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Development 1989 Supplement, 149-159Printed in Great Britain ©The Company of Biologists Limited 1989

149

Inducing factors and the control of mesodermal pattern in Xenopus laevis

J. C. SMITH, J. COOKE, J. B. A. GREEN, G. HOWES and K. SYMES*

Laboratory of Embryogenesis, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

* Present address: Department of Zoology, University of California, Berkeley, CA 94720, USA

Summary

The mesoderm of Xenopus laevis and other amphibia isformed through an inductive interaction during whichcells of the vegetal hemisphere act on cells of the animalhemisphere. Two groups of factors mimic the effects ofthe vegetal hemisphere. One group consists of membersof the fibroblast growth factor (FGF) family, while theother is related to transforming growth factor type B(TGF-B). In this paper we discuss the evidence that theFGF family represents 'ventral' mesoderm-inducingsignals, and the TGF-/J family 'dorsal' signals. The

evidence includes a discussion of the cell types formed inresponse to each type of factor, the fact that only XTC-MIF (a member of the TGF-/5 family) and not bFGF caninduce animal pole ectoderm to become Spemann'sorganizer, and an analysis of the timing of the gastru-lation movements induced by the factors.

Key words: mesoderm induction, mesoderm-inducingfactors, bFGF, XTC-MIF, thresholds, gastrulation,amphibian embryo, Xenopus laevis, developmental timers.

Introduction

The mesoderm of Xenopus laevis, and of other amphib-ian embryos, is formed through an inductive interactionin which cells of the vegetal hemisphere of the embryoact on blastomeres of the overlying marginal zone(Nieuwkoop, 1969, 1973; Sudarwati and Nieuwkoop,1971; reviewed by Smith, 1989; Fig. 1A). This interac-tion is usually demonstrated by juxtaposing tissue fromthe animal cap and the vegetal pole (Fig. IB). Nor-mally, the animal cap material is too far from vegetalblastomeres to receive an inductive signal and whencultured alone it forms 'atypical epidermis' - cells thatstain with antibodies to keratin but which do not adoptnormal epidermal morphology (see Smith etal. 1985).However, when animal cap cells are placed in contactwith vegetal cells a significant proportion (about 40%)differentiate as mesodermal cell types, including noto-chord and muscle (Dale et al. 1985; Gurdon et al. 1985).

Recently, significant progress has been made towardsidentifying the 'mesoderm-inducing factors' (MIFs)produced by vegetal pole cells. The candidates fall intotwo classes. One consists of members of the TGF-/3family and includes XTC-MIF (Smith, 1987; Smith et al.1988; Rosa et al. 1988), TGF-/32 (Rosa et al. 1988) andperhaps the protein encoded by the localized mRNAVgl (Rebagliati et al. 1985; Weeks and Melton, 1987).TGF-/31 is unusual in that it has no mesoderm-inducingactivity alone, but acts synergistically with members ofthe other class of MFFs, the FGF family (Kimelman andKirschner, 1987). Active members of this family includeaFGF, bFGF and embryonal carcinoma-derived growth

factor (ECDGF) as well as the protein products of thekFGF and INT-2 oncogenes (Slack et al. 1987; Kimel-man and Kirschner, 1987; Paterno et al. 1989; seepreceding paper by Slack and his colleagues).

In this paper, we compare the effects of the twoclasses of MIF and discuss how they might act togetherto establish the correct spatial pattern of cellulardifferentiation in the mesoderm of Xenopus. We thenreview what problems remain in coming to understandhow combinations of inducing factors might set up theremarkably constant mesodermal pattern of Xenopus(Cooke and Smith, 1987).

The three-signal model

A series of experimental embryological experimentshas led to the 'three-signal' model for the formation ofmesoderm in the early amphibian embryo (see Smith etal. 1985). This model is explained in detail in thepreceding paper in this volume by Slack et al. and willonly be described briefly here (Fig. 2). Thus duringoogenesis, which takes several months in Xenopus, theegg becomes polarized such that yolk, pigment and,most importantly, informational macromolecules, be-come localized to particular regions along the animal-vegetal axis (see Gerhart, 1980; Wylie et al. 1985;Melton, 1987; Yisraeli and Melton, 1988; Yisraeli etal.this volume). Until fertilization, however, the egg isradially symmetrical around the animal-vegetal axis:every meridian has the potential to form dorsal axialstructures.

