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Early development, pattern, and reorganization of the planula nervous system

in Aurelia (Cnidaria, Scyphozoa)

Article  in  Development Genes and Evolution · November 2008

DOI: 10.1007/s00427-008-0239-7 · Source: PubMed

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ORIGINAL ARTICLE

Early development, pattern, and reorganization of the planulanervous system in Aurelia (Cnidaria, Scyphozoa)

Nagayasu Nakanishi & David Yuan & David K. Jacobs &

Volker Hartenstein

Received: 6 May 2008 /Accepted: 11 July 2008# Springer-Verlag 2008

Abstract We examined the development of the nervoussystem in Aurelia (Cnidaria, Scyphozoa) from the earlyplanula to the polyp stage using confocal and transmissionelectron microscopy. Fluorescently labeled anti-FMRFamide,antitaurine, and antityrosinated tubulin antibodies were usedto visualize the nervous system. The first detectableFMRFamide-like immunoreactivity occurs in a narrowcircumferential belt toward the anterior/aboral end of theectoderm in the early planula. As the planula matures, theFMRFamide-immunoreactive cells send horizontal processes(i.e., neurites) basally along the longitudinal axis. Neuritesextend both anteriorly/aborally and posteriorly/orally, but thepreference is for anterior neurite extension, and neuritesconverge to form a plexus at the aboral/anterior end at thebase of the ectoderm. In the mature planula, a subset of cellsin the apical organ at the anterior/aboral pole begins to showFMRFamide-like and taurine-like immunoreactivity, suggest-ing a sensory function of the apical organ. During metamor-phosis, FMRFamide-like immunoreactivity diminishes in theectoderm but begins to occur in the degenerating primaryendoderm, indicating that degenerating FMRFamide-immunoreactive neurons are taken up by the primaryendoderm. FMRFamide-like expression reappears in the

ectoderm of the oral disc and the tentacle anlagen of thegrowing polyp, indicating metamorphosis-associated restruc-turing of the nervous system. These observations arediscussed in the context of metazoan nervous systemevolution.

Keywords Cnidaria . Scyphozoa . Neurogenesis . Planula .

Polyp

Introduction

A nervous system, defined as a network of interconnectedneurons, is shared by bilaterians, cnidarians, and cteno-phores in Metazoa. Phylogenetic relationships betweenthese taxa are becoming better resolved, favoring thehypothesis that cnidarians are the sister group to bilaterians(Dunn et al. 2008; Medina et al. 2001; Wallberg et al.2004). If this is the case, understanding the development ofthe nervous system in cnidarians is crucial in reconstructingthe ancestral condition of the nervous system from whichbilaterian and cnidarian nervous systems evolved. This,coupled with information on nervous system developmentin ctenophores, will be key to our understanding of theorigin and evolution of nervous systems in Metazoa.

Cnidarians consist of two sister clades, Anthozoa (e.g.,sea anemones and corals) and Medusozoa (e.g., jellyfishes),the latter one containing Staurozoa, Hydrozoa, Scyphozoa,and Cubozoa (Collins et al. 2006; Kim et al. 1999). For thepurpose of discussion in this paper, the cnidarian phylogeny,rooted with Bilateria, is assumed to be (Bilateria(Anthozoa(Staurozoa(Hydrozoa((Cubozoa)(Scyphozoa)))))). Cnidar-ians have often been considered radially symmetric anddiploblastic, consisting of ectoderm and endoderm separatedby a layer of extracellular matrix, the mesoglea (Brusca and

Dev Genes Evol (2008) 218:511–524DOI 10.1007/s00427-008-0239-7

Communicated by M.Q. Martindale

N. Nakanishi :D. Yuan :D. K. JacobsDepartment of Ecology and Evolutionary Biology, UCLA,621 Young Drive South,Los Angeles, CA 90095-1606, USA

V. Hartenstein (*)Department of Molecular, Cellular and Developmental Biology,UCLA,621 Young Drive South,Los Angeles, CA 90095-1606, USAe-mail: volkerh@mcdb.ucla.edu

Brusca 2003; Martindale 2005). However, several authorshave questioned whether ancestral cnidarians were radiallysymmetrical (Marques and Collins 2004; Salvini-Plawen andSplechtna 1979), and the view that they were bilaterallysymmetrical has recently received increasing support in lightof emerging molecular data on cnidarian development(Martindale 2005). A cnidarian life cycle consists of aswimming planula stage that locomotes in the aboral“anterior” direction, an asexually reproducing, polypoidstage, and a sexual, free-swimming medusa stage; anthozo-ans, staurozoans, and some hydrozoans sexually mature aspolyps and lack the medusa stage.

Histological, immunohistochemical, and electron micro-scopic techniques have been used to investigate the nervoussystems in planula larvae of several cnidarian species. Withthe exception of Cubozoa and Staurozoa, the presence of anervous system has been demonstrated at the planula stagein each cnidarian class. The cnidarian nervous systemgenerally consists of intra-ectodermal, epithelial sensorycells, basally located ganglion cells, and neurites that areformed by both sensory and ganglion cells and that runalongside the mesoglea (Thomas and Edwards 1991).Planulae of some anthozoan species have an aboral sensorystructure called the apical organ (Widersten 1968), whichhas been implicated to function in perception of chemicalcues for appropriate substrate selection for settlement andmetamorphosis (Chia and Bickell 1978; Chia and Koss1979). Parts of the planula nervous system are probablypeptidergic, as high levels of RFamide and GLWamideexpression have been identified in anteriorly/aborallylocated sensory cells and their neurites in several hydrozoanspecies (Groger and Schmid 2001; Leitz and Lay 1995;Martin 1992). Transmission electron microscopic analysesindicate that sensory cells are also enriched in the posterior/oral region of planulae in an anthozoan Anthopleuraelegantissima (Chia and Koss 1979) and a hydrozoanHydractinia echinata (Weis et al. 1985). In addition,rhabdomeric photoreceptor cells have been identified inthe posterior/oral region of the planula ectoderm in thecubozoan Tripedalia cystophora (Nordstrom et al. 2003),although (adult) cnidarian photoreceptor cells are generallyof the ciliary type (Eakin 1982).

