Genetic dissection of the hindbrain functions by the Gal4-UAS system in zebrafish

Kazuhide Asakawa1, Gembu Abe1, Koichi Kawakami1,2

1)National Institute of Genetics, 2)The Graduate University for Advanced Studies (SOKENDAI)

 In vertebrate, the hindbrain plays critical roles in regulating locomotion, postural control and orofacial behavior. In order to disclose specific roles of the hindbrain, we have been performing a screen for transgenic zebrafish line expressing Gal4FF, a variant of the yeast transcription factor Gal4, in the hindbrain by the Tol2 transposon-mediated Gal4 trap approaches. From these screens, we have identified two transgenic lines (g104 and g266) that expressed Gal4FF in the hindbrain neurons during early embryonic stages. Molecular characterization revealed that the both of these lines carried the Gal4FF trap construct in the cyp26c1 gene, which encodes a cytochrome p450 enzyme that is involved in hindbrain patterning. To inactivate the cyp26c1-positive hindbrain neurons, we crossed the cyp26c1-Gal4FF fish with the UAS:tetanus toxin light chain (TeTxLC) fish, which carried the TeTxLC gene downstream of the UAS (the Gal4 target site). First, we found that cyp26c1-Gal4FF;UAS:TeTxLC double transgenic embryos had defects in the touch-evoked escape behavior. When a tactile stimulus is given, wild type embryos exhibit an initial turn which is contralateral to the stimulus and then escape by swimming. In contrast, we found that the double transgenic embryos exhibit an ipsilateral turn at 48 hours post-fertilization (hpf). Furthermore, the double transgenic embryos showed the contralateral turn until 36 hpf and began to exhibit the ipsilateral turn after 40 hpf, suggesting that the hindbrain regulates the initial turn direction from 40 hpf. Second, we found that the double transgenic larvae failed to maintain posture. We also noticed that pectoral fin movement was abnormal in the double transgenic larvae, suggesting that regulation of pectoral fin movement by the hindbrain may be critical in postural control. Together, we propose these Gal4FF lines are useful to disclose the hindbrain functions. Efforts to further dissect the hindbrain functions are currently in progress.