Development and Engineering of Dopamine Neurons (Advances in Experimental Medicine and Biology, Vol. 651)

Development and Engineering of Dopamine Neurons

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY EditorialBoard: NATHAN BACK,State University ofNew York at Buffalo IRUNR. COHEN, The Weismann InstituteofScience ABELLAJTHA, N.S. KlineInstitutefor Psychiatric Research JOHN D. LAMBRIS, University ofPennsylvania RODOLFO PAOLETII, University ofMilan RecentVolumes in this Series Volume 643 TAURINE 7 Editedby JunichiAzuma Volume 644 TROPOMYOSIN Editedby PeterGunning Volume 645 OXYGEN TRANSPORT TO TISSUEXXX Editedby Per Liss, PeterHansell,DuaneF. Bruley, and DavidK. Harrison Volume 646 EARLY NUTRITION PROGRAMMING AND HEALTH OUTCOMES IN LATER LIFE Editedby BertholdKoletzko, Tamas Desci,DenesMolnar, and Anne De la Hunty Volume 647 THERAPEUTIC TARGETS OF THE TNF SUPERFAMILY Editedby IqbalGrewal Volume 648 ARTERIAL ANDALLIED CHEMORECEPTORS Editedby Constancio Gonzalez, ColinA. Nurse,and ChrisPeers Volume 649 MOLECULAR MECHANISMS OF SPONDYLOARATHROPATHIES Editedby CarlosLopez-Larrea, and RobertoDiaz-Peila Volume 650 V(D)JRECOMBINATION Editedby PierreFerrier Volume 651 DEVEOPLMENT AND ENGINEERING OF DOPAMINE NEURONS Editedby R. JeroenPasterkamp, MartenP.Smidt,and 1. Peter-H. Burbach A Continuation Order Plan is availablefor this series.A continuationorder will bring delivery of each new volume immediatelyupon publication.Volumes are billed only upon actual shipment.For further information please contact the publisher.

Development and Engineering of Dopamine Neurons Editedby R. Jeroen Pasterkamp, PhD Rudolf Magnus Institute ofNeuroscience Department ofNeuroscience and Pharmacology University MedicalCenter Utrecht Utrecht, The Netherlands Marten P. Smidt, PhD

Rudolf Magnus Institute ofNeuroscience Department ofNeuroscience and Pharmacology University MedicalCenter Utrecht Utrecht, TheNetherlands J. Peter H. Burbach, PhD

Rudolf Magnus Institute ofNeuroscience Department ofNeuroscience and Pharmacology University MedicalCenter Utrecht Utrecht, TheNetherlands

Springer Science+Business Media, LLC Landes Bioscience

Springer Science+Business Media, LLC Landes Bioscience Copyright <02009 LandesBioscience and Springer Science+Business Media,LLC All rightsreserved. Nopartofthisbookmaybe reproduced ortransmitted inanyformorbyanymeans, electronic ormechanical,including photocopy, recording, or anyinformation storageandretrievalsystem, withoutpermission in writingfromthepublisher, withthe exception of any material supplied specifically for the purposeof beingenteredand executedon a computer system; for exclusive use by the Purchaser of the work. Printedin the USA. SpringerScience+Business Media,LLC,233 SpringStreet,NewYork, NewYork10013, USA http://www.springer.com Pleaseaddress all inquiries to the publishers: LandesBioscience, 1002WestAvenue, Austin,Texas78701,USA Phone: 5121 637 6050;FAX: 5121 637 6079 http://www.landesbioscience.com The chapters in this bookare available in the Madame CurieBioscience Database. http://www.landesbioscience.com/curie Development andEngineering ofDopamineNeurons, editedby R. JeroenPasterkamp, MartenP.Smidt, J. PeterH.Burbach. LandesBioscience I Springer Science+Business Media,LLCdualimprintI Springer series: Advances in Experimental Medicine and Biology

ISBN: 978-1-4419-0321-1 Whilethe authors, editorsandpublisher believethatdrugselection anddosageandthe specifications and usageof equipment and devices, as set forth in this book, are in accordwith currentrecommendations and practiceat the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoingresearch, equipment development, changesin governmental regulations andtherapidaccumulation of information relatingto the biomedical sciences, the readeris urgedto carefully reviewand evaluatethe information provided herein.

Library of Congress Cataloging-in-Publication Data A C.I.P. catalogrecordfor thisbook is available fromthe Libraryof Congress.

PREFACE Theneurotransmitter dopamine hasjustcelebrated its50thbirthday. Thediscovery of dopamine as a neuronal entity in the late 1950sand the notion that it serves in neurotransmission has been a milestone in the field of neuroscience research. This milestone marked the beginning of an era that exploredthe brain as an integrated collectionof neuronalsystemsthat one coulddistinguish on basis of neurotransmitter identities, and importantly, in which one started to be able to pinpoint the seat of brain disease. Themesodiencephalic dopaminergic (mdDA) system, previously designated as midbraindopaminergic system,has receivedmuchattention sinceits discovery. The initialidentification of dopamine as a neurotransmitter in the centralnervoussystem (CNS)and its relevanceto psychiatric and neurological disorders have stimulated a plethoraof neurochemical, pharmacological and geneticstudiesinto the function of dopamine neurons andtheirprojections. In the lastdecade, studies on geneexpression and development have furtherincreasedthe knowledge of this neuronalpopulation and have unmaskeda new levelof complexity. The start of the moleculardissection of the mdDAsystemhas been markedby the cloningand characterization ofNurrl and Pitx3.Thesetranscription factorswere shownto have a criticalfunction during mdDAdevelopment. These initial studies have been followed by the identification of many other proteins,which have a crucialfunction in the creationof a dopamine neuronpermissive region,induction of precursors, induction of terminaldifferentiation and finallymaintenance of the mdDAneuronalpool. In addition, work showing thatthe historically distinguished regions of the substantia nigraparscompacta (SNc) andventraltegmental area(VTA) harbormolecularly distinctsetsof neuronal groups withspecific connectivity patterns hasaddedanew layerof complexity to howmdDA neurons are generatedand function in the adult CNS. The current challenge in the field of dopamine researchis to characterize the full extent of molecularprocesses that underliemdDAneuronprogramming and to translatethese findings into viable approaches for embryonic stem (ES)-cell engineering as an ultimate treatment of degenerative diseasesas Parkinson's disease. The chapterspresentedin this book providean overviewof the different stages that are distinguished duringmelDA neuronaldevelopment. Chapter 1 discusses the v

vi

Preface

dopamine systems of the zebrafish, being a powerful model organism for genetic intervention on the developmental programming of neuronal systems. Chapter 2 presents an overview of dopamine systems, which are present in the vertebrate CNS. Chapters 3-6 discuss the early specification of dopamine precursors and the programs that lead to terminal differentiation. In Chapters 7 and 8 the maintenance of dopamine neurons is discussed with a special emphasis on neurotrophic support. The specificconnectivity of the dopamine systemand the axon guidance rules that apply to developing dopamine neurons are described in Chapter9. An overview of ES-cellengineering of dopamine neurons is presented in Chapters 10 and 11. Theresearch directed towards unraveling themolecular programming of mdDA neuronscontinues to be highlyexciting. Onemay expectthat novelbiological principles will continue to emerge from this population of neurons. In the near future the field as a whole will mature towards a more comprehensive understanding of mdDAneuronal development and networkintegration, and will continue to apply knowledge of dopamine neuron development and function to the treatment of human disease. Ri jeroen Pasterkamp, PhD Marten P.Smidt, PhD J Peter H. Burbach, PhD

ABOUT THE EDITORS...

R. JEROEN PASTERKAMP is an Assistant Professor at the Rudolf Magnus Institute ofNeuroscience, Department ofNeuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, The Netherlands. The focus of his laboratory is directed towards understanding the molecular and intracellular signaling events involved in the formation of neuronal connections with a particular focus on the developing dopamine system. His research team concentrates on the developing mouse embryo using an integrated approach involving molecular biology, cell biology, in vivo functional proteomics, and mouse genetics. He received his PhD from the Netherlands Institute for Neurosciences (Amsterdam, The Netherlands) and did his Postdoctoral at the Department of Neuroscience, Johns Hopkins Univers ity School of Medicine , Baltimore, USA.

vii

ABOUT THE EDITORS...

MARTEN P. SMIDT is an Associate Professor at the Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, The Netherlands. The focus of his laboratory is directed towards understanding the developmental processes that underlie neuronal differentiation and specification. The main focus has been the development of mesodiencephalic dopamine neurons. The work includes mouse genetics, molecular genetics and molecular biology. Marten Smidt received his PhD from the University of Groningen (Groningen, The Netherlands) and did his Postdoctoral at the Department of Medical Pharmacology, Utrecht University, Rudolf Magnus Institute of Neuroscience (Utrecht, The Netherlands) .

viii

ABOUT THE EDITORS...

J. PETERH. BURBACH is a professorof Molecular Neuroscience at Utrecht University and headof the Department of Neuroscience and Pharmacology, Rudolf Magnus InstituteofNeuroscience, University Medical CenterUtrecht, Utrecht, The Netherlands. Hisresearch interests concerntheroleof transcription factors in developmentand regulation ofpeptidergicand dopaminergic neurons, and the molecular mechanisms of human neurodevelopmental disorders. He received his PhD from Utrecht University and did postdoctoral work at the Clinical Research Institute of Montreal, Canada. He obtained professorships in Molecular Biologyand Molecular Neuroendocrinology. Since 200I he is a Summer scientist at the Marine Biological Laboratory, Woods Hole,MA, USA.

ix

PARTICIPANTS Oliver von Bohlen und Halbach Interdisciplinary Center for Neurosciences Neuroanatomy University ofHeidelberg Heidelberg Germany

Dario Acampora CEINGE Biotecnologie Avanzate SEMM European School ofMolecular Medicine and Institute ofGenetics and Biophysics Naples Italy

J. Peter H. Burbach

Kambiz N. Alavian Department ofNeuroanatomy Interdisciplinary Center for Neuroscience University of Heidelberg Heidelberg Germany and Neuroregeneration Laboratories McLean Hospital Harvard Medical School Belmont, Massachusettes USA

John W.Cave Department ofNeurology and Neuroscience Weill Cornell Medical College Burke Medical Research Institute White Plains, New York USA

Siew-Lan Ang NIMR The Ridgeway London

UK

Harriet Baker Department ofNeurology and Neuroscience Weill Cornell Medical College Burke Medical Research Institute White Plains, New York USA

Rudolf Magnus Institute ofNeuroscience Department ofNeuroscience and Pharmacology University Medical Center Utrecht Utrecht The Netherlands

Wolfgang Driever Developmental Biology Institute Biology 1 University of Freiburg Freiburg Germany Luca Giovanni Di Giovannantonio CEINGE Biotecnologie Avanzate SEMM European School ofMolecular Medicine Naples Italy

xi

Participants

xii

Ole Isacson NeuroregenerationLaboratories Center for Neuroregeneration Research Harvard MedicalSchool Mclean Hospital Belmont,Massachusetts USA

AsheetaA. Prasad Rudolf MagnusInstitute of Neuroscience Department ofNeuroscience and Pharmacology UniversityMedicalCenter Utrecht Utrecht The Netherlands

Kerstin Krieglstein Institute for Anatomyand Cell Biology Department of MolecularEmbryology UniversityofFreiburg Freiburg Germany

Jan Pruszak NeuroregenerationLaboratories Center for Neuroregeneration Research Harvard MedicalSchool McLeanHospital Belmont,Massachusetts USA

SonjaKriks Department of Neurosurgery DevelopmentalBiologyProgram Sloan-Kettering Institute for Cancer Research New York, New York USA Pietro Mancuso CEINGE Biotecnologie Avanzate SEMM EuropeanSchoolof Molecular Medicine Naples Italy Daniela Omodei CEINGE Biotecnologie Avanzate SEMM EuropeanSchoolof Molecular Medicine Naples Italy R. jeroen Pasterkamp Rudolf MagnusInstitute of Neuroscience Department of Neuroscience and Pharmacology UniversityMedicalCenter Utrecht Utrecht The Netherlands

Eduardo Puelles Instituto de Neurociencias de Alicante CSIC and UniversidadMiguelHernandez SantJoan di\lacant Spain EleniRoussa Institute for Anatomyand Cell Biology Department of MolecularEmbryology Universityof Freiburg Freiburg Germany jorn Schweitzer

DevelopmentalBiology Institute Biology 1 UniversityofFreiburg Freiburg Germany

Antonio Simeone CEINGE Biotecnologie Avanzate SEMM EuropeanSchoolof Molecular Medicine and Institute of Genetics and Biophysics Naples Italy

xiii

Participants Horst H. Simon Department of Neuroanatomy Interdisciplinary Center for Neuroscience

(IZN)

University ofHeidelberg Heidelberg Germany Marten P. Smidt RudolfMagnus Institute ofNeuroscience Department ofNeuroscience and Pharmacology University Medical Center Utrecht Utrecht The Netherlands

Lorenz Studer Department ofNeurosurgery Developmental Biology Program Sloan-Kettering Institute for Cancer Research New York, New York USA Klaus Unsicker Interdisciplinary Center for Neurosciences Neuroanatomy University ofHeidelberg Heidelberg Germany

CONTENTS 1. DEVELOPMENT OF THE DOPAMINE SYSTEMS IN ZEBRAFISH ••••• 1 Jorn Schweitzer and Wolfgang Driever Abstract•.•.•..••••....••••••••••••••••.•.••••••••••.•••••••••.•••••••••.•••.•••••••.•.••••••.•.••••••••••.•••....•.•.•••••••••••...••••• 1 Introduction•••••••••••••••••••••••.•••••••••••.•...•••••••••••••••••••••••••••••.•••••••••.•••••••••••••••..••••...•.•••••••••••••••• 1 Overview of Dopaminergic Development in Zebrafish 2 Establishment of Dopaminergic Neuronal Connectivity 4 Genetic Approaches •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••..•.••••••.••••••••.•••••••• 6 Signaling Requirements for DA Differentiation••••••••••..•.••••....•.•.••••••••••.•..•.••••••••••......•.•.•• 6 9 Transcriptional Specification of Zebrafislt DA Neurons Integration of Pharmacology and Behavioral Analysis 11 Conclusions ••••••••••••••••••••••.•.•••••••••••••••••••••••••••••••••••••••••.•••.••••••.•.•••••.•.•.••.••••..•••••••••••....••••••• 11

2. DOPAMINE SYSTEMS IN THE FOREBRAIN

15

John W. Cave and HarrietBaker Abstract••••.•.............•••....•...•..•••....••••••••••.•••••••••.••••••••••.••••.••.••••••••••••••••••••••••••••••••••••••••••••••• 15 Introduction•••.••...•.•.•....•.......................•...........•.•••..•.....•.•....•....••••.•....••............•.•....••...•••.• 15 Anatomy and Function ofOB DANeurons •..••....••..••••.••.•.••..•.•......•.•••....••.....•••••••••....•... 16 08 DA Neurogenesis ....•...•......•••••••••••••••••••.•••.•.•..•..•.•••.••.•••.••••••....•....•••..•.•••..•.............••.•• 19 21 Molecular Genetic Mechanisms of08 DA Neuron Differentiation Expression and Function of Forebrain DA Receptors•••••••...•••..•...............•..••..••...........• 28 Prospective Directions for OB DA Neurobiology 28

3. THE ROLE OF OTX GENES IN PROGENITOR DOMAINS OF VENTRAL MIDBRAIN•••••.........................••...............................•... 36 AntonioSimeone, EduardoPuelles,DarioAcampora, DanielaOmodei, Pietro Mancusoand Luca Giovanni Di Giovannantonio Abstract••••••..•••••••••••.•.•••••••••••.......•.•..•••••••••••••••••••••...••...............•.•••..•••••••••••••••••..........•••.••• 36 Introduction••...•••••••..•.••••....•••.••••••.•••••••••••••••••••••••••..••••..•••••••••••••••••••••..••.••..•.•••••••..••••••••••• 36 Otx Genes in the Positioning of the Midbrain-IDndbrain Boundary (MHB) 37

xv

xvi

Contents

Ott-Dose Dependent Control of Anterior-Posterior (A-P) and Dorso-Ventral (D-V) Patterning of the Midbrain•••••••••••••••••••••••••••••••••••••••••••• 39 Ott2 Regulates Extent, Identity and Fate of Progenitor Domains in the Ventral Midbrain••••••••••••••••••••••••••••••.•••••••••••••••••••••••••••••••••••.•••••••••••••••••••.•••••• 41

4. TERMINAL DIFFERENTIATION OF MESODIENCEPHALIC DOPAMINERGIC NEURONS: THE ROLE OF NURRI AND PITX3 •••.•...........••••••••••••••••••••••••••.•••••••• 47 MartenP. Smidtand J. Peter H. Burbach Introduction•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 47 Terminal Differentiation of Substantia Nigra Neurons Depends on the Homeobox Gene Pitx3 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••.•• 47 Nurrl Is Essential for Generating the Full Dopaminergic Phenotype of MesodiencephaHc Dopaminergic Neurons 52 Concluding Remarks ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 54

5. FOXAI AND FOXA2 TRANSCRIPTION FACTORS REGULATE DIFFERENTIATION OF MIDBRAIN DOPAMINERGIC NEURONS •..•..•.•...•••.....•••••.•••••••••.••.....•••••.•.•......••.••••.••..•••••••....•..•.••..••••• 58 Siew-Lan Ang Abstract•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••.••••••••••••••••••••••••••••••••••••••••••• 58 Introduction•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 58 Expression of Foxa1/2 Proteins in the eNS ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••..•••••.• 59 Cross-Regulatory Roles of Foxa2 and Shh and Early Functions of Foxa2 in Dorsal-Ventral Patterning of the CNS ••••••••••••••••••••••••••••••••••••••••••••••.•••••••••••••••••• 59 A Role for Foxa1/2 in Neuronal Specification of the Midbrain Floor Plate .•••••••••••...•• 60 Foxa1/2 Are also Required for the Generation of Immature and Mature mDA Neurons ••••••••••••••••••••••••••.••••••••••••.•••••••..••••••••••.••••••••••••••••••••••••••••.•.•.•••••••••..••••• 60 Mechanims of Foxa Gene Regulation: Examples from Endodermal Organs •••••.•.••.••• 61 Concluding Remarks ••••••••••••••.••••••••••••••••••••••••••.••••...••••••.•••...•••.•.•••.••.•••••••••••.••••••.••••••..••• 63

6. TRANSCRIPTIONAL REGULATION OF THEIR SURVIVAL: THE ENGRAILED HOMEOBOX GENES

66

Horst H. Simonand Kambiz N. Alavian The Engralled Genes••••••••••.••..••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 66 Molecular Structure and Properties of the Engralled Proteins 66 The Engrailed Genes and Mesencephalic Dopaminergic (mesDA) Neurons (Early).•.67 The Engrailed Genes and Mesencephalic Dopaminergic Neurons (Later).•.•••••••••••••••• 68

Contents

xvii

7. NEUROTROPHIC SUPPORT OF MIDBRAIN OOPAMINERGIC NEURONS .••.••.....•..••..•.••••.••••.•• ~ ••••••.•••.•••....•.....•••••.....••...•....••................ 73 Oliver von Bohlen und Halbach and Klaus Unsicker Abstract•.•••••.....•••.•••.•••.......••••••••.•....•••.•••.•.•...••••••••...•••••••••••••.••••••.•...•••••.•••••.•••.••.•••..•........ 73 Introduction•••••••••••......•.•..•..•......•.•••••••••••••••••.•••.•.••.•••..••..••.••..••......•.••••.•.••..•••..•.••••••.••.•.••• 73 Neurotrophins•••••.••....••..•••••••.••••••••••••••.•••••••••..••.•••••••••••••••.••.••••••••••.•••••••••••••••••.......•.•.••..•• 74 Fibroblast Growth Factors (FGFs) •••••••••••••••••••••••••.••••...••.••.••..••••....•••••••••.••.•.•••••...•.....•• 75 Other Factors•••.••.•.•.........•.............•.•••••.•..•.•.••..•..........••....•.•.......•.....•.•..............••••••••...•.•.• 77 Future Directions .•...•••.......•.•...•.......•....••............•..•...•••..•••••••.•...•.•...••.•.•••••..•..•....•..........•. 77

8. TGF-f3 IN DOPAMINE NEURON DEVELOPMENT, MAINTENANCE AND NEUROPROTECTION.....••.••.••••......•..•..............•.......•.....•••.••..... 81 Eleni Roussa, Oliver von Bohlen und Halbach and Kerstin Krieglstein Abstract•••••.•..•••••••.•••••..............•.....••....•.•.•.••..••.••••••....•...••.....•...•.•.............•...•••.•..•.••••.•.••.•• 81 Introduction......•.•..••..•.•..•.••...•••....•.••••••••...••.•••......••...•................••.....•••.•.••••••••••...•..•......... 81 Evidence for TGF-~ Effects on the Induction of Dopaminergic Neurons in Vitro 82 Evidence for TGF-~ Effects on the Induction of Dopaminergic Neurons in Vivo •••.•.• 83 TGF-~ Superfamily Members and Induction of Dopaminergic Neurons •..••.•.••••••••.... 84 TGF-~ Promotes Survival of DAergic Neurons 85 GDNF Promotes Survival ofDAergic Neurons 85 TGF-~ and GDNF Cooperate to Promote Survival and Protection of DAergic Neurons••.••••••••••..•.•..••....••••••••••••••......................•.........•.....••.••...•..••....••.•• 86 Conserved Dopamine Neurotrophic Factor (CDNF) 86 Concluding Remarks ..•••.......•••..••.••••••••.•.•••••...•.......•...••........•...................•.•••..••.••••.•....•..• 86

9. AXON GUIDANCE IN THE DOPAMINE SYSTEM

91

AsheetaA. Prasad and R. Jeroen Pasterkamp Abstract..................................••.••..••............••.........•.........••........•••..••••.•.........•.•.•••.•.•.•...•••.. 91 Introduction•....................................................................................................................... 91 Mesencephalon ....................••.......•...............................................•.....................................92 Diencephalon ............••........................•••............................................................•..•••..•........ 93 Medial Forebrain Bundle ...................•..•............•.•..........................................................•. 93 Striatum ....................................••.................•................•.....................................................96 Cortex..•.•......•.........................................•..........................••...•...........•.........................•...... 97 Axon Guidance Molecules and Disease 97 Conclusions and Future Directions ......•.•..........•..............................................................98

xviii

Contents

10. PROTOCOLS FOR GENERATING ES-CELL-DERIVED DOPAMINE NEURONS ••••••••••••••••••••••••••••••••••••••••••••••••.•••••••••••••••••••.• 101 SonjaKriks and LorenzStuder Introduction•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 101 Neural Development •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 101 Derivation of Midbrain DA Neurons from Embryonic Stem Cells (ESCs) •••••••••••••••• 102 Remaining Key Challenges ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 106 New Developments ••••••••••••••••••••••••••••••••••••.•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••.•••••• 106 Human ESC Neural Intermediates 106 Cell Purification and Genetic Reporter Lines 107 The Use of Genetically Matched DA Neurons for Cell Therapy and Disease Modeling •••••••.•••••••••••••...••••••••••••.••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 107

11. MOLECULAR AND CELLULAR DETERMINANTS FOR GENERATING ES-CELL-DERIVED DOPAMINE NEURONS FOR CELL THERAPy•••.•..•••••••••.•••••••..••••••.••••.•••••••••••••..112 Jan Pruszakand Ole Isacson Abstract••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••.•••••••••••••••••••••••••••• 112 Introduction•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 112 Background and History ••••••••••••.••..•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••.•••••• 112 Principles of Engineering Dopamine Neurons in Vitro 114 Monitoring DA Differentiation in Vitro ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 117 ES-Cell Culture Conditions for DA Differentiation•••••••••••••••••••••••••••••••••••••••••••••••••••••• 117 Using Gene-Engineering to Specify DA Neurons in Vitro •••••••••••••••••••••.••••••••••.•.•.....••• 118 Selection of DA Neurons from ES-Cell Cultures••••••••••••••••••••.•.••••••••.•.••••••••••.•••.•••.•••••• 119 Future Perspective ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••.•••••••••••••••••••.•••••• 120

INDEX.•.••.....•...•......••••••••....•.••.•...•••.......•.••.•...•......•.•.......••••......•..•....••.•..••..•..••• 125

CHAPTERl

Development of the Dopamine Systems in Zebrafish Jorn Schweitzer and Wolfgang Driever*

Abstract

D

opaminergic neurons develop in several distinct regions of the vertebrate brain and project locally or send long axonal projections to distant parts ofthe eNS to modulate the activity ofa variety ofcircuits, controlling aspects ofphysiology, behavior and movement. The molecular control of dopaminergic differentiation and the evolution of the various dopaminergic systems are not well understood, as research has mostly focused on ascending mammalian dopaminergic systems of the substantia nigra and ventral tegmental area. Zebrafish have evolved as an excellent genetic and experimental embryological model to study specification and axonal projectivity of dopaminergic neurons. The large evolutionary distance between fish and mammals provides the opportunity to identify conserved core regulatory mechanisms that control differentiation and projection behavior ofthe various dopaminergic groups in vertebrates. Here, we present an overview of the formation of dopaminergic groups and their projections in zebrafish. We will further review the results from genetic analyses, which have revealed insights on signals as well as transcription factors contributing to dopaminergic differentiation. Together with recently established paradigms for behavioral analysis, dopaminergic systems are studied at all levels in zebrafish, from molecular and cellular to systems and behavioral.

Introduction

Biomedical research into molecular mechanisms ofParkinson's Disease and other diseasesofthe dopaminergic systems relies to a large degree on analysis ofdifferentiation and circuit formation of the dopaminergic system in animal models. Several features have made the zebrafish (Danio rerio) an excellent model organism to study neural development ofvertebrates. Zebrafish embryos develop rapidly and a functional larval nervous system is established within four days ofdevelopment.P As eggs are laid and fertilized externally, the embryos are well accessible to experimental manipulation at allstages. The short generation time ofthree months has enabled genetic analysis and mutagenesis screens. To facilitate molecular level analysis, the sequencing of the zebrafish genome is nearly complete (http://www.sanger.ac.uk/modelorgs/zebrafish.shtml) and a centralized database ofresources (www.zfin.org) has been established, providing structured information on genetics, mutations, gene expression and anatomy. The small embryos and larvae facilitate the combination of genetic analysis, cell biological manipulations and pharmacological interference in a single embryo. Further, the transparent embryos make possible visualization ofthe results on fixed tissue using whole embryo immunohistochemistry and in vivo using transgenic marking of cells with fluorescent proteins.' Taken together, these features make zebrafish an attractive model *Corresponding Author: Wolfgang Driever-Developmental Biology, Institute Biology 1, Faculty of Biology, University of Freiburg, Hauptstrasse 1, 79104 Freiburg, Germany. Email: [email protected]

Developmentand Engineering ofDopamine Neurons, edited by R.]. Pasterkamp, M.P. Smidt and J.P.H. Burbach. ©2009 Landes Bioscience and Springer Science+ Business Media.

Development and Engineering ofDopamineNeurons

2

system to studydopaminergic system development, However,astheevolutionary distance between zebrafish andhumanisabout350 millionyears, differences in neuroanatomy andcircuitformation needs to be carefully considered.

Overview ofDopaminergic Development in Zebrafish

The formation of dopaminergic groups has been studied by immunohistochemistry for Tyrosine hydroxylase (TH)~ aswellasbyanalysis of expression oftyrosine hydroxy!4se (th) in all catecholaminergic (CA) neurons,of dopamine transporter (dat) characteristic for dopaminergic (DA) neuronsand dopamine beta hydroxylase (dbh) for noradrenergic (NA) neurons?,s Absence ofDBH expression fromfore-and midbrainand coexpression with TH in the hindbrain revealed that NA neuronsare restrictedto the hindbrain and peripheralnervoussystem in zebrafish.

I

A

sc

rhombencephalon neuropil

noradrenerglc

I

---~

dopaminergic

diencephalon

PT H

Figure 1. Dopaminergic and noradrenergic cells groups of the 4 day old zebrafish larvae. A) Schematic drawing of 4 day old zebrafish brain with major brain subdivisions and locations of catecholaminergic groups highlighted. Anterior at left, dorsal at top. B,C) Detection of tyrosine hydroxylase expression in catecholaminergic neurons of 4 day old zebrafish larva. th mRNA was visualized using whole mount in situ hybridization. Images represent z-projections of two pictures each at different focal planes. B: Lateral view anterior at left, dorsal at top. The circumference of the head is emphasized by a black line. C: Dorsal view of same embryo as in A, magnified area of posterior tuberculum and hypothalamus, anterior at left. Abbreviations: Numbers 1-7 indicate DA subgroups of ventral diencephalon according to the numbering of (6); AAC : arch associated NA cells (carotid body); AP: area postrema; CE: cerebellum; H: hypothalamus; LC: locus coeruleus; MO: medulla oblongata ; OB: olfactory bulb; PA: pallium; po: preoptic area; PR: pretectum; PT: posterior tuberculum; TC: tectum; SC: spinal cord; SympG: sympathetic ganglia.

DevelopmentoftheDopamineSystems in Zebrafish

3

When comparingthe anatomicallocationsofzebrafish and mammalianDA groups,the exact anatomical correlates are not always clear, especially during embryogenesis, when morphology is not fullydeveloped. Therefore, instead of the AI-AI7 numberingestablishedfor mammalian DA groups,the anatomicalstructuresare usedfor zebrafish DA nornenclarure.v"Further,where distinct cellmorphologiesofDA neuronswithin an anatomicalregioncan be distinguished,DA cellclusters of the ventraldiencephalonhavebeen assigned numbers," Forcomparisonto mammalian DA systems, the forebrainprosomeremodelismore informative, because the geneexpression domains of zebrafish genesorthologues to those mammalianones that support this model have been esrablishedv":" (Fig. 1 and Table 1). The first dopaminergicneurons differentiatealreadyat about only 18 hours post fertilization (hpf) in the prospective posterior tuberculum (basal plate area of prosomere 3). Successively, additional groups of DA neurons are specified to build the full complement of earlylarval DA groups by four dayspost fertilization (dpf). The free swimmingzebrafish larvaehas formed the following DA groups (Fig. 1 and Table 1). i. Within the ventral diencephalon,several DA groups can be distinguished: the ventral portion ofthe posteriortuberculumcontainstwogroupsofDA cells with alargesomaand high levels ofDA expression, one closeto the alar-basal plate (group 2) boundary and one further basal(group4). Betweenthesegroups,a clusterof smallDA cellssituated closeto the ventriclecan be found (group 3). An additional group of smallDA cellsdevelops at the alar-basal horder and extendsfrom the posteriortuberculuminto the ventralthalamus (group 1). This group expands significantly in cell number during larval development.

Table 1. Comparison of catecho/aminergic groupsin zebrafish and mammals CA Mammals

lebrafish

NA Caudal rhombencephalic groups (Al-A3/Cl-C3)

Medulla oblongata area postrema

Rostral rhombencephalic groups (A4-A7) DA Mes-diencephalic groups (A8-Al0)

- (**)

Hindbrain

Locus coeruleus

Midbrain

- (*) Pretectum (ZF 8)

Pla

Forebrain

Diencephalic groups (All,A13) Ventral thalamus (ZF 0, 1) and P3ab posterior tuberculum (ZF 2, 3, 4, 6) Hypothalamic (A12, A14, retromammillary, mammillary and lateral hypothalamic)

Hypothalamic (ZF 5, 7, 10, 11 and posterior part of 6)

P4b-P6b?

Preoptic group (A15)

Preoptic group

P6b P6a

- (***)

Pallium-subpallium border

Olfactory bulb group (A16)

Olfactory bulb cluster

Retinal group (A17)

Retinal amacrine DA cells

Retina

Notes: Data are compiled from. 6 - 8,11,12,62,63 The zebrafish numbering system (ZF) follows". The column with the prosomere (P)assignment is tentatively where indicated by a question mark, a indicates alar and b basal portions. (*)No mesencephalic DA (ascending DA projections may derive from basal diencephalon posterior tuberculum/periventricular nucleus. (**)Small transient population in some mammalian embryos. (***)Cortical th expressing dispersed cells have been detected in primates, but unclear if DA transmission.

4

Development and EngineeringofDopamineNeurons

Another group developsat the ventral baseof the posterior tuberculum and extendsinto the hypothalamus(group 6). Within the hypothalamus,two DA groupscan be detected by 5 dpf(groups 5 and 7). ii. A cluster of DA cells that expands significantly during late larval development can be detected in the pretectum (group 8). iii. Groups ofDA cellsform in the preoptic and paraventricular region. iv. The olfactorybulb group. v. A group in the subpalliumat the pallial-subpallial border. Dopaminergic neurons are further present in the retina (dopaminergicamacrinecells).Based on the absenceof dat expression from areasthat express th in the hindbrain, it can be surmised that DA neurons do not developin the hindbrain. In contrast, noradrenergicneurons developin the locus coeruleusand in the medullaoblongata areapostremain the hindbrain. The description of the embryonic and larval DA systems is based on expression analysis of tyrosine hydroxylase-however, there are two th genesin zebrafish, tbl, which has been studied in detail and is the basisfor the current classification of zebrafish DA groups and th2, for which the expression has not been reported so farB and thus will be only brieflydiscussed here. These duplicate th geneslikelyoriginatefrom a genomeduplication at the baseof teleostevolution.The th2 gene is expressed late in zebrafish development starting at larval stagesand appearsto reveal additional DA populations in the preoptic,posterior tubercular and caudalhypothalamicregions (Mahler,Schweitzer, Filippi and Driever,unpublished). DA neuronsdo not developin the zebrafish mesencephalon. Thisestablishes a majordifference between fishand mammalsand has been attributed to a caudal-ward shift of dopaminergicactivity during evolutionfrom fishto mammals." Retrogradelabelingexperimentsrevealedascending projectionsfrom the posterior tuberculuminto the palliumand subpalliumin zebrafish,15,16 which led the authors to suggest that these DA groups maycontribute to ascendingregulatory circuits similar to those of the mammalian substantia nigra DA neurons. Work in white sturgeon also supports that ascendingprojections into the subpallium may be correlates of the nigrostriatal systemin mammals." In contrast to mammals, neurogenesis in teleostsextendsto all brain areasduringgrowth of the brain at adult stages. 18-20 UsingBrdU labelingexperimentsin adult zebrafish, it wasdemonstrated that new TH expressing neurons are added during adult stagesto all major DA regions: olfactory bulb,preoptic region,pretectum,posterior tuberculum and hypothalamus." Thusthe DA systems ofthe fishhavea significantcapacityfor growth and regenerationand makethe zebrafish an ideal model to study regenerationof specific DA subtypesfrom neural stem cellpopulations.

Establishment ofDopaminergic Neuronal Connectivity

Thedevelopmentof dopaminergictract formation in the zebrafish hasbeenanalyzedusingTH immunohistochemistryin developing"and adult zebrafish.4,5,15The overallpattern ofTH positive tracts and commissure in larvalfish correspondswell to those describedfor the adult fish.Here, wewillfocuson the formation of the majordopaminergictractsof 5 dpfzebrafish larvae." At this developmentalstage,zebrafish larvaeare free swimming,hunting and fully interactingwith their environment-however stillsmallenough to makeanti-TH wholemount immunohistochemistry possibleand thus enablinga precisedescription of dopaminergiccircuitry (Fig.2). The first TH positivefibersappear shortly afterthe firstDA neurons can be detected between 18 and 20 hpf in the prospective posterior tuberculum. Already by 24 hpf long TH positive descendingprocesses, which passedthe areaof the locuscoeruleusand project into the hindbrain and spinalcord can be detected. In 5 dayold zebrafish larvaethe following TH positivefibersand tracts can be distinguished. In the telencephalon DA neurons located in the olfactorybulb displayunipolar TH positive processes, which seemto project only locallywithin the olfactorylobe.The DA neurons residing between the pallium and subpallium also display unipolar processes projecting dorsally where

Development oftheDopamine Systems in Zebrajish

5

Figure 2. Visualization of catecholaminergic connectivity in the 3 day old zebrafish larvae. A z-projection of a confocal image stack of dorsal views is shown, anterior to left. Immunohistochemistry for TH reveals the distribution of catecholaminergic neurons and their respective axons in a 3 dpf old zebrafish. White arrows indicate some tracts with catecholaminergic contributions. Abbreviations: AC: anterior commissure; DF: decussating fibers; LB: lateral bundle; MLB: medial longitudinal bundle; SCP: spinal cord project ion.

they then contribute to other TH positive fibers within the lateral margin of the telencephalon and to the anterior commissure. In the diencephalon, TH positive fibers emerging from DA neurons located anterior within the preoptic DA group project dorsally,where they subsequently join the above-mentioned lateral TH positive fibers and also to the anterior commissure.The more caudal DA neurons ofthis group also project dorsally but then join other TH positive fibers within the postoptic commissure. The region at the lateral extremities and dorsal to the postoptic commissure seems to be an area, where TH positive fibers of different origin run together, which hampers to discern their point of origin. From that area, four tracts ofTH positive fibers could be followed. (1) Longitudinal bundles in the lateral margins ofthe rostral diencephalon, which terminate in the telencephalon. (2) A ventral tract to the posterior tuberculum and hypothalamus. (3) Dorsally located fibers, which project via the mesencephalon to the rhombencephalon and (4) a contralateral tract within the postoptic commissure. From the prerectal DA neurons TH positive processes can be followed that project ventrolaterally and ramify in the dorsal thalamus. Here they project either commissurally just caudal of the epiphysis or grow to the optic tectum to establish a dense network of rectal TH positive fibers. From the ventral diencephalic groups, projections contributing to the longitudinal bundle ofTH fibers passing via the mesencephalon to the rhombencephalon and even further into the spinal cord can be detected. Due to the heterogeneity ofDA neuronal groups within the ventral diencephalon, it is presently difficult to allocate defined projections to single groups using TH immunohistochemistry. In the larval zebrafish there are also TH positive fibers running in locations similar to those described for the preopticohypophyseal tract (PHT) as well as endohypothalamic tract (EHT).s

6

Development antiEngineering ofDopamineNeurons

Although zebrafish do not possess midbrain dopaminergicneurons,retrogradelabelingstudiesin the adult zebrafish revealed that DA neuronslocated in the ventraldiencephalonsend ascending projections to the telencephalon, into a region (subpallium) proposed to be homologousto the mammalianstriatum.IS TheseDA groupsmaycontribute to ascendingregulatorycircuitssimilar to those to which mammalianDA neurons of the SN and VTA contribute. In the rhombencephalon an extensive network ofTH reactivitycan be observed. Along the ventrolateralmedullaabundleof fibers positivefor TH courselongitudinallyand asecondbundle evenmorelaterally. In addition therearedecussating fibers, whichappearin asegmentalfashion. In the spinalcord TH positivefibers growingin aventrolateralposition and projectingto the caudalmost bodysegments can bedetected.It has to be consideredthat noradrenergic neuronslocatedin the locuscoeruleusand the medullaoblongataarealsopositivefor TH and haveascendingaswell as descendingprojections,22,23 which extend into the diencephalon and telencephalon, or spinal cord, respectively. Hence noradrenergicneurons mayalso contribute to someof the projections describedabovefor the DA neurons.

Genetic Approaches

Zebrafishpresentan idealsystem forso-called "forward" geneticmutagenesis screens to identify additional geneticcomponents contributing to specification and differentiationofDA neurons. The short generationtime and high fecundity allowlargescalegeneticscreens where thousands ofmutagenizedgenomescan be analyzedfor new mutations affecting DA systemdevelopment. Suchscreens havebeen performed usingeither Tyrosine hydroxylase immunohiseochemlstry" or th m-RNA in situ hybridization? However, the findingthat the majorcomplementofDA axonal trajectoriesare alreadypresent at 4-5 dpf at a time when the zebrafish is amenable to geneticmanipulations, makesit alsoan idealmodel systemto study the formation ofDA axonaltracts. Toevaluate the outcomeof suchgeneticscreens, wehavecompiledinformationon allmutations isolatedduring geneticscreens specifically designedto identifycatecholaminergic defectsand for whichthe affectedgeneshavealreadybeen identified(Table2).While this isonlya subsetof previouslyisolatedmutations,the comparisonreveals that sofar mainlyfour classes of geneshavebeen identifiedgivingriseto catecholaminergic defects: (i) Transcriptionfactorsspecifically involved in CA specification and differentiation, including Oep. Fezf2, Tfap2a, Foxil and Phox2a; (ii) Signalingpathways and transcriptionfactorsinvolved in patterning of the brain regionsin which DA neuronsdifferentiate, includingSmoothenedand PouSfl; (iii)Components of the transcription machinerywhichmayhavea differential requirementduringspecific aspectsofdevelopment, but arenot selectivelyinvolved in CA specification, includingSptS,Med 12 and Med27; (iv)Gene products which are provided maternally, such that earlydevelopmentand formation of the early DA groupsisrescuedin zygotic mutants bymaternalgeneproduct. mcmS isan example, in which late forming DA neurons are absent because proliferation of neural progenitor cellsduring late embryogenesis is impeded. In summary, genetic screens in zebrafish appear to efficiently identify novel factors contributing to DA and NA developmentand the implicationsfor mechanisms of neural subtype specification will be discussed below. It will be interestingto seewhether zebrafish genetics may be alsoproductiveto elucidatemechanisms of axonalpathfindingin the CA systems. In addition to genetic screens, it is possibleto interfere with gene function in zebrafish using translational knock-downor splice-inhibiting Morpholino antisense oligonucleotides. Thistechnique has also been extensively appliedin zebrafish to test potential functions of previously characterized genes in CA development.

Signaling Requirements for DA Differentiation

The availability of many mutations affectingcomponents of signaling pathways involved in brain patterning and celldifferentiation madeit possible to address signalingrequirementsfor DA neuronal specification in zebrafish (Table 3). In mammalianembryos,manymutations affecting major signalingpathways are lethal prior to DA system development. In contrast, most mutant

DevelopmentoftheDopamineSystems in Zebrafish

7

Table 2. Mutations isolatedduringgenetic screensbased on defects in catecho/aminergic development Affected Gene

Ref

Genetic Locus

Alleles

Phenotype

spielohne grenzen (spg)

m793

Locus coeruleus NA neurons absent pou5fl

64,26

foggy (fog)

mB06

Reduced numbers of DA and NA neurons

spt5 (Transcr. EF)

65

med27

mBB5

Late forming DA neurons

med27 (MED-C) 66

motionless (mot)

mB07

Reduced numbers of DA and NA neurons

medl2 (MED-C) 67

too few (tfu)

mBOB

Reduced numbers of DA neurons in fezl/fezf2 (ZF-TF) hypothalamus

38 68

no soul (nos)

mB09

Arch associated NA neurons absent foxil (FB-TF)

soulless (sou)

mBIO, mBII, mBl2

Locus coeruleus and arch associated NA neurons absent

phox2a (HD-TF) 69

otpa

mB66

DA groups 2,4,5 and 6 of ventral diencephalon reduced or absent

orthopedia a (HD-TF)

27,36, 37

smoothened (smo)

mB41

Reduced number of hypothalamic DA neurons

smoothened

26,70

mont blanc (mob)

mBl9

Locus coeruleus and medulla NA neurons absent

tfap2a (TF)

7

mcm5

mB50

Late forming DA groups

mcm5(CC)

71

Abbreviations: HD-TF: Homeodomain transcription factor; ZF- TF: Zinc finger transcription factor; MED- C: Subunit of transcriptional mediator complex; Transcr. EF: Transcription elongation factor; TF: Transcription factor; CC: Cell cycle/replication.

zebrafish embryos survive for at least two days, because a functional cardiovascular system is not required for early development and stores ofmaternally derived proteins enable cells to survive. Experiments in mammalian systems indicated a requirement of Shh and FGF8 signaling for dopaminergic neuron development." Analysis oface mutant zebrafish, which are devoid ofFGF8~ revealed that FGF8 contributes to specification ofLocus coeruleus NA neurons.f In contrast, all DA groups form, albeit some with developmental delay, e.g., the ventral thalamic DA neurons. Surprisingly, a negative effect ofFGF8 by suppressing Otp expression in the preoptic area has been observed." In zebrafish, for the Shh pathway, bothsyu (Shh) andsmu (Shh coreceptor Smoothened) mutant embryos have been analyzed.f In Shh signaling deficient embryos, the early ventral diencephalic and the olfactory bulb DA groups form, but prerectal and retinal amacrine DA cells are reduced or absent. Thus the floorplate-derived Shh signal may not contribute to specification of ventral diencephalic DA neurons, but Shh derived from the zona limitans intrathalamica at the border ofprosomers 2 and 3 may be involved in specification ofprecursors ofDA neurons in the pretectum. A prominent role, like the one proposed for Shh in mammalian midbrain DA neurons, thus does not appear to exist for zebrafish DA neurons that may form an ascending DA system. Nodal signals ofthe TGFbeta family play an important role in development ofthe ventral diencephalon." However, a complete depletion ofNodal signals in eye mutants (affecting Nodal related protein 2),or in oep mutant embryos (affecting the Oep Nodal coreceptor) also deletes a significant portion ofthe ventral diencephalon." This makes it difficult to distinguish whether the absence of ventral diencephalic DA neurons in these mutants is caused by early patterning defects or defects

Development and EngineeringofDopamineNeurons

8

Table 3. Impact of signaling pathways on specification and differentiation of zebrafish catecho/aminergic neurons Catecholaminergic Groups

Signal Affected

Nonaffected

Not Determined*

PT(-O), vDC(-), AC(-)

OB, LC,MO

PO, S~ HY

FGF8 (ace)

LC(O)

OB, PT, vDC, AC, MO

PO,HY

NodaIlTGF(MZsur)

vDC(-O)

LC,MO,AC

PT, OB, PO, SP, HY

Nodal/TGF(oep orcyc)

PT(O), vDC(O), HY(O), AC(cyc:O), OB(O)

LC, MO

PO, SP

Retinoic acid (>24hpf)

MO(O)

PT, OB, vDC, PO, S~ LC

HY,AC

Shh (smu, syu)

Modified from reference 7. Abbreviations for DA groups: PT: pretectal DA neurons; OB: olfactory bulb DA neurons; vDCa: anterior DA group ventral diencephalon (group 1 according to Rink and Wullimann); vDCp: posterior DA groups in the ventral diencephalon (groups 2-6 according to Rink and Wullimann); po: preoptic group; SP: DA group of subpallium; HY: hypothalamic DA groups; AC: amacrine DA cells of retina; LC: locus coeruleus NA neurons; MO: medulla oblongata area postrema NA neurons; (-): reduced number of cells; (0): absent; (*can not be determined because embryos develop global defects).

in specification of DA neurons. The analysis of the zebrafish sur mutation, which affects Fast 1/ FoxH I, a transcription factor and transducer ofNodal signals, was more informative. In MZsur mutant embryos, which lack both maternal and zygotic expression ofFastl/FoxH I, DA neurons are often completely absent from the ventral diencephalon. Analysis ofthe expression pattern of dbxla> a genes expressed in the basal plate ofprosomere 3, indicated that the posterior tuberculum still forms in MZsur embryos. This argues for the role ofNodal/TGFbeta famUysignals in specification ofthe ventral DA groups. Whether this involves direct induction ofventral diencephalic DA cells, or regulation ofthe prepattern ofthis region has been addressed experimentally. When mesendoderm development is experimentally rescued in MZoep mutant embryos, the formation ofdiencephalic th expressing neurons is also rescued," revealing that Nodal signals do not appear to act directlyon DA precursors, but that Nodal may be required for generation of a secondary signal in the mesendoderm to pattern the ventral diencephalon and induce DA development. Thus, some of the signals of the prechordal mesendoderm, which include Wnt8b, the Wnt antagonist Dkk, Shh, Twhh, Bmp4, Bmp2 and Adrnp,may be involved in ventral diencephalic DA specification. It has been proposed that Nodal may act through Shh to activate Otp, a diencephalic DA transcription factor, in the preoptic area, but not the posterior tuberculum." The retinoic acid (RA) synthesizing enzyme Ahd2/Aldh IaI is coexpressed with mammalian mesencephalic DA neurons." RA has been suggested to be required for proper establishment of mesencephalic DA Identity," Further, in the hindbrain RA has been directly involved in neuronal differentiation aswell as in patterning. DA development is not affected in zebrafish raldh2 mutants, while medullary NA cells are strongly reduced. When zebrafish embryos older than 24 hpf are exposed to retinoic acid, segment identity, as judged by establishment of the Hox gene code, is already complete and a shift in segment identities can not be induced. However, exogenous retinoic acid induces an expansion ofthe medullary NA group often well into rhombomere 3.7 These findings argue that within a dorsoventral domain competent to form NA neurons, the rostro-caudal retinoic acid gradient determines the anterior limit ofNA differentiation. Other eNS DA or NA

DevelopmentoftheDopamineSystems in Zebraftsh

9

groups are not affected by RA treatment under these conditions and thus RA does not appear to playa role in zebrafish DA development. Several other signaling systems play important roles in mammalian DA neural differentiation (reviewed in 32) However, for some, including the Wnt signaling pathway, their manipulation in the whole zebrafish embryo causes severe early anterioposterior patterning defects in the neural plate, which made it difficult to analyze their specific contributions to DA differentiation.

Transcriptional Speci6cation ofZebra6sh DA Neurons

The mammalian ascending mesencephalic DA systems have been extensively studied for transcription factors controlling early specification and late differentiation,32 including Pitx3, Lmxla, Lmxlb, Enl/2 and Nurrl/Nr4a2. In contrast, relatively little is known about diencephalic DA transcriptional control mechanisms.P'" which include Dlxl/2, Nkx2.1 and Msxl/2. Due to the ancient genome duplication, zebrafish have two paralogous genes each for several ofthe mammalian DA differentiation genes (often denoted as "a"and "b" paralogous genes).The nr4a2paralogue nr4a2ais co-expressed with TH in preoptic, pretectal and retinal amacrine dopaminergic neurons, while nr4a2bis only expressed in preoptic and retinal dopaminergic neurons." Both zebrafish nr4a2 paralogues are not expressed in ventral diencephalic dopaminergic neurons with ascending projections. Combined morpholino antisense oligo mediated knock-down ofboth nr4a2aand nr4a2b transcripts reveals that all zebrafish dopaminergic neurons expressing nr4a2a depend on Nr4a2 activity for th and datexpression.Thus Nr4a2 protein has a conserved evolutionary role in specification of the DA neurotransmitter phenotype, albeit it appears to be only one of several regulatory modules of dopaminergic differentiation, as most ventral diencephalic dopaminergic neurons do not express nr4a2 genes in zebrafish. Zebrafish lmxlb.l is expressed in noradrenergic neurons of the locus coeruleus and medulla oblongata, but knock-down revealsthat it is specificallyrequired for tyrosine hydroxylase expression only in the medulla oblongata area postrema noradrenergic neurons.35 The lmxl b.l and 1mxl b.2 genes as well as pitx3 are not expressed in dopaminergic neurons, but in a diencephalic territory that, based on sox2 expression, might contain precursor cellsfor ventral diencephalic dopaminergic neurons. Upon morpholino knock-down of both lmxlb paralogues, the number of neurons in diencephalic dopaminergic clusters which may send ascending projections appears specifically reduced. Thus lmxl b paralogues may contribute to generation ofdiencephalic dopaminergic precursors. Conversely, knock-down ofpitx3 does not specifically affect any diencephalic DA cluster. One may speculate that a di-mesencephalic longitudinal domain of lmxl b expression may be the basis for the expansion and posterior shift ofventral di-/ mesencephalic dopaminergic populations with ascending projections during evolution. Genetic analysis has provided information on the role in D A differentiation of transcription factors previously not connected to DA development in mammals. Molecular analysis of a zebrafish mutation affecting development of posterior tubercular and hypothalamic DA groups 2,4,5 and 6 revealed that the affected gene was ortbopedia a (otpa), which in wild-type embryos is coexpressed with TH in all neural groups affected in the mutant. 27,36,37 Combined mutant otpa and knockdown ofparalogous otpbcompletely eliminates all DA neurons ofgroups 2, 4, 5 and 6. Interestingly, Otp:': mouse embryos lack diencephalic dopaminergic neurons of the A 11 group, which constitute the diencephalospinal dopaminergic system. In both zebrafish and mouse, Otp is expressed in the affected dopaminergic neurons as well as in potential precursor populations and may contribute to dopaminergic cell specification and differentiation. In fish, overexpression ofOtp can induce ectopic th and dat expression, indicating that Otp can specify aspects ofdopaminergic identity. Thus Otp is one ofthe few known transcription factors that can determine aspects ofthe dopaminergic phenotype and the first known transcription factor to control the development of the diencephalospinal dopaminergic system. Genetic and experimental analysis has revealed that otp expression in zebrafish appears to be controlled by at least two parallel pathways, with a cell autonomous component represented by the transcription factor Fezl/Fezfl (forebrain embryo zinc finger-like protein) and a noncell-autonomous component, the G-protein coupled receptor

Adj

Co-expr.

Co-expr,

Adj

DC-6

Co-expr,

Adj

Adj

MO

Co-expr.

Adj

Adj

Adj

LC

~

~

;,.

I

~

b

1!

C!

~ lO

~.

f

~

~

;:I.

~:.I'

~

Co-expr,

Co-expr.

Adj

Adj

Adj

Adj

DC-5

t'l'1

Co-expr,

Co-expr.

Adj

Adj

Adj

Adj

DC·4

in adjacent cells but no co-expression. Abbreviations of the different CA groups: ACL: amacrine cell layer; DC-l/6: diencephalic groups from 1 to 6; LC: locus coeruleus; MO: medulla oblongata; OB: olfactory bulb; po: preoptic area; Pr: pretectum; SP: subpallium. Data compiled from. 35•36

Co-expr.

otpb

Adj

DC-3

'co-expr,' indicates overlapping expression in the considered area, '-' indicates no overlap, 'adj' indicates TH immunoreactivity and gene expression

Co-expr.

otpa

Adj

Adj

DC-2

Adj

Adj

Adj

DC-1

Imxlb.2

Co-expr,

Co-expr,

ACL

Adj

Co-expr.

Co-expr,

PO

lmxtb.t

pitxl

nr4a2b

Pr

Co-expr,

SP

nr4a2a

OB

Table 4. Overview of selected transcription factor expression pattern relative to each zebrafish catecholaminergic cluster at 96 hpf stage

..... <::>

Development oftheDopamineSystems in Zebrajish

11

PAC 1.37 Mutations in fezllfezfl have been characterized to affect the number of neurons in DA groups 2 to 7, while other DA groups are normal. 24,38,39 Thus, the DA phenotype offtzl/fezfl is consistent with it acting epistatic to otp. However, analysis ofmouse Fezl mutants indicates that it acts in a more global way during early forebrain patterning by repressing caudal diencephalic fate in the rostral diencephalon and thus mediates subdivision ofthe diencephalon and facilitates zona limitans organizer formation." Little is known so far on how DA neuronal differentiation is specifically controlled in zebrafish, but it has been shown that neurogeninl is expressed in diencephalic DA precursors before th expression is activated and that ngnl is required for proper neurogenesis and differentiation of forebrain DA neurons." Overexpression of ngnl induces formation of supernumerary DA neurons," which is consistent with a role of ngnl in lateral inhibition/Delta-Notch signaling during DA neurogenesis.

Integration ofPharmacology and Behavioral Analysis

The small size of the zebrafish and its ready uptake of many small molecules dissolved in its swimming water have led to it being utilized to analyze effects ofpharmacological agents as well as toxins on DA formation and survival, as well as fish behavior. Zebrafish react to administration of6-hydroxydopamine or MPTP by behavioral and neurochemical changes and under some conditions also with enhanced neural degeneration.P'" The generation of transgenic vmat2: GFP zebrafish with GFP marked dopaminergic neurons makes it even possible to assesseffects of drugs like MPTP on DA cell physiology and survival in living larvae.' Further, pharmacological experimental paradigms have been established to assay the effect ofdrugs on behaviors involving DA contriburions.V?" In parallel, dopamine receptor genes are being characterized. s1-s3There is rapid progress in the development ofrobust behavioral experimental paradigms,54-57 including some relevant to DA systems function, like prepulse inhibition PPI.58Recent developments in optical visualization of neural activity59,60 as well as optical manipulation of neural activity" will enable systems level understanding ofthe development ofdopaminergic systems in zebrafish.

Conclusions

Genetic analysis in zebrafish has just started to reveal the complexity ofmechanisms involved in DA subtype specification for the various DA groups developing in the brain. Similar to other systems studied in the hindbrain, the findings are consistent with local patterning of the dorsoventral and anterioposterior axis ofthe eNS generating a "prepattern~ which in combination with different local signals may serve to specify neural cells to take on a dopaminergic fate. As such, the regulatory input which controls DA differentiation may be convergent rather than following one or two instructive signals only. This is consistent with the idea that there are multiple regulatory modules, like the one including Nurr1, which may drive DA differentiation and which may have been used by evolution in a dynamic fashion to form the different DA subtypes. The rapid genetics and other experimental possibilities available in zebrafish will help to further shape our understanding of dopaminergic differentiation. A careful comparison with mammalian systems will reveal which aspects of DA differentiation are conserved among vertebrates. Since circuits from basal ganglia into striatum or subpallium are essential for movement control and circuits with similar function (albeit in different neuroanatomicallocations) exist from fish to mammals, one would expect that a significant portion of their molecular determinants may also be conserved. While this has not been substantiated so far for fish and mammalian ascending systems, the analysis of Otp function reveals conserved regulatory modules from fish to mammals for the descending diencephalospinal DA system.

Acknowledgements

We thankJulia Mahler for providing parts ofFigure 1 and Soojin Ryu for parts ofFigure 2. We are grateful to Alida Filippi andJochen Holzschuh for comments on the manuscript. The authors

12

Development and EngineeringojDopamineNeurons

work was supported byDFG grants SFB 780-B6 and EUZF-MODEL5 (WD) and DFG grant

5CHW 1404/1-1 (J5).

