Apluteal development of the sea urchin Holopneustes purpurescens Agassiz (Echinodermata: Echinoidea: Euechinoidea)

Zoological Journal of the Linnean Society (1995), 114: 349–364. With 5 figures

Apluteal development of the sea urchin Holopneustes purpurescens Agassiz (Echinodermata: Echinoidea: Euechinoidea) VALERIE B. MORRIS School of Biological Sciences, Zoology A08, University of Sydney, NSW 2006, Australia Received July 1994, accepted for publication October 1994

The external features of a shortened, apluteal development (lacking a pluteus larva) are described. Some features are unusual for echinoids. The large egg is distinctively marked by dark and pale coloured yolk. The sperm entry point is marked by a dark yolk spot and the first cleavage plane in most embryos is through the meridian on which the sperm entry point lies. Dark yolk in the animal hemisphere segregates largely to one blastomere in the two-cell embryo and pale yolk segregates to the other as a result of yolk movements during the first cell cycle. Progeny of the pale-yolk blastomere form adult oral structures and progeny of the dark-yolk blastomere form adult aboral structures. There is no feeding planktonic pluteus larva. The gastrula develops into a demersal vestibula larva with bilateral symmetry. The plane of symmetry is coincident with the Carpenter axis that defines a plane of symmetry through the madreporite in adult echinoderms. The coincidence shows that the anterior ambulacrum is vegetal with respect to egg polarity and the interradius originating at the madreporite is animal. The bilateral symmetry of the vestibula offers insight into the origin of radial symmetry in echinoderms and the body plan of an echinoderm ancestor. © 1995 The Linnean Society of London

ADDITIONAL KEY WORDS:*yolk marker – egg polarity – cytoplasmic movements – sperm entry point – first cleavage plane – oral : aboral lineage – demersal larva – bilateral symmetry – Carpenter axis – radial symmetry – echinoderm ancestry.

CONTENTS Introduction . . . . . . Material and methods . . . . Results . . . . . . . Fertilization and early cleavage Tracing blastomere fates . . Observations of single embryos The vestibula larva . . . Discussion . . . . . . Acknowledgements . . . . References . . . . . .

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INTRODUCTION

The shortened form of development in echinoids, one lacking a pluteus larva, was first described by Mortensen (1915) in Heliocidaris erythrogramma. 0024–4082/95/080349+16 $12.00/0

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At the time, he predicted correctly that a similar shortened development would occur in Phyllacanthus parvispinus, which he described later (Mortensen, 1921), and Holopneustes purpurescens, described here, on the basis of their large, intransparent eggs. The much later description of development in Heliocidaris erythrogramma by Williams & Anderson (1975) preceded the more recent sustained interest in apluteal development, as I call it here. The focus by Raff (1987, 1992) and co-workers (Bisgrove & Raff, 1989; Wray & Raff, 1990; Henry & Raff, 1990) has been on the evolution of the change in developmental mode from the pluteal to the apluteal type, particularly in the congeneric species Heliocidaris tuberculata and Heliocidaris erythrogramma. World patterns of developmental mode in echinoids have been described by Emlet (1990). Overall, about ten species of echinoids with modified development from six lineages have been investigated (Amemiya & Emlet, 1992). At a more general level, there is interest in the evolution and loss of feeding larval stages in marine invertebrates (Strathmann, 1978) and the impact of developmental mode on larval ecology (Emlet, McEdward & Strathmann, 1987). Development in H. purpurescens is apluteal, meaning that there is no feeding planktonic pluteus larva. The gastrula develops into a non-feeding demersal larva, here named the vestibula larva, in which the oral and aboral structures of the adult sea urchin soon start to form. The morphogenesis of adult structures can thus be traced from fertilization through cleavage and gastrulation to a juvenile urchin without the intervention of a pluteal stage. Tracing the morphogenesis was helped by an unusual feature of the H. purpurescens egg: this was a distribution of dark and pale coloured yolk that in the unfertilized egg marked animal-vegetal polarity. A putative sperm entry point (SEP) also was marked by a dark yolk spot that formed after fertilization. Using these natural markings, the relationships between egg polarity, the SEP, early cleavage planes and oral-aboral polarity were elucidated in the embryo and the vestibula. The vestibula has bilateral symmetry that can be related to egg polarity. The plane of this bilateral symmetry is coincident with the long-established Carpenter axis (Hyman, 1955), which specifies a plane of bilateral symmetry in the adult urchin, so the Carpenter axis can be related now to the animal-vegetal polarity of the egg. The external features of development in H. purpurescens are described in the present report. Some features have not been reported so far for other echinoids. These are a naturally marked SEP, an adult oral-aboral polarity that can be identified in the two-cell embryo, and a bilateral symmetry in the larva that is manifest in the adult urchin. Also not reported previously is the relationship between the embryonic animal-vegetal polarity and the axes of the adult sea urchin. The connexions between the embryonic, larval and adult axes and symmetries described here for H. purpurescens offer some insight into the form from which the adult echinoderm body plan might have evolved. MATERIAL AND METHODS

