Download Mesoderm induction and the control of gastrulation in Xenopus

Development 108, 229-238 (1990)
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229
Mesoderm induction and the control of gastrulation in Xenopus laevis: the
roles of fibronectin and integrins
J. C. SMITH1, K. SYMESH, R. O. HYNES2'3 and D. DeSIMONE3*
' Laboratory of Embryogenesis, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA,
UK
^Howard Hughes Medical Institute and ^Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, USA
* Present address: University of Virginia, Health Sciences Center, Department of Anatomy and Cell Biology, Box 439, School of Medicine,
Charlottesville, VA 22908, USA
t Present address: Department of Cell and Molecular Biology, 385 LSA, University of California, Berkeley, CA 94720, USA
Summary
Exposure of isolated Xenopus animal pole ectoderm to
the XTC mesoderm-inducing factor (XTC-MIF) causes
the tissue to undergo gastrulation-like movements. In
this paper, we take advantage of this observation to
investigate the control of various aspects of gastrulation
in Xenopus.
Blastomcres derived from induced animal pole regions
are able, like marginal zone cells, but unlike control
animal pole blastomeres, to spread and migrate on a
fibronectin-coated surface. Dispersed animal pole cells
are also able to respond to XTC-MIF in this way; this is
one of the few mesoderm-specific responses to induction
that has been observed in single cells.
The ability of induced animal pole cells to spread on
fibronectin is abolished by the peptide GRGDSP. However, the elongation of intact explants is unaffected by
this peptide. This may indicate that fibronectin-me-
diated cell migration is not required for convergent
extension.
We have investigated the molecular basis of XTCMIF-induced gastrulation-like movements by measuring
rates of synthesis of fibronectin and of the integrin f}y
chain in induced and control explants. No significant
differences were observed, and this suggests that gastrulation is not initiated simply by control of synthesis of
these molecules. In future work, we intend to investigate
synthesis of other integrin subunits and to examine
possible post-translational modifications to fibronectin
and the integrins.
Introduction
these molecules, and this idea is supported by studies of
the temporal expression of fibronectin and integrins.
The rate of synthesis of fibronectin increases dramatically at the mid-blastula transition (MBT; see Newport
and Kirschner, 1982), and the protein is first detectable
immunocytochemically about three hours later, at the
beginning of gastrulation (Boucaut and Darribere,
1983; Lee et al. 1984). This increase in fibronectin
synthesis does not require RNA synthesis, and must
involve activation of maternal message. The integTins
consist of a-and /S subunits (see Hynes, 1987). Analysis
of Xenopus Pi subunit mRNA shows that expression
begins around the gastrula stage (DeSimone and
Hynes, 1988); information is not yet available about the
a subunits.
These observations do not, .of course, prove that
gastrulation is controlled by synthesis of fibronectin and
its receptor. The similarity in timing may be coincidental, and indeed transcription of many genes first occurs
Two lines of evidence indicate that fibronectin plays an
important role in amphibian gastrulation. First, fibronectin is localised to the roof of the blastocoel, the
surface on which presumptive mesodermal cells migrate
(Boucaut and Darribere, 1983; Lee et al. 1984; Nakatsuji et al. 1985a). Second, if antibodies to fibronectin or
its receptors, integrins, are microinjected into the
blastocoels of amphibian embryos, gastrulation is inhibited (Boucaut et al. 1984a; Darribere et al. 1988).
Similar results are obtained by injecting synthetic peptides corresponding to a major cell-binding site of the
fibronectin molecule (Boucaut et al. 19846).
Although this work demonstrates that fibronectin
and its receptors are required for gastrulation to proceed, it does not address the question of how gastrulation is initiated and controlled. The simplest suggestion is that gastrulation is triggered by the synthesis of
Key words: gastrulation, fibronectin, integrins, mesoderm
induction, mesoderm-inducing factors, XTC-MIF,
amphibian embryo, Xenopus laevis.
230
J. C. Smith and others
at the MBT or soon afterwards (see, for example,
Sargent and Dawid, 1983; Kreig and Melton, 1985). In
this paper, we test the functions of fibronectin and
integrins more directly by taking advantage of the
recent observation that the XTC-mesoderm-inducing
factor (XTC-MIF; see Smith, 1987; Rosa et al. 1988;
Smith et al. 1988; Dawid et al. 1989; Smith, 1989)
induces gastrulation-like movements in isolated animal
pole regions (Symes and Smith, 1987; Cooke and
Smith, 1989). This observation makes it possible to
manipulate gastrulation and thus ask how its various
aspects are controlled.
