1 Department of Zoology, University of Toronto, 25 Harbord Street, Toronto,
Ontario M5S 3G5, Canada
2 Department of Biochemistry, Boston University School of Medicine, 715 Albany
Street, Boston, MA 02118, USA
* Author for correspondence (e-mail: winklbauer{at}zoo.utoronto.ca)
Accepted 16 February 2004
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SUMMARY |
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Key words: Xenopus, PDGF, Gastrulation, Cell migration
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Introduction |
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In Xenopus, migration of dorsal anterior mesoderm cells across the
ectodermal blastocoel roof (BCR) relies on their interaction with a network of
fibronectin (FN) containing fibrils
(Winklbauer and Keller, 1996),
the extracellular matrix of the BCR
(Nakatsuji and Johnson, 1983a
;
Winklbauer, 1998
). FN
contributes to the adhesion of mesoderm cells to the BCR, and, most
importantly, it elicits the formation of lamellipodia, the locomotory
organelles of these cells (Winklbauer,
1990
; Ramos and DeSimone,
1996
; Winklbauer and Keller,
1996
).
In pioneering work, Nakatsuji and Johnson
(Nakatsuji and Johnson, 1983b)
showed that in addition to serving as permissive substratum, the BCR
extracellular matrix contains guidance cues that determine the direction of
migration. When BCR is explanted to deposit its matrix on an inert surface,
dispersed mesodermal cells from several amphibian species migrate on this
conditioned substratum directionally, recognizing cues that guide them to the
animal pole region (Nakatsuji and Johnson,
1983b
). In Xenopus, migration in vitro is also
directional, although only when aggregates of mesodermal cells are used to
probe conditioned substratum, instead of single cells
(Fig. 1A). Moreover, in the
Xenopus embryo, anterior mesoderm cells extend locomotory processes
towards the animal pole, confirming that migration is directional in vivo
(Winklbauer and Nagel,
1991
).
|
PDGF signaling is involved in a variety of processes, including early
embryonic development, formation of the nervous system, heart and lung
development, and angiogenesis. It has also been implicated in several
pathological conditions, such as oncogenesis, atherosclerosis, lung and kidney
fibrosis, and wound healing. Major target processes of PDGF signaling are the
regulation of proliferation, and the control of cell motility and migration
(Ataliotis and Mercola, 1997;
Heldin and Westermark, 1999
;
Betsholtz et al., 2001
).
The PDGF family comprises four members: PDGFA and PDGFB, which form homo-
and heterodimers (Heldin and Westermark,
1999), and the CUB-domain containing PDGFC and PDGFD
(Li et al., 2000
;
Bergsten et al., 2001
). In the
Xenopus gastrula, PDGFA is expressed in the BCR, whereas PDGFB is
barely detectable, suggesting the exclusive presence of PDGFAA homodimers
(Mercola et al., 1988
). The
A-chain occurs in two splice variants, a long form (lfPDGFA) that contains a
C-terminal site involved in extracellular matrix binding, and a short form
(sfPDGFA) lacking the matrix-binding motif
(Raines and Ross, 1992
;
Andersson et al., 1994
). RNAs
of both variants are present in the Xenopus gastrula
(Mercola et al., 1988
). Of the
possible PDGF receptor dimers (PDGFR
, PDGFR
ß and
PDGFRßß), only PDGFR
recognizes PDGFAA
(Ataliotis and Mercola, 1997
;
Heldin and Westermark, 1999
),
and the PDGFR
isoform is in fact expressed in the Xenopus
mesoderm at gastrulation (Jones et al.,
1993
).
The complementary pattern of expression, with PDGFA ligand in the BCR and
its receptor in the adjacent mesoderm
(Ataliotis et al., 1995),
suggests a role for paracrine PDGFA signaling in mesoderm development.
Disruption of PDGF function by means of a dominant-negative PDGFR
leads
to aberrant movement of the mesoderm
(Ataliotis et al., 1995
), and
treatment of mesoderm aggregates with PDGFAA protein stimulates spreading on
FN in vitro (Symes and Mercola,
1996
). However, it is not known which of the several
mesoderm-driven gastrulation movements in Xenopus is actually
controlled by PDGFA.
