Differentiation of Embryonic Stem Cells

574 Pages·2003·4.564 MB·English

by  Wassarman P. (ed.), Keller G. (ed.)

Preface
Since their isolation over twenty years ago, embryonic stem (ES) cells
have come to play a prominent role in many different fields in biomedical
research.Whileusedextensivelyforgenetargetingstudiesforthegeneration
of‘‘knock-outmice,’’itistheircapacitytodifferentiateintoawidearrayof
lineages in culture that has most recently captured the attention of basic
scientists,clinicalresearchers,andthelaypublic.ThecapacityofanEScell
to differentiate into almost any cell type in a culture dish offers
unprecedented opportunities for studies in lineage commitment and
development, gene function, and cancer. In addition, the development of
human ES cells in 1998 has expanded the potential uses of the in vitro
differentiation approach to include the generation of specific cell types for
cell replacement therapy and regenerative medicine.
IthasbeenknownforsometimethatmouseEScellscandifferentiatein
culture and generate derivatives of the three primary germ cell layers:
ectoderm, endoderm, and mesoderm. In many of these early studies,
however, differentiation was not well controlled and the cultures often
consisted of mixtures of tissues. Significant advances in the methodologies
of ES cell differentiation have been made in recent years, and it is now
possible to generate relatively pure populations of cells from a number of
different lineages in a reproducible fashion. These new approaches have
enabled investigators to begin to use the system to probe the molecular
events regulating early lineage commitment, as well as to generate
populations appropriate for cell replacement therapy in preclinical models.
WiththeestablishmentofhumanEScells,thechallengeforthoseinthefield
of stem cell and developmental biology is to now translate the information
from the mouse to the human system.
Inthisvolume,wehavebroughttogetheracomprehensivecollectionof
the most up-to-date methods for the differentiation of both mouse and
human ES cells into a broad spectrum of lineages. Together with the
differentiation protocols, we have solicited a select set of protocols that
highlightapproachesforgenediscoveryandlineageselectionusingEScells.
The chapters are written by leaders in the field from the international
community and provide technical details that will enable the reader to
establishandmaintainEScellculturesandinducetheirdifferentiationintoa
large number of different lineages.
We hope that this volume on Differentiation of Embryonic Stem Cells
willfinditswayintomanylaboratoriesandprovestobeusefulatthebench
xv
xvi PREFACE
forinvestigatorsworld-wide.Weareextremelygratefultothemanyauthors
fortheirexcellentcontributionstothisvolumeandtheirpatienceindealing
with publication schedules. Finally, we extend our appreciation to Shirley
Light at Academic Press who organized the assembly of this and hundreds
of other volumes of Methods in Enzymology over several decades.
PAUL M.WASSARMAN
GORDONM.KELLER
Table of Contents
CONTRIBUTORSTOVOLUME365. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
VOLUMESINSERIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Section I. Differentiation of Mouse Embryonic Stem Cells
EarlyCommitmentStepsandGenerationofChimericMice
1. LineageSpecificDifferentiationofMouseESCells: JOYRATHJENAND
FormationandDifferentiationofEarlyPrimitive PETERD.RATHJEN 3
Ectoderm-like(EPL)Cells
2. Differentiation of F1 Embryonic Stem Cells into KEVINEGGANAND
Viable Male and Female Mice by Tetraploid RUDOLFJAENISCH 25
EmbryoComplementation
DifferentiationtoMesodermDerivatives:HematopoieticandVascular
3. HematopoieticCommitmentofESCellsin MARIONKENNEDYAND
Culture GORDONM.KELLER 39
4. InVitroDifferentiationofMouseEmbryonicStem STUARTT.FRASER,
Cells:HematopoieticandVascularCellTypes JUNYAMASHITA,
L.MARTINJAKT,
MITSUHIROOKADA,
MINETAROOGAWA,
SATOMINISHIKAWA,AND
SHIN-ICHINISHIKAWA 59
5. InVitroDifferentiationofMouseEmbryonicStem KENJIKITAJIMA,
CellstoHematopoieticCellsonanOP9Stromal MAKOTOTANAKA,
CellMonolayer JIEZHENG,
EIKOSAKAI-OGAWA,AND
TORUNAKANO 72
6. In Vitro Differentiation of Mouse ES Cells: JOSEPHB.KEARNEYAND
HematopoieticandVascularDevelopment VICTORIAL.BAUTCH 83
7. In Vitro Differentiation of Mouse ES Cells into MOTOKAZUTSUNETO,
Hematopoietic, Endothelial, and Osteoblastic TOSHIYUKIYAMANE,
Cell Lineages: The Possibility of In Vitro HIROMIOKUYAMA,
Organogenesis HIDETOSHIYAMAZAKI,AND
SHIN-ICHIHAYASHI 98
v
vi TABLEOFCONTENTS
8. Development of Hematopoietic Repopulating MICHAELKYBA,
CellsfromEmbryonicStemCells RITAC.R.PERLINGEIRO,
ANDGEORGEQ.DALEY 114
9. TheInVitroDifferentiationofMouseEmbryonic JONATHANG.LIEBER,
StemCellsintoNeutrophils GORDON M. KELLER, AND
G.SCOTTWORTHEN 129
10. Development and Analysis of Megakaryocytes KOJIETO,
fromMurineEmbryonicStemCells ANDREWL.LEAVITT,
TORUNAKANO,AND
SANFORDJ.SHATTIL 142
11. Development of Lymphoid Lineages from SARAHK.CHOAND
EmbryonicStemCellsInVitro JUANCARLOS
ZU´N˜IGA-PFLU¨CKER 158
12. Probing Dendritic Cell Function by Guiding the PAULJ.FAIRCHILD,
DifferentiationofEmbryonicStemCells KATHLEENF.NOLAN,AND
HERMANWALDMANN 169
13. Gene Targeting Strategies for the Isolation of WENJIEZHANG,
Hematopoietic and Endothelial Precursors from YUNSHINCHUNG,
DifferentiatedESCells BILLEADES,AND
KYUNGHEECHOI 186
14. Establishment of Multipotent Hematopoietic LEIFCARLSSON,
ProgenitorCellLinesfromESCellsDifferentiated EWAWANDZIOCH,
InVitro PERPE´TUAPINTODOO´ ,
ANDA˚ SAKOLTERUD 202
15. Vasculogenesis and Angiogenesis from In Vitro OLIVIERFERAUD,
DifferentiationofMouseEmbryonicStemCells MARIE-HE´LE`NE PRANDINI,
ANDDANIELVITTET 214
OtherDerivativesofMesoderm
16. ESCellDifferentiationtotheCardiacLineage KENNETHR.BOHELER 228
17. Bone Nodule Formation via In Vitro SARAHK.BRONSON 241
DifferentiationofMurineEmbryonicStemCells
18. In Vitro Differentiation of Mouse ES Cells: Bone JANKRAMER,
andCartilage CLAUDIAHEGERT,AND
JU¨RGENROHWEDEL 251
19. Development of Adipocytes from Differentiated BRIGITTEWDZIEKONSKI,
ESCells PHIVILLAGEOIS,AND
CHRISTIANDANI 268
Differentiation toEndodermDerivatives
20. In Vitro Differentiation of Embryonic Stem Cells TAKASHIHAMAZAKIAND
intoHepatocytes NAOHIROTERADA 277
TABLEOFCONTENTS vii
21. Differentiation of Mouse Embryonic Stem Cells GABRIELAKANIA,
