Il-6 cytokine family–mediated Stat3 signaling is proregenerative.

(A) Illustration of scarring in an adult mammalian heart in contrast to regeneration in an adult zebrafish heart. (B) Quantification of epicardial- and endothelial-derived αSMA⁺ cells after MI in mouse [fibroblasts (3); endothelial cells (4)] and 7 dpci in zebrafish [fibroblasts, Tg(tcf21:CreER), n = 4; endothelial cells, Tg(kdrl:Cre), n = 4]. (C) Schematics of the comparative transcriptional profiling. (D) Results from upstream regulator prediction analysis using Ingenuity Pathway Analysis. (E to H) Bright-field images of larval fin fold regeneration (E and G) [amputated at 48 to 60 hours post fertilization (hpf)] and their corresponding quantification of the fin fold area [(F) (il6st^sa1462, wt siblings, n = 11; mut, n = 9, 48 hpa; (H) stat3^stl27, wt siblings, n = 6; mut, n = 6, 72 hpa]. Data represent means ± SD (B, F, and H). Student’s t tests (F and H). n = larvae (F and H). Black dashed lines demarcate the amputation plane (E and G). Scale bars, 100 μm (E and G).

Il-11 signaling is essential for scar-free regeneration.

(A) Illustration of zebrafish Il-6 family cytokines and receptors and the downstream signaling pathways. (B) Heatmap showing RT-qPCR analysis of Il-6 family cytokine gene mRNA levels at 1 hpci (n = 6) compared with 1 hour post sham (hps; n = 5). (C) Illustration of wild-type and predicted mutant proteins and gross morphology of adult zebrafish siblings. (D and E) Bright-field images of larval fin fold regeneration (D) (wt siblings, n = 11; mut, n = 5; 72 hpa) and their corresponding quantification of the total fin fold area (E). (F) Wholemount images of Alizarin Red S stained regenerating adult scales [wt siblings, n = 6; mut, n = 5; 7 days post plucking (dpp)]. (G to I) Wholemount images of cardiac ventricles (G) (wt siblings, n = 5; mut, n = 5; 90 dpci), Acid Fuchsin Orange G (AFOG) staining on cryosections (H), and quantification of the scar area (I). (J to L) Wholemount images of caudal fins (J) [wt, n = 6; mut, n = 6, 14 days post amputation (dpa)], AFOG staining on longitudinal cryosections (K), and quantification of the regenerate area (L). SP, signal peptide; TMD, transmembrane domain; aa, amino acids (C). Data represent means ± SD (E, I, and L). Student’s t tests (E, I, and L). n, ventricles (B and G); n, larvae (D); n, adult zebrafish (F); n, caudal fins (J). Black dashed lines demarcate the injured area (G and H) and amputation plane (D and K); white dashed lines demarcate and white arrowheads point to regenerating scales (F); black arrowheads point to fused hemirays [insets in (J) and (K)]; red arrowheads point to the amputation plane (J). Ct values are listed in table S5. Scale bars, 5 mm (C), 100 μm (D), 500 μm (F), 200 μm (G and H), 1 mm (J), and 50 μm (K).

Il-11 signaling is required for the activation of global and tissue-specific regeneration gene programs.

(A and B) Differential expression of fin regeneration genes (13) in il11ra^−/− versus wild-type sibling caudal fin transcriptomic analysis, 24 hpa. (C to E) Confocal images of Tg(bglap:GFP) expression in wholemount caudal fins (C) (wt siblings, n = 4; mut, n = 6; 48 hpa), quantification of Tg(bglap:GFP) fluorescence intensity (D), and the number of distal osteoblasts (E). (F) Differential expression of known regulators of zebrafish cardiac regeneration (35) in il11ra^−/− versus wild-type sibling ventricle transcriptomic analysis, 96 hpci. (G) GSE analysis plot of KEGG Retinol metabolism from il11ra^−/− versus wild-type sibling ventricle transcriptomic analysis, 96 hpci. NE score, normalized enrichment score. (H and I) RT-qPCR analysis of aldh1a2 mRNA levels (H) (wt siblings, n = 6; mut, n = 6; 24 hpci) and immunostaining for Aldh1a2 expression on cardiac ventricle cryosections (I) (wt siblings, n = 5; mut, n = 4; 24 hpci). (J and K) RT-qPCR analysis of fn1b mRNA levels on dissected injured areas from cardiac ventricles (J) (wt siblings, n = 4; mut, n = 5; 96 hpci) and caudal fins (J) (wt siblings, n = 3; mut, n = 3; 48 hpa) and immunostaining on cryosections (K) (wt siblings, n = 5; mut, n = 4; 7 dpci) for Fibronectin1 (green) and myosin heavy chain (MHC; magenta) expression. Data represent means ± SD (E, H, and J); means ± SEM (D). Student’s t tests (E, H, and J). n, ventricles [(H), (I), (J) heart, and (K)]; n, caudal fins [(C) and (J) fin]. White arrows point to and white dashed lines demarcate the amputation plane (C); white arrowheads point to Tg(bglap:GFP) expression (C); yellow dashed lines demarcate the injured area (I and K) and bone rays (C); yellow arrowheads and asterisks indicate endocardial Aldh1a2 expression (I); red arrowheads point to epicardial Aldh1a2 expression (I). Ct values are listed in table S5. Scale bars, 100 μm (C and K) and 20 μm (I).

Il-11 signaling is required for CM repopulation after cardiac injury.

