研究者総覧

高橋 将文 (タカハシ マサノリ)

  • 博士課程人間生物学系専攻生体制御医学専攻分野分子細胞生物学専攻科 准教授
Last Updated :2021/12/07

研究者情報

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プロフィール

  • 大阪府出身。徳島大学工学部生物工学科卒業後、奈良先端科学技術大学院大学バイオサイエンス研究科博士前期課程、東北大学大学院医学系研究科医学履修博士課程修了。1) 器官形成における上皮間葉細胞相互作用、上皮(中皮)間葉転換、および組織幹細胞の振る舞い、2) 脳の発生発達過程における領域化、ニューロン分化、および神経回路形成、3) 哺乳全胚培養法とその応用、に興味をもっている。

研究キーワード

  • 神経堤細胞   線維芽細胞   中皮細胞   神経上皮細胞   Six ファミリー遺伝子   カドヘリン   歯の発生   上皮間葉相互作用   歯幹細胞   上皮間葉転換(EMT)   神経発生   脳幹   線条体   左右非対称性   哺乳類全胚培養   

研究分野

  • ライフサイエンス / 発生生物学
  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経科学一般 / 神経発生生物学
  • ライフサイエンス / 神経科学一般 / 神経発生生物学

経歴

  • 2020年06月 - 現在  自治医科大学大学院医学研究科基礎系大学院本務、分子病態治療研究センター循環病態・代謝学研究部(兼任)准教授
  • 2019年01月 - 2020年05月  自治医科大学(JMU)大学院医学研究科 基礎系大学院本務、分子病態治療研究センター 細胞生物研究部 (兼任)准教授
  • 2012年04月 - 2018年12月  自治医科大学大学院医学研究科 基礎系大学院本務、分子病態治療研究センター 細胞生物研究部 (兼任)講師
  • 2012年06月 - 2013年03月  東北大学医学部非常勤講師
  • 2007年04月 - 2012年03月  東北大学 大学院医学系研究科Graduate School of Medicine助教
  • 2011年02月 - 2012年02月  Division of Developmental Neurobiology, MRC National Institute for Medical Research, London, UKVisiting research fellow
  • 2003年04月 - 2007年03月  東北大学 大学院医学研究科助手
  • 2003年05月 - 2003年06月  Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Gottingen, GermanyGuest research fellow

所属学協会

  • 日本解剖学会   日本分子生物学会   日本発生生物学会   日本神経科学学会   日本細胞生物学会   国際発生生物学会   

研究活動情報

論文

  • Masanori Takahashi, Ryoji Fukabori, Hiroshi Kawasaki, Kazuto Kobayashi, Kiyoshi Kawakami
    The Journal of comparative neurology 2021年07月 [査読有り]
     
    The dorsolateral striatum (DLS) of rodents is functionally subdivided into somatotopic subregions that represent each body part along both the dorsoventral and anteroposterior (A-P) axes and play crucial roles in sensorimotor functions via corticostriatal pathways. However, little is known about the spatial gene expression patterns and heterogeneity of spiny projection neurons (SPNs) within somatotopic subregions. Here, we show that the cell adhesion molecule gene Cdh20, which encodes a Type II cadherin, is expressed in discrete subregions covering the inner orofacial area and part of the forelimb area in the ventral domain of the DLS (v-DLS) in rats. Cdh20-expressing cells were localized in the v-DLS at the intermediate level of the striatum along the A-P axis and could be classified as direct-pathway SPNs or indirect-pathway SPNs. Unexpectedly, comprehensive analysis revealed that Cdh20 is expressed in SPNs in the rat DLS but not in the mouse DLS or the ferret putamen (Pu). Our observations reveal that Cdh20 expression demarcates somatotopic subregions and subpopulations of SPNs specifically in the rat DLS and suggest divergent regulation of genes differentially expressed in the v-DLS and Pu among mammals.
  • Masanori Takahashi, Keiko Ikeda, Masaki Ohmuraya, Yoshiko Nakagawa, Tetsushi Sakuma, Takashi Yamamoto, Kiyoshi Kawakami
    Developmental dynamics : an official publication of the American Association of Anatomists 249 9 1098 - 1116 2020年04月 [査読有り][通常論文]
     
    BACKGROUND: The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown. RESULTS: A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos. CONCLUSION: Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme. This article is protected by copyright. All rights reserved.
  • Masanori Takahashi, Masaru Tamura, Shigeru Sato, Kiyoshi Kawakami
    Disease models & mechanisms 11 10 2018年10月 [査読有り][通常論文]
     
    Omphalocele is a human congenital anomaly in ventral body wall closure and may be caused by impaired formation of the primary abdominal wall (PAW) and/or defects in abdominal muscle development. Here, we report that mice doubly deficient in homeobox genes Six4 and Six5 showed the same ventral body wall closure defects as those seen in human omphalocele. SIX4 and SIX5 were localized in surface ectodermal cells and somatic mesoderm-derived mesenchymal and coelomic epithelial cells (CECs) in the PAW. Six4-/-;Six5-/- fetuses exhibited a large omphalocele with protrusion of both the liver and intestine, or a small omphalocele with protrusion of the intestine, with complete penetrance. The umbilical ring of Six4-/-;Six5-/- embryos was shifted anteriorly and its lateral size was larger than that of normal embryos at the E11.5 stage, before the onset of myoblast migration into the PAW. The proliferation rates of surface ectodermal cells in the left and right PAW and somatic mesoderm-derived cells in the right PAW were lower in Six4-/-;Six5-/- embryos than those of wild-type embryos at E10.5. The transition from CECs of the PAW to rounded mesothelial progenitor cells was impaired and the inner coelomic surface of the PAW was relatively smooth in Six4-/-;Six5-/- embryos at E11.25. Furthermore, Six4 overexpression in CECs of the PAW promoted ingression of CECs. Taken together, our results suggest that Six4 and Six5 are required for growth and morphological change of the PAW, and the impairment of these processes is linked to the abnormal positioning and expansion of the umbilical ring, which results in omphalocele.
  • Wataru Yamashita, Masanori Takahashi, Takako Kikkawa, Hitoshi Gotoh, Noriko Osumi, Katsuhiko Ono, Tadashi Nomura
    Development (Cambridge, England) 145 8 2018年04月 [査読有り][通常論文]
     
    The evolution of unique organ structures is associated with changes in conserved developmental programs. However, characterizing the functional conservation and variation of homologous transcription factors (TFs) that dictate species-specific cellular dynamics has remained elusive. Here, we dissect shared and divergent functions of Pax6 during amniote brain development. Comparative functional analyses revealed that the neurogenic function of Pax6 is highly conserved in the developing mouse and chick pallium, whereas stage-specific binary functions of Pax6 in neurogenesis are unique to mouse neuronal progenitors, consistent with Pax6-dependent temporal regulation of Notch signaling. Furthermore, we identified that Pax6-dependent enhancer activity of Dbx1 is extensively conserved between mammals and chick, although Dbx1 expression in the developing pallium is highly divergent in these species. Our results suggest that spatiotemporal changes in Pax6-dependent regulatory programs contributed to species-specific neurogenic patterns in mammalian and avian lineages, which underlie the morphological divergence of the amniote pallial architectures.
  • Takako Kikkawa, Masanori Takahashi, Noriko Osumi
    Current Protocols in Neuroscience 2017 3.30.1 - 3.30.16 2017年01月 [査読有り][通常論文]
     
