Optimized Gal4 genetics for permanent gene expression mapping in zebrafish

Combinatorial genetics for conditional transgene activation allows studying gene function with temporal and tissue specific control like the Gal4-UAS system, which has enabled sophisticated genetic studies in Drosophila. Recently this system was adapted for zebrafish and promising applications have been introduced. Here, we report a systematic optimization of zebrafish Gal4-UAS genetics by establishing an optimized Gal4-activator (KalTA4). We provide quantitative data for KalTA4-mediated transgene activation in dependence of UAS copy numbers to allow for studying dosage effects of transgene expression. Employing a Tol2 transposon-mediated KalTA4 enhancer trap screen biased for central nervous system expression, we present a collection of self-reporting red fluorescent KalTA4 activator strains. These strains reliably transactivate UAS-dependent transgenes and can be rendered homozygous. Furthermore, we have characterized the transactivation kinetics of tissue-specific KalTA4 activation, which led to the development of a self-maintaining effector strain "Kaloop.'' This strain relates transient KalTA4 expression during embryogenesis via a KalTA4-mediated autoregulatory mechanism to live adult structures. We demonstrate its use by showing that the secondary octaval nucleus in the adult hindbrain is likely derived from egr2b-expressing cells in rhombomere 5 during stages of early embryogenesis. These data demonstrate prolonged and maintained expression by Kalooping, a technique that can be used for permanent spatiotemporal genetic fate mapping and targeted transgene expression in zebrafish.