We identify a distinct in vivo function for PDGFA signaling in early Xenopus development. We show that interfering with PDGF function randomizes mesoderm migration on conditioned substratum in vitro, and disrupts the orientation of migratory cells in the embryo. We propose that matrix-bound PDGFAA could be instructive in mesoderm guidance during gastrulation.
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Materials and methods |
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Substratum conditioning
BCR explants were held against the bottom of Greiner tissue culture dishes
(TC grade) by strips of coverslip resting on both ends on silicone grease.
Before removing BCR explants by aspiration with buffer, their outlines and
orientation were marked on the dish. Substrata were saturated with 50 mg/ml
bovine serum albumin (BSA) and used in the migration assay
(Winklbauer and Nagel, 1991)
(Fig. 1A). FN-coated substratum
was prepared by incubating Greiner tissue culture dishes with bovine plasma
fibronectin solution (Sigma) at 200 µg/ml for 30 minutes, followed by
saturation of the substratum with 50 mg/ml BSA.
Directional migration assay
Displacement of anterior mesoderm explants (about 200 cells per explant) on
conditioned substratum (one explant per substratum) was observed on an
inverted microscope. Positions of explants were recorded at the start of the
experiment, and 1 hour later. The positions after 1 hour are indicated by
circles, with starting points all superimposed at the origin. Movement towards
the position of the animal pole of the BCR was assigned positive values on the
y-axis. Using a one-sided sign test, the null hypothesis that
explants move animally and vegetally with equal frequency was tested against
the alternative hypothesis, that aggregates move preferentially towards the
animal pole.
FN fibril and actin cytoskeleton staining
Embryos were fixed in 4% formaldehyde, BCRs were removed with needles,
blocked in 5% BSA and stained with rabbit antibody to Xenopus FN
(Winklbauer, 1998), and
FITC-conjugated secondary antibody. Anterior mesoderm cells on conditioned
substratum were fixed after 1 hour in 4% formaldehyde/0.1% Triton-X-100, and
stained with 5 µM TRITC-conjugated phalloidin (Sigma) for 30 minutes.
Scanning electron microscopy
Embryos were fixed in 2.5% glutaraldehyde in MBS, and BCRs were removed
with needles. Specimens were postfixed in 1% OsO4, dehydrated in a
graded ethanol series, critical point dried and sputter coated with
gold-palladium.
In situ hybridization
In situ hybridization was modified from the methods of Harland
(Harland, 1991). Alkaline
phosphatase substratum was BM Purple (Roche). For the antisense Xbra
digoxigenin probe, plasmid DB30 (gift of M. Sargent, London) was linearized
with BgIII and used as a template for transcription with T7 RNA
polymerase. chordin and gsc probes
(Sasai et al., 1994
;
Cho et al., 1991
) (gifts from
H. Steinbeisser, Heidelberg) were generated by linearizing clones 59 and H7,
respectively, in pBluescript SK() with EcoRI and transcription
with T7 polymerase.
Constructs, mRNA synthesis and injection
Plasmids pCS2 containing wild-type Xenopus PDGFR, a
dominant-negative version of this receptor, PDGFR37, or a PDGFR
missense construct (Ataliotis et al.,
1995
), were linearized with NheI and transcribed with T7
polymerase. pGHE2 containing the wild-type long form of Xenopus PDGFA
(Mercola et al., 1988
),
lfPDGFA, was linearized with NheI and transcribed with T7 polymerase.
A C-terminally truncated form of lfPDGFA lacking the matrix-binding motif
(amino acid residues 198-227) was generated by PCR using pGHE2-PDGFA
(Mercola et al., 1988
) as a
template (Fig. 1B). The
truncated form of PDGFA (trPDGFA) was subcloned into the
BamH1/EcoR1 site of pCS2 and used for in vitro transcription
with SP6 polymerase after linearization with XhoI. Plasmid pGHE2
harboring a processing defective mutant of mouse PDGFA that acts as a
dominant-negative mutant in Xenopus
(Mercola et al., 1990
) was
linearized with NheI and transcribed with T7 polymerase. Embryos were
injected at the four-cell stage into all four blastomeres, marginally with 250
pg mRNA per embryo (PDGFR
, PDGFR37, missense PDGFR
), or animally
with 400 pg mRNA per embryo (lfPDGFA, trPDGFA) or 200 pg mRNA per embryo
(dnPDGFA).
