<i>In vivo</i> zebrafish morpholino complementation assay showing the effect of <i>SIX6</i> nonsynonymous variants.

<p>Zebrafish embryos were microinjected with a translation blocking morpholino designed to target <i>six6a</i>. Total eye size (µm²) was measured 3 days post fertilization. Compared to the uninjected controls, morphants showed a significant reduction in eye size. Zebrafish were co-injected with the morpholino and a human <i>SIX6</i> allele (Glu93Gln, Glu129Lys, Asn141His, Leu205Arg, Thr212Met, or Ser242IIe). Results of each allele were compared to the <i>SIX6</i> non-risk allele (Ref). P-values are provided below the mean of each treatment.</p

<i>In vitro</i> luciferase assay results showing the effect of <i>SIX6</i> enhancer variants.

<p><i>SIX6</i> enhancer alleles were tested using a dual luciferase assay and the ratio of the experimental luciferase: control luciferase was calculated (DLR ratio). All vectors were co-transfected with NeuroD and E47. In this context, the <i>SIX6</i> enhancer is functioning to increase expression compared to the empty vector (pGL4.23), driven by a minimal promoter. Compared to the reference enhancer (Ref), one variant (Chr14:60974449_G) significantly increases the enhancer's activity.</p

Morpholino knockdown of <i>six6a</i>.

<p>Zebrafish were microinjected with a <i>six6a</i> translation blocking morpholino. Lateral images, taken 3 days post fertilization (3 dpf), of a wild-type zebrafish (left) and a morpholino injected zebrafish (right) are shown, highlighting the small eye phenotype (dashed circle).</p

Results from the <i>in vivo</i> zebrafish <i>six6a</i> morpholino complementation assay.

<p>N = sample size; SD = standard deviation.</p