Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA
Author for correspondence (e-mail:
skarnes{at}sanger.ac.uk)
Accepted 19 February 2004
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SUMMARY |
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Key words: Wnt, Lrp5, Lrp6, Gastrulation, Nodal, Fgf, Mouse
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Introduction |
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Analyses of Wnt gene mutations in mice have pointed to important roles for
Wnt signals during gastrulation (reviewed by
Yamaguchi, 2001). The first
morphological sign of gastrulation is the formation of the primitive streak on
the posterior side of the embryo. At the primitive streak, ectodermal epiblast
cells delaminate and either ingress between the ectoderm and the visceral
endoderm to become mesoderm, or displace the overlying visceral endoderm to
form the embryonic definitive endoderm (reviewed by
Tam and Behringer, 1997
).
Fate-mapping studies have shown that cells emerging from the posterior and
middle regions of the streak give rise to extra-embryonic mesoderm and trunk
mesoderm, respectively. Cells exiting the anterior primitive streak and later
the node contribute to the axial mesoderm and definitive endoderm. Opposite
the streak, a region known as the anterior visceral endoderm (AVE) imparts
anterior fates on the epiblast, principally by restricting posterior signals
(reviewed by Lu et al., 2001
;
Perea-Gomez et al., 2001
).
Subsequently, the anterior primitive streak and its derivatives are important
for inducing/maintaining anterior epiblast fates initiated by the AVE
(reviewed by Martinez-Barbera and
Beddington, 2001
). The loss of either ß-catenin or
Wnt3 function has severe effects on gastrulation
(Huelsken, 2000
;
Liu et al., 1999
). Both
mutants lack a primitive streak and mesoderm, although the AVE is specified.
Interestingly, the AVE fails to move anteriorly in ß-catenin mutants,
suggesting that proper positioning of the AVE either involves additional Wnt
signals or is independent of Wnt genes. Although defects in Wnt3a
mutants are less severe, these mutants also display perturbations of posterior
development. Wnt3a-/- embryos lose posterior somites, and
ectopic neural tissue forms at the expense of paraxial mesoderm
(Takada et al., 1994
;
Yoshikawa et al., 1997
). Thus,
Wnt signals are required for posterior patterning of the embryo during
gastrulation.
Recent genetic and biochemical studies have identified members of the LDL
receptor-related (Lrp) family, specifically Lrp5 and Lrp6 in vertebrates and
Arrow in Drosophila, as important components of the canonical Wnt
signaling pathway (Wehrli et al.,
2000; Pinson et al.,
2000
; Tamai et al.,
2000
) (reviewed by He et al.,
2004
). Lrp5/Lrp6 are single-pass transmembrane proteins that have
been proposed to act as Wnt co-receptors by binding to Wnt and frizzled in a
ternary complex (Tamai et al.,
2000
; Semenov et al.,
2001
; Schweizer and Varmus,
2003
), although a direct interaction between Drosophila
Wnt/Wingless and Arrow has not yet been demonstrated
(Wu and Nusse, 2002
). The
mechanism by which Wnt signals are transduced through Lrp5/6 appears to
involve binding of Lrp5/6 to axin, a downstream negative regulator of the
pathway that controls degradation of ß-catenin
(Mao et al., 2001b
;
Liu et al., 2003
;
Tolwinski et al., 2003
;
Tamai et al., 2004
). Wnt
signals stimulate phosphorylation of reiterated PPPSP motifs in the
intracellular domain of Lrp5/6, thereby recruiting binding of axin
(Tamai et al., 2004
) and
resulting in stabilization of ß-catenin. Lrp5/6 function can be inhibited
by binding to the secreted protein dickkopf
(Mao et al., 2001a
;
Bafico et al., 2001
;
Semenov et al., 2001
), which
is believed to disrupt Wnt-induced interactions between frizzled and Lrp5/6
(Semenov et al., 2001
). A
newly identified class of transmembrane proteins called kremens form a complex
with Lrp5/6 and dickkopf, resulting in rapid internalization and hence,
sequestering Lrp5/6 away from the frizzled receptor
(Mao et al., 2002
). Thus,
these biochemical studies demonstrate a key role for Lrp5/6 in modulating
cellular responses to Wnts.
