細胞移動、層・神経核の形成 Cell Migration and Layer/Nuclear Formation
P2-2-55 リーリンの特異的分解の機構と生理的意義 The mechanism and physiological significance of specific proteolytic cleavage of Reelin ○奥村恭子1, 久永有紗1, 鯉江真利1, 河野孝夫1, 服部光治1 ○Kyoko Okumura1, Arisa Hisanaga1, Mari Koie1, Takao Kohno1, Mitsuharu Hattori1 名古屋市立大学大学院　薬学研究科　病態生化学分野1 Dept. Biomed. Sci., Grad. Sch. Pharmaceuti. Sci., Nagoya City University, Aichi1 Reelin is a secreted protein that is essential for normal brain development and functions. Reelin has eight tandem repeats called Reelin repeats (RR) and is specifically cleaved around RR2 and RR3 (N-t site). We previously found that the N-t site cleavage virtually abolishes Reelin signaling activity in vitro. As dysfunction of Reelin caused by N-t site cleavage has been suggested to be involved in the pathogenesis of several neuronal diseases including Alzheimer's disease and schizophrenia, it is of great clinical importance to understand the N-t site cleavage mechanism. In this study, we try to identify the protease in charge of the N-t site cleavage and its physiological significance.We partially purified the protease in charge of the N-t site cleavage from the culture supernatant of cortical neurons and identified a candidate protease which belongs to metalloproteinase family. The recombinant candidate protease was able to cleave Reelin at the N-t site. Furthermore, its biochemical characteristics were quite similar to those of the protease in the culture supernatant of cortical neurons. These results suggested that the protease we identified is the major protease that cleaves Reelin in the brain. We utilized the active protease fraction from the culture supernatant of cortical neurons to determine the exact site of N-t cleavage. We then established a monoclonal antibody that recognizes only uncleaved Reelin by immunizing the peptide surrounding the cleavage site into Reelin-deficient mice. We now investigate the localization of uncleaved Reelin by using this antibody in brain to clarify the mechanism of N-t site cleavage. The identification of the protease and understanding of its regulatory mechanism will help establish novel methods for prevention and treatment of neurological diseases.	P2-2-57 胎生期海馬神経前駆細胞の移動解析 Cell-tracing analysis for progenitor cell migration in the embryonic dentate gyrus ○篠原広志1, 佐藤亨1, 戸田景子1, 塩田清二2, 石龍徳1 ○Hiroshi Shinohara1, Toru Sato1, Keiko Toda1, Seiji Shioda2, Tatsunori Seki1 東京医科大学 組織・神経解剖学1, 昭和大学・医・組織・第一解剖2 Department of Histology and Neuroanatomy, Tokyo Medical University, Tokyo1, Department of Anatomy, Showa University School of Medicine, Tokyo2 In general, neurogenesis occurs during embryonic and early postnatal stages, and ceases at adult stage. However, the dentate gyrus (DG) continues neurogenesis from embryonic to adult stages, although there is a distinct neurogenic pattern between two stages. In the adult DG, granule neurons are generated in the subgranular zone, while during embryonic period, dentate neural progenitors are initially produced in the ventricular zone (VZ) close to the fimbria, and then migrate through the suprafimbrial region to the subpial region (SP) where a new proliferative zone is formed to develop the presumptive dentate gyrus. During the migration, the progenitors differentiate into granule neurons or maintain property of neural progenitors that further contribute to perinatal and postnatal neurogenesis. Although the migration of the neural precursors and relocation of the region of neurogenesis are key processes for the formation of the dentate granule cell layer, the exact temporal and spatial patterns are still unknown. To address the problem, we performed cell-tracing analysis by in utero electroporation of RFP-expressing vectors that were introduced into the neuroepithelium of the medial cortex at E14.5 to 15.5. RFP-positive cells originated from the VZ migrated to the DG within 3 days. Immunohistochemical studies revealed that the RFP+/Tbr2+ cells were present in the SP, whereas the RFP+/Sox2+ cells were localized in both the SP and the hilus. These data indicate that the progenitors originated from the VZ migrate to different regions, and maintain the proliferation or differentiate into neurons. Moreover, we performed time-lapse imaging in cultured hippocampal slices and found at least two types of cell migration in the DG: the migration along the SP attaching the meninges and the migration into the hilus without attaching the meninges. Our results suggest that maintenance of progenitors and production of neurons are correlated with different modes of cell migration.
P2-2-58 DBZ KOマウスにおける大脳皮質発達の解析 Analysis of Cortical Development in Mice Lacking DBZ ○服部剛志1, 岡本昌之2, 駒田致和2, 小山佳久3, 高雄啓三7, 宮川剛6, 片山泰一4, 伊藤彰1, 佐藤真2, 遠山正彌5 ○Tsuyoshi Hattori1, Masayuki Okamoto2, Munekazu Komada2, Yoshihisa Koyama3, Keizo Takao7, Tsuyoshi Miyakawa6, Taiichi Katayama4, Akira Ito1, Makoto Sato2, Masaya Tohyama5 大阪大院・医・分子精神神経1, 福井大・医・形態機能2, 大阪大院・医・神経機能形態3, 大阪大院・小児発達・分子生物遺伝4, 近畿大・東洋医学・分子脳5, 藤田保健・総合医・システム医科学6, 生理研・行動代謝分子解析センター・行動様式解析7 Dept Mol Neuropsy, Univ of Osaka, Osaka1, Div Cell Biol and Neurosci, Dep of Mor and Phy Sci, Faculty of Med Sci, Univ of Fukui, Fukui2, Dep Anat and Neurosci, Univ of Osaka, Osaka3, Dep Child Dev and Mol Brain Sci, Univ of Osaka, Osaka4, Dep Trad Asian Med, Univ of Kinki, Osaka5, Div System Med Sci, Fujita Health Univ, Aichi6, NIPS, Behavior Patterns, Cent for Gene Anal of Behavior7 Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a (1;11) (q42.1;q14.3) translocation that segregates with major psychiatric disorders in a Scottish family. DISC1-Binding Zinc finger protein (DBZ) was found as a DISC1 interacting molecules by yeast-2-hybrid screening of a cDNA library. DISC1 and DBZ co-localize diffusely in the cytoplasm and centrosome, and involved in neurite extension and cell division. DBZ exclusively expresses in brain. Especially DBZ is expressed in the cerebral cortex, hippocampus, striatum and thalamus of adult rodent brain. To examine the functional role of DBZ in vivo, we have generated mice with a disrupted DBZ gene by homologous recombination in embryonic stem (ES) cells. Mice homozygous for the DBZ mutation were born from heterozygote intercrosses at approximately the frequency predicted by Mendel's law. The DBZ -/- mice were viable and fertile. However, the DBZ -/- mice showed disturbed cell migration and neurite extension of pyramidal neurons in the developing cerebral cortices. In addition, DBZ KO mice displayed abnormal emotional behavior as assessed by the novelty induced hypophagia test and fear conditioning test. In conclusion, lack of DBZ induces abnormal cortical development and alters emotional behavior.	P2-2-59 大脳新皮質層形成に必須のDab1はImportin依存性または非依存性に核移行する Dab1, an essential protein for the neocortical development, translocates into the nucleus by an Importin-dependent or -independent manner ○本田岳夫1, 仲嶋一範1 ○Takao Honda1, Kazunori Nakajima1 慶應・医・解剖1 Department of Anatomy, Keio University School of Medicine1 Six-layered cortical structure of mammalian neocortex is formed by accurate control of neuronal migration. One of the molecular mechanisms to regulate the precise neuronal migration is the Reelin-Dab1 signaling pathway. Reelin is a large glycoprotein secreted from Cajal-Retzius cells and received by ApoER2 or VLDLR, and induces Dab1 tyrosine phosphorylation. Mutation or deletion of the genes encoding these proteins causes almost the same neuroanatomical abnormalities such as a defect in the preplate splitting and inverted cortical lamination. Although Dab1 had been considered to be a cytoplasmic protein, we previously showed that Dab1 is a nucleocytoplasmic shuttling protein. In its steady state, Dab1 is mainly located in the cytoplasm. However, treatment with leptomycine B, a specific inhibitor of CRM1, resulted in nuclear accumulation of Dab1. By using deletion or substitutional mutants of Dab1, we have mapped a classic bipartite nuclear localization signal (cNLS) and two CRM1-dependent nuclear export signals. To reveal the functional significance of Dab1 shuttling, we examined whether mutation to the cNLS of Dab1 inhibits nuclear translocation of Dab1. Although the cNLS amino acid sequence (RKKGQDRSEATLIKRFK) solely can give an ability to proteins to translocate to the nucleus and mutation into the cNLS inhibits its ability, Dab1 mutant containing a mutation in the cNLS could unexpectedly translocate and accumulate in the nucleus, suggesting that the cNLS mutant of Dab1 is transported into the nucleus by an unidentified nuclear localization signal. Therefore, we examined the biochemical properties of nuclear import of Dab1, and found that cNLS sequence of Dab1 translocate into nucleus by an Importin-dependent and full-length Dab1 containing a mutation in the cNLS translocates into the nucleus by an Importin-independent pathway. We are trying to examine whether the nuclear translocation of Dab1 is involved in the neuronal migration mechanisms.
P2-2-60 前脳基底部における標識細胞群から生じる皮質抑制性介在ニューロンの多様性 Diversity of GABAergic interneurons originated from the same progenitor domain ○鳥越万紀夫1, 城崎航希1, 村上富士夫1 ○Makio Torigoe1, Koki Shirosaki1, Fujio Murakami1 大阪大学大学院　生命機能研究科　脳神経工学1 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan1 The diversity of GABAergic interneurons is crucial for information processing in the cortex. Elucidation of the mechanisms underlying the establishment of the diversity is thought to be a key to understanding the information processing in the cortex. However, its mechanisms are not fully understood. Birthdating and fate-mapping studies utilizing tissue transplantation as well as genetic fate-mapping analysis using transgenic mice indicated that differences in both spatial and temporal origins of interneuron progenitors in ganglionic eminences (GEs) cause their diversity. However, multiple subsets of interneurons were still labeled even under conditions in which temporally regulated induction of genes in the GEs was applied. Because both ventricular zone (VZ) and subventricular zone (SVZ) progenitiors were simultaneously labeled by previous inducible genetic fate-mapping studies, we speculated that neurons originating from the VZ and those that are subsequently generated from intermediate progenitors possibly located in the SVZ might give rise to distinct types of interneurons. To address this issue we labelled VZ progenitors in the GEs by in utero electroporation at embryonic day 9.5, 10.5 and 12.5 and observed labelled cells in mature cortex. To label neurons directly generated from VZ progenitors and those generated from intermediate progenitors in the same VZ progenitor domain, we co-electroporated conventional plasmids and plasmids that allow permanent integration of constructs to genome. We show evidence suggesting that the same progenitor domain generate distinct types of interneurons depending on whether they are generated from VZ progenitors or intermediate progenitors.	P2-2-61 PRドメインタンパク質Prdm8は大脳皮質神経発生を調節する PR domain protein Prdm8 regulates neocortical neurogenesis ○井上真悠子1,2, 黒田貴雄1, 加藤雅紀3, 眞貝洋一3, 水谷健一1,4 ○Mayuko Inoue1,2, Takao Kuroda1, Masaki Kato3, Yoichi Shinkai3, Ken-ichi Mizutani1,4 同志社大院　脳科学1, 京大院　生命2, 理研　基幹研3, JSTさきがけ4 Grad Sch of Brain Sci, Doshisha Univ, Kyoto1, Grad Sch of Bio, Kyoto Univ, Kyoto2, RIKEN ASI, Saitama3, PRESTO, JST4 During neocortical development, a multitude of cellular events must all occur with exquisite spatiotemporal control. Despite the critical nature of these prenatal events, the molecular and cellular mechanisms of neocortical development are far from fully understood. We have focused on PR domain protein Prdm8, because some members Prdm family are new candidate to control cortical neurogenesis. Immunostaining analysis showed that Prdm8 began to be expressed in postomitotic neurons of the cortical plate at E13.5, and expressed in layer II-V at P5. Also, we tried to analyze the detailed expression patterning of Prdm8 in the neocortex by using Prdm8 reporter mice (Prdm8-mVenus), and confirmed that the Prdm8 intensely expressed around intermediate zone at embryonic stages. And, differential mRNA expression profiling by Q-PCR between mVenus + and - was performed, and we confirmed that the genes which express in the migrating precursors and differentiating neurons were higher in the mVenus+ cells. In addition, we cultured Neuro-2a cell line in the presence or absence of retinoic acid, and found that Prdm8 mRNA is increased with neural differentiation. All together, the expression of Prdm8 is strictly regulated in migrating and differentiating neurons in the neocortex at embryonic stage, and in layer II-V neurons at postnatal stage. Next, we established Prdm8 KO mice (Prdm8-/-) by the disruption of Prdm8 locus completely, and found that weight loss of whole brain in the Prdm8-/-, compared with WT. Furthermore, CAG-EGFP reporter construct was introduced into the both Prdm8-/- and WT developing neocortex, and found that multipolar precursors stained with Unc5d was decreased in the Prdm8-/-. Also, we confirmed that relative neocortical thickness of upper layer was significantly decreased in Prdm8-/- at P6, compared with WT. These data indicate that Prdm8 plays an important role for neocortex in the regulation of the neurogenesis, and in the layer formation .
