Tegmentum Vasculosum Research Articles

The ultrastructure of a stria vascularis in the auditory organ of the cuban crocodile (Crocodylus rhombifer).

Background: An endocochlear potential (EP) exists in the mammalian cochlea generated by the stria vascularis and an associated fibrocyte network. It plays an essential role for sensory cell function and hearing sensitivity. In non-mammalian ectothermic animals the endocochlear potential is low and its origin somewhat unclear. In this study, we explored the crocodilian auditory organ and describe the fine structure of a stria vascularis epithelium that has not been verified in birds. Material and Methods: Three Cuban crocodiles (Crocodylus rhombifer) were analyzed with light and transmission electron microscopy. The ears were fixed in glutaraldehyde The temporal bones were drilled out and decalcified. The ears were dehydrated, and embedded and was followed by semi-thin and thin sectioning. Results: The fine structure of the crocodile auditory organ including the papilla basilaris and endolymph system was outlined. The upper roof of the endolymph compartment was specialized into a Reissner membrane and tegmentum vasculosum. At the lateral limbus an organized, multilayered, vascularized epithelium or stria vascularis was identified. Discussion: Electron microscopy demonstrates that the auditory organ in Crocodylus rhombifer, unlike in birds, contains a stria vascularis epithelium separate from the tegmentum vasculosum. It is believed to secrete endolymph and to generate a low grade endocochlear potential. It may regulate endolymph composition and optimize hearing sensitivity alongside the tegmentum vasculosum. It could represent a parallel evolution essential for the adaptation of crocodiles to their diverse habitats.

Expression of class III Semaphorins and their receptors in the developing chicken (Gallus gallus) inner ear.

Class III Semaphorin (Sema) secreted ligands are known to repel neurites expressing Neuropilin (Nrp) and/or Plexin (Plxn) receptors. There is, however, a growing body of literature supporting that Sema signaling also has alternative roles in development such as synaptogenesis, boundary formation, and vasculogenesis. To evaluate these options during inner ear development, we used in situ hybridization or immunohistochemistry to map the expression of Sema3D, Sema3F, Nrp1, Nrp2, and PlxnA1 in the chicken (Gallus gallus) inner ear from embryonic day (E)5-E10. The resulting expression patterns in either the otic epithelium or its surrounding mesenchyme suggest that Sema signaling could be involved in each of the varied functions reported for other tissues. Sema3D expression flanking the sensory tissue in vestibular organs suggests that it may repel Nrp2- and PlxnA1-expressing neurites of the vestibular ganglion away from nonsensory epithelia, thus channeling them into the sensory domains at E5-E8. Expression of Sema signaling genes in the sensory hair cells of both the auditory and vestibular organs on E8-E10 may implicate Sema signaling in synaptogenesis. In the nonsensory regions of the cochlea, Sema3D in the future tegmentum vasculosum opposes Nrp1 and PlxnA1 in the future cuboidal cells; the abutment of ligand and receptors in adjacent domains may enforce or maintain the boundary between them. In the mesenchyme, Nrp1 colocalized with capillary-rich tissue. Sema3D immediately flanks this Nrp1-expressing tissue, suggesting a role in endothelial cell migration towards the inner ear. In summary, Sema signaling may play multiple roles in the developing inner ear.

Molecular bases of K+ secretory cells in the inner ear: shared and distinct features between birds and mammals.

In the cochlea, mammals maintain a uniquely high endolymphatic potential (EP), which is not observed in other vertebrate groups. However, a high [K+] is always present in the inner ear endolymph. Here, we show that Kir4.1, which is required in the mammalian stria vascularis to generate the highly positive EP, is absent in the functionally equivalent avian tegmentum vasculosum. In contrast, the molecular repertoire required for K+ secretion, specifically NKCC1, KCNQ1, KCNE1, BSND and CLC-K, is shared between the tegmentum vasculosum, the vestibular dark cells and the marginal cells of the stria vascularis. We further show that in barn owls, the tegmentum vasculosum is enlarged and a higher EP (~+34 mV) maintained, compared to other birds. Our data suggest that both the tegmentum vasculosum and the stratified stria vascularis evolved from an ancestral vestibular epithelium that already featured the major cell types of the auditory epithelia. Genetic recruitment of Kir4.1 specifically to strial melanocytes was then a crucial step in mammalian evolution enabling an increase in the cochlear EP. An increased EP may be related to high-frequency hearing, as this is a hallmark of barn owls among birds and mammals among amniotes.

