Develop. Growth Differ. (2001) 43, 647–656

Introduction

Neurotransmitters such as the monoamines, acetylcho-line and GABA appear to be endogenous growth sig-nals in many animals, from protozoans to vertebrates.

5-Hydroxytryptamine (5-HT, serotonin) is a neuro-transmitter that plays an important role in a wide range of non-neural processes, from platelet aggre-gation to cell motility. Since Buznikov et al. (1968, 1970, 1972) detected serotonin in the eggs of the sea urchin, the intriguing question arose as to the role of 5-HT during the early phases of embryo development.

Much research has led to the suggestion that earlyembryos, at cleavage and gastrulation stages, use 5-HT, together with GABA and acetylcholine, to regu-late cell division and morphogenetic cell movements(Renaud et al. 1983; Buznikov 1984). A functional coupling of 5-HT with a second messenger systemthrough the regulation of protein kinase C (PKC) and a calcium mechanism has been found during

cleavage of sea urchin eggs (Buznikov et al. 1998;Shmukler et al. 1999). 5-Hydroxytryptamine and 5-HTantagonists administrated to chick embryos affect earlymorphogenesis, interfering with primitive streak form-ation, neurulation and somitogenesis (Palèn et al.1979). In vertebrate embryos after gastrulation, 5-HTis synthesized in the notochord, taken up by the overlaying neural plate and accumulates in the neuralfolds: in amphibian and in chick embryos, it has beenobserved that 5-HT binds to cytoskeletal elements ofthe neuroepithelial cells and may regulate changes incell shape and morphogenetic cell movements that areimportant for neural tube closure (Emanuelsson et al.1988; Lauder 1993). Moreover, it is known that 5-HTregulates mouse neural crest migration (Moiseiwitsch& Lauder 1995).

The origin of 5-HT-containing cells during the onto-geny of protochordates is not so well known as in verte-brates and sea urchins. Localization of 5-HT has beendemonstrated in 2-day-old larvae of the amphioxus(Holland & Holland 1993). The presence of 5-HT hasbeen reported in adults of a variety of ascidian species(Welsh & Loveland 1968; Sakharov & Salimova 1981;Pestarino 1982; Georges 1985). 5-Hydroxytryptaminehas been localized in the enterocromaffin cells of thegut, in the peripharyngeal band and in the endostyle

*Author to whom all correspondence should be addressed.Email: sirob@mailserver.unimi.itReceived 2 February 2001; revised 25 February 2001;

accepted 25 June 2001.

Serotonin localization in Phallusia mammillatalarvae and effects of 5-HT antagonists during larval development

Roberta Pennati,1* Silvia Groppelli,1 Cristina Sotgia,1 Simona Candiani,2

Mario Pestarino2 and Fiorenza De Bernardi1

1Department of Biology, Department of Biology, Section of Zoology SN 7B, University of Milano, via Celoria 26, 20133 Milano and 2Department of Experimental, Environmental and Applied Biology,University of Genova, viale Benedetto XV 5, 16132 Genova, Italy.

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an important role in a wide range of non-neural processes. Using immunofluorescence with an antiserotonin antibody, 5-HT was localized in thebrain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5-HT on development,we treated embryos with two different 5-HT receptor subtype antagonists. Treatment at the gastrula stage with 10 µM ondansetron, an antagonist of the 5-HT3 receptor, induced anterior truncation and a short tail. At 10 µM, ritanserin, a 5-HT2B receptor antagonist, induced larval phenotypes characterized by a roundish trunkregion with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system:their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We concludethat an appropriate level of 5-HT is necessary for correct development and morphogenesis. Moreover, a dif-ferent key role for multiple receptors in modulating the morphogenetic effects of 5-HT is suggested.

Key words: 5-hydroxytryptamine receptor, ondansetron, Phallusia mammillata, protochordates, ritanserin.

648 R. Pennati et al.

(Georges 1985). The only evidence of 5-HT localizationin ascidian larvae and embryos is its histochemicaldetection in the notochord and gut reported byBuznikov (1990).

The effects of 5-HT are mediated by an interactionwith multiple 5-HT receptor subtypes, which may acti-vate various intracellular signaling systems. Numerous5-HT receptors have been cloned and they have been classified into seven subtypes. Some of thesereceptors have been isolated in a large number ofinvertebrate and vertebrate species, from Mollusca (Li et al. 1995) to Drosophila (Saudou et al. 1992; Colas et al. 1995), Xenopus (Marracci et al. 1997) andmouse (Shuey et al. 1993; Choi et al. 1997), suggest-ing that the likely multiple role of 5-HT is widespreadin many animal phyla.

In the present study, endogenous 5-HT was localizedby immunofluorescence in the larvae of the ascidianPhallusia mammillata and the role of 5-HT was investi-gated during ascidian development by testing thesensitivity of embryos to pharmacological substancesknown to interfere with the availability of 5-HT and withthe activity of different 5-HT receptor subtypes.

