Development 105, 147-153 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

147

Presence of basic fibroblast growth factor in the early Xenopus embryo

J. M. W. SLACK and H. V. ISAACS

Imperial Cancer Research Fund, Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford,OX13PS

Summary

Mesoderm-inducing activity can be extracted fromXenopus embryos, eggs or whole ovary. It binds toheparin and can be neutralized by heparin or anti-bFGFbut not by anti-TGF/?. Two molecular forms can beidentified by Western blotting and have molecularweights of about 19 and 14K. The content in embryos isabout 7 units g~' (approximately 7 ng ml~*) which would

be sufficient for it to be acting as an endogenous inducerof ventral mesoderm. Attempts to detect TGF/5-likeinducing factors in embryos were not successful.

Key words: Xenopus oocyte, egg, blastula, heparin-bindinggrowth factors, fibroblast growth factor, transforminggrowth factor beta, mesoderm-including factors.

Introduction

Much interest has been aroused recently by the possi-bility of identifying the morphogens responsible formesoderm induction in the early amphibian embryo.This is the process by which the mesoderm is inducedfrom the animal hemisphere of the blastula by signalsfrom the vegetal region (Nieuwkoop, 1969; Dale et al.1985; Gurdon et al. 1985; Jones & Woodland, 1987).Recently we reported that a small group of heparin-binding growth factors (HBGFs) are active as meso-derm-inducing agents and the relatively high degree ofspecificity suggested to us that at least the ventralmesoderm induction within the embryo was mediatedby an HBGF (Slack et al. 1987). However, it has nowbeen found that another type of growth factor, TGF/J-2,is active (Rosa et al. 1988) and a mesoderm-inducingfactor recently purified from a Xenopus cell line is alsothought to belong to the TGF/3 family (Smith, 1987;Smith et al. 1988). So which, if any, of these factors isreally involved in mesoderm induction in the earlyembryo? A clue was provided by Kimelman &Kirschner (1987) who detected an mRNA of the bFGFtype in Xenopus embryos. We now show that a meso-derm-inducing factor can be isolated from Xenopusblastulae. It is similar to bFGF by biochemical andimmunological criteria and has a similar mesoderm-inducing activity in vitro. The quantity present is smallbut sufficient for it to be responsible for induction invivo. This is consistent with, although does not yetfinally prove, the involvement of bFGF as a morphogenin mesoderm induction.

Materials and methods

Extraction of HBGF from Xenopus materialEggs and embryos were dejellied with 2% cysteine hydro-chloride pH7-9 and stored at -70°C before use, as werewhole ovaries from adult females. The final protocol adoptedwas as follows: 25-100 g of ovary, unfertilized eggs or em-bryos were homogenized in 2-3 vols of 0-15M-ammoniumsulphate containing lmM-PMSF and 100 nin-pepstatin A.Homogenization was by Ultra turrax blender for 3x1 min atmaximum speed, the temperature being kept below 10°C withice. All subsequent steps up to the Amicon concentrationwere carried out at cold room temperature. The pH wasadjusted to 4-5 with acetic acid and the homogenate wasstirred for 1 h on ice. It was then centrifuged at 150000g forone hour and lipid was removed by straining through nylonmesh. The supernatant was neutralized with ammonia andfractionated by adding, successively, 0-24 g ml"1 (40%) and0'2gml"' (70%) solid ammonium sulphate followed bycentrifugation at 12000g for 10min. The 40-70% cut wasdialysed overnight against 0-6M-NaCl, 20mM-Tris pH7-0 andthen applied to a 2 ml column of heparin Sepharose (Sigma orPharmacia) equilibrated in the same buffer. The column waswashed in the same buffer and eluted with 2M-NaCl, 20 HIM-Tris-HCl pH7-0. Ovalbumin (Sigma, mol. wt std) was addedas carrier and the product was concentrated to 1 ml with anAmicon ultrafiltration cell and YM10 membrane. This ma-terial is referred to below as the 'heparin-bound fraction'.

