Figure 2. Cell cycle analysis of label retaining Notch expressing cells in first instar lymph gland of Drosophila.

(A–F) Transient activation of UAS-Histone2B::YFP by hs-Gal4 following scheme in (A) shows labeling of all cells of the embryonic lg at 2 hr post hs ([B–B""]; red; odd; n = 10). (C–F) Subsequent analyses of the rest of the embryos at 12 hr ([C]; n = 18), 13 hr ([D–E]; n = 20) and 18 hr (F; n = 10) AEH, reveals few high YFP cells near DV retaining label (shown by arrow-heads). (G) The dual Fucci system in fly. Cells fluoresce green in G1, red in S and yellow in G2-M phases of cell cycle. (H–H") At 8 hr AEH, N expressing cells are predominantly in G1 (only green) and S (only red) phases of cell cycle, whereas by 13 hr (I–I"), majority of these cells can be seen piling up in S phase (p=0.00121289, n = 28, two tailed unpaired Student’s t-test). Mitosis (cytoplasmic yellow, arrow, I) in these cells can also be detected from 13 hr onwards (p=0.00027463, n = 28, two tailed unpaired Student’s t-test). (J–J") show cell cycle status of N expressing cells at 15 hr, where they can now progress into G2 (yellow (J), p=1.79435E-05, n = 28, two tailed unpaired Student’s t-test) and M phases of mitosis (yellow, arrow head, p=8.49479E-09, two tailed unpaired Student’s t-test). (K–M) PH3 labeling in the lymph gland 13 hr and 15 hr AEH (M) No Fucci signal can be detected at 18 hr AEH (S phase, p=4.17476E-08; G2 phase, p=1.26203E-10; M phase, 2.30293E-07; n = 28; two tailed unpaired Student’s t-test). (N) Quantitative analysis of results from H–M.  Average number of cells in each phase of cell cycle was counted and plotted. DV= Dorsal Vessel. Scale bar = 5 μm. Error bars=SE. Genotypes are shown on top of corresponding panels. DAPI marks the nucleus. Hours after larval hatching are as indicated in each panel.

Figure 2—figure supplement 1. Cell cycle analyses of Notch expressing cells.

(A) Fluorescent ubiquitination based cell cycle indicator (Fucci), a fluorescent probe that labels cells in the S/G2/M phases (redrawn from Nakajima et al., 2011). Accumulation of GFP in the nucleus indicates that the cell is in the S/G2 phase while its distribution into the cytoplasm corresponds to the initiation of M phase. (B–D) At 8 hr AEH, Fucci (green) accumulates in nucleus of N expressing cells ([B]; red; E(spl); n = 31) indicating S/G2 phase of cell cycle (also see Figure 2H–H"). N expressing cells (red, E(spl)) undergo asynchronous division from around 13 hr AEH indicated by cytoplasmic GFP ([C]; Fucci, arrowhead, n = 31, p=8.18287E-09, two tailed unpaired Student’s t-test; also see Figure 2I–I"). (D) No Fucci signal is detectable at 18 hr AEH ([H]; n = 31; p=4.63838E-13, two tailed unpaired Student’s t-test; (also see Figure 2M). (E) Quantitative analyses of the data shown above. The numbers of N expressing cell at different cell cycle stages with respect to cytoplasmic and nuclear GFP (Fucci) are counted for each hour. Error bars=SE. (F–H) N expressing cells undergo mitotic division from 13 hr AEH. (F) shows no PH3 incorporation at 8 hr. (G) Cytoplasmic distribution of Fucci corresponding to PH3 positive cells, is detected at 13 hr AEH. (H) Although there is PH3 incorporation in the lymph gland but no Fucci signal can be detected at 18 hr AEH (anti-phospho-histone H3, red). Scale Bar is  μm . Genotypes are shown on top of corresponding panels. DAPI marks the nucleus. Hours after larval hatching are as indicated in each panel.

Figure 2—figure supplement 2. Absence of apoptosis in Notch expressing cells in first instar lymph gland of Drosophila.

(A–D") Time kinetics of N-Gal4, UAS-Apoliner show no apoptosis in N expressing cells at 8 hr (A–A"; n = 10), 13 hr (B–B"; n = 10), 18 hr (C–C"; n = 10) and 22 hr (D–D"; n = 10) AEH. Scale bar = 5 μm. Genotypes are shown on top of corresponding panels. DAPI marks the nucleus. Hours after larval hatching are as indicated in each panel.