Department of Biological Sciences, Graduate School of Science, University
of Tokyo, and Core Research for Evolutional Science and Technology (CREST),
Japan Science and Technology Corporation, Hongo 7-3-1, Bunkyo-ku, Tokyo
113-0033, Japan
* Present address: Department of Microbiology and Molecular Genetics, Harvard
Medical School, and Molecular Medicine Unit, Beth Israel Deaconess Medical
Center, 330 Brookline Avenue, Boston, MA 02215, USA
Author for correspondence (e-mail:
m_taira{at}biol.s.u-tokyo.ac.jp)
Accepted 14 July 2002
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SUMMARY |
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Key words: Xenopus laevis, Spemann organizer, Convergent extension, Neural plate closure, Planar cell polarity, Xlim-1, Receptor tyrosine kinase, Xror2, Xwnt11, Xfz7, Cdc42
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INTRODUCTION |
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Gastrulation in Xenopus involves a complex set of morphogenetic
movements. The main engine producing the driving force for gastrulation is
thought to be convergent extension that results from mediolateral
intercalation of the dorsal marginal zone (DMZ), including the Spemann
organizer region. While the cellular basis of convergent extension is well
documented, molecular mechanisms regulating this process remain poorly
understood. It was reported that Wnt5a and Wnt4 affect morphogenetic movements
of ectodermal and mesodermal tissues in whole embryos, and inhibit elongation
of animal caps treated with a mesodermalizing factor, activin
(Moon et al., 1993;
Ungar et al., 1995
). Wnt11, in
Xenopus and zebrafish, has been shown to be required for convergent
extension during gastrulation, and the regulation of convergent extension by
Wnt11 has been suggested to take place through a non-canonical pathway similar
to that involved in planar cell polarity (PCP) signaling in
Drosophila (Heisenberg et al.,
2000
; Tada and Smith,
2000
). Components of Wnt signaling for the PCP pathway include
Frizzled 7 (Xfz7), Strabismus (Stbm), Dishevelled, a Formin Homology Protein
called Daam1, and the Rho family GTPases, Rho, Rac and Cdc42 (all of which
have been suggested to mediate the regulation of convergent extension in
Xenopus) (Darken et al.,
2002
; Djiane et al.,
2000
; Habas et al.,
2001
; Heisenberg et al.,
2000
; Park and Moon,
2002
; Sokol, 1996
;
Tada and Smith, 2000
;
Wallingford and Harland, 2001
;
Wallingford et al., 2000
).
One of the organizer-specific transcription factors is the LIM class
homeodomain protein Xlim-1 (Taira et al.,
1992). The LIM domain mutant of Xlim-1, named Xlim-1/3m, or a
complex of Xlim-1 and the LIM domain-binding protein Ldb1, appears to behave
as an activated form of Xlim-1. Activated forms of Xlim-1 can promote the
formation of a partial secondary axis in whole embryos when expressed
ventrally, and can initiate expression of the organizer-specific genes
goosecoid (gsc), chordin and Xotx2, in animal caps
(Agulnick et al., 1996
;
Mochizuki et al., 2000
;
Taira et al., 1994
;
Taira et al., 1997
),
suggesting that Xlim-1 is involved in the functions of the organizer. Using
differential screening, we searched for genes that function downstream of
Xlim-1, and found that one such gene was the Xenopus ortholog of the
mammalian ror2 (Xror2), which is an orphan receptor tyrosine kinase
with an immunoglobulin domain, a Frizzled-like domain, and a kringle domain in
the ectodomain (Oishi et al.,
1999
; Rehn et al.,
1998
). Previous papers have reported that the ror gene,
cam-1/kin-8, in C. elegans is involved in asymmetrical cell
division and the migration of neural cells
(Forrester et al., 1999
), as
well as in dauer larva formation (Koga et
al., 1999
), and that mouse Ror2 is required for heart
development and skeletal patterning during cartilage development
(DeChiara et al., 2000
;
Takeuchi et al., 2000
).
However, the functions of the Ror family genes, Ror1 and
Ror2, in the early embryogenesis of vertebrates have not been
elucidated. In this study, we found that Xror2 was expressed mainly
in the dorsal mesoderm and posterior neuroectoderm, where dynamic
morphogenetic movements are observed
(Keller et al., 1992
), and
that Xror2 played a role in convergent extension through the PCP pathway of
Wnt signaling in Xenopus laevis.
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MATERIALS AND METHODS |
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Construction and screening of a subtracted library
About 500 animal cap explants from embryos pre-injected with 250 pg of
Xlim-1/3m mRNA or uninjected (negative control) explants were prepared at the
blastula stage and cultured until the early gastrula stage (stage 10.5).
Poly(A)+ RNA was purified from total RNA of animal caps using the
PolyATtract mRNA isolation system (Promega). cDNA synthesis and suppression
PCR for creating a subtracted cDNA library were performed using the PCR-Select
cDNA subtraction kit (Clontech). To avoid the concentration of cDNAs derived
from injected Xlim-1/3m mRNA, 30 ng of Xlim-1/3m mRNA was added to a 2 µg
poly(A)+RNA pool of negative controls before synthesizing cDNA.
Subtracted cDNA fragments were cloned into pT7 Blue (R) vector (Novagen) for
colony hybridization with the subtracted PCR cDNA pool and the non-subtracted
PCR cDNA pool. To obtain insert DNA fragments, each bacterial colony was
directly subjected to PCR with a T7 promoter primer and a U-19 primer with SP6
promoter sequences. PCR products were subjected to DNA sequencing using an ABI
PRISM 310 Genetic Analyser (Perkin Elmer) or used as templates for
digoxigenin-labeled RNA probes for whole-mount in situ hybridization.
Screening of a cDNA library and DNA sequencing
A Xenopus gastrulae cDNA library (stages 10.5 and 11.5; kindly
provided by Dr B. Blumberg) was screened by plaque hybridization with
PCR-amplified cDNA fragments as probes. Positive clones were sequenced for
both strands with the Thermo Sequenase Cycle sequencing kit (Amersham) or a
cDNA sequencing kit (Perkin Elmer) and analyzed with LONG READIR 4200 (Li-Cor)
or ABI PRISM 310, respectively. Amino acid sequences were aligned using the
PILEUP program of the Wisconsin Package, Version 10.0 (Genetic Computer Group,
GCG, Madison, Wisconsin).
RNA preparation and northern hybridization
Total RNA was extracted by the acid phenol method
(Chomczynski and Sacchi, 1987),
electrophoresed on agarose-formaldehyde gels and blotted onto a nylon membrane
(Nytran, Schleicher and Shuell) (Sambrook
et al., 1989
). Blots were hybridized with 32P-labeled
DNA probes, washed with 2x SSPE containing 0.1% SDS at 65°C, and
exposed to an imaging plate and measured using a BAS 2500 (Fuji).
Whole-mount in situ hybridization and histological studies
Whole-mount in situ hybridization was carried out according to Harland's
method (Harland, 1991) with or
without an automated system (Automated ISH System AIH-101, Aloka). For
hemisections, rehydrated embryos were cut with a razor blade in 1x PBS,
0.1% Tween 20 before hybridization. Probes were synthesized from pBluescript
II SK()-Xror2 (pSKXror2), which was the longest clone we
obtained, en2 (Hemmati-Brivanlou
et al., 1991
), nrp1
(Knecht et al., 1995
;
Richter et al., 1990
) and
XPA26 (Hikasa and Taira, 2001) using DIG or fluorescein RNA Labeling
Mix (Boehringer Mannheim). BM Purple (Boehringer Mannheim), BCIP (Boehringer
Mannheim) and Magenta phosphate (Sigma) were used for chromogenic reactions.
Some stained embryos were embedded in paraffin wax and sectioned at widths
between 10 and 15 µm.
Plasmid constructs for mRNA injection experiments
pCS2+MT1-GR-NA was constructed by inserting fragments encoding a Myc
tag, the hormone-binding domain (amino acids 511-777) of the human
glucocorticoid receptor (Hollenberg et
al., 1993
) and Xlim-1/
NA
(Taira et al., 1994
) into
pCS2+AdN (Mochizuki et al.,
2000
). pCS2-Xror2, pCS2-Xror2-TM or pCS2-Xror2-KR were constructed
by inserting a PCR fragment encoding full-length, amino acids 1-469 or amino
acids 1-399, respectively, of Xror2 into pCS2+. pCS2-Xwnt5a-Myc,
pCS2-Xwnt8-Myc and pCS2-Xwnt11-Myc were generated by inserting the coding
regions into pCS2+MT to connect five Myc tags at their C termini.
pCS2-Exfz7-FLAG was constructed by inserting PCR fragments (amino acids 1-209
of Xfz7) into pCS2+FTc, which encodes a FLAG tag at the C terminus (T.
