幹細胞、ニューロンとグリアの分化 Stem Cells, Neuronal and Glial Production/Differentiation
P3-1-43 多能性幹細胞からの神経分化におけるdopamineの役割 Role of dopamine in neural differentiation from pluripotent stem cells ○濱田祐輔1, 葛巻直子1,2, 須田雪明1, 成田道子1, 渡邉萌1, 志村絵理1, 岩澤千鶴1, 岡田洋平2, 赤松和土2, 岡野栄之2, 成田年1 ○Yusuke Hamada1, Naoko Kuzumaki1,2, Yukari Suda1, Michiko Narita1, Moe Watanabe1, Eri Shimura1, Chizuru Iwasawa1, Yohei Okada2, Wado Akamatsu2, Hideyuki Okano2, Minoru Narita1 星薬科大学薬理学教室1, 慶應義塾大学医学部生理学教室2 Dept. Pharmacol., Hoshi Univ. Sch. Pharm. Pharmaceut. Sci., Tokyo, Japan1, Dept. Physiol., Keio Univ. Sch. Med. Tokyo, Japan2 Pluripotent stem cells like ES and iPS cells have unlimited self-renewal ability and the potentials to differentiate into specialized cells. It has been reported that monoamines and neuropeptides appear in the embryo before cell differentiation, and have functions during embryogenesis such as differentiation and growth. Particularly, dopamine is a key modulator in the brain development by regulating cell cycle and proliferation. In the present study, we investigated the role of dopamine in mouse ES and iPS cell differentiation. At first, we found the progressive expression of many dopamine-related genes, such as tyrosine hydroxylase, dopamine receptors (D1, D2 and D3) and dopamine transporter, from undifferentiated ES and iPS cells to differentiated neural stem cells, while the mRNA of monoamine metabolic enzymes was high dense even in ES and iPS cells. Next, we profiled the influence of dopamine receptor stimulation located on ES and iPS cells on expressing the GPCR in neural stem cells. To profile the expression of mRNAs encoding 384 GPCRs in neural stem cells, a TaqMan GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between dopamine receptor-stimulated neural stem cells by dopamine and non-stimulated cells. These findings support the possibility that dopamine may play a role in regulating ES/iPS cell differentiation and transforming the differentiated neural stem cells. (1488/2000)	P3-1-44 Quaking isoform 5（Qki5）による細胞周期制御機構の解明 Quaking isoform 5 (Qki5) shows an unique cell cycle dependent expression pattern and regulates proliferation in fetal brain ○陶山智史1, 矢野真人1, 岡野栄之1 ○Satoshi Suyama1, Masato Yano1, Hideyuki Okano1 慶應義塾大学医学部生理学教室1 Dept Physiol, Keio Univ, Tokyo1 Neural Stem Cells (NSCs) produce their progeny and form a highly organized brain structure. Their proliferation is properly regulated to generate the correct number of specific cell types during developing brain. Major part of such regulation depends on the integration of multiple pathways including gene regulations, extrinsic factors, cell-cell interactions. Quaking (QK) protein, which is an RNA binding protein, plays a critical role of oligodendrocyte development. Qk gene transcripts are expressed in the germinal layer of the fetal brain where NSCs undergo mitosis. However, the function of QK in NSCs is still unknown.In this study, we investigated the function of QK in mouse fetal brain development. We showed that the expression of QK isororm1 (Qki5) depends on cell cycle and its expression was specifically enhanced in M-phase cell (pH3-positive mitotic cell) at the protein level. Moreover, in utero electroporation studies reveled that pH3-positive cells were increased in the Qki5 over-expression group compared with the control group, suggesting new link between Qki5-RNA regulation and cell cycle control in NSC. In addition, we used HITS-CLIP, High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, to generate genome-wide Qki5-RNA interaction map in the embryonic brain. Together, these results indicate that the mitotic phase specific expression of Qki5 promotes proliferation of NSCs and also that Qki5 dependent RNA regulation plays an important role of cell cycle regulation in the embryonic NSCs.
