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First published online October 26, 2007
doi: 10.1242/10.1242/dev.006858
1 Instituto Cajal, CSIC, Doctor Arce 37, 28002 Madrid, Spain.
2 Instituto de Neurociencias de Alicante, CSIC-UMH, Apartado 18, Sant Joan
d'Alacant, 03550 Spain.
3 Millennium Nucleus in Developmental Biology, Facultad de Ciencias, Universidad
de Chile, Santiago, Chile.
4 Department of Anatomy and Developmental Biology, University College London,
London, UK.
Author for correspondence (e-mail:
anieto{at}umh.es)
Accepted 29 August 2007
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: Extension, Prechordal plate, Axial mesendoderm, E-Cadherin, Epithelial-mesenchymal transition, Cell adhesion, Cell migration, DDC model
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). Moreover, as
EMT also occurs during pathological processes, Snail genes have been
implicated in tumour progression and renal fibrosis
(Nieto, 2002;
Thiery, 2002;
Boutet et al., 2006).
In amniotes, EMT is fundamental for the formation of tissues as important
as the mesoderm and the neural crest. However, no large-scale EMT has been
observed during gastrulation in anamniote embryos where a complex interplay of
different morphogenetic movements gives rise to a mass sheet-like migration
and the cells maintain their contacts while moving. These morphogenetic
movements can be described as: (1) epiboly towards the vegetal pole over the
yolk cell; (2) internalisation of the mesendoderm at the margin; and (3)
dorsal convergence and extension along the anteroposterior axis (C&E)
(Keller et al., 2000;
Solnica-Krezel, 2006). In
contrast to the Xenopus gastrula, C&E movements in zebrafish seem
to be independent of both epiboly and mesendoderm internalisation
(Myers et al., 2002), and
convergence can even be separated from extension
(Myers et al., 2002;
Glickman et al., 2003).
Indeed, the interdependence of these two processes in Xenopus may in
part be due to the strength of the adhesive forces that maintain cells
together during involution and convergent-extension. However, during
internalisation in fish, cells can be found in groups or even as individual
cells (Carmany-Rampey and Schier,
2001; Montero et al.,
2005).
Although Snail genes have been implicated in the formation of the neural
crest and the mesoderm in different vertebrate species (reviewed by
Nieto, 2002), functional
analyses in zebrafish have only been carried out on snail1a. Four
Snail genes have been described in teleosts: snail1, snail2, snail3
and slug (Thisse et al.,
1993; Hammerschmidt and
Nüsslein-Volhard, 1993;
Thisse et al., 1995;
Locascio et al., 2002;
Manzanares et al., 2004).
Phylogenetic analyses have shown that snail1 and snail2 are
duplicates of the Snail1 gene that arose after gene duplication in
the teleost lineage (Postlethwait et al.,
1998; Manzanares et al.,
2001). snail1 and snail2 have been renamed
snail1a and 1b, respectively. Similarly, slug has
been renamed snail2, and the most recently described family member is
snail3 (Barrallo-Gimeno and Nieto,
2005).
The patterns of snail1a and 1b expression in the neural
crest suggest that as in other species, Snail genes are fundamental for the
development of this tissue (Thisse et al.,
1993; Hammerschmidt and
Nüsslein-Volhard, 1993;
Thisse et al., 1995). However,
in the absence of massive EMT at gastrulation these genes were considered to
be less important in convergence and extension
(Locascio and Nieto, 2001).
Nevertheless, snail1a has since been implicated in the anterior
movement of the axial mesendoderm in the fish embryo
(Yamashita et al., 2004) where
elegant time-lapse studies demonstrated that individual cells delaminate from
the embryonic shield (Montero et al.,
2005). Here we show that snail1b is important for the
movements that take place during extension, with snail1a and
1b fulfilling non-redundant functions in the movements that direct
the migration of the axial mesendoderm.
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MATERIALS AND METHODS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). AB and
Tup-Lof wild-type strains were used in all experiments.
