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First published online 2 October 2008
doi: 10.1242/dev.023416
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Division of Cell and Developmental Biology, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Author for correspondence (e-mail:
c.j.weijer{at}dundee.ac.uk)
Accepted 9 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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, follow to form somites. Both expression of a
dominant-negative PDGFR and depletion of endogenous PDGFR
ligands through injection of PDGFR-Fc fragments, inhibit the migration
of mesoderm cells after their ingression through the primitive streak.
siRNA-mediated downregulation of Pdgfa expression in the epiblast on
one side of the streak strongly blocks the migration of mesoderm cells into
that side. Beads soaked in PDGFA elicit a directional attractive movement
response in mesoderm cells, showing that PDGFA can provide directional
information. Surprisingly, however, PDGF signalling is also required for
directional movement towards other attractants, such as FGF4. PDGF signalling
controls N-cadherin expression on mesoderm cells, which is required for
efficient migration. PDGF signalling activates the PI3 kinase signalling
pathway in vivo and activation of this pathway is required for proper
N-cadherin expression.
Key words: Gastrulation, Cell movement, N-cadherin, PDGF signalling, PI3 kinase signalling
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Psychoyos and Stern, 1996).
The signals that guide the migration of the epiblast cells towards the
primitive streak followed by their ingression and migration away from the
primitive streak are not yet understood in detail, nor are the mechanisms by
which these cells move. FGF signalling plays a major role in mesodermal cell
migration. We have provided evidence that migration away from the primitive
streak involves FGF8 signalling, and that movement back in towards the midline
involves chemotaxis towards factors produced by the forming headprocess and
notochord, notably FGF4 (Yang et al.,
2002). It is, however, to be expected that a wider array of
guidance molecules is required to guide the different mesodermal cell
populations to their correct targets. Indeed, experiments in other organisms,
especially frogs, fish and mice, have implicated PDGF signalling in particular
in the control of mesodermal cell movement during early development.
In Xenopus it has been shown that PDGFA is expressed in the
ectoderm, whereas the PDGF receptor (PDGFR) is expressed in the deeper
mesodermal cells migrating anterior along the blastocoel roof. Expression of a
dominant-negative PDGFR, with a single-point mutation that prevents activation
of the receptor, results in severe gastrulation defects
(Ataliotis et al., 1995). The
embryos showed a loss of anterior structures and failure of proper closure of
the neural tube resulting in spina bifida, suggesting a defect in the anterior
migration of mesodermal cells. Explanted prechordal plate mesoderm cells are
able to follow a directional signal, in a conditioned extracellular matrix
deposited on a plastic surface by animal cap cells, in a direction that
corresponds to anterior (Nagel and
Winklbauer, 1999). This directional migration is blocked by
expression of dominant-negative PDGFR or by morpholino knockdown of
Pdgfr in the migrating prechordal plate cells
(Nagel et al., 2004). PDGF
signalling is also required for the correct attachment of the prechordal plate
cells to the matrix lining the blastocoel roof and for anterior migration in
vivo (Van Stry et al., 2004).
Furthermore, it has been shown that isolated zebrafish mesodermal cells orient
their protrusive activity towards an ectopic PDGF source, which involves
localised PI3 kinase signalling in the leading edge of the migrating cells.
Inhibition of PI3 kinase signalling in vivo results in the markedly slower,
but still directed, movement of mesoderm cells
(Montero et al., 2003).
In higher vertebrates there are two PDGF receptors, PDGFR and
PDGFRβ, that form both homo- and heterodimers, and there are at least
four PDGF genes, Pdgfa, Pdgfb, Pdgfc and Pdgfd, that give
rise to PDGFAA, PDGFAB, PDGFBB, PDGFCC and PDGFDD ligands. PDGFAA, PDGFAB,
PDGFCC and PDGFBB are ligands for the PDGFR homodimer, whereas PDGFAB,
PDGFCC, PDGFBB and PDGFDD are ligands for the PDGFR-PDGFRβ dimer,
and PDGFBB and PDGFDD for the PDGFRβ homodimer
(Heldin and Westermark, 1999;
Tallquist and Kazlauskas,
2004). Owing to this complexity, the effects of PDGF signalling on
early development, especially gastrulation, are not yet well characterised. In
mice, PDGFA is expressed in the epiblast and PDGFR in the mesoderm
(Mercola et al., 1990).