Reagents
Human recombinant PDGFAA protein, long form (TEBU, Germany) was used at 50
ng/ml. Tyrphostin AG 1296, an inhibitor of PDGF receptor tyrosin kinases, and
AG 43 control tyrphostin (Calbiochem-Novabiochem) were dissolved in DMSO (10
mg/ml) and used at a final concentration of 5 µM, Wortmannin (Sigma) at
100-200 nM. Anti-PDGFA morpholino antisense oligonucleotide (GeneTools,
5'-AGAATCCAAGCCCAGATCCTCATTG-3')
and 5 bp mispaired control morpholino (exchanged bases underlined) were
injected at 60 ng per embryo.
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Results |
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The inhibitory PDGFR37 does not impair mesoderm induction in
Xenopus (Ataliotis et al.,
1995). However, in order to ensure that its effect on migration is
not due to subtle alterations of mesoderm development, PDGFR
signaling
was blocked directly in migrating explants taken from normally developed
mesoderm, by treatment with the cell permeable inhibitor, AG1296
(Kovalenko et al., 1997
). This
inhibition (Fig. 2G), but not
treatment with a similar control substance, AG43
(Fig. 2H), also abolished
directionality, suggesting that PDGFR signaling is required during actual
migration, and not during formation or patterning of the mesoderm. As the
previously reported effect of PDGFR
signaling on mesoderm motility was
mediated by PI3 kinase (Symes and Mercola,
1996
), aggregates on conditioned substratum were also treated with
an inhibitor of phosphatidylinositol 3 (PI3) kinase, Wortmannin. In its
presence, migration was non-directional
(Fig. 2I), implicating this
kinase in the pathway that regulates directionality of movement.
PDGFA expression in the BCR is essential for directional migration
PDGF signaling was also disrupted by blocking PDGFA in the BCR. Expression
of the dominant negative dnPDGFA (Mercola
et al., 1990) in the BCR leads to non-directional migration of
normal mesoderm on substratum conditioned with injected BCR
(Fig. 3A). Likewise, impeding
translation of endogenous PDGFA mRNA in the BCR by morpholino antisense
oligonucleotide randomizes translocation
(Fig. 3B), whereas injection of
a 5-mispaired control morpholino has no such effect
(Fig. 3C). To further
demonstrate specificity of the morpholino, we employed the fact that mesoderm
differentiation can be induced by exogenous PDGFA in animal caps expressing
PDGFR
(Ataliotis et al.,
1995
). In contrast to untreated caps, those co-injected with
receptor and with Xenopus PDGFA mRNA containing the morpholino target
site develop transparent bulges indicative of ventral-type mesoderm.
Additional injection of anti-PDGFA morpholino completely blocks this effect,
but it can be rescued by treatment of explants with PDGFA protein
(Fig. 3I). This suggests that
the morpholino specifically inhibits Xenopus PDGFA function.
|
To guide cells on conditioned substratum, PDGFA should be associated with the extracellular matrix. To see whether the matrix-binding capability of lfPDGFA is essential for its interference with directional migration, we deleted the C-terminal matrix-interacting motif from the wild-type lfPDGFA. When this truncated form, trPDGFA, is expressed in the BCR, directionality of migration on respective conditioned substratum is not affected (Fig. 3F). Apparently, only matrix-binding PDGFA can interfere with mesoderm guidance. This implies that it is the endogenous matrix-binding lfPDGFA that is involved in vivo in determining the direction of migration on the BCR.
