検索詳細｜MF大学 (2024)

Pcgf1 gene disruption reveals primary involvement of epigenetic mechanism in neuronal subtype specification in the enteric nervous system.

Bayu Pratama Putra, Keisuke Ito, Carla Cirillo, Mukhamad Sunardi, Haruhiko Koseki, Toshihiro Uesaka, Hideki Enomoto

The enteric nervous system (ENS) regulates gut functions independently from the central nervous system (CNS) by its highly autonomic neural circuit that integrates diverse neuronal subtypes. Although several transcription factors are shown to be necessary for the generation of some enteric neuron subtypes, the mechanisms underlying neuronal subtype specification in the ENS remain elusive. In this study, we examined the biological function of Polycomb group RING finger protein 1 (PCGF1), one of the epigenetic modifiers, in the development and differentiation of the ENS by disrupting the Pcgf1 gene selectively in the autonomic-lineage cells. Although ENS precursor migration and enteric neurogenesis were largely unaffected, neuronal differentiation was impaired in the Pcgf1-deficient mice, with the numbers of neurons expressing somatostatin (Sst+ ) decreased in multiple gut regions. Notably, the decrease in Sst+ neurons was associated with the corresponding increase in calbindin+ neurons in the proximal colon. These findings suggest that neuronal subtype conversion may occur in the absence of PCGF1 and that epigenetic mechanism is primarily involved in specification of some enteric neuron subtypes. This article is protected by copyright. All rights reserved.

Jul. 2023, Development, growth & differentiation, English, Domestic magazine

Scientific journal

Link to Kobe Univ. Repository (Kernel)

A single RET mutation in Hirschsprung disease induces intestinal aganglionosis via a dominant-negative mechanism

Mukhamad Sunardi, Keisuke Ito, Yuya Sato, Toshihiro Uesaka, Mitsuhiro Iwasaki, Hideki Enomoto

Elsevier BV, Dec. 2022, Cellular and Molecular Gastroenterology and Hepatology

Scientific journal

Link to Kobe Univ. Repository (Kernel)

Uts2b is a microbiota-regulated gene expressed in vagal afferent neurons connected to enteroendocrine cells producing cholecystokinin.

Yuta Yoshioka, Yoshihisa Tachibana, Toshihiro Uesaka, Hiroyuki Hioki, Yuya Sato, Takumi f*ckumoto, Hideki Enomoto

Enteroendocrine cells (EECs) are the primary sensory cells that sense the gut luminal environment and secret hormones to regulate organ function. Recent studies revealed that vagal afferent neurons are connected to EECs and relay sensory information from EECs to the brain stem. To date, however, the identity of vagal afferent neurons connected to a given EEC subtype and the mode of their gene responses to its intestinal hormone have remained unknown. Hypothesizing that EEC-associated vagal afferent neurons change their gene expression in response to the microbiota-related extracellular stimuli, we conducted comparative gene expression analyses of the nodose-petrosal ganglion complex (NPG) using specific pathogen-free (SPF) and germ-free (GF) mice. We report here that the Uts2b gene, which encodes a functionally unknown neuropeptide, urotensin 2B (UTS2B), is expressed in a microbiota-dependent manner in NPG neurons. In cultured NPG neurons, expression of Uts2b was induced by AR420626, the selective agonist for FFAR3. Moreover, distinct gastrointestinal hormones exerted differential effects on Uts2b expression in NPG neurons, where cholecystokinin (CCK) significantly increased its expression. The majority of Uts2b-expressing NPG neurons expressed CCK-A, the receptor for CCK, which comprised approximately 25% of all CCK-A-expressing NPG neurons. Selective fluorescent labeling of Uts2b-expressing NPG neurons revealed a direct contact of their nerve fibers to CCK-expressing EECs. This study identifies the Uts2b as a microbiota-regulated gene, demonstrates that Uts2b-expressing vagal afferent neurons transduce sensory information from CCK-expressing EECs to the brain, and suggests potential involvement of UTS2B in a modality of CCK actions.

Jun. 2022, Biochemical and biophysical research communications, 608, 66 - 72, English, International magazine

Scientific journal

Link to Kobe Univ. Repository (Kernel)

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

Mathys Grapotte, Manu Saraswat, Chloé Bessière, Christophe Menichelli, Jordan A. Ramilowski, Jessica Severin, Yoshihide Hayashizaki, Masayoshi Itoh, Michihira Tagami, Mitsuyoshi Murata, Miki Kojima-Ishiyama, Shohei Noma, Shuhei Noguchi, Takeya Kasukawa, Akira Hasegawa, Harukazu Suzuki, Hiromi Nishiyori-Sueki, Martin C. Frith, Imad Abugessaisa, Stuart Aitken, Bronwen L. Aken, Intikhab Alam, Tanvir Alam, Rami Alasiri, Ahmad M. N. Alhendi, Hamid Alinejad-Rokny, Mariano J. Alvarez, Robin Andersson, Takahiro Arakawa, Marito Araki, Taly Arbel, John Archer, Alan L. Archibald, Erik Arner, Peter Arner, Kiyoshi Asai, Haitham Ashoor, Gaby Astrom, Magda Babina, J. Kenneth Baillie, Vladimir B. Bajic, Archana Bajpai, Sarah Baker, Richard M. Baldarelli, Adam Balic, Mukesh Bansal, Arsen O. Batagov, Serafim Batzoglou, Anthony G. Beckhouse, Antonio P. Beltrami, Carlo A. Beltrami, Nicolas Bertin, Sharmodeep Bhattacharya, Peter J. Bickel, Judith A. Blake, Mathieu Blanchette, Beatrice Bodega, Alessandro Bonetti, Hidemasa Bono, Jette Bornholdt, Michael Bttcher, Salim Bougouffa, Mette Boyd, Jeremie Breda, Frank Brombacher, James B. Brown, Carol J. Bult, A. Maxwell Burroughs, Dave W. Burt, Annika Busch, Giulia Caglio, Andrea Califano, Christopher J. Cameron, Carlo V. Cannistraci, Alessandra Carbone, Ailsa J. Carlisle, Piero Carninci, Kim W. Carter, Daniela Cesselli, Jen-Chien Chang, Julie C. Chen, Yun Chen, Marco Chierici, John Christodoulou, Yari Ciani, Emily L. Clark, Mehmet Coskun, Maria Dalby, Emiliano Dalla, Carsten O. Daub, Carrie A. Davis, Michiel J. L. de Hoon, Derek de Rie, Elena Denisenko, Bart Deplancke, Michael Detmar, Ruslan Deviatiiarov, Diego Di Bernardo, Alexander D. Diehl, Lothar C. Dieterich, Emmanuel Dimont, Sarah Djebali, Taeko Dohi, Jose Dostie, Finn Drablos, Albert S. B. Edge, Matthias Edinger, Anna Ehrlund, Karl Ekwall, Arne Elofsson, Mitsuhiro Endoh, Hideki Enomoto, Saaya Enomoto, Mohammad fa*ghihi, Michela fa*giolini, Mary C. Farach-Carson, Geoffrey J. Faulkner, Alexander Favorov, Ana Miguel Fernandes, Carmelo Ferrai, Alistair R. R. Forrest, Lesley M. Forrester, Mattias Forsberg, Alexandre Fort, Margherita Francescatto, Tom C. Freeman, Martin Frith, Shinji f*ckuda, Manabu Funayama, Cesare Furlanello, Masaaki Furuno, Chikara Furusawa, Hui Gao, Iveta Gazova, Claudia Gebhard, Florian Geier, Teunis B. H. Geijtenbeek, Samik Ghosh, Yanal Ghosheh, Thomas R. Gingeras, Takashi Gojobori, Tatyana Goldberg, Daniel Goldowitz, Julian Gough, Dario Greco, Andreas J. Gruber, Sven Guhl, Roderic Guigo, Reto Guler, Oleg Gusev, Stefano Gustincich, Thomas J. Ha, Vanja Haberle, Paul Hale, Bjrn M. Hallstrom, Michiaki Hamada, Lusy Handoko, Mitsuko Hara, Matthias Harbers, Jennifer Harrow, Jayson Harshbarger, Takeshi Hase, Akira Hasegawa, Kosuke Hashimoto, Taku Hatano, Nobutaka Hattori, Ryuhei Hayashi, Yoshihide Hayashizaki, Meenhard Herlyn, Kristina Hettne, Peter Heutink, Winston Hide, Kelly J. Hitchens, Shannon Ho Sui, Peter A. C. ’t Hoen, Chung Chau Hon, Fumi Hori, Masafumi Horie, Katsuhisa Horimoto, Paul Horton, Rui Hou, Edward Huang, Yi Huang, Richard Hugues, David Hume, Hans Ienasescu, Kei Iida, Tomokatsu Ikawa, Toshimichi Ikemura, Kazuho Ikeo, Norihiko Inoue, Yuri Ishizu, Yosuke Ito, Masayoshi Itoh, Anna V. Ivshina, Boris R. Jankovic, Piroon Jenjaroenpun, Rory Johnson, Mette Jorgensen, Hadi Jorjani, Anagha Joshi, Giuseppe Jurman, Bogumil Kaczkowski, Chieko Kai, Kaoru Kaida, Kazuhiro Kajiyama, Rajaram Kaliyaperumal, Eli Kaminuma, Takashi Kanaya, Hiroshi Kaneda, Philip Kapranov, Artem S. Kasianov, Takeya Kasukawa, Toshiaki Katayama, Sachi Kato, Shuji Kawaguchi, Jun Kawai, Hideya Kawaji, Hiroshi Kawamoto, Yuki I. Kawamura, Satoshi Kawasaki, Tsugumi Kawashima, Judith S. Kempfle, Tony J. Kenna, Juha Kere, Levon Khachigian, Hisanori Kiryu, Mami Kishima, Hiroyuki Kitajima, Toshio Kitamura, Hiroaki Kitano, Enio Klaric, Kjetil Klepper, S. Peter Klinken, Edda Kloppmann, Alan J. Knox, Yuichi Kodama, Yasushi Kogo, Miki Kojima, Soichi Kojima, Norio Komatsu, Hiromitsu Komiyama, Tsukasa Kono, Haruhiko Koseki, Shigeo Koyasu, Anton Kratz, Alexander Kukalev, Ivan Kulakovskiy, Anshul Kundaje, Hiroshi Kunikata, Richard Kuo, Tony Kuo, Shigehiro Kuraku, Vladimir A. Kuznetsov, Tae Jun Kwon, Matt Larouche, Timo Lassmann, Andy Law, Kim-Anh Le-Cao, Charles-Henri Lecellier, Weonju Lee, Boris Lenhard, Andreas Lennartsson, Kang Li, Ruohan Li, Berit Lilje, Leonard Lipovich, Marina Lizio, Gonzalo Lopez, Shigeyuki Magi, Gloria K. Mak, Vsevolod Makeev, Riichiro Manabe, Michiko Mandai, Jessica Mar, Kazuichi Maruyama, Taeko Maruyama, Elizabeth Mason, Anthony Mathelier, Hideo Matsuda, Yulia A. Medvedeva, Terrence F. Meehan, Niklas Mejhert, Alison Meynert, Norihisa Mikami, Akiko Minoda, Hisashi Miura, Yohei Miyagi, Atsushi Miyawaki, Yosuke Mizuno, Hiromasa Morikawa, Mitsuru Morimoto, Masaki Morioka, Soji Morish*ta, Kazuyo Moro, Efthymios Motakis, Hozumi Motohashi, Abdul Kadir Mukarram, Christine L. Mummery, Christopher J. Mungall, Yasuhiro Murakawa, Masami Muramatsu, Mitsuyoshi Murata, Kazunori Nagasaka, Takahide Nagase, Yutaka Nakachi, Fumio Nakahara, Kenta Nakai, Kumi Nakamura, Yasukazu Nakamura, Yukio Nakamura, Toru Nakazawa, Guy P. Nason, Chirag Nepal, Quan Hoang Nguyen, Lars K. Nielsen, Kohji Nishida, Koji M. Nishiguchi, Hiromi Nishiyori, Kazuhiro Nitta, Shuhei Noguchi, Shohei Noma, Cedric Notredame, Soichi Ogishima, Naganari Ohkura, Hiroshi Ohno, Mitsuhiro Ohshima, Takashi Ohtsu, Yukinori Okada, Mariko Okada-Hatakeyama, Yasushi Okazaki, Per Oksvold, Valerio Orlando, Ghim Sion Ow, Mumin Ozturk, Mikhail Pachkov, Triantafyllos Paparountas, Suraj P. Parihar, Sung-Joon Park, Giovanni Pascarella, Robert Passier, Helena Persson, Ingrid H. Philippens, Silvano Piazza, Charles Plessy, Ana Pombo, Fredrik Ponten, Stéphane Poulain, Thomas M. Poulsen, Swati Pradhan, Carolina Prezioso, Clare Pridans, Xiang-Yang Qin, John Quackenbush, Owen Rackham, Jordan Ramilowski, Timothy Ravasi, Michael Rehli, Sarah Rennie, Tiago Rito, Patrizia Rizzu, Christelle Robert, Marco Roos, Burkhard Rost, Filip Roudnicky, Riti Roy, Morten B. Rye, Oxana Sachenkova, Pal Saetrom, Hyonmi Sai, Shinji Saiki, Mitsue Saito, Akira Saito, Shimon Sakaguchi, Mizuho Sakai, Saori Sakaue, Asako Sakaue-Sawano, Albin Sandelin, Hiromi Sano, Yuzuru Sasamoto, Hiroki Sato, Alka Saxena, Hideyuki Saya, Andrea Schafferhans, Sebastian Schmeier, Christian Schmidl, Daniel Schmocker, Claudio Schneider, Marcus Schueler, Erik A. Schultes, Gundula Schulze-Tanzil, Colin A. Semple, Shigeto Seno, Wooseok Seo, Jun Sese, Jessica Severin, Guojun Sheng, Jiantao Shi, Yishai Shimoni, Jay W. Shin, Javier SimonSanchez, Asa Sivertsson, Evelina Sjostedt, Cilla Soderhall, Georges St Laurent, Marcus H. Stoiber, Daisuke Sugiyama, Kim M. Summers, Ana Maria Suzuki, Harukazu Suzuki, Kenji Suzuki, Mikiko Suzuki, Naoko Suzuki, Takahiro Suzuki, Douglas J. Swanson, Rolf K. Swoboda, Michihira Tagami, Ayumi Taguchi, Hazuki Takahashi, Masayo Takahashi, Kazuya Takamochi, Satoru Takeda, Yoichi Takenaka, Kin Tung Tam, Hiroshi Tanaka, Rica Tanaka, Yuji Tanaka, Dave Tang, Ichiro Taniuchi, Andrea Tanzer, Hiroshi Tarui, Martin S. Taylor, Aika Terada, Yasuhisa Terao, Alison C. Testa, Mark Thomas, Supat Thongjuea, Kentaro Tomii, Elena Torlai Triglia, Hiroo Toyoda, H. Gwen Tsang, Motokazu Tsujikawa, Mathias Uhlén, Eivind Valen, Marc van de Wetering, Erik van Nimwegen, Dmitry Velmeshev, Roberto Verardo, Morana Vitezic, Kristoffer Vitting-Seerup, Kalle von Feilitzen, Christian R. Voolstra, Ilya E. Vorontsov, Claes Wahlestedt, Wyeth W. Wasserman, Kazuhide Watanabe, Shoko Watanabe, Christine A. Wells, Louise N. Winteringham, Ernst Wolvetang, Haruka Yabukami, Ken Yagi, Takuji Yamada, Yoko Yamaguchi, Masayuki Yamamoto, Yasutomo Yamamoto, Yumiko Yamamoto, Yasunari Yamanaka, Kojiro Yano, Kayoko Yasuzawa, Yukiko Yatsuka, Masahiro Yo, Shunji Yokokura, Misako Yoneda, Emiko Yoshida, Yuki Yoshida, Masahito Yoshihara, Rachel Young, Robert S. Young, Nancy Y. Yu, Noriko Yumoto, Susan E. Zabierowski, Peter G. Zhang, Silvia Zucchelli, Martin Zwahlen, Clément Chatelain, Piero Carninci, Michiel J. L. de Hoon, Wyeth W. Wasserman, Laurent Bréhélin, Charles-Henri Lecellier

