Fig. 6. Early proliferating mesenchymal cells contribute to the blastema. Fin sections from BrdU (red) pulse-chase experiments are shown. Fish were pulsed with BrdU for 2 hours at (A) 16 h.p.a., (B) 36 h.p.a. and (C) 70 h.p.a. and chased until harvesting at 72 hours. Fluorescent sections were counterstained with DAPI (blue) to reveal nuclei. (A) When a 2-hour BrdU pulse was given during early blastema formation (16 h.p.a.), staining is most apparent in the DMB and apical epidermis (arrows), including the basal epidermal layer. Note that BrdU label is diluted, indicating that blastemal cells divided before segregating into the DMB. BrdU label is even more diluted in the proximal parts of the regenerate (small dots, indicated by arrowheads) during outward growth of the regenerate. (B) When fish were pulsed at the end of blastema formation (36 h.p.a.) BrdU label in the DMB is mostly undiluted, while it is diluted in the proximal blastema. (C) By contrast, a late pulse of BrdU did not result in staining in the DMB (72 h.p.a.), indicating the absence of DNA synthesis during outgrowth. (D) A bar graph of the fraction of BrdU-positive cells in the DMB (0-20 µm) for the various 2-hour pulse times, 12 h.p.a., 16 h.p.a., 28 h.p.a., and 36 h.p.a.. Note that the fraction of BrdU-positive DMB cells is greatest when a pulse occurs late in blastema formation (36-38 h.p.a.), indicating that blastemal cells segregate into DMB late in blastema formation. For each case, the number of cells in the DMB from pulse-chase experiments was compared to 70-72 hour BrdU-treated fins using a Mann-Whitney test. P and Z values are: P<0.05, Z=1.649; P<<0.001, Z=7.558; P<<0.001, Z=9.518; P<<0.001, Z=11.249 for pulses administered at 12, 16, 28, and 36 hours respectively (Zcritical=1.645). Error bars indicate s.e.m. Scale bars: 50 µm.
