Foxg1 is required for proper separation and formation of sensory cristae during inner ear development

The vestibular portion of the inner ear, the three semicircular canals and their sensory cristae, is responsible for detecting angular head movements. It was proposed that sensory cristae induce formation of their non‐sensory components, the semicircular canals. Here, we analyzed the inner ears of Foxg1^?/? mouse mutants, which display vestibular defects that are in conflict with the above model. In Foxg1^?/? ears, the lateral canal is present without the lateral ampulla, which houses the lateral crista. Our gene expression analyses indicate that at the time when canal specification is thought to occur, the prospective lateral crista is present, which could have induced lateral canal formation prior to its demise. Our genetic fate‐mapping analyses indicate an improper separation between anterior and lateral cristae in Foxg1^?/? mutants. Our data further suggest that a function of Foxg1 in the inner ear is to restrict sensory fate. Developmental Dynamics 238:2725–2734, 2009. © 2009 Wiley‐Liss, Inc.     

Expression and function of FGF10 in mammalian inner ear development.

We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10's main receptors.     

Atoh1 null mice show directed afferent fiber growth to undifferentiated ear sensory epithelia followed by incomplete fiber retention.

Inner ear hair cells have been suggested as attractors for growing afferent fibers, possibly through the release of the neurotrophin brain-derived neurotrophic factor (BDNF). Atoh1 null mice never fully differentiate hair cells and supporting cells and, therefore, may show aberrations in the growth and/or retention of their innervation. We investigated the distribution of cells positive for Atoh1- or Bdnf-mediated beta-galactosidase expression in Atoh1 null and Atoh1 heterozygotic mice and correlated the distribution of these cells with their innervation. Embryonic day (E) 18.5 Atoh1 null and heterozygotic littermates show Atoh1- and BDNF-beta-galactosidase-positive cells in comparable distributions in the canal cristae and the cochlea apex. Atoh1-beta-galactosidase-positive but only occasional Bdnf-beta-galactosidase-positive cells are found in the utricle, saccule, and cochlea base of Atoh1 null mutant mice. Absence of Bdnf-beta-galactosidase expression in the utricle and saccule of Atoh1 null mice is first noted at E12.5, a time when Atoh1-beta-galactosidase expression is also first detected in these epithelia. These data suggest that expression of Bdnf is dependent on ATOH1 protein in some but does not require ATOH1 protein in other inner ear cells. Overall, the undifferentiated Atoh1- and Bdnf-beta-galactosidase-positive cells show a distribution reminiscent of that in the six sensory epithelia in control mice, suggesting that ear patterning processes can form discrete patches of Atoh1 and Bdnf expression in the absence of ATOH1 protein. The almost normal growth of afferent and efferent fibers in younger embryos suggests that neither fully differentiated hair cells nor BDNF are necessary for the initial targeted growth of fibers. E18.5 Atoh1 null mice have many afferent fibers to the apex of the cochlea, the anterior and the posterior crista, all areas with numerous Bdnf-beta-galactosidase-positive cells. Few fibers remain to the saccule, utricle, and the base of the cochlea, all areas with few or no Bdnf-beta-galactosidase-positive cells. Thus, retention of fibers is possible with BDNF, even in the absence of differentiated hair cells.     

Tissue-specific roles of Tbx1 in the development of the outer, middle and inner ear, defective in 22q11DS patients

Most 22q11.2 deletion syndrome (22q11DS) patients have middle and outer ear anomalies, whereas some have inner ear malformations. Tbx1, a gene hemizygously deleted in 22q11DS patients and required for ear development, is expressed in multiple tissues during embryogenesis. To determine the role of Tbx1 in the first pharyngeal pouch (PPI) in forming outer and middle ears, we tissue-specifically inactivated the gene using Foxg1-Cre. In the conditional mutants, PPI failed to outgrow, preventing the middle ear bone condensations from forming. Tbx1 was also inactivated in the otic vesicle (OV), resulting in the failure of inner ear sensory organ formation, and in duplication of the cochleovestibular ganglion (CVG). Consistent with the anatomical defects, the sensory genes, Otx1 and Bmp4 were downregulated, whereas the CVG genes, Fgf3 and NeuroD, were upregulated. To delineate Tbx1 cell-autonomous roles, a more selective ablation, exclusively in the OV, was performed using Pax2-Cre. In contrast to the Foxg1-Cre mutants, Pax2-Cre conditional mutant mice survived to adulthood and had normal outer and middle ears but had the same inner ear defects as the Tbx1 null mice, with the same gene expression changes. These results demonstrate that Tbx1 has non-cell autonomous roles in PPI in the formation of outer and middle ears and cell-autonomous roles in the OV. Periotic mesenchymal markers, Prx2 and Brn4 were normal in both conditional mutants, whereas they were diminished in Tbx1-/- embryos. Thus, Tbx1 in the surrounding mesenchyme in both sets of conditional mutants cannot suppress the defects in the OV that occur in the null mutants.     

