Methods in
Molecular Biology 1467
Nicholas Hastie Editor
The Wilms’
Tumor (WT1)
Gene
Methods and Protocols
,METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
,
,The Wilms’ Tumor (WT1) Gene
Methods and Protocols
Edited by
Nicholas Hastie
MRC Human Genetics Unit, University of Edinburgh, Edinburgh, UK
,Editor
Nicholas Hastie
MRC Human Genetics Unit
University of Edinburgh
Edinburgh, UK
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-4021-9 ISBN 978-1-4939-4023-3 (eBook)
DOI 10.1007/978-1-4939-4023-3
Library of Congress Control Number: 2016943848
© Springer Science+Business Media New York 2016
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Cover illustration: Section of the kidney of an adult Wt1-GFP knock-in mouse (Hosen et al., Leukemia 21:1783–1791,
2007) showing green fluorescence in the Wt1-expressing podocytes of the glomeruli. Label was enhanced with an anti-
GFP antibody and an Alexa480-conjugated secondary antibody. Endothelial cells are labelled in red with an anti-Pecam1
antibody and a Cy5-conjugated secondary antibody. Nuclei are counterstained with DAPI. Author: Dr. Rita Carmona,
University of Málaga (Spain).
Printed on acid-free paper
This Humana Press imprint is published by Springer Nature
The registered company is Springer Science+Business Media LLC New York
,Preface
I was delighted when asked by John Walker whether I would be interested in editing a
volume on the Wilms’ tumor gene (WT1), for the distinguished Methods in Molecular
Biology Series. However my first thought was to question whether a single gene would
cover sufficient ground for a complete volume. I was heartened by the fact that similar suc-
cessful volumes in the series had been compiled on other genes, notably p53. Also on fur-
ther consideration I realized WT1 would be ideal. It is a multifunctional protein, mutations
in which may lead to a variety of disorders in humans, including the eponymous pediatric
kidney cancer, leukemia, gonadal dysgenesis, and occasionally diaphragmatic hernia and
heart disease. WT1 is a key regulator of the development of a number of tissues, particularly
those involving switches between mesenchymal-epithelial switches; in addition to the kid-
ney, gonads, and heart it has been shown to mark and regulate stem/progenitors for vis-
ceral fat. Beyond its role in development, WT1 has been shown to be required for the
homeostasis of a number of adult tissues and to be activated in tissue repair. WT1 is a zinc
finger protein that clearly in part regulates all these cellular processes by functioning as a
transcription factor. However an increasing body of evidence suggests that WT1 also regu-
lates post-transcriptional processes by binding to RNA. What is more, two major WT1
splice isoforms differing by only three amino acids appear to have different relative roles in
transcription and post-transcriptional processes. Finally, as WT1 is expressed in a number of
adult epithelial tumors but not the healthy tissue counterparts immune cancer therapies
against the protein are being trialed. All these facets ensure that WT1 is a rich source for
methods chapters. The volume starts with three review chapters to set the scene. These
cover the involvement of WT1 in pediatric cancer, kidney disease, and tissue development
and homeostasis. These are followed by methods chapters, firstly on tools for studying
developmental and cellular processes. These include chapters on cell marking and lineage
tracing, epicardial cell methodology, colony forming assays for bone marrow stem cells,
isolation of adipocyte progenitors using Fluorescence Activated Cell Sorting, methods for
studying angiogenesis, and multiphoton imaging of lipids. All these chapters deal exclu-
sively with mice and mouse tissues/cells. Zebrafish provide another valuable organism for
studying Wt1 biology and function, so there is an overview of Wt1 in zebrafish followed by
two valuable methods chapters on immunohistochemistry in zebrafish and isolation of kid-
ney podocytes. The remaining methods chapters cover some of the latest tools in Genomics,
Molecular Biology, and Biochemistry. These begin with methods for dissecting transcrip-
tion factor function in cell-free systems and for measuring the binding constants of protein-
nucleic acid interaction. These are followed by chapters on ChIP-Seq to identify
transcriptional targets and methods for identifying WT1-interacting RNA and proteins.
The final methods chapter describes bioinformatic approaches for analyzing Next Generation
Sequence data. To round the volume off there is a chapter on Cancer Immune Therapy
v
,vi Preface
based on antibodies to WT1. This is a combination of overview and some methodological
detail. All the chapters are by experts in the field and I was delighted when everyone
approached agreed to write a chapter. I thank the authors and editors and do hope the read-
ers find this volume helpful.