H. purpurescens is an endemic Australian species that was collected from coastal waters near Sydney, New South Wales. Eggs were released from

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excised ovaries by washing in several changes of sea water. Some were secondary oocytes that then underwent a second maturation division, releasing a polar body. Eggs were fertilized with a suspension of sperm prepared from excised testes. Development was observed in live embryos under a stereomicroscope. Observations were made on samples of embryos reared in covered 70 mm diameter crystallizing dishes or on single embryos reared alone in 35 mm diameter petri dishes, from various fertilizations. The fates of early blastomeres were traced in the single embryos by drawing the cleavage patterns and yolk distribution at various times. The early cleavage divisions left marks that could be identified later in cleavage and in gastrulae and larvae. The times taken to reach particular developmental stages were for embryos reared at 20°C. For photography, live embryos were held still on a poly-llysine coated surface. Some embryos and larvae were also fixed in 2% paraformaldehyde in sea water, dehydrated in graded ethanols, cleared in xylene and mounted in DPX. For scanning electron microscopy, larvae were fixed in 2.5% glutaraldehyde in sea water, postfixed in osmium tetroxide in sea water, dehydrated in a graded acetone series, critical point dried and observed in a JSM 35C (JEOL) microscope.

RESULTS

The mature egg of H. purpurescens was large (580 mm mean diameter) and distinctively coloured by yolk (Fig. 1A). Much of the animal hemisphere, identified from the extrusion of a polar body, was marked by a dark brown yolk, while the vegetal pole was marked by a small aggregation of dark yolk (Fig. 1A, B). Between the two dark yolk regions, the egg was pale creamy white (Fig. 1A, B). The egg floated just beneath the water surface, with the animal hemisphere uppermost. Fertilization and early cleavage After fertilization, a dark yolk spot that was assumed to mark the SEP became visible on the embryo surface (Fig. 2A). The spot was obvious 20 min after fertilization and a sperm channel leading from it was identified in fixed preparations (Fig. 2B). SEPs were observed in 77% of a sample of fertilized embryos (n  349) from various parents. SEPs were best seen against the pale creamy white region of the embryo. SEPs in the animal or vegetal dark yolk regions were less easy to identify. Two apparent movements of yolk were observed in live embryos during the first cell cycle. In the first, which was obvious by 60 min after fertilization, the animal dark yolk and the vegetal dark yolk were drawn towards the SEP (Fig. 2A, B). The vegetal dark yolk formed a distinct point directed towards the SEP (Fig. 2A, B). In the second and later movement, the animal dark yolk rotated approximately about an equatorial axis through the meridian on which the SEP lay (Fig. 2A, C). The animal dark yolk rotated from an approximately polar position towards an equatorial position (cf. Figs 1B and

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2A). The rotation was inferred from a displacement of dark yolk in the animal and vegetal regions, with streaking in the supposed plane of rotation. Largely as a result of the second yolk movement, the animal dark yolk was segregated mostly to one blastomere of the two-cell embryo at the first cleavage division (Fig. 2C, D), which occurred about 100 min after fertilization. The cytokinetic constriction of the first division passed through or close to the meridian on which the SEP lay in most embryos (Fig. 2C), so the first cleavage plane was largely to one side of the animal dark yolk which had rotated towards an equatorial position. The first cleavage plane was through or close to the SEP meridian in 80% of a sample of embryos with an identifiable SEP (n  368) from various parents. The animal dark yolk was segregated to one or other blastomere, left or right of the SEP in equatorial view (Fig. 2C). It was to the left in 36% and to the right in 48% of embryos in the sample above in which the first cleavage plane passed through the SEP meridian (n  294). In the remaining 16%, the animal dark yolk was distributed approximately equally between the two blastomeres. The blastomere to which the animal dark yolk segregated may have been influenced by a slightly eccentric position of the dark yolk in the animal hemisphere of the unfertilized egg. The animal dark yolk was eccentric with respect to the animal-vegetal polar axis in 79% of a sample of unfertilized eggs (n  111) from various females. The rotation of animal dark yolk after sperm entry may have been towards the side where more of it happened to lie. At the second cleavage divisions, the two cleavage planes were usually tilted oppositely to the polar axis so as to form cross furrows at the animal and vegetal poles. The third cleavage planes, which were approximately equatorial, were also each tilted forming cross furrows at junctions with previous planes. Such cross furrows are a general feature of cleavage patterns (Morris, Dixon & Cowan, 1989). In some embryos, the orientations of second and third cleavage planes were reversed: one of the second planes was approximately equatorial while the next (third) planes in the progeny were approximately meridional. The fourth and fifth cleavage divisions subdivided each blastomere of the eight-cell embryo into four cells, making a 32-cell embryo, but rarely did cleavage planes contact the animal pole as generally

