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First published online 7 March 2007
doi: 10.1242/dev.02834
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Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Author for correspondence (e-mail:
mbronner{at}caltech.edu)
Accepted 5 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Snail2 (Slug), cadherin6B, Neural crest, Epithelial-to-mesenchymal transitions, E boxes
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; LaBonne and
Bronner-Fraser, 2000), a zinc-finger transcription factor that
plays a key role in regulating epithelial-to-mesenchymal transitions (EMTs),
resulting in neural crest delamination from the neural tube.
Snail2 is expressed in premigratory and early migrating neural crest cells.
Overexpression of Snail2 results in expansion of the neural crest domain in
Xenopus and an increase in the number of migratory neural crest cells
from the chick hindbrain (LaBonne and
Bronner-Fraser, 1998; del
Barrio and Nieto, 2002); conversely, knock-down using antisense
Snail2 RNA in Xenopus results in inhibition of neural crest
migration and a decrease or loss of many neural crest derivatives
(Carl et al., 1999). As a
member of the Snail superfamily of transcriptional repressors, Snail2
functions as an important regulator through its ability to modulate the
activity of other genes. However, surprisingly little is known about the
downstream targets of Snail2 during neural crest development.
Here, we have combined the techniques of morpholino antisense
oligonucleotide (MO) knock-down and quantitative PCR to identify possible
downstream target genes of Snail2 in avian embryos. In particular, we have
assessed the response of several transcripts in order to better describe the
molecular basis of Snail2-mediated neural crest EMT. Our results identify a
number of genes that are upregulated upon alleviation of Snail2 repression in
the premigratory chick neural crest cell population, thus representing
potential targets of Snail2 repression during the process of EMT. We further
show that one putative target of Snail2 is cadherin6B (Cad6B), which
has been previously reported to be expressed in the premigratory neural crest
and to be downregulated as neural crest cells delaminate and migrate away from
the dorsal neural tube in the avian trunk
(Nakagawa and Takeichi, 1995;
Nakagawa and Takeichi, 1998).
Through the identification of Snail2 binding sites (E boxes) within the
Cad6B regulatory region using the available chick Cad6B
genomic sequence, together with chromatin immunoprecipitation, electrophoretic
mobility shift and luciferase assays, we demonstrate that Snail2 directly
represses Cad6B transcription in a highly dynamic manner that is
required for neural crest EMT. Furthermore, our studies show that the
regulation of neural crest EMT by Snail2 occurs in a Cad6B-mediated manner.
Our results are the first to reveal a direct downstream target of Snail2
repression during avian neural crest EMT.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Briefly, the MO was injected into the lumen of the neural
tube and two 25 volt, 25 msecond pulses were applied across the embryo. To
determine the reduction in numbers of cells containing Snail2 protein on the
electroporated compared with the non-electroporated side, we examined the
presumptive neural crest domain in transverse sections through the dorsal
neural tube of embryos unilaterally transfected with the Snail2 MO (five
embryos by counting cells in 
four sections per embryo). We then took the
ratio of the number of Snail2-positive cells on the electroporated side to the
number of Snail2-positive cells on the control side. To determine the
efficiency of the Snail2 MO to knock-down Snail2 protein translation in these
same sections, we examined the number of Snail2 protein-positive cells (green)
on the electroporated side that also contained the Snail2 MO (red) and thus
were yellow. This number was then divided by the total number of Snail2
MO-positive (red) cells in the same sections, with the efficiency of reduction
expressed as a percentage.
Chick embryo culture
Fertilized chicken eggs were obtained from AA Enterprises (Ramona, CA) and
incubated on their sides at 38°C in a humidified incubator (Lyon Electric,
Chula Vista, CA). Tissue was dissected in PB-1 standard medium. Chamber slides
(LabTek) were coated with 10 µg/ml recombinant fibronectin (BD Biosciences)
and 16.6 µg/ml poly-L-lysine (Sigma). Neural folds were explanted to slides
and cultured in serum-free DMEM plus N2 supplement (Gibco-BRL) for 4 hours
prior to fixation and imaging. To determine the number of cells that have
undergone EMT in explanted tissue, the number of cells containing the MO (red)
that have undergone EMT (i.e. mesenchymal in appearance) and have emigrated
from the explants were counted, using DAPI staining and phase-contrast
microscopy to identify individual cells. This number was divided by the total
number of cells that have undergone EMT. Emigrating cells were counted in this
manner from at least thirty explants over three independent experiments.
