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ZFIN ID: ZDB-FIG-170914-38
Nguyen-Chi et al., 2017 - TNF signaling and macrophages govern fin regeneration in zebrafish larvae. Cell Death & Disease   8:e2979 Full text @ Cell Death Dis.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Conditions:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Protruding-mouth to Day 4
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagent:
Observed In:
Stage Range: Protruding-mouth to Day 6

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 6hpA. White arrows indicate the wound. (b) Number of GFP-F+ cells in the wound region in indicated conditions (Nlarvae=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 3dpf 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 3dpA after DMSO and PTX treatments. (e) Regenerated fin length at 3dpA in indicated conditions (mean±S.E.M., three independent experiments, **P<0.005). (f) Blastema cell proliferation at 6, 24, 48 and 72hpA in indicated conditions. Mitotic cells were detected in the fin region using an anti-PH3 antibody (Nlarvae=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 3dpf and cells were collected at 6hpA using fluorescence-activated flow cytometry and qRT-PCR used to quantify tnfr1 mRNA steady state levels relative to ef1a in the following cell populations: mpeg1tnfa (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 24hpA. (i) Macrophages (mCherry-F+) recruitment in the wound region at indicated time points in Ctrl and tnfr1 morphants (Nlarvae=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 3dpA. (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 72hpA in indicated conditions was detected using an anti-PH3 antibody (Nlarvae=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=100μm

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP ump2Tg; ump5Tg amputation: caudal fin, chemical treatment: Pentoxifylline Protruding-mouth regenerating tissue macrophage IFL
ump2Tg; ump5Tg amputation: caudal fin Protruding-mouth regenerating tissue macrophage IFL
mCherry ump2Tg amputation: caudal fin Day 4 regenerating fin macrophage IFL
ump2Tg + MO3-tnfrsf1a amputation: caudal fin Day 4 regenerating fin macrophage IFL
ump2Tg; ump5Tg amputation: caudal fin, chemical treatment: Pentoxifylline Protruding-mouth regenerating tissue macrophage IFL
ump2Tg; ump5Tg amputation: caudal fin Protruding-mouth regenerating tissue macrophage IFL
tnfa AB amputation: caudal fin, chemical treatment: Pentoxifylline Protruding-mouth caudal fin RTPCR
AB amputation: caudal fin Protruding-mouth caudal fin RTPCR
tnfrsf1a ump2Tg; ump5Tg amputation: caudal fin Protruding-mouth cell RTPCR
Protruding-mouth macrophage RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
AB amputation: caudal fin, chemical treatment: Pentoxifylline Protruding-mouth caudal fin tnfa expression amount, ameliorated
Day 4 blastema cell population proliferation decreased occurrence, abnormal
Day 5 blastema cell population proliferation decreased occurrence, abnormal
Day 6 fin regeneration decreased efficacy, abnormal
Day 6 regenerating fin decreased length, abnormal
AB amputation: caudal fin Protruding-mouth caudal fin tnfa expression increased amount, abnormal
AB + MO3-tnfrsf1a amputation: caudal fin Day 4 blastema cell population proliferation decreased occurrence, abnormal
Day 5 blastema cell population proliferation decreased occurrence, abnormal
Day 6 fin regeneration decreased efficacy, abnormal
Day 6 regenerating fin decreased length, abnormal
ump2Tg + MO3-tnfrsf1a amputation: caudal fin Protruding-mouth - Day 6 regenerating fin macrophage decreased amount, abnormal
Day 4 regenerating fin macrophage decreased amount, abnormal
ump2Tg; ump5Tg amputation: caudal fin, chemical treatment: Pentoxifylline Protruding-mouth regenerating fin macrophage decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Cell Death & Disease for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Death Dis.