細胞移動、層・神経核の形成 Cell Migration and Layer/Nuclear Formation
P1-1-57 歯状回の顆粒細胞を産生するGfap発現神経前駆細胞の性質は周生期に変化する The property of Gfap-expressing dentate neural progenitors producing granule cells is altered during perinatal period ○石龍徳1, 皆川史織2, 佐藤亨1, 戸田景子1, 岩室祥一2, 塩田清二3 ○Tatsunori Seki1, Shiori Minakawa2, Toru Sato1, Keiko Toda1, Shoichi Iwamuro2, Seiji Shioda3 東京医科大・組織神経解剖1, 東邦大・理・生物2, 昭和大・医・解剖3 Dept Histol Neuroanat, Tokyo Med Univ, Tokyo1, Dept Biol, Toho Univ, Chiba2, Dept Anat, Showa Univ, Tokyo3 In the adult hippocampus, granule cells continue to be generated from stem cells that are astrocyte-like cells expressing glial fibrilar acidic protein (GFAP). Previously we have studied the embryonic origin of the granule cells by use of Gfap-Green fluorescent protein (GFP) transgenic mice, and have demonstrated that distinctive Gfap-expressing progenitor cells initially originate in a focal region in the neuroepithelium of the medial pallium at the dorsal edge of the fimbria, called the dentate notch, and later travel through the dorsal portion of the fimbria and the subpial region to the anlage of the dentate gyrus where Gfap-expressing progenitors form the granule cell layer, implying that astrocyte-like neural progenitors continue to generate granule neurons from the beginning of dentate development, throughout life. To further know the property of Gfap-expessing progenitor cells, here we examined the expression of brain lipid-biding protein (BLBP) in the Gfap-GFP mice, because BLBP is a well-known marker for neocortical stem cells. Although in the embryonic neocortex, BLBP+ radial glial cell bodies densely lined the lateral ventricle wall and expanded their radial processes throughout the entirety of the neocortical layer, BLBP+ cells were scarcely detected in the hippocampus at E15.5 and 17.5. During these stages, exceptional strong BLBP-expressing cells were present in the fimbria and subpial region, and a very few BLBP+ cells with long processes are scattered in the dentate gyrus. Most GFP+ cells in the hippocampus were devoid of BLBP. Only GFP+ cells in the fimbria expressed BLBP. However, after birth, GFP+ cells in the developing dentate gyrus gradually became BLBP+, and most GFP+ radial progenitors in the subgranular zone expressed BLBP by postnatal day 14. These results suggest that stem /primary progenitor cells of the dentate granule cells are distinct from those of the neocortex and alter their property after birth.	P1-1-58 発生期大脳皮質においてリーリンはそのシグナル下流分子を通して神経細胞の凝集を誘導する Reelin induces neuronal aggregation through its downstream molecules in the developing cortex ○久保健一郎1, 関根克敏1, 山川眞以1, 石井一裕1, 野田万理子1, 廣田ゆき1, 本田岳夫1, 林周宏1, 仲嶋一範1 ○Ken-ichiro Kubo1, Katsutoshi Sekine1, Mai Yamakawa1, Kazuhiro Ishii1, Mariko Noda1, Yuki Hirota1, Takao Honda1, Kanehiro Hayashi1, Kazunori Nakajima1 慶應義塾大学医学部解剖学1 Dep. of Anatomy, Keio Univ. Sch. of Med., Tokyo, Japan1 During neocortical development in mammals, most of cortical projection neurons are born near the ventricle and migrate radially towards the brain surface to be aligned in precisely defined patterns in the cortical plate. These patterns of neuronal alignment are regulated by an extracellular matrix protein, Reelin, which is secreted from Cajal-Retzius neurons in the marginal zone of the developing cortex. When Reelin binds to its receptors, tyrosine phosphorylation of the intracellular adaptor protein Disabled 1 (Dab1) is induced. The molecular cascade of Reelin signal is being uncovered, including the intracellular Dab1-Crk/CrkL-C3G-Rap1-Integrin α5β1 pathway. However, the biological response of neurons to the Reelin signal is yet to be fully understood. To elucidate the biological role of Reelin signal in the developing cortex, we used the in utero electroporation system. Ectopically expressed Reelin caused the leading processes of migrating neurons to assemble in the Reelin-rich region, which in turn induced their cell bodies to form cellular aggregates around Reelin. The ectopic Reelin-rich region became cell-body-sparse and dendrite-rich, resembling the MZ, and the late-born neurons migrated past their predecessors toward the central Reelin-rich region within the aggregates, resulting in a birthdate-dependent "inside-out" alignment even ectopically. Interestingly, Downstream molecules were found to be necessary for formation of the aggregates. Underlying mechanisms will be discussed.