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).
The antisense morpholinos (MOs) blocking snail1b translation
hybridise to bases -28 to -4 (MO1) and -2 to +23 (MO2) with respect to the
start codon, and the snail1a MOs are as previously described
(Yamashita et al., 2004).
Likewise, the cdh1 MOs were as described by Babb and Marrs
(Babb and Marrs, 2004), and the
standard control morpholino was used for control injections (Gene Tools, LLC).
All oligonucleotides were injected into the yolk of one- or two-cell embryos
(3-20 ng/embryo) using a pressure injector (PicoSpritzer). Rescue and overexpression analyses were performed with snail1b mRNA that lacked the 5'UTR region to which MO1 binds, and that were synthesised and capped using the mMessage mMachine Kit (Ambion). Rescue was achieved by co-injecting 3 ng of morpholino plus 250 pg mRNA per embryo, and 150 pg of mRNA per embryo was injected for overexpression studies. Phenotypic analyses involved at least 50 embryos for each specific marker studied under each experimental condition.
In situ hybridisation
In situ hybridisation of whole-mount zebrafish embryos was performed as
described by Nieto et al. (Nieto et al.,
1996), with minor modifications, and fluorescent whole-mount in
situ hybridisation was carried out as described by Denkers et al.
(Denkers et al., 2004). In
these experiments, the following probes were used: snail1a
(previously snail1) and snail1b [previously snail2
(Thisse et al., 1993;
Thisse et al., 1995)];
cdh1 [cadherin 1; also known as E-cadherin
(Babb and Marrs, 2004)];
hgg1 (Vogel and Gerster,
1997) [also known as ctsl1b (cathepsin L) -
ZFIN]; spt (Griffin et al.,
1998) [also known as tbx16 (T-box gene 16) -
ZFIN]; myoD (Weinberg et al.,
1996); papc (Yamamoto
et al., 1998) [also known as pcdh8 (protocadherin
8) - ZFIN]; dlx3 (Akimenko et
al., 1994); and ntl [no tail
(Schulte-Merker et al.,
1992)]. The zebrafish anti-Cdh1 antibody
(Babb and Marrs, 2004) was used
at a dilution of 1/500. Embryos were embedded in gelatin to obtain vibratome
sections (50 µm).
Cell transplantations
Donor embryos were injected at the one- to two-cell stage with
fluorescein-dextran (10,000 kDa; Molecular Probes), alone or together with the
appropriate dose of the snail1b morpholino. At the shield stage,
10-20 cells were transplanted into the shield of an unlabelled sibling host.
Alternatively, donor embryos were also injected with mRNA encoding a
membrane-tagged RFP. After an incubation of 1 hour and at approximately 70%
epiboly, grafted embryos were mounted dorsal side up in 0.8% low-melting point
agarose in Danieau's solution, and images were obtained on a Leica SP2
confocal microscope using a 40x water immersion objective. Embryos were
allowed to develop to the tail-bud stage, when they were photographed on a
Leica FLIII dissecting microscope. The distances that three individual cells
moved in each experiment (wt
wt, n=7, mowt, n=5)
were assessed using the Manual Tracking plug-in for ImageJ 1.37v software.
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), kindly provided by Reinhard Köster]. The embryos
were then embedded at the appropriate stage in low gelling 1% agarose in
Danieau buffer. Imaging was performed on a Leica SP2 confocal system mounted
on an inverted microscope using a 10x objective, and z-stacks
were obtained every 5 minutes and processed for maximum intensity projection
using ImageJ software. |
RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). To
disrupt snail1b activity, an oligonucleotide against the 5'UTR
region (MO1) and another that overlapped the start codon of the
snail1b cDNA (MO2; Fig.
