Language：English   Publishing type：Research paper (scientific journal)  

The centrohelid heliozoan Raphidocystis contractilis has many radiating axopodia, each containing axopodial microtubules. The axopodia show rapid contraction at nearly a video rate (30 frames per second) in response to mechanical stimuli. The axopodial contraction is accompanied by cytoskeletal microtubule depolymerization, but the molecular mechanism of this phenomenon has not been elucidated. In this study, we performed de novo transcriptome sequencing of R. contractilis to identify genes involved in microtubule dynamics such as the rapid axopodial contraction. The transcriptome sequencing generated 7.15-Gbp clean reads in total, which were assembled as 31,771 unigenes. Using the obtained gene sets, we identified several microtubule-severing proteins which might be involved in the rapid axopodial contraction, and kinesin-like genes that occur gene duplication. On the other hand, some genes for microtubule motor proteins involved in the formation and motility of flagella were not found in R. contractilis, suggesting that the gene repertoire of R. contractilis reflected the morphological features of non-flagellated protists. Our transcriptome analysis provides basic information for the analysis of the molecular mechanism underlying microtubule dynamics in R. contractilis.

DOI： 10.1111/jeu.12955

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.24480/bsj-review.13a3.00221

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Language：English   Publishing type：Research paper (scientific journal)  

d-Serine, a free amino acid synthesized by serine racemase, is a coagonist of N-methyl-d-aspartate-type glutamate receptor (NMDAR). d-Serine in the mammalian central nervous system modulates glutamatergic transmission. Functions of d-serine in mammalian peripheral tissues such as skin have also been described. However, d-serine's functions in nonmammals are unclear. Here, we characterized d-serine-dependent vesicle release from the epidermis during metamorphosis of the tunicate Ciona. d-Serine leads to the formation of a pocket that facilitates the arrival of migrating tissue during tail regression. NMDAR is the receptor of d-serine in the formation of the epidermal pocket. The epidermal pocket is formed by the release of epidermal vesicles' content mediated by d-serine/NMDAR. This mechanism is similar to observations of keratinocyte vesicle exocytosis in mammalian skin. Our findings provide a better understanding of the maintenance of epidermal homeostasis in animals and contribute to further evolutionary perspectives of d-amino acid function among metazoans.

DOI： 10.1126/sciadv.abn3264

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Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

Vasopressin/oxytocin (VP/OT)–related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the “platytocin” system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an “antidiuretic hormone” and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

DOI： 10.1126/sciadv.abk0331

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

The dynamics of microscopic marine plankton in coastal areas is a fundamental theme in marine biodiversity research, but studies have been limited because the only available methodology was collection of plankton using plankton-nets and microscopic observation. In recent years, environmental DNA (eDNA) analysis has exhibited potential for conducting comprehensive surveys of marine plankton diversity in water at fixed points and depths in the ocean. However, few studies have examined how eDNA analysis reflects the actual distribution and dynamics of organisms in the field, and further investigation is needed to determine whether it can detect distinct differences in plankton density in the field. To address this, we analyzed eDNA in seawater samples collected at 1 km intervals at three depths over a linear distance of approximately 3.0 km in the Seto Inland Sea. The survey area included a location with a high density of Acoela (Praesagittifera naikaiensis). However, the eDNA signal for this was little to none, and its presence would not have been noticed if we did not have this information beforehand. Meanwhile, eDNA analysis enabled us to confirm the presence of a species of Placozoa that was previously undiscovered in the area. In summary, our results suggest that the number of sequence reads generated from eDNA samples in our project was not sufficient to predict the density of a particular species. However, eDNA can be useful for detecting organisms that have been overlooked using other methods.

DOI： 10.2108/zs210073

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Bombesin is a putative antibacterial peptide isolated from the skin of the frog, <italic>Bombina bombina</italic>. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (<italic>Xenopus tropicalis</italic>), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in <italic>Xenopus</italic>. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of <italic>Xenopus</italic>. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the <italic>‘gut-brain peptide’</italic> systems.