Referencess

1. Kimmel CB, Ballard ~ Kimmel SR et al. Stages of embryonic development of the zebrafish. Dev Dyn 1995; 203(3):253-310. 2. Kuwada]Y. Development of the zebrafish nervous system: Genetic analysis and manipulation. Curr Opin Neurobiol1995; 5(1):50-4. 3. Wen L, Wei ~ Gu W et al. Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev Bioi 2008; 314(1):84-92. 4. Kaslin J, Panula P. Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio). J Comp Neuro12001; 440(4):342-77. 5. Ma PM. Catecholaminergic systems in the zebrafish. IV. Organization and projection pattern of dopaminergic neurons in the diencephalon. J Comp Neurol 2003; 460(1):13-37. 6. Rink E, Wullimann ME Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study. Brain Res Dev Brain Res 2002; 137(1):89-100. 7. Holzschuh J, Barrallo-Gimeno A, Ettl AK et al. Noradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid and require tfap2a for expression of the neurotransmitter phenotype. Development 2003; 130(23):5741-54. 8. Holzschuh J, Ryu S, Aberger F et al. Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo. Mech Dev 2001; 101(1-2):237-43. 9. Wullimann MF, Rupp B, Reichert H. Neuroanatomy of the zebrafish brain. Birkhaeuser Switzerland: Verlag, Basel, 1996. 10. Mueller T, Wullimann ME Atlas of Early Zebrafish Brain Development. Amsterdam: Elsevier B~ 2005. 11. Puelles L, Verney C. Early neuromeric distribution of tyrosine-hydroxylase-immunoreactive neurons in human embryos. J Comp Neuro11998; 394(3):283-308. 12. Ryu S, Holzschuh J, Mahler J et ale Genetic analysis of dopaminergic system development in zebrafish. J Neural Transm 2006; (70}:61-6. 13. Candy J, Collet C. Two tyrosine hydroxylase genes in teleosts. Biochim Biophys Acta 2005; 1727(1):35-44. 14. Smeets W], Marin 0, Gonzalez A. Evolution of the basal ganglia: new perspectives through a comparative approach. J Anat 2000; 196(Pt 4):501-17. 15. Rink E, Wullimann MF. The teleosrean (zebrafish) dopaminergic systemascendingto the subpalliurn (striatum) is located in the basal diencephalon (posterior tuberculum). Brain Res 2001; 889(1-2):316-30. 16. Rink E, Wullimann ME Connections of the ventral telencephalon and tyrosine hydroxylase distribution in the zebrafish brain (Danio rerio) lead to Identification of an ascending dopaminergic system in a teleost. Brain Res Bull 2002; 57(3-4):385-7. 17. Pinuela C, Northcutt RG. Immunohistochemical organization of the forebrain in the white sturgeon, Acipenser transmontanus. Brain Behav Evo12007; 69(4):229-53. 18. Adolf B, Chapouton P, Lam CS et ale Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon. Dev BioI 2006; 295( 1):278-93. 19. Chapouton P, Adolf B, Leucht C et al. her5 expression reveals a pool of neural stem cells in the adult zebrafish midbrain. Development 2006; 133(21):4293-303. 20. Grandel H, Kaslin J, Ganz J et al. Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate. Dev Bioi 2006; 295(1):263-77. 21. McLean DL, Fetcho JR. Ontogeny and innervation patterns of dopaminergic, noradrenergic and serotonergic neurons in larval zebrafish.J Comp NeuroI2004; 480(1):38-56. 22. Ma PM. Catecholaminergic systems in the zebrafish, II. Projection pathways and pattern of termination of the locus coeruleus. J Comp Neuro11994; 344(2):256-69. 23. Ma PM. Catecholaminergic systems in the zebrafish. III. Organization and projection pattern of medullary dopaminergic and noradrenergic neurons. J Comp Neuro11997; 381(4):411-27. 24. Guo S, Wilson S~ Cooke S et al. Mutations in the zebrafish unmask shared regulatory pathways controlling the development of catecholaminergic neurons. Dev Bioi 1999; 208(2):473-87. 25. Ye ~ Shimamura K, Rubenstein JLR et al. FGF and Shh Signals Control Dopaminergic and Serotonergic Cell Fate in the Anterior Neural Plate. Cell 1998; 93:755-66. 26. Holzschuh J, Hauptmann G, Driever W. Genetic analysis of the roles of Hh, FGF8 and nodal signaling during catecholaminergic system development in the zebrafish brain. J Neurosci 2003; 23(13):5507-19.

Development ofthe Dopamine Systems in Zebrajish

13

27. Del Giacco L, Sordino P, Pistocchi A et al. Differential regulation of the zebrafish orthopedia 1 gene during fate determination of diencephalic neurons. BMC Dev Bioi 2006; 6:50. 28. Rohr KB, Barth KA, Varga ZM et al. The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity. Neuron 2001; 29(2):341-51. 29. Mathieu J, Barth A, Rosa FM et al. Distinct and cooperative roles for Nodal and Hedgehog signals during hypothalamic development. Development 2002; 129(13):3055-65. 30. McCaffery P, Drager UC. High levelsof a retinoic acid-generatingdehydrogenasein the meso-telencephalic dopamine system. Proc Nat! Acad Sci USA 1994; 91(16):7772-6. 31. Jacobs FM, Smits SM, Noorlander CW et al. Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency. Development 2007; 134(14):2673-84. 32. Smidt MP, Burbach JP. How to make a mesodiencephalic dopaminergic neuron. Nat Rev 2007; 8(1):21-32. 33. Andrews GL, Yun K, Rubenstein JL et al. Dlx transcription factors regulate differentiation of dopaminergic neurons of the ventral thalamus. Mol Cell Neurosci 2003; 23(1):107-20. 34. Ohyama K, Ellis P, Kimura S et ale Directed differentiation of neural cells to hypothalamic dopaminergic neurons. Development 2005; 132(23):5185-97. 35. Filippi A, Durr K, Ryu S et al. Expression and function of nr4a2, lmxlb and pitx3 in zebrafish dopaminergic and noradrenergic neuronal development. BMC Dev BioI 2007; 7:135. 36. Ryu S, Mahler J, Acampora D et al. Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development. Curr Bioi 2007; 17(10):873-80. 37. Blechman J, Borodovsky N, Eisenberg M et al. Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia. Development 2007; 134(24):4417-26. 38. Levkowitz G, Zeller J, Sirotkin HI et al. Zinc finger protein too few controls the development of monoaminergic neurons. Nature Neurosci 2003; 6(1):28-33. 39. Rink E, Guo S. The too few mutant selectively affects subgroups of monoaminergic neurons in the zebrafish forebrain. Neuroscience 2004; 127(1):147-54. 40. Hirata T, Nakazawa M, Muraoka 0 er al. Zinc-6nger genes Fez and Fez-like function in the establishment of diencephalon subdivisions. Development 2006; 133(20):3993-4004. 41. Jeong JY, Einhorn Z, Mercurio S et al. Neurogenin 1 is a determinant of zebrafish basal forebrain dopaminergic neurons and is regulated by the conserved zinc finger protein Tof/Fezl. Proc Nat! Acad Sci USA 2006; 103(13):5143-8. 42. Anichtchik O~ Kaslin J, Peitsaro N et ale Neurochemical and behavioural changes in zebrafish Danio rerio after systemic administration of 6-hydroxydopamine and I-methyl-4-phenyl-l,2,3,6-tetrahydropyridine. J Neurochem 2004; 88(2):443-53. 43. Bretaud S, Lee S, Guo S. Sensitivity of zebra6sh to environmental toxins implicated in Parkinson's disease. Neurotoxicol TeratoI2004; 26(6):857-64. 44. Lam CS, Korzh ~ Strahle U. Zebra6sh embryos are susceptible to the dopaminergic neurotoxin MPTP. Eur J Neurosci 2005; 21(6):1758-62. 45. McKinley ET, Baranowski TC, Blavo DO et al. Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons. Brain Res Mol Brain Res 2005; 141(2):128-37. 46. Pamg C, Roy NM, Ton C et al. Neurotoxicity assessment using zebrafish.J Pharmacol Toxicol Methods. 2007; 55(1):103-12. 47. Bretaud S, Li Q Lockwood BL et al. A choice behavior for morphine reveals experience-dependent drug preference and underlying neural substrates in developing larval zebrafish. Neuroscience 2007; 146(3):1109-16. 48. Cahill GM. Circadian melatonin rhythms in cultured zebrafish pineals are not affected by catecholamine receptor agonists. Gen Comp Endocrinol1997; 105(2):270-5. 49. Darland T, DowlingJE. Behavioral screening for cocaine sensitivity in mutagenized zebrafish. Proc Nat! Acad Sci USA 2001; 98(20):11691-6. 50. Lau B, Bretaud S, Huang Y et al. Dissociation of food and opiate preference by a genetic mutation in zebrafish. Genes Brain Behav 2006; 5(7) :497-505. 51. Boehmler ~ Carr T, Thisse C et al. D4 Dopamine receptor genes of zebra6sh and effects of the antipsychotic clozapine on larval swimming behaviour. Genes Brain Behav 2007; 6(2):155-66. 52. Boehmler ~ Obrecht-Pflumio S, Canfield V et al. Evolution and expression ofD2 and D3 dopamine receptor genes in zebrafish. Dev Dyn 2004; 230(3) :481-93. 53. Li P, Shah S, Huang L et al. Cloning and spatial and temporal expression of the zebrafish dopamine Dl receptor. Dev Dyn 2007; 236(5):1339-46. 54. Burgess HA, Granato M. Modulation of locomotor activity in larval zebrafish during light adaptation. J Exp BioI 2007; 210(Pt 14):2526-39. 55. Bally-Cuif L. Teleosts: simple organisms? Complex behavior. Zebrafish 2006; 3(2):127-30.

Development and EngineeringofDopamineNeurons

14

56. Ninkovic J, Bally-Cuif L. The zebrafish as a model system for assessing the reinforcing properties of drugs of abuse. Methods San Diego: Calif, 2006; 39(3):262-74. 57. Ninkovic J, Folchert A, Makhankov YV et ale Genetic identification of AChE as a positive modulator of addiction to the psychostimulant D-amphetamine in zebrafish. J NeurobioI2006; 66(5):463-75. 58. Burgess HA, Granato M. Sensorimotor gating in larval zebrafish, J Neurosci 2007; 27(18):4984-94. 59. Friedrich RW: Habermann CJ, Laurent G. Multiplexing using synchrony in the zebrafish olfactory bulb. Nature Neurosci 2004; 7(8):862-71. 60. Meyer MP, Smith SJ. Evidence from in vivo imaging that synaprogenesis guides the growth and branching of axonal arbors by two distinct mechanisms. J Neurosci 2006; 26(13):3604-14. 61. Zhang F, Wang LP, Brauner M et at Multimodal fast optical interrogation of neural circuitry. Nature 2007; 446(7136):633-9. 62. Arenzana FJ, Arevalo R, Sanchez-Gonzalez R et al. Tyrosine hydroxylase immunoreactivity in the developing visual pathway of the zebrafish. Anat Embryol 2006; 211(4):323-34. 63. Bjorklund A, Dunnett SB. Dopamine neuron systems in the brain: an update. Trends Neurosci 2007; 30(5):194-202. 64. Belting H-G, Hauptmann G, Meyer D et al spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer. Development 2001; 128(21):4165-76. 65. Guo S, Yamaguchi ~ Schilbach S et al. A regulator of transcriptional elongation controls vertebrate neuronal development. Nature 2000; 408(6810):366-9. 66. Durr K, Holzschuh J, Filippi A et al. Differential roles of transcriptional mediator complex subunits Crsp34/Med27, Crsp150/Med14 and Trapl00/Med24 during zebrafish retinal development. Genetics 2006; 174(2):693-705. 67. Wang X, Yang N, Uno E et al. A subunit of the mediator complex regulates vertebrate neuronal development. Proc Natl Acad Sci USA 2006; 103(46):17284-9. 68. Lee SA, Shen EL, Fiser A er al. The zebrafish forkhead transcription factor Foxil specifies epibranchial placode-derived sensory neurons. Development 2003; 130(12):2669-79. 69. Guo S, Brush J, Teraoka H et al. Development of noradrenergic neurons in the zebrafish hindbrain requires BMP, FGF8 and the homeodomain protein soulless/Phox2a. Neuron 1999; 24(3):555-66. Burgess S, Hopkins N. Analysis of the zebrafish smoothened mutant reveals conserved and 70. Chen divergent functions of hedgehog activity. Development 2001; 128:2385-96. 71. Ryu S, Holzschuh J, Erhardt S et ale Depletion of minichromosome maintenance protein 5 in the zebrafish retina causes cell-cycle defect and apoptosis. Proc Natl Acad Sci USA 2005; 102(51):18467-72.

w:

CHAPTER 2

Dopamine Systems in the Forebrain John ~ Cave and Harriet Baker"

Abstract

T

he brain contains a number ofdistinct regions that share expression ofdopamine (DA) and its requisite biosynthetic machinery, but otherwise encompass a diverse array offeatures and functions. Across the vertebrate family, the olfactory bulb (0B) contains the major DA system in the forebrain. OB DA cells are primarily periglomerular interneurons that define the glomerular structures in which they receive innervation from olfactory receptor neurons as well as mitral and tufted cells, the primary OB output neurons. The OB DA cells are necessary for both discrimination and the dynamic range over which odorant sensory information can be detected. In the embryo, OB DA neurons are derived from the ventricular area ofthe evaginating telencephalon, the dorsal lateral ganglionic eminence and the septum. However, most OB DA interneurons are generated postnatally and continue to be produced throughout adult life from neural stem cells in the subventricular zone of the lateral ventricle and rostral migratory stream. Adult born 0 B DA neurons are capable ofintegrating into existing circuits and do not appear to degenerate in Parkinson's disease.Severalgenes have been identified that regulate the differentiation ofOB DA interneurons from neural stem cells. These include transcription factors that modify the expression of tyrosine hydroxylase, the first enzyme in the DA biosynthetic pathway and a reliable marker of the DA phenotype. Elucidation of the molecular genetic pathways ofOB DA differentiation may advance the development ofstrategies to treat neurological disease.

Introduction

The dopaminergic (DA) neuronal systems of the brain exhibit substantial diversity. All DA neurons express the requisite enzymes for dopamine biosynthesis, but there are regional differences in the morphology and co-expression ofother neuroactive substances, as well as the capacity for regeneration and the susceptibility to neurodegenerative diseases. For example, substantia nigra DA neurons co-express glutamate and CCK and have long projections into the striatum that are essential for control of movement. These midbrain DA neurons also selectively degenerate in Parkinson's disease (PD).l By contrast, olfactory bulb (OB) DA neurons co-express GABA and have short axonal projections that remain within the main 0 B that are necessary for processing of odorant sensory information from olfactory receptor neurons.r" Furthermore, the 0 B DA neurons are continuously generated through out the lifespan of the adult"? and do not degenerate in PD.81he molecular and genetic mechanisms responsible for the common DA phenotype (that is, the production ofdopamine) as well as the wide variety of associated features remain an area ofintensive study. This chapter will focus primarily on the anatomical, molecular genetic and physiological characteristics ofthe 0 B DA neurons. These neurons are the major endogenous DA-producing system in the forebrain."? The OB DA neurons are a sub-group ofa diverse population ofinterneurons in the *Corresponding Author: Harriet Baker-Department of Neurology and Neuroscience, Weill Cornell Medical College, Burke Medical Research Institute, 785 Mamaroneck Ave, White Plains, New York 10606, USA. Email: [email protected]

Development and Engineering ofDopamine Neurons, edited by R.]. Pasterkamp, M.P. Smidt and J.P.H. Burbach. ©2009 Landes Bioscience and Springer Science+ Business Media.

Development and Engineering ofDopamineNeurons

16

o Bthat havebeenintensivdystudiedin an effon to understandthe mechanisms regulatingneurogenesis and the generation ofneuronaldiversity,u-13 TheOB DAneurons areanintegralcomponent ofcircuitrythat serves asapowerfulmodd for neuralnetworklearning,memoryconsolidation and behavioral plasticity.14-16 Much of the informationpresentedin thischapterisderivedfrom studies with the rodent OB (specifically, the mouseand rat),but a growingnumberof studieshaverevealed that the data derivedfrom rodent studiesextendinto primates, includinghumans," Anatomy and Function ofOB DA Neurons

In somevertebratespecies, includingmonkeysand humans,DA-producingcells are found in forebrain regionssuch as the striamm.18.19 However, the main OB contains the major forebrain DA systemcommon to all verrebraees," Thus, this chapter will focus primarilyon the OB DA neurons. Approximately 5% of neurons in the main OB are DA intemeurons, They show a distinct laminardistributionthat islimitedprimarily to the glomerularlayer. 21 Most OB DA cells aresmall, periglomerular(PG) interneurons(about 5-10 urn in diameter),although somearelargerexternal tufied cells (about 10-15 urnin diameter;Fig. 1).2.20.22 Several studiesindicatethat 10-16% of allPG neurons are DA cells,23-25 Glomeruliare distinctivespheroidalneuropilstructures (50-150urn in diameterin rodents) that aredefinedbya layerofPG and glial cdIs.20 Thesestructuresserveasthe initial processingcenterofsensoryinformationfrom the olfactoryreceptorneurons.Theneuropil within the glomeruliis composedof the axon terminalsfrom the olfactoryreceptor neurons,the apicaldendrites from mitral and tufted (MIT) projection neurons,dendritic processes from OB juxtaglomerularneurons (including the DA cells) and terminalsfrom centrifugalinnervation of both basalforebrain cholinergicneurons and dorsal raphe cellserotoninergic projections.26-28 Within the glomeruli, OB DA interneuronsreceive region-specific axo-dendrieic innervation from the axonterminalsof the olfactoryreceptornervefibers and make dendro-dendriticcontacts with the apical dendrites of OB MIT-cells (Fig. 2).29.30 Thesesynapticconnections are distinct from other groupsofPG interneurons.Forexample, both calretinin-and calbindin-expressinginterneurons,whichdo not co-express DA, onlymakedendro-dendriticconractswiththe MIT-cells within the 0 B glomeruli.23.3 1,321his heterogeneityin synapticorganizationwithin the glomeruli

A

Open

9

Figure 1. Laminardistribution ofTH-immunoreactivedopamine neurons in a horizontal section of olfactory bulbs taken from an adult mouse with unilateral naris closure. A) A low magnification image shows the normal distribution of PG DA neurons in the glomerular layer of the OB contralateral to naris closure (open). The OB ipsilateral to the closure (closed) displays a drastic reduction in the number of TH-immunoreactive cells and processes. B) A higher magnification micrograph illustrates the processes (arrows) of the PG DA neurons entering the glomeruli. Bar = 200 11m in A and 20 11m in B. Abbreviations: epl, external plexiform layer; gl, glomerular layer; gr, granule cell layer; m, mitral cell layer; on, olfactory nerve layer.

Dopamine Syst~ms in the Forebrain

17

Granule Cell Mitral Cell External Plexiform Tufted Cell Glomerular Olfactory Nerve Cribriform Plate Receptor cells Cilia

Nasal cavity

Figure 2. Schematic representation of selected synaptic connections in the olfactory dopaminergic system. Axons from olfactory receptor neurons (small hatched circles) make axo-dendritic synapses w ith apical dendrites of the mitral cells (large hatched cells), tufted cells (medium pear-shaped cell) and processes of PG cells, including the DA neurons (white) . Axons from glutamatergic mitral/tufted cells are the primary output neurons of the OB through the lateral olfactory tract (LOT). DA interneurons are stimulated by both olfactory receptor neurons and mitral/tufted neurons. Mitral/tufted neurons also make dendro-dendritic synapseswith granule cell interneurons. Both DA PG and granule cells express GABA (dark grey). Question mark (7) indicates a population of granule cells in the mitral cell layer (dark grey and white cell) that express GABA and TH mRNA, but not TH protein.

suggests that OB DA interneuronshavea function in the processing of olfactorysensoryinformation distinct from the other sub-groupsofOB interneurons. Across all vertebratespecies, 0 BDA neuronsarereadily identifiedbythe expression of tyrosine hydroxylase (TH), the firstenzymein the DA biosyntheticpathway.33.34The OB doesnot contain noradrenergic(NE)-producing neurons, but centrifugalNE afferents from the locus ceruleusto the OB alsoexpress TH.z.3S However, TH expression in the NE terminationsisverylowand docs not complicateanalysis ofOB DA neuronal function.' In contrast to TH,expression of aromaticamino aciddecarboxylase (AADC), the secondand lastenzymein the DA biosyntheticpathway, exhibitscross species variation. Forexample, AADC isreadilyobservable in the rat 0 B, but it isdetectableat only lowlevels in the mouse0 B.36 Other markers of functional DA cells, such as dopamine transporter (DAT) and both D, and D z DA receptors, areexpressed at either low or variable levels. 37-4O Thus, TH expression is consideredthe most reliable markerofOB DA neurons. TH expression in OB PG interneuronsisdependenton afferent synaptic activity in theolfactory receptorneurons.z,z1,22,41 BothTH mRNAandproteinexpression aredramaticallydown-regulated in the 0 Bbyperturbationsthat compromise eitherodorant access to the olfactoryepitheliumor cyclic nucleotide-gated channelfunctionin theolfactory receptorcells (Fig. 1).22.4Z Studies in whichanimals weresubjected to odordeprivation byeithernarisocclusion, chemical or surgical deafferentation have shownthat theloss ofTH isconcomitantwithaloss ofdetectable DN.43aswellasadramaticincrease in Dz receptorexpression.f Asdiscussed below, the activity dependence ofTH expression and DA production islikely critical for both odorant identification and detectionof odorant intensity.

Development andEngineering ofDopamine Neurons

18

Almost all OB DA lnterneurons also co-express GABA.23,31,45 GABA, the major inhibitory neurotransmitter in brain, is found in about 55% ofthe interneuronsin the glomerularlayerand almost all inremeurons in the granule celllayer.23.25Jl OB GABAergic interneurons are typically sub-dividedbythe co-expression ofother neuroactive substances suchasDA,calbindin,calretinin and CCK.31.46 ActivationofDA receptorsarereported to modulate the response of GABA receptors within the samecell," Theseresultssuggest that the corelease ofDA with GABAmaymodify the responseof both the olfactory receptor neurons and Mff-cells to the inhibitory effects of GABA. OB DA interneurons are a necessary dement in the processing of afferentsensoryinformation from the olfactory epithelium (Fig. 3). Within the glomeruli, the axons of glutamatergic olfactoryreceptorneuronsprovideexcitatoryinput to both MIT and PG neurons,includingDA cdls,4S-50 Glutamatereleased from MIT-cellsisalsoexcitatoryon DA neuronsand other PG cells," StimulationofPG GABAergic interneurons resultsin the release of GABA which inhibits both olfactoryreceptorand MIT neurons52-55 aswellasother PG neurons.56.57 0 BDA intemeurons also release dopamine thatactspresynapticallyon D 2receptors to modulatethe release ofglutamate from

PG dendrite

\

OA~

r---------'_G---,ABA-02 0 GABA-A GABA-B <j1

~

1!

Inhibitory

Figure 3. Schematic representation of selected neurotransmitters and their cognate receptors in OB glomeruli. Olfactory receptor axon terminals release glutamate that excite mitral/tufted and PG cells through AMPA, NMDA and mGluRl receptors. PG DA neurons release both DA and GABA that inhibit both olfactory receptor and mitral/tufted neurons through D2 and GABA-B receptors. GABA can also inhibit PG neurons through GABA -A receptors.

DopamineSystems in theForebrain

19

olfactory receptor neurons.51,58.59 Although somewhat controversial, several studies have reported that MIT neurons are also presynaptically inhibited by DA through D 2 receptorsf'()-62 Together, GABA and DA modify the output of sensory information from the OB by directly modulating the excitation ofboth the olfactory receptor and MIT neurons. The activity dependent expression ofTH suggests that DA is essential for the regulation of odorant information processing in response to either high or low levels ofafferent odor-induced synaptic activity. When odorant access to the OE is prevented by naris occlusion, the MIT-cell responses to odor stimulation show enhanced sensitivity.3.4 The finding that expression ofthe isoforms ofthe GABA biosynthetic enzyme, glutamic acid decarboxylase, are not activity dependent suggests that this enhanced MIT-cell sensitivity is likely the result of diminished DA-mediated inhibition. 63.64 Furthermore, on restoration ofsensory input following prolonged odorant sensory deprivation, MIT neurons show impaired discrimination of individual odorants." Thus, the OB DA system is critical for both discrimination and the dynamic range over which odorant sensory information can be relayed from olfactory receptor neurons to other brain regions.

OB DA Neurogenesis

Embryonically (E)-derived DB interneurons are predominantly generated from progenitor cells located in the subventricular zone (SVZ) of the dorsal lateral ganglionic eminence (dLGE) beginning at about EI4 in the mouse (Fig. 4).65 The dLGE is a proliferative zone in the developing telencephalon that is defined by the expression of transcription factor proteins such as Pax6, Gsh2, ErSI and Dlx-I,2,5,6.66.67 Immature OB intemeurons tangentially migrate from the dLGE to the developing 0 B and then radially migrate to their final glomerular or granule cell layer positions.7.68.69 Although the dLGE is considered the primary source of embryonic DB DA interneurons, additional sites oforigin have been proposed. A recent study suggested that precursor cells localized to the ventricular layer of the evaginating telencephalon may also contribute neurons to the embryonic DB, including DA interneurons (Fig. 4).70 These OB neural stem cells have molecular features distinct from 0 B progenitors originating from the dLGE. A second alternative embryonic origin may be the medial septum (Fig. 4).71 Neural progenitors in the medial septum also have molecular features distinct from the dLGE, including the expression of the Zic I and Zic3 transcription factor proteins. The consequences of these alternative embryonic origins and their distinct molecular features are not clear, but the OB DA neurons derived form these alternative origins may have functional properties that differ from those cells ofthe dLGE lineage. Although the generation of OB DA interneurons is initiated during mid-embryonic development, the majority of these interneurons are born during late embryonic and neonatal time periods.65.72.73 These late-embryonic and postnatal neurons are generated in the rostral migratory stream (RMS) and subventricular zone (SVZ) of the lateral ventricle, which is believed to be, in part, a remnant of the embryonic LGE. Nearly all of the transcription factors that define the embryonic dLGE are also expressed in the postnatal SVZ.66.67.74-79 In the mouse, neurogenesis of DB interneurons peaks between £18 and postnatal (P) day 5.65 These late embryonic and postnatally generated neurons migrate tangentially through the RMS before moving radially to their final positions in the granule and glomerular layers of the 0 B. Although their proliferation rate decreases after P5, neurogenesis of 0 B intemeurons, including DA cells, continues throughout the lifetime of the adult, including humans.F'" The predominant hypothesis is that late-embryonic and postnatally generated 0 B interneurons, including DA cells,are derived from slowlydividing neural stem cellslocated in SVZ and RMS (for a comprehensive review, see refs. 5,81-84). These neural stem cells have several features attributed to astrocytes, such as the expression of GFAP, but they can also be cultured in the presence of £GF to generate both neurons and glia. In both the RMS and SVZ, these slowly dividing stem cells produce transit amplifying cells that express markers such as N G2 and Olig2. The transit amplifying cells give rise to migrating neuroblasts, which can be identified by the expression of such genes as PSA-NCAM, doublecortin and neuron-specific type III-tubulin (Tu]l). These

Development andEngineering ofDopamineNeurons

20

c

Figure 4. Schematic representation of embryonic and postnatal origins of DB DA neurons. A) The earliest reported origin of DA cells are those interneurons derived from stem cells in the evaginating telencephalon (dark/red layer), at approximately El3 .S in the mouse. B) Mid-embryonically derived DB DA neurons originate from the dorsal lateral ganglionic eminence (dark/blue regions) and the medial septum (light/yellow regions), at about E16.5 in the mouse. C) Post-natally and adult derived DB DA neurons are generated from progenitors in the subventricular zone of the lateral ventricle. These progenitors migrate to the DB through the rostral migratory stream, which is also a putative source for some DB DA neurons. Abbreviations: CX, cortex; dLGE, dorsal lateral ganglionic eminence; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCX, neocortex; DB, olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular zone. A color version of this figure is available at www.landesbioscience.com/curie.

Dopamine Systemsin the Forebrain

21

neuroblasts (precursor cells) tangentially migrate through the SVZ and RMS in chains that are enclosed in tubes formed by transit amplifying cells, slowly dividing neural stem cells and glia. It has been estimated that approximately 30,000 progenitors per day enter the adult OB, but only a small percentage of these cells mature and differentiate into functional 0 B neurons. There is a growing consensus that progenitors for specific 0 B interneuron sub-types, such as DA cells, are generated in distinct regions within the SVZ and RMS. Consistent with this idea is the observation that the location of stem cells in the SVZ and RMS reflect different embryonic origins." For example, the majority of cells lining the lateral ventricle are derived from the Gsh2 expressing regions in the LGE, whereas the ventral and dorsal regions of the SVZ contain stem cells derived from the Nkx2.1 expressing region ofthe MGE and the Emxl expressing regions of the embryonic cortex, respectively. There is a general agreement that the majority ofpostnatally derived OB DA neurons are generated from the more dorsal neurogenic regions in the SVZ.85.86 However, there is controversy as to whether there is a preferential rostral-caudal origin of0 B D A cells. Some studies have suggested that PG interneurons, including DA neurons, are preferentially derived from stem cells in the RMS.16 By contrast, other studies have suggested that OB DA cells are generated from stem cells within a long neurogenic region that includes the dorsal SVZ and subcallosal zone." Generation ofneuronal diversity may also show temporal-dependence, suggestingthat distinct PG interneuron sub-types are produced during different developmental windows." Current controversies surrounding the spatial and temporal origins of0 B neurons may result from the use of different experimental techniques, but there is a clear consensus that stem cells within the neurogenic SVZ and RMS regions are not a homogenous population. Identification of the specific origin for DA progenitors in both the embryonic and postnatal animal is also complicated by the fact that there are no known markers specific to DA cells prior to terminal differentiation. Both requisite biosynthetic enzyme proteins for DA production, TH and AADC, are expressed only in the differentiated neurons in the glomerular layer and not in migrating immature DA precursor cells.36 The transcription factors Pax6, Er81, Meis2 and Dlx-l,2,S,6 are co-expressed in both the immature and terminally differentiated neurons.16,66,75,77,87-92 However, these transcription factor proteins are expressed in other OB interneuron sub-types that do not express TH and, thus, these proteins do not specifically label DA precursor cells. Although TH protein is expressed only in the glomerular layer (Fig. SA), several studies have shown that the upstream gene regulatory region ofTH is transcriptionally active in areas outside of the glomerular layer. TH mRNA is expressed in the superficial granule cell layer, even though TH protein is not detectable in this layer (Fig. SB).88,93 Transgenic mice containing either GFP or LacZ reporter genes under the control ofeither 9 kb or 4.5 kb ofthe TH upstream gene regulatory region also exhibited transgene expression in the superficial granule cell layer as well as in the RMS (Fig. SC).93-95 Together, these studies suggest that there are spatially dependent translational regulatory mechanisms that limit the expression ofTH protein and consequently DA biosynthesis, to the B glomerular layer. It is possible that the cells in the superficial granule cell layer which contain TH promoter activity, but lack TH protein, are immature DA neurons. However, these cells do not appear to migrate and express NeuN, a marker ofterminally differentiated neurons." Although the OBis the major 0 A system in the forebrain, there is TH gene activity in other regions. In mice, TH mRNA and reporter gene expression under the control ofthe TH promoter has been detected in both the cortex and striatum." The human and primate striata contain a small number ofprojection neurons that express TH protein.18,19 Although the origin ofthese cells with TH gene activity is not presently known, it is interestingto note that some cortical interneurons and . striatal projection neurons are also derived from the LGE.97,98The functional role ofthese non-OB neurons with TH mRNA and/or TH protein in the forebrain remains to be determined.

o

Molecular Genetic Mechanisms ofOB DA Neuron Differentiation

The underlying molecular genetic pathway of midbrain DA neuron differentiation is well established (for details see chapters on molecular development ofDA neurons.) Briefly,midbrain DA neurons originate in the ventral mes-diencephalic neuroepithelium where Sonic hedgehog

Development and Engineering ofDopamineNeurons

22

A

TH protein

Figure 5. Patterns of both TH protein and TH mRNA expression as well as TH/LacZ reporter gene activity in the adult DB . A) TH protein immunoreactivity is restricted to the glomerular layer. B) High level expression of TH mRNA is seen both in the glomerular layer and in cells scattered in the external plexiform layer. Lightly-labeled cells are found in the mitral and superficial granule cell layers. C) An X-gal stained section reveals expression of the LacZ reporter gene under the control of the 9 kb upstream TH gene regulatory region. X-gal activity can be detected in the same layers as the TH mRNA. Bar = 50 urn. Abbreviations: epl, external plexiform layer; gl, glomerular layer; gr, superficial granule cell layer; m, mitral cell layer.

DopamineSystems in theForebrain

23

(Shh) and FGF8 signaling pathways cooperatively interact. This interaction between Shh and FGF8 initiates expression of the transcription factors Otxl, Nkx2.2 and Sox2 in neuroblasts. Midbrain neuralprogenitor cells developfrom theseneuroblastsand express transcriptionfactors such as Lmxla, Msxl and Ngn2. The committed midbrain DA neuronal precursor cellsexpress AADC and the transcription factorsLmxl b and EnI. Subsequently, terminal differentiationof midbrain DA neurons occurswith the expression of genessuch as TH, VMAT2, DAT and the transcription factorsNurr I and Pitx3. Bycontrast, the moleculargeneticpathways that regulateOB DA differentiationare not well defined.A significant challenge associated with definingthe moleculargeneticpathways necessary for OB DA neurons isthat there is no singlespatialand temporaloriginspecific to theseneurons. As discussed above, there is evidence for multiple embryonic origins of these neurons and the origin of postnatallyderivedneuronswithin either the RMS or SVZ is ambiguous. Furthermore, it is not clearwhether thesevariousoriginshaveeither the same, partiallyoverlapping or unique moleculargeneticpathways for differentiationof 0 B DA neurons. Despite the ambiguitysurroundingtheir spatialand temporal origins,several genesinvolved in the differentiationofOB DA neurons havebeen identifiedand are summarizedin Tables 1-3. One such gene, Er81, is expressed in both the embryonic dLGE and postnatal SVZ, RMS and oB.66,77 Almost all 0 B TH immunoreactive cellsalsocontain Er81 and TH expression isdrasticallyreduced in Er81 deficientmice (Fig.6). LikeTH, Er81 expression levels are alsodependent on afferentsynapticactivityof olfactoryreceptor neurons." However, Er81 is not specific for DA differentiationsinceit is alsoexpressed in some OB interneurons that do not contain TH, such as calretinin containing neurons." The transcription factor Pax6is alsocriticalfor OB DA differentiation. The Pax6Sey mutation is embryonic lethal when homozygous, but heterozygous Pax6Sey mutant mice are viable and havean almost total lossofTH expression in the OB. In wild-typemice,nearly all OB DA cells co-express TH and Pax6.87However, a significant fraction ofPax6 immunoreactive cells lackTH expression, suggesting that Pax6is not specific to OB DA neurons.Also,Pax6 expression in the OBis not dependent on afferent synapticactivityof the olfactoryreceptor neurons (Fig. 7). The molecular geneticmechanism bywhichPax6regulates differentiation ofOB DA neuronsisunclear, in part, becausethe Pax6geneencodesat leastthree differentDNA-binding protein isoforms that eachhavea unique consensus target DNA binding sequence.?"!" The relevantPax6isoforms and the target genesof these isoforms necessary for 0 B DA differentiationhavenot been identified. Furthermore, Pax6has been reported to influenceneuronal progenitor migration and proliferation.l04.107 Thesenonspecific, generalneurogenicfunctions ofPax6 complicateanalysis of specific contributions to OB DA differentiation. The immediate early gene (lEG) family is likely to be essential for mediating the synaptic activity-dependent expression ofTH in OB DA precursor cells. The homologous lEG family members Nurr I and NGFI-Bareorphan nuclearreceptortranscriptionfactors, whichareexpressed in the OB in asynaptic activity-dependent manner.Nurr 1, but not NGFI-B,isalsoexpressed in the midbrain.108,109 However, there isno evidencethat eitherTH or Nurr I expression in the midbrain is activitydependent. Nurr I can alsomodulate TH geneexpression through binding sitesin the TH proximal prornoter.l'v"! In Nurr I deficientmice,TH expression is absent in the midbrain, but still present in the OB as a likelyconsequence ofNGFI-B functional redundancy.l08,1l2,1l3 The TH proximal promoter also contains evolutionarily conserved binding sites for the lEG basic-leucine zipper (bZip) transcription factor proteins CREB and AP-I (the latter is a heterodimer formed by membersof the Fosandjun protein families) .114,115 In vivomousestudies haveshownthat mutation of either the AP-I or CREB bindingsitein the TH proximalpromoter can disrupt reporter geneexpression under the control of the 9kb TH promoter in the 0 B.94,116 However, there are several lEG bZip proteins expressed in the OB glomerularlayerin a synaptic activitydependent manner that can bind these consensus sites.!'? Thus,like Nurr l and NGFI-B, there is likelyto be redundancy in the regulation ofTH expression by bZip lEGs. For example, expression of the bZip FosB protein in the OB glomerularlayeris activitydependent and FosB

24

Development and EngineeringofDopamineNeurons

Table 1. Potential key regulators of DB DA interneuron differentiation AP-l

CREB

Dlx-family

Er81

Gsh2

Meis2 Nurrl/NGFI-B

Pax6

licl,3

Heterodimer formed by members of the Fos and jun basic-leucine zipper transcription factors; several Fosand [un family members are expressed in the DB and the expression of some members is dependent on olfactory neuron afferent synaptic activity (Liu 1999); mutation of AP-l binding site in TH proximal promoter eliminates reporter gene expression in the DB of transgenic mice;94 DB expression of FosB and junD is activity dependent and both proteins in DB-cell Iysatesbind an AP-l binding site in the TH promoter!" Basic-leucine zipper transcription factor; CREB is expressed in the postnatal DB granule and glomerular layers:'" mutation of an evolutionarily conserved CRE binding site in TH proximal promoter eliminates reporter gene expression in the DB of transgenic mice.!" Homeodomain transcription factors; Dlxl,2,S,6 are expressed in the LGEand postnatal SVl, RMS and OB;66,74-76,93,142-144 Dlx2 is expressed in transit amplifying cells and migrating neuroblasts within the SVl and RMS;91 mice lacking Dlxl or Dlx2 have a modest or strong reduction of DB TH+ cells, respectively, whereas mice lacking both Dlxl and Dlx2 have a near total loss of DB TH+ cells;145,146 mice lacking DlxS have a strong reduction in the number of DB TH+ cells:" TH+ cells in the DB overlap with reporter gene expression driven by a DlxS/6 gene regulatory fragment.90,92 ETS-DNA binding domain transcription factor is expressed in the dLGE, SVl, RMS and OB;66,77 Er81 is co-expressed with TH in periglomerular interneurons;" Er81 expression in the DB is activity dependent:" there is a major loss of TH+ cells in homozygous Er81 mutant mice (Cave and Baker, unpublished). Homeodomain transcription factor protein; expressed in the embryonic LGE and postnatal SVl, RMS and 08 67,79,147; mice lacking Gsh2 have a large loss of DB TH+ cells. TALE homeodomain protein; Meis2 is expressed in the embryonic LGEas well as the postnatal SVl, RMS and OB;90 Meis2 is co-expressed in DB TH+ cells." Homologous orphan nuclear receptor transcription factors; both proteins are expressed in the superficial granule cell and glomerular layers of DB in a synaptic activity-dependent manner.'" TH proximal promoter contains a functional Nurr1 binding response elernent.t'v'" Paired box and homeodomain transcription factor; Pax6 is expressed in the embryonic dLGE and postnatal SVl, RMS and OB;78,87-69 nearly all TH+ cells in DB also co-express Pax6 and there is an almost total loss of DB TH+ cells in mice heterozygous for Pax6 Sey mutation." line finger transcription factors; licl,3 are expressed in the embryonic septum as well as the glomerular and granule cell layers of the postnatal 08;71 mice lacking both Zicl and lic3 have a significant loss of DB interneurons, including TH+ cells."

can bind the AP-l binding site,117 but TH immunoreactivity and enzymatic activity are normal in mice lacking FosB (Fig. 8). There is a dearth ofknowledge regarding the membrane channels and receptors as well as their cognate intra-cellular signaling pathways in the DA progenitor cells that mediate DA differentiation in response to afferent synaptic activity. Studies with primary cultures of 0 B and forebrain organotypic slice cultures indicate that L-type calcium channels are critical for activity dependent expression ofTH.118.119 It is tempting to speculate that the activation ofcalcium channels induces

Dopamine Systems in the Forebrain

25

Figure 6. TH immunoreactivity in 10 day-old Er81-mutant and wild-type mice. Homozygous mutation of the Er81 gene drastically reduces TH immunoreactivity in the DB glomerular layer of the mutant (A) as compared to the wild-type mouse (B). Bar = 30 prn.

Figure 7. Pax6 and TH expression in the glomerular layer of an adult mouse with unilateral naris closure . In the DB contralateral to naris closure, almost all neurons with perikaryal TH immunofluorescence (F, green) also contain Pax6 (C, red) as indicated by the yellow cells in the merged image (A). However, there are several cells with Pax6 that lack TH. As shown in B, D and E, Pax6 immunofluorescence in the DB ipsilateral to the naris closure is unchanged even though only a few cells express TH . Bar = 100 urn.

Developmentand EngineeringofDopamineNeurons

26

Table 2. Phenotype-independent regulators of OB interneuron differentiation Arx

Dcx

Ephrins

Mash1

Myst4

Notch1

Homeodomain transcription factor; Arx is expressed in the embryonic LGEas well as the postnatal SVl, RMS and OB,128,148 mice lacking Arx have impaired SVl-progenitor proliferation and migration as well as a substantial loss of TH+ cells in the OB.128 Microtubule-associated phosphoprotein; migrating neuroblasts in the postnatal SVl, RMS and OB express DCX,149-151 knock down of Dcx diminishes migration of SVl-derived neuroblasts.l" Ephrins are membrane-bound ligands for Eph receptor tyrosine kinases; ephrins A2, B2 and B3 and Eph receptors A4, A7 and B1-3 are expressed in the postnatal SVl and RMS where they regulate progenitor proliferation and migration. 153,154 Basic-helix-Ioop-helix transcription factor; Mash1 is expressed in the embryonic LGE76,155-157 as well as the postnatal SVl and RMS;158 transit amplifying precursor cells express Mash1 and loss of Mash1 substantially reduces the number of TH+ cells in the OB.158 Histone acetyltransferase; Myst4 is expressed in the embryonic LGEand postnatal SVl;159 Myst4 is critical for progenitor proliferation and mice lacking Myst4 have a progressive loss of TH+ cells in the OB.159 Transmembrane receptor that proteolytically releases a transcription co-activator upon ligand binding; Notch1 and its ligands Jagged1 and Delta1 as well as its downstream target gene HesS are expressed in the postnatal SVZ and RMS160-164; Notch1 is critical for proliferation of SVZ progenitors and over-expression of activated Notch1 drastically reduces the number of SVl-derived migrating progeni-

tors.!"

0lig2

PK2

Basic helix-loop-helix transcription factor; 0lig2 is expressed in the transit amplifying precursor cell;16,165 positive regulator of oligodendritic cell fates and negative regulator of neuronal lineages in precursor cells." Cysteine-rich secreted protein; PK2 and its receptors are expressed in complementary patterns within embryonic and postnatal DB where PK2 acts as a chemoattractant for migrating SVZ-derived progenitors.?" periglomerular layer is indiscernible or malformed in mice lacking PK2.166

PSA-NCAM Polysialylated neural cell adhesion molecule; PSA-NCAM is expressed in migrating neuroblasts and is critical for tangential migration i~ the SVZ and RMS.167-170 Reelin Secreted glycoprotein that promotes shift from tangential to radial migration of SVZ-derived progenitors in the OB;171 in the OB, Reelin is highly expressed in the olfactory nerve layer, mitral cell layer and in a descending gradient through the granule cell layer;": the Reelin receptor ApoER2 is strongly expressed in the RMS.171 Shh

Secreted signaling protein; Shh is expressed in the slowly dividing neural stem cell and the transit amplifying cell (type Band C cells, respectively) in the postnatal SVZ and Shh is critical for maintenance and proliferation of these cells.172-174 Slit1,2 Secreted ligand proteins that bind Robo receptors; SIit1,2 are expressed in both embryonic and postnatal septum whereas the Rob01,2 receptors are expressed in the postnatal SVl and RMS;175-178 Slit proteins are chemorepellants that guide migrating SVl-derived progenitors. 175,178-180 Tenascin-R Extracellular matrix glycoprotein that promotes radial migration of progenitor cells by initiating detachment of tangentially migrating SVl-derived neuroblasts.'" in the postnatal DB, Tenascin-R is expressed in the granule cell and internal plexiform layer and is dependent on olfactory receptor neuron synaptic activity.": Vax1 Homeodomain transcription factor; Vax1 is expressed in the embryonic LGEl82 as well as the postnatal SVZ and RMS;183 loss of Vax1 results in disorganization of the RMS and impaired OB interneuron progenitor rnigration.!"

27

Dopamine Systems in theForebrain

lEG expression, and consequently TH expression, through wellestablished calcium secondmessengersignaling pathways.P?Forebrain slice cultureshavealso suggested that OB TH expression is modulatedby GABA(unpublishedobservation), Asstated above. a majorityof the PG interneuronsare GABAergic and the DA interneuronsalsocontain GABA-A receptors. GABAplays well documentedrolesin regulating proliferation. migrationand geneexpression in neuralprogenitors

A

"-s:.:.

'. , .,

.-

: ..

,

'

..-

. . ,. .

.. ; I.

,

.

\ -

... ~ :

"

-

~

.'

,

,

.

...

... '

.,- . :. i

B Supershifted... Boun d

1......

C

.z-

~ 1.0 co Q)

E

Ki'

53 0.5 ::I: I-

Free AP-1...._ . . Probe openOBNE c1osedOBNE a.- FosB -

~ co

~

+

+ + + + +

a;

0:::

+/+ -/FosB genotype

Figure 8. Functional redundancy of FosB in the regulation of TH within the OB . A) Immunohistochemistry in an adult mouse with unilateral naris closure reveals that FosB expression in the glomerular layer (gil is dependent on olfactory neuron afferent synaptic activ ity (d. open versus closed). B) FosB antibody super-shift electromobility gel-shift assays reveal that FosB is present in OB nuclear extracts (OB NE) and can bind a probe containing the AP-l binding site in the TH proximal gene promoter. The FosB supershift with OB NE ipsilateral to unilateral naris closure (closed) presumably results from residual FosB expression. C) Relative TH enzyme activity in the OB is not significantly different in mice lacking FosB relative to wild-type mice.

28

Development and EngineeringofDopamineNeurons

Table 3. Regulators of DB DA differentiation through oHactory neuron innervation of the DB Arx

DlxS

CNG2

Zic1,3

Homeodomain transcription factor; Arx is expressed in the embryonic LGEas well as the postnatal SVZ, RMS and OB;128 mice lacking Arx have a loss of olfactory neuron innervation of the OB and a substantial decrease of TH+ cells in the OB.128,184 Homeodomain transcription factor; DlxS is expressed in the LGE, SVZ, OB as well as olfactory epithelium and olfactory placode.'" in mice lacking DlxS, olfactory receptor neurons fail to properly innervate the OB and there is a strong reduction in the number of OB TH+ cells.!" Transmembrane cyclic AMP gated channel; CNG2 (OCNC1) is expressed in the olfactory epithelium and is required for signal transduction in olfactory receptor neurons; loss of CNG2 results in abnormal pruning of olfactory receptor neuron fibers, as well as a block of afferent olfactory receptor neuron synaptic activity in the OB which dramatically reduces TH expression." Zinc finger transcription factors; olfactory receptor neurons fail to properly innervate the OB in mice lacking both Zic1 and Zic3. 71

in both the SVZ and hippocampus.24.121-126 It is possible that the modulation ofTH expression by GABA is necessary for the terminal differentiation ofDA progenitor cells. There are alsogenes that modulate the OB DA phenotype through either general aspects of neurogenesis (Table 2) or olfactory receptor neuron function (Table 3), rather than specifically regulating 0 B DA differentiation. For example, the loss ofeither Notch1 or Arx impairs proliferation and migration of 0 B interneuron progenitors.127.128 Alternatively, the loss of DlxS disrupts olfactory receptor neuron innervation ofthe OB,75 and mutations in the cyclic nucleotide gated channel 2 (CNG2) gene blocks signal transduction in olfactory receptor neurons.F? Thus, the regulation of DB DA neuron differentiation is complex and requires the convergence ofdiverse molecular genetic pathways.

Expression and Function ofForebrain DA Receptors

Dopamine acts through fivereceptor variants, D 1-D5,that are expressed in distinct and partially overlapping patterns within the forebrain (for an extensive review, see refs. 130,131). D 1. O 2 and DS receptors are widely expressed in the striatum, limbic system and DB as well as the prefrontal, premotor, cingulate and entorhinal cortices. D 5 receptor levels are notably lower than either the D 1 or D 2 receptors in most regions. Both D 3 and 0 4 receptor expression is largely limited to the limbic system, although D4receptors are also highly expressed in the frontal cortex. Forebrain neurons expressing DA receptors are innervated primarily by midbrain DA cell groups. The mesostriatal DA projections from the substantia nigra, ventral tegmentum and retrorubral nucleus (area A9, AI0 and A8, respectively) innervate several regions within the striatum as part of the neural circuitry that controls movement.P' As stated above, loss of the substantia nigra DA neurons and their associated projections is the hallmark ofParkinson's Disease (PD). In addition to the nigrostriatal system, the mesolimbic/mesocortical DA projections that originate largely from the ventral tegmentum (area AIO) innervate limbic system regions that include the hippocampus and amygdala as well as cortical regions that include the cingulate and prefrontal cortex.i" These mesolimbic/mesocortical DA projections have been implicated in several neurological conditions, including drug addiction (reward and reinforcement mechanisms) 134 and schizophrenia.l" as well as learning and rnemory.-"

Prospective Directions for OB DA Neurobiology

The mechanisms for DB DA differentiation may be important for advancing cell-replacement therapeutic strategies to treat neurodegenerative disorders, such as PD. DB DA neurons have several

DopamineSystems in theForebrain

29

advantageous properties that include a capacity to readily integrate into pre-existing circuitry" and a resistance to degeneration in PD.8 Emerging cell-transplant therapeutic strategies use replacement DA neurons generated from stem cells (either embryonic or adult-derived), but efficient production of functional replacement DA neurons remains elusive.137-139 Also, DA production alone is not sufficient and other neuronal properties are also critical, to generate cells suitable for transplant.l40.l4l Thus, it is important to not only delineate the various molecular genetic pathways that afford DA production, but also the pathways that generate the diverse array offeatures and functions ofDA neurons in the brain. Elucidation ofthese diverse pathways may enable the engineering ofreplacement neurons that incorporate the unique, advantageous properties of OB DA neurons in order to improve the clinical effectiveness ofreplacement-cells.

Acknowledgements

This work was supported by the NIH (ROI-DCOO8955) and the Burke Medical Research Institute.

References

1. Hornykiewicz O. Parkinson's disease: from brain homogenate to treatment. Fed Proc 1973; 32(2):183-190. 2. Baker H, Kawano T, Margolis FL et al. Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat. J Neurosci 1983; 3:69-78. 3. Wilson DA, Sullivan RM. The D2 antagonist spiperone mimics the effects of olfactory deprivation on mitral/tufted cell odor response patterns. J Neurosci 1995; 15(8):5574-5581. 4. Wilson DA, Wood JG. Functional consequences of unilateral olfactory deprivation: time course and age sensitivity. Neurosci 1992; 49: 183-192. 5. Lledo PM, Alonso M, Grubb MS. Adult neurogenesis and functional plasticity in neuronal circuits. Nat Rev Neurosci 2006; 7(3):179-193. 6. Lledo PM, Saghatelyan A, Lemasson M. Inhibitory interneurons in the olfactory bulb: from development to function. Neuroscientist 2004; 10(4):292-303. 7. Luskin MB. Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 1993; 11:173-189. 8. Huisman E, Uylings HB, Hoogland PV. A 100% increase of dopaminergic cells in the olfactory bulb . may explain hyposmia in Parkinson's disease. Mov Disord 2004; 19(6):687-692. 9. Halasz N, Hokfelt T, Ljungdahl A et al. Dopamine neurons in the olfactory bulb. Adv Biochem Psychopharmacol1977; 16:169-177. 10. Lidbrink P, Jonsson G, Fuxe K. Selective reserpine-resistant accumulation of catecholamines in central dopamine neurones after DOPA administration. Brain Res 1974; 67(3):439-456. 11. Lledo PM, Saghatelyan A. Integrating new neurons into the adult olfactory bulb: joining the network, life-death decisions and the effects of sensory experience. Trends Neurosci 2005; 28(5):248-254. 12. Marin 0, Rubenstein JL. A long, remarkable journey: tangential migration in the telencephalon. Nat Rev Neurosci 2001; 2(11):780-790. 13. Wonders CP, Anderson SA. The origin and specification of cortical interneurons. Nat Rev Neurosci 2006; 7(9):687-696. 14. Gheusi G, Cremer H, McLean H et al. Importance of newly generated neurons in the adult olfactory bulb for odor discrimination. Proc Nat! Acad Sci USA 2000; 97(4):1823-1828. 15. Gheusi G, Lledo PM. Control of early events in olfactory processing by adult neurogenesis. Chern Senses 2007; 32(4):397-409. 16. Hack MA, Saghatelyan A, de Chevigny A et al. Neuronal fate determinants of adult olfactory bulb neurogenesis, Nat Neurosci 2005; 8(7):865-872. 17. Ortega-Perez I, Murray K, Lledo PM. The how and why of adult neurogenesis. J Mol Histo12007; 99 (6):555-562. 18. Cossette M, Lecomte F, Parent A. Morphology and distribution of dopaminergic neurons intrinsic to the human striatum. J Chern Neuroanat 2005; 29(1):1-11. 19. Cossette M, Levesque D, Parent A. Neurochemical characterization of dopaminergic neurons in human striatum. Parkinsonism Relat Disord 2005; 11(5):277-286. 20. Farbman AI. Cell Biology of Olfaction. New York: Cambridge University Press, 1992: 21. Baker H, Farbman AI. Olfactory afferent regulation of the dopamine phenotype in the fetal rat olfactory system. Neurosci 1993; 52:115-134. 22. Baker H, Morel K, Stone DM et al. Adult naris closure profoundly reduces tyrosine hydroxylase expression in mouse olfactory bulb. Brain Res 1993; 614:109-116.

30

Development ana EngineeringofDopamineNeurons

23. Kosaka K, Aika ~ Toida K et al. Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb. Neurosci Res 1995; 23(1):73-88. 24. De Marchis S, Bovetti S, Carletti B et ale Generation of distinct types of periglomerular olfactory bulb inrerneurons during development and in adult mice: implication for.intrinsic properties of the subventricular zone progenitor population. J Neurosci 2007; 27(3):657-664. 25. Parrish-Aungst S, Shipley MT, Erdelyi F et al. ~titative analysis of neuronal diversity in the mouse olfactory bulb. J Comp NeuroI2007; 501(6):825-836. 26. Macrides F, Davis BJ, Youngs WM et al. Cholinergic and catecholaminergic afferents to the olfactory bulb in the hamster: a neuroanatomical, biochemical and histochemical investigation. J Comp Neurol 1981; 203(3):495-514. 27. McLean JH, Shipley MT. Serotonergic afferents to the rat olfactory bulb: II. Changes in fiber distribution during development. J Neurosci 1987; 7(10):3029-3039. 28. McLean JH, Shipley MT. Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat. J Neurosci 1987; 7(10):3016-3028. 29. Kasowski HJ, Kim H, Greer CA. Compartmental organization of the olfactory bulb glomerulus.] Comp Neurol 1999; 407(2):261-274. 30. White EL. Synaptic organization of the mammalian olfactory glomerulus: new findings including an intraspecific variation. Brain Res 1973; 60(2):299-313. 31. Kosaka K, Toida K, Aika Y et al. How simple is the organization of the olfactory glomerulus? The heterogeneity of so-called periglomerular cells. Neurosci Res 1998; 30(2): 101-110. 32. Toida K, Kosaka K, Aika Y er al. Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb-IV. Intraglomerular synapses of tyrosine hydroxylase-immunoreactive neurons. Neuroscience 2000; 101(1):11-17. 33. Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase: The initial step in norepinephrine biosynthesis. J BioI Chern 1964; 239:2910-2917. 34. ]oh TH, Geghman C, Reis DJ. Immunochemical demonstration of increased accumulation ofTH protein in sympathetic ganglia and adrenal medulla elicited by reserpine.] Neurochem 1973; 39:342-348. 35. McLean ]H, Shipley MT. Postnatal development of the noradrenergic projection from locus coeruleus to the olfactory bulb in the rar. ] Comp Neuro11991; 304(3):467-477. 36. Baker H, Abate C, Szabo A et al. Species specific distribution of aromatic I-amino acid decarboxylase in rodent adrenal gland, cerebellum and olfactory bulb.] Comp Neuro11991; 305:119-129. 37. Cerruti C, Walther DM, Kuhar M] ee al. Dopamine transporter mRNA expression is intense in rat midbrain neurons and modest outside midbrain. Brain Res Mol Brain Res 1993; 18(1-2):181-186. 38. Coronas V; SrivastavaLK, Liang JJ er al. Identification and localization of dopamine receptor subtypes in rat olfactory mucosa and bulb: a combined in situ hybridization and ligand binding radioautographic approach.] Chern Neuroanat 1997; 12(4):243-257. 39. Koster NL, Norman AB, Richtand NM et al. Olfactory receptor neurons expressD2 dopamine receptors. J Comp Neuro11999; 411(4):666-673. 40. Meador-Woodruff]H, Mansour A, Bunzow ]R et al. Distribution of D2 dopamine receptor mRNA in rat brain. Proc Nat! Acad Sci USA 1989; 86(19):7625-7628. 41. Baker H. Unilateral, neonatal olfactory deprivation alters tyrosine hydroxylase expression but not aromatic amino acid decarboxylase or GABA immunoreactivity. Neurosci 1990; 36:761-771. 42. Baker H, Cummings DM, Munger SD et al. Targeted deletion of a cyclic nucleotide-gated channel subunit (OCNCl): Biochemical and morphological consequences in adult mice. ] Neurosci 1999; 19:9313-9321. 43. Philpot BD, Men D, McCarty R er al. Activity-dependent regulation of dopamine content in the olfactory bulbs of naris-occluded rats. Neuroscience 1998; 85(3):969-977. 44. Guthrie KM, Pullara ]M, Marshall ]F et al. Olfactory deprivation increases dopamine D2 receptor density in the rat olfactory bulb. Synapse 1991; 8:61-70. 45. Gall CM, Hendry SCH, Seroogy KB et al. Evidence for co-existence of GABA and dopamine in neurons of the rat olfactory bulb. J Comp Neurol 1987; 266:307-318. 46. Waclaw RR, Allen Z] 2nd, Bell SM et al. The zinc finger transcription factor Sp8 regulates the generation and diversity of olfactory bulb interneurons. Neuron 2006; 49(4):503-516. 47. BrunigI, SommerM, Han H et al. Dopamine receptorsubtypesmodulateolfactorybulb gamma-aminobutyric acid type A receptors. Proc Nat! Acad Sci USA 1999; 96(5):2456-2460. 48. Aroniadou-Anderjaska V, Ennis M, Shipley MT. Glomerular synaptic responses to olfactory nerve input in rat olfactory bulb slices. Neuroscience 1997; 79(2):425-434. 49. Berkowicz DA, Trombley PQ, Shepherd GM. Evidence for glutamate as the olfactory receptor cell neurotransmitter.] Neurophysiol1994; 71(6):2557-2561. 50. Ennis M, Zimmer LA, Shipley MT. Olfactory nerve stimulation activates rat mitral cells via NMDA and non NMDA receptors in vitro [see comments]. Neuroreport 1996;7(5):989-992.