Mochizuki and M. T.). A point mutant (pCS2-Xror2-3I), small deletion mutants
(pCS2-Xror2-FZ
1 and pCS2-Xror2TM-FZ
1) and a C-terminal
FLAG-tagged construct (pCS2-Xror2KR-FLAG) were generated using an in vitro
site-directed mutagenesis system (GeneEditor, Promega). All constructs were
verified by sequencing. For mRNA injection, plasmid constructs were linearized
with appropriate restriction enzymes, and transcribed using the MEGAscript kit
(Ambion) and a 7mG(5')ppp(5')G CAP analog (New England Biolabs).
mRNA (20 pg/embryo) encoding nuclear ß-galactosidase (nß-gal) was
co-injected as a lineage tracer, and the enzyme activity of nß-gal was
visualized using Red-Gal (Research Organics) as substrate. Some embryos
stained with ß-gal reaction were subjected to whole-mount in situ
hybridization or embedded in paraffin wax for sectioning.
Immunoprecipitation and western blotting
Immunoprecipitation was carried out as described previously
(Djiane et al., 2000) with
some modifications. Embryos were injected with mRNAs in the animal pole region
at the two-cell stage. Nine injected embryos at the mid-gastrula stage were
homogenized with 900 µl of extraction buffer (150 mM NaCl, 5 mM EDTA, 0.5%
NP-40, 10 mM Tris-HCl pH 7.5, 2 mM PMSF, 25 µM leupeptin and 0.2 units/ml
aprotinin). Cell extracts (900 µl) were incubated with anti-FLAG M2
antibody (Sigma) for 2 hours at room temperature and further incubated at
4°C for 3 hours after adding 40 µl of protein G agarose beads (Roche).
Proteins attached to the beads were washed with extraction buffer four times,
subjected to SDS-PAGE and blotted to an Immobilon membrane (Millipore).
Blotted membranes were exposed to anti-Myc 9E10 monoclonal antibody conjugated
with peroxidase (BioMol Research Lab) and were developed by ECL+plus reagents
(Amersham).
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RESULTS |
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The longest Xror2 cDNA clone we isolated is 3924 bp long and encodes a
predicted protein of 930 amino acids that is highly homologous to human and
mouse Ror2, a receptor tyrosine kinase
(Masiakowski and Carroll,
1992; Oishi et al.,
1999
) (Fig. 1A).
Ror family proteins contain an immunoglobulin-like domain, a Frizzled-like
domain, a kringle domain, a transmembrane domain and a tyrosine kinase domain
(Masiakowski and Carroll,
1992
; Oishi et al.,
1999
). The Frizzled-like domain of Xror2 has a motif containing 10
conserved cysteines (Fig. 1A,
asterisks) that is characteristic of the ectodomain of the Wnt receptor
Frizzled family (Rehn et al.,
1998
). However, the ligand of the Ror receptor family has not yet
been identified. Within the tyrosine kinase domain, Xror2 has a predicted
ATP-binding motif (GXDXXGAIK) that is conserved among the Ror2
proteins but not the Ror1 proteins (GXCXXGAIK)
(Fig. 1A,B)
(Oishi et al., 1999
).
|
For the functional analysis of Xror2 as described below, we made five
mutant constructs (Fig. 1B).
Xror2-3I is a kinase domain point-mutant in which three lysines at position
504 (in the putative ATP-binding motif), 507 and 509 were all replaced with
isoleucine. Xror2-TM is a kinase domain-deleted mutant in which the
intracellular region, including the tyrosine kinase domain, was deleted.
Xror2-KR is a putative secreted type construct that contains only the
ectodomain. Xror2-FZ1 and Xror2TM-FZ
1 are Frizzled-like
domain-deleted mutants in which 20 amino acid residues (positions 175-194),
including the second cysteine in the Xror2 Frizzled-like domain, were deleted
from wild-type Xror2 and Xror2-TM, respectively.
Xror2 is upregulated by Xlim-1 plus Ldb1 and BMP antagonists
Northern blot analyses showed that expression of Xror2 is
activated by co-expression of Xlim-1 and Ldb1, and by the BMP antagonists
chordin and noggin in animal caps (Fig.
2A,B). Because the chordin gene is upregulated by Xlim-1
in animal caps (Fig. 2A),
Xror2 expression may be mediated by chordin expression.