To investigate the function of Lrp5 and Lrp6, mutational
analyses have been performed in mice. Mice homozygous for a targeted mutation
in Lrp5 are viable and exhibit subtle defects in bone ossification
and eye vasculature that recapitulate aspects of the human disease,
osteoporosis-pseudoglioma syndrome (Kato
et al., 2002; Gong et al.,
2001
). Although there is ample evidence to support the role of
Wnts in osteoblast proliferation and bone formation, the specific Wnt proteins
involved have not been identified by genetic analysis (reviewed by
Patel and Karsenty, 2002
). In
addition, Lrp5 mutant mice have impaired glucose tolerance
(Fujino et al., 2003
). In
mice, disruption of Lrp6 causes severe developmental defects
(Pinson et al., 2000
). These
abnormalities include a deletion of caudal midbrain, axis truncation and limb
patterning defects. This constellation of phenotypes is strikingly similar to
those seen in embryos lacking Wnt1, Wnt3a and Wnt7a
function, respectively (McMahon and
Bradley, 1990
; Takada et al.,
1994
; Parr and McMahon,
1995
). The defects in Lrp6 mutants reflect a composite of
some, but not all, Wnt mutant phenotypes.
Several explanations could account for the fact that neither Lrp5-nor Lrp6-deficient embryos exhibit a gastrulation phenotype, as observed in Wnt3 mutants. For example, functional redundancy may exist between these two closely related proteins. Alternatively, Lrp5 and Lrp6 may each be required for signaling by only a subset of Wnts. To address these issues, we examined the phenotype of mice carrying null mutations in both Lrp5 and Lrp6. Our data clearly demonstrate that these two proteins compensate for one another during embryonic development. In addition, our analysis reveals a crucial role for both Lrp5 and Lrp6 in patterning of the embryo during gastrulation.
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Materials and methods |
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PCR genotyping of Lrp5/6-deficient mice and embryos
Weanlings were genotyped at 3 weeks of age by PCR of DNA prepared from tail
samples. Tail tissue (1 mm) was boiled at 100°C for 20-30 minutes in
50-150 µl of 1 N NaOH/1mM EDTA under mineral oil. After neutralization with
an equal volume of 40 mM Tris-HCl pH 7.5-8.0, 1-5 µl of the DNA extract was
used for PCR amplification. For both Lrp5 and Lrp6 PCR
genotyping, an annealing temperature of 60°C and 30-35 cycles of
amplification were used. The Lrp5 mutant allele was detected by the
amplification of a 350 bp product using a forward primer in neo (neo:
5'-GCAGCGCATCGCCTTCTATC) and a reverse primer in the Lrp5
genomic sequence (Lrp5-D1: 5'-CTTCTCTCCAGACTCCCAAAGC). The Lrp5
wild-type allele was detected by the amplification of a 520 bp product using
the Lrp5-D1 primer and a forward primer from sequence within the genomic
region deleted during homologous recombination (Lrp5-U1:
5'-GAGCTCTCAAGCTCAGCCAG). Using genomic DNA from wild-type and
Lrp6-/- embryos, PCR was performed with overlapping primer
sets within intron 5 of the Lrp6 gene and the gene-trap insertion
site was mapped to within the first 650 bp of the intron. Two primers from
this region (Lrp6-U1: 5'-CAGGCATGTAGCCCTTGGAG and Lrp6-D1:
5'-ACTACAAGCCCTGCACTGCC) were used to PCR amplify a 285 bp DNA product
corresponding to the wild-type allele. A vector-specific primer (en2:
5'-GTAGAGTTCCCAGGAGGAGCC) complementary to the En2 intron
sequence of the gene trap vector (Skarnes
et al., 1995) in combination with primer Lrp6-U1 amplified a
fragment of approximately 385 bp from the mutant allele.
Timed matings were used to collect embryos at desired stages with noon on the day of the copulation plug designated as 0.5 dpc. Unfixed embryos and embryos following whole-mount RNA in situ hybridization were genotyped by PCR after removal of maternal tissues. Normal littermates from several genotypic classes were used as controls for comparisons with the Lrp5+/-;Lrp6-/- and Lrp5-/-;Lrp6-/- mutant embryos.
Northern blot analysis
Total RNA from adult kidney and brain of wild-type and Lrp5
homozygous animals was prepared using Trizol reagent (Gibco-BRL). Northern
blot analysis was performed using the following probes: Lrp5, a 400
bp EcoRI/NotI cDNA fragment from the 3' untranslated
region (mouse expressed sequence tag, GenBank #AI119858); and actin, a 610 bp
PstI fragment of the mouse ß-actin cDNA fragment.
Expression analysis of whole-mount embryos
For detection of ß-galactosidase activity, embryos were stained with
X-Gal (5-bromo-4-chloro-3-indolyl ß-D-galactoside) as previously
described (Skarnes, 2000). RNA
in situ hybridization was performed on embryos according to the protocol
developed by Henrique and Ish-Horowicz (see
http://iprotocol.mit.edu/protocol/59.htm),
partly described by Henrique et al.
