P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster

Abstract

We have developed a method for turning on and off the expression of transgenes within Drosophila in both time and space. Two different enhancer detector elements carrying an RU486-inducible form of the yeast transcription factor GAL4 were constructed and used to generate enhancer detector lines. These lines were screened for RU486-inducible reporter gene expression in the adult head. We identified lines that exhibit inducible expression in many cell and tissue types, verifying that the elements respond to nearby enhancers. No expression was detected in the absence of the ligand. The P[Switch1] element responded to genomic enhancers less efficiently than P[Switch2] but produced more specific patterns of expression. Two P[Switch] lines were used to ablate fat body tissue in adult females through the induced expression of diphtheria toxin. These females were sterile, which correlates with fat body loss, and they died prematurely.

Figure 1

Structure of the P{Switch} enhancer detector elements. (A) P{Switch1}. The Gene-Switch cassette was inserted downstream of the P-transposase promoter and transcriptional start site. The translational start site is within the Gene-Switch cassette. The positions of the LoxP sites are shown below the transposon and are marked by arrows. The 5′ and 3′ P-element inverted repeats are illustrated as triangles. (B) P{Switch2}. The Gene-Switch cassette was cloned in frame at an EcoRI site within the P-transposase gene.

Figure 2

P{Switch} elements provide inducible gene expression. Frontal head sections of S₁32/P{UAS-lacZ.B}, uninduced (A) and induced (C). Robust fat body expression is observed after induction. The third antennal segment of S₁155/+; P{UAS-lacZ.B}, uninduced (B) and induced (D).

Figure 3

P{Switch} enhancer detectors induce reporter expression in a variety of tissues and cell types. (A) Line S₂6 shows expression specifically in the medulla. (B) S₂1 shows expression near the antennal nerve (arrow). (C) S₁44 drives expression in the ocellar ganglion. (D) S₁13 expression is found within the neurolemma. (E) S₁6 is expressed in a diffuse pattern throughout the central brain and optic lobes. (F, G, and K) Gene-Switch in S₁6, S₁145, and S₂33 is expressed in nonneuronal cell types. Shown are 20 μM cyrosections in which all neurons are labeled by anti-embryonic lethal abnormal vision (ELAV) in red. Nuclear localized β-gal, corresponding to P{Switch} expression, is labeled in green. (F) S₁6 frontal section. The β-gal positive nuclei lie just inside the cellular cortex. (G) S₁145 optic lobes. Several large nuclei are labeled on the perimeter of the optic lobes with smaller nuclei just inside the layer of neuronal cell bodies. (K) S₂33 third antennal segment. The β-gal-positive nuclei are distinct from neurons in the antenna. S₁86 is expressed within both the maxillary palps (H) and the third antennal segment (I). (J) S₂33 exhibits broad expression in the third antennal segment as detected by β-gal histochemistry

Figure 4

The induction of β-gal in P{Switch1} line S₁6. In A, S₁6/P{UAS-lacZ.B} flies were fed RU486 for the indicated times (blue portion of timeline) and then transferred to food without RU486 (yellow portion of timeline). All heads were cut at 24 h. In B, all flies were fed for 1 h and then transferred to food without RU486. Heads were sectioned at the time indicated.

Figure 5

Induction of diphtheria toxin (DTI) within adult fat bodies produces tissue ablation. Sagittal cryosections of P{Switch} abdomens are shown. Arrowheads point to areas that contain the dispersed fat body tissue. A–D are stained for β-gal activity. E–H are stained with hematoxylin and eosin. I and J are fixed and stained with osmium tetroxide. (A) S₁106/P{UAS-lacZ.B}, uninduced (B) P{UAS-lacZ.B}/+; S₂30/+ uninduced. (C) S₁106/P{UAS-lacZ.B} induced. (D) P{UAS-lacZ.B}/+; S₂30/+ induced. (E) S₁106/P{UAS-DTI} uninduced. (F) P{UAS-DTI}/+; S₂30/+ uninduced. (G) S₁106/P{UAS-DTI } induced. (H) P{UAS-DTI}/+; S₂30/+ induced. (I) S₁106/P{UAS-DTI} uninduced. (J) S₁106/P{UAS-DTI} induced.