However, while chordin expression was reduced in animal caps injected
with lower doses of Xlim-1/3m or Xlim-1 plus Ldb1
(Fig. 2A), Xror2
expression was maintained under the same conditions, suggesting that
Xror2 expression by Xlim-1 is not solely mediated by
chordin.
|
To elucidate whether or not protein synthesis is required for the
activation of Xror2 gene by Xlim-1 in animal caps, we constructed a
hormone-inducible construct of an active form of Xlim-1 (GR-NA)
(Gammill and Sive, 1997
;
Tada et al., 1998
;
Tada and Smith, 2000
;
Taira et al., 1994
). As shown
in Fig. 2C, induction of
Xror2 expression by GR-
NA in the presence of DEX was inhibited
by CHX, suggesting that Xror2 expression is indirectly activated by
Xlim-1 in animal caps. Conversely, in agreement with our previous report
(Mochizuki et al., 2000
),
activation of the gsc gene by GR-
NA was not inhibited by CHX,
emphasizing that gsc is a direct target of Xlim-1.
Spatiotemporal expression of Xror2 in Xenopus
embryos
Northern blot analysis was performed to analyze the temporal expression of
Xror2. While maternal transcripts of Xror2 were not detected
during cleavage stages, expression of Xror2 was first detected at the
early gastrula stage (stage 10), and its expression peaked from the early
neurula stage (stage 13) to the mid-neurula stage (stage 15)
(Fig. 2D). Up to the tailbud
stage (stage 28), the expression of Xror2 was maintained at high
levels (Fig. 2D).
Whole-mount in situ hybridization showed that Xror2 transcripts are first observed in the DMZ at the early gastrula stage (stage 10.25) with laterally expanding expression (Fig. 3A). In early gastrula embryos bisected along the midline (stage 10.25), Xror2 transcripts were detected mainly in the dorsal mesoderm and ectoderm above the dorsal lip (Fig. 3B). As shown in Fig. 3C, the expression of both Xror2 (left) and Xlim-1 (right) was observed strongly in the dorsal mesoderm and also faintly in the ventral mesoderm, overlapping with each other except for the expression of Xror2 in the dorsal ectoderm. As gastrulation proceeded, Xror2 transcripts were intensely detected in the mesoderm and the posterior portion of the overlying dorsal ectoderm, but not in the dorsal endomesoderm (Fig. 3D).
|
Expression of Xror2 in the epithelial and sensorial layers of the
neuroectoderm is maintained in late gastrula to early neurula embryos (stages
12 to 14, Fig. 3E,F), whereas
the expression in the mesoderm is restricted to the notochord at stage 14
(Fig. 3F). In the neuroectoderm
of neurula embryos (stages 15 to 17), Xror2 transcripts were detected
with a clear border at the anterior limit of the expression domain
(Fig. 3G,H).
Fig. 3I shows that the anterior
border of Xror2 expression (magenta) is posterior to the
midbrain-hindbrain boundary indicated by the en2 expression
(turquoise), although the lateral stripes of Xror2 expression
corresponding to the neural crest extends more anteriorly than does
en2 expression. At this stage, the transverse sections showed that
Xror2 expression was detected in both epithelial and sensorial layers
of the neuroectoderm and also faintly in the notochord
(Fig. 3J). At tailbud stages,
the anterior border of Xror2 expression in the neural tube became
obscure, but the expression in pharyngeal arches 1 to 4 was clearly observed
(Fig. 3K)
(Sadaghiani and Thiebaud,
1987).
Xror2 and its intracellular domain mutant constructs cause a
shortened anteroposterior axis accompanied by head defects
If Xror2 is a downstream gene of Xlim-1 in the organizer, it is
possible that Xror2 takes part in the functions of Xlim-1. We therefore tested
first whether or not ectopic expression of Xror2 in the ventral marginal zone
(VMZ) initiates secondary axis formation. As a result, Xror2 did not elicit
any apparent ectopic axis even at high doses (1-3 ng/embryo), but instead
caused malformation in posterior structures (48%, n=159;
Fig. 4B), whereas Xlim-1/3m
initiated secondary axis formation (data not shown), as reported
(Taira et al., 1994).
|
We next overexpressed Xror2 constructs in the DMZ, where putative ligands for Xror2 may exist. Embryos injected with wild-type Xror2 showed a shortened body axis with dorsal bending and abnormalities in head structures, which included one-eyed phenotypes (73%, n=112; Fig. 4D). The frequency of these phenotypes was reduced, but not abolished completely, when the kinase domain point mutant Xror2-3I was expressed (30%, n=70; Fig. 4E), implying that the kinase activity may not be essential for this phenotype. This possibility was supported by the results that phenotypes with the kinase domain deletion mutant Xror2-TM were similar to those with Xror2-3I in terms of short stature and head defects (50%, n=58; Fig. 4F). In contrast to Xror2-3I and Xror2-TM, Xror2-KR showed much weaker phenotypes, even at a twofold molar ratio to that of wild-type (5%, n=93; Fig. 4G), suggesting that the phenotypes with wild-type, Xror2-3I and Xror2-TM are not due to the depletion of its putative ligands. Thus, these data suggest that the membrane-anchored ectodomain of Xror2 per se appears to have some role for cell-cell communications, which could be enhanced by its kinase activity.
