Xenopus ectodermal cells have previously been shown to respond to acidic and basic FGF by differentiating into mesodermal tissue. In the present study, ectodermal explants from Xenopus blastulae were shown to have high affinity binding sites for 125I-aFGF (Kd = l·4× 10−10M). The total number of sites, determined by Scatchard analysis, was 3× 108 per explant (surface area of approximately 1mm2). Two putative receptors of relative molecular mass 130 000 and 140 000 were identified by chemical crosslinking to 125I-aFGF. Both acidic and basic FGF, but not TGFβ2, could compete for affinity labelling of these bands. The receptor density at the cell surface parallels the developmental competence of Xenopus animal pole cells to respond to FGF. Receptors are present at highest density in the marginal zone but are not restricted to cells in this region.

The formation of mesoderm in the early amphibian embryo is thought to occur by an inductive mechanism whereby cells of the marginal zone form mesoderm under the influence of a signal emanating from the cells of the vegetal hemisphere. This inductive signal can be mimicked by members of the fibroblast growth factor (FGF) (Slack et al. 1987; Kimelman & Kirschner, 1987) and transforming growth factor beta (TGFβ) families of growth factors (Smith et al. 1988; Rosa et al. 1988). Recently, a protein homologous to basic FGF (bFGF) has been identified in oocytes and developing embryos of Xenopus laevis suggesting that this molecule may be an endogenous inducer (Kimelman et al. 1988; Slack & Isaacs, 1989).

It is generally considered that in any instructive induction process the signal acts to select one developmental pathway out of two or more possible pathways that the responding cells are capable of following. The ability to respond to a signal(s) is known as the competence of the responding tissue (Waddington, 1940). Until now the molecular basis of competence has been obscure but the identification of certain inducing factors with growth factors enables us to predict that a necessary component of competence will be possession of the appropriate growth factor receptor.

We report here the characterization, developmental expression and spatial distribution of a putative receptor for FGF in the developing Xenopus laevis embryo.

Embryos

Xenopus laevis embryos were obtained by artificial fertilization, maintained and dissected as described by Godsave et al. (1988) at indicated developmental stages according to Nieuw-koop & Faber (1967).

Cells

C3H10T1/2 murine fibroblast cells were maintained in Dul-becco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf serum (Gibco) at 37 °C in a humidified 5 % CO2 incubator.

Growth factors

aFGF and bFGF were prepared from bovine brain according to the methods of Gospodarowicz et al. (1984), Esch et al. (1985) and Lobb et al. (1986). TGFβ2 was purchased from R&D systems.

Iodination of aFGF

Due to the quantity of ligand required to optimize binding conditions and subsequently characterize ligand-receptor interactions, our experiments were performed with acidic FGF which was available in milligram amounts. The meso derm-inducing activity of aFGF has previously been shown to be equivalent to that of basic FGF (Slack et al. 1988).

aFGF was iodinated to a specific activity of 105 cts min-1 ng-1 and purified on heparin Sepharose (Pharmacia) according to the methods described by Olwin & Hauschka (1986). The structural homogeneity of the iodinated aFGF was verified routinely by SDS-polyacrylamide gel electrophoresis followed by autoradiography. The biological activity of the l25I-aFGF was assessed in a standard mesoderminducing factor assay (Godsave et al. 1988) and was found to be indistinguishable from that of native aFGF.

Surface labelling of explants

Embryos were dissected into three regions: animal cap, marginal zone and vegetal pieces as shown in Fig. 6 and placed in an agar-fined Petri dish containing 3 ml of NAM. Half a millicurie of Na125I was added and a 22 × 22mm glass coverslip that had been coated with lodogen (Pierce) according to the manufacturer’s instructions, was floated on top. After a 15 min incubation at 22 °C, the explants were washed with NAM containing 100 mw-KI until no radioactivity could be detected in the wash medium. The explants were counted in a Beckman gamma 4000 counter.

