Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel
Author for correspondence (e-mail:
kalcheim{at}nn-shum.cc.huji.ac.il)
Accepted 31 August 2004
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SUMMARY |
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Key words: Avian embryo, Cell cycle, Cyclin D1, Epithelial to mesenchymal transition, Neural tube, Somite
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Introduction |
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EMT of NC cells requires the coordinated action of transcription factors,
extracellular matrix, cytoskeletal and cell-adhesion proteins
(Nieto, 2001;
Halloran and Berndt, 2003
).
The identity and mechanism of action of upstream factors that initiate the
process remained, however, elusive. In previous studies, we have shown that a
high rostral to low caudal gradient of BMP4 activity is established along the
dorsal neural tube by a reciprocal gradient of expression of its inhibitor
noggin. BMP then triggers emigration of NC progenitors in a manner that is
independent of initial specification events. Noggin downregulation is
in turn carried out by the developing somites, which consequently determine
the timing of NC delamination
(Sela-Donenfeld and Kalcheim,
1999
; Sela-Donenfeld and
Kalcheim, 2000
; Sela-Donenfeld
and Kalcheim, 2002
). More recently, the cell cycle was found to
play a pivotal role in EMT of NC cells. We showed that trunk-level avian NC
cells synchronously emigrate in the S-phase of the cell cycle. Next, we
reported that the transition from G1 to S is a necessary event for NC
delamination because specific inhibition of the transition from G1 to S
blocked NC emigration, whereas arrest at S or G2 phases had no immediate
effect (Burstyn-Cohen and Kalcheim,
2002
).
These findings raise the question of whether BMP signaling and G1/S
transition independently regulate EMT of NC cells or, alternatively, whether
these processes interact with each other. In the present study, we report that
noggin overexpression prevents the entry of neuroepithelial cells into the S
phase of the cycle at axial levels corresponding to NC emigration (epithelial
and dissociated somites) but not at levels opposite the segmental plate where
NC cells have not yet initiated delamination. Moreover, mimosine, which
specifically blocks G1/S transition, inhibits BMP-induced NC cell
delamination. Hence, BMP regulates NC delamination in a cell cycle-dependent
manner. We then examined the hypothesis that Wnt signaling, which controls
G1/S transition by regulating cyclin D1 transcription
(Tetsu and McCormick, 1999)
mediates BMP activity on both G1/S transition and NC delamination. We find
that expression of Wnt1 along the dorsal tube, but not that of
Wnt3a, is reciprocal to the pattern of noggin transcription
and depends upon BMP activity. In addition, grafting dissociating somites in
place of segmental plate mesoderm, a procedure that results in premature
downregulation of noggin mRNA, upregulates Wnt1
transcription and leads to precocious NC emigration from the caudal tube.
Furthermore, abrogating canonical Wnt signaling downregulates transcription of
various BMP-dependent genes in the dorsal tube. Thus, in the context of NC
delamination, BMP acts upstream of Wnt1. Consistent with this notion,
inhibition of the canonical branch of Wnt activity, but not of the
non-canonical pathway, prevents both G1/S transition and NC delamination and
overexpression of ß-catenin rescues NC delamination in noggin-inhibited
neural primordia. Thus, BMP-dependent Wnt signaling is necessary for NC
delamination.
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Materials and methods |
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In ovo grafting of noggin-secreting cells
CHO cells producing Xenopus noggin and dhfr-CHO control cells were
grown as previously described (Lamb et
al., 1993; Sela-Donenfeld and
Kalcheim, 2002
). To establish confluent monolayers, cells were
replated on eight-well chamber slides (Lab-Tek), grown in serum-containing
medium for 2 days and then transferred to serum-free medium until explantation
of neural primordia. For grafting purposes, confluent cultures were harvested
and pelleted. The vitelline membrane of 16- to 20-somite stage chick embryos
was removed. A slit was performed along the dorsal edge of the neural tube at
levels corresponding either to the rostral segmental plate and two last formed
epithelial somites, or along the caudal half of the segmental plate.
Concentrated cell suspensions were applied on top of the neural tube with a
micropipette. Cell implants were performed under an Olympus dissecting
microscope with a x40 total magnification. Embryos were further
incubated for 8-10 or 20-24 hours and then fixed for immunocytochemistry
and/or in situ hybridization.
Grafting of BMP-4-coated beads
Heparin-acrylic beads (Sigma) were immersed in a solution of BMP4 [R&D,
50 ng/ml in phosphate buffered saline (PBS)/1% fetal calf serum (FCS)] for 1.5
hours followed by repeated washings in PBS. To graft the beads, a slit was
made along the dorsal aspect of the neural tube at the segmental plate level
of the axis. A single BMP-coated bead per embryo was then inserted being held
between the neural folds. Embryos were further incubated for 6-8 hours.
Explants of neural primordia
Neural tubes containing premigratory NC were excised from levels
corresponding to the segmental plate and three most recently formed somites of
16-20 somite stage-quail embryos, and then explanted onto eight-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 medium (Gibco-BRL)
to which the following factors were added: L-mimosine (Calbiochem) and BMP4
(R&D). Mimosine was dissolved to a concentration of 0.05 M in 0.12 M HCl;
BMP4 was reconstituted in PBS/1% FCS to a stock solution of 100 ng/µl.