Our knowledge of cnidarian nervous system develop-ment mostly derives from work on hydrozoans. Thedevelopmental origin of the nervous system has beenstudied in several hydrozoan species, namely, Hydra,Pennaria tiarella, and Phialidium gregarium by electronmicroscopy and experimental manipulations. In Hydra,ultrastructural analyses of stages of differentiation indicatethat sensory and ganglion cells differentiate exclusivelyfrom interstitial cells (Davis 1974; Lentz 1965). Interstitialcells, also called “I-cells,” are basophilic pluripotent stemcells capable of differentiating into many other cell types

including epitheliomuscular cells, gland cells, and cnido-blasts (Lentz 1965). Corroborating the above conclusion,Hydra polyps rendered free of I-cells, nerve cells, andnematoblasts by treatment with colchicine failed to developany nerve cells, and were thus unable to display anyspontaneous behavior (Campbell et al. 1976). By contrast,in another hydrozoan, Pennaria tiarella, colchicine-treatedplanula larvae without I-cells developed sensory cells in theectoderm, suggesting that sensory cells can differentiatefrom the ectodermal epithelium in this species (Martin andThomas 1981). This conclusion is supported by a similarobservation on another hydrozoan, Phialidium gregarium,in which planula larvae whose endoderm was surgicallyremoved at early gastrulation became devoid of I-cells,ganglion cells, and cnidocytes but developed sensory cellsin the ectoderm (Thomas et al. 1987).

Metamorphosis from planulae to polyps follows settlementof the swimming planula onto the substrate, during which thenervous system becomes reorganized in some hydrozoans.For instance, sensory cells in the ectoderm of planulaedegenerate in the hydrozoans Mitrocomella polydiademata(Martin et al. 1983) and H. echinata (Weis and Buss 1987) atmetamorphosis. In addition, anterior/aborally biased expres-sion of RFamides in a subset of neurons of planulaedisappears during metamorphosis and reappears in the oralregion of primary polyps in H. echinata (Plickert 1989) andPennaria tiarella (Martin 2000), suggesting the occurrenceof repatterning of the nervous systems associated withmetamorphosis.

In nonhydrozoan groups, we have little information onthe embryonic origin and development of the nervoussystem, and it is therefore not clear how representative nervoussystem development of hydrozoans is for cnidarians ingeneral. A transmission electron microscopy (TEM) studyhas failed to identify neural elements in the planulae of thescyphozoan Cassiopea xamachana (Martin and Chia 1982),although histological observations suggested the presence ofneurons in planulae of another scyphozoan Aurelia (Korn1966). Here, we used neuronal markers anti-FMRFamide,antitaurine, and antityrosinated tubulin (tyrTub) antibodies,coupled with confocal microscopy and TEM, (1) to confirmthe presence of neurons and neurites in the planula larvae ofthe scyphozoan Aurelia and (2) to examine the pattern anddevelopment of the nervous system from an early planulathrough metamorphosis into a polyp. Our results indicate thatAurelia planulae possess an anterior/aboral ectodermalnervous system, formed by ectodermally derived, epithelialsensory neurons with simple T-shaped neurites. Duringmetamorphosis, the aboral planula nervous system degener-ates and is transiently taken up by the endoderm, which alsoundergoes apoptosis (Yuan et al. 2008). The settled polypthen develops an orally concentrated ectodermal nervoussystem around the mouth opening and in the tentacles.

512 Dev Genes Evol (2008) 218:511–524

Materials and methods

Animals

The embryos and planula larvae of Aurelia were collectedand allowed to develop as described elsewhere (Yuan et al.2008).

Immunohistochemistry and confocal microscopy

Procedures for fixation and immunohistochemistry for Aureliaembryos were as described previously (Yuan et al. 2008).Primary antibodies that were used for this study wereantibodies against FMRFamide (rabbit; 1:500, Chemicon),taurine (rabbit; 1:200, Chemicon), and tyrosinated tubulin(mouse; 1:800, Sigma). Immunoreactivity against themolluscanneuropeptide Phe–Met–Arg–Phe–NH2 (FMRFamide) occurswidely in the cnidarian nervous systems (Grimmelikhuijzen1983), and the anti-FMRFamide antibody likely immunoreactswith endogenous neuropeptides with the C-terminal sequenceGly–Arg–Phe–NH2 in cnidarians. Nuclei were labeled usingfluorescent dyes Sytox (1:2,000) or TOTO (1:5 in Vectashieldmounting medium); filamentous actin was labeled usingrhodamine phalloidin (1:25) or phalloidin conjugated toAlexaFluor 568. Secondary antibodies that were used for thisstudy were AlexaFluor 568 (rabbit; 1:200), AlexaFluor 488(mouse; 1:200), Cy3 (rabbit; 1:500, Jackson Laboratory), andCy5 (rabbit; 1:200). The specimens were mounted inVectashield (Vector Laboratories Inc.) and then examined andrecorded using a Biorad Model MRC1024ES ConfocalMicroscope and Laser Sharp version 3.2 software. Theconfocal stacks were viewed using the program Image J.

