Pulsation waves along the Ciona heart tube reverse by bimodal rhythms expressed by a remote pair of pacemakers.

The heart of ascidians (marine invertebrate chordates) has a tubular structure, and heartbeats propagate from one end to the other. The direction of pulsation waves intermittently reverses in the heart of ascidians and their relatives; however, the underlying mechanisms remain unclear. We herein performed a series of experiments to characterize the pacemaker systems in isolated hearts and their fragments, and applied a mathematical model to examine the conditions leading to heart reversals. The isolated heart of Ciona robusta autonomously generated pulsation waves at ∼20 to 25 beats min-1 with reversals at ∼1 to 10 min intervals. Experimental bisections of isolated hearts revealed that independent pacemakers resided on each side and also that their beating frequencies periodically changed as they expressed bimodal rhythms, which comprised an ∼1.25 to 5.5 min acceleration/deceleration cycle of a beating rate of between 0 and 25 beats min-1. Only fragments including 5% or shorter terminal regions of the heart tube maintained autonomous pulsation rhythms, whereas other regions did not. Our mathematical model, based on FitzHugh-Nagumo equations applied to a one-dimensional alignment of cells, demonstrated that the difference between frequencies expressed by the two independent terminal pacemakers determined the direction of propagated waves. Changes in the statuses of terminal pacemakers between the excitatory and oscillatory modes as well as in their endogenous oscillation frequencies were sufficient to lead to heart reversals. These results suggest that the directions of pulsation waves in the Ciona heart reverse according to the changing rhythms independently expressed by remotely coupled terminal pacemakers.

Heterologous functional expression of ascidian Nav1 channels and close relationship with the evolutionary ancestor of vertebrate Nav channels.

Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary ß subunits. Detailed information on the structure-function relationships of Nav1 subunits has been obtained through heterologous expression experiments and analyses of protein structures. The basic properties of Nav1s, including their gating and ion permeation, were classically described in the squid giant axon and other invertebrates. However, heterologous functional expression of Nav1s from marine invertebrates has been unsuccessful. Ascidians belong to the Urochordata, a sister group of vertebrates, and the larval central nervous system of ascidians shows a similar plan to that of vertebrates. Here, we report the biophysical properties of ascidian Ciona Nav1 (CiNav1a) heterologously expressed in Xenopus oocytes. CiNav1a exhibited tetrodotoxin-insensitive sodium currents with rapid gating kinetics of activation and inactivation. Furthermore, consistent with the fact that the Ciona genome lacks orthologous genes to vertebrate ß subunits, the human ß1 subunit did not influence the gating properties when coexpressed with CiNav1a. Interestingly, CiNav1a contains an ankyrin-binding motif in the II-III linker, which can be targeted to the axon initial segment of mammalian cortical neurons. Our findings provide a platform to gain insight into the evolutionary and biophysical properties of Nav1s, which are important for the development of targeted therapeutics.

The trunk-tail junctional region in Ciona larvae autonomously expresses tail-beating bursts at ∼20 second intervals.

Swimming locomotion in aquatic vertebrates, such as fish and tadpoles, is expressed through neuron networks in the spinal cord. These networks are arranged in parallel, ubiquitously distributed and mutually coupled along the spinal cord to express undulation patterns accommodated to various inputs into the networks. While these systems have been widely studied in vertebrate swimmers, their evolutionary origin along the chordate phylogeny remains unclear. Ascidians, representing a sister group of vertebrates, give rise to tadpole larvae that swim freely in seawater. In the present study, we examined the locomotor ability of the anterior and posterior body fragments of larvae of the ascidian Ciona that had been cut at an arbitrary position. Examination of more than 200 fragments revealed a necessary and sufficient body region that spanned only ∼10% of the body length and included the trunk-tail junction. 'Mid-piece' body fragments, which included the trunk-tail junctional region, but excluded most of the anterior trunk and posterior tail, autonomously expressed periodic tail-beating bursts at ∼20 s intervals. We compared the durations and intervals of tail-beating bursts expressed by mid-piece fragments, and also by whole larvae under different sensory conditions. The results suggest that body parts outside the mid-piece effect shortening of swimming intervals, particularly in the dark, and vary the burst duration. We propose that Ciona larvae express swimming behaviors by modifying autonomous and periodic locomotor drives that operate locally in the trunk-tail junctional region.

An α7-related nicotinic acetylcholine receptor mediates the ciliary arrest response in pharyngeal gill slits of Ciona.