Restricted expression of Fgf16 within the developing chick inner ear.

Fibroblast growth factor (FGF) signaling is required for otic placode induction and patterning of the developing inner ear. We have cloned the chick ortholog of Fgf16 and analyzed its expression pattern in the early chick embryo. Expression is restricted to the otic placode and developing inner ear through all the stages examined. By the closed otocyst stage, expression has resolved to anterior and posterior domains that partially overlap with those of bone morphogenetic protein 4 (Bmp4), a marker of the developing sensory patches, the cristae of the anterior and posterior semicircular canals. Platelet-derived growth factor alpha (PDGFA), another growth factor with restricted otic expression, also overlaps with Fgf16 expression. The restricted expression pattern of Fgf16 suggests a role for FGF signaling in the patterning of the sensory cristae, together with BMP signaling.     

Comparative assessment of Fgf's diverse roles in inner ear development: A zebrafish perspective

Progress in understanding mechanisms of inner ear development has been remarkably rapid in recent years. The research community has benefited from the availability of several diverse model organisms, including zebrafish, chick, and mouse. The complexity of the inner ear has proven to be a challenge, and the complexity of the mammalian cochlea in particular has been the subject of intense scrutiny. Zebrafish lack a cochlea and exhibit a number of other differences from amniote species, hence they are sometimes seen as less relevant for inner ear studies. However, accumulating evidence shows that underlying cellular and molecular mechanisms are often highly conserved. As a case in point, consideration of the diverse functions of Fgf and its downstream effectors reveals many similarities between vertebrate species, allowing meaningful comparisons the can benefit the entire research community. In this review, I will discuss mechanisms by which Fgf controls key events in early otic development in zebrafish and provide direct comparisons with chick and mouse.     

The organogenesis of the membranous labyrinth in Polypterus senegalus Cuvier, 1829 (Pisces, Holostei, Polypteridae)

The organogenesis of the membranous labyrinth of Polypterus senegalus is described. 1. The otocyst seems to be formed by the invagination of a thick portion of the deeper layer of the epidermis. 2. The 3 semi-circular canals are formed by 3 pairs of invaginations of the wall of the otocyst and at the expense of its volume; the anterior vertical canal is completed first, followed by the horizontal and then the posterior vertical one; the ampullae, not very developed, appear a long time after the formation of their corresponding canals: that of the horizontal canal appears first, followed by that of the posterior and finally that of the anterior one. 3. The sensory areas derive from a common macula which subsequently divides into 2 zones, the anterior one giving rise to the utricular macula and the anterior and horizontal cristae, the posterior one giving rise to the posterior crista and the saccular macula; from the latter subsequently develops the lagenar macula. 4. The otoconiae appear as soon as the otocyst forms; the otoliths are agglomerations of otoconias brought together by an interstitial cement. 5. The endolymphatic primordium is formed before that of the semi-circular canals; the endolymphatic sack becomes voluminous and spreads over the brain as far as the sagittal plane.     

Inner ear formation during the early larval development of Xenopus laevis.

The formation of the eight independent endorgan compartments (sacculus, utricle, horizontal canal, anterior canal, posterior canal, lagena, amphibian papilla, and basilar papilla) of the Xenopus laevis inner ear is illustrated as the otic vesicle develops into a complex labyrinthine structure. The morphology of transverse sections and whole-mounts of the inner ear was assessed in seven developmental stages (28, 31, 37, 42, 45, 47, 50) using brightfield and laser scanning confocal microscopy. The presence of mechanosensory hair cells in the sensory epithelia was determined by identification of stereociliary bundles in cryosectioned tissue and whole-mounts of the inner ear labeled with the fluorescent F-actin probe Alexa-488 phalloidin. Between stages 28 and 45, the otic vesicle grows in size, stereociliary bundles appear and increase in number, and the pars inferior and pars superior become visible. The initial formation of vestibular compartments with their nascent stereociliary bundles is seen by larval stage 47, and all eight vestibular and auditory compartments with their characteristic sensory fields are present by larval stage 50. Thus, in Xenopus, inner ear compartments are established between stages 45 and 50, a 2-week period during which the ear quadruples in length in the anteroposterior dimension. The anatomical images presented here demonstrate the morphological changes that occur as the otic vesicle forms the auditory and vestibular endorgans of the inner ear. These images provide a resource for investigations of gene expression patterns in Xenopus during inner ear compartmentalization and morphogenesis.     

