1 Millennium Nucleus in Developmental Biology, Facultad de Ciencias, Universidad
de Chile, Casilla 653, Santiago, Chile
2 Instituto Cajal, CSIC, Doctor Arce 37, 28002, Madrid, Spain
* Author for correspondence (e-mail: rmayor{at}uchile.cl)
Accepted 23 October 2002
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SUMMARY |
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We show that Snail is able to induce the expression of Slug and all other neural crest markers tested (Zic5, FoxD3, Twist and Ets1) at the time of specification. This activation is observed in whole embryos and in animal caps, in the absence of neural plate and mesodermal markers. We show that Snail is required for neural crest specification and migration and that it works as a transcriptional repressor. These functions have been previously attributed to Slug. However, Slug alone is unable to induce other neural crest markers in animal cap assays, and we show that Snail and Slug can be functionally equivalent when tested in overexpression studies. This suggests that, in Xenopus embryos, at least some of the functions previously attributed to Slug can be carried out by Snail. This is additionally supported by rescue experiments in embryos injected with dominant-negative constructs that indicate that Snail lies upstream of Slug in the genetic cascade leading to neural crest formation and that it plays a key role in crest development.
Key words: Snail, Slug, Neural crest, Crest specification, Crest migration, Zic5, FoxD3
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INTRODUCTION |
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The function of one of the vertebrate Snail family members, Slug,
has been studied in neural crest development by gain- and loss-of-function
experiments in chick and Xenopus. In the chick embryo, antisense
oligonucleotides directed against Slug mRNA can prevent neural crest
migration (Nieto et al.,
1994), whereas overexpression of Slug induces an increase
in the production of the neural crest (Del
Barrio and Nieto, 2002
). Similarly, injections of Slug
antisense RNA or of a dominant-negative form of Slug in
Xenopus embryos reduces the expression of neural crest markers and
inhibits the migration of the crest from the neural tube
(Carl et al., 1999
;
LaBonne and Bronner-Fraser,
2000
; Mayor et al.,
2000
). Conversely, overexpression of Slug led to an
enlargement of the neural crest territory
(LaBonne and Bronner-Fraser,
2000
; Mayor et al.,
2000
). These results support the idea that Slug plays a
key role in neural crest development in chick and amphibian embryos. However,
mice homozygous for a null mutation in Slug are viable and display no
obvious defects in neural crest formation, migration or development
(Jiang et al., 1998
). This can
be explained by the fact that, in the mouse, Slug is not expressed in
the premigratory neural crest but rather the other vertebrate family member,
Snail, is expressed in this population
(Jiang et al., 1998
;
Sefton et al., 1998
). Indeed,
Snail is capable of inducing a complete EMT in mammalian epithelial
cells (Cano et al., 2000
), and
it should be noted that Snail null mutant mice die at gastrulation
due to defects in EMT at the stage when the early mesoderm delaminates from
the primitive streak (Carver et al.,
2001
). Thus, in neural crest development, it seems that the role
played by Slug in the chick, may be performed by Snail in
the mouse. However, owing to the early lethality of the mutants, the direct
role of Snail in neural crest specification remains to be tested in
the mouse.
We have previously shown that, in Xenopus, Snail is one of the
earliest genes expressed in the prospective neural crest
(Essex et al., 1993;
Mayor et al., 1993
). However,
no direct comparison between the expression of Snail and
Slug has been performed. In addition, Snail function has not
been fully analysed in this population. In this study we have generated
functional derivatives of Xenopus Snail in order to study its role in
neural crest development. Our results show that Snail acts as a
transcriptional repressor, whose activity is required for the early
specification and migration of the neural crest. Interestingly, the activation
of Snail is sufficient to trigger the expression of all the neural
crest markers tested, including Slug. Expression of these markers
could be induced both in whole embryos and in animal caps in the absence of
neural and mesodermal markers. We propose that Snail lies upstream of
Slug in the genetic cascade responsible for neural crest
specification.
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MATERIALS AND METHODS |
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Plasmid constructs
Inducible DNA constructs were prepared by fusing the entire coding regions
of Xsnail (amino acid residues 1-259), Xslug (amino acid
residues 1-266), chick Snail (amino acid residues 1-256) and
Slug (amino acid residues 1-268) to the ligand binding domain of the
human glucocorticoid receptor (GR, amino acid residues 512-777)
(Fig. 1). Coding sequences were
amplified by PCR using the following primers (see
Fig. 1A-D):
|
GR was obtained by PCR from pSP64T-MyoD-GR
(Kolm and Sive, 1995) using
the primers 5'-GGCGCCGAGCTCCCCTCTGAAAATCCTGG-3' and
5'GCGGGCTCGAGCCACTTTTGATGAAACAGAAC-3'.
The PCR products were purified and cloned into pGEM-T Easy vector (Promega). EcoRI/SacI sites in the Snail and Slug genes and, SacI/XhoI sites from GR were used to fuse the two, and ligate them into an EcoRI/XhoI-digested pCS2+ vector (donated by Dr D. Turner). The Xsnail (amino acid residues 134-259) and Xslug (amino acid residues 72-266) zinc-finger coding regions were amplified using the primers 5'-GAATTCCAAGCACAAACAGTTGCACTG-3' and 5'-GAGCTCCGTGGGCCACCGTGCACC-3', and 5'-CCCAGTGAATTCATGCCACGATCTTTTCTG-3' and 5'-TACTGGAGCTCCATGTGCTACACAGCA-3' respectively, and cloned into pGEM-T Easy vector. The EcoRI and SacI sites (underlined) were used to fuse them to GR (Fig. 1E,F). To generate the XsnailN or XslugN constructs (Fig. 1G, H), the N-terminal part of Xsnail (amino acid residues 1-145) and Xslug (amino acid residues 1-151) were amplified by PCR using the primers 5'-GAATTCCATGCCCCGGTCATTTCTGG-3' and 5'-GAGCTCTGGGAGTCACAGTGCAACTG-3', and 5'-GAATTCAATGACCCGATCTTTTCTGG-3' and 5'-GAGCTCTGGGCGTCGCAATGCAGCTG-3',respectively. The PCR products were purified and cloned into pGEM-T Easy vector, EcoRI/SacI-digested and ligated with a SacI/XhoI-digested GR fragment into pCS2+ vector digested with EcoRI/XhoI.
To generate the transcriptional activator and repressor chimeras, the Xsnail and Xslug zinc-finger DNA-binding domains obtained as described above were fused to the engrailed repressor domain (EnR) or to the E1A transactivator domain in the pCS2+EnR and pCS2+E1A plasmids (donated by N. Papalopulu).
To obtain the E1A fusion protein (Fig.
1J), the pCS2+E1A vector and Xsnail zinc-finger fragment
fused to GR were digested with EcoRI/XhoI and ligated. The
EnR fusion construct (Fig. 1I)
was generated by exchanging the E1A domain, excised with XhoI and
KpnI, from the pCS2+ZnFXsnailGR-E1A or pCS2+ZnFXslugGR-E1A with the
EnR-coding sequence, excised with the same enzymes, from the pCS2+EnR vector.
All fusion constructs were sequenced on both strands at junction sites by
automated DNA sequencing (BRC, Cornell University, Ithaca, NY). All cDNAs were
linearized and transcribed with a GTP cap analog (New England Biolabs) using
SP6, T3 or T7 RNA polymerases, as described elsewhere
(Harland and Weintraub, 1985).
After DNAse treatment, RNA was phenolchloroform extracted, ethanol
precipitated and resuspended in DEPC-treated distilled water.
RNA microinjection, lineage tracing and dexamethasone induction
Dejellied 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; FDX, Molecular Probes) as a lineage tracer.
For animal cap assays, mRNA was injected into the animal side of the two
bastomeres 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 12.5 or 16 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 hybridisation.
In situ hybridisation and immunohistochemistry
Antisense probes containing Digoxigenin-11-UTP (Roche Biochemicals) were
prepared for FoxD3 (Sasai et al.,
2001), Xtwist
(Hopwood et al., 1989
),
Sox2 (Dr RM Grainger, personal communication) and cytokeratin
Xk81A (Jonas et al., 1985) by in vitro transcription. In order to
avoid cross hybridisation between Snail and Slug, and to
distinguish between endogenous expression and exogenous mRNA, the probes for
Xsnail (Essex et al.,
1993
) and Xslug
(Mayor et al., 1995
) were
synthesised from 3' untranslated regions prepared with the following
primers: 5'-GCACAATGGACTCCTTAAATTCCTG-3' (upstream) and 5'-
GTGACCGGGTGCTCATTGTG-3' (downstream), and 5'-GTTTACCA-
GGACTTAACACCTCC-3' (upstream) and 5'-GCATTCCCTT-
AAACCCTTCTTGG-3' (downstream), respectively.
