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First published online 21 December 2006
doi: 10.1242/dev.02742
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1 Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical
School, Jerusalem 91120 - PO Box 12272, Israel.
2 Institute for Molecular Cardiovascular Research, University Hospital, RWTH
Aachen, 52074, Germany.
* Author for correspondence (e-mail: kalcheim{at}nn-shum.cc.huji.ac.il)
Accepted 9 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Adherens junctions, ß-catenin, Cell cycle, Epithelial to mesenchymal transition, Wnt, Quail
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The molecular network underlying NC specification is being
elucidated (Basch et al., 2004;
Gammill and Bronner-Fraser,
2003). Following specification, NC cells successively emigrate
from the NT and follow stereotypic migrations throughout the embryo to reach
their homing sites and differentiate. To this end, premigratory cells undergo
a process of epithelial-to-mesenchymal transition (EMT), an indispensable
stage for generating cellular movement
(Halloran and Bendt, 2003;
Kalcheim and Burstyn-Cohen,
2005; Nieto,
2001).
Recent studies have demonstrated a role for dorsal NT-derived BMP and Wnt
signals in initiating NC delamination, independent of their prior
specification. We reported that a decreasing rostrocaudal gradient of BMP4
activity is established along the dorsal NT by a reciprocal gradient of its
inhibitor, noggin. BMP then induces EMT of NC progenitors. Noggin
downregulation is in turn triggered by the developing somites, which thus
determine the timing of NC emigration
(Sela-Donenfeld and Kalcheim,
1999; Sela-Donenfeld and
Kalcheim, 2000; Sela-Donenfeld
and Kalcheim, 2002). NC emigration can also be stimulated by Cv-2,
which promotes BMP activity in the avian NT
(Coles et al., 2004),
suggesting that BMP signaling is subject to both positive and negative
regulation. The cell cycle was also found to play a pivotal role. NC cells
synchronously emigrate in S phase, and the transition from G1 to S is
necessary for the process: specific inhibition of G1-S transition blocked NC
emigration, whereas arrest at S or G2 phases had no immediate effect
(Burstyn-Cohen and Kalcheim,
2002). A subsequent study integrated the above findings,
demonstrating that: (1) BMP regulates NC delamination in a cell
cycle-dependent manner; and (2) canonical Wnt signaling acts downstream of BMP
in the dorsal NT to mediate BMP activity in the context of both the G1-S
transition and NC delamination
(Burstyn-Cohen et al., 2004).
Additional studies suggested that EMT of NC cells is modulated by
transcription factors, cell adhesion molecules and other regulatory proteins
(Barrallo-Gimeno and Nieto,
2005; Halloran and Bendt,
2003; Kalcheim,
2000; Nieto,
2001). However, very little is known about their precise mechanism
of action or their possible relationship with the BMP/Wnt cascade described
above; this knowledge would prove invaluable in establishing the molecular
network underlying EMT of NC progenitors.
Here, we explored the mechanism by which N-cadherin acts on NC delamination
and its regulation by BMP. N-cadherin belongs to a family of
Ca2+-dependent cell adhesion molecules
(Hatta et al., 1988;
Hatta et al., 1987;
Hatta and Takeichi, 1986)
important for various developmental processes
(Gumbiner, 2000;
Nelson and Nusse, 2004).
N-cadherin is characterized by five extracellular cadherin-binding domains, a
transmembrane and an intracellular ß-catenin-binding domain
(Tepass et al., 2000). The
full-length 135 kDa protein is cleaved extracellularly by a rate-limiting
metalloproteinase, ADAM 10, generating a 40 kDa C-terminal fragment termed
CTF1, which is further processed by a 
-secretase-like activity into the
soluble 35 kDa intracellular CTF2, which is involved in the regulation of gene
expression (Fortini, 2002;
Marambaud et al., 2003;
Reiss et al., 2005). This
places N-cadherin within a category of cell surface receptors, including
Notch, amyloid precursor protein and Erb-B4, in which the intracellular domain
is liberated by -secretase-mediated cleavage
(Fortini, 2002). Whereas in
the latter cases, the cleaved intracellular domain translocates to the nucleus
to promote signaling events, there is no information as to whether a similar
mechanism operates in the case of N-cadherin.
N-cadherin is initially expressed in the entire NT. Presumptive
premigratory NC cells, however, lose the protein prior to departing from the
NT (Akitaya and Bronner Fraser,
1992; Duband et al.,
1988; Hatta et al.,
1987), suggesting that loss of N-cadherin is a prerequisite for NC
delamination. Functional studies using adenoviral-mediated overexpression of
N-cadherin reported a failure of melanoblast emigration, the last NC cells to
depart from the NT. Nevertheless, they showed a normal onset of cell
emigration followed by successful formation of neural derivatives, except for
a slight reduction in the number of dorsal root ganglion cells
(Nakagawa and Takeichi, 1998).
Reciprocally, loss of N-cadherin-mediated adhesion had no effect either on NC
emigration or NT morphology (Nakagawa and
Takeichi, 1998). Furthermore, injection of neutralizing antibodies
into the cranial tube resulted mainly in NT deformities and some ectopic
aggregates of NC cells, but the significance to EMT of NC was not studied
(Bronner-Fraser et al., 1992).
