CRB2 and CRB1 in Retinal Development and Maintenance

Abstract

Purpose: : In Leber congenital amaurosis vision is lost within the first year of life. In about 10% of the cases this is caused by mutations in the Crumbs homolog 1 or CRB1 gene. Mice lacking CRB1 show retinal degeneration in up to one quadrant of the retina, which might be due to overlapping functions with CRB2 in the other quadrants. CRB1 is localized in Müller glia cells, and CRB2 in Müller glia cells and photoreceptors, at a subapical region adjacent to adherens junctions at the outer limiting membrane. We generated and analyzed Crb2 conditional knockout (Crb2flox/flox) mice to study overlapping functions of CRB2. Moreover, we also studied the effect of removal of one allele of Crb1 in the Crb2-/- mice (Crb1+/-Crb2flox/flox). The aim of this project is to analyze the functions of CRB2 and CRB1 in retina development and maintenance.

Methods: : Crb2flox/flox mice were generated and crossed with Chx10-Cre mice. These animals were crossed with Crb1 mice. Animals from embryonic day 12.5 till 1.5 year of age were analyzed. In order to investigate the possible ectopic localization of proteins and cells we performed immunohistochemistry (IHC). Moreover, morphology of the retina was analyzed in plastic embedded sections and by electron microscopy. In vivo analysis of retinal function was performed using optical coherence tomography, scanning laser ophthalmoscopy and electroretinography (ERG).

Results: : Removal of only CRB2 resulted in retinal disorganization, starting, at the periphery, at embryonic day 18.5. At post-natal days 6 and 10, progressive disorganization was detected in all four quadrants of the retina, characterized by gaps at the outer limiting membrane (OLM) and many ectopic photoreceptor cell nuclei in the sub retinal space. At a later stage, retinal degeneration of the outer and inner retina was detected. IHC experiments showed loss of localization of members of the Crumbs complex at regions with disrupted OLM. We also observed increased number of phospho-H3 positive cells at postnatal day 3, increased number of c-caspase-3 positive cells in the outer nuclear layer from postnatal day 10, decreased length of cone photoreceptor segments, and increased levels of GFAP and CD11b suggesting gliosis and activated microglia. Crb1+/-Crb2flox/flox mice retinas showed more prominent phenotype with an earlier onset, compared with Crb2flox/flox retinas, characterized by many photoreceptor rosettes and misplaced cells in the sub retinal space and in the ganglion cell layer.

Conclusions: : The lack of CRB2 leads to early retinal disorganization and at a later stage to retinal degeneration. However, the severity and onset of the phenotype increased with additional loss of one allele of Crb1, suggesting a partial overlap of function between the CRB1 and CRB2.

Keywords: retinal development • retinal degenerations: cell biology • cell adhesions/cell junctions