Crb1 And Crb2 Controls Cell Division During Retina Development

Abstract

Purpose: : Mutations in the Crumbs homologue 1 (Crb1) gene are associated with Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP12). Mice lacking CRB1 demonstrate mild retinal degeneration. However, mice lacking both CRB1 and CRB2 display severe retinal disorganization (only 2 nuclear and 1 plexiform layers) and thickening. These pathological features are similar to CRB1 LCA patient retinas. In order to understand why Crb1-Crb2 double knockout retinas are so disorganized, we analyzed the phenotype during retinal development.

Methods: : Crb1 knockout mice were crossed with a retinal Crb2-Chx10Cre knockout mouse strain. Animals of different ages were analyzed. Morphological analysis was done with toluidine blue staining of plastic-embedded sections and electron microscopy. To study possible ectopic localizations and up-/down-regulations of protein expressions, immunohistochemistry (IHC) was done on retinas as well as Western blotting.

Results: : In adult retinas from mice lacking both CRB1 and CRB2, only the number of the latest born cells (rods, bipolar cells, Müller glia and the latest subtypes of amacrine cells) is increased and equivalently spread through the two nuclear layers. As early as E13.5, double knockout retinas show already gaps in the OLM, accompanied with ectopic localization of progenitor and postmitotic cells. Furthermore, mislocalisation of Chx10-positive progenitor nuclei increases throughout retinal development. From E15.5, increasing amounts of ectopic phospho-H3 positive cells are found through the entire thickness of the developing retina whereas Crumbs complex and adherens junction members disappeared progressively from the OLM during development. Islet1-positive precursor cells, for amacrine and ganglion cells, remain slightly affected whereas Otx2-positive precursor cells, for photoreceptors, are more ectopically localized and in higher proportion than control retinas at E17.5, corroborating our findings in adults. Total number of apoptotic cells is also increased during development as well as in adult double KO retinas.

Conclusions: : Loss of both CRB1 and CRB2 results in a more severe phenotype than in the Crb1 and Crb2 knockout mice, suggesting that CRB1 and CRB2 have overlapping functions in retina development. Removing CRB1 and CRB2 proteins induces an increased aberrant proliferation of progenitor cells during development leading to a disorganized and thicker retina in adult.

Keywords: retinal development • retinitis • retinal degenerations: cell biology