Zebrafish A Practical Approach

by Nusslein-Volhard, Christiane; Dahm, Ralf

• ISBN13: 9780199638086
• ISBN10: 019963808X
• Edition: 1st
• Format: Paperback
• Copyright: 2002-11-21
• Publisher: Oxford University Press

Summary

The zebrafish has become one of the most important model organisms to study biologial processes in vivo. As a vertebrate that has many of the strengths of invertebrate model systems, it offers numerous advantages to researchers interested in many aspects of embryonic development, physiologyand disease. The next few years will see the completion of large scale initiatives that exploit the zebrafish as a model system for the understanding of gene function in vertebrates, including the sequencing of the genome. The zebrafish will therefore play an increasingly important role in the future ofbiomedical research. Whole genome sequencing projects, such as the human genome project, have led to the isolation of tens of thousands of genes for which the in vivo function is unknown. It is therefore likely that an increasing number of researchers will turn to organisms such as the zebrafish tounderstand the in vivo requirement for the proteins these genes encode.Recent technical advances now allow the rapid testing of in vivo function of as yet uncharacterised genes in zebrafish in large numbers and at a speed that is impossible in other systems. This book not only provides a complete set of instructions that will allow researchers to establish thezebrafish in their laboratory. It also gives a broad overview of commonly used methods and a comprehensive collection of protocols describing the most powerful techniques.

