Molecular markers for cell types of the inner ear and candidate genes for hearing disorders

Abstract

To identify genes expressed in the vertebrate inner ear, we have established an assay that allows rapid analysis of the differential expression pattern of mRNAs derived from an auditory epithelium-specific cDNA library. We performed subtractive hybridization to create an enriched probe, which then was used to screen the cDNA library. After digoxigenin-labeled antisense cRNAs had been transcribed from hybridization-positive clones, we conducted in situ hybridization on slides bearing cryosections of late embryonic chicken heads, bodies, and cochleae. One hundred and twenty of the 196 clones analyzed encode 12 proteins whose mRNAs are specifically or highly expressed in the chicken's inner ear; the remainder encode proteins that occur more widely. We identified proteins that have been described previously as expressed in the inner ear, such as ␤-tectorin, calbindin, and type II collagen. A second group of proteins abundant in the inner ear includes five additional types of collagens. A third group, including Coch-5B2 and an ear-specific connexin, comprises proteins whose human equivalents are candidates to account for hearing disorders. This group also includes proteins expressed in two unique cell types of the inner ear, homogene cells and cells of the tegmentum vasculosum.Developmental analysis of the inner ear is hindered by the paucity of molecular markers for specific cell types. At present, most cells in the cochlea and vestibular organs cannot be unambiguously identified before they display their characteristic mature forms. The cloning of cDNAs that encode proteins specific to hair cells, supporting cells, and other cells of the inner ear is, in turn, retarded by the limited numbers of these cells in an animal. This disadvantage potentially can be circumvented through enrichment of specific cDNAs by subtractive hybridization (1-3) and library normalization (4). That only a few inner ear-specific cDNAs have been obtained from libraries constructed by this approach (2, 3), however, suggests that the efficient cloning of cDNAs specific to sensoryepithelial cell types requires libraries constructed from more abundant sources than whole cochleae. To address this problem, we constructed a cDNA library from sensory epithelia of the chicken's cochlea and developed an enriched probe with which to identify cDNAs encoding proteins expressed by specific cell types of the inner ear.The identification of proteins specific to the internal ear is also potentially useful in the study of heritable human deafness, which afflicts about one individual in a thousand. Several proteins that have been identified as markers for specific aural cell types recently have been advanced as candidate genes for human hearing disorders (for reviews see refs. 5-8). Some of the genes identified by the strategy described in this paper are likely to have human equivalents that cause hearing disorders when mutated. MATERIALS AND METHODSLibrary Construction and Screening. Basilar papillae from 250 late-embryonic (E, embryonic day) (8% E14...

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