1 Millennium Nucleus in Developmental Biology, Faculty of Science, University of
Chile, Casilla 653, Santiago, Chile
2 Department of Anatomy and Developmental Biology, University College London,
London WC1E 6BT, UK
* Author for correspondence (e-mail: rmayor{at}uchile.cl)
Accepted 22 October 2003
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SUMMARY |
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Activation of Xiro1 or Notch signaling led to an enlargement of the neural crest territory, whereas blocking their activity inhibited the expression of neural crest markers. It is known that BMPs are involved in the induction of the neural crest and, thus, we assessed whether these two elements might influence the expression of Bmp4. Activation of Xiro1 and of Notch signaling upregulated Hairy2A and inhibited Bmp4 transcription during neural crest specification. These results, in conjunction with data from rescue experiments, allow us to propose a model wherein Xiro1 lies upstream of the cascade regulating Delta1 transcription. At the early gastrula stage, the coordinated action of Xiro1, as a positive regulator, and Snail, as a repressor, restricts the expression of Delta1 at the border of the neural crest territory. At the late gastrula stage, Delta1 interacts with Notch to activate Hairy2A in the region of the neural fold. Subsequently, Hairy2A acts as a repressor of Bmp4 transcription, ensuring that levels of Bmp4 optimal for the specification of the neural plate border are attained in this region. Finally, the activity of additional signals (WNTs, FGF and retinoic acid) in this newly defined domain induces the production of neural crest cells. These data also highlight the different roles played by BMP in neural crest specification in chick and Xenopus or zebrafish embryos.
Key words: Xenopus, Iroquois, Notch signaling, BMP, Neural crest, Msx, Hairy2, Delta1, Snail
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Introduction |
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The generation of neural crest precursors is dependent on the interaction
between the neural plate and the non-neural ectoderm
(Moury and Jacobson, 1990;
Selleck and Bronner-Fraser,
1995
; Mancilla and Mayor,
1996
; Mayor et al.,
1997
). From studies in chick, amphibian and zebrafish embryos,
some of the signals involved in the induction of the neural crest have been
identified, for example, BMPs, Wnts, FGF and retinoic acid
(Liem et al., 1995
;
Selleck et al., 1998
;
Streit and Stern, 1999
;
Mayor et al., 1995
;
Mayor et al., 1997
;
LaBonne and Bronner-Fraser,
1998
; Deardorff et al.,
2001
; García-Castro et
al., 2002
; Saint-Jeannet et
al., 1997
; Villanueva et al.,
2002
). However, the molecular interactions that are involved in
these induction processes seem to be different in the chick to those in
Xenopus and zebrafish embryos.
In the chick, blocking BMP activity inhibits neural crest development, and
augmenting BMP activity, or its ectopic application, expands the neural crest
population (Liem et al., 1995;
Selleck et al., 1998
).
However, in Xenopus and zebrafish it appears that the early induction
of neural crest cells depends on a gradient of BMP activity (reviewed by
Chitnis, 1999
;
Aybar and Mayor, 2002
). As
such, neural crest cells are specified at the border between the neural plate
and the epidermis, where intermediate concentrations of BMPs are established,
i.e. where the BMP4 concentration is lower than that required to induce
epidermis formation and above that which induces neural tissue
(Morgan and Sargent, 1997
;
Marchant et al., 1998
;
Wilson et al., 1997
;
LaBonne and Bronner-Fraser,
1998
; Villanueva et al.,
2002
; Nguyen et al.,
1998
).
The molecular mechanisms that underlie the differences in the way that BMP
acts during neural crest induction in the chick and in Xenopus or
zebrafish are not understood. Thus, in order to study the role of BMP
signaling on neural crest induction in Xenopus, and to compare it
with what it is known in the chick, we have analyzed two different molecules
implicated in the control of BMP4 transcription. The Notch/Delta signaling
pathway is thought to influence neural crest development in zebrafish and
chick by controlling BMP transcription
(Endo et al., 2002;
Cornell and Eisen, 2000
;
Cornell and Eisen, 2002
).
