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First published online 29 March 2006
doi: 10.1242/dev.02343
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1 Department of Genetics, Stanford University Medical School, Stanford, CA
94062, USA.
2 Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel
Institute of Technology, Haifa 31096, Israel.
* Author for correspondence (e-mail: jbaker{at}stanford.edu)
Accepted 27 February 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Convergent extension, NF-AT, Xenopus, Neural CE
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In
Xenopus, the dorsal mesoderm and posterior neural ectoderm undergo a
specialized type of cellular movement called convergent extension (CE),
whereby a field of cells narrows (converges) mediolaterally and then lengthens
(extends) anteroposteriorly (Keller,
2002; Wallingford et al.,
2002). These movements are the driving force that elongates the
anteroposterior axis of the embryo and closes the neural tube
(Keller and Danilchik, 1988;
Keller et al., 1992b).
Two types of non-canonical Wnt pathways have been described that mediate
both posterior neural ectodermal and dorsal mesodermal CE; the planar cell
polarity (PCP) pathway (Keller,
2002; McEwen and Peifer,
2000; Peifer and McEwen,
2002) and the Wnt/Ca2+ pathway
(Kuhl et al., 2000). These
pathways, under control of the secreted ligands Wnt11, Wnt5a and Wnt4, do not
activate ß-catenin signaling (Moon et
al., 1993; Torres et al.,
1996), but rather either function to release calcium
(Wnt/Ca2+ pathway) (Slusarski
et al., 1997a; Slusarski et
al., 1997b) or regulate PCP pathway members like strabismus,
prickle, JNK, Daam1 or dishevelled
(Wallingford et al., 2002).
Although the PCP and Wnt/Ca2+ pathways instruct morphogenesis in
both the neural ectoderm and dorsal mesoderm, the behavior and morphology of
cells undergoing CE is different within these cellular populations. It remains
unknown how these two pathways are integrated into coordinating distinct
cellular movements, although as these pathways share molecules and functions,
it is likely that there is extensive crosstalk between the various players as
well as possible subtle modifications of downstream targets
(Elul and Keller, 2000;
Elul et al., 1997;
Ezin et al., 2003;
Shih and Keller, 1992a;
Shih and Keller, 1992b).
The integration of pathways, like the Wnt/Ca2+ or PCP, to
coordinate cellular movements will most certainly involve the modification and
activation of different and diverse downstream transcriptional targets. The
nuclear effectors responsible for these events remain to be elucidated, but it
is likely that such effectors will need to integrate information from multiple
signaling events. NFAT, a well known transcriptional activator and modulator
of Ca2+, is important in several specialized morphological
movements (de la Pompa et al.,
1998; Graef et al.,
2001; Horsley et al.,
2001), including neuronal migration
(Graef et al., 2003). All
NF-AT proteins are characterized by calcineurin and rel-homology binding
domains (Rao et al., 1997).
Ca2+ activates the phosphatase, calcineurin, causing the
dephosphorylation of serines within the N terminus of the NF-AT protein, which
serves to transport NF-AT into the nucleus
(Beals et al., 1997;
Crabtree and Olson, 2002). In
the nucleus, NF-AT requires additional partners for DNA binding and
transcriptional activation (Hogan et al.,
2003; Im and Rao,
2004). This mechanism of action allows NF-AT to receive input from
multiple signaling pathways, including the Ca2+ and PCP pathway
(Chen et al., 1998;
Macian et al., 2001;
Yamanaka et al., 2002). This
crosstalk with known players of CE suggests that NF-AT could play a key role
in the coordination of cellular movements during early development.
In this paper, we provide evidence that a primary role of NF-AT in early
Xenopus development is to mediate CE in neural ectoderm. These
results are based on XNF-ATc3 expression and activity within the
Xenopus embryo, and an extensive analysis aimed at isolating the role
NF-AT plays in CE in both the neural ectoderm and dorsal mesoderm. Recent
published data support a role for NF-AT in CE movements in Xenopus
dorsal mesoderm and as a negative regulator of canonical Wnt signaling
(Saneyoshi et al., 2002).
Here, we add to this work and suggest that although XNF-ATc3 affects dorsal
mesodermal CE movements, it is also necessary for these movements within the
neural ectoderm. This is the first evidence showing that NF-AT signaling has a
role in neural CE movements and adds to a short list of molecules involved in
this process.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The plasmids CA XNF-AT, DN XNF-AT and WT XNF-AT used for
RNA synthesis are a kind gift of Dr K. Mikoshiba
(Saneyoshi et al., 2002). To
represent the identity of the constructs better, we refer to them as CA
XNF-ATc3, DN XNF-ATc3 and WT XNF-ATc3 in the text. For cyclosporin A (CsA)
treatment, embryos were incubated in 4 mM or 400 µM CsA (Bedford
Laboratories) at time points indicated in the text.
