M ETHODS IN MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Xenopus Protocols

Post-Genomic Approaches

Second Edition

Edited by Stefan Hoppler

Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK Peter D. Vize

Department of Biological Science, University of Calgary, Calgary, Canada Editors Stefan Hoppler Peter D. Vize Institute of Medical Sciences Department of Biological Science University of Aberdeen University of Calgary Foresterhill, Aberdeen, UK Calgary, Canada

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-61779-991-4 ISBN 978-1-61779-992-1 (eBook) DOI 10.1007/978-1-61779-992-1 Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2012943830

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Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Preface

The Xenopus system had a long and prominent history during the twentieth century as an accessible model system for studying vertebrate biology. Many discoveries in biochemistry, cell biology, and in particular in developmental biology have their roots in experimental investigation using Xenopus . Among the inherent advantages of the Xenopus model are the ease with which large numbers of eggs can be collected; the large size of those eggs and the embryos that develop from them after fertilization; a consistent fate map; the external development of these embryos and the speed with which the conserved vertebrate body plan is organized. The large cells in the early embryos were ideal for addressing cell biologi- cal questions but also allowed early embryonic patterning of the vertebrate embryo and the fate map to be unraveled. Many fundamental processes in vertebrate development were fi rst discovered in Xenopus in areas such as axial development, mesoderm induction, neural pat- terning, and more recently organogenesis and regeneration, to name but a few. The absence of well-developed genetics has often been cited as the one disadvantage of the Xenopus laevis model system compared to mouse or zebrafi sh for instance, but as this volume shows very clearly this criticism has been thoroughly put to rest. This volume of Methods in Molecular Biology on Xenopus comes at a crucial juncture for our model system. The recent publication of the draft genome for Xenopus tropicalis marks an important milestone. It reveals that the Xenopus genome is in itself an ideal model for vertebrate genomes since it manifests conserved vertebrate genomic organization and therefore reinforces the uniquely advantageous phylogenetic position of Xenopus as a gen- eral vertebrate model. The genome and the embrace of genomic approaches enabled by X. tropicalis as an experimental model have invigorated the entire Xenopus fi eld, not just those working with X. tropicalis . This volume seeks to focus on these new approaches. X. tropicalis offers tractable genetics that will complement the traditional strengths of Xenopus as a model system, as do the powerful transgenics methods pioneered in Xenopus . The genome and new deep sequencing approaches open up a new chapter in the analysis of gene expression on a whole transcriptome level and of protein expression and interaction on a proteome level; here in particular the traditional advantages of the Xenopus model system synergize very directly with the new information technology available to enable completely novel approaches. However, this impact is also felt in new methodology to image these expressed transcripts and proteins in embryos and in cells, particularly with sophisticated fl uorescence microscopy; and of course in new systems approaches and appli- cations for stem cell technology and regeneration. As a consequence, the computer and online databases will become as much a tool for Xenopus researchers in the future as the micropipette, the injection needle, and the dissection scalpel have been for many years.

v vi Preface

The new approaches inspired by the X. tropicalis genome combined with the unique experimental advantages of X. laevis and their distinctive phylogenetic position make them unique model systems for vertebrate embryonic development in the twenty-fi rst century. The protocols in this volume written by the leading experts in each method provide a tool- kit that will enable every laboratory to maximize the power of this extraordinary experi- mental system.

Aberdeen, UK Stefan Hoppler Calgary, Canada Peter D. Vize Contents

Preface...... v Contributors...... xi

PART I XENOPUS TROPICALIS

1 Xenopus tropicalis as a Model Organism for Genetics and Genomics: Past, Present, and Future...... 3 Robert M. Grainger 2 Husbandry of Xenopus tropicalis ...... 17 Alan Jafkins, Anita Abu-Daya, Anna Noble, Lyle B. Zimmerman, and Matthew Guille 3 Generating Diploid Embryos from Xenopus tropicalis ...... 33 Florencia del Viso and Mustafa Khokha 4 Navigating the Xenopus tropicalis Genome ...... 43 Ira L. Blitz