DopamineSystems in theForebrain

31

51. Ennis M, Zhou FM, Ciombor K] et al, Dopamine D2 receptor-mediated presynaptic inhibition of olfactory nerve terminals.] Neurophysiol Ztltlf, 86(6):2986-2997. 52. Aroniadou-Anderjaska V, Zhou FM, Priest CA et al. Tonic and synaptically evoked presynaptic inhibition of sensory input to the rat olfactory bulb via GABA(B) heteroreceptors. ] Neurophysiol 2000; 84(3): 1194-1203. 53. Keller A, Yagodin S, Aroniadou-Anderjaska V et al. Functional organization of rat olfactory bulb glomeruli revealed by optical imaging.] Neurosci 1998; 18(7):2602-2612. 54. Murphy G], Glickfeld LL, Balsen Z et ale Sensory neuron signaling to the brain: properties of transmitter release from olfactory nerve terminals.] Neurosci 2004; 24(12):3023-3030. 55. Palouzier-Paulignan B, Duchamp-Viret P, Hardy AB et al. GABA(B) receptor-mediated inhibition of mitral/tufted cell activity in the rat olfactory bulb: a whole-cell patch-clamp study in vitro. Neuroscience 2002; 111(2):241-2S0. 56. Murphy G], Darcy DP, Isaacson IS. Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit. Nat Neurosci 2005; 8(3):354-364. 57. Smith TC, ]ahr CEo Self-inhibition of olfactory bulb neurons. Nat Neurosci 2002; 5(8):760-766. 58. Berkowicz DA, Trombley PQ Dopaminergic modulation at the olfactory nerve synapse. Brain Res 2000; 8S5(1):90-99. 59. Hsia AY, Vincent]D, Lledo PM. Dopamine depressessynaptic inputs into the olfactory bulb.] Neurophysiol 1999; 82(2):1082-1085. 60. Davila NG, Blakemore L], Trombley PQ Dopamine modulates synaptic transmission between rat olfactory bulb neurons in culture.] Neurophysiol 2003; 90(1):395-404. 61. Gutierrez-Mecinas M, Crespo C, Blasco-Ibanez]M et al. Distribution of D2 dopamine receptor in the olfactory glomeruli of the rat olfactory bulb. Eur] Neurosci 2005; 22(6):1357-1367. 62. Davison IG, Boyd ]D, Delaney KR. Dopamine inhibits mitralltufted-> granule cell synapses in the frog olfactory bulb. ] Neurosci 2004; 24(37):80S7-8067. 63. Baker H, Towle AC, Margolis FL. Differential afferent regulation of the dopamine and gabaergic systems of the mouse main olfactory bulb. Brain Res 1988; 450:69-80. 64. Stone DM, Grillo M, Margolis FL et al. Differential effect of functional olfactory bulb deafferentation on tyrosine hydroxylase and glutamic acid decarboxylase messenger RNA levels in rodent juxtaglomerular neurons.] Comp Neuro11991; 311:223-233. 65. Hinds ]W Autoradiographic study of histogenesis in the mouse olfactory bulb. I. Time of origin of neurons and neuroglia. ] Comp Neurol 1968; 134(3):287-304. 66. Stenman ], Toresson H, Campbell K. Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis. ] Neurosci 2003; 23( 1):167-174. 67. Yun K, Garel S, Fischman S et al. Patterning of the lateral ganglionic eminence by the Gshl and Gsh2 homeobox genes regulates striatal and olfactory bulb histogenesis and the growth ofaxons through the basal ganglia. J Comp Neurol 2003; 461(2):IS1-16S. 68. Lois C, Garcia-Verdugo JM, Alvarez-Buylla A. Chain migration of neuronal precursors. Science 1996; 271 (525 1):978-981. 69. Luskin MB, Zigova T, Soteres BJ et al. Neuronal progenitor cells derived from the anterior subventricular zone of the neonatal rat forebrain continue to proliferate in vitro and express a neuronal phenotype. Mol and Cell Neurosci 1997; 8:351-366. 70. Vergano-Vera E, Yusta-Boyo MJ, de Castro F et al. Generation of GABAergic and dopaminergic interneurons from endogenous embryonic olfactory bulb precursor cells. Development 2006; 133(21):4367-4379. 71. Inoue T, Ora M, Ogawa M et al. Zicl and Zic3 regulate medial forebrain development through expansion of neuronal progenitors. J Neurosci 2007; 27(20):5461-5473. 72. Altman J, Das GD. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to postnatal neurogenesis in some brain regions. ] Comp Neurol 1966; 126(3):337-389. 73. Bayer SA. pH]Thymidine-radiographic studies of neurogenesis in the rat olfactory bulb. Exp Brain Res 1983; 50:329-340. 74. Bulfone A, Kim H], Puelles L et al. The mouse Dlx-2 (Tes-l) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos. Mech Dev 1993; 40(3):129-140. 7S. Long]E, Garel S, Depew MJ et al. DLXS regulates development of peripheral and central components of the olfactory system. ] Neurosci 2003; 23(2):S68-578.

32

Development and EngineeringojDopamineNeurons

76. Porteus MH, Bulfone A, LiuJK et ale DLX-2, MASH-l and MAP-2 expression and bromodeoxyuridine incorporation define molecularly distincT-cell populations in the embryonic mouse forebrain. J Neurosci 1994; 14(11 Pt 1):6370-6383. 77. Saino-Saito S, Cave ~ Akiba Y et al. ER81 and CaMKIV identify anatomically and phenotypically defined subsets of mouse olfactory bulb interneurons. J Comp Neuro12007; 502(4):485-496. 78. Stoykova A, Gruss P. Roles of Pax-genes in developing and adult brain as suggested by expression patterns. J Neurosci 1994; 14(3 Pt 2):1395-1412. 79. Toresson H, Campbell K. A role for Gshl in the developing striatum and olfactory bulb of Gsh2 mutant mice. Development 2001; 128(23):4769-4780. 80. Curtis MA, Kam M, Nannmark U et al. Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science 2007; 315(5816):1243-1249. 81. Doetsch F. 'Theglial identity of neural stem cells. Nat Neurosci 2003; 6(11):1127-1134. 82. Doetsch F. A niche for adult neural stem cells. Curr Opin Genet Dev 2003; 13(5):543-550. 83. Merkle FT, Alvarez-BuyllaA. Neural stem cells in mammalian development. Curr Opin Cell Bioi 2006; 18(6):704-709. 84. Luskin MB, Coskun V. 'The progenitor cells of the embryonic telencephalon and the neonatal anterior subventricular zone differentially regulate their cell cycle. Chem Senses 2002; 27(6):577-580. 85. Young KM, Fogarty M, Kessaris N et al. Subventricular zone stem cells are heterogeneous with respect to their embryonic origins and neurogenic fates in the adult olfactory bulb. J Neurosci 2007; 27(31) :8286-8296. 86. Merkle FT, Mirzadeh Z, Alvarez-Buylla A. Mosaic organization of neural stem cells in the adult brain. Science 2007; 317(5836):381-384. 87. Dellovade TL, Pfaff D~ Schwanzel-Fukuda M. Olfactory bulb development is altered in small-eye (Sey) mice. J Comp Neuro11998; 402(3):402-418. 88. Kohwi M, Osumi N, Rubenstein JL et al. Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb. J Neurosci 2005; 25(30):6997-7003. 89. Stoykova A, Fritsch R, Walther C er al. Forebrain patterning defects in Small eye mutant mice. Development 1996; 122(11):3453-3465. 90. Allen ZJ Znd, Waclaw RR, Colbert MC et al. Molecular identity of olfactory bulb interneurons: transcriptional codes of periglomerular neuron subtypes. J Mol HistoI2007;38(6):517-525 91. Doetsch F, Petreanu L, Caille I et al. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 2002; 36(6): 1021-1034. 92. Kohwi M, Petryniak MA, Long JE et al. A subpopulation of olfactory bulb GABAergic interneurons is derived from Emxl- and Dlx5/6-expressing progenitors. J Neurosci 2007; 27(26):6878-6891. 93. Saino-Saito S, Sasaki H, Volpe BT et al. Differentiation of the dopaminergic phenotype in the olfactory system of neonatal and adult mice. J Comp Neuro12004; 479(4):389-398. 94. Baker H, Liu N, Chun HS et ale Phenotypic differentiation during migration of dopaminergic progenitor cells to the olfactory bulb. J Neurosci 2001; 21(21):8505-8513. 95. Schimmel JJ, Crews L, RofBer-Tarlov S er al. 4.5 kb of the rat tyrosine hydroxylase 5' flanking sequence directs tissue specific expression during development and contains consensus sites for multiple transcription factors. Brain Res Mol Brain Res 1999; 74(1-2):1-14. 96. Baker H, Kobayashi K, Okano H et al. Cortical and striatal expression of tyrosine hydroxylase mRNA in neonatal and adult mice. Cell Mol Neurobiol 2003; 23(4-5):507-518. 97. Anderson SA, Marin 0, Horn C et ale Distinct cortical migrations from the medial and lateral ganglionic eminences. Development 2001; 128(3):353-363. 98. Wichterle H, Turnbull DH, Nery S et al. In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain. Development 2001; 128(19):3759-3771. 99. Chi N, Epstein JA. Getting your Pax straight: Pax proteins in development and disease. Trends Genet 2002; 18(1):41-47. 100. Czerny T, Busslinger M. DNA-binding and transactivation properties of Pax-6: three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5). Mol Cell Bioi 1995; 15(5):2858-2871. 101. Epstein JA, Glaser T, Cai J et al. Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. Genes Dev 1994; 8(17):2022-2034. 102. Kozmik Z, Czerny T, Busslinger M. Alternatively spliced insertions in the paired domain restrict the DNA sequence specificity of Pax6 and Pax8. Embo J 1997; 16(22):6793-6803. 103. Mishra R, Gorlov IP, Chao LY et ale PAX6, paired domain influences sequence recognition by the homeodomain. J BioI Chem 2002; 277(51):49488-49494. 104. Carie D, Gooday D, Hill RE et al. Determination of the migratory capacity of embryonic cortical cells lacking the transcription factor Pax-6. Development 1997; 124(24):5087-5096.

DopamineSystems in the Forebrain

33

105. Haubst N, Berger J, Radjendirane V et al. Molecular dissection of Pax6 function: the specific roles of the paired domain and homeodomain in brain development. Development 2004; 131(24) :6131-6140. 106. Quinn JC, Molinek M, Martynoga BS et al. Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism. Dev Biol 2007; 302( 1):50-65. 107. Talamillo A, Quinn JC, Collinson JM et al. Pax6 regulates regional development and neuronal migration in the cerebral cortex. Dev Bioi 2003; 255(1):151-163. 108. Liu N, Baker H. Activity-dependent Nurrl and NGFI-B gene expression in adult mouse olfactory bulb. Neuroreport 1999; 10(4):747-751. 109. Zetterstrom RH, Williams R, Perlmann T et al. Cellular expression of the immediate early transcription factors Nurrl and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system. Brain Res Mol Brain Res 1996; 41(1-2):111-120. 110. Kim KS, Kim CH, Hwang DY et al. Orphan nuclear receptor Nurr 1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell-specific manner. J Neurochem 2003; 85(3):622-634. 111. Sakurada K, Ohshima-Sakurada M, Palmer TO et al. Nurrl, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development 1999; 126(18):4017-4026. 112. Le W, Conneely OM, Zou L et al. Selective agenesis of mesencephalic dopaminergic neurons in Nurr l-deficient mice. Exp Neuro11999; 159(2):451-458. 113. Zetterstrom RH, Solomin L, Jansson L et al. Dopamine neuron agenesisin Nurr l-deficient mice. Science 1997; 276:248-250. 114. Fung BP, Yoon SO, Chikaraishi OM. Sequences that direct rat tyrosine hydroxylase gene expression. J Neurochem 1992; 58(6):2044-2052. 115. Nagamoto-Combs K, Piech KM, BestJA et al. Tyrosine hydroxylase gene promoter activity is regulated by both cyclic AMP-responsive dement and AP1 sitesfollowing calciuminflux, Evidence for cyclic amp-responsive element binding protein-independent regulation.J BioI Chem 1997; 272(9):6051-6058. 116. Trocme C, Sarkis C, Hermel JM et al. CRE and TRE sequences of the rat tyrosine hydroxylase promoter are required for TH basal expression in adult mice but not in the embryo. Eur J Neurosci 1998; 10(2):508-521. 117. Liu N, Cigola E, Tinti C et al. Unique regulation of immediate early gene and tyrosine hydroxylase expression in the odor-deprived mouse olfactory bulb. J Biol Chem 1999; 274:3042-3047. 118. Akiba Y, Sasaki H, Saino-Saito S et al. Temporal and spatial disparity in cFOS expression and dopamine phenotypic differentiation in the neonatal mouse olfactory bulb. Neurochem Res 2007; 32(4-5):625-634. 119. Cigola E, Volpe BT, Lee JW et al. Tyrosine hydroxylaseexpression in primary cultures of olfactory bulb: role of L-type calcium channels. J Neurosci 1998; 18:7638-7649. 120. West AE, Chen WG, Dalva MB et al. Calcium regulation of neuronal gene expression. Proc Nat! Acad Sci USA 2001; 98(20):11024-11031. 121. Behar TN, Smith SV, Kennedy RT et al. GABA(B) receptors mediate motility signals for migrating embryonic cortical cells. Cereb Cortex 2001; 11(8):744-753. 122. Bolteus AJ, Bordey A. GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone. J Neurosci 2004; 24(35):7623-7631. 123. Cancedda L, Fiumelli H, Chen K et al. Excitatory GABA action is essential for morphological maturation of cortical neurons in vivo. J Neurosci 2007; 27(19):5224-5235. 124. Carleton A, Petreanu LT, Lansford R er al. Becoming a new neuron in the adult olfactory bulb. Nat Neurosci 2003; 6(5):507-518. 125. Gascon E, Dayer AG, Sauvain MO et al. GABA regulates dendritic growth by stabilizing lamellipodia in newly generated interneurons of the olfactory bulb. J Neurosci 2006; 26(50):12956-12966. 126. Ge S, Pradhan DA, Ming GL et al. GABA sets the tempo for activity-dependent adult neurogenesis. Trends Neurosci 2007; 30(1):1-8. 127. Chambers CB, Peng Y, Nguyen H et al. Spatiotemporal selectivity of response to Notch l signals in mammalian forebrain precursors. Development 2001; 128(5):689-702. 128. Yoshihara S, Omichi K, YanazawaM et al. Arx horneobox gene is essential for development of mouse olfactory system. Development 200S; 132(4):751-762. 129. Baker H, Cummings DM, Munger SD et al. Targeted deletion of a cyclic nucleotide-gated channel subunit (OCNCl): biochemical and morphological consequences in adult mice. J Neurosci 1999; 19(21):9313-9321. 130. Missale C, Nash SR, Robinson SWet al. Dopamine receptors: from structure to function. Physiol Rev 1998; 78(1):189-225.

34

Development ana Engineering ofDopamineNeurons

131. Mansour A, Watson SJ Jr. Dopamine receptor expression in the central nervous system. In: Bloom F, Kupfer D, eds, Pyschopharmacology: The Fourth Generation of Progress. New York City: Raven Press Ltd, 1995:207-219. 132. Mink J. Basal Ganglia. In: Zigmond M, Bloom F, Landis S, Roberts J, Squire L, eds, Fundamental Neuroscience. New York:Academic Press, 1999:951-972. 133. Fallon J, Loughlin S. Substantia nigra. In: Paxinos G, ed. The rat nervous system.New York:Academic Press, 1995:215-237. 134. Nestler EJ. Molecular mechanisms of drug addiction. Neuropharmacology 2004; 47 (Suppll):24-32. 135. Gray JA, Joseph MH, Hemsley DR et ale The role of mesolimbic dopaminergic and retrohippocampal afferentsto the nucleus accumbensin latent inhibition: implications for schizophrenia.BehavBrain Res 1995; 71(1-2):19-31. 136. Pezze MA, Feldon J. Mesolimbic dopaminergic pathways in fear conditioning. Prog Neurobiol 2004; 74(5):301-320. 137. Goldman S. Stem and progenitor cell-based therapy of the human central nervous system. Nat Biotechnol 2005; 23(7):862-871. 138. Lindvall 0, Kokaia Z. Stem cells for the treatment of neurological disorders. Nature 2006; 441(7097):1094-1096. 139. Lindvall 0, KokaiaZ, Martinez-Serrano A. Stem cell therapy for human neurodegenerative disorders-how to make it work. Nat Med 2004; 10 (Suppl):S42-50. 140. Lindvall 0, Bjorklund A. Cell therapy in Parkinson'sdisease. NeuroRx 2004; 1(4):382-393. 141. Winkler C, Kirik D, Bjorklund A. Cell transplantation in Parkinson's disease: how can we make it work? Trends Neurosci 2005; 28(2):86-92. 142. Saine-Saito S, Berlin R, Baker H. Dlx-I and DIx-2 expressionin the adult mouse brain: relationship to dopaminergic phenotypic regulation. Journal of Comparative Neurology 2003; 461(1):18-30. 143. Eisenstat DO, Liu JK, Mione M et al. DLX-l, DLX-2 and DLX-5 expressiondefine distinct stages of basal forebrain differentiation.] Comp Neuro11999; 414(2):217-237. 144. Liu JK, Ghattas I, Liu S et al. DIx genes encode DNA-binding proteins that arc expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev Dyn 1997; 210(4):498-512. 145. Bulfone A, Wang F, Hevner R et al. An olfactory sensory map developsin the absenceof normal projection neurons or GABAergic interneurons. Neuron 1998; 21(6):1273-1282. 146. Qiu M, Bulfone A, Martinez S er ale Null mutation of DIx-2 results in abnormal morphogenesis of proximalfirst and second branchial arch derivatives and abnormal differentiationin the forebrain. Genes Dev 1995; 9(20):2523-2538. 147. Corbin ]G, Gaiano N, Machold RP er al. The Gsh2 homeodomain gene controls multiple aspects of telencephalic development. Development 2000; 127(23):5007-5020. 148. Colombo E, Collombat P, Colasante G et al. Inactivation of Arx, the murine ortholog of the X-linked lissencephaly with ambiguousgenitalia gene, leads to severe disorganization of the ventral telencephalon with impaired neuronal migration and differentiation. J Neurosci 2007; 27(17):4786-4798. 149. Brown JP, Couillard-Despres S, Cooper-Kuhn CM et al. Transient expression of doublecortin during adult neurogenesis. J Comp Neuro12003; 467(1):1-10. 150. GleesonJG, Lin PT, FlanaganLA et ale Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 1999; 23(2):257-271. 151. NacherJ, Crespo C, McEwen BS.Doublecortin expression in the adult rat telencephalon.EurJ Neurosci 2001; 14(4):629-644. 152. Ocbina PJ, Dizon ML, Shin L et ale Doublecortin is necessaryfor the migration of adult subventricular zone cells from neurospheres. Mol Cell Neurosci 2006; 33(2):126-135. 153. Conover JC, Doetsch F, Garcia-Verdugo JM et al. Disruption of Eph/ephrin signalingaffectsmigration and proliferation in the adult subventricular zone. Nat Neurosci 2000; 3(11):1091..1097. 154. Holmberg], Armulik A, Senti KA et ale Ephrin-A2 reverse signalingnegatively regulates neural progenitor proliferation and neurogenesis, Genes Dev 2005; 19(4):462-471. 155. Guillemot F, Joyner AL. Dynamic expression of the murine Achaete-Scute homologue Mash-l in the developing nervous system. Mech Dev 1993; 42(3):171-185. 156. Guillemot F, Lo LC, Johnson JE et ale Mammalian achaete-scutehomolog 1 is required for the early development of olfactory and autonomic neurons. Cell 1993; 75(3):463-476. 157. Lo LC, Johnson JE, Wuenschell CW et al. Mammalian achaete-scute homolog 1 is transiently expressedby spatially restricted subsets of early neuroepithelial and neural cresT-cells. Genes Dev 1991; 5(9):1524-1537. 158. Parras CM, Galli R, Britz 0 et al. Mashl specifies neurons and oligodendrocytesin the postnatal brain. EMBO J 2004; 23(22):4495-4505. 159. Merson TO, Dixon MP, Collin C et al. The transcriptional coactivator Querkopf controls adult neurogenesis. J Neurosci 2006; 26(44):11359-11370.

Dopamine Systemsin the Forebrain

35

160. Givogri MI, de Planell M, Galbiati F et al. Notch signaling in astrocytes and neuroblasts of the adult subventricular zone in health and after cortical injury. Dev Neurosci 2006; 28(1-2):81-91. 161. Higuchi M, Kiyama H, Hayakawa T et al. Differential expression of Notch1 and Notch2 in developing and adult mouse brain. Brain Res Mol Brain Res 1995; 29(2):263-272. 162. Irvin DK, Nakano I, Paucar A et al. Patterns ofJagged1, Jagged2, Delta-like 1 and Delta-like 3 expression during late embryonic and postnatal brain development suggest multiple functional roles in progenitors and differentiated cells. J Neurosci Res 2004; 75(3):330-343. 163. Irvin DK, Zurcher SD, Nguyen T et al. Expression patterns of Notch 1, Notch2 and Notch3 suggest multiple functional roles for me Notch-DSL signaling system during brain development. J Comp Neurol 2001; 436(2):167-181. 164. Stump G, Durrer A, Klein AL et al. Notch l and its ligands Delta-like and Jagged are expressed and active in distincT-cell populations in the postnatal mouse brain. Mech Dev 2002; 114(1-2):153-159. 165. Hack MA, Sugimori M, Lundberg C et al. Regionalization and fate specification in neurospheres. the role of Olig2 and Pax6. Mol Cell Neurosci 2004; 25(4):664-678. 166. Ng KL, Li JD, Cheng MY et al. Dependence of olfactory bulb neurogenesis on prokineticin 2 signaling. Science 2005; 308(5730):1923-1927. 167. Cremer H, Lange R, Christoph A et al. Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning. Nature 1994; 367(6462):455-459. 168. Ono K, Tomasiewicz H, Magnuson T et al. N-CAM mutation inhibits tangential neuronal migration and is phenocopied by enzymatic removal of polysialic acid. Neuron 1994; 13(3):595-609. 169. Tomasiewicz H, Ono K, Yee D et al. Genetic deletion of a neural cell adhesion molecule variant (N-CAM-180) produces distinct defects in the central nervous system. Neuron 1993; 11(6):1163-1174. 170. Wichterle H, Garcia-VerdugoJM, Alvarez-Buylla A. Direct evidence for homotypic, glia-independent neuronal migration. Neuron 1997; 18(5):779-791. 171. Hack I, Bancila M, Loulier K et al. Reelin is a detachment signal in tangential chain-migration during postnatal neurogenesis, Nat Neurosci 2002; 5(10):939-945. 172. Balordi F, Fishell G. Hedgehog signaling in the subventricular zone is required for both the maintenance of stem cells and the migration of newborn neurons. J Neurosci 2007; 27(22):5936-5947. 173. Machold R, Hayashi S, Rudin M et al. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 2003; 39(6) :937-950. 174. Palma ~ Lim DA, Dahmane N et al. Sonic hedgehog controls stem cell behavior in the postnatal and adult brain. Development 2005; 132(2):335-344. 175. Hu H. Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 1999; 23(4):703-711. 176. Li HS, Chen JH, Wu W et al. Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons. Cell 1999; 96(6):807-818. 177. Marillat ~ Cases 0, Nguyen-Ba-Charvet KT et al. Spariotemporal expression patterns of slit and robo genes in the rat brain. J Comp Neurol2oo2; 442(2):130-155. 178. Wu ~ Wong K, Chen J et al. Directional guidance of neuronal migration in the olfactory system by the protein Slit. Nature 1999; 400(6742):331-336. 179. Chen JH, Wen L, Dupuis S et al. The N-terminalleucine-rich regions in Slit are sufficient to repel olfactory bulb axons and subventricular zone neurons. J Neurosci 2001; 21(5):1548-1556. 180. Sawamoto K, Wichterle H, Gonzalez-Perez 0 et al. New neurons follow the flow of cerebrospinal fluid in the adult brain. Science 2006; 311(5761):629-632. 181. Saghatelyan A, de Chevigny A, Schachner M et al. Tenascin-R mediates activity-dependent recruitment of neuroblasts in the adult mouse forebrain. Nat Neurosci 2004; 7(4):347-356. 182. Hallonet M, Hollemann T, Wehr Ret al. Vaxl is a novel homeobox-containing gene expressed in the developing anterior ventral forebrain. Development 1998; 125(14):2599-2610. 183. Soria JM, Taglialatela P, Gil-Perotin S et al. Defective postnatal neurogenesis and disorganization of the rostral migratory stream in absence of the Vaxl homeobox gene. J Neurosci 2004; 24(49):11171-11181. 184. Levy NS, Bakalyar HA, Reed RR. Signal transduction in olfactory neurons. J Steroid Biochem Mol Bioi 1991; 39(4B):633-637. 185. LongJE, Garel S, Alvarez-Dolado M et al. Dlx-dependent and -independent regulation of olfactory bulb interneuron differentiation. J Neurosci 2007; 27(12):3230-3243.

CHAPTER 3

The Role of Otx Genes in Progenitor Domains ofVentral Midbrain

Antonio Simeone, * Eduardo Puelles, Dario Acampora, Daniela Omodei, Pietro Mancuso and Luca Giovanni Di Giovannantonio

Abstract

T

he mesencephalicdopami.nergic (mesDA) neurons playa relevant role in the control of movement,behaviourand cognition. Indeed lossand/or abnormaldevelopmentofmesDA neurons is responsible for Parkinson'sdiseaseas well as for addictive and psychiatric disorders. A wealth of information has been provided on gene functions involved in the molecular mechanism controllingidentity,fate and survivalofmesDA neurons. Collectively, thesestudiesare contributing to a growingknowledgeofthe geneticnetworks required for proper mesDAdevelopment, thus disclosingnew perspectivesfor therapeutic approaches of mesDA disorders. Here we will focus on the control exerted by Otx genes in earlydecisions regulating the differentiation of progenitors located in the ventral midbrain. In this context, the regulatory network involving Otx functional interactions with signallingmolecules and transcription factors required to promote or prevent the development ofmesDA neurons will be analyzed in detail.

Introduction

Dopaminergic (DA) neurons represent collectively a specialized population of neurons distributed in the forebrain and midbrain and responsible for the synthesisof dopamine, one of the neurotransmitters ofthe vertebrate central nervous system. In the midbrain, DA neurons are located in typical positions corresponding to the ventral tegmental area (VTA), the substantia nigra (SN) and the retrorubral field (RRF). DA neurons ofthe VTA project to the ventromedial striatum, nucleus accumbens,temporal lobe and olfactory tubercle; those ofthe SN innervate the dorsolateral striatum and so do those ofthe RRF.1-3 Mesencephalic DA (mesDA)neuronsplaya crucialrolein the controlof motor,sensorimotorand motivatedbehaviours. Impairmentin survival and/or developmentof mesDAneuronsisresponsible for abnormal control of voluntary movement and cognition.r" Degeneration of the DA neurons in the SN leadsto the characteristic symptomsof Parkinson's disease, while abnormal synaptictransmissionofVTA DA neurons is involvedin psychiatricdisorderssuch as drug addiction (increased DA transmission)and anhedonia (decreased DA transmission), whereasan exaggerated forebrain (mesocorticolimbic) DA transmissiongeneratespsychoticsymptomsofschizophrenia. Thesedisorderscausedbydegeneration,abnormal developmentor dysfunction of mesDA neurons, highlight the relevanceofthis population ofneurons and the enormous effort to understand the molecular basis controlling identity and fate of mesDA progenitors as well as the survival, functioning and sensitivityto neurodegeneration of mature mesDA neurons. *Corresponding Author: Antonio Simeone-CEINGE Biotecnologie Avanzate and SEMM European School of Molecular Medicine, via Comunale Margherita 482, 80145 Naples, Italy. Email: [email protected]

Developmentand Engineering ofDopamine Neurons, edited by R.]. Pasterkamp, M.P. Smidt and J.P.H. Burbach. ©2009 Landes Bioscienceand Springer Science+Business Media.

TheRoleofOtx Genes in Progenitor Domains ofVentralMidbrain

37

During development, the anterior neuroectoderm is first regionalized in forebrain, midbrain and hindbrain and soon after this process neural cell-types with a specific fate emerge from these areas.10-1 3These events are controlled by inducing signals and depend on the responding ability of target progenitor cells. Thus, patterning signals are converted into positional identity by specific transcription factors whose regionally-restricted expression provides neuronal precursors with a precise molecular identity. 13-1 5 Induction ofventral mesencephalic progenitors depends on the inducing properties ofFgf8 and Shh signals emitted at the isthmic organizer (IsO) and floor plate, respectively, while competence in interpreting these inducing signals is provided by the molecular code defined by the expression ofdifferent transcription factors. IS-IS Several gene functions have been proven to be relevant for different aspects of mesDA differentiation. In particular, the transcription factors Pitx3, Lmxl b and En and the orphan nuclear receptor Nr4a2 (Nurr1) playa relevant role in mesDA terminal differentiation. Is-27 Here, we will focus on Otxl and Ow, two transcription factors containing a bicoid-like homeodomain and homologous to the Drosophila orthodentide (otd) gene. 2S,29 Otx genes are required for specification ofthe anterior neuroectoderm (Otx2),30 corticogenesis (Otx1),31 regionalization ofthe early anterior neural plate (Otxl and Otx2)32,33 and neural differentiation. 34-40Among these functions, we will concentrate this review on the role exerted by Otx genes on the differentiation ofventral midbrain progenitors.

Otx Genes in the Positioning ofthe Midbrain-Hindbrain Boundary (MHB)

At early somite stage the expression of the signalling molecule FgfS is activated in a relatively wide territory around the presumptive MHB and soon sharpened at the so called IsO. On the mesencephalic side ofthe IsO and adjacent to the FgfS ring ofexpression, another signalling molecule, Wnt1, is transcribed. Therefore, at the MHB the juxtaposition ofdifferently specified territories can generate an organiser centre at their interface, where cellular interactions result in the production ofsignalling molecules with inducing properties.41,42 Midbrain- and cerebellum-inducingproperties ofthe IsO have so far been demonstrated only for FgfS.43,44 In this context and, importantly, it has been shown that Fgffi and Shh are both required to induce mesDA neurons in cultured midbrain explants." Proper development and functioning ofthe IsO is determined by a complex cascade of genetic interactions. I7,IS,4S-47 In this process, an important role of Otx gene products is to control the positioning of the IsO (Fig. 1). As shown by mutant mice carrying only one Otx2 functional allele in an Otxl null background (Otxl- I-; Otx2!I-), a drastic reduction in the amount ofOtx gene products generates molecular and morphological transformation ofthe caudal forebrain and midbrain into an enlarged rostral hindbrain. 32 This territorial repatterning is the primary consequence ofan abnormal rostral repositioning ofthe Fg/8 expression domain.P A complete transformation offorebrain and midbrain territories into rostral hindbrain is exhibited by homozygous hOtx1 2/ hOtx1 2 embryos which lacked most of the Otx protein in the neuroectoderm." Conversely, ectopic expression of Otx2 in the rostral hindbrain results into a corresponding and posterior expansion of the midbrain (Fig. 1).50 Together these findings indicate a crucial dose-dependent requirement of Otx proteins that is necessary for maintenance offorebrain and midbrain identities throughout a continued repression acting on the posteriorizing genetic determinants such as the homeobox-containing gene Gbx2. Indeed, Gbx2 is required to maintain the metencephalic identity by defining its anterior border with the midbrain." Gbx2 null mutants lacked the cerebellum and exihibited caudal enlargement ofthe midbrain (Fig. 1) preceded bya corresponding posterior expansion ofthe Otx2 expression domain. Moreover, by ectopically expressing Gbx2 in the midbrain, the posterior domain of Otx2 becomes repressed (Fig. 1).52 This has suggested that Gbx2 is a posteriorising factor required to confine Otx2 activity to the anterior neural plate. This Otx2 and Gbx2 antagonism has been investigated in detail. 3s,s3 Indeed mutant embryos deficient in the neuroectoderm for both Otx2 and Gbx2 proteins (hOtx1 2/hOtx1 2; Gbx2:'I-) exhibited broad co-expression ofmidbrain and rostral hindbrain markers and, subsequently, failed to activate

38

Deuelopmen: and Enginuring ofDopamjn~Neurons

Forebrain P6-P4

P3",J

Midbra in

P2

Hindbra in

Pl

~

rn '6

'i

········ ···············F~~··················

-m ~;.:.:.···.···.·_. .

Hb

Gbx2

.~.~ .-. -Otx If~_. ~~" " " " '" . lJJ

'GbX2

Fgf

m-~,~~- - --- - - -

Hb

Gbx2

..............................

........ ...

.

~

Otx2

.-

• •_

.= ....' (\1 <\l • •

.c:

~

u ~

_

@) i

• • •_ .

·

.

0tx2

.

··-··--cF ,.1,

.. ' Fgf8

Fgf8

.

~

g

__

.. . .. . ... _--_ .. _--.. ----"-- .- . --.-

.

• • • • • • •Gbx2

Figure 1. Schematic representation of the expression pattern of Otx2 (grey), Gbx2 (dark grey) and Fgf8 (pale grey) in wild type, Otxr"; Otx2+1- , hOtxF/hOtxF, Enl0tx2!+, Gbx2-l - and hOtxF/hOtxF; Gbx2-1- mutant embryos. In general, reduction of Otx proteins as in Otxl:"; Otx2+1- or hOtxF/hOtx1 2 mutants results in anterior expansion of rostral hindbrain at the expense of midbrain or midbrain and forebrain. In contrast, lack of Gbx2 protein (Gbx2-I - ) or ectopic expression of Otx2 throughout the rostral hindbrain (EnlO tx2!+) results in a posterior expansion of the midbrain at the expense of the hindbrain. Mutant embryos lacking in the neuroectoderm both Gbx2 and Otx2 (hOtxF/hOtxF; Gbx2-1-) fail to segregateearly forebrain, midbrain and rostral hindbrain territory and do not activate regionally specific markers. Abbreviations: MHB, midbrain-hindbrain border; Hb, Hindbrain; Mb, Midbrain; Pl-6, prosomeres 1 to 6; ZLI, zona limitans intrathalamica. A color version of this image is available at www.landesbioscience .com/curie.

The RoleofOtx Genes in Progenitor DomainsofVentral Midbrain

39

forebrain and midbrain-specific geneexpression (Fig. l). In thesemutant embryos, Fgf8isexpressed throughout the entire anterior neural plate, thus indicating that its activationis independent of both Ow and Gbx2 functions and that FgfB cannot repress Otx2 without the contribution of Gbx2.The analysis of this double mutant also supports the concept that regionalsegregation of early midbrain-hindbrain markers and competence of anterior neural tissue in responding to forebrainand midbrain inducingactivities (FgfB) requiresOw. Furthermore,it hasbeen studied if the Otx2-Gbx2 dose-dependentantagonismis determined bythe relative amount of these two transcription factorsand can be reversible in appropriateand predictablegenetic conditions.P To addressthis issue, mutant embryoscarryinga singlehypomorphic Otx2 allele(Otx2A.) in an Otx2 and Gbx2 null background (Otx2"A1-; GbX~/-) havebeen compared to mutant embryoscarrying the Otx? hypomorphicallele in an Otx2 null background(Otx2/J-). Otxl'iJ- embryosdisplayed an almosthead-less phenorype/"whileOtx2/J-; Gbx~/- doublemutantsexhibitedregionalsegregation of earlymid-hindbrain markers as well as forebrain and midbrain-specific gene expression. This finding suggests that the Otx2-Gbx2 antagonism is defined by the relative amount of these two factors, sincelow levelof Otx2 protein in a Gbx2 wild type background resultsinto a head-less phenotype (posterior prevalence), whilst me samelevelof Otx2 in a Gbx2 null backgroundleads to regionalisation and maintenanceofforebrain and midbrain identities (anterior prevalencej.P A further mutant inactivatingOtx2 by the Cre-recombinase under the Enl transcriptionalcontrol has revealed additional aspectsof the MHB control exerted by Otx genes." In this mutant (Enl CW/+; Otx2floxljllJx), Otx2 waslost anteriorlyonlyin the ventralmidbrain whilemoreposteriorly it wasinactivatedin both dorsaland ventralmidbrain. MHB and FgfB expression wereanteriorly shifted,but this shift wascharacterized by a dorsal-anteriorrotation of the MHB resultingin the repattemingofthe caudaldorsalmidbraininto cerebellum. No obviousabnormalitywasdetected in the caudalventral midbrain. Sincein this mutant Otxl expression wasunaffectedand, in the caudalventralmidbrain Otxl exhibitedhigh levelof expression, it wasinvestigated whether Otxl alone was sufficient to maintain the positioning of the ventral site of the MHB. In conditional mutantslackingalsoOtxl (Enl creI+; Otx2floxljkJx; Otxl- I- ) , the expression ofFgf8but not that of Gbx2 resultedrostrallyexpandedalongthe entiremidbrain.Thesefindings have providednovelfunctional information on the control exertedby Otxgeneson MHB positioningand Fgf8 expression after £9.5. Indeed, i) this control is not uniform alongthe dorso-ventral (D-V) axisof the MHB and, due to the cooperative effectof Otxl and Otx2, it appearsmore efficient latero-ventrally; and ii) Gbx2 expression in the ventral midbrain is prevented after£9.5 byan Otx-independent negative mechanism,sincefailedanteriorisationof ventral expression of Gbx2 is insensitive to the lackof Otxl and 00:2 proteins.Interestingly, in conditionalmutantsinactivating Gbx2byEnl-driven Cre recombinase, Otx2 expression wasslightlyexpandedand only on the dorsal sideof rhombomere 1, even though Gbx2 was ablated from the entire rhombornere." One possible explanation, as suggested by the authors,is the existence of a Gbx2..independent negative and late (after8 somite stage)regulation of Otx2 expression in the rostral hindbrain. Thus,these studies35,55 suggest that the identity of ventralmidbrain and rostralhindbrain ismaintained after£9-E9.5byan Otx- and Gbx2-independentnegative control of Gbx2 and Otx2 expression, respectively.

Orx-Dose Dependent Control ofAnterior-Posterior (A-P) and Dorso-Ventral (D-V) Patterning ofthe Midbrain

Positioningof the IsO and Fgf8 expression at the MHB is an important processrelevantfor maintenanceand proper specification offorebrain and midbrain. In this process, the generalrole of Otxl and Otx2 proteins is to antagoniseposteriorizingdeterminants such asGbx2.Alongthe D-Vaxisofthe midbrain Otx2 isexpressed throughout the entireneuroepithelium whiletheventral limit of Otxl slightlyoverlaps with Shhexpression at £10.5,34 thus suggesting that Otxl and Otx2 mayhavea repressive effecton Shh expression and, as for FgfB, may be involved in determining the correct positioning of its dorsal border. This would support the attracting possibilitythat they control D-V and A-P patterning of the midbrain by definingthe contemporarypositioning of Shh and FgfB signalling molecules. Depletion of Otxl and Otx2 in the lateral midbrain of

40

Development and EngineeringofDopamineNeurons

Otxl crtl+; Otx~x/- conditional mutants resulted in a significant dorsal expansionof the Shh do-

main and a contemporaryanterior enlargementof Fg/8 expression. ThisA-P and D-Vexpansion is more pronounced in conditional mutants but alreadyevident in Otxl+I - ; OtxZ"l- embryosand is independent on Otxl or Otx2 specific-properties. This suggests that, as for Fg/8 at the MHB, a minimal threshold of Ott gene products is required also at the alar-basal boundary (ABB)to definethe correctpositioningof Shh expression."In this mutant the expression domain of genes required to confercell-type diversityalongthe D-Vaxisof the midbrain in response to Boorplate derived Shh activitywas investigated. 13-151his expression analysis indicated that gradualreduction of Ott proteins in the lateral midbrain is responsible for a proportional severity in dorsal expansionof the Shh pathway.34 Moreover, this findinghas alsoprovidedfurther evidence that a gradeddistribution of Shh controlsD-V patterning of midbrain in vivo,aspreviously reported.56 Although the mechanism controlling Shh positioning has been not yet elucidated,expression and moleculardata suggest a potential explanation. In conditional mutants the Shh expansionis invariably accompainedby a similar dorsal de-repression of Foxa2 expression and, noteworthy, in wild type embryosthe ventral border of Otxl expression coincideswith that of the corepressor Grg4.Thus Grg4 and Otxl are co-expressed at the dorsalborder of the Foxa2 domain. It has been shown that in chick and medaka embryos Otx2 and Gbx2 refine the position of Fg/8 by respectively activatingand repressing the expression of the corepressor Grg4.47.57 On this basis it is likely that the Otx-dependent antagonism operating at the MHB through the repressive effecton Gbx2 and Pax2expression, might be effective also at the ABBwhere it would control the positioning of Shh expression through the antagonisticrepression of Foxa2 (Fig. 2). In this context, since Grg4 expression is not affectedin conditional mutants, it can be argued that its specific DNA-binding partner should be down-regulatedby the Ott reduction and, in principle, it cannot be excludedthat the partner might correspondto the Ott geneproducts.Thispossibility wassupported bycell-culture experiments showingthat Grg4 mayinteract with Otx proteins and modulate their transactivating ability. Indeed, in these experiments the transactivating abilityof Otxl and Otx2 wasstronglyreducedby the addition of increasingamounts ofthe Grg4 expressing plasmid.Moreoverand importantly, Ott and Grg4 proteins mayphysically interact at least on cell-transfection extracts." Finally, the comparisonbetweenthe Ott domain interactingwith Grg4 and the Grg4 binding domain identifiedin insect and vertebratetranscriptionfactors35•58-62 revealed significant homology. Therefore, these findings strengthen the possibilitythat dorsal antagonism on Shh/Foxa2 expression at the ABB might require direct or indirect interaction with the Otx-Grg4repressing complex. Thus,sinceGrg4isableto interactwith differentclasses of transcription factorsincludingPaxand Nkx homeodomain proteins,47.58-62 thesefindingsprovide further support to the generalideathat a combinatorialseries of interactionsbetweencorepressor molecules and transcription factors belongingto differentgene families, defines a sophisticated regulatorynetworkcontrollingthe transcriptionof signallingmolecules and cellulardeterminants. It has been proposed that A-P and D-V signalling centers at specific positions along the neural tube mayinstruct the generationand the number of distinct classes of neurons accordingto their position and accessibility to the morphogenetic signal. 16.63 Basedon experimental evidence, we havesuggested that the Otx dose-dependentmechanismmight control midbrain morphogenesis through the integratedpositioningof two signalingsources operatingalongthe A-P (FgfB) or the D-V (Shh) axisat the sametime" (Fig.2). This mechanismmaycontribute to providepositional cues to neuronal precursorswithin the midbrain. Finally, based on the fact that Otxl has so far beenisolatedonlyin gnatostornes, whichhaveawelldefinedmidbrain,thisevidence alsosuggested that the Otx geneduplicationeventmight haveinfluencedbrain morphogenesis during evolution byalteringthe Ott dosageand, consequently, the neuronalresponse to the positionalcuesemitted by the IsO (Fgf8)and Boorplate (Shh) organisingtissues.

41

The RoleofOtx Genes in Progenitor Domainso/VentralMidbrain

• • •

Foxa2 + Shh Foxa2 Midbrain Hindbrain

MHB

+

Otx1 Otx2

Figure 2. Model of Otxl and Otx2 functioning in A-P and D-V patterning of the midbrain. Otxl and Otx2 are requ ired for proper positioning of signalling molecules such as Fgf8 at the MHS and Shh in the floor plate region of the midbrain. Through this dose-dependent antagonism, Otxl and Otx2 contribute to the establishment of the identity, fate and extent of midbrain progenitors. In this process positioning of Fgf8 and Shh express ion may require Otx-dependent repression of Gbx2 and Pax2 at the MHS and, pe rhaps , Foxa2 in proximity of the ASS. In this process Otxl and Otx2 might require the corepressing activity of Grg4. Abbreviations as in the legend to Figure 1 plus A, P, D, V which stand for anterior, posterior, dorsal and ventral, respectively; and ASS, alar-basal boundary.

Otx2 Regulates Extent, Identity and Fate ofProgenitor Domains in the Ventral Midbrain

Previous studies indicated that in the spinal cord and hindbrain, graded distribution of Shh activity is interpreted by class II Nkx factors, which, in turn , are critical intermediaries in the assignment ofthe identity and fate ofneuronal progenitor domains.l-" However, in the spinal cord, rostral hindbrain and midbrain the molecular code defined by Shh and Nkx expression patterns exhibits a characteristic, regionally-restricted profile. In particular, in the rostral hindbrain Nkx2.2 is coexpressed with Shh and is ventral to the Nkx6.1 domain, while in the ventral midbrain the Nkx6.l domain partially overlapped with Shh and is ventral to the Nkx2.2 expressing domain (Fig. 3). In the midbrainthe Nla6.l progenitordomaingenerates the red nucleus (RN) and oculomotor

42

Developmentand EngineeringofDopamineNeurons

(OM) neurons. More ventrally the Shh expressing domain produces mesDA neurons. On the other hand in the rostral hindbrain serotonin (Ser) neurons originate from the progenitor domain expressing Shh and Nkx2.2. Therefore, the progenitor domains for mesDA and Ser neurons appear to be different in molecular identity, since they share the expression of Shh but differ at least for the presence of Nkx2.2 and 0tx2. This suggests that the different expression code of these two progenitor domains may be relevant in the establishment ofthe Ser and mesDA neuronal phenotype. In this context, it has been shown that Nkx2.2 is essential for the coordinated generation of hindbrain Ser neurons. 64,65 Therefore, a crucial issue is to elucidate the regulatory mechanism(s) and factor (s) controlling the identity code ofmidbrain and hindbrain progenitor domains. Studies on Otx genes provided in vivo evidence that Otx2 is a major genetic determinant of this process in the ventral midbrain.v" Indeed, lack of Otx2 from E9.5 produces relevant abnormalities in the expression pattern of Sbb, Nkx6.1 and Nkx2.2 and generates a major change in the identity and fate of mesDA and RN progenitors, which, in turn, exhibit a molecular code similar to that observed in the rostral hindbrain. This strongly suggests that Otx2 is required to provide midbrain neuronal precursors with a specific differentiation code suppressing that ofthe anterior hindbrain. To perform this role, 0tx2 exerts a dual control, that is, repression of Nkx2.2 in the ventral midbrain and maintenance of the Nkx6.1 expression domain through dorsal antagonism on Shh expression. Indeed, lack of 0tx2 in the ventral and lateral midbrain of Enl'r"; Otxlflox/~x causes dorsal expansion of Sbb, loss of Nkx6.1 expression and ventral de-repression of Nkx2.2. 35 Thus, the molecular code ofventral midbrain, although anterior to FgfBand MHB shows strong similarity with that ofthe rostral hindbrain and, Ser neurons are generated in place ofmesDA and RN neurons (Fig. 3 ). In a second mutant (Otxl cre/+; OtXJ!oxl-) lacking 0tx2 in lateral but not ventral midbrain, Shh expression is dorsally expanded, the ventral domain ofNkx6.1 is lost and a remarkable increase in mesDA neuronal generation is observed'? suggesting that in this case presumptive RN precursors (normally positive for Nkx6.1 and negative for Nkx2.2) acquired the identity and fate of presumptive mesDA progenitors (positive for Shh and negative for Nkx6.1 and Nkx2.2) (Fig. 3). In sum, in Enl cre/+; Otx2floxl~x embryos where Otx2 is inactivated in ventral and lateral midbrain, progenitor domains undergo an anterior into posterior change of identity and fate (midbrain into hindbrain), while in Otxl'r", OtxJ!loxl- mutants." where 0tx2 is inactivated only in the lateral midbrain, they undergo a dorsal into ventral transformation (RN into mesDA). However in En1crd+; Otx2floxljWx embryos, OM neurons are not severelyaffected and mesDA neurons, although heavily reduced in number, are never completely abolished. For OM neurons, a likely explanation is based on the fact that, as revealed by BrdU experiments and Islet 1 (Isll) immunodetection, this neuronal cell-type is generated quite early (between E9.5 and £10) and, thereby, should be not severely affected by the Otx2 inactivation. For mesDA neurons, our data suggest that the ventralmost fraction ofprecursors is excluded from the Nkx2.2 ventralisation and thereby, retain the proper identity and fate. Why these mesDA precursors are not permissive to express Nkx2.2 remains to be answered. Complete suppression ofthe mesDA phenotype is observed only in Otx mutants exhibiting full transformation ofmidbrain into rostral hindbrain and coordinated anterior shift of both MHB and expression of Fg/8 and Gbx2 at early somite stage. 32,66 To assess whether (i) the repressive effect ofOtx2 on Nkx2.2 expression represents a permissive event required for mesDA differentiation; and (ii) mesDA into Ser neurotransmitter fate switch depends on Nkx2.2 activation in progenitor cells expressing Shh, the effect of Otx2 inactivation has been studied in the ventral midbrain ofNkx2.2 null mutants. In this triple mutant (Enl Cre/ +; OtXJ!oxljWx; Nkx2.2-/-), the mesDA population is rescued and no ectopic Ser neurons are detected. Thus, this indicates that Otx2 is required in the ventral midbrain to suppress the hindbrain differentiation program and that the repression of Nkx2.2 is a crucial event ofthis process necessary to promote the generation of mesDA neurons. These findings also suggest that in the absence of Otx2 the ventral neural tube anterior to the MHB would generate Ser neurons independendy. Interestingly, in conditional mutants lacking Otx2 in ventral midbrain, also the expression of the signalling molecule Wntl islost in this area while in triple mutants lacking Otx2 in a Nkx2.2 null background, the mesDA neurons are recovered together with the expression of Wntl suggesting that Wntl

43

'IbeRol«ofOtx Genes in Progenitor Domainso/VentralMidbrain

wild type

Midbrain

Otx1

Cre/+

Otx2

Hindbrain

floxl-

En1

Cre/+

Otx2

floxlflox

Midbrain

RN

~

DA

RN and dorsal DA

!

Ser

Figure 3. Progenitor domains and neuronal cell populations in the ventral midbrain and ventral hindbrain of wild-type and Otx2 conditional mutants . In wild-type embryos the combined analysis of Shh, Nkx6 ./ and Nkx2.2 reveals that Nkx2.2 is expressed in a restricted population of progenitors around the presumptive ABB; ventrally, the Nkx6 .1 expression domain is subdivided in two areas by Shh expression; Shh, in turn, is expressed alone only in the floor plate region . The domain expressing Nkx6.1 generates RN and OM neurons and the doma in expressing Shh alone mesDA neurons. Compared to the midbrain, in the rostral hindbrain, the main difference in the subventricular zone is represented by the ventrally expanded doma in of Nkx2 .2 which is largely co-expressed with Shh. The Nk x2.2 expressing domain generates Ser neurons. In Otxlc",I+; Otx2 floxl- conditional mutants, depletion of Otx gene products in the lateral, but not in the ventral midbrain, causes a dorsal expansion of Shh expression which might be responsible for Nkx6 .1 repression. This results in a dorsal to ventral transformation of RN into mesDA progenitors . In th is context OM neurons are marginally affected . In En/c,el+; Otx2floxlfloxmutants, lack of Otx2 in ventral and lateral midbra in results in the dorsal expansion of Shh, repression of Nk x6.1 and, importantly, ventral de-repression of Nkx2 .2. Thus, this indicates that Otx2, besides to antagonize Shh , is also required to suppress Nkx2 .2 ventrally. In this mutant , the molecular code of progenitors in the ventral midbrain is very similar to that of progenitors located in the ventral hindbra in . Indeed, presumptive RN and dorsal mesDA progenitors generate Ser neurons thus undergoing an anterior into posterior transformation of their fate and identity eventhough the ventral MHB is not affected . Only progenitors in the floor plate region where Nkx2.2 is not expressed, still retains a mesOA identity and fate. Abbreviations as in previous Figures plus OM, oculomotor nucleus; RN, red nucleus; OA, dopaminergic neurons; Ser, serotonergic neurons.

44

Development and EngineeringofDopamineNeurons

may be involved in the molecular network required to promote the generation ofmesDA neurons. This aspect has been studied in detail by analyzing mutant embryos expressing Wntl under the transcriptional control of Enl (Enl Wnt1/+) .36 In this mutant Wntl expression is activated in the floor plate ofthe anterior hindbrain and in this territory Gbx2 was repressed and Otx2 was activated, thus suggesting that Wnt 1 is sufficient to induce Otx2 expression, which in turn repressed Gbx2. To investigate whether also Otx2 is able to induce Wnt 1,a further mutant expressing Otx2 under the Enl transcriptional control (EnlOtx2l+)36 has been analyzed. In this mutant hindbrain, ectopic expression of Otx2 correlates with a corresponding activation of Wntl in the same territory. Together these data suggested that Otx2 and Wntl may be engaged in a positive feedback loop in which Otx2 is required for Wntl expression and viceversa. However the fact that, as previously mentioned, in Enl Cre/+; OtxPxljIox; Nkx2.l,-/- triple mutants Wntl expression is recovered in the ventral midbrain lacking Otx2, suggests that Otx2 is not required to maintain Wntl expression or that other factors in addition to Otx2, may compensate for lack of Otx2 and promote Wntl expression in the ventral midbrain. Moreover, to provide further support to previous findings indicating the existence ofa mesDA permissive network requiring Nkx2.2 suppression by Otx2, it has been studied ifin Enl Wntl/+ mutants, ectopic expression of Wntl in the rostral hindbrain was able to promote Otx2-mediated repression of Nkx2.2 and a DA fate switch of Ser progenitors. Strikingly, Wnt-l mediated activation of Otx2 in the ventral hindbrain resulted in repression of Nkx2.2 and in generation ofDA neurons.v These data suggest that Wntl may represent a third signalling molecule required together with Shh and FgfB to promote the generation ofDA neurons. Finally, all the mutant strains here discussed also provide evidence that the mesDA differentiation program may be affected in its identity or induced ectopically by altering the expression of Otx2 or that of Wntl. In particular these examples suggest that alteration in fate and identity ofventral midbrain or ventral hindbrain progenitors seems not to depend on the relative position of Fg/8 rather it appears that signalling molecules required for mesDA differentiation invariably require Otx2 to be properly interpreted by target progenitor cells. This implies that Otx2 should playa role in the establishment ofthe cellular competence to respond to these inducing signals and supports the idea that midbrain-polarised activity ofShh and FgfBdepends on the molecular identity of the responding tissue and may not represent an intrinsic property of these inducing molecules.

References

1. Dahlstrom A, Fuxe K. Localization of monoamines in the lower brain stem. Experientia 1964; 20:398-399. 2. Hokfelt T, Matensson A, Bjorklund S et al. Distributional maps of tyrosine hydroxylase-immunoreactive neurons in the rat brain. In: Bjorklund A, Hokfelt T, eds. Handbook of Chemical Neuroanatomy: Classical Transmitters in the CNS. Amsterdam: Elsevier, 1984; 2:227-379. 3. Bjorklund A, Lindvall O. Dopamine-contianing systems in the CNS. In: Biorklund A, Hokfelt eds. Handbook of Chemical Neuroanatomy: Classical Transmitters in the CNS. Amsterdam: Elsevier, 1984; 2:55-121. 4. ]ellinger KA. The pathology of parkinsons disease. Adv Neuro12001; 86:55-72. 5. Egan MF, Weinberger DR. Neurobiology of schizophrenia. Curr Opin Neurobiol1997; 7:701-707. 6. Klockgether T. Parkinson's disease: clinical aspects. Cell Tissue Res 2004; 318:115-120. 7. von Bohlen und Halbach 0, Schober A, Krieglstein K. Genes, proteins and neurotoxins involved in Parkinson's disease. Prog Neurobiol 2004; 73: 151-177. 8. Kelley AE, Berridge KC. The neuroscience of natural rewards: relevance to addictive drugs.] Neurosci 2002; 22:3306-3311. 9. Isacson O. On neuronal health. Trends Neurosci 1993; 16:306-308. 10. Rubenstein ]L, Shimamura K, Martinez S et al. Regionalization of the prosencephalic neural plate. Annu Rev Neurosi 1998; 21:445-477. 11. Lumsden A, Krumlauf R. Patterning the vertebrate neuraxis. Science 1996; 274: 1109-1115. 12. Wurst ~ Bally-Cuif L. Neural plate patterning: upstream and downstream of the isthmic organizer. Nat Rev Neurosci 2001; 2:99-108. 13. ]essell TM. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet 2000; 1:20-29.

