Driever, W. et al. A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37-46

Cardiovascular Research Center, Massachusetts General Hospital, Charlestown 02129, USA.
Development (Impact Factor: 6.46). 01/1997; 123:37-46. DOI: 10.5167/uzh-215
Source: PubMed


Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for developmental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according to their phenotypes. The analyses and genetic complementation of mutations from several categories are reported in separate manuscripts. Mutations affecting pigmentation, motility, muscle and body shape have not been extensively analyzed and are listed here. A total of 331 mutations were tested for allelism within their respective categories. This defined 220 genetic loci with on average 1.5 alleles per locus. For about two-thirds of all loci only one allele was isolated. Therefore it is not possible to give a reliable estimate on the degree of saturation reached in our screen; however, the number of genes that can mutate to visible embryonic and early larval phenotypes in zebrafish is expected to be several-fold larger than the one for which we have observed mutant alleles during the screen. This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish.

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Available from: Salim Abdelilah-Seyfried, Sep 29, 2015
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    • "The zebrafish has become a central model system to investigate vertebrate development. Early foundational studies utilized zebrafish in large-scale forward genetic screens to identify mutants affecting different aspects of embryonic development (Driever et al., 1996; Haffter et al., 1996). These studies took advantage of the many benefits of zebrafish husbandry and embryogenesis. "
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    ABSTRACT: The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses. Copyright © 2015 Elsevier Inc. All rights reserved.
    Developmental Cell 12/2014; 32(1). DOI:10.1016/j.devcel.2014.11.018 · 9.71 Impact Factor
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    • "Some of the most predominant methods used to gain information are through the use of forward and reverse genetic approaches (Lawson and Wolfe, 2011), as well as chemical genetics (Lessman, 2011). Large-scale forward diploid genetic screens have led to the identification of many mutants that recapitulate human congenital disorders (Amsterdam and Hopkins, 2006; Driever et al., 1996; Haffter et al., 1996), and alternate strategies, such as those using haploids, enable moderate genome coverage in small-scale screening efforts (Kroeger et al., in 2014). Although the identification of chemically induced genetic lesions by positional cloning with meiotic mapping has historically been laborious (Zhou and Zon, 2011), the advent of next-generation sequencing technologies have enabled whole-genome sequencing and whole-exome sequencing (Bowen et al., 2012; Gupta et al., 2010; Kettleborough et al., 2013; Leshchiner et al., 2012; Obholzer et al., 2012; Ryan et al., 2013; Voz et al., 2012). "
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    ABSTRACT: During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. © 2014 Wiley Periodicals, Inc.
    genesis 09/2014; 52(9):771-792. DOI:10.1002/dvg.22798 · 2.02 Impact Factor
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    • "Importantly, in mutagenesis screens environmental variables such as light cycle, temperature, water quality, density, and feeding are controlled thus limiting variance that might obscure identification of the genetic control of phenotype. Large-scale screens have been carried out in both zebrafish and medaka for mutations affecting early development to find genes essential for development (Driever et al., 1996; Haffter et al., 1996b; Loosli et al., 2000; Furutani-Seiki et al., 2004). As a consequence of their phenotypic effect, many of these mutations are lethal early in life with only a small percentage leading to phenotypes in adults (Haffter et al., 1996b). "
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    ABSTRACT: Fishes are wonderfully diverse. This variety is a result of the ability of ray-finned fishes to adapt to a wide range of environments, and has made them more specious than the rest of vertebrates combined. With such diversity it is easy to dismiss comparisons between distantly related fishes in efforts to understand the biology of a particular fish species. However, shared ancestry and the conservation of developmental mechanisms, morphological features and physiology provide the ability to use comparative analyses between different organisms to understand mechanisms of development and physiology. The use of species that are amenable to experimental investigation provides tools to approach questions that would not be feasible in other ‘non-model’ organisms. For example, the use of small teleost fishes such as zebrafish and medaka has been powerful for analysis of gene function and mechanisms of disease in humans, including skeletal diseases. However, use of these fish to aid in understanding variation and disease in other fishes has been largely unexplored. This is especially evident in aquaculture research. Here we highlight the utility of these small laboratory fishes to study genetic and developmental factors that underlie skeletal malformations that occur under farming conditions. We highlight several areas in which model species can serve as a resource for identifying the causes of variation in economically important fish species as well as to assess strategies to alleviate the expression of the variant phenotypes in farmed fish. We focus on genetic causes of skeletal deformities in the zebrafish and medaka that closely resemble phenotypes observed both in farmed as well as natural populations of fishes.
    Journal of Applied Ichthyology 08/2014; 30(4). DOI:10.1111/jai.12533 · 0.87 Impact Factor
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