Whole-mount in situ hybridization
Linearized cDNAs were used to synthesize digoxigenin-labeled antisense RNA
probes, and whole-mount in situ hybridizations were performed using `Protocol
Four' as previously described (Xu and
Wilkinson, 1998). Stained embryos were imaged on a Zeiss Stemi
SV11 microscope and processed using Photoshop 7.0 (Adobe Systems).
Immunohistochemistry
The distribution of Snail2 protein was assessed using anti-Snail2 antisera
[Developmental Studies Hybridoma Bank (DSHB), Iowa University; clone 62.IE6].
Briefly, whole embryos were fixed for 15 minutes in 4% paraformaldehyde at
room temperature and washed with PBS containing 0.1% Tween 20 (PBTw). Embryos
were then blocked in 10% sheep serum in PBTw before adding the anti-Snail2
antibody (1:100, in 5% sheep serum in PBTw) and incubating at 4°C
overnight. The primary antibody was washed off with PBTw, and replaced by an
Alexa-Fluor 488 secondary antibody (Molecular Probes, 1:500, diluted in 5%
sheep serum in PBTw) with incubation overnight at 4°C. The embryos were
then washed, mounted and imaged. Immunohistochemical detection of Cad6B (DSHB,
clone CCD8B-1; 1:100) was performed as described
(Nakagawa and Takeichi, 1998)
with an Alexa-Fluor 488 secondary antibody (Molecular Probes, 1:500). If
required, embryos were cryostat-sectioned at 14 µm, and sections were
DAPI-stained.
RNA and cDNA preparation
Total RNA was isolated using the RNAqueous Total RNA Isolation Kit
(Ambion), and cDNA was synthesized using random hexamers and the Superscript
II RT-PCR system (Invitrogen) according to the manufacturer's
instructions.
Quantitative polymerase chain reaction (QPCR)
QPCR was performed using the ABI 7000 in a TaqMan (Applied Biosciences) or
SYBR Green (Bio-Rad) assay as described
(Taneyhill and Bronner-Fraser,
2005). Briefly, 50 µl reactions were performed using the
2xTaqMan mix in the presence of cDNA, 100-300 nM of each primer, and 450
nM of each probe; 25 µl reactions were performed using the 2xiTaq
SYBR Green mix in the presence of cDNA and 50-100 nM of each primer. After
normalization to a standard (chick 18S rRNA), fold upregulation or
downregulation was determined by dividing the relative expression value for
the gene of interest in the presence of the Snail2 MO by that
obtained for the gene of interest in the presence of the control MO.
Chicken Cad6B sequence identification, isolation and construct preparation
The genomic location of chick Cad6B was identified using the
Ensembl
(www.ensembl.org)
web browser by searching with the complete chick Cad6B cDNA sequence
(Genbank accession number D42149). Sequence reads were assembled using
AssemblyLIGN and analysis was performed using MacVector 7.1.1.
The Cad6B genomic sequence was divided in silico into portions containing two E-box motifs. Fragments were amplified by PCR from BAC clone CH261-110K4 using Expand Long Template Polymerase (Roche) with appropriate primers:
E4_5', 5'-TTCATCCACCAGTAACCCCTTG-3';
E3_3', 5'-GCCCTTTTCCCTTCATTCTCC-3';
E2_5', 5'-GCAAAGATACCCTGGACTTCGG-3';
E1_3', 5'-CCAACATCCCTCCTTGTAACCC-3';
EA_5', 5'-TTCTCCACCACACATACCAGTGC-3'; and
EB_3', 5'-AGACACAGTCCCATTTGAGTTTGG-3'.
Fragments were inserted into the KpnI (5') and XhoI (3') cloning sites of the pGL2-Promoter Luciferase vector (Promega). All constructs were sequenced to confirm PCR and cloning accuracy.
