Development and Distribution of thePeptidergic System in Larval and Adult

Patiriella: Comparison of Sea StarBilateral and Radial Nervous Systems

MARIA BYRNE* AND PAULA CISTERNAS

Department of Anatomy and Histology, University of Sydney,New South Wales 2006, Australia

ABSTRACTDevelopment of the larval peptidergic system in the sea star Patiriella regularis and

structure of the adult nervous system in Patiriella species were documented in an immuno-fluorescence investigation using antisera to the sea star neuropeptide GFNSALMFamide 1(S1) and confocal microscopy. P. regularis has planktotrophic development through bipinna-ria and brachiolaria larvae. In early bipinnaria, two groups of immunoreactive cells appearedon either side of the anterior region and proliferated to form a pair of dorsolateral ganglia.The ganglia gave rise to fine varicose fibres that innervated the preoral and adoral ciliatedbands. Peptidergic cells also innervated the postoral ciliated band, and a nerve tract con-nected the pre- and postoral bands. Fully developed bipinnaria had a well-developed pepti-dergic system, the organisation of which reflected the bilateral larval body plan. As thebrachiolar attachment complex differentiated at the anterior end, the ganglia became posi-tioned on either side of the anterior projection, from which they innervated the complex. It issuggested, based on the distribution of S1-like immunoreactivity in association with ciliaryand attachment structures, that the peptidergic system functions in modulation of feeding,swimming, and settlement. The larval peptidergic system degenerates as the larval body isresorbed during metamorphosis. In adults, S1-like immunoreactivity was intense in theaxonal region of the ectoneural nervous system and in hyponeural perikarya. Immunoreac-tive cells in the neuroepithelium connected with the surface and may be sensory. Examina-tion of immunoreactivity in several Patiriella species attests to the highly conserved organi-sation of the peptidergic system in adult asteroids. J. Comp. Neurol. 451:101–114, 2002.© 2002 Wiley-Liss, Inc.

Indexing terms: neuropeptide; neurogenesis; sea star; echinoderm

Since the discovery of FMRFamide from a bivalve mol-lusc 25 years ago, FMRFamide related peptides have beenfound in most invertebrate phyla (Price and Greenberg,1977; Greenberg and Price, 1992; Muneoka et al., 2000).Many of these peptides reside in neuronal cells and gan-glia, where they function as neuromodulators and trans-mitters. The number of neuroactive peptides greatly ex-ceeds the number of conventional neurotransmitters, andhundreds have been characterised. The long evolutionaryhistory and conserved nature of some peptide familieshave generated interest in their phylogenetic affiliationswithin the protostomes (Dickinson et al., 1999; Muneokaet al., 2000). The major deuterostome invertebrate phy-lum, the Echinodermata, possess the SALMFamide familyof neuropeptides, and four of these have been isolated, two

from asteroids and two from holothuroids (Elphick et al.,1991a,b; Dıaz-Miranda et al., 1992; Muneoka et al., 2000).Other deuterostome taxa possess a suite of peptides, in-cluding FMRFamides but not SALMFamides (Muneoka etal., 2000). In considering the position of the Echinoder-

Grant sponsor: Australian Research Council.*Correspondence to: Maria Byrne, Department of Anatomy and Histol-

ogy, F13, University of Sydney, New South Wales, 2006 Australia.E-mail: mbyrne@anatomy.usyd.edu.au

Received 8 May 2001; Revised 4 December 2001; Accepted 7 May 2002DOI 10.1002/cne.10315Published online the week of July 29, 2002 in Wiley InterScience (www.

interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 451:101–114 (2002)

© 2002 WILEY-LISS, INC.

mata near the base of the chordate line, it is interestingthat SALMFamides appear unique to this phylum.

SALMFamides are widespread in the echinoderm ner-vous system and have been localised in the neural tissueof most of the echinoderm classes (Moore and Thorndyke,1993; Ghyoot et al., 1994; Moss et al., 1994; Dıaz-Mirandaet al., 1995; Newman et al., 1995a; Byrne et al., 1999,2001; Beer et al., 2001; Byrne, 2001; Cisternas et al.,2001). Antisera generated against these peptides wereused as markers in the studies cited above to investigatethe distribution of neuronal tissue in developing and adultechinoderms. The neurobiology of the ciliated bands ofechinoderm larvae, particularly that of echinoplutei, hasbeen investigated in numerous studies, which show thatthe associated neurons are strongly serotonergic (Burke etal., 1986; Bisgrove and Burke 1987; Nakajima et al., 1993;Chee and Byrne, 1999a,b). This neurochemical appears tobe absent in the adult nervous system. The SALMFamidesby contrast are present in the nervous systems of bothechinoderm life stages (Moore and Thorndyke, 1993; Mosset al., 1994; Newman et al., 1995a; Byrne et al., 1999,2001; Beer et al., 2001). It has been suggested that thepresence of GFNSALMFamide 1 (S1)-like immunoreactiv-ity in larval and adult asteroids might provide a tool withwhich to trace development of the juvenile nervous systemthrough metamorphosis (Moss et al., 1994; Byrne et al.,1999). Among invertebrates, the change from the bilater-ally symmetrical nervous system of free-swimming echi-noderm larvae to the radial nervous system of benthicadults during metamorphosis is striking.

