Refined LexA transactivators and their use in combination with the Drosophila Gal4 system

Abstract

The use of binary transcriptional systems offers many advantages for experimentally manipulating gene activity, as exemplified by the success of the Gal4/UAS system in Drosophila. To expand the number of applications, a second independent transactivator (TA) is desirable. Here, we present the optimization of an additional system based on LexA and show how it can be applied. We developed a series of LexA TAs, selectively suppressible via Gal80, that exhibit high transcriptional activity and low detrimental effects when expressed in vivo. In combination with Gal4, an appropriately selected LexA TA permits to program cells with a distinct balance and independent outputs of the two TAs. We demonstrate how the two systems can be combined for manipulating communicating cell populations, converting transient tissue-specific expression patterns into heritable, constitutive activities, and defining cell territories by intersecting TA expression domains. Finally, we describe a versatile enhancer trap system that allows swapping TA and generating mosaics composed of Gal4 and LexA TA-expressing cells. The optimized LexA system facilitates precise analyses of complex biological phenomena and signaling pathways in Drosophila.

Fig. 1.

(A) Three examples of experiments that require two independent binary transcriptional systems. (i) The areas A (green) and B (blue) represent two interacting cell populations defined and manipulated by a-Gal4 and b-LexA TA, respectively. Variations in the balance between the activities of Gal4 and a LexA TA allow manipulation of the interaction. (ii) If the a promoter activity is influenced by the manipulation of A then a-Gal4 (green) could be affected. This problem can be circumvented if the a-Gal4 activity is irreversibly converted to a constitutive c-LexA TA activity by using the Flp/FRT technique: e.g., Flp driven by a-Gal4 removes the FRT-flanked transcriptional termination cassette (>stop>) from c>stop>LexA TA, giving rise to expression of c-LexA TA (orange) in the area A independent of the a-Gal4 activity. (iii) Flp driven by d-LexA TA removes the >stop> from UAS>stop>X in D (magenta). The UAS>X is activated in E (yellow) at the intersection of A ∩ D because of Gal4 expression in A. (B) Schematics of the Gal4 (G4) and LexA TAs used in this study. GAD (G), G4 AD; GDBD, G4 DBD; H, G4 hinge region; L, LexA; TP, Thr860 to Pro modification in Gal4; VPcAD, VP16 complete AD; VPmAD, VP16 minimal AD; V_n, n tandem copies of the VPmAD; V₁, one copy of the VPmAD flanked by two mutated VPmADs (VPmAD^FG and VPmAD^FY indicate substitutions of phenylalanine to a glycine or a tyrosine) (13).

Fig. 2.

(A–K) The activity of the different dpp-LexA TAs. Representative fluorescence images showing expression of rCD2::GFP (mGFP; green) in a wing disc heterozygous for lexO-rCD2::GFP; dpp-LexA TA at 18 °C. Nuclei were stained with DAPI (DNA; blue). (Scale bars: 50 μm.) (L and M) dpp-LV16-86Fb heterozygous animals display a morphological defect, whereas dpp-LHV₃-86Fb heterozygotes do not. Arrows show leg truncation. (N) Relative activities of less detrimental LexA TAs and G4 compared with LG, analyzed by dual luciferase assay in wing discs. Activities of LexA TAs at 25 °C and that of G4 at 18 °C are shown. Numbers above the bars indicate activity relative to LG. Gal80-suppressible LexA TAs are orange.

Fig. 3.

Using the LexA system in conjunction with Gal4. (A–C) A screening system for genes involved in Dpp signaling/transport. At the restrictive temperature for tub-Gal80^ts, dpp-LHG drives lexO-Egfp::dpp and en-G4 drives UAS-X^IR (IR, inverted repeat to induce RNAi). (B and C) Third instar wing discs from larvae with the indicated genotypes. The system was activated by a temperature shift to 29 °C at the early larval stages. Egfp::Dpp (green) driven by dpp-LHG causes overgrowth of the wing discs. pMad (red) reports the status of Dpp signaling. En (yellow) defines P compartment. (C) tkv^IR driven by en-G4 results in reduction of pMad levels in the P compartment and of size in the pouch. (D and E) Using the CONVERT technique to fix an expression pattern. In the conventional G4-based system, expression of tkv^QD by brk-G4 decreases brk promoter activity, leading to suppression of the brk-G4 activity and variable tkv^QD levels due to an artificial feed back loop. The CONVERT system converts brk-G4 activity to constitutive LHV₂ activity. At the restrictive temperature for tub-Gal80^ts, Flp expressed by brk-G4 irreversibly removes the >y⁺> from act >y⁺>LHV₂. The resulting act>LHV₂ clones in the brk domain will now constitutively express tkv^QD, irrespective of the later status of brk-G4. (E) Third instar wing discs in which Dpp signaling in the brk domain is manipulated. mGFP (mCD8::GFP; green) and mRFP (mCherry::CAAX; red) are expressed under control of brk-G4 and act>LHV₂, respectively. Genotypes of the larvae, and the conditions for the temperature shift and cycles are shown. In the temperature shift conditions, most cells of the brk domain with tkv^QD lost mGFP expression because of suppression of brk-G4 activity. Nuclei were stained with DAPI (DNA; blue). Schematics in A and D: red and blue lines represent positive and negative signals, respectively. (Scale bars: 50 μm.)

Fig. 4.

The G-MARET system. (A) Schematics of the G-MARET system. P{≥G4}^mr, a P element insertion of the G (Gal4)-MARET (mr), which contains a loxP site (≥) and an attP site. G4 expression is driven by an enhancer in the genomic vicinity. P{≥G4,y⁺≥LexA TA}^mr, a derivative of P{≥G4}^mr in which a LexA TA together with another loxP site and yellow gene (y⁺) has been introduced by the ϕC31 site-specific integration system. The ≥G4,y⁺≥ constitutes a loxP cassette, which can be removed by a Cre recombinase. In the modified P element, G4 but not the LexA TA, is still under control of the genomic enhancer. P{≥LexA TA}^mr, the P element after removal of the loxP cassette in the P{≥G4,y⁺≥LexA TA}^mr. (B) Expression of GFP and RFP that is under the control of a G-MARET insertion, mr43 ≥ G4, and its derivatives in the wing discs. A UAS-mGFP (mCD8::GFP; green) lexO-mRFP (mCherry::CAAX; red) was crossed to the mr43≥G4 or its derivatives, with or without hsp70-cre, as indicated. mr43≥LHG was established from a progeny of the cross of mr43≥G4≥LHG with hsp70-cre, followed by heat shocking. The magnified dotted area highlights the mosaicism of G4 and LHG clones. Nuclei were stained with DAPI (DNA; blue). (Scale bars: 50 μm.)