Further dilutions to 600 µM and 100 ng/ml, respectively, were performed in
culture medium, and controls consisted of the appropriate diluents. Explants
were cultured for a total of 10 hours and a pulse of BrdU (10 µg/ml, Sigma)
was administered 1 hour before fixation.
In another experimental series, hemi-neural tubes were co-electroporated with Wnt1/GFP or ß-catenin/GFP encoding DNAs (see below). Two hours later, the neural primordia were enzymatically isolated and plated either on fibronectin-coated wells or onto monolayers of control CHO or CHO-noggin cells previously grown for 17 hours in serum-free medium. Cultures were incubated for 14 hours and co-cultures for 16-20 hours and pulsed with BrdU for 1 hour before fixation.
In ovo manipulations
Grafting of dissociating somites
Host embryos aged 16-18 somites were windowed and their vitelline membrane
was removed. A fragment of caudal segmental plate corresponding to four or
five prospective somites was removed unilaterally as previously described
(Kalcheim and Teillet, 1989).
Donor embryos aged 25-27 somites were pinned ventral side down on
Sylgard-coated dishes. The fourth to ninth to last formed somites,
corresponding to the onset of dissociation into dermomyotome and sclerotome,
were excised in one piece along with a narrow strip of intermediate and
lateral plate mesoderm. Donor somites were then grafted in the gap left after
removal of the unsegmented mesoderm of the younger hosts. Special care was
taken in keeping the correct dorsoventral and mediolateral orientations. In
control experiments, sclerotomal fragments devoid of dermomyotome were excised
from more rostral levels and similarly grafted. Embryos were incubated for
additional 10 hours, fixed in 4% formaldehyde and processed for in situ
hybridization.
In ovo electroporation
DNA (3-4 µg/µl) was microinjected into the lumen of the neural tube
of 18- to 23-somite stage chick embryos with micropipettes, and electrodes
were placed on either side of the embryos at the level of the segmental plate
and recently formed somites. A square wave electroporator (BTX, San Diego, CA)
was used to deliver four pulses of current at 25 volts, 10 mseconds each.
Embryos were reincubated for 9 hours or 14-16 hours followed by a 1 hour pulse
of BrdU (10 mM) or by processing for in situ hybridization. Another series was
reincubated for 2 hours followed by explantation of isolated tubes on
fibronectin, CHO or CHO-noggin monolayers. DNA expression vectors employed
were: pCAGGS-AFP (Momose et al.,
1999); pCAdelLEF-1 (Kubo et
al., 2003
); ß-catenin fused to the engrailed repressor domain
(ß-Eng/pcDNA3.1+MT) (Montross et al.,
2000
); pEFBOSS/xNoggin (Endo
et al., 2002
), full-length mouse ß-catenin/pCAGGS and
Wnt1/pCAGGS (Nishihara et al.,
2003
). Truncated forms of Xdishevelled (from S. Sokol) were
pCS2-Xdd1, pCS2-DEP+ and pCS2-D2-GFP
(Sokol, 1996
;
Rothbacher et al., 2000
;
Tada and Smith, 2000
).
pCS2-Xdd1 and pCS2-DEP+ were fused to YFP (from eYFP-N1, Clontech) and
subcloned into the pCAGGS electroporation vector.
Tissue processing and immunocytochemistry
Explants and embryos were fixed in 4% formaldehyde/PBS. Embryo fragments
were embedded in paraffin wax and sectioned at 5 or 10 µm. Immunostaining
for BrdU was as described (Burstyn-Cohen
and Kalcheim, 2002). Rabbit anti-GFP antibodies (Molecular Probes)
were used at 1:200 in combination with monoclonal anti-BrdU antibodies. Nuclei
were visualized with Hoechst (Sigma).
In situ hybridization
In-situ hybridization was performed as previously described
(Burstyn-Cohen and Kalcheim,
2002) with the following chick-specific probes: Wnt1 and Wnt3a
(from A. P. McMahon), Cyclin D1 (from F. Pituello), RhoB
(Liu and Jessell, 1998
), Cad
6B (Nakagawa and Takeichi,
1995
), Msx1 and Pax3
(Monsoro-Burq et al., 1996
),
FoxD3 (Dottori et al., 2001
;
Kos et al., 2001
), Slug
(Nieto et al., 1994
), and Sox9
(Cheung and Briscoe, 2003
).
Measurements of cell proliferation and NC delamination
Neural tube explants
The number of emigrating NC cells was counted using phase-contrast optics
along a 900 µm length of tube explant. Cells (0-300/900 µm length of
tube) were scored as +1; 300-600 cells/900µm length were scored as +2; more
than 600 cells/900 µm length were scored as +3. The average scores were
normalized to control values. In electroporated hemi-tubes, the total number
of GFP+ NC cells was similarly counted along a length of 500 µm of tube
explants. Five to eight explants were assayed per treatment. Results represent
the average fold increase in NC delamination or the mean number of emigrated
GFP+ NC cells (±s.d. of three or four similar experiments).
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. Incorporation of BrdU into
neural tube cells was taken as a general measure of drug efficacy.
Embryo sections
In electroporated embryos that received Xdd1-GFP or DEP+-GFP, the number of
GFP+ and BrdU+ cells was monitored in 10-30 alternate sections. Between 100
and 130 GFP+ cells/hemi-tube were scored for BrdU incorporation. The
proportion of BrdU-incorporating nuclei in all treatments described was found
to be similarly affected in the dorsal region of the tube (when considering
only eight or nine nuclei from the dorsal midline) as well as at more ventral
areas. Therefore, to increase the sample size per embryo, counts included the
entire dorsoventral extent of the epithelium. Results are expressed as
percentage±s.d. of BrdU+ nuclei out of the GFP+ population. When
electroporating the other constructs, assessment of cell proliferation was
monitored as the number of total nuclei (Hoechst+) that incorporated BrdU.
