Figure 7. Growth failure and ciliopathy phenotypes are separable in a dose dependent manner.

a-d) Morphological ciliopathy phenotypes are evident at higher levels of plk4 depletion. a) 1.5 ng plk4 morphants at 5 dpf exhibit ciliopathy phenotypes, including dilated brain ventricles (arrow), pronephric duct cysts (arrow head) and ventral body axis curvature. b) Penetrance of ciliopathic phenotypes (scored by presence of ventral curvature) in 2 dpf plk4 morphant embryos injected with 0.5 – 3ng MO. c) Heart laterality defects are observed at 3 dpf in Tg(bre:egfp) zebrafish embryos injected with 3 ng plk4 MO, with loss of asymmetry (midline) or inversion of ventricle-atrium asymmetry (reversed) (A, atrium; V, ventricle). Right, quantification of heart laterality defects (exp=2, n>40 embryos).

d) The proportion of ciliated cells is reduced in the Kupffer’s vesicles of 3 ng plk4 morphants. Left, representative images from control and 3 ng plk4 morphants at 16 hpf. aPKC, atypical protein kinase C (Kupffer’s vesicle marker, green); Ac-tubulin, acetylated tubulin, cilia (red). Right, quantification of cells ciliated in the Kupffer’s vesicle.

e,f) Model of disease pathogenicity. e) Autoregulation of Plk4 results in a narrow window in which mutations impair enzymatic activity without resulting in embryonic lethality. At 50% transcript levels protein levels are normal³⁴. Further depletion of PLK4, as seen in PLK4 patients, leads to protein loss and growth defects. Additional loss of cellular PLK4 activity results in cilia-related phenotypes. f) Centriole duplication becomes inefficient at reduced PLK4 levels, with reduced centriole number impairing mitotic spindle formation and cell cycle progression. With severe reduction in PLK4 activity, cells completely lacking centrioles are generated, which are unable to form cilia, leading to ciliopathy phenotypes.