Download - Anterior neural centres in echinoderm bipinnaria and auricularia larvae: cell types and organization

Acta Zoologica

(Stockholm)

83

: 99–110 (April 2002)

© 2002 The Royal Swedish Academy of Sciences

Abstract

Lacalli, T.C. and Kelly, S.J. 2002. Anterior neural centres in echinoderm bipinnaria and auricularia larvae: cell types and organization. —

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Serial and interval electron micrograph series were used to examine theanterior part of the ciliary band system in the bipinnaria larva of

Pisasterochraceus

and the auricularia larva of

Stichopus californicus

for evidence ofganglion-like organization. The bipinnaria has paired concentrations of Multi-polar with Apical Processes (MAP) cells in this region that correspond inposition with previously identified clusters of serotonergic and peptidergicneurones. MAP cells located in the centre of the band have well-developedapical processes, but no cilium. Those at the sides of the band have fewerprocesses, but some have recumbent cilia that extend under the glycocalyx,suggesting a sensory function. Comparable cell types are not found elsewherein the band, a clear indication that the apical parts of the ciliary band systemare organized in a distinctive fashion. Two neuronal cell types were identifiedin the apical region of the auricularia larva, a conventional bipolar neurone thatcorresponds with previously described serotonergic apical cells, and morenumerous MAP cells for which there is no previous record and hence, noknown transmitter. Previous immunocytochemical studies are summarizedand re-examined in the light of these results. Relevant evolutionary issues arealso discussed, but the data fail to provide strong evidence either for or againstGarstang’s hypothesis that the chordate brain and spinal cord derive fromlarval ciliary bands resembling those of modern echinoderms.

Thurston Lacalli, Department of Biology, University of Saskatchewan, Saskatoon, Sask., Canada S7N 5E2. E-mail: [email protected]

Blackwell Science Ltd

Anterior neural centres in echinoderm bipinnaria and auricularia larvae: cell types and organization

Thurston C. Lacalli and Samantha J. Kelly

Biology Department, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E2

Keywords:

bipinnaria, auricularia, ciliary band innervation, apical organ, larval nervous systems

Accepted for publication:

4 July 2001

Introduction

Planktotrophic echinoderm larvae are supplied with an elab-orate set of ciliary bands that are used for both locomotionand feeding. The bands have a ciliary nerve running basallyalong their length, and this is assumed to be involved incontrolling ciliary beat. Electron microscopy (EM) has beenused to identify neurones in the band, but a better under-standing of the overall organization of the larval nervoussystem has been obtained from studies of whole larvaestained by chemical or immunocytochemical methodsfor specific transmitters, e.g. catecholamines, serotonin, orneuropeptides (Burke 1983; Burke

et al

. 1986; Bisgrove andBurke 1987; Nakajima 1987a,b; Thorndyke

et al

. 1992;Moss

et al

. 1994; Chee and Byrne 1999). These methodsreveal a diffuse system of neurites derived from cells scattered

along the band, often containing catecholamines, and fromganglion-like neuronal clusters at specific locations, notablyin the oral region and the apical plate. Both serotonergic andpeptidergic neurones have been identified in the latter (e.g.Thorndyke

et al

. 1992; Moss

et al

. 1994), and these typicallyform a loose plexus of neurites of strictly local extent. Thebipinnaria differs from other echinoderm larvae in having asymmetrical pair of ganglion-like neuronal clusters locatedslightly behind the apex (Nakajima 1987b; Moss

et al

. 1994),rather than a single apical centre.

Despite these studies, our understanding of how ciliarybeat is controlled in echinoderm larvae is still very fragmen-tary. Only some of the larval neurotransmitters have so farbeen identified and localized, and comparatively few behavi-oural studies have been done to test those that are known. Inaddition, the larvae are too large for a thorough EM analysis

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Nerve cells in echinoderm larvae

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using serial sections, which is the only way to catalogue all ofthe neurones present, irrespective of their contents. Func-tional interpretation is complicated by the absence of con-ventional synapses, a characteristic of echinoderms generally(Cobb 1987), and of their larvae as well.

