Fig. 3

Impaired left-right asymmetry development upon Mcm2 knockdown is accompanied by shorter cilia. (A) Expression of mcm2in the tailbud of zebrafish embryos. Scale bars: 150 μm (tailbud and 4ss), 200 μm (8 ss). (B) Injection strategy to target KV cells. (C) Randomized heart looping after KV-specific Mcm2 ablation. P = 0.0007, two-tailed Fisher's exact test. n = 4 experiments. Number of embryos: NI = 121; splCTRL = 115; Mcm2 splMO = 118. (D) Confocal stacks of motile cilia (green) in the KV of 6–8 ss embryos injected with Mcm2 control or Mcm2 translation blocking MO. Cilia were labelled using an anti-acetylated tubulin antibody. Apical cell borders for visualization of the KV area were stained with an anti-PKCζ antibody (red). Scale bar: 10 μm. (E) Cilia numbers are not changed in the KV of Mcm2 morphants. P = 0.1403 (CTRL MO vs Mcm2 MO), p>0.9999 (Mcm2 MO/Ctrl Mo vs Rescue). Kruskal–Wallis test with Dunn's multiple comparisons test. In four experiments 29 CTRL MO, 28 Mcm2 MO and 12 Rescue KVs were assessed. (F) Depletion of Mcm2 results in shorter KV cilia, which can be rescued by co-injection of MO-insensitive RNA encoding Mcm2. P < 0.0001. Kruskal–Wallis test with Dunn's multiple comparisons test. n = 4 with 920 CTRL MO and 870 Mcm2 MO and 243 Rescue cilia. (G) KV outlines at 6–8 ss. Darker colour reflects larger areas. Scale bar: 25 μm. (H) KV is not significantly altered upon Mcm2 knockdown or its rescue. n = 19 CTRL MO, 22 Mcm2 MO and 10 Rescue KVs. P = 0.7952 (Ctrl MO vs Mcm2 MO), P = 0.1425 (Mcm2 MO vs Rescue). Kruskal–Wallis test with Dunn's multiple comparisons test. (I) Confocal stacks of immunostained pronephric ducts (cilia: acetylated tubulin, red, duct: PKCζ, green) at 24 hpf. Dilatation of the pronephric duct as well as disorganization of cilia was seen in the majority of Mcm2 depleted zebrafish. Scale bar: 10 μm. (J) Distal cilia length in the pronephros is reduced upon Mcm2 knockdown. n = 10 CTRL MO and 8 Mcm2 MO embryos with 368 and 299 cilia, respectively. P < 0.0001. Two-tailed Mann–Whitney test. (K) Primary cilia in the tailbud of 6–8 ss embryos. Scale bar: 5 μm. (L) Primary cilia length is reduced in the presence of Mcm2 MO and restored, when co-injected with RNA encoding Mcm2. n = 375 (CTRL MO), 413 (Mcm2 MO) and 171 (Mcm2 rescue) cilia. P < 0.0001. Kruskal–Wallis test with Dunn's multiple comparisons test. (M) Hh pathway activity is dampened in Mcm2 depleted zebrafish as shown for the Hh target genes gli1 and nkx2.2a. P = 0.0018 (gli1) and P = 0.0013 (nkx2.2a), unpaired, two-tailed t-test with Welch's correction. n = 5 experiments.