Download - Apical organs in echinoderm larvae: insights into larval evolution in the Ambulacraria

Apical organs in echinoderm larvae: insights into larval evolution in the

Ambulacraria

Maria Byrne,a,� Yoko Nakajima,b Francis C. Chee,a and Robert D. Burkec

aDepartment of Anatomy and Histology, F13, University of Sydney, NSW 2006, AustraliabDepartment of Biology, Keio University, Yokohama 223-8521, JapancDepartments of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Road, Victoria, BC, Canada V8W 3N5�Author for correspondence (email: [email protected])

SUMMARY The anatomy and cellular organization ofserotonergic neurons in the echinoderm apical organexhibits class-specific features in dipleurula-type (auricularia,bipinnaria) and pluteus-type (ophiopluteus, echinopluteus)larvae. The apical organ forms in association with anteriorciliary structures. Apical organs in dipleurula-type larvae aremore similar to each other than to those in either of the pluteusforms. In asteroid bipinnaria and holothuroid auricularia theapical organ spans ciliary band sectors that traverse theanterior-most end of the larvae. The asteroid apical organ alsohas prominent bilateral ganglia that connect with an apicalnetwork of neurites. The simple apical organ of the auriculariais similar to that in the hemichordate tornaria larva. Apicalorgans in pluteus forms differ markedly. The echinopluteus

apical organ is a single structure on the oral hood betweenthe larval arms comprised of two groups of cells joined by acommissure and its cell bodies do not reside in the ciliaryband. Ophioplutei have a pair of lateral ganglia associatedwith the ciliary band of larval arms that may be the ophiuroidapical organ. Comparative anatomy of the serotonergicnervous systems in the dipleurula-type larvae of theAmbulacraria (Echinodermata1Hemichordata) suggests thatthe apical organ of this deuterostome clade originated asa simple bilaterally symmetric nerve plexus spanningciliary band sectors at the anterior end of the larva. Fromthis structure, the apical organ has been independentlymodified in association with the evolution of class-specificlarval forms.

INTRODUCTION

Nervous system organization forms the basis for description

of body plans across the metazoa. This has generated long-

standing interest in the links between neural anatomy, neuro-

genesis, phylogeny, and metazoan evolution (Garstang 1894;

Lacalli 1994, 2005; Hay-Schmidt 2000; Nielsen 2005). Here,

we assess evolution of the echinoderm serotonergic nervous

system with respect to larval morphology and phylogenetic

relationships in the Ambulacraria (Echinodermata1Hem-

ichordata), a monophyletic clade sister to the other major

deuterostome clade (Tunicata1Chordata) (Zeng and Swalla

2005; Swalla 2006). In echinoderms, serotonin is a conspic-

uous marker for the bilateral larval nervous system that de-

velops in association with the ciliary bands (Cisternas and

Byrne 2003; Nakajima et al. 2004a; Byrne et al. 2006; Nakano

et al. 2006).

Our current understanding of the serotonergic system of

echinoderm larvae is largely based on studies of seven

echinoid (Bisgrove and Burke 1986, 1987; Bisgrove and Raff

1989; Thorndyke et al. 1992; Nakajima et al. 1993, 2004a;

Yaguchi et al. 2000; Beer et al. 2001) and five asteroid (Burke

1983a; Nakajima 1988, 2004a; Moss et al. 1994; Chee and

Byrne 1999a) species. These studies provide an extensive da-

taset encompassing larvae from major echinoid and asteroid

taxa. We also have information on the serotonergic system of

three holothuroid and one ophiuroid species (Burke et al.

1986; Cisternas et al. 2001; Cisternas and Byrne 2003; Byrne

et al. 2006; Nakano et al. 2006). The serotonergic system of

the tornaria larva from the sister taxa, the Hemichordata, is

also described (Nakajima et al. 2004b; Nezlin and Yushin

2004; Nielson and Hay-Schmidt 2007).

Despite their disparate adult body plans, echinoderms and

hemichordates are united by possession of similar dipleurula-

type larvae (Fig. 1), a feeding larval form considered to be a

synapomorphy for the Ambulacraria (Zeng and Swalla 2005).

Echinoderms have a diverse array of dipleurula and pluteal

larval forms (review, Raff and Byrne 2006). Holothuroids,

asteroids, and crinoids have dipleurula-type larvae whereas

echinoids and ophiuroids have pluteal-type larvae with larval

arms supported by skeletal rods (Fig. 1). The Ambulacraria is

in an informative phylogenetic position to contribute to our

understanding of neural evolution in the invertebrate deut-

erostomes and in the chordates. Here, we determined if the

EVOLUTION & DEVELOPMENT 9:5, 432 –445 (2007)

& 2007 The Author(s)

Journal compilation & 2007 Blackwell Publishing Ltd.

