A systematic genome-wide analysis of zebrafish protein-coding gene function

• Published in: Nature (London) Vol. 496; no. 7446; pp. 494 - 497
• Main Authors: Kettleborough, Ross N. W., Busch-Nentwich, Elisabeth M., Harvey, Steven A., Dooley, Christopher M., de Bruijn, Ewart, van Eeden, Freek, Sealy, Ian, White, Richard J., Herd, Colin, Nijman, Isaac J., Fényes, Fruzsina, Mehroke, Selina, Scahill, Catherine, Gibbons, Richard, Wali, Neha, Carruthers, Samantha, Hall, Amanda, Yen, Jennifer, Cuppen, Edwin, Stemple, Derek L.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25.04.2013; Nature Publishing Group
• Subjects: 631/208/135; Alleles; Animals; Data mining; Disease; DNA sequencing; Exome - genetics; Female; Gene Knockout Techniques; Genes; Genetic aspects; Genetic Complementation Test; Genetics; Genome - genetics; Genomes; Genomics; Humanities and Social Sciences; letter; Male; Molecular Sequence Annotation; multidisciplinary; Mutagenesis; Mutation; Mutation - genetics; Nucleotide sequencing; Phenotype; Polymorphism, Single Nucleotide - genetics; Proteins; Reagents; Science; Zebra fish; Zebrafish; Zebrafish - genetics; Zebrafish - physiology; Zebrafish Proteins - genetics; Zebrafish Proteins - metabolism; United States
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature11992

A project to identify the phenotypes of disruptive mutations in every zebrafish protein-coding gene has so far revealed potentially disruptive mutations in more than 38% of the protein-coding genes, and the phenotypic consequences of each allele can be assessed using a novel multi-allelic phenotyping scheme. The zebrafish genome The genome of the zebrafish — a key model organism for the study of development and human disease — has now been sequenced and published as a well-annotated reference genome. Zebrafish turns out to have the largest gene set of any vertebrate so far sequenced, and few pseudogenes. Importantly for disease studies, comparison between human and zebrafish sequences reveals that 70% of human genes have at least one obvious zebrafish orthologue. A second paper reports on an ongoing effort to identify and phenotype disruptive mutations in every zebrafish protein-coding gene. Using the reference genome sequence along with high-throughput sequencing and efficient chemical mutagenesis, the project's initial results — covering 38% of all known protein-coding genes — they describe phenotypic consequences of more than 1,000 alleles. The long-term goal is the creation of a knockout allele in every protein-coding gene in the zebrafish genome. All mutant alleles and data are freely available at go.nature.com/en6mos . Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches 1 , 2 , 3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes 3 , 4 , this number falls considerably short of the more than 22,000 mouse protein-coding genes 5 . Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning 6 , insertional mutagenesis 7 , 8 , 9 , antisense morpholino oligonucleotides 10 , targeted re-sequencing 11 , 12 , 13 , and zinc finger and TAL endonucleases 14 , 15 , 16 , 17 have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes 18 . Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence 18 , 19 , high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.