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation of Snail2 protein associated with the
Cad6B regulatory region was carried out using described procedures
and buffers (Upstate). Briefly, dorsal neural tube tissue dissected from the
premigratory chick trunk or midbrain region was fixed in 1% formaldehyde and
then subjected to sonication (ten times, 15-second pulse and 1 minute recovery
on ice) using a microtip attachment on a Branson 450 Sonifier set at 50%
output. After sonication, 20 µl of chromatin was taken as a standard curve
template for QPCR. Equivalent amounts of chromatin were used in three
different immunoprecipitations carried out at 4°C overnight: an
anti-rabbit GFP antibody control (5 µg of GFP antibody; Molecular Probes),
a no antibody control, and an anti-Snail2 antibody (150-250 µl of Snail2
antibody). QPCR was then performed on the immunoprecipitated chromatin as
described above to detect association of Snail2 to each of the E-box pairs, as
well as to the 3' region of the Cad6B transcript in exon 11 (a
region with no predicted E-box motifs) as a negative control. The results
reported for the Snail2 antibody were corrected for any background
immunoprecipitated chromatin by subtracting the averaged value obtained in the
presence of the GFP (or IgG) and no antibody immunoprecipitates (both usually
undetectable). Because the amount of chromatin obtained from the tissue prior
to immunoprecipitation differs between experiments, the results are presented
as a representative ChIP experiment, with the s.d. reported for association of
Snail2 with each E-box pair.
Electrophoretic mobility shift assay (EMSA)
Snail2 or control nuclear extract was isolated from transiently transfected
293T cells according to standard techniques. 10 µg of nuclear extract was
incubated with double-stranded E box-containing oligonucleotides end-labeled
with 33P (GE Healthcare) using T4 polynucleotide kinase (Promega).
Protein-DNA binding reactions were incubated with or without unlabeled
competitor DNA for 20 minutes at room temperature in the presence of an EMSA
buffer, prior to addition of the labeled probe
(Wu et al., 2005). Poly(dI-dC)
(GE Healthcare) was used as a non-specific competitor. E1,
5'-TAGGTTCACGACAGGTATGCAGTTAGAT-3'; E2,
5'-ACAAATCCTGTCAGGTAGTTCTGCTCCA-3'; all mutated
oligonucleotides had the E-box core consensus sequence (underlined) altered
from CAGGTA to TTGGTA. Incubated reaction mixtures were separated on a
high-ionic-strength non-denaturing 4% PAGE gel. Dried gels were exposed to
phosphorimaging screens, and signal was detected using a STORM840
Phosphorimager (GE Healthcare).
Construction of mutant Cad6B E box-luciferase reporters and QPCR-based luciferase assays
Primers were designed to change the E-box core consensus sequence from
CAGGTA to TTGGTA (for E boxes 1, 2, 3 and 4; sequences available on request).
Site-directed mutagenesis was carried out using the QuikChange II
Site-Directed Mutagenesis Kit according to the manufacturer's instructions
(Stratagene), utilizing the wild-type Cad6B E box-luciferase
reporters as the template. All constructs were sequenced to ensure accuracy of
mutagenesis.
200 ng of wild-type or mutant luciferase reporter construct [Cad6B E box-luciferase or control (pGL2-Promoter)] and 240 ng of either a Snail2 or empty vector control construct [Tag-2B-Snail2 or Tag-2B vector (Stratagene)] were transiently transfected into 293T cells using the Lipofectamine2000 reagent and Opti-MEM I media (Gibco-BRL) according to the manufacturer's instructions (Invitrogen). Cells were harvested 24 hours post-transfection and total RNA was isolated. SYBR Green QPCR was carried out using primers designed to amplify both the luciferase and ampicillin amplicons (sequence available upon request). A normalized value of luciferase expression for each construct was obtained by dividing the average luciferase quantity obtained for each construct by the corresponding amount of ampicillin. The effect of Snail2 on this normalized luciferase expression was then calculated by dividing the normalized value obtained in the presence of Snail2 by the normalized value obtained in the presence of the empty vector. The reported results are an average of at least three independent transfection experiments, with each QPCR reaction performed in triplicate. Experimental results for the mutant E-box reporters represent the mode value obtained from three independent transfection experiments.
Immunoprecipitation and immunoblotting
Cells utilized for immunoprecipitation and/or immunoblotting were lysed in
Lysis Buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT
plus Complete Protease Inhibitors from Roche) by vortexing, followed by a 20
minute incubation on ice and 10 minute centrifugation at 20,800
g, 4°C. Total protein concentration (cytoplasmic and
nuclear fractions) was determined by performing a Bradford assay (Bio-Rad).
Immunoprecipitations and immunoblotting were carried out using a Flag-M2
peroxidase antibody (Sigma) and/or the Snail2 antibody using standard
methods.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A single lissaminated MO to Snail2 was electroporated into neural tube cells at two different stages in order to target premigratory trunk neural crest (embryos with 21-23 somites) or premigratory midbrain neural crest (embryos with 5-7 somites). Importantly, the MO was introduced just at the onset of Snail2 expression at each axial level. At selected times after MO electroporation, the neural folds were dissected on the electroporated side at the level adjacent to somites +4 to -1 for the trunk region, or in the midbrain for the cranial region (Fig. 1A,B). The dissected tissue was then assayed by quantitative PCR (QPCR) using primers for candidate target genes (Fig. 1C,D).
Taking advantage of the known role of Snail2 as a transcriptional repressor
in multiple systems (Nieto,
2002; Barrallo-Gimeno and
Nieto, 2005), we examined genes whose transcript levels were
increased as a result of knocking-down Snail2 translation. We
selected a number of good candidates based on their relative expression
patterns compared with Snail2. These included FoxD3, Msx1, Pax3,
Ncad, Cad6B, and RhoB.
Four and 2 hours after electroporation of the Snail2 MO in the trunk, several candidate transcripts, including FoxD3 and Cad6B (2 and 4 hours), Ncad (2 hours), and Msx1 (4 hours) (Fig. 1D), were upregulated in Snail2 MO-treated compared with control MO-treated embryos. Further characterization revealed that Cad6B was upregulated 3-fold in the presence of the Snail2 MO as early as 30 minutes post-electroporation in the trunk (Fig. 1D). In addition, upregulation of at least 1.5-fold (mode value of 3.6) of Cad6B was observed 30 minutes after electroporation of the midbrain region with the Snail2 MO (Fig. 1D). Although detectable by QPCR, these changes in Cad6B transcript levels were below the threshold of detection possible by in situ hybridization and/or immunohistochemistry. Interestingly, no upregulation of a related cadherin, Ncad, was observed by QPCR after 30 minutes, showing the specificity of Snail2 for Cad6B (data not shown).
At later times (e.g. 15 hours) after MO electroporation in the trunk, some genes (Ncad, Cad6B, RhoA) exhibited decreased transcript levels in the presence of the Snail2 MO, whereas others were unchanged, making it likely that their repression is not mediated directly by Snail2 at this time point (see Fig. S2 in the supplementary material).
Snail2 and Cad6B have largely non-overlapping expression patterns during chick neural crest emigration
The rapid upregulation of Cad6B transcripts in the presence of the
Snail2 MO after only 30 minutes raises the intriguing possibility
that it may be a direct target of Snail2 repression. Consistent with this
hypothesis, Cad6B has been shown to be downregulated upon neural
crest emigration in the trunk (Nakagawa
and Takeichi, 1995; Nakagawa
and Takeichi, 1998). To gain further insight into the relationship
between Snail2 and Cad6B at stages corresponding to EMT of
the neural crest, we compared their mRNA and protein expression patterns at
midbrain and trunk levels using whole-mount in situ hybridization and
immunostaining (Figs 2,
3).