PART II GENETICS IN XENOPUS

5 Genetic Analysis of Xenopus tropicalis ...... 69 Timothy J. Geach, Derek L. Stemple, and Lyle B. Zimmerman 6 Forward Genetic Screens in Xenopus Using Transposon-Mediated Insertional Mutagenesis ...... 111 Donald A. Yergeau, Clair M. Kelley, Haiqing Zhu, Emin Kuliyev, and Paul E. Mead 7 Targeted Gene Disruption with Engineered Zinc-Finger Nucleases (ZFNs) ...... 129 John J. Young and Richard M. Harland 8 Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides . . . . . 143 Yanan Zhao, Shoko Ishibashi, and Enrique Amaya 9 Chemical Genetics and Drug Discovery in Xenopus ...... 155 Matthew L. Tomlinson, Adam E. Hendry, and Grant N. Wheeler 10 Maternal mRNA Knock-down Studies: Antisense Experiments Using the Host-Transfer Technique in Xenopus laevis and Xenopus tropicalis ...... 167 David J. Olson, Alissa M. Hulstrand, and Douglas W. Houston

vii viii Contents

PART III XENOPUS TRANGENESIS

11 Generating Transgenic Frog Embryos by Restriction Enzyme Mediated Integration (REMI)...... 185 Shoko Ishibashi, Kristen L. Kroll, and Enrique Amaya 12 A Simple Method of Transgenesis using I-SceI Meganuclease in Xenopus ...... 205 Shoko Ishibashi, Nick R. Love, and Enrique Amaya 13 Using FC31 Integrase to Mediate Insertion of DNA in Xenopus Embryos ...... 219 You E. Li, Bryan G. Allen, and Daniel L. Weeks 14 Xenopus Transgenics: Methods Using Transposons ...... 231 Clair M. Kelley, Donald A. Yergeau, Haiqing Zhu, Emin Kuliyev, and Paul E. Mead 15 Comparative Genomics-Based Identification and Analysis of Cis-Regulatory Elements...... 245 Hajime Ogino, Haruki Ochi, Chihiro Uchiyama, Sarah Louie, and Robert M. Grainger 16 Tet-On Binary Systems for Tissue-Specific and Inducible Transgene Expression ...... 265 Daniel R. Buchholz

PART IV GENE EXPRESSION: FROM GENE TO TRANSCRIPT TO PROTEIN

17 Chromatin Immunoprecipitation Analysis of Xenopus Embryos ...... 279 Robert C. Akkers, Ulrike G. Jacobi, and Gert Jan C. Veenstra 18 Transcriptomics Using Next Generation Sequencing Technologies ...... 293 Dasfne Lee-Liu, Leonardo I. Almonacid, Fernando Faunes, Francisco Melo, and Juan Larrain 19 Databases of Gene Expression in Xenopus Development ...... 319 Michael J. Gilchrist and Nicolas Pollet 20 Investigating Alternative RNA Splicing in Xenopus ...... 347 Agnès Mereau and Serge Hardy 21 Immunoisolation of Protein Complexes from Xenopus...... 369 Frank L. Conlon, Yana Miteva, Erin Kaltenbrun, Lauren Waldron, Todd M. Greco, and Ileana M. Cristea 22 Complementary Proteomic Analysis of Protein Complexes ...... 391 Todd M. Greco, Yana Miteva, Frank L. Conlon, and Ileana M. Cristea

PART V IMAGING XENOPUS DEVELOPMENT

23 Antibody Development and Use in Chromogenic and Fluorescent Immunostaining ...... 411 Eamon Dubaissi, Niki Panagiotaki, Nancy Papalopulu, and Peter D. Vize Contents ix

24 Multiple Fluorescent In Situ mRNA Hybridization (FISH) on Whole Mounts and Sections...... 431 Robert Lea, Boyan Bonev, Eamon Dubaissi, Peter D. Vize, and Nancy Papalopulu 25 Methods to Analyze microRNA Expression and Function During Xenopus Development...... 445 Boyan Bonev and Nancy Papalopulu 26 A Bromodeoxyuridine (BrdU) Based Protocol for Characterizing Proliferating Progenitors in Xenopus Embryos...... 461 Hélène Auger, Raphaël Thuret, Warif El Yakoubi, and Nancy Papalopulu 27 Microscopy Tools for Quantifying Developmental Dynamics in Xenopus Embryos ...... 477 Sagar D. Joshi, Hye Young Kim, and Lance A. Davidson