432

Fig. 1. Phylogenetic relationships in the Ambulacraria (Echinodermata1Hemichordata) (after Smith et al. 2004) and larval forms. Theancestral dipleurula larva proposed by Muller (1853) and drawn by Dawydoff (1928) is placed in a basal position. Hemichordates, crinoids,asteroids, and holothuroids have dipleurula-type larvae. Ophiuroids and echinoids have pluteal-type larvae. Serotonergic apical organs (inred) of feeding larvae and associated ciliary bands (gray bands) are shown in the schematics on the right. These sketches are made from aperspective of looking at the anterior end of the larva with the larval body projecting into the plane of the page. Crinoids do not havefeeding larvae but have a dipleurula-like ciliary band (Nakano et al. 2003) and the doliolaria, with its apical catecholaminergic cells (Chia etal. 1986), are included to complete the tree. In the holothuroid auricularia, asteroid bipinnaria and hemichordate tornaria apical organ cellbodies are located in the ciliary band nerve and give rise to fibers that span between the bands. The bipinnaria also has lateral ganglia(arrows). The cell bodies of the echinopluteal apical organ are not located in the ciliary band nerve. The echinoid apical organ has twogroups connected by a bundle of nerves. In ophiuroids the apical organ of the ophiopluteus has two lateral ganglia with few cell bodieswithin the ciliary band nerve.

Echinoderm apical organ evolution 433Byrne et al.

morphological similarities among dipleurula larvae extend to

their serotonergic nervous systems and apical organs. Interest

in the echinoderm and hemichordate larval nervous systems

has focussed on Garstang’s (1894) hypothesized evolutionary

link between the dipleurula larva and the chordate nervous

system. This hypothesis, that the dipleurula ciliary band was a

forerunner of the chordate nerve cord, now has little support,

but as the chief site of larval neurogenesis, their potential

significance to chordate neural evolution remains of interest

(Lacalli 2003, 2005).

Anterior serotonergic structures, variously called apical

ganglia, anterior ganglia, or apical organs, are prominent in

Ambulacraria larvae and are focal structures for comparative

anatomy (Lacalli 1994; Lacalli and Kelly 2002; Nakajima et

al. 2004b; Nezlin and Yushin 2004). We use the term apical

organ. Echinoderm apical organs are well placed to serve as

sensory structures as the larvae swim anterior end foremost

(Burke 1983b; Bisgrove and Burke 1986, 1987; Chee and By-

rne 1999a; Beer et al. 2001; Yaguchi and Katow 2003; Nielsen

2005). Functional data are sparse, but some physiological and

pharmacological studies indicate a role for serotonin in mod-

ulation of the ciliary beat and metamorphosis (Chee and By-

rne 2001; Yaguchi and Katow 2003; Katow et al. 2004, 2007).

With the improved resolution afforded by confocal mi-

croscopy, detailed comparative data on the serotonergic sys-

tems are available for larvae of echinoderm classes that

diverged over 500Ma (Chee and Byrne 1999a, b; Byrne and

Cisternas 2002; Nakajima et al. 2004a; Byrne et al. 2006;

Nakano et al. 2006). Here we review the apical organs of

larval Echinodermata, comparing anterior serotonergic struc-

tures in each of the classes with those in the Hemichordata.

New data are presented for the nervous system of asteroids

and ophiuroids. Although a focus on the serotonergic system

considers just one subset of larval neurons, this is the only

neurochemical for which we have good comparative data.

Serotonin immunoreactivity has been an important anatom-

ical tool to visualize nervous systems across the metazoa. This

evolutionary ancient neurotransmitter is present in the ner-

vous systems of most bilaterians from lower invertebrates to

craniates and has received particular attention for phyloge-

netic comparisons (Hay-Schmidt 2000; Page and Parries 2000;

Page 2002; Nielsen 2005; Stach 2005).

Morphological evolution of the serotonergic neurons of

the apical organs of echinoderm and hemichordate larvae is

assessed within a phylogenetic framework (Fig. 1). We con-

sider the relationships among the echinoderm larval types and

features of the putative ancestral-type neuronal organization

in the Ambulacraria and how this organization may have

been modified through evolution of the larval form. For the

Echinodermata the apical organs of the asteroid bipinnaria

and holothuroid auricularia are modern representatives of

the ancestral-type dipleurula larva, whereas the apical organs

of the ophiopluteus and echinopluteus represent the pluteus

larva (Fig. 1, Table 1). The serotonergic apical organ of the

hemichordate tornaria (Nakajima et al. 2004b; Nezlin and

Yushin 2004; Nielson and Hay-Schmidt 2007) is also con-

sidered. With the assumption that the dipleurula is an Am-

bulacraria synapomorphy, we hypothesized that apical

organs in dipleurula-type larvae should be more similar to

each other than to these structures in pluteal forms and that

the ancestral-type echinoderm serotonergic system was mod-

ified independently in the different classes through evolution

of larval form.

DIPLEURULA-TYPE LARVAE

Class Asteroidea

Development of the asteroid serotonergic nervous system

is first evident in gastrulae where immunoreactive cells are

scattered through the ectoderm of the animal half of the

embryo (Fig. 2, A and B). In early bipinnaria these immuno-

reactive cells appear to migrate to the mid-larval region and

appear to line up along the developing pre- and postoral

ciliary bands (Figs. 2, C and 3A). Bipinnaria have two ciliary

bands (Figs. 1 and 3, A and F). Serotonin immunoreactivity

is most evident in the anterior half and oral region of the

larva (Fig. 3, A–D). The asteroid apical organ comprises a

network of neurites at the anterior end of the larva (larval

apex) and two lateral ganglia at the base of the preoral hood

(Figs. 1 and 3, C, D, and F). This structure is described for

bipinnaria from two major asteroid orders (Fig. 3, A–G),

although the presence of the apical plexus varies among

species. At the anterior end of the larva the pre- and postoral

ciliary bands traverse the preoral hood parallel to each other

on the dorsal and ventral sides of the larva (Fig. 3, A and F).

