Role of hindbrain in inner ear morphogenesis: analysis of Noggin knockout mice

Signaling from rhombomeres 5 and 6 of the hindbrain is thought to be important for inner ear patterning. In Noggin −/− embryos, the gross anatomy of the inner ear is distorted and malformed, with cochlear duct outgrowth and coiling most affected. We attributed these defects to a caudal shift of the rhombomeres caused by the shortened body axis and the kink in the neural tube. To test the hypothesis that a caudal shift of the rhombomeres affects inner ear development, we surgically generated chicken embryos in which rhombomeres 5 and 6 were similarly shifted relative to the position of the inner ears, as in Noggin mutants. All chicken embryos with shifted rhombomeres showed defects in cochlear duct formation indicating that signaling from rhombomeres 5 and 6 is important for cochlear duct patterning in both chicken and mice. In addition, the size of the otic capsule is increased in Noggin −/− mutants, which most likely is due to unopposed BMP signaling for chondrogenesis in the peri-otic mesenchyme.     

Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions

The mammalian inner ear comprises the cochleovestibular labyrinth, derived from the ectodermal otic placode, and the encasing bony labyrinth of the temporal bone. Epithelial-mesenchymal interactions are thought to control inner ear development, but the modes and the molecules involved are largely unresolved. We show here that, during the precartilage and cartilage stages, Fgf9 is expressed in specific nonsensory domains of the otic epithelium and its receptors, Fgfr1(IIIc) and Fgfr2(IIIc), widely in the surrounding mesenchyme. To address the role of Fgf9 signaling, we analyzed the inner ears of mice homozygous for Fgf9 null alleles. Fgf9 inactivation leads to a hypoplastic vestibular component of the otic capsule and to the absence of the epithelial semicircular ducts. Reduced proliferation of the prechondrogenic mesenchyme was found to underlie capsular hypoplasticity. Semicircular duct development is blocked at the initial stages, since fusion plates do not form. Our results show that the mesenchyme directs fusion plate formation and they give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. In addition to the vestibule, in the cochlea, Fgf9 mutation caused defects in the interactions between the Reissner's membrane and the mesenchymal cells, leading to a malformed scala vestibuli. Together, these data show that Fgf9 signaling is required for inner ear morphogenesis.     

Distinct roles for hindbrain and paraxial mesoderm in the induction and patterning of the inner ear revealed by a study of vitamin-A-deficient quail

The hindbrain and cranial paraxial mesoderm have been implicated in the induction and patterning of the inner ear, but the precise role of the two tissues in these processes is still not clear. We have addressed these questions using the vitamin-A-deficient (VAD) quail model, in which VAD embryos lack the posterior half of the hindbrain that normally lies next to the inner ear. Using a battery of molecular markers, we show that the anlagen of the inner ear, the otic placode, is induced in VAD embryos in the absence of the posterior hindbrain. By performing grafting and ablation experiments in chick embryos, we also show that cranial paraxial mesoderm which normally lies beneath the presumptive otic placode is necessary for otic placode induction and that paraxial mesoderm from other locations cannot induce the otic placode. Two members of the fibroblast growth factor family, FGF3 and FGF19, continue to be expressed in this mesodermal population in VAD embryos, and these may be responsible for otic placode induction in the absence of the posterior hindbrain. Although the posterior hindbrain is not required for otic placode induction in VAD embryos, the subsequent patterning of the inner ear is severely disrupted. Several regional markers of the inner ear, such as Pax2, EphA4, SOHo1 and Wnt3a, are incorrectly expressed in VAD otocysts, and the sensory patches and vestibulo-acoustic ganglia are either greatly reduced or absent. Exogenous application of retinoic acid prior to 30 h of development is able rescue the VAD phenotype. By performing such rescue experiments before and after 30 h of development, we show that the inner ear defects of VAD embryos correlate with the absence of the posterior hindbrain. These results show that induction and patterning of the inner ear are governed by separate developmental processes that can be experimentally uncoupled from each other.     

