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Papers - Hirota, Yuki

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Heterozygous Dab1 Null Mutation Disrupts Neocortical and Hippocampal Development

Honda T., Hirota Y., Nakajima K.

eNeuro 10 ( 4 ) 2023.04

Loss-of-function mutations in Reelin and DAB1 signaling pathways disrupt proper neuronal positioning in the cerebral neocortex and hippocampus, but the underlying molecular mechanisms remain elusive. Here, we re-port that heterozygous yotari mice harboring a single autosomal recessive yotari mutation of Dab1 exhibited a thinner neocortical layer 1 than wild-type mice on postnatal day (P)7. However, a birth-dating study suggested that this reduction was not caused by failure of neuronal migration. In utero electroporation-mediated sparse labeling revealed that the superficial layer neurons of heterozygous yotari mice tended to elongate their apical dendrites within layer 2 than within layer 1. In addition, the CA1 pyramidal cell layer in the caudo-dorsal hippo-campus was abnormally split in heterozygous yotari mice, and a birth-dating study revealed that this splitting was caused mainly by migration failure of late-born pyramidal neurons. Adeno-associated virus (AAV)-medi-ated sparse labeling further showed that many pyramidal cells within the split cell had misoriented apical den-drites. These results suggest that regulation of neuronal migration and positioning by Reelin-DAB1 signaling pathways has unique dependencies on Dab1 gene dosage in different brain regions.

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A Unique “Reversed” Migration of Neurons in the Developing Claustrum

Oshima K., Yoshinaga S., Kitazawa A., Hirota Y., Nakajima K., Kubo K.I.

Journal of Neuroscience 43 ( 5 ) 693 - 708 2023.02

ISSN 02706474

The claustrum (CLA) is a cluster of neurons located between the insular cortex and striatum. Many studies have shown that the CLA plays an important role in higher brain function. Additionally, growing evidence suggests that CLA dysfunction is associated with neuropsychological symptoms. However, how the CLA is formed during development is not fully understood. In the present study, we analyzed the development of the CLA, especially focusing on the migration profiles of CLA neurons in mice of both sexes. First, we showed that CLA neurons were generated between embryonic day (E) 10.5 and E12.5, but mostly at E11.5. Next, we labeled CLA neurons born at E11.5 using the FlashTag technology and revealed that most neurons reached the brain surface by E13.5 but were distributed deep in the CLA 1 d later at E14.5. Time-lapse imaging of GFP-labeled cells revealed that some CLA neurons first migrated radially outward and then changed their direction inward after reaching the surface. Moreover, we demonstrated that Reelin signal is necessary for the appropriate distribution of CLA neurons. The switch from outward to “reversed” migration of developing CLA neurons is distinct from other migration modes, in which neurons typically migrate in a certain direction, which is simply outward or inward. Future elucidation of the characteristics and precise molecular mechanisms of CLA development may provide insights into the unique cognitive functions of the CLA.

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Erratic and blood vessel-guided migration of astrocyte progenitors in the cerebral cortex

Tabata H., Sasaki M., Agetsuma M., Sano H., Hirota Y., Miyajima M., Hayashi K., Honda T., Nishikawa M., Inaguma Y., Ito H., Takebayashi H., Ema M., Ikenaka K., Nabekura J., Nagata K.i., Nakajima K.

Nature Communications 13 ( 1 ) 2022.12

Astrocytes are one of the most abundant cell types in the mammalian brain. They play essential roles in synapse formation, maturation, and elimination. However, how astrocytes migrate into the gray matter to accomplish these processes is poorly understood. Here, we show that, by combinational analyses of in vitro and in vivo time-lapse observations and lineage traces, astrocyte progenitors move rapidly and irregularly within the developing cortex, which we call erratic migration. Astrocyte progenitors also adopt blood vessel-guided migration. These highly motile progenitors are generated in the restricted prenatal stages and differentiate into protoplasmic astrocytes in the gray matter, whereas postnatally generated progenitors do not move extensively and differentiate into fibrous astrocytes in the white matter. We found Cxcr4/7, and integrin β1 regulate the blood vessel-guided migration, and their functional blocking disrupts their positioning. This study provides insight into astrocyte development and may contribute to understanding the pathogenesis caused by their defects.

