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First published online April 13, 2007
doi: 10.1242/10.1242/dev.02830
1 Department of Anatomy I, Fujita Health University School of Medicine, Toyoake,
Aichi 470-1192, Japan.
2 Laboratory of Molecular and Genetic Information, Institute for Molecular and
Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032,
Japan.
Author for correspondence (generation of Dlgh1 mutant mice) (e-mail:
akiyama{at}iam.u-tokyo.ac.jp)
Author for correspondence (other issues) (e-mail:
tsenda{at}fujita-hu.ac.jp)
Accepted 8 February 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Dlgh1 (Dlg1), Gene targeting, Kidney, Ureter, Vagina, Müllerian duct, Wolffian duct
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Dlgh1 is expressed in mammalian epithelial and neuronal cells, and has been
reported to be involved in cell-cell adhesion of intestinal epithelial cells
(Laprise et al., 2004), cell
cycle inhibition in cervical cancer and cultured cells
(Ishidate et al., 2000;
Watson et al., 2002), cellular
polarization in migrating astrocytes
(Etienne-Manneville et al.,
2005), and localization and clustering of glutamate receptors at
the synaptic membrane (Leonard et al.,
1998). Furthermore, Naim et al. showed that Dlgh1 is involved in
nephrogenesis (Naim et al.,
2005).
Dlgh1 is considered to function as a scaffold protein and is known to
interact with various proteins. The L27 domain in the N-terminal region of
Dlgh1 interacts with calmodulin-associated serine/threonine kinase (CASK),
another member of the MAGUK proteins, and this interaction recruits Dlgh1 to
regions of cell-cell contact (Lee et al.,
2002). Furthermore, Dlgh1 contains three PDZ domains, an SH3
domain and a GK-like domain, which are involved in protein-protein
interactions.
During ontogeny, epithelial tissues with functional characteristics
appropriate for their developmental stage and environmental circumstances are
organized. In this process, the cells acquire two important epithelial
features: cell-cell adhesion and apicobasal polarity. In addition, epithelial
tissues change their shape during early developmental stages in order to form
organs. During these processes, many functional proteins must be localized at
precisely regulated subcellular locations, and scaffolding proteins appear to
play important roles in these protein localizations. Since many binding
partners of Dlgh1 have been identified and as Dlgh1 is widely distributed in
the body (Caruana and Bernstein,
2001), we hypothesized that Dlgh1 might be involved in the
development of epithelial tissues.
The roles of Dlgh1 in mammalian development have previously been analyzed
using gene-targeted mice lacking the C-terminal half of the Dlgh1 protein
(Caruana and Bernstein, 2001).
In these mice, a lacZ gene is inserted into Dlgh1, such that
the gene product contains the N-terminal three PDZ domains, but lacks the SH3,
protein 4.1 and GK-like domains. The homozygous mice exhibit growth
retardation in utero, hypoplasia of the premaxilla and mandible and a cleft
secondary palate, and die perinatally. In addition, the nephron number is
decreased by 30% (Naim et al.,
2005). In that study, however, the functions of Dlgh1 that are
attributed to intermolecular interactions via its PDZ domains could not be
analyzed in the gene-targeted mice. Therefore, the physiological significance
of Dlgh1 functions in epithelial cells, which have been analyzed in
invertebrate and vertebrate cultured cells, has not yet been confirmed.
