Development 107, 229-241 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

229

Mesoderm-inducing factors and Spemann's organiser phenomenon in

amphibian development

JONATHAN COOKE

Laboratory of Embryogenesis, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK

Summary

Certain proteins from 'growth factor' families caninitiate mesodermal development in animal cap cells ofthe amphibian blastula. Cells that are in early stages oftheir response to one such factor, XTC-MIF (Smith etal.1988), initiate the formation of a new axial body planwhen grafted to the ventral marginal zone of a similarlyaged host embryo (Cooke et al. 1987). This replicates thenatural control of this phase of development by thedorsal blastoporal lip when similarly grafted; the clas-sical 'organiser' phenomenon. I have explored system-atically the effect, upon the outcome of this patternformation using denned inducing factors, of varyinggraft size, XTC-MIF concentration to which graft cellswere exposed, length of exposure before grafting, andhost age. The 'mesodermal organiser' status, evoked bythe factor, appears to be stable, and the variables mostinfluencing the degree of completeness and orderliness ofsecond patterns are graft size and factor concentration.

Inappropriately large grafts are not effective. A Xenopusbasic fibroblast growth factor homologue, present in theembryo and known to be a strong inducer but ofmesoderm with a different character from that inducedby XTC-MIF, produced no episode of pattern formationat all when tested in the procedure described in thispaper. Organiser status of grafts that have been exposedto mixtures of the two factors is set entirely by thesupplied XTC-MIF concentration. Lineage labelling ofthese grafts, and of classical dorsal lip grafts, revealsclosely similar though not identical patterns of contri-bution to the new structure within the host. Implicationsof the results for the normal mechanism of body patternformation are discussed.

Key words: organiser, body pattern, XTC-MIF, bFGF,dorsal blastoporal lip, lineage labelling, Xenopus.

Introduction

Spemann and Mangold (1924) first described the graft,from the dorsal blastoporal lip of an early newt gastrulainto the presumptive ventral region of a similar host,that led to formation of an extra set of axial body partscentred on the graft region. Donor cells contributed inan integrated way to the anterior and dorsal regions ofnew mesodermal pattern, while the associated nervoussystem was very largely host-derived. These conclusionshave been extended to other amphibian types, notablythe frog Xenopus, and it is understood that the twinningis based not on extra growth with local intercalation ofnew structure, but upon widespread alteration of themap that describes cells' allocation to particular 'fates'within an embryo of normal tissue extent (Cooke,1972a,/), 1973a, 1979, 1981; Gimlich and Cooke, 1983;Smith and Slack, 1983; Jacobson, 1984). Since theoriginal work, it has usually been proposed that thisprocess has two separate components; the graft firstinteracts with its surroundings to complete a new mapfor dorsal axial mesodermal differentiations, and theresulting midline mesoderm, migrating beneath ecto-derm via a blastoporal lip of it own, then delivers signals

that elicit a coordinated pattern of neural developmentthere (e.g. Nieuwkoop, 1973; Spemann, 1931). ButSpemann remained convinced that he was witnessing amore unified episode of interactions (see 1938 for self-review and Hamburger, 1988 for an intellectual biogra-phy).

The classical 'organiser' graft is of tissue whosenormal fate in the donor would have been to becomethose same dorsal-anterior mesodermal structures thatit now contributes to the new pattern. At least inXenopus, however, the most profound and symmetricaltwinning results instead from surgical or other manipu-lations at much earlier stages (Gerhart et al. 1981;Gimlich and Gerhart, 1984; Black and Gerhart, 1986).These cause doubling with respect to a normally singleregional specialisation, situated within the egg's yolkyhemisphere that contributes the endodermal layer tothe body. Mesoderm, and the overall pattern develop-ing within it, result from subsequent inductive interac-tion. Signals originating in the endodermal regionsrecruit cells from the overlying equatorial region intomesodermal development during roughly the 102- to104-cell, or blastula stages (Nieuwkoop, 1969, 1973;Sudarwati and Nieuwkoop, 1971; Gurdon et al. 1985;

230 J. Cooke

Warner and Gurdon, 1987). Although mesoderm isinduced all around the meridians of the normal embryo,these signals vary in character such that a relativelyrestricted sector has the pattern-organising power atearly stages, and transfers this to the mesodermal zoneabove because it induces mesodermal tissue with aspecial character. Thus twinning occurs whenever or-ganising potential is made to appear at two positions,either by early endodermal grafting (Gimlich and Ger-hart, 1984), later mesodermal grafting (e.g. Cooke,\912b), or other, mechanical or biochemical manipu-lations (Black and Gerhart, 1986; Cooke, 1987; Kaoetal. 1986).

It has been proposed on independent grounds, bothfrom morphogenetic theory (Dale and Slack, 1987) andfrom close observation of cellular behaviour duringnormal gastrulation (Keller and Danilchik, 1988), thatonly two types of mesoderm are induced at the outset (-three if prechordal head mesoderm is counted asdistinct). Mesodermal body patterns would then formin relation to juxtapositions of these initially inducedmesodermal territories, on the basis of gradients in oneor more further morphogen signals that could be set upand maintained if the 'dorsal axial' territory were asource and the remaining type of mesoderm a dif-fusional sink for such molecules. Further evidence forsuch an arrangement in normal development, i.e. for anearly interaction between mesoderms of differing initialspecifications rather than direct quantitative or qualitat-ive gradation in one inductive activity, comes fromultraviolet irradiation of eggs before the midpoint of thefirst cell cycle (e.g. Scharf and Gerhart, 1983; Cookeand Smith, 1987). This prevents the normal creation ofwhat appears to be a quite small special sector withinthe endodermal source region for inducers, and isfollowed by development of radially symmetrical bodiespossessing essentially normal amounts of mesoderm,but mesoderm only of lateroventral type.

