Knockdown of PIP5k3 Fleck Corneal Dystrophy-Linked Gene in Zebrafish

Purpose: Mutations in PIP5K3 gene are associated with François–Neetens Mouchetée Fleck Corneal Dystrophy (CFD) (Li et al. 2005). This rare autosomal dominant corneal dystrophy is characterized by small white flecks in the stroma which contain complex lipids. PIP5k3 which belongs to the phosphoinositide 5-kinase family, is localized in the internal membranes, maintains vesicles integrity and regulates endosomes trafficking containing lysosomaly directed fluid phase cargo. Our aim is to investigate the role of PIP5k3 during eye development, using the zebrafish as model. Methods: Hundreds of zebrafish embryos at stage 1 to 4 cells were injected with 1 nl of an antisense morpholino oligonucleotide covering -8 to +17 of the pip5k3 mRNA to knockdown translation. The sense morpholino was used for control injections. Morpholino concentrations of 1000 µM, 500 µM and 100µM were tested which correspond to 8 ng, 4 ng and 0.8 ng respectively. Larvae morphology was observed at 5dpf and morpholino toxicity assessed. Then, they were separated according to their phenotype severity, embedded in Technovit 7100, cut at 5µm and stained with toluidine blue for eye histology analysis. Results: Injected larvae with no visible morpholino toxic effect at the macroscopic level look like uninjected larvae. The eyes of these morphants have a normal macroscopic morphology. At histology, all retina layers are present and well developed except for the inner nuclear layer where between 4 to 8 small white vesicles or swollen cells per eyes are visible. Other more severe phenotypes in the morphant eyes and entire body were also observed, but these are probably related to morpholino toxicity as they were also observed on larvae injected with control morpholino. Conclusions: In contrary to the human phenotype where flecks are observed in the cornea, knockdown experiments in zebrafish reveal \"flecks\" in the INL. PIP5k3 mutations in mammalian cells lead to endocytic vacuolation and this could be correlated with the zebrafish knockdown. Further analysis of the treated zebrafish will be necessary to elucidate the physiological role of PIP5k3 in the development of the eye.