(A) At 48 hr post fertilization (hpf), the ventricle (V) of the two-chambered zebrafish heart is placed left and above the atrium (A). Adsl-depleted embryos more frequently develop inversely looped hearts or developed unlooped hearts (no loop) (as scored by whole-mount in situ hybridization for cardiac myosin light chain 2 [cmlc2]). N = 6 experiments with a total of 266 embryos (NI), 176 embryos (CTRL MO), and 188 embryos (Adsl ATG MO). Scale bar = 100 μm. (B) Whole-mount in situ hybridization for angiopoietin-like 3 (angptl3) to assess liver placement in 48 hpf embryos. Dorsal view. Scale bar = 200 μm. 185 NI, 121 CTRL MO, and 99 Adsl ATG MO embryos. (A, B) Two-tailed Fisher’s exact test; **p<0.0015, ****p<0.0001. (C) Whole-mount in situ hybridization for the left lateral plate mesoderm marker southpaw (spaw) at 20 somite stage (ss). Spaw is normally expressed in the left lateral plate mesoderm. When LR asymmetry is disturbed, spaw can be detected on the right side or on both sides. Aberrant expression of spaw in Adsl morphants. Co-injection of RNA encoding zebrafish Adsl restores proper spaw expression. Two-tailed Fisher’s exact test; *p=0.0451, **p=0.0016, ****p<0.0001. Results from five experiments with 121 embryos (NI), 142 (CTRL MO), 128 (Adsl ATG MO), and 105 (Rescue) are shown. Scale bar = 200 μm. (D) Confocal z-stacks of the Kupffer’s vesicle (KV) of six ss embryos. Cilia are stained red (acetylated tubulin), while apical cell borders were stained for PKCζ (green). Scale bar = 10 μm. (E) No significant changes in the size of the KV upon Adsl depletion. n = 25 (CTRL MO), 25 (Adsl ATG MO), and 18 embryos (rescue with zebrafish adsl RNA). Each circle is one embryo, line indicates median. Kruskal–Wallis test with Dunn’s correction. p-values: CTRL MO vs. Adsl ATG MO: 0.2582; CTRL MO vs. Rescue: >0.9999; Adsl ATG MO vs. Rescue: 0.1684. (F) No significant changes in the number of cilia per KV. n = 32 (CTRL MO), 30 (Adsl ATG MO), and 20 embryos (rescue with zebrafish adsl RNA). Each circle is one embryo, lines show means. One-way ANOVA with Sidak’s multiple comparison test. p=0.5538 (CTRL MO vs. Adsl ATG MO), 0.2844 (CTRL MO vs. Rescue), and 0.9225 (Adsl ATG MO vs. Rescue). (G) Shorter cilia in Adsl morphants can be partially elongated by co-injection of RNA encoding zebrafish Adsl. n = 960 cilia (CTRL MO), 798 (Adsl ATG MO), and 540 (Rescue). Kruskal–Wallis test with Dunn’s correction, lines indicate medians; ***p=0.0008.
