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Last Updated :2024/09/18

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Master

Affiliation (Master)

  • Faculty of Science　Biological Sciences　Reproductive and Developmental Biology

Affiliation (Master)

  • Faculty of Science　Biological Sciences　Reproductive and Developmental Biology

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Degree

  • (BLANK)（Hokkaido University）

Profile and Settings

  • Name (Japanese)

    Kotani

  • Name (Kana)

    Tomoya

  • Name

    200901033482069243

Alternate Names

Achievement

Research Areas

  • Life sciences / Cell biology
  • Life sciences / Molecular biology
  • Life sciences / Morphology, anatomy

Research Experience

  • 2012/10 - Today Hokkaido University Associate Professor
  • 2010/04 - 2012/09 Hokkaido University Assistant Professor
  • 2007/04 - 2010/03 Hokkaido University Assistant Professor
  • 2004/04 - 2007/03 National Institute of Genetics

Education

  • 1999/04 - 2004/03  Hokkaido University
  •        - 2001  Hokkaido University  Graduate School, Division of Natural Science
  • 1995/04 - 1999/03  Hokkaido University
  •        - 1999  Hokkaido University  Faculty of Science

Published Papers

  • Takahiro Sanada, Tomoya Kotani
    Biological Procedures Online 26 (1) 2024/07/10 [Refereed]
     
    Abstract Mammalian oocytes accumulate more than ten thousand mRNAs, of which three to four thousand mRNAs are translationally repressed. The timings and sites of translational activation of these dormant mRNAs are crucial for promoting oocyte maturation and embryonic development. How these mRNAs are accumulated and distributed in oocytes is therefore a fundamental issue to be explored. A method that enables visualization of mRNA molecules with high resolution in a simple manner would be valuable for understanding how oocytes accumulate and regulate the dormant mRNAs. We have developed a highly sensitive whole-mount in situ hybridization method using in vitro-synthesized RNA probes and the tyramide signal amplification (TSA) system optimized for mouse oocytes and embryos. By using this method, Pou5f1/Oct4, Emi2, and cyclin B1 mRNAs were detected in immature oocytes and 2-cell stage embryos. Confocal microscopy showed that these mRNAs formed granular structures in the oocyte cytoplasm. The structures of Pou5f1/Oct4 and cyclin B1 mRNAs persisted in 2-cell stage embryos. Pou5f1/Oct4 RNA granules exhibited a solid-like property in immature oocytes and became liquid-like droplets in 2-cell stage embryos. Double-staining of cyclin B1 mRNA with Emi2 or Pou5f1/Oct4 mRNA revealed that these mRNAs were distributed as different RNA granules without overlapping each other and that the size of cyclin B1 RNA granules tended to be larger than that of Emi2 RNA granules. The structures and distribution patterns of these mRNAs were further analyzed by N-SIM super-resolution microscopy. This analysis revealed that the large-sized RNA granules consist of many small-sized granules, suggesting the accumulation and regulation of dormant mRNAs as basal-sized RNA granules. The method established in this study can easily visualize the structure and distribution of mRNAs accumulated in mammalian oocytes and embryos with high sensitivity and super-resolution. This method is useful for investigating the cellular and molecular mechanisms of translational control of mRNAs by which maturation and early developmental processes are promoted.
  • Keisuke Sato, Tomoya Kotani
    STAR protocols 5 (2) 102951 - 102951 2024/03/15 [Refereed]
     
    Fertilized eggs initiate translation of stored mRNAs in spatially and temporally controlled manners. Here, we present a protocol for visualizing spatial and temporal translation in zebrafish embryos by fluorescence in situ hybridization and proximity ligation assay. We describe steps for labeling newly synthesized proteins and mRNA, visualizing mRNA translation and mRNA, sample mounting, and observation. Coupling detection of mRNA molecules with their translation sites is useful for understanding the molecular and cellular mechanisms that drive embryo development. For complete details on the use and execution of this protocol, please refer to Sato et al.1 and Takada et al.2.
  • Yuki Takada, Ludivine Fierro, Keisuke Sato, Takahiro Sanada, Anna Ishii, Takehiro Yamamoto, Tomoya Kotani
    Science Advances 9 (47) 2023/11/24 [Refereed]
     
    Eggs accumulate thousands of translationally repressed mRNAs that are translated into proteins after fertilization to direct diverse developmental processes. However, molecular mechanisms underlying the translation of stored mRNAs after fertilization remain unclear. Here, we report a previously unknown RNA processing of 3′ end sequences of mature mRNAs that activates the translation of stored mRNAs. Specifically, 9 to 72 nucleotides at the 3′ ends of zebrafish pou5f3 and mouse Pou5f1 mRNAs were deleted in the early stages of development. Reporter assays illustrated the effective translation of the truncated forms of mRNAs. Moreover, promotion and inhibition of the shortening of 3′ ends accelerated and attenuated Pou5f3 accumulation, respectively, resulting in defective development. Identification of proteins binding to unprocessed and/or processed mRNAs revealed that mRNA shortening acts as molecular switches. Comprehensive analysis revealed that >250 mRNAs underwent this processing. Therefore, our results provide a molecular principle that triggers the translational activation and directs development.
  • Keisuke Sato, Moeko Sakai, Anna Ishii, Kaori Maehata, Yuki Takada, Kyota Yasuda, Tomoya Kotani
    iScience 25 (6) 104344 - 104344 2022/06/17 [Refereed]
     
    Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that pou5f3 mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of pou5f3 granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein became colocalized with pou5f3 granules after fertilization and, moreover, nascent Pou5f3 was shown to be synthesized in the granules. This functional change was accompanied by changes in the state and internal structure of granules. Dissolution of the granules reduced the rate of protein synthesis. Similarly, nanog and sox19b mRNAs in zebrafish and Pou5f1/Oct4 mRNA in mouse assembled into granules. Our results reveal that subcellular compartments, termed embryonic RNA granules, function as activation sites of translation after changing physical properties for directing vertebrate development.
  • Natsumi Takei, Keisuke Sato, Yuki Takada, Rajan Iyyappan, Andrej Susor, Takehiro Yamamoto, Tomoya Kotani
    Current Research in Cell Biology 100009 - 100009 2021/06 [Refereed]
  • Kai Otsuka, Shin Matsubara, Akira Shiraishi, Natsumi Takei, Yui Satoh, Miho Terao, Shuji Takada, Tomoya Kotani, Honoo Satake, Atsushi P Kimura
    Frontiers in endocrinology 12 665874 - 665874 2021 [Refereed]
     
    The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start (Steroidogenesis activating lncRNA in testis), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start-knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start-KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1, while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start-KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1, Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start-KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs.
  • Natsumi Takei, Yuki Takada, Shohei Kawamura, Keisuke Sato, Atsushi Saitoh, Jenny Bormann, Wai Shan Yuen, John Carroll, Tomoya Kotani
    Journal of cell science 2020/11/04 [Refereed]
     
    Temporal and spatial control of mRNA translation has emerged as a major mechanism for promoting diverse biological processes. However, the molecular nature of temporal and spatial control of translation remains unclear. In oocytes, many mRNAs are deposited as a translationally repressed form and are translated at appropriate timings to promote the progression of meiosis and development. Here, we show that changes in subcellular structures and states of the RNA-binding protein Pumilio1 regulate the translation of target mRNAs and progression of oocyte maturation. Pumilio1 was shown to bind to Mad2 and Cyclin B1 mRNAs, assemble highly clustered aggregates, and surround Mad2 and Cyclin B1 RNA granules in mouse oocytes. These Pumilio1 aggregates were dissolved prior to the translational activation of target mRNAs possibly by phosphorylation. Stabilization of Pumilio1 aggregates prevented the translational activation of target mRNAs and progression of oocyte maturation. Together, our results provide an aggregation-dissolution model for the temporal and spatial control of translation.
  • Yuki Takada, Rajan Iyyappan, Andrej Susor, Tomoya Kotani
    Histochemistry and cell biology 2020/09/15 [Refereed]
     
    Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.
  • Ahmed Z Balboula, Karen Schindler, Tomoya Kotani, Manabu Kawahara, Masashi Takahashi
    Molecular human reproduction 26 (9) 689 - 701 2020/09/01 [Refereed]
     
    As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus-oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.
  • Satoh R, Bando H, Sakai N, Kotani T, Yamashita M
    Zygote (Cambridge, England) 27 (6) 423 - 431 0967-1994 2019/12 [Refereed][Not invited]
  • Satoh Y, Takei N, Kawamura S, Takahashi N, Kotani T, Kimura AP
    Biology of reproduction 100 (3) 833 - 848 0006-3363 2019/03 [Refereed][Not invited]
  • Saitoh A, Takada Y, Horie M, Kotani T
    Zygote (Cambridge, England) 1 - 9 0967-1994 2018/10 [Refereed][Not invited]
  • Natsumi Takei, Takuma Nakamura, Shohei Kawamura, Yuki Takada, Yui Satoh, Atsushi P. Kimura, Tomoya Kotani
    Biological Procedures Online 20 (1) 6  1480-9222 2018/03/01 [Refereed][Not invited]
     
    Background: Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method. Results: The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs. Conclusions: This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.
  • Tomoya Kotani, Kaori Maehata, Natsumi Takei
    Results and Problems in Cell Differentiation 63 297 - 324 1861-0412 2017 [Refereed][Invited]
     
