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First published online 17 September 2008
doi: 10.1242/dev.019760
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1 Pharmacology Institute, Im Neuenheimer Feld 366, University of Heidelberg,
69120 Heidelberg, Germany.
2 Institute of Developmental Genetics, Helmholtz Center Munich,
Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
3 Group of Neurobiology, School of Allied Health Sciences, Osaka University
Faculty of Medicine, Yamadaoka 1-7, Suita, Osaka 565-0871, Japan.
Author for correspondence (e-mail:
rohini.kuner{at}pharma.uni-heidelberg.de)
Accepted 12 August 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Kidney development, RhoA, ROCK, Epithelial-mesenchymal interaction, Transgenic mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Shah et al.,
2004). These cell-cell interactions are postulated to be mediated
by signals or cues that are secreted or expressed in a locally discrete
manner. Several candidates for secreted cues have been described in specific
organs [e.g. transforming growth factor beta (TGFβ), bone morphogenetic
proteins (BMPs), glial-derived nerve growth factor (GDNF), hepatocyte growth
factor (HGF), etc. (reviewed by Moustakas
and Heldin, 2007; Shah et al.,
2004; Zhang and Vande Woude,
2003)].
Semaphorins constitute a family of secreted or cell-specific cues, the
cellular effects of which are achieved via activation of transmembrane
proteins called plexins (Tamagnone et al.,
1999). Although semaphorins were first described as important axon
guidance molecules (Liu and Strittmatter,
2001), several recent studies have established them as key
regulators of invasive growth, apoptosis and the immune system
(Chédotal et al., 2005;
Fiore and Puschel, 2003;
Suzuki et al., 2008). Plexin
B1, the prototypic member of B-family plexins, and its ligand semaphorin 4D
(Sema4d) have been functionally implicated in diverse processes such as
migration and proliferation of neuronal, endothelial and tumour cells, and
angiogenesis and axonal navigation, among others
(Basile et al., 2004;
Conrotto et al., 2005;
Giordano et al., 2002;
Masuda et al., 2004;
Suzuki et al., 2008;
Swiercz et al., 2002;
Swiercz et al., 2004).
In recent studies, we and others have elucidated the composition of the
plexin B1 signalling complex. Plexin B1 activation leads to an activation of
the RhoGTPase, RhoA (Aurandt et al.,
2002; Hirotani et al.,
2002; Perrot et al.,
2002; Swiercz et al.,
2002) and inactivation of R-Ras
(Oinuma et al., 2004) and Rac
(Vikis et al., 2000). Plexin
B1 also physically associates with receptor tyrosine kinases (RTKs), such as
Met (Giordano et al., 2002)
and Erbb2 (Swiercz et al.,
2004), leading to their activation upon binding to Sema4d.
Moreover, plexin B1 activation has been recently associated with the
activation of diverse intracellular pathways, involving FAK, Src, Pyk2,
p190RhoGAP and others (Basile et al.,
2005; Basile et al.,
2007; Barberis et al.,
2005).
Very little is known about the role of plexin B family members in
developmental processes in vivo. Using genetically modified mice as tools, we
have recently addressed the functions of B-type plexins in neuronal migration
and patterning of the brain in vivo (Deng
et al., 2007; Friedel et al.,
2007). However, not much is known about whether and how B-type
plexins modulate the development and maturation of organs outside of the
nervous system.
Based upon our finding that plexin B1 and its ligand Sema4d are expressed in a complementary pattern in epithelial and mesenchymal compartments, respectively, over crucial developmental time periods, we hypothesize that this ligand-receptor pair plays a role in organogenesis. Using the developing metanephros as a model system, we have elucidated here the functional contribution of Sema4d and plexin B1 in developmental mesenchyme-epithelial interactions, and we show that plexin B1-mediated RhoA activation is important in shaping the architecture of the developing kidney ex vivo as well as in vivo.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
|
). At 24 hours or 48 hours in vitro, cultured metanephric
kidneys were fixed in 100% ice-cold methanol. In some cases, morphology was
documented with photomicrographs using a phase-contrast inverted microscope.
In a majority of experiments, fixed kidneys were subjected to whole-mount
immunofluorescence as described below.
