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.
Neuronal secretion of peptides that act as intercellular signaling molecules (neuropeptides) is an evolutionarily ancient characteristic of nervous systems, which is reflected in the diversity of neuropeptides that have been discovered in bilaterian and non-bilaterian phyla. Furthermore, evidence of a pre-metazoan origin of neuropeptide signaling has been reported. Neuropeptides are derived from larger precursor proteins that have an N-terminal signal peptide, which targets these molecules to the lumen of the endoplasmic reticulum for secretion. The precursor proteins can comprise one or more neuropeptides, which are bounded by monobasic or dibasic cleavage sites. Furthermore, during neuropeptide precursor processing post-translational modifications of neuropeptides can occur, which include most commonly the conversion of a C-terminal glycine residue to an amide group that is protective against carboxypeptidases. Other post-translational modifications of neuropeptides include conversion of an N-terminal glutamine to pyroglutamate, which is protective against aminopeptidases, tyrosine sulfation and intramolecular and/or intermolecular formation of disulphide bridges between cysteine residues. Neuropeptides typically exert effects on other cells by binding to specific G-protein coupled receptors (GPCRs), acting locally as modulators of synaptic transmission and/or systemically as hormones. The actions of neuropeptides at a cellular level then manifest at the organ/organismal level to cause changes in physiological processes and/or behavior. Thus, neuropeptides are important regulators of, for example, feeding and digestion, osmoregulation, growth, locomotor activity and reproductive processes.
Investigation of the evolution of neuropeptide signaling systems has been greatly facilitated by transcriptome/genome sequencing. Initially, this was restricted to widely studied “model” species such as humans, mice, the nematode Caenorhabditis elegans and the insect Drosophila melanogaster, with comparison and functional characterization of neuropeptide signaling systems in these species providing key insights into neuropeptide relationships and the evolutionary origins of different neuropeptide types. However, as transcriptome/genome sequencing has been applied to an ever-growing wider variety of animal taxa, important new insights into neuropeptide evolution have been obtained. One of the animal phyla that has been important for reconstruction of the evolutionary history of neuropeptide signaling systems is the phylum Echinodermata (e.g., starfish, brittle stars, sea urchins, sea cucumbers, feather stars). As ambulacrarian deuterostomes, echinoderms, together with hemichordates, are positioned in a sister clade to the phylum Chordata (vertebrates, urochordates, and cephalochordates) and therefore they provide a key evolutionary link between research on neuropeptide systems in protostome invertebrates (e.g., arthropods, nematodes, mollusks, and annelids) and vertebrates. Visualization of neuropeptide expression in echinoderm nervous systems was first enabled by use of antibodies to neuropeptides discovered in other phyla (e.g., the molluscan cardioactive peptide FMRFamide). More recently, insights into the neuropeptide repertoire of echinoderms were enabled by sequencing of the transcriptome/genome of the sea urchin Strongylocentrotus purpuratus (class Echinoidea). Subsequently, analysis of transcriptome/genome sequence data has enabled discovery of neuropeptide precursor genes in other echinoderms, including sea cucumbers (class Holothuroidea), starfish (class Asteroidea), and brittle stars (class Ophiuroidea). Furthermore, molecular characterization of neuropeptide signaling systems in selected echinoderm species has provided important insights into neuropeptide relationships and neuropeptide evolution. For example, the discovery of corazonin-type signaling in the starfish Asterias rubens and in other echinoderms revealed the urbilaterian origin of a neuropeptide system that hitherto had been thought to be unique to arthropods or protostomes. Similarly, the discovery of both somatostatin-type and allatostatin-C-type neuropeptides in A. rubens and other echinoderms revealed that neuropeptides that hitherto were thought to be orthologs are in fact paralogs. Additionally, functional characterization of neuropeptides in A. rubens and other echinoderms has revealed evolutionarily ancient and conserved roles in regulation of, for example, feeding processes and reproduction.
Whilst much has been learnt in recent years about neuropeptide signaling systems in echinoderms, there remains one echinoderm class that has received little attention—the Crinoidea (feather stars and sea lilies). Crinoids are of particular interest for evolutionary studies on echinoderms because they are a sister clade to the sub-phylum Eleutherozoa, which comprises the Asterozoa (Asteroidea and Ophiuroidea) and Echinozoa (Echinoidea and Holothuroidea). Therefore, comparative analysis of crinoids and eleutherozoans may facilitate reconstruction of the neuropeptide systems that existed in the common ancestor of extant echinoderms. Furthermore, anatomical studies have investigated crinoid neuroarchitecture and revealed homology between different regions of crinoid and eleutherozoan nervous systems. In the lecithotrophic doliolaria larvae of feather stars, the nervous system comprises an anterior apical organ and a diffuse basi-epidermal nerve plexus. The larval nervous system degenerates at metamorphosis and new neural cell populations develop in the post-metamorphic pentacrinoid stage. On the oral surface neurons are found associated with the tube feet, while on the aboral surface the aboral nerve center (ANC) forms at the base of the calyx. The ANC projects aborally into the stalk, forming the stalk nerve, and orally into five thin nerves. In the adult, the oral nervous system comprises ectoneural and hyponeural systems that are homologous to the corresponding components in eleutherozoans. A third conspicuous component of the nervous system in crinoids is the aboral entoneural system, which comprise