Binding assays

Animal pole explants taken from Xenopus laevis blastulae were incubated individually at 4 °C in Terasaki culture dishes (Falcon) lined with 1 % agarose and containing 15 μl of binding medium (normal amphibian media (NAM; Slack & Forman, 1980), l mg ml-1 haemoglobin (Sigma) and 125I-aFGF) with or without excess unlabelled aFGF. After 45 min, the explants were washed thoroughly with 10 ml of NAM and counted in a Beckman gamma 4000 counter.

Crosslinking analysis

Chemical crosslinking of 125I-aFGF to C3H10T1/2 cells was performed exactly as described by Olwin & Hauschka (1986).

Crosslinking of 125I-aFGF to explants was performed at 4°C in 24-well dishes (Falcon). Ten explants were placed in each well in a total volume of 300 μl of NAM containing 1 mg ml-1 haemoglobin and 120 ng ml-1 125I-aFGF. After a 45 min incubation period, the explants were washed with 3 ml of NAM. Bis-sulfosuccinimidylsuberate (BS3; Pierce) was added to a final concentration of 50 μg ml-1 in NAM. After an additional 45 min incubation, the explants were washed further with 2 ml of NAM and the residual sites were blocked with 40mm-glycine in NAM. The explants were collected into 100 μl of Triton medium (1% Triton X-100, 10mw-EDTA, Imm-PMSF, 0 ·02% sodium azide in phosphate buffered saline, pH 7·3) and extracted for 30 min on ice. Insoluble material was pelleted at 12000g in a microfuge at 4°C and the soluble supernatant fraction was precipitated with 10 volumes of ice-cold acetone. The acetone precipitate was dissolved in Laemmli sample buffer (Laemmli, 1970) and electrophoresed using the discontinuous SDS-polyacrylamide gel system of Laemmli and 8% gels. After electrophoresis, the gels were fixed and stained in 0·15% Coomassie Blue R250 in 45% methanol, 10% acetic acid for 15 min and then destained overnight in 20% methanol, 6% acetic acid before drying. The gels were autoradiographed at —70 °C using Kodak X-Omat AR film and appropriate intensifying screens.

Characterization of the Xenopus embryo FGF receptor In order to examine the expression of FGF receptors in Xenopus embryos, we first characterized the binding of aFGF to ectodermal explants.

Incubation of ectodermal tissue with 125I-aFGF and increasing concentrations of unlabelled aFGF (Fig. 1) revealed that a significant proportion (70−75 % ) of the 125I-aFGF bound was displaceable, as one would expect of a ligand-receptor interaction. Competition for binding of 125I-aFGF to specific sites was complete with a fivefold molar excess of unlabelled aFGF.

Fig. 1.

Specificity of binding of l25I-aFGF to ectodermal explants. Animal cap explants excised as illustrated in Fig. 6B were incubated for 45 min with 7-2 nM 125I-aFGF in the presence of 0, 7×2, 36, 72 or 144 HM of unlabelled aFGF. The radioactivity bound is expressed as a percentage of the radioactivity bound in the absence of unlabelled aFGF. Each point represents the average value from eight explants.

Fig. 1.

Specificity of binding of l25I-aFGF to ectodermal explants. Animal cap explants excised as illustrated in Fig. 6B were incubated for 45 min with 7-2 nM 125I-aFGF in the presence of 0, 7×2, 36, 72 or 144 HM of unlabelled aFGF. The radioactivity bound is expressed as a percentage of the radioactivity bound in the absence of unlabelled aFGF. Each point represents the average value from eight explants.

Binding analysis (Fig. 2) indicated that specific sites were saturated at approximately 7 nM (120 ng ml-1) and Scatchard analysis (Fig. 2, inset) revealed a single class of binding sites with a dissociation constant of 1·4×10−10M. This value is similar to that reported for mammalian cells (0-5X10−10M for Swiss 3T3, Olwin & Hauschka, 1986; 2·5×10−10M for BHK21, Neufeld & Gospodarowicz, 1986). The number of binding sites, determined from the x-intercept of the Scatchard plot, was 3×108 per explant, equivalent to about 1mm2 of exposed blastocoelic membrane.