Two distinct types of mesoderm-inducing factor(MIFs) that may function in development have nowbeen identified and characterised. On the basis of theireffects upon competent Xenopus blastula ectoderm invitro and, after their microinjection into the blastocoel,in vivo, it has seemed likely that they respectivelyspecify initial mesodermal states corresponding to thetwo proposed above. XTC-MIF, a protein secreted by aXenopus cell line, shows evidence of belonging to thetransforming growth factor - /? family (Smith, 1987;Smith et al. 1988; .Rosa et al. 1988). At picomolarconcentrations, it induces mesoderm resembling thatnaturally produced in the dorsal sector of the embryo inthe timing and character of its gastrulation movements(Symes and Smith, 1987; Cooke and Smith, 1989) and inits later differentiations as notochord and somite musclewith associated neuralisation of adjacent ectoderm.Like the natural signaling of Xenopus mesoderm for-mation (Warner and Gurdon, 1987), it respecifiesectoderm only as an extracellular signal via the cellsurface (Cooke et al. 1987). MIFs of the other class aremembers of the heparin-binding fibroblast growth fac-tor (FGF) family (Slack etal. 1987, 1988). They induce

mesoderm, which resembles in its mechanical behav-iour during gastrulation the natural mesoderm of lat-eroventral type (Keller and Danilchik, 1988; Cooke andSmith, 1989), and which rarely differentiates as axialformations of notochord and segmenting somite withneural inductive capacity. The gene for a Xenopushomologue of basic FGF, present in eggs and earlyembryos, has now been cloned (Kimelman andKirschner, 1987; Kimelman et al. 1988, henceforth inthis paper called XbFGF). This is effective in givingmesoderm of lateroventral or 'mesenchymal' differen-tiation capacities at very low picomolar concentrationsin vitro. In response to much higher concentrationssome muscle is seen, but it seems unlikely on the basisof at least one estimate of the protein's concentration inembryo that much muscle would be directly induced(Slack and Isaacs, 1989).

It seemed likely that mesoderm having organiserstatus might be distinctively specified by an in situcounterpart of XTC-MIF, possibly that molecule itself.A grafting operation has provided striking support forthis hypothesis (Cooke et al. 1987). Shortly after injec-tion of the soluble inducer into the blastocoel of ablastula stage donor embryo, a small (approx. 102 cell)piece of its blastocoel roof tissue is cut out, washed andthen integrated into the ventral subequatorial region(marginal zone) of a host like a classical dorsal blasto-pore lip 'organiser' graft. Second axial body patternsdevelop that can be as well organised and complete asare those due to the dorsal lip graft. The scheme of suchoperations is shown in Fig. 1A. This procedure hasbeen chosen rather than the more obvious in vitrotreatment of competent tissue with MIF before graft-ing, because pilot experiments have revealed that itallows selection of more standardised, homogeneouslystimulated graft tissue pieces. Variation in the rate ofrounding up of explants in vitro, and perhaps inresponse rate to the factor, causes great variation inintensity of response in material from different eggbatches. It also, we believe, leads in itself to spatialorganisation within the newly induced tissue (Cookeetal. 1987). In the present paper I investigate in detailthe conditions affecting this formation of 'second bodypatterns', and compare the natural organiser tissue withMIF - activated grafts of blastocoel roof, henceforthreferred to as experimental organiser tissue. I have alsoassessed the relative role of the FGF class of inducer.Conclusions are based on more than 350 larval patternsresulting from grafts after donor injections with purifiedXTC-MIF, 200 after injection with cloned purifiedXbFGF or with XbFGF/XTC - MIF mixtures, and 20after injection with saline - the control operation.Operations were also made to graft either supra-blastoporal mesodermal pieces or the subjacent endo-dermal cells, from the natural 'organiser' region ofXenopus early gastrulae, into blastula hosts. 70 of theoperations in all were accompanied by lineage labellingof the grafted cells, after initial injection of donor eggswith rhodamine-lysine-dextran (RLDX).

The results of these experiments do furnish strongevidence that a morphogenetic gradient system within

Inducing factors and the organiser 231

B

wash

Fig. 1. Experimental grafting operation. (A) The scheme of the operations. Major variables are MIF concentrations inblastocoel of blastula donor (injected in 2/3 strength saline, pH8.0+0.5 mg ml BSA), time from donor injection tografting, size of graft and absolute age of host blastula (usually lh younger than donor). Operation in 2/3 saline pH7.2,lowered to 1/5 before onset of host gastrulation. Lower left shows an enlarged section of trie normal mid-dorsal marginalregion. This is opposite the experimental graft implantation site in hosts, and is also the origin of 'natural organiser' graftsfrom donors. The site of bottle cell formation separates the suprablastoporal mesodermal region, the most effectiveorganiser when grafted with preservation of animal-vegetal (a-v) orientation, from yolky endoderm cells that are by thisstage ineffective as organiser (see Results). (B) Ventrovegetal view (as at operation) of early stage 8 host with newly healedgraft of optimal size, shape and position. Insets below show views, from the side, of natural organiser (right) and XTC-MIFexperimental grafts in position at onset of donor's gastrulation. Invagination and underlying involution are spatiallysequenced in dorsal lip but not in ectopically XTC-MIF-induced tissue.

mesoderm is the basis of the organiser effect. Theycannot yet help us understand the further cellularmechanisms, however, whereby such an initial gradientis utilised in controlling the pattern and proportions forcellular differentiation. This could be by direct (Wol-pert, 1971; cp Summerbell, 1979; Tickle et al. 1985), orindirect (Cooke, 1983; Pate, 1984; Smith, 1989) re-sponses to morphogen concentration levels. The rel-evance of the quantitative observations to the idea of asignal gradient in axial pattern formation is explained inthe discussion.

Materials and methods

Synchronously fertilised embryos of Xenopus were obtainedby standard procedures. They were dejellied with 2% cys-teine HCL at pH7.9, washed with 3/4 normal amphibianmedium (NAM) and placed animal pole up in fitting wellsunder 1/10 NAM containing 5% Ficoll (SIGMA) at early

blastula stages immediately prior to injection of graft donorswith factors. Relative stages of donors and hosts for individualversions of the operations were manipulated by culture attemperatures of 15, 18 or 23°C from early cleavage stages.

Donors were injected with a standard 200 (stage 7 and early8) or 300 (stage late 8 and 9) nannolitres of 3/4 NAM,ph8.0+0.5 mgml~L fraction V bovine serum albumin, con-taining purified factors at concentrations calculated to givethose required in the blastocoel, using glass micropipettes andan oil-filled AGLA micrometer system. Injection over a fewseconds caused effective subirrigation of the animal capregion and homogeneous exposure of the roof tissue (Cookeand Smith, 1989). Donor injections for the control operationcontained no factors.

