RRC ID 4115
Author Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa K, Urasaki A, Horikawa K, Ikeo K, Takeda H, Kawakami K.
Title Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like.
Journal Development
Abstract Gene trap and enhancer trap methods using transposon or retrovirus have been recently described in zebrafish. However, insertional mutants using these methods have not been reported. We report here development of an enhancer trap method by using the Tol2 transposable element and identification and characterization of insertional mutants. We created 73 fish lines that carried single copy insertions of an enhancer trap construct, which contained the zebrafish hsp70 promoter and the GFP gene, in their genome and expressed GFP in specific cells, tissues and organs, indicating that the hsp70 promoter is highly capable of responding to chromosomal enhancers. First, we analyzed genomic DNA surrounding these insertions. Fifty-one of them were mapped onto the current version of the genomic sequence and 43% (22/51) were located within transcribed regions, either exons or introns. Then, we crossed heterozygous fish carrying the same insertions and identified two insertions that caused recessive mutant phenotypes. One disrupted the tcf7 gene, which encodes a transcription factor of the Tcf/Lef family mediating Wnt signaling, and caused shorter and wavy median fin folds and pectoral fins. We knocked down Lef1, another member of the Tcf/Lef family also expressed in the fin bud, in the tcf7 mutant, and revealed functional redundancy of these factors and their essential role in establishment of the apical ectodermal ridge (AER). The other disrupted the synembryn-like gene (synbl), a homolog of the C. elegans synembryn gene, and caused embryonic lethality and small pigment spots. The pigment phenotype was rescued by application of forskolin, an activator of adenylyl cyclase, suggesting that the synbl gene activates the Galpha(S) pathway leading to activation of adenylyl cyclase. We thus demonstrated that the transposon-mediated enhancer trap approach can indeed create insertional mutations in developmental genes. Our present study provides a basis for the development of efficient transposon-mediated insertional mutagenesis in a vertebrate.
Volume 135(1)
Pages 159-69
Published 2008-1
DOI 10.1242/dev.009050
PII 135/1/159
PMID 18065431
MeSH Animals Cloning, Molecular Colforsin / pharmacology DNA Transposable Elements / genetics* Embryo, Nonmammalian / embryology Embryo, Nonmammalian / metabolism Enhancer Elements, Genetic / genetics* Gene Expression Regulation, Developmental* / drug effects Mutagenesis, Insertional / genetics* Mutation / genetics* Nuclear Proteins / genetics* Nuclear Proteins / metabolism Phenotype Phylogeny Trans-Activators / genetics* Trans-Activators / metabolism Transcription Factors / genetics Transcription Factors / metabolism Transcription, Genetic / genetics Zebrafish / embryology Zebrafish / genetics Zebrafish / metabolism Zebrafish Proteins / genetics* Zebrafish Proteins / metabolism
IF 5.413
Times Cited 89
WOS Category DEVELOPMENTAL BIOLOGY
Resource
Zebrafish HG2A? HG3A? HG6A? HG6B? HG6C? HG6D? HG9B? HG9C? HG10A? HG10B? HG10C? HG21A? HG21B? HG21C? HG21E? HG21K? HGn6A? HGn8E? HGn8H? HGn12B? HGn12C? HGn12F? HGn12H? HGn12J? HGn14C? HGn15A? HGn15B? HGn16A? HGn20A? HGn20C? HGn20D? HGn20E? HGn21A? HGn22A? HGn23A? HGn25B? HGn25D? HGn26A? HGn26B? HGn28A? HGn30A? HGn31A? HGn31B? HGn32A? HGn32B? HGn34A? HGn34B? HGn36A? HGn39B? HGn39C? HGn39D? HGn42A? HGn42D? HGn43A? HGn44B? HGn46C? HGn46E? HGn46F? HGn50A? HGn50B? HGn50D? HGn50E? HGn50G? HGn53D? HGn54B? HGn62A? HGn62B? HGn63B?