Kiyokazu Agata

Kyoto University, Kioto, Kyōto, Japan

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Publications (192)648.78 Total impact

  • 12/2015; 1(1). DOI:10.1186/s40851-014-0010-z
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    ABSTRACT: Pluripotency is an important feature of early embryonic cells of multicellular organisms. Recent advances in stem cell research have shown that Nanog and Pou5f1 (Oct3/4) play important roles in mammalian pluripotency. However, whether these molecules exert conserved functions in other species remains unknown. Although the epiblast of the early chicken embryo would provide a useful experimental model, a lack of antibodies against chicken Nanog (cNanog) and chicken PouV/Pou5f3 (cPouV) proteins has hampered intensive investigation. Here we report newly raised polyclonal antibodies that specifically recognize cNanog and cPouV proteins. The specificity and sensitivity of the antibodies were validated by both western blotting and immunostaining with transfected 293T cells and chicken embryonic tissues. Immunohistochemistry using these antibodies revealed that cNanog protein was specifically localized in epiblastic cells and germ cells. In contrast, cPouV expression was seen almost ubiquitously. We also found that chicken epiblast-derived colony-forming cells that morphologically resemble mouse embryonic stem cells were cNanog-positive, implying that these colony-forming cells possess pluripotency. The anti-cPouV antibody further enabled us to identify a previously unknown region at the N-terminus of the cPouV protein containing a characteristic motif that is absent in mammalian Pou5f1. Thus, the antibodies raised in this study are useful tools for studying the functions of cNanog and cPouV at the protein level and the molecular mechanisms of chicken pluripotency. © 2015 Japanese Society of Developmental Biologists.
    Development Growth and Regeneration 03/2015; 57(3). DOI:10.1111/dgd.12205 · 2.18 Impact Factor
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    ABSTRACT: A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements.
    PLoS ONE 02/2015; 10(2):e0116997. DOI:10.1371/journal.pone.0116997 · 3.53 Impact Factor
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    ABSTRACT: Adult planarians possess somatic pluripotent stem cells called neoblasts that give rise to all missing cell types during regeneration and homeostasis. Recent studies revealed that the Yorkie (Yki)/Yes-associated protein (YAP) transcriptional coactivator family plays an important role in the regulation of tissue growth during development and regeneration, and therefore we investigated the role of a planarian yki-related gene (termed Djyki) during regeneration and homeostasis of the freshwater planarian Dugesia japonica. We found that knockdown of the function of Djyki by RNA interference (RNAi) downregulated neoblast proliferation and caused regeneration defects after amputation. In addition, Djyki RNAi caused edema during homeostasis. These seemingly distinct defects induced by Djyki RNAi were rescued by simultaneous RNAi of a planarian mats-related gene (termed Djmats), suggesting an important role of Djmats in the negative regulation of Djyki, in accordance with the conservation of the functional relationship of these two genes during the course of evolution. Interestingly, Djyki RNAi did not prevent normal protonephridial structure, suggesting that Djyki RNAi induced the edema phenotype without affecting the excretory system. Further analyses revealed that increased expression of the D. japonica gene DjaquaporinA (DjaqpA), which belongs to a large gene family that encodes a water channel protein for the regulation of transcellular water flow, promoted the induction of edema, but not defects in neoblast dynamics, in Djyki(RNAi) animals. Thus, we conclude that Djyki plays two distinct roles in the regulation of active proliferation of stem cells and in osmotic water transport across the body surface in D. japonica. © 2015 The Authors Development, Growth & Differentiation published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Society of Developmental Biologists.
    Development Growth and Regeneration 02/2015; 57(3). DOI:10.1111/dgd.12195 · 2.18 Impact Factor
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    ABSTRACT: Urodele amphibians, such as newts, can regenerate a functional limb, including joints, after amputation at any level along the proximal-distal axis of the limb. The blastema can regenerate the limb morphology largely independently of the stump after proximal-distal identity has been established, but the remaining and regenerated tissues must be structurally reintegrated (matched in size and shape). Here we used newt joint regeneration as a model to investigate reintegration, because a functionally interlocking joint requires structural integration between its opposing skeletal elements. After forelimbs were amputated at the elbow joint, the joint was regenerated between the remaining and regenerated skeletal elements. The regenerated cartilage was thick around the amputated joint to make a reciprocally interlocking joint structure with the remaining bone. Furthermore, during regeneration, the extracellular matrix (ECM) of the remaining tissues was lost, suggesting that the remaining tissues might contribute to the morphogenesis of regenerating cartilage. Our results showed that the area of the regenerated cartilage matched the area of the apposed remaining cartilage, thus contributing to formation of a functional structure.This article is protected by copyright. All rights reserved.
