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The Motor Ganglion (MG) is a small collection of neurons that control the swimming movements of the tunicate tadpole larva. Situated at the base of the tail, molecular and functional comparisons suggest that may be a homolog of the spinal cord and/or hindbrain ("rhombospinal" region) of vertebrates. Here we review the most current knowledge of the development, connectivity, functions, and unique identities of the neurons that comprise the MG, drawn mostly from studies in Ciona spp. The simple cell lineages, minimal cellular composition, and comprehensively mapped "connectome" of the Ciona MG all make this an excellent model for studying the development and physiology of motor control in aquatic larvae. Research Highlights: We review the latest research on the development, circuitry, and function of the Motor Ganglion of tunicate larvae, focused primarily on the most well‐studied species in the Ciona genus. [ABSTRACT FROM AUTHOR]

Metamorphosis is the dramatic and irreversible reconstruction of animal bodies transitioning from the larval stage. Because of the significant impact of metamorphosis on animal life, its timing is strictly regulated. Invertebrate chordate ascidians are the closest living relatives of vertebrates. Ascidians exhibit metamorphosis that converts their swimming larvae into sessile adults. Ascidian metamorphosis is triggered by a mechanical stimulus generated when adhesive papillae adhere to a substrate. However, it is not well understood how the mechanical stimulus is generated and how ascidian larvae sense the stimulus. In this study, we addressed these issues by a combination of embryological, molecular, and genetic experiments in the model ascidian Ciona intestinalis Type A, also called Ciona robusta. We here showed that the epidermal neuronal network starting from the sensory neurons at the adhesive papillae is responsible for the sensing of adhesion. We also found that the transient receptor potential (TRP) channel PKD2 is involved in sensing the stimulus of adhesion. Our results provide a better understanding of the mechanisms underlying the regulation of the timing of ascidian metamorphosis. [ABSTRACT FROM AUTHOR]

Wnt signaling is essential during animal development and regeneration, but also plays an important role in diseases such as cancer and diabetes. The canonical Wnt signaling pathway is one of the most conserved signaling cascades in the animal kingdom, with the T‐cell factor/lymphoid enhancer factor (TCF/LEF) proteins being the major mediators of Wnt/β‐catenin‐regulated gene expression. In comparison with invertebrates, vertebrates possess a high diversity of TCF/LEF family genes, implicating this as a possible key change to Wnt signaling at the evolutionary origin of vertebrates. However, the precise nature of this diversification is only poorly understood. The aim of this study is to clarify orthology, paralogy, and isoform relationships within the TCF/LEF gene family within chordates via in silico comparative study of TCF/LEF gene structure, molecular phylogeny, and gene synteny. Our results support the notion that the four TCF/LEF paralog subfamilies in jawed vertebrates (gnathostomes) evolved via the two rounds of whole‐genome duplications that occurred during early vertebrate evolution. Importantly, gene structure comparisons and synteny analysis of jawless vertebrate (cyclostome) TCFs suggest that a TCF7L2‐like form of gene structure is a close proxy for the ancestral vertebrate structure. In conclusion, we propose a detailed evolutionary path based on a new pre‐whole‐genome duplication vertebrate TCF gene model. This ancestor gene model highlights the chordate and vertebrate innovations of TCF/LEF gene structure, providing the foundation for understanding the role of Wnt/β‐catenin signaling in vertebrate evolution. [ABSTRACT FROM AUTHOR]

The geographical expansion of invasive species usually leads to temporary and/or permanent changes at multiple levels (genetics, epigenetics, gene expression, etc.) to acclimatize to abiotic and/or biotic stresses in novel environments. Epigenetic variation such as DNA methylation is often involved in response to diverse local environments, thus representing one crucial mechanism to promote invasion success. However, evidence is scant on the potential role of DNA methylation variation in rapid environmental response and invasion success during biological invasions. In particular, DNA methylation patterns and possible contributions of varied environmental factors to methylation differentiation have been largely unknown in many invaders, especially for invasive species in marine systems where extremely complex interactions exist between species and surrounding environments. Using the methylation‐sensitive amplification polymorphism (MSAP) technique, here we investigated population methylation structure at the genome level in two highly invasive model ascidians, Ciona robusta and C. intestinalis, collected from habitats with varied environmental factors such as temperature and salinity. We found high intrapopulation methylation diversity and significant population methylation differentiation in both species. Multiple analyses, such as variation partitioning analysis, showed that both genetic variation and environmental factors contributed to the observed DNA methylation variation. Further analyses found that 24 and 20 subepiloci were associated with temperature and/or salinity in C. robusta and C. intestinalis, respectively. All these results clearly showed significant methylation divergence among populations of both invasive ascidians, and varied local environmental factors, as well as genetic variation, were responsible for the observed DNA methylation patterns. The consistent findings in both species here suggest that DNA methylation, coupled with genetic variation, may facilitate local environmental adaptation during biological invasions, and DNA methylation variation molded by local environments may contribute to invasion success. Epigenetic variation is often involved in response to diverse local environments, thus representing one potential adaptive mechanism to promote invasion success. Here, we investigated population epigenomic structure in two highly invasive model ascidians collected from habitats with varied environmental factors. Our results showed that epigenetic modifications, coupled with genetic variation, play crucial roles in rapid adaptive microevolution during biological invasions, and epigenetic variation molded by local environments may contribute to invasive species' rapid adaptation to different local habitats. [ABSTRACT FROM AUTHOR]

The basic mechanisms controlling mitosis are highly conserved in animals regardless of cell types and developmental stages. However, an exceptional aspect of mitosis is seen during early animal embryogenesis in which a large fertilized egg is quickly divided into smaller blastomeres according to the reproducible spatiotemporal pattern that does not rely on the cell-cycle arrest or growth. This mitosis, referred to as cleavage, overlaps in the timeframe with the specification of cell fate. The precise spatiotemporal regulation of cleavages is therefore essential to the creation of the appropriate cell number and to the morphology of an embryo. To achieve the reproducibility of cleavage during embryogenesis, the relative timing of mitosis between cells, which we refer to as synchrony, must be properly regulated. Studies in model organisms have begun to reveal how the synchrony of mitosis is regulated by the developmental modulation of cell-cycle machineries. In this review, we focus on three such mechanisms: biochemical switches that achieve the synchrony of mitosis, the nucleo-cytoplasmic ratio that provokes the asynchrony of mitosis, and the transcriptional mechanisms coupled with cell fate control that reestablish the synchrony of mitosis in each fate-restricted compartment. Our review is an attempt to understand the temporal patterns of cleavages in animal embryos created by the combinations of these three mechanisms. [ABSTRACT FROM AUTHOR]

Variation in density of early stages, that is, larvae and juveniles, is a major determinant of the distribution and abundance of the adult population of most marine invertebrates. These early stages thus play a key role in competitive interactions, and, more specifically, in invasion dynamics when biologically similar native and non-native species ( NNS) come into contact in the same habitat. We examined the settlement dynamics and settlement rate of two important members of the fouling community that are common on human-made infrastructures around the world: Ciona robusta (formerly known as Ciona intestinalis type A) and C. intestinalis (formerly known as C. intestinalis type B). In the western English Channel, the two species live in close syntopy following the recent introduction of C. robusta in the native European range of C. intestinalis. Using settlement panels replaced monthly over 2 years in four marinas (including one studied over 4 years) and species-diagnostic molecular markers to distinguish between juveniles of both species ( N = 1,650), we documented similar settlement dynamics of both species, with two settlement periods within a calendar year. With one exception, settlement times were highly similar in the congeners. Although the NNS showed lower settlement density than that of the native congener, its juvenile recruitment was high during the second settlement period that occurs after the warm season, a pattern also observed in adult populations. Altogether, our results suggest that species' settlement dynamics do not lead to the dominance of one species over the other through space monopolization. In addition, we showed that changes over time are more pronounced in the NNS than in the native species. This is possibly due to a higher sensitivity of the NNS to changes of environmental factors such as temperature and salinity. Environmental changes may thus eventually modify the strength of competitive interactions between the two species as well as species dominance. [ABSTRACT FROM AUTHOR]

Biological introductions bring into contact species that can still hybridize. The evolutionary outcomes of such secondary contacts may be diverse (e.g. adaptive introgression from or into the introduced species) but are not yet well examined in the wild. The recent secondary contact between the non-native sea squirt Ciona robusta (formerly known as C. intestinalis type A) and its native congener C. intestinalis (formerly known as C. intestinalis type B), in the Western English Channel, provides an excellent case study to examine. To examine contemporary hybridization between the two species, we developed a panel of 310 ancestry-informative SNPs from a population transcriptomic study. Hybridization rates were examined on 449 individuals sampled in eight sites from the sympatric range and five sites from allopatric ranges. The results clearly showed an almost complete absence of contemporary hybridization between the two species in syntopic localities, with only one-first-generation hybrid and no other genotype compatible with recent backcrosses. Despite the almost lack of contemporary hybridization, shared polymorphisms were observed in sympatric and allopatric populations of both species. Furthermore, one allopatric population from SE Pacific exhibited a higher rate of shared polymorphisms compared to all other C. robusta populations. Altogether, these results indicate that the observed level of shared polymorphism is more probably the outcome of ancient gene flow spread afterwards at a worldwide scale. They also emphasize efficient reproductive barriers preventing hybridization between introduced and native species, which suggests hybridization should not impede too much the expansion and the establishment of the non-native species in its introduction range. [ABSTRACT FROM AUTHOR]

Understanding the factors that cause population divergence has long been of interest to marine biologists in their attempts to interpret the effect of humanmediated vectors. Broadcast-spawning species with limited dispersal capability are excellent candidates to measure the present-day patterns of genetic diversity. The tunicate Ciona intestinalis (Ascidiacea) is comprised of a complex of morphologically cryptic species that form vigorous aggregates in eutrophic habitats (harbors, gulfs and lagoons) where they can compete with the epibenthic community and cause biofouling problems. This study investigated biogeographic variability and migration patterns of C. intestinalis sp. A along Northeast Atlantic and Mediterranean coasts using microsatellite markers. Data presented here on 371 specimens collected from 17 populations reveal high genetic polymorphism, but with a deficit of heterozygote deficiency. Absence of evidence for isolation by distance suggests that the genetic patterns do not reflect the geographic distribution of sampled populations. Substantial gene flow and artificial potential for dispersal boost high levels of within-population genetic variability and prevent genetic differentiation within and between seas. A predominant eastward migration pattern was revealed by the data set, with very limited opportunity for C. intestinalis sp. A to travel westward. This directional movement indicates that other properties (e.g. habitat quality, genetic traits, mating system, life cycle) may cause adaptive divergence at a large biogeographic scale. [ABSTRACT FROM AUTHOR]