(Henrique et al., 1995
), with
the following modifications: (1) the hybridization mix contained SSC at a pH
of 7.0; (2) hybridization and subsequent high temperature washes were carried
out at 67°C; (3) the anti-DIG-AP antibody (Roche #1 093 274) was used at a
dilution of 1:4000; (4) post-antibody washes consisted of four one hour washes
at room temperature and one overnight wash at 4°C; and (5) embryos were
stained in 2.25 µl/ml NBT (75 mg/ml) and 1.75 µl/ml BCIP (50 mg/ml).
Antisense riboprobes were synthesized using a DIG RNA labeling kit (Roche,
#1 175 025) for the following genes: Bmp4
(Winnier et al., 1995),
Cer1 (Belo et al.,
1997
), Fgf8 (Mahmood
et al., 1995
), Foxa2
(Sasaki and Hogan, 1993
);
Gbx2 (Wassarman et al.,
1997
), Hex (Bedford et
al., 1993
), Hesx1
(Thomas et al., 1995
),
Ebaf (previously Lefty2)
(Meno et al., 1999
),
Lhx1 (Shawlot and Behringer,
1995
), Lrp5 (Gong et
al., 2001
), Nodal
(Meno et al., 1998
),
Pou5f1 (A. Smith), sprouty 2
(Minowada et al., 1999
),
Six3 (Oliver et al.,
1995
), Shh (Echelard
et al., 1993
), T
(Wilkinson et al., 1990
),
Tbx6 (Image clone #599230) and Wnt3
(Roelink et al., 1990
).
Skeletal preparations and histology
Skeletal preparations were made using standard methods
(Parr and McMahon, 1995).
Histological sections of embryos were processed as previously described
(Pinson et al., 2000
).
Following in situ hybridization, selected embryos were fixed in 60%
ethanol/30% formaldehyde/10% acetic acid, dehydrated in ethanol, embedded in
paraffin wax, sectioned at 8 µm and counterstained with nuclear fast red
(Vector, #H-3403) for 1 minute.
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Results |
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Lrp6 is expressed ubiquitously during embryonic development
(Pinson et al., 2000) and
Lrp5 is expressed in many adult tissues
(Kato et al., 2002
). The early
lethality of Lrp5+/-;Lrp6-/- and
Lrp5-/-;Lrp6-/- embryos prompted us to
examine Lrp5 and Lrp6 expression during gastrulation
(Fig. 1E-N). Insertion of the
gene-trap vector into the 5th intron of Lrp6 creates a fusion protein
with ß-galactosidase (Mitchell et
al., 2001
), which was used as a reporter of Lrp6
expression. Lrp6 is broadly expressed in the embryo, although expression is
stronger in the embryonic region (Fig.
1F,G). Lrp6 is expressed throughout the embryonic ectoderm and in
nascent mesoderm and endoderm emerging from the primitive streak
(Fig. 1H,I). Although the
targeted allele of Lrp5 is tagged with IRES-ß-galactosidase, the
reporter is not functional, as we failed to detect ß-galactosidase
activity in Lrp5+/- or Lrp5-/-
embryos. Instead, we used whole-mount RNA in situ hybridization to examine
Lrp5 expression in wild-type embryos. Expression is strongest in
visceral endoderm overlying the extra-embryonic ectoderm, but is excluded from
the visceral endoderm surrounding the epiblast
(Fig. 1K,L, data not shown).
Lrp5 is also expressed throughout the embryonic and extra-embryonic
ectoderm. Unlike Lrp6, Lrp5 expression is not detected in mesoderm or
definitive endoderm exiting the primitive streak
(Fig. 1M,N). Thus, the
phenotypes associated with
Lrp5+/-;Lrp6-/- and
Lrp5-/-;Lrp6-/- mutants are consistent
with the overlapping expression of both genes in the early embryo.
Limb defects in Lrp5+/-;Lrp 6+/- and Lrp 5-/-;Lrp6+/- mutants
Mice homozygous for the published Lrp5tm1Kry mutation
have defects in phalanx ossification at P4
(Kato et al., 2002). Neonatal
limbs of pups from intercrosses of double heterozygous mutant animals were
examined for similar defects (Fig.