PART VI NOVEL APPROACHES IN XENOPUS

28 Mathematical Modeling of Gene Regulatory Networks in Xenopus Development...... 497 Yasushi Saka 29 Stem-Cell-Like Embryonic Explants to Study Cardiac Development ...... 515 Boni A. Afouda 30 Studying Regeneration in Xenopus ...... 525 Caroline W. Beck 31 On-Line Resources for Xenopus...... 541 Jeff Bowes

Index...... 563

Contributors

ANITA ABU-DAYA • National Institute for Medical Research, London , England, UK BONI A. AFOUDA • Institute of Medical Sciences, University of Aberdeen, Scotland , UK ; MRC Centre for Regenerative Medicine, University of Edinburgh, Scotland , UK ROBERT C. AKKERS • Department of Molecular Biology, Nijmegen Center of Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands BRYAN G. ALLEN • Department of Biochemistry, The University of Iowa, Iowa City, IA , USA LEONARDO I. ALMONACID • Molecular Bioinformatics Laboratory , Millennium Institute on Immunology and Immunotherapy, Ponti fi cia Universidad Catolica de Chile, Santiago, Chile ENRIQUE AMAYA • Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, England, UK HÉLÈNE AUGER • Faculty of Life Sciences, University of Manchester, Manchester, England, UK CAROLINE W. BECK • Department of Zoology, University of Otago, Dunedin, New Zealand IRA L. BLITZ • Department of Developmental and Cell Biology and the Developmental Biology Center, University of California, Irvine , CA , USA BOYAN BONEV • Faculty of Life Sciences, University of Manchester, Manchester, England, UK JEFF BOWES • Department of Biological Sciences, University of Calgary , Calgary , AB , Canada DANIEL R. BUCHHOLZ • Department of Biological Sciences, University of Cincinnati, Cincinnati, OH , USA FRANK L. CONLON • Department of Genetics, University of North Carolina, Chapel Hill, NC , USA ILEANA M. CRISTEA • Department of Molecular Biology , Princeton University, Princeton, NJ , USA LANCE A. DAVIDSON • Department of Bioengineering and Developmental Biology, University of Pittsburgh, Pittsburgh, PA , USA EAMON DUBAISSI • Faculty of Life Sciences, University of Manchester, Manchester, England, UK FERNANDO FAUNES • Center for Aging and Regeneration and Millennium Nucleus in Regenerative Biology, Ponti fi cia Universidad Catolica de Chile, Santiago , Chile TIMOTHY J. GEACH • National Institute for Medical Research, London, England , UK MICHAEL J. GILCHRIST • Division of Systems Biology, MRC National Institute for Medical Research, London , UK ROBERT M. GRAINGER • Department of Biology, University of Virginia, Charlottesville, VA, USA TODD M. GRECO • Department of Molecular Biology , Princeton University, Princeton, NJ , USA