P3-1-45 マウスES細胞由来運動神経細胞に対する骨格筋細胞の効果―共培養方法を用いた解析― Effects of skeletal muscle cells on the growth of motor neurons derived from mouse ES cells -analyzed by a unique co-culture system- ○川端佑果1, 小林麻美1, 蜷川菜々1, 鳥橋茂子1 ○Yuka Kawabata1, Mami Kobayashi1, Nana Ninagawa1, Shigeko Torihashi1 名古屋大学大学院　医学系研究科　リハビリテーション療法学専攻1 Dept of Rehabilitation Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan1 Embryonic stem (ES) cells differentiate into motor neurons when treated with retinoic acid and sonic hedgehog. In the embryo, the development of motor neurons in the brain and spinal cords extend axons to skeletal muscle fibers and form neuromuscular junctions. Thereafter, functions of skeletal muscle cells are achieved. Although various factors are involved in the axonal growth and formation of neuromuscular junctions, it has been remained unclear yet. In previous studies of interactions between motor neurons derived from ES cells and skeletal muscle cells, co-culture systems that two cell types are maintained at the same time in the same chamber, have been usually used. Also, such in vitro co-culture of motor neurons and skeletal muscle cells is a popular model system to analyze neuromuscular junction formation and their function. However, it does not mimic embryonic development in vivo. We then, developed new co-culture system using pair chambers in which motor neurons derived from mouse ES cells and C2C12 myoblasts were incubated independently. After a certain time period, walls of the pair chambers were removed and two gropes of cells were finally co-cultured. We analyzed the effects of the co-culture system and found that the number of survival motor neurons increased and axonal growth of them were prolonged. The results indicate that skeletal muscle cells release certain growth factor to promote neuronal growth.	P3-1-46 神経系細胞への低濃度ビスフェノールAの長期曝露は神経分化に影響を及ぼす Long-term exposure of neuronal precursor cells to bisphenol A alters neurodifferentiation ○福嶋伸之1, 西村侑華1, 長尾哲二1 ○Nobuyuki Fukushima1, Yuka Nishimura1, Tetsuji Nagao1 近畿大学理工学部生命科学科1 Dept Life Sci. Kinki Univ1 Bisphenol A (BPA), an endocrine disruptor, has been shown to influence the endocrine and nervous system functions. In more recent years the research focus has turned to the effects of lower doses of BPA in the development of an organism, particularly brain. However, the detail actions of BPA in the nervous system remains unclear. In the present study, we examined whether BPA exposure influenced neuronal differentiation of cortical neuroepithelial (NP) cells or PC12 cells. Cortical NP cells were cultured as neurospheres in the absence or presence of 0.01 to 100 nM BPA for 7 days, followed by culturing on poly-lysine-coated substratum without BPA for further 7 days. BPA treatment resulted in stimulation of neuronal differentiation in a bell shape manner with the maximal effect at 1 nM. By contrast, similar 7-day exposure of PC12 cells to BPA inhibited neuronal differentiation induced by nerve growth factor in the absence of BPA. The effect of BPA was not observed when PC12 cells were treated with BPA only for 1 day. RT-PCR analyses revealed that cortical NP cells expressed genes of Esrα, Esrβ, GPR30 and Errγ, whereas PC12 cells expressed Esrβ and GPR30. Thus, the opposed effects of BPA in cortical NP and PC12 cells might be due to the difference in the expression of BPA-targeting receptors. Interestingly, the inhibitory effect of BPA pre-treatment in PC12 cells was still observed when BPA was withdrew from PC12 cells for 7 days following 7-day exposure, indicating that the BPA's actions were intracellularly memorized. Because BPA is known to induce epigenetic alterations in several types of cells or tissues, similar changes might occur in PC12 cells during long-time exposure to BPA. Elucidation of the molecular mechanisms is currently underway.