Specimens were prepared, hybridised and stained according to Harland
(Harland, 1991) with
modifications (Mancilla and Mayor,
1996
). Detection of labelled antisense probes was performed using
alkaline-phosphatase conjugated anti-digoxigenin Fab fragments (Roche
Biochemicals) and with NBT/BCIP (purple) as substrate. We have designed a new
protocol to detect the lineage tracer in combination with the in situ
hybridisation procedure. First, the in situ hybridisation alkaline-phosphatase
reaction was stopped by incubation in methanol at 65°C for 1 hour, then
embryos were rehydrated and blocked with 2% Roche blocking-reagent before
incubating with alkaline phosphatase conjugated anti-Fluorescein Fab fragments
(Roche Biochemicals). The phosphatase activity resulting from the lineage
tracing was detected using BCIP (green) as substrate. Rabbit Polyclonal
anti-phosphohistone-3 from Upstate Biotechnology was used to analyse mitotic
cells according the method described elsewhere
(Turner and Weintraub,
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 synthesised using AMV reverse transcriptase (Roche
Biochemicals) and oligo(dT) primer. The primers designed for this study were:
Xslug, 5'- GTTTACCAGGACTTATCACCTCC-3' (upstream) and
5'-GCATTCCCTTAAACCCTTCTTGG-3' (downstream); Xsnail,
5'-GCACAATGGACTCCTTAAATTCCTG-3' (upstream) and
5'-GTGACCGGGTGCTCATTGTG-3' (downstream).
The Ets1 primer sequences used were those previously
(Meyer et al., 1997).
Xtwist, Ncam, Sox2, Xbra, H4 and BMP4 primer sequences were obtained
from the website of Dr Eddy De Robertis
(http://www.hhmi.ucla.edu/derobertis/protocol_page/oligos.PDF).
Zic5 primer sequences were as described previously
(Nakata et al., 2000
). 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.
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RESULTS |
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In Xenopus, the ectoderm is formed of two layers: the superficial or sensorial layer, and the deep layer where the neural crest is generated. At the mid neurula stage (Fig. 2G), the cells expressing Snail in the outer band correspond to the crest cells, while the cells in the thin inner band (white arrowhead in Fig. 2G) will end up in the roof plate of the neural tube. In summary, Snail is the earliest known gene to be expressed in the prospective neural crest, preceding Slug expression. Snail early expression is restricted to the neural plate border, including the prospective neural crest region and the anterior neural folds, which are fated to become the forebrain (Fig. 2H,I).
Snail promotes neural crest specification
In order to analyse the influence that Snail might have on neural
crest development, we have used an inducible Snail construct. The use
of such an inducible construct is important as Snail is expressed in
the mesoderm at early stages, and any effects on this tissue could indirectly
influence the neural crest. Thus, one blastomere of two-cell stage embryos was
injected with 50 to 700 pg of XsnailGR mRNA, and the expression of several
neural crest, neural plate and epidermal markers was analysed at the neurula
stage. A reproducible phenotype was observed when >100 pg of mRNA were
injected. This phenotype involved the expansion of the area in which neural
crest markers were expressed [Snail, 85% of the embryos showed
expansion (n=107); Slug, 76% (n=96); Zic5,
63% (n=97, not shown); Twist, 67% (n=86) and
FoxD3, 62%, (n=85)], and a reduction in the area over which
the neural plate marker Sox2 (87%, n=92) and the epidermal
marker cytokeratin (89%, n=65) were expressed
(Fig. 3). It is interesting to
note that the expression of cytokeratin almost completely disappeared from the
injected side of the embryo. This observation suggests that the expansion of
the neural crest domain results from a transformation of the epidermis and
some of the neural plate region into prospective neural crest. This is similar
to the mechanism that has been proposed to explain the increase in neural
crest markers observed after Slug overexpression
(LaBonne and Bronner-Fraser,
2000; Mayor et al.,
2000
). However, it is also possible that as a result of the
injection, an increase in the rate of neural crest cell proliferation was
triggered. In order to rule out this possibility, we repeated the
Snail and Slug injections in the presence of the inhibitors
of cell proliferation hydroxyurea and aphidicolin (HUA)
(Harris and Hartenstein, 1991
;
Sharpe and Goldstone, 1997
).
This inhibition did not affect the expression or the expansion of the
territory in which the neural crest markers were observed after injecting the
SnailGR or SlugGR construct: Snail (92%, n=42; 82%,
n=45) or Slug (85%, n=49; 85%, n=42)
(Fig. 4). Cell proliferation
can be assessed by the detection of a phosphorylated form of H3 histone
(Fig. 4F). In the presence of
HUA, its signal is barely detectable (Fig.
4E). Thus, we conclude that the expansion in the neural crest
territory is not due to an increase in cell proliferation but instead is the
consequence of generating additional neural crest cells.
|
|
To address if Snail expression was sufficient to induce neural crest markers we performed animal caps experiments. Embryos were injected at the one-cell stage with 1 ng of Snail mRNA and at the blastula stage (stage 9), the animal caps were dissected out and cultured until they reached the equivalent of stage 20, when the expression of different markers was analysed by RT-PCR. In these experiments, the expression of H4 histone was used as a loading control. In whole embryos, the normal expression of all the markers tested was observed, while in uninjected control animal caps the expression of specific markers could not be detected (Fig. 5A). The injection of 1 ng of Snail mRNA was sufficient to trigger the expression of the neural crest markers Snail, Slug, Twist, Zic5 and Ets1, but not that of neural plate markers, such as NCAM or Sox2, or mesodermal markers, such as Xbra (Fig. 5A). This result suggests that Snail itself is capable of converting ectodermal cells into neural crest cells, and that it could lie upstream of the genetic cascade required for neural crest specification.
|
Because our results show that Snail produces a strong reduction of epidermal markers, and it is known that the development of epidermis requires high levels of BMP activity, we tested whether Snail overexpression had an effect on BMP4 transcription. The expression of BMP4 was analysed in animal caps as described above and a clear inhibition of BMP4 expression was observed when the animal caps were injected with Snail (Fig. 5B).
Inhibition of Snail activity blocks early neural crest
specification
In order to test whether Snail activity is required for neural
crest development, we tested the effect of a dominant-negative construct in
which the zinc-finger domain of Snail was fused to the GR element
(XsnailZnFGR). This construct was designed to bind to the appropriate
sequences in the promoter of target genes, but with the idea that it will be
incapable of regulating transcription, as has been shown for this type of
construct using the Slug gene
(LaBonne and Bronner-Fraser,
2000; Mayor et al.,
2000
). Its injection blocked the expression of Snail (82%
of the embryos, n=57), Slug (77%, n=67),
FoxD3 (83%, n=55), Twist (83%, n=53) and
Zic5 (77%, n=43, not shown)
(Fig. 6A-D). In order to show
the specificity of this dominant negative, we performed the following rescue
experiment. Embryos were co-injected with the same amount of mRNA that codes
for XSnailZnfGR (dominant negative) and XsnailGR (wild type) and the
expression of several neural crest markers was analysed. A strong rescue in
the expression of the neural crest markers was observed
(Fig. 6E,F). The injected
embryos show a normal expression of Slug (83% of normal expression in
the injected side, n=54), Snail (96%, n=27),
Zic5 (91%, n=33, not shown) and FoxD3 (84%,
n=31). Thus, by inhibiting Snail function the early
specification of the neural crest was blocked as determined by the analysis of
five different markers.
|
Owing to the sequence similarity between Snail and Slug
in the finger region (Manzanares et al.,
2001), the possibility exists that the injection of XsnailZnFGR
could affect the function of both genes. Thus, we decided to generate another
dominant-negative construct by fusing the highly divergent N-terminal domain
of Snail to the GR element (XsnailNGR). This construct will not be
able to bind DNA but should be capable of binding other proteins required for
the transcriptional activity of these factors, as shown for a similar FoxD3
construct (Sasai et al.,
2001
). We considered that XsnailNGR would serve as a more specific
construct to test the effects of expressing dominant-negative Snail
constructs. Indeed, injection of the N-terminal dominant-negative version also
blocked the expression of Slug (89%, n=47) and
FoxD3 (83%, n=46) (Fig.
6G,H). Nevertheless, and in order to check unambiguously the
specificity of this construct, we carried out a rescue experiment similar to
that described for XsnailZnFGR. The co-injection of the XsnailNGR (dominant
negative) together with XsnailGR (wild type) produces a rescue in the
expression of the neural crest markers analysed (61% of normal Slug
expression, n=26; and 56% of normal FoxD3, n=25)
(Fig. 6I,J). Thus, taken
together our results show that Snail activity can be specifically
blocked by two types of dominant negatives and that Snail is required
to control the expression of all the neural crest markers tested.