Transmission electron microscopy

Preparation of planula specimens for transmission electronmicroscopy was performed as previously described (Yuanet al. 2008). Samples were sectioned by the MicroscopicTechniques Laboratory and examined using the JEOL100CX transmission electron microscope at the ElectronMicroscope Laboratory of the UCLA Brain ResearchInstitute.

Results

The nervous system of the planula larva is formed by apopulation of regularly spaced ectodermal neurons whoseaxons extend preferentially in longitudinal direction. Bothglobal markers such as the antityrosinated tubulin antibodyas well as markers for specific neuronal subtypes, such asanti-FMRFamide and antitaurine antibodies, visualize thenervous system of the planula. The ratio of FMRFamide-

immunoreactive neurons/fibers relative to the overallnumber of fibers that stain with the anti-tyrTub antibodyis in the order of 50% (see below). In the following, we willprimarily focus on the set of FMRFamide-immunoreactiveneurons, which can be easily quantified and followedthroughout their development.

The nervous system is formed by sensory neurons that formwithin the planula ectoderm

We characterized four stages of planula development, basedon the density and distribution of neurons that roughlycorrelated with the gradual elongation of the maturingplanula. During the first stage (ovoid stage; Fig. 1a–c),FMRFamide-like immunoreactivity appears in a smallnumber of ectodermal cells that are restricted in theirdistribution to a narrow circumferential belt extending from20–40% embryo length (anterior/aboral tip is 0%). Cellbodies are localized in the apical stratum of the ectoderm;short processes extend basally where they thicken intogrowth cones. During the second stage (ellipsoid planula;Fig. 1d–f), FMRFamide-immunoreactive neurons havestarted to form horizontal processes that extend along thelongitudinal axis of the planula and represent the onset ofthe nervous system. During the third stage (rectangularplanula), neurites have lengthened both aborally (“anteriorly”)and orally, but the preference is for anterior neurite extension.Underneath the anterior ectoderm, neurites form an apicalnerve plexus (Figs. 1h and 2c). The number and density ofneurons has increased considerably; from stage threeonward, neurons occupy a domain that extends from about15% to more than 60% of the body length. Significantly,none of the cells of the apical organ, which at this stagebegin to stand out from the rest of the ectoderm by theirgreater apicobasal length and long cilia (Fig. 1i; see alsoYuan et al. 2008), show FMRFamide-like immunoreactivity.FMRFamide-like expression in a subset of cells of the apicalorgan defines the fourth stage (mature planula; Fig. 1j–l).Thus, at this stage, one can distinguish a (small) populationof “apical neurons” from a much larger group of “lateralneurons”.

Pattern and morphology of FMRFamide-immunoreactiveneurons of the mature planula

Details of the morphology and pattern of the FMRFamide-immunoreactive nervous system of the mature planula areillustrated in Figs. 2, 3, and 4. Laterally, most, if not all,neuronal cell bodies are restricted to the superficial stratumof the ectodermal epithelium (Fig. 2a); neuronal nucleiform part of the superficial layer of nuclei (Fig. 2b).Neurites extend basally before branching into a T-shaped,longitudinal fiber. These fibers show numerous varicosities,

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which may represent synapses (Fig. 2a). Apical neurons aresimilar in morphology and distribution to their lateralcounterparts (Fig. 2c,d). Cell bodies and nuclei of apicalneurons are also restricted to the superficial realm of theectoderm, which is noteworthy because the bulk of cells of

the apical organ have nuclei that are located basally(Fig. 2d; Yuan et al. 2008).

Within their domain of occurrence, FMRFamide-immunoreactive neurons are quite regularly distributed. Inthe transverse axis, neurons are separated by 3–5 cell

Fig. 1 Development ofthe planula nervous system.Z-projections of confocalsections of Aurelia planulae la-beled with antibody againstFMRFamide. Panels on the left(a, d, g, j) show superficialplanes of section (level at thebase of ectoderm and nerve net).Panels in the middle and right(b, c, e, f, h, i, k, l) arelongitudinal sections throughcenter of specimen. In rightcolumn (c, f, i, and l),antityrosinated tubulin (tyrTub)and Sytox (Syt) labeling ofsections depicted in middlecolumn are shown. a–c Early(ovoid) planula. Nerve cellbodies (ne) have started to ap-pear in the ectoderm (ecp), oc-cupying a cylindrical domainnear the aboral pole. Nerve cellprocesses (neurites; np) at thebase of the ectoderm are short orabsent. d–f Midstage (ellipsoid)planula. Number of neurons hasincreased and T-shaped, longi-tudinally oriented neurites forma sparse nerve net (nn) at thebase of the ectoderm. g–i Late(“rectangular”) planula. Nervecell processes have furtherelongated and cross the midlineat the anterior, aboral pole(apical nerve plexus, ap).Elongated ectodermal cells,expressing high tyrTub levels,form the apical organ (ao).j–l Mature planula. FMRFamide-immunoreactive sensory neuronsare present in the apical organ(arrows in j and k). Bar, 25 μm

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diameters (Fig. 2e,f). Along the anteroposterior axis,neuronal cell density follows a gradient in both directions(Fig. 2g). Peak densities (averaged from ten specimens) arefound at 40% larval length (from the anterior), reachingapproximately one neuron per 100 μmsq (Fig. 2i). Densi-

ties decline anteriorly and posteriorly; the smaller peak atthe anterior tip of the larva is caused by the apical neurons.Similar to the even distribution of cell bodies along thetransverse axis, the spacing of longitudinal FMRFamide-immunoreactive neurites in the nervous system is fairly