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TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF)

Morimoto Y., Tokumitsu A., Sone T., Hirota Y., Tamura R., Sakamoto A., Nakajima K., Toda M., Kawakami Y., Okano H., Ohta S.

Neurochemical Research 47 ( 9 ) 2741 - 2756 2022.09

ISSN 03643190

One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF–CHD7–TPT1–SMO signaling axis in regulating hESC–NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC–NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway. Graphical Abstract: [Figure not available: see fulltext.]

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Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone in mice

Ito N., Riyadh M.A., Ahmad S.A.I., Hattori S., Kanemura Y., Kiyonari H., Abe T., Furuta Y., Shinmyo Y., Kaneko N., Hirota Y., Lupo G., Hatakeyama J., Felemban Athary Abdulhaleem M., Anam M.B., Yamaguchi M., Takeo T., Takebayashi H., Takebayashi M., Oike Y., Nakagata N., Shimamura K., Holtzman M.J., Takahashi Y., Guillemot F., Miyakawa T., Sawamoto K., Ohta K.

Science Translational Medicine 13 ( 587 ) 2021.03

ISSN 19466234

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.

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The secreted glycoprotein reelin suppresses the proliferation and regulates the distribution of oligodendrocyte progenitor cells in the embryonic neocortex

Ogino H., Nakajima T., Hirota Y., Toriuchi K., Aoyama M., Nakajima K., Hattori M.

Journal of Neuroscience (Journal of Neuroscience) 40 ( 40 ) 7625 - 7636 2020.09

ISSN 02706474

Copyright © 2020 the authors. Oligodendrocyte (OL) progenitor cells (OPCs) are generated, proliferate, migrate, and differentiate in the developing brain. Although the development of OPCs is prerequisite for normal brain function, the molecular mechanisms regulating their development in the neocortex are not fully understood. Several molecules regulate the tangential distribution of OPCs in the developing neocortex, but the cue molecule(s) that regulate their radial distribution remains unknown. Here, we demonstrate that the secreted glycoprotein Reelin suppresses the proliferation of OPCs and acts as a repellent for their migration in vitro. These functions rely on the binding of Reelin to its receptors and on the signal transduction involving the intracellular protein Dab1. In the late embryonic neocortex of mice with attenuated Reelin signaling [i.e., Reelin heterozygote-deficient, Dab1 heterozygote-deficient mutant, or very low-density lipoprotein receptor (VLDLR)-deficient mice], the number of OPCs increased and their distribution shifted toward the superficial layers. In contrast, the number of OPCs decreased and they tended to distribute in the deep layers in the neocortex of mice with abrogated inactivation of Reelin by proteolytic cleavage, namely a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3)-deficient mice and cleavage-resistant Reelin knock-in mice. Both male and female animals were used. These data indicate that Reelin–Dab1 signaling regulates the proliferation and radial distribution of OPCs in the late embryonic neocortex and that the regulation of Reelin function by its specific proteolysis is required for the normal development of OPCs.

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VLDLR is not essential for reelin-induced neuronal aggregation but suppresses neuronal invasion into the marginal zone.

Hirota Y, Nakajima K.