We generated mutant mice null for the Dlgh1 gene. In these mice, a neo cassette sequence is inserted into exon 3 of Dlgh1, such that the protein product does not contain any PDZ domains, even if it is translated. In the present study, we analyzed the phenotypes of these targeted mice to elucidate the functions of full-length Dlgh1 protein in developing epithelial tissues. We focused on the development of the urogenital tracts, in which the correct organization of epithelial tissues is of particular importance for morphogenesis. In normal development of the urinary and genital tracts, a ureteric bud sprouts from the Wolffian duct and then penetrates the metanephric mesenchyme. After such penetration, reciprocal interactions between the ureteric bud tips and the metanephric mesenchyme induce a series of ureteric branches that form the collecting duct system, and differentiation of the condensed mesenchymal cells into epithelial cells that compose the urinary tubules. The lower part of the ureteric bud elongates out of the metanephric mesenchyme and becomes the ureter. Beside the Wolffian duct, the Müllerian duct (also called the paramesonephric duct) is formed by invagination of the coelomic epithelium. The Wolffian and Müllerian ducts differentiate into male and female reproductive tracts, respectively. Developmental analyses of the urogenital organs in homozygous (Dlgh1-/-) mice revealed structural abnormalities of the kidneys and urogenital tracts derived from the Wolffian and Müllerian ducts, indicating the involvement of Dlgh1 in the morphogenesis of these organs. In order to identify the factors responsible for the abnormal development of these organs, we compared the proliferative indexes and localizations of cell-cell junctional proteins in the ureteric epithelium of Dlgh1+/+ and Dlgh1-/- mice. Contrary to previous hypotheses, the results revealed that epithelial cell proliferation was decreased in Dlgh1-/- mice, and that the subcellular distribution of the junctional proteins was not affected by the loss of Dlgh1 protein. These results provide novel insights into the functions of Dlgh1 and suggest that it plays crucial roles in the development of urogenital organs.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). The other primary antibodies used in this study were as
follows: anti-glyceraldehyde 3-phosphate dehydrogenase (G3PDH) antibody
(MAB374; Chemicon), anti-pancytokeratin antibody (C2562; Sigma), anti-Pax2
antibody (71-600; Zymed), anti-E-cadherin antibody (C20820; BD Transduction),
anti-ZO-1 antibody (61-7300; Zymed) and anti-occludin antibody (71-1500;
Zymed). All Alexa Fluor-conjugated secondary antibodies were purchased from
Molecular Probes and used at 1:200 dilution. For western blotting,
peroxidase-conjugated secondary antibodies were purchased from Jackson
ImmunoResearch Laboratories.
Gene targeting
A genomic Dlgh1 DNA clone was isolated from a TT2 genomic library,
and used to generate a Dlgh1-targeting vector containing a short
homologous arm (0.85 kb) on the 5' side of exon 3, and a long homologous
arm (8.5 kb) on the 3' side of exon 3. A neomycin resistance (neo)
cassette without a promoter or polyadenylation signal was inserted in-frame
between the two homologous arms at the ClaI site in exon 3
(Fig. 1A). In this mutant
allele, the DNA sequence encoding the N-terminal 18 amino acids of Dlgh1 was
followed by the neo cassette. Therefore, the protein encoded by the mutant
allele would only contain a small part of the L27 domain, and none of the
other domains of Dlgh1, such as the PDZ or SH3 domains. TT2 embryonic stem
(ES) cells were electroporated and clones selected by standard procedures.
Surviving clones were screened for homologous recombination by PCR using the
following primers: 5' Dlg-forward primer (F),
5'-GCTGTCAGTCCACAGCTAACACAGGCTACT-3'; 3' Dlg-reverse primer
(R), 5'-TGTCCTAAGTTAAGGACCATCTAGAGAGCC-3'; neo gene primer (N),
5'-TCGTGCTTTACGGTATCGCCGCTCCCGATT-3'. Correctly targeted clones
were used for aggregation with 8-cell embryos and chimeric males were mated
with C57BL/6 females to generate heterozygous offspring. In this study, two
strains of mice derived from different recombinant ES clones were used and the
phenotypes were similar between the two strains. Both strains were maintained
through strict brother-sister mating for more than ten generations. To
minimize any possible impact of the undefined genetic background, we used
littermates as controls in all experiments. For western blotting, tissue
lysates were prepared from the brain and kidney of E15.5 mice. Aliquots of
total protein (5 µg) were electrophoresed in individual lanes and then
transferred onto a PVDF membrane. Dglh1 protein was immunodetected using the
SuperSignal WestFemto Maximum Sensitivity Substrate (Pierce). RT-PCR was
performed on RNA samples from the kidney of E15.5 mice using Omniscript
reverse transcriptase (Qiagen) and ExTaq (Takara), following the
manufacturers' protocols. All animal protocols were carried out according to
the Guidelines for the Management of Laboratory Animals in Fujita Health
University. The numbers of mice used in this study are indicated in
Table 1.
