mechanism of anteroposterior axis specification in vertebrates · but vertebrates differ from...

Development 114, 285-302 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

Review Article 285

Mechanism of anteroposterior axis specification in vertebrates

Lessons from the amphibians

J. M. W. SLACK and D. TANNAHILL

ICRF Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

Contents

SummaryIntroductionResults from experimental embryology

Possible commitments for anteroposterior levels atpregastrula stagesFate mapMesoderm induction

Formation of the anteroposterior mesodermal patternduring gastrulation

MethodsNeural inductionRegional specificity of neural inductionReciprocal effectsPosterior dominance within the neural plateHow many territories?

Preliminary conclusions

The molecular eraMolecular markers

Non-homeobox markersHomeobox expression patternsAre homeobox genes coding factors?

Reinvestigation of problems using molecular methodsMesoderm induction and axial patterningNeural induction

Neural inducing factorsDorsal biasTangential inductionRegional specificity of neural induction

Retinoic acidConclusions

Summary

Interest in the problem of anteroposterior specificationhas quickened because of our near understanding of themechanism in Drosophila and because of the homologyof Antennapedia-like homeobox gene expression pat-terns in Drosophila and vertebrates. But vertebratesdiffer from Drosophila because of morphogenetic move-ments and interactions between tissue layers, bothintimately associated with anteroposterior specification.The purpose of this article is to review classical findingsand to enquire how far these have been confirmed,refuted or extended'by modern work.

The "pre-molecular" work suggests that there areseveral steps to the process:

(i) Formation of anteroposterior pattern in mesodermduring gastrulation with posterior dominance.

(ii) Regional specific induction of ectoderm to formneural plate.

(iii) Reciprocal interactions from neural plate tomesoderm.

(iv) Interactions within neural plate with posteriordominance.

Unfortunately, almost all the observable markers arein the CNS rather than in the mesoderm where the initialspecification is thought to occur. This has meant that the

specification of the mesoderm has been assayed in-directly by transplantation methods such as the Ein-steckung.

New molecular markers now supplement morphologi-cal ones but they are still mainly in the CNS and not themesoderm. A particular interest attaches to the genes ofthe Antp-like HOX clusters since these may not only bemarkers but actual coding factors for anteroposteriorlevels.

We have a new understanding of mesoderm inductionbased on the discovery of activins and fibroblast growthfactors (FGFs) as candidate inducing factors. Thesefactors have later consequences for anteroposteriorpattern with activin tending to induce anterior, and FGFposterior structures.

Recent work on neural induction has implicatedcAMP and protein kinase C (PKC) as elements of thesignal transduction pathway and has provided newevidence for the importance of tangential neural induc-tion. The regional specificity of neural induction hasbeen reinvestigated using molecular markers and pro-vides conclusions rather similar to the classical work.

Defects in the axial pattern may be produced byretinoic acid but it remains unclear whether its effectsare truly coordinate ones or are concentrated in certainregions of high sensitivity.

286 J. M. W. Slack and D. Tannahill

In general the molecular studies have supported andreinforced the "pre-molecular ones". Important ques-tions still remain:

(i) How much pattern is there in the mesoderm (howmany states?)

(ii) How is this pattern generated by the invaginatingorganizer?

(iii) Is there one-to-one transmission of codings to theneural plate?

(iv) What is the nature of the interactions within theneural plate?

(v) Are the HOX cluster genes really the anteropos-terior codings?

Key words: anteroposterior specification, neural induction,homeobox genes, retinoic acid, Xenopus, Cynops, Triturus.

Introduction

All multicellular animals have some sort of anteropos-terior polarity at some stage in their life cycle. In thecase of the fruit fly Drosophila we now understand, inoutline, how different body regions are specified alongthe anteroposterior axis and we know that the endresult of this process is the activation of the homeoboxgenes of the Antennapedia (ANT-C) and Bithoraxcomplexes (BX-C) at different body levels (reviews:Akam, 1987; Ingham, 1988; Slack, 1991). Immenseexcitement has resulted from the discovery that thereare homeobox (HOX) clusters in the mouse and othervertebrates which show a definite evolutionary hom-ology to the ANT-C/BX-C in Drosophila such thatindividual genes have retained their relative positions inthe cluster, and also that their position in the cluster isdirectly related to the spatial domain of expression inthe anteroposterior axis of the body (Duboule andDoll6, 1989; Graham et al., 1989). Because of thisevolutionary homology of HOX cluster gene ex-pression, high hopes have been generated of solving theproblem of the anteroposterior axis, perhaps thegreatest problem of embryology, in a single giant leapfrom Drosophila to Man.

But vertebrates differ from insects in three importantways. Firstly, in most insects, including Drosophila, theearly events of regional specification occur while theembryo is a multinucleate syncytium, whereas invertebrates most regional specification occurs in amulticellular embryo. Secondly, at about the time thatthe main regions of the body plan are specified, theDrosophila embryo consists of a simple ellipsoidalmonolayer of cells. In vertebrates, extensive morpho-genetic movements take place before the stage at whichthe body plan has become specified, which is around theend of gastrulation. Thirdly, induction is critical fromthe earliest stages in setting up regional identities invertebrate embryos. In Drosophila, interactions be-tween tissue layers do seem to take place but only at alater stage.

In this article we shall review what experimentalembryology has to say about the problem of anteropos-terior specification and then enquire to what extent the

questions have been answered by the application ofmolecular techniques over the last few years. Theembryological work has overwhelmingly concentratedon amphibian embryos and so we have focused onthem, only including occasional comparisons withmammalian data where it is available.

Results from experimental embryology

Possible commitments for anteroposterior-posteriorlevels at pre-gastrula stagesIt is well known that various experimental manipu-lations carried out between fertilization and the begin-ning of gastrulation have effects on the anteroposterioraxis; for example, irradiation of fertilized eggs withultraviolet light causes dose-dependent reductions ofthe anterior end (Malacinski et al., 1975; Scharf andGerhart, 1983). Because of this, some authors havesuggested or implied that at least some anteroposteriorcodings become specified before gastrulation (egCooke, 1989). But of course early treatments may havelate effects that are indirect and involve severalintervening causal steps, so what is the real evidencebearing on this question?

Fate mapWe start with the fate map of the blastula. In amphibianembryos, fate mapping studies have all shown that thenotochord and adjacent part of the somites arise innormal development from the dorsal marginal zone, aregion known as the organizer (Vogt, 1929; Pasteels,1942; Keller, 1976; Dale and Slack, 1987). Although theolder fate maps based on vital staining showedprospective regions for individual somites on the lateblastula, it is now known from studies with injectablelineage labels that there is too much cell mixing duringgastrulation for such small territories to remain coher-ent and distinct from one another (eg Dale and Slack,1987). Particularly among the dorsal midline cellsforming the notochord, there is a high degree of activecellular intercalation which provides the driving forcefor the extreme anteroposterior extension of this region(Wilson et al., 1989). So there cannot be a detailedtopographic projection from regions in the blastula toparticular anteroposterior levels in the later axis, andthis necessarily means that there cannot be a detailedmosaic of regions committed to particular anteropos-terior levels at the blastula stage. In the mouse a similarsituation exists insofar as the anterior part of theprimitive streak tends to populate the notochord andsomites along the entire length of the body (Tarn andBeddington, 1987; Lawson and Pedersen, 1987), andthere seems to be even more cell mixing than in theamphibians.

