Trophic Factor Gene Expression in Supporting Cells of the Crista Ampullaris:Implications for Hair Cell Regeneration

Current global gene expression techniques allow the evaluation and comparison of the expression of thousands of genes in a single experiment, providing a tremendous amount of information. However, the data generated by these techniques are context-dependent, and minor differences in the individual biological samples, methodologies for RNA acquisition, amplification, hybridization protocol and gene chip preparation, as well as hardware and analysis software, lead to poor correlation between the results. In the inner ear the acquisition of RNA from individual cell populations remains a challenge due to the high density of the different cell types and the paucity of tissue. Consequently laser capture microdissection was used to selectively collect individual cells and regions of cells from cristae ampullares followed by extraction of total RNA and amplification to amounts sufficient for high throughput analysis. To demonstrate hair cell specific gene expression, myosin VIIA, calmodulin, and alpha9 nicotinic acetylcholine receptor subunit mRNAs were amplified using RT-PCR. To demonstrate supporting cell specific gene expression, cyclin-dependent kinase inhibitor p2kip1 mRNA was amplified using RT-PCR. Subsequent experiments with alpha9 RT-PCR demonstrated phenotypic differences between type I and type II hair cells, with expression only in type II hair cells. Using the laser capture microdissection technique, microarray expression profiling demonstrated 408 genes with more than a five-fold difference in expression between the hair cells and supporting cells, of these 15 were well annotated. There were 9 annotated genes with greater than a five-fold expression difference in the hair cells relative to the supporting cells, and 8 annotated genes with greater than a five-fold expression difference in the supporting cells relative to the hair cells. Keywords: hair cell specific gene expression, laser capture microdissection Using laser capture microdisection, the more superficial layer of the cristae ampullaris sensory epithelium encompassing types I and II hair cells, and the supporting cell cytoplasm interspersed between them was captured in the first pass, and the underlying supporting cell bodies resting on the basal lamina were captured in a second pass. Individual hair cells were identified under light microscopy at 200X magnification by their stereociliary tuft and the calyceal nerve ending surrounding them and the presence of their nucleus in the outermost portion of the epithelia.