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First published online 16 October 2008
doi: 10.1242/dev.029025
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Division of Developmental Biology, Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
Author for correspondence (e-mail:
heabq9{at}chmcc.org)
Accepted 17 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Dkk1, Wnt11, Wnt5a, Xenopus, Complex
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Further
studies suggested that the two classes also showed differences in their
targets: the Wnt1 group stabilized β-catenin (the canonical Wnt pathway),
and the second group activated either or both Jun NH2-terminal
kinase (JNK) and calcium/calmodulin dependent kinase 2, and caused convergent
extension movements and/or cellular polarization (the non-canonical group)
(Kikuchi et al., 2007).
Recent studies suggest that the above classification of Wnts is too rigid,
as individual Wnts can activate both canonical and non-canonical pathways,
depending on context. Wnt11 regulates cell movements in Xenopus and
zebrafish gastrulation and neurulation by non-canonical Wnt pathways
(De Calisto et al., 2005;
Tada et al., 2002;
Tada and Smith, 2000), but
also activates a canonical β-catenin-dependent pathway in establishing
the dorsal/ventral axis (Tao et al.,
2005). Wnt5a activates non-canonical signaling in rat cardiac
myocytes and Xenopus gastrulation, whereas it activates canonical
signaling for self-renewal in mouse embryonic stem cells
(Katoh and Katoh, 2007). In
addition, purified Wnt5a activates canonical signaling in the presence of the
frizzled 4 receptor, and suppresses it in the presence of the Ror2 receptor
(Mikels and Nusse, 2006). One
possibility that has not been considered previously is that Wnt proteins may
act together to activate signaling pathways.
The current model for Wnt function in dorsal/ventral axis signaling in
Xenopus embryos is that, after fertilization, vegetally localized
Wnt11 mRNA is moved by cortical rotation movements and becomes
enriched on the dorsal versus the ventral side of the fertilized egg
(Tao et al., 2005). During
cleavage, Wnt11 is secreted by dorsal vegetal cells and activates signaling
through a canonical Wnt pathway, so that on the dorsal side, newly synthesized
β-catenin is more stable, interacts with XTcf3 in dorsal nuclei and
activates Wnt target gene expression at MBT. However, although there is more
Wnt11 mRNA dorsally at the 32-cell stage, there is a significant
amount of ventral Wnt11 mRNA (Tao
et al., 2005), which raises the question why is there no
Wnt11 target gene activation on the ventral side of the embryo? This
study was initiated to determine whether the maternally encoded Wnt antagonist
Dkk1 plays an important role in limiting the site and amount of activation of
maternal Wnt target genes.
Dkk1 belongs to a small family of 24-29 kDa secreted glycoproteins
(Niehrs, 2006). Zygotic
Dkk1 mRNA is necessary and sufficient for head formation in
Xenopus (Glinka et al.,
1998), and is implicated as a tumor suppressor in mammals
(Niehrs, 2006). Dkk1 acts as a
Wnt antagonist indirectly, by preventing the Wnt11-dependent interaction of
LRP and frizzled (Bafico et al.,
2001; Semenov et al.,
2001). Hitherto, Dkk1 function has been considered to be specific
for canonical rather than non-canonical Wnt signaling
(Semenov et al., 2001),
although recently it was shown to bind glypican 4 and to activate
non-canonical signaling (Caneparo et al.,
2007). Here, we demonstrate that maternal Dkk1 antagonizes both
canonical and non-canonical signaling to regulate the dorsal/ventral
patterning of the early Xenopus embryo.
As Wnt5a has been shown to affect non-canonical Wnt signaling during
gastrulation in Xenopus (Moon et
al., 1993), we reasoned that maternal Wnt5a may be the
non-canonical Wnt regulated by maternal Dkk1. Surprisingly, depletion of
maternal Wnt5a resulted in the same ventralized phenotype as that we reported
previously for maternal Wnt11 depletion
(Tao et al., 2005). This
result suggested that both Wnt11 and Wnt5a are required for axis formation in
the early embryo. We show here that Wnt5a acts together with Wnt11 in the
initial signaling event that activates canonical, β-catenin-dependent,
Xnr3, siamois and Xnr5 expression, as well as in
non-canonical JNK activation of the morphogenetic movements of gastrulation.
