Structure and function in the saccule of the goldfish (Carassius auratus): a model of diversity in the non-amniote ear.

The vertebrate inner ear is comprised of a remarkable diversity of cell types, including several types of sensory hair cells. In amniotes (reptiles, birds, and mammals), the morphological and physiological characteristics that distinguish these cell types have been well documented, while cellular variation in the ears of non-amniotes (all other vertebrate groups) has remained underrecognized. Since non-amniotes have become increasingly popular models for developmental and genetic research, a more comprehensive understanding of structure and function in the inner ears of these species is warranted. This paper first reviews the large body of data describing the morphology and physiology of hair cells and afferent neurons in the inner ear of the goldfish (Carassius auratus). In particular, we examine the structure of the goldfish saccule, an endorgan that has been the subject of numerous investigations on audition. New data on the structural variation of synaptic bodies in saccular hair cells are also presented, and the functional implications of these data are discussed. Finally, we conclude that hair cell structure varies along the length of the goldfish saccule in a manner consistent with known physiological characteristics of the endorgan. The saccule provides an excellent model for investigating structure-function relationships in the vertebrate inner ear, as well as the development of auditory and vestibular sensory epithelia.

Expression of Math1 and HES5 in the cochleae of wildtype and Jag2 mutant mice.

The sensory epithelium within the mammalian cochlea (the organ of Corti) is a strictly ordered cellular array consisting of sensory hair cells and nonsensory supporting cells. Previous research has demonstrated that Notch-mediated lateral inhibition plays a key role in the determination of cell types within this array. Specificallly, genetic deletion of the Notch ligand, Jagged2, results in a significant increase in the number of hair cells that develop within the sensory epithelium, presumably as a result of a decrease in Notch activation. In contrast, the downstream mediators and targets of the Notch pathway in the inner ear have not been determined but they may include genes encoding the proneural gene Math1 as well as the HES family of inhibitory bHLH proteins. To determine the potential roles of these genes in cochlear development, in situ hybridization for Math1 and HES5 was performed on the cochleae of wild-type vs. Jagged2 mutants (Jag2deltaDSL). Results in wild-type cochleae show that expression of Math1 transcripts in the duct begins on E13 and ultimately becomes restricted to hair cells in the sensory epithelium. In contrast, expression of HES5 begins on E15 and becomes restricted to supporting cells in the epithelium. Results in Jag2 mutant cochleae suggest that Math1 transcripts are ultimately maintained in a larger number of cells as compared with wild-type, while transcripts for HES5 are dramatically reduced throughout the epithelium. These results are consistent with the hypothesis that activation of Notch via Jagged2 acts to inhibit expression of Math1 in cochlear progenitor cells, possibly through the activity of HES5.

Notch signalling pathway mediates hair cell development in mammalian cochlea.

The mammalian cochlea contains an invariant mosaic of sensory hair cells and non-sensory supporting cells reminiscent of invertebrate structures such as the compound eye in Drosophila melanogaster. The sensory epithelium in the mammalian cochlea (the organ of Corti) contains four rows of mechanosensory hair cells: a single row of inner hair cells and three rows of outer hair cells. Each hair cell is separated from the next by an interceding supporting cell, forming an invariant and alternating mosaic that extends the length of the cochlear duct. Previous results suggest that determination of cell fates in the cochlear mosaic occurs via inhibitory interactions between adjacent progenitor cells (lateral inhibition). Cells populating the cochlear epithelium appear to constitute a developmental equivalence group in which developing hair cells suppress differentiation in their immediate neighbours through lateral inhibition. These interactions may be mediated through the Notch signalling pathway, a molecular mechanism that is involved in the determination of a variety of cell fates. Here we show that genes encoding the receptor protein Notch1 and its ligand, Jagged 2, are expressed in alternating cell types in the developing sensory epithelium. In addition, genetic deletion of Jag2 results in a significant increase in sensory hair cells, presumably as a result of a decrease in Notch activation. These results provide direct evidence for Notch-mediated lateral inhibition in a mammalian system and support a role for Notch in the development of the cochlear mosaic.

Expression of proneural and neurogenic genes in the embryonic mammalian vestibular system.

One of the most striking aspects of all auditory and vestibular sensory epithelia is the mosaic pattern of hair cells and supporting cells. The factors that are required for the development of this mosaic have not been determined, however the results of recent studies have demonstrated that components of the neurogenic (Notch) signaling pathway are expressed in the developing inner ears of a number of different vertebrate species. To examine whether this signaling pathway may play a similar role in the development of the hair cell mosaic in the mammalian vestibular system, the expression patterns of proneural (Math1) and neurogenic (Notch1, Jagged2, HES5) genes were examined in the developing mouse inner ear. Results indicate that Notch1 is initially expressed throughout the developing inner ear and becomes restricted to non-sensory cells within the developing sensory epithelia. In contrast, initial expression of Math1 and Jagged2 is localized to the developing sensory epithelia and ultimately becomes restricted to hair cells. Interestingly, transcripts for HES5, a target of Notch activation, are expressed in the developing cristae but not in the saccule or utricle. These results are consistent with the hypothesis that formation of the hair cell mosaic is regulated through the neurogenic pathway. However the differential expression of HES5 within the ear indicates that the downstream targets of Notch1 activation are not consistent across all of the sensory epithelia and suggests that the effects of activation of Notch1 in the saccule and utricle must be regulated through alternate target genes.