Immunohistochemistry
Following in situ hybridization, some sections were processed for
immunohistochemistry with an anti-WT1 antibody (1:300, Santa Cruz
Biotechnology) as described before
(Worzfeld et al., 2004). For
whole-mount staining, cultured mouse kidneys were fixed using 100% ice-cold
methanol, blocked in PBS/1% Triton X-100/1% BSA for 1 hour and incubated with
primary antibodies (anti-calbindin-D-28K antibody; 1:300; Sigma-Aldrich,
Germany and anti-WT1 antibody) overnight at 4°C and stained further using
standard procedures.
Confocal analysis and quantification of metanephric morphological parameters: metanephroi stained as wholemounts were imaged using a confocal laser-scanning microscope (Leica TCS AOBS) in stacks spanning the entire thickness of the metanephroi. Ureteric bud branch tips immunostained with the anti-calbindin antibody were counted blindly in each frame and expressed as mean±s.e.m. Sister kidneys were compared using Student's paired t-test. When more than two treatment groups were compared, analysis of variance (ANOVA) with random measures followed by post-doc Fischer's test was employed. Metanephric condensates, resulting in comma-shaped bodies were counted and analysed in a similar manner.
Pharmacological reagents
ROCK activity was inhibited via treatment with Y27632 (Sigma Aldrich). The
medium was supplemented with K252a (Sigma Aldrich) or a specific inhibitor of
Met signalling, PHA-665752 (Tocris)
(Christensen et al., 2003).
Importantly, experiments comparing the effects of pharmacological inhibitors
between mock- and Sema4d-treated kidneys were always carried out on sister
kidneys derived from the same embryo.
Preparation of Sema4d
HEK 293T cell were transfected with Sema4d-AP or empty AP expression
plasmids in serum-free medium, Sema4d-AP was purified from supernatants and
its activity was assessed via alkaline phosphatase activity assays as
described previously (Deng et al.,
2007). The working concentration of Sema4d-AP was standardized at
150 mU/ml in medium (approximately equivalent to 1 nM).
Bacterial expression and protein purification: TAT-C3 was produced and
purified in Escherichia coli strain DE3 as GST fusion using standard
protocols (Brandt et al.,
2003).
Determination of activated Rho: the amount of activated RhoA was determined
as described previously (Swiercz et al.,
2002). For the determination of activated RhoA in kidney, kidneys
were extracted from E14 mice, homogenized in RIPA buffer and GTP-bound RhoA
was precipitated and detected via immunoblotting using a monoclonal anti-RhoA
antibody (BD, Heidelberg, Germany).
Genetically-modified mice
The generation of mice with constitutive global deletion of the plexin
B1-encoding gene, Plxnb1 (Plxnb1-/-) has been
described previously (Deng et al.,
2007). Furthermore, heterozygous knock-in mutant mice expressing a
cDNA encoding β-galactosidase (lacZ) targeted into the
Plxnb1 locus (Plxnb1lacZ/+) were used in studies on
expression analysis of plexin B1 via β-galactosidase staining
(Friedel et al., 2007).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Shah et al., 2004). At E13.5,
we found that plexin B1 was expressed in round epithelial structures, which
resembled ureteric buds (arrows in Fig.
1A). We reasoned that if these plexin B1-expressing cells indeed
represented ureteric epithelium, they ought to be surrounded by a cap of
mesenchyme, which is known to condense and aggregate around ureteric buds at
E13.5 in mice (Saxen, 1987).
Indeed, using mRNA in situ hybridization for plexin B1 and co-staining for a
marker for metanephrogenic mesenchyme, WT1, we found a clear complementarity
of plexin B1 expression and WT1-stained condensing mesenchyme
(Fig. 1E). To confirm our
results further, particularly in the light of a recent contradictory
publication (Fazzari et al.,
2007) that has reported plexin B1 expression in the mesenchyme, we
took advantage of a mouse line expressing the reporter gene product
β-galactosidase under the control of the plexin B1 promoter
(Plxnb1lacZ). This knock-in reporter reflects closely the
developmental expression pattern of plexin B1 in the nervous system, because
the entire promoter elements of the Plxnb1 gene are available to
direct lacZ expression (Friedel
et al., 2007). At E17.5, when epithelial-mesenchymal interactions
between ureteric buds and condensing mesenchyme still take place in the outer
cortex of the kidney, tissue sections of Plxnb1lacZ reporter embryos
also revealed lacZ-staining in ureteric buds in the outer cortex
(arrows in the inset of Fig.