    From the beginning of oogenesis, oocytes accumulate tens of thousands of mRNAs for promoting oocyte growth and development. A large number of these mRNAs are translationally repressed and localized within the oocyte cytoplasm. Translational activation of these dormant mRNAs at specific sites and timings plays central roles in driving progression of the meiotic cell cycle, axis formation, mitotic cleavages, transcriptional initiation, and morphogenesis. Regulation of the localization and temporal translation of these mRNAs has been shown to rely on cisacting elements in the mRNAs and trans-acting factors recognizing and binding to the elements. Recently, using model vertebrate zebrafish, localization itself and formation of physiological structures such as RNA granules have been shown to coordinate the accurate timings of translational activation of dormant mRNAs. This subcellular regulation of mRNAs is also utilized in other animals including mouse. In this chapter, we review fundamental roles of temporal regulation of mRNA translation in oogenesis and early development and then focus on the mechanisms of mRNA regulation in the oocyte cytoplasm by which the activation of dormant mRNAs at specific timings is achieved.
  • Mayu Horie, Tomoya Kotani
    EUROPEAN JOURNAL OF CELL BIOLOGY 95 (12) 563 - 573 0171-9335 2016/12 [Refereed][Not invited]
     
    Many translationally repressed mRNAs are deposited in the oocyte cytoplasm for progression of the meiotic cell cycle and early development. mos and cyclin B1 mRNAs encode proteins promoting oocyte meiosis, and translational control of these mRNAs is important for normal progression of meiotic cell division. We previously demonstrated that cyclin B1 mRNA forms RNA granules in the zebrafish and mouse oocyte cytoplasm and that the formation of RNA granules is crucial for regulating the timing of translational activation of the mRNA. However, whether the granule formation is specific to cyclin B1 mRNA remains unknown. In this study, we found that zebrafish mos mRNA forms granules distinct from those of cyclin B1 mRNA. Fluorescent in situ hybridization analysis showed that cyclin B1 RNA granules were assembled in dense clusters, while mos RNA granules were distributed diffusely in the animal polar cytoplasm. Sucrose density gradient ultracentrifugation analysis showed that the density of mos RNA granules was partly lower than that of cyclin B1 mRNA. Similar to cyclin B1 RNA granules, mos RNA granules were disassembled after initiation of oocyte maturation at the timing at which the poly(A) tail was elongated. However, while almost all of the granules of cyclin B1 were disassembled simultaneously, a fraction of mos RNA granules firstly disappeared and then a large part of them was disassembled. In addition, while cyclin B1 RNA granules were disassembled in a manner dependent on actin filament depolymerization, certain fractions of mos RNA granules were disassembled independently of actin filaments. These results suggest that cytoplasmic regulation of translationally repressed mRNAs by formation of different RNA granules is a key mechanism for translational control of distinct mRNAs in the oocyte. (C) 2016 Elsevier GmbH. All rights reserved.
  • Ryoma Yoneda, Yui Satoh, Ikuya Yoshida, Shohei Kawamura, Tomoya Kotani, Atsushi P. Kimura
    MOLECULAR REPRODUCTION AND DEVELOPMENT 83 (6) 541 - 557 1040-452X 2016/06 [Refereed][Not invited]
     
    Spermatogenesis is regulated by many meiotic stage-specific genes, but how they coordinate the many individual processes is not fully understood. The Prss/Tessp gene cluster is located on mouse chromosome 9F2-F3, and the three genes at this site (Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4) are specifically activated during meiosis in pachytene spermatocytes. We searched for DNase I hypersensitive sites (HSs) and long noncoding RNAs (lncRNAs) at the Prss/Tessp locus to elucidate how they are activated. We found eight DNase I HSs, three of which were testis germ cell-specific at or close to the Prss42/Tessp-2 promoter, and a testisspecific lncRNA, lncRNA-HSVIII, that was transcribed from a region adjacent to the Prss42/Tessp-2 gene. lncRNA-HSVIII transcripts localized to nuclei of most pachytene spermatocytes and the cytosol of stage-X pachytene spermatocytes and spermatids. Chromosome conformation capture revealed that the lncRNA-HSVIII locus specifically interacted with the Prss42/Tessp-2 promoter in primary and secondary spermatocytes. A 5.8-kb genome sequence, encompassing the entire lncRNA-HSVIII sequence and its flanking regions, significantly increased Prss42/Tessp-2 promoter activity using a reporter-gene assay, yet this construct did not change lncRNA-HSVIII expression, indicating that the elevated promoter activity was likely through enhancer activity. Indeed, both upstreamand downstream regions of the lncRNA-HSVIII sequence significantly increased Prss42/Tessp-2 promoter activity. Our data therefore identified the direct interaction of a genomic region in the lncRNA-HSVIII locus with the Prss42/Tessp-2 promoter in spermatocytes, and suggested that sequences adjacent to the lncRNA function as enhancers for the Prss42/Tessp-2 gene. (C) 2016 Wiley Periodicals, Inc.
  • Yumiko Nukada, Mayu Horie, Akimasa Fukui, Tomoya Kotani, Masakane Yamashita
    CYTOSKELETON 72 (9) 491 - 501 1949-3584 2015/09 [Refereed][Not invited]
     
    Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis. (C) 2015 Wiley Periodicals, Inc.
  • Kazuki Takahashi, Tomoya Kotani, Yoshinao Katsu, Masakane Yamashita
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 448 (1) 22 - 27 0006-291X 2014/05 [Refereed][Not invited]
     
    In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3' untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3' UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 riaRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumiliol and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation. (C) 2014 Elsevier Inc. All rights reserved.
  • Kyota Yasuda, Tomoya Kotani, Masakane Yamashita
    DEVELOPMENTAL BIOLOGY 382 (2) 517 - 529 0012-1606 2013/10 [Refereed][Not invited]
     
    Subcellular localization of messenger RNAs (mRNAs) to correct sites and translational activation at appropriate timings are crucial for normal progression of various biological events. However, a molecular link between the spatial regulation and temporal regulation remains unresolved. In immature zebrafish oocytes, translationally repressed cyclin B1 mRNA is localized to the animal polar cytoplasm and its temporally regulated translational activation in response to a maturationAnducing hormone is essential to promote oocyte maturation. We previously reported that the coding region of cyclin B1 mRNA is required for the spatio-temporal regulation. Here, we report that a sequence, CAGGAGACC, that is conserved in the coding region of vertebrate cyclin B1 mRNA is involved in the regulation. Like endogenous cyclin B1 mRNA, reporter mRNAs harboring the sequence CAGGAGACC were localized to the animal polar cytoplasm of oocytes, while those carrying mutations in the sequence (with no change in the coding amino acids) were dispersed in the animal hemisphere of oocytes. Furthermore, translational activation of the mutant mRNAs was initiated at a timing earlier than that of endogenous and wild-type reporter mRNAs during oocyte maturation. Interaction of CAGGAGACC with proteins in vitro suggests that this sequence functions in collaboration with a trans-acting protein factor(s) in oocytes. These findings reveal that the sequence in the coding region of cyclin B1 mRNA plays an important role as a cis-acting element in both subcellular localization and translational timing of mRNA, providing a direct molecular link between the spatial and temporal regulation of mRNA translation. (C) 2013 Elsevier Inc. All rights reserved.
  • Tomoya Kotani, Kyota Yasuda, Ryoma Ota, Masakane Yamashita
    Journal of Cell Biology 202 (7) 1041 - 1055 0021-9525 2013 [Refereed][Not invited]
     
    Temporal control of messenger RNA (mRNA) translation is an important mechanism for regulating cellular, neuronal, and developmental processes. However, mechanisms that coordinate timing of translational activation remain largely unresolved. Full-grown oocytes arrest meiosis at prophase I and deposit dormant mRNAs. Of these, translational control of cyclin B1 mRNA in response to maturation-inducing hormone is important for normal progression of oocyte maturation, through which oocytes acquire fertility. In this study, we found that dormant cyclin B1 mRNA forms granules in the cytoplasm of zebrafish and mouse oocytes. Real-time imaging of translation revealed that the granules disassemble at the time of translational activation during maturation. Formation of cyclin B1 RNA granules requires binding of the mRNA to Pumilio1 protein and depends on actin filaments. Disruption of cyclin B1 RNA granules accelerated the timing of their translational activation after induction of maturation, whereas stabilization hindered translational activation. Thus, our results suggest that RNA granule formation is critical for the regulation of timing of translational activation. © 2013 Shigeoka et al.
  • Tomoya Kotani
    VITAMINS AND HORMONES: HEDGEHOG SIGNALING 88 273 - 291 0083-6729 2012 [Refereed][Invited]
     
    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. (C) 2012 Elsevier Inc.
  • Ryoma Ota, Tomoya Kotani, Masakane Yamashita
    BIOCHEMISTRY 50 (25) 5648 - 5659 0006-2960 2011/06 [Refereed][Not invited]
     
    Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.
  • Akira Muto, Masamichi Ohkura, Tomoya Kotani, Shin-ichi Higashijima, Junichi Nakai, Koichi Kawakami
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 (13) 5425 - 5430 0027-8424 2011/03 [Refereed][Not invited]
     
    Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS: GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS systemis a powerful tool to study functional neural circuits in zebrafish.
  • Ryoma Ota, Tomoya Kotani, Masakane Yamashita
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 (4) 2853 - 2863 0021-9258 2011/01 [Refereed][Not invited]
     
    Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.
  • Kyota Yasuda, Tomoya Kotani, Ryoma Ota, Masakane Yamashita
    DEVELOPMENTAL BIOLOGY 348 (1) 76 - 86 0012-1606 2010/12 [Refereed][Not invited]
     