DOI： 10.1038/s41598-021-92528-x

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Other Link： http://www.nature.com/articles/s41598-021-92528-x

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Various <italic>Hydra</italic> species have been employed as model organisms since the 18^th century. Introduction of transgenic and knock-down technologies made them ideal experimental systems for studying cellular and molecular mechanisms involved in regeneration, body-axis formation, senescence, symbiosis, and holobiosis. In order to provide an important reference for genetic studies, the <italic>Hydra magnipapillata</italic> genome (species name has been changed to <italic>H. vulgaris</italic>) was sequenced a decade ago (Chapman <italic>et al.</italic>, 2010) and the updated genome assembly, Hydra 2.0, was made available by the National Human Genome Research Institute in 2017. While <italic>H. vulgaris</italic> belongs to the non-symbiotic brown hydra lineage, the green hydra, <italic>Hydra viridissima</italic>, harbors algal symbionts and belongs to an early diverging clade that separated from the common ancestor of brown and green hydra lineages at least 100 million years ago (Schwentner and Bosch 2015; Khalturin <italic>et al.</italic>, 2019). While interspecific interactions between <italic>H. viridissima</italic> and endosymbiotic unicellular green algae of the genus <italic>Chlorella</italic> have been a subject of interest for decades, genomic information about green hydras was nonexistent. Here we report a draft 280-Mbp genome assembly for <italic>Hydra viridissima</italic> strain A99, with a scaffold N50 of 1.1 Mbp. The <italic>H. viridissima</italic> genome contains an estimated 21,476 protein-coding genes. Comparative analysis of Pfam domains and orthologous proteins highlights characteristic features of <italic>H. viridissima</italic>, such as diversification of innate immunity genes that are important for host-symbiont interactions. Thus, the <italic>H. viridissima</italic> assembly provides an important hydrozoan genome reference that will facilitate symbiosis research and better comparisons of metazoan genome architectures.

DOI： 10.1534/g3.120.401411

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Metamorphosis, a widespread life history strategy in metazoans, allows dispersal and use of different ecological niches through a dramatic body change from a larval stage [1, 2]. Despite its conservation and importance, the molecular mechanisms underlying its initiation and progression have been characterized in only a few animal models. In this study, through pharmacological and gene functional analyses, we identified neurotransmitters responsible for metamorphosis of the ascidian Ciona. Ciona metamorphosis converts swimming tadpole larvae into vase-like, sessile adults. Here, we show that the neurotransmitter GABA is a key regulator of metamorphosis. We found that gonadotropin-releasing hormone (GnRH) is a downstream neuropeptide of GABA. Although GABA is generally thought of as an inhibitory neurotransmitter, we found that it positively regulates secretion of GnRH through the metabotropic GABA receptor during Ciona metamorphosis. GnRH is necessary for reproductive maturation in vertebrates, and GABA is an important excitatory regulator of GnRH in the hypothalamus during puberty [3, 4]. Our findings reveal another role of the GABA-GnRH axis in the regulation of post-embryonic development in chordates.

DOI： 10.1016/j.cub.2020.02.003

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Since its initial publication in 2002, the genome of Ciona intestinalis type A (Ciona robusta), the first genome sequence of an invertebrate chordate, has provided a valuable resource for a wide range of biological studies, including developmental biology, evolutionary biology, and neuroscience. The genome assembly was updated in 2008, and it included 68% of the sequence information in 14 pairs of chromosomes. However, a more contiguous genome is required for analyses of higher order genomic structure and of chromosomal evolution. Here, we provide a new genome assembly for an inbred line of this animal, constructed with short and long sequencing reads and Hi-C data. In this latest assembly, over 95% of the 123 Mb of sequence data was included in the chromosomes. Short sequencing reads predicted a genome size of 114-120 Mb; therefore, it is likely that the current assembly contains almost the entire genome, although this estimate of genome size was smaller than previous estimates. Remapping of the Hi-C data onto the new assembly revealed a large inversion in the genome of the inbred line. Moreover, a comparison of this genome assembly with that of Ciona savignyi, a different species in the same genus, revealed many chromosomal inversions between these two Ciona species, suggesting that such inversions have occurred frequently and have contributed to chromosomal evolution of Ciona species. Thus, the present assembly greatly improves an essential resource for genome-wide studies of ascidians.

DOI： 10.1093/gbe/evz228

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Cnidarians are astonishingly diverse in body form and lifestyle, including the presence of a jellyfish stage in medusozoans and its absence in anthozoans. Here, we sequence the genomes of Aurelia aurita (a scyphozoan) and Morbakka virulenta (a cubozoan) to understand the molecular mechanisms responsible for the origin of the jellyfish body plan. We show that the magnitude of genetic differences between the two jellyfish types is equivalent, on average, to the level of genetic differences between humans and sea urchins in the bilaterian lineage. About one-third of Aurelia genes with jellyfish-specific expression have no matches in the genomes of the coral and sea anemone, indicating that the polyp-to-jellyfish transition requires a combination of conserved and novel, medusozoa-specific genes. While no genomic region is specifically associated with the ability to produce a jellyfish stage, the arrangement of genes involved in the development of a nematocyte-a phylum-specific cell type-is highly structured and conserved in cnidarian genomes; thus, it represents a phylotypic gene cluster.