Fig. 2.

Saturation of binding of 125I-aFGF to ectodermal explants. Animal cap explants excised as illustrated in Fig. 6B were incubated for 45 min at 4°C with increasing concentrations of 125I-aFGF in NAM, washed extensively with cold NAM and counted directly in a gamma counter. Non-specific binding was determined in the presence of an excess (72 RM) of unlabelled aFGF and these values were subtracted from total binding. Each point represents the average value from 48 explants and six separate experiments. The coefficient of variance for each point was between 5 and 10 %. The inset is a Scatchard plot of the binding data.

Fig. 2.

Saturation of binding of 125I-aFGF to ectodermal explants. Animal cap explants excised as illustrated in Fig. 6B were incubated for 45 min at 4°C with increasing concentrations of 125I-aFGF in NAM, washed extensively with cold NAM and counted directly in a gamma counter. Non-specific binding was determined in the presence of an excess (72 RM) of unlabelled aFGF and these values were subtracted from total binding. Each point represents the average value from 48 explants and six separate experiments. The coefficient of variance for each point was between 5 and 10 %. The inset is a Scatchard plot of the binding data.

Further characterization of the Xenopus embryo receptor for aFGF was carried out by affinity labelling (Fig. 3). The crosslinker used (BS3) was water soluble to ensure that only proteins on the external surface of the cells would be labelled. Crosslinking of 125I-aFGF to ectodermal explants revealed two affinity-labelled bands (Fig. 3, lane 2): a major product of relative molecular mass = 130 000 and a minor product of 140 000 Mr which was sometimes not observed. These labelled bands did not appear in the absence of crosslinker (Fig. 3, lane 1). The specificity of the interaction between 125I-aFGF and these two proteins was verified by demonstrating that the label could be competed out by the addition of a tenfold excess of unlabelled aFGF (Fig. 3, lane 3). The relative molecular masses of these two labelled proteins are similar to those reported by Neufeld & Gospodarowicz (1986) for mammalian cells (125×103 and 145×103) and indeed, when samples of 125I-aFGF crosslinked to a murine fibroblast cell line, C3H10T1/2, were run alongside crosslinked Xenopus explants, the affinity-labelled bands migrated to the same position (Fig. 4). In this particular experiment, the 140×103Mr, labelled band was not observed, even on longer exposures. The reason for this is not currently known.

Fig. 3.

Affinity labelling of FGF receptors in Xenopus blastulae. Explants were incubated for 45 min with 7·2nM-125I-aFGF in the presence (lane 3) or in the absence (lane 1 and 2) of 72 nM-unlabelled aFGF and in the presence (lane 2 and 3) or absence (lane 1) of crosslinker as described in the Materials and methods. The molecular masses of the two crosslinked products are indicated and were determined using protein standards electrophoresed on the same gel.

Fig. 3.

Affinity labelling of FGF receptors in Xenopus blastulae. Explants were incubated for 45 min with 7·2nM-125I-aFGF in the presence (lane 3) or in the absence (lane 1 and 2) of 72 nM-unlabelled aFGF and in the presence (lane 2 and 3) or absence (lane 1) of crosslinker as described in the Materials and methods. The molecular masses of the two crosslinked products are indicated and were determined using protein standards electrophoresed on the same gel.

Fig. 4.

Affinity labelling of FGF receptors in the presence of excess TGFβ2, acidic or basic FGF. Explants were incubated for 45 min at 4°C with 7·2DM-I-aFGF alone (lane 1) or with a tenfold molar excess of aFGF (lane 2), bFGF (lane 3) or TGFβ2 (lane 4). Crosslinking was performed as described in Methods. Crosslinking to C3H10T1/2 (lane 5) was performed in parallel and loaded onto the same gel. Arrows indicate the labelled products.

