|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 3 January 2007
doi: 10.1242/dev.02739
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Division of Developmental Biology, Cincinnati Children's Research Foundation,
3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
2 Department of Biological Sciences, The University of Iowa, 257 Biology
Building, Iowa City, IA 52242-1324, USA.
* Author for correspondence (e-mail: heabq9{at}chmcc.org)
Accepted 14 November 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Xenopus, ß-catenin, Wnt11, LRP6, Axis formation
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). LRP6 is required in the mouse for midbrain, axis and limb
patterning (Kelly et al.,
2004), and a mis-sense mutation in LRP6 causes
folatesensitive neural tube defects in the Crooked tail mutant
(Carter et al., 2005). LRP6 is
upregulated in several human tumor cell lines
(Li et al., 2004;
Logan and Nusse, 2004).
Despite its importance in development and disease, the mechanism of LRP6
function is incompletely understood. LRP6 activity in Wnt signaling requires
binding of the intracellular protein, axin, to its cytoplasmic domain through
a phosphorylationdependent process involving GSK-3ß and CK1
(Davidson et al., 2005;
Tamai et al., 2000;
Tamai et al., 2004;
Wehrli et al., 2000;
Zeng et al., 2005). Axin is an
essential component of the canonical Wnt signaling pathway, and modulates the
level of cytoplasmic ß-catenin by targeting it for phosphorylation and
degradation. When axin function is blocked by Wnt-pathway activation or by
antisense depletion of axin mRNA, ß-catenin is stable and enters
nuclei to initiate the transcription of target genes
(Kofron et al., 2001;
Nusse, 2005;
Willert et al., 1999a).
However, the regulation of axin function by LRP proteins is not clear. LRP may
bind to axin and sequester it from the ß-catenin-destruction complex
(Nusse, 2005), or may promote
its degradation, thus inactivating the ß-catenin-degradation complex
(Mao et al., 2001;
Tolwinski et al., 2003;
Willert et al., 1999b).
Alternatively, in the absence of Wnt signaling, axin may constitutively
shuttle ß-catenin out of the nucleus; Wnt signaling blocks shuttling by
binding LRP to axin, allowing nuclear ß-catenin to rise
(Cong and Varmus, 2004;
Wiechens et al., 2004). To
determine which mechanism works in axis formation requires an analysis of
endogenous axin in wild-type and LRP6-depleted embryos.
Here, we analyze the function of LRP6 in the dorsal axisspecifying pathway
in early Xenopus embryos. In Xenopus, the most recent model
of the mechanism of axis formation involves Wnt signaling during the cleavage
stage (Tao et al., 2005). This
model suggests that the axis-forming pathway is activated by Wnt11 because the
protein is necessary and sufficient for dorsal-axis formation, is stored as a
maternal transcript in the vegetal cortex of the oocyte, and is enriched in
dorsal cells of the embryo at the 32-cell stage
(Schroeder et al., 1999;
Tao et al., 2005). However,
the timing and mechanism of signal transduction is not known. The frizzled
family member Xfz7, the EGF-CFC protein FRL1 and heparan sulphate proteoglycan
activity are required downstream of Wnt11
(Sumanas et al., 2000;
Tao et al., 2005). LRP6 has
previously been implicated in this pathway because it is maternally expressed
(Houston and Wylie, 2002) and
because over-expression of LRP6 on the ventral side of the 4-cell-stage embryo
causes the formation of a partial second axis
(Tamai et al., 2000). However,
when injected into early embryos, a dominant-negative form of LRP6, lacking
the cytoplasmic domain, does not interfere with endogenous axis formation,
suggesting either that this Wnt signaling pathway does not require LRP6, or
that signaling occurs earlier in development and cannot be blocked by the
expression of the dominant-negative construct
(Tamai et al., 2000).
Axin is an essential negative regulator of canonical Wnt signaling
pathways, acting by causing the degradation of ß-catenin
(Hamada et al., 1999;
Lee et al., 2003;
Salic et al., 2000;
Willert et al., 1999a). In
Xenopus, depletion of axin mRNA causes an increased nuclear
localization of ß-catenin, increased expression of Wnt target genes,
including siamois and Xnr3, and radially dorsalized
phenotypes (Kofron et al.,
2001). In Xenopus egg extracts, axin is estimated to be
present at low levels (picomolar quantities) compared with ß-catenin,
GSK3ß and disheveled (nanomolar quantities), and ß-catenin
degradation is extremely sensitive to small changes in axin concentration
(Lee et al., 2003;
Salic et al., 2000). Injection
of only 6 pg of axin mRNA is sufficient to rescue the dorsalized
phenotype of axin-depleted Xenopus embryos
(Kofron et al., 2001).