Our results indicate that animal pole blastomeres
exposed to XTC-MIF, whether as intact explants or as
isolated cells, are able to spread and migrate on a
fibronectin-coated substrate. In this respect, they resemble prospective mesodermal cells, but differ from
uninduced animal pole cells (Nakatsuji, 1986). However, acquisition of the ability to spread on fibronectin
is not accompanied by an increase in synthesis of
fibronectin or of the integTin ft chain. We conclude
from this that initiation of gastrulation is not controlled
through synthesis of these proteins, although it is
possible that existing molecules are redistributed within
cells or undergo post-translational modification.
The ability of both XTC-MIF-induced animal pole
blastomeres and of presumptive mesodermal cells to
spread on fibronectin is abolished by the peptide
GRGDSP, which contains the fibronectin cell attachment site (Pierschbacher and Ruoslahti, 1984; Yamada
and Kennedy, 1984). However, the elongation of
induced animal pole explants is unaffected by this
peptide. This suggests that the 'convergent extension'
movements of gastrulation (see Keller, 1986; Keller and
Danilchik, 1988; Keller and Tibbetts, 1989) do not
depend on interactions involving RGD sites in fibronectin or other matrix molecules.
Materials and methods
Embryos
Embryos of Xenopus laevis were obtained by artificial fertilization as described by Smith and Slack (1983). They were
chemically dejellied using 2% cysteine hydrochloride
(pH7.8-8.1), washed and transferred to Petri dishes coated
with 1% Noble agar and containing 10% normal amphibian
medium (NAM: Slack, 1984). The embryos were staged
according to Nieuwkoop and Faber (1967).
XTC-mesoderm-inducing factor
XTC-MIF was partially purified from heated XTC-cell-conditioned medium by DEAE-Sepharose chromatography followed by phenyl Sepharose chromatography, as described by
Cooke et al. (1987) and Smith et al. (1988). One unit of
mesoderm-inducing activity is defined as the minimum quantity that must be present in lml medium for induction to
occur. The sample of partially purified XTC-MIF used in
these experiments contained approximately 7.7X103 units
rng"1 protein.
Preparation of cell substrates
Tissue culture dishes (35 mm diameter, Nunc), or the wells of
multiwell or microtitre plates (Nunc), were treated at room
temperature for 4-18 h with 20-100/tg ml"1 bovine or rat
plasma fibronectin, kindly supplied by Dr Heather Streeter
(NIMR) or Mr Terry Butters and Dr Colin Hughes (NIMR).
The surfaces were then rinsed and 'blocked' with NAM
containing 0.5 % bovine serum albumin (BSA) for 20min.
Embryo dissections
Mid-blastula Xenopus embryos (stages 8-9) were dissected
into animal, marginal zone, and vegetal pole regions using
electrolytically sharpened tungsten needles.
Cell spreading assays
Animal pole or dorsal marginal zone regions were transferred
to calcium- and magnesium-free medium (CMFM: 100 ITIMNaCl, 5mM-KCl, lmM-NaHCO3, 2.5mM-sodium phosphate,
pH7.5 and 0.25 % (w/v) gentamycin sulphate; Sargent et al.
1986). The outer layer of cells, which is difficult to dissociate,
was discarded and the inner layer was disaggregated into a
single-cell suspension. These cells were seeded onto fibronectin-coated surfaces in NAM containing 0.5% BSA and,
where appropriate, XTC-MIF, and they were scored 1-2 h
later.
Cells could be divided into three classes (see Fig. 1 for
examples). Non-adherent cells are spherical and move about
the dish when it is disturbed. Adherent cells cannot easily be
dislodged by disturbing the dish, but like non-adherent cells
they are roughly spherical. They do not flatten but frequently
undergo 'circus movements', during which cytoplasmic protrusions move slowly around the circumference of the cell.
Spread cells adhere to the substrate, flatten, and send out
processes. They can sometimes be seen to migrate over the
fibronectin-coated substrate (see Fig. 4). Spread cells are
clearly distinct from adherent cells.
Cultures were scored independently by two observers
(J.C.S. and K.S.), one of whom was ignorant of the coding of
the experiment. The two assessments did not differ significantly.
Antibodies and inhibitory peptides
These experiments used a rabbit antiserum raised against
Xenopus plasma fibronectin (Heasman et al. 1981) and a
rabbit antiserum raised against a 39-amino acid peptide
corresponding to a COOH-terminal portion of vertebrate
integrin /Si subunits (Marcantonio and Hynes, 1988). The
synthetic peptides GRGDSP and GRGESP were synthesized
on an Applied Biosystems Peptide Synthesizer and purified by
HPLC by Gene Yee, to whom we are very grateful.