Earlier preparations differed in that the initial homogenatewas centrifuged less vigorously (25 OOOg, 30 mins), a 40-80 %ammonium sulphate precipitate was taken, and also that aCM-Sephadex column at pH6 was employed before theheparin column.

Western blotsThe samples were separated using 15 % SDS-polyacrylamidegels and transferred to nitrocellulose using standard tech-

148 J. M. W. Slack and H. V. Isaacs

niques. The blots were blocked for lh in 0-15M-NaCl, 0-1 M-Tris-HCl pH7-4, 1% milk powder. They were stainedovernight in the same solution with 30[igm\~l anti-bFGF(protein A purified) or 2-5 ,ugml"1 anti-bFGF 16-30 (affinitypurified) with or without 25^gml~1 of BSA-peptide conju-gate or free peptide. Visualization was with the VectorAlkaline phosphatase ABC kit and number 2 substrate,following the manufacturer's protocol.

Silver staining0-75 mm thick SDS gels were silver stained by the method ofHeukshoven & Dernick (1985).

Heparin HPLCThe heparin-bound fraction was diluted to 0-5M-NaCl with20mM-Tris-HCl pH7-5, applied to a Heparin 5PW HPLCcolumn (Anachem) and eluted with a gradient of NaCl from0-5 to 1-5M in 20mM-Tris-HCl pH7-5. A portion of eachfraction was assayed and another portion run on a gel,transferred to nitrocellulose and reacted with one of theantibodies.

BioassayAssays were carried out by the end-point dilution method forinducing factors described by Godsave el al. (1988). Briefly,explants of ectoderm from the animal pole region of Xenopusblastulae are exposed to serial dilutions of the preparation.Induction is assessed by the formation of swollen vesicles after2-3 days culture (such vesicles always contain mesodermaltissues). The titre of the preparation is the reciprocal of thehighest dilution that retains activity, i.e. if 1/64 is the highestactive dilution then the activity is 64 units ml"1

Embryological methodsMethods for fertilization, composition of salines, histologicaltechniques and inducing factor treatments are all given inGodsave et al. (1988).

Materialsa and bFGF were prepared in the laboratory from bovinebrain, using methods described previously (Slack et al. 1988).kFGF was an in vitro translation mixture prepared by G.Paterno in this laboratory (see Paterno, Gillespie, Slack andHeath, submitted). Antibodies to a and bFGF were protein Apurified IgG donated by Dr D. Gospodarowicz (UC MedicalCenter, San Francisco). The anti-bFGF(16-30) was preparedin the laboratory against a thyroglobulin conjugate of thepeptide HFKDPKRLYCKNGGF, and was affinity purifiedusing a column carrying a BSA conjugate of the peptide.Although the peptide is from the bovine sequence (Abrahamet al. 1986), it differs from the Xenopus sequence only in thatit has H instead of S at the first position (D. Kimelman,personal communication). TGF/3-2 and anti-TGF/3 were pur-

chased from R&D Systems Inc. Anti-kFGF was donated byDr D. Rogers (Genetics Inst.). Heparin was type I purchasedfrom Sigma.

Results

Extraction of endogenous inducing factor fromXenopus ovary, eggs and embryos.Preliminary experiments using ovary showed that sol-uble mesoderm-inducing activity could be extracted byhomogenizing in a suitable buffer and stirring in thecold room for lh . Activity was assayed by observingmesoderm induction in explants of ectoderm isolatedfrom Xenopus blastulae and exposed to serial dilutionsof the fractions (Godsave et al. 1988). Similar yieldswere obtained using low (0-15 M) or high (1 M) salt andlow (4-5) or neutral (7-4) pH and the standard methodchosen was that previously used for HBGFs, namely0-15M-ammonium sulphate at pH4-5 (Gospodarowiczet al. 1984; Esch et al. 1985; Lobb et al. 1986). Thesolubilized activity is relatively stable in crude extractsand is only reduced by about fourfold by acidification(0-1% trifluoroacetic acid) or boiling. Although thereare losses during subsequent steps, most or all of theremaining activity was found to bind to CM-Sephadexat pH6 and, more significantly, to bind to heparinSepharose in 0-6M-NaCl at pH7, thus satisfying thedefinition of an HBGF.

