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URASAKI Akihiro
Graduate School of Science, Technology and Innovation / Department of Science, Technology and Innovation
Associate Professor

Researcher basic information

■ Research Keyword
  • 難治性血管病
  • 血管・リンパ管形成
  • ゼブラフィッシュ
  • トランスポゾン
■ Research Areas
  • Life sciences / Genomics
  • Life sciences / Genetics
  • Life sciences / Molecular biology
  • Life sciences / Developmental biology
■ Committee History
  • Sep. 2022 - Sep. 2022, The 28th Japanese Medaka and Zebrafish Meeting, Organizing Committee

Research activity information

■ Paper
  • Toru Iwasa, Akihiro Urasaki, Yuki Kakihana, Nami Nagata-Akaho, Yukihiro Harada, Soichi Takeda, Teruhisa Kawamura, Isao Shiraishi, Kenichi Kurosaki, Hiroko Morisaki, Osamu Yamada, Osamu Nakagawa
    Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease caused by the defects of ALK1/ACVRL1 receptor signaling. In this study, we evaluated 25 recently identified ACVRL1 missense variants using multiple computational pathogenicity classifiers and experimentally characterized their signal transduction capacity. Three extracellular residue variants showed no detectable cell surface expression and impairment of bone morphogenetic protein 9 (BMP9) responsiveness of SMAD-dependent transcription in luciferase assays. Four variants with amino acid replacement in the motifs essential for the intracellular kinase function lost SMAD-dependent signaling. Most of other variations in the kinase domain also caused marked downregulation of signaling; however, two variants behaved as the wild-type ACVRL1 did, while computational classifiers predicted their functional abnormalities. Three-dimensional structure prediction using the ColabFold program supported the significance of the L45 loop and NANDOR domain of ACVRL1 for its association with SMAD1 and BMPR2, respectively, and the variations in these motifs resulted in the reduction of SMAD signaling. On the other hand, two of the GS domain variants maintained high signal transduction capacity, which did not accord with their computational pathogenicity prediction. These results affirm the requirement of a combinatory approach using computational and experimental analyses to accurately predict the pathogenicity of ACVRL1 missense variants in the HHT patients.
    MDPI AG, Jul. 2023, Journal of Clinical Medicine, 12(15) (15), 5002 - 5002
    Scientific journal

  • Yukihiro Harada, Toru Tanaka, Yuji Arai, Yoshie Isomoto, Atsushi Nakano, Shu Nakao, Akihiro Urasaki, Yusuke Watanabe, Teruhisa Kawamura, Osamu Nakagawa
    Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.
    WILEY, Jun. 2021, GENES TO CELLS, English, International magazine
    [Refereed]
    Scientific journal

  • Yumi Kinugasa-Katayama, Yusuke Watanabe, Takashi Hisamitsu, Yuichiro Arima, Norika M. Liu, Ayaka Tomimatsu, Yukihiro Harada, Yuji Arai, Akihiro Urasaki, Teruhisa Kawamura, Yoshihiko Saito, Osamu Nakagawa
    Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell populations are fundamental in studying its detailed mechanisms using in vivo experimental models. We previously demonstrated that Tmem100 was a novel endothelial gene encoding a small transmembrane protein, and that Tmem100 null mice showed embryonic lethality due to severe impairment of vascular formation. In the present study, we generated an EGFP reporter mouse line using a 216 kb genomic region containing mouse Tmem100 gene. A novel line designated as Tmem100-BAC-EGFP mice precisely recapitulated the Tmem100 expression profile at the mid-gestational stage, which was highly enriched in endothelial cells of large caliber arteries in mouse embryos. FACS experiments demonstrated that Tmem100-BAC-EGFP mice served to selectively purify a specific population of arterial endothelial cells, indicating their usefulness not only for the research concerning Tmem100 expression and function but also for comparative analysis of multiple endothelial cell subgroups in embryonic vascular development.
    WILEY, Apr. 2021, GENESIS, 59(4) (4), e23416, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiro Urasaki, Seiya Morishita, Kosuke Naka, Minato Uozumi, Kouki Abe, Liguo Huang, Emiko Watase, Osamu Nakagawa, Koichi Kawakami, Takaaki Matsui, Yasumasa Bessho, Naoyuki Inagak
    The zebrafish sensory posterior lateral line is an excellent model system to study collective cell migration and organogenesis. Shootin1 is a cytoplasmic protein involved in neuronal polarization and axon guidance. Previous studies have shown that shootin1 couples actin filament retrograde flow with extracellular adhesive substrates at the leading edge of axonal growth cones, thereby producing mechanical force for the migration and guidance of axonal growth cones. However, the functions of shootin in peripheral cells remain unknown. Here we identified two novel shootin family members, shootin1 and shootin3. In zebrafish, shootin1 and shootin3 are expressed in the posterior lateral line primordium (PLLP) and neuromasts during embryonic development. A shootin1 mutant displayed a reduced speed of PLLP migration, while shootin1;shootin3 double mutation inhibited cell proliferation in the PLLP. Furthermore, our results suggest that shootin1 and shootin3 positively regulate the number of neuromasts and the number of cells in deposited neuromasts. Our study demonstrates that shootins mediate collective cell migration of the posterior lateral line primordium and formation of neuromasts in zebrafish.
    Lead, NATURE PUBLISHING GROUP, Aug. 2019, SCIENTIFIC REPORTS, 9(1) (1), 12156 - 12156, English, International magazine
    [Refereed]
    Scientific journal

  • Liguo Huang, Akihiro Urasaki, Naoyuki Inagaki
    Rab small GTPases play key roles in intracellular membrane trafficking. Rab33a promotes axon outgrowth of cultured rat hippocampal neurons by mediating the anterograde axonal transport of Golgi-derived vesicles and the concomitant exocytosis of these vesicles at the growth cone. However, the functions of Rab33 in vivo are unclear. Here, we show that zebrafish rab33a and rab33ba are orthologs of mammalian Rab33a and Rab33b, respectively. They are expressed in the developing brain, including in neurons of the telencephalic dorsorostral cluster and the diencephalic ventrorostral cluster, which project axons to form the anterior and postoptic commissures, respectively. Although rab33a single mutant and rab33ba single mutant fish did not show remarkable defects, fish carrying the rab33a; rab33ba double mutations displayed dysgenesis of the anterior and postoptic commissures. Single-cell labeling in the telencephalic dorsorostral cluster demonstrated that the rab33a; rab33ba double mutation inhibits axonal extension in the anterior commissure. These results suggest that Rab33a and Rab33ba mediate axon outgrowth and the formation of the forebrain commissures in the zebrafish brain in a cooperative manner.
    NATURE PUBLISHING GROUP, Feb. 2019, SCIENTIFIC REPORTS, 9(1) (1), 1799 - 1799, English, International magazine
    [Refereed]
    Scientific journal