Fig. 2 Pattern of the FMRFamide-immunoreactive nervous system inplanulae. Z-projections of confocal sections of mature Aurelia planulaelabeled with antibody against FMRFamide. Panels of the left column(a, c, e, g) are double labeled with antityrosinated tubulin (tyrTub);panels of the middle column (b, d, f, h) represent the same sections,double labeled with Sytox (Syt). a, b and c, d depict lateral ectoderm(ecp) and apical organ (ao), respectively; apical surface is to the top,basal surface at the bottom. e, f and g, h are tangential sections of theectoderm at a superficial level and basal level, respectively. Notelocation of cell bodies of FMRFamide-immunoreactive neurons (ne) insuperficial stratum of ectoderm (a, e). Neurites radiate basally wherethey bifurcate into branches that project posteriorly and anteriorly;these branches form the nerve net (nn). i Distribution of FMRFamide-immunoreactive neurons along oral–aboral axis. Photograph at top

indicates subdivisions (1–10) of planula. Number of neuronal cellbodies falling in these subdivisions were counted for five half animals(only the one side facing towards the microscope lens was counted);averages are plotted in histogram at the bottom. Note concentration ofcell bodies falling within interval between 30% and 50% of the bodyaxis, as well as peak in apical organ. j Distribution of FMRFamide-immunoreactive axons in the nerve net. Cross sections of halves ofplanula nerve net at about 50% body length are represented byhemicircles for six animals. Positions of labeled axons visible inconfocal stacks were projected onto the cross sections (blue dots).Note that axons are quite regularly distributed around the entireperimeter, rather than falling into symmetrically positioned bundles.Bar, 10 μm (a–h); 25 μm (i)

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regular (Fig. 2h–j). The comparison of tyrTub-positiveneurites (presumably the sum total of all neurites) and theFMRFamide/tyrTub double-labeled neurites reveals that thelatter neurites represent approximately one half (Fig. 2h).FMRFamide-immunoreactive neurites are spaced apart 1–2 cell diameters (Fig. 2i). Plotting these neurites on thecircle representing a cross section of the nervous system(Fig. 2j) reveals that neurites occur at equal frequencies allaround the larva, indicating that they have a uniformcircumferential distribution. In other words, there is noapparent differentiation of the nervous system along the“dorsoventral” axis.

Pattern and morphology of taurine-immunoreactive neuronsin the late/mature planula

The taurine-immunoreactive nervous system in the late/mature planula consists of apical neurons and neuritesoriented longitudinally along the mesoglea, but lacks lateralneurons (Fig. 3). The taurine-immunoreactive neurites areconcentrated anteriorly/aborally, forming an apical nerveplexus underneath the anterior ectoderm (Fig. 3b). As inFMRFamide-immunoreactive apical neurons, the taurine-immunoreactive neuronal cell bodies in the apical organ arerestricted to the superficial stratum of the ectoderm (Fig. 3b,c).The spatial distribution of the taurine-immunoreactive apicalneurons is more clustered than that of the FMRFamide-immunoreactive neurons, as the taurine-immunoreactivecells appear adjacent to each other (Fig. 3c). Differences inthe pattern of spatial distributions between FMRFamide- andtaurine-immunoreactive apical neurons suggest that there aremultiple populations of apical neurons with distinct bio-

chemical properties. Whether coexpression of FMRFamideand taurine occurs in any cells in the apical organ remains tobe investigated.

The relationship of neurites and myoepithelial fibersin the nervous system

The Aurelia planula possesses a network of mostlylongitudinally oriented myofibrils located in basal exten-sions of the ectoderm. These actin-rich myofibrils can bevisualized light microscopically with a fluorescent dyephalloidin (Fig. 4a–d). The highest level of phalloidinlabeling is seen in the cytoplasm of endoderm cells,surrounding the densely packed, irregularly sized vacuolesthat are found in the endoderm (Fig. 4a,b). Phalloidinlabeling in the ectoderm is relatively weak, but closeinspection reveals a thin layer of labeling running along thebasal membrane of ectodermal cells, which we assume tocorrespond to the myofibrils. A tangential confocal section(Fig. 4f) clearly shows the spatial organization of theclosely packed, longitudinally oriented myofibrils. Doublelabeling with phalloidin and the anti-FMRFamide antibodyindicates that the neurites forming the nervous systemextend alongside the myofibrils (Fig. 4c,d).

Electron microscopically, myofibrils appear as inconspic-uous, slender bundles of filaments in the basal cytocortex ofectoderm cells (Fig. 4i,j). The mesoglea, a band of electron-dense material of 100–200 nm in thickness, separatesectoderm and endoderm (Fig. 4i). Ectodermal cells splitup into processes, or “lobes”, of variable diameter as theyapproach the mesoglea. Myofibrils occur in some of theselobes, always within a distance of less than 200 nm from

Fig. 3 Pattern of the taurine-immunoreactive nervous system inplanulae. Z-projections of confocal longitudinal sections through thecenter of late/mature Aurelia planulae labeled with antibody againsttaurine. a Late/mature planula labeled with TOTO and antityrosinatedtubulin (tyrTub). Note the fully developed apical organ (ao). b Samesections as a labeled with TOTO and antitaurine (Tau). Note the higherlevel of taurine immunoreactivity in the cells of the apical organ and

neurites in the apical nerve plexus (ap) and longitudinally orientednerve processes (np). Particularly high levels of taurine are detected inthe cytoplasm of the apical necks of cells (arrow). c Highermagnification of the apical organ of the same specimen, labeled withTOTO, antityrosinated tubulin (tyrTub) and antitaurine (Tau), showingtaurine immunoreactivity in apical cytoplasm of cells of the apicalorgan (arrow). Bar, 50 μm (a, b); 10 μm (c)

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the mesoglea. Spaces between these basoectodermal lobescontain the neurites. Typically, neurites occupy a positionfurther away from the mesoglea relative to associatedmyofibrils, a finding that has also been reported for theplanula of a hydrozoan Pennaria tiarella (Martin andThomas 1980). In rare cases, figures that might representneuromyoepithelial junctions can be observed between themembranes of neurites and ectoderm cells (Fig. 4j).

