Neural crest lineage in the protovertebrate model Ciona.

Neural crest cells are multipotent progenitors that produce defining features of vertebrates such as the 'new head'1. Here we use the tunicate, Ciona, to explore the evolutionary origins of neural crest since this invertebrate chordate is among the closest living relatives of vertebrates2-4. Previous studies identified two potential neural crest cell types in Ciona, sensory pigment cells and bipolar tail neurons5,6. Recent findings suggest that  bipolar tail neurons are homologous to cranial sensory ganglia rather than derivatives of neural crest7,8. Here we show that the pigment cell lineage also produces neural progenitor cells that form regions of the juvenile nervous system following metamorphosis. Neural progenitors are also a major derivative of neural crest in vertebrates, suggesting that the last common ancestor of tunicates and vertebrates contained a multipotent progenitor population at the neural plate border. It would therefore appear that a key property of neural crest, multipotentiality, preceded the emergence of vertebrates.

Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

The CNS of the protovertebrate Ciona intestinalis contains a single cluster of dopaminergic (DA) neurons, the coronet cells, which have been likened to the hypothalamus of vertebrates. Whole-embryo single-cell RNA sequencing (RNA-seq) assays identified Ptf1a as the most strongly expressed cell-specific transcription factor (TF) in DA/coronet cells. Knockdown of Ptf1a activity results in their loss, while misexpression results in the appearance of supernumerary DA/coronet cells. Photoreceptor cells and ependymal cells are the most susceptible to transformation, and both cell types express high levels of Meis Coexpression of both Ptf1a and Meis caused the wholesale transformation of the entire CNS into DA/coronet cells. We therefore suggest that the reiterative use of functional manipulations and single-cell RNA-seq assays is an effective means for the identification of regulatory cocktails underlying the specification of specific cell identities.

Functional diversification process of opsin genes for teleost visual and pineal photoreceptions.

Most vertebrates have a rhodopsin gene with a five-exon structure for visual photoreception. By contrast, teleost fishes have an intron-less rhodopsin gene for visual photoreception and an intron-containing rhodopsin (exo-rhodopsin) gene for pineal photoreception. Here, our analysis of non-teleost and teleost fishes in various lineages of the Actinopterygii reveals that retroduplication after branching of the Polypteriformes produced the intron-less rhodopsin gene for visual photoreception, which converted the parental intron-containing rhodopsin gene into a pineal opsin in the common ancestor of the Teleostei. Additional analysis of a pineal opsin, pinopsin, shows that the pinopsin gene functions as a green-sensitive opsin together with the intron-containing rhodopsin gene for pineal photoreception in tarpon as an evolutionary intermediate state but is missing in other teleost fishes, probably because of the redundancy with the intron-containing rhodopsin gene. We propose an evolutionary scenario where unique retroduplication caused a "domino effect" on the functional diversification of teleost visual and pineal opsin genes.

Neuronal identities derived by misexpression of the POU IV sensory determinant in a protovertebrate.

The protovertebrate Ciona intestinalis type A (sometimes called Ciona robusta) contains a series of sensory cell types distributed across the head-tail axis of swimming tadpoles. They arise from lateral regions of the neural plate that exhibit properties of vertebrate placodes and neural crest. The sensory determinant POU IV/Brn3 is known to work in concert with regional determinants, such as Foxg and Neurogenin, to produce palp sensory cells (PSCs) and bipolar tail neurons (BTNs), in head and tail regions, respectively. A combination of single-cell RNA-sequencing (scRNA-seq) assays, computational analysis, and experimental manipulations suggests that misexpression of POU IV results in variable transformations of epidermal cells into hybrid sensory cell types, including those exhibiting properties of both PSCs and BTNs. Hybrid properties are due to coexpression of Foxg and Neurogenin that is triggered by an unexpected POU IV feedback loop. Hybrid cells were also found to express a synthetic gene battery that is not coexpressed in any known cell type. We discuss these results with respect to the opportunities and challenges of reprogramming cell types through the targeted misexpression of cellular determinants.

