1
Department of Genetics, Stanford University, Stanford, CA 94305-5120,
USA
2
Department of Biochemistry, University of Ulm, Germany
*
Author for correspondence at present address: Institute of Zoology II,
University Karlsruhe (e-mail:
doris.wedlich{at}zi2.uni-karlsruhe.de
Accepted 21 May 2001
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SUMMARY |
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Migration of transplanted cranial neural crest cells was blocked when
full-length Xcad-11 or its mutant lacking the ß-catenin-binding site
(cXcad-11) was overexpressed. In addition, the expression of neural
crest markers (AP-2, Snail and twist) diminished within the
first four hours after grafting, and disappeared completely after 18 hours.
Instead, these grafts expressed neural markers (2G9, nrp-1 and
N-Tubulin). ß-catenin co-expression, heterotopic transplantation
of CNC cells into the pharyngeal pouch area or both in combination failed to
prevent neural differentiation of the grafts.
By contrast, eXcad-11 overexpression resulted in premature
emigration of cells from the transplants. The AP-2 and Snail
patterns remained unaffected in these migrating grafts, while twist
expression was strongly reduced. Co-expression of
eXcad-11 and
ß-catenin was able to rescue the loss of twist expression,
indicating that Wnt/ß-catenin signalling is required to maintain
twist expression during migration.
These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/ß-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.
Key words: Cadherin, Neural crest, Migration, Xenopus
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INTRODUCTION |
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It was previously assumed that cadherin function is restricted to
non-migrating, predominantly polarised epithelial tissues where cadherins are
found in adherens junctions (Kemler,
1992). The most thoroughly
studied member of the cadherin gene superfamily, E-cadherin, was
characterised as a tumour suppressor gene because loss of its expression
correlated with increased invasiveness of tumours (Birchmeier and Behrens,
1994
). Interestingly,
downregulation of cadherin expression in early development correlates with the
start of migration. For example, when chicken neural crest cells delaminate
from the neural folds, Ca2+-dependent adhesion decreases, and
N-cadherin and c-cad6B are downregulated (Newgreen and Gooday,
1985
; Akitaya and
Bronner-Fraser, 1992
; Nakagawa
and Takeichi, 1995
).
However, the identification of the type II classical cadherins,
cadherin-11, cadherin-6 and cadherin-7 (Tanihara et al.,
1994; Hoffmann and Balling,
1995
; Nakagawa and Takeichi,
1995
; Nakagawa and Takeichi,
1998
; Inoue et al.,
1997
; Hadeball et al.,
1998
; Vallin et al,
1998
), and of the
protocadherins PAPC and AXPA (Kim et al.,
1998
) led to an exciting
discovery: these cadherins are upregulated in migrating cells, such as neural
crest cells, as well as in invasive tumour cells and mesodermal cells
undergoing convergent extension movements. Still, despite the correlation of
their expression profiles with cell movement, little is known about their
function in cell migration or cell differentiation.
The cranial neural crest (CNC) is ideal to study these questions because it
exhibits highly migratory cell behaviour while simultaneously undergoing cell
specification. At the beginning of the 20th century, classical grafting and
ablation experiments in amphibians had already revealed that these cells gave
rise to craniofacial cartilage, the peripheral nervous system (PNS) and
pigment cells (Landacre, 1921;
Stone, 1921
; Raven,
1933
). The identification of
neural crest marker genes (Hopwood et al.,
1989
; Winning et al.,
1991
; Essex et al.,
1993
) and novel molecular
labelling and microscopic techniques allowed the confirmation of these
morphological observations in different organisms (Le Dourain,
1982
; Sadaghiani and Thiebaud,
1987
; Hall and
Hörstadius,
1988
).
The presence of some common progenitor cells for the different neural crest
derivatives at the migratory stage is still in discussion (Groves and
Bronner-Fraser, 1998; Mayor et
al., 1998
; LaBonne and
Bronner-Fraser, 1999
). Single
cell tracking (Collazo et al.,
1993
) and neural crest cell
culture studies (LeDourain and Smith,
1988
; Anderson et al.,
1997
) support the existence of
multipotent neural crest progenitors that become committed to different fates
during migration. There is strong evidence that neural crest is induced by
inhibition of BMP, followed by activation of canonical Wnt/ß-catenin or
e/bFGF signalling (Mayor et al.,
1995
; Saint-Jeannet et al.,
1997
; Chang and
Hemmati-Brivanlou, 1998
;
LaBonne and Bronner-Fraser,
1998
). This leads to
activation of marker genes Slug/Snail, twist and AP-2 at the
premigratory stage. Recent reports (Carl et al.,
1999
; LaBonne and
Bronner-Fraser, 2000
) have
revealed a function for Slug in CNC migration. However, neither the
control of movement at the cellular level nor the process of cell
specification during migration is understood.