    This unit describes basic methods formammalianwhole embryo culture (WEC) using embryonic day 10.5 mouse embryos, including the preparation of highquality immediately centrifuged (IC) rat serum that is commonly used for WEC and is essential for normal growth and development of cultured mouse and rat embryos in vitro. An alternative protocol for different stages of rodent embryos is also introduced. Since embryos for WEC are dissected out of the uterus and manipulated under the microscope, one can overcome many of the difficulties of gene delivery encountered using in utero electroporation. A description for a gene transfer method to label neural stem/progenitor cells of the cortical primordium in a highly region-specific manner is also included.
  • Tatsuki Kawasaki, Masanori Takahashi, Hiroshi Yajima, Yoshiyuki Mori, Kiyoshi Kawakami
    DEVELOPMENT GROWTH & DIFFERENTIATION 58 6 530 - 545 2016年08月 [査読有り][通常論文]
     
    The periodontal ligament (PDL) is a connective tissue that attaches the tooth cementum to the alveolar bone and is derived from dental follicle cells (DFCs). The DFCs form fibroblasts, osteoblasts, cementoblasts, and PDL stem cells (PDLSCs). We previously reported homeobox transcription factor Six1 expression in mouse DFCs. However, the role of Six1 in periodontal tissue development is largely unknown. In this study, we analyzed SIX1 expression in mouse periodontal tissue cells during postnatal development and adulthood. We also addressed the role of SIX1 in mouse periodontium development and in human cultured PDL-derived cells (PDLCs). In mouse development, SIX1 production was abundant in DFCs and PDL cells by 2 weeks, but it was greatly diminished in the PDL at 4 weeks and in adults. Although the SIX1-positive cell distribution was sparse in the adult PDL, SIX1-positive cells were observed with low expression levels. We used 5-ethynyl-20-deoxyuridine (EdU) for cell labeling to reveal numerous EdU/SIX1-double positive cells at 2 weeks; however, a few EdU-positive cells remained at 4 weeks. The proportion of DFCs that incorporated EdU was significantly lower in Six1deficient mice compared with wild-type mice at E18.5. In human PDLCs, SIX1 was intensely expressed, and SIX1-knockdown using siRNA reduced proliferating PDLCs. Our results suggest that SIX1 is a key proliferation regulator in mouse DFCs and human PDLCs, which provides novel insight into Six family gene function in mammals.
  • Keiko Ikeda, Masanori Takahashi, Shigeru Sato, Hiroyuki Igarashi, Toru Ishizuka, Hiromu Yawo, Satoru Arata, E. Michelle Southard-Smith, Kiyoshi Kawakami, Hiroshi Onimaru
    PLOS ONE 10 7 e0132475  2015年07月 [査読有り][通常論文]
     
    The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO(2)/pH and pO(2) levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Botzinger complex group (preBotC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O-2 sensor molecules in the adult central and peripheral nervous systems.
  • Masanori Takahashi, Sayaka Makino, Takako Kikkawa, Noriko Osumi
    JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 90 2014年08月 [査読有り][通常論文]
     
    Mammalian whole embryo culture ( WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged ( IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos ( i. e., E8.0- 12.5 for the mouse, and E10.0- 14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.
  • Takako Kikkawa, Takeshi Obayashi, Masanori Takahashi, Urara Fukuzaki-Dohi, Keiko Numayama-Tsuruta, Noriko Osumi
    GENES TO CELLS 18 8 636 - 649 2013年08月 [査読有り][通常論文]
     
    Pax6 balances cell proliferation and neuronal differentiation in the mammalian developing neocortex by regulating the expression of target genes. Using microarray analysis, we observed the down-regulation of Dmrta1 (doublesex and mab-3-related transcription factor-like family A1) in the telencephalon of Pax6 homozygous mutant rats (rSey(2)/rSey(2)). Dmrta1 expression was restricted to the neural stem/progenitor cells of the dorsal telencephalon. Overexpression of Dmrta1 induced the expression of the proneural gene Neurogenin2 (Neurog2) and conversely repressed Ascl1 (Mash1), a proneural gene expressed in the ventral telencephalon. We found that another Dmrt family molecule, Dmrt3, induced Neurog2 expression in the dorsal telencephalon. Our novel findings suggest that dual regulation of proneural genes mediated by Pax6 and Dmrt family members is crucial for cortical neurogenesis.
  • Hiroshi Shinohara, Nobuyuki Sakayori, Masanori Takahashi, Noriko Osumi
    BIOLOGY OPEN 2 7 739 - 749 2013年07月 [査読有り][通常論文]
     
    The mammalian cerebral cortex develops from proliferative apical progenitor cells (APs) that exhibit cell cycle-dependent nuclear movement (interkinetic nuclear migration; INM), which may be important for efficient and continuous production of neurons. The Pax6 transcription factor plays a major role in INM by regulating various downstream molecules. We have previously observed abnormal INM and unstable localization of the centrosome in APs of the Pax6 homozygous mutant rat embryo. To understand the mechanisms of INM, we focused on the centrosomes of APs. One of the centrosomal proteins, ninein, is specifically localized in the centrosome of APs. We observed a dramatic downregulation of ninein in APs of the Pax6 mutant. Moreover, knockdown of ninein by RNAi induced ectopic distribution of reduced numbers of BrdU-positive (S-phase) and PH3-positive (M-phase) cells. Furthermore, time-lapsed imaging demonstrated that knockdown of ninein in vivo induced abnormal INM. Finally, we observed impaired microtubule regrowth in neural progenitors taken from Pax6 homozygous mutant rat embryos, which was recovered by via ninein overexpression. We also found that ninein knockdown enlarged the surface size area of apical endfeet of the APs. Our results suggest that ninein plays a role in the molecular machinery essential for INM by connecting microtubules to the centrosome. (C) 2013. Published by The Company of Biologists Ltd.
  • Emiko Yamanishi, Masanori Takahashi, Yumiko Saga, Noriko Osumi
    DEVELOPMENT GROWTH & DIFFERENTIATION 54 9 785 - 800 2012年12月 [査読有り][通常論文]
     
    Neural crest (NC) cells originate from the neural folds and migrate into the various embryonic regions where they differentiate into multiple cell types. A population of cephalic neural crest-derived cells (NCDCs) penetrates back into the developing forebrain to differentiate into microvascular pericytes, but little is known about when and how cephalic NCDCs invade the telencephalon and differentiate into pericytes. Using a transgenic mouse line in which NCDCs are genetically labeled with enhanced green fluorescent protein (EGFP), we observed that NCDCs started to invade the telencephalon together with endothelial cells from embryonic day (E) 9.5. A majority of NCDCs located in the telencephalon expressed pericyte markers, that is, PDGFR beta and NG2, and differentiated into pericytes around E11.5. Surprisingly, many of the NC-derived pericytes express p75, an undifferentiated NCDC marker at E11.5, as well as NCDCs in the mesenchyme. At the same time, a minor population of NCDCs that located separately from blood vessels in the telencephalon were NG2-negative and some of these NCDCs also expressed p75. Proliferation and differentiation of pericytes appeared to occur in a specific mesenchymal region where blood vessels penetrated into the telencephalon. These results indicate that (i) NCDCs penetrate back into the telencephalon in parallel with angiogenesis, (ii) many NC-derived pericytes may be still in pre-mature states even though after differentiation into pericytes in the early developing stages, (iii) a small minority of NCDCs may retain undifferentiated states in the developing telencephalon, and (iv) a majority of NCDCs proliferate and differentiate into pericytes in the mesenchyme around the telencephalon.
  • Yuji Tsunekawa, Joanne M. Britto, Masanori Takahashi, Franck Polleux, Seong-Seng Tan, Noriko Osumi
    EMBO JOURNAL 31 8 1879 - 1892 2012年04月 [査読有り][通常論文]
     
    Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3' untranslated region (3'UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved. The EMBO Journal (2012) 31, 1879-1892. doi:10.1038/emboj.2012.43; Published online 6 March 2012 Subject Categories: cell & tissue architecture; neuroscience
  • Masanori Takahashi, Noriko Osumi
    MECHANISMS OF DEVELOPMENT 128 5-6 289 - 302 2011年05月 [査読有り][通常論文]
     
    The vertebrate neuroepithelium can be subdivided into non-boundary and boundary regions. The boundary regions act as signaling centers for regional specification and neurogenesis in the neighboring non-boundary regions. An important question is how boundary regions are specified and maintained during brain development. In this study, we report that Pax6, a homeodomain transcription factor, regulates boundary-cell specification between rhombomeres of the developing rat hindbrain. We compared expression patterns of four boundary-cell markers, including PLZF (Zbtb16), Ring1A (Ring1), Wnt5a, and cadherin7 (Cdh7), in wild-type and Pax6 loss-of-function mutant hindbrains and found that the expression of Zbtb16, Ring1, and Wnt5a was down-regulated in the rhombomere boundaries, while Cdh7 expression was up-regulated in the non-boundary regions of the Pax6 mutant hindbrain. Morphological observations revealed that the boundary regions were larger and that the interface between the boundary and non-boundary regions was obscured in the Pax6 mutant hindbrain. We also found ectopic neuronal differentiation in the boundary cells of the Pax6 mutant hindbrain. In addition, we observed that Hes5 was down-regulated and that Neurogenin2 (Neurog2) was up-regulated in the boundary regions of the Pax6 mutant hindbrain. Because Hes genes have been shown to inhibit neuronal differentiation by repressing proneural genes, Pax6 may act through this pathway to prevent neurogenesis in the boundary cells. Taken together, Pax6 seems to be required for coordinating boundary-cell specification and reducing neurogenesis within the hindbrain boundary region. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Kenta Kobayashi, Tomoyuki Masuda, Masanori Takahashi, Jun-ichi Miyazaki, Masahiro Nakagawa, Motokazu Uchigashima, Masahiko Watanabe, Hiroyuki Yaginuma, Noriko Osumi, Kozo Kaibuchi, Kazuto Kobayashi
    EUROPEAN JOURNAL OF NEUROSCIENCE 33 4 612 - 621 2011年02月 [査読有り][通常論文]
     
    Cranial motor neurons, which are divided into somatic motor (SM), branchiomotor (BM) and visceral motor (VM) neurons, form distinct axonal trajectories to innervate their synapse targets. Rho GTPase regulates various neuronal functions through one of the major effector proteins, Rho-kinase. Here, we addressed the in vivo role of the Rho/Rho-kinase signaling pathway in axon patterning of cranial motor neurons. We performed conditional expression of a dominant-negative mutant for RhoA or Rho-kinase in transgenic mice by using the Cre-loxP system to suppress the activity of these molecules in developing cranial motor neurons. Blockade of the Rho/Rho-kinase signaling pathway caused defects in the patterning of SM axons but not in that of BM/VM axons, in which defects were accompanied by reduced muscle innervation and reduced synapse formation by SM neurons. In addition, blockade of the signaling pathway shifted the trajectory of growing SM axons in explant cultures, whereas it did not appear to affect the rate of spontaneous axonal outgrowth. These results indicate that the Rho/Rho-kinase signaling pathway plays an essential role in the axon patterning of cranial SM neurons during development.
  • Keiko Numayama-Tsuruta, Yoko Arai, Masanori Takahashi, Makiko Sasaki-Hoshino, Nobuo Funatsu, Shun Nakamura, Noriko Osumi
    BMC DEVELOPMENTAL BIOLOGY 10 6  2010年01月 [査読有り][通常論文]
     
    Background: The transcription factor Pax6 is essential for the development of the central nervous system and it exerts its multiple functions by regulating the expression of downstream target molecules. To screen for genes downstream of Pax6, we performed comprehensive transcriptome profiling analyses in the early hindbrain of Pax6 homozygous mutant and wild-type rats using microarrays. Results: Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up-or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally. Conclusions: These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.
  • Koji Nonomura, Masanori Takahashi, Yoshio Wakamatsu, Teruko Takano-Yamamoto, Noriko Osumi
    JOURNAL OF ANATOMY 216 1 80 - 91 2010年01月 [査読有り][通常論文]
     
    Six family transcription factor genes play multiple and crucial roles in the development of the vertebrate sensory system including the eye, olfactory epithelium and otic vesicle, and these genes are highly expressed in the neural crest-derived cranial mesenchymal cells in the mouse embryo. However, expression patterns have yet to be determined for the Six family genes in the developing tooth germ. In this study, we examined expression of six members of the Six family genes in the dental mesenchyme and the dental epithelium of the developing tooth germs in mice by in situ hybridization. We found dynamic expression patterns for Six1, Six2, Six4 and Six5 in the oral epithelium and mesenchymal cells with distinct expression patterns at the early stage before invagination of the dental epithelium. In addition, expression of Six1 and Six4 was observed in the inner enamel epithelium of the incisor and molar tooth germs at the cap stage. Expression of Six5 was maintained in the bell stage tooth germs, and intense expression of Six1 and Six4 was detected not only in the mesenchyme-derived dental follicle but also in the proliferating inner enamel epithelium of the labial cervical loop of the incisor tooth germ. Taken together, our results suggest that dynamic expression of Six family genes represents specific stages of the developing tooth germ. This dynamic expression is embodied in changes in both space and over time, and these changes in expression suggest that Six family genes may participate in tooth germ morphogenesis and the proliferation and/or differentiation of the incisor ameloblast stem/progenitor cells.
  • Masanori Takahashi, Noriko Osumi
    Journal of Visualized Experiments 42 2010年 [査読有り][通常論文]
     
    Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft, and introduce various applications of WEC technique for studies in mammalian developmental biology. © JoVE 2006-2011 All Rights Reserved.
  • Masanori Takahashi, Noriko Osumi
    BMC DEVELOPMENTAL BIOLOGY 8 87  2008年09月 [査読有り][通常論文]
     
    Background: Vertebrate classic cadherins are divided into type I and type II subtypes, which are individually expressed in brain subdivisions (e.g., prosomeres, rhombomeres, and progenitor domains) and in specific neuronal circuits in region-specific manners. We reported previously the expression of cadherin19 (cad19) in Schwann cell precursors. Cad19 is a type II classic cadherin closely clustered on a chromosome with cad7 and cad20. The expression patterns of cad7 and cad20 have been reported previously in chick embryo but not in the developing and adult central nervous system of mammals. In this study, we identified rat cad7 and cad20 and analyzed their expression patterns in embryonic and adult rat brains. Results: Rat cad7 protein showed 92% similarity to chick cad7, while rat cad20 protein had 76% similarity to Xenopus F-cadherin. Rat cad7 mRNA was initially expressed in the anterior neural plate including presumptive forebrain and midbrain regions, and then accumulated in cells of the dorsal neural tube and in rhombomere boundary cells of the hindbrain. Expression of rat cad20 mRNA was specifically localized in the anterior neural region and rhombomere 2 in the early neural plate, and later in longitudinally defined ventral cells of the hindbrain. The expression boundaries of cad7 and cad20 corresponded to those of region-specific transcription factors such as Six3, Irx3 and Otx2 in the neural plate, and Dbx2 and Gsh1 in the hindbrain. At later stages, the expression of cad7 and cad20 disappeared from neuroepithelial cells in the hindbrain, and was almost restricted to postmitotic cells, e. g. somatic motor neurons and precerebellar neurons. These results emphasized the diversity of cad7 and cad20 expression patterns in different vertebrate species, i.e. birds and rodents. Conclusion: Taken together, our findings suggest that the expression of cad7 and cad20 demarcates the compartments, boundaries, progenitor domains, specific nuclei and specific neural circuits during mammalian brain development.
  • Masanori Takahashi, Tadashi Nomura, Noriko Osumi
    Development, growth & differentiation 50 6 485 - 97 2008年08月 [査読有り][招待有り]
     