  • Yasuna Higashiguchi, Kazuhiro Katsuta, Takunori Minegishi, Shigenobu Yonemura, Akihiro Urasaki, Naoyuki Inagaki
    Shootin1 is a brain-specific cytoplasmic protein involved in neuronal polarity formation and axon outgrowth. It accumulates at the leading edge of axonal growth cones, where it mediates the mechanical coupling between F-actin retrograde flow and cell adhesions as a clutch molecule, thereby producing force for axon outgrowth. In this study, we report a novel splicing isoform of shootin1 which is expressed not only in the brain but also in peripheral tissues. We have renamed the brain-specific shootin1 as shootin1a and termed the novel isoform as shootin1b. Immunoblot and immunohistochemical analyses with a shootin1b-specific antibody revealed that shootin1b is distributed in various mouse tissues including the lung, liver, stomach, intestines, spleen, pancreas, kidney and skin. Interestingly, shootin1b immunoreactivity was widely detected in epithelial cells that constitute simple and stratified epithelia; in some cells, it colocalized with E-cadherin and cortactin at cell-cell contact sites. Shootin1b also localized in dendritic cells in the spleen. These results suggest that shootin1b may function in various peripheral tissues including epithelial cells.
    SPRINGER, Oct. 2016, CELL AND TISSUE RESEARCH, 366(1) (1), 75 - 87, English, International magazine
    [Refereed]
    Scientific journal

  • Dorien M. A. Hermkens, Andreas van Impel, Akihiro Urasaki, Jeroen Bussmann, Henricus J. Duckers, Stefan Schulte-Merker
    SoxF family members have been linked to arterio-venous specification events and human pathological conditions, but in contrast to Sox17 and Sox18, a detailed in vivo analysis of a Sox7 mutant model is still lacking. In this study we generated zebrafish sox7 mutants to understand the role of Sox7 during vascular development. By in vivo imaging of transgenic zebrafish lines we show that sox7 mutants display a short circulatory loop around the heart as a result of aberrant connections between the lateral dorsal aorta (LDA) and either the venous primary head sinus (PHS) or the common cardinal vein (CCV). In situ hybridization and live observations in flt4: mCitrine transgenic embryos revealed increased expression levels of flt4 in arterial endothelial cells at the exact location of the aberrant vascular connections in sox7 mutants. An identical circulatory short loop could also be observed in newly generated mutants for hey2 and efnb2. By genetically modulating levels of sox7, hey2 and efnb2 we demonstrate a genetic interaction of sox7 with hey2 and efnb2. The specific spatially confined effect of loss of Sox7 function can be rescued by overexpressing the Notch intracellular domain (NICD) in arterial cells of sox7 mutants, placing Sox7 upstream of Notch in this aspect of arterial development. Hence, sox7 levels are crucial in arterial specification in conjunction with hey2 and efnb2 function, with mutants in all three genes displaying shunt formation and an arterial block.
    COMPANY BIOLOGISTS LTD, May 2015, DEVELOPMENT, 142(9) (9), 1695 - 1704, English, International magazine
    [Refereed]
    Scientific journal

  • Norihito Kishimoto, Clara Alfaro-Cervello, Kohei Shimizu, Kazuhide Asakawa, Akihiro Urasaki, Shigenori Nonaka, Koichi Kawakami, Jose Manuel Garcia-Verdugo, Kazunobu Sawamoto
    In the brain of adult mammals, neuronal precursors are generated in the subventricular zone in the lateral wall of the lateral ventricles and migrate into the olfactory bulbs (OBs) through a well-studied route called the rostral migratory stream (RMS). Recent studies have revealed that a comparable neural stem cell niche is widely conserved at the ventricular wall of adult vertebrates. However, little is known about the migration route of neuronal precursors in nonmammalian adult brains. Here, we show that, in the adult zebrafish, a cluster of neuronal precursors generated in the telencephalic ventricular zone migrates into the OB via a route equivalent to the mammalian RMS. Unlike the mammalian RMS, these neuronal precursors are not surrounded by glial tubes, although radial glial cells with a single cilium lined the telencephalic ventricular wall, much as in embryonic and neonatal mammals. To observe the migrating neuronal precursors in living brain tissue, we established a brain hemisphere culture using a zebrafish line carrying a GFP transgene driven by the neurogenin1 (ngn1) promoter. In these fish, GFP was observed in the neuronal precursors migrating in the RMS, some of which were aligned with blood vessels. Numerous ngn1:gfp-positive cells were observed migrating tangentially in the RMS-like route medial to the OB. Taken together, our results suggest that the RMS in the adult zebrafish telencephalon is a functional migratory pathway. This is the first evidence for the tangential migration of neuronal precursors in a nonmammalian adult telencephalon. J. Comp. Neurol. 519:3549-3565, 2011. (C) 2011 Wiley-Liss, Inc.
    WILEY-BLACKWELL, Dec. 2011, JOURNAL OF COMPARATIVE NEUROLOGY, 519(17) (17), 3549 - 3565, English, International magazine
    [Refereed]
    Scientific journal

  • Norihito Kishimoto, Kohei Shimizu, Hideto Nagai, Kazuhide Asakawa, Akihiro Urasaki, Holger Knaut, Shigenori Nonaka, Koichi Kawakami, Kazunobu Sawamoto
    ELSEVIER IRELAND LTD, 2011, NEUROSCIENCE RESEARCH, 71, E74 - E74, English
    [Refereed]

  • Koichi Kawakami, Gembu Abe, Tokuko Asada, Kazuhide Asakawa, Ryuichi Fukuda, Aki Ito, Pradeep Lal, Naoko Mouri, Akira Muto, Maximilliano L. Suster, Hitomi Takakubo, Akihiro Urasaki, Hironori Wada, Mikio Yoshida
    Background: We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4F-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS: TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology.Description: To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish.Conclusion: zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.
    BMC, Oct. 2010, BMC DEVELOPMENTAL BIOLOGY, 10, 105 - 105, English, International magazine
    [Refereed]
    Scientific journal

  • Jeroen Bussmann, Frank L. Bos, Akihiro Urasaki, Koichi Kawakami, Henricus J. Duckers, Stefan Schulte-Merker
    The endothelial cells of the vertebrate lymphatic system assemble into complex networks, but local cues that guide the migration of this distinct set of cells are currently unknown. As a model for lymphatic patterning, we have studied the simple vascular network of the zebrafish trunk consisting of three types of lymphatic vessels that develop in close connection with the blood vasculature. We have generated transgenic lines that allow us to distinguish between arterial, venous and lymphatic endothelial cells (LECs) within a single zebrafish embryo. We found that LECs migrate exclusively along arteries in a manner that suggests that arterial endothelial cells serve as the LEC migratory substrate. In the absence of intersegmental arteries, LEC migration in the trunk is blocked. Our data therefore demonstrate a crucial role for arteries in LEC guidance.
    COMPANY BIOLOGISTS LTD, Aug. 2010, DEVELOPMENT, 137(16) (16), 2653 - 2657, English, International magazine
    [Refereed]
    Scientific journal

