Zebrafish A Practical Approach

by Nusslein-Volhard, Christiane; Dahm, Ralf

• ISBN13: 9780199638093
• ISBN10: 0199638098
• Format: Hardcover
• 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	xiii
Abbreviations	xvii
Introduction: zebrafish as a system to study development and organogenesis	1	(212)
Christiane Nusslein-Volhard
Darren T. Gilmour
Ralf Dahm
History	1	(1)
Phylogeny	2	(1)
The zebrafish genome	2	(2)
Advantages of the zebrafish as a model system	4	(1)
Purpose of this book	4	(3)
Acknowledgements	5	(1)
References	5	(2)
Keeping and raising zebrafish	7	(32)
Michael Brand
Michael Granato
Christiane Nusslein-Volhard
Introduction	7	(1)
Aquaria systems and water conditions	7	(5)
Aquaria systems	7	(3)
Aquaria	10	(2)
Water	12	(3)
Fresh fish water	12	(2)
Filtering	14	(1)
Room conditions and maintenance	15	(3)
Room conditions	15	(1)
Temperature	16	(1)
Illumination	16	(1)
Cleaning	16	(2)
Safety	18	(1)
Fish care	18	(10)
Keeping adult fish	18	(1)
Raising fish	19	(9)
Maintaining stocks	28	(6)
Maintaining wild-type stocks	28	(1)
Maintaining mutant stocks	28	(1)
Identifying and maintaining adult heterozygous mutant carriers	29	(1)
Maintaining mutant stocks as frozen sperm	30	(4)
Fish health	34	(5)
Fish diseases	34	(1)
Fish handling in systems with latent TB	35	(1)
Quarantine	36	(1)
References	37	(2)
Looking at embryos	39	(20)
Stefan Schulte-Merker
Introduction	39	(1)
In situ hybridization	39	(6)
Single probe	40	(3)
Detecting two differently labelled probes in situ	43	(2)
Antibody staining	45	(6)
Detecting antigens in early embryos using biotinylated secondary antibodies	45	(2)
Antibody staining of zebrafish larvae	47	(1)
Double in situ hybridization combined with antibody staining (triple stain)	48	(2)
Pre-absorption of antibodies	50	(1)
Mounting	51	(2)
Viewing chambers for observing embryos	51	(1)
Methyl cellulose mounting	51	(1)
Araldite mounting	52	(1)
Mounting in benzyl benzoate/benzyl alcohol	53	(1)
Preparing sections	53	(4)
Plastic sections using Technovit	54	(1)
Paraffin sections combined with antibody staining	54	(1)
Embedding and sectioning using JB-4 resin	55	(1)
Toluidine Blue staining of semi-thin sections	56	(1)
PTU treatment to prevent melanization of embryos	57	(1)
Endothelial cell staining	57	(2)
Acknowledgements	58	(1)
References	58	(1)
The morphology of larval and adult zebrafish	59	(36)
Thomas F. Schilling
Introduction	59	(1)
Body proportions and surface features	59	(6)
Adult	59	(2)
Larvae and juveniles	61	(4)
Skeleton and musculature	65	(11)
Adult skeleton	65	(4)
Larval and juvenile skeletal development	69	(4)
Adult musculature	73	(1)
Larval and juvenile muscle development	74	(2)
Nervous system	76	(11)
Adult central nervous system	76	(3)
Larval and juvenile neural development	79	(4)
Adult peripheral nervous system and sense organs	83	(3)
Larval and juvenile development of the PNS and sense organs	86	(1)
Cardiovascular, digestive, and reproductive organs	87	(8)
Adult	87	(4)
Larval and juvenile organogenesis	91	(1)
Acknowledgements	92	(1)
References	92	(3)
Cell labelling and transplantation techniques	95	(26)
Donald A. Kane
Yasuyuki Kishimoto
Introduction	95	(1)
Cell labelling techniques	96	(7)
Labelling whole embryos with lineage tracer dyes	97	(4)
Labelling individual blastomeres after 256-cell stage	101	(1)
Labelling groups of cells by photo-activation	102	(1)