We demonstrate that Wnt11 and Wnt5a interact in both biochemical and
functional tests. The results suggest that complexes formed between Wnt5a
homodimers and Wnt11 homodimers are required to activate the dorsal signaling
pathway in the early embryo.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Antisense
oligos were injected on day 1, oocytes were matured on day 3 and fertilized on
day 4. For rescue experiments, mRNA was injected on day 3. Antisense oligos
were: Dkk1 AS,
5'-g*g*a*gttggtcatcat*g*a*c-3';
Wnt11 AS,
5'-g*t*c*ggagccattggt*a*c*t-3';
Wnt5a AS,
5'-c*c*a*gacagttggctg*c*a*c-3';
and β-catenin morpholino, 5'-tttcaaccgtttccaaagaaccagg-3'.
Where * indicates a phosphorothioate bond.
Quantitative RT-PCR and in situ hybridization
Total RNA from oocytes, explants and early embryos was isolated using the
protocol of Tao et al. (Tao et al.,
2005). Real-time RT-PCR were performed using a LightCycler
(Roche). Water-blank and RT-minus controls were included in all runs. All
RT-PCR results are presented as percentage compared with the level in
uninjected embryos after normalization to the expression of ornithine
decarboxylase (ODC). Whole-mount in situ hybridization was as described
(Birsoy et al., 2006).
Luciferase assay
TOPflash DNA (Upstate; 50 pg), together with 25 pg pRLTK DNA, was injected
into two dorsal or ventral cells at the four-cell stage. Three replicate
samples each of three embryos were frozen for each group at the early gastrula
stage and assayed using Promega luciferase assay system.
Western blot
Western blots were carried out as described previously
(Birsoy et al., 2006).
Antibodies concentrations were: rabbit anti-pJNK, 1/750; rabbit anti-total
JNK, 1/750; rabbit anti-pSmad1, 1/500; rabbit anti-pSmad2, 1/500 (all from
Cell Signaling); and mouse anti-tubulin (DM1A, Neomarker), 1/5000.
Dimerization assays
Tagged mRNAs were injected at doses 100 pg-1 ng, and embryos were frozen at
stage 10 in batches of five, lysed in 50 µl (10 µl/embryo) of ice-cold
PBS-Triton buffer (1xPBS, pH 7.4; 1% Triton X-100) supplemented with
Iodoacetamide (IAA, 10 mM). Clear lysate was obtained by a 10-minute
centrifugation (14,400 g at 4°C), and was mixed either
with sample buffer without β-ME (non-reducing condition) or with sample
buffer containing 10% β-ME (reducing condition). Samples were processed
by standard SDS-PAGE and western blotting. Antibodies: rat anti-HA (clone
3F10, Roche), 1/2000; rabbit anti-Myc (Cell Signaling), 1/1000; rabbit
anti-Flag (Sigma), 1/2000; mouse anti-GFP (clone B-2, Santa Cruz
Biotechnology), 1/500.
Co-immunoprecipitation
Embryos were frozen at stage 10 in batches of 50 and lysed with 1 ml (20
µl/embryo) ice-cold PBS-Triton buffer. The homogenate was spun at 500
g for 5 minutes at 4°C; the supernatant was collected in a
new tube and spun at 14,400 g for 10 minutes at 4°C. The
clear lysate was mixed with protein-A or protein-G agarose beads coated with
the antibody of interest and incubated for 2 hours at 4°C. The beads were
pelleted, washed four times with ice-cold lysis buffer, mixed with minimum
volume of SDS-PAGE sample buffer and processed through standard
electrophoresis and western blot protocol using ice-cold CAPS buffer (0.2%
CAPS, 15% methanol, pH 10.5) for wet transfer.