  • Kazuhiro Yagita, Kyoji Horie, Satoshi Koinuma, Wataru Nakamura, Iori Yamanaka, Akihiro Urasaki, Yasufumi Shigeyoshi, Koichi Kawakami, Shoichi Shimada, Junji Takeda, Yasuo Uchiyama
    The molecular oscillations underlying the generation of circadian rhythmicity in mammals develop gradually during ontogenesis. However, the developmental process of mammalian cellular circadian-oscillator formation remains unknown. In differentiated somatic cells, the transcriptional-translational feedback loops (TTFL) consisting of clock genes elicit the molecular circadian oscillation. Using a bioluminescence imaging system to monitor clock gene expression, we show here that the circadian bioluminescence rhythm is not detected in the mouse embryonic stem (ES) cells, and that the ES cells likely lack TTFL regulation for clock gene expression. The circadian clock oscillation was induced during the differentiation culture of mouse ES cells without maternal factors. In addition, reprogramming of the differentiated cells by expression of Sox2, KIf4, Oct3/4, and c-Myc genes, which were factors to generate induced pluripotent stem (iPS) cells, resulted in the re-disappearance of circadian oscillation. These results demonstrate that an intrinsic program controls the formation of the circadian oscillator during the differentiation process of ES cells in vitro. The cellular differentiation and reprogramming system using cultured ES cells allows us to observe the circadian clock formation process and may help design new strategies to understand the key mechanisms responsible for the organization of the molecular oscillator in mammals.
    NATL ACAD SCIENCES, Feb. 2010, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107(8) (8), 3846 - 3851, English, International magazine
    [Refereed]
    Scientific journal

  • Tetsuya Koide, Nobuhiko Miyasaka, Kozo Morimoto, Kazuhide Asakawa, Akihiro Urasaki, Koichi Kawakami, Yoshihiro Yoshihara
    In fish, amino acids are food-related important olfactory cues to elicit an attractive response. However, the neural circuit underlying this olfactory behavior is not fully elucidated. In the present study, we applied the Tol2 transposon-mediated gene trap method to dissect the zebrafish olfactory system genetically. Four zebrafish lines (SAGFF27A, SAGFF91B, SAGFF179A, and SAGFF228C) were established in which the modified transcription activator Gal4FF was expressed in distinct subsets of olfactory sensory neurons (OSNs). The OSNs in individual lines projected axons to partially overlapping but mostly different glomeruli in the olfactory bulb (OB). In SAGFF27A, Gal4FF was expressed predominantly in microvillous OSNs innervating the lateral glomerular cluster that corresponded to the amino acid-responsive region in the OB. To clarify the olfactory neural pathway mediating the feeding behavior, we genetically expressed tetanus neurotoxin in the Gal4FF lines to block synaptic transmission in distinct populations of glomeruli and examined their behavioral response to amino acids. The attractive response to amino acids was abolished only in SAGFF27A fish carrying the tetanus neurotoxin transgene. These findings clearly demonstrate the functional significance of the microvillous OSNs innervating the lateral glomerular cluster in the amino acid-mediated feeding behavior of zebrafish. Thus, the integrated approach combining genetic, neuroanatomical, and behavioral methods enables us to elucidate the neural circuit mechanism underlying various olfactory behaviors in adult zebrafish.
    NATL ACAD SCIENCES, Jun. 2009, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(24) (24), 9884 - 9889, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiro Urasaki, Koichi Kawakami
    The Tol2 transposon system can create insertions in the zebrafish genome efficiently. By using this system, the gene trap and enhancer trap methods have been developed. The gene trap and enhancer trap constructs contain the green fluorescent protein (GFP) reporter gene or the yeast Gal4 transcription activator gene. By creating random integrations of these constructs in the genome, transgenic fish expressing the GFP gene or the Gal4 gene in specific cells, tissues or organs are generated. These fish are valuable resources for developmental biology. Especially, the Gal4-expressing transgenic fish can be used to ectopically express any gene of interest placed downstream of the Gal4 recognition sequence, UAS, and thereby allow visualization, modification or ablation of the Gal4-expressing cells. In this chapter, we will describe how the gene trap and enhancer trap screens can be performed and how the transposon insertions created by these methods can be analyzed.
    Lead, 2009, Methods in molecular biology (Clifton, N.J.), 546, 85 - 102, English, International magazine
    [Refereed]
    Scientific journal

  • Maximiliano L Suster, Hiroshi Kikuta, Akihiro Urasaki, Kazuhide Asakawa, Koichi Kawakami
    The zebrafish (Danio rerio) is a useful model for genetic studies of vertebrate development. Its embryos are transparent and develop rapidly outside the mother, making it feasible to visualize and manipulate specific cell types in the living animal. Zebrafish is well suited for transgenic manipulation since it is relatively easy to collect large numbers of embryos from adult fish. Several approaches have been developed for introducing transgenes into the zebrafish germline, from the injection of naked DNA to transposon-mediated integration. In particular, the Tol2 transposable element has been shown to create insertions in the zebrafish genome very efficiently. By using Tol2, gene trap and enhancer trap vectors containing the GFP reporter gene or yeast transcription activator Gal4 gene have been developed. Here we outline methodology for creating transgenic zebrafish using Tol2 vectors, and their applications to visualization and manipulation of specific tissues or cells in vivo and for functional studies of vertebrate neural circuits.
    2009, Methods in molecular biology (Clifton, N.J.), 561, 41 - 63, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiro Urasaki, Taro Mito, Sumihare Noji, Ryu Ueda, Koichi Kawakami
    The Tol2 element is a transposon found from a genome of a vertebrate, a small teleost medaka fish. Tol2 encodes a gene for a transposase which is active in vertebrate animals so far tested; for instance, in fish, frog, chicken and mammals, and transgenesis methods using Tol2 have been developed in these model vertebrates. However, it has not been known whether Tol2 can transpose in animals other than vertebrates. Here we report transposition of Tol2 in an invertebrate Drosophila melanogaster. First, we injected a transposon donor plasmid containing a Tol2 construct and mRNA encoding the Tol2 transposase into Drosophila eggs, and found that the Tol2 construct could be excised from the plasmid. Second, we crossed the injected flies, raised the offspring, and found that the Tol2 construct was integrated into the genome of germ cells and transmitted to the next generation. Finally, we constructed a Tol2 construct containing the white gene and injected the transposon donor plasmid and the transposase mRNA into fertilized eggs from the white mutant. We analyzed their offspring, and found that G1 flies with wild type red eyes could be obtained from 35% of the injected fly. We cloned and sequenced 34 integration loci from these lines and showed that these insertions were indeed created through transposition and distributed throughout the genome. Our present study demonstrates that the medaka fish Tol2 transposable element does not require vertebrate-specific host factors for its transposition, and also provides a possibility that Tol2 may be used as a new genetic tool for transgenesis and genome analysis in Drosophila. (c) 2008 Elsevier B.V. All rights reserved.
    Lead, ELSEVIER SCIENCE BV, Dec. 2008, GENE, 425(1-2) (1-2), 64 - 68, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiro Urasaki, Kazuhide Asakawa, Koichi Kawakami
    The Tol2 transposable element is a powerful genetic tool in model vertebrates and has been used for transgenesis, insertional mutagenesis, gene trapping, and enhancer trapping. However, an in vivo transposition system using Tol2 has not yet been developed. Here we report the in vivo Tol2 transposition system in a model vertebrate, zebrafish. First, we constructed transgenic zebrafish that carried single-copy integrations of Tol2 on the genome and injected transposase mRNA into one-cell stage embryos. The Tol2 insertions were mobilized efficiently in the germ lineage. We then mobilized an insertion of the Tol2 gene trap construct in the nup214 gene, which caused a recessive lethal mutant phenotype, and demonstrated that this method is applicable to the isolation of revertants from a transposon insertional mutant. Second, we constructed transgenic fish carrying the transposase cDNA under the control of the hsp70 promoter. Double-transgenic fish containing the transposase gene and a single-copy Tol2 insertion were treated with heat shock at the adult stage. We found that transposition can be induced efficiently in the male germ cells. We analyzed new integration sites and found that the majority (83%) of them were mapped on chromosomes other than the transposon donor chromosomes and that 9% of local hopping events mapped less than 300 kb away from the donor loci. Our present study demonstrates that the in vivo Tol2 transposition system is useful for creating genome-wide insertions from a single-copy donor and should facilitate functional genomics and transposon biology in vertebrates.
    Lead, NATL ACAD SCIENCES, Dec. 2008, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105(50) (50), 19827 - 19832, English, International magazine
    [Refereed]
    Scientific journal