This paper uses serial EM to examine neural organizationin the bipinnaria larva of

Pisaster ochraceus

and the auricularialarva of


, concentrating on the anteriorpart of the dorsal bands where the dorsal ganglia are located.This area is of special interest from a phylogenetic perspec-tive, because of its possible relation to the anterior part of thecentral nervous system (CNS) in chordates, as discussedbelow. We are especially interested in a cell type described byLacalli

et al

. (1990) from the preoral and postoral transversebands of

P. ochraceus

. Among the more conventional neu-rones and sensory cells, these authors described a class ofmultipolar neurones with unusual apical processes (Fig. 1).The processes traverse the surface of the ciliary band, form-ing periodic surface junctions with the band cells. Cells ofthis general type, which we refer to here as Multipolar withApical Processes (MAP) cells, were first reported from theciliary bands of pluteus larvae (Ryberg and Lundgren 1977;Nakajima 1986b). Nakajima (1986a) has described similarcells with recumbent cilia and branched apical processesfrom the apical and oral field epithelium of pluteus larvae,and concluded that they are sensory in nature. MAP cells arenow known to occur in the ciliary bands of bipinnaria, auric-ularia and tornaria larvae (Lacalli and West 1993; Lacalli andGilmour 2001), but their function is not known. To assessthis, it would be useful to know how widespread MAP cellsare in the nervous system, whether there are multiple types,and how these are distributed. Our data are not complete,but provide some useful new information on the subject. Weuse this also as an opportunity to review the existing histo-chemical and immunocytochemical data, in part to showhow limited the current understanding of larval innervationreally is, and how much still remains to be done.

Methods

Larvae of

Pisaster ochraceus

(Brandt 1835) were obtained,cultured and processed for EM as previously described byLacalli

et al

. (1990). Three 20-day bipinnaria larvae(Fig. 2A) were examined. The first was cut in the sagittalplane, and serial series were obtained through the preoraland postoral transverse bands; the results are described byLacalli

et al

. (1990). A second larva was sectioned in thetransverse plane, from the apex to just beyond the anterodorsalridge, a distance of about 320

µ

m, and sections were collectedat 0.5-

µ

m intervals. Photographs were taken of the bandevery 0.5

µ

m on the left side in the region of the dorsalganglion, and at 1-

µ

m or greater intervals elsewhere.Sections were cut at a thickness of 80–85 nm, which facili-tates cell identification by accentuating differences betweencells. Such sections are less than optimal for examining

ultrastructural details at high power, however, as is evidentin the micrographs. A third larva was sectioned transverselyat 60–65 nm, and series were obtained of the anterodorsalridges, for comparison with thicker sections.

Larvae of


(Stimpson 1857), also knownas

Parastichopus californicus

, were obtained and cultured (byT.H.J. Gilmour), and prepared for EM as described byLacalli and West (2000). Two 21-day-old larvae were exam-ined, by which stage they were 1–2 days from the onset ofmetamorphosis. Both were sectioned in the sagittal planeto obtain various views of the ciliary bands. A serial series of350 sections was obtained from one specimen showing thepreoral and postoral ciliary bands and the vertical portion ofthe anterodorsal ridge on the left side (Fig. 2B). Photographswere taken of both the transverse bands and the anterodorsalridge every second section. Cutting the apical parts of theband in the longitudinal rather than the transverse planemakes the task of reconstruction more manageable in termsof the number of sections required, but the nerve fibres aremuch more difficult to trace when cut this way. This severelylimited our ability to follow individual fibres any distancewithin the nerve itself.

Results

Pisaster ochraceus

The dorsal ciliary band (Fig. 3A) is a zone of thickened epi-thelium with a central ciliary field derived from a columnararray of uniciliate ciliary band cells. Flanking these is a tran-sitional zone of cells that bridge to the flattened oral andaboral epithelium on either side. Large mucus cells lie atirregular intervals within the band along its aboral side. Flat-tened extensions from the base of each mucus cell projectinto the nerve plexus, which, in the past, has led to theirbeing mistaken for neurones. On the oral side, adjacent tothe ciliary field, there is a file of cells with apical projectionsresembling microvilli, some of which branch. From a com-parison of adjacent sections, the projections appear to besomewhat flattened, or at least not as slender as microvilli.We refer to them here as apical folds, and to the cells as apicalfold cells. Whether the processes really branch is not clear;the appearance of branching may be due more to their ten-dency to curl and/or fold back on themselves. The cells forma file one cell wide, but the cells’ apices are significantlylonger than they are wide, and those from adjacent cells aretypically found side-by-side in any given section (e.g. as inFig. 3B). This is particularly noticeable in the ganglionicregion and more caudally, where up to four apices may bepresent in each section. The apical fold cells occur also in theanterodorsal ridges, and may well be a feature of the dorsalband as a whole. They are, however, absent from the trans-verse bands.

