Fig 3

(A) The 2 injections label the same cells in S-phase for small-labeling intervals, leading to NSCs that are both EdU and BrdU positive, denoted as double-labeled S-phase (DLS). (B–E) Example DLS (yellow arrow) for a labeling interval Δt = 9 h. (F) The DLS proportion is high for Δt = 9 h and decreases rapidly with increasing Δt. Each dot represents the value for 1 brain hemisphere. (G) After a division, 1 of the daughter cells already labeled by the Time 1 label can enter in a new S-phase and incorporate a second label. This cell thereby redivides. (H–S) Three examples of redividing NSCs with labeling intervals of 24 h (H–K), 48 h (L–O), and 72 h (P–S). Scale bar: 10 μm. (T) The proportions of redividing NSCs within the dividing NSCs at Time 1 remain high from Δt = 24 h to Δt = 72 h labeling intervals. Each dot represents 1 brain hemisphere. (U) Only 1.9±1.7% of randomly drawn divisions (same amount as observed per hemisphere) from all NSCs would be redrawn at random, while 14±8% of observed NSCs in S-phase reenter S-phase (p = 9.4 ×·10⁻¹⁰, 2-sample Kolmogorov–Smirnov test). Box plots range from the 25th to the 75th percentile, and the central mark indicates the median and whiskers include points that are not more than 1.5 times the interquartile range away from the top or bottom of the box. BrdU, 5-bromo-2′-deoxyuridine; DLS, double-labeled S-phase; EdU, 5-Ethynyl-2′-deoxyuridine; GFP, green fluorescent protein; NSC, neural stem cell.