Complex Neural Architecture in the Diploblastic Larvaof Clava multicornis (Hydrozoa, Cnidaria)

Stefano Piraino,1* Giuliana Zega,2 Cristiano Di Benedetto,3 Antonella Leone,1,4 Alessandro Dell’Anna,1,2

Roberta Pennati,2 Daniela Candia Carnevali,3 Volker Schmid,5 and Heinrich Reichert5

1Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita del Salento, 73100 Lecce, Italy2Dipartimento di Biologia, Functional and Reproductive Biology, Universita di Milano, 20133 Milano, Italy3Dipartimento di Biologia, Zoologia e Citologia, Universita di Milano, 20133 Milano, Italy4CNR-ISPA, Campus Ecotekne, 73100 Lecce, Italy5Biozentrum, Neurobiology, University of Basel, 4056 Basel, Switzerland

ABSTRACTThe organization of the cnidarian nervous system has

been widely documented in polyps and medusae, but

little is known about the nervous system of planula

larvae, which give rise to adult forms after settling and

metamorphosis. We describe histological and cytologi-

cal features of the nervous system in planulae of the

hydrozoan Clava multicornis. These planulae do not

swim freely in the water column but rather crawl on the

substrate by means of directional, coordinated ciliary

movement coupled to lateral muscular bending move-

ments associated with positive phototaxis. Histological

analysis shows pronounced anteroposterior regionaliza-

tion of the planula’s nervous system, with different

neural cell types highly concentrated at the anterior

pole. Transmission electron microscopy of planulae

shows the nervous system to be unusually complex,

with a large, orderly array of sensory cells at the ante-

rior pole. In the anterior half of the planula, the basiec-

todermal plexus of neurites forms an extensive

orthogonal network, whereas more posteriorly neurites

extend longitudinally along the body axis. Additional

levels of nervous system complexity are uncovered

by neuropeptide-specific immunocytochemistry, which

reveals distinct neural subsets having specific molecular

phenotypes. Together these observations imply that the

nervous system of the planula of Clava multicornis man-

ifests a remarkable level of histological, cytological, and

functional organization, the features of which may be

reminiscent of those present in early bilaterian animals.

J. Comp. Neurol. 519:1931–1951, 2011.

VC 2011 Wiley-Liss, Inc.

INDEXING TERMS: Cnidaria; Hydrozoa; neuroanatomy; planula; CNS; cephalization; early central nervous system

evolution

A shared feature of Cnidaria and Bilateria is the devel-

opment of a nervous system composed of interconnected

neural cells. In all animal groups, the number of neural

cells, the complexity of their connectivity, and their orga-

nization into regionalized neural assemblies show a great

deal of variation. The evolutionary origin of neuroanatomi-

cal complexity and diversity in eumetazoan nervous

systems represents one of the fundamental problems of

neurobiology. A number of recent comparative develop-

mental and genetic analyses suggest that the nervous

systems of protostomes and deuterostomes arose from a

common evolutionary origin (Lichtneckert and Reichert,

2005, 2008; Denes et al., 2007; Arendt et al., 2008) and

that the urbilaterian nervous system was already a

complex, centralized structure (De Robertis, 2008). Alter-

native views propose multiple, independent evolutionary

origin of central nervous systems (Holland, 2003; Lowe

et al., 2003) and of neuronal types (Moroz, 2009). Since

cnidarians are the evolutionary sister clade of bilaterians

(Collins, 1998; Medina et al., 2001; Ryan et al., 2006;

Dunn et al., 2008), a key question concerns the nature of

a prototype nervous system from which both cnidarian

and bilaterian nervous systems might have evolved. This

Additional Supporting Information may be found in the online version ofthis article.

Grant sponsor: Italian Ministry for Research and Education; Grantnumber: 2007-5WCPWM (to S.P.); Grant sponsor: Swiss National ScienceFoundation (to H.R.).

*CORRESPONDENCE TO: Stefano Piraino, Dipartimento di ScienzeTecnologie Biologiche Ambientali, Universita del Salento, 73100 Lecce,Italy. E-mail: stefano.piraino@unisalento.it

VC 2011 Wiley-Liss, Inc.

Received April 24, 2010; Revised September 18, 2010; AcceptedDecember 30, 2010

DOI 10.1002/cne.22614

Published online February 24, 2011 in Wiley Online Library(wileyonlinelibrary.com)

The Journal of Comparative Neurology | Research in Systems Neuroscience 519:1931–1951 (2011) 1931

RESEARCH ARTICLE

issue is especially interesting in view of recent molecular

developmental data supporting the notion that ancestral

cnidarians already possessed the molecular toolkit nec-

essary to establish a triploblastic and bilateral Bauplan

(Boero et al., 1998, 2007; Finnerty, 2003; Spring et al.,

2002; Seipel and Schmid, 2005; Matus et al., 2006). To

address this question, and thus elucidate the evolutionary

origin of nervous system centralization, new studies on

cnidarian nervous systems that integrate comparative

developmental and neuroanatomical approaches are

needed.

In cnidarians, a great deal of information on structural

organization of the nervous system has been obtained for

adult stages. The nervous systems of both polyps and

medusae are located in both ectodermal and endodermal

cell layers and contain motoneurons, ganglionic inter-

neurons, and sensory cells (the latter mostly within the

ectoderm; for recent reviews see Watanabe et al., 2009;

Galliot et al., 2009). Polyps generally display a ‘‘diffuse’’

epithelial nerve net without morphologically discrete sen-

sory or motor processing centers, except for a localized

ring-like concentration of neural elements around the

mouth (often called the ‘‘head’’ in reports on Hydra). In

swimming jellyfish, a more sophisticated, radially organ-

ized nervous system occurs as single or double nerve

ring at the margin of the umbrella, interconnecting multi-

ple neural cell types and incorporating complex sense

organs (statocysts, ocelli, rhopalia) to form the advanced

neural circuitry required for coordinated movement,

vision, and complex behavioral control (Garm et al.,

2007a,b; Parkefelt and Ekstrom, 2009). The functions of

these neural structures resemble those of the central

nervous system of bilateral animals (Mackie, 2004; Garm

et al., 2006).

In contrast to the detailed knowledge of the nervous

systems in adult cnidarians, little is known about neural

organization in early developmental stages. Cnidarian

ontogeny usually involves a swimming planula larva that,

in response to specific stimuli, settles on the substrate

and metamorphoses into the polyp stage. In Medusozoa,

the polyp stage may give rise to the medusa stage. Many

cnidarian taxa may lack either the polyp or the medusa,

but only in few cases showing direct embryonic develop-

ment into the polyp has the planula stage been sup-

pressed. The presence of neurons in the larva has been

demonstrated in three major cnidarian taxa, namely, the

Anthozoa (Chia and Koss, 1979; Miller and Ball, 2000;

Hayward et al., 2001; Marlow et al., 2009), Hydrozoa

(Martin and Thomas, 1980, 1981; Thomas et al., 1987;

Plickert et al., 1988; Martin, 1992, 2000), and Scyphozoa

(Korn, 1966; Nakanishi et al., 2008; Yuan et al., 2008). In

planulae, the nervous system is located in the ectoderm

and comprises epithelial sensory cells and basally located

ganglionic cells, whose neurites project in a net overlying

the mesoglea (Martin, 1988a,b). In hydrozoans, endoder-

mal stem cells (interstitial cells or i-cells) give rise to

nematoblasts and neuroblasts, which differentiate into

nematocytes and ganglionic cells, respectively, during

their migration toward their final location in the ectoderm.

During the gradual (direct) development of the anthozoan

Nematostella vectensis embryo, a second, endodermal

network of ganglionic cells also appears in late develop-

mental stages (Marlow et al., 2009), linked to the early

onset of polyp-specific structures (pharynx, mesenteries,

and gastric cell populations).

The swimming planulae of some anthozoans and scy-

phozoans possess a specialized ectodermal population of

elongated ciliated cells at the anterior pole, with basal

nuclei, forming the apical organ or apical tuft, a sensory

structure probably involved in perception of chemical

cues at the site of metamorphosis (Widersten, 1968; Chia

and Bickell, 1978; Chia and Koss, 1979; Nakanishi et al.,

2008; Yuan et al., 2008; Marlow et al., 2009). In hydro-

zoan planulae, this structure does not occur.