The RoleofOtx Genes in Progenitor DomainsofVentral Midbrain

45

14. Edlund T, jessell TM. Progression from extrinsic to intrinsic signaling in cell fate specification: a view from the nervous system. Cell 1999; 96:211-224. 15. Briscoe J, Ericson J. Specification of neuronal fates in the ventral neural tube. Curr Opin Neurobiol 2001; 11:43-49. 16. Ye ~ Shimamura K, Rubenstein JL et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998; 93:755-766. 17. Simeone A. Positioning the isthmic organizer where Otx2 and Gbx2 meet. Trends Genet 2000; 16:237-240. 18. Prakash N, Wurst ~ Development of dopaminergic neurons in the mammalian brain. Cell Mol Life Sci 2006; 63:187-206. 19. Simeone A. Genetic control of dopaminergic neuron differentiation. Trends Neurosci 2005; 28:62-65; discussion 65-66. 20. Smidt MP, Asbreuk CH, Cox JJ er al. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmxlb. Nat Neurosci 2000; 3:337-341. 21. Saucedo-Cardenas 0, ~intana-Hau JD, Le WD et al. Nurrl is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Nat! Acad Sci USA 1998; 95:4013-4018. 22. Semina EV, Ferrell RE, Mintz-Hittner HA et al. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD. Nat Genet 1998; 19:167-170. 23. Smidt MP, van Schaick HS, Lanctot C et al. A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Nat! Acad Sci USA 1997; 94:13305-13310. 24. Smidt MP, Smits SM, Bouwmeester H et al. Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene pitx3. Development 2004; 131:1145-1155. 25. van den Munckhof P, Luk KC, See-Marie L et al. Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons. Development 2003; 130:2535-2542. 26. Nunes I, Tovmasian LT, Silva RM et ale Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Nat! Acad Sci USA 2003; 100:4245-4250. 27. Simon HH, Saueressig H, Wurst W et al. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J Neurosci 2001; 21:3126-3234. 28. Simeone A, Acampora D, Gulisano M et al. Nested expression domains of four homeobox genes in developing rostral brain. Nature 1992; 358:687-690. 29. Simeone A, Acampora D, Mallamaci A et ale A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EMBO J 1993; 12:2735-2747. 30. Acampora D, Mazan S, Lallemand Y et al. Forebrain and midbrain regions are deleted in 0tx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. Development 1995; 121:3279-3290. 31. Acampora D, Mazan S, Avantaggiato V et al. Epilepsy and brain abnormalities in mice lacking the Otxl gene. Nat Genet 1996; 14:218-222. 32. Acampora D, Avantaggiato ~ Tuorto F et ale Genetic control of brain morphogenesis through Otx gene dosage requirement. Development 1997; 124:3639-3650. 33. Martinez-Barbera JP, Signore M, Boyl PP et al. Regionalisation of anterior neuroectoderm and its competence in responding to forebrain and midbrain inducing activities depend on mutual antagonism between OTX2 and GBX2. Development 2001; 128:4789-4800. 34. Puelles E, Acampora D, Lacroix E et al. Otx dose-dependent integrated control of antero-posterior and dorso-ventral patterning of midbrain. Nat Neurosci 2003; 6:453-460. 35. Puelles E, Annino A, Tuorto F et al. Otx2 regulates the extent, identity and fate of neuronal progenitor domains in the ventral midbrain. Development 2004; 131:2037-2048. 36. Prakash N, Brodski C, Naserke T et al. A Wntl-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo. Development 2006; 133:89-98. 37. Borgkvist A, Puelles E, Carta Met al. Altered dopaminergic innervation and amphetamine response in adult Otx2 conditional mutant mice. Mol Cell Neurosci 2006; 31:293-302. 38. Puelles E, Acampora D, Gogoi R er al. Otx2 controls identity and fate of glutamatergic progenitors of the thalamus by repressing GABAergic differentiation. J Neurosci 2006; 26:5955-5964. 39. Acampora D, Simeone A. The TINS Lecture. Understanding the roles of Otxl and Otx2 in the control of brain morphogenesis. Trends Neurosci 1999; 22:116-122. 40. Simeone A, Puelles E, Acampora D. The Otx family. Curr Opin Genet Dev 2002; 12:409-415. 41. Lawrence PA, Struhl G. Morphogens, Compartments and Pattern: Lessons from Drosophila? Cell 1996; 85:951-961.

46

Development andEngineering ofDopamine Neurons

42. Martinez S, WassefM, Alvarado-MalIan RM. Induction of a mesencephalic phenotype in the 2 day-old chick prosencephalon is preceded by the early expression of the homeobox gene en. Neuron 1991; 6:971-981. 43. Crossley PH, Martinez S, Martin GR. Midbrain development induced by FGF8 in the chick embryo. Nature 1996; 380:66-68. 44. Shimamura K, Rubenstein JL. Inductive interactions direct early regionalization of the mouse forebrain. Development 1997; 124:2709-2718. 45. Joyner AL, Liu A, Millet S. Otx2, Gbx2 and FgfS interact to position and maintain a mid-hindbrain organizer. Curr Opin Cell BioI 2000; 12:736-741. 46. Rhinn M, Brand M. The midbrain-hindbrain boundary organizer. Curr Opin Neurobiol 2001; 11:34-42. 47. Ye ~ Bouchard M, Stone D et at Distinct regulators control the expression of the mid-hindbrain organizer signal FGF8. Nature Neurosc 2001; 4: 1175-1181. 48. Suda ~ Matsuo I, Aizawa S. Cooperation between Otxl and Otx2 genes in developmental patterning of rostral brain. Mech Dev 1997; 69:125-141. 49. Acampora D, Avantaggiato V, Tuorto F et at Visceral endoderm-restricted translation of 00:1 mediates recovering of Oa2 requirements for specification of anterior neural plate and proper gastrulation. Development 1998; 125:5091-5104. 50. Broccoli V, Boncinelli E, Wurst W. The caudal limit of Otx2 expression positions the isthmic organizer. Nature 1999; 401:164-168. 51. Wassarmann KM, Lewandoski M, Campbell K et at Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer dependent on Gbx2 gene function. Development 1997; 124:2923-2934. 52. Millet S, Campbell K, Epstein OJ et at A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer. Nature 1999; 401:161-164. 53. Li YH, Joyner AL. Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression. Development 2001; 128:4979-4991. 54. Pilo BoylP, Signore M, Acampora D et al. Forebrain and midbrain development requires epiblast-restricted Otx2 translational control mediated by its 3' UTR. Development 2001; 128:2989-3000. 55. Li J~ Lao Z, Joyner AL. Changing requirements for Gbx2 in development of the cerebellum and maintenance of the mid/hindbrain organizer. Neuron 2002; 36:31-43. 56. Agarwala S, Sanders TA, Ragsdale cwo Sonic hedgehog control of size and shape in midbrain pattern formation. Science 2001; 291:2147-2150. 57. Heimbucher T, Murko C, Bajoghli B et al. Gbx2 and Otx2 interact with the WD40 domain of Groucho/ Tie corepressors, Mol Cell Biol 2007; 27:340-51. 58. Eberhard D, Jimenez G, Heavey B et at Transcriptional repression by Pu5 (BSAP) through interaction with corepressors of the Groucho family. EMBO J 2000; 19:2292-2303. 59. Fisher AL, Candy M. Groucho proteins: transcriptional corepressors for specificsubsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev 1998; 12:1931-1940. 60. Fisher AL, Ohsako S, Caudy M. The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain. Mol Cell BioI 1996; 16:2670-2677. 61. Muhr J, Andersson E, Persson M et at Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 2001; 104:861-873. 62. Zhu CC, Dyer MA, Uchikawa M et at Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of corepressors. Development 2002; 129:2835-2849. 63. Wolpert L. Positional information and the spatial pattern of cellular differentiation. J Theor BioI 1969; 25:1-47. 64. Briscoe J, Sussel L, Serup P et al. Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 1999; 398:622-627. 65. Pattyn A, Vallstedt A, Dias JM et al. Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors. Genes Dev 2003; 17:729-737. 66. Brodski C, Weisenhorn DM, Signore M et at Location and size of dopaminergic and serotonergic cell populations are controlled by the position of the midbrain-hindbrain organizer. J Neurosci 2003; 23:4199-4207.

CHAPTER 4

Terminal Differentiation ofMesodiencephalic Dopaminergic Neurons: The Role ofNurrl and Pitx3

Marten P. Smidt" and J. Peter H. Burbach

Introduction

T

he orphan nuclear hormone receptor Nurr 1 and the homeobox Pitx3 were the first two transcription factors that were implicated in the development ofmesodiencephalic dopaminergic (mdDA) neurons.P'Ihese factors have their own expression profile in the brain: Nurr1 is expressed in many forebrain regions, whereas Pia3 is exclusively expressed in mdDA neurons. Functional analysis of the respective mouse mutants have emphasized the importance of both factors for mdDA development and their difference in mode of action: Nurrl has been implicated particularly in specifying the dopaminergic neurotransmitter phenotype and in neuronal maintenance, while Pia3 is essential for the development of a subset of mdDA neurons encompassing the SNc. Recent data on molecular mechanisms ofaction and regulation oftarget genes reveal a large complexity and suggest that Nurr1 and Pitx3 are part ofextended regulatory networks. In this chapter we highlight the molecular programming ofmdDA neurons'" from the viewpoint ofPitx3 and Nurr1.

Terminal Differentiation ofSubstantia Nigra Neurons Depends on the Homeobox Gene Pitx3

Identification ofPitx3

The first member ofthe pituitary homeobox family, Pitxl (Ptxl ), was cloned from AtT-20 cells and was identified as putative key component of pituitary development and function," Shortly, after the discovery ofPit xl, a second family member was cloned and named Pitx2 (Rieg, Ptx2),6-8 involved in a craniofacial dysmorphology called Rieger's syndrome. Pitx3 (Pa3) was identified shortly after by two different groups at about the same time. 2,9 Pitx3 is expressed in the eye lens, muscle and the central nervous system in different temporal domains (Fig. 1). During mouse development (-£11-£18 2) Pia3 is present in the eye lens, in skeletal muscles (not in the heart) and in mdDA neurons. After birth only in the mdDA neurons expression is maintained. In the embryonic eye-lens Pia3 is expressed in the outer rim of the emerging lens from Ell until approximately E 18.9 The important role ofPitx3 in eye development is evident in aphakia mice.9,l O-14 The aphakia allele contains 2 deletions in the Pitx3 gene proximal *Corresponding Author: Dr. Marten P. Smidt- Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Email: [email protected]

Developmentand Engineering ofDopamine Neurons, edited by R.]. Pasterkamp, M.P. Smidt and J.P.H. Burbach. ©2009 Landes Bioscience and Springer Science+ Business Media.

48

Development and Engineering ojDopamine Neurons

•

Pitx3 gene

o

••

Figure 1. Specificity of Pitx3 transcripts over the three expression domains, eye lens, skeletal muscle and mdDA neurons in the central nervous system.22.33

promoter" which leads to a lack ofPitx3 expression in the brain and eye lensl6. 17 which causes recessive arrested eyedevelopmentat the lens stalk stage.Human Picx3 mutations are causative of autosomal-dominantcataractsand dysgenesis of the anterior segment mesenchym and cause recessive neurodevelopmental abnormalitles.P'" The second embryonal expression site, the skeletal muscle,2O·21 has similar temporal Picx3 expression as the eye lens. Recently, the muscle-specific expression was linked to the use of a muscle-specific promoter in the Picx3 genelocated betweenexon 1 and 2 and initiatestranscription of an alternativeexon 1.22Thereforein the muscle a transcript isgeneratedthat consists of an alternative exon 1 and exon 2, 3 and 4 (Fig. 1). Thethird siteof expression isin the brain.The moststrikingfeatureofbrain expression ofPitx3 is the strict limitation to mdDA neurons. In double-labeling studies usingin situ hybridization and immunohistochemistrya consistentoverlap ofPitx3 and Th expression in the SNc and VTA was observed.F" Moreover, a study was published that described the knock-in of a Picx3-Gfp construct in the Picx3 locus." In this study, using a different approach, it was confirmed2.25.26 that Th and Picx3 overlapcompletelyin mdDA neurons in the adult stage." Interestingly, in the samestudy it wasshown that the expression ofTh and Pitx3 does not completelyoverlapduring development. Therearecells that express onlyTh and cells that express only Pitx3. Ishasbeen suggested that Pitx3 itselfcan be responsible for Th activationin cells werePitx3 is expressed before Th. The direct activationofTh by Picx3 has been suggested from in-vitro experiments.F'" Other studies argued against a direct activationof the Th gene by Pitx3. It has been describedthat in Lmxlb knock-out animalsPicx3 negative cells stillexpressed Th in the ventraltegmentum during development." Recently, it hasbecomeapparent that the mdDA neuronalpopulation maynot be homogeneousand that differences in molecularcodingmight causesubset-specific dependencies towardsPitx3.30.31 Thismayalsosuggest that in specific subsets Th expression maydependon Pitx3 whereas other subsetsare independent.

Consequences ofPitx3 Ablation Abnormal Development ofmdDA Neurons in the Aphakia Mouse Therestrictedexpression ofPitx3 in mdDA neuronssuggested the importanceof this homeobox genein neuronaldevelopmentand adult function. Earlyin vitro analysis on the possible function

TerminalDifferentiation ofMesodiencephalic Dopaminergic Neurons

49

ofPitx3 hinted towards a role in the regulation ofTh gene expression.F'" Analysis ofthe Lmxlb null mutant indicated that, in vivo, Th could be expressed in the absence of Pitx3 in developing dopamine neurons in the ventral tegmentum." More insight in the in vivo function ofPitx3 was gained by the analysis ofaphakia mice 26,32-35 (a natural Pin3 mutant) and Pitx3/Gfp knock-outl knock-in mice. 36,37 The aphakia mutant was discovered in the 1960s based on a eye lens defect." Initial genetic analysisofthe aphakia mouse showed a deletion in the upstream enhancer region ofthe Pitx3 gene," Later it was shown that a second deletion is present which spans the promoter area, exon 1 and part ofintron 1.39 Since the Pin3 transcription initiation site is deleted, next to a part ofthe upstream promoter region, the aphakia mouse may be considered a null mutant for the Pitx3 gene. It should be noted however that the muscle-specific transcript is still present in these mice due to the use of an alternative promoter and transcription start site (see above, Fig. 1;21). Interestingly, analysis of Pin3 expression indicated that eye expression was missing in these animals.P Furthermore, Pitx3 expression was not detectable in mdDA neurons of the aphakia mouse, both in the adult brain and in E12.5 embryos. 26,32-35 The genesis and developmental fate ofmdDA neurons in the absence ofPitx3 was determined by analyzing the expression ofTh throughout the entire midbrain from ElLS onwards." At the onset ofTh expression (ElLS), the distribution ofTh-positive neurons in the ventral midbrain is indistinguishable between wild-type and aphakia mice. The first differences in neuroanatomy are observed at E12.5 and concern the rostral part ofthe Th expression domain. At this position, the most lateral Th-positive neurons are absent in aphakia mice. In wild-type mice, Th-positive neurons accumulate in a more lateral position, fonning the SNc. This distinct cell group is absent in E12.5 aphakia embryos and onwards. As a consequence ofthe developmental defect in aphakia mice, marked changes in neurochemical and anatomical architecture of adult mdDA neurons are present.26,32-35 Th-positive neurons are virtually absent in the SNc, with the exception of the most lateral tip. In the area where the SNc and VTA overlap, as well as in part of the VTA itself: extensive cell loss was found. The loss ofTh-positive neurons is not due to loss ofTh transcripts but is a consequence ofneuronal loss as was shown by Nissl staining in adjacent sections. 26,34,40 The neurons that are present in the altered mdDA neuronal population expressthe known developmental and dopaminergic neurotransmitter synthesis genes." This suggeststhat spared neurons ofthe VTA display a normal mdDA phenotype. Furthermore, none of these genes appear to depend on Pitx3 for their expression. This supports earlier suggestions" that Pitx3 is not required for Th expression in this specific mdDA subset. However, molecular differences are present, exemplified by altered electrophysiological properties of the remaining mdDA neurons in aphakia mice." The action potential (AP) width was smaller, the 1h had a faster activation time constant, the action potential width at halfmaximum was reduced, the AP depolarisation and repolarisation rate are faster and finally, the amount of spontaneous active neurons is increased. These changes suggest that the neurons may have a different molecular make-up, although it should be noted that these differences may rely on adaptation ofthe neurons to the alterations in the network as a consequence ofmdDA neuronal loss in the SNc. Since expression data suggested that Pitx3 is expressed in all mdDA neurons.r" the specific defect detected in the SNc could not easily be explained. Initially, it was suggested that Pitx3 is expressed in only those mdDA neurons that are lost in the aphakia mouse." However, data from two other groups showed that this is not the case.26,36 Therefore, it has been suggested that the specific vulnerability of the SNc in aphakia mice as a consequence ofPitx3 ablation results from molecular differences between specific subsets of mdDA neurons within the SNc and VTA.26.30 Understanding the molecular mechanisms of Pitx3 action will ultimately unravel aspects of Pitx3-function related to SNc vulnerability and might provide new clues in understanding SNc degeneration in Parkinson's disease. Interestingly, a recent publication described the characterization ofa family with two consanguineous members that are homozygous for a deletion mutation in the Pin3 gene." This study describes, in addition to the well known eye phenotype," neurological abnormalities in these patients which might be expectedfrom the described Pitx3-I- brain phenotype in mice.The neurological

50

Development and EngineeringojDopamineNeurons

abnormalities arestaticin timeand includechoreiformmovementof the headand to alesser extent the trunk. A more in depth analysis ofthese patients might enlight the role of Pitx3 in relation to mdDA function in humans. mdDA neurons are generated in the mesodiencephalic neuroepithelium at the ventricular zone.Dopaminergicprecursorsmigratefrom the neuroepitheliumto the ventralmidbrainwhere they adopt the full dopaminergicphenotype." An earlymarkerfor these migratingprecursorsis L-amino aciddecarboxylase (AadC),26 expressed two daysearliercomparedto Th.42 In the ventral positionTh and Pia3 are lnduced.P'Iherestrictedexpression ofPitx3 at the ventralpositionof the developingmldbrain'" suggests that Pitx3 is not directlyinvolved in proliferationnor migration of dopaminergicprecursors.Although, it cannot be excludedthat Pitx3 inducesgenesinvolved in the generation of guidance cuesfor migratingprecursors. Taken together, the mechanismby which Pitx3influences survival ofSNc dopaminergicneuronsisunknown and intriguing.mdDA progenitors arrivingat the primordial SNc mayneed signals to be vitalized. Suchsignals maybe providedbythe localgrowthfactorenvironmentor through direct cell-cell contacts." Pitx3isapparentlyrequiredin a crucialdifferentiationand/or vitalizationstep,maybebyinducingreceptors for suchsignals. Through analysis of direct and indirect transcriptionaltargetsofPitx3 the actual differentiatingactivityofPitx3 maybe elucidated(seebelow).

Nigro-Striatal Connectivity As a consequence of neuronal lossin the SNc and the VTA, connectionsto the striatalareas are severely affected.26,32-35 Projectionsto the caudate putamen are virtually absent whereasthe nucleus accumbens and the olfactory tubercle seem normally innervated. Dopamine measurements of dissectedbrain areasby high pressureliquid chromatographysuggest that the dorsal striatum is depleted of dopamine (93%decrease) and that the ventral striatum has a reduction of 69 %.3S Similaranalysis byothers showedthat the dorsalstriatum showsan -80-909634•40 and the ventral striatum is not reduced decline in dopamine content at all.34 Theseexperiments indicate the total dopamine content and are strictlydependent on the sampleposition. Fast scan cyclic voltametryanalysis, a measureof locallyevokeddopamine release,44.45 has shownthat the evoked release is severely reduced in the caudateputamen whereasthe nucleusaccumbens showedonly a minor decrease." All the available data clearly indicate that the dopaminergicinnervation by the mdDA systemis loweredespecially in the caudateputamen as a consequence ofearlylossof a subsetof mdDA neurons. Theapparentlackof connectivityof the mdDA neuronsto the caudateputamenwasconfirmed by retrogradetracingstudies.26•4O In wild-typemice,fluorogold-labeled neurons weredetected in the SNc and the VTA after electrochemical injectionsin the caudateputamen. Double labeling studiesusingPitx3 antibodies confirmedthe identity of the traced neurons as mdDA neurons." It wasshown that aphakiamicelackanyretrogradetracingsignalafterdorsalstriatalinjectionsof fluorogold. It is unknown at present whether any rewiringhas occurredbetween remainingdopamine neurons in the aphakiamidbrain and target areasas the nucleusaccurnbens, the olfactory tubercleand the prefrontal cortex.Takentogether,connectionsbetween mdDA neurons and the caudateputamen are lost, most likelyas a consequence of initial neuronalloss. Behavioral Consequences ofPitx3 Mutation The aphakiamouseisviableand capable of reproduction without specialmeasures. This indicates that the defect in vision and brain function is not impairingvital functions of the animal. However, specific deficitsin mdDA function havebeen described.26.35.41.46.47 Initially, Pitx3mutant mice(aphakia)werepresentedasParkinsonmicebasedon the neuroanatomicaldefectand initial behavioraltesting.35•47In a 24hour recordingsetup it wasshownthat thesemicedisplayed a lower overallactivityas compared to control. It should be noted, however, that the experiments were performed with normal C 57/B16 control mice. In this waystrain differences mayhaveattributed to the describedphenotype. The effects of the Pitx3 ablation on walkingpatterns wereanalyzed in comparisonto C 57/B16 control mice.26Theseanalyzes indicated no abnormalposture and no aberrant walkingsequence.PAdditional analysis of climbingbehavior" showeda clearincrease

TerminalDiJfirentiation ofMesodiencephalic Dopaminergic Neurons

51

in performance.P Although this increasewas also observed in a blind control group, the aphakia animals scored clearlyhigher.26 It was concluded that the dopaminergic output to the nucleus accumbens may be increased or less well controlled partly due to the abnormally low levelsof DAT.26 The possibility of treatment of the aphakia phenotype was extensively studied and it was shown that the behavioralphenotype could partially be rescued through the treatment with L-Dopa.47•49 This suggestedthat, despite the lost innervation of the dorsal striatum by Th fibers, L-Dopa isconverted to dopamine and releasedin the striatum to overcomethe hypoactivestate in the light phase.A recent paper has compared Pitx3-1- mice with littermate controls (Pin3+/- and +/+).46 Interestingly, the initial observation ofhypoactivitywaslost and wastherefore due to strain differences caused by extensive inbred breeding of aphakia mice. The data of 24 hour activity recordings ofaphakia compared to control CS7/BI6 animalsconfirmed earlierdata, whereasthe Pitx3-1- compared to littermate Pitx3+/+ showedthat the activitydifferences, especiallyin the dark phase are not present. These data together with neurochemical data showing the compensation ofthe system41.47 suggestthat the nigrostriatal systemis damaged by the neuronal loss in aphakia mice, but is able to compensate this loss in terms of overall activity. More detailed analysis of the behavioralphenotype has shown that Pin3 ablation induces (I) increasedwakefulness with increasedspontaneous behaviorduring the sleepcycle(light phase) and (2) reduced frequencyof rearing and general horizontal movement. The increased spontaneous behavior during the sleep cyclehas been attributed to increasedserotonin signaling.A recent study described the increased amount of Sert binding and the lossof lightphase-hyperactivity afteradministration ofthe 5-HT blocker (inhibitor oftryptophane hydroxylase) p-chlorophenylalanin (PCPA) to aphakiamice." The apparent hyperinnervation by 5-HT neurons ofthe striatum provides the mechanism ofthe describedbeneficialeffectsof Ldopa." The S-HT neurons can convert the L-Dopa to dopamine by means ofAadc and corelease dopamine with serotonin. In conclusion, Pitx3 ablation in mice causesdis-regulation ofDA signalingover the 24 hour cycleresultingin hyperactivityduring the sleepphase and an increasein consolidation ofspecific movement components.

Transcriptional Targets ofPitx3

TransfectionofES cellswith Pitx3induced neurons that expressed the aldehydedehydrogenase 2 (Ahd2, Raldh l, Raldh la) gene." Thesedata suggestedthat the Ahd2 gene maybe a transcriptional target ofPitx3. However,Ahd2 isalreadyexpressed during earlydevelopment in proliferating DA progenitor cells,s2 whereas Pitx3 is expressedlater in development, indicating that Pitx3 is not required for the initial expressionofAhd2. Recent data have shown that there is a second Ahd2-activating phase in a specific subset of mdDA neurons that relieson Pitx3,31 It was shown that a direct interaction between the Pitx3 protein and the Ahd2 promoter existsin MN9D cells and that Pitx3 activatesthe Ahd2 gene in vitro and in vivo (Table I). Moreover,the conversion of retinal to retinoic acid (RA) catalyzedby Ahd2 s3.sSdrives the terminal differentiation of the Ahd2 expressingsubset of mdDA neurons. Moreover,the mdDA neuronal lossphenotype, as a consequence of Pitx3 ablation, can be rescued by the application of RA. This suggests that RA, as an enzymaticproduct, is a keymoleculein earlyand late mdDA development and that the late function is established through the expressionofPitx3. A functional associationwas shown between Pitx3 and expressionof a member of the small leucinerich proteoglycansgenefamily, mimecan.The high levelof mimecan expressionwasfound in human osteoblasticMG-63 cellsin which Pitx3 is bound closeto the mimecan gene as shown by chromatin immunoprecipitation (ChIP). These data suggestthat Pitx3 may act as a positive regulator of mimecan transcription.56 Although the Th gene can be activated without Pitx3, the apparent roleofPitx3 in the activationof the Th genewassuggested."Theauthors showedthat in a homozygousPitx3-Gfp knock-in/Pitx3 knock-out animalGfppositivecells arepresent that do not express Th. From that experiment it wasconcluded that, since mdDA neurons activatethe Pitx3 locus as shown by the expressionof Gfp and fail to express Th, Pitx3 is involvedin activatingTh in those neurons. In addition, it wasshown that the co-expression ofPitx3 and Th is not complete

52

Development and EngineeringofDopamineNeurons

Table 1. Overview of described Pitx3 transcriptional targets TargetGene

Tissue

Ahd2

Pitx3 transfected ES-cells51and mdDA neurons" mdDa neurons" MG-G3 cells" SH-SY5Y cells and ventromidbrain culture (e14.5)57 SH-SY5Y cells and ventromidbrain culture (e14.5)57

Th

Mimecan Gdnf Bdnf

in developmentalstages (EI2-EI4), indicatingthat the activationofTh is may be dependingon initial activationby Pitx3 in specific subsetsof mdDA neurons. Finally, it wasrecentlysuggested that Pitx3 mayhavea rolein the activationofBdnfand Gdnf. In SHSYSYcellsand in transfectedprimaryculturesofventralmidbrain isolates the introduction ofPitx3leads to increasedendogenousexpression ofBDNF and Gdn£57

Molecular Mechanism ofPitx3 Activity

Expression data showthat Pitx3 isexpressed at the ventralsideof the mesodiencephalon. The neurons that are present at this location are youngmdDA neurons that undergo terminal differentiation. Thissuggests that Pitx3 isinvolved in terminaldifferentiationand, maybe, initiation of axonaloutgrowth and/or late migration events.4,26 The recentdata suggesting that the Ahd2 gene is a direct transcriptionaltarget ofPitx3 in a subsetof mdDA neurons indicate that RA-initiated terminaldifferentiationisdependingon Pitx3in the Ahd2 expressing subset.Carefulcomparison of the neuronsthat lack Th expression in Pitx3-I- ; Gfp+l+ micesuggests that thissubsetof neuronsis similarto the Ahd2 subsetof neurons.Thissuggests that theseneuronsaredistinct from the other mdDA neurons in the fact that Pitx3 is both involved in the activationof Ahd2 and Th, whereas the formerisessential to driveterminaldifferentiationof this subset.In addition, the re-activation of terminaldifferentiationand the appearance ofTh, in this subsetbythe applicationofexogenous RA31 suggest that both genesareactivatedbyPitx3.Although,the possibilitythat Th activationis indirectlycausedbyPitx3and thereforea resultofRA-signalingcannot be discarded. In conclusion, the Pitx3targetsAhd2 and Th areprobablydirect and indirect transcriptionaltargetsrespectively. Thisalsoprovidesinsight in Pitx3 targetsto be uncoveredin the future, sincemanygenesmaybe activatedupon stimulationby RA in the Ahd2 subsetof mdDA neurons.

Nurrl Is Essential for Generating the Full Dopaminergic Phenotype ofMesodiencephalic Dopaminergic Neurons Cloning andExpression ofNurrl

The nuclearhormone receptor Nurr 1 (Nr4a2) wascloned from a mousebrain eDNA library and identified as a paralog of Nurr77 (Nr4al) and Nor l (Nr4a3).58 In contrast to most other nuclear receptors,no ligand has been identifiedfor Nurr L'" Moreover, the protein can act fully activewithout anyligandand maybe modulated through intracellularsignaling and phosphorylation events." DifferentisoformofNurrrI exist,one usesan alternativeexonl and an other usesan alternativeexon7, this latter form iscalledNurr2.60,61Theusage of an alternativeexon 1wasfound through the existence of EST sequences. Nurr I caninteractwith other nuclearhormone receptors as Rxr and canactasamonomer. Detailedexpression analysis showedthat NurrI ishighlyexpressed in the olfactorybulb, the enthorhinal cortex,the posteriorhypothalamicnucleus, the centralgray matter, the motor nucleusof the vagusnerve,the substantianigra,the ventral tegmentalareathe retrorubral fieldand rostral-caudal linear nucleusraphe.62-64

TerminalDifferentiation ofMesoJiencephalic Dopaminergic Neurons

53

Nurrl Is Essential ofthe TerminalDifferentiation ofmtiDA Neurons

Initial analysis ofthe Nurr 1 knock out animal suggestedthat ablationofNurr 1 causedagenesis of mdDA neurons.1 Analysis of a different Nurrl knock-out suggested that the young mdDA neurons were present but failed to activate the 1h enzyme.The presence of the mdDA neurons was established through the expressionofPitx3.65 This indicated that Nurrl is essentialfor the formation of dopamine neuronsin the ventralmesodiencephalon. Analysis oflater stagesof mdDA developmentin thesemiceindicated that the neurons aregraduallylost and aremostlydisappeared at PO.65.661he other brain systems that expressNurr 1 are spared after Nurr 1 ablation.f The initial differencein interpretation ofthe different knock-outs is merelycausedby the definitions that are used to define a dopamine neurons. The agenesis that was discussedis based on the fact that no fullymatured dopamine neurons arisein the ventral tegmentum. At this point there isno evidence that supports the possibilitythat the different knock-outs havedifferent phenotypes.

Transcriptional Targets anJMechanism ofAction ofNurrl

The neurotransmitter phenotype is,amongst others,determined byNurr 1,sinceTh,Vmat,Dat and Ret are regulated through Nurr 1.1•67-69 In addition, recent studies have increased the list of identified transcriptional targets (Table2). It becomesclear,from this list, that Nurrl is essential for the activation of many genes that are involved in mdDA development and adult function. These include maintenance through Ret and specificationof the transmitter phenotype through Th,Vmat2,Aadc and Dat, In addition, the interestinginteraction with- and activationofpS7Kip2 byNurr 170suggestthat Nurr 1 isassociatedwith other proteins to exert its mode ofaction (Fig.2). Ablation ofpS7Kip2 results on dramatic reduction ofTh expressionsuggestingthat Nurr 1 and PS7Kip2 cooperate in drivingexpressionofTh, although it should be noted that in these animals also Nurr 1 is diminished. Elegant experimentsto unravelthe function ofRxr-Nurr 1 heterodimers":" asputative downstream effectors of RA (9-cis-RA) signalingwere performed by using chimeric forms of Nurr 1 coupled to Ga14. This experimental approach revealed the Rxr-ligand-bound-dependence of Nurr 1 signalingin the ventral midbrain during mdDA development." After isolation of the most activefraction in a Nurr 1-Rxr reporter assay derived from £15.5 midbrain tissue,the Rxr ligand was shown to be a docosahexaenoic acid {DHA)73.74 instead of9-cis-RA. Signalingthrough the Rxr-Nurr1 heterodimer was found to be involved in mdDA neuronal survival, which might be crucial in relation to the described mdDA survivaldefect in Nurr 1-1- mice. Interestingly, activation ofthe Rar pathwaythrough Rxr /Rar heterodimers and 9-cis-RA, negativelyinfluenced.The question remains whether direct signaling ofall-trans RA through Rar plays a part in developing mdDA neurons, as suggestedby the rescueofthe Pitx3-1- phenotype by RA (see above).

Table 2. Overview of reported Nurr1 transcriptional targets Transcriptional Target Gene

Tissue

Ret Vmat2 Aadc

mdDA neurons" mdDA neurons" mdDA neurons"?", MN9D cells" mdDA neurons1,67 mdDA neurons" MN9D cells" midbrain cultures" ventral midbrain" mdDA neurons'?"

Th

Oat Vip Bdnf Gtp Cyclohydrolase p57kip2 Os teocaIcin CypllB2 Neuropilin 1

Osteoblast" H295R adrenocortical cells" developing brain stem'"

Development andEngineering ofDopamine Neurons

54

High lranscriptlonal aetlvity

rumK~'\ N<JIrl

J

CcHepressor complex

Low transcriptional aetlvi1y

mdOA neuronal developmenl _

Figure 2. Molecular circuit of co -activators and repressors of Nurrl that converge signaling towards the differentiation and maintenance of mdDA neurons.

Another level of regulation of Nurr1 is provided by beta-catenin throughWnt signaling."

In the absence ofWnt signaling Nurrl is bound by Lef-l in a so-called corepressor complex.

After Wnt signaling and subsequent stabilization of beta-catenin Lef-l is released from Nurr1 and beta-carenin forms a complex with Nurrl and Lef-I . Both complexes can then exert their transcriptional activity on their specific promoters (Fig. 2) . In SK-N-MC cells the activity of the Nurrl/beta-carenin complex in addition to regulation by Lef-l/betacatenin was confirmed by the transc riptional activation of the Kcnip4 gene. In the promoter ofthis gene both recognition sequences were found and the gene was suggested to be regulated by both complexes.Ylt remains to be determined whether these type ofinteractions are involved in mdDA neurons and which players ofthe Wnt signaling cascade are present. The authors suggested a model in which Wnt signaling activates both pathways, the Tcf/Lefpathway and the NBRE (Nurrl responsive element) driven targets. In another study protein-protein interactions ofNurr1were analyzed by using a pull-down assaywith extracts of the dopaminergic cell line CSM 14.176 Complexes with Erk2 or -5 showed high transcriptional activity and Nurr 1was able to recruit ErkS to an NBRE suggesting that Nurr 1 itself may be a substrate for ErkS. On the other hand, the identified interaction of Nurr1 with Lim-kinase (LimK) resulted in a lower transcriptional activity. This type ofinteractions suggests that Nurr1 may act to converge many cellular signals and translate those to specific transcriptional events. It is at present unknown whether these interactions with Erk2/S and LimK are relevant for the regulation ofgene expression in in-vivo mesodiencephalic dopaminergic neurons.

Concluding Remarks

From the above described data it is clear that both Pitx3 and Nurr1 are crucial regulators of gene expression in the development and maintenance of mdDA neurons. Moreover, the initial studies did not reveal any of the real complexities of the transcriptional events that underlie development of these neurons and more specifically development of subsets of these dopamine neurons." Through the analysis of transcriptional targets and interactors of Nurr1 and Pitx3 it will become more clear how these two transcription factors drive terminal differentiation and how identified mechanisms ofaction can provide new clues for drug development to cure pathologies as Parkinson's disease.

TerminalDifferentiation ofMesodie»cephalic Dopaminergic Neurons

References

55

1. Zetterstrom R, Solomin L, Jansson L ec al. Dopamine neuron agenesis in Nurr l-deficient mice. Science 1997; 276(5310):248-250. 2. Smidt M, vanSchaick H, Lanctot C et al. A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Nad Acad Sci USA 1997; 94(24):13305-10. 3. Smits SM, Smidt MP. The role of Pitx3 in survival of midbrain dopaminergic neurons. J Neural Transm Suppl 2006; 70:57-60. 4. Smidt MP, Burbach JPH. How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci 2007; 8(1):21-32. 5. Lamonerie T, Tremblay JJ, Lanctot C et al. Ptxl, a bicoid-related homeo box transcription factor involved in transcription of the pro-oplomelanocortin gene. Genes Dev 1996; 10(10):1284-95. 6. Semina E, Reiter R, Leysens N et al. Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in rieger syndrome. Nat Genet 1996; 14(4):392-9. 7. Gage PJ, Camper SA. Pituitary homeobox 2, a novel member of the bicoid-related family of homeobox genes, is a potential regulator of anterior structure formation. Hum Mol Genet 1997; 6(3):457-64. 8. Smidt M, Cox J, vanSchaick H et al. Analysis of three Ptx2 splice variants on transcriptional activity and differential expression pattern in the brain. J Neurochem 2000; 75(5):1818-25. 9. Semina E~ Reiter RS, Murray JC. Isolation of a new homeobox gene belonging to the pitx/rieg family: expression during lens development and mapping to the aphakia region on mouse chromosome 19. Hum Mol Genet 1997; 6(12):2109-2116. 10. Varnum DS, Stevens LC. Aphakia, a new mutation in the mouse. J Hered 1968; 59(2):147-50. 11. Grimm C, Chatterjee B, Favor er al. Aphakia (ak), a mouse mutation affecting early eye development: fine mapping, consideration of candidate genes and altered pax6 and six3 gene expression pattern. Dev Genet 1998; 23(4):299-316. 12. Graw J. Mouse models of congenital cataract. Eye 1998; 13(Pt 3b):438-44. 13. Graw J. Cataract mutations and lens development. Prog Retin Eye Res 1999; 18(2):235-67. 14. Graw J, Loster J. Developmental genetics in ophthalmology. Ophthalmic Genet 2003; 24(1):1-33. 15. Rieger DK, Reichenberger E, McLeanW et aI. A double-deletion mutation in the pitt3 gene causes arrested lens development in aphakia mice. Genomics 2001; 72( 1):61-72. 16. Smidt MP, Smits SM, Burbach JP. Molecular mechanisms underlying midbrain dopamine neuron development and function. Eur J Pharmacol 2003; 480(1-3):75-88. 17. SmidtMP, Smits SM, Bouwmeester H et al. Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene pitx3. Development 2004; 131(5):1145-55. 18. Semina EY: Ferrell RE, Mintz-Hittner HA et aI. A novel homeobox gene pitx3 is mutated in families with autosomal-dominant cataracts and asmd. Nat Genet 1998; 19(2):167-170. 19. Bidinost C, Matsumoto M, Chung D et al. Heterozygous and homozygous mutations in PITX3 in a large Lebanese family with posterior polar cataracts and neurodevelopmental abnormalities. Invest Ophthalmol Vis Sci 2006; 47(4):1274-1280. 20. Smidt MP, Smits SM, Burbach JPH. Homeobox gene Pitx3 and its role in the development of dopamine neurons of the substantia nigra. Cell Tissue Res 2004; 318(1):35-43. 21. L'Honore A, Coulon Y: Marcil A et al. Sequential expression and redundancy of pitx2 and pitx3 genes duringmuscle development. Dev Biol 2007; 307(2):421-433. 22. Coulon V, L'Honore A, Ouimette JF et al. A muscle-specific promoter directs pitx3 gene expression in skeletal muscle cells. J BioI Chern 2007; 282(45):33192-33200. 23. Smidt MP, Asbreuk CH, Cox JJ et al. A second independent pathway for development of mesencephalic dopaminergic neurons requires lmxlb. Nat. Neurosci 2000; 3(4):337-41. 24. Zhao S, Maxwell S, Jimenez- Beristain A et al. Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. Eur J Neurosci 2004; 19(5):1133-40. 25. Smidt M, Asbreuk C, Cox J et al. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmxlb. Nat Neurosci 2000; 3(4):337-41. 26. Smidt MP, Smits SM, Bouwmeester H et al. Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3. Development 2004; 131(5):1145-55. 27. Cazorla P, Smidt M, O'Malley K et al. A response element for the homeodomain transcription factor Ptx3 in the tyrosine hydroxylase gene promoter. J Neurochem 2000; 74(5):1829-37. 28. Lebel M, Gauthier Y, Moreau A er al. Pitx3 activates mouse tyrosine hydroxylase promoter via a high-affinity binding site. J Neurochem 2001; 77(2):558-67. 29. Messmer K, Remington MP, Skidmore F et al. Induction of tyrosine hydroxylase expression by the transcription factor pitx3. Int J Dev Neurosci 2007; 25(1):29-37. 30. Smits SM, Burbach JPH, Smidt MP. Developmental origin and fate of mesodiencephalic dopamine neurons. Prog Neurobiol 2006; 78(1):1-16.

56

Development and EngineeringofDopamineNeurons

31. Jacobs FMJ, Smits SM, Noorlander CW et ale Retinoic acid counteracts developmental defects in the substantia nigra caused by pitx3 deficiency, Development 2007; 134(14):2673-2684. 32. Burbach JPH, Smits S, Smidt MP. Transcription factors in the development of midbrain dopamine neurons. Ann N Y Acad Sci 2003; 991:61-8. 33. Smidt MP, Smits SM, Burbach JPH. Molecular mechanisms underlying midbrain dopamine neuron development and function. Eur J Pharmacol2003; 480(1-3):75-88. 34. Hwang D~ Ardayfio P, Kang UJ er al. Selective loss of dopaminergic neurons in the substantia nigra ofPitx3-de6.cient aphakia mice. Brain Res Mol Brain Res 2003; 114(2):123-31. 35. van denMunckhof P, Luk KC, Ste-Marie L et al. Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons. Development 2003; 130(11):2535-42. 36. Zhao S, Maxwell S, Jimenez-Beristain A et al. Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. Eur J Neurosci 2004; 19(5): 1133-40. 37. Maxwell SL, Ho H~ Kuehner E et ale Pitx3 regulates tyrosine hydroxylase expression in the substantia nigra and Identifies a subgroup of mesencephalic dopaminergic progenitor neurons during mouse development. Dev Bioi 2005; 282(2):467-79. 38. Semina EV: Murray JC, Reiter Ret al. Deletion in the promoter region and altered expression of pitx3 homeobox gene in aphakia mice. Hum Mol Genet 2000; 9(11):1575-1585. 39. Rieger 0, Reichenberger E, McLean W et al. A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice. Genomics 2001; 72(1):61-72. 40. Nunes I, Tovmasian LT, Silva RM et al. Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Nat! Acad Sci USA 2003; 100(7):4245-50. 41. Smits SM, Mathon OS, Burbach JPH et al. Molecular and cellular alterations in the Pitx3-de6.cient midbrain dopaminergic system. Mol Cell Neurosci 2005; 30(3):352-63. 42. 'Ieirelman G, Jaeger CB, Albert V et al. Expression of amino acid decarboxylase in proliferating cells of the neural tube and notochord of developing rat embryo. J Neurosci 1983; 3(7):1379-88. 43. Hynes M, Poulsen K, Tessier-Lavigne M et al. Control of neuronal diversity by the floor plate: contact-mediated induction of midbrain dopaminergic neurons. Cell 1995; 80(1):95-101. 44. Davidson C, Ellinwood EH, Douglas SB et ale Effect of cocaine, nomifensine, gbr 12909 and win 35428 on carbon fiber microelectrode sensitivity for voltammetric recording of dopamine. J Neurosci Methods 2000; 101(1):75-83. 45. Zhuang X, Oosting RS, Jones SR et al. Hyperactivity and impaired response habituation in hyperdopaminergic mice. Proc Nad Acad Sci USA 2001; 98(4):1982-7. 46. Kas MJH, van derLinden AJA, Oppelaar H et al. Phenotypic segregation of aphakia and pitx3-null mutants reveals that pitx3 deficiency increases consolidation of specific movement components. Behav Brain Res 2008; 186(2):208-214. 47. van denMunckhofP, Gilbert F, Chamberland Met ale Striatal neuroadaptation and rescue of locomotor deficit by I-dopa in aphakia mice, a model of parkinson's disease.} Neurochem 2006; 96(1):160-170. 48. Costall B, Naylor R], Nohria V. Climbing behaviour induced by apomorphine in mice: a potential model for the detection of neuroleptic activity. Eur] Pharmacol1978; 50(1):39-50. 49. Hwang DY: Fleming SM, Ardayfio P et al 3,4-dihydroxyphenylalanine reverses the motor deficits in Pitx3-deficient aphakia mice: behavioral characterization of a novel genetic model of Parkinson's disease. J Neurosci 2005; 25(8):2132-7. 50. Smits SM, Noorlander C'W: Kas M]H et al. Alterations in serotonin signalling are involved in the hyperactivity of pitx3-deficient mice. Eur J Neurosci 2008; 27(2):388-395. 51. Chung S, Hedlund E, Hwang M et al. The homeodomain transcription factor Pia3 facilitates differentiation of mouse embryonic stem cells into AHD2-expressing dopaminergic neurons. Mol Cell Neurosci 2005; 28(2):241-52. 52. Wallen A, Zettersrrom R, Solomin L et ale Fate of mesencephalicAHD2-expressing dopamine progenitor cells in NURRI mutant mice. Exp Cell Res 1999; 253(2):737-46. 53. Blentic A, Gale E, Maden M. Retinoic acid signalling centres in the avian embryo identified by sites of expression of synthesising and catabolising enzymes. Dev Dyn 2003; 227(1):114-127. 54. Fan X, Molotkov A, Manabe SI et al. Targeted disruption of Aldhlal (Raldhl) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina. Mol Cell Biol 2003; 23(13):4637-4648. 55. Molotkov A, Dnester G. Genetic evidence that retinaldehyde dehydrogenase raldh l (aldhlal) functions downstream of alcohol dehydrogenase adhl in metabolism of retinol to retinoic acid. ] Biol Chern 2003; 278(38) :36085-36090. 56. Tasheva ES, Klocke B, Conrad GW Analysis of transcriptional regulation of the small leucine rich proteoglycans. Mol Vis 2004; 10:758-72.

TerminalDifferentiation ofMesodiencephalic Dopaminergic Neurons

57

57. Peng C, Fan S, Li X et al. Overexpression of pitx3 upregulates expression of bdnf and gdnf in sh-sy5y cells and primary ventral mesencephalic cultures. FEBS Lett 2007; 581(7):1357-1361. 58. Law S, Conneely 0, DeMayo F et al. Identification of a new brain-specific transcription factor, NURR1. Mol Endocrinol 1992; 6(12):2129-35. 59. Shiau AK, Coward P, Schwarz M et al. Orphan nuclear receptors: from new ligand discovery technologies to novel signaling pathways. Curr Opin Drug Discov Devel2001; 4(5):575-590. 60. Michelhaugh SK, Vaitkevicius H, Wang J et ale Dopamine neurons express multiple isoforms of the nuclear receptor nurrl with diminished transcriptional activity. J Neurochem 2005; 95(5):1342-1350. 61. Ohkura N, Hosono T, Maruyama K et al. An isoform of nurrl functions as a negative inhibitor of the ngfi-b family signaling. Biochim Biophys Acta 1999; 1444(1):69-79. 62. Saucedo-Cardenas 0, Conneely OM. Comparative distribution of nurrl and nur77 nuclear receptors in the mouse central nervous system. J Mol Neurosci 1996; 7(1):51-63. 63. Xiao ~ Castillo S, Nikodem V. Distribution of messenger RNAs for the orphan nuclear receptors Nurrl and Nur77 (NGFI-B) in adult rat brain using in situ hybridization. Neuroscience 1996; 75(1):221-30. 64. Backman C, Perlmann T, Wallen A et al. A selective group of dopaminergic neurons express nurr1 in the adult mouse brain. Brain Res 1999; 851(1-2):125-132. 65. Saucedo-Cardenas 0, Quintana-Hau JD, Le WD et al. Nurrl is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Nat! Acad Sci USA 1998; 95(7):4013-8. 66. LeW, Conneely 0, Zou L et al. Selective agenesis of mesencephalic dopaminergic neurons in Nurr l-deficient mice. up Neuro11999; 159(2):451-8. 67. Smits SM, Ponnio T, Conneely OM et al. Involvement of Nurr1 in specifying the neurotransmitter identity of ventral midbrain dopaminergic neurons. Eur Jneurosci 2003; 18(7):1731-8. 68. A WA, Castro D, Zetterstrom Ret al. Orphan nuclear receptor Nurrl is essential for Ret expression in midbrain dopamine neurons and in the brain stem. Mol Cell Neurosci 2001; 18(6):649-63. 69. Saucedo-Cardenas 0, Q uintana-Hau J, Le W et al. Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Nat! Acad Sci USA 1998; 95(7):4013-8. 70. Joseph B, Wallen-Mackenzie A, Benoit G er al p57(Kip2) cooperates with Nurrl in developing dopamine cells. Proc Nat! Acad Sci USA 2003; 100(26):15619-24. 71. Perlmann T, Jansson L. A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev 1995; 9(7):769-82. 72. Aarnisalo P, Kim CH, Lee JW et al. Defining requirements for heterodimerization between the retinoid X receptor and the orphan nuclear receptor Nurr l. J BioI Chern 2002; 277(38):35118-23. 73. Wallen-Mackenzie A, deUrquiza AM, Petersson S et al. Nurrl-RXR heterodimers mediate RXR ligand-induced signaling in neuronal cells. Genes Dev 2003; 17(24):3036-47. 74. deUrquiza AM, Liu S, Sjoberg M et ale Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain. Science 2000; 290(5499):2140-2144. 75. Kitagawa H, Ray WJ, Glantschnig H et al. A regulatory circuit mediating convergence between nurr 1 transcriptional regulation and wnt signaling. Mol Cell Bioi 2007; 27(21):7486-7496. 76. Sacchetti P, Carpentier R, Segard P et al. Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor nurr1. Nucleic Acids Res 2006; 34(19):5515-5527. 77. Gil M, McKinney C, Lee MK et al. Regulation of gtp cyclohydrolase i expression by orphan receptor nurrl in cell culture and in vivo. J Neurochem 2007; 101(1):142-150. 78. Jankovic J, Chen S, Le WD. The role of nurrl in the development of dopaminergic neurons and parkinson's disease. Prog Neurobiol 2005; 77(1-2):128-138. 79. Hermanson E, Joseph B, Castro D et al. Nurrl regulates dopamine synthesis and storage in MN9D dopamine cells. Exp Cell Res 2003; 288(2):324-34. 80. Luo Y, Henricksen LA, Giuliano RE er al. Vip is a transcriptional target of nurr1 in dopaminergic cells. Exp Neurol 2007; 203(1):221-232. 81. Volpicelli F, Caiazzo M, Greco D et al. Bdnf gene is a downstream target of nurr 1 transcription factor in rat midbrain neurons in vitro. J Neurochem 2007; 102(2):441-453. 82. Pirih FQ Tang A, Ozkurt Ie et al. Nuclear orphan receptor Nurrl directly trans activates the osteocalcin gene in osteoblasts. J Biol Chern 2004; 279(51):53167-74. 83. Bassett MH, Suzuki T, Sasano H er ale The orphan nuclear receptors NURRI and NGFIB regulate adrenal aldosterone production. Mol Endocrinol2004; 18(2):279-90. 84. Hermanson E, Borgius L, Bergsland Met al. Neuropilinl is a direct downstream target of Nurrl in the developing brain stem. J Neurochem 2006; 97(5):1403-11.

CHAPTERS

Foxal and Foxa2 Transcription Factors Regulate Differentiation ofMidbrain Dopaminergic Neurons Siew-Lan Ang*

Abstract

M

idbrain dopaminergicneurons (mDA),comprisingthe substantianigrapars compacta (A8), the ventral tegmental area (A9) and the retrorubal field (AIO) subgroups, are generated from floor plate progenitors, rostral to the isthmic boundary. Floor plate progenitors are specified to become mDA progenitors between embryonic days 8.0 and 10.5. Subsequentlytheseprogenitorsundergo neuronaldifferentiationin two phases, termed earlyand late differentiation to generate immature and mature neurons respectively. Genes that regulate specification, earlyand late phasesof differentiationof mDA cellshaverecentlybeen identified. Among them, the forkheadwingedhelix transcription factors Foxal and Foxal (Foxal/2), have been shown to have essential and dose dependent roles at multiple phasesof development. In this chapter, I will summarize recent studies demonstratinga role for Foxal/2 in regulatingthe neuronal specification and differentiationof mDA progenitorsand concludewith projectionson future directionsof this areaof research.

Introduction

Theforkheadbox (Fox) geneisnamedafterthe Drosophilageneforkhead (fkh), the founding memberof this new class of transcription factors.Thefkh mutation causes homeotic transformation of ectodermalportion of the gut: foregutand hindgut arereplacedbyectopicheadstructures in Drosophila fkh mutant embryos.' More that 100 forkheadproteins havebeen isolatedwhich share a IIO-amino acid DNA- binding motif (forkhead motif) that is conservedfrom yeast to man. Foxgeneshavebeen classified into distinct families and havebeen shown to be required for developmentof manyorgansaswellasfor regulatinghomeostasis in adults (reviewed in references 2 and 3). ThevertebrateFoxasubfamily ofFoxtranscriptionfactors,comprisingof FoxaI, Foxa2, Foxa3, is most closely relatedto the Drosophilafkh protein. Foxaproteins wereisolatedinitially asliver-specific transcriptionfactors. Hence,theywereknownashepaticnuclearfactor-3 (HNF3) a, ~ and y. Foxaproteins haveimportant rolesduringdevelopmentof multipletissues and organs as well as in regulatingglucose metabolismand homeostasis of adipocytesin adult mice.These functions ofFoxaproteins havebeen extensively reviewed." Foxaproteins regulatelongevityin C. elegans S as wellas celldeath in Drosophila/ However, relatively little is known about the rolesof Foxal/2 in the centralnervoussystem (eNS). In this chapter,I will focuson recentfindings demonstrating cellintrinsicrolesfor FoxaI /2 proteins in the ventralmidbrain. In particular,Foxa1/2 are required for the neuronalspecification and differentiationof dopaminergicprogenitorsin the midbrain.' In addition, Foxal/2 are required for the developmentof oculomotor and red nuclei *Siew-Lan Ang-NIMR, The Ridgeway, London, NW7 1AA. Email: [email protected]

Developmentand Engineering ojDopamine Neurons, edited by R]. Pasterkamp, M.P.Smidt and J.P.H. Burbach.©2009 LandesBioscience and SpringerScience+Business Media.

59

Foxal and Foxa2 Transcription Factors

neurons'?Recent studiesalsoreveal a rolefor Foxa2in regulatinghindbrain serotonergicneuron developrnenc,"Together, thesestudiesidentify novel rolesofFoxa1l2 proteins within the CNS.

Expression ofFoxal/2 Proteins in the CNS

Foxa2expression is observedin the floor plate of CNS starting asearlyasembryonicdays8.0 (£8.0) in the rostralCNS.9-11 Foxa2expression occursonlyin medialcells ofthe midbrain at £8.5 and then subsequently spread to include more lateral cells. Foxal expression is initiated about half a day later than Foxa2in floor plate cells. In contrast, expression ofFoxa3 is not detected in the CNS. By£9.5, Foxal and Foxa2show similarexpression in the midbrain and are expressed in allventral midbrain progenitors (Fig. 1 and Ferriet al7) . In addition, Foxa1l2 are expressed in tyrosinehydroxylase" [Th") mDA and Brn3a· red nucleipostmitotic neurons, but not in Isletl" oculomotor neurons (Fig.1and Ferriet al7) at £12.5. Caudalto the midbrain, Foxa2expression is alsoinitiated in the p3 domainof neuralprogenitorsin the hindbrain that gives riseto serotonergic neurons," Foxa1l2 expression persiststhroughout developmentand evenposmatallyin the adult midbrainY·13 Foxa1l2 are expressed continuouslyin mDA progenitors and neurons, suggesting multiple rolesfor theseproteins during developmentand in adults.

Cross-Regulatory Roles ofFoxa2 and Shh and Early Functions ofFoxa2 in Dorsal-Ventral Patterning ofthe CNS

How is Foxa1l2 expression induced in the CNS? Promoter studies on the transcriptional regulatorysequences of the Foxa2genehaveprovidedevidence that Shh regulateFoxa2expression in the floor plate, via the zinc fingerproteins Glil and Gli2.14 Reciprocally, Foxa2has also been shown to regulatesonic hedgehog (Shh) expression through Foxa2 binding sitesin its promoter," Hence a positivecross-regulatory loop exists between Foxa2and Shh. This cross-regulationhas madeit difficult to dissociate the functionsofShh and Foxa2 in the CNS. Lossoffunction studies ofFoxa2 null mutant embryosindicatea rolefor Foxa2in the developmentof the floor plate due to a failureof notochord developmentand consequentlylossofShh signalling." However, Foxa2 likely alsohas cellautonomous rolesin the specification of the mousefloorplate.Consistent with this idea, a recent study in zebrafish embryosdemonstrated that the zebrafish Foxa2homolog, axial, is required for the maintenanceof the floor plate." Less is known about upstream genes that regulate Foxal expression. Using transgenicmice, Clevidenceet allS demonstrated that 4 kilobases of upstream regulatorysequences are sufficient to driveexpression in the floor plate region of the thorax. In the endoderm of Xenopus embryos,

Foxa2

TH

Foxa2 TH

Figure 1. Double-labelling of Foxa2 and TH in coronal sections of the midbrain at E12.5 by immunocytochemistry using specific antibodies (only the ventral region is shown). Foxa2 is expressed in all ventral midbrain progenitors and in TH+ mDA neurons. Scale bar: 100 11m.

60

Development and EngineeringojDopamineNeurons

Sox17and ~-catenin havebeen suggested to regulatethe expression ofFoxal and Foxa2. 19 Foxal alsohas distinct domainsof expression in the endoderm tissue(reviewed in reference 4). It would be interesting to determine the molecular mechanisms responsible for the distinct spatial and temporal expression ofFoxal and Foxa2.

