A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in Drosophila

Abstract

The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (LexAop) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.

PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine

“…3a,b ). To carry out a neuron-specific assay of the effects of increased DA expression, we induced extra TH production, and hence more DA synthesis, within DAergic neurons in 35-day-old males by feeding drug-sensitive mutant flies engineered with the LexPR / LexAop gene expression technique 21 ( UAS-LexPR;TH-Gal4 > LexAop-TH ) ( Fig. 3c ) 1.5 mM RU486 for 5 days.…”

“… 18 ) (gift from Mark Wu, Johns Hopkins University), NP3024-Gal4 and NP5945-Gal4 (#113066, #105062; purchased from the Drosophila Genetic Resource Centre, Kyoto Institute of Technology, Japan), UAS-thRNAi (#108879; purchased from the Vienna Drosophila Resource Centre, Vienna, Austria). The UAS - and LexAop -effector strains used were UAS–TH 52 , UAS-LexPR-attp40 21 , UAS – mCD8 :: GFP , UAS-ReaChR , LexAop-ReaChR (#5137, #5130, #53748, #53747; purchased from the Bloomington Drosophila Stock Centre, University of Indiana, IN, USA). The LexAop-mKO and fru-Gal4 (a gift from Ann-Shyn Chiang, NTHU, Taiwan) and the LexAop-GeneSwitch-attp40 (ref.…”

“…The LexAop-mKO and fru-Gal4 (a gift from Ann-Shyn Chiang, NTHU, Taiwan) and the LexAop-GeneSwitch-attp40 (ref. 21 ) were obtained from the laboratories that generated them. Our laboratory generated the LexAop-TH-attp2 , LexAop-TH-attp40 , LexAop-thRNAi-attp2 , LexAop-thRNAi-attp40 , LexAop-FLP-attp3 , UAS-frt-stop-frt-TH-attp40 transgenic lines used in this study.…”

“…Whole-mount immunolabelling of the adult brain was performed as previously described 21 . In brief, the dissected brains were fixed in 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) left on ice for 30 min before beginning the dissection.…”

Parallel circuits control temperature preference in Drosophila during ageing

“…The pP[UAS-AI-TH]: pUAST-TH clone was provided by Sean B. Carroll (HHMI). It contained EcoRI and KpnI cutting sites to release the full TH gene, after which this region was cloned into the EcoRI and KpnI sites of the pP[UAST-AI] vector 37 to create the pP[UAS-AI-TH] transgene.…”