Fig. 2. Fin regeneration blastema consists of two functionally distinct regions, a non-proliferating distal and proliferating proximal blastema. Fish were incubated with BrdU for 6 hours before harvesting their fins. Longitudinal sections were stained with BrdU antibody (brown signal indicated by black arrowheads) and then counterstained with Hematoxylin. (A) 6 hours post-amputation (h.p.a.), a thin layer of wound epidermis covers the stump. The region immediately proximal to the amputation plane shows basal levels of epidermal proliferation; no BrdU-positive cells are detected in the intraray mesenchyme. (B) Early signs of mesenchymal disorganization are seen 12 h.p.a. (bracket). The number of proliferating epithelial cells is at basal levels, but there is no proliferation in the mesenchyme. (C) BrdU-positive mesenchymal cells were first detected 18 h.p.a. (arrowheads). Wound epidermis 24 h.p.a. thickens and the basal epidermal layer is initiated. (D) The number of BrdU-positive mesenchymal cells continues to increase, with some positive cells detected distal to the amputation plane. In addition to fibroblast-like cells, proliferating scleroblasts (D,E and insets) are seen (red arrowheads). (E) 2 days post-amputation, a functional blastema is formed. Note that the number of BrdU-positive cells is high in the proximal blastema, while the distal blastema remains unlabeled. Proliferating scleroblasts are observed immediately distal to bone stumps. (F) 3 days after amputation, newly formed bone is visible distal to old bone stumps. High levels of BrdU incorporation are seen in the proximal blastema, while the distal blastema remains unlabeled. Note that newly formed, BrdU-positive scleroblasts were apparent immediately behind the central zone of proliferation. The amputation plane is indicated by a dashed line. b, blastema; be, basal epidermal layer; e, wound epidermis; dmb, distal-most blastema; l, lepidotrichia; m, intraray mesenchyme; pb, proximal blastema; s, scleroblasts. Original magnifications: (A) 400x, (B) 400x, (C) 250x, (D) 200x, (E) 126x, (F) 126x and (G) 100x.