Cell adhesion assay
For cell adhesion assays stage 12.5-13 embryos were transferred into
Ca2+/Mg2+-free medium CMFM
(Sive et al., 2000) and neural
plates were removed. After removal of the mesoderm, the neural tissue was
transferred to a new dish containing CMFM and cells were manually dissociated
using an eyebrow knife. If cells did not dissociate they were further agitated
for 30 minutes. For re-aggregation single cells were transferred to 1/3
NMR.
ß-Galactosidase staining and whole mount in situ hybridization
ß-Galactosidase staining and whole-mount in situ hybridization were
performed as previously described (Borchers
et al., 2002; Harland,
1991). Antisense probes were generated using the following
plasmids: p33 En2 for engrailed (Brivanlou
and Harland, 1989), pG1s for HoxB9
(Sharpe et al., 1987), XKrox20
for Krox20 (Bradley et al.,
1993), psp6nucßgal for lacZ
(Smith and Harland, 1991),
pNPG152 for Nrp1 (Knecht et al.,
1995), XPax3 for Pax3 (Bang et
al., 1999), XSix1-pBSII for Six1
(Pandur and Moody, 2000),
pCS2-Sox2 for Sox2 (Mizuseki et al.,
1998), and pXAG-1 for XAG-1
(Sive et al., 1989). For the
XNF-ATc3 in situ hybridization, we generated two XNF-ATc3 probes by RT-PCR
from a Clontech Xenopus MATCHMAKER cDNA library. Probes were designed
against regions of the XNF-ATc3 sequence that are most divergent from
NF-ATc1-c4. The first probe targets a fragment that includes the N-terminal
transcription activation domain (TAD) and the calcineurin docking side (CDS).
This fragment was cloned by using the following primers:
5'-CATTGCGGTTGGGAAGATTTG-3' and
5'-CAAGATGAAGGCAGAGATGGTCC-3'. The second probe targets a fragment
that contains the CDS, two SP boxes and part of the first nuclear localization
signal. Primers that were used to clone this fragment are:
5'-CAGTTCAACCCATTCTTCCTGC-3' and
5'-TGCCGTCTTTTCCCACAAGG-3'. The resulting PCR products were cloned
into pCRII-TOPO (Invitrogen). Antisense probes generated from both plasmids
produced identical results in whole-mount in situ hybridization.
Analysis of CE in the mesoderm
Ectodermal explants were prepared as described by Wallingford and Harland
(Wallingford and Harland,
2001). To induce mesodermal CE, ectodermal explants were incubated
in 35 ng/ml of human recombinant Activin A (R&D Systems). For cyclosporin
A (CsA) treatment, CsA (Bedford Laboratories) was added at concentrations as
stated in Fig. 5. FK506
(Prograf) was used at concentration of 100 ng/ml. Ectodermal explants were
cultured in individual agarose wells until control embryos reached stage 20.
Mesodermal CE was visible as elongation of the ectoderm.
For Keller explants, embryos were injected dorsally and animally at the
four-cell stage using GFP as a lineage tracer. From fluorescent embryos,
Keller open face explants (Shih and
Keller, 1992b) were prepared at stage 10.5 and cultured in
Steinberg solution [60 mM NaCl, 0.67 mM KCl, 0.34 mM
Ca(NO3)2, 0.83 mM MgSO4, 1 mM HEPES pH 7.4].
CE was assessed when unmanipulated sibling control embryos had reached stage
16.
Analysis of CE in neural tissue
To analyze NF-AT activity in neuralized tissue, animal caps were neuralized
in two different ways. Either XBF-2 RNA
(Mariani and Harland, 1998) or
dominant-negative BMP receptor RNA (Graff
et al., 1994) were injected in the animal pole of one-cell stage
embryos. Animal caps were cut at the blastula stage and cultured to stage
16/17 when CE was assessed and marker expression was analyzed by RT-PCR.
RT-PCR primers for EF1a, HoxB9, Krox20 and NCAM have been
described elsewhere (Hemmati-Brivanlou and
Melton, 1994). The primers for HoxD1 have been published
by Kolm and Sive (Kolm and Sive,
1995), the primers for XE10 (EphA2 receptor) by Weinstein et al.
(Weinstein et al., 1996).