Development (Development (Cambridge)) in press ( 12 ) 2020

Research paper (scientific journal), Joint Work, Accepted, ISSN 09501991

© 2020. Published by The Company of Biologists Ltd In the developing neocortex, radially migrating neurons stop migration and form layers beneath the marginal zone (MZ). Reelin plays essential roles in these processes via its receptors, apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). Although we recently reported that reelin causes neuronal aggregation via ApoER2, which is thought to be important for the subsequent layer formation, it remains unknown what effect reelin exerts via the VLDLR. Here, we found that ectopic reelin overexpression in the Vldlr-mutant mouse cortex causes neuronal aggregation, but without an MZ-like cell-sparse central region that is formed when reelin is overexpressed in the normal cortex. We also found that both the early-born and late-born Vldlr-deficient neurons invade the MZ and exhibit impaired dendrite outgrowth from before birth. Rescue experiments indicate that VLDLR suppresses neuronal invasion into the MZ via a cell-autonomous mechanism, possibly mediated by Rap1, integrin and Akt. These results suggest that VLDLR is not a prerequisite for reelin-induced neuronal aggregation and that the major role of VLDLR is to suppress neuronal invasion into the MZ during neocortical development.

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ApoER2 Controls Not Only Neuronal Migration in the Intermediate Zone But Also Termination of Migration in the Developing Cerebral Cortex

Hirota Yuki

Cereb Cortex (Cerebral Cortex) 28 ( 1 ) 223 - 235 2018.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 1460-2199

© The Author 2016. Published by Oxford University Press. All rights reserved. Neuronal migration contributes to the establishment of mammalian brain. The extracellular protein Reelin sends signals to various downstream molecules by binding to its receptors, the apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor and exerts essential roles in the neuronal migration and formation of the layered neocortex. However, the cellular and molecular functions of Reelin signaling in the cortical development are not yet fully understood. Here, to gain insight into the role of Reelin signaling during cortical development, we examined the migratory behavior of Apoer2-deficient neurons in the developing brain. Stage-specific labeling of newborn neurons revealed that the neurons ectopically invaded the marginal zone (MZ) and that neuronal migration of both early- and late-born neurons was disrupted in the intermediate zone (IZ) in the Apoer2 KO mice. Rescue experiments showed that ApoER2 functions both in cell-autonomous and noncell-autonomous manners, that Rap1, integrin, and Akt are involved in the termination of migration beneath the MZ, and that Akt also controls neuronal migration in the IZ downstream of ApoER2. These data indicate that ApoER2 controls multiple processes in neuronal migration, including the early stage of radial migration and termination of migration beneath the MZ in the developing neocortex.

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Control of Neuronal Migration and Aggregation by Reelin Signaling in the Developing Cerebral Cortex

Hirota Yuki

Frontiers in Cell and Developmental Biology 5 ( 40 ) 2017.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 2296-634X

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Roles of Wnt Signaling in the Neurogenic Niche of the Adult Mouse Ventricular-Subventricular Zone

Hirota, Yuki, Sawada, Masato, Huang, Shih-hui, Ogino, Takashi, Ohata, Shinya, Kubo, Akiharu, Sawamoto, Kazunobu

NEUROCHEMICAL RESEARCH 41 ( 1-2 ) 222 - 230 2016.02

Research paper (scientific journal), ISSN 0364-3190

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Reelin has a preventive effect on phencyclidine-induced cognitive and sensory-motor gating deficits

Ishii Kazuhiro, Nagai Taku, Hirota Yuki, Noda Mariko, Nabeshima Toshitaka, Yamada Kiyofumi, Kubo Ken ichiro, Nakajima Kazunori

Neuroscience Research 96 30 - 36 2015.07

ISSN 0168-0102

Reelin has recently attracted attention because of its connection to several neuropsychiatric diseases. We previously reported the finding that prior transplantation of GABAergic neuron precursor cells into the medial prefrontal cortex (mPFC) of mice significantly prevented the induction of cognitive and sensory-motor gating deficits induced by phencyclidine (PCP). The majority of the precursor cells transplanted into the mPFC of the recipient mice differentiated into members of a somatostatin/Reelin-expressing class of GABAergic interneurons. These findings raised the possibility that Reelin secreted by the transplanted cells plays an important role in preventing the deficits induced by PCP. In this study, we investigated whether Reelin itself has a preventive effect on PCP-induced behavioral phenotypes by injecting conditioned medium containing Reelin into the lateral ventricle of the brains of 6- to 7-week-old male mice before administrating PCP. Behavioral analyses showed that the prior Reelin injection had a preventive effect against induction of the cognitive and sensory-motor gating deficits associated with PCP. Moreover, one of the types of Reelin receptor was found to be expressed by neurons in the mPFC. The results of this study point to the Reelin signaling pathway as a candidate target for the pharmacologic treatment of neuropsychiatric diseases.