Materials and Methods

Embryos

Phallusia mammillata adults were collected in the gulfof Lerici, La Spezia, Italy, and kept in a natural sea wateraquarium at 12°C with a 12 h light– dark cycle.Embryos were obtained by in vitro fertilization of eggsdissected from the ovary and sperm collected in thedeferent duct of killed animals. Immediately after fertiliz-ation, embryos were rinsed, transferred to small Petridishes containing Millipore-filtered sea water (MFSW)and reared at 15°C. Post-metamorphosis larvae werefed daily with commercial coral food.

Chemicals

Ondansetron (1,2,3,9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one) mono-hydrochloride dihydrate is a selective antagonist of5-HT3 receptors. Because the drug is not availablecommercially, we used the chlorohydrated principle inbuffered solution present in Zofran (GlaxoWellcome,Verona, Italy). To obtain a working solution containing10 µM ondansetron, we diluted 147 µL Zofran in 100 mLsea water. At this dilution, the excipient of the drug(monohydrated citric acid, sodium citrate and sodiumchloride) may be considered ineffective, as shown bytest experiments with similar dilutions of human physio-logical solution.

Ritanserin (6-[2-(4-[bis(4-fluorophenyl)-methylene]-1-piperidinyl)-ethyl]-7-methyl-5H-thiazolo(3,2-alpyrimidin-

5one) and mianserin (1,2,3,4,10,14b-hexahydro-2-methyldibenzo [c,f]-pyrazino[1,2-a]azepine) are both 5-HT2B receptor antagonists (Sigma-Aldrich, Milano,Italy).

Treatments

When embryos reached the appropriate stage ofdevelopment for treatment, they were transferred tonew Petri dishes containing sea water in which thedrugs had been dissolved. We tested different concen-trations of the drugs: 0.1, 1, 5, 10 and 20 µM. Treatmentswere started just after fertilization (uncleaved eggs) or at the gastrula stage and were terminated at theswimming larva stage. The treatments lasted 24 and18 h, respectively. Larvae were allowed to continue their development in sea water. Malformations induced by the treatments were analyzed successively: (i) at the end of the treatment; (ii) 7 days after fertilization,corresponding to the beginning of metamorphosis; and (iii) 15 days after fertilization, corresponding to a completely metamorphosed juvenile.

Photographs of representative individuals were takenwith a Nomarski optic using a Leica microscope (LeicaMicrosystem SpA, Milano, Italy) equipped with a camera.

Immunofluorescence staining

Embryos and juveniles processed for immunostainingwere fixed for 1 h at room temperature in 4% para-formaldheyde dissolved in phosphate-buffered saline(PBS; pH = 7.2), rinsed twice for 10 min in PBS and conserved in 70% ethanol at 20°C. After rehydration,samples were washed in PBS, 0.25% Triton-X100 for20 min and incubated for 30 min with 50% normal goatserum (NGS) in PBS. Samples were then kept at 4°Cfor 48 h in a solution of 50% NGS, 50% PBS and different primary antibodies. After several washes inPBS, the samples were incubated in 1% bovine serumalbumin (BSA) in PBS for 1 h at room temperature,washed in PBS and incubated at 4°C overnight with differently labeled secondary antibodies. In someexperiments, 1 µg/mL phalloidin, tetramethylrhodamineisothiocyanate (TRITC) labeled (Sigma), was added to the secondary antibody solution. After washes, samples were mounted in 1,4-diazabicyclo[2,2,2]octane (DABCO; Sigma) and observed with a Leicaconfocal microscope equipped with an argon/kryptonlaser. The antibodies used were antihuman-5-HT(Medac, Hamburg, Germany) diluted 1:100, anti-�-tubulin, clone 2-28-33 (Sigma), diluted 1:250, anti-mouse IgG TRITC conjugate developed in sheep(Sigma) diluted 1:100 and fluorescein isothiocyanate(FITC)-conjugated antirabbit IgG developed in goat(Sigma) diluted 1:100.

5-HT in ascidian development 649

Histology

Juveniles developed from control and ritanserin-treatedlarvae were fixed and sectioned for detailed obser-vation of the heart. After staining in 1% carmin red for 3 h, specimens were embedded in Technovit 7100plastic (Heraeus Kulzer GmbH, Werheim, Germany)and sectioned at 5 µm. Sections were counterstainedwith 0.5% methylene blue in water for a few secondsand mounted in Entellan (Merck, Whitehouse Station,NJ, USA).