To assess neural CE in the neural plate, embryos were cultured until stage
12-12.5. Embryos were devitellinized and neural plate, including the
underlying mesoderm and endodermal epithelium, was removed. Neural plate
explants were cultured in Steinberg solution in petri dishes, which had been
blocked with 1% BSA (Fisher Scientific). The explants were immobilized using
glass coverslides (5x5 mm) with silicon grease-coated edges. To analyze
how CsA affects neural CE, explants were incubated in 400 nM CsA in Steinberg
solution. As CsA is dissolved in 33.2% ethanol, additional control explants
were incubated in 0.33% ethanol in Steinberg solution to test for toxicity or
other effects. Immediately after immobilizing the explants, images were taken
at 1-hour intervals using a Leica MZFLIII microscope and Leica DC500 camera
(Leica Fire Cam 1.2.0 software for Macintosh). Explants were monitored over
4-7 hours, until a closed neural tube was visible in control explants. The
area and lengths of the transplants was measured using the public domain
ImageJ program (developed at the US National Institute of Health and available
at
http://rsb.info.nih.gov/ij/).
The change in length (
L) was calculated as L=Lt:
Lo, whereby Lo is the length at the start of the
experiment and Lt is the length at the different time points (t=1
to 7). The change in area (A) and the change in neural tube length
(NT) were calculated in a similar way. The change in width (W)
was calculated by W=A: L. The control versus CsA
experiment was repeated three times with a total of 18 explants. The control
versus DN XNF-ATc3 experiment was performed twice with a total of 18 explants.
In two of our experiments, we analyzed a total of six explants for
ethanol-induced effects. These data were not combined, as individual
experiments continued over different time spans. However, the results of the
different experiments were identical and one representative experiment is
presented. Stars in the graphs indicate time points in which the CsA-treated
or DN XNF-ATc3 explants are significantly different in L or W
from the control and ethanol-treated explants in an unpaired Student's
t-test.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Hogan et al., 2003).
Therefore, we tested whether NF-AT signaling plays a role in CE movements
during Xenopus neural development. Our analysis of Xenopus
genomic and EST databases identified four Xenopus NFAT genes
(XNF-ATc1-4), each of which is orthologous to the four mouse NF-AT genes
(NF-ATc1-4; data not shown). The previously published Xenopus NF-AT
molecule (XNF-AT) is most closely related to mouse NF-ATc3
(Saneyoshi et al., 2002). For
clarity we will refer to XNF-AT as XNF-ATc3 (kind gift from K. Mikoshiba)
throughout this paper.
XNF-ATc3 has recently been shown to be involved in dorsoventral patterning
of the Xenopus embryo by inhibiting canonical Wnt signaling. Ventral
injection of a dominant-negative XNF-ATc3 (DN XNF-ATc3) induced the formation
of secondary axis or dorsalized embryos in over 80% of embryos
(Saneyoshi et al., 2002). We
saw a much smaller induction of dorsal fates and instead observed a severe
failure in neural tube formation. In our experiments, ventral injection of 1
ng DN XNF-ATc3 (kind gift of K. Mikoshiba) resulted in 2.3% partial axes and
3.4% double axes (0% axes in the control, n=194). Higher
concentrations of 2 ng or 4 ng DN XNF-ATc3 gave 27.6% partial and 5.2% double
axes, and 28.2% and 5.1% double axes, respectively (2.7% double axes in the
control, n=171).
In order to determine the role of XNF-AT during Xenopus
neurulation in the absence of any potential effects on earlier dorsoventral
patterning, we blocked XNF-ATc3 activity in a spatial and temporal manner.
Xenopus expresses three NF-AT paralogs (c1, c2 and c3) during
gastrulation (see Fig. S1 in the supplementary material). These molecules are
functionally redundant in mouse (Crabtree
and Olson, 2002), and therefore knockdown approaches using
morpholinos are not suitable in this instance. Therefore, to inhibit NF-AT
signalling, we used DN XNF-ATc3 or cyclosporin A (CsA), a calcineurin
inhibitor (Lin et al., 1991;
Liu et al., 1991) known to
effectively and specifically block NF-AT signaling
(Graef et al., 2001;
Graef et al., 2003). First, we
targeted injections of DN XNF-ATc3 specifically into the prospective
ectoderm. Second, we exposed embryos to CsA at gastrula stages, long after the
dorsoventral axis has been specified. These spatial and temporal approaches
bypass the effects of XNF-AT on dorsoventral patterning and mesodermal
specification, allowing the study of XNF-AT signaling only in the neural
ectoderm. DN XNF-ATc3 expressing or CsA-treated embryos were cultured until
neurula stage (19-21) when they were fixed and analyzed by whole-mount in situ
hybridization. To ensure that CsA treatment was effective, control embryos
were also cultured in CsA and analyzed for heart and gut defects
characteristic for CsA treatment (Yoshida
et al., 2004). Independent of the method used, embryos showed
wider neural tubes than the controls. This was seen along the whole
anteroposterior neural axis as markers were expanded, but not misplaced
(Fig. 1). Dorsoventral
patterning was not affected and we observed normal ventral marker patterning
(globin) in DN XNF-ATc3-injected embryos (data not shown).
