(A–C) A threshold level of Notch activation distinguishes stem cells and enteroblasts. (A) Single-cell measurements of the Notch reporter GBE-Su(H)-GFP:nls from live movies. Cells additionally co-express esg >his2ab::CFP (magenta) and ubi-his2ab::mRFP (gray). GBE-Su(H)-GFP:nls activation is quantified as GFP:RFP (see 'Materials and methods'). For the indicated cells, GFP:RFP = 0.94, 0.27, and 0.01, respectively. (B) GFP:RFP values correlate with visible GFP and nuclear volume. Progenitor (esg+) cells were scored by eye as either GFP-negative (top) or -positive (bottom). In cells without visible GFP, nearly all GFP:RFP values cluster between 0.0 and 0.2, and most nuclear volumes are small (<200 µm3). In cells with visible GFP, most GFP:RFP values are spread between 0.1 and 1.4, and large nuclear volumes (≥200 µm3), indicative of late enteroblasts, are associated with high GFP:RFPs. The blue dotted lines show the 0.17 enteroblast threshold from (C). (C) GFP:RFP values quantitatively distinguish stem cells and enteroblasts. Gray bars show real-time GFP:RFPs for all esg+ cells in two movies (29,102 GFP:RFPs from 251 cells). Two peaks (GFP:RFP = 0.015, 0.528) are separated by a local minimum (blue dotted line; GFP:RFP = 0.17). Purple bars (C’ inset) show real-time GFP:RFPs for ‘benchmark’ stem cells prior to an observed mitosis (1,294 GFP:RFPs from 18 pre-mitotic cells). The benchmark stem cell distribution matches the left peak of the esg+ cells, and 99.6% of ‘benchmark’ GFP:RFPs are less than 0.17. Data in (B) and (C) are aggregated from two movies. (D–E) Stem-like cells transition to enteroblasts over multiple hours. (D) Real-time activation of GBE-Su(H)-GFP:nls reveals a transition from a stem-like to an enteroblast state. During a transition period lasting 6.9 hr (gray background), GFP:RFP increases from a baseline of ~0.049 at t = 3.5 hr to the enteroblast threshold of 0.17 (blue dotted line) at t = 10.4 hr. After the transition, GFP:RFP continues to increase and reaches 0.364 at t = 15.0 hr. GBE-Su(H)-GFP:nls shown in green (top) and inverted gray (bottom); esg >his2ab::CFP, magenta; ubi-his2ab::mRFP, gray. See Video 15. (E) Kinetics of three additional enteroblast transitions. Initial baseline GFP:RFPs are <0.17. GFP:RFPs increase from baseline to 0.17 during transition periods lasting from 2.3 to 6.9 hr (gray backgrounds: t = 0.3–7.1 hr (top), 10.0–12.4 hr (middle), 9.5–11.8 hr (bottom)). Initial and final GFP:RFPs are as follows: 0.058, 0.426 (top); 0.069, 0.281 (middle); 0.022, 0.257 (bottom). All cells in (D) and (E) were born before imaging started. Genotype in all panels: esgGal4, UAS-his2b::CFP, Su(H)GBE-GFP:nls; ubi-his2av::mRFP. All scale bars are 5 µm.