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Reelin receptors ApoER2 and VLDLR are expressed in distinct spatiotemporal patterns in developing mouse cerebral cortex

Hirota Yuki, Kubo Ken ichiro, Katayama Kei ichi, Honda Takao, Fujino Takahiro, Yamamoto Tokuo T., Nakajima Kazunori

Journal of Comparative Neurology 523 ( 3 ) 463 - 478 2015.02

ISSN 0021-9967

In mammalian developing brain, neuronal migration is regulated by a variety of signaling cascades, including Reelin signaling. Reelin is a glycoprotein that is mainly secreted by Cajal-Retzius neurons in the marginal zone, playing essential roles in the formation of the layered neocortex via its receptors, apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). However, the precise mechanisms by which Reelin signaling controls the neuronal migration process remain unclear. To gain insight into how Reelin signaling controls individual migrating neurons, we generated monoclonal antibodies against ApoER2 and VLDLR and examined the localization of Reelin receptors in the developing mouse cerebral cortex. Immunohistochemical analyses revealed that VLDLR is localized to the distal portion of leading processes in the marginal zone (MZ), whereas ApoER2 is mainly localized to neuronal processes and the cell membranes of multipolar cells in the multipolar cell accumulation zone (MAZ). These different expression patterns may contribute to the distinct actions of Reelin on migrating neurons during both the early and late migratory stages in the developing cerebral cortex. J. Comp. Neurol. 523:463-478, 2015.

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Roles of planar cell polarity signaling in maturation of neuronal precursor cells in the postnatal mouse olfactory bulb

Hirota Yuki, Sawada Masato, Kida Yasuyuki S., Huang Shih Hui, Yamada Osamu, Sakaguchi Masanori, Ogura Toshihiko, Okano Hideyuki, Sawamoto Kazunobu

Stem Cells 30 ( 8 ) 1726 - 1733 2012.08

ISSN 1066-5099

Neuronal precursor cells generated by stem cells in the subventricular zone (SVZ) migrate and differentiate into mature interneurons in the olfactory bulb (OB). The mechanisms responsible for the dynamic morphological changes in cells during this process are largely unknown. Wnt/planar cell polarity (PCP) signaling regulates various developmental events, including neuronal migration and neurite formation. Here, we studied the function of two components of the PCP pathway, Dishevelled2 and Van Gogh like-2, in the newborn neurons in the postnatal mouse OB. Electroporation- or lentivirus-mediated introduction of vectors carrying a knockdown or dominant-negative construct of these genes into the SVZ altered the distribution and dendrite formation of newborn neurons in the OB, suggesting that PCP signaling is involved in regulating the maturation of new neurons in the OB. © AlphaMed Press.

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Cellular Composition and Organization of the Subventricular Zone and Rostral Migratory Stream in the Adult and Neonatal Common Marmoset Brain

Sawamoto, Kazunobu, Hirota, Yuki, Alfaro-Cervello, Clara, Soriano-Navarro, Mario, He, Xiaoping, Hayakawa-Yano, Yoshika, Yamada, Masayuki, Hikishima, Keigo, Tabata, Hidenori, Iwanami, Akio, Nakajima, Kazunori, Toyama, Yoshiaki, Itoh, Toshio, Alvarez-Buylla, Arturo, Manuel Garcia-Verdugo, Jose, Okano, Hideyuki

JOURNAL OF COMPARATIVE NEUROLOGY 519 ( 4 ) 690 - 713 2011.03

Research paper (scientific journal), ISSN 0021-9967

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Expression and Proliferation-Promoting Role of Diversin in the Neuronally Committed Precursor Cells Migrating in the Adult Mouse Brain

