Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

B RIEFINGS IN FUNC TIONAL GENOMICS . VOL 13. NO 2. 82^94 doi:10.1093/bfgp/elt042 Mutagenesis and phenotyping resources in zebrafish for studying development and human disease Gaurav Kumar Varshney and Shawn Michael Burgess Advance Access publication date 26 October 2013 Abstract Keywords: zebrafish; mutagenesis; phenotyping; resources; knockouts INTRODUCTION In the age of the sequenced human genome, diseases and phenotypes can be rapidly mapped by genomewide association studies (GWAS) to potential candidate genes [1] (http://www.genome.gov/ gwastudies/) and candidates are increasingly identified by exome sequencing [2], but in both cases these merely represent correlations with diseases and cannot prove disease causation alone. A key issue still remains in determining genetic causes of disease: the functions of the vast majority of human genes have only been predicted computationally and have never been tested or verified in vivo. It is essential that functional testing of every gene be carried out so that better predictions for candidate disease genes from GWAS or exome/genome sequencing can be made. For decades, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster have been favorite model systems for geneticists to carry out functional genetic studies, these models have contributed immensely to our understanding of signaling pathways, metabolism, the cell cycle, embryonic patterning, aging, homeostasis and many other areas. Their utility was enhanced even more when their genomes were sequenced, opening new avenues for systematic testing of gene function. Although these nonvertebrates are excellent model systems to study conserved developmental pathways, many aspects of vertebrate embryonic development such as tissue patterning and morphogenesis have features unique to the vertebrate lineage. Mouse (Mus Musculus) is the most commonly used vertebrate model organism with a high-quality reference genome, with nearly all genes having been identified. There are many powerful genetic tools available (e.g. targeted conditional knockouts) to study gene function in mouse, however, the maintenance of large mouse colonies is expensive, making it difficult for large-scale genetic screens and phenotyping studies. Three decades ago, George Streisinger and colleagues Corresponding author. Shawn Michael Burgess, Developmental Genomics Section, Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA. Tel: 301-594-8224; Fax: 301-496-0474; E-mail: DrsVarshney and Burgess are employees of the National Human Genome Research Institute, in the Genome Technology Branch, and are currently working on large-scale mutagenesis in zebrafish. Published by Oxford University Press 2013. This work is written by US Government employees and is in the public domain in the US. The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. Mutagenesis and phenotyping resources in zebrafish RANDOM MUTAGENESIS APPROACHES Chemical-mediated mutagenesis ENU (N-ethyl-N-nitrosourea) is the most commonly used chemical mutagen in zebrafish and was used for the two largest forward genetic screens that identified thousands of mutants with embryonic developmental phenotypes [15, 16]. The identification of mutated genes by positional cloning is still laborious, although the positional cloning methods have simplified over the years [17]. Recent advances in genomic technologies and next-generation sequencing further improved these methods and the mutated genes can now often be identified by whole-genome sequencing at low coverage (3–8X). Voz et al. developed a fast mapping method using the whole-genome sequencing (8-fold coverage); in this method the affected locus can be identified by the analysis of single-nucleotide polymorphism (SNP) homozygosity. Compared with traditional positional cloning, this method requires many fewer mutant embryos and can be performed in a few weeks [18]. However, given the high number of variations within or between different strains of zebrafish, it could be challenging to distinguish a homozygous mutation-causing variant with linked but low-frequency SNPs. Bowen et al. documented and developed an extensive SNP database in zebrafish that can be useful in mapping mutations with low-coverage whole-genome sequencing [19]. Two additional cloning strategies have been developed using the new sequencing platforms: bulk segregant-based linkage analysis (BSFseq) and homozygosity mapping (HMFseq). In BSFseq, a carrier of the mutant is out-crossed to a different wild-type strain, and the resulting F1 hybrid pairs are repeatedly crossed to generate a few hundred progeny, which are pooled and sequenced. The HMFseq is faster as it does not involve a mapping cross but relies on the inherently high SNP rate present in most of the lab zebrafish lines. Multiple carrier pairs are crossed and mutant larvae are collected, pooled and sequenced. Both strategies use sequences from a pool of mutants and analysis of whole-genome sequence for causative mutations using a similar bioinformatics pipeline. An open source tool MegaMapper (https://wikis.utexas.edu/ display/bioiteam/MegaMapper) is also available for the analysis of both the HMFseq and BSFseq approaches [20, 21]. Two approaches based on transcriptome sequencing have been developed: Mutation Mapping [3] introduced the small, freshwater teleost fish the zebrafish (Daniorerio) as a model organism to geneticists. Since then, zebrafish has gained significant momentum as a model for studying vertebrate development and modeling human disease. The zebrafish genome is only the third vertebrate genome to be ‘finished’ [4] recently joining human [5–7] and mouse [8] in having a high quality reference genome sequence. Annotations show that zebrafish has the largest number of genes (26 000) of any sequenced vertebrate [4]. Comparison of the zebrafish genome to the human genome revealed that 70% of all human g (...truncated)