However, even if there is too much mixing for adetailed topographic projection from pregastrula topostgastrula anteroposterior levels, it remains possiblethat there is some projection; for example, separatecohorts of cells destined to become head (prechordalmesoderm), trunk (notochord) and tail (tailbud bias-

Anteroposterior specification 287

Fig. 1. Fate of the dorsal marginal zone in Cynops, afterOkada and Hama (1945). An early gastrula is shown onthe left and 1,2,3 represent three zones of tissue above thedorsal lip which were stained with vital dyes. On the rightis shown the approximate projection of these zones ontothe dorsal side of the neurula. The dotted line shows theanterior limit of the notochord.

tema) might involute in sequence and thus be derivedfrom territories arranged in a vegetal-to-animal sense inthe blastula. The highest resolution studies addressingthis question were carried out long ago with Cynopsusing vital stains (Okada and Hama, 1945). These alsoshow the dramatic elongation of small dye marks overmuch of the axial length but the results do suggest thatthere is a projection of about "head-trunk-tail" resol-ution (Fig. 1). Whether this is also true for Xenopus,where the corresponding region is much smaller and liesinternally, remains to be seen.

Mesoderm inductionEven if there is a limited or statistical fate mapprojection, this does not necessarily mean that there isany anteroposterior commitment at the early stage. Weknow that during the blastula stages, mesoderm-inducing factors (MLFs) are released from the vegetalcells and induce a belt of cells around the equator (themarginal zone) to a mesodermal character. This processis called mesoderm induction (reviewed Smith, 1989;Slack, 1991). Around most of the circumference theinduced mesoderm is of ventral character but in a smallregion it is of dorsal character and this region is calledthe organizer. It has been proposed that the organizer-inducing signal consists of activin or something withsimilar properties to activin. If prospective regions forhead, trunk and tail mesoderm had different states ofspecification in the blastula then this would presumablyreflect the response to a vegetal-to-animal gradient ofthe activin-like factor such that head (closest theblastopore) was formed in response to high levels andtail (furthest from the blastopore) to low levels (Fig. 2).

Direct studies on the specification of the dorsalmarginal zone do not show much difference in antero-posterior quality between regions normally forminghead, trunk and tail. The best studies were done withCynops (Hama et al., 1985; Fig. 3). When explantssimilar to the regions 1, 2, 3 of Fig. 1 are wrapped inectoderm and cultured, pieces 1 and 2 form predomi-nantly trunk structures, and piece 3 predominantly

headtrunktailventral mesoderm

Fig. 2. Diagram showing how a potential specification ofanteroposterior states would have to occur at themesoderm induction stage. DV, dorsal mesoderm-inducingzone; W , ventral mesoderm-inducing zone.

epidermis

notochord and somites

notochord and somites

Fig. 3. Specification of dorsal marginal zone in Cynops,showing that it is not as expected from the model of Fig. 2.Explants were removed, wrapped in ectoderm and culturedin isolation until differentiation (see Hama et al 1985).Pieces 1 and 2 both produce mainly trunk-like structures(notochord and somites) while piece 3 produces mainlyepidermis.

epidermis. Piece 1 does not form heads as might beexpected from the model of Fig. 2. In this series theimplants and jackets were not labelled so we do notknow what was formed by self-differentiation and whatby induction. The difference between pieces 1 and 2(mesodermalized) and 3 (still epidermal) is probablydue to the fact that the response to the activin-likefactor spreading up from the vegetal hemisphere hasnot yet reached its final frontier. This conclusion issupported by other studies in which it is shown thatregion 3 becomes mesodermalized as it approaches theUp (Kan6da and Hama, 1979; Kan6da, 1980,1981). Thegeneral absence of head structures formed by any of theexplants argues against a mechanism of the type shownin Fig. 2 and suggests that the formation of rudimentsfor different levels of the anteroposterior axis occursafter the blastula stage.

Formation of the anteroposterior mesodermal patternduring gastrulation

MethodsUnfortunately there are no histological markers ofposition along the anteroposterior axis of the mesodermexcept for the difference between the prechordal regionand the trunk. This means that it is necessary to make

288 /. M. W. Slack and D. Tannahill

CD . _ CD

Fig. 4. Procedures for assaying anteroposterior character.(A) Einsteckung: The explant is grafted into the blastocoelof the host, is moved to the anteroventral region by thegastrulation movements, and may interact with hostpharyngeal endoderm, ventral mesoderm and ventralectoderm. (B) Surface graft: The explant is grafted to aparticular position on the embryo surface, often the ventralmarginal zone. (C) Sandwich: The explant is wrapped inearly gastrula ectoderm.

use of structures induced by the mesoderm such as partsof the central nervous system. The validity of suchresults depends on an assumption of a one-to-oneregional specificity of neural induction which we shallconsider more carefully below.

The early (pre-Second World War) studies employedmainly the "Einsteckung" procedure introduced by O.Mangold, in which the test tissue is introduced into theblastocoel of an early gastrula through a hole in theectoderm (Fig. 4A). The gastrulation movements carrythe graft to a ventral position and bring it into contactwith ventral ectoderm. Neural structures formed in thisposition are regarded as having been "induced" by thegraft although in most cases there was no attempt tolabel the grafts and thus find the contribution of graftand host tissue to the structures. More seriously thegraft may interact with the advancing pharyngealendoderm, or with ventral mesoderm of the hostgastrula. There may also be mechanical effects on theforming host axis leading to splitting, particularly at theanterior end. All these factors make Einsteckungexperiments difficult to interpret. An improvement is toimplant the graft in the surface of the host. This at leastcontrols its position more accurately and hence the hostregions with which it can interact (Fig. 4B). A bettermethod still is to culture the test tissue in a jacket ofgastrula ectoderm (Fig. AC). This removes most of theuncertainties due to morphogenetic movements and tointeractions with non-ectodermal parts of the host. Itstill requires the assumption of one-to-one neuralinduction, but could only be improved on by directobservation of anteroposterior character using molecu-lar markers.

Fig. 5. (A) Fate map of the urodele open neural plate afterJacobson (1959). (B) Fate map for Xenopus open neuralplate, after Eagleson and Harris (1990). SC, spinal cord.

Neural inductionBecause most of the observable anteroposterior patternscored in experiments consists of neural or evenplacodal structures, it is essential to be aware of thepotential for complexity in the results arising fromneural induction itself. Fate maps of the neural plate areshown in Fig. 5. It should be noticed that the head isgrossly overrepresented in relation to the trunk and tailand much of the posterior part of the body is formedpost-neurulation by the expansion of the tailbud. In factthe most posterior part of the neural plate is notexclusively neural but contributes heavily to tail somites(Woodland and Jones, 1988).

Workers on neural induction have often scored theirspecimens into three classes: archencephalic, meaningforebrain-like and also containing derivatives of thecephalic placodes; deuterencephalic, or mid/hindbrainlike; and spinocaudal, or containing simple neural tube,notochord, somites and tailfin. Spinocaudal inductionscontain a lot of tissue conventionally classified asmesodermal. This has in the past led to confusionbetween the mesoderm induction which occurs in theblastula and a possible spinocaudal induction leading tothe formation of the most posterior part of the neuralplate. But the distinction between these two eventsshould now be clear in both time, space and in the logicof developmental decision making. We shall returnbelow to the question of whether they might be due tothe same chemical inducing factor.

