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Dennis Defoe, Whitney Rich, Theresa Harrison; Morphological Complexity of Mouse Corneal Endothelial Cells Revealed by Mosaic Analysis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1668.
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© ARVO (1962-2015); The Authors (2016-present)
In a previous investigation, we examined how individual corneal endothelial cells with distinct p27 gene mutations differ in their proliferative behavior (Defoe et al., ARVO 2011). As a result of these experiments, we noticed unusual structural features of the cells that required further analysis. In the present study, we have begun to examine the detailed morphology of single cells in situ, after marking them by high cytosolic expression of green or red fluorescent proteins (GFP or RFP).
To visualize small numbers of widely distributed cells filled with fluorescent label, we used mosaic analysis with double markers (MADM; Zong et al., 2005). For MADM, two mouse lines, each with reciprocally chimeric transgenes consisting of partial coding sequences for GFP and RFP, separated by an intron-embedded LoxP site, were interbred with an Hprt-Cre-expressing strain. Following the limited occurrence of Cre-mediated interchromosomal recombination during mitosis, functional GFP and RFP were reconstituted and each expressed in one of the two daughter cells. Corneas from MADM mice were fixed, flat-mounted and visualized by fluorescence confocal microscopy.
Individual cells filled with GFP or RFP appear multipolar, with many tapered pseudopodial processes radiating from their cell body. Such extensions are indicative of a complex and highly elaborate plasma membrane. Examination of rare cases where red and green cells lie directly adjacent to one another reveals that processes of the two cells undergo extensive interdigitation. Because of this overlap, individual cells are observed to cover a greater area than might be expected if their boundaries were mutually exclusive.
Our results give a picture of corneal endothelial cell morphology very different from the polygonal outlines observed after staining for actin filaments or intercellular junction proteins. The data may help explain the discontinuous tight junction pattern seen in both light and electron micrographs. More importantly, they suggest that cell density alone may be an insufficient indicator of the overall condition of the endothelium in health and disease.
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