1F), thereby entirely corroborating the results obtained by in
situ hybridization and co-staining experiments. At E15.5, in agreement with
previous results (Perälä et al.,
2005), plexin B1 is found in developing glomeruli (arrow in
Fig. 1C), indicating that,
after the mesenchyme-to-epithelium transition has taken place, plexin B1
starts to be expressed in epithelial cells that had been derived primarily
from mesenchymal cells. These observations could be confirmed via
β-galactosidase staining of E17.5 embryos derived from
Plxnb1lacZ reporter mice, which showed expression of plexin B1 in
glomerular structures of the inner cortical layer. When analyzing the
expression of Sema4d by in situ hybridization at E13.5, we found staining for
Sema4d in a complementary pattern to plexin B1 in the metanephrogenic
mesenchyme (Fig. 1B). This is
consistent with the observation that Sema4d mRNA is found in comma- and
S-shaped tubular structures at E15.5, which are of mesenchymal origin
(asterisks in Fig. 1D).
|
). In
mouse, at E13.5 (bud stage), the oral epithelium invaginates into the
underlying condensing mesenchyme. During this period, we found plexin B1 to be
strongly expressed in the ectoderm-derived epithelial layer
(Fig. 2A). Strikingly, Sema4d
was selectively expressed in the mesenchymal layer, which is aggregated
directly under the epithelial layer (Fig.
2A). This expression pattern persisted during the cap stage and
bell stage (not shown), and was also seen at later stages of progressive
differentiation, where plexin B1 was found in the enamel epithelium (arrows in
Fig. 2A) and Sema4d was
expressed in odontoblasts (arrowheads in
Fig. 2A).
During the development of the olfactory primordia, the interaction between
the olfactory placodal epithelium and the associated mesenchyme is essential
to guide olfactory patterning, morphogenesis and differentiation
(Balmer and La Mantia, 2005).
At E13.5 and E15.5, plexin B1 was found to be strongly expressed throughout
the olfactory epithelium (arrows in Fig.
2B), supporting previous results
(Perälä et al.,
2005). Complementary to the distribution of plexin B1, we found an
expression of Sema4d in the mesenchymatous shelves that are covered by the
olfactory epithelium (arrowheads in Fig.
2B).
|
).
At E13.5, when segmental bronchi progressively branch, plexin B1 was strongly
expressed in the bronchial epithelium (Fig.
1C). Plexin B1 mRNA continued to be highly expressed in the
epithelium after E15.5 during the formation of terminal bronchioles (arrows in
Fig. 1C, E15.5). Sema4d was
found on the inner aspect of plexin B1-expressing epithelium, rather than in
the lung mesenchyme (Fig.
1C). Thus, plexinB1 and Sema4d were observed to show complementary expression patterns in several but not all developing non-neuronal organs, with plexin B1 being typically found in the epithelium and Sema4d in the surrounding mesenchymal tissue, suggesting a role for this ligand-receptor pair in epithelial-mesenchymal interactions during organogenesis.
Effects of Sema4d on metanephroic development
To directly test this hypothesis, we used a well-established mammalian
model of mesenchyme-epithelial interactions: mesenchyme-induced epithelial
branching in the developing urinary collecting duct
(Shah et al., 2004). We
employed the ex vivo organ culture system of kidneys derived from mice at E12,
which enables testing effects of individual molecules on branching
morphogenesis and nephron formation over 2-4 days
(Saxen, 1987)
(Fig. 3A-C). Three-dimensional
reconstruction of anti-calbindin-stained ureteric trees following confocal
microscopy on whole cultured metanephroi revealed normal development in terms
of number of ureteric tips and the length and area of developing kidneys,
consistent with previous reports (Saxen,
1987; Woolf et al.,
1995) (Fig. 3D-G).
