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C. Rivera, A.E. Griep; Requirement for the PDZ Domain Containing Protein Dlg–1 for Mouse Lens Development in vivo . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2940.
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© ARVO (1962-2015); The Authors (2016-present)
PDZ domain containing proteins are scaffold molecules that have been shown to play a role in maintaining proper cell–cell adhesion, polarity and cell growth in Drosophila. Previous studies from our lab have shown that the expression of the viral oncoprotein E6 leads to increased proliferation, defects in cell adhesion, polarity, and differentiation. The purpose of this study is to understand the role of PDZ protein Dlg–1 in lens development. To do so, we analyzed lenses expressing E6 throughout the lens epithelium. Although E6 has been shown to be a powerful developmental tool, it has the potential of disrupting multiple PDZ proteins that are found in the lens. Therefore, to understand the individual contribution of Dlg–1, we generated mice with a lens specific deletion of Dlg–1 beginning in the lens vesicle stage by crossing Dlg–1flox mice to MLR10Cre mice.
Paraffin sections from P5 NT and E6 lenses were stained for Dlg–1 and analyzed by confocal microscopy. The subcellular localization of Dlg–1 was assessed by immunoblotting the cytoskeletal and cytoplasmic associated protein fractions from NT and E6 lenses at different developmental time points. Paraffin sections from P1 Dlg–1+/+ and Dlg–1flox/flox;Cre lenses were stained by hematoxylin and eosin (H&E) to assess lens morphology. Sections were also immunostained for BrdU to study the levels of DNA synthesis. The organization of N–cadherin, α –catenin and ß –actin were analyzed by immunofluorescence and confocal microscopy.
Confocal microscopy and western blot data showed that E6 expression caused Dlg–1 to shift from the cytoplasmic membrane to the cytoplasm in the fiber cells at the lens periphery, indicating that interference with Dlg–1 may contribute at least in part to the lens phenotype in these mice. H&E analysis of Dlg–1flox/flox;Cre lenses showed retention of nuclei into the center of the fiber cell and epithelial cell division in the wrong polarity plane, generating a multilayered epithelium. Increased BrdU positive cells were observed throughout the lens epithelium and fiber cell compartment of the Dlg–1flox/flox;Cre as compared to the NT lenses. Finally, the sutures of Dlg–1flox/flox;Cre lenses failed to completely close and the organization of N–cadherin, α –catenin and ß –actin were highly affected, especially at the apical and basal tips of the fiber cells.
These results show that disruption of Dlg–1 in the lens is sufficient to affect proper cell cycle regulation and fiber cell differentiation. The mechanism through which Dlg–1 affects these processes may at least in part be associated with its role in modulating cadherin containing adhesion–complexes.
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