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Proc Natl Acad Sci U S A. Aug 19, 1997; 94(17): 9445–9450.
PMCID: PMC23217
Neurobiology

Essential role of POU–domain factor Brn-3c in auditory and vestibular hair cell development

Abstract

The Brn-3 subfamily of POU–domain transcription factor genes consists of three highly homologous members—Brn-3a, Brn-3b, and Brn-3c—that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes plays distinctive roles in the somatosensory, visual, and auditory/vestibular systems.

Keywords: inner ear development

A number of transcription factors have been implicated in decisions related to neuronal vs. nonneuronal cell fate, regional specification in the nervous system, or determination of the terminally differentiated phenotype. For example, bHLH factors such as neuroD and the achaete–scute family control neural vs. ectodermal cell fates (1, 2), Hox genes control regional specification along the neuraxis (3), and several POU–domain genes act at late stages to control the survival and final differentiated phenotype of particular neuronal subtypes (4). The POU–domain family was intially defined by the mammalian pituitary-specific transcription factor Pit-1/GHF-1, the octamer binding proteins Oct-1 and Oct-2, and the Caenorhabditis elegans gene Unc-86 (5). Genetic studies in mice and humans indicate that many POU–domain genes function in the terminal stages of central nervous system development. SCIP/Tst-1/Oct-6 controls the differentiation of Schwann cells (68), Pit-1/GHF-1 is required for the normal development of the anterior pituitary (9), Brn-4/RHS2/POU3F4 is required for the normal development of the middle ear (10), and Brn-2 is required for the specification of subsets of neurons in the hypothalamus (11, 12).

The class IV POU–domain group is defined by the Unc-86 gene (13), the Drosophila I-POU gene (14, 15), and the three vertebrate Brn-3 genes (1620). The Unc-86 protein is found exclusively within a subset of neurons and neuroblasts, and Unc-86 loss-of-function mutations affect some of these cells by causing a daughter cell to assume the fate of its mother or by altering cell phenotypes postmitotically (2123). In mammals, the three highly homologous class IV POU–domain genes, Brn-3a, Brn-3b, and Brn-3c (also referred to as Brn-3.0, Brn-3.2, and Brn-3.1, respectively), are expressed in distinct but overlapping patterns in the developing and adult brainstem, retina, and dorsal root and trigeminal ganglia (16, 1820, 24, 25).

Targeted mutations in Brn-3a and Brn-3b have revealed a high degree of functional specificity in vivo. Targeted deletion of the Brn-3a gene results in a decreased number of neurons in the trigeminal ganglia, altered gene expression in the dorsal root and trigeminal ganglia, and defects in brainstem nuclei involved in sensory processing and motor control (26, 27). Targeted deletion of the Brn-3b gene results in a selective absence of ≈70% of retinal ganglion cells with little or no effect on other neurons (28, 29). In this paper we show that Brn-3c is expressed in auditory and vestibular hair cells and that targeted deletion of Brn-3c leads to defective inner ear function secondary to a complete absence of sensory hair cells. Results similar to those described here have been reported by Erkman et al. (29).

MATERIALS AND METHODS

Targeted Deletion of Brn-3c.

To generate the gene targeting construct, the 4.8-kb BamHI–EcoRI fragment containing the Brn-3c gene was cloned into the BamHI site of pMCTK (L.G., unpublished work). Subsequently, the 1.8-kb XhoI–XhoI fragment containing the first exon and the majority of the second exon, and encompassing most of the coding region of Brn-3c, was replaced with a 1.6-kb XhoI–XhoI segment containing the PGKneo cassette. The targeting vector was linearized at a unique NotI site and 25 μg of linearized DNA was electroporated into 107 AB1 embryonic stem (ES) cells that were then selected for resistance to G418 and 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-5-iodouracil (FIAU). Among the 288 ES colonies resistant to G418 and FIAU, 7 contained the expected homologous recombination event, as indicated by Southern blot analysis using flanking probes. Two targeted ES cell clones were injected into C57BL/6J blastocysts to generate chimaeric mice, and the chimaeric mice were bred to C57BL/6J mice to produce heterozygotes and homozygotes for further analysis.