Fig. 4.

Affinity labelling of FGF receptors in the presence of excess TGFβ2, acidic or basic FGF. Explants were incubated for 45 min at 4°C with 7·2DM-I-aFGF alone (lane 1) or with a tenfold molar excess of aFGF (lane 2), bFGF (lane 3) or TGFβ2 (lane 4). Crosslinking was performed as described in Methods. Crosslinking to C3H10T1/2 (lane 5) was performed in parallel and loaded onto the same gel. Arrows indicate the labelled products.

Acidic and basic FGF interact with the same receptors, but TGFβ2 does not

A second class of molecules that has been shown to possess mesoderm-inducing activity is that of the TGF/3 family (Rosa et al. 1988; Smith et al. 1988). We examined the possibility that both classes of molecules interact with the same receptors. l25I-aFGF was crosslinked to explants in the presence of excess aFGF, bFGF or TGFβ2 (Fig. 4). Only aFGF and bFGF could compete for the affinity-labelled band, suggesting that TGFβ2 acts through a different receptor system.

Expression of FGF receptors is developmentally regulated but not restricted to presumptive mesodermal cells

We wished to examine whether the presence of FGF receptors corresponded to the ability of Xenopus embryonic cells to become induced to form mesoderm. Therefore, we determined the receptor density in animal hemisphere explants taken at different times during the period of competence (Smith et al. 1985; Gurdon et al. 1985; Jones & Woodland, 1987; Slack et al. 1988) as well as the receptor density in different regions of the embryo.

Embryos were maintained at 23°C and explants were taken at various times after fertilization. For these experiments, the entire animal hemisphere was used in order to ensure that the values determined represented receptor numbers for a constant surface area. Nonspecific binding was measured in each experiment and subtracted from the total. The receptor density was found to increase from the earliest stage examined (4h after fertilization; stage 7: 128 cells) to a maximum density sevenfold higher at 6h postfertilization (stage 8: 1024 cells) (Fig. 5). The receptor density gradually decreases after 6 h to reach a level that was only 10 % of maximum by early gastrula (stage 10). This pattern of expression corresponds reasonably well to the onset and cessation of competence previously determined using exogenous FGF (Slack et al. 1988).

Fig. 5.

Stage-specific binding of 125I-aFGF to ectodermal explants. Embryos were maintained at 23°C and animal hemisphere explants were dissected at various times after fertilization. Explants were incubated with 7·2 nM-125I-aFGF ± 72 nM unlabelled aFGF for 45 min 4°C, washed extensively with NAM and counted. Specific binding was calculated by subtracting non-specific binding from the totals. Each column represents the average value from 40 explants and five separate experiments. Standard deviations are indicated.

Fig. 5.

Stage-specific binding of 125I-aFGF to ectodermal explants. Embryos were maintained at 23°C and animal hemisphere explants were dissected at various times after fertilization. Explants were incubated with 7·2 nM-125I-aFGF ± 72 nM unlabelled aFGF for 45 min 4°C, washed extensively with NAM and counted. Specific binding was calculated by subtracting non-specific binding from the totals. Each column represents the average value from 40 explants and five separate experiments. Standard deviations are indicated.

Fig. 6.

Regional distribution of FGF receptors in midblastula embryos. Midblastula embryos were dissected into animal, marginal zone and vegetal pieces as indicated in B or the marginal zone was subdivided into dorsal and ventral pieces as in C. The future dorsal side of the embryo was identified by the vital dye mark (hatched). Explants were incubated with 7·2nM-125T-aFGF ± 72 nM-unlabelled aFGF for 45 min at 4°C, washed extensively with NAM and counted. Specific binding was calculated by subtracting nonspecific binding from the totals. The results are presented as ratios of specific binding with respect to that bound to pieces of the marginal zone or the dorsal marginal zone ‘with similar surface area, as described in the text. The columns on the left hand side of A represent the average value from 64 embryos and four separate experiments; the columns on the right hand side of A represent the average value from 28 embryos and two separate experiments. Standard deviations are indicated.