Generation of Wnt11
C and Wnt11-GFP
Wnt11C was generated as a flag-tagged EcoRI/XhoI
fragment by PCR amplification of nucleotides 1-846 (amino acids 1-282) of
Wnt11 ORF and inserted in the pCS107 vector. For Wnt11-GFP, the full Wnt11 ORF
was amplified as an EcoRI/BamHI fragment and inserted behind
and in frame with the GFP gene in the pEGFP-N1 vector. Wnt11 and GFP were
excised as a single fragment by EcoRI/NotI digestion, and
inserted into pCS107 vector. Wnt11C and Wnt11-GFP constructs were
linearized (NsiI) and transcribed with Sp6 polymerase.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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An antisense oligonucleotide was effective in depleting Dkk1 mRNA to less than 20% of control levels in stage 6 oocytes (Fig. 1D). In a temporal expression series comparing sibling control and Dkk1-depleted oocytes and embryos, Dkk1 mRNA expression remained low in oligo-injected embryos until the onset of zygotic transcription, when zygotic Dkk1 mRNA was expressed in both control and oligo-injected groups (Fig. 1A).
When sibling control oocytes and oocytes injected with 5, 7.5 and 10 ng of Dkk1AS oligo were fertilized, they developed normally to the late blastula stage, when Dkk1-depleted embryos underwent abnormal shape changes. During gastrulation and neurulation, devitellined embryos cultured on agar became extremely elongated compared with uninjected sibling controls (Fig. 2A). These effects were specifically due to Dkk1 mRNA depletion, as they were partially rescued by injecting human Dkk1 mRNA (20 pg) into Dkk1-depleted oocytes before maturation (Fig. 2B). As Dkk1 is a Wnt antagonist, we tested whether its depletion upregulated the maternal canonical Wnt signaling pathway. Fig. 2C shows that Dkk1 depletion caused the upregulation of direct Wnt target genes expression (Xnr3 and Xnr5) compared with their expression in sibling control uninjected embryos, which was rescued by human Dkk1 mRNA (20 pg) (Fig. 2C).
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Increased expression of Xnr3 and Xnr5 in Dkk1-depleted embryos could be due to enhanced Wnt signaling in the correct site of expression, or to Wnt signal to ectopic sites, or both. Whole-mount in situ hybridization analysis at the early gastrula stage showed that Xnr5 mRNA was enriched in the usual dorsal vegetal location, and was also ectopically expressed in the ventral vegetal area in Dkk1-depleted embryos (Fig. 2F). Xnr3 mRNA was present in a narrower longer area of the dorsal equatorial and vegetal region compared with sibling uninjected control early gastrulae (see Fig. S1 in the supplementary material). Confirming the ectopic activation of canonical signaling, TOPflash reporter activity was enhanced in ventral half embryos at the late blastula stage (see Fig. S2 in the supplementary material) and Xnr5 was increased on the ventral side by RT-PCR (see Fig. S3 in the supplementary material).
Increased activation of the direct Wnt target genes should cause enhanced
activation of secondary targets (Xanthos
et al., 2002), which was seen for the dorsal markers chordin,
goosecoid and Hex (Fig.
2G and data not shown). These experiments show that Dkk1 regulates
both the level and position of expression of canonical Wnt target genes.
Xnrs activate each other's expression, increasing Smad2 signaling
(Hyde and Old, 2000;
Takahashi et al., 2000). We
therefore analyzed the Smad2 phosphorylation state of Dkk1-depleted and
sibling control embryos at the late blastula and early gastrula stages, using
dissected dorsal and ventral halves. P-Smad2 was increased in both dorsal and
ventral half embryos compared with controls
(Fig. 2H).
Increased levels of chordin expression in Dkk1-depleted embryos might abrogate signaling in the BMP pathway. Fig. 2H shows that the level of phosphorylated Smad1 was decreased in ventral and dorsal Dkk1-depleted half embryos compared with controls. These experiments show that the loss of Wnt antagonism caused by maternal Dkk1 depletion has profound effects on other signaling networks within 2 hours of MBT.