After the required response periods in donors, donor andhost embryos were washed in 3/4 NAM, demembranatedusing fine forceps and placed on 2 % Agar beds that had beenequilibrated overnight with 2/3 NAM, pH7.2 for operations.Blastocoel roof grafts, two to four per donor from around theanimal pole, were cut out, washed for several seconds byaspiration in a Spemann pipette and transferred to the vicinity

232 /. Cooke

of host embryos for implantation in their ventral marginalzones as depicted in Fig. 1. Operations were performed insmall groups such that grafts remained free in the bathingsolution for several minutes between excision and implan-tation, in addition to the washing and transfer procedures.Ionic strength of the solution was lowered to 1/5 NAM after30min, or at least 30min before the expected onset ofgastrulation movements in hosts. Batches of embryos wereselected as hosts in which the pigmentation and cell sizegradations predicting the dorsoventral orientation were ratherreliably visible during blastula stages, and operations provingto have placed grafts at much less than 180° to the host-controlled dorsal midline (Cooke 1972a,b) were rejected.

Fixation of larval bodies resulting from operations was atmid 30s stages (Nieuwkoop and Faber, 1967). Most secondaxial patterns were assessed structurally in relation to the full'host' pattern (see Results) by dissection under the micro-scope after a few minutes fixation in K2Cr2O7/acetic acid(Cooke and Smith, 1989). This allowed visualisation of eachlandmark structure of the body plan. Other examples wereembedded in 58 °C wax and sectioned at 7 fim for staining withFeulgen, light green and orange G (Cooke, 1979, 1981). Forlineage labelling of graft contributions, donors were injectedat onset of second cleavage with 15 nl of rhodamine-lysine-dextran at SOmgml"1 in water. Fixation of operated embryoswas in 4% fresh formalin in 0.08M-phosphate buffer, over-night at room temperature, followed by graded dehydrationin butanol, and embedment in wax at 58 °C via a butanol/waxmixture. Sections were cut at 10 fim and examined withappropriate filter sets in the Zeiss epifluorescence micro-scope.

Results

Classification of individual resultsIndividual examples of a body plan, while being coher-ent and well-ordered, can vary in completeness. Incom-plete Xenopus plans, possessing only partial sets ofmesodermal and induced neural structure, are seenfollowing nonsurgical interventions that diminish thesize or intensity of the centre for dorsal-anteriorspecification (Scharf and Gerhart, 1983; Cooke andWebber, 1985; Cooke, 1985). They form a 'nested'series, in that omission first occurs at one extreme ofpattern, i.e. for dorsal and anterior structures directlyassociated with the organiser region, and then pro-gresses in a posterior and ventral direction. Secondinitiations of pattern formation, in response to grafts oftissue that has what I here refer to as organiser status,result in the same series of partial plans as well as thecomplete one. Total tissue available for morphogenesisis essentially unaltered after the operation (Cooke,1979), but second patterns can be relatively extensive,embracing almost half the tissue into their structure, orrelatively small. Extent and structural completenesstend to correlate in examples, as they would if deter-mined by the profiles and spatial extents of gradients,attained on particular occasions by the activity of somemorphogen system. This is outlined in Fig. 2, a classifi-cation of the patterns seen into a series from 1, theminimal recognisable formation, to 5 the completeplan, in relation to the 'host-centred' plan in twinnedlarval bodies. Mean scores for second body patterns of

twins, attained after particular variations of the oper-ating procedure, thus represent their relative effective-ness in setting up instances of this 'primary' morpho-genetic gradient field. The small inset diagrams inFig. 2, interpreting the normal and examples of twinnedbody patterns in terms of hypothetical profiles for theprimary gradient, will be referred to more in theDiscussion section.

XTC-MIF specifies a special cell stateI have conducted experiments with electrophoreticallypure XTC-MIF to assess its role in relation to XbFGF,and any possible synergy with that molecule, in in-ducing organiser status in target cells that are after allcompetent to respond to both factors. XbFGF is anextremely potent inducer, causing animal cap tissue tobehave as mesoderm during gastrulation after exposureto considerably lower concentrations (approx.0.1 ngml"1) than are required in the case of XTC-MIF(unpublished results). Both Xenopus-derived factorscause stable induction after well under an hour'sexposure in vitro, and the altered cellular texture andthickness of blastocoel roof tissue such as is used forgrafts, after injection of either factor into donors,already indicates respecification of this tissue as meso-derm before the donor age of onset of gastrulation (seeCooke and Smith, 1989). But in 60 operations usingXbFGF, involving groups of 100-400 donor cells fromblastocoel roofs l i -2h after injection to give exposureat 2-500ngmP1 (approx. 20-5000 of its 'units'ml"1),no host embryo developed even a grade 1 secondpattern. This range of conditions embraces those opti-mal for the twinning result after XTC-MIF injection(see below). Furthermore, as shown in Table 1, XbFGFgives no evidence for synergy with XTC-MIF in in-ducing organiser status in blastocoel roof, whetherpresent as the major or the minor component in amixture of the factors at relevant concentrations. The'intensity' of organiser status in grafts from blastocoelroofs is dictated solely by XTC-MIF concentrationpreviously injected beneath them (Table 1).

The 'organiser graft', found in earlier Xenopus workto give an incidence of large and complete secondpatterns, contains the presumptive mesoderm (andfuture archenteron roof) just above the earliest externalblastoporal lip at Nieuwkoop and Faber stage 10(Fig. 1A, lower left), but also an equal mass of thesubjacent, larger yolky endoderm cells (- future archen-teron floor). The precursors of these latter cells, at theyoung blastula stage, are those most effective in in-ducing new dorsal axial mesoderm when cultured incombination explants (Nieuwkoop, 1973; Dale andSlack, 1987) or grafted into host embryos (Gimlich andGerhart, 1984). It is of importance to be sure that thecellular state caused by response to XTC-MIF corre-sponds to that of organiser mesoderm, rather than onlyits distinctive inducing endoderm. I have accordinglygrafted these anatomical components of the naturalorganiser region separately, from beginning gastrulaeinto younger hosts. The results confirm that the dorsalendoderm cells of donors are almost past their period of