Indeed, Notch/Delta signaling has already been shown to be involved in a wide
variety of other developmental processes, including neurogenesis, gliogenesis,
somitogenesis, compartment boundary formation and eye development (reviewed by
Artavanis-Tsakonas et al.,
1999
; Chitnis et al.,
1995
; Cho and Choi,
1998
; Domínguez and de
Celis, 1998
; Kehl et al.,
1998
; Cavodeassi et al.,
1999
; Scheer et al.,
2001
). The Iro protein has been shown to control BMP transcription
in the ectoderm and mesoderm of Xenopus embryos
(Gómez-Skarmeta et al.,
1998
; Glavic et al.,
2001
; Glavic et al.,
2002
; Gómez-Skarmeta et
al., 2001
), and has been implicated in the development of the
neural crest in zebrafish (Itho et al., 2002). The Iroquois genes participate
in several developmental processes, including sensory organ development,
compartment boundary formation in Drosophila, dorsal mesoderm
formation, neural plate induction, dorsoventral patterning of the neural tube
and midbrainhindbrain development
(Bürglin, 1997
;
Cavodeassi et al., 2001
;
Gomez-Skarmeta and Modolell,
2002
; Leyns et al.,
1996
; Gomez-Skarmeta and
Modolell, 1996
;
Papayannopoulos et al., 1998
;
Diez del Corral et al., 1999
;
Glavic et al., 2001
;
Kudoh and Dawid, 2001
;
Gomez-Skarmeta et al., 1998
;
Gomez-Skarmeta et al., 2001
;
Bellefroid et al., 1998
;
Bosse et al., 1997
;
Briscoe et al., 2000
;
Cohen et al., 2000
;
Glavic et al., 2002
;
Itoh et al., 2002
).
Through conditional Notch/Delta and iro1 gain- and loss-of-function strategies, we demonstrate that Notch/Delta signaling and the iro1 protein in Xenopus play a direct role in neural crest induction by downregulating BMP4 transcription. Furthermore, a series of rescue experiments indicate that iro1 acts upstream of Notch/Delta in the cascade of neural crest induction. We also show that iro1 positively regulates Delta1 transcription, in contrast to Snail, a gene that is specifically expressed in the neural crest and which negatively regulates Delta1. It should be mentioned that our experiments were performed using neural crest markers that are initially expressed only in the anterior neural crest. As a result, we discuss a model in which the interaction between iro1, Delta/Notch and Snail generates a pattern of gene expression in the anterior neural crest region that is required for the specification of these cells. Finally, our findings regarding the repression of BMP transcription through the activity of Notch/Delta signaling, and the ensuing induction of the neural crest, is in contrast to what has been observed in the chick, providing us with an explanation for the apparent differences between neural crest induction in chick and Xenopus embryos.
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Materials and methods |
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Plasmid constructs and in vitro RNA synthesis
Inducible DNA constructs of Xmsx1 were prepared by fusing the
entire coding region of Xmsx1 (amino acid residues 1-294) to the
ligand-binding domain of the human glucocorticoid receptor (GR; amino acid
residues 512-777). A dominant-negative DNA construct (dnXmsx1) was
prepared by fusing the homeodomain region of Xmsx1 (amino acid
residues 156-294) to the GR domain. Coding sequences were amplified by PCR,
using a high fidelity polymerase (Roche Molecular Biochemicals, Mannheim,
Germany) and the following primers:
The PCR products were purified and cloned into pGEM-T Easy vector (Promega), digested with EcoRI/SacI, and ligated with a SacI/XhoI-digested GR fragment into a pCS2+ vector digested with EcoRI/XhoI. Both fusion constructs were automatically sequenced on both strands at the junctions (BRC, Cornell University, Ithaca, NY, USA).
The Xiro1, Notch, Delta, Su(H), SnailGR and
Snail dominant-negative (SnailNGR) constructs have all been described
previously (Gomez-Skarmeta et al.,
2001; McLauglin et al., 2000;
Aybar et al., 2003
). All cDNAs
were linearized and transcribed as described by Harland and Weintraub
(Harland and Weintraub, 1985
),
using a GTP cap analog (New England Biolabs), and SP6, T3 or T7 RNA
polymerases. After DNAse treatment, RNA was extracted with phenol-chloroform
and precipitated with ethanol. GFP mRNA was used as a control for
injections. For injection, mRNA was resuspended in DEPC-water and injected
into two-cell stage embryos using 8-12 nl needles.