In this region, serotonergic cells are scattered along the cili-

ary band and fibers span between the bands (Fig. 3, A and

F). Nerve fibers from cells at the larval apex extend poste-

riorly along the ciliary band nerve to connect with the lateral

ganglia (Figs. 1 and 3, A and D).

In apical view, the similar development of serotonergic

neurons along the two ciliary bands conveys a dorso-ventral

symmetry to the apical organ (Fig. 3, A and F). The lateral

ganglia are the most conspicuous feature of the asteroid apical

organ (Fig. 3, B–E). Fully developed ganglia have 30–50 cells

that give rise to projecting fibers with a beaded appearance

(Fig. 3, D and E). These connect with serotonergic cells in the

oral region (Fig. 3A). Confocal imaging revealed that some of

the apical organ cells in Patiriella regularis had cilia-like pro-

cesses extending from their apex. These processes, about

15mm long (SE50.03mm; n510), extend beyond the epi-

thelium (Fig. 3, F and G). They may correspond to the sen-

sory-like process identified in ultrastructural studies of the

apical organs of the bipinnaria of Pisaster ochraceus (Lacalli

et al. 1990). At their base, these cells give rise to fibers with a

beaded, varicose appearance (Fig. 3G).

434 EVOLUTION & DEVELOPMENT Vol. 9, No. 5, September^October 2007

As the bipinnaria develop to the settlement-stage bra-

chiolaria larva, the anterior serotonergic cells appear to

disperse along the ciliary band nerve tracts. Brachiolaria

have three brachial arms and an adhesive disk for benthic

attachment (Fig. 3H). The lateral ganglia seen in the bipinn-

aria are conspicuous in the brachiolaria of Asterina pectinif-

era, but are absent in the competent brachiolaria of

P. regularis (Fig. 3H). Settlement-stage larvae of P. regularis

have serotonergic neurons in the epithelium of the bra-

chial arms. There are no traces of the apical organ in

lecithotrophic asteroid larvae and these larvae also lack

ciliary bands (Fig. 3I). Lecithotrophic brachiolaria have

serotonergic cells in the adhesive papillae of the brachiolar

arms (Fig. 3, I and J) that have cilia-like apical processes and

basal neurites (Chee 2000).

Class Holothuroidea

In holothuroid development, serotonin-positive cells first

appear at the animal pole of late gastrulae where they

develop into the apical organ (Byrne et al. 2006; Nakano et al.

2006). Early serotonergic cells remain at the anterior end of

the developing auricularia and become aligned along the an-

terior ciliary band region. Holothuroid auriculariae have

a single ciliary band that loops around the body and two

portions of the band traverse the right and left sides of the

larval apex (Figs. 1 and 4A). These two bands are offset 901

compared with those that cross this region in the asteroid

bipinnaria (cf. Figs. 3A and 4A). The arrangement of the

ciliary band of the auricularia is similar to that in the tornaria

larva of hemichordates (Fig. 1).

Unlike asteroids, early serotonergic cells do not migrate

in the auricularia as the ciliary band forms. Serotonin

immunoreactivity remains restricted to the anterior-most

region of the auricularia (Fig. 4, A–D). The apical organ of

Chirodota gigas develops as two groups of serotonergic neu-

rons associated with the right and left anterior ciliary band

sections at the larval apex with a few processes connecting the

bands (Fig. 4, A–D). Similar apical nerve connections are

described for the auriculariae of Parastichopus californicus

and Stichopus japonicus (Burke et al. 1986; Nakano et al.

2006). These three species represent two major holothuroid

orders. Ganglionic clusters of serotonergic cells do not devel-

op in auriculariae. In C. gigas, six to 22 serotonergic cells

develop along the apical ciliary band sectors and a few fibers

span between them in the short stretch where the bands run

parallel to each other (Fig. 4, C and D). The cells are bipolar

and multipolar and are interconnected by processes. A few

immunoreactive cells present in the oral hood connect with

the ciliary band nerve (Fig. 4, A and B). Bipolar sensory

cells that may correspond to serotonergic cells are present in

the anterior ciliary band region of auriculariae (Lacalli and

Kelly 2002).

Table1.Comparativecellularstructure

andanatomyoftheserotonergic

system

andapicalorgansofhemichordate

andechinoderm

larvae

Dipleurula

Larvae

PluteusLarvae

Feature

Tornaria

Auricularia

Bipinnaria

Echinopluteus

Ophiopluteus

Bilateral

Sym

metry

Yes

Yes

Yes

Yes

Yes

Ciliary

ban

dorientation

Right–left

Right–left

Dorso-ventral

Right–left

Right–left

Originofserotonergicprogenitorcells

FAnim

alectoderm

ingastrulae

Anim

alectoderm

ingastrulae

Anim

alectoderm

ingastrulae

FSerotonergiccellsmigrate

No

No

Yes

No

No

Apical

organ

cellbodiesin

theciliaryban

dYes

Yes

Yes

No

Yes

Apical

organ

form

sanetwork

ofneurites

betweenciliaryban

ds

Yes

Yes

Yes

No

No

Apical

organ

has

bilateral

ganglionic

groupsofcells

No

No

Yes

Yes

Yes

Apical

organ

pairedorsinglestructure

Paired

Paired

Paired

Single

Paired

Sensory-likecellspresent

Yes

Yes

Yes

Yes

FSerotonergicganglia

inad

oralciliaryban

dNo

No

Yes

Yes

Yes

F,nodata.