Knockouts exist for both Pdgfa and Pdgfr. Knockout of
Pdgfr results in developmental defects including incomplete
cephalic closure and increased apoptosis on the pathways followed by migrating
neural crest cells. Furthermore, alterations in the formation of the sternum
and ribs appear to result from deficiencies in the formation of the myotome
(Hoch and Soriano, 2003;
Soriano, 1997). Knockout of
Pdgfa leads to the death of half of the embryos before E10; the other
half die shortly after birth owing to a failure of alveolar septation and lung
emphysema (Bostrom et al.,
1996). The variable phenotype has been attributed to differences
in genetic background, compensation by expression of Pdgfb, maternal
factors, placental transfer of PDGF and/or embryonic position in the uterus.
The cause of the early developmental lethality at E10 has not been analysed in
detail.
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;
Yang et al., 2002;
Yue et al., 2008). In this
study, we characterise the expression of Pdgfa, Pdgfb and
Pdgfc and of their receptor Pdgfr and show that
signalling through PDGFA and PDGFR is required for the migration of
mesoderm cells in vivo, and that the effects are mediated through the control
of N-cadherin expression and PI3 kinase signalling. |
MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), then early chick (EC) cultures prepared
(Chapman et al., 2001) followed
by electroporation-based transfection as described
(Leslie et al., 2007). Imaging
of cell movement during early gastrulation was performed as described
(Yang et al., 2002).
Brightfield and fluorescence images were taken every 3 minutes for periods of
up to 25 hours. A computer-controlled motorised stage allowed us to record the
development of six embryos simultaneously. Typically, we recorded three
experimental embryos and three controls; the remaining surviving embryos were
photographed at the beginning and end of the experiment. Each experiment was
repeated at least three to four times.
Molecular biology
cDNA probes for in situ hybridisation were obtained from the Chick EST
Consortium (Boardman et al.,
2002). We used the following cDNAs: Pdgfa (ChEST460a12,
linearised with SacII), Pdgfb (ChEST115n16, linearised with
BglII), Pdgfc (ChEST270m4, linearised with NheI),
Pdgfd (ChEST74b4, linearised with SacII),
Pdgfr (ChEST404l3, linearised with HpaI). cDNAs were
transcribed with T3 RNA polymerase. In situ hybridisation was performed
according to standard procedures
(Wilkinson and Nieto,
1993).
The pCAβ-PDGFR37-IRES-GFP expression construct was made by amplifying
the coding sequence from the original vector, containing Xenopus
PDGFR37 in PCS2, with gene-specific primers: PDGFR37 forward,
5'-GAATATATTAGCGGCCGCATGATGCCTGCCATGAGG-3'; PDGFR37 reverse,
5'-GAATATATTAGAATTCTCACAGGAAACTGTCCTC-3'. The fragment was
inserted into the pCAβ/GFP expression vector, which has an internal
ribosome entry site (IRES) directing the expression of GFP
(Yue et al., 2008). The
dominant-negative N-cadherin receptor was constructed by cloning the
extracellular and transmembrane domains of chick N-cadherin in pEGFP-N1
(Invitrogen) as a BglII-SalI fragment, amplified using
primers 5'-TTGATATAGATCTCGGGCCATGTGCC-3' and
5'-TTATCACGGCGCGTCGACCATACTACGAACATCA-3' from a full-length
N-cadherin cDNA. To label cells with GFP, they were transfected with pEGFP-N1.
Pdgfa knockdown was achieved by electroporation of a chick
Pdgfa siRNA Smartpool (10 µg/ml) custom designed by Dharmacon
using previously described procedures
(Leslie et al., 2007). The
full-length N-cadherin expression construct was made by PCR amplification of
N-cadherin with the primers 5'-TTGATATAGATCTCGGGCCATGTGCC-3' and
5'-CGACCGCGGTGGCGGCCGCTCTAGAACT-3' and cloning as a
BglII-NotI fragment into pCAβ/GFP. Proper expression of
these constructs was tested by transfection into COS7 cells followed by
western analysis of cell lysates.