Table of Contents

List of protocols	p. xiii
Abbreviations	p. xvii
Introduction: zebrafish as a system to study development and organogenesis	p. 1
History	p. 1
Phylogeny	p. 2
The zebrafish genome	p. 2
Advantages of the zebrafish as a model system	p. 4
Purpose of this book	p. 4
Acknowledgements	p. 5
References	p. 5
Keeping and raising zebrafish	p. 7
Introduction	p. 7
Aquaria systems and water conditions	p. 7
Aquaria systems	p. 7
Aquaria	p. 10
Water	p. 12
Fresh fish water	p. 12
Filtering	p. 14
Room conditions and maintenance	p. 15
Room conditions	p. 15
Temperature	p. 16
Illumination	p. 16
Cleaning	p. 16
Safety	p. 18
Fish care	p. 18
Keeping adult fish	p. 18
Raising fish	p. 19
Maintaining stocks	p. 28
Maintaining wild-type stocks	p. 28
Maintaining mutant stocks	p. 28
Identifying and maintaining adult heterozygous mutant carriers	p. 29
Maintaining mutant stocks as frozen sperm	p. 30
Fish health	p. 34
Fish diseases	p. 34
Fish handling in systems with latent TB	p. 35
Quarantine	p. 36
References	p. 37
Looking at embryos	p. 39
Introduction	p. 39
In situ hybridization	p. 39
Single probe	p. 40
Detecting two differently labelled probes in situ	p. 43
Antibody staining	p. 45
Detecting antigens in early embryos using biotinylated secondary antibodies	p. 45
Antibody staining of zebrafish larvae	p. 47
Double in situ hybridization combined with antibody staining (triple stain)	p. 48
Pre-absorption of antibodies	p. 50
Mounting	p. 51
Viewing chambers for observing embryos	p. 51
Methyl cellulose mounting	p. 51
Araldite mounting	p. 52
Mounting in benzyl benzoate/benzyl alcohol	p. 53
Preparing sections	p. 53
Plastic sections using Technovit	p. 54
Paraffin sections combined with antibody staining	p. 54
Embedding and sectioning using JB-4 resin	p. 55
Toluidine Blue staining of semi-thin sections	p. 56
PTU treatment to prevent melanization of embryos	p. 57
Endothelial cell staining	p. 57
Acknowledgements	p. 58
References	p. 58
The morphology of larval and adult zebrafish	p. 59
Introduction	p. 59
Body proportions and surface features	p. 59
Adult	p. 59
Larvae and juveniles	p. 61
Skeleton and musculature	p. 65
Adult skeleton	p. 65
Larval and juvenile skeletal development	p. 69
Adult musculature	p. 73
Larval and juvenile muscle development	p. 74
Nervous system	p. 76
Adult central nervous system	p. 76
Larval and juvenile neural development	p. 79
Adult peripheral nervous system and sense organs	p. 83
Larval and juvenile development of the PNS and sense organs	p. 86
Cardiovascular, digestive, and reproductive organs	p. 87
Adult	p. 87
Larval and juvenile organogenesis	p. 91
Acknowledgements	p. 92
References	p. 92
Cell labelling and transplantation techniques	p. 95
Introduction	p. 95
Cell labelling techniques	p. 96
Labelling whole embryos with lineage tracer dyes	p. 97
Labelling individual blastomeres after 256-cell stage	p. 101
Labelling groups of cells by photo-activation	p. 102
Labelling groups of cells by lipophilic membrane dyes	p. 103
Transplantation techniques	p. 103
Precision transplantation of small groups of cells	p. 104
Large-scale transplantations of early blastomeres	p. 108
Transplantation of pieces of embryos	p. 109
Procedures for observing labelled cells	p. 110
Procedures for observing live material	p. 111
Procedures for preparing fixed material	p. 114
Procedures for analysing data	p. 117
References	p. 119
Manipulating gene expression in the zebrafish	p. 121
Introduction	p. 121
Microinjection of zebrafish embryos	p. 121
Equipment required	p. 122
The microinjection procedure	p. 125
Transient expression approaches	p. 128
DNA constructs and mRNA transcripts give different expression profiles upon injection	p. 129
RNA injection	p. 129
DNA microinjection and its applications	p. 131
Promoter analysis	p. 132
Co-injection	p. 133
Rescue of mutants using transgenesis	p. 134
Artificial chromosome transgenesis in zebrafish	p. 134
Generation of stable germ line transgenic lines	p. 137
Identifying transgenic founder fish by PCR	p. 138
Identification of transgenic founder fish by reporter gene expression	p. 139
Generation of homozygous transgenic fish	p. 140
Morpholino 'knockdown' of gene activity	p. 141
References	p. 143
Mutagenesis	p. 145
Introduction	p. 145
Mutagenic agents	p. 145
Chemical mutagens: N-ethyl-N-nitrosourea	p. 145
Radiation sources	p. 149
Insertional mutagenesis	p. 150
Selection of background genetic lines	p. 152
General criteria for selecting genetic backgrounds	p. 152
Selection of lines amenable for in vitro fertilization and parthenogenesis	p. 1520
Selection of lines free of lethal or sterile mutations	p. 153
Genetic screening strategies	p. 153
Inbreeding of families carrying non-mosaic germ lines	p. 153
Screens involving ploidy manipulation	p. 156
Screens for recessive mutations affecting adult traits and maternal-effect mutations	p. 167
Screens for dominant mutations	p. 167
Allele screens	p. 167
Screen tests	p. 172
Morphological screens	p. 172
Screens using molecular markers and tissue stains	p. 172
Locomotion and behavioural screens	p. 172
Conclusions	p. 172
Acknowledgements	p. 173
References	p. 173
Mapping and cloning	p. 175
Maps of the zebrafish genome	p. 175
Mapping mutations using SSLP markers	p. 177
Mapping approaches	p. 177
Selection of zebrafish lines and SSLPs	p. 178
Mapping strategy	p. 180
Set-up for high-throughput mapping	p. 183
Evaluating pooled PCR gels	p. 185
Evaluating single-embryo PCR gels	p. 186
Calculation of a map position	p. 189
Fine mapping	p. 192
Radiation hybrid mapping of candidate genes	p. 192
Matching mutations with candidate genes	p. 195
Searching maps and databases	p. 195
Evidence for the identity of a mutant locus	p. 196
Demonstrating a direct linkage by SNP genotyping	p. 196
Positional cloning of mutations	p. 199
Genomic libraries	p. 199
Screening for genomic clones	p. 201
Analysing a genomic clone	p. 207
Acknowledgements	p. 210
References	p. 210
List of suppliers	p. 213
Atlas of embryonic stages of development in the zebrafish	p. 219
Table of zebrafish mutations	p. 237
Index	p. 293
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