Fig. 6.

Regional distribution of FGF receptors in midblastula embryos. Midblastula embryos were dissected into animal, marginal zone and vegetal pieces as indicated in B or the marginal zone was subdivided into dorsal and ventral pieces as in C. The future dorsal side of the embryo was identified by the vital dye mark (hatched). Explants were incubated with 7·2nM-125T-aFGF ± 72 nM-unlabelled aFGF for 45 min at 4°C, washed extensively with NAM and counted. Specific binding was calculated by subtracting nonspecific binding from the totals. The results are presented as ratios of specific binding with respect to that bound to pieces of the marginal zone or the dorsal marginal zone ‘with similar surface area, as described in the text. The columns on the left hand side of A represent the average value from 64 embryos and four separate experiments; the columns on the right hand side of A represent the average value from 28 embryos and two separate experiments. Standard deviations are indicated.

The final question examined was whether all cells have FGF receptors and, if so, can we detect a regional difference in the receptor density? In the first part of this experiment, 125I-aFGF was bound to midblastula embryos that had been divided into three regions: animal cap (AC), marginal zone (MZ) and vegetal (V) pieces, as illustrated in Fig. 6B. Since each piece has a different shape and size, it was necessary to correct for differences in the available binding area. Darlington (1989) had previously shown by autoradiography of sectioned explants that, at 4°C, 125I-aFGF does not penetrate into the explanted tissue but binds only to the cells at the blastocoelic surface. Therefore, estimates of the surface area of the three regions were made by cell surface iodination. The number of counts per min of 1251 incorporated into each piece was assumed to be a direct measure of the available surface area. The average of six determinations (data not shown) gave a ratio of 1·0:2·0:2·7 for AC:MZ:V. All bound aFGF values obtained were corrected according to these ratios.

The labelled aFGF specifically bound to each region of the embryo is expressed as a ratio of that bound to the marginal zone. This was done to correct for variations among batches of embryos and also slight variations in embryo staging since the important point was the relative numbers of receptors in different parts of the same embryo. The highest receptor density in all of four experiments was found in the marginal zone while animal caps and vegetal pieces contained 70% and 40 %, respectively (Fig. 6A). The presence of receptors on vegetal cells was verified by affinity-labelling (not shown). Thus, cells in all regions of the stage 8 blastula express FGF receptors on their cell surface. This may be particularly relevant to possible models of mesoderm formation since vegetal cells do not form mesoderm in vivo and are not induced to differentiate by FGF in vitro (unpublished results).

Different mesodermal structures are formed along the dorso ventral axis of the embryo, with notochord and muscle arising from the dorsal marginal zone and mesenchyme and blood from the ventral marginal zone. We wished to determine whether we could detect a difference in the number of receptors in these two regions. The dorsal side of the embryo which can be identified at the 4-cell stage (Dale & Slack, 1987b) was marked with Nile Blue in 5 % agarose. The accuracy of the marking was verified by examining control embryos at gastrulation. In 50 out of 51 embryos (98%) the dorsal side was marked. Dorsal and ventral marginal zone pieces were excised at midblastula stage as illustrated in Fig. 6C and the number of receptors determined by 125I-aFGF binding. As can be seen in Fig. 6A, there was no detectable difference in the number of receptors in these two regions.

