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.
Distel
Martin
M
Helmholtz Zentrum München, Institute of Developmental Genetics, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany.
Wullimann
Mario F
MF
Köster
Reinhard W
RW
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation; D013194:Staining and Labeling; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
D004268:DNA-Binding Proteins; C542436:Gal4 protein, zebrafish; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins