The role of WNT signaling genes in the optic fissure and eye morphogenesis in a zebrafish model

Ocular coloboma is a condition caused by a malformation in optic fissure formation during early eye morphogenesis. It is characterized by visual impairment and in some instances, blindness in some children. The prevalence of ocular coloboma is estimated to range from 2 to 14 per 100,000 children. Although many pathways are known to play an important role in optic fissure closure, the mechanisms and genetic regulation driving optic fissure closure during development are still not widely understood. Different factors are associated with ocular coloboma, including genetic predisposition and environmental influences. The Wingless and Int1 (WNT) signalling pathway is conserved among vertebrates and plays an important role in regulating different developmental processes, including eye development. When the pathway is in a stimulated state, presence of WNT ligand binding to its receptor, results in deactivation of the complex consisting of APC, Axin and GSK3. This deactivation leads to accumulation of the cytoplasmic β-catenin that is translocated into the nucleus and activates transcription of target genes. Fzd5 gene plays an important role in eye morphogenesis during early stages of development. We hypothesized that abnormal levels of WNT signalling is driving the ocular coloboma phenotype. Here, we over-activated the WNT signalling pathway using the small molecule Bromoindirubin-3′-oxime (BIO) and lithium chloride (LiCl), which have been previously shown to impact this pathway. Multiple doses of LiCl treatment has a sensitivity period of disrupting WNT signalling pathway that lead to coloboma phenotype. On the other hand, BIO treatment lead to molecular disruption of WNT signalling pathway with normal eye development. Over-activation of WNT signalling was confirmed by the expression level of GSK3 and ß-catenin using western blot. Secondly, we generated a stable WNT mutant zebrafish line (fzd5) using CRISPR/Cas9 technology and observed coloboma phenotype by brightfield microscopy. This study demonstrated that overexpression of WNT signalling had a dose-dependent response and sensitivity period effect that lead to abnormal eye development both coloboma and microphthalmia phenotype in zebrafish model. Moreover, we demonstrated that zebrafish is an excellent model for CRISPR gene knockout of ocular developmental genes.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat