1 Millennium Nucleus in Developmental Biology, Facultad de Ciencias, Universidad
de Chile, Casilla 653, Santiago, Chile
2 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104-6058, USA
3 Department of Anatomy and Developmental Biology, University College London,
UK
Author for correspondence (e-mail:
rmayor{at}uchile.cl)
Accepted 22 September 2003
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SUMMARY |
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As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region.
In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.
Key words: Neural crest, Msx genes, Bmp gradient, slug, snail, foxd3, Xenopus, Zebrafish
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Introduction |
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Diffusible proteins such as Bmp, Wnt, FGF and retinoic acid play an
important role in neural crest induction (for a review, see
Aybar and Mayor, 2002;
Knecht and Bronner-Fraser,
2002
). It has been shown in chick and amphibians that an
interaction between neural plate and epidermis is able to induce neural crest
cells (Moury and Jacobson,
1990
; Selleck and
Bronner-Fraser, 1995
; Mancilla
and Mayor, 1996
). There is strong evidence that supports the role
of Wnt factors in neural crest induction (reviewed by
Yanfeng et al., 2003
). In
chick embryos, Wnt could be one of the inductive signals produced by the
epidermis (García-Castro et al.,
2002
). It has also been shown that the addition of Bmp to chick
neural plate is able to induce neural crest cells
(Liem et al., 1995
).
Experiments in Xenopus and zebrafish support a role for Bmp signals
during neural crest induction. It has been shown in these animal models that a
gradient of Bmp is able to specify the neural plate border, including neural
crest cells (Wilson et al.,
1997
; Marchant et al.,
1998
; Nguyen et al.,
1998
; LaBonne and
Bronner-Fraser, 1998
). However, in order to fully induce neural
crest additional signals are required. These additional signals are Wnts, Fgf
and retinoic acid (Saint-Jeannet et al.,
1997
; LaBonne and
Broner-Fraser, 1998
; Lekven et
al., 2001
; Deardorff et al.,
2001
; Mayor et al.,
1997
; Villanueva et al.,
2002
). A recent model proposes that the threshold value of a Bmp
gradient specifies the anterior neural fold, which in a second step is
transformed into neural crest cells by the posteriorizing signals of Wnt, FGF
and retinoic acid (Villanueva et al.,
2002
; Aybar and Mayor,
2002
).
Once the neural crest is specified by these secreted molecules, a genetic
cascade of transcription factors is activated in the prospective neural crest
cells. Several genes that code for transcription factors have been found to be
expressed in the neural crest (for a review, see
Mayor and Aybar, 2001;
Aybar et al., 2002
), including
snail (Mayor et al.,
1993
; Essex et al.,
1993
; Linker et al.,
2000
), slug (Mayor et
al., 1995
), zic5
(Nakata et al., 2000
),
foxd3 (Sasai et al.,
2001
; Dirksen and Jamrich,
1995
), twist (Hopwood
et al., 1989
), sox9
(Spokony et al., 2002
) and
sox10 (Aoki et al.,
2003
; Honoré et al.,
2003
). Although the hierarchical relationship between these
transcription factors has not been established, snail seems to lie
furthest upstream in the genetic cascade of neural crest development
(Aybar et al., 2003
).
The connection between the inductive molecules and the transcription
factors activated in the neural crest has not been elucidated. As described
above, one of the molecules that has a clear role in neural crest induction is
Bmp. To link the inductive molecules to the transcription factors, we have
started to analyse the role that downstream targets of Bmp could have on
neural crest specification. Most of the Bmp target genes identified to date
encode homeobox proteins, including msx1
(Suzuki et al., 1997),
msx2, vent1 (Gawantka et al.,
1995
), vent2
(Onichtchouk et al., 1996
) and
dlx5 (Miyama et al.,
1999
). In this work, we have studied how Msx gene expression is
controlled in the neural folds and the role of msx1 in neural crest
development.
Msx genes, vertebrate homologues of Drosophila msh (muscle segment
homeobox) genes, are good candidates for Bmp targets. Three different Msx
genes have been identified in the mouse
(Shimeld et al., 1996), and at
least two of them, msx1 and msx2, have been isolated from
human (Jabs et al., 1993
),
Xenopus (Su et al.,
1991
) and zebrafish (Ekker et
al., 1997
). Bmp4 protein can induce or upregulate expression of
Msx genes in the epidermis, dental mesenchyme, hindbrain, neural tube, limb
bud, paraxial ectomesoderm, facial primordia and ventral mesoderm
(Vainio et al., 1993
;
Graham et al., 1994
;
Liem et al., 1995
;
Wang and Sassoon, 1995
;
Shimeld et al., 1996
;
Watanabe and Le Douarin, 1996
;
Barlow and Francis-West, 1997
;
Suzuki et al., 1997
;
Yamamoto et al., 2000
).