We have investigated the function of Xcad-11, which is expressed in migrating CNC cells. We found that endogenous Xcad-11 expression restrains cranial neural crest migration. However, preventing the migration of CNC cells results in a change from CNC marker expression (AP-2, Snail and twist) to neural marker expression (2G9, nrp-1 and N-Tubulin). This switch was independent of both Wnt/ß-catenin signalling and transplant localisation, suggesting that an increase in cell adhesion promotes neural differentiation. Wnt/ß-catenin signalling, however, was found essential for twist expression in migrating CNC.
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MATERIALS AND METHODS |
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GST-pull-down assay
Xcad-11 and cXcad-11 protein were expressed from circular plasmids
using the transcription and translation kit (TNT) from Promega (Mannheim,
Germany) according to the manufacturer's instructions. GST-ß-catenin
harbouring amino acids 1-284 of ß-catenin (Bauer et al.,
1998
) was expressed in
Escherichia coli XL-1-blue. Protein expression and pull-down assay
were performed as stated previously (Giehl et al.,
2000
). After SDS-PAGE, the
precipitated protein was detected by the monoclonal 9E10 Myc antibody (10
hours, 4°C), and peroxidase-coupled goat anti-mouse antiserum (2 hours,
room temperature). Immunoreactive proteins were visualised using the
ECLTM western blotting detection system (Amersham, Braunschweig,
Germany).
Injection of Xenopus laevis embryos
In vitro transcribed mRNA of Xcad-11 (250 pg, 0.6 ng, 0.8 ng, 1 ng, 1.6
ng), eXcad-11 (250 pg, 0.8 ng, 1 ng, 2.3 ng) and
cXcad-11 (0.8
ng, 1 ng, 2.5 ng) were co-injected with 100 pg GFP-RNA into one blastomere of
a two-cell stage embryo. Embryos were obtained by in vitro fertilisation,
cultivated and injected as described previously (Geis et al.,
1998
), and staged according to
the normal table of Nieuwkoop and Faber (Nieuwkoop and Faber,
1975
). Embryos at stage 14
exhibiting GFP-fluorescence were sorted in terms of left or right side
fluorescence using an Olympus epifluorescence microscope. Embryos were used
either for transplantation or further cultivated until stage 28 and analysed
by whole-mount in situ hybridisation.
Transplantation assay
Transplantation of CNC was performed as previously described (Borchers et
al., 2000). To trace the
transplanted cells Myc-tagged GFP-RNA was injected into one blastomere of
two-cell embryos. The epidermis covering the cranial neural crest area was
peeled off from the GFP-positive side. Part of the underlying CNC was removed
at the premigratory stage and inserted in an uninjected control, the host
embryo, which was treated accordingly. Transfer of neuroepithelial cells was
avoided, which was controlled by in situ hybridisation.
The migration pattern of transplanted embryos was analysed by GFP
fluorescence from stage 14 to 48 using an Axiophot microscope (Zeiss, Jena,
Germany), and documented on Kodak Ektachrome 160T film. To compare the
velocity of eXcad-11-expressing and GFP-control transplants, 111
transplants were prepared using three egg batches, which were continuously
monitored over a timespan of 48 hours. The migration patterns of transplants
exhibiting migration 18 hours after grafting (86% of the GFP and 85% of the
eXcad-11 transplants) were compared immediately after transplant
healing and at later time points. For further analysis, transplanted embryos
of different stages were fixed for 2 hours (room temperature) in 3.7%
formaldehyde in MEMFA (0.1 M MOPS, 2 mM EGTA, 1 mM MgSO4), and used
for whole-mount in situ hybridisation or immunohistochemistry. The transplants
were identified in transverse sections by detection of the Myc-tagged GFP
protein (monoclonal 9E10 Myc antibody).
Whole-mount in situ hybridisation and immunohistochemistry
Standard and double-staining whole-mount in situ hybridisation were
performed according to Hollemann et al. (Hollemann et al.,
1999). In the case of
AP-2/Snail double in situ hybridisation, colour images were taken immediately
after Fast Red staining for AP-2. After removing the red signal by washing in
100% ethanol, the embryos were incubated in digoxigenin antibody and stained
for Snail with NBT/BCIP. The following plasmids were used to generate
antisense probes: AP-2 (Winning et al.,
1991
), Snail (Essex
et al., 1993
), twist
(Hopwood et al., 1989
), NeuroD
(Lee et al., 1995
), sox2
(Streit et al., 1997
), nrp-1
(Knecht et al., 1995
),
N-Tubulin (Richter et al.,
1988
), sox3 (Zygar et al.,
1998
) and Xcadherin-6 (David
and Wedlich, 2000
). The
template for the neural cell adhesion molecule (N-CAM; Kintner and Melton,
1987
) antisense probe was
generated via our recently described PCR approach (David and Wedlich,
2001
) from Xenopus
stage 30 total cDNA. Primers were: N-CAM up, 5'-
GTCAAGTAAGCGGAGAAGCC-3'; T3/N-CAM lo,
5'-AATTAACCCTCACTAAAGGGTCCATCCTCAATTGGTTCAC-3'. The Xcad-11
whole-mount probe is directed against base pairs 40-1222, and ranges from the
untranslated region to the EC3 domain. The plasmid was linearised with
SacI and transcribed using T7 polymerase. All antisense probes were
generated from linearised plasmids using the SP6 or T7 transcription Kits
(Boehringer, Mannheim, Germany) with DIG RNA Labeling Mix (Boehringer,
Mannheim, Germany) according to the manufacturer's instructions. The
fluorescein-labelled twist probe was created using the Fluorescein
RNA Labeling Mix (Boehringer, Mannheim, Germany). The embryos were either
examined as whole mounts or serially sectioned, and immunohistochemically
analysed. Sectioning, immunohistochemistry and confocal analysis were
performed as described previously (Borchers et al.,
2000
).