00000000000000000000000000000000000000000000000 3 Figure 1. Unfertilized eggs and embryos of H. purpurescens, photographed live. A, unfertilized egg with the animal-vegetal axis tilted away from the observer to show the disc of dark yolk at the vegetal pole and part of the extensive cap of dark yolk in the animal hemisphere. B, unfertilized egg with the animal-vegetal axis parallel to the observer showing a small pale bleb (arrow) at the animal pole associated with polar body extrusion. C–F and H, embryos in animal-pole view, G in vegetal-pole view, oriented with the site of the first cleavage plane vertical and the future oral region to the left and the aboral region to the right. The dark yolk is largely to the right of the site of the first cleavage plane. C, 16-cell embryo. D, blastula composed of many cells; the domains of blastomeres of the 16-cell and 32-cell embryos are still identifiable in places, for example in the dark upper right quadrant (c. 12 h). E, shortly before hatching, with the domains of blastomeres of the 32-cell embryo identifiable in the upper right quadrant (c. 16 h). F, after hatching, when the infolds marking the domains of early blastomeres have smoothed out (c. 18 h). G, early gastrula showing the archenteron (c. 19 h). H, early gastrula with the infold of the first cleavage plane smoothed out but with the site of the first cleavage plane still visible (c. 19 h). The magnifications in A–H are the same; scale bar  100 mm.

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depicted for radial cleavage. No micromeres were formed at the fourth divisions, as they are not in other echinoids lacking a pluteal stage (Mortensen, 1921; Raff, 1987). Tracing blastomere fates The first, second and third cleavage planes were distinctly marked by infoldings of the hyaline layer into the cytokinetic furrows (Fig. 2C, D). The marks left by the infolds remained visible for some time, enabling the sites of these cleavage planes to be identified at later developmental stages (Figs 1C–F, H, 2E–G). The fourth and fifth cleavage planes were also sometimes identifiable (Fig. 1D, E). The marks of the early cleavage planes and the distribution of dark and pale yolk helped in tracing the fates of early blastomeres. In the late unhatched blastula (Fig. 2E), the site of the first cleavage plane was marked by a deep infold separating a region of mostly pale yolk from a region of dark yolk (Fig. 2E). The pale and dark yolk regions were descended respectively from the blastomeres to which the pale and dark yolk had been segregated in the two-cell embryo. Evidence of this and of the deep infold in the late blastula marking the site of the first cleavage plane was obtained by observing the development of single embryos. At hatching, the infold of the first cleavage plane opened out but still separated pale and dark yolk regions (Fig. 2F). The infold of the first cleavage plane separated a region of mostly pale yolk from a region of dark yolk in 83% of a sample of embryos selected at hatching where the infold was meridional (n  315), that is, where it bisected the shallow archenteric cup at the vegetal pole (v.i.). This frequency was similar to that (84% v.s.) at which pale yolk