Results

5-HT localization in the larvae of P. mammilata

In the swimming larvae of P. mammillata 5-HT-contain-ing cells were detected by immunofluorescence withan anti-5-HT antibody under a confocal microscope(Fig. 1A,C–F). In the central nervous system (CNS),bright fluorescence was present in 11 pear-shapedcells surrounding the ocellus, corresponding to the retinal cells of the photoreceptor complex (Barnes

Fig. 1. Immunofluorescence local-ization of 5-hydroxytryptamine (5-HT;serotonin) in a swimming larva of Phallusia mammillata by anti-5-HT fluorescein isothiocyanate-conjugated antibody, observed with a confocal microscope. Allphotos are composite images of 20 optical sections, taken at 1 µmintervals. (A) Whole-mount immuno-fluorescence of a swimming larva.Bar, 100 µm. (B) Schematic drawingof (A). The localization of 5-HT isindicated in green. (C) Higher magnification of the anterior part of the trunk region; 5-HT is presentin the papillae, in two epidermalneurons of the rostral trunk (arrows).Bar, 20 µm. (D) Optical section onthe confocal microscope of thesame region as in (C), at a deeperlevel, showing 5-HT localization insome ventral endodermal cells(arrow). Bar, 20 µm. (E) Light micro-scope image of sensory organs pigment cells superimposed on fluorescent localization of 5-HT-positive cells surrounding the ocellus. Bar, 10 µm. (F) Magnifi-cation of a portion of the tail. Thesignal is present in the pericharyaof two dorsal neurons and in thefibers connecting them (arrow). Bar,10 µm. ap, adhesive papillae; b,brain vesicle; n, notochord; rten,rostral trunk epidermal neuron; t,tunic; vec, ventral endodermal cells.

650 R. Pennati et al.

1974; Fig. 1E). A less intense signal appeared diffusedin the ventral part of the trunk region. At a highermagnification, the signal was found in some ventral

endodermal cells of the anterior trunk region (Fig. 1D).At least one 5-HT-positive cell was present in eachpapilla (Fig. 1C). A faint signal was present in the

Fig. 2. Effects of all treatmentswith antagonists of 5-HT receptorson Phallusia mammillata embryosat the concentrations indicated.Early treatments were started justafter fertilization and stopped atthe gastrula stage. Late treat-ments were started at the gastrulastage and stopped at the swim-ming larva stage. The effects wereevaluated on swimming larvaeand are reported in percentage.The numbers at the top of eachcolumn indicate the numbers ofscored larvae. (A) Ondansetronearly treatment; (B) ondansetronlate treatment; (C) ritanserin early treatment; (D) ritanserin late treatment. (�), normal; (�),malformed; ( ) not developed.

Fig. 3. Images taken with aNomarski optic of the larvaescored in Fig. 2 and of 15-day-oldjuveniles developed from theselarvae. (A) Control swimminglarva. (B) 15-day-old control juve-nile. (C) Larva developed from 1 µM ondansetron-treated gas-trula. (D) Larva developed from 10 µM ondansetron-treated gas-trula. (E) Juvenile metamorphosedfrom a larva treated as in (D). (F)Larva developed from 10 µM

ritanserin-treated gastrula. (G)Juvenile metamorphosed from alarva treated as in (F). Bar, 100 µm.

5-HT in ascidian development 651

pericharia of two epidermal neurons of the rostral trunk,described by Takamura (1998), which are present onthe axon-like fibers connecting the papillae with thesensory vesicle (Fig. 1C). In the tail, 5-HT was presentin the pericharia of two groups of neurons in the ventral and dorsal epidermis, connected by immuno-positive fibers (Fig. 1A,F). These neurons were oftenpaired, but their distribution and number varied amonglarvae.

Treatments

We treated ascidian embryos with two substances,ondansetron and ritanserin, known to be antagonistsof 5-HT3 and 5-HT2B receptors, respectively. The effectsof the treatments with both substances were dosedependent. The lower dose of ritanserin and ondan-setron, 0.1 µM, was almost ineffective in inducingmalformations and the number of undeveloped eggsin all treatments was similar to that of controls (data notshown). At the highest dose, 20 µM, both substancesproved to be toxic; in fact, almost all embryos wereseverely affected or died.

By starting the treatments at different developmentalstages, we detected some differences in the sensitivityof ascidian embryos to the action of the drugs.Treatments had no significant effects when undertakenjust after fertilization and were more efficient from the gastrula stage onwards. In fact, both 1 and 10 µM

ondansetron and ritanserin applied soon after fertiliz-ation caused an incidence of malformed embryos andundeveloped eggs similar to that of controls (Fig. 2A,C).At 1 µM, ondansetron, an antagonist of 5-HT3 recep-tors, caused mild phenotypes in 22.4% of larvae whengiven at the gastrula stage (Fig. 2B). The larvaeshowed flat adhesive papillae and regular pigmentedcells. The tail was slightly shortened (Fig. 3C). Almostall larvae (92%) developed from 10 µM ondansetron-

treated embryos were severely affected (Fig. 2B). Theywere unable to hatch and showed truncation of theanterior end. The trunk region appeared enlarged andthe adhesive papillae were absent. The tail was bentand hardly extended half the length of the trunk region(Fig. 3D). Despite the severe malformations, 50% of larvae were able to metamorphose inside the chorionand to develop into almost normal juveniles, whichshowed only developmental delay and size reduction(Fig. 3E).