150 J. C. Smith and others

B

Fig. 1. Mesoderm induction. (A) At early blastula stagesthe Xenopus embryo can be considered to consist of twocell types: presumptive ectoderm in the animal hemisphere(light stippling) and presumptive endoderm in the vegetalhemisphere (no stippling) (Jones and Woodland, 1986).During mesoderm induction a signal from the vegetalhemisphere induces overlying equatorial cells to formmesoderm (heavy stippling). This view is somewhatsimplified for it is not possible to draw an accurate linebetween vegetal inducing cells and animal pole respondingcells. Indeed, it may be that some cells both produce andrespond to the signal. (B) The classical demonstration ofmesoderm induction. Blastomeres from the animal pole of alineage-labelled blastula-staged embryo are placed incontact with cells from the vegetal pole of an unlabelledembryo.

This symmetry is broken at fertilization, when thesperm entry point defines the direction of rotation ofthe cortex of the egg with respect to the subcorticalcytoplasm. The orientation of this rotation in turnaccurately predicts the future dorsoventral axis of theembryo (see Gerhart et al. this volume). The mechan-ism by which this occurs is unknown, but it is clear thatmost of the dorsoventral patterning information thuscreated is in the vegetal region of the egg. This can bedemonstrated using animal-vegetal combinations;when animal pole cells are combined with dorsal

Fig. 2. The three-signal model. Two mesoderm inductionsignals are assumed to derive from the vegetal region of theearly blastula. The dorsal-vegetal (DV) signal inducesdorsal mesoderm, or 'organizer' tissue (O) while the ventralvegetal signal (VV) induces general ventral mesoderm(VM). The ventral mesoderm then receives a signal fromthe organizer, probably during gastrulation, which results inthe formation of additional muscle (Ml) and perhapspronephros (M2); only the most remote tissue (M3)remains as ventral blood-forming mesoderm.

vegetal blastomeres they form notochord, a dorsalmesodermal cell type, while ventral vegetal blastomeresinduce blood, mesenchyme and mesothelium which areusually regarded as ventral mesodermal cell types(Boterenbrood and Nieuwkoop, 1973; Dale et al. 1985;Dale and Slack, 19876; see Smith, 1989). An analogousexperiment was performed by Gimlich and Gerhart(1984), who showed that embryos made radially sym-metrical by UV irradiation of the vegetal hemisphere ofthe egg shortly after fertilization could be 'rescued' bydorsal vegetal, but not ventral vegetal, blastomeres.

It should be noted that the terms 'dorsal' and 'ventral'are somewhat misleading when applied to the preneur-ula-stage embryo. As a result of the earlier and morevigorous gastrulation movements on the 'dorsal' side ofthe embryo, these cells form most of the head andanterior of the larva, including structures which in thefinal morphology are 'ventral', as well as the notochordand some somitic tissue of the trunk. Consequently,blastomeres on the 'ventral' side tend to form ventralstructures in the trunk region of the embryo, but axialstructures more posteriorly (Cooke and Webber, 1985;Keller, 1975, 1976; Smith and Slack, 1983; Cleine andSlack, 1985; Dale and Slack, 1987a).

Thus, at the blastula stage one can distinguish, byoperational criteria, at least two types of mesoderminduction signal. One of these induces predominantlydorsal cell types and one ventral. But two signals, oreven three or four, from the vegetal hemisphere areunlikely to be sufficient to establish directly a complexand reproducible pattern of mesodermal cell types. Oneindication that more signalling subsequently takes placeis that the fate map of Xenopus shows that at least halfof the muscle of the embryo arises from the ventral sideof the embryo (Cooke and Webber, 1985; Keller, 1976;Dale and Slack, 1987a), whereas the ventral vegetalblastomeres induce very little muscle from respondinganimal pole tissue (Dale et al. 1985; Dale and Slack,19876). This paradox can be resolved by the third signalof the model, which is produced by dorsal mesodermcells and which acts on adjacent ventral mesoderm tocause it to form muscle instead of ventral tissues. Thisinteraction can be demonstrated by juxtaposing dorsaland ventral marginal zones in vitro. Normally, in

Mesoderm induction in Xenopus 151

isolation, the ventral tissue forms blood, mesenchymeand mesothelium, but in response to the dorsal tissue itforms muscle instead. The dorsal tissue, meanwhile,forms notochord as usual (Slack and Forman, 1980;Smith et al. 1985; Dale and Slack, 19876). An alterna-tive demonstration of this phenomenon of dorsalizationis to implant dorsal marginal zone tissue into the ventralmarginal zone of a host early gastrula (Smith and Slack,1983). This is the famous organizer graft of Spemannand Mangold (1924), and results in an embryo withmirror-image symmetry in the mesoderm which is alsoreflected in the induction of two neural tubes (Gimlichand Cooke, 1983; Smith and Slack, 1983; Jacobson,1984).