Degeneration of the planula nervous system during earlymetamorphosis

Following larval settlement, the primary nervous system ofthe planula degenerates. This process was followed in regard

to the subpopulation of FMRFamide-immunoreactiveneurons. Initially, prior to formation of the secondaryendoderm, neurons and axons are still visible in the anterior(aboral) ectoderm of the animal but show irregular trajectoriesand decrease in number (Fig. 5a–c). Slightly later, still prior toformation of the secondary endoderm, cell bodies as well asaxons appear to break down into small fragments (Fig. 5d–f).At this stage, FMRFamide-immunoreactive bodies appear inlarge numbers in the primary endoderm. We infer based onthis observation that fragments of apoptotic neurons thatretain FMRFamide-like immunoreactivity are taken up bythe cells of the primary endoderm. By the following stage,when the secondary endoderm becomes visible at the oralpole (Fig. 5j–l), FMRFamide-immunoreactive material has

Fig. 4 Neuromuscular system of the planula. a–h Confocal sectionsof late planula labeled with phalliodin (actin filaments) and antibodiesagainst tyrosinated tubulin and FMRFamide. In upper panels (a–d)section was taken near center of animal. The primary endoderm showsstrong concentration of phalloidin-positive microfilaments (a, b). Notein b and d the presence of a thin layer of phalloidin label adjacent toendoderm. This layer corresponds to actin-rich myofilaments(mf) located in basal end feet of ectodermal cells. FMRFamide-immunoreactive axons (c, d) are found at the same level as myofila-ments. e–g Tangential sections at basal level of ectoderm, showingrelationship of nerve net and myofilaments. In e, ectodermal cells andaxons of nerve net (axtT) are labeled with tyrTub. Subset of these axons

also express FMRFamide (g, h), whereas others are FMRFamidenegative (arrows in h). f Densely packed myofilaments which areoriented preferentially longitudinally. Merging the phalloidin channelwith the other channels (h) demonstrates close apposition of myofila-ments and axons. i–k Electron micrographs of sections of planula,showing endoderm (enp), mesoglea (mg), and basal ectoderm (ecp).Myofilaments (mf) are found within the basal end feet of ectoderm cells,adjacent to basal membrane. Profiles of microtubule-rich axons (ax) arelocated in channels in between ectodermal end feet. Arrowhead in jhints at possible contact zone between axon and ectoderm cell; notemembrane densities and vesicles in axon. Bars, 25 μm (a); 10 μm(b–h); 0.5 μm (i–k)

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Fig. 5 Degeneration of planula nerve net after larval settlement. Z-projections of confocal sections of settled larvae labeled with antibodyagainst FMRFamide. Panels on the left (a, d, g) show superficialplanes of section (level at the base of ectoderm and nerve net). Panelsin the middle and right (b, c, e, f, h, i, k, l), as well as panel j, arelongitudinal sections through center of specimen. In right column (c, f,i, and l), antityrosinated tubulin (tyrTub) and Sytox (Syt) labeling ofsections depicted in middle column are shown. Panels of the first threerows (a–i) show early settled larva (ovoid shape; prior to formation ofmouth opening). j–l Midstage settled larva (ovoid shape; mouthopening has formed). In larva shown in top row (a–c) FMRFamide-immunoreactive nervous system is still intact; axons of nerve net (nn

in a) are irregularly oriented, possibly due to contraction of body inoral–aboral axis. In larva shown in second row (d–f), mostFMRFamide label is seen in granules located within the primaryendoderm (enp). These granules are interpreted as apoptotic fragmentsof neurons (cd) that had moved out of the ectoderm into the primaryendoderm. Some irregularly shaped cell bodies and axons are stillremaining in ectoderm. g–i FMRFamide label is confined to theendoderm. j–l The FMRFamide-immunoreactive remnants (cd) arecleared from the primary endoderm, presumably during the same waveof apoptosis that affects the endoderm cells themselves (see Yuan et al.2008). Other abbreviations: ao apical organ/attachment site; enssecondary endoderm; mo mouth opening. Bar, 25 μm

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disappeared from the ectoderm completely; it is foundexclusively in the primary endoderm. At this point, theprimary endoderm itself becomes “compressed” bythe secondary endoderm into ovoid cluster of cells fillingthe aboral pole of the animal. Cell death appears to remove alarge fraction, if not all, of the primary endoderm cells, asrevealed by the prevalence of caspase-positive cell frag-ments in the anterior/aboral endoderm of the settled larva(Yuan et al. 2008).

Formation of the secondary nervous system of the tentaclesand oral disc

Metamorphosis of the settled larva begins with theappearance of the mouth opening, flanked by secondaryendoderm that soon expands to fill the interior of thedeveloping polyp. The ectoderm around the mouth openingforms the oral disc, and the ectoderm surrounding the discforms the anlagen of tentacles. FMRFamide-immunoreactiveneurons appear in the ectoderm of the oral disc and thetentacle anlagen (Fig. 6a–d) and differentiate into a basiepi-thelial nervous system that pervades the growing tentaclesand the oral disc (Fig. 6e–h). The overall patterning andmorphology of FMRFamide-immunoreactive neuronsappears similar to that of the primary neurons encounteredin the planula. Neurons are evenly distributed and spaced 3–5 cell diameters apart (Fig. 6i,j,l); individual cell bodies areintegrated in the ectoderm (epithelial neurons) and produceT-shaped axons that project along the longitudinal axis of thetentacles (Fig. 6j,l), or form circular fibers around the mouth(Fig. 6h), respectively. Some neurons of mature polypsappear to be multipolar, forming four or more neurites thatextend out directly from the cell body (Fig. 6o). The ratio ofFMRFamide-immunoreactive neurons/axons to overall neu-rons is also similar, if somewhat less, to the one establishedfor the planula nervous system: double labeling of tentaclesof mature polyps with tyrTub and anti-FMRFamide anti-bodies reveals that 30–40% of the tyrTub-positive fibers alsolabel with the anti-FMRFamide antibody (Fig. 6l).