P3-1-47 タウリンはマウス発生期大脳新皮質においてGABAA受容体のリガンドとして神経系前駆細胞の内在的性質制御に関与する Taurine is involved in the regulation of the intrinsic properties of the neural progenitors as a possible ligand for GABAA receptors in the mouse developing neocortex ○栃谷史郎1, 古川智範2, 福田敦夫2 ○Shiro Tochitani1, Tomonori Furukawa2, Atsuo Fukuda2 徳島大学大学院ヘルスバイオサイエンス研究部・機能解剖学分野1, 浜松医科大学・神経生理学講座2 Dept Anatomy and Dev Neurobiol, Inst of Health Biosci, The Univ of Tokushima Grad Sch, Tokushima, Japan1, Dept of Neurophysiol, Hamamatsu Univ Sch of Medicine, Hamamatsu, Japan2 Spatial and temporal regulation of the intrinsic properties of neural progenitors to generate the diverse type of neurons and glial cells underlies the development of complex structure of the central nervous system. Our previous data demonstrate that GABAA receptors mediate the signals to regulate the properties of the neural progenitors in the early phase of neocortical development. GABA and taurine are known as endogenous ligands for GABAA receptors. Immunohistochemical analyses on E10.5, E12.5 and E14.5 neocortical slices revealed that taurine was enriched in the cells in the preplate at E10.5, although the apparent signals for GABA were firstly detected at E14.5. Quantification of GABA and taurine using high performance liquid chromatography showed that the molar ratio of GABA to taurine present in the E13.5 telencephalon was 1:516. Injection of D-cysteine sulfinic acid (DCSA; 15 mmol/kg body weight), an inhibitor of taurine synthesis, into the pregnant mice on E9-13 resulted in the decrease in the taurine level in the telencephalon of their E13.5 embryos by 23.6%. The decrease in the frequency of the Tbr2-positive basal progenitors, the increase in the number of Pax6-positive apical progenitors and the decrease in the thickness of Doublecortin-positive layers were observed in the neocortices of the E13.5 embryos obtained from the DCSA-treated dams. The differentiation into Satb2-positive upper-layer neurons was suppressed, while the differentiation into Tbr1-positive deep-layer neurons was enhanced in the neocortices of the E13.5 embryos which had been exposed to the hypo-taurine condition. These features the embryos from the DCSA-treated dams exhibited resembled those observed in the embryos fetally exposed to GABAA antagonists. These results totally suggest the possibility that taurine may function as a regulator of the intrinsic cellular properties of neural progenitors as a ligand for GABAA receptors in the developing neocortex.	P3-1-48 Dmrta1は哺乳類大脳皮質発生過程における神経細胞産生を制御する The doublesex homolog Dmrta1 regulates the production of early-born neurons in the mammalian cerebral cortex ○吉川貴子1, 高橋将文1, 勝山裕1, 大隅典子1 ○Takako Kikkawa1, Masanori Takahashi1, Yu Katsuyama1, Noriko Osumi1 東北大院・医・発生発達1 Div. of Dev. Neurosci., Grad. Sch. of Med., Tohoku Univ., Sendai, Japan1 The transcription factor Pax6 balances cell proliferation and neuronal differentiation in the neocortex by regulating the expression of target genes. During the course of searching downsteam targets of Pax6, we identified Dmrta1 (double-sex and mab-3 related transcription factor-like family A1, Dmrt4) as a gene specifically expressed in the developing cortex. Members of Dmrt genes encode a large family of transcription factors involved in sexual development. Although the roles of the Dmrt family genes have been characterized in the development of vertebrate sexual organs, the precise function of Dmrta1 in the brain remains largely unknown. In this study, we found that Dmrta1 expression was restricted in the neural stem/progenitor cells of the dorsal telencephalon. Overexpression of Dmrta1 induced the expression of the proneural gene Neurogenin2 (Neurog2) and conversely repressed Ascl1 (Mash1), another proneural gene expressed in the ventral telencephalon. In Dmrta1 knockout (KO) mice, corticogenesis was unexpectedly normal except for a reduction in the number of Cajal-Retzius cells. Because CR cells are derived from specific regions within the telencephalon, i.e., the pallial septum (PS), ventral pallium (VP), and cortical hem (CH), we carefully examined the expression of Dmrta1 in these regions. We found that Dmrta1 was expressed in the PS, VP, and the dorsal part of the CH. Therefore, Dmrta1 may function to produce CR cells that are crucial in mammalian corticogenesis, from their progenitor domains. Our novel findings suggest that dual regulation of proneural genes mediated by Pax6 and Dmrt family members is crucial in regional specification and early neurogenesis in the mammalian cortex.
P3-1-49 α4/β2ニコチン型アセチルコリン受容体による神経系前駆細胞の分化制御 Promotion by alpha4/beta2 nicotinic acetylcholine receptor subtype of neuronal differentiation ○藤川晃一1, 宝田剛志1, 福森良1, 米田幸雄1 ○Koichi Fujikawa1, Takeshi Takarada1, Ryo Fukumori1, Yukio Yoneda1 金沢大院・薬・薬物学1 Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School, Kanazawa, Japan1 In our previous studies, activation of ionotropic NMDA and GABA receptors led to positive and negative regulation of subsequent neuronal differentiation of undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of ionotropic nicotinic acetylcholine receptor (nAChR) by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains. On RT-PCR analysis, mRNA expression was seen for particular nAChR subunits in undifferentiated rat and mouse progenitors. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and MTT reduction activity without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells. Both effects of nicotine were significantly prevented by a heteromeric alpha4beta2 nAChR subtype antagonist, but not by a homomeric alpha7 nAChR subtype antagonist. Sustained exposure to nicotine preferentially increased the expression of Math1 among different bHLH proneural genes examined in undifferentiated progenitors. These results suggest that alpha4beta2 nAChR subtype would be functionally expressed to play a role in the mechanism underlying the determination of proliferation and subsequent differentiation into a neuronal lineage in association with preferential promotion of Math1 expression in undifferentiated neural progenitors of developing rodent neocortex.	P3-1-50 DNAポリメラーゼβの神経前駆細胞における作用が大脳皮質における神経細胞分化に必要である DNA polymerase β function in neural progenitors is required for cortical cell differentiation ○大西公平1, 菅生紀之1, 豊田峻輔1, 平山晃斉1, 八木健1, 山本亘彦1 ○Kohei Ohnishi1, Noriyuki Sugo1, Shunsuke Toyoda1, Teruyosi Hirayama1, Takesi Yagi1, Nobuhiko Yamamoto1 大阪大院・生命機能1 Graduate School of Frontier Biosciences, Univ of Osaka, Osaka1 DNA repair as well as chromatin remodeling and transcription regulation is thought to be crucial for neuronal differentiation. Lack of DNA polymerase β (Polβ), a component of the base excision repair (BER) pathway, has been shown to induce apoptosis of postmitotic cortical cells in the cortical plate (CP). This suggests that Polβ is involved in cortical cell differentiation from progenitors to neurons. However, the role of Polβ in this process is totally unknown. We addressed this issue by investigating phenotypic differences between Polβ conditional knockout mice, Emx1-CRE/Polβfl/fl and Nex-CRE/Polβfl/fl mice. In Emx1-CRE/βfl/fl embryos, in which the floxed Polβ allele is deleted in all excitatory cortical cells including neural progenitor cells, part of cortical neurons in CP showed cleaved caspase3-positive apoptosis in E14.5, similarly to the phenotype of Polβ-/- embryos. In addition, the nuclei in the majority of cortical neurons showed several γH2AX foci that indicate DNA double strand breaks in this mutant mice. In contrast, E14.5 Nex-CRE/Polβfl/fl embryos in which the floxed allele is deleted in postmitotic neurons did not show such abnormalities in the developing cortex. These results demonstrate that lack of Polβ in neuronal progenitor cells but not in postmitotic neurons induces abnormal apoptosis and DNA double strand breaks in postmitotic cortical cells. This indicates that Polβ function in differentiation from progenitors to neurons is required for neuronal progenitors.
P3-1-51 形態依存的な神経前駆細胞の核移動は神経上皮の混雑を防ぎ、大脳組織形成を確実にする Interkinetic nuclear migration through TAG-1 assisted progenitor elongation prevents neuroepithelial overcrowding and ensures neocortical histogenesis ○岡本麻友美1, 難波隆志2, 篠田友靖1, 佐合健1, 斉藤加奈子1, 榊原明1, 川口綾乃1, 仲嶋一範3, 上田昌宏4,5, 林茂生6, 藤森俊彦7, 貝淵弘三2, 宮田卓樹1 ○Mayumi Okamoto1, Takashi Namba2, Tomoyasu Shinoda1, Ken Sagou1, Kanako Saito1, Akira Sakakibara1, Ayano Kawaguchi1, Kazunori Nakajima3, Masahiro Ueda4,5, Shigeo Hayashi6, Toshihiko Fujimori7, Kozo Kaibuchi22, Takaki Miyata1 名大院・医・細胞生物1, 名大院・医・神経情報薬理2, 慶應院・医・解剖3, 阪大院・理・1分子4, 理研・QBiC・細胞シグナル動態5, 理研・CDB・形態形成シグナル6, 基生研・初期発生7 Dept Anat and Cell Biol, Nagoya Univ, Grad Sch of Med, Aichi, Japan1, Dept Cell Pharmacol, Nagoya Univ, Grad Sch of Med, Aichi, Japan2, Dept Anat, Keio Univ, Grad Sch of Med, Tokyo, Japan3, Dept Biol Sci, Osaka Univ, Grad Sch of Sci, Osaka, Japan4, Lab. for Cell Signaling Dynamics, QBiC,RIKEN, Osaka, Japan5, Lab. for Morphogenetic Signaling, CDB, RIKEN, Kobe, Japan6, Div Embryology, NIBB, Aichi, Japan7 Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1, which is enriched basally in early mouse cerebral walls, causes retraction of progenitors' basal processes. Basally disconnected stem-like progenitors failed to undergo basalward INM and overcrowded in the periventricular space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with in toto live imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although the heterotopic progenitors unexpectedly remained stem-like, sequentially producing neurons until late embryonic period, histogenesis was severely disrupted. Thus, INM is essential to prevent nuclear/somal overcrowding, thereby ensuring normal brain histogenesis.	P3-1-52 分裂期神経幹細胞特異的に限定的に発現する糖転移酵素GnT-V Focal expression of n-acetylglucosaminyltransferase V in proliferating stem/progenitor cells in mouse brain ○浜之上誠1,2, 池田義孝3, 高松研1,2 ○Makoto Hamanoue1,2, Yoshitaka Ikeda3, Ken Takamatsu1,2 東邦大学医学部生理学講座細胞生理学分野1, 東邦大学慢性炎症性疾患の先進医療技術開発センター2, 佐賀大学医学部分子生命科学科細胞生物学3 Dept. Physiol., Toho Univ. Sch. of Med., Tokyo1, Div. of Chr. Inflam. Dis., Adv. Med. Res. Center, Toho Univ. Grad. Sch. of Med., Tokyo2, Dept. of Biomol., Physiol, Saga Med. Sch, Saga3 Neural stem/progenitor cells (NSCs) express variety of asparagine-liked oligosaccharide chains, called N-glycans, on cell surface. In the NSCs, N-glycans is mainly composed of hybrid-type and complex-type N-glycans, however, its expression of N-acetylglucosaminyltransferase-V (GnT-V), regulating enzyme of hybrid-type N-glycans, remains fully unknown. In this study, cultured NSCs were prepared from adult or embryo cortex, and maintained these cell either as proliferating cells or differentiated cells in vitro. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and Western blot analysis of these cells revealed that GnT-V focally expressed in proliferating cells, and its expression markedly diminished in differentiated cells. In addition, focal expression of GnT-V was detected in nestin-positive cells at the subventricular zone in the mouse brain. These results indicate that specific GnT-V expression in NSCs, and suggest that GnT-V could participate in maitainance of NSCs including cell proliferation and/or cell migration.
P3-1-53 外側脳室下帯のbasal radial gliaはマーモセット脳の脳回脳溝形成に寄与している BASAL RADIAL GLIA IN OUTER SUBVENTRICULAR ZONE CONTRIBUTES TO GYRUS-SULCAL FORMATION IN THE MARMOSET CEREBRAL CORTEX ○村山綾子1, 岡野栄之1,2 ○Ayako Murayama1, Hideyuki Okano1,2 慶應義塾・医・生理1, 理研, BSI, Wako2 Dept Physiol, Keio Univ, Tokyo1, RIKEN, BSI, Wako2 The progenitors in the subventricular zone (SVZ) significantly contribute to thicken cerebral cortex. Recent studies revealed a novel type of SVZ progenitor, referred to as basal radial glia (bRG), which retains a basal process to the pial surface, sustains expression of radial glial markers and is capable of self-renewal. bRGs occur at high relative abundance in the outer SVZ (OSVZ) of gyrencephalic animals, but lower of lissencephalic rodents, suggesting that the division of bRG in OSVZ is important to generate gyrencephalic brain. The common marmoset, Callithrix jacchus, is a primate which has few gyri/sulci but the abundance of bRG cells is similar to that in human neocortex. In this study, we characterized bRG cells in developing marmoset OSVZ using the technique of sequential BrdU and EdU labeling by intraperitoneal injection to pregnant mother, in combination with immunofluorescent staining of brain slice. We found that the cell cycle length of bRG of marmoset may be longer than that of human. Furthermore, by histological analysis, the OSVZ in the presumptive sulcal region at E92 (embryonic day 92) was found to be thinner than that in non-sulcal regions. The result suggested that the bRG in OSVZ contributes to the girus-sulcal formation in marmoset cerebral cortex.	P3-1-54 分化発生期と成体期における小脳バーグマングリアの系譜解析 Lineage tracing of Sox9-expressing cerebellar Bergmann glial cells after brain injury ○増山典久1, 橋本了哉1, 川口義弥2, 秋山治彦3, 星野幹雄1 ○Norihisa Masuyama1, Ryouya Hashimoto1, Yoshiya Kawaguchi2, Haruhiko Akiyama3, Mikio Hoshino1 国立精神・神経医療研究センター1, 京大・iPS細胞研究所2, 京大・医学部付属病院・整形外科3 National Center of Neurology and Psychiatry, Kodaira1, Center for iPS cell research and application, Kyoto Univ, Kyoto2, Dept Orthopaedic Surgery, Kyoto Univ Hospital, Kyoto3 Although constitutive neurogenesis exclusively occurs in the restricted brain regions, it has been reported that, in many other regions including the cerebral cortex, striatum and spinal cord, the resident progenitor cells facilitate proliferation and differentiation to neurons in response to brain injury. However, it is unclear whether proliferative cells exist in the adult cerebellum or whether some cell types in the adult cerebellar tissue induce neural stem cell properties in response to stress conditions. Here we found that Sox9 is expressed in the germinal zone of the developing cerebellar primordium and its expression persists in Bergmann glial cells until adult stages. In the lineage tracing analyses using mice with ires-CreER knock-in allele at the Sox9 locus and Rosa26-EYFP knock-in allele, we found that the progeny of Sox9 expressing cells at the perinatal stages give rise to Pax2-positive GABAergic interneurons, astrocytes and maturating oligodendrocytes. In the adult cerebellum, only Bergmann glial cells express Sox9, suggesting that the multipotent differentiation property of Sox9+ cells is diminished along the cerebellar development in normal conditions. On the other hand, in stress conditions with stab wound injury, genetically traced Sox9+ progeny promote cell proliferation and expression of neuronal markers. Bergmann glia specific conditional knock-out revealed that Sox9 is required to induce cell proliferation in response to brain injury. Our data support that Bergmann glia facilitates stem cell properties in the stress condition and Sox9 plays a central role in this process.