The expression of Msx genes is complex. In Xenopus embryos,
msx1 is initially expressed in ventral mesoderm and ectoderm, but
later becomes restricted to the neural folds and dorsal neural tube
(Suzuki et al., 1997). It has
been shown that msx1 can act as a ventralizing factor of the mesoderm
and as an inhibitor of nodal signaling
(Yamamoto et al., 2000
;
Yamamoto et al., 2001
).
Despite its expression in the neural fold, its function in neural crest
development has not been analyzed.
In order to understand how msx1 expression is regulated in the prospective neural crest, we proceeded to inhibit Bmp activity and then analyzed the expression of msx1. We inhibited Bmp activity by grafting into Xenopus embryos beads soaked with noggin, by expressing a dominant-negative form of Bmp or a dominant negative of its receptor in Xenopus embryos, or by using several Bmp/Smad zebrafish mutants. Our results show that inhibition of Bmp activity leads to an increase in msx1 expression in Xenopus and zebrafish embryos. In addition, by generating different levels of Bmp activity in animal cap ectoderm, we show that there is a specific concentration of Bmp that can induce msx1 expression. To study msx1 function in neural crest specification and development, we carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element. Our results show that activation of msx1 induces an expansion of the neural crest territory, as analyzed by the expression of snail, slug and foxd3, whereas expression of an msx1 dominant negative suppresses all neural crest markers analyzed. By performing rescue experiments of the Msx genes dominant-negative with inducible forms of snail and slug, we show that msx1 lies upstream of the Snail family of genes in the genetic cascade of neural crest specification. We propose a model whereby Msx genes are induced by a gradient of Bmp activity and that this induction is essential for neural crest specification.
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Materials and methods |
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Morpholino treatment in zebrafish
In order to inhibit mesoderm formation, the following combination of two
different spadetail (spt) and notail (ntl)
morpholinos (MO) was injected at the one-cell stage zebrafish embryo. The
spt MO was a kind gift of Sharon Amacher and Bruce Draper. The mix of
MO was a kind gift from Kate Lewis. The mix used had the final concentrations
of: ntl MO 1 mg/ml; spt MO#2 0.075 mg/ml; spt MO#1
0.75 mg/ml. The ntl MO sequence has been previously published
(Nasevicius and Ekker, 2000)
and the spt MO sequences are: spt MO 1,
5'-AGCCTGCATTATTTAGCCTTCTCTA-3'; and spt MO 2
5'-GATGTCCTCTAAAAGAAAATGTCAG-3'.
Plasmid constructs
Inducible DNA constructs of Msx genes were prepared by fusing the entire
coding regions of msx1 (amino acid residues 1-294,
Fig. 5A) to the ligand-binding
domain of the human glucocorticoid receptor (GR, amino acid residues 512-777).
A dominant-negative DNA construct (dnmsx) was prepared by fusing the
homeodomain region of msx1 (amino acid residues 156-294) to the GR
element (Fig. 5A). Coding
sequences were amplified by PCR with a high fidelity polymerase (Roche
Molecular Biochemicals, Mannheim, Germany) and the following primers:
msx1, 5'-ATGGGGGATTCGTTGTATGGATCGC-3' and
5'-GAGCTCCGGACAGATGGTACATGCTGTATCC-3'; and
DNmsx1, 5'-GAATTCATGAGCCCACCCGCCTG-3' and
5'-GAGCTCCGGACAGATGGTACATGCTGTATCC-3'.
|
RNA microinjection, lineage tracing and dexamethasone induction
Dejellied Xenopus embryos were placed in 75% NAM containing 5%
Ficoll and one blastomere of two-cell stage embryos was injected with
differing amounts of capped mRNA containing 1-3 µg/µl lysine fixable
fluorescein dextran (40,000 Mr; FLDX, Molecular Probes) as
a lineage tracer. For the inhibition of Bmp activity, the dominant negatives
were injected in one animal blastomere of eight- to16-cell stage embryo. For
animal cap assays, mRNA was injected into the animal side of the two
blastomeres of two-cell stage embryos. Approximately 8-12 nl of diluted RNA
was injected into each embryo. Ethanol-dissolved dexamethasone (10 µM) was
added to the culture medium at stages 12 or 17, and maintained until the
embryos were fixed. To control the possible leakage of inducible chimeras, a
sibling batch of embryos were cultured without dexamethasone and processed for
in situ hybridization. One-cell stage swr/bmp2b mutant zebrafish
embryos derived from crosses of swr mutant homozygous adults
(Nguyen et al., 1998) were
microinjected with either chordin mRNA
(Miller-Bertoglio et al.,
1997
) or a dominant negative Bmp type I receptor (
Bmpr)
mRNA (Graff et al., 1994
), as
previously described (Nguyen et al.,
2000
).