Concentrated supernatants of HEK293T cells expressing AP-tagged Sema4d were
used to test the effects of plexin B1 activation via exogenously supplied
Sema4d on developing kidneys, and supernatants from vector-transfected HEK293
cells served as controls (mock), as described previously
(Deng et al., 2007). Sema4d or
mock treatments were always performed on sister kidneys derived from
individual embryos at E12 (Fig.
3H,I), in order to circumvent differences arising from dissimilar
development and maturation of embryos within and between litters. The
functional integrity of Sema4d-enriched medium was confirmed by testing its
ability to activate RhoA in a RBD-GST-based pull-down assay
(Fig. 3J)
(Swiercz et al., 2002).
E12 kidneys cultured with Sema4d over 48 hours showed a striking reduction
in the extent of ureteric branching when compared with sister kidneys cultured
in the presence of mock medium (see Fig.
3H,I for typical examples and
Fig. 3K for summary). Both the
number of the developed, calbindin-positive ureteric tips, as well as the
number of branch points in the ureteric tree were found to be consistently
reduced upon Sema4d treatment when compared with mock treatment
(P=0.001 and P=0.01, respectively;
Fig. 3K). Sema4d-induced
deceleration of kidney growth was also evident as a decrease in the length
(P
0.001) and area (P=0.002) of developing kidneys
(Fig. 3L). To address whether
activation of plexin B1 also affects the differentiation of metanephrogenic
mesenchyme, we addressed the effects of Sema4d treatment on the number of
comma-shaped bodies identified via WT1 staining, which result from the
transition of metanephric mesenchyme into tubular epithelium. This was not
affected by Sema4d (see Table S1 in the supplementary material), suggesting
therefore that activation of plexin B1 leads to selective changes in the
branching morphogenesis of ureteric tips in the developing kidney.
|
). When
compared with pre-immune serum (control), treatment with the anti-Sema4d
antibody led to a partial reduction in Sema4d-induced RhoA activation in
HEK293T cells (Fig. 4A),
showing that this approach can at least partially attenuate Sema4d-induced
activation of plexin B1. E12 kidneys cultured with the anti-Sema4d antibody
showed a small, but significant, increase in the number of ureteric tips
(P=0.004), as well as in the number of branch points
(P=0.04), when compared with sister kidneys cultured with pre-immune
serum (Fig. 4B-D). The length
and area of anti-Sema4d-treated kidneys showed only a trend towards increase,
which did not reach statistical significance (P=0.15 and
P=0.11 for length and area, respectively;
Fig. 4E). Albeit the magnitude
of the observed differences in ureteric branching was small, given that the
anti-Sema4d antibody may have only partially impeded Sema4d-plexin B1
signalling, the above results suggest that Sema4d, synthesized and released
endogenously by the metanephrogenic mesenchyme at E12, functions to repress
branching morphogenesis of ureteric tips.
Signalling mechanisms mediating effects of Sema4d on ureteric branching
The nature of molecular pathways that mediate the inhibitory effects of
Sema4d on ureteric branching raises an important and complex issue. Among the
plexin B1 signalling mediators, RTKs and, in particular, Met have been best
studied with respect to kidney development
(Woolf et al., 1995;
Santos et al., 1994;
Zhang and Vande Woude, 2003).
To assess their potential involvement in plexin B-mediated cellular effects in
the developing kidney, we cultured sister kidneys in the presence of
pharmacological inhibitors with either Sema4d-containing medium or control
medium (see Fig. 5A-D for
typical examples; see Fig. 5E,F
for a summary). K252a is known to block RTKs such as Met and Trks
(Morotti et al., 2002).
Consistent with previous transgenic studies reporting a requirement for RTKs
such as Met and Ret in ureteric branching, we observed that 200 nM K252a in
the absence of Sema4d led to an inhibition of ureteric branching: the numbers
of branch points and ureteric tips, as well as the length and area of the
metanephroi, were significantly reduced
(Fig. 5E,F). Furthermore, the
number of comma-shaped bodies arising from the mesenchyme-derived epithelium
was also significantly reduced (see Table S1 in the supplementary material).