The frog inner ear: picture perfect?

Many recent accounts of the frog peripheral auditory system have reproduced Wever's (1973) schematic cross-section of the ear of a leopard frog. We sought to investigate to what extent this diagram is an accurate and representative depiction of the anuran inner ear, using three-dimensional reconstructions made from serial sections of Rana pipiens, Eleutherodactylus limbatus and Xenopus laevis. In Rana, three discrete contact membranes were found to separate the posterior otic (=endolymphatic) labyrinth from the periotic (=perilymphatic) system: those of the amphibian and basilar recesses and the contact membrane of the saccule. The amphibian 'tegmentum vasculosum' was distinguishable as a thickened epithelial lining within a posterior recess of the superior saccular chamber. These features were also identified in Eleutherodactylus, but in this tiny frog the relative proportions of the semicircular canals and saccule resemble those of ranid tadpoles. There appeared to be a complete fluid pathway between the right and left periotic labyrinths in this species, crossing the cranial cavity. Xenopus lacks a tegmentum vasculosum and a contact membrane of the saccule; the Xenopus ear is further distinguished by a lateral passage separating stapes from periotic cistern and a more direct connection between periotic cistern and basilar recess. The basilar and lagenar recesses are conjoined in this species. Wever's diagram of the inner ear of Rana retains its value for diagrammatic purposes, but it is not anatomically accurate or representative of all frogs. Although Wever identified the contact membrane of the saccule, most recent studies of frog inner ear anatomy have overlooked both this and the amphibian tegmentum vasculosum. These structures deserve further attention.

Molecular cloning, characterization, and expression of pannexin genes in chicken

Pannexins (Panx) are a family of proteins that share sequences with the invertebrate gap junction proteins, innexins, and have a similar structure to that of the vertebrate gap junction proteins, connexins. To date, the Panx family consists of 3 members, but their genetic sequences have only been completely determined in a few vertebrate species. Moreover, expression of the Panx family has been reported in several rodent tissues: Panx1 is ubiquitously expressed in mammals, whereas Panx2 and Panx3 expressions are more restricted. Although members of the Panx family have been detected in mammals, their genetic sequences in avian species have not yet been fully elucidated. Here, we obtained the full-length mRNA sequences of chicken PANX genes and evaluated the homology of the amino acids from these sequences with those of other species. Furthermore, PANX gene expression in several chicken tissues was investigated based on mRNA levels. PANX1 was detected in the brain, cochlea, chondrocytes, eye, lung, skin, and intestine, and PANX2 was expressed in the brain, eye, and intestine. PANX3 was observed in the cochlea, chondrocytes, and bone. In addition, expression of PANX3 was higher than PANX1 in the cochlea. Immunofluorescent staining revealed PANX1 in hair cells, as well as the supporting cells, ganglion neurons, and the tegmentum vasculosum in chickens, whereas PANX3 was only detected in the bone surrounding the cochlea. Overall, the results of this study provide the first identification and characterization of the sequence and expression of the PANX family in an avian species, and fundamental data for confirmation of Panx function.

Regional expression of the ADAMs in developing chicken cochlea

The expression patterns of five members of the ADAM (a disintegrin and metalloprotease) family including ADAM9, ADAM10, ADAM17, ADAM22, and ADAM23 were analyzed in different anatomical structures of the developing chicken cochlea by in situ hybridization and immunohistochemistry. Results show that ADAM9, ADAM10, and ADAM17 are widely expressed in the sensory epithelium of the basilar papilla, by homogene cells, spindle-shaped cells, and acoustic ganglion cells, and in the tegmentum vasculosum, each with a different pattern. ADAM22 expression is restricted to spindle-shaped cells and acoustic ganglion cells, while ADAM23 is prominently expressed by hair cells and acoustic ganglion cells. Furthermore, ADAM10 protein is coexpressed with several members of the classic cadherins, including cadherin-7, N-cadherin, and R-cadherin in distinct anatomical regions of the cochlea except for acoustic ganglion cells. The expression of the ADAMs in the developing cochlea suggests a contribution of the ADAMs to the development of distinct cochlear structures.

Avian dark cells.

