There are known differences in the properties of hair cells along the tonotopic axis of the avian auditory epithelium, the basilar papilla (BP). To determine the genetic basis of these differences, we compared gene expression between the high-(HF), middle-, and lowfrequency (LF) thirds of 0-day-old chick auditory epithelia. RNA amplified from each sample was hybridized to whole-genome chicken arrays and GeneSpring software was used to identify differentially expressed genes. Two thousand six hundred sixty-three genes were found to be differentially expressed between the HF and LF segments, using a fold-change cutoff of 2 and a p value of 0.05. Many ion channel genes were differentially expressed between the HF and LF regions of the BP, an expression pattern that was previously established for some but not all of these genes. Quantitative PCR was used to verify tonotopic expression of 15 genes, including KCNMA1 (Slo) and its alternatively spliced STREX exon. Gene set enrichment analyses (GSEA) were performed on the microarray data and revealed many microRNA gene sets significantly enriched in the HF relative to the LF end, suggesting a tonotopic activity gradient. GSEA also suggested differential activity of the kinases protein kinase C and protein kinase A at the HF and LF ends, an interesting corollary to the observation that there is tonotopic expression of the STREX exon that confers on Slo sensitivity to the activity of kinases. Taken together, these results suggest mechanisms of induction and maintenance of tonotopicity and enhance our understanding of the complex nature of proximal-distal gene expression gradients in the chicken BP.

BackgroundAuditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients.Methodology/Principal FindingsGene expression was profiled in forskolin treated (i.e., proliferating) and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6), 48 (n = 6), and 72 (n = 12) hours in culture. In the forskolin-treated epithelia there was significant (p<0.05; >two-fold change) upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a), which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells.Conclusions/SignificanceThese studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells.

Specialized sensory-transducing hair cells regenerate in response to injury in non-mammalian vertebrates such as birds and fish but not in mammals. Previous work has shown that overexpression of microRNA181a (miR181a) in cultured chicken basilar papillae, the avian counterpart of the cochlea, is sufficient to stimulate proliferation with production of new hair cells. The present study investigates the role of miR181a in hair cell regeneration after injury in explants of chicken auditory epithelia. Basilar papillae were explanted from 0-day-old chickens and transfected with either anti-miR181a, which knocks down endogenous miR181a, or a non-targeting miRNA and cultured with streptomycin to eliminate all hair cells from the epithelium. Labeling with BrdU was used to quantify proliferation. Explants exposed to streptomycin and transfected with anti-miR181a had significantly fewer BrdU positive cells than basilar papillae treated with streptomycin and transfected with a non-targeting miRNA. Activated caspase-3 and myosin VI labeling were used to show that the pattern of hair cell death and loss, respectively, were not affected by anti-miR181a transfection. MiR181a downregulation therefore seems to dimish the proliferative component of hair cell regeneration rather than prevent hair cell death following ototoxic injury.