Chemical transmission in the sea anemone Nematostella vectensis: A genomic perspective

Comparative Biochemistry and Physiology, Part D 4 (2009) 268–289

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Comparative Biochemistry and Physiology, Part D j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p d

Chemical transmission in the sea anemone Nematostella vectensis: A genomic perspective Michel Anctil ⁎ Département de sciences biologiques and Centre de recherches en sciences neurologiques, Université de Montréal, Case postale 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7

a r t i c l e

i n f o

Article history: Received 26 March 2009 Received in revised form 30 June 2009 Accepted 7 July 2009 Available online 16 July 2009 Keywords: Anthozoa Cnidaria Genomic analysis Hormone signalling Nematostella Neuropeptides Neurotransmitters Starlet sea anemone

a b s t r a c t The sequencing of the starlet sea anemone (Nematostella vectensis) genome provides opportunities to investigate the function and evolution of genes associated with chemical neurotransmission and hormonal signaling. This is of particular interest because sea anemones are anthozoans, the phylogenetically basal cnidarians least changed from the common ancestors of cnidarians and bilaterian animals, and because cnidarians are considered the most basal metazoans possessing a nervous system. This analysis of the genome has yielded 20 orthologues of enzymes and nicotinic receptors associated with cholinergic function, an even larger number of genes encoding enzymes, receptors and transporters for glutamatergic (28) and GABAergic (34) transmission, and two orthologues of purinergic receptors. Numerous genes encoding enzymes (14), receptors (60) and transporters (5) for aminergic transmission were identified, along with four adenosine-like receptors and one nitric oxide synthase. Diverse neuropeptide and hormone families are also represented, mostly with genes encoding prepropeptides and receptors related to varying closeness to RFamide (17) and tachykinin (14), but also galanin (8), gonadotropin-releasing hormones and vasopressin/ oxytocin (5), melanocortins (11), insulin-like peptides (5), glycoprotein hormones (7), and uniquely cnidarian peptide families (44). Surprisingly, no muscarinic acetylcholine receptors were identified and a large number of melatonin-related, but not serotonin, orthologues were found. Phylogenetic tree construction and inspection of multiple sequence alignments reveal how evolutionarily and functionally distant chemical transmitter-related proteins are from those of higher metazoans. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Cnidarians are regarded as the most basal metazoans possessing nervous systems. Consequently, they have attracted considerable interest among neurobiologists as the evolutionary implications of studying their nervous systems became increasingly apparent. Cnidarian neurons are largely organized into planar nerve nets of varying density that are distributed in the ectoderm and, especially within the anthozoan class, in the endoderm (Pantin, 1952; Batham, 1965). As the neuronal processes cross over each other in the nerve net, they form more or less specialized synaptic junctions in which synaptic vesicles were identified (Anderson and Grünert, 1988; Westfall and Grimmelikhuijzen, 1993; Westfall et al., 1995). A large body of electrophysiological investigations showed that cnidarian neurons fundamentally work like neurons of physiologically more complex animals and that mechanisms of chemical neurotransmis-

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sion in cnidarians are similar to those of higher invertebrates and of vertebrates (Anderson and Spencer, 1989; Mackie, 1990). Although many forms of neuronal activity were documented in cnidarian nervous systems, the lack of strong evidence for classical transmitters (Martin and Spencer, 1983) coupled with evidence of widespread use of epithelial conduction and of electrical synapses between neurons in hydrozoan experimental models (Mackie, 2004), led to skepticism about the nature and extent of transmitter use in these animals. However, the last 25 years have witnessed the emergence of a large body of biochemical, immunohistochemical, molecular and physiological investigations demonstrating the presence in neurons and the biological activity of neuropeptides, biogenic amines and fastacting small transmitters such as glutamic acid and GABA (Grimmelikhuijzen et al., 1996, 2002; Kass-Simon and Pierobon, 2007 for reviews). Yet, while the evidence for the role of both neuropeptides and classical transmitters in effector activities of all cnidarian classes is persuasive, the body of existing data is too incomplete and fragmented among several species to gain a satisfactory picture of the set of transmitter systems available to cnidarians. The recent sequencing of the genome of the starlet sea anemone Nematostella vectensis (Putnam et al., 2007) provides a unique opportunity to explore the full repertoire of putative gene products known to be

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involved in transmitter biosynthesis, transport and receptors, all within a single cnidarian species. Recently, the morphological organization of the nervous system of this anemone was described (Marlow et al., 2009). The starlet sea anemone is a representative of the Anthozoa, the most basal cnidarian class which includes also corals and sea pens. Representatives of this class are considered to be closer to the ancestor of bilaterian animals than are other cnidarians (Bridge et al., 1992, 1995). Therefore, any analysis of protein families from the genome of N. vectensis is likely to provide insights on the evolution of protein structure and function in the context of the eumetazoan ancestry of cnidarians and of their predating the emergence of bilaterian animals. In addition, it is the intention of this genomic approach to provide a resource for investigators to explore new avenues and hypotheses in their efforts to understand various aspects of transmitter function in cnidarians. Transmitter-associated proteins belong to different classes of proteins (enzymes, transporters, receptors), all of which contributing to transmitter function while potentially exhibiting distinctive features that reflect their evolutionary history. The following analysis of the repertoire of putative transmitter-related genes in the genome of N. vectensis aims at assessing for the first time the range, functional capability and evolutionary implications of transmitter systems in a cnidarian. For the purpose of this analysis, the word «transmitter» is used in a broad sense to include all substances that may be released from neurons and that act as bona fide neuroactive substances (triggering a post-synaptic response), modulators (modulating a pre- or post-synaptic event) or hormones (triggering a response significantly away from the release site). 2. Methods Protein sequences were searched from the US Department of Energy Joint Genome Institute website for N. vectensis (http:// genome.jgi-psf.org/Nemve1/Nemve1.home.html). Searches were conducted using annotation keywords or search engines such as KOG and BLAST available on the site. PHI-BLAST was also used to improve hit returns on queries of neuropeptide precursor proteins, using PHI patterns for the various neuropeptide families. All hits with an e-value below e− 10 were selected. The selected sequences were downloaded through the MEGA v.4 software (Tamura et al., 2007) and directed to the RPS-BLAST alignment tool for inspection. Sequences of interest that were deemed too short or that included incomplete expected conserved domains were discarded. Duplicates and splice variants were identified by manually inspecting all aligned sequences and were removed from the pool of analysed sequences. For phylogenetic analyses each protein transcript of interest was aligned with BLAST against the entire nr database. Sequences among the first 100 hits were selected and used to construct phylogenetic trees. The hits and related N. vectensis sequences were next aligned using ClustalW with MegAlign (Lasergene, DNASTAR) and trees were constructed with the MEGA implementation of distance neighborjoining with complete deletion of gaps. Manual deletions were also performed to emphasize conserved domains. For sequences of membrane proteins, the vast majority of which are G protein-coupled receptors (GPCR), the N- and C-tails were removed. Consensus trees were obtained by bootstrapping the data (1000 replicates). To further validate some of the phylogenies, maximum likelihood analyses were conducted with PUZZLE (Strimmer and von Haeseler, 1996). In addition, sequences were selected from the constructed trees of each protein class to create alignments designed to assess the extent to which the sea anemone proteins retained aa residues important for functional features of the corresponding protein class. For this purpose multiple alignments of proteins and their putative sea anemone orthologues were generated with MegAlign using the ClustalW algorithm and were displayed with the GeneDoc editor program (version 2.7; http://www.psc.edu/biomed/genedoc). The Statistics report function of GeneDoc was also used to evaluate

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percentages of residue similarity and identity in pairwise alignments. For this purpose the N- and C-tails of all membrane protein sequences were removed. SignalP was used to detect signal peptides of neuropeptide precursor proteins and NeuroPred for prediction of cleavage sites. 3. Results and discussion 3.1. An overview of the repertoire Nearly 280 N. vectensis sequences of appropriate length and/or including integral functional domains were retained for analysis. Receptors represent nearly 70% of these sequences. The remaining sequences are distributed among biosynthetic or inactivating enzymes, cell membrane or vesicular membrane transporters and neuropeptide precursors. Although every effort was made to cull from the genome all transcripts relevant to transmitters, it is likely that some were missed due to oversight or to their belonging to hitherto unknown transmitter categories. For convenience candidate genes are classified in three categories: small transmitters acting through both ionotropic and metabotropic receptors such as acetylcholine, amino acid and purinergic transmitters (Table 1), aminergic and other small transmitters such as adenosine and nitric oxide (Table 2) and neuropeptides/hormones Table 1 Genes of N. vectensis predicted to code for proteins associated with acetylcholine, amino acids and ATP. Transmitter type Acetylcholine Choline acetyltransferase Acetylcholinesterase Nicotinic receptors

Amino acids Glutamate AMPA receptors Kainate receptors NMDA receptors Metabotropic receptors Glutamate transporters Vesicular transporters GABA Glutamate decarboxylase GABAa receptors

Glycine receptor GABAb receptors

Protein ID number

EST ID number

Nv_416, Nv_95805, Nv_203043

Nv_163430

Nv_31599, Nv_87444, Nv_119959, Nv_209664 Nv_211382 Nv_40919, Nv_85091, Nv_91696, Nv_91941 Nv_110265, Nv_198343, Nv_198927, v_199721 Nv_200917, Nv_205808, Nv_205855, Nv_214990

Nv_160761, Nv_171912 Nv_239659, Nv_244109 Nv_240779, Nv_247410

Nv_13877, Nv_24412, Nv_50912, Nv_104623 Nv_117160, Nv_132356 Nv_141731 Nv_11315, Nv_31895, Nv_51517, Nv_211456 Nv_31331, Nv_40374, Nv_105783, Nv_197524 Nv_198894, Nv_201378, Nv_218792, Nv_229374 Nv_110362, Nv_210965, Nv_230013

Nv_241281, Nv_244506

Nv_173595

Nv_10128, Nv_11440, Nv_81701, Nv_123866 Nv_138860, Nv_231086 Nv_60452, Nv_60834, Nv_70014 Nv_40863, Nv_60804, Nv_93322, Nv_103931 Nv_111552, Nv_114921, Nv_201724, Nv_204447 Nv_211643, Nv_215047, Nv_230093 Nv_22284 Nv_86565, Nv_87697, Nv_206093, Nv_223171

GABA transporters Nv_60521, Nv_79255, Nv_79785, Nv_81637 Nv_109800, Nv_228010 Vesicular Nv_1064, Nv_22306, Nv_33294, transporters Nv_60758 Nv_96724, Nv_142801, Nv_206710, Nv_214632 Nv_222356 ATP Purinergic receptors (P2X)