Metabolic labelling of embryonic tissue and
immunoprecipitation
Dissected pieces of mid-blastula Xenopus embryos were
incubated in NAM, with or without XTC-MIF as appropriate,
in the presence of 0.3-0.9 mCimP 1 [35S]methionine at room
temperature (19-22°C). At the end of the incubation period,
the tissues were rinsed three or four times in NAM and frozen
on dry ice in a minimum volume of fluid.
Samples for immunoprecipitation using the anti-fibronectin
antibody were homogenized in 2 M-urea as described by Lee et
al. (1984). Samples for analysis using the anti-integrin antibody were homogenized in O.lM-NaCl, 1% Triton X-100,
lmM-PMSF, 0.22 trypsin inhibitor unit (TIU) ml"' aprotinin,
and 20mM-Tris, pH7.6. In both cases, aliquots were then
removed to determine total radioactivity incorporated into
acid-insoluble material, and the extracts were diluted five-fold
with 'immunoprecipitation buffer' (O.IM-KCI, 5mM-MgCl2,
1% Triton X-100, 1% sodium deoxycholate, 2mM-PMSF,
Mesoderm induction and gastrulation
0.22 TIU ml"1 aprotinin, and O.lM-Tris, pH8.2) containing
0.4 % pre-immune serum. After 15 min at 4°C, a 10 % volume
of Protein A-Sepharose slurry was added, and after a further
15 min the mixtures were centrifuged and the supernatents
divided equally between two or three fresh tubes. To one such
tube immune serum was added to 0.4%, and to another preimmune serum was added to the same concentration. As a
further control for integrin immunoprecipitations, samples
contained 0.4% immune serum plus 100/igml""1 of the
original peptide antigen as competitor.
These tubes were incubated at 4°C for 1 h with gentle
rocking, and then a 20% volume of Protein A-Sepharose
slurry was added and the incubation repeated. Finally the
tubes were centrifuged and the pellets washed four times with
immunoprecipitation buffer before solubilizing proteins in
50/d of gel sample buffer (Laemmli, 1970).
Samples were analysed by polyacrylamide gel electrophoresis using the buffer system of Laemmli (1970) with a 7%
separating gel to detect integrins and a 5 % separating gel to
detect fibronectin. Fluorography was carried out according to
Bonner and Laskey (1974). Sample loading was normalised
according to total incorporation of radioactivity into acidprecipitable material.
Results
As in previous work, treatment of stage 8-9 Xenopus
animal pole regions with 5-20 units ml~l XTC-MIF
resulted in dramatic elongation of the explants, and
eventually the formation of dorsal mesodermal cell
types, including notochord and muscle (data not shown,
but see Smith, 1987; Cooke et al. 1987; Symes and
Smith, 1987; Smith et al. 1988).
Blastomeres from induced ectoderm can spread on
fibronectin
We were first interested to discover whether the cells of
animal pole explants exposed to XTC-MIF could
spread and migrate on fibronectin-coated surfaces.
Accordingly, explants were exposed to 20 units ml"1
XTC-MIF or a control solution from stage 8 to stage 10
in NAM containing 10% of the normal divalent cation
concentration to prevent them 'rounding up'. The
explants were then placed, blastocoel-facing surface
down, onto surfaces coated with 50 ng ml" 1 fibronectin,
and observed at intervals. Dorsal marginal zone
explants were dissected from early gastrula embryos
and cultured in the same way, essentially as described
by Shi et al. (1989).
As expected, cells from uninduced animal pole regions did not adhere to the fibronectin-coated substrate, while some blastomeres from the dorsal marginal zone regions adhered and began to migrate away
from the body of the explant (data not shown). Cells
from explants exposed to XTC-MIF behaved like dorsal
marginal zone blastomeres and like them adhered to
the substrate, spread and began to migrate. In some
cases virtually all of the cells seemed to stick to the
fibronectin-coated surface.
To obtain a more quantitative analysis, induced and
uninduced explants and dorsal marginal zone regions
were disaggregated into single cell suspensions at stage
10 and the blastomeres were seeded onto substrates
231
coated with 20, 50 or lOOjigmr1 fibronectin. Three
different concentrations of fibronectin were used to
allow for possible variation in spreading behaviour of
blastomeres from different egg batches. After 1-2 h the
proportions of non-adherent, adherent and spread cells
were determined (see Materials and methods). The
proportions of adherent and non-adherent cells varied
between different experiments, depending both on egg
batch and on the concentration of fibronectin used (see
Winklbauer, 1988). However, when the proportion of
spread cells to unspread (non-adherent plus adherent)
cells was considered, the results obtained with all three
concentrations of fibronectin were similar and consistent from experiment to experiment. Figs 1 and 2 show
data obtained with 50^gml -1 fibronectin, in which
37% of cells from the dorsal marginal zone region
spread on fibronectin, as compared with only 1 % from
uninduced animal pole explants. Treatment of animal
pole explants with XTC-MIF resulted in 45 % of the
cells becoming spread after disaggregation.