RNA purification and RT-PCR
Total RNA was isolated from half heads of stage 28 embryos using the
Purescript kit from Biozym (Oldendorf, Germany). For reverse transcription,
SUPERSSCRIPTTM RNase H- Reverse Transcriptase (GibcoBRL,
Karlsruhe, Germany) was used, and PCR was performed as described in the
manual. The primers corresponding to twist (Hopwood et al.,
1989) and H4 (Gradl
et al., 1999a
) were used as
previously described. Primers for AP-2 and Snail were as
follows: AP-2 forward, 5'-CTCAATCCCAACGAGGTGTTC-3';
AP-2 reverse, 5'-CAGAATAGGATTTGGTCTGGAG-3';
Snail forward, 5'-GTGTGTATCACTATTGGGTAGG-3';
Snail reverse 5'-TGTCTTTGTGATCATCATTGGG-3'.
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RESULTS |
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The different Xcad-11 constructs were tested in a transplantation assay
(Borchers et al., 2000). In
vitro transcribed wild-type or mutant Xcad-11 RNA was co-injected with
Myc-tagged green fluorescence protein (GFP) RNA into one blastomere of
two-cell stage Xenopus laevis embryos. Before onset of neural crest
migration, part of the GFP-positive CNC was transplanted into uninjected
GFP-negative host embryos (Fig.
1C). As controls, CNC grafts from embryos injected with only
GFP-RNA were used. The migration behaviour of the transplants was evaluated by
examining whole mounts for GFP fluorescence 18 hours after grafting.
Overexpression of wild-type or cytoplasmically truncated Xcad-11
(cXcad-11) inhibits migration of CNC cells
Xcad-11 overexpression led to inhibition of CNC cell migration in a
dose-dependent manner (Fig. 1D,
Table 1). Injection of 1 ng of
full-length Xcad-11 RNA resulted in inhibition of migration in 34% of the
transplants. Higher doses of injected full-length RNA completely blocked
migration but were lethal in the majority of transplanted embryos (e.g. 1.6 ng
Xcad-11 RNA, Table 1).
Surprisingly, injecting cXcad-11 RNA lacking the ß-catenin binding
site also blocked migration (Fig.
1D, Table 1). This
mutant was less toxic to embryos, allowing the injection of higher RNA doses
(2.5 ng). When non-migrating grafts were analysed in transverse sections, the
majority of cells were tightly clustered in close proximity to the brain
(Fig. 1E). No differences in
cell shape or cell behaviour were seen between full-length or
cXcad-11
expressing grafts. These results demonstrate that Xcad-11 confers adhesiveness
to the injected cells independently of ß-catenin.
|
eXcad-11-expressing transplants start migration earlier than
GFP control transplants
As the homophilic binding site is deleted in the extracellular Xcad-11
mutant (eXcad-11, Fig.
1A), we expected a decrease in cell adhesion. Grafts expressing
eXcad-11 show a migration pattern similar to that of the GFP control
(Fig. 1D). Transverse sections
revealed that
eXcad-11-expressing cells migrate as a cohort of loosely
associated cells indistinguishable from the GFP controls
(Fig. 1E). However, when
eXcad-11 and GFP control transplants were continuously monitored over
the first 18 hours, we observed that cells from
eXcad-11-expressing
transplants emigrated earlier than those of the GFP controls
(Fig. 1F,G). The strongest
effect was observed 4 to 7 hours post-grafting, when 70% of the
eXcad-11-expressing transplants (n=46) showed cell emigration,
while 50% of the controls (n=49) were still as compact as at the
beginning of the experiment (Fig.
1F). After 18 hours, however, the GFP controls showed the same
100% migration as the
eXcad-11-expressing grafts. The
eXcad-11-expressing grafts were indistinguishable from the controls
after 48 hours when the cranial crest gave rise to cartilage
(Fig. 1F,G).
We also co-injected eXcad-11 with full-length Xcad-11 RNA, and found
that inhibition of migration by Xcad-11 was partially restored
(Table 1). In contrast,
co-expression of
cXcad-11 with wild-type RNA led to an increase in the
non-migrating phenotype (Table
1). Thus,
eXcad-11 acts in dominant-negative manner in
terms of adhesiveness, while
cXcad-11 behaves like the wild-type
cadherin.