This indicates that although neural tube morphogenesis was seriously affected,
cell fate decisions have remained intact. Furthermore, embryos injected with
DN XNF-ATc3 had few gastrulation defects when compared with controls
(4.7% n=85 for DN XNF-ATc3; 2.3% n=127 for controls).
To confirm that the Xenopus CsA-phenotype we observe is specific
for an inhibition of endogenous NF-AT signaling, we performed rescue
experiments using wild-type XNF-ATc3 (WT XNF-ATc3, gift of K. Mikoshiba). CsA
is known to inhibit NF-AT signaling specifically in vitro and during mouse
development (Graef et al.,
2001; Graef et al.,
2003). Indeed, injection of 2 ng WT XNF-ATc3 could rescue
the CsA-induced neural tube closure defect
(Fig. 1B).
CE defects mediated by cell adhesion changes cause the embryonic neural tube closure defects
Proper neural tube closure is dependent on a variety of morphogenetic
movements, including neural fold formation and CE movements
(Schoenwolf and Smith, 1990).
To distinguish whether inhibition of XNF-ATc3 is involved in either neural
fold formation or CE movements, we analyzed whether neural fold hinge points
exists in embryos either expressing DN XNF-ATc3 or treated with CsA. This was
achieved by staining effected embryos with phalloidin, which detects f-actin
within the hinge points of the neural folds. Our analysis showed that hinge
point and neural fold formation were not affected by inhibition of NF-AT
signaling (Fig. 2). However,
the neural plate area was wider in these embryos and neural tube closure was
delayed, strongly indicating a defect in neural CE. Therefore, we will refer
to this phenotype as neural CE defect throughout the text.
As CE is known to be mediated by the Wnt/Ca2+ pathway, and as NF-AT is a crucial regulator of Ca2+ signals, we hypothesized that DN XNF-ATc3 might inhibit CE by changing the adhesive properties of the neural ectodermal cells. To this end, neural ectoderm injected with DN XNF-ATc3 was dissected at stage 12.5-13 and dissociated in Ca2+/Mg2+-free medium (CMFM). Neural tissue expressing DN XNF-ATc3 immediately dissociated into single cells (Fig. 2I), while the control neural cells needed further agitation to dissociate (Fig. 2H). To induce re-aggregation, single cells were transferred to a Ca2+-containing medium. The control cells aggregated well (Fig. 2K), while only some DN XNF-ATc3-expressing neural cells showed aggregation (Fig. 2L). This data supports the hypothesis that the convergent extension defect is a consequence of a change in cell adhesion and points towards an involvement in cell adhesion processes regulated by the Wnt/Ca2+ pathway.
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; Sasai et al.,
2004; Wallingford and Harland,
2001; Wallingford et al.,
2000). Therefore, we tested whether overexpression of XNF-ATc3
also affects neural CE. We employed a calcineurin-independent active mutant of
XNF-ATc3 (CA XNF-ATc3) to address this issue. To avoid effects on mesoderm,
CA XNF-ATc3 was targeted by injection into the prospective ectoderm
of one-cell stage Xenopus embryos. Activation of XNF-ATc3 signaling
resulted in neural CE defects. Although CA XNF-ATc3 leads to severe neural CE
defects, the embryos remained normal throughout gastrulation, with the
exception that at the highest concentrations (500 pg RNA) there was a slight
delay in closure of the blastopore lip. This is seen in
Fig. 3A, which shows embryos
expressing different concentrations of CA XNF-ATc3 during gastrula and neurula
stages. To analyze the effect of NF-AT activation on neural marker expression, we injected very low doses of CA XNF-ATc3 (50 pg RNA) to avoid severe neural CE defects. The embryos were analyzed by in situ hybridization with probes specific for different neural regions. The expression of the cement gland marker XAG-1, the forebrain marker otx2 (data not shown), the midbrain-hindbrain boundary marker engrailed, and the hindbrain and neural crest marker, Krox20, were reduced (Fig. 3B). The expression of the neural tube markers Pax3, Sox2 and HoxB9 was wider, indicating mild neural CE defects even at this low concentration. At higher doses of CA XNF-ATc3 we see the same severe neural CE defects as seen with the DN XNF-ATc3 (data not shown). These observations suggest that activation of XNF-ATc3 causes defects in anteroposterior patterning and in neural CE.
