Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new ENU-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, while homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, oncomodulin, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric p-value analysis showed hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally.

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system. deafness | mouse model | sensory system | currents | action potentials I n the mammalian cochlea, inner hair cells (IHCs) and outer hair cells (OHCs) transduce sound into electrical responses. IHCs are the primary sensory receptors that relay sound stimuli to the brain with high temporal precision via the release of neurotransmitter from their ribbon synapses onto type I spiral ganglion neurons (1). Synaptic ribbons are specialized organelles able to tether a large number of synaptic vesicles at the cell's active zones and are thought to allow sensory cells to mediate high rates of sustained synaptic transmission, coordinated release of multiple vesicles, and temporally precise transfer of information (2). OHCs provide electromechanical amplification of the cochlear partition to enhance the sensitivity and frequency selectivity of the mammalian cochlea via voltage-dependent electromotility, which is mediated by the motor protein prestin (3) and modulated by the inhibitory efferent cholinergic system (4). Before sound-induced responses begin at the onset of hearing, which occurs at around postnatal day 12 (P12) in most rodents, hair cells undergo a precise developmental program. Although hair cell maturation is known to be influenced by many proteins (5-9), we know little about the mechanisms underlying their biophysical and morphological development, especially those involved in the functional differentiation of IHCs and OHCs that occurs from around birth (10).MicroRNAs (miRNAs) regulate posttranscriptional gene expression programs by decreasing the level of target mRNA in mammals (11) and are involved in tissue development, cell fate specification, morphogenesis, and a range of diseases (12-16). Members of the miR-183 family (miR-96, miR-182, and miR-183) are specific to sensory organs (17, 18) and are highly expressed in the inner ear (19,20), eye (17), and nose (21). In the inner ear...

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2 , was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.

SummaryWHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter stereocilia, at the level of inter-stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.