Xcad-11 constructs altered expression of cranial neural crest
markers
As it is clear that Xcad-11 RNA injections alter the migration of
transplanted cells, we asked whether the CNC pattern was disturbed. Therefore,
we analysed the expression patterns of Xcad-11 and the CNC marker genes
AP-2, twist and Snail in embryos 18 hours after grafting.
The expression of those markers on the untreated side of each embryo served as
a control.
The Xcad-11 pattern resembles the migration behaviour of the grafts
(compare Fig. 2A with
Fig. 1D). Non-migrating grafts
expressing either the full-length or cXcad-11 were identified at the
site of implantation (Fig. 2A,
arrows), and only few Xcad-11-positive migrating neural crest cells were
found. These migrating cells were GFP negative and derived from residual host
neural crest. As expected, overexpression of
eXcad-11, which resulted
in premature migration, led to an increased Xcad-11 signal in the migrating
crest streams (Fig. 2A).
|
Like Xcad-11, AP-2 expression was also reduced in cephalic crest
streams of embryos containing non-migrating transplants (Xcad-11 and
cXcad-11, Fig. 2B, red
arrowheads). This indicates that a part of the migrating AP-2 cell
population was retained or lost. AP-2 expression in embryos with
eXcad-11 expressing grafts showed no effect compared with the control
side (Fig. 2B). Thus, as with
Xcad-11, the expression pattern of AP-2 correlated with the migration
behaviour of the grafts.
The analysis of Snail expression revealed no differences between the transplant-containing and untreated sides of the embryos, regardless of which Xcad-11 construct was injected (Fig. 2C). We did not, however, observe a reduction of Snail-positive cephalic crest streams in embryos containing non-migrating grafts, which would be expected if parts of the host population are removed and replaced by non-migrating donor tissue.
The effect of the different Xcad-11 constructs on twist expression
was more dramatic than on AP-2 or Xcad-11. twist expression
was strongly reduced in grafts that expressed eXcad-11
(Fig. 2D, arrowhead), although
cell migration was not inhibited by this mutant. In non-migrating
cXcad-11 grafts, there was no difference in twist expression
visible between the grafted and the untreated sides. By contrast,
overexpression of the full-length Xcad-11, which contains the
ß-catenin-binding site, led to reduced twist expression
(Fig. 2D, arrowhead). Thus,
presence of the intracellular Xcad-11 domain led to a reduced twist
expression on the side containing the transplant.
Taken together, the AP-2, Snail and twist expression patterns of the transplanted embryos were affected by the Xcad-11 constructs in different ways.
Lineage tracing, injection experiments and RT-PCR confirm the
transplantation results
Owing to the transplantation procedure, the in situ hybridisation patterns
reflect a mixture of host and donor CNC at the transplant-containing side (+
in Fig. 2A-D). However,
transverse sections (Fig. 3A-C)
allowed the distinction of donor and host CNC as the donor tissue could be
identified by immunostaining of the Myc-tagged GFP. As expected, the
non-migrating (Xcad-11- or cXcad-11-expressing) grafts were always
clearly separated from the migrating neural crest cells. An example for
Xcadherin-11-overexpressing donor tissue and twist-expressing, migrating host
CNC cells is shown in Fig. 3A. The migrating donor cells, on the other hand, intermingled with host cephalic
crest cells (Fig. 3B): cells
from a GFP control transplant are twist positive and found among
twist-expressing host cells. By contrast, most of the
eXcad-11-expressing cells were twist-negative and adjacent to
twist-positive host cells (Fig.
3C).
|
The grafting results were further confirmed by single-sided RNA injections.
The RNA of the different Xcad-11 constructs was co-injected with GFP-RNA into
a single blastomere at the two-cell stage, and the embryos were analysed by
whole-mount in situ hybridisation at stage 28. In these experiments, not only
the CNC, but also the surrounding tissues express the injected cadherin
constructs. Nevertheless, we obtained the same results as in the grafting
experiments. The Snail subpopulation was not affected by the
different injected constructs (data not shown). The AP-2 signal was
reduced by wild-type Xcad-11 or cXcad-11, while
eXcad-11 had no
effect (Fig. 4A).
twist expression was strongly reduced by
eXcad-11 or
full-length RNA injection at the injected side
(Fig. 4A). In the case of the
eXcad-11 construct, twist expression was decreased, on
average, in 75% of the embryos (87 embryos, five experiments). Most
strikingly, endogenous twist expression was recovered in 99% of the
embryos (88 embryos, three experiments,
Fig. 4B) after co-injection of
ß-catenin. Thus, downregulation of twist in migrating,
eXcad-11-expressing CNC was most probably caused by ß-catenin
depletion from canonical Wnt signalling.