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Histology
For histological analyses, either the kidneys or urogenital organs were
dissected from E18.5 embryos and fixed overnight in 4% paraformaldehyde (PFA)
in PBS. Next, the tissues were embedded in paraffin and serially sectioned at
5 µm. The sections were stained with Hematoxylin and Eosin. For measurement
of the renal area, longitudinal sections were prepared and microscopic images
were captured using a BX50 (Olympus) and DC200 (Leica) digital camera system.
The renal area, except for the cavity of the renal pelvis, was measured in
each section using an imaging application software package (Adobe Photoshop
version 7), and the largest area among serial sections was used for
comparisons among genotypes.
Whole-mount immunofluorescence staining
To visualize the embryonic urogenital organs, dissected organs were
immunofluorescently stained with an anti-Pax2 antibody. Urogenital tissues
were dissected from fetuses in PBS and fixed in 4% PFA in PBS for 10-30
minutes. The tissues were incubated with an anti-Pax2 antibody diluted in PBS
containing 0.1% Tween 20 (PBST) and 2% normal goat serum (2% NGS/PBST) at
4°C overnight. In some experiments, an anti-pancytokeratin antibody was
added to this solution. After the incubation, the tissues were washed with
PBST (1x3 minutes, 2x30 minutes, then overnight) and then
incubated with Alexa Fluor 488-conjugated anti-rabbit IgG diluted in 2%
NGS/PBST. For detection of the anti-pan-cytokeratin antibody, Alexa Fluor
568-conjugated anti-mouse IgG was used. Finally, the tissues were washed as
described above, immersed in glycerol and observed under a fluorescence
microscope (SteREO Lumar V12; Carl Zeiss).
Immunohistochemistry
E11.5 embryos from pregnant wild-type mice were fixed overnight in 4% PFA
in PBS at 4°C. For immunohistochemistry of occludin and ZO-1, urogenital
organs dissected from E15.5 embryos from pregnant heterozygous mice were fixed
for 30 minutes in 4% PFA in PBS at room temperature and frozen in OCT compound
(Sakura). The frozen tissues were then sectioned and immunostained as
described previously (Iizuka-Kogo et al.,
2005). For detection of Dlgh1 and E-cadherin in E14.5 embryos,
embryos were fixed overnight in 4% PFA in PBS at 4°C. After embedding in
paraffin, the tissues were sectioned at 5 µm, rehydrated and stained as for
the frozen sections. The stained sections were observed under a laser scanning
confocal microscope (LSM 510; Carl Zeiss).
Detection of cell proliferation by 5-bromo-2'-deoxyuridine (BrdU) labeling
E12.5 pregnant heterozygous mice were injected with 50 µg of BrdU per g
body weight. 2 hours after the injection, the embryos were collected, fixed
overnight in 4% PFA in PBS and paraffin-embedded. Horizontal serial
cross-sections of the embryos were cut at 5 µm thickness and 20 µm
intervals from the ureteropelvic junction to the lower end of the Wolffian
duct at the urogenital sinus. Thereafter, BrdU incorporated into the ureteric
epithelium was detected using an anti-BrdU antibody
(5-Bromo-2'-deoxyuridine Labeling and Detection Kit; Roche) in
combination with a Histofine Mouse Stain Kit (Nichirei), according to the
manufacturers' protocols. The number of immunopositive nuclei per unit area of
each ureteric cross-section was calculated in every section and the mean
values were compared between Dlgh1+/+ and
Dlgh1-/- mice.
Statistical analysis
F-tests and t-tests were used for statistical evaluation of the
data. All statistical analyses were performed using Microsoft Excel 2003
(Microsoft Corporation). Values of P<0.05 were considered to
indicate statistical significance.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Abnormal structures of urogenital organs in prenatal Dlgh1-/- mice
Since Dlgh1-/- mice die soon after birth, prenatal
E18.5 embryos were used for the following observations. The mean body length
(crown-to-rump length, CRL) was significantly shorter in
Dlgh1-/- mice than in Dlgh1+/+ or
Dlgh1+/- mice (see Fig. S2A in the supplementary
material). In addition, a cleft palate was observed in
Dlgh1-/- mice, as reported previously
(Caruana and Bernstein, 2001).