2). Lrp5-/- mutants also demonstrate a loss of
middle phalanx ossification at 18.5 dpc
(Fig. 2B). Interestingly,
Lrp 5/6 double heterozygotes are more severely affected than
Lrp5 homozygotes, exhibiting the absence of multiple ossification
centers (Fig. 2C). This result
clearly shows that Lrp5 and Lrp6 have redundant functions in the limb. In
addition to osteogenic defects, aberrant limb patterning is apparent in these
animals. Approximately 39% of
Lrp5+/-;Lrp6+/- animals display
postaxial digit loss. Typically one digit on the right forelimb is missing
(Fig. 2E). By contrast,
Lrp5-/-;Lrp6+/- animals show more
severe limb defects, which exhibit a loss of one to two postaxial digits in
the forelimb, missing carpals, shorter metacarpals
(Fig. 2G,J) and in more severe
cases a missing zeugopod element (Fig.
2G,I). Hindlimbs are generally more affected than forelimbs,
displaying only one to two digits and loss of the fibula
(Fig. 2G,L,M). The defects
observed in Lrp5-/-;Lrp6+/- limbs are
similar to those seen in Lrp6-/- mice
(Pinson et al., 2000
),
providing further evidence that Lrp5 and Lrp6 share overlapping functions in
the limb. As previously noted for Lrp6-/- mutants
(Pinson et al., 2000
), the
defects in limb patterning in
Lrp5-/-;Lrp6+/- mutants resemble
Wnt7a mutants (Parr and McMahon,
1995
). More recently it has been shown that conditional removal of
Wnt3 function in the limb ectoderm and of ß-catenin function in
the ventral limb ectoderm result in similar limb phenotypes
(Barrow et al., 2003
;
Soshnikova et al., 2003
).
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The accumulation of cells at the primitive streak and their failure to
properly migrate away from the streak is characteristic of Fgf8 and
Fgfr1 mutant embryos (Sun et al.,
1999; Deng et al.,
1994
; Yamaguchi et al.,
1994
). In addition, the expression of Tbx6 is absent in
Fgf8-/- (Sun et al.,
1999
) embryos and significantly reduced in
Fgfr1-/- mutants
(Ciruna and Rossant, 2001
).
This similarity in phenotypes could be explained if Lrp5/6 is required to
maintain the expression of Fgf8 and/or its receptor in the primitive
streak. To address this issue, we examined the expression of Fgf8 and
sprouty 2 (Spry2), a downstream target of Fgf signaling
(Crossley and Martin, 1995
;
Mahmood et al., 1995
;
Maruoka et al., 1998
;
Minowada et al., 1999
). Both
Spry2 and Fgf8 and are readily detected at the primitive
streak of wild-type and
Lrp5+/-;Lrp6-/- embryos
(Fig. 3M,N and data not shown),
suggesting that Fgf signaling is intact in these embryos.
Anterior primitive streak derivatives are expanded in Lrp5+/-;Lrp6-/- embryos
The expression of Lhx1 in the anterior region of
Lrp5+/-;Lrp6-/- embryos
(Fig. 3F) suggests that unlike
the paraxial mesoderm, the axial mesendoderm, a derivative of the anterior
primitive streak, is specified. The prolonged expression of Lhx1 at
the primitive streak (Fig.
3F,H) and the reduction in the domain of Ebaf expression
(Fig. 3J), however, suggest
that the anterior primitive streak may be expanded. Thus, we next examined the
expression of various molecular markers that delineate different populations
of axial mesendoderm cells that emerge from the anterior primitive streak/node
in Lrp5+/-;Lrp6-/- embryos
(Fig. 4). Indeed, there is a
dramatic expansion of Foxa2 expression at 7.5 dpc, a marker of the
anterior primitive streak and axial mesendoderm
(Fig. 4A) (Ang et al., 1993;
Monaghan et al., 1993
;
Sasaki and Hogan, 1993
), in
mutant embryos (Fig. 4B).
Moreover, Hex expression is broader in 7.5 dpc
Lrp5+/-;Lrp6-/- embryos
(Fig. 4D), indicative of an
increase in anterior definitive endoderm (ADE)
(Fig. 4C)
(Thomas et al., 1998
).
Examination of Shh and T expression at 8.5 dpc, however,
shows that although axial mesoderm is specified in the
Lrp5+/-;Lrp6-/- embryo, it is not
expanded (Fig. 4E-J)
(Echelard et al., 1993
;
Wilkinson et al., 1990
). In
fact, in more severely affected embryos, axial mesoderm appears reduced
(Fig. 4F). Together, these
results demonstrate that derivatives of the anterior primitive streak, in
particular the ADE, are expanded in
Lrp5+/-;Lrp6-/- embryos.
