3-OST-5 is required in whole embryos and cell autonomously in Kupffer’s vesicle for LR development. (A,B) Differential interference contrast images of 48 hpf embryos. Uninjected (A) and 3-OST-5 MO-injected (B) embryos at 48 hpf showing dose-dependent curly down tail phenotype. Both 3-OST-5 MOs showed a dose-dependent increase in both heart and gut reversals. (C,D) cmlc2-gfp transgenic fish (expressing GFP in cardiomyocytes) were used to visualize normal left looping (C) and reversed right looping (D) in 3-OST-5 MO1 embryos at 48 hpf. (E) Percentages of reversed heart and gut looping in 3-OST-5 morphants. Injection of 3-OST-5 MO1 (n=120; **P<0.001) or 3-OST-5 MO2 (n=121; **P<0.001) resulted in significant heart and gut looping compared with uninjected embryos (n=361). Co-injection of 3-OST-5 MO1 and 3-OST-5 ‘rescue’ mRNA resulted in a significant rescue of heart and gut orientation (n=137; **P<0.001). Injection of 3-OST-5 ‘rescue’ mRNA alone (n=171) or 3-OST-3Z MO (n=106) did not alter normal heart and gut looping. Error bars represent s.d. for all heart and gut looping treatments. (F) Representative images of normal (left-sided), reversed (right-sided), absent and bilateral spaw expression in 3-OST-5 morphants. Percentages of normal (left-sided), reversed (right-sided), absent, and bilateral spaw expression in the LPM of 3-OST-5 morphants (MO1, n=183; MO2, n=206), 3-OST-3Z morphants (n=240) or uninjected control embryos (n=220). 3-OST-5 morphants had a preponderance of bilateral expression, in comparison with predominantly left-sided expression in uninjected and 3-OST-3Z MO1-injected embryos. Embryos injected for DFC/KV-targeted knockdown, DFC3-OST-5 MO1 (n=112), showed a high rate of bilateral spaw expression. This is in contrast to DFCcontrol MO (n=97), Yolk3-OST-5 MO1 (n=61) and Yolkcontrol MO (n=90) injected embryos showing left-sided spaw expression.