Behavioral Testing.

Hearing was tested by observing the startle response. Mice were placed individually in a small Plexiglas restrainer in a sound-insulated room. At 10-sec intervals, each animal was exposed five times to a sharp sound produced by striking a metal rod. Ten Brn-3c (+/+), 10 Brn-3c (+/−), and 11 Brn-3c (−/−) animals were tested. Balance was tested by placing mice individually on a soft rubber-coated horizontal drum 6 cm in diameter and 5.5 cm deep, positioned 15 cm above a cushioned pad. In 1 set of 10 trials the drum was stationary, and in a second set of 10 trials the drum was rotated by a variable speed motor at 7 rpm. In each trial the mouse was observed for 60 sec after it was placed on the drum and the time elapsed until it fell from the drum was recorded. Seven Brn-3c (+/+), 7 Brn-3c (+/−), and 7 Brn-3c (−/−) animals were tested.

Auditory Brainstem Responses.

Auditory-evoked brainstem responses were recorded using a Nicolet Compact Four (Nicolet). Mice were anesthetized with Avertin by intraperitoneal injection, and electrodes were placed at the vertex (active), bilaterally in the neighborhood of the postauricular bullae (reference), and in the forehead (ground). The acoustic stimulus consisted of a click of approximately 0.1 msec duration, presented at a rate of 11.4 per second. Responses were averaged over 1,000 stimuli. Auditory thresholds were determined by visual inspection of response traces obtained at stimulus intervals of 5 dB. Absolute stimulus intensities were calibrated to obtain the sound pressure level (SPL).

Semi-Thin Plastic Sections.

Deeply anesthetized mice were perfused transcardially with PBS, 2% paraformaldehyde, 2% gluteraldehyde, 1 mM CaCl2, 1 mM MgCl2. Following dissection of the temporal bone, the stapes was removed from the oval window, the round window was perforated, and a small hole was made in the apex of the cochlea. The cochlea and the vestibular system were gently perfused with the same fixative, immersion fixed on ice for 2 hr, and then gently perfused with PBS, 1% osmium tetroxide. After immersion fixation in osmium tetroxide for 2 hr on ice in the dark, the temporal bones were rinsed with PBS, fixed overnight in PBS, 2% paraformaldehyde, 2% gluteraldehyde, and decalcified for 7–10 days in PBS, 0.1 M EDTA, 0.1% gluteraldehyde. Fixed and decalcified temporal bones were dehydrated and embedded in Spurr’s resin (30). One micrometer sections were stained with methylene blue.

Scanning Electron Microscopy.

A detailed description is presented in ref. 31.

Whole Mounts of the Organ of Corti.

Temporal bones were harvested, locally perfused, and immersion fixed for 2 hr in 4% paraformaldehyde/PBS. Following dissection of the bony cochlea as described (31), the organ of Corti and immediately adjacent tissue were stained with 0.5% cresyl violet and mounted in glycerin. Acetylcholine esterase histochemistry (32) was performed in the presence of ethoxypropazine hydrochloride at 0.1 mM to inhibit pseudocholinesterase (33). Cochleas were locally perfused, dissected at the apex to reveal one turn of the organ of Corti, fixed overnight in PBS, 4% paraformaldehyde, and reacted for 4 hr at 4°C to reveal acetylcholinesterase activity.

Immunostaining.

Affinity-purified rabbit anti-Brn-3a, anti-Brn-3b, and anti-Brn-3c antibodies have been described (20). Each of these antibodies specifically recognizes the Brn-3 family member against which it was raised as determined by the distinctive patterns of immunostaining obtained with each antibody and by the selective elimination of immunostaining in mice lacking the corresponding gene (refs. 20, 26, and 28; this work). For immunostaining of cochlear whole mounts, temporal bones were dissected as described above and the cochlea was locally perfused with 4% paraformaldehyde/PBS, opened at the apex to reveal one turn of the organ of Corti, fixed for 24 hr in PBS/4% paraformaldehyde, and processed for immunostaining as previously described for retina whole mounts (20). Following immunostaining, the tectorial membrane was removed, and the organ of Corti was isolated from the bony cochlea, stained with 4′,6-diamidino-2-phenylindole (DAPI), and mounted in glycerin. Immunostaining of the developing auditory and vestibular system was performed on animals after up to 5 days of postnatal development without decalcification as described (20).