The three-signal model is summarized in Fig. 2. It isimportant to note that the model does not address theacquisition of anteroposterior positional values. This israther poorly understood although the current modelsare discussed in this volume by Gerhart and his col-leagues. One suggestion these authors make, however,is that the completeness of the anteroposterior axisdepends upon the size of the organizer, the regiondesignated 'O' in Fig. 2. We present some results belowwhich are consistent with this view.

Mesoderm-induclng factors

As outlined in the Introduction, mesoderm-inducingfactors in Xenopus fall into two families: FGF and TGF-fi. The members of these families are summarized inTable 1. At present, no member of either family satis-fies all the criteria for being a true morphogen (seeSlack and Isaacs, 1989; Wolpert, this volume). Thus forthe TGF-/3 family the most active inducer is XTC-MIF(Smith et al. 1988; Green et al., in preparation) but it isnot yet known whether the molecule is present in theearly embryo. One member of the TGF-/3 family whichis present in the embryo is the Vgl protein (Dale et al.1989; Tannahill and Melton, 1989), but it is not yetknown whether this has mesoderm-inducing activity.

The situation is slightly clearer with the FGF family,where it is known that bFGF is present in the embryo insufficient quantity to act as a mesoderm inducer (Kimel-man etal. 1988; Slack and Isaacs, 1989). It is not known,however, if the protein is localized to the vegetal regionof the embryo, and indeed this seems unlikely becausebFGF is synthesized throughout oogenesis and wouldbe expected to be free to diffuse throughout the oocyte.In addition, Xenopus bFGF, like bovine bFGF, carriesno secretion signal sequence (Abraham et al. 1986;Kimelman et al. 1988), so it is not clear how it mightescape from vegetal blastomeres. To confirm that bFGFplays a role in mesoderm induction it is necessary,therefore, to ablate the protein from the embryo andshow that mesoderm formation is disrupted.

Thus it is not clear whether the two Xenopus-derivedinducing factors available in pure form - XTC-MIF andbFGF - play a role in normal development. Neverthe-less, it has been possible to carry out important exper-iments with the molecules, and as we discuss below the

results are consistent with bFGF being the ventralvegetal inducing signal and XTC-MIF, or somethinglike it, the dorsal signal.

XTC-MIF and bFGF: dorsal and ventral signals

We outline below three types of experiment thatindicate that XTC-MIF and bFGF mimic, respectively,the dorsal and ventral mesoderm-inducing signals rep-resented in Fig. 2. This conclusion is important in itself,but, as we show, the work should lead to improvedunderstanding of thresholds in development, of Spe-mann's organizer, of the control of cell motility, and oftiming in development.

Concentration-dependent effects of XTC-MIF andbFGFThe first series of experiments investigates the effects ofexposing Xenopus animal pole regions to differentconcentrations of XTC-MTF or Xenopus bFGF(XbFGF). Animal caps were dissected from embryos atthe midblastula stage and cultured in inducing factoruntil sibling embryos reached the swimming tadpolestage. In order that all the cell types present could berecognized, and to preserve the three-dimensionalstructure of the explants, the specimens were fixed,sectioned and stained by conventional histological pro-cedures, before being examined under the microscope.

Two conclusions could be drawn from this exper-iment. First, only XTC-MIF, and not XbFGF, inducednotochord from animal pole tissue (Smith et al. 1988;Green et al. in preparation; see Godsave et al. 1988 andSlack et al. 1988 for results with bovine bFGF). Thisstrongly suggests that XTC-MIF more resembles thedorsal mesoderm-inducing signal and XbFGF the ven-tral. The second conclusion is that the type of inductionobserved depends upon the concentration of inducingfactor. Thus for XbFGF, 1 ngml"1 causes all explants tobecome induced, but the cell types formed only rarelyinclude muscle; most of the induced tissue is bestdescribed as mesenchyme and mesothelium (Fig. 3E).These cell types are often regarded as 'ventral meso-derm' but it is worth emphasizing that without molecu-lar markers this classification is tentative at best, andmay result from a regrettable tendency to classify anytissue formed in response to a 'mesoderm-inducingfactor' as 'mesoderm'. The best justification for the viewis that the cells resemble mesoderm in their socialbehaviour and adhesive preferences (Cooke and Smith,1989) and that they resemble the cell types formed byanimal pole tissue after juxtaposition with ventralvegetal pole tissue (Dale et al. 1985; see Smith, 1989).Higher concentrations of XbFGF begin to inducemuscle, but 25-50ngml"1 is required for the formationof significant amounts (Fig. 3F) (Green et al., in prep-aration; see Godsave et al. 1988 and Slack et al. 1988 forresults with bovine bFGF). As stated above, notochordis never observed.