00000000000000000000000000000000000000000000000 3 Figure 2. Embryos of H. purpurescens from fertilization to the early gastrula, photographed live except for B. A, equatorial view shortly before the first cleavage. The vegetal dark yolk (white arrow) is pointed towards the SEP (black arrow) and the animal dark yolk has rotated right relative to the SEP. A slight flattening at the animal pole precedes the first cleavage. B, equatorial view of a fixed wholemount before the first cleavage but turned to the left with respect to A. SEP (white arrow) with a sperm channel (black arrow) leading from it; tip of the vegetal dark yolk is pointed towards the SEP. C, the same embryo and view as A with the first cleavage furrow (arrowhead) now distinct but slightly to one side of the SEP (arrow). The animal dark yolk is segregated largely to the blastomere to the right of the SEP. D, animal-pole view towards the end of the second cleavage cycle (140 min). The animal dark yolk has segregated largely to one blastomere; the first (one arrowhead) and second (two arrowheads) cleavage furrows are evident. E, animal-pole view of a late blastula shortly before hatching (16 h). The animal dark yolk is largely on one side of the first cleavage infold (small arrowhead); infolds marking probably the second (two arrowheads) and fourth (large arrowhead) cleavage planes persist. F, the same embryo and a view as E just after hatching (17 h). The gastrula has spread out along the oral-aboral axis, no longer constrained by the fertilization membrane, and the infold of the first cleavage plane is marked by prominent indentations (arrowheads) in the profile. G, early gastrula (19 h) in lateral view. The site of the first cleavage plane lies between the two arrowheads; the upper one also marks the animal pole and the lower one the vegetal pole; the site of a third cleavage plane in one aboral blastomere is arrowed. The region to the right of the first cleavage plane will become aboral while the pale yolk region to the left will become oral; the pale yolk region occupies less of the gastrula surface because most of the progeny of the vegetal oral blastomeres of the eight-cell embryo have become part of the archenteric invagination. Scale bar  100 mm.

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was separated from dark yolk by the first cleavage plane, in embryos where the plane passed through the SEP meridian. The infolds of the early cleavage planes in the vegetal region contributed to gastrulation by transforming into a shallow archenteric cup at hatching. Later, in the early gastrula, the marks of the first and third cleavage planes (Fig. 2G) showed that the archenteric invagination (Fig. 1G) formed largely from the progeny of two vegetal blastomeres of the eight-cell embryo that were descendants of the pale-yolk blastomere of the two-cell embryo. The region of pale yolk that remained visible on the surface of the early gastrula was thus reduced to less than half of the total surface (Fig. 2G). The cells of this pale yolk region were descended largely from the two animal blastomeres of the eight-cell embryo that were progeny of the pale-yolk blastomere of the two-cell embryo. An asymmetric gastrulation has also been reported in Heliocidaris erythrogramma (Wray & Raff, 1990), where six of the eight vegetal blastomeres of the 16-cell embryo contributed to internal cell types. The late gastrula was elongate and demersal. The pale yolk region still occupied less than half the surface area, extending from a small blastopore to part way along one side of the gastrula. It was from this pale yolk region that the vestibule and oral structures developed in the vestibula larva. Observations of single embryos The fates of the two blastomeres of the two-cell embryo, one marked by pale yolk and the other by dark yolk, were traced by observing single embryos from fertilization to the early vestibula where oral and aboral structures were evident. In 26 out of 27 embryos so observed, the vestibule and oral structures formed from the pale-yolk blastomere, while aboral structures formed from the dark-yolk blastomere. The fate of the pale-yolk blastomere was independent of its position to the left (13 embryos) or right (13 embryos) of the SEP, as described above, in these 26 embryos. In the other embryo, the dark yolk did not segregate clearly to either blastomere at the two-cell stage but lay across the first cleavage plane. The vestibule in this embryo developed across the first cleavage plane in the region marked by pale yolk opposite the region of dark yolk. In a supporting study, observations were started at hatching rather than at fertilization. For this, samples of embryos were selected at hatching for (a) segregation of dark yolk to one side of the infold marking the first cleavage plane (n  81) and (b) equal distribution of dark yolk across the infold (n  16). In both (a) and (b) the vestibule developed most often in the region lacking the dark yolk. The vestibule thus developed to one side of the first plane in embryos selected for (a) (76 embryos) but across it in those selected for (b) (14 embryos). In the few remaining embryos, five from (a) and two from (b), the vestibule spanned the pale-dark boundary between the regions of pale and dark yolk, showing that oral-aboral fate can be displaced from a strict alignment with the pale and dark yolk distribution. However, the vestibule never formed solely on the side marked by the dark yolk. Oral-aboral polarity in H. purpurescens can thus be identified at least as early as the two-cell embryo. The observations of single embryos and the

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data from the supporting study of hatched embryos showed that yolk segregation, or the cytoplasmic rotation possibly causing it, is more important in determining oral-aboral fate than the site of the first cleavage plane. The lack of importance of cleavage-plane sites in determining cell fate was also evident from the reversed order of second and third cleavage planes, described above, which did not alter cell fates from those expected from the yolk distribution. In Heliocidaris erythrogramma, a segregation of developmental potential during the first cell cycle has also been shown not to be tied to the first cleavage plane (Henry, Wray & Raff, 1990).