Dkk1 inhibits non-canonical Wnt signaling in axis formation
To determine whether the observed effects of Dkk1 depletion were due only
to the hyper-activation of the canonical Wnt signaling pathway, we compared
embryos depleted of both Dkk1 and β-catenin with those depleted of each
alone. If the phenotype caused by Dkk1 depletion were completely dependent on
canonical signaling, double-depleted embryos should phenocopy the ventralized
β-catenin-depleted phenotype. However,
Fig. 3A shows that some aspects
of the Dkk1 depletion phenotype were β-catenin independent. Specifically,
at the late blastula and early gastrula stages, Dkk1/β-catenin-depleted
embryos underwent the same abnormal shape changes as Dkk1-depleted siblings
(Fig. 3A). The embryos began to
elongate along the animal-vegetal axis compared with wild-type or
β-catenin depleted siblings, and formed a constricted region in the
equatorial zone, such that they developed a `mushroom' shape at stage 9. By
contrast, loss of canonical Wnt target gene expression occurred in
Dkk1/β-catenin-depleted embryos, as it did in β-catenin-depleted
siblings (Fig. 3C). In
addition, by the neurula and tailbud stages, the Dkk1/β-catenin-depleted
embryos phenocopied their ventralized β-catenin-depleted siblings,
suggesting that later cell movements were dependent on signaling through
β-catenin (Fig. 3B).
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The fact that Dkk1-depleted embryos continued to undergo abnormal
gastrulation movements in the absence of β-catenin suggested that Dkk1
may also regulate non-canonical Wnt signaling. As JNK activation is a key
component in non-canonical signaling
(Schambony and Wedlich, 2007;
Yamanaka et al., 2002), we
next investigated JNK activity. We found that there was a sustained increase
in the level of phospho-JNK1 compared with controls in the late blastula and
early gastrula stages in Dkk1-depleted embryos
(Fig. 3E). This increase was
localized to the equatorial zone (presumptive mesoderm) compared with the
vegetal mass (presumptive endoderm) in equatorial and vegetal explants
(Fig. 3F).
As Dkk1/β-catenin-depleted embryos and explants underwent extreme morphogenetic movements, we asked whether they maintained higher levels of p-JNK compared with controls. Fig. 3G shows that Dkk1/β-catenin-depleted embryos have increased levels of p-JNK compared with uninjected sibling controls. To confirm the dependence on enhanced JNK1 activity, Dkk1-depleted equatorial zones were cultured with or without the JNK inhibitor SP600125. In the presence of SP600125, the excessive elongation of Dkk1-depleted equatorial zones was significantly reduced (Fig. 3H). Together, these experiments show that Dkk1 acts to limit the extent of non-canonical Wnt signaling, leading to morphogenetic movement via JNK-1 activity.
Maternal Wnt5a and Wnt11 are both required for canonical and non-canonical signaling in the early Xenopus embryo
As Wnt5a is involved in non-canonical Wnt signaling during gastrulation in
Xenopus (Moon et al.,
1993; Schambony and Wedlich,
2007), we reasoned that maternal Wnt5a may be the non-canonical
Wnt regulated by maternal Dkk1. We designed an antisense oligonucleotide that
was effective in depleting Wnt5a mRNA, and did not degrade maternal
Wnt11, β-catenin or Dvl2 mRNA
(Fig. 4A). We confirmed that
zygotic Wnt5a mRNA does not begin to accumulate until after
gastrulation in control uninjected embryos
(Fig. 4B)
(Moon et al., 1993), such that
any loss-of-function phenotypes caused before this time must be the result of
the loss of maternal Wnt5a. Sibling control oocytes and maternal
Wnt5a-depleted oocytes were fertilized and developed normally to the gastrula
stage. Wnt5a-depleted embryos gastrulated without forming axial structures,
showing the `ventralized' phenotype typical of maternal Wnt11- and
β-catenin-depleted embryos (Fig.