Most cnidarians have relatively simple, radially sym-

metrical planulae that drift (‘‘swim’’) in the water column

by rotating along the main body axis before settlement

and metamorphosis occur. Within several taxa, however,

non-swimming planulae that display positive geotaxis and

crawl on the bottom have been reported (Otto, 1976;

Hartnoll, 1977; Gerrodette, 1981; Sommer, 1992;

Hellberg, 1995; Orlov, 1996; Heltzel and Babcock, 2002;

Bouillon et al., 2006). Both the ‘‘swimming’’ and the

‘‘crawling’’ movements of planulae are based on ciliary

locomotion (Werner, 1984; Bouillon et al., 2006). The be-

havioral repertoire required for oriented locomotion on

the substrate, however, differs significantly from the rota-

tory activity of ‘‘swimming’’ planulae. The differences in

coordinated behavioral activity of the two types of planula

may be reflected in corresponding differences in the

organization of the nervous system. Indeed, given the

common behavioral constraints encountered by any

animal with benthic (bottom-related) locomotory activity,

it is conceivable that the neurobiological organization of

‘‘crawling’’ cnidarian planulae might be comparable to

that of simple bilaterians with similar behavior.

To investigate both morphological and functional

aspects of this, we focused on the nervous system of the

planula of Clava multicornis (Hydrozoa), a larva with pro-

nounced benthic locomotory activity. Here we report

behavioral observations as well as morphological and neu-

rochemical features of its nervous system. First, behavioral

studies document that the planula shows directed locomo-

tion, with the anterior/aboral pole oriented forward,

accompanied by alternating sideways bending movements.

Second, neuroanatomical observations indicate marked

Piraino et al.

1932 The Journal of Comparative Neurology |Research in Systems Neuroscience

anteroposterior regionalization of the nervous system char-

acterized by a large array of sensory cells at the anterior

pole. Third, immunocytochemical analyses reveal an un-

usual complexity of neural-specific components revealing

distinct cell populations with different molecular proper-

ties. Overall, these findings imply that the presumptive

‘‘simple’’ nervous system of these planulae displays a

surprisingly high level of histological, cytological, and bio-

chemical complexity. Further investigations are needed to

clarify whether the similarities in neural architecture

between cnidarians, including the planulae reported here,

and early bilaterian animals are conserved characters that

were shared by their last common ancestor or whether

they arose from parallel, independent evolution of nervous

systems.

MATERIALS AND METHODS

AnimalsFertile colonies of Clava multicornis (Forskal 1775)

were collected at low tide along Roscoff (France) rocky

shores and transferred to the laboratory. Animals were

reared in aquaria with an open-sea circulation system

(14�C). Daily, some female colonies were isolated,

exposed to light to stimulate planula release, and

checked every hour to collect larvae. Prehatching stages

were obtained by dissection of female gonophores.

The phototropic behavior of planulae was documented

by an image-recording system consisting of a dissecting

microscope (Leica MZ12) equipped with digital photo-

videocamera (Olympus C5050). Newly hatched planula

larvae (12 hours) were kept overnight in the dark before

observation. To observe their behavior, planulae were

transferred into 30-mm glass Petri dishes kept in a water

bath maintained at 14�C. Directed light from a halogen

50-W lamp was provided on one side of the Petri dish

through a fiberglass cable. Images of planula larvae were

taken every 5 seconds to show modifications of body

shape and arranged in series to create a single image.

Light and electron microscopyPlanulae were first anesthetized by short incubation

with 7% MgCl2 and then fixed with 2% glutaraldehyde in

0.1 M cacodylate buffer, pH 7.2. Samples were then post-

fixed with 1% osmium tetroxide in 0.1 M cacodylate

buffer, dehydrated in a graded ethanol series, and em-

bedded in an Epon-Araldite mixture according to standard

transmission electron microscopy (TEM) methods (Maun-

nsbach and Afzelius, 1999). Semithin (1-lm) and ultrathin

(50-nm) sections were cut with a Reichert-Jung Ultracut E

using diamond knives (Diatome Histo and Ultra 45�).

Semithin sections, collected on standard slides and

stained with crystal violet and basic fuchsin, were con-

trolled under a Jenaval light microscope. Ultrathin sec-

tions, collected on 300-mesh copper grids, were stained

with aqueous uranyl acetate and lead citrate (Reynolds,

1963), carbon coated under an Emitech K400X carbon

coater, and observed with a Jeol 100 SX electron micro-

scope. Micrographs were taken directly under the micro-

scope with Kodak 4489 photographic films for TEM.

ImmunohistochemistryPlanulae were fixed in 4% paraformaldehyde (PFA) in

0.1 M phosphate-buffered saline (PBS), pH 7.2, for 1 hour

at room temperature. After rinsing with 0.1 M PBS, fol-

lowed by triplicate washes in 0.4 M glycine, the samples

were stored in 70% ethanol at –20�C. After rehydration,

specimens were incubated with 0.1% Tween-20, 0.25%

Triton X-100 in PBS (PBT) for 30 minutes and then

washed three times for 10 minutes each with PBS. Subse-

quently, samples were blocked with 10% horse serum in

PBT (PBT-HS) for 30 minutes and finally incubated over-

night at 4�C with the different primary antibodies (see

Table 1) diluted in PBT-HS.

After several washes in PBS, samples were incubated

in PBT-HS for 1 hour and then overnight at 4�C with

appropriate secondary (goat anti-rabbit or goat anti-

mouse) antibodies (Alexa Fluor 488 or Alexa Fluor 594

TABLE 1.

Primary Antibodies Used in This Study

Antigen Immunogen Manufacturer, species, type, catalog No. Dilution

Tyrosinatedtubulin

Synthetic peptide (C-terminal amino acids of a-tubulinfrom porcine brain plus an additional N terminallysine and a C-terminal tyrosine; KVEGEGEEEGEEY)

Sigma (Italy), clone TUB-1A2, murine monoclonal,T9028

1:500

Acetylatedtubulin

Purified protein from Strongylocentrotus purpuratus Sigma (Italy), clone 6-11B-1, murine monoclonal,T6793

1:500

GLWamide Synthetic C-terminal-GLWamidated peptide(CAAPPGLW�NH2) coupled via succinimidylm-maleimido-benzoate (MBS) to heyhole limpethemocyanin (KHL)

Gift from Prof. Thomas Leitz (Germany), clone1676IIIp, rabbit polyclonal

1:700

RFamide RF-NH2 coupled via carbodiimide to bovinethyroglobulin

Gift from Prof. Thomas Leitz (Germany), clone1773IIIp, rabbit polyclonal

1:700

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1933

IgG; Invitrogen, Carlsbad, CA), diluted 1:200–400 in PBT-

HS. Next, the specimens were washed four times in PBS

and mounted in 1,4-diazabicyclo[2,2,2]octane (Dabco,

Sigma Italy) on microscope slides. Immunolabeled speci-

mens were observed via laser scanning confocal

microscopy systems (Leica TCS-NT, Zeiss LSM 510, Zeiss

LSM 5 Pascal). Fluorescence was detected with the com-

binations of 488-nm excitation filter and 530/30-nm

bandpass filter or 530–560 nm excitation filter and 590/

50-nm bandpass filter. Multiple optical sections (0.7–1

Figure 1. Life cycle of C. multicornis. A: Hydroid colony growing on Ascophyllum nodosum (brown seaweed). B: Closeup view of male

polyp: whitish gonophores at different stages of development encircle the hydranth column, just below the tentacles. C: Female polyps

with bright bluish gonophores (embryos at different stages of development). D,E: Female gonophores and embryos at different stages of

development. D: One or two eggs are contained within each gonophore, where fertilization and development take place. E: Sixteen- to

thirty-two-cell embryos (DAPI staining). F: Early larva, 24 hours postfertilization. The anteroposterior axis is already established. G: Hatched

planula. Scale bars ¼ 2 mm in A; 400 lm in B,C; 100 lm in D,G; 60 lm in E,F.

Piraino et al.

1934 The Journal of Comparative Neurology |Research in Systems Neuroscience

lm) were taken through the depth of the specimen

(Z axis), in order to reconstruct 3-D images of the whole

planula. Confocal stack images were recorded and post-

processed with dedicated software. The number and step

size of ‘‘optical sections’’ are given for each image in the

figure legends. Series of optical sections obtained by scan-

ning whole-mount specimens were projected into one

image with greater focal depth. In control samples incu-

bated either with the primary or the secondary antibody

alone, no fluorescent signal or staining was observed.

Antibody characterizationPrimary antibodies (Table 1) raised against tyrosinated

a-tubulin and acetylated a-tubulin were used as pan (gen-

eralized)-neuronal markers, because a-tubulin is a major

components of axonal processes (see Kreis, 1987; Siddi-

qui et al., 1989; Jellies et al., 1996). The tyrosinated

a-tubulin antibody was raised against a synthetic peptide

(T13) containing 11 C-terminal amino acids of a-tubulin

from porcine brain plus an additional N terminal lysine

and a C-terminal tyrosine at the C-terminus (Kreis, 1987).