Ikeda, Makiko, Hirota, Yuki, Sakaguchi, Masanori, Yamada, Osamu, Kida, Yasuyuki S., Ogura, Toshihiko, Otsuka, Takanobu, Okano, Hideyuki, Sawamoto, Kazunobu

STEM CELLS 28 ( 11 ) 2017 - 2026 2010.11

Research paper (scientific journal), ISSN 1066-5099

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Reelin-Nrp1 interaction regulates neocortical dendrite development in a context-specific manner

2. Kohno, T., Ishii, K., Hirota, Y., Honda, T., Makino, M., Kawasaki, T., Nakajima, K., Hattori, M

Journal of Neuroscience (Journal of Neuroscience) 40 ( 43 ) 8248 - 8261 2010.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 02706474

© 2020 the authors Reelin plays versatile roles in neocortical development. The C-terminal region (CTR) of Reelin is required for the correct formation of the superficial structure of the neocortex; however, the mechanisms by which this position-specific effect occurs remain largely unknown. In this study, we demonstrate that Reelin with an intact CTR binds to neuropilin-1 (Nrp1), a transmembrane protein. Both male and female mice were used. Nrp1 is localized with very-low-density lipoprotein receptor (VLDLR), a canonical Reelin receptor, in the superficial layers of the developing neocortex. It forms a complex with VLDLR, and this interaction is modulated by the alternative splicing of VLDLR. Reelin with an intact CTR binds more strongly to the VLDLR/Nrp1 complex than to VLDLR alone. Knockdown of Nrp1 in neurons leads to the accumulation of Dab1 protein. Since the degradation of Dab1 is induced by Reelin signaling, it is suggested that Nrp1 augments Reelin signaling. The interaction between Reelin and Nrp1 is required for normal dendritic development in superficial-layer neurons. All of these characteristics of Reelin are abrogated by proteolytic processing of the six C-terminal amino acid residues of Reelin (0.17% of the whole protein). Therefore, Nrp1 is a coreceptor molecule for Reelin and, together with the proteolytic processing of Reelin, can account for context-specific Reelin function in brain development.

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Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II

Hirota Yuki, Meunier Alice, Huang Shihhui, Shimozawa Togo, Yamada Osamu, Kida Yasuyuki S., Inoue Masashi, Ito Tsubasa, Kato Hiroko, Sakaguchi Masanori, Sunabori Takehiko, Nakaya Masa Aki, Nonaka Shigenori, Ogura Toshihiko, Higuchi Hideo, Okano Hideyuki, Spassky Nathalie, Sawamoto Kazunobu

Development 137 ( 18 ) 3037 - 3046 2010.09

ISSN 0950-1991

Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.

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New Neurons Clear the Path of Astrocytic Processes for Their Rapid Migration in the Adult Brain

Kaneko, Naoko, Marin, Oscar, Koike, Masato, Hirota, Yuki, Uchiyama, Yasuo, Wu, Jane Y., Lu, Qiang, Tessier-Lavigne, Marc, Alvarez-Buylla, Arturo, Okano, Hideyuki, Rubenstein, John L. R., Sawamoto, Kazunobu

NEURON 67 ( 2 ) 213 - 223 2010.07

Research paper (scientific journal), ISSN 0896-6273

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Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia

Guirao, Boris, Meunier, Alice, Mortaud, Stephane, Aguilar, Andrea, Corsi, Jean-Marc, Strehl, Laetitia, Hirota, Yuki, Desoeuvre, Angelique, Boutin, Camille, Han, Young-Goo, Mirzadeh, Zaman, Cremer, Harold, Montcouquiol, Mireille, Sawamoto, Kazunobu, Spassky, Nathalie

NATURE CELL BIOLOGY 12 ( 4 ) 341 - U86 2010.04

Research paper (scientific journal), ISSN 1465-7392

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Subventricular zone-derived neural progenitor cells migrate along a blood vessel scaffold toward the post-stroke striatum