P2-2-62 新皮質を構成するサブプレートニューロンの発生起源・分布・運命 The origin, distribution and fate of subplate neurons in the neocortex ○片山悠司1, 萩本和也1, 村上富士夫1, 田辺康人1 ○Yuji Katayama1, Kazuya Hagimoto1, Fujio Murakami1, Yasuto Tanabe1 大阪大院・生命機能・脳神経工学1 Grad Sch Frontier Biosci, Osaka Univ, Osaka, Japan1 All the brain functions depend on the spatially and temporally-controlled generation of distinct subtypes of neurons during development of the CNS. At the onset of corticogenesis, subplate (SP) neurons and Cajal-Retzius (CR) cells are generated and form the preplate (PP). Many previous studies including those that led to the proposal of the concept of dual-origin of neocortex provided a prevailing view that PP neurons are generated from the underlying neocortical ventricular zone (VZ) prior to the onset of cortical plate (CP) generation. However, this idea on the PP origins, especially those of CR cells, has been challenged by several studies, and CR cells have been shown to be generated from extracortical regions and migrate into the neocortex during the development. In contrast, there has been no clear answer yet as to the SP neuron ontogeny. Moreover, whether SP neurons persist to exist throughout the neocortical development remained controversial. Here we focused our analyses on the origin, distribution and fate of SP neurons. We asked whether SP neurons are generated from the underlying neocortical VZ cells by performing in vivo electroporation-mediated gene transfer at embryonic day (E) 10. Our study showed that, at E18, most of the fluorescently-labeled gene-incorporated descendants were located in the SP, but not in the marginal zone, and some of them were positive for Nurr1, a subplate marker. Moreover, our study also showed that those Nurr1-positive gene-incorporated descendants were still present at postnatal day 21 and distributed as they were at E18. These data demonstrated that a subpopulation of SP neurons, unlike CR cells, are at least generated from the underlying neocortical VZ and persist even in the matured neocortex. Our ontogenic analyses suggested that SP neurons can be categorized as CP neurons that migrate radially from the VZ and locate their cell bodies in an inside-out manner during development of the neocortex.	P2-2-63 発生・発達期海馬歯状回におけるProx1発現細胞の分子的特徴による分割 Molecular subdivision of Prox1 expressing cells in the developing dentate gyrus ○杉山拓1, 勝山裕1, 大隅典子1 ○Taku Sugiyama1, Yu Katsuyama1, Noriko Osumi1 東北大学大学院　医学系研究科　発生発達神経科学分野1 Div. Dev. Neurosci., Tohoku Univ. Grad. Sch. Med.1 The dentate gyrus (DG) of the hippocampal formation is involved in learning and memory. Differentiation of DG granule cells starts during embryogenesis, and production of the DG granule cells persists throughout life. Expression of a homeodomain protein Prox1 marks differentiated DG granule cells, whereas Prox1 expression was reported in the dentate neuroepithelium (DNE) at the embryonic day 14 (E14). Prox1 knockout experiment showed that defects of the DG were observed in Prox1 knockout after E16, exhibiting ectopic cell death. However, it is not clear whether Prox1 is essential for maturation of DG granule cells or determination of the cell fate. Here we immunohistochemically examined Prox1 expression in embryonic and postnatal stages. We detected Prox1 expression in the DNE from E12, in which stem cell markers were coexpressed with Prox1. At E16, we found two cell groups expressing high or low level of Prox1. Expression of stem cell markers and a proliferative marker PCNA was undetectable in Prox1(high) cells but observed in Prox1(low) cells. Double immunostaining with neurogenic markers indicated that the transition of Prox1 expression from low to high takes place during Tbr2 expressing stage. Prox1(high) and Prox1(low) cells were observed at postnatal day 5 (P5). Prox1(low) cells decreased at P10, and were not observed at P20. These observations suggest that there is a transition of Prox1 expression from low to high during Tbr2 expressing stage. Interestingly, Prox1 was not expressed in the neural stem cells in the SGZ of mature DG, and Prox1 was detectable from Tbr2 expressing stage. Thus, our observations suggest a molecular difference in the neural stem cells and in regulation of Prox1 expression between developmental and adult stages. Because Prox1(high) cells were observed after E16, it is possible that higher expression level of Prox1 is essential for proper development of DG, including cell survival.

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