Updated on 2026/03/10

写真a

 
IKEDA KEIKO
 
Organization
Institute of Integrated Research Cell Biology Center Visiting Associate Professor (Lecturer)
Title
Visiting Associate Professor (Lecturer)
External link

Degree

  • Doctor ( Jichi Medical University )

Research Interests

  • 脳神経節

  • 転写制御

  • 包括脳ネットワーク

  • 嗅上皮発生

  • パイオニアニューロン

  • K-ATPase

  • Na

  • Neuro

  • Chromatin

  • Gene Transcriptional Regulation

Research Areas

  • Life Science / Molecular biology

  • Life Science / Neuroscience-general

  • Life Science / Clinical pharmacy

  • Life Science / Physiology

  • Life Science / Anatomy

  • Life Science / Developmental biology

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Education

  • Jichi Medical University   Graduate School, Division of Medicine

    - 1994

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  • Jichi Medical University

    - 1994

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    Country： Japan

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  • Jichi Medical University   Faculty of Medicine

    - 1987

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  • Jichi Medical University

    - 1987

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    Country： Japan

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Research History

  • Tokyo Institute of Technology   Institute of Innovative Research   Visiting Associate Professor (Lecturer)

    2021.4

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  • Kagawa Nutrition University   Nutritional Science Institute   Visiting Professor

    2019.5

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  • Showa University   School of Dentistry

    2019.4

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  • Jichi Medical University

    2018.6 - 2021.3

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  • International University of Health and Welfare   School of Medicine   Professor

    2017.4 - 2020.3

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  • Hyogo College of Medicine   Faculty of Medicine

    2011.4 - 2017.3

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  • National Institutes of Natural Sciences

    2010.7 - 2011.3

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  • Jichi Medical University

    2003

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  • Jichi Medical University

    1996 - 2003

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  • Jichi Medical School, Research Assistant

    1994 - 1996

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  • Jichi Medical University

    1994 - 1996

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Professional Memberships

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Committee Memberships

  •   - 自治医科大学 医学部 学生副部長  

    2008   

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Papers

  • Intrinsic responses to hypoxia and hypercapnia of neurons in the cardiorespiratory center of the ventral medulla of newborn rats. International journal

    Hiroshi Onimaru, Yui Koyanagi, Kamon Iigaya, Keiko Ikeda, Masahiko Izumizaki

    Pflugers Archiv : European journal of physiology   477 ( 5 )   685 - 705   2025.5

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    Language：English   Publishing type：Research paper (scientific journal)  

    The rostral ventrolateral medulla (RVLM) includes a variety of neurons essential for cardiorespiratory control. Although some of these neurons are thought to be intrinsically sensitive to hypercapnia and/or hypoxia, relationships between types of neurons and responses to hypoxia and/or hypercapnia are not well understood. Tyrosine hydroxylase (TH) is one of the cell-type markers of the RVLM neurons. Here, we report effects of hypoxia and hypercapnia on TH-positive or -negative neurons in the RVLM of newborn rats. Brainstem-spinal cord preparations were isolated from 0-3-day-old Wistar rats and superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4 at 25-26 °C. Membrane potential responses to hypoxia (95% → 0% O2) and/or hypercapnia (2% → 8% CO2) were examined in the presence of tetrodotoxin (TTX) after identification of the firing pattern. We found that TH-positive C1 neurons in the RVLM were sensitive to hypoxia with membrane depolarization but less sensitive to hypercapnia. TH-negative neurons in the C1 area showed responses similar to those of C1 neurons. Moreover, C1 area neurons remained depolarized by hypoxia in the presence of TTX plus gliotransmitter blockers. In contrast, Phox2b-positive and TH-negative neurons in the parafacial respiratory group were intrinsically sensitive to CO2 but not sensitive to hypoxia. Respiratory-related neurons (Phox2b and TH negative) showed a variable response to hypoxia: unchanging, depolarizing, or hyperpolarizing. Our findings suggest that C1 area neurons in the RVLM are intrinsically sensitive to hypoxia and belong to one of the elements constituting central hypoxic sensors.

    DOI： 10.1007/s00424-025-03077-5

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  • Cellular mechanisms of synchronized rhythmic burst generation in the ventromedial hypothalamus

    Kamon Iigaya, Hiroshi Onimaru, Keiko Ikeda, Makito Iizuka, Masahiko Izumizaki

    Pflügers Archiv - European Journal of Physiology   2024.10

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    Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

    DOI： 10.1007/s00424-024-03031-x

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    Other Link： https://link.springer.com/article/10.1007/s00424-024-03031-x/fulltext.html

  • Cell Responses of the Ventrolateral Medulla to PAR1 Activation and Changes in Respiratory Rhythm in Newborn Rat En Bloc Brainstem-Spinal Cord Preparations

    Hiroshi Onimaru, Isato Fukushi, Keiko Ikeda, Itaru Yazawa, Kotaro Takeda, Yasumasa Okada, Masahiko Izumizaki

    Neuroscience   528   89 - 101   2023.9

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    Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

    DOI： 10.1016/j.neuroscience.2023.08.002

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  • Activation of Phox2B-positive neurons in the dorsal medulla induced sucking and hiccup(タイトル和訳中)

    Iizuka Makito, Ikeda Keiko, Igarashi Hiroyuki, Kobayashi Kazuto, Onimaru Hiroshi, Izumizaki Masahiko

    The Journal of Physiological Sciences   73 ( Suppl.1 )   113 - 113   2023.5

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    Language：English   Publisher：(一社)日本生理学会  

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  • Activation of astrocytes in the ventrolateral medulla via PAR1 and modulation of respiratory rhythm in newborn rat brainstem-spinal cord preparations(タイトル和訳中)

    Onimaru Hiroshi, Fukushi Isato, Ikeda Keiko, Yazawa Itaru, Takeda Kotaro, Okada Yasumasa, Izumizaki Masahiko

    The Journal of Physiological Sciences   73 ( Suppl.1 )   109 - 109   2023.5

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  • Minocycline prevents hypoxia-induced seizures Reviewed International coauthorship International journal

    Isato Fukushi, Keiko Ikeda, Kotaro Takeda, Masashi Yoshizawa, Yosuke Kono, Yohei Hasebe, Mieczyslaw Pokorski, Yasumasa Okada

    Frontiers in Neural Circuits   17   1006424   2023.3

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    Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

    Severe hypoxia induces seizures, which reduces ventilation and worsens the ictal state. It is a health threat to patients, particularly those with underlying hypoxic respiratory pathologies, which may be conducive to a sudden unexpected death in epilepsy (SUDEP). Recent studies provide evidence that brain microglia are involved with both respiratory and ictal processes. Here, we investigated the hypothesis that microglia could interact with hypoxia-induced seizures. To this end, we recorded electroencephalogram (EEG) and acute ventilatory responses to hypoxia (5% O2 in N2) in conscious, spontaneously breathing adult mice. We compared control vehicle pre-treated animals with those pre-treated with minocycline, an inhibitory modulator of microglial activation. First, we histologically confirmed that hypoxia activates microglia and that pre-treatment with minocycline blocks hypoxia-induced microglial activation. Then, we analyzed the effects of minocycline pre-treatment on ventilatory responses to hypoxia by plethysmography. Minocycline alone failed to affect respiratory variables in room air or the initial respiratory augmentation in hypoxia. The comparative results showed that hypoxia caused seizures, which were accompanied by the late phase ventilatory suppression in all but one minocycline pre-treated mouse. Compared to the vehicle pre-treated, the minocycline pre-treated mice showed a delayed occurrence of seizures. Further, minocycline pre-treated mice tended to resist post-ictal respiratory arrest. These results suggest that microglia are conducive to seizure activity in severe hypoxia. Thus, inhibition of microglial activation may help suppress or prevent hypoxia-induced ictal episodes.

    DOI： 10.3389/fncir.2023.1006424

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  • Effects of PAR1 activation on respiratory rhythm generation in the ventrolateral medulla of newborn rats(タイトル和訳中)

    Onimaru Hiroshi, Fukushi Isao, Ikeda Keiko, Yazawa Itaru, Takeda Kotaro, Okada Yasumasa, Izumizaki Masahiko

    The Journal of Physiological Sciences   72 ( Suppl.1 )   118 - 118   2022.12

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    Language：English   Publisher：(一社)日本生理学会  

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  • Intrinsic properties and synaptic connectivity of Phox2b-expressing neurons in rat rostral parvocellular reticular formation. International journal

    Risa Kajiwara, Shiro Nakamura, Keiko Ikeda, Hiroshi Onimaru, Atsushi Yoshida, Yumi Tsutsumi, Kiyomi Nakayama, Ayako Mochizuki, Masanori Dantsuji, Akiko Nishimura, Satoshi Tachikawa, Takehiko Iijima, Tomio Inoue

    Neuroscience research   178   41 - 51   2022.5

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    Language：English   Publishing type：Research paper (scientific journal)  

    The paired-like homeobox 2b gene (Phox2b) is critical for the development of the autonomic nervous system. We have previously demonstrated the distinct characteristics of Phox2b-expressing (Phox2b+) neurons in the reticular formation dorsal to the trigeminal motor nucleus (RdV), which are likely related to jaw movement regulation. In this study, we focused on Phox2b+ neurons in the rostral parvocellular reticular formation (rPCRt), a critical region for controlling orofacial functions, using 2-11-day-old Phox2b-EYFP rats. Most Phox2b+ rPCRt neurons were glutamatergic, but not GABAergic or glycinergic. Approximately 65 % of Phox2b+ rPCRt neurons fired at a low frequency, and approximately 24 % of Phox2b+ rPCRt neurons fired spontaneously, as opposed to Phox2b+ RdV neurons. Stimulation of the RdV evoked inward postsynaptic currents in more than 50 % of Phox2b+ rPCRt neurons, while only one Phox2b+ rPCRt neuron responded to stimulation of the nucleus of the solitary tract. Five of the 10 Phox2b+ neurons sent their axons that ramified within the trigeminal motor nucleus (MoV). Of these, the axons of the two neurons terminated within both the MoV and rPCRt. Our findings suggest that Phox2b+ rPCRt neurons have distinct electrophysiological and synaptic properties that may be involved in the motor control of feeding behavior.

    DOI： 10.1016/j.neures.2021.12.009

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  • Novel oxygen sensing mechanism in the spinal cord involved in cardiorespiratory responses to hypoxia. International journal

    Nicole O Barioni, Fatemeh Derakhshan, Luana Tenorio Lopes, Hiroshi Onimaru, Arijit Roy, Fiona McDonald, Erika Scheibli, Mufaddal I Baghdadwala, Negar Heidari, Manisha Bharadia, Keiko Ikeda, Itaru Yazawa, Yasumasa Okada, Michael B Harris, Mathias Dutschmann, Richard J A Wilson

    Science advances   8 ( 12 )   eabm1444   2022.3

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    Language：English   Publishing type：Research paper (scientific journal)  

    As blood oxygenation decreases (hypoxemia), mammals mount cardiorespiratory responses, increasing oxygen to vital organs. The carotid bodies are the primary oxygen chemoreceptors for breathing, but sympathetic-mediated cardiovascular responses to hypoxia persist in their absence, suggesting additional high-fidelity oxygen sensors. We show that spinal thoracic sympathetic preganglionic neurons are excited by hypoxia and silenced by hyperoxia, independent of surrounding astrocytes. These spinal oxygen sensors (SOS) enhance sympatho-respiratory activity induced by CNS asphyxia-like stimuli, suggesting they bestow a life-or-death advantage. Our data suggest the SOS use a mechanism involving neuronal nitric oxide synthase 1 (NOS1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We propose NOS1 serves as an oxygen-dependent sink for NADPH in hyperoxia. In hypoxia, NADPH catabolism by NOS1 decreases, increasing availability of NADPH to NOX and launching reactive oxygen species-dependent processes, including transient receptor potential channel activation. Equipped with this mechanism, SOS are likely broadly important for physiological regulation in chronic disease, spinal cord injury, and cardiorespiratory crisis.

    DOI： 10.1126/sciadv.abm1444

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  • Role of microglia in blood pressure and respiratory responses to acute hypoxic exposure in rats Reviewed

    Masashi Yoshizawa, Isato Fukushi, Kotaro Takeda, Yosuke Kono, Yohei Hasebe, Keiichi Koizumi, Keiko Ikeda, Mieczyslaw Pokorski, Takako Toda, Yasumasa Okada

    The Journal of Physiological Sciences   72 ( 1 )   2022

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    Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

    Abstract

    Microglia modulate cardiorespiratory activities during chronic hypoxia. It has not been clarified whether microglia are involved in the cardiorespiratory responses to acute hypoxia. Here we investigated this issue by comparing cardiorespiratory responses to two levels of acute hypoxia (13% O2 for 4 min and 7% O2 for 5 min) in conscious unrestrained rats before and after systemic injection of minocycline (MINO), an inhibitor of microglia activation. MINO increased blood pressure but not lung ventilation in the control normoxic condition. Acute hypoxia stimulated cardiorespiratory responses in MINO-untreated rats. MINO failed to significantly affect the magnitude of hypoxia-induced blood pressure elevation. In contrast, MINO tended to suppress the ventilatory responses to hypoxia. We conclude that microglia differentially affect cardiorespiratory regulation depending on the level of blood oxygenation. Microglia suppressively contribute to blood pressure regulation in normoxia but help maintain ventilatory augmentation in hypoxia, which underscores the dichotomy of central regulatory pathways for both systems.