Nv_239847 Nv_171792

Nv_102596, Nv_104653

Nv_158857, Nv_176489 Nv_239821, Nv_243252 Nv_244104 Nv_161287, Nv_187148 Nv_236066, Nv_247614 Nv_241391, Nv_241392 Nv_247861

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Table 2 Genes of N. vectensis predicted to code for proteins associated with biogenic amines and other non-peptidergic transmitters. Transmitter type

Protein ID Number

EST ID Number

Monoamines Enzymes Tyrosine/tryptophan hydroxylase

Nv_203775, Nv_216737

Nv_237576 Nv_240046 Nv_245974 Nv_164399 Nv_164601, Nv_179511 Nv_191334

Tyrosinase DOPA decarboxylase Dopamine-β-hydroxylase PNMT-like (adrenaline) HIOMT (melatonin) Monoamine oxidase Receptors Monoamine-like (DA/5-HT) Monoamine-like (adrenergic) Monoamine-like (histamine) Melatoninergic

Transporters Monoamine transporters Vesicular amine transporters Adenosine Adenosine-like receptors Nitric oxide Nitric oxide synthase (NOS) NOS binding protein

Nv_98001, Nv_204120 Nv_79900, Nv_93717 Nv_209258 Nv_34681, Nv_91623, Nv_123672 Nv_94865, Nv_136792, Nv_196827, Nv_229539 Nv_13440, Nv_14501 Nv_24241, Nv_41866

Nv_164564, Nv_239384 Nv_247298 194295 Nv_175230

Nv_85851, Nv_117699, Nv_118705, Nv_122716, Nv_207168, Nv_214347, Nv_222805, Nv_222806, Nv_223523, Nv_1848, Nv_1944, Nv_82546, Nv_85943 Nv_85995, Nv_97538, Nv_119248, Nv_131304, Nv_148199, Nv_212680, Nv_197923, Nv_1897, Nv_23357, Nv_97861, Nv_109382 Nv_123316, Nv_136356, Nv_199298, Nv_200221, Nv_204979, Nv_212860, Nv_1499, Nv_1642, Nv_2167, Nv_6197 Nv_13917, Nv_33192, Nv_34809, Nv_52597, Nv_105626, Nv_113440, Nv_113494, Nv_196476, Nv_197968, Nv_198502, Nv_198600, Nv_202553, Nv_203795, Nv_208482, Nv_209463, Nv_209464 Nv_209465 Nv_211467, Nv_211963, Nv_212429 Nv_209450, Nv_209454, Nv_229612 Nv_213661, Nv_214544

Nv_172825, Nv_189286 Nv_241669, Nv_244057

Nv_171569

Nv_1683, Nv_82495, Nv_169811, Nv_216997 Nv_110559 Nv_44036

Table 3 Genes of N. vectensis predicted to code for proteins associated with neuropeptides. Transmitter type

Transcript ID number

EST ID number

RFamide-related Precursors Antho-RFamide Other RFaPs Amidating enzymes Receptors RFa/NPFF/GnIH/NPY

Nv_1374 Nv_9531, Nv_16904/97952 Nv_101809, Nv_230646

Nv_172604

Nv_13858, Nv_24147, Nv_33569, Nv_34324, Nv_34835, Nv_61644, Nv_81082, Nv_84747, Nv_99552, Nv_113178, Nv_210664

Nv_238927, Nv_241688 Nv_244873, Nv_245559

Other cnidarian peptides Antho-RIamide-like precursor Antho-RNamide-like precursor Antho-RPamide-like precursors Antho-RWamide-like precursor LWamide precursor Unique cnidarian neuropeptide receptors (unclassifiable)

Galanin-related Galanin-like precursors Galanin-like receptors Tachykinin-related Putative precursors Receptors Tachykinin/SIFamide receptors GnRH/vasopressin-related GnRH-like precursor Vasopressin-like precursor GnRH/vasopressin-like receptors Melanocortin-related α-MSH-like precursor Melanocortin-like receptors Insulin-like peptides Peptides Receptors Glycoprotein hormone receptors (LGR type A)

Nv_65111 Nv_244953⁎ Nv_200817 Nv_37852 Nv_141747 Nv_126270 Nv_1668, Nv_1720, Nv_1919, Nv_1951, Nv_1990, Nv_13425, Nv_13739, Nv_21366, Nv_24914, Nv_33886, Nv_237937 Nv_34191, Nv_34333, Nv_34364, Nv_34518, Nv_41807, Nv_52016, Nv_79851, Nv_83657, Nv_86966, Nv_87779 Nv_90581, Nv_91081, Nv_98771, Nv_100423, Nv_101072, Nv_105690, Nv_107948, Nv_111898, Nv_112480, Nv_119626, Nv_119692, Nv_127852, Nv_157068, Nv_196320, Nv_197583, Nv_198963, Nv_200814 Nv_209920, Nv_212697 Nv_96465, Nv_128410, Nv_142194 Nv_108333, Nv_133576, Nv_206354, Nv_206604, Nv_211853 Nv_88765, Nv_94714 Nv_13531, Nv_22729, Nv_34692, Nv_41719, Nv_61697, Nv_87682, Nv_103257, Nv_106757, Nv_122333, Nv_133598, Nv_196253, Nv_207006

Nv_244378

Nv_216820 Nv_65450, Nv_206388

Nv_241190

Nv_201906, Nv_209131

Nv_8156, Nv_38399 Nv_199985, Nv_204373, Nv_205839, Nv_207495, Nv_208724, Nv_210358, Nv_211188, Nv_219411, Nv_244403 Nv_246236 Nv_199266, Nv_207484 Nv_50070, Nv_85808, Nv_198971 Nv_80429, Nv_204412, Nv_212618, Nv_197977, Nv_200697, Nv_217594, Nv_221138

Nv_238729

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Fig. 1. Relationships of the ACh-like enzymes of N. vectensis. (A) Rooted neighbor-joining (NJ) tree showing that the choline acetyltransferase (ChAT)-like sea anemone transcripts (shaded box) cluster with carnithine acetyltransferases (CrAT) of higher metazoans rather than with ChATs. Numbers on the nodes in this and subsequent tree figures represent the percentage of bootstrap replicates supporting this topology. Scale for branch lengths at bottom represents the number of substitutions per site in this and subsequent tree figures. (B) Alignment of ChAT β strands 8 (upper panel) and 12 (lower panel) showing residues important for catalytic activity (⁎) and substrate interaction (+). (C) Unrooted tree in which acetylcholinesterase (AChase)-like sea anemone sequences (shaded box) stand as an outgroup to various cholinesterases. (D) Alignment of Chase β strand 9/helix 10 (upper panel) and of segment between β strand 10 and helix 14 (lower panel) showing conserved active site residues (⁎) and conserved residues in active site gorge (+).

Fig. 2. Relationship of the nicotinic-like receptors of N. vectensis. (A) Rooted NJ tree showing the sea anemone subclades (shaded) forming an outgroup to invertebrate and vertebrate counterparts. Note that the GABA-A and glycine receptors are not nested with the nicotinic and sea anemone sequences. (B) Alignment of β strands 4 and 6 (upper panel) and of sequence fragments flanking β strand 10 (lower panel) showing residues important for ligand binding (o), for binding pocket boundary formation (+) and for contacting docked ACh and nicotine (⁎).

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(Table 3). In addition to the candidate genes, Tables 1–3 also provide a list of available EST transcripts in order to gain an appreciation for the distribution of expressed proteins among the transmitter categories. 3.2. Acetylcholine Evidence so far for acetylcholine as transmitter in cnidarians is weak and controversial (Scemes, 1989; Kass-Simon and Pierobon, 2007). Cholinergic agonists were reported to induce muscle contractions and increase bioelectric activity in hydrozoans and sea anemones (Romanes, 1885; Mendes and Freitas, 1984; Kass-Simon and Passano, 1978).

Although early histochemical studies reported acetylcholinesterase (AChE) activity in Hydra (Lentz and Barnett, 1961), later attempts failed to detect AChE or choline acetyltransferase (ChAT) activity in other hydrozoans and in sea anemones (Scemes, 1989; Van Marle, 1977). In view of this background, it may seem surprising that 20 sequences apparently related to cholinergic function were found among the transcripts of N. vectensis: 3 ChATs, 5 AChEs and 12 nicotinic receptors (Table 1). Phylogenetic analysis suggests that the putative ChAT sequences appear closer to the carnithine-AT (CrAT) clade than to the ChAT clade (Fig. 1A). This is consistent with the higher residue identity score of transcripts against CrAT (40%) than against ChAT (35%). In

Fig. 3. Relationships of the glutamate-like receptors of N. vectensis. (A) Unrooted NJ tree showing some sea anemone sequences clustering with AMPA/kainate receptors and others with NMDA receptors. (B) Alignment of sea anemone sequences with corresponding AMPA receptor (upper panel) and with NMDA receptor sequences (lower panel). The represented sequence segment is in the N-terminal part and just precedes the first transmembrane region. The conserved motif PLTxxxxR is important for glutamate interaction at the binding site. (C) Unrooted NJ tree showing some sea anemone sequences clustering with metabotropic glutamate receptors and others with calcium sensors. (D) Alignment of sea anemone sequences with corresponding metabotropic glutamate receptor sequences in the N-terminus. Consensus residue for agonist binding (⁎) and residues important for binding pocket formation (+) in groups I (red), II (green) and III (violet) receptors are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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contrast, multiple alignment shows retention of conserved residues for catalytic activity and substrate interaction consistent with both ChAT and CrAT function (Govindasamy et al., 2004; Cai et al., 2004) (Fig. 1B). An arginine residue critical for electrostatic interaction with carnithine in helix 18 is conserved by the sea anemone enzymes (not shown). Tree analysis shows that sea anemone transcripts form outgroups to AChEs and butyrylChEs (BChE) from both vertebrates and invertebrates (Fig. 1C). Residue identity scores of transcripts against AChEs or BChEs range from 32 to 37%. Multiple alignment of these sequences revealed that some of the residues important for catalytic activity or for the formation of the substrate interaction gorge (Bourne et al., 2003; Lazari et al., 2004) are preserved in the sea anemone orthologues (Fig. 1D). That these enzymes are expressed and potentially functional in N. vectensis is hinted by the presence of several EST sequences related to AChE and ChAT (Table 1). The transcripts annotated as nicotinic receptor subunits showed a range of 44–59% similarity and 24–37% identity with several vertebrate and invertebrate counterparts. NJ analyses suggest that the putative sea anemone nicotinic receptor subunits cluster sep-