Snail lies upstream of Slug in the genetic cascade leading
to the neural crest development
The temporal appearance of Snail and Slug in the neural
crest together with the ability of Snail to upregulate Slug
expression in whole embryos and in animal caps suggest that Snail
could be upstream of Slug in the genetic cascade that specifies the
neural crest cells. If this is the case, the effect of a dominant-negative
Snail could be rescued by Slug, but that of a
dominant-negative Slug should not be rescued by Snail. To
test this prediction, embryos were co-injected with XsnailZnFGR (dominant
negative) and SlugGR (wild type) and the expression of different neural crest
markers was analysed. An almost complete rescue of the neural crest expression
was observed (Fig. 7A,B).
Injected embryos showed a normal expression of Slug (94%;
n=69), Snail (85%; n=41), Zic5 (91%;
n=46) and FoxD3 (88%; n=42). A similar rescue was
observed when the XsnailNGR dominant negative was co-injected with SlugGR (not
shown). This suggests that Slug activity is downstream of
Snail. To further confirm this finding, we developed a new
dominant-negative Slug construct similar to XsnailNGR. The highly
divergent N-terminal domain of Slug was fused to the GR element
(XslugNGR) and this construct was injected into one blastomere of a two-cell
stage embryo. This injection blocked the expression of Slug itself
(86% of the embryos, n=43) and that of FoxD3 (74%,
n=46) (Fig. 7C,D) in a
similar manner to that previously described for the dominant-negative
zinc-finger Slug construct
(LaBonne and Bronner-Fraser,
2000; Mayor et al.,
2000
). However, this inhibition in the expression of the neural
crest markers was not efficiently rescued by co-injection of SnailGR. Only 30%
of the injected embryos showed a normal expression of Slug
(n=23; Fig. 7E) and
38% of them showed normal FoxD3 expression (n=38;
Fig. 7F). By contrast, the
rescue of the Slug dominant-negative by SlugGR was apparent in 77%
and 72% of the embryos with respect to Slug (n=44) and
FoxD3 expression (n=43), respectively. Thus, the rescue of
the Snail dominant-negative by Slug, and the difficulty to
rescue Slug activity by Snail indicates that Snail
is upstream of Slug in the genetic cascade that specifies the neural
crest in the ectoderm.
|
Snail functions as a transcriptional repressor
In order to better understand the molecular mechanisms that underlie the
activity of Snail during neural crest development, the zinc-finger
region of Snail was fused to the activation domain of E1A or the
repressor domain from Drosophila engrailed. Both these constructs
were fused to the GR element to make them inducible and they were called
XsnailZnFGRE1A and XsnailZnFGrEnR, respectively. One blastomere of a two-cell
stage embryo was injected with one or other of these constructs and the
expression of several neural crest markers analysed at the neurula stage. The
injection of the repressor construct resulted in an enlargement of the
territory expressing Slug (81% of expansion, n=44),
Snail (88%, n=44), FoxD3 (85%, n=41),
Zic5 (78%, n=46, not shown) and Twist (78%,
n=46) (Fig. 8A-D).
Conversely, injecting the activator construct led to an inhibition of the
expression of Slug (86% of inhibition, n=45), Snail
(82%, n=58), FoxD3 (81%, n=48), Zic5 (80%,
n=56, not shown) and Twist (76%, n=50)
(Fig. 8E-H). Thus, as the
repressor construct produced the same phenotype as wild-type Snail,
and the opposite effect was produced by the activator construct, we concluded
that Snail probably functions as a transcriptional repressor in this
system.
|
Snail has a role on neural crest migration
As Snail is also expressed during the migration of neural crest
cells, we analysed whether it might also influence this process. Embryos
injected with XsnailGR and with XsnailZfGR (dominant negative) were allowed to
develop to the midneurula stage (stage 16-17) when the neural crest is
specified and expresses both Slug and Snail
(Mayor et al., 1995;
Mancilla and Mayor, 1996
).
When the injected embryos were treated with dexamethasone at stage 16, and the
expression of neural crest markers was analysed at stage 22-23, a prominent
effect on neural crest migration was observed. The population of migrating
crest expressing Slug (81% of embryos with an increase in the
injected side, n=43; Fig.
9A,D) and Snail (82%, n=51, not shown) increased
following injection of XsnailGR. In addition, injection of XsnailZfGR resulted
in a reduction in the migration of the crest cells (67% of embryos with a
reduction in the migration in the injected side, n=48;
Fig. 9B,E) or Snail
(74%, n=46, not shown). These results are similar to those observed
when the Slug dominant-negative construct was used previously in
equivalent experiments (LaBonne and
Bronner-Fraser, 2000
). As Slug overexpression had not
been examined previously at stages during which the neural crest is migrating,
we also injected embryos with XslugGR and treated them in a similar way.