Neural induction has long been regarded as anappositional induction, that is a process involvingcommunication between two separate tissue layers,although it has long been known that neural inductioncan also propagate within a sheet of competentectoderm (homeogenetic induction: Mangold and Spe-mann, 1927). In normal development neural inductionoccurs in a sequential manner as gastrulation proceeds,the most anterior part of the invaginating tissuemigrating under the entire prospective neural platefrom posterior to anterior.

Regional specificity of neural inductionThe first studies indicating the regional specificity of

Fate


Specification Homeogeneticinduction

Archenteron roofinduction

nasal placodttforebrain eye»

balancersneural tube

Fig. 6. Comparison of normal fate, specification (ie self- differentiation behaviour of explants), homeogenetic inducingcapacity (ie what is induced by each level from gastrula ectoderm) and inductions by the archenteron roof, based on thedata of ter Horst (1948). In this diagram structures occurring with a frequency of >40% are shown, "neural tube"represents the three original categories "neural", "neural tube" and "spinal cord".

neural induction date back to the 1930s, using Triturus.They employed either archenteron roof explants,presumed to be the normal inducer of the neural plate,or explants of the neural plate itself.

The best study is that of ter Horst (1948) whocompared the specification of each level of the neuralplate of Triturus embryos with its homeogeneticinductive capacity and also with the inductive capity ofthe archenteron roof from the same level (Fig. 6). Sheimplanted the archenteron roof or neural plate pieces injackets of ectoderm from early gastrulae and stainedone member of the combination with Nile Blue. Thevital staining meant that she could gain at least someidea which parts of the resulting structures were derivedfrom the ectoderm and which from the implant. Theneural plate pieces give homeogenetic inductionsappropriate to the fate map; brain, eyes and nasalplacodes from the most anterior fifth, hindbrain fromthe second and third fifth, and tails from the mostposterior fifth. There is also considerable induction ofotocysts by all levels. On the other hand, with thearchenteron roof pieces the maximal induction ofbrains, eyes and otocysts was by the second piece, andmaximum induction of hindbrain by the fourth piece(Fig. 6). The archenteron roof thus seemed rather moreanterior in character than its own normal fate, or thenormal fate of its overlying ectoderm, would suggest.As we shall see, the reason for this is probably that theinduced tissue also receives signals through the plane ofthe ectoderm. Another study by Sala (1955) using theaxolotl gives similar results, as does the originalEinsteckung study of Mangold (1933).

The conclusion from these studies is that there areindeed regional differences in the inductive character ofdifferent anteroposterior levels of the archenteron roof.To some extent these correspond to normal fates, but

there tends to be a anterior shift in character relative tofate.

Reciprocal effectsAlthough the discussion of neural induction is normallypredicated on the assumption that the signals pass inone direction, from the archenteron roof to theectoderm, this may be an oversimplification since thereare several papers describing reciprocal effects. Katoand Okada (1956) claimed that early dorsal lips fromCynops would only produce notochord and somites ifcombined with gastrula ectoderm which itself formedflorid neural structures. Muchmore (1958) showed thatcoculture of prospective Ambystoma somite with neuralplate could greatly boost the amount of muscle formed.Nieuwkoop and Weijer (1978) found that twice as muchnotochord was formed by posterior archenteron roofcultured with gastrula ectoderm in which florid neuralinduction occurred, rather than with neurula epidermis,in which it did not.

Specification of anteroposterior codingsThe acquisition of distinct anteroposterior characterappears to be progressively determined during gastru-lation. A recent experiment on Xenopus which supportsthis idea involves the inhibition of gastrulation move-ments at different times by the injection of polysulpho-nated compounds such as suramin or trypan blue(Gerhart et al., 1989). These agents cause truncationsfrom the anterior end of the embryo the extent of whichis dependent on the time of injection. Injections at theonset of gastrulation result in a massive deletion of theaxis all the way from the head to tail whereas injectionsat the mid-gastrula stage result in deletions of only themost anterior structures and injections into the lategastrula have little effect. These observations suggest


EDL causes spinocaudalinductions

aged EDL causesarchencephalic inductions

invaginated EDL (AAR) causesarchencephalic inductions

| [ LDL causes spinocaudalinductions

AAR causes archencephalicinductions

PAR causes spinocaudalinductions

Both together causespinocaudal inductions

Fig. 7. Summary of the results of experiments by Okadaand Takaya (1942a,b). EDL, early dorsal lip; LDL, latedorsal lip; AAR, anterior archenteron roof; PAR,posterior archenteron roof.

that axis determination happens progressively duringgastrulation such that the length of time of gastrulationand/or the extent of axial elongation determines theability to produce succesively more anterior structures.

Most of the earlier experiments on anteroposteriorspecification were caired out on Cynops. The crucialobservation, made by Okada and Takaya (1942a,b) wasthat the inducing effect of the dorsal lip region changedon invagination (Fig. 7A,B). Early dorsal lip. tissuefrom the surface, grafted to the ventral side of anotherembryo, induced trunk/tails, while the same tissue iftaken after invagination induced heads. The change ininducing capacity also occurred during in vitro cultureof the tissue for 12-24 hours. Several of the originalexperiments were repeated with similar results bySuzuki et al. (1984). When the inductive capacity ofexplants of archenteron roof were examined (Fig. 7C),there was the same sort of regional specificity notedabove, although these studies are of lower resolutionthan those of ter Horst or Sala. But significantly, whenboth anterior and posterior archenteron roof were usedtogether as the inducer, the inductions were oftrunk/tail character. The same result was obtainedwhen recently invaginated material from early and lategastrulae were used in combination as an inducer.Although as cephalized creatures our natural inclina-tion is to suppose that the head should be the dominant

region, or "high point" of the axis, these results suggestthat it is actually the posterior which is dominant.

This surprising conclusion is also supported byexperiments of Hall (1937) using Triturus in which earlyand late dorsal lips were interchanged. If a late lip wasremoved and replaced with an early lip, the resultingembryos were fairly normal, ie the early lip both formedand induced posterior structures. If a late lip wassubstituted for an early lip then the anterior end of theresulting embryos very much resembled an ectopic tail.In both series of experiments the juxtaposition ofanterior with posterior leads to the posterior remainingposterior and to the anterior becoming posterior. Inother words posterior dominates over anterior. Themain problem with both series is that they did not use acell label and so could not reliably distinguish graft fromhost tissue. Repeating these critical experiments with alineage label must now be regarded as an importantpriority.

Posterior dominance within the neural platePosterior dominance in the neural plate is most clearlyshown in a series of experiments using urodeles in whichfolds of gastrula ectoderm were implanted verticallyinto the open neural plate of a host embryo (Nieuw-koop, 1952a,b,c; Fig. 8). Neural induction proceeds upthe folds; the structure induced at the base is the sameas that formed by the surrounding neural plate, whilethose formed further up the fold are successively moreanterior in character. This suggests that the anteropos-terior series of structures formed by the neural plate arecoded in the early neurula by some gradient-like set ofstates which can propagate in a decremental waythrough the ectoderm and does not require theimmediate proximity of the archenteron roof. A rathersimilar set of data has recently been produced from aseries in which prospective forebrain instead of gastrulaectoderm was used for the folds (Nieuwkoop andAlbers, 1990).