Abstract

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

Springer Science and Business Media LLC, Dec. 2021, Nature Communications, 12(1) (1)

[Refereed]

Scientific journal

Enhanced enteric neurogenesis by Schwann cell precursors in mouse models of Hirschsprung disease.

Toshihiro Uesaka, Mitsumasa Okamoto, Mayumi Nagashimada, Yoshihiro Tsuda, Miho Kihara, Hiroshi Kiyonari, Hideki Enomoto

Hirschsprung disease (HSCR) is characterized by congenital absence of enteric neurons in distal portions of the gut. Although recent studies identified Schwann cell precursors (SCPs) as a novel cellular source of enteric neurons, it is unknown how SCPs contribute to the disease phenotype of HSCR. Using Schwann cell-specific genetic labeling, we investigated SCP-derived neurogenesis in two mouse models of HSCR; Sox10 haploinsufficient mice exhibiting distal colonic aganglionosis and Ednrb knockout mice showing small intestinal aganglionosis. We also examined Ret dependency in SCP-derived neurogenesis using mice displaying intestinal aganglionosis in which Ret expression was conditionally removed in the Schwann cell lineage. SCP-derived neurons were abundant in the transition zone lying between the ganglionated and aganglionic segments, although SCP-derived neurogenesis was scarce in the aganglionic region. In the transition zone, SCPs mainly gave rise to nitrergic neurons that are rarely observed in the SCP-derived neurons under the normal condition. Enhanced SCP-derived neurogenesis was also detected in the transition zone of mice lacking RET expression in the Schwann cell lineage. Increased SCP-derived neurogenesis in the transition zone suggests that reduction in the vagal neural crest-derived enteric neurons promotes SCP-derived neurogenesis. SCPs may adopt a neuronal subtype by responding to changes in the gut environment. Robust SCP-derived neurogenesis can occur in a Ret-independent manner, which suggests that SCPs are a cellular source to compensate for missing enteric neurons in HSCR.

Nov. 2021, Glia, 69(11) (11), 2575 - 2590, English, International magazine

Scientific journal

Live visualization of a functional RET‐EGFP chimeric receptor in hom*ozygous knock‐in mice

Mukhamad Sunardi, Keisuke Ito, Hideki Enomoto

Wiley, Aug. 2021, Development, Growth & Differentiation, 63(6) (6), 285 - 294

Scientific journal

Increased RET Activity Coupled with a Reduction in the RET Gene Dosage Causes Intestinal Aganglionosis in Mice

Mitsumasa Okamoto, Toshihiro Uesaka, Keisuke Ito, Hideki Enomoto

Society for Neuroscience, May 2021, eneuro, 8(3) (3), ENEURO.0534 - 20.2021

Scientific journal

Link to Kobe Univ. Repository (Kernel)

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene.

Taichi Nakatani, Mitsuhiro Iwasaki, Atsuhiro Yamamichi, Yuta Yoshioka, Toshihiro Uesaka, Yuko Bitoh, Kosaku Maeda, Takumi f*ckumoto, Tatsuya Takemoto, Hideki Enomoto

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (RetM919T , RetN396K and RetY792F ) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. RetM919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in RetM919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.

May 2020, Development, growth & differentiation, 62(4) (4), 214 - 222, English, Domestic magazine

[Refereed]

Scientific journal

Link to Kobe Univ. Repository (Kernel)

Generation of conditional ALK F1174L mutant mouse models for the study of neuroblastoma pathogenesis.

Sachie Ono, Takeshi Saito, Keita Terui, Hideo Yoshida, Hideki Enomoto

Neuroblastoma, an embryonal tumor arising from the sympathetic ganglia and adrenal medulla, is among the most intractable pediatric cancers. Although a variety of genetic changes have been identified in neuroblastoma, how they contribute to its pathogenesis remains largely unclear. Recent studies have identified alterations of the anaplastic lymphoma kinase (ALK) gene in neuroblastoma; ALK F1174L (a phenylalanine-to-leucine substitution at codon 1174) represents one of the most frequent of these somatic mutations, and is associated with amplification of the MYCN gene, the most reliable marker for the poor survival. We engineered the mouse Alk locus so that ALK F1174L is expressed by its endogenous promoter and can be induced in a spatiotemporally controlled fashion using Cre-loxP system. Although expression of ALK F1174L resulted in enhanced proliferation of sympathetic ganglion progenitors and increased the size of the sympathetic ganglia, it was insufficient to cause neuroblastoma. However, lethal neuroblastoma frequently developed in mice co-expressing ALK F1174L and MYCN, even in a genetic background where MYCN alone does not cause overt tumors. These data reveal that physiological expression of ALK F1174L significantly potentiates the oncogenic ability of MYCN in vivo. Our conditional mutant mice provide a valuable platform for investigating the pathogenesis of neuroblastoma.

Oct. 2019, Genesis (New York, N.Y. : 2000), 57(10) (10), e23323, English, International magazine

[Refereed]

Scientific journal

Mice conditionally expressing RET(C618F) mutation display C cell hyperplasia and hyperganglionosis of the enteric nervous system.

Mitsumasa Okamoto, Yuta Yoshioka, Kosaku Maeda, Yuko Bito, Takumi f*ckumoto, Toshihiro Uesaka, Hideki Enomoto

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.

May 2019, Genesis (New York, N.Y. : 2000), 57(5) (5), e23292, English, International magazine

[Refereed]

Link to Kobe Univ. Repository (Kernel)

Technologies for Live Imaging of Enteric Neural Crest-Derived Cells.

Hao MM, Bergner AJ, Newgreen DF, Enomoto H, Young HM

2019, Methods in molecular biology (Clifton, N.J.), 1976, 97 - 105

[Refereed]

Dual origin of enteric neurons in vagal Schwann cell precursors and the sympathetic neural crest

Isabel Espinosa-Medina, Ben Jevans, Franck Boismoreau, Zoubida Chettouh, Hideki Enomoto, Thomas Mueller, Carmen Birchmeier, Alan J. Burns, Jean-Francois Brunet

Most of the enteric nervous system derives from the "vagal" neural crest, lying at the level of somites 1-7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the "vagal crest" subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations. Crest cells adjacent to somites 1 and 2 produce Schwann cell precursors that colonize the vagus nerve, which in turn guides them into the esophagus and stomach. Crest cells adjacent to somites 3-7 belong to the crest streams contributing to sympathetic chains: they migrate ventrally, seed the sympathetic chains, and colonize the entire digestive tract thence. Accordingly, enteric ganglia, like sympathetic ones, are atrophic when deprived of signaling through the tyrosine kinase receptor ErbB3, while half of the esophageal ganglia require, like parasympathetic ones, the nerve-associated form of the ErbB3 ligand, Neuregulin-1. These dependencies might bear relevance to Hirschsprung disease, with which alleles of Neuregulin-1 are associated.

NATL ACAD SCIENCES, Nov. 2017, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 114(45) (45), 11980 - 11985, English

[Refereed]

Scientific journal

Catenins Steer Cell Migration via Stabilization of Front-Rear Polarity

Vassil Vassilev, Anna Platek, Sylvain Hiver, Hideki Enomoto, Masatoshi Takeichi

Cell migration plays a pivotal role in morphogenetic and pathogenetic processes. To achieve directional migration, cells must establish a front-to-rear axis of polarity. Here we show that components of the cadherin-catenin complex function to stabilize this front-rear polarity. Neural crest and glioblastoma cells undergo directional migration in vivo or in vitro. During this process, alpha E-catenin accumulated at lamellipodial membranes and then moved toward the rear with the support of a tyrosine-phosphorylated beta-catenin. This relocating alpha E-catenin bound to p115RhoGEF, leading to gathering of active RhoA in front of the nucleus where myosin-IIB arcs assemble. When catenins or p115RhoGEF were removed, cells lost the polarized myosin-IIB assembly, as well as the capability for directional movement. These results suggest that, apart from its well-known function in cell adhesion, the beta-catenin/alpha E-catenin complex regulates directional cell migration by restricting active RhoA to perinuclear regions and controlling myosin-IIB dynamics at these sites.

CELL PRESS, Nov. 2017, DEVELOPMENTAL CELL, 43(4) (4), 463 - +, English

[Refereed]

Scientific journal

FANTOM5 CAGE profiles of human and mouse samples.

Shuhei Noguchi, Takahiro Arakawa, Shiro f*ckuda, Masaaki Furuno, Akira Hasegawa, Fumi Hori, Sachi Ishikawa-Kato, Kaoru Kaida, Ai Kaiho, Mutsumi Kanamori-Katayama, Tsugumi Kawashima, Miki Kojima, Atsutaka Kubosaki, Ri-Ichiroh Manabe, Mitsuyoshi Murata, Sayaka Nagao-Sato, Kenichi Nakazato, Noriko Ninomiya, Hiromi Nishiyori-Sueki, Shohei Noma, Eri Saijyo, Akiko Saka, Mizuho Sakai, Christophe Simon, Naoko Suzuki, Michihira Tagami, Shoko Watanabe, Shigehiro Yoshida, Peter Arner, Richard A Axton, Magda Babina, J Kenneth Baillie, Timothy C Barnett, Anthony G Beckhouse, Antje Blumenthal, Beatrice Bodega, Alessandro Bonetti, James Briggs, Frank Brombacher, Ailsa J Carlisle, Hans C Clevers, Carrie A Davis, Michael Detmar, Taeko Dohi, Albert S B Edge, Matthias Edinger, Anna Ehrlund, Karl Ekwall, Mitsuhiro Endoh, Hideki Enomoto, Afsaneh Eslami, Michela fa*giolini, Lynsey Fairbairn, Mary C Farach-Carson, Geoffrey J Faulkner, Carmelo Ferrai, Malcolm E Fisher, Lesley M Forrester, Rie Fujita, Jun-Ichi Furusawa, Teunis B Geijtenbeek, Thomas Gingeras, Daniel Goldowitz, Sven Guhl, Reto Guler, Stefano Gustincich, Thomas J Ha, Masahide Hamaguchi, Mitsuko Hara, Yuki Hasegawa, Meenhard Herlyn, Peter Heutink, Kelly J Hitchens, David A Hume, Tomokatsu Ikawa, Yuri Ishizu, Chieko Kai, Hiroshi Kawamoto, Yuki I Kawamura, Judith S Kempfle, Tony J Kenna, Juha Kere, Levon M Khachigian, Toshio Kitamura, Sarah Klein, S Peter Klinken, Alan J Knox, Soichi Kojima, Haruhiko Koseki, Shigeo Koyasu, Weonju Lee, Andreas Lennartsson, Alan Mackay-Sim, Niklas Mejhert, Yosuke Mizuno, Hiromasa Morikawa, Mitsuru Morimoto, Kazuyo Moro, Kelly J Morris, Hozumi Motohashi, Christine L Mummery, Yutaka Nakachi, Fumio Nakahara, Toshiyuki Nakamura, Yukio Nakamura, Tadasuke Nozaki, Soichi Ogishima, Naganari Ohkura, Hiroshi Ohno, Mitsuhiro Ohshima, Mariko Okada-Hatakeyama, Yasushi Okazaki, Valerio Orlando, Dmitry A Ovchinnikov, Robert Passier, Margaret Patrikakis, Ana Pombo, Swati Pradhan-Bhatt, Xian-Yang Qin, Michael Rehli, Patrizia Rizzu, Sugata Roy, Antti Sajantila, Shimon Sakaguchi, Hiroki Sato, Hironori Satoh, Suzana Savvi, Alka Saxena, Christian Schmidl, Claudio Schneider, Gundula G Schulze-Tanzil, Anita Schwegmann, Guojun Sheng, Jay W Shin, Daisuke Sugiyama, Takaaki Sugiyama, Kim M Summers, Naoko Takahashi, Jun Takai, Hiroshi Tanaka, Hideki Tatsukawa, Andru Tomoiu, Hiroo Toyoda, Marc van de Wetering, Linda M van den Berg, Roberto Verardo, Dipti Vijayan, Christine A Wells, Louise N Winteringham, Ernst Wolvetang, Yoko Yamaguchi, Masayuki Yamamoto, Chiyo Yanagi-Mizuochi, Misako Yoneda, Yohei Yonekura, Peter G Zhang, Silvia Zucchelli, Imad Abugessaisa, Erik Arner, Jayson Harshbarger, Atsushi Kondo, Timo Lassmann, Marina Lizio, Serkan Sahin, Thierry Sengstag, Jessica Severin, Hisashi Shimoji, Masanori Suzuki, Harukazu Suzuki, Jun Kawai, Naoto Kondo, Masayoshi Itoh, Carsten O Daub, Takeya Kasukawa, Hideya Kawaji, Piero Carninci, Alistair R R Forrest, Yoshihide Hayashizaki

