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First published online December 7, 2007
doi: 10.1242/10.1242/dev.009050
1 Division of Molecular and Developmental Biology, National Institute of
Genetics, Mishima, Shizuoka 411-8540, Japan.
2 Department of Biological Sciences, University of Tokyo, Tokyo 113-0033,
Japan.
3 Center for Information Biology and DNA Data Bank of Japan, National Institute
of Genetics, Mishima, Shizuoka 411-8540, Japan.
4 Department of Genetics, The Graduate University of Advanced Studies
(SOKENDAI), Mishima, Shizuoka 411-8540, Japan.
Author for correspondence (e-mail:
kokawaka{at}lab.nig.ac.jp)
Accepted 19 October 2007
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
G
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.
Key words: Zebrafish, tcf7, synembryn, Enhancer trapping, Insertional mutagenesis
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