Between 250 and 300 nuclei were counted per embryo. Results represent the
percentage±s.d. of BrdU+ cells from 4-14 embryos/treatment. NC
delamination was similarly monitored in alternating sections. Results are
expressed either as percentage of GFP+/total emigrating nuclei in embryos that
received GFP alone versus Xdd1-GFP or DEP+-GFP or as number of Hoechst+ nuclei
located up to the migration staging area
(Weston, 1991) in intact
versus transfected sides of co-electroporated embryos.
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Results |
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Inhibition of G1/S transition with mimosine prevents BMP-induced BrdU incorporation and NC delamination
To further examine the relationship between BMP activity and the cell
cycle, G1/S transition was specifically blocked with mimosine. This inhibitor
was previously shown to upregulate levels of the cdk inhibitor p27
(Wang et al., 2000), causing
its translocation into the nuclei of cultured NC cells and inhibiting NC
delamination (Burstyn-Cohen and Kalcheim,
2002
). Neural primordia containing premigratory NC were explanted
onto fibronectin. Ten hours later, the first NC cells were present on the
substrate of control cultures and treatment with BMP enhanced the number of
emigrating mesenchymal cells by 72% (Fig.
2A,C,E). Consistent with previous results, mimosine alone
inhibited the onset of NC delamination by 62% compared with controls and
blocked DNA synthesis (Fig.
2B,E). The combination of mimosine and BMP resembled the effect of
mimosine alone, with significant reduction of NC delamination to 50% of
control values and to 29% of BMP-treated values
(Fig. 2D,E). Furthermore, BMP
co-treatment did not overcome the inhibition of G1/S transition caused by
mimosine (Fig. 2D). These
results suggest that BMP signaling is upstream of events controlling G1/S
transition. As both BMP activity and G1/S transition are required for NC
delamination (Sela-Donenfeld and Kalcheim,
1999
; Sela-Donenfeld and
Kalcheim, 2000
; Burstyn-Cohen
and Kalcheim, 2002
), our results suggest that these processes are
epistatically related.
|
Transcription of Wnt1, but not of Wnt 3a, is inhibited by short-term noggin treatment
Wnt1 mRNA is apparent along the rostral dorsal neural tube of
avian embryos roughly to the level of the last formed somite pair/rostralmost
portion of the segmental plate (Fig.
3A, Fig. 4B). This
pattern is positively correlated with both the rostrocaudal gradient of NC
delamination and with previously reported levels of BMP activity along the
tube, and is reciprocal to that of noggin mRNA, which is high in the
dorsal tube opposite segmental plate levels and decreases rostralwards
(Sela-Donenfeld and Kalcheim,
1999; Sela-Donenfeld and
Kalcheim, 2000
; Sela-Donenfeld
and Kalcheim, 2002
). Localized grafts of CHO-noggin cells
completely abolished transcription of Wnt1 as early as 6-10 hours
following implantation (Fig.
3B,C; n=20), while control cells had no effect
(n=9, not shown). Likewise, electroporation of a noggin-encoding
vector, but not of GFP-encoding DNA, downregulated Wnt1 along
transfected hemi-tubes (n=9 for each construct,
Fig. 3E and data not shown).
Conversely, grafts of a BMP4-coated bead on the dorsal tube at the level of
the segmental plate resulted in premature upregulation of Wnt1 mRNA
(arrow in Fig. 3F,
n=5). Hence, BMP regulates transcription of Wnt1 in the
dorsal tube.
|
|
Wnt1 transcription in the dorsal tube is modulated by the developing somites
We have previously shown that the dorsomedial region of developing somites
inhibits noggin transcription in the dorsal neural tube. Loss of
noggin activity releases BMP4 from inhibition, resulting in NC emigration
(Sela-Donenfeld and Kalcheim,
2000). We reasoned that if Wnt1 is part of the
BMP/noggin-dependent signaling cascade, then experimentally induced
downregulation of noggin should result in a corresponding and
premature upregulation of Wnt1. Dissociating somites (opposite which
the tube is noggin/Wnt1+) were unilaterally grafted in the
place of the unsegmented mesoderm (opposite which the tube is
noggin+/Wnt1). This procedure precociously upregulated
expression of Wnt1 in the caudal hemi-tube adjacent to the graft
(Fig. 4A, n=8). At a
slightly more anterior level, opposite the rostral segmental plate where early
Wnt1 transcription is already apparent in the intact side, grafting
of dissociating somites resulted in expansion of the domain of Wnt1
expression. This was associated with premature emigration of NC cells, which
retained Wnt1 expression following delamination
(Fig. 4B). By contrast,
grafting of mesenchymal sclerotomal fragments devoid of dermomyotome, which
has previously been shown to have no effect on noggin transcription
(Sela-Donenfeld and Kalcheim,
2000
), had no effect either on expression of Wnt1 mRNA
(Fig. 4C, n=5). Thus,
similar to BMP/noggin, graded production of Wnt1 along the dorsal
tube is regulated by the dorsomedial region of developing somites. Moreover,
Wnt1 expression is directly correlated with BMP activity and
reciprocally related to noggin production further supporting the
notion that Wnt1 is a downstream effector of BMP signaling.