In this study, antisera to S1 were used to documentdevelopment of the peptidergic nervous system in thelarvae of the sea star Patiriella regularis and the distri-bution of S1 immunoreactivity in the nervous system inadult Patiriella species. P. regularis has planktotrophicdevelopment through feeding larvae, a mode of develop-ment considered to represent the ancestral state for echi-noderms (Strathmann, 1978; Byrne and Barker, 1991). Aswith many asteroids, P. regularis has two larval stages,the bipinnaria, followed by the brachiolaria (Fig. 1a–c).The bipinnaria is characterised by its ciliated band sys-tem, which is used for feeding and swimming (Fig. 1a,b).The brachiolaria stage is characterised by the anteriorattachment complex, which is used for benthic attachmentduring metamorphosis (Fig. 1c). The rudiment of the de-veloping juvenile is located in the posterior region. Duringmetamorphosis, the larval body degenerates as the youngsea star develops (Byrne and Barker, 1991). Confocal mi-croscopy of the larvae of P. regularis was used to deter-mine the pattern of differentiation of the peptidergic sys-tem with respect to changes in larval anatomy,particularly the increasing complexity of the ciliated bandsystem during the bipinnaria stage and morphogenesis ofthe attachment complex. We also examined the nervoussystem of the larval gut, because this structure serves asa rudiment for the adult digestive system and may containneurons that persist through metamorphosis. The adultgut is known to have a well-developed peptidergic system(Newman et al. 1995b). Immunoreactivity in the adultnervous system was examined in several Patiriella speciesand compared with that documented for distantly relatedasteroids (Moore and Thorndyke, 1993; Newman et al.,1995a) to determine the level of conservation of the aster-oid peptidergic system. Although both life history stagesin Paririella have well-developed peptidergic systems

with some morphological similarities, we found that thestructure and potential functions of these systems wereunrelated. They are composed of two different sets ofneurons.

MATERIALS AND METHODS

Larval culture

Patiriella regularis were obtained from the DerwentRiver Estuary, Tasmania. Ovaries were dissected and in-cubated in 10–5 M 1-methyladenine in 0.45 �m filtered seawater (FSW) to induce oocyte maturation. Mature eggswere collected, placed in FSW, and fertilised with spermat a concentration of 105 sperm/ml. After 15 minutes,fertilised eggs were rinsed in FSW to remove excesssperm. Approximately 1 ml of the egg suspension wastransferred to 250-ml glass beakers filled with FSW toachieve a final concentration of one to three embryos/ml.Cultures were stirred intermittently by a rack system ofmotor-driven paddles (Strathmann, 1987). The sea waterwas replaced twice per week. From day 3 of development,the larvae were fed the diatom Chaetoceros calcitrans(strain CS-178; CSIRO Marine Laboratories) every 4 days.Cultures were reared at 18–25°C.

Larval stages examined

The developmental stages of P. regularis examinedranged from ages 1 to 63 days (see Byrne and Barker,1991). From each of 10 independent cultures, 15–25 gas-trulae (1–2 days, n � 150) and early bipinnaria (3–9 days,n � 250) were fixed in 4% paraformaldehyde in FSW for 1hour. From three cultures 10–20 advanced bipinnaria(3–4 weeks, n � 50) and brachiolaria (up to 8 weeks, n �50) were relaxed in 7% MgCl2 solution for approximately5 seconds prior to fixation. After fixation, the specimenswere rinsed in FSW and phosphate-buffered saline (PBS;pH 7.4) before being processed for immunocytochemistry.

For immunocytochemistry of the adult nervous system,arm portions were dissected from P. regularis, P. calcar, P.gunnii, and P. exigua. Specimens of the latter three spe-cies were collected in the Sydney area. The arm portionswere fixed in Bouin’s solution until the skeleton was de-calcified (3–5 days) and processed for wax embedding (Ki-ernan, 1999). Arm sections 5 �m thick were examined.

Immunocytochemistry

The specimens were washed in three rinses of 0.1 MPBS buffer (pH 7.4) for 10 min each to remove seawater.Nonspecific antibody binding was blocked by incubatinglarvae in PBS containing 0.3% Triton X-100, 5% normalgoat serum, and 0.1% bovine serum albumen (BSA) for 1hour at room temperature. Triton X-100 was added in thisstep to permeabilise larvae. Excess blocking solution wasremoved by three 10 minute rinses in PBS. Specimenswere then incubated for 16 hours at 4°C in rabbit anti-S1polyclonal antibody (BLIII/anti-GFNSALMFamide;kindly provided by Dr. M. Thorndyke, University of Lon-don) at a dilution of 1:2,000 in 0.1% BSA/PBS. The optimalprimary antibody dilution was determined from a titra-tion series (1:100–1:5,000) tested on sections of adult ra-dial nerve cord. After incubation in primary antibody, thelarvae were rinsed with PBS for 3 � 10 minutes to removeunbound primary antibody. The specimens were incu-bated for 1.5 hours in either 1) biotinylated goat anti-

102 M. BYRNE AND P. CISTERNAS

Fig. 1. Planktotrophic larvae of P. regularis. a: Bipinnaria (2.5days); side view of a slightly flexed (dorsally) larva showing its spa-cious buccal cavity. The preoral ciliated band follows the contours ofthe oral hood. The postoral ciliated band cavity loops around thelarval body. b: Bipinnaria (18 days); ventral view. The ciliated bandsare prominent. Arrows on either side of the buccal opening indicatethe transverse regions of preoral (upper arrow) and postoral (lowerarrow) ciliated bands. The anterior projection has developed and isbordered dorsally by the preoral ciliated band and ventrally by the

postoral ciliated band. c: Brachiolaria (8 weeks); ventral view. Theattachment complex is at the anterior end, and the developing juve-nile is at the posterior end (from Byrne and Barker, 1991, withpermission). The anterior projection is behind the central brachium.A, adoral ciliated band; AD, adhesive disc; An, anus; AP, anteriorprojection; BC, buccal cavity; CBr, central brachium; E, esophagus; H,five-lobed hydrocoel of developing juvenile; LBr, lateral brachium; M,mouth; OH, oral hood; PO, postoral ciliated band; PR, preoral ciliatedband; S, stomach. Scale bars � 70 �m in a, 80 �m in b, 160 �m in c.