Inducing factors and the organiser 233

Fig. 2. The classification of degree of completeness in second body plans. The normal larval body plan (0) and bodiesshowing second plans of increasing (1-5) completeness and relative extent within their tissue mass. Major body partsrepresented symbolically, and labelled for the complete 'host' plan, can accordingly be followed as subsets present in secondplans of increasing grade. Neural axis is omitted for clarity, except for landmark positions that diagnose pattern grades,ev—ear vesicle; ec, eyecup; fb, forebrain field; eg, cement gland; pc, prechordal mesoderm; nc, notochord; s, somitesegments; h, heart; pn, pronephros; g, gill structure; bp, blastopore site. Notochords seldom fuse but tail somites do, with atendency to terminalise the blastopore. Numbers of segments anterior to the level of fusion are usually equal, counting tolandmark positions of pn or ev, in host and second patterns (Smallcombe and Cooke unpublished). Insets below and to rightof certain drawings represent profiles proposed for a primary morphogen gradient system, to account for typical lower andhigher grade twinned bodies in relation to the normal single one, with the relative extents of sources (i.e. numbers ofeffectively localised 'organiser' cells, see Discussion) shown as heavy baseline.

234 J. Cooke

Table 1. Effectiveness of tissue grafts as organiser,after exposure to XTC-MIF/ XbFGF mixtures and to

their XTC-MIF component alone

Factor mixture(Units ml"1 total activityin blastocoel)

Response grades(see text and Fig. 2)

0

2

1

1

1

13141214

2

766738

3

968

4

376

5

1

Meai

2.92.93.01.351.051.401.70

XTC-MIF 900: XbFGF 90XTC-MIF 900: XbFGF 180XTC-MIF 900 ALONEXTC-MIF 180: XbFGF 900XTC-MIF 90: XbFGF 900XTC-MIF 180 ALONEXTC-MIF 90 ALONE

Grafts of c. 250 stage 9 donor cells were used, excised fromblastocoel roofs 2 h after injection to allow for a possible slowerrecording of cellular response to FGF. 1 unit of activity ml"1 offactors is the concentration just causing an in vitro inductiveresponse in Xenopus animal cap tissue (Keller and Danilchik,1988).

inductive signalling by onset of gastrulation (Jones andWoodland, 1987), causing minimal disturbance to pat-tern when transferred alone to a ventral marginalposition even in young blastula hosts (30 operations).The mesoderm component immediately above the lip isat this stage the efficacious organiser. Even whendivided longitudinally and used as smaller grafts to twohosts, this tissue initiates patterns that compare in sizeand completeness with those due to XTC-MIF exper-imental grafts. Blastocoel roof exposed to appropriateinjected concentrations of XTC-MIF begins a subtlechange of cellular anatomy that results in close simi-larity to the natural suprablastoporal organiser by stage10+ (Cooke and Smith, 1989). Strikingly, both thesetissues are most effective when placed in midblastulaventral marginal zones some hours before onset ofgastrulation activity in the latter (Table 3, and seeCooke, 1973ft), as if specific signalling between 'or-ganiser' cells and others can begin before the time ofmorphogenetic movements with some benefit. It isclearly the status of organiser mesoderm that is speci-fied in competent ectoderm by XTC-MIF. Onceevoked, this status is stable.

Optimal conditions for initiation of body patternThere is a reduced probability of obtaining the anteriorheadparts that mark the complete (grade 5) body planin development controlled by the experimental, asopposed to natural organiser, grafts. Experimentalgraft-organised second axes are more likely than dorsallip graft-organised ones to run behind the host axis intheir schedule of morphogenesis (convergent exten-sion, neural plate and tube formation etc.). This mayaccount for their tendency to gain a smaller ultimateshare in the host tissue. Dorsal lip tissue must be inhomopolar orientation with its host, as regards theanimal-to-vegetal dimension (presumptive head-tail,see Fig. 1), in order to set up second patterns (Cooke,1972c; Keller, 1986). Reversed implantation leads tomechanical disturbance to gastrulation but not to any

new axial structure. This can be related to a quite fine-grained and durable organisation among the cells of thisregion even before gastrulation, that actively controlstheir temporal order of involution and is often main-tained even if the grafted tissue is reversed relative to itsnew surroundings. The graft's movements then create abarrier that leads to mechanical conflict. But appropri-ately orientated dorsal lip mesoderm grafts recreate thelip-like invagination overlying a site of involution attheir lower edge. This new centre for gastrulationmovements comes to involve considerable host tissue,and a second pattern develops with a morphogeneticschedule nearly up to that in the 'host' axis. By contrast,brick-shaped pieces of the experimental organiser tissuehave no ascertainable axes other than 'inside-outside'polarity, and when implanted tend to produce a moreradially symmetrical, placode-like formation in thehost's marginal zone when their donor reaches stage10+. For such 'MIF' grafts that are of near-optimal size,however, shape in relation to the host structure isrelevant. A 'brick' whose width, subtending about 40°around the annular marginal zone of the host, is twiceits height tends to give the best second patterns. Thesecomparative features of natural and experimental orga-niser tissue are represented in Fig. IB.

The variables most dramatically influencing meansize and completeness scores of second patterns are theoriginal concentration of XTC-MIF in donor blastocoeland graft size. 200 ng (approx. 103 'units') ml"1 XTC-MIF has been, overall, the optimal blastocoel concen-tration for graft donors. The optimum graft is then atissue piece as in Fig. IB, and corresponding to some250 cells of a stage 9 (approx. 4xlO3 cell) donor. Cellnumber progresses very rapidly in the cleaving butnongrowing blastula, but it is graft size that matters.Second patterns after optimal versions of the exper-imental operation are most commonly of grades 3 and4, while after comparable natural organiser mesodermgrafts, grade 4 is the commonest result. Grade 5 (i.e.complete) second patterns, a minor class in Xenopuseven after natural organiser grafts of these sizes,nevertheless occur three times as often as with theexperimental grafts. As few as 50 grafted stage 9 cells,after exposure to 20ngml~1 XTC-MIF, can lead to agrade 1 or 2 axial formation.