    Temporal translation control of localized mRNA is crucial for regulating various cellular and developmental processes. However, little is known about the mechanisms of temporal translation control of localized mRNA due to the limitation in technology. cyclin B1 mRNA at the animal polar cytoplasm of immature zebrafish oocytes is translationally repressed, and its activation is temporally regulated during maturation. Mechanisms of cyclin B1 translation in oocytes were analyzed using transgenic zebrafish in which reporter mRNAs are produced from transgenes introduced into the genome through transcription in the nucleus followed by transport to the cytoplasm, as in endogenous mRNAs. Real-time imaging of the site and timing of translation showed that mRNAs containing the full-length cyclin B1 sequence precisely mimic the localization and translation of endogenous cyclin B1 mRNA. However, mRNAs containing cyclin B1 3' untranslated region but lacking open reading frame (ORF) underwent abnormal localization and precocious translational activation, indicating the significance of the ORF in translational control of cyclin B1 mRNA. Our genetic approach in combination with real-time imaging of the translation site and timing provides a novel insight into the mechanisms of temporal control of translation. (C) 2010 Elsevier Inc. All rights reserved.
  • Kotani Tomoya, Yasuda Kyota, Ota Ryoma, Yamashita Masakane
    GENES & GENETIC SYSTEMS 85 (6) 416 - 416 1341-7568 2010/12 [Refereed][Not invited]
  • Tomoya Kotani, Shun-ichiro Iemura, Tohru Natsume, Koichi Kawakami, Masakane Yamashita
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (7) 5106 - 5116 0021-9258 2010/02 [Refereed][Not invited]
     
    During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type I alpha subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells.
  • Kotani Tomoya, Iemura Shun-ichiro, Natsume Tohru, Kawakami Koichi, Yamashita Masakane
    MECHANISMS OF DEVELOPMENT 126 S324  0925-4773 2009/08 [Refereed][Not invited]
  • Toshiharu Iwai, Shinya Inoue, Tomoya Kotani, Masakane Yamashita
    ZOOLOGICAL SCIENCE 26 (1) 9 - 16 0289-0003 2009/01 [Refereed][Not invited]
     
    As with zebrafish, attention has focused on the teleost medaka Oryzias latipes as an experimental animal representative of non-mammalian vertebrates in various fields of biological science. To enable real-time analyses of the dynamics of nuclei and chromosomes in living medaka cells, we produced a transgenic medaka expressing a fusion protein between histone H2B and green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter. Since the nuclei and chromosomes of transgenic medaka cells are labeled with GFP, their morphological changes can be instantly monitored throughout the mitotic cell cycle progression under a fluorescent microscope without any fixation and staining of samples. However, GFP-labeling of nuclei and chromosomes is not successful during early embryonic development until zygotic expression begins and during the meiotic cell cycle progression, because the CMV promoter does not work in these stages. In addition, histone H2B-GFP fusion proteins are expressed in an organ-specific manner; strong and ubiquitous expression occurs in cells comprising the gut and fin, whereas the expression is restricted to certain types of cells in the liver and brain. These findings suggest that the CMV-driven expression of the histone H2B-GFP transgene is modified depending on the integration site of the transgene in the genome. Nevertheless, easy and precise monitoring of cytological changes in nuclei and chromosomes in the majority of mitotic cells by using the transgenic medaka will greatly contribute to a better understanding of control mechanisms of nuclear and chromosomal behaviors in vertebrate cells.
  • Ryoma Ota, Kaori Suwa, Tomoya Kotani, Koich Mita, Masakane Yamashita
    ZOOLOGICAL SCIENCE 25 (7) 773 - 781 0289-0003 2008/07 [Refereed][Not invited]
     
    It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3 beta (GSK3 beta) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3 beta are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3 beta, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3 beta is a component of the PI3K signal transduction pathway.
  • Tomoya Kotani, Koichi Kawakami
    DEVELOPMENTAL BIOLOGY 316 (2) 383 - 396 0012-1606 2008/04 [Refereed][Not invited]
     
    Somite boundary formation is crucial for segmentation of vertebrate somites and vertebrae and skeletal muscle morphogenesis. Previously, we developed a Tol2 transposon-mediated gene trap method in zebrafish. In the present study, we aimed to isolate transposon insertions that trap maternally-expressed genes. We found that homozygous female fish carrying a transposon insertion within a maternally-expressed gene misty somites (mys) produced embryos that showed obscure somite boundaries at the early segmentation stage (12-13 hpf). The somite boundaries became clear and distinct after this period and the embryos survived to adulthood. This phenotype was rescued by expression of mys cDNA in the homozygous adults, confirming that it was caused by a decreased mys activity. We analyzed a role of the mys gene by using morpholino oligonucleotides (MOs). The MO-injected embryo exhibited severer phenotypes than the insertional mutant probably because the mys gene was partially active in the insertional mutant. The MO-injected embryo also showed the obscure somite boundary phenotype. Fibronectin and phosphorylated FAK at the intersomitic regions were accumulated at the boundaries at this stage, but, unlike wild type embryos, somitic cells adjacent to the boundaries did not undergo epithelialization, suggesting that Mys is required for epithelialization of the somitic cells. Then in the MO-injected embryos, the boundaries once became clear and distinct, but, in the subsequent stages, disappeared, resulting in abnormal muscle morphogenesis. Accumulation of Fibronectin and phosphorylated FAK observed in the initial stage also disappeared. Thus, Mys is crucial for maintenance of the somite boundaries formed at the initial stage. To analyze the mys defect at the cellular level, we placed cells dissociated from the MO-injected embryo on Fibronectin-coated glasses. By this cell spreading assay, we found that the mys-deficient cells reduced the activity to form lamellipodia on Fibronectin while FAK was activated in these cells. Thus, we demonstrate that a novel gene misty somites is essential for epithelialization of the somitic cells and maintenance of the somite boundary. Furthermore, Mys may play a role in a cellular pathway leading to lamellipodia formation in response to the Fibronectin signaling. We propose that the Tol2 transposon mediated gene trap method is powerful to identify a novel gene involved in vertebrate development. (C) 2008 Elsevier Inc. All rights reserved.
  • Kazuhide Asakawa, Maximiliano L. Suster, Kanta Mizusawa, Saori Nagayoshi, Tomoya Kotani, Akihiro Urasaki, Yasuyuki Kishimoto, Masahiko Hibi, Koichi Kawakami
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (4) 1255 - 1260 0027-8424 2008/01 [Refereed][Not invited]
     
