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 beta-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.

Figure 1

In situ hybridization analysis of cDNAs expressed in the inner ear. (A) On a cross-section of the basilar papilla, β-tectorin mRNA occurs in clear cells (CC), supporting cells (SC), and cuboidal cells (CuC). (B) Calbindin mRNA is abundant in supporting cells (SC) and detectable in hair cells (HC). (C) Strong signals for type II collagen mRNA are visible in the superior fibrocartilaginous plate (SFP) and inferior fibrocartilaginous plate (IFP) and, as indicated by the arrow, between the auditory ganglion (AG) and sensory epithelium (SE). All collagen mRNAs found in the inner ear were expressed in a similar manner. (D) Coch-5B2 mRNA occurs in spindle-shaped cells (arrow) marking the path of neurites between the auditory ganglion (AG) and sensory epithelium (SE). Weaker expression is detectable in the superior and inferior fibrocartilaginous plates (SFP and IFP). (E and F) Coch-5B2 mRNA displays strong labeling of muscle spindles in the gastrocnemius muscle; note the encapsulation and the nuclear chain in (F). (G) Connexin 31 mRNA is expressed robustly in cells of the tegmentum vasculosum (TV), cuboidal cells (CuC), supporting cells (SC), and clear cells (CC). (H) Homogenin mRNA is detectable in homogene cells (HoC). (I) The intense yellow fluorescence of rhodamine-coupled phalloidin signals a high abundance of filamentous actin in homogene cells (HoC) as well as in hair bundles of the sensory epithelium (SE). (J) Otokeratin mRNA occurs in cells of the tegmentum vasculosum (TV).

Figure 2

(A) Amino acid sequence of chicken Coch-5B2. A predicted signal peptide at the amino terminus is underlined; a putative N-glycosylation site (N-X-[S/T]) at amino acid positions 218–220 is printed in bold. (B) Comparison of the deduced chicken connexin 31 (cCx31) amino acid sequence with those of murine connexins 26 (mCx26) and 30 (mCx30). Identical amino acid residues are marked with dots, and gaps are indicated by dashes. Potential transmembrane domains of chicken connexin 31 at amino acid positions 23–39, 76–92, 138–154, and 189–205 are indicated by lines. (C) Comparison of the chicken homogenin amino acid sequence with those of human gelsolin and bovine adseverin. Identical residues are marked with dots, and gaps are indicated by dashes. Note that the similarities of homogenin to gelsolin and to adseverin are distributed nearly equally throughout the protein. (D) Amino acid sequence of chicken otokeratin. Two putative cAMP- and cGMP-dependent protein kinase phosphorylation sites ([R/K]-[R/K]-X-[S/T]) are underlined. Eight putative protein kinase C phosphorylation sites ([S/T]-X-[R/K]) are printed in bold. The intermediate-filament signature motif ([I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E]) is boxed.

Figure 3

Northern blot analysis of the expression of connexin 31 (A) and homogenin and otokeratin (B) transcripts in hatchling tissues. Total RNA isolated from the tissues indicated was hybridized to ³²P-labeled cDNA fragments covering the complete coding sequences and the 3′ untranslated regions of connexin 31, homogenin, and otokeratin. As a control for RNA loading, the blots were rehybridized to a chicken β-actin probe.