Exploring Regeneration Mechanisms in Drosophila Melanogaster: Testing a Novel 3rd Ablation System with Glur1

Drosophila melanogaster, known as fruit flies, are widely studied in genetics due to their ability to regenerate in the larval stage. The wing imaginal disc of Drosophila has been instrumental in understanding the complex process of tissue regeneration. To study tissue regeneration, damage can be induced in the disc through genetic manipulation, physical damage, or irradiation. Genetic methods have been deployed by expressing genes capable of inducing various cell death modalities, such as apoptosis or necrosis. The GAL4/UAS system allows for precise, tissue-specific expression of such genes upon exposure to an increase in temperature. A second system, known as Duration and Location (DUAL) control, was developed that allows for both UAS expression and cell death to occur simultaneously by using ablation tools that are activated by a heat shock. At the same time, DUAL control uses similar tools from the first system to allow for UAS manipulation to occur in the tissue around the dying cells. While the current genetic methods provide powerful tools for studying cell ablation and regeneration in Drosophila wing discs, they come with notable limitations. The first system is inherently limited in that it does not facilitate the simultaneous expression of RNAi during the time of cell ablation. The primary limitation of DUAL control lies in its inability to separate the timing of cell ablation from UAS expression. The activation of both processes at the same time means that we cannot temporally separate the induction of UAS manipulation from the onset of cell death. Their limitations create a need for a newer system that offers control over the timing and specificity of gene manipulation during regenerative processes. In this work, we attempt to create a third ablation system that allows for separation of ablation and gene expression. Utilizing both a heat shock and heat shift, this new system could potentially allow for a higher level of control in regenerative experiments. The system was built by crossing Drosophila stocks containing the necessary components, incorporating Gal80ts and Gal4tp to allow for two ways of temporal control. We tested the system to determine a protocol and confirm that all components were working correctly. Our results found that the system is viable when the heat shock is done prior to the heat shift, however the long exposure to higher temperatures when the heat shift is done first causes the system to express genes of interest prematurely. Despite this, the development of this third ablation system still offers promising potential for achieving greater temporal control and enhancing the precision of regenerative studies.