Embryonic Cochlea Research Articles

Follistatin regulates the specification of the apical cochlea responsible for low-frequency hearing in mammals

The cochlea's ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region's frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, weprovide invivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.

The acquisition of positional information across the radial axis of the cochlea.

The mammalian cochlea detects sound and transmits this information to the brain. A cross section through the cochlea reveals functionally distinct epithelial domains arrayed around the circumference of a fluid-filled duct. Six major domains include two on the roof of the duct (Reissner's membrane medially and the stria vascularis laterally) and four across the floor of the duct, including the medial and lateral halves of the sensory domain, the organ of Corti. These radial domains are distinguishable in the embryonic cochlea by differential expression of transcription factors, and we focus here on a subset of the factors that can influence cochlear fates. We then move upstream of these genes to identify which of five signaling pathways (Notch, Fgf, Wnt, Bmp, and Shh) controls their spatial patterns of expression. We link the signaling pathways to their downstream genes, separating them by their radial position, to create putative gene regulatory networks (GRNs) from two time points, before and during the time when six radial compartments arise. These GRNs offer a framework for understanding the acquisition of positional information across the radial axis of the cochlea, and to guide therapeutic approaches to repair or regenerate distinct cochlear components that may contribute to hearing loss.

Early Development of Resident Macrophages in the Mouse Cochlea Depends on Yolk Sac Hematopoiesis.

Resident macrophages reside in all tissues throughout the body and play a central role in both tissue homeostasis and inflammation. Although the inner ear was once believed to be “immune-privileged,” recent studies have shown that macrophages are distributed in the cochlea and may play important roles in the immune system thereof. Resident macrophages have heterogeneous origins among tissues and throughout developmental stages. However, the origins of embryonic cochlear macrophages remain unknown. Here, we show that the early development of resident macrophages in the mouse cochlea depends on yolk sac hematopoiesis. Accordingly, our results found that macrophages emerging around the developing otocyst at E10.5 exhibited dynamic changes in distribution and in situ proliferative capacity during embryonic and neonatal stages. Cochlear examination in Csf1r-null mice revealed a substantial decrease in the number of Iba1-positive macrophages in the spiral ganglion and spiral ligament, whereas they were still observed in the cochlear mesenchyme or on the intraluminal surface of the perilymphatic space. Our results demonstrated that two subtypes of resident macrophages are present in the embryonic cochlea, one being Csf1r-dependent macrophages that originate from the yolk sac and the other being Csf1r-independent macrophages that appear to be derived from the fetal liver via systemic circulation. We consider the present study to be a starting point for elucidating the roles of embryonic cochlear resident macrophages. Furthermore, resident macrophages in the embryonic cochlea could be a novel target for the treatment of various inner ear disorders.

A spontaneous mouse deletion in Mctp1 uncovers a long-range cis-regulatory region crucial for NR2F1 function during inner ear development

Hundreds of thousands of cis-regulatory DNA sequences are predicted in vertebrate genomes, but unlike genes themselves, few have been characterized at the functional level or even unambiguously paired with a target gene. Here we serendipitously identified and started investigating the first reported long-range regulatory region for the Nr2f1 (Coup-TFI) transcription factor gene. NR2F1 is temporally and spatially regulated during development and required for patterning and regionalization in the nervous system, including sensory hair cell organization in the auditory epithelium of the cochlea. Analyzing the deaf wanderer (dwnd) spontaneous mouse mutation, we traced back the cause of its associated circling behavior to a 53 kb deletion removing five exons and adjacent intronic regions of the poorly characterized Mctp1 gene. Interestingly, loss of Mctp1 function cannot account for the hearing loss, inner ear dysmorphology and sensory hair cell disorganization observed in dwnd mutants. Instead, we found that the Mctp1dwnd deletion affects the Nr2f1 gene located 1.4 Mb away, downregulating transcription and protein expression in the embryonic cochlea. Remarkably, the Mctp1dwnd allele failed to complement a targeted inactivation allele of Nr2f1, and transheterozygotes or Mctp1dwnd homozygotes exhibit the same morphological defects observed in inner ears of Nr2f1 mutants without sharing their early life lethality. Defects include improper separation of the utricle and saccule in the vestibule not described previously, which can explain the circling behavior that first brought the spontaneous mutation to attention. By contrast, mice homozygous for a targeted inactivation of Mctp1 have normal hearing and inner ear structures. We conclude that the 53 kb Mctp1dwnd deletion encompasses a long-range cis-regulatory region essential for proper Nr2f1 expression in the embryonic inner ear, providing a first opportunity to investigate Nr2f1 function in postnatal inner ears. This work adds to the short list of long-range regulatory regions characterized as essential to drive expression of key developmental control genes.

