Fig. 3. G₂ shortens dramatically with the shift from blastema formation to regenerative outgrowth. (A-C) Longitudinal sections obtained from fins that were regenerating for (A) 24 hours (n=5), (B) 48 hours (n=3), and (C) 72 hours (n=4). Fins were treated with BrdU for 6 hours, followed by detection of BrdU (red) and anti-phosphohistone (H3P) (green), and counterstaining with DAPI (blue) to reveal nuclei. Double labeling appears yellow (arrowheads). (A) The first mesenchymal mitotic cells are seen 24 hours after amputation. (B) The number of cells undergoing mitosis continues to increase by 48 hours. Note that only a small fraction of H3P-positive cells (arrows) are also BrdU-positive 24 or 48 h.p.a., indicating that the duration of the median blastemal G₂ is greater than 6 hours. (C) By 72 hours post-amputation, almost all H3P-positive cells are also BrdU-positive, indicating that the median blastemal G₂ is less than 6 hours. (D) The fraction of labeled mitoses is shown for blastema formation (48 hours h.p.a.; 252 H3P-positive cells counted from 3 regenerates, 0.087±0.033) and during regenerative outgrowth (72 h.p.a.; 337 H3P-positive cells counted from 4 regenerates, 0.991±0.005). Note that the fractions are significantly different. Error bars indicate s.e.m. Z value for Mann-Whitney test is 26.759 (Z_critical is 1.645), P<<0.001.