PDZ-Domain Containing Proteins are Required for Proper Development of the Lens

Abstract

Abstract: : Purpose: In Drosophila and C. elegans embryos, the membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain appear to be necessary for proper maintenance of cell junctions, cell polarity and proliferation in epithelial tissues. However, the roles of these proteins, some of which are tumor suppressors in Drosophila, in mammalian development are very poorly understood. Interestingly, the lenses of transgenic mice that express a viral oncoprotein, E6, that can bind to and inactivate several MAGUKs, show a phenotype similar that seen in epithelia in Drosophila mutants in these genes. In this study, we sought to characterize the expression of MAGUKs in the lens and determine whether they may be necessary for lens epithelial development and/or maintenance. Methods: RT-PCR, in situ hybridization, western blotting, and immunohistochemistry were used to determine the presence of message and protein for MAGUKs in lenses from nontransgenic animals. Lenses from mice carrying an insertional mutation in one gene, dlg (dlg^gt), were analyzed by histological and BrdU analyses. Lenses from transgenic mice expressing E6^wt, E6^I128T, an E6 mutant that retains the ability to bind MAGUKs, or E6^Δ146-151, an E6 mutant that loses the ability to bind PDZ proteins were examined using histological, BrdU, and differentiation marker analyses. Results: Expression studies indicated that dlg, scrib, mgl, magi1, and magi3 are expressed in the lens at the RNA level. To date, DLG and Scribble protein have also been detected. Lenses from dlg^gt mice show increased numbers of BrdU positive cells and incorporation of BrdU into cells in the transition zone. Lenses from transgenic mice that express the E6^Δ146-151 mutant are indistinguishable from control nontransgenic mice whereas lenses from mice that express E6^I128T retain the E6^wt phenotype. Conclusions: Together, these results indicate that multiple MAGUKs including but not limited to dlg and scrib are expressed in the lens and that at least one of these, dlg, is required in the epithelium and transition zone for maintaining the normal pattern of growth and differentiation in the lens. However, because the phenotype of the dlg^gt lens is not as severe as that observed in the lenses of the E6^wt transgenic mice, it is likely that MAGUKs in addition to DLG play an essential role in lens development.

Keywords: proliferation • cell adhesions/cell junctions • transgenics/knock-outs