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Papers - Hotta, Kohji

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Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos

Yamada Shigehiro, Hotta Kohji, Yamamoto Takamasa S., Ueno Naoto, Satoh Nori, Takahashi Hiroki

Developmental Biology 328 ( 1 ) 1 - 12 2009.04

ISSN 0012-1606

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS. © 2009 Elsevier Inc. All rights reserved.

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Genome-wide searching with base-pairing kernel functions for noncoding RNAs

Morita Kensuke, Saito Yutaka, Sato Kengo, Oka Kotaro, Hotta Kohji, Sakakibara Yasubumi

Nucleic Acids Research 37 ( 3 ) 999 - 1009 2009

ISSN 0305-1048

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).

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Improved genome assembly and evidence-based global gene model set for the chordate Ciona intestinalis

Satou Yutaka, Mineta Katsuhiko, Ogasawara Michio, Sasakura Yasunori, Shoguchi Eiichi, Ueno Keisuke, Yamada Lixy, Matsumoto Jun, Wasserscheid Jessica, Dewar Ken, Wiley Graham B., Macmil Simone L., Roe Bruce A., Zeller Robert W., Hastings Kenneth E M, Lemaire Patrick, Lindquist Erika, Endo Toshinori, Hotta Kohji, Inaba Kazuo

Genome Biology 9 ( 10 ) 2008.10

ISSN 1474-7596

Background: The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly. Results: We have prepared a less-fragmented assembly on the basis of scaffold-joining guided by paired-end EST and bacterial artificial chromosome (BAC) sequences, and BAC chromosomal in situ hybridization data. The new assembly (115.2 Mb) is similar in length to the initial assembly (116.7 Mb) but contains 1,272 (approximately 50%) fewer scaffolds. The largest scaffold in the new assembly incorporates 95 initial-assembly scaffolds. In conjunction with the new assembly, we have prepared a greatly improved global gene model set strictly correlated with the extensive currently available EST data. The total gene number (15,254) is similar to that of the initial set (15,582), but the new set includes 3,330 models at genomic sites where none were present in the initial set, and 1,779 models that represent fusions of multiple previously incomplete models. In approximately half, 5′-ends were precisely mapped using 5′-full-length ESTs, an important refinement even in otherwise unchanged models. Conclusion: Using these new resources, we identify a population of non-canonical (non-GT-AG) introns and also find that approximately 20% of Ciona genes reside in operons and that operons contain a high proportion of single-exon genes. Thus, the present dataset provides an opportunity to analyze the Ciona genome much more precisely than ever. © 2008 Satou et al.; licensee BioMed Central Ltd.

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A novel biological role of tachykinins as an up-regulator of oocyte growth: Identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis

Aoyama, Masato, Kawada, Tsuyoshi, Fujie, Manabu, Hotta, Kohji, Sakai, Tsubasa, Sekiguchi, Toshio, Oka, Kotaro, Satoh, Nori, Satake, Honoo

ENDOCRINOLOGY 149 ( 9 ) 4346 - 4356 2008.09

Research paper (scientific journal), Joint Work, ISSN 0013-7227

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Ca²⁺ influx through P2X receptors induces actin cytoskeleton reorganization by the formation of cofilin rods in neurites