ABSTRACT Background: Ascidian larval epidermal sensory neurons (ESNs) each extend a single cilium into the outer body structure, or tunic, to form a unique external sensory network, the ASNET (ascidian dendritic network in tunic). The functions of ESNs and the ASNET are unknown, though it has been suggested that they play important roles in swimming larvae. In this study, we used transgenic larva expressing the photoconvertible protein Kaede pan-neuronally to identify the orientation and morphology of single ESN external processes within the ASNET. Results: When individual ESN cell bodies were UV-irradiated, the photoconverted Kaede protein diffused into the external processes that projected to the edge of the tunic, indicating that these processes are cytoplasmically connected to the cell bodies of ESNs. We were, therefore, able to comprehensively catalog the morphology and orientation of each ESN external process. Most trunk ESNs appeared to extend neurites to the palp, but no processes were observed to emanate from palp neurons. ESN processes differed systematically in their morphology and orientation, suggesting that the ASNET is formed in a regulated, non-random fashion. Conclusions: This study reveals the organization of ESN sensory fields in the ascidian larval tunic. Developmental Dynamics 243:1362-1373, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: In the simple ascidian chordate Ciona, the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non-compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape, and behavior vary consistently along the anterior-posterior (AP) axis. Results: Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non-uniform expression in the notochord. Ezrin/radixin/moesin ( ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin-like gene ( BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF-β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL, and TGF-β expression are evident even before the notochord cells have intercalated into a single-file column. Conclusions: We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. Developmental Dynamics 243:612-620, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channels (ASICs) constitute a family of neuron-specific voltage-insensitive sodium channels gated by extracellular protons. Functions of ASICs in mammals include nociception, mechanosensation, and modulation of synaptic transmission. However, the role protons play in mediating the effects of ASICs remains elusive. We have examined ASICs from the ascidian Ciona intestinalis, a simple chordate organism whose nervous system in the larval stage exhibits high similarity to that of higher vertebrates. We found that the ascidian genome contains a single ASIC gene that gives rise to two splice forms analogous to the mammalian ASIC1 and ASIC2. CiASIC is expressed in most neurons of the larva but is absent in the adult. Despite high sequence similarity with mammalian counterparts, CiASIC is proton-insensitive when examined in heterologous systems or in larval neurons; the latter rules out the possibility that proton sensitivity is conferred by accessory proteins or particular factors present only in Ciona neurons. Down-regulation of the CiASIC transcript by double-stranded RNA disrupted the regular pattern of larval swimming, implying that proton-independent mechanisms mediate the effects of ASIC in vivo. Together the data identify ASIC as a highly conserved channel distinctive of chordate nervous systems and show that protons are not essential for ASIC function. [ABSTRACT FROM AUTHOR]

Developmental processes can change during evolution at many levels of the ontogeny of an individual. Embryos of solitary ascidians have a largely invariant mode of development, with fixed cleavage patterns and fate maps. Thus the cell lineages and final body plan of the two quite distantly related species considered in this review, Ciona intestinalis and Halocynthia roretzi, are highly similar. However, close comparison of the developmental mechanisms used by these two species provide examples of evolutionary changes and help pinpoint which aspects of development are evolutionarily flexible. Examples of both similarity and diversity are observed in the mechanisms used to generate the full complement of larval muscle. We will describe the changes in muscle-cell lineage, as well as some striking differences in the intercellular signalling pathways used to induce muscle fate. The somewhat surprising conclusion is that in ascidians, as in nematode vulval development, different signalling mechanisms have been adopted to mediate similar interactions between equivalently positioned cells. [ABSTRACT FROM AUTHOR]

The larval nervous system of the ascidian Ciona intestinalis exhibits an abstract form of the vertebrate nervous system. The Ci-Gαi1 gene, which encodes a G-protein α subunit, is specifically expressed in distinct sets of neurons in C. intestinalis larvae, including papillar neurons of the adhesive organ, ocellus photoreceptor cells, and cholinergic and GABAergic neurons in the central nervous system (CNS). A GFP reporter gene driven by the 4.2-kb 5′ flanking region of Ci-Gαi1 recapitulated the endogenous gene expression patterns. Comparative genomic analysis of the Gαi1 gene between C. intestinalis and Ciona savignyi identified an 87-bp highly conserved non-coding sequence located between –3176 and –3090 bp upstream of the gene. Deletion of this conserved upstream sequence resulted in the complete loss of reporter expression in the central nervous system, while reporter expression in the adhesive organ and mesenchyme cells remained unaffected. The conserved upstream sequence can activate gene expression from basal promoters in the brain vesicle, although it requires additional cis-regulatory sequences to fully activate the CNS-specific gene expression. These results suggest that different types of central neurons share a common transcriptional activation mechanism that is different from that of papillar neurons. [ABSTRACT FROM AUTHOR]

