Published papers

Excess administration of glutamate is known to induce Ca2+ overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg2+ in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg2+ in the excitotoxicity, we simultaneously measured intracellular Ca2+ and Mg2+, using fluorescent dyes, Fura red, a fluorescent Ca2+ probe, and KMG-104, a highly selective fluorescent Mg2+ probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg2+ concentration ([Mg2+]i). Extracellular Mg2+ was not required for this glutamate-induced increase in [Mg2+]i, and no increase in intracellular Ca2+ concentration ([Ca2+]i) or [Mg2+]i was observed in neurons in nominally Ca2+-free medium. Application of 5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca2+]i and [Mg2+]i. Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg2+]i but did induce an additive increase in [Ca2+]i. Moreover, the glutamate-induced increase in [Mg2+]i was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg2+ efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, prevented this [Mg2+]i increase. These results indicate that glutamate-induced increases in [Mg2+]i result from the Mg2+ release from mitochondria and that Ca2+ accumulation in the mitochondria is required for this Mg2+ release. © 2010 Wiley-Liss, Inc.

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions. © 2010 by Cold Spring Harbor Laboratory Press.

In ascidian Ciona intestinalis, a subset of trunk epidermal neurons were shown to possess external network of neural projections. To characterize a more complete network in naturally hatched (chorionated) larvae, we visualized the structure with a confocal laser scanning microscope. High resolution images revealed the huge network consisting of several subnetworks in whole-larval tunic. We named this network the ASNET (ascidian dendritic network in tunic). The ASNET was dynamically generated and collapsed during larval stages. Interestingly, one of the subnetworks found around apical trunk epidermal neurons was bilaterally asymmetric. In caudal epidermal neurons, transmission electron microscopy revealed that 9+2 axonemes were accompanied by a vesicle-containing mass in the ASNET arbor, but the distal end of the arbor contained only the vesicle-containing fibrous mass and no 9+2 axonemes. The characteristics of the ASNET suggest that it forms a unique outer body network in the ascidian larval tunic. © 2010 Wiley-Liss, Inc.

Brachyury, a T-box transcription factor, is expressed in ascidian embryos exclusively in primordial notochord cells and plays a pivotal role in differentiation of notochord cells. Previously, we identified ∼450 genes downstream of Ciona intestinalis Brachyury (Ci-Bra), and characterized the expression profiles of 45 of these in differentiating notochord cells. In this study, we looked for cisregulatory sequences in minimal enhancers of 20 Ci-Bra downstream genes by electroporating region within ∼3 kb upstream of each gene fused with lacZ. Eight of the 20 reporters were expressed in notochord cells. The minimal enchancer for each of these eight genes was narrowed to a region ∼0.51.0-kb long. We also explored the genome-wide and coordinate regulation of 43 Ci-Bra-downstream genes. When we determined their chromosomal localization, it became evident that they are not clustered in a given region of the genome, but rather distributed evenly over 13 of the 14 pairs of chromosomes, suggesting that gene clustering does not contribute to coordinate control of the Ci-Bra downstream gene expression. Our results might provide Insights Into the molecular mechanisms underlying notochord formation in chordates. © 2010 Zoological Society of Japan.

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca 2+. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca2+ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca 2+, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks. © 2010 Niino et al.

Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9 + 0 structure similar to vertebrate primary cilia, a 9 + 0 structure with electron-dense materials in the center, a 9 + 2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan. © 2009 Elsevier Inc. All rights reserved.

Despite the accumulating research on noncoding RNAs (ncRNAs), it is likely that we are seeing only the tip of the iceberg regarding our understanding of the functions and the regulatory roles served by ncRNAs in cellular metabolism, pathogenesis and host-pathogen interactions. Therefore, more powerful computational and experimental tools for analyzing ncRNAs need to be developed. To this end, we propose novel kernel functions, called base-pairing profile local alignment (BPLA) kernels, for analyzing functional ncRNA sequences using support vector machines (SVMs). We extend the local alignment kernels for amino acid sequences in order to handle RNA sequences by using STRAL's; scoring function, which takes into account sequence similarities as well as upstream and downstream base-pairing probabilities, thus enabling us to model secondary structures of RNA sequences. As a test of the performance of BPLA kernels, we applied our kernels to the problem of discriminating members of an RNA family from nonmembers using SVMs. The results indicated that the discrimination ability of our kernels is stronger than that of other existing methods. Furthermore, we demonstrated the applicability of our kernels to the problem of genome-wide search of snoRNA families in the Caenorhabditis elegans genome, and confirmed that the expression is valid in 14 out of 48 of our predicted candidates by using qRT-PCR. Finally, highly expressed six candidates were identified as the original target regions by DNA sequencing. © 2009 The Author(s).