Importantly, however, inhibition of Met via K252a did not change the nature or
the magnitude of the effects of Sema4d on developing metanephroi
(Fig. 5C-F). Thus, inhibition,
rather than activation, of RTKs mimicked the effects of Sema4d, suggesting
that Sema4d-induced repression of ureteric branching is not associated with
the activation of RTKs via plexin B1.
Because K252a can block several kinases in addition to inhibiting Met (e.g.
Morotti et al., 2002), we used
PHA-665752, which has been recently identified as an active-site competitive
inhibitor of Met with over 50-fold selectivity for Met when compared with a
broad panel of diverse tyrosine- and serine-threonine kinases
(Christensen et al., 2003). We
observed that pre-treatment with PHA-665752 (1 µM) significantly reduced
ureteric branching (Fig. 5I,J)
and decreased area and length in mouse metanephroi cultured at day E12
(Fig. 5K,L). Importantly,
treatment with PHA-665752 did not alter Sema4d-induced reduction in ureteric
branching (Fig. 5I-L). We
therefore inferred that Met activation is not required for Sema4d-induced
inhibition of branching morphogenesis in the developing metanephros.
Using a Rhotekin-GST-based pull-down assay, we observed that, similar to
developing neurons, application of Sema4d indeed leads to an activation of
RhoA in developing metanephroi at E12 when compared with treatment with mock
medium (Fig. 6A). To assess the
role of Rho-dependent mechanisms in Sema4d-induced effects on developing
metanephroi, we used lower concentrations of Y27632 (1 µM and 3 µM),
which dose-dependently and specifically inhibit the Rho kinase (ROCK)
(Narumiya et al., 2000;
Davies et al., 2000).
Treatment with 1 µM Y-27632 tended to increase the number of ureteric tips
in mock-treated kidneys (P=0.01) and produced a small, but
statistically significant, increase in the number of branch points
(P=0.013), suggesting that endogenous ROCK mediates an inhibitory
tone on ureteric branching during development under the conditions used in
this study (typical examples are in Fig.
6B,C; see summary in Fig.
6J). Importantly, 1 µM Y27632 fully reversed the inhibitory
effects of Sema4d on ureteric branching
(Fig. 6D,J). Furthermore,
Sema4d produced a striking increase in the ureteric branching in the presence
of 1 µM Y27632 (84.4±2.6% increase in branch points and
29.8±3.4% increase in ureteric tips), which was significantly higher
than that observed in metanephroi treated with 1 µM Y27632 and mock medium
(36.3±2.7% increase in branch points and 6.0±3.1% increase in
ureteric tips; Fig. 6D,J)
(P<0.001 in both cases).
|
;
Michael et al., 2005), we used
doses of Y27632 that specifically block ROCK
(Davies et al., 2000) to study
whether structural changes are linked with ureteric branching and how these
are modulated by Sema4d-plexin B1 interactions. We observed that both 1 µM
and 3 µM concentrations of Y27632 were associated with a rapid increase in
the size of the metanephroi (Fig.
6C,F,K). However, unlike 1 µM Y27632, a concentration of 3
µM Y27632 produced a blatant deformation of the developing ureteric tree
with a pronounced swelling of ureteric tips (see
Fig. 6E for typical examples).
Staining of the actin cytoskeleton with TRITC-labelled phalloidin revealed a
flattening of wedge cells, as well as an increase in the lumen of ureteric
tips in metanephroi cultured in the presence of 3 µM Y27632
(Fig. 6G,H). Furthermore,
unlike 1 µM Y27632, treatment with 3 µM Y-27632 did not increase
ureteric branching over mock medium (Fig.