A similar transformation in cell types is seen withXTC-MIF. Low concentrations (less than lngmP1)

152 /. C. Smith and others

tend to induce mesenchyme and mesothelium (Fig. 3B)while higher concentrations result in muscle formation(Fig. 3C). However, with XTC-MIF muscle formationis maximal at about 5ngml~' and in some experimentsmuscle differentiation falls off beyond this point to bereplaced by notochord and neural tissue (Fig. 3D)(Smith el al. 1988). The transformation from muscledifferentiation to notochord formation is, however,somewhat variable (Green et al. in preparation) andthis is currently under investigation.

Rather than considering XbFGF as a weaker meso-

derm inducer than XTC-MIF, it may be more accurateto regard the two factors as producing qualitativelydifferent types of mesoderm rather than different'grades' of the same type. Some cell-biological evidencefor this point of view is provided by recent studies onMIF-induced gastrulation movements (below) but ad-ditional molecular evidence comes from recent work byRosa (1989) which shows that the gene Mix.l is stronglyactivated by XTC-MIF but not by XbFGF, at concen-trations where both induce mesoderm.

The observation that different concentrations of

Fig. 3. Cell types formed in response to bFGF and XTC-MIF. (A) Animal pole regions cultured in the absence ofmesoderm-inducing factors form "atypical epidermis". (B) 0.2ngml XTC-MIF causes the formation of •mesenchyme'(arrow) and •mesothelium' (arrowhead). (C) l-2ngml~' XTC-MIF is sufficient to induce quite large amounts of musclefrom animal pole regions (arrow). (D) In some experiments with high concentrations of XTC-MIF, notochord is formed(arrow). Notice the cement gland at the opposite side of the explant. (E) l-10ngml~' Xenopus bFGF causes the formationof mesenchyme and mesothelium. (F) Concentrations of approximately 50ngml~' bFGF are required to induce muscle(arrow). Scale bar in (F) is 50^m, and also applies to frames (A) to (E).

Thresholds in mesoderm induction


XTC-MIFMany

thresholdsOne

threshold Result

25 ng/ml Neural tissueNotochord

3 ng/ml Muscle

0.5 ng/mlMesenchymeMesothelium

Zero Epidermis

XTC-MIF tend to cause the formation of different celltypes raises the question of thresholds in development(Lewis etal. 1977). What form might these thresholdstake? According to one view, each responding animalpole cell would have many thresholds, each activated ata certain concentration of XTC-MIF (Fig. 4; see Smith,1989). Thus at O.SngmP1 XTC-MIF the first thresholdwould be exceeded and mesenchyme and mesotheliumwould form. Higher concentrations would cause furtherthresholds to be exceeded so that at, say, 25 ngmP1 thecells would be programmed to form notochord andneural tissue. The following experiment, however,argues against such a model, and we go on to suggest analternative view that the threshold phenomenon rep-resents a cell population effect.

It is possible to disaggregate animal pole blastomeresby culturing animal caps in calcium- and magnesium-free medium. Such cells can be kept as a small heap orthey can be dispersed over the culture dish, essentiallyas described by Sargent et al. (1986). If either heaped ordispersed cells are reaggregated at the early gastrulastage, by addition of divalent cations, they go on toform epidermis, their normal fate. If heaped cells areexposed to XTC-MIF, washed, and then reaggregatedat the early gastrula stage, when mesodermal com-petence is almost over, they form muscle, epidermis,and sometimes notochord (Fig. 5; Symes, 1988). This

Fig. 4. Two models for thresholds in mesoderminduction. In one model (left), each individualcell has a qualitatively different response todifferent concentrations of XTC-MIF; that is,each cell has many thresholds. The levels ofresponse directly determines how the celleventually differentiates. In the other model(right) each cell has only one threshold, and thecell types that eventually form depend on theproportion of cells that exceed that threshold.This model requires that there be communicationbetween cells during, or shortly after inductionby XTC-MIF.

indicates that deprivation of divalent cations does notaffect the primary response to XTC-MIF. By contrast,when cells are dispersed during exposure to XTC-MIF,muscle formation does not occur after reaggregation,although epidermal differentiation is supressed (Fig. 5;Symes, 1988). Several experiments, including measure-ments of rates of RNA and protein synthesis, indicatethat this response is not due to toxicity of XTC-MIF(Symes et al. 1988).

These results suggest that mesoderm formation afterexposure to XTC-MIF is not a direct response, but onewhich requires contact-dependent cell-cell interactionsduring induction. If this is so, the changes in patterns ofcell differentation with different concentrations ofXTC-MIF might occur through these interactions, andit may only be necessary for blastomeres to have onethreshold to XTC-MIF. Indirect evidence for a singlethreshold is that the time of onset of gastxulationmovements in response to XTC-MIF, one of the earliestresponses to induction yet observed (Symes and Smith,1987; Cooke and Smith, 1989), and one that relatesdirectly to mesodermal cell type, does not depend onthe concentration of factor (Cooke and Smith, 1989; seebelow).

An alternative view of thresholds in mesoderm induc-tion is that the phenomenon represents a cell popu-lation effect, with the proportion of cells responding to


XTC-MIF does not act on single cellsto induce mesoderm

Stage 8 animal capcells disaggregatedIn calcium-freemedium

si/ \ /

Earlygastrulastage:wash andaggregate

MUSCLE EPIDERMISepidermis

EPIDERMIS

induction determining which cell types eventually form.Each cell, for example, could have a single binaryon/off switch which is tripped by the primary inducingfactor. Secondary signals, whose local strength woulddepend on the proportion of nearby 'switched on' cells,would then determine cell fate. Thus if 10 % of the cellsrespond to the primary signal, the population as a wholemight form mesenchyme and mesothelium; if 50%respond, muscle might arise; and, if 90%, the cellsmight form notochord and neural tissue (Fig. 4). Infuture work, we hope to test this idea by mixing inducedand uninduced cells in different proportions.

One way in which the proportion of cells respondingto induction might influence cell differentiation couldinvolve a timing mechanism. The sequence of celldifferentiation in the mesoderm of Xenopus seems tofollow the dorsoventral axis. Thus the notochord, themost dorsal mesodermal cell type, becomes visible as aseparate group of cells at the late gastrula stage (Kelleret al. 1985), and presumably this is preceded by tran-scription-dependent changes in the cell surfaces of thesecells. Activation of muscle-specific actin genes occurs atthe late gastrula stage (Gurdon et al. 1985), whiletranscription of globin genes, which characterize themost ventral cell type, blood, does not start until thetailbud stage (Banville and Williams, 1985). Accordingto one model, similar to Cooke's (1983) 'serial diversiontheory1, cells induced by XTC-MIF or FGF might passthrough phases of development during which they are

Fig. 5. Design of experiment to show that cellcontact is required during treatment with XTC-MIF if muscle (and probably other mesodermalcell types) is to form. Animal caps aredissociated at the early blastula stage andcultured as indicated, either in a small heap ordispersed over the surface of an agar-coated Petridish. Some cultures contain XTC-MIF. At thebeginning of gastrulation, near the end of theperiod of competence to respond to XTC-MIF,the cultured blastomeres are thoroughly washed,reaggregated, and cultured until sibling embryosreached stage 40. The results were obtained byimmunofluorescence analysis of specimens. Notethat in the absence of XTC-MIF both 'heaped'and "dispersed" cultures form epidermis and nomuscle. When "heaped' cells are exposed toXTC-MIF they eventually form epidermis andmuscle, but neither cell type is detected ifblastomeres are exposed to XTC-MIF whiledispersed.

capable of differentiating first as notochord, then asmuscle, and then as successively more ventral celltypes. For differentiation to occur there must be athreshold level of a second signal, whose concentrationdepends upon the proportion of cells that are induced.If a small number of cells initially responded to induc-tion, the concentration of second signal would be slowto build up, so that the phases during which notochordor muscle pathways can be entered will have passed,and their only option is to form ventral cell types. Ifmany cells responded to induction, the second signalwould accumulate rapidly, so that notochord could beformed. One reason why XTC-MIF but not bFGF caninduce notochord might be that they produce differentsecond signals which build up at different rates or havedifferent specific activities. Alternatively, the accumu-lation of second signal in response to FGF may startlater in development. Recent results on the timing ofgastrulation movements induced by XTC-MIF andbFGF, described below, may support the latter idea(Cooke and Smith, 1989).

The requirement for cell contact between animal poleblastomeres during induction by XTC-MIF, if meso-derm is to form, bears upon some recent results ofGurdon (1988). Gurdon finds that the ability of ananimal pole cell to form muscle in response to vegetalpole cells is dependent on the presence of other,similarly induced, animal pole cells. He calls thisphenomenon the 'community effect". It is possible that


the effect is merely permissive; that is, that cellsspecified as muscle simply cannot express their appro-priate phenotype as isolated individuals. Alternatively,cell contact could be instructive in the sense that thedisposition and abundance of induced cells could decidewhich and how much of several fates eventually appear.

