|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online October 26, 2007
doi: 10.1242/10.1242/dev.010272
1 Department of Cell and Developmental Biology, Vanderbilt University Medical
Center, Nashville, TN 37232, USA.
2 Department of Medicine, Vanderbilt University Medical Center, Nashville, TN
37232, USA.
3 Department of Cell Biology, Harvard Medical School, Boston, MA 02115,
USA.
* Author for correspondence (e-mail: ethan.lee{at}vanderbilt.edu)
Accepted 5 September 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Lrp6, Wnt, Planar cell polarity, Convergent extension, Gastrulation, Xenopus
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). In the
absence of a Wnt signal, cytoplasmic ß-catenin is normally maintained at
low levels due to its degradation by a complex consisting of Axin, the tumor
suppressor APC (adenomatous polyposis coli), and the kinases CKI
and
GSK3. Activation of Wnt/ß-catenin signaling occurs upon binding of Wnt
ligands to members of the Frizzled (Fz) and low-density lipoprotein
receptor-related protein family of receptors
(Tamai et al., 2000;
Wehrli et al., 2000). Wnt
binding initiates a series of events resulting in ß-catenin stabilization
and consequent activation of gene transcription by binding of ß-catenin
to members of the TCF/LEF family of transcription factors.
Wnt/PCP signaling controls convergent-extension movements of the axial and
paraxial mesoderm and neural ectoderm during embryonic development in
Xenopus, zebrafish and mice
(Jones and Chen, 2007). In
Xenopus embryos, the Wnt/PCP pathway affects cell morphology and
motility largely through its modulation of the actin cytoskeleton. Cells
acquire distinct polar morphologies that promote directed migration and
intercalation during the morphogenetic movements of gastrulation and
neurulation. Although numerous genes that regulate Wnt/PCP signaling have been
identified, mechanisms by which the signal is transduced are not well
understood. Assembly of the actin cytoskeleton as a consequence of Wnt/PCP
signaling is thought to occur via cortical recruitment of Dishevelled (Dsh;
also known as Dvl - Xenbase) and activation of Jun-N-terminal kinase (JNK) and
the Rho family of GTPases (Habas et al.,
2001; Yamanaka et al.,
2002).
Lrp6 has been shown to play a critical role in activating
Wnt/ß-catenin signaling. Its intracellular domain contains five PPP(S/T)P
motifs, each of which is phosphorylated upon Wnt stimulation
(Davidson et al., 2005;
Zeng et al., 2005). Each of
the five PPP(S/T)P motifs of Lrp6, when phosphorylated, can bind to Axin and,
through an as yet undefined mechanism, inhibit ß-catenin degradation
(Tamai et al., 2004). In the
current model of Wnt signaling, Lrp6 signals exclusively in a
ß-catenin-dependent manner (Tamai et
al., 2000; Wehrli et al.,
2000). Recently, Dkk1, a secreted Wnt/ß-catenin antagonist
that binds Lrp6, was shown to regulate convergent-extension movements in
zebrafish via activation of Wnt/PCP signaling
(Caneparo et al., 2007).
Our studies indicate that Lrp6, a previously characterized core Wnt/ß-catenin component, is an essential regulator of convergent-extension movements in Xenopus embryos via its inhibition of Wnt/PCP signaling. Furthermore, we show that Lrp6 is asymmetrically distributed in mesodermal cells that participate in convergent extension, suggesting a role for Lrp6 in the establishment and maintenance of cell polarity during embryogenesis.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Embryos were staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1994).
For microinjections, embryos were kept in 3% Ficoll in 0.2x Marc's
modified Ringer's (MMR) medium and fixed at stage 26 for analysis. In
vitro-transcribed, capped mRNA was synthesized using the mMessage mMachine Kit
(Ambion, Austin, TX, USA). Lrp6MO was from Gene Tools LLC (Philomath, OR,
USA). Detection of notochord with monoclonal antibody Tor70
(Kushner, 1984) was performed
as described previously (Lane and Keller,
1997).
Keller sandwiches of uniform width and length were prepared from dorsal
marginal zone (DMZ) explants at stage 10+
(Keller and Danilchik, 1988)
and cultured in 1 x DFA medium
(Sater et al., 1993) until
stage 18. To determine explant length-to-width ratios, the longest axis of
each explant was divided by its widest perpendicular aspect (short axis).
Animal pole ectodermal explants (animal caps) were dissected at stage 9 and
cultured in 75% MMR in the presence of 10 ng/ml activin (R&D Systems,
Minneapolis, MN, USA) until stage 20 when elongation was assessed. At least 28
caps were assessed for each set of injections.
For Dsh localization studies, caps were co-injected with 150 pg of Xenopus Dsh-GFP and visualized at stage 11 by confocal microscopy (Zeiss Axiovert LSM 510 META; Carl Zeiss, Jena, Germany). Cells with >90% of staining at their cortex were scored as having cortical Dsh-GFP. JNK (also known as Mapk8) activation was assessed in animal caps (stage 11) using phospho-JNK specific antibody (Promega V7931; Promega, Madison, WI, USA). Picture intensity was normalized to control uninjected and ß-catenin-injected caps. Cells with exclusive nuclear phospho-JNK staining were counted as positive.
|
TOPFLASH reporter assay
For TOPFLASH reporter assays, HEK293 cells were transiently co-transfected
and luciferase activity assessed according to the manufacturer's instructions
(Promega, Madison, WI, USA). Assays were performed in triplicate with samples
normalized to ß-galactosidase.