Discussion

The nervous system of Aurelia planulae

Korn (1966) made histological observations of the spatialdistribution andmorphology of neurons in scyphozoan Aureliaplanulae. Our immunohistochemical and TEM studiesconfirm Korn’s fundamental inference that scyphozoanAurelia planulae possess a nervous system. FMRFamide-immunoreactive cells in the Aurelia planula ectoderm arebipolar and spindle-shaped with apically located nuclei andextend from the mesoglea to the external surface. These cells

closely resemble sensory cells described in other cnidarians(e.g., Pennaria tiarella; Martin and Thomas 1980). Inaddition, these cells extend fibers with numerous varicositiesbasally along the mesoglea (Fig. 2a), indicative of neuriteswith chemical synapses. Furthermore, the TEM images showneuroepitheliomuscular junctions (Fig. 3k) as well as neuriteslocated just apical to myofibrils in the ectoderm (Fig. 3i,j),similar in position to the neuronal processes in a hydrozoanplanula (Martin and Thomas 1980). This work and theaccompanying paper (Yuan et al. 2008) document the firstimmunohistochemical and ultrastructural evidence for thepresence of neurons in a scyphozoan planula.

Embryonic origin of the Aurelia nervous system

In Aurelia, FMRFamide-immunoreactive neurons are firstdetected in the anterior/aboral region of the early planulaectoderm, but not in the endoderm (Fig. 1a–c), suggestingthat the first planula FMRFamide-immunoreactive neuronsdifferentiate in the anterior/aboral ectoderm in Aurelia. Thework on Hydra polyps showed that interstitial cells gaverise to all types of neurons (Davis 1974; Campbell et al.1976), leading to the supposition that this might be thegeneral case in all cnidarians. However, in the planula stageof other hydrozoans, such as Pennaria tiarella andPhialidium gregarium, ectodermal epithelial cells, and notthe I-cells in the endoderm, have been demonstrated to bethe source of ectodermal sensory cells (Martin and Thomas1981; Thomas et al. 1987). In the planulae of thescyphozoan Aurelia, the endoderm is unlikely to be thesource of the ectodermal sensory cells, as there has been noobservation of endodermal cells migrating into the ecto-derm in Aurelia, although the opposite situation can occur(Yuan et al. 2008). We therefore suggest that the firstFMRFamide-immunoreactive sensory cells develop fromthe ectodermal cells in Aurelia. However, further experi-ments, such as dye injection and TEM analyses of thedevelopment of nerve cells, may be conducted to furthertest this hypothesis.

If first-born cnidarian neurons are ectodermal in origin,differentiation of neural tissue from ectoderm would be anadditional shared feature of bilaterian and cnidariandevelopment beyond those involving axial developmentalregulation that have been recently discussed (Martindale2005). In bilaterian embryos, neural progenitor cells areborn after gastrulation in a specialized region of theectoderm, the neurectoderm, and they differentiate intoneurons and glial cells to generate the nervous systems(Harris and Hartenstein 1998; Hatten and Heintz 1998).Based on the observations on the pattern of neurogenesis inmedusozoan cnidarians and bilaterians, we hypothesize thatthe first-born neurons and their progenitor cells in the lastcommon ancestor of cnidarians and bilaterians were also

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Fig. 6 Formation of the perioral and tentacular nerve net. Z-projections of confocal sections of late settled larvae (a–d) and polyps(e–o) labeled with antibody against FMRFamide, tyrosinated tubulin(tyrTub), and Sytox (Syt). Panels on the left (a, b, e, f, i, l) showsuperficial planes of section (level at the base of ectoderm and nervenet). Panels on the right (c, d, g, h, j, k, m–o) are longitudinal sectionsthrough center of specimen or tentacle, respectively. a–d After theplanula nerve net has disappeared (see Fig. 4), but prior to outgrowthof tentacles, FMRFamide immunoreactivity appears at the oral pole.Labeled cells and their processes (secondary nerve net, nns) arelocated within the ectoderm in a ring-shaped domain surrounding themouth (perioral nerve ring). e–g Early polyp (four tentacle stage).Tentacle buds grow out from the perioral ectoderm, the same domainthat also contains the nerve cells. As ectoderm cells elongate and form

tentacle, they take nerve cells along with them. These cells form thetentacular nerve net (nnt in f). A second nerve net, called pharyngealnerve net (nnph), appears at the base of the pharyngeal epithelium thatlines the mouth opening (mo in f, g). Radial nerve fibers (perioralnerve fibers, pon in h) connect the tentacular nerve net and thepharyngeal nerve net. i, j Eight tentacle polyp; k–o 16 tentacle polyp(k tentacle contracter; l, o tentacle extended). Neurons labeled withanti-FMRFamide and tyrTub are exclusively intraectodermal, sensoryneurons (net). Neurites form a net of longitudinally oriented fibers,very similar to the nerve net of the planula, between ectoderm andtentacular endoderm (ent; n, o). Many neurons appear multipolar, withtwo or more neurites extending from the cell body towards proximallyand distally (o). Bars, 25 μm (a–h); 10 μm (i–o)

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derived from the ectoderm. This hypothesis predicts that (1)the first-born neurons in other cnidarian species includingthose belonging to Anthozoa and Staurozoa are likely todevelop in the ectoderm and (2) the process of neurogenesisin the ectoderm should be under the control of a conservedancestral developmental genetic program in both cnidariansand bilaterians, if neurogenesis in the ectoderm hasexperienced strong purifying selection throughout theevolution of Metazoa.