Edinburgh, UK Nicholas Hastie
,Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 WT1 Mutation in Childhood Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Jocelyn Charlton and Kathy Pritchard-Jones
2 Clinical Aspects of WT1 and the Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Eve Miller-Hodges
3 The Role of WT1 in Embryonic Development and Normal
Organ Homeostasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Bettina Wilm and Ramon Muñoz-Chapuli
4 Tools and Techniques for Wt1-Based Lineage Tracing. . . . . . . . . . . . . . . . . . . 41
Bettina Wilm and Ramon Muñoz-Chapuli
5 Biological Systems and Methods for Studying WT1 in the Epicardium . . . . . . 61
Víctor Velecela, Janat Fazal-Salom, and Ofelia M. Martínez-Estrada
6 Isolation and Colony Formation of Murine Bone and Bone Marrow Cells. . . . 73
Sophie McHaffie and You-Ying Chau
7 Isolation and Fluorescence-Activated Cell Sorting
of Murine WT1-Expressing Adipocyte Precursor Cells. . . . . . . . . . . . . . . . . . . 81
Louise Cleal and You-Ying Chau
8 In Vivo Assays for Assessing the Role of the Wilms’ Tumor
Suppressor 1 (Wt1) in Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Richard J. McGregor, R. Ogley, PWF Hadoke, and Nicholas Hastie
9 Multiphoton Microscopy for Visualizing Lipids in Tissue . . . . . . . . . . . . . . . . 105
Martin Lee and Alan Serrels
10 Function and Regulation of the Wilms’ Tumor Suppressor 1 (WT1)
Gene in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Birgit Perner, Thomas J. D. Bates, Uta Naumann, and Christoph Englert
11 Immunofluorescence Staining of WT1 on Sections of Zebrafish Embryos
and Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Birgit Perner and Christoph Englert
12 Fluorescence-Activated Cell Sorting (FACS) Protocol for Podocyte
Isolation in Adult Zebrafish. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Thomas J.D. Bates, Uta Naumann, and Christoph Englert
13 In Vitro Transcription to Study WT1 Function . . . . . . . . . . . . . . . . . . . . . . . . 137
Stefan G.E. Roberts
14 Measuring Equilibrium Binding Constants for the WT1-DNA Interaction
Using a Filter Binding Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Paul J. Romaniuk
vii
,viii Contents
15 Identifying Direct Downstream Targets: WT1 ChIP-Seq Analysis . . . . . . . . . . 177
Fabio da Silva, Filippo Massa, and Andreas Schedl
16 WT1 Associated Protein-Protein Interaction Networks . . . . . . . . . . . . . . . . . . 189
Ruthrothaselvi Bharathavikru and Alex von Kriegsheim
17 Methods to Identify and Validate WT1–RNA Interaction . . . . . . . . . . . . . . . . 197
Ruthrothaselvi Bharathavikru and Tatiana Dudnakova
18 Bioinformatic Analysis of Next-Generation Sequencing Data
to Identify WT1-Associated Differential Gene and Isoform Expression . . . . . . 211
Stuart Aitken and Ruthrothaselvi Bharathavikru
19 Immunotherapy Targeting WT1: Designing a Protocol
for WT1 Peptide-Based Cancer Vaccine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Sumiyuki Nishida and Haruo Sugiyama
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
, Contributors
STUART AITKEN • Medical Research Council—Human Genetics Unit, Institute of Genetics
and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh,
Scotland, UK
THOMAS J.D. BATES • Leibniz Institute for Age—Fritz Lipmann Institute, Jena, Germany
RUTHROTHASELVI BHARATHAVIKRU • Medical Research Council-Human Genetics Unit,
Institute of Genetics and Molecular Medicine, Western General Hospital, University
of Edinburgh, Edinburgh, Scotland, UK
JOCELYN CHARLTON • UCL Institute of Child Health, London, UK
YOU-YING CHAU • MRC Human Genetics Unit, MRC Institute of Genetics and Molecular
Medicine, University of Edinburgh, Edinburgh, UK; British Heart Foundation Centre
for Cardiovascular Science, The Queen’s Medical Research Institute, University
of Edinburgh, Edinburgh, UK
LOUISE CLEAL • MRC Human Genetics Unit, MRC Institute of Genetics and Molecular
Medicine, University of Edinburgh, Edinburgh, UK
TATIANA DUDNAKOVA • Wellcome Trust Centre for Cell Biology, The University of Edinburgh,
Edinburgh, UK
CHRISTOPH ENGLERT • Leibniz Institute for Age—Fritz Lipmann Institute, Jena, Germany;
Friedrich Schiller University, Jena, Germany
JANAT FAZAL-SALOM • CELLTEC-UB, Cellular Biology Department, University
of Barcelona, Barcelona, Spain
PWF HADOKE • University/BHF Centre for Cardiovascular Science, The Queen’s Medical
Research Institute, University of Edinburgh, Edinburgh, Scotland, UK
NICHOLAS HASTIE • MRC Human Genetics Unit, Institute of Genetics and Molecular
Medicine, Western General Hospital, University of Edinburgh, Edinburgh, Scotland, UK
ALEX VON KRIEGSHEIM • Systems Biology Ireland, Conway Institute, Belfield, Ireland
MARTIN LEE • Institute of Genetics and Molecular Medicine, University of Edinburgh,
Edinburgh, Scotland, UK
OFELIA M. MARTÍNEZ-ESTRADA • CELLTEC-UB, Cellular Biology Department, University
of Barcelona, Barcelona, Spain
FILIPPO MASSA • Institute of Biology Valrose, Université de Nice-Sophia, Nice, Cedex 2,
France; Inserm, UMR1091, Nice, France; CNRS, UMR7277, Nice, France
RICHARD J. MCGREGOR • University/BHF Centre for Cardiovascular Science, The Queen’s
Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, UK; MRC
Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General
Hospital, University of Edinburgh, Edinburgh, Scotland, UK
SOPHIE MCHAFFIE • MRC Human Genetics Unit, MRC Institute of Genetics and Molecular
Medicine, University of Edinburgh, Edinburgh, Scotland, UK
EVE MILLER-HODGES • ECAT Clinical Lecturer—Nephrology, IGMM Human Genetics
Unit, Western General Hospital, University of Edinburgh, Edinburgh, Scotland, UK
RAMON MUÑOZ-CHAPULI • Department of Animal Biology, University of Malaga,
Malaga, Spain
ix
