Optimized Gal4 genetics for permanent gene expression mapping in zebrafish

Abstract

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.enhancer trap ͉ fate mapping ͉ Gal4-UAS ͉ secondary octaval nucleus

Transcriptional Activity and DNA Methylation Dynamics of the Gal4/UAS System in Zebrafish

“…Previously, an optimized Gal4/UAS system was established in zebrafish (Distel et al 2009). They compared the activator activity and toxic effect of three Gal4-VP16 activators, GalTA4, TA3, and TA2, which are different in the repeats of VP16 trans-activation domain (13-amino acid core sequence), and it was suggested that the TA4 is a relative highly efficient activator with low toxicity compared with TA2.…”

“…To generate the mylpfa:EGFP transgenic construct, the mylpfa regulatory region was subcloned into the 4xKGFP plasmid (Distel et al 2009) containing the Tol2 transposon and the EGFP coding sequence using the SalI and EcoRI sites. To generate the mylpfa:Gal4 construct, the KalTA4 coding sequence from the 4xKaloop plasmid (Distel et al 2009) was subcloned into the mylpfa:EGFP construct using the EcoRI and NotI sites.…”

“…To generate the mylpfa:Gal4 construct, the KalTA4 coding sequence from the 4xKaloop plasmid (Distel et al 2009) was subcloned into the mylpfa:EGFP construct using the EcoRI and NotI sites. To generate the mylpfa:Gal4;UAS:EGFP construct, a UAS:EGFP fragment was generated by overlap extension PCR (Heckman and Pease 2007) using the 4xKGFP plasmid as the template and a set of four primers (U1, U2, U3, and U4) (Supplemental Table S1); this fragment was subsequently cloned into the mylpfa:Gal4 construct using the BglII and XhoI sites.…”

“…Another remarkable advantage of the Gal4/UAS system is its ability to act as a transcriptional enhancer. The Gal4-VP16 fusion protein and its variant KalTA4 have been shown to be potent transcriptional enhancers that can amplify the expression of UASlinked genes, especially when a weak promoter is used (Iyer et al 2001;Distel et al 2009;Xiong et al 2013).…”

Glycine is able to induce both a motility speed in- and decrease during zebrafish neuronal migration

“…All experiments were carried out using fertilized eggs from the fish line Tg(atoh1a:Gal4TA4) (line hzm2Tg) [6], crosses of Tg(atoh1a:Gal4TA4) with Tg(4xUAS-GFP) (hzm3Tg) [30], or wt brass.…”