Department of Molecular and Cellular Biology, 16 Divinity Avenue, Harvard University, Cambridge MA 02138, USA
* Author for correspondence (e-mail: amcmahon{at}mcb.harvard.edu)
Accepted 12 August 2002
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SUMMARY |
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Key words: Kidney, Sonic hedgehog, Shh, Proliferation, Smooth muscle, Mouse, Ureter, Hydroureter
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INTRODUCTION |
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The kidney is a classic model for the study of epithelial-mesenchymal
interactions. The ureteric bud is essential for metanephric mesenchyme
survival (Grobstein, 1953;
Grobstein, 1955
), and the
metanephric mesenchyme is required for the growth and branching of the
ureteric bud (Ekblom, 1992
;
Erickson, 1968
;
Grobstein, 1953
;
Grobstein, 1955
). The
developmental roles (if any) of the distal collecting ducts and the ureteric
epithelium are unknown. Because smooth muscle forms adjacent to these regions,
it seems likely that these tissues might regulate smooth muscle
development.
Sonic hedgehog (Shh), a Drosophila Hedgehog (Hh)
homolog, is expressed in the urothelium
(Bitgood and McMahon, 1995;
Karavanova et al., 1996
) (this
study). Shh has previously been shown to be involved in cell
survival, proliferation, differentiation and pattern formation in various
embryonic tissues (for reviews, see Ingham
and McMahon, 2001
; McMahon et
al., 2002
). Interestingly, Shh is located on human
chromosome 7q36; deletions within this region, which may include Shh,
are associated with kidney defects such as hydroureter
(Lurie et al., 1990
;
Nowaczyk et al., 2000
).
Mutations in the Shh signaling pathway have been linked to renal
anomalies in humans such as the VACTERL syndrome
(Kim et al., 2001
). In the
mouse, removal of Shh generates a spectrum of defects (reviewed by
McMahon et al., 2002
),
including kidney hypoplasia (A. P. M., unpublished). However, deciphering the
possible role of Shh in kidney development directly in Shh
mutants is hindered by the fusion of the paired kidney primordia, a secondary
consequence of midline defects in early somite stage embryos (A. P. M.,
unpublished). To address the role of Shh in kidney development, we
have developed a conditional loss of function genetic approach that removes
Shh signaling from the kidney primordium. These studies indicate that
Shh is a crucial paracrine factor for the control of proliferation
and differentiation in the subjacent mesenchyme that underlies the
urothelium.
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MATERIALS AND METHODS |
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To further characterize the expression of HoxB7/Cre, lines were
established that were transgenic for both HoxB7/Cre and either the
ROSA or Z/AP reporter alleles
(Lobe et al., 1999). Males
carrying both HoxB7/Cre and one of the reporter transgenes were
crossed to SW females and embryos were dissected at 24-hour intervals from
E9.5 to P1. Either whole embryos (E9.5-E11.5) or dissected urogenital systems
(E12.5-P1) were analyzed for lacZ (ROSA) or lacZ and
alkaline phosphatase (Z/AP) expression according to Lobe et al.
(Lobe et al., 1999
). For the
Z/AP embryos, salmon galactoside (Biosynth) was used as the
ß-galactosidase substrate. For histology, processed kidneys were
post-fixed in 4% paraformaldehyde, embedded in paraffin wax and sectioned at 6
um.
The HoxB7/Cre mice were mated to Shhn/+ mice
(St-Jacques et al., 1998) to
generate HoxB7/Cre, Shhn/+ males. These males were mated
to homozygous Shh conditional females (Shhc/c)
(Dassule et al., 2000
) to
generate HoxB7/Cre, Shhc/n progeny in which Shh signaling
was specifically removed from the kidney urothelium. These kidneys are
referred to as `Shh mutant kidneys' in the text.
Histological, in situ, histochemical and immunological analysis of
kidneys
Kidneys were fixed in 4% paraformaldehyde at 4°C, dehydrated through a
graded ethanol series, embedded in paraffin wax and sectioned at 6 µm
(embryonic and newborn kidneys) or 7 µm (adult kidneys). Sections were
stained with Hematoxylin and Eosin for histological analysis. In situ
hybridization with 35S-labeled probes was performed according to
Wilkinson et al. (Wilkinson et al.,
1987a; Wilkinson et al.,
1987b
), with minor modifications for the Shh probe (the
hybridization temperature was reduced from 55°C to 50°C, and the
washing temperature from 65°C to 60°C). To assay for
ß-galactosidase activity on sections, tissues were fixed in 2%
paraformaldehyde for 50 minutes at 4°C, washed with PBS extensively,
cryoprotected in 30% sucrose overnight and embedded in OCT. Cryosections (12
µm) were prepared and stained with X-gal according to the protocol
of Oberdick et al. (Oberdick et al.,
1994
). Images were captured with a JVC KY-F70 digital camera on a
Leitz DMRD microscope or by a Nikon digital camera DXM1200 on a Nikon SMZ1500
stereoscope.