    Mammalian whole embryo culture (WEC) was developed long before transgenic and gene targeted animals are widely used. Electroporation (EP) into cultured rodent embryos has expanded the potential to analyze gene functions in mammalian embryos by transferring exogenous plasmid vectors or small nucleotides in region- and stage-specific ways. This method is quite simple, and therefore enables us to analyze gene functions more quickly than genetic manipulation. In this review, we introduce combinatorial methods of WEC and EP, and summarize various applications in developmental neurobiology.
  • Tadashi Nomura, Masanori Takahashi, Yoshinobu Hara, Noriko Osumi
    PLOS ONE 3 1 e1454  2008年01月 [査読有り][通常論文]
     
    The mammalian neocortex is characterized as a six-layered laminar structure, in which distinct types of pyramidal neurons are distributed coordinately during embryogenesis. In contrast, no other vertebrate class possesses a brain region that is strictly analogous to the neocortical structure. Although it is widely accepted that the pallium, a dorsal forebrain region, is specified in all vertebrate species, little is known of the differential mechanisms underlying laminated or non-laminated structures in the pallium. Here we show that differences in patterns of neuronal specification and migration provide the pallial architectonic diversity. We compared the neurogenesis in mammalian and avian pallium, focusing on subtype-specific gene expression, and found that the avian pallium generates distinct types of neurons in a spatially restricted manner. Furthermore, expression of Reelin gene is hardly detected in the developing avian pallium, and an experimental increase in Reelin-positive cells in the avian pallium modified radial fiber organization, which resulted in dramatic changes in the morphology of migrating neurons. Our results demonstrate that distinct mechanisms govern the patterns of neuronal specification in mammalian and avian pallial development, and that Reelin-dependent neuronal migration plays a critical role in mammalian type corticogenesis. These lines of evidence shed light on the developmental programs underlying the evolution of the mammalian specific laminated cortex.
  • Kosei Sato, Yutaka Koizumi, Masanori Takahashi, Atsushi Kuroiwa, Koji Tamura
    DEVELOPMENT 134 7 1397 - 1406 2007年04月 [査読有り][通常論文]
     
    Pattern formation along the proximal-distal (PD) axis in the developing limb bud serves as a good model for learning how cell fate and regionalization of domains, which are essential processes in morphogenesis during development, are specified by positional information. In the present study, detailed fate maps for the limb bud of the chick embryo were constructed in order to gain insights into how cell fate for future structures along the PD axis is specified and subdivided. Our fate map revealed that there is a large overlap between the prospective autopod and zeugopod in the distal limb bud at an early stage (stage 19), whereas a limb bud at this stage has already regionalized the proximal compartments for the prospective stylopod and zeugopod. A clearer boundary of cell fate specifying the prospective autopod and zeugopod could be seen at stage 23, but cell mixing was still detectable inside the prospective autopod region at this stage. Detailed analysis of HOXA11 AND HOXA13 expression at single cell resolution suggested that the cell mixing is not due to separation of some different cell populations existing in a mosaic. Our findings suggest that a mixable unregionalized cell population is maintained in the distal area of the limb bud, while the proximal region starts to be regionalized at the early stage of limb development.
  • T Yokoo, T Ohashi, JS Shen, K Sakurai, Y Miyazaki, Y Utsunomiya, M Takahashi, Y Terada, Y Eto, T Kawamura, N Osumi, T Hosoya
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 9 3296 - 3300 2005年03月 [査読有り][通常論文]
     
    The use of stem cells has enabled the successful generation of simple organs. However, anatomically complicated organs such as the kidney have proven more refractory to stem-cell-based regenerative techniques. Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish self-organs from autologous stem cells and transplant them as syngrafts back into donor patients. To this end, we have striven to establish an in vitro organ factory to build up complex organ structures from autologous adult stem cells by using the kidney as a target organ. Cultivation of human mesenchymal stem cells in growing rodent embryos enables their differentiation within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. We show that a combination of whole-embryo culture, followed by organ culture, encourages exogenous human mesenchymal stem cells to differentiate and contribute to functional complex structures of the new kidney.
  • M Takahashi, N Osumi
    DEVELOPMENTAL DYNAMICS 232 1 200 - 208 2005年01月 [査読有り][通常論文]
     
    To identify a novel type II classical cadherin, we searched the genome database and found rat cadherin19 (cad19) with high similarity to human cadherin19. We also found nucleotide sequences corresponding to rat cad19 in mouse and chicken genomes. In situ hybridization of rat cad19 revealed that rat cad19 mRNA was initially expressed in cephalic neural crest cells, and then in the cranial ganglia, migrating trunk neural crest cells, the nascent dorsal root ganglia, and the sympathetic ganglia. Expression of cad19 overlapped with that of neural crest markers, including Sox10 and AP-2, but cad19 expression was confined to subpopulations of the neural crest-derived cells, those typically observed in the satellite glia at the periphery of the ganglia and Schwann cell precursors along the peripheral nerves. cad19 mRNA was not detected in cells expressing Phox2b, an epibranchial placode-derived neurons, nor in those expressing neuronal markers such as Hu protein. These observations suggest that cad19 is expressed in neural crest-derived, non-neuronal cells. Although the expression of cad19 mRNA persisted in Schwann cell precursors at E14.5, it was no longer detected in maturing Schwann cells at later stages. These results suggest that cad19 is an evolutionarily conserved cadherin and may be involved in the early development of Schwann cells in the peripheral nervous system. (C) 2004 Wiley-Liss, Inc.
  • Y Ogawa, H Takebayashi, M Takahashi, N Osumi, Y Iwasaki, K Ikenaka
    DEVELOPMENTAL NEUROSCIENCE 27 6 364 - 377 2005年 [査読有り][通常論文]
     
