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Fig. 3 Cardiac function promotes OFT growth. (A,F,K,P,U,V,W) Lateral views of three-dimensional reconstructions comparing the OFT and ventricle in wild-type (WT) (A,K) and tnnt2a mutant embryos (F,P), labeled by immunofluorescence for Alcama (red) and Tg(fli1a:negfp) (green). As Alcama is found in the basolateral boundaries of myocardial cells, the endocardium is visible through the myocardial layer. Closer views of the OFT (A′,F′,K′,P′) demonstrate relatively similar morphology in WT (A′) and tnnt2a mutant (F′) embryos at 36 hpf (n=5, 4; N=2). See also Movies 1 and 3. However, by 51 hpf, whereas the WT OFT endocardial and myocardial volumes (K′) have expanded significantly (P<0.0005, Student's t-test), the OFT endocardium in tnnt2a mutants (P′) has failed to expand, as quantified in U and V, resulting in a significantly higher ratio of myocardial to endocardial volume in tnnt2a mutants by 51 hpf (W) (n=7, 6; N=2; **P<0.01, ***P<0.001, difference from WT at same stage, Student's t-test). See also Movies 2 and 4. (B,C,G,H,L,M,Q,R) Lateral (B,G,L,Q) and frontal (C,H,M,R) views of smoothed OFT endocardial surfaces generated from three-dimensional reconstructions of cytoplasmic GFP localization in Tg(kdrl:grcfp) embryos (Fig. S3) display relatively similar OFT morphologies in WT (B,C) and tnnt2a mutants (G,H) at 36 hpf and the different OFT morphologies in WT (L,M) and tnnt2a mutants (Q,R) at 51 hpf. Lateral and frontal surfaces are oriented with their proximal end at the bottom. Of note, the lateral side of the WT OFT at 36 hpf (B) appears to transition into the frontal side at 51 hpf (M), suggesting that the OFT rotates 90° during this time. (D,E,I,J,N,O,S,T,X,Y) Middle cross-sections through the OFTs shown in A′, F′, K′, P′ indicate that the OFT endocardium collapses in tnnt2a mutants between 36 (I) and 51 hpf (S), in contrast to its broadening in WT (D,N). These contrasts are also evident in comparisons between middle (E,J,O,T), proximal (E′,J′,O′,T′) and distal (E″,J″,O″,T″) cross-sections through the smoothed surfaces of the OFT endocardium in Tg(kdrl:grcfp)-expressing embryos (Fig. S3), as quantified in X and Y (n=6, 4 for 36 hpf; 5, 5 for 51 hpf; N=2; **P<0.01, ***P<0.001, ****P<0.0001; differences from WT at same positions, Student's t-test). The broadening of the WT endocardium between 36 and 51 hpf is especially striking at its distal end (E″,O″,X,Y). Proximal, middle and distal sections are oriented with the OC of the OFT to the left. (U-Y) Bar graphs and line graphs indicate mean±s.e.m. Scale bars: 5 μm.