At midbrain levels, both Snail2 and Cad6B transcripts and proteins were found to be expressed initially in a small population of cells (Fig. 2). Snail2 transcripts and protein accumulated when the embryo had 6-7 somites, just prior to neural crest emigration. Cad6B transcripts were initially expressed in the neural tube but were subsequently downregulated at the 5-6 somite stage (ss), prior to initiation of neural crest emigration. However, Cad6B protein persisted until the 6-7 ss, just one stage prior to the onset of neural crest cell emigration from the midbrain (8 ss). After this stage, Cad6B protein was downregulated. Thus, in the midbrain region, we initially observed somewhat overlapping expression of Snail2 and Cad6B, but this expression became largely reciprocal (high Snail2 protein, low Cad6B mRNA) immediately prior to and during neural crest EMT at the 8 ss. A similar correlation was noted at truncal levels, with Snail2 protein levels high in the premigratory neural crest when Cad6B levels were low (Fig. 3). Thus, the expression profiles of Snail2 protein and Cad6B transcripts in the midbrain and trunk regions are consistent with a possible regulatory relationship between Snail2 and Cad6B.
Snail2 binds to the Cad6B regulatory region via E boxes
The Snail family of transcriptional repressors interacts with a core E-box
motif CANNTA/G to mediate repression of their target genes
(Giroldi et al., 1997;
Nieto, 2002). To determine if
the Cad6B sequence contains E-box motifs, we analyzed the genomic
locus 130 kb upstream and downstream of the coding region in silico. Chick
Cad6B is composed of 12 exons that are spread over 
102 kb on
chromosome 2 (Ensembl Gene ID; ENSGALG00000012917). Sequence analysis
identified a number of clustered E boxes containing the sequence CAGGTA
surrounding the second exon of Cad6B, the first coding exon
(Fig. 4A). To confirm the
accuracy of the deposited sequence, the sequence was compared with a BAC clone
(CH261-110K4) containing the region of interest. Approximately 8 kb of the
region surrounding the ATG of Cad6B was sequenced in both directions,
and six E-box motifs were found to be clustered in this putative regulatory
region. The E-box motifs are grouped in three distinct regions and have been
termed E4 and E3 (265 bp apart and 6.5 kb upstream of exon 2), E2 and E1 (305
bp apart and 2343 bp upstream of exon 2), and EA and EB (133 bp apart and 1.1
kb downstream of exon 2) (Fig.
4A). It should be noted that we identified several errors in the
assembly of the deposited genomic sequence.
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In vivo ChIP assay
To demonstrate in vivo association of Snail2 protein with the putative
Cad6B regulatory region, we performed ChIP experiments with an
antibody to Snail2 in conjunction with QPCR, using primers and probes designed
to amplify genomic sequence between the E boxes comprising an E-box pair, on
tissue isolated from the premigratory region of the chick trunk (+4 to -1
somite region) and the chick midbrain. The nature of this experiment allowed
us to quantitatively determine the amount of Cad6B genomic sequence
(represented by each clustered pair of E boxes) with which Snail2 potentially
associates, generating a profile that describes the affinity of Snail2 for
different E-box pairs (Fig.
4B). In the premigratory region of the avian trunk, Snail2 was
found to preferentially associate with E1 and E2, with the overall interaction
affinity described as E1 and E2>E3 and E4>EA and EB. This preferential
association also held true for the midbrain, except that little to no Snail2
was found to associate with EA and EB at this axial level. No association of
Snail2 was observed in a control QPCR reaction using Cad6B sequence
from exon 11 that lacks any predicted E-box motifs, and no E boxes were
immunoprecipitated in the absence of antibody or in the presence of a
non-specific antibody, such as GFP or IgG. These in vivo results are the first
to describe Snail2 association with the regulatory region of a bona fide
target gene in a biological system.
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).
Luciferase assays
QPCR-based luciferase assays were performed to assess the ability of Snail2
to regulate luciferase expression driven by various Cad6B E
box-luciferase reporters (Fig.
4A,D). Co-transfection of Snail2 and Cad6B E
box-luciferase reporters into 293T cells resulted in a statistically
significant decrease in luciferase expression in the presence of E1
and E2, and E3 and E4, when compared with co-transfection of these reporters
with an empty vector control (Fig.