Preparations were made from ovary, unfertilizedeggs and blastula-stage embryos and, in all cases,virtually all of the recoverable activity was found tobind to heparin. A protocol from a 50 g embryopreparation is shown in Table 1. The total content ofinducing activity, judged from the activity of crudeextracts, was 7 units g"1 wet weight in packed dejelliedembryos (average of 5 preparations), compared toabout 1000unitsg"1 of bovine brain, the standardsource for HBGFs. The actual values for the fivepreparations were: 16, 13, 5, 2 and 1 units g"1 wet wt,the two lowest figures coming from small scale prep-arations using about 0-25-0-5 g embryos. If these twowere excluded on the grounds that the scaled-downprotocol might promote losses, then the average wouldbe 11 units g"1 wet wt. The serial dilution assay has aninherent uncertainty of a factor of 2 but from theseresults it seems unlikely that the true figure wouldexceed 30 or be less than 5 units g"1 wet wt. The contentin unfertilized eggs was about the same as for embryos,but in ovary was substantially higher (109 units

Table 1. Purification of mesoderm-inducing activity from Xenopus blastulae

FractionTotal protein

(nig)Total activity Specific activity Recovery

(units) (units mg"1) (%)

1. 40-80% ammonium sulphate precipitate2. CM-Sephadex bound3. Heparin-Sepharose bound

40 % ammonium sulphate precipitateCM-Sephadex unboundHeparin-Sepharose unbound

2107-50-072-75

1907

6404801028-84020

3064

14573-20-22-8

(100)7516

Steps 1, 2 and 3 are sequential. Protein was determined by the Folin method.

Presence of basic fibroblast growth factor in the early Xenopus embryo 149

g wet wt, average of 8 preparations of which max. 197and min. 68 unitsg wetwt). It should be rememberedthat ovary contains follicle cells, connective tissue,nerves, blood vessels etc. in addition to oocytes, and atpresent we do not know how much of the activity ispresent in the oocytes and how much in the other celltypes.

Although the evidence (see below) suggested that allthe inducing activity was attributable to bFGF, wethought it possible that TGF/3-like factors might not beextracted or might not be active under these conditions.Accordingly, we extracted both blastulae and also thepellets left by the aqueous buffer extraction with acidethanol, following the protocol of Assoian et al. (1983)for TGF/?. However, we were unable to detect anyinducing activity in these preparations or any synergismwith bovine bFGF. Furthermore, there was no growthinhibition activity using CCL64 mink lung cells whichare sensitive to both TGF/3-1 and TGF/3-2 (data notshown). These measurements place an upper limit onTGF/3 content of 0-8 inducing units g""1 wetwt, aboutone tenth of the total observed activity.

Neutralization experimentsBoth crude extracts and those partially purified byheparin affinity chromatography were tested against apanel of reagents to characterize them. At the sametime the specificity of the reagents was checked bytesting them against pure factors. The factors wereaFGF, bFGF, kFGF and TGF/3-2, and the reagentswere antibodies to aFGF, bFGF, kFGF, TGF/3, andalso heparin. Conditioned medium from XTC cells wasalso tested. In each case a concentration of about10 units ml"1 of the factor was tested against twofoldserial dilutions of the reagents. If there is any inhibitionthis becomes lifted below a certain dilution thus provid-ing a neutralization titre. These were reasonably repro-ducible, except for TGF/3-2/heparin, and showed thatthere was no cross inhibition of pure factors by anti-bodies to other factors and that the neutralization titreswere in the range 1-50 /xg of IgG per unit of factor. Thecollected results are shown in Fig. 1. It is clear that the

NEUTRALIZATION DATA

Xenopus preparations behave like bFGF and not likethe other factors and since complete neutralization canbe achieved with anti-bFGF it seems probable thatthere are no other active factors present either in thecrude extracts or in the heparin-purified material.

We considered the possibility that the residual ac-tivity remaining after acidification of crude extractsmight be TGF/3-like. However, this too was inhibitableby anti-bFGF but not by anti-TGF/?, suggesting that thepartial acid resistance simply represents protection byother materials in the extract.