A Role for Foxal/2 in Neuronal Specification ofthe Midbrain Floor Plate

mDA neurons,unlikeother neurons in the CNS arisefrom Boorplate (ventralmidline) progenitors.Earlystudiesby Hynesand Rosenthal" suggested that dopamine neuronsarisefrom the vicinity of Boorplate cells. Initially, it wasassumed that Boorplate cells only providepatterning signals, such as Shh and Wnt, that regulatethe specification of mDA fate in neuralprogenitors." However, more recentlyOno et al22 demonstratedthat BoorplateprogenitorscangeneratemDA neurons usingin vitro cellculture experiments. Generationof neurons by Boorplate progenitors is a unique featureof rostralBoorplate cells, sincethe floorplate from hindbrain and spinalcord regions has so far not been shown to generatepostmitotic neurons. The ability of rostral Boor plate cells to undergo neurogenesis is most likelygovernedby the expression of the proneural basic-helix-loop-helix transcriptionfactorNgn2,whichendowsprogenitorcells with the abilityto undergo neuronaldifferentiation. Thishypothesisissupported by the fact that lossofNgn21eads to severely reducednumbersof mDA neuronsin mice.23,24 Expression ofNgn2 starts aroundE10.5, coincidingwith the onset of neurogenesis in the midbrain. Hence, Ngn2 presumably regulates the timing of neurogenesis in the midbrain. Besides Ngn2, Mashl isalsoexpressed and functions cooperatively with Ngn2 to regulateneurogenesis in the midbrain." Insightsinto the regulationofNgn2 expression in mDA progenitorshavecomefrom lossand gain of function studiesin chickand mouseembryos.Lossof function of the Lim homeodomain transcriptionfactorLmxla and the muscle segmenthomeodomaintranscriptionfactorMsxlleads to a reduction but not completeabsence ofNeurogenin2 expression in mDA progenitors.P'" The partial effecton Ngn2 expression in Lmxla mutant micemaybe due to compensationbyLmxlb. Likewise the residualNgn2 expression in Msxl- I- mutant wasexplainedby potential redundant function of the closely related Msx2gene.Future studiesrequiringthe generationof Lmxla and Lmx:lbdouble and Msxl and Msx2double mutant micewillbe necessary to determinewhether redundancy of gene function is the main reason for the incompletelossof Ngn2 expression. In addition,other transcriptionfactors, suchasOtx226and Foxa1 12, alsocontributeto the regulation ofNgn2 expression in mDA progenitors (seebelow). The rolesofFoxa1 and Foxa2in regulatingneuronaldifferentiationof mDA progenitorshave been studiedusingmoleculargeneticapproaches in mice.A conditional Foxa2mutant mouseline Foxa2Hoxwasused,because Foxa2 null mutant embryosdie by E9.527 precludingthe possibilityof studying the role of Fox112 in mDA neuron developmentthat occurs normallybetween E10.5 and E14.5.In contrast, Foxa1 null mutant mouseembryosdie postnatallyfrom hypoglycemia." Ngn2 expression wasseverely reducedin Nestin-Cre; Foxa2HoxlHox; Foxal"' doublemutant mouse embryos (referred to henceforth as Foxal/2 double mutants), indicating a role for Foxal/2 in regulatingits expression.'Interestingly, Lmxl a, Lmxlband Msxl arenormallyexpressed in these mutant embryos,suggesting that Foxal/2 regulates neurogenesis by an independent mechanism or at the samelevelasthesegenes. It isstillunresolved how Foxa2 regulatesneurogenesis. Wnt and TgfJ3 molecules arepossible candidates, sincestudiesin other parts of the eNS has suggested that these signallingmolecules can regulatethe expression of proneural genesin certain contett. 29•31

Foxal/2 Are also Required for the Generation ofImmature and Mature mDA Neurons

Neuronal differentiationof mDA progenitors, occurring between the ventricular zone and marginal zone, can be divided into two distinct phases, termed early and late differentiation (Fig. 2A-A'''). During early differentiation, progenitor cells exit cell cycle to form immature neurons that migrate just outside the ventricular zone to reach the intermediate zone (zone 2

Foxa1 and Foxa2 Transcription Factors

61

in Fig. 2A"). Subsequently, immature neurons continue migrating to the marginal zone (zone 3 in Fig. 2A") to become mature neurons during the late phase of differentiation. These immature neurons express generic neuronal differentiation markers, such as ~-tubulin, similar to neurons in other regions of the CNS. In addition, the expression of a neuronal subtype specific marker Nurr I, is also initiated in immature mDA neurons. NurrI, a transcription factor belonging to the orphan nuclear receptor family,is required for the development and maintenance ofdopaminergic neurons (reviewed by Wallen and Perlmann 32). Gain offunction studies in chick embryos has suggested that Lmxla is sufficient to induce the formation ofectopic NurrI + mDA neurons from lateral midbrain progenitors," In addition, transfection ofLmxla into mouse embryonic stem cells efficiently promoted their differentiation into mDA neurons." These results suggest that Lmxla regulates the expression ofNurrI in mDA neurons. Loss offunction studies ofFoxal /2 in mice has demonstrated that Foxal /2 are required for the expression ofNurrI in immature neurons? In Foxal/2 double mutant embryos, immature mDA neurons expressing Lmxla and Lmxlb are generated, but these neurons do not express Nurrl. Hence, like Lmxla, Foxal/2 are potential upstream regulators ofNurrl. Whether Lmxla/b and Foxal/2 act together or in parallel pathways to regulate Nurr I expression remains to be determined. The homeodomain transcription factors Engrailedl and Engrailed2 (En I /2) are also expressed in immature neurons, while the expression in mDA progenitors begins to be downregulated from EII.5 onwards.v" In Foxal/2 double mutants, expression ofEn I is also missing in the immature mDA neurons, suggesting that Foxal/2 may be required for the maintenance ofEn I expression. Altogether, these results demonstrate that there is a block in the generation of immature mDA neuron during the early phase ofmDA neurogenesis (Fig. 2 A"'). Foxal /2 is required in this phase ofneuronal differentiation for regulating the expression ofNgn2 and the expression ofNurr I and Enl in mDA progenitors and immature neurons respectively. In contrast, phenotypic analyses of Foxa I and Foxa2 single mutant embryos have revealed a later role for Foxa genes in regulating differentiation of immature to mature neurons due to the fact that Foxal/2 function cooperatively in a dose dependent manner to regulate different phases ofmDA neuron development? In contrast to the block in early differentiation in Foxal/2 double mutants, normal numbers ofpostmitotic mDA neurons are found in Nestin-cre: Foxa2flox/ftox single conditional mutants and Foxa 11acZ/lacZ mutant embryos, but there is a delay in the transition from immature to mature neurons during the late phase ofneurogenesis (Fig. 2A"'). Specifically,expression ofTH and AADC is significantly reduced at EI2.5. However, the number ofTH neurons is almost completely recovered by EI8.5. Recovery of mature neurons occurs slower in embryos with loss of 3 copies than those with loss of2 copies ofFoxa genes, supporting the idea that Foxal/2 function cooperatively to regulate the late differentiation step ofmDA neurons. This finding also supports the hypothesis that compensation by Foxa I and Foxa2 is responsible for the recovery of mDA neurons in single mutant embryos. The ability of Foxal to compensate for Foxa2 functions suggests similar roles for these genes in regulating mDA neuron development. Foxal/2 have also been shown to have similar roles in the development ofthe lung and hepatic endoderm.r-" In contrast, Foxa 1/2 also appears to have distinct domains of gene expression" and cannot substitute for each other's function in other tissues." The generation and analysis of mice in which Foxal is substituted for Foxa2 by gene targeting in embryonic stem cells could provide further insight into redundant and unique functions for Foxal/2 in vivo.

Mechanims ofFoxa Gene Regulation: Examples from Endodermal Organs

Foxa proteins are thought to function as 'pioneer factors' by binding to nucleosome core particles and enabling chromatin access for other transcription factors." In addition, in vitro assays demonstrate that Foxa proteins function as transcriptional activators by binding DNA in a sequence specific manner and transactivating genes. Numerous Foxa targets have been identified through DNA binding studies, chromatin immunoprecipitation and transcriptional reporter assay

Development andEngineering ofDopamine Neurons

62

A"

A'

A

A'" 1) mDA progenitor

WT

2) immature mDAneuron

early

~

Foxa1f2, Lmx1 alb Ngn2

Foxa OM

3) mature mDA neuron

late

Foxa1f2, Lmx1 alb, Nurr1, En1

Foxa1/2, Lmx1aJb, Th,AADC

x~

Foxa 8M

Figure 2. Molecularly distinct DA cell populations in the ventral midbrain. (A-A"') An E12.5 mouse embryo indicating the plane of the section shown in N. An enlarged view of the ventral midbra in (square box in N) illustrating the position occupied by mitotic mDA progenitors in the ventricular zone (zone 1), immature mDA neurons in the intermediate zone (zone 2) and mature mDA postmitotic neurons in the marginal zone (zone 3). (A"') Distinct phases of mDA neurons generated by an early and late differentiation step. The step of differentiation at which Foxa1/2 single mutant (Foxa SM) and Foxa1l2 double mutants (Foxa DM) mouse embryos are blocked is shown by an X. The curved arrow indicates cycling cells. Panels A, N and A" are reprinted from Figures 2Aa and 2Ab. 44

in tissue culture cells or via analysis ofgene expression in Foxa mutant genetic models (reviewed in Friedman and Kaestner, ref 4). Within the nervous system, Foxa2 has been shown to regulate the expression ofShh in transgenic animals," the expression ofvitronectin in Neuro2a cells" and activity of microtubule associatedprotein-IB promoter in primary cultures of mes-metencephalic neurons." Aslessis known about how Foxaproteins modulate transcriptional activity in the nervous system, we will review here some general features of Foxa transcriptional mechanisms through a comparison ofstudies carried out in the endoderm in C. elegam and mammals.

Foxal and Foxa2Transcription Factors

63

Foxa proteins have been shown to play multiple roles during the development ofendodermal organs in mouse and C. elegans from genetic studies. Hence, a key question is how diverse transcriptional responses are orchestrated by a single transcription factor. Evidence for three distinct mechanisms have been obtained primarily from studies of the PHA-4 gene {C. elegans homolog ofFoxa) and also from mammalian studies. In vertebrates, Foxa2 associates with genes long before they are transciptionallyactive and is thought to promote transcriptional potentiation." Foxa transcriptional factors are capable ofinitiating chromatin opening in vitro and enable chromatin access for other transcription factors, hence they have been termed "pioneer factors".38 Recent studies suggest that a histone variant H2A.Z/HTZ-l contributes to Foxa modulation of chromatin in C. elegans.42 A second well established mechanism employed by PHA-4 and Foxa in gene regulation is functional cooperation or interaction with other transcription factors (reviewed in Friedman and Kaestner, ref 4). A third mechanism has been proposed from results ofa microarray screen in C. elegans that identified direct target genes ofPHA-4 in pharyngeal cells. Analyses of the upstream regulatory sequences ofsome ofthese genes have led to the conclusion that temporal regulation can be modulated by the affinity ofPHA-4 for its DNA binding site: promoters with. high-affinity sites are competent to be expressed early whereas those with low affinity sites are typically activated at later times." Given the evolutionary conservation offunction ofFoxa gene family members, it is tempting to speculate that Foxal /2 proteins may use similar molecular mechanisms to regulate gene activity in the CNS. Our genetic studies have demonstrated that higher gene dosage ofFoxal/2 is required for later differentiation compared to the earlier differentiation targets? This finding raises the possibility that Foxal/2 regulate mDA neuronal differentiation using enhancers that have different binding affinities like in C. elegans. Alternatively, Foxal/2 might interact with different cofactors to regulate distinct targets.

Concluding Remarks

In this chapter, I have summarised recent progress in our understanding ofthe role ofFoxa1/2 in the development ofmDA neurons. Foxal/2 play multiple roles during mDA neuron development. The challenge for the next fiveyears is to understand the molecular and cellular mechanisms underpinning these distinct roles.An important direction is to identify direct transcriptional targets ofFoxal/2 in mDA cells and to understand mechanistically how Foxal /2 regulates different genes at distinct phases ofmDA neuron development. Since Foxal/2 are required for mDA neuron differentiation, future insights into their functions in mDA neurons will contribute to a better understanding oftranscriptional control ofneuronal differentiation. Besides gene dosage dependence, Foxal /2 likely also cooperate with other cofactors in a combinatorial manner to regulate neuronal differentiation, as it is required for the development ofother neuronal subtypes such as serotonergic, oculomoter and red nuclei neurons. Unravelling the genetic networks specifying the development ofmDA neurons will enable the differentiation ofpure neuronal populations from stem cells. Importantly, basic research into the biology ofmDA neurons will likely have implications for several human neurological diseases.

Acknowledegements

I wish to acknowledge the support of the Medical Research Council UK and the Parkinson's Disease Society UK and to thank Simon Stott for helpful comments on the manuscript.

References

1. Weigel D, Jurgens G, Kuttner F et al. The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the drosophila embryo. Cell 1989; 57:645-58. 2. Kaestner KH, Knochel ~ Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev 2000; 14:142-6. 3. Pohl BS, Knochel W Of Fox and Frogs: Fox (fork head/winged helix) transcription factors in xenopus development. Gene 2005; 344:21-32.

4. Friedman JR, Kaestner KH. The Foxa family of transcription factors in development and metabolism. Cell Mol Life Sci 2006; 63:2317-28.

64

Development and Enginttring ofDopamintNeurons

5. Panowski SH, Wolff S, Aguilaniu H er al. PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans. Nature 2007; 447:550-5. 6. Cao C, Liu ~ Lehmann M. Fork head controls the timing and tissue selectivity of steroid-induced developmental cell death. J Cell Bioi 2007; 176:843-52. 7. Ferri AL, Lin ~ Mavromatakis YE et ale Foxal and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner. Development 2007; 134:2761-9. 8. Jacob J, Ferri AL, Milton C et ale Transcriptional repression coordinates the temporal switch from motor to serotonergic neurogenesis. Nat Neurosci 2007. 9. Ang SL, Wierda A, Wong D et ale The formation and maintenance of the definitive endoderm lineage in the mouse: Involvement ofHNF3/forkhead proteins. Development 1993; 119:1301-15. 10. Monaghan AP, Kaestner KH, Grau E er al. Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm. Development 1993; 119:567-78. 11. Sasaki H, Hogan BL. HNF-3 beta as a regulator of Boor plate development. Cell 1994; 76:103-15. 12. Thuret S, Bhatt L, O'Leary DD et al. Identification and developmental analysis of genes expressed by dopaminergic neurons of the substantia nigra pars compacta. Mol Cell Neurosci 2004; 25:394-405. 13. Wijchers PJ, Hockman MF, Burbach JP et al. Identification of forkhead transcription factors in cortical and dopaminergic areas of the adult murine brain. Brain Res 2006; 1068:23-33. 14. Sasaki H, Hui C, Nakafuku M et ale A binding site for Gli proteins is essential for HNF-3beta Boor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 1997; 124:1313-22. 15. Epstein DJ, McMahon AP, Joyner AL. Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-depcndent and -independent mechanisms. Development 1999; 126:281-92. 16. Weinstein DC, Ruiz i Altaba A, Chen WS et al. The winged-helix transcription factor HNF-3 beta is required for notochord development in the mouse embryo. Cell 1994; 78:575-88. 17. Norton WH, Mangoli M, Lele Z et al. Monorail/Foxa2 regulates Boorplate differentiation and specification of oligodendrocytes, serotonergic raphe neurones and cranial motoneurones. Development 2005; 132:645-58. 18. Clevidence DE, Zhou H, Lau LF et al. The -4 kilobase promoter region of the winged helix transcription factor HNF-3alpha gene elicits transgene expression in mouse embryonic hepatic and intestinal diverticula. Int J Dev Bioi 1998; 42:741-6. 19. Sinner D, Rankin S, Lee M et al. Sox17 and beta-carenin cooperate to regulate the transcription of endodermal genes. Development 2004; 131:3069-80. 20. Hynes M, Poulsen K, Tessier-Lavigne M et al. Control of neuronal diversity by the floor plate: contact-mediated induction of midbrain dopaminergic neurons. Cell 1995; 80:95-101. 21. Ye ~ Shimamura K, Rubenstein JL er al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998; 93:755-66. 22. Ono Y et al. Differences in neurogenic potential in Boor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic Boor plate cells. Development 2007; 134:3213-25. 23. Andersson E, Jensen JB, Parmar M et al. Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2. Development 2006; 133:507-16. 24. Kele J, Simplicio N, Ferri AL et al. Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons. Development 2006; 133:495-505. 25. Andersson E, Tryggvason U, Deng Q et ale Identification of intrinsic determinants of midbrain dopamine neurons. Cell 2006; 124:393-405. 26. Vernay B, Koch M, Vaccarino F et al. Otx2 regulates subtype specification and neurogenesis in the midbrain. J Neurosci 2005; 25:4856-67. 27. Ang SL, Rossant J. HNF-3 beta is essential for node and notochord formation in mouse development. Cell 1994; 78:561-74. 28. Kaestner KH, Katz J, Liu Y et al. Inactivation of the winged helix transcription factor HNF3alpha affects glucose homeostasis and islet glucagon gene expression in vivo. Genes Dev 1999; 13:495-504. 29. Hirabayashi ~ Itoh Y, Tabata H et al. The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 2004; 131:2791-801. 30. Lee JE, Wu SF, Goering LM et al. Canonical Wnt signaling through Lefl is required for hypothalamic neurogenesis. Development 2006; 133:4451-61. 31. Garcia-Campmany L, Marti E. The TGFbeta intracellular effector Smad3 regulates neuronal differentiation and cell fate specification in the developing spinal cord. Development 2007; 134:65-75. 32. Wallen A, Perlmann T. Transcriptional control of dopamine neuron development. Ann NY Acad Sci 2003; 991:48-60.

Foxal and Foxa2Transcription Factors

65

33. Alberi L, Sgado P, Simon HH. Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons. Development 2004; 131:3229-36. 34. Wan H, Dingle S, Xu Y et al. Compensatory roles of Foxal and Foxa2 during lung morphogenesis. ] BioI Chern 2005; 280:13809-16. 35. Lee CSt Friedman ]Rt Fulmer ]T et al. The initiation of liver development is dependent on Foxa transcription factors. Nature 2005; 435:944-7. 36. Besnard ~ Wert SEt Hull WM et al. Immunohistochemical localization of Foxal and Foxa2 in mouse embryos and adult tissues. Gene Expr Patterns 2004; 5:193-208. 37. Wolfrum C, Stoffel M. Coactivation of Foxa2 through Pgc-l beta promotes liver fatty acid oxidation and triglyceride/VLDL secretion. Cell Metab 2006; 3:99-110. 38. Cirillo LA et aleOpening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 2002; 9:279-89. 39. Shimizu S, Kondo M, Miyamoto Y et al. Foxa (HNF3) up-regulates vitronectin expression during retinoic acid-induced differentiation in mouse neuroblastoma Neuroza cells. Cell Struct Funct 2002; 27:181-8. 40. Foucher I, Montesinos ML, Volovitch Met ale Joint regulation of the MAPIB promoter by HNF3beta/ Foxa2 and Engrailed is the result of a highly conserved mechanism for direct interaction of homeoproteins and Fox transcription factors. Development 2003; 130:1867-76. 41. Shim EY, Woodcock C, Zaret KS. Nucleosome positioning by the winged helix transcription factor HNF3. Genes Dev 1998; 12:5-10. 42. Updike DL, Mango SEeTemporal regulation of foregut development by HTZ-l/H2A.Z and PHA-41 FoxA. PLoS Genet 2006; 2:eI61. 43. Gaudet], Mango SEe Regulation of organogenesis by the Caenorhabditis elegans Foxa protein PHA-4. Science 2002; 295:821-5. 44. Ang SL. Transcriptional control of midbrain dopaminergic neuron development. Development 2006; 133:3499-506.

CHAPTER 6

Transcriptional Regulation oflheir Survival: The Engrailed Homeobox Genes Horst H. Simon" and Kambiz N. Alavian

The Engrailed Genes

T

he mammalian Engrailed genes'? were originally cloned by their sequence homology to the Drosophila engrailed. Orthologous genes ofthese homeobox transcription factors are found throughout the animal kingdom includingall investigated vertebrate species.t" They take part in regionalization ofthe early embryo and later participate in neuronal specificadon.P'" Engrailed was first mentioned as a spontaneously occurring mutation in Drosophila melanogaster 1920: 22disrupting the normal development ofthoracic segments, leading to scutellar modifications, an addition sex comb and malformation of the wings.23.24 Differences in protein length between orthologues and paralogues are significant reaching from 552 amino acids in drosophila to 261 for the zebrafish engrailed-2. Despite these large differences on the sequence level the genes are functional conserved even over different phyla. When the mouse En 1 coding sequence is replaced by its paralogue En2 or by its drosophila homologue, the otherwise lethal phenotype of the En] null mutation with large midbrain deficiencies is rescued resulting in a viable mouse. 25,26 These experiments suggest a irreplaceable role for the engrailed genes in bilateria.

Molecular Structure and Properties ofthe Engrailed Proteins

Sequence comparison ofengrailed homologues revealed fivedistinct subregions within the En protein, designatedEHI-5 (engrailed homology regions) (Fig.l).13Thehomeodomain (EH4) is the largest and most conserved part. This domain is constituted ofapproximately 60 amino acids and is shared with all classes of homeodomain proteins." It consists of three alpha helices that recognize specific sequences in the large groove ofdouble stranded DNA.28-30 The other domains are involved in interactions with regulatory cofactors. At least 12 distinct soluble nuclear proteins have been described to interact with the Engrailed proteins, thereby modulating their function and binding specificity.31 The EH 1 and EH5 domains participate in the repression oftranscription. The remaining two domains, EH2 and EH3, mediate the molecular association to .Pbx/extradenticle proteins.32,33 The latter has a significant impact on the functionality by changing the affinity of the engrailed proteins to DNA and/or redirecting them to different target. This can determine whether the proteins act as activators or repressors of transcription. 34-36 Additionally, there is a common serine rich phosphorylation site N -terminal to EH2, which is posttranslationally modified.31,37 The phosphorylation of this site increases DNA binding of recombinant engrailed by several fold 38but also seems to modulate in mammalian cells a rather peculiar properry/" Despite being transcriptional regulators located in the nucleus, a small proportion «5%) ofthe intracellular *Corresponding Author: Horst H Simon-Department of Neuroanatomy, Interdisciplinary Center for Neuroscience (IZN), University of Heidelberg, 1m Neuenheimer Feld 307, 69120 Heidelberg. Email: [email protected]

DevelopmentandEngineering ofDopamine Neurons, edited by RJ. Pasterkamp, M.P. Smidt andJ.P.H. Burbach. ©2009 Landes Bioscience and Springer Science+ Business Media.

67

Transcriptional Regulation ofThtir Survival: The Engrailed Homeobox Genes

Conserved Domains EHl Groucho ·binding domain

p

EH2 EH3

EH4

PbxlExd Homeobox Binding Domains

EH5 Repressor of Transcription

Figure 1. Molecular structureof the engrailed proteins. EH1-S - engrailed homology regions, P - phosphorylation site.

engrailed protein is found associated to membrane vesicles," The vesicle-associated protein is secretedand actsin paracrinemanneron other cells.37.41 The secretiondepends,in addition to the phosphorylation site, on a short region in the homeodomain that is essential for nuclearexpon and extracellular release of the protein." Expression of two mammalian Engraileds (Engrailed-] andEngrailed-2;En] andEn2, respectively) changesmarkedlybetweenearlyembryogenesis and the adult. In the CNS, the two genes start to be expressed at E8.0of mousedevelopmentin two discretedomainsin the midbrain. Later the two domainsfuseto forma ringofcellsrostraland caudalto the isthmus(mid-/hindbrain border).43.4S Theisthmusgives laterriseto anlagenofcerebellum, pons,colliculi and the periaqueductal gray, where the expression of the genesis maintained. Additionally, two ventrolateralstripes of En1extend from the rostralhindbrain down the length of the spinalcord, madeup of cellsin the germinalzone aswellasdifferentiatedneurons.18.44 The zoneof expression gives riseto a subset of association neuronsthat projectlocally to somaticmotor neurons.Thisexpression ceases at around E17. Up to rnidgestation, the two genes arealsofound outsideof the neuraltube. likesomitesand their derivatives, in abdominalregions, Rathke's pouch and the limbs.FromE15onwards,the two genesare restricted to the CNS with exceptionof the limb expression. In the adult CNS, Enl is expressed in the Purkinjecell layer, the colliculi, the superiorolive,periaqueductalgrayand the ventral midbrain. En2 is detectablein the colliculi, the periaqueductalgray, the ventralmidbrain and extensively in the granular layerof the cerebellum.

TheEngrailedGenesandMesencephalic Dopaminergic (mesDA) Neurons (Early)

In respect to the mesDAneurons,En] andEn2haveadualfunction. Duringearlydevelopment they regulatethe expression domainsofone ofthe diffusible factorsthat isneededfor the specification of their precursorcellsin the ventral midbrain (Fig. 2A).As anyother neuronal population, mesDAneurons are generatedfrom an originaluniform neuroepithelium.When the basicbody plan has been laid down, they are region-specifically induced by the combinatorialinteraction of sonic hedgehog (Shh) and the fibroblast growth factor 8 (FgfS). The isthmus releases the latter. En] and En2 playa crucialin the formation of this tissueand therebydetermine the sizeof the Fgf8expression domain. The earlyinitiation of Fgf8 in the dorsal midbrain does not require the expression of the two transcription factors, howeverthe appearance in the isthmus depends on their expression. Experimental evidencesuggests that En] andEn2 directly regulateFgf8. Ectopic expression of En] in chicken,zebrafish and xenopusembryosleadsto ectopic Fgf8 domains.46.47 This regulationof Fgf8by the EngraUed proteins isthe resultof a direct interaction with the promoter. A conservedEn] binding site exists in the firstintron site of the Fgf8locus in the genome of man, mouse, rat and chicken." Electrophoretic mobility shitt assays using nuclearextractsof EScellembryoidbodies demonstrated that En] can bind to this site.Directed mutation reduces the activityof this enhancer significantly. As a result, the significant smallerFfgS domain in the EngraUed double (En]-/-;En2:'I-) mutant embryos at E8/9 43 could be the reason for the lower number of postmitotic mesDAneurons at E12.19.48

68

Deoelopment andEngineering ofDopamine Neurons

Figure 2. Engrailed expression in the mammalian brain. A) E9 immunostaining of E9 whole mount embryo. The tauLacZ expression marker reveals the expression domains of Enl and En2. S,C) In situ hybridization on adult sagittal brain sections. Arrow points to DA neurons in ventral midbrain. t - telencephalon, m z midbrain, cb = cerebellum, sc = superior colliculus, ic - inferior colliculus.

There is an alternativeexplanationfor this low cellcount. The Engrailedgenesmayprovidea subsetof mesDAprecursorcellswith secondorder positionalinformation that is requirefor their induction in the ventral midbrain by the Shh and Fgfll. Direct evidence for this does not exist, howeverthe expression domain of En1 corresponds with the areawheretheseprecursorcellsare located.3.49 The earlyidentity of midbrain and the first two rhombomeresof the hindbrain are defined by Otx2 and Gbx2, respectively. If the expression domain of Otx2 is expanded caudally, the anterior hindbrain is transformed into midbrain and viceversa. so In experiments, where the Otx2 expression wasinactivatedregionspecific byCre-recornbinase in the En1locus, the numbers ofDA neuron wasmarkedlyreduced.stronglysuggesting thatEn1 is expressed bymesDAprecursor cells," If the Otx2 expression is movedmore rosrrally outsidethe En1 expression domain. the mesDAneurons are mis-positionedbut otherwisenormal in numbers.S

The EngrailedGenes and Mesencephalic Dopaminergic Neurons (Later)

MesDA neurons belong to one of the first neuronal population generatedin the developing CNS.53 Experiments using tritiated thymidine on rat fetuses 54showed that the neurons become

Transcriptional Regulation ofTheir Survival: The Engrailed Homeobox Genes

69

postrnitotic in mice between ElO to E13. 12 to 24 hours later, tyrosinehydroxylase (TH), the rate limiting enzymeofdopamine synthesis, is detectableby immunohistochemistry.t! Initially. the neuronsdo not express the Engrai/ed genes. En} andEn2 appeargradually at E11.5and from E14onwards, eachofthe tyrosinehydroxylase (TH) positive neuronsin the midbrainpossesses an Engrailedpositivenucleus. Thisexpression continuesinto adulthood (Fig.2 B,C).48The relatively late onset of expression suggests that En} and En2 do not participate in the earlypostmitotic specification. Thisis probablya function ofthe ligand-independent nuclearreceptorNurr1.56.57 The most pronounced featureof the Engrai/ed genes istheir cell-autonomous requirementfor the survival and long-term maintenance ofmesDAneurons.In mutant micehomozygous double null mutants for En} and En2 (En}-!-; En~/-) , the neuronsare generatedin the midbrain neuroepithelium,becomepostmitotic and start to express genes that aretypicalfor their neurotransmitter phenotype like,for example, tyrosine hydroxylase. However. shortlythereafter, the mutant neuronsbegin to die and are completely lost by E14.19.48During this time,the cells showsigns of apoptosis like the activation of caspase-3 and fragmented, pyknoticnuclei. Classical cellmixing experiments, silencing by RNA interference and the generationof chimericmice, composedof a mixtureof Enlr"; En~/- and wild type cells48•58demonstratedthat that the requirementfor the survival ofmesDAneuronsisof cell-autonomous nature and is not rooted in the largedeficiency in the mutant midbrain. In total three of the eight Engrailed genotypes die at birth and exhibit a gene-dose dependent lossof mesDAneurons (seeTable1). Theseare allgenotypes which are at leasthomozygote null for En} (En}-!-; En2+/+, Enl "; En2'"I-; En}-!-; En~/-) . All others are viable and fertileand no alterationisobservable at birth. Mostinterestingly, fortwo of theseviable Engrailed mutant genotypes,a slowprogressive postnatal degeneration of mesDAneurons has been reported.59.60 Mice heterozygote null for En} in aEn2 wildtype background(Enj+I-;En2'"I+) exhibita slowdecrease in the numberofmesDAneuronsbetween8 and 24 weeks afterbirth resultingin a 38%and 23% reductionin substantianigraparscompacta(SNpc,A9) and ventraltegmenatal area(VTA,A10) respectively and impairedmotor performance.P The other studyreported, in contrast,no alterations in Ent": mice up to 24 months, but a slowprogressive specific nigraldegeneration during the first three months after birth in another En} heterozygote genotype, En} heterozygous null in aEn2 null background (Enj+I-;En~/-). Finally, 70%ofDA neuronsin the SNPC neuronsare lost, however, neither their counterparts in the VTA nor those in the retrorubral field (A8) are affected. The remainingnigral DA neurons maintain their projection to the caudateputamen,

0

substantia nigra

E120%

..-----

gl 00%

1"':-

_.

J!l 80%

B6O%

1:1,-=

040%

8. 20%

~

0%

PO

PIS

-

I-=-

11 It

P30

>3010

E

c -

E

ventral tegmentum 120% ..__._- retrorubral field ..__._._....

i

gl 00%

i

'

!

.!!!. 80%

i

8 60% o

40%

J: ....

0%

8.

20% PO

PIS

P30

>3mo

Figure 3. Slow progressive loss of nigral DA neurons in Enl'"; En£!- (EnHT) mice during the first three months after birth, (A-C) TH immunohistochemistry on sections . D,E) Count of mesDA neurons in SNpc (D) and VTA (E).

Development and Engineering ofDopamineNeurons

70

Table 1. Engrailed phenotypes. Summary of Engrailed mutantphenotypes in respect to mesDA neurons En1

En2

PhenotypemesDA Neurons

Survival

Citation

+/+ +/+ +/+ +/-

+/+ +/-i+/+

Viable, Viable, Viable, Viable,

Simon et al 2001 Simon et al 2001 Sonnier et al 2007

+/+/-

+/-/-

-/-/-t-

+/+ +/-t-

Wild type Wild type-like Wild type-like Progressive loss in SN/VTA (38%/23% , respectively) between 8 and 24 weeks Wild type-like Progressive loss> PO, specific to SN (70%) during the first three months Distribution disturbed Small residue left None; lost by E14

fertile fertile fertile fertile

Viable, fertile Viable, fertile

Sgado et al2006 Sgado et al 2006

PO lethal PO lethal PO lethal

Simon et al 2001 Simon et al 2001 Alberi et al 2004

but dopamine content is diminished and release of the neurotransmitter reduced. This leads to concurrent molecular change in striatal GABAergic neurons ofthe direct and indirect pathways/" Consequently, motor performance of the mutant mice is markedly affected. The mice exhibit significant reduction in forward locomotion and a four-fold increase in freezing episodes when swimming, reminiscent of akinesia and bradykinesia in PD patients. Additionally, these mutant mice share another feature with PD patients. Weight loss is common amongst PD patients starting prior to diagnosis and continuing with disease progression.62,63 After initial normal weight gains, a differences between heterozygote animals and their littermate controls of 20% builds up. This is attributable to a lower food up-take. Since there is no Engrailed expression detectable at this age in the gastrointestinal tract nor associated tissues, this lower weight is likely the result ofDA depletion in the basal ganglia. The described analyses and the functional conservation ofthe engrailed genes over hundred of millions ofyears ofevolution suggests that a tight regulation ofthe En} andEn2 genes is essential for the generation, survival and maintenance ofmesDA neurons. Intriguingly, a recent association study into PD indicated an SNP variation in the intron ofEn} gene as a potential risk factor for the sporadic forms of this disease." It remains to be seen whether variation in the sequences of En} andEn2 can be associated to familiar or sporadic forms of PD.

References

1. Joyner AL, Skarnes WC, Rossant J. Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells. Nature 1989; 338:153-156. Herrup K et al. Abnormal embryonic cerebellar development and patterning of 2. Millen, KJ, Wurst postnatal foliation in two mouse Engrailed-2 mutants. Development 1994; 120:695-706. 3. Wurst Auerbach AB, Joyner AL. Multiple developmental defects in Engrailed-l mutant micel an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development 1994; 120:2065-2075. 4. Wedeen CJ, Weisblat DA. Segmental expression of an engrailed-dass gene during early development and neurogenesis in an annelid. Development 1991; 113:805-814. 5. Wanninger At Haszprunar G.·The expression of an engrailed protein during embryonic shell formation of the tusk-shell, Antalis entalis (Mollusca, Scaphopoda). Evol Dev 2001; 3:312-321. 6. Duman-Scheel M, Patel NH. Analysis of molecular marker expression reveals neuronal homology in distantly related arthropods. Development 1999; 126:2327-2334. 7. Patel NH, Martin-Blanco E, Coleman KG et al. Expression of engrailed proteins in arthropods, annelids and chordates. Cell 1989; 58:955-968. 8. Scholtz G, Patel NH, Dohle W Serially homologous engrailed stripes are generated via different cell lineages in the germ band of amphipod crustaceans (Malacostraca, Peracarida). Int J Dev BioI 1994; 38:471-478.

w:

w:

Transcriptional Regulationof Their Survival:TheEngrailed Homeobox Genes

71

9. Lowe CJ, Wray GA. Radical alterations in the roles of homeobox genes during echinoderm evolution. Nature 1997; 389:718-721. 10. Holland LZ, Kene M, Williams NA et al. Sequence and embryonic expressionof the amphioxus engrailed gene (AmphiEn)/the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development 1997; 124:1723-1732. 11. Ekker M, Wegner J, Akimenko MA er al. Coordinate embryonic expression of three zebrafish engrailed genes. Development 1992; 116:1001-1010. 12. Joyner AL, Kornberg T, Coleman KG er al. Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene. Cell 1985; 43:29-37. 13. Logan C, Hanks MC, Noble-Topham S et al. Cloning and sequence comparison of the mouse, human and chicken engrailed genes reveal potential functional domains and regulatory regions. Dev Genet 1992; 13:345-358. 14. Lopez-Corrales NL, Sonstegard TS, Smith TP. Comparative gene mapping: cytogenetic localization of PROC, EN1, ALPI, TNPI and ILIB in cattle and sheep reveals a conserved rearrangement relative to the human genome. Cytogenet Cell Genet 1998; 83:35-38. IS. Koster JG, Eizema K, Peterson-Maduro LJ et al. AnalysisofWnt/Engrailed signaling in Xenopus embryos using biolistics. Dev Bioi 1996; 173:348-352. 16. Zec N, Rowitch DH, Bitgood MJ et al. Expression of the homeobox-containing genes ENI and EN2 in human fetal midgestational medulla and cerebellum. J Neuropathol Exp Neuro11997; 56:236-242. 17. Barak 0, Lazzaro MA, Lane WS et al. Isolation of human NURF: a regulator of Engrailed gene expression. EMBO J 2003; 22:6089-6100. 18. SaueressigH, Burrill J, Goulding M. Engrailed-l and netrin-l regulate axon pathfinding by association interneurons that project to motor neurons. Development 1999; 126:4201-4212. 19. Simon HH, SaueressigH, Wurst W et al. Fate of Midbrain Dopaminergic Neurons Controlled by the Engrailed Genes J Neurosci 2001; 21:3126-3134. 20. Condron BG, Patel NH, Zinn K. Engrailed controls glial/neuronal cell fate decisions at the midline of the central nervous system. Neuron 1994; 13:541-554. 21. Lundell MJ, Chu-LaGraff Q Doe CQ et al. The engrailed and huckebein genes are essential for development of serotonin neurons in the Drosophila CNS. Mol Cell Neurosci 1996; 7:46-61. 22. Eker R. The recessive mutant engrailed in Drosophila melanogaster, Hereditas 1929; 12:217-222. 23. Brasted A. An analysisof the expression of the mutant "Engrailed" in Drosophila Melanogaster. Genetics 1941; 26:347-373. 24. Tokunaga C. The differentiation of a secondary sex comb under the influence of the gene engrailed in Drosophila melanogaster, Genetics 1961; 46:157-176. 25. Hanks M, Wurst ~ Anson-Cartwright L et al. Rescue of the En-l mutant phenotype by replacement of En-l with En-2. Science 1995; 269:679-682. 26. Hanks MC, Loomis CA, Harris E et al. Drosophila engrailed can substitute for mouse Engrailed 1 function in mid-hindbrain, but not limb development. Development 1998; 125:4521-4530. 27. Manak ]R, Scott MP. A class act/conservation of homeodomain protein functions. Development 1994; (Suppl): 61-77. 28. Kissinger CR, Liu BS, Martin-Blanco E et al. Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution/a framework for understanding homeodomain-DNA interactions. Cell 1990; 63:579-590. 29. Desplan C, TheisJ, O'Farrell PH. The sequence specificityof homeodomain-DNA interaction. Cell 1988; 54:1081-1090. 30. Ades SE, Sauer RT. Differential DNA-binding specificity of the engrailed homeodornain/the role of residue SO. Biochemistry 1994; 33:9187-9194. 31. Gay NJ, Poole S, Kornberg T. Association of the Drosophila melanogaster engrailed protein with specific soluble nuclear protein complexes. EMBO J 1988; 7:4291-4297. 32. Peltenburg LT, Murre C. Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins. Development 1997; 124:1089-1098. 33. van Dijk MA, Murre C. Extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell 1994; 78, 617-624. 34. Kobayashi M, Fujioka M, Tolkunova EN et al. Engrailed cooperates with extradenticle and hemothorax to repress target genes in Drosophila. Development 2003; 130:741-751. 35. Gemel J, Jacobsen C, MacArthur CA. Fibroblast growth factor-S expression is regulated by intronic engrailed and Pbxl-binding sites. J Biol Chern 1999; 274:6020-6026. 36. Serrano N, Maschat F. Molecular mechanism of polyhomeotic activation by Engrailed. EMBO J 1998; 17:3704-3713.

72

Development andEngineering ofDopamine Neurons

37. Cosgaya JM, Aranda A, Cruces J et al. Neuronal differentiation of PC12 cells induced byengrailed homeodomain is DNA-binding specific and independent of MAP kinases.J Cell Sci 1998; 111(Pt 16): 2377-2384. 38. Bourbon HM, Martin-Blanco E, Rosen D et ale Phosphorylation of the Drosophila engrailed protein at a site outside its homeodomain enhances DNA binding. J BioI Chern 1995; 270:11130-11139. 39. Maizel A, Tassetto M, Filhol 0 et al. Engrailed homeoprotein secretion is a regulated process. Development 2002; 129:3545-3553. 40. joliot A, Trembleau A, Raposo G er al. Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties. Development 1997; 124:1865-1875. 41. Joliot A, Maize!A, Rosenberg D et al. Identification of a signal sequence necessaryfor the unconventional secretion of Engrailed homeoprotein. Curr BioI 1998; 8:856-863. 42. Maizel A, Bensaude 0, Prochiantz A et al. A short region of its homeodomain is necessary for engrailed nuclear export and secretion. Development 1999; 126:3183-3190. 43. Liu A, Joyner AL. EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse midi hindbrain region. Development 2001; 128:181-191. 44. Davis CA, Joyner AL. Expression patterns of the homeo box-containing genes En-l and En-2 and the proto-oncogene int-l diverge during mouse development. Genes Dev 1988; 2:1736-1744. 45. Davidson D, Graham E, Sime C et al. A gene with sequence similarity to Drosophila engrailed is expressed during the development of the neural tube and vertebrae in the mouse. Development 1988; 104:305-316. 46. Shamim H, Mahmood R, Logan C et al. Sequential roles for Fgf4, Enl and FgfB in specification and regionalisation of the midbrain. Development 1999; 126:945-959. 47. Risroratore F, Carl M, Deschet K et ale The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-engz, Development 1999; 126:3769-3779. 48. Alberi L, Sgado P, Simon HH. Engrailed genes are cell-autonomously required to prevent apoptosis in mesencephalic dopaminergic neurons. Development 2004; 131:3229-3236. 49. Ye ~ Shimamura K, Rubenstein JL et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998; 93:755-766. 50. Joyner AL, Liu A, Millet S. Otx2, Gbx2 and FgfS interact to position and maintain a mid-hindbrain organizer. Curr Opin Cell Bioi 2000; 12:736-741. 51. Puelles E, Annino A, Tuorto F et al. Otx2 regulates the extent, identity and fate of neuronal progenitor domains in the ventral midbrain. Development 2004; 131:2037-2048. 52. Puelles E, Acampora D, Lacroix E et al. Otx dose-dependent integrated control of antero-posterior and dorso-ventral patterning of midbrain. Nat Neurosci 2003; 6:453-460. 53. Sechrist J, Bronner-Fraser M. Birth and differentiation of reticular neurons in the chick hindbrain: ontogeny of the first neuronal population. Neuron 1991; 7:947-963. 54. Altman J, Bayer SA. Development of the brain stem in the rat. V. Thymidine-radiographic study of the time of origin of neurons in the midbrain tegmentum. J Comp Neuro11981; 198:677-716. 55. Foster GA, Schulrzberg M, Kokfelt T er al. Ontogeny of the dopamine and cyclic adenosine-3':5'-monop hosphate-regulated phosphoprotein (DARPP- 32) in the pre and postnatal mouse central nervous system. lot J Dev Neurosci 1988; 6:367-386. 56. Wang Z, Benoit G, Liu J et ale Structure and function of Nurr 1 identifies a class of ligand-independent nuclear receptors. Nature 2003; 423:555-560. 57. Martinat C, Bacci JJ, Leete T et al. Cooperative transcription activation by Nurrl and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype. Proc Nat! Acad Sci USA 2006; 103:2874. 58. Simon HH, Thuret S, Alberi L. Midbrain dopaminergic neurons: control of their cell fate by the engrailed transcription factors. Cell Tissue Res 2004; 318:53-61. 59. Sonnier L, Le Pen G, Hartmann A et ale Progressive loss of dopaminergic neurons in the ventral midbrain of adult mice heterozygote for Engrailed 1. J Neurosci 2007; 27:1063-1071. 60. Sgado P, Alberi L, Gherbassi D et al. Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice. Proc Nat! Acad Sci USA 2006; 103:15242-15247. 61. Gerfen CR. Molecular effects of dopamine on striatal-projection pathways. Trends Neurosci 2000; 23:S64-70. 62. Beyer PL, Palarino ~ Michalek D et al. Weight change and body composition in patients with Parkinson's disease. J Am Diet Assoc 1995; 95:979-983. 63. Chen H, Zhang SM, Hernan MA et al. Weight loss in Parkinson's disease. Ann Neurol 2003; 53:676-679. 64. Fuchs J, Mueller JC, Lichtner P et al. The transcription factor PITX3 is associated with sporadic Parkinson's disease. Neurobiol2007 Aging doi:l0.l016Ij.neurobiolaging.2007.08.014.

CHAPTER 7

Neurotrophic Support ofMidbrain Dopaminergic Neurons Oliver von Bohlen und Halbach" and Klaus Unsicker

Abstract

I

n this chapter we review work on neurotrophic factors for midbrain dopaminergic neurons mainly from the past decade, with a focus on neurotrophins and fibroblast growth factors. We summarize data obtained from animal models of Parkinson's disease, review analyses of neurotrophin, neurotrophin receptor and FGF-2 knockout mice and put these into context with data obtained from patients with Parkinson's disease and from postmortem studies. We provide a brief overview on several other factors (EGF, TGF-a, IGF, CNTF, PDGF, inrerleukins) and their capacity to promote survival and protect lesioned DAergic neurons. TGF-f3s are reviewed in a separate chapter (Roussa et al, this volume).

Introduction

. Neurotrophic factors are operationally defined as proteins, which are synthesized and released by neural and nonneural cells and required for the development, differentiation and maintenance ofneurons in the developing and adult central nervous system (CNS). Considerable efforts have been invested over the years in the search for proteins and small molecule analogues that can promote the survival ofembryonic neurons and protect postnatal neurons from lesion-mediated cells death, with the perspective to develop such factors into therapeutic tools for the treatment of neurodegenerative disorders. Parkinson's disease (PD) is the most frequent movement disorder. Its hallmark symptoms, bradykinesia, resting tremor and rigidity, are caused by lossesofdopaminergic (DAergic) cell bodies in the substantia nigra pars compacta (SN) and their axonal projections to the striatum.' The pathogenesis ofPD is currently unknown, but both environmental and genetic factors have been implicated in the neurodegenerative process leading to neuron death.' Currently, there is no treatment available to cure PD; allavailable therapies can only ameliorate the symptoms ofPD. One therapeutic strategy aims at enhancing the survival ofthe remaining DAergic neurons in the diseased SN and promote axonal regeneration by applying "dopaminotrophic" factors, either by infusion or by grafting genetically engineered cells. Neurotrophic factors with well established survival promoting effects on DAergic neurons in vitro and in vivo include members of the neurotrophin, fibroblast-growth factor (FGF) ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), insulin-like growth factor (IGF) and interleukin families. Reviews on these factors and their applicability to lesioned nigrostriatal DAergic neurons have been published.t" The present review summarizes the most substantial findings with a focus on the past decade.

*Corresponding Author: Oliver von Bohlen und Halbach-Interdisciplinary Center for Neurosciences, Neuroanatomy, University of Heidelberg, 1m Neuenheimer Feld 307 0-69120 Heidelberg, Germany. Email: [email protected]

Developmentand Engineering ofDopamine Neurons, edited by R.J. Pasterkamp, M.P. Smidt andJ.P.H. Burbach. ©2009 Landes Bioscience and Springer Science+Business Media.

74

Development andEngineering ofDopamine Neurons

Neurotrophins

This family of neurotrophic factors comprises the paradigmatic neurotrophic factor NGF. brain-derived neurotrophicfactor(BDNF) and the neurotrophins (NT)-3. -4and _6.1- 10 TrkAis the functional receptorfor NGF.TrkB serves asa receptorfor BDNF and NT-4. whereas NT-3 signals primarily throughTrkC. In addition.neurotrophins canact throughp75NTR (p75 neurotrophin receptor. seeFig. I) with approximately equallowaffiniry. BDNF and NT-3,alongwith their cognate receptors trkB and trkC. are expressed. inter alia.in the developing and adult SN and striaturn.l':" suggesting responsiveness ofDAergicneuronsin the nigrostriatal system to the corresponding trkBand trkC ligands. BDNF and NT-3 have both been found to playa significant role in promotingsurvival and differentiation ofSN DAergic neurons in vitroand in viVO. 15•16 While NT-4 hasalsobeenshown to act as a survival factor for embryonic midbrain DAergic neurons," NGF is apparently not relevant in this system."Recently. it has alsobeen demonstrated that BDNF is requiredfor the establishment of the correctnumberofDAergicneuronsin the SNpC. 19

Data from Anima/MoJe/s ofPD

Several animalmodels ofPD replicate some. but not allfeatures of humanPD. Suchmodels include. amongst others.unilateral6-hydroxydopamine (6-0HDA) or systemic l-methyl-s-phenyl-I.2.3.6-tetrahydropyridine (MPTP) lesions.' Lesions ofthe DAergic nigrostriatal system with 6-hydroxydopamine (6-0HDA) havebeen shown to reduce BDNF mRNAlevels in the SN.20 6-0HDA-injectedanimals treatedwithNT-3orBDNF showed areduction of6-0HDA-induced behavioral deficits.21,22 Likewise, 6-0HDA-induced rotational behavior can be prevented by BDNF somatic genetransfer into neurons of theSN.23 Moreover.intrastriatal grafts offibroblasts, genetically engineered to produceBDNF,preventneurodegeneration of the 6-0HDA-Iesioned nigrostriatal system." Likewise. implantation of immortalized BDNF synthesizing fibroblasts largely prevents MPTP-inducedDAergic neuronaldegeneration andenhances DAlevels.25.26 Together. thesedata suggest that BDNF isan important factorfor the maintenance and survival ofDAergicneurons and thatdysfunctions in neurotrophinsignaling maycause pathological alterations in the DAergic nigrostriatal system.27.28

-J

~

trkA

-?

~

trkC

Figure1. Schematic overview: Neurotrophinscanactthroughspecific receptors of thetrk-family as well as through p75NTR receptors.

Neurotrophic SupportofMidbrain Dopaminergic Neurons

75

Datafrom Knockout Mice

Sincein humans a majorityofDAergic neuronsof the SN reveals immunoreactivities for trkB and trkC,13 it isconceivable that reducedlevels of thesereceptorsmaybe implicatedin the degeneration of nigrostriatalDAergicneurons. Along this line, youngadult hypomorphic trkB mice, which express only approximately 25% of wild type levels of trkB, havebeen reponed to display a significantloss ofDAergic SN neurons." Moreover, aged trkB singleand trkB/trkC double heterozygous knockout mice displaylosses of DAergicneurons in the SN and striatal DAergic fibers." However, the reduction in SN DAergicneurons in agedtrkB heterozygous mutant mice maynot exclusively result from a deficitof trkB in the SN, sinceconditional trkB knockout mice (DAT-trkBmice,displaying a 65%lossof trkB mRNA in DAergiccells) do not showan obvious reduction in DAergicneuron numbers." This maysuggest that extra-nigrostriatal trkB-positive circuits,which areconnectedto the trkB nigrostriatalsystemmayaccountfor the phenotype seen in agedheterozygous trkB mutant mice. Most of the available data indicate that the proper functioning of the BDNF/trkB signaling pathwayis important for the survival and maintenance of DAergicneurons, but little is known concerning the other members of the neurotrophin famUy. This may suggest that the other neurotrophins are not crucialin regulatingthe maintenanceof adult DAergicneurons. Evenso, trkC is apparently important for adult DAergic neurons, since aged heterozygous trkC knockout mice displaymild reductions in TH-positive neurons of the SN and mild reductions in the densityof catecholaminergic fibers in the striatum," amygdala and hippocampus.P whereas mice over-expressingtrkC havehigher numbersofTH-positive cells in the SN.33

Datafrom PostMortem Studies and PD Patients

Postmortem analyses ofPD-diseased human SN haverevealed reductions in BDNF mRNA and protein levels,27.28.34 raisingthe possibilitythat there is a link between reduced BDNF levels and PD. This notion is supported bydata obtained from BDNF mRNA antisenseinfusionsinto the rat SN, which causesubstantiallosses ofTH-positive neurons (-40%),reduced densitiesof DA uptake sites (-34%) and altered behavior," In clinically and neuropathologically typicalPD, BDNF mRNA expression in the SN is reduced by70%.This reduction is due, in part, to lossof DAergicneurons which express BDNF. However, surviving DAergicneurons in the diseasedSN alsoexpressed lessBDNF mRNA (20%,) than their normal counterparts." Speculations that mutations in the BDNF gene might be a risk factor for PD are supported the observationthat in a japanese population homozygosity for the V66M polymorphismof the BDNF gene occurs more frequently in patients with PO than in unaffected healthy subjects/" This polymorphismwas alsofound to be associated with PO in the so-called GenePO Study (an internationalconsortiumof30 participatingsites). Fromthisstudy, however, it isnot clearwhether this association is found only in a specific subsetofPD patientsof a specific ethnic group 37. Two single nucleotidepolymorphisms at position C270T of the BONF gene havebeen identified in patientswith familial PO, suggestingthat BDNF mayplayarolein thedevelopment offamilialPD.38 However, therearealsoseveral other studiesthat havenot found associations betweenmutationsin the BDNF geneand PO, ase.g., in a Chinese," Finnish." Greekand North American" population. Thus,mutationsin the BDNF genecan hardlybe considered asa strongriskfactor for developing PD. Nevertheless, the data from the Japanese group at least indicate that proper functionality of BDNF is important for the maintenanceof the DAergicneuronsin the adult human SN.

Fibroblast Growth Factors (FGFs)

The famUy of fibroblastgrowth factors comprises 23 membersthat signalvia four receptors, whose gene and protein structures and intracellular signaling cascades have been extensively investigated. 6•42-44 FGF-2 is one prominent member of this famUy and widelyexpressed in the developing, neonatal and postnatal mammalian nervous system including the substantia nigra, striatum and several limbicareas.45-47

76

Development and EngineeringofDopamineNeurons

Starting with the discovery that FGF-2 promotes survival and fiber outgrowth of cultured embryonicmidbrain DAergicneurons,48.49 numerousfurther studieshaveconfirmedand further elaboratedon the notion that FGF-2 is a potent trophic factor for midbrain DAergicneurons. Most notably,FGF-2 protects DAergicneurons againstMPTP toxicitynot only in vitro but alsoin vivo.50-54.54 Additional studieshavecorroborated the notion that FGF-2 also protects neurons againsta widerspectrumoftoxins,including6-0HDA and glutamate-induced lesions.55.56 Theobservationthat striatalFGF-2 mRNAwasupregulatedin responseto an MPTP-lesion,lend support to the ideathat FGF-2 mayhavephysiological significance in the nigrostriatalsystem. 57.58

Theseand other resultsraisedexpectationsthat FGF-2 might be a target in the searchfor novel therapeutic approaches to treat PD. Along this line, it was found that FGF-2 can enhance DAergicfiber formation from nigral graftsand that FGF-2 treated graftsfrom E16 rats contained alargernumber ofDAergic neurons than the controls.59 In addition, intracerebralinfusion or pretreatment of transplantedDAergic neuronswith FGF-2resultedin increased survival ofthe graftedDAergic neurons/" Unfortunately, repeated intracerebralinfusionsof FGF-2 induced an inflammatoryreaction in the striatum." compromising the usabilityof this route of FGF-administration for promoting graft survival. Possibly, cograftingofDAergic neurons with FGF-2 producing fibroblasts" or Schwanncells62 mayofferan approachto enhancethe survival and function ofDAergic neurons graftedinto the damaged brain. However, it is crucialto know whether on the long run, these cografts mayalso induce inflammatoryreactions. While exogenous FGF-2 isindeed beneficial for the survival ofDAergic neurons,endogenous FGF-2 is apparentlynot essential for the developmentand survival ofDAergic neurons as concludedfrom the analysis ofFGF-2 knockout mice.In one studyit has been describedthat FGF-2 deficient mice do not displayany significant alterations, as compared to age-matched controls, in the density of DAergicneurons within the SN or in the density of DAergicfibers within the striatum. Likewise, the DA-Ievels (as determined by HPLC-ED) within the striatum were not different between these groupS.63 Moreover, no difference in the density of DAergicneurons in the SN, DAergicfiberdensitiesor DA-levels in the striatumwerefound in MPTP-tteated FGF-2 knockout miceas comparedto MPTP-treated controls/" In another study it has been reported that FGF-2 deficientmice did not displayalterations in density ofDAergic neurons in the SN.64 Moreover, no significant alterationsin the tyrosine hydroxylase protein levels (as determined by Western-blotting)in the SN werefound in caseof the FGF-2 deficientmice.64 However, the total number ofDAergic neurons in the SN ofFGF-2 deficientmicewasfound to be about 15%higher than in the correspondingcontrols.Thiseffect seemedto beattributedto anincreasedvolumeof the SN in the FGF-2knockoutmice.Thisincrease in the numbers of DAergicneurons in the SN is somewhatsurprising,sinceone would expect a reduction in the number ofDAergic neurons in the FGF-2 deficientmice.A possiblemechanism explainingthis findingcouldbe that the lackofFGF-2 is (over)compensatedduringdevelopment by other trophic factors, which could be upregulaeed/" In the samestudy the effectof 6-0HDA treatmentwasanalyzed and it wasfoundthat that 6-0HDA-treated FGF-2knockoutmiceshowed lessremaining DAergicneurons in the SN than the 6-0HDA-treated control animals.64 From these experiments it wasconcluded that other trophic factorsare not ableto compensatefor the missingendogenousFGF-2 afterlesion.64 Together, both studiesindicatethat lackofendogenous FGF-2doesnot inducealossofDAergic neurons in untreated animals. It is likelythat other membersof the FGF-family or other trophic factors maycompensatefor the lossofFGF-2. Depending on the paradigmused,treatment with toxinsthat areusedas"animalmodelsof Parkinson's disease~65 endogenousFGF-2mayplay, under certain conditions, a role in protection or lesion repair during adulthood. The different animal modelsused mayactivateor suppress differentially other FGF-family members. FGF-20, for example, might be of specific interest in this context.FGF-20 is expressed in the adult and 6-0HDA-lesionedstriatumand SN66.67 and it hasbeendocumentedthat recombinantrat FGF-20enhances the survival ofmidbrainDAergic neurons." In a recentgain-andloss-of-function

Neurotrophic Support ofMidbrainDopaminergic Neurons

77

in vitro study, it has further been demonstrated that FGF-20 promotes survival and stimulates DA release in the 5N; moreover FGF-20 rescues mature DA neurons from 6-0HDA toxicity.68 It would be ofinterest to know whether reduced endogenous levels ofFGFs might be involved in the development ofPD, or whether mutations in the genes for different FGF members might be associated with PD. 50 far, only for FGF-20 such an association has been documented in one study from the US69 and from a Japanese cohort," but not in Finnish or Greek PD patients." It could be speculated that FGF-20 compensates the loss of FGF-2 in the FGF-2 knockout mice. It may be possible that the beneficial effect ofFGF-20 is mediated through FGF receptor 3 (FGFR3), since in FGFR3 deficient mice a loss of about 20% ofthe DAergic neurons in the SN has been documented/"

Other Factors

In addition to neurotrophins, FGFs and TGF-f3s (for the latter, see review by Roussa et al, this volume), several other factors including ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), transforming growth factor-a (TGF-a), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and members of the interleukin (IL) family have been implicated in regulating development, survival and differentiation ofmidbrain DAergic neurons. Literature on these factors in the context of trophic functions on DAergic neurons are relatively scarce and have been reviewed previously to a large extent," Following the discovery that EGF protects DAergic neurons from glutamate toxicity in culrure." analysis ofTGF-a knockout mice" revealed a 50% loss ofboth neonatal and adult nigra! DAergic neurons, while neurons in the ventral tegmental area were not affected. These findings were interpreted in terms ofa TGF-a requirement for the normal proliferation or differentiation ofDAergic neurons within the SN. The PDGF BB isoform was reported to stimulate survival of rat and human mesencephalic DAergic neurons in culture." Moreover, PDGF as well as BDNF have been found to induce striatal neurogenesis in adult rats with 6-0HDA lesions." The probably most important study on CNTF reports that following axotomy of rat nigrostriatal DAergic neurons infusions of CNTF close to the lesioned neurons prevented cell losses, but not the loss ofTH?5 Two members of the IL, IL-l f3 and IL-6 have been shown to act as neurotrophic factors on DAergic neurons and protect DAergic neurons from MPP+ toxicity, respectively,"

Future Directions

Following more than a decade of screening a broad spectrum of growth factors for putative trophic functions on midbrain DAergic neurons, there is now clearly a focus on GDNF, neurturin and MANF (see review by Roussa et al, this volume), cytokines which hold great promises for the treatment of PD. While clinical studies with GDNF have failed to consistently document beneficial effects in patients with PD, neurturin and MANF still remain to be clinically tested. Current attempts to develop such factors into therapeutical tools also include the generation of small molecule-analogues. Finally, what should never be forgotten in this context: therapeutic biological efficacy of a molecule is as important as the technology underlying its application.

