(a) The small cells with high RFP and low dGFP signals are positive for the enteroendocrine (EE) cell marker Pros, suggesting that these cells are differentiated cells that turn off esg. (b) Patterns of the dynamic reporter driven by esg-Gal4 in the Drosophila intestine at different developmental stages. In the second instar, the adult midgut progenitors (AMPs) exist as individual cells and disperse throughout the intestine. Using TransTimer, we observed considerable variation in the dGFP/RFP ratio, indicating an unexpected heterogeneity in this cell population. In the third instar intestine, the AMPs proliferate and form clusters called midgut imaginal islands (MIIs). Some AMPs with high Notch activity start to differentiate into peripheral cells (PCs), which surround the AMPs to create a transient stem cell niche. Consistent with this model, cells at the periphery of MII showed more red color, suggesting that these cells started to differentiate and lost their stemness. During the early pupal stage, the MIIs break down, while PCs and a large portion of AMPs differentiate into pupal intestine cells, which will be degraded at the end of the pupal stage. Consistent with this, many esg +AMPs turn red as these cells start to differentiate. During metamorphosis, the larval intestine is completely turned over, and the late pupal intestine, which will become the future adult intestine, is newly generated from AMPs. Therefore, except for the ISCs that still maintain the dGFP signal, differentiated gut cells all show the RFP signal, suggesting that they were recently derived from esg +AMPs. During the adult stage, esg +ISCs and enteroblast (EBs) are mitotically inactive and predominately show a yellow color. However, in the presence of the damaging reagent Bleomycin, which triggers strong cell proliferation and differentiation, a significant separation between dGFP and RFP signal is observed. Scale bar: (a, b) 50 μm.