Kojima Takuro, Hirota Yuki, Ema Masatsugu, Takahashi Satoru, Miyoshi Ichiro, Okano Hideyuki, Sawamoto Kazunobu

Stem Cells 28 ( 3 ) 545 - 554 2010.03

ISSN 1066-5099

The subventricular zone (SVZ) of the adult brain contains neural stem cells that have the capacity to regenerate new neurons after various insults. Brain ischemia causes damage to brain tissue and induces neural regeneration together with angiogenesis. We previously reported that, after ischemic injury in mice, SVZ-derived neural progenitor cells (NPCs) migrate into the striatum, and these NPCs are frequently associated with blood vessels in the regenerating brain tissue. Here we studied the role of blood vessels during the neural regeneration in more detail. BrdU administration experiments revealed that newly generated NPCs were associated with both newly formed and pre-existing blood vessels in the ischemic striatum, suggesting that the angiogenic environment is not essential for the neuron-blood vessel interaction. To observe migrating NPCs and blood vessels simultaneously in damaged brain tissue, we performed live imaging of cultured brain slices after ischemic injury. In this system, we virally labeled SVZ-derived NPCs in Flk1-EGFP knock-in mice in which the blood vessels are labeled with EGFP. Our results provide direct evidence that SVZ-derived NPCs migrate along blood vessels from the SVZ toward the ischemic region of the striatum. The leading process of the migrating NPCs was closely associated with blood vessels, suggesting that this interaction provides directional guidance to the NPCs. These findings suggest that blood vessels play an important role as a scaffold for NPCs migration toward the damaged brain region. © AlphaMed Press.

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Various facets of vertebrate cilia: motility, signaling, and role in adult neurogenesis

Huang, Shihhui, Hirota, Yuki, Sawamoto, Kazunobu

PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES 85 ( 8 ) 324 - 336 2009.10

Research paper (scientific journal), ISSN 0386-2208

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Efhc1 deficiency causes spontaneous myoclonus and increased seizure susceptibility

Suzuki Toshimitsu, Miyamoto Hiroyuki, Nakahari Takashi, Inoue Ikuyo, Suemoto Takahiro, Jiang Bin, Hirota Yuki, Itohara Shigeyoshi, Saido Takaomi C., Tsumoto Tadaharu, Sawamoto Kazunobu, Hensch Takao K., Delgado-Escueta Antonio V., Yamakawa Kazuhiro

Human Molecular Genetics 18 ( 6 ) 1099 - 1109 2009

ISSN 0964-6906

Mutations in EFHC1 gene have been previously reported in patients with epilepsies, including those with juvenile myoclonic epilepsy. Myoclonin1, also known as mRib72-1, is encoded by the mouse Efhc1 gene. Myoclonin1 is dominantly expressed in embryonic choroid plexus, post-natal ependymal cilia, tracheal cilia and sperm flagella. In this study, we generated viable Efhc1-deficient mice. Most of the mice were normal in outward appearance, and both sexes were found to be fertile. However, the ventricles of the brains were significantly enlarged in the null mutants, but not in the heterozygotes. Although the ciliary structure was found intact, the ciliary beating frequency was significantly reduced in null mutants. In adult stages, both the heterozygous and null mutants developed frequent spontaneous myoclonus. Furthermore, the threshold of seizures induced by pentylenetetrazol was significantly reduced in both heterozygous and null mutants. These observations seem to further suggest that decrease or loss of function of myoclonin1 may be the molecular basis for epilepsies caused by EFHC1 mutations. © The Author 2009. Published by Oxford University Press. All rights reserved.

