Kiyokazu Agata

Kyoto University, Kioto, Kyōto, Japan

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Publications (219)674.76 Total impact

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    ABSTRACT: The robust regenerative ability of planarians is known to be dependent on adult pluripotent stem cells called neoblasts. One of the morphological features of neoblasts is cytoplasmic ribonucleoprotein granules (chromatoid bodies: CBs), which resemble germ granules present in germline cells in other animals. Previously, we showed by immuno-electron microscopic analysis that DjCBC-1, a planarian Me31B/Dhh1/DDX6 homologue, which is a component of ribonucleoprotein granules, was localized in CBs in the planarian Dugesia japonica. Also, recently it was reported using another planarian species that Y12 antibody recognizing symmetrical dimethylarginine (sDMA) specifically binds to CBs in which histone mRNA is co-localized. Here, we showed by double immunostaining and RNA interference (RNAi) that DjCBC-1-containing CBs and Y12-immunoreactive CBs are distinct structures, suggesting that CBs are composed of heterogeneous populations. We also found that the Y12-immunoreactive CBs specifically contained a cytoplasmic type of planarian PIWI protein (DjPiwiC). We revealed by RNAi experiments that Y12-immunoreactive CBs may have anti-transposable element activity involving the DjPiwiC protein in the neoblasts.
    No preview · Article · Feb 2016 · Development Growth and Regeneration
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    ABSTRACT: Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the “germ-line multipotency program” and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells.
    No preview · Article · Dec 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: While olfaction is one of the most important senses in most terrestrial mammals, it is absent in modern toothed whales (Odontoceti, Cetacea). Furthermore, behavioral evidence suggests that gustation is very limited. In contrast, their aquatic sistergroup, baleen whales (Mysticeti) retain small but functional olfactory organs, and nothing is known about their gustation. It is difficult to investigate mysticete chemosensory abilities because experiments in a controlled setting are impossible. Here, we use the functional regionalization of the olfactory bulb (OB) to identify the loss of specific olfactory functions in mysticetes. We provide the whole-genome sequence of a mysticete and show that mysticetes lack the dorsal domain of the OB, an area known to induce innate avoidance behavior against odors of predators and spoiled foods. Genomic and fossil data suggest that mysticetes lost the dorsal domain of the OB before the Odontoceti-Mysticeti split. Furthermore, we found that all modern cetaceans are revealed to have lost the functional taste receptors. These results strongly indicate that profound changes in the chemosensory capabilities had occurred in the cetacean lineage during the period when ancestral whales migrated from land to water.
    Full-text · Article · Dec 2015
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    ABSTRACT: Planarians belong to an evolutionarily early group of organisms that possess a central nervous system including a well-organized brain with a simple architecture but many types of neurons. Planarians display a number of behaviors, such as phototaxis and thermotaxis, in response to external stimuli, and it has been shown that various molecules and neural pathways in the brain are involved in controlling these behaviors. However, due to the lack of combinatorial assay methods, it remains obscure whether planarians possess higher brain functions, including integration in the brain, in which multiple signals coming from outside are coordinated and used in determining behavioral strategies. In the present study, we designed chemotaxis and thigmotaxis/kinesis tracking assays to measure several planarian behaviors in addition to those measured by phototaxis and thermotaxis assays previously established by our group, and used these tests to analyze planarian chemotactic and thigmotactic/kinetic behaviors. We found that headless planarian body fragments and planarians that had specifically lost neural activity following regeneration-dependent conditional gene knockdown (Readyknock) of synaptotagmin in the brain lost both chemotactic and thigmotactic behaviors, suggesting that neural activity in the brain is required for the planarian's chemotactic and thigmotactic behaviors. Furthermore, we compared the strength of phototaxis, chemotaxis, thigmotaxis/kinesis, and thermotaxis by presenting simultaneous binary stimuli to planarians. We found that planarians showed a clear order of predominance of these behaviors. For example, when planarians were simultaneously exposed to 400 lux of light and a chemoattractant, they showed chemoattractive behavior irrespective of the direction of the light source, although exposure to light of this intensity alone induces evasive behavior away from the light source. In contrast, when the light intensity was increased to 800 or 1600 lux and the same dose of chemoattractant was presented, planarian behaviors were gradually shifted to negative phototaxis rather than chemoattraction. These results suggest that planarians may be capable of selecting behavioral strategies via the integration of discrete brain functions when exposed to multiple stimuli. The planarian brain processes external signals received through the respective sensory neurons, thereby resulting in the production of appropriate behaviors. In addition, planarians can adjust behavioral features in response to stimulus conditions by integrating multiple external signals in the brain.
    Preview · Article · Dec 2015