Although this series of experiments showed no inhi-bition of aFGF or kFGF by heparin, there may be aslight effect at high heparin concentrations as thesehave sometimes been apparent in series performed byother workers in this laboratory. Furthermore, two outof four experiments suggested that TGF/3-2 could beinhibited by heparin as effectively as bFGF, while theother two experiments showed no inhibition. We haveno explanation for this unreproducible behaviour.

It should be noted that none of the reagents testedhad any effect on the activity of the XTC-conditionedmedium. The anti-TGF/3 used is supposed to neutralizeboth TGF/3-1 and -2 and this may suggest that XTCfactor is less similar to TGF/3-2 than has been proposedby Rosa et al. (1988).

Biochemical characterizationTwo antibodies were used as Western blotting reagentsin this study. The first was an anti-peptide, anti-bFGF(16-30), prepared in this laboratory, which iscapable of detecting Ing of bovine bFGF with highspecificity. The second is the anti-bFGF used for theneutralization experiments. This is not so specific,showing a number of cross-reactive bands in the highermolecular weight range, and could detect about 20 ng ofbovine bFGF.

When the anti-bFGF(16-30) was used to stain blots ofheparin-bound material from ovary or embryos, a bandwas always seen at a molecular weight of about 19K,slightly larger than bovine bFGF. It sometimes ap-

aFCF bFGF kFGF TGFJ.-2 XTC-MIF X-OU-H X-EM X-EM-H

anti-aFGF

anti-bFGF

anti-kFGF

anti-IGFb

hepapin

H::S!:i «;;

Titre fop neutralization of one unit

mo neutralization observed

variable results

111 - SB Micpogpans/Ml/unit

§1 - 18 Microgrrans/Ml/unit

Fig. 1. Neutralization of Xenopusmesoderm-inducing factor by variousreagents.

150

58 -49 -

3 7 -27 -

20 -

14 -

J. M. W. Slack and H. V. Isaacs

1 2 3 4 5 6 8

1 4 -

1 2 3 4

A BFig. 2. Western blots of heparin-bound fractions. (A) Lane 1: Molecular weight markers, lane 2: bovine bFGF, lanes 3, 4:Xenopus ovary material, lanes 5, 6: Xenopus egg material, lanes 7, 8: Xenopus blastula material. Lanes 2-8 have beenstained with anti-bFGF (16-30) and alternate lanes (4, 6, 8) with excess free peptide included. (B) Lanes 1 and 5: Molecularweight markers, lane 2 Xenopus ovary material, lane 3: bovine bFGF, lane 4: Xenopus blastula material. Lanes 2-4 werestained with anti-bFGF IgG.

peared as a single band (lanes 3 and 5) and sometimesas a doublet (lane 7). It was completely suppressed byinclusion of a 10-fold excess of free peptide or BSA-peptide conjugate in the reaction mixture, showing thatthe antibody binding was specific (Fig. 2A). Whenovary preparations were further fractionated bychromatography on a heparin 5PW column, whichpermits gradient elution, the 19K reactive specieseluted at about l-25M-NaCl, coinciding with a peak ofinducing activity. However, larger amounts of inducingactivity also eluted at lower salt concentrations and itwas clear that the 19K band could not represent all of it.

When the neutralizing anti-bFGF was used to stainWestern blots of heparin-bound material from ovary orembryos, the 19K band was again seen but now it wasaccompanied by another band at about 14K (Fig. 2B).Using this antibody, there was quite a good fit betweenimmunoreactivity and biological activity on the heparin5PW fractionation, with the 14K band corresponding toa peak of activity eluting at about 0-8 M-salt and the 19Kband to the peak eluting at 1-25 M (Fig. 3). After theHPLC separation, it was possible to identify a silver-staining band corresponding to each of the immuno-reactive bands, although the 14K silver band extendedover more fractions than the biological activity and soprobably still does not represent a pure product(Fig. 4). The intensities of the blot and silver bandsallowed us to estimate that the specific activities of thepure factors must be in the region of 106 units mg"1

which is the same as for bovine bFGF.Because of the chemical and immunological simi-

larities of our factor to bFGF, it is tempting to assume

that it is the Xenopus homologue of mammalian bFGF.It will accordingly be referred to as such.