Dark cells (DCs) of mammalian and non-mammalian species help to maintain the homeostasis of the inner ear fluids in vivo. Although the avian cochlea is straight and the mammalian cochlea is coiled, no significant difference in the morphology and/or function of mammalian and avian DCs has been reported. The mammalian equivalent of avian DCs are marginal cells and are located in the stria vascularis along a bony sheet. Avian DCs hang free from the tegmentum vasculosum (TV) of the avian lagena between the perilymph and endolymph. Frame averaging was used to image the fluorescence emitted by several fluorochromes applied to freshly isolated dark cells (iDCs) from chickens (Gallus domesticus) inner ears. The viability of iDCs was monitored via trypan blue exclusion at each isolation step. Sodium Green, BCECF-AM, Rhodamine 123 and 9-anthroyl ouabain molecules were used to test iDC function. These fluorochromes label iDCs ionic transmembrane trafficking function, membrane electrogenic potentials and Na+/K+ ATPase pump's activity. Na+/K+ ATPase pump sites, were also evaluated by the p-nitrophenyl phosphatase reaction. These results suggest that iDCs remain viable for several hours after isolation without special culturing requirements and that the number and functional activity of Na+/K+ ATPase pumps in the iDCs were indistinguishable from in vivo DCs. Primary cultures of freshly iDCs were successfully maintained for 28 days in plastic dishes with RPMI 1640 culture medium. The preparation of iDCs overcomes the difficulty of DCs accessability in vivo and the unavoidable contamination that rupturing the inner ear microenvironments induces.

Na +,K +-ATPase activity and ultrastructural localization in the tegmentum vasculosum in the cochlea of the duckling

The tegmentum vasculosum of the avian cochlear duct mimics the stria vascularis of the mammalian cochlear duct in both location and structure, and previous studies indicate that it may be its functional counterpart with regard to endolymph synthesis. In the present study, we report on the enzymatic activity and ultrastructural localization of the Na +,K +-ATPase in the tegmentum vasculosum of the duckling. Na +,K +-ATPase activity was determined by measuring K +-dependent, ouabain-sensitive p-nitrophenyl phosphatase ( p-NPPase) activity in homogenates of dissected regions of the cochlear duct. The ultrastructural localization of the Na +,K +-ATPase was identified using K +-dependent, ouabain-sensitive, p-NPPase cytochemistry. Specific enzyme activity was localized primarily in homogenates of the tegmentum vasculosum (2.27 μmol p-nitrophenyl phosphate/mg protein/min) when compared to homogenates of the entire cochlear duct (0.69 μmol p-nitrophenyl phosphate/mg protein/min). Reaction product for p-NPPase was localized primarily along the basolateral plasma membrane folds of the dark cells. The cytochemical deposits appeared to be located exclusively on the cytoplasmic side of the plasma membrane. The light cells were devoid of reaction product. Biochemical and cytochemical localization of p-NPPase activity on the basolateral plasma membrane folds of the dark cells of the tegmentum vasculosum in conjunction with the ultrastructural morphology of these cells is compatible with a Na +,K +-ATPase-dependent ion transport function related to endolymph synthesis.

Ultrastructure and blood supply of the tegmentum vasculosum in the cochlea of the duckling

The tegmentum vasculosum of the duckling consists of a highly folded epithelium which extends over the dorsal and lateral walls of the cochlear duct, separating the scala media from the scala vestibuli. This epithelium consists of two distinct cell types, dark cells and light cells, and is well vascularized. The surface of the epithelium is formed by a mosaic of alternating dark and light cells. The goblet-shaped dark cells have an electron-dense, organelle-rich cytoplasm, and are expanded basally by extensive basolateral plasma membrane infoldings, within which are numerous mitochondria. Dark cells are isolated from each other and from the capillaries within the epithelium by intervening light cells. In contrast, columnar light cells exhibit an electron-lucent, organelle-poor cytoplasm and may extend from the underlying capillaries to the endolymphatic surface. Light cells contain abundant, coated endocytic vesicles on their apical surfaces and are bound, apically, to other light cells or to dark cells by tight junctions and desmosomes. Laterally, light cells are linked to each other either by complex, fluid-filled membrane interdigitations or by extensive gap junctions. Plasma membrane interdigitations and obvious, fluid-filled intercellular spaces characterize the lateral borders between light and dark cells. Vascular corrosion casting reveals the three-dimensional anatomy of the cochlear vasculature. A continuous arteriolar loop fed by anterior and posterior cochlear arterioles encircles the cochlear duct. The rich capillary beds of the tegmentum vasculosum are supplied by arching arterioles arising from this loop. These capillaries are the continuous type and are situated primarily within the core of the epithelium or along its border with the scala vestibuli. The structure and blood supply of the tegmentum vasculosum are characteristic of an epithelium involved in active transport.