4C) (Heasman et al.,
1994; Tao et al.,
2005). Axis formation was partially rescued by the reintroduction
of Wnt5a mRNA before fertilization
(Fig. 4D). Wnt5a-depleted
embryos had reduced expression of the canonical Wnt target genes Xnr3
and Xnr5, which was rescued by the reintroduction of Wnt5a
mRNA, showing the specificity of the phenotype
(Fig. 4E). We confirmed that
Wnt5a depletion affected canonical Wnt signaling as endogenous TOPflash
activity was reduced at the late blastula stage after Wnt5a depletion
(Fig. 4F). Furthermore,
Wnt5a mRNA overexpression caused an upregulation of TOPflash, and
this was completely dependent on the presence of β-catenin protein
(Fig. 4G). These experiments
show that maternal Wnt5a, like maternal Wnt11, is essential for activation of
the canonical Wnt pathway required for axis formation.
We next asked whether maternal Wnt11 or Wnt5a is required for the non-canonical Wnt pathway. Embryos depleted of either Wnt11 or Wnt5a were analyzed at the early and late blastula stages for activated JNK activity. Fig. 4H shows that p-JNK1 levels were reduced by either Wnt11 or Wnt5a depletion, suggesting that both are required for non-canonical pathway activity.
To confirm whether both maternal Wnt11 and Wnt5a are essential for activating the signaling pathways leading to axis formation, we examined the effect of double depletions of Dkk1 together with either Wnt11 or Wnt5a. If Wnt11 and Wnt5a are both required, then depleting either should abrogate Dkk1 hyperactivation activity and cause ventralization and loss of p-JNK. If one Wnt signaling pathway is separate from the other, and antagonized by Dkk1, Dkk1 depletion should enhance the activity of the remaining Wnt. As shown in Fig. 4H-J, the double depletions of both Dkk1/Wnt11 and Dkk1/Wnt5a caused the same phenotype as the single Wnt depletion: i.e. reduced p-JNK1 and loss of expression of canonical Wnt target genes. The Dkk/Wnt-depleted embryos developed with a ventralized phenotype (see Fig. S5 in the supplementary material). This indicates that both maternal Wnt11 and 5a are required for activating the signaling pathways leading to axis formation.
As Pol2 transcription is not active until MBT
(Dunican et al., 2008), and
maternal Wnt11 is expressed in Wnt5a-depleted oocytes
(Fig. 4A) and vice versa (data
not shown), maternal Wnt5a is not upstream or downstream of maternal Wnt11 in
a transcriptional sense before MBT. In addition, after MBT, the maternal
depletion of Wnt5a does not affect the level of Wnt11 at the early gastrula
stage (data not shown). One possible explanation of the similarity in
phenotype caused by their depletion is that Wnt5a mRNA could maintain
the localization of Wnt11 mRNA in the vegetal cortex of the oocyte
(Ku and Melton, 1993) or in
its translocation to the dorsal side after fertilization
(Schroeder et al., 1999).
However, neither Wnt11 mRNA distribution in the oocyte nor its dorsal
enrichment at the 32-cell stage was altered by Wnt5a depletion
(Fig. 4K,L; data not
shown).
Wnt11 and Wnt5a interact in physical and functional complexes
Next we tested whether Wnt11 and Wnt5a physically interact in
co-immunoprecipitation assays after co-injection of Wnt11-HA and
Wnt5a-Myc mRNA. Fig.
5A shows that Wnt11-HA protein was detected in complexes with
Wnt5a-Myc and that the immunoprecipitation of Wnt11-HA by the anti-Myc
antibody was Wnt11 specific, as the Nodal-related protein Xnr2-HA did not
interact. To test whether Wnt11 and Wnt5a non-specifically interacted during
synthesis, we repeated the immunoprecipitation after co-injecting Wnt11-HA and
Wnt5-Myc either into the same blastomere or into two adjacent blastomeres of
four-cell stage embryos (Fig.
