Poc1b Knockdown Results in Visual Impairment in Zebrafish
Poc1b morphants display the typical ciliopathy phenotypes previously described by Pearson et al.,15 including pericardial edema, small eyes, pigment mislocalization, and a shortened and curved body axis (Figure S4A). Small eyes were already observed in larvae treated with 2 ng MO and became more frequent as the dose increased (Figure 4B; Figure S4B). At a dose of 6 ng MO, smaller eyes occurred in 39.8% of morphants and were on average significantly smaller than wild-type eyes (Figure 4B; Figure S4B), and 92.5% of morphants displayed one or more of the phenotypes described above without higher mortality than in controls. Specificity of the MO knockdown was previously verified with a second MO targeting the 5′ splice site of poc1b exon 2, which resulted in 59.1% of morphants with small eyes (Figure S4B).
The OKR was assessed in larvae injected with control and poc1b MOs. OKR was absent or lower in morphants with small eyes than in wild-type or control-MO-injected larvae (Figure 4C; Movies S1 and S2). Morphants that received the same dose of MO but had normal-sized eyes responded normally to the OKR stimulus. This phenotype was confirmed in larvae treated with a splice-site-blocking MO (data not shown). Outer-segment length was not affected in control-MO-injected larvae and morphants that did show an OKR. Histological analysis of the retina of the morphants subjected to OKR measurement revealed shortened or absent outer segments of the photoreceptors, whereas lamination appeared normal (Figures 4D and 4E). We observed a perfect correlation between the small-eye phenotype and a diminished or absent OKR. The size of the eyes appeared to correlate with outer-segment length and responsiveness to visual stimuli. As such, we could quantify the size of the eye to measure the effects of loss of Poc1b function. Indeed, coinjection of 100 pg human wild-type POC1B mRNA, but not of c.199_201del (p.Gln67del) or c.317C>G (p.Arg106Pro) mutant POC1B mRNA, significantly rescued poc1b knockdown (Figure 4B; Figure S4B).
Immunohistochemical staining for typical rod (rhodopsin) and cone (zpr-1) markers was absent from a subset of cells in morphants with smaller eyes (Figure S4C). High-magnification pictures showed that whereas immunostaining was absent in certain regions of the morphant retina, the nuclei of the photoreceptor cells were still present.