The acetylated a-tubulin antibody was raised from the

purified protein from the outer arm of the S. purpuratus

(sea urchin) axoneme. Both a-tubulin antibodies recog-

nized a single band at 55 kD on Western blots of total

protein extracts from Clava multicornis polyps. The cross-

phylum specificity of the two antibodies was confirmed

by their consistent pattern of staining in a large number

of metazoan taxa, including cnidarians (Thomas and

Edwards, 1991). In particular, the antiacetylated a-tubulin

antibody has been used as marker of an interphyletically

conserved epitope present in cilia (Piperno and Fuller,

1985) but also for labeling nerve cells and fibers specifi-

cally in different cnidarian taxa (see, e.g., Anthozoa:

Rentzsch et al., 2008; Scyphozoa: Kozmik et al., 2008;

Hydrozoa: Chiori et al., 2009). Similarly, the antityrosi-

nated tubulin antibody has been systematically used as a

neuronal marker in Cnidaria (Groger and Schmid, 2000;

Galliot et al., 2009; Watanabe et al., 2009), with strong

reactivity against motoneuron populations (Nakanishi

et al., 2009, 2010).

Two additional primary polyclonal antibodies directed

against two amidated neuropeptides, GLWamide and

RFamide peptides, were also used (Grimmelikhuijzen,

1985; Leitz et al., 1994). GLWamide and RFamide

antisera were raised and kindly provided by J. Schmich

and T. Leitz (Kaiserslautern). GLWamide controls

(Schmich et al., 1998a,b) were performed by using antise-

rum preadsorbed with He-LWamide II coupled to Hi-Trap

NHS-activated beads (Pharmacia, Freiburg, Germany),

which abolished any staining. The RFamide antiserum

was preincubated with 2 mg/ml bovine thyroglobulin, fol-

lowed by centrifugation to remove antibody populations

specific for the carrier protein (Grimmelikhuijzen, 1985).

Specificity of the polyclonal anti-RFamide antibody has

also been demonstrated in several cnidarian species

(Plickert, 1989; Plickert and Schneider, 2004). In

addition, we have observed colocalization of the 1773IIIp

RFamide antibody staining pattern with the in situ hybrid-

ization labeling pattern for the mRNA of the precursor

protein for neuropeptide Pol-RFamide II in Turritopsis

dohrnii and Hydractinia (Podocoryne) carnea, two hydro-

zoans phylogenetically related to Clava multicornis

(Piraino et al., unpublished data).

Photomicrograph productionDigital images were imported as TIFF files from Leica

or Zeiss software (confocal microscopy) and JEOL TEM

software (electron microscopy) into Adobe Photoshop

and adjusted for optimal brightness and contrast and for

conversion of red-green fluorescent figures into magenta-

green ones.

RESULTS

Development and locomotory behavior ofplanula larvae

The hydroid Clava multicornis (C. multicornis) is a pro-

genetic hydroid without a medusa stage. The embryonic

development of larval planulae takes place on the polyp

colony. When hatched, the planula is worm-like, cigar-

shaped, and approximately 600–800 lm long and has a

prominent anterior pole (club-shaped ‘‘head’’) that tapers

to the posterior pole (‘‘tail’’; Fig. 1G). In contrast to most

planulae, these larvae do not swim in the water column.

C. multicornis planulae instead display a smooth, gliding

movement on the benthos (seaweeds, rocky bottom)

while searching for a suitable site for settlement, where

Figure 2. Phototropic and locomotory behavior of a planula. The hatched larva exhibits positive phototaxis while crawling on the substrate

by alternate left–right bending along the main A-P body axis. Scale bar ¼ 150 lm.

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1935

within a few days they will ultimately stop, attach by the

anterior end, and develop into primary polyps.

To characterize this locomotory behavior in detail, 50

planulae were placed in Petri dishes individually over 0.5-

mm-graph paper and their movements recorded. The pla-

nulae always moved forward, with the larger, club-shaped

pole ahead. In the presence of light, the planulae were

positively phototactic (Fig. 2). The smooth, anteriorly

directed gliding movement of the planulae was based on

ciliary activity, and it was consistently accompanied by

lateral bending movements with alternate arching along

the main body axis resulting from coordinated myoepithe-

lial contractions (Fig. 2, Supp. Info. Movie 1).

As a result of bending movements propagated from

the anterior end, the planulae displayed consistent

exploratory crawling behavior with alternating sweeps to

the left and right. Occasionally, the front end was transi-

ently lifted off the substrate. Planulae would overcome

small obstacles (e.g., chalk granules) by alternating left-

right bending movements of increasingly wider range

(Supp. Info. Movie 2). When crawling on the substrate,

planulae did not exhibit rotation along the anteroposterior

body axis. For diploblastic planula larvae, these

substrate-bound, forward movements coupled with

alternating lateral bending movements represent com-

plex behavior, which is reminiscent of the undulatory

locomotion patterns of simple bilaterians. These behav-

ioral observations suggest that the nervous system in C.

multicornis planula larvae might be more complex than

that in other cnidarian larval forms. To investigate this,

Figure 3. Microscopic anatomy of C. multicornis planula. A–E: Semithin sections (0.9 lm) A: Longitudinal frontal section at the level of

the endodermal cavity showing diploblastic organization of the planula larva with ectoderm (ec) and endoderm (en) separated by thin mes-

oglea (m). Cnidocytes are visible in the ectoderm and are most numerous at the posterior body pole (asterisks). Anterior pole is to the

left (ic, inner cavity). Boxes I and II correspond to TEM sections shown in Figure 4A,B, respectively. B,C and D,E: Pairs of transverse sec-

tions corresponding to the levels indicated by dashed lines. In each pair, transverse sections are separated by 5 lm, whereas the distance

between pairs is about 30 lm. B,C: Anterior sections showing the initial appearance of an inner lumen; at this level, cirumferential ecto-

derm thickness varies slightly. D,E: At the level of maximum larval circumference, the lumen widens, and ectoderm thickness appears

uniform. In contrast, basiectodermal lacunae associated with the neural plexus are not distributed symmetrically over the mesoglea

(arrows, compare also with Fig. 5D). Scale bars ¼ 50 lm in A; 20 lm in B (applies to B–E).

Piraino et al.

1936 The Journal of Comparative Neurology |Research in Systems Neuroscience

we first carried out a histological analysis of C. multicornis

planulae.

Anterior polarization of the planulabody plan

Histological analysis of planulae revealed their typical

two-layered anatomy, consisting of an external pseudos-

tratified ectoderm and an inner vacuolated endoderm

separated by a thin, acellular mesogleal boundary

(Fig. 3A). A distinctive feature of the ectoderm was its

different thickness at the anterior and posterior poles,

ranging from 55 lm at the anterior to 22 lm at the poste-

rior (Fig. 3A). In addition, transverse sections taken

through the anteriormost part of the planula also revealed

differences in the ectodermal thickness along the body

circumference, whose mean values vary from 41.0 6 1.2

lm to 52.5 6 2.5 lm (Fig. 3B,C). This ectoderm asymme-

try is no longer present at the level at which the anterior

pole attains its maximum width (Fig. 3D,E).

The ectoderm contains several types of specialized cel-

lular elements, including mucous cells and other putative

secretory cells, scattered columnar support cells, and pre-

sumptive sensory cells (Figs. 4A,B, 5A), together with

functional cnidocytes distributed mostly at the posterior

larval end (Fig. 3A). Secretory and sensory cell types were

tightly packed in the anterior ectoderm and showed typi-

cal junctional complexes (Figs. 4A, 5A). Large mucous

cells filled with electron-transparent vesicles together with

numerous other secretory cells were arranged in a dense,

pseudostratified pattern (Figs. 4A,B, 5A). These latter cell

types showed clusters of electron-dense vesicles differing

in shape and size and comparable to the type I and type II

granules described for the larva of Pennaria disticha

Figure 4. Sensory cells in the anterior ectoderm of C. multicornis planula. A: Transmission electron microscopy (TEM) longitudinal section

of the ectoderm corresponding to box I in Figure 3A. In the apical portion of the pseudostratified ectoderm, different cell types are pres-

ent: presumptive supportive cells (sp), large mucous cells (mc) filled with typical granules, a presumptive sensory cell with a dendritic-like

protrusion (sc I; arrow), and a ganglionic cell (gc) in the basal portion of the ectoderm with a large nucleus and a cytoplasm rich in small

granules. This latter cell lies above aligned bundles of neuronal processes of the basiepithelial neural plexus (p). Typical apical junctional

complexes (arrowheads) are evident between different cells. Clear evidence for apocrine secretion is visible distally. B: TEM detail showing

the anterior-lateral area of the ectoderm in longitudinal section corresponding to box II in Figure 3A. Scattered secretory and presumptive

sensory elements (sc II, sc III) are present. The latter form small clusters of two or three sensory cells characterized by an elongated,

polarized shape; heterochromatic nuclei; apical processes (microvilli); scattered cilia (arrowhead); and different types of electron-dense

granules ranging from 150 to 700 nm. Typical secretory cells contain granules and empty vacuoles (asterisks), and some show apoptotic

nuclei (arrow). Scale bars ¼ 4 lm in A; 2 lm in B.