Tissue-autonomous requirement of XNF-AT for neural CE
The temporal and spatial inhibition experiments performed above strongly
indicate that XNF-ATc3 does affect the morphology of the neural ectoderm. As
we only found gastrulation defects when using higher concentrations of CA
XNF-ATc3, we analyzed to what degree XNF-ATc3 influences dorsal mesodermal
movements. We activated and inhibited XNF-ATc3 signals specifically in either
the neural ectoderm or the dorsal mesoderm by targeting CA XNF-ATc3
or DN XNF-ATc3 with GFP specifically either into the
presumptive neural ectoderm or the dorsal mesoderm (see cartoon in
Fig. 4). At gastrula stages, we
analyzed the GFP fluorescence and confirmed that the constructs were targeted
correctly (Fig. 4A). When
overexpressed in the neural ectoderm, both CA XNF-ATc3 and DN
XNF-ATc3 resulted in neural CE defects at concentrations as low as 50 pg
and 250 pg, respectively. Overexpression at the same concentrations in the
dorsal mesoderm did not affect either neural CE or dorsal mesodermal movements
(Fig. 4B). However, expression
of higher concentrations of CA XNF-ATc3 in the dorsal mesoderm led to delays
in blastopore lip closure (100 pg CA XNF-ATc3 shows 9% delay, 500 pg
CA XNF-ATc3 results in 36% delayed embryos).
Inhibition of XNF-AT signaling does not inhibit mesodermal CE
We observed that activation, but not inhibition, of XNF-ATc3 within the
dorsal mesoderm can alter gastrulation movements. To confirm this finding, we
tested whether XNF-ATc3 was necessary and/or sufficient to affect dorsal
mesodermal movements in a rigorously defined explant system. Treatment of
ectodermal explants with activin leads to the formation of dorsal mesoderm,
which elongates as a result of CE (Fig.
5A,B). Inhibition of this elongation, without a change in cell
fate, has been the gold standard by which to judge whether a protein can alter
CE movements in the mesoderm. Therefore, we repeated the activin-treated
ectoderm assay to ascertain whether our observations with XNF-ATc3 in the
whole embryo were consistent with those of the explant system. CA
XNF-ATc3 and DN XNF-ATc3 were injected into the prospective
ectoderm of one-cell Xenopus embryos. Ectoderm was explanted at stage
9 and cultured in the presence or absence of activin. As in the whole embryos,
we did observe inhibition of elongation in activin-treated explants expressing
active CA XNF-ATc3 (Fig. 5C,D).
This activity is consistent with that previously described
(Saneyoshi et al., 2002).
Inhibiting XNF-ATc3, either with DN XNF-ATc3 or CsA, never inhibited
activin-induced elongation of explants
(Fig. 5E,F), even at high
concentrations and in combination with the calcineurin inhibitor FK506
(Flanagan et al., 1991;
Liu et al., 1991) (data not
shown).
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NF-AT activity affects CE in neuralized explants
In whole embryos, NF-AT is necessary for CE in the neural ectoderm. As
NF-AT appears to also play a role in elongation of dorsal mesoderm, we sought
to further tease apart neural and mesodermal morphogenesis by examining NF-AT
signaling in neural explants. To this end, we expressed active or
dominant-negative XNF-ATc3 in explants composed of different types of neural
ectoderm, which are void of mesoderm.
To determine whether XNF-ATc3 can affect the CE of posterior neural tissue,
we overexpressed the transcription factor XBF-2 in ectodermal explants. XBF-2
induces general neural, hindbrain and spinal cord cell fates without inducing
mesoderm. These explants elongate as a result of CE in the posterior neural
ectoderm (Mariani and Harland,
1998; Wallingford and Harland,
2001). As expected, two-thirds of the XBF-2 expressing animal caps
elongated in our experiments, while the uninjected controls did not
(Fig. 6A). Repressors of neural
CE, like Xdsh-D2, have been shown to inhibit elongation of XBF-2 expressing
explants (Wallingford and Harland,
2001). Indeed, co-expression of CA XNF-ATc3 blocks XBF-2 induced
CE. RT-PCR analysis on these explants confirmed that the effects are not the
result of a dramatic cell fate change (Fig.