References

1. Chase TN, Oh]D, Blanchet PJ. Neostriata!mechanisms in Parkinson's disease. Neurology 1998; 51:530-535. 2. von Bohlen und Halbach 0, Schober A, Krieglseeln K. Genes, proteins and neurotoxins involved in Parkinson's disease. Prog Neurobiol 2004; 73: 151-177. 3. Krieglstein K. Factors promoting survival of mesencephalic dopaminergic neurons. Cell Tissue Res 2004; 318:73-80. 4. Unsicker K. Growth factors in Parkinson's disease. Prog Growth Factor Res 1994; 5:73-87. 5. Nagatsu T, Mogi M, Ichinose H et al. Cytokines in Parkinson's disease. J Neural Transm 5uppI2000; 143-151. 6. Reuss B, von Bohlen und Halbach 0. Fibroblast growth factors and their receptors in the central nervous system. Cell Tissue Res 2003; 313:139-157.

78

Development and Engineering ofDopamineNeurons

7. Barde YA. Neurotrophins: a family of proteins supporting the survival of neurons. Prog Clin Biol Res 1994; 390:45-56. 8. Frade JM, Barde YA. Nerve growth factor: two receptors, multiple functions. Bioessays 1998; 20:137-145. 9. Barbacid M. The Trk family of neurotrophin receptors. J Neurobiol1994; 25:1386-1403. 10. Chao MY. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci 2003; 4:299-309. 11. Altar CA, Siuciak JA, Wright P et ale In situ hybridization of trkB and trke receptor mRNA in rat forebrain and association with high-affinity binding of [125I]BDNF, [125I)NT-4/5 and [125I]NT-3. Eur J Neurosci 1994; 6:1389-1405. 12. Katoh-Semba R, Semba R, Takeuchi IK et al. Age-related changes in levelsof brain-derived neurotrophic factor in selected brain regions of rats, normal mice and senescence-accelerated mice: a comparison to those of nerve growth factor and neurotrophin-3. Neurosci Res 1998; 31:227-234. 13. Nishio T, Furukawa S, Akiguchi I et al. Medial nigral dopamine neurons have rich neurotrophin support in humans. NeuroReport 1998; 9:2847-2851. 14. Numan S, Seroogy KB. Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study. J Comp Neurol 1999; 403:295-308. 15. Hyman C, Hofer M, Barde YA et al. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 1991; 350:230-232. 16. Hagg T. Neurotrophins prevent death and differentially affect tyrosine hydroxylase of adult rat nigrostriatal neurons in vivo. Exp Neurol 1998; 149:183-192. 17. Hynes MA, Poulsen K, Armanini Met ale Neurotrophin-4/5 is a survival factor for embryonic midbrain dopaminergic neurons in enriched cultures. J Neurosci Res 1994; 37:144-154. 18. Hyman C, Juhasz M, Jackson C et ale Overlapping and distinct actions of the neurotrophins BDNF, NT-3 and NT-4/5 on cultured dopaminergic and GABAergic neurons of the ventral mesencephalon. J Neurosci 1994; 14:335-347. 19. Baquet ZC, Bickford PC, Jones KR. Brain-derived neurotrophic factor is required for the establishment of the proper number of dopaminergic neurons in the substantia nigra pars compacta. J Neurosci 2005; 25:6251-6259. 20. Venero JL, Beck KD, Hefti F. 6-Hydroxydopamine lesions reduce BDNF mRNA levels in adult rat brain substantia nigra. NeuroReport 1994; 5:429-432. 21. Altar CA, BoylanCB, FritscheM et ale Efficacy of brain-derivedneurotrophic factor and neurotrophin-3 on neurochemical and behavioral deficits associatedwith partial nigrostriatal dopamine lesions.J Neurochem 1994; 63:1021-1032. 22. Singh 5, Ahmad R, Mathur D et al. Neuroprotective effect of BDNF in young and aged 6-0HDA treated rat model of Parkinson disease. Indian J Exp Biol 2006; 44:699-704. 23. Klein RL, Lewis MH, Muzyczka N et al. Prevention of 6-hydroxydopamine-induced rotational behavior by BDNF somatic gene transfer. Brain Res 1999; 847:314-320. 24. LevivierM, Przedborski S, BencsicsC er ale Intrastriatal implantation of fibroblasts genetically engineered to produce brain-derived neurotrophic factor prevents degeneration of dopaminergic neurons in a rat model of Parkinson's disease. J Neurosci 1995; 15:7810-7820. 25. Frim DM, Uhler TA, Galpern WR et al. Implanted fibroblasts genetically engineered to produce brain-derived neurotrophic factor prevent I-methyl-4-phenylpyridinium toxicity to dopaminergic neurons in the rat. Proc Nat! Acad Sci USA 1994; 91:5104-5108. 26. Galpern WR, Frim DM, Tatter SB er ale Cell-mediated delivery of brain-derived neurotrophic factor enhances dopamine levelsin an MPP+ rat model of substantia nigra degeneration. Cell Transplant 1996; 5:225-232. 27. Howells D~ Porritt MJ, Wong JY et at. Reduced BDNF mRNA expression in the Parkinson's disease substantia nigra. Exp Neuro12000; 166:127-135. 28. Mogi M, Togari A, Kondo T et al. Brain-derived growth factor and nerve growth factor concentrations are decreased in the substantia nigra in Parkinson's disease. Neurosci Lett 1999; 270:45-48. 29. Zaman V, Nelson ME, Gerhardt GA et al. Neurodegenerative alterations in the nigrostriatal system of trkB hypomorphic mice. Exp Neuro12004; 190:337-346. 30. von Bohlen und Halbach 0, Minichiello L, Unsicker K. Haploinsufficiency for trkB and trke receptors induces cell loss and accumulation of alpha-synuclein in the substantia nigra. FASEB J 2005; 19:1740-1742. 31. Kramer ER, Aron L, Ramakers GM et al. Absence of Ret Signaling in Mice Causes Progressive and Late Degeneration of the Nigrostriatal System. PLoS BioI 2007; 5:e39. 32. von Bohlen und Halbach 0, Minichiello L. Neurotrophin receptor heterozygosity causes deficits in catecholaminergic innervation of amygdala and hippocampus in aged mice. J Neural Transm 2006; 113:1829-1836.

Neurotrophic SupportofMidbrain Dopaminergic Neurons

79

33. Dierssen M, Gratacos M, Sahun I et ale Transgenic mice overexpressing the full-length neurotrophin receptor TrkC exhibit increased catecholaminergic neuron density in specific brain areas and increased anxiety-like behavior and panic reaction. Neurobiol Dis 2006; 24:403-418. 34. Parain K, Murer MG, Yan Q et al. Reduced expression of brain-derived neurotrophic factor protein in Parkinson's disease substantia nigra. NeuroReport 1999; 10:557-561. 35. Porritt MJ, Batchelor PE, Howells DW: Inhibiting BDNF expression by antisense oligonucleotide infusion causes loss of nigral dopaminergic neurons. Exp Neuro12005; 192:226-234. 36. Momose ~ Murata M, Kobayashi K et ale Association studies of multiple candidate genes for Parkinson's disease using single nucleotide polymorphisms. Ann Neurol2oo2; 51:133-136. 37. Karamohamed S, Latourelle JC, Racette BA et ale BDNF genetic variants are associated with onset age of familial Parkinson disease: GenePD Study. Neurology 2005; 65:1823-1825. 38. Parsian A, Sinha R, Racette B et al. Association of a variation in the promoter region of the brain-derived neurotrophic factor gene with familial Parkinson's disease. Parkinsonism Relat Disord 2004; 10:213-219. 39. Hong CJ, Liu HC, Liu TY et al. Brain-derived neurotrophic factor (BDNF) Val66Met polymorphisms in Parkinson's disease and age of onset. Neurosci Lett 2003; 353:75-77. 40. Saarela MS, Lehtimaki T, Rinne JO et ale No association between the brain-derived neurotrophic factor 196 G > A or 270 C > T polymorphisms and Alzheimer's or Parkinson's disease. Folia Neuropathol 2006; 44: 12-16. 41. Xiromerisiou G, Hadjigeorgiou GM, EerolaJ et ale BDNF tagging polymorphisms and haplotype analysis in sporadic Parkinson's disease in diverse ethnic groups. Neurosci Lett 2007; 415:59-63. 42. Omitz DM, Itoh N. Fibroblast growth factors. Genome Bioi 2001; 2:REVIEWS3005. 43. Powers CJ, McLeskey S~ Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 2000; 7:165-197. 44. Unsicker K, Reuss B, von Bohlen und Halbach O. Fibroblast growth factors in brain functions. In: Lajtha A, Lim R, eds. Handbook of Neurochemistry and Molecular Neurobiology. Neuroactive Proteins and Peptides, New York, Heidelberg: Springer; 2006:93-122. 45. Bean AJ, Elde R, Cao YH et al. Expression of acidic and basic fibroblast growth factors in the substantia nigra of rat, monkey and human. Proc Nad Acad Sci USA 1991; 88:10237-10241. 46. Cintra A, Cao YH, Oellig C et al. Basic FGF is present in doparninergic neurons of the ventral midbrain of the rat. NeuroReport 1991; 2:597-600. 47. Bean A]. Oellig C, Pettersson RF et al. Differential expression of acidic and basic FGF in the rat substantia nigra during development. NeuroReport 1992; 3:993-996. 48. Ferrari G, Minozzi MC, Toffano G et al. Basic fibroblast growth factor promotes the survival and development of mesencephalic neurons in culture. Dev Bioi 1989; 133:140-147. 49. Ferrari G, Minozzi MC, Toffano G et ale Basic fibroblast growth factor affects the survival and development of mesencephalic neurons in culture. Adv Exp Med Bioi 1990; 265:93-99. SO. Otto D, Unsicker K. Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTP-treated mice. J Neurosci 1990; 10:1912-1921. 51. Park TH, Mytilineou C. Protection from I-methyl-4-phenylpyridinium (MPP+) toxicity and stimulation of regrowth of MPP{+)-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF. Brain Res 1992; 599:83-97. 52. Chadi G, Moller A, Rosen L et al. Protective actions of human recombinant basic fibroblast growth factor on MPTP-Iesioned nigrostriatal dopamine neurons after intraventricular infusion. Exp Brain Res 1993; 97:145-158. 53. Date I, Yoshimoto Y, Imaoka T et al. Enhanced recovery of the nigrostriatal dopaminergic system in MPTP-treated mice following intrastriatal injection of basic fibroblast growth factor in relation to aging. Brain Res 1993; 621: 150-154. 54. Otto D, Unsicker K. FGF-2-mediated protection of cultured mesencephalic dopaminergic neurons against MPTP and MPP+: specificity and impact of culture conditions, nondopaminergic neurons and astroglial cells. J Neurosci Res 1993; 34:382-393. 55. Casper D, Blum M. Epidermal growth factor and basic fibroblast growth factor protect dopaminergic neurons from glutamate toxicity in culture. J Neurochcm 1995; 65:1016-1026. 56. Shults C~ Ray J, Tsuboi K et al. Fibroblast growth factor-2-producing fibroblasts protect the nigrostriatal dopaminergic system from 6-hydroxydopamine. Brain Res 2000; 883:192-204. 57. Leonard S, Luthman D, Logel J et al. Acidic and basic fibroblast growth factor mRNAs are increased in striatum following MPTP-induced dopamine neurofiber lesion: assay by quantitative PCR. Brain Res Mol Brain Res 1993; 18:275-284. 58. Rufer M, Wirth SB, Hofer A et al. Regulation of connexin-43, GFAP and FGF-2 is not accompanied by changes in astroglial coupling in MPTP-Iesioned, FGF-2-treated parkinsonian mice. J Neurosci Res 1996; 46:606-617.

80

Developmentand EngineeringofDopamineNeurons

59. Giacobini MM, Stromberg I, Almstrom S ee al. Fibroblast growth factors enhance dopamine fiber formation from nigral grafis, Brain Res Dev Brain Res 1993; 75:65-73. 60. Mayer E, Fawcett ~ Dunnett SB. Basic fibroblast growth factor promotes the survival of embryonic ventral mesencephalic dopaminergic neurons-II. Effects on nigral transplants in vivo. Neuroscience 1993; 56:389-398. 61. Takayama H, Ray J, Raymon HK et al. Basic fibroblast growth factor increases dopaminergic grafi survival and function in a rat model of Parkinson's disease. Nat Med 1995; 1:53-58. 62. Timmer M, Muller-Ostermeyer F, Kloth Vet ale Enhanced survival, reinnervation and functional recovery of intrastriatal dopamine grafis cotransplanted with Schwann cells overexpressing high molecular weight FGF-2 isoforms. Exp NeuroI2004; 187:118-136. 63. Zechel S, Jarosik J, Kiprianova I et aleFGF-2 deficiency does not alter vulnerability of the dopaminergic nigrostriatal system towards MPTP intoxication in mice. Eur J Neurosci 2006; 23:1671-1675. 64. Timmer M, Cesnulevicius K, Winkler C et al. Fibroblast growth factor (FGF)-2 and FGF receptor 3 are required for the development of the substantia nigra and FGF-2 plays a crucial role for the rescue of dopaminergic neurons atter 6-hydroxydopamine lesion. J Neurosci 2007; 27:459-471. 65. von Bohlen und Halbach, O. Modeling neurodegenerative diseases in vivo review. Neurodegenerative Dis. 2006; 2:313-320. 66. Ohmachi S, Watanabe Y, Mikami T et al. FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain. Biochem Biophys Res Commun 2000; 277:355-360. 67. Grothe C, Timmer M, Scholz T et al. Fibroblast growth factor-20 promotes the differentiation of Nurrl-overexpressing neural stem cells into tyrosine hydroxylase-positive neurons. Neurobiol Dis 2004; 17:163-170. 68. Murase S, McKay RD. A specific survival response in dopamine neurons at most risk in Parkinson's disease. J Neurosci 2006; 26:9750-9760. 69. van der Walt ]M, Noureddine MA, Kittappa R et al. Fibroblast growth factor 20 polymorphisms and haplorypes strongly influence risk of Parkinson disease. Am J Hum Genet 2004; 74: 1121-1127. 70. Satake ~ Mizuta I, Suzuki S et al. Fibroblast growth factor 20 gene and Parkinson's disease in the Japanese population. NeuroReport 2007; 18:937-940. 71. Clarimon J, Xiromerisiou G, Eerola J et al. Lack of evidence for a genetic association between FGF20 and Parkinson's disease in Finnish and Greek patients. BMC Neuro12005; 5:11. 72. Blum M. A null mutation in TGF-alpha leads to a reduction in midbrain dopaminergic neurons in the substantia nigra. Nat Neurosci 1998; 1:374-377. 73. Nikkhah G, Odin P, Smits A et al. Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture. Exp Brain Res 1993; 92:516-523. 74. Mohapel P, Frielingsdorf H, Haggblad J et al. Platelet-derived growth factor (PDGF-BB) and brain-derived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions. Neuroscience 2005; 132:767-776. 75. Hagg T, Varon S. Ciliary neurotrophic factor prevents degeneration of adult rat substantia nigra dopaminergic neurons in vivo. Proc Nat! Acad Sci USA 1993; 90:6315-6319. 76. Akaneya ~ Takahashi M, Hatanaka H. Interleukin-1 beta enhances survival and interleukin-6 protects against MPP+ neurotoxicity in cultures of fetal rat dopaminergic neurons. Exp Neurol 1995; 136:44-52.

CHAPTER 8

TGF-~ in Dopamine Neuron

Development, Maintenance and Neuroprotection

Eleni Roussa, Oliver von Bohlen und Halbach and Kerstin Krieglstein*

Abstract

T

ransforminggrowthfactorbetas(TGF-(3s) aremultifunctionalcytokineswith widespread distribution.In the nervoussystem the biological effects ofTGF-(3coverregulationofproliferation,migration,differentiation, survival and death. Specifically, the effects ofTGF-f3 on mesencephalic DAergicneuronsextendfrom induction and specification of the dopaminergic phenotype via promotion of survival to neuroprotection in animal models of parkinsonism. Experimentalin vitro and in vivomodelshavecontributed to a better understandingofthe putativemechanisms underlyingthe effects ofTGF-f3on DAergicneurons and unravelled synergisms betweenmembersof theTGF-f3superfamily. In this chapter,we will reviewthe literatureavailable with focuson TGF-f3 proper and glialcell-line-derived neurotrophic factor (GDNF).

Introduction

TGF-f3s aremultifunctionalcytokines with widespread distribution.1 Theisolationand characterizationofTGF-f}byAnita Robertsand MikeSporn"haveleadto the identification of more than 30 relatedproteins that arenowsummarized asthe TGF-f} superfamily.' Within this superfamily, TGF-f3 proper build a subfamily of three membersTGF-f31, TGF-f32 and TGF-f33. TGF-f3s are synthesized aspreproproteinscontaininga signalpeptide and a C-terminallylocatedmaturepart. TGF-(3s form disulfide bridged homodimers and are folded in a cystine-knot likemotif.' During processing the mature protein stays noncovalently bound to its proprotein building a latent biologically inactiveform.5•6 TGF-fls signalviaa heteromerictransmembraneserin-threoninekinase receptor,wherebythe signalmaybe mediatedintracellularviaSmad-proteins that, in combination with other components,translocateinto the nucleusto form the transcriptionalcomplex." The biological effects of TGF-(3 in the nervous system cover regulation of proliferation, migration, differentiation, survival and death."!' GDNF hasbeen identifiedasa glialcellline-derived neurotrophic factorfor midbraindopaminergicneurons.P It containsthe sevenconservedand typically spacedcysteinresidues found in all membersof the TGF-(3 superfamily; however, it sharesless than 20%homologywith anyof the known TGF-f3 familymember.'? The effects of TGF-f3 in the development of mesencephalic DAergic neurons extend from induction and specification of the DAergicphenotype viapromotion of survival to neuroprotection in animal modelsof parkinsonism. *Corresponding Author: Kerstin Krieglstein-Institute for Anatomy and Cell Biology, Department of Molecular Embryology, University of Freiburg, Albertstrasse 17, 0-79104 Freiburg, Germany. Email: [email protected]

Developmentand Engineering ofDopamine Neurons, edited by RJ. Pasterkamp, M.P.Smidt andJ.P.H. Burbach.©2009 LandesBioscience and SpringerScience+Business Media.

82

Development and EngineeringofDopamineNeurons

DAergic neuronsarelocatedin theventralmesencephalon, a brainareacontainingtissue-spedfic stem cells, in spatialproximityto the signalingcenterfloor plate and isthmus.A commitment on the part of mesencephalic progenitors to the acquisitionof a dopaminergiccellfate is thought to implytwo complementarysources of inductivesignals: extrinsic, representedbydiffusible factors from the neighbouringsignalling centresalongthe dorsoventraland anteriorposterioraxisof the neuraltube that dictatepositionalinformationand intrinsic,reflectedbythe expression ofa master setof transcriptionfactorsthat restrictcellfatechoices."Exploringputativehierarchies, sequences and intersections ofintrinsicand extrinsic determinantsishowever, achallenging task. Earlystudies identified sonic hedgehog (Shh), the inductive signalfrom the floor plate and fibroblast growth factor 8 (FGF8), the correspondingsignalof the isthmus,as keypatterning molecules along the dorsoventraland anteriorposterioraxisof the neuraltube,respectively, involved in the inductionof mesencephalic DAergicneurons.15-19 However, several studieshaveproposedadditionalmolecules to be essential for the generationof mesencephalic DAergicneurons,amongthem TGF-fJS.20-23 During recent yearsthe scientific focus in elucidatingthe molecularequipment of progenitor cells committed to differentiate into DAergic neurons has shifted.The transcriptional code of precursor cellsthat triggers these cells to acquire a neuronal cell fate and, more specifically, a dopaminergic neurotransmitter phenotype has certainlynot yet been completed.Nevertheless, genetic mouse models have contributed to the identification of transcription factors, such as Nurr L'" Pitx3,25 Enl/2,26Lmxlb" and Lmxl a." as components ofthe dopaminergictranscriptional network. The requirement for TGF-f3 in the induction of midbrain DAergicneurons has been proved in several in vitro and in vivomodels.

Evidence for TGF-~ Effects on the Induction ofDopaminergic Neurons in Vitro

Thestartingpoint for thesestudieswasthe observation that duringearlymidbraindevelopment the spatialand temporal expression pattern ofTGF-(3sand TGF-f3 receptorscoincidedwith the distribution ofTH immunoreactivity. Indeed, besides TGF-f32 and TGF-(33 expression in the notochord and floor plate,both ligandsand receptorsare additionallyexpressed in the mesencephalic floorfrom E12 onwards.29lhe apparent and strikinglocationofTGF-fls and their cognate receptor in the regionwhereDAergicneurons are born hasled to the considerationthat TGF-f3s might contribute to the induction processof this neuronalpopulation. Treatment of low-densityculturesfrom rat EI2 ventral mesencephalon with a singledose of exogenous TGF-fJ3 increases TH immunopositive cells twofold within 24 h, whereasneutralization of endogenous TGF-f3 completelyabolishes the induction ofDAergic neurons, even in the presenceof exogenous Shh.20 Viceversa,TGF-f3 cannot cope with the Shh-dependent loss ofDAergic neurons in vitro, as shown by neutralization of endogenous Shh in the presenceof exogenous TGF-fJ.2O Thesedata suggest that induction of midbrain DAergicneurons does not depend solely on Shh function, but alsoon TG F-f3 signaling. It isastonishingthat this important contribution ofTGF-(3in the developmentof midbrain dopaminergic neuronshas escapednotice for manyyears. Shh isthe prominent moleculemediatingthe inductiveeffectof the floorplateon DAergic neurons.P'" In Shh mutant mice" a floorplate is lacking, the notochord isdegenerated and the ventral celltypesof the neural tube are absent.However, the keyexperiments that led to the establishmentof Shh as the inductivesignalfor DAergicneurons wereperformed in in vitro explants." In retrospect,it canbe assumed that TGF-f3 wasendogenously presentin theseexplants and consequentlyavailable as a cofactor for Shh to accomplish its differentiationpromoting effects.Thesedata alongwith the observationthat neither factor in the absence of the other hasthe capacityto induce TH argue againsta sequentialmode of action and favora cooperative model for actionsofShh and TGF-f3. The scenarioof a cooperative Shh/TGF-f3action for the induction of dopaminergicneurons hasbeenalsotestedin the neurospheres invitromodelderivedfrom EI2 mouseventralmidbrain." Treatment of the cells with TGF-(3 inducesNurr I and TH, but not Pitx3. Combined treatment

TGF-fJin DopamineNeuron Development, Maintenanceand Neuroprotection

83

with TGF-~, Shh and FGF8 causes an additional increase in Nurr1 and TH expression, whereas neutralization of each endogenously expressed individual factor dramatically reduces Nurr 1, Pitx3 and TH. Moreover, TGF-f3 is able to induce ectopically dopaminergic neurons in the dorsal mesencephalon. Together, the in vitro studies show: 1) TGF-f3s, in addition to Shh and FGF8, are required for the induction ofventral midbrain dopaminergic neurons, 2) neither TGF-f3, nor Shh, nor FGF8 are sufficient to induce dopaminergic neurons and 3) an interdependency ofTGF-f3, Shh and FGF8 in the differentiation ofdopaminergic neurons takes place, suggesting cooperation ofthese factors to induce dopaminergic cell fate. Analysis ofthe TGF-f3 differentiation signalling pathway shows that this process is receptor-mediated, involving the Smad pathway and the p38 mitogen-activated protein kinase pathway (MAPK). As discussed later, TGF-f3 signalling is also involved in promoting survival of mesencephalic dopaminergic neurons. This dual function ofthe TGF-f3 pathway in dopaminergic neurons occurs at different stages ofdevelopment and is thought to imply a distinct mechanistic basis.The question often asked as to whether the role ofTGF-f3s in the generation ofdopaminergic neurons might rely on a potential neurotrophic support has been addressed experimentally. Here, it should be noted that the experimental design and approach to study differentiation of dopaminergic neurons in vitro is completely different from and by no means interchangeable with that used for studying survival of dopaminergic neurons. The in vitro experiments identifying the survival promoting effects ofTGF-f3 on dopaminergic neurons are performed in cultures derived from dissociated mesencephalic tissue at later developmental stages (rat E14) without previous expansion. These cultures are treated several times with TGF-fl for 6-7 days. The TGF-f3 survival promotingeffeas on seeded dopaminergic neurons is then documented by a reduced decline ofTH positive neurons throughout the culture period with factor treatment as compared to controls. In the experiments addressing the role ofa given factor in the induction and differentiation ofdopaminergic neurons, dissociated rodent ventral mesencephalic tissue on E12 is directly plated onto coated cover slips and factor effects are assessed within 24 h ofa single dose. Alternatively, the established protocol to enrich eNS precursor cells, according to Reynolds and Weiss,31 can be applied: rodent E12 ventral mesencephalon selected for mesencephalic "neural stem cells"/ progenitor cells based on their capacity to survive in non-adherent suspension cultures, expanded these mesencephalic progenitor cells and generated neurospheres. After seeding dissociated neurospheres on coated culture dishes (now allowing cell attachment and differentiation), the effects of a single dose of factor treatment in differentiation ofprogenitors on the dopaminergic fate can be assessedwithin a short period of time, Le., 3 days. Parallel monitoring of cell proliferation and apoptosis events between different treatments ascertain that the observed effects are indeed inductive signals on mesencephalic progenitors towards dopaminergic cell fate rather than neurotrophic support on already differentiated dopaminergic neurons.

Evidence for TGF-fl Effects on the Induction ofDopaminergic Neurons in Vivo

Dopaminergic neurons in the midbrain represent only one population of catecholaminergic neurons in the midbrain, hindbrain and diencephalon. Are all catecholaminergic neurons ofthe same transmitter specified by the same developmental signals? This question has been addressed in several in vivo models. In the zebrafish, subgroups of basal diencephalic dopaminergic neurons may be considered to be homologous to some of the A8-AlO dopaminergic neurons of higher vertebrates. 32-34 Elegant forward genetics have challenged the dominant role ofShh and FGF8 in the induction ofdopaminergic neurons and provided compelling evidence for the importance ofTGF-f3 in this process. Agenesis of dopaminergic neurons is observed in mutants with affected TGF-f3/Nodal signalling, such as eye (endoding the Nodal-related protein Ndr2)35 and oep," In contrast, in the mutants with impaired Hh signaling syu (syu is the othologofthe mammalian Shhgene)37 andsmu (encoding Smo),38 as well as in ace (acerebellar/fgf8),39 a mutant with the MHB/FGF8 signaling affected, early dopaminergic neuron differentiation appears normal. Ocher catecholaminergic

84

Development and EngineeringofDopamineNeurons

groups, such as the pretectal group and the locus coeruleus apparently require Nodal/Shh and FGF8 signaling, respectively," Data obtained in the chickembryo have also led to the conclusion that TGF-~s are important players in the induction ofmidbrain dopaminergic neurons." A major advantage ofthe developing chick as an experimental model is that the processes ofinduction, differentiation and maintenance ofdopaminergic neurons take place within defined, specific time slots during development. The developmental period E2-7 spans the critical time for the induction of dopaminergic neurons, E4-7 definitively lacks the critical period of phenotype induction but includes a long period of development toward a dopaminergic phenotype and E6-10 represents the time frame in which phenotype stabilization and maintenance ofdopaminergic neurons are regulated. Consequently, the role ofa factor ofinterest within the developmental cascade ofevents can be specificallyand reliably analyzed in each and every frame ofdevelopment. Neutralization ofendogenous TGF-~ by systemic application of antibodies against TGF-~ to the chorionic-allantoic membrane of chick embryos on E2-7 but not E4-7 selectivelyreduces ventral dopaminergic neurons, but has no effect on locus coeruleus and diencephalic TH immunoreactive neurons. Because neutralization of endogenous TGF-~ does not interfere with floor plate development and Shh expression, the scenario of a TGF-~/Shh cooperation is again very attractive. What could the putative underlying molecular mechanisms of such factor cooperation be? It is possible that a signaling crosstalk between TGF-~/Smad2 and Shh via TGF-~-induced factor (TGIF), a transcriptional repressor of the TGF-~ pathway that may act upstream and/or downstream ofShh or altemativdyvia truncated Gli3 on Smad occurs," This hypothesis is based on the fact that mutations in human Tgifhave been associated with holoprosencephaly in humans, as seen in mutations in the Shh gene.41 On the other hand, Tgi/I-/Shh-I- double mutant embryos are indistinguishable from Shb": embryos, supporting the view that there is no genetic interaction between Shh and Tgif42 It is also conceivable that Smads, known to form complexes with other transcription factors, may either directly interact with Gli, or be involved in its transcriptional regulation. In mice, the role ofTGF-~ isoforms in the induction ofmesencephalic dopaminergic neurons has recently been evaluated using embryos lacking one allele of TGF-fJ2 or TGF-fJ3 and double 1-) mice embryos on E14.5 reveal knockout mice. 22 TGF-~ double knockout (TgfPZ'-/TgfP3reduced number ofmidbrain TH immunoreactive cells,aswell as TH/Nurr 1 double labeled cells, whereas the number of TH immunopositive cells in the locus coeruleus is not affected. In mice carrying one allele of TGF-fJ2 (Tgf-fJ2;+I-/Tgf-fJ3-I-j, or TGF-~3 (Tgf{Jl-I-/TgfP3+I-j determination ofTH positive cells reveals that TGFf3-2 is, specifically for the midbrain, relatively more important than TGF-f33.22

TGF-~ Superfamily Members and Induction ofDopaminergic

Neurons

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), artemin (ART) and persephin (PSPN), also named the GDNF family ligands (GFLs), are distant members of the TGF-~ superfamily. GFLs signal through a multicomponent receptor system comprised of a high-affinity binding component, a GPI-linked GFRa subunit and a common signaling component, the transmembrane tyrosine kinase Ret. 43•44 GFLs differentially promote survivaland regulate differentiation in peripheral and central neuronal populations. Interestingly, gdnf'-,45-47pspn-I- ,48 ntn- I- 49 andartn- I- 50 mice lack apparent developmental deficits in dopaminergic neurons, suggesting that considerable redundancy offactors with similar actions is present in this central neuronal population. As discussed below, GDNF is an established survival promoting factor for midbrain dopaminergic neurons in vitro and in vivo. In addition, GDNF induces Nurr1 and Pin:3, but not TH in vitro." Surprisingly, although GDNF requires TGF-f3 to exert its survival promoting effects,S2.53 Tgf{Jl-I-/Gdnf'- double mutant mice embryos on £14.5 lack a dopaminergic phenotype (unpublished data), suggesting that TGF-f3 isoforms may compensate for the loss ofeach other. In vitro, unpublished data from our laboratory strongly suggest the combination of TGF-f3 together with PSPN to be a potent dopaminergic inductive signal.The TH positive cellsgenerated

TGF-fJin DopamineNeuronDevelopment, Maintenance and Neuroprotection

85

are proper dopaminergic neurons expressing the whole molecular machinery and are additionally less vulnerable to MPP+ toxicity. The data availableso far also suggest that phenotype induction and survivaloffully differentiated neurons are accomplished through distinct pathways and requirement for individual factors. TGF-f3 is required for the induction and survival of dopaminergic neurons, whereas GDNF is required for differentiated dopaminergic neurons rather than developing ones, suggesting that GDNF may play an essential role in regulating and/or maintaining a differentiated neuronal phenotype. In all, the current state of research clearly indicates that none of the molecules is sufficient to induce dopaminergic neurons. Interplays and dependences between extrinsic and intrinsic components represented by diffusible secreted factors and transcription factors are rather the putative underlying mechanisms that still need to be elucidated.

TGF-~ Promotes Survival ofDAergic Neurons

TGF-f3 has been shown to promote neuron survival ofseveral neuron populations in vitro, including cultured motoneurons, sensory and mesencephalic dopaminergic neurons, respectively.54-58 In vitro, allthree mammalian TGF-f3 isoforms promoted the survivalofdopaminergic neurons and prevented MPP+ toxicity.55.56.59 Furthermore, in vivo experiments using immunoneutralization of TGF-f3 in developing chick (£6-10), a time period in development where phenotype induction and specification is already completed, resulted in reduced numbers of mesencephalic dopaminergic neurons." In rodents, following a period ofneurogenesis DAergic neurons undergo ontogenetic celldeath in early postnatal stages." During this period ofdevelopment the transcriptional cofactor homeodomain interactingprotein kinase 2 (HIPK2) has been shown to function as a mediator of TGF-f3-dependent regulation ofsurvival ofmidbrain dopaminergic neurons.f HIPK2 function depends on its interaction with receptor-Smad, Smad3, to regulate TGF-f3 target genes and seems to reflect TGF-f33 functions in this scenario.f These data strongly support the notion that TGF-f3 serves a role in regulating DAergic neuron survival in vertebrates in vivo as well. However, further analyses are required to unravel the role of distinct TGF-f3 isoforms in time, space and function during DAergic neuron development and maintenance. Several other members of the TGF-f3 superfamily were also shown to promote survival of mesencephalic DAergic neurons, including BMP, GDF as well as GDNF family ligands. 12.63-65 However, it should be noted that the mode of promoting survival is certainly different for individual signaling factors. TGF-f3 and GDNF have been shown to act directly on DAergic neurons, while GDNF is thought to act from the striatum in a target-derived manner and TGF-f3locally at the level ofthe cell bodies in the substantia nigra. In contrast, BMPs act indirectly on DAergic neurons, mediated via stimulation ofastrocytes. 20,64

GDNF Promotes Survival ofDAergic Neurons

Following the discovery of GDNF as a "dopaminotrophic" factor for cultured midbrain neurons,12.55.66 it was essential to establish its potency as a trophic factor for toxically impaired dopaminergic neurons ofthe nigrostriatal system in vivo. Indeed, it was found that GDNF applied exogenously to unilateral6-hydroxydopamine (6-0HDA) lesioned67-70 or to I-methyl-4-phenyl-l,2,3,6- tetrahydropyridine (MPTP)-lesioned rodents" largely prevented cell losses in the substantia nigra and restored striatal DA levels and DAergic fiber densities. Likewise, viral based application ofGDNF in animal models ofparkinsonism involving6-0HDA rats or MPTP monkeys was found to reverse functional deficits and to prevent nigrostriatal degeneration.F:" These results raised hopes that GDNF may be beneficial in the treatment of PD. However, it seemed to be crucial that GDNF be applied to the striatum and not to the SN. Injection ofGDNF into the striatum has a significant protective effect on nigrostriatal function at the behavioral level and on the integrity of the nigrostriatal pathway, whereas injection of GDNF into the SN has a protective effect on the nigral cell bodies, but not on the striatal innervation and thus failed to provide any functional benefit. Furthermore, GDNF, intraventricularly injected, does not improve the outcome after 6-0HDA lesion." One of the beneficial effects ofGDNF administred to the

86

Development and Engineering ojDopamineNeurons

striatum in vivois the suppression of apoptosisin DAergicneurons of the SN. Consistent with a role for striatal GDNF in regulatingthis event, applicationof GDNF neutralizingantibodies to the striatum augmentedcelldeath in the substantianigra." Clinicaltrialsof deliveryof GDNF directlyinto the putamen of human PD patientshasbeen found to significantly improvemotor functions and qualityof life,without severe sideeffects.77•78 However, despitethe positiveoutcome of these smallopen-labelclinicaltrials77•79•80 other studies includinga recent randomized controlled double-blindplacebo-controlledclinicaltrial failedto demonstrate efficacy and safetyof GDNF.81-83 TGF-~ and GDNF Cooperate to Promote Survival and Protection ofDAergic Neurons

Although TGF-fl doesnot fallinto the categoryof neurotrophicfactors,it iscrucially involved in the regulationof neuron survival in vitro and in vivo.When neurons aregrownin the presence oflow and subthreshold concentrations of establishedtrophic factors, i.e.,neurotrophins, neurokines and fibroblast growth factors,52.84 TGF-fl clearly shiftsthe dose-response curve to lower neurotrophic factor concentrations.52 Under such conditions, evensubthresholdconcentrations of a neurotrophic molecule canelicitaprominent survival promotingeffect. Thebiological significanceofTGF-(3 in modulatingneurotrophic factor efficacy becomesevenmorestrikingwhen the endogenousTGF-fl, which is synthesized by neurons,is neutralizedby TGF-fJ antibodies.~2.84 It may, therefore,be possible that it is not sufficient to applyonly GDNF, sincedata suggesting that the neurotrophic effects of GDNF requiresthe presenceofTGF-f3,which activates the transport ofGFRal to the cellmembrane44.85.86 are accumulating. Alongthis line,it hasbeen reported that for examplein cultured dopaminergicneurons, GDNF is not trophicallyactive unlesssupplemented with TGF-beta. Moreover, immunoneutralization of endogenous TGF-beta abolishes the neurotrophic effectof GDNF in culture." Moreover, this has not only been demonstratedin cellcultures,but alsoin animalmodelsofPD, as,for example, in MPTP treated animals. MPTP intoxication induces a loss of dopaminergic neurons in the substantia nigra and a decline in striatal dopamine contents. GDNF protects against the destructiveeffects of MPTP, including losses ofnigral neuronsand striataldopamine.V" Applicationof antibodiesneutralizingallthree TGF-f3 isoformsto the MPTP-lesioned striatum howeverabolishes the neurotrophic effect of GDNF, suggesting that striatal TGF-f3 maybe essential for permitting exogenous GDNF to act as a neuroprotectivefactor," In summary, thesedata suggest that trophic effects ofGDNF in the MPTP-Iesioned nigrostriatal dopaminergicsystem require endogenous TGF-f3. Consequently, co-applicationof GDNF and TGF-(3 is proposed for preclinicaland clinicaltrials for the treatment of human PD.

Conserved Dopamine Neurotrophic Factor (CDNF)

In 2007,Lindholmand collaborators haveidentifiedaconserved dopamineneurotrophicfactor (CD NF) asa trophicfactorfor DAergicneurons." CD NF and itshomologue "formesencephalic astrocyte-derived neurotrophic factor" (MANF)88 form a new familyof secretedproteins. It was shown that MANF selectively and potently promoted survival ofDAergic neurons at low (0.052.5 ng/mL) concentrations in vitr088.89 and CDNF was shown to prevent 6-0HDA induced degenerationof rat DAergicneurons usinga singledoseof CDNF injectionin a rat experimental model of Parkinson's disease."

Concluding Remarks

TGF-f3 is a contextualactingcytokine showinga broad rangeof functions on mesencephalic DAergicneuronsthat extendfrominduction and specification of the dopaminergic phenotypevia promotion of survival to neuroprotection in animal modelsof parkinsonism. Although TGF-(3 is not sufficient to inducea mesencephalic DAergicphenotype or to promote its survival, TGF-fl still serves an essential function in the lifeof a dopamine neuron.

TGF-f3 in Dopamine NeuronDevelopment, Maintenance andNeuroprotection

References

87

1. Roberts AB, Sporn MB. The transforming growth factor-Bs, In: Sporn MB, Roberts AB, eds. Handbook of Experimental Pharmacology. Heidelberg: Springer Verlag, 1990; 95:419-472. 2. Roberts AB, Anzano MA, Lamb LC et al. New class of transforming growth factors potentiated by epidermal growth factor: isolation from nonneoplastic tissues. Proc Natl Acad Sci USA 1981; 78:5339-5343. 3. Miyazono K, Suzuki H, Imamura T. Regulation of TGF-beta signaling and its roles in progression of tumors. Cancer Sci 2003; 94:230-234. 4. McDonald NQ, Hendrickson WA. A structural superfamily of growth factors containing a cystine knot motif. Cell 1993; 73:421-424. 5. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFbeta activation. J Cell Sci 2003; 116:217-224. 6. Rifkin DB. Latent transforming growth factor-beta (TGF-beta) binding proteins: orchestrators of TGF-beta availability. J BioI Chem 2005; 280:7409-7412. 7. Miyazono K, ten Dijke P, Heldin CH. TGF-beta signaling by Smad proteins. Adv Immunol 2000; 75:115-157. 8. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003; 113:685-700. 9. Krieglstein K. Transforming growth factor-betas in the brain. Handbook of Neurochemistry and Molecular Neurobiology 2006: 123-141. 10. Bortner M, Krieglstein K, Unsicker K. The transforming growth factor-betas: structure, signaling and roles in nervous system development and functions. J Neurochem 2000; 75:2227-2240. 11. Flanders KC, Ren RF, Lippa CF. Transforming growth factor-betas in neurodegenerative disease. Prog Neurobiol 1998; 54:71-85. 12. Lin LF, Doherty DH, Lile JD et al. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993; 260: 1130-1132. 13. Unsicker K, Suter-Crazzolara C, Krieglstein K. Neurotrophic roles of GDNF and related factors. In: Hefti F, ed. Handbook of Experimental Pharmacology 134: Neurotrophic Factors. Heidelberg: Springer, 1999: 189-224. 14. Smidt MP, Burbach PH. How to make a mesodiencephalic dopaminergic neuron. Nature Rev Neurosci 2007; 8:21-32. 15. Hynes M, Porter JA, Chinag C et al. Induction of midbrain dopaminergic neurons by Sonic Hedgehog. Neuron 1995a; 15:35-44. 16. Hynes M, Poulsen K, Tessier-Lavigne M et al. Control of neuronal diversity by the Boor plate: contact-mediated induction of midbrain dopaminergic neurons. Cell 1995b; 80:95-101. 17. Hynes M, Rosenthal A. Specification of dopaminergic and serotonergic neurons in the vertebrate CNS. Curr Opin Neurobiol1999; 9:26-36. 18. Ye 'W: Shimamura K, Rubenstein JR et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998; 93:755-766. 19. Crossley PH, Martinez S, Martin GR. Midbrain development induced by FGF8 in the chick embryo. Nature 1996; 380:66-68. 20. Farkas LM, Diinker N, Roussa E et al. Transforming growth factor-Bs are essential fort he development of midbrain dopaminergic neurons in vitro and in vivo. J Neurosci 2003; 23:5178-5186. 21. Holzschuh J, Hauptmann G, Driever W. Genetic analysis of the roles of Hh, FGF8 and Nodal signaling during catecholaminergic system development in the zebrafish brain. J Neurosci 2003; 23:5507-5519. 22. Roussa E, Wiehle M, Diinker N et al. Transforming growth factor ~ is required for differentiation of mouse mesencephalic progenitors into dopaminergic neurons in vitro and in vivo: ectopic induction in dorsal mesencephalon. Stem Cells 2006; 24:2120-2129. 23. Roussa E, Krieglstein K. Induction and specification of midbrain dopaminergic cell: focus on SHH, FGF8 and TGF-~. Cell Tisssue Res 2004; 318:23-33. 24. Saucedo-Cardenas 0, ~intana-Hau JD, Le WD et al. Nurrl is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic neurons. Proc Nat! Acad Sci USA 1998; 95:4013-4018. 25. Smidt MP, Smits SM, Bouwmeester H et al. Early developmental failure of substantia nigra dopamine neurons in mice lacking the hom eodomain gene pitx3. Development 2004; 131: 1145-1155. 26. Simon HH, Saueressig H, Wurst W et al. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J Neurosci 2001; 21:3126-3134. 27. Smidt MP, Asbreuk CH, Cox JJ et al. A second pathway for development of mesencephalic dopaminergic neurons requires Lmxlb. Nat Neurosci 2000; 3:337-341. 28. Andersson E, Tryggvason U, Deng Q et al. Identification of intrinsic determinants of midbrain dopamine neurons. Cell 2006; 124:393-405.

88

Development and EngineeringofDopamineNeurons

29. Unsicker K, Meier C, Krieglstein K et ala Expression, localization and function of transforming growth factor-betas in embryonic chick spinal cord, hindbrain and dorsal root ganglia. J Neurobiol 1996; 29:262-276. 30. Chiang C, Litingtung ~ Lee E et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 1996; 383:407-413. 31. Reynolds BA, Weiss S. Clonal and population analysisdemonstrate that an EGF-responsive mammmlian embryonic CNS precursor is a stem cell. Dev Bioi 1996; 175:1-13. 32. Kaslin J, Panula P. Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio). J Comp Neurol2oo1; 440:342-377. 33. Rink E, Wullimann MF. Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study. Brain Res Dev Brain Res 2002; 137:89-100. 34. Rink E, Wullimann MF. The teleostean (zebrafish) dopaminergic system ascending to the subpallium (striatum) is located in the basal diencephalon (posterior tuberculum). Brain Res 2001; 889:316-330. 35. Sampath K, Rubinstain AL, Chang AM et al. Induction of the zebrafish ventral brain and floorplate requires Cyclops/nodal signaling. Nature 1998; 395:185-189. 36. Zhang J, Talbot WS, Schier AF. Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 1998; 92:241-251. 37. Schauerte HE, van Eeden FJ, Fricke C et al. Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish, Development 1998; 125:2983-299. 38. Varga ZM, Amores A, Lewis KE et al. Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development 2001; 128:3497-3509. 39. Reifers F, Bohli H, Walsh EC et al. M FgfS is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 1998; 125:2381-2395. 40. Gripp ~ Wotton D, Edwards MC et ale Mutations in TGIF causeholoprosencephaly and link NODAL signalling to human neural axis determination. Nat Genet 2000; 25:205-208. 41. Roessler E, Belloni E, Gaudenz K et al. Mutations in the human Sonic hedgehog gene cause holoprocencephaly. Nat Genet 1996; 357-360. 42. Shen J, Walsch CA. Targeted disruption of Tgif the mouse otholog of a human holoprocencephaly gene, does not result in holoproncencephaly in mice. Mol Cel Bioi 2005; 25:3639-3647. 43. Airaksinen MS, Saarma M. The GDNF family: signalling, biological functions and therapeutic value. Nature Rev 2002; 3:383-394. 44. Sariola H, Saarma M. Novel functions and signaling pathways for GDNE J Cell Sci 2003; 116:3855-3862. 45. Moore ~ Klein RD, Farinas I er al. Renal and neuronal abnormalities in mice lacking GDNF. Nature 1996; 382:76-79. 46. PichelJG, Shen L, Hui SZ et ale Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 1996; 382:73-76. 47. Sanchez MP, Silos-Santiago I, Frisen J et al. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 1996; 382:70-73. 48. Tomac AC, Agulnick AD, Haughey N et al. Effects of cerebral ischemia in mice deficient in Persephin. Proc Nat! Ac Sci USA 2002; 99:9521-9526. 49. Heuckeroth RO, Enomoto H, Grider JR er al. Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory and parasympathetic neurons. Neuron 1999; 22:253-263. 50. Honma Y, Araki T, Gianino S et al. Artemin is a vascular-derived neurotrophic factor for developing sympathetic neurons. Neuron 2002; 35:267-282. 51. Roussa E, Krieglsteln K. GDNF promotes neuronal diffrentiation and dopaminergic development of mouse mesencephalic neurospheres. Neurosci Let 2004; 361:52-55. 52. Krieglstein K, Henheik P, Farkas L et al. Glial cell line-derived neurotrophic factor requires transforming growth factor-beta for exerting its full neurotrophic potential on peripheral and CNS neurons. J Neurosci 1998; 18:9822-9834. 53. Schober A, Peterziel H, von Bartheld CS et al. GDNF applied to the MPTP-Iesioned nigrostriatal system requires TGF-beta for its neuroprotective action. Neurobiol Dis 2007; 25:378-391. 54. Krieglsrein K. Factors promoting survival of mesencephalic dopaminergic neurons. Cell Tissue Res 2004; 318:73-80. 55. Krieglstein K, Suter-Crazzolara C, Fischer WH et al. TGF-beta superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity. EMBO J 1995; 14:736-742. 56. Poulsen KT, Armanini MP, Klein RD et al. TGF beta 2 and TGF beta 3 are potent survival factors for midbrain dopaminergic neurons. Neuron 1994; 13:1245-1252.

TGF-f:J in DopamineNeuronDevelopment, Maintenanceand Neuroprotection

89

57. Chalazonitis A, KalbergJ, Twardzik DR et al. Transforming growth factor beta has neurotrophic actions on sensory neurons in vitro and is synergistic with nerve growth factor. Dev Bioi 1992; 152:121-132. 58. Martinou JC, Le Van Thai A, Valette A et al. Transforming growth factor beta 1 is a potent survival factor for rat embryo motoneurons in culture. Dev Brain Res 1990; 52:175-181. 59. Krieglstein K, Unsicker K. Transforming growth factor-beta promotes survival of midbrain dopaminergic neurons and protects them against N-methyl-4-phenylpyridinium ion toxicity. Neuroscience 1994; 63:1189-1196. 60. Roussa E, Farkas LM, Krieglstein K. TGF-~ promotes survival on mesencephalic dopaminergic neurons in cooperation with Shh and FGF-8. Neurobiol Dis 2004; 16:300-310. 61. 00 TF, Burke RE. The time course of developmental cell death in phenotypically defined dopaminergic neurons of the substantia nigra. Brain Res Dev Brain Res 1997; 98:191-196. 62. Zhang J, Pho V, Bonasera SJ et al. Essential function of HIPK2 in TGF-beta-dependent survival of midbrain dopamine neurons. Nat Neurosci 2007; 10:77-86. 63. Krieglstein K, Suter-Crazzolara C, Hotten G er al. Trophic and protective effects of growth/differentiation factor 5, a member of the transforming growth factor-beta superfamily, on midbrain dopaminergic neurons. J Neurosci Res 1995; 42:724-32. 64. Jordan J, Bortner M, Schluesener HJ et ala Bone morphogenetic proteins: neurotrophic roles for midbrain dopaminergic neurons and implications of astroglial cells. Eur J neurosci 1997; 9:1699-1709. 65. Strelau J, Schober A, Sullivan A er al. Growth/differentiation factor-IS (GDF-15), a novel member of the TGF-beta superfamily, promotes survival of lesioned mesencephalic dopaminergic neurons in vitro and in vivo and is induced in neurons following cortisal lesioning. J Neural Transm 2003; 65 (Suppl):197-203. 66. Beck KD, Valverde J, Alexi T et al. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain. Nature 1995; 373:339-341. 67. Bowenkamp KE, Hoffman AF, Gerhardt GA et al. Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons. J Comp Neuro11995; 355:479-489. 68. Hoffer BJ, Hoffman A, Bowenkamp K et al. Glial cell line-derived neurotrophic factor reverses toxin-induced injury to midbrain dopaminergic neurons in vivo. Neurosci Lett 1994; 182:107-111. 69. Kearns CM, Gash DM. GDNF protects nigral dopamine neurons against 6-hydroxydopamine in vivo. Brain Res 1995; 672:104-111. 70. Sauer H, Rosenblad C, Bjorklund A. Glial cell line-derived neurotrophic factor but not transforming growth factor beta 3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion. Proc Nat! Acad Sci USA 1995; 92:8935-8939. 71. Tomac A, Lindqvist E, Lin LF et al. Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. Nature 1995; 373:335-339. 72. Gash DM, Zhang Z, Ovadia A et al. Functional recovery in parkinsonian monkeys treated with GDNF. Nature 1996; 380:252-255. 73. Kordower JH, Emborg ME, Bloch J et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 2000; 290:767-773. 74. Mandel R], Spratt SK, Snyder RO et al. Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats. Proc Nat! Acad Sci USA 1997; 94:14083-14088. 75. Kirik D, Rosenblad C, Bjorklund A. Preservation of a functional nigrostriatal dopamine pathway by GDNF in the intrastriata16-0HDA lesion model depends on the site of administration of the trophic factor. Eur] Neurosci 2000; 12:3871-3882. 76. 00 TF, Kholodilov N, Burke RE. Regulation of natural cell death in dopaminergic neurons of the substantia nigra by striatal glial cell line-derived neurotrophic factor in vivo. ] Neurosci 2003; 23:5141-5148. 77. Gill SS, Patel NK, Horton GR et al. Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 2003; 9:589-595. 78. Slevin JT, Gash DM, Smith CD et al. Unilateral intraputamenal glial cell line-derived neurotrophic factor in patients with Parkinson disease: response to 1 year of treatment and 1 year of withdrawal. J Neurosurg 2007; 106:614-620. 79. Patel NK, Bunnage M, Plaha P et al. Intraputamenal infusion of glial cell line-derived neurotrophic factor in PD: a two-year outcome study. Ann Neuro12005; 57:298-302. 80. Slevin JT, Gerhardt GA, Smith CD et al. Improvement of bilateral motor functions in patients with Parkinson disease through the unilateral intraputaminal infusion of glial cell line-derived neurotrophic factor. J Neurosurg 2005; 102:216-222. 81. Nutt JG, Burchiel KJ, Comella CL et al. Randomized, double-blind trial of glial cell line-derived neurotrophic factor (GDNF) in PD. Neurology 2003; 60:69-73.

90

Development and EngineeringofDopamineNeurons

82. Lang AE, Gill S, Patel NK et al. Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease. Ann Neuro12006; 59:459-466. 83. Sherer TB, Fiske BK, Svendsen CN et al. Crossroads in GDNF therapy for Parkinson's disease. Mov Disord 2006; 21:136-41. 84. Krieglseein K, Unsicker K. Distinct modulatory actions ofTGF-beta and LIF on neurotrophin-mediated survival of developing sensory neurons. Neurochem Res 1996; 21:843-50. 85. Peterziel H, Unsicker K, Krieglstein K. TGF~ induces GDNF responsiveness in neurons by recruitment of GFRal to the plasma membrane. J Cell Bioi 2002; 159:157-167. 86. Peterziel H, Paech T, Strelau J et al. Specificity in the crosstalk of TGF~/GDNF family members is determined by distinct GFR alpha receptors. J Neurochem Online.accepted Articles Accepted article online: 2007 doi: 10.l111/j.1471-4159.2007.04962.x. 87. Lindholm P, Vouwainen MH, Lauren J et al. Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature 2007; 448:73-77. 88. Petrova P, Raibekas A, Pevsner J et al. MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J Mol Neurosci 2003; 20: 173-188. 89. Petrova PS, Raibekas A, Pevsner J et al. Discovering novel phenotype-selective neurotrophic factors to treat neurodegenerative diseases. Prog Brain Res 2004; 146:168-183.

CHAPTER 9

Axon Guidance in the Dopamine System Asbeeta A. Prasad and R. Jeroen Pasterkamp*

Abstract

M

eso-diencephalicdopamineneurons (mdDA) neuronsarelocatedin the retrorubral field (RRF), substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) and giverise to prominent ascendingaxon projections. Theseso-calledmesotelencephalic projectionsareorganizedinto three main pathways: the mesostriatal, mesocorticaland mesolimbic pathways.Mesotelencephalicpathways in the adult nervous system have been studied in much detail as a result of their important physiologicalfunctions and their implication in psychiatric, neurological and neurodegenerativedisease. In comparison, relatively little is known about the formation of these projection systems during embryonic and postnatal development. However, understanding the formation of mdDA neurons and their projections is essentialfor the design of effective therapies for mdDA neuron-associated neurological and neurodegenerative disorders. Here we summarizeour current knowledge of the ontogeny of mdDA axon projections in subsystems of the developingrodent central nervous system (CNS) and discussthe cellularand molecular mechanisms that mediate mdDA axon guidance in these CNS regions.

Introduction

Meso-diencephalicdopamine neurons (mdDA) neurons acquiredtheir name from their origin within the mes-and diencephalonand from their characteristicexpressionofthe neurotransmitter dopamine.P Anatomicallyand functionally distinct groups ofmdDA neurons havebeen identified in the retrorubral field(RRF or A8), the substantia nigra pars compacta (SNc or A9) and the ventral tegmental area (VTA or AIO).3-s MdDA neurons give rise to prominent ascending axon projections.Theseso-calledmesotelencephalic projectionsareorganizedinto three main pathways: the mesostriatal, mesocorticaland mesolimbicpathways.I SNc neurons project axonsto the dorsal striatum forming the mesostriatalpathway.The mesostriatalpathwayis involvedin the coordination of voluntary movement, which is highlighted by the symptoms ofParkinson'sdisease(PD). In PD patients, mdDA neurons in the SNcdegenerateleadingto impaired motor control and even completelossofmovementin extremecases," Distinct from SNcprojections,VTA and RRFaxons prominently innervatethe ventromedialstriatum and prefrontal cortex (PFC), contributing to the mesolimbicand mesocorticalpathways,respectively. Mesocorticolimbicprojections are involved in the regulationofemotions and rewardand defects in mesocorticolimbicconnectivityhavebeen implicatedin addictivebehavior,depressionand schizophrenia.i" The anatomical,functional and molecularpropertiesof mesotelencephalicpathwaysin the adult nervoussystemhavebeen studied intensely as a result of their important physiologicalfunctions and their implication in human *Corresponding Author: R. leroen Pasterkamp-Departmentof Neuroscience and Pharmacology, RudolfMagnusInstituteof Neuroscience, UniversityMedical CenterUtrecht, Universiteitsweg 100, 3584 CG, Utrecht,The Netherlands. Email: r.j.pasterkarnpwurncutrecht.nl

Developmentand Engineering ofDopamine Neurons, edited by R.]. Pasterkarnp,M.P.Smidt and J.P.H. Burbach. ©2009 Landes Bioscience and Springer Science+Business Media.

92

Development'and Engin~~ring ofDopamjn~ Neurons

disease. In comparison, relatively little isknownabout the formation of theseprojection systems duringembryonic and postnataldevelopment." Here wesummarize our current understanding of the development of mdDA projections in rodents, whichhave beenstudiedin mostdetail In eachof the sections outlined below, a briefdescription of the ontogenyof mDA projections in a specific part of the developing centralnervous system (CNS) is followed bya summary of our presentknowledge of the cellular and molecular mechanisms that regulate mdDAaxonguidance in theseCNS regions.

Mesencephalon

In rodents, mdDA neuronsareborn around ElO.5 (mouse) or E12 (rat) and beginto extend neurites at Ell.S (mouse) or E13(rat).2 Initially, theseneurites follow adorsaltrajectory within the mesencephalon but thendeflectrostrally towards thediencephalon (Fig. lA, B).12'14 Chemotropic factors in thefloorplate,caudal brainstemanddorsalmesencephalon (DM) may contribute to this reorientation ofmdDAaxons (Fig. 2A). Tissue culturestudies showthat repulsive cuesformdDA axonsemanate from the floorplateand caudal brain stemregion in vitro.!2·15,16 Interestingly, the secretedaxonrepellent Slit3isexpressed at high levels in the caudal mesencephalon (Fig. 2B).J7 MdDAneuronsexpress theSlitreceptors Robo1andRobo217•18andSlid robustly repels embryonic mdDA axons in collagen matrixassays in vitro (Table 1).19 It is therefore temptingto speculate

B.

A.

E12.5

E13.5

E14.5

E15.5

c.

Figure 1. The ontogeny of rat mesodiencephalic dopamine (mdDA) axon projections during early to midembryonic development. Initially mdDA axons follow a dorsal trajectory (A) but then deflect rostrally towards the diencephalon (B). C) In the diencephalon, mdDA axons course ventrally and then reorient into a rostral direction at the border of the diencephalon and telencephalon. D) Around E14, mdDA axons reach an area ventrolateral to the develop ing striatum. Over the next few days the number ofaxons in this region increases without advancing notably beyond or into the overlying striatum. Around E17, the first mdDA axons begin to enter the developing striatum (not shown).

Axon Guidance in theDopamine System

93

that the caudalbrainstem region secretes Slit3 to repel mdDA axonstowards the diencephalon. An elegantstudybyNakamuraand colleagues further reveals a rostrocaudalor caudorostralgradient of short-rangedirectional cuesin the DM that mayalsocontribute to the stereotypicrostral trajectory ofmdDA axonsin the mesencephalon (Fig.2A).14The DM region expresses another memberof the Slit family, Slitl (Fig.2B).17 LikeSlit3,Slit1is a potent repellentfor mdDA axons actingthrough Robo receptors(Table1).19 Although Slitsaresecretedmolecules, they areknown to form tight interactionswith the extracellular matrix.20Therefore, Slit1mayserveasashort-range repulsive guidance cue for mdDA axons in the DM. It should be noted, however, that in addition to Slit1 other axon repellents are expressed in the DM (e.g., Sema3F) that mayimpose the rostraltrajectoryof mdDA axons(Fig.2B).13.21 Furthermore,the resultsof Nakamuraet aldo not excludethe possibilitythat a gradient of chemoattractivemolecules enforces the reorientationof mdDA axonsin the DM. Supportively, mRNA expression data suggest that Sema3C labelsthe future trajectoryof mdDA axonsin the mesencephalon. Sema3Cisa strong chemoattractantfor embryonic mdDA fibers (Table 1) and may attract these axons towards the rostral part of tAe mesencephalon in vivo (Fig.2B).13 Future studies employing genetically modified mice and immunohistochemicalstains,to establishSlit and Sema3protein expression in the DM, are needed to determine if the combined actionsof Slitsand Sema3s dictate to the stereotypictrajectoryof mdDA axonsin the mesencephalon.

Diencephalon

In the diencephalon,mdDA axonscourseventrallyand then reorient into a rostraldirectionat the border of the diencephalonand telencephalon(Fig.1C).12-14,22 Toidentifyfactorsthat control the trajectory of mdDA projections in the diencephalon,explants derived from different parts of the embryonicdiencephalonwerecoculturedwith mdDA neuron-containingmesencephalon explants." Explantsfrom the rostralpretectum (PT) werefound to exert growth-promotingand attractiveeffects on mdDA axons, whereasdorsal and ventral thalamustissues did not influence mdDA axon growth and guidance (Fig.2A).13The rostral part of the PT expresses two secreted semaphorins, Sema3Cand Sema3F, with opposingeffectson mdDA directionalgrowth in vitro. While Sema3Cattracts mdDA axons in culture, Sema3Fstronglyrepelsthese axons(Table1).A subset of mdDA neurons expresses neuropilin (Npn)-1 and/or Npn-2, obligatory components of Sema3receptorsand the axon attractiveeffectof the rostral PT can be neutralizedbyblocking Npn-l and Npn-2 in vitro.13 Overall,theseresultssuggest that axonalattractant Sema3Cis (partly) responsible for the attractiveeffectof the rostral PT on mdDA axons(Fig.2B).The observation that the PT alsocontains the axon repellentSema3Fraises the question why the PT has a net attractiveand not repulsive effect. A possibleexplanationfor this observationis that the attractive effectof Sema3C masksSema3F repulsion becauseof higher expression levels of the former or through mechanisms involving competitive agonism, as has been shown for Sema3A, Sema3B and Sema3C.23 Future analyses of Npn or Sema3-deficient micewill help to establishthe role of Sema3Cand other Sema3s in mdDA pathfinding in the diencephalon.

Medial Forebrain Bundle

Within the diencephalonand telencephalonmdDA axonsdiverge into two tightlyfasciculated axonaltracts,formingthe medialforebrain bundles (MFBs).The molecularcuesthat control the fasciculation of mdDA axonsinto two ipsilateral MFBsare unknown but the MFB region itself is known to exert potent chemotropic effects on mdDA axons. Coculture studies indicate that MFBexplantsderivedfrom E12 and £15 rat embryoshavean attractiveeffecton mdDA axons." This suggests that the MFB regionproduceschemoattractant molecules that guide mdDA axons toward the rostral telencephalon. Remarkably, £19 MFB explants no longer attract (or repel) mdDA axons." This specific regulation of the chemotropic properties of the MFB region may allowmdDA axonsto exit the MFB at mid-to-lateembryonicstagesand to proceedtowardstheir synaptictargets.Similarmolecularmechanisms havebeenreported for axonalprojectionsin other regionsof the CNS includingthe spinalcord."

Development andEngineering ojDopamine Neurons

94

A. Cellular

+

B. Molecular

+

Figure 2. Axon guidance of mesodiencephalic dopamine (mdDA) neurons in the embryonic mesencephalon and diencephalon. A) Schematic representation indicating the different brain regions that provide chemotropic signals for embryonic mdOA axons as determined by in vitro experiments . Regions displaying chemorepulsive (-) factors and chemoattractive (+) effects are ind icated. The floor plate (FP) and caudal brainstem (CB) regions produce chemorepellent molecules that may help to reorient mdDA axons rostrally. This rostral path is further enforced by a rostrocaudal or caudorostral gradient of chemoattractants or chemorepellents, respectively, in the dorsal mesencephalon (OM). The rostral part of the pretectum (PT) attracts mdDA axons in vitro. B) Schematic representation indicating the axon guidance molecules that have been proposed to mediate guidance events in the mesencephalon and diencephalon. Slit3 is expressed in the CB and may function to repel mdDA axons rostrally. This reorientation into a rostral direction may furthermore be controlled by a caudorostral gradient of the axon repellents Slitl and/or Sema3F. Sema3C is expressed along the mdOA trajectory in the mesencephalon and in the PT where it mediates chemoattractive responses. OT, dorsal thalamus; VT, ventral thalamus.

Robo

Robo

nd

abnormal ventral trajectory"

no defects reported *

nd

nd

nd

nd

nd

nd

nd

KO Mouse Analysis of mdDA Axon Pathways

Lin and Isacson, 2006

Lin et al, 2005

Bagri et al, 2002; Lin and lsacson, 2005, 2006

Bagri et al, 2002; Lin and Isacson, 2006

Hernandez-Montiel et ai, 2008

Hernandez-Montiel et al, 2008

Hernandez-Montiel et ai, 2008

Lin et al, 2005

Lin et al, 2005

Vue et al, 1999

Vue et al, 1999

References

*In Slttt: Slit2 double mutant mice the MFB is split, mdDA axons descend aberrantly into the hypothalamus and cross the midline. Abbreviations: DeC: deleted in corectal cancer; nd: not determined; Npn: neuropilin; Robo: roundabout.

Axon repulsion

Robo

Axon repulsion

Axon branching

SIit2

SIit3

Robo

Axon repulsion

SIit1

Npn-1/Npn-2

Npn-2

Axon attraction

Axon repulsion

Sema3C

Sema3F

Npn-1

DCC

Axon growth promotion

Axon growth promotion

DCC

nd

nd

Receptor In Vitro

Axon attraction

Sema3A

Netrin-1

Neuronal cell death

EphrinB2 Axon growth inhibition

AGM

In Vitro Effect on mdDA Axons

Table 1. Axon guidance molecules for mesodiencephalic dopamine (mdDA) neurons

b

~

II

~

~ ...

i·...

~

...So

;;.

~

;lS

~

~

~

~

96

Development and EngineeringofDopamineNeurons

Brain regions flanking the presumptive trajectory ofthe MFB such as the thalamus and hypothalamus have been proposed to dictate the characteristic ventrolateral position of the MFBs in the telencephalon. Although thalamic explants have no long-range chemotropic effects on mdDA axons in vitro, mdDA axons do not enter thalamic explants.P This observation hints at the presence ofcontact-dependent inhibitors ofmdDA axon outgrowth in the embryonic thalamus. The idea that medial brain structures contribute to the positioning ofthe MFBs is further supported by the disorganization ofthe caudal hypothalamus and concomitant aberrant midline crossing of MFB fibers in Nkx2.l mutant mice. 25,26 Based on the reduced expression ofSema3A in the caudal hypothalamus of Nkx2.l mutant mice it was postulated that Sema3A acts as a midline repellent for mdDA axons. 25 However, the finding that Sema3A functions a chemotrophic rather than a chemotropic cue for mdDA axons in vitro contrasts this idea." It should be noted, however, that individual Sema3s are bifunctional and can exert repulsive, attractive or axon growth promoting effects depending on the biological context in which they are encountered." Since the biological context ofcultured neurons is likely to be different from in vivo conditions further work is needed to study the role of Sema3A in vivo. Alternative explanations for the abnormal crossing ofMFB fibers in the absence of Nkx2.1 include the loss of a physical instead of a molecular barrier for axon growth and the decreased expression ofrepulsive cues other thanSema3A such as Slit2. Slit2 repels mdDA axons in vitro (Table 1)18 and mdDA axons are displaced ventrally as they course through the diencephalon of Slit2 mutant mice." In addition, in Slitl; Slit2 double mutants, the MFB splits into two components and mdDA axons descend ventrally into the hypothalamus towards the midline. Furthermore, many fibers abnormally cross the midline at the level of the basal telencephalon in the absence ofSlit 1 and Slit2.28 The observation that Nkx2.l mutant mice display changes in Slit expression" and wiring defects resembling those observed in Slit mutants suggests that abnormal Slit function may underlie the pathfinding errors observed in mice lacking Nkx2.1. However, mdDA pathways are more severely and in part also differently affected in Nkx2.l as compared to Slit mutants. In addition, many mdDA axons project normally in Slitl; Slit2 double mutants. Thus multiple distinct guidance cues are needed for the proper formation ofmdDA pathways.

Striatum

Around E14 in rat, mdDA axons in the MFB reach and invade the region ventrolateral to the developing ganglionic eminence (GE)/ striatum. Over the next few days the number ofaxons in this region increases without advancing into a rostral or dorsal direction (Fig. ID). Around E17, mdDA axon bundles begin to enter the developing striatum coincident with the emergence of a chemoattractive activity from the striatal region. Intriguingly, both late embryonic and postnatal but not early embryonic striatal explants attract mdDA explants in vitro.I2.13.22.29~31 This suggests that the aforementioned 'waiting period' for mdDA axons may be a consequence of the lack of chemoattraction by the early embryonic striatum. The molecules that mediate these chemoattractive effects are unknown. Interestingly, Sema3A is expressed by the embryonic striatum at the stage when mdDA axons enter the striatum. 13,32,33 However, function-blocking antibodies against the Sema3A receptor component Npn-l do not neutralize the attractive effect ofstriatal explants on mdDA axons." Another brain structure that may help to enforce the 'waiting period' for mdDA axons ventrolateral to the striatum is the neocortex. Cortical explants exert a repulsive effect on mdDA axons in vitro" and molecular cues emanating from the cortex could inhibit the rostral and dorsal progression ofmdDA fibers. From E17 onwards, rat mdDA axons penetrate the developing striatum. From their initial ventrolateral position mdDA, axons start to extend into dorsal, medial, lateral and rostral directions to establish the topographic connections that are found in the adult. In the adult, mdDA neurons in the SNc densely innervate the dorsal striatum, while VTA neurons predominantly target the ventral striatum. 1 The formation ofthese topographic connections is controlled by specific axonal pruning events during late embryonic and early postnatal development." In contrast to the adult situation, axon collaterals from mdDA neurons in the embryonic VTA and SNc (El 5,E17) innervate both

Axon Guidancein the DopamineSystem

97

the dorsal and ventral striatum. Topographic specificity is achievedduring late embryonic and earlypostnatal developmentthrough the selective elimination of axon collaterals from the SNc and VTA targeting the ventral and dorsal striatum, respectively." Although the molecularbasis of these axonalpruning eventsremainsto be established, Eph and ephrins havebeen implicated in the formation of topographicconnectionsbetween the dopaminergicmesencephalon and the striatum." EphB1 is expressed at high levels in the SN, but only weakly in the VTA. In the embryonic striatum, the EphB1 ligand ephrinB2 is stronglyexpressed in the ventromedialstriatum (targeted by VTA axons)but only weakly in the dorsolateralstriatum (targeted by SNc axons). Thus, SNc neurons with high levels of EphB1 project to the dorsolateralstriatum which wealdy expresses ephrinB2. In contrast,VTA neurons,which express low levels of EphB1, innervate the ventromedialstriatum wherehigh levels of ephrinB2are found. Tissueculture studiesshow that ephrinB2 inhibits the growth of EphBl-positive SNc neurons in vitro." Furthermore, ephrins can induce axonaldegenerationof cultured hippocampal neurons." Overall, these data suggest that ephrinB2mayregulatethe formationof topographic mdDA projectionsbyactingasan axon guidance and/or pruning factor for SN collaterals. It should be noted, however, that analysis of EphBl mutant miceisincoherentto the ideathat EphB1functionsin the formationof mesostriatal connections." Thissuggests that in SNcneuronsEph receptorsother than EphB1 maybeinvolved in detecting ephrinB2expression in the striatum. Genetic manipulation ofanother Eph, EphA5, results in prominent defects in mesostriatal projections. Mice overexpressing an extracellular fragmentofEphAS (EphAS-Fe),known to antagonizeephrinA signaling."displaya reduction in the number of mesostriatal projectionsin adulthood.39•4OThe observationthat neuronal survival is unaffectedin EphAS-Fe mice supports the idea that EphAS-ephrinAsignalingis required for the maintenanceand/or formation of mesostriatal projections.

Cortex

Theprefrontalcortex(PFC) receives adensedoparninergic innervation.ThefirstmdDA axons reach the rat PFC around EIS and at first remain confined to the subplate (SP). Over the nen fewdays, the number mdDA axonsin the SP increases but no penetration of the overlying cortical plate can be observed until EI7-EI8. Thus, similar to abovementioned 'waiting period' for mesostriatalprojections,mesocorticalaxonsstallfor several daysbeforeenteringmore superficial layers of the developingcortex." Themolecularcuesthat regulatethe spatiotemporalinnervation of the PFC bymdDA axonsremainto be identified.Following their initial penetration of the CP, mdDA axonscontinue to establishlayer-specific and topographic connectionswith the PFC.41.42 Remarkably and as stated above, corticaltissuestronglyrepels mdDA axonsin vitro.P However, the cortex is a large and heterogeneousstructure and the origin of the cortical explants used in this studywasnot specified. Other work,employingcoculturesof dissociated mesencephalic and cortical cells, showsthat neurons from different cortical regionsexert differentialeffects on the maturation of mdDA neurons." Theseresultsindicatethat characterization of chemotropicinfluencesexerted by (nonltarget regionsin the cortex on mdDA axonswill require microdissection and testingof corticalsubregions.

Axon Guidance Molecules and Disease

Studyingthe cellularand molecularbasisof mdDA neural connectivityallows understanding the factorsunderlyingdisease onset,progression and furthers the developmentof new therapeutic strategies, particularlywithin regenerative medicine. Forexample, insightinto the molecular control of mesotelencephalic pathwayformation and maintenancemayhelp to repair the mdDA system ofPD patients and could alsoprovideinsight into the onset and progression of this disorder.PD was originallyidentified byJames Parkinsonin 1817 on the basisof severe motor dysfunction. Succeeding analysis of postmortem tissue revealed a characteristic loss of SN mdDA neurons. Current therapies for treating PD aim to restore cerebral dopamine levels by administrating levodopa, a prodrug that is converted to dopamine by the enzymetyrosinehydroxlyase in dopaminergicneurons.However, due to the progressive neurodegenerative nature ofPD, the effectof

98

Development and EngineeringofDopamineNeurons

levodaisonly transient."Long-termgoalsof recovery striveto maintain stabledopaminelevels by transplantingdopamine-producing cells into the brainofPD patients.44 Thepitfallsof this approach arethe quantitiesof high-qualitycellsrequiredfor transplantationand the inability of manyof the grafted neurons to establishfunctional neural connections.The vital role of axon guidancemoleculesin cellreplacementthereapiesiswellrecognized. A recent studyshowsthat EScell-derived mdDA neurons are responsive to guidance cues such as Slits and netrin-I." Furthermore, the axonsof transplanted neurons can be guided in vivoby ectopic expression of guidancecues.45,46 Therefore, axonguidancemolecules mayserveto assist graftedmdDA neuronsin makingsuccessful new connections and in preventingthem from forming the inappropriate connectionsbelieved to underlie someof the sideeffects of cellreplacement strategies. In addition to the role of axon guidancemolecules in guidingnewlyformed projectionsof transplanted mdDA neurons,several studieshaveshownsignificant differences in axonguidancegeneexpression betweencontrol and PD patients or PD mousemodels.47-5oFurthermore,genomicpathwayapproaches identifypolymorphismsin the axonguidancepathways ofPD patients." Although further work is needed to establishthe roleof abnormalaxonguidancecuefunctionin thepathophysiologyofPD, changes in the expression of axonguidancemolecules couldleadto alteredpatterns of neuronalconnectivity in the mdDA system and as a consequence to neuronal dysfunctionand loss. Themesocorticolimbic system isinvolved in rewardseekingbehavior andisaffected duringdrug addiction.v" Drug inducedeffects canbe becomeperpetualwheredevelopmentand maintenance of addiction causechanges in neuralmorphologyand synapticactivity.52 Interestingly, the expression of axonguidancemolecules is modulated during the developmentof drug addiction.35,39,53,54 Cocaine induces Significant changes in gene expression of several membersof semaphorin and Eph/ephrin families in the NAc and VTA region." Additionally, disruption ofEphA/ephrin-A signalinginduceschanges in the behavioral response to psychostimulants," It isthereforetempting to speculatethat drug-induced changesin the expression of axon guidancemolecules contribute to the structural adaptationsthat underliethe long-termeffects of prolonged drug exposure. Changesin dopaminergic neurotransmission havealsobeenimplicatedin neurological diseases such asdepressionand schizophrenia.7,g,55,56 Although it remainsto be determinedwhether structural changes in dopaminergic connectionsunderliethesealterationsin neurotransmission, genetic studieslink axonguidancecuesto several of thesedisorders. Whether or not dysregulation of axon guidancemoleculeexpression is coupledwith thesedisordersremainsto be investigated.

Conclusions and Future Directions

Mesotelencephalic projections mediate a wide range of physiological functions and are affected in variousneurological, psychiatric and neurodegenerative disorders. It is wellrecognized that insight into the mechanisms that control the formation and maintenanceof mesotelencephalicprojectionsisessential for understandingand treatmentsof perturbed mdDA connectivity. Unfortunately, relatively little is known about the molecular signals that control the wiring of the mdDA system. Work during the past fewyearshas identifiedseveral differentaxon guidance molecules that may control the formation of mesotelencephalic projections. However, most of our current knowledge of mdDA axon guidancederives from geneexpression analysis and in vitro studiesand validationin in vivomodelswillbe required to establishhow axon guidancecues function in concert to establishfunctional mesotelencephalic connections.

Acknowledgements

The authors thank Marten Smidt en Peter Burbachfor critically readingthe manuscript and membersof the Pasterkamp labforhelpfuldiscussions. Workin the authors'laboratoryissupported by grants from Netherlands Organization of Scientific Research, Dutch Brain Foundation, the International ParkinsonFoundation,the Human FrontierScience Programand ABC Genomics Center Utrecht (to RJP). RJP is a NARSAD Henry and William TestInvestigator.

Axon Guidance in theDopamine System

References

99

1. Bjorklund A, Dunnett SB. Dopamine neuron systems in the brain: an update. Trends Neurosci 2007; 30: 194-202. 2. Smidt MP, BurbachJP. How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci 2007; 8:21-32. 3. Carlsson A, Falck B, Hillarp NA. Cellular localization of brain monoamines. Acta Physiol Scand Suppl 1962; 56:1-28. 4. Dahlstrom A, Fuxe K. Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of rnonoamines in me cell bodies of brainstem neurons. Acta Physiol Scand SuppI1964; 232:1-55. 5. Hokfelt T, Martensson R, Bjorklund A et ale Distribution of tyrosine hydroxylase-immunoreactive neurons in the rat brain. Bjorklund A, Holdlet T, eds, In: Handbook Chemical Neuroanatomy (Classical Transmitters in the CNS, Part I). Amsterdam: Elsevier 1984; (2):409-440. 6. Savitt JM, Dawson VL, Dawson TM. Diagnosis and treatment of parkinson disease: molecules to medicine. J Clin Invest 2006; 116:1744-1754. 7. Dunlop B~ Nemeroff CB. The role of dopamine in the pathophysiology of depression. Arch Gen Psychiatry 2007; 64:327-337. 8. Guillin 0, Abi-Dargham A~ Laruelle M. Neurobiology of dopamine in schizophrenia. Int Rev Neurobiol 2007; 78:1-39. 9. Nestler E]. Genes and addiction. Nat Genet 2000; 26:277-281. 10. Robinson TE, Berridge KC. The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Brain Res Rev 1993; 18:247-291. 11. Van den Heuvel DMA, Pasterkamp RJ. Getting connected in the dopamine system. Progress Neurobiol 2008; in press. 12. Gates MA, Coupe VM, Torres EM et ale Spatially and temporally restricted chemoattractive and chemorepulsive cues direct the formation of the nigro-striaral circuit. Eur J Neurosci 2004; 19:831-844. 13. Hernandez-Montiel HL, Tamariz E, Sandoval-Minero MT et al. Semaphorins 3A, 3C and 3F in mesencephalic dopaminergic axon pathfinding. J Comp NeuroI2008; 506:387-397. 14. Nakamura S, Ito ~ Shirasaki R et al. Local directional cues control growth polarity of dopaminergic axons along the rostrocaudal axis. J Neurosci 2000; 20:4112-4119. 15. Holmes C, Jones SA, Greenfield SA. The influence of target and nontarget brain regions on the development of mid-brain dopaminergic neurons in organotypic slice culture. Brain Res Dev Brain Res 1995; 88:212-219. 16. Tamada A, Shirasaki R, Murakami F. Floor plate chemoattracts crossed axons and chemorepels uncrossed axons in the vertebrate brain. Neuron 1995; 14:1083-1093. 17. Marillat V, Cases 0, Nguyen-Ba-Charvet KA et al. Spatiotemporal expression patterns of slit and robo genes in the rat brain. J Comp Neuro12002; 442:130-155. 18. Lin L, Rao Y, Isacson 0. Netrin-l and slit-2 regulate and direct neurite growth of ventral midbrain dopaminergic neurons. Mol Cell Neurosci, 2005; 28:547-555. 19. Lin L~ Isacson 0. Axonal growth regulation of fetal and embryonic stem cell-derived dopaminergic neurons by Netrin-I and Slits. Stem Cells 2006; 24:2504-2513. 20. de Wit J~ Verhaagen]. Proteoglycans as modulators of axon guidance cue function. Adv Exp Med BioI. 2007; 600:73-89. 21. Funaro H~ Saito-Nakazato ~ Takahashi H. Axonal growth from the habenular nucleus along the neuromere boundary region of the diencephalon is regulated by semaphorin 3F and netrin-l. Mol Cell Neurosci. 2000; 16:206-220. 22. Voorn P~ Kalsbeek A~ Jorritsma-Byham B et al. The pre and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum of the rat. Neuroscience 1988; 25:857-887. 23. Takahashi T, Nakamura F, Jin Z et al. Semaphorins A and E act as antagonists of neuropilin-l and agonists of neuropilin-2 receptors. Nat Neurosci, 1998; 1:487-493. 24. Dickson B]. Molecular Mechanisms of Axon Guidance. New York, NY. Science 2002; 298:1959-1964. 25. Kawano H, Horie M, Honma S et al. Aberrant trajectory of ascending dopaminergic pathway in mice lacking Nkx2.1. Exp Neurol, 2003; 182:103-112. 26. Marin 0, Baker J, Puelles Let al. Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections. Development 2002; 129:761-773. 27. Zhou Y, Gunput RF, Pasterkamp RJ. Semaphorin signaling: progress made and promises ahead. Trends BioI Sci. 2008 Apr;33(4):161-70. 28. Bagri A, Marin 0, Plump AS et al. Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 2002; 33:233-248.

100

Development andEngineering ofDopamine Neurons

29. Johansson S, Stromberg I. Fetal lateral ganglionic eminence attracts one of two morphologically different types of tyrosine hydroxylase-positive nerve fibers formed by cultured ventral mesencephalon. Cell Transplant 2003; 12:243-255. 30. Ostergaard K, Schou JP, Zimmer J. Rat ventral mesencephalon grown as organotypic slice cultures and cocultured with striatum, hippocampus and cerebellum. Exp Brain Res 1990; 82:547-565. 31. Plenz D, Kitai ST. Organotypic cortex-striatum-mesencephalon cultures: the nigrostriatal pathway. Neurosci Lett 1996; 209:177-180. 32. Marin 0, Yaron A, Bagri A et al. Sorting of striatal and cortical internenrons regulated by semaphorin-neuropilin interactions. New York. Science 2001; 293:872-875. 33. Pascual M, Pozas E, Soriano E. Role of class 3 semaphorins in the development and maturation of the septohippocampal pathway. Hippocampus 2005; 15:184-202. 34. Hu Z, Cooper M, Crockett DP et al. Differentiation of the midbrain dopaminergic pathways during mouse development. J Comp NeuroI2004; 476:301-311. 35. Yue ~ Widmer DA, Halladay AK et al. Specification of distinct dopaminergic neural pathways: roles of the Eph family receptor EphBl and ligand ephrin-B2. J Neurosci 1999; 19:2090-2101. 36. Gao PP, Yue ~ Cerretti DP et ale Ephrin-dependent growth and pruning of hippocampal axons. Proc Natl Acad Sci USA. 1999; 96:4073-4077. 37. Richards AB, Scheel TA, Wang K et al. EphBl null mice exhibit neuronal loss in substantia nigra pars reticulata and spontaneous locomotor hyperactivity. Eur J Neurosci 2007; 25:2619-2628. 38. Gale ~ Holland SJ, Valenzuela D M et al. Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 1996; 17:9-19. 39. Halladay AK, Tessarollo L, Zhou Ret al. Neurochemical and behavioral deficits consequent to expression of a dominant negative EphA5 receptor. Brain Res Mol Brain Res 2004; 123:104-111. 40. Sieber BA, Kuzmin A, Canals JM et al. Disruption of EphA/ephrin-a signaling in the nigrostriatal system reduces dopaminergic innervation and dissociates behavioral responses to amphetamine and cocaine. Mol Cell Neurosci 2004; 26:418-428. 41. Kalsbeek A, Voorn P, Buijs RM et al. Development of the dopaminergic innervation in the prefrontal cortex of the rat. J Comp Neurol 1988; 269:58-72. 42. Van Eden CG, Hoorneman EM, Buijs RM et al. Immunocytochemical localization ofdopamine in the prefrontal cortex of the rat at the light and electron microscopical level. Neuroscience 1987; 22:849-862. 43. Hemmendinger LM, Garber BB, Hoffmann PC et al. Target neuron-specific process formation by embryonic mesencephalic dopamine neurons in vitro. Proc Nat! Acad Sci USA. 1981; 78:1264-1268. 44. Winkler C, Kirik D, Bjorklund A. Cell transplantation in Parkinson's disease: how can we make it work? Trends Neurosci 2005; 28:86-92. 45. Jin ~ Ziemba KS, Smith GM. Axon growth across a lesion site along a preformed guidance pathway in the brain. Exp Neurol, 2008 Apr;210(2):521-30 46. Ziemba KS, Chaudhry N, Rabchevsky AG et al. Targeting axon growth from neuronal transplants along preformed guidance pathways in the adult CNS. J Neurosci 2008; 28:340-348. 47. Griinblatt E, Mandel S, Jacob-Hirsch J er al. Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin-proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes. J Neural Transm 2004; 111:1543-1573. 48. Grunblatt E, Mandel S, Maor Get al. Gene expression analysis in N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine mice model of Parkinson's disease using cDNA microarray: effect of R-apomorphine. J Neurochem 2001; 78:1-12. 49. Hauser MA, Li Yj, Xu H et al. Expression profiling of substantia nigra in parkinson disease, progressive supranuclear palsy and frontotemporal dementia with parkinsonism. Arch Neurol2005; 62:917-921. SO. Miller RM, Callahan LM, Casaceli C et ale Dysregulation of gene expression in the I-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-Iesioned mouse substantia nigra. J Neurosci 2004; 24:7445-7454. 51. Lesnick TG, Papapetropoulos S, Mash DC et al. A genomic pathway approach to a complex disease: axon guidance and Parkinsons disease. PLoS Genet 2007; 3:e98. 52. Robinson TE, Kolb B. Structural plasticity associated with exposure to drugs of abuse. Neuropharmacology 2004; 47 (Suppll):33-46. 53. Bahi A, Dreyer JL. Cocaine-induced expression changes of axon guidance molecules in the adult rat brain. Mol Cell Neurosci, 2005; 28:275-291. 54. Jassen AK, Yang H, Miller GM et al. Receptor regulation of gene expression of axon guidance molecules: implications for adaptation. Mol Pharmacol2006; 70:71-77. 55. Dailly E, Chenu F, Renard CE et al. Dopamine, depression and antidepressants. Fundam Clin Pharmacol 2004; 18:601-607. 56. Sesack SR, Carr DB. Selective prefrontal cortex inputs to dopamine cells: implications for schizophrenia. Physiol Behav 2002; 77:513-517.

CHAPTER 10

Protocols for Generating ES Cell-Derived Dopamine Neurons Sonja Kriks* and Lorenz Studer

Introduction

S

tem cells are defined by their ability to self-renew and to differentiate into specific specialized cell types. Pluripotent stem cells such as embryonic stem cells are capable of differentiatinginto all cell types of the three germ layers. Self-renewal and differentiation potentialarepropertiesthat make stemcells an attractive source for celltherapeuticeffonsincluding the treatment of neurologicaldiseases such as Parkinson's disease (PD). Parkinson's disease isone of the most common neurological disordersand is characterizedby the selective degenerationof dopamine (DA) neurons in the ventral midbrain. The midbrain region contains three groupsof DA neurons, the retrorubral field (A8), the tegmental areaof the ventral midbrain (VTA, AIO) and the substantia nigra pars compacta (A9). Only the latter subgroup is primarily affected in PD and responsible for most of the motor dysfunction. Due to this rather selective loss of DA neurons in the substantianigra, PD is considereda neurological disease amenableto cellreplacement. Cell replacementtherapyin PD has been attempted in several hundred patientsworldwide usingfetalhuman DA neurons.While promisingresultshavebeen reported in several open label studies(e.g., 1,2) placebo-controlled clinicaltrialsusinghumanfetaldopamineneuronshaveyielded modestclinicalimprovementat best.3,4 Furthermore,a subsetof thesepatientsdisplayed disabling graft-induceddyskinesias. There are many potential reasonsfor this relatively poor outcome as discussed in detailelsewhere.' However, the limited availability ofdonor tissue, the lowpercentage ofDA neuronswithin fetalgraftsand ethicalconcernsassociated with the useofhuman fetaltissue suggest that alternativecellsources are required for successful clinicaltranslation. Thecurrentlymostpromisingcellsourcefor generatingauthenticmidbraindopamineneurons in vitro are embryonic stem cells (ESCs). The main advantage of a stem cell based strategy, in contrast to fetal tissue, is the availability of potentially unlimited sources of defined DA neurons at anystageofdifferentiation. While recentdevelopments and noveldifferentiation protocolshave brought the stem cellfieldcloserto this goal,considerable challenges remain in translatingthese potential advantages of ESCsinto safeand efficacious celltherapy. ~euralJ)evelopmnent

Theformationof the nervoussystem beginswith neuralinduction, the processbywhichdorsal ectodermalcellsof the gastrula-stage embryoaredirected towardsa neural identity in response to signals from the underlyingmesoderm.Thesesignals compriseNoggin, Chordin and Follistatin, which act on the overlying dorsalectoderm by blockingBMP signaling, leadingto the formation of the neuralplate.6-8 Initially, theneuralplateis thoughttohave ananterior character, astheinhibition *Corresponding Author: Sonja Kriks-Developmental Biology Program and Department of Neurosurgery, Sloan-Kettering Institute for Cancer Research, 1275 York Ave, Box 256, New York, New York 10065. Email: [email protected]

Developmentand Engineering ofDopamine Neurons, edited by R]. Pasterkamp, M.P.Smidt and J.P.H. Burbach.©2009 LandesBioscience and SpringerScience+Business Media.

102

Development andEngineering ofDopamine Neurons

ofBMP signalingbyBMPinhibitorsinducesthe expression ofanteriormarkerproteins,but not of posterior markersand is thereforereferredto asthe anterior ground stateofthe CNS. To develop a moreposteriorcharacter, suchasmidbrain,hindbrain and spinalcord,this anterior ground state is modified by posteriorizingsignals including retinoic acid (RA) and membersofthe fibroblast growth factor (FGF) and Wnt families. 9.13 Patterning cues for midbrain induction include fibroblast growth factor 8 (FGF8), a factor criticalfor the induction and maintenanceof the midbrain-hindbrainorganizer" and sonichedgehog (SHH), 15 a ventralizingmorphogen,secretedbythe underlyingnotochord. The intersection of these two signals at the time of neural induction is essential in the formation of the ventral midbrain domain.P whereDA neurons are born.

Derivation ofMidbrain DA Neurons from Embryonic Stem Cells (ESCs) GeneralProperties andDifferences between Mouse andHuman ESCs

Embryonic stem cellswere first isolated from the inner cell mass (ICM) of a mouse blastocyst-stage embryo in the early 1980s.17.18It has been shown that culture of the inner cellmassin the presenceof mouseembryonicfibroblasts (MEFs)can resultin clonalpopulationsof cellswith extensive proliferation capacity and pluripotent differentiation properties as demonstrated by their ability to generatechimericmice.The abilityof mouseESCs to contribute to the germ-line ofchimericmice has been the basisof modem mousegeneticssuch as gene targeting in mice.19.20 However, mouseESCscannot differentiate into trophectoderm under normalconditions.Mouse ESCscanbe identifiedbya setoftranscriptionfactorscharacteristic of the pluripotentstateincluding Oct4, Nanog and Sou or surface markerssuch asSSEAl. MouseESCscan be propagatedon MEFsor under feeder-free conditions in the presenceof leukemiainhibiting factor (LIF). More recent studies haveshown that BMPs can substitute for serum-derived factors" and that under certain conditions mouseESCscan be propagatedin the absence of anygrowthfactorsor feeders upon inhibition ofFGF, Erk and GSK3signaling (3i protocolj.f In 1998the firstsuccessful isolation of human ESCs has been reported." Similarto mouseESCs,human ESCs alsoexpress a set of characteristic surface markers includingSSEA3 and SSEA4 and express a nearlyidenticalset of transcriptionfactorsassociated with pluripotencyincludingOct4, Sou and Nanog.Furthermore, both mouseand human ESCscanbe propagatedon MEFs.However, definedsignals essentialfor human ESC self-renewal are distinct from those in mouse ESCs.For example, human ESCs are not dependent on LIF signals,23.24 but requireFGF2 for maintainingan undifferentiatedstate." In contrast,in mouseESC, the MEKpathway, whichisactivated byFGFspromotesdifferentiation.26 Similarly, TGFf3 signals areimportantforhuman27.28but not mouseESCself-renewal. Furthermore, BMPs causedifferentiationof human ESCs along extraembryonic lineages29.30 while promoting self-renewal in mouseESCS.21 Recentstudiesin the mousesuggest that thesesurprisingdifferences in growth requirementsmayreflectdifferentdevelopmental stages asmouseepiblastderivedpluripotent stem cells mimic the growth conditions of human ESCS.31,32 One particular challenge for human ESC studiesis the lackof an appropriatein vivoassay as the generationof chimericmice, germ-linecontribution or tetraploid complementationassays are not available giventhe ethical and biological constraints.While teratoma formation has been used as a surrogateassay,33 it is important to includefunctionalassays in human ESC derivatives in vitro or upon transplantation in vivosuch as in the caseof ESC derivedmidbrain dopamine neurons.

How to Define a Midbrain DA Neuron in Vitro

The first step in definingmidbrain dopamine neuron identity in vitro is the demonstration of neural and neuronalidentity followed byco-expression ofdopamine relatedbiochemicalmarkers such as tyrosine-hydroxylase (TH), aromatic acid decarboxylase (AADC), VMAT2 (vesicular monoamine transporter) and the high affinitydopamine transporter (DAT). In addition to positivebiochemicalmarkers, it isequallyimportant to ascertainthe absence of markersexpressed in

Protocolsftr GeneratingES Cell-Derived DopamineNeurons

103

nondopamine neurons and in dopamine neurons outsideof the midbrain. Lackofnoradrenergic markerssuch as dopamine beta hydroxylase (DBH) and GABAergic markerssuch as glutamate acid decarboxylase (GAD) are of particular importance. Analysis of biochemicalmarkers should be complementedwith region specific markers such as the transcription factors FoxA2,34.35 Nurr 1,36 Pitx3,37 Lmxla." Otx2 39and Lmxlb." Regional markers that distinguish midbrain dopamine neurons from DA neurons of forebrain and diencephalicidentity include Pax6and Nkx2.141 respectively. Midbrain dopamine neuron morphology42 and ultrastructure'Y' havebeen describedin greatdetail and ESC derivedDA neurons should approximatethesecharacteristic features of primary midbrain dopamine neurons.Finally, assays are required to demonstrate neuronal and dopaminergic function. Electrophysiological measurements should be used comparingphysiological behaviorof ESC derived45-48 to primary midbrain dopamine neurons," Biochemical function can be readilyassessed in vitro bymeasuring DA release via HPLC analysis46.47.49.5o or in vivovia microdialysis."

ProtocolsfOrDA Neuron Derivation

Most in vitro differentiation protocols for the derivation of midbrain dopamine neurons from ESCs can be subdividedinto at least three sequentialsteps.The first step is neural induction of ESCs followed by exposure to midbrain patterning factors including SHH and FGF8 (seeabove)during which neural precursorsadopt midbrain/hindbrain identity.49The midbrain patterning step is followedby terminal differentiationduringwhich ventral midbrain precursors differentiateinto maturedopamine neuronsin the absence of anymitogensbut in the presence of neurotrophic factorsand in someprotocolsadditionaldifferentiationagentssuchasascorbic acid and dibutyrylcyclic AMP.47 Both in mouseand human ESCsat the end of thisfinaldifferentiation stepabout 50%of allESC progenyhasdifferentiatedinto postmitotic neuronsand a considerable percentageof theseESC derivedneuronsexpress tyrosinehydroxylase, the rate limitingenzymein DA synthesis, aswellasother knownDA neuron markers includingAADC andVMAT2.Overthe last fewyears it has becomeclearthat this basicthree stepdifferentiation strategyrequiresfurther refinementwith regard to monitoring floor plate markerexpression during ESC differentiation, directing midbrain versus hindbrain induction and synchronizing the differentiationof uncommitted and midbrain committed precursorstowardsDA neuron fate. The keychallenge in usingESC progenyfor cellreplacement therapiesin PD is the generation of authentic midbrain dopamine neuronsexpressing the completetranscription,biochemicaland functional profileof a mature midbrain dopamine neuron in vitro and in vivo. While manyavailableprotocolsincludethe useofSHH/FGF8 followed bya terminaldifferentiationstepthere are considerable differences in the strategies used to achieve neural induction. One important question is how these differences in neural induction and earlypatterning strategies affectoutcome. Most available protocols for neural induction and early patterning can be grouped into three main categories: a) embryoid body (EB) mediated differentiation, b) differentiationinduced via cocultureon stromalfeederlayers suchasPA-6and MS-5and neuralinduction through "default" in the absence of inducing signals. EB-Based Protocol The classic strategyof neural induction in mouse ESCs is basedon embryoid body (EB)formation induced by aggregation of ESCs on non-adherent plates.EBsare thought to mimic the environment of the peri-implantation embryo? where cell-cell interactions facilitate inductive eventsleadingto the formation of the three germlayers. The first generationofEB basedneuron induction protocols werebased on exposureto retinoic acid (RA) such as the 4- /4+ protocol.53 However, retinoicacidbasedneural induction biases neuralprogenytowardshindbrain/anterior spinal cord identity which is not suitable for midbrain dopamine neuron derivation.EB-based protocols that obviatethe useof retinoic acidareoften basedon multistepapproaches that select, expandand differentiate neural precursorsfrom EBsunder serumfree condirions.lt The first successful demonstration of generatingmidbrain dopamine neuronsfrom mouseESCswasbasedon such a multi-step approach (5 step protocol)." This study demonstrated that timed exposure to

104

Development and EngineeringofDopamineNeurons

SHH/FGF8 will bias mouse ESC derivedprecursorstowardsmidbrain/hindbrain identity and lead to the efficient generation of midbrain dopamine neurons (-34% of all neurons, -22% of total cells). EBbasedprotocolshavebeensubsequently adaptedto human ESC differentiation.ss.s6 Several EB basedneural differentiationprotocols havebecomethe basisfor current strategies to generatehumanESCderivedmidbrainDA neuronsviadefinedfaetors48•s7or throughan additional midbrain astrocytemediated inductiveeffect. 58

Feeder-Based Protocol Another strategy for the directed differentiation of ESC into the neural lineage includes coculturingof ESC with neural inducingbone marrow-derived stromalcelllinessuch as PA-6or MS5.Kawasaki et alS9 developeda differentiationprotocol in which they cocu1tured mouseESC with PA6feeders exhibiting"stromalcellderivedinducing activity" (SDIA).After two weeksof differentiation the investigators found that ESC progeny spontaneouslydifferentiate into 52% neuronsand 30%ofwhichwereDA neurons.Interestingly, DA neurondifferentiation wasachieved in the absence ofanyextrinsicpatterning molecules such as SHH and FGF8.60 Our group demonstrated that combining neural induction through MS5 stromal cellswith appropriate patterning and growth factors leads to the highly efficient derivation of midbrain dopamine neurons.f The first step of the protocol was plating of undifferentiatedESCs at low density (100 cells/cm2) on MS5in serum-replacement medium (KSR).At day5of differentiation the medium was supplementedwith SHH and FGF8 to induce midbrain patterning followed by continued exposure to SHH and FGF8 in N2 medium supplementedwith FGF2 for further expansionof committed midbrain precursors. After 11 daysof differentiation, allmitogenswere withdrawn and the final differentiationwas induced in the presenceof ascorbic acid (AA) and brain-derived-neurotrophic factor (BDNF). This differentiationparadigm has been used in cell transplantation settings including the proof of concept demonstration of therapeutic cloning in using nuclear transfer (nt) ESC derivedDA neurons in mouse models" and subsequently in individuallypatched PD mice." The useof stromalfeedercoculturealsoled to the firstsuccessful protocols for generatingprimatef and eventually human ESC derivedmidbrain DA neurons." The human ESC protocols are based on neural induction on MS5 for up to 28 days(Passage 0, 16-28 days)followed by isolation of neural rosettes and culture in the presenceofSHH/FGF8 (Passage 1,additional7-10days),singlecelldissociation and continued midbrainpatterning in the presenceofSHH/FGF8 (Passage 2, expansion, additional 5-7days). Thefinaldifferentiationstep is induced upon withdrawalof SHH and FGF8 and addition of several differentiation, specification and survival factorsincludingascorbic acid (AA), BDNF,GDNF, dbcAMP and TGF(33 (P2 differentiation,additional 7-14 days). The aboveprotocol yields -30-50% postmitotic neurons and 70% of these neurons co-express TH. The remainingcells are mostlynestin+ precursorsthat differentiatein a more protracted manner. While the initial studies suggested that stromal feeder mediated induction may bias cells exclusively towards a ventral midbrain fate, subsequentwork showedclearly that other regional neural subtypescan be induced in response to appropriate patterning cuesin mouse," primate'" and in human ESCs.64 Default Pathway The neuraldefaulthypothesisisbasedon work in the frogembryowhereit wasshownthat the neural"inducing"activityof the organizer, asdefinedbythe Spemannand Mangoldnearly80 years ago,65 is mediated by BMP antagonists(seeintroduction). Thereforeit wasproposed that neural induction occursby default in the absence of any signals actively preventingit such as BMPs. Effortsat translatingthishypothesis into ESC differentiation protocolsyieldeda setofEB-free, feeder-free and serum-free culture conditions,therebydeprivingESC from most cell-cell interactions and extrinsicsignals. Spontaneousdifferentiation ofESCs alongthe neurallineagehas been reported in someof theveryearliest human ESCbasedneuralinduction protocols'"though induction occurredat rather high celldensities following overgrowthof undifferentiatedhuman ESCs under serum-free culture conditions.f Variations of the default model include the generation of

ProtocolsfOrGeneratingES Cell-DerivedDopamineNeurons

105

ESC derivedprimitiveneuralstemcells,67,68 definedasan Oct4+/Nestin+ intermediategivingrise to definitive neuralstemcells in the presence ofFGF2. Anotherwiddy usedapproachisthe generation ofNeuralStem(NS)linesbyculturingundifferentiatedESCsin Neurobasal/B27mediumin the presenceofFGF2/EGF.69 While the protocol wasdevelopedfor mouseESCsit wasproposed that the protocol can be readily adapted to human ESCS.69 Finallythe useof Noggin can improve neural induction under serum-free adherent cell culture conditions particularly for the neural differentiationof human ESCS.29 The useof Noggin is not limited to default differentiationprotocolsand alsoenhancesneuralinduction in EB70 and stromal-feeder basedprotocols," While the developmentof definedneuralinduction protocolsiscriticalfor translatingESC research towards future clinicaluse,the question remainswhether default protocols perform at levels comparable to EBor stromal feederbasedprotocols in the caseof midbrain DA neurons differentiation. It is obviousthat SHH/FGF8 baseddefault protocols can yieldTH+ neuron progeny,69 but subtype characterizationstudiessuggest that fewof those TH+ cells co-express authentic midbrain mark.ers.However, midbrain specific DA neuron induction wasreported usingadefaultprotocol upon forced expression of the intrinsic midbrain DA neuron determinant Lmxla."

Key Considerations Comparing DA Neuron Induction from Mouse vs Human ESCs Allthree neuralinduction strategies havebeensuccessfully adaptedfor the generationof neural progenyand DA neuron induction from human ESCs.As discussed above, there are remarkable differences in the signal controllingthe maintenance of undifferentiated mouseversus humanESCs. However, the signals responsible for neuralinduction, midbrain specification and DA differentiation appearsimilarin mouseand human ESC protocols.One potential difference betweenmouse and human ESCs relates to the dependence of active BMP inhibition during neural induction. While under most conditions the addition of noggin is dispensable for the neural differentiation of mouse ESCs,human ESC differentiationtowards neural fatesis greatlyenhanced by noggin. Furthermore,there are obviousdifferences in the timelineof in vitro differentiationbetween human and mouseESCsthat likelyreflectintrinsicdifferences in developmental speedbetweenthe two species during ontogeny. While most mouseESC basedprotocolsyieldDA neuron progeny within about 2 weeksof differentiation," human ESC basedprotocols often require 1-2months of in vitro differentiationperiods." Another finalmajor difference is the emergence of a distinct early neuroepithelial intermediate, termed neural rosettes. Rosettes are observed during neural differentiationof human ESCsbut moredifficultto observeand ofientransientduringneuraldifferentiationof mouseESCs.Neuralrosetteshavebeencharacterized recentlyasanovelearlyneural stem cell type (R-NSCs).64 R-NSCs express distinct molecular markers from classic FGF/EGF expandedneuralstemcelllinesand retain the abilityto respondto patterning cuesthat specific AP and DV identityincludingthe induction of midbraindopamineneurons/" A strongforebrainbias hasalsobeen observedwhen usingserum-free EB(SFEB) basedprotocolsthat selectfor forebrain progenyin both mouseand humanESCprogeny.72,73 Persistence offorebrain rosette-like structures following SHH/FGF8 exposure are one of the keychallenges for translationalmedicineas such structures can induce severe neural overgrowthupon grafting.58.64.74.75 Genetic Strategies to Promote DA Neuron Differentiation from ESCs Thereisclearevidence that extrinsic factor basedprotocolscanyieldlargenumber of authentic midbrain DA neurons particularly from mouse ESCS.46 However, genetic strategies are being developedto further enhance midbrain DA neuron yieldand specificity in ESC cultures.Some of the first successful genetic strategies were based on the overexpression of Nurr1, which was shown to enhance yield,biochemicaldifferentiationand in vivofunction in mouse ESC derived midbrain DA neurons.v-" Overexpression of variousmidbrain specific transcription factors in human ESC culturessuggested that combined actionofextrinsic Pitx3 and Nurr1 enhancesyield of human ESC derivedmidbrain DA neurons." However, characterization of midbrain identity and evidence for in vivofunction wasverylimited in this study.More recentlyoverexpression of Lrnxla has been shown to dramatically enhance midbrain dopamine neuron yieldat leastwhen using a neural default induction protocol that includes exposure to SHH and FGF8. Under

106

Development ana EngineeringofDopamineNeurons

these conditions most ESC progeny adopted dopaminergicfeatures and expressed appropriate midbrain specific markers," Current studies are aimed at confirmingthese data in human·ESCs and at demonstratingthat Lmxl mediated improvementin midbrain DA neuron yieldtranslates into improvedin vivoperformancein animalmodelsof PD.

Remaining Key Challenges

The main current challenge for the field is the demonstration that human ESC derived DA neurons can be generated under conditions that allow safeand efficacious use for cell therapy. Unfortunately, most in vivostudiesusinghuman ESC derivedmidbrain DA neuronsto date show poor invivosurvival andfunctionor completeloss ofphenotypeupon transplantation (e.g.,57.71.78.79). These findings were surprising given the robust in vivo function of mouse ESC derived DA neurons.45.46 Furthermore there isconsiderable concernabout the possibilityoftissueovergrowth either via teratoma formation in protocols that retain smallnumbers ofundifferentiatedESCs80 or the advent of neural overgrowthdue to remaininguncommitted neural precursors.58,74,75 It is likelythat concernsregardingtumor formation will requiresolutionsthat includecellselectionat appropriatedifferentiationstagebasedon geneticor surfacemarkers. Evidence of robust survival ofhuman ESC derivednonDA neural cells" and the successful useof human fetal midbrain DA neuronsin comparable transplantationparadigms suggest that poor survival ofhumanESCderived DA neuronscannot be readily explainedbyan immunological response againstxenografted cells. It rather mayreflecta specificvulnerabilityof human ESC derivedDA neuronswithinthe adult host striatum or the incompletespecification ofDA neuron phenotype resultingin lossofDA neuron phenotype in vivo. Recent studiesin micehighlighted that several transcription factorsessential for DA neuron developmentsuch as FoxAI/2, Nurr I, Enl/2 and Pitx3 alsoplaycriticalrolesin postmitotic midbrain DA neuron survivaland maintenanceof phenotype.3435.82.83 Therefore an important hypothesis concerningpoor in vivosurvival isthe possibilitythat insufficient expression levels for someof these transcriptionfactorsin postmitotic human ESC derivedDA neurons are responsible for compromisedsurvival, phenotype stabilityor function." A final important challenge for the future is the selective generation of specific DA neuron subtypeswithin the ventral midbrain. Currently available differentiationprotocols are unableto enrichfor nigra!or VTA type DA neuronsin acontrolledfashion. Forcelltherapeuticapplications in PD it would be desirable to graft nigral DA neurons only.VTA neurons compriseup to 50% of all DA neurons in most PD grafiingparadigms.s' VTA-typeDA neurons are generally located in the center of the graftand do not efficiently reinnervatethe host striatum. Furthermore,VTA neurons exhibit clear differences with regard to vulnerability and growth factor requirements. Thereforethe presenceofVTA type DA neurons will interferenot only for celltherapybut also for effortsaimed at disease modelingand drug development.

New Developments

There are number of novel developments in the ESC fieldthat could become usefulfor the derivation and purificationof unlimited numbers of midbrain DA neurons from human ESCs. We briefly discuss three majoradvances includingthe identification ofdefinedintermediatestages during ESC differentiation, the availability of novelreporter lines to purify DA neuron progeny and the availability of nucleartransferESCsand inducedpluripotent stemcells to address concerns relatedto tissuematchingand to providenovelopportunities in disease modeling.

Human ESC Neural Intermediates

Recently, a novel early neural stem cell stage has been identified, called the rosette neural stem cells (R-NSCs).64 Unlike classic FGF/EGF expanded neural stem cell types, rosette-stage cellsrespond readilyto appropriatepatterning cues.Bydefault,R-NSCs adopt a forebrainfate as evidencedby the expression of the forebrainmarkerBF-I, but can be respecified into manyother lineages including a dopaminergic fate. Therefore, R-NSCs may represent the first NSC type capableof generatingthe full neuronal diversity. One of the potential advantages of R-NSCs it

Protocolsfor GeneratingES Cell-Derived DopamineNeurons

107

the possibilityof amplifyingcells at the R-NSC stage. This should allow increasingoverallcell yieldwhilereducingthe riskof retaininganycontaminatingundifferentiatedhuman ESCs.Sucha strategymayalsobeusefulforsynchronizingthe neuralprecursorcellpopulationprior to midbrain DA neuron specification. Finally, understandingthe biologyofR-NSCs should reducethe riskfor neural overgrowthat the time of transplantation.Two recent studiesproposed that proliferation of human ESC derivedneural intermediatescan be usedfor the highlyefficient derivationofDA neuron progeny. While two independent studies used attached monolayer culture expansionof neuralprecursors84•85another studyproposed proliferationof human ESC derivedneuralprecursors as "spherical neural masses"86 WhUe there is clear evidence that these neural populations generateTH+ cells, it is lessclearwhether the TH+ cells in these studiesdo indeed correspond to authentic midbrain DA neurons.

Cell Purification and Genetic Reporter Lines

ES reporter cell lines allow the purification and enrichment of distinct neural cell types by fluorescent activatedcellsorting (FACS)at defineddevelopmentalstages, which isimportant for the identificationof the optimal celltype and stagein cellreplacement paradigms. Such reporter linesarealso apowerfultool forlineage characterization and screeningforextrinsic factorsinvolved in fate specification. Recently, the Studer lab has succeededin generatingseveral BAC-transgenic GFP reporter mouseESC linesto labelcells committed to a neuraland dopaminergic fate,includingHes5::GFP neuralstemcellreporterline,DIll ::GFPlinemarkingneuroblasts, Nurr-l ::GFPcells markingearly postmitotic neurons and Pia3::YFP cells for the identificationof more differentiatedmidbrain specific DA neurons" (Y. Ganat, S. Kriks unpublisheddata). Wealsoadapted the sameapproach for human ESC and generated several human ESC GFP reporter lines that mark neural and midbrain DA neurons at specific stages. The useof genetargetingin mouseESCsyieldeda Pia3 knockin ESC line" that hasbeenused to isolateand graftmidbrain DA neuronsinto PD rats.89WhUe transplantationofFACS purified Pia3::eGFP+ cellsresulted in the restoration of behavioral deficits in some animals, the yield of survivingDA neurons waspoor and many animalsdid not show any survivinggrafts.89These data suggest that Pia3 marksa stagesuboptimalfor transplantation sincePia3 isexpressed only in mature dopamine neurons several daysaftercellcycle exit.The useof geneticreporters should allow us to identify the stage most suitable for grafting. Subsequentgenetic characterization of cells at such an optimized stagemayultimatelyyieldsurface markers for usein human ESC lines without geneticmodification.

The Use ofGenetically Matched DA Neurons for Cell Therapy and Disease Modeling

Recent work from our group has highlighted the fact that genetically matched DA neurons show significantly improved survival and function upon transplantation into animal modelsof PD. The successful treatment of individualPD miceusingtherapeuticcloningwasa majorbreakthrough for the field. 61However, nucleartransfertechnologyhasnot yet been usedsuccessful: for generatinghuman nucleartransferESCs.Therecentsuccess in monkeys suggests that thereshould be no major biologicalhurdles in humans, but practicalconsiderations and the lack of sufficient oocyte donor cells hamper the prospect of this approach for celltherapy. The breakthrough studiesby Yamanka and colleagues'??' and several other groupS92.94 on the directed reprogrammingof somaticcells backto an ES-like stage(induced pluripotent stemcells, iPSCs) providean excitingalternativeto the useof somaticcellnuclear transfer. Most recentlyit hasbeen shownthat mouseiPSC derivedDA neuron can be usedto improvebehavioral function in a rat PD model." For translational applications it will be essential to develop strategies that yieldiPSC cells that do not retain anystablegeneticmodifications following the reprogramming process. Furthermore, it needs to be shown that the approach works in an autologous setting treating individual PD mice using their matched iPSC lines. However, it is obvious that iPSe

108

Developmentand EngineeringofDopamineNeurons

technology will open up novelexcitingdirectionsin the useof in vitro generatedDA neuronsfor celltherapyand disease modeling.

References

1. Piccini P, Brooks DJ, Bjorklund A et al. Dopamine release from nigral transplants visualized in vivo in a Parkinson's patient. Nature Neuroscience 1999; 2:1137-1140. 2. Lindvall 0, Brundin P, Widner H et ale Grafts of fetal dopamine neurons survive and improve motor function in Parkinson's disease. Science 1990; 247:574-577. 3. Freed CR, Greene PE, Breeze RE et al. Transplantation of embryonic dopamine neurons for severe Parkinson's disease. N Eng! J Med 2001; 344:710-719. 4. Olanow CW: Goetz CG, Kordower JH er al. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson's disease. Ann Neuro12003; 54:403-414. 5. Bjorklund A, Dunnett SB, Brundin P et ale Neural transplantation for the treatment of Parkinson's disease. Lancet Neurol 2003; 2:437-445. 6. Smith WC, Harland RM. Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 1992; 70:829-840. 7. Sasai ~ Lu B, Steinbeisser H et al. Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 1995; 377:757. 8. Hemmati Brivanlou A, Kelly OG, Melton DA. Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 1994; 77:283-295. 9. Cox WG, Hemmati-Brivanlou A. Caudalization of neural fate by tissue recombination and bFGF. Development 1995; 121:4349-4358. 10. Doniach T. Basic FGF as an inducer of anteroposterior neural pattern. Cell 1995; 83:1067-1070. 11. McGrew LL, Lai CJ, Moon RT. Specification of the anteroposterior neural axis through synergistic interaction of the Wnt signaling cascade with noggin and follistarin, Dev Bioi 1995; 172:337-342. 12. Bang AG, Papalopulu N, Kintner C et al. Expression of Pax-3 is initiated in the early neural plate by posteriorizing signals produced by the organizer and by posterior non-axial mesoderm. Development 1997; 124:2075-2085. 13. Blumberg B, Bolado J Jr, Moreno TA et a!' An essential role for retinoid signaling in anteroposterior neural patterning. Development 1997; 124:373-379. 14. Crossley PH, Martinez 5, Martin GR. Midbrain development induced by FGF8 in the chick embryo. Nature 1996; 380:66-68. 15. Hynes M, Porter JA, Chiang C et al. Induction of midbrain dopaminergic neurons by sonic hedgehog. Neuron 1995; 15:35-44. 16. Ye WL, Shimamura K, Rubenstein JR et ale FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998; 93:755-766. 17. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292:154-156. 18. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Nat! Acad Sci USA 1981; 78:7634-7638. 19. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 1987; 51:503-512. 20. Doetschman T, Gregg RG, Maeda N et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 1987; 330:576-578. 21. Ying QL, Nichols J, Chambers I et al. BMP induction of id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003; 115:281-292. 22. Ying QL, Wray J, Nichols} et al. The ground state of embryonic stem cell self-renewal. Nature 2008; 453:519-523. 23. Thomson JA, Itskovitz-Eldor }, Shapiro 55 et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282:1145-1147. 24. Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000; 18:399-404. 25. Xu CH, Inokuma MS, Denham J et a!' Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotechnology 2001; 19:971-974. 26. Nichols}, Chambers I, Taga T et ale Physiological rationale for responsivenessof mouse embryonic stem cells to gp130 cytokines, Development 2001; 128:2333-2339. 27. Amit M, Shariki C, Margulets V et al. Feeder layer and serum-free culture of human embryonic stem cells. BioI Reprod 2004; 70:837-845. 28. Vallier L, Reynolds D, Pedersen RA. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Bioi 2004; 275:403-421.

Protocolsfor GeneratingES Cell-Derived DopamineNeurons

109

29. Pera MF, Andrade J, Houssami S er aL Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci 2004; 117:1269-1280. 30. Xu RH, Chen X, Li DS er ale BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol2oo2; 20:1261-1264. 31. Tesar PJ, Chenoweth JG, Brook FA et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 2007; 448:196-199. 32. Brons IG, Smithers LE, Trotter MW et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 2007; 448:191-195. 33. Lensch M~ Schlaeger TM, Zon LI et ale Teratoma formation assayswith human embryonic stem cells: a rationale for one type of human-animal chimera. Cell Stem Cell 2007; 1:253-258. 34. Ferri AL, Lin ~ Mavromatakis YE et al. Foxal and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner. Development 2007; 134:2761-2769. 35. Kittappa R, Chang ~ Awatramani RB et al. The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age. PLoS Bioi 2007; 5:e325. 36. Zetterstrom RH, Solomin L, Jansson L et al. Dopamine neuron agenesis in Nurrl-deficient mice. Science 1997; 276:248-250. 37. Smidt MP, Van Schaick HA, Lanctot C et al. A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Nat! Acad Sci USA 1997; 94:13305-13310. 38. Andersson E, Tryggvason U, Deng Q et ale Identification of intrinsic determinants of midbrain dopamine neurons. Cell 2006; 124:393-405. 39. Vernay B, Koch M, Vaccarino F et al. Otx2 regulates subtype specification and neurogenesis in the midbrain. J Neurosci 2005; 25:4856-4867. 40. Smidt MP, Asbreuk CH, Cox JJ et al. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmxlb, Nat Neurosci 2000; 3:337-341. 41. Ohyama K, Ellis P, Kimura S et al. Directed differentiation of neural cells to hypothalamic dopaminergic neurons. Development 2005; 132:5185-5197. 42. Grace AA, Onn SP. Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. J Neurosci 1989; 9:3463-3481. 43. Nirenberg MJ, Vaughan RA, Uhl GR et al. The dopamine transporter is localized to dendritic and axonal plasma membranes of nigrostriatal dopaminergic neurons. J Neurosci 1996; 16:436-447. 44. Sesack SR, Aoki C, Pickel VM. Ultrastructural localization of D2 receptor-like immunoreactivity in midbrain dopamine neurons and their striatal targets. J Neurosci 1994; 14:88-106. 45. Kim JH, Auerbach JM, Rodriguez-Gomez JA et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 2002; 418:50-56. 46. Barberi T, Klivenyi P, Calingasan NY et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 2003; 21:1200-1207. 47. Perrier AL, Tabar V, Barberi T er al. From the cover: Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Nat! Acad Sci USA 2004; 101:12543-8. 48. Yan Y: Yang D, Zarnowska ED et al. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 2005; 23:781-790. 49. Lee SH, Lumelsky N, Studer L et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000; 18:675-679. SO. Perrier AL, Tabar V, Barberi T et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Nat! Acad Sci USA 2004; 101:12543-12548. 51. Rodriguez-Gomez JA, Lu JQ, Velasco I et al. Persistent dopamine functions of neurons derived from embryonic stem cells in a rodent model of Parkinson disease. Stem Cells 2007; 25:918-928. 52. Doetschman TC, Eisretter H, Katz M er al. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985; 87:27-45. 53. Bain G, Kitchens D, Yao M et al. Embryonic stem cells express neuronal properties in vitro. Dev BioI 1995; 168:342-357. 54. Okabe S, Forsberg-Nilsson K, Spiro AC et al. Development of neuronal precursor cells and functional postmitoric neurons from embryonic stem cells in vitro. Mech Dev 1996; 59:89-102. 55. Zhang SC, Wernig M, Duncan ID er al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol2001; 19:1129-1133. 56. Carpenter MK, Inokuma MS, Denham J et al. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neuro12001; 172:383-397. 57. Yang D, Zhang ZJ, Oldenburg M et al. Human embryonic stem cell-derived dopaminergic neurons reverse functional deficit in parkinsonian rats. Stem Cells 2008; 26:55-63.

110

Development and Engineering ofDopamineNeurons

58. Roy NS, Cleren C, Singh SK et al. Functional engrafiment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes, Nature Med 2006; 12:1259-1268. 59. Kawasaki H, Mizuseki, Nishikawa S et at. Induction of midbrain dopaminergic neurons from es cells by stromal cell-derived inducing activity. Neuron 2000; 28:31-40. 60. Morizane A, Takahashi J, Shinoyama M et al. Generation of graftable dopaminergic neuron progenitors from mouse ES cells by a combination of coculture and neurosphere methods. J Neurosci Res 2006; 83:1015-1027. 61. Tabar V, Tomishima M, Panagiotakos G et ale Therapeutic cloning in individual Parkinsonian mice. Nature Med 2008; 14:379-381. 62. Kawasaki H, Suernori H, Mizuseki K et at. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Nat! Acad Sci USA 2002; 99:1580-1585. 63. Mizuseki K, Sakamoto T, Watanabe K et al. Generation of neural crest-derived peripheral neurons and Aoor plate cells from mouse and primate embryonic stem cells. Proc Nat! Acad Sci USA 2003; 100:5828-5833. 64. Elkabetz Y, Panagiotakos G, AlShamy G et aleHuman ES cell-derived neural rosettes reveal a functionally dinstinct early neural stem cell stage. Genes Dev 2008; 22:152-165. 65. Speamann H, Mangold H. Induktion von embryonanlagen durch implantation artfremder organisaroren, Wilhelm Roux Arch Entw Mech Organ 1924; 100:599-638. 66. Reubinoff BE, Itsykson P, Turetsky T et ale Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001; 19:1134-1140. 67. Tropepe V, Hitoshi S, Sirard C et ale Direct neural fate specification from embryonic stem cells: A primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 2001; 30:65-78. 68. Smukler SR, Runciman SB, Xu S et al. Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences,J Cell BioI 2006; 172:79-90. 69. Conti L, Pollard SM, Gorba T et al. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS BioI 2005; 3:e283. 70. Itsykson P, Ilouz N, Turetsky T et al. Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Mol Cell Neurosci 2005; 30:24-36. 71. Sonntag KC, Pruszak ], Yoshizaki T et al. Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the BMP antagonist noggin. Stem Cells 2007; 25:411-418. 72. Watanabe K, Kamiya 0, Nishiyama A et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci 2005; 8:288-296. 73. Watanabe K, Ueno M, Kamiya D et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol2007; 25:681-686. 74. Ferrari D, Sanchez-Pernaute R, Lee H et al. Transplanted dopamine neurons derived from primate ES cells preferentially innervate DARPP-32 striatal progenitors within the graft. Eur J Neurosci 2006; 24: 1885-1896. 75. Sanchez-Pernaure R, Studer L, Ferrari D et al. Long-term survival of dopamine neurons derived from parthenogenetic primate embryonic stem cells (Cyno1) in rat and primate striatum. Stem Cells 2005; 23:914-922. 76. Chung S, Sonntag KC, Andersson T et al. Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons. Eur J Neurosci 2002; 16:1829-1838. 77. Martinat C, Bacci JJ, Leete T et al. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype. Proc Nat! Acad Sci USA 2006; 103:2874-2879. 78. Park CH, Minn YK, Lee JY et ale In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons. J Neurochem 2005; 92:1265-1276. 79. Ben-Hur T, Idelson M, Khaner H et al. Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells 2004; 22:1246-1255. 80. Brederlau A, Correia AS, Anisimov SV et al. Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells 2006; 24:1433-1440. 81. Tabar V, Panagiotakos G, Greenberg ED et al. Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat Biotechnol2005; 23:601-606. 82. Sgado P, Alberi L, Gherbassi D et al. Slow progressive degeneration of nigral dopaminergic neurons in postnatal engrailed mutant mice. Proc Natl Acad Sci USA 2006; 103:15242-15247.

Protocolsfor GeneratingES Cell-Derived Dopamine Neurons

111

83. Saucedo-Cardenas 0, Q!!.intana-Hau JD, Le WD et a1. Nurrl is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Nat! Acad Sci USA 1998; 95:4013-4018. 84. Ko ~ Park CH, Koh HC et a!' Human embryonic stem cell-derived neural precursors as a continuous, stable and on-demand source for human dopamine neurons. J Neurochem 2007; 103:1417-1429. 85. Hong 5, Kang UJ, Isacson 0 et a1. Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons. J Neurochem 2008; 104:316-324. 86. Cho MS, Lee YE, Kim JY et a1. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc Nat! Acad Sci USA 2008; 105:3392-3397. 87. Tomishima MJ, Hadjantonakis AK, Gong 5 er a1. Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library. Stem Cells 2007; 25:39-45. 88. Zhao SL, Maxwell 5, Jimenez-Beristain A et a1. Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. Eur J Neurosci 2004; 19:1133-1140. 89. Hedlund EM, Pruszak J, Lardaro T er a1. Embryonic stem (ES) cell-derived Pitx3-eGFP midbrain dopamine neurons survive enrichment by FACS and function in an animal model of parkinson's disease. Stem Cells 2008; 26:1526-1536. 90. Takahashi K, YamanakaS. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-676. 91. Takahashi K, Tanabe K, Ohnuki M er a1. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861-872. 92. Wernig M, Meissner A, Foreman R et a1. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007; 448:318-324. 93. Maherali N, Sridharan R, Xie W et a1. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 2007; 1:55-70. 94. Yu J, Vodyanik MA, Smuga-Otto K et a1. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917-1920. 95. Wemig M, Zhao JP, Pruszak J et a1. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci USA 2008; 105:5856-5861.

CHAPTERll

Molecular and Cellular Determinants for Generating ES-Cell Derived Dopamine Neurons for Ceillherapy Jan Pruszak and Ole Isacson*

Abstract

E

mbryonic stem (ES)cellscangeneratemidbrain dopaminergic(DA) neuronal phenotypes in vitroandhavebeensuccessfully appliedto restorefunction in animal modelsof Parkinson's disease (PD). How canwe best integrateour growinginsight into the regulatorycascade of transcription factorsguidingmidbrain specification to further improvethe in vitro differentiation ofmidbrain DA neurons for celltherapy of PD ?To characterizethe differentiationofauthentic DA neurons in vitro,expression patterns of the numerousmidbrain-characteristic markersneed to be investigated. When usingforced geneexpression, such factors haveto be closely monitored to avoidgenerationof nonphysiological celltypes.Fluorescent markerssuchasPitx3-GFP,TH-GFP, Soxl-GFP or surfaceantigens haveproven useful for elimination of unwanted cell types by cell sorting, thereby avertingtumors and increasingthe DA fraction for transplantation studies.The importanceofappropriatetimingduringapplicationofextrinsic factorsand the influence of cell-cell interactions in the dish has to be taken into account. This conceptual synopsis outlines current objectives, progress, but alsochallenges, in derivingmidbrain DA neurons from pluripotent stem cellsfor clinicaland scientific applications.

Introduction

Previouschapters in this volume describe processes that are involvedin the specification of midbrain dopaminergicneurons during normal embryological development in vivo. Protocols of embryonic stem celldifferentiation, as describedin the preceding section, have been developedand provided proof-of-principlethat dopaminergic (DA) neurons can indeed be generated from mouse,human and other embryonic stem cells.'? Closer investigation, however, revealed that a high yielddifferentiationofES cells into authenticmidbrain-like dopaminergicneurons ableto restorefunction in animalmodelsstillposesspecific challenges'v (Fig.1).Fundamental questionsremainin how to organize,integrateand applythe growingknowledgeofembryological midbrain developmentin ways that enablegeneration of dopaminergicneurons from embryonic stem cellsfor scientific and celltherapeutic applications.

Background and History

Clinical trials grafting midbrain tissue obtained from aborted fetuses have demonstrated, in principle the feasibility of restoring function in Parkinson's disease patients.r" With some *Corresponding Author: Ole lsacson-i-Neuroregeneration Laboratories, Center for Neuroregeneration Research, Harvard Medical School, McLean Hospital, Belmont, MA 02478, USA. Email: [email protected]

Developmentand Engineering ojDopamine Neurons, edited by RJ. Pasterkamp, M.P.Smidt and J.P.H. Burbach. ©2009 LandesBioscience and SpringerScience+Business Media.

Molecular and Cellular Determinantsfor GeneratingES-Cell

113

DA differentiation

A

proliferative potential & pluripotency comm itment towar

time

B

in vivo

~

positional information

c

L-::tE::::==:J--

-

in vitro

-

-

-

-

-

-.../

Figure 1. A) In the developing embryo and in the differentiation from embryonic stem cells, neural cell fate determination is represented by a restriction of proliferative capacity and differentiation potential, which concurs with increasing approx imation to the specific phenotype of interest. Compared to immature ES cells, advanced-stage committed neural precursors or adult neural stem cells show a more limited capacity to self-renew and to differentiate towards various lineages of therapeutic interest. Reprogramming approachess'<' may help to modulate this restriction, but are so far poorly understood. B) Normal in vivo development represents an evolutionarily fine -tuned three-dimensional inductive system.Very specific spatial arrangements, gradients and cell-cell communication ensure that cells developing in the midbrain dopaminergic lineage are correctly specif ied. The timing of these complex processes appears to be precisely controlled and synchronized . C) Surprisingly enough, it is possible to guide ES cells along the path of midbrain DA differentiation in vitro. 2,14.16.52.66 However, it is clear that such attempts have so far been rather poorly defined and are not yet a perfect imitation of normal development. Consequently, only a subset of ES cells in the dish may be induced and patterned equivalently and the yield may vary. Given the challeng ing complexity, the numerous steps and the final outcome are not entirely controlled.

methodologies, the transplantationoffetalmidbrain tissue has beendemonstratedto be aclinically beneficial and safeoption with stableresults? Recentdata demonstratesthe survival and integration of fetalcells for more than a decadeafter transplantationin Parkinsonpatients (Isacson et al, unpublished data). Some patients exhibited improvement of symptoms to the extent that they could be takenoffdrugs.Thisencourages ongoingtranslationalresearch effons in this paradigm," As human ventral midbrain tissuefrom aborted fetuses has been difficultto obtain and ethically controversial, earlieralternative efforts used xenotransplantation. Graftingpig ventral midbrain cells in PD patients, although with variable and high tissue rejection rates, demonstrated that engraftmentof xenogeneic dopamine neurons in the human host brain is possible."!' With the derivation ofhuman embryonicstemcelllines,12 suchsources havebeenexploredfor applications in celland tissue replacement. Transplantationstudiesprovidedevidencethat dopaminergicneurons generated from immature mouseembryonic stem cellscould restore function in rodent models of Parkinson's disease:13•14 After grafting relatively undifferentiated mouse ES cells, a fraction

114

Development and Engineering ojDopamineNeurons

of those differentiatedinto DA neurons of the appropriate midbrain phenotypes and animals bearingsufficient numbersofDA neurons showedimprovementin functional motor behavioral tests." Interestingly, a natural propensity (so called default neural pathway) was observed of graftedES-derived (blastulastage)cells to differentiatetoward mid- and hindbrain phenotype.IS In addition, asexpectedupon graftingpartiallyundifferentiatedEScells, evenin low doses, some of the animals developedtumors containing progeny of all germ layers (teratoma). Such work exemplified the vastpotential, but alsothe challenges of usinga truly pluripotent cellsource(Fig. 1). In recent years, further refinement and protocol development has occurred in experiments transplantingprimate,includinghuman,EScells into Parkinsonrodent2.16 and monkeyl7.18 models in an attempt to restore function using experimentalpreclinicalcell therapeutic strategies (see Fig.2A,B). For the future, human embryonic stem cellscurrentlyrepresent the most promising cell source to obtain sufficient amounts of medicalgrade,standardized,normallydifferentiated cellsfor celltherapeuticapproaches in variousfields of regenerative medicine." However, cellular heterogeneityleadingto formation of tumors and generation of unwanted cell types is a major problem that must be overcome'r" (Fig.2C). The goal,therefore,is to generatethe equivalentof a physiologically functional dopaminergic neuron from human pluripotent stem cellsin high purity. The advantages of this approach include a potentiallyunlimited sourceof cells, a biomedically controlled production of specific cell types and the ability to bypass the ethical and technical hurdles of obtaining midbrain material from aborted human fetuses at a specific gestationalage.The specific challenges to be met are the stabledevelopmentof pluripotent stem cells into dopaminergicneurons of the specific midbrain phenotype (A9) of interest and their application to diseasedbrain without introduction of any unwanted effects.

Principles ofEngineering Dopamine Neurons in Vitro

While differentiationstudies of embryonic stem cellscan serveas a valuable tool to further increaseour understanding of midbrain development, we do not necessarily need to fully understand everydetail in the sequenceand developmental cascade that result in the development of midbrain DA neurons in order to utilize ES cellsfor scientific or therapeutic discovery and applications. However, the specificity and safetyof the midbrain-likeDA cells generatedmust be ascertainedprior to use in clinicalcellreplacement applications. Theaccess to human EScellsnow enablesthe study and targeted manipulation of midbrain dopaminergicdevelopmentin the human species. Forexample, differences in human and rodent TH promoter sequences are known," exemplifying differences in human versus rodent midbrain DA developmentand regulation.ES cellsrepresenta system that isparticularlyamenable for potential comparative analyses in mouse versus human DA differentiation. Furthermore,genetically engineeredor patient-derived celllines with specific genetic defects22.23 will be very helpful for studies of genesinvolved in phenotype maintenance or specific vulnerability.24.25 Therefore, ES cell-derived dopamine neurons can be considered a useful tool for cell therapeutic, basic developmental and pharmacological studies alike.Given the potentially unlimited supplyof cells, such research work can be conducted in a high-throughput manner. On the other hand, one has to be aware that artificial in vitro conditions can representa confoundingfactorin testingmolecules withpotentialinductive effects on dopaminergic development. Platingdensity, handling of the cells and cell-cell interactionsin the micro-environmentof a dish mayoverruleany appliedpatterning factors (Fig.3 A,B).Consequently, function and phenotype stability of such cellsmay be affectedand only a minor fraction of the differentiatedcells may represent an authentic equivalentto the midbrain DA neurons generated in fetal development. Similarproblemshavebeenobservedin the differentiation ofhematopoieticcelltypesfrombESe, which have so far failed to functionally reconstitute the hematopoietic system in vivo,despite mimicking marker expression in vitro.26 Such findings exemplify that continued refinement of protocols is required to further approximate the differentiationof ES cells to a true imitation of normal development,creatingan authentic equivalentof fetalcells. So far,it is unknown whether

Molecular and Cellular Determinantsfor GeneratingES-Cell

A

neural induction

B

Heslin

c

115

DA differentiation

TH

neuronal maturation Figure 2. A) Neural induction of human ES cells can be achieved by coculture with stromal feeder cell lines':' in combination with the BMP-antagonist Noggln .' Typical neuroepithelial " rosette" structures can be observed, co-expressing the neural precursor marker nestin (left panel; 21 div). Those precursors are mechanically or enzymatically selected for further patterning and differentiation. A video article demonstrating the neural induction procedure in detail is available at www.jove.com (Karki et al 2006 67) . B) At later stages of in vitro development, a network of neuronal processes and immunoreactivity for neuronal (Tu)l) and dopaminergic (TH) markers can be observed (right panel; 42 div). Phasecontrast images and immunocytochemical stainings of the hESC cell line H9 (WA-09; W iCeIl™) are shown. C) Cellular heterogeneity of differentiating ES cell culture : An inherent feature of immature ES cells (to; left panel) is pluripotency, the potential to develop into cell types of all germ layers. Upon differentiation (t mid panel), this potential may lead to heterogeneity with regard to " developmental stage(x-axis)and to cell lineage (z-axis). Subsets of cells in the dish escape the extrinsic patterning factors and differentiation signals, which results in remaining immature proliferative cell types (mid panel, white arrows) and the development of cells of nonneural lineages (second panel, arrow head). Optimized derivat ion of midbra in DA neurons from plur ipotent cells w ill exclude such unwanted cell types and further increase the fract ion of the specific phenotype of interest (right panel) (modified from Pruszak et al 2007 5 ) .

the transplantationof postmlrotic dopamine neuronsor that of patterned proliferative dopaminergicprecursorcellswill be of greateradvantage in this paradigm. In fetal transplantation studies usingmidbrain DA cells, postmitotic cells haveshown to be the effective agenr." Novellabeling strategies using for example Neurogenin-z-Gf'P" and Pitx-3-GFP,29,30 or surface antigens, such as Corin" and other novelneural lineagemarkers,'willhelp to further refineand selectthe most appropriate DA cellfor grafiing, from both primary tissueand EScellcultures.

Monitoring DA Differentiation in Vitro

What characterizes the DA phenotype that we intend to differentiate in vitro?Analogousto normal development, precursorsto DA neurons generatedfrom ES cells in vitro should express ventraland midbrain regionspecific markers. Tight control of markerexpression overtime could

Development andEngineering ofDopamineNeurons

116

hdp in ensuring that the cascade of eventsmimics the normal in vivosituationduringdevelopment. Midbrain-region markers that need to be carefully monitored at multipletimeduring ES cell differentiation include the various stage-characteristic transcriptionfactors such as EnIl2, Pitx3,Lmxlb, Lmxla and Nurr1.Dopaminergic neuronsexpress tyrosinehydroxylase (TH), the rate-limiting enzyme of dopamine neurotransmittersynthesis converting tyrosineto L-DOPA. As other catecholaminergic neuronsalsoexpress this enzyme, positivityof the aromaticaciddecarboxylase (AADC) or absence of dopaminebeta-hydroxylase (DBH) needsto becharacterized aswell. Additionalfunctionally relevant markers of differentiated DA neuronsarethe dopamine

A

B

0, ' - - - - --:==1;

Figure 3. A) In vitro differentiation of ES cells utilizes media supplements and inductive substrates to direct ES cells toward the phenotype of interest. In addition, secreted factors and cell-cell interactions w ithin the dish playa major role. B) Overall, the goal has been to mimic the highly regulated steps of normal, in vivo development to achieve a similarly orchestrated sequence of gene expression during these artificial in vitro conditions. C) Given the complexity of these processes, such biomedical approaches require a synthesized "bird's eye" view of the known transcriptional interactions. Identifying key factors and particularly relevant developmental cascades, for example by using gene regulatory network diagrams, is helpful not only for improving protocols generating DA neurons for experimental cell therapy but also for approaching an integrated understanding of midbrain DA development. In addition, ES cell differentiation experiments represent a useful system to test, modulate and validate such developmental theories . Panel (C) illustrating a sequence of events leading to the development of DA phenotypes was generated using BioTapestry Editor 2.1.0 software [www. biotapestry.orgl from data presented in recent review and or iginal articles 4,3s,s o,68-76 (modified from Pruszak & Isacson, 2008) .

Molecular and CellularDeterminantsfOrGeneratingES-Cell

117

transporter (DAT), recycling DA from the synapticcleftback into the presynapticterminal, or the vesicular monoamine transporter (VMAT2),packagingDA in the cytoplasminto vesicles for storage and release. Markers that are particularlypresent in the substantia nigra pars compacta (A9 region, as opposed to the DA neurons of the ventral tegmental area,AIO), include the inwardlyrectifyingpotassiumchannel GIRK-2 and aldehydedehydrogenase-2 (ADH-2; Raldhl). Ideally, cellsgeneratedduring in vitro differentiationof embryonicstem cellsshould express the aforementionedmarkers, to ensurethat the blueprint of a fetal DA neuron has been matched as closely as possible. Other markers, e.g., common neuronal markers such as beta-tubulin III also need to be monitored in order to ensurethat a true neural subtype and not an artificial in vitro derivative, for exampleexpressing tyrosinehydroxylase and other DA markers in nonneural cells," wasgenerated.

ES-Cell Culture Conditions for DA Differentiation

How is midbrain DA differentiationfrom ES cells beingapproached? In vitro protocolswere developedto enablethe targeteddifferentiationof dopaminergicneuronsfrom mouseembryonic stem cells. 14 Asdescribedin detailearlierin this volume,keydeterminantsfor this regionalization are rostro-caudal and ventro-dorsal patterning gradients (Fig. IB). To enhance the fraction of dopaminergicdifferentiationfrom ES cells, media is supplementedwith sonic hedgehog (Shh) and fibroblast growthfaetor-8(FGF-8),ascriticalfactorsfor normaldevelopmentof the midbrain region." In addition, retinoic acid signalingis important for rostro-caudalpositioning, thereby definingthe mid-hindbrain boundary and the isthmicorganizers. In an elegantattempt to mimic the specificity of rostro-caudalpatterning in the dish," someprotocolshaveused retinoicacidto calibratethe generationof midbrain-typeDA neuronsin EScellculture.Additional retinoid signalingpathways important for midbrain DA specification remainto be investigated in detail." Startingwith thesestepsof neural induction and positioning,EScelldifferentiationprotocols have aimed to recapitulate the time course seen in normal rodent development.This could be achieved, for example, bya sequence of stepsrangingfrom initial formationof clusterscontaining cells of all germ layers ("embryoidbodies"),a selectionfor neurallyinduced cells, the expansion and patterning of neural precursor cells and finally the differentiationand maturation into dopaminergicneurons appropriate for transplantation studies." In other cases, empiricaladdition of supplementalfactors,which had shown to be protectivein ventral midbrain cellculture paradigms, such as the GFRalpha ligands GDNF or Neurturin,36,37 the transforminggrowth factor beta3,38 interleukin-Ibeta" and cAMp40 has been applied in midbrain DA differentiationfrom ESce11s. 1.2,41 Similarly, the fibroblast growthfactor-20hasbeen shownto promote DA survival4 2,43 and differentiation" and has been applied in ES celldifferentiationparadigms." Toincrease the yieldof neuralprecursorcellsbasicfibroblast growthfactor (bFGF)/fibroblast growth faetor-2 (FGF-2) wasadded at the expansion stage." As a side note, parallelapproaches wereintended to exploit the presenceof fetal midbrain neural precursor cells for the generation of dopaminergic neurons.v Long-term expansion of such developmentally more restricted cell sources resulted in decreaseddopaminergic and neuronal differentiation, which illustrated, in principle,the advantage of usingembryonicstemcells asa trulypluripotent sourcewith the capacity to givecontinuallygiveriseto any celllineageof therapeutic interest.46,47 Other sources, such asmesenchymal stem cells or corticalneural stemcells havenot provenusefulso far. Interestingly, the developmental programin vitro appearsto recapitulatethe species-specific temporal courseof normal midbrain dopaminergicneuron specification in mouse,monkeyand human ES cells.I,2,32 The complexityof the system, namely the targeted developmentof a very specific neuronal subtype from a pluripotent unpatterned cell,entails numerous challenges. Still,empiricism and guidedintuition haveshownthat suchendeavors canbeexperimentally fruitful at the proof-of-principlelevel, 1,2 In the future, screeningand high-throughput approaches are likelyto yieldfurther insight into how to optimize such protocols for the larger scaleproduction of a varietyof stem cell-derived celltypesfor regenerative medicine.In addition, detaileddevelopmental studieswill

118

Development and EngineeringofDopamineNeurons

further elucidatethe sequentialinterplayof transcriptionalregulatorsduring DA differentiation in vitro and in vivo. Recently, optimizingthedifferentiation protocolsforembryonicstemcell-deriveddopaminergic neurons for cell therapeutic studieshas focusedon two major hurdles preventingtranslation to a clinicalsetting: (i) the specification of the precisedopaminergicphenotype of interest, (ii) the prevention of any unwanted effects (seesection below),such as the generation of tumors from remainingundifferentiatedcells(seesection SelectionofDA Neurons from ES Cell Cultures).

Using Gene-Engineering to Specify DA Neurons in Vitro

Generatingthe dopaminergicsubtypelost in Parkinson's disease, the nigrostriatalneurons in the Substantianigra,pars compacta (A9),is of particular interest.Applyingknowledge from developmentalstudiesin micecan help in identifyingcandidatekeyregulatorsof the dopaminergic phenotype(Table1).Forexample, Pitx3hasbeenutilizedin EScellsystems, particularlypromoting the development of AHD2-positive cells, suggesting a specific involvement in A9 DA neurons while overallTH-positivity remained unaffected.48 Thisis in contrast to studiesusingexpression of the nuclear transcription factor Nurr 1, as a potent driver and apparent master regulator of TH-positivity.Itsexpression resultsin an overallincrease ofmidbrain dopaminergicmarkerssuch asDAT and VMAT-2, with no specific effecton A9 development/"Nurr 1 isimportant for development and maintenanceof the midbrain dopaminergicneuronal phenotype. It wasshown in a study usinga tetracyclin-inducible mouseEScellline that Nurr 1 expression at the nestin-positive precursor cellstagecan induce gene expression of dopaminergicmarkerssuch as AADC, DAT and AHD2 evenin nonneuronal cells.32In theseexperiments, other transcriptionfactorsrelevant for midbrain developmentsuchas Otx2, En-I, GBX2,Pitx3 and LmxIb remainedunaffectedby the induced Nurr 1 expression. Such experiments illustrate that ES cellscan be a useful tool to identify/confirm the function of certain transcriptional interactions. It also illustrates that the strategyof forcedexpression of certainkeymodulatorsrequirescarefulfinetuning-one example couldbe the combinedapproach,for example usingexpression of both Pitx3and Nurr 1 together, as recendydone in human ES cells." Overall,the challenges encounteredin generatingthe appropriateDA phenotypefrom EScells illustratethat additional factorsincludingcell-cell interactionsare likelyimportant in specifying the DA phenotype of interest. Furthermore, the design of protocols for midbrain dopamine neuron differentiationutilizesnot only the translation of knowledge from developmental studies,but alsorequiresattention to empirically proven determinants. Obviously, toxiccompounds of tissueculture media need to be excluded. For example, the widelyapplied HEPES bufferwas shown to havea negative impact on DA differentiationfrom ES cells,"Another exampleof such rather ill-defined measures is the usageof astroglial" or bone-marrow-derived stromalfeedercell lines,suchas PA63.51or MSScells,I,2,52,53 which havebeen shownto enhanceneural induction and dopaminergicdifferentiationin vitro.Theprecisenatureof this effectisunclear, but it exemplifies the importance of cell-cell communicationby adhesionmolecules and/or released factors.

Selection ofDA Neurons from ES-Cell Cultures

Formation of teratoma and/or neural tumors has been describedafier transplantation of ES cell-derived neural cells into rat Parkinsonmodels.2,16,54-56 In contrast to normal development, ES cellculturesrepresenta rather crude mix of cells at variousdevelopmental stages, of a number of celllineages, of cellcycle duration, etc.' (Fig.2C). Suchcellularcacophonyisnot onlyan issuefor therapeutic applications, but also for developmental and cellbiological studies,underlining the necessity of cellselectionapproaches, that can eliminateunwanted cells and increase the homogeneity of developmental stageand celllineage. This can be addressed by usinggene-engineered embryonic stem cells undergoing neural/dopaminergic differentiation that express fluorescent markerssuch asgreenfluorescent protein (GFP) in specific cellpopulationsof interest (seeTable 1). For example, presence of the transcription factorSox-l in cells undergoing neurallineage specification can be exploitedto select for neuralcells usinga Soxl-GFP construct,therebyeliminating

Molecular and Cellular DeterminantsfOrGeneratingES-Cell

119

Table 1. Gene-engineered stem cellsin dopaminergic cell transplantation paradigms Gene

Species Approach

Effect

References

BcI-XL

Mouse

Overexpression

t DA diff

Shim et al. J Neurosci 2004;79 Liste

Nurrl

Mouse

Overexpression

t DA diff

Chung et al, EJN 2002;49 Sonntag et al. EJN 2004;32 Marti nat et al, PNAS 2006 50

Pitx3-GFP Mouse

Cell sorting

Pitx3

Mouse

Overexpression

t DA fraction t DA diff

Smad-/-, Cripto-/-

Mouse

Knock-out

t Neural diff

Soxl-GFP

Mouse

Cell sorting

SynapsinGFP

Human

Cell sorting

t Neurons

TH-GFP

Mouse

Cell sorting

t DA fraction

et al. J Neurosci 2004 80

Hedlund et al. in submission" Chung et al. Mol Cell Neurosci 2005;48 Martinat et al. PNAS 2006 50 Sonntag et al. Mol Cell Neurosci 2005;81 Parish et al. Stem Cells

2005 82 ~ Tumor formation

Chung et al. J Neurochem 2006 57 Pruszak et al. Stem Cells 2007 5 Hedlund et al. Stem Cells 2007;77 Yoshizaki et al. Neurosci Lett 2004 78

Factors important for midbrain DA neuronal specification are used to optimize ES cell differentiation protocols. The examples shown here demonstrate how fluorescent labeling enables the analysis and selection of specific cellular subsets.5,29,57,77,78 Furthermore, overexpression or inducible expression systems enable studies of transcriptional regulatory networks and also can be helpful for increasing the yield of the phenotype of interest. 32,48-50,79-82 Caveats of genetic engineering approaches include the generation of nonphysiological expression patterns, which can result in phenotypes that represent a mere caricature of normal development

immature, tumor-generating stem cells." At later stages of differentiation, constructs such as Synapsin-GFP for neuronal cells? or Pitx3-GFP specifically for dopaminergic cells (Hedlund et al in submission) were used to select for postrnitotic neuronal cells for transplantation and in vitro studies. In addition to genetic fluorescent markers, the identification of specific neural and dopaminergic surface antigens':" will be critical for studies of developmental potential and for translation to clinical applications.

Future Perspective

The generation of authentic midbrain-like dopaminergic neurons from human pluripotent stem cells will enable a variety ofdevelopmental, cell biological and pharmacological studies and assayson human neurons in culture and in animal models. DA neurons generated from ES cells can be modulated for and utilized in drug discovery paradigms, genetic assaysmodeling human diseases and in detailed developmental studies. Compared to primary tissue, DA cells created from ES cells are particularly amenable to manipulation and analysis in high throughput systems and recent advances suggest that transgenic strategies will be feasible and fruitful also in human ES cells.58•59 Conceptually, it is most reasonable to trigger and/or induce genes in the normal sequence required to maintain the phenotype of interest. So far this sequential, «chronological" approach (mimicking the sequence of normal development) has been helpful, although we do not always know when the entire cell population (or subsets thereof) generated in vitro are responsive to

Development and EngineeringofDopamineNeurons

120

specific patterningcues. Questions of timingarise. Forexample, whenisprogenitorcellfate already too restricted by intrinsicepigeneticfactors?It is alsopossiblethat someprogenitor cellsmaybe more responsive to localsignals from surroundingcells, therebyoverridingintended homogenous signalingfrom additives in the cellculture induction protocol. Hopefully,in the future enough willbe known about the transcriptionalregulatorycascades and epigeneticconditions to induce a midbrain DA phenotype, such that we can produce desiredneurons even from prepatterned/ more restricted cell types. Our understanding of alternative cell sources, such as nuclear transfer-derived23.60 or other reprogrammed cells islikelyto increase, includinga moreprecisecontrolof their pluripotency.Recentsignificant advances in generatinginducedpluripotent stem (iPS)cells from mouse and human fibroblasts, in which "sternness" wasinduced by retroviralintroduction of only a handful of key regulators,61-65 indicate that the de-differentiation and reprogramming of many cell types may be possibleand that such celllines may be useful, if controllablefrom a tumorigenesis perspective.

References

1. Perrier Ai, Tabar V, Barberi T, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Nat! Acad Sci USA 2004; 101:12543-12548. 2. Sonntag KC, Pmszak J, YoshizakiT et al Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin. Stem Cells 2007; 25:411-418. 3. Kawasaki H, Mizuseki K, Nishikawa S et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000; 28:31-40. Stem cells may reshape the prospect of parkinsons disease therapy. 4. Sonntag KC, Simantov R, lsacson Brain Res Mol Brain Res 2005; 134:34-51. 5. Pruszak J, Sonntag KC, Aung MH et al. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells 2007; 25:2257-2268. 6. lsacson 0, Bjorklund LM, Schumacher JM. Toward full restoration of synaptic and terminal function of the dopaminergic system in parkinsons disease by stem cells. Ann Neurol 2003; 53 Suppl 3: SI35-S146. 7. Mendez I, Sanchez-Pernaute R, Cooper 0 et al. Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with parkinsons disease. Brain 2005; 128:1498-1510.

o.

8. Isacson O. The production and use of cells as therapeutic agents in neurodegenerative diseases. Lancet

Neurol 2003; 2:417-424. 9. Deacon T, Schumacher J, Dinsmore J et al. Histological evidence of fetal pig neural cell survival after transplantation into a patient with Parkinson's disease. Nat Med 1997; 3:350-353. 10. Dinsmore JH, Pakzaban P, Deacon TW et al. Survival of transplanted porcine neural cells treated with F(ab')2 antibody fragments directed against donor MHC class-I in a rodent model. Transplant Proc 1996; 28:817-818. 11. Isacson 0, Deacon T~ Pakzaban P et al. Transplanted xenogeneic neural cells in neurodegenerative disease models exhibit remarkable axonal target specificity and distinct growth patterns of glial and axonal fibres. Nat Med 1995; 1:1189-1194. 12. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem ceHlines derived from human blastocysts. Science 1998; 282:1145-1147. 13. Bjorklund LM, Sanchez-Pernaute R, Chung S et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Nat! Acad Sci USA 2002; 99:2344-2349. 14. Lee SH, Lumelsky N, Studer L et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol2000; 18:675-679. 15. Deacon T, Dinsmore J, Costantini LC et al. Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation. Exp Neuro11998; 149:28-41. 16. Roy NS, Cleren C, Singh SK et al. Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med 2006; 12:1259-1268. 17. Takagi Y: Takahashi J, Saiki H et al. Dopaminergic neurons generated from monkey embryonic stem cells function in a parkinson primate model. J Clin Invest 2005; 115:102-109. 18. Sanchez-Pernaute R, Studer L, Ferrari D et al. Long-term survival of dopamine neurons derived from parthenogenetic primate embryonic stem cells (cyno-l) after transplantation. Stem Cells 2005; 23:914-922.

Molecular and Cellular Determinantsfor GeneratingES-Cell

121

19. Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 2005; 19:1129-1155. 20. Carson CT, Aigner S, Gage FH. Stem cells: the good, bad and barely in control. Nat Med 2006; 12:1237-1238. 21. Kelly BB, Hedlund E, Kim C et al. A tyrosine hydroxylase-yellow fluorescent protein knock-in reporter system labeling dopaminergic neurons reveals potential regulatory role for the first intron of the rodent tyrosine hydroxylase gene. Neuroscience 2006; 142:343-354. 22. Verlinsky ~ Strelchenko N, Kukharenko V et ale Human embryonic stem cell lines with genetic disorders. Reprod Biomed Online 2005; 10:105-110. 23. Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells and the potential for cell therapy. N Engl J Med 2003; 349: 275-286. 24. Chung CY: Seo H, Sonntag KC et al. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet 2005; 14:1709-1725. 25. Chung C~ Koprich JB, Endo S et al. An endogenous serine/threonine protein phosphatase inhibitor, G-substrate, reduces vulnerability in models of Parkinson's disease. J Neurosci 2007; 27:8314-8323. 26. McKinney-Freeman SL, Daley GQ Towards hematopoietic reconstitution from embryonic stem cells: a sanguine future. Curr Opin HematoI2007; 14:343-347. 27. Sinclair SR, Fawcett J~ Dunnett SB. Dopamine cells in nigral grafts differentiate prior to implantation. Eur J Neurosci 1999; 11:4341-4348. 28. Thompson LH, Andersson E, Jensen JB et al. Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon. Exp Neuro12006; 198:183-198. 29 Hedlund E, Pruszak ], Lardaro T et ale Embryonic stem (ES) cell-derived Pitx3-eGFP midbrain dopamine neurons survive enrichment by FACS and function in an animal model of Parkinson's disease. 2008. in submission 30. Zhao S, Maxwell S, jimenez-Beristain A et al. Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. Eur. J. Neurosci 2004; 19: 1133-1140. 31. Ono Y, Nakatani T, Sakamoto Y et al. Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells. Development 2007;134: 3213-3225. 32. Sonntag KC, Simantov R, Kim KS et al. Temporally induced Nurr1 can induce a nonneuronal dopaminergic cell type in embryonic stem cell differentiation. Eur J Neurosci 2004; 19:1141-1152. 33. Ye ~ Shimamura K, Rubenstein JL et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998; 93:755-766. 34. Okada ~ Shimazaki T, Sobue G et al. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev BioI 2004; 275:124-142. 35. Smidt MP, Burbach JP. How to make a mesodiencephalic dopaminergic neuron. Nat Rev Neurosci 2007; 8:21-32. 36. Horger BA, Nishimura MC, Armanini MP et al. Neurturin exerts potent actions on survival and function of midbrain dopaminergic neurons. J. Neurosci 1998; 18: 4929-4937. 37. Rosenblad C, Kirik D, Bjorklund A. Neurturin enhances the survival of intrastriatal fetal dopaminergic transplants. Neuroreport 1999; 10:1783-1787. 38. Krieglstein K, Reuss B, Maysinger D et al. Short communication: transforming growth factor-beta mediates the neurotrophic effect of fibroblast growth factor-2 on midbrain dopaminergic neurons. Eur J Neurosci 1998; 10:2746-2750. 39. Ling ZD, Potter ED, Lipton JW et al. Differentiation of mesencephalic progenitor cells into dopaminergic neurons by cytokines. Exp Neurol 1998; 149:411-423. 40. Branton RL, Love RM, Clarke DJ. cAMP included during cell suspension preparation improves survival of dopaminergic neurons in vitro. Neuroreport 1998; 9:3223-3227. 41. Rolletschek A, Chang H, Guan K et al. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors. Mech. Dev. 2001; 105: 93-104. 42. Ohmachi S, Watanabe Y, Mikami T et al. FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain. Biochem. Biophys. Res. Commun. 2000; 277: 355-360. 43. Murase S, McKay RD. A specific survival response in dopamine neurons at most risk in Parkinson's disease. J Neurosci 2006; 26:9750-9760. 44. Grothe C, Timmer M, Scholz T et al. Fibroblast growth factor-20 promotes the differentiation of Nurr l-overexpressing neural stem cells into tyrosine hydroxylase-positive neurons. Neurobiol. Dis. 2004; 17:163-170

Development and Engineering ofDopamineNeurons

122

45. Studer L, Tabar V, McKay RD. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1998; 1:290-295. 46. Hong S, Kang UJ, Isacson 0 er al. Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons. J Neurochem 2007; 47. Chung S, Shin BS, Hwang M et al. Neural precursors derived from embryonic stem cells, but not those from fetal ventral mesencephalon, maintain the potential to differentiate into dopaminergic neurons after expansion in vitro. Stem Cells 2006; 24: 1583-1593. 48. Chung S, Hedlund E, Hwang M et ale The homeodomain transcription factor Pitx3 facilitates differentiation of mouse embryonic stem cells into AHD2-expressing dopaminergic neurons. Mol. Cell Neurosci, 2005; 28: 241-252. 49. Chung S, Sonntag KC, Andersson T et al. Genetic engineering of mouse embryonic stem cells by Nurrl enhances differentiation and maturation into dopaminergic neurons. Eur. J. Neurosci, 2002; 16: 1829-1838. 50. Martinat C, Bacci JJ, Leete T et al. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype. Proc. Natl. Acad. Sci. U. S. A 2006; 103:2874-2879. 51. Kim DW; Chung S, Hwang M et al. Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells. Stem Cells 2006; 24:557-567. 52. Barberi T, Klivenyi P, Calingasan NY et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat. Biotechnol, 2003; 21:12001207. 53. Pruszak J, Isacson In vitro differentiation of dopaminergic neurons in human embryonic stem cells-a practical handbook (eds, Sullivan S, Cowan C, Eggan K.) 2007; 54. Brederlau A, Correia AS, Anisimov SV et al. Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells 2006; 24, 1433-1440. 55. Yang D, Zhang ZJ, Oldenburg M et al. Human embryonic stem cell-derived dopaminergic neurons reverse functional deficit in parkinsonian rats. Stem Cells 2008 Jan;26(1):55-63. 56. Ben Hur T, Idelson M, Khaner H et al. Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells 2004; 22:1246-1255. 57. Chung S, Shin BS, Hedlund E er al. Genetic selection of soxl GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation. J. Neurochem 2006; 97: 1467-1480. 58. Zwaka TP, Thomson JA.Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003; 21:319-321. 59. Xia X, Ayala M, Thiede BR et al. In vitro and in vivo induced transgene expression in human embryonic stem cells and derivatives. Stem Cells 2007; 60. Hochedlinger K, Jaenisch R. Nuclear reprogramming and pluripotency, Nature 2006; 441:1061-1067. 61. Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861-872. 62. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-676. 63. Wernig M, Meissner A, Foreman R et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007; 448:318-324. 64. Yu J, .Vodyanik M.A, Smuga-Otto K et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917-1920. 65. Maherali N, Sridharan R, Xie W et al. Directly Reprogrammed Fibroblasts Show Global Epigenetic Remodeling and Widespread Tissue Contribution. Cell Stem Cells 2007; 1: 55-70. 66. Yan Y, Yang D, Zarnowska ED et al. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 2005; 23: 781-790. 67. Karki S, Pruszak J, Isacson 0 et al. ES cell-derived neuroepithelial cell cultures. Journal of Visualized Experiments, www.myjove.com 2006. 68. Smidt MP, Asbreuk CH, Cox JJ et al. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmxlb. Nat Neurosci 2000; 3:337-341. 69. Maxwell SL, Li M. Midbrain dopaminergic development in vivo and in vitro from embryonic stem cells. J Anat 2005; 207:209-218. 70. Prakash N, Wurst w: Genetic networks controlling the development of midbrain dopaminergic neurons. J Physiol 2006; 575:403-410.

o.

Molecular and CellularDeterminantsfOrGeneratingES-Cell

123

71. Prakash N, Wurst W Development of dopaminergic neurons in the mammalian brain. Cell Mol Life Sci 2006; 63:187-206. 72. Andersson E, Tryggvason U, Deng Q et aI. Identification of intrinsic determinants of midbrain dopamine neurons. Cell 2006; 124,393-405. 73. Ferri AL, Lin ~ Mavromatakis YE et al. Foxal and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner. Development 2007; 134:2761-2769. 74. Ang SL. Transcriptional control of midbrain dopaminergic neuron development. Development 2006; 133:3499-3506. 75. Vitalis T, Cases 0, Parnavelas]G. Development of the dopaminergic neurons in the rodent brainstem. Exp NeuroI2005; 191 Suppll:Sl04-S112. 76. Smits SM, Ponnio T, Conneely OM et al. Involvement of Nurrl in specifying the neurotransmitter identity of ventral midbrain dopaminergic neurons. Eur] Neurosci 2003; 18:1731-1738. 77. Hedlund E, Pruszak], Ferree A et al. Selection of embryonic stem cell-derivedenhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations. Stem Cells 2007; 25: 1126-1135. 78. Yoshizaki T, Inaji M, Kouike H et al. Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells. Neurosci Lett 2004; 363: 33-37. 79. Shim Koh HC, Chang MY et al. Enhanced in vitro midbrain dopamine neuron differentiation, dopaminergic function, neurite outgrowth, and I-methyl-4-phenylpyridium resistance in mouse embryonic stem cells overexpressing Bel-XL.] Neurosci 2004; 24: 843-852. 80. Liste I, Garcia-Garcia E, Martinez-Serrano A. The generation of dopaminergic neurons by human neural stem cells is enhanced by Bel-XL, both in vitro and in vivo.] Neurosci 2004; 24:10786-10795. 81. Sonntag KC, Simantov R, Bjorklund L et al. Context-dependent neuronal differentiation and germ layer induction of Smad4-/- and Cripto-/- embryonic stem cells. Mol Cell Neurosci 2005;28: 417-429. 82. Parish CL, Parisi S, Persico MG et al. Cripto as a target for improving embryonic stem cell-based therapy in Parkinson's disease. Stem Cells 2005; 23:471-476.

.rw:

INDEX A Alar basalboundary (ABB) 40,41,43 Addiction 28, 36, 98 Anatomy 1, 16 Animal models ofParkinsonism 81,85,86 Aphakia 47-51 Apoptosis 69, 83, 86 Aromatic amino acid decarboxylase (AADC) 17,21,23,61,102,103,116,118 Ascendingprojection 4, 6, 9 Axon branching 95 Axon growth 93, 95, 96 Axon guidance 91, 92, 94, 95, 97, 98

B 1,11,51,74,75,91,98,103 BMP 85, 101, 102, 104, 105, 115

Beha~or

c Cell sorting 107, 112, 119 Cell therapy 101, 106-108, 112, 116 Chimeric 53,69, 102 Clinical application 119 Conserved dopamine neurotrophic factor (CDNF) 86 Cortex 20,21,28, 50, 52, 91, 96, 97 CREB 23,24

D Dj receptor 17-19,28 Deleted in colorectal cancer (DCC) 95 Development 2-4,6-9, 11, 15, 19,21,36,37, 47,48,51,53,54,56-61,63,66,67,73, 75-77,81-85,91,92,96-98,.101, 105, 106, 112-119 Diencephalon 2, 3, 5-8, 11, 83, 91-94, 96 Diencephalospinal 9, 11 Dopamine 2,11,15-18,28,36,49-51,53, 54,60,69,70,81,86,91,92,94,95,97, 98,101-105,107,112-116,118

Dopaminergic (DA) 2-9, 11, 15- 21, 23, 24, 27-29,36,43,44,51,62,68-70,74-77, 85,101-108,112-120 Dopaminergic differentiation 1, 9, 11, 117, 118 Dorsal lateral ganglionic eminence (dLGE) 15,19,20,23,24 Drosophila 37, 58, 66

E Embryo 1-3,6-9, 15,37-40,42- 44, 49, 58-62,66-68,84,93,101,102,104,113 Embryoid body 67, 103, 117 Embryonic development 19 Embryonic stem cell (ESC) 61, 101, 102, 112-114,117,118, Eph 26, 97, 98 Ephrin 26,97,98 Er81 19,21, 23-25 Evolution 1,4,9,11,40,70

F FGF2 102, 104, 105 FGF8 7,8,23,82-84, 102-105 Flourescentactivated cellsorting (FACS) 107 Fluorescentmarkers 112, 118, 119 Forebrain embryo zinc finger-like protein (Fezl/FezfL.) 9 Forced expression 105, 118 FosB 23, 24, 27

G GABA 15,17-19,27,28 Gbx2 37-42, 44, 68 GDF 85 Gene regulatory network 116 Genetic screen 6, 7 GFP 11,21, 107, 112, 115, 118, 119 Glial cell-line-derived neurotropic factor (GDNF) 77,81,84-86, 104, 117

126

Glutamate aciddecarboxylase (GAD) 19, 103 Glutamatergic 17, 18 Granule cell 16-19,21,22,24,26 Green fluorescent protein 118 Grg4 40,41 Gsh2 19,21, 24

H Hindbrain 2-4,8, 11,37,38,39,41- 44,59, 60,67,68,83,102-104,114,117 Homeodomain 7,24,26,28, 37,40,60,61, 66,67,85 Homeodomain interactivityprotein kinase2 (HIPK2) 85 Human embryonicstem cell 113-115, 117-119

I Immediateearlygene (lEG) 23, 27 Induced pluripotent stem 106, 107, 120 In vitro differentiation 103, 105, 112, 116, 117 iPS cell 107 Isthmicorganizer 37, 117 Isthmus 67,82

L LacZ 21,22 Leukemiainhibiting factor 102 Lmxla 9,23,60,61,82, 103, lOS, 116 Lmxl b 9,23, 37,48,49,60,61, 103, 116, 118

M MANF 77,86 Marker 15,17,19,21,37-39,50,61,68, 102,103,105-107,112,114-119 Medial forebrain bundle 93 Meis2 21,24 Mesencephalon 4,5,82,83,92-94,97 Mesodiencephalon 52, 53, 92 Midbrain 2,3,6, 7, 15,21,23,28,36-44,49, 50,52,53,58-62,66-69, 73, 74, 76, 77, 81-85, 101-107,112-120

Developmentand EngineeringofDopamineNeurons

Midbrain- hindbrain boundary (MHB) 37-43,83 Migration 23,26,27,28, 50, 52, 81 Mitral cell 16, 17, 22, 26 Mouseembryonicstem cell 61, 113, 117 Mousemodel 82, 98, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) 11,74,76,85,86 MS5 104,118 Mutagenesis 1,6

N Netrin 95,98 Network 5,6, 16, 36,40,44,47,49,63, 82, 115,116, 119, Neural differentiation 9,37, 104, 105 Neuralinduction 101-105,115,117,118 Neural lineagemarker 115 Neural stem cell (NSC) 4, 15, 19,21,26,83, 105-107,113,117 Neural transplantation Neuroblast 19,21,23,24,26, 107 Neurodegenerative disease 15,91 Neurogenesis 4,11, 16, 19,28,60,61,77,85 Neurological disorder 101 Neuropilin 53, 93, 95 Neuroprotection 81, 86 Neurospheres 82, 83 Neurotransmitter 9, 18, 36,42,47,49, 53, 69, 70,82,91, 116 Neurotrophic factors 73,74,77,86, 103 Nkx2.2 23,41-44 Nkx6.1 41-43 Nodal 7, 8, 83, 84 Nr4a2 9, 37, 52 Nurr1 9, 11,23,24, 37,47, 52-54,61,69, 82-84,103,105,106,116,118,119

o Odor deprivation 17 Olfactory bulb (OB) 2-4,7,8, 10, 15-29,52 Olfactory receptor neuron 15-19,23,26,28 Orthopedia 7, 9 otp 6-9, 11 otx genes 36, 37, 39,40,42,43

127

Index

P PA-6 104 Parkinson's disease 1, 15,28, 36,49, 54,63, 73,76,86,91,101,112,113,118 Patterning 6-9, 11, 37, 39-41, 59,60, 82, 102-106,114,115,117,120 Pax6 19,21,23,24,25, 103 Pharmacology 11 Pitx3 9,23,37,47-54,82,83,84,103, 105, 106,107,112,116,118,119 Pluripotency 102,115,120 Posteriortuberculum 2-5,8 Promoter 21,23,24,27,48,49, 51, 54, 59, 62, 63, 67, 114, Prosomere 3, 8, 38 Ptx3 47

R Red nucleus 41, 43 Regenerative medicine 97, 114, 117 Retinoic acid 8, 51, 102, 103, 117 RNA interference 69 Robo 26, 93, 95 Rosette 104-106, 115 Rostralmigratory stream (RMS) 15, 19,20, 21,23,24,26,28

s Secretion 67 Semaphorin 93, 98 Septum 15, 19,20,24,26 Serotonin 42, 51 Serum-free EB (SFEB) 105 Slit 26, 92, 93, 96, 98 Sonichedgehog (Shh) 21,59,67,82, 102-105,117 SSEA3 102 SSEA4 102 Stem cell 4, 15, 19,20,21,26,29,61,63,82, 83,101,102,105,106,107,112-114, 117,118,119

Striatalconnectivity SO Striatum 6, 11, 15, 16,21,28,36, SO, 51, 73-76,85,86,91,92,96,97,106 Substantianigra (SN) 1,4,6, 15,28,36,47, 52,58,69,70,73-77,85,86,91,101, 117,118 Subventricular zone (SVZ) 15, 19-21, 23, 24,26,28,43 Surface antigen 112, 115 Survival 11,36,50,53,66,69,70,73-77,81, 83-86,97,104,106,107,113

T

Telencephalon 4-6, 15, 19,20,68,92,93,96 Transcriptionfactor (TF) 1,6,7,8,9, 10, 15,19,21,23,24,26,28,36,37,39,40, 47,54,58,60,61,63,66,67,82,84,85, 102,103,105,106,112,116,118 Transit amplifyingcell 19,21, 24, 26, Transplantation 98, 102, 104, 106, 107, 112, 113,115,117-119, Tufted cell 15, 16, 17 Tyrosine hydroxylase (TH) 2,4-6,9, 10, 1517, 19,21-28, 59,61,69, 75-77,82-84, 102-105,107,112,114-119

V

Ventralmesencephalon 82, 83 Ventralthalamus 3, 93, 94 Ventraltegmentalarea (VTA) 6,36,48-50, 69,70,91,96-98,101,106

w Weight 70 Wntl 37,42,44

z Zic transcription factor protein (Zic) 19, 24,28