Cardiac injury triggers a localized increase in tbx20 expression. (A–H) Representative images of ISH for tbx20 on the heart sections of uninjured (A,B) and injured ventricles and atriums at different time points as indicated (C–H). Red arrowheads indicate atrial epicardium. (I,J) Confocal images of uninjured (I) and 7 dpa (J) heart sections from Tg(tbx20:moxGFP) zebrafish immunostained for GFP and α-actinin (red). DAPI marks nuclei. Boxed areas are magnified on the right panels. Dashed lines in (J) demarcate amputation planes. Scale bar: 100 μm (A–J); 20 μm (inset images).

Myocardial tbx20 overexpression in adult hearts promotes CM proliferation and reduces fibrotic scars. (A) Schematic diagram of transgenes of the control line Tg(TRE3G:tbx20) and the inducible myocardial tbx20 overexpression line Tg(TRE3G:tbx20^CMOE) with DOX treatment. (B) Experimental strategy employed to induce myocardial tbx20 overexpression during heart regeneration. (C) Representative images of ISH with tbx20-E2A-mCherry probe on 7 dpa heart sections from DOX treated Tg(TRE3G:tbx20) and Tg(TRE3G:tbx20^CMOE) fish. Scale bar: 100 μm. (D) Upper panels: Representative confocal fluorescence images of heart sections from 7 dpa zebrafish immunostained for PCNA (green) and Mef2 (red). Insets showing high-magnification images of proliferating cardiomyocytes, arrowheads indicate PCNA⁺Mef2⁺ cells. Lower panels: Representative images of heart sections from 21 dpa fish stained with AFOG, muscle stained brown, collagen is blue and fibrin is red. The genotypes of fish were indicated above the images. Scale bar: 100 μm. (E) Quantification of CM proliferation at border zone and injury site on 7 dpa heart sections (n = 7 in each group). (F) Quantification of scar area at 21 dpa (n = 9 in each group). The fish in (C–F) were treated with DOX as indicated in (B). Each value in (E,F) represents mean ± SEM, **p < 0.01, ***p < 0.001.

tbx20 overexpression in the adult myocardium enhances CM dedifferentiation after injury. (A–D,G,H,J,K) Representative confocal fluorescence images of sections of injured ventricles from Tg(TRE3G:tbx20) and Tg(TRE3G:tbx20^CMOE) zebrafish co-stained with antibodies against ZO-1 (green) and α-actinin (red) (A,B), cTnT (green) and N-cadherin (red) (C,D), α-SMA (red) and α-actinin (green) (G,H), and Runx1 (red) and α-actinin (green) (J,K). DAPI was used to stain nuclei. Boxed areas in (A,B,J,K) are magnified on the right with split channels. Insets in (C,D) show enlarged images of the dashed boxes. Arrowheads in (A-I, B-I) indicate CMs with disassembled sarcomeric structure in the border zone adjacent to the injury site. Arrowheads in (C,D) point to CMs adjacent to the injury site. Arrows and arrowheads in (G,H) indicate α-SMA⁺ cells in the regenerating compact layer and trabecular layer, respectively. Arrowheads in (J,K) indicate Runx1⁺α-actinin⁺ CMs. (E) Quantification of organized sarcomeric units in cTnT-labeled myocardium (80 × 80 pixels) in border zone from 7 dpa ventricle sections of Tg(TRE3G:tbx20) (C, n = 5) and Tg(TRE3G:tbx20^CMOE) (D, n = 6) zebrafish. (F) Statistical analyses of qPCR for α-SMA, runx1, nppb and nppa in the injured ventricle apices from Tg(TRE3G:tbx20) and Tg(TRE3G:tbx20^CMOE) zebrafish at 7 dpa. (I) Representative images of ISH with α-SMA on 7 dpa heart sections from Tg(TRE3G:tbx20^CMOE) fish. (L) Dot plot showing the area of α-SMA stained on heart sections in (G, n = 7) and (H, n = 5). (M) Dot plot showing the percentage of Runx1⁺α-actinin⁺ cells in the border zone in (J,K), n = 6 in each group. All fish were treated with DOX as described in Figure 2B and hearts were harvested at 7 dpa. Dashed lines delineate injured area. The values in (E,F,L,M) are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Scale bar: 50 μm (A,B); 20 μm (A-I,B-I,C-D’,J-I-K-II”); 100 μm (G–K).