Microinjection of mRNAs and lineage tracing
Dejellied embryos were placed in 75% NAM containing 5% Ficoll. One
blastomere of two-cell stage embryos was injected with different amounts of
capped mRNA in a solution containing 1-3 µg/µl of lysine fixable
fluorescein dextran, as previously described
(Aybar et al., 2003)
RNA isolation and RT-PCR analysis
Total RNA was isolated from embryonic tissue by the guanidinethiocyanate
phenol-chloroform method (Chomczynski and
Sacchi, 1987), and cDNA was synthesized using AMV reverse
transcriptase (Roche Biochemicals) and an oligo(dT) primer. For PCR analysis,
the primers for H4 used were those described previously
(Aybar et al., 2003
). The
primers used to analyze Xenopus Delta1 expression amplify a 331 bp
product corresponding to the 3'UTR region:
5'-GTCCTGGAGAGCAATATGCTCCAG-3' and
5'-CCATTGTACTGTGAACACAGCATGC-3'.
PCR amplification with these primers was performed over 30 cycles and the PCR products were analyzed on 1.5% agarose gels. PCR was performed simultaneously with RNA that had not undergone reverse transcription to control for genomic DNA contamination. Quantification of PCR bands was performed using ImageJ software (NIH, USA) on 8-bit grayscale JPG files. The values were normalized to the levels of H4 from the same sample and expressed as relative intensities for comparison (sample/H4x10).
Whole-mount in situ hybridization, immunohistochemistry and Myc staining
Antisense RNA probes for Xiro1
(Gómez-Skarmeta et al.,
1998), Xslug (Mayor
et al., 1995
), Foxd3
(Sasai et al., 2001
),
Hairy2A (Wettstein et al.,
1997
), Bmp4
(Hemmati-Brivanlou and Thomsen,
1995
), Xmsx1 (Suzuki
et al., 1997
), Serrate
(Kiyota et al., 2001
) and
Notch (Coffman et al.,
1990
) were synthesized from cDNAs incorporating digoxigenin or
fluorescein (Boehringer Mannheim) tags. Embryo specimens were prepared,
hybridized and stained according to the method of Harland
(Harland, 1991
). The alkaline
phosphatase substrates used were NBT/BCIP, or BCIP alone.
Antibody staining after in situ hybridization of the embryos was performed
according to the method described by Turner and Weintraub
(Turner and Weintraub, 1994),
using a mouse anti-Myc monoclonal antibody from BabCo. The 12/101 polyclonal
antiserum from the Developmental Studies Hybridoma Bank was used to label
somites (Griffin et al.,
1987
).
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Results |
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The specific effect of Notch signaling on the neural crest
Based on the pattern of Notch expression and its ligands, we set out to
determine whether Notch signaling might be involved in the induction of the
neural crest. It has become clear that an interaction between the neural plate
and the epidermis, and signals from the paraxial mesoderm, are involved in the
induction of the neural crest (Selleck and
Bronner-Fraser, 1995; Mancilla
and Mayor, 1996
; Bonstein et
al., 1998
; Marchant et al.,
1998
; Monsoro-Burq, 2003). It has also been established that Notch
signaling is involved in the development of the neural plate and mesoderm
(Coffman et al., 1993
). Thus,
we took care not to interfere with the development of the mesoderm and the
neural plate when studying the role of Notch signaling in the induction and
development of the neural crest. It is known that the mesoderm is specified
earlier than the neural tissues, and it has been reported that the neural
plate is specified earlier than the neural crest
(Smith and Slack, 1983
;
Servetnick and Grainger, 1991
;
Mancilla and Mayor, 1996
;
Woda et al., 2003
). Therefore,
in order to specifically study neural crest development, Notch signaling was
interfered after the mesoderm and the neural plate had already been specified.
For this reason, inducible constructs that activated or inhibited Notch
signaling were used to control the timing of intervention.