|
We used RT-PCR to validate the effects of our cadherin constructs on neural
crest marker gene expression. The expression levels of AP-2, twist
and Snail were compared between the RNA-injected and non-injected
sides of tadpole heads. Fig. 4C
shows a representative RT-PCR of one tadpole head for each injected Xcad-11
construct that was tested for all three neural crest markers. Twist was not
detected when full-length and eXcad-11 were overexpressed, while
cXcad-11 had no effect on twist expression. There was no
reduction of AP-2 RNA in Xcad-11- or
eXcad-11-injected embryos, while
cXcad-11 injection resulted in loss of the AP-2 band. The level of AP-2
RNA in embryos overexpressing
cXcad-11 or full-length Xcad-11 varied
between undetectable and normal levels in different experiments, while the
results of twist expression were reproducible. This was most probably
due to different molecular effects; interference with Wnt/ß-catenin
signalling in the case of twist exhibits a stronger phenotype than inhibition
of crest migration alone, as seen for AP-2. We also analysed the expression of
Snail after RNA injections of the different Xcad-11 constructs, but
were unable to detect significant alterations in RT-PCR analyses
(Fig. 4C). This confirmed the
in situ hybridisation results.
In summary, Xcadherin-11 affected the AP-2-, Snail- and twist-expressing CNC domains in different ways: the twist subpopulation was diminished, predominantly owing to Xcad-11 interfering with Wnt/ß-catenin signalling, while the AP-2 subpopulation was reduced via cadherin-mediated adhesion. Snail expression was not affected in our various experimental systems.
The cranial neural crest consists of heterogeneous cell
subpopulations
As the Xcad-11 constructs in transplantation and injection experiments
affected CNC marker expression differently, we addressed the question of
whether the cephalic crest represents a heterogeneous cell population. To
analyse this, double in situ hybridisation was performed. When the Xcad-11 and
twist domains were compared directly in the same embryo,
Xcad-11-expressing cells (Fig.
5A, blue) were found more dorsally located than the
twist-expressing ones (Fig.
5A, red). The AP-2 and Snail expression patterns
were compared by single and double in situ hybridisation from stage 20 up to
stage 27. The temporal and spatial expression of both markers differed
slightly during CNC migration: at stage 20, the mandibular stream of the AP-2
pattern had already separated from the emigrating hyoid stream
(Fig. 5B, arrowhead), while the
Snail expression was also found between these streams
(Fig. 5D). Later, at stage 26,
the branchial stream showed expression of AP-2
(Fig. 5C, asterisk) but not
Snail (Fig. 5E,
asterisk). This indicates that the Snail expression domains diverge
from the AP-2 domains. The main differences were observed around
stage 26-27, while in later stages, Snail was also present in the branchial
stream (see Fig. 2).
|
Taken together, the expression patterns of Xcad-11, AP-2, twist and Snail only partially overlap, indicating that these markers may temporally form separate CNC subpopulations.
Inhibition of migration abrogates the undifferentiated neural crest
state and results in neural differentiation
The next issue to resolve was whether the non-migrating CNC transplants
maintained their neural crest character. Therefore, we analysed these
transplants in later stages for expression of twist, Snail and
AP-2, which are markers for undifferentiated migrating CNC cells.
Although a twist signal was detected directly after healing in
Xcad-11 expressing grafts (Fig.
6A,D), the signal began to fade 4 hours after grafting
(Fig. 6B,E) and was completely
lost 18 hours after grafting (Fig.
6C,F). This was also observed when cXcad-11 was
overexpressed in the grafts (Fig.
6G,H,J,K). As this mutant does not bind ß-catenin
(Fig. 1B), loss of the twist
signal in non-migrating crest cells did not result from inhibition of
Wnt/ß-catenin signalling. Snail expression also decreased in
non-migrating transplants expressing either full-length Xcad-11
(Fig. 6I,L) or
cXcad-11
(data not shown). Expression was completely lost 18 hours post-grafting
(Fig. 6I,L). AP-2 was not
suitable for this assay, as it is expressed in specific domains of the brain,
making a clear distinction between neural crest cells and neural epithelium
impossible.
|
The loss of CNC marker expression raised the question of what tissue types
differentiate in these non-migrating transplants 18 hours after grafting. Most
strikingly, all non-migrating grafts were positive for the neural marker 2G9
(Fig. 7A), which is specific
for brain, spinal chord and lateral line (Jones and Woodland,
1989). In migrating cephalic
crests streams of GFP controls (Fig.
7B) and
eXcad-11-expressing transplants (data not shown),
2G9 staining was not detected. Moreover, the non-migrating transplants became
positive for nrp-1 (Fig.
7E,F). This is a general neural marker (Knecht et al.,
1995
), which is expressed in
the central nervous system (CNS), and transiently in developing cranial
ganglia and nerves at stage 20-24, but not in migrating CNC cells from stage
24 onwards.
|
Because ß-catenin signalling is required to maintain twist
expression in migrating crest cells (Figs
2,4),
we attempted to sustain twist expression and block nrp-1 and
2G9 expression in non-migrating grafts. This was done by co-injection of
ß-catenin RNA either with full-length or cXcad-11 RNA. As
cXcad-11 RNA is unable to bind and deplete ß-catenin from the
canonical Wnt-signalling pathway, this co-injection experiment would resemble
an overexpression of ß-catenin. As seen in the case of
cXcad-11
(Fig. 7G), twist
expression could not be rescued by ß-catenin and the grafts became
positive for the 2G9 marker (Fig.
