一般口演（若手道場）
若手道場 神経発生
Wakate Dojo: Neurogenesis
座長：一瀬 宏（東京工業大学）・松田 泰斗（九州大学医学研究院基盤幹細胞学分野） |
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| 2022年6月30日 16:10～16:25 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06e1-01
Fut9 deficient cause abnormal neuronal migration in the developing brain
*Asmaa Abdullah(1), Yoshitaka Hayashi(1), Naoko Morimura(1), Mariko Omatsu(2), Seiji Hitoshi(1)
1. Dept Integrative Physiology, Shiga Univ of Med Sci, Shiga, Japan, 2. Dept Cell Physiology, Shiga Univ of Med Sci, Shiga, Japan
Keyword: FUCOSYLTRANSFERASE, BRAIN LAYER, NEURONAL MIGRATION
Fucosyltransferase (Fut) is an enzyme responsible for fucosylation of N-glycans that bind to proteins or lipids. Using in situ hybridization (ISH), we found that two fucosylation enzymes, Fut9 and Fut10, showed a spatiotemporal expression across embryogenesis in the brain and the retina. In the developing brain, neural stem/progenitor cells are enriched in the ventricular zone/subventricular zone (VZ/SVZ) of the cortex and the ganglionic eminence. Fut9 is expressed in the dorsomedial portion of VZ/SVZ and upper cortical layer of cortex in E15.5 but can be detected only in the cortex in E19.5. Meanwhile, across embryogenesis, Fut10 is detected in VZ/SVZ. The developing mouse retina has two prominent layers; the inner neuroblast layer (INBL), which consists of migrated differentiating cells, and the outer neuroblast layer (ONBL), which consists of mitotic progenitor cells. Fut9 is expressed at INBL in E15.5 and E19.5, while Fut10 is expressed in the retina surface in E12.5, and the expression becomes prominent in ONBL in E15.5 and E19.5. To characterize Fut9 and Fut10 expressing cells in the brain, we established a co-detection method of mRNA localization and protein expression using ISH followed by immunohistochemistry. Fut9 is colocalized with Ctip2, marker for layer V/VI and TLE4, marker for corticothalamic projection neurons in the layer V/VI in E19.5 cortex, while Fut10 is colocalized with Sox2, marker for stem/progenitor cells in VZ/SVZ of cortex and ONBL of the retina. Fut9 KO mice showed significant decrease of Satb2 (a marker for Layer II to VI) negative and Ctip2 strongly positive cells by flow cytometry analysis in the adult cortex. To study whether the decreased number of these cells is associated with neuron production, we performed a neuronal birthdating analysis by labeling newborn neurons in E11.5, E12.5 and E14.5 using 5-ethynyl-2`-deoxyuridine (EdU), 5-bromo-2'-deoxyuridine (BrdU), and in utero electroporation using fluorescent plasmid, respectively. We found decrease in percentage of EdU+ cells distribution at Layer VI in Fut9 KO cortex and tendency to increase in percentage of EdU+ cells in subplate layer. In the developing retina, there was a tendency to decrease in percentage of EdU+ cells in the INBL. We could not find any abnormalities in Fut10 KO mice from these analyses. Therefore, these results suggested that Fut9 is associated with the maturation and guide of neuronal migration during early stage of development. | | 2022年6月30日 16:25～16:40 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06e1-02
分泌型タンパク質Ahkirinは脳発生時の自然免疫応答に関与し、神経幹細胞ニッチの恒常性の維持に働く
Akhirin, a secreted protein, is involved in innate immune responses during brain development and maintains homeostasis in the neural stem cell niche.
*工藤 三希子(1)、松尾 直毅(1)、太田 訓正(1)
1. 九州大学
*Mikiko Kudo(1), Naoki Matsuda(1), Kunimasa Ohta(1)
1. Kyushu University
Keyword: Neuroinflammation, Brain innate immunity, Microglia, Akhirin
Previously, we identified Akhirin (AKH) as a novel secretory molecule expressed in the chick embryo’s lens epithelium. AKH contains one LCCL domain (involved in the innate immune response from bacterial infections in the inner ear) and two vWF domains (one of the famous blood coagulation factor), exhibits heterophilic cell adhesion property (Ahsan et al., 2005). AKH is expressed explicitly in the neural stem cell (NSC) niche region (microenvironment where NSCs are present) of the central nervous system (eye, spinal cord, and brain) (Ahsan et al., 2005, Athary et al., 2015, Anam et al., 2020). Our findings suggest that AKH is one of NSCs niche regulator and plays a crucial role in their development, however the detail molecular function of AKH in brain development is still unclear. The timing of NSC proliferation and differentiation is controlled by the interaction of NSCs/ neural progenitor cells with glial cells and various other cells in the NSC niche. In the mouse brain, there is a NSC niche in the subventricular zone facing lateral ventricles (LV). Here, using an anti-AKH polyclonal antibody, we show that AKH is expressed in the neuronal ependymal cells layer, choroid plexus ependymal cells layer, and the cerebral spinal fluid at embryonic brain. Compared to wild-type mice, AKH knock out mice (AKH-/-) have aberrant LV expansion and suppressed NSC proliferation, as well as behavioral abnormalities. Furthermore, we revealed the abnormal blood vessels formation and the increase of activated microglia in the AKH-/- brain. Recently, microglial association has been reported for maintenance of homeostasis in the NSC niche. To elucidate the reason behind microglial activation induced by AKH deficiency, we observed that AKH is involved in the innate immune response of the developing mouse brain. In the developing brain, the blood-brain barrier (BBB) is immature, and the maturation of the BBB is complex and remains unclear because it involves factors such as vascularization and glial maturation. We propose that AKH regulates innate immunity in the brain during development, when the brain’s immune system is still underdeveloped, and is involved in maintaining homeostasis of NSC niche regulation to maintain normal brain development. | | 2022年6月30日 16:40～16:55 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06e1-03
発生期の大脳皮質に形成される細胞外マトリクスは神経細胞移動に必要である
The extracellular matrix formed in the developing cerebral cortex is required for neuronal migration
*武渕 明裕夢(1)、武智 美奈(2)、佐藤 ちひろ(3)、北島 健(3)、北川 裕之(4)、宮田 真路(1)
1. 東京農工大学農学府、2. 名古屋大学大学院生命農学研究科、3. 名古屋大学糖鎖生命コア研究所、4. 神戸薬科大学生化学研究室
*Ayumu Mubuchi(1), Mina Takechi(2), Chihiro Sato(3), Ken Kitajima(3), Hiroshi Kitagawa(4), Shinji Miyata(1)
1. Grad Sch Agr, Tokyo Univ Agr and Tech, Fuchu, Japan, 2. Grad Sch Bioagr Sci. Nagoya Univ, Nagoya, Japan, 3. iGCORE, Nagoya Univ, Nagoya, Japan, 4. Lab Biochem, Kobe Pharma Univ, Kobe, Japan
Keyword: Neuronal migration, Cerebral cortical development, Extracellular matrix, Tenascin-C
認知や記憶などの脳の高次機能を司る大脳皮質は、発生期に神経幹細胞から分化した神経細胞が脳表層へ向かって移動することで形成される。脳室帯で生まれた未熟な神経細胞は、複数の短い突起を持つ多極性の形態を示す。その後、中間帯の上部に移動すると、先導突起と軸索を持つ双極性の形態に変化し、神経幹細胞が伸ばす線維に沿って皮質板を放射状に移動する。中間帯の上部は他の領域と異なり細胞密度が低く細胞外マトリクスが豊富に存在する。しかし、大脳皮質形成における細胞外マトリクスの生理学的役割はよく分かっていない。我々は以前、中間帯には多糖であるヒアルロン酸が豊富に存在することを報告した。そこで本研究では、ヒアルロン酸が形成する細胞外マトリクスの機能を調べることを目的とした。 まず、ヒアルロン酸結合ドメインを持つ分子に着目し、ヒアルロン酸の結合相手を探索した。その結果、発生期の神経組織に高発現するプロテオグリカンであり、双極性障害のリスク因子としても報告されているニューロカンのN末端領域が同定された。次に、免疫沈降と質量分析法を組み合わせた解析から、ニューロカンのC末端領域には、テネイシンCが結合することが示された。マウス胎仔の脳室にヒアルロン酸分解酵素を注入すると、中間帯におけるニューロカンとテネイシンCの集積が減少したことから、生体内でヒアルロン酸/ニューロカン/テネイシンCの三者複合体が形成されることが明らかとなった。そこで、この細胞外マトリクスの機能を解明するために、ニューロカンとテネイシンCの両者を欠損したダブルノックアウト (DKO) マウスを作製した。脳室帯で生まれた神経細胞を蛍光分子で標識し神経細胞の移動を比較したところ、野生型に比べDKOマウスでは脳表層への移動が遅れ、中間帯に神経細胞が蓄積していた。ヒアルロン酸分解酵素の注入によっても、神経細胞移動の遅延が確認された。また、子宮内エレクトロポレーション法により神経細胞を可視化し形態を詳細に解析したところ、DKOマウスでは、多極性神経細胞から双極性神経細胞への形態変化が遅れていることが分かった。さらに、細胞外マトリクスのどの成分が上記の現象に関与するのか調べるために、ヒアルロン酸、ニューロカン、テネイシンCをそれぞれコーティングしたガラス上で大脳皮質神経細胞を培養した。その結果、テネイシンCは、神経細胞の突起を伸長させ、形態変化を促進させる作用をもつことが明らかとなった。 以上の結果から、中間帯に形成されるヒアルロン酸/ニューロカン/テネイシンCの三者複合体は、神経細胞の移動と形態変化を促進させることで、大脳皮質形成において重要な役割を果たすことが示唆された。 | | 2022年6月30日 16:55～17:10 沖縄コンベンションセンター 会議場B3・4 第6会場
1WD06e1-04
哺乳類発生期大脳新皮質で移動神経細胞が辺縁帯直下で移動を停止するメカニズム
The mechanism of how migrating neurons stop just beneath the marginal zone in the developing mammalian neocortex
*林 光太郎(1)、林 周宏(1)、本田 岳夫(1,2)、Nadia Bahi-Buisson(3)、Alessandra Pierani(3)、仲嶋 一範(1)
1. 慶應義塾大学医学部、2. 岐阜薬科大学、3. パリ大学
*Kotaro Hayashi(1), Kanehiro Hayashi(1), Takao Honda(1,2), Nadia Bahi-Buisson(3), Alessandra Pierani(3), Kazunori Nakajima(1)
1. Keio University School of Medicine, Tokyo, Japan, 2. Gifu Pharmaceutical University, Gifu, Japan, 3. Université de Paris, Paris, France
Keyword: Reelin, Cajal-Retzius cell, Cortical development, Marginal zone (MZ)
The mammalian cerebral cortex consists of six neuronal layers, each of which has a distinctive type of neurons. During development of the cerebral cortex, excitatory neurons are born near the ventricle, migrate toward the surface of the brain, and stop just beneath the marginal zone (MZ). When neurons stop migrating, they extend their leading processes to develop dendrites into the MZ, whereas their soma do not invade the MZ, forming a cell body-dense primitive cortical zone (PCZ). Reelin is an extracellular protein secreted from Cajal Retzius (CR) cells located in the MZ and plays a critical role in the cortical layering, because the Reelin-deficient mice, reeler, show basically inverted cortical layers. Ectopic overexpression of Reelin in the developing neocortex causes formation of a neuronal aggregate, which possesses a process-rich and cell-body sparse region in its center surrounded by a cell-body-rich region. Therefore, Reelin is thought to have an important role in the formation of the MZ and PCZ, but how Reelin regulates the formation of these structures remains unknown. In this study, we analyzed a Reelin mutant found in a human pachygyria patient. Ectopic expression of this mutant Reelin into the mouse developing neocortex by using in utero electroporation caused neuronal aggregation without the central cell body-sparse region, implying that this mutated residue is critical for the appropriate neuronal alignment. Thus, we then generated a knock-in (KI) mouse in which this point mutation was introduced into the Reelin gene using the CRISPR-Cas9 system. This KI mouse exhibited abnormal alignment and orientation of CR cells in the neonatal period. In addition, the secreted amount of Reelin in the MZ seemed decreased in the KI mice. The defective secretion of the mutant Reelin was confirmed using cultured cells that had been transfected with the mutant Reelin. On the other hand, biochemical experiments showed that the mutant Reelin could normally induce phosphorylation of Dab1, a well-known downstream adaptor protein in the Reelin signaling. These results suggest that the mutated residue of Reelin, originally found in a human patient, is important for the protein function, independently of the cannonical Reelin pathway mediated by Dab1 phosphorylation. | |
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