Myocardial tbx20 overexpression affects expression of various injury responsive genes following heart injury. (A) Schematic of DOX treatment, heart injury and tissue collection procedures. RNA samples were extracted from the ventricular apices (below the dashed line). For each group, 6–8 samples were collected, and three independent biological replicates were carried out. (B) Significantly enriched gene ontology (GO) analysis based on RNA-seq results revealed upregulated gene categories (upper panel), and downregulated gene categories (lower panel). (C–F) Bar graphs indicate qPCR analyses of relative expression levels of genes associated with “Cell cycle regulators” (C), “ECM function” (D), “Immune response” (E) and “Sarcomere related genes” (F) in ventricular apices from DOX-treated Tg(TRE3G:tbx20^CMOE) and Tg(TRE3G:tbx20) at 7 dpa. Data represents three biological replicates. Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. (G–J) Representative images of ISH analyses for hbegfb(G,H) and mylk3(I,J) on 7 dpa heart sections from Tg(TRE3G:tbx20^CMOE) and Tg(TRE3G:tbx20) zebrafish with DOX treatment. Scale bar: 100 μm.

Myocardial tbx20 overexpression enhances endocardial activation and regeneration. (A) Schematic of experimental procedures for DOX treatment, cryoinjury and immunostaining experiments. (B,C) Representative confocal fluorescence images of heart sections from DOX-treated Tg(TRE3G:tbx20; flk:GFP) (B) and Tg(TRE3G:tbx20^CMOE; flk:GFP) (C) zebrafish at 5 dpci immunostained for cTnT (red), GFP and DAPI. Boxed areas (I and II) indicate locations of the magnified insets, respectively. Box-I in (B,C) indicate the remote (uninjured) region of heart sections, and Box-II in (B,C) display the endocardial cells in injured site. Arrows in (B-II) and (C-II) point to the endocardial cell protrusions. (D,E) Representative confocal fluorescence images of heart sections from DOX-treated Tg(TRE3G:tbx20) (D) and Tg(TRE3G:tbx20^CMOE) fish (E) at 5 dpci immunostained for Fli1 (green) and PCNA (red). Boxed areas indicate locations of the magnified insets. Arrowheads point to the Fli1⁺PCNA⁺ proliferating endocardial/endothelial cells. (F) Scatter plot showing the percentage of GFP⁺ area within the wound area of DOX treated Tg(TRE3G:tbx20) (n = 8) and Tg(TRE3G:tbx20^CMOE) (n = 11) hearts at 5 dpci. Mean ± SEM, ***p < 0.001. (G) Dotted diagram indicates the proliferation indices of endocardial/endothelial cells with Fli1⁺PCNA⁺ signals in the border zone and injury site from 5 dpci Tg(TRE3G:tbx20) (n = 5) and Tg(TRE3G:tbx20^CMOE) (n = 6) cardiac sections. Mean ± SEM, ***p < 0.001. (H–M) Representative images of ISH analysis for nfatc1(H,I), aldh1a2(J,K), and lepb(L,M) expression at 5 dpci or 7 dpa hearts from DOX-treated Tg(TRE3G:tbx20) and Tg(TRE3G:tbx20^CMOE) fish. Dashed lines delineate the injured area. Red arrowheads indicate endocardium with nfatc1(I), aldh1a2(K) and lepb(M) signal in the injury site. Scale bar: 100 μm.

