Abstract
Abstract: :
Purpose: We have carried out a study of GFP–positive cells in alpha 3 (Cx46) and alpha 8 (Cx50) knockouts and double knockout lenses to investigate how the loss of different connexin subunits alters the properties of GFP+ cells in the lens and to examine whether connexin subunits contribute to the macromolecular diffusion pathway in mature fibers. Using GFP+ cells, we have also evaluated lens cell structures in the living lens at different developmental stages. Methods: A GFP transgenic mouse line was used to breed with different connexin knockout mice to produce GFP positive cells in their lenses. Lens GFP–positive cells, the cells’ fluorescent intensity, and cellular structures were imaged by a Leica confocal microscopy. Results: These mouse lenses show a mosaic pattern of GFP–positive and GFP–negative lens epithelial cells. Wild type and alpha 3 (–/–) knockout lenses have similar expression patterns of GFP+ epithelial cells at different ages, while the alpha 8 (–/–) knockout lenses show a reduced number of GFP+ epithelial cells with variable intensity of GFP fluorescence. A diffuse GFP signal appears in inner mature fibers of wild type, alpha 3 (–/–) and alpha 8 (–/–) lenses. The double knockout lenses contain abnormal GFP–positive epithelial and lens fibers cells and a dark triangle area, underneath the epithelium that is never observed in either alpha 3 (–/–) or alpha 8 (–/–) lenses. Three–dimensional view of GFP+ epithelial cells shows more cellular extrusions in the basal side than in the apical side of lens epithelial cells. Conclusions: These results suggest that a loss of alpha 8 connexin subunits, but not a loss of alpha 3 connexin, alters GFP expression in lens epithelial cells. It seems that the macromolecular diffusion pathway in the mature fibers is not affect by a loss of either alpha 3 or alpha 8 subunits in the lens. However, a loss of both alpha 3 and alpha 8 abolishes this macromolecular diffusion pathway. Moreover, the appearance of a dark triangle area underneath epithelium indicates that anterior suture formation is disrupted in the double knockout lenses.
Keywords: gap junctions/coupling • microscopy: confocal/tunneling • cell-cell communication