103PEPTIDERGIC SYSTEM IN LARVAL AND ADULT SEA STARS

rabbit antibody (Sigma, St. Louis, MO) and diluted 1:200in PBS or 2) goat anti-rabbit antibody conjugated to Alexared (Molecular Probes, Eugene, OR) and diluted 1:500 inPBS. Unbound secondary antibody (for incubation 1above) was removed with three 10 minute rinses in PBS.Specimens incubated in biotinylated secondary antibodywere then incubated in streptavidin-fluorescein isothio-cyanate (FITC)-conjugated antibody (Vector Laboratories,Burlingame, CA) diluted 1:200 for 30 minutes in the dark.After a final rinse in PBS, the specimens were mounted onslides with Vectashield (Vector Laboratories), and cover-slips were sealed with nail polish.

Paraffin sections (5.0 �m thick) of the adult nervoussystem were treated using the above-described protocol,except for the absence of Triton X-100 in the blockingsolution. Arm sections were also processed for immuno-chemistry by using the 3,3�-diaminobenzidine (DAB)method (Kiernan, 1999). After removal of wax, the sec-tions were dehydrated in graded ethanols to 100% meth-anol and blocked for endogenous oxidases in 100% meth-anol containing 3% H2O2 for 45 minutes. This wasfollowed by three rinses each in 100% methanol, 50%methanol in PBS, and PBS. Nonspecific binding wasblocked, and the sections were incubated in primary an-tibody as described above. The sections were then rinsedthree times in PBS to remove unbound antibody, followedby incubation in a 1:200 dilution of goat anti-rabbit horse-radish peroxidase (Sigma) in 1% BSA in PBS for 1.5 hourat room temperature in a moist chamber. Sections werethen rinsed three times in PBS and incubated in 0.046 MDAB-1 DAB tablet (Sigma) in 15 ml Tris buffer, pH 7.6,containing 12 �l 30% H2O2. The sections were then rinsedin distilled water and the coverslips mounted. Paraffinsections of the nerve cord were also stained with haema-toxylin and eosin (H/E) for light microscopy (Kiernan,1999). Controls for each experiment included omission of1) primary antibody, 2) secondary antibody, and 3) pri-mary and secondary antibodies. A further control involvedreplacement of primary antibody with preimmune serum(rabbit).

S1-like immunoreactivity in whole larvae and radialnerve cord sections was visualised using a laser scanningconfocal microscope (Bio-Rad MRC 600). Specimens la-beled with FITC were excited at a wavelength of 488 nmand detected over a wavelength of 520 nm. Those labeledwith Alexa Red were excited at 568 nm and detected overa wavelength of 585 nm. Localisation of labeled cells wasdetermined with respect to larval anatomy in 3D recon-structions of serial optical images generated by ConfocalAssistant v4.02 (Bio-Rad). Serial sections of larva werecaptured at 0.2–2.0 �m intervals. The number and thick-ness of sections for each reconstruction are provided in thefigure legends.

RESULTS

Gastrula

Early and late gastrula (24–48 hours) of P. regulariswere the first developmental stages examined (n � 150).No S1-like immunoreactivity was observed (data notshown).

Bipinnaria

Early bipinnaria (2–3 days old) have a complete diges-tive tract and start to feed. In these larvae, two groups of

flask-shaped cells (5–6 � 12 �m diameter), with one tothree processes and an eccentric nucleus appeared in thedorsolateral region of the larva, anterior to the mouth(Fig. 2a–d). These cells, two to three per group, were thefirst S1-like immunoreactive cells observed and repre-sented developing paired ganglia (see below). Serial sec-tions revealed that the cell bodies were located at variousdepths within the larval epithelium. At this stage, thepeptidergic cells were loosely arrayed and were linked toeach other by thin fibres dotted with varicosities (Fig. 2c).

Bipinnaria larva (5 days) had a well-developed oral hoodand ciliated band system (Fig. 1a). The ciliated band sys-tem has three regions, the preoral ciliated band anterior tothe mouth, the postoral ciliated band below the mouth,and the adoral ciliated band along the lower edge of themouth opening (Fig. 1a). Along the upper and lower rim ofthe buccal cavity, the pre- and postoral ciliated bands havetransverse segments (Fig. 1b). Additional S1-like immu-noreactive cells and fibres appeared in the developingganglia. By this stage, each ganglion contained 5–10 neu-rons, many of which were multipolar. As detected in con-trols (data not shown), the round structures in the rightand left coelom and the gut wall were autofluorescent (Fig.2a). The structures in the coelom were diffuse and did notappear cellular. The surface epithelium also exhibited apunctate autofluorescence of an unknown nature.

In older bipinnaria larva (6 days and older), the numberof multipolar S1-positive cells and processes innervatingthe anterior dorsal region continued to increase (Fig.3a,b). By this stage, 15–20 S1-like immunoreactive cellswere present in each ganglion (Fig. 3c). Because of theclose stacking of cell bodies in the ganglia, it was notpossible to accurately count cell number as the larvaeaged. Fibres from the ganglion extended anteriorly alongthe dorsal region and also laterally and ventrally towardthe oral hood to connect the two ganglionic networks (Fig.3a). The continuity and symmetry of the ganglionic net-work were evident around the oral region (Fig. 3a). Theciliated bands were innervated by peptidergic cells andfibres that connected to the ganglia. In the oral region,fibres from each ganglion intermingled with developingfibres and cells in the epithelium of the preoral and theadoral ciliated bands (Fig. 3b,d). A few fibres were alsopresent in the epithelium of the postoral ciliary band (Fig.3b). Fibres from the postoral ciliary band extended ven-trally to connect with fibres in the adoral ciliary band andthe ganglionic fibres. A fine nerve tract along the outeredge of the buccal cavity connected the pre- and postoralciliated bands (Fig. 3b). Most of the immunopositiveperikarya were bipolar, ovoid cells (�10 �m long).

As the bipinnaria developed through the middle andlate bipinnaria (2–3 weeks) stages, the ciliated band sys-tem became more prominent (Fig. 1b), and the gangliacontinued to add cells and fibres (Fig. 4a,b). S1-like im-munoreactivity was particularly conspicuous in the epi-thelia of the ciliated bands (Fig. 4a,c,d). By week 3, ananterior projection started to grow as an extension of thepreoral hood (Figs. 1b, 4a) in preparation for developmentof the brachiolar attachment complex. As this projectiongrew, the bilateral ganglia moved anteriorly and becamepositioned on either side of its base (Fig. 4a). In Figure 4b,approximately 11 ganglion perikarya are evident in a 1.0-�m-thick projection of a 3-week-old bipinnaria. From dor-sally to ventrally, the ganglia were approximately 5 �mthick, indicating that they contained a total of 40–60

104 M. BYRNE AND P. CISTERNAS

perikarya. The associated network of fibers spanning be-tween the two ganglia was a conspicuous feature of theanterior end of the larvae (Fig. 4a). As is characteristic ofthe peptidergic system, the fibres were dotted with vari-cosities. The ganglia retained their connection to the pep-tidergic cells in the preoral ciliary band through fine nervefibres that traversed along the lateral sides of the oralhood (Fig. 4a).

In late bipinnaria (Fig. 1b), the peptidergic networkassociated with the ciliated bands was well developed.Multipolar, ovoid, and spindle-shaped cells (�12 �m long)dotted the epithelium of the preoral ciliated band. A sec-ond row of multipolar cells with long, varicose processes

was located immediately behind the preoral ciliated bandin the roof of the buccal cavity (Fig. 4c,d). These two rowsof cells and their fibres formed a conspicuous preoralnerve plexus innervating the preoral ciliated band andoutermost region the roof of the buccal cavity (Fig. 4a,c).The peptidergic cells and fibres associated with the adoralciliated band along the lower edge of the mouth openingalso formed an extensive nerve plexus. In the postoralciliated band, a few cell bodies communicated through anetwork of fibres (Fig. 4a). By this stage, the nerve tractconnecting the pre- and postoral ciliated bands was welldeveloped (Fig. 4a). The ciliated band nerve network alsogave rise to fibres that extended posteriorly. The rest of

Fig. 2. Confocal reconstructions of 2-day-old P. regularis showingS1-like immunoreactivity. a: Dorsal view showing developing rightand left ganglia (arrows). Autofluorescent structures are located inthe developing coeloms on either side of the stomach and in thestomach wall (84 sections, 1.08 �m thick). b: Side view showing adeveloping ganglion (arrow), oral hood, and buccal cavity (136 sec-

tions, 0.5 �m thick). c,d: Developing ganglion with S1-like-immunoreactive neurons and their fibres, which are dotted with var-icosities (a, 31 sections, 0.9 �m thick; b, 16 sections, 0.36 �m thick).Dots on surface are autoflourescent structures. BC, buccal cavity; Co,coelom; N, neuron; NF, nerve fibre; OH, oral hood; S, stomach; V,varicosity. Scale bars � 50 �m in a,b, 10 �m in c,d.

105PEPTIDERGIC SYSTEM IN LARVAL AND ADULT SEA STARS

the bipinnaria stage, through development of the earlybrachiolaria, involved an increase in the complexity of thepeptidergic system associated with the ciliated bands(data not shown). The fully differentiated bipinnarial ner-vous system included bipolar and multipolar peptidergiccells.

Brachiolaria

The brachiolaria stage (8 weeks) is reached when thelarval attachment complex forms as an extension of theanterior projection (Figs. 1c, 5a,b). This larval organ com-prises a central brachium, two lateral brachia, and anadhesive disc (Fig. 1c). The brachia and disc are adhesive,and their epithelia contain a battery of secretory cells thatare the source of material that cements settling larvae tothe substratum (Byrne et al., 2001). Cells bearing short,sensory-like cilia or hairs also extend from the surface ofbrachia (Byrne et al., 2001). The brachia are highly mus-cular structures and are flexed and relaxed as the larvaeswam anterior end down along the surface of the substra-tum.

The peptidergic network innervating the ciliated bandsof brachiolaria was well developed and gave rise to alateral nerve net on either side of the larva (Fig. 5d–f). Inassociation with morphogenetic growth of the anteriorregion, the ganglia were shifted anteriorly, retaining theirposition at the base of the anterior projection (Fig. 5a–d).Although the ganglia retained their association with theciliated bands through the general nerve network, most oftheir fibres now innervated the attachment complex. Asthe brachia and adhesive disc developed, S1-positive fibresfrom the ganglia extended ventrally to innervate thesestructures (Fig. 5b). Within the brachiolar complex, pep-tidergic fibres connected the brachia and adhesive disc.The adhesive disc possessed an extensive network of cellsand fibres that traversed to the lateral brachia (Fig. 5b,d).Some fibres from the adhesive disc extended dorsallythrough the oral hood (Fig. 5b,d). In the oral region, fibresfrom the adoral nerve plexus extended posteriorly alongthe gut to innervate the esophagus (Fig. 5e). Immunopo-sitive cells and processes were seen for the first time in thestomach epithelium (Fig. 5c).