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Neurogenesis in the adult subventricular zone

Hirota Yuki, Sawamoto Kazunobu

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 7 ) 863 - 869 2008.06

ISSN 0039-9450

Cyclin-dependent kinase 5 is required for control of neuroblast migration in the postnatal subventricular zone

Hirota Yuki, Ohshima Toshio, Kaneko Naoko, Ikeda Makiko, Iwasato Takuji, Kulkarni Ashok B., Mikoshiba Katsuhiko, Okano Hideyuki, Sawamoto Kazunobu

Journal of Neuroscience 27 ( 47 ) 12829 - 12838 2007.11

ISSN 0270-6474

At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone. Copyright © 2007 Society for Neuroscience.

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Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum

Yamashita Toru, Ninomiya Mikiko, Acosta Pilar Hernández, García-Verdugo Jose Manuel, Sunabori Takehiko, Sakaguchi Masanori, Adachi Kazuhide, Kojima Takuro, Hirota Yuki, Kawase Takeshi, Araki Nobuo, Abe Koji, Okano Hideyuki, Sawamoto Kazunobu

Journal of Neuroscience 26 ( 24 ) 6627 - 6636 2006

ISSN 0270-6474

Recent studies have revealed that the adult mammalian brain has the capacity to regenerate some neurons after various insults. However, the precise mechanism of insult-induced neurogenesis has not been demonstrated. In the normal brain, GFAP-expressing cells in the subventricular zone (SVZ) of the lateral ventricles include a neurogenic cell population that gives rise to olfactory bulb neurons only. Herein, we report evidence that, after a stroke, these cells are capable of producing new neurons outside the olfactory bulbs. SVZ GFAP-expressing cells labeled by a cell-type-specific viral infection method were found to generate neuroblasts that migrated toward the injured striatum after middle cerebral artery occlusion. These neuroblasts in the striatum formed elongated chain-like cell aggregates similar to those in the normal SVZ, and these chains were observed to be closely associated with thin astrocytic processes and blood vessels. Finally, long-term tracing of the green fluorescent-labeled cells with a Cre-loxP system revealed that the SVZ-derived neuroblasts differentiated into mature neurons in the striatum, in which they expressed neuronal-specific nuclear protein and formed synapses with neighboring striatal cells. These results highlight the role of the SVZ in neuronal regeneration after a stroke and its potential as an important therapeutic target for various neurological disorders. Copyright © 2006 Society for Neuroscience.

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The transmembrane protein, Tincar, is involved in the development of the compound eye in Drosophila melanogaster

Hirota Yuki, Sawamoto Kazunobu, Takahashi Kuniaki, Ueda Ryu, Okano Hideyuki

Development Genes and Evolution 215 ( 2 ) 90 - 96 2005.02

ISSN 0949-944X

We previously cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. Here, we have studied the expression pattern and functions of tinc during developmental processes. Tinc mRNA is expressed in the central and peripheral nervous systems, and midgut during embryogenesis. In the third-instar larval eye disc, tinc mRNA is strongly expressed in all the differentiating ommatidial cells within and in the vicinity of the morphogenetic furrow. Loss-of-function analysis using the RNA-interference method revealed severe defects of eye morphogenesis during the late developmental stages. Our results suggested that tinc may have an indispensable role in the normal differentiation of ommatidial cells. © Springer-Verlag 2005.

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tincar encodes a novel transmembrane protein expressed in the Tinman-expressing cardioblasts of Drosophila

Hirota Yuki, Sawamoto Kazunobu, Okano Hideyuki

Mechanisms of Development 119 ( SUPPL. 1 ) 2002.12

ISSN 0925-4773

We cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. The tinc mRNA was expressed specifically in four of the six pairs of cardioblasts in each segment, in a pattern identical to that of tinman (tin), a homeobox gene required for the specification of the dorsal vessel. In the non-Tin-expressing pairs of cardioblasts, tinc transcription seemed to be repressed by Seven-up. © 2003 Elsevier Science Ireland Ltd. All rights reserved.