xi xii Contributors

MATTHEW GUILLE • European Xenopus Resource Centre, School of Biological Sciences, University of Portsmouth, Portsmouth, England, UK SERGE HARDY • UMR 6061 CNRS-Université de Rennes 1, Rennes , France RICHARD M. HARLAND • Department of Molecular and Cell Biology , University of California, Berkeley , CA , USA ADAM E. HENDRY • School of Biological Sciences, University of East Anglia, Norwich , England, UK DOUGLAS W. HOUSTON • Department of Biology, The University of Iowa, Iowa City, IA , USA ALISSA M. HULSTRAND • Department of Biology, The University of Iowa, Iowa City, IA , USA SHOKO ISHIBASHI • The Healing Foundation Centre, The Faculty of Life Sciences, University of Manchester, England, UK ULRIKE G. JACOBI • Department of Molecular Biology, Nijmegen Center of Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands ALAN JAFKINS • European Xenopus Resource Centre, School of Biological Sciences, University of Portsmouth, England, UK SAGAR D. JOSHI • Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA , USA ERIN KALTENBRUN • Department of Genetics, University of North Carolina, Chapel Hill, NC , USA CLAIR M. KELLEY • Department of Pathology, St. Jude Children’s Research Hospital, Memphis , TN , USA MUSTAFA KHOKHA • Department of Pediatrics and Genetics, Yale University School of Medicine, New Haven, CT , USA HYE YOUNG KIM • Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA , USA KRISTEN L. KROLL • Department of Developmental Biology , Washington University School of Medicine, St Louis , MO , USA EMIN KULIYEV • Department of Pathology, St. Jude Children’s Research Hospital, Memphis , TN , USA JUAN LARRAIN • Center for Aging and Regeneration and Millennium Nucleus in Regenerative Biology, Ponti fi cia Universidad Catolica de Chile, Santiago , Chile ROBERT LEA • The Faculty of Life Sciences, University of Manchester, Manchester, England, UK DASFNE LEE-LIU • Center for Aging and Regeneration and Millennium Nucleus in Regenerative Biology, Ponti fi cia Universidad Catolica de Chile, Santiago , Chile YOU E. LI • Department of Biochemistry, The University of Iowa, Iowa City, IA , USA SARAH LOUIE • Department of Biology, University of Virginia, Charlottesville, VA, USA NICK R. LOVE • The Healing Foundation Centre, The Faculty of Life Sciences, University of Manchester, England, UK PAUL E. MEAD • Department of Pathology, St. Jude Children’s Research Hospital, Memphis , TN , USA FRANCISCO MELO • Molecular Bioinformatics Laboratory , Millennium Institute on Immunology and Immunotheraphy, Ponti fi cia Universidad Catolica de Chile, Santiago, Chile Contributors xiii

AGNÈS MEREAU • UMR 6061 CNRS-Université de Rennes 1, Rennes, France YANA MITEVA • Department of Molecular Biology , Princeton University, Princeton, NJ , USA ANNA NOBLE • European Xenopus Resource Centre, School of Biological Sciences, University of Portsmouth, Portsmouth, England, UK HARUKI OCHI • Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama, Ikoma, Nara , Japan HAJIME OGINO • Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama, Ikoma, Nara , Japan DAVID J. OLSON • Department of Biology, The University of Iowa, Iowa City, IA , USA NIKI PANAGIOTAKI • The Faculty of Life Sciences, University of Manchester, Manchester, England, UK NANCY PAPALOPULU • The Faculty of Life Sciences, University of Manchester, Manchester, England, UK NICOLAS POLLET • Institute of Systems and Synthetic Biology, Genopole, CNRS, Université d’Evry Val d’Essonne, Evry , France YASUSHI SAKA • Institute of Medical Sciences, University of Aberdeen, Scotland, UK DEREK L. STEMPLE • Wellcome Trust Sanger Genome Institute, Hinxton, England, UK RAPHAËL THURET • University of Manchester, Manchester, England, UK MATTHEW L. TOMLINSON • School of Biological Sciences, University of East Anglia, Norwich , England, UK CHIHIRO UCHIYAMA • Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama, Ikoma, Nara , Japan GERT JAN C. VEENSTRA • Department of Molecular Developmental Biology, Nijmegen Center of Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands FLORENCIA DEL VISO • Department of Pediatrics and Genetics, Yale University School of Medicine, New Haven, CT , USA PETER D. VIZE • Department of Biological Science, University of Calgary, Calgary, Canada LAUREN WALDRON • Department of Genetics, University of North Carolina, Chapel Hill, NC , USA DANIEL L. WEEKS • Department of Biochemistry, The University of Iowa, Iowa City, IA , USA GRANT N. WHEELER • School of Biological Sciences, University of East Anglia, Norwich , England, UK WARIF EL YAKOUBI • University of Manchester, Manchester, England , UK DONALD A. YERGEAU • Department of Pathology, St. Jude Children’s Research Hospital, Memphis , TN , USA JOHN J. YOUNG • Department of Molecular and Cell Biology , University of California, Berkeley , CA , USA YANAN ZHAO • The Healing Foundation Centre, The Faculty of Life Sciences, University of Manchester, England, UK HAIQING ZHU • Department of Pathology, St. Jude Children’s Research Hospital, Memphis , TN , USA LYLE B. ZIMMERMAN • National Institute for Medical Research, London , England, UK