We first analyzed the effect of activating Notch signaling at different
developmental times on the formation of the mesoderm, neural plate and neural
crest. Ligand activation of Notch results in the proteolytic cleavage of its
transmembrane domain and the release of the cytoplasmic region (NICD)
(Struhl and Adachi, 2000).
NICD can then translocate to the nucleus, where it interacts with the
transcriptional repressor Suppressor of Hairless (Su(H)), forming a
transcriptional activator complex
(Artavanis-Tsakonas et al.,
1999
). Here, we have used an inducible form of NICD
(NICDGR) in order to control the time of its activation. We injected
mRNA encoding NICDGR into one blastomere of a two-cell stage embryo,
and induced its expression, by exposure to dexamethasone, immediately after
the injection (stage 2), at the blastula stage (stage 6-8) or at the gastrula
stage (stage 12). The development of the mesoderm was assessed by analyzing
the expression of the somite antigen 12/101; development of the neural plate
and neural crest induction were assessed by analyzing Sox2 and
Xslug expression, respectively. As for non-inducible forms of
activated Notch (Coffman et al.,
1993
), early activation of NICDGR provoked both the
expansion of the somites and neural plate on the injected side
(Fig. 2A,C), as well as the
inhibition of the anterior neural crest
(Fig. 2E). Similar results were
obtained when NICDGR was activated prior to stage 8. By contrast,
when induced at stage 12, NICDGR had no effect on somite or neural
plate development (Fig. 2B,D),
but rather a clear expansion of the neural crest markers was observed
(Fig. 2F). These results
indicated that to study the specific effects of Notch signaling on neural
crest development, and to avoid any influence on the mesoderm or neural plate,
all the Notch signaling constructs should be activated at stage 12. Indeed,
using inducible constructs of Dlx proteins, an early effect was observed on
neural plate and neural crest development, whereas a later induction produced
alterations specific to the neural crest
(Woda et al., 2003
). Thus, in
all the following experiments inducible constructs were activated at stage
12.
|
|
Finally, to confirm that these constructs were indeed acting on the Notch
signaling pathway, we analyzed their effects on the expression of
Hairy2A, a known target gene of Notch
(Dawson et al., 1995). Each of
the constructs that augmented Notch signaling provoked an expansion of the
Hairy2A expression domain (Fig.
3Q,R). By contrast, those that inhibited Notch signaling
diminished the expression of Hairy2A
(Fig. 3S,T). Thus, we concluded
that the activation of Notch signaling enlarges the neural crest territory and
the domain of Xmsx1 expression, while inhibiting BMP4
transcription. Conversely, inhibition of Notch signaling produces exactly the
opposite effect.
The Notch target gene Hairy2A is sufficient to induce neural crest cells in Xenopus embryos
Hairy2A is a vertebrate target of Notch signaling that belongs to
the Enhancer of Split complex. This bHLH transcription factor can act
as a transcriptional repressor and has been implicated in the repression of
neuronal differentiation (Dawson et al.,
1995; Wettstein et al.,
1997
). We analyzed whether overexpression of Hairy2A also
influenced the expression of neural crest markers. Overexpression of
Hairy2A repressed N-tubulin expression, a control for the
activity of Hairy2A mRNA, at the sites where primary neurons form
(Fig. 4A). As we had previously
shown that an early activation of Notch signaling leads to an expansion of the
somites and, in turn, to an indirect effect on neural crest induction, we took
care of injecting the Hairy2A mRNA specifically into the blastomeres
fated to become ectoderm. We performed the injection of Hairy2A mRNA
into two animal blastomeres of an eight-cell stage embryo. In order to show
that there was no effect on mesodermal development, the somite antigen 12/101
was analyzed. No effect on 12/101 was observed in the injected side
(Fig. 4B). Interestingly, the
same group of embryos that exhibited normal somite development showed an
increase in Xslug expression (Fig.
4C). In addition, the expression of Bmp4 was also
decreased in these embryos, although the expression of Xmsx1
augmented (Fig. 4D-F). These
results suggest that the expansion of the neural crest population upon the
activation of Notch signaling may be a consequence of the increase in
Hairy2A expression provoked in these embryos.
|
It has been shown that Xiro1 acts as a transcriptional repressor
(Glavic et al., 2001;
Gomez-Skarmeta et al., 2001
).