6B). Surprisingly, even though inhibition of XNF-ATc3 results in
neural tube closure defects in the whole embryo, explants co-expressing DN
XNF-ATc3 and XBF-2 elongate at least as well as those expressing only XBF-2
(Fig. 6A).
Furthermore, we analyzed the effect of activating and inhibiting XNF-ATc3
only within anterior neural tissue. To this end, we generated anterior neural
tissue by expressing the dominant-negative BMP receptor (BMP DNR)
(Graff et al., 1994) in
ectodermal explants. As these explants do not contain posterior neural
ectoderm, they do not undergo CE. Co-expression of CA XNF-ATc3 and BMP DNR did
not lead to elongation of explants. However, if DN XNF-ATc3 was co-expressed
with BMP DNR these explants showed some elongation
(Fig. 6C). Analysis of neural
marker expression by RT-PCR showed an increase in HoxB9 expression compared to
explants expressing only BMP DNR or co-expressing both BMP DNR and CA XNF-ATc3
(Fig. 6D). This indicates an
increase in posterior marker expression in the DN XNF-ATc3 expressing explants
and thus may explain their ability to elongate.
Inhibition of XNF-AT signaling blocks neural CE
Our data shows that CA XNF-ATc3 and DN XNF-ATc3 affect neural CE in
opposite ways. Activation of XNF-ATc3 always inhibits CE, whether dorsal
mesoderm or neural ectoderm, in vivo or in vitro. Inhibition of XNF-ATc3 is a
much more complex story and its effects on neural CE are contradictory. In
vivo, DN XNF-ATc3 blocks neural CE. In vitro, it stimulates extension of
neuralized explants. We hypothesized that this difference was the result of
neighboring non-neural cells within the whole embryo that, when present, can
change the competence of the neural tube to respond to inhibition of NF-AT
signaling. Therefore, we analyzed how inhibition of NF-AT signaling shapes CE
in explants of the neural plate that contain the underlying mesoderm
(Elul and Keller, 2000). We
injected one-cell stage embryos with DN XNF-ATc3 and GFP
RNA, and removed the GFP-fluorescent neural plates at late gastrula stages
(Fig. 7A). These DN XNF-ATc3
expressing neural plates and control neural plates were cultured in vitro
until complete neural tube closure was visible in control explants. Images
were taken in hourly intervals and CE was evaluated by the change in length
(L) and the change in width (W) over the course of the
experiment. The length of control explants increased faster than the length of
the DN XNF-ATc3 injected explants. Additionally, DN XNF-ATc3
expressing neural plates remained wider than the controls. We also noted that
the DN XNF-ATc3 explants developed a curled shape and their neural tubes were
significantly shorter than the ones in control explants. The curling may
result from an unequal expression of DN XNF-ATc3. To circumvent this problem,
we removed the neural plates of late gastrula stage embryos and treated them
with CsA (Fig. 7B) or ethanol
as a vehicle control. As the DN XNF-ATc3 explants, the CsA-treated neural
plates showed an inhibition in CE compared with the control and
ethanol-treated explants. In contrast to the DN XNF-ATc3-overexpressing
explants, the CsA-treated neural plates did not curl up and the whole explant
appears evenly shorter and wider than the controls. To verify that inhibition
of NF-AT signaling is not the result of major cell fate changes, we also
analyzed the CsA-treated explants by in situ hybridization using the cement
gland marker XAG-1, the rhombomere r3 and r5 marker Krox20, and the spinal
cord marker HoxB9. Although XAG-1 expression varied depending on the amount of
anterior tissue that was included in the explants, the expression pattern of
the CsA-treated explants was identical to the controls
(Fig. 7C).
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; Im and Rao,
2004). To determine whether XNF-AT signaling is active within the
neural tube, we expressed a reporter plasmid containing a minimal IL-2
promoter followed by green fluorescent protein (GFP) in Xenopus
embryos [kind gift of G. Crabtree (Graef
et al., 1999)]. The reporter is correctly activated by NF-AT and
repressed by CsA in Xenopus embryos (see Fig. S3 in the supplementary
material). As NF-AT binding sites control the expression of GFP, we were able
to visualize NF-AT-induced transcription in regions of the embryo that
contained any active, dephosphorylated NFAT, along with the relevant
co-activator. This reporter assay provides a read-out for all NF-AT activity,
including XNF-ATc3 and other Xenopus NF-AT genes. To ensure
that we obtained a read-out for all embryonic regions, we targeted the
reporter plasmid to animal, medial and vegetal regions of one-cell stage
embryos. The first detectable GFP fluorescence was seen at stage 12 in the
developing neural plate (see Fig. S2A, parts a-c in the supplementary
material). During neurula stages (15-21), fluorescence increased significantly
with the most prominent staining present in the anterior neural regions and in
the neural tube (see Fig. S2A, parts d-i in the supplementary material). As
development progressed, we also observed fluorescence within the somites and
ventral regions (see Fig. S2A, parts j-n in the supplementary material).