In preliminary work, mentioned in Symes and Smith
(1987), we found that uninduced animal pole blastomeres were able to spread on fibronectin-coated surfaces almost as well as marginal zone cells. This may
have been due to use of a glass, rather than a plastic,
substrate, or to inadequate 'blocking' of free substrate
with bovine serum albumin (see Winklbauer, 1988).
Single cells can respond to XTC-MIF and spread on
fibronectin
We next tested whether dispersed animal pole cells
could respond to XTC-MIF by spreading on fibronectin. Dissected animal pole regions were disaggregated
into single cell suspensions at stage 9 (late blastula
stage) and cultured in 20 units ml" 1 XTC-MIF or a
control solution for 1 h, until control embryos reached
stage 10, the beginning of gastrulation. Dorsal marginal
zone regions were then dissected from early gastrulae
and disaggregated, and the three populations of cells
were seeded onto substrates coated with 20, 50, or
100^gml"1 fibronectin, as before. The results were
similar with all three concentrations of fibronectin;
Fig. 3 shows data obtained with 100 ng ml" ] . Over 60 %
of blastomeres from the dorsal marginal zone spread on
the fibronectin-coated surface, whereas none from
uninduced animal pole regions did so. However, over
95 % of cells exposed to XTC-MIF while in a dispersed
state spread on the substrate. This result indicates first
that single cells are able to respond to XTC-MIF by
spreading on a fibronectin-coated surface. Second, the
data show that virtually all the deep cells of the animal
pole are able to respond to XTC-MIF by spreading on
fibronectin, and that the smaller percentage of spread
cells observed when intact explants were exposed to the
factor (Fig. 2) is probably due to inaccessibility of the
inner cells.
Continuous observation of cells that had been exposed to XTC-MIF showed further that they could
migrate on the fibronectin-coated surface (Fig. 4).
232
J. C. Smith and others
Fig. 1. Individual cells from XTC-MIF-treated Xenopus
animal pole regions, like those from the dorsal marginal
zone, adhere to a fibronectin-coated surface; those of
uninduced animal pole regions do not. (A) The dorsal
marginal zone region of a Xenopus early gastrula was
disaggregated into a single cell suspension and seeded onto
tissue-culture plastic coated with 50 j/g ml" 1 fibronectin.
Many of the cells attach to the substrate, flatten, and send
out processes. (B) Animal pole regions exposed to XTCMIF from the mid-blastula to the early gastrula stage were
disaggregated into a single cell suspension which was seeded
onto a similar fibronectin-coated substrate. Many of the
cells have spread. The arrow indicates a spread cell, and the
arrow-head a non-adherent cell. (C) Cells disaggregated
from uninduced animal pole regions do not spread on a
fibronectin-coated surface. The arrow indicates an adherent
cell. Scale bar in C is 50urn, and also applies to A and B.
100
CH Non-adherent
[I] Adherent
H Spread
60-
Fig. 2. Blastomeres derived from the dorsal marginal zone
and from animal pole regions treated with XTC-MIF spread
on a fibronectin-coated substrate; cells from uninduced
animal pole regions do not. Blastomeres were disaggregated
from dorsal marginal zone regions (DMZ), uninduced
animal pole regions (AP) or animal pole regions that had
been treated with XTC-MIF (AP-MIF). They were seeded
onto tissue-culture plastic which had been treated with 50 /ig
ml" 1 fibronectin, and the results were scored after 2 h as
described in the Materials and methods. An average of 171
cells was counted for each treatment. Similar results were
obtained with cells seeded onto 100/ig ml"' and 20/Jg ml" 1
fibronectin. For 100 fig ml" 1 , the proportions of cells
derived from DMZ, AP, and AP-MIF regions that spread
on the substrate were, respectively, 4 3 % , 3 % and 34%
(average of 200 cells per point). For 20pg ml" 1 , the
proportions were 27%, 0.4% and 40% (average of 206
cells per point).