Ciliary movement is a fundamental process to support animal life, and the movement pattern may be altered in response to external stimuli under the control of nervous systems. Juvenile and adult ascidians have ciliary arrays around their pharyngeal gill slits (stigmata), and continuous beating is interrupted for seconds by mechanical stimuli on other parts of the body. Although it has been suggested that neural transmission to evoke ciliary arrest is cholinergic, its molecular basis has not yet been elucidated in detail. Here, we attempted to clarify the molecular mechanisms underlying this neurociliary transmission in the model ascidian Ciona Acetylcholinesterase histochemical staining showed strong signals on the laterodistal ciliated cells of stigmata, hereafter referred to as trapezial cells. The direct administration of acetylcholine (ACh) and other agonists of nicotinic ACh receptors (nAChRs) onto ciliated cells reliably evoked ciliary arrest that persisted for seconds in a dose-dependent manner. While the Ciona genome encodes ten nAChRs, only one of these called nAChR-A7/8-1, a relative of vertebrate α7 nAChRs, was found to be expressed by trapezial cells. Exogenously expressed nAChR-A7/8-1 on Xenopus oocytes responded to ACh and other agonists with consistent pharmacological traits to those observed in vivo Further efforts to examine signaling downstream of this receptor revealed that an inhibitor of phospholipase C (PLC) hampered ACh-induced ciliary arrest. We propose that homomeric α7-related nAChR-A7/8-1 mediates neurociliary transmission in Ciona stigmata to elicit persistent ciliary arrest by recruiting intracellular Ca2+ signaling.

AMPA glutamate receptors are required for sensory-organ formation and morphogenesis in the basal chordate.

AMPA-type glutamate receptors (GluAs) mediate fast excitatory transmission in the vertebrate central nervous system (CNS), and their function has been extensively studied in the mature mammalian brain. However, GluA expression begins very early in developing embryos, suggesting that they may also have unidentified developmental roles. Here, we identify developmental roles for GluAs in the ascidian Ciona intestinalis Mammals express Ca2+-permeable GluAs (Ca-P GluAs) and Ca2+-impermeable GluAs (Ca-I GluAs) by combining subunits derived from four genes. In contrast, ascidians have a single gluA gene. Taking advantage of the simple genomic GluA organization in ascidians, we knocked down (KD) GluAs in Ciona and observed severe impairments in formation of the ocellus, a photoreceptive organ used during the swimming stage, and in resorption of the tail and body axis rotation during metamorphosis to the adult stage. These defects could be rescued by injection of KD-resistant GluAs. GluA KD phenotypes could also be reproduced by expressing a GluA mutant that dominantly inhibits glutamate-evoked currents. These results suggest that, in addition to their role in synaptic communication in mature animals, GluAs also have critical developmental functions.

Evolutionary History of Voltage-Gated Sodium Channels.

Every cell within living organisms actively maintains an intracellular Na+ concentration that is 10-12 times lower than the extracellular concentration. The cells then utilize this transmembrane Na+ concentration gradient as a driving force to produce electrical signals, sometimes in the form of action potentials. The protein family comprising voltage-gated sodium channels (NaVs) is essential for such signaling and enables cells to change their status in a regenerative manner and to rapidly communicate with one another. NaVs were first predicted in squid and were later identified through molecular biology in the electric eel. Since then, these proteins have been discovered in organisms ranging from bacteria to humans. Recent research has succeeded in decoding the amino acid sequences of a wide variety of NaV family members, as well as the three-dimensional structures of some. These studies and others have uncovered several of the major steps in the functional and structural transition of NaV proteins that has occurred along the course of the evolutionary history of organisms. Here we present an overview of the molecular evolutionary innovations that established present-day NaV α subunits and discuss their contribution to the evolutionary changes in animal bodies.

Morphology and Physiology of the Ascidian Nervous Systems and the Effectors.

Neurobiology in ascidians has made many advances. Ascidians have offered natural advantages to researchers, including fecundity, structural simplicity, invariant morphology, and fast and stereotyped developmental processes. The researchers have also accumulated on this animal a great deal of knowledge, genomic resources, and modern genetic techniques. A recent connectomic analysis has shown an ultimately resolved image of the larval nervous system, whereas recent applications of live imaging and optogenetics have clarified the functional organization of the juvenile nervous system. Progress in resources and techniques have provided convincing ways to deepen what we have wanted to know about the nervous systems of ascidians. Here, the research history and the current views regarding ascidian nervous systems are summarized.

