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Title: Evaluation of cellular processes and identification of candidate genes critical to corneal epithelial development
Author: Nowak-Musial, Magdalena
ISNI:       0000 0004 2748 7015
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2009
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The overall aim of this study was to determine factors and mechanisms that underlie the regulation of epithelial patterning and homeostasis during corneal development. Histological staining was performed in chick corneas, embryonic day (ED) 4 to 21, to evaluate changes in the overall epithelial cell morphology, in particular cell shape, cell size and the number of epithelial cell layers. Epithelial differentiation patterns were identified in frozen sections of chicken corneas after immunolocalisation of pan-cytokeratins (Pan-CK) and cytokeratin 3 (CK3). Proliferating Cell Nuclear Antigen (PCNA) and caspase 3 (active) immunolocalisation studies, as well as, TUNEL-labelling (Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labelling) were performed to assess temporal and spatial localisation of cell proliferation and death in the developing corneal epithelium respectively. The expression of PCNA and CK3 were later confirmed by immunoblotting. Total RNA was isolated from epithelia at selected developmental time points and collected for microarray analysis. Gene expression profiles were analysed by appropriate mathematical methods. The sensitivity of arrays in producing data trends was validated by quantitative RT-PCR. Histological findings included changes in stratification an increase in the number of cell layers, change in cell morphology. In this study it was demonstrated that after becoming two layered by ED4, the epithelium underwent further stratification to form intermediate cell layers at about EDM. These changes were accompanied by changes in cell shape commencing at ED10. Cell proliferation appeared high throughout corneal development, with peak proliferation between ED12 and ED14 in the limbal, peripheral and central epithelium, respectively, thereafter the level of proliferation decreased. The above coincided with changes in epithelial morphology (stratification) and changes in expression of cytokeratin (CK) epithelial markers. The appearance of pan-CK labelling was first observed at ED10 and the presence of CK3 immunolabelling appeared in epithelial cells at ED12. TUNEL-labelling and caspase 3 (active) immunolocalisation demonstrated only few TUNEL-positive cells, mostly restricted in the limbal region of the corneal epithelium, in the mid and later developmental stages. Microarray analyses identified gene families and their members (including these involved in stem cell biology) likely to be relevant in the regulation of homeostasis during corneal epithelial development, as well as, differentially expressed genes that reveal changes in biological processes due to the change in time. RT-qPCR confirmed the differential expression patterns of seven genes of interest following analysis of microarray data. Patterns of cell proliferation and differentiation showed changes during the development of the corneal epithelium that reflect the interaction of a complex network of mitogenic, apoptotic and differentiation agents. The changes in gene expression profiles, detected by the microarray analyses, were consistent with the phenotypic changes in the developing chick corneal epithelium. The microarray data provided the first study to present a good overall picture of genes expression in the developing chick corneal epithelium.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available