P3-1-55 視細胞における錐体特異的な転写因子の機能解析 Investigation of the role for cone-specific transcription factors in photoreceptor differentiation ○小川洋平1, 白木知也1, 小島大輔1,2, 深田吉孝1 ○Yohei Ogawa1, Tomoya Shiraki1, Daisuke Kojima1,2, Yoshitaka Fukada1 東京大学大学院・理・生物化学1, 科学技術振興機構さきがけ2 Dept Biophys and Biochem, Grad Sch Sci, Univ Tokyo, Tokyo1, JST, PRESTO, Japan2 In vertebrates, rods and cones are retinal photoreceptor cells required for forming vision. Rods are sensitive to dim light and responsible for twilight vision, while cones function under daylight condition. Furthermore, a combination of multiple cone subtypes having various spectral sensitivities confers color vision. Those spectral sensitivities are based on exclusive expression of a single type of opsin with a unique absorption spectrum in each cone subtype. For normal color vision, therefore, it is necessary to precisely regulate expression of opsins. Previous studies on mice identified important transcription factors governing the expression of visual opsins. In mice, however, two cone opsins are co-expressed in a subset of single cones, and hence studies on the mouse retinal photoreceptors may not provide enough information about general mechanisms regulating cone opsin expressions in vertebrates. As an alternative animal model, we chose zebrafish, which is a diurnal animal and has four cone subtypes. Our recent microarray analysis of the zebrafish rods and cones has identified genes that are expressed predominantly in the cones. Among these genes, we focused on those encoding transcription factors to understand molecular mechanisms underlying photoreceptor differentiation. We first performed overexpression experiments of the transcription factors in the zebrafish retina under the regulation of fugu crx (cone-rod homeobox) promoter. In many vertebrates, crx is the master regulator of photoreceptors and is known to be expressed strongly in the retinal photoreceptor layer. We are generating knock-out zebrafishes of those genes with TAL effector nuclease to investigate transcriptional regulatory networks of mature cones in adult zebrafish.	P3-1-56 興奮毒性感受性を備えたヒト iPS 細胞由来神経細胞を用いた神経毒性評価系開発の試み An attempt to develop a neurotoxicity evaluation system using human induced pluripotent stem cell-derived neurons vulnerable to exicitotoxicity ○大津香苗1, 高橋華奈子1, 重本 -最上由香里1, 岡田洋平2, 岡野栄之2, 佐藤薫1, 関野祐子1 ○Ohtsu Kanae1, Kanako Takahashi1, Yukari Shigemoto-Mogami1, Yohei Okada2, Hideyuki Okano2, Kaoru Sato1, Yuko Sekino1 国立衛研・薬理1, 慶應大・医・生理2 Div Pharmacol, NIHS,Tokyo1, Dept Physiol, Sch Med, Keio Univ, Tokyo2 Human induced pluripotent stem cells (hiPSCs) hold potent possibility in drug discovery process. However, to what extent hiPSCs-derived neurons reproduce the synaptic functions and whether they are vulnerable to excitotoxicity remains to be elucidated. In this study, we investigated the functional receptors of commercially-available hiPSC-derived neurons and 253G1-derived neurons by fura-2 Ca2+ imaging method. The commercially-available hiPSC-derived neurons showed Ca2+ responses to L-Glu, high K+ at the differentiation day 1 and until day 21. The Ca2+ responses were mainly mediated by AMPA or kainate receptors but not by NMDA receptors. 253G1-derived neurons showed Ca2+ responses to L-Glu, high K+ from day 10 but did not respond to NMDA until day 40. The results so far suggest that hiPSCs-derived neurons express little NMDA receptors and need additional factors to get sufficient synaptic maturation. Currently we are searching for the essential factors for synaptic maturation.