    The central nervous system of the mammalian embryo is organized according to the expression of region-specific transcription factors along the anteroposterior and/or the dorsoventral axis. For example, the dorsal ventricular zone (VZ) of the embryonic spinal cord expresses Pax3 and Pax7, the ventral VZ expresses Pax6, and the more ventral VZ expresses Nkx2.2. Properties of neuronal precursors located in the VZ are determined by the characteristic expression patterns of these transcription factors, leading to the generation of distinct classes of neurons. Recent studies demonstrated that radial glial cells produce neurons in addition to glia during central nervous system development. Thus, neuronal precursor diversity may be dependent upon the diversity of radial glial cells. To investigate this hypothesis, we analyzed the expression of radial glial cell markers and transcription factors in the mouse embryonic spinal cord. We show that radial glial cells indeed express domain-specific transcription factor. Moreover, they varied in expression of the astrocyte-specific glutamate transporter. The region where the astrocyte-specific glutamate transporter is strongly expressed in the ventral radial glial cells is closely related to the Pax6-expressing domain, and the weakly expressing region corresponding to the Nkx2.2-expressing domain. Furthermore, dorsal radial fibers expressed ephrin-B1. Thus, different types of radial glial cells exist in different domains defined by the transcription factor expression at E12.5. We also show that this diversity continues to the gliogenic stage of radial glial cells. This raises the idea that astrocytes generated from different domains along the dorsoventral axis in the mouse spinal cord have distinct characteristics. Copyright (c) 2005 S. Karger AG, Basel.
  • K Kobayashi, M Takahashi, N Matsushita, J Miyazaki, M Koike, H Yaginuma, N Osumi, K Kaibuchi, K Kobayashi
    JOURNAL OF NEUROSCIENCE 24 14 3480 - 3488 2004年04月 [査読有り][通常論文]
     
    A variety of neurons generated during embryonic development survive or undergo programmed cell death (PCD) at later developmental stages. Survival or death of developing neurons is generally considered to depend on trophic support from various target tissues. The small GTPaseRho regulates diverse cellular processes such as cell morphology, cell adhesion, cell motility, and apoptosis. Rho-dependent serine-threonine protein kinase (Rho-kinase-ROK-ROCK), one of the effector proteins, transmits signals for some Rho-mediated processes. Here, we report the in vivo role of the Rho signaling pathway through Rho-kinase during development of motor neurons (MNs) in the spinal cord. We performed conditional expression of a dominant-negative form for RhoA(RhoADN) or for Rho-kinase (Rho-KDN) in transgenic mice by using the Cre-loxP system to suppress the activity of these signaling molecules in developing MNs. Expression of RhoA DN reduced the number of MNs in the spinal cord because of increased apoptosis while preserving the gross patterning of motor axons. Expression of Rho-K DN produced developmental defects similar to those observed in RhoA DN expression. In addition, analysis of transgenic mice expressing Rho-K DNshowed that the increased apoptosis of MNs was induced at the early embryonic stages before the initiation of PCD, and that MN death at the late embryonic stages corresponding to the period of PCD was moderately enhanced in the transgenic mice. These findings indicate that the Rho signaling pathway, primarily through Rho-kinase, plays a crucial role in survival of spinal MNs during embryogenesis, particularly at the early developmental stages.
  • K Kasai, M Takahashi, N Osumi, S Sinnarajah, T Takeo, H Ikeda, JH Kehrl, G Itoh, H Arnheiter
    GENES TO CELLS 9 1 49 - 58 2004年01月 [査読有り][通常論文]
     
    Sonic hedgehog (Shh) is a secreted morphogen crucial for cell fate decision, cellular proliferation, and patterning during vertebrate development. The intracellular Shh signalling is transduced by Smoothened (Smo), a seven-transmembrane spanning protein that belongs to the G-protein coupled receptor family. Among four families of Galpha subunits, Galphai has been thought to be responsible for transducing Shh signalling, while several lines of evidence indicated that other signalling pathways may be involved. We found that the G12 family of heterotrimeric G proteins and the small GTPase RhoA are involved in Shh/Smo-mediated cellular responses, including stimulation of target gene promoter and inhibition of neurite outgrowth of neuroblastoma cells. We also found that the G12/RhoA pathway is responsible for Smo-induced nuclear import of GLI3 which is thought to transduce Shh signals to nucleus. Furthermore, misexpression of a G12-specific GTPase-activating protein in rat neural tubes leads to pertubation of motor neurone and interneurone development, mimicking the effects of decreased Shh signalling. These results show that Shh signalling is mediated in part by activating G12 family coupled signalling pathways. The participation of RhoA, a pivotal molecular switch in many signal transduction pathways, may help explain how Shh can trigger a variety of cellular responses.
  • M Takahashi, K Sato, T Nomura, N Osumi
    DIFFERENTIATION 70 4-5 155 - 162 2002年06月 [査読有り][招待有り]
     
    One of the goals of developmental neuroscience in the post-genomic era is to clarify functions of a huge number of anonymous genes of which only DNA sequences are identified. More convenient methods for genetic manipulation in vertebrates, especially mammals, could help us to identify functions of the novel genes. Here we introduce a novel gene transfer technology using electroporation (EP), which is a simple and powerful strategy for genetic analysis. We have applied this method to cultured mammalian embryos in order to understand the function of specific genes in the developing brain. We have also performed EP in developing fetuses in utero guided by ultrasound image. The combination of these techniques in addition to analysis of genetic mutants will clarify functions of individual genes, gene interactions, and the molecular mechanisms underlying the brain development.
  • M Takahashi, N Osumi
    DEVELOPMENT 129 6 1327 - 1338 2002年03月 [査読有り][通常論文]
     
    Recent studies have shown that generation of different kinds of neurones is controlled by combinatorial actions of homeodomain (HD) proteins expressed in the neuronal progenitors. Pax6 is a HD protein that has previously been shown to be involved in the differentiation of the hindbrain somatic (SM) motoneurones and V1 interneurones in the hindbrain and/or spinal cord. To investigate in greater depth the role of Pax6 in generation of the ventral neurones, we first examined the expression patterns of HD protein genes and subtype-specific neuronal markers in the hindbrain of the Pax6 homozygous mutant rat. We found that Islet2 (SM neurone marker) and En1 (V1 interneurone marker) were transiently expressed in a small number of cells, indicating that Pax6 is not directly required for specification of these neurones. We also observed that domains of all other HD protein genes (Nkx2.2, Nkx6.1, Irx3, Dbx2 and Dbxl) were shifted and their boundaries became blurred. Thus, Pax6 is required for establishment of the progenitor domains of the ventral neurones. Next, we performed Pax6 overexpression experiments by electroporating rat embryos in whole embryo culture. Pax6 overexpression in the wild type decreased expression of Nkx2.2, but ectopically increased expression of Irx3, Dbxl and Dbx2. Moreover, electroporation of Pax6 into the Pax6 mutant hindbrain rescued the development of Islet2-positive and En1-positive neurones. To know reasons for perturbed progenitor domain formation in Pax6 mutant, we examined expression patterns of Shh signalling molecules and states of cell death and cell proliferation. Shh was similarly expressed in the floor plate of the mutant hindbrain, while the expressions of Ptc1, Gli1 and Gli2 were altered only in the progenitor domains for the motoneurones. The position and number of TUNEL-positive cells were unchanged in the Pax6 mutant. Although the proportion of cells that were BrdU-positive slightly increased in the mutant, there was no relationship with specific progenitor domains. Taken together, we conclude that Pax6 regulates specification of the ventral neurone subtypes by establishing the correct progenitor domains.

講演・口頭発表等

  • 疾患マウスモデルを用いた腹壁閉鎖不全の分子細胞機序の解析  [通常講演]
    高橋 将文
    第18回自治医科大学シンポジウム 2019年09月 シンポジウム・ワークショップパネル(公募)
  • Six4/Six5二重遺伝子変異マウスを用いた臍帯ヘルニア発症機序の解析  [通常講演]
    高橋 将文
    第123回日本解剖学会総会・全国学術集会 2018年03月 口頭発表(一般)
  • 歯および歯周組織形成におけるSix1の機能  [通常講演]
    高橋 将文
    文部科学省私立大学戦略的研究基盤形成支援事業シンポジウム2017-非感染性疾患(NCD)の病態解明と診断・治療法の開発拠点の形成 2017年07月
  • The distribution of Cdh20 mRNA demarcates a novel subdomain corresponding to somatotopic representation and brain asymmetry in the dorsolateral striatum.  [通常講演]
    高橋 将文
    12th Nikko International Symposium 2015 Frontier in Translational Neuroscience 2015年11月 シンポジウム・ワークショップパネル(公募)
  • Current State of Tohoku University and Sendai.  [招待講演]
    高橋将文
    Symposium: Japan Earthquake. 2011年03月 シンポジウム・ワークショップパネル(指名)
  • 哺乳類菱脳における境界の維持のメカニズム  [招待講演]
    高橋将文
    シンポジウム「仙台動物発生コロキウム」 2010年01月 シンポジウム・ワークショップパネル(指名)
  • Imaging analysis of the apical structure of neuroepithelial cells: evidence of hindbrain cytonemes.  [通常講演]
    Takahashi, M, Osumi, N
    41st Annual Meeting for the Japanese Society of Developmental Biologists. 2008年05月 シンポジウム・ワークショップパネル(公募)
  • Boundaries in the hindbrain.  [招待講演]
    高橋将文
    Symposium: Developmental biology of compartment and signaling center, Toward innovative research: Lessons from the Kornbergs. 2007年07月 シンポジウム・ワークショップパネル(指名)
  • The Analysis of Domain Formation and Progenitor Cell Behavior in the Mammalian Neural Tube.  [通常講演]
    Takahashi, M, Osumi, N
    The 2 nd Internatinal Symposium on Future Medical Engineering based on Bio-nanotechmology (21st Century COE program), 2003年09月 シンポジウム・ワークショップパネル(公募)
  • The role of Pax6 in specification of neurons in the hindbrain.  [招待講演]
    Takahashi, M, Osumi, N
    日本神経科学大会 2003年07月 シンポジウム・ワークショップパネル(指名)
  • The role of Pax6 in progenitor domains formation in the hindbrain.  [招待講演]
    Takahashi, M, Osumi, N
    International Mini-Symposium: Multiple Roles of Pax6 in Development. 2003年06月 シンポジウム・ワークショップパネル(指名)

MISC

  • Masanori Takahashi, Takako Kikkawa, Noriko Osumi Neuromethods 102 141 -157 2015年 [査読有り][招待有り]
     
    Electroporation has been widely used in various animals to introduce transgenes into their tissues and organs. We have previously developed a gene transfer method into the developing brain of rat and mouse embryos by applying electroporation to a mammalian whole embryo culture technique. We can directly transfer exogenous genes and small nucleic acids, e.g., double strand RNAs, siRNAs, and Morpholino oligos, into desirable regions of the developing brain of cultured embryos at different stages by easily adjusting the direction of electrodes. This method enables us to provide simple and convenient gain-of- function and loss-of-function studies to explore novel understanding of molecular and cellular mechanisms underlying mammalian brain development.
  • 高橋将文 脳科学事典 web 2014年01月 [査読無し][通常論文]
  • 高橋将文 脳科学事典 web 2013年03月 [査読無し][通常論文]
  • 高橋将文 脳科学事典 web 2013年02月 [査読無し][通常論文]
  • Tadashi Nomura, Masanori Takahashi, Noriko Osumi Electroporation and Sonoporation in Developmental Biology 129 -141 2009年 [査読無し][通常論文]
     
    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell--cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use. Whole-embryo mammalian culture system was established by New and colleague in the 1970s, and was modified thereafter by several researchers (reviewed by New, 1971, 1978 Sturm and Tam, 1993 Hogan et al., 1994 Eto and Osumi-Yamashita, 1995 Tam, 1998). At first, the whole-embryo culture system was used in the field of teratology, and then applied in a variety of developmental biology fields, because this system well maintains the embryonic growth and morphogenesis that are comparable to those in utero, and dramatically improves accessibility to the early fetal stages. Furthermore, in combination with gene-delivery techniques such as electroporation, gene expression can be manipulated in a tissue- and region-specific manner (Osumi and Inoue, 2001 Takahashi et al., 2002). Here we introduce this combinatorial proce dure applying of the electroporation technique to whole-embryo culture system. We then illustrate the use of this approach in the developmental neurobiology by present ing our findings on molecular mechanisms controlling stem cell maintenance and neuronal migration in the embryonic cortex. Finally, we will discuss future directions and a potential widespread value of this method in developmental biology. © 2009 Springer Japan.
  • 生命科学を志して-研究する人々-
    高橋将文 Brain & Mind (Crest Osumi Project) 4 11 -11 2006年09月 [査読無し][通常論文]
  • 写真撮影とデータ処理
    高橋将文, 大隅典子 注目のバイオ実験シリーズ「免疫染色&in situ ハイブリダイゼーション最新プロトコール」 165 -197 2006年09月 [査読無し][通常論文]
  • 免疫染色法の実際
    高橋将文, 大隅典子 注目のバイオ実験シリーズ「免疫染色& in situ ハイブリダイゼーション最新プロトコール」 103 -127 2006年09月 [査読無し][通常論文]
  • Yuji Tsunekawa, Masanori Takahashi, Noriko Osumi FUTURE MEDICAL ENGINEERING BASED ON BIONANOTECHNOLOGY, PROCEEDINGS 203 -+ 2006年 [査読無し][通常論文]
     
    To produce precise number of neurons and glial cells from neuroepithelial cells, the progeression and exit of the cell cycle should accurately be coordinated. In mammalian neuroepithelial cells, the molecular and cellular mechanisms coordinating the cell cycle progression and neuronal differentiation is yet unknown. Recently, we noticed that a certain cell cycle regulator protein localized in the basal endfeet of the mouse neuroepithelial cells. However, it is difficult to know in which cell cycle phase the protein localizes, because suitable cell cycle markers expressed at the endfeet of the neuroepithelial cells are missing. To address this issue, we performed sequential labeling of neuroepithelial cells by introducing EGFP-F cDNA into cultured mouse embryos using electroporation and short pulse labeling of S-phase cells by incorporating BrdU, and analyzed the cell cycle phase of EGFP-F labeled cells by using antibodies to BrdU and PH3 (a M-phase marker). We found that EGFP-F-labled cells were in S-phase to G1-phase, but not in G2-phase to M-phase 6 hours after electroporation, and that both of the nucleus and the whole outline of neuroepithelial cell including bipolar processes were clearly visualized. These results suggest that our labeling strategy makes it possible to characterize the cell cycle of neuroepithelial cells, and therefore the cell cycle-dependent distribution of cytoplasmic proteins at the endfeet would be elucidated in the future.
  • Masanori Takahashi, Norjko Osumi FUTURE MEDICAL ENGINEERING BASED ON BIONANOTECHNOLOGY, PROCEEDINGS 211 -+ 2006年 [査読無し][通常論文]
     