The effects of these variables, though strong, arehard to tabulate. There is inevitably a unique responselevel to the whole procedure among the embryos ofdifferent ovulations, and only a limited sample ofindividual operations can be done on each experimentaloccasion. Table 2 nevertheless shows pooled means ofsecond pattern scores in 3 experiments that have usedvaried MIF concentrations and graft sizes. Other vari-ables were kept optimal at 1-2 h from factor injection tothe grafting of early stage 9 cell groups into stage 8 hosts(see below). An optimal combination of factor concen-tration and graft size emerges. Appropriately smallgrafts that have responded to saturating concentrationsof the factor, placed exactly midventrally in hosts, causeall the grade 4 and the occasional grade 5 results(Fig. 3A,B). Results from smaller dose x size products

Inducing factors and the organiser 235

Table 2. Effectiveness of blastocoel roof tissue asorganiser, related to graft size and to concentration of

XTC-MIF experiencedXTC-MIFconc.(units ml"1 in donorblastocoel)

Graft size (st. 9 cells)

500+

1.72.11.1

N.D.N.D.N.D.4.4

200-250

3.43.61.81.71.3

Zero3.9

50-100

2.72.60.60.80.6

N.D.2.9

3000100030020010050

Dorsal lip

Entries are mean pattern grades for pooled samples of operationstotalling around 25 per entry. Thus, after middle-sized grafts and1000 units ml"1, 12 grade 3, 12 grade 4 and 1 grade 5 patternresulted. After middle-sized grafts and 100 units ml"1, 4 zeroresponses, 12 grade 1 and 10 grade 2 patterns resulted. Othervariables, notably those of timing (see Table 3 and text) were nearoptimal in the present experiments.

tend to be small and of lower score though internallywell-ordered, while those from much larger than opti-mal grafts, regardless of MIF concentration, are usuallylarge but ill-organised, poorly differentiated and low-scoring disturbances in the host body plan (seeFig. 4D). Natural organiser tissue grafted with properorientation shows a contrasting property, in that thelarger the piece that is implanted, the larger and morevigorous is the new set of cellular movements andultimately the more complete (frequently grade 5) is thesecond body pattern formed. This contrast in behaviourbetween dorsal lip and ectopically MIF-induced tissueconstitutes striking evidence that further spatial organi-sation must be an early part of natural 'dorsal axial'induction.

Donor tissue reaches the full potential as organiserthat is to be evoked by any particular concentration ofthe factor 40min to lh after injection. No appreciablechange then occurs in this potential even if grafting isdelayed for the further 3h that may elapse before itdisplays new cellular behaviour resembling that in thenatural stage 10 organiser (Table 3). Advancing hostage at grafting, between large-celled blastula (st.7) andonset of gastrulation, exerts only a mild downgradingeffect (Table 3), suggesting that the relevant signallinginteractions occur largely during the gastrula period,though they may begin beforehand. Ambient tempera-ture between healing-in of grafts and the end ofgastrulation is kept near 18°C, when Xenopus developsat near half its 'optimal' rate (- mid 20s°C). It has beenthe author's overall experience that such cool ambientsduring gastrulation optimise extent and completeness ofsecond patterns after organiser grafting, and this re-mains true of XTC-MIF experimental grafting.

Cross-species recognition of the inductive function ofXTC-MIFI have asked whether Ambystoma (the axolotl), whichmay be distant from the frog by something closer toclass than to subclass status taxonomically (Nieuwkoop

Fig. 3. Twinned larval bodies in Xenopus and Ambystoma.(A, B) Xenopus larvae with second axes of grades 4 and 5,respectively. Small terminal retinal formation in upper axisof A is hidden, but forebrain field and cement glands areapparent in two right-hand examples in B. (Remainingexample in B is of grade 3). Grades of structure routinelydiagnosed on fix-dissection. (C, D) Normal, and equallypartitioned anteriorly complete twinned bodies ofAmbystoma (earlier stage of morphogenesis than A,B). Dresulted after placement of small XTC-MIF(1000 units ml"1) graft from early gastrula midventrally tosynchronous host, 4h after factor injection.

236 /. Cooke

Table 3. Time and stage related variables and theeffectiveness of dorsal lip and of XTC-MIF

experimental tissue grafts

Dorsal lip graftsExperimental grafts;

time from XTC-MIFinjection to grafting (hrs)

<0.5ca. 0.75ca. 1.0ca. 1.752.5-3.5

(donor gastrulation)

Time

2.5-4.0(st. 7-8

host)

3.9

N.D.2.93.53.4

N.D.

to host gastrulation (h)

Around 1.5(early st.9 host)

N.D.

0.92.83.33.33.1

<0.5(st. 9+ host)

3.6

N.D.N.D.3.13.13.0

Entries are mean pattern grades (see Table 1 last column andFig. 2) for second axes of 15-20 operations, pooled from 3-5experimental occasions (i.e. ovulations of eggs) in which operationswere performed with the stated combination of variables. Graftsizes was always near optimum. Dorsal lip grafts were similar-sizedlateral halves of suprablastoporal lip, with proper orientation, 18operations per entry.

and Satasurya, 1983), is responsive to XTC-MIF whenthe molecule is used as in the present Xenopus exper-iments. The answer was strikingly positive, with 19examples of second axes resulting from 25 operationseven though graft placement, after late blastular donorinjections and a four hour intervening period, was notuntil host early gastrula stage. The response was also'better' in that the newly induced axial pattern wasanteriorly complete in nearly half the cases (neverequalled in a Xenopus experiment) and in 5 casesappeared to achieve equal partitioning of the availabletissue with the host field. An example is seen in Fig. 3D,and the histological appearance of second patternelements in Fig. 4A,B.

