FIGURE 6

FIGURE 6

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’).