To examine the role of the amino acid GABA in the locomotion of basal chordates, we investigated the pharmacology of swimming and the morphology of GABA-immunopositive neurones in tadpole larvae of the ascidians Ciona intestinalis and Ciona savignyi. We verified that electrical recording from the tail reflects alternating muscle activity during swimming by correlating electrical signals with tail beats using high-speed video recording. GABA reversibly reduced swimming periods to single tail twitches, while picrotoxin increased the frequency and duration of electrical activity associated with spontaneous swimming periods. Immunocytochemistry for GABA revealed extensive labelling throughout the larval central nervous system. Two strongly labelled regions on either side of the sensory vesicle were connected by an arc of labelled fibres, from which fibre tracts extended caudally into the visceral ganglion. Fibre tracts extended ventrally from a third, more medial region in the posterior sensory vesicle. Two rows of immunoreactive cell bodies in the visceral ganglion extended neurites into the nerve cord, where varicosities were seen. Thus, presumed GABAergic neurones form a network that could release GABA during swimming that is involved in modulating the time course and frequency of periods of spontaneous swimming. GABAergic and motor neurones in the visceral ganglion could interact at the level of their cell bodies and/or through the presumed GABAergic fibres that enter the nerve cord. The larval swimming network appears to possess some of the properties of spinal networks in vertebrates, while at the same time possibly showing a type of peripheral innervation resembling that in some protostomes. [ABSTRACT FROM AUTHOR]

Simple cupular organs similar to those described in Ciona intestinalis were observed in Corella eumyota. They consist of a macula containing the celt bodies of 20-30 primary sensory neurons whose cilia project into a dome- or finger-shaped structure, the cupula. Rather than being found in the mantle lining as in C. intestinalis, the organs were located on the atrial surface of the branchial sac. The sensory innervation was examined in whole-mount preparations using anti-tubulin immunohistochemistry. Sensory neurons in C. eumyota showed no immunoreactivity with antisera raised against gonadotropin-releasing hormone (GnRH). A novel, elongated sense organ termed the cupular strand was found in Corella inflata. It has the same basic components as the simple type of cupular organ but consists of a single, long structure containing ∼1500 sensory cells. Located on the atrial surface of the branchial sac, it extends along the midline of the dorsal fold, from the gonoduct openings almost as far as the brain. Preparations were examined using optical and electron microscopy. Nerves and cilia were visualized by anti-tubulin immunofluorescence microscopy. It was possible to follow the sensory axons from the macula of the cupular strand to points where they joined branches of the visceral nerve, which enters a nerve root at the back of the brain. In C. inflata the sensory cell bodies and their axons were immunoreactive not only With anti-tubulin but also with an antiserum raised against Tunicate I GnRH. There was no immunoreactivity, however, with Chicken II and catfish GnRH antisera. All three GnRH antisera labeled the dorsal strand plexus, a structure associated with production of GnRH in its role as a reproductive hormone. We concluded that the GnRH-like molecule labeled in sensory neurons differs from the form of GnRH found in the dorsal strand plexus, and may have a different function, perhaps in the neural control of ciliary activity. The... [ABSTRACT FROM AUTHOR]

SUMMARY All vertebrates have directional asymmetries in the organization of their internal organs. In jawed vertebrates, development of asymmetry is controlled by a conserved molecular pathway that includes Pitx2 , which is expressed by lateral plate mesoderm cells on the left side of the embryo. Pitx2 is a member of the Pitx homeobox gene family, the expression of which also marks stomodeal ectoderm and the adenohypophysis. Here we report the characterization of Pitx genes from Branchiostoma floridae (an amphioxus) and Ciona intestinalis (a urochordate), representatives of two basal chordate lineages and successively deeper outgroups to the vertebrates. Expression of B. floridae Pitx is similar to that reported from B. belcheri , a different amphioxus species. Expression of the Ciona Pitx ortholog in the embryonic primordial pharynx and adult neural complex leads us to propose the Ciona primordial pharynx and ciliated funnel are homologous to the adenohypophyseal placode and adenohypophysis, respectively. Additionally, in both species we identify asymmetrical left-sided expression of Pitx genes during embryonic development. This shows that asymmetrical Pitx gene expression, and by inference directional asymmetry, evolved before the radiation of living chordates and should be considered a chordate character. [ABSTRACT FROM AUTHOR]