Xror2 and its intracellular domain mutants interfere with convergent
extension during gastrulation and affect neural plate closure during
neurulation
To examine whether overexpression of Xror2 constructs on the dorsal side
leads to abnormalities in morphogenetic movements, nß-gal mRNA was
co-injected as a tracer into one blastomere on the right side at the four-cell
stage. As shown in Fig. 5A,
globin-expressing control cells were restricted in their distribution along
the midline in the trunk region as a result of normal convergent extension,
and not in the head region as expected. By contrast, cells expressing Xror2,
Xror2-3I or Xror2-TM expanded laterally on the right side of both the mesoderm
and ectoderm layers (Fig.
5B-D). The ability of wild-type Xror2 to interfere with convergent
extension was higher than that of the Xror2 kinase domain mutants (Xror2-3I
and Xror2-TM) at similar molar levels
(Table 1). Xror2-KR seemed to
slightly affect convergent extension at higher doses, but most
Xror2-KR-expressing embryos showed normal movements, similar to
globin-expressing embryos (Fig.
5A,E; Table 1).
When open yolk plug phenotypes were observed at a low frequency in embryos
expressing globin and Xror2-KR, nß-gal-positive cells converged to the
midline at a significant frequency (Fig.
5F; Table 1),
implying that convergent extension and yolk plug closure are separable events
(see also Fig. 5B').
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|
We also noticed that Xror2-TM initiates the condensation of pigmented cells at early neurula stages in nß-gal-positive regions of dorsal ectoderm (Fig. 5D, black arrows and arrowheads; Table 1; see also Fig. 6C). This activity differs from that of wild-type and Xror2-3I. At the late neurula stage (stage 18), Xror2 or Xror2-3I mRNA-injected embryos still failed to close the neural plate in nß-gal-positive regions (Fig. 5H,I, white arrows) compared with globin- and Xror2-KR-expressing embryos (Fig. 5G,K; Table 2), whereas Xror2-TM mRNA-injected embryos showed the closing neural plate with pigmented cells (Fig. 5J, white arrowheads; Table 2). These data imply that Xror2 has distinct roles in neurulation of the pigmented epithelial layer and the sensorial layer.
|
|
To examine whether or not the Frizzled-like domain of Xror2 is involved in
its functional activities, we constructed mutants with a small deletion in the
Frizzled-like domain, based on the report that the same mutation in
Drosophila Frizzled 2 inhibits Wnt binding
(Hsieh et al., 1999)
(Fig. 1B). We observed that
Xror2-FZ
1 and Xror2TM-FZ
1 produced much less inhibition on
convergent extension and neural plate closure during gastrulation and
neurulation in comparison with Xror2 and Xror2-TM, respectively
(Table 3 and data not shown).
These results indicate that the effect of Xror2 on morphogenetic movements is
significantly dependent on its Frizzled-like domain, raising the possibility
that Xror2 might interact with a Wnt pathway.
|
Xror2 does not affect gene expression of molecular markers for neural
and notochordal differentiation
To test whether wild type and mutant versions of Xror2 affect the cell fate
of neural tissue and the notochord, we analyzed expression of nrp1 as
a pan-neural marker (Knecht et al.,
1995) and XPA26 as a notochord marker (Hikasa and Taira,
2001) in embryos co-injected with Xror2 constructs and nß-gal mRNA. In
Xror2- (Fig. 6B,E), Xror2-3I-
(not shown) or Xror2-TM- (Fig.
6C,F) expressing embryos, nß-gal-positive cells overlapped
with nrp1 and XPA26 expression, similar to control cells
(Fig. 6A,D). It should also be
noted that the notochord is stacked near the unclosed blastopore and failed to
elongate anteriorly in Xror2- (Fig.
6E), Xror2-3I- (not shown) and Xror2-TM-
(Fig. 6F) expressing embryos.
This is clearly different from the open yolk plug phenotype caused by the
inhibition of mesoderm formation by a dominant-negative FGF receptor, in which
two columns of notochord extend posteriorly around the blastopore
(Isaacs et al., 1994
).
Moreover, we observed that these Xror2 constructs do not affect the staining
with the muscle-specific antibody 12/101 (data not shown). Thus, we conclude
that the failure of convergent extension caused by Xror2 constructs was not
due to changes in cell fate.
