Characterisation of the Morphology and Genetic Determinants of Retinal Vasculature in Zebrafish

Abstract

Purpose:: Many human eye diseases are linked with abnormalities in the retinal blood supply. The advantages of zebrafish as a model for genetic, developmental and pharmacological studies has not been exploited in this area. Our objectives are to: i) characterise the morphology of the developing, adult and senescent zebrafish intraocular vasculature; and ii) identify novel genetic and chemical determinants for hyaloid/retinal vasculature

Methods:: Vessels were visualised under bright-field and fluorescence microscopy after staining by the following techniques: i) fli-1 transgenic line expressing EGFP in endothelial cells, ii) antibodies specific for blood vessels and glial cells, iii) DAPI nuclear staining and iv) endogenous alkaline phosphatase staining. For ultrastructural analysis of the vessels larval, adult and senescent retinas were processed for electron microscopy. Analyses for genetic determinants of intraocular vasculature utilises collections of described vascular and lens mutants, morphant screens and an ongoing mutagenesis screen. For chemical screens a library of 5000 small molecules is being screened to ascertain drugs which induce specific intraocular vascular phenotypes.

Results:: In zebrafish, blood vessels are initially found attached to the lens from 2.5 days post fertilisation, but progressively lose contact with the lens during development (at 20-30 dpf) to finally overlay the inner surface of the adult retina. Ultrastructural analysis shows the vessels share many features with mammalian intraretinal blood supplies, including pericytes and junctional complexes. Vesicle vacuolar organelles emanating from the vessels are observed fusing to the inner limiting membrane, suggesting an active transfer of substances between the vessels and the retina. Mutants and morphants with abnormal intraocular vasculature morphology will be presented.

Conclusions:: Zebrafish retinal blood supply is easily and reproducibly observed during development and abnormalities in these intraocular vessels are clearly detected in mutant backgrounds. Therefore zebrafish can be applied in genetic and chemical approaches to identify molecular regulators of intraocular vasculature development.

Keywords: vascular cells • retinal development • blood supply