1A) were designed. The vast majority of embryos that were injected
with 1-1.5 ng of the morpholino survived, although at higher doses (3 ng) only
80% of the embryos reached the tail bud stage and only 60% survived for 24
hours after the injection. Unexpectedly, we found that injecting
snail1b MOs induced an early phenotype at gastrulation stages and the
shortening of the body axis at later stages
(Fig. 1B-E,H,I). The phenotype
we observed was somewhat surprising given the pattern of snail1b
expression in the mesoderm, where snail1a rather than
snail1b is more prominently expressed
(Fig. 1K-R). Indeed, the main
sites of snail1a expression in the mesoderm at gastrulation stages
are the margin, the adaxial cells (Fig.
1K,L) and the paraxial mesoderm
(Fig. 1N) (see
Thisse et al., 1993). By
contrast, the main mesodermal sites for snail1b are the converging
cephalic mesoderm (Fig. 1Q),
the adaxial cells and a population of the axial mesendodermal cells
(Fig. 1O,R)
(Thisse et al., 1995). In all cases, the phenotypes were highly penetrant with more than 90% of the living embryos developing a similar phenotype at each concentration of morpholino. No defects were observed in uninjected embryos, in embryos injected with buffer alone (Danieau), or in those embryos injected with a negative control MO.
The injection of either morpholino alone (MO1 or MO2) induced similar
defects and with a similar dose response. Since MO1 was used in all our
control experiments (see also Fig. S1 in the supplementary material), it was
used in most of the experiments shown at a dose of 3 ng per embryo unless
otherwise indicated. To confirm the specificity of the phenotype, we
co-injected MO1 along with the coding region of snail1b mRNA.
Significantly, both the phenotypic and lethality effects observed after MO1
injection could be rescued in up to 80% of embryos (n>100;
Fig. 1F,G,J), indicating that
the shortening of the body axis was induced specifically by the
snail1b MOs. In order to assess whether we were dealing with
morphogenetic defects or with a failure to specify the mesoderm, we assessed
the expression of axial [no tail
(Schulte-Merker et al., 1992)]
and paraxial [paraxial protocaherin
(Yamamoto et al., 1998)]
mesoderm markers at gastrulation stages. Both these markers were detected in
the embryos, indicating that fate specification was not significantly
affected. In fact, somites still formed in the axial mesoderm, although they
had an abnormal shape, and evidence of the compression was detected by
myoD expression at segmentation stages
(Fig. 2). Together, these data
indicated that snail1b MOs induced a severe impairment in
anteroposterior extension without affecting mesodermal fate.
Interfering with snail1b induces defects in extension movements by augmenting cell adhesion in the axial mesendoderm
Since the anterior migration of the axial mesendoderm is an important
component of the extension movements and snail1b is expressed in this
territory, we analysed the alterations in the snail1b MO-injected
embryos using the prechordal plate markers, hatching gland gene 1
[hgg1 (Vogel and Gerster,
1997)] and spadetail [spt
(Griffin et al., 1998)].
Injection of snail1b MO expanded the expression domains of both these
markers, extending spt expression more than hgg1
(Fig. 3A-C,F-H). By contrast,
ectopic overexpression of snail1b mRNA induced the anterior
compression of the hgg1-domain and altered the distribution of the
spt-positive cells but not the number of expressing cells
(Fig. 3K-M and data not shown).
These two complementary phenotypes are compatible with snail1b being
involved in the regulation of cell-cell adhesion during anterior migration.
Snail is a strong repressor of cdh1 (E-cadherin)
(Cano et al., 2000;
Batlle et al., 2000) and as
expected, injection of the snail1b MO enlarged the domain of
cdh1 expression in the axial mesendoderm
(Fig. 3J), as well as the
hgg1 expression domain. Furthermore, less cdh1 was detected
throughout the embryos than overexpressed snail1b
(Fig. 3N,O). Snail1b did indeed
appear to be acting as a repressor of cadherin in these embryos, as the
morphant phenotype could be ameliorated by reducing cadherin function with the
injection of cdh1 MOs (Fig.