For immunohistochemistry and immunofluorescence, paraffin wax embedded
sections were dewaxed in xylenes and rehydrated through a graded ethanol
series. Slides were microwaved for 15 minutes in 1 mM Tris-HCl (pH 8.0), 5 mM
EDTA to unmask antigens. Sections were blocked in 2% sheep serum in PBS+0.1%
Triton X-100 for 30 minutes. Endogenous peroxidase activity was quenched by
incubating sections in 3% H2O2 for 10 minutes when
peroxidase-conjugated secondary antibodies were used. The sections were then
incubated with any of the following: monoclonal anti-smooth muscle
-actin antibody (1:2000, Sigma), biotin-conjugated Dolichos
bifloris agglutinin (DBA) (1:200, Sigma) or polyclonal
anti-phospho-histone H3 antibodies (1:50, Upstate Biotechnology) at 4°C
overnight. Alexa 568-conjugated anti-mouse IgG (Molecular probes), Alexa
568-conjugated anti-rabbit IgG (Molecular probes) or Alexa 488-conjugated
Streptavidin (Molecular probes) were used for immunofluorescent detection of
the binding of the primary reagents. Nuclei were counterstained with DAPI.
Sections were mounted in Vectashield mounting media (Vector laboratories) and
visualized with a Zeiss LSM510 Axioplan 2 confocal microscope. Frozen sections
stained with X-gal were post-fixed with 4% paraformaldehyde for 20
minutes, and stained with antibodies as above. For immunohistochemistry,
peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch) was used as the
secondary antibody and its binding was visualized by histochemical staining in
3,3'-Diaminobenzidine (DAB) (Sigma). These sections were counterstained
with Methyl Green, dehydrated and mounted in Permount mounting media (Fisher).
Images were collected with a JVC KY-F70 digital camera on a Leitz DMRD
microscope. Mitotic indices were calculated as the percentage of nuclei that
were phospho-histone H3-positive in three to four adjacent sections in the
same region of the proximal and distal ureter of Shh mutant and
wild-type kidneys.
Calculation of kidney volume and the number of glomeruli
Glomeruli were identified by the presence of a Bowman's capsule. The volume
of kidneys, of the cortex and of the medulla was measured according to Bertram
et al. (Bertram et al., 1992)
with the following modifications: 6 µm sections were stained with
Hematoxylin and Eosin. The area of the tissue section of interest was measured
with NIH image 1.62 and multiplied by the thickness to obtain the
volume/section. The volume for each kidney is the sum of the volume for each
section.
Ureteral mesenchymal cell primary culture and BrdU staining
Mesenchyme of E12.5 ureters was mechanically separated from the epithelium
in sterile D-PBS (BioWhittaker) and cultured on fibronectin (Sigma)-coated
Lab-Tek glass chamber slides (Nalge Nunc International) or fibronectin-coated
48-well tissue culture plates in DMEM supplemented with 10 ng/ml recombinant
human TGF (Sigma) and 50 ng/ml recombinant human FGF2 (R&D
Systems). No epithelial pieces were observed in the culture. Thus, there was
no major contamination of urothelium in the ureteral mesenchymal cell culture.
Palmitic acid-modified recombinant human sonic hedgehog protein (N-SHH;
Biogen), recombinant human noggin protein (Regeneron) and recombinant human
bone morphogenetic protein 4 (BMP4, Genetics institute) were added to the
culture as indicated in the text. Fresh culture media (50% volume change) was
added every 2 days. At day 5, cells were labeled with 10 µM BrdU for 11
hours, and then processed according to the Becton Dickinson immunocytometry
system manual. Cells were incubated with monoclonal anti-BrdU antibody (1:100,
Becton Dickinson) at 4°C overnight, antibody binding was visualized by
incubating with Alexa 568-conjugated anti-mouse IgG. Cells were counterstained
with DAPI, mounted in Vectashield mounting media and visualized with a Zeiss
LSM510 Axioplan 2 confocal microscope.
