Development of the GAL4-enhancer trap system using the Tol2 transposable element and its application to inhibition of synaptic transmission in zebrafish.

Asakawa, K.1 , Mizusawa, K.1 , Urasaki, A.1 , Kotani, T.1 , Nagayoshi, S.1 , Kishimoto, Y.1 , Hibi, M.2 , Kawakami, K.1

1. National Institute of Genetics, 2. RIKEN CDB

 The GAL4-enhancer trap system is a powerful method to modulate cellular functions in living animals by targeted gene expression. By using the Tol2 transposon system, we aimed to develop a GAL4-enhancer trap system in zebrafish.
 First, we constructed the enhancer trap Tol2 construct hsp-gal4, which contains a modified GAL4 gene downstream of the zebrafish hsp70 promoter, and the reporter Tol2 constructs, UAS-EGFP and UAS-DsRED. Then, we created transgenic fish carrying these transposon constructs by Tol2-mediated transgenesis. Embryos obtained from crosses between the hsp-gal4 fish and these reporter fish could express EGFP or DsRED ubiquitously upon heat shock, indicating that the Gal4 protein can activate expression of a gene downstream of UAS.
 Next, to determine whether Gal4-enhancer trapping is feasible, we created random insertions of the hsp-gal4 construct in the genome by Tol2-mediated transgenesis. In a pilot screen, we successfully isolated various Gal4 expression patterns during embryonic stages by crossing the hsp-gal4 fish with the UAS-EGFP fish. In the hsp-gal4 fish, the expression pattern of the GAL4 mRNA was consistent with the GFP expression, indicating that the hsp-gal4 construct is regulated by locus-specific enhancers.
 Finally, to test if the GAL4-enhancer trap lines isolated in this study can be used to modulate neuronal functions, we constructed the transgenic fish carrying a gene for tetanus toxin light chain (TeTxLC), which blocks release of neurotransmitter via synaptic vesicles, downstream of UAS. We found that the embryos from the cross between UAS-TeTxLC fish and the hsp-gal4 fish expressing the Gal4 in the brain and the spinal cord failed to respond to a tactile stimulation in the touch response test during embryonic stages. We think that, in the double transgenic embryos, the Gal4 was expressed in neurons that are involved in the touch response and synaptic transmission of such neurons was blocked by induction of the TeTxLC expression.