Cellular contributions to pattern by the graftsContributions of lineage-labelled graft descendents areexemplified in Fig. 4. Grafts of normal blastocoel roof(as in the control operation) leave a small patch in thehost epidermis or gut wall, or both, near the junction ofthese at the larval blastopore site. A larger but morescattered population is found to have entered lateralplate and the more laterally derived somite mesodermof the trunk and tailbud. These sites are predictablefrom the normal fate map of the graft surround (Cookeand Webber, 1985), the pluripotent status of the graft attime of implantation and the presence of 'ventrolateral'inducing signals at the graft site until gastrula stages(Dale and Slack, 1987), and the phenomenon of dorsali-sation whereby initially nonaxial mesoderm can sub-sequently be recruited into somite structure (Smith andSlack, 1983). Surprisingly, XbFGF experimental grafts,which undoubtedly have mesodermally respecified tis-sue in them at the outset, are indistinguishable fromcontrol grafts in their contribution on an individualbasis (18 experimental and 20 control-grafted larvae

analysed). More descendents may tend to populatemidtrunk levels and lateral somite while fewer remainin mesoderm near blastopore and tailbud., but likecontrol cells they never enter anterior structure, noto-chord or parachordal parts of the somite cross-sectionof the normal, singly organised host pattern. In situinductive signals at the graft site therefore seem ablelargely to substitute for XbFGF experienced before-hand by implanted cells, in controlling their long-termfate. Their 'preinduction' within the donor embryo maynevertheless ensure that experimental graft cells areamong earlier-involuting, thus somewhat more an-teriorly fated, mesoderm.

Descendents of 18 XTC-MIF experimental and of 18natural mesodermal organiser grafts were in all casescentred upon the midlines of substantial second axialmesodermal patterns. They by no means constitutethese mesodermal axes, however. They are typicallyintermixed with host-derived mesoderm in the anteriorregions, and also form the roof of a new (endodermal)archenteron or else give rise to an integral strip of theendoderm beneath the new mesodermal dorsal midline.They give a progressively more scattered minor contri-bution to somite, or are absent, as progressively moreposterior levels of the somite segment series are exam-ined in the new axis that has been initiated by theirimplantation. The anteriorly confined distribution isparticularly seen for experimental graft-produced pat-terns of low grade, after grafting both small numbersand suboptimally large numbers of mesodermised cells.Notochord, in patterns that include it, is the onlystructure essentially made of graft cells even at pos-terior axial levels, and also the only occasion when ananatomical and a graft-host lineage boundary coincideover any distance in pattern. A case from an XTC-MIFgraft is shown in Fig. 4F. The compact contribution oflabelled cells to notochord is also seen in the morefrequent occurrences of this structure after graftingdorsal lip tissue. Whether dorsal lip grafts cause noto-chord formation or not, their contribution tends to bemore compact and dorsally centred in the cross-sectionof second patterns, as well as more completely rostro-caudally distributed, than is that of experimental MIFgrafts.

XTC-MIF grafts leave a substantial contribution inthe non-neural ectoderm overlying the anterior end ofthe mesodermal pattern they have organised, sugges-ting that they contain a variable proportion of cells thathave not been diverted from their epidermal pre-specification at time of implantation. Grafted dorsal lipsin the present series contributed only occasionally andto a small extent to the ectodermal pattern of hosts.This was entirely in ventral spinocaudal parts of secondnervous systems. Such a contribution might be explic-able in terms of our conventional view of neuralinduction, according to which this occurs in response tonewly involuting dorsal axial mesoderm. Alternatively,it might indicate that dorsal lip grafts contain cells thatwere already part of a pattern of biass or 'induction'towards membership of the CNS at the onset ofgastrulation in the donor (see Keller, 1986; Sharpe etal.

Inducing factors and the organiser 237

Fig. 4. Structure and graft contributions in second axes. (A-C) Feulgen/light green/Orange G-stained wax histology,transverse sections. (A, B) Eye/diencephalic and pronephric levels, respectively, of the second axial pattern in theAmbystoma embryo of Fig. 3D. (C) Trunk level section showing equal partitioning of mesoderm and sizes of nervoussystems in XTC-MIF graft-organised twin body of Xenopus. (D-L) Rhodamine-lysine-dextran lineage-labelled grafts inXenopus. Buffered paraformaldehyde fixation and wax histology, 10 ̂ m sections transverse to single bodies and of equaloblique angles to T.S. for axes of twins. 'Host' dorsal midline (off-frame except in C and K) is towards top for D-K andright for L. (D) Neighbouring anterior levels populated by suboptimally large XTC-MIF graft from which little organisationresulted. Note extent and solid distribution of labelled tissue. (E, F) Ear vesicle/hindbrain and pronephric levels,respectively, of grade 4 XTC-MIF graft-organised second pattern. Extensive head mesenchyme, the notochord and a mid-dorsal endodermal strip populated, and parachordal somite contributed to, by graft. (G-J) Ear vesicle/hindbrain,pronephric and successive trunk levels, respectively, of grade 3 pattern after XTC-MIF grafting. Disorganised headmesoderm and the anterior somite axis largely populated by graft, but contribution rapidly decays posteriorly to give well-organised but unlabelled somite. (K, L) Unilateral tailbud somite and symmetrical midventral mesodermal contribution to asingle normal axis after XbFGF grafting. This contribution, according with normal fate of graft surroundings, is notdistinguishable from that of untreated blastocoel roof grafts. Scale bar=100^m approx. Labelling as Fig. 2.

1987; Spemann, as related in Hamburger, 1988). Thedifference in character of ectodermal contribution asbetween experimental and natural organiser tissuetends to support the latter idea, and to suggest once

again that the normal dorsal marginal zone achievesmore early spatial organisation into subdomains thancan be achieved by the homogeneously stimulatedexperimental tissue.

238 /. Cooke

Fig. 5. Sequence of intercellular signalling proposed in the'3 signal' class of model. Pregastrula stage represented inlateral view, dorsal at right. In the downward-hangingvegetal hemisphere, a sector emitting inductive signal for'dorsal axial' mesoderm abutts against or is superimposedon the more widely distributed source of 'ventrolateral'vegetal inductive activity (signals 1 and 2). The minor andmajor mesodermal sectors of different character which arethus induced in the marginal zone act respectively as sourceand sink for subsequent intramesodermal morphogengradients (signals 3). Although shown on the same diagram,this intramesodermal signalling may occur largely duringgastrulation. Dorsal axial sector is shown stippled withdensity decreasing from vegetal border (first - gastrulating,future anterior) into animal cap (later - gastrulating, moreposterior), a, animal pole; v, vegetal pole. Shorterarrowheads=initial inductive signals, longerarrowheads=gradient fields within mesoderm occurringbefore and during gastrulation, either in signals 3 or by self-organising interactions within dorsal axial sector (seeDiscussion).