7H). This was also observed when wild-type Xcad-11 was
co-expressed with ß-catenin (data not shown). Thus, neuralisation of
non-migrating grafts could not be prevented by adding ß-catenin.
To rule out the possibility that neural marker expression was caused by contamination of tissue with neural epithelium, we controlled the accuracy of our transplantations. This was done by in situ hybridisation using probes for nrp-1, N-Tubulin, sox2 and sox3 1 hour after graft insertion. As shown for sox3 (Fig. 7C,D), and summarised in Table 2, the transplants were negative for neural markers briefly after grafting. Note that CNC is distinguishable from the neural epithelium at the time of transplantation by its lateral position, translucent appearance and loose packing.
|
Heterotopic grafting did not prevent neuralisation of non-migrating
grafts
The next approach to rescue the neural crest marker expression in
non-migrating transplants was heterotopic transplantation. To examine whether
the deficit of extrinsic signals, which are normally present along the cranial
migratory routes, resulted in the switch to neural marker expression, we
transplanted non-migrating grafts into the presumptive pharyngeal pouch area.
To our surprise, these non-migrating grafts expressing full-length Xcad-11
(Fig. 8A,B) or cXcad-11
(data not shown) and were positive for the neural marker 2G9, but negative for
twist expression. Additionally, we tried to rescue twist
expression by co-injection of ß-catenin RNA. As shown in
Fig. 8C,D, ß-catenin RNA
co-injection was unable to sustain twist expression. Furthermore, the
heterotopic transplants expressed nrp-1
(Fig. 8E-G). Interestingly, in
all these heterotopic transplants 2G9, or nrp-1 staining was most
prominent in the centre of the graft.
|
Non-migrating CNC grafts express CNS-specific neural markers
As neural crest cells contribute to the formation of peripheral nerves and
ganglia, we tried to define the neural character of the transplants more
closely. The embryos containing grafts were subjected to in situ hybridisation
at tailbud stage, using probes for marker genes which are predominantly
expressed either in CNS or PNS (Table
2). It is noteworthy that genes exclusively expressed in CNS or
PNS are not known in vertebrates. In spite of this limitation, we found that
the transplants express neural markers as 2G9, nrp-1 and
N-Tubulin (Fig. 8A-I),
but not NeuroD (Fig.
8J,K) and Xcadherin-6, which are most prominent in
ganglia and nerves of the PNS (see summary in
Table 2).
Xcad-11 and deletion constructs do not affect induction of neural
plate and cranial neural crest
Overexpression of cadherin constructs often results in abnormal phenotypes
with altered gene expression, owing to interference with Wnt/ß-catenin
signalling or changes in adhesion. One important question is whether the
injected Xcad-11 constructs affect the specification of neural epithelium
versus neural crest or the physical segregation of these tissues.
To this end, we analysed the morphology of the neural plate, CNC, placodes
and peripheral nerves by in situ hybridisation in embryos that were injected
with various Xcad-11 RNAs into one blastomere at two-cell stage. N-CAM and
nrp-1 were used as markers for neural plate, twist, AP-2 and snail for CNC,
sox2 and sox3 for neural plate and placodes, NeuroD and Xcadherin-6 for PNS.
Embryos were co-injected with GFP-RNA. Before the in situ hybridisation, the
embryos were selected for proper single-sided GFP distribution. Expression of
neural markers was found to be unchanged
(Fig. 9A,C,D,H,I). The nerves
and ganglia of the PNS were formed normally
(Fig. 9F,G). Transverse
sections demonstrate the proper localisation of the GFP
(Fig. 9I). Importantly, the
induction of CNC was not inhibited by expression of cXcad-11
(Fig. 9B),
eXcad-11
(Fig. 9E) or full-length
Xcad-11 (data not shown). The strongest effect observed was a slight reduction
of twist signal on the injected side (Fig.
9E). Thus, the dramatic downregulation of twist
expression in
eXcad-11 and wild-type Xcad-11 RNA injected embryos (see
Fig. 5) takes place at a later
stage, during CNC migration.
|
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DISCUSSION |
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Xcad-11 regulates migration of cranial neural crest cells by its
adhesive function
Overexpression of wild-type Xcad-11 and also, surprisingly, its
cytoplasmically deleted mutant, led to an increase in cell-cell contacts and
inhibition of migration. Although cXcad-11 completely lacks the
ß-catenin binding site, it acts as a dominant-active mutant like the
wild-type cadherin. Therefore, anchorage of Xcad-11 to the cytoskeleton via
ß-catenin seems to play only a minor role in mediating cell-cell adhesion
between neural crest cells. The binding of p120ctn to Xcad-11 also
appears not to be necessary for the adhesive function of Xcad-11: based on
sequence alignment to classical type I E-cadherin, only 14 amino acids of the
juxtamembrane region that interacts with p120ctn (Provost and Rimm,
1999
) are preserved in
cXcad-11. Furthermore, the conserved core binding sequence (Thoreson et
al., 2000
) was completely
deleted, making p120ctn binding to this mutant unlikely. The
results of this work are consistent with previous findings that clustering
effects of the transmembrane or extracellular E-cadherin domain could be
sufficient to mediate cell adhesion (Ozawa and Kemler,
1998
; Huber et al.,
1999
). In contrast to
cXcad-11, the extracellular deletion mutant (
eXcad-11) behaved
as a dominant-negative form, and this was demonstrated by premature cell
emigration out of the transplant. Interestingly, Nakagawa and Takeichi
(Nakagawa and Takeichi, 1998
)
produced similar results when they introduced N-cadherin deletion mutants into
chicken neural crest using an adenoviral expression system. Paralleling our
results, they showed that the mutant lacking the ß-catenin-binding site
partially inhibited migration of melanocyte precursors while the extracellular
deletion mutant did not.
