Discovery and functional characterization of neuropeptides in crinoid echinoderms.

Neuropeptides are one of the largest and most diverse families of signaling molecules in animals and, accordingly, they regulate many physiological processes and behaviors. Genome and transcriptome sequencing has enabled the identification of genes encoding neuropeptide precursor proteins in species from a growing variety of taxa, including bilaterian and non-bilaterian animals. Of particular interest are deuterostome invertebrates such as the phylum Echinodermata, which occupies a phylogenetic position that has facilitated reconstruction of the evolution of neuropeptide signaling systems in Bilateria. However, our knowledge of neuropeptide signaling in echinoderms is largely based on bioinformatic and experimental analysis of eleutherozoans-Asterozoa (starfish and brittle stars) and Echinozoa (sea urchins and sea cucumbers). Little is known about neuropeptide signaling in crinoids (feather stars and sea lilies), which are a sister clade to the Eleutherozoa. Therefore, we have analyzed transcriptome/genome sequence data from three feather star species, Anneissia japonica, Antedon mediterranea, and Florometra serratissima, to produce the first comprehensive identification of neuropeptide precursors in crinoids. These include representatives of bilaterian neuropeptide precursor families and several predicted crinoid neuropeptide precursors. Using A. mediterranea as an experimental model, we have investigated the expression of selected neuropeptides in larvae (doliolaria), post-metamorphic pentacrinoids and adults, providing new insights into the cellular architecture of crinoid nervous systems. Thus, using mRNA in situ hybridization F-type SALMFamide precursor transcripts were revealed in a previously undescribed population of peptidergic cells located dorso-laterally in doliolaria. Furthermore, using immunohistochemistry a calcitonin-type neuropeptide was revealed in the aboral nerve center, circumoral nerve ring and oral tube feet in pentacrinoids and in the ectoneural and entoneural compartments of the nervous system in adults. Moreover, functional analysis of a vasopressin/oxytocin-type neuropeptide (crinotocin), which is expressed in the brachial nerve of the arms in A. mediterranea, revealed that this peptide causes a dose-dependent change in the mechanical behavior of arm preparations in vitro-the first reported biological action of a neuropeptide in a crinoid. In conclusion, our findings provide new perspectives on neuropeptide signaling in echinoderms and the foundations for further exploration of neuropeptide expression/function in crinoids as a sister clade to eleutherozoan echinoderms.

Receptor deorphanization in an echinoderm reveals kisspeptin evolution and relationship with SALMFamide neuropeptides.

BACKGROUND: Kisspeptins are neuropeptides that regulate reproductive maturation in mammals via G-protein-coupled receptor-mediated stimulation of gonadotropin-releasing hormone secretion from the hypothalamus. Phylogenetic analysis of kisspeptin-type receptors indicates that this neuropeptide signaling system originated in a common ancestor of the Bilateria, but little is known about kisspeptin signaling in invertebrates. RESULTS: Contrasting with the occurrence of a single kisspeptin receptor in mammalian species, here, we report the discovery of an expanded family of eleven kisspeptin-type receptors in a deuterostome invertebrate - the starfish Asterias rubens (phylum Echinodermata). Furthermore, neuropeptides derived from four precursor proteins were identified as ligands for six of these receptors. One or more kisspeptin-like neuropeptides derived from two precursor proteins (ArKPP1, ArKPP2) act as ligands for four A. rubens kisspeptin-type receptors (ArKPR1,3,8,9). Furthermore, a family of neuropeptides that act as muscle relaxants in echinoderms (SALMFamides) are ligands for two A. rubens kisspeptin-type receptors (ArKPR6,7). The SALMFamide neuropeptide S1 (or ArS1.4) and a 'cocktail' of the seven neuropeptides derived from the S1 precursor protein (ArS1.1-ArS1.7) act as ligands for ArKPR7. The SALMFamide neuropeptide S2 (or ArS2.3) and a 'cocktail' of the eight neuropeptides derived from the S2 precursor protein (ArS2.1-ArS2.8) act as ligands for ArKPR6. CONCLUSIONS: Our findings reveal a remarkable diversity of neuropeptides that act as ligands for kisspeptin-type receptors in starfish and provide important new insights into the evolution of kisspeptin signaling. Furthermore, the discovery of the hitherto unknown relationship of kisspeptins with SALMFamides, neuropeptides that were discovered in starfish prior to the identification of kisspeptins in mammals, presents a radical change in perspective for research on kisspeptin signaling.