RT-PCR
Total RNA was prepared from either freshly dissected ureteral mesenchyme or
mesenchyme after 5 days of culture using an RNAqueous-4-PCR kit (Ambion).
Samples were treated with DNaseI, and reverse transcribed according to the
manual for 5'RACE for rapid amplification of cDNA ends (Invitrogen). To
ensure the amount of PCR products reflects the abundance of the specific cDNA
being amplified in total cDNA sample, the PCR cycle number for the linear
amplification range was determined with the most abundant cDNA sample, and PCR
with all samples was performed with the PCR cycle number identified.
ß-actin PCR primers and conditions were as specified in the QuantumRNA
ß-actin kit (Ambion). Primers for smooth muscle -actin PCR were as
described (Yang et al., 1999
),
and PCR conditions were 94°C for 1 minute, 55°C for 1 minute and
72°C for 1 minute for 35 cycles. Primers and conditions for Bmp4
PCR were according to Oxburgh and Robertson (L. Oxburgh and E. J. Robertson,
unpublished).
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RESULTS |
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The HoxB7/Cre transgenic mouse line
Analysis of the role of Shh in metanephric kidney formation in
Shh-null mutants has been hindered by the midline defects in these
animals, which causes the fusion of the two kidneys (J. Y., T. J. C. and A. P.
M., unpublished). This problem was circumvented by generation of a mouse
strain that drives Cre recombinase under the control of the HoxB7
promoter/enhancer specifically within the mesonephric duct and its derivatives
(the Wolffian duct, the collecting duct epithelium of the kidney and the
ureteral epithelium). Cre activity could be detected with the ROSA
reporter in the mesonephric duct as early as E9.5
(Fig. 2A), well before
Shh expression initiates in the ureteric bud epithelium. HoxB7/Cre
activity was detected throughout the ureteric bud from its initiation at
E10.25 (Fig. 2B), and was
clearly able to initiate recombination-mediated expression of the
ROSA reporter gene in all ureteric bud epithelial cells by E12.5
(Fig. 2E,F). This is further
confirmed by the Z/AP reporter
(Fig. 2G-I). Consequently,
intercrossing this transgenic line with one carrying a conditional
Shh allele (Shhc)
(Dassule et al., 2000) that
requires Cre-mediated recombination to remove essential sequences in exon2
thereby generating a null allele (Shhn)
(Lewis et al., 2001
)
allows the complete removal of Shh signal production prior to the normal
activation of Shh in the ureteric epithelium.
|
Removal of Shh activity from the urothelium causes renal
hypoplasia, hydronephrosis and hydroureter
HoxB7/Cre, Shhc/n newborn pups were viable. However,
their kidneys (hereafter referred to as `Shh mutant kidneys' for
simplicity) were smaller and displayed a prominent hydroureter when compared
with sex- and age-matched wild-type or heterozygous littermates
(Fig. 3, compare 3A with 3B,
and 3E with 3F). Hydroureter is usually more severe in the proximal region.
Other than size, the gross anatomy of Shh mutant kidneys was not
affected in newborn mutants (Fig.
3C,D). Consistent with this observation, the expression patterns
of regional collecting duct markers, Wnt15 and Wnt7b, were
unaltered in mutants (data not shown). In addition to hydroureter (compare
Fig. 3I with 3J),
hydronephrosis (distention of the pelvis of a kidney) was detected in half of
the adult mutant kidneys (5/10; Fig.
3H). Histological examination revealed that the hydronephric
kidneys lost most of their inner medulla and the inner stripe of the outer
medulla (Fig. 3G,H).
Hydronephrosis in the mutant adult kidneys was probably secondary to the
hydroureter, as severe hydronephrosis is always associated with severe
hydroureter and no hydronephrosis was detected in newborn pups.
|
To determine whether Shh activity was effectively removed from the kidney, we examined Ptch expression (Fig. 4A-D). At E14.5, a time before significant differentiation of ureteral mesenchyme initiates (see below), Ptch expression was reduced to basal levels, indicating that no Shh signaling was occurring at this stage. Consistent with this observation, RT-PCR of Shh expression using exon 2 primers indicated a complete absence of a functional Shh transcript at E14.5 (data not shown).
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Shh signaling is required for mesenchymal cell proliferation
The mutant kidneys in newborn pups were 52% smaller than those in their
wild-type littermates (Fig. 5A;
wild type, n=3; mutant, n=4; P=0.002), and the
glomerular number was reduced by 40% (P=0.004). However, the
glomerular density in the mutant kidneys increased by 26%
(Fig. 4B, P=0.03). To
determine if this increase in glomerular density is due to differential
effects of Shh on cortical and medullary regions of the kidney
(Shh expression is primarily in the medulla), we further quantified
the cortical and medullary volume. The reduction of the cortical and the
medullary volume of Shh mutants is similar, 51% (P=0.003)
and 46% (P=0.002), respectively. The cortical glomerular density in
mutant kidneys increased by 24% (P=0.02), similar to that of the
whole mutant kidneys. These data suggest that the higher glomerular density in
the entire mutant kidney is not due to the underdevelopment of the medullary
region relative to the rest of the kidney. No gross size differences were seen
between the glomerulus of the mutant kidneys and that of the wild type.
|
Consistent with the hypoplasia in the kidney, the length of the E14.5 mutant ureter was about 21% shorter than that of the wild-type littermates (n=5, P=0.03), and fewer mesenchymal cells lined the ureteral epithelium of mutants (data not shown). Taken together, these data suggest that Shh is involved in survival and/or proliferation of mesenchymal cells in the kidney and ureter.
Ptch expression indicates that mesenchymal cells adjacent to the Shh expression domain are the target of Shh signaling. We therefore focused our analysis on these cells, in particular, the condensed mesenchymal cells surrounding the epithelium of the ureter that are a morphologically distinct population of cells that respond strongly to Shh signaling at E14.5 (cells within the dotted circle, Fig. 5C,D). We examined the effect of removal of Shh signaling on cell proliferation and apoptosis in this cell population. Ureter sections were immunostained for phospho-histone H3, in order to quantify cells in M phase of the cell cycle and thereby measure the mitotic index (Fig. 5C-E). In the proximal ureter of the mutants, the mitotic index of cells in the subjacent mesenchyme that normally demarcates the Shh-responsive zone was 52% that of wild-type littermates (Fig. 5E proximal, n=4, P=0.004). In the distal ureter, the mitotic index of this population of cells was 48% that of the wild-type littermates (Fig. 5E distal, P=0.05). Thus, cell proliferation is greatly reduced in Shh mutants. TUNEL analysis of the ureteral mesenchyme detected no significant difference in cell death between mutant and wild-type tissues (data not shown), indicating that apoptosis was unlikely to play a role in the generation of the mutant phenotype.
We further examined the role of Shh in cell proliferation with
primary ureteral mesenchymal cell cultures. Mesenchymal cells dissected from
E12.5 ureter were cultured in the absence of serum. Each culture was
supplemented with 50 ng/ml FGF-2 and 10 ng/ml TGF- to maintain ureteral
mesenchymal cells which rapidly die in the absence of these factors (SHH alone
did not support ureteral mesenchymal cell survival in the culture without
these factors; J. Y. and A. P. M., unpublished). Approximately 4.8% of cells
cultured in FGF-2 and TGF-
alone (control) were actually proliferating
(in S phase), while this number significantly increased to 14.0% of
mesenchymal cells cultured with 0.8 nM N-SHH protein (SHH;
Fig. 5F, P=0.007). The
observation that Shh can promote ureteral mesenchymal cell
proliferation in vitro correlates well with the reduction of proliferation of
this Shh-responsive cell population in Shh mutant
kidneys.
Bmp4 expression has been shown to be regulated by Shh signaling in
several tissues and its Drosophila counterpart Dpp is a
target of Drosophila Hedgehog (for reviews, see
Ingham and McMahon, 2001;
McMahon et al., 2002
).
Bmp4 was co-expressed with Ptch in mesenchymal cells
surrounding the Shh-expressing collecting ducts
(Fig. 4E) and the ureter
(Fig. 4G) in wild-type embryos.
Bmp4 expression in this domain was abolished in Shh mutant
kidneys at E14.5, indicating that Bmp4 expression depended on Shh
signaling (Fig. 4F,H). As
expected, expression of Bmp4 in glomeruli was not affected in
Shh mutants (Fig. 4F,
inset).