The vestibula larva The vestibula larva, named for its large vestibule, developed from the late gastrula. A flattening appeared on the oral side over the pale yolk region descended from the two animal blastomeres of the eight-cell embryo, that were descended from the pale-yolk blastomere of the two-cell embryo. The flattening encompassed the blastopore, which was subterminal on the oral side. At this stage, the larva swam oral side down, aboral side up, and, in the fashion of a protostomate, with the blastopore leading. The flattening on the oral side formed a vestibular plate (Fig. 3A). A fold developed around the edge of the plate (Fig. 3A), then drew together (Fig. 3B) to form a vestibule with an external opening. Five primary tube feet formed on the plate within the vestibule. Two pairs formed in an animal to vegetal series, with the members of each pair sited symmetrically either side the plane of bilateral symmetry of the larva (Fig. 3C). The fifth primary tube foot, the pentapod, formed in the midline and was the most vegetal of the series, closest to the blastopore (Fig. 3C). On the aboral side of the vestibula, the genital plates formed. Genital plate 2, identified from the hydropore channel, was in the midline in the most animal position furthest from the pentapod (Fig. 3D, E). The plane of bilateral symmetry of the vestibula was thus through genital plate 2 and the pentapod, as well as through the animal pole and the blastopore. The plane of bilateral symmetry of the vestibula is coincident with the Carpenter axis of bilateral symmetry (Fig. 5A) used in descriptions of the adult morphology of all classes of echinoderms (Hyman, 1955). The coincidence shows that ambulacrum A of the Carpenter system (Hyman, 1955) originates at the pentapod and so is vegetal with respect to egg polarity, while the interradius originating at genital plate 2 is animal with respect to egg polarity. Since the plane of bilateral symmetry of the vestibula also usually coincides with the plane of rotation of the animal dark yolk orthogonal to the first cleavage plane, described above, the Carpenter axis also has a relationship with early cytoplasmic movements. Scanning electron micrographs of the vestibula (Fig. 4) show the vestibular opening and tube feet within the vestibule (Fig. 4A, B). The surface of the vestibula is uniformly ciliated without a prominent ciliary band (Fig. 4A, C, D). Cilia are surrounded at their bases by villi (Fig. 4E). Metamorphosis in H. purpurescens was simple. The rim of the vestibular opening relaxed outwards to the ambitus and then contracted over it towards

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Figure 3. Vestibula larvae of H. purpurescens, A and B photographed live, C, D, E fixed wholemounts, vegetal region lowermost. A, oral view of an early vestibula (33 h) shows the flattening of the vestibular plate with a fold (black arrow) developing around its edge. The plate forms over the pale yolk region descended from the oral blastomere of the two-cell embryo that remains on the surface after gastrulation (cf. Fig. 2G). The site of the first cleavage plane is a line visible in places (white arrow); approximate position of the animal pole (arrowhead). B, oral view at later stage (35 h) where the fold around the edge of the plate has begun to draw together. C, oral view of a late vestibula (8 d) with two pairs of tube feet either side of the sagittal plane and the pentapod (arrow) sited medially and vegetally. D, aboral view of a late vestibula (8 d) showing the site of the hydropore channel (white arrow) which is at higher magnification in E; pentapod (black arrow). E, skeleton of the hydropore channel (*) is at the edge of genital plate 2, whose central spicule is arrowed. Scale bars: A–D  100 mm; E  10 mm.

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Figure 4. Scanning electron micrographs of the vestibula (A–D, 5 days; E, 2 days). A, oral view, vegetal region lowermost, scale bar  100 mm. B, tube feet within the vestibule of A at higher magnification, scale bar  50 mm. C, profile of the ciliated surface, bar  10 mm. D, cilia on the surface near the vestibular opening, scale bar  10 mm. E, cilia with their bases surrounded by villi, scale bar  1 mm.

the aboral pole, everting the structures of the vestibule before relaxing back to the ambitus. DISCUSSION

Three features of the development of H. purpurescens are of particular significance. The first is the coloured yolk, which acts as a marker of egg polarity and of blastomeres during early and late development. The second is the absence of a pluteus larva which allows the fates of early blastomeres to be traced to adult structures. The third is the bilateral symmetry of the vestibula which enables adult symmetry to be related to egg polarity. The bilateral symmetry leads to speculation about the origin of radial symmetry in a bilaterally symmetric echinoderm ancestor. The distinctive distribution of dark yolk marking the animal hemisphere and the vegetal pole of the unfertilized egg is so far unique for echinoids.

