The functions of the hindbrain revealed by the Gal4-UAS system

Kazuhide Asakawa1, Gembu Abe2, Koichi Kawakami1

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

 The hindbrain plays critical roles in regulating locomotion, postural control and orofacial behavior. In order to disclose specific roles of the hindbrain in these behaviors, 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 gene trap and enhancer 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 but not in the spinal neurons. 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:TeTxLC effector fish, which carried the tetanus toxin light chain (TeTxLC) gene downstream of the UAS (the Gal4 target site). Then, we investigated the behaviors of the resulting cyp26c1-Gal4FF;UAS:TeTxLC double transgenic embryos. 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 when the tactile stimuli was given at 48 hpf. Furthermore, we found that 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 direction of the initial turn later than 40 hpf. Second, we found that the cyp26c1-Gal4FF;UAS:TeTxLC double transgenic larvae failed to maintain posture at 84 hpf and later. 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.