Defining functional specificity of stress responses in Drosophila melanogaster

Survival of an organism depends on its perception and response to external stressors such as infection, osmotic stress (ionic or desiccation) or xenobiotic stress. At the cellular level, stress is perceived and relayed via signal transduction pathways that alter transcription or establish new transcriptional programmes that modulate physiology at the whole-organism level to regain homeostasis and promote survival. The vital function of epithelial tissues (e.g., kidneys in vertebrates and Malpighian tubules in insects) is systemic balance of nutrient, solute and water levels. Additionally, epithelial tissues act in sensing stress and relaying signals for adaptation and tolerance to stress. The PhD work presented here is to delineate the roles of Relish and cGMP-dependent kinases in mechanisms of epithelial stress handling using a genetically tractable epithelium, the Drosophila Malpighian tubule, as an in vivo model for stress sensing and response. Relish, a transcription factor, is the insect orthologue of the mammalian NF-kB. It regulates the insect’s innate immune pathway and is highly expressed in D. melanogaster tubules. We show that Relish expressed in Malpighian tubules modulates organismal tolerance to osmotic stress caused due to a high salt diet (salt stress). In order to determine the genes that are involved in salt stress tolerance, Affymetrix Drosophila GeneChips (microarrays) were run with RNA isolated from the wild-type and Relish mutant tubules from flies fed either on normal food, or on ‘salt food ‘. The transcriptomic data was analysed to find genes that were dependent and independent of Relish in response to stress. Additionally, the data also revealed that during salt stress, with the loss of Relish related signalling pathway, the other stress response pathways, in particular, the c-Jun kinase pathway is hyper-activated. This suggests (1) a potential cross-talk occurring between Relish and other stress response pathways, and (2) a redundancy in stress response pathways, for adapting to salt stress. These data demonstrate a novel role for Relish in salt tolerance in Drosophila melanogaster. Moreover, under unstressed conditions, expression of 448 genes was significantly altered and a reduced basal fluid secretion rates were observed in Relish mutant tubules. This suggests that basal Relish activity is required for optimal working of the tissue. In addition, a study to elucidate the immune and osmotic stress-associated roles of cyclic guanosine monophosphate (cGMP)-dependent kinases - Dg1 and Dg2 - in tubules was carried out. Salt stress and desiccation stress survival assays in flies with targeted knock down of each of Dg1 and Dg2 genes in tubule principal cells showed an opposing stress phenotype for these two stressors. No immune phenotype was observed on infection with non-lethal gram negative bacteria. This showed that the cGMP-associated osmotic stress response mechanisms were beneficial or detrimental to survival of organism, depending on the type of stressor and downstream effectors. The understanding gained from this in vivo approach of studying stress pathways in Drosophila Malpighian tubules can be further explored through a systems biology approach . This, together with combinatorial gene knockdown studies to reveal stress network “hubs”, may be applied to development of potential targets of insecticides and in biomedical sciences.