3P-S). For these rescue experiments, embryos were injected with
the usual dose of snail1b MO and a low dose of cdh1 MO (500
pg) to avoid the severe phenotype of strong cdh1 knockdown
(Babb and Marrs, 2004), and
thus, generating a partial loss of Cdh1 function. The prechordal plate adopts
a normal shape in embryos injected with both snail1b and
cdh1 MOs (3 ng and 500 pg, respectively), and its shape in cadherin
morphants is similar to that observed in embryos with Snail1b gain of function
(compare Fig. 3V with O). Cells
still disaggregate in the posterior region of double morphants as observed in
the cdh1 MO-injected embryos (asterisks in
Fig. 3R,S, and not shown). It
therefore appears that increased cell adhesion most probably accounts for the
defects in extension movements in snail1b morphants.
|
) (Fig. 1R).
Moreover, as a transcription factor, snail1b should act in a
cell-autonomous manner. To better define the territory of snail1b
expression, we contrasted the distribution of hgg1 and confirmed that
these two markers identified independent cell populations that partially
intermingle at their interface as they migrate during gastrulation
(Fig. 4A-C). By the tailbud
stage, cells that express hgg1 and snail1b were completely
separated such that a sharp boundary had formed
(Fig. 4D). We also compared the
distribution of the posterior axial mesodermal marker no tail
(ntl) with the snail1b-expressing population and we were
unable to identify double-labelled cells. As such, the posterior limit of
snail1b expression abuts with the anterior limit of ntl
expression (see Fig. S2 in the supplementary material).
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Snail1a and 1b cooperate to regulate the directed migration of the axial mesendoderm
Snail1a has been implicated in anterior movement of the axial mesendoderm
in the zebrafish embryo, since the prechordal plate in morphant embryos was
located at a more posterior position
(Yamashita et al., 2004).
However, snail1a is not expressed in the prechordal plate
(Thisse et al., 1993), again
suggesting that, like snail1b, this transcription factor seems to act
in a cell non-autonomous manner to direct the migration of the prechordal
plate cells. Accordingly, we carefully analysed snail1a expression
sites in the gastrulating fish, identifying a previously neglected domain of
extraembryonic snail1a expression in the yolk syncytial layer YSL
located underneath and anterior to the prechordal plate cells at 70% epiboly
(compare Fig. 6A with B, in
territories anterior to the dotted line encircling the hgg1-positive
region, and Fig. 6D). This
expression domain can be better assessed in sections
(Fig. 6D). Higher levels of
snail1a expression surrounding the hgg1-expressing cells are
already evident at 60% epiboly, where the previously described high levels of
expression in the margin are also observed
(Fig. 6E,F).
When the expression patterns of the two snail1 genes were compared with that of hgg1 (Fig. 6), their distribution was mutually exclusive, as they are with respect to cdh1 (Figs 4 and 6). Indeed, as shown above, hgg1-expressing cells also express cdh1 (Fig. 4E), and both gene transcripts are excluded from snail1a or snail1b expressing cells (Fig. 4 and Fig. 6G,H). Thus, a dynamic and complementary pattern of snail1 and Cdh1 expression divides the embryo into territories with different intercellular adhesion properties (summarised in Fig. 6I with data from Figs 2, 4 and 6).
Since snail1a and 1b are expressed in independent
populations and they generate a distinct phenotype when disturbed
individually, we wondered whether they cooperate to promote extension
movements during fish gastrulation. We co-injected snail1a and
snail1b MOs and found that the defects in the axial mesendoderm were
compounded (Fig. 7D;
n>20). Hence, at the end of gastrulation the double morphants show
the defects produced in both single morphants
(Fig. 7B-D). The anterior limit
to axial mesendoderm migration in these morphant embryos was more posterior
than in control embryos [as in snail1a morphants in Yamashita et al.
(Yamashita et al., 2004) and
Fig. 7B,J; n>20],
and they developed an abnormally elongated prechordal plate, similar to the
snail1b morphants (Fig.
