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First published online 23 May 2007
doi: 10.1242/dev.02861
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1 Developmental Genetics, DKBW Centre for Biomedicine, University of Basel
Medical Faculty, Mattenstrasse 28, CH-4058 Basel, Switzerland.
2 Department of Medical Biochemistry and Molecular Biology, Biocenter Oulu,
Laboratory of Developmental Biology, Aapistie 5A, PO Box 5000, University of
Oulu, F-90570 Oulu, Finland.
3 Department of Histology, Anatomy Institute, Pestalozzistrasse 20, CH-4056
Basel, Switzerland.
Author for correspondence (e-mail:
Rolf.Zeller{at}unibas.ch)
Accepted 5 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Antagonist, BMP, gremlin 1, Kidney, Mouse, Signalling
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yu et al., 2004). In mouse
embryos, the ureteric bud forms around embryonic day (E) 10.5 as an epithelial
swelling in the caudal-most part of the Wolffian duct. Development of the
definitive kidney is initiated when the ureteric bud elongates to invade and
induce the metanephric mesenchyme. In turn, the induced metanephric mesenchyme
produces signals that initiate nephrogenesis. During ureteric bud outgrowth,
the epithelial tip region thickens to form an ampulla just prior to appearance
of the first epithelial branch (Shakya et
al., 2005). In addition, this process induces condensation of the
metanephric mesenchyme, which is the first morphological sign of nephrogenesis
(Saxén, 1987). Many of
the human congenital anomalies of the kidney and urinary tract (CAKUT) are
caused by defects in these early inductive events, but their aetiology remains
poorly understood (Batourina et al.,
2002; Pope et al.,
1999).
During the last decade, significant parts of the molecular networks and e-m
feedback signalling interactions that regulate mammalian kidney organogenesis
have been identified (Costantini,
2006; Vainio and Lin,
2002). It is established that ureteric bud formation and the
induction of its branching require GDNF (glial cell line derived neurotrophic
factor), a secreted growth factor that is expressed by the metanephric
mesenchyme. The GDNF ligand interacts with its cognate receptor RET (ret
proto-oncogene), which is first expressed by the Wolffian duct and then by the
ureteric epithelial tips as branching morphogenesis progresses
(Schuchardt et al., 1996). The
current view is that establishment of signalling between GDNF, RET and its
co-receptor GFR
1 (glial cell line derived neurotrophic factor family
receptor 1) is essential for ureteric bud formation and initiation of
outgrowth and branching (Costantini and
Shakya, 2006). As the rostral part of the Wolffian duct initially
expresses Ret, it is competent to form supernumerary buds and
branches upon exposure to GDNF (Shakya et
al., 2005). Therefore, a mechanism must exist that restricts
ureteric bud formation to the caudal-most part of the Wolffian duct. Indeed,
supernumerary epithelial buds form in mouse embryos lacking either SLIT2 or
ROBO2 functions, the SPRY1 intra-cellular antagonist or the FOXC1
transcriptional regulator. Molecular analysis showed that SLIT2 and/or ROBO2
signalling is required to restrict Gdnf expression to caudal
mesenchyme (Grieshammer et al.,
2004). FOXC1 is also required for caudal restriction of
Gdnf (Kume et al.,
2000), but is not a target of SLIT2/ROBO2 signalling
(Grieshammer et al., 2004). By
contrast, SPRY1, an intra-cellular antagonist of tyrosine kinase receptors,
reduces the sensitivity of the Wolffian duct to GDNF, such that only one
ureteric bud forms (Basson et al.,
2005; Chi et al.,
2004). In Spry1-deficient mouse embryos, ectopic
epithelial buds form and multiple- and hydroureters are formed, which results
in phenotypes identical to the human CAKUT syndrome
(Basson et al., 2005). However,
ectopic expression of Spry2 in the Wolffian duct sensitises the
epithelium to GDNF signalling, which again results in formation of
supernumerary epithelial buds (Chi et al.,
2004). Hence, the current view is that the interaction of these
different signal transduction cascades restricts GDNF expression and activity
such that only one ureteric bud forms in the caudal-most part of the Wolffian
duct (Basson et al., 2005;
Grieshammer et al., 2004).
