamine solution (TEA), plus 0.025
ml acetic anhydride/liter of TEA. The slides were then hybridized with 4.0
µg/ml of DIG-labeled RNA probes (DIG labeling mix; Roche) in a
formamide/sodium citratesodium chloride (SSC) buffer in a 55°C oven
overnight. The slides were then washed with a 5xSSC/formamide solution.
and treated with RNAse-A, 2xSSC and 0.2xSSC before being blocked
with blocking reagent (Roche). Anti-DIG alkaline phosphatase antibody (Roche)
was then applied, followed by BM purple AP substrate (Roche).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Np63 is the predominant isoformNp63
(Nylander et al., 2002).
Expression of the Np63 isoform (detected by
anti-Np63 antibody) was found to be similar to that of the
pan-p63 antibody (detected by 4A4), suggesting that
Np63 represents the predominant isoform during bladder
development. Np63 expression began in the ventral UGS
epithelium at E11.5 and extended to the rest of the epithelium later in
development (Fig. 2E-H).
Np63 was also expressed in the epithelium overlying the
urogenital tubercle (Fig.
2E,F). By contrast, TAp63 (detected by
anti-TAp63) was expressed only transiently from E11.5 to E12.5 in the
epithelium of the distal hindgut and in its communication with the UGS
(Fig. 2I,J). TAp63
expression decreased markedly in the distal hindgut after E14.5
(Fig. 2K,L and data not shown)
and was not expressed in the skin overlying the urogenital tubercle.
To verify the dominant p63 N-terminal isoform expressed in
wild-type bladders, RT-PCR was performed on RNA extracted from E15.5 wild-type
bladders using primers specific to Np63 and TAp63,
respectively. This analysis confirmed that the predominant N-terminal isoform
of p63 during early bladder development was Np63
(Fig. 2M). Unlike in the skin,
where p63 is the predominant C-terminal isoform
(Westfall et al., 2003;
Yang et al., 1998), RT-PCR
detected only p63 and p63ß in the bladder
epithelium (Fig. 2M). Thus, we
concluded that Np63 and Np63ß are
the predominant isoforms of p63 expressed in bladder epithelium
during development.
p63 expression is ventrally restricted during early bladder development
To understand better why p63-/- embryos develop ventral
midline defects, the p63 expression pattern during early bladder
development was studied using immunohistochemistry. Although there was
widespread p63 expression throughout the skin and urothelium in E18.5
embryos (data not shown), sagittal sections of E11.5 embryos showed that
p63 expression was restricted to the ventral epithelia of the
urogenital tubercle and UGS, the tail bud, the oral epithelium
(Fig. 3A, arrows), and the
apical ectodermal ridge (data not shown). Horizontal pelvic sections of E11.5
embryos confirmed that p63 expression was present in the skin
overlying the urogenital tubercle (Fig.
3B) and ventral UGS epithelium
(Fig. 3C). In later
gestational-stage embryos, p63 expression was stronger and epithelial
stratification was more advanced in the ventral skin compared with that of the
dorsal skin (Fig. 3D-F). In
summary, p63 expression in early bladder- and skinepithelia
development is ventrally restricted.
p63-deficient bladder epithelium is abnormal along the dorso-ventral axis
To determine whether the stratification of the endoderm-derived urothelium
is affected similarly to that of the ectoderm-derived epithelium in the
absence of p63, E18.5 p63-/- bladders were
stained with hematoxylin and Eosin. This analysis revealed that, whereas
wild-type bladder epithelium differentiates into stratified transitional
urothelium (Fig. 4A,C), the
bladder epithelium of p63-/- mutants fails to stratify and
remains as a single layer (Fig.
4B,D). Differences in epithelial morphology were also noted along
the dorso-ventral axis. The dorsal epithelium of p63-/-
bladder consisted mainly of simple cuboidal cells
(Fig. 4D), whereas the ventral
epithelial cells were primarily simple squamous cells
(Fig. 4B, arrow).