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arately from those of bilateral animals (Fig. 2A). The tree also shows that the sea anemone sequences are orthologues of nicotinic receptor subunits and not of GABA-A and glycine receptor subunits to which they are related. Most of the residues considered important for ligand binding and for the formation of the ligand binding pocket (Le Novère et al., 1999; Schapira et al., 2002) are preserved in the predicted sea anemone nicotinic receptors (Fig. 2B). While all the proteins necessary for nicotinic neurotransmission are apparently present in the sea anemone, no muscarinic receptor was detected. All 10 transcripts assigned to the muscarinic class in the JGI Genome Assembly of N. vectensis turned out to belong to other classes of metabotropic GPCRs. Data supporting effects of muscarinic agents on muscle contraction and bioelectric activity exist for cnidarians (see Kass-Simon and Pierobon, 2007 for review), but these are insufficient to establish the existence of muscarinic receptors. A blast search apparently yielded Hydra genes for muscarinic receptors (Watanabe et al., 2009), but none is identified and there is no evidence that the gene sequences were subjected to scrutiny for functional domains. Without more reliable data, it can only be assumed that muscarinic

Fig. 4. Relationships of the GABAergic-like receptors of N. vectensis. (A) Unrooted NJ tree showing that sea anemone sequences form outgroups in relation with GABA-A receptors whereas one sequence clusters clearly with a glycine receptor. (B) Alignment of sea anemone sequences with corresponding GABA-A receptors. The represented sequence segments are on the C-terminal side of loop E (upper panel) and in loops B and C (lower panel) of the ligand-binding domain. Residues important for GABA binding in α1 (orange) and β2 subunits (red), and residues important for benzodiazepine binding in γ subunit (green) are shown. A cysteine residue involved in a sulfhydryl bridge is also shown. (C) Unrooted NJ tree showing that a few sea anemone sequences are nested with GABA-B2-like receptors but not with GABA-B1 receptors. (D) Alignment of sea anemone sequences with corresponding GABA-B receptors. The represented sequence segments are in the first extracellular loop (upper panel) and in the area of the fifth transmembrane region (lower panel). Residues important for agonist binding (⁎) and affinity (+) are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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receptors may have evolved in the common ancestors of protostomes and deuterostomes, representatives from both of which possess muscarinic receptor genes. 3.3. Amino acids Excitatory (glutamate/aspartate) and inhibitory (GABA/glycine) transmitters have long been considered of paramount importance in the nervous system of both vertebrates and invertebrates (Hall et al., 1979; Bowery and Smart, 2006). In cnidarians these transmitters were reported to be involved in activities as diverse as nematocyst discharge (Kass-Simon and Scappaticci, 2004), pacemaker networks associated with motor activity (Kass-Simon et al., 2003; Ruggieri et al., 2004) and in

the feeding response (Pierobon et al., 1995, 2001, 2004). Predicted proteins for amino acid transmitters are well represented in N. vectensis. A large number of ionotropic glutamate receptors were identified (Table 1). Several AMPA-like and NMDA-like receptors are present, but only one kainate-like. While AMPA/kainate-like sea anemone receptors do not cluster readily with specific vertebrate subtypes, the NMDA-like sea anemone proteins appear closer to the NMDA2 than to the NMDA1 subtype (Fig. 3A). The AMPA orthologues show higher similarity/ identity with their bilaterian counterparts (up to 61/40%) than the NMDA orthologues (up to 40/22%). Only some of the residues considered important for glutamate binding in mammalian AMPAs and NMDAs (Lampinen et al., 1998; Furukawa and Gouaux, 2003; Chen and Wyllie, 2006) are present in the sea anemone orthologues (Fig. 3B).

Fig. 5. Relationships of the amino acid-like transporters of N. vectensis. (A) Unrooted NJ tree showing that sea anemone sequences form an outgroup in relation to various excitatory amino acid transporters (EAAT). (B) Alignment of sea anemone sequences with corresponding EAATs. The represented sequence segments are in the third extracellular loop (upper panel), seventh (middle panel) and tenth (lower panel) transmembrane regions. Consensus residues for the EAAT family (⁎) and residues implicated in glutamate transport (+) are shown. (C) Unrooted NJ tree showing that some sea anemone sequences form an outgroup in relation with various GABA (GAT) and glycine (GlyT) transporters whereas others are nested with GATs. (D) Alignment of sea anemone sequences with corresponding GATs, taurine (TauT) and creatine (CreaT) transporters. The represented sequence segments are in the first (upper panel) and sixth (lower panel) transmembrane regions. Consensus residues for the SLC6 family of transporters (⁎) and a residue involved in sodium ion interaction (o) are shown.

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In contrast to the cholinergic suite of receptors, many metabotropic glutamate receptors (mGlutR), members of the GPCR superfamily, were clearly identified among the sea anemone transcripts (Table 1). In addition, calcium-sensing receptors (CaSR), which are closely related to mGlutR but are not involved in chemical transmission, were detected. Phylogenetic analyses show a clear cleavage between CaSR and mGlutR, although the relationship of the sea anemone CaSR clade with that of vertebrates is poorly resolved (Fig. 3C). The position of the sea anemone mGlutR clade outside of the vertebrate mGlutR classification is strongly supported by bootstrap validation and by the level of transcript residue identity with vertebrate orthologues (27–38%). It is also supported by the poor conservation of the signature residues for the various vertebrate mGlutR subtypes even though residues important for glutamate binding by the sea anemone sequences are conserved (Fig. 3D; Rosemond et al., 2002; Kuang et al., 2006; Muto et al., 2007). The large number of ionotropic and metabotropic receptors suggests an important role of glutamic acid as transmitter in sea anemones and hints at the diversity of responses possible. The few identified ESTs displaying homology with these receptors indicate that the latter are expressed and functional in N. vectensis.

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Glutamate dehydrogenase (GAD) is involved in the synthesis of gamma-aminobutyric acid (GABA). GABA and GAD immunoreactivities were reported in neurons of the anthozoan Eunicella cavolini (Girosi et al., 2007) and GABA immunoreactivity in the ectodermal nerve net of the starlet sea anemone (Marlow et al., 2009). There are at least two apparent splice variants of GAD in the sea anemone genome (Table 1). However, they exhibit more conserved residues with prokaryote (32% residue identity) than with eukaryote GADs (13%). There are twice as many GABAa as GABAb receptors in the sea anemone genome and one unambiguous glycine receptor was also found (Table 1). The sea anemone GABAa receptors appear to be distantly related to their various vertebrate and cephalopod orthologues whereas one sea anemone transcript appeared to nest with a glycine receptor (Fig. 4A). This distant relationship to GABAa receptors is also reflected in the shared residue conservation scores (up to 52% similarity, 32% identity) and in the several residues important for GABA binding (Galvez et al., 1999; Ci et al., 2008) that are not present among sea anemone transcripts (Fig. 4B). The sea anemone metabotropic GABAb receptors, in contrast, appear to form sister clades with vertebrate and invertebrate type 2 GABAb orthologues, but not

Fig. 6. Relationships of the vesicular amino acid-like transporters of N. vectensis. (A) Rooted NJ tree showing that sea anemone sequences form a clade with a mammalian vesicular glutamate transporter (vGluT1) but not with other vertebrate (vGlut2-6) and invertebrate glutamate transporters. (B) Alignment of sea anemone sequences with corresponding vGluTs. The represented sequence segments are in helices 5 (upper panel) and 10 (lower panel). Residues facing the center of the pore separating the N- and C-tail domains are shown (⁎). (C) Rooted NJ tree showing that sea anemone sequences form outgroups in relation to various vesicular inhibitory amino acid transporters (VIAAT). (D) Alignment of sea anemone sequences with corresponding VIAATs. The domains in the represented sequence segments are identified above the panels and include highly conserved residues.

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with type 1 receptors (Fig. 4C). However, examination of residues important for ligand binding or affinity shows that the sea anemone transcripts have conserved both type 1 and type 2 specific residues (Fig. 4D). Glutamate can be inactivated by reuptake from the synaptic cleft through excitatory amino acid transporters (EAAT; Palacin et al., 1998), members of the solute carrier superfamily (SLC). Three transporters related to this superfamily, showing the typical 10 transmembrane domains, were identified in the sea anemone genome (Table 1). They show significant similarity with vertebrate glial and neuronal EAAT, but also with other EAATs, sharing 59–66% residue similarity and 42–46% identity with various vertebrate EAATs. Tree analysis shows that the sea anemone sequences form an outgroup in relation to a host of vertebrate EAATs (Fig. 5A). Therefore, it is unclear if any of these sequences are related to EAATs involved in transport at neuronal membranes as opposed to less selective, epithelial amino acid transports. Signature residues and those involved in glutamate binding (Yernool et al., 2004; Pedretti et al., 2008) are largely shared by the sea anemone EAAT (Fig. 5B). Glutamate is also stored in synaptic vesicles through vesicular transporters (SLC17 family). Five transcripts from the sea anemone genome (Table 1) are nested with a rat vesicular glutamate transporter (vGlut1) which is remote from other types of vertebrate and invertebrate vGlut orthologues (Fig. 6A). However, the consensus residues shared by the rat vGlut1 and sea anemone transcripts are within the score range of those shared with other vGluts (48–64% similarity, 27–43% identity). There are divergences in the extent to which the various sea anemone transcripts show conservation of the residues important for binding pocket formation and for glutamate binding (Almqvist et al., 2008) (Fig. 6B). A surprisingly large number of inhibitory amino acid transporters were found (Table 1). Among them, five are plasma membrane transporters (SLC6 family) and nine are vesicular inhibitory amino acid transporters (SLC32, VIAAT). Immunoreactivity to GABA vesicular transporters was previously reported in the sea fan (Girosi et al., 2007). Some of the SLC6-like sea anemone sequences cluster with vertebrate GABA GAT1 proteins while others cluster with unclassified vertebrate and invertebrate SLC6 members (Fig. 5C). Their sequences are highly conserved, with up to 70% residue similarity and 49% identity with various GAT1 orthologues. None clustered with glycine transporters. Signature residues of the SLC6 family and residues important for substrate binding (Palacin et al., 1998) are highly conserved in most transcripts (Fig. 5D). The VIAAT transcripts are distributed in two clades, one of which clusters with A1 members of the vertebrate SLC32 family and with a few invertebrate VIAATs whereas the other forms a separate, more distantly related clade (Fig. 6C). The sea anemone transcripts