Noggin treatment
Acrylic beads (Sigma) were incubated overnight with 100 µg/ml of noggin
protein (a kind gift from R. Harland). The beads where grafted into embryos at
the appropriate stage and the expression of several markers was later analyzed
by in situ hybridization. PBS-soaked beads were used as controls.
In situ hybridisation
For Xenopus embryos, antisense probes containing
digoxigenin-11-UTP (Roche Biochemicals) were prepared for msx1
(Suzuki et al., 1997),
XSnail (Essex et al.,
1993
; Aybar et al.,
2003
), foxd3 (Sasai
et al., 2001
), XSlug
(Mayor et al., 1995
),
sox2 (Kishi et al.,
2000
) and cytokeratin Xk81A
(Jonas et al., 1985
) by in
vitro transcription. Specimens were prepared, hybridized, and stained
according to Harland (Harland,
1991
) with the modifications described in Mancilla and Mayor
(Mancilla and Mayor, 1996
).
Zebrafish embryos were fixed overnight in 4% paraformaldehyde. Antisense RNA
probes for zebrafish gene msxb
(Ekker et al., 1997
) was
synthesized from cDNAs using digoxigenin (Boehringer Mannheim) as a label.
Hybridization was done as previously described
(Jowett and Lettice,
1994
).
RNA isolation and RT-PCR analysis
Total RNA was isolated from embryonic tissue by the guanidine
thiocyanate/phenol/chloroform method
(Chomczynski and Sacchi, 1987),
and cDNAs were synthesized using AMV reverse transcriptase (Roche
Biochemicals) and oligo(dT) primer.
Primers for XSlug and H4 have previously been described
(Aybar et al., 2003). The
primers used to analyse Xenopus msx1 expression that amplify a 156 bp
product were 5'-GCTAAAAATGGCTGCTAA-3' and
5'-AGGTGGGCTGTGTAAAGT-3'. PCR amplification with these primers was
performed over 28 cycles, and the PCR products were analysed on 1.5% agarose
gels. As a control, PCR was performed with RNA that had not been
reverse-transcribed to check for DNA contamination. Quantitation of PCR bands
was performed using ImageJ software (NIH, USA) on 8-bit greyscale JPG files
and the values were normalized to the H4 levels from the same sample and
expressed for comparison as relative intensities (sample/H4x10).
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Results |
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So far, we have shown that there is no a direct correlation between the level of Bmp activity and the expression of the Msx genes, as a reduction in the first leads to an increase in the expression of the second. As we show in Fig. 1, there are several differences in the expression of Msx genes and bmp4. First, strong expression of bmp4 is detected in the anterior neural plate border, whereas almost no expression of msx1 is observed in that region (Fig. 1, arrowhead). The ventral epidermis shows a clear expression of bmp4 (Fig. 1B, arrow), and almost no Msx gene expression (Fig. 1E, arrowhead). The highest level of Msx gene expression can be observed in the posterior neural folds (Fig. 1D,E, asterisk), while the level of Bmp4 expression in that region is intermediate to the expression in the anterior neural plate border and the epidermis (Fig. 1A,B, asterisk). In conclusion, the levels of Bmp4 expression do not match exactly those of msx1. However, the levels of bmp4 expression do not necessarily correlate with Bmp activity levels. Furthermore, it is known that Bmp binding molecules are secreted from the dorsal mesoderm, and in consequence the levels of Bmp activity around the neural plate and neural folds could be lower than those suggested by the levels of Bmp4 mRNA. Taken together, these results prompted us to analyse directly the possibility that msx1 transcription is induced at a certain threshold concentration of a Bmp gradient.