As p63 has previously been shown to be essential in ectodermal
epithelial commitment and/or differentiation
(Mills et al., 1999), the role
of p63 in bladder development was examined using wellestablished
markers for epithelial differentiation in E18.5 embryos. K18, a marker of the
endoderm or uncommitted epithelium, is not expressed in stratified or
differentiated epithelia (Koster et al.,
2004). We noted that, in mature wild-type bladder urothelium, K18
expression was absent (Fig.
4E,G). In the p63-/- bladder, whereas the
dorsal epithelium did not express K18 (Fig.
4H), the ventral epithelium retained K18 expression, indicating
that it was uncommitted to stratification
(Fig. 4F). To further determine
the status of epithelial differentiation in bladder tissue, the expression of
uroplakin 3, a marker for terminally differentiated urothelium, was studied
(Wu et al., 1999). Uroplakin 3
was strongly expressed in mature wild-type bladder urothelium
(Fig. 4I,K). In the
p63-/- bladder, uroplakin expression was reduced in the
dorsal epithelium (Fig. 4L) and
undetectable in the ventral epithelium
(Fig. 4J). This suggests that,
whereas the p63-/- ventral bladder epithelium is
undifferentiated, the dorsal epithelium is capable of differentiation, even in
the absence of p63 (Fig.
4K,L). As null mutation of p63 has been reported to be
associated with intestinal metaplasia
(Signoretti et al., 2005;
Yang et al., 1999), intestinal
markers were also examined in the p63-/- bladder
epithelium. This analysis revealed that intestinal transformation does not
occur in the p63-/- bladder epithelium (Fig. S1 in the
supplementary material). In summary, null mutation of p63 was noticed
to affect the development of bladder epithelium differentially along the
dorso-ventral axis, ultimately resulting in uncommitted and undifferentiated
ventral bladder epithelium.
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Np63 is known to act as a naturally occurring dominant
negative. It has been shown to counteract the pro-apoptotic actions of
TAp63 and p53 in vitro
(Yang et al., 1998). We have
shown that Np63 is the major isoform of p63 expressed
in the developing ventral bladder, and that both the mesenchyme and epithelium
of the ventral UGS develop abnormally in the absence of Np63
(Fig. 5A,B). As such, we
hypothesized that p63 acts as a pro-survival protein in the
developing bladder, thus preventing the apoptosis of ventral UGS epithelium
during development. To directly test this hypothesis, we examined the amount
of apoptosis in p63-/- bladders by TUNEL assay and cleaved
caspase-3 expression. The number of TUNEL-positive cells in the ventral UGS
epithelium of E11.5 p63-/- mutants was significantly
higher than that of wild-type controls
(Fig. 5C,D) (44% versus 9%,
Student's t-test, P<0.05). This increase in apoptosis was
further corroborated by an observed increase in cleaved caspase-3 expression
in p63-/- ventral UGS epithelium (35% versus 5%, Student's
t-test, P<0.05) (Fig.
5E,F). In comparison, we noted minimal apoptotic activity in the
dorsal epithelia of both wild-type and p63-/- bladders, as
determined by TUNEL assay and cleaved caspase-3 expression
(Fig. 5C-F). We also compared
the apoptotic activities (percentage of cleaved caspase-3-positive cells) of
skin overlying the p63-/- and wild-type urogenital
tubercles (12.8±2.7% and 2.7±1.7%) and found the difference
between them was statistically significant (Student's t-test,
P<0.01). This phenomenon of increased apoptosis in the absence of
p63 does not appear to be restricted to the ventral UGS. Oral-cavity
epithelium, which also expresses high levels of p63 during early
development (Fig. 3A), was
noted to have a statistically significant increase in apoptosis in the
p63-/- embryo (Fig.
5G,H, 49.0±1.0% versus 9.0±1.0%, Student's
t-test, P<0.05). In summary, our data show that apoptosis
is increased in the epithelia of the ventral UGS, as well as in other
epithelial structures where p63 expression is normally high during
early development.