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals.

Characterization of NGFFYamide Signaling in Starfish Reveals Roles in Regulation of Feeding Behavior and Locomotory Systems.

Neuropeptides in deuterostomian invertebrates that have an Asn-Gly motif (NG peptides) have been identified as orthologs of vertebrate neuropeptide-S (NPS)-type peptides and protostomian crustacean cardioactive peptide (CCAP)-type neuropeptides. To obtain new insights into the physiological roles of NG peptides in deuterostomian invertebrates, here we have characterized the NG peptide signaling system in an echinoderm-the starfish Asterias rubens. The neuropeptide NGFFYamide was identified as the ligand for an A. rubens NPS/CCAP-type receptor, providing further confirmation that NG peptides are orthologs of NPS/CCAP-type neuropeptides. Using mRNA in situ hybridization, cells expressing the NGFFYamide precursor transcript were revealed in the radial nerve cords, circumoral nerve ring, coelomic epithelium, apical muscle, body wall, stomach, and tube feet of A. rubens, indicating that NGFFYamide may have a variety of physiological roles in starfish. One of the most remarkable aspects of starfish biology is their feeding behavior, where the stomach is everted out of the mouth over the soft tissue of prey. Previously, we reported that NGFFYamide triggers retraction of the everted stomach in A. rubens and here we show that in vivo injection of NGFFYamide causes a significant delay in the onset of feeding on prey. To investigate roles in regulating other aspects of starfish physiology, we examined the in vitro effects of NGFFYamide and found that it causes relaxation of acetylcholine-contracted apical muscle preparations and induction of tonic and phasic contraction of tube feet. Furthermore, analysis of the effects of in vivo injection of NGFFYamide on starfish locomotor activity revealed that it causes a significant reduction in mean velocity and distance traveled. Interestingly, experimental studies on mammals have revealed that NPS is an anxiolytic that suppresses appetite and induces hyperactivity in mammals. Our characterization of the actions of NGFFYamide in starfish indicates that NPS/NG peptide/CCAP-type signaling is an evolutionarily ancient regulator of feeding and locomotion.

The evolution of neuropeptide signalling: insights from echinoderms.

Neuropeptides are evolutionarily ancient mediators of neuronal signalling that regulate a wide range of physiological processes and behaviours in animals. Neuropeptide signalling has been investigated extensively in vertebrates and protostomian invertebrates, which include the ecdysozoans Drosophila melanogaster (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda). However, until recently, an understanding of evolutionary relationships between neuropeptide signalling systems in vertebrates and protostomes has been impaired by a lack of genome/transcriptome sequence data from non-ecdysozoan invertebrates. The echinoderms-a deuterostomian phylum that includes sea urchins, sea cucumbers and starfish-have been particularly important in providing new insights into neuropeptide evolution. Sequencing of the genome of the sea urchin Strongylocentrotus purpuratus (Class Echinoidea) enabled discovery of (i) the first invertebrate thyrotropin-releasing hormone-type precursor, (ii) the first deuterostomian pedal peptide/orcokinin-type precursors and (iii) NG peptides-the 'missing link' between neuropeptide S in tetrapod vertebrates and crustacean cardioactive peptide in protostomes. More recently, sequencing of the neural transcriptome of the starfish Asterias rubens (Class Asteroidea) enabled identification of 40 neuropeptide precursors, including the first kisspeptin and melanin-concentrating hormone-type precursors to be identified outside of the chordates. Furthermore, the characterization of a corazonin-type neuropeptide signalling system in A. rubens has provided important new insights into the evolution of gonadotropin-releasing hormone-related neuropeptides. Looking forward, the discovery of multiple neuropeptide signalling systems in echinoderms provides opportunities to investigate how these systems are used to regulate physiological and behavioural processes in the unique context of a decentralized, pentaradial bauplan.

Localization of Neuropeptide Gene Expression in Larvae of an Echinoderm, the Starfish Asterias rubens.