Overexpression of Slug produced an increase in the migratory
population of crest cells as seen by the expression of Slug (77% of
embryos with stronger migration in the injected side, n=47,
Fig. 9C,F) and Snail
(75%, n=51, not shown). Thus, both Snail and Slug
overexpression during the stages of crest migration gives rise to an increase
in the migration of this population of cells.
|
Ectopic expression of chick and mouse Snail and
Slug in Xenopus embryos
Slug has been shown to be important in triggering EMT during crest
migration in the chick (Nieto et al.,
1994; Del Barrio and Nieto,
2002
). In the mouse, Snail rather than Slug is
the gene expressed in this population, and Snail has also been shown
to induce a complete EMT in mammalian epithelial cells
(Cano et al., 2000
). Thus,
during neural crest development, it seems likely that in the mouse
Snail might fulfil the role played by Slug in the chick.
This suggests that these genes may be functionally equivalent and that both
are capable of triggering EMT when expressed at the appropriate time and place
(Del Barrio and Nieto, 2002
).
Because in Xenopus both genes are expressed in the premigratory
neural crest, we wanted to know whether they are functionally equivalent in
these cells and whether the chick genes have a similar effect when ectopically
expressed in the frog. After injection of mRNA encoding the inducible forms of
Slug and Snail from Xenopus and chick into one
blastomere of a two-cell stage Xenopus embryos, the expression of the
neural crest marker Slug was analysed at the late neurula stage
(stage 25). The expression of Slug was enhanced by the injection of
500 pg of mRNA for XsnailGR (83% of enlargement of Slug in the
injected side, n=53), XslugGR (88% n=55), chick SnailGR
(67%, n=45) and chick SlugGR (82%, n=46)
(Fig. 10). Thus, these
injections produced an expansion of the neural crest territory and, an
enhanced migration of the crest migration on the injected side. Similar
results (Fig. 10) were
obtained after injection of mouse Slug (65%, n=60) and
Snail (72%, n=51) (not shown). Thus, Snail and
Slug from Xenopus, chick or mouse are functionally
equivalent when overexpressed in Xenopus embryos.
|
![]() |
DISCUSSION |
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Snail is the earliest marker for the neural crest
Snail is the earliest marker of the neural crest described to date
in Xenopus embryos, as it can be detected in the prospective neural
crest from stage 11 onwards. All the other early neural crest markers such as
Slug, FoxD3 and members of the Zic and Meis families are not detected
in the neural folds until later on in development
(Sasai et al., 2001;
Nakata et al., 2000
;
Linker et al., 2000
;
Maeda et al., 2002
). We show
that, in addition, the initial expression of Snail in the ectoderm
constitutes an arc that surrounds the neural/nonneural territory, including
the anterior neural plate border. Just before the other early neural crest
markers appear, or become confined to the crest-producing neural folds,
Snail expression becomes weaker in the anterior region that will
become part of the forebrain and does not form neural crest
(Le Douarin and Kalcheim,
1999
). This expression pattern is unique for Snail and
perfectly compatible with the recently proposed induction-posteriorisation
model for neural crest formation
(Villanueva et al., 2002
;
Aybar and Mayor, 2002
).
Snail is required for the early specification of neural
crest cells
Having illustrated the intriguing expression pattern of Snail, we
examined its role in neural crest development. Because Snail is also
involved in mesoderm development, one must take care in interpreting the
phenotypes generated by overexpressing this gene from the two-cell stage of
development. A phenotype relevant to the neural crest could be the result of a
previous effect on mesoderm development. For this reason, we have used
inducible constructs that were activated around the time the neural crest is
specified (Mayor et al., 1995;
Mancilla and Mayor, 1996
) and
long after mesoderm specification (Smith
et al., 1985
).
Overexpression of Snail in whole embryos augmented the domain of
expression of all the neural crest markers analysed at the expense of the
adjacent ectoderm and neural plate. The amplification of this domain was not
the result of an increase in cell proliferation as it was not inhibited when
cell division was blocked. Thus, we infer that Snail has the ability
to transform ectodermal cells into neural crest cells. This observation was
further confirmed in animal cap assays, where expressing Snail mRNA
alone was sufficient to trigger the expression of a series of early and late
neural crest markers, in the absence of the expression of neural plate and
mesodermal markers. This differs from results obtained when other early neural
crest markers are expressed. The expression of genes such as Meis, Pbx,
FoxD3 and Zic family members not only triggers the expression of
neural crest markers, but also induces 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
).
Furthermore, Slug overexpression was unable to trigger the expression
of neural crest markers in animal cap assays as analysed by in situ
hybridisation (LaBonne and Bronner-Fraser,
1998
). Thus, it seems that Snail is the only gene that
has been described to date that is capable of specifically transforming
ectodermal cells into neural crest cells.
In support of Snail performing a central role in neural crest
development, blocking Snail activity through the injection of
dominant negative constructs produced a complete inhibition in the expression
of neural crest markers. One of these constructs involved fusing the DNA
binding domain to the GR element to make it inducible. A similar construct has
been used to study Slug function
(LaBonne and Bronner-Fraser,
2000). We were aware of the possibility that this construct could
affect the function of both genes because of the sequence similarity between
Snail and Slug in the zinc-finger region (Manzanares et al.,
2002). It is worth noting that we were able to show that in the mesoderm,
where there is a complementary expression of Slug and Snail,
the inhibition of Slug function by this Slug dominant-negative
version could only be rescued by Slug and not by co-injection of
Snail (Mayor et al.,
2000
). This suggests that it specifically works inhibiting
Slug function. However, the limitation of this approach is
particularly relevant when studying the function of Snail family
members in neural crest development in Xenopus, as both genes are
co-expressed in this tissue. Therefore, we generated another dominant negative
construct that contained the N-terminal region of Snail, which is
highly divergent from that of Slug. Using this construct, a similar
inhibition of neural crest markers was observed, indicating that the activity
of Snail is indeed required for the early specification of the neural
crest. Furthermore, we were able to rescue the effect of the Snail
dominant-negative using Slug co-injection, but we were not able to
rescue the effect of Slug dominant-negative by using Snail
co-expression. Taken together, these results strongly support the conclusion
that Snail is upstream of Slug in the specification of the
neural crest cells.
We have also shown that Snail probably functions as a
transcriptional repressor, as the injection of a repressor construct produces
the same phenotype as that of the wild-type Snail, and has the
opposite effect to that of an activator construct. This is not surprising, as
Snail family members have been shown to act as repressors in many species from
Drosophila to humans (Nieto,
2002). Thus, the simplest interpretation of our data is that
Snail represses the expression of a factor that prevents the
expression of the other neural crest markers. The ability of Snail to
transform animal caps into cells expressing neural crest markers and the
inhibition of epidermal markers in the whole embryo suggest that such an
unknown factor should be expressed in the ectoderm. Candidate molecules
include BMP4 and the genes downstream of it, such as Msx1 and the D1x
genes (Bendall and Abate-Shen,
2000
). Indeed, the observation that Snail is able to
repress BMP4 expression in animal caps supports the idea that this factor
could be the target of Snail repression during neural crest
specification. A similar function has been attributed to Slug in the
dorsal mesoderm, where this gene influences the development of the Spemann
organiser by repressing BMP4 expression in this tissue
(Mayor et al., 2000
). Thus,
our results suggest that Snail is also a repressor of BMP4, and that
this repression is required for the specification of the neural crest
territory. This conclusion is supported by the model in which the neural crest
is induced by a gradient of BMP activity
(Marchant et al., 1998
;
Morgan and Sargent, 1997
;
Nguyen et al., 1998
). Thus, if
Snail were to repress BMP4, Snail injections will cause the
levels of BMP4 to be reduced over a more extended area, thereby displacing the
threshold level necessary to specify neural crest to the epidermal region and
leading to the conversion of ectodermal cells to neural crest.
Snail is involved in neural crest migration
As Snail is expressed in migratory neural crest cells, we
investigated what function it may fulfill during migration. Because we have
shown that Snail interferes with neural crest specification, we
adopted the same approach to study the role of Snail in migration by
activating the inducible constructs only once neural crest precursors had
already been formed. Overexpression of Snail at the midneurula stage
produced an increase in the number of migrating neural crest cells. This is in
agreement with results obtained by overexpressing Slug in the chick
(Del Barrio and Nieto, 2002).
Blocking Snail activity with dominant-negative constructs led to a
reduction in neural crest migration as has also been described for
Slug in Xenopus (LaBonne
and Bronner-Fraser, 2000
). However, some migratory cells were
observed in the manipulated embryos, suggesting that other factors probably
cooperate with Snail in the migratory process. Among the possible
factors, Slug is a good candidate because it has already been
proposed to act as a maintenance factor of the mesenchymal phenotype
(Ros et al., 1997
). Moreover,
it has been suggested that these two factors cooperate during crest migration
in the chick and mouse, as a subpopulation of migratory crest express both
genes (Sefton et al., 1998
;
Cano et al., 2000
).