To further test the dependence of Bmp4 expression on Shh signaling in ureteral mesenchyme, we examined Bmp4 expression in cultured ureteral mesenchymal cells in the presence or absence of N-SHH protein (Fig. 4I). Bmp4 levels increased on addition of N-SHH protein, lending support to the conclusion that Bmp4 is a potential target of Shh signaling to the ureteral mesenchyme. Furthermore, the levels of Bmp4 expression observed in cultures in the absence of N-SHH (Fig. 5I, lane2) were lower than freshly dissected ureteral mesenchyme (Fig. 5I, lane1) is consistent with Shh-mediating maintenance of Bmp4 expression.
The fact that Bmp4 appears to be downstream of Shh signaling raised the possibility that the proliferative function of Shh is mediated through Bmp4. However, when ureteral mesenchyme was cultured with 0.8 nM N-SHH and the potent BMP4 antagonist, 300 ng/ml noggin, to block Bmp4 signaling, there was no significant difference in the stimulation of proliferation than that observed in cells cultured with N-SHH alone (Fig. 5F). Furthermore, addition of 100 ng/ml BMP4 completely blocked proliferation, indicating that BMP4 is antimitogenic for this cell population (Fig. 5F). The anti-proliferative effects of BMP4 were antagonized by addition of 300 ng/ml Noggin, the same concentration used in the SHH/Noggin co-culture experiment, demonstrating that Noggin was active in these assays (Fig. 5F). Noggin alone had no statistically significant proliferative effect on the cultures (Fig. 5F; noggin, P=0.12). Taken together, these results suggest that the proliferative function of Shh was not mediated by Bmp4, supporting a more direct action of Shh signaling. Furthermore, that Shh stimulated cell proliferation while at the same time inducing Bmp4 expression suggests that Shh can overcome the inhibitory effect of Bmp4.
Smooth muscle differentiation in the ureteral mesenchyme is delayed
in Shh mutant kidneys
Smooth muscle forms from condensed mesenchyme that underlies the urothelium
in the ureter and the renal pelvis
(McHugh, 1995). The
peristaltic movement of smooth muscles propels urine from the renal pelvis to
the bladder and relieves the kidney paranchyma from the damaging pressure that
fluid build-up causes. Bmp4 has been shown to promote formation of
smooth muscle in the kidney and ureter
(Raatikainen-Ahokas et al.,
2000
). The hydroureter phenotype and the expression pattern of
Shh and Ptch in the ureter and the renal pelvis along with
the lack of Bmp4 expression prompted us to examine smooth muscle
formation in the mutant kidneys.
The timing and pattern of smooth muscle differentiation in the mouse kidney
and ureter was not well documented. Initially, we characterized normal smooth
muscle differentiation in wild-type kidneys. At E13.5, mesenchymal cells
condense around the epithelium. However, the absence of smooth muscle
-actin protein (SMA), an early marker of smooth muscle differentiation,
indicated that smooth muscle differentiation had not occurred (data not
shown). At E14.5, SMA was detected in scattered condensed mesenchymal cells of
the proximal ureter (closer to the kidney,
Fig. 6A,B, arrow) and the
future renal pelvis (data not shown), but not the distal ureter (closer to the
bladder, Fig. 6C,D). At E15.5,
distal ureteral mesenchymal cells started to produce SMA
(Fig. 6G,H), while the majority
of condensed mesenchymal cells in the proximal ureter
(Fig. 6E,F) and the future
pelvis (data not shown) show abundant SMA protein. By E16.5, SMA was present
along the entire length of the ureter (Fig.
6I-L) and the renal pelvis (data not shown). Unlike the situation
in the rat (Baker and Gomez,
1998
), but similar to that in humans
(Tacciuoli et al., 1975
;
Matsuno et al., 1984
), smooth
muscle differentiation in the mouse ureter forms in a descending direction
(from the kidney to the bladder) along the proximodistal axis of the
ureter.
|
In Shh mutant kidneys at E15.0, no SMA was detected at any axial level of the ureter in contrast to wild-type embryos where SMA production was detected in the proximal ureter (Fig. 7A-D). At the newborn stage, SMA was detected in the proximal ureter of Shh mutants (Fig. 7E,F), but in contrast to wild-type littermates, the number of mesenchymal cells that produced SMA decreased in more distal regions such that almost no SMA was detected in the distal-most part of the ureter, closest to the bladder (Fig. 7G,H). Furthermore, mesenchymal cells in the distal ureter were not as condensed as those of wild-type siblings. Histological examination at the newborn stage indicates that SMA-positive cells in wild-type and mutants are smooth muscle cells. Taken together, these data indicate that some smooth muscle formation occurs in Shh mutant kidneys, but formation was delayed and the number of SMA-producing cells was greatly reduced at birth. The decrease in the number of SMA-producing cells from proximal to distal end of the ureter in mutants is in good agreement with the sequence of smooth muscle differentiation in the wild type, reflecting a general delay in smooth muscle differentiation along the length of the ureter. That some smooth muscle differentiation occurred in the HoxB7/Cre, Shhc/n mutants is not due to the ineffective removal of Shh activity by the Cre-mediated recombination, as ureteral mesenchyme from kidneys of Shh null mice also produced SMA (data not shown).
