Investigative Ophthalmology & Visual Science, Vol 37, 1815-1825, Copyright © 1996 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Cell lineage and the differentiation of corneal epithelial cells

DC Beebe and BR Masters
Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.

PURPOSE. Studies were designed to determine whether cell division and cell differentiation are linked directly in the corneal epithelium. To obtain these data, corneal basal epithelial cells were labeled during DNA synthesis, and the resultant daughter cells were followed for as long as 2 weeks. METHODS. Adult rats were injected with 5-bromo- deoxyuridine (BrdU), a thymidine analog, and were killed 6 hours to 14 days later. Corneas were fixed and permeabilized, and BrdU-labeled nuclei were detected with a monoclonal antibody to BrdU and a fluorescent-labeled secondary antibody. Fluorescent nuclei were visualized in three dimensions in corneal whole-mounts using a laser scanning confocal microscope. Optical sections were collected and displayed as serial images, three-dimensional anaglyphs, color-encoded projections, or three-dimensional reconstructions. Data were confirmed using 3H-thymidine autoradiography of epithelia sectioned parallel to the corneal surface. RESULTS. Cells synthesizing DNA at the time of injection incorporated BrdU into their DNA. Pairs of labeled nuclei were produced by the division of cells that had been labeled with BrdU. These daughter cells remained in the basal layer of the epithelium for a variable period of time. Some daughter cells continued to divide, producing clusters of labeled basal cells. When labeled daughter cells left the basal layer and began the process of terminal differentiation, they nearly always did so together. The synchronous differentiation of daughter cells was evident from the pairs of labeled nuclei seen throughout the depth of the epithelium from 2 to 14 days after labeling. CONCLUSIONS. Cell division and differentiation are not linked directly in the corneal epithelium. After cell division, daughter cells either remain in the basal layer, where they may undergo additional rounds of cell division, or both cells differentiate synchronously. When the daughter cells of a mitosis differentiate, the time between the previous cell division and differentiation is highly variable. This suggests that the coordination of cell division and differentiation in the corneal epithelium involves a complex regulatory network.

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