Here, we test the hypothesis that LRP6 is the necessary receptor for Wnt11 signal transduction and that it acts by degrading axin. We show first that LRP6 is essential for Wnt11-activated dorsal-axis formation in Xenopus. Second, we show that axin protein levels are increased when LRP6 is depleted, indicating that LRP6 regulates the degradation of axin. Third, we identify a specific time in early development, the 8-cell stage, when axin levels are reduced on the dorsal side of the wild-type embryo, indicating the time of transduction of the Wnt signal. Finally, we present evidence that Wnt11 and LRP6 also act in oocytes to regulate axin, maintaining ß-catenin in a low, steady-state level. This suggests that canonical Wnt signaling pathways may have three states (off, steady-state and transcriptional activation) rather than the generally accepted two (off and transcriptional activation).
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Collagenase treatment
Oocytes were washed in Ca2+-free 1xMMR (100 mM NaCl, 2 mM
KCl, 15 mM HEPES) and nutated in 0.02 mg/ml collagenase (Sigma Type 1) in
Ca2+-free MMR +1.6 mM CaCl2. Denuded oocytes were washed
in 1xMMR and then in OCM.
Inhibition of proteasomal-mediated protein degradation
Oocytes were treated with MG132 (Sigma) as described by Dupont et al.
(Dupont et al., 2005). A
concentration of 1:100 MG132 was made in sterile water from a stock of 10 mM
MG132 in DMSO and 10 nl was injected into oocytes. Injected oocytes were
cultured in OCM containing 10 µM MG132 for 2-3 days. Oocytes were harvested
for western blots in lysis buffer containing 10 µM MG132.
Oligos and mRNAs
Antisense oligo for LRP6
5'-T*C*G*AGGCTGATCCAG*C*T*C-3';
for Wnt11
5'-G*T*C*GGAGCCATTGGT*A*C*T-3';
and for Axin
5'-T*T*C*CTCGCCAGGAAC*T*G*G-3',
where phosphorothioatemodified residues are indicated by an asterisk
(*).
Oligos were resuspended in sterile distilled water and injected in doses of
4-5 ng per oocyte. Mouse LRP6 was subcloned into pRN3, linearized
with SfiI and transcribed with T3 message machine (Ambion).
Xenopus pCS2+ Myc-ß-catenin plasmid was linearized with
NotI, and pSP64T-Wnt11 with XbaI, and transcribed with SP6
message machine kit (Ambion). Analysis of gene expression using real-time
RT-PCR was as described by Tao et al. (Tao
et al., 2005). The ß-catenin constructs used for
Fig. 7F,G,H were either
wild-type or stabilized ß-catenin in pCS2-MT
(Yost et al.1996). The
stabilized ß-catenin lacks the four N-terminal Gsk3ß phosphorylation
sites because of their mutation from serine to alanine.
Western blotting
In total, five embryos or oocytes were homogenized in 200 µl of ice-cold
PBS plus protease inhibitors (PIC P8340 Sigma 1:100, PMSF 10 µg/ml) and
spun for 5 minutes at 700 g, 4°C. Supernatants were
precipitated with acetone for 20 minutes on ice. Samples were spun for 5
minutes at 3000 g, 4°C. Pellets were resuspended in 50
µl 2xSDS sample buffer and boiled for 5 minutes. A total of 18 µl
was loaded onto 7.5% Tris-glycine ready gel (Bio-Rad) and electrophoresed for
2.5 hours at 75 V. Protein was transferred to nitrocellulose membranes and
blocked overnight in PBS-Tween 0.1%, 5% dry milk at 4°C. Antibody
conditions were anti-axin 1067 AP 1:400 (gift from Peter Klein, University of
Pennsylvania, Philadelphia, PA), anti-ß-catenin: Sigma C2206 1:2000,
Roche anti-c-myc clone 9E10 1:2000. Membranes incubated with HRP-conjugated
antibodies were incubated with ECL developing solution (Amersham) and exposed
to X-ray film at variable times to avoid the saturation of bands (Hyperfilm,
Amersham). Quantification was performed using IPlab densitometry software,
normalized to 
-tubulin levels and values expressed as the percentage
change relative to controls. Blots were re-probed with a FITC-conjugated
anti--tubulin antibody (Sigma F2168) at 1:4000 for loading control, and
imaged on a Typhoon variable mode imaging system.
Co-immunoprecipitation
Animal cells of embryos were injected with 500 pg Wnt11-HA mRNA
and 1.5 ng LRP6-N-myc RNA (lacking the carboxyl intracellular domain
(Tamai et al., 2000) at the 4-
to 8-cell stage. Adjacent blastomeres were injected such that only secreted
protein could interact in order to avoid non-specific interactions between the
two proteins in the ER/golgi complex. Embryos were harvested at stage 10.5 and
centrifuged to remove yolk protein. Co-immunoprecipitation was carried out as
described previously (Tao et al., 2005). The anti-HA high-affinity rat
antibody (3F10, Roche) was used to pull-down Wnt11-HA protein. Lrp6-N-myc
protein was detected using the anti-c-myc antibody (9e10, Roche). Detection of
proteins in western blots used HRP-conjugated anti-rat or -mouse antibodies
and ECL plus (GE healthcare, RPN2132). Blots were visualized with ECL
fluorescence on a Molecular Dynamics typhoon phosphor imager.