Fig. 2. Serotonin immunoreactivity in the gastrulae and early larvae of asteroids (A–C) and echinoids (D–F). (A–C) Patiriella regularis.Serotonergic cells (arrows) are scattered over the animal hemisphere of early (A) and late (B) gastrulae. (C) The serotonergic cells (arrows)appear to move to the mid-body region of the early bipinnaria where their position matches that of the developing ciliary bands. (D–E) Seaurchin gastrulae (D) Strongylocentrotus droebachiensis and (E) Heliocidaris tuberculata show the first serotonergic cells of the developingapical organ (arrows). (F) In the early larva of Strongylocentrotus purpuratus the apical organ (AO) has eight to nine cells interconnected byprocesses. AE, archenteron; BP, blastopore; M, mouth; S, stomach. A, C from Chee and Byrne (1999a); D from Bisgrove and Burke (1987);E from Bisgrove and Raff (1989); F from Bisgrove and Burke (1986). Scale bars: 50mm.

Fig. 3. Asteroidea: Patiriella regularis (A, C, D–H, J); Pisaster ochraceus (B),Meridiastra calcar (I). (A) Confocal reconstruction of the midregion of a bipinnaria showing the serotonergic ciliary band nerves (CBN) traversing the larval apex along the base of the pre- and post-oralciliary bands and immunopositive cells in the oral region around the mouth (M). In addition to the apical network of neurites the asteroidapical organ has two lateral ganglia (arrows) (B). Epifluorescence and (C, D). Confocal images of the lateral ganglia (LG). (E) Detail of oneganglion showing cell bodies (arrow) and thin fibers dotted with varicosities (arrowheads). (F) Confocal reconstruction looking down on theapex of a bipinnaria showing cell bodies (arrows) of the ciliary band nerves (CBN) and the connecting fibers (F) at the base of the pre- (top)and post (bottom)-oral ciliary band epithelia. Sensory-like cells bear apical processes (arrowhead). (G) Detail of sensory-like (SL) cells withapical processes (arrowheads) and basal axons with a beaded appearance (arrows). (H, I) Confocal reconstruction of a competentbrachiolaria of species with planktotrophic (H) and lecithotrophic (I) development. Serotonergic cells are scattered along the ciliary bandand along the edge of the arms (arrows). (J) Serotonergic cells along the edge of the brachiolar arms (arrows). A, adhesive disk; BA,brachiolar arms; S, stomach. (A) from Chee and Byrne (1999a); (B) FromMoss et al. (1994); (J) From Chee and Byrne (1999b). Scale bars:(A–D, F) 50mm; (E) 20mm; (G) 10mm; (H, I) 150mm; (J) 25mm.


Class Crinoidea

Nonfeeding larvae of stalked crinoids have a reduced dip-

leurula-like ciliary band (Fig. 1; Nakano et al. 2003). No

data have been published on the serotonergic system of

this larva, but inferences on the crinoid larval nervous system

are available from results obtained with the doliolaria larva of

Florometra serratissima using the glyoxylic acid method for

catecholamines (Chia et al. 1986). Doliolaria larvae have a

single catecholaminergic structure at the anterior end (Fig. 1)

that might be the apical organ. This multicellular structure is

associated with the larval adhesive organ (Chia et al. 1986). It

does not develop in association with a ciliary band.

Phylum Hemichordata

The hemichordate tornaria is a dipleurula-type larva and has

a single ciliary band similar to the holothuroid auricularia. As

for the auricularia, the serotonergic nervous system is largely

located in the anterior-most region of the larva (Fig. 1;

Fig. 4. Holothuroidea: Chirodota gigas. (A–B) The apical organ (AO) in the auricularia consists of cells in the ciliary band nerve along theventral (VB) and dorsal (DB) regions of the ciliary band that traverse the larval apex. These cells give rise to fibers (arrow) span between theciliary bands. A few cells (arrowhead) may be present in the oral hood. (C–D) Confocal 3-D reconstruction looking down on the apex ofthe auriculariae showing the right (RN), left (LN), dorsal (DN), and ventral (VN) portions of the ciliary band nerve. Where the right andleft portions of the ciliary band run in parallel at the larval apex fibers (arrows) span between the bands. H, hydropore; M, mouth;S, stomach. Scale bars (A, B, C) 100mm; (D) 50mm.


Nakajima et al. 2004b; Nezlin and Yushin 2004). The apical

organ cell bodies and processes are in a restricted region ad-

jacent to the anterior ciliary bands (Fig. 1, Nakajima et al.

2004b).

PLUTEUS-TYPE LARVAE

Class Echinoidea

The sea urchin serotonergic system is first evident in the an-

imal plate epithelium of gastrulae where a few immunoreac-

tive cells form (Fig. 2, D–F) and these give rise to basal

branched axons (Bisgrove and Burke 1986; Yaguchi et al.