These transplantations and fold experiments clearlyshow that posterior dominates over anterior in theneural plate as it does in the mesoderm. This mightsuggest that the regional specification of the neuralplate could be achieved by a single posterior-to-anteriorgradient, but Nieuwkoop and his colleagues havealways favoured a two-step process consisting of an

Fig. 8. Nieuwkoop's (1952a-c) "fold" experiments, in whicha fold of gastrula ectoderm is implanted into the openneural plate. Anterior lies to the left and the numbers Nl-N4 and O represent territories forming different neuralstructures (see also Fig. 9). The anterior inductions containfew structures but are large, while the posterior onescontain more structures but are smaller.


initial activation and a later transformation. Activationmeans a simple induction of neural tissue, and withoutfurther stimulus this will lead to forebrain. Transform-ation represents the further stimulus required to inducemore posterior structures and, because of the numberof such structures, it would have to represent a complexsignal of a graded character, or some graded set ofstates with the high point or maximum number of activecomponents present at the posterior end. The originalreasons for postulating the existence of two separatesteps were rather complex and depend on consider-ations which may no longer be valid today. But two ofthem still seem reasonable: firstly, the volume ofinduced tissue in the fold experiments decreases fromanterior to posterior while the degree of posterioriza-tion increases. This behaviour would not be expectedfrom a single posterior-to-anterior gradient in which thesize of inductions should correlate with the number ofstructures produced (see Slack, 1991, Chapter 3).Secondly, the spread of the neural induction intomediolaterally oriented ectodermal grafts which are incontact with the archenteron roof does not result in aposterior-anterior set of structures but simply a lateralextension of the appropriate AP level, as it does duringnormal development (Albers, 1987).

A comparable "two-signal" model for neural induc-tion was been produced by Yamada (1950, 1990). Herethe activating signal is called "dorsalization" (unfortu-nately this could be confused with dorsalization of themesoderm by the organizer) and the transformingsignal is called "caudalizing". Saxen and Toivonen alsoproduced a two-signal model which was based on theeffects of heterologous inducers on gastrula ectoderm(Toivonen and Saxen, 1955; Saxen and Toivonen,1961).

How many territories?We have seen that there is good evidence for at leastsome regional specificity in the archenteron roof. Theresults of Nieuwkoop's fold experiments and ter Horst'sjacket experiments also show the potential for substan-tial regional specification by interactions within theneural plate. (This is really the "tangential induction"much stressed recently and discussed further below). Sowhat is the balance of these processes in normaldevelopment? It might be that the archenteron roofcontains as few as two specificities, representingroughly the activating and transforming principles ofNieuwkoop. The activating principle might be all overand the transforming principle just in the extremeposterior. The appositional induction would theninduce the neural plate as a whole and also initiate aposteriorizing centre within the neural plate from whichwould propagate a graded signal (or set of states)towards the anterior. All the resulting pattern withinthe neural plate would depend on this signal. At theother extreme it might be that there is an appositionalinduction of all the territories in the neural plate one-to-one by corresponding territories in the archenteronroof. The tangential interactions within the neural platewould not then be a part of normal development, but

only demonstrable in experimental circumstances inwhich the two tissue layers had been separated. We donot know which of these models is nearer the truth andindeed the most likely possibility is that it is somemixture of the two: a certain complexity of pattern inthe archenteron roof would be communicated byappositional induction and then this would becomeelaborated to a more complex pattern by interactionswithin the neural plate.

Preliminary conclusionsThe results reviewed thus far, although some are quiterecent in time, represent a tradition of work stemmingfrom the late 1920s and can fairly be described as "pre-molecular". We shall now briefly summarise theseresults as a model and below we shall ask how well it canstand up to recent molecular data.

The essential features of the model are:(i) Anteroposterior specification occurs during gas-

trulation, not before.(ii) Anteroposterior codings are both transmitted

from the mesoderm to the neural plate and within theplane of the neural plate.

(iii) Both in the mesoderm and in the neural plate,posterior dominates over anterior.

This model is illustrated in Fig. 9. The organizer isformed at the blastula stage in response to a "DV"signal from the dorsovegetal blastomeres. It initially hasa posterior specification but the first cohorts of cells toinvaginate (or involute, in the Xenopus deep marginalzone), acquire an extreme anterior specification, andprogressively later cohorts acquire progressively lessanterior codings. We do not know whether this changeoccurs in response to an environmental change, such asexposure to the blastocoelic fluid, or occurs auton-omously with time. We also do not know to what extentinteractions within the forming archenteron roof arenecessary to establish the territories in the correctsequence or how many such territories there are. As theendo-mesodermal axis is formed, the surroundingectoderm begins to become regionalized in response tosignals emitted from it. The neural plate is formed astwo or more territories appropriate to the AP codings ofthe underlying mesoderm. Further pattern complexityarises through a gradient-like signal emitted from theposterior and transmitted in the plane of the neuralplate. The neural crest is probably also formed as partof this process. The epidermis becomes subdivided intohead and trunk level, the head level embodying acompetence to form the various sensory placodes of thehead (otic, nasal, lens, lateral line) in response to laterinduction from appropriate parts of the neuraxis.

The molecular era

Molecular markersNon-homeobox markers

Along the axial mesoderm there are no qualitativehistological differences except for the prechordal/noto-chord boundary. In the experiments described in the


Fig. 9. Summary of mechanism of anteroposterior specification based on "pre-molecular" work. Arrows representinductions. DV, dorsovegetal territory; VV, ventrovegetal; O, organizer; VM, ventral mesoderm; C, cement glandterritory; O1-O4, territories in dorsal mesoderm; Nl- N4, territories in neural plate; be, blastocoel; arch, archenteron.

previous section, the assessment of anteroposteriorcoding was necessarily based on markers in ectodermalstructures. The forebrain, midbrain, hindbrain andspinal cord are distinguished on the basis of thickness ofdifferent parts of the tube and the disposition of thefibre tracts (Fig. 10). Other commonly used markers arethe eye, the otocyst, cement gland (anura) andbalancers (urodeles). This method of scoring the resultspresents several problems. Firstly, since the visiblemarkers are neural or epidermal they depend oninductive processes after the initial anteroposteriorspecification in the mesoderm. Secondly, the differen-tiation markers appear quite late after the events ofanteroposterior specification themselves; in the case ofurodeles several days of culture may be required.Thirdly, the more subtle distinctions, for examplebetween different parts of the brain, take some practiceto make reliably, especially if the resulting tissue isdisorganized.

For all these reasons, molecular markers for differentanteroposterior levels are highly desirable. In principle

they may be direct, expressed early, and objective.They can be made quantitative, in Northern blots orprobe protection assays, or combined with conventionalhistology by in situ hybridization. Unfortunately it isstill the case that the recently introduced molecularmarkers are predominantly neural or epidermal, so westill cannot observe directly the anteroposterior codingof mesoderm which is probably the leading tissue layerin formation of the anteroposterior pattern.

The disposition of several molecular markers isshown in Fig. 11. Three pan-neural markers have beendescribed: N-CAM (Kintner and Melton, 1987) isexpressed from gastrulation onwards. XIF-6, a homol-ogue of mammalian neurofilament-M, (Sharpe, 1988;Sharpe and Gurdon, 1990) is expressed from the time ofneural tube closure. A glycoprotein identified by amonoclonal antibody 2G9, is expressed from about thesame stage (Jones and Woodland, 1989). XIF3 (Sharpeet al., 1989) is an intermediate filament gene homolo-gous to mammalian peripherin. It is expressed at a lowlevel from the onset of gastrulation in the whole animal

epiphysis

mesencephalon

eye

rhombencephalon

otocyst

cranial ganglia IX,X

telencephalon


nasal placode

notochord

cranial ganglia V—VIII

otocyst

cranial ganglia IX,X

dorsal view ventral viewposterior

rhombencephalon

otocyst

notochord ganglion

mesencephalon

Infundibulum

lens

nasal placode

telencephalon

B anterior

Fig. 10. Structures in urodele CNS commonly used to score specimens with respect to anteroposterior level. (A) Wholebrains. (B) Transverse sections. Adapted from von Woellwarth (1952).