DOI： 10.1038/s41559-019-0853-y

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Many multicellular organisms rely on symbiotic associations for support of metabolic activity, protection, or energy. Understanding the mechanisms involved in controlling such interactions remains a major challenge. In an unbiased approach we identified key players that control the symbiosis between Hydra viridissima and its photosynthetic symbiont Chlorella sp. A99. We discovered significant up-regulation of Hydra genes encoding a phosphate transporter and glutamine synthetase suggesting regulated nutrition supply between host and symbionts. Interestingly, supplementing the medium with glutamine temporarily supports in vitro growth of the otherwise obligate symbiotic Chlorella, indicating loss of autonomy and dependence on the host. Genome sequencing of Chlorella sp. A99 revealed a large number of amino acid transporters and a degenerated nitrate assimilation pathway, presumably as consequence of the adaptation to the host environment. Our observations portray ancient symbiotic interactions as a codependent partnership in which exchange of nutrients appears to be the primary driving force.

DOI： 10.7554/eLife.35122

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the long-standing use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2015.07.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Hox cluster genes play crucial roles in development of the metazoan antero-posterior axis. Functions of Hox genes in patterning the central nervous system and limb buds are well known. They are also expressed in chordate endodermal tissues, where their roles in endodermal development are still poorly understood. In the invertebrate chordate, Ciona intestinalis, endodermal tissues are in a premature state during the larval stage, and they differentiate into the digestive tract during metamorphosis. In this study, we showed that disruption of a Hox gene, Ci-Hox10, prevented intestinal formation. Ci-Hox10-knock-down larvae displayed defective migration of endodermal strand cells. Formation of a protrusion, which is important for cell migration, was disrupted in these cells. The collagen type IX gene is a downstream target of Ci-Hox10, and is negatively regulated by Ci-Hox10 and a matrix metalloproteinase ortholog, prior to endodermal cell migration. Inhibition of this regulation prevented cellular migration. These results suggest that Ci-Hox10 regulates endodermal strand cell migration by forming a protrusion and by reconstructing the extracellular matrix. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2015.03.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans.

DOI： 10.1002/bies.201400065

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Maternal mRNAs play crucial roles during early embryogenesis of ascidians, but their functions are largely unknown. In this study, we developed a new method to specifically knockdown maternal mRNAs in Ciona intestinalis using transposon-mediated transgenesis. We found that GFP expression is epigenetically silenced in Ciona intestinalis oocytes and eggs, and this epigenetic silencing of GFP was used to develop the knockdown method. When the 59 upstream promoter and 59 untranslated region (UTR) of a maternal gene are used to drive GFP in eggs, the maternal gene is specifically knocked down together with GFP. The 5' UTR of the maternal gene is the major element that determines the target gene silencing. Zygotic transcription of the target gene is unaffected, suggesting that the observed phenotypes specifically reflect the maternal function of the gene. This new method can provide breakthroughs in studying the functions of maternal mRNAs.

DOI： 10.1038/srep05050

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Background Dinoflagellates are known for their capacity to form harmful blooms (e.g., "red tides") and as symbiotic, photosynthetic partners for corals. These unicellular eukaryotes have permanently condensed, liquid-crystalline chromosomes and immense nuclear genome sizes, often several times the size of the human genome. Here we describe the first draft assembly of a dinoflagellate nuclear genome, providing insights into its genome organization and gene inventory. Results Sequencing reads from Symbiodinium minutum were assembled into 616 Mbp gene-rich DNA regions that represented roughly half of the estimated 1,500 Mbp genome of this species. The assembly encoded ∼42,000 protein-coding genes, consistent with previous dinoflagellate gene number estimates using transcriptomic data. The Symbiodinium genome contains duplicated genes for regulator of chromosome condensation proteins, nearly one-third of which have eukaryotic orthologs, whereas the remainder have most likely been acquired through bacterial horizontal gene transfers. Symbiodinium genes are enriched in spliceosomal introns (mean = 18.6 introns/gene). Donor and acceptor splice sites are unique, with 5′ sites utilizing not only GT but also GC and GA, whereas at 3′ sites, a conserved G is present after AG. All spliceosomal snRNA genes (U1-U6) are clustered in the genome. Surprisingly, the Symbiodinium genome displays unidirectionally aligned genes throughout the genome, forming a cluster-like gene arrangement. Conclusions We show here that a dinoflagellate genome exhibits unique and divergent characteristics when compared to those of other eukaryotes. Our data elucidate the organization and gene inventory of dinoflagellates and lay the foundation for future studies of this remarkable group of eukaryotes. © 2013 Elsevier Ltd.

