Smad3 inhibition reduces twist expression and Snail-positive CM number during regeneration. (A–D) Whole-mount in situ hybridization showing the expression level change of the EMT marker twist1b during regeneration. Smad3 inhibition via SIS3 treatment reduced the upregulation of twist1b at 5 dpf/2 dpt. Dashed lines outline the hearts. (E–H″) Representative fluorescent images of Tg(vmhc:mCherry-NTR; flk:GFP) hearts with immunostaining of Snail (white), MF20 (red), and DAPI (blue) in the control or ablated hearts without or with SIS3 treatment at 5 dpf/2 dpt. (E–H) Maximal projections of z-stack images, overlay of red and white channels; yellow dashed lines outline the ablated area. (E′–H′) Optical sections of panels (E–H), overlay of four channels. (E″–H″) Enlargement of box area in panels (E′–H′); arrowheads point to extruding Snail+ CMs into the outer layer. (I) Quantification of Snail+ CM number in the ablated hearts without or with SIS3 treatment at 5 dpf/2 dpt. N = 6 and 8, respectively. Mean ± s.e.m., Student’s t-test, two-tailed, *P < 0.05. Scale bars, (A–D) 50 μm, (E–H′) 20 μm, and (E″–H″) 10 μm. dpf, days post-fertilization, dpt, days post-treatment; atr., atrium; oft., out flow tract; vent., ventricle; CM, cardiomyocyte; EMT, epithelial–mesenchymal transition.
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