294 J. M. W. Slack and D. Tannahill

em gland \ ^

XCG-1, XAG-1C=I N-CAM, XIF-6, NF-3, 2G9

XIF-3Xhox-3(late)

En-2

XIHbox-1 CZXIHbox-6

I Xhox-3(early)

Fig. 11. Approximate levels ofexpression of molecularmarkers used to studyanteroposterior specification inXenopus. The proportions ofthe axial levels shown areabout what they would be atthe time of neural tube closurealthough the markers are notall necessarily expressed at thisstage.

hemisphere and becomes enriched in various neuralstructures: the cranial ganglia, the cranial neural crest,the hindbrain and motor nuclei along the neural tube.Quantitatively this expression pattern means that itpredominates in the anterior. Other neural-specificantibodies have been described by Itoh and Kubota(1989). Numerous pan-epidermal markers have beendescribed (eg Slack, 1985; Akers et al., 1986; London etal., 1988) and a number of markers specifically for thecement gland (Jamrich and Sato, 1989; Sive et al.,1989).

Homeobox expression patternsIn Drosophila the activation of the homeobox genes ofthe Antennapedia and Bithorax complexes is the laststep in a long and complex process of anteroposteriorspecification which starts with the maternal factorsbicoid and nanos and works through the zygotic gap andpair-rule genes (reviewed Slack, 1991). Other insects,such as Tribolium, have a single HOM gene clustercontaining genes homologous to both the ANT-C andBX-C (Beeman et al., 1989). It is thought that thisrepresents the primordial condition with the complexhaving become split in Drosophila. As is well known,the order of the genes on the chromosome is the sameas the order of expression in the embryo, with the genescentromere-proximal in the cluster being expressed inthe anterior of the germ band and the genes centro-mere-distal in the cluster being expressed posterior.

We now know that vertebrates possess clusters ofhomeobox genes (HOX clusters) showing a distincthomology to the ANT-C/BX-C of Drosophila (or HOMcomplex of other insects) and that for each cluster thereis also the correlation between gene position andanteroposterior level of expression such that theanterior limit of expression in the neural tube correlateswith the order of the genes from the 3' to the 5' end ofthe cluster (Duboule and Dolle", 1989; Graham et al.,1989). The different HOX clusters in vertebratesappear to have arisen from the primordial HOM clusterby gene duplication, and the corresponding members ofdifferent clusters have been called paralogs. There are

also a number of genes outside the HOX clusters whichcontain recognisable homeoboxes with a lower degreeof amino acid identity and presumably diverse func-tions. A recent review (Shashikant et al., 1991)provides extensive discussion on the molecular biologyand expression patterns of the homeobox genes,particularly in the mouse, and this material will not berepeated here.

A number of homeobox genes have been cloned fromXenopus over the last few years and those published todate are collected in Table 1, which also lists theirrelationship to the mouse genes. In general the 3'-anterior: 5'-posterior correlation seems to hold up forXenopus, although the expression data is somewhatincomplete and some genes are known to have havedifferent expression patterns at different developmen-tal stages.

Are homeobox genes coding factors?From the discovery of the first homeobox gene outsideDrosophila (Carrasco et al., 1984), there has been astrong presumption that the HOX clusters represent akind of biological Rosetta stone that would enable us toread the epigenetic code of the vertebrates (Slack,1984). Evidence for this view has steadily accumulated(De Robertis et al., 1989, 1991). The expressionpatterns (Shashikant et al., 1991) are, to a firstapproximation, what would be expected if genes wereturned on at different concentrations of a posterior-to-anterior morphogen gradient. So each gene has a sharpanterior expression limit and its domain of expressionextends from there to the posterior end. Expression ofthe same gene is frequently found in both mesodermand neural tube suggesting an association with antero-posterior level rather than tissue type.

More direct evidence comes from experiments inwhich homeobox gene expression has been caused tooccur ectopically or has been reduced below normal.Ectopic expression in Xenopus is achieved by injectionof a synthetic mRNA prepared from a vector bearing aphage RNA polymerase promoter and, usually, variousfeatures to optimise stability and translational efficiency


Table 1.

Name

putativemouse

Synonyms homologue Normal expression pattern References

HOX cluster genes (listed approximately in anteroposterior sequence of expression domains)Xhox.lab 1.6 Up late gastrula in ectoderm and mesoderm.XhoxlA 2.6 Up from early gastrula. Trunk-tail axis,

mainly somitesXlHbox 4 Xhox IB 2.1 Up from late neurula, peak in tailbud stage.

Xhox 2.1 Stripe in hindbrain.

Sive and Cheng, 1991Harvey et al., 1986

Harvey et al., 1986Fritz and De Robertis, 1988Jegalian and De Robertis, 1990

XlHbox 5XlHbox 1

XlHbox 2

XlHbox 7XlHbox 6

XlHbox 3

3.4ACl, Xebl 3.3

MM3 2.3

2.42.5

Xhox 36

Other homeogenes (listed approximately inMixGoose coidXhox 3

Xhox 7.1

En 2

XlHbox 8

Evx

7.1

En 2

Up late neurula, down tailbud stage.Up late gastrula, spinal cord, and post.mesoderm. In tadpole, cervical spinal cordand adjacent mesoderm. Two proteins.

Ovary. Up early gastrula.

Up neurula, band in spinal cord.Up early neurula, post, neural plate.

Up gastrula, peaks late neurula, post, ectoand mesoderm.

temporal order of expression)Up late blastula in endo and mesoderm.Up late blastula, in dorsal lip.Up early gastrula, posterior mesoderm. Intailbud, ant. neural crest and ventrolat. cellsfrom midbrain-ant.sp.cord.

Up gastrula in dorsal mesoderm of whole AP axis,later in neural tube, neural crest, lateral plate.

Up early neurula, stripe at mid-hind brainjunction.

Up tailbud stage as band in endoderm level ofsomite 3-5.

Fritz and De Robertis, 1988Carrasco and Malacinski, 1987Oliver et al., 1988

Milller et al., 1984Fritz and De Robertis, 1988Fritz and De Robertis, 1988Sharpe et al., 1987Wright et al., 1990Condie and Harland, 1987Fritz and De Robertis, 1988

Rosa (1989)Blumberg et al., 1991Ruiz I Altaba and Melton, 1989aRuiz i Altaba, 1990

Su et al., 1991

Hemmati Brivanlou and Harland, 1989Hemmati Brivanlou et al., 1991Wright et al., 1988

(Moon and Christian, 1989). Overexpression of Xhox1A, normally expressed in the somites, causes adisruption of somite segmentation (Harvey and Melton,1988). Overexpression of Xhox-3, normally expressedearly on in the posterior, causes a loss of anterior parts(Ruiz i Altaba and Melton, 1989c). Overexpression ofXlHbox 1, initially expressed in the posterior and laterin the cervical region of the spinal cord, also disruptssegmentation and CNS pattern (Wright et al., 1989).There are also some relevant data on this question fromthe mouse. Overexpression of Hox 1.1, normallyexpressed in the thoracic region spinal cord andsomites, causes malformations in the hindbrain region(Kessel et al., 1990). This dominant posteriorizationwould be expected if the region modified normallydiffered only in that it lacked Hox 1.1 expression.Several groups are currently attempting to ablate theactivity of HOX genes by homologous recombination ofinactive gene copies into ES cells followed by thecreation of germ line chimeras, and hence a strain ofheterozygous mice from which the homozygote em-bryos may be produced by mating. An early exampleconcerns Hox 1.5 (Chisaka and Capecchi, 1991) whichis normally expressed in the neural tube anterior to theotocysts and in the prevertebral somites. The homo-zygous nulls have defects in the branchial arch region.A cheaper but less direct way of altering HOX geneexpression in mouse embryos is to administer retinoicacid to the mothers at 7-8 days of incubation. This

causes ectopic (anterior) expression of some HOXgenes and repression of others and also leads to avariety of vertebral transformations (Kessel and Grass,1991). Of course in this case it cannot be guaranteedthat the retinoic acid is actually acting via the HOXgenes.