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|
). As
such, we analyzed the expression of the mitochondrial apoptotic mediators
Bax and Apaf1 in E12.5 and E13.5 wild-type and
p63-/- bladders by qPCR. In the p63-deficient
bladders, the relative expressions of Bax and Apaf1 were
increased at both E12.5 and E13.5 (Fig.
5I,J). Taken together, our data showed that, in the absence of
Np63, there was increased mitochondrial apoptotic activity in
the developing bladder.
ßNp63 is anti-apoptotic during bladder development
To confirm the anti-apoptotic role of Np63 during bladder
development, E13.5 p63-/- bladder primary cell cultures
were infected with bicistronic adenoviruses expressing TAp63,
Np63ß, Np63 and green fluorescent
protein (GFP), or GFP alone, for 24 hours. The cells were then harvested and
the expressions of Bax and Apaf1 were quantified with
immunoblots. Their gel tensiometry readings were compared. Compared with the
GFP-transfected control, transfection with Np63ß or
Np63 adenoviruses significantly reduced the expressions
of both Bax (P<0.05) and Apaf1
(P<0.01), whereas transfection with TAp63
adenovirus led to an increase in the expressions of both Bax and
Apaf1 (P<0.05) (Fig.
6A,B). To confirm the anti-apoptotic role of Np63,
organ cultures of E13.5 p63-/- bladders were infected with
bicistronic adenoviruses expressing Np63 and GFP or GFP
alone for 24 hours. Bax expression was examined by qPCR. We observed
a more than 50% reduction of Bax relative expression in the
Np63-infected p63-/- bladder. These
data suggest that Np63, the predominant isoform of
p63 in the bladder, plays an anti-apoptotic role during bladder
development.
Apoptosis of bladder cells of E12.5 p63-/- animals is associated with upregulation of p53 and p73 expression
We then examined whether the expressions of p53 and p73
were affected in p63-/- bladders. Immunohistochemical
staining showed an upregulation of p53 expression in the ventral aspect of
E11.5 p63-/- UGS (Fig.
6C,D). Additionally, there also appeared to be an upregulation of
p73 expression in p63-/- UGS
(Fig. 6E,F). We proceeded to
quantify the p53 and p73 mRNA expressions in E12.5 and E13.5
wild-type and p63-/- bladders using real-time PCR
analysis. There was a compensatory upregulation of the relative expressions of
both p53 and p73 in E12.5 p63-/-
bladders, compared with those of the wild-type controls (Student's
t-test, P<0.05). Interestingly, in E13.5
p63-/- bladders, the expressions of both p53 and
p73 were downregulated (Fig.
6G,H, Student's t-tests, P<0.01). The
transient upregulation of p53 and p73 expression in
p63-/- bladders co-incides temporally with increased
apoptosis (Fig. 5C-F).
|
Np63). To examine how the p63-null epithelium
affects the adjacent mesenchyme, we studied the expression of Msx1, a
homeobox gene that is induced in the mesenchyme by an epithelium-derived
signal. In the wild-type control embryos, Msx1 was expressed in the
ventral subepithelial mesenchyme of E13.5 bladders, suggesting expression of
Msx1 in the mesenchyme by the ventral UGS epithelium
(Fig. 7A, arrow). By contrast,
the expression of Msx1 is greatly reduced or absent in the
sub-epithelial mesenchyme of E13.5 p63-/- bladders
(Fig. 7B). To assess whether
the p63-positive epithelium release mediator(s) that are crucial for
homeostasis of the mesenchyme, we studied, by mRNA in situ hybridization, the
expression of Fgf8, another marker normally induced in the apical
ectodermal ridge by p63 (Mills et
al., 1999; Yang et al.,
1999). Fgf8 is normally expressed in the mesenchyme of
E11.5 wild-type UGS (Maruoka et al.,
1998) (Fig. 7C). We
found that the expression of Fgf8 was downregulated in the
p63-/- ventral UGS
(Fig. 7D). These results
suggest that p63 deficiency in UGS epithelium is associated with a
failure of induction in the adjacent UGS mesenchyme, especially ventrally.