2000). These cells form along the aboral margin of the animal

plate that has the ciliary tuft, a cluster of nonmotile cilia

characteristic of echinoid embryos. The animal plate is neu-

rogenic ectoderm that forms the apical organ and the cells are

derived from ciliary band ectoderm (Yaguchi et al. 2000,

2006, 2007). Unlike the morphogenetic movement that ap-

pears to occur in asteroids, early serotonergic cells in echinoid

development remain at the animal pole where they form the

apical organ (Figs. 2F and 5, A–C). By the early prism stage,

the apical organ connects with axons that extend to the pos-

terior region of the larva (Nakajima et al. 2004a).

Fig. 5. Echinoidea: Strongylocen-trotus droebachiensis (A, C), Psam-mechinus miliaris (B), Heliocidariserythrogramma (D, E). (A–C) Inechinoplutei the apical organ (AO)is formed by two groups of cellsand by a joining commissure (C)of fibers. (D) The apical organ(AO) of the lecithotrophic-reducedpluteus consists of two clusters ofcells. (E) Enlarged view of one ofthe cell clusters. AD, adoral gan-glion; AL, anterolateral arm;CBN, ciliary band nerve; M,mouth; PP, primary podium. (A,C) from Bisgrove and Burke(1987); (B) from Beer et al.(2001). (D, E) from Bisgrove andRaff (1989). Scale bars (A, B)50mm; (C) 25mm; (E) 25mm.


The anatomy and cellular structure of the apical organ

of echinoplutei is conserved in species across major

echinoid orders (Bisgrove and Burke 1986, 1987; Thorn-

dyke et al. 1992; Nakajima et al. 1993, 2004a; Yaguchi et

al. 2000; Beer et al. 2001). In the early four-armed pluteus

of Hemicentrotus pulcherrimus, the apical organ is a single

linear structure with the right and left ends having two

branches (Yaguchi et al. 2000). This bilateral structure is

part of the ciliary band of the early larva (Yaguchi et al.

2000). The apical organ subsequently develops as a

distinctive structure on the oral hood between the preoral

arms (Fig. 5, A and B). The cell bodies of the apical

organ do not reside within the ciliary band epithelium,

although they send neurites to the ciliary band nerve

tracts (Figs. 1 and 5B). Three major serotonergic

tracts emerge from the apical organ innervating

the antero-lateral arms, the adoral ciliary band, and the

esophagus.

The fully differentiated echinoid apical organ, although a

single structure, retains its bilateral symmetry. The cell

bodies are located in two clusters connected by a dense

neuropil (Figs. 1 and 5, B and C). In advanced echinoplutei,

the apical organ has 40–50 cells in two clusters (Fig. 5, B and

C). Sensory-like processes from apical organ cells extend be-

yond the epithelium (Beer et al. 2001). The apical organ

also has unusual sensory cells with coiled cilia that lie within

the hyaline layer (Nakajima 1986, 1993). The echinopluteal

apical organ remains a prominent structure through the

late larval stage and is lost during metamorphosis (Bisgrove

and Burke 1986, 1987; Beer et al. 2001). Adoral serotonergic

ganglia are conspicuous along the posterior rim of the mouth

(Fig. 5A).

Interestingly, the apical organ in the lecithotrophic larva of

Heliocidaris erythrogramma is also bilaterally symmetrical. It

is formed by two clusters of cells that are not connected and

which are positioned on the oral side of the larva anterior to

the primary podia (Fig. 5, D and E). They might be homol-

ogous to the two cell clusters in the apical organ of plank-

totrophic echinoplutei, as seen in the congener, Heliocidaris

tuberculata (Bisgrove and Raff 1989), except that they differ in

location. Both ganglia in H. erythrogramma are anterior to

the rudiment. They persist longer in development than the

apical organ in echinoplutei.

Class Ophiuroidea

In ophiuroids, serotonergic cells are not evident in gastrulae

and do not appear in the apical region at any stage of de-

velopment in the three species (representing three families)

examined. Ophioplutei have a single ciliary band that traces

the edge of the larval arms (Fig. 1). In the early four-armed

ophioplutei of Amphipholis kochii and Ophiothrix ciliaris, a

few serotonergic cells form a pair of ganglia in the dorsal

epithelium at the base of the anterolateral arms (Figs. 1 and 6,

A–C). Although not placed in an apical position, these bilat-

erally symmetrical ganglia appear to be the ophiuroid apical

organ. These ganglia, however, were not evident in the

ophiopluteus of Ophiactis resiliens (Fig. 6G).

The serotonergic system of ophioplutei is most prominent

in the antero-lateral region of the larvae. In eight-armed lar-

vae, serotonergic processes positioned at the junction between

the anterolateral and postoral arms extend processes part way

along the ciliary epithelium of these arms (Fig. 6, B–F). A

cross-section of the base of these arms inO. ciliaris shows the

ciliary band nerve tract in the epithelium along both sides of

the arm (Fig. 6, C–E). Serotonergic cell bodies are sparse and

include bipolar and multipolar cells. Immunoreactivity was

not seen in the posterodorsal or posterolateral arms of any

ophiopluteus examined. In the advanced pluteus of O. resi-

liens, clusters of serotonergic cells were dotted along the an-

terolateral arms but were not seen in structures that could be

considered to be ganglia (Fig. 6, G and H). Ophioplutei of all

three species have a conspicuous serotonergic immunoreac-

tivity along the posterior rim of the mouth in an adoral gan-

glion (Fig. 6, F and G).