Metanephric kidney development is then initiated by the onset of ureteric bud
outgrowth, and invasion and induction of the metanephric mesenchyme under the
influence of GDNF-RET signalling
(Costantini and Shakya,
2006).
Wnt11 expression is activated in the epithelial tip of the
ureteric bud and WNT11 signalling is in turn required to propagate mesenchymal
GDNF signalling, which results in establishment of an autoregulatory e-m
feedback signalling loop (Majumdar et al.,
2003). In Wnt11-deficient mouse embryos, Gdnf
expression remains lower and the number of epithelial branches is reduced.
Conversely, the disruption of GDNF signal reception in Ret-deficient
embryos reduces Wnt11 expression. Therefore, autoregulatory
GDNF-WNT11 feedback signalling co-ordinately controls the progression of
metanephric branching morphogenesis after initiation of ureteric bud outgrowth
(Majumdar et al., 2003).
During branching, the ureteric epithelial tips secrete additional signals
(e.g. WNT9b) (Carroll et al.,
2005), which induce nephrogenesis. Nephrogenesis is regulated by
transcriptional activation of another WNT signal, WNT4 in the condensing
mesenchyme. Mouse embryos that lack Wnt4 fail to form metanephric
kidneys due to disruption of the mesenchyme to epithelial transition of the
nephrogenic precursors (Stark et al.,
1994).
Several BMP ligands and their receptors are expressed during metanephric
kidney organogenesis, but relatively little is known about their essential
roles in these processes as loss-of-function mutations often cause early
embryonic lethality. However, genetic inactivation of Bmp7 in mouse
embryos leads to premature depletion of the nephrogenic mesenchyme, which
manifests itself in a dysplastic kidney phenotype
(Dudley et al., 1995;
Luo et al., 1995). A recent
study shows that BMP4 can compensate for the lack of Bmp7 during
metanephric kidney development, which indicates that BMPs could potentially
replace one another during kidney organogenesis
(Oxburgh et al., 2005).
Furthermore, a small fraction of mice heterozygous for a Bmp4
loss-of-function allele display CAKUT-like phenotypes and/or multicystic
dysplastic kidneys (Miyazaki et al.,
2000). This phenotype is likely caused by defects in ureteric
stalk elongation, but treatment of metanephric kidney primordia with
recombinant BMP4 also interferes with epithelial branching morphogenesis in
culture, which indicated that BMP4 activity may require dynamic modulation
(Miyazaki et al., 2000;
Raatikainen-Ahokas et al.,
2000). Gremlin 1 (GREM1) is a member of the CAN domain family of
extra-cellular BMP antagonists that binds BMP2 and BMP4 with highest affinity
in vitro (Hsu et al., 1998).
However, the ligands antagonised by GREM1 in vivo during vertebrate
embryogenesis remained elusive to date. We recently showed that Grem1
is expressed by the mesoand metanephric mesenchyme and that metanephric kidney
organogenesis is disrupted in Grem1-deficient mouse embryos,
resulting in bilateral renal agenesis and neo-natal lethality
(Michos et al., 2004). In
particular, metanephric kidney development is blocked at the stage of
initiating of ureteric bud outgrowth. However, Gdnf expression is
initially normal in the metanephric mesenchyme and the epithelium continues to
express Ret. The block in initiating ureteric bud outgrowth causes
progressive loss of Gdnf expression and elimination of the
metanephric mesenchyme by apoptosis.