    02/2015; 2(1). DOI:10.1002/reg2.28
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    ABSTRACT: In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development. © 2015. Published by The Company of Biologists Ltd.
    Development 01/2015; 142(5). DOI:10.1242/dev.116996 · 6.27 Impact Factor
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    ABSTRACT: Animal evolution is closely linked to the emergence of the nervous system. At present it is unknown how the basic mechanisms of neural induction and formation of central nervous systems evolved. We addressed this question in Nematostella vectensis, a member of cnidarians, the ancient sister group of bilaterians. We found that β-catenin signalling is crucial for the early induction of the embryonic nervous system. β-Catenin activity at the blastopore induces specific neurogenic genes required for development of the oral nervous system. β-Catenin signalling induces also Bmp signalling, which, at later larval stages, becomes indispensible for the maintenance and asymmetric patterning of the oral nervous system along the primary and secondary (directive) axes. We hypothesize that the consecutive and functionally linked involvement of β-catenin and Bmp signalling in the formation of the cnidarian oral nervous system reflects an ancestral mechanism that evolved before the cnidarian/bilaterian split.
    Nature Communications 12/2014; 5:5536. DOI:10.1038/ncomms6536 · 10.74 Impact Factor
  • Takeshi Inoue, Taiga Yamashita, Kiyokazu Agata
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    ABSTRACT: For most organisms, sensitive recognition of even slight changes in environmental temperature is essential for adjusting their behavioral strategies to ensure homeostasis and survival. However, much remains to be understood about the molecular and cellular processes that regulate thermosensation and the corresponding behavioral responses. Planarians display clear thermotaxis, although they have a relatively simple brain. Here, we devised a quantitative thermotaxis assay and unraveled a neural pathway involved in planarian thermotaxis by combinatory behavioral assays and RNAi analysis. We found that thermosensory neurons that expressed a planarian Dugesia japonica homolog of the Transient Receptor Potential Melastatin family a (DjTRPMa) gene were required for the thermotaxis. Interestingly, although these thermosensory neurons are distributed throughout their body, planarians with a dysfunctional brain due to regeneration-dependent conditional gene knockdown (Readyknock) of the synaptotagmin gene completely lost their thermotactic behavior. These results suggest that brain function is required as a central processor for the thermosensory response. Therefore, we investigated the type(s) of brain neurons involved in processing the thermal signals by gene knockdown of limiting enzymes for neurotransmitter biosynthesis in the brain. We found that serotonergic neurons with dendrites that were elongated toward DjTRPMa-expressing thermosensory neurons might be required for the processing of signals from thermosensory neurons that results in thermotaxis. These results suggest that serotonergic neurons in the brain may interact with thermosensory neurons activated by TRPM ion channels to produce thermotaxis in planarians. Copyright © 2014 the authors 0270-6474/14/3415701-14$15.00/0.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2014; 34(47):15701-14. DOI:10.1523/JNEUROSCI.5379-13.2014 · 6.75 Impact Factor
  • Shigeru Kuratani, Kiyokazu Agata
    ZOOLOGICAL SCIENCE 10/2014; 31(10):623. DOI:10.2108/066.031.1001 · 0.88 Impact Factor
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    ABSTRACT: The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D1 or D2 receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.
    Biochemical and Biophysical Research Communications 07/2014; 449(4):412–418. · 2.28 Impact Factor