Furthermore, the whole-intestine length and whole-lung weight of
Dlgh1-/- mice were significantly reduced compared with
those of Dlgh1+/+ or Dlgh1+/- mice,
whereas the length of the left-right and sagittal axes of the cerebrum and the
whole-liver weight were not affected in Dlgh1-/- mice
(A.I.-K., T.A. and T.S., unpublished). In the present study, we focused on the
morphology of the urogenital organs. In contrast to
Dlgh1+/+ mice (Fig.
2A-C), hypoplasia of the kidney and ureter
(Fig. 2D), megaureter
(Fig. 2E), duplicated ureter
(Fig. 2F), hydronephrosis
(Fig. 2G), malposition of the
gonads and vaginal aplasia (Fig.
2H) were found in Dlgh1-/- mice at various
incidences. These abnormalities were not seen in Dlgh1+/+
mice (Fig. 2A-C,I,J).
First, in order to clarify whether the kidney hypoplasia was associated with the impaired growth of the whole body, relative kidney length (ratio of the longitudinal kidney length to the CRL) was compared among the genotypes. As shown in Fig. 2I, the relative kidney length was significantly shorter in Dlgh1-/- mice than in Dlgh1+/+ mice. Given these results, the kidney hypoplasia does not appear to be a result of the decrease in body size. Histological observation did not reveal any significant differences in the number of glomeruli per unit area or histological dysplasia of the renal tissues. However, hydronephrosis was seen in 15% of Dlgh1-/- mouse kidneys, but not in Dlgh1+/+ mice (Fig. 2B,G).
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Regarding the reproductive organs, malposition of the ovaries and testes were often seen in Dlgh1-/- mice (five of eight Dlgh1-/- mice). In these mice, the uteri were located on the abdominal side of the kidneys with the ovaries positioned between the adrenal glands and the kidneys in the females, and the testes were found in the abdominal cavity at irregular positions in the males (data not shown). In Dlgh1+/+ and Dlgh1+/- female mice, the vaginal cavity formed downward behind the urethra (Fig. 2C). By contrast, no such downward formation of the vaginal cavity was seen in Dlgh1-/- female mice (Fig. 2H).
Development of hypoplastic kidneys and ureters in Dlgh1-/- mice
To clarify the nature of the abnormalities in the urinary organs in
prenatal Dlgh1-/- mice, we followed their development. To
visualize the structures of the urinary organs, we performed whole-mount
immunostaining for cytokeratin (an epithelial marker) and Pax2 (a
transcription factor). Since Pax2 is expressed in the Wolffian and
Müllerian ducts, metanephric mesenchyme and ureters
(Torres et al., 1995), their
shapes can be observed by means of whole-mount immunofluorescence staining.
Immunofluorescence staining of the urinary organs revealed that the
Pax2-positive mesenchyme, denoting the metanephric mesenchyme, was contracted
in Dlgh1-/- mice during early development of the kidney
(Fig. 3A-C; see Fig. S2B-E in
the supplementary material). Regarding the ureteric structure, budding and
invasion of the ureter from the duct into the metanephric mesenchyme were seen
in both Dlgh1+/+ and Dlgh1-/- mice at
E11.2 (Fig. 3A). In normal
embryos, the ureteric bud divides for the first time and forms a T-shaped
structure by E11.5 (Fig. 3B,
upper, inset). However, among the E11.5 Dlgh1-/- embryos,
bilateral ectopic caudal branching of the ureters was observed in one of seven
Dlgh1-/- mice (Fig.
3B, lower, inset). This ectopic branching of the ureter might
cause the ureteric duplication seen at E18.5. At E12.5, the numbers of
ureteric bud tips in the metanephric mesenchyme, which are formed through
ureteric branching, were statistically equivalent among the different
Dlgh1 genotypes. In addition, the normal ureteric branching pattern
was observed in all Dlgh1+/+ and
Dlgh1+/- kidneys and in some of the
Dlgh1-/- kidneys (Fig.
