Inhibition of nitric oxide synthesis impedes zebrafish ventricle regeneration. A DAF-FM DA staining showed that NO (green) was primarily present in the bulbus arteriosus (BA), cleithrum (C), pharyngeal jaw bone (P), notochord (N) and caudal fin (CF) in larvae at 4 dpf. Scale bar, 150 μm. B–D DAF-FM DA staining of Tg(vmhc:mCherry-NTR) or Tg(kdrl:mCherry-ras) hearts at 4 dpf showed that NO was enriched in the smooth muscle layer of the BA. Scale bars, 10 μm. E Schematic timeline diagram of MTZ treatment to induce ventricle ablation and L-NMMA treatment to inhibit NO production. F–I″ The NO level was significantly decreased in ablated hearts at 24 hpt and gradually increased to a level comparable to control hearts at 96 hpt (F–G″). L-NMMA treatment dramatically reduced NO level in control and ablated hearts (H–I″). Dashed lines outline the hearts. Scale bars, 50 μm. J Quantification of relative DAF-FM DA intensity of BA in control or ablated hearts with or without L-NMMA treatment. N = 5 for each group. Mean + s.e.m. ANOVA analysis, ***P < 0.001, ****P < 0.0001 as compared with control group; ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 as compared with ablated group. K Quantification of the heart recovery rate in the ablated and ablated + L-NMMA-treated groups at 96 hpt. The numbers of larvae analyzed for each condition are indicated. Binomial test, ***P < 0.001, ****P < 0.0001. dpf days post fertilization, hpt hours post treatment, V ventricle, BA bulbus arteriosus, NO nitric oxide

Reduced blood flow suppresses NO production in the BA. A, B DAF-FM DA staining of Tg(vmhc:mCherry-NTR) hearts showed that reduced blood flow via tnnt2a MO injection or tricaine treatment markedly suppressed NO production. SNP treatment could supply NO. C–G′′′ DAF-FM DA staining showed that tricaine treatment for 0–24 or 48–72 hpt significantly reduced NO level in ablated hearts at 96 hpt. H Quantification of relative DAF-FM DA intensity of BA in the control, ablated, ablated + Tric-treated (0–24 hpt) and ablated + Tric-treated (48–72 hpt) groups. N = 5 for each group. Mean + s.e.m. ANOVA analysis, *P < 0.05, ***P < 0.001, ****P < 0.0001 as compared with control group; ^###P < 0.001, ^####P < 0.0001 as compared with ablated group. I Quantification of the heart recovery rate in the ablated, ablated + Tric-treated (0–24 hpt) and ablated + Tric-treated (48–72 hpt) groups at 96 hpt. The numbers of larvae analyzed for each condition are indicated. Binomial test, ****P < 0.0001. J Quantification of the heart recovery rate in ablated groups at 96 hpt treated with tricaine for 0–24 or 48–72 hpt, with or without co-treatment of SNP. The numbers of larvae analyzed for each condition are indicated. Binomial test; ns, not significant; ***P < 0.001. K DAF-FM DA staining of ablated hearts at 96 hpt treated with tricaine for 0–24 or 48–72 hpt, with or without co-treatment of the NO donor SNP. Scale bars, 50 μm. Dashed lines outline the hearts. dpf days post fertilization, hpt hours post treatment, NO nitric oxide, MO morpholino, Tric tricaine, SNP sodium nitroprusside dihydrate