The ciliary nerve in the ganglionic region is more a broadplexus than a single nerve, consisting in most places of a


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number of branching and anastomosing tracts as describedby Burke (1983). The majority of neurones in the ganglionicregion resemble those shown in Fig. 3 (A) and (B). Theyhave a coarsely granular cytoplasm and flattened basalprocesses that project into the plexus. Their basal processesform smaller neurites that, by our estimation, account forabout half of the fibres in the plexus. Two subtypes of

granular neurones, both with apical specializations, couldbe distinguished. Those in the ganglionic region had pro-cesses containing clumps of dense material, but no vesicles.These were replaced near the back of the ganglionic regionby cells whose basal processes were filled with closely packedclear vesicles, 35–50 nm in diameter. The latter appear to beidentical (though denser, due to section thickness) to the

Fig. 1—A, B. The apex and cell body, respectively (*s) of a type 1 MAP (MAP1) cell from the anterodorsal ridge of a P. ochraceus larva; a thinner section than in Fig. 3 to show more ultrastructural detail. Parts of one of the apical processes are indicated (arrowheads in A); basal neurites belonging to this cell type are visible in the ciliary nerve (small arrows in B). Scale bars 2 µm. —C. A stereo three-dimensional computer reconstruction of a MAP1 cell from the preoral transverse band, to show the apical processes; based on data of Lacalli et al. (1990).



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MAP cells identified elsewhere in the band and illustrated inFig. 1. They occur in all parts of the band we have examinedexcept in the anterior region. We refer to them here as MAP1cells, and the granular cells lacking visible vesicles, whichappear to be restricted to the ganglionic region, as MAP2cells.

The MAP2 cells are irregularly distributed in the gan-glionic region, but are often clustered (Fig. 4). Assumingeach cell retains its subapical attachments to the epitheliumthroughout development, its site of origin within the bandcan be determined by the point at which the cell apexemerges at the band surface. Some MAP2 cells emergewithin the ciliary field and, like MAP1 cells, these have com-paratively slender apical processes, a basal body, or remnantof one, and no cilium. However, a significant number ofMAP2 cells emerge in the flanking transitional zone thatseparates the ciliary field from the adjacent epithelium. Thesehave broader apices, usually with a raised edge that projectsabove the surface of adjacent cells, like the rim of a platter.The rim itself appears to produce one or more blunt projec-tions, though the detailed morphology of these structureswas difficult to determine from our interval series. On theaboral side of the band, MAP2 cells are typically arranged inclusters of four, and many have cilia. The cilia are recumbent,i.e. they project below the glycocalyx (as in Fig. 3A) ratherthan penetrating through it, as locomotory cilia do. On the

oral side, MAP2 cells occur singly or in pairs, and some ofthese also have recumbent cilia. Besides the differences justnoted in apical morphology, we could not distinguish anyfurther subtypes among MAP2 cells based on morphologicalcriteria. Clearly, however, the MAP2 category, as definedhere, could include more than one cell type.

A third type of neurone was encountered sporadicallyalong the anterior ciliary band that contained scattered 45–65 nm dense-core vesicles in both its perinuclear cytoplasmand neurites (Fig. 3C). In this respect the cells closely re-semble sensory cells described from the transverse bands byLacalli

et al

. (1990). They were bipolar in the few cases wherethis could be determined, and those whose apices extendedto the surface had cilia but no other apical specializations. Itis not clear, however, whether all of them projected to theband surface. Some may not, and occasional neurite-likestructures containing a ciliary axoneme were encounteredwithin the apical band, either in the nerve plexus or passingbetween cells, which suggests that there may be a class ofneurones that detach from the band surface, yet retain theircilium.

