Identification of differentiating cochlear hair cells in vitro.

Abstract

The expression of hair cell-specific genes involved in differentiation was studied in the cell line UB/OC-1 (University Bristol/Organ of Corti). Studies of gene expression in cochlear hair cells are restricted by the small number of cells available and by their experimental inaccessibility. The cell line was derived from the H2K(b)tsA58 transgenic mouse, which harbors a conditionally expressed immortalizing gene. Two genes that are characteristic of hair cells were upregulated during differentiation of UB/OC-1 cells in vitro. They are the transcription factor Brn3.1, which is essential for hair cell differentiation, and the alpha9 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 analyzed by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR). Western blot analysis and immunofluorescence analysis were performed on the cell line with anti-Brn3.1 antibody. Reaction products for alpha9 were detected after 3 to 6 days under differentiating conditions. Low levels of Brn3.1 were detectable under proliferating conditions and increased under differentiating conditions. All cells expressed Brn3.1 under differentiating conditions. This temporal pattern of gene expression is very closely similar to that found in vivo. The cochlear hair cell line UB/OC-1 provides a valuable experimental system because it conditionally expresses genes essential for normal differentiation and electrophysiology. It should prove valuable in the identification and characterization of genes involved in development and may provide material for screening new therapeutic methods of stimulating recovery and regeneration of hair cells.