Fig. 3. Confocal reconstructions of a 6-day-old P. regularis bipin-naria showing S1-like immunoreactivity. a: Dorsal view showing bi-lateral ganglia and their processes, which extend around the buccalregion. The mouth and stomach are evident (84 sections, 1 �m thick).b: Ventral view of a dorsally flexed larva showing the spacious buccalcavity and mouth opening at its base. S1-like-immunoreactive cellsand fibres are associated with the adoral and postoral ciliated bands.The preoral band is out of view above the plane of section. A thinimmunoreactive tract (arrow) is present along the edge of the buccal

cavity. The fibres along the roof of the buccal cavity (arrowhead) arederived from the ganglia (84 sections, 1 �m thick). c: Projection of theleft ganglion showing neuronal cell bodies and fibres (33 sections, �mthick). d: Buccal region showing fibres innervating the adoral ciliaryband and the roof of the buccal cavity (arrowhead; 54 sections, 0.54�m thick). A, adoral ciliated band; M, mouth; PO, postoral ciliatedband; BC, buccal cavity: G, ganglion; M, mouth; N, neuron; NF, nervefibre; S, stomach. Scale bars � 100 �m in a,b,d, 10 �m in c.

106 M. BYRNE AND P. CISTERNAS

The developing juvenile star is located in the posteriorregion of the brachiolaria (Fig. 1c). After permanent at-tachment to the substratum, metamorphosis ensues, andthe larval body is resorbed into the oral region of thedeveloping juvenile (see Fig. 5; Byrne and Barker, 1991).Because of the opacity and autofluorescence of the devel-oping juvenile, we were unable to resolve cellular struc-tures or S1-like immunoreactivity (data not shown). Itappeared, however, that there was no immunoreactivityin metamorphosing larvae.

Adult nervous system

The adult CNS of adult Pateriella consisted of a radialnerve cord positioned along each arm (�radius), whichmerged with the circumoral nerve ring (Fig. 6a,b). The

radial nerve cord extended down the oral surface betweenthe tube feet and had a V-shaped profile. As is character-istic of asteroids, the nervous system was divided into tworegions, the outer ectoneural system and the inner hypo-neural system (Fig. 6b). These two portions of the nervecords were separated by a thin basal lamina. The ectoneu-ral portion is composed of two layers, an outer,pseudostratified neuroepithelium and the inner, through-conducting axonal layer (Fig. 6b). The hyponeural systemformed a thin layer of tissue along the innermost region ofthe nerve cord (Fig. 6b). Along its length, the radial nervecord gave rise to lateral nerves supplying the longitudinalnerve of the tube feet (Fig. 6a).

Examination of S1-like immunoreativity in the nervoussystem of four Pateriella species revealed a consistent

Fig. 4. Confocal reconstructions of advanced bipinnaria of P. regu-laris (3 weeks). a: Ventral view of a larva with the ganglia positionedat the base of the anterior projection. Nerve fibres extend from theganglia down the sides of the oral hood to connect with fibres inner-vating the preoral ciliated band. An immunoreactive tract (arrow-head) on either side of the buccal cavity connects the pre- and postoralciliated bands (64 sections, 2 �m thick). b: Reconstruction of the rightganglion showing ganglion cells, fine nerve fibres that extend ven-trally to the epithelium to the oral hood (7 sections, 1 �m thick).c: Ventral view. A row of cells and nerve fibres in the roof of the buccal

cavity (arrow) connect with the preoral ciliary band. An extensivenerve net is associated with the postoral ciliary band (5 sections, 1 �mthick). d: Preoral nerve plexus. A multipolar cell in the roof of thebuccal cavity with at least three processes (arrowheads) extendingtoward cells in the preoral ciliated band. The preoral ciliated band isbelow the plane of section (20 sections, 0.2 �m thick). AP, anteriorprojection; BC, buccal cavity; C, multipolar cell; G, ganglion; GC,ganglion cells; OH, oral hood; PO, postoral ciliated band; PR, preoralciliated band; NF, nerve fibre; NN, nerve net; V, varicosity. Scalebars � 100 �m in a, 20 �m in b, 50 �m in c, 10 �m in d.

107PEPTIDERGIC SYSTEM IN LARVAL AND ADULT SEA STARS

Figure 5

distribution of peptidergic cells, in the radial and circu-moral nerves. Immunoreactivity was conspicuous in theectoneural system, where it was intense throughout theaxonal region (Fig. 7a–c) and localised in punctate vari-cose structures (Fig. 8a,b). Immunoreactive cell bodieswere not evident in the axonal region. Elongate S1-

positive cells spanned the neuroepithelium, and the im-munoreactivity was concentrated in discrete intracellulargranules (Figs. 7a,d, 8a,b). These cells gave rise to apicalprocesses that projected toward the cuticle and basal pro-cesses that merged with the axonal region (Fig. 8b). Im-munoreactive cells were also scattered along the hyponeu-ral layer (Figs. 7a, 8a). The lateral nerve merged with thebasiepithelial nerve plexus, a thin immunoreactive layerat the base of the tube foot epithelium (Fig. 7d). Immuno-reactivity, as seen in the tube foot plexus, was character-istic of the basiepithelial plexus throughout the body ofPateriella (not illustrated).

DISCUSSION

The nervous systems of the planktotrophic larvae ofmarine invertebrates have many similarities, includingthe presence of anterior ganglia, innervation of ciliarystructures, and expression of a similar suite of neuro-chemicals (Barlow and Truman, 1991; Moss et al., 1994;Lin and Leise 1966; Kempf et al., 1997; Marois and Carew,1997a–c; Byrne et al., 1999, 2001; Dickinson et al., 1999;Hay-Schmidt, 2000). These commonalties across phyloge-netically disparate taxa suggest strong convergence in theevolution of nervous system development in response toselection pressures specific to the planktonic environment(Kempf et al., 1997; Marois and Carew, 1997a; Hadfield,2000). Interest in the neuronal architecture of echinoderm

Fig. 6. LM cross sections (H/E stained) of the adult nervous sys-tem of P. gunnii. a,b: The radial nerve cord extends down the oral sideof the arm between the tube feet. The ectoneural system is composedof the inner axonal region and the outer neuroepithelium. The hypo-neural system is a thin layer along the inner side of the nerve cord. On

either side of the nerve cord, the ectoneural system sends a lateralnerve to the tube feet (arrow). Ax, axonal region; H, haemal sinus, Hn,hyponeural system; M, tube foot muscle; NE, neuroepithelium; R,radial nerve cord; S, skeleton; TF, tube foot. Scale bars � 40 �m in a,100 �m in b.

