Closing the gap: development of a novel zebrafish-based tool to assess optic fissure closure

Abstract

Purpose: Proper closure of the optic fissure during early embryogenesis is critical for normal eye formation. Failure of optic fissure closure results in coloboma and related ocular defects. Mutations in several developmentally important genes are known to cause colobomas; however, the genetic etiology for most patients remains unknown. As a means to characterize novel genes and pathways that contribute to coloboma formation, we developed a method to evaluate optic fissure closure in zebrafish.

Methods: The genetic and developmental similarities to the mammalian eye make zebrafish (Danio rerio) an ideal model to study early vertebrate eye development. To evaluate optic fissure closure, we assayed for changes in Pax2a expression, a gene transiently expressed in the ventral optic cup prior to optic fissure closure. The quantity of green fluorescent protein (GFP) in the enhancer trap mp204a:GFP transgenic zebrafish line was used as a proxy for Pax2a expression. As proof of principle, we tested whether sema3e, a gene expressed in ventral mesenchyme that exists between the edges of the optic fissure, affected GFP levels in the enhancer trap mp204a:GFP transgenic line. As a second test of optic fissure closure, we evaluated basement membrane dissolution by observing changes in laminin expression at the edges of the closing optic fissure.

Results: Transgenic expression of GFP in the mp204a:GFP enhancer trap line recapitulates endogenous Pax2a expression in the eye field, midbrain-hindbrain boundary, otic placode and pronephric mesoderm. Quantitative three-dimensional analysis using ImageJ software revealed increased expression of Pax2a in sema3e knockdown embryos compared to uninjected controls at 48 hours post fertilization (hpf), indicating aberrant optic fissure closure. Moreover, a reduction in overall eye size was observed at 48 hpf. We also observed that in sema3e knockdown zebrafish, laminin expression was increased compared to uninjected wild-type and the optic fissure edges exhibited delayed closure.

Conclusions: We demonstrate that quantitative three-dimensional analysis of the enhancer trap mp204a:GFP transgenic zebrafish line can facilitate the identification and screening of candidate genes that impact optic fissure closure.

Keywords: 440 candidate gene analysis • 599 microscopy: light/fluorescence/immunohistochemistry • 551 imaging/image analysis: non-clinical