3 and Fig. 7C,K;
n>100). Significantly, the cells expressing snail1b in
the snail1a morphants migrate normally and often continue migrating
on top of the hgg1-expressing cells
(Fig. 7F).
The cells that express snail1a located anterior to the prechordal plate are unaffected when the function of snail1b is compromised, explaining how the anterior limit of the prechordal plate is established correctly (Fig. 7C). However, the polster is formed before epiboly has been completed (see Fig. S3 in the supplementary material) and when the morphant embryo reaches the tailbud stage, the prechordal plate has already adopted an abnormal shape (Fig. 2H and Fig. 7C). Since the snail1b MO used does not induce mRNA degradation, we can follow the cells in which Snail1b function has been knockdown since they still express its mRNA (Fig. 7C,G). In these embryos, time-lapse studies of the movements of the cells containing snail1b transcripts (the translation of which is impaired by the morpholinos) indicated that their anterior movement was much slower than in control embryos (not shown). When we observed the movements of the prechordal plate cells, we found that the anterior cells move forward normally while those at its posterior edge appeared to struggle to advance and lagged behind (for an example, see Movies 3 and 4 in the supplementary material). At the tail bud stages the lagging hgg1 cells were still intermingled with cells that contained snail1b transcripts (Fig. 7G), explaining the aberrant elongated shape of the prechordal plate (Fig. 2H and Fig. 7C,K). We did not find any double-labelled cells, indicating that snail1b knockdown is not accompanied by a change in cell fate towards the hgg1-expressing prechordal plate cells. Nevertheless, the enlargement of the area occupied by the prechordal plate (hgg1-expressing cells) in embryos injected with snail1b MOs could be due to increased proliferation. This does not seem to be the case because we were unable to detect a significant change in the total number of hgg1-positive cells in wild-type, snail1a, snail1b and snail1 double morphants (n=3 for each condition) when their nuclei were counted (Fig. 7M-P). In addition, even in the most compact areas, the number of nuclei per field in the hgg1-positive territory in snail1b or in snail1a plus 1b morphants was around 50% of that observed in wt or snail1a morphants. (Fig. 7M-P). These data indicate that the same number of cells occupied a much larger area, explaining the elongated aberrant shape of the polster region observed in these embryos (Fig. 3 and Fig. 7C,D).
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DISCUSSION
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), and
their preservation may reflect the appearance of new functions associated to
either gene or their subfunctionalisation. According to the
duplication-degeneration-complementation model (DDC)
(Lynch and Force, 2000), their
subfunctionalisation would imply the complementary loss of expression between
the two duplicates. Our functional analysis in the axial mesendoderm
highlights the translation of this complementarity into functional
cooperation.
Mutually exclusive expression of the two snail1 genes and Cdh1 provides a framework for dynamic adhesion in the axial mesendoderm
The mutually exclusive expression of snail1 and Cdh1 generates an
interesting framework of transient and dynamic adhesion that somehow
reconciles different models of the regulation of cell adhesion during the
directed migration of prechordal mesoderm cells (see
Solnica-Krezel, 2006). At the
beginning of gastrulation, the internalisation of the first axial
mesendodermal progenitors occurs through the delamination of individual cells
(Montero et al., 2005). This
process is reminiscent of the EMT that occurs during mesodermal delamination
in amniotes as they do not express cadherin. In agreement with this view, in
the shield, these individual cells express goosecoid and
snail1a (Thisse et al.,
1993). Similarly, the first axial mesodermal cells to ingress
during gastrulation in Xenopus embryos delaminate as individual cells
that transiently express Snail1, as discussed previously (see
Morales and Nieto, 2004). Upon
delamination, cells very quickly re-express Cdh1 and migrate as a cohesive
anterior group. Cdh1 re-expression can be seen as a rapid mesenchymal to
epithelium transition (MET, the reverse of EMT), which occurs concomitant with
the loss of snail1a and appears to be necessary for the proper
migration of the mesendodermal cells
(Montero et al., 2005). As
such, injection of cdh1 MO
(Montero et al., 2005) impairs
anterior migration, as occurs in different cdh1 hypomorphic mutants
(Kane et al., 2005;
Shimizu et al., 2005).