Additional loss-of-function approaches focused on its role in maintaining NT
integrity without addressing the problem of NC development
(Ganzler-Odenthal and Redies,
1998; Lele et al.,
2002). Hence, the longstanding hypothesis proposing that
N-cadherin prevents NC delamination has, at best, been only partially
substantiated. Furthermore, the mode of N-cadherin activity on NC remains
unexplored.
Using electroporation of the NT, we show that full-length N-cadherin is sufficient to completely block NC delamination while downregulating cyclin D1 and the G1-S transition. Transfection of mutant forms of N-cadherin reveals that NC delamination requires the combined activity of both cell adhesion and ß-catenin-binding domains. Hence, N-cadherin is required for maintaining all potential NC in a premigratory state and this function can be explained both by its adhesive properties and by inhibiting canonical Wnt-mediated signaling. Is N-cadherin part of the BMP-dependent cascade leading to NC delamination? We report that N-cadherin protein is expressed along the dorsal tube in a decreasing caudal-to-rostral gradient similar to that exhibited by noggin. Furthermore, we show that its normal downregulation is prevented by noggin and, reciprocally, is stimulated by BMP4. Thus, N-cadherin is part of the BMP-dependent pathway leading to NC delamination. We propose that BMP affects N-cadherin stability via ADAM10, as treatment of explanted neural primordia with GI254023X, a specific inhibitor of endogenous ADAM10, maintains membrane-bound cadherin and inhibits NC delamination when added either alone or in combination with BMP4. Most importantly, soluble CTF2, the end product of N-cadherin degradation, translocates into nuclei, stimulates transcription of cyclin D1 and promotes delamination of NC cells. This is likely to result, at least partially, from enhanced ß-catenin transcription. Taken together, we suggest that BMP-mediated downregulation of N-cadherin in the dorsal tube serves both to reduce intercellular adhesion and to facilitate transcriptional activity leading to the generation of NC cell movement.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Electroporation and expression vectors
DNA (3-5 µg/µl) was microinjected into the lumen of the NT of 15- to
18-somite-stage embryos at the level of the segmental plate and two recently
formed somites. A four parameter PulseAgile square wave
electroporator (PA-4000, Cyto Pulse Sciences) was used to deliver three groups
of sequential pulses as follows: 3x18V of 20 mseconds each; 3x26V
of 15 mseconds each; 3x18V of 20 mseconds each. Embryos were then
incubated for various times ranging from 8-24 hours, some followed by a 1-hour
pulse of Brdu (10 mM) or by processing for in situ hybridization. Another
series of embryos were incubated for 2 hours followed by explantation of
isolated tubes on fibronectin (see below).
The expression vector pCAGGS-AFP
(Momose et al., 1999) was used
as a control (referred to as control-GFP). DNAs encoding full-length chicken
N-cadherin, N-cadherin lacking part of the extracellular domain (cN390
)
and N-cadherin lacking either the intracellular ß-catenin-binding domain
(CBR-) or the juxtamembrane domain (JMD-)
(Fujimori and Takeichi, 1993;
Horikawa and Takeichi, 2001;
Nakagawa and Takeichi, 1998);
N-cadherin tail (CTF2) (Sadot et al.,
1998); Xenopus noggin and mouse BMP4
(Endo et al., 2003) were
subcloned into the pCAGGS vector and fused in-frame to a GFP-encoding
sequence. Experimental details are available upon request.
Grafting of BMP4-coated beads
Heparin-acrylic beads (Sigma) were immersed in BMP4 (R&D, 50 ng/ml in
1% fetal calf serum in PBS) or BSA for 1.5 hours followed by repeated washings
in PBS. To graft the beads, a slit was made along the dorsal aspect of the NT
at the caudal segmental plate level of the axis. A single BMP-coated bead per
embryo was then inserted, being held between the neural folds.
Explants of neural primordia
Neural tubes containing premigratory NC were excised from segmental plate
levels of 16- to 20-somite-stage embryos and then explanted onto 8-well
chamber slides (Lab-Tek) pre-coated with fibronectin (Sigma, 50 µg/ml), as
described (Burstyn-Cohen and Kalcheim,
2002). Culture medium consisted of CHO-S-SFM II (Gibco-BRL) to
which BMP4 (100 ng/ml), or the ADAM10-inhibitor GI254023X (12 µM), or both
were added.
Tissue processing, immunocytochemistry and in situ hybridization
Embryos were fixed with 4% formaldehyde, embedded in paraffin wax and
sectioned at 5 or 10 µm. Rabbit anti-GFP (Molecular Probes) was used at
1:500 in combination with HNK-1, N-cadherin, or Brdu immunolabelings or with
in situ hybridization for cyclin D1. Additional in situ
hybridizations were performed with noggin, ß-catenin or
with a chicken N-cadherin probe encompassing nucleotides 816 to 1523
of the coding domain. Monoclonal antibodies against the intracellular domain
of N-cadherin (Zymed) were applied following antigen retrieval by boiling the
slides in 0.1 M Tris buffer (pH 9.5) for 5 minutes. The GC4 antibody against
the extracellular region of N-cadherin was obtained from Sigma. Nuclei were
visualized with Hoechst. Whole-mount embryo preparations and sections were
photographed using a DP70 cooled CCD digital camera (Olympus) mounted on a
BX51 microscope (Olympus).