|
Shh inhibits smooth muscle formation in a dose-dependent
manner
Smooth muscle formation in the ureter has been reported to occur in cells
immediately adjacent to the urothelium, a similar position to smooth muscle in
the respiratory system (McHugh,
1995), which is also Shh dependent
(Pepicelli et al., 1998
).
Close observation of newborn ureters detected 1-2 layers of ureteral
mesenchymal cells that are SMA negative between the SMA-positive smooth muscle
and the ureteral epithelium (arrow, Fig.
8A). We refer to these cells as the subepithelial ureteral
mesenchymal cells for simplicity. No such cells are detected in the renal
pelvis (data not shown). In the proximal ureter of Shh mutants, this
cell population was completely absent (Fig.
8B), suggesting that these cells required Shh signaling for their
establishment and/or maintenance.
|
To determine if the sub-epithelial ureteral mesenchymal cells were directly
Shh responsive, we examined ureters from
Ptch-lacZ+/- newborn mice in which lacZ was
knocked into the Ptch locus
(Goodrich et al., 1997). X-gal
staining of these mice faithfully recapitulated the endogenous Ptch
expression pattern in many tissues including the kidney and ureter
(Goodrich et al., 1997
)
(Fig. 8C and data not shown).
The ß-gal-positive cells in the ureter were SMA negative and lay directly
adjacent to the epithelium (Fig.
8D, arrow). Therefore, the subepithelial ureteral mesenchymal
cells appear to be the subset of ureteral mesenchymal cells that respond to
high-level Shh signaling at the newborn stage. As Ptch was expressed
in all the likely smooth muscle progenitor cells that appear as condensed
mesenchyme around the ureteral epithelium before the initiation of SMA
production at E14.5 (Fig. 1C),
but not in SMA-producing cells later in development, it would appear that
smooth muscle progenitor cells, but not differentiated smooth muscle cells,
normally respond to Shh signaling. The subepithelial ureteral mesenchymal
cells, closest to the Shh signaling source, responded to Shh
signaling but did not produce SMA, suggesting that high levels of Shh
signaling may in fact inhibit smooth muscle differentiation. To address this
issue, we assayed smooth muscle
-actin expression in ureteral
mesenchymal cultures in response to N-SHH
(Fig. 8E). Smooth muscle
-actin expression could be detected by RT-PCR as early as E12.5 in the
ureteral mesenchyme (Fig. 8E,
lane 1), and cells cultured for 5 days without N-SHH protein still expressed
smooth muscle
-actin (Fig.
8E, lane 2). However, addition of 0.8 nM N-SHH greatly reduced,
and addition of 80 nM N-SHH completely abolished, the expression of smooth
muscle
-actin (Fig. 8E,
lanes 3 and 4). Thus, Shh inhibits smooth muscle differentiation in
ureteral mesenchyme cultures in a dose-dependent fashion.
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DISCUSSION |
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Shh and mesenchymal cell proliferation
Collectively our analyses of Shh mutants and the response of
ureteral mesenchyme in vitro indicated that Shh served as a mitogen to promote
proliferation of ureteral mesenchymal cells. This effect is likely to extend
to the mesenchyme in the kidney proper, which abutted the
Shh-expressing collecting duct epithelium and had elevated level of
Ptch expression. The decreased proliferation of medullary mesenchyme
is likely to play a major role in the observed hypoplasia of Shh
mutant kidneys. The proliferative effect of Shh was not mediated by
its downstream target gene, Bmp4, but was probably direct, as
Ptch expression was upregulated in the proliferative zone. Other
proliferative factors, such as Fgf7, have been reported to play a
role in proliferation of kidney tissues
(Qiao et al., 1999). However,
Fgf7 acts primarily on the collecting duct epithelium, and it is
therefore unlikely to mediate Shh-directed stimulation of mesenchymal cell
proliferation. Shh has been shown to play important roles in
regulating cell proliferation in many tissues, such as the gut mesencyme, the
early hair follicles and the central nervous system (for reviews, see
Ingham and McMahon, 2001
;
McMahon et al., 2002
). The
finding that Shh stimulates cell proliferation in kidney formation
adds to the evidence for a mitogenic action of Shh in developing
target tissues, a role shared by Ihh, which has a mitogenic function
in the developing endochondral skeleton
(Long et al., 2001
;
St-Jacques et al., 1999
).
