Methods for reverse genetic screening in zebrafish by resequencing and TILLING

Sood, Raman; English, Milton A.; Jones, MaryPat; Mullikin, James C.; Wang, Duen-Mei; Anderson, M.; Wu, Dongying; Chandrasekharappa, Settara C.; Yu, Jun; Zhang, Jinghui; Liu, P. Paul

doi:10.1016/j.ymeth.2006.04.012

Cited by 90 publications

(59 citation statements)

References 27 publications

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“…16 and 32. grl vu59 mutants were generated by TILLING method (33) (SI Appendix, section III). grl and gata5 deletion constructs were generated by PCR-based subcloning technique (SI Appendix, section VIII).…”

Section: Methodsmentioning

confidence: 99%

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Jia

King²,

Chopra³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (Grl), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in grl cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression.cardiomyocyte ͉ proliferation ͉ size control ͉ transcription T he embryonic heart grows through a combination of cardiomyocyte proliferation and an increase in cell mass due to the generation of myofibrillar arrays (1). Cardiac proliferation diminishes progressively after birth, and postmitotic hypertrophy in the adult heart provides most of the adaptive responses necessary to supply increased cardiac output (2). Despite recent progress that has been made in the regulation of postnatal cardiac hypertrophy, the mechanisms and pathways that control embryonic heart growth are poorly delineated. It is not known what molecular signals restrain cardiomyocyte proliferative growth in the embryonic heart. The GATA zinc-finger transcription factors promote myocardial differentiation and expansion. Among the three gata genes (gata4, gata5, and gata6) in zebrafish, gata5 plays the most prominent role in heart growth and development (3). Mutations in gata5 in zebrafish cause a reduction in expression of early and late myocardial genes and a decrease in cardiac progenitor cells and proliferative cardiomyocytes, resulting in small hearts. Forced gata5 expression in the zebrafish embryo increases heart size and occasionally produces ectopically beating myocardial tissue (4). The phenotype of zebrafish gata5 mutants closely resembles the cardiac phenotypes in gata4 mutant mice. Myocardium-restricted deletion of murine gata4 or gata4/gata6 double heterozygote causes a marked reduction in cardiomyocyte proliferation and results in hypoplastic hearts (5-7). Although it seems to be clear that certain levels of GATA activity are required to drive myocardial proliferative growth, opposing signals might also be necessary to constrain the excessive cardiac growth during development.grl encodes a hairy-related basic helix-loop-h...

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Section: Methodsmentioning

confidence: 99%

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Jia

King²,

Chopra³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…2 A; Sood et al, 2013). In addition, we screened a zebrafish DNA library of ENU-induced mutations (Sood et al, 2006) and uncovered a mutant fish line carrying a T371C/L124P missense mutation within exon 4 of the AK2 gene. The mutated amino acid in this mutant ( Fig.…”

mentioning

confidence: 99%

Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress

Rissone¹,

Weinacht²,

Marca³

et al. 2015

J Cell Biol

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“…3,74 The offspring of each uniquely mutagenized individual can be screened for mutations in genes of interest and are used to establish a mutant line. 3,103 Mutant zebrafish lines that have been established by this method harbor mutations in important cancer susceptibility genes, including zebrafish orthologues of the human tumor suppressor genes TP53, APC, PTEN, and BRCA2, among others. 9,24,39,85,93,99 Creation of transgenic zebrafish lines is also possible, since 1-cell stage zebrafish embryos can be injected with plasmid or bacterial artificial chromosome (BAC) gene constructs with relative ease.…”

Section: Mechanisms For Developing Cancer Models In Zebrafishmentioning

confidence: 99%

Zebrafish Models for Human Cancer

Shive

2012

Vet Pathol

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For decades, the advancement of cancer research has relied on in vivo models for examining key processes in cancer pathogenesis, including neoplastic transformation, progression, and response to therapy. These studies, which have traditionally relied on rodent models, have engendered a vast body of scientific literature. Recently, experimental cancer researchers have embraced many new and alternative model systems, including the zebrafish (Danio rerio). The general benefits of the zebrafish model for laboratory investigation, such as cost, size, fecundity, and generation time, were quickly superseded by the discovery that zebrafish are amenable to a wide range of investigative techniques, many of which are difficult or impossible to perform in mammalian models. These advantages, coupled with the finding that many aspects of carcinogenesis are conserved in zebrafish as compared with humans, have firmly established a unique niche for the zebrafish model in comparative cancer research. This article introduces methods for generating cancer models in zebrafish and reviews a range of models that have been developed for specific cancer types. Keywords animal disease models, cancer, genetic modification, zebrafishThe zebrafish (Danio rerio) emerged as a model for scientific research in the 1930s, with many early studies devoted to the investigation of developmental processes in embryos. 53 The use of zebrafish in research has subsequently expanded into a broad range of disciplines. As a research species, zebrafish offer certain advantages over traditional vertebrate models, including rapid generation time, high fecundity, external embryonic development, a capacity for high stocking density in relatively small areas, and lower maintenance costs. 64 In recent years, the zebrafish has come into its own as a relevant comparative model for a diverse spectrum of human diseases.Although zebrafish are relative newcomers to the field of comparative cancer research, these small animals have emerged as a powerful tool for investigators. As vertebrate organisms, they share many features of embryonic development and adult anatomy that are critical for comparative animal models. 64,71 In the world of cancer research, this has important implications ranging from conserved signaling pathways and transcriptional regulatory mechanisms, to organogenesis and tissue differentiation, to cellular transformation and tumor initiation. Zebrafish cancer models have been produced by a variety of methods, leading to a broad spectrum of cancer susceptibilities in animals of multiple age groups. Flexibility in genetic manipulation, adaptability to current tools for cancer analysis, and capacity to define cellular and molecular interactions in vivo provide the zebrafish with its own very unique set of advantages for the investigation of cancer biology. Mechanisms for Developing Cancer Models in ZebrafishZebrafish cancer models have been developed by a number of mechanisms (Table 1). Although initial studies relied on chemical carcinogens for t...

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Methods for reverse genetic screening in zebrafish by resequencing and TILLING

Cited by 90 publications

References 27 publications

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress

Zebrafish Models for Human Cancer

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