An analysis of DRONC function and its regulation of expression during Drosophila development.

Abstract

Correct development of multicellular organisms requires the programmed removal of supernumerary, redundant, or damaged cells, a process achieved by apoptosis. Apoptosis, or Programmed Cell Death (PCD) is executed by caspases, a highly conserved family of cysteine proteases. The removal of redundant larval tissues during metamorphosis is controlled by the steroid hormone ecdysone. Ecdysone signalling is mediated by the nuclear receptor heterodimer EcR/Usp, which in turn transcriptionally activates a host of transcription factors which then go on to regulate genes essential for PCD, like caspases. The apical caspase dronc is upregulated in the larval midgut and salivary glands prior to their destruction and is dependent on the BRC and E93 transcription factors. To further understand the role of dronc in development, a dronc mutant fly was generated and a transgenic promoter-reporter strategy was employed to investigate dronc regulation. Larval organs from dronc mutants lack dying cells and, when irradiated, fail to show a radiation-induced PCD response. The midguts from dronc mutants undergo apoptosis and have high caspase activity. These data indicate that a droncindependent caspase activation pathway is active in the midgut. Salivary glands from dronc mutants failed to be removed and have reduced caspase activity. Consequently it is clear that the role of DRONC differs significantly between the midgut and salivary glands. The employment of a transgenic dronc promoter-LacZ reporter system identified promoter regions essential for the correct temporal and spatial expression of dronc. A region of the dronc promoter between 1.1kb and 2.8kb has elements essential for LacZ expression in salivary glands. This region was also dependent on the BR-C and E93 transcription factors for salivary gland expression. A functional ecdysone receptor binding element (EcRBE) was identified in the dronc proximal promoter. The EcR-B1 isoform directly binds this EcRBE and is necessary for correct dronc expression in the larval salivary glands. This work revealed some novel findings regarding the role of DRONC in development and the availability of a specific dronc mutant now makes it possible to explore some of the recently published non- poptotic roles of dronc. This work aids in understanding how nuclear hormones control transcription and shows dronc to be an ideal model gene to explore these molecular and genetic processes.