XTC-MIF-treated animal cap cells behave asSpemann's organizerThe above results suggest that animal pole cells exposedto XTC-MIF should be regarded as dorsal mesoderm,because they form notochord. A further property ofdorsal mesoderm, however, is that it should also pro-duce the dorsalizing signal mentioned above (seeFig. 2). We have tested this prediction by microinject-ing XTC-MIF, bFGF or a control solution into theblastocoels of Xenopus embryos at the early blastulastage. Two hours later, small pieces of animal poletissue are removed from these embryos, washed care-fully, and grafted to the ventral marginal zones of hostembryos (Fig. 6). The results show that ectodermtreated with XTC-MIF, but not with bFGF or a controlsolution, does indeed behave as Spemann's organizerand therefore produces a dorsalization signal (Cooke etal. 1987; Cooke, 1989). Experiments using lineage-labelled grafts confirm that the secondary axes in the

Inject MIF

Wash

Fig. 6. Experiment to show that XTC-MIF-treated animalcaps behave like Spemann's organizer. XTC-MIF, bFGF, ora control solution are injected into the blastocoel of aXenopus embryo at the mid-blastula stage. 1-2 h later smallpieces of animal cap tissue are dissected from the treatedembryo, washed, and grafted to the ventral side of a hostembryo.

The drawing at bottom left illustrates the architecture ofthe dorsal lip of the blastopore, the natural organizer. Onlytissue above the bottle cells, consisting of dorsal mesodermand suprablastoporal endoderm, has organizer activity;more vegetal endoderm lacks activity. Presumably XTC-MIF induces tissue of the former type.

double-dorsal embryos do consist largely of host tissueand have not arisen through self-differentiation of thegraft (Cooke, 1989).

Interestingly, as would be predicted by the work ofGerhart et al. (this volume), the more 'powerful' theorganizer (whether in terms of the concentration ofXTC-MIF injected into the blastocoel or in terms of thesize of the gTaft), the more complete the secondary axisformed, provided the cells having organizer activity areadequately localized within the tissue. Thus, presump-tive ectoderm that has been exposed to a high concen-tration of XTC-MIF induces head, trunk and tailstructures, while that exposed to lower concentrationsmight only induce trunk and tail. Finally, at the lowestconcentrations of factor only tail is formed (Cooke,1989). As with the concentration 'threshold' effects forXTC-MIF-induced cell differentiation, we do not yetknow whether this gradation is mediated by a series ofdistinct organizer states in the cells, or through vari-ation in the total number of cells in the graft that havebeen induced into an organizer 'state'.

These observations are of interest for three reasons.First, they confirm that XTC-MIF-treated ectodermdoes indeed resemble dorsoanterior mesoderm, whilebFGF-treated tissue is more similar to ventroposteriormesoderm, which lacks organizer activity (Smith andSlack, 1983). Second, the results offer an opportunity toscreen for factors that represent the elusive organizersubstance. It should be possible to conduct a differen-tial screen to identify secreted molecules that areproduced in response to XTC-MIF but not to bFGF.And finally, it may be possible to design experiments toexplain how positional values are established along theanteroposterior axis of the embryo.

Inducing factors, gastrulation and the developmentaltimerThe first visible manifestation of mesoderm induction isgastrulation. In Xenopus, as in other Amphibia, gastru-lation is under precise temporal and spatial control.This control is exerted through the cells of the meso-derm, which provide the motive force for gastrulation(Keller, 1986). Mesoderm-inducing factors, such asXTC-MIF and bFGF, induce gastrulation-like move-ments in animal pole tissue (Fig. 7) (Symes and Smith,1987; Cooke et al. 1987; Rosa et al. 1988; Cooke andSmith, 1989), and this provides a new approach to thestudy of gastrulation (Smith, Symes, Hynes and DeSi-mone, in preparation).

One particularly interesting aspect of MIF-inducedgastrulation movements concerns their timing. In apreliminary study Symes and Smith (1987) estimatedthe time of onset of gastrulation movements in isolatedanimal pole regions exposed to XTC-MIF. Within thelimits of observation, the explants started to elongate atthe equivalent of the early gastrula stage, irrespective ofthe stage at which they were exposed to the factor. Thissuggested that cells of the animal hemisphere contain agastrulation 'timer', which is running even in those cellsthat would not normally need to refer to it.

