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Background: Ascidians significantly change their body structure through metamorphosis, but the spatio-temporal cell dynamics in the early metamorphosis stage has not been clarified. A natural Ciona embryo is surrounded by maternally derived non-self-test cells before metamorphosis. However, after metamorphosis, the juvenile is surrounded by self-tunic cells derived from mesenchymal cell lineages. Both test cells and tunic cells are thought to be changed their distributions during metamorphosis, but the precise timing is unknown., Results: Using a metamorphosis induction by mechanical stimulation, we investigated the dynamics of mesenchymal cells during metamorphosis in a precise time course. After the stimulation, two-round Ca 2+ transients were observed. Migrating mesenchymal cells came out through the epidermis within 10 min after the second phase. We named this event "cell extravasation." The cell extravasation occurred at the same time as the backward movement of posterior trunk epidermal cells. Timelapse imaging of transgenic-line larva revealed that non-self-test cells and self-tunic cells temporarily coexist outside the body until the test cells are eliminated. At the juvenile stage, only extravasated self-tunic cells remained outside the body., Conclusions: We found that mesenchymal cells extravasated following two-round Ca 2+ transients, and distributions of test cells and tunic cells changed in the outer body after tail regression., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

The endostyle is a ventral pharyngeal organ used for internal filter feeding of basal chordates and is considered homologous to the follicular thyroid of vertebrates. It contains mucus-producing (glandular) and thyroid-equivalent regions organized along the dorsoventral (DV) axis. Although thyroid-related genes (Nkx2-1, FoxE, and thyroid peroxidase (TPO)) are known to be expressed in the endostyle, their roles in establishing regionalization within the organ have not been demonstrated. We report that Nkx2-1 and FoxE are essential for establishing DV axial identity in the endostyle of Oikopleura dioica. Genome and expression analyses showed von Willebrand factor-like (vWFL) and TPO/dual oxidase (Duox)/Nkx2-1/FoxE as orthologs of glandular and thyroid-related genes, respectively. Knockdown experiments showed that Nkx2-1 is necessary for the expression of glandular and thyroid-related genes, whereas FoxE is necessary only for thyroid-related genes. Moreover, Nkx2-1 expression is necessary for FoxE expression in larvae during organogenesis. The results demonstrate the essential roles of Nkx2-1 and FoxE in establishing regionalization in the endostyle, including (1) the Nkx2-1-dependent glandular region, and (2) the Nkx2-1/FoxE-dependent thyroid-equivalent region. DV axial regionalization may be responsible for organizing glandular and thyroid-equivalent traits of the pharynx along the DV axis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The representative characteristic of ascidians is their vertebrate-like, tadpole shape at the larval stage. Ascidians lose the tadpole shape through metamorphosis to become adults with a nonmotile, sessile body and a shape generally considered distinct from that of vertebrates. Solitary ascidians including Ciona species are extensively studied to understand the developmental mechanisms of ascidians, and to compare these mechanisms with their counterparts in vertebrates. In these ascidian species, the digestive and circulatory systems are not well developed in the larval trunk and the larvae do not take food. This is in contrast with the inner conditions of vertebrate tadpoles, which have functional organs comparable to those of adults. The adult organs and tissues of these ascidians become functional during metamorphosis that is completed quickly, suggesting that the ascidian larvae of solitary species are a transient stage of development. We here discuss how the cells and tissues in the ascidian larval body are converted into those of adults. The hearts of ascidians and vertebrates use closely related cellular and molecular mechanisms that suggest their shared origin. Hox genes of ascidians are essential for forming adult endodermal structures. To fully understand the development and evolution of chordates, a complete elucidation of the mechanisms underlying the adult tissue/organ formation of ascidians will be needed. WIREs Dev Biol 2018, 7:e304. doi: 10.1002/wdev.304 This article is categorized under: Comparative Development and Evolution > Body Plan Evolution Early Embryonic Development > Development to the Basic Body Plan., (© 2017 Wiley Periodicals, Inc.)

The chordate ascidians, the major group of tunicate s, is the best animal group for studying molecular and cellular processes underlying formation of a chordate body plan. For these studies, transgenic technologies are powerful. Transgenesis of ascidians has a long history of more than 20 years, and many practical tips have been accumulated. This book is aimed at summarizing the accumulated techniques in ascidians in addition to concrete research in which transgenic techniques have played pivotal roles. This book is useful for fast assimilation of the techniques and for learning the unique devices developed by the enthusiasm of ascidian researchers.

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GC-rich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event., (© 2016 The Author(s).)

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Embryonic and postembryonic development in ascidians have been studied for over a century, but it is only in the last 10 years that the complex molecular network involved in coordinating postlarval development and metamorphosis has started to emerge. In most ascidians, the transition from the larval to the sessile juvenile/adult stage, or metamorphosis, requires a combination of environmental and endogenous signals and is characterized by coordinated global morphogenetic changes that are initiated by the adhesion of the larvae. Cloney was the first to describe cellular events of ascidians' metamorphosis in 1978 and only recently elements of the molecular regulation of this crucial developmental step have been revealed. This review aims to present a thorough view of this crucial developmental step by combining recent molecular data to the already established cellular events., (© 2014 Wiley Periodicals, Inc.)

Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans, and appendicularians. With the advent of inexpensive high-throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines., (© 2014 Wiley Periodicals, Inc.)

The larvae of non-vertebrate chordate ascidians consist of countable numbers of cells. With this feature, ascidians provide us with excellent models for studying cellular events in the construction of the chordate body. This review discusses the recent observations of morphogenetic movements and cell cycles and divisions along with tissue specifications during ascidian embryogenesis. Unequal cleavages take place at the posterior blastomeres during the early cleavage stages of ascidians, and the structure named the centrosome-attracting body restricts the position of the nuclei near the posterior pole to achieve the unequal cleavages. The most-posterior cells differentiate into the primordial germ cells. The gastrulation of ascidians starts as early as the 110-cell stage. During gastrulation, the endodermal cells show two-step changes in cell shape that are crucial for gastrulation. The ascidian notochord is composed of only 40 cells. The 40 cells align to form a single row by an event named the convergent extension, and then the notochord cells undergo vacuolation to transform the notochord into a single hollowed tube. The strictly restricted number of notochord cells is achieved by the regulated number of cell divisions coupled with the differentiation of the cells conducted by a key transcription factor, Brachyury. The dorsally located neural tube is a characteristic of chordates. During the closure of the ascidian neural tube, the epidermis surrounding the neural plate moves toward the midline to close the neural fold. This morphogenetic movement is allowed by an elongation of interphase in the epidermal cell cycles.

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.

The ascidian larva is an excellent model for studies of the functional organization and neuronal circuits of chordates due to its remarkably simple central nervous system (CNS), comprised of about 100 neurons. To date, however, the identities of the various neurons in the ascidian larva, particularly their neurotransmitter phenotypes, are not well established. Acetylcholine, GABA, and glycine are critical neurotransmitters for locomotion in many animals. We visualized putative cholinergic neurons and GABAergic/glycinergic neurons in the ascidian larva by immunofluorescent staining using antibodies against vesicular acetylcholine transporter (VACHT) and vesicular GABA/glycine transporter (VGAT), respectively. Neurons expressing a cholinergic phenotype were found in the brain vesicle and the visceral ganglion. Five pairs of VACHT-positive neurons were located in the visceral ganglion. These putative cholinergic neurons extended their axons posteriorly and formed nerve terminals proximal to the most anterior muscle cells in the tail. VGAT-positive neurons were located in the brain vesicle, the visceral ganglion, and the anterior nerve cord. Two distinct pairs of VGAT-positive neurons, bilaterally aligned along the anterior nerve cord, extended axons anteriorly, near to the axons of the contralateral VACHT-positive neurons. Cell bodies of the VGAT-positive neurons lay on these nerve tracts. The neuronal complex, comprising motor neurons with a cholinergic phenotype and some of the GABA/glycinergic interneurons, has structural features that are compatible with a central pattern generator (CPG) producing a rhythmic movement of the tail. The simple CPG of the ascidian larva may represent the ancestral state of the vertebrate motor system.

