Fig. 5

TNF signaling is required for caudal fin regeneration. (a) Tg(mpeg1:mCherry-F/tnfa:eGFP-F) larvae were amputated and treated with either DMSO or pentoxifylline (PTX). Representative fin images show single fluorescence channels and overlay of fluorescence (Merge) at 6?hpA. White arrows indicate the wound. (b) Number of GFP-F⁺ cells in the wound region in indicated conditions (N_larvae=8?10, mean±S.E.M., two independent experiments, ***P<0.001). (c) qRT-PCR of tnfa mRNA relative to ef1a in DMSO or PTX treated larvae. Caudal fins were uncut or cut at 3?dpf and larvae were immediately treated with DMSO or PTX. RNA was extracted from tails (18 larvae per sample, mean±S.E.M. of three experiments, *P<0.05). (d) Representative transmitted light images of fins at 3?dpA after DMSO and PTX treatments. (e) Regenerated fin length at 3?dpA in indicated conditions (mean±S.E.M., three independent experiments, **P<0.005). (f) Blastema cell proliferation at 6, 24, 48 and 72?hpA in indicated conditions. Mitotic cells were detected in the fin region using an anti-PH3 antibody (N_larvae=13?23, average of cut/uncut ratio±S.E.M., two independent experiments, *P<0.05, **P<0.005). (g) Steady state levels of tnfr1 mRNA in sorted macrophages. Tg(mpeg1:mCherry-F /tnfa:eGFP-F) were amputated at 3?dpf and cells were collected at 6?hpA using fluorescence-activated flow cytometry and qRT-PCR used to quantify tnfr1 mRNA steady state levels relative to ef1a in the following cell populations: mpeg1^?tnfa^? (neg), mpeg1⁺tnfa^? (mpeg1⁺) and mpeg1⁺tnfa⁺ (mean values of four independent experiments±S.E.M. *P<0.05). (h) Fin images are representative overlays of mCherry fluorescence with transmitted light of Tg(mpeg1:mCherry-F) control morphants (Ctrl MO) or tnfr1 morphants (tnfr1 MO) at 24?hpA. (i) Macrophages (mCherry-F⁺) recruitment in the wound region at indicated time points in Ctrl and tnfr1 morphants (N_larvae=5?18 per group, mean±S.E.M., two independent experiments, *P<0.05, **P<0.005 respect to Ctrl MO condition). (j) Fin images are representative transmitted light images of Ctrl morphants and tnfr1 morphants at 3?dpA. (k) Corresponding regenerated fin length (mean±S.E.M. from three independent experiments, ***P<0.001). (l) Blastema cell proliferation at 6, 24, 48 and 72?hpA in indicated conditions was detected using an anti-PH3 antibody (N_larvae=5?17, cut/uncut ratio±S.E.M., two independent experiments, **P<0.01). (a, d, h and j) Dotted lines outline the fin, dashed arrows indicate the position of the initial amputation; scale bar=100?m