  • Saori Nagayoshi, Eriko Hayashi, Gembu Abe, Naoki Osato, Kazuhide Asakawa, Akihiro Urasaki, Kazuki Horikawa, Kazuho Ikeo, Hiroyuki Takeda, Koichi Kawakami
    Gene trap and enhancer trap methods using transposon or retrovirus have been recently described in zebrafish. However, insertional mutants using these methods have not been reported. We report here development of an enhancer trap method by using the Tol2 transposable element and identification and characterization of insertional mutants. We created 73 fish lines that carried single copy insertions of an enhancer trap construct, which contained the zebrafish hsp70 promoter and the GFP gene, in their genome and expressed GFP in specific cells, tissues and organs, indicating that the hsp70 promoter is highly capable of responding to chromosomal enhancers. First, we analyzed genomic DNA surrounding these insertions. Fifty-one of them were mapped onto the current version of the genomic sequence and 43% (22/51) were located within transcribed regions, either exons or introns. Then, we crossed heterozygous fish carrying the same insertions and identified two insertions that caused recessive mutant phenotypes. One disrupted the tcf7 gene, which encodes a transcription factor of the Tcf/Lef family mediating Wnt signaling, and caused shorter and wavy median fin folds and pectoral fins. We knocked down Lef1, another member of the Tcf/Lef family also expressed in the fin bud, in the tcf7 mutant, and revealed functional redundancy of these factors and their essential role in establishment of the apical ectodermal ridge (AER). The other disrupted the synembryn-like gene (synbl), a homolog of the C. elegans synembryn gene, and caused embryonic lethality and small pigment spots. The pigment phenotype was rescued by application of forskolin, an activator of adenylyl cyclase, suggesting that the synbl gene activates the G(alpha s) pathway leading to activation of adenylyl cyclase. We thus demonstrated that the transposon-mediated enhancer trap approach can indeed create insertional mutations in developmental genes. Our present study provides a basis for the development of efficient transposon-mediated insertional mutagenesis in a vertebrate.
    COMPANY BIOLOGISTS LTD, Jan. 2008, DEVELOPMENT, 135(1) (1), 159 - 169, English, International magazine
    [Refereed]
    Scientific journal

  • Kazuhide Asakawa, Maximiliano L. Suster, Kanta Mizusawa, Saori Nagayoshi, Tomoya Kotani, Akihiro Urasaki, Yasuyuki Kishimoto, Masahiko Hibi, Koichi Kawakami
    Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.
    NATL ACAD SCIENCES, Jan. 2008, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105(4) (4), 1255 - 1260, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiro Urasaki, Ghislaine Morvan, Koichi Kawakami
    The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (5') and the right (3') ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of > 11 kb could transpose, but a certain length of spacer, < 276 but > 18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 5'-(A/G)AGTA-3', is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.
    Lead, GENETICS SOCIETY AMERICA, Oct. 2006, GENETICS, 174(2) (2), 639 - 649, English, International magazine
    [Refereed]
    Scientific journal

  • Tomoya Kotani, Saori Nagayoshi, Akihiro Urasaki, Koichi Kawakami
    The Tol2 transposon system can create chromosomal insertions in the zebrafish germ lineage very efficiently. We constructed a Tol2-based gene trap vector, T2KSAG, which contains a splice accepter, the GFP gene and the polyA signal. In the pilot screen for gene trapping using T2KSAG, we identified 38 fish lines expressing GFP in specific organs and tissues. In the SAGp53A line, GFP is expressed in the forebrain and midbrain, and the insertion of the gene trap construct captured a transcript of the kab gene encoding a zebrafish homolog of the human KARP (Ku86 autoantigen related protein)-binding protein (KAB). In the SAGm18B line, GFP is expressed in the central nervous system, and the insertion captured a transcript of a gene for succinyl CoA:3-oxoacid CoA-transferase (SCOT). Here, we describe how we performed the gene trap screen and characterized the gene trap insertions and will discuss the outcome of the pilot screen. (c) 2006 Elsevier Inc. All rights reserved.
    ACADEMIC PRESS INC ELSEVIER SCIENCE, Jul. 2006, METHODS, 39(3) (3), 199 - 206, English, International magazine
    [Refereed]
    Scientific journal

  • Shannon Fisher, Elizabeth A. Grice, Ryan M. Vinton, Seneca L. Bessling, Akihiro Urasaki, Koichi Kawakami, Andrew S. McCallion
    Evaluating the biological relevance of the myriad putative regulatory noncoding sequences in vertebrate genomes represents a huge challenge. Functional analyses in vivo have typically relied on costly and labor-intensive transgenic strategies in mice. Transgenesis has also been applied in nonrodent vertebrates, such as zebrafish, but until recently these efforts have been hampered by significant mosaicism and poor rates of germline transmission. We have developed a transgenic strategy in zebrafish based on the Tol2 transposon, a mobile element that was recently identified in another teleost, Medaka. This method takes advantage of the increased efficiency of genome integration that is afforded by this intact DNA transposon, activity that is mediated by the corresponding transposase protein. The approach described in this protocol uses a universal vector system that permits rapid incorporation of DNA that is tagged with sequence targets for site-specific recombination. To evaluate the regulatory potential of a candidate sequence, the desired interval is PCR-amplified using sequence-specific primers that are flanked by the requisite target sites for cloning, and recombined into a universal expression plasmid (pGW_ cfosEGFP). Purified recombinant DNAs are then injected into 1-2-cell zebrafish embryos and the resulting reporter expression patterns are analyzed at desired timepoints during development. This system is amenable to large-scale application, facilitating rapid functional analysis of noncoding sequences from both mammalian and teleost species.
    NATURE PUBLISHING GROUP, 2006, NATURE PROTOCOLS, 1(3) (3), 1297 - 1305, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiro Urasaki, Yasuhiko Sekine, Eiichi Ohtsubo
    The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant. A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family. To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision. Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100 transposes in E. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.
    AMER SOC MICROBIOLOGY, Sep. 2002, Journal of bacteriology, 184(18) (18), 5104 - 12, English, International magazine
    [Refereed]
    Scientific journal