DISCUSSION

Larval apical organs in the Ambulacraria have serotonergic

cells that develop as bilaterally organized structures that differ

in ontogeny, cellular organization, and anatomy (Fig. 1, Ta-

ble 1). They range from a loosely organized network of neu-

rites to ganglionic groups of cells. Apical organ cell bodies

reside in the ciliary band epithelium of all larvae with the

exception of the echinopluteus. The apical organ is most

complex in asteroid and echinoid larvae where it forms

Fig. 6. Ophiuroidea: Amphipholis kochii (A), Ophiothrix ciliaris (B–F), Ophiactis resiliens (G, H). (A) Epifluorescence image of an earlyophiopluteus showing a developing lateral ganglion (arrow) at the base of the anterolateral arms (AL) and the ciliary band nerve (CBN).(B–E) Deconvolution microscopy image of an eight-armed ophiopluteus. The lateral ganglia (arrows) at the junction of anterolateral (AL)and postoral (PO) arms are formed by a few cells and processes in the basal epithelium (arrows). (F) In advanced eight-arm ophioplutei, thelateral ganglia (LG) connect with the ciliary band nerve along the anterolateral arm (AL). Serotonergic immunoreactivity is also present inthe preoral ciliary band nerve (PRO) and adoral ganglion (AD). (G, H) O. resiliens larvae lack lateral ganglia, but have a prominent adoralganglion (AD) and the ciliary band nerve (CBN) along the anterolateral arm. Arrow, cell bodies of the CBN. M, mouth; PL, posterolateralarm; S, stomach. Scale bars (A) 100mm; (B, C) 50mm; (D, E) 20mm; (F) 80mm; (G) 35mm; (H) 15mm.


extensive innervation of the anterior region, ciliary bands, and

the oral region. The apical organ is least complex in ho-

lothuroids and hemichordates, largely limited to a cell bodies

and connectives at the anterior end of the larva. Where pres-

ent, the apical organ in ophiuroids is restricted to the lateral

ganglia at the base of the arms.

Across the Ambulacraria the apical organ differentiates in

association with ciliary structures; the animal plate ciliary tuft

in echinoids, and anterior ciliary bands in asteroids, ho-

lothuroids, ophiuroids, and hemichordates (Table 1). In dip-

leurula-type larval development, serotonergic progenitor cells

arise in the apical ectoderm of holothuroid and hemichordate

embryos and remain restricted to the anterior-most region,

whereas those in asteroid embryos appear to migrate to the

developing ciliary band. This difference in neuroblast behav-

ior is likely to be linked to the more-extensive serotonergic

nervous system in asteroid larvae. Morphogenetic migration

associated with ciliary band formation is also reported for

synaptotagmin immunoreactive cells in asteroid bipinnaria

(Nakajima et al. 2004a) and catecholaminergic cells in ho-

lothuroids auricularia (Chen et al. 1995). In the pluteus-type

larvae of echinoids, early serotonergic cells are restricted to

the animal plate ectoderm (Nakajima et al. 2004a: Yaguchi et

al. 2006). The origin of serotonergic cells in ophiuroid devel-

opment has not been described.

In asteroids and holothuroids, the apical organ is formed

by serotonergic cells along ciliary band sectors at the larval

apex and fibers that cross between the bands. The apical or-

gan of the bipinnaria also has lateral ganglia that connect with

the apical plexus (Fig. 1). In both these dipleurula-type larvae,

the apical organ is immunopositive for a number of neural

markers including neuron-specific synaptotagamin and echi-

noderm-specific neuropeptide (S15GNLSFamide 1) and

also stains for catecholamine (Moss et al. 1994; Chen et al.

1995; Byrne et al. 2001; Byrne and Cisternas 2002; Nakajima

et al. 2004a: Burke et al. 2006). These markers also show a

network of neurites spanning between apical ciliary bands.

The organization of apical organs in the two plutei differs

from that seen in the dipleurula forms and from each other.

Echinoplutei have an apical organ, formed by two ganglionic

groups of cells joined by a commissure. It is a single structure

and the cell bodies do not reside within the ciliary band ep-

ithelium (Fig. 1). In contrast, the two lateral ganglia in the

apical organ of the ophiopluteus are part of the ciliary band

(Fig. 1), although their development varies among ophiuroid

species. Cells of the echinoid apical organ are immunopositive

for synaptotagamin and S1 and some cells express more than

one neurochemical (Thorndyke et al. 1992; Beer et al. 2001;

Nakajima et al. 2004a). There are no published data on other

neural markers for the ophiuroid apical organ.