When trPDGFA is expressed in the mesoderm, migration on normal conditioned substratum is randomized (Fig. 3G). This shows that the trPDGFA construct is functional, and that its ectopic expression in the mesoderm interferes with the orientation of aggregates on normal substratum. In addition, exposure of mesoderm aggregates to human recombinant PDGFAA protein during migration on normal conditioned substratum abolishes directionality (Fig. 3H). This confirms that proper PDGF signaling is required during migration, and not earlier, as, for example, during patterning. Furthermore, as in the previous experiment, it suggests that the abundant, uniform presence of soluble PDGFAA interferes with the ability of migrating cells to read the cues on the conditioned substratum, arguing also in favor of an instructive role of matrix-binding lfPDGFA in cell guidance.
PDGF signaling is required for orientation and protrusive activity of migrating cells in the embryo
By the mid-gastrula stage, vegetal rotation
(Winklbauer and Schurfeld,
1999) has moved the anterior dorsal mesoderm against the BCR. The
blastocoel floor is concave, and the mesoderm exhibits a pointed leading edge
that is apposed to the BCR (Fig.
4A). Both the expression of inhibitory dnPDGFA
(Fig. 4B) and the
overexpression of wild-type lfPDGFA (Fig.
4C) in the BCR affect gastrula morphology in a similar way: the
mesoderm does not develop a pointed leading edge, and the blastocoel floor
does not become concave (Fig.
4B,C). This morphology is consistent with mesoderm movement across
the BCR being impeded while vegetal rotation continues to internalize vegetal
cell mass.
|
|
To exclude the possibility that disorientation or absence of protrusions
are due to defective FN fibril network formation
(Winklbauer and Nagel, 1991;
Winklbauer and Keller, 1996
),
the BCR was stained with FN antibody. A normal FN fibril network
(Fig. 4G) is present in dnPDGF
(Fig. 4H) or lfPDGFA
overexpressing embryos (Fig.
4I), or after anti-PDGF morpholino injection (not shown).
Together, these results suggest that orientation of migrating mesodermal cells
and normal protrusive activity in the embryo depend on proper PDGFA
signaling.
PDGF signaling controls mesoderm translocation during gastrulation
To determine what consequences disoriented migration has on gastrulation,
mesoderm translocation was inferred from the expression pattern of the
mesoderm markers Xbra (Smith et
al., 1991,Smith et al.,
1991
), chordin (Sasai
et al., 1994
) and goosecoid (gsc)
(Cho et al., 1991
)
(Fig. 6). Xbra is
initially expressed in a subequatorial ring
(Fig. 6A), and
chordin, a marker of head and axial mesoderm, in the dorsal lip
(Fig. 6D). Expression of
dnPDGFA or overexpression of lfPDGFA in the BCR does not abolish this pattern
(Fig. 6B,C,E,F). This
supplements the finding that inhibition of PDGF receptor function does not
affect mesoderm regionalization (Ataliotis
et al., 1995
).
|
Deficient mesoderm movement most probably contributes to the aberrant later
development of PDGF signaling-defective embryos. As previously described
(Ataliotis et al., 1995),
expression of inhibitory PDGFR37 results in diminished head structures, a
shortened, bent axis and split tails (Fig.
7A). A comparable phenotype was obtained after expression of
dnPDGFA or wild-type lfPDGFA in the BCR
(Fig. 7C,D). Phenotypes were
less pronounced after injection of PDGFA morpholino
(Fig. 7B), 5-mismatch control
morpholino had no effect (Fig.
7E).
|
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Discussion |
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We have determined that mesodermal expression of the same dominant negative
PDGFR-37 used by Ataliotis et al.
(Ataliotis et al., 1995)
interferes with directional movement on conditioned substratum. This suggests
that the previously observed gastrulation defects can at least in part be
explained by an impaired guidance mechanism. Further, our results show that
for directional translocation, PDGFA/PDGFR
signaling is required during
actual migration, and not during mesoderm development: treatment of mesoderm
explants with various agents during the 1 hour period of migration on
conditioned substratum is sufficient for interference with directionality, and
normal mesoderm explants move non-directionally on substratum conditioned with
PDGFA-defective BCR.