Neurites packed with dense-core vesicles, evidently fromthe bipolar cell-type just described, were encountered allalong the ciliary nerve. They were especially common nearthe apex of the larva and in small branches from the ciliarynerve that traverse the oral field at various points. This

Fig. 2—A. A lateral view of a bipinnaria larva of P. ochraceus, ≈ 20 days old. The apex (a) of the larva is on the left and the ventral surface faces up. The mouth lies in the cleft between the preoral (pr) and postoral (po) transverse bands. The region occupied by the dorsal ganglion (*) lies just forward of the anterodorsal ridge (ar). Scale bar 300 µm. —B. A dorsal view of the apex of a 21-day auricularia of S. californicus showing the region between the apex (a) and the anterodorsal ridges (ar). Reconstructions (e.g. Figure 7) were prepared of the region enclosed by the box. Scale bar 100 µm.


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Fig. 3—The ciliary band of a P. ochraceus larva in the ganglionic region, in transverse section. —A. A typical section, orientated with the oral margin and cells with apical folds (af ) on the left; the direction of ciliary beat is from left to right. Shows two MAP2 cells (large *s), one whose apical surface emerges in this section (large arrow) surrounded by recumbent cilia (small arrows) from other such cells. The aboral MAP2 cell is one of a cluster of four cells, parts of which are visible around it (small *s). Scale bar 4 µm. —B. A detail of the oral margin near a cluster of three MAP2 cells (*s). The apical parts of all three are visible in this section (large arrows, and surface processes emerge from two of these; note that the one on the right is confined within the ciliary band, the one on the far left

emerges between the apices of two apical fold (af ) cells. A flattened basal process extends from the left MAP cell (small arrows) into the basal plexus (n). Scale bar 4 µm. —C–E. The ciliary nerve at three different anteroposterior levels: in front of the ganglionic region (C), within it (D), and behind it (E). Note the prevalence of fibres with dense-core vesicles (small arrows) in (C), and a neuronal cell body (*) also with scattered dense-core vesicles. Lightly stained neurites of unknown origin and dense processes from the MAP2 cells predominate in (D) whereas, in (E), a number of the processes (small arrows) are packed with clear vesicles, indicating that they belong to MAP1 cells; the base of one such cell is visible at the top of the figure. Scale bars 1 µm.



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suggests that such fibres are comparatively long, i.e. that theytravel some distance from their point of origin. In contrast,the basal processes derived from the MAP cells appear to bemainly local in extent. This can be seen in the changingappearance of the basal nerve plexus as one moves throughthe ganglionic region. Anterior to this region, the plexuscontains numerous lightly stained fibres of unknown origin,but few dense fibres of the type produced by MAP2 cells(Fig. 3D). The latter predominate in the ganglionic region,but largely disappear, to be replaced by MAP1 fibres morecaudally (Fig. 3E).


The anterior part of the band in this species is similar in over-all organization to the bipinnaria band, but is narrower, andthe aboral mucus cells are developed into a contiguous row,one to two cells in width (Figs 5, 6A). The mucus cells areupright and columnar in shape where they flank the band.Assuming a degree of turgidity, they probably providemechanical support for the ciliary band, which projectsabove the surrounding epithelium as a distinct ridge overmuch of its length (e.g. Fig. 2B). In contrast, the basal partsof mucus cells located near the apex project beneath thesurrounding cells (Fig. 6B,C), and the epithelium is conse-quently thinner, again consistent with a support function. Asecond, more irregularly shaped type of mucus cell contain-ing large, dense granules occurs on both sides of the band;examples are visible in Fig. 6(B).

Two nerve cell types could be distinguished in the vicinityof the apex in

S. californicus

, one a MAP-type cell with apicalprocesses, the other a flask-shaped neurone lacking suchprocesses. The MAP cells (Fig. 6B–D) have a very fine-grained cytoplasm of medium density containing sparselyscattered dense-core vesicles 40–65 nm in diameter. Eachcell has basal processes that expand to form flattened sheetsalong the top and bottom of the nerve (Fig. 6D). Thesebranch at various points, so processes on both the top andbottom of the nerve can, in fact, belong to the same cell. Thecytoplasm contained in the processes matches that in thecells themselves; it is of medium density with scattereddense-core vesicles, and so is easily distinguished from othertypes of nerve fibres. MAP cells occur outside the apicalregion (e.g. several were found in the preoral transverseband), and 10–15 MAP processes were found in randomlychosen sections through the ciliary nerve, in nerves with45–55 fibres in total. This suggests that MAP cells occurregularly along the length of the band. We have not assayedthe band outside the apical region in any detail, however, inorder to confirm this conclusion.

