An image analysis method for regionally defined cellular phenotyping of the Drosophila midgut

The intestine is divided into functionally distinct regions along the anteroposterior (A/P) axis. How the regional identity influences the function of intestinal stem cells (ISCs) and their offspring remain largely unresolved. We introduce an imaging-based method, “Linear Analysis of Midgut” (LAM), which allows quantitative, regionally defined cellular phenotyping of the whole Drosophila midgut. LAM transforms image-derived cellular data from three-dimensional midguts into a linearized representation, binning it into segments along the A/P axis. Through automated multivariate determination of regional borders, LAM allows mapping and comparison of cellular features and frequencies with subregional resolution. Through the use of LAM, we quantify the distributions of ISCs, enteroblasts, and enteroendocrine cells in a steady-state midgut, and reveal unprecedented regional heterogeneity in the ISC response to a Drosophila model of colitis. Altogether, LAM is a powerful tool for organ-wide quantitative analysis of the regional heterogeneity of midgut cells.