intoPancreaticandHepaticCells PRZEMYSLAW BLYSZCZUK,
JAROSLAWCZYZ,
ANNENAVARRETE-SANTOS,
ANDANNAM.WOBUS 287
DifferentiationtoEctodermDerivatives
22. GeneratingCNSNeuronsfromEmbryonic,Fetal, JONG-HOONKIM,
andAdultStemCells DAVIDPANCHISION,
RAJAKITTAPPA,AND
RONMCKAY 303
23. Defined Conditions for Neural Commitment and QI-LONGYINGAND
Differentiation AUSTING.SMITH 327
24. DevelopmentofMelanocytesfromESCells TAKAHIROKUNISADA,
TOSHIYUKIYAMANE,
HITOMIAOKI,
NAOKOYOSHIMURA,
KATSUHIKOISHIZAKI,AND
TSUTOMUMOTOHASHI 341
Section II. Differentiation of Mouse Embryonic Germ Cells
25. IsolationandCultureofEmbryonicGermCells MARIAP.DEMIGUELAND
PETERJ.DONOVAN 353
Section III. Gene Discovery by Manipulation of
Mouse Embryonic Stem Cells
26. GeneTrapMutagenesisinEmbryonicStemCells WEISHENG V. CHEN AND
PHILIPPESORIANO 367
27. Gene Trap Vector Screen for Developmental HEIDISTUHLMANN 386
GenesinDifferentiatingESCells
28. Gene-Based Chemical Mutagenesis in Mouse YIJINGCHEN,
EmbryonicStemCells JAYL.VIVIAN,AND
TERRYMAGNUSON 406
Section IV. Differentiation of Monkey and Human
Embryonic Stem Cells
29. Growth and Differentiation of Cynomolgus HIROFUMI SUEMORI AND
MonkeyESCells NORIONAKATSUJI 419
30. Isolation,Characterization,andDifferentiationof MARTINF.PERA,
HumanEmbryonicStemCells ADAMA. FILIPCZYK,
SUSANM.HAWES,AND
ANDREWL.LASLETT 429
viii TABLEOFCONTENTS
31. FactorsControllingHumanEmbryonicStemCell MAYA SCHULDINER AND
Differentiation NISSIMBENVENISTY 446
32. DevelopmentofCardiomyocytesfromHumanES IZHAKKEHAT,
Cells MICHALAMIT,
AMIRAGEPSTEIN,
IRITHUBER,
JOSEPH ITSKOVITZ-ELDOR,
ANDLIORGEPSTEIN 461
AUTHOR INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
SUBJECT INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
Contributors to Volume 365
Articlenumbersareinparenthesesfollowingthenamesofcontributors.
Affiliationslistedarecurrent.
MICHAL AMIT (32), Department of Sciences,FredHutchinsonCancerResearch
Physiology and Biophysics, Bruce Rappa- Center, 1100 Fairview Avenue N, A2-025,
port Faculty of Medicine, Technion-Israel P.O. Box 19024, Seattle, Washington
Institute of Technology, 2 Efron Street, 98109-1024
POB9649,Haifa31096,Israel
YIJING CHEN (28), Department of Genetics,
HITOMIAOKI(24),DepartmentofTissueand University of North Carolina at Chapel
Organ Development, Regeneration and Hill,ChapelHill,NorthCarolina27599
AdvancedMedicalScience,GifuUniversity
Graduate School of Medicine, Gifu, 500- SARAH K. CHO (11), Division of Biological
8705,Japan Sciences, University of California at San
Diego, 9500 Gilman Drive, La Jolla,
VICTORIA L. BAUTCH (6), Department of California92093
Biology and Program in Genetics, Univer-
sity of North Carolina at Chapel Hill, KYUNGHEECHOI(13),DepartmentofPathol-
CB#3280, Chapel Hill, North Carolina ogy&Immunology,WashingtonUniversity
27599 SchoolofMedicine,660S.EuclidAvenue,
Box8118,St.Louis,Missouri63110
NISSIM BENVENISTY (31), Department of
Genetics, The Silberman Institute of Life YUN SHIN CHUNG (13), Department of
Sciences, The Hebrew University, Jerusa- Pathology & Immunology, Washington
lem91904,Israel University School of Medicine, 660 S.
