Neurotrophins are trophic and mitogenic proteins that play a role in the development, differentiation, connectivity and survival of neurons, acting via their specific receptors in the central and peripheral nervous system, including the retina. The preferred receptor for brain derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4) is TrKB. Muller cells may play an important role in neurotrophin function in the nervous system. It has been suggested that neurotrophins exert their effects on photoreceptors by acting indirectly through activation of Muller cells. The aims of the present work were to characterise the expression of BDNF, NT4 and TrkB by human Muller cells in vitro, and to examine whether changes in the expression of these molecules occur in retina from patients with proliferative vitreoretinopathy (PVR) when compared with normal retina. In addition, changes in the expression of BDNF, NT4 and glial fibrillary acidic protein (GFAP) were also investigated in melanoma affected human retina following laser photocoagulation. A variety of techniques were employed to investigate the expression of the above neurotrophins and TrkB at the RNA and protein levels. These included cell culture and mRNA extraction, RT-PCR, Western blot, and Immunocytochemistery. Confocal laser scanning microscopy and light microscopy were used for imaging. The results showed that cultured human Muller cells express BDNF and NT4 as indicated by both mRNA and protein expression. A truncated isoform of TrkB was also shown to be expressed by a spontaneously immortalized human Muller cell line used in the study (MIO-M1). Staining for NT4 was greatly increased in retinal sections from eyes with PVR, compared with normal retina. NT4 expression by Muller cells in situ was confirmed by confocal imaging observations that cells staining for this neurotrophin co-stained for GFAP. By contrast, there was a decrease in TrkB immunostaining in PVR retinectomy sections compared with controls. NT4 staining was also reduced at the site of laser burns in melanoma affected retina. Possible explanations are discussed in the thesis.

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ChxlO is a homeobox transcription factor essential for the development of the vertebrate eye. A mutation in the human gene CHX10 causes microphthalmia and a similar phenotype is observed in the ocular retardation, ChxlO null mouse. Lack of ChxlO causes a reduction in proliferation of neural retinal progenitor cells (RPCs) in the developing retina, resulting in a reduction in the size of the eye. The aim of this study was threefold: to identify downstream targets of the ChxlO gene and discover more about the role of ChxlO at a molecular level, to further characterise the ChxlO'1' phenotype and the behaviour of the mutant RPCs, and to investigate the possibility that the ChxlO''" retina harbours stem cell-like cells. Firstly, Affymetrix GeneChip technology was used to compare the gene expression profile of wild type retina with that of the ChxlO null retina during early development. Significant differential expression was observed for 34 characterised genes genes involved in neuronal differentiation were down-regulated whereas genes involved in retinal pigmented epithelium (RPE) specification were up- regulated. These findings highlight a novel role for ChxlO in maintaining the neural retina/RPE boundary. Secondly, experiments aimed at characterising ChxlO''' RPCs showed that they exhibit altered cycling properties compared to wild type RPCs and that their differentiation to neural retinal cells is delayed. The data resulting from these studies support a model in which the transition from Gl phase of the cell cycle to S phase or to differentiation is disrupted in the ChxlO''' retina. Finally, a population of proliferating cells was discovered in the adult ChxlO''' retina, which showed neurogenic properties and formed neurospheres in culture, a point of interest in the context of retinal stem cell research. The study conducted here has offered valuable clues as to how ChxlO affects retinal progenitor cell proliferation and differentiation at both a molecular and a cellular level. It is hoped this work will contribute to the current knowledge of eye development in general, and ChxlO's involvement in this process in particular.

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