Bilaterian neuronal precursor cells are specified andsegregated from nonneural cells in the neurectoderm by ahighly conserved, lateral inhibition mechanism involvingthe transmembrane receptor Notch, its membrane-boundligand Delta, and “proneural” genes, which encode thetranscription factors of the basic-helix-loop-helix class,such as Achaete–Scute proteins (Wolpert 2007). Notchand Delta homologs have been identified in cnidariangenomes (Putnam et al. 2007; Technau et al. 2005), buttheir role in the nervous system development has not beeninvestigated. The expression pattern of an achaete–scute-like gene in adult Hydra suggested a role in differentiationof sensory neurons but not in specifying them, as noexpression was detected in interstitial cells (Hayakawa et al.2004). The reason for the lack of equivalence in the precisefunction of achaete–scute genes between a cnidarian andbilaterians is unclear. This could be due to independentevolution of neural functions in the two lineages from anonneural ancestral function of the achaete–scute gene inthe last common ancestor of cnidarians and bilaterians. Itcould also have resulted from divergence in functions viagene duplication or relaxation of the ancestral functionalconstraint from an ancestral function of the achaete–scutegene in nervous system development. It is important tonote, however, that the pattern of neurogenesis in Hydramay not be representative of all cnidarians as discussedearlier. Thus, it will be critical to examine the systems thatdisplay other modes of cnidarian neurogenesis, such asPennaria (a nonhydra hydrozoan), Hydractinia (a nonhydrahydrozoan), and/or Aurelia (a scyphozoan), if we are togain further insights into the ancestral developmentalgenetic mechanism of cnidarian neurogenesis.

Pattern of the planula nervous system

Mature Aurelia planulae possess an anteriorly/aborallyconcentrated, radially symmetric, ectodermal nervous system,consisting of (1) “apical neurons,” FMRFamide- and taurine-immunoreactive cells in the apical organ (arrows in Figs. 1j,kand 2c,d; arrows in Fig. 3b,c), (2) “lateral neurons,”FMRFamide-immunoreactive cells that are regularly spaced(3–5 cells apart from each other) in the anterior/aboraldomain of 15–60% body length (Fig. 2e-j), and (3)FMRFamide-, taurine-, and tyrTub-immunoreactive neurites

from these neurons, forming a plexus at the base of theapical organ and extending longitudinally along mesogleatowards the posterior/oral region (Figs. 2c,d, g,h and 3b).

An aboral concentration of nerve cells appears to be typicalfor most planulae investigated so far. For instance, RFamide/FMRFamide-immunoreactive sensory cells and their neuritesare enriched in the anterior/aboral domain in the ectoderm ofplanulae in the anthozoans Acropora millepora (Hayward etal. 2001) and Nematostella vectensis (our unpublishedobservation). In addition, planulae of many anthozoanspecies have an aboral apical organ (Widersten 1968) thatcontains a cluster of long columnar sensory cells with abundle of cilia forming an apical tuft, which is probablyhomologous to the apical organ of Aurelia (Yuan et al.2008). In some species, abundant ganglion cells are locatedat the basal surface of the apical organ (Chia and Koss1979). In hydrozoan planulae, antibodies against RFamideand GLWamide also revealed an anteriorly/aborally biaseddistribution of neurons (Leitz and Lay 1995; Martin 2000;Plickert 1989; Plickert and Schneider 2004), closely resem-bling the FMRFamide-like expression pattern in Aurelia, andneurites form both transversally and longitudinally orientedprocesses (Groger and Schmid 2001; Martin and Thomas1980; Weis et al. 1985). The above observations on thestructure of anthozoan, hydrozoan, and scyphozoan planulanervous systems suggest that planulae in the last commoncnidarian ancestor probably had the anteriorly/aborallyconcentrated ectodermal nervous system with apical organ-like structures and longitudinally oriented neurites.

The anteriorly/aborally concentrated ectodermal nervoussystem in cnidarian planulae probably function to transmitthe external chemical stimuli and to trigger metamorphosis.Treatment of H. echinata planulae with RFamides andLWamides, which are expressed in the apical and lateralneurons of the planulae, has been shown to cause inhibitionand induction of metamorphosis, respectively (Katsukuraet al. 2003; Leitz et al. 1994). Taurine also inhibitsmetamorphosis of H. echinata planulae when appliedexternally (Berking 1988). These observations may indicatethat a release of LWamides from GLWamide-immunoreactiveneurons into the target cells and a loss/reduction of RFamidesand taurine in (FM)RFamide- and taurine-immunoreactiveneurons, respectively, are crucial for execution of meta-morphosis into a polyp. Indeed, neuron-specific immuno-reactivity against anti-GLWamide and anti-(FM)RFamideantibodies is lost upon metamorphosis (Martin 2000;Plickert 1989; Schmich et al. 1998), and a large quantityof taurine is released into the surrounding medium fromH. echinata planulae upon induction of metamorphosis(Berking 1988). Thus, the existing evidence stronglysuggests that the anteriorly/aborally concentrated (FM)RFamide-, GLWamide-, and taurine-immunoreactive ner-vous systems in cnidarian planulae must respond to the

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external chemical cue, likely via the immunoreactive sensorycells, in order to initiate the process of metamorphosis.