    DOI： 10.1186/s12576-022-00848-y

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    Other Link： https://link.springer.com/article/10.1186/s12576-022-00848-y/fulltext.html

  • 呼吸リズムにおける脳性ナトリウム利尿ペプチドとデクスメデトミジンの作用

    香月 姿乃, 鬼丸 洋, 池田 啓子, 土肥 謙二, 泉崎 雅彦

    日本救急医学会雑誌   32 ( 12 )   1743 - 1743   2021.11

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    Language：Japanese   Publisher：(一社)日本救急医学会  

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  • Effects of acetylcholine on hypoglossal and C4 nerve activity in brainstem-spinal cord preparations from newborn rat. International journal

    Shino Katsuki, Keiko Ikeda, Hiroshi Onimaru, Kenji Dohi, Masahiko Izumizaki

    Respiratory physiology & neurobiology   293   103737 - 103737   2021.11

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    Effects of acetylcholine (ACh) on respiratory activity have been an intriguing theme especially in relation to central chemoreception and the control of hypoglossal nerve activity. We studied the effects of ACh on hypoglossal and phrenic (C4) nerve activities and inspiratory and pre-inspiratory neurons in the rostral ventrolateral medulla in brainstem-spinal cord preparations from newborn rats. ACh application increased respiratory rhythm, decreased inspiratory hypoglossal and C4 nerve burst amplitude, and enhanced pre-inspiratory hypoglossal activity. ACh induced membrane depolarization of pre-inspiratory neurons that might be involved in facilitation of respiratory rhythm by ACh. Effects of ACh on hypoglossal and C4 nerve activity were partially reversed by a nicotinic receptor blocker, mecamylamine. Further application of a muscarinic receptor antagonist, oxybutynin, resulted in slight increase of hypoglossal (but not C4) burst amplitude. Thus, ACh induced different effects on hypoglossal and C4 nerve activity in the brainstem-spinal cord preparation.

    DOI： 10.1016/j.resp.2021.103737

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  • 孤束核のPhox2b陽性ニューロンは吸啜リズムの発現に必須である(Phox2b-positive neurons located in the solitary nucleus is essential to trigger sucking)

    Iizuka Makito, Ikeda Keiko, Igarashi Hiroyuki, Kobayashi Kazuto, Onimaru Hiroshi, Izumizaki Masahiko

    The Journal of Physiological Sciences   71 ( Suppl.1 )   165 - 165   2021.8

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  • Decreased content of ascorbic acid (Vitamin C) in the brain of knockout mouse models of Na+,K+-ATPase-related neurologic disorders

    Keiko Ikeda, Adriana A. Tienda, Fiona E. Harrison, Kiyoshi Kawakami

    PLoS ONE   16 ( 2 )   2021.2

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    Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

    DOI： 10.1371/journal.pone.0246678

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  • Optogenetic Approach to Local Neuron Network Analysis of the Medullary Respiratory Center. International journal

    Hiroshi Onimaru, Keiko Ikeda

    Advances in experimental medicine and biology   1293   449 - 458   2021

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    Using an optogenetic approach, we analyzed a local neuron network of the respiratory center in the medulla of a brainstem-spinal cord preparation isolated from neonatal rat. We developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch) or one of the step-function channelrhodopsin variants (ChRFR) under the control of Phox2b promoter-enhancer regions. Then, in en bloc preparations from 0- to 2-day-old Tg neonatal rats, we analyzed membrane potential changes of medullary respiratory-related neurons in response to photostimulation of the rostral ventral medulla. The photostimulation-induced inhibition or facilitation of the respiratory rhythm in Arch-expressing or ChRFR-expressing Tg rat preparations, respectively. Selective photoactivation of Phox2b-positive neurons expressing ChRFR in the rostral ventrolateral medulla of a neonatal rat en bloc preparation induced membrane potential changes of respiratory-related neurons that were dependent on heterogeneous properties of synaptic connections in the respiratory center. We concluded that the optogenetic approach is a powerful method of verifying a hypothetical model of local networks among respiratory-related neurons in the rostral ventrolateral medulla of neonatal rat.

    DOI： 10.1007/978-981-15-8763-4_29

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  • Characteristics of cortical spreading depression and c-Fos expression in transgenic mice having a mutation associated with familial hemiplegic migraine 2. International journal

    Chunhua Tang, Miyuki Unekawa, Mamoru Shibata, Yutaka Tomita, Yoshikane Izawa, Hiroki Sugimoto, Keiko Ikeda, Kiyoshi Kawakami, Norihiro Suzuki, Jin Nakahara

    Cephalalgia : an international journal of headache   40 ( 11 )   1177 - 1190   2020.10

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    BACKGROUND: Cortical spreading depression is thought to be the underlying mechanism of migraine aura. In 2006, three relatives having the point mutation E700K in ATP1A2 exon 15 were diagnosed with familial hemiplegic migraine 2 characterized by complicated forms of aura. Here, we generated a transgenic mouse model having the human E700K mutation in the Atp1a2 orthologous gene. OBJECTIVE: To investigate the characteristics of cortical spreading depression in a mouse model with E700K mutation in the Atp1a2. METHODS: Cortical spreading depression was induced by applying stepwise increases of KCl concentration or electrical stimulation intensity to C57BL/6J-Tg(Atp1a2*E700K)9151Kwk mice (Tg, both sexes) and corresponding wild-type animals. Under urethane anesthesia, the responsiveness and threshold to cortical spreading depression were examined and the distribution of c-Fos expression, a neuronal activity marker, was immunohistochemically determined. RESULTS: Overall, Tg mice showed significantly faster propagation velocity (p < 0.01) and longer full-width-at-half-maximum (p < 0.01) than wild-type animals, representing a slower recovery from direct current potential deflection. The cortical spreading depression threshold tended to be lower in Tg, especially in females. c-Fos-positive cells were significantly enhanced in the ipsilateral somatosensory cortex, piriform cortex, amygdala and striatum (each p < 0.05 vs. contralateral side). Numbers of c-Fos positive cells were significantly higher in the ipsilateral amygdala of Tg, as compared with wild-type animals (p < 0.01). CONCLUSION: The effect of cortical spreading depression may be greater in E700K transgenic mice than that in wild-type animals, while the threshold for cortical spreading depression shows little change. Higher c-Fos expression in the amygdala may indicate alterations of the limbic system in Tg, suggesting an enhanced linkage between cortical spreading depression and amygdala connectivity in familial hemiplegic migraine 2 patients.

    DOI： 10.1177/0333102420929028

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  • Optogenetic Analysis of Respiratory Neuronal Networks in the Ventral Medulla of Neonatal Rats Producing Channelrhodopsin in Phox2b-positive Cells Reviewed

    Keiko Ikeda, Hiroyuki Igarashi, Hiromu Yawo, Kazuto Kobayashi, Satoru Arata, Kiyoshi Kawakami, Masahiko Izumizaki, Hiroshi Onimaru

    Pflugers Arch   471 ( 11-12 )   2019.12

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  • 家族性片麻痺性片頭痛2型のマウスモデルにおける皮質拡延性抑制の影響(Effects of cortical spreading depression in a mouse model of familial hemiplegic migraine 2)

    唐 春花, 畝川 美悠紀, 柴田 護, 冨田 裕, 伊澤 良兼, 杉本 大樹, 池田 啓子, 川上 潔, 鈴木 則宏, 中原 仁

    脳循環代謝   31 ( 1 )   110 - 110   2019.11

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  • Different impacts on brain function depending on the mode of delivery Reviewed

    Keiko Ikeda, Hiroshi Onimaru, Tohru Matsuura, Kiyoshi Kawakami

    Brain Research   1720   146289 - 146289   2019.10

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    Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

    DOI： 10.1016/j.brainres.2019.06.008

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  • Responses to hypercapnia and hypoxia of neurons in the cardio-respiratory center of the ventral medulla of newborn rats

    Hiroshi Onimaru, Keiko Ikeda, Hiroyuki Igarashi, Hiromu Yawo, Kazuto Kobayashi, Satoru Arata, Kiyoshi Kawakami, Masahiko Izumizaki

    IBRO Reports   6   S409 - S409   2019.9

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    Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

    DOI： 10.1016/j.ibror.2019.07.1303

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  • Structural and functional defects of the respiratory neural system in the medulla and spinal cord of Pax6 mutant rats. Reviewed International journal

    Ikeda K, Onimaru H, Inada H, Tien Lin S, Arata S, Osumi N

    Brain research bulletin   152   107 - 116   2019.7

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    Pax6 is an important transcription factor expressed in several discrete domains of the developing central nervous system. It has been reported that Pax6 is involved in the specification of subtypes of hindbrain motor neurons. Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth, probably due to impaired respiratory movement. To determine whether the respiratory center in the medulla functions normally, we analyzed the histological and neurophysiological properties of the medulla and spinal cord in fetal rats with this mutation. First, the medulla of rSey2/rSey2 at embryonic (E) 21.5-E22.5 tended to be smaller than those from heterozygous mutant (rSey2/+) and wild-type (+/+) littermates. Through immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the parafacial respiratory group (pFRG) of rSey2/rSey2 rats. Expression of neurokinin-1 receptor (NK-1R) was weak and dispersed in rSey2/rSey2 rats. In addition, rSey2/rSey2 rats have a defect of the hypoglossal nerve root. Electrophysiological analysis using brainstem-spinal cord preparations (E21.5-E22.5) revealed that rSey2/rSey2 rats showed larger fluctuation of the amplitude of inspiratory activity monitored from the fourth cervical root although there was no significant difference in the respiratory rate among rSey2/rSey2, rSey2/+, and +/+ littermates. The response of respiratory rhythm to high CO2 was similar among all genotypes. Optical recordings of neuronal activity revealed that the activity of the pFRG tended to be weaker and inspiratory activity appeared in more scattered areas in the caudal ventral medulla in the rSey2/rSey2 rats. These results suggest that the basal activity of the respiratory system was preserved with mild impairment of the inspiratory activity in the rSey2/rSey2 rats and that the Pax6 gene is involved in the functional development of the neuronal system producing effective inspiratory motor outputs for survival.

    DOI： 10.1016/j.brainresbull.2019.07.007

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  • Analysis of the neuronal network of the medullary respiratory center in transgenic rats expressing archaerhodopsin-3 in Phox2b-expressing cells. Reviewed International journal

    Ikeda K, Kaneko R, Yanagawa Y, Ogawa M, Kobayashi K, Arata S, Kawakami K, Onimaru H

    Brain research bulletin   144   39 - 45   2019.1

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    Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) comprise one of the respiratory rhythm generators in the medulla of the neonatal rat. A subgroup of pFRG/Pre-I neurons expresses the transcription factor Phox2b. To further analyze detailed neuronal mechanisms of respiratory rhythm generation in the neonatal rat, we developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch). Brainstem-spinal cord preparations were isolated from 0-2-day-old Tg newborn rats and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25-26 °C. Inspiratory fourth cervical ventral root (C4) activity was monitored, and membrane potentials of neurons in the pFRG including Pre-I and inspiratory neurons were recorded. Phox2b-positive cells in the Tg rats were essentially positive for enhanced green fluorescent protein (EGFP) signals (reporter for Arch) in the pFRG. Continuous photo-stimulation of the rostral ventral medulla for up to 90 s by covering the pFRG with green laser light (532 nm) induced a decrease of respiratory rate measured at C4 accompanied by membrane hyperpolarization of Phox2b-positive pFRG/Pre-I neurons. In contrast, Phox2b-negative inspiratory neurons were not hyperpolarized during the photo-stimulation. Our findings showed that Phox2b-expressing pFRG/Pre-I neurons are involved in the maintenance of the basic respiratory rhythm in neonatal rat.

    DOI： 10.1016/j.brainresbull.2018.11.005

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  • Targeted expression of step-function opsins in transgenic rats for optogenetic studies Reviewed

    Hiroyuki Igarashi, Keiko Ikeda, Hiroshi Onimaru, Ryosuke Kaneko, Kyo Koizumi, Kaoru Beppu, Kayo Nishizawa, Yukari Takahashi, Fusao Kato, Ko Matsui, Kazuto Kobayashi, Yuchio Yanagawa, Shin-Ichi Muramatsu, Toru Ishizuka, Hiromu Yawo

    Scientific Reports   8 ( 1 )   5435   2018.12

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    Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

    DOI： 10.1038/s41598-018-23810-8

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  • Expressions of VGLUT1/2 in the inspiratory interneurons and GAD65/67 in the inspiratory Renshaw cells in the neonatal rat upper thoracic spinal cord. Reviewed

    Iizuka M, Ikeda K, Onimaru H, Izumizaki M

    IBRO reports   5   24 - 32   2018.12

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  • Na+,K+-ATPase α2サブユニット欠損マウスにおける大脳皮質拡延性抑制後の病態特性(Property of behavior after cortical spreading depression in Na+, K+-ATPase α2 subunit defective mice)

    唐 春花, 畝川 美悠紀, 柴田 護, 冨田 裕, 伊澤 良兼, 池田 啓子, 川上 潔, 鈴木 則宏, 中原 仁

    脳循環代謝   30 ( 1 )   142 - 142   2018.10

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  • The Expression of Galanin in the Parafacial Respiratory Group and its Effects on Respiration in Neonatal Rats Reviewed

    Tara G. Bautista, Angelina Y. Fong, Paul M. Pilowsky, Keiko Ikeda, Kiyoshi Kawakami, Darko Spirovski, Hiroshi Onimaru

    Neuroscience   384   1 - 13   2018.8

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    Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

    DOI： 10.1016/j.neuroscience.2018.05.009

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  • Enhanced susceptibility to cortical spreading depression in two types of Na+,K+-ATPase α2 subunit-deficient mice as a model of familial hemiplegic migraine 2. Reviewed International journal

    Unekawa M, Ikeda K, Tomita Y, Kawakami K, Suzuki N

    Cephalalgia : an international journal of headache   38 ( 9 )   1515 - 1524   2018.8

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    Background Patients with familial hemiplegic migraine type 2 (FHM2) have a mutated ATP1A2 gene (encoding Na+,K+-ATPase α2 subunit) and show prolonged migraine aura. Cortical spreading depression (CSD), which involves mass depolarization of neurons and astrocytes that propagates slowly through the gray matter, is profoundly related to aura. Methods In two types of Atp1a2-defective heterozygous mice, Atp1a2tm1Kwk (C-KO) and Atp1a2tm2Kwk (N-KO), the sensitivity and responsiveness to CSD were examined under urethane anesthesia. Results In both cases, heterozygotes exhibited a low threshold for induction of CSD, faster propagation rate, slower recovery from DC deflection, and profound suppression of the electroencephalogram, compared to wild-type mice. A high dose of KCl elicited repeated CSDs for a longer period, with a tendency for a greater frequency of CSD occurrence in heterozygotes. The difference of every endpoint was slightly greater in N-KO than C-KO. Change of regional cerebral blood flow in response to CSD showed no significant difference. Conclusion Heterozygotes of Atp1a2-defective mice simulating FHM2 demonstrated high susceptibility to CSD rather than cortical vasoreactivity, and these effects may differ depending upon the knockout strategy for the gene disruption. These results suggest that patients with FHM2 may exhibit high susceptibility to CSD, resulting in migraine.

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  • Confocal calcium imaging analysis of respiratory-related burst activity in the parafacial region Reviewed

    Hiroshi Onimaru, Shiro Nakamura, Keiko Ikeda, Kiyoshi Kawakami, Tomio Inoue

    Brain Research Bulletin   139   16 - 20   2018.5

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    DOI： 10.1016/j.brainresbull.2018.01.013

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  • Atp1a3 deficient heterozygous mice show lower rank in the hierarchy and altered social behavior. Reviewed

    Sugimoto H, Ikeda K, Kawakami K

    Genes, brain, and behavior   17 ( 5 )   e12435   2017.10

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  • DISTINCTIVE FEATURES OF PHOX2B-EXPRESSING NEURONS IN THE RAT RETICULAR FORMATION DORSAL TO THE TRIGEMINAL MOTOR NUCLEUS Reviewed

    Kouta Nagoya, Shiro Nakamura, Keiko Ikeda, Hiroshi Onimaru, Atsushi Yoshida, Kiyomi Nakayama, Ayako Mochizuki, Masaaki Kiyomoto, Fumihiko Sato, Kiyoshi Kawakami, Koji Takahashi, Tomio Inoue

    NEUROSCIENCE   358   211 - 226   2017.9

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    DOI： 10.1016/j.neuroscience.2017.06.035

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  • Knockout of sodium pump alpha 3 subunit gene (Atp1a3(-/-)) results in perinatal seizure and defective respiratory rhythm generation Reviewed

    Keiko Ikeda, Hiroshi Onimaru, Kiyoshi Kawakami

    BRAIN RESEARCH   1666   27 - 37   2017.7

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    DOI： 10.1016/j.brainres.2017.04.014

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  • Effects of a TRPV1 agonist capsaicin on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats Reviewed

    Mariho Tani, Sayumi Kotani, Chikara Hayakawa, Shih-Tien Lin, Saki Irie, Keiko Ikeda, Kiyoshi Kawakami, Hiroshi Onimaru

    PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY   469 ( 2 )   327 - 338   2017.2

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    DOI： 10.1007/s00424-016-1912-8

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  • The respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology Reviewed

    Keiko Ikeda, Kiyoshi Kawakami, Hiroshi Onimaru, Yasumasa Okada, Shigefumi Yokota, Naohiro Koshiya, Yoshitaka Oku, Makito Iizuka, Hidehiko Koizumi

    JOURNAL OF PHYSIOLOGICAL SCIENCES   67 ( 1 )   45 - 62   2017.1

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  • A novel reporter rat strain that conditionally expresses the bright red fluorescent protein tdTomato Reviewed

    Hiroyuki Igarashi, Kyo Koizumi, Ryosuke Kaneko, Keiko Ikeda, Ryo Egawa, Yuchio Yanagawa, Shin-Ichi Muramatsu, Hiroshi Onimaru, Toru Ishizuka, Hiromu Yawo

    PLoS ONE   11 ( 5 )   e0155687   2016.5

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    DOI： 10.1371/journal.pone.0155687

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  • Activation of Six1 Expression in Vertebrate Sensory Neurons Reviewed

    Shigeru Sato, Hiroshi Yajima, Yasuhide Furuta, Keiko Ikeda, Kiyoshi Kawakami

    PLOS ONE   10 ( 8 )   e0136666   2015.8

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    DOI： 10.1371/journal.pone.0136666

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  • Long-lasting facilitation of respiratory rhythm by treatment with TRPA1 agonist, cinnamaldehyde Reviewed

    Mariho Tani, Itaru Yazawa, Keiko Ikeda, Kiyoshi Kawakami, Hiroshi Onimaru

    JOURNAL OF NEUROPHYSIOLOGY   114 ( 2 )   989 - 998   2015.8

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    DOI： 10.1152/jn.00282.2015

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  • A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry Reviewed

    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   2015.7

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  • Effects of ouabain on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats and in decerebrate and arterially perfused in situ preparation from juvenile rats. Reviewed International journal

    Tsuzawa, K, Yazawa, I, Shakuo, T, Ikeda, K, Kawakami, K

    Neuroscience   286   404 - 411   2015.1

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    The significance of Na/K-ATPase on respiratory rhythm generation is not well understood. We investigated the effects of the Na/K-ATPase blocker, ouabain, on respiratory rhythm. Experiments were performed with brainstem-spinal cord preparation from 0 to 3-day-old Wistar rats and with decerebrate and arterially perfused in situ preparation from juvenile rats (postnatal day 11-13). Newborn rat preparations were superfused at a rate of 3.0 ml/min with artificial cerebrospinal fluid, equilibrated with 95% O2 and 5% CO2, pH 7.4, at 26-27 °C. Inspiratory activity was monitored from the fourth cervical ventral root (C4). Application of ouabain (15-20 min) resulted in a dose-dependent increase in the burst rate of C4 inspiratory activity. After washout, the burst rate further increased to reach quasi-maximum values under each condition (e.g. 183% of control in 1 μM, 253% in 10 μM, and 303% in 20 μM at 30 min washout). Inspiratory or pre-inspiratory neurons in the rostral ventrolateral medulla were depolarized. We obtained similar results (i.e. increased phrenic burst rate) in an in situ perfused preparation of juvenile rats. Genes encoding the Na/K-ATPase α subunit were expressed in the region of the parafacial respiratory group (pFRG) in neonatal rats, suggesting that cells (neurons and/or glias) in the pFRG were one of the targets of ouabain. We concluded that Na/K-ATPase activity could be an important factor in respiratory rhythm modulation.

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  • Heterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress Reviewed

    Hiroki Sugimoto, Keiko Ikeda, Kiyoshi Kawakami

    BEHAVIOURAL BRAIN RESEARCH   272   100 - 110   2014.10

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    DOI： 10.1016/j.bbr.2014.06.048

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  • Six1 is a key regulator of the developmental and evolutionary architecture of sensory neurons in craniates Reviewed

    Hiroshi Yajima, Makoto Suzuki, Haruki Ochi, Keiko Ikeda, Shigeru Sato, Ken-ichi Yamamura, Hajime Ogino, Naoto Ueno, Kiyoshi Kawakami

    BMC BIOLOGY   12   40   2014.5

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    DOI： 10.1186/1741-7007-12-40

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  • Cytoarchitecture and CO2 Sensitivity of Phox2b-Positive Parafacial Neurons in the Newborn Rat Medulla Reviewed

    Hiroshi Onimaru, Keiko Ikeda, Tani Mariho, Kiyoshi Kawakami

    CENTRAL NERVOUS SYSTEM CONTROL OF RESPIRATION   209   57 - 71   2014

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    DOI： 10.1016/B978-0-444-63274-6.00004-7

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  • Enhanced inhibitory neurotransmission in the cerebellar cortex of Atp1a3-deficient heterozygous mice. Reviewed International journal

    Keiko Ikeda, Shin'Ichiro Satake, Tatsushi Onaka, Hiroki Sugimoto, Naoki Takeda, Keiji Imoto, Kiyoshi Kawakami

    The Journal of physiology   591 ( 13 )   3433 - 49   2013.7

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    Dystonia is characterized by excessive involuntary and prolonged simultaneous contractions of both agonist and antagonist muscles. Although the basal ganglia have long been proposed as the primary region, recent studies indicated that the cerebellum also plays a key role in the expression of dystonia. One hereditary form of dystonia, rapid-onset dystonia with parkinsonism (RDP), is caused by loss of function mutations of the gene for the Na pump α3 subunit (ATP1A3). Little information is available on the affected brain regions and mechanism for dystonia by the mutations in RDP. The Na pump is composed of α and β subunits and maintains ionic gradients of Na(+) and K(+) across the cell membrane. The gradients are utilized for neurotransmitter reuptake and their alteration modulates neural excitability. To provide insight into the molecular aetiology of RDP, we generated and analysed knockout heterozygous mice (Atp1a3(+/-)). Atp1a3(+/-) showed increased symptoms of dystonia that is induced by kainate injection into the cerebellar vermis. Atp1a3 mRNA was highly expressed in Purkinje cells and molecular-layer interneurons, and its product was concentrated at Purkinje cell soma, the site of abundant vesicular γ-aminobutyric acid transporter (VGAT) signal, suggesting the presynaptic localization of the α3 subunit in the inhibitory synapse. Electrophysiological studies showed that the inhibitory neurotransmission at molecular-layer interneuron-Purkinje cell synapses was enhanced in Atp1a3(+/-) cerebellar cortex, and that the enhancement originated via a presynaptic mechanism. Our results shed light on the role of Atp1a3 in the inhibitory synapse, and potential involvement of inhibitory synaptic dysfunction for the pathophysiology of dystonia.

