Light and electron microsope techniques have been used to investigate the normal stage 4-5 epiblast periphery and wound closure at the epithelial margin. The stage 4-5 epiblast is attached to the vitelline membrane by an association of flattened 'edge cells'. Basal 'edge cells' possess lamellipodia oriented in the direction of epiblast expansion. Adjacent cells are connected by junctions and appear to retain their respective positions during the 'gliding' movement of the attached periphery. Fixation at low temperatures produces an alteration in 'edge cell' morphology consistent with a retraction of the epiblast margin. After the excision of approximately 200?m of epiblast periphery, the wound gapes due to tensions within the proximal epithelium. The epiblast cells respond to the trauma of wounding by rounding. Wound closure commences within 1 hour of 'edge cell' excision as epiblast cells at the margins attach to the membrane in distal-proximal sequence. The attached wound margins migrate toward each other across the membrane and close the wound at around 10 hours reincubation. Junctions are not observed at the wound margins and the cells appear to employ a 'rolling and sliding' form of locomotion. These results suggest that 'edge cells' are intrinsically different from those of the proximal epiblast. The normal stage 4-5 epiblast is overlain by a basement membrane. This structure is not observed at the early wound margin. The migrating wound margins deposit extracellular materials on the vitelline membrane and these resemble substances associated with the normal stage 4-5 epiblast basement membrane. Similar materials are also produced by explants and isolated cells of the epiblast periphery when cultured on the vitelline membrane. It is suggested that these materials may represent an attempt to reconstitute a basement membrane during wound closure.

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The airway epithelium demonstates the ability to quickly repair following physical injury. The morphologic features of this dynamic repair process have been well characterised at the anatomic and cellular level using a number of animal model systems and these studies have provided a solid foundation upon which our understanding of normal repair is build. With the advent of molecular and bioinformatic tools and resources the opportunity exists to extend the value of these models in defining the molecular pathways and interactions that underlay the normal repair process. This thesis represents a realisation of this opportunity. A large animal model was developed in which selected areas of airway epithelium were subjected to bronchial brush biopsy as part of routine bronchoscopic examination prcedures in anaethetised sheep. The process resulted in a physical perturbation of the normal pseudostratified structure of the sheep airwway epithelium at specific locations. By careful experimental design it was possible, within the same animals. to identify and sample from sites undergoing repair at different intervals subsequent in injury. To supplement the histological evaluation of the repair process and align findings with extablished small animal models of airway epithelial repair proliferative cell labelling strategies were implemented in order to study the location and extent of cellular proliferation occurring duringthe repair process. Molecular approaches towards defining the transcriptional response to physical injury comprised application of microarray technology using a commercially sources array platform. Such approach demanted preliminary effort directed towards optimising RNA integrity and yield from airway samples. Following preliminary studies directed towards optimising the model conditions patterns of airway epithelial repair following bronchial brush biopsy were studies in eight sheep at degined time points (6 hours, 1,3, & 7 days) post-injury. Bronchial brush biopsy resulted in the acute removal of the epithelial cell layer and components of the underlying structures. repair processes were rapidly implemented through initial epithelial dedifferentiation, proliferatino and migration at the wound margins and subsequent time-depentend changes in the proportion of subepithelial structures, including smooth muscle and blood vessels, as the epithelial surface moved towards repair. Transcriptional analysis revewaled that over 13,000 probes showed evidence of differential expession at some point during the repair process (p<0.05), whilst of these, 1491 probes had in excess of a two-fold change in expression. array results were validated against conventional semi-quantitative RT-PCR for selected genes. Differentially expressed genes with previously characterised roles in epithelial migration, prolifereation and differentiation were identified during the repair process. The relative emphasis of gene products with particular functional roles varied during the course of repair. Indeed gene ontology (GO) terms identified included those associated with the inflammatory response, cellular migration, extracellular matrix activities, differentiation, proliferation, cellular development, cell cycle activities, cellular adhesion, apoptosis and mitosis. In addition the Kyoto Encyclopedia of Genes and Gneomes (KEGG) databases were queried and such process indicated the involvement of cell communication, 053 and complement and coagulation cascade pathways throughout the repair process, initial (6h) Toll-like receptor and cytokine-cytoine receptor interaction pathways, and the progressive involement of cell cycle, focal adhesion and extracellulaar matrix (ECM)-receptor, and cytokine interaction pathways as the epithelium repaired. The model of airway epithelial injury developed in this thesis generated features broadly consistent with those previosly described in relation to various small animal model systems. Importantly, and in addition, this thesis defines the molecular features associated with repair in this model system and provides a useful resource with which to assess the comparative fetures of the airway transcriptional response to physical injury, It is through such comparison, using analogous methodology, that the fundamental pathways and interactions that underlay normal repair and regeneration can be identified and therafter extended towards inderstanding the basis for variation associated with natural and experimental disease.

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Jawed vertebrates (gnathostomes) consist of two clades with living representatives, the chondricthyans (cartilaginous fish including sharks, rays, and chimaeras) and the osteichthyans (bony fish and tetrapods), and two fossil groups, the "placoderms" and "acanthodians". These extinct forms were thought to be monophyletic, but are now considered to be paraphyletic partly due to the discovery of early chondrichthyans and osteichthyans with characters that had been previously used to define them. Among these are fin spines, large dermal structures that, when present, sit anterior to both median and/or paired fins in many extant and fossil jawed vertebrates. Making comparisons among early gnathostomes is difficult since the early chondrichthyans and "acanthodians", which have less mineralized skeleton, do not have large dermal bones on their skulls. As a result, fossil fin spines are potential sources for phylogenetic characters that could help in the study of the gnathostome evolutionary history. This thesis examines the development and internal structure of fin spines in jawed vertebrates using two-dimensional (2D) thin sections and three-dimensional (3D) synchrotron datasets. The development of the dorsal fin spine of the holocephalan, Callorhinchus milii, was described from embryos and compared to that of the neoselachian, Squalus acanthias, whose spine has been the model for studying fossil shark spines. It was found that the development of the C. milii fin presents differences from S. acanthias that suggest it might be a better candidate for studying "acanthodian" fin spines. The 3D histology of fossil fin spines was studied in Romundina stellina, a "placoderm"; Lophosteus superbus, a probable stem-osteichthyan; and sever­­al "acanthodians". The 3D vascularization reconstructed from synchrotron radiation microtomographic data reveal that "acanthodian" and Lophosteus spines grew similarly to what is observed in chondrichthyans, which differs slightly from the growth of the Romundina spine. Chondrichthyans and "acanthodians" also share similarities in their internal organization. Overall, Lophosteus and Romundina spines are more similar in terms of morphology and histology compared to chondrichthyans and "acanthodians". These results support the current hypothesis of gnathostome phylogeny, which places "acanthodians" on the chondrichthyan stem. They also emphasize the need for further study of vertebrate fin spines using 3D approaches.

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