Luciferase assays
TOPflash DNA (60 pg) containing three copies of the TCF-binding site
upstream of a minimal TK promoter and luciferase open reading frame, together
with 20 pg pRLTK DNA (Renilla reniformis luciferase used as an internal
control), was injected into two dorsal cells at the 4-cell stage of control
and LRP6-depleted embryos. Three replicate samples, each of four embryos, were
frozen at the late blastula stage and luciferase assays were carried out using
the Promega luciferase assay system.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
When LRP6-depleted oocytes were fertilized, they developed normally until gastrulation, but then phenocopied maternal ß-catenin- or Wnt11-depleted embryos, displaying delayed gastrulation (data not shown) and the loss of axial structures at the tailbud stage - the so called `ventralized' phenotype (Fig. 1C,D; 121/132 cases [92%] had this appearance in ten experiments). Over-expression of mouse LRP6 mRNA in the oocyte had the opposite effect, causing dorsalization (Fig. 1C). The effect of antisense depletion was specific, because ventralization was rescued by the reintroduction of LRP6 mRNA into LRP6-depleted oocytes (Fig. 1D; 80/83 embryos [96%] rescued in this way in five experiments). The expression of the early zygotic Wnt11 target genes siamois, Xnr3, chordin and goosecoid was severely reduced in LRP6-depleted embryos at the early gastrula stage (Fig. 1E), and remained so throughout gastrulation (Fig. 1F, Fig. 2). Wnt target gene expression was rescued by the injection of LRP6 mRNA before fertilization, showing that the effect was specific (Fig. 1E). The expression of the endoderm marker XSox17 and the ventral marker Xwnt8 was delayed, but reached wild-type levels (Fig. 1F). Expression of the mesodermal marker Xbra was also reduced in LRP6-depleted embryos (Fig. 1E). Although embryos and molecular markers were not completely rescued to the wild-type state, Wnt target gene expression and dorsal-axis formation were consistently recovered by the injection of mouse LRP6 mRNA (Fig. 1G), showing the requirement for LRP6 in this process.
As further confirmation that LRP6 was required to activate the canonical Wnt signaling pathway, we used the TOPflash reporter to examine the effects of the loss of LRP6 activity on Tcf3- and/or LEF-mediated transcription. Fig. 1H shows that LRP6 depletion, at doses that cause ventralization, abolished the activity of this reporter in embryos at the late blastula stage.
|
). To analyze this further, we compared the expression of Wnt
target genes in sibling Wnt11- and LRP6-depleted embryos derived from the same
batch of oocytes. Fig. 2 shows
that, in both depletions, the expression of siamois and Xnr3
was severely reduced. Because ventralized phenotypes occur because of either
reduced dorsal gene expression, or the upregulation of ventral gene
expression, we examined the expression of the BMP target genes Xvent1
and Xvent2, as well as of BMP4. These markers were not
upregulated at stage 9.5-10.25, confirming that LRP6 depletion causes
ventralization by preventing dorsal gene activation.
To confirm that LRP6 acts in the maternal Wnt11 signaling pathway, we
tested whether LRP6 mRNA could rescue Wnt11-depleted embryos. Oocytes
were depleted of Wnt11 using an antisense oligo
(Tao et al., 2005), cultured
for 2 days and injected with mouse LRP6 mRNA before fertilization.
Mouse LRP6 mRNA (75 pg) rescued Wnt11-depleted embryos in producing
neural fold formation, although the size of the neural folds and head
formation were abnormal, whereas higher doses caused embryos to develop with a
dorsalized phenotype (Fig. 3A).
This abnormal morphology may be explained by the fact that Wnt11 depletion
causes a loss of a localized asymmetric signal, which is not replaced by LRP6,
so that the rescued axis is not normal. LRP6 mRNA also rescued the
expression of siamois and Xnr3 in Wnt11-depleted embryos at
the gastrula stage (Fig.
3B).
By contrast, the effects of maternal LRP6 depletion were not rescued by the injection of 100-300 pg Wnt11 mRNA (Fig. 3C,D), although these doses of Wnt11 RNA caused the dorsalization of wild-type siblings (data not shown). Thus, LRP6 lies downstream of Wnt11 in the signaling pathway.
To place ß-catenin relative to LRP6 in the pathway, we asked whether LRP6-depleted embryos could be rescued by the injection of ß-catenin mRNA. ß-catenin mRNA (50 pg) was injected into one dorsal cell in LRP6-depleted embryos at the 4-cell stage, and rescued the formation of axial structures as well as siamois and Xnr3 expression (Fig. 3E,F). Thus, LRP6 is upstream of ß-catenin in the axis-forming pathway.