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

Aug. 2017, Scientific data, 4, 170112 - 170112, English, International magazine

[Refereed]

Scientific journal

Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor

Hidekazu Sotoyama, Yuriko Iwakura, Kanako Oda, Toshikuni Sasaoka, Nobuyuki Takei, Akiyoshi Kakita, Hideki Enomoto, Hiroyuki Nawa

Glial cell line-derived neurotrophic factor (GDNF) positively regulates the development and maintenance of in vitro dopaminergic neurons. However, the in vivo influences of GDNF signals on the brain dopamine system are controversial and not fully defined. To address this question, we analyzed dopaminergic phenotypes of the transgenic mice that overexpress GDNF under the control of the glial Gfap promoter. Compared with wild-type, the GDNF transgenic mice contained higher levels of GDNF protein and phosphorylated RET receptors in the brain. However, there were reductions in the levels of tyrosine hydroxylase (TH), dopamine, and its metabolite hom*ovanillic acid in the striatum of transgenic mice. The TH reduction appeared to occur during postnatal development. Immunohistochemistry revealed that striatal TH density was reduced in transgenic mice with no apparent signs of neurodegeneration. In agreement with these neurochemical traits, basal levels of extracellular dopamine and high K+-induced dopamine efflux were decreased in the striatum of transgenic mice. We also explored the influences of GDNF overexpression on lomomotor behavior. GDNF transgenic mice exhibited lower stereotypy and rearing in a novel environment compared with wild-type mice. These results suggest that chronic overexpression of GDNF in brain astrocytes exerts an opposing influence on nigrostriatal dopamine metabolism and neurotransmission. (C) 2017 Elsevier B.V. All rights reserved.

ELSEVIER IRELAND LTD, Jul. 2017, NEUROSCIENCE LETTERS, 654, 99 - 106, English

[Refereed]

Scientific journal

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome

Laurence D. Hurst, Avazeh T. Ghanbarian, Alistair R. R. Forrest, FANTOM consortium, Lukasz Huminiecki, Michael Rehli, J. Kenneth Baillie, Michiel J. L. de Hoon, Vanja Haberle, Timo Lassmann, Ivan V. Kulakovskiy, Marina Lizio, Masayoshi Itoh, Robin Andersson, Christopher J. Mungall, Terrence F. Meehan, Sebastian Schmeier, Nicolas Bertin, Mette Jørgensen, Emmanuel Dimont, Erik Arner, Christian Schmidl, Ulf Schaefer, Yulia A. Medvedeva, Charles Plessy, Morana Vitezic, Jessica Severin, Colin A. Semple, Yuri Ishizu, Robert S. Young, Margherita Francescatto, Intikhab Alam, Davide Albanese, Gabriel M. Altschuler, Takahiro Arakawa, John A.C. Archer, Peter Arner, Magda Babina, Sarah Baker, Piotr J. Balwierz, Anthony G. Beckhouse, Swati-Bhatt Pradhan, Judith A. Blake, Antje Blumenthal, Beatrice Bodega, Alessandro Bonetti, James Briggs, Frank Brombacher, A. Maxwell Burroughs, Andrea Califano, Carlo V. Cannistraci, Daniel Carbajo, Yun Chen, Marco Chierici, Yari Ciani, Hans C. Clevers, Emiliano Dalla, Carrie A. Davis, Michael Detmar, Alexander D. Diehl, Taeko Dohi, Finn Drabløs, Albert S.B. Edge, Matthias Edinger, Karl Ekwall, Mitsuhiro Endoh, Hideki Enomoto, Michela fa*giolini, Lynsey Fairbairn, Hai Fang, Mary C. Farach-Carson, Geoffrey J. Faulkner, Alexander V. Favorov, Malcolm E. Fisher, Martin C. Frith, Rie Fujita, Shiro f*ckuda, Cesare Furlanello, Masaaki Furuno, Jun-ichi Furusawa, Teunis B. Geijtenbeek, Andrew Gibson, Thomas Gingeras, Daniel Goldowitz, Julian Gough, Sven Guhl, Reto Guler, Stefano Gustincich, Thomas J. Ha, Masahide Hamaguchi, Mitsuko Hara, Matthias Harbers, Jayson Harshbarger, Akira Hasegawa, Yuki Hasegawa, Takehiro Hashimoto, Meenhard Herlyn, Kelly J. Hitchens, Shannan J. Ho Sui, Oliver M. Hofmann, Ilka Hoof, Fumi Hori, Lukasz Huminiecki, Kei Iida, Tomokatsu Ikawa, Boris R. Jankovic, Hui Jia, Anagha Joshi, Giuseppe Jurman, Bogumil Kaczkowski, Chieko Kai, Kaoru Kaida, Ai Kaiho, Kazuhiro Kajiyama, Mutsumi-Katayama Kanamori, Artem S. Kasianov, Takeya Kasukawa, Shintaro Katayama, Sachi Kato, Shuji Kawaguchi, Hiroshi Kawamoto, Yuki I. Kawamura, Tsugumi Kawashima, Judith S. Kempfle, Tony J. Kenna, Juha Kere, Levon M. Khachigian, Toshio Kitamura, S. Peter Klinken, Alan J. Knox, Miki Kojima, Soichi Kojima, Naoto Kondo, Haruhiko Koseki, Shigeo Koyasu, Sarah Krampitz, Atsutaka Kubosaki, Andrew T. Kwon, Jeroen F.J. Laros, Weonju Lee, Andreas Lennartsson, Kang Li, Berit Lilje, Leonard Lipovich, Alan-sim Mackay, Riichiroh Manabe, Jessica C. Mar, Benoit Marchand, Anthony Mathelier, Niklas Mejhert, Alison Meynert, Yosuke Mizuno, David A. de Lima Morais, Hiromasa Morikawa, Mitsuru Morimoto, Kazuyo Moro, Efthymios Motakis, Hozumi Motohashi, Christine L. Mummery, Mitsuyoshi Murata, Sayaka-Sato Nagao, Yutaka Nakachi, Fumio Nakahara, Toshiyuki Nakamura, Yukio Nakamura, Kenichi Nakazato, Erik van Nimwegen, Noriko Ninomiya, Hiromi Nishiyori, Shohei Noma, Tadasuke Nozaki, Soichi Ogishima, Naganari Ohkura, Hiroko Ohmiya, Hiroshi Ohno, Mitsuhiro Ohshima, Mariko-Hatakeyama Okada, Yasushi Okazaki, Valerio Orlando, Dmitry A. Ovchinnikov, Arnab Pain, Robert Passier, Margaret Patrikakis, Helena Persson, Silvano Piazza, James G.D. Prendergast, Owen J.L. Rackham, Jordan A. Ramilowski, Mamoon Rashid, Timothy Ravasi, Patrizia Rizzu, Marco Roncador, Sugata Roy, Morten B. Rye, Eri Saijyo, Antti Sajantila, Akiko Saka, Shimon Sakaguchi, Mizuho Sakai, Hiroki Sato, Hironori Satoh, Suzana Savvi, Alka Saxena, Claudio Schneider, Erik A. Schultes, Gundula G. Schulze-Tanzil, Anita Schwegmann, Thierry Sengstag, Guojun Sheng, Hisashi Shimoji, Yishai Shimoni, Jay W. Shin, Christophe Simon, Daisuke Sugiyama, Takaaki Sugiyama, Masanori Suzuki, Naoko Suzuki, Rolf K. Swoboda, Peter A.C. 't Hoen, Michihira Tagami, Naoko Takahashi, Jun Takai, Hiroshi Tanaka, Hideki Tatsukawa, Zuotian Tatum, Mark Thompson, Hiroo Toyoda, Tetsuro Toyoda, Eivind Valen, Marc van de Wetering, Linda M. van den Berg, Roberto Verardo, Dipti Vijayan, Ilya E. Vorontsov, Wyeth W. Wasserman, Shoko Watanabe, Christine A. Wells, Louise N. Winteringham, Ernst Wolvetang, Emily J. Wood, Yoko Yamaguchi, Masayuki Yamamoto, Misako Yoneda, Yohei Yonekura, Shigehiro Yoshida, Suzan E. Zabierowski, Peter G. Zhang, Xiaobei Zhao, Silvia Zucchelli, Kim M. Summers, Harukazu Suzuki, Carsten O. Daub, Jun Kawai, Peter Heutink, Winston Hide, Tom C. Freeman, Boris Lenhard, Vladimir B. Bajic, Martin S. Taylor, Vsevolod J. Makeev, Albin Sandelin, David A. Hume, Piero Carninci, Yoshihide Hayashizaki

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X’s gene content, gene expression, and evolution.

Public Library of Science, Dec. 2015, PLoS Biology, 13(12) (12), English

[Refereed]

Scientific journal

Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System

Toshihiro Uesaka, Mayumi Nagashimada, Hideki Enomoto

Elucidation of the cellular identity of neuronal precursors provides mechanistic insights into the development and pathophysiology of the nervous system. In the enteric nervous system (ENS), neurogenesis persists from midgestation to the postnatal period. Cellular mechanism underlying the long-term neurogenesis in the ENS has remained unclear. Using genetic fate mapping in mice, we show here that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate in the postnatal period and contributes to the ENS. We found SCP-derived neurogenesis in the submucosal region of the small intestine in the absence of vagal neural crest-derived ENS precursors. Under physiological conditions, SCPs comprised up to 20% of enteric neurons in the large intestine and gave rise mainly to restricted neuronal subtypes, calretinin-expressing neurons. Genetic ablation of Ret, the signaling receptor for glial cell line-derived neurotrophic factor, in SCPs caused colonic oligoganglionosis, indicating that SCP-derived neurogenesis is essential to ENS integrity. Identification of Schwann cells as a physiological neurogenic source provides novel insight into the development and disorders of neural crest-derived tissues.

SOC NEUROSCIENCE, Jul. 2015, JOURNAL OF NEUROSCIENCE, 35(27) (27), 9879 - 9888, English

[Refereed]

Scientific journal

Link to Kobe Univ. Repository (Kernel)

The neurotrophic factor receptor RET drives haematopoietic stem cell survival and function

Diogo Fonseca-Pereira, Silvia Arroz-Madeira, mariana Rodrigues-Campos, Ines A. M. Barbosa, Rita G. Domingues, Teresa Bento, Afonso R. M. Almeida, Helder Ribeiro, Alexandre J. Potocnik, Hideki Enomoto, Henrique Veiga-Fernandes

Haematopoiesis is a developmental cascade that generates all blood cell lineages in health and disease. This process relies on quiescent haematopoietic stem cells capable of differentiating, self renewing and expanding upon physiological demand(1,2). However, the mechanisms that regulate haematopoietic stem cell homeostasis and function remain largely unknown. Here we show that the neurotrophic factor receptor RET (rearranged during transfection) drives haematopoietic stem cell survival, expansion and function. We find that haematopoietic stem cells express RET and that its neurotrophic factor partners are produced in the haematopoietic stem cell environment. Ablation of Ret leads to impaired survival and reduced numbers of haematopoietic stem cells with normal differentiation potential, but loss of cell-autonomous stress response and reconstitution potential. Strikingly, RET signals provide haematopoietic stem cells with critical Bcl2 and Bcl2l1 surviving cues, downstream of p38 mitogen-activated protein (MAP) kinase and cyclic-AMP-response element binding protein (CREB) activation. Accordingly, enforced expression of RET downstream targets, Bcl2 or Bcl2l1, is sufficient to restore the activity of Ret null progenitors in vivo. Activation of RET results in improved haematopoietic stem cell survival, expansion and in vivo transplantation efficiency. Remarkably, human cord-blood progenitor expansion and transplantation is also improved by neurotrophic factors, opening the way for exploration of RET agonists in human haematopoietic stem cell transplantation. Ourwork shows that neurotrophic factors are novel components of the haematopoietic stem cell microenvironment, revealing that

NATURE PUBLISHING GROUP, Oct. 2014, NATURE, 514(7520) (7520), 98 - +, English

[Refereed]

Scientific journal

Mouse Spermatogenic Stem Cells Continually Interconvert between Equipotent Singly Isolated and Syncytial States

Kenshiro Hara, Toshinori Nakagawa, Hideki Enomoto, Mikiko Suzuki, Masayuki Yamamoto, Benjamin D. Simons, Shosei Yoshida

The identity and behavior of mouse spermatogenic stem cells have been a long-standing focus of interest. In the prevailing "A(s) model,'' stem cell function is restricted to singly isolated (As) spermatogonia. By examining single-cell dynamics of GFR alpha 1+ stem cells in vivo, we evaluate an alternative hypothesis that, through fragmentation, syncytial spermatogonia also contribute to stem cell function in homeostasis. We use live imaging and pulse labeling to quantitatively determine the fates of individual GFR alpha 1+ cells and find that, during steady-state spermatogenesis, the entire GFR alpha 1+ population comprises a single stem cell pool, in which cells continually interconvert between As and syncytial states. A minimal biophysical model, relying only on the rates of incomplete cell division and syncytial fragmentation, precisely predicts the stochastic fates of GFR alpha 1+ cells during steady state and post-insult regeneration. Thus, our results define an alternative and dynamic model for spermatogenic stem cell function in the mouse testis.