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    ABSTRACT: Planarians have strong regenerative abilities derived from their adult pluripotent stem cell (neoblast) system. However, the molecular mechanisms involved in planarian regeneration have long remained a mystery. In particular, no anterior-specifying factor(s) could be found, although Wnt family proteins had been successfully identified as posterior-specifying factors during planarian regeneration (Gurley et al., 2008; Petersen and Reddien, 2008; Hayashi et al., 2011). A recent textbook of developmental biology therefore proposes a Wnt antagonist as a putative anterior factor (Gilbert, 2013). That is, planarian regeneration was supposed to be explained by a single decreasing gradient of the β-catenin signal from tail to head. However, recently we succeeded in demonstrating that in fact the extracellular-signal regulated kinases (ERK) form a decreasing gradient from head to tail to direct the reorganization of planarian body regionality after amputation (Umesono et al., 2013).
    Zoology 06/2014; 117(3). DOI:10.1016/j.zool.2014.04.001 · 1.60 Impact Factor
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    ABSTRACT: The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonicatyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D1 or D2 receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.
    Biochemical and Biophysical Research Communications 05/2014; 449(4). DOI:10.1016/j.bbrc.2014.05.059 · 2.28 Impact Factor
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    ABSTRACT: The majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples. We found that there is only about 1% of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs. We propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression.
    BMC Genomics 01/2014; 15(1):35. DOI:10.1186/1471-2164-15-35 · 4.04 Impact Factor
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    ABSTRACT: Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator-like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single-cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.
    Development Growth and Regeneration 12/2013; DOI:10.1111/dgd.12103 · 2.18 Impact Factor
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    ABSTRACT: In natural forests, hundreds of fungal species colonize plant roots. The preference or specificity for partners in these symbiotic relationships is a key to understanding how the community structures of root-associated fungi and their host plants influence each other. In an oak-dominated forest in Japan, we investigated the root-associated fungal community based on a pyrosequencing analysis of the roots of 33 plant species. Of the 387 fungal taxa observed, 153 (39.5%) were identified on at least two plant species. Although many mycorrhizal and root-endophytic fungi are shared between the plant species, the five most common plant species in the community had specificity in their association with fungal taxa. Likewise, fungi displayed remarkable variation in their association specificity for plants even within the same phylogenetic or ecological groups. For example, some fungi in the ectomycorrhizal family Russulaceae were detected almost exclusively on specific oak (Quercus) species, whereas other Russulaceae fungi were found even on "non-ectomycorrhizal" plants (e.g., Lyonia and Ilex). Putatively endophytic ascomycetes in the orders Helotiales and Chaetothyriales also displayed variation in their association specificity and many of them were shared among plant species as major symbionts. These results suggest that the entire structure of belowground plant-fungal associations is described neither by the random sharing of hosts/symbionts nor by complete compartmentalization by mycorrhizal type. Rather, the colonization of multiple types of mycorrhizal fungi on the same plant species and the prevalence of diverse root-endophytic fungi may be important features of belowground linkage between plant and fungal communities.
    Ecology and Evolution 09/2013; 3(9):3112-3124. DOI:10.1002/ece3.706 · 1.66 Impact Factor
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    ABSTRACT: The planarian Dugesia japonica can regenerate a complete individual from a head, trunk or tail fragment via activation of somatic pluripotent stem cells. About a century ago, Thomas Hunt Morgan attempted to explain the extraordinary regenerative ability of planarians by positing two opposing morphogenetic gradients of formative "head stuff" and "tail stuff" along the anterior-posterior axis. However, Morgan's hypothesis remains open to debate. Here we show that extracellular signal-related kinase (ERK) and Wnt/β-catenin signalling pathways establish a solid framework for planarian regeneration. Our data suggest that ERK signalling forms a spatial gradient in the anterior region during regeneration. The fibroblast growth factor receptor-like gene nou-darake (which serves as an output of ERK signalling in the differentiating head) and posteriorly biased β-catenin activity negatively regulate ERK signalling along the anterior-posterior axis in distinct manners, and thereby posteriorize regenerating tissues outside the head region to reconstruct a complete head-to-tail axis. On the basis of this knowledge about D. japonica, we proposed that β-catenin signalling is responsible for the lack of head-regenerative ability of tail fragments in the planarian Phagocata kawakatsui, and our confirmation thereof supports the notion that posterior β-catenin signalling negatively modulates the ERK signalling involved in anteriorization across planarian species. These findings suggest that ERK signalling has a pivotal role in triggering globally dynamic differentiation of stem cells in a head-to-tail sequence through a default program that promotes head tissue specification in the absence of posteriorizing signals. Thus, we have confirmed the broad outline of Morgan's hypothesis, and refined it on the basis of our proposed default property of planarian stem cells.