At 1 µM, ritanserin, a substance with a high affinity for the 5-HT2B receptor subtype, caused malformationsin 31.9% of treated embryos when treatment started atthe gastrula stage (Fig. 2D). At a higher concentration(10 µM), ritanserin induced a higher incidence ofmalformations (50.1%; Fig. 2D). The phenotypesinduced by the two doses of ritanserin were similar and consisted of a slightly shortened axis. All larvaehatched simultaneously with the controls. The trunkregion in ritanserin-treated larvae was roundish with flatpapillae. The brain vesicle was not affected and thepigment cells appeared in the normal position. In thetail, the notochord and the muscle cells were normallydifferentiated, but some discontinuities were presentamong the cells (Fig. 3F). Despite their unhealthyappearance, the larvae could swim and 50% of larvaewere able to metamorphose. At 15 days after fertiliz-ation, the juveniles showed a highly reproduciblephenotype characterized by size reduction and anabnormal cardiocirculatory system (Fig. 3G). All juve-niles observed showed no blood circulation, their heartcontractions were ineffective and blood cells accumu-lated in the heart cavity. Malformed juveniles survivedfor 3 months with a very low growth rate and then died.To confirm the in vivo observations, we undertook histo-logical sections of 15 control and 15 ritanserin-treatedjuveniles 1.5 months after metamorphosis. The heartcavity of juveniles developed from ritanserin-treated

Fig. 4. Histological sections ofthe heart region of (A) a 15-day-old control juvenile and (B) a 15-day-old ritanserin-treated juve-nile; the heart cavity is threefoldenlarged and filled with bloodcells. ht, heart; pc, pericardialcavity. Bar, 20 µm.

652 R. Pennati et al.

larvae was approximately threefold larger than in con-trols and it was filled with blood cells. The myocardialwall appeared thinner than in control juveniles (Fig. 4B).In order to better identify both the circulating hemocytesand the cells contained in the heart cavity, we per-formed a double-immunofluorescence experiment:TRITC-labeled phalloidin identified filamentous actin inthe blood cells of the juveniles and FITC-conjugatedanti-�-tubulin antibody highlighted the ciliary crown ofthe gills (Fig. 5A,B). This experiment enabled us toobserve the scarce number of hemocytes in ritanserin-

treated animals compared with controls (Fig. 5B) and confirmed the presence of a high number of bloodcells in the heart of treated specimens as observed in histological sections (Fig. 5C,D).

In order to observe the presence and distribution ofendogenous 5-HT in metamorphosed juveniles, we per-formed double-immunofluorescence experiments on15-day-old juveniles with anti-5-HT and anti-�-tubulinantibodies, marking the position of the gill-slits (Fig. 6).Control juveniles showed a localization of 5-HT in somecells of the peripharyngeal band, in a double rod along

Fig. 5. Immunofluorescence on a 15-day-old control juvenile (A,C) and a ritanserin-treated juvenile (B,D), observed by confocal micro-scope. (A,B) Whole juveniles; composite images of 20 optical sections taken at 1 µm intervals. Bar, 50 µm. (C,D) Details of the heartregion; composite images of 10 optical sections taken at 0.5 µm intervals. Bar, 20 µm. TRITC-labeled phalloidin (red) identifies filamentous actin in the blood cells of the juveniles and the anti-�-tubulin antibody-fluorescein isothiocyanate-conjugate (green) marksthe cilia of the gills. The number of blood cells (asterisks) is very low in the juvenile developed from the ritanserin-treated embryo and the blood cells accumulated in the heart. (E) Schematic drawing of the anatomy and labeling of a 15-day-old juvenile. asm, atrial siphon muscles; en, endostyle; gs, gill slits; h, heart; ng, nervous ganglion; oe, esophagus; osm, oral siphon muscles; pb, peripharyngeal band; pc, peripharyngeal cavity; rt, cells of the reabsorbed tail; st, stomach.

5-HT in ascidian development 653

the endostyle and in the gut (Fig. 6A,C). In juvenilesdeveloped from ritanserin-treated larvae, 5-HT localiz-ation was similar to that seen in controls (Fig. 6B,C). In both control and treated animals, a dozen cells positive for 5-HT immunostaining were present in theperipharingeal band and a similar number of positivecells were observed in the endostyle (Fig. 6D). Even if a reduction of 5-HT-containing cells in the gut is possible, more experiments are necessary to confirmthese data.

Some preliminary experiments were performed treat-ing embryos with mianserin, another 5-HT2B receptorantagonist. The results of these experiments were similar to those obtained with ritanserin, even if mian-serin seemed to be less effective than ritanserin.

Discussion

Since Buznikov et al. (1968, 1970, 1972) describedthe presence of 5-HT in the eggs of sea urchin beforecleavage, the role of this neuroamine has been emerg-

ing as a regulator in a wide variety of developmentalprocesses. While for vertebrates a lot of information isnow available on this topic, little is known about the roleof 5-HT during the development of ascidians, despitetheir key position as a group related to the ancestralchordate stock.