The complete cell lineage and MAPK- and Otx-dependent specification of the dopaminergic cells in the Ciona brain.

The Ciona larva has served as a unique model for understanding the development of dopaminergic cells at single-cell resolution owing to the exceptionally small number of neurons in its brain and its fixed cell lineage during embryogenesis. A recent study suggested that the transcription factors Fer2 and Meis directly regulate the dopamine synthesis genes in Ciona, but the dopaminergic cell lineage and the gene regulatory networks that control the development of dopaminergic cells have not been fully elucidated. Here, we reveal that the dopaminergic cells in Ciona are derived from a bilateral pair of cells called a9.37 cells at the center of the neural plate. The a9.37 cells divide along the anterior-posterior axis, and all of the descendants of the posterior daughter cells differentiate into the dopaminergic cells. We show that the MAPK pathway and the transcription factor Otx are required for the expression of Fer2 in the dopaminergic cell lineage. Our findings establish the cellular and molecular framework for fully understanding the commitment to dopaminergic cells in the simple chordate brain.

The pre-vertebrate origins of neurogenic placodes.

The sudden appearance of the neural crest and neurogenic placodes in early branching vertebrates has puzzled biologists for over a century. These embryonic tissues contribute to the development of the cranium and associated sensory organs, which were crucial for the evolution of the vertebrate "new head". A previous study suggests that rudimentary neural crest cells existed in ancestral chordates. However, the evolutionary origins of neurogenic placodes have remained obscure owing to a paucity of embryonic data from tunicates, the closest living relatives to those early vertebrates. Here we show that the tunicate Ciona intestinalis exhibits a proto-placodal ectoderm (PPE) that requires inhibition of bone morphogenetic protein (BMP) and expresses the key regulatory determinant Six1/2 and its co-factor Eya, a developmental process conserved across vertebrates. The Ciona PPE is shown to produce ciliated neurons that express genes for gonadotropin-releasing hormone (GnRH), a G-protein-coupled receptor for relaxin-3 (RXFP3) and a functional cyclic nucleotide-gated channel (CNGA), which suggests dual chemosensory and neurosecretory activities. These observations provide evidence that Ciona has a neurogenic proto-placode, which forms neurons that appear to be related to those derived from the olfactory placode and hypothalamic neurons of vertebrates. We discuss the possibility that the PPE-derived GnRH neurons of Ciona resemble an ancestral cell type, a progenitor to the complex neuronal circuit that integrates sensory information and neuroendocrine functions in vertebrates.

Spatio-temporal regulation of Rx and mitotic patterns shape the eye-cup of the photoreceptor cells in Ciona.

The ascidian larva has a pigmented ocellus comprised of a cup-shaped array of approximately 30 photoreceptor cells, a pigment cell, and three lens cells. Morphological, physiological and molecular evidence has suggested evolutionary kinship between the ascidian larval photoreceptors and vertebrate retinal and/or pineal photoreceptors. Rx, an essential factor for vertebrate photoreceptor development, has also been suggested to be involved in the development of the ascidian photoreceptor cells, but a recent revision of the photoreceptor cell lineage raised a crucial discrepancy between the reported expression patterns of Rx and the cell lineage. Here, we report spatio-temporal expression patterns of Rx at single-cell resolution along with mitotic patterns up to the final division of the photoreceptor-lineage cells in Ciona. The expression of Rx commences in non-photoreceptor a-lineage cells on the right side of the anterior sensory vesicle at the early tailbud stage. At the mid tailbud stage, Rx begins to be expressed in the A-lineage photoreceptor cell progenitors located on the right side of the posterior sensory vesicle. Thus, Rx is specifically but not exclusively expressed in the photoreceptor-lineage cells in the ascidian embryo. Two cis-regulatory modules are shown to be important for the photoreceptor-lineage expression of Rx. The cell division patterns of the photoreceptor-lineage cells rationally explain the generation of the cup-shaped structure of the pigmented ocellus. The present findings demonstrate the complete cell lineage of the ocellus photoreceptor cells and provide a framework elucidating the molecular and cellular mechanisms of photoreceptor development in Ciona.