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  • Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods Reviewed

    Shigeru Sato, Keiko Ikeda, Go Shioi, Kazuki Nakao, Hiroshi Yajima, Kiyoshi Kawakami

    DEVELOPMENTAL BIOLOGY   368 ( 1 )   95 - 108   2012.8

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    DOI： 10.1016/j.ydbio.2012.05.023

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  • RELATIONSHIP BETWEEN THE DISTRIBUTION OF THE PAIRED-LIKE HOMEOBOX GENE (PHOX2B) EXPRESSING CELLS AND BLOOD VESSELS IN THE PARAFACIAL REGION OF THE VENTRAL MEDULLA OF NEONATAL RATS Reviewed

    H. Onimaru, K. Ikeda, K. Kawakami

    NEUROSCIENCE   212   131 - 139   2012.6

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    DOI： 10.1016/j.neuroscience.2012.03.037

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  • Postsynaptic mechanisms of CO2 responses in parafacial respiratory neurons of newborn rats Reviewed

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    JOURNAL OF PHYSIOLOGY-LONDON   590 ( 7 )   1615 - 1624   2012.4

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    DOI： 10.1113/jphysiol.2011.222687

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  • Development of gustatory papillae in the absence of Six1 and Six4 Reviewed

    Yuko Suzuki, Keiko Ikeda, Kiyoshi Kawakami

    JOURNAL OF ANATOMY   219 ( 6 )   710 - 721   2011.12

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    DOI： 10.1111/j.1469-7580.2011.01435.x

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  • Menthol inhibits the respiratory rhythm in brainstem preparations of the newborn rats Reviewed

    Mariho Tani, Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami, Ikuo Homma

    NEUROREPORT   21 ( 17 )   1095 - 1099   2010.12

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    DOI： 10.1097/WNR.0b013e3283405bad

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  • Opn5 is a UV-sensitive bistable pigment that couples with Gi subtype of G protein Reviewed

    Takahiro Yamashita, Hideyo Ohuchi, Sayuri Tomonari, Keiko Ikeda, Kazumi Sakai, Yoshinori Shichida

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   107 ( 51 )   22084 - 22089   2010.12

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    DOI： 10.1073/pnas.1012498107

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  • Expression of Six1 and Six4 in mouse taste buds Reviewed

    Yuko Suzuki, Keiko Ikeda, Kiyoshi Kawakami

    JOURNAL OF MOLECULAR HISTOLOGY   41 ( 4-5 )   205 - 214   2010.10

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    DOI： 10.1007/s10735-010-9280-8

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  • Six family genes control the proliferation and differentiation of muscle satellite cells Reviewed

    Hiroshi Yajima, Norio Motohashi, Yusuke Ono, Shigeru Sato, Keiko Ikeda, Satoru Masuda, Erica Yada, Hironori Kanesaki, Yuko Miyagoe-Suzuki, Shin'ichi Takeda, Kiyoshi Kawakami

    EXPERIMENTAL CELL RESEARCH   316 ( 17 )   2932 - 2944   2010.10

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    DOI： 10.1016/j.yexcr.2010.08.001

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  • Conserved expression of mouse Six1 in the pre-placodal region (PPR) and identification of an enhancer for the rostral PPR Reviewed

    Shigeru Sato, Keiko Ikeda, Go Shioi, Haruki Ochi, Hajime Ogino, Hiroshi Yajima, Kiyoshi Kawakami

    DEVELOPMENTAL BIOLOGY   344 ( 1 )   158 - 171   2010.8

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    DOI： 10.1016/j.ydbio.2010.04.029

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  • Phox2b Expressing Neurons in the Most Rostral Medulla of Newborn Rats Reviewed

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    NEW FRONTIERS IN RESPIRATORY CONTROL   669   87 - 90   2010

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    DOI： 10.1007/978-1-4419-5692-7_18

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  • Six1 is indispensable for production of functional progenitor cells during olfactory epithelial development Reviewed

    Keiko Ikeda, Ryoichiro Kageyama, Yuko Suzuki, Kiyoshi Kawakami

    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY   54 ( 10 )   1453 - 1464   2010

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    DOI： 10.1387/ijdb.093041ki

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  • Six1 is essential for generating pioneer neurons, differentiation of olfactory sensory neurons, and production of sustentacular cell progenitors in olfactory epithelium. Reviewed

    Ikeda K, Ando Z, Ookawara S, Sato S, Kageyama R, Kawakami K

    Dev. Biol. 311, 53-68.   2007

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    DOI： 10.1016/j.ydbio.2007.08.020

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  • Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarrays Reviewed

    K Ikeda, H Nakazawa, A Shimo-Oka, K Ishio, S Miyata, Y Hosokawa, S Matsumura, H Masuhara, S Belloncik, R Alain, N Goshima, N Nomura, K Morigaki, A Kawai, T Kuroita, B Kawakami, Y Endo, H Mori

    PROTEOMICS   6 ( 1 )   54 - 66   2006.1

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    DOI： 10.1002/pmic.200500022

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  • [Novel function of sodium pump]. Reviewed

    Ikeda K, Onimaru H, Kawakami K

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   50 ( 5 )   434 - 439   2005.5

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  • SP1 AND AN E-BOX BINDING-PROTEIN REGULATE NA+/K+-ATPASE ALPHA-2-SUBUNIT GENE Reviewed

    K IKEDA, K NAGANO, K KAWAKAMI

    SODIUM PUMP   33 - 36   1994

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  • TRANSCRIPTION FACTORS REGULATING THE NA+/K+-ATPASE GENES Reviewed

    K KAWAKAMI, Y SUZUKIYAGAWA, Y WATANABE, K IKEDA, K NAGANO

    SODIUM PUMP   1 - 10   1994

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  • Enhanced susceptibility to cortical spreading depression and different degree in two-types of Na plus , K plus -ATPase alpha2 subunit-deficient mice

    Miyuki Unekawa, Keiko Ikeda, Yutaka Tomita, Kiyoshi Kawakami, Norihiro Suzuki

    CEPHALALGIA   37   204 - 204   2017.9

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  • Relationship between distribution of Phox2b expressing neurons and capillary blood vessels in the parafacial region of the rostral ventrolateral medulla of rat

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    NEUROSCIENCE RESEARCH   71   E160 - E161   2011

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    DOI： 10.1016/j.neures.2011.07.693

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  • Developmental switch of primary sensory system from Rohon-Beard cells to Dorsal root ganglia

    Kiyoshi Kawakami, Hiroshi Yajima, Makoto Suzuki, Haruki Ochi, Keiko Ikeda, Shigeru Sato, Hajime Ogino, Naoto Ueno

    DEVELOPMENTAL BIOLOGY   344 ( 1 )   531 - 531   2010.8

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    DOI： 10.1016/j.ydbio.2010.05.395

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  • Six1 is indispensable for production of functional apical and basal progenitors during olfactory epithelial development

    Keiko Ikeda, Ryoichiro Kageyama, Kiyoshi Kawakami

    DEVELOPMENTAL BIOLOGY   344 ( 1 )   498 - 498   2010.8

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    DOI： 10.1016/j.ydbio.2010.05.301

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  • Regulatory role of Six1 in the development of taste papillae

    Yuko Suzuki, Keiko Ikeda, Kiyoshi Kawakami

    CELL AND TISSUE RESEARCH   339 ( 3 )   513 - 525   2010.3

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  • Postsynaptic mechanisms of CO2 responses in parafacial neurons of newborn rats

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    NEUROSCIENCE RESEARCH   68   E167 - E167   2010

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    DOI： 10.1016/j.neures.2010.07.2315

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  • Regulatory role of Six1 in the development of taste papillae.

    Yuko Suzuki, Keiko Ikeda, Kiyoshi Kawakami

    Cell Tissue Res in press   339 ( 3 )   513 - 525   2010

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  • Phox2b, RTN/pFRG neurons and respiratory rhythmogenesis

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    RESPIRATORY PHYSIOLOGY & NEUROBIOLOGY   168 ( 1-2 )   13 - 18   2009.8

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  • Preplacodal region marked by Six1 in mice

    Keiko Ikeda, Kiyoshi Kawakami

    DEVELOPMENTAL BIOLOGY   331 ( 2 )   458 - 459   2009.7

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    DOI： 10.1016/j.ydbio.2009.05.271

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  • Identification and characterization of Six1 enhancers

    Kiyoshi Kawakami, Shigeru Sato, Keiko Ikeda, Hiroshi Kiyonari

    DEVELOPMENTAL BIOLOGY   331 ( 2 )   520 - 521   2009.7

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    DOI： 10.1016/j.ydbio.2009.05.498

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  • CO(2)-Sensitive Preinspiratory Neurons of the Parafacial Respiratory Group Express Phox2b in the Neonatal Rat

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    JOURNAL OF NEUROSCIENCE   28 ( 48 )   12845 - 12850   2008.11

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  • Multiple Evolutionarily Conserved Enhancers Control Expression of Eya1

    Tadashi Ishihara, Shigeru Sato, Keiko Ikeda, Hiroshi Yajima, Kiyoshi Kawakami

    DEVELOPMENTAL DYNAMICS   237 ( 11 )   3142 - 3156   2008.11

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  • Differential expression of Eya1 and Eya2 during chick early embryonic development

    Tadashi Ishihara, Keiko Ikeda, Shigeru Sato, Hiroshi Yajima, Kiyoshi Kawakami

    GENE EXPRESSION PATTERNS   8 ( 5 )   357 - 367   2008.5

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  • Early neurogenesis in the development of olfactory epithelium

    Keiko Ikeda, Kiyoshi Kawakami

    NEUROSCIENCE RESEARCH   61   S23 - S23   2008

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  • Six1 is essential for early neurogenesis in the development of olfactory epithelium

    Keiko Ikeda, Shigeo Ookawara, Shigeru Sato, Zen-ichi Ando, Ryoichiro Kageyama, Kiyoshi Kawakami

    DEVELOPMENTAL BIOLOGY   311 ( 1 )   53 - 68   2007.11

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  • Defective interaction between dual oscillators for respiratory rhythm generation in Na+, K+-ATPase α2 subunit-deficient mice

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    Journal of Physiology   584 ( 1 )   271 - 284   2007.10

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  • Defective interaction between dual oscillators for respiratory rhythm generation in Na+,K+-ATPase alpha 2 subunit-deficient mice

    Hiroshi Onimaru, Keiko Ikeda, Kiyoshi Kawakami

    JOURNAL OF PHYSIOLOGY-LONDON   584 ( 1 )   271 - 284   2007.10

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  • Roles of Six1 and Six4 in development of cranial ganglia

    M. Yamakado, Z. -Ic. Ando, K. Kawakami, K. Ikeda

    INTERNATIONAL JOURNAL OF DEVELOPMENTAL NEUROSCIENCE   24 ( 8 )   510 - 510   2006.12

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    DOI： 10.1016/j.ijdevneu.2006.09.098

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  • Six1 is a key regulator of multiple cell type differentiation in olfactory epithelium

    K. Ikeda, Z. -Ic. Ando, R. Kageyama, K. Kawakami

    INTERNATIONAL JOURNAL OF DEVELOPMENTAL NEUROSCIENCE   24 ( 8 )   510 - 510   2006.12

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    DOI： 10.1016/j.ijdevneu.2006.09.097

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  • Six1 and Six4 promote survival of sensory neurons during early trigeminal gangliogenesis

    Yoshiyuki Konishi, Keiko Ikeda, Yoichiro Iwakura, Kiyoshi Kawakami

    Brain Research   1116 ( 1 )   93 - 102   2006.10

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  • Six1 and Six4 promote survival of sensory neurons during early trigeminal gangliogenesis

    Yoshiyuki Konishi, Keiko Ikeda, Yoichiro Iwakura, Kiyoshi Kawakami

    BRAIN RESEARCH   1116   93 - 102   2006.10

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  • Role of homeodomain transcription factor Six1 in olfactory sensory neuron development

    Keiko Ikeda, Zen-ichi Ando, Shigeo Oowakara, Kiyoshi Kawakami

    DEVELOPMENTAL BIOLOGY   295 ( 1 )   408 - 408   2006.7

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    DOI： 10.1016/j.ydbio.2006.04.252

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  • Role of homeobox transcription factor Six1 in mouse basal development

    K Ikeda, ZI Ando, S Ookawara, K Kawakami

    CHEMICAL SENSES   31 ( 1 )   J21 - J21   2006.1

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  • MODULATION OF NEURAL ACTIVITIES BY Na, K-ATPase alpha 2 SUBUNIT THROUGH FUNCTIONAL COUPLING WITH TRANSPORTERS

    Kiyoshi Kawakami, Keiko Ikeda

    CELLULAR AND MOLECULAR BIOLOGY   52 ( 8 )   92 - 96   2006

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  • Hyperphagia and obesity in Na,K-ATPase alpha 2 subunit-defective mice

    K Kawakami, T Onaka, M Iwase, Homma, I, K Ikeda

    OBESITY RESEARCH   13 ( 10 )   1661 - 1671   2005.10

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  • Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4.

