In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila

Abstract

The recent establishment of high-throughput methods for culturing Drosophila provided a unique ability to screen compound libraries against complex disease phenotypes in the context of whole animals. However, as compound studies in Drosophila have been limited so far, the degree of conservation of compound activity between Drosophila and vertebrates or the effectiveness of feeding as a compound delivery system is not well known. Our comprehensive in vivo analysis of 27 small molecules targeting seven signaling pathways in Drosophila revealed a high degree of conservation of compound activity between Drosophila and vertebrates. We also investigated the mechanism of action of AY9944, one of the Hh pathway antagonists that we identified in our compound feeding experiments. Our epistasis analysis of AY9944 provided novel insights into AY9944's mechanism of action and revealed a novel role for cholesterol transport in Hh signal transduction.

Electronic supplementary material: The online version of this article (doi:10.1007/s12154-010-0051-5) contains supplementary material, which is available to authorized users.

Keywords: Chemical genetics; Compound feeding; Drosophila; Drug discovery; Signal transduction.

Fig. 1

Signaling pathway components used to test gene–compound interactions. Key components of Hh (a), insulin/PI3K (b), Ras/MAPK (c), JNK (d), Wnt (e), cell cycle (f), and apoptosis (g) are depicted. Arrows indicate activation and T bars represent inhibition. For simplicity, only the core components of each pathway are shown. Pathway components used for gene–compound interaction experiments are in blue and compounds are in red

Fig. 2

Summary of observed gene–compound interactions. Phenotypic read-outs tested for modification by compound feeding for Hh (a), insulin/PI3K (b), Ras/MAPK (c), JNK (d), and Wnt (e) pathways, as well as cell cycle (f) and apoptosis (g), are shown. The enhancement of a phenotype by a compound is shown in yellow and the suppression in green (n > 50 for each experiment). Phenotypes that are not modified by compound feeding are in gray and interactions that are not tested are in white. The phenotypes used in control experiments to test for specificity are in blue

Fig. 3

Hh pathway antagonists inhibit the expression of the Hh target gene engrailed in the wing imaginal disk. a Schematic representation of the expression patterns of Hh pathway components and domain of Hh signaling activity in the wing imaginal disk. Hh is expressed in the posterior compartment [12] (P, green) and travels into the anterior compartment (A, yellow) to activate signaling [47] (blue arrows). There is no Hh signaling in the posterior compartment as the transcriptional effector Ci is only expressed in the anterior compartment [35]. Hh signaling activates the expression of en in a narrow domain (yellow/green stripes) just anterior to the A/P boundary [47] (red line). en expression in the posterior compartment is independent of Hh signaling. b Graphic representation of the Hh misexpression assay. Misexpression of Hh throughout the wing imaginal disk (blue bar) leads to the ectopic activation of en expression throughout the anterior compartment (green arrows). c–len expression in wing disks from larvae of genotype 69B-gal4, gal80ts>UAShh. c, d No ectopic en is detectable in disks from larvae raised at 18 °C (gal80ts active). d–len expression in the wing pouch area of the wing imaginal disk (outlined by a yellow rectangle in c). e Ectopic en is induced in the anterior compartment as a result of Hh misexpression after a temperature shift to 29 °C for 48 h. f–len expression in disks misexpressing Hh incubated with DMSO (f), AY9944 (g), Hh(Ant)-1 (h), SANT-1 (i), as well as IBMX and forskolin in combination (j) for 24 h. AY9944, Hh(Ant)-1, and SANT-1 all lead to a strong reduction of ectopic en induced by Hh misexpression (g–i) while incubation with DMSO alone has no effect on ectopic en expression (f). IBMX and forskolin can only inhibit en expression together

Fig. 4

Cholesterol transport inhibitors inhibit Hh-induced Ptc internalization and en expression. a Cholesterol distribution in compound-treated salivary glands monitored by filipin staining. b Wild-type salivary gland cells show membrane-localized Ptc (green, left-most panel, top) and no en expression (green, left-most panel, bottom). Right panels, Ptc localization (top) and en expression (bottom) in compound-treated salivary glands misexpressing Hh. Nuclei are shown in red

Fig. 5

Epistasis analysis between AY9944 and Hh pathway components. a, b Wing imaginal disks carrying ptc^-/- (a) or pka^-/- (b) clones treated with AY9944 or Hh(Ant)-1 as control and labeled with anti-En antibody (red). Mutant cells are marked by the absence of GFP (green). c, d Wing imaginal disks misexpressing ptc.DN (c) or Hh.N (d) are treated with AY9944 or Hh(Ant)-1 and labeled with anti-En antibody. Red arrows indicate where en expression is ectopically activated. e Ptc localization (green, top panels) and en expression (green, bottom panels) in compound-treated salivary glands misexpressing Hh.N. Nuclei are shown in red

Fig. 6

Cholesterol transport inhibitors chloroquine and imipramine behave identically to AY9944. aen expression in wing imaginal disks misexpressing Hh (top), hh.N (middle), and Ptc.DN (bottom) treated with DMSO, chloroquine, or imipramine. b Ptc localization (green top panels) and en expression (green, bottom panels) in salivary glands misexpressing Hh.N treated with DMSO, chloroquine, or imipramine. Nuclei are shown in red