Determination and morphogenesis in the sea urchin embryo · urchin eggs forms one of the...

Development 100, 559-575 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

Review Article 559

Determination and morphogenesis in the sea urchin embryo

FRED H. WILT

Department of Zoology, University of California, Berkeley, CA 94720, USA

Summary

The study of the sea urchin embryo has contributedimportantly to our ideas about embryogenesis. Thisessay re-examines some issues where the concerns ofclassical experimental embryology and cell and mol-ecular biology converge. The sea urchin egg has aninherent animal-vegetal polarity. An egg fragmentthat contains both animal and vegetal material willproduce a fairly normal larva. However, it is notclear to what extent the oral-abpral axis is specifiedin embryos developing from meridional fragments.Newly available markers of the oral-aboral axis allowthis issue to be settled. When equatorial halves, inwhich animal and vegetal hemispheres are separated,are allowed to develop, the animal half forms aciliated hollow ball. The vegetal half, however, oftenforms a complete embryo. This result is not in accordwith the double gradient model of animal and vegetalcharacteristics that has been used to interpret almostall defect, isolation and transplantation experimentsusing sea urchin embryos. The effects of agents usedto animalize and vegetalize embryos' are also duefor re-examination. The classical animalizing agent,Zn2+, causes developmenal arrest, not expression ofanimal characters. On the other hand, Li ' , a vegeta-lizing agent, probably changes the determination ofanimal cells. The stability of these early determinativesteps may be examined in dissociation-reaggregationexperiments, but this technique has not beenexploited extensively.

The morphogenetic movements of primary mesen-chyme are complex and involve a number of interac-tions. It is curious that primary mesenchyme isdispensable in skeleton formation since in embryosdevoid of primary mesenchyme, the secondary mes-enchyme cells will form skeletal elements. It is likelythat during its differentiation the primary mesen-chyme provides some of its own extracellular micro-environment in the form of collagen and proteogly-cans. The detailed form of spicules made by primarymesenchyme is determined by cooperation betweenthe epithelial body wall, the extracellular materialand the inherent properties of primary mesenchymecells.

Gastrulation in sea urchins is a two-step process.The first invagination is a buckling, the mechanism ofwhich is not understood. The secondary phase inwhich the archenteron elongates across the blastocoelis probably driven primarily by active cell repacking.The extracellular matrix is important for this repack-ing to occur, but the basis of the cellular-environ-mental interaction is not understood.

There are new tools, especially well-defined specificantibodies and recombinant DNA clones, that may beapplied to these problems and should help illuminatesome of the underpinnings of embryogenesis in anorganism that relies so heavily on cellular interactionsas a develomental strategy.

Key words: sea urchin, determination, axis, gastrulation.

Introduction

The subject of this essay is to explore some selectedaspects of determination and morphogenesis in thedeveloping sea urchin embryo. Some explanation isnecessary to justify this undertaking. Many readerswill know that there are a number of reviews, somevery recent, that consider various aspects of thedevelopment of sea urchin embryos. Although there

are some papers on various aspects of sea urchindevelopment that have just appeared or are in pressand therefore do not figure in any of these accounts,most of what I shall discuss has been cited by others.Rather than presenting a review, I propose to recon-sider and re-examine some important older exper-iments and some more recent information bearing onthe same problems, in the hope of highlighting whatopportunities and challenges there are for the student

560 F. H. Wilt

of development. There are limitations of space as wellas my own limitations and I shall restrict consider-ation to two broad issues: the determination of earlylineages in the sea urchin embryo, especially the roleof cell interactions in establishing the overall patternof the embryo; and the cellular and molecular basesof gastrulation which lead to the formation of theorgans of the pluteus larva.

Practitioners of molecular biology, cellular biologyor embryology tend to have their own sets of biases orassumptions particular to a given field. One mayoccasionally detect a hint of arrogance among adher-ents of one approach or another. My own personalbias is that the time is ripe for a concerted applicationof the joint tools and approaches of cell and molecu-lar biology and experimental embryology to problemsof development. I believe that nowhere is this moreapparent than in the developing sea urchin.

Though most biologists realize that the sea urchinembryo has had an important role in the history ofphysiology, genetics and even molecular biology, it isnot so well remembered that information from thesecreatures played a crucial role in some present ideasabout embryology, per se. It was here that thedifference between the prospective fate and potencyof a cell was clearly understood and underlined(Horstadius, 1939). Our ideas and language about thenature of determination and commitment of cellslargely come from work on sea urchins and amphib-ians. It is well known that the ideas of gradients thatled to modern ideas on positional information werefirst applied in a detailed way to sea urchin embryos(Runnstrom, 1929). Some of the first penetratinganalyses of the cellular basis of morphogenetic move-ments were carried out in sea urchins (Gustafson &Wolpert, 1967).

The ability to isolate mutants in C. elegans andD. melanogaster affords great power in the analysis ofdevelopment. On the other hand, development inthese two organisms depends heavily on stereotypedcytoplasmic-nuclear interactions to establish lin-eages. This contrasts with the situation in sea urchinsand amphibians where cell interactions are exten-sively used to found lineages and establish pattern.Sea urchins, like amphibians, are accessible for analy-sis of the cellular basis of morphogenesis. Whilestandard diploid genetic analysis is not presentlypractical in sea urchins (Hinegardner, 1969), thepower of paragenetic analysis afforded by recombi-nant DNA technology is now applicable (Flytzanis etal. 1985). It is in the area of the mechanisms of theorigin of lineages and the role of cell interactions inthis process, as well as the relationship of changingcell phenotype to morphogenesis, that the study ofthe sea urchin embryo may be able to make generalcontributions to the study of development.

The reader may wish to consult some excellentreviews of different aspects of development of seaurchins. In addition to Horstadius' masterly treat-ment of the classical embryology of the sea urchinembryo, (1939, 1973), there are excellent analyses ofclassical work in the book edited by Czihak (1975),especially the chapter by Okazaki (1975). The thirdedition of Wilson's 'The Cell in Development andHeredity' (1925) is still a valuable source. The orig-inal (1973) and new installment of the book byGiudice treats the literature in detail (1986). Solurshhas written an excellent account of morphogenesisand cell motility (1986), and Gustafson & Wolpert'sstimulating analysis of morphogenesis (1967) treatsmany problems. Spiegel & Spiegel (1986) and McClay(1987; McClay & Ettensohn, 1987) have also writtenrecent reviews of selected aspects of morphogenesis.Eric Davidson and his colleagues have publishedseveral reviews on gene expression in early sea urchindevelopment (Angerer & Davidson, 1984); the newthird edition of 'Gene Expression in Early Develop-ment' by Davidson (1986) treats both molecularbiological and embryological issues in sea urchindevelopment in a penetrating way.

Determination

Cell interactions, embryo patterning and earlydetermination of cell lineage

The study of cell determination and patterning of theembryo has been dominated by the double gradienttheory of Runnstrom (1929). These ideas, derived inlarge part from experiments and theories of Child(see Child, 1940, for a review), have a strikinglymodern ring, though they lack the mathematicalfoundation of newer theoretical work (e.g. Mein-hardt, 1982) and lack the insight of Lewis Wolpert(1971) that gradients have to be 'interpreted' by thecells of the embryo if they are to amount to anything.

