トップページ研究室紹介(基礎医学領域)機能形態学 細胞生物学/超微形態学(解剖学第三)

機能形態学

細胞生物学/超微形態学(解剖学第三)

研究室概要

Our Researches

We are studying the mechanism of brain development, especially focusing on the cerebral cortex and the cerebellum. Our main approach is looking at cells in developing brain tissues. We use various tissue culture techniques such as slice culture.

Miyata, T., Kawaguchi, A., Okano, H., and Ogawa, M. Asymmetric inheritance of radial glial fibers by cortical neurons. Neuron 31, 727-741 (2001)

Miyata, T., Kawaguchi, A., Saito, K., Kawano, M., Muto, T., and Ogawa, M.: Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells. Development 131, 3133-3145 (2004)

Miyata, T.and Ogawa, M.: Twisting of neocortical progenitor cells underlies a spring-like mechanism for daughter cell migration. Curr.Biol. 17, 146-151 (2007)

Okamoto, M., Namba, T., Shinoda, T., Kondo, T., Watanabe, T., Inoue, Y., Takeuchi, K., Enomoto, Y., Ota, K., Oda, K., Wada, Y., Sagou, K., Saito, K., Sakakibara, A., Kawaguchi, A., Nakajima, K., Adachi, T., Fujimori, T., Ueda, M. Hayashi, S., Kaibuchi, K., Miyata, T. TAG-1–assisted progenitor elongation streamlines nuclear migration to optimize subapical crowding. Nat. Neurosci.,16: 1556-1566 (2013) DOI: 10.1038/nn.3525

Cells are fluoresently labelled by introducing plasmid vectors designed to express fluorescent proteins (GFP, Venus, mCherry, etc), by using lipophilic dyes (DiI etc), or by making transgenic mice carrying transgenes under tissue-or cell-type-specific promoters. Fluorescently labelled slices, which are prepared manually using microknives under dissection microscopes, are time-lapse monitored using confocal microscopes. This approach enebles us to observe morphology and behavior of single cells. How cells of interest migrate, migrate, or extend processes in the three-dimensional condition can be directly monitored. This technique can be coupled with gene-manipulating experiments using in utero electroporation methods or pharmacological tests. Fully describing and understanding normal and abnormal behaviors of cells and underlying mechanisms in the developing brain is important for (1) establishing a basis of understanding the function of adult brains, (2) elucidating pathogenesis of human brain anomalies and understanding brain diseases from a developmental viewpoint, and (3) contributing to regenerative medicine that uses stem cells to construct artificial three-dimensional tissue-like structures.

宮田卓樹 研究室 HP

研究実績

Recent Publications

Okamoto, M., Shinoda, T., Kawaue, T., Nagasaka, A., Miyata, T. Ferret-mouse differences in interkinetic nuclear migration and cellular densification in the neocortical ventricular zone. Neurosci. Res. 83, 25-32, 2014

PubMed

The thick outer subventricular zone (OSVZ) is characteristic of the development of human neocortex. How this region originates from the ventricular zone (VZ) is largely unknown. Recently, we showed that over-proliferation–induced acute nuclear densification and thickening of the VZ in neocortical walls of mice, which lack an OSVZ, causes reactive delamination of undifferentiated progenitors and invasion by these cells of basal areas outside the VZ. In this study, we sought to determine how VZ cells behave in non-rodent animals that have an OSVZ. A comparison of mid-embryonic mice and ferrets revealed: (1) the VZ is thicker and more pseudostratified in ferrets. (2) The soma and nuclei of VZ cells were horizontally and apicobasally denser in ferrets. (3) Individual endfeet were also denser on the apical (ventricular) surface in ferrets. (4) In ferrets, apicalward nucleokinesis was less directional, whereas basalward nucleokinesis was more directional; consequently, the nuclear density in the periventricular space (within 16 μm of the apical surface) was smaller in ferrets than in mice, despite the nuclear densification seen basally in ferrets. These results suggest that species-specific differences in nucleokinesis strategies may have evolved in close association with the magnitudes and patterns of nuclear stratification in the VZ.