It is well known that, in vertebrates, 5-HT is found in the neurons of the CNS, the neurons of the entericnervous system and in enterochromaffin cells of the gut lining. However, the distribution of 5-HT-containingcells is not so well known in ascidians.

In ascidians, the presence of 5-HT was first dis-covered in gastrointestinal extracts of Ciona adults by Erspamer (1946). 5-Hydroxytryptamine has beenfound in the endostyle between zones 7 and 8, in 3-month-old Ciona, Ascidia, Ascidiella and Phallusia,and in the digestive tracts of Ciona and Styela adults(Fritsch et al. 1982; Pestarino 1982; Georges 1985). 5-Hydroxytryptamine has also been reported by histochemistry in the CNS of Halocynthia roretzi(Fujii & Takeda 1988) and it was quantified by

Fig. 6. Double immunofluores-cence on 15-day-old juvenileswith fluorescein isothiocyanateconjugate antihuman 5-hydroxy-tryptamine (5-HT; serotonin) anti-body and with TRITC-conjugateanti-�-tubulin antibody, observedby confocal microscope. All photos are composite images of 20 optical sections taken at 1 µm intervals. (A) Control juvenileshows localization of endo-genous 5-HT in the peripharyn-geal band, along the endostyleand in the gut. (B) In the juveniledeveloped from a ritanserin-treated embryo, cells producingendogenous 5-HT have the samelocalization but are lower in number than in control. Bar, 50 µm. (C) Schematic drawing of a 15-day-old juvenile. Greenspots indicate the localization of5-HT. (D) Schematic drawing of aportion of the endostyle to showthe localization of 5-HT (greenspots; between zones 7 and 8)and �-tubulin (red strip; zones 1and 2). In some specimens, afterfixing, the two external lips of theendostyles (zones 7 and 8) over-lap and the 5-HT-containing cellsappear displaced in a single row(see Fig. 6A). asm, atrial siphonmuscles; en, endostyle; gs, gillslits; h, heart; ng, nervous ganglion; oe, esophagus; osm, oral siphon muscles; pb, peripharyngeal band; pc, peripharyngeal cavity; rt, cells of the reabsorbedtail; st, stomach.

654 R. Pennati et al.

three-dimensional high-pressure liquid chromatog-raphy in the neural complex of Ciona (Takeda 1992).The only evidence of 5-HT localization in ascidian larvae and embryos is its histochemical detection in the notochord and gut reported by Buznikov (1990).

The immunofluorescent localization of 5-HT in the ascidian larvae is shown here for the first time. 5-Hydroxytryptamine is localized in some neurons ofthe tail, connected by immunopositive fibers. Thesecells have the same shape and the same distributionas the primary neurons reported by Torrence & Cloney(1982) in Diplosoma macdonaldi and recentlydescribed by Takamura (1998) in the larvae of Cionaintestinalis. 5-Hydroxytryptamine is present in a rod oftrunk cells in the anterior ventral position. These cellsmay contribute to the formation of the posterior half ofthe adult endostyle, as described by Hirano & Nishida(2000) in H. roretzi. In the CNS, 5-HT is concentratedin the anterior dorsal part, near the ocellus, the photo-receptive organ of the larva. This localization suggestsa photoreceptive or a photoneuroendocrine function of5-HT for ascidian larvae similar to that observed in amphioxus and in vertebrates (Terio 1964; Nunez et al. 1981). The serotonergic system may control thecomplex larval behavior in response to light. In fact, the larvae of the solitary ascidians show a positive phototaxis during the dispersal phase of their life, but,at the time of settlement, most ascidian larvae avoidlight and prefer to settle on dark or shaded surfaces(Cloney 1982). Metamorphosis transforms a swimminglarva into a sessile adult and the early photoreceptivefunction of the serotonergic system may be lost,together with a reduction of sense organs, and 5-HTbecomes restricted to other systems. In fact, after meta-morphosis, we were not able to detect 5-HT in theneural complex of the juveniles. However, 5-HT is local-ized in numerous cells of the gut, possibly the entero-chromaffin cells already described in the adult of Ciona(Gerzeli 1964). 5-Hydroxytryptamine is also present inthe lateral part of the endostyle and in the peripharyn-geal band (Georges 1985). Georges (1985) reportedthat 5-HT-containing cells first appeared in the 3-month-old juvenile. We detected 5-HT also in younger juve-niles, 15 days after metamorphosis. In the endostyle,5-HT-containing cells occupy the position betweenzone 7 and 8 as calcitonin-like cells, described byThorndyke & Probert (1979), and cells with iodinatingcapacity (Nilsson et al. 1988). At this time, we cannotadvance any argument about the role of 5-HT in theperipharyngeal band.

Until now, no 5-HT receptor has been cloned inascidians and nothing is known about the kind of recep-tor present. We used substances known to be 5-HTreceptor antagonists in vertebrates as pharmaco-

logical tools to investigate whether multiple 5-HTreceptor subtypes are present in ascidian embryos.Teratogenic effects were observed with a range ofantagonist concentrations from 1 to 10 µM. In all cases,the severity of malformations induced and the inci-dence of the malformations increased with the doseapplied (Fig. 2).

