Sea Anemone Nematostella Vectensis Research Articles

Cellular pathways during spawning induction in the starlet sea anemone Nematostella vectensis

In cnidarians, long-term ecological success relies on sexual reproduction. The sea anemone Nematostella vectensis, which has emerged as an important model organism for developmental studies, can be induced for spawning by temperature elevation and light exposure. To uncover molecular mechanisms and pathways underlying spawning, we characterized the transcriptome of Nematostella females before and during spawning induction. We identified an array of processes involving numerous receptors, circadian clock components, cytoskeleton, and extracellular transcripts that are upregulated upon spawning induction. Concurrently, processes related to the cell cycle, fatty acid metabolism, and other housekeeping functions are downregulated. Real-time qPCR revealed that light exposure has a minor effect on expression levels of most examined transcripts, implying that temperature change is a stronger inducer for spawning in Nematostella. Our findings reveal the potential mechanisms that may enable the mesenteries to serve as a gonad-like tissue for the developing oocytes and expand our understanding of sexual reproduction in cnidarians.

Functional Characterization of Hexacorallia Phagocytic Cells.

Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore a pillar of innate immunity, whereby foreign particles are engulfed and degraded in lysolitic vesicles. In hexacorallians, phagocytic mechanisms are poorly understood, though putative anthozoan phagocytic cells (amoebocytes) have been identified histologically. We identify and characterize phagocytes from the coral Pocillopora damicornis and the sea anemone Nematostella vectensis. Using fluorescence-activated cell sorting and microscopy, we show that distinct populations of phagocytic cells engulf bacteria, fungal antigens, and beads. In addition to pathogenic antigens, we show that phagocytic cells engulf self, damaged cells. We show that target antigens localize to low pH phagolysosomes, and that degradation is occurring within them. Inhibiting actin filament rearrangement interferes with efficient particle phagocytosis but does not affect small molecule pinocytosis. We also demonstrate that cellular markers for lysolitic vesicles and reactive oxygen species (ROS) correlate with hexacorallian phagocytes. These results establish a foundation for improving our understanding of hexacorallian immune cell biology.

The Tentacular Spectacular: Evolution of Regeneration in Sea Anemones.

Sea anemones vary immensely in life history strategies, environmental niches and their ability to regenerate. While the sea anemone Nematostella vectensis is the starlet of many key regeneration studies, recent work is emerging on the diverse regeneration strategies employed by other sea anemones. This manuscript will explore current molecular mechanisms of regeneration employed by non-model sea anemones Exaiptasia diaphana (an emerging model species for coral symbiosis studies) and Calliactis polypus (a less well-studied species) and examine how these species compare to the model sea anemone N. vectensis. We summarize the field of regeneration within sea anemones, within the greater context of phylum Cnidaria and in other invertebrate models of regeneration. We also address the current knowledge on two key systems that may be implemented in regeneration: the innate immune system and developmental pathways, including future aspects of work and current limitations.

Characterization of the dynamics and variability of neuronal subtype responses during growth, degrowth, and regeneration of Nematostella vectensis

BackgroundThe ability to regenerate body parts is a feature of metazoan organisms and the focus of intense research aiming to understand its basis. A number of mechanisms involved in regeneration, such as proliferation and tissue remodeling, affect whole tissues; however, little is known on how distinctively different constituent cell types respond to the dynamics of regenerating tissues. Preliminary studies suggest that a number of organisms alter neuronal numbers to scale with changes in body size. In some species with the ability of whole-body axis regeneration, it has additionally been observed that regenerates are smaller than their pre-amputated parent, but maintain the correct morphological proportionality, suggesting that scaling of tissue and neuronal numbers also occurs. However, the cell dynamics and responses of neuronal subtypes during nervous system regeneration, scaling, and whole-body axis regeneration are not well understood in any system. The cnidarian sea anemone Nematostella vectensis is capable of whole-body axis regeneration, with a number of observations suggesting the ability to alter its size in response to changes in feeding. We took advantage of Nematostella’s transparent and “simple” body plan and the NvLWamide-like mCherry fluorescent reporter transgenic line to probe the response of neuron populations to variations in body size in vivo in adult animals during body scaling and regeneration.ResultsWe utilized the previously characterized NvLWamide-like::mCherry transgenic reporter line to determine the in vivo response of neuronal subtypes during growth, degrowth, and regeneration. Nematostella alters its size in response to caloric intake, and the nervous system responds by altering neuronal number to scale as the animal changes in size. Neuronal numbers in both the endodermal and ectodermal nerve nets decreased as animals shrunk, increased as they grew, and these changes were reversible. Whole-body axis regeneration resulted in regenerates that were smaller than their pre-amputated size, and the regenerated nerve nets were reduced in neuronal number. Different neuronal subtypes had distinct responses during regeneration, including consistent, not consistent, and conditional increases in number. Conditional responses were regulated, in part, by the size of the remnant fragment and the position of the amputation site. Regenerates and adults with reduced nerve nets displayed normal behaviors, indicating that the nerve net retains functionality as it scales.ConclusionThese data suggest that the Nematostella nerve net is dynamic, capable of scaling with changes in body size, and that neuronal subtypes display differential regenerative responses, which we propose may be linked to the scale state of the regenerating animals.