Fig. 5. Confocal reconstructions of the brachiolaria of P. regularis(8 weeks). a: Ventral view with the central brachium extending fromthe anterior projection and developing lateral brachia. Immunoreac-tivity is associated with the pre- and postoral ciliated bands (94sections, 2 �m thick). b: Fibres from the ganglion extend ventrally toconnect with fibres innervating the adhesive disc. The lower edge ofthe mouth is lined by fibres of the adoral nerve plexus (75 sections, 1�m thick). c: Junction between the esophagus and stomach (arrow). Afew immunoreactive fibres are scattered in the stomach epithelium (5sections, 1 �m thick). d: Ventral (left) and dorsal (right) views of abrachiolaria with a well-developed brachiolar complex showing exten-sive immunoreactivity in the adhesive disc and brachia. The gangliaare positioned at the base of the anterior projection (94 sections, 2 �mthick). e,f: Ventral view of the buccal region of the larva shown in d.The adoral nerve plexus along the lower edge of the mouth extendsfibres along the esophagus (arrow). An interconnecting nerve netextends laterally from the ciliated bands (e � 43 sections, 1 �m thick;f � 1 section, 1 �m thick). A, adoral nerve plexus; AD, adhesive disc;AP, anterior projection; B, brachium; E, esophagus; G, ganglion; M,mouth; NN, nerve net; OH, oral hood; PO, postoral ciliated band; PR,preoral ciliated band; S, stomach. Scale bars � 100 �m in a–e, 50 �min f.

109PEPTIDERGIC SYSTEM IN LARVAL AND ADULT SEA STARS

ciliated bands has focussed on Garstang’s hypothesis(Garstang, 1894) that the chordate nerve cord was derivedfrom the ciliated band of an ancestral larva similar to theechinoderm dipleurula (Lacalli, 1994; Hay-Schmidt,

2000). Immunocytochemical studies show an extensivearray of neurochemicals associated with echinoderm cili-ated bands, including serotonin, �-aminobutyric acid(GABA), dopamine, and SALMFamides (Burke et al.,

Fig. 7. LM sections (DAB immunocytochemistry) of the adult ner-vous system of Pateriella species. a: P. regularis. The axonal region isintensely S1 positive. Bipolar cells span the neuroepithelium (ar-rows). A few hyponeural perikarya and the basiepithelial nerve plexusare also immunoreactive. b,c: Longitudinal sections of the radialnerve cord (b, P. calcar; c, P. regularis) showing S1-positive axonalregion, lateral nerve to tube feet, basiepithelial nerve plexus, and

S1-positive cells in the neuroepithelium (arrows). d: P. calcar. Thebasiepithelial plexus of the tubefoot is positioned between the epithe-lium and the muscle layer. Ax, axonal region; B, basiepithelial nerveplexus; E, epithelium; Hn, hyponeural perikaryon; L, lateral nerve; M,tube foot muscle; NE, neuroepithelium; T, tube foot. Scale bars � 100�m in a,c,d, 200�m in b.

110 M. BYRNE AND P. CISTERNAS

1986; Bisgrove and Burke, 1987; Nakajima et al., 1993;Moss et al., 1994; Chen et al., 1995; Chee and Byrne,1999a,b; Beer et al., 2001; Cisternas et al., 2001). S1-positive cells, such as those associated with the ciliatedbands of P. regularis, are also associated with the ciliatedbands of ophiuroid and echinoid plutei (Thorndyke et al.,1992; Beer et al., 2001; Cisternas et al., 2001).

Bipinnaria peptidergic system

The peptidergic system of the bipinnaria of P. regularishad the expected suite of characters, with a concentrationof neurons in anterior ganglia and innervation of ciliatedbands. As in P. regularis, S1-like immunoreactivity in thebipinnaria of the asteroids Asterias rubens and Pisasterochraceus is localised in paired anterior dorsolateral gan-glia. The resolution conferred by confocal microscopy inthis study of P. regularis provided previously unresolveddetail on the development of the ganglia. They were thefirst peptidergic structures to develop, and the number ofperikarya in the ganglia increased as the larvae devel-oped. Contrary to the suggestion that these paired gangliaarise by splitting of a single apical ganglion (Lacalli,1994), the ganglia of P. regularis were a bilateral pairfrom the outset of their development. As the digestive andciliated band systems differentiated during week 1, pro-cesses from the ganglia intermingled around the buccalcavity and extended nerve fibres to the ciliated bands.These early events coincided with the onset of feedingcompetence.

Asteroid bipinnaria have been observed to retain cap-tured particles in the buccal cavity before they are rejectedor propelled into the oesophagus by ciliary activity(Strathmann, 1971). The concentration of S1-like immu-noreactivity in the roof of the buccal cavity (preoral nerveplexus) and along the posterior rim of the mouth (adoralnerve plexus) suggests that the peptidergic system may beinvolved in this behaviour, perhaps through modulation of

the ciliary activity that affects acceptance or rejection ofparticles. Although peptidergic neurons innervating theciliated organs of other marine larvae have also beensuggested to modulate ciliary activity (Barlow and Tru-man, 1991; Dickinson et al., 1999), there are no neuro-physiological data to support this suggestion. In adultTritonia, some neuropeptides (TPep-like peptides) modu-late ciliary beat frequency, but others (FMRFamides) donot (Willows et al., 1997). Serotonergic neurons in the pre-and adoral nerve plexus of P. regularis have also beensuggested to modulate ciliary activity (Chee and Byrne1999a). Serotonin is known to have a cilioexcitatory influ-ence in echinoderm larvae (Soliman, 1983).

