Intravital imaging strategy FlyVAB reveals the dependence of Drosophila enteroblast differentiation on the local physiology

Aging or injury in Drosophila intestine promotes intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation. However, the manner the local physiology couples with dynamic EB differentiation assessed by traditional lineage tracing method is still vague. Therefore, we developed a 3D-printed platform “FlyVAB” for intravital imaging strategy that enables the visualization of the Drosophila posterior midgut at a single cell level across the ventral abdomen cuticle. Using ISCs in young and healthy midgut and enteroendocrine cells in age-associated hyperplastic midgut as reference coordinates, we traced ISC-EB-enterocyte lineages with Notch signaling reporter for multiple days. Our results reveal a “differentiation-poised” EB status correlated with slow ISC divisions and a “differentiation-activated” EB status correlated with ISC hyperplasia and rapid EB to enterocyte differentiation. Our FlyVAB imaging strategy opens the door to long-time intravital imaging of intestinal epithelium. Tang et. al. demonstrate a 3Dprinted platform, FlyVAB, for intravital imaging and visualization of the Drosophila posterior midgut at a single-cell level. This method enables tracking of the stem cell lineage in the midgut of the flies constantly for up to 10 days.