Open-faced explants, time-lapse imaging, phalloidin staining and Lrp6 localization
Open-faced `shaved' Keller DMZ and ventral marginal zone (VMZ) explants
(Shih and Keller, 1992b) were
dissected at stage 10.5 from embryos co-injected with mRNA encoding
myristoylated GFP (mGFP) plus Xenopus strabismus (stbm)
mRNA, Lrp6MO, or lrp6-B mRNA. Explants were filmed by time-lapse
confocal microscopy (Zeiss Axiovert LSM 510 META; Carl Zeiss, Jena, Germany)
by capturing one frame every 15 seconds over 15 minutes. Randomly chosen cells
from each movie were traced and their motility was measured as described
(Tahinci and Symes, 2003).
Cell orientations, length-to-width ratios, and protrusion distributions were
calculated using ImageJ software
(http://rsb.info.nih.gov/ij/)
for the middle frame of each movie. Cells from DMZ or VMZ explants were
dissociated in 1 x modified Barth's saline (MBS) without Ca2+
or Mg2+ and plated on 200 µg/ml fibronectin (R&D Systems,
Minneapolis, MN, USA). After adhering for 30 minutes, cells were fixed and
stained with Alexa Fluor 594 phalloidin (Invitrogen, Carlsbad, CA, USA).
GFP-positive cells were used for determining morphologies. Length-to-width
ratios and protrusion distributions were measured using ImageJ. At least 216
protrusions were measured for each set of injected explants.
For Lrp6 immunolocalization in animal caps, embryos were co-injected with
vesicular stomatitis virus G (VSVG)-Lrp6 and mGFP. Embryos were dissected at
stage 9 (animal caps) or stage 10.5 (DMZs), fixed at stage 16 (animal caps) or
11 (DMZs; explants did not undergo appreciable elongation from stage 10.5 to
11), and processed for immunostaining as described previously
(Lane and Keller, 1997). For
Lrp6 staining in dissociated cells, DMZ explants were dissociated in 1 x
MBS in the absence of Ca2+ or Mg2+, plated on
fibronectin-coated slides (200 µg/ml; R&D Systems, Minneapolis, MN,
USA), and allowed to adhere for 30 minutes. Cells were fixed in 4%
formaldehyde and stained with anti-VSVG-FITC (Bethyl Laboratories, Montgomery,
TX, USA) and Alexa Fluor 594 phalloidin (Invitrogen, Carlsbad, CA, USA) as
described previously (Sawin et al.,
1992).
|
Embryo cryosections
Embryos (stage 10.5) were placed in embedding medium (OCT) with their
suprablastoporal regions (prospective chordamesoderm) facing the embedding
resin, such that these regions would be sectioned first. Embedded embryos were
frozen on dry ice and 10 µm sections were made using a Leica CM3050 S
cryostat microtome (Leica Microsystems, Bensheim, Germany). Anti-VSVG (1:200)
was applied to sections overnight at 4°C. Sections were incubated with Cy3
secondary antibody (1:200), mounted in antifade (90% glycerol, 4% n-propyl
gallate), and examined using the FluoView 100 confocal microscope (Olympus,
Tokyo, Japan). Cells with Lrp6 staining extending over less than half of their
periphery were scored as having polarized Lrp6 distribution.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
For gain of Lrp6 function, we injected mRNA encoding the intracellular
domain of Lrp6 fused to an N-terminal myristoylation sequence
(Tamai et al., 2004). This
construct constitutively transduces the Wnt/ß-catenin signal and causes
anterior duplication when injected ventrally whereas full-length Lrp6 has only
weak canonical activity (Fig.
1A). Unless otherwise indicated, this constitutively active
construct was used in all subsequent studies. Embryos injected in their
presumptive neural ectoderm (dorsoanimal blastomeres) fail to fully elongate
their anteroposterior (AP) axes and develop with dorsal flexure; they have
bifurcated, bent notochords, suggestive of defects in neural convergent
extension (Wallingford and Harland,
2001). Embryos injected in their presumptive axial mesoderm
(dorsovegetal blastomeres) develop with closed neural tubes, but have
shortened AP axes with thicker notochords, suggestive of defective mesodermal
convergent extension (Fig. 1A).
Similar phenotypes are observed when embryos are injected with Xdd, a dominant
negative mutant of Dishevelled that affects convergent extension
(Sokol, 1996). The ability of
Lrp6 to block gastrulation or neurulation cannot be attributed to
Wnt/ß-catenin pathway activation because functionally equivalent amounts
of ß-catenin mRNA that cause a similar percentage of anterior duplication
(injected ventrally) do not block AP axis elongation when injected into dorsal
blastomeres (Fig. 1A). These
results are reminiscent of studies of Wnt/PCP components in that either loss
or gain of Lrp6 function causes gastrulation and/or neurulation defects
(Ueno and Greene, 2003),
depending on the injection site.
|
). These results show that either gain or loss of Lrp6
causes defects in morphogenesis rather than mesendodermal tissue
patterning.
We next assessed the role of Lrp6 in regulating convergent extension using
Xenopus explants, animal caps and Keller sandwiches
(Symes and Smith, 1987;
Keller and Danilchik, 1988).
