Lrp4 Regulates Initiation of Ureteric Budding and Is
Crucial for Kidney Formation – A Mouse Model for
Courtney M. Karner1., Martin F. Dietrich2., Eric B. Johnson2., Natalie Kappesser2, Christian Tennert2,
Ferda Percin3, Bernd Wollnik4, Thomas J. Carroll1, Joachim Herz2*
1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 2Department of Molecular Genetics,
University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 3Department of Medical Genetics, Faculty of Medicine, Gazi University, Ankara,
Turkey, 4Center for Molecular Medicine Cologne (CMMC) and Institute of Human Genetics, University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses
in Aging-Associated Diseases (CECAD), Cologne, Germany
Background: Development of the kidney is initiated when the ureteric bud (UB) branches from the Wolffian duct and
invades the overlying metanephric mesenchyme (MM) triggering the mesenchymal/epithelial interactions that are the basis
of organ formation. Multiple signaling pathways must be integrated to ensure proper timing and location of the ureteric
Methods and Principal Findings: We have used gene targeting to create an Lrp4 null mouse line. The mutation results in
early embryonic lethality with a subpenetrant phenotype of kidney agenesis. Ureteric budding is delayed with a failure to
stimulate the metanephric mesenchyme in a timely manner, resulting in failure of cellular differentiation and resulting
absence of kidney formation in the mouse as well as comparable malformations in humans with Cenani-Lenz syndrome.
Conclusion: Lrp4 is a multi-functional receptor implicated in the regulation of several molecular pathways, including Wnt
and Bmp signaling. Lrp42/2mice show a delay in ureteric bud formation that results in unilateral or bilateral kidney
agenesis. These data indicate that Lrp4 is a critical regulator of UB branching and lack of Lrp4 results in congenital kidney
malformations in humans and mice.
Citation: Karner CM, Dietrich MF, Johnson EB, Kappesser N, Tennert C, et al. (2010) Lrp4 Regulates Initiation of Ureteric Budding and Is Crucial for Kidney
Formation – A Mouse Model for Cenani-Lenz Syndrome. PLoS ONE 5(4): e10418. doi:10.1371/journal.pone.0010418
Editor: Rory Edward Morty, University of Giessen Lung Center, Germany
Received January 28, 2010; Accepted April 9, 2010; Published April 29, 2010
Copyright: ? 2010 Karner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: M.F.D. is supported by a fellowship from the Boehringer Ingelheim Foundation. B.W. was supported by the German Federal Ministry of Education and
Research (BMBF) by grant number 01GM0880 (SKELNET) and 01GM0801 (E-RARE network CRANIRARE). J.H. is supported by grants from the National Institutes of
Health, the American Health Assistance Foundation, and the Perot Family Foundation. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
The definitive kidney forms as a result of inductive interactions
between the metanephric mesenchyme and the UB . In the
mouse, signals from the metanephric mesenchyme stimulate the
ureteric bud to branch from the Wolffian duct around embryonic
stage E10.5 . The UB subsequently invades the overlying
metanephric mesenchyme and produces signals that are necessary
for survival, proliferation and differentiation of the mesenchyme
. The timing and location of ureteric budding are critical factors
in kidney organogenesis. Genetic and surgical manipulations have
revealed that the mesenchyme is only competent to respond to
signals from the bud for a narrow time window . Failure of the
bud to reach the mesenchyme in this narrow window results in
apoptosis of the mesenchyme and subsequent kidney agenesis
[5,6]. Defects in secondary branching of the ureteric bud can
result in a range of phenotypes ranging from congenital anomalies
like hypoplastic kidneys to cystic dysplasia .
Defects in kidney formation constitute some of the most
common birth defects in humans . Multiple signaling pathways
have been implicated in UB branching. The GDNF/Ret, FGF
and Wnt signaling pathways are necessary for normal branching
[4,9,10] while the BMP pathway appears to act as a branching
inhibitor . As would be expected, tight regulation of these
pathways is essential to insure the proper timing and location of
branching. Although we have gained a great deal of information
on the molecular mechanism regulating ureteric bud branching in
mice, there has been surprisingly little correlation between these
major pathways and congenital defects in man .