However, when mRNA encoding both Xiro1 (not shown) and its inducible
repressor fusion (HDGREnR) was injected and then activated at stage
12, Xslug expression was augmented
(Fig. 5A). Conversely,
activation, at stage 12, of both the inducible dominant-negative fusion
(HDGR) and the inducible activator fusion (HDGRE1A)
inhibited Xslug expression (Fig.
5B,C). By contrast, transcription of Bmp4 at the neural
plate border was repressed in embryos injected with HDGREnR
(Fig. 5D) but increased in
embryos overexpressing HDGRE1A and HDGR
(Fig. 5E,F). It should be noted
that Bmp4 has a complex and dynamic pattern of expression in the
neural folds, and that the inhibition of Xiro1 not only affects the
levels of Bmp4 expression but also its distribution. The expression
of Xmsx1 was augmented and expanded when Xiro1 and
HDGREnR was injected into embryos
(Fig. 5G), whereas the levels
of transcripts diminished and its expression pattern was disrupted in embryos
injected with the mRNAs encoding for the activator and dominant-negative
constructs (Fig. 5H,I).
Finally, overexpression of HDGREnR de-repressed Hairy2A
expression in the neural fold (Fig.
5J), whereas injecting HDGRE1A and HDGR
decreased Hairy2A expression (Fig.
5K,L). Thus, Xiro1, in addition to being involved in the
expression of neural crest markers, also influences Bmp4 and
Hairy2A expression in the neural crest precursor domain.
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Discussion |
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Notch signaling in neural crest specification
In Xenopus embryos, the expression patterns of Notch, the
Notch ligand Delta1 and the Notch downstream gene Hairy2A
suggest that these molecules might be implicated in the formation of the
neural crest. Interestingly, in contrast to the homogenous expression
described previously (Kiyota et al.,
2001), we observed that another Notch ligand, Serrate, is
expressed in a complex pattern very similar to that of Delta1. Thus,
both ligands are expressed in cells that surround those expressing
Xslug and hence they could activate Notch and, thus, Hairy2A
in the neural folds. The restricted pattern of Hairy2A expression
overlaps that of Xslug, suggesting that other elements either repress
Hairy2A transcription in the adjacent epidermis and neural plate, or
permit the expression of this gene in the neural fold region. One of these
elements could be Notch itself.
In Xenopus, Notch is detected in neural tissue and is excluded
from the non-neural ectoderm, thereby accounting for the absence of
Hairy2A expression in the epidermis
(Coffman et al., 1990) (this
work). Our analysis of Notch signaling demonstrates that increasing Notch
activity at the early gastrula stage produces an expansion of the neural crest
territory. Interestingly, the increase in Xslug and Foxd3
expression produced by Notch activation is in contrast to the repression of
Slug upon changes in Notch activity previously described in the chick
(Endo et al., 2002
). In
addition, inhibition of Notch signaling by DeltaStu, or by
a dominant-negative form of Suppressor of Hairless, produces a
reduction in the number of Xslug- and Foxd3-positive cells.
Furthermore, direct overexpression of the Notch target gene Hairy2A
leads to the induction of neural crest cells. Thus, our results provide
evidence of a role for Notch and its downstream elements in the specification
of Xenopus neural crest.
The molecular mechanism by which Notch signaling controls the induction of
the neural crest in the chick appears to involve the upregulation of
BMP4 expression, necessary for neural crest induction
(Liem et al., 1995;
Endo et al., 2002
). However,
in Xenopus, the activity of BMP is opposite to that of the chick, and
a decrease in BMP activity relative to that seen in the non-neural ectoderm
induces neural crest cells. Therefore, the observed increase of Xslug
and Foxd3 expression is most likely due to the repression of
Bmp4 transcription. Indeed, here we show that the activation of Notch
represses Bmp4 expression in Xenopus embryos. In addition,
inhibition of Notch signaling by DeltaStu, or by a
dominant-negative form of Suppressor of Hairless, produces an
increase in Bmp4 transcription. Our analysis of the influence of
Notch signaling on the BMP pathway further showed that the precise pattern of
Xmsx1 expression, a BMP target gene, is finely regulated in the
neural crest precursor domain.