Epithelial-mesenchymal transition (EMT) is a cellular mechanism during which
certain cells switch from an epithelial to a mesenchymal status. During
development, EMT is involved in neural-crest migration, heart morphogenesis
and formation of palate mesenchymal cells from the oral epithelium on E13.5 in
mice (Larue and Bellacosa,
2005). We questioned whether bladder epithelial cells undergo EMT,
as this might explain why the failure of epithelial development is associated
with failure of mesenchymal development. We studied the expression of Snai1
(m-snail), which induces the epithelial-mesenchymal transition
(Barrallo-Gimeno and Nieto,
2005; Cano et al.,
2000). We could not detect any Snai1 expression in either the
wild-type or p63-/- E14.5 bladders by in situ
hybridization (Fig. 7E,F).
These results failed to demonstrate the role of EMT at this stage of bladder
development.
To explain the paucity of smooth muscle in the ventral bladder wall,
mesenchymal cell proliferation was studied with the incorporation of BrdU.
Msx1 is commonly expressed in regions of rapid proliferation
(Bendall and Abate-Shen, 2000)
and Fgf8 regulates survival and proliferation in the anterior heart field
(Park et al., 2006). In the
absence of epithelial Np63 expression, mesenchymal expression
of both Msx1 and Fgf8 were decreased. This was accompanied
by a reduction in cell proliferation in both the epithelium and mesenchyme,
especially ventrally (Fig.
7G,H). The difference in cell proliferation between
p63-/- and the wild-type control was statistically
significant (ANOVA, dorsal epithelium and mesenchyme: P<0.01;
ventral epithelium and ventral mesenchyme: P<0.0001)
(Fig. 7I). Taken together, in
the absence of Np63, the ventral bladder epithelium fails to
induce expression of Msx1 and Fgf8 in the adjacent
mesenchyme. This is associated with a decreased mesenchymal cell
proliferation.
Smooth-muscle differentiation is disturbed in p63-deficient bladders
Msx1 is known to repress terminal differentiation
(Bendall and Abate-Shen, 2000).
To determine the effect of Msx1 downregulation on smooth-muscle
differentiation, the expression of smooth-muscle heavy-chain myosin, which is
present only in mature smooth-muscle cells
(Owens, 1995), was studied
immunohistochemically. In E14.5 wild-type bladders, smooth-muscle heavy-chain
myosin expression was absent or very weak, whereas, in the
p63-/- bladder, its expression was strong in the thin
ventral bladder wall (Fig.
8A,B). This suggests that the absence of Msx1 in the ventral
mesenchyme allowed premature smooth-muscle differentiation in the adjacent
mesenchyme. Despite premature smooth-muscle differentiation, the E18.5
p63-/- bladder contained little or no smooth muscle
ventrally, but did retain a thin layer of smooth muscle dorsally
(smooth-muscle -actin). Moreover, the lamina propria was either greatly
reduced or absent in the p63-/- bladder
(Fig. 8C,D). In addition,
unlike the wild-type bladder detrusor muscle, which displayed well-organized
smooth-muscle stratification, the dorsal smooth muscle in the
p63-/- bladder was disorganized and non-stratified
(Fig. 8E,F).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Np63 is preferentially expressed in the ventral UGS during
early bladder development. Second, in the absence of Np63,
ventral bladder epithelial development is abnormal because of an increase in
ventral bladder epithelial apoptosis. Third, Np63 prevents
this ventral bladder epithelial apoptosis by, at least partially,
downregulating the mitochondrial apoptotic pathway. Finally, in the absence of
Np63, there is decreased cell proliferation in the UGS
mesenchyme. The increased epithelial apoptosis and decreased cell
proliferation in both epithelium and mesenchyme ultimately results in bladder
exstrophy in p63-/- embryos.
|
) and
ventral UGS (Fig. 3C)
(Kurita et al., 2004a). In the
absence of p63, development of these ventral structures is defective,
and is manifested as a truncated maxilla, cleft palate, ventral pelvic-wall
defect (Ince et al., 2002),
bifid genitalia and bladder exstrophy. We have established that
Np63 is the predominant p63 isoform throughout
bladder development. In a zebrafish model, Np63 has been noted
to be required for ventral specification, with loss of Np63
resulting in a reduction of ventral (non-neural) ectoderm, whereas
overexpression of Np63 expands the ventral ectoderm
(Bakkers et al., 2002).