To determine whether Wnt11 physically interacts with LRP6, HA-tagged Wnt11
(500 pg) and LRP6 lacking the C-terminus
(Tamai et al., 2000)
(LRP6-N-myc 1.5 ng) were injected into adjacent cells at the 4- to 8-cell
stage, and were frozen at the early gastrula stage for co-immunoprecipitation
analysis. Fig. 3G shows that
LRP6-N-myc interacts with Wnt11-HA.
|
;
Salic et al., 2000), such that
small changes can dramatically alter Wnt signal transduction. This finding
supports the model that LRP6 transduces Wnt signals by causing axin
degradation. Because axin is known to cause ß-catenin degradation, such increased axin levels would be predicted to result in reduced ß-catenin protein in LRP6-depleted embryos. Blots were stripped and re-probed using a ß-catenin-specific antibody, and this prediction was confirmed; the trend for ß-catenin levels was opposite to that for axin, particularly at the early blastula stage (Fig. 4A).
Fig. 3A shows that Wnt11 is upstream of LRP6 in the axis-forming pathway. Therefore, we asked whether Wnt11 depletion also affects axin levels in the early embryo. Wnt11 depletion enhances axin levels in the 8-cell-stage embryo (Fig. 4B), with increases ranging from 19-46% in three experiments.
The most recent model of Wnt11 function in the axis-forming pathway
proposes that sperm activates the cortical rotation movements of the first
cell cycle, which leads to a dorsal asymmetry of Wnt11 mRNA and
protein, causing increased Wnt11 signaling dorsally during the cleavage to
blastula stages (Heasman, 2006;
Larabell et al., 1997;
Schneider et al., 1996;
Tao et al., 2005). The work
here suggests the following model (Fig.
4): by the 8-cell stage, Wnt11 protein is secreted, mostly by
dorsal vegetal cells, and binds to LRP6
(Fig. 4Cii). Association of
Wnt11 with the extracellular domain of LRP6 causes LRP6 phosphorylation
(Davidson et al., 2005;
Tamai et al., 2000;
Tamai et al., 2004;
Wehrli et al., 2000;
Zeng et al., 2005) and axin
degradation. Because less axin is present to degrade ß-catenin,
ß-catenin is stabilized, binds to Xenopus Tcf3 and enters nuclei
on the dorsal side. On the ventral side, low levels of Wnt11 signaling occur,
maintaining a level of axin that degrades sufficient ß-catenin to reduce
nuclear accumulation. In LRP6-depleted embryos, Wnt11 molecules are unable to
activate the pathway, so axin increases in concentration and ß-catenin is
degraded on the dorsal as well as the ventral side
(Fig. 4Ciii). Next, we tested
four predictions of this model, as follows:
(1) LRP6 is not a dorsally localized mRNA
To determine whether LRP6 mRNA is asymmetrically localized in
embryos, we examined LRP6 mRNA levels in dorsal versus ventral
half-embryos at the 32-cell stage (Fig.
5A). Fig. 5A shows
that LRP6 mRNA is not enriched dorsally.
(2) Axin protein is less abundant dorsally than ventrally in wild-type 8-cell-stage embryos
We examined total axin protein in western blots of dorsal and ventral
wild-type half embryos at the 8-cell stage. Axin levels were reduced dorsally
compared with ventral levels in three repeats of the experiment
(Fig. 5B; 32% and 59% less than
the ventral levels of expression). Blots were stripped and re-probed for
ß-catenin levels, and ß-catenin expression was found to be increased
on the dorsal side compared with ventral levels
(Fig. 5B; 14% and 20% more than
the ventral side).
To determine whether low levels of axin were maintained dorsally, we
examined a series of embryos from the 8- to the 64-cell stage. Only at the
8-cell stage was axin at a low level dorsally compared to ventrally.
ß-catenin was enriched dorsally compared to ventrally throughout the 16-
to 64-cell stages (Fig. 5C).
This correlates with the reported time of nuclear localization of
ß-catenin protein in dorsal cells
(Larabell et al., 1997).
|
|
), whereas LRP6 depletion causes their downregulation
(Fig. 1E). In embryos depleted
of both LRP6 and axin, the loss of LRP6 would be predicted to allow remaining
axin to be stabilized, thus attenuating the up-regulation of siamois
and Xnr3. Fig. 5E
shows that this was the case. Depletion of both axin and LRP6 partially
rescued the over-expression of siamois and Xnr3 caused by
axin depletion. However, sibling LRP6- and axin-depleted embryos did not
develop normal axes, most probably due to the lack of asymmetry of dorsal gene
expression (data not shown). Together, these results are consistent with the model shown in Fig. 4C, in which LRP6 transduces Wnt11 signals dorsally by causing axin degradation at the 8-cell stage. When this process is blocked by LRP6 depletion, axin levels rise, leading to increased ß-catenin degradation and reduced expression of Wnt target genes.
LRP6 regulates axin levels in stage-6 oocytes
The Wnt pathway is assumed to be inactive in full-grown oocytes, because
transcription is repressed, and chromatin is methylated and hypo-acetylated
(Bird and Wolffe, 1999;
Landsberger and Wolffe, 1995).