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Anterior neurectoderm is expanded in Lrp5+/-;Lrp6-/- embryos
As axial mesendoderm is required for maintenance of anterior neural fates
in the epiblast (reviewed by
Martinez-Barbera and Beddington,
2001), we next assayed the expression of neurectoderm markers in
Lrp5+/-;Lrp6-/- embryos
(Fig. 5). Expression of
Six3, a marker of anterior neurectoderm
(Fig. 5A,C)
(Oliver et al., 1995
), is
broader both posteriorly and laterally in mutant embryos at 8.0 dpc
(Fig. 5B). The consequence of
this broader domain is clearly evident by 8.5 dpc by the formation of a
greatly enlarged forebrain in Lrp
5+/-;Lrp6-/- embryos
(Fig. 5D). To determine if more
posterior neurectoderm forms, we examined Gbx2 expression, a marker
of anterior hindbrain at 8.5 dpc (Fig.
5E) (Bouillet et al.,
1995
; Wassarman et al.,
1997
). In Lrp5+/-;Lrp6-/-
embryos there is a stripe of Gbx2 expression in the head region, indicating
the formation, but lack of expansion, of anterior hindbrain
(Fig. 5F). Thus, in
Lrp5+/-;Lrp6-/- embryos, there is a
specific expansion of anterior neurectoderm.
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Posterior patterning is absent in Lrp5/6-deficient embryos
At the onset of gastrulation, double homozygous mutant embryos appear
normal (data not shown), but by 7.5 dpc, they are visibly smaller and abnormal
compared with their littermates. In normal gastrulating embryos, mesoderm
exits the primitive streak on the posterior side of the embryo and migrates
between the ectoderm and endoderm layers, and the definitive endoderm
displaces the overlying visceral endoderm
(Fig. 6A). Sagittal sections of
double homozygous embryos show a complete absence of mesoderm and definitive
endoderm, while the ectoderm appears slightly thickened and collapsed
(Fig. 6B). In addition,
development of extra-embryonic tissue is severely compromised and structures
such as the amnion and allantois are not visible.
|
We noted a patch of Lhx1 expression on the presumptive anterior
side of double homozygous mutant embryos
(Fig. 6N, arrow), suggesting
that AVE is formed. This was not surprising as the AVE is specified prior to
gastrulation and forms independently of the primitive streak (reviewed by
Lu et al., 2001). Other
markers of the AVE, Hex and Cer1
(Fig. 6Q)
(Thomas et al., 1998
;
Belo et al., 1997
;
Biben et al., 1998
;
Pearce et al., 1999
;
Shawlot et al., 1998
), are
also expressed in double homozygous mutant embryos, but in fewer cells than
normal (Fig. 6P,R). Although
both Hex and Cer1 are also normally expressed in the
definitive endoderm (Fig. 6O,Q)
(Thomas et al., 1998
;
Belo et al., 1997
;
Biben et al., 1998
;
Pearce et al., 1999
;
Shawlot et al., 1998
), the
remaining Hex and Cer1-expressing cells in double homozygous
mutants most probably correspond to the AVE as the definitive endoderm is lost
subsequent to the failure to establish a primitive streak
(Fig. 6B,P,R). Together, these
results show that while the AVE is specified in double homozygous mutant
embryos, the primitive streak and its mesodermal and endodermal derivatives do
not form.
It has been suggested that the AVE is not sufficient to specify anterior
fates in the epiblast, instead requiring additional signals from the anterior
primitive streak (Tam and Steiner,
1999) (reviewed by
Martinez-Barbera and Beddington,
2001
; Lu et al.,
2001
). Surprisingly, however, an expanded domain of Hesx1
expression, a marker of anterior axial mesendoderm and overlying anterior
neurectoderm (Fig. 6S)
(Thomas et al., 1995
;
Hermesz et al., 1996
), is
observed in double homozygous mutant embryos at 7.5 dpc
(Fig. 6T) and at 8.5 dpc (data
not shown). Because these embryos fail to form primitive streak derivatives,
the domain of Hesx1 expression most probably corresponds to anterior
neurectoderm. We therefore hypothesize that AVE cells are sufficient to
specify anterior neurectodermal fate in the absence of a primitive streak. The
expanded domain of Hesx1 expression may reflect the absence of
posterior signals that normally counteract anterior signals (reviewed by
Lu et al., 2001
).