In the present study, we established that culturing early Grem1-deficient kidney primordia in medium supplemented with recombinant GREM1 restores ureteric bud outgrowth and supernumerary epithelial buds are induced. Multiple epithelial buds invade the metanephric mesenchyme and initiate branching morphogenesis. Wnt11 expression in the epithelial tips and Gdnf expression in the metanephric mesenchyme around the epithelium is restored, which is indicative of establishment of e-m feedback signalling in GREM1-treated mutant kidney primordia. We identify excessive BMP activity in the metanephric mesenchyme around the ureteric bud as the primary signalling defect in Grem1-deficient mouse embryos. As a consequence, the invasion of the mutant metanephric mesenchyme by the ureteric bud and concurrent establishment of the autoregulatory GDNF/WNT11 feedback signalling loop are disrupted. Therefore, it was important to identify the BMP ligand(s) antagonised by GREM1 in the mesenchyme. BMP4 was identified as a relevant BMP signal by its partially overlapping expression with Grem1 during initiation of ureteric bud outgrowth and by genetic complementation: inactivation of only one copy of the Bmp4 gene in Grem1-deficient mouse embryos completely restores metanephric kidney organogenesis and functions. We conclude that GREM1-mediated reduction of BMP4 activity in the mesenchyme around the nascent ureteric bud is essential to (1) initiate ureteric bud outgrowth and invasion of the metanephric mesenchyme, and (2) enable autoregulatory e-m feedback signalling that regulates the dynamics of epithelial branching morphogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
Bmp4 gene was inactivated in the germline by inter-crossing the
floxed Bmp4loxP-lacZ allele
(Kulessa and Hogan, 2002) with
the Cre deleter mouse strain
(Schwenk et al., 1995). The
resulting Bmp4
-lacZ allele was
used to generate compound mutant mice for analysis in a mixed 129/C57BL6
background. Compound mutants carrying the Hoxb7-GFP transgene to mark
the ureteric epithelium (Srinivas et al.,
1999) were used for analysis. PCR genotyping of mice and embryos
was done as described previously (Kulessa
and Hogan, 2002; Michos et
al., 2004; Srinivas et al.,
1999). All animal experiments were performed in accordance with
Swiss law and were approved by the regional veterinary authorities.
Molecular and morphological analysis of embryos and organs
Embryos were accurately staged by determining their somite numbers.
Whole-mount and section RNA in situ hybridisation and detection of
ß-galactosidase activity were performed as described previously
(Zuniga et al., 2004;
Grieshammer, 2004). For
histological analysis, 7-10 µm sections were prepared from
paraffin-embedded samples fixed in 4% paraformaldehyde. Dewaxed sections were
either stained with haematoxylin and Eosin or Periodic Acid Schiff (PAS) to
reveal the brush border (microvilli) of the distal and proximal tubules.
Phosphorylated SMAD (pSMAD1/5/8; SMAD8 is also known as SMAD9 - Mouse Genome
Informatics) proteins were detected on 10 µm sagittal sections of stretched
and paraffin-embedded embryos. Dewaxed sections were incubated with pSMAD1/5/8
antibodies (1:250, Cell Signaling) following the manufacturer's instructions,
with the exception that endogenous peroxidases were inactivated with 3%
H2O2 in PBS for 30 minutes. Signal amplification was
performed using the appropriate ABC kit (Vector Laboratories) and the
immunocomplexes were detected with DAB staining. Sections were counterstained
with Hoechst 33258 (5 µg/ml) to reveal the nuclei containing no pSMAD
antigen.
Kidney primordia cultures
Kidney primordia were isolated from somite-staged wild-type and mutant
embryos and cultured in DMEM supplemented with 10% foetal bovine serum and
0.5% penicillin-streptomycin (Invitrogen) on Nucleopore filters (0.1 µm
pore size, Corning) as previously described
(Lin et al., 2001) with the
following modifications. Experiments were performed using E10.75-11.25 (38-44
somites) embryos instead of the classic T-shape stages (E11.5, 48-50 somites).
It is important to realise that it is not possible to efficiently rescue
kidney primordia from Grem1-deficient embryos older than E11.25. A
piece of Gelfoam (Pharmacia) was soaked in medium and the Nucleopore filter
with the kidney primordia was placed on top in a 6-well plate and incubated in
a humidified atmosphere at 37°C with 5% CO2. Recombinant GREM1
(R&D Systems) was added to the culture medium at 2-5 µg/ml and changed
every 48 hours. noggin-conditioned medium was produced using the B3-CHO cell
line (Smith et al., 1993) and
used at a dilution of 1:4. Recombinant GDNF (R&D Systems) was used at 100
ng/ml final concentration. After culture, the kidney primordia were fixed in
4% paraformaldehyde (PFA) and processed for molecular analysis. The
metanephric mesenchyme was separated from the ureteric bud as described
previously (Lin et al., 2001)
and isolated mesenchyme was cultured in the same way as metanephric primordia.
LiCl (15 µM) treatment was for 72 hours to allow for sufficient early
tubulogenesis (Davies et al.,
1995; Oxburgh and Robertson,
2002).
