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Background: The dorsally located central nervous system (CNS) is an important hallmark of chordates. Among chordates, tunicate ascidians change their CNS remarkably by means of a metamorphosis from a highly regionalized larval CNS to an oval-shaped juvenile CNS without prominent morphological features. The neuronal organization of the CNS of ascidian tadpole larvae has been well described, but that in the CNS of postmetamorphosis juveniles has not been characterized well. Results: We investigated the number of neural cells, the number and position of differentiated neurons, and their axonal trajectories in the juvenile CNS of the ascidian Ciona intestinalis. The cell bodies of cholinergic, glutamatergic, and GABAergic/glycinergic neurons exhibited different localization patterns along the anterior-posterior axis in the juvenile CNS. Cholinergic neurons extended their axons toward the oral, atrial and body wall muscles and pharyngeal gill to regulate muscle contraction and ciliary movement. Conclusions: Unlike its featureless shape, the juvenile CNS is highly patterned along the anterior-posterior axis. This patterning may be necessary for exerting multiple roles in the regulation of adult tissues distributed throughout the body. This basic information of the juvenile CNS of Ciona will allow in-depth studies of molecular mechanisms underlying the reconstruction of the CNS during ascidian metamorphosis. Developmental Dynamics 244:1375-1393, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Knockout of genes with CRISPR/ Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/ Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sg RNA) and Cas9 m RNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sg RNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sg RNA dramatically reduced mutation efficiency at the on-target site. CRISPR/ Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases. [ABSTRACT FROM AUTHOR]

Background: Transposon-mediated transgenesis is an excellent method for creating stable transgenic lines and insertional mutants. In the chordate Ciona intestinalis, Minos is the only transposon that has been used as the tool for germline transformation. Adding another transposon system in this organism enables us to conduct genetic techniques which can only be realized with the use of two transposons. Results: In the present study, we found that another Tc1/ mariner superfamily transposon, sleeping beauty (SB), retains sufficient activity for germline transformation of C. intestinalis. SB shows efficiencies of germline transformation, insertion into gene coding regions, and enhancer detection comparable to those of Minos. We have developed a system for the remobilization of SB copies in the C. intestinalis genome by using transgenic lines expressing SB transposase in the germ cells. With this system, we examined the manner of SB mobilization in the C. intestinalis genome. SB shows intrachromosomal transposition more frequently than Minos. Conclusions: SB-based germline transformation and the establishment of a new method that uses its frequent intrachromosomal transposition will result in breakthroughs in genetic approaches that use C. intestinalis together with Minos. Developmental Dynamics 242:30-43, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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.)

Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis., (© 2014 Wiley Periodicals, Inc.)

Ascidians have been providing a unique experimental system for a variety of fields, including reproductive biology, developmental biology, neurobiology, immunology, and evolutional biology. Recent progress in the genome sequencing of Ciona intestinalis has led to the development of a great tool for investigating the gene functions and expressions involved in several biological events in ascidians. The disclosure of genomic information has ushered in the postgenomic era, spearheaded by extensive protein analysis. The characterization of the function, localization, and molecular interaction of cellular proteins results in a more direct description of the molecular mechanism underlying several biological processes. Proteomics in ascidians, however, has just recently appeared and is not well established yet. In this study, we give an outline of the technical processes used in proteomics and review the recent status of ascidian proteomics., (Copyright 2007 Wiley-Liss, Inc.)

Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2., (Copyright 2006 Wiley-Liss, Inc.)