Ciliary neurotrophic factor stimulates cardioprotection and the proliferative activity in the adult zebrafish heart

Figures

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  Fig. 1. Transcriptional changes after thoracotomy suggest the activation of cardioprotective and EMT genes in the epicardium. a Experimental design. High throughput sequencing (HTPS) of ventricles collected at 0 (control), 1 and 7 days post-thoracotomy (dpt). For each group, RNA was extracted from a pool of 8 ventricles. The encircled numbers indicate differentially expressed genes at each time point. The middle number depicts differentially expressed genes common for both time points. b Heat-map representation of the common 53 differentially expressed genes at 1 and 7 dpt. Genes are grouped according to biological function. Fold changes are represented in log2 scale (blue: log2 < 0; red: log2 > 0). c In-situ hybridization of ventricular transversal sections reveals upregulation of several candidate genes (purple) in the epicardial and sub-epicardial region at 7 dpt, compared to control hearts at 0 dpt. The frames indicate the part of the section that is magnified on the right side of each image. n ≥ 3 hearts. Scale bar for the whole section, 100 μm; for the magnified area, 50 μm. d Immunofluorescence staining of ventricular sections of transgenic fish ET27:EGFP (red) with antibodies…

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  Fig. 2. The cell-cycle entry and the deposition of ColXII after thoracotomy are dependent on the JAK/STAT3 pathway. a Experimental design. The requirement of different pathways for the increased CM mitotic activity and the ColXII deposition observed after preconditioning was tested at 7 dpt by using specific inhibitors of JAK/STAT (1 µM Ruxolitinib), TGF-β (20 µM SB431542) or FGF (10 µM PD173074) signaling. b, c Transversal sections of hearts treated with different drugs indicated at the left side. Scale bar for the whole section, 500 μm; for the magnified area, 100 μm. b Ventricle of transgenic fish cmlc2:DsRed2-nuc (red) immunostained for the G1/S-phase marker MCM5 (green). Some double positive cells are indicated with arrows. Treatment with Ruxolitinib markedly reduces cells proliferation in the ventricle. c Ventricle of wild type fish immunostained against Tropomyosin (red) and ColXII (green). In the presence of Ruxolitinib, intramyocardial ColXII is reduced. d Proportion of MCM5 + nuclei among cmlc2:DsRed2-nuc + nuclei. e Proportion of MCM5 + cmlc2:DsRed2-nuc-negative nuclei among DAPI + nuclei. f Proportion of the ColXII-positive area within the surface of ventricular section. n ≥…

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  Fig. 3. Single injection of zCNTF elevates the mitotic activity and ColXII deposition. a Experimental design to assess effects of a single intrathoracic injection of zCNTF in the presence of the JAK/STAT inhibitor, 1 µM Ruxolitinib, at 7 dpi. b, c Immunofluorescence staining of heart sections. Scale bar for the whole section, 500 μm; for the magnified area, 100 μm. b Transversal heart sections of transgenic fish cmlc2:DsRed2-nuc (red) to demarcate CM nuclei, immunostained with the G1/S-phase marker MCM5 (green) display enhanced CM proliferation (arrows) after zCNTF injection in the absence of the JAK/STAT inhibitor. c Transversal heart sections of wild type fish immunostained with of ColXII (green) and Tropomyosin (red). Intramyocardial ColXII is increased in zCNTF-injected hearts. JAK/STAT inhibition abolished this effect. d Proportion of MCM5-positive cells among cmlc2:DsRed2-nuc/DAPI-positive CM nuclei. e Proportion of MCM5 + cmlc2:DsRed2-nuc-negative nuclei among DAPI + nuclei. f Proportion of the ColXII-positive area within the surface of ventricular section. n ≥ 4 hearts; ***P < 0.001

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  Fig. 4. Exogenous zCNTF recruits leucocytes into the zebrafish uninjured heart. a–d Immunofluorescence staining of heart sections from different conditions as indicated at the left side of the images. a Hearts of wild type fish stained for L-plastin (green) and DAPI (blue). Single injection of zCNTF results in higher accumulation of L-plastin in the heart at 7 dpi. This effect is abolished in the presence of the JAK/STAT inhibitor, 1 µM Ruxolitinib. Scale bars, 100 μm. b Hearts of transgenic fish mpeg1:EGFP (green) stained for L-plastin (red) and F-actin (Phalloidin, blue). The number of mpeg1:EGFP/L-plastin-expressing cells is increased at 7 days after zCNTF injection. Some double positive cells are indicated with arrows. Scale bers 50 μm. c, d Ventricles of wild type fish stained for Mpx (c, green) or Lyz (d; green) and F-actin (Phalloidin, blue). The number of double positive leucocytes is increased at 7 days after zCNTF injection (arrows). Scale bars, 50 μm. e Quantification of L-plastin + area in ventricular sections. f Quantification of cells expressing mpeg1:EGFP, Mpx and Lyz normalized to the ventricle area at different conditions. n ≥ 4 hearts; ≥ 2 sections per heart; *P < 0.05;…

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  Fig. 5. Administration of zCNTF reduces apoptosis and boosts initiation of heart regeneration after cryoinjury. a Experimental design for the apoptosis assay. zCNTF was injected into the pericardial cavity 72 h before cryoinjury. Hearts were collected at 6 and 12 h post-cryoinjury (hpci). b Transversal heart sections at 6 hpci stained for apoptotic cells using the TUNEL assay (green) and a myocyte nuclear marker Mef2 (red). Cryoinjured zone is encircled with a dashed line. Scale bar for the whole section, 500 μm; for the magnified area, 100 μm. c Quantification of TUNEL-positive nuclei at 6 and 12 hpci, in hearts injected with control proteins and zCNTF. n ≥ 4 hearts; ≥ 2 sections per heart; *P < 0.05; **P < 0.01. d Experimental design for assessment of the initiation of regeneration. zCNTF was injected into the pericardial cavity at 3 days before cryoinjury (dpci). Hearts were collected at 7 dpci. e, f Immunofluorescence staining of heart sections. Scale bar for the whole section, 500 μm; for the magnified area, 100 μm. e Transversal heart sections of transgenic fish cmlc2:DsRed2-nuc immunstained for MCM5 (green) display an enhanced number of proliferating CMs (arrows) after zCNTF…

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  Fig. 6. Cntf mutants fail to enhance CM proliferation after thoracotomy. a Schematic drawing of the cntf locus containing 4 exons. UTR (white); translated sequences (orange boxes). The sequence of 154-174 nucleotides (in red) flanking PAM sequence (in blue) in the 3rd exon was targeted by the CRISPR/Cas9-sgRNA RNP complex. b Schematic drawing of the two deletions induced by CRISPR/Cas9. cntfdel207 contains a deletion of 207 bp in the 3rd exon spanning the exon/intron boundary. cntfdel7 comprises a deletion of 7 bp in the middle of the 3rd exon leading to a frameshift and a premature stop codon. c Genotypes and images of wild type and CNTF mutant siblings. F0 mosaic heterozygous candidates were crossed to obtain F1 trans-heterozygous mutants, which are viable without visible phenotype. Scale bar, 5 mm. d Experimental design. e Quantification of PCNA-positive cells among Mef2/DAPI-positive CM nuclei. n ≥ 3 hearts; ≥ 3 sections per heart; *P < 0.05, **P < 0.01. f Quantification of BrdU-positive cells among Mef2/DAPI-positive CM nuclei. n ≥ 3 hearts; ≥ 3 sections per heart; *P < 0.05. g, h Transversal heart sections of wt and cntf mutant fish at 7 dpt, immunostained against Mef2 (green, a…