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.

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.

Zebrafish kif20a knockdown studies.

Bright-field 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.

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