Developmental Defects in Nuc1 Retina Affect Normal Physiological Function

Abstract

Abstract: : Purpose: We have discovered a spontaneous mutation in the Sprague Dawley rat with a novel and unusual eye phenotype that we have named Nuc1. In this study we show that the Nuc1 mutation suppresses programmed cell death during development compared to a wild–type retina. Morphological and functional tests suggest Nuc1 also affects the maturation process of the neuronal cells in the retina. Methods: Immunofluorescence was performed on frozen sections using postnatal day 9, 25, 70 and 87 Nuc1 homozygous and wild–type Sprague Dawley rats with antibodies to neurofilament 70, syntaxin, rhodopsin kinase 1α, GFAP, PKC–α, calbindin, JH455 and JH492. TUNEL staining was performed for apoptotic cells. Whole isolated retinas were flat–mounted and observed under fluorescence microscopy after cardiac injection of FITC–conjugated dextran to compare retinal vascular development in Nuc1 homozygous with the wild–type. Dark and light–adapted eletroretinography (ERG) were recorded to observe the function of rod and cone pathways. Results: In Nuc1 homozygotes, the transient layer of Chievitz persists in adult retinas, indicative of delayed development, and the retinas are thicker than normal. Semi–quantitative measurements of TUNEL positive cells, which have begun to degrade their genomic DNA, indicate approximately two–fold more apoptotic cells in the wild–type retina as compared to Nuc1 homozygotes. Our immunohistochemical studies indicate delayed differentiation of amacrine, horizontal, bipolar and ganglion cells in Nuc1. This developmental abnormality activates Muller cells, believed to be involved in the protection and/or repair of retinal neurons, and their processes span the Nuc1 retina. The ERGs show dysfunction of the rod pathway while the cone system function remains intact in Nuc1 homozygotes. Immunocytochemical studies indicate abnormal rods but morphologically normal cones in Nuc1 homozygotes. Our studies also indicate that the retinal vasculature and neuronal cells are coordinately affected in Nuc1. Conclusions: The Nuc1 retina is an excellent model to study vertebrate central nervous system development. Nuc1 may provide important clues as to how developmental deficits affect normal functions of the retina.

Keywords: retinal development • apoptosis/cell death • electroretinography: non-clinical