Development 1989 Supplement, 141-148(1989)Printed in Great Britain © T h e Company of Biologists Limited 1989

141

The role of fibroblast growth factor in early Xenopus development

J. M. W. SLACK, B. G. DARLINGTON, L. L. G1LLESPIE, S. F. GODSAVE, H. V. ISAACS

and G. D. PATERNO

Imperial Cancer Research Fund, Developmental Biology Unit, Department of Zoology, South Parks Road, Oxford 0X1 3PS

Summary

In early amphibian development, the mesoderm isformed around the equator of the blastula in response toan inductive signal from the endoderm. A screen ofcandidate substances showed that a small group ofheparin-binding growth factors (HBGFs) were active asmesoderm-inducing agents in vitro. The factors aFGF,bFGF, kFGF and ECDGF all show similar potency andcan produce inductions at concentrations above about100 pM. The product of the murine int-2 gene is alsoactive, but with a lower specific activity. Above theinduction threshold there is a progressive increase ofmuscle formation with dose. Single blastula ectodermcells can be induced and will differentiate in a definedmedium to form mesodermal tissues. All inner blastulacells are competent to respond to the factors but outercells, bearing oocyte-derived membrane, are not.

Inducing activity can be extracted from Xenopusblastulae and binds to heparin like the previouslydescribed HBGFs. Antibody neutralization and Westernblotting experiments identify this activity as bFGF. Theamounts present are small but would be sufficient toevoke inductions in vivo. It is not yet known whether thebFGF is localized to the endoderm, although it is knownthat inducing activity secreted by endodermal cells canbe neutralized by heparin.

The competence of ectoderm to respond to HBGFsrises from about the 128-cell stage and falls again by theonset of gastrulation. This change is paralleled by a rise

and fall of binding of 125I-aFGF. Chemical cross-linkingreveals that this binding is attributable to a receptor ofrelative molecular mass about 130 xlO3. The receptor ispresent both in the marginal zone, which responds to thesignal in vivo, and in the animal pole region, which is notinduced in vivo but which will respond to HBGFs invitro.

In the embryo, the induction in the vicinity of thedorsal meridian is much more potent than that aroundthe remainder of the marginal zone circumference.Dorsal inductions contain notochord and will dorsalizeventral mesoderm with which they are later placed incontact. This effect might be due to a local high bFGFconcentration or, more likely, to the secretion in thedorsal region of an additional, synergistic factor. It isknown that TGF-/J-1 and -2 can greatly increase theeffect of low doses of bFGF, although it has not yet beendemonstrated that they are present in the embryo.Lithium salts have a dorsalizing effect on whole embryosor on explants from the ventral marginal zone, and alsoshow potent synergism when applied together withHBGFs.

Key words: Xenopus laevis, mesoderm induction,mesoderm-inducing factors, fibroblast growth factor,fibroblast growth factor receptor, transforming growthfactor beta, competence, morphogens.

Introduction

Work in experimental embryology has given us a fairlydetailed picture of the processes of regional specifi-cation occurring in the Xenopus embryo prior togastrulation. These processes are collectively called'mesoderm induction' because they lead to the forma-tion of a ring of mesodermal tissue around the equatorof the blastula (Nieuwkoop, 1969; Dale et al. 1985;Gurdon et al. 1985; Jones and Woodland, 1987). Thisknowledge has made it possible to ask meaningfulbiochemical questions about the nature of the signalsand the responses and about how they can lead to the

formation of a spatial pattern of specified regions in twoor three dimensions.