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DN-cadherin is required for spatial arrangement of nerve terminals and ultrastructural organization of synapses

Iwai, Y, Hirota, Y, Ozaki, K, Okano, H, Takeichi, M, Uemura, T

MOLECULAR AND CELLULAR NEUROSCIENCE 19 ( 3 ) 375 - 388 2002.03

Research paper (scientific journal), ISSN 1044-7431

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Musashi and Seven in absentia downregulate Tramtrack through distinct mechanisms in Drosophila eye development

Hirota Yuki, Okabe Masataka, Imai Takao, Kurusu Mitsuhiko, Yamamoto Atsuyo, Miyao Sachiyo, Nakamura Makoto, Sawamoto Kazunobu, Okano Hideyuki

Mechanisms of Development 87 ( 1-2 ) 93 - 101 1999.09

ISSN 0925-4773

We have examined the roles played by the Drosophila neural RNA-binding protein Musashi (MSI) in eye development. MSI expression was observed in the nuclei of all photoreceptor cells (R1-R8). Although a msi loss-of-function mutation resulted in only weak abnormalities in photoreceptor differentiation, we found that the msi eye phenotype was significantly enhanced in a seven in absentia (sina) background. sina is known to be involved in the degradation of the Tramtrack (TTK) protein, leading to the specification of the R7 fate. We demonstrated that MSI also functions to regulate TTK expression. The sina msi mutants showed significantly high ectopic expression of TTK69 and failure in the determination of the R1, R6, and R7 fates. Other photoreceptor cells also failed to differentiate with abnormalities occurring late in the differentiation process. These results suggest that MSI and SINA function redundantly to downregulate TTK in developing photoreceptor cells. Copyright (C) 1999 Elsevier Science Ireland Ltd.

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The Drosophila Ral GTPase regulates developmental cell shape changes through the Jun NH₂-terminal kinase pathway

Sawamoto Kazunobu, Winge Per, Koyama Shinya, Hirota Yuki, Yamada Chiharu, Miyao Sachiyo, Yoshikawa Shingo, Jin Ming Hao, Kikuchi Akira, Okano Hideyuki

Journal of Cell Biology 146 ( 2 ) 361 - 372 1999.07

ISSN 0021-9525

The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRal(G20V) and DRal(S25N), were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRal(G20V) and DRal(S25N) act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRal(G20V) and DRal(S25N) mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH₂-terminal kinase kinase (JNKK) and Jun NH₂-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.

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Ectopic expression of constitutively activated Ral GTPase inhibits cell shape changes during Drosophila eye development

Sawamoto Kazunobu, Yamada Chiharu, Kishida Shosei, Hirota Yuki, Taguchi Akiko, Kikuchi Akira, Okano Hideyuki

Oncogene 18 ( 11 ) 1967 - 1974 1999.03

ISSN 0950-9232

The small GTP-binding protein Ral is activated by RalGDS, one of the effector molecules for Ras. Active Ral binds to a GTPase activating protein for CDC42 and Rac. Although previous studies suggest a role for Ral in the regulation of CDC42 and Rac, which are involved in arranging the cytoskeleton, its in in vivo function is largely unknown. To examine the effect of overexpressing Ral on development, transgenic Drosophila were generated that overexpress wild-type or mutated Ral during eye development. While wild-type Ral caused no developmental defects, expression of a constitutively activated protein resulted in a rough eye phenotype. Activated Rat did not affect cell fate determination in the larval eye discs but caused severe disruption of the ommatidial organization later in pupal development. Phalloidin staining showed that activated Ral perturbed the cytoskeletal structure and cell shape changes during pupal development. This phenotype is similar to that caused by RhoA overexpression. In addition, the phenotype was synergistically enhanced by the coexpression of RhoA. These results suggest that Ral functions to control the cytoskeletal structure required for cell shape changes during Drosophila development.

Argos induces programmed cell death in the developing Drosophila eye by inhibition of the Ras pathway (vol 5, pg 262, 1998)

Sawamoto, K, Taguchi, A, Hirota, Y, Yamada, C, Jin, MH, Okano, H

CELL DEATH AND DIFFERENTIATION 5 ( 6 ) 548 - 548 1998.06

Research paper (scientific journal), ISSN 1350-9047

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