Considering Xcadherin-11, our data reveals that the adhesive function of
this cadherin is important in regulating the onset and migration velocity of
the cephalic crest. This was demonstrated by the enhanced migration of the
dominant-negative eXcad-11 mutant, when compared with the GFP control,
and by the block of cell migration by dominant-active forms (wild-type
Xcad-11,
c-Xcad-11). As Xcad-11 confers adhesiveness independently of
catenin binding in our assay system, an influence of catenins or
post-translational modifications of Xcad-11 on the adhesive strength remains
elusive. Schneider et al. have reported a potential connection as they
observed a reduced catenin expression in migrating neural crest cells
(Schneider et al., 1993
).
Other factors, like small GTPases of the Rho family, seem unlikely to modulate
Xcadherin adhesiveness. For example, RhoB, which has been assigned to the
delamination process, fades in its expression at the time Xcad-11 is expressed
in migrating neural crest cells (Liu and Jessell,
1998
).
The question of why migrating cephalic crest cells require adhesive
properties still remains. Expression of different cadherins may guide the
homing of migrating neural crest cells (Nakagawa and Takeichi,
1995). Proof of this idea
remains elusive because of the lack of markers for specific crest derivatives.
However, a crude analysis of the cranial skeleton in tadpoles that had been
injected with wild-type Xcad-11 and deletion mutants in one blastomere of a
two-cell stage embryo, showed no cranial defects on the injected compared with
control sides (data not shown). Independent of any effect on the crest
derivatives, the typical cranial migration pattern (Sadaghiani and
Thiébaud,
1987
) remained unaffected.
Overexpressing either wild-type Xcad-11 or
cXcad-11 at very low doses
or
eXcad-11 at high doses, all of which show a migratory phenotype, did
not disturb the pattern. We conclude from this data that endogenous Xcad-11
reduces the migratory velocity and plays no role in separating the mandibular,
hyoid and branchial stream. Thus, Xcadherin-11 function is distinct from that
of ephrin receptors, whose dominant-negative expression leads to fusion of
crest streams (Helbling et al.,
1998
).
Xcad-11 function and neural crest specification
The existence of different cell subpopulations with different migratory
behaviour has been discussed for Xsnail, Xslug and Xtwist
(Linker et al., 2000).
Moreover, subpopulations can also exhibit overlapping domains as seen by
double in situ hybridisation for twist and Xcad-11
(Fig. 5A), and AP-2 and Snail
(Fig. 5D-E). Interestingly, the
expression of Xcad-11 and deletion mutants had different effects on the twist,
AP-2 and Snail subpopulations, confirming the heterogeneous character of the
CNC. One drawback of the assay system used here is that whole-mount in situ
hybridisation does not resolve expression profiles of single cells. Therefore,
the role of Xcad-11 in segregating cell clusters within one cephalic crest
stream will remain elusive until detection of the markers can be improved.
Clustering and restraining CNC cells on their migratory routes probably
alters their specification. Although, based on inhibition of BMP and
activation of Wnt/ß-catenin and/or e/bFGF signalling, the neural crest
fate is defined at the premigratory stage (Mayor et al.,
1995; LaBonne and
Bronner-Fraser, 1998
), further
specification depends on exposure to extrinsic factors along the migratory
routes (Hall and Hörstadius,
1988
; LaBonne and
Bronner-Fraser, 1999
). Some of
these molecules have been identified for trunk neural crest using cell
cultivation (Le Douarin and Smith,
1988
; Anderson et al.,
1997
), while those important
for the differentiation of CNC are still unknown. By preventing CNC cells from
migration, we were able to abolish the undifferentiated state of CNC
prematurely. This was seen in non-migrating grafts, which started to lose
Snail, AP-2 and twist expression 4 hours after
transplantation. However, this was not caused by blocking Wnt/ß-catenin
signalling via depletion of ß-catenin, because expression of
cXcad-11 had the same effect as the full-length form. In addition,
ß-catenin co-expression in these non-migrating grafts did not prevent
fading of the neural crest marker twist, and upregulation of neural markers.