Spreading of induced cells on fibronectin is inhibited
by the peptide GRGDSP
To discover whether the spreading of induced animal
pole blastomeres depends on a specific interaction
between the cells and fibronectin, we used the synthetic
peptide GRGDSP, which corresponds with the primary
fibronectin cell attachment site (Pierschbacher and
Ruoslahti, 1984; Yamada and Kennedy, 1984). Control
cultures contained the peptide GRGESP, in which Asp
is replaced by Glu. Animal pole blastomeres were
disaggregated from Xenopus embryos at the late blastula stage and treated with XTC-MIF in the presence of
different concentrations of peptide. The proportions of
spread cells were determined 1.5 h later. MIF-induced
spreading was completely inhibited by 5 ITIM-GRGDSP,
whereas the same concentration of GRGESP reduced
the proportion of spread cells by only 46 %. The same
experiment showed that spreading of dorsal marginal
zone cells is completely inhibited by 2mM- and 5mMGRGDSP, as has been reported for prospective meso-
Mesoderm induction and gastrulation
Fig. 3. Single animal pole blastomeres treated with XTCMIF are able to spread on a fibronectin-coated substrate.
Blastomeres were disaggregated from animal pole regions at
the late blastula stage and cultured in a control solution
(AP) or XTC-MIF (AP-MIF) until the early gastrula stage.
The cells were then seeded onto tissue-culture plastic which
had been treated with 100ng ml"' fibronectin, along with
cells from the dorsal marginal zone of normal embryos
(DMZ). The results were scored after 2 h as described in
the Materials and methods. An average of 286 cells was
counted for each treatment. Similar results were obtained
with cells seeded onto 50^g ml"' and 20 fig ml" 1
fibronectin. For 50^g ml" 1 , the proportions of DMZ, AP,
and AP-MIF cells that spread on the substrate were,
respectively, 25 %, 1 % and 67 % (average of 287 cells per
point). For 20/ig m l " ' , the proportions were 19%, 0% and
84 % (average of 206 cells per point).
derm from the urodele Pleurodeles waltl (Shi et al.
1989).
The slight inhibition of spreading observed with the
peptide GRGESP has been noted in other studies
(Dufour et al. 1988) and may reflect a lower affinity
interaction of this peptide with the integrin receptor
binding site (Hautanen et al. 1989).
.
233
• Non-adherent
• Adherent
• Spread
DMZ
AP
Cell type
AP-MIF
The peptide GRGDSP does not inhibit elongation of
induced animal pole explants
The ability of induced animal pole blastomeres to
spread on fibronectin reflects the role of cell migration
in amphibian gastrulation (see Keller, 1986). However,
an equally important aspect of gastrulation, particularly
in Xenopus, is convergent extension, which is driven
more by cell rearrangement and intercalation than by
cell migration (Keller, 1986), and which is probably
responsible for the elongation of induced animal pole
explants (Symes and Smith, 1987). We have investigated whether fibronectin-mediated interactions are
Fig. 4. Disaggregated animal pole blastomeres treated with XTC-MTF migrate on a fibronectin-coated substrate. A and B
show animal pole blastomeres prepared as described in the legend to Fig. 3. They were photographed at intervals of lOmin,
beginning at the equivalent of stage 11; the figure in the top right hand corner of each frame represents the time in minutes.
Scale bar in the final frame of B is 30^m, and applies to all frames.
234
J. C. Smith and others
•I
Fig. 5. Elongation of XTC-MIF-treated animal pole regions
is not inhibited by the peptide GRGDSP. Animal pole
regions were dissected from Xenopus embryos at the midblastula stage and cultured in XTC-MIF (A-C) or a control
solution (D) in the absence of peptide (A) or in the
presence of 10mM-GRGDSP (B) or 10mM-GRGESP (C).
The explants were examined 15 h later, when controls were
at stage 13. Neither peptide has affected the elongation of
induced explants. Scale bar in D is 200 fan, and applies to
all frames.
required for convergent extension by incubating
explants in the peptides GRGDSP and GRGESP in the
presence of XTC-MEF or a control solution. The results
of two experiments using a total of 160 explants show
that even 10 mM-GRGDSP has no effect whatsoever on
convergent extension of induced explants (Fig. 5).
Further incubation of such explants also showed that
the peptides had no effect on mesodermal cell differentiation (data not shown). These results suggest that the
RGD cell attachment site is required for migration of
prospective mesodermal cells, but is not necessary for
convergent extension.
Induced ectoderm does not synthesize fibronectin or
the integrin ft chain at an increased rate
The molecular basis of the ability of single induced
animal pole blastomeres to spread on fibronectin, and
of a population of such cells to cooperate in convergent
extension movements, is unknown. To investigate this
we have studied rates of fibronectin and integrin ft
chain synthesis in induced explants compared with
uninduced tissue.