The MAP cells in the apical region are distributed asshown in Fig. 7(A). Their apices emerge both within theciliary field and at the edges of the band among the cells ofthe flanking transitional zone. In the case of the former, theapical processes are comparatively well developed, and runsome distance along the band. MAP apices emerging else-where generally have much reduced processes, sometimes asingle short one, and often, but not always, a short cilium.Other than the position and appearance of the apex, the cellsall appear to be of one type, i.e. we cannot identify clear sub-types among the MAP cells on morphological criteria alone.

The second nerve cell type had simple cilia, sunken in ashallow pit in some instances, and no apical processes. Thecytoplasm was more coarsely granular than that of the MAPcells, with extensive endoplasmic reticulum, multiple stacksof Golgi cisternae (Fig. 6F), and occasional dense-corevesicles. All had well-developed basal processes, and all were

Fig. 4 —A. Dorsal view of a bipinnaria larva of P. ochraceus showing the location (arrow) of the left dorsal ganglion. —B. A reconstruction of the region indicated in (A) from a single larva; shows the extent of the band as a whole (between the outer lines), mucus cells (light shading), the zone occupied by the ciliary field (darker shading), and numerous MAP2 cells, which form clusters in many places. Cell morphology, including the shapes of the apical processes, is simplified and somewhat schematic. One MAP1 (arrow) cell occurs at the back of this region, and more caudally, MAP1 cells predominate.


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bipolar with one exception, so we will refer to them as bipolarcells. They form a small plexus at the apex of the larva(Fig. 7), and are distributed along both sides of the adjacentanterior part of the band. No similar cells were encounteredin a random sample of sections through other parts of theciliary band, except for one example in the oral regionforward of the mouth. Processes from the apical bipolarcells could be traced some distance within the nerve. Thenerve contains a number of fibres with significant concentra-tions of vesicles of several types, including dense-corevesicles (Fig. 6F). However, in no case could we connect afibre containing such vesicles with a bipolar cell, though theirfibres otherwise look like neurites.

Discussion

Comparing the EM and fluorescence data

Fluorescence studies of band innervation in dipleurula-typelarvae (i.e. the bipinnaria, auricularia, pluteus and tornaria)reveal a number of common features. The ciliary nerve ismost reliably stained by the glyoxylic acid method for cate-cholamines (Burke 1983; Burke

et al

. 1986; Nakajima1987a; Dautov and Nezlin 1992; Chen

et al

. 1995), and thisis thought to be due to the presence of dopamine (Bisgroveand Burke 1987). The fibres revealed by this method gener-ally travel some distance within the nerve, and arise fromcells scattered along the band and from cell clusters in theoral region and the apex. Clusters of serotonergic neuronesare reported from the oral region as well, where they formeither adoral clusters or a circumoral ring, and also from theapex (Burke

et al

. 1986; Bisgrove and Burke 1987; Nakajima1987a; Chee and Byrne 1999). In all cases their fibres areinvariably short, forming at most a local plexus. The bipin-naria is unusual in having paired serotonergic centres locatedjust behind the apex. These have been referred to as ‘dorsalganglia’ though they appear to be no more than simple clus-ters of neurones of a few specific types. The fact that they areinvariably paired appears to be due to the continued growthof the central part of the apex to form a distinct apical lobe.This splits the initial rather diffuse population of anterior ser-otonergic cells into two, a process documented by Nakajima(1987b). Peptidergic neurones, containing the S1 (GFN-SALMFamide) peptide, are known from both pluteus andbipinnaria larvae (Thorndyke

et al

. 1992; Moss

et al

. 1994).The peptidergic cells occupy roughly the same location asthe paired anterodorsal clusters of serotonergic neurones,and it is not clear whether the two transmitters are co-localized,i.e. whether the staining is due to the presence of two sepa-rate cell types or one type containing two transmitters.