 

Kawaue T, Sagou K, Kiyonari H, Ota K, Okamoto M, Shinoda T, Kawaguchi A, Miyata T. Neurogenin2-d4Venus and Gadd45g-d4Venus transgenic mice: Visualizing mitotic and migratory behaviors of cells committed to the neuronal lineage in the developing mammalian brain. Dev Growth Differ. 56, 293-304, 2014

PubMed To achieve highly sensitive and comprehensive assessment of the morphology and dynamics of cells committed to the neuronal lineage in mammalian brain primordia, we generated two transgenic mouse lines expressing a destabilized (d4) Venus controlled by regulatory elements of the Neurogenin2 (Neurog2) or Gadd45g gene. In mid-embryonic neocortical walls, expression of Neurog2-d4Venus mostly overlapped with that of Neurog2 protein, with a slightly (1 h) delayed onset. Although Neurog2-d4Venus and Gadd45g-d4Venus mice exhibited very similar labeling patterns in the ventricular zone (VZ), in Gadd45g-d4Venus mice cells could be visualized in more basal areas containing fully differentiated neurons, where Neurog2-d4Venus fluorescence was absent. Time-lapse monitoring revealed that most d4Venus+ cells in the VZ had processes extending to the apical surface; many of these cells eventually retracted their apical process and migrated basally to the subventricular zone, where neurons, as well as the intermediate neurogenic progenitors that undergo terminal neuron-producing division, could be live-monitored by d4Venus fluorescence. Some d4Venus+ VZ cells instead underwent nuclear migration to the apical surface, where they divided to generate two d4Venus+ daughter cells, suggesting that the symmetric terminal division that gives rise to neuron pairs at the apical surface can be reliably live-monitored. Similar lineage-committed cells were observed in other developing neural regions including retina, spinal cord, and cerebellum, as well as in regions of the peripheral nervous system such as dorsal root ganglia. These mouse lines will be useful for elucidating the cellular and molecular mechanisms underlying development of the mammalian nervous system.

 

Hashimoto, M., Hata, A., Miyata, T., Hirase, H.  Programmable wireless light-emitting diode stimulator for chronic stimulation of optogenetic molecules in freely moving mice. Neurophoton. 1(1), 011002 (May 28, 2014). doi:10.1117/1.NPh.1.1.011002

 

Namba, T., Kibe, Y., Funahashi, Y., Nakamuta, S., Takano, T., Ueno, T., Shimada, A., Kozawa, S., Okamoto, M., Shimoda, Y., Oda, K., Wada, Y., Masuda, T., Sakakibara, A., Igarashi, M., Miyata, T., Faivre-Sarrailh, C., Takeuchi, K., Kaibuchi, K. Pioneering axons regulate neuronal polarization in the devveloping cerebral cortex. Neuron 81, 814-829, 2014

PubMed

 

Ageta-Ishihara, N., Miyata, T., Ohshima, C., Watanabe, M., Sato, Y., Hamamura, Y., Higashijima, T., Mazitschek, R., Bito, H., Kinoshita, M. Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation. Nat. Commun. 4: 2532, DOI: 10.1038/ncomms3532

PubMed

 

Okamoto, M., Namba, T., Shinoda, T., Kondo, T., Watanabe, T., Inoue, Y., Takeuchi, K., Enomoto, Y., Ota, K., Oda, K., Wada, Y., Sagou, K., Saito, K., Sakakibara, A., Kawaguchi, A., Nakajima, K., Adachi, T., Fujimori, T., Ueda, M. Hayashi, S., Kaibuchi, K., Miyata, T. TAG-1–assisted progenitor elongation streamlines nuclear migration to optimize subapical crowding. Nat. Neurosci., 16: 1556-1566 (2013) DOI: 10.1038/nn.3525

プレスリリース

PubMed

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 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