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The only other known externally visible markers of polarity in unfertilized eggs of echinoids are Boveri’s pigment band in Paracentrotus eggs, and the jelly canal and polar bodies, which are rarely seen (Schroeder, 1980a). The dark yolk spot that forms after fertilization was assumed to mark the SEP. This assumption was based on the time of appearance of the dark yolk spot and its connexion internally to a channel of dark yolk through which the sperm may move. A naturally marked SEP is also unique for echinoids. With the markers for the SEP and egg polarity, the relationship between the first cleavage plane, the SEP and egg polarity could be observed in a large sample of embryos. The observations established that in most embryos the first cleavage plane passes through or close to the SEP meridian. Previously, Ho¨rstadius (1973) wrote that the balance of evidence was against a relationship between the SEP and the first cleavage plane in echinoids, and Schroeder (1980b) has also argued against a relationship. Schatten (1979), however, reported that the first mitotic apparatus is oriented perpendicular to the egg radius passing through the SEP in Lytechinus variegatus. The location of the first cleavage plane in H. purpurescens is similar to that in Xenopus, where the first cleavage plane is commonly close to the SEP meridian (Danilchik & Black, 1988). The first cleavage plane in H. purpurescens is most often orthogonal to the adult body axis, which is the oral-aboral axis, as it also is in Heliocidaris erythrogramma (Wray & Raff, 1990). The movements of yolk during the first cell cycle in H. purpurescens lead to the segregation of dark yolk largely to one blastomere and pale yolk to the other in the two-cell embryo. The segregation appears to have morphogenetic significance since the fates of the two blastomeres can be predicted from their yolk content. The second yolk movement, which is an apparent rotation of dark yolk about an equatorial axis, is reminiscent of the subcortical cytoplasmic rotation during the first cell cycle in Xenopus embryos which specifies dorso-ventral polarity (Vincent, Oster & Gerhart, 1986). The plane of rotation in Xenopus is coincident with the plane of bilateral symmetry of the Xenopus larva, just as the plane of rotation in H. purpurescens is coincident with the plane of bilateral symmetry of the vestibula. So, the rotation in H. purpurescens may have a role in specifying oral-aboral polarity. The development of H. purpurescens is apluteal, progressing after gastrulation to the vestibula, which has the body plan of the adult urchin. Because the egg and the vestibula are large, development can be followed in live embryos and larvae at the magnifications available in a stereomicroscope. Using such methods of direct observation, the progeny of the two blastomeres of the two-cell embryo were traced to their fate in the adult body plan. The orally sited vestibule and five primary podia form from progeny of the pale-yolk blastomere, while the aborally sited apical system of genital and ocular plates forms from progeny of the dark-yolk blastomere. A separation of oral and aboral fate between the blastomeres of the two-cell stage in Heliocidaris erythrogramma was demonstrated by injecting fluorescent-labelled markers (Wray & Raff, 1990). The oral and aboral blastomeres could not be identified as such, however, as they can in H. purpurescens. The reliable prediction of oral-aboral fate from the distribution of pale and dark yolk in blastomeres of the two-cell embryo shows that such fate is specified before the first cleavage plane forms, a conclusion also reached by