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the “pro-hair cell” transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea: Lgr4/5 in the developing cochlea

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferationof precursors and promotes the formation of hair cells by up-regulating Atoh1expression. Not much, however, is known about the regulation of Wnt/β-catenin activityin the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated byan interaction between LGR receptors and their ligands from the R-spondin family. Thedeficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality.Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increasesWnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction ofinner and outer hair cells (OHC). Supernumerary hair cells are likely formed due toan up-regulation of the “pro-hair cell” transcription factors Atoh1, Nhlh1, and Pou4f3.Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs inthe heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolongedthe proliferation in the mid-basal turn of E13 cochleae, causing an increase in thenumber of SOX2-positive precursor cells within the pro-sensory domain. The prematuredifferentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficientembryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion.Altogether, our findings suggest that LGR4 and LGR5 play an important role in theregulation of hair cell differentiation in the embryonic cochlea.

Investigation of the GJB6 Deletion Mutations Del (GJB6-D13s1830) and Del (GJB6-D13s1854) in Iranian Patients with Autosomal-Recessive Non-Syndromic Hearing Loss (ARNSHL)

Hearing loss (HL) is the most common inherited sensory disorder affecting about 1 in 1000 births. The first locus for nonsyndromic autosomal recessive HL is on chromosome 13q11-22. The two genes, GJB2 and GJB6, are closely located on chromosome and are known to be co-expressed in the embryonic cochlea. Deletion mutations involving GJB6 were associated with autosomal-recessive nonsyndromic hearing loss (ARNSHL) and in combination with a GJB2 mutation with digenic ARNSHL. The objective of this study was to screen for the del (GJB6-D13S1830) and del (GJB6-D13s1854) mutations in GJB6 gene in patients with ARNSHL from Iran, using multiplex PCR and direct sequencing methods. Agarose gel electrophoresis and DNA sequencing of amplified fragment of the PCR reaction showed none of the patients was found to carry deletion in GJB6 gene which indicates that these deletions are restricted to certain populations and indicating a founder effect regarding these deletions.

HMGA2, the Architectural Transcription Factor High Mobility Group, Is Expressed in the Developing and Mature Mouse Cochlea

Hmga2 protein belongs to the non-histone chromosomal high-mobility group (HMG) protein family. HMG proteins have been shown to function as architectural transcription regulators, facilitating enhanceosome formation on a variety of mammalian promoters. Hmga2 are expressed at high levels in embryonic and transformed cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, Hmga2. Our previous affymetrix array data showed that Hmga2 is expressed in the developing and adult mammalian cochleas. However, the spatio-temporal expression pattern of Hmga2 in the murine cochlea remained unknown. In this study, we report the expression of Hmga2 in developing and adult cochleas using immunohistochemistry and quantitative real time PCR analysis. Immunolabeling of Hmga2 in the embryonic, postnatal, and mature cochleas showed broad Hmga2 expression in embryonic cochlea (E14.5) at the level of the developing organ of Corti in differentiating hair cells, supporting cells, in addition to immature cells in the GER and LER areas. By postnatal stage (P0–P3), Hmga2 is predominantly expressed in the hair and supporting cells, in addition to cells in the LER area. By P12, Hmga2 immunolabeling is confined to the hair cells and supporting cells. In the adult ear, Hmga2 expression is maintained in the hair and supporting cell subtypes (i.e. Deiters’ cells, Hensen cells, pillar cells, inner phalangeal and border cells) in the cochlear epithelium. Using quantitative real time PCR, we found a decrease in transcript level for Hmga2 comparable to other known inner ear developmental genes (Sox2, Atoh1, Jagged1 and Hes5) in the cochlear epithelium of the adult relative to postnatal ears. These data provide for the first time the tissue-specific expression and transcription level of Hmga2 during inner ear development and suggest its potential dual role in early differentiation and maintenance of both hair and supporting cell phenotypes.