Discussion

First steps in regionalisation of mesodermEctopic mesoderm in the blastocoel roof of XTC-MIF-injected embryos resembles, in the timing within gastru-lation of its 'involution' behaviour and in the mode ofintercellular adhesion it adopts, the mesoderm beneaththe early dorsal lip. The differences in character be-tween it and experimentally XbFGF-specified meso-derm are independent of the concentrations and priortime of application of the inducers. The hypothesisrepresented in Fig. 5 is supported by these and theother findings reviewed in the Introduction and bythose reported in this paper. It is a version of the '3-signal model' first proposed by Slack and explicitly laidout by Dale and Slack (1987), and follows the generalnotion that pattern is realised in response to a morpho-

gen gradient system. Initial inductive signals from theendodermal region of two types (i.e. signals 1 and 2) areproposed, with their effective sources so distributed asto give rise to a restricted sector of 'dorsal axial' typemarginal zone and a larger remaining sector of thelateroventral type. Intramesodermal gradients in afurther morphogen or morphogens (i.e. 'signals 3') thenarise because the dorsal axial sector is a source for themolecules involved, and the remaining tissue acts as asink by actively sequestering/destroying, and/or pass-ively providing diffusion space. This model, and all thepresent work, relate only to proposed mechanismsunderlying the reliability of normal development. It isnow clear (e.g. Kao et al. 1986; Slack et al. 1988) thatdistortion of intracellular mechanisms of signal trans-duction, as presumably with the Lithium ion, can erodethe normal distinction between cell states achieved viathe heparin-binding and the expected TGF-/3-likeclasses of factor and their distinctive cellular receptors.This piece of cell biology will only be unravelled withfurther understanding of the mechanisms of the signaltransductions themselves.

The present work strengthens belief in some versionof the overall model depicted in Fig. 5, for the initialorganisation of pattern in the mesoderm. XTC-MIFand XbFGF might be, or might closely resemble, theinitial signals 1 and 2 that set up source and sink statesfor the signals 3. The evidence for an actual, corre-sponding in situ role for XbFGF is currently somewhatstronger than is that for XTC-MIF, since it is present inthe egg and blastula although its distribution is not yetknown to be appropriately localised (Kimelman andKirschner, 1987; Kimelman et al. 1988; Slack andIsaacs, 1989). The vegetal region of the Xenopus eggand blastula appears to contain RNAs for several TGF-f3 relatives, of which the best characterised, Veg. 1(Weeks and Melton, 1987), does not code for XTC-MIF. Furthermore, various TGF-/3 family members areineffective as mesoderm inducers of animal cap tissueon their own but can potentiate the action of factors ofthe FGF family so that mesoderm of dorsal character isproduced (e.g. Kimelman and Kirschner, 1987). In thepresent experiments, all groups of animal cap cellsprobably receive a certain intensity of natural ventro-lateral inductive signal after their grafting to the hostventral margin, and this could conceivably replicate anadditional and necessary signal found earlier on in thedorsal marginal zone where the natural organiser orig-inates. The mechanical behaviour and differentiationsinduced in animal cap tissue in vitro by pure XTC-MIF,however (Smith et al. 1988; Rosa et al. 1988; Symes andSmith, 1987), indicate that this particular signal mol-ecule, whether or not it is the relevant in vivo one,induces the mesodermal organiser status in cells in theschema of Fig. 5 when acting alone. Work of thepresent type aims not so much to add to the evidencesabout which particular molecules are the in vivo com-ponents, but to help in understanding of the logicalsequence of signals and cell states whereby pattern isbuilt up. A distinctive experimental prediction of the 3signal class of model, with the presently postulated

Inducing factors and the organiser 239

candidates for signals 1 and 2, concerns expected resultsof experiments where pieces of animal cap tissue withdiffering histories of exposure to inducers in vitro arejoined together (Cooke et al. 1987). Such combining ofpieces that have recently received XTC-MIF andXbFGF should result in significantly greater spatialorganisation of mesodermal structure than occurs ineither 'homotypic' combination, or in combinationswhere one component piece had not previouslyreceived any mesoderm-inducing signal. This predic-tion is under test.

Alternative mechanisms to the above for the primaryarrangement of the body pattern would be (a) direct useof a gradient in the XTC-MIF signal, from its restrictedsource, to order pattern, (b) direct responses to differ-ent 'source intensities' of a mesodermal inductivestimulus, graded around the endodermal zone (Cookeand Webber, 1985), or (c) a variety of schemes wherebyboth receptor and potential FGF class of inductor wereubiquitously present in competent tissue, but this'autocrine' mechanism of mesoderm formation wasonly made active, in a graded way, by encounter with anXTC/TGF-/3 class signal. But against (a) and (b), thereis evidence that the XTC-MIF molecule's concentrationand time of delivery does not in itself set 'position value'of mesoderm in the whole embryo (Cooke and Smith,1989), and that any spread of signal from new dorsalaxial mesoderm to adjacent but as yet not mesodermallyinduced tissue is a slow and thus local process (Cookeet al. 1987). Whereas it is straightforward in principle tosee, from our understanding of transcriptional controlprocesses, how quantitative gradation in an intracellu-lar mms-acting factor might determine more than oneswitch (thus, two states) within previously equivalentcells (e.g. Driever and Nusslein-Volhard, 1988), areceptor-binding intercellular signal protein does notappear apt for such direct control of several cell states.Alternative (c) above has difficulty in accounting for theoccurrence of extensive mesoderms of radially sym-metrical 'lateroventral' character, in embryos whichhave been prevented from forming any dorsal-axial-type inductive sector (Scharf and Gerhart, 1983; Cookeand Smith, 1987).