We have identified and characterized receptors for FGF that are present on the cell surface of developing Xenopus laevis embryos. These receptors are similar in relative molecular mass and bind aFGF with an affinity similar to that reported for mammalian cells (Neufeld & Gospodarowicz, 1986; Olwin & Hauschka, 1986). Like the mammalian receptors, they interact with both aFGF and bFGF, but not with TGFβ2, another growth factor which can induce mesoderm in Xenopus animal pole explants (Rosa et al. 1988). The receptor density per mm2 of blastocoelic membrane is about 3 x 108 which is within the range of figures reported for various mammalian tissue culture cells (Gospodarowicz et al. 1987). We have often observed that one of the crosslinked bands corresponding to 140 × 103Mr. is not always present on explant cell surfaces. This receptor form may represent an alternative receptor, a post-transcriptional or post-translational modification of the receptor such as glycosylation. We are currently investigating whether the appearance of these forms are developmentally regulated and whether or not one receptor form is specific for induction of mesoderm by FGF.

Once growth factors had been identified as embryonic morphogens, it was natural to propose that the competence of the tissue to respond to a signal would correspond to possession of the appropriate specific receptor. Our results suggest that receptor occupancy is a limiting step for mesoderm induction by FGFs. This is because the dose-response curve in terms of muscle formation provoked by aFGF (Slack et al. 1988) parallels closely the binding curve for aFGF given here, with a half-maximal response at 3−4 nM. For the time course, there is an almost exact parallel between the rise and fall of receptor density in the animal hemisphere and the rise and fall of inducibility by exogenous FGF, both qualitatively (Slack et al. 1988) and quantitatively in terms of muscle formation (Darlington, 1989). Inducibility is very low at stage 6 (32 cells), rises to a maximum by stage 8 and falls to a very low level again by stage 10; the maximum inducibility and receptor count is at about the 1024 cell stage. Competence to respond to the natural inducing signal(s) has been determined by Gurdon et al. (1985) and Jones & Woodland (1987) as extending approximately from stage 6×5 (64 cells) to stage 10 inclusive. This is somewhat longer than the duration of FGF-inducibility since there are about two hours from stage 9 to stage 10 at 23°C. Although no active factors other than bFGF have yet been detected in early embryos, it is possible that the natural signal includes some TGF/Llike component and it is this that is responsible for the later inductions. We have shown that TGFβ2 does not cross react with the FGF receptor and so it is probable that this substance acts through its own receptor. Competence to respond to XTC-MIF, a TGFμ-like molecule, has been shown to extend to stage 11 (Symes et al. 1988; Darlington, 1989) which would suggest that TGFβ receptors persist to a later stage than FGF receptors.

As far as the spatial extent of mesoderm induction is concerned, we have already formed a fairly precise picture from embryological experiments. In normal development, about 30% of the animal hemisphere becomes incorporated into the mesoderm (Dale & Slack, 1987a), but we know that all parts are capable of being induced, either by vegetal cells or by treatment with MIFs. The type of mesoderm formed from animal pole tissue (axial or ventral) can be controlled by the position of origin of the inducing tissue. Only the most dorsal sector of the vegetal hemisphere is capable of provoking axial inductions (Gimlich, 1986; Dale & Slack, 1987b). These results suggest that all parts of the animal hemisphere are competent to form any mesodermal tissue and that both the extent of the mesoderm and the spatial pattern of tissues within it are determined by the spatial distribution of the signal(s). However, this is a qualitative assessment and we have not until now had any idea about the relative degree of competence of marginal zone versus animal pole tissue. Although the measurements presented here show that the receptor density is slightly higher in the marginal zone compared with the animal pole region, this difference is not sufficient to significantly alter the effective dose of FGF. In addition, there was no detectable difference in the receptor density along the dorsoven-tral axis of the marginal zone, a difference which might have accounted for the different mesodermal structures formed along this axis. Our results also show that the vegetal cells which do not normally form mesoderm and which cannot be caused to do so by treatment with FGF also contain FGF receptors. Thus, the expression of FGF receptors on the cell surface may be a requirement for competence, but the presence of receptors in itself is not sufficient to specify cells that can differentiate into mesoderm. We presume that the signal transduction pathway required for mesoderm induction is not active in vegetal cells. It is also important to realize that the distribution of one type of receptor may tell only part of the story. Mesoderm induction may involve two or even more morphogens, as originally proposed on the basis of embryological experiments by Smith et al. (1985) and supported by the discovery of two active groups of factors: FGF and TGFβ