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).
Antibodies used were phosphospecific (S473) AKT (PKB) (Cell Signaling
Technologies), β-catenin (15B8, Sigma), chick N-cadherin (6B3), chick
integrin 6 (P2C6C4) and chick integrin β1 (CSAT), chick laminin
(310/31-2) and chick fibronectin (B3/D6, Developmental Studies Hybridoma
Bank). Secondary antibodies were HRP-conjugated (Promega) and detection was
performed using the Tyramide Signal Amplification System (Molecular Probes).
For neutralising PDGFR ligands, 0.5 µl 100 µg/ml recombinant
mouse PDGFR-Fc chimera (R&D Systems, Cat. 1062-PR-050) was
microinjected into each embryo. For PDGF stimulation experiments, heparin
beads were soaked in recombinant rat PDGFAA short form (R&D Systems, Cat.
1055-AA-050) or human PDGFAA (R&D Systems, Cat. 221-A) long form at 20
µg/ml at room temperature for 3 hours, followed by several washes with PBS
before implantation into the embryo. FGF4 beads were prepared as described
(Yang et al., 2002). |
RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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and its ligands Pdgfa, Pdgfb and Pdgfc.
Pdgfa expression was detected in the epiblast from HH stage 3 onwards, on
both sides of the primitive streak. The intensity of expression increased from
posterior to anterior, as far as a region just anterior of the node where
expression stopped abruptly at the anterior border of the future neural plate
(Fig. 1A-F'). Expression
of Pdgfr was detected from HH3 onwards in cells in the
primitive streak that were about to ingress, and in migrating mesoderm cells
(Fig. 1G-L). Expression
appeared to be upregulated in the forming somites, especially in the
sclerotome, from HH7-8 onwards (Fig.
1G'-L'). The Pdgfr message in the
forming somites was highly localised in the side lining the somatic cavity
(Fig. 1K'). Expression of
Pdgfb was very weak up to HH8
(Fig. 1M,N), making it unlikely
that PDGFAB or PDGFBB are major signalling molecules during these early stages
of development. Expression of Pdgfd was undetectable (not shown).
Pdgfc, however, showed weak expression in the early streak stages
(Fig. 1O-Q), predominantly in
the forming brain, neural tube and in the forming somites
(Fig. 1Q'-Q''').
Blocking signalling through PDGFR blocks migration of mesoderm cells away from the primitive streak
The expression pattern of Pdgfr in the mesoderm and the
strong expression of Pdgfa in the ectoderm suggested that they might
be the major signalling partners involved in the control of migration of
mesoderm cells. We therefore investigated the effects of interfering with
PDGFR and PDGFA signalling on mesodermal cell migration. Expression of
a dominant-negative (dn) PDGFR from a vector with an internal ribosome
entry site driving the simultaneous expression of GFP allowed us to track the
movements of transfected GFP-expressing cells during gastrulation. In
experiments in which a control construct expressing GFP was electroporated in
one half of the epiblast of a HH3 embryo, we observed that the transfected
epiblast cells migrated towards the primitive streak, ingressed, then moved
out on both sides of the streak to form paraxial mesoderm (5/5 embryos)
(Fig. 2A). Expression of
dn-PDGFR in one half of the epiblast resulted in the migration of transfected
cells towards the primitive streak, but the ability of these cells to migrate
away from the streak was severely compromised (8/8 embryos)
(Fig. 2B). To investigate
whether this effect might be due to a failure of the transfected cells to
undergo EMT, we sectioned some of the embryos. This revealed that most of the
cells expressing higher levels of the dn-Pdgfr construct
accumulated in the upper layers of the streak
(Fig. 2B'). Some cells
expressing lower levels of the dn-Pdgfr construct managed to
ingress and migrate a little distance away from the streak. In control
embryos, most cells ingressed through the streak and then migrated away from
the streak (Fig.
