Tecta is a modular, non-collagenous protein of the tectorial membrane, an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of nonsyndromic deafness and a genotype-phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the TectaL1820F, G1824D/+ mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the TectaC1837G/+ mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family, and the TectaC1619S/+ mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the tectorial membrane. ABR thresholds in the 8-40 kHz range are elevated by 30-40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20-30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in tectorial membrane structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

Alpha- and beta-tectorin (Tecta and Tectb) are major non-collagenous components of
the tectorial membrane (TM). The presence of a zona pellucida (ZP) domain in both
tectorins suggests that Tecta and Tectb can form hetero- or homopolymers. It is unclear,
however, how these proteins assemble to form the TM matrix. The mechanisms of
apical targeting, secretion and processing of the tectorins are also unexplored. I used
fluorescently-tagged tectorin constructs for stable transfection into polarised epithelial
MDCK cells or transient expression in mouse cochlear cultures to develop an in vitro
model of TM matrix assembly. Significant amounts of matrix were not observed with
stable tectorin expression in monolayer cultures of MDCK cells. In contrast, I observed
substantial amounts of dense extracellular matrix on the apical surfaces of outgrowth
zone cells when cochlear cultures were transiently transfected with either Tecta or
Tectb. When ectopically expressed in hair cells, Tecta and Tectb locate to the distal tips
of the hair bundle.

To study the role of the inner-ear protein Ceacam16 in hearing, we generated a
Ceacam16 functional null mouse model. The Ceacam16 gene was inactivated by
targeted replacement of exons 2-5 with the bacterial lacZ gene. β-gal staining I
performed reveals that Ceacam16 is expressed in the epithelial cells of the spiral limbus
and inner sulcus, and in both the pillar cells and Deiter’s cells. I first detected the presence of Ceacam16 in the TM at P12, four days before the defined striated-sheet
matrix is observed. Transmission electron microscopy reveals a complete loss of
striated-sheet matrix in Ceacam16 null mice in comparison to the wild-type.

The results of this thesis suggest neonatal mouse cochlear cultures as a model for
studying tectorin-based extracellular matrix production and also reveal that Ceacam16 is
required for normal formation and/or maintenance of striated-sheet matrix.

Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), which specifically immunolabels hair cells in at least five vertebrate classes, including sharks and rays, bony fish, amphibians, birds, and mammals. We used HCS-1 to immunoprecipitate the cognate antigen and identified it as otoferlin, a member of the ferlin protein family. Mutations in otoferlin underlie DFNB9, a recessive, nonsyndromic form of prelingual deafness characterized as an auditory neuropathy. Using immunocytochemistry, we find that otoferlin is associated with the entire basolateral membrane of the hair cells and with vesicular structures distributed throughout most of the hair cell cytoplasm. Biochemical assays indicate that otoferlin is tightly associated with membranes, as it is not solubilized by alterations in calcium or salt concentrations. HCS-1 immunolabeling does not co-localize with ribeye, a constituent of synaptic ribbons, suggesting that otoferlin may, in addition to its proposed function in synaptic vesicle release, play additional roles in hair cells.