Genetic Dissection of Neural Circuits by ToL2 Transposon- mediated Gal4 Gene and Enhancer Trapping in Zebrafish

Kazuhide Asakawa1, Maximiliano L. Suster1, Kanta Mizusawa1, Saori Nagayoshi1, Tomoya Kotani1, Akihiro Urasaki1, Yasuyuki Kishimoto1, Masahiko Hibi2, and Koichi Kawakami1

1) Division of Molecular and Developmental Biology, National Institute of Genetics, and Department of Cenetics, The Craduate University for Advanced Studies (SOKENDAI), Japan, 2) Laboratory for Vertebrate Axis Formation, RIKEN Center for Developmental Biology, Japan

We report here the development of a novel targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS). We showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, we performed gene trap and enhancer trap screens by creating random integrations of these Gal4FF trap constructs into the zebrafish genome by Tol2-mediated transgenesis. Fish injected with these trap constructs were crossed with UAS:GFP reporter fish and the resulting F1 embryos were screened for unique GFP expression. By this scheme, we generated 185 fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which blocks synaptic transmission. By crossing the UAS:TeTxLC fish with Gal4FF transgenic fish, we identified lines that exhibited defects in touch response behavior. The expression patterns of the toxin in these embryos were further analyzed by constructing and crossing with the UAS:TeTxLC:CFP transgenic line. From these analyses, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.