    The vertebrate nervous system consists of a huge number of neurons and glial cells. These cell types are generated from neuroepithelial cells at precise positions during development. However, little is known about how kinetics of neuroepithelial cells is regulated, and how the balance of proliferation and differentiation are genetically coordinated. To elucidate cellular mechanisms underlying such complex cell behaviors in the neuroepithelium, we established time-lapse imaging system by applying confocal laser-scanning microscopy for the rat spinal cord slice culture. By electroporation the spinal cord with expression plasmids of fluorescent proteins, nuclear movement of neuroepithelial cells and morphological change on their radial fibers were clearly visualized. Four-dimensional (4-D) time-lapse analyses allowed us to investigate the spatiotemporal dynamics of neuroepithelial cells. These approaches make it possible to analyze functions of genes that regulate cell kinetics and cell morphology.
  • 神経系における細胞増殖・分化の制御
    高橋将文, 大隅典子 発生における細胞増殖制御 77 -85 2004年02月 [査読無し][通常論文]
  • 笠井謙次, 高橋将文, 西巻春明, 益田健史, 竹尾友宏, 楊朝隆, 池田洋, 大隅典子, 伊藤元 生化学 74 (11) 1384 2002年11月 [査読無し][通常論文]
  • 哺乳類胚神経管および胎児脳への遺伝子導入法
    高橋将文, 佐藤健一, 大隅典子 遺伝子医学別冊:図で観る発生・再生医学実験マニュアル 22 -35 2002年07月 [査読無し][通常論文]

受賞

  • 2019年11月 自治医科大学 自治医科大学医学部優秀論文賞
     
    受賞者: 高橋 将文
  • 2019年09月 自治医科大学 JMUシンポジウム講演優秀賞
     
    受賞者: 高橋 将文
  • 2006年05月 東北大学バイオサイエンスシンポジウム ポスター賞
     
    受賞者: 高橋将文
  • 2003年03月 東北大学 総長賞
     
    受賞者: 高橋将文
  • 1997年01月 財団法人三木康楽会 康楽賞
     
    受賞者: 高橋将文

共同研究・競争的資金等の研究課題

  • 腹側閉鎖の細胞機構ーSIX転写因子による腹壁細胞の増殖、移動、上皮間葉転換の制御
    文部科学省:学術研究助成基金助成金(基盤研究 (C))
    研究期間 : 2019年 -2021年 
    代表者 : 川上 潔、高橋将文、佐藤滋
  • 歯形成における細胞動態と歯上皮幹細胞の維持分化を司る制御機構の解明
    文部科学省:学術研究助成基金助成金(基盤研究(C))
    研究期間 : 2019年 -2021年 
    代表者 : 高橋 将文
  • 腹壁閉鎖不全モデルマウスを用いた臍帯ヘルニア発症の分子細胞機序の解明
    母子健康協会:小児医学研究助成
    研究期間 : 2019年12月 -2020年11月 
    代表者 : 高橋 将文
  • 神経堤に由来する歯および歯周組織細胞の分化・再生機構の解明
    徳島大学先端酵素学研究所:共同利用ゲノム編集支援
    研究期間 : 2017年04月 -2018年03月 
    代表者 : 高橋 将文
  • 線条体の機能局在化を制御する分子機構の解明
    文部科学省:学術研究助成基金助成金(基盤研究 (C))
    研究期間 : 2016年 -2018年 
    代表者 : 高橋 将文
  • ヒト疾患モデルマウスを用いた臍帯ヘルニア発症の新規分子機序の解明
    自治医科大学 医学部研究奨励金
    研究期間 : 2013年04月 -2014年03月 
    代表者 : 高橋 将文
  • 文部科学省:学術研究助成基金助成金 (基盤研究(C))
    研究期間 : 2012年 -2014年 
    代表者 : 高橋 将文
  • ラット線条体外側部投射ニューロンに発現するカドヘリンサブ タイプの機能解析
    包括型脳科学研究推進支援ネットワーク:脳機能プロービング研究支援活動 ウイルスベクター
    研究期間 : 2014年 
    代表者 : 高橋 将文
  • ラット線条体腹外側領域投射ニューロンに発現するカドヘリンサブタイプの細胞内局在様式の解明
    包括型脳科学研究推進支援ネットワーク:脳分子プロファイリング開発支援活動 脳機能分子発現解析
    研究期間 : 2013年 
    代表者 : 高橋 将文
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2010年 -2011年 
    代表者 : 高橋 将文
     
    本研究では、Pax6遺伝子変異ラット胚の菱脳境界形成過程を詳細に解析し、Pax6が境界領域においてNotch シグナル関連遺伝子の発現を誘導または維持することで、境界の未分化性維持に寄与している可能性を見いだした。また、ゼブラフィッシュ胚を用いた菱脳境界付近細胞の高解像度タイムラプスイメージング解析により、EphA4/ephrinB3bが境界細胞の移動方向および極性を制御することで、正常な境界が確立されることを見いだした。
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2008年 -2009年 
    代表者 : 高橋 将文
     
    本研究では、ラット線条体側方領域および大脳皮質体性感覚野におけるカドヘリン20遺伝子の左右非対称発現細胞の種類を解析した。線条体のカドヘリン20発現細胞はすべて淡蒼球に投射する間接路および、黒質網様部に投射する直接路の投射ニューロンであることが明らかになった。さらに、ラットにおけるカドヘリン20遺伝子の左優位発現と利き手と関係を明らかにするために、food reachingテスト装置を独自に作製し、成体ラットの利き手の同定に成功した。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2005年 -2009年 
    代表者 : 大隅 典子, 野村 真, 高橋 将文
     
    脳が発生・発達する過程において、「神経幹細胞」というタネのような細胞が分裂して数を増やし(増殖)、神経機能を営むのに特殊化した細胞に変化する(分化)することが必要である。本研究では神経幹細胞の増殖と分化において重要な働きをする分子であるPax6の下で働く分子群を同定し、その機能について解析した。また、神経幹細胞を維持する機構として、細胞周期を制御するCyclin D2という因子の細胞内局在が重要であることを明らかにした。
  • 成体脳における左右非対称性形成の分子基盤の解明
    財団法人 艮陵医学振興会:医学研究助成金(研究B)
    研究期間 : 2007年 -2007年 
    代表者 : 高橋将文
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2005年 -2006年 
    代表者 : 高橋 将文
     
    神経細胞(ニューロン)やグリア細胞を生み出す神経幹細胞として機能する神経上皮細胞は、細胞周期に依存した核運動を行なう極性細胞であるが、細胞周期や核運動を統合する機構や形態を維持する制御機構については未だ不明の点が多い。本研究課題では、Pax6変異ラット胚における神経上皮細胞の挙動のイメージング解析を行ない、神経上皮細胞の細胞周期および運動性の制御におけるPax6遺伝子の役割を明らかにすることを目的としている。18年度は、まず前年度に確立したラット胚脊髄神経管のスライス培養とレーザー顕微鏡を用いた細胞の挙動のイメージング法(Takahashi and Osumi,2007)により、Pax6変異ホモ胚神経上皮細胞の挙動を解析した。ニューロン産生からアストロサイト産生の移行期の神経上皮細胞を蛍光タンパク質により標識し、5分間隔での経時観察を行った。Pax6変異ホモ胚の後期神経上皮細胞では、前年度に野生型胚において解析した神経上皮細胞の細かいスパイク様の構造の数がやや増加しており、神経上皮細胞の微細構造に変化が生じていると考えられた。また前年の解析からPax6変異胚神経上皮細胞内において、分子の輸送や局在化に変化があることが示唆されていたことから、Pax6変異胚神経上皮細胞において、細胞骨格タンパク質および細胞骨格の制御に関わる因子の発現を解析した。Nestinやα-tubulin, Kinesin, β-catenin,δ-cateninの発現様式は野生型と比較して変化は見られなかったが、微小管形成に関与することが報告されているS100βの発現が神経上皮細胞の基底膜側末端で認められなかった。以上のことから、Pax6は神経上皮細胞の形態や細胞内輸送などを何らかの方法で制御しており、増殖および分化における神経上皮細胞の維持に重要な因子であることが示唆された。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2005年 -2005年 
    代表者 : 高橋 将文, 大隅 典子, 沼山 恵子
     
    脊推動物の神経発生においては、ペアードドメインとホメオドメインを持つ転写因子Pax6が前脳・菱脳・脊髄の神経上皮細胞で領域特異的に発現している。自然発症のPax6変異動物の表現型から、Pax6は非常に多岐に渡る神経系の発生事象に関わることが知られており、様々な標的遺伝子の発現とその下流の遺伝子カスケードを統率し、脳の発生を司ると予想されるが、中枢神経系における標的遺伝子はほとんど明らかになっていない。そこで我々は、神経発生におけるPax6標的遺伝子と下流遺伝子ネットワークを解明するため、野生型とPax6ホモ変異型ラットの初期脳における網羅的な遺伝子発現比較解析を行った。ラット11.5日胚の菱脳及び終脳の領域よりRNAを抽出し、約30,000転写産物に対応するプローブセットを搭載した高密度オリゴヌクレオチドアレイGeneChip(Affymetrix)を用いて得られた遺伝子発現プロファイルを比較し、Pax6の下流遺伝子群を同定した。さらに、リアルタイム定量PCRにより発現量の比を検証するとともに、in situハイブリダイゼーションで発現様式の確認を行い、Pax6標的候補としてFabp7,Unc5h1,Cyp26b1,Dmrt4などの遺伝子を同定した。Pax6変異胚で最も顕著な発現減少を示した遺伝子は脂肪酸結合タンパク質Fabp7であった。電気穿孔法と全胚培養系を組み合わせた過剰発現による機能獲得実験や野生型胚でのRNA干渉による機能欠失実験などの機能解析を行い、Fabp7遺伝子が転写因子Pax6の標的候補であり、神経新生に重要な役割を担うことをJ. Neurosci.誌に発表した。また、LacZレポータートランスジェニックラットの作製により、Fabp7遺伝子発現における領域特異的エンハンサーが転写開始点上流5.7kb以内に存在することを見いだした。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2004年 -2004年 
    代表者 : 大隅 典子, 高橋 将文
     
    野生型およびPax6ホモ変異型ラット胚(胎齢11.5日)各40匹から菱脳領域を切り出してpoly(A)^+RNAを調整し、逆転写酵素反応とDNA合成酵素反応により得られた二本鎖cDNAを鋳型にin vitro転写反応でビオチン標識cRNAを合成し、数10〜200塩基程度に断片化してターゲットとして用いた。ほぼ全ての遺伝子を網羅する約30,000の転写産物に対するプローブセットが載ったGeneChip Rat Expression Set 230 (Affymetrix)とハイブリダイズ後、SAPE (Streptavidin Phycoerythrin)染色を行ってその蛍光をスキャンし、各プローブセットの検出シグナルをMicroarray Suiteで演算処理し遺伝子発現シグナル値を求めた。誤差を収束させるために、RNA調整から4回実験を繰り返した。得られたシグナル値を解析ソフトウェアGeneSpring上でチップ毎のメジアンを1に規準化してから平均を取り、野生型とPax6変異胚での比較解析を行った。シグナル値に大きな増減を示した遺伝子群についてはリアルタイム定量PCRにより発現量の比を検証したが、非常に相関が良く、この方法の有効性が確信された。最も顕著な発現変動を示した遺伝子は脂肪酸結合タンパク質Fabp7で1/30以下にまで発現が減少していた。Unc5h1やCyp26b1遺伝子などでもPax6変異体での著しい発現減少を明らかにした。Fabp7については、発現様式の確認、電気穿孔法と全胚培養系を組み合わせた野生型胚でのRNA干渉による機能欠失実験や過剰発現による機能獲得実験などの機能解析から、Fabp7遺伝子が転写因子Pax6の標的候補であり、神経新生に極めて重要な役割を担うことを明らかにした(論文投稿中)。また、胎齢11.5日の終脳領域についても同様に2回の実験を行い、遺伝子発現比較解析を行った。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2004年 -2004年 
    代表者 : 大隅 典子, 高橋 将文
     
    1)神経上皮細胞の挙動のイメージング解析神経上皮細胞は細胞周期に伴い非常にダイナミックな動態を示す。すなわち、基底膜側と脳室側を結ぶ細長い細胞質を細胞核がエレベーターのように往復し、先端側すなわち神経管の内側に到達したときに分裂期に入り、そこで2つの娘細胞に分裂する。神経上皮細胞の核はこのエレベーター運動を繰り返しながら、幹細胞としての性質を保持すると同時に、より分化した神経前駆細胞や神経細胞を生じる。そこで細胞分裂と分化の様相をタイムラプス観察するためのシステムを、蛍光顕微鏡および2光子顕微鏡を用いて確立した。このシステムを用いて菱脳および脊髄神経上皮細胞の挙動を観察した結果、神経上皮細胞から微細な突起が出たり引っ込んだりしているという様相が初めて認められた。また、終脳神経上皮細胞を観察した結果、Pax6変異ラットではエレベーター運動の異常と中心体の挙動の異常が見られた。2)神経幹細胞の増殖と分化を統合する分子機構の網羅的解析野生型脳とPax6遺伝子変異動物脳で発現する遺伝子のプロファイルをマイクロアレイ法を用いて比較し、定量RT-PCRおよびin situハイブリダイゼーション法により発現様式を確認することにより、Pax6の下流遺伝子として脂肪酸結合タンパク質をコードするFabp7を見いだした。電気穿孔法による遺伝子導入を行い、RNA干渉法(siRNA法)によるFABP7の機能阻害を行ったところ、神経上皮の形態が異常になり、細胞増殖の減少とニューロン分化の亢進が認められた。したがって、Pax6はFABP7を介し、神経上皮細胞の性質の維持に深く関わることが示された。
  • 転写因子Pax6が制御する菱脳腹側神経上皮の区画化と ニューロンサブタイプの決定
    菅原医学財団:
    研究期間 : 2003年 
    代表者 : 高橋 将文

委員歴

  • 2020年01月 - 現在   国際歯科研究学会及び米国歯科研究学会   Journal of Dental Research, Editorial Board

担当経験のある科目

  • 生物学実習(医学部1年次)自治医科大学
  • 大学院カリキュラム講義II (脳の発生・発達期における領域化のメカニズム)(修士・博士課程)自治医科大学大学院医学研究科
  • 人体構造学 (中枢神経系の発生と機能、脳の構築と神経幹細胞)(修士課程)自治医科大学大学院医学研究科
  • 人間生物学系2 (脳の発生とヒト脳の進化)(博士課程)自治医科大学大学院医学研究科
  • 基礎ゼミナール(共通教育科目 1年次)東北大学
  • 医療工学技術者創生のための再教育システム(分子生物学および細胞生物学実習講師)東北大学 REDEEM プロジェクト
  • 基礎チュートリアル教育(医学部3年次)東北大学
  • 脳解剖実習(医学部2年次)東北大学
  • 発生学(医学部2年次、歯学部2年次、鳥類胚実習を含む)東北大学
  • 細胞生物学(医学部1年次)東北大学
  • 神経解剖学実習(医学部2年次)自治医科大学
  • 発生生物学入門(医学部1年次)自治医科大学
  • 細胞生物学 (医学部1年次)自治医科大学


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