    Ando Z, Sato S, Ikeda K, Kawakami K

    FEBS   272 ( 12 )   3026 - 3041   2005

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  • Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4. FEBS(Ando Z, Sato S, Ikeda K, Kawakami K)(Academic Journal、2005)272/,3026-3041

    Ando Z, Sato S, Ikeda K, Kawakami K

    FEBS   272 ( 12 )   3026 - 3041   2005

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  • Malfunction of respiratory-related neuronal activity in Na(+), K(+)-ATPase alpha 2 subunit-deficient mice is attributable to abnormal Cl(-) homeostasis in brainstem neurons

    K Ikeda, H Onimaru, J Yamada, K Inoue, S Ueno, T Onaka, H Toyoda, A Arata, T Ishikawa, MM Taketo, A Fukuda, K Kawakami

    JOURNAL OF NEUROSCIENCE   24 ( 47 )   10693 - 10701   2004.11

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  • Malfunction of respiratory-related neuronal activity in Na(+), K(+)-ATPase alpha 2 subunit-deficient mice is attributable to abnormal Cl(-) homeostasis in brainstem neurons

    K Ikeda, H Onimaru, J Yamada, K Inoue, S Ueno, T Onaka, H Toyoda, A Arata, T Ishikawa, MM Taketo, A Fukuda, K Kawakami

    JOURNAL OF NEUROSCIENCE   24 ( 47 )   10693 - 10701   2004.11

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  • Lack of respiratory neural network in sodium pump alpha2 subunit-deficient mice

    K Ikeda, K Kawakami

    DEVELOPMENTAL BIOLOGY   271 ( 2 )   592 - 592   2004.7

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  • Roles of Six1 in patterning of otic and nasal primordia.

    K Kawakami, K Nakamura, H Ozaki, S Sato, K Ikeda

    DEVELOPMENTAL BIOLOGY   271 ( 2 )   586 - 587   2004.7

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  • Six1 controls patterning of the mouse otic vesicle

    H Ozaki, K Nakamura, J Funahashi, K Ikeda, G Yamada, H Tokano, H Okamura, K Kitamura, S Muto, H Kotaki, K Sudo, R Horai, Y Iwakura, K Kawakami

    DEVELOPMENT   131 ( 3 )   551 - 562   2004.2

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  • Facilitative role of endogenous oxytocin in noradrenaline release in the rat supraoptic nucleus

    T Onaka, K Ikeda, T Yamashita, K Honda

    EUROPEAN JOURNAL OF NEUROSCIENCE   18 ( 11 )   3018 - 3026   2003.12

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  • Degeneration of the amygdala/piriform cortex and enhanced fear/anxiety behaviors in sodium pump alpha 2 subunit (Atp1a2)-deficient mice

    K Ikeda, T Onaka, M Yamakado, J Nakai, T Ishikawa, MM Taketo, K Kawakami

    JOURNAL OF NEUROSCIENCE   23 ( 11 )   4667 - 4676   2003.6

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  • Degeneration of the amygdala /piriform cortex and enhanced fear/anxiety behaviors in sodium pump a2 subunit (Atp1a2) deficient mice.

    Ikeda K, Onaka T, Yamakado M, Nakai J, Ishikawa T, Taketo MM, Kawakami K

    J. Neurosci.   23   4667 - 4676   2003

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  • Molecular interaction and synergistic activation of a promoter by Six, Eya, and Dach proteins mediated through CREB binding protein

    K Ikeda, Y Watanabe, H Ohto, K Kawakami

    MOLECULAR AND CELLULAR BIOLOGY   22 ( 19 )   6759 - 6766   2002.10

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  • The H1 and H2 regions of the activation domain of herpes simplex virion protein 16 stimulate transcription through distinct molecular mechanisms

    K Ikeda, T Stuehler, M Meisterernst

    GENES TO CELLS   7 ( 1 )   49 - 58   2002.1

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  • Impaired interactions between mouse Eya1 harboring mutations found in patients with branchio-oto-renal syndrome and Six, Dach, and G proteins

    H Ozaki, Y Watanabe, K Ikeda, K Kawakami

    JOURNAL OF HUMAN GENETICS   47 ( 3 )   107 - 116   2002

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  • Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex

    T Otsuki, Y Furukawa, K Ikeda, H Endo, T Yamashita, A Shinohara, A Iwamatsu, K Ozawa, JM Liu

    HUMAN MOLECULAR GENETICS   10 ( 23 )   2651 - 2660   2001.11

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  • Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex.

    T Otsuki, Y Furukawa, K Ikeda, H Endo, T Yamashita, A Shinohara, A Iwamatsu, K Ozawa, JM Liu

    BLOOD   98 ( 11 )   782A - 783A   2001.11

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  • Six family genes - structure and function as transcription factors and their roles in development

    K Kawakami, S Sato, H Ozaki, K Ikeda

    BIOESSAYS   22 ( 7 )   616 - 626   2000.7

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    Language：English   Publishing type：Book review, literature introduction, etc.  

    DOI： 10.1002/1521-1878(200007)22:7<616::AID-BIES4>3.0.CO;2-R

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  • Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes

    K Ikeda, DJ Steger, A Eberharter, JL Workman

    MOLECULAR AND CELLULAR BIOLOGY   19 ( 1 )   855 - 863   1999.1

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  • Transcriptional activators direct histone acetyltransferase complexes to nucleosomes

    RT Utley, K Ikeda, PA Grant, J Cote, DJ Steger, A Eberharter, S John, JL Workman

    NATURE   394 ( 6692 )   498 - 502   1998.7

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  • Tissue and developmental distribution of Six family gene products

    H Ohto, T Takizawa, T Saito, M Kobayashi, K Ikeda, K Kawakami

    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY   42 ( 2 )   141 - 148   1998.3

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  • Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6

    K Ikeda, JP Halle, G Stelzer, M Meisterernst, K Kawakami

    MOLECULAR AND CELLULAR BIOLOGY   18 ( 1 )   10 - 18   1998.1

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  • Regulation of transcription by multisubunit complexes that alter nucleosome structure

    DJ Steger, RT Utley, PA Grant, S John, A Eberharter, J Cote, T Owen-Hughes, K Ikeda, JL Workman

    COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY   63   483 - 491   1998

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  • In vivo footprinting analysis of the Na,K-ATPase alpha3 subunit gene promoter in rat cardiocytes

    Murakami Y, Ikeda K, Ikeda U, Shimada K, Kawakami K

    Jichi Medical School   20   35 - 43   1997

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  • Cis-elements involved in differential expression of Na+-K+-ATPase alpha 2 subunit gene in muscle differentiation

    K Ikeda, K Kawakami

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1308 ( 1 )   67 - 73   1996.7

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  • Structure, function and expression of a murine homeobox protein AREC3, a homologue of Drosophila sine oculis gene product, and implication in development

    K Kawakami, H Ohto, K Ikeda, RG Roeder

    NUCLEIC ACIDS RESEARCH   24 ( 2 )   303 - 310   1996.1

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  • DNA-BINDING THROUGH DISTINCT DOMAINS OF ZINC-FINGER-HOMEODOMAIN PROTEIN AREB6 HAS DIFFERENT EFFECTS ON GENE-TRANSCRIPTION

    K IKEDA, K KAWAKAMI

    EUROPEAN JOURNAL OF BIOCHEMISTRY   233 ( 1 )   73 - 82   1995.10

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  • POSSIBLE IMPLICATIONS OF SP1-INDUCED BENDING OF DNA ON SYNERGISTIC ACTIVATION OF TRANSCRIPTION

    K IKEDA, K NAGANO, K KAWAKAMI

    GENE   136 ( 1-2 )   341 - 343   1993.12

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  • ANOMALOUS INTERACTION OF SP1 AND SPECIFIC BINDING OF AN E-BOX-BINDING PROTEIN WITH THE REGULATORY ELEMENTS OF THE NA,K-ATPASE ALPHA-2-SUBUNIT GENE PROMOTER

    K IKEDA, K NAGANO, K KAWAKAMI

    EUROPEAN JOURNAL OF BIOCHEMISTRY   218 ( 1 )   195 - 204   1993.11

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  • ANALYSIS OF CIS-ELEMENTS AND TRANSACTING FACTORS OF NA, K-ATPASE ALPHA2 SUBUNIT GENE

    K IKEDA, K KAWAKAMI, K NAGANO

    JOURNAL OF CELLULAR BIOCHEMISTRY   161 - 161   1993.1

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  • FUNCTIONAL REORGANIZATION OF ADULT CAT SOMATOSENSORY CORTEX IS DEPENDENT ON NMDA RECEPTORS

    M KANO, K LINO, M KANO

    NEUROREPORT   2 ( 2 )   77 - 80   1991.2

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  • OPTOKINETIC RESPONSE OF CELLS IN THE NUCLEUS-RETICULARIS TEGMENTI PONTIS OF THE PIGMENTED RABBIT

    M KANO, K IINO, K MAEKAWA, MS KANO

    EXPERIMENTAL BRAIN RESEARCH   87 ( 2 )   239 - 244   1991

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Awards

  • 公正かつ公平な審査員

    2018.8   日本学術振興会  

    池田 啓子

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  • 医学研究賞受賞

    2013.11   兵庫県医師会  

    池田 啓子

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  • 学術研究助成賞受賞

    2010.5   日本女医会  

    池田 啓子

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  • 国際細胞生物学会議第9回大会（開催地 ソウル）優秀ポスター賞

    2008.10  

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  • 自治医科大学医学部 平成19年度 最優秀教員賞 受賞

    2007  

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    Country：Japan

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  • 日本医師会医学研究奨励賞受賞

    2006.11   日本医師会  

    池田 啓子

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  • 日本女性科学者の会奨励賞

    2005.6  

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    Country：Japan

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  • 自治医科大学奨励賞（自治医科大学）受賞

    1999.4  

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    Country：Japan

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  • 地域保健医療研究助成賞（地域医療振興財団）受賞

    1994.5  

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Research Projects

  • Ｎａポンプ神経疾患の症状発現トリガー物質、アスコルビン酸が神経系に及ぼす基盤解明

    Grant number：22K07377  2022.4 - 2025.3

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    池田 啓子

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    Grant amount：\4290000 （ Direct Cost: \3300000 、 Indirect Cost：\990000 ）

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  • Molecular mechanisms of responses to hypoxic stimulation in medullary cardio-respiratory center neurons

    Grant number：22K06474  2022.4 - 2025.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

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    Grant amount：\2600000 （ Direct Cost: \2000000 、 Indirect Cost：\600000 ）

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  • 膵癌リンパ管侵襲の病態解明とそれを抑制する新規治療法の開発

    Grant number：20K08364  2020.4 - 2024.3

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    大城 久, 池田 啓子, 福嶋 敬宜, 仲矢 丈雄

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    Grant amount：\4290000 （ Direct Cost: \3300000 、 Indirect Cost：\990000 ）

    Stage IIの膵頭部原発浸潤性膵管癌の手術症例を後方視的に解析した．癌の最大割面におけるリンパ管侵襲の本数を免疫組織化学的に調べた．術前化学療法施行例（n=9）と術前化学療法未施行例（n=51）との間で，癌の最大割面におけるリンパ管侵襲本数に差があるか否かを検討した．その結果，術前化学療法施行例では未施行例に比べてリンパ管侵襲本数が有意に少ないことが明らかにされた(P<0.05，Mann-WhitneyのU検定)．

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  • Increased oxidative stress in brains of pathophysiological model mice for Na pump gene related neurologic disorders

    Grant number：18K06968  2018.4 - 2021.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IKEDA KEIKO

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    Grant amount：\4550000 （ Direct Cost: \3500000 、 Indirect Cost：\1050000 ）

    Mutations in the genes encoding Na,K-ATPase (Na pump) alpha2 and alpha3 subunit (ATP1A2 and ATP1A3, respectively) are associated with different neurologic disorders, such as rapid onset dystonia parkinsonism, alternating hemiplegia of childhood, and so on. However, pathophysiological bases of these disorders are still unknown and no cure has yet to be found. A preliminary experimental study shows that the level of ascorbic acids is decreased in the brains of Atp1a2 and Atp1a3 knockout mice (pathological model mice). The present study investigated 1. Functional coupling between Na pump alpha subunit and a transporter of ascorbic acid (SVCT2); 2. The level of oxidative stress of Na pump knockout mice; 3. Genetical link between Atp1a2/Atp1a3 and the gene encoding SVCT2. The results indicate increased level of oxidative stress is one of underlying pathological mechanisms in the Na pump related neurological disorders.

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  • Crosstalk between the neural circuit for controlling mastication and the autonomic nervous system

    Grant number：17K19775  2017.6 - 2019.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Research (Exploratory)

    INOUE TOMIO, IKEDA Keiko

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    Grant amount：\6370000 （ Direct Cost: \4900000 、 Indirect Cost：\1470000 ）

    The supratrigeminal nucleus and the parabrachial nucleus are located dorsal to the trigeminal motor nucleus (RdV). The former is related to controlling jaw movements and the latter receives autonomic inputs. Furthermore, PHOX2B is essential for autonomic nervous system development and the neurons expressing Phox2b (Phox2b+ neurons) are abundantly found in the RdV. In this study, we examined the properties of Phox2b+ neurons located in the RdV. Phox2b+ neurons were glutamatergic, whereas Phox2b-negative (Phox2b-) neurons are GABAergic and glycinergic. Phox2b+ neurons showed low-frequency firing, while Phox2b- neurons exhibited high-frequency firing. About half of the Phox2b+ and Phox2b- neurons send their axons to the trigeminal motor nucleus. These results suggest that Phox2b+ neurons have clearly different properties from Phox2b- neurons and might play important roles in feeding-related functions under the crosstalk control between motor and autonomic systems.

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  • Analysis of sodium pump knockout mice as a pathophysiological model for familial hemiplegic migraine type 2

    Grant number：26461284  2014.4 - 2017.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IKEDA KEIKO, KAWAKAMI KIYOSHI, SATAKE SHIN-ICHIRO

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    Grant amount：\5070000 （ Direct Cost: \3900000 、 Indirect Cost：\1170000 ）

    The sodium pump α2 subunit is expressed in central nervous tissue, particularly astrocytes and pyramidal cells in the hippocampus. Mutations in ATP1A2 encoding the;sodium pump α2 subunit cause Familial hemiplegic migraine type 2 (FHM2), an autosomal dominant inheritance migraine with aura and motor weakness and in some cases with seizures and cerebellar symptoms. To understand pathophysiology of migraine and develop novel therapeutics and drugs, we examined the Atp1a2 knockout heterozygous mice by histological and electrophysiological analyses. We found that increased sensitivity of induction of CSD (cortical spreading depression) response in the knockout heterozygous mice compared with wild-type littermate mice. We also established the novel transgenic mouse line harboring homologous mutations found in patients. We have examined their sensitivities to seizures and behaviors, and evaluated them as migraine-model mice.

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  • Analysis of respiratory center using transgenic rats in which EYFP or photo-sensitive ion channels are introduced in Phox2b-expressing cells

    Grant number：25430012  2013.4 - 2016.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    Onimaru Hiroshi, IKEDA KEIKO

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    Grant amount：\4290000 （ Direct Cost: \3300000 、 Indirect Cost：\990000 ）

    We have analyzed neuron networks of the medullary respiratory center using transgenic (Tg) rats in which fluorescent protein (EYFP) or photosensitive ion channels, archaerhodopsin (Arch) were expressed under control of Phox2b gene expression. First, we confirmed specific expression of EYFP in Phox2b positive cells of Tg rats. The central respiratory activity in the brainstem-spinal cord preparation from Tg rats did not differ from that of wild type rats. Using calcium imagings, we confirmed that many of EYFP positive cells in the pFRG showed Pre-I neuron like activity. We also confirmed that optogenetics is useful for analysis of respiratory rhythm generation in the en bloc newborn rat preparation from Arch expressing Tg rats.

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  • Molecular mechanism of cellular interaction between placode and neural crest

    Grant number：23590224  2011 - 2013

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IKEDA Keiko, KAWAKAMI Kiyoshi

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    Grant amount：\5460000 （ Direct Cost: \4200000 、 Indirect Cost：\1260000 ）

    The cranial peripheral nervous system is unique in the sense that it is derived from both ectodermal placodes and neural crest cells (NCCs). The interdependent relationship exists between placodes and NCCs all through development. Considering obvious importance of understanding the interactions between placodes and NCCs, it is not fully uncovered about the ligands and receptors governing their interactions, especially in the mammals. The aim of this research was to identify molecules for the interaction. We have already reported that transcription factor encoding genes, Six1 and Six4 expressed solely in the placodes, but not in the NCCs. Knockout mouse embryos targeting these genes showed defects not only placodal, but also NCCs derived organs. Using the gene targeting mice and wild type one, here we searched candidate molecules essential for the interaction between placodes and NCCs. We also examined the effects of inhibiting the expression of candidate molecules during development.

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  • Ionic mechanisms of CO2 sensitivity in Phox2b neurons as the medullary central chemoreceptor

    Grant number：22500296  2010 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    ONIMARU Hiroshi, IKEDA Keiko

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    Grant amount：\3380000 （ Direct Cost: \2600000 、 Indirect Cost：\780000 ）

    It has been suggested that Phox2b expressing cells including pFRG/Pre-I neurons in the rostral ventrolateral medulla function as the central chemoreceptors (i.e. CO2 sensor). In the present study, we investigated neuronal mechanisms of high CO2 reception of pFRG/Pre-I neurons, using the brainstem-spinal cord preparation from newborn rats. The CO2 sensitivity of pFRG/Pre-I neurons was preserved even after blockade of Na+channels and Ca2+channels, and the membrane depolarization induced by hypercapnic stimulation was mainly due to the closing of K+channels. We also found that Phox2b-expressing cells including pFRG/Pre-I neurons in the parafacial region of the rostral ventral medulla tended to assemble around capillary blood vessels. We confirmed that TASK1,2 and 3 channels were less significantly involved in hypercapnic responses of pFRG/Pre-I neurons. Our findings demonstrate that the pFRG/Pre-I neurons possess postsynaptic mechanisms that are directly responsible to high CO2 stimulation.

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  • Physiological function of Na pumpα3 subunit gene and involvement in pathophysiology of dystonia parkinsonism.

    Grant number：21590239  2009 - 2011

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    KAWAKAMI Kiyoshi, IKEDA Keiko, ONAKA Tatsushi

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    Grant amount：\4680000 （ Direct Cost: \3600000 、 Indirect Cost：\1080000 ）

    Dystonia is characterized by excessive involuntary and prolonged simultaneous contractions of both agonist and antagonist muscles. One hereditary form of dystonia, DYT12, is caused by loss of function mutations of the gene forα3 isoform of Na pump(ATP1A3). To provide insight into the molecular etiology of DYT12, we generated and analyzed knockout heterozygous mice(Atp1a3^<+/->). Atp1a3^<+/-> mice showed increased sensitivity to kainate-induced dystonia than wild-type littermates(WT). Electrophysiological studies showed enhanced response of Purkinje cells in Atp1a3^<+/-> at cerebellar inhibitory synapses. Our results shed light on the role of Atp1a3 in inhibitory synapse, and its potential involvement in the expression of dystonia symptoms of DYT12.

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  • Role of pioneer neurons in development of peripheral nervous sysytem

    Grant number：20590177  2008 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IKEDA Keiko

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    Grant amount：\4680000 （ Direct Cost: \3600000 、 Indirect Cost：\1080000 ）

    The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, "early neurogenesis" occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Pioneer neurons migrate out OP/OE and localize between OP/OE and the forebrain. The importance of pioneer neurons are important the first connection between peripheral and central nervous system during the development. To uncover the molecular mechanism that govern this connection, we investigated the genes involved in the production of pioneer neurons using Six1^<-/->, because Six1^<-/-> shows total absence of pioneer neurons and their derivatives. We identified several candidate genes working under Six1. Interestingly, some of them are involved in retinoic acid signaling pathways. Others are genes whose products are important for cell-cell communication, such as Notch and Jagged. In parallel, we found that Six1 is expressed in both apical and basal progenitors at later stages. In Six1^<-/->, apical proliferating cells were absent and no morphologically identifiable sustentacular cells were observed. Consistently, the expression of Notch2 and Jagged1 in the apical layer was absent in Six1^<-/->. Basal proliferating cells were observed in Six1^<-/->, but the expression of Ngn1, NeuroD, Notch1, and Jagged2 in the basal layer was absent. In sum, Six1 regulates the expression of Notch, Jagged, and other neurogenic bHLH transcription factors for production of pioneer neurons, apical progenitors, and basal progenitors.

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  • Principle of organogenesis derived from neural crest cells

    Grant number：18390061  2006 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    KAWAKAMI Kiyoshi, YAJIMA Hiroshi, IKEDA Keiko

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    Grant amount：\17060000 （ Direct Cost: \14600000 、 Indirect Cost：\2460000 ）

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  • Role of Sixl in development of olfactory epithelium

    Grant number：18590172  2006 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IKEDA Keiko

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    Grant amount：\4020000 （ Direct Cost: \3600000 、 Indirect Cost：\420000 ）

    The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, "early neurogenesis" occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows "established neurogenesis," in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mashl at E10.5 in the Six1-deficient mice (Six1^<-/->). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^<-/-> at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^<-/->. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5. Currently, to analyze gene regulatory network essential for production of pioneer neurons more detail, we are planning to do microarray analysis of the relative gene expression profiles in E9.5 wild-type v.s. Six1^<-/-> OP identified candidate genes for differentiation of pioneer neurons.

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  • Role of Homeobox transcription factor encoding gene, Six in peripheral neural network

    Grant number：17590172  2005 - 2006

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    YAMAKADO Makoto, IKEDA Keiko, KAWAKAMI Kiyoshi

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    Grant amount：\3400000 （ Direct Cost: \3400000 ）

    Six genes are homologues of the Drosophila sine oculis (so), which plays an essential role in the development of compound eye. There are six members of Six genes, from Six1 to Six6 in mice. Of these, Six1 and Six4 are expressed in sensory placodes, trigeminal ganglion, epibranchial ganglia, and neural crest cells during mouse development. To understand roles of Six genes in formation of sensory peripheral nervous system, we analyzed double homozygous knockout mice (Six1^<-/->Six4^<-/->). During the development of placode-derived and neural crest-derived cranial sensory ganglia, an early arrest of neurogenesis was observed in Six1^<-/->Six4^<-/->. The epibranchial progenitor cells failed to express Neurogenin1 and Neurogenin2, that are normally expressed and required for the determination of neuronal precursors in placodal-derived trigeminal and epibranchial ganglion, respectively at embryonic day (E) 9.5. NeuroD as well as Phox2b genes that are essential for neural differentiation and maintenance, were also dramatically decreased. Moreober, failure to activate normal differentiation program in placodal-derived neurons resulted in abnormal apoptosis of the progenitor cells in Six1^<-/->Six4^<-/->. We also observed that the delamination of placode epithelial cells to form ganglia were disturbed in Six1^<-/->Six4^<-/->. As for neural crest cell, the expression of Sox10 at E8.5 was not altered in wild-type and Six1^<-/->Six4^<-/->, while its expression at E10.5 was severely reduced in Six1^<-/->Six4^<-/->. These results indicated that production and migration of neural crest cells were not disturbed, but differentiation to glial cells were disrupted in Six1^<-/->Six4^<-/->.In sum, our data suggest that Six1 and Six4 play roles for early differentiation, delamination, and survival of the placodally derived cranial sensory neurons and differentiation of neural crest cells.

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  • 感覚器形成におけるパターニング

    Grant number：16027246  2004 - 2005

    日本学術振興会  科学研究費助成事業  特定領域研究

    川上 潔, 小西 慶幸, 佐藤 滋, 池田 啓子

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    Grant amount：\6000000 （ Direct Cost: \6000000 ）

    Six1/Six4二重欠損マウスではE10.5において三叉神経節内にアポトーシスが原因だと考えられる凝縮した核が多数検出されたが、E13.5ではこのような凝縮した核の増加は観察されなかった。抗一本鎖DNA抗体、抗活性化カスパーゼ抗体を用いた解析により、アポトーシスの増加がSix1/Six4二重欠損マウスで観察される三叉神経節の萎縮の要因の一つであることが示唆された。Six1/Six4二重欠損マウスの三叉神経節から得られた神経細胞を培養すると神経栄養因子の存在下でもアポトーシスを起こすことから、細胞内在的な要因の存在が示唆された。Six1/Six4欠損マウス三叉神経節では、生存因子であるBcl-xや神経栄養因子の受容体であるTrkCの発現低下が観察されたため、Six1/Six4によるこれらの因子の発現維持が三叉神経節の発生に関与することが示唆された。
    Six1と隣接するSix4を含む180kbpのゲノム領域を種々の脊椎動物で比較した。ヒト-マウス間で保存され、かつ哺乳類以外の脊椎動物のいずれかでも保存された23の非エクソン配列を同定した。マウスゲノムよりPCRにて増幅した保存配列をptkEGFPベクターに挿入し、ニワトリ胚を用いてエンハンサー活性の検索を行った。その結果、プラコード原基(PPR)、種々のプラコードとプラコードに由来する感覚器と脳神経節においてEGFPの発現を活性化する6種類の独立したエンハンサーを同定した。これら6種類のエンハンサー活性を重ね合わせると、マウス及びニワトリ胚の種々のプラコードにおけるSix1とSix4本来の発現パターンがほぼ再現される。
    嗅神経細胞の初期に発現するNeurogenin1、Lhx2、NeuroD等の発現の低下がSix1/Six4二重欠損マウスにおいて観察された。また、GAP43,Tuj1陽性の細胞は形成されるが嗅神経の成熟マーカーであるOMPの陽性細胞は全く見られなかった。加えて、嗅繊毛が観察されず嗅上皮における配向が消失していた。一方、支持細胞のマーカーであるサイトケラチン陽性の細胞は存在するが、支持細胞の嗅上皮での配向は消失していた。神経分化を抑制することが知られているHes1およびHes5の発現がホモマウスの嗅上皮では亢進していた。これらの観察から、Six1は嗅上皮の嗅神経細胞や支持細胞の発生・分化を司り、上皮構造の形成維持にもかかわることが明らかとなった。

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  • Functional coupling of Sodium pump and KCC2 in respiratory neural network.