Homma Kohei, Niino Yusuke, Hotta Kohji, Oka Kotaro

Molecular and Cellular Neuroscience 37 ( 2 ) 261 - 270 2008.02

ISSN 1044-7431

In physiological and pathological events, extracellular ATP plays an important role by controlling several types of purinergic receptors and changing cytoskeleton dynamics. To know the process of ATP-dependent cytoskeleton remodeling, we focused on cofilin, a key regulator of actin cytoskeleton, and investigated the dynamics of cofilin in PC12 cells through fluorescent protein-labeled cofilin and actin, Ca²⁺ imaging, and fluorescence resonance energy transfer (FRET) techniques. As a result, ATP induced intracellular Ca²⁺ increase, following cofilin rods' formation. ATP-induced cofilin rods' formation was not observed in cells expressing unphosphorylatable variant of cofilin. A P2X receptor agonist, but not P2Y, induced the formation of cofilin rods, whereas calmodulin and calcineurin inhibitors suppressed it. These results indicate that Ca²⁺ influx through P2X receptors induces the formation of cofilin rods via calcineurin-dependent dephosphorylation of cofilin. This pathway might be one candidate to explain the effects of ATP on neuronal development and injury. © 2007 Elsevier Inc. All rights reserved.

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Brachyury-downstream gene sets in a chordate, Ciona intestinalis

Hotta Kohji, Takahashi Hiroki, Satoh Nori, Gojobori Takashi

Evolution and Development 10 ( 1 ) 37 - 51 2008.01

ISSN 1520-541X

In vertebrates, Brachyury, a T-box transcription factor gene, seems to have a dual role in the differentiation of axial midline mesoderm cells into notochord and gastrulation cell movements regulated by non-canonical Wnt/planar cell polarity (Wnt/PCP) signaling. To understand the function of Brachyury-downstream genes in chordate embryos, from a series of our survey on differential expression, including subtractive hybridization, dot-blot assays, EST sequences and the expression patterns in whole-mount in situ hybridization at embryonic stages, we developed a knowledge database called "CINOBI: Ciona Notochord and Brachyury-downstream gene Index" to create comprehensive catalogues of Brachyury-downstream gene sets in Ciona intestinalis. Combining genome and large-scale cDNA data, we were able to characterize 450 non-redundant Brachyury-downstream genes: Twenty-four genes were newly annotated as notochord-expressed genes. Several genes are components of signaling pathways such as Wnt/PCP, NfκB and TGF-β signaling. We propose that Brachyury is linked to these pathways regulating the expression of each component, and such a regulatory mechanism might be conserved among chordates. © 2008 The Author(s).

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A web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva

Hotta, Kohji, Mitsuhara, Kenta, Takahashi, Hiroki, Inaba, Kazuo, Oka, Kotaro, Gojobori, Takashi, Ikeo, Kazuho

DEVELOPMENTAL DYNAMICS 236 ( 7 ) 1790 - 1805 2007.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 1058-8388

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Brachyury-downstream notochord genes and convergent extension in Ciona intestinalis embryos

Hotta Kohji, Yamada Shigehiro, Ueno Naoto, Satoh Nori, Takahashi Hiroki

Development Growth and Differentiation 49 ( 5 ) 373 - 382 2007.06

ISSN 0012-1592

Formation of the chordate body is accomplished by a complex set of morphogenetic movements including convergent extension of notochord cells. In the ascidian Ciona intestinalis, Brachyury plays a key role in the formation of the notochord, and more than 30 Bra-downstream notochord genes have been identified. In the present study, we examined the effects of functional suppression of nine Bra-downstream notochord genes, which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Ci-trop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When the function of the first two genes (Ci-PTP and Ci-ACL) was suppressed with specific morpholinos, the notochord cells failed to converge, while functional suppression of Ci-prickle resulted in a failure of intercalation, and therefore the cells in these three types of embryo remained in the mid-dorsal region of the embryo. Functional suppression of the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and Ci-ASAK) resulted in the partial defect of intercalation, and the notochord did not consist of a single row. In addition, when the function of the last two genes (Ci-ERM and Ci-pellino) was suppressed, notochord cells failed to elongate in the embryo, even though convergence/extension took place normally. These results indicate that many Bra-downstream notochord genes are involved in convergence/extension of the embryo. © 2007 The Authors.

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T-box genes in the Ascidian Ciona intestinalis

Takatori Naohito, Hotta Kohji, Mochizuki Yasuaki, Satoh Gouki, Mitani Yasuo, Satoh Nori, Satou Yutaka, Takahashi Hiroki

Developmental Dynamics 230 ( 4 ) 743 - 753 2004.08

ISSN 1058-8388

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.