6B,E,J). Interestingly, addition of Sema4d to the medium could
rescue some, but not all, the effects of 3 µM Y27632. Swelling of ureteric
tips, as well as an increase in the size of lumen, was less prominent in
Sema4d + 3 µM Y27632-treated metanephroi
(Fig. 6F,I). Furthermore,
Sema4d produced a striking increase in the ureteric branching in the presence
of 3 µM Y27632 (55.7±3.1% increase in branch points and
15.1±3.1% increase in ureteric tips), which was significantly higher
than that observed in metanephroi treated with mock medium, Sema4d medium
alone or 3 µM Y27632 with mock medium (see
Fig. 6F for a typical example;
see Fig. 6J for a summary)
(P0.05 in all cases). However, kidney hyperplasia elicited by 3
µM Y27632 was not changed by Sema4d treatment
(Fig. 6F,K), suggesting that
mechanisms underlying Y27632-induced metanephroic hyperplasia differ from
those associated with deformation of ureteric tips and changes in ureteric
branching in the developing kidney. Furthermore, we observed that treatment
with a TAT-fusion protein of recombinant C3 exoenzyme (TAT-C3, 0.5 µM), a
known specific direct inhibitor of RhoA, reversed the inhibitory effects of
Sema4d and increased ureteric branching over control levels (see Fig. S1 in
the supplementary material; P<0.001), which is consistent with
effects of Y27632 at doses of 1 µM and 3 µM.
|
36%
more ureteric tips than their Plxnb1+/- littermates
(Fig. 7F). In order to
ascertain whether this truly reflects enhanced ureteric branching, we analyzed
the number of ureteric tips in a region of interest of a defined area (white
boxes in Fig. 7B,C). The number
of ureteric tips per unit area was consistently higher in
Plxnb1-/- mice than in their corresponding
Plxnb1+/- littermates at E13.5
(Fig. 7G). At E14.5,
Plxnb1-/- embryos continued to show larger kidneys with a
significantly greater area than Plxnb1+/- embryos
(Fig. 7H,I). However, starting
from E15.5, the kidneys of Plxnb1-/- embryos did not
differ from those of Plxnb1+/- embryos
(Fig. 7H,I). Furthermore,
analysis of Nissl-stained paraffin sections showed that the number of
developed nephrons did not differ between Plxnb1-/- and
Plxnb1+/- embryos at E17.5
(Fig. 7J). Taken together,
these data suggest that an activation of plexin B1 is indeed functionally
linked to the inhibition of epithelial branching morphogenesis during early
stages of ureteric development, which is compensated over later stages of
kidney development in mice that lack plexin B1. Interestingly, in E12 metanephroi isolated from mice lacking plexin B1, Sema4d failed to inhibit ureteric branching, although inhibition was observed in metanephroi of wild-type littermate embryos. Furthermore, in these ex vivo culture experiments, we observed that metanephroi derived from plexin B1 knockout mice were consistently larger than those from wild-type littermates, and they failed to respond to Sema4d with respect to kidney size (Fig. 8), which is entirely consistent with our in vivo analysis of plexin B1 knockout mice (Fig. 7). These results thus show that plexin B1 can fully account for the observed effects of Sema4d on branching morphogenesis in the kidney.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and, very
recently, also in the kidney (Tufro et
al., 2008). In the developing salivary gland, however, signalling
via Class III semaphorins has recently been reported to facilitate, rather
than to inhibit, cleft formation (Chung et
al., 2007). A functional role for Class IV semaphorins in
branching morphogenesis during organogenesis had not been experimentally
demonstrated so far. We observed that both endogenously expressed as well as
exogenously applied Sema4d suppresses the branching morphogenesis of the
ureteric bud epithelium, which is the first and crucial important step in the
establishment of a collecting duct system for enabling kidney function.
Entirely consistent with this finding, we observed that mice lacking plexin B1
demonstrate exaggerated ureteric branching and increased kidney size.
Importantly, these processes were detected over early stages of kidney
development, i.e. between E12.5-E14.5, during which crucial functional
interactions between the ureteric bud epithelium and metanephrogenic
mesenchyme take place (Saxen,
1987).
The finding that the major functional outcome of the activation of the
Sema4d-plexin B1 axis in the developing kidney entails an inhibition, rather
than stimulation, of branching morphogenesis is unexpected in the light of the
ability of Sema4d-plexin B1 to signal via RTKs, such as Met, which are
recognized as activators of branching morphogenesis
(Santos et al., 1994;
Woolf et al., 1995;
Zhang and Vande Woude, 2003).