Lrp4 is a member of the low-density lipoprotein (LDL) gene
family . Mutations in this membrane receptor have been
implicated in neuromuscular junction , limb and tooth
development where it appears to integrate signaling from multiple
pathways including Wnts and Bmps [15,16,17]. Here, we describe
an additional role for Lrp4 in the formation of the UB. Loss of
Lrp4 results in a delay in UB formation and a subpenetrant kidney
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agenesis phenotype. We also identified mutations in Lrp4 in
humans with congenital kidney defects . These studies
establish Lrp4 as a critical regulator of ureteric budding in both
mice and humans.
Materials and Methods
HoxB7-Cre and Catnbexon3floxmouse lines have previously been
described [19,20]. The Lrp4 knockout (KO) mouse was generated
by replacing the first exon with a neomycin resistance cassette
using techniques described previously . The long arm of
homology upstream of the first exon of Lrp4 was generated by
PCR using primers MEJ23 (59-GCGGCCGCCAGGTCAT-
short arm of homology downstream of the first exon of Lrp4
was generated by PCR amplification using the primers MEJ33 (59-
39) and MEJ35(59-CTCGAGGGTTACAGACTCTGCAA-
CTGCTCTACCTCATTG-39). The long arm and short arm of
homology were cloned into pJB1 using the NotI and XhoI
restriction sites, respectively.
Mice were maintained on a mixed 129/C57 background. All
animal work was conducted according to the relevant national and
international guidelines and in accordance with the recommen-
dations of the Weatherall report, ‘‘The use of non-human primates
in research’’ (no primates were used in this study). Animal
experiments conducted in Dallas were also reviewed and approved
by the Institutional Committee on Animal Use and Care (IACUC)
at UT Southwestern Medical Center.
KO Mice were genotyped by PCR as follows: MEJ358 (59-
C-39) were used to selectively amplify the wild-type allele and
-39) and KOT12 were used to amplify the knockout allele. The
HoxB7Cre allele was amplified using the primers 59-CCAT-
GAGTGAACGAACCTGG-39 and TGATGAGGTTCGCAA-
GAACC to give a 400 base pair band using the conditions
previously described. The b-catenin exon3flox allele was amplified
using the primers: 59-AACTGGCTTTTGGTGTCGGG-39 and
59-TCGGTGGCTTGCTGATTATTTC-39. Using a 55uC ex-
tension temperature, the wild type allele yields a 291 base pair
band while the exon 3 floxed allele yields a 400 base pair band.
In situ hybridization
Whole-mount in situ hybridization was performed as previously
described using the following antisense probes: cRet, Wnt11,
Pax2, Wnt9b, Lrp4 and GDNF [15,19]. Briefly, embryos were
harvested and fixed in 4% paraformaldehyde in PBS at 4uC
overnight. Embryos were treated with 10 mg/ml proteinase K in
PBST for 20 minutes at room temperature and hybridized
overnight at 72uC with digoxigenin-UTP labeled probes. Embryos
were then incubated overnight at 4uC with alkaline phosphatase-
coupled anti-digoxigenin antibody (Roche Applied Science). Color
reaction was developed using BM Purple (Roche).
Kidneys from P0 pups were emersion fixed with 10% formalin
and embedded in paraffin. The kidneys were then sectioned and
stained with H&E using standard techniques.
Whole mount antibody staining
Embryonic day 10.5 embryos were dissected in PBS and staged
according to somite number. Embryos at the 38 somite stage
were fixed overnight in 4% PBS (Electron microscopy services)
overnight at 4uC. After fixation embryos were dehydrated and
rehydrated through a graded ethanol series. Embryos were then
washed four times for 30 minutes at room temperature with
heavy agitation in PBS +0.1% Triton-X (PBStx). Embryos were
blocked for at least 3 hours at room temperature in 10% FBS/
PBStx. Embryos were incubated with antibodies to E-Cadherin
(Rat 1:400 Zymed) and Pax2 (Rabbit 1:400 Covance) overnight
at 4 degrees Celsius, then washed six times 30 minutes each wash
at room temperature in PBStx. Embryos were incubated with
fluorescently coupled secondary antibodies (Molecular probes)
overnight at 4uC followed by extensive washing in PBStx.