Briefly, we believe that the egg is divided into threecytoplasmic zones by the onset of the first cleavage:animal, ventrovegetal and dorsovegetal. The animalhemisphere will form epidermis in the absence ofinductive signals, but also has the competence to formmesodermal and probably endodermal tissues in re-sponse to such signals. The vegetal hemisphere consistsof a large 'ventral inducing' zone and a small 'dorsalinducing' zone comprising less than 90° of latitudearound the dorsal meridian (Dale and Slack, 19876).During the blastula stages, these two regions emit

142 J. M. W. Slack and others

signals which induce, respectively, an extended regionof ventral mesoderm around most of the equator, and asmall organizer region on the dorsal side. The signalsare quite short range, their influence extending only afew cell diameters (Gurdon, 1989), but, because ofsimultaneous migration of cells down into the equa-torial zone, about 40% of the animal hemisphereeventually becomes recruited into the mesoderm (Daleand Slack, 1987a). These signals are the first two of the'three-signal model' which our group has advanced toexplain mesodermal patterning, the third being a dor-salization of the mesoderm as a function of distancefrom the organizer (Slack and Forman, 1980; Smith andSlack, 1983; Dale and Slack, 1987b). This model mayneed to be revised as new data come in but at presentwe believe that it still provides the best unified accountof the known facts.

This understanding naturally leads us to ask threequestions: (1) What is the molecular nature of theinducing substances? (2) Are dorsal and ventral signalsqualitatively or quantitatively different? (3) What is themolecular nature of the competence of the animalhemisphere cells? Two critically important clues wereprovided by recent experiments on signal transmission.Grunz and Tacke (1986) showed that the signals couldpass through a nucleopore filter in the absense of cellprocesses, and Warner and Gurdon (1987) showed thatthe signals could pass from vegetal to animal cells evenwhen gap junction communication had been blocked.These biological experiments greatly narrowed thepossible range of mechanisms and firmly pointedtowards signals that consisted of secreted extracellularsubstances. In this paper, we describe our recent workon the role of fibroblast growth factor in mesoderminduction. Work on TGF/5-like factors is described inthe accompanying paper by Smith and his colleagues.

Which factors are active?

Although sources of mesoderm-inducing factors (MIFs)were discovered many years ago, they tended to excitelittle interest. This was for three reasons: they camefrom heterologous sources; they were assayed asgrafted pellets, a method that precludes quantitativebiochemistry; and most were very crude extracts. Thebest characterized was the 'vegetalizing factor' ofTiedemann (1982) isolated from late chick embryos, buteven this did not inspire confidence in the widerscientific community. We started work on the subject in1984, following our reinvestigation of the basic meso-derm induction phenomenon, and commenced byestablishing an assay procedure for MIFs which wasquantitative and which worked in solution. Briefly, thisconsists of treating animal pole explants with serialdilutions of the test substance and defining the mini-mum concentration required to provoke an induction as1 unit ml"'. The full procedure is described in Godsaveet al. (1988; see also Cooke et al. 1987). We thenattempted to extend Tiedemann's work on the chickembryo factor using our improved assay, but, following

the report by Smith (1987) of inducing activity secretedby a Xenopus cell line, we turned our attention to aninvestigation of known growth factors. In our initialscreen, we tested a wide range of factors and found onlythree that were active. These were basic fibroblastgrowth factor (bFGF), embryonal carcinoma derivedgrowth factor (ECDGF) and acidic fibroblast growthfactor (aFGF), all of which belonged to a small group ofheparin-binding growth factors (Slacker al. 1987). Morerecently, we have examined some of the FGF-likeoncogenes that have recently been discovered (Paternoet al. 1989). We have done this by in vitro transcriptionof cDNAs from plasmids containing SP6/T7 bacterio-phage promoters followed by translation in a rabbitreticulocyte lysate. The lysate can then be assayeddirectly by treating ectoderm explants with a series ofdilutions, and the specific activity determined bymeasurement of the concentration of the translatedprotein. So far, we have examined kFGF, which is theproduct of the human ks and hst oncogenes (Delli-Boviet al. 1987; Taira et al. 1987), and INT-2, the product ofthe murine int-2 oncogene (R. Smith et al. 1988). Bothare active as mesoderm-inducing factors. The specificactivity of the kFGF is very similar to that of the a andbFGF, while the specific activity of INT-2 is very muchlower. Considering the factors as a group, there is agood correlation between their mesoderm-inducingactivity and their mitogenic activity when tested onmammalian fibroblasts. This suggests that similar signaltransduction machinery is being used for the twoprocesses. It should be emphasized that MIFs do nothave any mitogenic effect on Xenopus blastula ecto-derm cells, which are already cleaving every 30min inthe absence of growth factors and are probably in-capable of further stimulation.

