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

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  • Acta Zoologica

    (Stockholm)

    83

    : 99110 (April 2002)

    2002 The Royal Swedish Academy of Sciences

    99

    Abstract

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

    Acta Zoologica

    (Stockholm)

    83

    : 99110

    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 againstGarstangs 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: lacalli@usask.ca

    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

    AZO_103.fm Page 99 Wednesday, March 6, 2002 3:17 PM

  • Nerve cells in echinoderm larvae

    Lacalli and Kelly Acta Zoologica

    (Stockholm)

    83

    : 99110 (April 2002)

    100

    2002 The Royal Swedish Academy of Sciences

    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

    Stichopus californicus

    , 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

    m

    m, and sections were collectedat 0.5-

    m

    m intervals. Photographs were taken of the bandevery 0.5

    m

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

    m

    m or greater intervals elsewhere.Sections were cut at a thickness of 8085 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 6065 nm, and series were obtained of the anterodorsalridges, for comparison with thicker sections.

    Larvae of

    Stichopus californicus

    (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 12 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

    AZO_103.fm Page 100 Wednesday, March 6, 2002 3:17 PM

  • Acta Zoologica

    (Stockholm)

    83

    : 99110 (April 2002)

    Lacalli and Kelly

    Nerve cells in echinoderm larvae

    2002 The Royal Swedish Academy of Sciences

    101

    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, 3550 nm in diameter. The latter appear to beidentical (though denser, due to section thickness) to the

    Fig. 1A, 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 m m. 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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  • Nerve cells in echinoderm larvae

    Lacalli and Kelly Acta Zoologica

    (Stockholm)

    83

    : 99110 (April 2002)

    102

    2002 The Royal Swedish Academy of Sciences

    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 4565 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. 2A...

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