Ascidians, or sea squirts, are primitive chordates, and their tadpole larvae share a basic body plan with vertebrates, including a notochord and a dorsal tubular central nervous system (CNS). The CNS of the ascidian larva is formed through a process similar to vertebrate neurulation, while the ascidian CNS is remarkably simple, consisting of about 100 neurons. Recent identification of genes that are specifically expressed in a particular subtype of neurons has enabled us to reveal neuronal networks at single-cell resolution. Based on the information on neuron subtype-specific genes, different populations of neurons have been visualized by whole-mount in situ hybridization, immunohistochemical staining using specific antibodies, and fluorescence labeling of cell bodies and neurites by a fluorescence protein reporter driven by neuron-specific promoters. Neuronal populations that have been successfully visualized include glutamatergic, cholinergic, gamma-aminobutyric acid/glycinergic, and dopaminergic neurons, which have allowed us to propose functional regionalization of the CNS and a neural circuit for locomotion. Thus, the simple nervous system of the ascidian larva can serve as an attractive model system for studying the development, function, and evolution of the chordate nervous system.

Ascidian is a good model to understand the cellular and molecular mechanisms responsible for mRNA localization with the discovery of a large family of localized maternal mRNAs, called postplasmic/PEM RNAs, which includes more than 40 members in three different ascidian species (Halocynthia roretzi, Ciona intestinalis, and C. savignyi). Among these mRNAs, two types (Type I and Type II) have been identified and show two different localization patterns from fertilization to the eight-cell stage. At the eight-cell stage, both types concentrate to a macromolecular cortical structure called CAB (for Centrosome Attracting Body) in the posterior-vegetal B4.1 blastomeres. The CAB is responsible for unequal cleavages and the partitioning of postplasmic/PEM RNAs at the posterior pole of embryos during cleavage stages. It has also been suggested that the CAB region could contain putative germ granules. In this review, we discuss recent data obtained on the distribution of Type I postplasmic/PEM RNAs from oogenesis to late development, in relation to their localization and translational control. We have first regrouped localization patterns for Type I and Type II into a comparative diagram and included all important definitions in the field. We also have made an exhaustive classification of their embryonic expression profiles (Type I or Type II), and analyzed their functions after knockdown and/or overexpression experiments and the role of the 3'-untranslated region (3'UTR) controlling both their localization and translation. Finally, we propose a speculative model integrating recent data, and we also discuss the relationship between postplasmic/PEM RNAs, posterior specification, and germ cell formation in ascidians., (Copyright 2007 Wiley-Liss, Inc.)

An ascidian, Ciona savignyi, is regarded as a good experimental animal for genetics because of its small and compact genome for which a draft sequence is available, its short generation time and its interesting phylogenic position. ENU-based mutagenesis has been carried out using this animal. However, insertional mutagenesis using transposable elements (transposons) has not yet been introduced. Recently, one of the Tc1/mariner superfamily transposons, Minos, was demonstrated to cause germline transgenesis in the related species Ciona intestinalis. In this report, we show that Minos has the ability to transpose from DNA to DNA in Ciona savignyi in transposition assays. Although the activity was slightly weaker than in Ciona intestinalis, Minos still caused germline transgenesis in Ciona savignyi. In addition, one insertion seemed to have caused an enhancer trapping. These results indicate that Minos provides a potential tool for transgenic techniques such as insertional mutagenesis in Ciona savignyi.

During ascidian embryogenesis, some mRNAs show clear localization at the posterior-most region. These postplasmic mRNAs are divided into two groups (type I and type II) according to their pattern of localization. To elucidate how these localization patterns are achieved, we attempted to identify the localization elements of these mRNAs. When in vitro synthesized postplasmic mRNAs were introduced into eggs, these mRNAs showed posterior localization similar to the endogenous mRNAs. The posterior localization of these mRNAs was mediated by their 3' untranslated regions (3' UTRs), as is the case for several localized Drosophila and Xenopus mRNAs. We identified smaller fragments of the 3' UTRs of HrWnt-5 and HrPOPK-1 mRNAs (type I) and HrPet-3 mRNA (type II) that were sufficient to direct green fluorescent protein mRNA to the posterior pole. For the localization of HrWnt-5 mRNA, two UG dinucleotide repetitive elements were essential. Motifs similar to these small elements also exist within the HrPOPK-1 mRNA localization element and 3' UTR of HrZF-1 mRNA, suggesting the conservation of localization elements among type I mRNAs. In contrast, the smallest sequence that suffices for the posterior localization of HrPet-3 (a type II mRNA) has different features from those of type I mRNAs; indeed, it does not have an identifiable critical element. This difference may distinguish type II mRNAs from type I mRNAs. These findings, especially the identification of the small localization element of HrWnt-5 mRNA, provide new insights into the localization of mRNAs during ascidian embryogenesis.

The ascidian egg is a well-known mosaic egg. In order to investigate the molecular nature of the maternal genetic information stored in the egg, we have prepared cDNAs from the mRNAs in the fertilized eggs of the ascidian, Halocynthia roretzi. The cDNAs of the ascidian embryo were sequenced, and the localization of individual mRNA was examined in staged embryos by whole-mount in situ hybridization. The data obtained were stored in the database MAGEST (http://www.genome.ad.jp/magest) and further analyzed. A total of 4240 cDNA clones were found to represent 2221 gene transcripts, including at least 934 different protein-coding sequences. The mRNA population of the egg consisted of a low prevalence, high complexity sequence set. The majority of the clones were of the rare sequence class, and of these, 42% of the clones showed significant matches with known peptides, mainly consisting of proteins with housekeeping functions such as metabolism and cell division. In addition, we found cDNAs encoding components involved in different signal transduction pathways and cDNAs encoding nucleotide-binding proteins. Large-scale analyses of the distribution of the RNA corresponding to each cDNA in the eight-cell, 110-cell and early tailbud embryos were simultaneously carried out. These analyses revealed that a small fraction of the maternal RNAs were localized in the eight-cell embryo, and that 7.9% of the clones were exclusively maternal, while 40.6% of the maternal clones showed expression in the later stages. This study provides global insights about the genes expressed during early development.

In vertebrate embryogenesis, many Wnt genes are expressed in the neural tube and play important roles in regional specifications. There are many subfamilies of Wnt, and each subfamily shows distinct expression patterns in the neural tube. Ascidian larvae have a dorsal hollow neural tube similar to that of vertebrates. To date, the degree of correspondence between regionality of the neural tubes of ascidians and vertebrates remains unclear. To compare cellular differences in neural tubes, Wnt genes can be used as molecular probes. We report here that a new member of the ascidian Wnt gene family, HrWnt-7, was expressed in the tail neural tube at the early tailbud stage. Moreover, in cross-section, HrWnt-7 was expressed in the dorsal and ventral ependymal cells.

A cDNA library prepared from fertilized eggs of the ascidian Halocynthia roretzi was screened for prelocalized mRNAs in the early embryo by means of whole-mount in situ hybridization using a digoxigenin-labeled antisense RNA of each clone. Random mass screening of 150 cDNAs in a fertilized egg yielded six different clones which showed mRNA localization in the posterior-vegetal cytoplasm of the 8-cell embryo. An in situ hybridization study of the detailed spatial distribution of each mRNA in embryos of various stages revealed that there are, in contrast to the identical localization in embryos after the 16-cell stage, two distinct patterns of RNA distribution at earlier stages. One is colocalization with the myoplasm from the prefertilization stage to the 8-cell stage (type I postplasmic RNAs). The other is delayed accumulation of RNA at the posterior-vegetal cytoplasm after fertilization (type II postplasmic RNAs). We found that both types of RNAs associate with the cytoskeleton, but that they show different sensitivities to inhibitors of the cytoskeleton; translocation of the type I RNAs is dependent upon microfilaments during the first phase of ooplasmic segregation and dependent upon microtubules during the second phase of segregation, whereas translocation of the type II RNAs is dependent upon microfilaments throughout ooplasmic segregation. These results show that there are two pathways for the localization of the RNAs at the posterior-vegetal cytoplasm in the 8-cell embryo of the ascidian H. roretzi., (Copyright 2000 Academic Press.)