Gastrulation movements induced by bFGF are less


Fig. 7. XTC-MIF induces gastrulation-like movements in animal pole explants. (A) A control explant. (B) An inducedexplant 15 h after treatment with XTC-MIF. Scale bar in (B) is 200jtm, and also applies to (A).

dramatic (Slack et al. 1987), making it harder toestimate their time of onset. However, Cooke et al.(1987) and Cooke and Smith (1989) have introduced atechnique to overcome this problem. This involvesmicroinjecting MIFs into the blastocoels of Xenopusembryos so that the entire blastocoel roof becomesconverted to mesoderm. As a result the whole of thistissue undergoes changes in cell shape and adhesionthat mimic those occurring in the marginal zone of theembryo. The timing of these events can be estimatedvery precisely, by dissection of living and fixed em-bryos, and the first results confirmed those of Symesand Smith (1987) in showing that the time of onset ofgastrulation-like movements is independent of the timeof receipt of inducing factor. The data showed, how-ever, that the time of onset was also independent of theconcentration of factor and that the response could bevery rapid. Thus if microinjection of the factor wasdelayed until just before the onset of gastrulation in thehost embryo, the minimum possible interval betweenmicroinjection and onset of gastrulation movements inthe ectopically induced mesoderm was at most 30min(Cooke and Smith, 1989).

Microinjection of bFGF into the blastocoels of Xeno-pus embryos confirmed that the time of onset of theresulting ectopic gastrulation movements is, similarly,independent both of time of injection and of concen-tration of factor. However, the time at which bFGF-induced movements commence is significantly later,within the progress of the embryos 'own' gastrulationmovements, than the time at which XTC-MlF-inducedmovements start. The interval, about 60 min, resemblesthe difference in time of gastrulation of dorsal andventral mesoderm (Gerhart and Keller, 1986).

Here, then, is the third piece of evidence that XTC-MIF induces dorsal mesoderm and bFGF ventral; thegastrulation movements induced by the two factorsdiffer in timing in a way that corresponds to the dorsaland ventral mesoderm of the early gastrula. But theseresults are also of great interest in the more generalquestiqn of 'developmental timers'. Gurdon (1987) hasemphasized that a significant difference between em-bryonic induction and other cellular interactions is

indeed that in induction the time of response dependsupon the responding tissue and not on the time ofreceipt of the signal. The example used by Gurdon wasactin gene activation; Gurdon et al. (1985) showed thatin animal-vegetal combinations this always occurs atthe midgastrula stage, irrespective of the stage at whichthe combination was made. Thus, if the tissues wereapposed at the early blastula stage, transcription oc-curred some 9.5 h later, but, if they were placed incontact at the early gastrula stage, the interval was only5-7 h. In the molecular analysis of developmental timemeasurement, it may be more profitable to study thecontrol of gastrulation than that of actin synthesis,because this is a much earlier response and there arelikely to be fewer intervening steps between signal andresponse. In particular, it is noteworthy that onegastrulation-specific response, the ability to spread on afibronectin-coated substrate, can occur in single cells(Smith, Symes, Hynes and DeSimone, in preparation),whereas muscle formation may require interactionsbetween groups of cells (Gurdon, 1988; Symes et al.1988). In the analysis of induction, it is a great advan-tage to be able to study single cells (Gurdon, 1987).

Conclusions

The work described in this paper summarizes theevidence that, of the two classes of mesoderm-inducingfactor that have been discovered, members of the FGFfamily are likely to constitute the ventral vegetal signal,and members of the TGF-/J family the dorsal vegetalsignal. We think this is an important conclusion whichshould assist the design of future experiments aimed atunderstanding pattern formation in the mesoderm ofXenopiis and perhaps other organisms.

Much, however, remains to be done. First, andseemingly most straightforward, it is necessary to estab-lish just which of the inducing factors listed in Table 1are present and active in the Xenopus embryo. It is thennecessary to discover which of these are required formesoderm formation, by inactivating the endogenousfactors. At present it is not completely clear how this


Table 1. Mesoderm-inducing factors in Xenopus

Factor

Present in early embryo?

Family

TGF-/3TGF-/3TGF-/3TGF-/3

FGFFGFFGFFGFFGF

Active?

UnknownYes

With FGFYesYesYesYesYesYes

RNA

YesUnknownUnknownUnknownUnknown

YesUnknownUnknownUnknown

Protein

YesUnknownUnknownUnknown

NoYes

UnknownUnknownUnknown

Localized?