P3-1-57 活性化ミクログリアは炎症性サイトカインにより神経新生、オリゴデンドロサイト新生を促進する Activated microglia enhance neurogenesis and oligodendrogenesis via inflammatory cytokines ○干川和枝1, 重本-最上由香里1, 大野泰雄1, ジェームスゴールドマン2, 関野祐子1, 佐藤薫1 ○Kazue Hoshikawa1, Yukari Shigeoto-Mogami1, Yasuo Ohno1, James E Goldman2, Yuko Sekino1, Kaoru Sato1 国立衛研・薬理1, コロンビア大学医学部神経病理2 Lab Neuropathol, Div Pharmacol, NIHS, Tokyo1, Div Neuropathol, Columbia Univ, NY, USA2 Although microglia had long been considered as brain resident immune cells, increasing evidences have suggested that they also have physiological roles in the normal central nervous system (CNS). In this study, we found that a large number of active-form of microglia accumulated in the subventricular zone (SVZ) from P1 to P10. When microglial activation was suppressed by minocycline, the number of cells positive for KI67, Doublecortin(DCX), O1, significantly decreased. In this condition, minocycline decreased the levels of inflammatory cytokines, i.e., IL-1β, IL-6, IFN-γ, TNF-α in SVZ tissue lysates. We confirmed that activated microglia facilitated the neurogenesis and oligodendrogenesis using in vitro neurosphere differentiation assay. When we applied each recombinant cytokine, IL-1β and IFN-γenhanced neurogenesis, while IL-1β and IL-6 enhanced oligodendrogenesis. Moreover, the effects of activated microglia on neurogenesis and oligodendrogenesis were significantly suppressed by the mixture of function blocking antibodies to these cytokines. These results strongly suggest that microglia facilitate neurogenesis and oligodendrogenesis in the early postnatal SVZ via inflammatory cytokines.	P3-1-58 神経幹細胞の自己複製能を維持するシグナルの空間的制御 Spatial and temporal control of signaling pathways for neural stem cell self-renewal in the developing mammalian brain ○下向敦範1, 後藤明弘2, 松田道行2, 松崎文雄1 ○Atsunori Shitamukai1, Akihiro Goto2, Michiyuki Matsuda2, Fumio Matsuzaki1 理研・発生再生科学総合研究センター・非対称細胞分裂1, 京大院・生命科学・生体制御2 Lab. for Cell Asymmetry, RIKEN CDB, Kobe1, Lab. of Bioimaging, Grad. School of Biostudies, Kyoto Univ., Kyoto2 Although the vertebrate brain commonly develops from a single layer of neuroepithelial tube, the brain organization is highly diversified among spices, as typically seen in the huge and highly folded human brains vs. small and smooth rodent brains. Developmental control of neural stem cell divisions underlies such a diversity of brain organization. In the developing brain, the neural stem cells divide asymmetrically to self-renew and produce differentiated cells. Recently, many molecules have been identified to regulate neural stem cell self-renewal. However, it remains to be elucidated how the inheritance of the epithelial structure during neural stem cell division is involved in signaling mediated by those molecules. To clarify this, we performed the live imaging of neural stem cells and reveled a tight correlation between inheritance of the basal epithelial process and stem cell self-renewal, suggesting the importance of the epithelial structure for maintaining neural stem cell identity. We are currently addressing the issue of how spatially the epithelial structure of neural stem cells contributes to the control of stem cell self-renewal. I would like to introduce our recent results about the spatial and temporal control of these signaling pathways during neural stem cell division.
P3-1-59 Withdrawn	P3-1-60 Involvement of Cav1.2 and Cav1.3, L-type Ca2+ channel subclasses in postnatal neurogenesis ○Daniel Teh1,2, Toru Ishizuka1,2, Hiromu Yawo1,2 Tohoku University, Graduate School of Life Science1, JST (CREST)2 The dentate gyrus of hippocampus is one of regions where neurogenesis is present even in the adult animals. Calcium influx through L-type Ca2+ channels (LTCCs) appears to regulate neurogenesis as it activates several mitogenic and survival signaling pathways. Here we tested if LTCCs are involved in neurogenesis using PZ5 neural stem cells, which are homogenous undifferentiated Type-1 neural progenitor cells isolated from the adult rat hippocampus. The differentiation of PZ5 cells were evaluated immunocytochemically after 12-days incubation with differentiating medium. The proliferation rate was evaluated using trypan blue exclusion method and BrdU staining. We found that the proliferation rate of undifferentiated PZ5 was affected neither by the general LTCC agonist, BayK 8644, Cav1.2/1.3 specific agonist, FPL64176, nor by the LTCC antagonist, nimodipine. However, our data demonstrated that BayK and FPL significantly increased the fraction of cells expressing neuronal markers (βIIItubulin+/MAP2+) after differentiation. Nimodipine, on the other hand, reduced the neuronal population. Between these two groups, the astrocytic population remained the same, but reduction in the oligodendrocytic population was notable in the BayK and FPL group. We have found also that only Cav1.2 and Cav1.3 LTCC subsets were detected in the undifferentiated PZ5 cells.It is suggested that the activation of Cav1.2 and Cav1.3 promote the PZ5 neural stem cells to be differentiated to the neuronal population at the expense of the oligodendrocytic population.

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