P1-1-59 海馬歯状回顆粒細胞層形成におけるLOTUSの役割 Role of LOTUS in granular layer formation of dentate gyrus ○池谷真澄1, 横山高玲4, 栗原裕司1, 山本亘彦3, 五嶋良郎2, 川原信隆4, 竹居光太郎1 ○Masumi Iketani1, Takaakira Yokoyama4, Yuji Kurihara1, Nobuhiko Yamamoto3, Yoshio Goshima2, Nobutaka Kawahara4, Kohtaro Takei1 横浜市大・医・生命医科学1, 横浜市大院・医・分子薬理神経生物2, 大阪大院・生命機能・細胞分子神経生物3, 横浜市大院・医・脳神経外科4 Div. of Med. Life. Sci., Sch. of Med., Yokohama City Univ., Yokohama, Japan1, Dept. of Mol. Pharmacol. and Neurobiol., Grad. Sch. of Med., Yokohama City Univ., Yokohama, Japan2, Cell. Mol. Neurobiol. Lab., Grad. Sch. of Frontier Biosci., Osaka Univ.3, Dept. of Neurosurgery, Grad. Sch. of Med., Yokohama City Univ., Yokohama, Japan4 The granular layer of dentate gyrus is formed by migration of progenitor cells through the region of hilus. We found that LOTUS, an endogenous Nogo receptor-1 (NgR1) antagonist, was expressed in developing mouse dentate gyrus. NgR1 has been well studied as a molecule to inhibit nerve regeneration in the adult central nervous system. However, little is known about its function in neural development. We found that abnormal formation of dentate gyrus in neonatal LOTUS-knockout mice. The granule cells of dentate gyrus were distributed in a scattered matter in the hilus region. Such abnormal distribution of the granule cells suggested that lack of LOTUS inducing Nogo signaling gives rise to cell migration disturbance. Recently, it has been reported that Nogo and NgR1 play an important role in regulation of neural migration in developing neocortex. Therefore, it is suspected that LOTUS may contribute to cell migration in the dentate gyrus formation through down-regulation of NgR1 function.	P1-1-60 マウス扁桃体のトランスクリプトーム解析 Spatio-temporal transcriptome of the mouse amygdala ○相馬美歩1,2, 茂櫛薫3, 田中博3, 田中光一1,2 ○Miho Soma1,2, Kaoru Mogushi3, Hiroshi Tanaka3, Kohichi Tanaka1,2 東京医歯大・難研・分子神経1, 東京医歯大・脳統合機能研セ2, 東京医歯大・難研・生命情報3 Dept Mol Neurosci, Med Res Inst, Tokyo Med & Dent Univ, Tokyo, Japan1, Cent Brain Integ Res, Tokyo Med & Dent Univ, Tokyo, Japan2, Dept Bioinform, Med Res Inst, Tokyo Med & Dent Univ, Tokyo, Japan3 The amygdala is composed of several subdivisions and is involved in the cognitive function and emotional behavior. Amygdala dysfunction has been indicated in various psychiatric disorders including autism, depression, and schizophrenia. However, pathological changes of amygdala in these diseases are not yet fully described. In addition, the molecular pathways that mediate amygdala development and function are largely unknown. In order to analyze the molecular basis related to the amygdalar function, we examined the spatio-temporal dynamics of the mouse amygdala transcriptome. Using a laser capture microdissection method, we prepared tissue samples from five amygdala subnuclei including lateral nucleus (La), basolateral nucleus (BL), basomedial nucleus (BM), medial nucleus (Me), and central nucleus (Ce) of male and female mice spanning the periods from embryonic development to late adulthood. In neonatal amygdala, expression level of genes that regulate the neuronal development, neuronal differentiation, axonal guidance, and myelination were specifically changed. During the postnatal period, gene expression profiles were classified into two major clusters: La/BL cluster and BM/Me/Ce cluster. Each amygdalar subdivision had distinct gene expression pattern, and we found several marker genes that are useful for distinguish each amygdalar subdivision. We also identified some genes that differ in expression by sex or age. This study provides a comprehensive data set on the mouse amygdala transcriptome and insights into the molecular basis of mouse amygdalar development and function.