A mechanism for graded motor control encoded in the channel properties of the muscle ACh receptor.

The larva of the invertebrate chordate Ciona intestinalis possesses only 36 striated muscle cells and lacks body segmentation. It can swim, however, like a vertebrate tadpole, and how its simple body achieves such sophisticated motor control remains puzzling. We found that muscle contractions in Ciona larvae are variable and can be changed by sensory stimuli, so that neuromuscular transmission can convert the variable neural inputs into graded muscle activity. We characterized the molecular nature of the nicotinic acetylcholine receptor (nAChR) at neuromuscular synapses. When heterologously expressed in Xenopus oocytes, this nAChR channel exhibited two biophysical features resembling vertebrate neuronal nAChRs rather than the muscle type: inward rectification and high Ca(2+) permeability. Both of these properties were abolished by a simple mutation at the channel pore in one of the non-α subunits, called BGDE3, so as to adopt the sequence of related subunits in vertebrates, γ and ε. In vivo exchange of native BGDE3 with this mutant severely disrupted graded motor control, producing instead sporadic all-or-none-like flexions. The graded nature of excitation-contraction (E-C) coupling in this organism is based on the traits of the nAChR channel pore, which confer fine controllability on such a coarse motor architecture.

RNA interference in the appendicularian Oikopleura dioica reveals the function of the Brachyury gene.

The appendicularian Oikopleura dioica is a chordate that has a remarkably simple adult body with small cell number. Its transparency, stereotyped cell lineages, short life cycle, and small genome make it a promising new experimental model of chordate developmental biology. However, the functions of its various genes are still poorly understood due to lack of a tool for suppression of gene expression. Here, we applied a double-stranded RNA (dsRNA)-based-RNA interference (RNAi) method in O. dioica. For introducing dsRNA into eggs and embryos, we injected dsRNAs into the ovary. dsRNA, which is specific to EGFP or mCherry mRNA, decreased the exogenous mRNA-derived fluorescence in both eggs and embryos. dsRNA specific to the Brachyury gene of O. dioica, which is a homologous gene of a key notochord transcriptional factor in ascidians, triggered degradation of endogenous Brachyury mRNA and induced malformation or loss of the notochord in the tail. This effect was Brachyury sequence specific, as three dsRNAs covering different sequences produced the same phenotype. The result is in accordance with its expression site and also with the key regulatory function of Brachyury in notochord formation in other chordates. RNAi in O. dioica would be a useful tool for gaining insight into the oogenesis and early developmental processes in chordates.

The crystalline phase of cellulose changes under developmental control in a marine chordate.

The native form of cellulose is a fibrillar composite of two crystalline phases, the triclinic I(α) and monoclinic I(ß) allomorphs. Allomorph ratios are species-specific, and this gives rise to natural structural variations in cellulose crystals. However, the mechanisms contributing to crystal formation remain unknown. We show that the two crystalline phases of cellulose are tailored to distinct structures during different developmental stages of the tunicate chordate Oikopleura dioica. Larval cellulose consisting of I(α) allomorph constitutes the body cuticle fin, whereas adult cellulose consisting of I(ß) allomorph frames a mucous filter-feeding device, the "house." Both structures are secreted from the epidermis in accordance with the mutually exclusive expression patterns of two distinct putative cellulose synthase genes. We discuss a possible linkage between structural variations of the crystalline phases of cellulose and the underlying evolutionary genetics of cellulose biosynthesis.

Forming a tough shell via an intracellular matrix and cellular junctions in the tail epidermis of Oikopleura dioica (Chordata: Tunicata: Appendicularia).

A postanal tail is a major synapomorphy of the phylum Chordata, which is composed of three subphyla: Vertebrata, Cephalochordata, and Tunicata (Urochordata). Among tunicates, appendicularians are the only group that retains the tail in the adult, and the adult tail functions in locomotion and feeding in combination with a cellulose-based house structure. Given the phylogenetic position of tunicates, the appendicularian adult tail may possess ancestral features of the chordate tail. We assess the ultrastructural development of the tail epidermis of the appendicularian Oikopleura dioica. The epidermis of the larval tail is enclosed by the larval envelope, which is a thin sheet similar to the outer tunic layer of ascidian larvae. The epidermis of the adult tail seems to bear no tunic-like cellulosic integuments, and the tail fin is a simple folding of the epidermis. Every epidermal cell, except for the triangular cells at the edge of the tail fin, has a conspicuous matrix layer of fibrous content in the apical cytoplasm without enclosing membranes. The epidermis of the larval tail does not have a fibrous matrix layer, suggesting the production of the layer during larval development and metamorphosis. Zonulae adhaerentes firmly bind the epidermal cells of the adult tail to one another, and the dense microfilaments lining the cell borders constitute a mechanical support for the cell membranes. The intracellular matrix, cell junctions, and cytoskeletons probably make the tail epidermis a tough, flexible shell supporting the active beating of the oikopleuran adult tail.