Biological effects of Xenopus bFGFA number of ectoderm explants were examined histolo-gically after treatment with various doses of XenopusbFGF and culture for three days to allow differen-tiation. The results were identical whether embryo-,egg- or ovary-derived material was used, the prep-arations tested being heparin-bound fraction from eachand, in the case of the ovary, also the two active peaksfrom the heparin 5PW gradient separation. At lowdoses, of about 1-4 units ml"1, the inductions weretypically ventral in pattern, containing loose mesen-chyme surrounding a mesothelial layer with a fewpycnotic cells in the centre and sometimes a small wispof muscle (Fig. 5A). At higher doses, from8-256 units ml"1, the inductions were mainly 'inter-mediate' (see Dale & Slack, 1987), containing abundantloose mesenchyme and large muscle blocks (Fig. 5B).However, two cases out of 20 treated with high dosescontained notochord, which is perhaps more thanwould be expected from comparable experiments withbovine FGFs. The external appearance of the explantsafter induction was the same as previously described forbovine bFGF, usually consisting of swollen vesicles withsome contents (Fig. 5C,E). Cases inhibited by anti-bodies or heparin were indistinguishable fromuntreated ones (Fig. 5D).

Discussion

In order to qualify as a morphogen a molecule has to

Presence of basic fibroblast growth factor in the early Xenopus embryo 151

Fig. 3. Heparin 5PW separation of heparin-bound materialfrom Xenopus ovary.

satisfy a number of criteria. These have recently beenthe subject of much discussion in the developmentalbiology community and a consensus set might read asfollows. Numbers 1-6 apply to any instructive inducingfactor, and nos 7-9 specifically to cases where amorphogen gradient is thought to specify a spatialpattern.

(1) The candidate molecule should produce the ap-propriate types of induction when tested on isolatedresponding tissue.

(2) It should be present in the embryo at the stageswhen the normal process is taking place.

(3) The amount should be adequate for the biologi-cal activity observed in vivo.

(4) It should be exported from the region identifiedas the signalling centre.

(5) It should be transmitted to the responding tissue.(6) Inhibition of its synthesis, transport or mode of

action by mutation or by specific chemical antagonistsshould inhibit the response in vivo.

(7) A gradient of concentration should exist withinthe responding tissue.

(8) Different structures should be induced by differ-ent concentrations in vitro.

(9) The order of structures in terms of decreasingdistance from the signalling centre shall be the same as

2 0 -

1 4 -

F18 F25Fig. 4. Silver stain and Western blot using anti-bFGF IgGof material from peak fractions shown on Fig. 3. Fraction18 is from the 0-8 M peak and fraction 25 from the 1-25Mpeak.

the order in terms of increasing concentration requiredto induce.

So far, only three substances approach the satisfac-tion of all these criteria. These are the bicoid product inthe egg of Drosophila (Driever & Niisslein-Volhard,1988), retinoic acid in the developing limb bud of thechick (Thaller & Eichele, 1987) and the slime mouldmorphogen, DIF (Morris et al. 1987). In each of thesecases, a number of uncertainties remain about their rolein vivo, but these would take too long to discuss here.

How does Xenopus bFGF rate as a candidate mor-phogen for the process of mesoderm induction? It isactive in vitro, and we have now shown that it is presentin the embryo at the appropriate stage. Since, bydefinition, a concentration of 1 unit ml is the mini-mum required for induction we can conclude that theamount in the embryo, although low, is adequate for itto be functioning as a morphogen in normal develop-ment, at least as an inducer of ventral mesoderm andparticularly if it is concentrated within the equatorialbelt of responding tissue. We may conclude that it haspassed tests 1, 2 and 3.