Hemodynamic-responsive Trpv4 modulates NO and Notch signaling during ventricle regeneration. A Trpv4 immunostaining (red) in Tg(kdrl:eGFP) larvae at 3 dpf showed ubiquitous expression of Trpv4 in the hearts. Bottom panels, enlargement of boxed areas of the bulbus arteriosus (BA) and atrioventricular canal (AVC). B Trpv4 immunostaining (intensity gradient) indicated that Trpv4 upregulation after ventricle ablation was blocked in tricaine-treated hearts at 24 and 48 hpt. C DAF-FM DA staining of Tg(vmhc:mCherry-NTR) larvae showed that Trpv4 agonist 4α-pdd treatment for 48–72 hpt partially rescued the NO signal which was suppressed by tricaine in ablated hearts. D Confocal images of Tg(vmhc:mCherry-NTR; tp1:d2GFP) hearts showed that Trpv4 agonist 4α-pdd treatment for 0–24 hpt partially rescued Notch signaling which was suppressed by tricaine in ablated hearts. E, F Quantification of the heart recovery rate in ablated groups treated with DMSO, DMSO + Tric, 4α-pdd + Tric (48–72 hpt) or 4α-pdd + Tric (0–24 hpt) at 96 hpt. The numbers of larvae analyzed for each condition are indicated. Binomial test, **P < 0.01. G NO production in ablated hearts was significantly inhibited in trpv4^−/− mutants. H Notch signaling activation in ablated hearts was significantly inhibited in trpv4^−/− mutants. Scale bars, 50 μm. Dashed lines outline the hearts. dpf days post fertilization, hpt hours post treatment, NO nitric oxide, Tric tricaine

Temporal inhibition of Trpv4, Notch and NO signaling differentially affects ventricle regeneration. A–C Quantification of the heart recovery rate at 96 hpt in ablated groups treated with HC-067047 (TRPV4 antagonist), DAPT (Notch signaling inhibitor) or L-NMMA (NO synthase inhibitor) at different stages. The numbers of larvae analyzed for each condition are indicated. Binomial test; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. D–H′ Immunostaining of anti-phospho-histone H3 (pH3, green) and anti-myosin heavy chain (MF-20, red) in the hearts of control and ablated groups at 48 or 72 hpt with DMSO, HC-067047, DAPT or L-NMMA treatment for 0–24 or 48–72 hpt. I, J Quantification of pH3⁺ cardiomyocyte numbers in control and ablated hearts treated with DMSO, HC-067047, DAPT or L-NMMA for 0–24 or 48–72 hpt. N = 11 for each group. Mean + s.e.m. ANOVA analysis; ns, not significant; *P < 0.05, ****P < 0.0001. K–O′ Confocal images of Tg(vmhc:mCherry-NTR; amhc:CreERT2; β-act2:RSG) control and ablated hearts at 72 hpt with DMSO, HC-067047, DAPT or L-NMMA treatment for 0–24 or 48–72 hpt. P, Q Quantification of the percentages of GFP-positive area in control or ablated ventricles with DMSO, HC-067047, DAPT or L-NMMA treatment for 0–24 or 48–72 hpt. N = 10 for each group. Mean + s.e.m. ANOVA analysis; *P < 0.05, ****P < 0.0001. Scale bars, 50 μm. Dashed lines outline the hearts. hpt hours post treatment, HC HC-067047

Trpv4 and NO signaling play essential roles in the late stage of ventricle regeneration. A Schematic timeline diagram of MTZ treatment to induce ventricle ablation and HC-067047 treatment to inhibit Trpv4 or L-NMMA treatment to inhibit NO synthesis. B DAF-FM DA staining of Tg(vmhc:mCherry-NTR) larvae at 96 hpt revealed that HC-067047 or L-NMMA treatment for 48–72 hpt blocked NO production. C Confocal images of control and ablated hearts treated with DMSO, HC-067047 or L-NMMA for 48–72 hpt in Tg(vmhc:mCherry-NTR; myl7:H2B-eGFP) larvae at 96 hpt. D Quantification of H2B⁺ CM numbers in DMSO-, HC-067047- and L-NMMA-treated ablated hearts. N = 19, 15, 10, respectively. Mean + s.e.m. ANOVA analysis; **P < 0.01, ***P < 0.001. E Confocal images of control and ablated hearts treated with DMSO, HC-067047 or L-NMMA for 48–72 hpt in Tg(vmhc:mCherry-NTR; myl7:mAG-zGeminin) larvae at 72 hpt. F Quantification of zGeminin⁺ CM numbers in DMSO-, HC-067047- and L-NMMA-treated ablated hearts. N = 17, 10, 15, respectively. Mean + s.e.m. ANOVA analysis; *P < 0.05, ***P < 0.001, ****P < 0.0001. G–I Whole-mount in situ hybridizations showed reduced expression of nkx2.5, gata4 and hand2 at 72 hpt in control and ablated hearts treated with DMSO, HC-067047 or L-NMMA for 48–72 hpt. Numbers indicate the ratio of representative staining observed. Scale bars, 50 μm. Dashed lines outline the hearts. hpt hours post treatment, NO nitric oxide, CM cardiomyocyte, V ventricle, A atrium, HC HC-067047