    Nature 07/2013; DOI:10.1038/nature12359 · 42.35 Impact Factor
  • Shota Nakanoh, Kenji Okazaki, Kiyokazu Agata
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    ABSTRACT: As amniotes diversified, mammals may have modified mechanisms of cellular pluripotency along with the acquisition of a placenta. What then defined pluripotent states in the ancestral amniotes? To study the evolutionary background of pluripotency in amniotes, we tested the effects of extracellular effectors on primary culture cells from avian and reptile embryos in serum-free medium. When treated with a combination of a MEK inhibitor and a GSK3 inhibitor (2i condition), chicken early embryos formed domed colonies (DCs), which were morphologically indistinguishable from the colonies formed by mouse and rat naïve embryonic stem cells. However, no DCs formed when cells from further-developed embryos were cultured in the 2i condition, indicating that there is a clear boundary of DC-forming ability at around the stage of primitive streak elongation. Quail embryos at the blastoderm and cleavage stages also formed DCs in the 2i condition, which is consistent with the notion that the appearance of DCs corresponds with the presence of pluripotent cells in embryos. Gecko blastoderms also formed DCs in the 2i condition, but gastrulas did not. ERK activation by bFGF caused an effect opposite to that of the 2i condition, namely, it dispersed colonies of cells even from early embryos in all species examined. These results suggest that the regulation of pluripotency by FGF/ERK signaling may date back at least to the common ancestor of mammals, birds, and reptiles. However, gene expression analysis indicated the possibility that mammalian pluripotency transcription factors function differently in non-mammalian amniotes.
    ZOOLOGICAL SCIENCE 07/2013; 30(7):543-52. DOI:10.2108/zsj.30.543 · 0.88 Impact Factor
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    ABSTRACT: COVER PHOTOGRAPH: Low magnification image of planarian flatworms (Schmidtea mediterranea) fed with liver paste containing either red, green, yellow, or blue food dye. Uptake of colored liver paste containing in vitro synthesized double-stranded RNA is readily visualized in the planarian gut. Ingestion and processing of double-stranded RNA in the planarian gut leads to systemic RNA-interference. Image credit: Labib Rouhana and Phillip A. Newmark, Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign. From RNA Interference by feeding in vitro-synthesized double-stranded RNA to planarians, Labib Rouhana, Jennifer A. Weiss, David J. Forsthoefel, Hayoung Lee, Ryan S. King, Takeshi Inoue, Norito Shibata, Kiyokazu Agata, and Phillip A. Newmark, Developmental Dynamics 242:718-730, 2013.
    Developmental Dynamics 06/2013; 242(6):C1. DOI:10.1002/dvdy.23982 · 2.67 Impact Factor
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    ABSTRACT: Background: The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced via injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results: We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time, and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions: This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Developmental Dynamics, 2013. © 2013 Wiley Periodicals,Inc.
    Developmental Dynamics 06/2013; 242(6). DOI:10.1002/dvdy.23950 · 2.67 Impact Factor

Publication Stats

5k Citations
648.78 Total Impact Points

Institutions

  • 2006–2015
    • Kyoto University
      • • Department of Biophysics
      • • Department of Molecular and Developmental Biology
      Kioto, Kyōto, Japan
  • 2012
    • Institut Marqués, Spain, Barcelona
      Barcino, Catalonia, Spain
  • 2010
    • RIKEN
      Вако, Saitama, Japan
  • 2003–2009
    • Kyoto Pharmaceutical University
      • Laboratory of Clinical and Translational Physiology
      Kioto, Kyōto, Japan
    • National Institute of Genetics
      • Center for Information Biology
      Mishima, Shizuoka-ken, Japan
  • 1994–2009
    • Himeji Institute of Technology
      • Faculty of Science
      Himezi, Hyōgo, Japan
    • Shinshu University
      • Faculty of Agriculture
      Matsumoto, Nagano-ken, Japan
  • 1988–2008
    • National Institute for Basic Biology
      Okazaki, Aichi, Japan
  • 2002
    • Okayama University
      • Department of Biology
      Okayama, Okayama, Japan
  • 1991
    • National Institute of Animal Health
      Ibaragi, Ōsaka, Japan