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1937

Figure 5

Piraino et al.

1938 The Journal of Comparative Neurology |Research in Systems Neuroscience

(as P. tiarella; Martin and Thomas, 1980; Thomas and

Edwards, 1991). In some cases, morphological evidence

of apocrine secretion could also be detected (Fig. 4A).

A plexus-like, basiepithelial aggregation of putative

neuronal processes was evident in longitudinal sections

of the anterior pole (Figs. 4A, 5B,D,E, 6A). This prominent

anterior plexus was located beneath the ectodermal cell

body layer and was separated from the mesoglea by irreg-

ular basal protrusions of the ectodermal cells (Fig. 5D). In

the basal region, close to the plexus, both the support

cells and the mucous cells showed large phagosomes

containing degenerating organelles and apoptotic nuclei

(Fig. 4A,B). The support cells themselves were not easily

distinguishable either because they were hidden and

compressed by other large and more prominent cellular

elements or because they often shared some of the mor-

phological features of gland cells. Myoepithelial cells

whose basally elongated contractile elements lie above

the mesogloea were also found in the larval ectoderm.

Their contractile apparatus showed aligned thin and thick

filaments and scattered Z-elements but did not appear to

be organized in a regular pattern (Fig. 5F).

Presumptive sensory cells were elongated, showing a

heterochromatic nucleus located centrally and a promi-

nent nucleolus. These cells had a rather dense cytoplasm

rich in organelles, including mitochondria, a well-

developed Golgi, numerous parallel RER cisternae, and

abundant glycogen granules. The cytoplasm was charac-

terized by numerous granules, differing in electrondensity

from strong to moderate, and of three size ranges (small,

�150–200 nm; medium, �400 nm; large, �750 nm;

Figs. 4B, 5A). A first type of presumptive sensory cell (sc

I), although rarely observed, was characterized by a club-

shaped, prominent swelling (Fig. 4A), resembling the mor-

phology of the bulb-shaped dendritic portion of the olfac-

tory cells in mammals (see Donini et al., 1998). A second

type of vesicle-rich, presumptive sensory cell (sc II) pos-

sessed a long cilium arising from a pit with an apical open-

ing (Figs. 4B, 5G). In the apical cytoplasm of these cells,

several granules were concentrated sometimes close to

the outer surface and suggesting exocytosis to the pit

lumen. A third type of presumptive sensory cell (sc III)

had apical portions with protrusions or short microvilli,

(Figs. 4B, 5A,C). These sensory cells were spaced regu-

larly, either singly or in characteristic multicellular associ-

ations, in which at least one cell with small granules was

associated with one cell with large granules between the

tightly packed glandular cells (Fig. 5A). The slender basal

processes of these presumptive sensory cells extended

into the anterior basiectodermal plexus (Fig. 4A,B).

Scattered nonciliated ganglion cells were detected in a

basal position above the mesoglea among packed and

aligned bundles of putative neuronal processes (Fig. 4B).

They were filled with microtubules, mitochondria, and elec-

tron-dense neurosecretory granules (about 8–10 nm in

diameter; Fig. 5E). These cells were characterized by a large

nucleus with a prominent nucleolus and finely granular

cytoplasm. The ectoderm was characterized by the pres-

ence of typical junctional complexes (septate junctions

associated with zonulae adherentes) between adjacent

cells (Figs. 4A, 5A,C,G). The occurrence of gap junctions on

lateral cell membranes, located halfway through the thick-

ness of ectoderm, was indicated by microinjections and

incubation assays with fluorescent dyes of different molec-

ular weights (Leone, unpublished data).

The endoderm of planulae consisted of highly vacuo-

lated cells with a loose epithelial organization (Fig. 6B).

Differentiating cnidoblasts (Fig. 6C) were more abundant

near the posterior pole. These endodermal cells were on

the whole comparable in morphology to the other hydro-

zoan larvae (Thomas and Edwards, 1991), with abundant

cytoplasmic inclusions (phagosomes, secretory and yolk

Figure 5. TEM section of planula ectoderm pattern and structure. A: Longitudinal section of pseudostratified anterior ectoderm. Putative

sensory cells (sc) are clustered among large cells (mc) and are characterized by large heterocromatic nuclei localized in their midapical por-

tion and several mitochondria. Sensory cell clusters contain cells with granules of three different sizes. In the apical portion of the ecto-

derm, junctional complexes are clearly visible (arrowheads). B: Semithin longitudinal horizontal section of the anterior pole. A prominent

neural plexus is detected as an arc at the base of the ectoderm (arrows); a narrow gap in the mesoglea is often observed at the anterior

pole of the larva (arrowhead). C: Apical portion of presumptive sensory cells (sc III), with protrusions by short microvilli emerging from the

ectodermal surface. The cytoplasm contains several dark granules and some mitochondria. D: Electron micrograph showing the arrangement

of the nerve plexus (p) with respect to the basal processes of ectodermal cells and the mesoglea (m). A thick bundle of neuronal processes

(seen both in longitudinal and in transversal sections) lies over a series of thin columnar cell protrusions, interspersed by lacunae (l). Mito-

chondria are present in both neurites and lacunae. The basal processes of ectodermal cells are in contact with the mesogleal layer, which

acts as a basal lamina. E: Enlargement of neuronal processes in longitudinal section showing bundles of microtubules (arrowhead). F: Detail

of the basal portion of the ectoderm showing the presence of clear contractile elements (arrow) forming a wide domain of a myoepithelial

cell; thin and thick filaments are visible. G: Detail of the apical portion of a sensory cell (sc II) showing a prominent cilium arising from

within a cleft or pit, surrounded by granule-rich cytoplasm. The basal apparatus consisting of a typical cnidarian rootlet and of proximal and

distal centrioles (arrows) is readily distinguishable. The cells are connected by junctional complexes formed by septate junctions followed

by zonulae adherents (arrowheads). Scale bars ¼ 5 lm in A; 25 lm in B; 1 lm in C; 2 lm in D,F; 500 nm in E,G.

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1939

granules, empty vacuoles; Fig. 6B,C). In transverse and

longitudinal sections, the endoderm showed a narrow

central lumen throughout the length of the planula (Figs.

4A, 6A–C). Longitudinal sections revealed a gap in the

mesoglea close to the anterior pole corresponding to an

interruption of the ectodermal layer, leaving the endo-

derm cells in contact with the outer environment

(Fig. 6D,E). Near this mesogloeal discontinuity, the endo-

dermal lumen became distinguishable (Fig. 6D,E). Loose

cell contacts within the central epithelium, allowing

exchange of fluid and small particles (<100 lm) between

the inner cavity and the external medium via a funnel-

shaped ectodermal area, were revealed by short-term

(2 hours) incubation experiments of planulae with dyes or

GFP-conjugated particles (Leone et al., unpublished data).

To investigate the organization of the presumptive sen-

sory cells and neural processes of the planula in more

detail, we adopted a complementary immunocytochemical

approach. Both pan-neuronal and nerve cell-specific

markers were used in this study.

Figure 6. Planula ectoderm and endoderm. A: Semithin transverse section at the anterior pole. The main neural plexus (arrowheads) lies

above the basal cytoplasmic processes of ectodermal cells. In transverse sections, it forms an arc, extending for perhaps one-third of the

anterior circumference. The thin mesoglea and vacuolated endoderm (en) encircle the inner cavity (ic). B: Thin transverse section showing

details of the developing inner cavity (ic). Many ciliary processes can be seen still enclosed in cytoplasmatic portions, and the apical cyto-

plasm (ac; arrowheads) may be close to being discharged. Junctional complexes between the cells (arrows) are clearly seen in the subapi-

cal region. C: Thin transverse section. Typical vacuolated and ciliated cells line the planula’s internal cavity, in which several cilia are

visible in cross-section (arrowheads). Ciliogenesis has been completed, and the cavity is much more pronounced. Differentiation of endo-

dermal cnidoblasts (arrow) is complete. Various dense granules and phagosomes (ph) are interspersed within the endoderm cells. D: Semi-

thin longitudinal horizontal section through the anterior pole of a planula. The mesoglea (delimited by dashed lines) presents a gap, and

here endodermal cells reach the outer surface (arrow). E: TEM detail of the area in D showing the gap area in the mesoglea and ectoderm.

A few endodermal cells are directly exposed to the environment here (arrow). Scale bars ¼ 25 lm in A,D; 4 lm in B,C; 10 lm in E.

Piraino et al.

1940 The Journal of Comparative Neurology |Research in Systems Neuroscience

Architecture of the planula nervous systemThe architecture of the entire nervous system of the

planula was studied first by immunocytochemical labeling

of whole mounts with antisera against two pan-neuronal

markers, the tyrosinated a-tubulin (TT) and the acetylated

a-tubulin (AT; Piperno and Fuller 1985; Kreis, 1987; Siddi-

qui et al., 1989; Jellies et al., 1996). In both cases, whole-

mount analyses revealed a striking polarization of the

nervous system in that sensory cells and nerve fibers

were restricted almost exclusively to the anterior half of

the planulae (Fig. 7A,B). Spindle-shaped sensory cells

were located in a spatially restricted, cap-like arrange-

ment at the anterior pole (Fig. 7A), with apical processes

reaching the planula’s outer surface and basal neurites

projecting toward an extensive basiectodermal neural

plexus (Fig. 7B,C). The anteriormost part of this plexus

consisted of densely arranged fiber bundles that pro-

jected in an orthogonal, rectilinear manner, whereas, in

Figure 7. Immunohistochemical analysis of planula nervous system with antityrosinated tubulin (TT) and antiacetylated a-tubulin antibod-

ies (AT). A: Whole mount of a TT-immunostained planula (sum of 12 optical sections) revealing a highly developed neural network at the

anterior pole. The TT-immunoreactive system shows ectodermal sensory elements concentrated in the anterior first one-fourth of the

larva together with orthogonal commissural pathways, which in the second one-fourth of the body crosses obliquely the main A-P axis at

a 45� angle. Additional TT-immunoreactive elements located in the posterior half of the larva were identified via light microscopy as cni-

docysts. B,C: Planulae labeled by AT immunoreactivity. Labeling is seen in the outer cilia (oc), localized all over the planula ectodermal

surface, and in the internal endodermal cilia (ic) within the lumen. B: Sum of five optical horizontal sections at the level of the inner

lumen. Several elongated cells are labeled in the anterior ectoderm, partially clustered in lateral groups (circled; lc, lateral cells). Gangli-

onic cells are also scattered over the median and posterior regions (gc). A basiectodermal nerve plexus lines the mesoglea and appears

thicker at the anterior pole (first one-fourth of planula). Note the anterior discontinuity of mesoglea (arrowhead). C: Sum of five optical

horizontal sections at the base of the ectoderm. Numerous spindle-shaped cells labeled in the anterior ectoderm connect to the nerve

plexus, whose fibers are organized in an orthogonal network. The anteriormost gap in the mesoglea is paralleled by a looser organization

of the nerve plexus. D: AT immunoreactivity in a prehatching planula (36 hours postfertilization; sum of five optical horizontal sections)

shows the early appearance of sensory elements and commissural elements orthogonal to the main body axis. The gonophore peduncle

(at the top of the embryo shows a large number of protective cnidocytes and ciliated cells. Scale bars ¼ 45 lm in A,B; 40 lm in C; 30

lm in D.

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1941

the rearmost part of the plexus, these bundles formed a

more oblique arrangement (Fig. 7A,C). The dense neural

plexus ended at approximately half the distance along the

anteroposterior axis. In the posterior half of the planula,

only a few longitudinal TT-immunoreactive fibers were

seen. The only other TT-immunoreactive cells in the pos-

terior half were nonneuronal cnidocytes, which were

more concentrated at the posterior pole of the planula

(Fig. 7A), where the mouth of the primary polyp develops

at metamorphosis.

The analysis of optical sections of planulae immunola-

beled with antiacetylated a-tubulin revealed sensory cells

restricted to the larval anterior pole and neural processes

that projected into the neural plexus (Fig. 7B,C). The

arrangement of the sensory cells and the organization of

the associated neural plexus were regular and highly or-

dered, with two notable exceptions. First, narrow, denser

clusters of AT-immunoreactive cells were detected in the

anterior ectoderm, where these cells appeared to be

more concentrated than elsewhere (Fig. 7B, dashed

Figure 8. GLWamide-immunoreactive nervous system of planulae. Localization of GLWamide peptides in whole mounts. A: Color-depth

code localization (sum of 12 optical horizontal sections) of GLWamide-immunoreactive cells. A dense three-dimensional array of ectoder-

mal sensory cells characterizes the anterior pole of the planula. From each peripheral cell body, a thin cytoplasmatic process extends

proximally. B: In the posterior part of the planula, GLWamide-immunoreactive neurons run longitudinally along the border between ecto-

derm and endoderm. These presumptive ganglionic interneurons might also have a sensory function, insofar as their cell bodies may

extend to the surface. C,D: Anterior region of planula enlarged. C: A single optical section close to the top surface of the planula.

D: Same specimen as in C. A single optical section of the planula head (top view) at the median point along the Z-axis shows that GLW-

amide cells are absent from the centralmost area of the ectoderm, i.e., at the mesogleal disruption shown in Figures 5 and 6. E: Enlarge-

ment of a bipolar ganglion cell from the posterior region of the planula. Punctate immunolabeling F: C. multicornis embryo 24 hours post-

fertilization with low or no signals of GLWamide immunoreactivity (same as Fig. 1F). G: C. multicornis embryo 36 hours postfertilization.

After establishment of the A-P body axis, differentiation of GLWamide-immunoreactive cells is seen at the anterior pole of the future

planula. Scale bars ¼ 40 lm in A,F; 35 lm in B; 30 lm in C,D; 5 lm in E; 45 lm in G.

Piraino et al.

1942 The Journal of Comparative Neurology |Research in Systems Neuroscience

circles). These clusters appeared not to be labeled by the

TT antiserum. Second, the neural plexus had a fascicular

organization at its most anterior tip, which was disrupted

at the narrow area where the discontinuity in the meso-

glea was detected by TEM microscopy (Fig. 7B,C). This

feature was revealed only by AT immunolabeling. AT-im-

munoreactive sensory cells and neural fibers were rarely

observed in the posterior half of the planula (Fig. 7B), con-

firming the findings obtained in the whole-mount assays

using the anti-TT antibody.

Because acetylated a-tubulin is a major component of

axonemes, immunolabeled cilia were also observed

arising from the ectoderm cell layer or into the inner

endoderm lumen (Fig. 7B). AT-immunoreactive cells were

detectable in the embryonic nervous system soon after

the establishment of the primary body axis (from 18 to 24

hours postfertilization; Fig. 7D).

Localization and morphology of GLWamide-immunoreactive neurons

The use of specific antibodies for cnidarian neuropepti-

des showed that the global pan-neural pattern obtained

by the TT and AT immunolabeling can be separated mor-

phologically into component subpatterns covering differ-

ent neural types. We screened first for cells that were

immunolabeled by polyclonal antibodies for the GLWa-

mide neuropeptide family, which are characterized by

Figure 9. RFamide-immunoreactive nervous system of planulae. A: Whole-mount localization of RFamide-immunoreactive system (sum of

12 optical horizontal sections) reveals presumptive sensory neurons encircling the anterior end of the planula and bipolar ganglionic cells

with longitudinally oriented neurites showing punctate immunolabeling. B: Enlargement of the anterior end of the planula. RF-immunoreac-

tive sensory cells appear in a narrow belt, mainly as two opposing clusters and with a few located at the anterior pole. Their cell bodies

are situated basally or within the ectoderm and have cytoplasmic processes extending distally and proximally. A commissural plexus of

RFamide-positive neurite bundles is situated between the ectoderm and the extracellular matrix, linking the two lateral clusters of sensory

cells transversely. C: Enlargement of RFamide-immunoreactive sensory cell, with typical club-shaped, short apical process and basal neu-

rite. D: Enlargement of a bipolar ganglionic cell from the posterior pole. E: Differentiating RFamide-immunoreactive cells in C. multicornis

embryo 36 hours postfertilization, when A-P polarity is already established. Posterior ganglionic cells project a single neurite anteriorly.

Scale bars ¼ 40 lm in A; 25 lm in B; 10 lm in C; 3 lm in D; 30 lm in E.

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1943

the C-terminal aminoacid sequence -Gly-Leu-Trp-NH2

(Leitz et al., 1994). These peptides play an important role

as neurotransmitters in cnidarians, but they have been

also found in mammals, in which they have a presumptive

role in sensory mechanisms (Hamaguchi-Hamada et al.,

2009). Two distinct types of GLWamide-positive cells were

found in C. multicornis planulae (Fig. 8A–E).

The first consists strikingly of up to 80 GLWamide-

immunoreactive cells densely packed at the anterior

pole of the planula (Fig. 8A). In the anteriormost part

of the ectoderm, the pattern of these GLWamide-

immunoreactive cells shows an overlap with TT- and

AT-immunoreactive cells (Fig. 7A–C). A depth-coded pro-

jection series of confocal sections indicates that a signif-

icant number of GLWamide-immunoreactive cells are

regularly arranged in a dome-shaped array at the front

end of the planula (Fig. 8A). The posterior border of this

neuronal cupola of GLWamide-immunoreactive cells

corresponds approximately to the level of the anterior

neural plexus. The elongated, club-shaped cells show

features typical of sensory cells observed by TEM; they

extend apically toward the outer surface and project

a long basal neurite into the anterior neural plexus

(Fig. 8C,D). At the apex of the GLWamide-immunoreac-

tive dome, a small gap in the array of sensory cells corre-

sponds to the mesogleal gap beneath the funnel-shaped

area described above (Fig. 8D).

A second type of GLWamide-immunoreactive neurons

is observed in the middle and posterior parts of the pla-

nula behind the anterior neural plexus (Fig. 8B,E). These

cells are less abundant, and their distribution within the

ectoderm is rather sparse. They become rare toward the

posterior pole of the planula and are hardly detectable in

the tail end (Fig. 8B). In contrast to the anterior GLWa-

mide cell population, these bipolar neurons have short

somata that are basally embedded in the ectoderm and

are associated with two longitudinal neurites, which pro-

ject anteriorly to the neural plexus and posteriorly to the

planula tail (often manifesting punctate immunolabeling;

Fig. 8E). These cells are also detected by the TT and AT

antisera. Based on their number, arrangement, and

ectodermal location, as well as form of perykarya, neurite

morphology, and projection patterns, this second type of

GLWamide-immunoreactive cell most likely corresponds to

the basally located ‘‘ganglionic’’ cells previously described

from other hydrozoan planulae (Martin, 1988a,b).

The GLWamide-immunoreactive neural cells of the pla-

nula are first detected in the embryonic ectoderm 24–36

hours after fertilization, when the anterior-posterior

polarization in the embryo is already clearly established

(Fig. 8F,G). The GLWamide neural system is already fully

organized when the planula detaches from the mother

polyp and begins its locomotory activity.

Localization and morphology ofRFamide-immunoreactive neurons

RFamide immunolabeling also reveals two morpho-

logically distinct types of neural cells in the planula

ectoderm: presumptive sensory neurons and neurons with

longitudinally oriented fiber processes (Fig. 9A). However,

the number and the distribution pattern of these RFamide-

immunoreactive cells are distinctly different from those of

GLWamide-immunoreactive cells. First, RFamide-immuno-

reactive neurons are markedly fewer (20–25) than GLWa-

mide-immunoreactive cells (Fig. 9A,B). Second, most

RFamide-immunoreactive cells are localized in a narrow

belt encircling the ‘‘head’’ of the planula (Fig. 9A,B).

Furthermore, they appear to be clustered mostly into two

main groups on opposing sides; few scattered cells are

oriented toward the forefront of the planula or between

the two main clusters along the narrow belt (Fig. 9B).

These cells apparently are not labeled by the TT antise-

rum; however, they may correspond to the narrow clusters

labeled by the AT antiserum. The most anterior RFamide-

immunoreactive cells show typical features of sensory

neurons. They are club-shaped with a short apical process

extending to the outer surface of the planula and a longer

basal neurite-like process projecting toward the anterior

plexus (Fig. 9C). RFamide-immunoreactive cells of the sec-

ond type (Fig. 9D) have short somata scattered along the

main body axis with two opposing basal neurites running

above the mesoglea toward the anterior and posterior

poles. They show an orderly pattern of parallel distribution

along the anteroposterior axis (Fig. 9A). Like the

GLWamide-immunoreactive cell population, cells of the

RFamide-immunoreactive neural system are apparent in

the embryonic ectoderm (Fig. 9E) by the time when the

main planula body axis is established at 24 hours postfer-

tilization. Both anteriorly clustered sensory neurons and

neurons with longitudinal projecting fibers are detected

in 24–36 hours postfertilization embryos (Fig. 9E). At

the time of release from the mother polyp, the RFamide-

positive neural system is fully differentiated in the planula.

DISCUSSION

In contrast to the planktonic planulae of many cnidar-

ians that perform simple ciliary swimming behavior

(Werner, 1984; Bouillon et al., 2006), the benthic planu-

lae of C. multicornis carry out relatively complex, sub-

strate-bound, directed locomotion. This crawling behavior

is characterized by the forward orientation of the planu-

la’s anterior end, usually accompanied by lateral bending

movements that help the animal to progress on the sub-

strate. Such coordinated locomotory activity requires a

relatively complex nervous system, which must develop

during embryogenesis, insofar as it is completed and

already functional when a planula hatches. The anterior

pole of the larva is especially important for rapid sensing

Piraino et al.

1944 The Journal of Comparative Neurology |Research in Systems Neuroscience

of the structure and suitability of the substrate. The

anterior end is highly polarized anatomically and corre-

spondingly displays dense aggregates of specific types of

neural cells. The ultrastructural analysis revealed in the

C. multicornis planula the occurrence of chemical

synapse-like structures with asymmetric distribution of

synaptic vescicles (not shown), suggesting a polarized

conduction of signal in one direction, as observed in

anthozoan planula larvae (Chia and Koss, 1979). More-

over, functional and molecular evidence demonstrated

the occurrence of gap junction communication channels

with critical roles both in the sensory-motor integration/

homeostasis of C. multicornis larvae and in the control of

signaling pathways driving larval morphogenesis (Leone

et al., unpublished data).

Our analysis of the planula larva of C. multicornis

reveals a remarkably high degree of anterior neuroana-

tomical specialization. This is expressed in 1) the number

and types of sensory neurons concentrated in the ante-

rior ectoderm and 2) the characteristic localization of a

prominent anterior neural plexus. Both of these morpho-

logical features are likely to be prerequisites for the for-

ward-directed locomotory behavior of the planula. The

complementary concentration of mucous gland cells at

the anterior pole provides a useful specialization for tran-

sient adhesion of the larva as it crawls on the substrate

and for its final settlement at metamorphosis (Bouillon,

1995). Given that the gland cells are in basal contact with

the neural plexus or embedded between sensory

elements, the extent of mucous secretion could possibly

be regulated by neural control.

Anterior polarization of the larval nervoussystem

A striking feature of anterior neural polarization in pla-

nulae of C. multicornis is the large number of sensory

cells in a densely arranged, dome-shaped domain. This

orderly arrangement of sensory cells is especially appa-

rent in GLWamide-immunoreactive labeled preparations.

A comparable degree of neural concentration has yet not

been observed in any other cnidarian planulae.

In terms of the anatomical complexity, the nervous sys-

tems of most cnidarian planulae comprise a radially

organized, diffuse network that is distributed throughout

the swimming larva (see Galliot et al., 2009). Integrated

into this network are scattered groups of sensory cells

(mainly at the anterior pole) and nematocytes (mainly at

the posterior or ‘‘oral’’ end, where the polyp mouth will be

formed at metamorphosis). In planulae of Aurelia, an RFa-

mide neural network has been shown to be regionalized

along the A-P axis in a graded plexus with regularly

spaced neurons in the anterior domain and longitudinal

neurites extending toward the aboral pole (for up to 60%

of the body length), whereas TT-immunoreactive cells

appear quite homogeneously distributed (Nakanishi

et al., 2008; Galliot et al., 2009). In planulae of Acropora,

RFamide-immunoreactive cells were found to be more

densely distributed along the anterior larval half but did

not manifest any recognizable clustered organization

(Hayward et al., 2001). In the early planula of Nematos-

tella, RFamide-immunoreactive cells appear to be

scattered in the ectoderm, with a higher concentration

localized near the ciliary apical tuft, and later in develop-

ment corresponding cells are manifest at the base of the

developing tentacles of the primary polyp (Marlow et al.,

2009; Galliot et al., 2009).

According to Thomas and Edwards (1991), ‘‘. . . neither

sensory organs [n]or a concentration of neural elements

has been demonstrated in the hydrozoan planulae.’’ A few

studies of nervous system development in hydrozoan pla-

nulae have been carried out. In the hydrozoan Podocor-

yne carnea, modular units composed of ring-like belts of

ectodermal nerve cells begin to differentiate from the

anterior toward the posterior larval pole starting at

24 hours postfertilization to form a diffuse network

(Groger and Schmid, 2001). The 24-hour planula of the

hydrozoan Pennaria disticha has been shown to possess a

subpopulation of ganglionic FMRFamide-immunoreactive

cells (Martin, 1988a,b), whose cell bodies and neurites

form a basal plexus in the ectoderm confined to the ante-

rior half of the planula. Sensory cells appear initially at

the anterior pole and later in development extend along

the entire length of the planula (Martin, 1988b). Both

LWamide neurosecretory and RFamide-immunoreactive

nerve cells were observed in the anterior parts of both

developing and mature larvae of Hydractinia echinata

(Plickert, 1989; Leitz and Lay, 1995; Gajewski et al.,

1996). The spatial distribution pattern of the GLWamide-

immunoreactive system, however, has been documented

so far only in Hydractinia echinata polyps and larvae

(Schmich et al., 1998a,b). In this species GLWamide-

immunoreactive sensory cells are scattered radially in the

ectoderm; in the anterior part of the planula, they are

localized subapically in a belt 150 lm wide (see Gajewski

et al., 1996: Fig. 3A,B; and Schmich et al., 1998b:

Fig. 2C). This arrangement contrasts with the majority

of GLWamide-immunoreactive cells in the planulae of

C. multicornis, which are markedly polarized in a

restricted domain in the foremost part of the planula

head (Fig. 8A).