RESULTS

Defective Hearing and Balance in Mice Homozygous for a Targeted Deletion of the Brn-3c Gene.

To assess the role of Brn-3c in vivo, a targeted deletion of this gene was constructed in ES cells and introduced via blastocyst fusion into the mouse germ line. Brn-3c heterozygotes [(+/−)] are indistinguishable from the wild-type [(+/+)] in viability, fertility, and readily observable behaviors. They are also indistinguishable by all of the histologic criteria described below. Brn-3c homozygotes [(−/−)] have normal viability, but are 10–20% smaller than wild type, have low fertility, and spend much of their time running in circles. We think that the low weight and fertility of Brn-3c (−/−) mice may be secondary to the circling behavior and the resulting higher energy expenditure.

Circling behavior has been described for a number of mouse lines with inner ear defects (34). As a first step in examining inner ear function, we determined whether a sharp sound could elicit a startle response in Brn-3c (+/+), (+/−), and (−/−) mice. Brn-3c (+/+) and (+/−) mice show a robust startle response (10 mice for each genotype), whereas Brn-3c (−/−) mice show no response (11 mice). To begin to localize the site of the defect and to quantitatively assess audition, auditory brainstem responses were recorded from Brn-3c (+/+), (+/−), and (−/−) mice. Fig. Fig.11A shows representative auditory brainstem responses from Brn-3c (+/−) and (−/−) mice. As with the wild type, Brn-3c (+/−) mice have an auditory threshold of approximately 50 dB SPL. In contrast, Brn-3c (−/−) mice show no response at any stimulus level, including 114 dB SPL, the highest level tested.

Figure 1
Auditory brainstem responses and balancing defects in Brn-3c (−/−) mice. (A) Representative auditory brainstem responses to a click stimulus are shown at different stimulus intensities. Brn-3c (+/−) mice ...

To assess vestibular function, two quantitative tests were performed. In one test, mice were placed individually on a horizontal drum, and the time elapsed until they fell from the drum was recorded (Fig. (Fig.11B). Brn-3c (+/+) and (+/−) animals rarely fell from the drum, even when it was slowly rotated. In contrast, Brn-3c (−/−) mice exhibit extremely poor balance, typically falling from a stationary drum within 10 sec. When the drum was slowly rotated, forcing the Brn-3c (−/−) animals to walk, none remained on the drum after 5 sec. As a second test of vestibular function, we monitored the ability of mice to remain upright and swim effectively in a tub of water. Twelve Brn-3c (+/+) and 12 Brn-3c (+/−) mice remained upright and swam, whereas each of 5 Brn-3c (−/−) mice tested tumbled about when placed in the water.

Expression of Brn-3c in the Developing and Adult Inner Ear.

The behavioral and electrophysiological defects described above are consistent with a primary defect in the inner ear of Brn-3c (−/−) mice. Therefore, as an initial step in examining the possibility that Brn-3c might play a role in inner ear development or function, we immunostained the developing and adult inner ear with affinity-purified anti-Brn-3c antibodies. In our earlier immunocytochemical analysis of Brn-3c, all regions of the adult central nervous system were examined except for the inner ear, leaving open the possibility that Brn-3c might be expressed there (20).

During late embryonic and early postnatal development, Brn-3c immunolabeling was observed specifically in developing auditory and vestibular hair cells (Fig. (Fig.2).2). From this analysis, it appears that all hair cells within the organ of Corti, otolith organs, and cristae express Brn-3c by embryonic day (E)15.5, the earliest time examined. As expected for a transcription factor, immunolabeling is confined to nuclei. Immunoreactivity was not observed in nonneuronal cells or in the spiral or vestibular ganglia at any developmental stage. The peak times during which auditory and vestibular hair cells undergo their final mitoses are at E14 and E14–E16, respectively (35), suggesting that Brn-3c is involved in early steps in hair cell development. In contrast, Brn-3a and Brn-3b are present in many neurons within the developing and adult spiral and vestibular ganglia, but are absent from hair cells (data not shown).