In all these cases, migration is not inhibited, but randomized with respect
to direction, ultimately resembling translocation on an artificial isotropic
FN substratum. Coupled with the fact that blocked PDGFA/PDGFR signaling
has the same effect on directionality as a spatially homogeneous, artificial
PDGF signal, this argues that PDGFA/PDGFR
activity is instructive, and
directly involved in establishing the direction of migration. However, we
cannot strictly rule out the possibility that PDGF signaling is permissive,
but is required at exactly the normal level of activity. As expected for a
matrix-dependent guidance cue, only the lfPDGFA that contains the
matrix-binding motif is able to disrupt directional migration when
overexpressed in the BCR.
Several possibilities exist to explain how substratum-dependent cues may
guide translocation. One set of mechanisms involves the trapping of randomly
migrating cells at a target site. For example, haptotaxis leads to the
accumulation of cells in regions of increased substratum adhesiveness
(Carter, 1965). However,
BCR-conditioned substratum exhibits no spatial differences in adhesiveness
(Winklbauer and Nagel, 1991
).
Other trapping mechanisms can be envisioned, for example, mechanisms based on
the differential stimulation of motility by the substratum. However, these
options are all restrained by the fact that in Xenopus, the mesoderm
migrates as a coherent mass, where cells have limited freedom for independent
random movement. On conditioned substratum, whole explants move persistently
towards the animal pole, without random excursions to explore the substratum.
By contrast, single mesoderm cells migrate randomly, but they are unable to
accumulate in the animal pole area on conditioned substratum
(Winklbauer et al., 1992
).
Together, these observations argue against a trapping mechanism of mesoderm
guidance.
A different type of guidance mechanism relies on orienting each migrating
cell towards its target, as in eukaryote chemotaxis. The hallmark of such a
mechanism, the presence of polarized cells all pointing with their locomotory
processes towards the target of migration, is actually observed in the
Xenopus gastrula, in the shingle arrangement of anterior dorsal
mesoderm cells (Winklbauer and Nagel,
1991). This arrangement also forms when explants migrate
directionally on conditioned substratum, but not when they move randomly on FN
(Winklbauer et al., 1992
). In
the embryo, the shingle arrangement disappears when PDGFA function is
compromised by inhibition or overexpression. In both cases, protrusions point
with equal frequency towards and away from the animal pole. Apparently, a
precisely controlled mode of PDGFA/PDGFR
signaling is essential for
correct mesoderm cell orientation, such as when a gradient of matrix-bound
PDGFA serves as a chemoattractant for the migratory cells. In situ
hybridization data showing that PDGFA expression in the BCR is strongest at
the animal pole, and decreases towards the marginal zone
(Ataliotis et al., 1995
), are
consistent with this hypothesis.
Chemotaxis through PDGFR signaling is well documented (e.g.
Westermark et al., 1990
;
Kundra et al., 1994
;
Hansen et al., 1996
;
Ronnstrand et al., 1999
), and
PDGFR
promotes a chemotactic response at least in some cells (e.g.
Hosang et al., 1989
;
Ferns et al., 1990
;
Rosenkranz et al., 1999
;
Yu et al., 2001
), although
probably through a different mechanism (for reviews, see
Hayashi et al., 1995
;
Ronnstrand and Heldin, 2001
).
A great deal of work on PDGF-stimulated chemotaxis relates to cultured cells
in vitro. Recently, an in vivo role for the Drosophila PDGF/VEGF
receptor in guiding border cell migration during oogenesis has been
demonstrated (Duchek et al.,
2001
). By showing that PDGFA/PDGFR
signaling is required
for directional migration of mesoderm on its endogenous matrix substratum, as
well as for cell orientation in the embryo, we demonstrate such a guidance
function for this receptor-ligand pair for the vertebrate embryo.
