ltbp1, ltbp3 DKO larvae develop OFT aneurysm with hyperplastic and hypertrophic features. (A,B,D,E,G,H) Single optical sections through the OFTs of 5 dpf control (CTRL; A,D,G) and ltbp1−/−; ltbp3−/− (B,E,H) larvae double immunostained for striated muslce (A,B; MF20, red) and Eln2-positive OFT smooth muscle (A,B,D,E,G,H; green) and counterstained with DAPI to visualize nuclei (D,E; blue). The white lines in A and B highlight the maximal OFT diameters between the walls of Eln2-positive smooth muscle. DKO OFTs required more optical sections than CTRL OFTs to capture all Eln2-positive cells. (C,F,I) Dot plots showing the maximal OFT diameters, Eln2-positive OFT smooth muscle cell (SMC) numbers or SMC areas in 5 dpf CTRL (n=5 in C, n=5 in F, n=34 total cells from three hearts in I) and ltbp1−/−; ltbp3−/− (n=5 in C, n=6 in E, n=30 total cells from three hearts in I) larvae. SMC number was quantified by counting DAPI-stained nuclei surrounded by Eln2-positive fluorescence. SMC size was measured by quantifying the area within the cell perimeter (areas encircled by dashed lines in G and H). Single optical sections shown in G and H are from z-stacks presented in Fig. 1K,L. (J,K) Single optical sections of OFTs from 5 dpf CTRL and ltbp1−/−; ltbp3−/− larvae carrying the endothelial/endocardial fli1a:nGFP transgene, double immunostained for striated muscle (MF20, red) and GFP (magenta) and counterstained with DAPI (blue) to visualize nuclei. (L) Dot plot showing the number of endocardial cells in OFTs of 5 dpf CTRL (n=8) and ltbp1−/−; ltbp3−/− (n=7) larvae. For all dot plots, statistical significance was determined with an unpaired t-test. Error bars show one standard deviation. ***P<0.001. ****P<0.0001. Scale bars: 20 µm.
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