Meanwhile, work in other laboratories has shownthat some factors belonging to the TGF/3 family are alsoactive. These are TGF0-2 (Rosa el al. 1988) and theXTC-MIF of Smith (Smith, 1987; J. C. Smith et al. 1988)and so at the time of writing we have a total of sevenactive factors.

Which factors are present in the embryo?

Obviously the minimum requirement for identificationof an endogenous morphogen is that the substanceshould be present in the embryo at the developmentalstage when the relevant events are happening, and inamounts that are capable of exhibiting the observeddegree of biological activity. We have approached thisproblem directly by asking whether a MIF can beobtained from the Xenopus blastula, and which of theseven or more candidates it is. Our results show that it ispossible to purify a MIF from Xenopus blastulae usingheparin-affinity chromatography and that it consists oftwo proteins of Mr 19 and 14xlO3 which react withantibodies against bFGF (Slack and Isaacs, 1989). Thequantity in blastulae is about lOngmT1 which is suf-ficient to account for the ventral but not the dorsalinduction. The biological properties and specific activity

FGF in Xenopus development 143

of the Xenopus bFGF seem similar to the bovine bFGFwhich has been used for most of our experiments on theresponses of animal cells. All the MIF activity in a crudeembryo or ovary extract can be inhibited by a neutraliz-ing antibody to bFGF, but not by antibodies to a or kFGF or TGF/5-2. Parallel work by Kimelman et al.(1988) has also shown the presence of bFGF mRNAand protein in Xenopus blastulae. Their estimate ofquantity is much greater than ours but, unlike ours, it isnot based on the use of quantitative biological assaymethods.

We would obviously predict that the bFGF would besecreted by the cells of the vegetal hemisphere. So far,immunolocalization on embryo sections has not provedsuccessful, probably because of the small quantitiespresent. We have shown that the MIF released byvegetal cells in transfilter experiments can be neutral-ized by heparin, as can both Xenopus and bovinebFGF, but not by anti-bFGF antibodies. This maymean that the bFGF is secreted as part of some complexnot recognised by our neutralizing antibody, but furtherwork is necessary to prove beyond doubt that thevegetal cells really secrete bFGF. One problem in thisregard is the well-known fact that bFGF lacks a classicalsignal sequence for secretion (Abraham et al. 1986),and so there remains some uncertainty about its mech-anism of release from cells.

see increasing amounts of mesenchyme and increasingamounts of muscle (Fig. 1G,H; Fig. 2B). Notochord issometimes observed following the higher dose treat-ments, particularly when in vitro translated bFGF isused, but its formation is not very predictable. Thisdose-response curve is significantly different from thatobtained with XTC-MIF, which will induce notochordreliably at a low multiple of the minimum inducingconcentration (J. C. Smith et al. 1988), however, it isprobably rather similar to that of Xenopus bFGF(Fig. 3).

We have examined the location of I-labelled FGFin explants and find that it binds mainly to those plasmamembranes that are exposed at the blastocoelic surface.There is little binding to the plasma membrane of theexternal surface (oocyte-derived or O-membrane) andlittle penetration into the cell mass (Darlington, 1989).The maximal response to the high doses consists ofabout 20% muscle by cell composition with an ad-ditional 10-20% of mesenchyme and this probablyrepresents all the cells that were exposed on theblastocoelic surface of the explant at the time oftreatment. The fact that many cells in induced explantsare still epidermal may be entirely due to the limitedpenetration of the FGF since our studies of single cellsleads us to believe that all cells without O-membraneare potentially inducible (see below).

Effects of FGF on ectoderm explants

In this work, it has been found that the properties of aand bFGF in their capacity as MIFs are very similarindeed. In what follows, 'FGF' will be used to refer toeither form indifferently.