share a large number of conserved residues with the A1 type of VIAAT (Fig. 6D). 3.4. ATP P2X receptors are cation channels gated by ATP released from purinergic neurons (see Burnstock, 2007 for review). ATP was shown to trigger circular muscle contraction more potently than other nucleotides in the sea anemone Actinia equina (Hoyle et al., 1989). In addition, evidence for a role of ATP, presumably released from sensory neurons, in sea anemone sensory hair cell repair was presented (Watson et al., 1999). The case for a transmitter role for ATP is bolstered by the identification of two P2X receptors in the N. vectensis genome (Table 1) sharing strong sequence homology with vertebrate P2X4 (54% residue identity) and, to a lesser extent, with a Hydra P2X-like receptor (48% identity). Phylogenetic analysis provides strong support for the sea anemone receptors forming a clade with invertebrate (plathelminth and Aplysia) P2X receptors that is separate from a variety of vertebrate P2X receptors (Fig. 7A). Interestingly, inclusion of the Hydra P2X in the sea anemone clade was not validated by bootstrapping. Examination of alignments shows that the residues important for ligand binding and ion channel activities (Silberberg et al., 2005) are largely preserved in the sea anemone transcript (Fig. 7B). 3.5. Biogenic amines Next to neuropeptides the most compelling case for the physiological involvement of neurotransmitters in cnidarians comes from studies on monoamines (Anctil and Bouchard, 2004; Anderson, 2004). Although not all criteria for neurotransmitter identification have been met, studies on the sea pansy Renilla koellikeri, an anthozoan like N. vectensis, have converged to provide evidence of the presence in neurons (Umbriaco et al., 1990; Pani et al., 1995; Mechawar and Anctil, 1997; Anctil et al., 2002), biosynthesis (Pani and Anctil, 1994), exocytotic release (Gillis and Anctil, 2001), receptor binding (Awad and Anctil, 1993a,b; Hajj-Ali and Anctil, 1997) and inactivation (Anctil et al., 1984; Dergham and Anctil, 1998) of various monoamines. Dopaminergic transmission was also investigated in sea anemones and hydrozoans (Carlberg et al., 1984; Carlberg, 1992; Chung et al., 1989; Chung and Spencer, 1991a,b). As will become apparent below, proteins related to aminergic transmitters are heavily represented in the genome of N. vectensis (Table 2). The most striking feature of the suite of proteins (enzymes of the aminergic pathway, receptors, transporters) is that they belong to classical vertebrate types of biogenic amine proteins. For example,

Fig. 7. Relationship of the ATP-like receptor of N. vectensis. (A) Unrooted NJ tree showing that the sea anemone P2X-like sequence forms with plathelminth and mollusk orthologues a separate clade from vertebrate P2X receptors. (B) Alignment of sea anemone sequences with corresponding P2X receptors. The represented sequence segments are in the first (upper panel) and second (lower panel) transmembrane regions. Residues important for ligand binding affinity are shown (+). Red letters indicate residues which when substituted by tryptophan lead to non-conducting channels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. Relationships of biogenic amine-related enzymes of N. vectensis. (A) Unrooted NJ tree showing that a sea anemone EST clusters with various vertebrate phenylalanine hydroxylases (PaH) but not with tyrosine (TH) or tryptophan (TPH) hydroxylases. (B) Rooted NJ tree showing two sea anemone sequences clustering with monooxygenase X (moxd) sequences from various vertebrates instead of with related dopamine β hydroxylases (DBH). (C) Unrooted NJ tree in which several genomic and EST sea anemone sequences form an outgroup in relation to various vertebrate hydroxyindole-O-methyltransferases (HIOMT) and to related enzymes from microorganisms. (D) Rooted NJ tree showing two sea anemone sequences clustering with vertebrate and invertebrate monoamine oxidases (MAO), but not with amine oxidases from unicellular organisms.

neither enzymes of the octopamine biosynthetic pathway nor octopaminergic receptors were identifiable in the sea anemone genome, which is consistent with failure to detect octopamine by HPLC in the sea pansy (Pani and Anctil, 1993). Tyrosine (TH) and tryptophan (TPH) hydroxylases are key ratelimiting enzymes in the catecholaminergic and indolaminergic pathways. No transcript was found in the sea anemone genome that shares homology with these enzymes except one EST (Table 2). This sequence clustered robustly with phenylalanine hydroxylases rather than with TPHs or THs (Fig. 8A), which is consistent with a higher residue identity score with the zebrafish PaH (63%) than with the honeybee TH or human TPH (54%). This may account for the weak sea pansy TH-like and TPH-like activities relative to the vertebrate enzymes (Pani and Anctil, 1994). In contrast, a tyrosinase was extracted from the sea anemone Metridium senile that catalysed the hydroxylation of tyrosine (Carlberg et al., 1984). In this regard, the two tyrosinases present in the N. vectensis genome (Table 2) could be predicted to participate in the catecholaminergic pathway. The presence of a tyrosinase EST (Table 2) suggests that tyrosinases are expressed and functional in sea anemones.

Two transcripts each of dopa decarboxylases (DopaDC), which catalyse the conversion of Dopa into dopamine, and dopamine beta decarboxylases (DBH), which catalyse the formation of norepinephrine, were found (Table 2). Recently, Marlow et al. (2009) have demonstrated DBH expression by in situ hybridization in presumptive oral ectodermal neurons and pharyngeal ectodermal cells. While the DopaDC transcripts formed an outgroup clade to the vertebrate and invertebrate DopaDCs and to the histidine or tyramine counterparts (not shown), the DBH transcripts clustered with the moxd subfamily of monooxygenases (Fig. 8B). In support of this, the sea anemone transcripts possess in their copper-binding domain a motif (LxF; Xin et al., 2004) shared with moxd but not with DBH-related enzymes (DBHR) or DBHs. However, the residue identity score between transcripts and DBHRs was similar to that with moxd (35–37%). In contrast, the DopaDC transcripts share strong residue identity with vertebrate DopaDC counterparts (up to 50%) and possess nearly all the residues considered important for substrate binding and catalytic activity (not shown). No specific phenylethanolamine N-methyltransferase (PNMT) transcript was identified in N. vectensis. An N-methyltransferase transcript and a related EST sequence (Table 2) shared substantial residue identity with human

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PNMT (36%), but tree analysis placed them as a sister clade to a clade encompassing PNMTS, nicotinamide (NNMT) and indolethylamine Nmethyltransferases (INMT) (not shown). In addition, the sea anemone transcripts lack the PNMT residues critical for norepinephrine binding (Martin et al., 2001), thus making it unlikely that efficient synthesis of epinephrine may occur. Consistent with this, PNMT-like activity in the sea pansy showed little substrate specificity and yielded only minute amounts of epinephrine (Pani and Anctil, 1994). Hydroxyindolamine-O-methyltransferase (HIOMT) converts Nacetylserotonin to melatonin, the vertebrate pineal hormone. Melatonin immunoreactivity levels were reported to peak during the reproductive season in the sea pansy and immunoreactive melatonin was found in sea pansy neurons (Mechawar and Anctil, 1997). In addition, melatonin was found to have opposite actions to serotonin on sea pansy peristaltic activity (Anctil et al., 1991). Thus, although the presence of a HIOMT was expected, it is surprising that as many as three HIOMT gene transcripts and three related EST sequences were found in N. vectensis (Table 2). Multiple alignments show that the sea anemone transcripts share significant homology with vertebrate HIOMTs (28–40% residue identity). However, tree analysis suggests

Fig. 9. Relationships of biogenic amine-like receptors of N. vectensis to catecholaminergic and histaminergic receptors. (A) Rooted NJ tree showing that the sea anemone sequences form a sister clade of histaminergic H2 receptors while dopaminergic and adrenergic receptors fall outside the two sister clades. Note that two sea anemone sequences form a subclade with a receptor from another anthozoan, the sea pansy. (B) Alignment of sea anemone sequences with various catecholaminergic and histaminergic receptors. The represented sequence segments are in the fifth transmembrane region. Consensus residues for receptor interaction with biogenic amines (+) and for GPCRs (⁎) are shown.

that the transcripts form a clade separate from both vertebrate HIOMTs and O-methyltransferases of microorganisms (Fig. 8C). Monoamine oxidase (MAO) inactivates catecholamines and indoleamines by converting them into acid forms. Evidence of such conversions were reported in the sea pansy (Pani and Anctil, 1994). Four MAO transcripts were identified in the sea anemone genome (Table 2), two of which were shown by tree analysis to cluster with invertebrate and vertebrate MAOs, but not with amine oxidases from microorganisms (Fig. 8D). Residue identity in paired alignments with either vertebrate or invertebrate orthologues ranged from 23 to 48%. Although catecholaminergic-like and serotonergic-like receptor binding and monoaminergic modulation of behaviour are documented in cnidarians (Anctil and Bouchard, 2004; Kass-Simon and Pierobon, 2007), only two aminergic-like receptors from the sea pansy have been cloned and despite successful attempts to express them no ligand has been identified (Bouchard et al., 2003, 2004). It is bewildering, then, that as many as 54 GPCR sequences in the sea anemone genome appear to be orthologues of biogenic amine receptors (Table 2). Another surprise is that of these, twenty-four are melatonin-like receptors, which however is consistent with the presence of the HIOMT transcripts mentioned above. BLAST queries yielded a mixture of hits from different classes of aminergic receptors for all non-melatonergic sea anemone transcripts, in keeping with difficulties in classifying physiologically investigated and cloned sea pansy receptors. The classification arrived at in Table 2

Fig. 10. Relationship of indoleaminergic-like receptors of N. vestensis. (A) Rooted NJ tree showing that the sea anemone sequences form a sister clade with various vertebrate melatonin receptors, but not with serotonin receptors. (B) Alignment of sea anemone sequences with melatonergic and serotonergic receptors. The represented sequence segments overlap the third transmembrane region and second intracellular loop. Consensus residues (+) and motif (CxxCH) for melatonin receptors, and a conserved GPCR residue (⁎) are shown. Note that D in the DRY motif is substituted by N in both the sea anemone and melatonergic sequences.

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is tentative only and is based on the top hits of each transcript and on the clustering of the various transcripts in phylogenetic trees. Thus some of the transcripts present more similarity to dopaminergic or serotonergic receptors than to other aminergic receptor classes, others to adrenergic receptors, and still others to histaminergic receptors (Table 2). Vertebrate and protochordate orthologues largely dominate the hit lists, although occasional sequences from invertebrates such as the sea urchin are among the hits. Fig. 9A shows that many of the sea anemone transcripts, along with Ren 1 from the sea pansy (Bouchard et al., 2003), tend to cluster with histamine H2 receptors rather than with dopamine and adrenergic receptors. The best scores for residue identity are in the dopamine/serotonin group (40% for Nv_24241 against sea urchin D1B), followed by the histamine group (34%

Fig. 11. Relationships of biogenic amine-like transporters of N. vectensis. (A) Unrooted NJ tree showing that the two sea anemone sequences shown form a sister clade with various aminergic nerve terminal transporters, but not with GABA, taurine or creatine transporters. The panel below shows an alignment of sea anemone sequences with various aminergic transporters. The represented sequence segments overlap the sixth transmembrane region with residues considered to be involved in interaction with monoamines (⁎) and the fourth intracellular loop containing many residues where conformational changes occur during substrate binding and translocation (+). (B) Rooted NJ tree showing that the sea anemone sequences form an outgroup relative to aminergic vesicular transporters (VMAT) and members of the major facilitator superfamily (MSF) and related transporters. The tree is rooted with a VIAAT. The panel below shows an alignment of sea anemone sequences with aminergic vesicular transporters and a MFS. The represented sequence segments are in the second (left segment) and fourth (right segment) transmembrane regions. Two conserved motifs of VMATs are highlighted.