Ondansetron is a potent, highly selective drug thatbinds to the 5-HT3 receptor, a protein with four trans-membrane sites and a member of the ligand-gated ionchannel type, identified in vertebrates (Maricq et al.1991). When activated, this receptor causes fast depo-larizing responses in neurons. Functional 5-HT3 recep-tors or binding sites for 5-HT3 receptor ligands havebeen localized in the enteric nervous system, onsympathetic, parasympathetic and sensory nervefibers, in the CNS and on several mouse neuroblastomacell lines (Maricq et al. 1991). This receptor modulatesintestinal contractions and the transmission of pain, butits function in the CNS is not known (Richardson 1990).

Ondansetron causes no effects in ascidian embryoswhen administrated during cleavage, until the blastulastage, but larvae developed from embryos treated with ondansetron at the gastrula stage were severelymalformed. The incidence and severity of malfor-mations were dose dependent, thus corroborating ourobservations that the described malformations weredue to a specific action of the drug. Treated larvae werea little elongated, with a short tail and a round trunkregion, and were unable to hatch. This latter effect maybe due to incorrect differentiation of the cells secretingthe hatching enzyme, which are localized at the anter-ior and posterior ends of the larva (D’Aniello et al.1998). Truncation of anterior and posterior regions oftreated larvae suggests that a 5-HT action mediatedby the 5-HT3 receptor plays a role during morpho-genetic movements, starting at gastrulation, that leadto the elongation of the body along the cephalo-caudal axis. This hypothesis is supported by the obser-vation that treatment with ondansetron before gastru-lation was ineffective. The juveniles developed fromtreated larvae were normal. This suggests that, despitethe observed defects, the regions of the larva that giverise to the adult organs are not affected by the actionof ondansetron.

The other 5-HT receptors isolated in vertebrates andinvertebrates have been pharmacologically classifiedinto three groups, 5-HT1, 5-HT2 and 5-HT4. Thesereceptors transduce extracellular signals by activatingG-proteins and mediate slow modulatory responses viasecond messenger signaling pathways (Peroutka1988).

Ritanserin is a drug with a high binding affinity for 5-HT2B receptors in vertebrates and in invertebrates

5-HT in ascidian development 655

(Goldberg et al. 1994). The 5-HT2 receptor group is pharmacologically similar in increasing the intracellular level of inositol 1,4,5-tris-phosphate/diacylglycerol.

As observed for ondansetron, ritanserin was alsoineffective when given before gastrulation. Malform-ations induced by treatment at the gastrula stage withritanserin were less severe than those caused byondansetron and also the dose dependence of theeffects was less remarkable. In fact, only the incidenceof malformations in the larvae varied with the dose, butthe phenotypes induced by 1 and 10 µM ritanserin weresimilar. The larvae had a roundish trunk region with flatpapillae. The tail structure was disorganized, but thelarvae could swim and were able to metamorphose.Juveniles developed from treated larvae had severeheart defects: they showed no blood circulation, heartcontractions were ineffective and blood cells, low innumber, accumulated in the heart cavity. The myo-cardial wall appeared thinner than in control juveniles.These results suggest either a modification of the differ-entiation program of the cells that give rise to themyocardium and blood cells or a deficient migration ofthe precursor of heart muscle cells. These observationsare supported by similar results obtained by Choi et al. (1997). Treating mouse embryos at the stage offive somite pairs with ritanserin caused heart defectsand abnormal differentiation of myofilament of sarco-meres. Recently, Nebigil et al. (2000) showed that theknock-out of the 5-HT2B gene in mouse embryos leadsto heart defects, such as a severe reduction in the thick-ness of the ventricle coupled with a loss of the prolifer-ative capacity of myocytes. These data support ourobservation that, via the 5-HT2B receptor, 5-HT may alsoact as an important regulator of cardiac developmentin Protochordata. Our results are also consistent withthe findings on Xenopus laevis larvae, developed from embryos treated with ritanserin, which showedarrhythmic contractions and a dilated heart (I. Nardi et al., pers. comm., 1999).

Our results with ritanserin and ondansetron suggestthat 5-HT could be an endogenous regulator ofdevelopment, acting comparably with the much better-known retinoic acid. These two substances have thepresence of multiple receptors in common, which canact in different ways in the different tissues or embry-onic territories (Stratford et al. 1996). The 5-HT2B recep-tor homolog in Drosophila is expressed at the cellularblastoderm stage in a pattern similar to that of the pair-rule genes (Colas et al. 1995). This early embryonicexpression confirms the possibility that, in inverte-brates, these receptors are involved in 5-HT-dependentmorphogenetic processes.

In conclusion, the role of 5-HT as a regulator of

morphogenesis, already suggested for sea urchin and vertebrates, can also be confirmed for ascidianembryos: its action is prolonged throughout their larval life and in metamorphosed juveniles.