Our previous EM studies of

P. ochraceus

show that thetransverse bands contain three types of neurones (Fig. 8A):a row of sensory cells along the oral margin of the band,multipolar cells with apical processes (the MAP1 cellsreferred to above) within it, and a class of bipolar cells adjacent

Fig. 5—The anterior ciliary band in the auricularia of S. californicus. —A. Dorsal view of the front part of the reconstructed region, including one side of the apical plate. Shows the full extent of the band (between outer lines), the ciliary field (cb, darker shading), the mucus cells (mc, nuclei and surface exposure are shaded). Note that mucus cells along the band tend to be vertical (i.e. the surface exposure and cell outline coincide). In contrast, the basal parts of those in the apical region extend beneath the surrounding epithelium, and the latter is consequently flatter in these regions (e.g. as in Fig. 6B,C). —B. A typical section through the band to show the comparatively narrow file of cells responsible for the ciliary field, adjacent mucus cells (mc), the ciliary nerve (cn) and a subtrochal mesenchyme cell (st).



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Fig. 6—Selected views of the anterior ciliary band of S. californicus larvae, from longitudinal sections through the region indicated in Fig. 2(B). —A. A survey view, showing a file of ciliary band cells, the ciliary nerve (n), and one of the mucus cells (mc) lying forward of the ciliary band in this region; parts of two neurones (*s) are visible. The inset shows a detail of a MAP cell apex emerging at the surface (small arrow) and one of its apical branches (arrowhead). Scale bar 10 µm. —B, C. Sections of the apical region showing two nerve cells (*); a bipolar cell on the left, and a MAP cell on the right. The latter is also visible in (C), a nearby section; the basal parts of large mucus

cells (mc) occupy much of the thickness of the epithelium at this point. Scale bars 5 µm. —D. The basal part of a MAP cell. Its processes, along with those of other MAP cells (arrowheads) run along the top and bottom of the nerve. Scale bar 2 µm. —E. Detail of the nerve to show the variety of fibre types; many contain dense-core vesicles. Scale bar 1 µm. —F. Cell body of an apical bipolar cell. The perinuclear cytoplasm is voluminous and the Golgi is well developed; one of several stacks of cisternae (g) is visible in this section. The adjacent profiles (*s) are neurites from other apical cells of the same type. Scale bar 2 µm.


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to the ciliary nerve. Glyoxilic acid staining indicates that thesensory cells contain catecholamines (Burke 1983). Twoother classes of catecholamine-containing neurones occuralong the band (n1 and n2 cells; see Figs 31–33 in Burke1983), and these probably correspond with the bipolar cellscontaining dense-core vesicles described here. Glyoxilic acidtreatment shows no such cells in the transverse bands, how-ever, so it is unlikely that either the n1 or n2 cells, despitebeing bipolar, correspond with the bipolar cells reported byLacalli

et al

. (1990).In the anterior part of the dorsal band, as described here

(Fig. 8B), the oral margin of the band is occupied by a rowof non-neural apical fold cells rather than sensory cells and,in the ganglionic region, most of the neurones are MAP2cells. Moss

et al

. (1994) describe irregular clusters of sero-tonergic and peptidergic neurones from this same region in

P. ochraceus

. Their cells are therefore similar in distribution toour MAP2 cells, though less numerous, and in both cases theapices of many of the cells clearly project beyond the limitsof the ciliary field. The difference in cell numbers may bedue to a difference in the age of the larvae or to variabilitybetween specimens. It may also be that our results lumpseveral cell types including, for example, the serotonergicneurones, which we have not so far been able to distinguishby ultrastructural criteria. The serotonergic neurones in

S. californicus

appear not to be MAP-type cells, however (seebelow), which argues against this interpretation. We findcells containing dense-core vesicles in the apical region (asin Fig. 3C), but their distribution is inconsistent with theirbeing serotonergic, since they occur both forward of andbehind the ganglionic region.

It appears, from the above considerations, that at least asubset of our MAP2 cells correspond with the peptidergicneurones of Moss

et al

. (1994). This means also that atleast some peptidergic neurones belong to the MAP class.Catecholamine-containing neurones, in contrast, probablydo not. However, as one moves caudally out of the ganglionregion, the MAP2 cells are replaced by MAP1 cells, whichcontain clear vesicles, and these are common throughoutmuch of the rest of the band. Their distribution and mor-phology differ from any of the cell types so far demonstratedby histochemical or immunocytochemical methods, so theirtransmitter is unknown. Their numbers and ubiquity implyan important role in ciliary band function, yet their functionin the bipinnaria and the other dipleurula types in which theyoccur (e.g. the tornaria, Lacalli and Gilmour 2001) remainsa puzzle.