Results

・Heterogeneous apicobasal nuclear movements in the VZ

・TAG-1 knockdown induces progenitor shortening

・Shortened progenitors are periventricularly overcrowded

・Overcrowded cells are under excessive mechanical stress

・Overcrowded progenitors delaminate and form heterotopia

・Heterotopia maintains cytogenesis but disrupt histogenesis

Discussion

・TAG-1 and progenitors' histogenetic behaviors

・Morphology-based and synecological understanding of INM

・Mechanics underlying progenitors' behaviors

・Robust cytogenesis by shortened and heterotopic progenitors

 

Sapir, T., Levy, T., Sakakibara, A., Rabinkov, A., Miyata, T., Reiner, O. Shootin1 acts in concert with KIF20B to promote polarization of migrating neurons. (2013) J. Neurosci. 33:11932-11948 DOI: 10.1523/JNEUROSCI.5425-12.2013

PubMed

 

Wu, J Liu, L Matsuda, T , Zao, YRebane, A Drobizhev, M Chang, Y-F Araki, S Arai, Y March, K Thomas, HESagou, K , Miyata, TNagai, T , Li, W-H , and Campbell, RE  Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications. ACS Chem. Neurosci. (2013.3.1 on line出版)DOI: 10.1021/cn400012b

PubMed

 

Sakakibara, A.(corresponding author), Sato, T., Ando, R., Noguchi, N., Masaoka, M., Miyata, T. Dynamics of centrosome translocation and microtubule organization in neocortical neurons during distinct modes of polarization. Cereb. Cortex 24, 1301-1310, 2014 (doi:10.1093/cercor/bhs411)

PubMed

 

Xie, M.-J., Yagi, H., Kuroda, K., Wang, C.-C., Komada, M., Zhao, H., Sakakibara, A., Miyata, T. Nagata, K., Iguchi, T., Sato, M. WAVE2-Abi2 complex controls growth cone activity and regulates the multipolar-bipolar transition as well as the initiation of glia-guided migration. Cereb. Cortex 23:1410-1423 (2013) (doi: 10.1093/cercor/bhs123)

PubMed

 

Pérez-Martínez, F.J., Luque-Río, A., Sakakibara, A., Hattori, M., Miyata, T., Luque, J. M. Reelin-dependent ApoER2 downregulation uncouples newborn neurons from progenitor cells. Biol. Open 1:1258-1263 (2012) 

PubMed

 

Nakamuta, S., Funahashi, Y., Namba, T., Arimura, N., Picciotto, M.R., Tokumitsu, H., Soderling, T.R., Sakakibara, A., Miyata, T., Kamiguchi, H., Kaibuchi, K. Local application of neurotrophins specifies axons through inositol 1,4,5-trisphosphate, calcium, and ca2+/calmodulin-dependent protein kinases. Sci. Signal. 4(199):ra76 (2011)

PubMed

 

Natsume, S., Kato, T., Kinjo, S., Enomoto, A., Toda, H., Shimato, S., Ohka, F., Motomura, K., Kondo, Y., Miyata, T., Takahashi, M., Wakabayashi, T. Girdin maintains the stemness of glioblastoma stem cells. Oncogene 312715-2724 (2011)

PubMed

 

Miyata, T., Ono Y, Okamoto M, Masaoka M, Sakakibara A, Kawaguchi A, Hashimoto M, Ogawa M. Migration, early axonogenesis, and Reelin-dependent layer-forming behavior of early/posterior-born Purkinje cells in the developing mouse lateral cerebellum. Neural Dev. 5, 23 (2010) 

highly_accessed.gif PubMed

Purpose

How the young Purkinje cells migrate and initiate the layer formation in response to Reelin in the developing cerebellum? Although the dependence of Purkinje cells’ layer formation on the secreted protein Reelin is well known and a prevailing model suggests that Purkinje cells migrate along the “radial glial” fibers connecting the ventricular and pial surfaces, it is not clear how Purkinje cells behave in response to Reelin to initiate their layer. Furthermore, it is not known what nascent Purkinje cells look like in vivo. When and how Purkinje cells start axonogenesis must also be elucidated.