The similar ciliary band anatomy and apical organ struc-

ture in the auricularia and tornaria may reflect their shared

ancestry (Fig. 1). The right–left ciliary band organization with

two band sectors traversing each side of the oral hood is likely

to be the ancestral-type arrangement for the Ambulacraria. In

both larvae, the serotonergic system is largely restricted to the

apical region in a limited region of the ciliary band (Burke et

al. 1986; Tagawa et al. 2001; Nakajima et al. 2004b; Nezlin

and Yushin 2004). Their apical organs comprises serotonergic

cells and fibers along and spanning between the ciliary band

sectors at the anterior-most end of the larvae (Fig. 1). The

nervous systems in the auricularia and tornaria are also sim-

ilar in the cellular ultrastructure (Lacalli 1996; Lacalli and

Kelly 2002; Nakajima et al. 2004b; Nezlin and Yushin 2004).

The apical organ of the asteroid bipinnaria has a dorso-

ventral symmetry due to the path traversed by the two ciliary

bands across the larval apex (Figs. 1 and 3A). Lateral

serotonergic ganglia are a prominent feature of the asteroid

apical organ. As suggested by Lacalli (1994), these lateral

ganglia may have originated at the anterior end of an ances-

tral dipleurula and became laterally displaced during larval

evolution. It seems likely that they were derived from bilat-

erally symmetric cells along the two anterior ciliary bands.

In echinoplutei the apical organ is a single structure com-

prising two groups of serotonergic cells on the right and left

sides of the oral hood joined by a neuropil. This structure may

have originated by fusion of neurons derived from apical cili-

ary band sectors that traversed the right and left sides of a

dipleurula-like ancestor. The lateral serotonergic ganglia in

the ophiopluteus may have had a similar origin, but more

data are required to understand the larval neuronal architec-

ture of the Ophiuroidea.

Interestingly, two groups of apical organ cells in sea urchin

development have been retained through evolution of the re-

duced pluteus ofH. erythrogramma (Bisgrove and Raff 1989).

The orientation of these two groups of cells however is not

clear with respect to the position of the echinopluteus apical

organ. In echinoplutei, the apical organ persists to the late

larval stage, but its position relative to the developing juvenile

as in H. erythrogramma is not known. Alternatively, the or-

ganization and location of the apical organ in H. erythro-

gramma may be a novel feature associated with the evolution

of direct development.

Serotonergic immunoreactivity is absent in the oral re-

gion of the auricularia and tornaria (Nakajima et al. 2004b;

Nezlin and Yushin 2004; Byrne et al. 2006; Nakano et al.

2006). This suggests that the presence of serotonergic oral

ganglia along the posterior rim of the mouth of asteroid,

ophiuroid, and echinoid larvae is a derived and convergent

feature (Table 1). Oral serotonergic ganglia in these larvae

are suggested to have a gustatory function in feeding (Moss

et al. 1994; Chee and Byrne 1999a; Beer et al. 2001; Cis-

ternas and Byrne 2003). It seems likely that the auricularia

and tornaria have the ability to discriminate food particles,

as do bipinnaria and echinoplutei (Strathmann 1971). This

sensory ability may involve nonserotonergic neurons such


as those seen in the adoral ciliary bands of P. californicus

and Actinopyga miliaris (Burke et al. 1986; Chen et al. 1995).

The shared absence of serotonergic cells in the adoral ciliary

band in the auricularia and tornaria (Table 1) suggests that

the ancestral dipleurula larva may have employed other ne-

urochemicals to modulate feeding behavior.

Anterior serotonergic organs are prominent in marine

metazoan larvae and are suggested to serve a number of sen-

sory functions (Hay-Schmidt 2000; Nielsen 2005). The func-

tion of echinoderm apical organs in feeding, swimming, and

metamorphosis has been considered in several studies (Burke

1983b; Chee and Byrne 1999b; Beer et al. 2001; Cisternas and

Byrne 2003; Nakano et al. 2006). Pharmacological studies

with asteroid and echinoid larvae suggest that serotonin has a

role in control of ciliary beat (Chee and Byrne 2001; Yaguchi

and Katow 2003; Katow et al. 2004, 2007). The presence of

what appear to be sensory cells with a cilia-like apical process

and basal axons is consistent with the interpretation that api-

cal organ cells are sensory. Ascidian larvae have an anterior

serotonergic ganglion suggested to play a role in locomotion

and light detection (Stach 2005). An anterior or apical con-

centration of serotonergic cells is likely to be an ancestral

feature of deuterostome development. It is not clear, however,

what the significance of this feature might be with respect to

evolution of the chordate nervous system (Tagawa et al. 2001;

Lacalli and Kelly 2002; Lacalli 2005).

The persistence of the apical organ through develop-

ment differs among echinoderms. In echinoplutei, the api-

cal organ remains as a prominent feature to the competent

larval stage and is suggested to be involved in substrate

selection and modulation of metamorphosis (Burke

1983b; Beer et al. 2001). The presence of a well-

developed apical organ in the metamorphic larva of

H. erythrogramma, a nonfeeding larva, supports a role

for this structure in echinoid settlement. In contrast,

ophioplutei metamorphose in the plankton and their lar-

val serotonergic system is gone well before settlement

(Cisternas and Byrne 2003). In asteroids, sensory cells in

the attachment complex in feeding and nonfeeding bra-

chiolariae are likely to be involved in substrate selection,

settlement, and metamorphosis (Chee and Byrne 1999b;

Byrne 2001). Dissection experiments with the brachiolaria

of Asterina pectinifera indicate that the apical organ is not

required for settlement, but the serotonergic cells on the

brachia are involved in settlement (N. Murabe, personal

communication). The apical organ of holothuroids de-

generates in the doliolaria larva of Stichopus japonicus

(Nakano et al. 2006). Metamorphosis in echinoderms is

associated with a major change in neurochemistry. Little if

any of the larval nervous system contributes to the pent-

ameral nervous system of the juvenile (Byrne and

Cisternas 2002; Cisternas and Byrne 2003; Nakano et al.