Data analysis
Cell proliferation and NC delamination were monitored in at least five
embryos per treatment out of 8-21 embryos showing a similar phenotype, as
described (Burstyn-Cohen et al.,
2004). Briefly, the proportion of Brdu+/GFP+ cells or the number
of Hoechst+ nuclei located up to the migration staging area was measured in 25
sections of control versus experimental hemi-tubes, and expressed as the
mean±s.d. of total cases monitored. The number of NC cells with
mesenchymal morphology that exited from explanted neural tubes was counted in
33-43 microscopic fields per explant, each comprising an area of 2500
µm2. Results represent the average number of cells per explant
(±s.d. of 4-5 cultures counted out of at least 12 cultures per
treatment showing a similar phenotype) normalized to the length of the NT
fragment. Significance was examined using one-way analysis of variance
(ANOVA). When significant differences were indicated in the F ratio test
(P<0.005), the significance of differences between means of any
two of these groups was determined using the modified Tukey method for
multiple comparisons with an 
of 0.05.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). By contrast, no change in N-cadherin mRNA
expression was detected along the axis
(Fig. 1D-F), suggesting that
loss of N-cadherin is regulated post-transcriptionally.
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Membrane-anchored N-cadherin mutants have no effect on NC delamination but disrupt neuroepithelial morphology
To examine whether the inhibitory effect of N-cadherin on emigration of NC
cells can be mimicked by overexpressing selected domains of the protein, three
mutants were transfected at segmental plate levels. N-cadherin bearing a
deletion in the extracellular domain, N-cadherin lacking the
ß-catenin-binding domain, and N-cadherin lacking the juxtamembrane
domain, were fused to a GFP reporter to create cN390-GFP, CBR-GFP and
JMD-GFP, respectively (Fig.
3A). These mutants have been shown to act in a dominant-negative
fashion on cell adhesion and morphology in tissues expressing endogenous
N-cadherin (Fujimori and Takeichi,
1993; Horikawa and Takeichi,
2001; Nakagawa and Takeichi,
1998). Since most of the NT continuously expresses the protein, we
expected the mutant DNAs to similarly act in this region in a
dominant-negative fashion. By contrast, the dorsal-most region of the NT that
contains the premigratory NC downregulates N-cadherin by the time the
transgenes first become expressed (epithelial to dissociating somite levels,
not shown). Hence, regarding the dorsal NT, we asked whether the mutant
proteins were sufficient to compensate for the physiological loss of
endogenous N-cadherin. None of the mutants had any measurable effect on
delamination of NC cells as compared with control-GFP, with a similar number
of labeled cells emigrating from the NT in all cases
(Fig. 3B-I and data not shown).
Furthermore, we noticed that in cN390-GFP and CBR-GFP-treated embryos,
the delaminating cells were round and spread apart
(Fig. 3B,C,H and
Fig. 2E; and data not shown).
Hence, N-cadherin-mediated inhibition of NC delamination requires the
integrated activity of all its domains as this effect cannot be reproduced by
overexpressing individually any combination of two out of the three tested
domains. Nevertheless, both cN390-GFP and CBR-GFP provoked a loss of
the pseudostratified conformation in NT cells that continuously express
N-cadherin with a rounding up similar to that observed upon transfection of
wild-type N-cadherin (Fig.
2B,D,F and Fig.
3C,E,F,H; see also Fig.
4B,C). Surprisingly, transfection of JMD-had no effect on NT
morphology (Fig. 3G),
suggesting that maintenance of the epithelial conformation in this tissue is
not likely to result from events linked to the JMD, such as binding to p120
and the resulting changes in cytoskeletal assembly
(Gumbiner, 2000).
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|
; Burstyn-Cohen et al.,
2004). As previously documented, control-GFP had no effect on cell
proliferation (Fig. 4A,F) as
45±6.65% of GFP+ nuclei in the NT were Brdu+. By contrast, only
7.94±5.47% of GFP+ cells were Brdu+ in the wild-type N-cadherin-treated
tubes (Fig. 4A,B,D). Thus,
full-length N-cadherin causes an 83% reduction in DNA synthesis in
neuroepithelial cells. Next, we examined the effect of N-cadherin on
transcription of cyclin D1, a target of ß-catenin-dependent Wnt
signaling in the NT (Burstyn-Cohen et al.,
2004; Megason and McMahon,
2002). As expected from its effect on the G1-S transition,
cyclin D1 mRNA was downregulated in cells that received
N-cadherin-GFP as compared with control-GFP and to untransfected regions of
the NT (Fig. 4E-H). As a
control, we examined the expression of cyclin B2 mRNA, a G2-M cyclin,
and observed that in contrast to cyclin D1, levels of cyclin
B2 were increased (not shown); hence, N-cadherin caused a selective
inhibition of G1-specific gene transcription without producing a general
inhibition of transcriptional activity. These data suggest that N-cadherin
inhibits NC delamination by inhibiting the G1-S transition, a consequence of
canonical Wnt signaling in this system. This is consistent with studies
showing that cadherins antagonize ß-catenin signaling by binding and
sequestering it in catenin-cadherin complexes at the plasma membrane (see
Discussion).
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-GFP, CBR-GFP nor JMD-GFP had any effect
on the G1-S transition, with 40-50% of the Brdu+/GFP+ progenitors being found
in the NT, and with delaminating NC cells mostly in the S-phase of the cell
cycle (Fig. 4C (arrows),D and
data not shown). These data substantiate previous results showing that NC
delamination depends upon successful G1-S transition. In addition, taken
together with the observed delamination phenotypes, our data indicate that
maintenance of N-cadherin protein integrity is required in the NT for
effectively antagonizing transcriptional signaling; retention of the
ß-catenin-binding domain in membrane-anchored mutants is not sufficient,
however, contrary to what has been observed in several in vitro systems (see
Discussion).