Figure 2
Brn-3c in the developing mouse inner ear. Immunostaining with anti-Brn-3c antibodies of frozen sections at E17.5 (A and B) and E19.5 (C and D). Staining is present in the nuclei of developing hair cells at all stages shown, and is absent from other cell ...

In the adult cochlea, the distribution of Brn-3c protein was determined by whole-mount immunostaining. To visualize both labeled and unlabeled cells at high resolution, the organ of Corti was dissected, counterstained with DAPI, and examined under Nomarski optics (Fig. (Fig.3).3). Brn-3c immunolabeling is present exclusively in the nuclei of the single row of inner hair cells and the three rows of outer hair cells; the nuclei of supporting cells are unlabeled. In the adult cristae and otolith organs, Brn-3c is also present exclusively in hair cells (data not shown).

Figure 3
Brn-3c in the adult organ of Corti. One turn of a wild-type organ of Corti adjacent to the apex is shown. The cochlea was immunostained with affinity-purified anti-Brn-3c antibodies and the dissected organ of Corti was then incubated with DAPI. (A) DAPI ...

Histological Defects in the Inner Ear in Brn-3c (−/−) Mice.

In adult Brn-3c (+/+), (+/−), and (−/−) mice the external auditory canal, tympanic membrane, and middle ear, including the malleus, incus, and stapes, are normal in appearance. Dissection of the temporal bone revealed a normal arrangement of the semicircular canals and bony cochlea. In Brn-3c (−/−) mice, gentle movement of the malleus produced a corresponding movement of the footplate of the stapes, indicative of normal middle ear conduction. To determine if loss of Brn-3c altered the number or arrangement of auditory hair cells, the apical surface of the organ of Corti was exposed by removing the tectorial membrane and examined by scanning electron microscopy (Fig. (Fig.4).4). In Brn-3c (+/+) and (+/−) mice, the organ of Corti shows an identical arrangement of inner and outer hair cell ciliary bundles, which are flanked by the microvillus-bearing apical surfaces of supporting cells. However, in Brn-3c (−/−) mice the apical surface of the organ of Corti was completely devoid of ciliary bundles, consisting instead of a contiguous sheet of what appears to be supporting cells.

Figure 4
Scanning electron microscopy of the organ of Corti in Brn-3c (+/−) and (−/−) mice. (A and B) The apical surface of the organ of Corti in Brn-3c (+/−) mice shows the wild-type pattern ...

The absence of ciliary bundles in the Brn-3c (−/−) organ of Corti appears to reflect an absence of inner and outer hair cells as determined by staining whole mounts of the organ of Corti with cresyl violet (Fig. (Fig.5).5). In the Brn-3c (+/+) and (+/−) organ of Corti there is a single row of inner hair cell bodies and three rows of outer hair cell bodies, whereas the Brn-3c (−/−) organ of Corti contains only sparsely located cell bodies, presumably corresponding to the supporting cells of Hensen (Fig. (Fig.55 A and C). To identify outer hair cells more specifically, efferent cholinergic synapses were visualized histochemically. In the mouse, as in other mammals, outer hair cells receive massive cholinergic innervation, which is readily visualized histochemically (32, 33). As seen in Fig. Fig.55 B and D, in the Brn-3c (+/−) organ of Corti, acetylcholine esterase activity is localized to outer hair cells and to efferent nerve fibers emanating from the spiral ganglion, whereas in the Brn-3c (−/−) organ of Corti acetylcholine esterase activity is undetectable.

Figure 5
Whole-mount preparation of the organ of Corti in Brn-3c (+/+), (+/−), and (−/−) mice. (A) Organ of Corti from a Brn-3c (+/−) mouse stained with cresyl violet ...