NO signaling regulates CM migration during ventricle regeneration. A–C Whole-mount in situ hybridizations showed reduced expression of snail1a, twist1a and vimentin at 72 hpt in control and ablated hearts treated with DMSO, HC-067047 or L-NMMA for 48–72 hpt. Numbers indicate the ratio of representative staining observed. D–F Injection of myl7:lifeact-eGFP plasmid into Tg(vmhc:mCherry-NTR) embryos at the one-cell stage to monitor the migratory behavior of single eGFP⁺ CMs in the ventricles or atriums of ablated hearts treated with DMSO, HC-067047 or L-NMMA for 48–72 hpt. G Quantification of the cell migration rate at 96 hpt in ablated hearts treated with DMSO, HC-067047 or L-NMMA for 48–72 hpt. The numbers of larvae analyzed for each condition are indicated. Binomial test, ****P < 0.0001. H Time series images showed the migration process of an eGFP⁺ CM in the ablated heart at 48–96 hpt. Scale bars, 50 μm. Dashed lines outline the hearts. hpt hours post treatment, CM cardiomyocyte, V ventricle, A atrium, HC HC-067047

NO signaling mediates ventricle regeneration through TGF-β pathway. A, B Whole-mount in situ hybridizations showed the expression of tgfb1a and tgfb1b at 72 hpt in control and ablated hearts treated with DMSO, HC-067047 or L-NMMA for 48–72 hpt. Numbers indicate the ratio of representative staining observed. C Immunostaining of anti-myosin heavy chain (MF-20, red) and anti-phospho-Smad3 (green) in control and ablated Tg(vmhc:mCherry-NTR) hearts treated with DMSO, HC-067047 or L-NMMA alone or with SRI-011381 for 48–72 hpt. D Quantification of relative p-Smad3 intensity in control or ablated hearts with DMSO, HC-067047, L-NMMA or SRI-011381 treatment for 48–72 hpt. N = 7 for each group. Mean + s.e.m. ANOVA analysis; ***P < 0.001, ****P < 0.0001. E, F Confocal images of ablated hearts treated with DMSO, L-NMMA or L-NMMA + SRI-011381 for 48–72 hpt in Tg(vmhc:mCherry-NTR; myl7:H2B-eGFP) larvae at 96 hpt and in Tg(vmhc:mCherry-NTR; myl7:mAG-zGeminin) larvae at 72 hpt. G, H Quantification of H2B⁺ CM and zGeminin⁺ CM numbers in DMSO-, L-NMMA- and L-NMMA + SRI-011381-treated ablated hearts. N = 10 for each group. Mean + s.e.m. ANOVA analysis; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. I Confocal images of ablated hearts treated with DMSO, L-NMMA or L-NMMA + SRI-011381 for 48–72 hpt in Tg(vmhc:mCherry-NTR; amhc:CreERT2; β-act2:RSG) larvae at 72 hpt. J Quantification of the percentages of GFP-positive area in DMSO-, L-NMMA- and L-NMMA + SRI-011381-treated ablated hearts. N = 10 for each group. Mean + s.e.m. ANOVA analysis; ***P < 0.001, ****P < 0.0001. K Quantification of the heart recovery rate in ablated groups treated with DMSO, L-NMMA, L-NMMA + SRI-011381 at 96 hpt. The numbers of larvae analyzed for each condition are indicated. Binomial test, **P < 0.01. Scale bars, 50 μm. Dashed lines outline the hearts. hpt hours post treatment, CM cardiomyocyte, HC HC-067047, SRI SRI-011381