The gene family encoding RNA-binding proteins includes important regulators involved in the neurogenesis in both protostomes and deuterostomes. We isolated cDNAs of the ascidian homolog of one of the RNA-binding proteins, MUSASHI, from Halocynthia roretzi and Ciona intestinalis. The predicted amino acid sequences contained two RNA-recognition and RNA-binding motifs in the N-terminus and an ascidian-specific YG-rich domain in the C-terminus. Maternal transcripts of musashi were ubiquitous in early cleavage-stage embryos. Ascidian musashi had three domains of zygotic expression: the brain, nerve cord, and mesenchyma. The temporal order of the onset in these domains was highly divergent between the two species of ascidian examined.

Expressed Sequence Tag (EST) research on the ascidian Halocynthia roretzi revealed that Hrsmad1/5, a homolog of smad genes, is expressed in H. roretzi eggs. A comparison of amino acid sequences of smad family members showed that the isolated clone was a homolog of smad1 and smad5 of vertebrates. A molecular phylogenetic analysis showed that Hrsmad1/5 was separated from the common ancestor with the group containing smad1 and smad5. A northern blot analysis showed that transcript of Hrsmad1/5 was abundant in the fertilized egg. The amount of the transcript remained constant until the gastrulae and then rapidly decreased at the neurulae. The spatial expression of Hrsmad1/5 was investigated by means of whole-mount in situ hybridization. Maternal transcripts of Hrsmad1/5 were detected in the entire fertilized egg. The signals were localized preferentially to the animal blastomeres of the 8-, 16-, 32- and 64-cell stages. The zygotic expression of Hrsmad1/5 started in prospective epidermal blastomeres in the animal hemisphere at the 64-cell stage but not in cells of the central nervous system, and it decreased rapidly around the neural-plate stage. At the tail-bud stage, signals were detected broadly all through the trunk and in a small part of the epidermis in the tail region. This is the first report of a maternal RNA that preferentially accumulates in the animal hemisphere in early ascidian embryos. Animal blastomeres of ascidian embryos differentiate mainly into epidermis in a cell-autonomous manner and partly differentiate into neural tissues by induction. The Hrsmad1/5 gene may play a role in the signal transduction process in epidermal precursor cells of ascidian embryos.

Maternally localized cytoplasmic determinants play important roles in the embryogenesis of many animals, including ascidians. Cytoplasmic determinants are particularly important in the determination of cell fates, and in the establishment of the embryonic axes. Ascidians, which show mosaic development, are good models for the study of maternal cytoplasmic determinants. Here we report the isolation and characterization of HrPOPK-1 (Halocynthia roretzi posterior protein kinase-1), a putative protein serine/threonine kinase. HrPOPK-1 cDNA was obtained from a Halocynthia roretzi fertilized egg cDNA library by screening for localized RNAs using whole-mount in situ hybridization. HrPOPK-1 mRNA is strongly localized at the posterior pole of embryos. The pattern of HrPOPK-1 mRNA localization during early embryogenesis is identical to that of HrWnt-5 in Halocynthia roretzi, and to those of the posterior end mark (pem) transcripts of Ciona savignyi. In addition, HrPOPK-1 shows zygotic expression in neural tissues at the tailbud stage. These results show that the temporal regulation of HrPOPK-1 transcription is complex., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved.)

Ascidians show a highly determinate mode of development. In particular, components of the posterior-vegetal cytoplasm of fertilized eggs are responsible for the establishment of the embryonic axis. Recent studies have, however, also revealed significant roles of cell-cell interactions during embryogenesis. Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide range of developmental processes. Here we have isolated and characterized an ascidian Wnt gene, HrWnt-5, from Halocynthia roretzi. HrWnt-5 mRNA is present in the vegetal cortex in unfertilized eggs. After fertilization, HrWnt-5 mRNA moves to the equatorial region to form a crescent-like structure, after which the mRNA is concentrated in the posteriormost region of the embryo. This early pattern of HrWnt-5 mRNA localization coincides with another posterior-vegetally localized mRNA, pem, isolated from Ciona savignyi. In the gastrula, the zygotic HrWnt-5 mRNA is found in a variety of blastomeres, suggesting multiple roles of the gene.