■ MISC
  • CRMP
    Yusuke Kubo, Akihiro Urasaki, Naoyuki Inagaki
    2017, 脳科学辞典, https://bsd.neuroinf.jp/wiki/C, Japanese
    [Invited]
    Introduction commerce magazine

  • Identification of a shootin1 isoform expressed in peripheral tissues
    Higashiguchi Yasuna, Katsuta Kazuhiro, Minegishi Takunori, Yonemura Shigenobu, Urasaki Akihiro, Inagaki Naoyuki
    Shootin1 is a brain-specific cytoplasmic protein involved in neuronal polarity formation and axon outgrowth. It accumulates at the leading edge of axonal growth cones, where it mediates the mechanical coupling between F-actin retrograde flow and cell adhesions as a clutch molecule, thereby producing force for axon outgrowth. In this study, we report a novel splicing isoform of shootin1 which is expressed not only in the brain but also in peripheral tissues. We have renamed the brain-specific shootin1 as shootin1a and termed the novel isoform as shootin1b. Immunoblot and immunohistochemical analyses with a shootin1b-specific antibody revealed that shootin1b is distributed in various mouse tissues including the lung, liver, stomach, intestines, spleen, pancreas, kidney and skin. Interestingly, shootin1b immunoreactivity was widely detected in epithelial cells that constitute simple and stratified epithelia; in some cells, it colocalized with E-cadherin and cortactin at cell–cell contact sites. Shootin1b also localized in dendritic cells in the spleen. These results suggest that shootin1b may function in various peripheral tissues including epithelial cells.
    Springer Berlin Heidelberg, 13 May 2016, English

  • Baba Kentarou, Urasaki Akihiro, Inagaki Naoyuki
    Japanese Electrophoresis Society, Oct. 2014, SEIBUTSU BUTSURI KAGAKU, 58(2) (2), 49 - 52, Japanese

  • 馬場健太郎, 浦崎明宏, 稲垣直之
    Japanese Electrophoresis Society, 2014, 生物物理化学(Web), 58(2) (2), 49-52 (J-STAGE) - 52, Japanese

  • Tol2トランスポゾンによる子宮内膜腺癌株(イシカワ株)への効果的な遺伝子導入 着床障害不妊症の遺伝子治療に対する有益なDNAベクター(Efficient gene transfer to endometrial adenocarcinoma cell line (Ishikawa) by Tol2 transposable element : a possible DNA vector for gene therapy for implantation failure)
    鈴木 良知, 浦崎 明宏, 浅見 幸夫, 井坂 恵一, 川上 浩一
    生殖補助医療の進歩により、着床障害性不妊症の原因が子宮内膜内に存在すべきサイトカイン遺伝子の欠如およびそれらの発現の低下によるものであることが示唆され、子宮内膜細胞内にこれらの遺伝子を遺伝子導入する試みがなされてきている。今回我々は、メダカ由来のトランスポゾンによる遺伝子導入システム(Tol2ベクター)がヒト由来細胞(子宮内膜上皮細胞のモデル細胞株であるイシカワ株)において有効に機能するかについて検討をおこなった。Tol2ベクターにネオマイシン耐性遺伝子を組み込み、イシカワ株での遺伝子発現の有無を確認したところ、その発現が観察され、またTol2転移酵素存在下において非存在下の約4倍もの効率で遺伝子導入がなされていることが明らかとなった。さらに挿入されたTol2の両末端すべてに8塩基対の反復配列が観察された。このことより、Tol2ベクターによる遺伝子導入システムはウイルスベクターに比べてより有効なベクターとして今後汎用されることが期待される。本研究は着床障害に対する遺伝子治療の第一歩であるが、今後遺伝子異常によって引き起こされる疾患・癌治療・再生医療を含めた幅広い医療に臨床応用されることが期待される。(著者抄録)
    東京医科大学医学会, Oct. 2010, 東京医科大学雑誌, 68(4) (4), 396 - 402, English

  • Zebrafishを用いた神経のミエリネーション可視化による膜型プロテアーゼADAM19の機能の解明
    佐藤文規, 佐藤智美, 坂口和弥, 浦崎明宏, 和田浩則, 川上浩一, 瀬原淳子
    2010, 生化学

  • A new era of zebrafish research opened up by the Tol2 transposon technology
    浦崎 明宏, 浅川 和秀, 川上 浩一
    秀潤社, May 2009, Cell technology., 28(6) (6), 586 - 591, Japanese

  • ゲノム上を“動く遺伝子”トランスポゾン 脊椎動物におけるトランスポゾンを用いた遺伝学的方法論
    浦崎明宏, 川上浩一
    トランスポゾンを用いた遺伝学的方法論は微生物、植物、無脊椎動物の遺伝学的研究において非常に重要な役割を果たしてきた。しかしながら、脊椎動物においてはそのような方法論は長い間開発されてこなかった。その原因は、脊椎動物において活性のあるトランスポゾンが見つかっていなかったことにある。最近、脊椎動物で活性のあるトランスポゾンが見出され、遺伝子破壊や遺伝子導入のツールとして利用できるようになってきた。本稿では、脊椎動物で利用されているトランスポゾンの特徴を整理し、その応用の可能性について述べる。(著者抄録)
    (株)羊土社, Oct. 2007, 実験医学, 25(16) (16), 2507 - 2512, Japanese

  • トランスポゾンを用いたGal4-UAS法による細胞の可視化と機能改変
    川上浩一, 浅川和秀, 阿部玄武, 浦崎明宏, 菊田寛, 岸本康之, 武藤彩, SUSTER Maximiliano, 日比正彦
    2007, 生化学

■ Lectures, oral presentations, etc.
  • Vascular connection between newly formed intestinal and pre-existing circulatory systems during embryonic development in zebrafish
    Akihiro Urasaki, Osamu Nakagawa
    The 30th Japanese Vascular Biology and Medicine Organization (JVBMO2022), Dec. 2022

  • Physiological importance of ALK1 signaling for organotypic vascular formation in zebrafish
    Akihiro Urasaki, Yuki Kakihana, Nami Akaho Nagata, Yukihiro Harada, Osamu Nakagawa
    The 5th JCS Council Forum on Basic Cardio Vascular Research (BCVR2022), Dec. 2022