Untreated explants from around the animal pole ofXenopus blastulae develop into solid masses of epider-mal cells. It can be shown by using antibodies toepidermal markers that 100 % of cells become epider-mal (Fig. 1A-D). Mesoderm inductions can be pro-voked by FGF concentrations in excess of about 100 pM(Fig. 2A). After explants are exposed to FGF nothingmuch appears to happen for the first few hours, theexplants round up with their blastocoelic surface insideand the cells continue to cleave just like untreatedexplants. However, it is the first 90min or so ofexposure that are critical. After this time, the FGF canbe withdrawn without affecting the course of sub-sequent events. Then, while control embryos are under-going gastrulation, the explants elongate with the orig-inal closure point at one end and the original animalpole at the other (Fig. IE). Within a batch, the degreeof elongation depends on the applied dose, but betweenbatches there is considerable variation. After 24-36 h ofculture, the induced explants start to swell and soonbecome transparent (Fig. IF). These vesicles invariablycontain mesodermal tissues although the quantity andtype depends on the applied dose (Godsave et al. 1988;Slack et al. 1988). At low doses inductions consist ofsmall amounts of mesenchyme and mesothelium withthe occasional wisp of muscle while at higher doses we

Competence of the ectoderm

Using animal-vegetal combinations from differentstages, it has been shown that the competence of theectoderm to respond to the natural signal(s) extendsfrom about stage 6 (64 cells) to about stage 10i (Jonesand Woodland, 1987). We have studied the onset ofcompetence to FGF in the ectoderm by exposing for aperiod of 90 min explants taken from different stagesand this shows that competence begins at about stage 7.We have studied the loss of competence by permanentexposure of ectoderm explants taken from differentstages and this shows that competence is lost betweenstages 9 and 10 (Slack et al. 1988). Furthermore, thedegree of competence can be assessed by measurementof the amount of muscle formed by explants fromdifferent stages in response to a standard dose, and thisshows a rise and fall with the peak at stage 8 (Darl-ington, 1989). So the competence for FGF seems to riseat about the same time as competence for the naturalMIF(s) but falls rather earlier, since there are about 3 hbetween stage 9 and 10i at 22-24°C. Competence torespond to XTC-MEF seems to persist into gastrulation,until stage 101-11 according to our measurements(Darlington, 1989).

Since inductions arise in response to FGF concen-trations in the pM range we expected that an essentialmolecular component required for competence wouldbe a specific receptor. We have probed for a receptor onexplanted tissues using 125I-aFGF and the cross-linkingagent BS3. This has shown that a receptor is present andappears as two gel bands of MT about 130 and 140x 103,

144 /. M. W. Slack and others

#•

• If

Fig. 1. Mesoderm induction by FGF. (A) Untreated ectoderm explants after 16h. (B) Histological section of untreatedectoderm after 3 days. (C) Section stained with an antibody directed against cytokeratin XK70. All cells are stained. (D)Same section stained with DAPI to show cell nuclei. (E) FGF-treated explants after 16h. (F) FGF-treated explants after 3days ('vesicles"). (G) Section of induced explant stained with 12/101 anti-muscle antibody. (H) Same section stained withDAPI.

FGF in Xenopus development 145

2 4 8 16bFGF (ng/ml)

31 63 125

0-2 0-5 1 2 4 8 16bFGF (ng/ml)

31 63 125

Fig. 2. FGF dose-response curves. (A) Percentage ofexplants induced by different concentrations of bovinebFGF. (B) Amount of muscle formed in ectoderm explantsexposed to different concentrations of bFGF.

similar to the mammalian FGF receptor (Gillespie et al.1989). Binding studies show that about 70-80% ofbound I25I-FGF can be competed out by an excess ofunlabelled FGF. Assuming that this represents bindingto the specific receptor then the density is about 3xHrmolecules mm"2 of cell surface which is within therange of values measured for mammalian cells. Thebinding curve shows a half-maximal value of about3-4nM and a plateau at about 10nin, which is verysimilar to the dose-response curve for muscle forma-tion. This suggests that the receptor binding is a limitingstep in the response. If it were not, then a maximalresponse, in this case a maximal percentage of cellsinduced, would be obtained at an FGF concentrationbelow that required to saturate the receptors.