Shh and kidney smooth muscle differentiation
The mesenchymal cells in the ureter adopt two fates, correlating with their
position relative to the ureteral epithelium. The subepithelial ureteral
mesenchymal cells, which are closest to the Shh source and presumably receive
the highest level of Shh activity, do not differentiate into smooth muscle at
least by the newborn stage, smooth muscle differentiation is restricted to
cells further away from the epithelium.
The role of Shh in differentiation of smooth muscle progenitor
cells into smooth muscle in the ureter is complicated. Although we see that
smooth muscle forms in the absence of Shh signaling in vivo, as expected from
the in vitro data, smooth muscle differentiation is delayed. How can an
inhibitor of smooth muscle differentiation apparently also promote smooth
muscle formation? One possibility is that this promotion is a secondary effect
from the proliferative effect of Shh on smooth muscle progenitor
cells (Fig. 9). It is possible
that only when these progenitor cells proliferate to reach a certain cell
number/density will smooth muscle differentiation initiate. For example, a
specific cell mass may be required to establish sufficient levels of a smooth
muscle differentiation signal. Consequently, a reduced proliferation rate in
Shh mutants would be expected to reduce the production of such a
differentiation signal and delay smooth muscle differentiation even if the
inhibitory effect of Shh on smooth muscle differentiation is removed.
A similar observation has been made in Ihh mutants, where delay in
chondrocyte proliferation appears to retard the initiation of chondrocyte
differentiation even though Ihh normally inhibits the differentiation
process (Karp et al., 2000;
Long et al., 2001
;
St-Jacques et al., 1999
).
|
Shh may also promote smooth muscle formation in vivo through its induction
of some smooth muscle differentiation factors. Bmp4 is a vertebrate
homolog of Drosophila Decapentaplegic, which is a downstream target
gene of Hh signaling in the fly. In mouse, Bmp4 is expressed adjacent
to Shh expressing cells in many tissue types
(Bitgood and McMahon, 1995).
There is also some evidence that Bmp4 may also be a downstream target
of Shh (for reviews, see Ingham
and McMahon, 2001
; McMahon et
al., 2002
). In this study, we show that Bmp4 expression
is positively regulated by Shh signaling in renal mesenchyme.
Bmp4 has been shown to promote smooth muscle differentiation in the
ureter (Raatikainen-Ahokas et al.,
2000
). It is likely that one way Shh promotes smooth
muscle differentiation in smooth muscle progenitor cells is through its
induction of smooth muscle differentiation factors such as Bmp4 in
these cells, although the fact that smooth muscle formation still occurs in
the absence of Shh and Bmp4 suggests that Bmp4 is
not essential for renal smooth muscle formation. One can imagine several
possibilities to explain these results. For example, smooth muscle progenitor
cells may also receive (and produce) other smooth muscle differentiation
factors, the production of which is independent of Shh signaling.
The loss of the subepithelial ureteral mesenchymal cells in Shh mutants suggests that Shh signaling is required for establishment and/or maintenance of this population. Our in vitro data further showed that Shh inhibits smooth muscle formation in the ureteral smooth muscle progenitor cells in a dose-dependent manner. Together, these data are consistent with a model in which Shh establishes and/or maintains these subepithelial ureteral mesenchymal cells by active inhibition of their differentiation (Fig. 9). The simplest scenario is that in the absence of Shh signaling, these cells differentiate into smooth muscle. However, to prove this is the case would require a scheme whereby the fate of these cells could be tracked in vivo: this is, unfortunately, beyond our current capabilities.
By contrast, within the renal pelvis at the newborn stage, we do not detect a population of SMA-negative cells in the equivalent region, even though the subjacent mesenchymal cells also express Ptch, which is indicative of Shh signaling. One possible explanation of this regional difference could be that the actual levels of Shh signaling might be insufficient in the renal pelvis to inhibit all cells from adopting a smooth muscle fate.