DOI： 10.1016/j.cub.2013.05.062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We provide a new oligo-microarray for Ciona intestinalis, based on the NimbleGen 12-plex x 135k format. The array represents 106,285 probes, which is more than double the probe number of the currently available 44k microarray. These probes cover 99.2% of the transcripts in the KyotoHoya (KH) models, published in 2008, and they contain 81.1% of the entries in the UniGene database that are not included in the KH models. In this paper, we show that gene expression levels measured by this new 135k microarray are highly correlated with those obtained by the existing 44k microarray for genes common to both arrays. We also investigated gene expression using samples obtained from the ovary and the neural complex of adult C intestinalis, showing that the expression of tissue-specific genes is consistent with previous reports. Approximately half of the highly expressed genes identified in the 135k microarray are not included in the previous microarray. The high coverage of gene models by this microarray made it possible to identify splicing variants for a given transcript. The 135k microarray is useful in investigating the functions of genes that are not yet well characterized. Detailed information about this 135k microarray is accessible at no charge from supplemental materials, NCBI Gene Expression Omnibus (GEO), and http://marinegenomics.oist.jp. (c) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2013.01.042

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Innate immunity in corals is of special interest not only in the context of self-defense but also in relation to the establishment and collapse of their obligate symbiosis with dinoflagellates of the genus Symbiodinium. In innate immunity system of vertebrates, approximately 20 tripartite nucleotide oligomerization domain (NOD)-like receptor proteins that are defined by the presence of a NAIP, CIIA, HET-E and TP1 (NACHT) domain, a C-terminal leucine-rich repeat (LRR) domain, and one of three types of N-terminal effector domain, are known to function as the primary intracellular pattern recognition molecules. Surveying the coral genome revealed not only a larger number of NACHT- and related domain nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)-encoding loci (similar to 500) than in other metazoans but also surprising diversity of domain combinations among the coral NACHT/NB-ARC-containing proteins; N-terminal effector domains included the apoptosis-related domains caspase recruitment domain (CARD), death effector domain (DED), and Death, and C-terminal repeat domains included LRRs, tetratricopeptide repeats, ankyrin repeats, and WD40 repeats. Many of the predicted coral proteins that contain a NACHT/NB-ARC domain also contain a glycosyl transferase group 1 domain, a novel domain combination first found in metazoans. Phylogenetic analyses suggest that the NACHT/NB-ARC domain inventories of various metazoan lineages, including corals, are largely products of lineage-specific expansions. Many of the NACHT/NB-ARC loci are organized in pairs or triplets in the Acropora genome, suggesting that the large coral NACHT/NB-ARC repertoire has been generated at least in part by tandem duplication. In addition, shuffling of N-terminal effector domains may have occurred after expansions of specific NACHT/NB-ARC-repeat domain types. These results illustrate the extraordinary complexity of the innate immune repertoire of corals, which may in part reflect adaptive evolution to a symbiotic lifestyle in a uniquely complex and challenging environment.

DOI： 10.1093/molbev/mss213

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Ascidians are hyperaccumulators that have been studied in detail. Proteins and genes involved in the accumulation process have been identified, but regulation of gene expression related to vanadium accumulation remains unknown. To gain insights into the regulation of gene expression by vanadium in a genome-wide manner, we performed a comprehensive study on the effect of excess vanadium ions on a vanadium-rich ascidian, Ciona intestinalis, using a microarray. RT-PCR and enzyme activity assay were performed from the perspective of redox and accumulation of metal ions in each tissue. Glutathione metabolism-related proteins were significantly up-regulated by V-IV treatment. Several genes involved in the transport of vanadium and protons, such as Nramp and V-ATPase, were significantly up-regulated by V-IV treatment. We observed significant up-regulation of glutathione synthesis and degradation pathways in the intestine and branchial sac. In blood cells, expression of Ci-Vanabin4, glutathione reductase activity, glutathione levels, and vanadium concentration increased after V-IV treatment. V-IV treatment induced significant changes related to vanadium exclusion, seclusion, and redox pathways in the intestine and branchial sac. It also induced an enhancement of the vanadium reduction and accumulation cascade in blood cells. These differential responses in each tissue in the presence of excess vanadium ions suggest that vanadium accumulation and reduction may have regulatory functions. This is the first report on the gene regulation by the treatment of vanadium-rich ascidians with excess vanadium ions. It provided much information for the mechanism of regulation of gene expression related to vanadium accumulation.

DOI： 10.1007/s10534-012-9569-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Scleractinian corals are of fundamental ecological significance in tropical and sub-tropical shallow water. This ecological success is attributed to their ability of formation of obligate endosymbioses with dinoflagellates of the genus Symbiodinium. Nevertheless, approximately one-third of reef-building coral species are critically endangered and the remainder are under threat from the effects of climate change and local impacts. Molecular and cellular mechanisms involved in stress responses and the establishment and collapse of the symbiosis are therefore an urgent subject of research. Metazoans possess large numbers of genes that participate in response to environmental stressors, and chemical defense genes included P450 and other oxidases, various conjugating enzymes, ATP-dependent efflux transporters, oxidative detoxification proteins, as well as transcription factors that regulate these genes. Here we searched those genes in recently decoded the coral Acropora digitifera genome. We found that this genome contains a set of chemical defense genes in numbers comparable with other cnidarians and metazoans and that there are some lineage-specific gene family expansions in the coral genome. These provide information for future research into molecular mechanisms involved in coral stress responses.

DOI： 10.2108/zsj.29.510

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Housekeeping genes, widely expressed genes that are required for the basal function of most cell types, are clustered in the human and worm genomes. This arrangement suggests coordinate control of housekeeping gene expression at the chromosomal level. Here we examined whether this notion is applicable to a marine chordate, Ciona intestinalis. Using microarrays, we analyzed genes that were expressed in 11 organs of the adult, including the neural complex, branchial sac, esophagus, stomach, endostyle, intestine, body-wall muscle, heart, blood cells, ovary and testis. This analysis identified 158 genes that are expressed ubiquitously in these organs. These housekeeping genes could be classified into a range of Gene Ontology categories, in particular, ribosomal protein components. Of these 158 genes, we were able to map 141 genes onto the 14 pairs of the C. intestinalis chromosomes. They were distributed rather evenly over all the chromosomes, except for small clusters containing two or three genes. Therefore, the notion of chromosomal clustering of housekeeping genes is not applicable in this chordate. (C) 2011 Published by Elsevier B.V.

DOI： 10.1016/j.margen.2011.01.002

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Language：English   Publisher：8  

One of challenges in the field of developmental biology is to understand how spatially and/or temporally coordinated expression of genes is controlled at the chromosomal level. It remains controversial whether genes expressed in a given tissue are randomly distributed throughout a given animal genome, or instead resolve into clusters. Here we used microarray analysis to identify more than 1,700 genes that are expressed preferentially in each of 11 organs of the chordate Ciona intestinalis adult, and determined the location of these genes on the 14 pairs of Ciona chromosomes. In spite of extensive mapped gene analysis, we only confirmed small clusters containing two or three genes. Our result indicates that organ-specific genes are distributed rather evenly all over chromosomes, suggesting that the notion of clustering of organ-specific genes in animal genomes is not generally applicable to this chordate. genesis 49:662-672, 2011. © 2011 Wiley-Liss, Inc.

DOI： 10.1002/dvg.20730

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Despite the enormous ecological and economic importance of coral reefs, the keystone organisms in their establishment, the scleractinian corals, increasingly face a range of anthropogenic challenges including ocean acidification and seawater temperature rise(1-4). To understand better the molecular mechanisms underlying coral biology, here we decoded the approximately 420-megabase genome of Acropora digitifera using next-generation sequencing technology. This genome contains approximately 23,700 gene models. Molecular phylogenetics indicate that the coral and the sea anemone Nematostella vectensis diverged approximately 500 million years ago, considerably earlier than the time over which modern corals are represented in the fossil record (similar to 240 million years ago)(5). Despite the long evolutionary history of the endosymbiosis, no evidence was found for horizontal transfer of genes from symbiont to host. However, unlike several other corals, Acropora seems to lack an enzyme essential for cysteine biosynthesis, implying dependency of this coral on its symbionts for this amino acid. Corals inhabit environments where they are frequently exposed to high levels of solar radiation, and analysis of the Acropora genome data indicates that the coral host can independently carry out de novo synthesis of mycosporine-like amino acids, which are potent ultraviolet-protective compounds. In addition, the coral innate immunity repertoire is notably more complex than that of the sea anemone, indicating that some of these genes may have roles in symbiosis or coloniality. A number of genes with putative roles in calcification were identified, and several of these are restricted to corals. The coral genome provides a platform for understanding the molecular basis of symbiosis and responses to environmental changes.

DOI： 10.1038/nature10249

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Despite containing only approximately 330 cells, the central nervous system (CNS) of Ciona intestinalis larvae has an architecture that is similar to the vertebrate CNS. Although only vertebrates have a distinct hypothalamus-the source of numerous neurohormone peptides that play pivotal roles in the development, function, and maintenance of various neuronal and endocrine systems, it is suggested that the Ciona brain contains a region that corresponds to the vertebrate hypothalamus. To identify genes expressed in the brain, we isolated brain vesicles using transgenic embryos carrying Ci-beta-tubulin(promoter)::Kaede, which resulted in robust Kaede expression in the larval CNS. The associated transcriptome was investigated using microarray analysis. We identified 565 genes that were preferentially expressed in the larval brain. Among these genes, 11 encoded neurohormone peptides including such hypothalamic peptides as gonadotropin-releasing hormone and oxytocin/vasopressin. Six of the identified peptide genes had not been previously described. We also found that genes encoding receptors for some of the peptides were expressed in the brain. Interestingly, whole-mount in situ hybridization showed that most of the peptide genes were expressed in the ventral brain. This catalog of the genes expressed in the larval brain should help elucidate the evolution, development, and functioning of the chordate brain. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2011.01.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Our previous transcriptome analysis identified 565 genes that are preferentially expressed in the developing brain of Ciona intestinalis larvae. Here, we show by in-situ hybridization that the spatial expression patterns of these brain-specific genes fall into different categories depending on the regions where the gene is expressed. For example, Ci-opsin3 and Ci-Dkk3 are expressed in the entire brain, Ci-tyrosinase and Ci-TYRP1 in the dorsal region, and Ci-synaptotagmin3, Ci-ZF399, and Ci-PTFb in the ventral region. Other genes are specific to the posterior, anterior, central, posterior and ventral, or anterior-ventral region of the brain. This regional expression of genes in the Ciona brain is not always associated with cell lineage, suggesting that complex mechanisms control the regionalized expression of brain-specific genes.

DOI： 10.2108/zsj.27.103

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Specification of early embryonic cells of animals is established by maternally provided factors and interactions of neighboring cells. The present study addressed a question of autonomous versus non-autonomous specification of embryonic cells by using the Ciona intestinalis embryo, in particular the genetic cascade of zygotic expression of transcription factor genes responsible for notochord specification. To examine this issue, we combined the classic experiment of continuous dissociation of embryonic cells with the modern technique of oligonucleotide-based microarrays. We measured early zygotic expression of 389 core transcription factors genes and 118 major signal molecule genes in embryonic cells that were fully dissociated from the first cleavage. Our results indicated that even if cells are free from contact with neighbors, the major transcription factor genes that have primary roles in embryonic cell specification commence their zygotic expression at the same time as in normal embryos. Dissociation of embryonic cells did not affect extracellular signal-regulated kinases (ERK) activity. Although normal embryos treated with U0126 failed to express Bra and Twist-like-1, dissociated embryonic cells treated with U0126 expressed the genes. These results are discussed in relation to the grade of autonomous versus non-autonomous genetic cascades that are responsible for the specification of early Ciona embryonic cells.