4D). These results are in good agreement with those shown
previously for the regulation of the human
(Batlle et al., 2000) and mouse
(Bolós et al., 2003;
Conacci-Sorrell et al., 2003;
Peinado et al., 2004)
E-cadherin promoters by Snail1 and Snail2. Furthermore, we mutated the E boxes
in these luciferase reporters (making the same two point mutations in the
E-box core motif as in the EMSA experiments) to assess effects on
luciferase expression in the presence or absence of Snail2. Our
results showed that mutation of these E boxes abrogates the ability of Snail2
to repress luciferase expression. Our combined biochemical results
suggest that the binding of Snail2 to E boxes in the Cad6B regulatory
region mediates Snail2 repression of Cad6B transcription.
Snail2 affects neural crest EMT through a Cad6B-dependent mechanism
To examine whether Snail2 affects neural crest EMT in a Cad6B-mediated
manner, we designed a morpholino to target the 5' coding region of
Cad6B in order to knock-down Cad6B translation. Embryos were
electroporated with an equimolar mixture of either the Snail2 MO plus
a control MO, or the Snail2 MO plus the Cad6B MO. Dissected
neural folds from the electroporated side were placed in tissue culture, fixed
4 hours after explantation, and imaged. In the presence of the Snail2
MO alone, we observed only a small percentage of MO-positive neural crest
cells (13%; n=32 explants) emigrating from the explanted neural folds
(Fig. 5A), in keeping with
previous work in chick (Nieto et al.,
1994). The rescue of this decreased emigration, however, was
partially achieved by inhibiting Cad6B translation concomitantly with
the knock-down of Snail2 translation. Neural crest cells
electroporated with an equimolar mixture of the Snail2 and
Cad6B MOs were able to emigrate from cultured explants (51%;
n=36 explants; P<0.0001;
Fig. 5B,C). Collectively, these
results demonstrate that Snail2 affects neural crest EMT, in part, via a
Cad6B-dependent mechanism (Fig.
6).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Members of the Snail superfamily
(Sefton et al., 1998;
Hemavathy et al., 2000b;
Nieto, 2002;
Barrallo-Gimeno and Nieto,
2005) have been shown to control genes whose products function in
mediating cell movement both during metastasis
(Hemavathy et al., 2000a;
De Craene et al., 2005) and
embryonic cell migration (Barrallo-Gimeno
and Nieto, 2005). During oncogenesis, Snail1 and Snail2 play key
roles in facilitating tumor metastasis through their ability to directly
repress transcription of the adhesion molecule E-cadherin via an interaction
with the E-cadherin promoter (Cano et al.,
2000; Bolós et al.,
2003; Côme et al.,
2004).
A crucial developmental role for Snail family members is highlighted by the
mouse knockout of Snail1, which is embryonic lethal owing to
mesodermal defects during gastrulation
(Carver et al., 2001). This
process appears to be conserved during evolution because the mouse knockout
phenotype is highly reminiscent of the abnormal migration of mesodermal
precursor cells that occurs in Snail-deficient Drosophila
gastrulae (Grau et al., 1984;
Boulay et al., 1987). Previous
work, however, has shown that Snail2 is not essential for mesoderm
formation or neural crest development in mouse
(Jiang et al., 1998). To
address potential redundancies that might exist between mouse Snail family
members, a double knockout of Snail1 and Snail2 in the mouse
was recently generated; however, this mutant showed no defects in neural crest
development (up to E9.5) (Murray and
Gridley, 2006). These results raise the intriguing possibility
that there are distinct functional roles, both conserved and non-conserved,
for Snail family members in different organisms. For example, during mesoderm
formation, the function of Snail1 in mouse is highly conserved with
Xenopus Snail and chick Snail1 and Snail2.