The weight of evidence from use of several neuronalmarkers indicates that innervation of the buccal region ofasteroid larvae is extensive and complex. In this region,the ciliated bands of P. regularis are connected by pepti-dergic and serotonergic nerve tracts, indicating that allparts of the ciliated band system are in close communica-tion (Chee and Byrne, 1999a). The parallel distribution ofthe serotonergic and peptidergic systems in the buccalregion of P. regularis larvae suggests that their compo-nent neurons may interact (Chee and Byrne 1999a). Thesubsets of neurons recognised by antisera to serotonin andS1, however, appear to differ (Byrne et al., 1999; Chee andByrne, 1999a,b; Byrne, 2001). Serotonergic neurons arelarger than peptidergic ones. Nerve fibres dotted withvaricosities are characteristic of the peptidergic system,whereas they are less common in the serotonergic system.In echinoid larvae, S1- and serotonin-like immunoreactiveneurons also have a parallel distribution and appear to bedifferent cells (Beer et al., 2001). In the anteriormostregion of the bipinnaria of P. regularis, the distributions ofpeptidergic and serotonergic perikarya differ markedly.The apical ganglion (�apical organ) gives rise to an ex-tensive network of serotonergic fibres, which innervatesthe anterior sections of the pre- and postoral ciliated

Fig. 8. Confocal microscopy of the adult nervous system of Pateri-ella species. a: P. regularis. The ectoneural system is characterised byan immunoreactive axonal region and bipolar sensory-like cells (ar-rows). A few S1-positive hyponeural perikarya are evident. b: P.calcar. The bipolar cells span the neuroepithelium. They have a basal

process that merges with the axonal layer and immunoreactive gran-ules. Immunoreactivity in the inner axonal region is evident invaricose-like structures. G, granules; Hn, hyponeural perikaryon.Scale bars � 40 �m in a, 18 �m in b.

111PEPTIDERGIC SYSTEM IN LARVAL AND ADULT SEA STARS

bands (Chee and Byrne 1999a). This region did not showany S1-like immunoreactivity. A colocalisation study willbe required to determine the relationship of the peptider-gic and serotinergic systems of P. regularis.

Brachiolaria peptidergic system

Our study appears to be the only investigation of thepeptidergic system in advanced brachiolaria. In associa-tion with morphogenetic growth of the anterior region, thepaired ganglia became aligned with the attachment com-plex while maintaining their dorsolateral position at thebase of the anterior projection. Most of the fibres emanat-ing from the ganglia extended ventrally to innervate theattachment structure, although some connection to theciliated bands was maintained. The anterior shift in theposition of the ganglia presumably represents an ontoge-netic change in preparation for larval settlement andmetamorphosis. The paired ganglia of P. regularis persistthrough development. Their ontogeny and location aredistinct from those of the anterior ganglion, which disap-pears during brachiolaria development (Chee and Byrne,1999a,b; Chee, 2000). In Archaster typicus, each brachiumis associated with a ganglion containing catecholaminer-gic cells (Chen et al., 1995).

Paired anterior ganglia composed of peptidergic neu-rons may be characteristic of competent planktotrophicasteroid larvae. In contrast, a single apical ganglion(�apical organ) composed of serotonergic neurons ispresent in the competent larvae of echinoids and manyother marine invertebrates, in which it is considered to beinvolved in perception of surface cues for settlement andmetamorphosis (Bisgrove and Burke, 1987; Nakajima etal., 1993; Kempf et al., 1997; Marois and Carew, 1997a–c;Hadfield, 2000). In echinoplutei, the apical ganglion con-tains both peptidergic and serotonergic cells (Bisgrove andBurke, 1987; Thorndyke et al., 1992; Nakajima et al.,1993; Beer et al., 2001). The difference in the presence ofpaired anterior ganglia in the competent larvae of P. regu-laris and the single ganglion in competent echinopluteimay be associated with the contrasting mechanisms ofmetamorphosis in asteroids and echinoids. Substrate se-lection and settlement in asteroids are accomplished bythe brachiolar attachment complex, a specialised larvalorgan that functions as a sensor and effector for larvalsettlement. It seems likely that the peptidergic systemassociated with this structure in P. regularis and thecompetent brachiolaria of other Pateriella species (Byrneet al., 2001) plays a role in modulation of substrate selec-tion and attachment. In contrast, settlement in echinoidsis accomplished by nonlarval structures, the primary tubefeet of the juvenile. The apical ganglion of echinopluteidoes not innervate these tube feet (Beer et al., 2001).

Although the neurochemistry of marine invertebratelarvae is becoming increasingly well understood, ourknowledge of how these larvae’s nervous systems functionis fragmentary as a result of the difficulties in undertak-ing neurophysiological studies. Nonetheless, some generalinsights are emerging. Ciliated larval structures are usedfor swimming and feeding in phylogenetically diverse in-vertebrate taxa, and the rich innervation of these struc-tures provides evidence that a range of neurochemicalsdirectly or indirectly modulates ciliary behaviour. Neuralcontrol of ciliary beat has been demonstrated in electro-physiological studies of echinoplutei and veligers (Mackieet al., 1969; Arkett et al., 1987; Leise and Hadfield, 2000).

