Fig 1.
Echocardiography of index patient.
Postnatal echocardiography of patient II-2 showing a bipartite RV with agenesis of the apex. Marked dilatation of the RA due to severe tricuspid insufficiency (grade 3/4). RA, right atrium; RV, right ventricle; LV, left ventricle; LA, left atrium.
Fig 2.
KIF20A mutations affect Aurora B transport during cell division in patient fibroblasts.
Fig 2A shows quantification of KIF20A transcript level in unrelated controls and patients by qPCR done in duplicates. KIF20A expression was normalized to the expression of the house-keeping gene GAPDH. ** indicates p< 0,01. Fig 2B shows KIF20A levels in unrelated control and patient fibroblasts undergoing cell division. Fig 2C shows western blot analysis of KIF20A and other anaphase spindle protein levels in unrelated control and patient fibroblasts.
Fig 3.
KIF20A mutations affect Aurora B transport during cell division in patient fibroblasts.
(A) Localization of KIF20A in control (C2) and two patient (P1 and P2) fibroblasts undergoing cell division. (B) Cells were stained with antibodies for KIF20A, Aurora B and the Aurora B pS911 phosphorylation site on KIF23 (marked with arrows).
Fig 4.
Functional studies of the KIF20A R182W mutant.
(A) Microtubule stimulated ATPase assays for control wild type (WT) and patient (R182W) KIF20A proteins revealed a near complete loss-of-function. (B) Localization of wild type KIF20A (WT) and an engineered “rigor” mutant (E245A) and the patient-associated R182W mutant in HeLa cells revealed that Aurora B remains trapped on chromatin and is not present on the central spindle.
Fig 5.
Zebrafish kif20a knockdown studies.
(A) RT-PCR analysis of zebrafish kif20a gene expression during early stages. Gapdh was used as a housekeeping gene. (B) Western blot analysis of whole lysates from control and kif20a morphants showing a 74% protein reduction. Actin was used as a loading control. (C) Morphological analysis of zebrafish control and kif20a morphants at 3–4 dpf. Upper panel: Bright-field and fluorescence images of zebrafish control and kif20a morphants at 3 dpf. The white star indicates cerebral oedema in the morphants, the red arrows indicate the cardiac region where cardiac oedema is pronounced in the morphants and absent in controls. Lower panel: Bright-field and fluorescence images of zebrafish control and kif20a morphants at 4 dpf. The red arrows indicate the cardiac region where cardiac oedema is pronounced in the morphants and absent in controls. (D) Rescue experiments where embryos were injected with kif20a-MO only, or co-injected with human KIF20A WT cDNA or KIF20A R182W cDNA. The percentage of cardiac phenotype in each groups at 3 dpf is presented. Data are represented as mean ± SD. Stars represent the results of one-way ANOVA-Dunnett’s post hoc test (*p<0.05, **p < 0.01, ***p<0.001, ns is not significant). (E) Dose dependent effect of kif20a-MO with varying concentrations of kif20a-MO (range 0–2 mM), injection dose was 4.6 nl. The percentage of cardiac phenotype at 3 dpf is shown.
Fig 6.
Histology and cardiac function evaluation of zebrafish kif20a morphants.
H&E staining of zebrafish embryos injected with control MO and kif20a MO at 4 dpf. Upper panel: Bright-field images of control and kif20a morphants at 4 dpf showing the section locations 1 and 2. Middle panel: Representative H&E staining images of control and kif20a morphants at position 1 and 2 in 10x and 20x magnification. White arrows in position 1 indicate the aortic arch, and white arrows in position 2 indicate gut. Lower panel: Quantification of ventricle and atrium thickness using H&E staining data above. Data are represented as mean ± SD. Stars represent the results of one-way ANOVA-Dunnett’s post hoc test (*p<0.05, **p < 0.01, ns, not significant). (B) Cardiac function analysis. Left panel: Heart rate in beats per minute is compared in control and kif20a morphants at 3 dpf and 4 dpf. A significant increase in heart rate is observed in the morphants at 4 dpf, most likely due to progressive cardiac failure. Right panel: Fractional shortening measured at 4dpf is compared in control and kif20a morphants respectively in the atrium and ventricle. Although a significant increased fractional shortening is present in the morphants, more outliers are seen, suggesting systolic failure.
Table 1.
Functional data in zebrafish larvae.