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Loss of TNFα function comprises nonCM activation and heart regeneration. (A) Schematic of zebrafish tnfα gene, with a T-to-A substitution, which creates a premature stop truncating the protein right at the N terminus of its THD domain. (B) Immunohistochemistry for MF20 and GFP on 7-dpa sections from tcf21:nucGFP transgenic fish hearts (upper panel). Lower panels represent RNAscope in situ hybridization for postnb combined with immunostaining for MF20. White boxes represent the injury area, while orange boxes mark the peripheral area. Scale bar, 50 μm. (C) Quantification of the number of tcf21:nucGFP-positive cells and the area of postnb expression in the wound on sections shown in (B). n = 5. (D) Expression of fn1a and tcf21 in 7-dpa hearts determined by quantitative reverse transcription polymerase chain reaction. (E) RNAscope in situ hybridization for aEC marker fosl1a combined with immunohistochemistry for MF20 in the hearts. Scale bar, 50 μm. (F) Quantification of the number of fosl1a-positive dots in the white boxed areas in (E). n = 5. (G) AFOG staining of ventricles at 30 dpa to identify scar (blue) and fibrin (red). Black boxes indicate the injury areas. Scale bar, 100 μm. (H) Quantification of scar area at 30 dpa. n = 5. (I) Immunohistochemistry for PCNA and Nkx2.5 on 7-dpa sections. The white boxed regions are shown in zoom-in images. White arrows indicate PCNA/Nkx2.5 double-positive nuclei. Scale bar, 50 μm. (J) Quantification of PCNA-positive proliferating Nkx2.5-positive myocardial cells at 7 dpa. n = 5. White dashed lines indicate approximate resection plane. P value calculated with two-tailed Student’s t test. **P < 0.01.
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