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Mark Rosenblatt, Michael Sun, Victor H Guaiquil, Aihong Liu, Elaine Fuchs, Rachel Sartaj; Sox9 is required for corneal epithelial cell proliferation.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2605.
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© ARVO (1962-2015); The Authors (2016-present)
To identify the location and function of Sox9 in the cornea and understand the role of Sox9 in corneal stem cell maintenance and proliferation.
Antibodies against Sox9 (nuclear) and GFP (cytoplasmic) were used to immunostain adult corneal sections from Sox9eGFP transgenic mice, and the localization determined by wide field and confocal microscopy. Corneal epithelial cells were isolated from Sox9eGFP mice, separated into Sox9eGFP+ and Sox9eGFP- cells using FACS and then the RNA extracted for comparative RNA Seq of Sox9+ and Sox9- corneal cells. Candidate genes from the RNA Seq data was selected to immunostain on sections of corneal tissue from WT mice 1. To obtain novel genes that co-express with limbal and basal Sox9eGFP+ cells genes and 2. Identify pathways in which Sox9 is acting in the cornea. Antibodies tested were Amrcx2, Gantl14, Pamr1, Lrp2, Lgr5 and Col6a4. Lastly, to determine cell proliferative capacity of Sox9 in corneal cells, in vitro scratch assays were performed on WT murine corneal epithelial cells and stained using antibodies against Sox9 during wound healing.
In the mouse cornea, Sox9 and GFP proteins are restricted to the basal layer of the corneal epithelium. Sox9eGFP+ cells yielded between 0.5-1% of the entire corneal cell population. RNA Seq analysis produced approximately 7773 differential genes. Candidate genes significantly up-regulated in Sox9eGFP+ cells Amrcx2, Gantl14 and Lgr5, Pamr1, Lrp2 and down-regulated in Sox9eGFP- cells Col6a4 were tested on WT corneal sections to obtain novel corneal markers of limbal basal cells. Lastly, in cultured corneal cells, Sox9 was expressed in 80% of cells. After 18hrs of wounding, prior to closure, Sox9 was identified in cells along the wounded edge where cells were dividing, closing the wound, yet also included cells where scratches were not apparent.
Sox9 marks limbal basal corneal epithelial stem and progenitor cells in the central cornea. Given its localization to basal corneal epithelial layers and gene expression data obtained from our RNA Seq analysis comparing Sox9eGFP+ and Sox9eGFP- cells, this gene was investigated as a potential gene involved as a switch in proliferation. Our data, suggests that Sox9, a cell proliferation marker may be involved in mediating corneal epithelial stemness and maintaining correct corneal architecture.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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