The absence of auditory hair cells in Brn-3c (−/−) mice could represent an isolated defect or could reflect a more general disorganization of cochlear structure. To examine this question, the cochlea was analyzed using semi-thin sections stained with methylene blue (Fig. (Fig.6).6). The cochleas of Brn-3c (−/−) mice show no aberrations in overall architecture or in the structure of Reissner’s membrane or the stria vascularis, but differ from their wild-type counterparts in the absence of hair cells and the specialized architectural features immediately adjacent to hair cells. Pillar cells, which normally define the tunnel of Corti and the space of Nuel, are absent or rudimentary, and cells that resemble Deiter’s cells are variably present. A typical section, such as the one shown in Fig. Fig.66C, reveals only a single cell type, presumably the cells of Hensen, above the basilar membrane. A second difference in the Brn-3c (−/−) cochlea is seen in a large reduction in the number of neurons and myelinated fibers in the spiral ganglion (Fig. (Fig.66 B and D). Because Brn-3c is not expressed in supporting cells or in the spiral ganglion during development (see above), loss of supporting cells and spiral ganglion neurons is likely to be secondary to the loss of hair cells.

Figure 6
Absence of hair cells in the organ of Corti and defects in the spiral ganglion in Brn3c (−/−) mice. (A) Organ of Corti from a Brn-3c (+/−) mouse. The single inner hair cell, the three outer hair cells, and ...

A similar pattern of cell loss in Brn-3c (−/−) mice was observed in the vestibular organs and in the vestibular ganglion. Brn-3c (+/+) and (+/−) vestibular organs contain a high density of type 1 hair cells in which a lightly stained nerve chalice surrounds a darkly stained cell body (Fig. (Fig.77 A and B). Numerous stereocilia protrude from the apical surface and a single layer of darkly stained supporting cells lies beneath the sensory hair cells. In Brn-3c (−/−) mice, the bony structure of the vestibular system and the nonsensory epithelium within it appears to be unaffected, but the sensory epithelium contains only supporting cells (Fig. (Fig.77 C and D). Few or no myelinated axons are seen beneath the otolith organs in Brn-3c (−/−) mice.

Figure 7
Absence of hair cells in the otolith organs and cristae of Brn-3c (−/−) mice. (A) An otolith organ from a Brn-3c (+/−) mouse. Type I hair cells, the most abundant hair cell class, have a lightly stained ...

The absence of auditory and vestibular hair cells in adult Brn-3c (−/−) mice could reflect a failure of these cells to develop or survive at any stage prior to adulthood. To identify the time during development when hair cell loss occurs, inner ears of Brn-3c (+/+), (+/−), and (−/−) animals were examined at E17.5, E19.5, postembryonic day (P)1, and P5 by cresyl violet staining and by immunostaining with antibodies directed against Brn-3a and Brn-3b. This analysis revealed a progressive disorganization and loss of both auditory and vestibular hair cells as early as E17.5 with nearly complete loss by P5 (Fig. (Fig.8),8), suggesting that Brn-3c may not be required for the initial production of hair cells, but is required for their correct development and survival. Neuronal loss within the spiral and vestibular ganglia occurs later, with minimal cell loss at E17.5, clearly reduced numbers of neurons at P1 and P5, and severe depletion of neurons and myelinated fibers by several months of age (data not shown). Brn-3c is also expressed in approximately 30% of retinal ganglion cells, in a minority of neurons in the dorsal root and trigeminal ganglia, and in scattered cells within several nuclei in the midbrain (20). By cresyl violet staining we did not observe any differences in the size, number, or arrangement of neurons in the retina, dorsal root and trigeminal ganglia, and midbrain in Brn-3c (+/+), (+/−), or (−/−) mice at E17.5, E19.5, P1, and P5.

Figure 8
Defects during development of the cochlea of Brn-3c (−/−) mice. Cresyl violet-stained frozen sections showing the progressive development of the organ of Corti at P1 (A and B) and P5 (C and D). (A and C) Brn-3c (+/−). ...