3C, upper, inset). However, in 31.8% of cases, the
Dlgh1-/- ureters showed abnormal branching structures
(Fig. 3C, lower, inset). The
ureteric length from the lower end of the ureter to the upper end of the
T-shaped ureteric bud tip did not differ significantly between
Dlgh1-/- mice and the other genotypes at E11.5. By
contrast, by E12.5, the ureteric length relative to the CRL was significantly
shorter in Dlgh1-/- mice than in the other genotypes (see
Fig. S2F in the supplementary material).
Development of ectopic ureters in Dlgh1-/- mice
Megaureter and hydronephrosis were found in Dlgh1-/-
mice at E18.5. These disorders are caused by urinary obstruction, which is
induced by either constriction of the ureter, or by ectopic opening of the
ureter. Since the ureter sprouts from the Wolffian duct, the lower end of the
ureter does not open into the bladder immediately after budding. At this
stage, the lower part of the Wolffian duct beneath the budding site of the
ureter is known as the common nephric duct
(Fig. 3A-D, the region between
the two arrowheads). The common nephric duct is thought to become incorporated
into the bladder wall by E13.5, when the lower end of the ureter descends from
the Wolffian duct to the bladder wall. After E14.5, the lower ends of the
ureter and the Wolffian duct become separated from each other by proliferation
of the epithelial cells between the two ducts
(Batourina et al., 2002). These
successive changes were observed in Dlgh1+/+ mice
(Fig. 3, upper panels,
Fig. 5B). However, in all
Dlgh1-/- mice at E12.5, the lower end of the ureter was
connected to the Wolffian duct and did not descend to the bladder wall
(Fig. 3C, lower). Furthermore,
no Dlgh1-/- mice showed complete ureteric descent by E13.5
and they retained the common nephric duct
(Fig. 3D, lower). The common
nephric duct in normal development is equal to the lower part of the Wolffian
duct. We compared tissue sections around the remaining common nephric duct in
E13.5 Dlgh1-/- mice with the equivalent region containing
the base of the ureter and the Wolffian duct in Dlgh1+/+
mice at the same stage (Fig.
4). In Dlgh1+/+ mice, the ureter was
surrounded by sparse mesenchymal cells
(Fig. 4A), whereas the Wolffian
duct was surrounded by dense mesenchymal cells that showed no definite
orientation (Fig. 4B). In
Dlgh1-/- mice, the mesenchymal tissue on the ventral side
of the remaining common nephric duct (Fig.
4C, lower right) was dense and similar to that around the normal
Wolffian duct (Fig. 4B). By
contrast, the dorsal side of the duct was surrounded by a rather sparse
mesenchymal tissue in which the cells were orientated toward the duct
(Fig. 4C, upper left). This
sparse mesenchymal tissue was similar to that around the normal ureter
(Fig. 4A). Taken together, the
remaining common nephric duct in Dlgh1-/- mice was
surrounded by distinct mesenchymal tissues different from that around the
Wolffian duct in Dlgh1+/+ mice. Subsequently, at E15.5 or
later, some Dlgh1-/- ureters seemed to connect directly to
the bladder wall (Fig. 5H, left
side), even though the connecting structures among the ureter, bladder and
Wolffian duct were very different from those in Dlgh1+/+
mice, and these mice appeared to develop a passable urinary tract. However,
some Dlgh1-/- ureters were dilated owing to urinary
obstruction at E16.5 (Fig. 5I),
indicating their failure to connect to the bladder. Therefore, such ectopic
connection of the ureters seems to cause the megaureter and hydronephrosis
found in Dlgh1-/- mice at E18.5.
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in which the lower end of the ureter did not reach the bladder wall because
the budding site of the ureter was too rostral. By contrast, a comparison of
the lengths of the common nephric duct at E11.2, immediately after penetration
of the ureteric bud into the metanephric mesenchyme, indicated no significant
difference between Dlgh1+/+ and
Dlgh1-/- mice. As late as E11.5, the common nephric duct
length in Dlgh1-/- mice was 1.71-fold longer than that in
Dlgh1+/+ mice, a significant difference
(P<0.01). This indicates that the descent failure of the ureteric
end is not caused by ectopic budding of the ureter, but occurs during the
descent process itself.
