Methods to Characterize Drosophila Nuclear Receptor Activation and Function In Vivo

Abstract

Publisher Summary This chapter describes the use of transgenic animals that express a fusion of the yeast GAL4 DNA-binding domain (DBD) and the ligand binding domain (LBD) of a nuclear receptor, as a means of determining when and where the corresponding receptor is activated during development. This approach has provided new insights into ecdysteroid activation of the EcR/USP heterodimer and should, as well, facilitate the identification of ligands for orphan nuclear receptors. The chapter also discusses a new approach for functional genomics in Drosophila that has been used to characterize the stage-specific functions of nuclear receptors. This method utilizes the regulated expression of double-stranded RNA (dsRNA) corresponding to the gene of interest, directing targeted degradation of the corresponding mRNA by RNAi. Understanding the molecular mechanisms by which a systemic hormonal signal is refined into different stage- and tissue-specific biological responses requires characterization of the temporal and spatial patterns of nuclear receptor activation during development. With the completion of the Drosophila genome sequence, new methods are required that facilitate the transition from gene sequence to gene function. The use of GAL4-LBD fusions to characterize the temporal and spatial patterns of nuclear receptor activation and the selective inactivation of nuclear receptor gene function by inducible RNAi provide a powerful combination to dissect nuclear receptor-signaling pathways in Drosophila . It is likely that the patterns of nuclear receptor activation provide new insights into ecdysteroid signaling through the EcR/USP heterodimer, as well as new directions for understanding orphan receptor function. These methods greatly facilitate the ability to characterize nuclear receptor signaling pathways in this insect model system.