Auditory Hair Cell Centrioles Undergo Confined Brownian Motion Throughout the Developmental Migration of the Kinocilium

Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles’ confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them.

Gata3 Is a Critical Regulator of Cochlear Wiring

Spiral ganglion neurons (SGNs) play a key role in hearing by rapidly and faithfully transmitting signals from the cochlea to the brain. Identification of the transcriptional networks that ensure the proper specification and wiring of SGNs during development will lay the foundation for efforts to rewire a damaged cochlea. Here, we show that the transcription factor Gata3, which is expressed in SGNs throughout their development, is essential for formation of the intricately patterned connections in the cochlea. We generated conditional knock-out mice in which Gata3 is deleted after SGNs are specified. Cochlear wiring is severely disrupted in these animals, with premature extension of neurites that follow highly abnormal trajectories toward their targets, as shown using in vitro neurite outgrowth assays together with time-lapse imaging of whole embryonic cochleae. Expression profiling of mutant neurons revealed a broad shift in gene expression toward a more differentiated state, concomitant with minor changes in SGN identity. Thus, Gata3 appears to serve as an "intermediate regulator" that guides SGNs through differentiation and preserves the auditory fate. As the first auditory-specific regulator of SGN development, Gata3 provides a useful molecular entry point for efforts to engineer SGNs for the restoration of hearing.

Transfection of Mouse Cochlear Explants by Electroporation

The sensory epithelium of the mammalian inner ear, also referred to as the organ of Corti, is a remarkable structure comprised of highly ordered rows of mechanosensory hair cells and non-sensory supporting cells located within the coiled cochlea. This unit describes an in vitro explant culture technique that can be coupled with gene transfer via electroporation to study the effects of altering gene expression during development of the organ of Corti. While the protocol is largely focused on embryonic cochlea, the same basic protocol can be used on cochleae from mice as old as P5.

Expression of Pou3f3/Brn‐1 and its genomic methylation in developing auditory epithelium

In the mammalian cochlea, both the sensory cells-called hair cells (HCs)-and nonsensory cells such as supporting cells (SCs) and mesenchymal cells participate in proper auditory function through the expression of various functional molecules. During development, expression of certain genes is repressed through genomic methylation, one of the major epigenetic regulatory mechanisms. We explored the genomic regions that were differentially methylated in rat auditory epithelium at postnatal day 1 (P1) and P14 using amplification of intermethylated sites (AIMS). An AIMS fragment was mapped to the 3'-flanking region of Pou3f3/Brn-1. Bisulfite-converted PCR and quantitative methylation-specific PCR showed that the methylation frequency of the AIMS region and the adjacent CpG island was increased at P14, when the expression of Pou3f3 and the noncoding RNAs nearby decreased. Expression of de novo DNA methyltransferases 3a and 3b also suggests a role of epigenetic regulation during postnatal inner ear development. Immunohistochemical analysis showed that Pou3f3 was expressed specifically in the SCs and mesenchymal cells in the cochlea and established that Pou3f3 is a new cell-type marker for studying inner ear development. Mice deficient in Pou3f3 or Pou3f2 plus Pou3f3 did not exhibit any abnormality in the embryonic cochlea. Absence of Pou3f3 affected neither the proliferation nor the differentiation activities of HC progenitor cells. Pou3f3 may, however, be important for the maintenance or functional development of the postnatal cochlea. This is the first report to study involvement of an epigenetic regulatory mechanism in the developing mammalian auditory epithelium.

Prospective identification and purification of hair cell and supporting cell progenitors from the embryonic cochlea

Expression of the cyclin-dependent kinase inhibitor p27 Kip1 defines a post-mitotic population of cells in the embryonic mammalian cochlea that constitutes the nascent organ of Corti. Here, we describe techniques to purify these precursors using a transgenic p27/GFP reporter and fluorescence activated cell sorting (FACS). We demonstrate that these cells express other markers of the sensory lineage, such as Sox2, and when placed in dissociated cell culture differentiate as hair cells and supporting cells. The purified sensory progenitors thus obtained provide a means of studying the process of hair cell and supporting cell differentiation in vitro, as well as providing a means of analyzing the molecular and physiological properties of this unique population of cells.