Contrary to our expectations, activation of Notch often produced an
increase in Xmsx1 expression, even though Bmp4 transcription
was inhibited. Accordingly, treatments that blocked Notch signaling, and that
therefore activated Bmp4 expression, produced embryos where
Xmsx1 expression was impaired. These results support the conclusion
that Xmsx1 expression is induced at a specific level of BMP activity
(Tríbulo et al., 2003).
We also observed that, when overexpressed in embryos, Hairy2A
produced similar effects on Xslug, Bmp4 and Xmsx1
expression, and that it is able to rescue the effect of Su(H)DBMGR in
blocking Notch signaling.
In conclusion, Notch signaling activates the expression of Hairy2A
in the region of the neural folds, and thereby represses Bmp4
transcription. This effect of Notch signaling is dependent on Xmsx1
activity, as the inhibition of Notch by Su(H)DBMGR can be reversed by
Xmsx1, and the effects produced by activating Notch can be blocked by
a dominant-negative Xmsx1 construct. Our results also provide a
possible explanation for the apparent discrepancy in the role played by BMP in
chick and Xenopus or zebrafish neural crest induction. At the time of
neural crest induction, the levels of BMP at the neural plate border are high
in both Xenopus and zebrafish, and low in the chick. If we assume
that an intermediate level is required to induce neural crest in all these
vertebrates, then an increase in BMP levels in the chick would establish
similar levels to those generated by a decrease in Xenopus and
zebrafish. Thus, because of the initial differences in the levels of BMP in
these two groups of organisms, the molecular machinery that induces neural
crest formation (e.g. Notch/Delta, Xiro1) must adjust the specific
levels of BMP by producing opposing effects on BMP expression. Thus,
Notch/Delta signaling induces the neural crest by increasing BMP expression in
the chick (Endo et al., 2002),
and decreasing it in Xenopus.
The homeoprotein gene Xiro1 in neural crest specification
Genes of the Iroquois family have been implicated in a variety of
developmental processes, including dorsal mesoderm formation, neural
induction, compartment specification in the eye imaginal disc of
Drosophila and midbrain-hindbrain boundary formation
(Glavic et al., 2001;
Kudoh and Dawid, 2001
;
Papayannopoulos et al., 1998
;
Diez del Corral et al., 1999
;
Gomez-Skarmeta et al., 1998
;
Bellefroid et al., 1998
;
Bosse et al., 1997
;
Briscoe et al., 2000
;
Glavic et al., 2002
;
Itoh et al., 2002
). Our
results extend the role of Xiro1 during development to that of neural
crest specification. Indeed, it has already been demonstrated that
Xiro1 can bind to the Bmp4 promoter, and, by acting as a
repressor, it can inhibit Bmp4 transcription in both the Spemanns'
organizer and the neural plate
(Gomez-Skarmeta et al., 2001
;
Glavic et al., 2001
).
Our observations show that Xiro1 is expressed in the neural crest territory and that its activation produces an enlargement of this territory. By contrast, inhibition of Xiro1 leads to a reduction in the expression of neural crest markers. Like Notch signaling, Xiro1 also represses Bmp4 transcription and activates Hairy2A expression in the neural folds, as well as expanding the domain of Xmsx1 expression. The effects of inhibiting Xiro1 on neural crest specification can be reversed by activating Notch signaling, or by co-injecting the Notch target gene Hairy2A. Taken together, these results indicate that Xiro1 activity is upstream of Notch signaling.
Although the regulation of Notch activity by Xiro1 could operate at different levels, we have presented evidence that Xiro1 can upregulate Delta1 transcription. Activation of Xiro1 in animal caps or whole embryos, led to an upregulation of Delta1, whereas impairing Xiro1 produced an inhibition of Delta1 expression in the neural crest territory. Thus, Xiro1 seems to positively regulate Delta1 expression. However, as the expression of Delta1 and Xiro1 do not completely overlap, additional factors must be required either to activate Delta1 where Xiro1 is not expressed, or to inhibit its expression in those cells expressing Xiro1 but not Delta1.