P1-1-61 神経細胞移動と神経変性におけるリーリンシグナルとStk25の役割 The roles of Reelin-Dab1 and Stk25 signaling in the regulation of neuronal migration and neurodegeneration ○松木亨1,2 ○Tohru Matsuki1,2, Mariam Zaka3, Brian Howell2 愛知県コロニー・発達障害研究所　発生障害学部1, ニューヨーク州立大学アップステート医科大学2, 米国国立衛生研究所、神経変性疾患・脳卒中研究所3 Institute for Developmental Research, Kasugai, Aichi, Japan1, Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, USA2, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland USA3 The development of the brain is a complex process involving the regulation of neuronal positioning, axonogenesis, and dendritogenesis. Deviations from the normal program lead to the developmental diseases ranging in severity from epilepsy to mental retardation. Some known molecules related neuronal development also play important roles in neurodegenerative diseases, including Alzheimer's disease. We have been investigating the biological roles of Reelin-Dab1 signaling that are well known to govern the neuronal positioning during brain development. We previously reported that Stk25 regulates neuronal polarization and Golgi morphology by collaborating with Reelin-Dab1 signaling. However, two major questions still remain to be determined; 1) whether or not Reelin-Dab1 signaling has a critical role in neurodegeneration. Tau hyperphosphorylation is a hallmark of neurodegenerative disease including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. 2) Does Stk25 have roles in neuronal positioning and/or neurodegeneration? As shown in the role of Reelin-signaling, it has been estimated that Stk25 may be involved in the neuronal migration. Here, we show that dab1-gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. We also found that the knockdown of Stk25 reduces Tau phosphorylation in embryonic neurons. Furthermore, we found the implication that Stk25 may have an important role in neuronal migration. These results impact us to understand the biological roles of Reelin-Dab1 signal and Stk25 both in the mechanisms of neuronal development and neurodegeneration.	P1-1-62 低分子量G蛋白質Rap1活性化因子RA-GEF-1、RA-GEF-2のマウス大脳皮質形成時における神経前駆細胞分布の制御機構の解析 The Rap1 guanine nucleotide exchange factors, RA-GEF-1 and RA-GEF-2, are required for the proper distribution of neural progenitors in mice ○前田和宏1, , 西原香1, 藤川千恵1, 坪谷一樹1, 枝松裕紀1, 片岡徹1 ○Kazuhiro Maeta1, Shymaa Bilasy1, Kaori Nishihara1, Chie Fujikawa1, Kazuki Tsubotani1, Hironori Edamatsu1, Tohru Kataoka1 神戸大学大学院 医学研究科 分子生物学分野1 Div. of Mol. Biol., Kobe Univ. Grad. Sch. of Med., Kobe1 RA-GEF-1 (Rapgef2) and RA-GEF-2 (Rapgef6) are a family of guanine nucleotide exchange factors responsible for the activation of Rap1 small GTPases. We have reported that telencephalon-specific RA-GEF-1 conditional knockout (cKO) mice develop an ectopic cortical mass (ECM) underlying a relatively normal cerebral cortex and a severe defect of the major forebrain commissures (Bilasy et al., 2009, 2011). Additional disruption of RA-GEF-2 in cKO mice (dKO) results in further enlargement of the ECM. To obtain an insight into the function of these Rap1 GEFs in neural development, we further analyze the phenotypes of their mutant embryos. At embryonic day (E) 15, in the cortex of cKO mice both Pax6+ radial glial cells and Tbr2+ intermediate progenitors are mislocated in the intermediate zone and even in the cortical plate in addition to their normal locations, the ventricular and subventricular zones, respectively. Also, radial glial fibers are discontinuous and abnormally oriented. In dKO mouse embryos, such abnormalities in the progenitor locations and radial glial fibers appear much earlier and become more pronounced than those in cKO mice. Anti-beta-catenin immunostaining reveals that cells located at the ventricular surface exhibit an irregular shape in cKO and dKO embryos, suggesting the disruption of the adherence junctions. These results suggest that RA-GEF-1 and RA-GEF-2 play a crucial and cooperative role in the regulation of the distribution of neural progenitor cells through the maintenance of the adherens junctions in apical surface cells. Further analyses are underway to clarify the underlying molecular mechanisms.
P1-1-63 胎仔期ビスフェノールA曝露によるマウス大脳皮質形成障害と衝動行動 Perinatal Exposure to Bisphenol A Affects Cortical Development and Induces Impulsive Behavior in Mice 遠藤俊裕1, 宮崎航1, 木村栄輝1, 久保健一郎2, 仲嶋一範2, 掛山正心1, 遠山千春1 ○Wenting Ling1, Toshihiro Endo1, Wataru Miyazaki1, Eiki Kimura1, Ken-ichiro Kubo2, Kazunori Nakajima2, Masaki Kakeyama1, Chiharu Tohyama1 東京大院・医・健環医1, 慶大・医・解剖2 Lab Environ Health Sci, Univ of Tokyo, Tokyo1, Dept Anat, Keio Univ, Tokyo2 Bisphenol A (BPA), a polycarbonate material used in plastic dishes and bottles is one of the chemicals that have potentials to affect developing brain of children, but the mechanism of its adverse effects on brain development and behaviors is largely unknown. Thus, in this study we investigated whether and how maternal low-dose BPA exposure affects embryonic cortical development and behaviors later in adulthood.Neuronal migration, an essential process of cortical development, was investigated using an in utero electroporation (IUE) technique. Pregnant ICR mice were exposed to BPA by implanting osmotic pump at a dose equivalent to 0, 40 (BPA-40), 400 (BPA-400) μg/kg b.w./day from embryonic day 14.5 (E14.5) to E18.5. The mCherry expression plasmid was transfected into neural progenitor cells on E14.5 by IUE, and neuronal migration was analyzed on E18.5. Behavior of 10-week-old male offspring, born to C57BL/6J dams administered BPA at the same dose described above from E8.5 to E18.5, was tested using IntelliCage (IC). This behavior test allowed mice to receive a reward by a nose poke if they can wait a specified time (3 or 6 sec). The nose poke interval time was measured to evaluate the behavioral performance.The percentage of mCherry-labeled neurons in the cortical layer II/III of the somatosensory cortex of BPA-40 group was significantly decreased compared with that of the control group, indicating impairment of neuronal migration by BPA. Moreover, adult mice of the BPA-40 group exhibited a significant decrease in a nose poke interval in the IC test, indicating the occurrence of highly impulsive behavior. No significant alterations in neuronal migration or behavioral performance were found in the BPA-400 group. In conclusions, perinatal exposure to BPA disrupts cortical development during gestation and behavior later in adulthood in mice, but their relationship remains to be studied.	P1-1-64 神経細胞におけるDab1を介したCIN85とReelin受容体ApoER2・VLDLRの相互作用 Dab1-mediated interaction of multi-adaptor protein CIN85with Reelin-receptors, ApoER2 and VLDLR, in neurons ○渕上孝裕1, 佐藤裕1, 富田裕也1, 高野哲也1, 宮内伸也1, 土屋幸憲1, 斎藤太郎1, 久保健一郎2, 仲嶋一範2, 福田光則3, 服部光治4, 久永眞市1 ○Takahiro Fuchigami1, Yutaka Sato1, Yuya Tomita1, Tetsuya Takano1, Shin-ya Miyauchi1, Yukinori Tsuchiya1, Taro Saito1, Ken-ichiro Kubo2, Kazunori Nakajima2, Mitsunori Fukuda3, Mitsuharu Hattori4, Shin-ichi Hisanaga11 首都大・理工学・生命1, 慶應大・医学・解剖学2, 東北大・生命・脳科学3, 名古屋市大・薬学・病態生化学4 Dept Biol, Tokyo Metro Univ, Tokyo1, Dept Anatomy, Med, Keio Univ, Tokyo2, Dept Dev Biol & Neurosci, Tohoku Univ, Sendai3, Dept Biomed Sc, Pharm Sci, Nagoya City Univ, Nagoya4 Reelin-Dab1 signaling is indispensable for proper positioning of neurons in mammalian brain. Reelin is a glycoprotein secreted from Cajal-Reztuis cells in marginal zone of cerebral cortex, and its receptors are ApoER2 or VLDLR expressed on migrating neurons. When Reelin binds to ApoER2 or VLDLR, an adaptor protein Dab1 bound to the receptors undergoes Tyr-phosphorylation that is essential for Reelin signaling. We reported previously that Cdk5-p35 phosphorylates Dab1 at Ser400 and Ser491 and the phosphorylation regulates its binding to CIN85, which is an SH3-containing multiadaptor protein involved in endocytic downregulation of receptor tyrosine kinases. However, the interaction of CIN85 with Dab1 has not been addressed in neurons. We examined here a possibility that CIN85 has a role in Reelin-signaling. We found nonphosphorylated Dab1 mediated colocalization of CIN85 with ApoER2. The colocalization of CIN85 with ApoER2 was increased in neurons stimulated with Reelin repeats 3-6, an active Reelin fragment. The stimulation recruited CIN85 to domains in plasma membrane where it colocalized with ApoER2 and Dab1 and then to EEA1-labeled early endosomes in the cytoplasm. In addition, Tyr-phosphorylation of Dab1 strengthened the binding to CIN85. These results suggest that CIN85 participates in Reelin signaling through the binding to Dab1.

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