However, because axin protein levels act as an early indicator of LRP6
function, we examined the effects of LRP6 depletion on endogenous axin protein
levels in oocytes. Unexpectedly, we found that endogenous axin protein levels
were increased in LRP6-depleted oocytes compared to controls, suggesting that
maternal LRP6 has a role in maintaining oocyte axin at a steady-state level
(Fig. 6A; repeated in three
experiments).
This raises the query as to whether axin levels in the oocyte are controlled by Wnt signaling. Because the manually defolliculated oocytes used in these experiments retain some follicle cells, these cells could secrete a Wnt signal. We removed all remaining follicle cells using collagenase treatment, and compared endogenous axin levels in control and denuded oocytes after culturing for 3 days (Fig. 6B). No differences were seen between collagenase-treated oocytes compared to control oocytes, suggesting that follicle cells do not play a role in regulating oocyte axin levels.
|
We then investigated whether this mechanism regulates oocyte ß-catenin levels by examining endogenous ß-catenin levels in wild-type and LRP6-depleted oocytes. Levels were also compared 8 hours after progesterone-stimulated oocyte maturation. Fig. 6D shows that ß-catenin levels were low in oocytes compared with matured oocytes, and that new ß-catenin synthesis occurred during oocyte maturation. ß-catenin protein in matured oocytes decreased as a result of LRP6 depletion (by 23% compared with controls).
Because ß-catenin levels in non-matured oocytes are low, we next asked
whether maternal axin is required to maintain this state. We depleted
axin mRNA using an antisense oligo
(Kofron et al., 2001), and
cultured the oocytes for 3 days to reduce axin protein levels.
Fig. 6E shows that
axin-depletion caused an increase in ß-catenin levels in non-matured
oocytes compared with controls. Axin-depleted oocytes did not transcribe
siamois and Xnr3 even with these increased ß-catenin
levels (data not shown). These results suggest a role for maternal axin in
maintaining ß-catenin at low levels in the oocyte, and suggest that Wnt11
signals in an autocrine fashion through LRP6 to degrade sufficient axin to
maintain a steady-state level (Fig.
6F).
One prediction of the model that maternal axin regulates endogenous ß-catenin in oocytes is that exogenously introduced ß-catenin should also be degraded. To test this, we injected myc-tagged ß-catenin mRNA into oocytes, which were then cultured in the presence or absence of the proteasomal inhibitor MG132. The amount of myc-tagged ß-catenin was enhanced when proteasomal degradation was prevented, suggesting that exogenous protein is degraded (Fig. 7A). To test the role of LRP6, we co-injected LRP6 mRNA together with ß-catenin-myc mRNA and cultured oocytes for 3 days. Fig. 7B shows a substantial increase in the amount of myc-tagged ß-catenin protein in oocytes when LRP6 mRNA was co-injected with ß-catenin-myc mRNA, showing that LRP6 enhances the stability of the exogenous ß-catenin-myc protein.
A second test of whether ß-catenin is degraded in oocytes compared to embryos is to compare the ability of ß-catenin mRNA to rescue LRP6-depleted oocytes in which the mRNA was injected either before or after fertilization. We used a dose of ß-catenin mRNA (50 pg) that caused axis-duplication when injected into the ventral side of wild-type embryos at the 4-cell stage (data not shown). Fig. 3E and Fig. 7C,D show that ß-catenin mRNA rescued axis formation and dorsal marker expression when injected at the 4-cell stage, but the same dose did not rescue LRP6-depleted embryos when ß-catenin mRNA was injected into oocytes before maturation. This experiment was repeated three times with the same result. One trivial explanation of this difference might be that ß-catenin mRNA was broken down when injected into oocytes but not embryos. We confirmed that ß-catenin mRNA was not degraded when injected into oocytes (data not shown). Finally, to confirm that more degradation of ß-catenin protein occurs in oocytes than in embryos, we examined the levels of ß-catenin in western blots of siblings of the embryos shown in Fig. 7C, at the 64-cell stage (Fig. 7E). Embryos injected as oocytes with ß-catenin mRNA had less total ß-catenin protein at the 64-cell stage than those injected at the 4-cell stage. These findings are consistent with the hypothesis that, in the oocyte, axin degrades ß-catenin synthesized from the injected mRNA.
|
).
Fig. 7G shows that the
stabilized form of ß-catenin was indeed protected from degradation in
LRP6-depleted oocytes, and Fig.
7F,H shows that it rescued the expression of siamois and
Xnr3, and caused a dorsalized phenotype. Embryos derived from
LRP6-depleted oocytes injected with stabilized ß-catenin before
fertilization were radially dorsalized, unlike those injected with
ß-catenin after fertilization (Fig.