Wolffian ducts were then dissected away from the embryo and
imaged on a Zeiss NeoLumar stereoscope using an Olympus
Lrp4 is required for kidney formation
We have generated mice that harbor a null allele of Lrp4 by
replacing exon1 with a neomycin stop cassette (Lrp42/2). In the
examination of post-partum Lrp42/2mice (n=156) we found
51 percent bilateral and 22 percent unilateral kidney agenesis
(Fig. 1b, d, e). This distribution was gender-independent and
involved only structures derived from the UB and metanephric
mesenchyme (MM) (Fig. 1a–d). The small number of kidneys
that did form in Lrp4 knockouts were indistinguishable from
wild-type at both the histological and molecular level (Fig. 1f
and g). Functional analysis was not possible due to the
immediate post-partum lethality caused by neuromuscular
junction defects .
Lrp4 is widely expressed in the kidney during
To better understand its contribution to kidney formation, we
investigated the expressionof
Beginning at the initiation of kidney development, embryonic
day (E) 10.5, Lrp4 mRNA is visible in the mesonephric tubules
and the Wolffian duct adjacent to the MM (Fig. 2a). At E11.5,
Lrp4 is expressed throughout the ureteric epithelium and the
adjacent pre-tubular aggregates (Fig. 2b). Lrp4 continues to be
expressed in the ureteric bud derived epithelia and the pre-
tubular aggregates/renal vesicles throughout the embryonic
period (Fig. 2a–d).
Pax2 signaling remains intact in the absence of Lrp4
To gain insight into the nature of the mutant defect, we
evaluated the expression of a series of genes necessary for kidney
development. Pax2 is a critical regulator of kidney branching that
is normally expressed in the Wolffian duct, the ureteric bud/
collecting ducts and the metanephric mesenchyme throughout the
developmental period [21,22,23]. Pax2 is expressed normally in
the Wolffian duct and metanephric mesenchyme in both wild type
and Lrp4 mutants through E11.5 (Fig. 3a–d), although at E11.5
the mutant ureteric bud has not contacted the mesenchyme and
has not formed a T-shape (Fig. 3d). Failure of the ureteric bud to
invade the metanephric mesenchyme leads to a loss of the
metanephric mesenchyme and kidney agenesis . In support of
this hypothesis the mesenchymal expression of Pax2 is lost by
E12.5 in Lrp42/2animals (Fig. 3f).
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The GDNF/Ret/Wnt11 signaling network is unaffected by
Kidney development begins at E10.5 when the ureteric bud
branches from the Wolffian duct in response to GDNF secreted
from the mesenchyme. The apparent failure of the ureteric bud to
reach the mesenchyme in Lrp4 mutant kidneys is similar to what
has been observed in mice with defects in GDNF/Ret signaling
. GDNF is a ligand for c-Ret and a co-receptor, GFRa1 .
Mutations in any of these three genes results in partially penetrant
kidney agenesis. To examine potential defects in the GDNF/Ret
pathway, we first examined the expression of Ret and GDNF
mRNA. At E10.5, c-Ret is expressed in the ureteric bud at
equivalent levels in the Lrp4 knockout mice compared to their wild
type counterparts (Fig. 4a and b). At E11.5 the ureteric bud has
invaded the mesenchyme, bifurcated and upregulated c-Ret at the
ureteric tips while no bifurcation or upregulation of c-Ret occurs
in Lrp4 mutants (Fig. 4c and d). At E12.5, c-Ret levels are greatly
reduced in the knockout compared to wild type control (Fig. 4f). As
expected, GDNF is expressed in the metanephric mesenchyme at
normal levels at E10.5 and E11.5 (Fig. 4g–j). As was seen with
Pax2, by E12.5 mesenchymal expression of GDNF is completely
lost, consistent with the hypothesis that the ureteric bud has not
invaded the metanephric mesenchyme (Fig. 4k and l). We next
wanted to test whether Ret/GDNF signaling is intact in Lrp4
mutants. We examined the expression of Wnt11, a GDNF-
inducible downstream target of c-Ret . Similar to c-Ret,
Wnt11 expression is upregulated in the tips of the bud at E10.5
and 11.5. Importantly, Wnt11 is still expressed in Lrp42/2
ureteric buds at E11.5 that have failed to bifurcate, indicating that
Ret/GDNF signaling is active despite the apparent failure of
mesenchymal invasion. By E12.5 Wnt11 expression is absent,
presumably due to the loss of mesenchymal GDNF (Fig. 4a–f).
