Weir, Justin Neil MacDonald; (1999) Cochlear hair cell fate determination and differentiation in vitro. Doctoral thesis (M.D.), UCL (University College London).

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Abstract

Mammalian cochlear hair cells are relatively inaccessible and few in number. This hampers any research on their fate determination and differentiation. The production of conditionally immortal cell lines from the H2KbtsA58 transgenic mouse should overcome these difficulties. The aims of the present study were threefold. Firstly, to establish that the cell lines provide a viable in vitro system, by examining the pattern of molecular expression in the cochlear hair cell line UB/OC-1. Secondly, to examine differentiation by using clonal derivatives from the heterogeneous cell line UB/OC-l. Thirdly, to explore the process of lateral specification in the determination of cell fate and to explain the differentiation of hair cells and supporting cells from a common precursor. The methods used were cell culture, immunocytochemistry, reverse transcription polymerase chain reaction and western blot. The results demonstrated that firstly; the temporal expression pattern of Brn3.1, an essential transcription factor required for hair cell differentiation, and the a9 subunit of the nicotinic acetylcholine receptor, followed a similar pattern to that during normal development. Secondly, epithelial cell markers such as, vimentin, cytokeratin, actin and cadherin, and specific hair cell markers such as myosinVIIA and fimbrin were expressed when the hair cells differentiated. The pattern of expression suggested parallel pathways of gene expression during differentiation of hair cells. Thirdly, from the expression of Numblike, Notch 1, Jagged I and Jagged2, factors which are known to be involved in lateral specification, a model is proposed to explain hair cell fate determination. The results also demonstrate the much greater experimental flexibility offered by cell lines in understanding hair cell development. Future studies will focus on functional experiments that alter hair cell fate.

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