It is known that neural crest can be induced in Xenopus by an
intermediate level of Bmp and Wnt signalling
(LaBonne and Bronner-Fraser,
1998; Marchant et al.,
1998
; Saint-Jeannet et al.,
1997
; Villanueva et al.,
2002
). One-cell stage Xenopus embryos were injected with
a mixture of between 0 and 500 pg of dominant-negative Bmp4 (CM-Bmp4) mRNA and
50 pg of Wnt5a mRNA. Animal caps were dissected at stage 9, cultured until the
equivalent of stage 16 and the expression of Msx genes and the neural crest
marker XSlug were analysed by RT-PCR. As expected, XSlug
expression was induced at a specific concentration of CM-Bmp4 (100 pg,
Fig. 4A,B), lower or higher
amounts of CM-Bmp4 failed to induce strong XSlug expression,
confirming previous reports of induction of the neural crest by a gradient of
Bmp (Morgan and Sargent, 1997
;
Marchant et al., 1998
;
Nguyen et al., 1998
;
LaBonne and Bronner-Fraser,
1998
; Luo et al.,
2003
). Interestingly, the highest level of msx1
expression was also induced at 100 pg of CM-Bmp4
(Fig. 4A,B). Low levels of
msx1 can be detected at other concentrations, which probably
represent the normal epidermal expression observed in untreated animal caps (0
ng of CM-Bmp4 mRNA, Fig. 4A,B).
These results strongly support the idea that, like the neural crest markers,
msx1 transcription is activated by an intermediate concentration in a
Bmp gradient.
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When mRNA encoding msx-GR was injected into one blastomere of a two-cell stage embryo and then activated at stage 12, the expression of the neural crest markers, slug, snail and foxd3, was augmented in more than 70% of the injected embryos (Fig. 6A-C). Conversely, the activation at stage 12 of the inducible dominant negative fusion, which contains the Msx gene homeodomain (HDmsx-GR), impaired the expression of slug, snail and foxd3 in more than 75% of the injected embryos (Fig. 6F-H). These results indicate that msx1 is required for the expression of the neural crest markers. We then analyzed whether the expansion of the neural crest territory was made at the expense of the neural plate or the epidermis. The expression of the neural plate marker Sox2 and the epidermal marker XK81a was analyzed after the activation of msx-GR at stage 12. The results show that there was an inhibition in the expression of Sox2 in about 60% (Fig. 6D) and of XK81a in 63% (Fig. 6E) of the injected embryos. This result indicates that msx1 overexpression can transform the neural plate and epidermal cells that surround the prospective neural crest territory into neural crest cells. We should mention that although many injections were localized at the center of the neural plate or epidermis, we never observed ectopic expression of neural crest markers in those territories. These results suggest that msx1 cannot transform ectodermal cells by itself into neural crest cells, but probably requires additional co-factors that are present near the neural crest region. In support of this observation we never induced the expression of neural crest markers in animal caps injected with msx1 mRNA, as analyzed by RT-PCR (three independent experiments).
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To show that the msx1 dominant-negative (XHDmsx1-GR) specifically inhibits msx1 function, we performed rescue experiments. Embryos were injected in one blastomere at the two-cell stage. The inducible constructs were activated at stage 12, and the expression of the neural crest markers, XSlug, foxd3 and XSnail was analysed at stage 18. The injection of XHDmsx1-GR inhibited in greater than 65% of the embryos the neural crest marker expression (Fig. 6Q, for simplicity only the percentage of XSlug expression are indicated in the graphic, but similar percentages were obtained for foxd3 and XSnail expression). However, the co-injection of XHDmsx1-GR and msx-GR was able to rescue the expression of the neural crest markers in a dose-dependent manner. When XHDmsx1-GR and msx-GR were injected in a proportion of 2:1, the inhibition in the expression of the neural crest markers was reduced to 50% (Fig. 6K-M,Q), but when equal amounts of both constructs were co-injected, the inhibition of the neural crest markers was rescued in more than 80% (Fig. 6N-P,Q). As expected, the injection of msx-GR alone produced an expansion in the expression of the neural crest markers in more than 70% of the embryos (Fig. 6Q). Thus, we conclude that the phenotypic effects of the inducible msx1 dominant-negative reflect a modulation of the natural msx1 target genes.
Hierarchical relationship between msx1 and the Snail family
genes
Our results show that msx1 plays a role in the early specification
of the neural crest and, as it is a Bmp target that is involved in neural
crest induction, it is likely that msx1 is one of the earliest
transcription factors in the genetic cascade of neural crest specification.