The receptor density has been studied by binding of125I-aFGF to ectoderm explants taken from differentembryonic stages. The competable binding rises by afactor of 10 between the early and middle blastula, andfalls again to the starting level by the onset of gastru-lation. This closely parallels the rise and fall of com-petence to respond to FGF and suggests that com-petence is indeed controlled by receptor density.

Competition experiments have shown that both a andbFGF bind to the same receptor but TGF/3-2 does not.This again resembles the situation in mammalian cellsand makes it probable that the extended period ofcompetence that ectoderm explants show when treatedwith XTC-MIF is due to the presence of separate TGF/Jreceptors.

We have measured the regional distribution of FGFreceptors in stage 8 blastulae by binding studies onexplants (Gillespie et al. 1989). This shows, as pre-dicted, that FGF receptors are present both in themarginal zone region, which normally responds to thesignal in vivo, and in the animal pole region, which can

B

Fig. 3. Ectoderm explants induced by Xenopus bFGF and cultured for three days. (A) 4unitsml l. (B) 32 units ml '.bar, 100 fim.

Scale

146 J. M. W. Slack and others

respond in experimental situations but would not nor-mally do so in vivo. There is a slight excess of receptordensity in the marginal zone but this is only 50% morethan the animal pole value, so it would seem that thenormal extent of mesoderm induction is determined bythe extent of the signal and not by the presence of amore highly competent tissue in the marginal zone.There is no difference in receptor density betweendorsal and ventral regions of the animal hemisphere, sothis cannot account for the difference between dorsaland ventral inductions. FGF receptor is also present inthe vegetal region. We do not know whether these cellsneed FGF for their normal development since wecannot deprive them of it in the way that we can deprivethe animal cells. However, since they do not normallyturn into mesoderm, we can deduce that mesodermalcompetence consists of something more than the pres-ence of FGF receptors on the cell surface.

Competence of individual ectoderm cells

Some other workers have noticed that isolated ecto-derm cells will not differentiate into mesodermal celltypes after induction, although their differentiation intoepidermis may be suppressed (Symes et al. 1988). Thisphenomenon has been called the 'community effect'(Gurdon, 1988). We have found that this requirementcan be met by a few simple macromolecular additives tothe culture medium. Single internal blastula ectodermcells can be induced if they are treated with FGF andthen cultured in the presence of gamma-globulin on asurface coated with fibronectin and laminin. Usuallythey give rise to monotypic clones of muscle or an'epithelium' which is a non-muscle, non-epidermal celltype, possibly a form of kidney. Sometimes mixedcolonies are formed with more than one mesodermalcell type. When cells are treated for only 2 h with FGF,the colonies are always monotypic (Godsave and Slack,1989). We are presently using this culture system toexamine the specification of single cells isolated fromdifferent parts of the marginal zone of normal embryos,and have shown that mesodermal clones can be ob-tained from the marginal zone of midblastulae.

Further experiments involving the induction of singlecells have shown that cells bearing the oocyte-derivedmembrane (O-membrane) are non-inducible (Darl-ington, 1989). The most informative protocol has in-volved (1) labelling of donor embryos by injection withthe lineage label rhodamine-dextran-amine (RDA), (2)isolating single labelled cells in Ca2+-free medium, (3)wrapping these in ectodermal jackets from unlabelledembryos, (4) inducing the whole sandwich with FGF oranother MIF before it has sealed. When inner cellswholly surrounded by cleavage membrane (C-mem-brane) are used then many progeny of the labelled cellare found in the induction. However, when cells bear-ing O-membrane are used only a very few progeny arefound to be induced. Close examination of these fewshows that all of them are themselves wholly sur-rounded by C-membrane, and must therefore have

arisen from the original cell by tangential cleavage. Sothey do not represent exceptions but rather they areimportant positive controls, showing that the cultureconditions do not militate against mesoderm differen-tiation. A further control in these experiments isprovided by the fact that the cells that do not formmesoderm do form epidermis, showing that the failureto form mesoderm is not due to some damage inflictedon the cells in the course of the manipulations.

The nature of the dorsal induction

It is generally agreed that the signal near the dorsalmeridian differs from that around the remainder of theblastula circumference. Some workers have tended tothink that it is qualitatively similar but more intensewhile others have leaned towards the view that it isqualitatively different. If we accept that bFGF is theventral morphogen, then the quantitative view seemsunlikely since notochord inductions are not reliablyproduced even by very high concentrations of FGF, andthe uniform distribution of FGF receptor shows that thedorsal and ventral ectoderm will respond alike tosimilar concentrations of FGF. However, it has beenshown that the effect of FGF can be modified by otherfactors. There is strong synergism between FGF andTGF/3-1 (Kimelman and Kirschner, 1987) and betweenFGF and lithium ion (Slack et al. 1988). Neither TGF/3-1 nor Li are active as mesoderm-inducing factors ontheir own and the synergism is usually manifested as anexcess formation of muscle rather than by induction ofnotochord. TGF/J-2 does have mesoderm-inducing ac-tivity on its own, and like FGF does not usually inducenotochord. However, the synergism between FGF andTGF/3-2 is strong enough to give reliable induction ofnotochord (E. Amaya, pers. comm.). We have seenabove that the receptors for FGF and TGF/3 on Xeno-pus ectoderm are distinct but the synergistic effectssuggest that there is a common intermediate at somelevel in the signal transduction pathway. This inter-mediate is presumably one whose level can be elevatedby Li+.

A reasonable working hypothesis based on these datamight be that bFGF is the ventral morphogen andbFGF+TGF/3-2 the dorsal morphogen. We wouldfurther suppose that the FGF system is prelocalized inthe vegetal hemisphere of the egg while the TGF/Jsystem is activated on the dorsal side only as a result ofthe postfertilization cytoplasmic movements (see Fig. 4and Gerhart et al. this volume). This would then explainthe effects of UV radiation and Li+ on whole embryos.If the vegetal hemisphere of the egg is irradiated with asufficient dose of UV light then the cytoplasmic move-ments are inhibited and a radially symmetrical ventralembryo is formed (Grant and Wacaster, 1972; Cookeand Smith, 1987). The simplest interpretation of this isthat the FGF system is normally present in the vegetalhemisphere all around the circumference and is unaf-fected by the treatment while the TGF/3 system woulddepend on the postfertilization cytoplasmic movements

% • :

Oocyte Fertilized egg

m m

Early blastula Mesoderm induction

Late blastula

(dorsalization starting)Gastrula

Fig. 4. Diagram of current version of the three-signal model. The yellow dots are sources of bFGF, prelocalized in the oocyte.The red squares are sources of TGF/3-2 (or XTC-MIF) which become activated and localized on the dorsal side followingfertilization. The green colour represents FGF receptors rising and falling during the blastula stages. The arrows representshort-range diffusion of the morphogens. The open circles represent cells: red for organizer type, yellow for ventral mesodermtype, other colours for intermediate mesodermal types formed by dorsalization.

FGF in Xenopus development 147

which are blocked by the UV dose. Li+ treatment of theearly embryo produces a symmetrical dorsalization(Kao el al. 1986; Cooke and Smith, 1988). Here thepostfertilization movements have already happened butwe presume that the Li can elevate the concentration ofa signal transduction intermediate and so mimic theeffect of a uniform dorsal stimulus. It has been shownthat lithium will dorsalize isolated ventral marginalexplants to the level of large muscle masses (Slack et al.1988; Kao and Elinson, 1988).

In fact, we have no evidence at present that theXenopus homologue of TGF/J-2 is present in the earlyembryo and it may be that some other TGF/Mikemolecule is doing the job. An obvious candidate is theXTC-MIF of Smith since this has chemical propertiesresembling TGF/3 and is currently the most active of allthe MIFs and the only one that will induce notochordon its own. Another possibility is the Vgl product. Herewe know that the mRNA is present in the embryo andlocalized in the vegetal hemisphere (Weeks and Mel-ton, 1987; Yisrael et al., this volume). However, theredoes not appear to be any preferential localization onthe dorsal side, and perhaps more seriously there is asyet no indication of any biological activity shown by theprotein. Clearly more data is needed in this area and inparticular data on the presence, distribution and ac-tivity of TGF/J-like molecules in the early Xenopusembryo.

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