A gelatinous otolithic membrane (OM) couples a single calcified otolith to the sensory epithelium in the bluegill sunfish (Lepomis macrochirus) saccule, one of the otolithic organs in the inner ear. Though the OM is an integral part of the anatomic network of endorgan structures that result in vestibular function in the inner ear, the identity of the proteins that make up this sensory accessory membrane in teleosts, or in any vertebrate, is not fully known. Previously, we identified a cDNA from the sunfish saccular otolithic organ that encoded a new member of the collagen family of structural proteins. In this study, we examined biochemical features and the localization of the saccular collagen (SC) protein in vivo using polyclonal antisera that recognize the noncollagenous domains of the SC protein. The otolithic endorgans are part of the vestibular portion of the vertebrate inner ear and consist of a single or many calcified masses coupled to an underlying hair cell-containing sensory epithelium. A gelatinous otolithic͞otoconial membrane is attached to both the otolith and the underlying sensory epithelium and serves to couple the calcified mass(es) to the receptor epithelium. Displacement of the overlying otolith͞ otoconial mass(es) relative to the underlying receptor epithelium results in the displacement of hair cell stereociliary bundles that are critical to the mechanoelectrical transduction process in the inner ear (1).The teleost saccule is an otolithic organ involved in aspects of both vestibular (1) and acoustic function in certain fish (1, 2). The number of saccular sensory cells in certain teleosts (3) and cartilagenous fish (4) can be much larger than the number found in the human saccule (1). We took advantage of the large number of saccular hair cells found in certain teleosts to construct cDNA libraries in efforts to identify genes involved in vestibular function (5).Differential screening of the teleost saccular cDNA libraries led to the identification of candidate saccule-specific cDNAs. One of these was found to encode a unique form of collagen that was termed saccular collagen (SC). The 2.0-kb SC transcript was highly abundant in saccular RNA and appears unique to the saccule because it was not detected in several other sunfish tissues (6). A similar domain organization and homology of a conserved region within the carboxyl-terminal noncollagenous domain identified the SC as a new member of the family of collagens that includes collagens type VIII and type X (7-9). Expression of the SC gene was localized by in situ hybridization to secretory supporting cells located around the periphery of the saccular macula (6). In teleosts, supporting cells such as these have been correlated with the developmental appearance of the otolithic membrane (OM) (10) suggesting that the SC may be a component of the teleost OM. To date, the molecular identity of the proteins that comprise the teleost OM remains unknown.In this report, polyclonal SC-specific antisera were used to study the localization and...

ABSTRACTexpression two days later. Immunoreactivity appeared in abneural supporting cells days later than in hair cells, and not until E20 in neurally located supporting Mobile intracellular calcium buffers play an important cells. These results demonstrate that calbindin mesrole in regulating calcium flux into mechanosensory sage and protein levels are greater in high-frequency hair cells and calbindin D-28k is expressed at high hair cells. This "tonotopic" gradient may result from levels in the chick's basilar papilla. We have used RTthe stabilization of a basal-to-apical developmental gra-PCR, in situ hybridization, and immunohistology to dient and could be related at least in part to calcium demonstrate that calbindin expression varies systematchannel expression along this axis. ically according to hair cell position and develop-