Wnt signaling mediates BMP-dependent G1/S transition and NC delamination
Inhibition of the canonical pathway of Wnt signaling prevents G1/S transition and NC delamination
To examine whether Wnt activity is necessary for the above processes, we
overexpressed in the neural tube a truncated form of XDsh, a key component of
the Wnt signaling pathway (Veeman et al.,
2003; Wharton,
2003
) that harbors a partial deletion in the PDZ domain
(Xdd1-GFP). Previous studies have shown that Xdd1 acts in a dominant-negative
form to abolish both canonical as well as non-canonical Wnt signaling
(Sokol et al., 1995
;
Sokol, 1996
;
Tada and Smith, 2000
;
Rothbacher et al., 1995
;
Rothbacher et al., 2000
).
Electroporations were performed at segmental plate levels and embryos were
further incubated for 16 hours corresponding to the onset of NC delamination
at the treated levels of the tube. Hence, considering it takes 4-6 hours for
the transgene to be expressed, the net effect was measured for the length of
one cell cycle approximately. This limited incubation time allows us to assay
the direct effect of G1/S transition on NC behavior, as previously shown
(Burstyn-Cohen and Kalcheim,
2002
). Expression of a control GFP vector had no effect on DNA
synthesis (Fig. 5A-C) as
50.2±7.4% of GFP+ cells in transfected hemi tubes incorporated BrdU
into their nuclei, a similar value measured in control sides (data not shown)
(Burstyn-Cohen and Kalcheim,
2002
) (n=4 counted out of 10 similar embryos). By
contrast, only 4.2±2.6% of GFP+ cells were BrdU+ in Xdd1-treated tubes
(Fig. 5D-F, n=6
counted embryos of 12 embryos showing similar qualitative results). Thus, Xdd1
causes a 91.6% inhibition in DNA synthesis in neuroepithelial cells. This
striking inhibition is only mildly reflected 16 hours following
electroporation in the size of the neural tube; the amount of total nuclei in
the treated sides was 91.2±3% of that in contralateral sides of
Xdd1-treated embryos when compared with 100.2±4%, respectively, in
embryos that received control GFP.
|
To determine whether the canonical pathway accounts for the effects of
Xdd1, dominant-negative (dN)LEF1 [deleted in the ß-catenin-binding domain
(Kengaku et al., 1998;
Kubo et al., 2003
)] was
electroporated. The LEF/T-cell transcription factor (TCF) family members act
in conjunction with ß-catenin to influence transcription of
Wnt-responsive genes (Behrens et al.,
1996
; Molenaar et al.,
1996
; Cong et al.,
2003
). Overexpression of dNLEF1 resulted in 54% of total nuclei
that incorporated BrdU when compared with the contralateral side
(24.8±3.4% and 45.8±4.3%, respectively; n=5 embryos
counted out of nine showing a similar phenotype). A corresponding 60%
inhibition in NC delamination was measured (2.8±0.8 and 7.0±1.2
Hoechst+ NC cells/section, respectively). Likewise, inhibition of
ß-catenin activity by electroporation of ß-catenin fused to the
engrailed repressor domain reduced BrdU incorporation to 55% of control levels
(27.0±3.5% versus 49.1±6.2% in treated versus control sides,
respectively) and inhibited NC emigration by 56% (3.8±0.6 compared with
8.6±1.8 emigrating Hoechst+ NC cells/section) in four counted out of
nine embryos with a similar effect. Furthermore, both dNLEF1 and
ß-catenin-engrailed downregulated transcription of cyclin D1 in
the transfected hemi-tubes (see Fig.
7C,D and data not shown), confirming their effect through the
canonical pathway of Wnt signaling.
|
We next examined whether lack of NC emigration in Xdd1 and dNLef1-treated embryos could be explained by enhanced cell death. In control sides of Xdd1, dNLef1 and DEP+-electroporated embryos, the proportion of pyknotic/total Hoechst+ nuclei was 5.9±0.9%, 5.9±1.2% and 4.7±1.7%, respectively (n=3 embryos/treatment). In treated sides, it slightly increased to 10.8±1.7% and 10.1±4% in Xdd1 and dNLef1 embryos, respectively, but remained unchanged upon treatment with DEP+ (4.9±2.0%). In the latter embryos, however, numerous cells were present in the lumen of the neural tube and were therefore not considered in the quantifications. Taken together, we conclude that enhanced cell death cannot account for the inhibition in NC delamination measured upon treatment with Xdd1 and dNLef1. Hence, the failure of NC cells to delaminate is likely to result from the lack of G1/S transition in the premigratory progenitors that is specifically mediated by canonical Wnt signaling.