Expression of myosin VIIA in the developing chick inner ear neurons

The auditory-vestibular ganglion (AVG) is formed by the division of otic placode-derived neuroblasts, which then differentiate into auditory and vestibular afferent neurons. The developmental mechanisms that regulate neuronal cell fate determination, axonal pathfinding and innervation of otic neurons are poorly understood. The present study characterized the expression of myosin VIIA, along with the neuronal markers, Islet1, NeuroD1 and TuJ1, in the developing avian ear, during Hamburger–Hamilton (HH) stages 16–40. At early stages, when neuroblasts are delaminating from the otic epithelium, myosin VIIA expression was not observed. Myosin VIIA was initially detected in a subset of neurons during the early phase of neuronal differentiation (HH stage 20). As the AVG segregates into the auditory and vestibular portions, myosin VIIA was restricted to a subset of vestibular neurons, but was not present in auditory neurons. Myosin VIIA expression in the vestibular ganglion was maintained through HH stage 33 and was downregulated by stage 36. Myosin VIIA was also observed in the migrating processes of vestibular afferents as they begin to innervate the otic epithelium HH stage 22/23. Notably, afferents targeting hair cells of the cristae were positive for myosin VIIA while afferents targeting the utricular and saccular maculae were negative (HH stage 26–28). Although previous studies have reported that myosin VIIA is restricted to sensory hair cells, our data shows that myosin VIIA is also expressed in neurons of the developing chick ear. Our study suggests a possible role for myosin VIIA in axonal migration/pathfinding and/or innervation of vestibular afferents. In addition, myosin VIIA could be used as an early marker for vestibular neurons during the development of the avian AVG.     

Mechanics of the inner ear of the bullfrog (Rana catesbeiana): the contact membranes and the periotic canal

The frog inner ear consists of a complex of fluid-filled membranous sacs and canals containing eight distinct clusters of sensory hair cells. In this study we attempt to delineate the potential pathways for acoustic energy flow toward two of these clusters located within the amphibian papilla and the basilar papilla. Detailed morphological measurements of the periotic canal based on internal casts of the inner ear in the bullfrog (Rana catesbeiana) revealed that it is divided into a wide, tapered section and a narrower section comprised of two branches – one short and blind projecting into the endolymphatic space and another longer, terminating in the round window. Additionally, we used laser Doppler velocimetry to record the velocity responses of the contact membranes of the amphibian papilla and basilar papilla. We found that the acoustic energy flow through these two structures is frequency dependent such that the amphibian papilla contact membrane displays a peak velocity amplitude at frequencies less than 500 Hz, whereas the basilar papilla contact membrane velocity response exhibits a maximum above 1100 Hz. Our data advocate a mechanical substrate underlying the frequency segregation in the auditory nerve fibers innervating the amphibian papilla and the basilar papilla. Accepted: 9 March 2000     

Hoxa1 lineage tracing indicates a direct role for Hoxa1 in the development of the inner ear, the heart, and the third rhombomere

Loss of Hoxa1 function results in severe defects of the brainstem, inner ear, and cranial ganglia in humans and mice as well as cardiovascular abnormalities in humans. Because Hoxa1 is expressed very transiently during an early embryonic stage, it has been difficult to determine whether Hoxa1 plays a direct role in the precursors of the affected organs or if all defects result from indirect effects due to mispatterning of the hindbrain. In this study we use a Hoxa1-IRES-Cre mouse to genetically label the early Hoxa1-expressing cells and determine their contribution to each of the affected organs, allowing us to conclude in which precursor tissue Hoxa1 is expressed. We found Hoxa1 lineage-labeled cells in all tissues expected to be derived from the Hoxa1 domain, such as the facial and abducens nuclei and nerves as well as r4 neural crest cells. In addition, we detected the lineage in derivatives that were not thought to have expressed Hoxa1 during development. In the brainstem, the anterior border of the lineage was found to be in r3, which is more anterior than previously reported. We also observed an interesting pattern of the lineage in the inner ear, namely a strong contribution to the otic epithelium with the exception of sensory patches. Moreover, lineage-labeled cells were detected in the atria and outflow tract of the developing heart. In conclusion, Hoxa1 lineage tracing uncovered new domains of Hoxa1 expression in rhombomere 3, the otic epithelium, and cardiac precursors, suggesting a more direct role for Hoxa1 in development of these tissues than previously believed.     