Notably, both cN390-GFP and CBR-GFP, which caused the loss of the
pseudostratified morphology of NT cells (Figs
3,
4), also revealed an abnormal
pattern of interkinetic nuclear migration, defined as a cell cycle-dependent
radial migration of cell nuclei across the epithelium. In these embryos,
Brdu+/GFP+ nuclei were scattered throughout the apico-basal extent of the NT
(Fig. 4C and data not shown),
instead of being confined to its basal half where DNA replication normally
occurs (Fig. 4A). The finding
that cell proliferation occurs to a normal extent even in the disorganized
hemi-NT suggests that cell proliferation and interkinetic nuclear migration
are separable processes.
N-cadherin is a component of the BMP-noggin cascade underlying NC delamination
In previous studies, we demonstrated that the downregulation of
noggin in the dorsal NT at the level of epithelial somites (see also
Fig. 1G) relieves BMP activity
from the inhibition to which it is subjected along the caudal NT. BMP4 then
triggers NC delamination via the canonical Wnt pathway. Since N-cadherin
protein expression, but not mRNA, also disappears from the dorsal NT at
comparable axial levels (Fig.
1), we examined the hypothesis that BMP4 downregulates N-cadherin
immunoreactivity. The presence of BMP4-coated beads or the overexpression of a
BMP4-encoding DNA in the dorsal NT opposite the caudal segmental plate,
resulted in loss of N-cadherin 8 hours later, opposite both the caudal and
rostral segmental plate; at these levels, N-cadherin was still present in
control-treated and intact dorsal NTs (Fig.
5A-D, Fig. 1C and
data not shown). We next examined the physiological relevance of this effect
by inhibiting endogenous BMP. Transfection of control-GFP into the NT at
segmental plate levels revealed a normal loss of N-cadherin 15 hours later,
adjacent to dissociating levels of the somite
(Fig. 5E), a phenotype
accompanied by delamination of labeled NC cells
(Fig. 5E, arrows in insert). By
contrast, a similar overexpression of noggin-encoding DNA prevented the normal
downregulation of N-cadherin and inhibited NC emigration
(Fig. 5F). No change in
N-cadherin mRNA levels could be detected under these conditions (not shown),
consistent with the uniform pattern of N-cadherin mRNA apparent along the
neuraxis under normal conditions. Thus, the normal loss of N-cadherin protein
in the dorsal NT is initiated by BMP4, suggesting that BMP4 triggers NC
delamination in a N-cadherin-dependent manner.
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-secretase-like activity to
yield a soluble N-cadherin cytoplasmic tail, CTF2
(Fortini, 2002;
Marambaud et al., 2003;
Reiss et al., 2005).
Furthermore, ADAM10 has been shown to be expressed in the dorsal NT and in
emigrating avian NC in vitro (Hall and
Erickson, 2003), and also in the E9.5 mouse NT colocalized with
N-cadherin (Reiss et al.,
2005). We prevented ADAM10-dependent N-cadherin degradation by
treating neural primordia with the selective inhibitor GI254023X
(Hundhausen et al., 2003;
Ludwig et al., 2005). Neural
primordia containing premigratory NC were explanted onto fibronectin. Fifteen
hours later, NC cells were present on the substratum of control cultures; the
densely packed cells close to the NT still expressed N-cadherin, but cells
with a mesenchymal morphology had already lost membrane-bound immunoreactivity
as visualized with antibodies to extracellular or intracellular domains,
suggesting a complete cleavage of the protein
(Fig. 6A-C). Treatment with
BMP4 caused an 11-fold increase in the number of emigrating mesenchymal cells
with a complete loss of membrane-bound N-cadherin
(Fig. 6D-F,M;
P<0.002). Treatment with GI254023X alone inhibited emigration of
NC cells by 67±12% compared with controls
(Fig. 6G,M;
P<0.001). The combination of GI254023X and BMP4 allowed an initial
flattening of the dense epithelial sheet, but inhibited EMT of NC cells by
83±10% (P<0.002) and 98.0±1.3% (P<0.001)
of control and BMP-treated values, respectively
(Fig. 6J,M). We then verified
that GI254023X prevented the cleavage of N-cadherin. Indeed, staining with
antibodies to the extracellular and intracellular protein domains revealed
retention of membrane-associated immunostaining in GI254023X-treated explants
(Fig. 6H,I), and also in the
combined GI254023X+BMP4-treated explants
(Fig. 6K,L). Altogether, these
results suggest that BMP downregulates N-cadherin
(Fig. 5) by promoting its
degradation via ADAM10, and that cleavage of N-cadherin by ADAM10 is necessary
for NC EMT.
The soluble ß-catenin-binding cytoplasmic tail (CTF2) stimulates transcription of ß-catenin and cyclin D1 and enhances NC delamination
The end product of N-cadherin cleavage is a soluble cytoplasmic tail termed
CTF2 (Fig. 3A). Since this
fragment binds to ß-catenin, a key mediator of canonical Wnt signaling
that is necessary for NC delamination
(Burstyn-Cohen et al., 2004),
its effect on the above process was examined. We electroporated CTF2-GFP into
hemi-NTs and embryos were incubated for a further 16 hours. CTF2-GFP
stimulated the delamination of NC cells by 1.9±0.2-fold as compared
with controls; the emigrating NC cells were HNK-1+ and mostly Brdu+
(Fig. 7A,B), as previously
documented. Notably, this effect was preceded and accompanied by a prominent
upregulation of both ß-catenin and cyclin D1
transcription in the transfected hemi-tubes and in the emigrating NC cells
(Fig. 7C,D). In addition, CTF2
was highly enriched in the nuclei of transfected progenitors, a phenotype
clearly apparent in NC cells emigrating from explanted primordia
(Fig. 7E,F and data not shown).
These results indicate that CTF2 is likely to act through ß-catenin at
various levels, by enhancing ß-catenin synthesis and perhaps also by
translocating along with ß-catenin into the nucleus to promote
transcription of cyclin D1 and the G1-S transition.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
We therefore propose that BMP-dependent downregulation of N-cadherin in the
dorsal NT opposite the level of developing somites serves not only to relieve
intercellular adhesion and signaling inhibition, but also to enhance
transcriptional activity, leading to the onset of NC emigration
(Fig. 7G). Consequently, the
process of NC emigration illustrates the complexity of N-cadherin regulation,
as demonstrated here for the first time, involving opposite roles elicited by
the full-length protein and by its soluble degradation product.
We report that in the dorsal NT, full-length N-cadherin inhibits
transcription of ß-catenin-dependent genes such as cyclin D1 and
subsequent G1-S transition and NC delamination. Similar effects were reported
upon overexpression of ß-catenin-engrailed, dominant-negative Lef1 or
mutant dishevelled, which interfere with canonical Wnt activity
(Burstyn-Cohen et al., 2004).
Likewise, cyclin D1 transcription in the dorsal NT is low at caudal
axial levels where noggin and N-cadherin are active, and increases in
association with the downregulation of the these two proteins
(Burstyn-Cohen et al., 2004)
(see also this study). Hence, our data suggest that N-cadherin-dependent
inhibition of NC delamination results from the inhibition of
ß-catenin-dependent Wnt signaling
(Fagotto et al., 1996;
Funayama et al., 1995;
Gottardi and Gumbiner, 2001;
Heasman et al., 1994;
Sanson et al., 1996;
Wong and Gumbiner, 2003). This
is consistent with increasing evidence that ß-catenin is involved in
signaling through cadherins, given that ß-catenin has dual and mutually
exclusive functions in cadherin-mediated adhesion at the plasma membrane and
in transcriptional regulation as part of the canonical Wnt pathway
(Nelson and Nusse, 2004).
Increased cell proliferation has been observed upon loss of N-cadherin in
several in vivo systems (Ganzler-Odenthal
and Redies, 1998; Lele et al.,
2002), confirming that endogenous N-cadherin negatively modulates
the Wnt pathway.
|
; Sadot et al.,
1998; Stockinger et al.,
2001). By contrast, in the present in vivo context, we observed
that only full-length N-cadherin inhibited the G1-S transition and cyclin
D1 mRNA transcription, whereas membrane-anchored mutants retaining only
the ectodomain or the ß-catenin-binding domain were ineffective. This
suggests the need for functional coupling between adhesion and signaling
motifs, a situation likely to reflect the complexity of cell-to-cell
interactions that characterize the embryonic environment. Conversely, soluble
CTF2 has a prominent positive effect on signaling. Hence, our functional
assays suggest that the ß-catenin-binding domain of N-cadherin has either
a negative, positive or no effect on signaling followed by NC delamination,
depending on the structural context of the molecule (as full-length
N-cadherin, CTF2 or cN390, respectively).
The precise mechanism by which CTF2 stimulates cyclin D1
transcription and NC delamination remains to be investigated, yet it is likely
to involve several levels of regulation through ß-catenin. First, in our
system, CTF2 activates ß-catenin transcription, which is consistent with
recent in vitro data that in addition documented reduced ß-catenin
degradation (Uemura et al.,
2006). Increased cytoplasmic ß-catenin could facilitate
nuclear accumulation of the protein and activation of target genes. Indeed,
elevated cytoplasmic and nuclear ß-catenin were detected in vitro upon
overexpression of CTF2 (Uemura et al.,
2006). Second, because we detected CTF2 in the nucleus of
emigrating NC, it is possible that in addition to stimulating ß-catenin
transcription, CTF2 complexes with ß-catenin and the two undergo nuclear
translocation followed by cyclin D1 activation. FRET analysis of
SH-SY5Y cells expressing an inducible form of CTF2 revealed an interaction
between the two proteins (Uemura et al.,
2006). Notably, cadherins and TCF/Lef bind to the same sequence of
ß-catenin, hence predicting that CTF2 would abrogate rather than
stimulate signaling events. Yet, in vivo, this complex might have a more rapid
turnover than that of the isolated component proteins
(Huber et al., 2001), and the
released ß-catenin could then bind to TCF. Alternatively, nuclear
CTF2/ß-catenin could signal independently of TCF
(Olson et al., 2006;
Stadeli et al., 2006;
Wong and Gumbiner, 2003;
Xu et al., 2000). The
situation is further complicated in our system because BMP activates, in
parallel, both N-cadherin degradation to CTF2 and canonical Wnt signaling
(Fig. 7G), and possible
interactions between these pathways cannot be isolated from each other.
The cN390-GFP and CBR-GFP mutants distorted the normal
neuroepithelium, with misplaced S-phase nuclei observed across the apico-basal
thickness of the tube. This was followed by a failure of normal peripheral
gangliogenesis and disordered central neurogenesis (I.S. and C.K.,
unpublished). Consistent with these results, both cN390-GFP and CBR-GFP
have been shown to cause an initial loss of epithelial morphology in cells of
the avian dermomyotome, yet they had opposite effects in driving cell
segregation into dermal or myotomal domains, respectively
(Cinnamon et al., 2006). These
constructs also altered the morphology of epithelial cell lines
(Fujimori and Takeichi, 1993),
but, surprisingly, when misexpressed using an adenoviral approach, they had no
effect on NT morphology (Nakagawa and
Takeichi, 1998). These results suggest that adenovirus-mediated
gene delivery was less effective at the time points analyzed as compared with
electroporation. This assumption is further substantiated when considering the
effect of adenoviral-mediated overexpression of wild-type N-cadherin, which
mainly inhibited emigration of melanocyte progenitors
(Nakagawa and Takeichi, 1998),
which are the last NC cells to leave the NT, in contrast to the present study
in which electroporation of wild-type N-cadherin prevented emigration of all
potential NC cells and further gangliogenesis.
It is of interest to note that the dorsal NT continues expressing cadherin
6B, a type II cadherin (Liu and Jessell,
1998; Nakagawa and Takeichi,
1998; Sela-Donenfeld and
Kalcheim, 1999) after N-cadherin is downregulated. However, these
two cadherins share no structural or functional similarity
(Nakagawa and Takeichi, 1995)
and, moreover, they respond differently to BMP signaling. Whereas N-cadherin
protein is downregulated by BMP-mediated degradation (Figs
5,
6), maintenance of cadherin 6B
transcription is under positive regulation by BMP4
(Liu and Jessell, 1998;
Sela-Donenfeld and Kalcheim,
1999). Since BMP triggers NC delamination it is possible that, in
contrast to N-cadherin, cadherin 6B has a pro-delamination effect and/or
serves to segregate the premigratory NC from the adjacent CNS progenitors,
which continue expressing N-cadherin
(Nakagawa and Takeichi, 1995).
Together with the finding that cadherin 7 is expressed in migrating NC, these
results suggest that cadherins are not a generic property of epithelial cells
and that type II cadherins in particular may regulate morphogenetic
movements.
An intriguing finding of our study is that BMP4 activity triggers NC
delamination through an ADAM10-dependent-mechanism, as both the basal level of
cell emigration and that enhanced by exogenous BMP4 were inhibited by
GI254023X. This is expected because failure of ADAM10 activity, the
rate-limiting enzyme in cadherin cleavage, preserves N-cadherin in its
full-length conformation (Fig.
6); as such, N-cadherin actively antagonizes
ß-catenin-dependent signaling induced by BMP/Wnt and consequently
prevents NC emigration (Figs 2,
4). This inhibition can be
overcome upon treatment with CTF2, which on the one hand reduces cell adhesion
and on the other hand promotes signaling, further stressing the antagonistic
relationship between the two processes. Consistent with our data,
ADAM10-deficient cells exhibited reduced transcriptional activity and lower
expression levels of ß-catenin-dependent genes such as those encoding
cyclin D1, c-myc and c-jun (Reiss et al.,
2005). Hence, we propose that BMP4 stimulates at least two
distinct molecular pathways: (1) it triggers transcription of Wnt1 in the
dorsal NT, which in turn acts via its canonical pathway; and (2) it promotes
N-cadherin degradation, which releases the system from a pre-existing
inhibition and at the same time produces CTF2, a ß-catenin-binding
product with a pro-delamination activity. As a result, BMP-dependent Wnt and
CTF2 activities might converge to drive the onset of NC movement via a common
ß-catenin-dependent mechanism.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akitaya, T. and Bronner Fraser, M. (1992). Expression of cell adhesion molecules during initiation and cessation of neural crest cell migration. Dev. Dyn. 194, 12-20.[Medline]
Barrallo-Gimeno, A. and Nieto, M. A. (2005).
The Snail genes as inducers of cell movement and survival: implications in
development and cancer. Development
132,3151
-3161.
Basch, M. L., Garcia-Castro, M. I. and Bronner-Fraser, M. (2004). Molecular mechanisms of neural crest induction. Birth Defects Res. C Embryo Today 72,109 -123.[CrossRef][Medline]
Bronner-Fraser, M., Wolf, J. J. and Murray, B. A. (1992). Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube. Dev. Biol. 153,291 -301.[CrossRef][Medline]
Burstyn-Cohen, T. and Kalcheim, C. (2002). Association between the cell cycle and neural crest delamination through specific regulation of G1/S transition. Dev. Cell 3, 383-395.[CrossRef][Medline]
Burstyn-Cohen, T., Stanleigh, J., Sela-Donenfeld, D. and
Kalcheim, C. (2004). Canonical Wnt activity regulates trunk
neural crest delamination linking BMP/noggin signaling with G1/S transition.
Development 131,5327
-5339.
Cinnamon, Y., Ben-Yair, R. and Kalcheim, C.
(2006). Differential effects of N-cadherin-mediated adhesion on
the development of myotomal waves. Development
133,1101
-1112.
Coles, E., Christiansen, J., Economou, A., Bronner-Fraser, M.
and Wilkinson, D. G. (2004). A vertebrate crossveinless 2
homologue modulates BMP activity and neural crest cell migration.
Development 131,5309
-5317.
Duband, J. L., Volberg, T., Sabanay, I., Thiery, J. P. and Geiger, B. (1988). Spatial and temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during avian embryogenesis. Development 103,325 -344.[Abstract]
Endo, Y., Osumi, N. and Wakamatsu, Y. (2003).
Bimodal functions of Notch-mediated signaling are involved in neural crest
formation during avian ectoderm development.
Development 129,863
-873.
Fagotto, F., Funayama, N., Gluck, U. and Gumbiner, B. M.
(1996). Binding to cadherins antagonizes the signaling activity
of beta-catenin during axis formation in Xenopus. J. Cell
Biol. 132,1105
-1114.
Fortini, M. E. (2002). Gamma-secretase-mediated proteolysis in cell-surface-receptor signalling. Nat. Rev. Mol. Cell Biol. 3,673 -684.[CrossRef][Medline]
Fujimori, T. and Takeichi, M. (1993). Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin. Mol. Biol. Cell 4, 37-47.[Abstract]
Funayama, N., Fagotto, F., McCrea, P. and Gumbiner, B. M.
(1995). Embryonic axis induction by the armadillo repeat domain
of beta-catenin: evidence for intracellular signaling. J. Cell
Biol. 128,959
-968.
Gammill, L. S. and Bronner-Fraser, M. (2003). Neural crest specification: migrating into genomics. Nat. Rev. Neurosci. 4,795 -805.[CrossRef][Medline]
Ganzler-Odenthal, S. I. and Redies, C. (1998).
Blocking N-cadherin function disrupts the epithelial structure of
differentiating neural tissue in the embryonic chicken brain. J.
Neurosci. 18,5415
-5425.
Gottardi, C. J. and Gumbiner, B. M. (2001). Adhesion signaling: how beta-catenin interacts with its partners. Curr. Biol. 11,R792 -R794.[CrossRef][Medline]
Gottardi, C. J., Wong, E. and Gumbiner, B. M.
(2001). E-cadherin suppresses cellular transformation by
inhibiting beta-catenin signaling in an adhesion-independent manner.
J. Cell Biol. 153,1049
-1060.
Gumbiner, B. (2000). Regulation of cadherin
adhesive activity. J. Cell Biol.
148,399
-404.
Hall, R. and Erickson, C. (2003). ADAM 10, an active metalloprotease expressed during avian epithelial morphogenesis. Dev. Biol. 256,146 -159.[Medline]
Halloran, M. C. and Bendt, J. D. (2003). Current progress in neural crest cell motility and migration and future prospects for the zebrafish model system. Dev. Dyn. 228,497 -513.[CrossRef][Medline]
Hatta, K. and Takeichi, M. (1986). Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature 320,447 -449.[CrossRef][Medline]
Hatta, K., Takagi, S., Fujisawa, H. and Takeichi, M. (1987). Spatial and temporal expression pattern of N-cadherin cell adhesion molecules correlate with morphogenetic processes of chicken embryos. Dev. Biol. 120,215 -227.[CrossRef][Medline]
Hatta, K., Nose, A., Nagafuchi, A. and Takeichi, M.
(1988). Cloning and expression of cDNA encoding a neural
calcium-dependent cell adhesion molecule: its identity in the cadherin gene
family. J. Cell Biol.
106,873
-881.
Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994). Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell 79,791 -803.[CrossRef][Medline]
Horikawa, K. and Takeichi, M. (2001).
Requirement of the juxtamembrane domain of the cadherin cytoplasmic tail for
morphogenetic cell rearrangement during myotome development. J.
Cell Biol. 155,1297
-1306.
Huber, A. H., Stewart, D. B., Laurents, D. V., Nelson, W. J. and
Weis, W. I. (2001). The cadherin cytoplasmic domain is
unstructured in the absence of beta-catenin. A possible mechanism for
regulating cadherin turnover. J. Biol. Chem.
276,12301
-12309.
Hundhausen, C., Misztela, D., Berkhout, T. A., Broadway, N.,
Saftig, P., Reiss, K., Hartmann, D., Fahrenholz, F., Postina, R., Matthews, V.
et al. (2003). The disintegrin-like metalloproteinase ADAM10
is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates
CX3CL1-mediated cell-cell adhesion. Blood
102,1186
-1195.
Kalcheim, C. (2000). Mechanisms of early neural crest development: from cell specification to migration. Int. Rev. Cytol. 200,143 -196.[CrossRef][Medline]
Kalcheim, C. and Burstyn-Cohen, T. (2005). Early stages of neural crest ontogeny: formation and regulation of cell delamination. Int. J. Dev. Biol. 49,105 -116.[CrossRef][Medline]
Le Douarin, N. M. and Kalcheim, C. (1999). The Neural Crest. New York: Cambridge University Press.