Snail and Slug show functional equivalence
In this work, we show that Snail plays a key role in the
development of the neural crest in Xenopus embryos, similar to that
seen after Slug overexpression
(LaBonne and Bronner-Fraser,
1998). One explanation for this is that both genes are
functionally equivalent and that overexpression experiments cannot
discriminate between the two. This possibility was tested by comparing the
effect of injecting Snail or Slug mRNAs into
Xenopus embryos. No obvious differences could be detected in the
effects of either gene on the expression of neural crest markers or the
migration of the neural crest. Furthermore, overexpressing Snail or
Slug from chick and mouse also produced similar results, supporting
earlier data where it was suggested that both genes are functionally
equivalent in chick embryos (Del Barrio and
Nieto, 2002
). This also indicates that overexpression in
Xenopus cannot discriminate between their specific activities. Along
the same lines, the results obtained with dominant-negative constructs may
also reflect a lack of specificity. As mentioned above, to avoid such a
problem, we have used a dominant-negative construct, which is specific as it
contains the highly divergent region of the different family members that lies
outside of the zinc-finger domains
(Manzanares et al., 2001
) and
we were able to rescue the effect of Snail dominant negative by
co-expression of Snail wild type. In summary, considering that
Snail is expressed before Slug in the crest precursors, that
Snail but not Slug is able to induce neural crest markers in
animal cap assays and that Snail is able to induce Slug, we
believe that Snail may be responsible for at least some of the
functions previously associated to Slug. Thus, we propose that
Snail rather than Slug plays an early role in neural crest
development in Xenopus, as has been suggested in mammals
(Sefton et al., 1998
;
Jiang et al., 1998
;
Cano et al., 2000
;
Carver et al., 2001
). As
mentioned before, Slug may fulfil similar or additional functions in
the premigratory population at later stages and also during migration.
The relationship between Snail, Slug and other neural crest
markers
A number of different transcription factors have been shown to be expressed
in the prospective neural crest of Xenopus embryos (Meis1,
Pbx1, several Zic genes, FoxD3 and Slug), and
have been implicated in its early development
(Sasai et al., 2001;
Nakata et al., 2000
;
Pohl and Knöchel, 2001
;
Maeda et al., 2001
;
Maeda et al., 2002
;
LaBonne and Bronner-Fraser,
1998
; LaBonne and
Bronner-Fraser, 2000
; Mayor et
al., 2000
). We now show that Snail fulfils a similar
function. We show that Snail is the earliest specific marker of crest
precursors and we believe that it lies upstream of these other factors in the
genetic cascade of crest specification for the following reasons: (1)
Snail is able to induce all the above-mentioned neural crest markers;
(2) considering the onset of expression, the Zic genes are expressed
in the prospective neural plate from very early stages and become restricted
to the neural crest territory long after Snail; (3) Slug and
FoxD3 are expressed in the precursors of the neural crest after
Snail; (4) all the above-mentioned genes including Zic5
(Nakata et al., 2000
), with
the exception of Snail and Slug, induce neural plate markers
as well as neural crest markers when assayed in animal caps; and (5)
Slug is incapable of inducing any known crest markers in animal
caps.
Furthermore, we have recently proposed a model for neural crest induction
(Villanueva et al., 2002;
Aybar and Mayor, 2002
) in which
the entire neural plate border is induced first but later crest production is
restricted to the posterior regions of the neural fold. This model predicts
that the earliest crest-specific genes induced should be expressed in the
entire neural plate border, with later genes being restricted to the
definitive neural crest-forming region. Interestingly, Snail is the
only gene described to date whose expression fits in well with this model. Its
early expression along the entire length of the neural plate border correlates
with the first phase of crest specification
(Fig. 2H). However, definitive
crest will be only produced at more posterior levels upon the action of
signalling molecules (Wnt, FGF, retinoic acid) which will maintain high levels
of Snail expression (Fig.
2I). In agreement with this, FGFR signalling is needed for the
maintenance of Snail expression in the mouse primitive streak
(Ciruna and Rossant, 2001
).
This would justify that the low levels of Snail expression observed
in the anterior neural plate do not generate crest, and would also explain its
ability to induce all neural crest markers with the high dose used in animal
cap assays. Finally, Snail is the only gene described to date able to
specifically induce all neural crest markers in the absence of neural plate
markers. Altogether, our data indicate that Snail lies high and
upstream of Slug in the hierarchy of crest specification, being
activated very early in the territory competent to become crest.
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ACKNOWLEDGMENTS |
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