Translocation of the coherent mesodermal cell mass
Single mesoderm cells translocate vigorously in vitro. Their lamellipodia
continuously extend, retract, divide and shift along the cell margin. When
lamellipodia of two cells meet, they retract immediately, showing contact
inhibition; however, lamellipodia are not prevented from underlapping another
cell in a lamellipodia-free region
(Winklbauer and Selchow, 1992;
Winklbauer et al., 1992
). In
the embryo, the mesoderm forms a coherent mass, and random protrusive activity
should lead to frequent encounters between processes and to their retraction
as result of contact inhibition. This could explain the rarity of such
protrusions in explants on artificial FN substratum
(Winklbauer et al., 1992
), but
also in disoriented mesoderm on PDGFA-defective BCR. That cells extend
processes more frequently in control embryos would then be an indirect
consequence of their parallel orientation, which prevents collision of
lamellipodia. Consistent with this, protrusion formation of single cells does
not depend on PDGF signaling, and extension of cytoplasmic processes is not
increased, but decreased on BCR overexpressing lfPDGFA, arguing against a
direct role for PDGFA in the stimulation of protrusive activity.
The immediate function of the PDGF-mediated guidance mechanism would then be to orient cell processes into a parallel array, to ensure that protrusions can form on every cell, and that processes do not mutually neutralize their effects by pointing in random directions. In turn, to engage all cells at the interface with the substratum in active migration seems essential if the mesodermal mass is to move as a whole.
Facilitating the shear movement between mesoderm and BCR is crucial, as is
evident from the phenotypes of PDGF signaling-compromised embryos. That
phenotypes are similar, regardless of whether PDGFA function is diminished or
increased, argues that they are due to a common primary defect. Moreover,
similar phenotypes are obtained when migration is blocked by FN antibodies
(Ramos and DeSimone, 1996), or
when the shear between BCR and mesoderm is mechanically alleviated by the
fusion of the two layers (Winklbauer et
al., 2001
). This suggests that the common denominator is the
attenuation of mesoderm advance across the BCR, as betrayed by marker gene
expression patterns. Because in all cases, convergent extension
(Keller, 2002
) is not
deficient in blastopore lip explants, shortened axes and the lack of
blastopore closure must be secondary effects, caused by indirect inhibition of
convergent extension in the embryo through arrested anterior mesoderm
movement. PDGFA and its receptor are also expressed in a complementary pattern
at later stages, e.g. in the cranial neural crest
(Ho et al., 1994
), and
interfering with its late functions may contribute to the larval phenotype
observed, for example, to head reduction.
PDGFA/PDGFR signaling and mesoderm migration in other vertebrates
As in the frog, PDGFA and PDGFR show complementary expression in the
mouse gastrula, with the ligand in the epiblast and visceral endoderm, and the
receptor in the mesoderm (Palmieri et al.,
1992
; Orr-Urtreger and Lonai,
1992
). The role of PDGFA and PDGFR
in mouse mesoderm
migration is not clear, however. The mutant patch includes the
deletion of the PDGFR
(Smith et
al., 1991
,Smith et al.,
1991
; Stephenson et al.,
1991
). Homozygous patch embryos are defective in mesoderm
development, but the primary cause for this has not been revealed
(Orr-Urtreger et al., 1992
).
Embryos carrying a targeted null mutation for PDGFR
accumulate various
defects during early development, but again, data bearing directly on mesoderm
migration are lacking (Soriano,
1997
). The same is true for embryos homozygous for targeted null
alleles of PDGFA (Bostrom et al.,
1996
).
In the zebrafish, PDGFA and PDGFR are co-expressed in all cells of
the gastrula (Liu et al.,
2002a
; Liu et al.,
2002b
). Despite this striking difference in expression, which
suggests autocrine signaling, PDGFA is involved in the control of mesoderm
migration, as in Xenopus. However, the role of PDGFA differs. In the
fish, PDGFA signaling through PI3-kinase and PKB is needed for cell
polarization and protrusion formation; inhibition of this pathway decreases
protrusive activity and the velocity of migration, but cells are still
oriented towards the animal pole (Montero
et al., 2003
). By contrast, Xenopus anterior mesoderm
cells are intrinsically polarized
(Winklbauer and Selchow, 1992
;
Wacker et al., 1998
), and the
frequency of protrusion formation is only indirectly affected in PGDFA
misexpressing embryos. In Xenopus, PDGF signaling is required for the
orientation of cells towards the animal pole, and for directional
migration.
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ACKNOWLEDGMENTS |
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