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); Grem1 (Zuniga
et al., 2004)] located in two different exons to avoid potential
amplification of genomic DNA (PCR conditions are available upon request). The
PCR products were separated on 1.8% agarose gels and visualised by ethidium
bromide staining. Digital images were acquired to determine the relative
expression levels using the LI-COR Odyssey Imaging Software (version 2.0).
Three completely independent series of experiments were performed to assess
Gdnf expression. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
To gain insight into the functions of GREM1 during the inductive phase of
metanephric kidney organogenesis, wild-type and Grem1-deficient
metanephric kidney primordia (E11.0-11.25, 40-44 somites; expressing the
Hoxb7-GFP transgene) (Srinivas et
al., 1999) were cultured for up to 96 hours in the presence or
absence of soluble recombinant GREM1 protein. In contrast to wild-type
controls (Fig. 1A), the
development of Grem1-deficient kidney primordia arrests at the
ureteric bud stage (Fig. 1B,
n=13/16). Supplementation of the culture medium with GREM1 (5
µg/ml) restores ureteric epithelial outgrowth and branching
(Fig. 1C,D; n=12/16)
and induces several supernumerary epithelial buds (on average two to four;
indicated by red asterisks in Fig.
1C,D). Most supernumerary buds appear within 48 hours and by 96
hours the ureteric and ectopic epithelial buds have undergone between two and
four branching events (Fig.
1C,D). Epithelial tracings of the GREM1-treated Grem1
mutants (right-most panels in Fig.
1C,D) reveals that branching of the ureteric bud is delayed by at
least 24 hours in comparison to the wild-type (right-most panels in
Fig. 1A,E). Ureteric epithelial
outgrowth and branching are also restored and ectopic epithelial buds induced
when mutant kidneys are cultured in the presence of less recombinant GREM1 (2
µg/ml; data not shown) or the unrelated BMP antagonist noggin (see Fig. S1
in the supplementary material). In wild type, recombinant GREM1 also induces
ectopic epithelial buds (red asterisks) and ectopic branches of the ureteric
epithelium (blue asterisks, Fig.
1E). In general, treatment with soluble BMP antagonists induces
more ectopic epithelial buds in the rostral part of the mutant Wolffian duct
(red asterisks, Fig. 1C,D, and
see Fig. S1 in the supplementary material) than in wild-type kidney primordia
(red asterisks, Fig. 1E).
Therefore, the potential for ectopic epithelial bud formation appears
increased in the mutant, possibly due to the early developmental arrest of
Grem1-deficient kidney primordia.
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). By contrast, the expression of Wnt11 in the mutant
ureteric bud epithelium is lower than in the wild-type bud from the beginning
and is completely lost by E11.5 (see Fig. S2 in the supplementary material).
Therefore, Wnt11 and Gdnf provide good markers to monitor of
e-m feedback signalling in Grem1-deficient kidney primordia
(Fig. 2). Mutant kidney
rudiments (isolated from E10.75-11.0 mouse embryos; 38-40 somites) were
cultured for 48 hours to avoid the onset of massive apoptosis
(Fig. 2B,E and data not shown)
and to allow treatment with recombinant GREM1 to restore ureteric bud
outgrowth (white asterisks, Fig.
2C,F) and induce formation of ectopic buds (red asterisks,
Fig. 2C,F). Indeed, GREM1
treatment restores Gdnf expression in the distal mesenchyme around
the invading ureteric bud (white asterisk in
Fig. 2C) and expression is also
activated around the ectopic buds (red asterisks,
Fig. 2C). Semi-quantitative
RT-PCR analysis reveals that GREM1 treatment increases Gdnf
transcript levels by at least twofold (see Fig. S3 in the supplementary
material; 1.9±0.4, mean±s.e.m., n=3). In the ureteric
epithelium, the expression of Wnt11 is no longer detected in
Grem1-deficient kidney primordia after 48 hours
(Fig. 2E) in contrast to the
wild-type primordia (Fig. 2D).
Strikingly, GREM1 treatment of the mutant kidney primordia completely restores
Wnt11 expression in the epithelial tips of the invading ureteric
(white asterisk, Fig. 2F) and
all ectopic (red asterisks, Fig.
2F) buds. This molecular analysis establishes that recombinant
GREM1 is able to restore epithelial WNT11 signalling and thereby
autoregulatory e-m feedback signalling and metanephric branching
morphogenesis.