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for Nv_136356 against Amphioxus H2). Inspection of multiple alignments indicates that the sea anemone transcripts largely share with vertebrate orthologues the signature residues of GPCRs of the rhodopsin family. Residues important for histamine binding are conserved in some sea anemone transcripts (Fig. 9B) whereas other transcripts tend to conserve residues important for binding of amines other than histamine (Shi and Javitch, 2002). In contrast to non-melatonin aminergic receptors, all BLAST alignments of any melatonin receptor transcript of the sea anemone yielded first numerous hits for other sea anemone transcripts, followed by hits of much lower e-values for vertebrate melatonin receptors. No other aminergic or non-aminergic receptors were represented in the hits. This suggests that these transcripts form a tight assemblage of interrelated sea anemone receptors with some resemblance to vertebrate melatonin receptors. This is supported by the relatively low residue identity scores of the transcripts with a range of melatonin receptors (21–29%). Tree analysis shows that the sea anemone transcripts cluster robustly with vertebrate melatonin receptors but not with serotonin receptors (Fig. 10A). Alignment inspection revealed that while rhodopsin GPCR signature residues are largely shared by the sea anemone transcripts, only half the residues important for melatonin binding (Barrett et al., 2003) are retained (Fig. 10B). Melatonin receptor-specific motifs such as NRY/F at the exit

Fig. 12. Relationships of MECA receptors and nitric oxide synthase (NOS) of N. vectensis. (A) Rooted NJ tree showing that some sea anemone sequences cluster with adenosine receptors whereas others, together with coral sequences, cluster with melanocortin receptors. (B) Unrooted NJ tree showing that one sea anemone sequence forms with a coral NOS an outgroup to a broad variety of invertebrate and vertebrate NOSs.

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of TM3 and NaxxY in TM7 are largely preserved (not shown), but CxxCH in the second intracellular loop is absent (Fig. 10B). Pharmacological uptake experiments in the sea pansy (Anctil et al., 1984; Dergham and Anctil, 1998) suggested the existence of plasma membrane transporters for catecholamines and indoleamines in cnidarians. Three such transporters were identified in the sea anemone genome (Table 2). They cluster with aminergic rather than with amino acid transporters in tree analysis (Fig. 11A). Alignment inspection revealed that they show a high level of residue conservation with aminergic transporters (43–45% residue identity), including a residue involved in the interaction with monoamines in TM1 and leucine repeats in TM2 (not shown). A glycophorin-like motif in TM6 and residues involved in conformational change during substrate binding and translocation in the third intracellular loop (Torres et al., 2003) are also shared by the sea anemone transcripts (Fig. 11A). In addition, two vesicular monoamine transporters (VMAT) were found (Table 2). They formed a clade separate from vertebrate and invertebrate VMATs and from vertebrate members of the major facilitator superfamily (MFS) (Fig. 11B). Alignment inspection showed residue identity scores against vertebrate VMATs were low (11–15%) compared with those against MSFs and related transporters (22–30%). In addition, the sea anemone sequences lacked all conserved motifs and substrate-relevant aspartate residues of vertebrate VMATs (Fig. 11B). In contrast, they shared more conserved residues with insect VMATs than with MFSs and related transporters (not shown). 3.6. Adenosine So far there is no report of adenosine acting as transmitter in Cnidaria. Adenosine receptors are members of the melanocortinendoglin-cannabidoid-adenosine (MECA) family of GPCRs (Fredriksson et al., 2003; Schiöth and Fredriksson, 2005). Four sea anemone transcripts were identified that showed some homology with adenosine receptors (Table 2), two of which clustered with a vertebrate/invertebrate mix of adenosine receptors in tree analysis, whereas others clustered with melanocortin GPCRs (Fig. 12A). However, the residue identity scores against adenosine receptors are low (23–26%) and none of these transcripts share with adenosine receptors any of the consensus residues important for ligand binding (Fredholm et al., 2001). A similarly weak relationship to adenosine receptors was reported in four MECA-like GPCRs cloned from the coral Acropora millipora (Anctil et al., 2007). Thus the case for adenosine as a cnidarian transmitter is inconclusive at this point. 3.7. Nitric oxide The gaseous transmitter nitric oxide (NO) is a well-known modulator of blood vessel tone and gut muscles and is widespread across animal phyla (Moroz, 2001). Its involvement as neurotransmitter in cnidarians was bolstered by evidence of the involvement of NO in the feeding response of Hydra (Colasanti et al., 1997) and the subsequent localization of NADPH-diaphorase activity in Hydra neurons (Cristino et al., 2008). Similar findings in the jellyfish Aglantha digitale (Moroz et al., 2004) and the sea pansy (Anctil et al., 2005) were reported, in which NO was found to modulate swimming and fluid circulation, respectively. One NO synthase (NOS) transcript is present in N. vectensis (Table 2) and it forms with another cnidarian NOS an outgroup in relation to vertebrate neuronal and inducible NOS clades and to an invertebrate NOS clade (Fig. 12B). The transcript shares 55% residue identity with a previously cloned cnidarian NOS (mushroom coral) and 46–48% with vertebrate and invertebrate orthologues. All functional domains and residues important for iron binding are preserved in the sea anemone sequence. Residues important for substrate binding and pterin interaction in nNOS (Crane et al., 1998; Li and Poulos, 2005) are also preserved (not shown). The sea anemone sequence shares many consensus residues

Table 4 Neuropeptides predicted from putative preprohormones identified in the genome of N. vectensis. Peptide name

Transcript

Predicted peptide sequence

Antho-RFamide Nv-RFamide I Nv-RFamide II Nv-NPY Antho-RIamide II Nv-RNamide I Nv-RNamide II Nv-RPamide I Nv-RPamide II Nv-RPamide III

Nv_1374 Nv_16904 Nv_9531 Nv_9531 Nv_65111 Nv_200817 Nv_200817 Nv_37852 Nv_37852 Nv_244953

Nv-RPamide IV

Nv_244953

Nv-RWamide I Nv-RWamide II Nv-LWamide I Nv-LWamide II Nv-LWamide III Nv-LWamide IV Nv-LWamide V Nv-Galanin I Nv-Galanin II Nv-Tachykinin I Nv-Tachykinin II Nv-GnRH I Nv-GnRH II Nv-Vasopressin I Nv-Vasopressin II

Nv_141747 Nv_141747 Nv_126270 Nv_126270 Nv_126270 Nv_126270 Nv_126270 Nv_128410 Nv_96465 Nv_94714 Nv_88765 Nv_216820 Nv_216820 Nv_65450 Nv_206388

Nv-Vasopressin III

Nv_241190

Nv-α-MSH I Nv-α-MSH II Nv-α-MSH III

Nv_38399 Nv_38399 Nv_8156

b Glu-Gly-Arg-Phe-NH2 b Glu-Ile-Thr-Arg-Phe-NH2 Val-Val-Pro-Arg-Arg-Phe-NH2 Val-Val-Leu-Arg-Arg-Tyr-NH2 Tyr-Arg-Ile-NH2 Gly-Met-Asp-Gly-Arg-Asn-NH2 Gly-Met-Tyr-Arg-Arg-Asn-NH2 Trp-Ser-Cys-Ser-Leu-Arg-Pro-NH2 Trp-Ser-Cys-Cys-Leu-Arg-Pro-NH2 b Glu-Asp-Ala-Phe-Leu-Pro-LysPro-Arg-Pro-NH2 b Glu-Asp-Ser-Ser-Asn-Tyr-Glu-PhePro-Pro-Gly-Phe-His-Arg-Pro-NH2 Leu-Val-Gly-Arg-Trp-NH2 Asp-Arg-Trp-NH2 b Glu-Ala-Gly-Ala-Pro-Gly-Leu-Trp-NH2 b Glu-Ala-Gly-Pro-Pro-Gly-Leu-Trp-NH2 Gly-Pro-Pro-Gly-Leu-Trp-NH2 Gly-Ala-Pro-Gly-Leu-Trp-NH2 Asn-Ala-Pro-Gly-Leu-Trp-NH2 Gly-Asp-Thr-Gly-Ile-Thr-NH2 b Glu-Gly-Met-Thr-NH2 Tyr-Gln-Val-Ile-Phe-Glu-Gly-Val-Arg-NH2 Thr-Leu-Gln-Val-Gly-Arg-Arg-NH2 Gly-Ser-Ser-Ile-Pro-Arg-Pro-Gly-NH2 Asn-Tyr-Ser-Leu-Arg-Arg-Pro-Gly-NH2 Ala-Asn-Asp-Gly-Pro-Arg-Gly-NH2 b Glu-Glu-Glu-Gly-Val-Pro-Leu-ProArg-Gly-NH2 Pro-Gln-Pro-Gln-Arg-Ser-Met-Pro-ArgGly-NH2 Gly-Ala-Val-Pro-Val-NH2 Ser-Ala-Val-Pro-Val-NH2 b Glu-Tyr-Asn-Ile-His-Leu-Ala-Leu-Val-NH2

with iNOS as well, which is consistent with the dual nNOS/iNOS profile of NO action in the sea pansy (Anctil et al., 2005). An orthologue of NOS-binding protein was also found (Table 2). 3.8. Neuropeptides In contrast to classical neurotransmitters, several neuropeptides were identified and their presence in neurons demonstrated in cnidarians (Grimmelikhuijzen et al., 2004 for review). While some of them belong to the RFamide-related peptide superfamily (RFaP), which has representatives in every investigated metazoan phylum, others appear so far to be peptide families exclusive to cnidarians. Mature (secreted) peptides are generated by enzymatic cleavage of immature copies in larger precursor proteins (preprohormones). However, it is difficult to identify genes encoding precursors of the putative neuropeptides because of their peculiar organization and lack of recognizable functional domains in protein models. In spite of these hurdles, several precursor proteins were identified (Table 3). The identification of the putative mature peptides was based conservatively on the presence of the expected residues for cleavage at the N- and C-terminals of the immature copies. These include the conventional basic residues (lysine and/or arginine), but also acidic residues (aspartic/glutamic acid) or other unconventional pairs (X-proline, X-alanine) at the N-terminal that were documented in cnidarians (Grimmelikhuijzen et al., 2004). Other unusual residues may be involved (Wei et al., 2003), so that the number of predicted peptides listed in Table 4 is likely underestimated. Table 3 illustrates the diversity of neuropeptide families predicted to exist in the starlet sea anemone. Some of these have as yet no representative identified in cnidarians. Except for a glycoprotein hormone receptor in another sea anemone (Nothacker and Grimmelikhuijzen, 1993), no gene encoding neuropeptide or hormone receptors

