Temporal And Spatial Requirements Of Drop-Dead Expression For Adult Survival In Drosophila Melanogaster
Date of Award
Doctor of Philosophy (PhD)
Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, fragile tracheae, gut dysfunction, reduced body mass, and female sterility. The cause of early lethality and the function of the drd protein remain unknown. To elucidate a molecular function of DRD, the temporal and spatial requirements of the drd gene for survival were determined. drd expression was manipulated with the Gal4-UAS system, using UAS-RNAi transgenes and a UAS-drd transgene on a drd mutant background to knock down and rescue expression of drd, respectively.
To investigate the temporal requirements of drd expression for adult survival, the UAS-RNAi and UAS-drd rescue line were controlled with the Heat Shock Protein 70-Gal4 driver. Flies were heat-shocked at different stages of their lifecycle, and the survival of the resulting adult flies was assayed. Expression of drd during the second half of metamorphosis was necessary and sufficient to prevent rapid adult mortality.
To determine the spatial requirements of drd expression for adult survival, drd expression was knocked down with various tissue-specific Gal4 drivers and the progeny were screened for early lethality. Neurodegeneration and early lethality and gut dysfunction and early lethality were observed upon knockdown of drd in the tracheae and anterior cardia, respectively. The tracheal phenotype in drd mutants is hypothesized to cause hypoxia-induced neurodegeneration. Hypoxia-sensitive reporter transgenes were placed on a drd mutant background, but expression of these reporters was not observed, suggesting these flies are not hypoxic. Rescue of drd expression in the tracheae rescued neurodegeneration but not adult lethality. However, rescue of drd in the tracheae and anterior cardia rescued both causes of early lethality, neurodegeneration and starvation, indicating that drd expression in these two tissues is sufficient for survival.
From these data, it is hypothesized that drd is required for extracellular matrix (ECM) integrity. Both critical tissues produce an ECM; tracheae produce a cuticle to line the lumen and the anterior cardia synthesizes the peritrophic matrix, which lines the midgut. Additionally, cuticle processing occurs in metamorphosis, the critical period for drd expression. Future direction will focus on establishing a role for DRD in ECM synthesis.