Fig. 1

Spatiotemporal activation of S6K during zebrafish fin regeneration. (A-I′) Longitudinal sections of wild-type fin regenerates that were immunohistochemically stained with an antibody against p-S6K (green) at 0 (A), 6 (B), 12 (C), 18 (D), 24 (E), 36 (F), 48 (G), 72 (H), and 120 (I,I′) hpa (0 hpa, n = 3; 6 hpa, n = 4; 12 hpa, n = 3; 18 hpa, n = 4; 24 hpa, n = 5; 36 hpa, n = 4; 48 hpa, n = 3; 72 hpa, n = 4; 120 hpa, n = 3). The boxed area in I is enlarged in I′. DAPI fluorescent signal (blue) indicates the presence of nuclei. Dashed white lines indicate the amputation plane. The p-S6K fluorescent signals were barely detectable in the amputated fin at 0 hpa (A). At 6 hpa, p-S6K-positive cells were found in both intra-ray and epidermal cells (arrowheads in B), and the number of p-S6k-positive cells increased by 24 hpa (arrowheads in C, D, and E). Although p-S6K-positive cells were found in both the blastema and intra-ray region adjacent to and proximal to the amputation plane at 36 hpa (arrowheads in F), p-S6K-positive cells were mainly detected in the blastema at 48 hpa (arrowheads in G). At 72 and 120 hpa, p-S6K-positive cells were observed in the bilateral strip regions (arrows in H and I′), in the putative proximal medial blastema (arrowheads in H and I′), and in the wound epidermis, but not in the putative differentiated blastema cells (asterisks in H and I). Brackets indicate the p-S6K-negative cells in the tip of the putative proximal medial blastema domain and putative distal blastema (G,H,I′). It should be noted that both p-S6K and DAPI fluorescent signals did not overlap, as p-S6K and genomic DNA (DAPI specifically stains double-strand DNA) are localized in the cytosol and nucleus, respectively. Scale bars: 100 µm.