  • Physiological significance of ALK1 signaling in zebrafish organotypic vascular formation
    Akihiro URASAKI, Nami Akaho NAGATA, Yuki KAKIHANA, Yukihiro HARADA, Osamu NAKAGAWA
    The 5th JCS Council Forum on Basic CardioVascular Research (BCVR 2021), Sep. 2021
    Oral presentation

  • ACVRL1変異と受容体分子機能の関連解析
    岩朝徹, 長田菜美, 原田恭弘, 垣花優希, 浦崎明宏, 白石公, 黒嵜健一, 森崎裕子, 小宮山雅樹, 中川修
    HHT JAPAN 2021（第7回日本HHT研究会）, Jul. 2021
    Oral presentation

  • Tol2遺伝子導入システムの開発と生体ライブイメージングによる基礎血管研究
    浦崎明宏
    第57回日本小児循環器学会学術集会併催 第24回日本小児心血管分子医学研究会, Jul. 2021
    [Invited]
    Invited oral presentation

  • ACVRL1変異と受容体分子機能の関連解析
    岩朝徹, 長田菜美, 原田恭弘, 垣花優希, 浦崎明宏, 白石公, 黒嵜健一, 森崎裕子, 小宮山雅樹, 中川修
    第57回日本小児循環器学会学術集会併催 第24回日本小児心血管分子医学研究会, Jul. 2021
    Oral presentation

  • 胎生期血管形成におけるリン酸化酵素遺伝子SGK1の内皮特異的発現機構
    原田恭弘, 田中亨, 荒井勇二, 浦﨑明宏, 劉孟佳, 渡邉裕介, 川村晃久, 中川修
    第43回日本分子生物学会年会, Dec. 2020

  • 臓器特異的血管形成におけるALK1シグナルの生理学的重要性
    浦﨑明宏, 中川修
    第43回日本分子生物学会年会, Dec. 2020

  • Transcriptional regulation and physiological significance of ALK1 signal target genes in embryonic vascular endothelial cells
    Osamu NAKAGAWA, Yusuke WATANABE, Akihiro URASAKI, Toru TANAKA, Daiki SEYA, Norika LIU, Shoko TAMURA, Dai IHARA, Yukihiro HARADA, Teruhisa KAWAMURA
    International Vascular Biology Meeting 2020, Sep. 2020
    Oral presentation

  • 見えてきた微小環境依存的Notchシグナルの役割と病態生理 Notchシグナルと下流遺伝子の心血管形成機構と疾患における意義(Significance of Notch signaling and downstream genes in cardiovascular development and disease)
    中川 修, 渡邉 裕介, 浦崎 明宏
    日本薬学会年会要旨集, Mar. 2020, English, (公社)日本薬学会

  • 胚発生における心血管シグナル伝達系と環境因子の相互関係
    浦崎 明宏, 田中 亨, 原田 恭弘, 劉 孟佳, 川村 晃久, 渡邉 裕介, 中川 修
    日本内分泌学会雑誌, Feb. 2020, Japanese, (一社)日本内分泌学会

  • ゼブラフィッシュを用いた側線形成におけるshootin遺伝子の機能解析
    森下 誠也, 浦崎 明宏, 中公 甫, 魚住 海斗, 松井 貴輝, 別所 康全, 川上 浩一, 稲垣 直之
    生命科学系学会合同年次大会, Dec. 2017, Japanese, 生命科学系学会合同年次大会運営事務局

  • 新規shootinファミリーメンバーの同定と機能解析
    浦崎 明宏, 森下 誠也, 中公 甫, 魚住 海斗, 渡瀬 恵美子, 松井 貴輝, 別所 康全, 川上 浩一, 稲垣 直之
    生命科学系学会合同年次大会, Dec. 2017, Japanese, 生命科学系学会合同年次大会運営事務局

  • The Tol2 system opens up a new frontier in vertebrate vascular biology
    Akihiro Urasaki
    NCVC seminar, Jun. 2016, Japanese
    [Invited]
    Invited oral presentation

  • ゼブラフィッシュを用いた脳形成におけるshootin1の役割の解析
    浦崎明宏, 渡瀬恵美子, 松井貴輝, 川上浩一, 別所康全, 稲垣直之
    日本細胞生物学会大会要旨集, May 2014, Japanese

  • Tol2トランスポゾンシステムが拓く新たな脈管および神経研究
    浦﨑明宏
    奈良先端大セミナー, May 2013, Japanese
    [Invited]
    Invited oral presentation

  • Blood vessel formation by coordinated venous-fated angioblast migration
    Akihiro Urasaki
    5th Angioclub meeting, May 2011, English
    [Invited]
    Invited oral presentation

  • Zebrafishを用いた神経のミエリネーション可視化による膜型プロテアーゼADAM19の機能の解明
    佐藤文規, 佐藤智美, 坂口和弥, 浦崎明宏, 和田浩則, 川上浩一, 瀬原淳子
    生化学, 2010, Japanese

  • Toward Tol2-mediated knockouts of all zebrafish genes
    Akihiro Urasaki, Koichi Kawakami
    GENES & GENETIC SYSTEMS, Dec. 2009, English, GENETICS SOC JAPAN

  • Tol2を用いたゼブラフィッシュ遺伝子の網羅的破壊
    浦崎明宏, 川上浩一
    日本遺伝学会大会プログラム・予稿集, Aug. 2009, Japanese

  • ゼブラフィッシュGal4トラップ法を用いた神経回路の可視化と機能阻害
    浅川和秀, SUSTER Maximiliano L, 浦崎明宏, 小谷友也, 永吉さおり, 岸本康之, 日比正彦, 川上浩一
    Program Abstr Book Annu Meet Jpn Soc Dev Biol, 2008, Japanese

  • ゼブラフィッシュにおいて骨格特異的に発現する新規遺伝子の遺伝子トラップ法による同定
    水澤寛太, 浅川和秀, 浦崎明宏, 小谷友也, 永吉さおり, 岸本康之, 日比正彦, 近藤滋, 川上浩一
    日本水産学会大会講演要旨集, Sep. 2007, Japanese

  • New genetic techniques by excision of Tol2: isolation of revertants and creating frame shift mutations
    Akihiro Urasaki, Kazuhide Asakawa, Koichi Kawakami
    5th European Zebrafish Genetics and Development Meeting, Jul. 2007, English
    Poster presentation

  • テクニカルセッション ゼブラフィッシュにおけるJump starterトランスポゾン系:Tol2の熱誘導性転移はゲノムワイドな挿入を可能にする(The jump starter transposon system in zebrafish: heat-inducible transposition of a single integrated Tol2 can create genome-wide insertions)
    浦崎 明宏, 浅川 和秀, 川上 浩一
    日本発生生物学会・日本細胞生物学会合同大会要旨集, May 2007, English