2A').
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construct grafted in the streak
of an untransfected embryo also showed an almost complete inhibition of
migration away from the streak (Fig.
2D), whereas control cells expressing only GFP underwent EMT and
migrated out to form somites and lateral plate mesoderm
(Fig. 2C-C''). Sectioning
confirmed that the dn-Pdgfr-expressing cells were found, as
before, in the deeper layers of the embryo, but the majority of the cells
failed to migrate away (Fig.
2D'). Staining for β-catenin, a protein enriched in
epithelial adherens junctions, confirmed that many of the cells expressing
dn-Pdgfr had undergone EMT, as they did not express the high
levels of β-catenin associated with the apical adherens junctions seen
typically in epiblast cells (Fig.
2D'').
Depletion of PDGFR ligands blocks the migration of mesoderm cells
To investigate signalling through PDGFR in more detail, we depleted
PDGFR ligands by injection of recombinant PDGFR-Fc protein,
while monitoring the migration of GFP-labelled streak cells
(Fig. 2E). GFP-expressing
streak cells derived from a GFP-transfected donor embryo were grafted into a
homotypic position in an age-matched unlabelled host embryo, after which the
host embryo was injected with recombinant PDGFR-Fc protein. In these
experiments, very little migration of the GFP-expressing cells out of the
streak was observed (Fig.
2E',E''). Injection of the carrier protein BSA, used to
stabilise the PDGFR-Fc protein, had no effect on the migration of the
mesoderm cells (data not shown). Embryos injected with PDGFR-Fc protein
consistently developed much less well than the control embryos. Embryos
injected with BSA developed a recognisable head and somites (5/6), whereas
those injected with PDGFR-Fc did not form somites and showed only
limited head development (6/6) (Fig.
2E).
Knockdown of Pdgfa impairs cell migration away from the primitive streak
PDGFR can bind several PDGF ligands and therefore the observed
phenotypes could be caused by depletion of any one or a combination of
ligands. Since Pdgfa is the most prominent PDGF ligand expressed
during the early stages of streak development, we knocked down Pdgfa
expression by electroporation of Pdgfa siRNA to study the effects on
cell migration away from the streak. (Fig.
3A,A'). Our previous experiments established that siRNA is
transfected effectively in all cells of the transfected region. Although GFP
expression from transfected plasmid is more mosaic
(Leslie et al., 2007),
simultaneous transfection with GFP does allow us to track the movement of at
least some of the cells. Embryos transfected with GFP alone, or with a mixture
of GFP and a control siRNA (cyclophylin siRNA), served as controls
(Fig. 3B,B'). Embryos
transfected with control siRNA showed normal development and extensive
migration of GFP-expressing cells towards the streak, followed by ingression
and migration away form the streak to form paraxial mesodermal structures
(Fig. 3B,B'). The embryos
developed well and after 22 hours had reached HH8 or 9 (26.2% and 25%,
respectively; n=24 embryos) and showed normal Pdgfa
expression (Fig. 3B').
Embryos transfected with Pdgfa siRNA did not develop well
(Fig. 3B,B'). Development
was arrested at the extended streak stage at HH4, 5 and 6 (17.9%, 41% and
20.5%, respectively; n=39 embryos) and the GFP-expressing cells were
generally unable to migrate out of the streak, similar to what was observed
after expression of the dn-Pdgfr construct (compare
Fig. 2B with
Fig. 3A). In situ hybridisation
showed an almost complete absence of Pdgfa expression in these
embryos (Fig. 3A'). These
experiments show that PDGFA signalling is crucial in the control of mesodermal
cell movement away from the streak.
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To investigate whether the lack of mesodermal cells on the side of the streak where Pdgfa expression was reduced resulted from defective migration, or from a possible indirect effect on their differentiation, we assayed the effect of half-sided Pdgfa knockdown on the migration of GFP-labelled mesoderm cells derived from a normal donor embryo (Fig. 4E). In these experiments, streak cells are provided with a choice, either to migrate to the side of the embryo where Pdgfa expression is normal or to that where Pdgfa expression has been knocked down (Fig. 4C). In the majority of cases, the cells preferentially migrated to the side where Pdgfa expression was not affected, avoiding the side where Pdgfa expression had been knocked down (Fig. 4B,D,F).
