iguana mutant zebrafish have defects in ciliogenesis and left–right asymmetry. (A) Top, brightfield images of an igu mutant embryo and a wild-type sibling embryo (labeled below as “WT sib,” see Materials and methods), 48 h post-fertilization (hpf). The red line indicates the region of the pronephric duct that was analyzed for cilia. Anterior, left. Dorsal, up. Bar, 200 μm. Bottom, animals were labeled at 48 hpf with an anti-acetylated tubulin antibody. Bar, 20 μm. Right, the numbers of cilia within the posterior-most 100 μm of the pronephric duct were determined. The numbers of cilia in igu morphants and control morpholino-injected embryos were also determined. Data are means ± standard deviations. Asterisk, data were determined to be significantly different, P < 0.0001, unpaired t-test. (B) Animals were fixed at 28 hpf and labeled with a riboprobe from the cardiac myosin light chain 2 gene (cmlc2). Anterior, up; dorsal view. Bar, 100 μm. Asterisks indicate the labeled heart. A control morpholino-injected embryo displayed heart development on the left side of the embryo and an iguana morpholino-injected embryo displayed heart development on the right side. Right, percentages of embryos displaying left, medial, or right heart development are shown. 44 control morphants (control MO), 55 igu morphants (igu MO), 49 wild-type siblings of igutm79a/tm79a embryos (WT sib, see Materials and methods), and 9 igu mutant embryos were analyzed. (C) Cilia in Kupffer′s vesicle from control morpholino, iguana morpholino, and smoothened (smo) morpholino-injected embryos were labeled with an anti-acetylated tubulin antibody (red). Nuclei are labeled with DAPI (blue). White dotted circle, approximate boundary Kupffer′s vesicle. Embryos were fixed at the six somite stage. Bar, 10 μm. Right, mean numbers of cilia in Kupffer′s vesicle ± standard deviations. Asterisk, data were determined to be significantly different, P < 0.0001, unpaired t-test.
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