Fig. 5. Steep proliferation gradient is formed between distal-most and proximal blastema at the onset of regenerative outgrowth. (A-C) Longitudinal sections obtained from fins regenerating for (A) 36 hours, (B) 48 hours, and (C, E, F) 72 hours. BrdU incubations were for 6 hours (A-C), 140 minutes (E) and 40 minutes (F). BrdU labeling is red, DAPI is blue. A 20 µm scale bar is aligned with the most distal blastemal cells. (A) Distal portion of 36-hour regenerate showing proliferation in distal blastemal cells (n=4, 39 sections examined). (B) Distal-most blastema is established at the end of blastema formation and the beginning of regenerative outgrowth, 48 h.p.a. (n=3, 36 sections examined). Note the absence of BrdU labeling in the distal blastema. (C) During regenerative outgrowth, 72 h.p.a., the distal-most blastema is limited to 20 µm (n=4, 34 sections examined). Note the absence of proliferation within the distal 10 µm of DMB, and limited proliferation within the next 10 µm of DMB. (D) Differences (±s.e.m.) in the fraction of BrdU-positive cells of between 0-10 µm (left, blue bars) and 10-20 µm (red, right bar) indicate that DMB is established at the beginning of blastema formation and maintained throughout regenerative outgrowth. Z and P values for Mann-Whitney test are shown above each time point. (E,F) Representative fields of short BrdU treatments, (E) 140 minutes (n=4, 35 sections examined) and (F) 40 minutes (n=4, 36 sections examined) are shown. Note with progressively shorter BrdU treatment fewer distal blastema cells are labeled. (G) Fraction of BrdU-positive blastemal cells in 72-hour regenerates during 40 minutes, 140 minutes, and 6-hour treatments are plotted as a function of length from the distal tip of the blastema. Note the decrease in the zone of limited proliferation (double headed arrows of the corresponding color) with increased length of BrdU treatment. (H) Number of H3P-positive cells shown as a function of length from the distal tip of the blastema. Note that the trend closely follows that observed during BrdU incorporation experiments. Stars indicate the most distal scleroblasts. b, blastema; be, basal epidermal layer; dmb, distal-most blastema; m, intraray mesenchyme; pz, blastema proliferation zone; s, scleroblasts.