    Grant number：16500205  2004 - 2005

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IKEDA Keiko, KAWAKAMI Kiyoshi, ONIMARU Hiroshi

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    Grant amount：\3600000 （ Direct Cost: \3600000 ）

    Na^+, K^+-ATPase (Sodium pump) alpha2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2^<-/->) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity in Atp1a2^<-/-> was investigated using a brainstem-spinal cord en bloc preparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective in Atp1a2^<-/-> fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly in Atp1a2^<-/-> than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain of Atp1a2^<-/->. Lack of inhibitory neural activities in VLM of Atp1a2^<-/-> was observed by optical recordings. High intracellular Cl^- concentrations in neurons of the VLM of Atp1a2^<-/-> were detected in gramicidin-perforated patch-clamp recordings. The alpha2 subunit and a neuron-specific K-Cl cotransporter KCC2 were coimmunoprecipitated in a purified synaptic membrane fraction of wild-type fetuses. Based on these results, we propose a model for functional coupling between the Sodium pump alpha2 subunit and KCC2, which excludes Cl^- from the cytosolin respiratory center neurons. These results indicate that alpha2 subunit harbor a unique function that is not compensated by alphal subunit which express in the same neurons.

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  • Role of Six1 in nasal development

    2004

    ライフサイエンス基礎科学研究 

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    Grant type：Competitive

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  • NaポンプとCl輸送体(KCC2)の機能共役と、その破綻による脳の機能成熟不全

    Grant number：16015297  2004

    日本学術振興会  科学研究費助成事業  特定領域研究

    池田 啓子, 川上 潔

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    Grant amount：\3500000 （ Direct Cost: \3500000 ）

    ほ乳類のすべての細胞に存在するNa,K-ATPaseは、ATPの加水分解エネルギーを利用し、細胞膜内外のNaイオンとKイオンの濃度勾配を形成するポンプである。αとβの2つのサブユニットから構成され各々複数のアイソフォームが存在する。α1アイソフォームはすべての細胞に存在するのに対し、α2アイソフォームは神経、筋肉等の興奮性組織に存在する。後者の発生過程や生体内における特異的な機能は最近まで不明であったため、申請者はα2遺伝子ノックアウトマウスを作成し、生化学的、電気生理学的に解析を試みた。ホモマウスの外見は正常で野生型と同様に生まれてくるが、生直後から呼吸活動が開始せず10数分後に死亡する。摘出脳幹脊髄標本で呼吸活動を観察したところ、自発的な呼吸活動が欠如していた。電気刺激による応答は存在するため呼吸リズム活動を形成する素子としての神経細胞やネットワークは保持されていることがわかった。膜電位色素を用いて神経活動を観察したところ、野生型では抑制性の活動がでる領域で、ホモマウスでは興奮性神経活動に変換していた。さらに呼吸中枢が存在する部位のスライス標本にてグラミシジンパッチ法を行いGABAの逆転電位を測定し、細胞内クロライドイオン濃度を計算した。ホモマウスでは野生型に比し優位に上昇していた。その分子基盤として、神経細胞内クロライドイオンの排出に関わるK-Cl共輸送体(KCC2)とα2サブユニットが膜近傍で共局在することが明らかになった。また、GABAの再取込に関与するGABA輸送体との共役も明らかになった。以上の結果よりα2サブユニットは、種々の輸送体とカップルすることにより輸送体の機能を制御し、結果として周産期の呼吸中枢神経系の機能成熟に必須であること、この機能は同時に存在するα1サブユニットでは代償されないことが明らかにできた。

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  • ストレス性肥満を示すモデルマウスの性状

    Grant number：15659161  2003 - 2004

    日本学術振興会  科学研究費助成事業  萌芽研究

    川上 潔, 尾仲 達史, 池田 啓子

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    Grant amount：\3300000 （ Direct Cost: \3300000 ）

    私達の作成/解析したNa,K-ATPaseα2サブユニット遺伝子欠損ヘテロマウスは、恐怖/不安に関連した行動に異常を示すと同時に生後3ヶ月以降に肥満を呈する。前年度までにヘテロマウスのメスは肥満になることが明確となったが、その原因として、脂肪細胞の分化や機能が変化したためではなく、神経系の異常によって肥満が起きている可能性が高いことを明らかにした。本年度はマウスの摂食量、基礎代謝を測定して、肥満の機序を明確にする実験及び観察を行った。
    (1)Na,K-ATPaseα2サブユニット遺伝子欠損マウスと野生型マウスの摂食量を比較した。12時間ずつの明暗サイクルに分けて、メスの摂食量を測定したところ、ヘテロマウスはこ野生型よりも、明期での摂食量が増加していた。体重に有意差が現れないオスにおいても、ヘテロマウスの摂食量は野生型よりも増加していた。
    (2)Na,K-ATPaseα2サブユニット遺伝子欠損ヘテロマウスと野生型との体温、酸素消費量、二酸化炭素産生量及び呼吸商を比較したところ、二酸化炭素消費量がヘテロマウスで増加していた他は、有意差が見られなかった。従って、基礎代謝量の変化によって肥満が生じたとは考えられなかった。
    (3)明期での摂食量を制御する摂食ペプチドの発現に差があるかどうかを検証するために、視床下部からmRNAを単離し、オレキシン、POMC、NPY、MCHのmRNA量をRT-PCRにて比較検討した。ヘテロマウスメスにおいて、オレキシンの量が低下していた他は、mRNAの発現量に差が見られなかった。
    これらの結果から、ヘテロマウスのメスは明期の摂食量が増加することによって肥満になることが明確となったが、その原因は現在のところ明確にできていない。昼夜のリズム制御の異常などがひとつの原因とも考えられるが、今後の検討が必要である。

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  • Six-Eya-Dach遺伝子ネットワークによる器官形成

    Grant number：14034251  2002 - 2003

    日本学術振興会  科学研究費助成事業  特定領域研究

    川上 潔, 佐藤 滋, 尾崎 秀徳, 池田 啓子

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    Grant amount：\6800000 （ Direct Cost: \6800000 ）

    ホメオボックス遺伝子Six、その転写コアクチベーターEya、及びEyaとの共同作用因子Dachは互いの相互作用と遺伝子発現のフィードバックループを介して骨格筋、感覚器、腎臓などの器官形成を司る。本研究では、Six遺伝子欠損マウスの解析とSixタンパク質の標的遺伝子の同定を通じて、器官形成にいたる分子メカニズムを明確にする。本年度はSix1遺伝子破壊マウスでの内耳の異常形成機序、six1,Six4およびSix5の標的遺伝子同定を中心に研究を進めた。
    (1)six1遺伝子欠損ホモマウスは、内耳、鼻、胸腺、骨格筋および腎臓の形成不全がみられ、生直後に死亡する。耳胞での発生制御遺伝子群の発現を精査した。耳胞腹側の遺伝子発現が消失、背側の遺伝子発現領域が拡大しており、Six1の欠損が耳胞のパターン形成に重大な影響を与えたことを明確にした。
    (2)後腎間葉細胞を用いてSix1およびSix4タンパク質の標的遺伝子候補を同定した。Six1では活性化されるがSix4では活性化が弱い遺伝子、Six1では弱く抑制されるが、Six4では強く抑制される遺伝子、両者で同様に活性化される遺伝子がそれぞれ見つかった。標的遺伝子候補の中に、その欠損が腎臓や耳の形成異常につながることが知られた遺伝子も含まれていた。
    (3)VP16-Six5 (野生型)およびVP16-Six5W241R (変異型)の組換えアデノウイルスを、ヒト顆粒膜細胞株KGNに感染させ、マイクロアレーに対する競合ハイプリダイゼーションを行い、野生型と変異型とで2倍以上発現レベルが異なる遺伝子を標的候補として同定した。アレイ上にスポットされた12,814遺伝子の内の2.4%、310遺伝子が標的候補として同定できた。不妊症や遺伝子欠損マウスの表現型から卵胞の形成や機能に関与することが既に知られており、性腺機能低下症との関連が強く示唆される遺伝子が標的遺伝子として同定された。

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  • Analysis of the state of binding of tissue specific and general transcription factors to the promoter in vivo.

    Grant number：14580686  2002 - 2003

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IKEDA Keiko

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    Grant amount：\3100000 （ Direct Cost: \3100000 ）

    Recently, it has been gradually accepted that the mechanism of transcription should be understood with time concept viewpoint. Development, cell differentiation, and cell response to environmental stimuli can be interpreted to the process of the interaction between DNA and transcription factors with time. In this project, we focused on the interaction between tissue specific transcription factors and promoter DNA using myoblast differentiation system. The system was consist of 1. Mouse fibloblast of 10T1/2 which is stably transformed with ER-MyoD (ER for induction signal and MyoD is the master gene for muscle differentiation). 2. Transcription factors are MyoD, CBP, p300, and Six family protein. 3. Genes whose promoters have been suggested to cordinately function during muscle differentiation are muscle creatine kinase, myogenin, desmin, p21, myosin light chain, tublin alpha. We found that these genes are expressed at different time point of differentiation by Northern blot analysis. We next examined various transcription factor bindings to their promoter regions and found that first MyoD binds to their promoter in pararell with the transcriptional expression. Others are variable depending on the promoters. We further examined the gene dynamics using three dimentinal fluorescence in situ hybridization (3D FISH) technique (visualization of chromosomal territories and gene of interest with BAC probes) and observed the relaxing promoter region before MyoD binding. These results suggest that gene transcription is swithed on not only by specific transcription factor binding, but also chromatin-packed DNA, which contains promoter regions to be transcribed, is relaxed just before transcription factor binding. The molecular mechanism of the latter is now under investigation.

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  • ホメオドメイン転写因子SIX1の血液細胞周期および分化制御機構

    Grant number：13043045  2001

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    池田 啓子, 川上 潔

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    Grant amount：\2400000 （ Direct Cost: \2400000 ）

    転写因子Sixタンパク質の細胞周期への役割を明らかにすることを目的とした。
    成果(1)血球細胞分化Lineageに伴うSIX1発現の消長。成人骨髄から、造血幹細胞を採取し、種々の薬剤により分化を誘導した場合に内因性SIX1発現の変化をRT-PCR法にて検証した。SIX1は未分化細胞で少量発現し、赤血球、巨核球いずれの分化系列においても、分化刺激後2日目でその発現が増加し、4日目で多少低下した後、6日目で、再び発現が顕著に増大した。なお、細胞は分化刺激後6日目においては、分化系列が進行する一方、増殖能も増加していた。
    成果(2)血球系細胞株における発現。HL60(C)細胞株では増殖培地下で強発現が観察された。HL60(C)はPDBuまたはAraCにより分化を誘導し、誘導開始後1,2,3,4日目の細胞からmRNAを調製しノザン法にてSIX1の発現量を調べた。分化誘導・増殖能消失と同時に顕著にSIX1の発現レベルが顕著に低下した。この現象は赤血球、巨核球等の分化系譜にはよらなかった。これらの細胞株は、分化とともに増殖能が低下する。
    成果(3)血球系細胞株へのSIX1の強制発現による増殖能の変化。SIX1の強発現に用いる、野生型および、DNA結合能を欠く変異型を作成し、K562細胞に形質転換を行った。この際、SIX1遺伝子産物を発現する細胞は、同時にGFP遺伝子産物を発現させ、GFP陽性細胞群について、細胞周期を調べた。SIX1の強制発現により、S期の優位な増加が観察された。DNA結合能を欠く変異型SIX1も野生型と同程度のS期の優位な増加が観察された。さらに細胞同士の接着性の増加が観察された。
    成果(4)標的遺伝子の同定。野生型SIX1の強制発現株のマイクロアレイにより、種々のサイトカインや接着因子の有意な発現増強が観察された。

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  • Six-Eya-Dach遺伝子ネットワークによる器官形成

    Grant number：13045041  2001

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    川上 潔, 池田 啓子, 尾崎 秀徳

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    Grant amount：\3100000 （ Direct Cost: \3100000 ）

    Six遺伝子欠損マウスの解析、Sixタンパク質の標的遺伝子のスクリーニングおよびEyaとDachの分子機能の解析をとおして、器官形成に至る分子メカニズムを明確にすることを目標に研究を展開し、下記の5点の実績を得た。
    (1)Six4/Six5遺伝子二重変異マウスホモ個体のうち約半数は生後数時間以内に死亡するが、明らかな形態的組織学的異常は見いだせなかった。生き延びた個体は耳介の大きさが野生型に比して若干小さかった。
    (2)Six1遺伝子変異マウスホモ個体は内耳、鼻、腎臓および胸腺の形成異常が観察され、出生直後にすべて死亡した。Six1がこれらの器官の形成に必須の役割を果たしていることが示唆された。
    (3)Six5タンパク質の標的遺伝子として、中胚葉分化に伴って発現する転写因子やシグナル分子及びそのリセプター、また神経系で発現する転写因子やシグナル分子、さらに神経伝達物質の輸送体や受容体遺伝子などが同定された。Six5遺伝子はそれらの標的遺伝子群の制御を介して中胚葉由来の組織器官の形成や、神経組織の構築の発生にかかわることが示唆された。
    (4)Gal4-Eyaタンパク質とDach1タンパク質を293細胞に共発現するとGal4結合部位を複数持つ人工プロモーターのレポーター遺伝子転写活性が著しく上昇した。この、転写活性化にはCBPが関与することを明らかにした。
    (5)BOR症候群患者で検出されるEYA1の点突然変異のうちS486PとL504RにおいてはEya1とDach1、Gタンパク質及びSixタンパク質との相互作用の欠陥と、MyogeninプロモーターのSix5と協調した転写活性化の低下、さらに構造変化が見られた。Eyaドメインの変異がタンパク質間相互作用の欠陥を介してBOR症候群をひきおこことと、SIX、DACH及びGタンパク質もBOR症候群の病態過程と耳や腎臓の発生過程にかかわることが示唆された。

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  • Developmental Program and Genetic Disease by Six family genes.