    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.
  • Tomoya Kotani, Saori Nagayoshi, Akihiro Urasaki, Koichi Kawakami
    METHODS 39 (3) 199 - 206 1046-2023 2006/07 [Refereed][Not invited]
     
    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.
  • T Kotani, M Yamashita
    BIOCHEMICAL JOURNAL 389 (3) 611 - 617 0264-6021 2005/08 [Refereed][Not invited]
     
    Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. gamma-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of gamma-tubulin in spindle pole formation, we over-expressed GFP (green fluorescent protein)-fused gamma-tubulin (gamma-TuGFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. gamma-Tu-GFP associated with endogenous alpha-, beta- and gamma-tubulin, suggesting that it acts in the same manner as that of endogenous gamma-tubulin. During the process of aster formation, gamma-Tu-GFP aggregated as dots on microtubutes, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of gamma-tubulin by an anti-gamma-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of gamma-Tu-GFP, confirming the necessity of gamma-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated gamma-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of gamma-tubulin caused abnormal organization of microtubulles in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-gamma-tubulin antibody. The results indicate possible roles of gamma-tubutin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.
  • T Kotani, M Yamashita
    ZYGOTE 13 (3) 219 - 226 0967-1994 2005/08 [Refereed][Not invited]
     
    Vertebrate oocytes do not contain centrosomes and therefore form an acentrosomal spindle during oocyte maturation. gamma-Tubulin is known to be essential for nucleation of microtubules at centrosomes, but little is known about the behaviour and role of gamma-tubulin during spindle formation in oocytes. We first observed sequential localization of gamma-tubulin during spindle formation in Xenopus oocytes. gamma-Tubulin assembled in the basal regions of the germinal vesicle (GV) at the onset of germinal vesicle breakdown (GVBD) and remained on the microtubule-organizing centre (MTOC) until a complex of the MTOC and transient-microtubule array (TMA) reached the oocyte surface. Prior to bipolar spindle formation, oocytes formed an aggregation of microtubules and gamma-tubulin was concentrated at the centre of the aggregation. At the late stage of bipolar spindle formation, gamma-tubulin accumulated at each pole. Anti-dynein antibody disrupted the localization of gamma-tubulin, indicating that the translocation described above is dependent on dynein activity. We finally revealed that XMAP215, a microtubule-associated protein cooperating with gamma-tubulin for the assembly of microtubules, but not gamma-tubulin, was phosphorylated during oocyte maturation. These results suggest that gamma-tubulin is translocated by dynein to regulate microtubule organization leading to spindle formation and that modification of the molecules that cooperate with gamma-tubulin, but not gamma-tubulin itself, is important for microtubule reorganization.
  • Kotani T, Kawakami K
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 49 (13) 2111 - 2116 0039-9450 2004/10 [Refereed][Not invited]
  • S Nakahata, T Kotani, K Mita, T Kawasaki, Y Katsu, Y Nagahama, M Yamashita
    MECHANISMS OF DEVELOPMENT 120 (8) 865 - 880 0925-4773 2003/08 [Refereed][Not invited]
     
    Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In this study, we investigated the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, in the mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound to cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain but not to its phosphorylated form in mature oocytes. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. We propose that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation. (C) 2003 Elsevier Ireland Ltd. All rights reserved.
  • D. H.S. Shin, H. Matsubara, S. Kaneko, T. Kotani, M. Yamashita, S. Adachi, K. Yamauchi
    Fish Physiology and Biochemistry 28 (1-4) 519 - 520 0920-1742 2003 [Refereed][Not invited]
     