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Henry et al. (1990) for Heliocidaris erythrogramma after experimental manipulation of cleavage planes. The possibility that oral-aboral fate is, moreover, specified by a cytoplasmic rotation after sperm entry has been raised above, on the evidence (Vincent et al., 1986) that a subcortical cytoplasmic rotation specifies dorso-ventral polarity in Xenopus embryos. Maternal factors might also influence oral-aboral fate, as suggested for Heliocidaris erythrogramma (Henry et al., 1990), given the possibility that the slightly eccentric position of the dark yolk in the animal hemisphere of unfertilized eggs of H. purpurescens affects cytoplasmic movements in the first cell cycle. The lesser importance of the site of the first cleavage plane in determining cell fate, reported for Xenopus (Danilchik & Black, 1988) and Heliocidaris erythrogramma (Henry et al., 1990), is also evident in H. purpurescens, since the site of the first cleavage plane does not always coincide with the division between oral and aboral fates. The most significant feature of the vestibula is perhaps its bilateral symmetry. The relationship between the symmetry of the vestibula, the adult urchin and egg polarity became apparent when genital plate 2 was identified from the hydropore channel. The plane of bilateral symmetry of the vestibula turned out to be through the Carpenter axis of bilateral symmetry, which is through the madreporite (Hyman, 1955), so the anterior ambulacrum of the early literature (Hyman, 1955) or ambulacrum A of the Carpenter system (Hyman, 1955) has vegetal polarity and the interradius opposite it originating at genital plate 2 has animal polarity. The Carpenter axis is thus coincident with the fundamental animal-vegetal polarity of the egg. It is of interest then that ambulacrum A in the vestibula is behaviourally anterior, since the vestibula swims with the blastopore leading. The bilateral symmetry of the vestibula defines animal-vegetal polarity in the adult sea urchin and raises questions of antero-posterior polarity in sea urchin morphogenesis. The bilateral symmetry of the vestibula and particularly the arrangement of four of the primary podia in pairs either side of the plane of bilateral symmetry suggest how radial symmetry might have originated in echinoderms from ancestral bilateral symmetry. The five primary podia are common to juvenile rudiments in all echinoderm groups (Hyman, 1955). A possible scenario, therefore, is that the pentapod which lies in the plane of bilateral symmetry and bestows a radial symmetry on the arrangement of primary podia could have arisen by the fusion in the midline of a pair of podial primordia. The putative bilaterally symmetrical echinoderm ancestor might then have had numerous pairs of podia or tentacles serially arranged along an antero-posterior axis. Such speculation raises the further question of the ancestry of echinoderms. It has been strongly argued that the planktotrophic form of development is ancestral in echinoderms and that echinoids lacking a pluteus are derived (Strathmann, 1978; Raff, 1987; Emlet, 1990). For several echinoids, pluteal structures are described as reduced or lost (Raff, 1987; Amemiya & Emlet, 1992). H. purpurescens, in contrast, shows no traces of pluteal structures such as perhaps the prominent ciliary band of Heliocidaris erythrogramma (Wray & Raff, 1990) or the larval arms of Phyllacanthus imperialis (Olson, Cameron & Young, 1993). The vestibula appears to be homologous with only the juvenile rudiment of the pluteus. The structural relationship between the vestibula

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Figure 5. Two views of an outline of a pluteus fitted to a diagram of the vestibula. A, aboral view of the vestibula drawn from Fig. 3D overlaid with an outline of the pluteus of Echinus miliaris, simplified from Gordon (1926), with only four of the eight pluteal arms shown for clarity. Genital plates numbered G1 to G5, with the hydropore channel (hatched) at the edge of G2; vegetal region lowermost. The frontal plane (FP) of the pluteus passes through G4 and between G1 and G2 of the vestibula. The right posterodorsal (RPD) arm rod (solid line) connects with G3 and the right postoral (RPO) arm rod (solid line) connects with G5. Ocular plates 4 and 5 (04 and 05), which grow from the left posterodorsal (LPD) and left postoral (LPO) arm rods respectively (see B), are filled; they lie outside the ring of genital plates and deeper towards the oral side (see B). The Carpenter axis is through G2 and between G4 and G5, with the anterior ambulacrum lying vegetally at 05. The symmetry of the von Ubisch axis (Hyman, 1955) can be arrived at by displacing G4 towards the apex of the pluteus in line with the frontal plane and compressing G3 and G5 towards one another. B, lateral view of the vestibula, aboral to the right, overlaid with a dorsal view of the pluteus in A. The orientation of the pluteus was allowed to be determined by the positions of the genital and ocular plates to which the arm rods give rise. 04 grows from the LPD rod and 05 from the LPO rod; G3 and G5 grow as in A. The RPO arm rod (dashed line) is connected to G5 lying beneath G4. The pluteus sits most easily angled to the left and tilted upwards, as in A.

and the pluteus (Fig. 5) was derived by applying the data of Emlet (1985) on the origin of the apical plates from pluteal arm rods. The relationship shows that the pluteal arms can be viewed simply as extensions of the body wall supported by skeletal rods, creating a planktotrophic stage on the scaffold of the adult body plan. In this sense, the pluteus is an interpolation into the morphogenetic processes leading to the adult. The pluteus is not, as the feeding larvae of vertebrates are, a transitional stage with the same basic body plan as the adult. The search for the bilaterally symmetric echinoderm ancestor needs to extend beyond the form of the planktotrophic ancestor, as was attempted in constructing the dipleurula larva, reviewed in Holland (1988). The focus on the planktotrophic stage of development in echinoderm ancestry begs the question of the form of the adult from which the echinoderm group were derived. For the answer to this question, the focus needs to be on the origin of the echinoderm adult body plan. That the ancestor might have been a worm-like creature sporting muscularly controlled coelomic protrusions with terminal adhesive pads, like tube-feet, metamerically arranged along an antero-posterior axis, is a possibility. The development of H.