Myocardial Tbx20 mediates endocardial regeneration by activating Bmp6 signaling. (A,B) Myocardial tbx20 overexpression upregulates BMP target genes id1 and id2b as well as the BMP ligand bmp6 after heart resection (A) or cryoinjury (B), respectively. Data from three biological replicates. Mean ± SEM, **p < 0.01, ***p < 0.001. (C–E) Representative ISH images of heart sections from DOX-treated uninjured Tg(TRE3G:tbx20) (C), 5 dpci Tg(TRE3G:tbx20) (D) and Tg(TRE3G:tbx20^CMOE) zebrafish (E,E’) with bmp6 probe. Red arrowheads indicate endocardium with bmp6 signal. (F–I) Representative ISH images of heart sections from 5 dpci DOX treated Tg(TRE3G:tbx20) and Tg(TRE3G:tbx20^CMOE) fish with id1(F,G) and id2b(H,I) probe. Red arrowheads indicate endocardium with id1(G’) and id2b(I’) signal in the injury site. (J) Experimental procedures for DOX treatment, cryoinjury, FISH combined with immunostaining experiments. (K,L) Representative images of FISH analysis of bmp6 (red) combined with immunostaining for GFP and α-actinin (blue) on heart sections from 5 dpci Tg(TRE3G:tbx20;flk:GFP) (K) and Tg(TRE3G:tbx20^CMOE;flk:GFP) (L) zebrafish. Boxed areas indicate locations of the magnified and channel-separated panels below. White arrowheads point to GFP⁺ cells with bmp6 transcripts in border zone and injury site (K-I,K-I’,L-I,L-I’), yellow arrows in (L-I) and (L-I’) indicate non-endocardial cells with bmp6 transcripts. (M) Dotted diagram indicates the percentage of bmp6⁺ endocardial cells in injury site from 5 dpci DOX treated Tg(TRE3G:tbx20) (K, n = 5) and Tg(TRE3G:tbx20^CMOE) (L, n = 6) fish. Mean ± SEM, ***p < 0.001. (N,O) Representative images of FISH analysis of id2b (red) combined with immunostaining for GFP and α-actinin (blue) on heart sections from 5 dpci Tg(TRE3G:tbx20;flk:GFP) (N) and Tg(TRE3G:tbx20^CMOE;flk:GFP) zebrafish (O). Boxed areas indicate locations of the magnified and channel-separated panels below. White arrowheads point to GFP⁺ cells with id2b transcripts in border zone and injury site (N-I,N-I’,O-I,O-I’), yellow arrows in (O-I) and (O-I’) indicate non-endocardial cells with id2b transcripts. (P) Dotted diagram indicates the percentage of id2b⁺ endocardial cells in injury sites from 5 dpci DOX treated Tg(TRE3G:tbx20) (N, n = 5) and Tg(TRE3G:tbx20^CMOE) (O, n = 5) fish. Mean ± SEM, ***p < 0.001. Dashed lines delineate the injured area. Scale bar: 100 μm (C-I,K,L,N,O), 20 μm (K-I-L-I’,N-I-O-I’).

Inhibition of Bmp6 signaling restricts endocardial cell proliferation activated by myocardial tbx20 overexpression. (A–F) Representative ISH images showing expression of id1(A–C) and id2b(D–F) in DOX treated Tg(TRE3G:tbx20^CMOE) fish at 5 dpci after vehicles or inhibitors treatment. Red arrowheads indicate endocardium with id1(A) and id2b(D) signal in the injury site. (G–L) Representative confocal fluorescence images of heart sections immunostained for Fli1 (green) and PCNA (red) from vehicle, LDN-193189 and K02288 treated control (G–I) and myocardial tbx20 overexpressing fish (J–L) at 5 dpci. Boxed areas indicate locations of the magnified insets. Arrowheads point to the Fli1⁺PCNA⁺ proliferating endocardial cells. (M) Scatter plot showing the percentage of Fli1⁺ cell proliferation ratio in the border zone and injury site from (G–L). The values are mean ± S.E.M. Two-way ANOVA followed with Tukey’s multiple comparison test. n.s.: none significance, ***p < 0.001. (N) Model of myocardial tbx20 function during zebrafish heart regeneration: In CMs, myocardial tbx20 overexpression promotes CM dedifferentiation and proliferation following injury. In addition, myocardial tbx20 promotes endocardial regenerative progress by enhancing its proliferation via partially through Bmp6 signaling. Dashed lines delineate the injured area. Scale bar: 100 μm.