Np63 is also a direct target of Bmp4
(Bakkers et al., 2002), a
morphogen vital for correct ventral patterning
(Lemaire and Yasuo, 1998). In
light of these findings, our results suggest that Np63 may be
a ventralizing protein in mammalian development and absence of
Np63 may account for the ventral midline defects observed in
p63-/- embryos.
Msx1 is a mesenchymal marker, the expression of which is induced by
adjacent epithelial tissue (Jowett et al.,
1993). In p63-/- embryos, in which limb buds
are absent or vestigial, Msx1 expression in the progress zone beneath
the apical ectodermal ridge of the limb bud is greatly reduced or absent
(Mills et al., 1999;
Yang et al., 1999). Our
findings further suggest that epithelial-mesenchymal interaction also plays an
important role in ventral bladder development. Msx1 expression in the
ventral mesenchyme is deficient in the p63-/- bladder,
suggesting that p63-deficient epithelium fails to induce
appropriately the adjacent mesenchyme (Fig.
6A,B). This decreased Msx1 expression is associated with
a reduction in UGS mesenchymal proliferation and premature terminal
differentiation of the smooth muscle. Interestingly, Msx1 is also a
ventralizing signal responsible for mesoderm patterning under the regulation
of Bmp4 in Xenopus (Takeda et
al., 2000). In summary, a failure of mesenchymal induction may be
responsible for the changes observed in p63-/- ventral UGS
mesenchyme.
Notably, the specific epithelial signal to the UGS mesenchyme remains
undefined. A possible candidate protein for this role is the secreted
diffusible morphogen sonic hedgehog (Shh). Shh is
known to participate in numerous developmental processes involving the
epithelial-mesenchymal interaction (Ingham
and McMahon, 2001). It also promotes proliferation and inhibits
differentiation in renal mesenchymal cell development
(Yu et al., 2002).
Furthermore, Shh is expressed in the UGS epithelium during early
bladder organogenesis (Bitgood and McMahon,
1995; Mo et al.,
2001). We found that the expression of Shh was reduced in
the ventral p63-/- UGS, where the epithelium is squamous
and uncommitted (Fig. S2 in the supplementary material). The reduction of
Shh signaling may contribute to the reduction in cell proliferation
and premature terminal differentiation in ventral bladder mesenchymal
development. This remains to be determined.
Temporospatial restriction of p63 expression determines epithelial commitment to stratification and differentiation
The exact role of p63 during epithelial development is
controversial (McKeon, 2004).
p63 either commits the epithelium to stratification
(Mills et al., 1999) or
maintains epithelial proliferation (Yang
et al., 1999). Koster et al. suggested that TAp63
initiates epithelial commitment and that Np63 is responsible
for epithelial differentiation (Koster et
al., 2004). Our study shows that, during the developmental period
examined (E11.5-E17.5), Np63 expression began in the ventral
UGS and progressively extended to the remaining bladder urothelium. In the
p63-/- bladder, a clear phenotypic difference is noted in
the epithelium along the dorso-ventral axis of the bladder. The ventral
epithelium is squamous, with almost no adjacent smooth muscle, whereas the
dorsal epithelium is cuboidal with a thin layer of disorganized muscle. The
distal ventral UGS epithelium, which is destined to become the urethra,
transforms into an intestine-like epithelium
(Signoretti et al., 2005). The
ventral epithelium remains both uncommitted (positive for K18) and
undifferentiated (negative for uroplakin 3). The dorsal epithelium, however,
is both committed (negative for K18) and differentiated (positive for
uroplakin 3). These results suggest that the timing of p63 expression
in normal bladder development determines the extent of developmental delay.