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A genome-wide survey of the genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components

Hotta Kohji, Takahashi Hiroki, Ueno Naoto, Gojobori Takashi

Gene 317 ( 1-2 ) 165 - 185 2003.10

ISSN 0378-1119

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.

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The draft genome of Ciona intestinalis

Dehal Paramvir, Satou Yutaka, Campbell Robert K., Chapman Jarrod, Degnan Bernard, De Tomaso Anthony, Davidson Brad, Di Gregorio Anna, Gelpke Maarten, Goodstein David M., Harafuji Naoe, Hastings Kenneth E M, Ho Isaac, Hotta Kohji, Huang Wayne, Kawashima Takeshi, Lemaire Patrick, Martinez Diego, Meinertzhagen Ian A., Necula Simona, Nonaka Masaru, Putnam Nik, Rash Sam, Saiga Hidetoshi, Satake Masanobu, Terry Astrid, Yamada Lixy, Wang Hong Gang, Awazu Satoko, Azumi Kaoru, Boore Jeffrey, Branno Margherita, Chin-bow Stephen, DeSantis Rosaria, Doyle Sharon, Francino Pilar, Keys David N., Haga Shinobu, Hayashi Hiroko, Hino Kyosuke, Imai Kaoru S., Inaba Kazuo, Kano Shungo, Kobayashi Kenji, Kobayashi Mari, Lee Byung In, Makabe Kazuhiro W., Manohar Chitra, Matassi Giorgio, Medina Monica, Mochizuki Yasuaki, Mount Steve, Morishita Tomomi, Miura Sachiko, Nakayama Akie, Nishizaka Satoko, Nomoto Hisayo, Ohta Fumiko, Oishi Kazuko, Rigoutsos Isidore, Sano Masako, Sasaki Akane, Sasakura Yasunori, Shoguchi Eiichi, Shin-i Tadasu, Spagnuolo Antoinetta, Stainier Didier, Suzuki Miho M., Tassy Olivier, Takatori Naohito, Tokuoka Miki, Yagi Kasumi, Yoshizaki Fumiko, Wada Shuichi, Zhang Cindy, Hyatt P. Douglas, Larimer Frank, Detter Chris, Doggett Norman, Glavina Tijana, Hawkins Trevor, Richardson Paul, Lucas Susan, Kohara Yuji, Levine Michael, Satoh Nori, Rokhsar Daniel S.

Science 298 ( 5601 ) 2157 - 2167 2002.12

ISSN 0036-8075

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.

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Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

Hotta Kohji, Takahashi Hiroki, Asakura Tomomi, Saitoh Banjo, Takatori Naohito, Satou Yutaka, Satoh Nori

Developmental Biology 224 ( 1 ) 69 - 80 2000.08

ISSN 0012-1606

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.

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Brachyury downstream notochord differentiation in the ascidian embryo

Takahashi Hiroki, Hotta Kohji, Erives Albert, Di Gregorio Anna, Zeller Robert W., Levine Michael, Satoh Nori

Genes and Development 13 ( 12 ) 1519 - 1523 1999.06

ISSN 0890-9369

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.

Temporal expression patterns of 39 Brachyury-downstream genes associated with notochord formation in the Ciona intestinalis embryo

Hotta Kohji, Takahashi Hiroki, Erives Albert, Levine Michael, Satoh Nori

Development Growth and Differentiation 41 ( 6 ) 657 - 664 1999

ISSN 0012-1592

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.

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Repression of an ascidian gene in the tip of the tail of tail-bud-stage embryos

Hotta Kohji, Takahashi Hiroki, Satoh Noriyuki

Development Genes and Evolution 208 ( 3 ) 164 - 167 1998

ISSN 0949-944X

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.

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