5B). Fig. 5C shows
that Wnt11-HA protein was detected in complexes with Wnt5a-Myc, even when the
two mRNAs were injected into two separate cells (left panel). Similarly,
Wnt5a-HA was detected in complexes with Wnt11-GFP (right panel). This suggests
that Wnt11 and Wnt5a can interact extracellularly after secretion, as the Wnt
proteins were detected in complexes even when the mRNAs were injected in
different cells.
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). However,
when more physiological doses of the mRNAs were injected (2.5-10 pg), either
Wnt alone repressed endogenous TOPflash activity at the late blastula stage,
whereas co-injection of a mixture of 5 pg of Wnt11 and 2.5 pg Wnt5a mRNAs
doubled TOPflash activity compared with the endogenous Wnt signal
(Fig. 5E). This result was
repeated three times and suggests that, at physiological levels, complexes of
exogenous Wnt11+ 5a enhances endogenous canonical signaling, whereas exogenous
Wnt11 alone or Wnt5a alone interferes with the endogenous Wnt complex
stoichiometry and represses signaling.
Wnt11 and Wnt5a form homodimers but not heterodimers
Previous studies purifying Wnt1 (Int-1) and Wnt8 protein showed that they
form large complexes (>600 KkDa) in conditioned medium
(Dann et al., 2001;
Papkoff, 1989), and size
exclusion chromatography assays on Wnt3a suggested the active form was
monomeric (Willert et al.,
2003). However, no previous studies have tested the interaction of
two Wnts in functional and biochemical assays. In addition, the Wnt receptor
Frizzled forms dimers, and ligand-induced multimerization of Frizzled
receptors drives signal transduction
(Carron et al., 2003;
Dann et al., 2001). Therefore,
we asked whether Wnt ligands form homo- and heterodimers. Overexpression of
Wnt11-HA mRNA produced both a monomer-size band (50 kDa) and a
dimer-size band (100 kDa) when the gastrula lysate was analyzed in
non-reducing conditions (Fig.
6A). The dimer was lost when reducing agent β-mecaptoethanol
(β-ME) was added in the sample buffer, suggesting that the Wnt11 protein
dimerizes by forming disulphide bonds. To test whether the disulphide bonds
formed during the sample lysis, we supplemented the lysis buffer with 10 mM
iodoacetamide (IAA), which inactivates free cysteine side-chains.
Fig. 6B shows that the
dimer-sized band was still detected under these conditions. Wnt5a and Wnt8
proteins also formed dimers in non-reducing conditions
(Fig. 6C), and for Wnt5a,
dimers predominated over monomers.
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C, 30 kDa)
(Tada and Smith, 2000) and a
GFP-tagged Wnt11 (Wnt11-GFP, 75 kDa). As shown in
Fig. 6E, when Wnt11C was
overexpressed, homodimers still formed (suggesting that the C terminus is not
required for dimerization), but Wnt11C did not form heterodimers of the
expected size (marked by an asterisk in
Fig. 6E) with either
full-length Wnt11-HA or Wnt5a-Myc. To test whether GFP-tagged Wnt11 and Wnt5a-Myc heterodimerized, we injected various doses of Wnt5a-Myc mRNA together with 100 pg of Wnt11-GFP mRNA. Fig. 6D shows that Wnt11-GFP dimerizes when injected alone, but there was no change in the ratio of monomer to dimer or in their amounts in the presence of Wnt5a. Moreover, no band equivalent to Wnt11-GFP and Wnt5a-Myc heterodimer (125kDa) could be detected in co-injection samples (Fig. 6D). Thus, although dimerization may be a general property of Wnt ligands, we found no evidence suggesting that Wnt11 and Wnt5a form heterodimers. However, the fact that there was no cross-binding between Wnt11 and Wnt5a, even when the proteins were synthesized together in the same cells, strongly argues against the idea that Wnt proteins are `sticky' and interact non-specifically because of their many cysteine residues.
