Chamber identity disrupted at the AVC with loss and overexpression of Sema3f. A, B myh6 in 36 hpf mutant hearts expressed anterior (arrowheads) to the AVC (dotted line). C Discrete change in tnnt2a ISH signal at the atrial-ventricular border in WT but not mutant 48 hpf heart (arrowheads). D-G Confocal of 48 hpf hearts immunolabeled for pHH3 and MF20 (D,E), and DM-GRASP (F,G). Discrete expression border at the AVC (yellow arrowheads) in WT (D, N = 1, n = 5/5, and F, N = 3, n = 11/12) but not mutant hearts (E, N = 1, n = 8/9 and G (white arrowhead), N = 3, n = 10/12). H–K bmp4a mRNA restriction to the AVC (arrows) in WT at 48 hpf (H, N = 4, n = 21/30) and 72 hpf (J, N = 1, n = 8/9), but expansion beyond the AVC (arrowheads) in mutants (I, 48 hpf, n = 36/45; K, 72 hpf, n = 10/11). L, M myl7:sema3f plasmid-driven Sema3f expression in heart cardiomyocytes, with immunolabeling for myc-tagged Sema3f (L) and ISH for bmp4a (M) at 48 hpf. bmp4a mRNA expands beyond the AVC with Sema3f overexpression (arrowheads, M; N = 2, n = 24/37 expanded) but not control (bmp4a n = 17/20). N Average RGB MF-20 immunolabel intensity across the antero-posterior heart axis. High expression in WT (n = 5) ventricle sharply tapering at the atrial border (~ 150 arbitrary units (a.u.); arrowhead) is not seen in mutants (n = 9). Note sema3fb mutant hearts are small. O RT-qPCR for bmp4a mRNA (N = 4, p = 0.68). P-R Mean pHH3 + actively dividing MF20 + cardiomyocytes (P, p = 0.71), TUNEL + /MF20 + cardiomyocytes (Q, p = 0.99), and MF20 + ventricular cardiomyocytes (R, p = 0.72) at 48 hpf. S, T Mean ventricular cardiomyocyte area (S) and perimeter (T) in sema3fbca305 as compared to WT (p = 0.016 and p = 0.03, respectively). N = 3, n = 4–5 WT and n = 6 mutants, with 9–12 DM-GRASP + cells/embryo. Scale bar in C is 40 µm and in D is 25 µm (A,B,D-M)
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