Indeed, in several developing organs we studied, such as the kidney, lung and
teeth, the observed expression pattern of plexin B1 closely matches the
reported distribution of Met (Ohmichi et
al., 1998; Tabata et al.,
1996; Thewke and Seeds,
1996; Woolf et al.,
1995). Similarly, there are many parallels between the pattern of
expression of Sema4d reported here and that of the classical Met ligand HGF
reported in previous studies in developing tissues such as the lung, teeth and
olfactory conchae (Ohmichi et al.,
1998; Tabata et al.,
1996; Thewke and Seeds,
1996; Woolf et al.,
1995). Furthermore, there are many similarities between their
cellular functions. For example, both HGF and Sema4d have been reported to
trigger invasive growth in epithelial cells
(Giordano et al., 2002), a
process that uses the basic mechanisms of branching morphogenesis, with
considerable on-going crosstalk between the two ligand-receptor pairs
(Conrotto et al., 2004).
Similarly, both Sema4d and HGF have been reported to stimulate axonal
branching and growth in developing sensory neurons
(Maina et al., 1997;
Masuda et al., 2004).
Collectively, these findings indicate that the net modulation of branching
morphogenesis in biological systems by B-type plexins is highly context
dependent and that signalling via RTKs may play roles of varying prominence in
the different systems. Indeed, in addition to activating Erbb2 and Met, B-type
plexins can trigger multiple signalling events in parallel by modulating the
activity of Rho family GEFs and GAPs, which govern the activation status of
RhoA, R-Ras and several cytosolic kinases, including Pyk2, Src, FAK, PI3K and
Akt (Aurandt et al., 2002;
Basile et al., 2005;
Basile et al., 2007;
Giordano et al., 2002;
Hirotani et al., 2002;
Oinuma et al., 2004;
Perrot et al., 2002;
Swiercz et al., 2002;
Swiercz et al., 2004;
Vikis et al., 2000).
Furthermore, the cellular functions of several of these diverse mediators can
be mutually contradictory. For example, recent evidence suggests that,
depending upon whether Met or Erbb2 become recruited, plexin B1 elicits
entirely opposite effects upon cell motility via inactivation or activation of
RhoA, respectively (Swiercz et al.,
2008). These aspects can confer a tremendous level of complexity
and versatility to the biological functions of Sema4d-plexin B1 interactions
in developing organs.
|
;
Eisen et al., 2006;
Hasegawa et al., 1999;
Meyer et al., 2006; Micheal et
al., 2005; Rosário and Birchmeier,
2003). Consistent with the above, blockade of the RhoA effector
ROCK, via Y27632, in developing kidneys is associated with a diverse set of
phenotypic changes, and ROCK has been implicated in several distinct steps
during kidney development (Meyer et al.,
2006; Michael et al.,
2005). Here, we used doses that have been shown to block ROCK
specifically (Davies et al.,
2000; Narumiya et al.,
2000) and observed that partial blockade of ROCK, which did not
cause a marked deformation of ureteric tips, fully blocked the inhibitory
effects of Sema4d and increased ureteric branching. These results were
corroborated by experiments on RhoA inhibition via the C3 exoenzyme. This
suggests that RhoA signalling via ROCK acts as an endogenous brake on
branching morphogenesis during kidney development and that this function is
distinct from other functions of ROCK in maintaining the cytoskeletal
structure of the ureteric tree. This is supported by reports on the expression
of RhoA in the developing kidney in a pattern suggestive of a functional role
in epithelial-mesenchymal interactions
(Bianchi et al., 2003).
Furthermore, RhoA and ROCK have been associated with the inhibition of
branching morphogenesis in other model systems, such as the developing lung
(Moore et al., 2005) and
neurites of hippocampal neurons (Luo,
2000). Consistent with previous studies
(Michael et al., 2005;
Meyer et al., 2006), we
observed that a higher degree of ROCK blockade led to rapid structural
deformities in the entire developing metanephros, including swelling and
increased lumen in ureteric tips. The latter may be associated with a loss of
epithelial barrier function, which has previously been suggested to be
regulated by Rho GTPases (Fujita et al.,
2000; Hasegawa et al.,
1999). Interestingly, Sema4d was observed to partially rescue some
of these structural anamolies, either via an increase in RhoA activity and the
consequential drop in the degree of ROCK blockade, or through the actions of
other signalling mediators activated downstream of plexin B1.