Mesendodermal progenitors involute normally in these embryos
(Montero et al., 2005), as
expected considering that wild-type involuting cells lack Cdh1 because of the
expression of snail1a. Thus, cadherin is required for mesendodermal
extension in cells that lack snail1a and 1b, in the
prechordal plate and in the posterior regions but it is excluded from the
delaminating cells, which undergo a very transient EMT, and in those located
posterior to the hgg1-positive domain.
The migration of the prechordal plate cells is impaired in snail1a
and snail1b morphants due to the upregulation of Cdh1 in the
corresponding populations. Hence, the prechordal plate forms at an anomalous
posterior position in embryos deficient in snail1a function
(Yamashita et al., 2004). We
found similar phenotypes when 20 ng of snail1a MO was injected, an
unusually high dose when compared to that used for the snail1b MO
(1.5-3ng). Nevertheless, the expression of hgg1 showed that the
prechordal plate maintained its identity, again indicating that Snail1a is not
a fate determinant but rather an inducer of movement
(Barrallo-Gimeno and Nieto,
2005) and in agreement with the fact that the hgg1 domain
does not express any of the snail1 genes.
|
). The
correct movement of the prechordal plate cells depends on the expression and
motility of the snail1a cell population located underneath and in
front of them. In addition, the intermingling of the more posterior
hgg1-expressing prechordal plate cells and those expressing
snail1b at gastrulation stages in the wild-type embryo may explain
why the polster shows an abnormally elongated shape in embryos where snail1b
function is compromised. The increased cell to cell adhesion among the axial
mesendodermal cells that have aberrantly lost snail1b expression
disturbs the motility of the prechordal plate snail1-negative and
hgg1-positive cells that are forced to lag behind, and that sometimes
even detach from their pioneers. This effect would only be observed when a
significant proportion of axial mesendodermal cells have lost Snail1b
function, as occurs in snail1b morphants. As such, small grafts of
snail1b morphant cells into wild-type hosts only show a
cell-autonomous effect, being unable to perturb the behaviour of surrounding
normal axial mesendodermal cells. This suggests that a sort of `community
effect', i.e. certain number of abnormal cells, is required.
Our data indicate that the cell-autonomous function of snail1b is
necessary for the proper migration of the axial mesendoderm as observed by the
defects in migration of snail1b morphant cells transplanted into WT embryos.
In addition, the influence of snail1a and 1b loss of
function on snail1-negative, hgg1-positive prechordal plate
cells can be summarised as follows: the posterior cells push and the YSL is
likely to provide an active substrate for prechordal plate migration
(D'Amico and Cooper, 2001;
Rohde and Heisenberg, 2007).
The latter explains the abnormal posterior position of the prechordal plate in
snail1a morphants (Yamashita et
al., 2004) (this work). In summary, the precursors of the polster
require the activity of Snail1a, which exerts a cell-non-autonomous effect on
them, to reach their correct position, and they also require Snail1b function
to acquire a normal shape. Selective adhesion allows hgg1-expressing
prechordal plate cells to compact and separate from the more posterior cells
while migrating. Indeed, a clear morphological boundary between the polster
and the more posterior cells is evident in gastrulating fish embryos. Hence,
for the axial mesendoderm to migrate properly, cells located adjacent to the
hgg1-expression domain must migrate actively. Although this does not
exclude the need for prechordal plate cells to actively migrate
(Heisenberg and Tada, 2002),
it highlights the importance of a force driven by Snail1a in the YSL and of
the influence of actively migratory snail1b-expressing cells located
posteriorly.
|
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/22/4073/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Departamento de Anatomía y Embriología
Humana I, Facultad de Medicina, UCM, 28040 Madrid, Spain
Present address: Facultad de Ciencias de la Salud, Universidad Diego
Portales, Santiago, Chile
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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