Effects of Xror2 constructs on morphogenetic movements in animal
caps
Elongation of animal caps by treatment with activin provides a useful model
system for analyzing convergent extension in Xenopus
(Djiane et al., 2000;
Tada and Smith, 2000
). Xror2,
Xror2-3I and Xror2-TM strongly suppressed elongation of animal caps by activin
(Fig. 7A-D), consistent with
the observations in whole embryos (Fig.
5). Compared with globin-expressing animal caps, elongation was
slightly reduced by Xror2-KR, suggesting that Xror2-KR has a weak activity
that is not apparent in whole embryos (Fig.
7A,E). In Xror2-TM-expressing animal caps treated with activin,
neural groove-like structures with pigmentation were also observed
(Fig. 7D, arrowheads), as has
been seen in Xror2-TM-expressing whole embryos
(Fig. 5J).
|
Previous studies of Xwnt11 and Dishevelled have shown that both wild-type
and a dominant-negative form of these proteins have activity to inhibit the
morphogenetic movements of animal caps or DMZ explants, but coexpression of
both proteins can offset the phenotype from either of them
(Tada and Smith, 2000;
Wallingford et al., 2000
).
These phenomena imply that opposite effects on cell polarity can eventually
show similar phenotypes. To elucidate whether or not this relationship is
applied to wild-type Xror2 and Xror2-TM, we co-expressed these constructs in
animal caps treated with activin. Either wild-type Xror2 or Xror2-TM alone at
low doses moderately inhibited elongation of activin-treated animal caps
(Fig. 7F,G,I), whereas
co-expression of these two constructs inhibited the elongation more strongly
(Fig. 7H). These results
indicate that wild-type and Xror2-TM have the same activity in terms of
interference with the morphogenetic movements.
Functional interactions between Xror2 and Wnt signaling
components
As mentioned above, the results from overexpression of the Frizzled-like
domain mutants (Table 3)
implied the possibility that the activity of Xror2 could be involved in Wnt
signaling. Both Xwnt11 and Xfz7 have been shown to regulate convergent
extension (Djiane et al.,
2000; Ku and Melton,
1993
; Tada and Smith,
2000
), and exhibit significant overlapping expression with Xror2
in the dorsal marginal zone (Fig.
3). To assess involvement of Xror2 in Wnt signaling, we first
examined the effects of co-expression of Xror2, Xwnt11 and Xfz7 on convergent
extension (Table 4). A low dose
of Xror2 or Xwnt11 alone (25 or 50 pg/embryo, respectively) had a very small
effect on convergent extension, but a combination of Xror2 plus Xwnt11 showed
synergistic effects, which were higher than those of twofold doses of either
alone. Interestingly, such synergy was also observed between Xror2 and Xfz7.
Moreover, co-expression of the three proteins Xror2, Xwnt11 and Xfz7 exerted
stronger effects than those of any two of them, suggesting that Xror2, Xwnt11
and Xfz7 cooperate to function in Wnt signaling for convergent extension
(Table 4).
|
Synergistic effects of Xror2, Xwnt11 and Xfz7 raised the possibility that
Xror2 activates a PCP pathway of Wnt signaling, which has been suggested to
involve the Rho family GTPase Cdc42. A dominant-negative Cdc42 mutant
(Cdc42T17N) has been shown to offset the inhibitory effect of
Xwnt11 or Xfz7 on activin-induced elongation of animal caps
(Djiane et al., 2000). We
therefore tested whether inhibition of convergent extension by Xror2 was also
rescued by Cdc42T17N using animal cap assays. As shown in
Fig. 8, inhibition of
activin-induced elongation by Xror2 was rescued by co-expression of
Cdc42T17N to some extent (Fig.
8A,B,F), similar to that in the case of Xwnt11 and
Cdc42T17N (Fig.
8C,D,F). Interestingly we also noticed that the inhibition of
elongation by Xror2 and Xwnt11 is more effectively reversed by
Cdc42T17N in unpigmented cells rather than in pigmented cells.
These results suggest that the signal mediated by Xror2 activates Cdc42
through a PCP pathway of Wnt signaling, leading to inhibition of convergent
extension.
|
Physical interactions of Xror2 and Xwnt11
The existence of the Frizzled-like domain in Xror2, and the synergism
between Xror2 and Xwnt11 implied possible physical interactions between them.
Using co-immunoprecipitation experiments with epitope-tagged proteins, we
found that Xwnt11-Myc, Xwnt5a-Myc and Xwnt8-Myc all were co-immunoprecipitated
with FLAG-tagged Xror2-KR (Xror2KR-FLAG), similar to the FLAG-tagged
ectodomain of Xfz7 (Exfz7-FLAG) (Fig.