Development of the genital organs derived from the Wolffian and Müllerian ducts
Vaginal aplasia was observed in prenatal Dlgh1-/- mice.
To clarify the mechanism of this vaginal aplasia, we followed the development
of the Müllerian ducts, from which the upper vagina is thought to be
derived.
In Dlgh1+/+ mice, the lower part of the Müllerian duct became identifiable beside the Wolffian duct at E13.5, and was close to the bladder wall (Fig. 3D, upper). At E14.5, the lower ends of the Wolffian and ureteric ducts separated from each other (Fig. 5A,B). The lower ends of the Müllerian ducts were laterally fused in both sexes at E15.5 (Fig. 5C,D). At this stage, attenuation of the Wolffian and Müllerian ducts, indicating their degeneration, was observed in the female and male embryos, respectively. By E16.5, the female Wolffian ducts and male Müllerian ducts had almost completely disappeared (Fig. 5E,F). In addition, in the female embryos, the fusion part of the Müllerian ducts, which should form the uterine cervix and upper vagina, was seen to expand laterally and downward (Fig. 5E). In the male embryos, the primordium for the seminal vesicle emerged from the dorsal side of the lowest part of the Wolffian duct (Fig. 5F).
In contrast to Dlgh1+/+ mice, the lower end of the Müllerian duct in Dlgh1-/- mice had not elongated to the bladder wall by E13.5 (Fig. 3D, lower). Instead, in most embryos, it only reached to just above the branching point of the ureter from the Wolffian duct. At E14.5, the ureter and Wolffian duct remained connected with each other. In some embryos, the Müllerian duct was connected to the common nephric duct (Fig. 5G). Furthermore, the Müllerian ducts had not fused laterally by E15.5, and the lower end of the ureter did not separate from the Wolffian duct (Fig. 5H). As described above, some female embryos appeared to have a dilated ureter with a constriction (Fig. 5I), which might have been caused by the protracted ureteric connection to the degenerating Wolffian duct. In male embryos, primordia for the seminal vesicles could not be identified (Fig. 5J). The failure of lateral fusion and the obstruction of the Müllerian ducts in female Dlgh1-/- mice were confirmed by observation of serial cross-sections of the urogenital organs at E18.5. Fig. 6 shows cross-sections of Dlgh1+/+ and Dlgh1-/- mice at the level of the vesicoureteral junction. At this level, the uteri were fused laterally and a cervical lumen was present in Dlgh1+/+ mice (Fig. 6A). By contrast, the uterine lumens were separated laterally in Dlgh1-/- mice (Fig. 6B), and were seen to disappear in serial sections (data not shown).
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; Matsumine et al.,
1996; Muller et al.,
1995). Knockdown of Dlgh1 expression in Caco-2/15 cells has been
reported to decrease the accumulation of E-cadherin (also known as cadherin 1)
at cellcell contact sites, thereby inhibiting the structural integration of
adherens junctions (Laprise et al.,
2004). To examine whether the abnormal structure of the ureters in
Dlgh1-/- mice was caused by changes in the structure of
cell-cell junctional complexes, we determined the distribution of E-cadherin,
occludin and ZO-1 (also known as Tjp1) in the embryonic ureteric epithelium.
Contrary to our expectations, strong accumulation of E-cadherin at cell-cell
contact sites and accumulation of occludin and ZO-1 at the apicalmost portion
of the lateral membrane were also observed in Dlgh1-/-
mice (Fig. 7C-J). The ureteric or renal hypoplasia seen in Dlgh1-/- mice could be caused by defective cell proliferation. We therefore compared the proliferative index in the ureteric epithelium of Dlgh1-/- mice with that of Dlgh1+/+ mice (Table 2). The proliferative index of Dlgh1-/- mice at E12.5, when prominent elongation of the ureter occurs in normal mice, was significantly lower than that of Dlgh1+/+ mice. This finding suggests that decreased proliferation in the ureteric epithelium causes the short ureter of Dlgh1-/- mice.