    In order to identify maturational factors that can serve as indicators of egg quality in artificially matured Japanese eel, Anguilla japonica maturation-promoting factor (MPF consists of cdc2 and cyclin B) related proteins and chromosomes were examined. Mitogen-activated protein kinase (MAPK 44 kDa band) and cdc2 (35 kDa and 34 kDa band) were detected. Chromosomes aligned on a vertical spindle appeared normal however, chromosomes on a horizontal spindle and dispersed chromosomes seemed unusual. © 2004 Kluwer Academic Publishers.
  • T Kotani, M Yamashita
    DEVELOPMENTAL BIOLOGY 252 (2) 271 - 286 0012-1606 2002/12 [Refereed][Not invited]
     
    Maturing amphibian oocytes undergo drastic morphological changes, including germinal vesicle breakdown (GVBD), chromosome condensation, and spindle formation in response to progesterone. Two kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), are involved in these changes, but their precise roles are unknown. Unlike in Xenopus oocytes, discrimination of the functions of MAPK and MPF in Rana oocytes is easy owing to the lack of pre-MPF. We investigated the roles of these kinases by careful observations of chromosomes and microtubules in Rana oocytes. MPF and MAPK activities were manipulated by treatment with progesterone, c-mos mRNA, or cyclin B mRNA in combination with MAPK kinase inhibitors. Activation of one kinase without activation of the other induced only limited events; GVBD was induced by MPF without MAPK, and reorganization of microtubules at GVBD was induced by MAPK without MPF, but other events were not induced. In contrast, coactivation of MPF and MARK by injection of c-mos and cyclin B mRNA promoted almost all of the morphological changes that occur during maturation without progesterone, indicating that these are controlled by cooperation of MPF and MAPK. The results revealed the functions of MAPK and MPF in each process of sequential morphological changes during oocyte maturation. (C) 2002 Elsevier Science (USA).
  • T Kotani, N Yoshida, K Mita, M Yamashita
    MOLECULAR REPRODUCTION AND DEVELOPMENT 59 (2) 199 - 208 1040-452X 2001/06 [Refereed][Not invited]
     
    Cyclin B, the regulatory subunit of maturation-promoting factor (MPF), com prises several subtypes that are presumed to confer different functions on MPF although no direct evidence has been provided to date. To clarify the difference in the roles of cyclins B1 and B2, we used frog (Rana japonica) oocytes in which MPF is formed only after progesterone stimulation because it is possible to produce oocytes containing either cyclin B1-MPF or cyclin B2-MPF by antisense RNA-mediated translational inhibition of each mRNA. Using this advantage, we investigated the functions of cyclins B1 and B2 and obtained the following results: (a) oocytes synthesizing cyclin B2-MPF underwent meiosis I and II with formation of a bipolar spindle at each metaphase; (b) oocytes synthesizing cyclin B1-MPF formed a monopolar spindle at metaphase I and extruded an abnormal polar body; and (c) both oocytes underwent germinal vesicle breakdown (GVBD) and chromosome condensation. Immunocytochemical observations also revealed continuous localization of cyclin B2 on the spindle during meiosis. These results provide evidence of the requirement of cyclin B2, but not cyclin B1, for organizing the bipolar spindle, though either cyclin B1 or B2 is redundant for inducing GVBD and chromosome condensation. Mol. Reprod. Dev. 59:199-208, 2001. (C) 2001 Wiley-Liss, Inc.
  • T Kondo, T Kotani, M Yamashita
    DEVELOPMENTAL BIOLOGY 229 (2) 421 - 431 0012-1606 2001/01 [Refereed][Not invited]
     
    Cyclin B mRNA stored in immature zebrafish oocytes is translationally activated upon the stimulation of 17 alpha ,20 beta -dihydroxy-4-pregnen-3-one (17 alpha ,20 beta -DP), an event prerequisite for initiating oocyte maturation in this species. We investigated localization of cyclin B mRNA in zebrafish oocytes. Cyclin B mRNA was found to be exclusively localized as an aggregation along the cytoplasm at the animal pole of full-grown immature oocytes. When oocytes were treated with 17 alpha ,20 beta -DP, a meshwork of microfilaments in the oocyte cortex disappeared and the aggregation of cyclin B mRNA dispersed just prior to the initiation of cyclin B synthesis and germinal vesicle breakdown (GVBD). Cytochalasin B, but not nocodazole or taxol, deformed the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like 17 alpha ,20 beta -DP, cytochalasin B (10 mug/ml) induced both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without 17 alpha ,20 beta -DP. Conversely, disturbance of the aggregation of cyclin B mRNA with a low concentration (1 mug/ml) of cytochalasin B inhibited 17 alpha ,20 beta -DP-induced GVBD. These results suggest that the direct change in cyclin B mRNA from the aggregated form to the dispersed form is responsible for translational activation of the mRNA during zebrafish oocyte maturation. (C) 2001 Academic Press.