Evolutionary steps involving counterion displacement in a tunicate opsin.

Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, Ciona intestinalis This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.

Genome-wide identification and characterization of transcription start sites and promoters in the tunicate Ciona intestinalis.

The tunicate Ciona intestinalis, an invertebrate chordate, has recently emerged as a powerful model organism for gene regulation analysis. However, few studies have been conducted to identify and characterize its transcription start sites (TSSs) and promoters at the genome-wide level. Here, using TSS-seq, we identified TSSs at the genome-wide scale and characterized promoters in C. intestinalis. Specifically, we identified TSS clusters (TSCs), high-density regions of TSS-seq tags, each of which appears to originate from an identical promoter. TSCs were found not only at known TSSs but also in other regions, suggesting the existence of many unknown transcription units in the genome. We also identified candidate promoters of 79 ribosomal protein (RP) genes, each of which had the major TSS in a polypyrimidine tract and showed a sharp TSS distribution like human RP gene promoters. Ciona RP gene promoters, however, did not appear to have typical TATA boxes, unlike human RP gene promoters. In Ciona non-RP promoters, two pyrimidine-purine dinucleotides, CA and TA, were frequently used as TSSs. Despite the absence of CpG islands, Ciona TATA-less promoters showed low expression specificity like CpG-associated human TATA-less promoters. By using TSS-seq, we also predicted trans-spliced gene TSSs and found that their downstream regions had higher G+T content than those of non-trans-spliced gene TSSs. Furthermore, we identified many putative alternative promoters, some of which were regulated in a tissue-specific manner. Our results provide valuable information about TSSs and promoter characteristics in C. intestinalis and will be helpful in future analysis of transcriptional regulation in chordates.

The Use of cis-Regulatory DNAs as Molecular Tools.

Ascidians possess relatively small and compact genomes. This feature enables us to easily isolate cis-regulatory DNAs of genes of interest. Particularly, cis-regulatory DNAs of genes showing tissue- or cell-type-specific expression are routinely used for the artificial induction of gene expression. This strategy helps us to label cells, tissues, and organs of interest, and to investigate gene functions through overexpression, ectopic expression, and the disruption of functions by dominant-negative forms. Thus, cis-regulatory DNAs provide a powerful tool for tissue-specific genetic manipulation in studies of ascidian development and physiology. This chapter summarizes the types of cis-regulatory DNAs as a genetic manipulation tool, describes the methods used for isolating cis-regulatory DNAs, and provide reported examples of the use of cis-regulatory DNAs as molecular tools for investigating gene functions.

Revised lineage of larval photoreceptor cells in Ciona reveals archetypal collaboration between neural tube and neural crest in sensory organ formation.

The Ciona intestinalis larva has two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus, that are asymmetrically situated in the brain. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. Precise elucidation of the lineage of the photoreceptor cells will be key to understanding the developmental mechanisms of these cells as well as the evolutionary relationships between the photoreceptor organs of ascidians and vertebrates. Photoreceptor cells of the pigmented ocellus have been thought to develop from anterior animal (a-lineage) blastomeres, whereas the developmental origin of the nonpigmented ocellus has not been determined. Here, we show that the photoreceptor cells of both ocelli develop from the right anterior vegetal hemisphere: those of the pigmented ocellus from the right A9.14 cell and those of the nonpigmented ocellus from the right A9.16 cell. The pigmented ocellus is formed by a combination of two lineages of cells with distinct embryonic origins: the photoreceptor cells originate from a medial portion of the A-lineage neural plate, while the pigment cell originates from the lateral edge of the a-lineage neural plate. In light of the recently proposed close evolutionary relationship between the ocellus pigment cell of ascidians and the cephalic neural crest of vertebrates, the ascidian ocellus may represent a prototypic contribution of the neural crest to a cranial sensory organ.

Ependymal cells of chordate larvae are stem-like cells that form the adult nervous system.