Results

We found that Purkinje cells generated on embryonic day (E) 10 in the developing mouse cerebellum migrate tangentially towards the anterior, exhibiting an elongated morphology consistent with axonogenesis at E12. After their somata reach the outer/dorsal region by E13, they change “posture” by E14 through remodeling of non-axon (dendrite-like) processes and a switchback-like mode of somal movement towards a superficial Reelin-rich zone, while their axon-like fibers remain relatively deep, which demarcates the somata-packed portion as a plate (called the Purkinje plate). In the cerebellum of reelermice, which suffer from ataxic gait due to abnormal cerebellar cortical histogenesis, the early born posterior lateral Purkinje cells are initially normal during migration with anteriorly-extended axon-like fibers until E13, but then fail to form the plate due to the lack of the posture-change step. This is the first demonstration of the beginning of layer formation by Purkinje cells (summarized in the figure, published in Neural Development [2010] and selected as a “highly accessed paper”). This study provides a solid basis for further elucidation of Reelin’s function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives the overall brain morphogenesis. Our finding is relevant to future efforts to reconstruct the Purkinje cells layer in the cerebellum affected by degenerative diseases.

 

Miyata, T., Kawaguchi, D., Kawaguchi, A., Gotoh, Y. Mechanisms that regulate the number of neurons during mouse neocortical development. Curr. Opin. Neurobiol. 20, 22-28 (2010)

PubMed

 

Kato TM, Kawaguchi A, Kosodo Y, Niwa H, *Matsuzaki F. Lunatic fringe potentiates Notch signaling in the developing brain. Mol Cell Neurosci. 45, 12-25, 2010

PubMed

 

Uchida T, Baba A, Perez-Martinez FJ, Hibi T, Miyata T, Luque JM, Nakajima K, Hattori M. Downregulation of functional Reelin receptors in projection neurons implies that primary Reelin action occurs at early/premigratory stages. J Neurosci. 29:10653-62 (2009)

PubMed

 

Saito K, Dubreuil V, Arai Y, Wilsch-Brauninger M, Schwudke D, Saher G, Miyata T, Breier G, Thiele C, Shevchenko A, Nave KA, Huttner WB. Ablation of cholesterol biosynthesis in neural stem cells increases their VEGF expression and angiogenesis but causes neuron apoptosis. Proc Natl Acad Sci U S A 106(20):8350-5 (2009)

PubMed

 

Minobe, S., Sakakibara, A., Ohdachi, T., Kanda, R., Kimura, M., Nakatani, S., Tadokoro, R., Ochiai, W., Nishizawa, Y., Mizoguchi, A., Kawauchi, T., Miyata, T.: Rac is involved in the interkinetic nuclear migration of cortical progenitor cells. Neurosci. Res. 63, 294-301 (2009)

PubMed

Purpose

In the developing brain wall, neural progenitor cells exhibit nuclear migration during their cell cycle progression. After completing S phase in the basal (pial) side of the neuroepithelium or ventricular zone (VZ), their nuclei go to the apical (ventricular) surface of the VZ, where they undergo division. The nuclei of cells newly born at the apical surface of the VZ move toward the basal side during G1 phase of the cell cycle. This to-and-fro nuclear/somal movement or interkinetic nuclear migration (INM) was first suggested by Sauer in 1935 and was experimentally proven by pulse-and-chase experiments based on 3H-thymidine labeling. Our group previously established a time-lapse monitoring system of INM (Neuron, 2001). Although INM seems to be important for proper cytogenesis, the molecular mechanisms that underlie INM are not well understood. The small GTPase Rac functions in a number of cellular processes, including cytoskeletal regulation, cell-cell adhesion, and migration. Recent three-dimensional functional studies in the developing brain have shown that Rac regulates migration of neurons and extension of neuronal processes. Based on the function of Rac in neurons that exhibit dynamic morphological changes, we reasoned that Rac might also work in progenitor cells, which show highly dynamic behavior, such as INM and cell division. Involvement of Rac in INM has not yet been directly assessed at the single-cell level.

