Molecular biological studies of the mammalian inner ear have been limited by the relatively small size of the sensory endorgans contained within. The saccular otolithic organ in teleostian fish is structurally similar to its mammalian counterpart but can contain an order of magnitude more sensory cells. The prospect of the evolutionary conservation of proteins utilized in the vertebrate inner ear and the relative abundance of teleostian saccular sensory tissue made this an attractive system for molecular biological studies. A complementary DNA obtained by differential screening of a saccular complementary DNA library was identified that encodes an inner ear-specific collagen molecule.

To identify receptor tyrosine kinases (RTKs) present in the murine inner ear, a degenerate polymerase chain reaction (PCR) methodology was employed to clone partial cDNAs encoding RTKs from embryonic day-17.5 mouse whole inner ear RNA. At least 20 distinct TKs were identified within the first 50 subcloned PCR products obtained by this analysis (Davis/Lee et al., 1996). One of the receptor RTKs identified encoded an eph-related kinase not previously described in the mouse. Analysis of full-length cDNAs revealed that this RTK is the mouse homolog of the rat ehk-2 gene product (Maisonpierre et al., 1993). Differences in the carboxyl terminal of the mouse and rat ehk2 RTKs suggest that differential splicing of this gene may occur resulting in transcripts encoding truncated and nontruncated forms of the ehk2 RTK. Multiple transcripts corresponding to this RTK were detected by Northern blot analysis only in the mouse brain. RT-PCR analysis revealed the presence of transcripts encoding this kinase in adult mouse brain, inner ear, testes, ovary, thymus, and spleen. Transcripts encoding this kinase were localized using in situ hybridization to the postembryonic day 1 cochlear ganglion neurons in the inner ear and to neurons in discrete regions of the nervous system. This is the first report of eph-related RTK in inner ear tissue that is present in both the developing and adult inner ear tissue. Because this is a member of a family of RTKs that is implicated in establishing the specificity of neuron-target cell interactions (Garrity and Zipursky, 1995), additional studies to determine if the ehk-2 gene product is involved in such processes in the murine cochlea are warranted.

The vertebrate inner ear sensory epithelia contain different types of hair cells and supporting cells. The teleost saccule is anatomically similar to the mammalian saccule and is primarily involved in the detection of translational acceleration and orientation with respect to gravity. To facilitate molecular studies of the teleost saccule cDNA libraries were constructed from microdissected Lepomis macrochirus (bluegill sunfish) saccular maculae. To our knowledge, this is the first report of cDNA libraries constructed from the saccule. In one instance, a non-polymerase chain reaction-based method of amplifying a mRNA population from limited amounts of starting tissue was employed that allowed construction of cDNA libraries from nanogram amounts of tissue mRNA. Conventional cDNA libraries were constructed from the sunfish saccular maculae as well. These cDNA libraries enriched in hair cell and supporting cell transcripts should facilitate molecular biological studies of inner ear sensory epithelia. As an example of their utility, efforts to identify tyrosine kinases expressed in the saccular endorgan using low-stringency hybridization screening of these cDNA libraries and the partial sequence of a cDNA found to encode an erbB-2-related tyrosine kinase are also reported.

A chick cochlea cDNA library was constructed to clone molecules involved in peripheral auditory transduction and in the maintenance and regeneration of the sensory neuroepithelium following damage. Characterization of the library showed it to be of high complexity, to contain a high proportion of full-length cDNA inserts, and to contain a representative proportion of clones derived from hair cell transcripts. A cDNA clone encoding the chick homolog of the 40-kD subunit of the human replication factor C (also called activator 1) was isolated and the complete cDNA sequence determined. The predicted amino acid sequence is about 90% identical to that of the human homolog. Expression of the message for this replication factor was detected in brain and liver as well as in the cochlea. Expression levels in the brain are relatively high and are similar in developing and adult chicken nervous tissue. This suggests that replication factor C message expression, unlike that for the functionally associated factor proliferating cell nuclear antigen (PCNA), may be constitutive rather than cell cycle dependent. Although likely to be involved in DNA replication within the receptor neuroepithelium, expression of this replication factor message is not likely to constitute a marker for proliferation.