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Masters Degrees (Microbiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/8006

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Browsing Masters Degrees (Microbiology) by Subject "Anterior segment (Eye)"

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    Assessing the role of the transcription factor FOXC1 in the expression and regulation of the Adherens junction protein N-Cadherin during corneal endothelium development.
    (2011) Govender, Viveshree Shalom.; Sommer, Paula.
    The proper organization and differentiation of the anterior segment is pivotal for normal eye development. Neural crest-derived POM cells are key contributors to correct anterior segment formation, differentiating to form the monolayered corneal endothelium. Mice with homozygous null mutations in the forkhead transcription factor gene, Foxc1, fail to develop a proper corneal endothelium stabilized by adherens junctions, with the endothelium adhering to the lens, preventing anterior chamber separation. The aim of this study was to evaluate the interaction between Foxc1 and the adherens junction protein, N-cadherin, as well as an associated gene, Msx1, during key stages in corneal endothelium development. Foxc1 was over-expressed in E12.5 and E13.5 POM cells and qPCR was carried out to determine the effect of Foxc1 on N-cadherin and Msx1 gene expression. Data showed over-expression of Foxc1 in wildtype E12.5 and E13.5 POM cells to cause significant fluctuations in N-cadherin and Msx1 expression (p < 0.05). POM cells were then transfected with a Foxc1 knock-down plasmid or the Foxc1 overexpression plasmid to evaluate the effect of Foxc1 on N-cadherin protein expression by Western blot analysis, however, these results were inconsistent with the gene expression analyses with no significant differences in N-cadherin expression detected. N-cadherin protein expression and localization was then further assessed by means of immunocytochemistry (ICC) and confocal microscopy in monolayer and hanging-drop POM cell cultures. Both qPCR and confocal microscopy data showed consistency, indicating increased amounts of N-cadherin in E12.5 cells relative to E13.5 cells, with membrane-bound N-cadherin showing a clear lattice-work pattern in hanging drop culture. Foxc1 over-expression/knock-down studies on E12.5 and E13.5 POM cells together suggest that N-cadherin is transcriptionally regulated by Foxc1 and that Foxc1 has a threshold level at which it is able to exert control over N-cadherin in POM cells. Foxc1 expression is therefore essential in establishing N-cadherin adhesion junctions in the corneal endothelium. Preliminary data also suggests that Msx1 may directly interact with Foxc1 in POM cells, however, further studies must be undertaken to verify and establish the effects of Foxc1/N-cadherin/ Msx1 interaction in the development of a cohesive, integrated corneal endothelium and functional anterior segment.
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    The role of lens-derived signals in the development of the corneal endothelium.
    (2013) Silla, Zenzele.; Sommer, Paula.
    Corneal endothelial development is an intricate process driven by finely tuned gene expression. Its formation is necessary for the continued normal development of the anterior segment of the eye. The presence of an inductive lens able to secrete factors such as TGFβ2 as well as the expression of Foxc1 and Pitx2 is essential to corneal endothelial development, as in the absence of any of these; the corneal endothelium fails to form. Corneal endothelial development begins as peri-ocular mesenchyme (POM) cells migrate into the space between the lens and surface ectoderm at E11.5. From E12.5, these cells begin to transition from a mesenchymal to an epithelial/endothelial (MET) phenotype, differentiating into a monolayered endothelium by E15 characterised by inter-cellular junctions. To study the initial process of development, immortalised POM cell lines from E12.5 and E13.5 embryos were used. Expression of the key genes, the transcription factors, Foxc1 and Pitx2 and two genes involved in EMT/MET, Slug and Tsc22, were analysed at these stages to establish the developmental norm. The effect of the lens on these expression levels was then determined. To establish whether TGFβ2 is the lens secreted signal responsible for gene expression changes, cells were subjected to TGFβ2 treatment. In all these experiments, the role of Foxc1 in regulating gene expression was determined by Foxc1 overexpression and knockdown. The effect of the lens on cellular proliferation and on the expression and cellular arrangement of N-cadherin, a junction protein was also determined. The results showed that, at E12.5, the lens downregulates Foxc1 and Pitx2 expression, is a potent inducer of Tsc22 expression and is required for maintaining Slug levels. TGFβ2 was shown to play a role in Foxc1 and Pitx2 downregulation. Analysis suggests that Tsc22 expression is responsive to lens signals, but that TGFβ2 is not the signal responsible for its downregulation between E12.5 and E13.5. The lens has no effect on Slug expression in the presence of Foxc1, but when Foxc1 is silenced, Slug is induced. Thus, Foxc1 plays a crucial regulatory role in Slug expression. At E13.5, as differentiation is initiated, Foxc1 expression remains responsive to the lens and to TGFβ2. Pitx2 expression is still induced by the lens but, at this stage, TGFβ2 does not play a part in Pitx2 regulation suggesting involvement of other unknown lens secreted signals. Other lens secreted signal/s were also shown to downregulate Tsc22 and Slug at this stage. The lens was implicated in MET as it was shown to have an effect on N-cadherin localisation in 3-dimensional culture. E12.5 Spheroids exposed to E6 lenses formed a distinct lattice arrangement of N-cadherin compared to the uniform distribution in control cells. Although the 13.5 control cell aggregates also showed a lattice framework, it was more pronounced in the lens treated cells. The transcriptional role of Foxc1 was determined by overexpression and knockdown experiments where Foxc1 overexpression and knockdown upregulated Tsc22 and downregulated Pitx2 and Slug at E12.5. At E13.5, Pitx2 was downregulated and Slug was upregulated in response to aberrant expression of Foxc1. This was illustrative of the sensitivity these genes have to Foxc1 expression during development. It is known that the presence of a functioning lens and Foxc1 are essential for proper development of the corneal endothelium, which in turn is necessary for normal eye development. The understanding of the precise molecular mechanisms required for corneal endothelial development and the processes requisite for cell proliferation and differentiation has important consequences for providing further insight into the pathophysiology of anterior segment dysgenesis and glaucoma. Previous studies suggest that stem-cell like qualities are conferred in cells undergoing EMT. Such an investigation may lead to application in regenerative medicine such as the bioengineering of corneal tissue.