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J. Albon, P. J. Giles, M. Galay-Burgos, M. A. Wride, M. E. Boulton, M. M. Nowak-Musial; Microarray Analysis of Gene Expression in the Corneal Epithelium During Development. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3752.
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To identify genes that could potentially serve as developmental markers for corneal epithelial cells and play a role in the regulation of corneal epithelial cell homeostasis.
Total RNA was extracted from avian corneal epithelium at E6 to E21 (posthatch). After IVT labelling, RNA samples were fragmented and hybridised to Affymetrix Chicken GeneChip® Arrays with probes for over 39,000 transcripts (> 28,000 genes). Normalised data was visualised using MADRAS. Limma analysis, with FDR correction, identified differentially expressed genes throughout development. Gene clusters, associated with major biological processes (proliferation, differentiation and apoptosis) and stem cell biology, were identified by Principal Component Analysis (PCA) and Gene Ontology analysis. Differential expression of candidate genes was validated by real-time quantitative PCR.
361 genes (669 probe sets) involved in differentiation (162 genes), proliferation (62 genes) and apoptosis (143 genes), identified following microarray data analysis, were clustered into 10, 3 and 9 functional groups, respectively. Functional gene groups included: NGF-R, BMP, TNF-R, growth factors, caspase and fadd-like apoptosis regulators, Bcl-2 related apoptosis regulators, TGFβ-like, frizzled-related protein, semaphorin/CD100 antigen family, neurotrophins, transcription regulators and kinases. Within these clusters, 7 genes were involved in all three biological processes and 8 genes were annotated as stem cell biology-related. PCA identified predominant gene expression patterns in 2 discrete distributions: the first involved 2 genes important in differentiation and the second involved 6, 4 and 8 genes involved in apoptosis, proliferation and differentiation, respectively. Analysis of variance, with a control FDR (p<0.000000001), revealed 43 differentially expressed genes between postnatal and embryonic time points, 14 of which were stem cell-related. RT-qPCR was used to confirm differential expression patterns of 7 genes.
Microarray analyses identified genes and gene families (including stem cell biology-related) important in the regulation of epithelial homeostasis during corneal development. This expands our knowledge of mechanisms responsible for epithelial stem cell generation and regulatory processes ensuring continuous epithelial cell renewal throughout life.
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