DIFFERENTIATION OF COCHLEAR HAIR CELLS IN VITRO

Justin Weir*, Marcelo N. Rivolta¹, Matthew Holley¹

* University Department of ENT. Physiology Department,

1 University of Bristol, Bristol, BS2 lTD. England.

Studies of gene expression in cochlear hair cells are restricted by the small number of cells available and by their experimental inaccessibility. We are studying the expresssion of critical hair cell genes in the cell line UB/OC-1 (University Bnstol/Qrgan of Corti), derived from the H2K^btsA58 transgenic mouse which harbours a conditionally expressed immortalizing gene. Under differentiating conditions, after the immortalising gene has been inactivated, the cells upregulate two genes, the transcription factor Brn3.1 which is essential for hair cell differentiation and the alpha 9 subunit of the nicotinic acetylcholine receptor that is involved in olivocochlear efferent innervation. The expression of Brn3.1 and alpha9, at different time points under differentiating conditions, was analysed by semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCR). Reaction products for alpha9 were detected after 3-6 days under differentiating conditions. Low levels of Brn3.1 were detectable under proliferating conditions. Two sub-clones, UB/OC-1-14 and UB/OC-1-24, were then derived from the parental cell line UB/OC- 1. The former was entirely negative for Brn3.1 under proliferating conditions, but all cells expressed this gene under differentiating conditions. The latter was positive for Brn3.1 under both conditions.We hypothesise that the parental cell line UB/OC-1 is already committed to hair cell differentiation at the time of immortalisation and that this pattern of gene expression is very closely similar to that found in vivo.