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    ABSTRACT: Purpose: Following decapitation, the planarian Schmidtea mediterranea regenerates its head and eyes. The gene ovo is required for eye maintenance and regeneration in response to wounding. In this study, we investigated whether eye regeneration in S. mediterranea could occur absent a wound healing response. Methods: One hundred twenty S. mediterranea were treated with ovo RNA interference (RNAi) or control (unc-22) RNAi by feeding double-stranded RNA (dsRNA). Following eye loss, ovo RNAi treatment was halted and replaced with control RNAi treatment. Quantitative real-time PCR (qPCR) was used to monitor ovo expression. Eye functionality was monitored via a phototaxis assay. Photoreceptor neurons were visualized via immunofluorescence staining of arrestin. Results: Treatment with ovo RNAi caused eyes to gradually shrink until they were completely absent. One hundred percent of ovo RNAi-treated planarians lost both eyes within 137 days of treatment onset. ovo RNAi-treated planarians were unable to regenerate eyes in response to decapitation. Upon removal of ovo RNAi, eyes became visible as small pigmented spots in the head within 28 days. The eyes slowly developed, appearing to gain pigmented cells first and then nonpigmented photoreceptors. Phototaxis assays demonstrated functional eye loss and eye restoration. ovo mRNA was significantly decreased following treatment with ovo RNAi and significantly increased following removal of ovo RNAi. Arrestin staining was present in the eyes, optic nerves, and optic chiasm of worms with regenerated eyes but not in eyeless worms. Conclusions: S. mediterranea have the ability to generate functional eyes in the absence of a wound healing response. This ability requires the expression of ovo.
    No preview · Article · Nov 2015 · Investigative ophthalmology & visual science
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    ABSTRACT: We established a laboratory clonal strain of freshwater planarian (Dugesia japonica) that was derived from a single individual and that continued to undergo autotomous asexual reproduction for more than 20 years, and we performed large-scale genome sequencing and transcriptome analysis on it. Despite the fact that a completely clonal strain of the planarian was used, an unusually large number of mutations were detected. To enable quantitative genetic analysis of such a unique organism, we developed a new model called the Reference Gene Model, and used it to conduct large-scale transcriptome analysis. The results revealed large numbers of mutations not only outside but also inside gene-coding regions. Non-synonymous SNPs were detected in 74% of the genes for which valid ORFs were predicted. Interestingly, the high-mutation genes, such as metabolism- and defense-related genes, were correlated with genes that were previously identified as diverse genes among different planarian species. Although a large number of amino acid substitutions were apparently accumulated during asexual reproduction over this long period of time, the planarian maintained normal body-shape, behaviors, and physiological functions. The results of the present study reveal a unique aspect of asexual reproduction.
    Preview · Article · Nov 2015 · PLoS ONE
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    Full-text · Dataset · Nov 2015
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    Yoshitaro Akiyama · Kiyokazu Agata · Takeshi Inoue
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    ABSTRACT: The planarian Dugesia japonica tends to stay near the walls of its breeding containers and experimental dishes in the laboratory, a phenomenon called "wall preference". This behavior is thought to be important for environmental adaptation, such as hiding by planarians in nature. However, the mechanisms regulating wall-preference behavior are not well understood, since this behavior occurs in the absence of any particular stimulation. Here we show the mechanisms of wall-preference behavior. Surprisingly, planarian wall-preference behavior was also shown even by the head alone and by headless planarians. These results indicate that planarian "wall-preference" behavior only appears to be a "preference" behavior, and is actually an outcome of spontaneous behaviors, rather than of brain function. We found that in the absence of environmental cues planarians moved basically straight ahead until they reached a wall, and that after reaching a wall, they changed their direction of movement to one tangential to the wall, suggesting that this spontaneous behavior may play a critical role in the wall preference. When we tested another spontaneous behavior, the wigwag movement of the planarian head, using computer simulation with various wigwag angles and wigwag intervals, large wigwag angle and short wigwag interval reduced wall-preference behavior. This indicated that wigwag movement may determine the probability of staying near the wall or leaving the wall. Furthermore, in accord with this simulation, when we tested planarian wall-preference behavior using several assay fields with different curvature of the wall, we found that concavity and sharp curvature of walls negatively impacted wall preference by affecting the permissible angle of the wigwag movement. Together, these results indicate that planarian wall preference may be involuntarily caused by the combination of two spontaneous planarian behaviors: moving straight ahead until reaching a wall and then moving along it in the absence of environmental cues, and wigwag movements of the head.
    Preview · Article · Nov 2015 · PLoS ONE
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    Rio Tsutsumi · Shigehito Yamada · Kiyokazu Agata
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    ABSTRACT: A functional joint requires integration of multiple tissues: the apposing skeletal elements should form an interlocking structure, and muscles should insert into skeletal tissues via tendons across the joint. Whereas newts can regenerate functional joints after amputation, Xenopus laevis regenerates a cartilaginous rod without joints, a “spike”. Previously we reported that the reintegration mechanism between the remaining and regenerated tissues has a significant effect on regenerating joint morphogenesis during elbow-joint regeneration in newt (Tsutsumi et al., 2015). Based on this insight into the importance of reintegration, we amputated frogs’ limbs at the elbow joint, and found that frogs could regenerate a functional elbow joint between the remaining tissues and regenerated spike. During regeneration, the regenerating cartilage was partially connected to the remaining articular cartilage to reform the interlocking structure of the elbow joint at the proximal end of the spike. Furthermore, the muscles of the remaining part inserted into the regenerated spike cartilage via tendons. This study might open up an avenue for analyzing molecular and cellular mechanisms of joint regeneration using Xenopus. This article is protected by copyright. All rights reserved