Animal caps from Xenopus blastulae differentiate into round epidermal
tissue when cultured in vitro. In the presence of mesoderm-inducing activin,
however, their cells intercalate, resulting in cap elongation
(Symes and Smith, 1987).
Injection of activators or inhibitors of Wnt/PCP signaling blocks this
elongation process (Simons et al.,
2005; Takeuchi et al.,
2003). Animal caps from embryos injected with lrp6 mRNA
or Lrp6MO fail to elongate with activin treatment, resembling caps injected
with Wnt11, an activator of Wnt/PCP signaling
(Fig. 2A). Inhibition of
elongation in lrp6 mRNA- and Lrp6MO-injected caps occurs despite
proper induction of mesendodermal markers, confirming that loss and gain of
Lrp6 function result in inhibition of convergent extension
(Fig. 2B). Furthermore, this
inhibition induced by Lrp6 loss of function cannot be rescued by co-injection
of ß-catenin mRNA, confirming that Lrp6 blocks convergent-extension
movements in a ß-catenin-independent manner (see Fig. S1 in the
supplementary material).
Keller sandwiches consist of two apposed dorsal marginal zone (DMZ)
explants that elongate as a result of convergent extension of their neural and
mesodermal tissues (Keller and Danilchik,
1988). Keller sandwiches were formed from embryos injected with
either Lrp6MO or lrp6 mRNA, and their elongation was assessed at
stage 18. These explants were unable to fully elongate compared with control
explants (Fig. 2C), which
indicates defective convergent extension. Again, the effect of Lrp6MO in
blocking explant elongation was not rescued by co-injection of ß-catenin
mRNA (see Fig. S1 in the supplementary material). Taken together, our embryo
and explant data support a role for Lrp6 in controlling convergent-extension
movements during early Xenopus embryogenesis.
Lrp6 affects mesodermal cell morphology, motility and actin rearrangement
Cell intercalation drives mesodermal convergent extension during vertebrate
development. To intercalate, cells acquire an elongated shape and form
cytoplasmic processes along their long axes. These changes in morphology are
thought to be actin driven (Keller et al.,
2003).
To assess whether the overall morphology, arrangement and motility of
marginal zone cells is affected by loss of Lrp6 function, we performed live
cell analysis in open-faced `shaved' Keller explants
(Shih and Keller, 1992a) from
the marginal zone (Tahinci and Symes,
2003). Stbm, a Wnt/PCP signaling component, has been shown to
block convergent extension in dorsal explants
(Darken et al., 2002). Shaved
Keller explants of DMZ cells injected with Lrp6MO show slower motility and
resemble cells overexpressing Stbm (Fig.
3A). Cells in these explants have smaller length-to-width ratios
(i.e. rounder) and reduced ability to orient along the mediolateral embryonic
axis compared to control. Protrusions of these cells are predominantly
oriented along their short axes and are transient compared with those of
control cells, resembling cells overexpressing Stbm. Ventral marginal zone
(VMZ) cells injected with stbm mRNA or Lrp6MO show increased cell motility,
reflecting possible stimulation of Wnt/PCP signaling
(Fig. 3A).
|
|
). In
vertebrates, asymmetric distribution of several Wnt/PCP components has also
been reported: Dvl2 (Wang et al.,
2005), prickle (Ciruna et al.,
2006) and PKC (Kinoshita et
al., 2003).
We tested whether Lrp6 has a polarized distribution in intercalating
mesodermal cells. Animal caps injected with VSVG-lrp6 mRNA show a
uniform subcellular staining pattern (Fig.
4A). Strikingly, treatment of animal caps with activin at
concentrations that induce dorsal mesoderm and promote elongation (via a
mechanism that is believed to recapitulate convergent extension) causes,
indirectly, asymmetric relocalization of Lrp6
(Fig. 4A). Confirming these
findings, cryosections of whole embryos injected with VSVG-lrp6 show
that Lrp6 has a uniform distribution in cells of the animal pole and VMZ in
early gastrulae, but not the DMZ (see Fig. S2 in the supplementary material).
This spatial distribution of Lrp6 is reminiscent of the localization of
inhibitory PCP proteins in the Drosophila wing and eye
(Klein and Mlodzik, 2005).
We also observe a polarized subcellular distribution for Lrp6 in whole DMZ
explants. Embryos were injected with VSVG-tagged lrp6 mRNA (at a
concentration that did not perturb embryonic morphology), and open-faced
shaved Keller explants of the DMZ were stained with anti-VSVG antibody. To
more faithfully assess Lrp6 localization, explants were not cultured prior to
fixation and staining for Lrp6. VSVG-Lrp6 predominantly localized to the
posterior surface of intercalating DMZ cells throughout the whole explant
(Fig. 4B). This distribution of
VSVG-Lrp6 in DMZ cells is unlikely to be due to oriented anteroposterior cell
overlapping (Winklbauer and Nagel,
1991) because cortical actin staining can clearly be seen at
anterior and posterior surfaces of these cells.
