Fig. 9

Loss of MCM7 impairs cilia in zebrafish. (A) Confocal stacks of zebrafish KVs at 8ss. Cilia in red (acetylated tubulin) and apical borders of KV in green (PKCζ). Scale bar: 5 μm. (B) Cilia number is not significantly changed upon loss of Mcm2, but shows a tendency towards fewer cilia. Rescue embryos were consecutively injected with Mcm7 MO and capped RNA for human MCM7, which is insensitive to the MO. N = 14 (CTRL MO), 18 (Mcm7 MO) and 16 (Mcm7 MO+MCM7 RNA) KVs. P = 0.4035 (CTRL MO vs Mcm7 MO), 0.7541 (Mcm7 MO vs Rescue) and 0.0873 (CTRL MO vs Mcm7 Rescue). One-way ANOVA with Sidak's multiple comparison test. (C) Mcm7 knockdown leads to shorter KV cilia. Co-injection of RNA for human MCM7 rescues cilium length. P < 0.0001. Kruskal–Wallis test with Dunn's multiple comparisons test. n = 482 CTRL MO, 480 Mcm2 MO and 355 Rescue cilia. (D) Manipulation of Mcm7 levels does not change the area of the KV. Cartoon displays outlines of individual KVs with darker shading indicating larger areas. Scale bar: 25 μm. (E) Bar graph summarizing KV area measurements in control conditions and upon Mcm7 knockdown and rescue thereof. N = 14 (CTRL MO), 11 (Mcm7 MO) and 11 (Mcm7 MO+MCM7 RNA) KVs. P = 0.9269 (CTRL MO vs Mcm7 MO or Rescue). One-way ANOVA with Holm–Sidak's multiple comparison test. (F) The laterality gene southpaw (spaw) loses its leftward restriction upon knockdown of Mcm7 (22 ss). Reconstitution with human MCM7 restores expression left from the midline. ***P < 0.001 (CTRL MO vs Mcm7 MO and Mcm7 MO vs Mcm7 MO+hMCM7), two-tailed Fisher's exact test. Number of embryos: NI = 81; CTRL MO = 67; Mcm7 MO = 80, Mcm7 MO+hMCM7 = 66. (G) Confocal z-stacks of primary cilia in the tailbud of 8 ss embryos. Scale bar: 5 μm. (H) Knockdown of Mcm7 reduces primary cilium length in vivo. Co-injection of RNA for human MCM7 rescues the defect. n = 382 (CTRL MO), 334 (Mcm7 MO) and 272 (Mcm7 rescue) cilia. **P=0.0046, ****P < 0.0001. Kruskal–Wallis test with Dunn's multiple comparisons test.