The subepithelial ureteral mesenchymal cells receive the highest level of
Bmp4 and probably other smooth muscle differentiation factors. How
can Shh inhibit their response to these differentiation factors? It
is possible that the high levels of Shh that these cells receive
blocks their ability to respond to smooth muscle differentiation factors they
themselves and their neighbor cells produce. In the Drosophila wing
imaginal disc, Hh represses the expression of the Dpp receptor in
cells in the anterior compartment immediately neighboring the Hh-expressing
cells, thus blocking their response to the high level of Dpp they
produce, whereas cells farther away from Hh sources express
Dpp in response to Hh and also respond to Dpp
(Tanimoto et al., 2000). A
similar mechanism could exist in the ureter. Alternatively, Shh may
induce the expression of antagonists to these differentiation factors in these
cells. Unfortunately, we cannot examine these possibilities now because of the
lack of knowledge of all the smooth muscle differentiation factors that are
involved in kidney development. However we have ruled out the possible roles
of two of Bmp4 antagonists, noggin and gremlin, in this process, as their
expression was not detected in the kidney and ureter at E14.5, the time when
smooth muscle differentiation initiates and therefore inhibition of smooth
muscle formation in the subepithelial ureteral mesenchymal cells should be
active (J. Y. and A. P. M., unpublished).
The opposing effects of the promotion of proliferation of smooth muscle
progenitor cells by Shh together with the induction of a smooth
muscle differentiation signal, and the inhibitory effect of Shh on
smooth muscle differentiation may serve to ensure correct timing and pattern
of smooth muscle differentiation (Fig.
9). Our finding that Shh inhibits smooth muscle formation
in the kidney is in line with studies carried out in chick gut
(Sukegawa et al., 2000), where
it appears that gut epithelium-derived Shh inhibits smooth muscle
formation in mesenchymal cells closest to the epithelium, regulating the
radial pattern of mesenchymal differentiation. However, in that case, the
effect of Shh on the timing of smooth muscle differentiation in the
mesenchyme further away from the epithelium was not examined. It is tempting
to speculate that the role of Shh in visceral smooth muscle formation
is conserved in different organs.
Shh and hydroureter
One of the most obvious defects in Shh mutant kidneys is
hydroureter/hydronephrosis. Hydronephrosis is likely to be a secondary
consequence of hydroureter, as hydroureter was apparent first and the back
pressure produced from hydroureter is thought to trigger hydronephrosis.
Malformation and destruction of various urinary tract structures are
associated with hydroureter. As the dilation of the ureter was usually more
severe or only detected in the proximal ureter of Shh mutant kidneys,
the defect is unlikely to result from a UVJ (ureterovesical junction)
abnormality, which would be expected to result in a dilation of the distal
ureter. Furthermore, we always detected a lumen along the entire length of the
Shh mutant ureter, with no evidence of a ureteral valve (a
reduplication of the transitional epithelium folds that protrude into the
lumen), thus ruling out a possible anatomical obstruction. It is difficult to
speculate how the lack of the subepithelial ureteral mesenchymal cells may
contribute to hydroureter, before the fate of this cell population is
identified. These cells may give rise to smooth muscle cells, or they may
generate other cell types such as the lamina propria, a layer of connective
tissue between the urothelium and smooth muscle.
Whatever the exact fate of these subepithelial ureteral mesenchymal cells
is, the reduction of smooth muscle in the ureter is likely to play a causal
role in the observed hydroureter. Smooth muscle functions to propel urine from
the renal pelvis to the bladder. The reduced amount of smooth muscle in the
ureter of Shh mutant kidneys is likely to compromise the transport of
urine to the bladder, hence to cause the build-up of urine in the ureter. The
observed more dilated proximal ureter where fluid builds up is consistent with
smooth muscle formation in the distal ureter being more severely affected.
Malformation of smooth muscle is also associated with congenital ureteral
stricture in humans, which leads to hydroureter/hydronephrosis
(Culp, 1981;
Tanagho, 1981
). Interestingly,
hydroureter has been reported in some cases of human infants with a
chromosomal deletion of a Shh-encoding region
(Lurie et al., 1990
;
Nowaczyk et al., 2000
),
suggesting that a deficiency in SHH might lead to a similar phenotype in the
human kidney. The mouse Shh mutant kidney may therefore serve as an
animal model system for understanding the ontogeny of smooth muscle formation,
the pathogenesis of human congenital ureteral stricture, the hydroureter
associated with a deficiency in Shh signaling and for the possible development
of treatments for these defects in humans.
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ACKNOWLEDGMENTS |
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