The above loss-of-function experiments revealed that Wnt activity is
necessary for NC delamination. Next, we examined whether specific
overexpression of Wnt1 stimulates the process. To this end, hemi-neural tubes
were co-electroporated with full-length mouse Wnt1 and GFP-encoding DNAs or
with GFP alone. Two hours later, the transfected neural primordia were
isolated and seeded onto fibronectin-coated wells and grown for 14 hours in
serum-free medium. Assuming it takes 4-6 hours for the transgenes to be
significantly expressed, the net effect was measured over the length of one
cell cycle. Three different concentrations of the Wnt1 DNA were tested (0.2,
0.8 and 3.0 µg/µl) and the number of labeled cells that emigrated from
the hemi-tubes was counted as depicted in the Materials and methods. The
average number of GFP+ NC cells that exited a given length of control
hemi-tubes was 39.1±18 (about 20% of total NC cells that emigrated from
both sides of the tube at this time). Electroporation with 0.2 µg/µl of
Wnt1 DNA showed no significantly different emigration (43.7±19 GFP+
cells), but hemi-tubes that received 0.8 and 3.0 µg/µl of Wnt1 DNA
revealed a 1.66 and 2.6 fold stimulation in delamination of labeled cells when
compared with tubes that received GFP alone (64.7±8 and 101.8±23
GFP+ cells, P<0.005 and P<0.001, respectively,
Fig. 5J,L). Consistent with
previous findings (Burstyn-Cohen and
Kalcheim, 2002
), the delamination of transfected and untransfected
NC cells in both control and experimental treatments, was associated with
cells undergoing G1/S transition, as reflected by incorporation of BrdU into
their nuclei (Fig. 5K,M; data
not shown). Taken together, loss- and gain-of-function analysis substantiates
a role for Wnt signaling in regulating NC delamination and further suggests
that this effect may be exerted by Wnt1.
Inhibition of Wnt activity downregulates transcription of several BMP-dependent genes in the dorsal neural tube but has no effect on early genes associated with NC induction
Transcription of various dorsal neural tube-specific genes, including
Cad6B, Pax3, Msx1 and RhoB was found to depend upon BMP
activity from the dorsal tube
(Sela-Donenfeld and Kalcheim,
1999) (data not shown). If Wnt1 operates downstream of BMP, we
reasoned that inhibiting Wnt activity will similarly affect their
transcription. Unilateral electroporation of Xdd1, dNLEF1 or
ß-catenin-engrailed completely abolished expression of Cad6B, Pax3
and Msx1 along the electroporated region
(Fig. 6B-E and not shown),
whereas control GFP had no effect (Fig.
6A and not shown). Expression of cyclin D1, a direct
target of canonical Wnt signaling, reveals a dynamic pattern along the tube.
Opposite the segmental plate mesoderm, cyclin D1 mRNA is distributed
throughout the tube except for the floor plate and dorsal midline regions,
which reveal only low levels of transcription
(Fig. 7A). At more rostral
levels of the axis, concomitant with BMP and Wnt activation, cyclin
D1 expression also becomes apparent in the dorsal midline containing
premigratory NC and in emigrating NC cells
(Fig. 7B, arrowheads). Similar
to Cad6B, Pax3 and Msx1, transcription of Cyclin D1 was also
downregulated both by noggin overexpression, as well as by the Wnt-inhibitory
treatments Xdd1, dNLEF1 or ß-catenin-engrailed
(Fig. 7C,D; data not shown),
further stressing the epistatic link between the BMP and Wnt pathways. By
contrast, Dsh-DEP+ had no effect on transcription of either gene (data not
shown). Hence, only canonical Wnt signaling plays a role in transcriptional
activation of the above dorsal tube genes. Surprisingly, RhoB mRNA
levels were unaffected upon treatment with either Xdd1, dNLEF1 or
ß-catenin-engrailed (Fig.
6F), suggesting that RhoB acts either upstream of Wnt, affects
cyclin D1 production independently of Wnt activity, or is part of a separate
BMP-dependent pathway (see Discussion).
|
ß-Catenin overexpression rescues NC delamination in noggin-treated neural tubes
We showed that noggin transcription along the tube is reciprocal
to that of Wnt1 (Fig.
3). In addition, noggin overexpression inhibits Wnt1
transcription (Fig. 3), G1/S
transition (Fig. 1) and NC
delamination (Fig. 1)
(Sela-Donenfeld and Kalcheim,
1999). Furthermore, inhibiting canonical Wnt signaling had a
similar effect on cell cycle progression and NC emigration
(Fig. 5). Altogether, these
data support the notion that Wnt signaling acts downstream of BMP. In such a
case, forced expression of full-length ß-catenin is expected to rescue NC
delamination in neural primordia treated with noggin. To test this prediction,
hemi-neural tubes were co-electroporated in ovo with ß-catenin/GFP and 2
hours later the transfected neural primordia were isolated and explanted on
monolayers of control CHO or CHO-noggin cells.
When placed onto control CHO cells, GFP-positive NC cells exited the neural primordia that received either GFP alone or ß-catenin/GFP and the emigrating cells stained positive for BrdU which was delivered to the co-cultures 1 hour prior to fixation (Fig. 8A,B, n=7 out of eight and six out of seven explants, respectively). By contrast, when placed onto noggin/CHO monolayers, no NC emigration was observed from any of the tubes that received a GFP-encoding plasmid (Fig. 8C, n=7). This inhibition was reversed upon transfection with ß-catenin/GFP as both NC delamination and BrdU incorporation were observed in seven out of seven explants examined (Fig. 8D). Hence, canonical Wnt signaling acts downstream of BMP to stimulate NC delamination.