Members of the T-box family of transcription factors share an evolutionary conserved DNA-binding domain and play significant roles in various processes of embryonic development. Vertebrate T-box genes are categorized into the following five major subfamilies (eight groups), depending on sequence similarities: Brachyury, Tbx1 (Tbx1/10, Tbx15/18/22, Tbx20), Tbx2/3/4/5 (Tbx2/3 and Tbx4/5), Tbx6, and Tbr/Eomes/TBX21. Ascidians are primitive chordates, and their tadpole larva are considered to represent the simplified and basic body plan of vertebrates. In addition, it has been revealed that the ascidian genome contains the basic ancestral complement of genes involved in development. The present characterization of cDNAs and survey of the Ciona intestinalis draft genome demonstrated that the Ciona genome contains a single copy gene for each of the Brachyury, Tbx1/10, Tbx15/18/22, Tbx20, Tbx2/3, and Tbr/Eomes/TBX21 groups, and at least three copies of the Tbx6 subfamily. Each of the Ciona T-box genes shows a characteristic expression pattern, although that of Tbx20 was not determined in the present study. These results provide basic information that will be useful for future studies of the function of each gene, genetic cascades of different T-box genes, and genome-wide surveys of evolutionary changes in the T-box gene structure and organization in this primitive chordate. © 2004 Wiley-Liss, Inc.

Non-canonical Wnt signals similar to planar cell polarity (PCP) signaling in the fly control convergent extension (CE) of the dorsal mesoderm during gastrulation in vertebrates. Using the Ciona complete genome sequence and EST sequence data, we present here an initial and exhaustive search in non-vertebrate chordates, Ciona intestinalis for the family members as well as homologs or orthologs that are involved in PCP/CE signaling cascades. We clarified 7 cardinal gene families, including the MAPK, STE20 group kinase, Rho small GTPase, STAT, Glypican, Fz and Wnt gene families, as well as gene homologs or orthologs for known PCP/CE signaling components with their phylogenetic nature. As a result, we characterized 62 Ciona component genes. Among them, 59 genes were novel and functional genes which were supported by EST expressions and 15 genes belonged to PCP/CE component orthologs of other organisms or common ancestor genes. Moreover, from the phylogenetic point of view, we compared these components genome-widely with the PCP signaling components of fly and the CE signaling components of vertebrates. We then discovered not only that ascidians contain the basic ancestral signaling pathway components in chordates but also that several signaling components have not found in ascidian, indicating that ascidian CE pathway might have several gaps from vertebrate CE pathway. The present study provides an initial step for the subsequent analysis of CE in the non-vertebrate chordates, ascidians. In addition, this phylogenetic approach will help to facilitate understanding of the relationship between fly PCP signaling and the vertebrate CE pathway. © 2003 Elsevier B.V. All rights reserved.

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains ̃16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, β4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos. (C) 2000 Academic Press.

Expression of the Brachyury (Ci-Bra) gene of the ascidian Ciona intestinalis is initiated at the 64-cell stage. Gene expression is restricted to notochord precursor cells, and Ci-Bra plays a key role in notochord differentiation. In a previous study, nearly 50 cDNA clones for potential Ci-Bra-downstream genes that are expressed in notochord cells were isolated. The present determination, by whole-mount in situ hybridization, of the temporal expression patterns of 19 notochord-specific and 20 notochord-predominant genes demonstrated that the timings of initiation of the expression of various genes was not identical. The expression of several genes was initiated as early as the gastrula stage. However, the expression of most of the notochord- specific genes commenced at the neural plate stage. Partial nucleotide sequence data of these clones suggest that genes expressed earlier encode potential transcriptional factors and/or nuclear proteins, while those expressed later encode proteins implicated in cell adhesion, signal transduction, regulation of the cytoskeleton, and components of the extracellular matrix. These gene activities may be associated with changes in cell shape and adhesion during the intercalation and extension of the notochord cells.

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential Ci-Bra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation.

The occurrence of tadpole-type larvae is one of the key events used to understand the origin and evolution of chordates, and the rail bud of chordate embryos is thought to be associated with formation of the tip of the tail. Although some transcriptional factor genes including Brachyury are expressed in the tail bud, no structural genes have been reported to be expressed there. We report here that an ascidian gene HrTT-1 is expressed exclusively in the tip of elongating tail of the tail-bud embryo. This gene encodes a possible secreted protein of 415 amino acids with the SCP/TPX family consensus sequences.