PDGFA can act as an instructive ligand for the migration of mesoderm cells
The data in frogs and fish suggest that PDGFA could have an instructive
role in guiding mesoderm cells to more-anterior positions after their
migration out of the streak, acting as an attractive guidance cue. We tested
whether ectopically applied recombinant PDGFAA could act as a chemoattractant
in an in vivo chemotaxis assay (Yang et
al., 2002). We grafted GFP-expressing anterior or middle streak
cells into the area opaca of an unlabelled host embryo, in between two
heparin-coated beads soaked in recombinant rat PDGFAA (short form) or carrier
protein, respectively (Fig.
5A). In a significant number of cases, there was a preferential
migration in the direction of the beads soaked in recombinant rat PDGFAA
(Fig. 5B-E) (7 positive
responses in 12 experiments). Although many cells moved in the direction of
the PDGFAA-coated bead, we note that the migration of the cells was not as
strong or as directional as we had described previously for the movement of
these cells towards FGF4 in the same assays
(Yang et al., 2002). This
could indicate that the short form of PDGFAA used in the current experiment
diffused rapidly and did not form a stable gradient. However, similar
experiments performed with the long form of PDGFAA, which contains an
N-terminal matrix-binding motif and is assumed to be less diffusible, did not
show significantly different results (data not shown). The finding that
localised PDGF signalling elicits only a weak directional movement response,
but that blocking PDGF signalling results in a strong inhibition of migration
of mesoderm cells, strongly suggests that the role of PDGF signalling is not
confined to providing directional information, but that it might also be
required for another aspect of mesodermal cell movement. We therefore
investigated whether inhibition of PDGF signalling affects movement to ectopic
sources of other attractants, in this case FGF4
(Fig. 5F). Experiments in which
GFP and dn-Pdgfr-expressing cells were challenged with FGF4
confirmed that streak cells expressing GFP show a strong chemotactic response
to FGF4 (Fig. 5G-J). Cells
expressing the dn-Pdgfr construct, however, were severely
impeded in their ability to migrate towards the FGF4 source (7/8
experiments).
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(compare
Fig. 2B with
Fig. 6C). Furthermore,
expression of the dn-N-cadherin construct also abolished the ability of
mesoderm cells to migrate towards a localised FGF4 signal in the area opaca
(Fig. 6E,F), again mimicking
the effect of the expression of dn-PDGFR (compare
Fig. 5I with
Fig. 6F). These results show
that N-cadherin function is required for the migration of mesoderm cells and
that PDGF signalling plays a major role in the control of N-cadherin
expression on mesoderm cells. We noted that an ectopic FGF4 source strongly
attracts N-cadherin-expressing cells (Fig.
6G,G1,G2). We found little direct effect of FGF4 signalling on
N-cadherin RNA expression (data not shown). These experiments show that
mesoderm cells that respond to an FGF signal express N-cadherin and confirm
that FGF4 appears to be a much more potent attractant than PDGFA
(Fig. 6 compare B with G). An
important question is whether the inhibitory effects of decreased PDGF
signalling on cell migration can be attributed to reduced N-cadherin
expression? To investigate this, we co-transfected the dn-PDGFR with a
full-length N-cadherin expression construct. Cells co-expressing dn-PDGFR and
full-length N-cadherin migrated out of the streak on both sides of the embryo
(Fig. 7A,A1,A2), whereas cells
that were co-transfected with dn-PDGFR and a GFP expression construct remained
confined to the streak and ectoderm/neural tube
(Fig. 7B,B1,B2). Expression of
dn-PDGFR resulted in severe downregulation of endogenous N-cadherin expression
in the few cells that managed to ingress into the mesoderm in a
cell-autonomous manner (see Fig. S3 in the supplementary material). We found
no evidence for strong effects of PDGF signalling on the regulation of
integrins or extracellular matrix formation: we did not detect any effects of
siRNA-mediated knockdown of Pdgfa on integrin 6, integrin
β1, laminin or fibronectin expression (see Fig. S4 in the supplementary
material). Taken together, these experiments show that the major defects
caused by reduced PDGF signalling can be accounted for by its effects on
N-cadherin expression.
