細胞移動 Cell migration
O3-6-3-1 GDNF受容体・RETの細胞内トラフィッキングは神経発生に役割を持つか？ Intracellular trafficking of a GDNF receptor, RET - Roles in neural development? ○伊藤圭祐1, 榎本秀樹1 ○Keisuke Ito1, Hideki Enomoto1 理化学研究所 発生・再生科学総合研究センター 神経分化・再生研究室1 RIKEN CDB, Laboratory for Neuronal Differentiation and Regeneration1 Neurotrophic factors regulate neuronal development by binding to their cognate receptors, and accumulating evidence suggests that intracellular trafficking of the neurotrophic receptors plays a crucial role in development. However, little is known about the extent to which neurotrophic receptor trafficking is necessary in organogenesis. In particular, the mechanisms underlying the intracellular trafficking of RET tyrosine kinase, the signaling receptor for Glial cell line-derived neurotrophic factor (GDNF), remain largely unknown. Here, we report the generation and analyses of Ret51-EGFP knockin mice, which facilitate visualization of RET under physiological conditions. Unlike Ret-deficient mice, which die at birth due to the absence of the enteric nervous system (ENS) and kidneys, the Ret51-EGFP mice were viable, with normal development of RET-dependent organs, indicating that RET51-EGFP proteins exert physiological functions during development. Through timelapse imaging, we observed that punctate fluorescence of RET51-EGFP moved retrogradely within sensory and motor axons, both in vitro and in vivo. In ENS progenitors, the RET51-EGFP particles exhibited a polarized localization pattern and moved both antero- and retrogradely. We also found that RET51-EGFP particles colocalized with red fluorescent dye labeled-GDNF ligands. Endocytosis inhibitors abolished the colocalization of RET and GDNF and attenuated the motility of ENS progenitors, suggesting that internalization of GDNF and intracellular trafficking of GDNF-RET complexes are important for proper cell behavior during development. We also found that Rab11-mediated and Dynein-mediated pathways were required for the normal trafficking of RET51-EGFP proteins. These results reveal, for the first time, the properties of intracellular movement of the RET tyrosine kinase under physiological conditions, and suggest that polarized trafficking of RET is critical for directing the proper behavior of RET-dependent cells.	O3-6-3-2 マウス発生期における海馬神経細胞の移動様式 Migration mode of hippocampal neurons during mouse development ○林周宏1, 北澤彩子1, 久保健一郎1, 仲嶋一範1 ○Kanehiro Hayashi1, Ayako Kitazawa1, Ken-ichiro Kubo1, Kazunori Nakajima1 慶應義塾大学　医学部　解剖学教室1 Dept Anatomy, Keio University School of Medicine, Tokyo1 The hippocampus has a highly ordered structure and consists of distinct layers. Proper layer formation during development is necessary for higher brain function such as learning and memory. Projection neurons in the neocortex and the hippocampus are primarily generated near the ventricle. Then they move to their final locations, organize layers, and finally arrange neural circuits. Disruption of any of this process may cause mental illness. Thus neuronal migration is essential for the process of the hippocampal development. Although recent molecular analyses of neuronal migration have mainly been conducted in the cerebral neocortex, the cellular mechanisms underlying hippocampal development are not fully understood. Some studies on developing hippocampus have reported analyses of fixed brain sections by the method of Golgi staining, electron microscopy, and [3H] thymidine labeling. However, there still remain various questions about the migration of the hippocampal cells. Herein, we introduced a green fluorescent protein (GFP)-expression plasmid into ventricular zone cells in hippocampal CA1 region at various stages by using in utero electroporation and performed profile analyses of their migration. We also monitored cellular dynamics of GFP positive cells during migration with time-lapse imaging. The migration of hippocampal cells was characteristic and distinct from that of neocortical cells.
O3-6-3-3 異なる誕生日を有するプルキンエ細胞群は、相互作用し、小脳内に個別の領域を形成する Cohorts of Purkinje cells that share the same birthdate communicate each other and form individual compartments in the cerebellum ○橋本光広1, 宮田卓樹1 ○Mitsuhiro Hashimoto1, Takaki Miyata1 名古屋大学大学院　医学系研究科　細胞生物1 Dept Cell Biol, Nagoya Univ, Aichi, Japan1 We have reported that an adenoviral vector can introduce a foreign gene into neurons in a neuronal birthdate-specific manner. Using an adenoviral vector, we label Purkinje cells (PCs) born at embryonic day (E) 10.5, E11.5, and E12.5 and trace their fated positions in the mouse cerebellum. As a result, we find that each cohort of birthdate-related PCs forms longitudinal compartments in the cerebellum, and furthermore, these compartments formed by each cohort of birthdate-related PCs correlate strikingly with olivocerebellar projection. It suggests that there is close correlation between birthdate of PCs and the formation of cerebellar neuronal network. We hypothesized that each cohort of birthdate-related PCs had individual functions that is contributed to the regional specification and function in the cerebellum. To examine the hypothesis, we defined the genetic properties of each cohort of birthdate-related PCs with the adenoviral vector expressing enhanced yellow fluorescence protein, fluorescence-activated cell sorting, and DNA microarray analysis. As a result, we found that each cohort of birthdate-related PCs is characterized by the expression of a subset of genes involved in differentiation, regional specification and neuronal function in the cerebellum, and furthermore, we identified marker proteins for E10.5-born PCs and E11.5-born PCs, respectively. Using specific antibodies against these markers, we examined the forming process of the cerebellar compartments from E13.5 to E18.5. The result indicated that E10.5-born PCs did not intermingle with E11.5-born PCs and the distribution of them was drastically changed from linear formations to divided compartments. It indicates that PCs acquire their identity at their birthdate. Cohorts of PCs that share the same birthdate communicate each other and as a result they form individual compartments in the cerebellum.	O3-6-3-4 成体脳内を移動するニューロンの集団移動メカニズム: Rac1 FRETバイオセンサーを用いた時空間的解析 Rac1-mediated furrow formation in resting neurons is required for efficient collective migration of new neurons in rostral migratory stream of postnatal mouse brain ○匹田貴夫1, 大野彰久1, 澤田雅人1, 太田晴子1, 澤本和延1 ○Takao Hikita1, Akihisa Ohno1, Masato Sawada1, Haruko Ota1, Kazunobu Sawamoto1 名古屋市立大学　大学院医学研究科　再生医学分野1 Dept of Dev and Regen Biol, Nagoya City Univ, Nagoya1 New neurons generated in the ventricular-subventricular zone (V-SVZ) in the postnatal brain travel toward the olfactory bulbs using a collective cell migration mechanism called 'chain migration'. Here we investigated the activity and function of Rac1 in migration of these neurons. Inhibition of Rac1 activity resulted in migration defect and abnormal morphology of new neurons in vivo. Live cell imaging using FRET biosensor revealed that Rac1 is activated at the tip, proximal part of leading process and rear of cell soma, depending on the step in migration. During chain migration, Rac1 was activated at cell-cell contact site, where resting scaffold neurons formed furrows. Knockdown of Rac1 resulted in the prevention of furrow formation and migration along the resting neurons. These results suggest that Rac1 regulates not only the morphology of migrating neurons but also of resting scaffold neurons, thereby promoting the efficient chain migration of new neurons.

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