Results

 By cross-sectional and orthogonal immunofluorescence examination, we found that Rac1 is expressed in mid-embryonic mouse telencephalic progenitor cells. Localization is marked at the apical endfoot. Pharmacological inhibition of Rac in slice cultures during the adventricular phase of INM retards nucleokinesis and results in unsuccessful cytokinesis at the apical surface. Similar results were obtained by introducing a dominant-negative form of Rac1. These results suggest that Rac may play a role in INM in the developing mouse brain (Neuroscience Research, 2009).

 

Ochiai, W.,* Nakatani, S.,* Takahara, T., Kainuma, M., Masaoka, M., Minobe, S., Namihira, M., Nakashima, K., Sakakibara, A., Ogawa, M., Miyata, T.: Periventricular Notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells. Mol. Cell. Neurosci. 40, 225-233 (2009) (*Equal contribution)

PubMed

Purpose

Formation of the neocortex relies on the precise balance between neurogenesis and maintenance of a neural progenitor pool in the pallial primordium during embryonic development. The mechanism of this asymmetric daughter-cell production by the progenitor population has been extensively studied, but is still not fully understood In order to elucidate the intrinsic and extrinsic mechanisms regulating the asymmetric cell output of progenitor cells, a careful analysis of the spatiotemporal expression patterns of factors controlling cell fate is needed. Especially, how Notch is activated in neural progenitor cells and their daughter cells and how Notch ligand such as Delta-like 1 (Dll1) is expressed need to be elucidated. We previously found that differentiation of progenitor cells towards the neuronal lineage is regulated by Neurogenin2 (Ngn2) (Development, 2004). Then, Tbr2 was reported to be also important for the commitment to the neuronal lineage. Therefore, we extended our analysis to ask how nascent daughter cells start expressing Ngn2, and to determine the relationship between Ngn2 and Tbr2 during the course of lineage commitment of neural progenitor cells.

Results

Through collaboration with Dr. Yoon Kong (Pohang University of Science and Technology), we found that neural progenitor cells committed to the neuronal lineage expresse the Notch-ligand Delta-like 1 (Dll1). Further, our time-lapse observation directly showed that progenitor cells whose Notch activation is reduced by conditional knock-out of Mind bomb-1 (Mib1), an essential component of Notch ligand endocytosis, failed to maintain undifferentiated progenitor pool, leading to premature neuronal differentiation (Neuron, 2008). To track the developmental time course of Ngn2 and Tbr2 expression in VZ cells, time-lapse observation was performed on daughter cells of individually DiI-labeled progenitor cells in cultured neocortical slices, followed by immunostaining for Ngn2 or Tbr2. We found that Ngn2 protein expression was initiated asymmetrically in the surface-generated daughter cells as early as 2 h after birth, about 2 h earlier than the onset of Tbr2 expression. Luciferase and ChIP assays further revealed that Tbr2 is directly downstream of Ngn2. Daughter cells expressing Ngn2 or Tbr2 were connected to the ventricular surface and maintained expression of the two transcription factors after detaching from the ventricular surface. Inhibition of Notch signaling in nascent surface-born daughter cells by treatment with a γ-seceretase inhibitor strikingly increased the frequency of Ngn2 expression in daughter cells 2 h after birth. Activation of Notch was observed not only in the basal VZ, but also in the periventricular VZ containing nascent daughter cells. These results suggest that the periventricular area and the initial morphology of surface-born daughter cells may be important for the regulation of cell fate choice. (Molecular Cellular Neuroscience, 2009).