Unravelling the developmental and functional significance of an ancient Argonaute duplication

MicroRNAs (miRNAs) base-pair to messenger RNA targets and guide Argonaute proteins to mediate their silencing. This target regulation is considered crucial for animal physiology and development. However, this notion is based exclusively on studies in bilaterians, which comprise almost all lab model animals. To fill this phylogenetic gap, we characterize the functions of two Argonaute paralogs in the sea anemone Nematostella vectensis of the phylum Cnidaria, which is separated from bilaterians by ~600 million years. Using genetic manipulations, Argonaute-immunoprecipitations and high-throughput sequencing, we provide experimental evidence for the developmental importance of miRNAs in a non-bilaterian animal. Additionally, we uncover unexpected differential distribution of distinct miRNAs between the two Argonautes and the ability of one of them to load additional types of small RNAs. This enables us to postulate a novel model for evolution of miRNA precursors in sea anemones and their relatives, revealing alternative trajectories for metazoan miRNA evolution.

Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis.

The metabotropic gamma-aminobutyric acid B receptor (GABABR) is a G protein-coupled receptor that mediates neuronal inhibition by the neurotransmitter GABA. While GABABR-mediated signalling has been suggested to play central roles in neuronal differentiation and proliferation across evolution, it has mostly been studied in the mammalian brain. Here, we demonstrate that ectopic activation of GABABR signalling affects neurogenic functions in the sea anemone Nematostella vectensis. We identified four putative Nematostella GABABR homologues presenting conserved three-dimensional extracellular domains and residues needed for binding GABA and the GABABR agonist baclofen. Moreover, sustained activation of GABABR signalling reversibly arrests the critical metamorphosis transition from planktonic larva to sessile polyp life stage. To understand the processes that underlie the developmental arrest, we combined transcriptomic and spatial analyses of control and baclofen-treated larvae. Our findings reveal that the cnidarian neurogenic programme is arrested following the addition of baclofen to developing larvae. Specifically, neuron development and neurite extension were inhibited, resulting in an underdeveloped and less organized nervous system and downregulation of proneural factors including NvSoxB(2), NvNeuroD1 and NvElav1. Our results thus point to an evolutionarily conserved function of GABABR in neurogenesis regulation and shed light on early cnidarian development.

Transcriptomic Analysis in the Sea Anemone Nematostella vectensis.

The sea anemone Nematostella vectensis is an emerging research model to study embryonic development and regeneration at the molecular and global transcriptomic level. Transcriptomics analysis is now routinely used to detect differential expression at the genome level. Here we present the latest procedures for isolating high-quality RNA required for next generation sequencing, as well as methods and resources for quantifying transcriptomic data.

Experimental Tools to Study Regeneration in the Sea Anemone Nematostella vectensis.

Animal regeneration is a biological process leading to the reformation of injured or lost tissues/body parts. One of the most fascinating regenerative phenomena is the so-called whole-body regeneration, leading to the reformation of fully functional organisms within days after bisection. The sea anemone Nematostella vectensis is currently emerging as novel whole-body regeneration model. Here we describe the methods of inducing the regenerative process in this cnidarian as well as the fixation and staining protocols for morphological, molecular, and cellular analysis.