What Runnstrom proposed was that the early seaurchin egg and embryo were pervaded by two oppos-ing gradients; the one was a graded potentiality toform skeleton, gut, muscular elements and coelom,the so-called vegetal characteristics. This potentialitywas strongest and localized near the vegetal end ofthe egg, a region that could be identified in P. lividusbecause of a subequatorial pigment band in the cortexof eggs from some females of this species. The othergradient favoured a tendency to form very long ciliaopposite the site of blastopore formation. Someanimal halves will also form a thin epithelium typicalof gastrulae, a ventral ciliary band and sometimes astomodeum. These animal characters, especially thelong cilia and lack of internal organs, are somewhatdifficult to define in terms of specific differentiated

Determination and morphogenesis in the sea urchin embryo 561

characteristics. Nonetheless, the idea of Runnstromthat the two gradients of potentiality, animal andvegetal, run in opposite directions to one another,somehow interact and are occasionally 'mutuallyhostile' to one another, has been the basis forinterpretation of almost all subsequent experimentsin this area. The brilliant transplantation experimentsof Horstadius (1973) showed clearly that interactionsbetween blastomeres may cause spectacular trans-formation in cell fate; they have been primarilyinterpreted as a result of interaction of the twogradients.

Let us briefly look again at the evidence that theegg and early embryo have an inherent organizationthat specifies patterns; we shall examine both theoral-aboral axis and the animal-vegetal axis. Theevidence for a graded potential of development in seaurchin eggs forms one of the foundations of classicalexperimental embryology. If egg fragments or blasto-meres derived from eggs by cleavage are separatedalong the animal-vegetal axis, resulting in meridionalfragments, the separated parts form embryos withmore or less equal and complete representation of themajor organ systems of the embryo, consisting ofciliary tufts, skeleton, gut, coelomic rudiments andsurface epithelium. The result of the experiment isshown in cartoon fashion in Fig. 1A.

However, the total and equal developmental po-tential of meridional fragments or blastomeres is notreally securely established. The early experiments ofthis kind by Boveri (1901, 1918), Driesch (1894) andHebst (1900) did not examine blastomeres and frag-ments derived from a single egg, the result onlyshowing that a portion of an egg could give rise to alarva. In 1927, Plough reported that often one or bothmembers of a pair of isolated blastomeres from a2-cell stage developed poorly, slowly or abnormally.Horstadius & Wolsky (1936) also observed this; usingboth Lytechinuspictus and S. purpuratus, we have alsofrequently observed in our laboratory that bothblastomeres of the 2-cell stage may not each give riseto a perfect pluteus (unpublished; Maziu, 1958).Marcus (1979) carefully analysed development ofseparated blastomeres of A. punctulata at the 2-cellstage, in which about 60% survived to pluteus ascomplete pairs. Of the 10 % of the separated blasto-mere pairs that survived metamorphosis, a largeproportion showed abnormalities in size and in anat-omy of the ocular, genital and periproct plates of themature urchin.

If there is any predisposition to have localizeddevelopmental potential along an oral-aboral axis,this would be revealed by development of isolatedblastomeres or fragments from the 2- and 4-cellstages. However, the diagnosis of oral or aboral isdifficult to make by solely anatomical criteria until the

invagination of the archenteron is complete and thestomodeum forms. Manipulations that interfere withthe mechanics of gastrulation will affect the interpret-ation of the experimental result, regardless of theeffect on potential to form oral or aboral structures.Furthermore, this experiment can only assess fairlystable or irreversible biases in potential. Hence, thereis in the literature no decisive analysis of localizationof oral-aboral potentiality. In his masterly summaryof these experiments, Horstadius (1973) concludesthat the first two meridional divisions do distributepotential along the aboral-oral axis and Czihak'shistochemical staining results, showing localizedstaining of cytochrome oxidase (1963) along theoral-aboral axis at the 16-cell stage certainly sustainsthis point of view. The recent lineage tracer exper-iments of Cameron et al. (1987) reveal axis specifi-cation by the 8-cell stage.

The subject of the equipotentiality of early blasto-meres and fragments along the meridional axis is ripefor renewed analysis. Our understanding of gastru-lation is now better, allowing design of experiments in

ab

Fig. 1. The classical result of meridional (A) or equatorial(B) separation of egg fragments or blastomeres is depicted.As discussed in the text, meridional separations give rise toalmost normal larvae (A). Separation of animal (an) fromvegetal (veg) fragments along the equatorial plane (B) giverise to arrested blastulae from the animal half. Vegetalfragments may give rise to exogastrulae, as depicted.Vegetal fragments may also give rise to normal larvae, asdiscussed in the text, or, oral; ab, aboral; s, spicule;g, gut; an, animal; veg, vegetal.

562 F. H. Wilt

which abnormalities of gastrulation caused by alimited number of cells or purely mechanical con-siderations may be taken into account. Even moreimportant, there are easily applied, quantitativemarkers of potential along the oral-aboral axisthat have recently become available. Among thesemarkers are the Cy III genes of the actin family thatare exclusively expressed in aboral ectoderm cells,beginning at late blastula (Cox etal. 1986). The familyof genes termed 'spec' (Lynn et al. 1983), whichencode troponin-like proteins, are expressed in alocalized fashion in the aboral ectoderm cells. Thereis also an epitope recognized by a monoclonal anti-body (Ecto V) isolated in McClay's laboratory(McClay & Ettensohn, 1987) that is characteristic ofthe ectodermal cells in the stomodeal anlagen and theforegut endoderm. The expression of any of thesecharacters would have to be analysed during cases ofabnormal morphogenesis to determine if any of themare expressed if gastrulation is defective. If any of theabove indices of aboral differentiation pass this test,they will be admirable tools that may be applied toindividual embryos to re-examine this old question ofblastomere equivalence and determination of theoral-aboral axis.

The difference between results with the meridionalfragments that we have just discussed and equatorialfragments is strikingly illustrated in recent work byMaruyama, Nakaseko & Yagi (1985). They capita-lized on Schroeder's (1980a,b) important obser-vations, prefigured in much earlier work by Boveri(1901), that there are clear indications of the animal-vegetal axis in unfertilized eggs (see Sardef & Chang,1985). The position of the polar bodies is laterrevealed by a discontinuity in the jelly layer aroundthe egg, the jelly canal, unfortunately called a micro-pyle by some older workers. Sperm may activate thesea urchin egg anywhere on the egg surface. Theposition of the first cleavage plane bears no relation-ship to the site of sperm activation. Rather, in over90 % of the cases, the first cleavage plane traversesthe egg through the plane defined by the jelly canaland its opposite pole. The second cleavage traversesthe same jelly canal axis, thus producing four blasto-meres separated along the meridional axis. The thirdcell division is orthogonal to the first two, bisectingthe jelly canal axis, resulting in an equatorial separ-ation (Fig. 1). Thus, the jelly canal indicates theclassical animal-vegetal axis; the canal can be re-vealed by placing unfertilized eggs in a suspension ofvery fine ink particles, which penetrate the disconti-nuity of jelly that defines the canal.

When Maruyama et al. (1985) fertilized meridio-nally bisected eggs both halves formed gastrulaewhether they contained the female pronucleus or not.Though the androgenetic haploid merogons derived

from anucleate halves cleaved more slowly, they wereable to form imperfect plutei with the main organspresent. Separation of the egg along the equatorialplane gave a very different result. In 90% of thecases, the equatorial egg fragment from the animalhalf (where the jelly canal is located) never undergoesgastrulation, never forms pigment, never forms mes-enchyme or its derivatives and instead forms a 'per-manent' blastula. The long immobile cilia, character-istic of embryos of this half, do eventually becomereplaced with shorter beating cilia. This.is the classi-cal 'animal' development observed by many others.

Egg halves from the equatorial fragment oppositethe jelly canal formed fairly normal plutei in almostall of 160 cases; the photographs show that theresultant larvae are not quite normal, having less-extensive skeletal elements, but all major organsystems are present in the correct positions. Thepresence of the animal half is thus apparently notimportant for overall patterning or development ofany of the tissues of the embryo, playing at best only asubtle modulatory role. This is in accord with numer-ous observations from the time of Driesch thatvegetal egg halves may often develop into normallarvae (Wilson, 1925).