9) (Djiane et al.,
2000). This data suggests that the ectodomain, most likely the
Frizzled-like domain, of Xror2 can interact with several Xwnt proteins,
further emphasizing the possibility that Xror2 acts together with Xwnt11 for
the PCP pathway of Wnt signaling in the regulation of convergent
extension.
|
![]() |
DISCUSSION |
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From the gastrula to the neurula stages, Xror2 is expressed in the
involuting mesoderm and neural plate posterior to the midbrain-hindbrain
boundary (Fig. 3), where
convergent extension occurs (Keller et
al., 1992). Furthermore, Xror2 is probably expressed in
migrating neural crest cells in the pharyngeal arches at tailbud stages
(Fig. 3K). These Xror2
expression patterns imply a role of Xror2 in morphogenetic cell movements and
cell migration. Functional analyses support this possibility as discussed
below.
Roles of Xror2 in convergent extension and neural plate closure
Using mRNA injection experiments, we found that: (1) wild-type Xror2 as
well as its kinase domain mutants, Xror2-TM and Xror2-3I, cause disruption of
convergent extension in whole embryos and also in animal caps treated with
activin, whereas the secreted type construct Xror2-KR has a much weaker
activity; (2) Xror2-TM does not antagonize wild-type Xror2 in terms of the
inhibition of activin-induced elongation in animal caps; and (3) Xror2-TM
initiates condensation of pigmented cells in the closing neural plate, whereas
wild-type Xror2 and Xror2-3I inhibit neural plate closure and neural groove
formation. These data suggest that Xror2 has roles in convergent extension and
neural plate closure. Furthermore, inhibition of convergent extension by Xror2
does not appear to be dependent on the kinase domain, but dependent on the
ectodomain attached to the transmembrane region. This raises the possibility
that the ectodomain of Xror2 has a significant function without the tyrosine
kinase domain, when it exists on the cell membrane. This possibility is
consistent with the phenotype of C. elegans ror mutants. The C.
elegans ror gene, cam-1, is required for asymmetric cell
division, cell migration and axon outgrowth of a specific type of neuronal
cell, and a truncated kinase domain mutant shows only subtle effects on cell
migration and partial effects on asymmetric cell division. Moreover, the
defects of cell motility in null ror mutants can be rescued by
kinase-domain point-mutated constructs of the ror gene
(Forrester et al., 1999).
Thus, the ectodomain of both Xror2 and C. elegans Ror appears to have
a function similar to that of wild-type.
Kinase-independent functions of receptor kinases have been described not
only for C. elegans Ror, but also for other receptor tyrosine
kinases. For example, the ectodomain of MuSK (muscle-specific kinase), related
to the Ror family, mediates clustering of synaptic components via binding of
agrin, the MuSK ligand (Apel et al.,
1997). In the case of Xror2, how can the ectodomain itself have
functions without transducing signals via its kinase activity? One can
speculate that Xror2 works as a cell adhesion molecule, or interacts with some
membrane-anchored protein that is involved in convergent extension. Still, it
should be noted that the activities of kinase domain mutants Xror2-3I and
Xror2-TM, are weaker than those of wild-type Xror2 (Tables
1,
2), suggesting that the
tyrosine kinase activity of Xror2 does have some function in modulating
convergent extension.
It has been reported that neurulation takes place through two distinct
processes of cell movements in Xenopus
(Davidson and Keller, 1999).
The first visible morphogenetic cell movements in neurulation result in neural
fold fusion, in which superficial neural cells apically contract and roll the
neural plate to form the neural groove. After neural fold fusion, medial
migration of neural cells in a lateral sensorial layer occurs to form the
dorsal tube. During these processes, we found that wild-type and 3I mutant
proteins inhibit both neural fold fusion and convergent extension of medially
migrating sensorial layer cells, whereas Xror2-TM inhibits convergent
extension of sensorial cells but appears not to inhibit neural groove
formation (Fig. 5D,
Fig. 6C, Table 1). These data suggest
that the intracellular region of Xror2 is involved in the regulation of the
neural fold fusion of the epithelial layer.
Xror2 and Wnt signaling
In Drosophila, PCP signaling through Frizzled requires the
activity of a putative four transmembrane protein, Stbm and Dishevelled, and
activates the small GTPase RhoA and JNK
(Adler et al., 2000;
Axelrod et al., 1998
;
Boutros and Mlodzik, 1999
;
Eaton et al., 1996
;
Strutt et al., 1997
;
Taylor et al., 1998
;
Winter et al., 2001
;
Wolff and Rubin, 1998
).