Effects of streptomycin and amiloride on the function of the pigeon inner ear

In vitro, streptomycin and amiloride block the mechano-electrical transduction channels in isolated auditory hair cells of mammals and birds. The present in vivo experiments investigate their action in the intact pigeon inner ear. Streptomycin dissolved in artificial endolymph at concentrations between 1.0 and 6.2 mmol/l was applied into the scala media. After a bolus injection (0.09 to 0.36 l) the endocochlear potential (EP) was elevated from 6.8 to 7.9 mV. Similarly applied boli of 0.09 to 0.55 l of amiloride in concentrations of 1–2 mmol/l did not significantly elevate EP (8.7 versus 9.4 mV). Compound action potential (CAP) frequency threshold curves (FTCs) were elevated by both streptomycin and amiloride. The threshold elevation increased with frequency. With streptomycin injection, a complete loss of CAPs was found at frequencies above 1.2 kHz. With amiloride, the loss in sensitivity was 40 dB for the same frequency range. Single fibre recordings showed elevated thresholds and decreased sharpness of tuning with both drugs. With streptomycin, the evoked activity was affected regard-less of stimulation frequencies (above or below the fibre's characteristic frequency). Amiloride, however, preferentially elevated thresholds at frequencies above characteristic frequency. Thus amiloride seems to interfere with hair cell tuning mechanisms. Spontaneous activity decreased by about 30%, following the application of streptomycin. In contrast, injections of amiloride into the scala media were followed by an increase in spontaneous discharge rates.When the drugs were applied into scala tympani, streptomycin reduced CAP amplitudes within 2 min. Amiloride produced no effect if applied into the scala tympani.Abbreviations EP endocochlear potential - CAP compound action potential - FTC frequency threshold curve - CF characteristic frequency - ACh acetylcholin - ECG electro-cardiogram - CM cochlear microphonic - SPL sound pressure level     

Aescin and troxerutin as a successful combination for the treatment of inner ear perfusion disturbances

A fixed combination of aescin and troxerutin has been developed for treating inner ear perfusion problems of different aetiology. The efficacy of this combination is tested versus pentoxyfyllin in a randomized clinical study as group comparison with 34 patients for each group. The improvement of hearing after 40–44 days of treatment is determined as end point of treatment. Hearing was measured by threshold, whereby a difference of more than 10 dB is judged as a significant improvement. After the treatment with the combination of aescin and troxerutin hearing is significantly improved, in 23 of 34 patients the threshold is changed more than 10 dB, which is checked by sign-test with p<0.05. With pentoxyfyllin hearing is also improved, although to a lesser degree. Both drugs are well tolerated, major adverse drug effects are not observed with either treatment.     

Forced activation of Wnt signaling alters morphogenesis and sensory organ identity in the chicken inner ear

Components of the Wnt signaling pathway are expressed in the developing inner ear. To explore their role in ear patterning, we used retroviral gene transfer to force the expression of an activated form of beta-catenin that should constitutively activate targets of the canonical Wnt signaling pathway. At embryonic day 9 (E9) and beyond, morphological defects were apparent in the otic capsule and the membranous labyrinth, including ectopic and fused sensory patches. Most notably, the basilar papilla, an auditory organ, contained infected sensory patches with a vestibular phenotype. Vestibular identity was based on: (1) stereociliary bundle morphology; (2) spacing of hair cells and supporting cells; (3) the presence of otoliths; (4) immunolabeling indicative of vestibular supporting cells; and (5) expression of Msx1, a marker of certain vestibular sensory organs. Retrovirus-mediated misexpression of Wnt3a also gave rise to ectopic vestibular patches in the cochlear duct. In situ hybridization revealed that genes for three Frizzled receptors, c-Fz1, c-Fz7, and c-Fz10, are expressed in and adjacent to sensory primordia, while Wnt4 is expressed in adjacent, nonsensory regions of the cochlear duct. We hypothesize that Wnt/beta-catenin signaling specifies otic epithelium as macular and helps to define and maintain sensory/nonsensory boundaries in the cochlear duct.     

Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

Emx2 and early hair cell development in the mouse inner ear

Emx2 is a homeodomain protein that plays a critical role in inner ear development. Homozygous null mice die at birth with a range of defects in the CNS, renal system and skeleton. The cochlea is shorter than normal with about 60% fewer auditory hair cells. It appears to lack outer hair cells and some supporting cells are either absent or fail to differentiate. Many of the hair cells differentiate in pairs and although their hair bundles develop normally their planar cell polarity is compromised. Measurements of cell polarity suggest that classic planar cell polarity molecules are not directly influenced by Emx2 and that polarity is compromised by developmental defects in the sensory precursor population or by defects in epithelial cues for cell alignment. Planar cell polarity is normal in the vestibular epithelia although polarity reversal across the striola is absent in both the utricular and saccular maculae. In contrast, cochlear hair cell polarity is disorganized. The expression domain for Bmp4 is expanded and Fgfr1 and Prox1 are expressed in fewer cells in the cochlear sensory epithelium of Emx2 null mice. We conclude that Emx2 regulates early developmental events that balance cell proliferation and differentiation in the sensory precursor population.     

Primary culture of capillary endothelial cells from the spiral ligament and stria vascularis of bovine inner ear

Summary Methods for isolation and culture of microvascular endothelial cells of the inner ear were devised to provide an in-vitro system for studying endothelial functions in this tissue. Capillaries from the stria vascularis and spiral ligament were treated enzymatically to free them from surrounding tissue. Contamination by extraneous tissue was minimized by banding capillary segments in Percoll gradients and culture in plasma-derived serum on a fibronectin-coated substrate. Although only small amounts of inner ear tissue were available, tritiated thymidine autoradiography demonstrated that considerable growth in culture was possible. Addition of heparin and endothelial cell growth supplement to the medium enhanced proliferation. The endothelial origin of the cultured cells was confirmed by immunofluorescent demonstration of the presence of Factor VIII-related antigen and angiotensin-converting enzyme. In addition, tight junctions between cells were observed in both thin sections and platinum replicas obtained by freezefracture techniques. Endothelial cells from neither the stria vascularis nor the spiral ligament allowed passage of horseradish peroxidase across the monolayer during a 5-min period. However, endothelial cells from the stria vascularis exhibited a greater amount of pinocytotic activity than those of the spiral ligament, a difference that is also observed in vivo. Methods for expanding a small population of endothelial cells with retention of specialized properties into one of sufficient size for morphologic and biochemical studies have been demonstrated for the inner ear.     

Tropomyosin co-localizes with actin microfilaments and microtubules within supporting cells of the inner ear

Summary The distribution of tropomyosin, actin and tubulin in the supporting cells of the organ of Corti was studied by immunofluorescent localization of antibodies to these proteins. Tropomyosin colocalizes with actin and tubulin in the regions of the tunnel pillar and Deiters cells where actin microfilaments and microtubules had previously been observed ultrastructurally. Despite the implications of the presence of antiparallel actin filaments in the supporting cells, the presence of tropomyosin and the absence of myosin suggest that the role of tropomyosin may be to confer rigidity to the actin filaments. Thus the primary function of the cytoskeletal proteins in the supporting cells may be structural.