As the Lrp4 mutant phenotype does not appear to be the result
of defects in Ret/GDNF signaling, we examined the activity of
other pathways involved in ureteric bud branching. Lrp4 has been
implicated in the activity of both Bmp [26,27] and Wnt [28,29]
signaling and both of these pathways play roles in normal
branching morphogenesis. To test for defects in Bmp signaling, we
Figure 1. Unilateral and bilateral kidney agenesis in LRP4 knockout mice. Kidney agenesis in the Lrp4 knockout (b,d,e). Bilateral (b,d) or
unilateral (e) kidney agenesis with rudimentary ureters (red arrows). The lower urinary and genital systems of males and females remain intact.
Histological analysis (Hematoxylin-Eosin stain) does not reveal morphological defects in the kidneys that form in Lrp4 knockout animals (g) compared
to the wild-type kidneys (f).
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investigated the expression of phosphorylated Smads1, 4 and 8.
We were unable to detect differences in either the level or location
of p-Smad staining in either the mesenchyme or ureteric buds of
Lrp4 mutants at either E10.5 or 11.5 (data not shown). To assay
Wnt signaling, we examined the expression of Axin2 mRNA in the
Wolffian duct and ureteric bud. Similar to the situation with the p-
Smads, we were unable to detect significant differences in
transcript levels (data not shown).
Ureteric budding is delayed in Lrp4 null mice
The absence of metanephric mesenchyme at E12.5 is indicative
of a failure of the UB to reach these cells and provide survival
signals. The expression of Pax2 (Fig. 3d) indicated that the UB bud
is delayed in reaching the mesenchyme, which could be due either
to defects in growth of the ureteric bud or a delay in formation of
the bud. The complete lack of a phenotype in some mutants
seemed more in line with a delay in bud invasion. To investigate
the possibility of a budding delay as a possible explanation of our
phenotype, we examined ureteric bud formation at E10.5. Stage
and somite matched embryos were stained for the epithelial
markers Pax2 and E-cadherin to assess UB formation. E-cadherin
is a marker for the epithelial structures of the ureteric bud; Pax2 is
expressed in both epithelium and mesenchyme alike. We
proceeded with double-staining for a clear orientation within the
slide. Interestingly, although we noticed at least a partial ureter in
all newborn Lrp4 mutants, we found that the UB had formed in
only 12.5% (1/8) of 38 somite stage Lrp4 mutants (compared to
100% of cases for wild type controls) (Fig. 5a and b). These data
indicate that ureteric bud formation is delayed in mutants, and
that the failure to invade the mesenchyme in time to support
normal growth/survival is the cause for the frequent uni- or
bilateral kidney agenesis.
Wnt overexpression in the ureteric bud leads to kidney
Lrp4 is a negative regulator of the Wnt signaling pathway. This
lead us to hypothesize that overactive Wnt signaling in mutants
could be responsible for the delay in ureteric bud formation. We
therefore tested whether expression of a constitutively active b-
catenin transgene would result in a comparable phenotype to the
absence of Lrp4. Expression of this transgene under the control of
a HoxB7Cre promoter, which is restricted to the ureteric bud
epithelium indeed resulted in a comparable kidney agenesis
phenotype (Fig. 6a–c). The formation of the Wolffian duct and
distal ureters as well as bladder and adrenal glands remained
unaffected. The similarity of these two distinct animal models
suggests a role for deregulated Wnt signaling in the generation of
the Lrp4 knockout phenotype.
Lrp4 binds Gremlin1, a positive regulator of ureteric
Lrp4 has been established as a regulator of both the Wnt and
Bmp signaling pathways. This involves, at least in part, the binding
of signal modulating ligands to the extracellular domain. We tested
Gremlin1, a facilitator of ureteric budding, as a possible candidate.
Figure 2. Expression of Lrp4 in the developing kidney. At E10.5
Lrp4 is expressed throughout the Wolffian duct and the ureteric bud.
(a). At E11.5, Lrp4 is expressed in the ureteric bud and the pre-tubular
aggregates (b). At E12.5 and E 14.5, Lrp4 expression is maintained in the
ureteric bud and the renal vesicles (c and d, respectively). The Wolffian
duct and ureteric bud are outlined by dotted lines; the arrow points to
the early ureteric bud in (a) or the renal vesicles in (c), respectively.