2006). In contrast to its expression in the larval nervous

system, serotonin does not appear to be expressed in the

pentameral nerve cords of adult asteroids (M. Byrne

and F. Chee, unpublished data) although serotonin is

expressed in peripheral neurons of holothuroids (Inoue

et al. 2002).

Although serotonin immunocytochemisty provides excel-

lent insights into the anatomy of the larval nervous systems,

data for other neurochemicals and molecular markers are

needed to gain a better understanding of shared and derived

features of Ambulacraria larval nervous systems. Application

of the neuron-specific synaptotagamin antibody for instance

has revealed previously undetected complexity and details of

neural ontogeny (Nakajima et al. 2004a; Nakano et al. 2006).

We lack detailed studies of the neurophysiology of

echinoderm larvae and have a very poor understanding of

what larval nervous systems do. Through use of a number

of neural markers and advanced microscopy, we now have

good data on the cellular organization of the nervous system.

It would be useful to apply these data to functional studies.

The recent identification of neural genes in the sea urchin

genome and in library screens provides opportunities for new

approaches for comparative studies of neural specification

and gene expression (Sly et al. 2002; Burke et al. 2006). In-

vestigation of neural patterning genes in echinoderm and

hemichordate development show that some genes are ex-

pressed in the apical organs, but others are not (Tagawa et al.

2000, 2001; Taguchi et al. 2002; Takacs et al. 2002; Poustka et

al. 2004; Byrne et al. 2005; Lacalli 2005; Burke et al. 2006).

Recent research provides insights into the signaling that me-

diate specification of serotonergic neurons of the apical organ

in echinoid development (Yaguchi et al. 2006, 2007). Inves-

tigation of neural specification across larval Ambulacraria is

needed to address fundamental questions on neural ontogeny

and on the significance of differences such as the position of

apical organ cell bodies in or outside the ciliary band domain.

In particular, data on expression of a range of neural markers,

for the auricularia and tornaria, are needed to assess hypoth-

eses on the anatomy and development of the ancestral ner-

vous system in larval Ambulacraria.

The disparate ontogeny, cellular organization, and anat-

omy of serotonergic apical organs in echinoderm and hem-

ichordate larvae present a challenge to identify homologous

features and ancestral states. Table 1 lists the shared and lar-

va-specific features of these structures. The apical organs in

dipleurula-type larvae are more similar to each other than to

those in either of the pluteal forms (Fig. 1, Table 1). Mor-

phology thus reflects phylogeny. The similar bilateral orga-

nization of the apical organ in anterior ciliary band in the

tornaria, bipinnaria, and auricularia may reflect shared an-

cestry (Fig. 1). In contrast, the two plutei have markedly

different apical organs. The single apical organ of echinoplutei

with cell bodies positioned outside the band is an independent

elaboration of echinoid development, although its cell struc-


ture links it to the bilateral ancestral state. Ophiuroids have

two simple ganglia in ciliary band but these are not apical in

location. The dissimilar morphology of apical organs of

ophioplutei and echinoplutei supports the hypothesis that

these plutei are convergent larval forms (Strathmann 1988).

This hypothesis is supported by phylogeny (Fig. 1) and a

plethora of other differences in echinoid and ophiuroid de-

velopment (Primus 2005).

Shared features of larval form and serotonergic immuno-

reactivity provide insights into the putative apical organ

structure of the ancestral dipleurula (Fig. 1, Table 1). The

comparative data indicate that the ancestral apical organ

originated in apical ectoderm and developed into a bilateral

nerve plexus along and spanning between ciliary bands at the

anterior-most end of the larva. From this comparatively sim-

ple organization, the apical organ has been independently

modified during evolution of the distinctive class-specific lar-

val forms. Differences in the details of apical organ develop-

ment and structure in modern Ambulacraria are likely to have

arisen independently in the different clades though 500 million

years of evolution (Raff and Byrne 2006).

AcknowledgmentsThe research was supported by an Australian Research CouncilDiscovery Grant to M. B. Y. N. was supported by the Keio GijukuFukuzawa Memorial Fund for the Advancement of Education andResearch. R. D. B. was supported by an NSERC (Canada) Discov-ery Grant. We thank many colleagues and publishers for permissionto use previously published images. Drs. V. Morris, R. Raff, and L.Page are thanked for commenting on the manuscript. Drs. P. Cis-ternas, P. Selvakumaraswamy, and M. Orchard and Mr. T. Prowseprovided technical assistance. We thank the Electron MicroscopeUnit, University of Sydney, for the assistance with microscopy. TheWhiteley Centre, University of Washington, is thanked for supportduring preparation of this manuscript. The reviewers are thanked forhelpful comments. Contribution number 6 of the Sydney Institute ofMarine Science.

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