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signals through PI3 kinase to AKT activation is activation of PI3 kinase
(Klinghoffer et al., 2002;
Pickett et al., 2008). In
previous experiments, we showed that PIP3 signalling is necessary for the
directionality of the movement response of mesoderm cells during gastrulation
in the chick embryo (Leslie et al.,
2007). In the present study, we observed that the expression
pattern of PDGFR and the phosphorylation pattern of AKT (PKB) on serine
473 are very similar during the early stages of gastrulation
(Fig. 8, compare A with C).
Sectioning showed that AKT phosphorylation was essentially confined to
mesoderm cells, the same cells that also express PDGFR (compare
Fig. 8C' with
Fig. 1I').
High-magnification observations showed that activated AKT was, to a large
extent, localised at the plasma membrane of mesoderm cells (data not shown).
Inhibition of PDGF signalling through application of the PDGFR inhibitor
AG1296 resulted in severe depression of AKT phosphorylation in the embryo
(Fig. 8E), suggesting that the
PDGF signalling pathway is one of the major activators of PI3 kinase
signalling in vivo. Application of this inhibitor also resulted in a
significant block to development, similar to that observed after
downregulation of Pdgfa. Local application of the short form of
PDGFAA on beads resulted in the activation of the PI3 kinase pathway, as
measured by AKT phosphorylation some distance away from the beads
(Fig. 8D,D'), whereas
application of the long form resulted in activation of AKT phosphorylation in
the immediate vicinity of the beads (data not shown). These experiments show
that PDGF signalling is a major activator of the AKT pathway in vivo, and
through this it may not only control the directionality of cell movement but
also modulate N-cadherin expression. To investigate the latter directly, we
measured N-cadherin protein expression after incubation of embryos for 6 hours
in the presence of the PI3 kinase inhibitor LY294002. The results clearly
showed that inhibition of PDGF signalling through the PDGFR inhibitor AG1269,
as well as the PI3 kinase inhibitor LY294002, almost completely inhibited
N-cadherin expression as measured with N-cadherin antibody (see Fig. S5 in the
supplementary material), while there was no effect on N-cadherin RNA
expression over this period of time (data not shown). Together, these data
make it highly likely that PDGF signalling controls N-cadherin expression
through activation of the PI3 kinase pathway.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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are the main ligand and receptor expressed during early chick development.
PDGFA signalling through PDGFR is needed for the migration of cells
away from the primitive streak, as migration is blocked effectively by
transfection with dn-PDGFR or by depletion of its ligands. The results
of this study show that PDGF signalling is required for N-cadherin protein
expression. N-cadherin expression is known to be widespread on mesodermal
cells in the chick embryo, especially in presomitic and somitic mesoderm
(Hatta et al., 1987). Our
experiments show that N-cadherin function is required for efficient mesodermal
cell migration, even in the presence of strong guidance cues such as an
ectopic source of FGF4. N-cadherin expression has recently been shown to be
important in mesodermal cell migration in zebrafish
(Warga and Kane, 2007), and
disruption of N-cadherin in mice results in embryonic lethality E10 and in
severe adhesion defects in the primitive heart, as well as malformation and
fragmentation of somites (Horikawa et al.,
1999; Radice et al.,
1997), suggesting defective migration of mesodermal precursor
cells during the earlier stages of gastrulation, in agreement with our present
findings in chick.
It is likely that N-cadherin expression is required for the mesoderm cells
to gain traction from their neighbouring cells, as suggested by the strong
enrichment in cellular protrusions contacting other cells. Although cells also
need to gain traction from the extracellular matrix, and PDGF signalling has
been implicated in regulating this in other systems, our experiments have not
uncovered a role for PDGF signalling in the expression of integrins or
extracellular matrix components. However, it is likely that PDGF signalling,
possibly through N-cadherin expression, modulates integrin function rather
than expression and, thereby, cell-matrix interactions
(Chen and Gumbiner, 2006;
Czirok et al., 2004;
Nagel et al., 2004;
Symes and Mercola, 1996).
