Akiko Hozumi

Molecular cloning and characterization of a thioredoxin/nucleoside diphosphate kinase related dynein intermediate chain from the ascidian, Ciona intestinalis

Flagellar outer arm dynein from the ascidian, Ciona intestinalis, contains five intermediate chains (IC1-5). Molecular cloning of C. intestinalis IC3 shows significant sequence homology to the dynein intermediate chain (IC1) from sea urchin and human NM23-H8 protein. The N-terminal thioredoxin-related region is well conserved in the C. intestinalis IC3, sea urchin IC1, and human NM23-H8 protein. Three NDP kinase (NDPK)-related sequences are present in middle portions of both C. intestinalis IC3 and sea urchin IC1, but the human NM23-H8 protein had only two. A large part of the C-terminal glutamic acid-rich region present in sea urchin IC1 was greatly reduced in C. intestinalis IC3 and completely lost in human NM23-H8. Thus, thioredoxin/NDPK-related dynein intermediate chains (TNDK-DIC) would be a characteristic of metazoan flagella and they have become smaller in size and less acidic during evolution.

CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis

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 (sgRNA) and Cas9 mRNA, 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 sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA 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.

Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.

Molecular characterization of axonemal proteins and signaling molecules responsible for chemoattractant-induced sperm activation in Ciona intestinalis

Spermatozoa undergo dramatic physiological changes at fertilization. In the ascidian Ciona intestinalis, an egg-derived substance named SAAF induces both sperm activation and chemotaxis to the egg. To elucidate the molecular mechanism underlying these phenomena, whole sperm proteins before and after SAAF-treatment were analyzed by two-dimensional gel electrophoresis. By comparison of spot patterns before and after activation, we found twelve proteins that changed the isoelectric points. Seven proteins were shown to be axonemal proteins and others were suggested to be non-axonemal components. Analysis of these proteins by MS-based proteomic system revealed that components of several substructures of the axonemes underwent the changes in isoelectric point at sperm activation, including WD-repeat intermediate chains of outer and inner arm dyneins and a radial spoke protein LRR37, as well as novel axonemal proteins with armadillo repeats or SMC domain. Molecules for cell signaling such as 14-3-3 proteins, Skp1 and VCP/p97 also showed isoelectric changes at sperm activation. These results show a comprehensive feature for signaling mechanism of the activation of spermatozoa at fertilization and also shed new lights on the regulation of ciliary and flagellar movements.

Efficient transposition of a single Minos transposon copy in the genome of the ascidian Ciona intestinalis with a transgenic line expressing transposase in eggs.

Transgenesis with transposons is an important technique for studying genetic functions. In the ascidian Ciona intestinalis, methods for germline transformation with the Tc1/mariner transposon Minos have been established. A system to remobilize a single Minos copy in the genome is needed to refine this transgenic technique. In this study, such an experimental system was established with a transgenic line expressing Minos transposase in eggs. In the eggs of a double transgenic animal from a cross between the egg transposase line and a transgenic line having a single Minos insertion, the transposon was transposed into new positions of the Ciona genome, thus creating new insertions. Some of the new insertions caused enhancer detection. The majority of the new insertion sites were mapped on different chromosomes from that of the transposon donor. This characteristic of Minos is in contrast to that of the Sleeping Beauty transposon, which causes frequent intrachromosomal transposition. Developmental Dynamics 239:1076–1088, 2010.

Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases.

Summary: 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. genesis 52:431–439, 2014.

Maternal factor-mediated epigenetic gene silencing in the ascidian Ciona intestinalis

Epigenetic regulation of genes plays a critical role in achieving proper gene expression during development, and it has been reported that epigenetic modifications are associated with transposon silencing in many organisms. Here, we report a type of epigenetic gene silencing, maternal gfp/gene silencing (MGS), in the basal chordate Ciona intestinalis. A transgenic line of Ciona, Tg[MiTFr3dTPOG]45 (abbreviated as Tg45), which was created with the Minos transposon, has a tandemly arrayed insertion of gfp in the promoter region of Ci-CesA. Progeny of Tg45 showed a reduced level of GFP expression when eggs of Tg45 were fertilized with sperm of other gfp transgenic lines. Although the genotype is the same, animals developed from Tg45 sperm and the eggs of other transgenic lines did not exhibit this phenomenon, suggesting the involvement of a maternal cytoplasmic factor that influences GFP expression. The silencing starts during oogenesis and continues after fertilization without any tissue specificity. We found that post-transcriptional degradation of the gfp mRNA is involved in MGS.

Functional proteomics in Ciona intestinalis: a breakthrough in the exploration of the molecular and cellular mechanism of ascidian development.

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. Developmental Dynamics 236:1782–1789, 2007.

Neuronal map reveals the highly regionalized pattern of the juvenile central nervous system of the ascidian Ciona intestinalis

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.

Germline transgenesis of the chordate Ciona intestinalis with hyperactive variants of sleeping beauty transposable element.

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.