A more sophisticated variant of the overexpressionexperiment is to overexpress in one embryo and use thisas the donor of a graft into a normal embryo. Cho et al.(1991) have overexpressed XlHbox-6 (normally pos-terior) in blastulae and grafted animal caps into theblastocoel of normal hosts by the Einsteckung pro-cedure (Fig. 4A). This produces ectopic tails in whichmost of the mesoderm is graft derived and most of theneural structure and fin is host derived. Although thereare many objections to the Einsteckung procedurewhich have been listed above, it is quite clear that in thisexperiment the XlHbox-6 mRNA has a dramaticbiological activity, and there is at least a prima faciecase that, directly or indirectly, it confers posteriorcharacter on the graft.

Ablation of a Xenopus homeobox gene has beenattempted by Wright et al. (Wright et al., 1989) using anantibody to XlHbox 1. This does appear to transformthe cervical cord into a more hindbrain-like structure,the predicted result of the ablation of a gene whosefunction was to distinguish the hindbrain from theanterior cord. Here, however, the uncertainty iswhether the antibody actually inhibits the biochemical


activity of the gene product, since there is no indepen-dent assay for this.

At present, the evidence from Xenopus and from themouse that HOX cluster genes really code for antero-posterior levels is suggestive and encouraging, althoughthe case remains not fully proven.

Reinvestigation of problems using molecular methodsMesodermal induction and axial patterning

The problem of the anteroposterior axis intersects withthat of mesoderm induction because in amphibianembryos the gastrulation movements cause the antero-posterior and dorsoventral axes to be intimatelyassociated with each other, and not independent as theyseem to be in Drosophila.

The normal fate map of Xenopus shows that theentire dorsal midline arises from the dorsal marginalzone. However, quantitatively there is a preferentialcontribution of cells from the dorsal marginal zone tothe anterior of the later embryo and from the ventralmarginal zone to the posterior. In fact the dorsal lipregion forms both dorsal and ventral sides of theanterior. Tissue from the ventral marginal zone doesnot extend more than half way up the body from theblastopore in the ventral midline but makes a substan-tial contribution to the somites of the posterior trunkand tail (eg Dale and Slack, 1987). Because of thisnormal projection of tissues during gastrulation, treat-ments that alter the proportions of the marginal zonecircumference devoted to dorsal and ventral typemesoderm have consequential effects on the anteropos-terior axis. So eggs treated by irradiation of the vegetalhemisphere with ultraviolet light, which reduces theproportion of organizer tissue, later come to developwith anterior deficiencies (Malacinski et al., 1975;Scharf and Gerhart, 1983). Conversely, embryostreated with lithium at cleavage stages, which increasesthe proportion or organizer tissue, later come to haveposterior defects (Kao et al., 1986; Kao and Elinson,1988). The morphological findings have been reinforcedby study of molecular markers: thus UV embryosexpress elevated levels of posterior markers such asXhox 3 (Ruiz i Altaba and Melton, 1989a) whereaslithium embryos express elevated levels of anteriormarkers such as En-2 and XIF 3 (Hemmati Brivanlouand Harland, 1989; Sharpe et al., 1989).

The use of molecular markers that are normallyexpressed in restricted domains along the anteropos-terior axis has suggested that the cytokines provisio-nally identified as mediating mesoderm induction caninfuence patterning of the anteroposterior axis. Animalcaps treated with activin tend to behave in an anteriorway at later stages while those treated with FGF tend tobehave in a posterior way. For example, animal capstreated with FGF display increased expression of twoposterior markers, Xhox 3 and XlHbox 6 (Cho and DeRobertis, 1990; Ruiz i Altaba and Melton, 1989b),whereas activin-treated caps show increased expressionof a more anterior marker, XlHbox 1 (Cho and DeRobertis, 1990). The most dramatic demonstration ofanterior and posterior properties conferred by cytokine

treatment involves Einsteckung experiments withtreated animal caps (Ruiz i Altaba and Melton, 1989b).In these experiments activin-treated animal caps con-tribute to and induce secondary heads while FGF-treated caps contribute to and induce tails.

If we accept, as shown above, that there is not aspecification of territories for particular anteroposteriorvalues in the blastula, then how can these results beexplained? Perhaps the way to think of the treatedanimal cap is as a kind of "engine" for making bodyparts. The cap is not homogeneous and we know thatonly the exposed, formerly blastocoelic, layer of cellsinitially responds to the cytokines (Darlington, 1989).After the cap rolls up the induced cells are in a clump atone end of the ball with uninduced cells elsewhere.Activin-induced caps undergo a much more pro-nounced and earlier elongation than FGF caps, so itmay perhaps be the secondary interactions occuring indifferently shaped domains which bring about theanteroposterior differences. As far as Einsteckungexperiments are concerned, there is plenty of scope forfurther interactions to occur between the implanted capand the ventral mesoderm, the pharyngeal endodermand the ventral epidermis.

Neural inductionThe textbook picture of neural induction is of anappositional induction between the invaginating pro-spective archenteron roof and the overlying ectoderm,and it is tacitly assumed in anteroposterior studies thatthere is a one-to-one regional specificity of theinduction, each structure in the neural plate beinginduced by a corresponding territory in the mesoderm.Three issues that have received particular attentionrecently are the nature of the neural inducing signals,the existence of a neural "bias" in the dorsal part of theanimal hemisphere, and the importance of "tangential"as opposed to appositional neural induction.

Neural inducing factorsAlthough it is widely known that urodele gastrulaectoderm can be provoked to forming neuroepitheliumby a variety of stimuli, this is not the case for Xenopus.Neural structures are often induced by the activin groupof inducing factors but this is always in association withaxial mesoderm and is assumed to be secondary,resulting from neural induction within the explant. Sothere are still no candidates for the extracellularmediators of neural induction. It is however thoughtthat the process is mediated within the responding cellsby an activation of protein kinase C (PKC) togetherwith an elevation of cyclic AMP. A limited degree ofneural induction in dorsal gastrula ectoderm can beprovoked by phorbol esters (Davids et al., 1987) andthis is correlated with an activation of PKC (Otte et al.,1988). A more effective response can be obtained by acombination of phorbol ester with cAMP (Otte et al.,1989) suggesting that neural induction may involve theactivation of more than one second message pathway.

The cement gland is a structure derived from theectoderm just anterior to the neural plate. It appears to


be induced at the same time as. the neural plate since thefirst molecular markers appear at stage 12 (Jamrich andSato, 1989; Sive et al., 1989). It has been known forsome time that the cement gland, but not neuroepi-thelium, can be induced in ectoderm explants bytreatment with ammonium chloride (NH4CI; Picard,1975). Presumably this works by raising the intracellularpH, an event which, like activation of PKC or elevationof cAMP, is a familiar second message response in cellbiology.