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). In that study, however, no other morphological
abnormalities of the ureter and genital tract that resembled those observed in
our gene-targeted mice were described. If developmental abnormalities in the
lower urogenital tract occur only in our mutant mice, and not in mutant mice
lacking the Cterminus of Dlgh1, this would suggest that the N-terminal half of
Dlgh1, including the PDZ domains, is essential for development of the lower
urogenital tract. On the other hand, as renal hypoplasia was present in both
types of mutant mice, this phenotype appears to be due to the loss of the
C-terminus of Dlgh1.
Mechanism of the occurrence of renal hypoplasia
Pax2 is a transcription factor that plays important roles in the early
development of urogenital organs (Torres
et al., 1995). The Pax2-positive metanephric mesenchyme was
clearly contracted in Dlgh1-/- mice. Pax2-positive
mesenchymal cells have the ability to differentiate into convoluted tubules
(Rothenpieler and Dressler,
1993). Therefore, their number could contribute directly to the
size of the mature kidneys. Furthermore, a decrease in the total Pax2
expression level in the metanephric mesenchyme might repress the expression of
Gdnf, which is a target gene of Pax2 and essential for ureteric
branching. Consequently, both the differentiation of the convoluted tubules
and the ureteric branching might have been affected, which would lead to
hypoplasticity of the kidneys in Dlgh1-/- mice. The
mechanism regulating the expression of Pax2 in the metanephric primordium has
not yet been clarified. Elucidation of the mechanism by which Dlgh1 affects
this process would be of great interest.
Cause and effect of the disturbed development of the ureter
Prenatal hydronephrosis and megaureter are considered to arise owing to
failure of ureteric opening into the bladder wall, and are referred to as
CAKUT (congenital abnormalities of the kidney and urinary tract). In
Foxc1-/- (Kume et al.,
2000) and Bmp4+/-
(Miyazaki et al., 2000) mice,
the ureter buds from an ectopically anterior site, and therefore cannot
descend sufficiently or open into the bladder wall, thus resulting in
hydronephrosis and other correlated defects similar to those in
Dlgh1-/- mice. In addition, mice homozygous for the
retinoic acid receptor show hydronephrosis and megaureter
(Batourina et al., 2002). In
this case, the budding site and descent process of the ureter are normal, but
the lower end of the ureter does not separate from the Wolffian duct on the
bladder wall. By contrast, the descent process itself does not progress
completely in Dlgh1-/- mice, suggesting that Dlgh1
functions in a different context from the other genes described above. The
common nephric duct is thought to become incorporated into the bladder wall
during the descent of the ureteric end. BrdU analysis revealed that cellular
proliferation is decreased in the ureteric epithelium of
Dlgh1-/- mice at E12.5. Therefore, it might be possible
that elongation of the ureter caused by epithelial proliferation is required
for incorporation of the common nephric duct into the bladder wall. If this is
the case, then the loss of Dlgh1 and consequent decreased cellular
proliferation would result in impairment of the descent process of the
ureter.
Mechanism of the defects in Müllerian duct development
In addition to the abnormalities in the urinary organs, aplasia of the
uterine cervix and vagina was observed in Dlgh1-/- mice.
This is caused by fusion failure and obstruction of the Müllerian ducts.
Why were the Müllerian ducts obstructed in Dlgh1-/-
mice? Since lateral fusion of the Müllerian ducts can be impaired without
obstruction in humans (Gell,
2003), fusion failure does not seem to be a direct cause of the
obstruction. Rather, the impaired downgrowth of the Müllerian ducts
appears to cause the obstruction in Dlgh1-/- mice.
Kobayashi et al. confirmed that the Wolffian duct is required for
Müllerian duct formation by demonstrating that Wolffian ductspecific
knockout of the Lim1 gene (Lhx1 - Mouse Genome Informatics),
which is required for Wolffian duct development, inhibited the formation of
the Müllerian duct as well as that of the Wolffian duct itself
(Kobayashi et al., 2005). As
mentioned above, the mesenchymal tissues surrounding the Wolffian duct and
ureter are histologically very different. This indicates that the Wolffian
duct and ureter develop different characteristics through interactions with
their surrounding mesenchymal tissues, even though they are derived from the
same origin. By contrast, the common nephric duct in
Dlgh1-/- mice was surrounded by two types of mesenchymal
tissues, ventral Wolffian-type mesenchyme and dorsal ureter-type mesenchyme.