The results of Maruyama et al. (1985) are distinctlydifferent from the usual predictions of the doublegradient hypothesis. Fig. IB shows the classicalinterpretation of Horstadius (1939). Animal halvesgive rise to permanent blastulae, like those observedby Maruyama, while vegetal halves give rise tocreatures with little surface epithelium, overdeve-loped or exogastrulated archenterons, lack of a de-fined oral-aboral axis and abnormal spicules. Aspointed out by Horstadius (1939) and Wilson (1925)among others, there is considerable variation in theability of vegetal equatorial halves to give completelarvae, although this variation is relatively consistentin eggs from a given female. In any case, it is clearthat vegetal halves frequently give rise to completelarvae and the results of Maruyama et al. (1985) showconsistent near totipotency of the vegetal equatorialhalf.

The idea of a double gradient, as opposed to asingle centre of influence emanating from near thevegetal pole, is based on several considerations. First,the isolated animal half has accentuated developmentof one feature, the length of apical cilia, and so it isargued that this is an example of unrestrained animaltendency. This seems to be the main morphologicalindex of an 'animal tendency'. The second basis forthe idea comes from cell interactions revealed bytransplantation. If one isolates blastomeres arisingfrom the middle portion of a 32-cell-stage embryo, asshown in the cartoon of Fig. 2, normal embryos may


an 1 (8)

an 2 (8)J

veg 1 & 2 (8)

mic (8)

Fig. 2. The result of transplantation of blastomeres of the32-cell stage is shown schematically. If micromeres (black)are combined with blastomeres from the animal half(an 1 + an —2), a normal larva may result. If vegetalblastomeres are combined with animal blastomeres anormal larva also may result. The cartoon is meant toreflect interpretation of transplantations found in theclassical literature.

result. Or, if blastulae derived from animal blasto-meres combined with micromeres of the extremevegetal end are allowed to develop, a normal pluteusmay result. The classical interpretation of theseresults is that the manipulations still allow the devel-oping embryo to contain the correct ratio of animaland vegetal information.

It is very difficult to distinguish reciprocal tissueinteractions and inductions by micromeres or veg 2 onthe one hand, from a balance of opposing forces onthe other, as recently pointed out by Davidson(1986). There are, indeed, many observations that donot conform to the double-gradient hypothesis. Ihave just mentioned some of these, including thedevelopment of normal larvae from vegetal halvesand the inability of isolated micromeres to formanything other than skeleton. There has been noadequate way to distinguish a passive role of theanimal half from an active one. One of the moreprovocative experiments is that isolated animalhalves, when treated with the vegetalizing agent,Li+, do form nearly normal larvae. Both von Ubisch(1929) and later Horstadius (1939) showed that onecould obtain normal plutei from animal halves treatedwith Li+. The mechanism of action of Li+ is com-pletely elusive, but the complex set of epigeneticevents set in motion is equivalent to influencing theanimal half by micromere implantation. Clearly, theanimal half possesses regulative ability, may beinfluenced in its determination and this effect may becaused by an ionic imbalance!

It has been shown recently that Li ions also evokeappearance of dorsal and anterior structures in am-phibian eggs that have been exposed to u.v. irradia-tion and thereby rendered incapable of formingnormal dorsal-anterior structures (Kao, Masui &Elinson, 1986). This effect of Li+ on amphibian eggsis not displayed by a large range of other cations. Notheory of Li+ action has been proposed, but recent

work has shown that Li+ interferes with the regener-ation of phosphatidylinositol lipids, which are in-volved in the inositol phosphate secondary messengerpathway (Majerus etal. 1986). At any rate, the effectsof Li+ make theories of localization of morphogen-etic determinants in the vegetal egg half less compell-ing.

Another result bearing on the importance of gradi-ents and localized substances is a set of experimentsby Nemer, Wilkinson & Travaglini (1985) in whichthe classical, and most effective animalizing agent,Zn2+, is examined for its effects on gene expression.This is the first time an animalizing or vegetalizingagent has been examined directly for effects onexpression of a particular tissue-specific gene. Nemer,using embryos of S. purpuratus, examined effects ofZn2+ treatment on expression of the gene sped;sped encodes a troponin-C-like protein which occursonly in the aboral ectoderm of later-stage gastrulae.Spec 1 mRNA is localized only in aboral ectodermand thus acts as a good indication of differentiation ofcells of the animal hemisphere (Lynn et al. 1983).Nemer found that Zn2+ treatment causes inhibitionof transcription of Sped genes and, if Zn2+ isremoved by the chelator EDDA, normal develop-ment and sped expression resume. Hence, Zn2+

treatment of whole embryos causes a developmentalarrest, a kind of diapause, and not, apparently, achange in cell potential or fate. It will be interesting tolearn if surgically isolated animal halves display thesame kind of inhibition of sped gene expression; thefailure of sped expression in isolated animal halves,were this the result, would imply an inductive effectof the vegetal half on the animal portion of theembryo and it would strongly argue against an 'ani-malizing' gradient.

An alternative to the double-gradient hypothesis isthat separation of the animal half from materials inthe vegetal hemisphere causes developmental arrestof animal cells. The animal half cells are totipotentand may be provoked, either by induction or byexposure to Li+, to adopt new fates and form acomplete embryo. The distinction between gradientsof materials, somehow disposed in the vegetal half,and localized determinants may not be a usefuldistinction. Rather, one might emphasize the import-ance of early cell and tissue interactions, comparableto ideas about early amphibian development (Wylie,1986). The use of newly available tissue-specificmarkers of gene activity, in conjunction with iso-lation, defect and transplantation experiments, mayprovide a good way to re-examine the cell interac-tions in echinoderms.

If cell interactions are important, one would pre-dict that interruption of cell interactions would haveimportant consequences for expression of animal

564 F. H. Wilt

hemisphere lineage-specific markers, less so for veg-etal half markers like spicule matrix proteins (Bensonet al. 1987; Sucov et al. 1987) and collagen (Venkate-san, de Pablo, Vogeli & Simpson, 1986). Cameron,Hough-Evans, Britten & Davidson (1987) have re-cently published a careful analysis of the fate of theblastomeres of the 8-cell stage; aboral ectoderm isderived from blastomeres of both the animal andvegetal hemispheres and, therefore, aboral-ecto-derm-specific markers may not be adequate indi-cators of animal hemisphere differentiation. Oralectoderm is solely derived, however, from the animalhalf and the discovery of oral-ectoderm-specificmarkers would be welcome. Treatment of isolatedanimal half cells with Li+ should cause appearance ofvegetal-specific gene expression in animal half cells.Treatment of isolated cells or embryo parts with Zn2+

should suppress gene expression generally, and bereversible with EDDA. One can isolate cells ofdifferent determined types microsurgically. Micro-meres may be isolated en masse (Okazaki, 1975), ascan primary mesenchyme (Ettensohn & McClay,1986). It should be possible using these isolated celltypes to study mechanisms of their interactions. Forexample, the extracellular components synthesizedby these cells may be released into the medium ormay coat the culture substrate. Once these moleculesare characterized, their functions may be evaluatedby utilizing the medium or culture substrate as acomponent of the culture of either isolated cells orentire embryos.

