Inhibition of TGF-β/Smad3 signaling pathway impedes ventricular regeneration. (A–D) Representative fluorescence images showing the morphology of control Tg(myl7:lifeact-eGFP; vmhc:mCherry-NTR) hearts (A) and three types of ablated hearts at 7 dpf/4 dpt, fully regenerated ventricle (B, type 1), tiny ventricle (C, type 2), and partially regenerated ventricle (D, type 3). (E,F) Quantification of the heart regeneration ratio at 7 dpf/4 dpt in the ablated larvae with different length of SIS3 treatment. The numbers of larvae analyzed for each condition are indicated above the chart. Chi-square test, ****P < 0.0001. (G,H) Pie charts show cardiac morphology classification in the ablated larvae without (G) or with a 48-h SIS3 treatment (H) at 4 dpt. N = 221 and 119, respectively. (I,J) Representative immunostaining images of Tg(vmhc:mCherry-NTR) hearts showing that phospho-Smad3 signal was decreased in the ablated hearts at 5 dpf/2 dpt upon a 48-h SIS3 treatment (J). Green, anti-pSmad3; red, MF20 (anti-MHC). (K) Quantification of phospho-Smad3-positive cells in the ablated hearts without or with a 48-h SIS3 treatment at 5 dpf/2 dpt (N = 11 and 8, respectively). Mean ± s.e.m., Student’s t-test, two-tailed, ****P < 0.0001. (L–N) Transcriptomic analysis revealed differentially expressed genes involved in cell differentiation, cell cycle, and migration. Scale bars, 50 μm. dpf, days post-fertilization; dpt, days post-treatment; atr., atrium; oft., out flow tract; vent., ventricle.
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