Neuropeptides are an ancient class of neuronal signaling molecules that regulate a variety of physiological and behavioral processes in animals. The life cycle of many animals includes a larval stage(s) that precedes metamorphic transition to a reproductively active adult stage but, with the exception of Drosophila melanogaster and other insects, research on neuropeptide signaling has hitherto largely focused on adult animals. However, recent advances in genome/transcriptome sequencing have facilitated investigation of neuropeptide expression/function in the larvae of protostomian (e.g., the annelid Platynereis dumerilii) and deuterostomian (e.g., the urochordate Ciona intestinalis) invertebrates. Accordingly, here we report the first multi-gene investigation of larval neuropeptide precursor expression in a species belonging to the phylum Echinodermata-the starfish Asterias rubens. Whole-mount mRNA in situ hybridization was used to visualize in bipinnaria and brachiolaria stage larvae the expression of eight neuropeptide precursors: L-type SALMFamide (S1), F-type SALMFamide (S2), vasopressin/oxytocin-type, NGFFYamide, thyrotropin-releasing hormone-type, gonadotropin-releasing hormone-type, calcitonin-type and corticotropin-releasing hormone-type. Expression of only three of the precursors (S1, S2, NGFFYamide) was observed in bipinnaria larvae but by the brachiolaria stage expression of all eight precursors was detected. An evolutionarily conserved feature of larval nervous systems is the apical organ and in starfish larvae this comprises the bilaterally symmetrical lateral ganglia, but only the S1 and S2 precursors were found to be expressed in these ganglia. A prominent feature of brachiolaria larvae is the attachment complex, comprising the brachia and adhesive disk, which mediates larval attachment to a substratum prior to metamorphosis. Interestingly, all of the neuropeptide precursors examined here are expressed in the attachment complex, with distinctive patterns of expression suggesting potential roles for neuropeptides in the attachment process. Lastly, expression of several neuropeptide precursors is associated with ciliary bands, suggesting potential roles for the neuropeptides derived from these precursors in control of larval locomotion and/or feeding. In conclusion, our findings provide novel perspectives on the evolution and development of neuropeptide signaling systems and neuroanatomical insights into neuropeptide function in echinoderm larvae.

Transcriptomic identification of starfish neuropeptide precursors yields new insights into neuropeptide evolution.

Neuropeptides are evolutionarily ancient mediators of neuronal signalling in nervous systems. With recent advances in genomics/transcriptomics, an increasingly wide range of species has become accessible for molecular analysis. The deuterostomian invertebrates are of particular interest in this regard because they occupy an 'intermediate' position in animal phylogeny, bridging the gap between the well-studied model protostomian invertebrates (e.g. Drosophila melanogaster, Caenorhabditis elegans) and the vertebrates. Here we have identified 40 neuropeptide precursors in the starfish Asterias rubens, a deuterostomian invertebrate from the phylum Echinodermata. Importantly, these include kisspeptin-type and melanin-concentrating hormone-type precursors, which are the first to be discovered in a non-chordate species. Starfish tachykinin-type, somatostatin-type, pigment-dispersing factor-type and corticotropin-releasing hormone-type precursors are the first to be discovered in the echinoderm/ambulacrarian clade of the animal kingdom. Other precursors identified include vasopressin/oxytocin-type, gonadotropin-releasing hormone-type, thyrotropin-releasing hormone-type, calcitonin-type, cholecystokinin/gastrin-type, orexin-type, luqin-type, pedal peptide/orcokinin-type, glycoprotein hormone-type, bursicon-type, relaxin-type and insulin-like growth factor-type precursors. This is the most comprehensive identification of neuropeptide precursor proteins in an echinoderm to date, yielding new insights into the evolution of neuropeptide signalling systems. Furthermore, these data provide a basis for experimental analysis of neuropeptide function in the unique context of the decentralized, pentaradial echinoderm bauplan.

Identification of a neuropeptide precursor protein that gives rise to a "cocktail" of peptides that bind Cu(II) and generate metal-linked dimers.