Neither endocochlear potential nor Tegmentum vasculosum are affected in hearing impaired Belgian Waterslager canaries

We previously showed that the Belgian Waterslager canary strain is affected by a hereditary hearing loss that is associated with a reduced number of hair cells and hair cell pathologies in the basilar papilla. Since hair cell pathologies were also present in the sacculus, Weisleder et al. (1994) suggested that these birds are afflicted by Scheibe’s like dysplasia, a cochleo-saccular defect. In mammals, cochleo-saccular defects are characterized primarily by the lack of an endocochlear potential and abnormalities in the Stria vascularis which only secondarily lead to hair cell loss (Steel and Bock, 1983; Steel, 1994; 1995). Here we report the endocochlear potential of six ears from three non-Belgian Waterslager canaries and three ears of two Belgian Waterslager canaries to decide if Waterslager canaries are affected by a cochleo-saccular or by a neuroepithelial defect. The mean endocochlear potential was 17.6±2.5 mV in the non-Waterslager canaries and 20.3±0.6 mV in Waterslager canaries. In addition, and consistent with the presence of a normal endocochlear potential, light microscopy of the tegmentum vasculosum provided no evidence for pathology. These data show that Belgian Waterslager canaries are affected by a neuroepithelial rather than a cochleo-saccular inner ear defect.

Blood Supply and Function of the Tegmentum Vasculosum (Stria Vascularis) of the Duckling Cochlea

Abstract On the basis of ultrastructural, biochemical, cytochemical, and electro-physiological evidence, the stria vascularis, a highly vascular epithelium of the mammalian cochlea, has been implicated as one site of endolymph synthesis. It would seem natural to suspect that its highly developed counterpart in the avian cochlea, the tegmentum vasculosum (TV), might have a similar function. This study utilizes routine light and electron microscopy, biochemistry, cytochemistry, and vascular corrosion casting to describe the structure and blood supply to the (TV) in the duckling and provide evidence for its involvement in endolymph synthesis. Four to 16 week-old ducklings (Anas platyrhyncos)were anesthetized and anticoagulated and perfused via the common carotid arteries with buffered, 2% glutaraldehyde for routine light microscopy or transmission or scanning electron microscopy, or with Mercox resin for vascular corrosion casting. Casts were macerated in 5% KOH and warm water to remove tissue, cleaned in formic acid and water, dried by lyophilization, mounted on stubs and viewed at 10-20 kv by SEM.

Class III ?-tubulin expression in sensory and nonsensory regions of the developing avian inner ear

A previous study showed that class III beta-tubulin, a widely used neuron-specific marker, is expressed in mature and regenerating hair cells but not the support cells of the avian inner ear. We investigated the expression of this marker in the developing avian inner ear. We found that class III beta-tubulin is not neuron-specific in the avian embryo, but appears to accumulate in neuronal cell types, including hair cells, about the time of their differentiation. In the developing inner ear, some degree of class III beta-tubulin immunoreactivity is found in all regions of the otic epithelium from its formation as the otic placode (stage 10 [embryonic day, E1.5]) until about stage 21 (E3.5), when the prospective tegmentum vasculosum begins to lose its staining. By stage 35 (E8-9), most of the nonsensory epithelia have lost their class III beta-tubulin staining, leaving distinct regions of staining between the morphological compartments of the inner ear. Concurrent with the loss of staining from nonsensory regions, the hair cells of the sensory epithelia accumulate class III beta-tubulin, whereas the supporting cells decrease their staining. We also observed a similar pattern of development in another hair cell organ, the paratympanic organ. Double labeling with the 275 kD hair cell antigen (HCA) indicated that the majority of hair cells identifiable with class III beta-tubulin are HCA-positive. Additionally, presumptive hair cells were identified which were not within defined sensory epithelia. Our findings show that class III beta-tubulin can be used as an early marker for hair cell differentiation in all hair cell sensory epithelia in the chicken.