To estimate the rate of synthesis of fibronectin,
induced and uninduced animal pole regions, marginal
zone regions and vegetal pole regions were incubated in
[35S]methionine from stage 9 (late blastula) to stage
12-13 (late gastmla). Fibronectin synthesis was determined by immunoprecipitation and results from one of
two similar experiments are shown in Fig. 6A. There is
no enhancement of fibronectin synthesis in induced
animal pole regions compared with uninduced explants,
and this is consistent with the observation that animal
pole regions synthesize fibronectin at a similar rate to
the marginal zone. It was noteworthy, however, that in
two out of three experiments the vegetal pole region
was seen to synthesize significantly more fibronectin as
a fraction of its total protein synthesis than the other
regions (see Fig. 6A). This is currently under investigation. Overall, these results indicate that the ability of
induced animal pole blastomeres to spread on fibronectin is not due to an increased rate of synthesis of
fibronectin itself.
A more likely possibility is that the ability of induced
cells to spread on fibronectin is due to an increase in the
expression of fibronectin receptors. We therefore investigated the rate of synthesis of the integTin ft chain.
Induced and uninduced animal pole regions, marginal
zone regions, and vegetal pole regions were incubated
in [35S] methionine from stage 9 (late blastula) to stage
12-13 (late gastrula). Integrin J3 chain synthesis was
determined by immunoprecipitation. Five successful
experiments were carried out. In most, there was little
enhancement of the rate of synthesis of integrin ft chain
in induced explants compared with uninduced explants;
the largest increase, shown in Fig. 6B, was 2.8-fold. To
discover whether the slight increase observed in some
experiments reflected a brief period of a more marked
enhancement of synthesis, two time-course experiments were conducted, with integrin synthesis being
measured from stages 9-10, 10-11, 11-12 and 12-13.
No such 'burst' of synthesis was observed (data not
shown). In contrast to the measurements of fibronectin
synthesis, there was no enhancement of integrin ft
chain synthesis in the vegetal hemisphere of the embryo, but some experiments revealed a higher rate of
synthesis in the marginal zone than in the animal pole
region (Fig. 6B).
These results indicate that there can only be a very
slight increase in synthesis of the integrin ft chain in
response to mesoderm induction. It is not clear whether
this would be sufficient to allow animal pole blastomeres to spread on fibronectin. Another possibility,
however, is that changes in integrin a'subunit synthesis
might occur in response to XTC-MIF. Fig. 6B reveals
clear differences in a subunit synthesis between animal
and vegetal pole regions, and it is possible that more
subtle differences occur between induced and uninduced animal pole regions.
Mesoderm induction and gastrulation
12 3 4 5 6
78
235
1 2 3 4 5 6 7 8 9 10 11 12
-
ft
•
Fig. 6. XTC-MIF does not cause a stimulation of fibronectin or integrin p{ chain synthesis in Xenopus animal pole regions.
More fibronectin is synthesized in the vegetal hemisphere of the embryo. Animal pole regions, in the presence or absence of
XTC-MIF, marginal zone regions, and vegetal pole regions of Xenopus embryos were cultured in [35S] methionine from the
mid blastula stage to the late gastrula stage. Extracts were then subjected to immunoprecipitation using an anti-fibronectin
antibody, with normal rabbit serum (NRS) as a control (A), or using an antibody directed against the integrin /3i chain, with
pre-immune serum as a control (B). As an additional control in B, immunoprecipitations were also carried out in the
presence of the original peptide antigen. In both A and B, gel loading was normalized according to counts per minute
incorporated into total acid-insoluble material.
(A) Lanes 1, 3 and 5 show levels of fibronectin synthesis in animal pole, marginal zone, and vegetal pole regions
respectively. Lanes 2, 4 and 6 show NRS controls for lanes 1, 3 and 5 respectively. Lanes 7 and 8 compare fibronectin
synthesis in control (lane 7) and XTC-MIF-treated (lane 8) animal pole explants respectively. Arrow indicates fibronectin.
(B) Lanes 1, 4, 7 and 10 show levels of integrin /S, chain synthesis in animal pole regions treated with XTC-MIF, control
animal pole regions, marginal zone regions, and vegetal pole regions respectively. Lanes 2, 5, 8 and 11 show pre-immune
serum controls for lanes 1, 4, 7 and 10 respectively. Lanes 3, 6, 9 and 12 show controls in which the original peptide antigen
was included in the immunoprecipitation reaction. Arrow indicates integrin /3t chain. The 2.8-fold increase in synthesis of
the integrin jS, chain observed in animal pole explants treated with XTC-MIF (compare lanes 1 and 4) is the largest we have
observed in a total of 7 experiments. Bracket indicates integrin a chains that co-precipitate with the /3 chain. Note that these
differ in the animal pole and vegetal pole regions.