YesUnknownUnknownUnknownUnknownUnknownUnknownUnknownUnknown

Vg.' ,XTC-MIF2

TGF-/313

TGF-/K"aFGF5

bFGF*ECDGF7

INT-28

kFGF8

1 Rebagliati et at. (1985); Weeks and Melton (1987); Melton (L987); Yisraeli and Melton (1988); Yisraeli et al. (this volume); Dale et at.(1989); Tannahill and Melton (1989).

2Smith (1987); Rosa et al. (1988); Smith el al. (1988).3 Kimelman and Kirschner (1987).4Rosa era/. (1988).'Slack et al. (1987); Slack et al. (1988); Slack and Isaacs (1989).6Slack et al. (1987); Kimelman and Kirschner (1987); Kimelman el al. (1988); Slack and Isaacs (1989).'Slack eial. (1987).8 Paterno et al. (1989).

might best be done. The most obvious approach is tomicroinject antisense oligonucleotides into the matureoocyte to destroy endogenous inducing factor mRNA(Shuttleworth and Colman, 1988), followed by matur-ing the oocyte by passage through a female frog andthen fertilizing it (Holwill et al. 1987). However, thismay not be effective because most Vgl and bFGFprotein seems to be synthesized during earlieroogenesis (Kimelman et al. 1988; Dale et al. 1989; Slackand Isaacs, 1989; Tannahill and Melton, 1989). Analternative approach might involve microinjection ofantibodies against inducing factors into the fertilizedegg. However, experiments such as these are difficult tocontrol, and must be interpreted with care. When it isestablished which factors are involved in mesoderminduction it will be important to discover whether, likeVgl (see Yisraeli et al. this volume), they are localizedto the vegetal hemisphere, how that localization occurs,and, most importantly, how at least one of the factorsbecomes differentially activated on the dorsal side ofthe embryo in response to the postfertilization corticalrotation (see Gerhart et al. this volume).

It is also important to understand how factors likeXTC-MIF and Xenopus bFGF exert different embryo-logical, cellular and molecular effects, as outlined inthis paper and elsewhere (Rosa, 1989; Green et al. inpreparation). One possibility is that these differencesare caused by the two factors activating different secondmessenger pathways, although the fact that the activityof both XTC-MIF and bFGF is enhanced by treatmentof responding animal cap tissue with LiCl (Slack etal.1988; Cooke et al. 1989) might be thought to argue thatsome of the signal transduction pathways are shared:one effect of lithium is to inhibit inositol monophospha-tase (Hallcher and Sherman, 1980), thus blocking therecycling of InsP3 to inositol and perhaps modulatingthe effects of any factor acting through inositol lipid-linked signalling systems (Drummond, 1987). This viewof the effects of lithium is strengthened by the obser-

vation that the 'dorsalizing' effect of lithium, whenmicroinjected into ventral blastomeres of the 32-cellstage embryo, is prevented by coinjection of equimolarmyo-inositol (Busa and Gimlich, 1989). However,although these results are consistent with the suggestionthat XTC-MIF and bFGF act through a similar secondmessenger pathway, lithium has so many other effectson cells that other interpretations are possible (seeSlack etal. 1988).

A second aspect of the early response to inducingfactors is that, as we discussed above, this is unlikely tospecify cell differentiation directly; communication be-tween the cells of an induced population seems to berequired to define particular cell types (Symes et al.1988). A signalling process of this sort seems to occurduring gastrulation, and to be involved in establishingpositional values along the anteroposterior axis of theembryo (see Gerhart et al. this volume). The nature ofthe molecules involved in this interaction is unknown atpresent, but it is possible that one of the early genesactivated by XTC-MIF (Rosa, 1989) encodes a furthersignalling molecule.

Gastrulation itself may be controlled through a post-transcriptional response to mesoderm-inducing factors(Smith, Symes, Hynes and DeSimone, in preparation);animal pole cells can commence gastrulation-like move-ments in response to XTC-MIF within 30min (Cookeand Smith, 1989), during which time even the early geneMix.l is only weakly activated (Rosa, 1989). Post-transcriptional events following treatment with XTC-MIF and bFGF are currently under investigation in thislaboratory.

It is clear that mesoderm induction and mesodermalpatterning in Amphibia are complicated processes thatmust involve sequential inductive interactions, differentpatterns of cell movement, post-transcriptional andpost-translational controls, and the activation of manygenes. At present our understanding is limited, but theidentification of mesoderm-inducing factors, and the


suggestion that the two groups of factors are active indifferent regions of the embryo, should allow us toimprove our understanding quite rapidly.

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