BACKGROUND: Neuropeptides with an Amino Terminal Cu(II), Ni(II) Binding (ATCUN) motif (H2N-xxH) bind Cu(II)/Ni(II) ions. Here we report the novel discovery of a neuropeptide precursor that gives rise to a "cocktail" of peptides that bind Cu(II)/Ni(II) and form ternary complexes--the L-type SALMFamide precursor in the starfish Asterias rubens. METHODS: Echinoderm transcriptome sequence data were analysed to identify transcripts encoding precursors of SALMFamide-type neuropeptides. The sequence of the L-type SALMFamide precursor in the starfish Asterias rubens was confirmed by cDNA sequencing and peptides derived from this precursor (e.g. AYHSALPF-NH2, GYHSGLPF-NH2 and LHSALPF-NH2) were synthesized. The ability of these peptides to bind metals was investigated using UV/Vis, NMR, circular dichroism and EPR spectroscopy. RESULTS: AYHSALPF-NH2 and GYHSGLPF-NH2 bind Cu(II) and Ni(II) and generate metal-linked dimers to form ternary complexes with LHSALPF-NH2. Investigation of the evolutionary history of the histidine residue that confers these properties revealed that it can be traced to the common ancestor of echinoderms, which is estimated to have lived ~500 million years ago. However, L-type precursors comprising multiple SALMFamides with the histidine residue forming an ATCUN motif appears to be a feature that has evolved uniquely in starfish (Asteroidea). GENERAL SIGNIFICANCE: The discovery of a SALMFamide-type neuropeptide precursor protein that gives rise to a "cocktail" of peptides that bind metal ions and generate metal-linked dimers provides a new insight on ATCUN motif-containing neuropeptides. This property of L-type SALMFamides in the Asteroidea may be associated with a role in regulation of the unusual extra-oral feeding behaviour of starfish.

Phylogeny of Echinoderm Hemoglobins.

BACKGROUND: Recent genomic information has revealed that neuroglobin and cytoglobin are the two principal lineages of vertebrate hemoglobins, with the latter encompassing the familiar myoglobin and α-globin/ß-globin tetramer hemoglobin, and several minor groups. In contrast, very little is known about hemoglobins in echinoderms, a phylum of exclusively marine organisms closely related to vertebrates, beyond the presence of coelomic hemoglobins in sea cucumbers and brittle stars. We identified about 50 hemoglobins in sea urchin, starfish and sea cucumber genomes and transcriptomes, and used Bayesian inference to carry out a molecular phylogenetic analysis of their relationship to vertebrate sequences, specifically, to assess the hypothesis that the neuroglobin and cytoglobin lineages are also present in echinoderms. RESULTS: The genome of the sea urchin Strongylocentrotus purpuratus encodes several hemoglobins, including a unique chimeric 14-domain globin, 2 androglobin isoforms and a unique single androglobin domain protein. Other strongylocentrotid genomes appear to have similar repertoires of globin genes. We carried out molecular phylogenetic analyses of 52 hemoglobins identified in sea urchin, brittle star and sea cucumber genomes and transcriptomes, using different multiple sequence alignment methods coupled with Bayesian and maximum likelihood approaches. The results demonstrate that there are two major globin lineages in echinoderms, which are related to the vertebrate neuroglobin and cytoglobin lineages. Furthermore, the brittle star and sea cucumber coelomic hemoglobins appear to have evolved independently from the cytoglobin lineage, similar to the evolution of erythroid oxygen binding globins in cyclostomes and vertebrates. CONCLUSION: The presence of echinoderm globins related to the vertebrate neuroglobin and cytoglobin lineages suggests that the split between neuroglobins and cytoglobins occurred in the deuterostome ancestor shared by echinoderms and vertebrates.

Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family.

Neuropeptides are ancient regulators of physiology and behaviour, but reconstruction of neuropeptide evolution is often difficult owing to lack of sequence conservation. Here, we report that the receptor for the neuropeptide NGFFFamide in the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) is an orthologue of vertebrate neuropeptide-S (NPS) receptors and crustacean cardioactive peptide (CCAP) receptors. Importantly, this has facilitated reconstruction of the evolution of two bilaterian neuropeptide signalling systems. Genes encoding the precursor of a vasopressin/oxytocin-type neuropeptide and its receptor duplicated in a common ancestor of the Bilateria. One copy of the precursor retained ancestral features, as seen in highly conserved vasopressin/oxytocin-neurophysin-type precursors. The other copy diverged, but this took different courses in protostomes and deuterostomes. In protostomes, the occurrence of a disulfide bridge in neuropeptide product(s) of the precursor was retained, as in CCAP, but with loss of the neurophysin domain. In deuterostomes, we see the opposite scenario-the neuropeptides lost the disulfide bridge, and neurophysin was retained (as in the NGFFFamide precursor) but was subsequently lost in vertebrate NPS precursors. Thus, the sea urchin NGFFFamide precursor and receptor are 'missing links' in the evolutionary history of neuropeptides that control ecdysis in arthropods (CCAP) and regulate anxiety in humans (NPS).