Toxin-like neuropeptides in the sea anemone Nematostella unravel recruitment from the nervous system to venom

The sea anemone Nematostella vectensis (Anthozoa, Cnidaria) is a powerful model for characterizing the evolution of genes functioning in venom and nervous systems. Although venom has evolved independently numerous times in animals, the evolutionary origin of many toxins remains unknown. In this work, we pinpoint an ancestral gene giving rise to a new toxin and functionally characterize both genes in the same species. Thus, we report a case of protein recruitment from the cnidarian nervous to venom system. The ShK-like1 peptide has a ShKT cysteine motif, is lethal for fish larvae and packaged into nematocysts, the cnidarian venom-producing stinging capsules. Thus, ShK-like1 is a toxic venom component. Its paralog, ShK-like2, is a neuropeptide localized to neurons and is involved in development. Both peptides exhibit similarities in their functional activities: They provoke contraction in Nematostella polyps and are toxic to fish. Because ShK-like2 but not ShK-like1 is conserved throughout sea anemone phylogeny, we conclude that the two paralogs originated due to a Nematostella-specific duplication of a ShK-like2 ancestor, a neuropeptide-encoding gene, followed by diversification and partial functional specialization. ShK-like2 is represented by two gene isoforms controlled by alternative promoters conferring regulatory flexibility throughout development. Additionally, we characterized the expression patterns of four other peptides with structural similarities to studied venom components and revealed their unexpected neuronal localization. Thus, we employed genomics, transcriptomics, and functional approaches to reveal one venom component, five neuropeptides with two different cysteine motifs, and an evolutionary pathway from nervous to venom system in Cnidaria.

Hedgehog signaling is required for endomesodermal patterning and germ cell development in the sea anemone Nematostella vectensis.

Two distinct mechanisms for primordial germ cell (PGC) specification are observed within Bilatera: early determination by maternal factors or late induction by zygotic cues. Here we investigate the molecular basis for PGC specification in Nematostella, a representative pre-bilaterian animal where PGCs arise as paired endomesodermal cell clusters during early development. We first present evidence that the putative PGCs delaminate from the endomesoderm upon feeding, migrate into the gonad primordia, and mature into germ cells. We then show that the PGC clusters arise at the interface between hedgehog1 and patched domains in the developing mesenteries and use gene knockdown, knockout and inhibitor experiments to demonstrate that Hh signaling is required for both PGC specification and general endomesodermal patterning. These results provide evidence that the Nematostella germline is specified by inductive signals rather than maternal factors, and support the existence of zygotically-induced PGCs in the eumetazoan common ancestor.

Some like it hot: population-specific adaptations in venom production to abiotic stressors in a widely distributed cnidarian

BackgroundIn cnidarians, antagonistic interactions with predators and prey are mediated by their venom, whose synthesis may be metabolically expensive. The potentially high cost of venom production has been hypothesized to drive population-specific variation in venom expression due to differences in abiotic conditions. However, the effects of environmental factors on venom production have been rarely demonstrated in animals. Here, we explore the impact of specific abiotic stresses on venom production of distinct populations of the sea anemone Nematostella vectensis (Actiniaria, Cnidaria) inhabiting estuaries over a broad geographic range where environmental conditions such as temperatures and salinity vary widely.ResultsWe challenged Nematostella polyps with heat, salinity, UV light stressors, and a combination of all three factors to determine how abiotic stressors impact toxin expression for individuals collected across this species’ range. Transcriptomics and proteomics revealed that the highly abundant toxin Nv1 was the most downregulated gene under heat stress conditions in multiple populations. Physiological measurements demonstrated that venom is metabolically costly to produce. Strikingly, under a range of abiotic stressors, individuals from different geographic locations along this latitudinal cline modulate differently their venom production levels.ConclusionsWe demonstrate that abiotic stress results in venom regulation in Nematostella. Together with anecdotal observations from other cnidarian species, our results suggest this might be a universal phenomenon in Cnidaria. The decrease in venom production under stress conditions across species coupled with the evidence for its high metabolic cost in Nematostella suggests downregulation of venom production under certain conditions may be highly advantageous and adaptive. Furthermore, our results point towards local adaptation of this mechanism in Nematostella populations along a latitudinal cline, possibly resulting from distinct genetics and significant environmental differences between their habitats.