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Previously, Gremlin1 has been reported to antagonize Bmp4
signaling and its deletion in mice results in a renal phenotype with
skeletal involvement similar to the Lrp4 knockout. In co-
immunoprecipitation experiments, Gremlin1 binds to Lrp4
(Fig. 7). Although we failed to detect a direct difference in Bmp
pathway activation at the protein level, Lrp4 might function by
facilitating the presentation or integration of Gremlin1 into a
signaling complex that mediates the activation of ureteric budding.
Figure 3. Pax2 signaling remains intact in the absence of Lrp4. Pax2 is expressed normally in the metanephric mesenchyme and the ureteric
bud, indicated by the red arrows, at E10.5 in the wild type and Lrp4 knockout mice (a and b). At E11.5, Pax2 is expressed normally in both the ureteric
bud and metanephric mesenchyme, the latter indicated by black arrows, of wild type (c) and Lrp4 knockout animals (d). However, the ureteric bud
fails to invade the metanephric mesenchyme and does not undergo secondary branching in the Lrp4 knockout indicated by the yellow arrow (d). The
black arrows indicate mesenchymal expression of Pax2, which is present in the wild-type, but subsequently lost in the knock-out kidney mesenchyme
at E12.5 (e and f).
Figure 4. Expression of branching regulators in Lrp4 mutants. Expression of c-Ret (a–f), GDNF (g–l) and Wnt11 (m–r) in E10.5 (a,b,g,h,m and
n), E11.5 (c,d,i,j,o and p), and E12.5 (e,f,k,l,q,r) in wild-type (a,c,e,g,i,k,m,o, and q) and Lrp4 knockout (b,d,f,h,j,l,n,p and r) kidneys. C-Ret is expressed in
the ureteric bud at basal levels in the Lrp4 knockout mice at E10.5 (a,b). At E11.5, the Lrp4 knockout ureteric bud fails to bifurcate or upregulate c-Ret
expression at the tip of the ureteric bud (d) compared to wild-type embryos (c). At E12.5, the signal is greatly reduced in the knockout kidney (f).
GDNF is expressed normally in the metanephric mesenchyme at both E10.5 and 11.5 in wild type and Lrp4 knockout animals (g–j). By E12.5, GDNF
expression is completely lost from the Lrp4 knockout metanephric mesenchyme (k and l). Wnt11 is expressed normally at the tips of the ureteric bud
at both E10.5 (m and n) and 11.5 (o and p) in Lrp4 mutants compared to wildtype. By E12.5 Wnt11 is absent from the ureteric bud of Lrp4 knockout
animals (q and r). The Wolffian duct and ureteric bud are outlined by white dashed lines, mesenchyme (g, h, I, j) renal vesicles (e and q) and truncated
ureteric bud (f, l, r) are indicated by black arrows.
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Figure 5. Ureteric Budding is delayed in Lrp4 Mutants. 38 somite stage E10.5 embryos were stained with the epithelial markers Pax2 (red) and
E-cadherin (green) to label the Wolffian duct and developing ureteric bud. Representative images of kidney pairs for two wild-type and knock-out
animals are shown. In the wild-type (a–d), ureteric buds appear as expected while there is a frequent delay in ureteric bud outgrowth in the Lrp4
mutants (e,g,h). One Lrp4 mutant animal is shown with a unilateral outgrowth (f). In total, all 10 expected buds are formed at the 38 somite stage in
the wild-type background while only 1 out of 8 predicted buds is present in the knock-out (Panel b). P values (Student’s t-test) p,0.01 indicates
Figure 6. Wnt Overexpression in the Ureteric Bud Leads to Kidney Agenesis. Expression of a stabilized allele of b-catenin (Catnbexon3flox) in
the Wollfian duct using HoxB7Cre to activate transgene expression phenocopies the Lrp4 knockout phenotype with both uni- and bilateral kidney
agenesis (a-c). The formation of the Wolffian duct and distal ureters as well as bladder and adrenal glands remained unaffected. The asterisks (a and b)
indicate the position of regular kidneys. The arrows (b and c) indicate the predicted position of kidneys that have not formed.
