TGFβ signaling protects larval zebrafish from OFT aneurysm and ventricular dilation. (A) Experimental timeline for small-molecule-mediated inhibition of TGFβ signaling in wild-type animals. (B,C) Brightfield images of 5 dpf wild-type larvae treated with DMSO (B) or LY364947 (C). Arrowhead and * in C highlight a jaw protrusion and mild pericardial edema, respectively, observed in LY364947-treated animals. (D-F) Confocal images of hearts in 5 dpf wild-type animals treated with DMSO (D) or LY364947 (E,F) and double immunostained for striated muscle (MF20, red) and Eln2-positive OFT smooth muscle (green). E and F show representative hearts with moderate and severe phenotypes, respectively. Ventricular size was measured by quantifying the area within the perimeter of the chamber (areas encircled by dashed lines in D-F). (G) Dot plot showing the ventricular areas of 5 dpf wild-type larvae treated with DMSO (n=8) or LY364947 (n=11). (H-J) Single optical sections taken from the images shown in D-F through the OFTs of 5 dpf wild-type larvae treated with DMSO (H) or LY364947 (I,J). White lines show the maximal OFT diameters between the Eln2-positive walls of smooth muscle. (K) Dot plot showing the OFT diameters in DMSO-(n=5) and LY364947-(n=8) treated animals. (L-N) Confocal images of OFTs in 5 dpf wild-type animals treated with DMSO (L) or LY364947 (M,N) double immunostained for striated muscle (MF20, red) and phosphorylated Smad3 (pSmad3; green). (O) Dot plot showing the relative mean pSmad3 fluorescence intensities in the OFTs of DMSO-(n=9) or LY364947-(n=9) treated larvae. For all dot plots, statistical significance was determined with an unpaired t-test. Error bars show one standard deviation. **P<0.01. Scale bars: 20 µm.
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