DISCUSSION

This paper describes the consequences of targeted deletion of the POU–domain transcription factor Brn-3c. Brn-3c (−/−) mice exhibit severe defects in balance and hearing and they show a rapid and progressive loss of auditory and vestibular hair cells during late gestation and early postnatal life. Spiral and vestibular ganglion neurons degenerate over the ensuing weeks, but the middle ear and the overall architecture of the inner ear are unaffected. This phenotype is consistent with the observed pattern of Brn-3c immunoreactivity. In the inner ear, Brn-3c is found exclusively in auditory and vestibular hair cells beginning at least as early as E15.5. The degeneration of the spiral and vestibular ganglia in Brn-3c (−/−) mice is consistent with the previously described expression of brain-derived neurotrophic factor and neurotrophin-3 in auditory and vestibular hair cells (3639) and the essential role of these neurotrophic factors in the survival of spiral and vestibular ganglion neurons (4042).

The Brn-3c mutant phenotype in mice may be relevant to the study of some forms of human deafness. In humans, inherited congenital or early childhood deafness affects 1 person in 2,000 (43). In approximately 30% of the cases, inherited deafness is associated with other abnormalities (syndromic deafness). The remaining 70% of the cases are unaccompanied by other abnormalities (nonsyndromic deafness), and among this group approximately 85% are caused by mutations in any of a large number of autosomal recessive genes. The Brn-3c (−/−) phenotype in mice suggests the human Brn-3c gene as a candidate gene for autosomal recessive nonsyndromic deafness, and suggests that Brn-3c (−/−) mice may be useful as an animal model for some forms of inherited human deafness.

The highly specific pattern of neuronal loss in Brn-3c (−/−) mice is reminiscent of the specific loss of most retinal ganglion cells in Brn-3b (−/−) mice (28, 29) and of subsets of primary somatosensory neurons in the trigeminal ganglia and selected neurons in sensory and motor nuclei in the brainstem in Brn-3a (−/−) mice (26, 27). A striking feature of the three Brn-3 mutant phenotypes is the division of defects among the somatosensory, visual, and auditory/vestibular systems. In each of these three sensory systems, the most obvious cellular phenotype, neuronal loss, is only produced by deletion of one of the Brn-3 genes. Given the high degree of homology between the Brn-3 genes and the likelihood that they arose from a common ancestral Unc-86-like gene, it is tempting to speculate that the Brn-3 genes may act by a similar mechanism to promote cell survival. For example, neuronal loss in each of the Brn-3 mutants might be explained if the Brn-3 family controlled the expression of genes mediating the receipt of cell survival signals. This general possibility is suggested by the observations of McEvilly et al. (27) that transcripts derived from TrkB, TrkC, and the low-affinity nerve growth factor receptor (p75), are reduced in the E12.5 trigeminal ganglia of Brn-3a (−/−) mice, possibly presaging the loss of sensory neurons in these mice.

Thus far, descriptions of Brn-3 knockout mice—including the present one—have not extensively explored phenotypes other than cell loss. The possibility that additional, and more subtle, phenotypes remain to be identified in these mice is suggested by the variety of effects on neuronal lineage and identity produced by Unc-86 mutants in C. elegans (2123). In vertebrates, an additional level of complexity arises from the partially overlapping patterns of expression of the Brn-3 genes, suggesting that in some cells these genes may act in a combinatorial and/or partially redundant fashion. In this case, the earlier finding of Brn-3c in subsets of retinal ganglion cells and primary somatosensory neurons suggests that, in addition to its essential role in hair cell development and survival, Brn-3c may play a more subtle role in the development of these other neurons.

Acknowledgments

We thank J. Li and M. Delannoy for assistance with tissue sectioning and electron microscopy and P. Bhanot for helpful comments on the manuscript. This work was supported by the National Eye Institute and the National Institute of Child Health and Human Development (National Institutes of Health), the Robert A. Welch Foundation, the Retina Foundation, and the Howard Hughes Medical Institute.

ABBREVIATIONS

ES
embryonic stem
E
embryonic day
SPL
sound pressure level
DAPI
4′,6-diamidino-2-phenylindole
P
postembryonic day

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