Abstract
Purpose :
Uveal coloboma is a congenital eye disease caused by the incomplete closure of the optic fissure during early fetal eye development and accounts for up to 10% of childhood blindness. Disruptions in genes important for optic fissure closure (OFC) are predicted to cause coloboma. We hypothesize that a cross-species meta-analysis could be used to identify novel genes responsible for human coloboma. We validated our top candidate genes in zebrafish via duplex guide Crispr/Cas9 methodology.
Methods :
We compiled and matched gene expression profiles (of mice, zebrafish, and humans) from four independent research articles: Brown et al. 2009, Cao et al. 2018, Patel et al. 2020, Richardson et al. 2019. A time-course analysis comparing gene expression from before and during optic fissure fusion, and a spatial analysis comparing the optic fissure to the optic cup during OFC found several potential coloboma candidate genes. In situ expression studies of the top candidate genes identified were carried in zebrafish at 24 hours post-fertilization (hpf), 36hpf, 48hpf, and 72hpf, to observe spatial and temporal expression. Gene function is tested by inducing biallelic deletions via duplex guide multi-target CRISPR-Cas9 injections in zebrafish, as detailed by Wu et al. 2018 and Hoshijima et al. 2019, allowing us to knockdown the target genes and score the phenotype for coloboma in F0 stage.
Results :
Candidate genes lum, apod, wls, and anxa3 were localized to the developing wild-type zebrafish eye by in situ hybridization during the time of OFC. Notably, lum expression is seen around the eye, while apod and anxa3 are expressed prominently in the lens. Notably, wls is expressed along the optic fissure margins. CRISPR/Cas9 crispant knockdowns of candidate genes revealed coloboma phenotype (i.e., failure of OFC) in F0 zebrafish. Severity of phenotype varied slightly between each gene; they all presented a notable indentation and lack of pigmentation along the ventral region of the eye, a clear indication of improper OFC and coloboma.
Conclusions :
Our results are consistent with our hypothesis that cross-experiment analysis of gene expression data may help identify potential coloboma genes. These studies may improve our understanding of the molecular basis of OFC and assist in the identification of novel human disease genes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.