Project Summary Basement membranes are important structural components of most animal tissues, contributing structural support and architectural information to the adjoining cells and cell sheets. Basement membranes are the most ancient form of extracellular matrix, and their structure and composition are highly conserved throughout the animal kingdom. Like every part of a living organism, basement membranes get damaged and need to be repaired. Despite their conserved and ubiquitous nature, there is almost nothing known about how basement membranes are repaired, or how cells sense basement membrane damage to initiate repair. In large part, this paucity of information is caused by the difficulty of finding a suitable model. The ideal model for analyzing basement membrane repair would include conditional genetic approaches to studying gene function, reproducible damage, and a damaged tissue large enough for basic biochemical analysis. We have recently identified such a model for analyzing basement membrane repair, and it forms the basis for this proposal. We have found that feeding dextran sodium sulfate to adult Drosophila causes fast and reproducible damage to the basement membrane surrounding the gut tube, as measured by super-resolution microscopy, TEM, stress-strain analysis, and filtration capacity. The damaged basement membrane also leads to predictable and easily observed changes in gut morphology. Importantly, after withdrawal of dextran sodium sulfate, the basement membrane is repaired and the architecture is restored. Our preliminary data indicate that the basement membrane crosslinking enzyme peroxidase is required for basement membrane repair. In the two Aims of this R21 proposal, we examine the mechanisms by which peroxidase promotes repair, and how cells detect damage and initiate the repair response. These studies are highly novel and will provide some of the first information on the repair of basement membrane, and they will establish a new and powerful model for the continuing analysis of basement membrane repair.