Reorganization of the actin cytoskeleton is a major consequence of Wnt/PCP signaling. The mechanism by which Wnt/PCP signaling impinges on the cytoskeleton, however, is still poorly understood, and a direct link between Wnt/PCP components and the actin cytoskeleton has not been established. Thus, we sought to establish an association between the actin cytoskeleton and Lrp6, a potential regulator of Wnt/PCP signaling. We injected cells of the DMZ with VSVG-lrp6 mRNA, dissociated cells at stage 10.5, and stained them with anti-VSVG antibody and Rhodamine-phalloidin (Fig. 4C). Lrp6 was seen at the periphery of cells and was enriched adjacent to actin-rich cortical areas of cellular protrusions. This observation suggests a potential role for Lrp6 in regulating the actin cytoskeleton and is consistent with our proposed role for Lrp6 in polarized cell movement and regulation of convergent extension.
|
;
Takeuchi et al., 2003). To
determine whether Lrp6 is a positive or negative Wnt/PCP regulator, we tested
the ability of Lrp6 to oppose or reinforce the effect of sub-optimal amounts
of co-injected Wnt11 by tipping the balance towards increased Wnt11 signaling
(i.e. Lrp6 strengthens the Wnt11 effect) or decreased Wnt11 signaling (i.e.
Lrp6 weakens the Wnt11 effect; Fig.
5A). Injection of sub-optimal amounts of Lrp6 opposes
Wnt11-induced inhibition of animal cap elongation, indicating that Lrp6 is a
negative regulator of Wnt/PCP signaling. Similarly, sub-optimal amounts of
Wnt11 are reinforced by Lrp6MO in potentiating inhibition of animal cap
elongation, further indicating that Lrp6 downregulates Wnt/PCP signaling
(Fig. 5A).
|
). Upon
activation of Wnt/PCP signaling, Dsh-GFP localizes predominantly to the cell
cortex (Yanagawa et al.,
1995). Cortical localization of Dsh-GFP is readily observed in
animal caps expressing Wnt11 (Fig.
5B). Consistent with activation of the Wnt/PCP pathway, Lrp6MO
injection also results in Dsh-GFP localization to the cortex. As a control,
injection of animal caps with Wnt8, which signals through the
Wnt/ß-catenin pathway, does not cause cortical redistribution of
Dsh-GFP.
JNK is phosphorylated as a result of Wnt/PCP signaling
(Yamanaka et al., 2002).
Animal caps expressing Dsh or Lrp6MO show enhanced nuclear phospho-JNK
staining consistent with their roles as Wnt/PCP activators
(Fig. 5C). By contrast,
ß-catenin injections do not affect phospho-JNK. These results provide
molecular evidence that Lrp6 antagonizes Wnt/PCP signaling.
A 36-residue intracellular domain of Lrp6 is sufficient to mediate its effects on convergent extension
To determine the region of Lrp6 responsible for mediating its Wnt/PCP
activity, we performed structure-function analysis of its intracellular
domain. Previous studies have shown that the intracellular domain of Lrp6
contains five PPP(S/T)P motifs, each of which can activate Wnt/ß-catenin
signaling (Tamai et al.,
2004). C-terminal deletion mutants of Lrp6 lacking these motifs
were tested for their ability to perturb convergent extension in embryos
(Fig. 6A).
Embryos injected with various Lrp6 constructs were scored as normal, or
affected (mildly or severely) based on AP axes lengths as previously described
(Goto and Keller, 2002).
Surprisingly, progressive C-terminal deletions of Lrp6 result in an increasing
percentage of elongation defects until a 36-residue fragment (Lrp6-B) is
eliminated (Fig. 6A,B). Similar
to injections of Lrp6, injections of Lrp6-B result in severe mesodermal and
neural convergent-extension defects in embryos, animal caps and Keller
sandwiches (Fig. 6C). In
addition, Lrp6-B potentiates the effect of Lrp6 in blocking animal cap
elongation, indicating that Lrp6-B regulates convergent-extension activity in
the same direction as the full-length intracellular domain
(Fig. 6C). Finally, Lrp6-B
reverses the Lrp6MO-mediated block in elongation of animal caps (see Fig. S3A
in the supplementary material) and Keller sandwich explants (see Fig. S3B in
the supplementary material), indicating that it is sufficient to transduce a
signal to the PCP branch of the Wnt pathway. RT-PCR analysis of animal caps
and TOPFLASH reporter assays in HEK-293 cells reveal that Lrp6-B is unable to
activate ß-catenin-dependent transcription
(Fig. 6D,E) or to act in a
dominant-negative manner to inhibit Wnt-mediated induction of ß-catenin
targets (Fig. 6D,E). Thus, Lrp6
appears to mediate its convergent-extension activity through Lrp6-B,
independent of its role in regulating ß-catenin-mediated transcription
(Yokota et al., 2003). This
result is consistent with our observation that co-injection of ß-catenin
rescues the defects in Wnt/ß-catenin, but not Wnt/PCP signaling caused by
injection of Lrp6MOs into Xenopus embryos and explants
(Fig. 1B and see Fig. S1A,B in
the supplementary material).
Phalloidin staining of DMZ and VMZ cells expressing Lrp6-B reveals that this domain of Lrp6 alters the bipolar morphology of cells in the DMZ (see Fig. S2C in the supplementary material) without activating cell motility in the VMZ (see Fig. S2D in the supplementary material) or Wnt/PCP downstream events (e.g. Dsh localization or JNK phosphorylation; see Fig. S2E in the supplementary material). Injection of lrp6-B mRNA causes dramatic changes in the protrusive activity of DMZ and VMZ cells (see Fig. S2C in the supplementary material). Similar results were obtained by injection of the entire intracellular domain of Lrp6 (data not shown), further indicating that Lrp6-B can recapitulate the effects of Lrp6 on convergent extension.