  • 再生・組織形成・器官形成 ゼブラフィッシュにおけるトランスポゾン介在性Gal4遺伝子とエンハンサーとラップ法による標的遺伝子発現は単一の脊椎動物行動に必要なニューロンサブセットを決定する(Targeted gene expression by transposon-mediated Gal4 gene and enhancer trapping in zebrafish defines subsets of neurons required for simple vertebrate behaviors)
    浅川 和秀, 水澤 寛太, 永吉 さおり, 小谷 友也, 浦崎 明宏, 岸本 康之, 日比 正彦, 川上 浩一
    日本発生生物学会・日本細胞生物学会合同大会要旨集, May 2007, English

  • トランスポゾンを用いたGal4‐UAS法による細胞の可視化と機能改変
    川上浩一, 浅川和秀, 阿部玄武, 浦崎明宏, 菊田寛, 岸本康之, 武藤彩, SUSTER Maximiliano, 日比正彦
    生化学, 2007, Japanese

  • Remobilization of integrated transposons: the jump starter system in zebrafish
    Akihiro Urasaki, Kazuhide Asakawa, Koichi Kawakami
    7th International Conference on Zebrafish Development & Genetics, Jul. 2006, English
    Oral presentation

  • ゼブラフィッシュにおけるGal4エンハンサートラップ法によるファンクショナルゲノミクス
    浅川和秀, 浦崎明宏, 小谷友也, 永吉さおり, 岸本康之, 日比正彦, 川上浩一
    日本分子生物学会年会講演要旨集, Nov. 2005, Japanese

  • The Tol2-mediated genetic methodologies in zebrafish: gene trapping, enhancer trapping and remobilization
    Akihiro Urasaki, Kazuhide Asakawa, Tomoya Kotani, Saori Nagayoshi, Yasuyuki Kishimoto, Koichi Kawakami
    4th European Zebrafish Genetics and Development Meeting, Jul. 2005, English
    Oral presentation

  • トランスポゾンを用いた遺伝学的方法論によるゼブラフィッシュの初期発生研究
    小谷友也, 浦崎明宏, 永吉さおり, 浅川和秀, 岸本康之, 川上浩一
    日本発生生物学会大会発表要旨集, May 2005, Japanese

  • ゼブラフィッシュにおけるトランスポゾンTol2を用いた新しい発生遺伝学的方法論の開発
    浦崎明宏, 川上浩一
    日本分子生物学会年会プログラム・講演要旨集, Nov. 2004, Japanese

  • ゼブラフィッシュにおけるトランスポゾンを用いたGal4エンハンサートラップ法の構築
    浅川和秀, 伊藤安希, 浦崎明宏, MORVAN G, 小谷友也, 佐々木剛, 永吉さおり, 岸本康之, 日比正彦
    日本分子生物学会年会プログラム・講演要旨集, Nov. 2004, Japanese

  • シアノバクテリアの転移性遺伝因子ISY100転移の分子機構
    浦崎明宏, 大坪栄一
    日本分子生物学会年会プログラム・講演要旨集, Nov. 2003, Japanese

  • イネDNA型トランスポゾンTnr1/Osmarは大腸菌細胞内で転移可能である
    園田陽, 浦崎明宏, 土本卓, 大坪久子, 大坪栄一
    日本分子生物学会年会プログラム・講演要旨集, Nov. 2003, Japanese

■ Affiliated Academic Society
  • 小型魚類研究会

  • 日本分子生物学会

  • 日本血管生物医学会

■ Research Themes
  • 心臓発生期のゲノム網羅的エンハンサーアトラスと心臓内領域特異的遺伝子発現制御機構
    中川 修, 原田 恭弘, 川村 晃久, 白井 学, 高橋 篤, 浦崎 明宏, 橋本 大輝
    日本学術振興会, 科学研究費助成事業, 挑戦的研究(萌芽), 国立研究開発法人国立循環器病研究センター, 30 Jun. 2023 - 31 Mar. 2026

  • ALK1シグナルの臓器特異的血管形成における意義と難治性血管疾患の発症メカニズム
    浦崎 明宏, 中川 修, 渡邉 裕介, 垣花 優希, 原田 恭弘, 橋本 大輝, LAMRI LYNDA
    日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 国立研究開発法人国立循環器病研究センター, 01 Apr. 2022 - 31 Mar. 2025

  • Downstream genes of signaling pathways implicated in human cardiovascular diseases: in vivo significance and regulatory mechanisms
    中川 修, 浦崎 明宏, LAMRI LYNDA, 原田 恭弘, 垣花 優希, 橋本 大輝, 渡邉 裕介, 白石 公, 岩朝 徹, 能丸 寛子
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), National Cardiovascular Center Research Institute, 01 Apr. 2021 - 31 Mar. 2024

  • Integrated analysis of pathgenesis of hereditary hemorrhagic telangiectasia
    岩朝 徹, 渡邉 裕介, 中川 修, 浦崎 明宏, 黒嵜 健一, 石川 泰輔, 白石 公
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), National Cardiovascular Center Research Institute, 01 Apr. 2020 - 31 Mar. 2023
    過去に当センターで遺伝性出血性末梢血管拡張症の患者より検出した遺伝子変異と論文報告のあった同疾患患者の遺伝子変異について、変異プラスミドの作成と実験解析に向いたミスセンス変異のACVRL1変異及び野生型ACVRL1について、過去の論文を参考にマウス線維芽細胞株（本来ACVRL1の発現を持たないNIH-3T3細胞)にプラスミドを用いて導入した。 野生型を導入した細胞ではACVRL1をBMP9/10により刺激することで、その下流に位置する活性型(リン酸化)SMAD1/5といった蛋白の出現（ウエスタンブロットで解析）や、下流に位置する転写因子の一群の発現の亢進（ルシフェラーゼアッセイで解析）が見られることを確認した。一方で、変異型を導入した細胞の多くは下流の活性型(リン酸化)SMAD1/5、転写因子の発現が抑制されていたが、一部の変異では抑制されておらず、実際の症状を生じている患者との間にdiscrepancyが認められた。 また変異型を導入した細胞での免疫染色では、多くの変異では野生型ACVRL1と大きな変化は確認出来なかったが、一部の変異（膜通過部分の変異）においては細胞表面に存在するはずのACVRL1が消失していることが確認出来た。これらの細胞実験において生じる現象の差違が、実際の患者さんの症状の重篤さや発症様式に関連するか、今後検討を加えていく予定である。また合わせてゼブラフィッシュに変異型ACVRL1を導入し、実際の生体内で血管形成が変化していくかどうかを順次確認を加えていく予定である。