The responsiveness of the epithelium and mesenchyme in
Grem1-deficient kidney primordia was further studied as follows: (1)
By forced activation of canonical Wnt signal transduction in culture,
nephrogenesis is activated (see Fig. S4 in the supplementary material),
thereby establishing that the mutant metanephric mesenchyme remains responsive
to inductive signals; (2) By culturing Grem1-deficient kidney
primordia in an excess of recombinant GDNF (100 ng/ml;
Fig. 3) massive epithelial
overgrowth and branching are induced along the entire Wolffian duct similar to
what occurs in wild type (Fig.
3A,B) (see also Shakya et al.,
2005). These results indicate that the block to initiate ureteric
bud outgrowth and invasion of the mesenchyme is not caused by defective
epithelial signal reception, but rather by defects in signalling, i.e. the
failure to upregulate Gdnf expression in the mesenchyme and/or
Wnt11 in the ureteric epithelium.
The Grem1 deficiency causes aberrant nuclear accumulation of pSMAD proteins in the metanephric mesenchyme around the ureteric bud
The results so far reveal the importance of identifying the primarily
affected kidney compartment and relevant BMP ligand(s). With respect to the
latter, Bmp4 (in contrast to Bmp2 and Bmp7)
(Michos et al., 2004) is
expressed by the mesenchyme along the entire Wolffian duct, including the
region where the ureteric bud is forming
(Fig. 4C, compare with
Fig. 4A, see also
Fig. 4G). Therefore, the
Wolffian duct and nascent ureteric bud are `embedded' in Bmp4-
expressing mesenchyme. By contrast, the nascent metanephric mesenchyme is more
or less devoid of Bmp4 expression during ureteric bud outgrowth and
first branching (Fig. 4B,D).
Subsequently, Bmp4 expression is (re-) activated in metanephric
mesenchyme enveloping the ureteric stalk, consistent with its proposed
functions during stalk elongation
(Miyazaki et al., 2000) (and
data not shown). Just prior to initiation of ureteric bud outgrowth, the
highest levels of Grem1 transcripts are detected in the mesenchyme
around the caudal Wolffian duct and nascent ureteric bud
(Fig. 4E,H). Activation of
Grem1 expression does not seem to require GDNF signalling, as
Grem1 remains expressed in the metanephric kidney primordia of
Gdnf mutant mouse embryos (see Fig. S3 in the supplementary
material). After the first epithelial branching event, Grem1
expression is highest in the metanephric mesenchyme surrounding the ureteric
epithelial tips (Fig. 4F). The
transcriptional upregulation of Grem1 just prior to the onset of
ureteric bud outgrowth (Fig.
4E,H) would likely inhibit BMP4 locally and generate a region of
low mesenchymal BMP activity. Such regional lowering of mesenchymal BMP
activity likely relieves repression of ureteric bud outgrowth and enables its
invasion into the metanephric mesenchyme in response to GDNF signalling.
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) was analysed in wild-type and Grem1-deficient mouse
embryos (Fig. 5). During
ureteric bud formation in the wild type, the Wolffian duct and ureteric bud
are largely devoid of nuclear pSMAD proteins (brown stained nuclei,
Fig. 5A) as revealed by
abundant Hoechst fluorescence (light blue nuclei,
Fig. 5A). Whereas the pSMAD
antigen is abundant in the ventral mesenchyme, only a few pSMAD-positive
nuclei are apparent in the metanephric mesenchyme (demarcated region indicated
by `mm' in Fig. 5A). By
contrast, a significant fraction of the cells in the corresponding region of
Grem1-deficient embryos contain pSMAD proteins in their nuclei and
the metanephric mesenchyme appears overall less dense
(Fig. 5B). These results
indicate that BMP signal transduction is lowered in the wild-type metanephric
mesenchyme during initiation of ureteric bud outgrowth
(Fig. 5A), whereas it remains
excessively high in the mutant mesenchyme
(Fig. 5B). In the wild type,
the nuclear pSMAD levels remain low in both epithelium and condensing
metanephric mesenchyme during invasion and first branching
(Fig. 5C,E), while they
increase in stromal mesenchyme (Fig.