Dorsal biasIn the past it has often been assumed, without verygood evidence, that the competence of the animalhemisphere of the blastula/gastrula is uniform. We nowknow that at least in Xenopus there is some differencebetween dorsal and ventral regions of the animalhemisphere. This is shown by greater induction of N-CAM and XlHbox-6 in dorsal compared with ventralectoderm of gastrulae using the same dorsal mesodermas inducer (Sharpe et al., 1987). Conversely theepidermal marker Epi 1 is expressed better in isolatedventral compared with dorsal ectoderm (London et al.,1988; Savage and Phillips, .1989). Similar results havealso been obtained for mesoderm induction: activin-treated dorsal ectoderm from blastulae forms dorso-anterior structures whereas similarly treated ventralectoderm does not (Ruiz i Altaba and Jessell, 1991;Sokol and Melton, 1991). The molecular basis for thesedifferences in competence are not yet known, althoughit has been shown that different isozymes of PKCpredominate in the two regions (Otte et al., 1991).

These results do show that the dorsal ectoderm hassome predisposition to form axial mesoderm or neuralplate but they do not show that there is any anteropos-terior specificity within the sensitive region. For thereasons discussed above we would not think this likely.It must also be remembered that the pre-disposition ofventral ectoderm in favour of epidermal differentiationcan be over-ridden in certain experimental manipu-lations, and this tissue can be caused to form either axialmesoderm or neural plate.

Tangential inductionAn issue that has been raised in a rather sharp way bythe use of molecular markers is the possible contri-bution of "tangential" neural, induction to the forma-tion of the neural plate. Insofar as this involves a spreadof the induced state through the ectoderm this conceptresembles the older one of "homeogenetic induction",now known to occur in Xenopus (Grunz; 1990; Itoh andKubota, 1991). But "tangential induction" also impliesthat the initial signal from the mesoderm is passed in theplane of the tissue before gastrulation rather thanbetween the archenteron roof and the ectoderm duringgastrulation. The classical experiments with urodelessuggested that this process was not of significantimportance in normal development. For example theexplantation studies on Cynops, shown in Fig. 7, do notshow neural specification occurring in dorsal ectodermbefore it has been underlaid by archenteron roof. The

original exogastrula experiments of Holtfreter likewiseshowed that the ectodermal part of the exogastrula wasentirely epidermal rather than neural (Holtfreter,1933). However, explants from the dorsal animal regionof Xenopus blastulae can form neural tissue in theabsence of mesoderm (JS unpublished) and also theexpression of the epidermal marker Epi 1 is partiallysuppressed in such explants (London et al., 1988). Thissuggests that the situation in Xenopus may be different.

The proposal that neural induction can arise throughthe influence of tangential signals that spread throughthe plane of the ectoderm results from several types ofstudy. In Xenopus exogastrulae there does seem to besome neural development in the ectodermal sac. It hasbeen shown that the pan-neural markers N-CAM andNF-3, and the anterior neuronal marker Xhox 3 (not tobe confused with its incarnation as a posterior mesoder-mal marker at earlier stages!) are expressed (Kintnerand Melton, 1987; Dixon and Kintner, 1989; Ruiz iAltaba, 1990). Another way of looking at the putativetangential signal is to dissect explants consisting of earlygastrula ectoderm with dorsal marginal zone attached,culture them with blastocoelic surfaces apposed to stopthem rolling into balls ("Keller sandwiches": Keller etal., 1985) and then assay for neural markers. Exper-iments of this type have shown that N-CAM and NF-3are expressed, suggesting that the involution of dorsalmesoderm is not required for neural induction (Dixonand Kintner, 1989). These results suggest that weshould take seriously the idea that neural inductiondoes proceed to some extent tangentially in Xenopusand probably that neural patterning depends both onsignals from the archenteron roof and on those in theplane of the neural plate.

Tangential induction presumably occurs in responseto a signal from the newly induced axial mesoderm. It isunlikely to be the same as the mesoderm-inducingsignal from the vegetal cells because we would thenhave to suppose that neuralization represented theresponse to a weak (just above threshold) dorsal-mesoderm-inducing signal, and this is at variance withall the in vitro studies using cytokines.

Regional specificity of neural inductionWe have seen above that there is good evidence for adegree of regional specificity in neural induction. Thisconclusion has been generally supported by studiesusing molecular markers. For example Hemmati Bri-vanlou et al. (1990) showed that anterior notochordcould induce expression of the En-2 protein (midbrain-hindbrain boundary) more effectively than posteriornotochord while both regions induced N-CAM to asimilar extent. As we shall see below it is significant thatsome En-2 is induced by the posterior notochord. In thisstudy UV-irradiated embryos were used as ectodermdonors to remove the neural bias normally found on thedorsal side.

Sharpe and Gurdon (1990) have carried out a rathercomprehensive study employing the three markers XTF-6 (pan-neural) XIF-3 (anterior) and XlHbox-6 (pos-terior). As we would expect from the classical work,


neural specification, measured by synthesis of XIF-6 inexplants, occurs progressively from posterior to an-terior during gastrulation. The competence of ectodermto respond to dorsal mesoderm is lost by stage 14 (earlyneurula) for both anterior and posterior markers. Whencombinations are made of ectoderm with anterior orposterior archenteron roof XIF-3 is expressed in bothcases. XlHbox-6 is only expressed in the combinationcontaining the posterior mesoderm. The expression ofboth XIF-3 and XlHbox-6 in the posterior combinationis reminiscent of Nieuwkoop's fold experiments andrepresents support for the idea of posterior dominance.

As we have seen above, the induction of the cementgland is a process which seems to be allied to neuralinduction in general, although it differs in beingmimicked by NH4C1. A study by Sive et al. (1989)showed that there was a transient specification ofprospective neural plate to become cement gland. Inother words, when explants were taken at differentstages of gastrulation, the early region which self-differentiated cement gland later came to lie posteriorto the position of the cement gland. Further, whencombinations of gastrula ectoderm were made withdifferent parts of the archenteron roof, the region mostactive at inducing cement gland lay somewhat posteriorto the final cement gland position. This is exactly theeffect observed, but not really discussed, by Mangold(1933), ter Horst (1948) and indeed also by Sharpe andGurdon (1990).

To summarise: inductions in archenteron roof:ecto-derm combinations are more anterior than would beexpected from the normal fate of the mesoderm used.This is consistent with the idea of posterior dominancebecause a given level is able to induce structures moreanterior to itself but not those of its own level or moreposterior. It also implies the need for an additionalstimulus, perhaps the tangential signal, to achieve thedegree of posteriorization observed for each level innormal development.

Retinoic acidIf whole blastulae or gastrulae are incubated in retinoicacid then the resulting embryos are found to betruncated at their anterior ends (Durston et al., 1989;Sive et al., 1990- Ruiz i Altaba and Jessell, 1991). Athigh doses (10 M) forebrain- and midbrain-derivedstructures are lost and the hindbrain is disrupted, whileat low doses (10~7M) there is just a slight anteriorreduction. The period of sensitivity extends from theegg to the neurula, and exposure as short as 30 minutesat the blastula stage can produce truncations. Asexpected, the expression of a number of anteriorspecific genes is abolished in the most severelytruncated embryos (cement gland genes XCG-1 andXAG-1, anterior ectodermal gene XA-1 and theanterior neural homeobox gene En-2) (Sive et al.,1990).

Retinoic acid can also affect homeobox gene ex-pression in human embryonal carcinoma cells such thatit activates genes in the HOX-2 complex in a dose-dependent manner (eg Simeone et al., 1990). Low

concentrations of retinoic acid are sufficient to activategenes 3' (anterior) in the complex whereas highconcentrations are required to activate the 5' (pos-terior) genes. Given that Xenopus embryos are knownto contain endogenous retinoic acid (at a concentrationof 1.5 x 10"7M) (Durston et al., 1989) then theseresults might suggest that Xenopus embryos contain anendogenous gradient of retinoic acid from posterior toanterior which controls the body plan by activatingHOX genes in a sequential manner at differentanteroposterior levels. The addition of exogenousretinoic acid would then lead to the activation ofposterior genes at more anterior positions than normal,resulting in the observed transformations. Althoughthis model (Green, 1990) has an attractive simplicity,there are a number of problems. As far as the effects onHOX genes is concerned, the Xenopus data is notentirely consistent with the model. For example, whydoes the expression of XlHbox-6, which is moreposterior than XlHbox-1, increase sixfold on treatmentof whole embryos with retinoic acid whereas theexpression of XlHbox-1 remains unaffected (Cho andDe Robertis, 1990)? Why does XlHbox-3 (trunk-tail)remain constant while XlHbox-6 (trunk-tail) and XEF-3(head) increase (Sive et al., 1990)? Why is XlHbox-6not turned on at all in anterior explants (Sharpe, 1991) ?

The effects on the forebrain and rostral midbrain arenot found in other vertebrates and at least some of thisis probably due to a perturbation of gastrulationmovements. Although the anteroposterior extent of theinvaginated mesoderm is approximately normal, thereis a persistence of the blastocoel and failure of thearchenteron to expand fully (Sive et al., 1989).However there is also an apparently direct posterioriza-tion of the neural structures in ectoderm-mesodermcombinations based on a reduction of eyes (Durston etal., 1989) and of anterior molecular markers (Sive et al.,1989). Retinoic acid can suppress anterior differen-tiation in hitherto neuralized ectoderm dissected from amid-gastrula embryo (Durston et al., 1989) so this effectis probably a direct one on the neurectoderm ratherthan via the inducing capacity of the mesoderm.However additional effects on the mesoderm cannot beexcluded (Ruiz i Altaba and Jessell, 1991; Sive andCheng, 1991). In the anterior hindbrain, a detailedneuroanatomical study of treated embryos revealed acompression and disruption of structures rather than acoordinate posterior transformation (Papalopulu et al.,1991).

All the studies reveal defects in the tail as well as thehead but these are not made much of. UV irradiation offertilized eggs also reduces the tail but this is under-standable in terms of reduction of the amount oforganizer tissue, destined as it is to form the dorsalmidline of the whole body. In the case of retinoic acidthe tail reductions can arise from quite late treatments(stage 16-18: Ruiz i Altaba and Jessell, 1991) whichsuggest more of a specific toxic effect.

Ellinger-Ziegelbauer and Dreyer (1991) have shownthat in the neurula at least one retinoic acid receptor(RAR gamma) is preferentially localized at both


anterior and posterior ends of the body: in the anterior,it is expressed mainly in the prospective pharyngealendoderm/prechordal mesoderm, and in the posteriorin both the mesoderm and the overlying neural plate.The expression patterns of other retinoic acid receptorshave not yet been published, but are under activeinvestigation in several laboratories.

So at present it seems likely that the effects of retinoicacid are quite complex. There may be some shift ofepigenetic coding, particularly in the forebrain andmidbrain, but there is also some disruption of morpho-genetic movements and some toxicity which contributeto the overall morphological syndrome.

ConclusionsThis review will have shown that specification of theanteroposterior axis in vertebrates is a very complexprocess and probably occurs in a number of steps, eachof which involves different inductive signals andresponses. This should not really be surprising: Dros-ophila is very complicated and whether or not elementsof the mechanism are really homologous, vertebratesare not really going to be any simpler than Drosophila).

The pre-molecular work does provide us with a lot ofdata. Unfortunately nearly all of it refers to urodelessuch as Triturus and Cynops and how many of theconclusions hold up for Xenopus remains to beestablished, particularly on the critical issues of theacquisition of anteroposterior coding on imaginationand of posterior dominance. Xenopus has now becomethe world standard amphibian embryo and if urodelespecies are to retain their usefulness then someone willhave to generate a set of molecular markers for them.

However, if we can legitimately pool the pre-molecular and the molecular, then we are left with apicture not dissimilar from that already summarisedabove and shown in Fig. 9. It looks as though some sortof anteroposterior pattern is set up in the mesodermduring gastrulation and that the posterior end isdominant over the anterior. How complex this meso-dermal pattern is in terms of number of distinctterritories we do not know, although it must be at leasttwo. The pattern is then transmitted to the ectoderm ina neural induction process probably also involvingreciprocal effects of neural plate on archenteron roof.Further interactions probably occur within the neuralplate, also with posterior dominance to give the finalpattern.

The main innovations of the molecular era have beenthreefold: the introduction of molecular markers, thediscovery of the HOX cluster genes, and the introduc-tion of pure substances which can modify commitmentin a defined way (FGF, activin, retinoic acid). Molecu-lar markers are a good idea in principle, but have so farnot really extended the precision of what we canobserve by conventional histology. This is mainlybecause too many of the markers are in the CNS andderivatives of the epidermis and too few in themesoderm. However we can now at least think aboutthe possibility of observing formerly cryptic subdiv-

isions in the mesoderm and observing them soon afterthey are set up. This is progress.

After some years of uncertainty, the HOX clustergenes look like they really do represent the Rosettastone of regional specification. Further study of theirstructure, expression and regulation will undoubtedlyadvance our understanding of anteroposterior specifi-cation. The fact that more HOX genes are on in theposterior than in the anterior provides a simplemolecular correlate with the principle of posteriordominance, deduced from the embryological exper-iments. But vertebrates and insects have evolved a longway apart and even if the same overall system ofanteroposterior codings has been preserved, it does notcurrently look as though precise homologies can bedrawn between particular segments or structures ininsects and particular structures in vertebrates.

The effects of mesoderm-inducing factors and ofretinoic acid are rather baffling at present. If activinreally mimics the induction of the organizer then thisshould be capable of forming the whole of the axisrather than just the head. If posterior dominates overanterior then why do the highest doses of activin induceheads? The FGFs induce ventral rather than posteriorstructures in vitro and it probably needs the complexenvironment provided by the Einsteckung protocol forFGF-treated caps to produce high yields of tails. Theeffects of retinoic acid seem to be predominantly, butperhaps not entirely, on the ectoderm rather than themesoderm. The simple model of a posterior-to-anteriorgradient turning on the homeobox genes in 5' to 3'sequence is immensely appealing but the details of thedata do not really bear it out.

There is still some way to go before we understandvertebrates as well as we do Drosophila. But once againthe problem is in the forefront of scientific endeavour,and the tools are available for significantly extendingour view derived from the pre-molecular era.

We are grateful to Peter Holland, Jim Smith, VinceCunliffe, Robb Krumlauf and John Heath for reading andcommenting on various versions of this paper.

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{Accepted 29 October 1991)

mechanism of anteroposterior axis specification in vertebrates · but vertebrates differ from...

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