3E). This is consistent with the fact that RNA injected into
oocytes is able to diffuse widely, whereas that injected into a single site at
the 4-cell stage does not diffuse and allows a dorsal-ventral asymmetry to be
established. Finally, to determine whether, acting in parallel to LRP6, the co-receptor Xfz7 was able to activate the axis-forming pathway, we injected Xfz7 mRNA into LRP6-depleted oocytes before fertilization. LRP6-depleted embryos were not rescued by the injection of Xfz7 mRNA either phenotypically (not shown) or by the expression of siamois and Xnr3 (Fig. 7H).
Taken together, these data provide insight into a novel steady-state role for Wnt11 and LRP6 in regulating axin and ß-catenin in the oocyte.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), we found
no evidence of redundancy with LRP5, which continued to be expressed in
LRP6-depleted oocytes, but did not rescue the effect of LRP6 depletion.
How does maternal LRP6 function?
Both the sequestration (Nusse,
2005) and nuclear shuttling
(Cong and Varmus, 2004) models
of axin function predict that LRP6 depletion or over-expression in oocytes and
embryos would cause endogenous axin protein to change in localization rather
than in total amount. We show here that LRP6 loss of function caused increases
in axin levels in oocytes and embryos, supporting a role for LRP6 in the
degradation of axin. However, we cannot rule out that LRP6 function in early
embryos also involves the sequestration of axin.
When does Wnt11-LRP6 function in dorsal axis formation?
These experiments show that LRP6 is essential for axis formation. However,
because LRP6 is depleted by injecting antisense oligos into oocytes, we cannot
distinguish whether LRP6 is required for axis-formation after fertilization,
or before. The Wnt signaling event responsible for establishing the dorsal
axis is likely to occur after fertilization, because blocking ß-catenin
synthesis after fertilization using a translation-inhibitory morpholino oligo
causes ventralization if the morpholino is injected vegetally at the 2- to
4-cell stage (Heasman et al.,
2000; Yang et al.,
2002). If axis formation depended on oocyte-synthesized
ß-catenin that moved in vesicles to the dorsal side during the first cell
cycle (Rowning et al., 1997),
then blocking ß-catenin translation at the 2-to 4-cell stage should not
affect axis formation. Also, dorsal and/or ventral asymmetries of
Wnt11 mRNA and protein are evident after, but not before,
fertilization (Tao et al.,
2005; Schroeder et al.,
1999), and a dorsal reduction of axin occurs specifically at the
8-cell stage (Fig. 5B), whereas
ß-catenin becomes dorsally nuclearly localized by the 16-cell stage
(Larabell et al., 1997).
|
;
Yang et al., 2002). One
possibility is that ß-catenin is required for chromatin remodeling as a
prerequisite for transcriptional activation. If Wnt11-LRP6 fails to stabilize
dorsal ß-catenin early, then such remodeling may not be successfully
initiated. A second possibility is that a Wnt inhibitor, such as dickkopf, may
be synthesized after fertilization and may actively block all but the earliest
Wnt11-LRP6 interaction. Preliminary data suggest that Dkk mRNA is
synthesized during oogenesis and may be a candidate for this role. The fact that axin levels in wild-type embryos are not reduced dorsally after the 8-cell stage may be explained in any or all of three ways: first, synthesis of axin may outpace its degradation; second, axin may sequester, as well as degrade, ß-catenin; and third, Wnt signaling may be downregulated by a Wnt inhibitor. Further experiments are needed to distinguish between these possibilities.
Steady-state Wnt signaling
We have previously assumed that Wnt signaling is `activated' on the dorsal
side of the embryo during the cleavage stages, leading to increased
ß-catenin levels on the dorsal side. However, this begs the question of
what mechanism regulates the `steady state' levels of ß-catenin before
such an activation? In the current model of canonical Wnt signaling, the
assumption has been that expression of Wnt target genes or TCF reporter
constructs are indicators of Wnt signaling
(Boras-Granic et al., 2006;
De Langhe et al., 2005;
Hens et al., 2005), suggesting
that their lack of expression coincides with Wnt pathway inactivation. An
alternative model is that Wnt signaling regulates ß-catenin
constitutively (Fig. 6F), and
that the changes that lead to the nuclear accumulation of ß-catenin are
due to either increased levels of Wnt signaling
(Fig. 4C), or to the
involvement of other pathway components, rather than de novo activation. In
its simplest form, a constitutive low level of Wnt signaling would lead to low
levels of axin degradation by LRP6 (Fig.
6F). This in turn would lead to high levels of axin, and lower
ß-catenin levels. In the embryo, increased Wnt signaling on the dorsal
side would lead to increased levels of ß-catenin, through the same
mechanism. The data presented here provide evidence for the existence of a
steady-state role for Wnt signaling in modulating axin levels. Specifically,
Wnt11 depletion and LRP6 depletion both cause an increase in axin levels in
the full-grown oocyte. Our study has shown, in several ways, that the effects
on axin are important for ß-catenin regulation in the oocyte. First, the
amount of ß-catenin increased in oocytes when axin was depleted using an
antisense oligo (Fig. 6E).
Second, exogenous ß-catenin was degraded in oocytes in a
proteasome-dependent fashion (Fig.
