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M. M. Nowak, M. A. Wride, M. E. Boulton, J. Albon; Identification of Critical Stages in Development of Chicken Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3376. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To identify critical stages that define corneal epithelial development in the chick.
Chick eyes (embryonic day (ED) 4 to posthatching, n=3 per stage) were prepared for wax or frozen sectioning. Epithelial cell morphology (following H&E staining), differentiation (by immunolocalisation of cytokeratin (CK) 3 (AE5) and pan-CK (AE1/AE3)), proliferation (PCNA immunolabelling) and cell death (TUNEL assay) were assessed in triplicate sections in the limbus, periphery and central region of each corneal epithelium. Caspase 3 immunolocalisation was used to confirm cell death by apoptosis.
Two cell layers were present throughout the corneal epithelium from ED4 to ED10 and thereafter, the number of cell layers was greatest in the limbus (p<0.05). Epithelial stratification first appeared at ED10 with basal cuboidal cells identified solely in the limbus, while flattened superficial cells were observed in all regions. CK3 and pan-CK expression was first observed at ED12 in the limbal superficial cell layer and throughout the central and peripheral epithelium. Epithelial cell proliferation increased from ED4 (59.6% ± 2.29) to ED12 (81.4% ± 6.4), remained stable from ED12 - 16 (81.7% ± 3.82) and thereafter declined to 42% ± 9.7 at one day posthatch (p<0.05). From ED16, PCNA-positive cells were restricted to basal cells in the corneal limbus and suprabasal cells in the central region. Epithelial cell death was observed in the limbus from ED10 to posthatching, with peak levels of 10.6% at ED18 (p<0.05). Caspase 3 immunolocalisation confirmed corneal epithelial cell death by apoptosis.
Spatio - temporal changes in cell proliferation, differentiation and death are critical for chick corneal epithelial development.
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