Cell proliferation and hair cell addition in the ear of the goldfish, Carassius auratus.

Cell proliferation and hair cell addition have not been studied in the ears of otophysan fish, a group of species who have specialized hearing capabilities. In this study we used the mitotic S-phase marker bromodeoxyuridine (BrdU) to identify proliferating cells in the ear of one otophysan species, Carassius auratus (the goldfish). Animals were sacrificed at 3 h or 5 days postinjection with BrdU and processed for immunocytochemistry. The results of the study show that cell proliferation occurs in all of the otic endorgans and results in the addition of new hair cells. BrdU-labeled cells were distributed throughout all epithelia, including the primary auditory endorgan (saccule), where hair cell phenotypes vary considerably along the rostrocaudal axis. This study lays the groundwork for our transmission electron microscopy study of proliferative cells in the goldfish ear (Presson et al., Hearing Research 100 (1996) 10-20) as well as future studies of hair cell development in this species. The ability to predict, based on epithelial location, the future phenotype of developing hair cells in the saccule of the goldfish make that endorgan a particularly powerful model system for the investigation of early hair cell differentiation.

Hair cell precursors are ultrastructurally indistinguishable from mature support cells in the ear of a postembryonic fish.

The ultrastructure of S-phase cells in the postembryonic fish ear was compared with that of mature support cells. S-phase cells were identified by injecting animals with [3H]thymidine and sacrificing 3 h later. Sensory epithelia (saccules, utricles, and canals) were processed for light-level autoradiography. Sections containing thymidine-labeled cells were re-embedded and re-examined using transmission electron microscopy. The results indicate that S-phase cells differ from mature support cells only in nuclear position and shape. Otherwise their cytoplasmic characteristics are indistinguishable. Both cell types, on the other hand, are readily distinguishable from hair cells. These data provide ultrastructural evidence for the ability of mature support cells to enter the cell cycle in postembryonic vertebrates.

Novel afferent terminal structure in the crista ampullaris of the goldfish, carassius auratus.

Using transmission electron microscopy, we have identified a new type of afferent terminal structure in the crista ampullaris of the goldfish Carassius auratus. In addition to the bouton-type afferent terminals previously described in the ear of this species, the crista also contained enlarged afferent terminals that enveloped a portion of the basolateral hair cell membrane. The hair cell membrane was evaginated and protruded into the afferent terminal in a glove-and-finger configuration. The membranes of the two cells were regularly aligned in the protruded region of the contact and had a distinct symmetrical electron density. The electron-dense profiles of these contacts were easily identified and were present in every crista sampled. In some cases, efferent terminals synapsed onto the afferents at a point where the hair cell protruded into the terminal. The ultrastructural similarities of the goldfish crista afferents to calyx afferents found in amniotes (birds, reptiles, and mammals) are discussed. The results of the study support the hypothesis that structural variation in the vertebrate inner ear may have evolved much earlier in evolution than previously supposed.

Effects of low-frequency underwater sound on hair cells of the inner ear and lateral line of the teleost fish Astronotus ocellatus.

Fish (Astronotus ocellatus, the oscar) were subject to pure tones in order to determine the effects of sound at levels typical of man-made sources on the sensory epithelia of the ear and the lateral line. Sounds varied in frequency (60 or 300 Hz), duty cycle (20% or continuous), and intensity (100, 140, or 180 dB re: 1 muPa). Fish were allowed to survive for 1 or 4 days posttreatment. Tissue was then evaluated using scanning electron microscopy to assess the presence or absence of ciliary bundles on the sensory hair cells on each of the otic endorgans and the lateral line. The only damage that was observed was in four of five fish stimulated with 300-Hz continuous tones at 180 dB re: 1 muPa and allowed to survive for 4 days. Damage was limited to small regions of the striola of the utricle and lagena. There was no damage in any other endorgan, and the size and location of the damage varied between specimens. No damage was observed in fish that had been allowed to survive for 1 day poststimulation, suggesting that damage may develop slowly after exposure.

Hair cell heterogeneity in the goldfish saccule.

A set of cytological studies performed in the utricle and saccule of Astronotus ocellatus (Teleostei, Percomorphi, Cichlidae) identified two basic types of hair cells and others with some intermediate characteristics. This paper reports on applying the same techniques to the saccule of Carassius auratus (Teleostei, Otophysi, Cyprinidae) and demonstrates similar types of hair cells to those found in Astronotus. Since Carassius and Astronotus are species of extreme taxonomic distance within the Euteleostei, two classes of mechanoreceptive hair cells are likely to represent the primitive condition for sensory receptors in the euteleost inner ear and perhaps in all bony fish ears.