Effects of columella removal on inner ear morphology in the chick

We are currently removing the single middle ear bone (columella) in the domestic chick to introduce chemical agents directly into the inner ear. Since we are interested in the effect of these agents on neural structures within the avian basilar papilla (BP), we are concerned about any subtle changes that might result from the surgical procedure of columella removal alone. The purpose of this study was to use light and transmission electron microscopy to analyze morphological changes in the inner ear after columella removal. Fifteen-day-old chicks underwent a unilateral, bilateral or a sham removal of the columella. After columella removal, the oval window was either plugged with Gelfoam or Kimwipe (standard accepted procedure to prevent possible perilymph leak) or left uncovered. After a 5-day survival period, morphological changes were observed in the tegmentum vasculosum (TV) of all ears receiving a columella removal as compared to unoperated ears. Further, ears with Gelfoam plugging the oval window also had damage to the hair cells and support cells of the basilar papilla. In contrast, there were no observable differences in either auditory afferent or efferent nerve terminals on hair cells in the BP from any ears that had the columella removed compared to those from unoperated ears. These results suggest that columella removal alone may produce morphological changes to the TV within 5 days of surgery but not to structures within the BP. On the other hand, columella removal with a Gelfoam plug results in damage not only to the TV but also to cells within the basilar papilla during this same survival time. Despite damage to other structures within the inner ear, cochlear efferent and afferent terminals on surviving hair cells were unaffected by columella removal with or without plugging.

Molecular markers for cell types of the inner ear and candidate genes for hearing disorders.

To identify genes expressed in the vertebrate inner ear, we have established an assay that allows rapid analysis of the differential expression pattern of mRNAs derived from an auditory epithelium-specific cDNA library. We performed subtractive hybridization to create an enriched probe, which then was used to screen the cDNA library. After digoxigenin-labeled antisense cRNAs had been transcribed from hybridization-positive clones, we conducted in situ hybridization on slides bearing cryosections of late embryonic chicken heads, bodies, and cochleae. One hundred and twenty of the 196 clones analyzed encode 12 proteins whose mRNAs are specifically or highly expressed in the chicken's inner ear; the remainder encode proteins that occur more widely. We identified proteins that have been described previously as expressed in the inner ear, such as beta-tectorin, calbindin, and type II collagen. A second group of proteins abundant in the inner ear includes five additional types of collagens. A third group, including Coch-5B2 and an ear-specific connexin, comprises proteins whose human equivalents are candidates to account for hearing disorders. This group also includes proteins expressed in two unique cell types of the inner ear, homogene cells and cells of the tegmentum vasculosum.

Effects of kanamycin ototoxicity and hair cell regeneration on the DC endocochlear potential in adult chickens

High doses of aminoglycoside antibiotics cause massive damage to the avian basilar papilla. The resulting functional loss could conceivably arise from the reduction in the DC endocochlear potential (EP) due to impairment of the tegmentum vasculosum (TV) or to shunting of current through the damaged sensory epithelium. To test this hypothesis, the EP was measured in adult chickens after destroying hair cells in the basal half of the cochlea with a high dose (400 mg/kg per day for 10 days) of kanamycin (KM). KM treatment caused an increase in the steady-state EP from +18.1 to +23.3 mV and a decrease in the magnitude of the negative EP from −42.0 to −19.2 mV. The EP showed almost no change between 1 and 2 days and 1 week post-KM treatment. After 4 weeks of recovery, most hair cells had regenerated; however, the steady-state EP was still elevated by 13% and the negative EP was depressed by 37%. These results suggest that functional loss as shown by the large reduction in cochlear microphonic (CM) and the elevated thresholds of compound action potential (CAP) following KM treatment is not due to a reduction in the EP but may arise from functional deficits in the hair cells and/or the auditory nerve.

Recovery of noise-induced changes in the dark cells of the quail tegmentum vasculosum

Morphologic changes in the tegmentum vasculosum (TV) of adult quail after high intensity sound exposure were studied. Quail were continuously exposed to 115 dB SPL, 1500 Hz pure tone in a sound field for 12 h and either sacrificed immediately (0 day), 1, 2, 3, 4, 6 or 10 days later. Serial sections through the basilar papilla at 100μm intervals from base to apex were obtained for study with light microscopy and TEM. Significant morphologic changes were found within the TV of quail sacrificed on days 0–4. On a quantitative scale, the majority of recovery occurred within the first 24 h. After four days survival the tegmentum appeared nearly normal. This recovery correlates well with the temporal pattern of threshold shift recovery. These results demonstrate a temporal correlation between ultrastructural changes in the TV and functional recovery of hearing after intense sound exposure. A potential etiologic role of the TV in avian temporary threshold shift is suggested.