Another consideration is a closer examination ofthe inductive and differentiative capacities of the cellsderived from the vegetal half of the egg. The classicalview assigned a single potentiality, the vegetal influ-ence, to this hemisphere, but this surely cannot becorrect. The vegetal half normally selfdifferentiatesto form part (anal plate and aboral ectoderm) of theepithelial surface of the blastula (veg 1), the archen-teron, pigment cells of the surface epithelium, sec-ondary mesenchyme, and primary mesenchyme andits derived spicules. In addition, a special populationof cells in the coelomic sacs that resume division atmetamorphosis are derived from the original micro-meres (Pehrson & Cohen, 1986). Furthermore, inembryos devoid of micromeres, skeletons may formand, in this instance, Horstadius (1973) reports that itis secondary mesenchyme cells (that normally formmuscle) that transdifferentiate into skeletal elements.Others have noticed that in the absence of primarymesenchyme, the secondary mesenchyme will des-cend and assume the role of primary mesenchyme,forming a skeleton (von Ubisch, 1939; Langelan &Whiteley, 1985; Ettensohn & McClay, 1986). In anespecially interesting study apparently unknown inthe Occident, Fukushi (1962) showed that, if one

removed the primary mesenchyme cells from a mes-enchyme blastula, the secondary mesenchyme cells(derived from veg 2 tier) will change their morpho-genesis and differentiation and form a normal skel-eton. While cells from veg 2 and perhaps even veg 1have a wide potency, the extreme vegetal end, themicromeres, are limited in their potency, formingonly skeletal elements and cells destined for theechinus rudiment. There are no reported examples ofchange in micromere fate. Micromeres also havemorphogenetic influence on neighbouring cells,which will be discussed later.

There are indications that the fourth cleavageleading to separation of micromeres from macro-meres in the vegetal hemisphere is important inestablishment of lineages, thereby setting up partici-pants for the interactions that follow. For example,one may upset the normal time schedule of division ina number of ways, and thereby cause the thirddivision to form an 8-cell embryo which has an animalquartet of very large cells and a vegetal quartet ofsmall cells. Such small cells, so-called precociousmicromeres, behave as micromeres from a 16-cellembryo and may form spicules autonomously, inculture (Kitajima & Okazaki, 1980).

The normal cell division of the fourth cleavage isunusual in the vegetal quartet and has been describedby Dan and his associates (Dan, 1979; Dan, Endo &Uemura, 1983). First, the nuclei of the vegetalquartet of the 8-cell stage move toward the vegetalpole prior to prophase and the spindles that thenassemble are very close to the cortex of the vegetalpole, even apparently pushing some granules of thecortex in this region to one side (Dan et al. 1983). Ithas been suggested that this very close association ofthe spindle pole with the cortex may be important forformation of the micromere lineage. In support ofthis idea, the asymmetric fourth division may besuppressed with different agents; notably, treatmentwith SDS prevented nuclear migration and resulted inembryos that did not develop primary mesenchymeor spicules (Tanaka, 1976). Nonetheless, since this isa negative result, one must be cautious, especiallysince there are numerous examples in the classicalliteratures to which I have already alluded thatsupport the idea that the peculiar cleavage plane atthe fourth division is dispensible (Horstadius, 1939).Hence, the paper of Langelan & Whitely (1986)extending the experiments of Tanaka, using bothsand dollar and sea urchin embryos, probably rep-resents a more secure interpretation. They found thattreatment with SDS does suppress micromere forma-tion, but treated embryos were able to form primarymesenchyme and had normal spicules. Indeed, theywere also able to suppress the asymmetric fifthcleavage and in a large number of cases completely


suppressed ingression of primary mesenchyme intothe blastocoel. Nonetheless, these embryos under-went archenteron formation and secondary mesen-chyme cells descended from the archenteron tip,formed a syncytial array, and a larva replete with gutand skeleton formed in a high percentage of cases.Clearly, the unusual asymmetric divisions at thefourth and fifth cleavages are not essential in thesespecies, and any theory which posits that some specialcharacter of this division is essential for determi-nation seems incorrect, both on comparative grounds(Raff, 198) and on the basis of the experiment ofLangelan and Whiteley. The formation of spiculesfrom cells emanating from the archenteron tip is verylikely the same phenomenon observed in embryossurgically deprived of micromeres.

Manipulations of blastomeres of the early seaurchin embryo show that some cells have very greatpotentialities and cell interactions probably play arole in determination and origin of the normal patternof the embryo. The results do not seem really torequire a double gradient of opposing potentialities.In fact, because of the extremely wide differentiationpotential of the cells in the vegetal half, it is difficultto know if there is a gradient at all. What is requiredto make an embryo is some cells from veg 2 and someother material from the animal half (or even veg 1).The cells in veg 2 or micromeres probably causeresponding cells to adopt particular fates. It is likelythat both localized determinants in the vegetal hemi-sphere and cellular interactions serve to define pat-terns of determination and differentiation.

How early and how stable is cell lineage specification?The micromere lineage is apparently autonomous inmost of its further development from the moment ofits origin at the fourth cell division. Indeed, manycells derived from this lower vegetal portion of theegg may give rise to a cell lineage endowed withproperties of primary mesenchyme, as I have dis-cussed in the preceding section. There have beenextensive investigations to catalogue the biochemicaland cellular differences between micromeres and theremainder of the embryo, including histone types(Senger & Gross, 1978), complexity of RNA(Mizuno, Lee, Whiteley & Whitely 1974; Ernst,Hough-Evans, Britten & Davidson, 1980; Rodgers &Gross, 1978) and kinds of proteins synthesized(Tufaro & Brandhorst, 1979; Harkey & Whiteley,1983; Kitajima & Matsuda, 1982); these findings havebeen reviewed and analysed extensively by others onseveral occasions (Wilt, Benson & Uzman, 1985;Harkey, 1983). The difficulty with the interpretationof a catalogue of differences between different cells isthat it is impossible in the absence of a functional test

to know which differences, if any, are developmen-tally relevant. There is good evidence, though, thatthe factors of the egg that result in the eventualformation of the primary mesenchyme are not easilydislodged by stratification of organelles using centri-fugation (Wilson, 1925). Hence, some structural basisfor the origin of the lineage may reside in the plasmamembrane or the stiff submembraneous cortical re-gion. DeSimone & Spiegel (1985) have recentlylabelled cell surface proteins of micromeres from A.punctulata and 5. drobachiensis with 125I. The patternof labelled cell surface proteins is different in the twospecies, but in both cases there are distinct labelledbands in micromere cell surface proteins that are notpresent in surface proteins of the other blastomeretypes. Presumably, these labelled proteins exist in theegg and are distributed to the micromeres, thoughthis has not yet been directly shown. Sano (1977,1980) has shown that micromeres possess a differentagglutinability by Con A during cleavage and alsothat these cells have a somewhat different surfacecharge, as measured by their electrophoretic mo-bility. It has been reported that micromeres will 'cap'lectins, while mesomeres and macromeres will not(Roberson, Neri & Oppenheimer, 1975).

The stability of specification of the micromerelineage is probably strict, though this issue has notbeen adequately tested. By specification, we meanthe bias to form a given tissue when developmentoccurs in isolation. By determination, we mean thebias to form the same phenotype as the normal fatewhen cells develop in an ectopic location. The factthat micromeres, when isolated or transplanted, formskeleton does not entirely settle the issue, for if theirdescendants form other tissues, this could not havebeen detected. In a sense, changes in specification ordetermination can only be demonstrated, but not theconverse. One way to examine the stability of deter-mination is the dissociation-reaggregation exper-iment using marked cells. In this instance, micro-meres, or any other blastomeres, may be exposed to anew environment and the stability of early specifi-cation examined. Of course, transplantation isanother alternative, and one might use [3H]thymidineor vital lineage tracer dyes to check the results ofearlier work.