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of its presence in synaptic vesicles (Westfall and Grimmelikhuijzen, 1993). Recent studies showed numerous RFamide-immunoreactive neurons in the starlet sea anemone (Marlow et al., 2009; Watanabe et al., 2009). In the starlet sea anemone a precursor protein predicted to generate 23 copies of Antho-RFamide was found. In addition, two splice variants of a precursor protein were predicted to produce 27 copies of pEITRFamide (Nv-RFamide I), a putative new peptide (Tables 3 and 4). Finally, another transcript was predicted to generate 2 copies of NvRFamide II and of a related sequence of the NPY subfamily (Table 4). Peptidylglycine α-hydroxylating monooxygenases (PHM) convert a peptidylglycine into peptidylamide at the C-terminal of immature peptides and most cnidarian peptides are amidated. An amidating enzyme orthologue of the CP2 gene product of the sea anemone Calliactis parasitica (Williamson et al., 2000) was also found in N. vectensis (Table 3), with a residue identity score of 46%. The diversity of RFa peptides in N. vectensis is matched by the large number of putative RFa-related receptor orthologues (Table 3). The 11 receptors exhibited strong similarities to Neuropeptide FF (NPFF), RFa, gonadotropin-inhibiting hormone (GnIH) and Neuropeptide Y (NPY) receptors from both vertebrates and invertebrates. Paired alignments showed overall identity scores of 29–35% against human NPFF1 and pufferfish RFa receptors and of 27–32% against the salmon NPY7 receptor. Multiple alignments revealed that these sequences shared 72% of the conserved consensus residues identified in FMRFamide receptors (Cazzamali and Grimmelikhuijzen, 2002). Fig. 13A shows that some sea anemone transcripts cluster with RFaPs but not with NPY receptors, whereas others appear more or less related with galanin receptors. An example of conserved RFa-related receptor residues shared by sea anemone transcripts is shown in Fig. 13B. In contrast, few of the consensus residues for galanin receptors are preserved in the corresponding sea anemone transcripts (Fig. 13C) and the best residue identity score of the transcripts is 31% against the zebrafish galanin receptor.

Fig. 13. Relationship of RFamide peptide-related and galanin-like receptors of N. vectensis. (A) Unrooted NJ tree showing that sea anemone sequences cluster with diverse RFamiderelated receptors (RFaR), including neuropeptide FF (NPFF) and gonadotropin-inhibiting hormone (GnIH) receptors, whereas others cluster with a galanin-like receptor. (B) Alignment of sea anemone sequences with RFaRs. The represented sequence segment is in the sixth transmembrane region. The letters represent consensus aa residues of FMRFamide receptors. (C) Alignment of sea anemone sequences with galanin receptors. The represented sequence segment overlaps the sixth transmembrane region and third extracellular loop. Residues highlighted in red are consensus residues for galanin receptors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

has been identified in cnidarians. Over 80 putative neuropeptide receptors were identified in the genome of N. vectensis that appear to match the diversity of putative neuropeptides (Table 3). 3.8.1. RFamide-related peptides The first isolated neuropeptide to be sequenced in cnidarians was Antho-RFamide in a sea anemone (Grimmelikhuijzen and Graff, 1986) and in the sea pansy (Grimmelikhuijzen and Groeger,1987). Other RFaPs were later isolated in hydrozoans (Grimmelikhuijzen et al., 1988, 1992; Moosler et al., 1996) and scyphozoans (Moosler et al., 1997). In anthozoans, the role of Antho-RFamide as a neuromuscular transmitter was supported by its actions on muscle systems (McFarlane et al., 1987; Anctil and Grimmelikhuijzen,1989) and immunocytochemical evidence

3.8.2. Cnidarian-specific peptides A variety of new neuropeptides were isolated from the sea anemone Anthopleura elegantissima that displayed C-terminal signatures unique to cnidarians (Graff and Grimmelikhuijzen, 1988a,b; Grimmelikhuijzen et al., 1990; Nothacker et al., 1991a,b; Carstensen et al., 1992, 1993; Leitz et al., 1994). Putative counterparts for each of these new types of peptides except Antho-KAamide were identified in predicted precursor proteins of the starlet sea anemone (Table 3). A precursor was identified that contained 8 copies of Antho-RIamide II (Table 4), a tripeptide immunolocalized in endodermal sensory neurons and causing the inhibition of spontaneous sphincter contractions in the sea anemone C. parasitica (Nothacker et al., 1991a,b). The two N. vectensis members of the RNamide family (Table 4) are present as one copy each in their precursor protein (Table 3). They possess 3 more amino acids than Antho-RNamide, a tripeptide with known actions on antagonistic muscles of sea anemones (McFarlane et al., 1992). The RPamide family is represented in the starlet sea anemone by four predicted peptides, two of which (Nv-RPamide I and II) are heptapeptides encoded in 3 and 2 copies, respectively, in their precursor protein and differing by only one residue substitution. The two other members (Nv-RPamide III and IV) are longer sequences with a N-terminal bGlu residue represented by a copy each in another precursor protein (Table 4). They bear little resemblance to AnthoRPamides I–IV except that Nv-RPamide I and II share the C-terminal consensus LRPamide with Antho-RPamide II and Nv-RPamide III shares the sequence PRPamide with Antho-RPamide I (Grimmelikhuijzen et al., 2004). Antho-RPamide I was shown to enhance contractile activity and to increase the rate of spontaneous contractions in the sea anemone A. equina (Carstensen et al., 1992), an effect similar to that of Antho-RFamide on the sea pansy muscle systems (Anctil and Grimmelikhuijzen, 1989).

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Two putative peptides of the RWamide family (Table 4) were detected in a predicted precursor protein (Table 3). Both, LVGRWamide (Nv-Rwamide I) and DRWamide (Nv-Rwamide II), are present in three copies each. Their N-terminal residues differ from those of the Antho-RWamides (Grimmelikhuijzen et al., 2004), including the lack of a pyroglutamate. The Antho-RWamides have been immunolocalized in synaptic terminals of endodermal muscles of C. parasitica (Westfall et al., 1995) and induce contractions of endodermal muscles (McFarlane et al., 1991) presumably via calcium channel opening (Cho and McFarlane, 1996). Another family of peptides that generated much interest is the cnidarian metamorphosins, which are involved in the metamorphosis of planula larvae into mature animals (Leitz, 1998). Metamorphosin A (MMA) was first isolated from the sea anemone A. elegantissima (Leitz et al., 1994) and several more metamorphosins were later identified in the same species as well as in Hydra (Leviev and Grimmelikhuijzen, 1995; Takahashi et al., 1997; Leitz, 1998). They almost all share the C-terminal sequence GLWamide which is biologically active. These peptides were immunolocalized in larval sensory neurons and in endodermal neurons of more mature stages (Gajewski et al., 1996). In addition to the eight metamorphosins in A. elegantissima, the six in A. equina and the four in Anemonia sulcata (Leitz, 1998), five putative metamorphosins (Table 4) were identified in a single precursor protein (Table 3): four copies of pEAGAPGLWamide (Nv-LWamide I), three of pEAGPPGLWamide (Nv-LWamide II), six of GPPGLWamide (Nv-LWamide III), three of GAPGLWamide (Nv-LWamide IV) and four of NAPGLWamide (Nv-LWamide V). The N. vectensis peptides share best similarity with the metamorphosins of Hydractinia echinata and Hym331 of Hydra (two hydrozoans), and Ae-LWamide I of the sea anemone A. equina. The diversity of this peptide family is only matched by that of the RFarelated family. In addition to the uniquely cnidarian peptide families, there is an uncommonly large retinue of neuropeptide receptors (39) presenting homologies primarily with each other and, in some cases, weak similarities with a suite of RFa- and tachykinin-related receptors (Table 3). Therefore, they are listed here as unclassifiable. The presence of these receptors is not surprising since they may be associated with the various uniquely cnidarian peptides described above. It will be an important challenge to express them with a view to identify their ligands among the specific sea anemone peptides. 3.8.3. Galanin-related peptides Galanin is a highly conserved peptide found in the nervous system and gut of vertebrates (Tatemoto et al., 1983; Skofitsch and Jacobowitz, 1985; Chartrel et al., 1995). No galanin-like peptide has yet been identified in invertebrates, although galanin-like immunoreactivity was reported in the nervous system of a few invertebrates (Lundqvist et al., 1991; Diaz-Miranda et al., 1996), including Hydra (Yamamoto and Suzuki, 2001). Tree construction of RFa-related receptors revealed a subclade of sea anemone receptors that clustered with galanin receptors (Fig. 13A). These putative galanin-like receptors are listed in Table 3. This finding prompted a search for precursor proteins in which are encoded immature galanin-like peptides. As the C-terminal residues of galanin are required for high-affinity receptor binding of smooth muscle preparations (Rossowski et al., 1990), the C-terminal consensus G(L/I/M)Tamide was subjected to PHI-BLAST analysis. This yielded three predicted precursor proteins, one of which encoded 12 copies of the putative peptide GDTGITamide (Nv-Galanin I) and the other two 8 copies (Nv_142194) and one copy (Nv_96465) of pEGMTamide (Nv-Galanin II) (Table 4). Thus these sequences are much shorter than the 29–30 aa of mammalian galanins. This divergence of the sea anemone peptides is consistent with the lack in the sea anemone galanin-like receptors (Fig. 13C) of many of the key residues identified in mammalian galanin receptors (Church et al., 2002; Lundström et al., 2007).