DOI： 10.1111/j.1440-169X.2009.01124.x

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The Ras family small GTPases play a variety of essential roles in eukaryotes. Among them, classical Ras (H-Ras, K-Ras, and N-Ras) and its orthologues are conserved from yeast to human. In ascidians, which phylogenetically exist between invertebrates and vertebrates, the fibroblast growth factor (FGF)-Ras-MAP kinase signaling is required for the induction of neural system, notochord, and mesenchyme. Analyses of DNA databases revealed that no gene encoding classical Ras is present in the ascidians, Ciona intestinalis and Halocynthia roretzi, despite the presence of classical Ras-orthologous genes in nematode, fly, amphioxus, and fish. By contrast, both the ascidians contain single genes orthologous to Mras, Rras, Ral, Rap1, and Rap2. A single Mras orthologue exists from nematode to mammalian. Thus, Mras evolved in metazoans independently of other Ras family genes such as Rras. Whole-mount in situ hybridization showed that C. intestinalis Mras orthologue (Ci-Mras) was expressed in the neural complex of the ascidian juveniles after metamorphosis. Knockdown of Ci-Mras with morpholino antisense oligonucleotides in the embryos and larvae resulted in undeveloped tails and neuronal pigment cells, abrogation of the notochord marker brachyury expression, and perturbation of the neural marker Otx expression, as has been shown in the experiments of the FGF-Ras-MAP kinase signaling inhibition. Mammalian Ras and M-Ras mediate nerve growth factor-induced neuronal differentiation in rat PC12 cells by activating the ERK/MAP kinase pathway transiently and sustainedly, respectively. Activated Ci-M-Ras bound to target proteins of mammalian M-Ras and Ras. Exogenous expression of an activated Ci-M-Ras in PC12 cells caused ERK activation and induced neuritogenesis via the ERK pathway as do mammalian M-Ras and Ras. These results suggest that the ascidian M-Ras orthologue compensates for lacked classical Ras and plays essential roles in neurogenesis in the ascidian.

DOI： 10.1016/j.gene.2008.10.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

We report here characterization of five genes for novel components of the canonical Wnt/beta-catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss-of-function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci-PGAP1, a Ciona orthologue of human PGAP1, which encodes GPI (glycosylphosphatidylinositol) inositol-deacylase, Ci-ZF278, a gene encoding a C2H2 zinc-finger protein, Ci-C10orf11, a Ciona orthologue of human C10orf11 that encodes a protein with leucine-rich repeats, Ci-Spatial/C4orf17, a single counterpart for two human genes Spatial and C4orf17, and Ci-FLJ10634, a Ciona orthologue of human FLJ10634 that encodes a member of the J-protein family. Knockdown of each of the genes mimicked beta-catenin knockdown and resulted in suppression of the expression of beta-catenin downstream genes (Ci-FoxD, Ci-Lhx3, Ci-Otx and Ci-Fgf9/16/20) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild-type, of Ci-beta-catenin. Dosage-sensitive interactions were found between Ci-beta-catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci-beta-catenin in the Wnt/beta-catenin signaling pathway in early Ciona embryos.

DOI： 10.1111/j.1440-169x.2008.01012.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The sequenced genome of the urochordate ascidian Ciona intestinalis contains nearly 2,500 genes that have vertebrate homologues, but their functions are as yet unknown. To identify novel genes involved in early chordates embryogenesis, we previously screened 200 Ciona genes by knockdown experiments using specific morpholino oligonucleotides and found that suppression of the translation of 40 genes caused embryonic defects (Yamada et al. [2003] Development 130:6485-6495). We have since examined an additional 304 genes, that is, screening 504 genes overall, and a total of 111 genes showed morphological defects when gene function was suppressed. We further examined the role of these genes in the differentiation of six major tissues of the embryo: endoderm, muscle, epidermis, neural tissue, mesenchyme, and notochord. Based on the similarity of phenotypes of gene knockdown embryos, genes were categorized into several groups, with the suggestion that the genes within a given group are involved in similar developmental processes. For example, five were shown to be novel genes that are likely involved in beta-catenin-mediated endoderm formation. The type of large-scale screening used is, therefore, a powerful approach to identify novel genes with significant developmental functions, the details of which will be determined in future studies. Developmental Dynamics 236:1820-1831, 2007. (c) 2007 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.21181