Euclid Avenue, Box 8118, St. Louis,
PRZEMYSLAW BLYSZCZUK (21), In Vitro
Missouri63110
Differentiation Group, Institute of Plant
Genetics and Crop Plant Research, Cor- JAROSLAWCZYZ(21),InVitroDifferentiation
rensstr.3,Gatersleben,D-06466,Germany Group,InstituteofPlantGeneticsandCrop
Plant Research Corrensstr. 3, Gatersleben,
KENNETH R. BOHELER (16), Molecular Car-
D-06466,Germany
diologyUnit,LaboratoryofCardiovascular
Science, National Institute on Aging, NIH GEORGE Q. DALEY (8), Whitehead Institute
5600 Nathan Shock Drive, Baltimore, forBiomedicalResearch,HarvardMedical
Maryland21224 School, Cambridge, Massachusetts 02142-
SARAH K. BRONSON (17), Department of 1479
Cellular & Molecular Physiology, Penn
CHRISTIAN DANI (19), Centre de Biochimie,
State College of Medicine, The Milton
InstitutdeRecherchesSignalisation,Biolo-
S. Hershey Medical Center, H166, 500,
gieduDeveloppementetCancer,Universite´
University Drive, Hershey, Pennsylvania
de Nice-Sophia Antipolis, UMR 6543
17033-0850
CNRS,ParcValrose,Nicecedex2,06108
LEIFCARLSSON(14),Umea˚ CentreforMole- France
cular Medicine, Umea˚ University, 901 87
MARIA P. DE MIGUEL (25), Department of
Umea˚,Sweden
Microbiology, Kimmel Cancer Center,
WEISHENG V. CHEN (26), Program in Thomas Jefferson University, Philadelphia,
Developmental Biology, Division of Basic Pennsylvania19107
ix
x CONTRIBUTORS
PETER J. DONOVAN (25), Kimmel Cancer Medicine, P.O. Box 100275, Gainesville,
Center, Thomas Jefferson University, Phil- Florida326100275
adelphia,Pennsylvania19107
SUSAN M. HAWES (30) Monash Institute of
BILLEADES(13),DepartmentofPathology& Reproduction and Development, Monash
Immunology,WashingtonUniversitySchool University, 246 Clayton Road, Clayton,
of Medicine, 660 S. Euclid Avenue, Box Victoria,3168,Australia
8118,St.Louis,Missouri63110
SHIN-ICHIHAYASHI(7),DivisionofImmunol-
KEVIN EGGAN (2), Whitehead Institute for ogy,DepartmentofMolecularandCellular
BiomedicalResearch,9CambridgeCenter, Biology,SchoolofLifeScience,Facultyof
Cambridge,Massachusetts02142 Medicine, Tottori University, 86 Nishi-
Machi,Yonago,Tottori683-8503,Japan
KOJI ETO (10), Division of Vascular Biology,
Department of Cell Biology, Scripps CLAUDIA HEGERT (18), Department of Med-
Research Institute, 10550 North Torrey icalMolecularBiology,MedicalUniversity
Pines Road, VB-5, La Jolla, California ofLu¨beck,RatzeburgerAllee160,Lu¨beck,
92037 D-23538,Germany
PAUL J. FAIRCHILD (12), Sir William Dunn IRIT HUBER (32), Cardiovascular Research
SchoolofPathology,UniversityofOxford, Laboratory,Departmentof Biophysicsand
SouthParksRoad,Oxford,OX13RE,UK Physiology, The Bruce Rappaport Faculty
of Medicine, Technion-Israel Institute of
OLIVIERFERAUD(15),LaboratoireDeveloppe- Technology, 2 Efron Street, POB 9649,
ment et Vieillissement de L’endothelium,
Haifa31096,Israel
EMI INSERM 0219, DRDC/DVE, CEA
Grenoble, 17 rue des martyrs, 38054 KATSUHIKO ISHIZAKI (24), Department of
Grenoblecedex09,France Tissue and Organ Development, Regenera-
tion and Advanced Medical Science, Gifu
ADAMA.FILIPCZYK(30),MonashInstituteof University Graduate School of Medicine,
Reproduction and Development, Monash
Gifu500-8705,Japan
University, 246 Clayton Road, Clayton,
Victoria,3168,Australia JOSEPH ITSKOVITZ-ELDOR (32), Department
of Physiology and Biophysics, Bruce
STUART T. FRASER (4), Laboratory of Rappaport Faculty of Medicine, Technion-
Molecular Mouse Genetics, Institute for
Israel Institute of Technology, 2 Efron
Toxicology, Johannes Gutenberg-Univer-
Street,POB9649,Haifa31096,Israel