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Lrp4 mutations cause renal malformations in humans
In a cooperative effort, Li et al.  identified homozygous
LRP4 mutations in patients with Cenani-Lenz syndrome (CLS), a
congenital syndrome mainly characterized by musculoskeletal
malformations, analogous to the murine phenotype including
polysyndactyly and molar fusion. When evaluated for kidney
defects, Li and colleagues observed congenital kidney abnormal-
ities in homozygous carriers in more than half of the investigated
families, which was hitherto unknown. Renal agenesis, in
accordance with the murine phenotype, was observed in one-
third of the families, another 25% percent presented with ectopic
or hypoplastic kidneys. For one of the affected CLS patients of the
CL-6 family described by Li et al. , imaging and functional
studies revealed ectopic and hypoplastic kidneys on both sides
(Fig. 8a–d). Dynamic-static renal scintigraphy with Tc-99m DTPA
showed hypofunction of the right kidney, which contributed 26%
vs 74% (left kidney) to total renal function (Fig. 8e). Static renal
cortical scintigraphy with Tc-99m DMSA revealed increased
background activity (Fig. 8f). Creatinine in this patient was
elevated at 1.2 mg/dL. Both of these findings indicated impaired
renal function. Clinical variability of phenotypic expression
suggests that additional modifying factors that affect budding,
branching morphogenesis and organ maturation contribute to this
phenotype in humans.
In this study, we have shown that Lrp4 functions as a critical
regulator of kidney development in both mouse and human. In
mice, complete absence of functional Lrp4 leads to uni- or bilateral
kidney agenesis caused by a delay in the formation of the ureteric
bud. In other mouse models, e.g. the limb deformity (ld) mutation
or Danforth’s short tail (Sd) mice , delayed invasion of the ureteric
bud into the receptive mesenchyme results in mesenchymal
apoptosis and kidney agenesis . The fact that normal kidneys
do develop in a subset of Lrp4 null embryos suggests that the
Figure 7. Lrp4 binds the Bmp4 antagonist Gremlin1 in vitro.
Lrp4 has been implicated in modulating the Bmp signaling pathway
through binding of the Wnt and Bmp modulator Wise. Co-immuno-
precipitation reveals Gremlin1 binding to Lrp4 in vitro (Panel A lane 4);
we further confirmed the Lrp4 binding partners Wise, Dkk1 and SOST
(Panel A, lanes 6, 10 and 12). The Wnt agonist R-spondin 2 did not
interact with Lrp4 (Panel A lane 7 and 8). Transfection efficiency was
confirmed by immunoblot analysis (Panel B).
Figure 8. Hypoplastic and Hypofunctional Kidney in Human Lrp4 Mutations. CT scan reveals a severely hypoplastic kidney on the right and
mild hypoplasia on the left side (a–d). Both kidneys are ectopic with caudal and lateral shifts (a–d). Dynamic-static renal scintigraphy with Tc-99m
DTPA suggest right kidney dysfunction (e). Global renal functional participation; right kidney 26% and left kidney 74%. Static renal cortical
scintigraphy with Tc-99m DMSA background activity of radiopharmaceutical is higher than expected (f).