It is not even known if the properties of themicromere lineage are a necessarily coherent set ofbehaviours. Micromeres do give rise to another groupof cells that divide once, then remain quiescent, andlater resume division in the rudiments that will formthe adult urchin (Pehrson & Cohen, 1986). Theregulation of the formation of this sublineage is notunderstood. Furthermore, primary mesenchyme pre-cursors are entrained in a different rhythm of celldivision from their siblings (Dan, Tanaka, Yamazaki

566 F. H. Wilt

& Kato, 1980; Masuda & Sato, 1984). It is claimedthat the micromeres provide some mitotic pacemakerfor waves of cell division during the latter half ofcleavage (Filosa, Andreuccetti, Parisi & Monroy,1985). The importance of this pacemaker phenom-enon is not clear, especially since micromerelessembryos may form plutei. The progeny of micro-meres do change their affinities for one another andfor the hyalin layer and basal lamina (McClay & Fink,1982; Fink & McClay, 1985). And they become motileand display a complex set of behavioural qualities,including fusion, ectopic archenteron formation andinduction, as discussed before (Solursh, 1986).

The timing and stability of determination of othercells in addition to micromeres in the early embryo isalso not known. Presumably all layers other thanmicromeres have some lability in their specificationand determination during mid and late cleavage.When Horstadius transplanted various tiers of cells toectopic locations, the experiments were usually car-ried out on cells from the 32- to 64-cell stage. Theresults showed cells from the animal and vegetallayers participated in forming patterns of varioustypes in a regulative fashion. It is not so clear what thecellular basis for this regulation of pattern might be.Was there a respecification of cell fate, or were theremodifications of the rate and pattern of cell divisionsso that some cells reformed a pattern by differentialcell division, or were there changes in morphogeneticmovements that somehow led to regulation. Theseare, of course, not mutually exclusive possibilities.Probably the clearest case is the transplantation ofmicromeres to an isolated animal half in whichinvagination and archenteron formation take place inanimal half cells. This constitutes a change in cell fateof animal cells from surface epithelium to archen-teron lining, mesenchyme, etc. Data shown in linedrawings of the embryos formed from these trans-plants look like normal plutei; one presumes theyhave functional tissues and that lineage-specific genesare expressed, but this has not been definitivelyshown. It appears there is a genuine specification ofanimal cell fate due to induction by micromeres.Transplantation experiments that include any of theveg 1 or veg 2 tiers in the transplant recipient or donorare very difficult to interpret because isolated vegetalhalves that contain these cells may give rise to normalplutei.

Thus, even though the brilliant line of exper-imenters beginning with Boveri, Driesch and Herbst,and continuing with Horstadius and von Ubisch wereseminal, their investigations were limited, as are weall, by the tools of the time. Modern micromanipula-tors, stable lineage-tracer dyes, monoclonal anti-bodies and cell-lineage-specific genes that can be usedas markers open up new possibilities to investigate

lineage stability and transformation in a rigorous way.For example, animal half cells may be marked withtetramethylrhodamine isothiocyanate (Ettensohn &McClay, 1986). Unstained micromeres may be com-bined with such an animal half and the resultantregulated embryo examined by in situ hybridizationfor type IV collagen genes (Venkatesan et al. 1986) orcell surface proteins (Leaf et al. 1987) expressed inprimary and secondary mesenchyme to see if therehas been a respecification of the marked animal halfcells.

One method for testing stability of cell determi-nation mentioned previously is the dissociation andreaggregation of cells, a method easily carried outwith echinoderm embryos at a number of differentdevelopmental stages, as shown by the work ofGiudice and his colleagues. This work has beenreviewed extensively by others (Giudice, 1973, 1986;Spiegel & Spiegel, 1986). In some cases, even whendissociation is carried out at blastula stages, embryoswill form that have a surface epithelium, skeleton andgut. Experiments have not yet been performed to seeif these reconstructed tissues are correctly expressinglineage-specific genes. Surprisingly, there has beenvery little work to distinguish cell type transformationfrom sorting out. Nor has the influence of small nestsof incompletely dissociated cells been evaluated.Melvin & Evelyn Spiegel (1975) have demonstratedthat a mixture of dissociated cells from Arbacia andLytechinus sort out and reconstitute larva-like struc-tures. Spiegel (1978) also reaggregated micromeresstained with Nile blue sulphate with mesomeres andmacromeres from unstained embryos and showedthat the micromeres sort out within 4 h and go on toform primary mesenchyme. One could also test thedetermination of mesomeres, macromeres and othercell types in this kind of experiment, but it has not yetbeen done. Jenkinson (1911) cut mesenchyme blas-tulae and gastrulae into large pieces and found verylimited regulative abilities at these stages, though thistype of experiment might be useful if modern markerscould be used.

There is some evidence that suggests that theblastomeres of the early embryo possess a stableapicobasal polarity. Perhaps the original egg plasma-lemma defines a polarity for the blastomeres; newmembrane may appear on the blastocoel sides of theblastomeres and thereby provide a stable cell polaritymarker, much as is the case in amphibian blasto-meres. Marina Dan-Sohkawa and her co-workers(Kadokawa, Dan-Sohkawa & Eguchi, 1986) haverecently shown that dissociated starfish blastomeresdisplay an inherent apical-basal polarity and thatwhen the aggregates form a gastrula this polarity isintact: As cell division stops, septate desmosomesappear in the epithelium of the blastula of sea


urchins, as well as tight and gap junctions. Amemiya,Akasaka & Terayama (1982) examined the polarity ofarchenteron cells in the gut formed by exogastrula-tion. They found that the ciliated prospective luminalside, which is now the superficial side exposed to seawater in the exogastrula, still retained its originalpolarity. Spiegel & Howard (1985) have shown thatthe morphology of septate desmosomes in the gut ofexogastrulae have normal morphology and tissuedistribution in an exogastrula. It may be very in-formative to learn what properties of cells are auton-omous when maintained as dissociated cell popula-tions and what properties require cell associations.Though the stability of determination is difficult todiscern from the available evidence, it is likely thatuse of new molecular markers will be useful ininterpretation of embryological experiments.

Morphogenesis

The events resulting in specification of cell lineage area necessary prelude to the actual morphogenesis thatforms the pluteus. The activities of the individual cellsand their responses to cues that transcend domains ofsingle cells drive the formation of the embryo. Thesemorphogenetic activities in turn entrain further re-finements in the definition of the pattern of differen-tiation. It is appropriate to inquire how determinationin early stages brings about morphogenetic move-ments and how these movements in turn lead tofurther pattern specification and differentiation. Ishall discuss the morphogenetic movements of pri-mary mesenchyme cells and then turn to gastrulation.

Morphogenetic movements of primary mesenchymeSince the behaviour of primary mesenchyme cells hasbeen so well described in recent reviews, I wish to re-examine only a limited number of issues that seemespecially puzzling or ripe for analysis. The morpho-genetic movements of the sea urchin embryo were thesubject of a provocative review by Gustafson &Wolpert in 1947. They emphasized the role of celladhesion and changes in cell shape and motility asdriving forces in morphogenesis. The changing ad-hesive properties of primary mesenchyme cells havebeen the subject of many studies since then and thereare several recent reviews (McClay & Ettensohn,1987; Solursh, 1986; Spiegel & Spiegel, 1986; Wata-nabe, Bertolini, Kew & Turner, 1982). New ways oftesting the affinities of primary mesenchyme cells,and other types of cells, are consistent with the viewthat there are changes in the affinities of cells for oneanother, for hyalin and basal lamina, and that thesechanges help explain the ontogeny of behaviour ofprimary mesenchyme. Proof of these speculationswill come when the molecular basis of cell affinity and

adhesion is better understood. It might then bepossible to carry out experiments aimed to change theadhesive properties of cells by judicious use ofinhibitors, antibodies, anti-sense RNA of sequencesencoding putative adhesive components, or DNA-directed transformation of cells with sequencesencoding adhesive molecules fused to inappropriatepromoters.