Fibres from both the peptidergic and serotonergic neu-rones in

P. ochraceus

form a local plexus of restricted extent.In consequence, the ciliary nerve varies in character as onemoves caudally from the apex of the larva. This can be seenalso in section: the apical part of the nerve is a mixture oflightly stained fibres without vesicles and fibres with dense-core vesicles. Closer to the ganglionic region, the nerve isprogressively dominated by dense fibres from MAP2 cells.More caudally these are replaced by fibres with clear vesiclesfrom MAP1 cells, and the transverse bands are different yetagain. It is therefore clear, as the observations of Moss

et al

.(1994) imply, that at least the anterior part of the ciliary bandconsists of a series of zones of qualitatively different character.

The data from the auricularia larva of

S. californicus

give asomewhat different picture. Here the apex is more truncatedand contains, so far as we can distinguish, only two neuronalcell types. One is bipolar, lacks apical processes, and is dis-tributed in almost exactly the same way as the serotonergiccells reported by Burke

et al

. (1986). Our cells differ fromBurke’s in the length and regularity of their processes, butthis may be due to a difference in the age of the specimens.Ours are closer to metamorphosis by several days (R.D.Burke, personal communication), so they may be more fullydeveloped. In addition, in analysing interval series, small

Fig. 7—Dorsal view of the apical part of the auricularia band (as in Fig. 5A), showing the positions of all the neurones that could be identified in section. —A. A diagram showing the MAP cells (shaded nuclei); the complex morphology of their basal processes is much simplified. The ciliary nerve (cn) is shown and, for positional reference, partial outlines of seven bipolar cells (unshaded nuclei). —B. The same region, showing the positions of the bipolar cells in relation to the ciliary field (shaded).



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irregularities tend to be ignored, so the resulting tracings aresomewhat oversimplified. The differences may, nevertheless,be real: it has been suggested that apical serotonergic cellsplay a role in metamorphosis (Bisgrove and Burke 1987;Moss et al. 1994), and delaying their final development untilnear the end of the larval phase would be consistent with thisproposal.

The nature of the MAP cells identified in the auriculariais more problematic. They are similar to those in the bipin-naria bands but, unlike the latter, subtypes cannot be dis-tinguished among them. From their ultrastructure thereforethere appears to be only one type of MAP cell in the auricu-laria, and it produces neurites containing scattered dense-core vesicles. Comparison with the fluorescence data ofBurke et al. (1986) indicates that this cell type is neithercatecholamine-containing nor serotonergic. Possibly itcontains neuropeptides, but this is only conjecture. It isnevertheless a common cell type, especially in the apicalregion, which should facilitate its eventual identification byimmunocytochemical methods.

Functional and phylogenetic considerations

Previous work indicates a role for catecholamines in ciliarycontrol, specifically in producing arrests, though this may

be an indirect effect produced by the release of a secondneurotransmitter (Lacalli and Gilmour 1990; Lacalli et al.1990). Cholinergic agonists are consistently shown tohave a stronger behavioural effect than catecholamines,however, and assuming this is a normal physiological effect,it raises the question of where the cells responsible arelocated. Because of the difficulty of testing directly for thepresence of acetylcholine by immunocytochemical methods,no acetylcholine-containing neurones have yet been demon-strated in ciliary bands in any dipleurula-type larvae. Yet inthose examples tested to date, the bands stain strongly foracetylcholinesterase (Ryberg 1973; Dautov and Nezlin1992). It is tempting to suggest that the MAP1 cells of thebipinnaria, whose transmitter is as yet unknown, may becholinergic. The presence of clear vesicles suggests fasttransmission via either acetylcholine or an amino acid trans-mitter. An alternative explanation is that acetylcholine isreleased by cells in the band for some other purpose, e.g. aspart of a neuromuscular response, and cholinesterase ispresent in the band to protect its cells from a substance thatwould otherwise interfere with ciliary beat. Because theentire system lacks identifiable synapses, it is not possibleto be certain of any of the targets of transmitter release,whether muscle cells, mesenchyme, or ciliary band cells,and paracrine release followed by action at a distance could