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signaling capacity of the bud and the receptivity of the
mesenchyme is unaffected by loss of this gene. However, the
range of phenotypes observed in humans, from complete agenesis
to hypoplasia, along with the expression of Lrp4 mRNA in
multiple cell types of the kidney throughout the embryonic period
suggest this molecule may have additional roles in kidney
development, or that other factors exist, which can modify the
The precise mechanism for Lrp4 action during kidney
development is still unclear. During kidney development, tissue-
tissue interactions between the metanephric mesenchyme and the
UB are critical and rely on the integration and regulation of
several signaling pathways. Wnt signaling is crucial for UB
branching and has been shown to be regulated by Lrp4 in other
systems [15,16,17]. Intriguingly, a mouse model with UB-specific
overexpression of activated b-catenin presents with a very similar
phenotype to the Lrp4 mutant (Fig. 6). However, analysis of the
Wnt pathway activity has failed to reveal significant changes in
Lrp4 mutants, possibly due to high baseline activity in wildtype
An alternative yet equally plausible scenario is that Lrp4 is
involved in the modulation of Bmp signaling. We have found that,
like other members of the LDL receptor gene family, Lrp4 is
capable of modulating TGF-b related signaling [17,31]. In this
study, we have confirmed novel binding partners for Lrp4
including the Bmp regulating ligand Gremlin1 (Fig. 7). As
Gremlin1 knockout mice display a phenotype of bilateral kidney
agenesis (reportedly due to ectopic Bmp4 activity) , an
attractive model is that Lrp4 cooperates with Gremlin to inhibit
Bmp4 activity. However, similar to the case with b-catenin
signaling, we were unable to detect significant changes in the
expression of the Bmp targets, pSmad1, 4 and 8. It is therefore
possible that Lrp4 acts through an unrelated pathway or perhaps
through only partial modulation and integration of both Bmp and
Normal kidney formation occurs in a hypomorphic Lrp4
mutant, where only a secreted extracellular domain is expressed,
adding additional insight into the mechanism of Lrp4 during
ureteric budding [15,16]. These findings suggest that whatever
factor Lrp4 is normally interacting with in the kidney, it is
occurring extracellularly and most likely does not require
endocytosis of the receptor. Possible mechanisms include quench-
ing of Wnt and BMP modulators, such as Gremlin1 (Fig. 7) by the
secreted extracellular domain.
In summary, we have identified Lrp4 as a critical factor for UB
outgrowth and kidney formation in the mouse. We have also
shown that mutations in Lrp4 lead to the same or very similar
developmental malformations as seen in human LRP4 deficient
patients with Cenani-Lenz syndrome, further underscoring the
importance of Lrp4 for human genetics and medicine.
The authors are indebted to Wen-Ling Niu, Huichuan Reyna, Priscilla
Rodriguez and Isaac Rocha for superb technical assistance.
Conceived and designed the experiments: CMK MFD EBJ TC JH.
Performed the experiments: CMK MFD EBJ NK CT FP. Analyzed the
data: CMK MFD EBJ TC JH. Contributed reagents/materials/analysis
tools: CMK EBJ NK CT FP BW TC JH. Wrote the paper: CMK MFD
1. Shakya R, Jho EH, Kotka P, Wu Z, Kholodilov N, et al. (2005) The role of
GDNF in patterning the excretory system. Dev Biol 283: 70–84.
2. Saxen L, Sariola H (1987) Early organogenesis of the kidney. Pediatr Nephrol 1:
3. Maas R, Elfering S, Glaser T, Jepeal L (1994) Deficient outgrowth of the ureteric
bud underlies the renal agenesis phenotype in mice manifesting the limb
deformity (ld) mutation. Dev Dyn 199: 214–228.
4. Lipschutz JH (1998) Molecular development of the kidney: a review of the results
of gene disruption studies. Am J Kidney Dis 31: 383–397.
5. Gluecksohn-Schoenheimer S (1943) The Morphological Manifestations of a
Dominant Mutation in Mice Affecting Tail and Urogenital System. Genetics 28:
6. Gluecksohn-Schoenheimer S (1945) The Embryonic Development of Mutants of
the Sd-Strain in Mice. Genetics 30: 29–38.
7. Lu P, Sternlicht MD, Werb Z (2006) Comparative mechanisms of branching
morphogenesis in diverse systems. J Mammary Gland Biol Neoplasia 11:
8. Bates CM (2000) Kidney development: regulatory molecules crucial to both
mice and men. Mol Genet Metab 71: 391–396.
9. Kispert A, Vainio S, Shen L, Rowitch DH, McMahon AP (1996) Proteoglycans
are required for maintenance of Wnt-11 expression in the ureter tips.
Development 122: 3627–3637.
10. Kuro-o M (2006) Klotho as a regulator of fibroblast growth factor signaling and
phosphate/calcium metabolism. Curr Opin Nephrol Hypertens 15: 437–441.
11. Costantini F, Shakya R (2006) GDNF/Ret signaling and the development of the
kidney. Bioessays 28: 117–127.
12. Searle AG, Peters J, Lyon MF, Hall JG, Evans EP, et al. (1989) Chromosome
maps of man and mouse. IV. Ann Hum Genet 53: 89–140.
13. Tomita Y, Kim DH, Magoori K, Fujino T, Yamamoto TT (1998) A novel low-
density lipoprotein receptor-related protein with type II membrane protein-like
structure is abundant in heart. J Biochem 124: 784–789.
14. Weatherbee SD, Anderson KV, Niswander LA (2006) LDL-receptor-related
protein 4 is crucial for formation of the neuromuscular junction. Development
15. Johnson EB, Hammer RE, Herz J (2005) Abnormal development of the apical
ectodermal ridge and polysyndactyly in Megf7-deficient mice. Hum Mol Genet
16. Johnson EB, Steffen DJ, Lynch KW, Herz J (2006) Defective splicing of Megf7/
Lrp4, a regulator of distal limb development, in autosomal recessive mulefoot
disease. Genomics 88: 600–609.
17. Ohazama A, Johnson EB, Ota MS, Choi HY, Porntaveetus T, et al. (2008) Lrp4
modulates extracellular integration of cell signaling pathways in development.
PLoS One 3: e4092.
18. Li Y, Pawlik B, Elcioglu N, Aglan M, Kayserili H, et al. (2010) LRP4 receptor
mutations alter Wnt/b-catenin signalling causing limb and kidney malforma-
tions in Cenani-Lenz syndrome Am J Hum Gen, submitted.
19. Carroll TJ, Park JS, Hayashi S, Majumdar A, McMahon AP (2005) Wnt9b plays
a central role in the regulation of mesenchymal to epithelial transitions
underlying organogenesis of the mammalian urogenital system. Dev Cell 9:
20. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, et al. (1999) Intestinal
polyposis in mice with a dominant stable mutation of the beta-catenin gene.
EMBO J 18: 5931–5942.
21. Favor J, Sandulache R, Neuhauser-Klaus A, Pretsch W, Chatterjee B, et al.
(1996) The mouse Pax2(1Neu) mutation is identical to a human PAX2
mutation in a family with renal-coloboma syndrome and results in
developmental defects of the brain, ear, eye, and kidney. Proc Natl Acad
Sci U S A 93: 13870–13875.
22. Rothenpieler UW, Dressler GR (1993) Pax-2 is required for mesenchyme-to-
epithelium conversion during kidney development. Development 119: 711–720.
23. Dressler GR, Wilkinson JE, Rothenpieler UW, Patterson LT, Williams-
Simons L, et al. (1993) Deregulation of Pax-2 expression in transgenic mice
generates severe kidney abnormalities. Nature 362: 65–67.
24. Sampogna RV, Nigam SK (2004) Implications of gene networks for
understanding resilience and vulnerability in the kidney branching program.
Physiology (Bethesda) 19: 339–347.
25. Majumdar A, Vainio S, Kispert A, McMahon J, McMahon AP (2003) Wnt11
and Ret/Gdnf pathways cooperate in regulating ureteric branching during
metanephric kidney development. Development 130: 3175–3185.
26. Blank U, Seto ML, Adams DC, Wojchowski DM, Karolak MJ, et al. (2008) An
in vivo reporter of BMP signaling in organogenesis reveals targets in the
developing kidney. BMC Dev Biol 8: 86.
27. Michos O, Goncalves A, Lopez-Rios J, Tiecke E, Naillat F, et al. (2007)
Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and
GDNF/WNT11 feedback signalling during kidney branching morphogenesis.
Development 134: 2397–2405.
28. Bridgewater D, Rosenblum ND (2009) Stimulatory and inhibitory signaling
molecules that regulate renal branching morphogenesis. Pediatr Nephrol 24:
Lrp4 in Kidney Development
PLoS ONE | www.plosone.org8April 2010 | Volume 5 | Issue 4 | e10418
29. Merkel CE, Karner CM, Carroll TJ (2007) Molecular regulation of kidney Download full-text
development: is the answer blowing in the Wnt? Pediatr Nephrol 22:
30. Phelps DE, Dressler GR (1993) Aberrant expression of Pax-2 in Danforth’s short
tail (Sd) mice. Dev Biol 157: 251–258.
31. Choi HY, Dieckmann M, Herz J, Niemeier A (2009) Lrp4, a novel receptor for
Dickkopf 1 and sclerostin, is expressed by osteoblasts and regulates bone growth
and turnover in vivo. PLoS One 4: e7930.
Lrp4 in Kidney Development
PLoS ONE | www.plosone.org9 April 2010 | Volume 5 | Issue 4 | e10418