An indication that conditions 4, 5 and 6 may besatisfied is the fact that passage of the signal in atransfilter apparatus can be inhibited using concen-trations of heparin similar to those that inhibit bFGF invitro (Slack et al. 1987). However, it is unlikely thatheparin is a really specific reagent for bFGF as in theexperiments reported here we also noticed inhibition ofTGF/3-2 on some occasions. We therefore feel that

152 /. M. W. Slack and H. V. Isaacs

Fig. 5. Inductions produced by Xenopus bFGF after 3 days culture (control stage 40). (A) Ventral induction from4 units ml"1. (B) Intermediate induction from 32 units ml"1. Scale bar for A and B, 100 fim. (C) Induced explant produced by16 units ml"1. (D) as C, but with inclusion of 30^gml~' heparin, (E) as C but with inclusion of 200/igmn1 anti-TGF/3 (R&DSystems Inc.). Scale bar for C-E, 100 fm\.

more decisive evidence is required and we are continu-ing to work on this problem using the transfilterapparatus and other methods.

Xenopus bFGF approaches the satisfaction of con-dition 7 since low concentrations induce mesotheliumand mesenchyme only, higher concentrations inducemuscle, and the induction of notochord, although notcommon, has been observed. However, we have not sofar been able to localize the bFGF within the embryoeither by immunocytochemistry or by microscale ex-traction of dissected pieces, so we do not know whetherthere are any gradients in vivo. If we are correct in ourassessment from the gel bands that the specific activityof Xenopus bFGF is similar to that of bovine bFGF then7 units ml"1 translates into 7ngml~1 or 368 pM or221 molecules ̂ m"3. This is probably well below thedetection limit of any in situ method and will make

localization difficult. It is also unclear whether con-dition 9 is satisfied since the fate map data of Keller(1976) seems to suggest that the lateral plate arises fromnearer the vegetal tissue than the somites., and thiswould not correspond to the observed concentrationdependence of inductions in vitro, either for FGF or anyother mesoderm-inducing factor.

So, at present, Xenopus bFGF scores about 4 out of 9which in our opinion is not enough to be regarded as aproven morphogen. But the rapid pace of researcharound the world may enable us to reevaluate thesituation quite soon.

The occurrence of bFGF in the unfertilized eggsindicates that not only mRNA, as shown by Kimelman& Kirschner (1987), but also protein must be syn-thesized before it is needed for signalling. The pub-lished cDNA clone for bovine bFGF lacks a signal

Presence of basic fibroblast growth factor in the early Xenopus embryo 153

sequence for secretion (Abraham et al. 1986) and sincethere is no cell death during the early stages of Xenopusdevelopment we must presume that some novel mech-anism exists for getting it out of cells. Perhaps thereforeits export is controlled by the synthesis of some othermolecule at a later stage.

The difference of reactivity between our two anti-bodies is most easily explained by assuming that the14K form has been severely truncated at the JV-terminusand so lacks residues 16-30 against which the antipep-tide is directed. In some, but not all, preparations, somefainter immunoreactive bands were seen around15-16K with both antibodies, for example, see Fig. 2Alane 4. Also, the principal bands sometimes appearedas doublets and sometimes not. This sort of heterogen-eity is found also in FGF preparations from othersources but it is not known whether it representsmultiple endogenous forms or degradation during prep-aration .

As far as other factors are concerned we can place anupper limit on TGF/? content of about 10 % of bFGFactivity. However, this does not mean that TGF/Mikemolecules are definitely absent since we may still nothave found the optimal extraction procedures. Indeed,we expect other factors to be involved in mesoderminduction, partly because of the evidence advanced tosupport our own 'three signal model' (Dale & Slack,1987; Slack et al. 1988), and partly because the XTC-MIF (Smith, 1987; Smith et al. 1988) is a material ofimpressive potency obtained from a homologoussource, and it would be not be surprising if it too had arole to perform in the embryo.

We should like to thank Dr D. Gospodarowicz (UCMedical Center, San Francisco) for a gift of antibodies to aand bFGF; Dr G. Paterno (this laboratory) for the kFGF; DrD. Rogers (Genetics Institute, Cambridge, Mass.) for per-mission to use the anti-kFGF; and Dr J. Rothbard (ICRF,London) for making the peptide and peptide conjugates.

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{Accepted 15 November 1988)