Discussion
This paper describes two principal results. First, we
show that Xenopus animal pole blastomeres exposed to
XTC-MIF acquire the ability to spread and migrate on
fibronectin. This ability is abolished by incubating cells
in a peptide corresponding to the fibronectin cell
binding site, but this peptide does not inhibit the
convergent extension movements of intact XTC-MIFtreated explants. Convergent extension may instead
depend on active local cell mixing (Keller and Hardin,
1987; Keller and Tibbetts, 1989; our unpublished observations). In the second part of the paper, we demonstrate that the ability of induced blastomeres to spread
on fibronectin is not accompanied by an increase in the
rate of synthesis of fibronectin itself, or by a significant
increase in the rate of synthesis of one component of
potential fibronectin receptors, the integrin & chain.
We discuss these two points separately.
Animal pole cells exposed to XTC-MIF spread and
migrate on fibronectin
In the first part of this paper, we show that Xenopus
animal pole blastomeres exposed to XTC-MIF acquire
the ability to spread and migrate on a fibronectin-
coated surface. This spreading is completely inhibited
by the synthetic peptide GRGDSP, but less so by the
peptide GRGESP. Prospective mesodermal cells of the
marginal zone also have the ability to spread and
migTate on fibronectin, but uninduced animal pole cells
do not (Nakatsuji, 1986; see also Komazaki, 1988).
Interestingly, Winklbauer (1988) has recently shown
that endodermal cells of Xenopus also adhere strongly
to fibronectin; it is not possible, therefore, to decide
directly from these data whether XTC-MIF induces
animal pole cells to become mesoderm or to become
endoderm.
The role of fibronectin-mediated cell adhesion and
migration in amphibian gastrulation has been discussed
by Keller (1986) and Keller and Tibbetts (1989), who
suggest that the mesoderm of Xenopus can be divided
into two subpopulations. It is only the early-involuting
mesoderm, which differentiates into head, heart, blood
islands and lateral mesoderm, that migrates and spreads
on the blastocoel roof. The later-involuting mesoderm,
that differentiates into notochord and somite, does not
migTate but undergoes convergent extension, which
causes a dramatic elongation and narrowing of the
mesoderm through active cell rearrangement and intercalation. Keller (1986) suggests that one role of migTation may be to orientate the early convergent
236
J. C. Smith and others
extension movements, preventing the formation of an
external excresence; migration does not seem to be
required, however, for the convergent extension movements themselves, or for constriction of the blastopore
or the organization of the mesodermal mantle (see
Schechtman, 1942; Keller, 1984; Keller et al. 1985).
Our data indicate that blastomeres of animal pole
regions exposed to XTC-MIF are able to participate
both in migration (Fig. 4) and in convergent extension
(Fig. 5), and Keller's conclusion that cell migration is
not required for convergent extension is supported by
our observation that elongation of XTC-MIF-treated
animal pole explants is not inhibited by the synthetic
peptide GRGDSP (Fig. 5). Unequivocal interpretation
of this experiment is difficult because it is not possible to
be certain that the peptide gained access to all the cells
in the treated animal pole regions. Nevertheless, the
fact that explants treated with GRGDSP were indistinguishable from explants treated with GRGESP and
from untreated controls argues that fibronectin-mediated adhesive interactions are not required for convergent extension of induced animal pole regions. In
support of this, it is significant that microinjection of
RGD-containing peptides into the blastocoels of Xenopus embryos has little effect (J.C.S., unpublished observations), whereas this treatment completely blocks
gastrulation in the urodele Pleurodeles (Boucaut et al.
1984fc). This may reflect the fact that in urodeles
mesodermal cells tend to migrate more as individuals
after involution, while in Xenopus it is a cohesive
population of cells that undergoes convergent extension
movements (Keller, 1986).
The ability of XTC-MIF-treated animal pole blastomeres to spread and migrate on fibronectin is significant
because it occurs at the level of single cells, and, as
Gurdon (1987) has emphasized, this should simplify the
molecular analysis of induction. Until recently the only
mesoderm-specific response that has been observed in
single cells has been the suppression of epidermal
differentiation (Symes et al. 1988); positive responses,
such as muscle formation, have required cells to be
present in groups (Gurdon, 1988; Symes et al. 1988), or
at least to undergo several cell divisions (Godsave and
Slack, 1989). For this reason it is of great importance to
understand the molecular basis of the adhesion of
induced cells to fibronectin and discover how this is
linked with the activation of 'immediate early' genes
such as Mix. 1 (Rosa, 1989).