sity, Obere Zahlbacher Strasse 67, Mainz
55131,Germany RUDOLFJAENISCH(2),WhiteheadInstitutefor
BiomedicalResearch,9CambridgeCenter,
LIORGEPSTEIN(32),CardiovascularResearch Cambridge,Massachusetts02142
Laboratory, Bruce Rappaport Faculty of
Medicine,Technion-IsraelInstituteofTech- L. MARTIN JAKT (4), RIKEN Centre for
nology, 2 Efron Street, POB 9649, Haifa Developmental Biology, 2-2-3 Minatojima-
31096,Israel minamimachi, Chuo-ku, Kobe, Japan
650-0047
AMIRA GEPSTEIN (32), Cardiovascular
Research Laboratory, Bruce Rappaport GABRIELAKANIA(21),InVitroDifferentiation
FacultyofMedicine,Technion-IsraelInsti- Group,InstituteofPlantGeneticsandCrop
tute of Technology, 2 Efron Street, POB PlantResearch,Corrensstr.3,Gatersleben,
9649,Haifa31096,Israel D-06466,Germany
TAKASHI HAMAZAKI (20), Department of JOSEPHB.KEARNEY(6),PrograminGenetics
Pathology University ofFlorida Collegeof andMolecularBiology,UniversityofNorth
CONTRIBUTORS xi
Carolina at Chapel Hill CB#3280, Chapel University, 246 Clayton Road, Clayton,
Hill,NorthCarolina27599 Victoria,3168,Australia
IZHAK KEHAT (32), Cardiovascular Research ANDREW L. LEAVITT (10), Departments of
Laboratory, Bruce Rappaport Faculty of Laboratory Medicine and Internal Medi-
Medicine,Technion-IsraelInstituteofTech- cine,UniversityofCaliforniaatSanFran-
nology, 2 Efron Street, POB 9649 Haifa cisco,SanFrancisco,California94143
31096,Israel
JONATHAN G. LIEBER (9), Department of
GORDON M. KELLER (3,9), Carl C. Icahn Medicine, National Jewish Medical and
Center for Gene Therapy and Molecular
Research Center, 1400 Jackson Street,
Medicine,MountSinaiSchoolofMedicine,
Denver,Colorado80206
1425MadisonAvenue,Box1496,NewYork,
NewYork10029-6574 TERRY MAGNUSON (28), Department of
Genetics, University of North Carolina at
MARIONKENNEDY(3),CarlC.IcahnInstitute
Chapel Hill, Chapel Hill, North Carolina
forGeneTherapyandMolecularMedicine,
27599
Mount Sinai School of Medicine, 1425
Madison Avenue, Box 1496, New York, RON MCKAY (22), Laboratory of Molecular
NewYork10029-6574 Biology, NINDS, National Institutes of
Health,Bethesda,Maryland20892-4092
JONG-HOON KIM (22), Laboratory of Mole-
cular Biology, NINDS, National Institutes
TSUTOMU MOTOHASHI (24), Department of
ofHealth,Bethesda,Maryland20892-4092
Tissue and Organ Development, Regenera-
KENJIKITAJIMA(5),DepartmentofMolecular tion and Advanced Medical Science, Gifu
Cell Biology, Research Institute for University Graduate School of Medicine,
Microbial Diseases, Osaka University, 3-1 Gifu500-8705,Japan
Yamadaoka,SuitaOsaka,Osaka565-0871,
TORU NAKANO (5,10), Department of Mole-
Japan
cular Cell Biology, Research Institute for
RAJAKITTAPPA(22),LaboratoryofMolecular Microbial Diseases, Osaka University, 3-1
Biology, NINDS, National Institutes of Yamadaoka,SuitaOsaka,Osaka565-0871,
Health,Bethesda,Maryland20892-4092 Japan
A˚ SAKOLTERUD(14),Umea˚CentreforMole- NORIONAKATSUJI(29),DepartmentofDevel-
cular Medicine, Umea˚ University, 901 87 opment and Differentiation, Institute for
Umea˚,Sweden Frontier Medical Sciences, Kyoto Univer-
sity,Sakyo-ku,Kyoto,606-8507,Japan
JAN KRAMER (18), Department of Internal
Medicine I, University of Lu¨beck, Lu¨beck, ANNE NAVARRETE-SANTOS (21), Department
D-23538,Germany of Anatomy and Cell Biology, Martin
LutherUniversity,Halle-Wittenberg,Halle
TAKAHIRO KUNISADA (24), Department of
(Saale),D-06108,Germany
Tissue and Organ Development, Regenera-
tion and Advanced Medical Science, Gifu
SATOMI NISHIKAWA (4), RIKEN Centre for
University Graduate School of Medicine,