7A) and stabilized by LRP6-mRNA co-injection
(Fig. 7B). And third,
ß-catenin mRNA rescues LRP6-depleted embryos when injected after
fertilization, but not before (Fig.
7F). This work changes our assumption that Wnt signaling is an on
or off switch, and raises the query as to whether similar constitutive Wnt
regulation of axin occurs elsewhere in embryonic and adult tissues, in places
that would not be revealed by studying transcriptional target activation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bird, A. P. and Wolffe, A. P. (1999). Methylation-induced repression - belts, braces, and chromatin. Cell 99,451 -454.[CrossRef][Medline]
Boras-Granic, K., Chang, H., Grosschedl, R. and Hamel, P. A. (2006). Lef1 is required for the transition of Wnt signaling from mesenchymal to epithelial cells in the mouse embryonic mammary gland. Dev. Biol. 295,219 -231.[CrossRef][Medline]
Carter, M., Chen, X., Slowinska, B., Minnerath, S., Glickstein,
S., Shi, L., Campagne, F., Weinstein, H. and Ross, M. E.
(2005). Crooked tail (Cd) model of human folate-responsive neural
tube defects is mutated in Wnt coreceptor lipoprotein receptor-related protein
6. Proc. Natl. Acad. Sci. USA
102,12843
-12848.
Cong, F. and Varmus, H. (2004).
Nuclear-cytoplasmic shuttling of Axin regulates subcellular localization of
beta-catenin. Proc. Natl. Acad. Sci. USA
101,2882
-2887.
Davidson, G., Wu, W., Shen, J., Bilic, J., Fenger, U., Stannek, P., Glinka, A. and Niehrs, C. (2005). Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438,867 -872.[CrossRef][Medline]
De Langhe, S. P., Sala, F. G., Del Moral, P. M., Fairbanks, T. J., Yamada, K. M., Warburton, D., Burns, R. C. and Bellusci, S. (2005). Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung. Dev. Biol. 277,316 -331.[CrossRef][Medline]
Dupont, S., Zacchigna, L., Cordenonsi, M., Soligo, S., Adorno, M., Rugge, M. and Piccolo, S. (2005). Germ-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligase. Cell 121,87 -99.[CrossRef][Medline]
Hamada, F., Tomoyasu, Y., Takatsu, Y., Nakamura, M., Nagai, S.,
Suzuki, A., Fujita, F., Shibuya, H., Toyoshima, K., Ueno, N. et al.
(1999). Negative regulation of Wingless signaling by D-axin, a
Drosophila homolog of axin. Science
283,1739
-1742.
Heasman, J. (2006). Patterning the early
Xenopus embryo. Development
133,1205
-1217.
Heasman, J., Kofron, M. and Wylie, C. (2000). Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222,124 -134.[CrossRef][Medline]
Hens, J. R., Wilson, K. M., Dann, P., Chen, X., Horowitz, M. C. and Wysolmerski, J. J. (2005). TOPGAL mice show that the canonical Wnt signaling pathway is active during bone development and growth and is activated by mechanical loading in vitro. J. Bone Miner. Res. 20,1103 -1113.[CrossRef][Medline]
Houston, D. W. and Wylie, C. (2002). Cloning and expression of Xenopus Lrp5 and Lrp6 genes. Mech. Dev. 117,337 -342.[CrossRef][Medline]
Kelly, O. G., Pinson, K. I. and Skarnes, W. C.
(2004). The Wnt co-receptors Lrp5 and Lrp6 are essential for
gastrulation in mice. Development
131,2803
-2815.
Kofron, M., Klein, P., Zhang, F., Houston, D. W., Schaible, K., Wylie, C. and Heasman, J. (2001). The role of maternal axin in patterning the Xenopus embryo. Dev. Biol. 237,183 -201.[CrossRef][Medline]
Landsberger, N. and Wolffe, A. P. (1995). Chromatin and transcriptional activity in early Xenopus development. Semin. Cell Biol. 6,191 -199.[CrossRef][Medline]
Larabell, C. A., Torres, M., Rowning, B. A., Yost, C., Miller,
J. R., Wu, M., Kimelman, D. and Moon, R. T. (1997).
Establishment of the dorso-ventral axis in Xenopus embryos is presaged by
early asymmetries in beta-catenin that are modulated by the Wnt signaling
pathway. J. Cell Biol.
136,1123
-1136.