(A and B) Costaining for MEF2 (magenta) and PCNA (green) expression to determine CM mitotic index on cryosections from ventricles (A) (wt siblings, n = 7; mut, n = 6; 7 dpci) and quantification within the 100-μm wound border zone for 7 and 14 dpci (B). (C to E) F-actin staining on 50-μm-thick cryosections from il11ra^−/− versus wild-type ventricles, 7 dpci (C), and quantification of the number per ventricle (D) (wt siblings, n = 11; mut, n = 9) and length (E) (wt, n = 540; mut, n = 281) of CM protrusions. (F and G) Wholemount fluorescence images of Tg(gata4:EGFP) expression in ventricles (F) (wt siblings, n = 4; mut, n = 5; 14 dpci) and quantification of cortical CM wound coverage on the corresponding cryosections in fig. S8F (G). Data represent means ± SD (B, D, E, and G). Student’s t tests (B, D, and G); Mann-Whitney U test (E). n, ventricles (A, D, and F); n, CM protrusions (E). White dashed lines demarcate 100-μm injury border zone (A); yellow dashed lines demarcate the injured area (C and F). Scale bars, 50 μm (A and C) and 100 μm (F).

Il-11 signaling limits myofibroblast differentiation and profibrotic ECM remodeling after injury.

(A) Immunostaining for αSMA (white) and MHC (magenta) expression on cryosections from cardiac ventricles (wt siblings, n = 5; mut, n = 6; 7 dpci). (B) Quantification of αSMA⁺ cell density. (C) Immunostaining for αSMA (white), GFP (magenta), and Zns-5 antigen (scleroblasts, green) expression on longitudinal cryosections from Tg(fli1:EGFP) caudal fins (wt, n = 10; mut, n = 10; 14 dpa). (D) GSE analysis plots for Reactome and KEGG pathway terms from il11ra^−/− versus wild-type sibling adult ventricle transcriptomic analyses, 96 hpci. (E and F) RT-qPCR analysis on dissected injured areas from cardiac ventricles (E) (wt siblings, n = 4; mut, n = 5; 96 hpci) and caudal fins (F) (wt siblings, n = 4; mut, n = 4; 48 hpa) for selected profibrotic gene expression levels. (G and H) Immunostaining [(G) Elastin, green; MHC, magenta; (H) GFP, white] on cryosections from cardiac ventricles [(G) wt siblings, n = 11; mut, n = 10; (H) Tg(fli1:EGFP); mut, n = 6; 7 dpci]. Data represent means ± SD (B), and box plots (E and F) show median, interquartile range (IQR; box margins), and 5th and 95th percentiles (whiskers). Student’s t tests (B, E, and F). n, ventricles (A, E, G, and H); n, caudal fins (C); n, pools of two caudal fins (F). Yellow dashed lines demarcate the injured area (A, G, and H); yellow arrowheads point to αSMA⁺ myofibroblasts in il11ra mutant fins (C) and Elastin1 expression associated with endocardial cells in the injured area [inset in (H)]; white arrowheads point to vessel-associated αSMA⁺ smooth muscle cells (C); white arrows point to the amputation plane (C). Ct values are listed in table S5. Scale bars, 50 μm (A, C, G, and H), 20 μm [(C) left inset], and 10 μm [(C) right inset and (H) inset].

Il-11 signaling antagonizes EndoMT after cardiac injury.

Il-11 signaling in endothelial cells allows CM repopulation after cardiac injury.

IL-11 signaling feeds back to inhibit TGF-β–mediated scarring.

(A and B) Confocal images of immunostaining (A) (GFP, magenta; pSmad3, green; 7 dpci) on cryosections from il11ra^−/− versus wild-type Tg(fli1:EGFP) ventricles and quantification of percentage of pSmad3⁺ endocardial cells in the injured area (B) (wt siblings, n = 6; mut, n = 6). (C) RT-qPCR analysis for IL11RA, genes encoding TGF-β ligands, and TGF-β downstream target SNAI1 mRNA levels on HUVECs transfected with scrambled (n = 3) or IL11RA siRNAs (n = 3). (D) RT-qPCR analysis for genes encoding TGF-β ligands and for TGF-β downstream target SNAI1 mRNA levels on HUVECs treated with control (n = 4) or rhIL-11 (10 ng/ml; n = 4). (E) Experimental design and RT-qPCR analysis for IL11, genes encoding TGF-β ligands, and for TGF-β downstream target SNAI1 mRNA levels on HUVECs treated with control (n = 4) or rhTGFB2 (10 ng/ml; n = 4) or rhTGFB2 + rhIL-11 (10 ng/ml) (n = 4). (F) RT-qPCR analysis for genes encoding myofibroblast markers, TGF-β ligands, TGF-β downstream target SNAI1, and for fibrogenic ECM component mRNA levels on HUVECs transfected with scrambled (n = 4) or siIL11RA (n = 4) or siIL11RA + 10 μM TGFBR1 inhibitor (SB431542; n = 4). Data represent means ± SD (B); box plots (C to F) show median, IQR (box margins), and 5th and 95th percentiles (whiskers). Student’s t tests [(B) to (D) and (E) IL11]; one-way ANOVA (E and F). n, ventricles (B); n, biological replicates (C to F). Yellow dashed lines demarcate the injured area (A). Ct values are listed in table S5. Scale bars, 100 μm (A) and 20 μm (A, insets).