Delta1 is excluded from the center of the prospective neural crest
region, and its transcripts can only be seen at the border of the crest
region. This pattern of Delta1 expression suggests that a repressor
is acting in the crest region. Many transcriptional repressors are expressed
in the neural crest, including Snail
(Aybar et al., 2003),
Slug (LaBonne and Bronner-Fraser,
1999
; Mayor et al.,
2000
), Foxd3 (Sasai
et al., 2001
) and Zic5
(Nakata et al., 2000
).
Moreover, Snail appears to be upstream in this genetic cascade
(Aybar et al., 2003
). We show
here that Snail can repress Delta1 expression in animal caps
and in whole embryos, and that the inhibition of Snail activity
provokes an upregulation of Delta1 expression in the neural crest
territory. Our results strongly suggest that the expression of Delta1
in the neural crest could be patterned by the activity of Snail. It
is worth mentioning that the effect of Snail on Delta1
expression was not only seen in the ectoderm but also in the somites, where
Snail is also expressed (Essex et
al., 1993
). Thus, it seems feasible that Delta1
expression, which plays an important role in somite formation
(Jen et al., 1997
), could also
be under the control of Snail. Indeed in Drosophila, Snail
has been shown to represses Delta expression during the dorsoventral
patterning of the embryo (Cowden and
Levine, 2002
; Ip and Gridley,
2002
). It is also interesting to note that Snail is
weakly expressed in the anterior neural fold at the early gastrula stage, but
at the end of gastrulation, when Delta1 is strongly expressed in the
anterior neural fold, Snail expression is downregulated in that
region (Aybar et al., 2003
).
This complementary pattern of expression between Snail and
Delta1 also supports the idea that Snail is indeed a
repressor of Delta1 transcription. Finally, Snail may not
only serve to repress Delta1 in the neural crest, overexpression of
Snail induces the appearance of neural crest markers in animal caps
and in whole embryos (Aybar et al.,
2003
). Indeed, it is likely that the influence of Snail
on neural crest markers is independent of its repression of Delta1.
It is important to mention that Slug or Foxd3 are never
expressed in the anterior neural fold, being also putative inhibitors of
Delta1 in the crest region.
The role of the Iroquois genes in establishing embryonic boundaries seems
to be extended across this gene family. As mentioned before, Iroquois genes
participate in the development of the imaginal disc compartment in
Drosophila (Papayannopoulos et
al., 1998; Diez del Corral et
al., 1999
; Cavodeassi et al.,
1999
), and, in Xenopus, Xiro1 is involved in the
formation of the midbrain-hindbrain boundary
(Glavic et al., 2002
). It is
noteworthy that Notch signaling is also involved in both these processes
(Papayannopoulos et al., 1998
;
Domínguez and de Celis,
1998
). In Drosophila, the Iroquois genes influence Notch
signaling through the expression of Fringe, thereby defining the
dorsal and ventral compartments (Cavodeassi
et al., 1999
). In Xenopus, the Notch target genes
Hes1 and Hes3 (Hirata et
al., 2001
), and the Hes-related 1 gene (Xhr1)
(Shinga et al., 2001
), have
been implicated in establishing the midbrainhindbrain border, and in
particular in midbrain development. Recently, Xiro1 has been shown to
be involved in the establishment of this region by controlling Gbx2
and Otx2 expression (Glavic et
al., 2002
). It is thus tempting to speculate that Xiro1
might regulate Hes1, Hes3 and/or Xhr1 expression at the
midbrain-hindbrain boundary. Here, we present evidence that Xiro1 is
also involved in the establishment of the boundary between the neural plate
and the epidermis, i.e. the region in which the neural crest cells are
generated.
A molecular model for neural crest induction
The data generated over the past years, together with our present
observations, lead us to propose the following model for neural crest
induction (Fig. 8). It should
be noted that this model is predominantly based on data from the analysis of
neural crest markers that are initially expressed only in the anterior neural
crest. Therefore, additional studies using specific posterior neural crest
markers should be carried out to determine whether our model is also valid for
posterior neural crest cells.
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ACKNOWLEDGMENTS |
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REFERENCES |
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