The 3 signal class of model tallies best with thepresent results, particularly in accounting for the stabledevelopment of incomplete (Fig. 2,1-4) versions of thebody plan. But it can only do so if it explicitly proposesthat the cell states for the two initial mesodermalsectors, directly induced by signals 1 and 2, do not leaddirectly and uniquely to any particular differentiations.Instead, by their interaction, they define the polarityand extent of a morphogen landscape that will moredirectly order such differentiations. Thus XTC-MIFmay specify a single new state - the 'organiser' or signal3 source state, in an all-or-nothing manner at theindividual cell level. Maximum signal value attained inthe signal 3 gradients, thus the 'completeness score'(1-5) of patterns, should then be conditioned by theproduct of graft size and original XTC-MIF concen-tration. These will have set the effective number ofsignal 3 source cells present (cf. Summerbell, 1979;

Tickle et al. 1985). The influence of the effective extentof source territory in relation to surroundings, in settinga gradient profile from source tissue of constant proper-ties, is represented in the baselines below left-handparts of the profiles sketched in Fig. 2. These profileseach correspond to single or twinned body patterns thatare illustrated above and to their left. The source areamust nevertheless be suitably small within the sur-rounding 'sink' mesoderm to ensure a coherent gradi-ent; hence the low grade and disorganised patterns afterover-large grafts (see Fig. 4D). Naturally originatingbody pattern is assumed to be complete and wellproportioned insofar as earlier events ensure rightlyproportioned source and sink sectors.

It cannot be discounted that some of the effect ofgrafting both natural and experimental organiser tissuecomes from passive transfer, into the new surroundingintercellular spaces, of unbound factor of the samefunctional class as that which has induced dorsal specifi-cation in the grafted tissue. This would correspond tooperation of a version of mechanism (a) above for thenormal organisation of pattern within the dorsal axialsector, which is re-enacted after grafting. There is noreason to propose different classes of event, in thisregard, after experimental as opposed to natural or-ganiser tissue grafts, and in the future only experimentsinvolving antibody reagents unique for the fully charac-terised factors may be able rigorously to distinguishbetween the classes of mechanism. It is already knownthat ventral implantation of a small, non-living, initiallyhigh-density source of XTC-MIF, but one from whichthe molecule will rapidly diffuse (agarose or sephadex),does not simulate the organiser phenomenon. Suchimplantation merely causes the blastocoel-wide ectopicmesoderm formation seen when a comparable, lowtotal dose of factor is injected free as in the presentprocedure for creating donors (Cooke and Smith, 1989;J. Cooke and Emma J. Smith, unpublished work).

Spatial organisation within the natural organiser regionEctopically XTC-MIF-induced animal cap tissue clearlycorresponds, in general, to dorsal lip mesoderm. Whythen should it be appreciably less effective, mass formass, as organiser, and tend to make somewhat morerostrocaudally restricted and yet less compactly distrib-uted lineage contributions to second patterns that itorganises as a graft? Future head-to-tail and dorsoven-tral coordinates of the body plan are evident withinnormal mesoderm by gastrulation itself, as sequences oflocal time schedules of active involution. These pregas-trular gradations of mesodermal state are so laid outthat they must be conditioned by relative distances ofcells in the marginal zone from the endodermal sourcesof the inducers, as well as by spatial segregation of thosesources around the marginal zone. The endogenousMIF source is assumed to lie at the vegetal edge of theinduced dorsal axial tissue which is thereby able toacquire graded properties for a rostrocaudal sequenceof axial pattern which ectopic, homogeneously inducedtissue lacks (Cooke and Smith, 1989). This further

240 /. Cooke

refinement of spatial organisation must occur soon inthe normal development, because the dorsal lip tissuereveals itself in grafting experiments to be finely andimportantly polarised (Cooke, 1972c; Keller, 1986, seealso present Fig. IB). Signal circuitry having the formalproperties of local autocatalysis and then long-rangeinhibition, following initiation at one edge of a field,could mediate such an organisation (Gierer and Mein-hardt, 1972; Meinhardt, 1982; Cooke, 1989), and TGF-P biosynthesis shows the first of these dynamics (Van-Obberghen-Schilling et al. 1988). Properly orientatedgrafts of dorsal lip may retain their graded range ofpreliminary specifications. Hence their tendency forcompact but quite extended cellular contributions toaxes, with relatively little developmental delay. Newlyand homogeneously MIF-stimulated experimentalgrafts, by contrast, must start the new gradient systemab initio by selforganisation, as well as by being sourcefor signals 3. The success of the outcome, within thetime available, may depend upon the variable initialconditions as observed in this work. Hence also themore radially symmetrical arrangement of mechanicalactivity after implantation (Fig. IB), and the anteriorlyconfined lineage contribution with more posterior axialpattern organised, but not populated, by the graft.

The normal initiation of induction from one (vegetal)edge of the competent region, that is not properlysimulated by injection of soluble XTC-MIF beneath acell sheet, may also be required for reliable attainmentof the most 'anterior' specification within the generaldorsal axial region. See Meinhardt (1982) for fulltheoretical discussion of symmetry-breaking and spon-taneous pattern formation in morphogen systems. Thismost anterior tissue, of 'prechordaF specificity, isrequired for development of the complete plan in bothmesoderm and induced neural system (Nieuwkoop,1973), and in normal development it shows differentcell-mechanical behaviour from, and involutes soonerthan, dorsal axial tissue. But observation of even theoccasional complete (grade 5) axes that are seen inresponse to XTC-MIF stimulated implants does obviatethe need to postulate a third, as yet undiscovered typeof initial inducer, for dorsal prechordal mesoderm.

The completeness, extent and integration in thesecond organisations in Ambystoma are significant,confirming the folklore existing among experimentalembryologists that the pattern of differentiation inurodele embryo tissue is 'less determined' than is that ofanurans at each blastula and gastrula stage (Holtfreterand Hamburger, 1955). In Xenopus the negative effectsupon outcome of the operations of advancing host ageat grafting, and of increased ambient temperature, mayindicate that the necessary interactions across tissue canbe overtaken and limited by the onset of cell determi-nation. The inherently slower pace at which loss ofcompetence of tissue to be diverted in fate is occurringin Ambystoma would then allow superior results fromboth 'natural' and MIF-stimulated organiser grafting,and diminish the advantage of implanted but naturallypreorganised dorsal lip mesoderm over a homo-geneously induced cell group.

I thank Jim Smith of this laboratory for the supply of XTC-MIF, Dave Kimelman of the Department of Biochemistry,UCSF, California for that of the Xenopus bFGF, WendyHatton for skilled histology, John Gerhart, Jack Price andMartin Raff for invaluable comments during preparation ofthe manuscript, and the organisms themselves for theirintermittent supply of the requisite high quality eggs andsperm.

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(Accepted 5 July 1989)