3.8.4. Tachykinin-related peptides Vertebrate tachykinins (TKs) and invertebrate tachykinin-related peptides (TKRPs) are present in neurons or gut endocrine cells and are involved in various activities such as modulation of neuronal excitability, induction of intestinal and oviduct contractions, and developmental regulation (Otsuka and Yoshioka, 1993; Nässel, 1999; Satake et al., 2003). There is no known TK or TKRP in cnidarians, although substance P-like immunoreactivity was reported in Hydra (Grimmelikhuijzen et al., 1981). However, the C-terminal TK consensus sequence FxGLM typical of substance P was not found in N. vectensis. Instead a putative precursor protein was found that apparently encodes a single copy of a TKRP with the C-terminal FxGyR signature (Tables 3 and 4). In addition, a precursor protein was found in which are encoded 16 copies of a putative TKRP with the incomplete C-terminal motif GxR (Table 4). The N-terminal portion of the sea anemone TKRPs shows no sequence similarity with any of the known invertebrate TKRPs (Satake et al., 2003). In contrast to the paucity of putative ligands, numerous TK-like receptors were identified (Table 3). The 12 receptors showed homology, and tended to cluster with invertebrate TK (24–30% residue identity) and SIFamide receptors (27–33%) but slightly less with vertebrate TKs (22–28%). The sea anemone sequences cluster robustly with both invertebrate and vertebrate tachykinin receptors, but not with RFamide-related receptors (Fig. 14A). Multiple alignments showed that several of the consensus residues of TK receptors (Satake et al., 2003) are preserved in the TK-like sea anemone receptors, but some of the residues important for ligand binding are not (Fig. 14B). They are also absent in TK receptors from some of the other invertebrates.

Fig. 14. Relationship of tachykinin-like receptors of N. vectensis. (A) Rooted NJ tree showing that the sea anemone sequences cluster with known vertebrate and invertebrate tachykinin receptors. (B) Alignment of sea anemone sequences with tachykinin receptors. The represented sequence segment overlaps the third intracellular loop and sixth transmembrane region. Consensus motif (⁎) and residues involved in agonist binding to human neurokinin A receptor are shown (letters).

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3.8.5. Peptides related to gonadotropin-releasing hormone (GnRH) and vasopressin/oxytocin families GnRHs form a highly conserved family of decapeptides that play a major role in vertebrate reproduction (Seeburg et al., 1987). Several conserved GnRH isoforms have also been identified in protochordates, where they are also decapeptides (see Gorbman and Sower, 2003, for review), and in mollusks in which are encoded GnRHs with 12 amino acids (Iwakoshi et al., 2002; Zhang et al., 2008). Two forms of biologically active GnRH-immunoreactive material were extracted and partially purified in the sea pansy (Anctil, 2000), but the structure of these putative peptides was not elucidated. The latter study also reported the localization of immunoreactive GnRH in endodermal neurons of both the sea pansy and the starlet sea anemone. Some of these neurons were associated with gonad tissues and the sea pansy GnRH-like extracts as well as LHRH caused an inhibition of peristaltic contractions (Anctil, 2000). A precursor protein was detected in the starlet sea anemone (Table 3) in which two putative GnRH-like peptides appear to be encoded (Table 4). Both isoforms are octapeptides. Nv-GnRH I (4 copies) differs from Nv-GnRH II (one copy) by 4 aa in the N-terminal portion of the sequences. Both retain the C-terminal consensus RPGamide of mammalian GnRH and they share more similarity with LHRH than with other GnRH isoforms, which is consistent with the higher affinity of LHRH antibody compared with other (non-mammalian) antibodies against sea pansy immunoreactive materials (Anctil, 2000). It would be interesting to know whether the two sea anemone isoforms represent the two putative peptides extracted from the sea pansy. The vasopressin/oxytocin family of peptides shares with GnRH peptides the C-terminal Gamide. This is an important peptide family associated with the vertebrate neuro-hypophyseal axis and with roles in osmoregulation and reproduction (Acher, 1996). Several homologs were identified in invertebrates where they are expressed in the nervous system and may be involved in sexual behavior (Van Kesteren et al., 1995; Kanda et al., 2005). Vasopressin/oxytocin immunoreactive neurons have been visualized in Hydra (Grimmelikhuijzen et al., 1982; Koizumi and Bode, 1991) and more recently two vasopressin-related peptides were identified in Hydra that share the C-terminal tripeptide

Fig. 15. Relationship of GnRH-like/vasopressin-like receptors of N. vectensis. (A) Rooted NJ tree showing two sea anemone sequences that cluster with an ensemble of GnRH and vasopressin-related receptors. (B) Alignment of sea anemone sequences with GnRH and isotocin/vasotocin receptors. The represented sequence segment is in the sixth transmembrane region. Signature residue of rhodopsin GPCRs (⁎) and residues important for receptor structure and ligand pocket formation (+) are shown. A tyrosine residue involved that participates in ligand binding is also shown (Y).

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PRGamide with Arg-vasopressin but lack the N-terminal with the two cysteines connected by a disulfide bond that characterize the family (Morishita et al., 2003). The Hydra peptides appear to be involved in neuronal differentiation (Takahashi et al., 2000). In the starlet sea anemone, three putative prepropeptides were identified that encode each one predicted vasopressin-related peptide in one or several copies (Tables 3 and 4). As in the Hydra peptide sequences, the N-terminal cysteines are lacking, but the C-terminal PRGamide of Arg-vasopressin/vasotocin is preserved. Nv-vasopressin II extends the similarity with the Hydra peptides to LPRGamide, but similarities end there. It is apparent that the cnidarian peptides form a separate peptide family only remotely related with other invertebrate vasopressin/oxytocin members which all include the N-terminal cysteine residues found in the vertebrate orthologues (Kanda et al., 2005). BLAST alignments yielded two relatively weak hits with GnRH and vasopressin/oxytocin receptor families in N. vectensis (Table 3). Sequence residue identity scores were also low (16–20% against GnRH and vasotocin/isotocin receptors). Tree analysis shows that the two receptors cluster with an assemblage of GnRH and vasopressinrelated receptors (Fig. 15A). Multiple alignments reveal that the sea anemone sequences share some of the residues important for receptor structure and ligand binding with GnRH receptors (Mamputha et al., 2007) (Fig. 15B). Some of these residues are also shared by vasotocin/ isotocin receptors. The sea anemone sequences, therefore, may represent ancestral forms that later diverged to form specific GnRH and vasopressin/oxytocin receptors. 3.8.6. Melanocortin-related peptides Melanocortins in vertebrates are derived from the complex precursor proopiomelanocortin (POMC) (Hadley and Haskell-Luevano, 1999) and are involved in a broad variety of functions in addition to their wellknown roles in pigmentary control (α-MSH) and in adrenal cortex stimulation (ACTH) (Wikberg et al., 2000; Schioth, 2001). They are present in neurons as well as in a variety of cell types where their release leads to paracrine or autocrine responses. POMC precursors were reported in a leech and a mollusk (Salzet et al., 1997; Stefano et al., 1991), but their absence in the genome of Caenorhabditis elegans and of insects suggests that if they are indeed present in invertebrates, it is the result of lateral gene transfer from a chordate source (Dores and Lecaude, 2005). This view is supported in the present study as no POMC gene was identified in the genome of the starlet sea anemone. Instead, the transcripts of two putative preprohormones were found (Table 3) that encode peptides sharing C-terminal residues with α-MSH. The transcript Nv_38399 includes six copies of the predicted pentapeptide GAVPVamide and four copies of the variant SAVPVamide (Table 4), both of which share the last two C-terminal residues with α-MSH. The other transcript includes nine copies of a predicted nonapeptide sharing with α-MSH only the C-terminal residue Vamide (Table 4). BLAST searches yielded nine transcripts in the sea anemone genome that matched with melanocortin receptors (Table 3), especially with the MCR5 subtype with which they cluster in a MECA tree construct (Fig. 12A). Four coral GPCRs were recently found to form a sister clade to MECA members and to show more consensus residues for ligand binding with melanocortin than with other MECA receptors (Anctil et al., 2007). In the present study multiple alignments revealed that the sea anemone receptors share even more consensus residues with MCR5s (not shown). The finding that only α-MSH-like peptides are predicted in the sea anemone genome is consistent with reports that α-MSH binds with greater affinity on MCR5s than on other MCRs (Wikberg et al., 2000). 3.8.7. Insulin-related peptides Insulin is one of the best known hormones and it plays an important role in vertebrate energy metabolism. While the structure, function and location of synthesis of insulins are highly conserved in vertebrates, insulin-related peptides (ILPs) of higher invertebrates form a

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Fig. 16. Relationships of insulin-like growth factors and insulin-like peptide receptors of N. vectensis. (A) Unrooted NJ tree showing a clustering of sea anemone sequences with insulin-like growth factors but not with insulin-like peptides. (B) Unrooted NJ tree showing that a sea anemone sequence shares with a hydra sequence an outgroup position in relation to various insulin-related receptors. (C) Alignment of sea anemone sequences with B and A chains of insulin-related peptides. Conserved motifs and residues are highlighted below the multiple alignment. The cleavage sites are marked (DR...SR). (D) Alignment of sea anemone sequences with various insulin-related receptors. The represented sequence segment is on the C-terminal side of the leucine repeat region. Residues that form with tryptophan 176 of human IGF1 receptor a pocket for ligand interaction are shown (+).

structurally diverse group encoded by large multi-gene families that are expressed in the central nervous system (Smit et al., 1998). There is evidence that ILPs, in addition to a hormonal role, are synaptic modulators (Chiu et al., 2008). Some of the invertebrate ILPs include peptides related to the insulin-like growth factors (IGFs) which have widespread growth-promoting effects in vertebrates (Rotwein, 1991), especially in the nervous system (Ye and D'Ercole, 2006). In the fruitfly and the nematode C. elegans IGF signaling is also involved in the regulation of aging (Tatar et al., 2003; Honegger et al., 2008). An insulin-like receptor was cloned in Hydra that showed an expression pattern consistent with a role in promoting growth and morphogenetic patterning (Steele et al., 1996). This suggested that an insulin-related molecule was involved but none has been identified so far in any cnidarian species. Here two sea anemone transcripts appear to be insulin-related peptide orthologues that appear to be closer to IGFs from lower vertebrates than to vertebrate or invertebrate ILPs in phylogenetic analysis (Fig. 16A). Multiple alignment showed that, in spite of their low identity scores (15–23%), their organization is very similar to that shared by ILPs and IGFs (Smit et al., 1998), including the N-terminal signal sequence, the B and A chains separated by the DR/SR cleavage sites and the conserved motif sequences CGxxL in B chain and CCxxxC in A chain (Fig. 16C). Insulin and IGF receptors are closely related members of the tyrosine-kinase receptor superfamily. Three putative insulin-related receptors were identified in the sea anemone genome (Table 3) which showed surprisingly low residue identity scores against the Hydra orthologue (13–22%). A tree was constructed suggesting that the cnidarian receptors (Hydra and sea anemone) form an outgroup in relation to a broad range of vertebrate and invertebrate insulin receptors