Many transcription factors are expressed in the prospective neural crest, e.g.
snail, slug, zic5, foxd3, ap2, Dlx genes, sox9 and
sox10 and all of them may play a role in neural crest specification
(Mayor et al., 1993;
Essex et al., 1993
;
Linker et al., 2000
;
Mayor et al., 1995
;
Nakata et al., 2000
;
Dirksen and Jamrich, 1995
;
Hopwood et al., 1989
;
Luo et al., 2003
;
Woda et al., 2003
;
Aoki et al., 2003
;
Honoré et al., 2003
).
However, the hierarchical relationship between these factors has not been
elucidated. It was recently proposed that snail is an early gene
working upstream of the genetic cascade in neural crest specification
(Aybar et al., 2003
). Based on
these observations, we decided to analyse the relationship between
msx1 and snail and slug in neural crest
development.
A combination of HDmsx-GR and XSlug-GR was injected into one blastomere of
two-cell stage embryos, induced at stage 12 and the expression of XSlug,
foxd3 and XSnail was analysed. Different proportions of mRNA of
the two constructs were injected. As expected the injection of HDmsx-GR alone
produced a strong inhibition in the expression of the neural crest markers
(Fig. 7G); however,
co-injection of XSlug-GR rescued this effect in a dose-dependent manner
(Fig. 7A-G). The injection of
XSlug-GR leads to an expansion of the neural crest territory
(Fig. 7G), as previously
reported (Aybar et al., 2003).
Note that when equal amounts of HDmsx-GR and XSlug-GR are used, XSlug
expression is rescued in about 70% of the embryos
(Fig. 7A,G). Similar results
were obtained for other neural crest markers (foxd3,
Fig. 7B, 72% rescued;
XSnail, Fig. 7C, 68%
rescued). However, in the opposite experiment, co-injection of a dominant
negative slug (XSlugZnF-GR) (Aybar
et al., 2003
) with msx-GR, no significant rescue in the expression
of the neural crest markers was observed (XSlug expression was
rescued in less than 10% of the embryos, n=67). We conclude that
msx1 function lies upstream of XSlug function in the genetic
cascade of neural crest specification.
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Discussion |
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We present evidence that supports the idea that a specific level of Bmp activity leads to Msx gene transcription. In addition, we show that this level of Bmp activity corresponds exactly with the level that is able to induce neural crest cells. Thus, Msx gene transcription is dramatically increased in the ectoderm by a level of Bmp activity intermediate to the level required to induce neural plate or epidermis. This high level of Msx genes, induced at a precise and intermediate threshold of Bmp signaling, is required to induce neural crest cells.
There are many reports that support the idea that a gradient of Bmp activity divides the ectoderm into neural plate, neural crest and epidermis. The molecular mechanism by which different levels of Bmp are able to activate the transcription of different genes and in turn specify different tissues is unknown. One possibility is that Bmp activates all its downstream target genes in a linear way; thus, the gradient of Bmp should be transformed into a gradient of all of its targets genes, including Msx genes, with high and low levels in the ventral and dorsal sides, respectively. Then this gradient of Msx genes should specify the neural crest at an intermediate level by an unknown mechanism. In this work, we show that this is not the case for Msx genes, as this gene is induced by a precise and intermediate level of Bmp activity. Thus, we rule out the alternative that the neural crest is specified by an intermediate concentration of Msx protein within a gradient. Instead, we support the idea that the gradient of Bmp is immediately transformed into the activation of Msx genes at a precise threshold.
The molecular mechanism by which Msx genes are expressed only at intermediate levels of Bmp activity is unknown. One possibility is the presence of Bmp receptors with different affinities for its ligands and with different downstream targets, being Msx genes a specific target for a specific receptor. Another alternative is the activation at high levels of Bmp activity of a repressor of Msx gene transcription. This alternative is supported by the observation of an early and transient expression of msx1 and msxb in the ventral ectoderm, which could be inhibited at later stages by this hypothetical repressor. Additional experiments are required to distinguish between these and other alternatives.
Role of Msx genes in neural crest specification
Our gain- and loss-of-function experiments show that Msx genes are required
for the early specification of the neural crest. Inhibition of Msx gene
function at the time of neural crest specification by use of an inducible
dominant negative, leads to inhibition in the expression of the earliest
neural crest markers known like snail, slug and foxd3.
Activation of Msx genes just prior to neural crest specification leads to an
expansion of the endogenous neural crest territory; however, we never observed
isolated regions of neural crest marker induction within the neural plate or
epidermis. This result suggests that Msx genes work together with other
factors, present in the neural plate border, to specify neural crest cells.