CELL PRESS, May 2014, CELL STEM CELL, 14(5) (5), 658 - 672, English

[Refereed]

Scientific journal

Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation

Hiromasa Morikawa, Naganari Ohkura, Alexis Vandenbon, Masayoshi Itoh, Sayaka Nagao-Sato, Hideya Kawaji, Timo Lassmann, Piero Carninci, Yoshihide Hayashizaki, Alistair R. R. Forrest, Daron M. Standley, Hiroshi Date, Shimon Sakaguchi

Naturally occurring regulatory T (Treg) cells, which specifically express the transcription factor forkhead box P3 (Foxp3), are engaged in the maintenance of immunological self-tolerance and homeostasis. By transcriptional start site cluster analysis, we assessed here how genome-wide patterns of DNA methylation or Foxp3 binding sites were associated with Treg-specific gene expression. We found that Treg-specific DNA hypomethylated regions were closely associated with Treg up-regulated transcriptional start site clusters, whereas Foxp3 binding regions had no significant correlation with either up- or down-regulated clusters in nonactivated Treg cells. However, in activated Treg cells, Foxp3 binding regions showed a strong correlation with down-regulated clusters. In accordance with these findings, the above two features of activation-dependent gene regulation in Treg cells tend to occur at different locations in the genome. The results collectively indicate that Treg-specific DNA hypomethylation is instrumental in gene up-regulation in steady state Treg cells, whereas Foxp3 down-regulates the expression of its target genes in activated Treg cells. Thus, the two events seem to play distinct but complementary roles in Treg-specific gene expression.

NATL ACAD SCIENCES, Apr. 2014, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 111(14) (14), 5289 - 5294, English

[Refereed]

Scientific journal

A promoter-level mammalian expression atlas.

Alistair R R Forrest, Hideya Kawaji, Michael Rehli, J Kenneth Baillie, Michiel J L de Hoon, Vanja Haberle, Timo Lassmann, Ivan V Kulakovskiy, Marina Lizio, Masayoshi Itoh, Robin Andersson, Christopher J Mungall, Terrence F Meehan, Sebastian Schmeier, Nicolas Bertin, Mette Jørgensen, Emmanuel Dimont, Erik Arner, Christian Schmidl, Ulf Schaefer, Yulia A Medvedeva, Charles Plessy, Morana Vitezic, Jessica Severin, Colin A Semple, Yuri Ishizu, Robert S Young, Margherita Francescatto, Intikhab Alam, Davide Albanese, Gabriel M Altschuler, Takahiro Arakawa, John A C Archer, Peter Arner, Magda Babina, Sarah Rennie, Piotr J Balwierz, Anthony G Beckhouse, Swati Pradhan-Bhatt, Judith A Blake, Antje Blumenthal, Beatrice Bodega, Alessandro Bonetti, James Briggs, Frank Brombacher, A Maxwell Burroughs, Andrea Califano, Carlo V Cannistraci, Daniel Carbajo, Yun Chen, Marco Chierici, Yari Ciani, Hans C Clevers, Emiliano Dalla, Carrie A Davis, Michael Detmar, Alexander D Diehl, Taeko Dohi, Finn Drabløs, Albert S B Edge, Matthias Edinger, Karl Ekwall, Mitsuhiro Endoh, Hideki Enomoto, Michela fa*giolini, Lynsey Fairbairn, Hai Fang, Mary C Farach-Carson, Geoffrey J Faulkner, Alexander V Favorov, Malcolm E Fisher, Martin C Frith, Rie Fujita, Shiro f*ckuda, Cesare Furlanello, Masaaki Furino, Jun-ichi Furusawa, Teunis B Geijtenbeek, Andrew P Gibson, Thomas Gingeras, Daniel Goldowitz, Julian Gough, Sven Guhl, Reto Guler, Stefano Gustincich, Thomas J Ha, Masahide Hamaguchi, Mitsuko Hara, Matthias Harbers, Jayson Harshbarger, Akira Hasegawa, Yuki Hasegawa, Takehiro Hashimoto, Meenhard Herlyn, Kelly J Hitchens, Shannan J Ho Sui, Oliver M Hofmann, Ilka Hoof, Furni Hori, Lukasz Huminiecki, Kei Iida, Tomokatsu Ikawa, Boris R Jankovic, Hui Jia, Anagha Joshi, Giuseppe Jurman, Bogumil Kaczkowski, Chieko Kai, Kaoru Kaida, Ai Kaiho, Kazuhiro Kajiyama, Mutsumi Kanamori-Katayama, Artem S Kasianov, Takeya Kasukawa, Shintaro Katayama, Sachi Kato, Shuji Kawaguchi, Hiroshi Kawamoto, Yuki I Kawamura, Tsugumi Kawashima, Judith S Kempfle, Tony J Kenna, Juha Kere, Levon M Khachigian, Toshio Kitamura, S Peter Klinken, Alan J Knox, Miki Kojima, Soichi Kojima, Naoto Kondo, Haruhiko Koseki, Shigeo Koyasu, Sarah Krampitz, Atsutaka Kubosaki, Andrew T Kwon, Jeroen F J Laros, Weonju Lee, Andreas Lennartsson, Kang Li, Berit Lilje, Leonard Lipovich, Alan Mackay-Sim, Ri-ichiroh Manabe, Jessica C Mar, Benoit Marchand, Anthony Mathelier, Niklas Mejhert, Alison Meynert, Yosuke Mizuno, David A de Lima Morais, Hiromasa Morikawa, Mitsuru Morimoto, Kazuyo Moro, Efthymios Motakis, Hozumi Motohashi, Christine L Mummery, Mitsuyoshi Murata, Sayaka Nagao-Sato, Yutaka Nakachi, Fumio Nakahara, Toshiyuki Nakamura, Yukio Nakamura, Kenichi Nakazato, Erik van Nimwegen, Noriko Ninomiya, Hiromi Nishiyori, Shohei Noma, Shohei Noma, Tadasuke Noazaki, Soichi Ogishima, Naganari Ohkura, Hiroko Ohimiya, Hiroshi Ohno, Mitsuhiro Ohshima, Mariko Okada-Hatakeyama, Yasushi Okazaki, Valerio Orlando, Dmitry A Ovchinnikov, Arnab Pain, Robert Passier, Margaret Patrikakis, Helena Persson, Silvano Piazza, James G D Prendergast, Owen J L Rackham, Jordan A Ramilowski, Mamoon Rashid, Timothy Ravasi, Patrizia Rizzu, Marco Roncador, Sugata Roy, Morten B Rye, Eri Saijyo, Antti Sajantila, Akiko Saka, Shimon Sakaguchi, Mizuho Sakai, Hiroki Sato, Suzana Savvi, Alka Saxena, Claudio Schneider, Erik A Schultes, Gundula G Schulze-Tanzil, Anita Schwegmann, Thierry Sengstag, Guojun Sheng, Hisashi Shimoji, Yishai Shimoni, Jay W Shin, Christophe Simon, Daisuke Sugiyama, Takaai Sugiyama, Masanori Suzuki, Naoko Suzuki, Rolf K Swoboda, Peter A C 't Hoen, Michihira Tagami, Naoko Takahashi, Jun Takai, Hiroshi Tanaka, Hideki Tatsukawa, Zuotian Tatum, Mark Thompson, Hiroo Toyodo, Tetsuro Toyoda, Elvind Valen, Marc van de Wetering, Linda M van den Berg, Roberto Verado, Dipti Vijayan, Ilya E Vorontsov, Wyeth W Wasserman, Shoko Watanabe, Christine A Wells, Louise N Winteringham, Ernst Wolvetang, Emily J Wood, Yoko Yamaguchi, Masayuki Yamamoto, Misako Yoneda, Yohei Yonekura, Shigehiro Yoshida, Susan E Zabierowski, Peter G Zhang, Xiaobei Zhao, Silvia Zucchelli, Kim M Summers, Harukazu Suzuki, Carsten O Daub, Jun Kawai, Peter Heutink, Winston Hide, Tom C Freeman, Boris Lenhard, Vladimir B Bajic, Martin S Taylor, Vsevolod J Makeev, Albin Sandelin, David A Hume, Piero Carninci, Yoshihide Hayashizaki

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

Mar. 2014, Nature, 507(7493) (7493), 462 - 70, English, International magazine

[Refereed]

Scientific journal

Colonizing while migrating: how do individual enteric neural crest cells behave?

Heather M. Young, Annette J. Bergner, Matthew J. Simpson, Sonja J. McKeown, Marlene M. Hao, Colin R. Anderson, Hideki Enomoto

Background: Directed cell migration is essential for normal development. In most of the migratory cell populations that have been analyzed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance. Very little is known about how individual cells behave to achieve concomitant directional migration and population of the migratory route. We examined the behavior of enteric neural crest-derived cells (ENCCs), which must both advance caudally to reach the anal end and populate each gut region.Results: The behavior of individual ENCCs was examined using live imaging and mice in which ENCCs express a photoconvertible protein. We show that individual ENCCs exhibit very variable directionalities and speed; as the migratory wavefront of ENCCs advances caudally, each gut region is populated primarily by some ENCCs migrating non-directionally. After populating each region, ENCCs remain migratory for at least 24 hours. Endothelin receptor type B (EDNRB) signaling is known to be essential for the normal advance of the ENCC population. We now show that perturbation of EDNRB principally affects individual ENCC speed rather than directionality. The trajectories of solitary ENCCs, which occur transiently at the wavefront, were consistent with an unbiased random walk and so cell-cell contact is essential for directional migration. ENCCs migrate in close association with neurites. We showed that although ENCCs often use neurites as substrates, ENCCs lead the way, neurites are not required for chain formation and neurite growth is more directional than the migration of ENCCs as a whole.Conclusions: Each gut region is initially populated by sub-populations of ENCCs migrating non-directionally, rather than stopping. This might provide a mechanism for ensuring a uniform density of ENCCs along the growing gut.

BIOMED CENTRAL LTD, Mar. 2014, BMC BIOLOGY, 12, 23, English

[Refereed]

Scientific journal

Tissue Interactions in Neural Crest Cell Development and Disease

Yoshiko Takahashi, Douglas Sipp, Hideki Enomoto

The neural crest is a transient population of migratory cells in the embryo that gives rise to a wide variety of different cell types, including those of the peripheral nervous system. Dysfunction of neural crest cells (NCCs) is associated with multiple diseases, such as neuroblastoma and Hirschsprung disease. Recent studies have identified NCC behaviors during their migration and differentiation, with implications for their contributions to development and disease. Here, we describe how interactions between cells of the neural crest and lineages such as the vascular system, as well as those involving environmental signals and microbial pathogens, are critically important in determining the roles played by these cells.

AMER ASSOC ADVANCEMENT SCIENCE, Aug. 2013, SCIENCE, 341(6148) (6148), 860 - 863, English

[Refereed]

Scientific journal

Proliferation and Cell Cycle Dynamics in the Developing Stellate Ganglion

David G. Gonsalvez, Kylie N. Cane, Kerry A. Landman, Hideki Enomoto, Heather M. Young, Colin R. Anderson

Cell proliferation during nervous system development is poorly understood outside the mouse neocortex. We measured cell cycle dynamics in the embryonic mouse sympathetic stellate ganglion, where neuroblasts continue to proliferate following neuronal differentiation. At embryonic day (E) 9.5, when neural crest-derived cells were migrating and coalescing into the ganglion primordium, all cells were cycling, cell cycle length was only 10.6 h, and S-phase comprised over65% of the cell cycle; these values are similar to those previously reported for embryonic stem cells. At E10.5, Sox10(+) cells lengthened their cell cycle to 38 h and reduced the length of S-phase. As cells started to express the neuronal markers Tuj1 and tyrosine hydroxylase (TH) at E10.5, they exited the cell cycle. At E11.5, when >80% of cells in the ganglion were Tuj1(+)/TH+ neuroblasts, all cells were again cycling. Neuroblast cell cycle length did not change significantly after E11.5, and 98% of Sox10(-)/TH+ cells had exited the cell cycle by E18.5. The cell cycle length of Sox10(+)/TH- cells increased during late embryonic development, and similar to 25% were still cycling at E18.5. Loss of Ret increased neuroblast cell cycle length at E16.5 and decreased the number of neuroblasts at E18.5. A mathematical model generated from our data successfully predicted the relative change in proportions of neuroblasts and non-neuroblasts in wild-type mice. Our results show that, like other neurons, sympathetic neuron differentiation is associated with exit from the cell cycle; sympathetic neurons are unusual in that they then re-enter the cell cycle before later permanently exiting.

SOC NEUROSCIENCE, Apr. 2013, JOURNAL OF NEUROSCIENCE, 33(14) (14), 5969 - 5979, English

[Refereed]

Scientific journal

GDNF and endothelin 3 regulate migration of enteric neural crest-derived cells via protein kinase A and Rac1

Akihiro Goto, Kenta Sumiyama, Yuji Kamioka, Eiji Nakasyo, Keisuke Ito, Mitsuhiro Iwasaki, Hideki Enomoto, Michiyuki Matsuda

Enteric neural crest-derived cells (ENCCs) migrate from the anterior foregut in a rostrocaudal direction to colonize the entire gastrointestinal tract and to form the enteric nervous system. Genetic approaches have identified many signaling molecules regulating the migration of ENCCs however, it remains elusive how the activities of the signaling molecules are regulated spatiotemporally during migration. In this study, transgenic mice expressing biosensors based on Förster resonance energy transfer were generated to video the activity changes of the signaling molecules in migrating ENCCs. In an organ culture of embryonic day 11.25 (E11.25) to E13 guts, ENCCs at the rostral wavefront migrated as a cellular chain faster than the following ENCCs that formed a network. The faster-migrating cells at the wavefront exhibited lower protein kinase A (PKA) activity than did the slower-migrating trailing cells. The activities of Rac1 and Cdc42 exhibited an inverse correlation with the PKA activity, and PKA activation decreased the Rac1 activity and migration velocity. PKA activity in ENCCs was correlated positively with the distribution of GDNF and inversely with the distribution of endothelin 3 (ET-3). Accordingly, PKA was activated by GDNF and inhibited by ET-3 in cultured ENCCs. Finally, although the JNK and ERK pathways were previously reported to control the migration of ENCCs, we did not find any correlation of JNK or ERK activity with the migration velocities. These results suggest that external cues regulate the migration of ENCCs by controlling PKA activity, but not ERK or JNK activity, and argue for the importance of live imaging of signaling molecule activities in developing organs. © 2013 the authors.