Developmental Biology, 2-2-3 Minatojima-
Gifu500-8705,Japan
minamimachi, Chuo-ku, Kobe 650-0047,
MICHAELKYBA(8),CenterforDevelopmental Japan
Biology,UTSouthwesternMedicalCenter,
Dallas,Texas75390-9133 SHIN-ICHINISHIKAWA(4),RIKENCentrefor
Developmental Biology, 2-2-3 Minatojima-
ANDREWL.LASLETT(30),MonashInstituteof minamimachi, Chuo-ku, Kobe, Japan
Reproduction and Development, Monash 650-0047
xii CONTRIBUTORS
KATHLEENF.NOLAN(12),SirWilliamDunn University of Lu¨beck, Ratzeburger Allee
SchoolofPathology,UniversityofOxford, 160,Lu¨beck,D-23538,Germany
SouthParksRoad,Oxford,OX13RE,UK
EIKOSAKAI-OGAWA(5),DepartmentofMole-
MINETARO OGAWA (4), Department of Cell cular Cell Biology, Research Institute for
Differentiation, Institute of Molecular Microbial Diseases, Osaka University, 3-1
EmbryologyandGenetics,KumamotoUni- Yamadaoka,SuitaOsaka,Osaka565-0871,
versity,Japan Japan
MITSUHIRO OKADA (4), Department of Cell MAYA SCHULDINER (31), Department of
Differentitation, Institute of Molecular Genetics, The Silberman Institute of Life
EmbryologyandGenetics,KumamotoUni- Sciences, The Hebrew University, Jerusa-
versity,Kumamoto,Japan650-0047 lem91904,Israel
HIROMI OKUYAMA (7), Biotechnology SANFORDJ.SHATTIL(10),DivisionofVascular
Research Laboratories, Takara Bio Inc., Biology, Department of Cell Biology, The
Otsu,Siga520-2193,Japan Scripps Research Institute, 10550 North
Torrey Pines Road, VB-5, La Jolla,
DAVIDPANCHISION(22),LaboratoryofMole- California92037
cular Biology, NINDS, National Institutes
ofHealth,Bethesda,Maryland20892-4092 AUSTING.SMITH(23),InstituteforStemCell
Research, University of Edinburgh, King’s
MARTIN F. PERA (30), Monash Institute of Buildings, West Mains Road, Edinburgh,
Reproduction and Development, Monash ScotlandEH93JQ,UK
University, 246 Clayton Road, Clayton,
Victoria3168,Australia PHILIPPE SORIANO (26), Program in Develop-
mentalBiology,DivisionofBasicSciences,
RITAC.R.PERLINGEIRO(8),ViaCellInc,26 Fred Hutchinson Cancer Research Center,
LandsdowneSt,Cambridge,MA2139 1100FairviewAvenueN,A2-025,P.O.Box
PERPE´TUA PINTO DO O´ (14), Umea˚ Centre 19024,Seattle,Washington98109-1024
for Molecular Medicine, Umea˚ University, HEIDI STUHLMANN (27), Department of Cell
90187Umea˚,Sweden Biology, Division of Vascular Biology,
Scripps Research Institute, Mail CVN-26,
MARIE-HE´LE`NE PRANDINI (15), Laboratoire
10550NorthTorreyPinesRoad,LaJolla,
Developpement et Vieillissement de L’en-
California92037
dothelium, EMI INSERM 0219, DRDC/
DVE, CEA Grenoble, 17 rue des martyrs, HIROFUMISUEMORI(29),StemCellResearch
38054Grenoblecedex09,France Center, Institute for Frontier Medical
Sciences, Kyoto University, Sakyo-ku,
JOY RATHJEN (1), Department of Molecular
Kyoto,606-8507,Japan
Biosciences and ARC Special Research
CentrefortheMolecularGeneticsofDeve- MAKOTO TANAKA (5), Department of Mole-
lopment, Adelaide University, Molecular cular Cell Biology, Research Institute for
LifeSciences/335,Adelaide,5005,Australia Microbial Diseases, Osaka University, 3-1
Yamadaoka,SuitaOsaka,Osaka565-0871
PETERD.RATHJEN(1),DepartmentofMole-
Japan
cular Biosciences and ARC Special
ResearchCentrefortheMolecularGenetics NAOHIRO TERADA (20), Department of
ofDevelopment,AdelaideUniversity,Mole- Pathology, University of Florida College
cular Life Sciences/335, Adelaide, 5005, ofMedicine,P.O.Box100275,Gainesville,
Australia Florida32610-0275
JU¨RGEN ROHWEDEL (18). Department of MOTOKAZU TSUNETO (7), Division of Immu-
Medical Molecular Biology, Medical nology, Department of Molecular and