Targeted gene expression by transposon-mediated Gal4 gene and enhancer trapping in zebrafish defines subsets of neurons required for simple vertebrate behaviors


Kazuhide Asakawa, Kanta Mizusawa, Saori Nagayoshi, Tomoya Kotani, Akihiro Urasaki, Yasuyuki Kishimoto, Masahiko Hibi, and Koichi Kawakami

*Division of Molecular and Developmental Biology, National Institute of Genetics,
†Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), 1111 Yata, Mishima, Shizuoka 411-8540, Japan

Laboratory for Vertebrate Axis Formation, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

 We report here development of the Gal4 gene trap and enhancer trap methods using the Tol2 transposable element in zebrafish. We created random insertions of gene trap and enhancer trap constructs containing the modified yeast Gal4 transcription activator, Gal4FF, in the zebrafish genome. By crossing the Gal4FF transgenic fish with transgenic reporter fish carrying the GFP gene downstream of the Gal4 recognition sequence UAS, we isolated embryos expressing GFP in temporally and spatially restricted patterns. We identified more than 300 patterns and established 185 transgenic lines. To identify neural circuits regulating vertebrate behaviors, we constructed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene under the control of UAS. We crossed 121 Gal4FF lines with the UAS-TeTxLC effector fish, and found that nine of them exhibited defects in a response to a tactile stimulus. One of them responded to the stimulus but showed abnormality in the escape process, and another could not respond to the stimulus. These lines expressed Gal4FF in different types of neurons in the spinal cord, suggesting a differential role for such neurons in regulating early escape behaviors. Our experiments demonstrate that transposon-mediated Gal4FF gene trap and enhancer traps can be used efficiently to label and modify vertebrate neuronal functions in vivo. We propose fish lines expressing Gal4FF in specific cells, tissues, and organs, are a useful resource to study development, morphogenesis and organogenesis as well as neuronal functions.