To summarize, it is probable that the overall pattern of mesoderm induction in the embryo is defined by a combination of factors which would include a balance between diffusion and degradation of the inducer(s) (Slack, 1987), the presence of an endogenous inhibitor (Cooke et al. 1987) and the limited spread of the signal (Gurdon, 1989) coordinated with the timed expression by responding cells of receptors coupled to the appropriate intracellular signalling pathway.

Identification and characterization of the receptor for FGF in Xenopus embryos will allow us to investigate several aspects of the induction mechanism, such as the nature of the intracellular signalling pathway for FGF during differentiation and the interaction of other mesoderm inducers such as TGFμ2 with FGF in modulating the receptor densities of FGF and/or the response to FGF induction.

We thank Sue Godsave for critical reading of the manuscript. LLG is the recipient of a Medical Research Council of Canada Centennial Fellowship. GDP and JMWS are supported by the Imperial Cancer Research Fund.

Cooke
,
J.
,
Smith
,
J. C.
,
Smith
,
E.
&
Yaqoob
,
M.
(
1987
).
The organization of mesodermal pattern of Xenopus laevis: experiments using a Xenopus mesoderm inducing factor
.
Development
101
,
893
908
.
Dale
,
L.
&
Slack
,
J. M. W.
(
1987a
).
Fate map for the 32-cell stage of Xenopus laevis
.
Development
99
,
527
551
.
Dale
,
L.
&
Slack
,
J. M. W.
(
1987b
).
Regional specification within the mesoderm of early embryos of Xenopus laevis
.
Development
100
,
279
295
.
Darlington
,
B. G.
(
1989
).
The responses of ectoderm to mesoderm induction in early embryos of Xenopus laevis. D.Phil. thesis, University of Oxford
.
Esch
,
F.
,
Baird
,
A.
,
Ling
,
N.
,
Ueno
,
N.
,
Hill
,
F.
,
Geueroy
,
L.
,
Kleeper
,
R.
,
Gospodarowicz
,
D.
,
Bohlen
,
P.
&
Guillemin
,
R.
(
1985
).
Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino terminal sequence of bovine brain acidic FGF
.
Proc. natn. Acad. Sci. U.S A.
82
,
6507
6511
.
Gimlich
,
R. L.
(
1986
).
Acquisition of developmental autonomy in the equatorial region of the Xenopus embryo
.
Devi Biol.
115
,
340
352
.
Godsave
,
S. F.
,
Isaacs
,
H.
&
Slack
,
J. M. W.
(
1988
).
Mesoderm inducing factors: a small class of molecules
.
Development
102
,
555
566
.
Gospodarowicz
,
D.
,
Cheng
,
J.
,
Lui
,
G. M.
,
Baird
,
A.
&
Bohlen
,
P.
(
1984
).
Isolation of brain fibroblast growth factor by heparin sepharose affinity chromatography: Identity with pituitary fibroblast growth factor
.
Proc. natn. Acad. Sci. U.S.A.
81
,
6963
6967
.
Gospodarowicz
,
D.
,
Neufeld
,
G.
&
Schweigerer
,
L.
(
1987
).
Fibroblast growth factor: structural and biological properties
.
J. cell. Physiol. (Suppl)
5
,
15
26
.
Gurdon
,
J. B.
(
1989
).
The localization of an inductive response
.
Development
105
,
27
33
.
Gurdon
,
J. B.
,
Fairman
,
S.
,
Mohun
,
T. J.
&
Brennan
,
S.
(
1985
).
Activation of muscle-specific actin genes in Xenopus development by an induction between animal and vegetal cells of a blastula