Xenopus PCP signaling-related genes such as a class of Xwnt11, Xfz7,
Stbm, Dishevelled, Daam1 and small GTPases have been suggested to regulate
convergent extension during gastrulation
(Darken et al., 2002
;
Djiane et al., 2000
;
Habas et al., 2001
;
Park and Moon, 2002
;
Sokol, 1996
;
Tada and Smith, 2000
;
Wallingford and Harland, 2001
;
Wallingford et al., 2000
).
Interestingly, Xror2 has a Frizzled-like domain in the extracellular region,
which is expected to interact with Wnt proteins
(Rehn et al., 1998
). With
regard to interactions between Xror2 and Wnt signaling, our functional
analyses have led to the following conclusions: (1) Xror2 has the activity to
affect convergent extension, as do Xwnt11 and Xfz7; (2) the activity of Xror2
depends on its Frizzled-like domain and can be synergistic with Xwnt11 and
Xfz7; (3) the inhibitory effect of Xror2 on elongation of activin-treated
animal caps is modestly rescued by a dominant-negative Cdc42 mutant; and (4)
the ectodomain of Xror2 can bind to Xwnt11 and Xwnt5a. These results suggest
that Xror2 is involved in the non-canonical Wnt signaling for the PCP pathway.
Although the rescue of the animal cap elongation by a dominant-negative Cdc42
is weak, this may be explained by the involvement of other small GTPases such
as Rho and Rac in convergent extension, which have been suggested by Habas et
al. (Habas et al., 2001
). In
addition, it is also conceivable that Xror2 stimulates the Wnt signaling
through interaction with Frizzled, a seven transmembrane receptor, and perhaps
Stbm, a four transmembrane protein on the plasma membrane. These possibilities
are based on our findings of synergy between Xror2 and Xfz7, and the reported
observations that MuSK interacts with the ligand agrin
(Glass et al., 1996
), the four
transmembrane protein acetylcholine receptor
(Fuhrer et al., 1999
;
Fuhrer et al., 1997
), and the
cytoplasmic protein rapsyn through a putative transmembrane intermediate
(Apel et al., 1997
) to
stimulate clustering of acetylcholine receptors in the postsynaptic membrane.
It is therefore tempting to speculate that Xror2 mediates or modifies the PCP
signaling by complex formation with Wnt11 and the transmembrane proteins Xfz7
and Stbm to regulate convergent extension.
A loss-of-function study of Xfz7 by a morpholino approach has shown that
Xfz7-depletion leads to disruption of tissue separation between mesoderm and
ectoderm without affecting convergent extension
(Winklbauer et al., 2001).
Because Xfz8, which is closely related to Xfz7, has also been shown to be
expressed at the DMZ and to affect convergent extension
(Deardorff et al., 1998
;
Itoh et al., 1998
;
Wallingford et al., 2001
),
there might be functional redundancy between Xfz7 and Xfz8 in convergent
extension.
Comparison of Xror2 with mammalian Ror genes
In mice, targeted gene disruption of Ror2 has been shown to lead
to skeletal abnormalities with endochondrally derived foreshortened or
misshapen bones (DeChiara et al.,
2000; Takeuchi et al.,
2000
), and these phenotypes are significantly similar to those of
mice disrupted with Wnt5a (Yamaguchi et
al., 1999
). In humans, heritable dominant mutations in the
ROR2 gene cause brachydactyly type B, in which the thumbs and big
toes are spared (Oldridge et al.,
2000
). These data suggest that mammalian Ror genes play roles in
skeletal patterning and limb development in late embryogenesis.
In Xenopus, our results indicate that Xror2 has roles in
morphogenetic movements during gastrulation and neurulation at early
developmental stages without influencing cell fates. Although mouse
Ror2 is expressed in the primitive streak
(Matsuda et al., 2001), which
corresponds to the dorsal mesoderm of Xenopus, and has the same
effect as Xror2 on convergent extension when dorsally overexpressed in
Xenopus embryos (data not shown), it is not known whether or not Ror2
functions in morphogenetic movements in mice. As cell movements in
gastrulation and neurulation appear to be different between amphibians and
higher vertebrates, Xror2 may have unique functions in convergent extension
during gastrulation and neurulation in amphibians. Nevertheless, our data
provide the first evidence that Ror2 plays a role in morphogenetic movements
in relation to the PCP signaling pathway of Wnt in vertebrates, and that the
ligand of Ror2 is Wnts.
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ACKNOWLEDGMENTS |
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