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Bicd2 is required for ciliogenesis in zebrafish embryos. (A–H) Zebrafish embryos were injected with the indicated morpholinos (MOs) or mRNA at the one-cell stage and collected at different time points. Western blot analysis of Bicd2 protein (A). Actin was used as a loading control. Bright-field micrographs and statistical analysis of the ciliary phenotypes of embryos injected with the indicated MOs or mRNA (B, C). Scale bar, 1 mm. Body curvature, P (1, Ctrl MO vs. bicd2 MO) < 0.0001, P (2, bicd2 MO vs. bicd2 MO + bicd2 mRNA) < 0.0001; Pericardial edema, P (1) = 0.0002, P (2) = 0.0024; Abnormal otoliths, P (1) = 0.0022, P (2) = 0.0147; Hydrocephalus, P (1) = 0.0001, P (2) = 0.003. Confocal images of cilia stained with anti-acetylated-α-tubulin antibody in KVs at 10 somite stage (D). The borders of KVs are indicated by white circular dotted lines. Scale bar, 10 µm. Quantification analyses of cilia number and length in KVs (E, F). Cilia number, P (1, Ctrl MO vs. bicd2 MO) = 0.0026, P (2, bicd2 MO vs. bicd2 MO + bicd2 mRNA) = 0.0049; Cilia length, P (1) = 0.013, P (2) = 0.0486. Whole-mount in situ hybridization images of embryos hybridized with the cmlc2 probe (G). Scale bar, 100 µm. Quantification analysis of cmlc2 expression patterns (H). Left, P (1, Ctrl MO vs. bicd2 MO) = 0.0001, P (2, bicd2 MO vs. bicd2 MO + bicd2 mRNA) = 0.0008; Middle, P (1) = 0.0156, P (2) = 0.0383; Right, P (1) = 0.0042, P (2) = 0.0112. Ctrl, Control. hpf, hours post fertilization. n, number of embryo samples (C, E, H) or number of cilia (F). Data were presented as mean ± SD from three independent biological repeats. Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.
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