Mar. 2013, Journal of Neuroscience, 33(11) (11), 4901 - 4912, English

[Refereed]

Scientific journal

Transplanted progenitors generate functional enteric neurons in the postnatal colon

Ryo Hotta, Lincon A. Stamp, Jaime P.P. Foong, Sophie N. McConnell, Annette J. Bergner, Richard B. Anderson, Hideki Enomoto, Donald F. Newgreen, Florian Obermayr, John B. Furness, Heather M. Young

Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons they received synaptic inputs and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.

Mar. 2013, Journal of Clinical Investigation, 123(3) (3), 1182 - 1191, English

[Refereed]

Scientific journal

Development and developmental disorders of the enteric nervous system

Florian Obermayr, Ryo Hotta, Hideki Enomoto, Heather M. Young

The enteric nervous system (ENS) arises from neural crest-derived cells that migrate into and along the gut, leading to the formation of a complex network of neurons and glial cells that regulates motility, secretion and blood flow. This Review summarizes the progress made in the past 5 years in our understanding of ENS development, including the migratory pathways of neural crest-derived cells as they colonize the gut. The importance of interactions between neural crest-derived cells, between signalling pathways and between developmental processes (such as proliferation and migration) in ensuring the correct development of the ENS is also presented. The signalling pathways involved in ENS development that were determined using animal models are also described, as is the evidence for the involvement of the genes encoding these molecules in Hirschsprung disease-the best characterized paediatric enteric neuropathy. Finally, the aetiology and treatment of Hirschsprung disease in the clinic and the potential involvement of defects in ENS development in other paediatric motility disorders are outlined. Obermayr, F. et al. Nat. Rev. Gastroenterol. Hepatol. 10, 43-57 (2013); published online 11 December 2012; doi:10.1038/nrgastro.2012.234

NATURE PUBLISHING GROUP, Jan. 2013, NATURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY, 10(1) (1), 43 - 57, English

[Refereed]

Scientific journal

GDNF signaling levels control migration and neuronal differentiation of enteric ganglion precursors

Toshihiro Uesaka, Mayumi Nagashimada, Hideki Enomoto

Pleiotropic growth factors play a number of critical roles in continuous processes of embryonic development however, the mechanisms by which a single regulatory factor is able to orchestrate diverse developmental events remain imperfectly understood. In the development of the enteric nervous system (ENS), myenteric ganglia (MGs) form initially, after which the submucosal ganglia (SMGs) develop by radial inward migration of immature ENS precursors from the myenteric layer. Here, we demonstrate that glial cell line-derived neurotrophic factor (GDNF) is essential for the formation not only of the MGs, but the SMGs as well, establishing GDNF as a long-term acting neurotrophic factor for ENS development in a mouse model. GDNF promotes radial migration of SMG precursors. Interestingly, premigratory SMG precursors in the myenteric layer were distinguished from the surrounding neuronally differentiating cells by their lower activation of the GDNF-mediated MAPK pathway, suggesting that low activation of GDNF downstream pathways is required for the maintenance of the immature state. ENS precursors devoid of GDNF signaling during midgestation halt their migration, survive, and remain in an undifferentiated state over the long-term in vivo. Reactivation ofGDNFsignaling in these dormant precursors restores their migration and neuronal differentiation in gut organ culture. These findings suggest that pleiotropic function of GDNF is at least in part governed by modulating levels of intracellular activation of GDNF downstream pathways high activation triggers neuronal differentiation, whereas low activation is crucial for the maintenance of progenitor state. © 2013 the authors.

Society for Neuroscience, 2013, Journal of Neuroscience, 33(41) (41), 16372 - 16382, English

[Refereed]

Scientific journal

Trans-mesenteric neural crest cells are the principal source of the colonic enteric nervous system

Chihiro Nishiyama, Toshihiro Uesaka, Takayuki Manabe, Yohei Yonekura, Takashi Nagasawa, Donald F. Newgreen, Heather M. Young, Hideki Enomoto

Cell migration is fundamental to organogenesis. During development, the enteric neural crest cells (ENCCs) that give rise to the enteric nervous system (ENS) migrate and colonize the entire length of the gut, which undergoes substantial growth and morphological rearrangement. How ENCCs adapt to such changes during migration, however, is not fully understood. Using time-lapse imaging analyses of mouse ENCCs, we show that a population of ENCCs crosses from the midgut to the hindgut via the mesentery during a developmental time period in which these gut regions are transiently juxtaposed, and that such 'trans-mesenteric' ENCCs constitute a large part of the hindgut ENS. This migratory process requires GDNF signaling, and evidence suggests that impaired trans-mesenteric migration of ENCCs may underlie the pathogenesis of Hirschsprung disease (intestinal aganglionosis). The discovery of this trans-mesenteric ENCC population provides a basis for improving our understanding of ENS development and pathogenesis.

NATURE PUBLISHING GROUP, Sep. 2012, NATURE NEUROSCIENCE, 15(9) (9), 1211 - U64, English

[Refereed]

Scientific journal

Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression

Mayumi Nagashimada, Hiroshi Ohta, Chong Li, Kazuki Nakao, Toshihiro Uesaka, Jean-Francois Brunet, Jeanne Amiel, Delphine Trochet, Teruhiko Wakayama, Hideki Enomoto

The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse Phox2b locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward. the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine beta-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.

AMER SOC CLINICAL INVESTIGATION INC, Sep. 2012, JOURNAL OF CLINICAL INVESTIGATION, 122(9) (9), 3145 - 3158, English

[Refereed]

Scientific journal

Prox1 postmitotically defines dentate gyrus cells by specifying granule cell identity over CA3 pyramidal cell fate in the hippocampus

Tomohiko Iwano, Aki Masuda, Hiroshi Kiyonari, Hideki Enomoto, Fumio Matsuzaki

The brain is composed of diverse types of neurons that fulfill distinct roles in neuronal circuits, as manifested by the hippocampus, where pyramidal neurons and granule cells constitute functionally distinct domains: cornu ammonis (CA) and dentate gyrus (DG), respectively. Little is known about how these two types of neuron differentiate during hippocampal development, although a set of transcription factors that is expressed in progenitor cells is known to be required for the survival of granule cells. Here, we demonstrate in mice that Prox1, a transcription factor constitutively expressed in the granule cell lineage, postmitotically functions to specify DG granule cell identity. Postmitotic elimination of Prox1 caused immature DG neurons to lose the granule cell identity and in turn terminally differentiate into the pyramidal cell type manifesting CA3 neuronal identity. By contrast, Prox1 overexpression caused opposing effects on presumptive hippocampal pyramidal cells. These results indicate that the immature DG cell has the potential to become a granule cell or a pyramidal cell, and Prox1 defines the granule cell identity. This bi-potency is lost in mature DG cells, although Prox1 is still required for correct gene expression in DG granule cells. Thus, our data indicate that Prox1 acts as a postmitotic cell fate determinant for DG granule cells over the CA3 pyramidal cell fate and is crucial for maintenance of the granule cell identity throughout the life.

COMPANY OF BIOLOGISTS LTD, Aug. 2012, DEVELOPMENT, 139(16) (16), 3051 - 3062, English

[Refereed]

Scientific journal

Differential RET Signaling Pathways Drive Development of the Enteric Lymphoid and Nervous Systems

Amisha Patel, Nicola Harker, Lara Moreira-Santos, Manuela Ferreira, Kieran Alden, Jon Timmis, Katie Foster, Anna Garefalaki, Panayotis Pachnis, Paul Andrews, Hideki Enomoto, Jeffrey Milbrandt, Vassilis Pachnis, Mark C. Coles, Dimitris Kioussis, Henrique Veiga-Fernandes

During the early development of the gastrointestinal tract, signaling through the receptor tyrosine kinase RET is required for initiation of lymphoid organ (Peyer's patch) formation and for intestinal innervation by enteric neurons. RET signaling occurs through glial cell line-derived neurotrophic factor (GDNF) family receptor a co-receptors present in the same cell (signaling in cis). It is unclear whether RET signaling in trans, which occurs in vitro through co-receptors from other cells, has a biological role. We showed that the initial aggregation of hematopoietic cells to form lymphoid clusters occurred in a RET-dependent, chemokine-independent manner through adhesion-mediated arrest of lymphoid tissue initiator (LTin) cells. Lymphoid tissue inducer cells were not necessary for this initiation phase. LTin cells responded to all RET ligands in trans, requiring factors from other cells, whereas RET was activated in enteric neurons exclusively by GDNF in cis. Furthermore, genetic and molecular approaches revealed that the versatile RET responses in LTin cells were determined by distinct patterns of expression of the genes encoding RET and its co-receptors. Our study shows that a trans RET response in LTin cells determines the initial phase of enteric lymphoid organ morphogenesis, and suggests that differential co-expression of Ret and Gfra can control the specificity of RET signaling.

AMER ASSOC ADVANCEMENT SCIENCE, Jul. 2012, SCIENCE SIGNALING, 5(235) (235), ra55, English

[Refereed]

Scientific journal

Development of the Enteric Nervous System

Heather M. Young, Donald F. Newgreen, Hideki Enomoto

Elsevier Inc., 2012, Physiology of the Gastrointestinal Tract, 1, 475 - 488, English

[Refereed]

In book

Neural Precursor Death Is Central to the Pathogenesis of Intestinal Aganglionosis in Ret Hypomorphic Mice

Toshihiro Uesaka, Hideki Enomoto

The RET tyrosine kinase is required for the migration, proliferation, and survival of the enteric neural crest-derived cells (ENCCs) that form the enteric nervous system (ENS). Hypomorphic RET alleles cause intestinal aganglionosis [Hirschsprung disease (HSCR)], in which delayed migration and successive nonapoptotic ENCC death are considered to be major contributory factors. The significance of ENCC death in intestinal aganglionosis, however, has remained unclear. We show that elevated expression of Bcl-xL inhibits ENCC death in both Ret-null and hypomorphic states. However, the rescued Ret-null mice showed ENS malfunction with reduced nitric oxide synthase expression in colonic neurons, revealing the requirement of RET for neuronal differentiation. In contrast, the inhibition of cell death allows morphologically and functionally normal ENS formation in Ret hypomorphic mice. These results indicate that ENCC death is a principal cause of intestinal aganglionosis in a Ret hypomorphic state, and suggest that the inhibition of cell death is a route to the prevention of HSCR.

SOC NEUROSCIENCE, Apr. 2010, JOURNAL OF NEUROSCIENCE, 30(15) (15), 5211 - 5218, English

[Refereed]

Scientific journal

RET Signaling Is Required for Survival and Normal Function of Nonpeptidergic Nociceptors

Judith P. Golden, Masato Hoshi, Mohammed A. Nassar, Hideki Enomoto, John N. Wood, Jeffrey Milbrandt, Robert W. Gereau, Eugene M. Johnson, Sanjay Jain

Small unmyelinated sensory neurons classified as nociceptors are divided into two subpopulations based on phenotypic differences, including expression of neurotrophic factor receptors. Approximately half of unmyelinated nociceptors express the NGF receptor TrkA, and half express the GDNF family ligand (GFL) receptor Ret. The function of NGF/TrkA signaling in the TrkA population of nociceptors has been extensively studied, and NGF/TrkA signaling is a well established mediator of pain. The GFLs are analgesic in models of neuropathic pain emphasizing the importance of understanding the physiological function of GFL/Ret signaling in nociceptors. However, perinatal lethality of Ret-null mice has precluded the study of the physiological role of GFL/Ret signaling in the survival, maintenance, and function of nociceptors in viable mice. We deleted Ret exclusively in nociceptors by crossing nociceptor-specific Na(v)1.8 Cre and Ret conditional mice to produce Ret-Na(v)1.8 conditional knock-out (CKO) mice. Loss of Ret exclusively in nociceptors results in a reduction in nociceptor number and size, indicating that Ret signaling is important for the survival and trophic support of these cells. Ret-Na(v)1.8 CKO mice exhibit reduced epidermal innervation but normal central projections. In addition, Ret-Na(v)1.8 CKO mice have increased sensitivity to cold and increased formalin-induced pain, demonstrating that Ret signaling modulates the function of nociceptors in vivo. Enhanced inflammation-induced pain may be mediated by decreased prostatic acid phosphatase (PAP), as PAP levels are markedly reduced in Ret-Na(v)1.8 CKO mice. The results of this study identify the physiological role of endogenous Ret signaling in the survival and function of nociceptors.

SOC NEUROSCIENCE, Mar. 2010, JOURNAL OF NEUROSCIENCE, 30(11) (11), 3983 - 3994, English

[Refereed]

Scientific journal

Molecular Identification of Rapidly Adapting Mechanoreceptors and Their Developmental Dependence on Ret Signaling

Wenqin Luo, Hideki Enomoto, Frank L. Rice, Jeffrey Milbrandt, David D. Ginty

In mammals, the first step in the perception of form and texture is the activation of trigeminal or dorsal root ganglion (DRG) mechanosensory neurons, which are classified as either rapidly (RA) or slowly adapting (SA) according to their rates of adaptation to sustained stimuli. The molecular identities and mechanisms of development of RA and SA mechanoreceptors are largely unknown. We found that the "early Ret(+)" DRG neurons are RA mechanoreceptors, which form Meissner corpuscles, Pacinian corpuscles, and longitudinal lanceolate endings. The central projections of these RA mechanoreceptors innervate layers III through V of the spinal cord and terminate within discrete subdomains of the dorsal column nuclei. Moreover, mice lacking Ret signaling components are devoid of Pacinian corpuscles and exhibit a dramatic disruption of RA mechanoreceptor projections to both the spinal cord and medulla. Thus, the early Re(+) neurons are RA mechanoreceptors and Ret signaling is required for the assembly of neural circuits underlying touch perception.