Simple procedure for sperm cryopreservation in the larvacean tunicate Oikopleura dioica.

The larvacean tunicate Oikopleura dioica is an attractive organism for studies of the development, evolution, and physiology of chordates, showing considerable promise for genetic approaches given its short life cycle of five days. To facilitate future genetic studies, the development of protocols for the maintenance of individual strains is essential. Here we report a simple and practical protocol for the cryopreservation of sperm using liquid nitrogen (-196°C) and dimethyl sulfoxide (DMSO) as a protective agent. The quality of the frozen-thawed sperm was evaluated in terms of fertilizing ability and subsequent development of the fertilized eggs. We examined several parameters to optimize the efficiency of cryopreservation, such as the concentration of DMSO, the method for acclimation of sperm to DMSO before freezing, and for placing sperm in liquid nitrogen, as well as the pH of the seawater used in resuspending the thawed sperm. We confirmed that viable sperm were recovered after preservation for more than one year. In addition, mature animals, and even a subsequent generation, were obtained from eggs fertilized by the cryopreserved sperm. The present procedure seems to be simple and sufficiently practical for maintenance of future established lines of O. dioica using frozen sperm.

Ion channel clustering at the axon initial segment and node of Ranvier evolved sequentially in early chordates.

In many mammalian neurons, dense clusters of ion channels at the axonal initial segment and nodes of Ranvier underlie action potential generation and rapid conduction. Axonal clustering of mammalian voltage-gated sodium and KCNQ (Kv7) potassium channels is based on linkage to the actin-spectrin cytoskeleton, which is mediated by the adaptor protein ankyrin-G. We identified key steps in the evolution of this axonal channel clustering. The anchor motif for sodium channel clustering evolved early in the chordate lineage before the divergence of the wormlike cephalochordate, amphioxus. Axons of the lamprey, a very primitive vertebrate, exhibited some invertebrate features (lack of myelin, use of giant diameter to hasten conduction), but possessed narrow initial segments bearing sodium channel clusters like in more recently evolved vertebrates. The KCNQ potassium channel anchor motif evolved after the divergence of lampreys from other vertebrates, in a common ancestor of shark and humans. Thus, clustering of voltage-gated sodium channels was a pivotal early innovation of the chordates. Sodium channel clusters at the axon initial segment serving the generation of action potentials evolved long before the node of Ranvier. KCNQ channels acquired anchors allowing their integration into pre-existing sodium channel complexes at about the same time that ancient vertebrates acquired myelin, saltatory conduction, and hinged jaws. The early chordate refinements in action potential mechanisms we have elucidated appear essential to the complex neural signaling, active behavior, and evolutionary success of vertebrates.

Bilaterally Asymmetric Helical Myofibrils in Ascidian Tadpole Larvae.

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

A glycine receptor is involved in the organization of swimming movements in an invertebrate chordate.

BACKGROUND: Rhythmic motor patterns for locomotion in vertebrates are generated in spinal cord neural networks known as spinal Central Pattern Generators (CPGs). A key element in pattern generation is the role of glycinergic synaptic transmission by interneurons that cross the cord midline and inhibit contralaterally-located excitatory neurons. The glycinergic inhibitory drive permits alternating and precisely timed motor output during locomotion such as walking or swimming. To understand better the evolution of this system we examined the physiology of the neural network controlling swimming in an invertebrate chordate relative of vertebrates, the ascidian larva Ciona intestinalis. RESULTS: A reduced preparation of the larva consisting of nerve cord and motor ganglion generates alternating swimming movements. Pharmacological and genetic manipulation of glycine receptors shows that they are implicated in the control of these locomotory movements. Morphological molecular techniques and heterologous expression experiments revealed that glycine receptors are inhibitory and are present on both motoneurones and locomotory muscle while putative glycinergic interneurons were identified in the nerve cord by labeling with an anti-glycine antibody. CONCLUSIONS: In Ciona intestinalis, glycine receptors, glycinergic transmission and putative glycinergic interneurons, have a key role in coordinating swimming movements through a simple CPG that is present in the motor ganglion and nerve cord. Thus, the strong association between glycine receptors and vertebrate locomotory networks may now be extended to include the phylum chordata. The results suggest that the basic network for 'spinal-like' locomotion is likely to have existed in the common ancestor of extant chordates some 650 M years ago.

Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes.

Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so-called "prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate-unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome-wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment.

Is there intracellular cellulose in the appendicularian tail epidermis? A tale of the adult tail of an invertebrate chordate.

All tunicates produce a cellulosic extracellular matrix external to the epidermis; this is the most remarkable character of the subphylum (Tunicata = Urochordata). In Ascidiacea and Thaliacea, two of the three classes of Tunicata, cellulose is the main component of the tunic matrix that covers the whole body. In Appendicularia, however, the animals do not possess a tunic, and instead secrete a feeding apparatus called a "house" that is composed of cellulose. In the appendicularian Oikopleura dioica, we found a unique intracellular matrix in the apical cytoplasm of the adult tail epidermis that appears to form a tough shell supporting the tail. Although some experimental data suggest that this matrix may contain cellulose, the composition is still unknown. The significance of the tunicate capacity for cellulose synthesis is briefly discussed.

KCNQ1 subdomains involved in KCNE modulation revealed by an invertebrate KCNQ1 orthologue.

KCNQ1 channels are voltage-gated potassium channels that are widely expressed in various non-neuronal tissues, such as the heart, pancreas, and intestine. KCNE proteins are known as the auxiliary subunits for KCNQ1 channels. The effects and functions of the different KCNE proteins on KCNQ1 modulation are various; the KCNQ1-KCNE1 ion channel complex produces a slowly activating potassium channel that is crucial for heartbeat regulation, while the KCNE3 protein makes KCNQ1 channels constitutively active, which is important for K(+) and Cl(-) transport in the intestine. The mechanisms by which KCNE proteins modulate KCNQ1 channels have long been studied and discussed; however, it is not well understood how different KCNE proteins exert considerably different effects on KCNQ1 channels. Here, we approached this point by taking advantage of the recently isolated Ci-KCNQ1, a KCNQ1 homologue from marine invertebrate Ciona intestinalis. We found that Ci-KCNQ1 alone could be expressed in Xenopus laevis oocytes and produced a voltage-dependent potassium current, but that Ci-KCNQ1 was not properly modulated by KCNE1 and totally unaffected by coexpression of KCNE3. By making chimeras of Ci-KCNQ1 and human KCNQ1, we determined several amino acid residues located in the pore region of human KCNQ1 involved in KCNE1 modulation. Interestingly, though, these amino acid residues of the pore region are not important for KCNE3 modulation, and we subsequently found that the S1 segment plays an important role in making KCNQ1 channels constitutively active by KCNE3. Our findings indicate that different KCNE proteins use different domains of KCNQ1 channels, and that may explain why different KCNE proteins give quite different outcomes by forming a complex with KCNQ1 channels.

Gene expression profile of Ci-VSP in juveniles and adult blood cells of ascidian.

VSP is a transmembrane protein whose cytoplasmic region shows significant similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Notably, VSP exhibits a unique ability to transduce electrical signals into phosphoinositide turnover by coupling a transmembrane voltage sensor domain to the PTEN-like phosphoinositide phosphatase domain. Moreover, VSP gene is known to be widely conserved among deuterostome genomes, though the function of VSP in vivo remains largely unknown. In the present study, the expression pattern of ascidian VSP(Ci-VSP) was examined in embryos and juveniles of a marine invertebrate chordate, Ciona intestinalis. RT-PCR showed that Ci-VSP is expressed at the larval stage and that expression persists in juveniles. Whole mount in situ hybridization showed that Ci-VSP is expressed in cells of the stomach, intestine and blood cells of 2- to 3-week-old juveniles. Moreover, double staining blood cells from 2-month-old adults with Ci-VSP and Ci-PTEN probes showed that Ci-VSP-positive cells are a distinct population, separate from cells expressing Ci-PTEN. These findings suggest that in addition to its previously suggested roles in testis or sperm, Ci-VSP plays a key role in voltage-induced signal transduction in cells of the digestive system and blood.

Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate.

Genomes of animals as different as sponges and humans show conservation of global architecture. Here we show that multiple genomic features including transposon diversity, developmental gene repertoire, physical gene order, and intron-exon organization are shattered in the tunicate Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Ancestral architecture of animal genomes can be deeply modified and may therefore be largely nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain.