ltbp1, ltbp3 DKO zebrafish exhibit OFT aneurysm and ventricular dilation. (A-H) Brightfield images of hearts in wild-type zebrafish at 2 dpf (A,E), 3 dpf (B,F), 4 dpf (C,G), and 5 dpf (D,H) processed for double whole-mount in situ hybridization to detect ltbp3 (A-D, blue signal) or ltbp1 (E-H, blue signal) transcripts and the myocardial transcript cmlc2 (myl7; A-H; red signal). Little to no variation was observed between animals within each group (n>10/group). (I,J) Brightfield images of 5 dpf control (CTRL; I) and ltbp1^−/−; ltbp3^−/− (J) larvae. Arrowhead and * in J highlight jaw protrusion and mild pericardial edema, respectively, observed in double mutants. (K-N) Confocal images of hearts in 5 dpf CTRL (K,M) and ltbp1^−/−; ltbp3^−/− (L,N) larvae double immunostained for striated muscle (MF20, red) and Eln2-positive OFT smooth muscle (green). The single optical sections shown in M and N were taken from the images shown in K and L. V, ventricle. Scale bars: 20 µm.

ltbp1, ltbp3 DKO larvae develop ventricular dilation with molecular features of a stress response. (A,B) Confocal images of hearts in 5 dpf control (CTRL; A) and ltbp1^−/−; ltbp3^−/− (B) larvae immunostained for striated muscle (MF20, red). Ventricular size was measured by quantifying the area within the perimeter of the chamber (areas encircled by dashed lines). (C) Dot plot showing the ventricular areas of 5 dpf CTRL (n=22) and ltbp1^−/−; ltbp3^−/− (n=21) larvae. (D,E) Confocal images of hearts in 5 dpf CTRL (D) and ltbp1^−/−; ltbp3^−/− (E) larvae carrying the cmlc2:nucGFP transgene and immunostained for GFP. (F) Dot plot showing the number of ventricular cardiomyocytes in 5 dpf CTRL (n=7) and ltbp1^−/−; ltbp3^−/− (n=7) larvae. (G-H′) Single optical sections of hearts in 5 dpf CTRL (G,G′) and ltbp1^−/−; ltbp3^−/− (H,H′) larvae carrying the cmlc2:nucGFP transgene and immunostained for GFP. The boxed regions in G and H are shown enlarged in G′ and H′, respectively. Arrowheads highlight trabeculae, observed in seven out of seven ventricles for both experimental groups. (I,J) Dot plots showing the relative expression levels of nppa (I) and nppb (J) in 5 dpf CTRL and ltbp1^−/−; ltbp3^−/− larvae measured by qPCR. n=4 biological replicates and n=3 technical replicates per biological replicate. (K,L) Brightfield images of 5 dpf CTRL (K) and ltbp1^−/−; ltbp3^−/− (L) larvae processed for whole-mount in situ hybridization to detect nppa transcripts. Arrows in K and L highlight nppa expression in the heart. Little to no variation was observed between animals within each group (n>10/group). For all dot plots, statistical significance was determined with an unpaired t-test. Error bars show one standard deviation. ****P<0.0001; ***P<0.001; **P<0.01; ns, not significant. Scale bars: 20 µm.

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.

Hyperactivation of canonical TGFβ signaling in the distended OFTs of ltbp1, ltbp3 DKO larvae. (A,B,D,E) Confocal images of OFTs in 5 dpf (A,B) and 3 dpf (D,E) control (CTRL; A,D) and ltbp1^−/−; ltbp3^−/− (B,E) larvae double immunostained for striated muscle (MF20, red) and phosphorylated Smad3 (pSmad3; green). (C,F) Dot plots showing the relative mean pSmad3 fluorescence intensities in the OFTs of 5 dpf (C) or 3 dpf (F) CTRL (n=9 in C; n=6 in F) and ltbp1^−/−; ltbp3^−/− (n=9 in C; n=3 in F) larvae. For C and F, statistical significance was determined with an unpaired t-test. Error bars indicate one standard deviation. ***P<0.01; ns, not significant. Scale bars: 20 µm.

OFT aneurysm and ventricular dilation precede hyperactivation of TGFβ signaling in ltbp1, ltbp3 DKO larvae. (A,B) Confocal images of hearts in 4 dpf control (CTRL; A) and ltbp1^−/−; ltbp3^−/− (B) larvae double immunostained for striated muscle (MF20, red) and Eln2-positive OFT smooth muscle (green). (C) Dot plot showing the ventricular areas of 4 dpf CTRL (n=7) and ltbp1^−/−; ltbp3^−/− (n=7) larvae. (D,E) Single optical sections through the OFTs of 4 dpf CTRL (D) and ltbp1^−/−; ltbp3^−/− (E) larvae double immunostained for striated muscle (MF20, red) and Eln2-positive OFT smooth muscle (green). The white lines highlight the maximal OFT diameters between the walls of Eln2-positive smooth muscle. (F) Dot plot showing the maximal OFT diameters in 4 dpf CTRL (n=7) and ltbp1^−/−; ltbp3^−/− larvae (n=7). (G,H) Confocal images of OFTs in 4 dpf CTRL (G) and ltbp1^−/−; ltbp3^−/− (H) larvae double immunostained for striated muscle (MF20, red) and phosphorylated Smad3 (pSmad3, green). Dot plot showing the relative mean pSmad3 fluorescence intensities in the OFTs of 4 dpf CTRL (n=5) and ltbp1^−/−; ltbp3^−/− (n=7) larvae. For all dot plots, statistical significance was determined with an unpaired t-test. Error bars indicate one standard deviation. ****P<0.0001. ns, not significant. Scale bars: 20 µm.

Molecular similarities between disease tissue from ltbp1, ltbp3 DKO larvae and ascending aortic aneurysms from a mouse model of Marfan syndrome. (A) Volcano plot showing the distribution of log₂-fold changes and raw P-values for protein-coding RNAs isolated from co-dissected OFTs and ventricles of 5 dpf ltbp1^−/−; ltbp3^−/− larvae relative to control (CTRL) samples. Genes with raw P<10⁻¹⁰ are highlighted in purple and official gene symbols are provided. Genes meeting the inclusion criteria for GO term enrichment analysis – i.e. |log₂-fold change|>0.58496, equivalent to |fold change|>1.5, and adjusted P<0.05 – are plotted in purple and red (upregulated) or blue (downregulated). Gene symbols in bold highlight transcriptional alterations consistent with hyperactivated TGFβ signaling or cardiac stress. (B) Dot plots showing the relative expression levels of tgfb1b, tgfbr2a, thbs1a and serpine1 transcripts in 5 dpf CTRL and ltbp1^−/−; ltbp3^−/− larvae as determined by qPCR. Statistical significance was determined with an unpaired t-test. n=4 biological replicates and n=3 technical replicates. Errors bars show one standard deviation. *P<0.05; **P<0.01; ***P<0.001. (C-F) Brightfield images of 5 dpf (C,D) and 3 dpf (E,F) CTRL (C,E) and ltbp1^−/−; ltbp3^−/− (D,F) larvae processed for whole-mount in situ hybridization to detect thbs1a transcripts. Closed and open arrowheads highlight the atrioventricular canal (AVC) and outflow tract (OFT), respectively. Little to no variation was observed between animals within each group (n>10/group). (G) Coordinate plane showing the log₂-fold changes as in A plotted on the y-axis, and the log₂-fold changes for orthologous mouse genes in aneurysm tissue from Fbn1^mgR/mgR mice relative to control tissue plotted on the x-axis. Mouse data were sourced from Zilberberg et al. (2015). Orthologous gene pairs meeting the inclusion criteria for GO-term enrichment analysis – i.e. |log₂-fold change|>0.37851, equivalent to |fold change|>1.3, and adjusted P<0.1 in both datasets – are highlighted in red. For each quadrant, the probability (p) that the indicated number of gene pairs (n) fulfills the inclusion criteria by chance was determined using a hypergeometric test. ns, not significant. ****P<0.0001, *P<0.05.

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.