Sperm activating and -attracting factor (SAAF), derived from the egg of the ascidian Ciona, activates sperm motility through adenosine 3′:5′-cyclic monophosphate (cAMP)-synthesis. A demembranated preparation of intact immotile sperm without SAAF was shown to require cAMP for reactivation. However, a demembranated preparation of intact motile sperm treated with SAAF did not require cAMP for reactivation, suggesting that cAMP is a prerequisite factor for SAAF-dependent activation of sperm motility. Furthermore, a cAMP-dependent protein kinase (PKA) inhibitor, H-89, was found to inhibit sperm motility. During in vivo or in vitro activation of sperm motility by SAAF or cAMP, a 26 kDa axonemal protein and 21 kDa dynein light chain were phosphorylated, respectively, suggesting the involvement of PKA-dependent phosphorylation of these proteins in sperm activation. The calmodulin antagonist, W-7, and an inhibitor of calmodulin-dependent myosin light chain kinase, ML-7, also inhibited the activation of sperm motility. Inhibition was reversed by the addition of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Demembranated preparations of immotile sperm in the presence of W-7 or ML-7 were reactivated by cAMP, suggesting that calmodulin participated in sperm activation and that cAMP synthesis was followed by activation of a calmodulin-dependent mechanism. [ABSTRACT FROM AUTHOR]

Previous reports of tunicate endostyles have suggested that they contain little or no acidic glycoproteins in the glandular zones The endostyles of Ciona intestinalis and Styela plicata were examined after anhydrous fixation with cyanuric chloride PoIyanions were stained with alcian blue (AB) at pH 2.5 or azure A while sulfomucins were stained with high-iron diamine (HID) or AB at pH 1.0. Endostyles were also tested for sensitivity to acid hydrolysis (AU) and saponification. In Ciona zones 2 and 4 sometimes demonstrated positive HID and AB 10 responses. Almost invariably zone 6 was AB+ at pH 2. 5 zones 2 and 4 acre frequently responsive to AB hut less intense Each ot these 3 zones when AB+ was sensitive to AU Responses by zones 3 and 5 to AB (pH 2.5) azure A and saponification suggest that these zones contain mostly nuclear material In secretory zones 2.4 and 6 histological responses are consistent with the histochemistry of sialomucins Zones 1 and 8 had sulfated material in the apical edges in both animal groups. Among the fixatives used for only anhydrous fixation demonstrated most of the positive responses to polyanion-sensitive stains [ABSTRACT FROM AUTHOR]

Comments on an article on gene sequences from the tunicate Ciona intestinalis. Information on the Genomic Potential Hypothesis; Methods prone to contamination; Reason polymorphism would contradict the Genomic Potential Hypothesis.

The capacity for ascidians to inhabit coastal sea floor worldwide relies on their peculiar tolerance to environmental variables and pollution, which is considered, together with high levels of genetic diversity, among the main drivers of their invasive potential. In spite of the continued interest in the genetics of invasive species, little attention has been paid toward the microevolutionary processes that drive structure and fate of ascidian populations over time under chemically polluted conditions. Understanding the interplay between environmental and population dynamics is critical to predict the biodiversity of marine coastal ecosystems. In the present study, a local population of the ascidian Ciona robusta living in the Fusaro Lagoon has been monitored over a 13‐month period of sampling. Physico‐chemical parameters (temperature, salinity, turbidity, dissolved oxygen, heavy metals), genetic composition (microsatellites, ITS‐2), abundance and biomass (wet and dry weight) were assessed with the aim to infer fine‐scale temporal variation of population structure with respect to rapid environmental change. Analysis of biomass showed that C. robusta is highly sensitive to salinity and oxygen concentrations. Further, genetic analysis suggested a highly dynamic population structure, likely due to the strong clustering of temporal samples and distinct responses to environmental conditions, including bioaccumulation of heavy metals. Here, we hypothesize that rapid variation in allele frequencies of neutral markers in C. robusta populations may increase the ability of the species to colonize habitats that are subject to strong variation and are under heavy human pressure. [ABSTRACT FROM AUTHOR]