Evaluation of cellular processes and identification of candidate genes critical to corneal epithelial development

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

A New Quantitative Method for the Taxonomic Identification of Tetrapods

The rarity of good fossil samples throughout geologic time frequently makes fossil identification difficult. This dissertation presents a new, multivariate, statistically validated method to identify tetrapods based on quantification of the shapes of microstructural features in cortical bone of the postcranial skeleton. The ultimate goal is to reduce the reliance on rare, near-complete fossil skeletons. The method is validated on a set of 15,745 mammalian microstructural features from eleven diverse species. An additional set of 21,122 microstructural features from one species serve to examine microstructural variation within a single skeleton. Microstructural measurements were made on thin-sections using optical microscopy. Initial tests of the method were applied to extant mammalians whose taxonomic affinities were known. Three case studies comparing: 1) the left tibiae from 11 mammals,: 2) the mid-body of each left rib in Odocoileus virginianus, and: 3) five cross-sections from left rib seven of O. virginianus represented tests of inter-taxonomic, intra-skeletal, and intra-bone microstructural variation, respectively. Principal Component Analysis of measurements on the tibiae of 11 mammals was successful in discerning a taxonomic signal in the shape and size characteristics of primary vasculature, secondary osteons, Haversian canals, primary lacunae, and secondary lacunae. No single microstructure or measurement is sufficient to account for taxonomic variation. Rather, size, shape, and orientation of various microstructural features, in combination, define and distinguish the taxa. Soft Independent Modeling of Class Analogy properly reassigned test samples from several taxa. In contrast with the results from the multi-species set, analysis of the intra-skeletal and intra-bone case studies revealed no pattern of microstructural variation. The data suggest that the microstructural variation within a skeleton is small compared to variation between taxa and that intra-skeleton variation will not affect the overall taxonomic designation. All principal component analyses were tested and found to be significant at the 95% confidence level using Multiple Discriminant Analysis. This work establishes a methodology for using bone microstructural features as a means for reconstructing taxonomic identity and supports continued research on this methodology, with the goal of applying it to rare fossil specimens in order to enable a next-generation approach to paleoecological analysis