|
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Discussion |
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BMP signaling differentially regulates G1/S transition along the axis
We show that inhibition of BMP activity affects neuroepithelial cell
proliferation at somitic levels of the axis but not opposite the segmental
plate. These results suggest that G1/S transition depends upon BMP signaling
from segmented areas of the axis rostralwards, but not at caudal regions of
the tube. The latter is consistent with the presence of high levels of noggin
in the caudal dorsal tube which co-exist with normal proliferation rates
(Burstyn-Cohen and Kalcheim,
2002). By contrast, inhibition of canonical Wnt signaling blocked
cell cycle genes and G1/S transition along the entire length of the tube
(T.B.-C., J.S., D.S.-D. and C.K., unpublished). Hence, at somitic levels of
the axis, BMP-dependent Wnt signaling controls cell proliferation, whereas
opposite unsegmented regions Wnt activity is independent of BMP. This
differential regulation along the embryonic axis is, however, likely to be
exerted via distinct Wnt proteins and cyclins. For example, proliferation in
the caudal tube cannot depend upon Wnt1, because the latter is absent along
this area and so is transcription of Wnt3a until the 18th-20th somite stage,
when its expression becomes rostrocaudally homogeneous. Thus, Wnts other than
Wnt1 and Wnt3a (temporarily) are likely to regulate the progression of the
cell cycle in the caudal region of the dorsal tube. In addition, noggin
overexpression inhibited BrdU incorporation and Wnt1 but not
Wnt3a transcription following 10-12 hours of treatment. Hence,
residual endogenous Wnt3a does not appear to be sufficient for maintaining the
progression of the cell cycle. The possibility remains to be tested that at
segmented levels of the axis, the primary mechanism responsible for cell cycle
progression is Wnt1/cyclin D1 dependent (see below).
A second axial difference resides in the dynamic pattern of cyclin D1
expression, which is very low in the dorsal neural tube at segmental plate
areas and significantly increases from epithelial levels in the dorsal midline
and early emigrating NC cells onward concomitant with the onset of Wnt1
transcription. By contrast, cyclin D2 reveals a reciprocal axial pattern
(T.B.-C., J.S., D.S.-D. and C.K., unpublished). Given that somitic signals
downregulate transcription of noggin followed by BMP activation,
synthesis of Wnt1 and NC delamination (Fig.
4) (Sela-Donenfeld and
Kalcheim, 2000), we propose that the switch between differential
mechanisms of cell cycle regulation along the axis also depends upon dynamic
interactions with the paraxial mesoderm. Consistent with this notion, FGF8,
the levels of which are high along the segmental plate mesoderm but decrease
upon somitogenesis (Dubrulle et al.,
2001
; Dubrulle and Pourquie,
2004
) controls levels of cyclin D2 expression in the caudal neural
tube (F. Pituello, personal communication).
Wnt1 acts downstream of BMP in the dorsal neural tube
We have previously found that a balance between the activities of dorsal
tube-derived BMP and its inhibitor noggin regulates NC delamination. Here, we
report that BMP signaling also controls proliferation of neuroepithelial cells
at axial levels corresponding to NC delamination. As the transition between G1
and S phases of the cell cycle is necessary for initiating NC migration, we
hypothesized that BMP affects cell emigration by regulating the cell cycle.
The Wnt proteins present in the dorsal tube, Wnt1 and Wnt3a, are likely
candidates to mediate such an effect. This is because Wnt signaling directly
targets cyclin D1 production (Tetsu and
McCormick, 1999) and has been implicated in the control of cell
proliferation in the CNS (Dickinson et al.,
1994
; Megason and McMahon, 2003), including NC precursors
(Ikeya et al., 1997
). In the
present study, we find that Wnt1 acts downstream of BMP based on the following
results. First, expression of Wnt1 along the neural tube is
reciprocal to that of noggin, is downregulated shortly after exposure
to noggin and is prematurely upregulated by BMP overexpression. Likewise, in
rodents, a constitutively active form of BMP receptor I induced ectopic
expression of Wnt1 in the neural tube (Panchinsion et al., 2001) and
overexpression of constitutive active BMP receptor I in avian embryos led to
increased NC emigration (Liu et al.,
2004
) in agreement with our results
(Sela-Donenfeld and Kalcheim,
1999
).
It is worth mentioning that the expression of Wnt3a is more
dynamic, being absent from segmental plate levels of early embryos but present
homogeneously along the entire axis of embryos aged 20 somites and older.
Together with the finding that Wnt3a expression is downregulated
following overnight exposure to noggin
(Marcelle et al., 1997)
(T.B.-C., J.S., D.S.-D. and C.K., unpublished) but not shortly afterwards, we
infer that, unlike Wnt1, Wnt3a is not a direct target of BMP.
However, our functional interference assays do not discriminate between
different Wnt proteins and therefore do not rule out the possibility that
Wnt3a may also be involved in some aspects of the NC delamination process.
Second, we show that somitic signals that inhibit noggin
transcription in the dorsal tube resulting in activation of BMP
(Sela-Donenfeld and Kalcheim, 2001), also stimulate initial transcription of
Wnt1 and altogether cause premature emigration of NC cells from the
caudal tube. Third, inhibiting canonical Wnt signaling abrogates transcription
of several BMP-dependent genes in the dorsal tube, except for early
specification markers (see below). Fourth, overexpressing Wnt1 stimulates NC
delamination. Fifth, activating the canonical pathway under conditions in
which BMP signaling is inhibited by noggin overexpression, rescues both NC
delamination and G1/S transition. Thus, BMP is upstream of Wnt signaling in
the dorsal neural tube within the context leading to NC delamination. As BMPs
and Wnts continue to be expressed after NC cells accomplished emigration, this
hierarchical relationship between the two signaling systems may also remain
for later events, such as the specification of dorsal interneurons in the
spinal cord (Muroyama et al.,
2002; Liu et al.,
2004
).