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Discussion |
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Lrp5 and Lrp6 are very similar in their overall structure
(Brown et al., 1998), and the
intracellular portion of each receptor contains five PPPSP motifs important
for recruiting axin (Tamai et al.,
2004
). Previous work, however, has suggested that Lrp5 and Lrp6
are not equivalent in their ability to transduce Wnt signals. For example, in
Xenopus assays Lrp6 is sufficient to induce axis duplication on its
own, whereas Lrp5 is not. However, both Lrp5 and Lrp6 potentiate axis
duplication in the presence of sub-threshold levels of Wnt
(Tamai et al., 2000
). In
addition, Lrp6 is much more potent in activating a Wnt-responsive promoter in
cultured mammalian cells in response to a Wnt signal
(Holmen et al., 2002
). In
keeping with these studies, it is interesting that the loss of Lrp6
alleles consistently produces more severe phenotypes compared with the loss of
Lrp5 alleles (Table
1). Thus, an allelic series that presumably reflects the
progressive loss of Wnt signaling can be constructed as follows:
Lrp5+/- < Lrp6+/- <
Lrp5-/- <
Lrp5+/-;Lrp6+/- <
Lrp5-/-;Lrp6+/- <
Lrp6-/- <
Lrp5+/-;Lrp6-/- <
Lrp5-/-;Lrp6-/-. We conclude that Lrp5
and Lrp6 perform the same function during embryonic development, albeit with
different efficiencies. We note, however, that while Lrp5 and Lrp6 are both
widely expressed during embryonic development
(Fig. 1), they are not
coexpressed in all cells. Thus, it is formally possible that Lrp5 and Lrp6 may
have separate functions that we were not able to detect in our assays. We
found no evidence that would suggest qualitative differences in the
specificity of Lrp5 and Lrp6 for different subsets of Wnts, although further
genetic and biochemical studies will be required to settle this point.
Lrp5 and Lrp6 are required for primitive streak formation
Targeted disruption of Wnt3 has firmly established a role for Wnts
in posterior patterning of the early embryo. Wnt3 is initially
expressed in the proximal epiblast and overlying visceral endoderm of the
pre-gastrula embryo. Wnt3 mutants fail to form a primitive streak and
lack mesoderm (Liu et al.,
1999). Thus, Wnt3 is the earliest-acting of the mammalian Wnts
identified to date. Based on morphology and marker gene expression,
Lrp5/6 double homozygous mutant embryos are very similar to
Wnt3 mutant embryos. Like Wnt3 mutants,
Lrp5/6-deficient embryos fail to express markers of the primitive
streak (T, Fgf8 and Wnt3 itself), as well as markers of
nascent mesoderm (Lhx1, Tbx6, Wnt3) and definitive endoderm
(Cer1, Hex, Lhx1). By contrast, anterior patterning is correctly
established in both Wnt3 (Liu et
al., 1999
) and Lrp5/6 mutant embryos as indicated by the
expression and localization of AVE markers (Hex, Cer1 and
Lhx1). Thus, we conclude that Lrp5/6 function is probably required
for the reception of Wnt3 signals to establish posterior pattern in the
epiblast. The absence of Wnt3 expression in the Lrp5/6
double mutant gastrula embryo may reflect a failure to induce and/or maintain
the precursors of the primitive streak in the proximal epiblast of the
pre-gastrula embryo because of loss of signaling through Lrp5/6.
Alternatively, it is formally possible that Wnt3 is the target of another
unidentified Wnt with an earlier role in development that signals through
Lrp5/6, a hypothesis supported by slight differences in phenotypes between
Wnt3 and Lrp5-/-;Lrp6-/-
mutants, as discussed below.
Interestingly, the phenotype of the classical mutation
mesoderm-deficient (mesd) is very similar to Wnt3
and Lrp5/6-deficient mutants
(Holdener et al., 1994;
Liu et al., 1999
;
Hsieh et al., 2003
). The
mesd gene and its Drosophila homolog boca have been
recently identified as chaperones required for the proper expression of Lrp5/6
on the surface of cells (Hsieh et al.,
2003
; Culi and Mann,
2003
). The Lrp5/6 and mesd mutant embryos arrest
at a slightly earlier stage than Wnt3-deficient embryos. Signaling by
other Wnt genes expressed at gastrulation, such as Wnt3a
(Gavin et al., 1990
;
McMahon et al., 1992
) may
account for the continued growth of the epiblast in Wnt3 mutants.
However, considering the well-known role of LDL receptor family members in the
binding and uptake of macromolecules (reviewed by
Willnow, 1999
), it seems
reasonable that Lrp5/6 may have additional functions in the growth of the
embryo that are independent of Wnt signaling.
