Splice-blocking efficacy, primers used to assay splicing, left-right markers examined in 3-OST-5 morphants, tracks of fluorescent beads injected into the KV at 6 SS, 3-OST-5 RNA expression, midline marker expression. (A) Representative in situ images illustrating normal (black arrows) and reversed (red arrows) heart (cmlc2) and gut (fkd2) looping in 3-OST-5 morphants. (B) Table containing primers used for detecting spliced versus unspliced transcript when embryos were injected with translational splice blocking morpholinos. (C) Dose-dependent splice blocking efficacy of 3-OST-5 MO1. Increasing amounts of MO injected resulted in a 70±12% decrease of spliced mRNA. (D) Dose-dependent splice blocking efficacy of control 3-OST-3Z MO1. Increasing MO dose resulted in a 93±4% decrease of spliced mRNA. ImageQuant TL software was used to semi-quantitatively measure band intensity. The values listed are the percentage of spliced product compared with uninjected WT. (E,F) Whole-mount in situ analysis of 3-OST-5 mRNA, which was expressed ubiquitously throughout the embryo during epiboly (E) and early somitogenesis (F). Black arrow indicates staining in DFCs, white arrow indicates the position of KV. (G,H) Whole-mount in situ analysis of shh expression in uninjected (G) and 3-OST-5 morphants (H). (I,J) Whole-mount in situ analysis of lefty1 expression in the midline of uninjected (I, n=113) and 3-OST-5 morphants (J, n=82), 3-OST-5 morphants had normal expression of lefty1 expression in the midline. (K-M) Tracks of fluorescent beads injected into KV of uninjected (K) and 3-OST-3Z MO1 embryos (L) with normal bead flow, and 3-OST-5 MO1 embryos (M), displaying uncoordinated bead movement due to short but motile cilia.
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