As discussed above, BMP signaling regulates Wnt at the transcriptional
level. Whether Wnt protein activity in the dorsal neural tube is also
modulated by its known antagonists, such as BMP, is regulated by noggin,
remains to be clarified. In the trunk of avian embryos, Sfrp-1 and Sfrp-2 are
expressed in the neural tube but are excluded from the dorsal midline, where
the premigratory NC resides (Terry et al.,
2000) (see also Esteve et al.,
2000
). The expression of another inhibitor, frzb-1 (Sfrp-3), is
graded along the tube resembling that of noggin mRNA, with high levels being
transcribed in the neural folds opposite the segmental plate and turning to
low up to undetectable when advancing rostralward along the tube
(Baranski et al., 2000
;
Duprez et al., 1999
;
Jin et al., 2001
;
Ladher et al., 2000
). This
expression pattern is largely reciprocal to that of Wnt1, which is already
active at the level of epithelial somites based on the observed dorsalization
of one of its direct target genes, cyclin D1, at this axial level
(Fig. 7), and on the
corresponding onset of detectable NC delamination, a function here reported to
depend upon Wnt activity. Hence, it is possible that the low signal of frzb-1
mRNA still detected in 19-somite stage embryos at epithelial somite levels by
Jin et al. (Jin et al., 2001
)
is compatible with a degree of Wnt activity that permits initial NC
delamination to take place. Full activity of the protein is then enabled upon
complete frzb-1 downregulation during the progression of the emigration
process. Notably, in contrast to noggin, which titrates BMP activity at
segmental plate levels of the axis, the intense expression of frzb-1 along
this caudal region is likely to inhibit Wnt members other than Wnt1 or Wnt3a
(till the 18th-20th somite stage) because these are absent from the caudal
neuraxis.
Wnt signaling in initial NC specification versus subsequent delamination
Expression of Slug, FoxD3 and Sox9 is coincident with the
induction of NC and is upregulated de novo by upstream signals that induce NC,
including secreted molecules of the BMP and Wnt families that derive from the
ectoderm (Garcia-Castro et al.,
2002) (reviewed by Gammill and
Bronner-Fraser, 2003
). Here, we show that inhibiting canonical Wnt
signaling in the neural tube at later stages resulted in decreased G1/S
transition and impaired NC delamination but had no effect on transcription of
genes involved in earlier NC specification, such as Sox9, Foxd3 or
Slug. These results suggest that the latter gene products are not
involved in NC delamination mediated by Wnt. This notion is supported by
results of overexpressing FoxD3 and Sox9, which led to the
formation of ectopic NC but not to their delamination (Chung and Briscoe,
2003; Kos et al., 2001
) (but
see Dottori et al., 2001
). We
note that our experiments temporally and spatially separate between these
events because by the time of electroporation, the above transcripts are
already apparent at segmental plate levels of the tube and their subsequent
expression is independent of dorsal tube-derived Wnt activity. Likewise,
maintenance of Slug mRNA/protein was reported to be independent of BMP
(Sela-Donenfeld and Kalcheim,
1999
). In further support of this conclusion, a lack of
correspondence can be observed at post-specification stages between
Wnt1 expression along the tube and that of the early genes. For
example, Wnt1 is still absent from the caudal tube, which already
expresses highest levels of Slug, FoxD3 and Sox9.
Conversely, at more rostral levels, these genes are downregulated yet
BMP/Wnt genes remain expressed and NC delamination is still under
way.
Canonical Wnt signaling and EMT
Treatment of neural primordia with Xdd1 which blocks both canonical and
non-canonical branches efficiently inhibited both BrdU incorporation and NC
delamination. This was mimicked by treatment with dNLEF1 and
ß-catenin-engrailed, which specifically abrogate the canonical pathway.
By contrast, dishevelled mutants selectively interfering with non-canonical
Wnt signaling (DEP+ or D2) had no effect. Altogether, these data demonstrate
that the canonical pathway of Wnt signaling, which involves ß-catenin,
LEF/TCF and transcriptional activation of genes acting on G1/S transition and
execution of EMT, regulates the generation of NC cell migration. However, in
mice, conditional ablation of ß-catenin in Wnt1-expressing cells had no
apparent effect on either cell proliferation or NC emigration
(Hari et al., 2002), in
contrast to our results and to data from previous studies
(Ikeya et al., 1997
;
Megason and McMahon, 2002
).
Assuming that in mice, as in avian embryos, NC emigration begins immediately
following the onset of Wnt1 expression in the dorsal tube, it is
possible that ß-catenin protein persisted after the gene had been ablated
and its residual activity still accounted for the observed emigration. To
ensure early gene downregulation, our electroporations were performed at
segmental plate levels of the axis,
10 hours prior to initial Wnt1
expression and onset of NC delamination.
In line with our findings, de Melker et al.
(Melker et al., 2004) have
reported that ß-catenin and Lef-1 proteins translocate to the nucleus of
NC cells precisely during their delamination from explanted neural primordia.