Thus, the absence of p63 during early ventral bladder development
affects both epithelial commitment and differentiation, whereas, in dorsal
epithelium, where p63 is normally expressed later, p63
deletion does not appear to affect either commitment or differentiation.
Np63 is prosurvival in ventral bladder development
This study provides in vivo evidence that Np63 is
anti-apoptotic during bladder development. In vitro study showed that
Np63 can compete for the apoptotic target-gene site or form a
transactivationincompetent heterocomplex with p53 or TAp73,
thus inhibiting apoptosis (Yang et al.,
2002). In our study, apoptotic activity was increased in the
ventral UGS epithelium in the absence of Np63
(Fig. 5D,F). Expression of the
mitochondrial apoptotic mediators Bax and Apaf1 was also
elevated in p63-/- bladders; elevated Bax and
Apaf1 expressions in p63-/- bladders were rescued
by the overexpression of either Np63ß or
Np63. This rescue is corroborated by a previous study,
in which ectopic Np63 expression in the epidermis
reduces epidermal susceptibility to ultraviolet light-induced apoptosis
(Liefer et al., 2000). In
developing sympathetic neurons, where Np73 is the predominant
isoform, a p73 knockout leads to increased apoptosis in a fashion
similar to the p63 knockout in the ventral bladder. Overexpression of
Np73 rescues the sympathetic neurons from apoptosis induced by
withdrawal of the nerve growth factor
(Pozniak et al., 2000). Our
results not only confirm the functional consistency of N isoform
proteins of the p53 family, but also demonstrate the anti-apoptotic
role of the Np63 isoform during normal mammalian
development.
Np63 is also detected in oral carcinoma and the intensity
of its expression increases with the severity of dysplasia
(Nylander et al., 2002),
suggesting an oncogenic role or stem-cell pluripotency factor for the
Np63 isoform. The possible mechanism may involve a
p53 target gene, p21,as Np63 binds
the p21 promoter, represses its transcription and permits cell-cycle
progression (Westfall et al.,
2003). The scenario is similar to Np53, which is
tumorigenic (Mowat et al.,
1985). Overexpression of a C-terminal dominant-negative fragment
of p53 (Np53)
(Shaulian et al., 1992) in
human urothelial cells has been reported to increase the cell-proliferation
rate (Shaw et al., 2005). In
this study, p63-/- mutation has been shown to be
associated with a significant reduction of cell proliferation in the ventral
bladder epithelium. This suggests that Np63 also promotes
epithelial proliferation in mammalian bladder development. Autocrine
regulation of urothelial cell proliferation via the EGFR signaling loop
observed in urothelial regenerative response could also play a role in
urothelial development (Varley et al.,
2005).
Our data showed that deletion of p63 is associated with compensatory upregulation of p53 expression in the bladders of younger embryos (E11.5-E12.5). This p53 upregulation may further contribute to the apoptosis observed in the ventral p63-/- bladder, in addition to the protein-protein and protein-target gene interactions. Although the expression of p73 in p63-/- bladders is also upregulated, its role in inducing apoptosis is uncertain, as the predominant isoform of p73 expressed in bladder has not been studied.
In conclusion, we have established a p63-/- murine
model for BE. We have found that Np63 is expressed initially
in the ventral bladder urothelium and possesses a ventralizing property.
Although the complete bladder urothelium fails to stratify in the absence of
p63, ventral urothelial development is more delayed than that in the
dorsal epithelium, being both uncommitted and undifferentiated. We found that
Np63 is the predominant isoform in the bladder. Without
Np63, urothelial apoptosis is increased and cell proliferation
is reduced. We also noted a concurrent upregulation of p53
expression. Overexpression of Np63 and
Np63ß rescue the expression levels of the mitochondrial
apoptotic mediators Bax and Apaf1 in
p63-/- bladders. We conclude that Np63
plays a crucial anti-apoptotic role during ventral bladder development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4783/DC1
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ACKNOWLEDGMENTS |
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