Fig. 4 | European Journal of Human Genetics

Fig. 4

From: Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of Origin recognition complex factors

Fig. 4

Loss of additional ORC components provokes cilium dysfunction in zebrafish. a Live images of 48 hpf control and Orc4 and Orc6 depleted zebrafish embryos showing phenotypes reminding of MGS (shorter axis, smaller heads and eyes) and cilium dysfunction (pericardial oedema and body curvature). Scale bar: 500 µm. b KD of either Orc4 or Orc6 leads to cystic kidneys. Scale bar: 100 µm. c Percentage of embryos developing pronephric cysts. Co-injection of RNA encoding human ORC4 or ORC6 partially rescues cyst formation, while the MGS variants do not. n = 177 (NI), 172 (CTRL MO), 58 (Orc4 splMO), 63 (Orc4 splMO + ORC4), 63 (Orc4 splMO + ORC4 Y174C), 93 (Orc6 splMO), 108 (Orc6 splMO + ORC6) and 90 (Orc6 splMO + ORC6 K202R*) embryos. d Heart looping is randomised after Orc4 or Orc6 KD in zebrafish. n = 3 experiments with 79 NI, 93 CTRL MO, 89 Orc4 splMO and 102 Orc6 splMO embryos. e Orc4 and Orc6 deficient embryos display random placement of the endocrine pancreas. n = 3 experiments (79 NI, 93 CTRL MO, 89 Orc4 splMO and 102 Orc6 splMO embryos). f Embryos depleted of Orc4 and Orc6, respectively develop KVs with shortened cilia. Confocal stacks of KVs of 8 ss embryos stained for acetylated tubulin (green, cilia) and PKCζ (red, apical cell borders). Scale bar: 10 µm. g Cilia are shorter upon Orc4 or Orc6 KD. n = 194–292 cilia. Kruskal–Wallis test with Dunnett’s multiple comparison test. h Cilia number per KV was not affected by loss of Orc4 or Orc6. p = 0.3936 (CTRL MO vs. Orc4 splMO), p = 0.9050 (CTRL MO vs. Orc6 splMO). n = 9 (Orc4 splMO) and 10 KVs (CTRL MO, Orc6 splMO). One-way ANOVA with Dunnett’s multiple comparison test. i The KV area shows a tendency to be smaller upon KD of Orc4 and 6. p = 0.0551 (CTRL MO vs. Orc4 splMO), p = 0.03285 (CTRL MO vs. Orc6 splMO). n = 9–15 embryos in total. One-way ANOVA with Dunnett’s multiple comparison test. c–e Stacked bar graphs analysed using two-tailed Fisher’s exact tests. g–i Means ± SEM

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