CELL PRESS, Dec. 2009, NEURON, 64(6) (6), 841 - 856, English

[Refereed]

Scientific journal

Ret-Dependent Cell Rearrangements in the Wolffian Duct Epithelium Initiate Ureteric Bud Morphogenesis

Xuan Chi, Odysse Michos, Reena Shakya, Paul Riccio, Hideki Enomoto, Jonathan D. Licht, Naoya Asai, Masahide Takahashi, Nobutaka Ohgami, Masashi Kato, Cathy Mendelsohn, Frank Costantini

While the genetic control of renal branching morphogenesis has been extensively described, the cellular basis of this process remains obscure. GDNF/RET signaling is required for ureter and kidney development, and cells lacking Ret are excluded from the tips of the branching ureteric bud in chimeric kidneys. Here, we find that this exclusion results from earlier Ret-dependent cell rearrangements in the caudal Wolffian duct, which generate a specialized epithelial domain that later emerges as the tip of the primary ureteric bud. By juxtaposing cells with elevated or reduced RET activity, we find that Wolffian duct cells compete, based on RET signaling levels, to contribute to this domain. At the same time, the caudal Wolffian duct transiently converts from a simple to a pseudostratified epithelium, a process that does not require Ret. Thus, both Ret-dependent cell movements and Ret-independent changes in the Wolffian duct epithelium contribute to ureteric bud formation.

CELL PRESS, Aug. 2009, DEVELOPMENTAL CELL, 17(2) (2), 199 - 209, English

[Refereed]

Scientific journal

Regionalized Loss of Parvalbumin Interneurons in the Cerebral Cortex of Mice with Deficits in GFR alpha 1 Signaling

Alison J. Canty, Jule Dietze, Michael Harvey, Hideki Enomoto, Jeffrey Milbrandt, Carlos F. Ibanez

Inhibitory interneurons are crucially important for cerebral cortex function and behavior. The mechanisms controlling inhibitory interneuron diversification and allocation to distinct cortical areas remain poorly understood. GDNF (glial cell line-derived neurotrophic factor) and its receptor GFR alpha 1 have been implicated in the development of GABAergic precursors but, because of the early lethality of null mutants, their roles in postnatal maturation and function of cortical interneurons are unknown. "cis-only" mutant mice lack GFR alpha 1 only in cells that do not express the RET signaling receptor subunit and survive to adulthood. At birth, both null mutants and cis-only mice showed a specific loss of GABAergic interneurons in rostro- and caudolateral cortical regions but not in more medial areas. Unexpectedly, the adult cortex of cis-only mice displayed a complete loss of parvalbumin (PV)-expressing GABAergic interneurons in discrete regions (PV holes) interspersed among areas of normal PV cell density. PV holes predominantly occurred in the visual and frontal cortices, and their size could be affected by neuronal activity. Consistent with deficits in cortical inhibitory activity, these mice showed enhanced cortical excitability, increased sensitivity to epileptic seizure, and increased social behavior. We propose that GFR alpha 1 signaling guides the development of a subset of PV-expressing GABAergic interneurons populating discrete regions of the cerebral cortex and may thus contribute to the diversification and allocation of specific cortical interneuron subtypes.

SOC NEUROSCIENCE, Aug. 2009, JOURNAL OF NEUROSCIENCE, 29(34) (34), 10695 - 10705, English

[Refereed]

Scientific journal

Development of satellite glia in mouse sympathetic ganglia: Gdnf and Gfr alpha 1 are not essential

Thomas Callahan, Heather M. Young, Richard B. Anderson, Hideki Enomoto, Colin R. Anderson

The phenotypic development of satellite cells in mouse sympathetic ganglia was examined by localizing the transcription factors, Sox10 and Phox2b, the neuronal marker, tyrosine hydroxylase (TH), and brain-derived fatty acid binding protein (B-FABP), which identifies glial precursors and mature glia. In E10.5 mice, most cells in the sympathetic chain expressed both Sox10 and Phox2b, with a minority of cells expressing Sox10 only or Phox2b only. In E11.5 mice, the majority of cells expressed Sox10 only or Phox2b only. B-FABP was colocalized with Sox10 in satellite glial precursors, which were located on the periphery of the ganglion. There was no overlap between B-FABP and Phox2b or B-FABP and TH. During subsequent development, the number of B-FABP+ cells increased and they became more common deep within the ganglion. In E12.5 and E18.5 mice, there was no overlap between Sox10 and Phox2b, and 98% of Sox10 cells were also B-FABP+. Satellite glial precursors in E11.5-E15.5 mice also expressed the GDNF-binding molecule, GFR alpha 1. B-FABP immunoreactive cells did not express Ret or NCAM, two potential signaling molecules for GDNF/GFR alpha 1. In E12.5 and E18.5 mice lacking GFR alpha 1 or GDNF, the development of B-FABP immunoreactive satellite cells was normal, and hence neither GDNF or GFR alpha 1 are essential for the development of satellite glia in sympathetic ganglia. (c) 2008 Wiley-Liss, Inc.

WILEY-LISS, Oct. 2008, GLIA, 56(13) (13), 1428 - 1437, English

[Refereed]

Scientific journal

Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice

Toshihiro Uesaka, Mayumi Nagashimada, Shigenobu Yonemura, Hideki Enomoto

Mutations in the PET gene are the primary cause of Hirschsprung disease (HSCR), or congenital intestinal aganglionosis. However, how RET malfunction leads to HSCR is not known. it has recently been shown that glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 1 (GFR alpha 1), which binds to GDNF and activates RET, is essential for the survival of enteric neurons. In this study, we investigated Ret regulation of enteric neuron survival and its potential involvement in HSCR. Conditional ablation of Ret in postmigratory enteric neurons caused widespread neuronal death in the colon, which led to colonic aganglionosis. To further examine this finding, we generated a mouse model for HSCR by reducing Ret expression levels. These mice recapitulated the genetic and phenotypic features of HSCR and developed colonic aganglionosis due to impaired migration and successive death of enteric neural crest-derived cells. Death of enteric neurons was also induced in the colon, where reduction of Ret expression was induced after the period of enteric neural crest cell migration, indicating that diminished Ret expression directly affected the survival of colonic neurons. Thus, enteric neuron survival is sensitive to RET dosage, and cell death is potentially involved in the etiology of HSCR.

AMER SOC CLINICAL INVESTIGATION INC, May 2008, JOURNAL OF CLINICAL INVESTIGATION, 118(5) (5), 1890 - 1898, English

[Refereed]

Scientific journal

The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes

Thomas W. Gould, Shigenobu Yonemura, Ronald W. Oppenheim, Shiho Ohmori, Hideki Enomoto

Glial cell line-derived neurotrophic factor ( GDNF) regulates multiple aspects of spinal motoneuron ( MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha 1 ( GFR alpha 1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFR alpha 1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFR alpha 1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFR alpha 1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.

SOC NEUROSCIENCE, Feb. 2008, JOURNAL OF NEUROSCIENCE, 28(9) (9), 2131 - 2146, English

[Refereed]

Scientific journal

Conditional ablation of GFR alpha 1 in postmigratory enteric neurons triggers unconventional neuronal death in the colon and causes a Hirschsprung's disease phenotype

Toshihiro Uesaka, Sanjay Jain, Shigenobu Yonemura, Yasuo Uchiyama, Jeffrey Milbrandt, Hideki Enomoto

The regulation of neuronal survival and death by neurotrophic factors plays a central role in the sculpting of the nervous system, but the identity of survival signals for developing enteric neurons remains obscure. We demonstrate here that conditional ablation of GFR alpha 1, the high affinity receptor for GDNF, in mice during late gestation induces rapid and widespread neuronal death in the colon, leading to colon aganglionosis reminiscent of Hirschsprung's disease. Enteric neuron death induced by GFR alpha 1 inactivation is not associated with the activation of common cell death executors, caspase-3 or -7, and lacks the morphological hallmarks of apoptosis, such as chromatin compaction and mitochondrial pathology. Consistent with these in vivo observations, neither caspase inhibition nor Bax deficiency blocks death of colon-derived enteric neurons induced by GDNF deprivation. This study reveals an essential role for GFR alpha 1 in the survival of enteric neurons and suggests that caspase- independent death can be triggered by abolition of neurotrophic signals.

COMPANY OF BIOLOGISTS LTD, Jun. 2007, DEVELOPMENT, 134(11) (11), 2171 - 2181, English

[Refereed]

Scientific journal

Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis

Bhupinder P. S. Vohra, Keiji Tsuji, Mayumi Nagashimada, Toshihiro Uesaka, Daniel Wind, Ming Fu, Jennifer Armon, Hideki Enomoto, Robert O. Heuckeroth

Enteric nervous system (ENS) development requires complex interactions between migrating neural-crest-derived cells and the intestinal microenvironment. Although some molecules influencing ENS development are known, many aspects remain poorly understood. To identify additional molecules critical for ENS development, we used DNA microarray, quantitative real-time PCR and in situ hybridization to compare gene expression in E14 and PO aganglionic or wild type mouse intestine. Eighty-three genes were identified with at least two-fold higher expression in wild type than aganglionic bowel. ENS expression was verified for 39 of 42 selected genes by in situ hybridization. Additionally, nine identified genes had higher levels in aganglionic bowel than in WT animals suggesting that intestinal innervation may influence gene expression in adjacent cells. Strikingly, many synaptic function genes were expressed at E14, a time when the ENS is not needed for survival. To test for developmental roles for these genes, we used pharmacologic inhibitors of Snap25 or vesicle-associated membrane protein (VAMP)/synaptobrevin and found reduced neural-crest-derived cell migration and decreased neurite extension from ENS precursors. These results provide an extensive set of ENS biomarkers, demonstrate a role for SNARE proteins in ENS development and highlight additional candidate genes that could modify Hirschsprung's disease penetrance. (c) 2006 Elsevier Inc. All rights reserved.

ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2006, DEVELOPMENTAL BIOLOGY, 298(1) (1), 259 - 271, English

[Refereed]

Scientific journal

GFR alpha 1 expression in cells lacking RET is dispensable for organogenesis and nerve regeneration

H Enomoto, Hughes, I, J Golden, RH Baloh, S Yonemura, RO Heuckeroth, EM Johnson, J Milbrandt

The GDNF family ligands signal through a receptor complex composed of a ligand binding subunit, GFRalpha, and a signaling subunit, the RET tyrosine kinase. GFRalphas are expressed not only in RET-expressing cells, but also in cells lacking RET. A body of evidence suggests that RET-independent GFRalphas are important for (1) modulation of RET signaling in a non-cell-autonomous fashion (trans-signaling) and (2) regulation of NCAM function. To address the physiological significance of these roles, we generated mice specifically lacking RET-independent GFRalpha1. These mice exhibited no deficits in regions where trans-signaling has been implicated in vitro, including enteric neurons, motor neurons, kidney, and regenerating nerves. Furthermore, no abnormalities were found in the olfactory bulb, which requires proper NCAM function for its formation and is putatively a site of GDNF-GFRalpha-NCAM signaling. Thus RET-independent GFRalpha1 is dispensable for organogenesis and nerve regeneration in vivo, indicating that trans-signaling and GFRalpha-dependent NCAM signaling play a minor role physiologically.

CELL PRESS, Nov. 2004, NEURON, 44(4) (4), 623 - 636, English

[Refereed]

Scientific journal

The enteric nervous system is not essential for the propulsion of gut contents in fetal mice

RB Anderson, H Enomoto, JC Bornstein, HM Young

B M J PUBLISHING GROUP, Oct. 2004, GUT, 53(10) (10), 1546 - 1547, English

[Refereed]

Scientific journal

Dok-6, a novel p62 Dok family member, promotes Ret-mediated neurite outgrowth

RJ Crowder, H Enomoto, M Yang, EM Johnson, J Milbrandt

Activation of Ret, the receptor-tyrosine kinase for the glial cell line-derived neurotrophic factor ( GDNF) family ligands (GFLs), results in the recruitment and assembly of adaptor protein complexes that function to transduce signals downstream of the receptor. Here we identify Dok-6, a novel member of the Dok-4/5 subclass of the p62 Dok family of intracellular adaptor molecules, and characterize its interaction with Ret. Expression analysis reveals that Dok-6 is highly expressed in the developing central nervous system and is co-expressed with Ret in several locations, including sympathetic, sensory, and parasympathetic ganglia, as well as in the ureteric buds of the developing kidneys. Pull-down assays using the Dok-6 phosphotyrosine binding (PTB) domain and GDNF-activated Ret indicate that Dok-6 binds to the phosphorylated Ret Tyr(1062) residue. Moreover, ligand activation of Ret resulted in phosphorylation of tyrosine residue(s) located within the unique C terminus of Dok-6 predominantly through a Src-dependent mechanism, indicating that Dok-6 is a substrate of the Ret-Src signaling pathway. Interestingly, expression of Dok-6 potentiated GDNF-induced neurite outgrowth in GDNF family receptor alpha1 (GFRalpha1)-expressing Neuro2A cells that was dependent upon the C-terminal residues of Dok-6. Taken together, these data identify Dok-6 as a novel Dok-4/5-related adaptor molecule that may function in vivo to transduce signals that regulate Ret-mediated processes such as axonal projection.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Oct. 2004, JOURNAL OF BIOLOGICAL CHEMISTRY, 279(40) (40), 42072 - 42081, English

[Refereed]

Scientific journal

Neural cells in the esophagus respond to glial cell line-derived neurotrophic factor and neurturin, and are RET-dependent

H Yan, AJ Bergner, H Enomoto, J Milbrandt, DF Newgreen, HM Young

Glial cell line-derived neurotrophic factor (GDNF) is expressed in the gastrointestinal tract of the developing mouse and appears to play an important role in the migration of enteric neuron precursors into and along the small and large intestines. Two other GDNF family members, neurturin and artemin, are also expressed in the developing gut although artemin is only expressed in the esophagus. We examined the effects of GDNF, neurturin, and artemin on neural crest cell migration and neurite outgrowth in explants of mouse esophagus, midgut, and hindgut. Both GDNF and neurturin induced neural crest cell migration and neurite outgrowth in all regions examined. In the esophagus, the effect of GDNF on migration and neurite outgrowth declined with age between E11.5 and E14.5, but neurturin still had a strong neurite outgrowth effect at E14.5. Artemin did not promote neural migration or neurite outgrowth in any region investigated. The effects of GDNF family ligands are mediated by the Ret tyrosine kinase. We examined the density of neurons in the esophagus of Ret(-/-) mice, which lack neurons in the small and large intestines. The density of esophageal neurons in Ret(-/-) mice was only about 4% of the density of esophageal neurons in Ret(-/-) and Ret(-/-) mice. These results show that GDNF and neurturin promote migration and neurite outgrowth of crest-derived cells in the esophagus as well as the intestine. Moreover, like intestinal neurons, the development of esophageal neurons is largely Ret-dependent. (C) 2004 Elsevier Inc. All rights reserved.

ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2004, DEVELOPMENTAL BIOLOGY, 272(1) (1), 118 - 133, English

[Refereed]

Scientific journal

Dynamics of neural crest-derived cell migration in the embryonic mouse gut

HM Young, AJ Bergner, RB Anderson, H Enomoto, J Milbrandt, DF Newgreen, PM Whitington

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype. (C) 2004 Elsevier Inc. All rights reserved.

ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2004, DEVELOPMENTAL BIOLOGY, 270(2) (2), 455 - 473, English

[Refereed]

Scientific journal

GDNF availability determines enteric neuron number by controlling precursor proliferation

S Gianino, Grider, JR, J Cresswell, H Enomoto, RO Heuckeroth

To clarify the role of Ret signaling components in enteric nervous system (ENS) development, we evaluated ENS anatomy and intestinal contractility in mice heterozygous for Ret, GFRalpha1 and Ret ligands. These analyses demonstrate that glial cell line-derived neurotrophic factor (GDNF) and neurturin are important for different aspects of ENS development. Neurturin is essential for maintaining the size of mature enteric neurons and the extent of neuronal projections, but does not influence enteric neuron number. GDNF availability determines enteric neuron number by controlling ENS precursor proliferation. However, we were unable to find evidence of programmed cell death in the wild type ENS by immunohistochemistry for activated caspase 3. In addition, enteric neuron number is normal in Bax(-/-) and Bid(-/-) mice, suggesting that, in contrast to most of the rest of the nervous system, programmed cell death is not important for determining enteric neuron numbers. Only mild reductions in neuron size and neuronal fiber counts occur in Ret(+/-) and Gfra1(+/-) mice. All of these heterozygous mice, however, have striking problems with intestinal contractility and neurotransmitter release, demonstrating that Ret signaling is critical for both ENS structure and function.

COMPANY OF BIOLOGISTS LTD, May 2003, DEVELOPMENT, 130(10) (10), 2187 - 2198, English

[Refereed]

Scientific journal

RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons

H Enomoto, PA Crawford, A Gorodinsky, RO Heuckeroth, EM Johnson, J Milbrandt

Sympathetic axons use blood vessels as an intermediate path to reach their final target tissues. The initial contact between differentiating sympathetic neurons and blood vessels occurs following the primary sympathetic chain formation, where precursors of sympathetic neurons migrate and project axons along or toward blood vessels. We demonstrate that, in Ret-deficient mice, neuronal precursors throughout the entire sympathetic nervous system fail to migrate and project axons properly. These primary deficits lead to mis-routing of sympathetic nerve trunks and accelerated cell death of sympathetic neurons later in development. Artemin is expressed in blood vessels during periods of early sympathetic differentiation, and can promote and attract axonal growth of the sympathetic ganglion in vitro. This analysis identifies RET and artemin as central regulators of early sympathetic innervation.

COMPANY OF BIOLOGISTS LTD, Oct. 2001, DEVELOPMENT, 128(20) (20), 3963 - 3974, English

[Refereed]

Scientific journal

Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin

H Enomoto, RO Heuckeroth, JP Golden, EM Johnson, J Milbrandt

The neurotrophic factors that influence the development and function of the parasympathetic branch of the autonomic nervous system are obscure, Recently, neurturin has been found to provide trophic support to neurons of the cranial parasympathetic ganglion. Here we show that GDNF signaling via the RET/GFR alpha1 complex is crucial for the development of cranial parasympathetic ganglia including the submandibular, sphenopalatine and otic ganglia. GDNF is required early for proliferation and/or migration of the neuronal precursors for the sphenopalatine and otic ganglia. Neurturin exerts its effect later and is required for further development and maintenance of these neurons. This switch in ligand dependency during development is at least partly governed by the altered expression of GFR alpha receptors, as evidenced by the predominant expression of GFR alpha2 in these neurons after ganglion formation.

COMPANY OF BIOLOGISTS LTD, Nov. 2000, DEVELOPMENT, 127(22) (22), 4877 - 4889, English

[Refereed]

Scientific journal

Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification

AU Zaidi, H Enomoto, J Milbrandt, KA Roth

To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor alpha 2 (GFR alpha 2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFR alpha 2 mRNA and virtually all GFR alpha 2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFR alpha 2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization.

HISTOCHEMICAL SOC INC, Oct. 2000, JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, 48(10) (10), 1369 - 1375, English

[Refereed]

Scientific journal

Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons

RO Heuckeroth, H Enomoto, Grider, JR, JP Golden, JA Hanke, A Jackman, DC Molliver, ME Bardgett, WD Snider, EM Johnson, J Milbrandt

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha 1 or GFR alpha 2). Neurturin-deficient (NTN-/-) mice generated by hom*ologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons, GFR alpha 2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha 2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha 2/Ret receptor complexes as the critical mediators of NTN function in vivo.

CELL PRESS, Feb. 1999, NEURON, 22(2) (2), 253 - 263, English

[Refereed]

Scientific journal

Expression of MRP and cMOAT in childhood neuroblastomas and malignant liver tumors and its relevance to clinical behavior

T Matsunaga, H Shirasawa, T Hishiki, H Enomoto, K Kouchi, Y Ohtsuka, J Iwai, H Yoshida, M Tanabe, S Kobayashi, T Asano, T Etoh, Y Nishi, N Ohnuma

Advanced neuroblastoma and malignant liver tumor are representative childhood cancers for which combined chemotherapy including cisplatin and doxorubicin is routinely performed, The prognosis of patients with tumors which develop multiple drug resistance (MDR) is unfavorable. To elucidate the role of multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) in the clinical behavior of the tumors, we examined 42 neuroblastomas and 10 malignant liver tumors for the expressions of MRP and cMOAT by quantitative RNA-polymerase chain reaction (PCR). The amplification and expression of N-myc oncogene in the neuroblastomas were also investigated, We found a close association between MRP and N-myc expression in each neuroblastoma sample but no significant relationship between MRP expression and the patients' outcome, The forced expression of N-myc failed to enhance the expression of MRP in N-myc transfected neuroblastoma cell lines, cMOAT was rarely expressed in the neuroblastomas, but was frequently expressed in the malignant liver tumors. The expression of MRP and cMOAT in the childhood liver tumors was more common and higher, especially in advanced cases with a poor outcome, than that observed in normal liver or in 9 hepatocellular carcinomas from adult patients. The enhanced expression of these genes might be characteristic of childhood malignant liver tumors and related to their clinical chemoresistance.

ELSEVIER SCI IRELAND LTD, Dec. 1998, JAPANESE JOURNAL OF CANCER RESEARCH, 89(12) (12), 1276 - 1283, English

[Refereed]

Scientific journal

Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFR alpha 3-RET receptor complex

RH Baloh, MG Tansey, PA Lampe, TJ Fahrner, H Enomoto, KS Simburger, ML Leitner, T Araki, EM Johnson, J Milbrandt

The glial cell line-derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFR alpha 1-GFR alpha 4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFR alpha 3-RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFR alpha 1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFR alpha 1-RET) and NTN (GFR alpha 2-RET), Artemin has a preferred receptor (GFR alpha 3-RET) but that alternative receptor interactions also occur.

CELL PRESS, Dec. 1998, NEURON, 21(6) (6), 1291 - 1302, English

[Refereed]

Scientific journal

In vivo karyotypic evolution with altered biological characteristics in a case of neuroblastoma

H Enomoto, Y Nakamura, K Kondo, M Hirose, Y Kaneko, H Horie, H Takahashi, N Ohnuma, M Tanabe, A Nakagawara

Molecular and biological analyses of a neuroblastoma case in which the original tumor contained a nodular region are described. No significant difference was observed between the nodular and the surrounding tumor tissue with respect to histopathologic examination, N-myc amplification, and trkA expression. However, flow cytometric analysis demonstrated that the nodular region consisted of a hypertetraploid clone, whereas the surrounding tissue mostly contained a hyperdiploid clone. Chromosome analysis showed that each clone had a similar chromosome acquisition pattern, suggesting that the hypertetraploid cells of the nodular region arose from the hyperdiploid cells of the surrounding tissue. Moreover, primary culture findings of the tumor cells showed that the responses to nerve growth factor or retinoic acid were different between the two. Collectively, this case suggests the possibility that neuroblastoma acquires novel biological characteristics through karyotypic evolution in vivo. Copyright (C) 1998 by W.B. Saunders Company.

W B SAUNDERS CO, Sep. 1998, JOURNAL OF PEDIATRIC SURGERY, 33(9) (9), 1404 - 1407, English

[Refereed]

Scientific journal

GFR alpha 1-deficient mice have deficits in the enteric nervous system and kidneys

H Enomoto, T Araki, A Jackman, RO Heuckeroth, WD Snider, EM Johnson, J Milbrandt

Glial cell line-derived neurotrophic factor (GDNF) signals through a receptor complex composed of the Pet tyrosine kinase and a glycosylphosphatidylinositol(GPl-) anchored cell surface coreceptor, either GDNF family receptor alpha 1 (GFR alpha 1) or GFR alpha 2. To investigate the usage of these coreceptors for GDNF signaling in vivo, gene targeting was used to produce mice lacking the GFR alpha 1 coreceptor. GFR alpha 1-deficient mice demonstrate absence of enteric neurons and agenesis of the kidney, characteristics that are reminiscent of both GDNF- and Ret-deficient mice. Midbrain dopaminergic and motor neurons in GFR alpha 1 null mice were normal. Minimal or no neuronal losses were observed in a number of peripheral ganglia examined, including the superior cervical and nodose, which are severely affected in both Ret- and GDNF-deficient mice. These results suggest that while stringent physiologic pairing exists between GFR alpha 1 and GDNF in renal and enteric nervous system development, significant cross-talk between GDNF and other GFR alpha coreceptors must occur in other neuronal populations.

CELL PRESS, Aug. 1998, NEURON, 21(2) (2), 317 - 324, English

[Refereed]

Scientific journal

Antitumor vaccine effect of irradiated murine neuroblastoma cells producing interleukin-2 or granulocyte macrophage-colony stimulating factor

H Yoshida, H Enomoto, K Kawamura, K Takenaga, M Tanabe, N Ohnuma, S Sakiyama, M Tagawa

We have examined vaccination effects of cytokine-producing murine neuroblastoma cells (C1300). C1300 cells retrovirally transduced with interleukin-2 (IL-2) or granulocyte macrophage-colony stimulation factor (GMCSF) gene were established. Their in vitro proliferation rates and the class I expression of major histocompatibility complex were not different from those of wild-type cells. Five-Gy irradiation of the respective cytokine producers slightly reduced the in vitro cell growth but treatment with 15 Gy significantly impaired the proliferation. In contrast, the secretion of both cytokines from the respective transduced cells was retained compared with the cell growth. We immunized syngeneic mice with irradiated wild-type cells as a control or cytokine-producing cells and challenged the mice with unirradiated wild-type cells. The control mice developed tumors of the challenged wild-type cells, on the contrary, the mice which had received irradiated IL-2 or GM-CSF producers did not. Thus, IL-2 or GM-CSF-expressing syngeneic tumor cells can be potentially used as a tumor vaccine by inducing protective immunity against low immunogenic neuroblastomas in the inoculated hosts.

PROFESSOR D A SPANDIDOS, Jul. 1998, INTERNATIONAL JOURNAL OF ONCOLOGY, 13(1) (1), 73 - 78, English

[Refereed]

Scientific journal

Neuronal src and trk a protooncogene expression in neuroblastomas and patient prognosis

T Matsunaga, H Shirasawa, H Enomoto, H Yoshida, J Iwai, M Tanabe, K Kawamura, T Etoh, N Ohnuma

Neuroblastomas present a wide variety of clinical and biological behaviors, which are reflected by the heterogeneous expressions of protooncogenes related to the neuronal differentiation and amplification of the N-myc gene. High expression of trk A and Ha-ras in neuroblastomas has been shown to be associated with an excellent patient outcome. We have previously reported that neuron-specific src mRNA was increased in chemically differentiated neuroblastoma cell lines and in clinically observed neuroblastomas without N-myc amplification, In the present study, to clarify both the value of neuronal c-srcN2 expression as a prognostic indicator and the significance of the coexpression of these photooncogenes, we examined the expression of 3 alternatively spliced src, trk A and Ha-res in neuroblastoma tissues from 60 patients by competitive RNA-polymerase chain reaction (PCR). The restates indicate that protooncogene expression in neuroblastomas correlated with a favorable outcome for c-srcN2 and trk A. N-myc gene was amplified exclusively in tumors with low levels of trk A. Low expression of c-srcN2 and trk A might thus characterize different aggressive phenotypes due to different signal transduction pathways of neural differentiation in neuroblastoma. The combined analyses for c-srcN2 and trk A expression by RNA-BCR should provide information about the biological phenotype of a neuroblastoma within a short period of time after obtaining tumor material. (C) 1998 Wiley-Liss, Inc.