Reconstructing SALMFamide Neuropeptide Precursor Evolution in the Phylum Echinodermata: Ophiuroid and Crinoid Sequence Data Provide New Insights.

The SALMFamides are a family of neuropeptides that act as muscle relaxants in echinoderms. Analysis of genome/transcriptome sequence data from the sea urchin Strongylocentrotus purpuratus (Echinoidea), the sea cucumber Apostichopus japonicus (Holothuroidea), and the starfish Patiria miniata (Asteroidea) reveals that in each species there are two types of SALMFamide precursor: an L-type precursor comprising peptides with a C-terminal LxFamide-type motif and an F-type precursor solely or largely comprising peptides with a C-terminal FxFamide-type motif. Here, we have identified transcripts encoding SALMFamide precursors in the brittle star Ophionotus victoriae (Ophiuroidea) and the feather star Antedon mediterranea (Crinoidea). We have also identified SALMFamide precursors in other species belonging to each of the five echinoderm classes. As in S. purpuratus, A. japonicus, and P. miniata, in O. victoriae there is one L-type precursor and one F-type precursor. However, in A. mediterranea only a single SALMFamide precursor was found, comprising two peptides with a LxFamide-type motif, one with a FxFamide-type motif, five with a FxLamide-type motif, and four with a LxLamide-type motif. As crinoids are basal to the Echinozoa (Holothuroidea + Echinoidea) and Asterozoa (Asteroidea + Ophiuroidea) in echinoderm phylogeny, one model of SALMFamide precursor evolution would be that ancestrally there was a single SALMFamide gene encoding a variety of SALMFamides (as in crinoids), which duplicated in a common ancestor of the Echinozoa and Asterozoa and then specialized to encode L-type SALMFamides or F-type SALMFamides. Alternatively, a second SALMFamide precursor may remain to be discovered or may have been lost in crinoids. Further insights will be obtained if SALMFamide receptors are identified, which would provide a molecular basis for experimental analysis of the functional significance of the "cocktails" of SALMFamides that exist in echinoderms.

Discovery of a novel neurophysin-associated neuropeptide that triggers cardiac stomach contraction and retraction in starfish.

Feeding in starfish is a remarkable process in which the cardiac stomach is everted over prey and then retracted when prey tissue has been resorbed. Previous studies have revealed that SALMFamide-type neuropeptides trigger cardiac stomach relaxation and eversion in the starfish Asterias rubens. We hypothesized, therefore, that a counteracting neuropeptide system controls cardiac stomach contraction and retraction. Members of the NG peptide family cause muscle contraction in other echinoderms (e.g. NGFFFamide in sea urchins and NGIWYamide in sea cucumbers), so we investigated NG peptides as candidate regulators of cardiac stomach retraction in starfish. Generation and analysis of neural transcriptome sequence data from A. rubens revealed a precursor protein comprising two copies of a novel NG peptide, NGFFYamide, which was confirmed by mass spectrometry. A noteworthy feature of the NGFFYamide precursor is a C-terminal neurophysin domain, indicative of a common ancestry with vasopressin/oxytocin-type neuropeptide precursors. Interestingly, in precursors of other NG peptides the neurophysin domain has been retained (e.g. NGFFFamide) or lost (e.g. NGIWYamide and human neuropeptide S) and its functional significance remains to be determined. Investigation of the pharmacological actions of NGFFYamide in starfish revealed that it is a potent stimulator of cardiac stomach contraction in vitro and that it triggers cardiac stomach retraction in vivo. Thus, discovery of NGFFYamide provides a novel insight into neural regulation of cardiac stomach retraction as well as a rationale for chemically based strategies to control starfish that feed on economically important shellfish (e.g. mussels) or protected marine fauna (e.g. coral).