clustered with vertebrate IGF receptors (Fig. 16B). The general structure of the ILP/IGF receptors, including the leucine-rich repeats, the α and β chains, the single transmembrane domain and the tyrosine kinase domains, is preserved in the sea anemone receptors. Residues involved in formation of the receptor pocket for ligand interaction in IGF, but not in insulin, receptors (Garrett et al., 1998) are largely shared by one of the sea anemone orthologues (Fig. 16D). 3.8.8. Glycoprotein hormone-related receptors Vertebrate gonadotropins and thyrotropin are glycoprotein hormones that affect the differentiation and growth of gonads and the thyroid gland by binding to leucine-rich repeat-containing GPCRs (LGRs) (Pierce and Parsons, 1981; Braun et al., 1991; Van Loy et al., 2008). Although LGRs were cloned and characterized in a snail (Tensen et al.,1994), the fruitfly (Hauser et al.,1997) and the nematode C. elegans (Kudo et al., 2000), no natural ligand has been found in these invertebrates. Similarly, a LGR was cloned in the sea anemone A. elegantissima which shows extensive similarity to the mammalian LGRs (Nothacker and Grimmelikhuijzen, 1993), but no ligand has been identified. The localization of invertebrate LGR expression is unknown except in the snail where LGR expression was found in CNS neurons (Tensen et al., 1994). In the starlet sea anemone seven LGRs were identified in the genome (Table 3) but no ligand resembling vertebrate gonadotropins or thyrotropin was found. In tree analysis one sea anemone transcript clustered with thyrotropin and gonadotropin receptors and another with invertebrate glycoptotein receptors (Fig. 17A). Other transcripts failed to cluster with any of the receptor subclasses. Multiple alignments reveal that the structural features of LGRs are shared by the sea anemone

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Fig. 17. Relationship of glycoprotein-like receptors of N. vectensis. (A) Rooted NJ tree showing that two sea anemone sequences are nested either with vertebrate or invertebrate glycoprotein receptors whereas others fall outside of all receptor subclasses. (B) Alignment of sea anemone sequences with various glycoprotein receptors. The represented sequence segment overlaps the sixth transmembrane region and third extracellular loop. A classspecific motif for the different glycoprotein hormone receptors (+) and residues important for ligand pocket formation (letters) are shown.

receptors, such as the large N-terminal extracellular ectodomain involved in ligand binding and composed of tandem suites of leucinerich repeat motifs, and the 7-transmembrane domain typical of GPCRs at the C-terminal region (not shown). Residues important for ligand pocket formation (Moyle et al., 2004; Vassart et al., 2004) are conserved in some of the sea anemone sequences (Fig. 17B). Some of the sequences share, along with the glycoprotein hormone receptor of A. elegantissima, a conserved motif with fruitfly and sea urchin homologs (Fig. 17B). The transcript Nv_80429 shared 45% residue identity with the sea anemone A. elegantissima glycoprotein hormone, and Nv_204412 shared 37–40% with sea star and shrimp orthologues. 3.9. Evolutionary and functional implications Although this genomic survey predicts the occurrence in the starlet sea anemone of many of the sets of transmitter systems available to higher metazoans, it also reveals some unexpected findings. The major surprises are the presence of numerous gene transcripts related to cholinergic function but the absence of metabotropic acetylcholine receptors, the paradox between the dearth of specific biogenic aminesynthesizing enzymes and the large number of aminergic receptors, an indolaminergic system based on melatonin but lacking specific serotonergic contribution, and the greater diversity of represented

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neuropeptide families than anticipated from known cnidarian peptides previously identified by extraction and purification. The predicted presence of nicotinic, but not muscarinic, receptors suggests that acetylcholine or an acetylcholine-like substance functions only as a fast-acting transmitter by gating ion channels in sea anemones. Acetylcholine, while having no electrophysiological effect on jellyfish motor neurons (Spencer, 1989), induces muscle contractions in sea anemones (Mendes and Freitas, 1984), thus suggesting that it acts as a transmitter at neuromuscular junctions. It would be interesting to examine whether acetylcholine is released by sea anemone motor neurons and acts rapidly on individual muscle cells through nicotinic-like receptors. Whatever functional role acetylcholine may have, the range of transcripts associated with amino acid transmitters strongly indicate that the latter are more heavily involved in controlling activities in sea anemones than acetylcholine. In this cnidarians may not differ from other invertebrates. The failure to find transcripts for specific rate-limiting enzymes involved in the synthesis of catecholamines and serotonin suggests that if conventional biogenic amines are produced by sea anemones, their output must be sporadic and their biosynthetic pathways circuitous. Alternately, amine derivatives unique to cnidarians may be produced by hitherto undiscovered enzymes and act on receptors selective for them. The sea anemone genome includes a large number of aminergic-like receptors that defy inclusion within the vertebrate classification schemes. For example, none of the non-melatonin receptors could be unambiguously identified as dopamine, noradrenaline, serotonin or histamine receptors. This is consistent with earlier reports of cloned aminergic-like receptors in the sea pansy which, when expressed, failed to be activated by a wide range of aminergic compounds (Bouchard et al., 2003, 2004). The sum of evidence strongly suggests that aminergic-like transmitters unique to sea anemones act on a wide range of receptors to effect diverse biological responses. The large number of transcripts related to melatonin function is puzzling in view of the scarcity of literature on melatonin in invertebrates (Vivien-Roels and Pévet, 1993). Because many anthozoans harbor dinoflagellate symbionts and dinoflagellates are known to produce melatonin, the possibility arises that the presence of melatonin in the host is the result of interspecific exchange (Hardeland and Poeggeler, 2003). However, the specific presence of immunoreactive melatonin in sea pansy neurons (Mechawar and Anctil, 1997) and the absence of symbionts in the scarlet sea anemone argue against this view. The three isoforms of HIOMT in the genome and the three ESTs related to HIOMT strongly suggest that HIOMTs are expressed and melatonin is produced by sea anemones. On the other hand, why are there so many melatoninlike receptors? It is striking that these receptors are unambiguously matched with vertebrate melatonin receptors in BLAST alignments at the exclusion of other aminergic receptors, whereas no clear ligand candidate can be deduced for the other aminergic receptors, including the precursor of melatonin formation, serotonin. This suggests that melatonin as a transmitter and its receptors preceded the emergence of serotoninergic and other conventional aminergic systems in early metazoan evolution. While the large number of melatonin-like receptors may reflect melatonin's involvement in diverse biological activities of sea anemones, they may also serve as melatonin-binding proteins to stabilize melatonin for a role as anti-oxidant. No day–night rhythm of melatonin production was detected in the sea pansy (Mechawar and Anctil, 1997) and it has been proposed that the role of melatonin as an antioxidant preceded its involvement in circadian time-keeping early in evolution (Hardeland and Poeggeler, 2003). While 110 transcripts in the genome are associated with peptidergic transmitters, they are outnumbered by non-peptidergic transcripts (167). This may reflect the functional importance of nonpeptidergic transmitters in cnidarians, but as only a few of these transmitter candidates so far were demonstrated to be released from neurons, peptides still appear to be the dominant neurotransmitters. So far, of all the known vertebrate and invertebrate neuropeptide families, only

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RFamide-related peptides were identified in cnidarians. The other identified cnidarian peptide families possess carboxyterminal signatures that are unique to the phylum (Grimmelikhuijzen et al., 2004). While this survey of the starlet sea anemone genome confirms the existence of these peptides, it reveals also the existence in the sea anemone of peptides related to a wide range of vertebrate peptide families. Therefore, while it appears that some peptide families (RIamide, RNamide, RPamide, RWamide and LWamide) turned out to be evolutionary dead-ends, many others (related to galanins, tachykinins, GnRHs, vasopressins, melanocortins, insulins and glycoprotein hormones) had already emerged in various guises in cnidarians and radiated in higher metazoans. Despite the importance of neuropeptides in these animals, no peptidergic receptor has yet been characterized in cnidarians. This survey reveals the presence in the sea anemone genome of numerous receptor transcripts predicted to bind every class of putative peptide ligands belonging to known peptide families. It is particularly striking that RFamide-, tachykinin- and melanocortin-related receptors numerically outmatch their potential ligands (Table 3), suggesting the potential diversity of peptidergic signaling in sea anemones. Numerous other neuropeptide receptors are unclassifiable and may add to the diversity of signaling systems in cnidarians. There are many uniquely cnidarian neuropeptides the receptors of which are unknown, and even more neuropeptide receptors for which there is no clue on the nature of their ligands. A major research program will be required to match them together. When surveying phylogenetic trees constructed from the sea anemone transmitter-related transcripts it becomes clear that in most cases the latter form compact outgroups to other invertebrate and/or vertebrate orthologues. This is expected as sea anemones are considered to be representatives of the sister group to bilateral animals. Invertebrate orthologues are also expected to be closer to the corresponding sea anemone sequences than vertebrate counterparts, but in fact many sea anemone transmitter-related protein classes appear to be closer to vertebrate than to invertebrate counterparts. This is especially true of glutamate NMDA and metabotropic receptors, GABA ionotropic receptors, excitatory amino acid transporters, aminergic receptors, adenosine and melanocortin (MECA) receptors, RFamide-related and galanin receptors, and insulin-related growth factors. In these cases only protochordate and echinoderm sequences among invertebrates share the closeness with vertebrate sequences, thus suggesting homology of sea anemone sequences predominantly with deuterostome counterparts. This probably reflects gene loss by protostome descendants of the common ancestors of cnidarians and bilaterians, such as nematodes and arthropods (Technau et al., 2005), rather than any special evolutionary closeness between cnidarians and deuterostomes. Functionally important domains, motifs and residues of the predicted proteins also reflect the phylogenetic distance between sea anemones and higher metazoans. A leitmotiv of this survey is the limited extent to which these key sequences and residues are preserved in sea anemones. The best cases of preservation for some of the transcript isoforms are found in ionotropic receptors (nicotinic, GABAergic, P2X), amino acid transporters; to a lesser extent, DOPA decarboxylase (dopamine formation) and monoamine transporters, RFamide-related receptors, insulin growth factor-like receptors and leucine-repeat GPCRs. The highly conserved residues of ionotropic receptors and transporters may reflect severe constraints on membrane protein configurations associated with transmembrane molecular transport. In the case of the other sea anemone sequences where the usual vertebrate-like signatures are incomplete to large extents, it will remain to be investigated whether these sequences represent unique classes of cnidarian proteins with different substrate– or ligand–protein interactions from those known to exist in higher invertebrates and in vertebrates. The findings of this genomic survey of sea anemone transmitter systems suggest that chemical transmission was already complex in cnidarians, in agreement with a survey showing the diversity of developmental signaling genes in anthozoans (Technau et al., 2005).

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