Article

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

ABSTRACT

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
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    • "In addition, the complete genome sequence and its annotation are available , as well as a broad array of molecular and cellular tools. An increasing number of well characterized fish mutants has been derived from large scale mutagenesis screens (456; http://www.sanger.ac.uk/resources/zebrafish/zmp/), and many transgenic fish lines have been developed using fluorescent proteins (such as Green Fluorescent Protein - GFP) to report the expression of skeleton-related genes[7]. Recently, the development of reverse genetic approaches, such as TALE nucleases and Crispr/Cas9 systems, opened new horizons for targeted mutagenesis in zebrafish[8]. "
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    ABSTRACT: Background: The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results: Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions: The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration.
    Full-text · Article · Dec 2016 · BMC Developmental Biology
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    • "These reports justify the present work to further explore genome editing technology to model cardiomyopathy in adult zebrafish. Historically, most zebrafish stable mutants have been generated by either large scale mutagenesis screening or TILLING technology262728. Both strategies depend on generating random mutations in the whole genome, which is only feasible by collaborative efforts in large research centers. "

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    • "This is a highly potent mutagen that induces point mutations in the germ-line that are heritable in the offspring. Several genetic screens were performed in this fish revealing the presence of recessive mutations induced by ENU and their inheritance in the offspring (Mullins et al. 1994;SolnicaKrezel et al. 1994;Driever et al. 1996;Haffter et al. 1996). These genetic screens were fundamental because they demonstrated how mutations affecting a variety of developmental processes could be efficiently recovered from zebrafish; a vertebrate model that develops externally with a clear egg chorion. "
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    ABSTRACT: The mangrove killifish, Kryptolebias marmoratus, is unique among vertebrates due to its self-fertilizing mode of reproduction involving an ovotestis. As a result, it constitutes a simplistic and desirable vertebrate model for developmental genetics as it is easily maintained, reaches sexual maturity in about 100 days and provides a manageable number of relatively clear embryos. After the establishment and characterization of an initial mutagenesis pilot screen using N-ethyl-N-nitrosourea (Moore et al. 2012), a three-generation genetic screen was performed to confirm zygotic mutant allele heritability and simultaneously score for homozygous recessive mutant sterile F2 fish. From a total of 307 F2 fish screened, 10 were found to be 1° males, 16 were sterile, 92 wild type and the remaining 189, carriers of zygotic recessive alleles. These carriers produced 25% progeny exhibiting several zygotic phenotypes similar to those previously described in zebrafish (Mullins et al. 1994) and in the aforementioned pilot screen, as expected. Interestingly, new phenotypes such as golden yolk, no trunk and short tail, were observed. The siblings of sterile F2 mutants were used to produce an F3 generation in order to confirm familial sterility. Out of the 284 F3 fish belonging to 10 previously identified sterile families, 12 were found to be 1° males, 69 were wild type, 83 sterile and 120 were classified as */+ (either wild type or carriers) with undefined genotypes. This screen provides proof of principle that Kryptolebias marmoratus is a powerful vertebrate model for developmental genetics and can be used to identify mutations affecting fertility.
    Full-text · Article · Jan 2016 · G3-Genes Genomes Genetics
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