Mesoderm induction is not accompanied by an
increase in synthesis of fibronectin or the integrin ft
chain
The data presented here show that XTC-MIF does not
significantly affect rates of synthesis of fibronectin or of
the integrin ft chain (Fig. 6). We have not examined
levels of mRNA for these proteins, but it seems likely
that these are not affected by XTC-MIF either; this is
currently under investigation. Therefore, one can conclude that the biological effects of XTC-MIF are not
mediated by control of overall synthesis of fibronectin
or of the integrin ft chain. However, several other
possible mechanisms whereby XTC-MIF could affect
fibronectin-mediated adhesion and/or migration must
be considered.
In the case of fibronectin, it is possible that XTC-MIF
differentially affects the expression of the variously
spliced forms. Fibronectin synthesis occurs from both
maternal and embryonic transcripts, the latter appearing at around the time of gastrulation. (Lee et al. 1984;
DeSimone, Norton and Hynes, in preparation). The
maternal mRNA is already spliced and the predominant form contains all the alternatively spliced segments
(DeSimone, Norton and Hynes, in preparation). Barring some sort of selective translation induced by XTCMIF which, though possible, seems improbable, it
would appear that expression of different forms of
fibronectin from the maternal pool is an unlikely point
of regulation. However, the activation of fibronectin
transcription, which occurs shortly before gastrulation,
raises the possibility that XTC-MIF may regulate
splicing of these transcripts. This requires investigation,
especially since XTC-MIF shares many properties with
transforming growth factor /S (TGF-ft Smith et al. 1988)
and indeed TGF-ft has also been shown to induce
elongation with subsequent mesoderm formation in
Xenopus animal pole explants (Rosa et al. 1988). TGFft has been reported to affect splicing of fibronectin
RNA in cultured cells (Balza et al. 1988), and fibronectin splicing is also altered during wound healing
(ffrench-Constant et al. 1989) where TGF-/3 is thought
to play a role (Mustoe et al. 1987).
Another potential level of regulation of fibronectin
expression is at post-translational steps including export
and assembly into extracellular matrix. TGF-)9 promotes the latter process in cultured mammalian and
avian cells (Ignotz and Massagu6,1986,1987) and XTCMIF could do so as well.
However, the results presented in Figs 1-3 demonstrate that animal pole cells treated with XTC-MIF
show increased adhesion and spreading on exogenous
fibronectin and it seems more likely that this effect is
mediated through alterations in fibronectin receptors.
The fact that synthesis of the integrin ft chain is not
greatly affected by XTC-MIF may not be surprising.
TGF-/3 treatment of cultured mammalian and avian
cells elevates the surface level of several integrins
including fibronectin receptors (Ignotz and Massague\
1987; Roberts et al. 1988; Heino et al. 1989). However,
much of this effect arises by increases in synthesis of a
subunits, while the rate of synthesis of the ft subunit
changes only a little and rises only relatively late after
TGF-£ treatment (Roberts et al. 1988). Furthermore,
cells transformed by oncogenic viruses show reduced
fibronectin receptor function and alterations in a subunit expression (Plantefaber and Hynes, 1989) while
expression of the integrin ft chain at both mRNA and
protein levels is largely unaffected (Norton and Hynes,
1987; Plantefaber and Hynes, 1989). There are preliminary indications that the pattern of a subunits varies
throughout the embryo (Fig. 6B) and it is possible that
this occurs in response to XTC-MIF or other inducing
factors. Alterations in the pattern of integrin subunit
Mesoderm induction and gastrulation
expression could affect both the ability of cells to
adhere to and migrate on extracellular matrix and/or
the assembly of matrix itself. However, further investigation of these interesting possibilities will require aspecific reagents, which are currently not available for
amphibians.
In summary, our experiments appear to rule out
overall synthesis of fibronectin and the integrin fix chain
as points of regulation by XTC-MIF but more subtle
effects of this factor on the expression of fibronectin and
integrins remain possible. Furthermore, effects of
XTC-MIF on other matrix proteins such as laminin,
which is known to be present in the amphibian embryo
(Darribere etal. 1986; Nakatsuji etal. 19856), also need
examination.
We thank Jonathan Cooke, Tony Magee, Jack Price and
Fiona Watt for their helpful comments. We are also grateful
to Heather Streeter for bovine fibronectin, Terry Butters and
Colin Hughes for rat plasma fibronectin, and to Gene Yee for
synthetic peptides. This work was supported by the Medical
Research Council, by a short-term EMBO fellowship to
J.C.S. and by N1H grant RO1 CA17007. D.D. was a fellow of
USPHS, National Institute of General Medical Sciences,
F32GM10806.
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