  • 胸部大血管および心室筋形成における細胞分化の分子機構
    渡邉 裕介, 小柴 和子, 中川 修, 劉 孟佳, 浦崎 明宏, LAMRI LYNDA, 瀬谷 大貴, 田中 亨
    日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 国立研究開発法人国立循環器病研究センター, 01 Apr. 2019 - 31 Mar. 2022
    本研究は①「胸部大血管形成における内皮細胞発生・管腔構造形成の重要性と制御機構の解明」と、②「心室筋の異なる領域における転写制御機構と細胞運命の解明」を目的としている。 ①について、本年度は胸部大血管形成における内皮細胞でのHey1遺伝子の重要性を明らかにするため、出生直前の内皮細胞特異的Hey1欠損マウスの胸部大血管での表現型をmicroCTを用いて解析した。その結果、右大動脈弓などの構造異常を3次元的に観察することができた。また胸部大血管の由来となる胎生10.5日目胚での咽頭弓動脈の構造および遺伝子発現についても解析を進めている。さらに、内皮で発現するHey1遺伝子の転写制御機構を明らかにするため、Rbpj欠損マウス胚でのHey1内皮エンハンサー活性の解析を行った。その結果、Notch/Rbpjシグナル伝達経路が内皮でのHey1遺伝子発現に必須であることが明らかとなった。以上により、内皮で発現するHey1遺伝子の胸部大血管形成における重要性と転写制御機構の一端を明らかにした。 ②について、本年度は胎生期心室筋で発現するHey2遺伝子の心室筋エンハンサーの転写制御機構を明らかにするため、Hey2心室筋エンハンサー欠損マウスの作製および当該マウスでのHey2遺伝子発現の解析、ルシフェラーゼアッセイを用いたHey2心室筋エンハンサー活性化因子の検索、Tbx20欠損マウス胚心臓でのHey2心室筋エンハンサー活性の解析を行った。その結果、Hey2心室筋エンハンサーは心室筋におけるHey2遺伝子発現に必須であり、Tbx20およびGata因子により活性化されていることが明らかとなった。本研究実績はDev Biol誌に投稿し受理された（Ihara et al., 2020. 共責任著者）。

  • Role of vascular interaction in embryonic development
    Urasaki Akihiro
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), National Cardiovascular Center Research Institute, 01 Apr. 2019 - 31 Mar. 2022
    Vessels such as arteries, veins, and lymphatic vessels form anatomically distinct networks. The importance of contact, connection (anastomosis), and juxtaposition between different vessels in vertebrate development is still unclear. In this study, we investigated the role of interactions between different blood vessels during vascular formation. Using transparent zebrafish embryos, we analyzed the process of organ-specific vascular formation during embryogenesis. We found that defects in ALK1 signaling lead to cerebral arteriovenous malformations, in which arteries and veins connect directly without capillaries, and to a new type of vascular abnormality in another organ.

  • Significance of cellular signaling pathways in cardiovascular development and disease
    NAKAGWA Osamu
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), National Cardiovascular Center Research Institute, 01 Apr. 2018 - 31 Mar. 2021
    Cardiovascular development and morphogenesis are controlled by a variety of cellular signaling pathways, and their defects cause congenital or hereditary cardiovascular diseases. In this research project, we focused on the genes that functioned as downstream targets of Notch and ALK1 signaling pathways and identified their gene regulatory regions, which were called enhancers. We found that these enhancer regions were bound by different transcription factors, which were important for the cardiovascular expression of the downstream genes. We also demonstrated that these transcription factors and signaling molecules had distinct roles in various subregions of the heart and vasculature during development. These results help understand the molecular mechanisms of cardiovascular development and morphogenesis and the etiologies of human congenital and hereditary diseases.

  • Naoyuki Inagaki, URASAKI Akihiro
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Nara Institute of Science and Technology, 01 Apr. 2014 - 31 Mar. 2017
    Force interaction between cells and adhesive substreates induces cell migration that plays a key role in tissue morphogenesis. However, the molecular mechanics how cells generate forces between cells and substreates for cell migration and tissue morphogenesis remains unclear. Shootin1 is a brain-specific protein involved in axon formation. We found a novel splicing isoform of shootin1 which is expressed not only in the brain but also in peripheral tissues. We renamed the brain-specific shootin1 as shootin1a and termed the novel isoform as shootin1b. We found that shootin1 is involved in migration of olfactory neurons and formation of the olfactory bulb. Shootin1b interacts with actin filaments that undergo directional polymerization at the leading edge of migrating olfactory neurons. Our data suggest shootin1b mediates the linkage between the actin filaments and the cell adhesion molecule L1-CAM, thereby generating forces between cell’s leading and and substreates for migration.

  • Urasaki Akihiro
    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), Nara Institute of Science and Technology, 01 Apr. 2014 - 31 Mar. 2017
    In addition to shootin1, we found two novel shootin family members (shootin 2 and shootin 3). All three shootin genes were expressed in zebrafish embryos. In particular, shootin 1 and shootin 3 were expressed maternally and were expressed in the lateral line primordia in developing zebrafish embryos. Mutant analysis suggests that shootin 3 is involved in the early embryonic development and that shootin 1 and shootin 3 are involved in the formation of lateral lines, which are organs that sense water flow.

  • ゼブラフィッシュを用いた組織形態形成におけるshootin1の機能解析
    浦崎明宏
    文部科学省, 科学研究費補助金(若手研究(B)), 2014 - 2016, Principal investigator
    Competitive research funding

  • 浦崎 明宏
    日本学術振興会, 科学研究費助成事業 若手研究(B), 若手研究(B), 国立遺伝学研究所, 2010 - 2010, Principal investigator
    Competitive research funding

  • 浦崎 明宏
    日本学術振興会, 科学研究費助成事業 研究活動スタート支援, 研究活動スタート支援, 国立遺伝学研究所, 2009 - 2009, Principal investigator
    モデル脊椎動物ゼブラフィッシュを用いて、脈管形成におけるmekk3bの役割を明らかにすることを目的に研究を行った。独自に成したSAGFF27C遺伝子トラップ系統では、遺伝子トラップコンストラクトがmekk3b遺伝子のイントロンに挿入して、mekk3b遺伝子の転写産物をトラップしていた。まず、SAGFF27C ; UAS : GFP系統のGFP発現パターンの解析を行ったところ、発生初期には後主静脈で、発生後期には節管静脈およびリンパ管でGFPを発現することが分かった。次に、in situハイブリダイゼーションにより、後主静脈細胞でmekk3bが発現していることを確認した。さらに、モルフォリノオリゴヌクレオチドを用いてmekk3bの発現阻害による表現型の解析を行ったところ、mekk3b発現阻害胚では後主静脈が形成されなくなることが分かった。そして、mekk3b mRNAをmekk3b発現阻害胚に微量注入すると、異常表現型が回復した。このことから、mekk3bは後主静脈の形成に重要な役割を果たしていることが示された。後主静脈の形成過程の詳細を明らかにするために、血管内皮細胞でGFPを発現するflil:GFP系統や赤血球前駆細胞でRFPを発現するgata1:RFP系統を用いて、タイムラプス生体イメージングを行った。これまで、ヒトやマウスを使った研究では、体内で発生するため、発生初期の血管形成過程を捉えるのは困難であった。本研究では、胚が透明で、体外で発生するゼブラフィッシュを用いることにより、受精後最初に出来る血管の形成過程を捉えることに成功した。これらの結果は、「細胞の塊」から「血流のある血管」がいかにして形成されるかを細胞・分子レベルで理解する上で重要な知見であると考える。
    Competitive research funding

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