This is especially interesting, as the twist subpopulation needs
ß-catenin to maintain twist expression in migrating cells. Loss
of twist expression in migrating,
eXcad-11-expressing CNC
cells was rescued by co-expression of ß-catenin
(Fig. 4B). The unexpected
switch from undifferentiated CNC to the neural state in non-migrating grafts
can be explained in two different ways: (1) the initial neural crest induction
is reversible, and the CNC becomes neural epithelium once again; and (2) the
neural crest differentiates prematurely into neural crest derivatives, e.g.
nerves and ganglia of the PNS. Our extended in situ hybridisation study
(Table 2) promotes the idea
that the non-migrating grafts differentiate into neural CNS-like tissue rather
than PNS-specific structures. In addition, our results demonstrate that
increased cell-cell adhesion, which leads to compaction of CNC, induces neural
differentiation. As we could not observe the activation of neural marker genes
in migrating heterotopic grafts, the adhesion effect seems to be more
important than the influence of extrinsic factors. Support for our findings
comes from neural crest cell culture studies. It has been shown by Hagedorn et
al. (Hagedorn et al., 1999
)
that clusters of neural crest cells, in contrast to single cells,
differentiate into neural cells at the expense of non-neural derivatives,
independently of the type or concentration of added differentiation signal
(BMP-2, TGFß). In addition, ganglion formation in neural crest
derivatives correlates with upregulation of adhesion molecules (Akitaya and
Bronner-Fraser, 1992
).
Induction of neural crest is not disturbed by Xcad-11
overexpression
Overexpression of ß-catenin or Xwnt-1, Xwnt-8 and Xwnt 7B in
combination with noggin or chordin resulted in an increase and expansion of
the neural crest markers, while expression of gsk-3ß or dnXwnt-8 had the
opposite effect (Saint-Jeannet et al.,
1997; LaBonne and
Bronner-Fraser, 1998
; Chang
and Hemmati-Brivanlou, 1998
).
We expected that expression of Xcad-11 constructs with ß-catenin binding
sites at the time of neural crest induction should result in the same
phenotype, owing to interference with canonical Wnt signalling (Fagotto et
al., 1996
; Gradl et al.,
1999a
; Gradl et al.,
1999b
). This phenotype was not
observed. The proteins derived from the injected RNAs were strongly expressed
and correctly localised (data not shown). Most likely, gsk-3ß
overexpression is more efficient in blocking Wnt/ß-catenin signalling
than depletion of ß-catenin by cadherin expression. This, however, would
contradict our findings that binding of ß-catenin to Xcad-11 is
sufficient to repress twist expression in the migratory phase. The
discrepancies between the former reports and our data may result from the
different assay systems. While the induction of neural crest marker genes by
canonical Wnt signalling was analysed in animal caps injected with
noggin/chordin and Xwnt-1, -3A, or -7 RNA, our analysis focused on the in vivo
situation, which might include other putative inducers. Interestingly,
blocking canonical Wnt-signalling by gsk-3ß expression in the whole
embryo did not repress Krox-20 in the rhombomeres. Instead, the
stream of Krox-20-positive neural crest cells was lost (Saint-Jeannet et al.,
1997
). This could be explained
by a late Wnt/ß-catenin signalling defect at the migratory stage. Our
findings demonstrate that Wnt/ß-catenin signalling is also essential for
late events in CNC development, in particular to the maintenance of
twist expression during the period of CNC specification. Late
influence of ß-catenin, especially on neural crest specification, might
also play a role in pigment cell formation. This neural crest subpopulation
increased in presence of ß-catenin at the expense of neurones and glia
cells in zebrafish (Dorsky et al.,
1998
). Similar observations
were made in mice, showing that melanocyte formation was dependent on wnt-1
and wnt-3a signalling (Ikeya et al.,
1997
; Dunn et al.,
2000
). Interestingly, in
Xenopus, components of the canonical Wnt signalling cascade, e.g.
Xfz7, XLef-1, XTcf-3, are expressed in migrating cranial neural crest
(Molenaar et al., 1998
;
Wheeler and Hoppler, 1999
),
stressing a putative function in CNC specification.
Taken together, our data support the model of Mayor et al. in which the
decision between neural plate, neural fold and epidermis is made at the
premigratory stage (Mayor et al.,
1998). Wnt/ß-catenin
signalling is discussed to contribute to the induction of CNC at premigratory
stage. However, in this model the major role of Wnt factors is seen in
maintaining the neural crest differentiation program later, at the migratory
stage. Our findings confirm this idea, but also supplement the model, as we
identified additional factors important for CNC specification: increased
cell-cell adhesion and block of migration leads to neural differentiation,
while only migrating CNC cells are able to maintain the undifferentiated
neural crest state. The migrating CNC cells represent a heterogeneous cell
pool with a balanced Xcadherin-11 expression. Xcad-11-mediated adhesion
restrains CNC cells, and might allow their prolonged exposure to extrinsic
factors. Because Wnt/ß-catenin signalling is essential for the
twist-expressing CNC subpopulation, this signal must belong to the
group of extrinsic factors.
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ACKNOWLEDGMENTS |
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