WILEY-LISS, Jun. 1998, INTERNATIONAL JOURNAL OF CANCER, 79(3) (3), 226 - 231, English

[Refereed]

Scientific journal

Impaired tumorigenicity of IL-4-producing murine neuroblastoma cells in immunodeficient nude mice

H Yoshida, H Enomoto, M Miyauchi, K Takenaga, M Tanabe, N Ohnuma, S Sakiyama, M Tagawa

We have examined antitumor effect of murine neuroblastoma cells (C1300) retrovirally transduced with interleukin-4 (IL-4) gene in syngeneic A/J and nude mice. Although in vitro proliferation of IL-4-secreting C1300 cells (C1300/IL-4) was not different from that of wild-type cells, the in vivo tumor growth of C1300/IL-4 cells subcutaneously inoculated into immunocompetent A/J mice was retarded compared with that of wild-type cells and consequently, the survival of the A/J mice which received C1300/IL-4 cells was prolonged. In immunodeficient nude mice we observed accelerated growth rate of wild-type tumors in comparison with the tumors developed in A/J mice. In contrast, the tumor growth of C1300/IL-4 cells in nude mice was significantly suppressed and the growth was much slower than that of C1300/IL-4 cells inoculated in A/J mice. Thus, the secretion of IL-4 from tumor cells produced antitumor effect more efficiently in mature T cell-defective hosts than in immunocompetent mice. Our results suggest a possible clinical application of IL-4 expression in tumor cells via genetic manipulations especially in immunocompromised cancer patients.

PROFESSOR D A SPANDIDOS, May 1998, INTERNATIONAL JOURNAL OF ONCOLOGY, 12(5) (5), 1067 - 1071, English

[Refereed]

Scientific journal

Impaired tumorigenicity and decreased liver metastasis of murine neuroblastoma cells engineered to secrete interleukin-2 or granulocyte macrophage colony-stimulating factor

H Yoshida, H Enomoto, M Tagawa, K Takenaga, K Tasaki, A Nakagawara, N Ohnuma, H Takahashi, S Sakiyama

We have examined the antitumor effect of murine neuroblastoma cells (C1300) engineered to produce cytokines. Retrovirally transduced cells with human interleukin-2 (IL-2) or murine GM-CSF gene, but not murine IL-4 gene, abolished their tumorigenicity in syngeneic mice, although their in vitro growth rate and expression of class I antigens of the major histocompatibility complex were unchanged. Inoculation of wild-type cells into the mice, which had rejected IL-2 or CM-CSF producers, did not develop tumors, indicating that protective immunity was induced. In an experimental hematogenous metastasis model, we found that the numbers of metastatic foci in the liver caused by intravenous administration of IL-2 or GM-CSF producers were significantly reduced compared with those by the injection of wild-type or vector virus-transduced cells. No significant differences in their adhesiveness to extracellular matrices and ability to differentiate were observed among parent and transduced cells. Thus, these results indicate that IL-2 or CM-CSF secretion, in the vicinity of neuroblastoma cells, produced antitumor effect and reduced metastatic ability.

APPLETON & LANGE, Mar. 1998, CANCER GENE THERAPY, 5(2) (2), 67 - 73, English

[Refereed]

Scientific journal

B80 RNA-PCRによる神経特異的src, trk A, Ha-rasの発現と神経芽腫の予後

松永正訓, 田辺政裕, 吉田英生, 岩井潤, 榎本秀樹, 吉野薫, 大塚恭寛, 幸地克憲, 岡田忠雄, 川村健児, 江東孝夫, 大沼直躬

特定非営利活動法人日本小児外科学会, 1998, 日本小児外科学会雑誌, 34(3) (3), 697 - 697, Japanese

The genomic analysis of human DAN gene

T Ozaki, H Enomoto, Y Nakamura, K Kondo, N Seki, M Ohira, N Nomura, M Ohki, A Nakagawara, S Sakiyama

DAN gene is shown to be localized at human chromosome 1p36.11-p36.13, which resides within the consensus deletion observed in neuroblastoma, In the present study, we have isolated the genomic DNA containing the entire human DAN gene and determined its nucleotide sequence, Structural analysis revealed that DAN gene is composed of four exons and spans approximately 15 kb, Its overall structure was basically the same as that of rat DAN gene, Two distinct transcription initiation sites (major and minor) were identified by the primer extension experiment, Putative TATA and CAAT-like elements are present 38 and 366 bp upstream from the major transcription start site, respectively, however, no apparent TATA-like sequence was found in the upstream region of the minor transcription start site, The 400-bp region immediately upstream from the major transcription initiation site was strongly GC-rich (79% GC), Genomic Southern experiments demonstrated that the allelic loss of DAN gene might occur in neuroblastoma, Interestingly, there exist two dinucleotide repeats, (CA)(7) and (CA)(8), in the first intron of DAN gene, raising the possibility to distinguish two alleles of DAN gene in some of the cancer cells.

MARY ANN LIEBERT INC PUBL, Sep. 1997, DNA AND CELL BIOLOGY, 16(9) (9), 1031 - 1039, English

[Refereed]

Scientific journal

Chromosome mapping of the mouse and rat hom*ologs of the human DAN gene, D1S1733E

Y Matsuda, T Ozaki, H Enomoto, T Saito, S Sakiyama

SPRINGER VERLAG, Sep. 1996, MAMMALIAN GENOME, 7(9) (9), 709 - 710, English

[Refereed]

Scientific journal

A REGION OF CONSISTENT DELETION IN NEUROBLASTOMA MAPS WITHIN HUMAN-CHROMOSOME 1P36.2-36.3

PS WHITE, JM MARIS, C BELTINGER, E SULMAN, HN MARSHALL, M FUJIMORI, BA KAUFMAN, JA BIEGEL, C ALLEN, C HILLARD, MB VALENTINE, AT LOOK, H ENOMOTO, S SAKIYAMA, GM BRODEUR

Deletion of the short arm of human chromosome 1 is the most common cytogenetic abnormality observed in neuroblastoma. To characterize the region of consistent deletion, we performed loss of heterozygosity (LOH) studies on 122 neuroblastoma tumor samples with 30 distal chromosome 1p polymorphisms. LOH was detected in 32 of the 122 tumors (26%). A single region of LOH, marked distally by D1Z2 and proximally by D1S228, was detected in all tumors demonstrating loss, Also, cells from a patient with a constitutional deletion of 1p36, and from a neuroblastoma cell line with a small 1p36 deletion, were analyzed by fluorescence in situ hybridization, Cells from both sources had interstitial deletions of 1p36.2-36.3 which overlapped the consensus region of LOH defined by the tumors. Interstitial deletion in the constitutional case was confirmed by allelic loss studies using the panel of polymorphic markers. Four proposed candidate genes-DAN, ID3 (heir-1), CDC2L1 (p58), and TNFR2-were shown to lie outside of the consensus region of allelic loss, as defined by the above deletions, These results more precisely define the location of a neuroblastoma suppressor gene within 1p36.2-36.3, eliminating 33 centimorgans of proximal 1p36 from consideration, Furthermore, a consensus region of loss, which excludes the four leading candidate genes, was found in all tumors with 1p36 LOH.

NATL ACAD SCIENCES, Jun. 1995, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 92(12) (12), 5520 - 5524, English

[Refereed]

Scientific journal

OVEREXPRESSION OF DAN GENE-PRODUCT IN NORMAL RAT FIBROBLASTS CAUSES A RETARDATION OF THE ENTRY INTO THE S-PHASE

T OZAKI, Y NAKAMURA, H ENOMOTO, M HIROSE, S SAKIYAMA

Differential screening-selected gene aberrative in neuroblastoma (DAN) gene (previously named N03 gene), whose expression is significantly reduced in transformed cells, has recently been demonstrated to have a tumor-suppressive activity in vitro. In order to investigate biological roles of DAN gene product in normal rat fibroblasts (3Y1), marker-selected transfectants that expressed the high amount of DAN gene product were generated from 3Y1 cell lines. These clones did not exhibit morphological changes compared with parental 3Y1 cells; however, they showed a decrease in growth rate and a remarkable reduction in saturation density. Cell cycle analysis revealed that the overexpression of DAN gene product causes the retardation of the entry into the S phase. These results suggest that DAN gene product may have an important cole in regulation of the entry of cells into the S phase.

AMER ASSOC CANCER RESEARCH, Feb. 1995, CANCER RESEARCH, 55(4) (4), 895 - 900, English

[Refereed]

Scientific journal

IDENTIFICATION OF HUMAN DAN GENE, MAPPING TO THE PUTATIVE NEUROBLASTOMA TUMOR-SUPPRESSOR LOCUS

H ENOMOTO, T OZAKI, E TAKAHASHI, N NOMURA, S TABATA, H TAKAHASHI, N OHNUMA, M TANABE, J IWAI, H YOSHIDA, T MATSUNAGA, S SAKIYAMA

The expression of DAN gene (previously designated as N03 gene) is significantly reduced in a variety of transformed rat fibroblasts, including v-src- (SR-3Y1), SV40- and v-mos-transformed 3Y1 cells, compared with that in parental 3Y1 cells. Recently, DAN gene has been shown to possess a tumor suppressive activity when it is overexpressed in SR-3Y1 cells (Ozaki & Sakiyama, 1994). To assess the involvement of DAN gene,vith human neoplasms, we have isolated human DAN counterpart from a normal lung cDNA library by using rat DAN cDNA as a probe, and determined its chromosomal location. Human DAN gene mapped to chromosome 1p36.11-p36.13, which is well known to show highly significant linkage with the genesis and/or progression of human neuroblastoma. Southern blot analysis on tumor DNA from 26 patients with neuroblastoma has detected three patients showing genomic rearrangement or deletion within or closely linked to the DAN gene locus. Collectively, we propose that human DAN gene is a possible candidate for a tumor suppressor gene of human neuroblastoma.

STOCKTON PRESS, Oct. 1994, ONCOGENE, 9(10) (10), 2785 - 2791, English

[Refereed]

Scientific journal

COMPLEX-FORMATION BETWEEN LAMIN-A AND THE RETINOBLASTOMA GENE-PRODUCT - IDENTIFICATION OF THE DOMAIN ON LAMIN-A REQUIRED FOR ITS INTERACTION

T OZAKI, M SAIJO, K MURAKAMI, H ENOMOTO, Y TAYA, S SAKIYAMA

The retinoblastoma susceptibility gene product (pRB) has been known to function as a negative regulator of cell growth. Recent observations suggest that its biological activity might be modulated by an interaction with nuclear structures. By using in vitro binding assays, we have found that pRB can associate with lamin A, which has been known to be one of the major nuclear matrix proteins. A series of GST-lamin A deletion mutants was constructed to define the amino acid sequence required for binding to pRB. A GST-lamin A (247-355) contained an activity to associate with pRB, while the other constructs, such as GST-lamin A (37-244) or GST-lamin A (356-571), could not bind to pRB. Within the pRB-binding domain of lamin A, there exists the short amino acid sequence which is also present in the pRB-binding region of the transcription factor E2F-1. The similar experiments using a set of GST-RB deletion mutants revealed that a region containing the E1A-binding pocket B and the carboxy-terminal portion of pRB was responsible for binding to lamin A.

STOCKTON PRESS, Sep. 1994, ONCOGENE, 9(9) (9), 2649 - 2653, English

[Refereed]

Scientific journal

DEVELOPMENT OF CYCLOPHOSPHAMIDE RESISTANCE IN HUMAN NEUROBLASTOMA XENOGRAFTS

Tanabe M., Ohnuma N., Enomoto H., Iwai J., Yoshida H., Takahashi H.

Most patients with neuroblastoma (NB) achieve some response to aggressive chemotherapy. However, some of them have relapse and the tumors subsequently become refractory to drugs. The development of resistance to chemotherapy is a major problem in the treament of NB. We have examined the development of resitance to a high dose of Cyclophosphamide (CPM) in human NB growing as a xenograft (HNX) in nude mice. Three types of HNX (CH-NBI, CH-N-1, CH-NB-4) were treated in consecutive passages with a single dose of 250mg/kg CPM. At each passage the first treated tumor to regrow was transplanted into 10 fresh mice. Of these mice half were treated and half served as controls. This procedure was repeated six times. Growth curves were plotted and the time taken for treated and control groups to double in volume was obtained. Tumor growth delay (TGD) was calculated as the difference between these values. Various changes in TGD produced by repeated CPM treatments were observed in three HNX. TGD dropped from 10.7 to 7.1 days in CH-NB-4 (p < 0.05) at the 2nd transplant and from 9.6 to 7.5 days in CH-N-1 at the 5th transplant and no further decrease was observed by additional treatment in any tumor. TGD was 5.3 days in CH-NB-I at the 1st transplant and no decrease was observed by additional treatment. Treatment with CPM resulted in a rapid reduction in responsiveness to CPM in two HNX but no reduction was observed in one HNX which was considered to have primary resistance to CPM.

The Japanese Society of Pediatric Surgeons, 1994, Journal of the Japanese Society of Pediatric Surgeons, 30(7) (7), 1261 - 1266, Japanese

MOLECULAR-CLONING AND CHARACTERIZATION OF A CDNA SHOWING TUMOR-SUPPRESSIVE ACTIVITY IN V-SRC-TRANSFORMED 3Y1 RAT FIBROBLASTS

S SAKIYAMA, T OZAKI, H ENOMOTO

PERGAMON PRESS LTD, 1994, ADVANCES IN ENZYME REGULATION, VOL 34, 34, 247 - 255, English

[Refereed]

Scientific journal

PARATESTICULAR NEUROBLASTOMA WITH N-MYC ACTIVATION

T MATSUNAGA, H TAKAHASHI, N OHNUMA, M TANABE, H YOSHIDA, H ENOMOTO, H HORIE, H SHIRASAWA, B SIMIZU

W B SAUNDERS CO, Dec. 1993, JOURNAL OF PEDIATRIC SURGERY, 28(12) (12), 1612 - 1614, English

[Refereed]

Scientific journal

■ Lectures, oral presentations, etc.

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