    Full-text · Article · Oct 2015
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    ABSTRACT: Animal bodies are shaped by skeletons, which are built inside the body by biomineralization of condensed mesenchymal cells in vertebrates [1, 2] and echinoderms [3, 4], or outside the body by apical secretion of extracellular matrices by epidermal cell layers in arthropods [5]. In each case, the skeletons' shapes are a direct reflection of the pattern of skeleton-producing cells [6]. Here we report a newly discovered mode of skeleton formation: assembly of sponges' mineralized skeletal elements (spicules) in locations distant from where they were produced. Although it was known that internal skeletons of sponges consist of spicules assembled into large pole-and-beam structures with a variety of morphologies [7-10], the spicule assembly process (i.e., how spicules become held up and connected basically in staggered tandem) and what types of cells act in this process remained unexplored. Here we found that mature spicules are dynamically transported from where they were produced and then pierce through outer epithelia, and their basal ends become fixed to substrate or connected with such fixed spicules. Newly discovered "transport cells" mediate spicule movement and the "pierce" step, and collagen-secreting basal-epithelial cells fix spicules to the substratum, suggesting that the processes of spiculous skeleton construction are mediated separately by specialized cells. Division of labor by manufacturer, transporter, and cementer cells, and iteration of the sequential mechanical reactions of "transport," "pierce," "raise up," and "cementation," allows construction of the spiculous skeleton spicule by spicule as a self-organized biological structure, with the great plasticity in size and shape required for indeterminate growth, and generating the great morphological diversity of individual sponges.
    No preview · Article · Sep 2015 · Current biology: CB
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    ABSTRACT: Many amphibians can regenerate limbs, even in adulthood. If a limb is amputated, the stump generates a blastema that makes a complete, new limb in a process similar to developmental morphogenesis. The blastema is thought to inherit its limb-patterning properties from cells in the stump, and it retains the information despite changes in morphology, gene expression, and differentiation states required by limb regeneration. We hypothesized that these cellular properties are maintained as epigenetic memory through histone modifications. To test this hypothesis, we analyzed genome-wide histone modifications in Xenopus limb bud regeneration. The trimethylation of histone H3 at lysine 4 (H3K4me3) is closely related to an open chromatin structure that allows transcription factors access to genes, whereas the trimethylation of histone H3 at lysine 27 (H3K27me3) is related to a closed chromatin state that blocks the access of transcription factors. We compared these two modification profiles by high-throughput sequencing of samples prepared from the intact limb bud and the regenerative blastema by chromatin immunoprecipitation. For many developmental genes, histone modifications at the transcription start site were the same in the limb bud and the blastema, were stable during regeneration, and corresponded well to limb properties. These results support our hypothesis that histone modifications function as a heritable cellular memory to maintain limb cell properties, despite dynamic changes in gene expression during limb bud regeneration in Xenopus. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Aug 2015 · Developmental Biology
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    Full-text · Dataset · Jun 2015
  • Shota Nakanoh · Naoyuki Fuse · Yoshiko Takahashi · Kiyokazu Agata
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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.
    No preview · Article · Mar 2015 · Development Growth and Regeneration
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    Full-text · Dataset · Feb 2015
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    Yang An · Atsushi Toyoda · Chen Zhao · Asao Fujiyama · Kiyokazu Agata
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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.
    Full-text · Article · Feb 2015 · PLoS ONE
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    Byulnim Hwang · Yang An · Kiyokazu Agata · Yoshihiko Umesono
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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.
    Full-text · Article · Feb 2015 · Development Growth and Regeneration
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    Rio Tsutsumi · Takeshi Inoue · Shigehito Yamada · Kiyokazu Agata
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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.
    Full-text · Article · Feb 2015
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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.
    Full-text · Article · Jan 2015 · Development
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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.
    Full-text · Article · Dec 2014 · Nature Communications

  • No preview · Article · Dec 2014 · Genes & Genetic Systems

Publication Stats

6k Citations
674.76 Total Impact Points


  • 2006-2015
    • Kyoto University
      • • Department of Biophysics
      • • Department of Molecular and Developmental Biology
      Kioto, Kyōto, Japan
  • 2012
    • Institut Marqués, Spain, Barcelona
      Barcino, Catalonia, Spain
  • 2009-2010
    • RIKEN
      Вако, Saitama, Japan
  • 1988-2008
    • National Institute for Basic Biology
      Okazaki, Aichi, Japan
  • 2007
    • Kobe University
      • Faculty of Science
      Kōbe, Hyōgo, Japan
  • 2002-2003
    • Okayama University
      • Department of Biology
      Okayama, Okayama, Japan
  • 1993-2003
    • Himeji Institute of Technology
      • Faculty of Science
      Himezi, Hyōgo, Japan
  • 1991
    • National Institute of Animal Health
      Ibaragi, Ōsaka, Japan
  • 1989
    • Hokkaido University
      • Faculty of Pharmaceutical Sciences
      Sapporo, Hokkaido, Japan