The persistent common nephric duct, unlike the normal Wolffian duct, might not
induce the formation of the Müllerian duct. Determination of the
functional difference between the Wolffian duct and common nephric duct in
Dlgh1-/- mice might help to elucidate the induction
mechanism of the Müllerian duct by the Wolffian duct.
The mechanism for the lateral fusion of the Müllerian ducts is presently unclear. Because downward elongation of the Müllerian ducts was impaired in Dlgh1-/- mice, the Müllerian ducts might be unable to access each other adequately. Alternatively, there might be defects in the cell-cell recognition process that appears to be necessary during the integration of the two epithelial cell layers of the left and right Müllerian ducts.
Congenital absence of the uterus and vagina is known as
Rokitansky-Kuster-Hauser Syndrome in humans. In addition, and as mentioned
above, failure of lateral fusion of the Müllerian ducts with complete
formation of the vagina has been reported
(Gell, 2003). These disorders
are associated with abnormalities of the renal and urinary organs with various
incidences, similar to those in Dlgh1-/- mice, suggesting
that the functions of Dlgh1 are impaired at the onset of these congenital
abnormalities.
A possible function of Dlgh1 in promoting cellular proliferation in the ureter
In mature epithelial cells, Dlgh1 is localized at cell-cell contact sites.
Dlgh1 has previously been considered to facilitate cell-cell adhesion, thereby
inhibiting cellular proliferation. Regarding adhesion, Dlgh1 has been reported
to be involved in cellular polarization and/or integration of epithelial
cell-cell adhesion complexes in Nematoda and Drosophila and in human
Caco-2/15 cells (Firestein and Rongo,
2001; Laprise et al.,
2004; Woods and Bryant,
1991). Regarding proliferation, Dlgh1 has been reported to
cooperate with APC and negatively regulate the cell cycle in the mouse
fibroblast cell line NIH3T3 (Ishidate et
al., 2000). In addition, the onset of cervical cancer mediated by
the human papilloma virus is associated with virus-induced degradation of
Dlgh1 protein at cell-cell contact sites
(Watson et al., 2002).
However, our present results indicate that Dlgh1 is not required for the
proper subcellular localization and maintenance of E-cadherin, occludin and
ZO-1 in the developing ureter. Furthermore, the BrdU analysis revealed that
cellular proliferation was decreased in the ureteric epithelium of
Dlgh1-/- mice, and this probably underlies the shortening
of the ureters in Dlgh1-/- mice. Likewise, the hypoplasia
of the metanephros and maxillary process and the decreased body size of
Dlgh1-/- mice might also be due to the decreased cellular
proliferation. From the present results, we speculate that, under some
specific conditions during development, Dlgh1 might be involved in the correct
localization of intracellular signal transduction components and contribute to
efficient signal reception and transduction, resulting in positive regulation
of cellular proliferation. Recently, Frese et al. reported that Dlgh1 is
positively involved in human adenovirus type-9 E4-mediated oncogenesis, and
demonstrated a novel mechanism in which Dlgh1 changes its subcellular
distribution through an interaction with E4-ORF1 oncoprotein and subsequently
induces cell-autonomous proliferation
(Frese et al., 2006). These
results indicate that, according to the cellular conditions and through
interactions with other functional proteins, Dlgh1 can carry out various
functions that might sometimes be opposing, such as promoting and inhibiting
cellular proliferation, and are consistent with our present results and
hypothesis.
It will be important to clarify the developmental mechanisms underlying the urogenital disorders in Dlgh1-/- mice in order to fully understand the ontogenic functions of Dlgh1. The present results indicate that Dlgh1 is involved in regulating the structural organization of the epithelial ducts during ontogeny. Identification and characterization of the interactions between Dlgh1 and its binding partners in the urogenital organs and examination of the roles of Dlgh1 in other developmental processes will help to further elucidate its developmental functions.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1799/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Laboratory of Molecular Genetics, RIKEN Tsukuba Institute,
Japan
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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