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purpurescens through to a large bilateral symmetric larva thus contributes to the debate on echinoderm origins. ACKNOWLEDGEMENTS

The work was supported by research funds from the University of Sydney. I am indebted to R. Raff for stimulating my interest in echinoid development, M. Byrne and R. Emlet for reading the manuscript, J. Jeffery for drawing Figure 5 and H. Sowden for collecting sea urchins. Scanning electron microscopy was undertaken at the Electron Microscope Unit, University of Sydney, and I thank T. Romeo for assistance. REFERENCES Amemiya S, Emlet RB. 1992. The development and larval form of an echinothurioid echinoid, Asthenosoma ijimai, revisited. Biological Bulletin 182: 15–30. Bisgrove BW, Raff RA. 1989. Evolutionary conservation of the larval serotonergic nervous system in a direct developing sea urchin. Development, Growth and Differentiation 31: 363–370. Danilchik MV, Black SD. 1988. The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis I. Independence in undisturbed embryos. Developmental Biology 128: 58–64. Emlet RB. 1985. Crystal axes in recent and fossil adult echinoids indicate trophic mode in larval development. Science 230: 937–940. Emlet RB. 1990. World patterns of developmental mode in echinoid echinoderms. In: Hoshi M, Yamashita O, eds. Advances in invertebrate reproduction 5. Amsterdam: Elsevier, 329–335. Emlet RB, McEdward LR, Strathmann RR. 1987. Echinoderm larval ecology viewed from the egg. In: Jangoux M, Lawrence JM, eds Echinoderm studies 2. Rotterdam: Balkema, 55–136. Gordon I. 1926. The development of the calcareous test of Echinus miliaris. Philosophical Transactions of the Royal Society of London, Series B 214: 259–312. Henry JJ, Raff RA. 1990. Evolutionary change in the process of dorsoventral axis determination in the direct developing sea urchin, Heliocidaris erythrogramma. Developmental Biology 141: 55–69. Henry JJ, Wray GA, Raff RA. 1990. The dorsoventral axis is specified prior to first cleavage in the direct developing sea urchin Heliocidaris erythrogramma. Development 110: 875–884. Holland ND. 1988. The meaning of developmental asymmetry for echinoderm evolution: a new interpretation. In: Paul CRC, Smith AB, eds Echinoderm phylogeny and evolutionary biology. Oxford: Clarendon Press, 13–25. Ho¨rstadius S. 1973. Experimental embryology of echinoderms. Oxford: Clarendon Press. Hyman LH. 1955. The invertebrates: Echinodermata. The coelomate Bilateria IV. New York: McGraw-Hill. Morris VB, Dixon KE, Cowan R. 1989. The topology of cleavage patterns with examples from embryos of Nereis, Styela and Xenopus. Philosophical Transactions of the Royal Society of London, Series B 325: 1–36. Mortensen T. 1915. Preliminary note on the remarkable shortened development of an Australian seaurchin, Toxocidaris erythrogrammus. Proceedings of the Linnean Society of New South Wales 40: 203–206. Mortensen T. 1921. Studies of the development and larval forms of echinoderms. Copenhagen: Gad. Olson RR, Cameron JL, Young CM. 1993. Larval development (with observations on spawning) of the pencil urchin Phyllacanthus imperialis: a new intermediate larval form? Biological Bulletin 185: 77– 85. Raff RA. 1987. Constraint, flexibility, and phylogenetic history in the evolution of direct development in sea urchins. Developmental Biology 119: 6–19. Raff RA. 1992. Evolution of developmental decisions and morphogenesis: the view from two camps. Development 1992 Supplement: 15–22. Schatten G. 1979. Pronuclear movements and fusion at fertilization: time lapse video microscope observations. Journal of Cell Biology 83: 1989a. Schroeder TE. 1980a. The jelly canal marker of polarity for sea urchin oocytes, eggs, and embryos. Experimental Cell Research 128: 490–494. Schroeder TE. 1980b. Expressions of the prefertilization polar axis in sea urchin eggs. Developmental Biology 79: 428–443. Strathmann RR. 1978. The evolution and loss of feeding larval stages of marine invertebrates. Evolution 32: 894–906. Vincent J-P, Oster GF, Gerhart JC. 1986. Kinematics of gray crescent formation in Xenopus eggs: the

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