Once the primary mesenchyme cells have ingressedinto the blastocoel, the behaviour of the cells andtheir relationships to the extracellular environment isvery complex. The developmental autonomy of cellsderived from micromeres seems clear; the generalsequence and relative timing of cell division and ofchanges in adhesivity and motility all occur on sched-ule in isolated micromeres (Okazaki, 1975). How-ever, it is not known what effect cell associations haveon all these later behaviours of the micromere lin-eage. Do cells have to remain associated in order formotility to ensue or for spicule matrix genes to betranscribed? In other words, what, if any, are thehomotypic cell associations necessary for the mor-phogenesis of this lineage? The goal of research inthis area has been to describe the details of motilebehaviour of primary mesenchyme cells, to learnwhat molecules of the cell surface are important inmotility and in interactions of the cells with eachother and with the environment, and to describe thechanging nature of the extracellular environment inthe blastocoel. Solursh's (1986) recent review clearlydescribes the history and present status of work in thisarea. The experimental tasks are very difficult and weare only at the very beginning. Though primarymesenchyme cells are extremely motile (Karp &Solursh, 1985a,b), neither the internal machinery northe external molecules involved in motility or ad-hesion are known. The blastocoel and basal laminacontain sulphated glycosaminoglycans, collagen, fi-bronectin and laminin (Solursh & Katow, 1982;Spiegel, Burger & Spiegel, 1983; Katow & Hayashi,1985). The proportions and nature of the moleculeschange during gastrulation as primary mesenchymecells undergo their motile, then skeletogenic phases(Karp & Solursh, 1974). Interference with biosynthe-sis and processing of the various components resultsin changes in the matrix molecules and inhibitsprimary mesenchyme morphogenesis and differen-tiation. Reversal of treatments with sulphate-free seawater or /J-xylosides may allow development to re-sume. Since most of the treatments cause negativeeffects, e.g. cessation of morphogenesis, it is difficultto draw rigorous conclusions. There are some inter-esting recent studies that illustrate the complexity ofthis whole area.

Blankenship & Benson (1984) studied the forma-tion of skeletal spicules from isolated, cultured

568 F. H. Wilt

micromeres in the presence of inhibitors of collagenmetabolism. They found, as had others (Mintz,DeFrancesco & Lennarz, 1981), that exposure of thecultured cells to analogues of proline or inhibitors ofpost-translational modifications of collagen, resultedin inhibition of spicule formation. If, however, theculture dishes were coated with rat tail tendon col-lagen before use in the experiment, the same inhibi-tors had very little effect on the ability of micromeresto form spicules. This finding suggests that primarymesenchyme cells may synthesize collagen and thatcollagen then becomes part of a permissive environ-ment favouring differentiation into spicules. In linewith this finding, Venkatesan et al. (1986) haveisolated a portion of a collagen gene from a genomiclibrary of S. purpuratus using a mouse alpha 1 (IV)cDNA probe. The mRNA for this collagen in theembryo is 9 kb, appears first at the morula stage andbecomes prominent on RNA blots by the mesen-chyme blastula stage. This mRNA for a putative typeIV collagen is found localized in primary mesen-chyme cells (personal communication). It is possiblethat primary mesenchyme cells synthesize some col-lagen, which then forms part of the fibrous environ-ment of the blastocoel (Crise-Benson & Benson,1979) or even part of the basal lamina. Wessel &McClay (1985) have discovered an antigen (meso1)of the plasma membrane that appears on the surfaceof primary mesenchyme cells just as they ingress andthat later become evident in the basal lamina alongthe blastocoel. Kitajima & Matsuda (1982) havefollowed the synthesis of different proteins by cul-tured micromeres, as have Harkey & Whiteley(1983). More recently, Kitajima (1986) carried outsome of their experiments with micromeres on anagarose surface, which almost totally prevents attach-ment of cells to the substrate; the isolated micromeresdifferentiate while floating in the medium in grape-like clusters. Though definite proof is lacking, theidea that primary mesenchyme cells contribute in animportant, though perhaps permissive, way to theirown microenvironment is in accord with the facts.

As the primary mesenchyme cells undergo mor-phogenesis in vivo, the composition and morphologyof the extracellular material in the blastocoel changes(Akasaka, Amemiya & Terayama, 1980, 1982).Katow & Amemiya (1986) describe the appearance of15 nm fibrils during gastrulation (Kawabe, Armstrong& Pollock, 1981). Both the surface of the primarymesenchyme cell and the blastocoel side of the basallamina are associated with granules (15-30 nm) thatstain with ruthenium red and may be proteoglycan.Treatment of embryos with sulphate-free sea waterinhibits sulphation of the sulphated glycosaminogly-cans (predominantly chondroiton-6-sulphate, derma-tan sulphate and heparin sulphate) and results in a

reduction of the amount of the granular component(Katow & Solursh, 1979; Solursh & Katow, 1982).Treatment of sea urchin embryos with /?-D-xylosides,which interfere with synthesis of glycosaminoglycanattached to proteoglycan cores and may result inaccumulation of free glycosaminoglycans, also resultsin depression of appearance of the granular com-ponent (Solursh, Mitchell & Katow, 1986). Bothxylosides and sulphate-free sea water allow ingressionof primary mesenchyme cells into the blastocoel, butmigration of the ingressed mesenchyme is inhibited.Katow (1986) has studied the mobility of isolatedprimary mesenchyme cells in vitro on different sub-strates and gels in plastic dishes. Fibronectin, lami-nin, type IV collagen and dermatan sulphate were alltested as substrates for migration; only combinationsincluding fibronectin and collagen allowed migration,and a gel composed of fibronectin, collagen anddermatan sulphate was superior. Venkatasubrama-nian & Solursh (1984) and Katow & Solursh (1981)also studied migration of primary mesenchyme cellsin vitro and found that mesenchyme cells from em-bryos treated with sulphate-free sea water had aninherently lower capacity to migrate, even on com-petent substrata. The effects of sulphate deprivationmay, therefore, affect the migratory ability of pri-mary mesenchyme cells as much as or more thanaffecting the suitability of the substrate for motility. Itis appropriate to remember that during this period ofprimary mesenchyme cell morphogenesis there isintense synthesis of tunicamycin-sensitive N-glycosy-lations of protein, studied by Lennarz and his associ-ates (Heifitz & Lennarz, 1979; Lau & Lennarz, 1983;Schneider, Nguyen & Lennarz, 1978). Inhibitors ofglycosylation interfere with primary mesenchyme celldifferentiation and with gastrulation. Once again, allthe observations are consistent with the importanceof a multicomponent extracellular material, somecomponents of which may arise, in part, from themesenchyme cells themselves.

Morphology of spicules

The spicules that form in the living embryo often havea very complex morphology and the details of thearrangements of hooks, spurs, fenestrations andshape are species specific (Okazaki, 1975). The regu-lation of the formation of these beautiful patterns hasbeen a topic of interest to students of morphogenesisfor a long time. This topic has been studied by severalmethods and has been reviewed by Solursh (1986).The focus of attention has been directed to findingout whether the primary mesenchyme cells, theextracellular material or the epithelial wall contributeto the final detailed pattern. Of course, these rolesare not mutually exclusive and the accumulatedevidence suggests that all three of these participants


play some role. Von Ubisch (reviewed in 1939)showed this clearly in chimaeras formed from trans-plantation of micromeres of one species to embryosor animal halves of another species. Whether thespicule is simple or fenestrated is a property of theprimary mesenchyme cell. The overall position of thespicule and placement of rods seems to conform moreto the source of the epithelial wall. When primarymesenchyme cells of two species are enclosed in anepithelium from only one of the species, the result isoften so complex it is difficult to interpret. In someways, the interpretation of experiments on spiculepattern depend on which observations one chooses toemphasize. For example, in Okazaki's (1975) pio-neering work on development of isolated micro-meres, she pointed out that frequently rather normal-looking spicules would form in the absence of blasto-coel walls (but not of extracellular material). On theother hand, Harkey & Whitely (1980) concluded fromstudies of development of spicules in isolated bags ofbasal laminae that the presence of epithelium wasimportant to the final pattern. Undoubtedly bothviews are correct and the terminal differentiation ofspicule from the cells may represent an excellentopportunity to study how different tissues, cells andextracellular materials may interact to realize a com-plex pattern. Kinoshita & Okazaki (1984) carried outan important study in which details of the mor-phology of the spicules that form in vitro werestudied. They found that if micromeres adhere to thesubstrate, needle-like spicules form. Under con-ditions in which the micromeres formed cell aggre-gates the resultant spicule was three dimensional andsimilar to the spicule formed in vivo.

Ettensohn & McClay (1986) have recently deviseda way to vitally stain primary mesenchyme cells, thento inject them into the blastocoel of another embryoof the same species. Using this technique, they wereable to show that the migration and disposition of themesenchyme cells is dependent upon the stage of thehost. Interactions of primary mesenchyme with theblastocoel environment of the recipient embryo areimportant until the late gastrula stage. For instance,after placement of primary mesenchyme into theblastocoel of an early prehatching blastula, the im-planted mesenchyme cells remain quiescent until thehost cells begin their ingression, then the implantedcells assume their typical position in the blastocoeland integrate with the host primary mesenchymecells. The availability of culture methods and ourbetter understanding of the nature of the extracellularmaterial and cell surface makes molecular studies ofthese phenomena now possible.

DeSimone & Spiegel (1986a,fr) have carried outseveral studies characterizing the effects of injectionof concanavalin (con A) and wheat germ agglutinin

(WGA) into the blastocoel. WGA binds only aftermesenchyme cell ingression and binding sites areespecially prominent where primary mesenchymecells contact the basal lamina. Con A binds moregenerally to the basal lamina and causes the epithelialcells to round up. Treatment of embryos with sul-phate-free sea water alters the pattern of lectinbinding. Iwata & Nakano (1985a) have extracted apolysaccharide from sea urchin embryos that binds tosea urchin fibronectin. Treatment of embryos orcultured micromeres with antibody directed againstthe fibronectin-binding polysaccharide enhances spi-cule formation and alters the morphology of thespicules that form. Iwata & Nakano (1986) haverecently isolated a protein from sea urchin embryosthat may be involved with Ca2+ binding, as haveCarson et al. (1985). These proteins are distinct,however, from the structural protein of the spiculematrix recently characterized by Benson, Benson &Wilt (1986).

Primary mesenchyme and gastrulationThe micromere progeny supposedly play a role ingastrulation, though clearly primary mesenchymecannot be essential. There are many reports in theliterature in which sea urchin embryos deprived ofmicromeres gastrulate and form perfectly normalplutei (Horstadius, 1973). In cleavage-stage embryosdeprived of micromeres, new micromeres are notformed, nor is primary mesenchyme formed byingression. In fact, the primitive order of urchins, theEucidaroidea, have no micromeres or primary mes-enchyme, yet they gastrulate and form a skeleton(Raff, 1987). Embryos of the Asteroidea also have nomicromeres or primary mesenchyme (Kume & Dan,1968; Reverberi, 1971). It is curious, therefore, thattransplantation of sea urchin micromeres to a lateralaspect of a cleaving embryo supposedly induces asecondary archenteron (Horstadius, 1939). There areeven some cases where movement of a micromere tothe animal pole results in a small secondary archen-teron opposite to the host archenteron. These sec-ondary structures often fuse with the main invagina-tion of the embryo. This suggests that primarymesenchyme cells might be normally responsible forthe initial first phase of gastrulation. If so, themechanisms by which they act is completely un-known. Or is the power of the micromeres to inducean archenteron an erroneous interpretation and per-haps there is a polyingression of descendants of thetransplanted micromeres? Modern microscopic andcell marking methods ought to resolve this question.

When micromeres are removed there is no primarymesenchyme; the embryo gastrulates normally, thesecondary mesenchyme issues from the tip of thearchenteron and then some secondary mesenchyme

570 F. H. Wilt

cells move toward the blastopore region and assumethe behaviour of primary mesenchyme, subsequentlydifferentiating and forming skeletal spicules (Horsta-dius, 1973; Fukushi, 1962; Langelan & Whiteley,1985). This finding implies that secondary mesen-chyme cells, derived from the vegetal 2 tier, haveskeletogenic potential, completely concordant withHorstadius' isolation and transplantation exper-iments. It also implies that the pluripotential natureof secondary mesenchyme persists through the sec-ondary stages of gastrulation. It is interesting that acytoplasmic actin transcript, Cy Ha, is present insecondary mesenchyme cells between blastula andgastrula stages, a time when secondary mesenchymestill possesses skeletogenic potential (Cox etal. 1986).Secondary mesenchyme pluripotentiality seems topersist rather long. Furthermore, the observationimplies that primary mesenchyme somehow imposesa suppression of the skeletogenic potential of second-ary mesenchyme cells (Ettensohn & McClay, 1986).This seems to be an excellent situation to study long-range cell interactions; primary mesenchyme may beisolated (Ettensohn & McClay, 1987) and it might notbe too difficult to isolate populations of secondarymesenchyme as a population of outwandering cellsfrom archenterons (McClay & Marchase, 1979).Good markers of gene expression characteristic ofboth primary and secondary mesenchyme are avail-able and the influence of primary mesenchyme onskeletogenic potential of secondary mesenchymecould be studied in vitro.

GastrulationAn attempt to describe and understand gastrulationin terms of the behaviour of individual cells has beena long-standing effort for sea urchin development.Since Moore & Burt (1939) showed that isolatedvegetal halves could undergo gastrulation and Gus-tafson & Wolpert (1967) described changes in cellshape, motility and adhesivity, work on sea urchingastrulation has served as a model for attempts tounderstand gastrulation in other animals.

New advances have been made, both on thebiochemical and cellular fronts. Gastrulation pro-ceeds in two distinct phases: first, there is a bucklingof a thickened vegetal plate of the blastocoel wall,from which primary mesenchyme cells have alreadyemigrated. Ettensohn (1984) repeated the older ex-periment of Moore showing that this occurs in iso-lated vegetal halves. The process does not involverounding up of cells, as formerly predicted (Gustaf-son & Wolpert, 1967), nor is cell division or DNAsynthesis important in either this or the second phaseof gastrulation (Stephens, Hardin, Keller & Wilt,1986). My associates and I incorrectly attributed thehypothesis that cell division might play a role in

gastrulation to Wolpert and we regret this incorrectattribution. Ettensohn (1984) showed that less than100 cells of the approximate 500-cell mesenchymeblastula are involved in the invagination, and there isno further contribution of the epithelium of theblastula to the archenteron after the original vegetalplate has buckled. The signals for the beginnings ofinvagination, the role of primary mesenchyme ininvagination and the mechanics of the primary invagi-nation process are still a mystery.