Labelling groups of cells by lipophilic membrane dyes	103	(1)
Transplantation techniques	103	(7)
Precision transplantation of small groups of cells	104	(4)
Large-scale transplantations of early blastomeres	108	(1)
Transplantation of pieces of embryos	109	(1)
Procedures for observing labelled cells	110	(11)
Procedures for observing live material	111	(3)
Procedures for preparing fixed material	114	(3)
Procedures for analysing data	117	(2)
References	119	(2)
Manipulating gene expression in the zebrafish	121	(24)
Darren T. Gilmour
Jason R. Jessen
Shuo Lin
Introduction	121	(1)
Microinjection of zebrafish embryos	121	(7)
Equipment required	122	(3)
The microinjection procedure	125	(3)
Transient expression approaches	128	(9)
DNA constructs and mRNA transcripts give different expression profiles upon injection	129	(1)
RNA injection	129	(2)
DNA microinjection and its applications	131	(1)
Promoter analysis	132	(1)
Co-injection	133	(1)
Rescue of mutants using transgenesis	134	(1)
Artificial chromosome transgenesis in zebrafish	134	(3)
Generation of stable germ line transgenic lines	137	(4)
Identifying transgenic founder fish by PCR	138	(1)
Identification of transgenic founder fish by reporter gene expression	139	(1)
Generation of homozygous transgenic fish	140	(1)
Morpholino `knockdown' of gene activity	141	(4)
References	143	(2)
Mutagenesis	145	(30)
Francisco Pelegri
Introduction	145	(1)
Mutagenic agents	145	(7)
Chemical mutagens: N-ethyl-N-nitrosourea	145	(4)
Radiation sources	149	(1)
Insertional mutagenesis	150	(2)
Selection of background genetic lines	152	(1)
General criteria for selecting genetic backgrounds	152	(1368)
Selection of lines amenable for in vitro fertilization and parthenogenesis	152	(1)
Selection of lines free of lethal or sterile mutations	153	(1)
Genetic screening strategies	153	(19)
Inbreeding of families carrying non-mosaic germ lines	153	(3)
Screens involving ploidy manipulation	156	(11)
Screens for recessive mutations affecting adult traits and maternal-effect mutations	167	(1)
Screens for dominant mutations	167	(1)
Allele screens	167	(5)
Screen tests	172	(1)
Morphological screens	172	(1)
Screens using molecular markers and tissue stains	172	(1)
Locomotion and behavioural screens	172	(1)
Conclusions	172	(3)
Acknowledgements	173	(1)
References	173	(2)
Mapping and cloning	175	(38)
Robert Geisler
Maps of the zebrafish genome	175	(2)
Mapping mutations using SSLP markers	177	(15)
Mapping approaches	177	(1)
Selection of zebrafish lines and SSLPs	178	(2)
Mapping strategy	180	(3)
Set-up for high-throughput mapping	183	(2)
Evaluating pooled PCR gels	185	(1)
Evaluating single-embryo PCR gels	186	(3)
Calculation of a map position	189	(3)
Fine mapping	192	(1)
Radiation hybrid mapping of candidate genes	192	(3)
Matching mutations with candidate genes	195	(4)
Searching maps and databases	195	(1)
Evidence for the identity of a mutant locus	196	(1)
Demonstrating a direct linkage by SNP genotyping	196	(3)
Positional cloning of mutations	199	(14)
Genomic libraries	199	(2)
Screening for genomic clones	201	(6)
Analysing a genomic clone	207	(3)
Acknowledgements	210	(1)
References	210	(3)
Appendix 1 List of suppliers	213	(6)
Appendix 2 Atlas of embryonic stages of development in the zebrafish	219	(18)
Ralf Dahm
Appendix 3 Table of zebrafish mutations	237	(56)
Hans Georg Frohnhofer
Index	293