Functional regionalization of the adult zebrafish brain by the gene trap, enhancer trap and GAL4-UAS approaches

Pradeep Lal,1,2, Koichi Kawakami1,2

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

 Current understanding of the adult zebrafish brain is based on comparative neuroanatomy, histology and expression data of few genes. Although these techniques are useful in anatomical description of the overall adult brain, they cannot be used to visualize and manipulate different brain regions. Hence, the knowledge of relationship between anatomy and function of adult zebrafish brain is limited. The ability to selectively manipulate a small subset of neurons, can lead to significant insight into their role for a complex behavior of adult zebrafish. Therefore, it is important to regionalize the brain and also develop a method to visualize and manipulate the regionalized structures.
 We have developed the Gal4-UAS system and Gal4 gene trap and enhancer trap methods in zebrafish using the Tol2 transposon system. We isolated transgenic fish lines that expressed the Gal4 transactivator in specific tissues. By crossing these Gal4 lines with the UAS-GFP line, it is possible to visualize the Gal4-expressing cells. To identify transgenic lines showing Gal4 expression in a subset of neurons in adult zebrafish brain, we observed 351 adult Gal4 fish lines and selected 108 lines showing strong GFP fluorescence expression in the brain. In addition, we observed 115 GFP gene trap and enhancer trap lines and selected 34 lines with GFP fluorescence in the brain. These selected 142 fish lines were further analyzed by sectioning into 100 micron coronal slices of the brain. The observed GFP fluorescence patterns show unique patterns varying from ubiquitous whole brain patterns to very restricted expression patterns, such as a small region in dorsomedial telencephalon, lateral telencephalon, habenula, restricted region within hypothalamus, cerebellum etc. For most of these regions, anatomical description or marker genes have not been reported. Thus, these lines are useful for describing new regions in the brain and discovering new marker genes. These fish were further analyzed by Southern blot hybridization and inverse PCR to find the site of the Tol2 insertions. I will discuss how the expression patterns in embryos, those in the adult brain and the trapped genes are relevant.
 To deduce the role of specific Gal4 labeled neurons in adult zebrafish brain, we need to inhibit their function selectively and look for any resulting change in behavioral abnormality. This can be done by crossing these Gal4 lines with the UAS-tetanus toxin line which will selectively inhibit the neurotransmitter release in these Gal4 labeled neurons.