Metamorphosis

Metamorphosis in marine invertebrates is associatedwith major change in the nervous system, withdisappearance/cell death of some neurons, remodeling andincorporation of others, and differentiation of new cells(Barlow and Truman, 1991; Dickinson et al., 1999, 2000;Croll, 2000). Because of the contrasting life styles, it is notsurprising that the nervous systems of the planktoniclarvae and benthic adults of marine invertebrates aredistinct. In comparison to other marine invertebrates, inwhich neurogenesis through metamorphosis has been doc-umented, the radical change during echinoderm metamor-phosis from a bilateral nervous system to a radial onerepresents an extreme case. None of the larval nervoussystem would be expected to persist. In molluscan velig-ers, the velum, a specialised larval organ, is discardedalong with associated neuronal cells, whereas other sub-sets of neurons survive metamorphosis and serve as ascaffold for the adult CNS (Marois and Carew, 1990; Bar-low and Truman, 1991; Dickinson et al., 1999, 2000; Croll,2000). Although it was difficult to resolve immunoreactiv-ity in the developing rudiment of P. regularis, it appearsthat little of the larval peptidergic system survived meta-morphosis. Larval neurons degenerated as the larval bodywas resorbed into the developing juvenile gut. A recentstudy of the lecithotrophic brachiolaria of P. exigua re-vealed that S1-like immunoreactivity largely disappearsduring metamorphosis and that differentiation of newpeptidergic neurons does not occur until after the adultCNS has differentiated (Koop, 2000). The peptidergic neu-rons that appeared in the stomach of advanced larvae of P.regularis may contribute to development of the juvenileenteric plexus. Overall, our results support the contentionthat the larval and adult peptidergic systems of Pateriellaare largely composed of a different set of neurons, similarto that documented for the serotonergic systems of larvaland adult Aplysia (Marois and Carew, 1997c). The delay inappearance of peptidergic cells in juvenile P. exigua rep-resents de novo expression of S1-like immunoreactivity ata specific ontogenetic stage in neurogenesis (Koop, 2000).

Adult peptidergic system

In adult invertebrates, the distribution of peptidergicperikarya in identifiable ganglia is particularly well doc-umented for the molluscan and insect CNS (Too and Croll,1995; Elekes, 2000; Elekes et al., 2000). In comparisonwith with these studies, the dominance of neuropeptideimmunoreactivity in the axonal region of the fully differ-entiated CNS of Pateriella and other echinoderms is strik-ing (Moore and Thorndyke, 1993; Ghyoot et al., 1994;Dıaz-Miranda et al., 1995; Newman et al., 1995a; Inoue etal., 1999). In comparison with other invertebrate peptider-gic systems (Too and Croll, 1995; Elekes, 2000; Elekes etal., 2000), relatively few peptidergic perikarya were evi-dent in the CNS of Pateriella. Given the abundance ofimmunoreactivity in the axonal region, it is unlikely thatthe S1-immunopositive cells bodies spanning the outerneuroepithelium were the source of all the axons in theradial nerve cord. These cells have been reported for sev-eral asteroids and are likely to have a sensory role assurface receptors potentially providing information to theunderlying axonal system (Moore and Thorndyke, 1993;Newman et al., 1995a). Although their location supportsthis suggestion, their abundant large granules suggest

112 M. BYRNE AND P. CISTERNAS

that they may also be neurosecretory. Nerve fibres in thebasiepithelial plexus of Pateriella exhibited strong immu-noreactivity, a feature characteristic of the diffuse periph-eral nervous system of echinoderms (Moore andThorndyke, 1993; Dıaz-Miranda et al., 1995a; Newman etal., 1995a,b; Inoue et al., 1999).

The widespread distribution of S1-like immunoreactiv-ity in the adult nervous system of Pateriella is similar tothat found for Asterias, the genus from which the peptidewas identified (Elphick et al., 1991a,b). These findings forphylogenetically distant asteroids indicate that S1 is awidespread asteroid neuropeptide. The abundance of S1-like immunoreactivity in the ectoneural axonal region andin the peripheral basiepithelial nerve plexus suggests thatthis peptide has a multifunctional mode of activity, de-pending on location. SALMFamides modulate eversion ofthe asteroid stomach during feeding through control ofmuscle tone (Elphick and Melarange, 2001). A similarmode of action is reported for native SALMFamides in thesea cucumber digestive system (Dıaz-Miranda and Garcıa-Arraras, 1995). In ophiuroids, S1 modulates luminesence(De Bremaeker et al., 1999).

The prevalence of SALMFamides in the main nervetracts of larval and adult echinoderms and their strongexpression in axonal systems attest to the importance ofthese neurochemicals in the Echinodermata (Moore andThorndyke, 1993; Ghyoot et al., 1994; Dıaz-Miranda et al.,1995; Newman et al., 1995a,b; Byrne et al., 1999, 2001;Beer et al., 2001; Byrne, 2001; Cisternas et al., 2001). Ithas been suggested that these peptides may underlie thebasis of neuronal transmission in echinoderms (Dıaz-Miranda et al., 1995; Inoue et al., 1999). The enigmaticnature of neuronal activity in echinoderms, the generallack of identifiable synapses, and the difficulty in record-ing from neurons have made the echinoderm nervous sys-tem a difficult subject of study (Cobb, 1987). Perhaps afocus on neuropeptides will provide a productive avenue ofinvestigation into how the echinoderm nervous systemfunctions.

ACKNOWLEDGMENTS

Assistance was provided by A. Cerra, F. Chee, A. Feld-man, P. Selvakumaraswamy, and R. Smith. M. Thorndykekindly provided antisera to S1. The comments of two re-viewers greatly improved the article. We thank the Elec-tron Microscope Unit of the University of Sydney for theassistance of expert staff and for use of facilities.

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