This nuclear localization is only transient, as it was found to disappear from
cells undergoing advanced migration onto fibronectin substrates. These results
are indicative of a signaling activity of the canonical Wnt pathway that is
restricted to the delamination phase. As NC cells become synchronized to the S
phase of the cell cycle during delamination, but lose synchrony during
subsequent migration (Burstyn-Cohen and
Kalcheim, 2002
) and, moreover, upregulate cyclin D1 in the dorsal
tube during this process (this paper), it follows that under normal
conditions, the nuclear localization of ß-catenin and Lef-1 proteins
observed by de Melker et al. is positively associated with both G1/S
transition and NC delamination. This observation is consistent both with our
gain- and loss-of-function results. Surprisingly, this transient nuclear
translocation of ß-catenin and Lef-1 proteins reported by de Melker et
al. (de Melker et al., 2004
) to
occur during NC delamination contrasts with the authors own experimental data
showing that a pharmacological excess of Wnt1, achieved either by LiCl
treatment or by co-culturing neural primordia with the Wnt1-producing 2.69.23
cell line, inhibited both BrdU incorporation as well as NC emigration, perhaps
by reducing the ability of the cell to adhere to the culture substrate. These
data are unlikely to be of physiological significance because in the embryo,
as well as in explants, endogenous levels of Wnt activity in the dorsal tube
are compatible with NC proliferation and delamination. These data also differ
from our gain-of-function results (electroporation of full-length Wnt1 or
ß-catenin), which revealed a stimulation in cell delamination associated
with BrdU incorporation (this paper), as well as from those of others (e.g.
Megason and McMahon, 2002
;
Nishihara et al., 2003
), which
showed enhanced cyclin D1 expression followed by a stimulation of cell
proliferation upon Wnt overexpression. Although the exact reason for this
discrepancy remains unclear, it is possible that the paradigms used in de
Melker's study led to a particularly robust overexpression of the protein, up
to levels not attained upon Wnt1 electroporation, or, in the case of LiCl
treatment, to the activation of additional non-specific processes. These, in
turn, might have resulted in an adverse phenotype because they triggered
inhibitory feedback mechanisms or receptors (e.g.
Golan et al., 2004
). Even
under these conditions, the association between G1/S transition and NC
delamination that we have previously found
(Burstyn-Cohen and Kalcheim,
2002
) was maintained. Altogether, it is possible that the dose
and/or mode of presentation of Wnt proteins required for these processes have
to be tightly regulated.
The notion we put forward in this study that the ß-catenin/LEF
transcription factor complex is associated with EMT is supported by results of
several in vitro studies (Novak et al.,
1998; Eger et al.,
2000
). In addition, IGFII induced rapid ß-catenin
translocation to the nucleus of cultured cells during EMT as well as
transcription of target genes (Morali et
al., 2001
). Furthermore, activation of the ß-catenin pathway
directly induced EMT in normal corneal epithelium and DLD1 colon carcinoma
cells (Kim et al., 2002
).
Hepatocyte growth factor and epidermal growth factor induced ß-catenin
signaling, whereas stimulating cell motility and ectopic expression of various
forms of ß-catenin mimicked the process
(Muller et al., 2002
).
In addition to its role in canonical Wnt signaling, ß-catenin is also
a member of adherens junctions, where it links cadherins to the cytoskeleton,
thereby controlling intercellular interactions that characterize epithelial
adhesions (Ozawa et al., 1989;
Savagner, 2001
). We observed
that treatment with DEP+ and D2 perturbed epithelial integrity of the
transfected hemi-tubes, as monitored by the loss of the pseudostratified
structure, disorganized membrane n-cadherin and ß-catenin immunostaining
(T.B.-C., J.S., D.S.-D. and C.K., unpublished), but had no significant effects
on gene transcription, G1/S transition or NC delamination. Hence, the
non-canonical pathway mediated by specific domains of dishevelled (reviewed by
Veeman et al., 2003
) plays a
role in stabilizing the neuroepithelium. This pathway has also been implicated
in convergent extension, a type of cell movement that involves coordinated
migration of cohesive cell sheets (reviewed by
Locascio and Nieto, 2001
).
Instead, EMT of NC cells reflects the behavior of individual progenitors,
further suggesting that these two types of cell movement are differentially
regulated. Although results stemming mainly from in vitro studies suggest that
Wnt-dependent ß-catenin signaling and cadherin-ß-catenin-mediated
cell adhesion are possibly interrelated pathways (reviewed by
Nelson and Nusse, 2004
), our
results, performed in a physiological context, suggest that emigration of NC
cells is feasible as long as the ß-catenin transcriptional activation
pathway remains intact.
A model for NC delamination
The mechanisms underlying EMT differ among cell types and developmental
contexts, emphasizing the complexity of the pathways. Our studies, which are
focused on EMT of trunk-level NC cells stress the importance of two basic
signaling systems, BMP and Wnt, which co-exist in the dorsal neural tube
following initial NC specification. Previous results have shown that BMP4 is
required both for de novo transcription as well as for later maintenance of
RhoB (Liu and Jessell,
1998; Sela-Donenfeld and
Kalcheim, 1999
). In vitro inhibition also suggested that RhoB
activity is required for NC delamination
(Liu and Jessell, 1998
);
however, in vivo evidence is still missing. If found to be involved in NC
emigration in the embryo, our finding that transcription of RhoB is
under control of BMP but independent of canonical Wnt signaling
(Fig. 9) would suggest either
that RhoB activity is upstream of Wnt, that RhoB acts via a parallel pathway
independent of Wnt and/or that Wnt and RhoB pathways, though genetically
separate, interact at various levels, as shown for other systems (e.g.
Gumbiner, 2000
;
Roovers et al., 2003
).
|
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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