The observation of various types of malformationssuggests a different role for multiple receptors duringdifferent stages of development and, consequently,their inhibitors may be used to elucidate some develop-mental steps.

Acknowledgments

We are indebted to Dr U. Fascio (Department ofBiology, University of Milano, Italy) for assistance withthe confocal laser microscope and to Mr A. Maioli (La Spezia, Italy) for providing the animals. This workwas supported by grants from the Italian Ministerodell’Università e della Ricerca Scientifica (cofinanzia-mento 1999).

References

Barnes, S. N. 1974. Fine structure of the photoreceptor of theascidian tadpole during development. Cell Tissue Res. 155,27–45.

Buznikov, G. A. 1984. The action of neurotransmitters and relatedsubstances on early embryogenesis. Pharmacol. Ther. 25,23–59.

Buznikov, G. A. 1990. Neurotransmitters in embryogenesis InSoviet Scientific Reviews Supplement Series Physiology andGeneral Biology (Ed. T. M. Turpaev & N. K. Koltzov), pp. 10–73.Harwood Academic Publishers, Chur.

Buznikov, G. A., Chudakova, I. V., Berdysheva, L. V. & Vyazmina,N. M. 1968. The role of neurohumours in early embryogenesis.II Acetylcholine and catecholamine content in developingembryos of sea urchin. J. Embryol. Exp. Morphol. 20, 119–128.

Buznikov, G. A., Kost, A. N., Kucherova, N. F., Mndzhoyan, A. L.,Suvorov, N. N. & Berdysheva, L. V. 1970. The role of neuro-humours in early embryogenesis. III Pharmacological analysisof the role of neurohumours in cleavage divisions. J. Embryol.Exp. Morphol. 23, 549–569.

Buznikov, G. A., Marshak, T. L., Malchenko, L. A. et al. 1998.Serotonin and acetylcholine modulate the sensitivity of earlysea urchin embryos to protein kinase C activators. Comp.Biochem. Physiol. 120A, 457–462.

Buznikov, G. A., Sakharova, A. V., Manukhin, B. N. & Markova, L. N. 1972. The role of neurohumours in early embryogenesis.IV Fluorometric and hystochemical study of serotonin in cleav-ing eggs and larvae of sea urchins. J. Embryol. Exp. Morphol.27, 339–351.

Choi, D. S., Ward, S. J., Messaddeq, N., Launay, J. M. &Maroteaux, L. 1997. 5-HT2B receptor-mediated serotoninmorphogenetic functions in mouse cranial neural crest andmyocardiac cells. Development 124, 1745–1755.

Cloney, R. A. 1982. Ascidian larvae and the events of meta-morphosis. Am. Zool. 22, 817–826.

Colas, J. F., Launay, J. M., Kellermann, O., Rosay, P. & Maroteaux,L. 1995. Drosophila 5-HT2 serotonin receptor: Coexpressionwith fushi-tarazu during segmentation. Proc. Natl Sci. USA92, 5441–5445.

656 R. Pennati et al.

D’Aniello, A., De Bernardi, F., De Vincentiis, M. et al. 1998.Localization of hatching enzyme in embryos and larvae of the sea-squirt Ciona intestinalis. Invertebr. Reprod. Dev. 34,247–252.

Emanuelsson, H., Carlberg, M. & Lowkvist, B. 1988. Presence ofserotonin in early chick embryos. Cell Differ. 24, 191–199.

Erspamer, V. 1946. Presenza di enteramina o di una sostanzaenteraminosimile negli estratti gastrointestinali e splenici deipesci. e negli estratti gastroenterici delle Ascidie. Experientia2, 369–371.

Fritsch, H. A. R., Van Noorden, S. & Pearse, A. G. E. 1982. Gastro-intestinal and neurohormonal peptides in the alimentary tractand cerebral complex of Ciona intestinalis (Ascidiaceae). CellTissue Res. 223, 369–402.

Fujii, K. & Takeda, N. 1988. Phylogenetic detection of serotoninimmunoreactive cells in the central nervous system of inverte-brates. Comp. Biochem. Physiol. 89C, 233–239.

Georges, D. 1985. Presence of cells resembling serotoninergicelements in four species of tunicates. Cell Tissue Res. 242,341–348.

Gerzeli, G. 1964. The enterochromaffin cells in the oozooids and blastozooids of tunicates. Folia Histochem. Cytochem.2, 225–231.

Goldberg, J. I., Koehncke, N. K., Christopher, K. J., Neumann,C. & Diefenbach, T. J. 1994. Pharmacological characterizationof a serotonin receptor involved in an early embryonic behav-ior in Helisoma trivolvis. J. Neurobiol. 25, 1545–1557.

Hirano, T. & Nishida, H. 2000. Development fates of larval tissuesafter metamorphosis in the ascidian, Halocynthia roretzi. II.Origin of endodermal tissues of the juvenile. Dev. Genes Evol.210, 55–63.