Coupling of the Wnt/ß-catenin and Wnt/PCP pathways at the level of Lrp6
Identification of Lrp6-B as a region of Lrp6 that can inhibit Wnt/PCP
signaling without activating Wnt/ß-catenin targets indicates that these
two activities of Lrp6 require distinct domains. It is not clear, however,
whether they are physiologically coupled. To address this question, we used a
non-phosphorylatable mutant of the intracellular domain of Lrp6, Lrp6-m5, in
which all serine/threonine residues within the PPP(S/T)P motifs are replaced
by alanines. This mutant, which contains the Lrp6-B sequence, acts in a
dominant-negative manner to inhibit Wnt/ß-catenin signaling
(Tamai et al., 2004).
Interestingly, injection of Lrp6-m5 at concentrations that block
Wnt/ß-catenin signaling by RT-PCR (data not shown) failed to inhibit
animal cap elongation (Fig.
7A), suggesting that Lrp6 phosphorylation is necessary for Lrp6 to
mediate its Wnt/PCP activity (possibly by relieving steric inhibition to
expose the Lrp6-B region). This result is consistent with our observation of
increased Wnt/PCP activity upon progressive C-terminal truncations of Lrp6
regions necessary for Wnt/ß-catenin signaling
(Fig. 6A).
Activation of Wnt/PCP signaling has been suggested to antagonize
Wnt/ß-catenin signaling (Peters et
al., 1999; Yan et al.,
2001; Kuhl et al.,
2001; Schwarz-Romond et al.,
2002). To test whether inhibition of this antagonistic activity is
sufficient to promote Wnt/ß-catenin signaling, we co-injected Lrp6-B mRNA
with sub-optimal amounts of the Wnt/ß-catenin ligand, Wnt8, and scored
embryos for axis duplication, an in vivo read-out for Wnt/ß-catenin
activation. Lrp6-B injection potentiates the effects of Wnt8 in inducing axis
duplication (Fig. 7B),
indicating that inhibition of Wnt/PCP signaling at the level of Lrp6
potentiates Wnt/ß-catenin signaling. Based on these findings, we propose
a model in which Lrp6, through its interactions with cytoplasmic factors via
an intracellular 36-residue domain, acts as a switch between
Wnt/ß-catenin and Wnt/PCP signaling
(Fig. 7C).
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
The role of Lrp6 in mediating the switch from Wnt/PCP to Wnt/ß-catenin signaling
From this study we provide evidence for interaction between the
Wnt/ß-catenin and Wnt/PCP pathways at the level of Lrp6. We show that
injection of mRNA encoding a small intracellular domain of Lrp6, Lrp6-B,
inhibits Wnt/PCP signaling while potentiating Wnt/ß-catenin signaling.
Our data also indicate that Lrp6 activation (via activation of
Wnt/ß-catenin signaling) is a prerequisite for its ability to inhibit
Wnt/PCP signaling (Fig. 7C).
Our data suggest that coupling of Wnt/PCP inhibition with activation of
Wnt/ß-catenin is embedded at the structural level of Lrp6.
Because Lrp6 activation is necessary for inhibition of Wnt/PCP, it stands
to reason that canonical Wnt inhibitors that bind Lrp6 (e.g. Dkks, SOST) would
potentiate the Wnt/PCP pathway by relieving inhibition by Lrp6. Wnt-Fz-Lrp6
interactions can in turn be influenced by tissue-specific factors, depending
on the context (Tao et al.,
2005). This model is reminiscent of the Hedgehog signaling pathway
where signaling occurs through another seven-pass transmembrane protein,
Smoothened, upon relief of inhibition by the Hedgehog receptor, Patched
(Cohen, 2003).
Inhibition of Wnt/PCP by Lrp6 occurs through intracellular interactions
Recently, Dkk1 has been proposed to activate Wnt/PCP signaling by
interacting with glypican 4 (Caneparo et
al., 2007). Our observation that Lrp6 inhibits Wnt/PCP signaling
is unlikely to be a consequence of disruption of the Dkk1-glypican 4-mediated
activation of Wnt/PCP signaling because our gain-of-function studies were
conducted with the intracellular domain of Lrp6, which presumably cannot bind
to Dkk1, a secreted protein. Based on our results, it is possible that some of
the effect of Dkk1 on Wnt/PCP signaling may be indirectly due to its
interaction with Lrp6. Interestingly, Wise, a context-dependent activator or
inhibitor of Wnt signaling, can both bind Lrp6 and affect Wnt/PCP signaling,
as evidenced by its ability to block elongation in activin-treated animal caps
(Itasaki et al., 2003).
One obvious mechanism by which Lrp6 could inhibit Wnt/PCP signaling would
be through its interaction with the scaffold protein Axin. Axin can bind
directly to Dsh (Li et al.,
1999), and it is possible that, indirectly, Lrp6 could decrease
the activity of Dsh in the Wnt/PCP pathway and concomitantly increase its
activity in the Wnt/ß-catenin pathway. We believe that this scenario is
unlikely because the Lrp6 regions that have been shown to bind Axin and
mediate Wnt/ß-catenin signaling, the PPP(S/T)P motifs, are distinct from
the Lrp6 domain that we show inhibits Wnt/PCP signaling. It is possible that
Lrp6 directly interacts with Dsh to inhibit Wnt/PCP signaling. To our
knowledge, however, there has been no reported interaction between Lrp6 and
Dsh, and we have been unsuccessful in demonstrating interaction between either
the full intracellular domain of Lrp6 or Lrp6-B and Dsh (data not shown).
Thus, we favor a model in which Lrp6 mediates its Wnt/PCP activity through its
interaction with Wnt/PCP pathway components that function downstream of
Dsh.
Lrp6 localizes to the posterior edge of intercalating axial mesodermal cells
Core Fz/PCP components have been shown to have asymmetrical subcellular
localization patterns in Drosophila wing cells. Positive regulators
of Fz/PCP signaling are localized distally at the site of actin-rich hair
formation, whereas negative regulators are localized proximally (reviewed by
Jenny and Mlodzik, 2006).
Similar observations have been made in cochlea sensory hair cells of
vertebrates. For example, Dvl2 is localized to the site of actin-rich
stereocilia formation (Wang et al.,
2005) in the outer cell cortex, whereas Vangl2 [the mouse ortholog
of Stbm; Montcouquiol et al. (Montcouquiol
et al., 2006)] is localized to the opposing inner side.
Demonstration of asymmetric subcellular localization of Wnt/PCP components
during vertebrate convergent extension has been less straightforward. Initial
studies in cultured Xenopus explants indicated that Dsh and PKC are
enriched along the mediolateral axis of cells undergoing convergent extension
(Kinoshita et al., 2003).
Prickle, a Wnt/PCP regulator that interacts with Dsh, has recently been shown
to localize to the anterior end of notochord and neurectoderm cells during
zebrafish neurulation (Ciruna et al.,
2006). We have, however, been unable to observe polarized
localization of either Dsh-GFP or GFP-Prickle in the DMZ. Furthermore, their
localization is not altered by injection of lrp6 mRNA or Lrp6MO (data
not shown).
Our results show that Lrp6, a core component of the Wnt/ß-catenin
pathway, is localized to the posterior end of intercalating mesodermal cells
of the early Xenopus gastrula. It is tempting to speculate that Lrp6
may indirectly inhibit Dsh activity in the posterior end of these cells during
gastrulation. Interestingly, these mesodermal cells intercalate in a
mediolateral fashion, perpendicular to the axis of Lrp6 polarization. This
suggests that other signaling pathways (in addition to Wnt/PCP) may also act
to control cell polarity during convergent extension
(Hyodo-Miura et al., 2006).
Further characterization of the Wnt/PCP signaling cascade downstream of Lrp6
will probably provide clues as to the spatial and temporal activation of
Wnt/PCP components during vertebrate convergent extension.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/22/4095/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Caneparo, L., Huang, Y. L., Staudt, N., Tada, M., Ahrendt, R.,
Kazanskaya, O., Niehrs, C. and Houart, C. (2007).
Dickkopf-1 regulates gastrulation movements by coordinated modulation of
Wnt/betacatenin and Wnt/PCP activities, through interaction with the
Dally-like homolog Knypek. Genes Dev.
21,465
-480.
Ciruna, B., Jenny, A., Lee, D., Mlodzik, M. and Schier, A.
F. (2006). Planar cell polarity signalling couples cell
division and morphogenesis during neurulation. Nature
439,220
-224.[CrossRef][Medline]
Clevers, H. (2006). Wnt/beta-catenin signaling
in development and disease. Cell
127,469
-480.[CrossRef][Medline]
Cohen, M. M., Jr (2003). The hedgehog signaling
network. Am. J. Med. Genet. A
123, 5-28.[Medline]
Darken, R. S., Scola, A. M., Rakeman, A. S., Das, G., Mlodzik,
M. and Wilson, P. A. (2002). The planar polarity gene
strabismus regulates convergent extension movements in Xenopus.
EMBO J. 21,976
-985.[CrossRef][Medline]
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]
Glinka, A., Wu, W., Delius, H., Monaghan, A. P., Blumenstock, C.
and Niehrs, C. (1998). Dickkopf-1 is a member of a new family
of secreted proteins and functions in head induction.
Nature 391,357
-362.[CrossRef][Medline]
Goto, T. and Keller, R. (2002). The planar cell
polarity gene strabismus regulates convergence and extension and neural fold
closure in Xenopus. Dev. Biol.
247,165
-181.[CrossRef][Medline]
Habas, R., Kato, Y. and He, X. (2001).
Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires
a novel Formin homology protein Daam1. Cell
107,843
-854.[CrossRef][Medline]
Hyodo-Miura, J., Yamamoto, T. S., Hyodo, A. C., Iemura, S.,
Kusakabe, M., Nishida, E., Natsume, T. and Ueno, N. (2006).
XGAP, an ArfGAP, is required for polarized localization of PAR proteins and
cell polarity in Xenopus gastrulation. Dev. Cell
11, 69-79.[CrossRef][Medline]
Itasaki, N., Jones, C. M., Mercurio, S., Rowe, A., Domingos, P.
M., Smith, J. C. and Krumlauf, R. (2003). Wise, a
context-dependent activator and inhibitor of Wnt signalling.