MISC

Association Memberships

  • THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   日本動物学会   日本発生生物学会   

Research Projects

  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2024/04 -2027/03 
    Author : 小谷 友也
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2023/06 -2026/03 
    Author : 小谷 友也
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2021/04 -2024/03 
    Author : 小谷 友也, 山本 雄広
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
    Date (from‐to) : 2019/06 -2022/03 
    Author : Kotani Tomoya
     
    The proper production of eggs is crucial for almost all animal to promote development, since eggs accumulate almost all factors that are required for promoting developmental processes. The aim of this study was to develop a novel screening that enable us to identify maternal-effect mutants in order to understand functions of transcripts stored in eggs. In this study, we effectively isolated females homozygous for the insertions and identified a maternal-effect mutant using a small scale fish maintenance systems.
  • 卵形成から個体形成過程における翻訳の実態と役割の解明
    文部科学省：科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2016 -2018 
    Author : 小谷 友也
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013 -2015 
    Author : Kotani Tomoya
     
    In this study, we aimed to elucidate molecular mechanisms of evolutionally conserved control of cyclin B1 mRNA translation to understand the nature of translational machineries by which special and temporal translation is regulated. First, we demonstrated that binding of Pumilio1 to cyclin B1 mRNA is essential for the RNA granule formation, which was found in vertebrate oocytes, and that this Pumilio1-mediated RNA granule formation is important for specially and temporally regulated translational activation of the mRNA. Then, we identified IMP3 protein as a novel factor binding to cycle B1 mRNA, providing novel insight into the translational regulation of the mRNA. Furthermore, we generated transgenic animals visualizing dynamics of actin filaments and obtained results supporting the notion that assembly and depolymerization of actin filaments are involved in the translational regulation.
  • 文部科学省：科学研究費補助金(特定領域研究)
    Date (from‐to) : 2011 -2012 
    Author : 小谷 友也
     
    すべての動物の卵母細胞は、第一減数分裂前期で分裂を停止しmRNAと蛋白質を蓄積する。十分に成長した卵母細胞(未成熟卵)はホルモン刺激を受けて卵成熟を起こし、減数分裂の再開と第二分裂中期での再停止を経て受精可能な成熟卵となる。卵成熟の開始にともない遺伝子の転写は抑制されるため、成熟卵となるすべての現象は卵母細胞に蓄積された田RNAと蛋白質の修飾で制御される。サイクリンB1蛋白質は、卵成熟を推進する卵成熟促進因子(MPF)の調節サブユニットで、その翻訳は正常な卵成熟の進行に必須である。本研究において、我々は脊椎動物に普遍的なサイクリンB1翻訳制御機構の解明を目指し、ゼブラフィッシュとマウスを用いて解析を行った。その結果、次の成果を得てきた。1)ゼブラフィッシュとマウスの未成熟卵において、翻訳が抑制されたサイクリンB1 mRNAはRNA顆粒を形成する。2)これらRNA顆粒は、卵成熟過程における翻訳の活性化に伴って消失する。3)RNA結合蛋白質PumilioはサイクリンB1 mRNAに結合し、顆粒の形成に関わる。4)サイクリンB1 mRNAの顆粒形成はアクテン繊維に依存する。5)顆粒を形成しないmRNAはその翻訳時期が早まる。本年度は新たに、RNA顆粒の安定化が翻訳に及ぼす影響を解析し、次の成果を得た。1)PumilioのN末端領域の過剰発現によって顆粒が安定化し、翻訳の活性化が阻害される。2)アクチン繊維の安定化によってRNA顆粒が安定化し、同じく翻訳の活性化が阻害される。これらの結果から、RNA顆粒の形成とその消失がサイクリンB1の翻訳時期の制御に重要であることが示された。これらの成果は、成熟卵形成に必須のサイクリンB1翻訳の新規分子機構を明らかにしたのみでなく、RNA顆粒の形成が翻訳の活性化時期の制御に関わることを初めて示したものである。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2012 
    Author : KOTANI Tomoya
     
    Localization and translational control of cytoplasmic mRNAs play key roles in the regulation of site and timing of gene expression. We analyzed molecular mechanisms by which localization and translational control of cyclin B1 mRNA deposited in oocytes are regulated using transgenes and real-time imaging of the site and timing of translation. In this study, we revealed that a cis-acting element consisting of 9 nucleotides sequences in the coding region is necessary for the regulation of both localization and translational control of the mRNA. Our results suggest a functional link between mRNA localization and translational control.
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(若手研究(B))
    Date (from‐to) : 2009 -2010 
    Author : Tomoya KOTANI
     
    We previously identified a novel gene misty somites (mys), which encodes Mys protein, and found that almost all vertebrate genomes contain the gene encoding Mys protein homolog. In this study, we newly produced antibodies recognizing carboxy-terminus of human Mys protein, a region highly conserved in vertebrate Mys protein homologs. Mys protein homologs were found to be expressed in human and mouse cultured cells and Xenopus embryos. We then examined the localization of Mys protein in human cultured cells using the newly produced antibody and found that Mys protein was localized in Golgi ap...
  • 蛋白質間相互作用を用いた細胞分裂分子制御機構の解析
  • Molecular mechanism of protein-protein interaction in mitosis

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