The second phase of gastrulation, the elongation ofthe buckled plate across the blastocoel, is now betterunderstood. Ettensohn (1985) showed thatelongation was accompanied by repacking of cells. Ashort, squat cylinder with many cells along its per-imeter is converted into a longer, thinner cylinderwith a much smaller number of cells in the circumfer-ence. The archenteron epithelium behaves like afluid, cells repacking and changing neighbours duringelongation. There are junctional complexes betweenthe cells; different regions of the archenteron actuallypossess slightly different kinds of desmosomes, andthese desmosomes persist through gastrulation.Whether these junctional complexes undergo mech-anical and structural change, or form and reformat new positions during gastrulation, is unknown.Hardin & Cheung (1986) confirmed the conclusionson cell repacking as a mechanism of archenteronelongation. They carried out quantitative morpho-logical analysis and computer simulations and showedthat active cell repacking was probably a majorinfluence in elongation. They also adduced evidenceagainst an extensive role of filopodial contractionpulling the archenteron across the blastocoel. Theypointed out that pulling should also deform the apexof the blastula roof and produce shape changes at theblastopore end of the archenteron, but these are notobserved. Slight apical deformation is seen in somespecies of sea urchins from Japan, but not to theextent required by mechanical theory. More telling isa reanalysis of exogastrulation in S. purpuratus and L.Pictus by these workers. Perfectly normal but evertedarchenterons may form during exogastrulation andthese produce normal subdivision of the gut andelaborate esterases typical of gut. These guts areextended and as long as those in normal completegastrulae. In this instance, there can be no filopodialpulling. These exogastrulae do not form by eversionof normal invaginated archenterons, but developgradually with the same tempo of secondary gastru-lation as in the controls. Hence, even though filopo-dial exploration may be important in location of thestromodeum or play a subservient role in somespecies, cell repacking in the archenteron is probablythe primary driving force for elongation. It is interest-ing to note that cell repacking is also proposed as the


dominant motive force in amphibian gastrulation(Gerhart & Keller, 1986).

Hardin (1987) has recently shown that the primaryand secondary phases of gastrulation can take place inthe presence of sufficient levels of nocodazole todisrupt microtubules, while the colchicine analogue,/Mumi-colchicine, stops gastrulation but does notdisrupt microtubules. The microtubular assembly isprobably not involved in invagination or archenteronelongation. The way now seems open to examinechanges in cell shape, adhesivity and motility, andhow these properties bring about primary invagina-tion and cell repacking.

Another aspect of gastrulation that deserves com-ment is the role of the extracellular environment ofthe blastocoel and cell-surface-associated moleculesin the process. Just as in the case of studies withprimary mesenchyme cells, the changing compositionof basal lamina and the blastocoel contents arerelevant to gastrulation and are described in detailby Solursh (1986; see also Welply, Lau & Lennarz,1985; Wessel, Marchase & McClay, 1984; Yamaguchi& Kinoshita, 1985). As in primary mesenchyme cellingression, interference with metabolism of mol-ecules of the extracellular matrix often arrests gastru-lation. It would be helpful to know if such inhibitionstops both primary and secondary gastrulation, orjust secondary elongation, but this is not usuallynoted. I have noticed that embryos in sulphate-freesea water often undergo a limited form of invagina-tion; a small invagination may appear, resulting ina kind of button-like appearance, which then, how-ever, progresses no further. Tunicamycin, whichinhibits dolichol-phosphate-mediated AMinked glyco-sylations, and (5 xylosides, which inhibit processingand elaboration of polysaccharide components ofglycoproteins, both stop gastrulation (Schneider etal.1978; Solursh et al. 1986).

Several lines of recent evidence implicate collagenin gastrulation, though the description is incomplete.The essential finding is that interference with en-zymes that form hydroxyproline or hydroxylysine,also interfere with gastrulation (Mizoguchi & Yasu-masu, 1982a,fr, 1983a,b). Prolyl hydroxylase, anFe2+-requiring enzyme, may be inhibited by thechelator, a',O'-di-pyridyl, which also inhibits gastru-lation. Zn2+ also inhibits prolyl hydroxylase and theenzyme is stimulated, according to these authors, byascorbate and a--ketoglutarate. Ascorbate and a-ketoglutarate partially reverse effects of Zn2+ ongastrulation. But Zn2+ has many effects on cellularmetabolism, and the specificity of ascorbate and a-ketoglutarate action have not been satisfactorily de-termined. Hence, the evidence for involvement ofproline hydroxylation is only suggestive.

Evidence for involvement of collagen cross-linkingis somewhat stronger. Both Wessel & McClay (1987)and Butler, Hardin & Benson (1987) report that thelathyrogen /3-aminoproprionitrile (BAPN) preventscollagen cross-linking by inhibition of lysyl oxidase insea urchin embryos and prevents gastrulation. Theeffect is reversible, even after 96 h of developmentalarrest. Upon removal from BAPN, gastrulation iscompleted. Both groups of workers present evidencethat BAPN has little general effect on metabolism inthe embryo and that the BAPN actually does preventcollagen cross-linking. The completion of gastru-lation after 96 h arrest in the lathyrogen is remark-able, implying that the process that drives cell repac-king is very stable. There is no explanation, however,for why collagen might be necessary. When BAPNexposure begins after gastrulation starts, secondaryelongation continues, but the archenteron is flaccidand kinked, rather than turgid. Perhaps collagen onthe basal, blastocoel surface of the archenteron servesto stabilize consequences of other morphogeneticmanoeuvers.

Conclusion

There are opportunities to re-examine determi-nation, differentiation and morphogenesis in seaurchin embryos using modern tools of cellular andmolecular biology. Many of the questions and para-doxes that I have discussed are ready for experimen-tal attack. In the process of analysis many old ideasmay fall by the wayside, but we should soon gain amore complete understanding of sea urchin develop-ment. This will undoubtedly shed some light on howother embryos, like vertebrates, carry out develop-ment. I should like to briefly list some of the questionsthat have arisen for me as I have prepared this essay,as a way of summarizing some of the conclusions.

When is the oral-aboral axis first specified and howstable is this specification?

Does the expression of genes in animal half charac-teristic of surface epithelium depend upon interac-tions with cells from the vegetal hemisphere?

Can animal half cells be respecified by interactionswith micromeres or with lithium when modern mol-ecular markers are used as indices of differentiation?

Do all animalizing agents work by causing develop-mental arrest or is there a variety of mechanisms?

Is the vegetalizing influence seen in classical exper-iments a coherent response to some localized deter-minants) or can the various behaviours associatedwith 'vegetal' determinants be separated from oneanother?

Do dissociated cells from blastula and older stagessort out from one another to reconstruct the pluteus,or is respecification of cell fate involved?

572 F. H. Wilt

How do primary mesenchyme cells exert a negativeinfluence on secondary mesenchyme cells that sup-presses the skeletogenic potential of the latter?

Do primary mesenchyme cells truly play a role inprimary gastrulation?

What is the molecular mechanism by which cellrepacking drives archenteron elongation?

I appreciate the criticisms of my colleagues at Berkeley,especially Steve Benson, Jeff Hardin, Chris Killian, NickGeorge, Takashi Kitajima, and Laurie Stephens. I amgrateful to Hugh Woodland and Eric Davidson for theircriticisms. Work in my laboratory is supported by a grantfrom the National Institutes of Health.

References

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AMEMIYA, S., AKASAKA, K. & TERAYAMA, H. (1979).Reversal of polarity in ciliated cells of the isolated seaurchin pluteus gut. /. exp. Zool. 210, 177-182.

AMEMIYA, S. K. & TERAYAMA, H. (1982). Scanningelectron microscopical observations on the earlymorphogenetic processes in developing sea urchinembryos. Cell Diff. 11, 291- 293.

ANGERER, R. C. & DAVIDSON, E. H. (1984). Molecularindices of cell lineage specification in sea urchinembryos. Science 226, 1153-1160.

BENSON, S. C , BENSON, N. C. & WILT, F. (1986). The

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