Lee, E., Salic, A., Kruger, R., Heinrich, R. and Kirschner, M. W. (2003). The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol. 1,E10 .[CrossRef][Medline]
Li, Y., Lu, W., He, X., Schwartz, A. L. and Bu, G. (2004). LRP6 expression promotes cancer cell proliferation and tumorigenesis by altering beta-catenin subcellular distribution. Oncogene 23,9129 -9135.[CrossRef][Medline]
Logan, C. Y. and Nusse, R. (2004). The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20,781 -810.[CrossRef][Medline]
Mao, J., Wang, J., Liu, B., Pan, W., Farr, G. H., 3rd, Flynn, C., Yuan, H., Takada, S., Kimelman, D., Li, L. et al. (2001). Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol. Cell 7, 801-809.[CrossRef][Medline]
Nusse, R. (2005). Cell biology: relays at the membrane. Nature 438,747 -749.[CrossRef][Medline]
Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J. and Skarnes, W. C. (2000). An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407,535 -538.[CrossRef][Medline]
Rowning, B. A., Wells, J., Wu, M., Gerhart, J. C., Moon, R. T.
and Larabell, C. A. (1997). Microtubule-mediated transport of
organelles and localization of beta-catenin to the future dorsal side of
Xenopus eggs. Proc. Natl. Acad. Sci. USA
94,1224
-1229.
Salic, A., Lee, E., Mayer, L. and Kirschner, M. W. (2000). Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. Mol. Cell 5,523 -532.[CrossRef][Medline]
Schneider, S., Steinbeisser, H., Warga, R. M. and Hausen, P. (1996). Beta-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech. Dev. 57,191 -198.[CrossRef][Medline]
Schroeder, K. E., Condic, M. L., Eisenberg, L. M. and Yost, H. J. (1999). Spatially regulated translation in embryos: asymmetric expression of maternal Wnt-11 along the dorsal-ventral axis in Xenopus. Dev. Biol. 214,288 -297.[CrossRef][Medline]
Sumanas, S., Strege, P., Heasman, J. and Ekker, S. C. (2000). The putative wnt receptor Xenopus frizzled-7 functions upstream of beta-catenin in vertebrate dorsoventral mesoderm patterning. Development 127,1981 -1990.[Abstract]
Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J. P. and He, X. (2000). LDL-receptor-related proteins in Wnt signal transduction. Nature 407,530 -535.[CrossRef][Medline]
Tamai, K., Zeng, X., Liu, C., Zhang, X., Harada, Y., Chang, Z. and He, X. (2004). A mechanism for Wnt coreceptor activation. Mol. Cell 13,149 -156.[CrossRef][Medline]
Tao, Q., Yokota, C., Puck, H., Kofron, M., Birsoy, B., Yan, D., Asashima, M., Wylie, C. C., Lin, X. and Heasman, J. (2005). Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos. Cell 120,857 -871.[CrossRef][Medline]
Tolwinski, N. S., Wehrli, M., Rives, A., Erdeniz, N., DiNardo, S. and Wieschaus, E. (2003). Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity. Dev. Cell 4,407 -418.[CrossRef][Medline]
Wehrli, M., Dougan, S. T., Caldwell, K., O'Keefe, L., Schwartz, S., Vaizel-Ohayon, D., Schejter, E., Tomlinson, A. and DiNardo, S. (2000). arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407,527 -530.[CrossRef][Medline]
Wiechens, N., Heinle, K., Englmeier, L., Schohl, A. and Fagotto,
F. (2004). Nucleo-cytoplasmic shuttling of Axin, a negative
regulator of the Wnt-beta-catenin Pathway. J. Biol.
Chem. 279,5263
-5267.
Willert, K., Logan, C. Y., Arora, A., Fish, M. and Nusse, R. (1999a). A Drosophila Axin homolog, Daxin, inhibits Wnt signaling. Development 126,4165 -4173.[Abstract]
Willert, K., Shibamoto, S. and Nusse, R.
(1999b). Wnt-induced dephosphorylation of axin releases
beta-catenin from the axin complex. Genes Dev.
13,1768
-1773.
Yang, J., Tan, C., Darken, R., Wilson, P. and Klein, P. (2002). Beta catenin/TCF regulated transcription prior to the mid-blastula transition. Development 129,5743 -5752.[CrossRef][Medline]
Yost, C., Torres, M., Miller, J., Huang, E., Limelman, D. and
Moon, R. (1996). The axis-inducing activity, stability, and
subcellular localization of beta catenin is regulated in Xenopus
embryos by glycogen synthase kinase 3. Genes Dev.
10,1443
-1454.
Zeng, X., Tamai, K., Doble, B., Li, S., Huang, H., Habas, R., Okamura, H., Woodgett, J. and He, X. (2005). A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438,873 -877.[CrossRef][Medline]
Zuck, M. V., Wylie, C. C. and Heasman, J. (1998). Maternal mRNAs in Xenopus embryos: an antisense approach. In A comparative methods approach to the study of oocytes and embryos, (ed. J. D. Richter), pp.341 -354. Oxford: Oxford University Press.
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  C. S. Cselenyi, K. K. Jernigan, E. Tahinci, C. A. Thorne, L. A. Lee, and E. Lee LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of {beta}-catenin PNAS, June 10, 2008; 105(23): 8032 - 8037. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhao, K. Tanegashima, H. Ro, and I. B. Dawid Lrig3 regulates neural crest formation in Xenopus by modulating Fgf and Wnt signaling pathways Development, April 1, 2008; 135(7): 1283 - 1293. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||