.
Cell
41
,
913
922
.
Jones
,
E. A.
&
Woodland
,
H. R.
(
1987
).
The development of animal cap cells of Xenopus: a measure of the start of animal cap competence to form mesoderm
.
Development
101
,
557
563
.
Kimelman
,
D.
,
Abraham
,
J. A.
,
Haaparanta
,
T.
,
Palisi
,
T. M.
&
Kirschner
,
M. W.
(
1988
).
The presence of fibroblast growth factor in the frog egg: its role as a natural mesoderm inducer
.
Science
242
,
1053
1056
.
Kimelman
,
D.
&
Kirschner
,
M.
(
1987
).
Synergistic induction of mesoderm by FGF and TGF/3 and the identification of an mRNA coding for FGF in the early Xenopus embryo
.
Cell
51
,
869
877
.
Laemmli
,
U. K.
(
1970
).
Cleavage of structural proteins during assembly of the head of the bacteriophage T4
.
Nature, Land, m,
Lobb
,
R. R.
,
Harper
,
J. W.
&
Feit
,
J. W.
(
1986
).
Purification of heparin binding growth factors
.
Anal. Biochem.
154
,
1
14
.
Neufeld
,
G.
&
Gospodarowicz
,
D.
(
1986
).
Basic and acidic fibroblast growth factors interact with the same cell surface receptors
.
J. biol. Chem.
261
,
5631
5637
.
Nieuwkoop
,
P. D.
&
Faber
,
J.
(
1967
).
Normal Table of Xenopus laevis.
Amsterdam
:
North-Holland
.
Olwin
,
B. B.
&
Hauschka
,
S. D.
(
1986
).
Identification of the fibroblast growth factor receptor of Swiss 3T3 cells and mouse skeletal muscle myoblasts
.
Biochemistry
25
,
3487
3492
.
Rosa
,
F.
,
Roberts
,
A. B.
,
DanŒlpour
,
D.
,
Dart
,
L. L.
,
Sporn
,
M. B.
&
Dawid
,
I. B.
(
1988
).
Mesoderm induction in amphibians: the role of TGF/32-like factors
.
Science
239
,
783
785
.
Slack
,
J. M. W.
(
1987
).
Morphogenetic gradients-past and present
.
TIBS
12
,
200
204
.
Slack
,
J. M. W.
,
Darlington
,
B.
,
Heath
,
J. K.
&
Godsave
,
S. F.
(
1987
).
Mesoderm induction in early Xenopus embryos by heparin-binding growth factors
.
Nature, Lond.
326
,
197
200
.
Slack
,
J. M. W.
&
Forman
,
D.
(
1980
).
An interaction between dorsal and ventral regions of the marginal zone in early amphibian embryos
.
J. Embryol. exp. Morph.
56
,
283
299
.
Slack
,
J. M. W.
&
Isaacs
,
H. V.
(
1989
).
Presence of basic fibroblast growth factor in the early Xenopus embryo
.
Development
105
,
147
154
.
Slack
,
J. M. W.
,
Isaacs
,
H. V.
&
Darlington
,
B. G.
(
1988
).
Inductive effects of fibroblast growth factor and lithium ion on Xenopus blastula ectoderm
.
Development
103
,
581
590
.
Smith
,
J. C.
,
Dale
,
L.
&
Slack
,
J. M. W.
(
1985
).
Cell lineage labels and region specific markers in the analysis of inductive interactions
.
J. Embryol. exp. Morph. 89 Suppl., 317-331
.
Smith
,
J. C.
,
Yaqoob
,
M.
&
Symes
,
K.
(
1988
).
Purification, characterization and partial biological effects of the XTC mesoderm-inducing factor
.
Development
103
,
591
600
.
Symes
,
K.
,
Yaqoob
,
M.
&
Smith
,
J. C.
(
1988
).
Mesoderm induction in Xenopus laevis: responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells
.
Development
104
,
609
618
.
Waddington
,
C. H.
(
1940
).
Organisers and Genes. Cambridge University Press
.