In ascidian tunicates, the metamorphic transition from larva to adult is accompanied by dynamic changes in the body plan. For instance, the central nervous system (CNS) is subjected to extensive rearrangement because its regulating larval organs are lost and new adult organs are created. To understand how the adult CNS is reconstructed, we traced the fate of larval CNS cells during ascidian metamorphosis by using transgenic animals and imaging technologies with photoconvertible fluorescent proteins. Here we show that most parts of the ascidian larval CNS, except for the tail nerve cord, are maintained during metamorphosis and recruited to form the adult CNS. We also show that most of the larval neurons disappear and only a subset of cholinergic motor neurons and glutamatergic neurons are retained. Finally, we demonstrate that ependymal cells of the larval CNS contribute to the construction of the adult CNS and that some differentiate into neurons in the adult CNS. An unexpected role of ependymal cells highlighted by this study is that they serve as neural stem-like cells to reconstruct the adult nervous network during chordate metamorphosis. Consequently, the plasticity of non-neuronal ependymal cells and neuronal cells in chordates should be re-examined by future studies.

Transcriptional co-regulation of evolutionarily conserved microRNA/cone opsin gene pairs: implications for photoreceptor subtype specification.

The vertebrate retina contains two types of photoreceptor cells, rods and cones, which use distinct types of opsins and phototransduction proteins. Cones can be further divided into several subtypes with differing wavelength sensitivity and morphology. Although photoreceptor development has been extensively studied in a variety of vertebrate species, the mechanism by which photoreceptor subtypes are established is still largely unknown. Here we report two microRNAs (miRNAs), miR-726 and miR-729, which are potentially involved in photoreceptor subtype specification. In the medaka Oryzias latipes, the genes encoding miR-726 and miR-729 are located upstream of the red-sensitive opsin gene LWS-A and the UV-sensitive opsin gene SWS1, respectively, and are transcribed in the opposite direction from the respective opsin genes. The miR-726/LWS pair is conserved between teleosts and tetrapods, and the miR-729/SWS1 pair is conserved among teleosts. in situ hybridization analyses and fluorescence reporter assays suggest that these miRNAs are co-expressed with the respective opsins in specific cone subtypes. Potential targets of miR-726 and miR-729 predicted in silico include several transcription factors that regulate photoreceptor development. Functional analyses of cis-regulatory sequences in vivo suggest that transcription of the paired microRNA and opsin genes is co-regulated by common cis-regulatory modules. We propose an evolutionarily conserved mechanism that controls photoreceptor subtype identity through coupling between transcriptional and post-transcriptional regulations.

Nonreproductive role of gonadotropin-releasing hormone in the control of ascidian metamorphosis.

BACKGROUND: Gonadotropin-releasing hormones (GnRHs) are neuropeptides that play central roles in the reproduction of vertebrates. In the ascidian Ciona intestinalis, GnRHs and their receptors are expressed in the nervous systems at the larval stage, when animals are not yet capable of reproduction, suggesting that the hormones have non-reproductive roles. RESULTS: We showed that GnRHs in Ciona are involved in the animal's metamorphosis by regulating tail absorption and adult organ growth. Absorption of the larval tail and growth of the adult organs are two major events in the metamorphosis of ascidians. When larvae were treated with GnRHs, they completed tail absorption more frequently than control larvae. cAMP was suggested to be a second messenger for the induction of tail absorption by GnRHs. tGnRH-3 and tGnRH-5 (the "t" indicates "tunicate") inhibited the growth of adult organs by arresting cell cycle progression in parallel with the promotion of tail absorption. CONCLUSIONS: This study provides new insights into the molecular mechanisms of ascidian metamorphosis conducted by non-reproductive GnRHs.

Characterization of trans-spliced chimeric RNAs: insights into the mechanism of trans-splicing.