The expression of Hesx1 in the epiblast represents another
important difference between Lrp5/6- and mesd-deficient
embryos compared with Wnt3 mutants
(Fig. 6T). Hesx1
expression is reportedly absent in Wnt3 mutants, and this observation
has led to the hypothesis that the AVE is not sufficient to induce anterior
neurectoderm (Liu et al.,
1999). Based on transplant and explant studies, AVE signals are
instead believed to repress posteriorizing signals from the primitive streak,
thereby priming the epiblast to respond to additional anterior signals
emanating from the anterior primitive streak
(Tam and Steiner, 1999
;
Kimura et al., 2000
) (reviewed
by Lu et al., 2001
;
Perea-Gomez et al., 2001
). The
expression of Hesx1 in the epiblast of Lrp5/6 double mutants
and mesd mutants suggests that the AVE is, in fact, sufficient to
induce anterior neurectodermal cell fates in the absence of a primitive
streak. Presumably, the absence of posterior signals from the primitive streak
allows the AVE to induce anterior fates in a broader region of the
Lrp5/6 and mesd mutant epiblast. It is unclear why
Wnt3 mutants fail to express Hesx1; however, one possibility is that
another unidentified Wnt normally represses anterior fates in the epiblast and
that this inhibition is relieved in Lrp5/6 and mesd, but not
Wnt3, mutant embryos. It will be important to assay other markers of
anterior neurectoderm to determine whether Hesx1 expression in
Lrp5/6 double mutants reflects a `pre-neuralized' state or a complete
induction of anterior neural tissue.
Mesoderm defects in Lrp5+/-;Lrp6-/- mutants resemble mutants with defects in Fgf signaling
The elimination of all but one copy of Lrp5 in
Lrp5+/-;Lrp6-/- embryos has
interesting and dramatic effects on mesoderm formation and anterior
development. The characterization of this genotypic class allows us to examine
the effects of severely reducing, but not completely eliminating, Wnt
signaling in the early embryo. The morphology of these embryos, as well as
changes in marker gene expression, appear remarkably similar to the phenotypes
of Fgf8 and Fgfr1 mutant embryos
(Sun et al., 1999;
Deng et al., 1994
;
Yamaguchi et al., 1994
;
Ciruna and Rossant, 2001
). For
example, shortly after gastrulation
Lrp5+/-;Lrp6-/- mutants exhibit an
excess of cells accumulating at the primitive streak, forming a distinctive
bulge that protrudes into the amniotic cavity. This defect is characteristic
of Fgf8 and Fgfr1 mutant embryos in which mesoderm forms but
fails to properly migrate away from the primitive streak. The migration defect
is more severe in Fgf8 mutants, which fail to form any identifiable
tissues by 8.5 dpc. Thus, in this regard,
Lrp5+/-;Lrp6-/- mutants more closely
resemble Fgfr1 mutants. In both cases, expression of Tbx6 is
lost or significantly reduced and mutant embryos lack somites. Since the
expression of Spry2, a downstream target of Fgf signaling, is
maintained in Lrp5+/-;Lrp6-/- embryos
(Fig. 3N), the striking
similarity between Lrp5+/-;Lrp6-/-
embryos and Fgf8 and Fgfr1 mutants cannot be explained by a
downregulation of Fgf signaling. Thus, the gastrulation defects in
Lrp5+/-;Lrp6-/- embryos are probably
due to a reduction in Wnt signaling, rather than to an effect on Fgf
signaling.
A recent study has shown that Wnt signaling is attenuated in Frfr1
mutant embryos, establishing a molecular link between the Fgf and Wnt
signaling pathways (Ciruna and Rossant,
2001). In Fgfr1-/- embryos, cells of the
primitive streak express abnormally high levels E-cadherin, and as a
consequence, ß-catenin is sequestered at the membrane. Thus, one of the
normal responses to signaling through Fgfr1 is to downregulate
E-cadherin in cells undergoing the epithelial-to-mesenchymal transition,
thereby releasing ß-catenin from the membrane and priming these cells to
respond to Wnt signals. In this light, Wnt and Fgf signaling appear to define
parallel signaling pathways that converge on ß-catenin to regulate the
specification and migration of trunk mesoderm. In addition, it has recently
been shown that Wnts can downregulate E-cadherin expression in other tissues
(Jamora et al., 2003
). These
observations readily explain how a reduction in Wnt signaling in
Lrp5+/-;Lrp6-/- embryos can mimic the
loss of Fgf8/Fgfr1 function. The generation of hypomorphic alleles of
Wnt3 will be very useful in testing this hypothesis.
Expansion of anterior primitive streak derivatives and anterior neurectoderm in Lrp5+/-;Lrp6-/- embryos
Lineage studies have established that cells emerging from the anterior
region of the primitive streak and later the node, migrate anteriorly and give
rise to definitive endoderm and axial mesoderm
(Tam and Behringer, 1997).
Lrp5+/-;Lrp6-/- embryos have an excess
of anterior primitive streak derivatives, in particular the ADE, as judged by
an increase in the expression of Hex and Foxa2.
Interestingly, an abundance of anterior primitive streak derivatives is also
observed in Fgfr1-deficient embryos. In this case, an excess of axial
mesoderm rather than definitive endoderm is formed as judged by an increase in
expression of Shh and T
(Deng et al., 1994
;
Yamaguchi et al., 1994
).
Signals from the ADE and prechordal mesoderm are thought to be required to
induce and maintain anterior neurectoderm fates in the epiblast (reviewed by
Martinez-Barbera and Beddington,
2001
). Thus, the dramatic expansion of anterior neurectoderm
expressing the most rostral neural marker Six3 can be explained by
the overproduction of ADE in
Lrp5+/-;Lrp6-/- embryos. Inhibition of
Wnt signaling is known to be an important aspect of anterior development
(reviewed by Niehrs et al.,
2001
). Overexpression of the Wnt antagonist dickkopf1 leads to
enlarged heads in Xenopus (Glinka
et al., 1998
), and conversely, dickkopf 1 mutant mice lack
forebrains, failing to express Hesx1 and Six3 in the
neurectoderm (Mukhopadhyay et al.,
2001
). The expansion of the anterior neurectoderm is, therefore,
not an unexpected consequence of reducing Wnt signaling in
Lrp5+/-;Lrp6-/- embryos.
Although the mechanism by which attenuation of Wnt signals could lead to an
increase in anterior primitive streak derivatives is less clear, several
observations point to a role for Nodal in this process. Similar to
Lrp5+/-;Lrp6-/- embryos, an expansion
of anterior primitive streak derivatives coupled with a loss of trunk mesoderm
is observed in some classes of
Cer1-/-;Leftb-/- mutants and in
Drap1 mutants (Perea-Gomez et
al., 2002; Iratni et al.,
2002
). As Cer1 and Leftb are Nodal antagonists, and Drap1 is
probably a transcriptional corepressor for Nodal, these defects
appear to be a consequence of increased levels of Nodal signaling. In support
of this idea, mutant embryos with the loss-of-function of two Nodal signaling
modulators, Foxh1 and arkadia (Rnf111 - Mouse Genome
Informatics), fail to establish an anterior primitive streak/node and its
derivatives (Hoodless et al.,
2001
; Yamamoto et al.,
2001
; Episkopou et al.,
2001
). Finally, either conditional loss-of-function of
Smad2, a Nodal effector, in the epiblast or decreased Nodal signals
in the primitive streak results in a specific loss of the ADE and prechordal
mesoderm (Vincent et al.,
2003
). Consistent with these data and the defects observed in
Lrp5+/-;Lrp6-/- embryos,
Nodal expression is aberrantly maintained at elevated levels in the
primitive streak of Lrp5+/-;Lrp6-/-
mutants (Fig. 4K-P).
Interestingly, Nodal expression is also upregulated in Fgf8
mutant embryos (Sun et al.,
1999
). Thus, one important role for Fgf and Wnt signaling may be
to antagonize Nodal signaling in the primitive streak.
The mechanism by which Fgfs and Wnts antagonize anterior primitive streak
fates is unclear. The expression of Ebaf, a Nodal antagonist
expressed in nascent mesoderm, is decreased in
Lrp5+/-;Lrp6-/- embryos
(Fig. 3J). As Ebaf normally
acts to suppress the activity of Nodal in nascent mesoderm
(Meno et al., 1999), decreased
Ebaf expression in
Lrp5+/-;Lrp6-/- embryos may further
amplify Nodal activity in the primitive streak
(Fig. 4K-P) and could account
for the dramatic expansion of axial mesendoderm at the expense of paraxial
mesoderm. Whether Ebaf is a direct target of Wnt signaling will
require further study.
In summary, the generation of Lrp5+/-;Lrp6-/- mutants provides a unique opportunity to examine the effect of reducing, but not completely eliminating, Wnt signaling in the embryo. The defects observed in these embryos reveal interesting links between the Wnt, Fgf and Nodal signaling pathways. Future experiments will be directed towards understanding the complex interplay of these pathways in patterning and specification of cells fates in the mammalian embryo.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: University of California San Diego, School of Medicine,
2071 Cellular and Molecular Medicine Building East, 0674, 9500 Gilman Drive,
La Jolla, CA 92093-0674, USA
Present address: Wellcome Trust Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge CB10 1SA, UK
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