In this R21 high risk/high reward application, we propose to develop a new genetic tool with numerous potential applications in Drosophila research. We plan to develop a fly genetic circuit called FlpOn that will use transient photo-activation of user-selected cells to permanently alter gene expression in those selected cells. Because the FlpOn circuit harnesses components of the modular Gal4/UAS expression system that is already heavily used by the Drosophila community, it will integrate seamlessly with existing tools, allowing selective expression of literally tens of thousands of existing constructs promoting gene overexpression or RNAi knockdown. This tool will enable mosaic gene expression studies with unrivaled spatial resolution and experimental flexibility - harnessing the power of optogenetics in a modular format.
The first Aim i s devoted to developing and optimizing the FlpOn circuit.
The second Aim uses this new tool for pioneering studies in wound healing, allowing us to ask questions that were previously intractable. We will identify the upstream trigger and the downstream effects of a fast-acting calcium wave that is activated upon epithelial wounding. To do so, we will use the FlpOn circuit to manipulate gene expression in a small ring of cells, user laser ablation to wound normal cells in the interior of this ring, ad follow the outward propagation of the calcium wave through and beyond the altered ring of cells. This proposal stems from an ongoing collaboration between Andrea Page- McCaw (PI), an experienced Drosophila geneticist and cell biologist, and M. Shane Hutson (co-I), a physicist with expertise in optogenetics and tissue mechanics. The two labs share an interest in wound healing and each brings unique tools to the collaboration. At the end of the two-year study, we expect to have both generated a genetic tool that can be used widely throughout the fly community and demonstrated its unique effectiveness in a study of the epithelial wound response.
Much of our current understanding of cell and tissue function comes from genetic studies where genes are turned on or off in only some cells, followed by analysis of how the changed cells interact with surrounding normal tissue. Unfortunately, the tools for altering gene expression in a specific subset of cells are clunky and do not usually allow the researcher to choose exactly which cells will be affected. We propose to make a new light-activated genetic tool that will allow researchers to choose precisely which cells will have altered gene expression in a living animal (Aim 1) and to demonstrate the utility of this system in a study of wound-healing (Aim 2).
| Shannon, Erica K; Stevens, Aaron; Edrington, Westin et al. (2017) Multiple Mechanisms Drive Calcium Signal Dynamics around Laser-Induced Epithelial Wounds. Biophys J 113:1623-1635 |