Fig. 8—Sections through different parts of the bipinnaria band, to show regional differences; the oral margin is on the left in both. —A. The preoral transverse band, modified from Lacalli et al. (1990). A file of catecholamine-containing sensory cells (s) runs along the oral margin of the band. Scattered mucus cells (mc) occur on the aboral side, and the MAP1 cells (*) lie within the band itself. Lacalli et al. (1990) also found examples of bipolar cells (bp) associated with the ciliary nerve (cn), but these do not stain for catecholamines, whereas catecholamine-containing bipolar cells of two types were encountered regularly by Burke (1983) in other parts of the ciliary band. —B. The dorsal ciliary band in the region of the dorsal ganglion, from this account. Apical fold cells (af ) form a continuous file along the oral margin, and MAP2 cells (*) occur on both sides of the band and within it. The latter have apical processes. The MAP2 cells that emerge in flanking regions tend, in contrast, to have recumbent cilia but less extensive arrays of apical processes. Neurites of various types contribute to the basal nerve plexus, but there is no single well-defined ciliary nerve.

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Acta Zoologica (Stockholm) 83: 99–110 (April 2002) Lacalli and Kelly • Nerve cells in echinoderm larvae


well be the way that neural control is affected in some orall of these instances. Until the cells responsible for acetyl-choline release are identified, such questions will remainunanswered.

Identifying cholinergic cells is important also from an evo-lutionary perspective. Garstang (1894) originally suggestedthat the chordate nerve cord might derive from the ciliarybands of an ancestral dipleurula-type larva. If true, then thedorsal and ventral margins of the neural tube would beexpected to derive from the oral and aboral margins, respec-tively, of the ciliary band, and the various neuronal cell typesshould be positioned accordingly. Since cholinergic motone-urones are mainly ventral in the chordate nerve cord, a cor-responding class of cholinergic neurone would be expectedto occur on the aboral side of the ciliary band. In addition,since some neural crest derivatives release cathecholamines,catecholamine-containing cells should occur on the oral sideof the band. The latter appears to be the case in the transversebands in bipinnaria larvae (Lacalli et al. 1990), but the dorsalbands are organized somewhat differently, such that neuro-nal distributions show no preference for one side of the bandover the other. The present results therefore fail to provideobvious evidence either for or against Garstang’s hypothesis.It may, however, be significant that some of the translumenalcells in amphioxus are peptidergic (Uemura et al. 1997),since these are the closest thing to a MAP cell homologue yetencountered in chordates (Lacalli 1996), and some of theMAP cells are clearly peptidergic.

A second issue concerns regional specialization of theciliary nerve along the longitudinal axis of the body, especiallyin the apical region. Following Garstang, the precursor of thechordate brain should incorporate some or all of the antero-dorsal part of the ciliary band system. The dorsal ganglion ofthe bipinnaria is located in this region, and it contains sero-tonergic cells, which also occur in the anterior nerve cord inamphioxus (Holland and Holland 1993) and in the verte-brate hindbrain (Hay-Schmidt 2000). The amphioxus dataled Lacalli (1994, 1996) to suggest that the dipleurula apicalplate is incorporated into the chordate nerve cord at its ante-rior end. Nielsen (1999) has suggested an alternative: invert-ing the body dorsoventrally, he derives the dorsal nerve cordfrom the ventral postoral loop of the dipleurula band. Thisleaves the apical plate out of the nerve cord altogether.Recent data on the expression of the T-Brain gene in thetornaria larva supports the retention of the apical organ asa CNS component (Tagawa et al. 2000). However, anothergene characteristic of forebrain, Otx, is expressed in the oralregion in tornaria and auricularia larvae, including in thepostoral transverse band (Harada et al. 2000; Shoguchi et al.2000). If Otx is a reliable regional marker for forebrainhomologues, then its presence in the postoral region wouldsupport Nielsen’s hypothesis. There is a further complica-tion, in that Otx in some lower vertebrates is also expressedin some postoral structures (Tomsa and Langeland 1999), sothe whole issue, including the reliability of Otx as a marker

in dipleurula-type larvae, is far from resolved. There are, inconsequence, good reasons for wanting to know more aboutciliary band innervation in such larvae, especially in theapical and oral regions, so as to identify structural and organ-izational features with chordate counterparts. Whether any ofthe neuronal cell types so far identified in dipleurula larvaeare truly homologues of CNS neurones in chordates remainsto be determined, but the evidence to date is suggestiveenough to indicate the need for further research.

Acknowledgements

This study was supported by NSERC Canada. We thankT. H. J. Gilmour for providing most of the larvae.

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