In this context, another interesting observation was that pharmacological
blockade of ROCK unmasked stimulatory effects of Sema4d on ureteric branching
in developing metanephroi. This suggests that, upon plexin B1-mediated
activation, the RhoA-ROCK pathway inhibits branch-promoting signalling events
that are concurrently triggered by plexin B1. This finding is very interesting
because, so far, branching patterns are generally thought to be regulated by a
precise temporal and spatial balance between stimulatory branching morphogens,
such as HGF, and inhibitory morphogens, such as members of the TGFβ
superfamily, e.g. specific TGFβ isoforms and BMPs
(Shah et al., 2004;
Zhang and Vande Woude, 2003).
Via their unique ability to trigger activation of the branch-promoting RTKs,
as well as of inhibitory RhoA-dependent pathways, plexin B proteins harbour
the potential to fine-tune branching morphogenesis in a novel manner.
Furthermore, recent studies suggest that plexin B1 can switch between
molecular states that favour the activation of either Met or RhoA in a
context-dependent manner (Basile et al.,
2004; Conrotto et al.,
2005; Swiercz et al.,
2008). It is plausible, therefore, that plexin B proteins inhibit
branching morphogenesis at a specific stage of ureteric development and
counterbalance this via activation of alternative signalling pathways when
inhibition ceases to be required. Indeed, it has been hypothesized that,
during kidney development, negative-feedback processes between the
metanephrogenic mesenchyme and the ureteric buds are timed to a specific point
during mesenchyme-derived tubule formation and cease to exist following the
fusion of the metanephric tubule with the lateral ureteric branch as
nephrogenesis proceeds (Shah et al.,
2004).
The functional significance of inhibition of branching morphogenesis in
kidney development in vivo has been difficult to elucidate, as deletion mouse
mutants of the typical inhibitory morphogens or their receptors have yielded
mixed phenotypes that range from early embryonic lethality to no overt renal
anomalies (Shah et al., 2004;
Zhang and Vande Woude, 2003).
Although the phenotype of plexin B1 knockout mice closely matched our ex vivo
results at early stages in ureteric development, it appears that compensatory
mechanisms can overcome the effects of disturbances in the Sema4d-plexin B1 in
early development, consistent with the notion of functional redundancy of
branching-regulatory factors during embryonic kidney development
(Shah et al., 2004), probably
arising as a result of a convergence on common effector systems. Furthermore,
plexin B2, which can function as a low-affinity receptor for Sema4d, is also
expressed in the developing kidney.
In summary, this study extends the functional repertoire of the Sema4d-plexin B1 axis to modulation of branching morphogenesis during organogenesis outside the nervous system. Our results identify Sema4d as a novel negative morphogen in kidney development and suggest that activation of the RhoA-ROCK pathway by plexin B1 constitutes an important regulatory mechanism in the fine-tuning of epithelial branching via mesenchyme-derived cues.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/20/3333/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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13 was comparable
in the two samples (internal control). (B-D) Typical examples of
anti-calbindin-stained metanephroi cultured with either Sema4d or mock medium
in the presence or absence of the ROCK inhibitor Y27632 (1 µM). In the
presence of 1 µM Y27632, Sema4d did not attenuate ureteric branching.
(E,F) Y27632 (3 µM) led to a hyperplasia and distortion of
ureteric structures (arrowheads), to a lesser extent in Sema4d-treated kidneys
than in mock-treated kidneys. (G-I) Higher magnification views of
phalloidin-stained ureteric tips in Sema4d- or mock-treated kidneys cultured
in the presence of 3 µM Y27632. The Y27632-induced distortion of apical
wedge cells and actin bundles and the increase in tip lumen occurs to a lesser
extent in Sema4d-treated kidneys in comparison with mock-treated kidneys.
(J,K) Quantitative summary of the effects of Y27632 at either 1
µM or 3 µM on Sema4d- or mock-treated metanephroi.