Metamorphosis

During metamorphosis into a polyp, FMRFamide-likeexpression in the ectoderm of Aurelia planulae diminishes,shifts to the primary (i.e., planula-type) endoderm and islost (Fig. 5), in likely association with apoptosis (Yuanet al. 2008). In the developing polyp, FMRFamide-immunoreactive cells reappear in the ectoderm of the oraldisc and the tentacle anlagen, but the ectoderm in thenonoral region, except along the longitudinal cord musclefiber (data not shown), remains free of FMRFamide-likeimmunoreactivity. These observations indicate that theplanula-type anteriorly/aborally concentrated “primary”FMRFamide-immunoreactive ectodermal nervous systemdegenerates and the polyp-type posteriorly/orally concentrated“secondary” FMRFamide-immunoreactive ectodermal ner-vous system is generated de novo. Similar metamorphosis-associated repatterning of the (FM)RFamide-immunoreactivenervous systems occurs in hydrozoans (e.g., Plickert 1989;Martin 2000; also see “Introduction”), suggesting that thelast common ancestor of hydrozoans and scyphozoans alsounderwent the process of the (FM)RFamide-like ectodermalnervous system reorganization during metamorphosis into apolyp.

It is not known whether such repatterning of the (FM)RFamide-immunoreactive nervous system is common inanthozoans or staurozoans. In metamorphosing planulae ofthe anthozoan A. millepora, the decrease in the number ofectodermal cells expressing emx-Am and RFamide has beenreported (de Jong et al. 2006), suggesting the degenerationof the planula nervous system. de Jong et al. also reported adrastic reversal of Pax3/7-like Pax-Dam expression fromthe anterior/aboral to posterior/oral end associated with themetamorphosis. Pax3/7-related genes are involved in thespecification of a subset of neuroblasts in Drosophila(Skeath et al. 1995) and the dorsal neural tube in mice(Epstein et al. 1991). Therefore, the polarity reversal inPax-Dam expression in A. millepora might indicate thatmetamorphosis-associated repatterning of the nervous sys-tem also occurs in A. millepora, although whether thisreversal of “neural” gene expression indeed correlates withchanges in the nervous system architecture remains to beinvestigated.

Reconstruction of evolution of nervous system architectureand development

Based on the current data on the structure and developmentof nervous systems in cnidarians and bilaterians, the mostparsimonious interpretation would be that (1) the embryonic

origin of neurons in the last common ancestor of cnidariansand bilaterians was ectodermal; (2) the planulae of the lastcommon ancestor of cnidarians had anteriorly/aborallyconcentrated ectodermal nervous system with apical organ-like structure in the anterior/aboral end and longitudinallyoriented nerve fibers along mesoglea, which functioned torespond to external chemical cues for metamorphosis, and(3) in the last common ancestor of hydrozoans andscyphozoans, the planula ectodermal nervous system con-taining (FM)RFamide-like peptides degenerated and theposteriorly/orally concentrated ectodermal nervous systemcontaining (FM)RFamide-like peptides developed de novoduring metamorphosis into a polyp.

Clearly, more work is necessary to gain comprehensiveunderstanding of the early evolution of metazoan nervoussystems. Further comparative studies on the development ofcnidarian nervous systems involving diversely representedcnidarian taxa promise to improve our understanding of theevolutionary history of the nervous systems within Cnidaria.This, coupled with the data on bilaterian nervous systemdevelopment, will be critical in making more robustinferences on ancestral conditions of the nervous systemdevelopment in the last common ancestor of cnidarians andbilaterians.

Recent molecular phylogenetic analyses suggest spongeparaphyly, in which the demosponges are the sister group tothe clade that includes calcarious sponges, bilaterians, andcnidarians (Borchiellini et al. 2001; Halanych 2004).Demosponge genomes contain a number of homologs tobilaterian “nervous system” genes (Jacobs et al. 2007;Sakarya et al. 2007), functions of which remain to beinvestigated. As sponges do not have bona fide neurons(Jones 1962; Pavansde 1974), the characterization of the“nervous system” genes in demosponges will likely providekey insights into the origin and evolution of neurons thatcould have occurred in the lineage leading to cnidarians andbilaterians.

Another well-known “basal” metazoan group, whosephylogenetic position remains uncertain, is ctenophores.Although ctenophores possess true neurons as in cnidariansand bilaterians (Hernandez-Nicaise 1991), little is knownabout the development of their nervous system. The studyof ctenophore nervous system development will thus beimportant for assessing whether the nervous systemdevelopment in ctenophores shares a common evolutionaryorigin with that in bilaterians and/or cnidarians. Interest-ingly, recent phylogenomic analyses placed ctenophores asthe sister group to the rest of metazoans (Dunn et al. 2008).If true, information on the ctenophore nervous systemdevelopment, coupled with the data from sponges, cnidar-ians, and bilaterians, will allow us to infer whether therewas a single evolutionary origin of the metazoan nervoussystem at the base of the metazoan tree with the secondary

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loss of neurons in sponges. Hence, investigation ofctenophore nervous system development will also be keyto more complete understanding of the origin and evolutionof metazoan nervous systems.

Acknowledgements We thank Mike Schaadt and Kiersten Darrowof the Cabrillo Marine aquarium, San Pedro, CA, for providing uswith the Aurelia material. This work was supported by the UCLAEdwin W. Pauley fellowship (to N.N.) and the NASA AstrobiologyInstitute. We also thank anonymous reviewers for helping us toimprove the clarity of the manuscript.

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