A conditional tissue-specific transgene expression system using inducible GAL4

Abstract

In Drosophila, the most widely used system for generating spatially restricted transgene expression is based on the yeast GAL4 protein and its target upstream activating sequence (UAS). To permit temporal as well as spatial control over UAS-transgene expression, we have explored the use of a conditional RU486-dependent GAL4 protein (GeneSwitch) in Drosophila. By using cloned promoter fragments of the embryonic lethal abnormal vision gene or the myosin heavy chain gene, we have expressed GeneSwitch specifically in neurons or muscles and show that its transcriptional activity within the target tissues depends on the presence of the activator RU486 (mifepristone). We used available UAS-reporter lines to demonstrate RU486-dependent tissue-specific transgene expression in larvae. Reporter protein expression could be detected 5 h after systemic application of RU486 by either feeding or "larval bathing." Transgene expression levels were dose-dependent on RU486 concentration in larval food, with low background expression in the absence of RU486. By using genetically altered ion channels as reporters, we were able to change the physiological properties of larval bodywall muscles in an RU486-dependent fashion. We demonstrate here the applicability of GeneSwitch for conditional tissue-specific expression in Drosophila, and we provide tools to control pre- and postsynaptic expression of transgenes at the larval neuromuscular junction during postembryonic life.

Figure 1

The GeneSwitch/UAS expression system in Drosophila. Driver lines expressing the transcriptional activator GeneSwitch in a tissue-specific fashion are crossed to UAS-reporter lines with genomic inserts of a target gene fused to five GAL4-binding sites arrayed in tandem (5× UAS). In the absence of an activator, the GeneSwitch protein is expressed in target tissues but remains transcriptionally silent (black); Gene X is therefore not expressed. However, after systemic application of RU486 (red), the GeneSwitch protein becomes transcriptionally active (blue), mediating expression of gene X (green) in only those tissues expressing GeneSwitch. [Reproduced with permission from ref. (Copyright 1993, The Company of Bioligists Limited).]

Figure 2

RU486 activates GeneSwitch in the larval nervous system. (A–D) Confocal images of fixed bodywall preparations of Drosophila third instar larvae expressing cytosolic GFP (green channel) from a neuron-specific GeneSwitch driver. Larvae were raised in the presence (A, B) or absence (C, D) of RU486. Counterlabeling is for the nuclear localized even skipped (red channel). (E–G) Confocal images of unfixed, whole-mount first (E), second (F), or third (G) instar larvae expressing UAS-EGFP from ELAV-GeneSwitch. GeneSwitch was activated in embryos by feeding mothers RU486, but first instar larvae were transferred to, and developed on, normal food. (CNS, central nervous system; s, sensory neurons; n, nerves; sy, neuromuscular synapses.) (Bar = 100 μm.)

Figure 3

RU486-mediated expression starts rapidly and is dose-dependent. Quantitative Western blot analysis of GFP protein. Genotypes of the larvae are ELAV-GeneSwitch; UAS-EGFP if not otherwise indicated. UAS-EGFP;+ (UAS) and +;ELAV-GeneSwitch (GS) are parental lines; ELAV-GAL4;UAS-EGFP animals have constitutively active GAL4 in all neurons serve as a positive control (+ control, +C). (A) Timecourse of GFP protein (arrow) expression (hours after “larval bathing”) and (B) dose dependence of GFP protein expression on RU486 concentration (μg/ml) in larval food. (C) Qantitative analysis of Western blots in A (light gray, “Time”) and B (dark gray, “Dose”) normalized to positive control (ELAV-GAL4;UAS-EGFP, black).

Figure 4

Both basal transcriptional activity of ELAV-GeneSwitch and toxicity of RU486 are low. The viability of embryos expressing either a UAS-TNTE reporter (black bars, TnTx[e]) or a control UAS-EGFP reporter (gray bars, EGFP) from the ELAV-GeneSwitch driver is shown as a function of RU486 concentrations in the parents' food. The percentage of UAS-TNTE/ELAV-GeneSwitch first instar larvae surviving to adulthood in the absence of RU486 is shown as a hatched bar (TnTx[a]).

Figure 5

RU486 induces transgene expression in larval bodywall muscles. (A) Expression of UAS-EGFP driven by MHC-GeneSwitch in bodywall muscles of first (Left), early third (Center), and late third instar larvae (Right) in the presence (Bottom) or absence (Top) of RU486. The bodywall musculature was imaged through the cuticle in undissected larvae. Muscle fibers 7, 6, 13, 12, and 5 are labeled; first instar larva or muscles are outlined in red in uninduced animals for better visibility. (B) Schematic diagram for two electrode voltage-clamp analyses (Upper) and EKO-channel localization predominantly to NMJs in fixed bodywall preparations from third instar UAS-EKO/MHC-GeneSwitch larvae (Lower). (C) Representative current traces and (D) current–voltage relationship obtained under zero Ca²⁺ conditions for uninduced (blue traces and curves) or induced (red traces and curves) UAS-EKO/MHC-GeneSwitch animals (left column in C, squares in D) or UAS-EGFP/MHC-GeneSwitch controls (right column in C, circles in D).