Therefore, although NF-AT activity occurs throughout the embryo, it is clearly
active within the neural tissue at the time of neural tube closure. This
supports an endogenous function for XNF-AT signaling within the neural
ectoderm. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), leads to nuclear localization of XNF-ATc3 in ectodermal
explants (Saneyoshi et al.,
2002), resulting in neural tube closure defects in whole embryos.
Additionally, we know that disruption of Ca2+ signaling, which
activates NF-AT-dependent transcription, also blocks CE
(Wallingford et al., 2001),
suggesting that XNF-ATc3 may mediate Wnt/Ca2+ signals within the
nucleus during neural CE. Furthermore, multiple members of the PCP pathway are
known regulators of NF-AT in other contexts. NRH1, a recently identified
modulator of PCP signaling, leads to nuclear localization of XNF-ATc3
(Sasai et al., 2004).
Furthermore, JNK is known to phosphorylate c-Jun, a component of AP-1, which
is a well-studied NF-AT co-activator. Therefore, XNF-ATc3 may be a key
downstream regulator of neural CE by modifying both Wnt/Ca2+ and
PCP signals.
Several intriguing results need to be addressed concerning inhibition of
NF-AT signaling within the neural ectoderm. First, overexpression of active
NF-AT and dominant-negative NF-AT results in the same effect on neural tube
morphogenesis. From the literature on PCP signaling and its role in CE, it is
not surprising that inhibition and activation of NF-AT result in the same
phenotypic effect. Molecules involved in CE movements typically display
similar defects in morphogenesis when overexpressed or inhibited, indicating
that dose is absolutely crucial for the correct movements to ensue. This dose
effect has been observed for most of the known PCP molecules involved in
mesodermal CE, including dishevelled, frizzled 7 and NRH1
(Djiane et al., 2000;
Sasai et al., 2004;
Wallingford and Harland, 2001;
Wallingford et al., 2000).
|
) and
subsequently Ezin et al. (Ezin et al.,
2003) have analyzed the protrusive activity of the neural plate in
detail. Following gastrulation, the cells of the neural ectoderm develop an
organized monopolar character with medially directed motility that is
dependent upon the underlying mesoderm
(Elul and Keller, 2000). Upon
further dissection, this dependence was found to reside within the
notochord/notoplate region in the midline. Neural plate explants that lack the
midline fail to generate a monopolar character, having a bipolar and
mediolateral directed motility. These cells do not converge and extend as well
as explants that either contain the midline or have an ectopic midline added
(Ezin et al., 2003). These
results strongly suggest that signals from the midline instruct the overlying
neural ectoderm to obtain monopolar medially directed activities that lead to
coordinated and effective CE. Little is known on a cellular level about the
mechanisms of CE in neuralized explants. As no mesoderm is contained within
these explants, one cannot assume that the movements observed in elongating
ectoderm are similar to that of the endogenous situation. Indeed the explants
may reflect the weaker CE movements seen within neural plate explants that
lack mesoderm and contain bipolar mediolaterally directed protrusive
activities. This would be consistent with the observation that explants of
posterior neural tissue (including XBF-2 expressing explants) have typically
weak effects on elongation.
Why then do neuralized ectodermal explants converge and extend in the
presence of DN XNF-ATc3? One explanation is that NF-AT inhibits canonical Wnt
signaling. XNF-ATc3 has previously been implicated as a downstream target of
both the Wnt/Ca2+ pathway and as an inhibitor of canonical Wnt
signaling. It has been proposed that DN XNF-ATc3 could indeed activate
canonical Wnt signaling within ventral mesoderm
(Saneyoshi et al., 2002).
Although we do not observe the robust dorsalizing activity of DN XNF-ATc3
reported by Saneyoshi et al., we cannot completely discount this claim. In our
experiments, we do see a low level of dorsalization in whole embryos and can
also detect a slight induction of Xnr3 in ectoderm expressing DN XNF-ATc3
(data not shown). Thus, we believe that a mild activation of canonical Wnt
signaling does exist in response to DN XNF-ATc3. As GSK3ß is known to
interact directly with and actively phosphorylates NF-AT, this is the most
likely target of NF-AT within the canonical Wnt pathway. According to this
model, activating canonical Wnt signaling via GSK3ß within anterior
neural tissue would lead to posteriorization. This newly generated posterior
neural tissue would then converge and extend. Our data supports this
hypothesis in that we observe posteriorization and elongation of ectoderm
exposed to DN XNF-ATc3 (Saneyoshi et al.,
2002).
The question remains why this posteriorization does not occur within the whole embryo or within the neural plate explant environment. We propose two answers to this question. First, endogenous regulators, possibly secreted from the notochord, compete for the use of NF-AT as a downstream target and do not allow for an interaction with GSK3ß used for inhibition of canonical Wnt signaling. In the neuralized explants, these endogenous regulators are not present simply because mesoderm is not present and therefore XNF-ATc3 signaling may function in a distinctly different manner because of its own promiscuous nature. Second, the neuralized explants and whole embryos have been exposed to vastly different signals and certainly the whole embryo response is a superior read-out of function. Our explants were cut at blastula stage well before the complex cascades of signals sent and received during gastrulation and neurulation in Xenopus. It is simply possible that DN XNF-ATc3 is sufficient to posteriorize anterior ectoderm via the Wnt pathway within explants that have only responded to selective signals, whereas within the embryo, posterior patterning proceeds appropriately because the correct signals are present and do not co-opt other players. Certainly, the role of NF-AT is best demonstrated within these intact embryos, which receive appropriate endogenous signals and NF-AT signaling does indeed play an important role in regulating neural morphogenesis within this milieu.
Although XNF-AT can function as an inhibitor of the canonical Wnt pathway -
at least in neuralized ectodermal explants, the effects observed in whole
embryos on neural CE presented in this paper are not due to inhibition of the
canonical Wnt pathway. First, inhibition of NF-AT signaling with CsA after
dorsoventral and anteroposterior pattern has already been established, still
results in neural tube closure defects. Second, it is unlikely that Wnt
signaling is responsible for the neural tube defects generated by DN XNF-ATc3,
as they are not accompanied by posteriorization or cell fate changes within
the whole embryo. Third, the fate changes we observe after expression of CA
XNF-ATc3 are not consistent with an inhibition of canonical Wnt signaling by
NF-AT. Canonical Wnt signaling is known to posteriorize neural tissue.
Therefore, activation of NF-AT signaling, which according to the published
model (Saneyoshi et al., 2002)
inhibits canonical Wnt signaling, should anteriorize neural tissue. However,
overexpression of CA XNF-ATc3 in whole embryos decreases the expression of
both forebrain and midbrain markers, and in explants can induce ectopically
the posterior marker HoxD1. Taken together, we hypothesize that although
XNF-ATc3 can use the canonical Wnt pathway, this function cannot explain the
effects of XNF-ATc3 on neural tube morphogenesis. Indeed this specific
phenotypic effect is most surely caused by mediating PCP or
Wnt/Ca2+ pathways.
Cellular movements in neural and mesodermal CE are morphologically similar
(Keller et al., 1992a) and
mediated by conserved signaling pathways. However, there are differences in
cell behavior and cell polarity in these two events
(Elul and Keller, 2000;
Elul et al., 1997;
Ezin et al., 2003), which may
be controlled by different mechanisms and flavors of signaling targets. So
far, only mesodermal CE has been studied in depth at the molecular level and
only a few molecules have been shown to function in neural CE. This paper adds
XNF-ATc3 to the short list by demonstrating that XNF-ATc3 has a function in
neural CE. We suggest that XNF-AT plays a crucial role in the detection of
diverse signaling pathways and serves to activate downstream targets that will
determine morphogenetic movements within the neural tube. Certainly the
molecular nature of NF-AT is well suited to integrate information of multiple
signaling events, including Ca2+. The next challenge will be to
dissect these pathways and deduce how NF-AT relays the PCP and
Wnt/Ca2+ pathway to specify movements within the neural plate.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/9/1745/DC1
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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(loading
control). (C) One-cell stage embryos were injected into the animal
hemisphere with 0.2 ng of BMP DN receptor (BMP DNR), 0.2 ng of CA
XNF-ATc3- or 1 ng of DN XNF-ATc3. BMP-DNR was also co-injected with either CA
XNF-ATc3 or DN XNF-ATc3 at the same concentrations. (D) RT-PCR analysis
was performed with the markers HoxB9, HoxD1, XAG-1 and EF1