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Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried out a comprehensive survey for HSP70s and their cochaperones in the genome of Ciona intestinalis. We characterized all members of the CionaHSP70 superfamily, J-proteins, BAG family, and some other types of cochaperones. The Ciona genome contains 8 members of the HSP70 superfamily, all of which have human and protostome counterparts. Members of the STCH subfamily of the HSP70 family and members of the HSPA14 subfamily of the HSP110 family are conserved between humans and protostomes but were not found in Ciona. The Ciona genome encodes 36 J-proteins, 32 of which belong to groups conserved in humans and protostomes. Three proteins seem to be unique to Ciona. J-proteins of the RBJ group are conserved between humans and Ciona but were not found in protostomes, whereas J-proteins of the DNAJC14, ZCSL3, FLJ13236, and C21orf55 groups are conserved between humans and protostomes but were not found in Ciona. J-proteins of the sacsin group seem to be specific to vertebrates. There is also a J-like protein without a conserved HPD tripeptide motif in the Ciona genome. The Ciona genome encodes 3 types of BAG family proteins, all of which have human and protostome counterparts (BAG1, BAG3, and BAT3). BAG2 group is conserved between humans and protostomes but was not found in Ciona, and BAG4 and BAG5 groups seem to be specific to vertebrates. Members for SIL1, UBQLN, UBADC1, TIMM44, GRPEL, and Magmas groups, which are conserved between humans and protostomes, were also found in Ciona. No Ciona member was retrieved for HSPBP1 group, which is conserved between humans and protostomes. For several groups of the HSP70 superfamily, J-proteins, and other types of cochaperones, multiple members in humans are represented by a single counterpart in Ciona. These results show that genes of the HSP70 chaperone system can be distinguished into groups that are shared by vertebrates, Ciona, and protostomes, ones shared by vertebrates and protostomes, ones shared by vertebrates and Ciona, and ones specific to vertebrates, Ciona, or protostomes. These results also demonstrate that the components of the HSP70 chaperone system in Cionaare similar to but simpler than those in humans and suggest that changes of the genome in the lineage leading to humans after the separation from that leading to Ciona increased the number and diversity of members of the HSP70 chaperone system. Changes of the genome in the lineage leading to Cionaalso seem to have made the HSP70 chaperone system in this species slightly simpler than that in the common ancestor of humans and Ciona. © Cell Stress Society International 2006.

DOI： 10.1379/CSC-137R.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

A novel gene (Ci-Rga) essential for tissue differentiation during embryogenesis of the ascidian Ciona intestinalis is reported here. This gene was identified through functional screening of Ciona genes required for development by translational inhibition experiments with morpholino antisense oligonucleotides. The deduced protein of Ci-Rga contains two copies of a domain with unknown function called the MtN3/saliva domain. Phylogenetic analysis showed that Ci-Rga belongs to the MtN3/saliva family of genes conserved among metazoans and plants, and is an ortholog of mouse Rga (Recombination-activating gene 1 gene activation). During Ciona embryogenesis, both maternal and zygotic transcripts of Ci-Rga were expressed. Translational inhibition of Ci-Rga with specific morpholino resulted in abnormal embryos in which the cleavage pattern became atypical and expression of marker genes for each of the six major tissues, namely the endoderm, muscle, mesenchyme, notochord, neural tissue, and epidermis, was lost or suppressed at the tailbud stage. Although differentiation of all the six major tissues was affected by Ci-Rga knock-down, the degree of abnormalities and the timing of appearance of abnormalities were different among tissues. Expression analysis of developmentally important genes involved in the fate specification, such as Ci-Bra, Ci-Twist-like1a, Ci-Otx, Ci-Fgf9/16/20, Ci-Lhx3, Ci-FoxD, and Ci-Tbx6b, showed that an initial step of the fate specification of notochord, mesenchyme, and neural tissue, but not of endoderm or muscle, is impaired in the knock-down embryo. These results showed that Ci-Rga is a multifunctional gene essential for tissue differentiation during embryogenesis, and is primarily required for the fate specification of notochord, mesenchyme, and neural tissue, and provide some insights into the function of this little-known group of genes.

DOI： 10.1111/j.1432-0436.2005.00037.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

Primary mesenchyme cells (PMC), the skeletogenic cells derived from the micromeres of the sea urchin embryo, are involved in the differentiation of the gut. When PMC were deleted from the mesenchyme blastula, both formation of the constrictions in the gut and expression of endoderm-specific alkaline phosphatase were significantly delayed. Therefore, the correct timing of gut differentiation depends on the existence of PMC, probably via a type of promotive signal. To date, the only role of PMC in other tissue differentiation has been a suppressive signal for the conversion of secondary mesenchyme cells (SMC) into skeletogenic cells. The present experiments using PMC ablation and transplantation showed that both signaling processes occurred in the same short period during gastrulation, but the embryos kept their competence for gut differentiation until a later stage. Further investigations indicated that conversion of SMC did not cause delay in gut differentiation and that SMC did not mediate the PMC signal to the endoderm. Therefore, the effect of PMC on gut differentiation could be a new role that is independent of the suppressive effect for SMC conversion.

DOI： 10.1046/j.1440-169X.2003.00702.x

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