To determine which molecular form of Wnt11 complexes with Wnt5a, we analyzed the immunoprecipitate of Wnt 11-HA and Wnt5a-myc in the presence or absence of reducing agent. Fig. 6F shows that, in non-reducing conditions, Wnt5a-Myc mainly immunoprecipitates dimers and oligomers of Wnt11-HA, and not monomers. When disulphide bonds were broken by β-ME addition, only monomeric Wnt11-HA was seen. This suggests that the co-IP between Wnt11 and Wnt5a occurs between Wnt11 and Wnt5a dimers or oligomers, and not between monomers.
Finally, as maternal Dkk1 acts as a Wnt antagonist, we asked how Dkk1
depletion affected the levels of dimer and/or monomer form of Wnt11-HA or
Wnt5a-Myc protein when each mRNA was overexpressed in oocytes. Here, matured,
non-matured uninjected and Dkk1-depleted oocytes were compared, as oocyte
maturation often stimulates protein synthesis
(Richter et al., 1982). When
Wnt11-HA mRNA and Wnt5a-myc mRNA were injected into
Dkk1-depleted oocytes, more Wnt protein, particularly dimeric Wnt protein
(*) was detected in non-reducing conditions compared with injection
into control oocytes (Fig. 6G).
Thus, loss of Dkk1 caused an increase in the total amount of Wnt dimer,
presumably by increasing Wnt protein stability or reducing its turnover. This
was not due to increased Wnt transcription as the stage 6 oocyte is
transcriptionally inactive.
Taken together, Figs 5 and 6 suggest that secreted homodimers of Wnt11 and homodimers of Wnt5a form non-covalently linked higher molecular weight complexes to activate the canonical and non-canonical processes involved in axis formation (Fig. 7).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Previous studies have suggested similarities in Wnt11 and Wnt5a function.
When Wnt11 was depleted from mesodermal stem cells, Wnt5a was able to
substitute for Wnt11 and rescue a broad range of blood cell types
(Brandon et al., 2000).
Overexpressed Wnt11 and Wnt5a activate canonical or non-canonical signaling in
different co-receptor contexts (Heisenberg
et al., 2000; Mikels and
Nusse, 2006; Tao et al.,
2005). In addition, both Wnt11 and Wnt5a physically interact with
the frizzled 7 receptor and FRL1 co-receptor
(Djiane et al., 2000;
Tao et al., 2005). Here, we
suggest that that, at least in the signaling pathway involved in dorsal axis
formation, Wnt11 and Wnt5a form a complex and function together.
The extent to which the effects of maternal RNA depletion of Dkk1, Wnt5a and Wnt11 last beyond the onset of zygotic transcription
For canonical signaling, the earliest effects caused by Dkk1, Wnt5a and
Wnt11 depletions were on the stabilization of β-catenin (stage 8), on
Tcf3-reporter activity (TOPflash activity; stage 8.5), and on siamois,
Xnr3 and Xnr5 expression (stage 9). These changes are known to
be dependent on Wnt signaling during the cleavage stages
(Xanthos et al., 2002;
Yang et al., 2002;
Tao et al., 2005). Thus, Dkk1,
Wnt5a and Wnt11 depletion first causes effects pre-MBT. At MBT, once the
XTcf-regulated transcriptional network is activated, the indirect effects of
the up- (in the case of Dkk1 depleted) and downregulation (for Wnt11/5a) of
these canonical Wnt targets become complex, including effects on Nodal
signaling and on BMP antagonists (chordin), which in turn affect gastrulation
movements. In addition, after the depletion of the maternal pools of Dkk1,
Wnt5a and Wnt11, the total amount of mRNA (zygotic+ maternal) during
gastrulation remains lower than in wild-type sibling embryos. Thus, some
effects may be due to the lack of direct maternally encoded Wnt
signaling/signaling antagonism during gastrulation. This is particularly true
for Wnt5a, as zygotic transcripts do not begin to accumulate until the end of
gastrulation (Moon et al.,
1993), compared with early gastrula for Wnt11 and Dkk1. Here, we
have concentrated on examining further the mechanism of the interaction of
Wnt11 and Wnt5a with regards to canonical signaling, as the TOPflash assay is
an early and direct measure. However, it will be equally important to
establish whether, when maternal Wnt5a signals during gastrulation, it is also
acting in a complex with Wnt11.