Development 130,4295
-4305.
Jenny, A. and Mlodzik, M. (2006). Planar cell
polarity signaling: a common mechanism for cellular polarization.
Mt. Sinai J. Med. 73,738
-750.[Medline]
Jones, C. and Chen, P. (2007). Planar cell
polarity signaling in vertebrates. BioEssays
29,120
-132.[CrossRef][Medline]
Kao, K. R. and Elinson, R. P. (1988). The
entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior
enhanced Xenopus laevis embryos. Dev. Biol.
127, 64-77.[CrossRef][Medline]
Keller, R. and Danilchik, M. (1988). Regional
expression, pattern and timing of convergence and extension during
gastrulation of Xenopus laevis. Development
103,193
-209.
Keller, R., Davidson, L. A. and Shook, D. R.
(2003). How we are shaped: the biomechanics of gastrulation.
Differentiation 71,171
-205.[CrossRef][Medline]
Kinoshita, N., Iioka, H., Miyakoshi, A. and Ueno, N.
(2003). PKC delta is essential for Dishevelled function in a
noncanonical Wnt pathway that regulates Xenopus convergent extension
movements. Genes Dev.
17,1663
-1676.
Klein, T. J. and Mlodzik, M. (2005). Planar
cell polarization: an emerging model points in the right direction.
Annu. Rev. Cell Dev. Biol.
21,155
-176.[CrossRef][Medline]
Kuhl, M., Geis, K., Sheldahl, L. C., Pukrop, T., Moon, R. T. and
Wedlich, D. (2001). Antagonistic regulation of convergent
extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca2+
signaling. Mech. Dev.
106, 61-76.[CrossRef][Medline]
Kushner, P. D. (1984). A library of monoclonal
antibodies to Torpedo cholinergic synaptosomes. J.
Neurochem. 43,775
-786.[CrossRef][Medline]
Lane, M. C. and Keller, R. (1997). Microtubule
disruption reveals that Spemann's organizer is subdivided into two domains by
the vegetal alignment zone. Development
124,895
-906.[Abstract]
Li, L., Yuan, H., Weaver, C. D., Mao, J., Farr, G. H., 3rd,
Sussman, D. J., Jonkers, J., Kimelman, D. and Wu, D. (1999).
Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated
regulation of LEF-1. EMBO J.
18,4233
-4240.[CrossRef][Medline]
Montcouquiol, M., Sans, N., Huss, D., Kach, J., Dickman, J. D.,
Forge, A., Rachel, R. A., Copeland, N. G., Jenkins, N. A., Bogani, D. et
al. (2006). Asymmetric localization of Vangl2 and Fz3
indicate novel mechanisms for planar cell polarity in mammals. J.
Neurosci. 26,5265
-5275.
Nieuwkoop, P. D. and Faber, J. (1994).
Normal Table of Xenopus laevis (Daudin). New York:
Garland.
Peng, H. B. (1991). Solutions and Protocols. In
Xenopus laevis: Practical uses in Cell and Molecular
Biology. Vol. 36 (ed. B. K. Kay and H. B.
Peng), pp. 657-662. San Diego: Academic
Press.
Penzo-Mendez, A., Umbhauer, M., Djiane, A., Boucaut, J. C. and
Riou, J. F. (2003). Activation of Gbetagamma signaling
downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus
gastrulation. Dev. Biol.
257,302
-314.[CrossRef][Medline]
Peters, J. M., McKay, R. M., McKay, J. P. and Graff, J. M.
(1999). Casein kinase I transduces Wnt signals.
Nature 401,345
-350.[CrossRef][Medline]
Sater, A. K., Steinhardt, R. A. and Keller, R.
(1993). Induction of neuronal differentiation by planar signals
in Xenopus embryos. Dev. Dyn.
197,268
-280.[Medline]
Sawin, K. E., Mitchison, T. J. and Wordeman, L. G.
(1992). Evidence for kinesin-related proteins in the mitotic
apparatus using peptide antibodies. J. Cell Sci.
101,303
-313.
Schwarz-Romond, T., Asbrand, C., Bakkers, J., Kuhl, M.,
Schaeffer, H. J., Huelsken, J., Behrens, J., Hammerschmidt, M. and Birchmeier,
W. (2002). The ankyrin repeat protein Diversin recruits
Casein kinase Iepsilon to the beta-catenin degradation complex and acts in
both canonical Wnt and Wnt/JNK signaling. Genes Dev.
16,2073
-2084.
Shih, J. and Keller, R. (1992a). Cell motility
driving mediolateral intercalation in explants of Xenopus laevis.
Development 116,901
-914.
Shih, J. and Keller, R. (1992b). Patterns of
cell motility in the organizer and dorsal mesoderm of Xenopus
laevis. Development 116,915
-930.
Simons, M., Gloy, J., Ganner, A., Bullerkotte, A., Bashkurov,
M., Kronig, C., Schermer, B., Benzing, T., Cabello, O. A., Jenny, A. et
al. (2005). Inversin, the gene product mutated in
nephronophthisis type II, functions as a molecular switch between Wnt
signaling pathways. Nat. Genet.
37,537
-543.[CrossRef][Medline]
Sokol, S. Y. (1996). Analysis of Dishevelled
signalling pathways during Xenopus development. Curr.
Biol. 6,1456
-1467.[CrossRef][Medline]
Symes, K. and Smith, J. C. (1987). Gastrulation
movements provide an early marker of mesoderm induction in Xenopus
laevis. Development 101,339
-349.
Tahinci, E. and Symes, K. (2003). Distinct
functions of Rho and Rac are required for convergent extension during Xenopus
gastrulation. Dev. Biol.
259,318
-335.[CrossRef][Medline]
Takeuchi, M., Nakabayashi, J., Sakaguchi, T., Yamamoto, T. S.,
Takahashi, H., Takeda, H. and Ueno, N. (2003). The
prickle-related gene in vertebrates is essential for gastrulation cell
movements. Curr. Biol.
13,674
-679.[CrossRef][Medline]
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]
Ueno, N. and Greene, N. D. (2003). Planar cell
polarity genes and neural tube closure. Birth Defects Res. C Embryo
Today 69,318
-324.[CrossRef][Medline]
Wallingford, J. B. and Harland, R. M. (2001).
Xenopus Dishevelled signaling regulates both neural and mesodermal
convergent extension: parallel forces elongating the body axis.
Development 128,2581
-2592.
Wang, J., Mark, S., Zhang, X., Qian, D., Yoo, S. J.,
Radde-Gallwitz, K., Zhang, Y., Lin, X., Collazo, A., Wynshaw-Boris, A. et
al. (2005). Regulation of polarized extension and planar cell
polarity in the cochlea by the vertebrate PCP pathway. Nat.
Genet. 37,980
-985.[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]
Winklbauer, R. and Nagel, M. (1991).
Directional mesoderm cell migration in the Xenopus gastrula.
Dev. Biol. 148,573
-589.[CrossRef][Medline]
Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M.,
Hanafusa, H., Takada, R., Takada, S. and Nishida, E. (2002).
JNK functions in the non-canonical Wnt pathway to regulate convergent
extension movements in vertebrates. EMBO Rep.
3, 69-75.[CrossRef][Medline]
Yan, D., Wallingford, J. B., Sun, T. Q., Nelson, A. M.,
Sakanaka, C., Reinhard, C., Harland, R. M., Fantl, W. J. and Williams, L.
T. (2001). Cell autonomous regulation of multiple
Dishevelled-dependent pathways by mammalian Nkd. Proc. Natl. Acad.
Sci. USA 98,3802
-3807.
Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J. and
Nusse, R. (1995). The dishevelled protein is modified by
wingless signaling in Drosophila. Genes Dev.
9,1087
-1097.
Yokota, C., Kofron, M., Zuck, M., Houston, D. W., Isaacs, H.,
Asashima, M., Wylie, C. C. and Heasman, J. (2003). A novel
role for a nodal-related protein; Xnr3 regulates convergent extension
movements via the FGF receptor. Development
130,2199
-2212.
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]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  R. van Amerongen and R. Nusse Towards an integrated view of Wnt signaling in development Development, October 1, 2009; 136(19): 3205 - 3214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Bryja, E. R. Andersson, A. Schambony, M. Esner, L. Bryjova, K. K. Biris, A. C. Hall, B. Kraft, L. Cajanek, T. P. Yamaguchi, et al. The Extracellular Domain of Lrp5/6 Inhibits Noncanonical Wnt Signaling In Vivo Mol. Biol. Cell, February 1, 2009; 20(3): 924 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Shnitsar and A. Borchers PTK7 recruits dsh to regulate neural crest migration Development, December 15, 2008; 135(24): 4015 - 4024. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. S. Cselenyi and E. Lee Context-Dependent Activation or Inhibition of Wnt-{beta}-Catenin Signaling by Kremen Sci. Signal., February 26, 2008; 1(8): pe10 - pe10. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
) between the long axis of the cell and the
mediolateral axis (purple line) of the explant was used to calculate its
orientation in the explant. Protrusion stability was measured by counting the
number of stable (present throughout the 15 minute movie) and transient
protrusions (appearing after the first frame and/or disappearing before the
last). (B) Similar cellular changes were observed in isolated DMZ cells
of embryos injected with Lrp6MO. DMZ and VMZ cells from stage 10.5 embryos
were dissociated, plated on fibronectin, immediately fixed and stained with
Rhodamine-phalloidin. DMZ cells from wnt11 mRNA (Xwnt; 160 pg)-,
stbm mRNA (Xstbm; 200 pg)-, or Lrp6MO (LRP6MO; 40 ng)-injected
embryos have decreased length-to-width ratios, increased number of total
cytoplasmic protrusions, and increased numbers of cytoplasmic protrusions
along the short axes versus the long axes compared to GFP-injected (200 pg)
control. VMZ cells from wnt11-, stbm-, or Lrp6MO-injected
embryos show no obvious length-to-width ratio changes, but show a greater
number of protrusions along their long axes and fewer protrusions along their
short axes compared to controls. Statistical analyses in A and B were done
using Student's t-test. Error bars indicate standard deviation.
Asterisks mark differences that are statistically significant from control
(P<0.01). Numbers of cells analyzed are indicated in parentheses.
DMZ and VMZ cells used to determine length-to-width ratios were also used to
determine number of protrusions per cell. Cells and explants were co-injected
with myristoylated GFP (200 pg) to highlight plasma membranes and trace
injected cells. Scale bars: 25 µm in A; 20 µm in B.