The effects of exposure to intense sound on the DC endocochlear potential in the chick

Chicks were exposed to an intense pure tone (0.9 kHz, 120 dB SPL) for 48 h. The DC endocochlear potential was measured with a microelectrode inserted into scala media in six separate groups of animals between 0 and 12 days post exposure. Similar data were obtained from seven groups unexposed control of chicks between 2 and 15 days of age. One to nine animals were tested in each control or exposed group. The results in the control chicks revealed that the EP at 2 days of age (6.7 mV) was 36% of the mature value (15.2 mV) noted at 6 days of age. In the exposed animals, at 0-days recovery, the EP showed a 63% reduction relative to that measured in age matched control chicks. The level of EP in the exposed animals quickly recovered, and by 3 and 6 days post exposure exhibited a loss of only 7 and 3 percent relative to the age-matched controls. The recovery of EP was plotted against the recovery of evoked potential thresholds reported by others, and the time-course of the recovery functions were very similar. This relationship suggested that recovery of auditory function in the chick was highly correlated with the restoration of the EP. Damage to the tegmentum vasculosum and its capacity to secrete potassium, as well as the shunting of current through the damaged basilar papilla, are considered as mechanisms for the reduction of EP loss in the exposed ear.

Distribution of gentamicin to the cochlea of the chicken embryo.

Aminoglycoside antibiotics are ototoxic in mammals and birds, including recently hatched chicks, but chicken embryos are insensitive to the ototoxicity of gentamicin, kanamycin, and streptomycin. To determine whether or not the insensitivity is due to a lack of antibiotic distribution to the avian cochlea, the distribution of gentamicin to the cochlea of the White Leghorn chicken embryo was compared to the distribution to the cochlea of the recently hatched White Leghorn chick. Fertile eggs were injected with a maximally tolerated dose of gentamicin sulfate (0.1 mg/egg/day) on incubation days 10-18, and the chicks were injected subcutaneously with either 5 mg (non-ototoxic) or 100 mg (ototoxic) gentamicin sulfate/kg body weight on days 1-9 after hatching. Gentamicin sulfate was histochemically detected within the basilar papilla (the avian equivalent of the organ of Corti) in all treated chicken embryos and chicks by 1 day after the first injection, and the staining was intense after 3 days of treatment. By ultrastructural immunocytochemistry, mild, diffuse labeling for gentamicin sulfate was detected within the endoplasmic reticulum of short and tall hair cells of chicken embryos by incubation day 17. Moderate labeling of gentamicin sulfate was detected in the infracuticular region of lysosomes of hair cells in chicks receiving 5 treatments of gentamicin sulfate at 5.0 mg/kg body weight and after 1 treatment of gentamicin sulfate at 100 mg/kg body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

The potassium concentration in the cochlear fluids of the embryonic and post-hatching chick

The potassium concentration was measured in the perilymphatic and endolymphatic spaces of the chick's cochlea using ion-specific microelectrodes. The mean values in post-hatching chicks were 8.1 and 161 mM/l, respectively and are thus the same as in mammals. The high potassium concentration is reached at the latest at stage E42, and thus before the tegmentum vasculosum is fully developed. The endocochlear potential of chicks was maximally +13 mV and thus somewhat below the published values for birds. Positive potentials in the endolymphatic space of 5 mV or more were already present in animals of age E41 and E42 and the developmental process is complete at the latest one day after hatching.

Retinoic acid modulation of the early development of the inner ear is associated with the control of c-fos expression

The effects of retinoic acid (RA) on the early development of the inner ear were studied in vitro using isolated chick embryo vesicles. Low concentrations of RA (1-50 nM) inhibited vesicular growth in stage 18 otic vesicles that were made quiescent and then reactivated by either serum or bombesin. Growth inhibition was concentration-dependent and was paralleled by a reduction in the rate of DNA synthesis as measured by [3H]thymidine incorporation. Half-inhibition occurred between 1 and 10 nM RA, and the full effect at 20 nM. Retinoic acid, in the presence of serum, induced the precocious differentiation of (1) secretory epithelium, the tegmentum vasculosum and endolymphatic sac and (2) early sensory and supporting epithelia. These structures were positioned in their corresponding normal presumptive areas. The overall direction of growth was reversed by RA and the ratio of the internal to the external vesicular surface area increased with RA concentration. The expression of the nuclear proto-oncogene c-fos in the developing otic vesicle was transient and stage-dependent. High levels of c-fos mRNA were positively correlated with cell proliferation. Incubation of growth-arrested otic vesicles with bombesin plus insulin at concentrations that induced cell proliferation produced a strong induction of c-fos. This mitogen-induced expression was suppressed by 25 nM RA. The results suggest (1) a role for retinoic acid in controlling the early development of the inner ear and (2) that this control is effected through the regulation of the proto-oncogene c-fos.