This explanation is also supported by our inability to induce neural crest
markers in animal caps injected with Msx genes. Taken together, we propose
that the Bmp gradient induces at the neural plate border the expression of Msx
genes and another factor, and that both are required to activate the genetic
cascade of neural crest specification. This additional factor could also be
activated by the Wnts, Fgf or retinoic acid signaling, as it is known that
they are required for neural crest induction
(LaBonne and Bronner-Fraser,
1998; Villanueva et al.,
2002
; Saint-Jeannet et al.,
1997
; Luo et al.,
2001a
; Luo et al.,
2001b
). One possible candidate for this additional factor could be
pax3, as it is known that it is expressed at the neural plate border
in a domain slightly broader than the neural crest territory, and it is able
to activate the expression of neural crest markers
(Bang et al., 1999
;
Mayor et al., 2000
). In our
animal cap gradient experiment, Wnt signaling was required to induce neural
crest and msx1 expression (Fig.
4E), as widely reported
(LaBonne and Bronner-Fraser,
1998
; Villanueva et al.,
2002
; Saint-Jeannet et al.,
1997
; Luo et al.,
2001a
; Luo et al.,
2001b
; Honoré et al.,
2003
). In addition, it has been observed that there is a
synergistic effect between Bmp and Wnt signaling in the induction of Msx genes
in culture cells (Willert et al.,
2002
). The ability of Msx genes to induce neural crest is time
dependent. When the activation of Msx genes is performed before gastrulation,
it does not promote neural crest development, but instead promotes epidermal
development (Suzuki et al.,
1997
); when Msx genes are activated after gastrulation (stage 17),
once the neural crest is specified
(Mancilla and Mayor, 1996
;
Aybar and Mayor, 2002
), no
increase in neural crest markers is observed (not shown).
Once the neural crest genetic cascade is activated by Msx genes, the
expression of specific genes that are able to confer neural crest identity is
induced. One of the earliest genes in this cascade seems to be snail
(Aybar et al., 2003), as it is
the only gene identified so far whose expression in animal caps is able to
specifically induce the expression of early and late neural crest markers. The
expression of genes such as Meis, Pbx, foxd3 and Zic family members,
not only trigger the expression of neural crest markers, but also induce the
expression of neural plate markers (Sasai
et al., 2001
; Nakata et al.,
2000
; Mizuseki et al.,
1998
; Nagai et al.,
1997
; Nakata et al.,
1997
; Nakata et al.,
1998
; Maeda et al.,
2002
). In addition, snail seems to lie upstream of
slug in the neural crest genetic cascade
(Aybar et al., 2003
). In this
work, we rescued the effect of an Msx gene dominant negative by snail
or slug co-injection, but we were not able to rescue the effect of a
slug or snail dominant negative by msx1
co-expression. Taken together, these results strongly support the conclusion
that Msx genes are upstream of snail/slug in the specification of the
neural crest cells. This conclusion is consistent with the fact that Msx genes
are a direct target of Bmp, which is one inducer of the neural crest.
It should be noted that the expression of Msx genes includes the prospective neural crest territory, but also encompasses cells adjacent to the neural crest. Those adjacent cells could be placodal and dorsal neural tube cells. Thus, although we have demonstrated a clear role for Msx genes in early neural crest specification, other experiments remain to be performed to investigate the role of Msx genes in other neural fold cells types. Interestingly, there is evidence that indicates that the preplacodal field, which is adjacent to the neural crest, is also specified by a precise threshold concentration of the Bmp gradient (A. Glavic and R.M., unpublished)
Loss of function of Msx genes in the mouse, by knocking out the gene or use
of antisense oligonucleotides, produced a wide range of phenotypes, many of
them related to the development of neural crest derivatives
(Foerst-Potts and Sadler,
1997; Jumlongras et al.,
2001
; Satokata and Maas,
1994
). However, analysis of these results is complicated by the
presence of three Msx genes, which could operate in a redundant manner. In
addition to the early role of Msx genes in neural crest specification, these
genes play a role in the control of apoptosis
(Gomes and Kessler, 2001
;
Marazzi et al., 1997
). The
more complex pattern of expression within the neural folds observed at neurula
stages is probably related to its apoptotic function in the neural crest
(C.T., M.J.A. and R.M., unpublished).
In conclusion, the dynamic expression of Msx genes during embryonic development is probably a consequence of a complex system of transcriptional regulation and reflects the multiple functions that this gene plays in several developmental processes. We have unravelled one of its roles in early neural crest development and have shown how its expression is controlled in these cells.
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ACKNOWLEDGMENTS |
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