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A novel inducible mutagenesis screen enables to isolate and clone both embryonic and adult zebrafish mutants.

ABSTRACT: Conventional genetic screens for recessive mutants are inadequate for studying biological processes in the adult vertebrate due to embryonic lethality. Here, we report that a novel inducible mutagenesis system enables to study gene function in both embryonic and adult zebrafish. This system yields genetic mutants with conditional ectopic over- or under-expression of genes in F₁ heterozygotes by utilizing inducible Tet-On transcriptional activation of sense or anti-sense transcripts from entrapped genes by Tol2 transposase-meditated transgenesis. Pilot screens identified 37 phenotypic mutants displaying embryonic defects (34 lines), adult fin regeneration defects (7 lines), or defects at both stages (4 lines). Combination of various techniques (such as: generating a new mutant allele, injecting gene specific morpholino or mRNA etc) confirms that Dox-induced embryonic abnormalities in 10 mutants are due to dysfunction of entrapped genes; and that Dox-induced under-expression of 6 genes causes abnormal adult fin regeneration. Together, this work presents a powerful mutagenesis system for genetic analysis from zebrafish embryos to adults in particular and other model organisms in general.

SUBMITTER: Ma Z

PROVIDER: S-EPMC5583359 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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A novel inducible mutagenesis screen enables to isolate and clone both embryonic and adult zebrafish mutants.

Ma Zhipeng Z Zhu Peipei P Pang Meijun M Guo Liwei L Chang Nannan N Zheng Jiyuan J Zhu Xiaojun X Gao Ce C Huang Honghui H Cui Zongbin Z Xiong Jing-Wei JW Peng Jinrong J Chen Jun J

Scientific reports 20170904 1

Conventional genetic screens for recessive mutants are inadequate for studying biological processes in the adult vertebrate due to embryonic lethality. Here, we report that a novel inducible mutagenesis system enables to study gene function in both embryonic and adult zebrafish. This system yields genetic mutants with conditional ectopic over- or under-expression of genes in F<sub>1</sub> heterozygotes by utilizing inducible Tet-On transcriptional activation of sense or anti-sense transcripts fr ...[more]

PMID: 28871129

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Project description:BackgroundFetal alcohol spectrum disorders (FASD) are a highly variable set of phenotypes caused by fetal alcohol exposure. Numerous factors influence FASD phenotypes, including genetics. The zebrafish is a powerful vertebrate model system with which to identify these genetic factors. Many zebrafish mutants are housed at the Zebrafish International Resource Center (ZIRC). These mutants are readily accessible and an excellent source to screen for ethanol (EtOH)-sensitive developmental structural mutants.MethodsWe screened mutants obtained from ZIRC for sensitivity to EtOH teratogenesis. Embryos were treated with 1% EtOH (41 mM tissue levels) from 6 hours postfertilization onward. Levels of apoptosis were evaluated at 24 hours postfertilization. At 4 days postfertilization, the craniofacial skeleton, peripheral axon projections, and sensory neurons of neuromasts were examined. Fish were genotyped to determine whether there were phenotype/genotype correlations.ResultsFive of 20 loci interacted with EtOH. Notable among these was that vangl2, involved in convergent extension movements of the embryonic axis, interacted strongly with EtOH. Untreated vangl2 mutants had normal craniofacial morphology, while severe midfacial defects including synophthalmia and narrowing of the palatal skeleton were found in all EtOH-treated mutants and a low percentage of heterozygotes. The cell cycle gene, plk1, also interacted strongly with EtOH. Untreated mutants have slightly elevated levels of apoptosis and loss of ventral craniofacial elements. Exposure to EtOH results in extensive apoptosis along with loss of neural tissue and the entire craniofacial skeleton. Phenotypes of hinfp, mars, and foxi1 mutants were also exacerbated by EtOH.ConclusionsOur results provide insight into the gene-EtOH interactions that may underlie EtOH teratogenesis. They support previous findings that EtOH disrupts elongation of the embryonic axis. Importantly, these results show that the zebrafish is an efficient model with which to test for gene-EtOH interactions. Understanding these interactions will be crucial to understanding of the FASD variation.

Dataset Information

A novel inducible mutagenesis screen enables to isolate and clone both embryonic and adult zebrafish mutants.

Publications

A novel inducible mutagenesis screen enables to isolate and clone both embryonic and adult zebrafish mutants.

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