Trans-splicing is a post-transcriptional processing event that joins exons from separate RNAs to produce a chimeric RNA. However, the detailed mechanism of trans-splicing remains poorly understood. Here, we characterize trans-spliced genes and provide insights into the mechanism of trans-splicing in the tunicate Ciona. Tunicates are the closest invertebrates to humans, and their genes frequently undergo trans-splicing. Our analysis revealed that, in genes that give rise to both trans-spliced and non-trans-spliced messenger RNAs, trans-splice acceptor sites were preferentially located at the first functional acceptor site, and their paired donor sites were weak in both Ciona and humans. Additionally, we found that Ciona trans-spliced genes had GU- and AU-rich 5' transcribed regions. Our data and findings not only are useful for Ciona research community, but may also aid in a better understanding of the trans-splicing mechanism, potentially advancing the development of gene therapy based on trans-splicing.

Monoaminergic modulation of photoreception in ascidian: evidence for a proto-hypothalamo-retinal territory.

BACKGROUND: The retina of craniates/vertebrates has been proposed to derive from a photoreceptor prosencephalic territory in ancestral chordates, but the evolutionary origin of the different cell types making the retina is disputed. Except for photoreceptors, the existence of homologs of retinal cells remains uncertain outside vertebrates. METHODS: The expression of genes expressed in the sensory vesicle of the ascidian Ciona intestinalis including those encoding components of the monoaminergic neurotransmission systems, was analyzed by in situ hybridization or in vivo transfection of the corresponding regulatory elements driving fluorescent reporters. Modulation of photic responses by monoamines was studied by electrophysiology combined with pharmacological treatments. RESULTS: We show that many molecular characteristics of dopamine-synthesizing cells located in the vicinity of photoreceptors in the sensory vesicle of the ascidian Ciona intestinalis are similar to those of amacrine dopamine cells of the vertebrate retina. The ascidian dopamine cells share with vertebrate amacrine cells the expression of the key-transcription factor Ptf1a, as well as that of dopamine-synthesizing enzymes. Surprisingly, the ascidian dopamine cells accumulate serotonin via a functional serotonin transporter, as some amacrine cells also do. Moreover, dopamine cells located in the vicinity of the photoreceptors modulate the light-off induced swimming behavior of ascidian larvae by acting on alpha2-like receptors, instead of dopamine receptors, supporting a role in the modulation of the photic response. These cells are located in a territory of the ascidian sensory vesicle expressing genes found both in the retina and the hypothalamus of vertebrates (six3/6, Rx, meis, pax6, visual cycle proteins). CONCLUSION: We propose that the dopamine cells of the ascidian larva derive from an ancestral multifunctional cell population located in the periventricular, photoreceptive field of the anterior neural tube of chordates, which also gives rise to both anterior hypothalamus and the retina in craniates/vertebrates. It also shows that the existence of multiple cell types associated with photic responses predates the formation of the vertebrate retina.

Cell lineage and cis-regulation for a unique GABAergic/glycinergic neuron type in the larval nerve cord of the ascidian Ciona intestinalis.

The tunicate Ciona intestinalis larva has a simple central nervous system (CNS), consisting of fewer than 400 cells, which is homologous to the vertebrate CNS. Recent studies have revealed neuronal types and networks in the larval CNS of C. intestinalis, yet their cell lineage and the molecular mechanism by which particular types of neurons are specified and differentiate remain poorly understood. Here, we report cell lineage origin and a cis-regulatory module for the anterior caudal inhibitory neurons (ACINs), a putative component of the central pattern generator regulating swimming locomotion. The vesicular GABA/ glycine transporter gene Ci-VGAT, a specific marker for GABAergic / glycinergic neurons, is expressed in distinct sets of neurons, including ACINs of the tail nerve cord and others in the brain vesicle and motor ganglion. Comparative genomics analysis between C. intestinalis and Ciona savignyi and functional analysis in vivo identified the cis-regulatory module responsible for Ci-VGAT expression in ACINs. Our cell lineage analyses inferred that ACINs derive from A11.116 cells, which have been thought to solely give rise to glial ependymal cells of the lateral wall of the nerve cord. The present findings will provide a solid basis for future studies addressing the molecular mechanism underlying specification of ACINs, which play a critical role in controlling larval locomotion

Retinal Cone Mosaic in sws1-Mutant Medaka (Oryzias latipes), A Teleost.