We do not yet know exactly how PDGF signalling controls N-cadherin
expression, but it most likely involves a post-translational mechanism as we
do not detect a direct effect of PDGF signalling on N-cadherin RNA expression
(see Fig. S2 in the supplementary material). Although there are many
possibilities for post-translational control of N-cadherin expression, it has
been shown that the extracellular domain is proteolytically cleaved by
metalloproteases, notably ADAM10 (Maretzky
et al., 2005; Reiss et al.,
2005; Uemura et al.,
2006), and that after further processing the resulting
intracellular domain of N-cadherin may have a nuclear signalling function in
early development (Shoval et al.,
2007).
In our experiments, PDGFA acted as a weak attractant and therefore we
cannot rule out the possibility that it has an instructional role in
mesodermal cell migration. The RNA expression pattern would suggest that
gradients of PDGFA exist that could direct migration of mesoderm cells in an
anterior direction, as proposed in frogs and fish
(Ataliotis et al., 1995;
Montero et al., 2003;
Nagel et al., 2004). Ectopic
sources of both the long and short form of PDGFA can attract mesoderm cells
(Fig. 5), and the experiments
in which Pdgfa expression in the epiblast was reduced in one half of
the embryo tended to show less anterior migration in the half with reduced
PDGFA expression (Fig. 4B,D),
compatible with the hypothesis that PDGFA acts as an instructive signal.
The effects of manipulating PDGF signalling in the chick embryo appear to
be stronger than those described in the mouse embryo after deletion of
Pdgfr or Pdgfa
(Bostrom et al., 1996;
Soriano, 1997). The strong
effects observed with dn-PDGFR in the chick embryo could be due to its
ability to form dimers with PDGFRβ and thus to effectively inhibit all
PDGF signalling by inactivating all possible receptor complexes. Likewise,
injection of PDGFR-Fc fragments will bind all ligands for this receptor
(the PDGFAA, PDGFAB and PDGFCC dimers) and thus potentially block signalling
by multiple ligands (Heldin and Westermark,
1999; Tallquist and
Kazlauskas, 2004). However, the very strong inhibitory effects of
Pdgfa knockdown on the migration of mesoderm cells in the chick
embryo suggest that this is a major in vivo signal, in agreement with the low
expression levels of Pdgfb and Pdgfc observed in the early
chick embryo.
The expression pattern of PDGFA in the chick shows considerable similarity
to the pattern of AKT S437 phosphorylation. PDGF stimulation induces AKT
phosphorylation, whereas application of the PDGFR inhibitor AG1296 reduces in
vivo AKT phosphorylation on S473 almost to zero, as well as blocking
development. These findings strongly suggest that signalling through PDGFR is
a major in vivo signal activating the PI3 kinase pathway during gastrulation
and that this pathway is crucial for normal development. Our data strongly
suggest that the PDGF signalling-dependent regulation of N-cadherin expression
is mediated by the PI3 kinase signalling pathway, which therefore not only
controls the directionality but also the speed of cell migration. These data
are in good agreement with findings in zebrafish in which application of
AG1295 resulted in slow and defective anterior migration of prechordal plate
mesoderm cells (Montero et al.,
2003).
In conclusion, it is evident that PDGF signalling is required for the
proper migration of mesoderm cells during the early stages of gastrulation in
the chick embryo. PDGFA is the major form of PDGF expressed during the early
stages of chick embryo development. We propose that the PDGFA signalling
domain outlines a route along which paraxial mesoderm cells move efficiently,
a process that is likely to involve control of the expression of N-cadherin on
mesodermal cells as well as the provision of guidance information for the
cells to move forward. This guidance system has to cooperate with other
signalling systems, such as the FGF8-mediated repulsion that is required to
send cells away from the streak and the FGF4-mediated attractive movement
towards the anterior and back towards the midline
(Fig. 9)
(Yang et al., 2002).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/21/3521/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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