 

Yoon, K.-J., Koo, B.-K., Jeong, H.-W., Ghim, J., Kwon, M.-C., Moon, J.-S., Miyata, T.Kong, Y.-Y.: Mind bomb 1-experssing intermediate progenitors generate Notch signaling to maintain radial glial cells. Neuron 58, 519-531 (2008)

PubMed

 

Sunabori, T., Tokunaga, A., Nagai, T., Sawamoto, K., Okabe, M., Miyawaki, A., Matsuzaki, Y., Miyata, T., Okano, H.: Cell-cycle-specific nestin expression coordinates with morphological changes in embryonic cortical neural progenitors. J. Cell Sci. 121, 1204-1212 (2008)

PubMed

 

Koyasu T, Kondo M, Miyata K, Ueno S, Miyata T, Nishizawa Y, Terasaki H.  Photopic electroretinograms of mGluR6-deficient mice. Curr Eye Res. 33, 91-99 (2008) 

PubMed

 

Miyata, T.: Development of three-dimensional architecture of the neuroepithelium: Role of pseudostratification and cellular 'community'. Dev. Growth Differ. 50, S105-S112 (2008)

PubMed

 

Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S., Fukami, K., Miyata, T., Miyoshi, H., Imamura, T., Ogawa, M., Masai, H. and Miyawaki, A.: Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487-498 (2008).

PubMed

 

Konno, D., Shioi, G., Shitamukai, A., Mori, A., Kiyonari, H., Miyata, T. and Matsuzaki, F.: Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Nat. Cell Biol. 10, 93-101 (2008)

PubMed

 

Nishizawa, Y., Imafuku, H., Saito, K., Kanda, R., Kimura, M., Minobe, S., Miyazaki, F., Kawakatsu, S., Masaoka, M., Ogawa, M. and Miyata, T.: Survey of the morphogenetic dynamics of the ventricular surface of the developing mouse cortex. Dev. Dyn.  236, 3061-3070 (2007)

PubMed

 

Tamai, H., Shinohara, H., Miyata, T., Saito, K., Nishizawa, Y., Nomura, T. and Osumi, N.: Pax6 transcription factor regulates interkinetic nuclear movement in cortical progenitor cells via centrosomal stabilization. Genes Cells 12, 983-996 (2007)

PubMed

 

Miyata, T.: Morphology and mechanics of daughter cells "delaminating" from the ventricular zone of the developing neocortex. Cell Adh. Migr. 1, 99-101(2007)

PubMed

 

Miyata, T.and Ogawa, M.: Twisting of neocortical progenitor cells underlies a spring-like mechanism for daughter cell migration. Curr.Biol. 17, 146-151 (2007)

PubMed

 

Ochiai, W., Minobe, S., Ogawa, M., Miyata, T.: Transformation of pin-like ventricular zone cells into cortical neurons. Neurosci. Res. 57, 326-329 (2007)

PubMed

 

Miyata, T.: Asymmetric cell division during brain morphogenesis. Prog. Mol. Subcell. Biol. 452, 121-142 (2007)

PubMed

 

大学院入学案内

脳が形成される原理に興味を持ちその解明に挑んでくれる修士課程,博士課程の学生を募集します.神経前駆細胞とその娘細胞の三次元組織中での挙動をとらえるための新しいイメージング法あるいは細胞機能を問うための新しい実験法などの開発には多角的で柔軟な発想で挑む必要がありますので,いろいろなバックグラウンドの方を広く歓迎します.他分野で最先端の研究を進める国内施設(連携的研究者の所属施設)における長期滞在型の技術取得など,さまざまな支援を行なっていく予定です.
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構成員名/英名表記 役職 所属
宮田卓樹/MIYATA Takaki
教授細胞生物学分野
川口綾乃/KAWAGUCHI Ayano
准教授細胞生物学分野
篠田友靖/SHINODA Tomoyasu
助教細胞生物学分野
服部祐季/HATTORI Yuki
特任助教細胞生物学分野
齋藤加奈子/SAITO Kanako
学振研究員(RPD)細胞生物学分野

研究分野紹介

専攻 機能構築医学
大講座 機能形態学
分野 細胞生物学

研究キーワード

中枢神経系,発生,細胞運命決定,細胞移動,層形成,細胞極性,イメージング,単一細胞遺伝子発現プロファイリング