Purpose: Ablation of short single cones (SSCs) expressing short-wavelength-sensitive opsin (SWS1) is well analyzed in the field of regenerative retinal cells. In contrast with ablation studies, the phenomena caused by the complete deletion of SWS1 are less well-understood. To assess the effects of SWS1 deficiency on retinal structure, we established and analyzed sws1-mutant medaka. Methods: To visualize SWS1, a monoclonal anti-SWS1 antibody and transgenic reporter fish (Tg(sws1:mem-egfp)) were generated. We also developed a CRISPR/Cas-driven sws1-mutant line. Retinal structure of sws1 mutant was visualized using anti-SWS1, 1D4, and ZPR1 antibodies and coumarin derivatives and compared with wild type, Tg(sws1:mem-egfp), and another opsin (lws) mutant. Results: Our rat monoclonal antibody specifically recognized medaka SWS1. Sws1 mutant retained regularly arranged cone mosaic as lws mutant and its SSCs had neither SWS1 nor long wavelength sensitive opsin. Depletion of sws1 did not affect the expression of long wavelength sensitive opsin, and vice versa. ZPR1 antibody recognized arrestin spread throughout double cones and long single cones in wild-type, transgenic, and sws1-mutant lines. Conclusions: Comparative observation of sws1-mutant and wild-type retinas revealed that ZPR1 negativity is not a marker for SSCs with SWS1, but SSCs themselves. Loss of functional sws1 did not cause retinal degeneration, indicating that sws1 is not essential for cone mosaic development in medaka. Our two fish lines, one with visualized SWS1 and the other lacking functional SWS1, offer an opportunity to study neural network synapsing with SSCs and to clarify the role of SWS1 in vision.

Profiling ascidian promoters as the primordial type of vertebrate promoter.

BACKGROUND: CpG islands are observed in mammals and other vertebrates, generally escape DNA methylation, and tend to occur in the promoters of widely expressed genes. Another class of promoter has lower G+C and CpG contents, and is thought to be involved in the spatiotemporal regulation of gene expression. Non-vertebrate deuterostomes are reported to have a single class of promoter with high-frequency CpG dinucleotides, suggesting that this is the original type of promoter. However, the limited annotation of these genes has impeded the large-scale analysis of their promoters. RESULTS: To determine the origins of the two classes of vertebrate promoters, we chose Ciona intestinalis, an invertebrate that is evolutionarily close to the vertebrates, and identified its transcription start sites genome-wide using a next-generation sequencer. We indeed observed a high CpG content around the transcription start sites, but their levels in the promoters and background sequences differed much less than in mammals. The CpG-rich stretches were also fairly restricted, so they appeared more similar to mammalian CpG-poor promoters. CONCLUSIONS: From these data, we infer that CpG islands are not sufficiently ancient to be found in invertebrates. They probably appeared early in vertebrate evolution via some active mechanism and have since been maintained as part of vertebrate promoters.

A single motor neuron determines the rhythm of early motor behavior in Ciona.

Recent work in tunicate supports the similarity between the motor circuits of vertebrates and basal deuterostome lineages. To understand how the rhythmic activity in motor circuits is acquired during development of protochordate Ciona, we investigated the coordination of the motor response by identifying a single pair of oscillatory motor neurons (MN2/A10.64). The MN2 neurons had Ca2+ oscillation with an ~80-s interval that was cell autonomous even in a dissociated single cell. The Ca2+ oscillation of MN2 coincided with the early tail flick (ETF). The spikes of the membrane potential in MN2 gradually correlated with the rhythm of ipsilateral muscle contractions in ETFs. The optogenetic experiments indicated that MN2 is a necessary and sufficient component of ETFs. These results indicate that MN2 is indispensable for the early spontaneous rhythmic motor behavior of Ciona. Our findings shed light on the understanding of development and evolution of chordate rhythmical locomotion.