5C,E). This increase is consistent with a role of BMP4 in ureteric
stalk elongation (Miyazaki et al.,
2000). In Grem1 mutant kidneys, the aberrant pSMAD levels
persist throughout the mutant metanephric mesenchyme, while the arrested
ureteric bud epithelium remains largely devoid of nuclear pSMAD proteins
(Fig. 5D,F). The low pSMAD
levels in the wild-type metanephric mesenchyme
(Fig. 5A,C) overlap well with
the area normally expressing Grem1
(Fig. 4E,F). In summary, the
aberrant and persistently high BMP(4) activity in the mutant metanephric
mesenchyme reveals a primary defect in Grem1-deficient kidney
primordia (Fig. 5B,D,F) and
sharply contrasts with the dynamic changes of BMP activity in the wild type
(Fig. 5A,C,E).
Genetic reduction of BMP4 activity in Grem1-deficient mouse embryos rescues metanephric kidney organogenesis and postnatal kidney functions
To test the hypothesis that excess mesenchymal BMP4 activity blocks the
initiation of ureteric bud outgrowth, compound mutant embryos and mice
carrying both Grem1 and Bmp4 loss-of-function mutations were
generated. The early lethality of Bmp4-deficient mouse embryos
(Winnier et al., 1995) is not
rescued by additional inactivation of Grem1, which precludes analysis
of double homozygous embryos. However, inactivation of one copy of the
Bmp4 gene in Grem1-deficient
(G1/; B4/+) mouse
embryos restores ureteric bud morphology
(Fig. 6C, compare with
Fig. 6A,B). In
G1/; B4/+ mutant
embryos (Fig. 6C,F,I,L),
ureteric bud outgrowth and branching are initiated with kinetics similar to
that in the wild type (Fig.
6A,D,G,J). Consistent with this rescue of epithelial outgrowth and
branching, the expression of Gdnf in the mesenchyme
(Fig. 6F,L, compare with
Fig. 6E,K) and Wnt11
in the ureteric bud epithelium are restored (see Fig. S2 in the supplementary
material and data not shown), which is indicative of intact autoregulatory e-m
feedback signalling during branching and normal progression of metanephric
branching morphogenesis.
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/; B4/+ embryos
by E14.5 (Fig. 7C, compare with
Fig. 7A). By contrast, in
Grem1-deficient embryos at this stage both kidneys have been
eliminated by apoptosis (as revealed by the complete lack of Pax2
expression; Fig. 7B). All
G1/; B4/+ mice
are born with two fully developed and functional kidneys
(Fig. 7G, compare with
Fig. 7D; n=20),
whereas complete bilateral renal agenesis is observed in about 90% of all
Grem1-deficient litter mates (n=22/25; the other three
manifested unilateral renal aplasia). Histological analysis of
G1/; B4/+ mice at
1 and 8 months old reveals that their kidneys are morphologically
indistinguishable from wild-type kidneys
(Fig. 7E-I). No signs of
congenital malformations such as CAKUT, glomerulosclerosis or polycystic
kidney disease are observed (Fig.
7G,H,I). Taken together, these results show that the genetic
reduction of BMP4 activity in Grem1-deficient mouse embryos
completely restores metanephric kidney organogenesis and functions.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Formation of the ureteric bud, initiation of its outgrowth and branching
appear as distinct processes (Fig.
8). In the metanephros, a single ureteric bud forms in the caudal
part of each of the bilateral Wolffian ducts at the level of the mid-hind limb
bud. GDNF is essential for ureteric bud formation, as ureteric buds fail to
form in most Gdnf-deficient mouse embryos
(Pichel et al., 1996). As
Grem1 remains expressed in Gdnf mutant metanephric kidney
rudiments, the activation of its expression does not depend on GDNF signalling
and seems not to require the presence of an intact ureteric bud.