Lele, Z., Folchert, A., Concha, M., Rauch, G. J., Geisler, R., Rosa, F., Wilson, S. W., Hammerschmidt, M. and Bally-Cuif, L. (2002). parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube. Development 129,3281 -3294.[Medline]
Liu, J. P. and Jessell, T. M. (1998). A role for rhoB in the delamination of neural crest cells from the dorsal neural tube. Development 125,5055 -5067.[Abstract]
Ludwig, A., Hundhausen, C., Lambert, M. H., Broadway, N., Andrews, R. C., Bickett, D. M., Leesnitzer, M. A. and Becherer, J. D. (2005). Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules. Comb. Chem. High Throughput Screen 8, 161-171.[CrossRef][Medline]
Marambaud, P., Wen, P. H., Dutt, A., Shioi, J., Takashima, A., Siman, R. and Robakis, N. K. (2003). A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell 114,635 -645.[CrossRef][Medline]
Megason, S. G. and McMahon, A. P. (2002). A
mitogen gradient of dorsal midline Wnts organizes growth in the CNS.
Development 129,2087
-2098.
Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K. and Yasuda, K. (1999). Efficient targeting of gene expression in chick embryos by microelectroporation. Dev. Growth Differ. 41,335 -344.[CrossRef][Medline]
Nakagawa, S. and Takeichi, M. (1995). Neural crest cell-cell adhesion controlled by sequential and subpopulation-specific expression of novel cadherins. Development 121,1321 -1332.[Abstract]
Nakagawa, S. and Takeichi, M. (1998). Neural crest emigration from the neural tube depends on regulated cadherin expression. Development 125,2963 -2971.[Abstract]
Nelson, W. J. and Nusse, R. (2004). Convergence
of Wnt, beta-catenin, and cadherin pathways. Science
303,1483
-1487.
Nieto, A. M. (2001). The early steps in neural crest development. Mech. Dev. 105, 27-35.[CrossRef][Medline]
Olson, L. E., Tollkuhn, J., Scafoglio, C., Krones, A., Zhang, J., Ohgi, K. A., Wu, W., Taketo, M. M., Kemler, R., Grosschedl, R. et al. (2006). Homeodomain-mediated beta-catenin-dependent switching events dictate cell-lineage determination. Cell 125,593 -605.[CrossRef][Medline]
Reiss, K., Maretzky, T., Ludwig, A., Tousseyn, T., de Strooper, B., Hartmann, D. and Saftig, P. (2005). ADAM10 cleavage of N-cadherin and regulation of cell-cell adhesion and beta-catenin nuclear signalling. EMBO J. 24,742 -752.[CrossRef][Medline]
Sadot, E., Simcha, I., Shtutman, M., Ben-Ze'ev, A. and Geiger,
B. (1998). Inhibition of beta-catenin-mediated
transactivation by cadherin derivatives. Proc. Natl. Acad. Sci.
USA 95,15339
-15344.
Sanson, B., White, P. and Vincent, J. P. (1996). Uncoupling cadherin-based adhesion from wingless signalling in Drosophila. Nature 383,627 -630.[CrossRef][Medline]
Sela-Donenfeld, D. and Kalcheim, C. (1999). Regulation of the onset of neural crest migration by coordinated activity of BMP4 and Noggin in the dorsal neural tube. Development 126,4749 -4762.[Abstract]
Sela-Donenfeld, D. and Kalcheim, C. (2000). Inhibition of noggin expression in the dorsal neural tube by somitogenesis: a mechanism for coordinating the timing of neural crest emigration. Development 127,4845 -4854.[Abstract]
Sela-Donenfeld, D. and Kalcheim, C. (2002). Localized BMP4-noggin interactions generate the dynamic patterning of noggin expression in somites. Dev. Biol. 246,311 -328.[CrossRef][Medline]
Stadeli, R., Hoffmans, R. and Basler, K. (2006). Transcription under the control of nuclear Arm/beta-catenin. Curr. Biol. 16,R378 -R385.[CrossRef][Medline]
Stockinger, A., Eger, A., Wolf, J., Beug, H. and Foisner, R.
(2001). E-cadherin regulates cell growth by modulating
proliferation-dependent b-catenin transcriptional activity. J. Cell
Biol. 154,1185
-1196.
Tepass, U., Truong, K., Godt, D., Ikura, M. and Peifer, M. (2000). Cadherins in embryonic and neural morphogenesis. Nat. Rev. Mol. Cell Biol. 1, 91-100.[CrossRef][Medline]
Uemura, K., Kihara, T., Kuzuya, A., Okawa, K., Nishimoto, T., Bito, H., Ninomiya, H., Sugimoto, H., Kinoshita, A. and Shimohama, S. (2006). Activity-dependent regulation of beta-catenin via epsilon-cleavage of N-cadherin. Biochem. Biophys. Res. Commun. 345,951 -958.[CrossRef][Medline]
Wong, A. S. and Gumbiner, B. M. (2003).
Adhesion-independent mechanism for suppression of tumor cell invasion by
E-cadherin. J. Cell Biol.
161,1191
-1203.
Xu, L., Corcoran, R. B., Welsh, J. W., Pennica, D. and Levine,
A. J. (2000). WISP-1 is a Wnt-1- and beta-catenin-responsive
oncogene. Genes Dev. 14,585
-595
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12 explants per treatment
stained with each antibody). The broken white line (B,C) delineates the border
between the explant and emigrated cells. E and F exhibit only emigrated cells,
whereas H,I,K and L exhibit only densely packed but non-mesenchymal cells.
Scale bar: 100 µm in A,D,G,J; 10 µm in B,C,E,F,H,I,K,L.