Feeding-dependent tentacle development in the sea anemone Nematostella vectensis

In cnidarians, axial patterning is not restricted to embryogenesis but continues throughout a prolonged life history filled with unpredictable environmental changes. How this developmental capacity copes with fluctuations of food availability and whether it recapitulates embryonic mechanisms remain poorly understood. Here we utilize the tentacles of the sea anemone Nematostella vectensis as an experimental paradigm for developmental patterning across distinct life history stages. By analyzing over 1000 growing polyps, we find that tentacle progression is stereotyped and occurs in a feeding-dependent manner. Using a combination of genetic, cellular and molecular approaches, we demonstrate that the crosstalk between Target of Rapamycin (TOR) and Fibroblast growth factor receptor b (Fgfrb) signaling in ring muscles defines tentacle primordia in fed polyps. Interestingly, Fgfrb-dependent polarized growth is observed in polyp but not embryonic tentacle primordia. These findings show an unexpected plasticity of tentacle development, and link post-embryonic body patterning with food availability.

The genetic basis for PRC1 complex diversity emerged early in animal evolution

Polycomb group proteins are essential regulators of developmental processes across animals. Despite their importance, studies on Polycomb are often restricted to classical model systems and, as such, little is known about the evolution of these important chromatin regulators. Here we focus on Polycomb Repressive Complex 1 (PRC1) and trace the evolution of core components of canonical and non-canonical PRC1 complexes in animals. Previous work suggested that a major expansion in the number of PRC1 complexes occurred in the vertebrate lineage. We show that the expansion of the Polycomb Group RING Finger (PCGF) protein family, an essential step for the establishment of the large diversity of PRC1 complexes found in vertebrates, predates the bilaterian-cnidarian ancestor. This means that the genetic repertoire necessary to form all major vertebrate PRC1 complexes emerged early in animal evolution, over 550 million years ago. We further show that PCGF5, a gene conserved in cnidarians and vertebrates but lost in all other studied groups, is expressed in the nervous system in the sea anemone Nematostella vectensis, similar to its mammalian counterpart. Together this work provides a framework for understanding the evolution of PRC1 complex diversity and it establishes Nematostella as a promising model system in which the functional ramifications of this diversification can be further explored.

Decoupling behavioral and transcriptional responses to color in an eyeless cnidarian

BackgroundAnimals have specific molecular, physiological, and behavioral responses to light that are influenced by wavelength and intensity. Predictable environmental changes – predominantly solar and lunar cycles – drive endogenous daily oscillations by setting internal pacemakers, otherwise known as the circadian clock. Cnidarians have been a focal group to discern the evolution of light responsiveness due to their phylogenetic position as a sister phylum to bilaterians and broad range of light-responsive behaviors and physiology. Marine species that occupy a range of depths will experience different ranges of wavelengths and light intensities, which may result in variable phenotypic responses. Here, we utilize the eyeless sea anemone Nematostella vectensis, an estuarine anemone that typically resides in shallow water habitats, to compare behavioral and molecular responses when exposed to different light conditions.ResultsQuantitative measures of locomotion clearly showed that this species responds to light in the blue and green spectral range with a circadian activity profile, in contrast to a circatidal activity profile in the red spectral range and in constant darkness. Differences in average day/night locomotion was significant in each condition, with overall peak activity during the dark period. Comparative analyses of 96 transcriptomes from individuals sampled every 4 h in each lighting treatment revealed complex differences in gene expression between colors, including in many of the genes likely involved in the cnidarian circadian clock. Transcriptional profiling showed the majority of genes are differentially expressed when comparing mid-day with mid-night, and mostly in red light. Gene expression profiles were largely unique in each color, although animals in blue and green were overall more similar to each other than to red light.ConclusionsTogether, these analyses support the hypothesis that cnidarians are sensitive to red light, and this perception results in a rich transcriptional and divergent behavioral response. Future work determining the specific molecular mechanisms driving the circadian and potential circatidal rhythms measured here would be impactful to connect gene expression variation with behavioral variation in this eyeless species.

Selective profiling of N- and C-terminal nucleotide-binding sites in a TRPM2 channel.

Transient receptor potential melastatin 2 (TRPM2) is a homotetrameric Ca2+-permeable cation channel important for the immune response, body temperature regulation, and insulin secretion, and is activated by cytosolic Ca2+ and ADP ribose (ADPR). ADPR binds to two distinct locations, formed by large N- and C-terminal cytosolic domains, respectively, of the channel protein. In invertebrate TRPM2 channels, the C-terminal site is not required for channel activity but acts as an active ADPR phosphohydrolase that cleaves the activating ligand. In vertebrate TRPM2 channels, the C-terminal site is catalytically inactive but cooperates with the N-terminal site in channel activation. The precise functional contributions to channel gating and the nucleotide selectivities of the two sites in various species have not yet been deciphered. For TRPM2 of the sea anemone Nematostella vectensis (nvTRPM2), catalytic activity is solely attributable to the C-terminal site. Here, we show that nvTRPM2 channel gating properties remain unaltered upon deletion of the C-terminal domain, indicating that the N-terminal site is single-handedly responsible for channel gating. Exploiting such functional independence of the N- and C-terminal sites, we selectively measure their affinity profiles for a series of ADPR analogues, as reflected by apparent affinities for channel activation and catalysis, respectively. Using site-directed mutagenesis, we confirm that the same N-terminal site observed in vertebrate TRPM2 channels was already present in ancient cnidarians. Finally, by characterizing the functional effects of six amino acid side chain truncations in the N-terminal site, we provide first insights into the mechanistic contributions of those side chains to TRPM2 channel gating.

NvPOU4/Brain3 Functions as a Terminal Selector Gene in the Nervous System of the Cnidarian Nematostella vectensis

Terminal selectors are transcription factors that control the morphological, physiological, and molecular features that characterize distinct cell types. Here, we show that, in the sea anemone Nematostella vectensis, NvPOU4 is expressed in post-mitotic cells that give rise to a diverse set of neural cell types, including cnidocytes and NvElav1-expressing neurons. Morphological analyses of NvPOU4 mutants crossed to transgenic reporter lines show that the loss of NvPOU4 does not affect the initial specification of neural cells. Transcriptomes derived from the mutants and from different neural cell populations reveal that NvPOU4 is required for the execution of the terminal differentiation program of these neural cells. These findings suggest that POU4 genes have ancient functions as terminal selectors for morphologically and functionally disparate types of neurons and they provide experimental support for the relevance of terminal selectors for understanding the evolution of cell types.

Expression Analysis of Cnidarian-Specific Neuropeptides in a Sea Anemone Unveils an Apical-Organ-Associated Nerve Net That Disintegrates at Metamorphosis.

Neuropeptides are ancient neuronal signaling molecules that have diversified across Cnidaria (e.g., jellyfish, corals, and sea anemones) and its sister group Bilateria (e.g., vertebrates, insects, and worms). Over the course of neuropeptide evolution emerged lineage-specific neuropeptides, but their roles in the evolution and diversification of nervous system function remain enigmatic. As a step toward filling in this knowledge gap, we investigated the expression pattern of a cnidarian-specific neuropeptide—RPamide—during the development of the starlet sea anemone Nematostella vectensis, using in situ hybridization and immunohistochemistry. We show that RPamide precursor transcripts first occur during gastrulation in scattered epithelial cells of the aboral ectoderm. These RPamide-positive epithelial cells exhibit a spindle-shaped, sensory-cell-like morphology, and extend basal neuronal processes that form a nerve net in the aboral ectoderm of the free-swimming planula larva. At the aboral end, RPamide-positive sensory cells become integrated into the developing apical organ that forms a bundle of long cilia referred to as the apical tuft. Later during planula development, RPamide expression becomes evident in sensory cells in the oral ectoderm of the body column and pharynx, and in the developing endodermal nervous system. At metamorphosis into a polyp, the RPamide-positive sensory nerve net in the aboral ectoderm degenerates by apoptosis, and RPamide expression begins in ectodermal sensory cells of growing oral tentacles. In addition, we find that the expression pattern of RPamide in planulae differs from that of conserved neuropeptides that are shared across Cnidaria and Bilateria, indicative of distinct functions. Our results not only provide the anatomical framework necessary to analyze the function of the cnidarian-specific neuropeptides in future studies, but also reveal previously unrecognized features of the sea anemone nervous system—the apical organ neurons of the planula larva, and metamorphosis-associated reorganization of the ectodermal nervous system.

Initial Virome Characterization of the Common Cnidarian Lab Model Nematostella vectensis.

The role of viruses in forming a stable holobiont has been the subject of extensive research in recent years. However, many emerging model organisms still lack any data on the composition of the associated viral communities. Here, we re-analyzed seven publicly available transcriptome datasets of the starlet sea anemone Nematostella vectensis, the most commonly used anthozoan lab model, and searched for viral sequences. We applied a straightforward, yet powerful approach of de novo assembly followed by homology-based virus identification and a multi-step, thorough taxonomic validation. The comparison of different lab populations of N. vectensis revealed the existence of the core virome composed of 21 viral sequences, present in all adult datasets. Unexpectedly, we observed an almost complete lack of viruses in the samples from the early developmental stages, which together with the identification of the viruses shared with the major source of the food in the lab, the brine shrimp Artemia salina, shed new light on the course of viral species acquisition in N. vectensis. Our study provides an initial, yet comprehensive insight into N. vectensis virome and sets the first foundation for the functional studies of viruses and antiviral systems in this lab model cnidarian.

Dynamic Evolution of the Cthrc1 Genes, a Newly Defined Collagen-Like Family.

Collagen triple helix repeat containing protein 1 (Cthrc1) is a secreted glycoprotein reported to regulate collagen deposition and to be linked to the Transforming growth factor β/Bone morphogenetic protein and the Wnt/planar cell polarity pathways. It was first identified as being induced upon injury to rat arteries and was found to be highly expressed in multiple human cancer types. Here, we explore the phylogenetic and evolutionary trends of this metazoan gene family, previously studied only in vertebrates. We identify Cthrc1 orthologs in two distant cnidarian species, the sea anemone Nematostella vectensis and the hydrozoan Clytia hemisphaerica, both of which harbor multiple copies of this gene. We find that Cthrc1 clade-specific diversification occurred multiple times in cnidarians as well as in most metazoan clades where we detected this gene. Many other groups, such as arthropods and nematodes, have entirely lost this gene family. Most vertebrates display a single highly conserved gene, and we show that the sequence evolutionary rate of Cthrc1 drastically decreased within the gnathostome lineage. Interestingly, this reduction coincided with the origin of its conserved upstream neighboring gene, Frizzled 6 (FZD6), which in mice has been shown to functionally interact with Cthrc1. Structural modeling methods further reveal that the yet uncharacterized C-terminal domain of Cthrc1 is similar in structure to the globular C1q superfamily domain, also found in the C-termini of collagens VIII and X. Thus, our studies show that the Cthrc1 genes are a collagen-like family with a variable short collagen triple helix domain and a highly conserved C-terminal domain structure resembling the C1q family.

Cytoskeletal and synaptic polarity of LWamide-like+ ganglion neurons in the sea anemone Nematostella vectensis.

The centralized nervous systems of bilaterian animals rely on directional signaling facilitated by polarized neurons with specialized axons and dendrites. It is not known whether axo-dendritic polarity is exclusive to bilaterians or was already present in early metazoans. We therefore examined neurite polarity in the starlet sea anemone Nematostella vectensis (Cnidaria). Cnidarians form a sister clade to bilaterians and share many neuronal building blocks characteristic of bilaterians, including channels, receptors and synaptic proteins, but their nervous systems comprise a comparatively simple net distributed throughout the body. We developed a tool kit of fluorescent polarity markers for live imaging analysis of polarity in an identified neuron type, large ganglion cells of the body column nerve net that express the LWamide-like neuropeptide. Microtubule polarity differs in bilaterian axons and dendrites, and this in part underlies polarized distribution of cargo to the two types of processes. However, in LWamide-like+ neurons, all neurites had axon-like microtubule polarity suggesting that they may have similar contents. Indeed, presynaptic and postsynaptic markers trafficked to all neurites and accumulated at varicosities where neurites from different neurons often crossed, suggesting the presence of bidirectional synaptic contacts. Furthermore, we could not identify a diffusion barrier in the plasma membrane of any of the neurites like the axon initial segment barrier that separates the axonal and somatodendritic compartments in bilaterian neurons. We conclude that at least one type of neuron in Nematostella vectensis lacks the axo-dendritic polarity characteristic of bilaterian neurons.