Interestingly, recombinant GREM1 is itself able to induce supernumerary
epithelial buds in Grem1-deficient kidney primordia. These results
indicate that in spite of the early developmental arrest, the competence of
the rostral Wolffian duct to form supernumerary epithelial buds remains intact
in Grem1-deficient embryos. In wild-type embryos, the signalling
interactions mediated by SLIT2/ROBO2 and FOXC1 restrict Gdnf
expression to the caudal Wolffian duct (see Introduction and references
therein). We provide evidence that BMP4 signalling by the mesenchyme
enveloping the Wolffian duct is part of a safeguard mechanism that inhibits
formation of supernumerary epithelial buds
(Fig. 8A). Local upregulation
of Grem1 expression reduces pSMAD-mediated BMP signal transduction in
the mesenchyme and relieves this repression around the ureteric bud, which
enables ureteric bud outgrowth and invasion of the metanephric mesenchyme
(Fig. 8B). Taken together, the
successful initiation of ureteric bud outgrowth likely requires both
antagonism of BMP4 by GREM1 in mesenchyme and signalling by GDNF from the
metanephric mesenchyme to RET in the ureteric epithelium
(Fig. 8A,B).
Subsequently, autoregulatory feedback signalling between GDNF in the
mesenchyme and WNT11 in the epithelial tips is established to regulate
branching morphogenesis (Fig.
8C) (Majumdar et al.,
2003). As GREM1 is required for upregulation of Wnt11 in
the ureteric epithelium and Gdnf expression in the mesenchyme, it
appears to be crucial for establishment of e-m feedback signalling
(Fig. 8C). As BMP signal
transduction is increased in the metanephric mesenchyme of
Grem1-deficient embryos, one might expect a direct effect of GREM1
and/or BMP4 on mesenchymal Gdnf expression. However, treatment of
isolated metanephric mesenchyme with either GREM1 or BMP4 did not alter
Gdnf expression significantly within 18-24 hours (O.M. and R.Z.,
unpublished). Therefore, we favour an alternative explanation, by which the
primary defect (elevated BMP4 activity) in the Grem1-deficient
metanephric mesenchyme blocks initiation of ureteric epithelial outgrowth and
signalling as evidenced by the loss of Wnt11 expression.
GREM1-mediated reduction of BMP activity in the mesenchyme may act via
epithelial signalling to propagate mesenchymal Gdnf expression,
analogous to the requirement of GREM1 for Shh expression in the limb
bud mesenchyme (see below). Further (genetic) studies are required to
understand how excess BMP signal transduction in the mesenchyme blocks
initiation of ureteric bud outgrowth. In agreement with our studies, others
have already shown that (1) addition of recombinant BMPs to metanephric kidney
primordia in culture partially inhibits epithelial branching morphogenesis
(Bush et al., 2004;
Piscione et al., 1997), and
(2) overexpression of the BMP receptor type 1A (ALK3; also known as
BMPR1A-Mouse Genome Informatics) in the ureteric epithelium causes renal
aplasia or dysplasia in a fraction of mice
(Hu et al., 2003).
Last but not least, the present study reveals striking mechanistic
similarities in the way GREM1-mediated e-m feedback signalling controls limb
and kidney organogenesis. During limb bud development, GREM1-mediated BMP4
antagonism is key to establishing and propagating the feedback signalling loop
between SHH (expressed by the posterior mesenchyme) and FGF (in the apical
ectodermal ridge, AER), which enables progression of limb bud morphogenesis
(Panman et al., 2006;
Michos et al., 2004;
Zuniga et al., 1999). Our
previous studies showed that GREM1 is not required to initiate SHH signalling
by the limb bud organiser in the posterior mesenchyme, but is essential to
initiate the dynamic phase of SHH/FGF e-m feedback signalling. In particular,
GREM1-mediated SHH/FGF feedback signalling regulates the temporally and
spatially coordinated propagation of both signalling centres during limb bud
outgrowth and patterning (Panman et al.,
2006). Similarly, GREM1 is not required to activate GDNF
signalling in the metanephric mesenchyme and for formation of the ureteric
bud, but for initiation of epithelial outgrowth and establishment of
autoregulatory GDNF/WNT11 e-m feedback signalling. In both the limb and kidney
primordia, GREM1 acts in the mesenchyme to reduce BMP4 signal transduction and
thereby relieve the inhibitory effect on the ureteric and AER epithelium (this
study and J.-D. Bénazet and R. Z., unpublished). As a consequence, the
expression of Wnt11 in the ureteric bud and Fgfs in AER are
upregulated and this epithelial signalling in turn propagates GDNF in the
metanephric, and SHH in the limb bud, mesenchyme, respectively.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2397/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Genetics and Development, Columbia
University, HHSC-1416, 701 West 168 Street, New York, NY 10032, USA
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