Cloning and developmental expression of Shaker potassium channels in the cochlea of the chicken

Signal coding by the receptor and neuronal cells of the auditory system involves various ion channels that modulate a sound stimulus. The genes that encode a number of these ion channels and their accessory subunits are presently unknown for channels found in the sensory epithelium and cochlear nerve. Among these genes are those that encode delayed rectifier and transient type potassium channels found in both the sensory cells and the ganglion. Here, we report the cloning and developmental expression of Shaker family members that include cKv1.2, cKv1.3, cKv1.5, and the Shaker-related cGMP-gated potassium channel cKCNA10. Clones were obtained by screening a chicken embryonic cochlea cDNA library using, as a probe, a mixture of two DNA fragments of cKv1.2 and cKv1.3 obtained by the reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed chicken homologues of Kv1.2, Kv1.3, Kv1.5 and cGMP-gated potassium channels with a deduced amino acid homology of 96–98%, 82–84%, 67–71% and 67–79% to correspondent mammalian homologues. During development of chicken inner ear, RT-PCR studies show expression of cKv1.2, cKv1.3 and cKv1.5 as early as Embryonic Day (ED) 3, while cKCNA10 was detected at low levels beginning on ED6 and was highly expressed by ED9. Additionally, analysis of expression in different parts of the cochlea showed that these genes were co-expressed in different regions of the cochlea, including the cochlear ganglion, sensory epithelium, lagena, and tegmentum. This expression pattern suggests the potential for the formation of heteromeric channels from the corresponding α-subunits in these various tissues.

Retinoic Acid Signaling Is Necessary for the Development of the Organ of Corti

The cellular mosaic of the mammalian organ of Corti represents one of the most highly ordered structures in any vertebrate system. A single row of inner hair cells and three or four rows of outer hair cells extend along the basal-to-apical axis of the cochlea. The factors that play a role in the development of specific cell types within the cochlea are largely unknown; however, the results of previous studies have strongly suggested that retinoic acid plays a role in the development of cells as hair cells. To determine whether cochlear progenitor cells can respond directly to retinoic acid, the expression patterns for each of the RAR and RXR receptors within the embryonic cochlear duct were determined by in situ hybridization. Results indicate that RARα, RXRα, and RXRγ are initially expressed throughout the cochlear duct. As development continues, the expression of each receptor becomes more intense in cells that will develop as hair cells. At the same time, receptor expression is down-regulated in cells that will develop as nonsensory cell types. To determine the effects of retinoic acid signaling during the development of the organ of Corti, activation of retinoid receptors was blocked in cultures of the embryonic cochlea through receptor-specific antagonism or inhibition of retinoic acid synthesis. Results indicate that inhibition of retinoic acid signaling induces a significant decrease in the number of cells that develop as hair cells and a disruption in the development of the organ of Corti. These results demonstrate that cells within the developing cochlea can respond to retinoic acid and that signaling by retinoic acid is necessary for the normal development of the organ of Corti.

Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice

This study examined the potential for hair cell regeneration in embryonic and neonatal mouse organs of Corti maintained in vitro. Small numbers of hair cells were killed by laser microbeam irradiation and the subsequent recovery processes were monitored by differential interference contrast (DIC) microscopy combined with continuous time-lapse video recordings. Replacement hair cells were observed to develop in lesion sites in embryonic cochleae and on rare occasions in neonatal cochleae. In embryonic cochleae, replacement hair cells did not arise through renewed proliferation, but instead developed from preexisting cells that changed from their normal developmental fates in response to the loss of adjacent hair cells. In cochleae established from neonates, lost hair cells usually were not replaced, but 11 apparently regenerated hair cells and a single hair cell labeled by 3H-thymidine were observed as rare responses to the creation of hair cell lesions in these organs. The results indicate that the organ of Corti can replace lost hair cells during embryonic and on rare occasions during early neonatal development. The ability of preexisting cells to change their developmental fates in response to hair cell death is consistent with the hypothesis that during embryonic development hair cells may inhibit neighboring cells from specializing as hair cells. In neonatal cultures, the rare occurrence of apparently regenerated hair cells indicates that some cells in the postembryonic organ of Corti retain response mechanisms that can lead to self-repair.