Thus while a modest degree of regionalization has

been reported for the nervous system of other planulae,

in none of these is the neural architecture comparable to

the highly clustered organization observed in C. multicor-

nis. Moreover, none of the cnidarian planulae investigated

so far show the circumferential distribution of sensory

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1945

cells around a frontal gap in the ectoderm (Figs. 6D,E,

8A,C,D). Although an anterior enrichment of sensory and

ganglionic cells is known for most planulae, the types and

number of sensory cells and their degree of clustered

localization in C. multicornis planulae are unparalleled.

The array of anterior sensory cells appears to comprise

diverse types. Based on their ultrastructural features, at

least three putative sensory cell types can be identified

with distinct apical structures and differing cytoplasmic

content (granules of different sizes and electron density).

Some of these are also characterized by the expression

of specific neuropeptides. Indeed, a clear subdivision of

the large, seemingly homogeneous population of anterior

sensory neurons becomes apparent in comparing GLWa-

mide-immunoreactive and RFamide-immunoreactive cells

(Figs. 8, 9). Whereas GLWamide immunoreactivity reveals

a large apical dome of sensory cells, immunoreactivity for

the RFamide peptide is shown only by a restricted belt of

sensory neurons that mostly form two contralateral cell

groups at the front pole of the planula. Given the cylindri-

cal appearance of the planula and the current notion of

radial symmetry in cnidarians, this latter observation is

remarkable. A comparison of the two sets of GLWamide-

labeled and RFamide-labeled cells with the total cell

population labeled by pan-neural cell markers indicates in

addition the existence of further, as yet unidentified neu-

ral cell subpopulations. For example, the neural elements

that are responsible for the extensive rectilinear network

of (TT-immunoreactive) commissural fibers in the anterior

part of the planula remain to be identified.

The arrays of sensory cells are probably chemo- or

photoreceptors involved in key events such as settlement

and metamorphosis. Neuropeptides secreted from sen-

sory cells are thought to represent important internal

signals for the coordination of the metamorphic process

in cnidarians (Schwoerer-Bohning et al., 1990; Leitz,

1993; Schmich et al., 1998a,b; Plickert et al., 2003) as

well as for the synaptic transmission of neuronal activity

(Watanabe et al., 2009). High levels of neuropeptide

immunoreactivity have been found in nervous systems of

planulae in a number of cnidarian species. In the hydro-

zoan Hydractinia echinata, small, anterior neural clusters

immunoreactive for GLWamide and/or RFamide are

thought to be involved in the detection of chemical cues,

in the control of locomotory and phototropic behavior,

and in the initiation of metamorphosis (Plickert, 1989;

Leitz and Lay, 1995; Schmich et al., 1998a,b; Muller and

Leitz, 2002; Katsukura et al., 2004). After discovery of

metamorphosin A, various other GLWamides have

been isolated from adult hydrozoans and anthozoans

(for review see Leitz, 1998), and the occurrence of

GLWamide-immunoreactive cell populations has been

documented in the neural system of several cnidarians,

including anthozoans, scyphozoans, and hydrozoans

(Leitz et al., 1994; Leitz and Lay, 1995). Their putative

function is in chemical reception and as an activation

signal for the metamorphic process. The existence of a

novel mammalian neuropeptide related to the cnidarian

GLWamide family and perhaps involved in sensory mech-

anisms has recently been documented (Hamaguchi-

Hamada et al., 2009). Several reports indicate the ability

of GLWamide peptides to induce metamorphosis in planu-

lae belonging to different taxa, from corals to hydroids

(Leitz and Lay, 1995; Iwao et al., 2002; Muller and Leitz,

2002; Plickert et al., 2003). In view of these findings, it is

conceivable that the GLWamide-immunoreactive cells of

C. multicornis might be directly involved in the initiation

and/or signaling regulation of this process. Both

GLWamide-immunoreactive and RFamide-immunoreactive

neurites exhibited punctate immunolabeling patterns

(Figs. 8A,B,E, 9A), a common feature of cnidarian peptider-

gic systems that is thought to reflect multiple releasing

sites of neuropeptides along the length of the neurites

(see Parkefelt and Ekstrom, 2009, and references therein).

Given the pronounced positive phototactic behavior of

planulae of C. multicornis, at least some of the anterior

sensory cells are likely to be photoreceptors. In particular,

the RFamide-immunoreactive cells might be involved in

light detection. Although the detailed mode of function of

the RFamide peptide family in cnidarians remains to be

clarified (Watanabe et al., 2009), reports on the larvae of

Hydractinia echinata and young medusae of Tripedalia

cystophora suggest a role in transmission of photic

stimuli (Katsukura et al., 2004; Plickert and Schneider,

2005). Martin (2004) has suggested an interaction

between opsins and RFamide peptides in photoreceptors

of Cubozoa. This last observation seems to be confirmed

in Hydrozoa by the colocalization of RFamide peptides in

cells expressing the CropJ opsin in Cladonema radiatum

medusae (Suga et al., 2008).

RFamide-immunoreactive sensory cells and their

neurites have been reported to be diffusely distributed in

the anterior half of ‘‘swimming/rotating’’ anthozoan pla-

nulae (Hayward et al., 2001; Marlow et al., 2009). Mature

‘‘swimming/drifting’’ planulae of the scyphozoan Aurelia

furthermore possess an anteriorly concentrated ectoder-

mal nervous system with an apical, lateral distribution

pattern of RF-immunopositive cells and an apical plexus

extending toward the posterior end (Nakanishi et al.,

2008). Compared with the FMRFamide-immunoreactive

nervous system of the Aurelia planula, however, the num-

ber of apical sensory cells in that of C. multicornis

appears to be greater, and these are located in a nar-

rower belt and are clustered mostly into two contralateral

domains. Phototactic behavior comparable to that of

C. multicornis planulae has been reported in the planula

Piraino et al.

1946 The Journal of Comparative Neurology |Research in Systems Neuroscience

of H. echinata, which, while creeping forward, bends the

front end through the axis of light direction, exposing the

previously shaded side toward the light source and stimu-

lating bending in the opposite side. In this context, the

two lateral clusters of RFamide-immunoreactive cells in

C. multicornis planulae might be involved in photorecep-

tion and/or transmission of the light stimuli to the myoe-

pithelial cells that mediate bending of the body. Further

experiments will shed light on the presumptive sensory

functions of these cells and the molecular basis for photo-

reception in C. multicornis planulae.

Whereas the role of RFamides has been associated

with specific sensory functions such as photoreception

and with specific behaviors such as phototaxis, the neural

signaling related to phototactic behavior and the role of

GLWamide in planulae of C. multicornis and in cnidarians

generally are not yet understood. The marked differences

in spatial pattern and number of GLWamide-immunoreac-

tive vs. RFamide-immunoreactive cells in planulae of

C. multicornis support the notion that the nervous system

of these planulae is remarkably highly developed, com-

prising multiple populations of neuronal cells with differ-

ent morphological and biochemical properties. Whether

coexpression of GLWamide and RFamide occurs in a

restricted subset of these cells has not yet been deter-

mined and remains to be investigated, but the distinctive

patterns of distribution of these two neural cell popula-

tions suggest a discrete localization for each peptide.

An unusually complex neural plexus is found in the

anterior part of this planula (Fig. 10). It comprises a highly

Figure 10. Diagram of the nervous system of C. multicornis planulae. The anterior pole is at the top in both the drawing and the picture.

GLWamide immunoreactive (GLWamide-ir) cells are in magenta, RFamide immunoreactive (RFamide-ir) cells in green. Nonpeptidergic, tyro-

sin-/tubulin-immunoreactive nervous fibers are in blue. Scale bar ¼ 100 lm in photograph.

Neural complexity in hydrozoan larvae

The Journal of Comparative Neurology | Research in Systems Neuroscience 1947

ordered array of circumferential, oblique, and longitudinal

neurite fiber bundles. The neurites of the sensory cells

project into this network of bundles, so many of the

processes in the plexus may convey sensory information.

Neurites that arise from scattered ganglionic cells in the

posterior part of the planula appear also to project into

the anterior plexus. This implies that the anterior plexus

may be involved in the integration of neural information

from different parts of the planula. In planulae of other

hydrozoans, a diffuse ectodermal neural plexus extending

to the entire length of the planula has already been

described (Martin, 1988a,b; Leitz, 1993; Groger and

Schmid, 2001), but, in terms of neurite number, diversity,

polarization, and pattern of arrangement, the level of

structural and organizational complexity of the neural

plexus of the larvae of C. multicornis seems to be

unmatched.

Evolutionary considerationsIn its overall arrangement, the regularly spaced and

highly concentrated array of diverse sensory cells at the

front end of the planula is reminiscent of the sensory

neuronal arrays of simple bilaterians (Raikova et al., 1998;

Gaerber et al., 2007; Kotikova and Raikova, 2008;

Schmidt-Rhaesa, 2009). Moreover, the ordered array of

neural processes in this plexus has morphological features

that are also found in the central neuropiles of bilaterian

ganglia (Meinertzhagen, 2010). For example, the occur-

rence near the anterior (cephalic) pole of multiple longitu-

dinal fiber bundles intersected orthogonally by commis-

sural fiber bundles is a characteristic feature of the central

nervous system of some simple protostomes (Nielsen,

1994). Given its high level of morphological complexity,

the anatomical restriction of the anterior neural plexus to

one side of the planula along a secondary body axis

(orthogonal to the AP main axis) in theory would be suffi-

cient to result in the formation of a structure with most of

the features of a simple bilaterian central nervous system.

It is currently not known whether this type of hypothetical

‘‘dorsoventral’’ restriction of the anterior plexus occurs in

any cnidarian planulae, but the circumferential asymmetry

in thickness observed in the ectoderm of the anteriormost

region of planulae of C. multicornis might be considered to

support the hypothesis of an early establishment of a

primordial dorsoventral patterning mechanism.

A further feature of the anterior neural plexus in the

planulae of C. multicornis planula is reminiscent of the

properties of anterior central ganglia in protostomes. In

the planula, circumferential GLWamide-immunoreactive

neurite bundles of the anterior neural plexus are distrib-

uted around a frontal gap in the ectoderm and mesoglea,

where the endoderm bulges out is in contact with the

external environment. This condition is comparable to the

circumferential organization of RFamide-immunoreactive

and GLWamide-immunoreactive cells and neurites sur-

rounding polyp hypostomes (Grimmelikhuijzen, 1985;

Schmich et al., 1998a; Martin, 2000). In protostomes,

neurite bundles of the anterior neural ganglia form

circumferential connectives encircling the anterior esoph-

agus. Hence, in both cases, elements of the anterior

nervous system are formed and assembled around a

cylindrical nonneural domain. To our knowledge, this is

the first observation of an anatomical orifice in the ante-

rior pole of a hydrozoan planula larva at the opposite end

from the so-called oral pole, where the mouth of the

primary polyp will be formed on metamorphosis. The

functional role of this histological discontinuity requires

further investigation. The observation of a ciliated endo-

dermal epithelium lining the inner cavity prompted the

hypothesis that effective communication with the outer

environment might exist. If so, the cilia might be required

for the circulation of water, comparable to circulatory

mechanisms observed within the endodermal canals of

small hydromedusae (Roosen-Runge, 1967). Preliminary

experiments (Leone, unpublished observations) corrobo-

rate the hypothesis of a functional orifice by demonstrat-

ing exchange of fluids and particles between the inner

planula cavity and the surrounding environment.

Taken together, all of these organizational features

imply that the nervous system of C. multicornis planula is

characterized by a remarkable level of complexity whose

features are reminiscent of those found in simple bilater-

ian animals. What might be the evolutionary origin of this

complexity? Two possibilities can be envisaged. First, the

relative complexity of the nervous system in planulae

might have evolved as an adaptation to a benthic life style

and in response to the needs of substrate-based direc-

tional locomotion. In this case, the nervous system of

C. multicornis planulae would reveal the evolutionary and

developmental potential of a cnidarian-specific radial Bau-

plan to generate nervous system complexity. Second, the

relative complexity of the nervous system of C. multicornis

planulae might indicate that the common ancestor of cni-

darians and bilaterians had already attained a degree of

ancestral neural ‘‘cephalization.’’ In this case, the nervous

system of C. multicornis planulae might reflect organiza-

tional and developmental features that in extant bilater-

ians characterize the assembly of a brain-like centralized

nervous system. The two hypotheses are not mutually

exclusive: the adaptation of nervous systems to a benthic

life style might follow in consequence of the activation of

preexisting genetic mechanisms for anterior nervous sys-

tem polarization and concentration. Given the limited

knowledge about the ecology and neuroanatomy of the

larval stages in most cnidarians, further investigations of

cnidarian larvae displaying a benthic locomotory behavior

Piraino et al.

1948 The Journal of Comparative Neurology |Research in Systems Neuroscience

comparable that of the planulae of C. multicornis should

indicate whether the occurence of an anterior concentra-

tion (‘‘cephalization’’) of the nervous system is a plesio-

morphic or apomorphic larval character.

Although the notion that cnidarians are derived from a

bilateral ancestor is controversial, there is undisputed evi-

dence that cnidarians and bilaterians share an extensive

developmental genetic toolkit leading to axial patterning,

neural specification, and triploblasty in protostome and

deuterostome lineages (Kusserow et al., 2005; Boero

et al., 2007; Jacobs et al., 2007). Whether this common

developmental toolkit is used in a cnidarian-specific way

to build the cnidarian nervous system or it is used accord-

ing to a basic ur-Eumetazoan pattern to generate the

nervous systems of all animals remains an open question.

There has been considerable debate in the literature

about the origin of the cnidarian oral–aboral and directive

body axes and how they relate to bilaterian anteroposte-

rior and dorsoventral axes (Martindale, 2005; De Jong

et al., 2006; Rentzsch et al., 2006; Ball et al., 2007). A

major difficulty lies in the uncertain equivalence of Hox/

Parahox gene expression patterns in different cnidarians

(Yanze et al., 2001; Finnerty et al., 2003). On the other

hand, the finding of a conserved role of the Wnt family sig-

naling in axis formation supports the idea of oral–aboral

determination having common ancestry in cnidarians and

bilaterians (Guder et al., 2006; Momose et al., 2008).

Planulae of the hydrozoan C. multicornis possess

several morphological specializations that differ from

those of the larval stages of traditional cnidarian model

systems. Among these are an anterior opening, directed

bilateral locomotion, and primitive ‘‘cephalization’’ at the

anterior end. Its overall morphology presents key features

that have been suggested as plesiomorphic requisites of

a planuloid ancestor of cnidarians and bilaterians

(Hyman, 1951; Holland, 2003). In this respect and in view

of the occurrence of a mouth-like anterior discontinuity, it

is difficult to define the functional anterior pole of a C.

multicornis planulae as the ‘‘aboral’’ pole. The current

oral–aboral controversy is probably linked to a polyp-

focused anatomical terminology, which should probably

be abandoned. It has already been noted that the ecto-

derm and endoderm of planulae cannot be assumed to

be developmentally equivalent in different species of

cnidarians and also that gene expression patterns in

planulae of different cnidarian taxa must be compared

with caution (Yuan et al., 2008). Moreover, modes of

gastrulation and developmental timing are so diversified

in cnidarian embryos and larvae (Bouillon, 1995) that

class- or phylum-level generalizations should not be

based on single model observations. Similarly, the

clustered anterior nervous system in planulae of C.

multicornis should not be interpreted per se as a

‘‘protobrain’’-equivalent structure. However, one should

not rule out the possibility that, by anticipation of sexual

maturity (progenesis) in a cnidarian-prototype ancestral

larva, further C. multicornis-like anatomical adaptations

for creeping bilateral locomotion (including a centralized

nervous system) might have been retained and become

specialized further, thus breaking down most evolutionary

constraints linked to the anatomies of both polyp and

medusa. Additional comparative studies on new models

of cnidarian/early bilaterian relationships will be required

to address these issues and to test the hypothesis that

the prototypal neuroanatomical condition of a common

ancestor from which extant cnidarian and bilaterian nerv-

ous systems derived had already attained a high level of

organizational complexity.

ACKNOWLEDGMENT

This article is dedicated to the late Volker Schmid

(Basel), who deeply inspired this work. Ferdinando Boero

(Lecce) encouraged and offered valuable advice on earlier

versions of the manuscript. We thank T. Leitz (Heidelberg)

for kindly providing the neuropeptide antibodies, Elisa

Giangrande (Lecce) for laboratory assistance, and Elaine

A. Robson (Reading) for English revision. Fabio Tresca

(Lecce) offered technical support for preparation of

figures. We appreciate the hospitality offered by Patrick

Cormier at the Station Biologique de Roscoff. We also

thank the anonymous reviewers whose detailed com-

ments improved this article.

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Neural complexity in hydrozoan larvae

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