The absence of a neural crest phenotype in the mouse
Snail1/Snail2 double knockout indicates that significant
differences exist with respect to the function of Snail family members in the
neural crest of different vertebrates. Xenopus Snail
(Carl et al., 1999;
LaBonne and Bronner-Fraser,
2000) and chick Snail2
(Nieto et al., 1994) (present
study) have been shown to have important functions in neural crest
development. In particular, knock-down of Xenopus Snail not only
affects neural crest emigration and migration, but also the formation of some
neural crest derivatives (Carl et al.,
1999). It is possible that neural crest development in the mouse
proceeds through a redundant pathway that compensates for the loss of both
Snail family members, perhaps reflecting a change in placental mammals. In
this scenario, additional neural crest specifier genes may play a more
dominant role in mouse versus chick or Xenopus. Consistent with
species-specific roles for different transcription factors, the expression
patterns of mouse and chick Snail1 and Snail2 genes appear
to be `swapped' during neural crest development
(Sefton et al., 1998). Taken
together, these results suggest that there is conservation of some, but not
all, functions of Snail family members in the vertebrate lineage. Therefore,
the molecular mechanisms controlling neural crest EMT in mouse and chick are
likely to differ based on changes in the expression of both Snail and cadherin
family members in these organisms.
Snail2 regulates the expression of many genes, including the cell adhesion molecule cadherin6B, during the EMT that characterizes neural crest development
The process of EMT is elegantly exemplified during embryonic development by
the delamination of neural crest cells from the dorsal neural tube. Using this
model system, we delineate the molecular role of Snail proteins during chick
neural crest development by identifying target genes regulated by Snail2, the
avian functional homolog of Snail1 (Sefton
et al., 1998; del Barrio and
Nieto, 2002). To this end, we have knocked-down the expression of
Snail2 in the developing neural crest region and have identified a number of
genes that are sensitive to depleted levels of Snail2 protein. We provide the
first quantitative insight into the dynamic regulation of gene transcription
during neural crest EMT in vivo. We find that depletion of Snail2 causes rapid
and robust changes in the transcript levels of many genes in the premigratory
neural crest (Msx1, FoxD3, Ncad, Cad6B) at many different time
points.
|
). Snail1 undergoes extensive phosphorylation that
promotes its degradation and results in its localization to the cytoplasm,
thus abrogating its function as a transcriptional repressor
(Zhou et al., 2004). Given
these biochemical properties that characterize the Snail superfamily, it is
probable that complete repression of Cad6B in these regions cannot
occur until a sufficient level of Snail2 protein has accumulated and has been
dephosphorylated.
|
|
;
Cano et al., 2000;
Bolós et al., 2003). In
chick, E-cadherin is not expressed in the neural tube at the stages under
consideration, ruling it out as a possible Snail2 target in the neural crest.
Furthermore, our in vivo explantation results suggest that proper neural crest
EMT in the avian embryo proceeds in a Cad6B-dependent manner, with repression
of Cad6B achieved by Snail2 prior to neural crest cell emigration.
Our combined results are the first to be obtained in vivo that describe a
direct target for Snail2 repression during avian neural crest EMT
(Fig. 6).
The important developmental function of Snail2 has been well characterized
during gastrulation (Nieto et al.,
1994; Ciruna et al.,
1997), somitogenesis (Dale et
al., 2006) and neural crest cell development
(Nieto et al., 1994;
LaBonne and Bronner-Fraser,
1998; LaBonne and
Bronner-Fraser, 2000; Sakai et
al., 2006). Increasingly, a number of genes regulated by Snail
family members have been identified with roles in development and disease in
the control of key EMT processes (De
Craene et al., 2005). The EMT that characterizes neural crest cell
emigration from the dorsal neural tube is a highly coordinated event that
involves the upregulation of mesenchymal markers and molecules that promote
morphological changes and migration (such as Bmp4, Msx1/2, RhoA/B and
Cad7), as well as the repression of genes that function to maintain
the epithelial, non-motile state of the cell (such as Cad6B). Indeed,
alterations in the expression of some of these same molecular EMT players also
render tumor cells invasive, further emphasizing the shared molecular
properties ascribed to the neural crest and metastasizing cancer cells
(Hemavathy et al., 2000a;
Côme et al., 2004;
De Craene et al., 2005).
Collectively, our results demonstrate that repression of Cad6B by
Snail2 is highly dynamic and one of the significant events during the
epithelial-to-mesenchymal conversion of a non-motile dorsal neural tube cell
into a migratory neural crest cell.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/8/1481/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Barrallo-Gimeno, A. and Nieto, M. A. (2005).
The Snail genes as inducers of cell movement and survival: implications in
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