N-myc expression in the embryonic cochlea of the mouse

N-myc expression in the mouse embryo was examined during the late cochlear organogenesis. Tissue distribution of N-myc expression was histologically analyzed by in situ hybridization of the transcript in the cochlea between 15 and 18 days of gestation. At 15 days of gestation, N-myc expression was found very conspicuous in nervous structure of the cochlea such as the auditory nerve and the spiral ganglion. Moreover, N-myc was also present in the Köllikers organ and in the epithelium surrounding the cochlear canal. A few days later, N-myc expression was still clearly present in the Köllikers organ but less so in nervous structures. This study shows that cochlear tissues derived from the otic placode present a significant level of N-myc transcript during late embryogenesis. N-myc expression seems to be related to cell differentiation in the inner ear.

The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture.

The mammalian organ of Corti has one of the most highly ordered patterns of cells in any vertebrate sensory epithelium. A single row of inner hair cells and three or four rows of outer hair cells extend along its length. The factors that regulate the formation of this strict pattern are unknown. In order to determine whether retinoic acid plays a role during the development of the organ of Corti, exogenous retinoic acid was added to embryonic mouse cochleae in vitro. Exogenous retinoic acid significantly increased the number of cells that developed as hair cells and resulted in large regions of supernumerary hair cells and supporting cells containing two rows of inner hair cells and up to 11 rows of outer hair cells. The effects of retinoic acid were dependent on concentration and on the timing of its addition. Western blot analysis indicated that cellular retinoic acid binding protein (CRABP) was present in the sensory epithelium of the embryonic cochlea. The amount of CRABP apparently increased between embryonic day 14 and postnatal day 1, but CRABP was not detectable in sensory epithelia from adults. A retinoic acid reporter cell line was used to demonstrate that retinoic acid was also present in the developing organ of Corti between embryonic day 14 and postnatal day 1, and was also present in adult cochleae at least in the vicinity of the modiolus. These results suggest that retinoic acid is involved in the normal development of the organ of Corti and that the effect of retinoic acid may be to induce a population of prosensory cells to become competent to differentiate as hair cells and supporting cells.

Human embryonic organ of Corti in tissue culture and NSE expression.

For the first time ever, the cochlear tissue of the human embryo has been successfully grown in vitro in two cases, and neuro-specific enolase (NSE) immunoreactivity was studied in one of these tissues. The outer hair cells were arranged in three rows, and the inner hair cells in one row and in better order than the outer hair cells. After NSE immunostaining, the outer spiral bundle, tunnel fiber, outer hair cells, inner hair cells, and the spiral ganglion cells showed positive staining. This data suggests that the human embryonic cochlea has nearly reached complete maturation by 16 weeks, and that the tissue of the Corti organ can be differentiated and matured in vitro.

Hair cell differentiation in the developing chick cochlea and in embryonic Cochlear organ culture

We have defined a method for growing chick embryonic cochleae in organ culture that preserves many aspects of hair cell differentiation. Cochlear ducts were isolated from embryonic day 8 chicks, placed in organ culture, and incubated for 48 hours (to a point equivalent to embryonic day 10). The cultured ducts were then fixed and processed for scanning electron microscopy. As controls, cochlear ducts at embryonic days 8 and 10 were dissected and immediately fixed and processed for scanning electron microscopy. We chose this period to culture cochleae because at the corresponding time in vivo hair cells undergo a dynamic phase of differentiation. During this time, the number of stereocilia in the stereociliary bundle increases, and two to three rows of stereocilia nearest the kinocilium elongate, initiating the staircase pattern of the bundle. Also, the orientation of many hair cells shifts from nonpolarized at embryonic day 8 to polarized toward the inferior edge of the basilar papilla at embryonic day 10. Many of these aspects of hair cell differentiation proceed normally in organ culture. The appropriate distal-to-proximal gradients of hair cell density, apical surface area, and stereociliary number are preserved. Elongation of the 1-2 stereociliary rows next to the kinocilium continues, and more stereociliary bundles are oriented toward the inferior edge in cultured cochleae than in embryonic day 8 chicks. It appears that cochlear organ culture can serve as an effective method with which to study how hair cell differentiation is regulated.