Holland, N. D. & Holland, L. Z. 1993. Serotonin-containing cells in the nervous system and other tissue during ontogeny of a lancelet, Branchiostoma floridae. Acta Zool.74, 195–204.

Lauder, J. M. 1993. Neurotransmitters as growth regulatory signals: Role of receptors and second messengers. TrendsNeurosci. 16, 233–239.

Li, X. C., Giot, J. F., Kuhi, D., Hen, R. & Kandel, E. R. 1995. Cloningand characterization of two related serotonergic receptors fromthe brain and the reproductive system of Aplysia that activatephospholipase C. J. Neurosci. 15, 7585–7591.

Maricq, A. V., Peterson, A. S., Brake, A. J., Myers, R. M. & Julius, D. 1991. Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel. Science254, 432–437.

Marracci, S., Cini, D. & Nardi, I. 1997. Cloning and developmentalexpression of 5-HT1A receptor gene in Xenopus laevis. Mol.Brain Res. 47, 67–77.

Moiseiwitsch, J. R. D. & Lauder, J. M. 1995. Serotonin regulatesmouse cranial neural crest migration. Proc. Natl Acad. Sci. USA92, 7182–7186.

Nebigil, C. G., Choi, D. S., Dierich, A. et al. 2000. Serotonin 2Breceptor is required for heart development. Proc. Natl Acad.Sci. USA 97, 9508–9513.

Nilsson, O., Fredriksson, G., Ofverholm, T. & Ericson, L. E. 1988.Electron-microscopic immunocytochemistry of 5-hydroxy-tryptamine in ascidian endostyle. Cell Tissue Res. 253,137–143.

Nunez, E. A., Geshon, M. D. & Silverman, A. 1981. Uptake of 5-hydroxytryptamine by gonadotrophs of the bat’s pituitary: A combined immunocytochemical radioautographic analysis.J. Histochem. Cytochem. 29, 1336–1346.

Palèn, K., Thorneby, L. & Emanuelsson, H. 1979. Effects of sero-tonin antagonist on chick embryogenesis. Wilhelm Roux’s Arch.187, 89–103.

Peroutka, S. J. 1988. 5-Hydroxytryptamine receptor subtypes.Annu. Rev. Neurosci. 11, 45–60.

Pestarino, M. 1982. Occurence of different secretin-like cells inthe digestive tract of the ascidian Styela plicata (Urochordata,Ascidiacea). Cell Tissue Res. 226, 231–235.

Renaud, F., Parisi, E., Capasso, A. & De Prisco, P. 1983. On the role of serotonin and 5-methoxy-tryptamine in the regulation of cell division in sea urchin eggs. Dev. Biol.98, 37–46.

Richardson, B. P. 1990. Serotonin and nociception. Ann. N.Y.Acad. Sci. 600, 511–519.

Sakharov, D. A. & Salimova, N. 1981. Serotonin-containing cellsin the ascidian endostyle. Experientia 38, 802–803.

Saudou, F., Boschert, U., Amlaiky, N., Plassat, J. L. & Hen, R.1992. A family of Drosophila serotonin receptors with distinctintracellular signaling properties and expression patterns.EMBO J. 11, 7–17.

Shmukler, Y. B., Buznikov, G. A. & Whitaker, M. J. 1999. Action ofserotonin antagonists on cytoplasmic calcium levels in earlyembryos of sea urchin Lytechinus pictus. Int. J. Dev. Biol. 43,179–182.

Shuey, D. L., Sadler, T. W., Tamir, H. & Lauder, J. M. 1993.Serotonin and morphogenesis. Transient expression of serotonin uptake and binding protein during craniofacial morphogenesis in the mouse. Anat. Embryol. 187, 75–85.

Stratford, T., Horton, C. & Maden, M. 1996. Retinoic acid isrequired for the initiation of outgrowth in the chick limb bud.Curr. Biol. 6, 1124–1133.

Takamura, K. 1998. Nervous network in larvae of the ascidianCiona intestinalis. Dev. Genes Evol. 208, 1–8.

Takeda, N. 1992. Biogenic monoamine system detected simul-taously in the neural complex of the ascidian, Ciona intestinalis.Comp. Biochem. Physiol. 103C, 489–493.

Terio, B. 1964. Intorno ai fotorecettori nell’anfiosso. Atti Soc.Peloritana Sci. Fis. Matemat. Nat. 10, 111–125.

Thorndyke, M. C. & Probert, L. 1979. Calcitonin like cells in thepharynx of the ascidian Styela clava. Cell Tissue Res. 203,301–309.

Torrence, S. A. & Cloney, R. A. 1982. The nervous system of ascid-ian larvae. Primary sensory neurons in the tail. Zoomorphology99, 103–115.

Welsh, J. H. & Loveland, R. E. 1968. 5-Hydroxytryptamine in theascidian, Ciona intestinalis L. Comp. Biochem. Physiol. 27,719–722.