The mechanism of interaction of Wnt11 and Wnt5a
Unlike Wnt11 mRNA, Wnt5a mRNA is not a localized mRNA in
oocytes or early embryos. The simplest hypothesis to explain the dependence of
dorsal signaling on both Wnt11 and Wnt5a is that, in the cleavage stage
embryo, the dorsal area where Wnt11 is enriched and Wnt5a is co-expressed, is
the only quadrant where a threshold is passed allowing transduction of the Wnt
signal (Fig. 7). In this model,
the crucial asymmetry that sets up the dorsal dominance of gastrulation
movement and of organizer gene expression is the cortical rotation movement of
the first cell cycle that sweeps vegetally localized Wnt11 mRNA away
from the sperm entry point, leading to more Wnt11 protein (shown in yellow)
being secreted by the dorsal vegetal quadrant. Wnt5a protein (shown in blue)
is a necessary but constitutive signaling partner in the dorsal axis pathway.
The size of the dorsal Wnt signaling zone is further limited by the activity
of secreted Dkk1 protein. We assume here that Wnt 11/Wnt5a interaction and Dkk
antagonism occur extracellularly, as they are all secreted proteins. In
addition, we show that Wnt11 and Wnt5a physically interact by
co-immunoprecipitation studies, where the mRNAs are injected into different
cells, so that the proteins can only come together after secretion.
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As dimerization is important for the receptor Frizzled to activate downstream signaling, proper dimerization of Frizzled may be a prerequisite for such a multimeric complex to be fully active. One suggestion is that the minimum active Wnt signaling unit during endogenous dorsal signaling at the cleavage stage consists of a multimeric complex of four Wnts (a homodimer of Wnt11 and a homodimer of Wnt5a) bound to eight frizzled proteins (four dimers) (Fig. 7).
When either Wnt11 or Wnt5a is overexpressed (100-500 pg), each
hyperactivates TOPflash and causes hyperdorsalization, showing that either
protein alone acting in excess can activate the canonical pathway. It is
likely that the excess exogenous protein dominates over the endogenous
Wnt11/5a mechanism, which is likely to be acting with much smaller amounts of
protein. Frizzled 7 is ubiquitous in the early Xenopus embryo, and it
interacts with both Wnt11 and Wnt5a
(Witzel et al., 2006) (and
data not shown). The endogenous Wnt pool is very sensitive to exogenously
supplied Wnt protein, as 2.5-10 pg of injected Wnt11 or
Wnt5a mRNA inhibited endogenous signaling, whereas a mixture of 2.5
pg Wnt5a + 5 pg of Wnt11 mRNA increased TOPflash above
endogenous levels. This suggests that, at physiological doses, the exogenous
individual Wnt may act in an inhibitory fashion on the endogenous signaling
complex, whereas exogenous complexes containing both Wnt11 and Wnt5a
homodimers synergize with the endogenous signal.
In summary, we have shown here a novel aspect of Wnt signaling: two Wnts required for both canonical and non-canonical signaling in a pathway that is essential for establishing the dorsal asymmetry of the embryo. We have suggested that the mode of activity is via non-covalently linked tetrameric and oligomeric complexes made up of homodimers of Wnt11 together with homodimers of Wnt5a. An important issue remaining is whether this is a paradigm for Wnt signaling activity in many other embryonic and adult tissues where two Wnts are expressed in overlapping territories.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/22/3719/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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-tubulin as a loading control. Quantitation is shown on the right.
(F,G) In situ hybridization of sibling control and Dkk1-depleted
early gastrulae (Xnr5, F; chordin, G). (H) Western
blot of phospho-Smad2 and -Smad1 proteins in control and Dkk1-depleted sibling
at late blastulae (stage 9.5) and early gastrulae (stage 10.5). Before
freezing, embryos were hemisected into batches of four dorsal (dor) and four
ventral (ven) halves.
