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D. M. Defoe, L. B. Bundon, P. D. Moore, J. D. Sword, T. A. Harrison; Visualization of Corneal Epithelium Turnover Using Mosaic Analysis With Double Markers (MADM). Invest. Ophthalmol. Vis. Sci. 2010;51(13):3741.
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The view that the limbus is the exclusive source of stem cells for regeneration of the adult corneal epithelium has been challenged in recent studies that suggest an additional local corneal source of these cells. In the present study, we have applied a new fate mapping technique to trace epithelial renewal at single-cell resolution in unperturbed corneas.
Two mouse lines, each with chimeric genes consisting of partial coding sequences for green and red fluorescent proteins (GFP and RFP), separated by a LoxP site, were interbred with Cre recombinase-expressing strains (Zong et al., 2005, Cell 121, 479). 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.
Whole-mounted corneas, examined by fluorescence stereoscopy, exhibit a relatively high density of labeled cells in an annular zone at the level of the limbus. Emanating from this zone are linear arrays of cells extending from the tissue periphery to the central region. While reminiscent of radial stripes seen in X chromosome-inactivation mosaics, these uninterrupted arrays consist of much smaller groups of cells arranged either in single-file or organized into thicker bands. In frozen sections, labeled cells are often seen across all layers of the epithelium. Similar labeling patterns are observed with either a ubiquitously-expressed Cre driver (Hprt-Cre) or a recombinase targeting long-term progenitors (Krt14-Cre).
These studies establish the efficacy of the MADM technique as a means to follow the life cycle of regenerating epithelial cells. While they support a role for limbal stem/progenitor cells during normal corneal development and maintenance, they do not exclude a parallel involvement of putative corneal stem cells. The relative contribution of these two cell sources will be addressed in subsequent studies using inducible Cre drivers to achieve clonal analysis.
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