    Grant number：12470029  2000 - 2002

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    KAWAKAMI Kiyoshi, IKEDA Keiko, SATO Shigeru, OZAKI Hidenori

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    Grant amount：\14200000 （ Direct Cost: \14200000 ）

    This study aims to reveal roles of Six family genes in development, to elucidate gene network including Six and involvement of Six genes in pathology of myotonic dystrophy (DM1). We performed analyses of Six gene defective mice, screening of target genes of Six proteins and analyses of molecular function of Eya and Dach protein that are cooperative factors of Six protein.
    1 Six4/Six5 double knockout mice die within several hours after birth but we could not find any apparent anatomical anomalies. Six1 gene defective mice die just after birth and showed defective formation of inner ear, nose, kidney and thymus. The morphological abnormalities were noted from E10-11. Six1 gene is suggested to be essential for the formation of these organs.
    2 Target genes of Six5 proteins were identified. Transcription factors, signaling molecules and its receptors that are expressed during mesoderm differentiation were identified as putative target in P19 cells. Transcription factors and signaling molecules in nervous systems, transporters and receptor proteins of neural transmitters. In myoblasts, genes including myogenin, myosin, troponin, acetylcholine receptors that arespecific to skeletal muscle were identified. In lens epithelial cells, genes that had been shown to be involved in cataractogenesis were identified. It is suggested that altered regulation of these target genes leads to some symptoms of DM1.
    3 We revealed that cooperative activation by GAL4-Eya and Dach is mediated through CBP. CBP bound to an immobilized chromatin template only in the presence of both GAL4-Eya and Dach. We also found that Dach can bind to chromatin as well as DNA regardless of the presence of GAL4-Eya protein. The binding affinity to chromatin was higher than that to naked DNA. The conserved DD1 domain of Dach is responsible for the DNA binding activity.

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  • 生化学的アプローチによる難聴遺伝子の同定と病態の解明

    Grant number：12877273  2000 - 2002

    日本学術振興会  科学研究費助成事業  萌芽研究

    川上 潔, 池田 啓子

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    Grant amount：\2100000 （ Direct Cost: \2100000 ）

    EYA1遺伝子は難聴や腎形成不全を伴うBOR (Branchio-oto-renal)症候群の原因遺伝子である。Eyaタンパク質はSixタンパク質やDachタンパク質、Gタンパク質と相互作用し、遺伝子の発現制御に関与することが知られている。本研究ではEyaタンパク質を含む複合体やDachとの相互作用が示されたタンパク質を含む複合体を精製し、遺伝子の発現制御にかかわる機能解析と新規なEya相互作用タンパク質の同定を行い、新規難聴遺伝子の発見につなげることを目指している。本年度は次のような研究実績を得た。
    (1)昨年度樹立したTagを付加したEyaタンパク質を発現するHeLa細胞株を大量に培養し、核抽出液を調製した。イオン交換カラムクロマトグラフィーを用いた後にtagを用いたアフィニティー精製を行うことで、Eya1と複合体を形成していると考えられる複数のタンパク質を確認できた。質量分析での同定にはさらに量を増やした精製が必要であった。
    (2)EyaとDachによる協同的な活性化にはCBPが関与する。Dachタンパク質はEyaの存在がなくても裸のDNAやクロマチンDNAに結合できることを見いだした。このDNA結合活性は、Dachの保存領域であるDD1ドメインによることを明らかにした。DD1ドメインはForkhead-Winged Helixであることが他のグループによって示され、我々の結果が構造的にも支持された。
    (3)Dachタンパク質を含む新規複合体の同定に用いるために、Dachを発現するHeLa細胞株を樹立しつつある。

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  • 試験管内転写系を用いた新規コアクチベーターの解析

    Grant number：12028231  2000

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    池田 啓子

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    Grant amount：\2100000 （ Direct Cost: \2100000 ）

    <研究背景>
    転写は、基本転写因子群によって行われる基本転写と、上流域結合因子群と基本転写因子群との協同作用による制御転写にわけて考えることができる。細胞内で観察される転写活性化機構の分子的実体を明らかにするべく、1)転写活性化モデルとして使用されている強力な転写活性化因子であるヘルペスウイルス蛋白質VP16活性化ドメインの、コアクチベーターの精製2)ほ乳類生体内に存在し目や筋肉の発生に関係するEyaコアクチベーターの複合体の精製を行った。
    <実施状況>
    1)VP16の活性化ドメインをさらにH1ドメインとH2ドメインに分けた。2つのサブドメインは培養細胞をいた一過性形質転換法による転写活性化様式に違いがあることから、ドメイン特異的な因子またはメカニズムを通じた転写活性化であると示唆された。従来のDignam法によらず、Low Salt法(Ikeda,K.＆ Meisterernst,M)により調製されたHeLaS核抽出液にVP16H1ドメインに依存的な活性が再現性よく観察された。裸およびクロマチンDNA鋳型の試験管内転写系を用いてそれぞれのサブドメインを通じて作用するコアクチベーターを探索した。前者のドメインにはMediator ComplexであるSMCCが結合し裸のDNA鋳型からの転写を担う。後者のドメインにはSMCCとCBP/p300が結合しクロマチンDNA鋳型からの転写を担うことが明らかになった。
    2)HeLa細胞の核抽出液300mlから、イオン交換カラム、ゲル濾過カラム等を用いて、Eyaを含む複合体を精製した。同複合体は、SDS-PAGEにて6つのポリペプチドから成ることが示唆された。終精製画分は、Sixタンパク質存在下、裸およびクロマチンDNAを鋳型にした試験管内転写系では転写活性化能を有しなかった。

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  • ホメオボックス蛋白質Sixの核-細胞質移行と、細胞周期制御・ガン化との関係

    Grant number：12215135  2000

    日本学術振興会  科学研究費助成事業  特定領域研究(C)

    川上 潔, 池田 啓子

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    Grant amount：\3000000 （ Direct Cost: \3000000 ）

    SIX1は乳がん細胞株において広く発現が見られ、悪性度の強いがん細胞ほどその発現が昂進している。また、X線照射時のG2/Mチェックポイントによる増殖停止が、SIX1の強制発現によって回避されることから、細胞周期の調節および増殖制御に深く関与していることが示唆された。分化の道筋が明確で、種々の分化制御因子によって、分化をコントロールできる血球細胞に注目し、SIX1の細胞周期と細胞増殖における役割と、細胞がん化のコントロール因子としての可能性を明らかにするために、血球細胞分化Lineageに伴うSIX1発現の消長とUT-7細胞の分化に伴う発現変動を検討した。SIX1は未分化血球細胞で発現し、赤芽球、骨髄球、巨核球いずれの分化系列においても、分化刺激後2日でその発現が増加し、刺激後4日でいったん低下した後、分化刺激後6日で、再び発現が顕著に増大した。UT-7細胞株においては、増殖培地でのSIX1の発現は顕著であるが、エリスロポイエチンおよびトロンボプラスチンによる分化刺激後2日でSIXの発現が著しく低下した。これらの結果から、血球細胞分化によって、SIX1が一元的に増加ないし減少するのではなく、分化の道筋によって発現量を調節することで、分化状態での増殖をコントロールしている可能性が示された。そのことを実験的に示すために、HL-60細胞にSIX1を強制発現させたときの分化増殖への影響と、UT-7にアンチセンスSIX1を発現させたときの効果を検証している。

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  • クロマチン鋳型からの転写に必須なCBP/p300を含む複合体の精製とその機能

    Grant number：11770067  1999 - 2000

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    池田 啓子

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    Grant amount：\1800000 （ Direct Cost: \1800000 ）

    <研究目的>
    転写は、基本転写因子群によって行われる基本転写と、上流域結合因子群と基本転写因子群との協同作用による制御転写にわけて考えることができる。筆者は試験管内再構成転写系を用いて制御転写の転写活性化および抑制化機構の解析を行ってきた。その中で今までに報告されている再構成転写系における転写活性化は、因子の活性化ドメインの特異性が反映されないという点において、形質転換法等で観察される細胞内での転写活性化とは明らかに異なることを発見した。細胞内で観察される転写活性化機構を明らかにするべく、核抽出液の調製法から検討し、強力なVP16活性化ドメインに特異的なコアクチベーターの精製を試みた。
    <実施状況>
    VP16の活性化ドメインをさらにN末部分(以下H1ドメイン;アミノ酸412-456)とC末部分(以下H2ドメイン;アミノ酸453-490)に分けた。2つのサブドメインは培養細胞を用いた一過性形質転換法による転写活性化様式に違いがあることから、ドメイン特異的な因子またはメカニズムを通じた転写活性化であると示唆された。従来のDignam法によらず、Low Salt法(Ikeda,K.＆ Meisterernst,M)により調製されたHeLaS核抽出液にVP16H1ドメインに依存的な活性が再現性よく観察された。コンベンショナルなカラムとアフィニティカラムを使用し、裸およびクロマチンDNA鋳型の試験管内転写系を用いてそれぞれのサブドメインを通じて作用するコアクチベーターを探索した。前者のドメインにはMediator ComplexであるSMCCが結合し裸のDNA鋳型からの転写を担う。後者のドメインにはSMCCとCBP/p300が結合しクロマチンDNA鋳型からの転写を担うことが明らかになった。

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  • CBP/p300を含む新規複合体のクロマチン高度再構成転写系を用いた機能解析

    Grant number：11154222  1999

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    池田 啓子

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    Grant amount：\2100000 （ Direct Cost: \2100000 ）

    細胞内で観察される転写活性化を反映する核抽出液の調製法と試験管内転写系を用いて、強力なVP16活性化ドメインに特異的なコアクチベーターの精製を試みた。従来のDignam法によらずLow Salt法(Ikeda, K. & Meisterernst, M)により調製されたHeLas核抽出液にVP16H1ドメイノ(転写活性化ドメインN末部分)に依存的な活性が再現性よく観察された。コンベンショナルなカラムとアフィニティカラムを使用し、その活性を担う因子を同定、PC6-MOVE(Positive Cofactor No.6-Mediator of VP16 enhancer)と名付けた。PC6-MOVEは既知のgenaral positive cofactor (USA fractionから精製、クローニングされたPC1, PC2, PC3, PC4, PC5, NC1, NC2)ではないこと、SRC-1等の既知の転写因子特異的コアクチベーターやコアクチベーター複合体であるARCおよびTRAP/SMCC6との異同を生化学的・免疫化学的に明らかにした。一方、VP16H2ドメイン(転写活性化ドメインC末部分)にはヒストンアセチルトランスフェラーゼ活性を有するコアクチベーターp300/CBPが直接相互作用することにより、特異的転写活性化がおこることを明らかにした。p300/CBPとの相互作用に必須のVP16側の3つのアミノ酸と、VP16H2ドメイノとの相互作用するp300/CBP側の部位を同定した。P300/CBPはクロマチン鋳型DNAからの転写に必須であることを確認した。以上の結果よりVP16の強力な転写活性化は、従来報告されている基本転写因子とPC6-MOVE、p300/CBPとの協同作用によると結論した。

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  • In vivo function of Na,K-ATPase alpha subunit genes

    1994

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    Grant type：Competitive

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  • Health consciousness of homeless at Sanya

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    Grant type：Competitive

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  • The strcture and its function of transcription factor AREB6

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    Grant type：Competitive

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  • Transcriptional Regulation of Na, K-ATPase α1 and α2 subunit gene

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    Grant type：Competitive

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  • Role of Six gene family in cranial ganglia development

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    Grant type：Competitive

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