Mutational ataxia resulting from abnormal vestibular acquisition and processing is partially compensated for

Due to the multisensory input into the balance system, the loss of one input, such as an ear, can generally be compensated for. However, when a mismatch or incomplete loss of inputs occurs, the ability to compensate for the stimulus misrepresentation may be compromised. The inner ear and cerebellum are important input and processing centers for balance but no genetic models have been generated to assess balance or compensation in the abnormal development of both these organs/brain areas. Important to their formation is regulation of proliferation mediated by the proto-oncogene N-Myc. Conditional knockouts (CKOs) of N-Myc using Tg(Pax2-Cre) have a misshapen and smaller ear with a fused utricle, saccule, and cochlea and absent horizontal canal, aberrant cochlear and vestibular innervations, and a size reduction in the cerebellum. CKOs are viable with obvious behavioral deficits, including circling behavior and unstable gait. To test the degree of ataxia and possible compensation of vestibular defects in these mutant mice, we use the Noldus Catwalk System to assess the gait of Tg(Pax2-Cre) N-Myc CKOs over five months. N-Myc CKOs perform worse than control littermates, in particular, in step regularity. We show that disrupting one member of the Myc family during embryonic development coincides with a differential loss of function in the cochlea compared to the vestibular apparatus. In addition, we show that the distortion in the ear morphology combined with a reduction of the cerebellum, rather than a complete loss of the vestibular-cerebellar pathway, leads to partial behavioral compensation that remains unchanged over time.     

Capsaicin stimulation of the cochlea and electric stimulation of the trigeminal ganglion mediate vascular permeability in cochlear and vertebro-basilar arteries: a potential cause of inner ear dysfunction in headache

Trigeminal neurogenic inflammation is one explanation for the development of vascular migraine. The triggers for this inflammation and pain are not well understood, but are probably vasoactive components acting on the blood vessel wall. Migraine-related inner ear symptoms like phonophobia, tinnitus, fluctuation in hearing perception and increased noise sensitivity provide indirect evidence that cochlear blood vessels are also affected by basilar artery migraine. The purpose of this investigation was to determine if a functional connection exists between the cochlea and the basilar artery. Neuronally mediated permeability changes in the cochlea and basilar artery were measured by colloidal silver and Evans Blue extravasation, following orthodromic and antidromic stimulation of the trigeminal ganglion innervating the cochlea. Capsaicin and electrical stimulation induced both dose- and time-dependent plasma extravasation of colloidal silver and Evans Blue from the basilar artery and anterior inferior cerebellar artery. Both orthodromic and antidromic activation of trigeminal sensory fibers also induced cochlear vascular permeability changes and significant quantitative differences between the treated and control groups in spectrophotometric assays.These results characterize a vasoactive connection between the cochlea and vertebro-basilar system through the trigeminal sensory neurons. We propose that vertigo, tinnitus and hearing deficits associated with basilar migraine could arise by excitation of the trigeminal nerve fibers in the cochlea, resulting in local plasma extravasation. In addition, cochlear "dysfunction" may also trigger basilar and cluster headache by afferent input to the trigeminal system.     

Regional regulation of palatal growth and patterning along the anterior-posterior axis in mice

Cleft palate is a congenital disorder arising from a failure in the multistep process of palate development. In its mildest form the cleft affects only the posterior soft palate. In more severe cases the cleft includes the soft (posterior) and hard (anterior) palate. In mice a number of genes show differential expression along the anterior-posterior axis of the palate. Mesenchymal heterogeneity is established early, as evident from Bmp4-mediated induction of Msx1 and cell proliferation exclusively in the anterior and Fgf8-specific induction of Pax9 in the posterior palate alone. In addition, the anterior palatal epithelium has the unique ability to induce Shox2 expression in the anterior mesenchyme in vivo and the posterior mesenchyme in vitro. Therefore, the induction and competence potentials of the epithelium and mesenchyme in the anterior are clearly distinct from those in the posterior. Defective growth in the anterior palate of Msx1-/- and Fgf10-/- mice leads to a complete cleft palate and supports the anterior-to-posterior direction of palatal closure. By contrast, the Shox2-/- mice exhibit incomplete clefts in the anterior presumptive hard palate with an intact posterior palate. This phenotype cannot be explained by the prevailing model of palatal closure. The ability of the posterior palate to fuse independent of the anterior palate in Shox2-/- mice underscores the intrinsic differences along the anterior-posterior axis of the palate. We must hitherto consider the heterogeneity of gene expression and function in the palate to understand better the aetiology and pathogenesis of non-syndromic cleft palate and the mechanics of normal palatogenesis.     

人内耳半规管和壶腹嵴的组织发生

目的：较全面地了解人内耳半规管和壶腹嵴的组织发生。方法：取人胚胎内耳，光镜和电镜下观察。结果：第6w胚胎半规管上皮为单层柱状，第7w上皮增至3～4层，第9w为单层立方上皮；第11w后，立方上皮逐渐变矮，第23w膜半规管为单层扁平上皮，第31w半规管的扁平细胞内可见线粒体、粗面内质网和吞饮小泡等结构。第6w胚胎壶腹嵴已出现，由两层细胞组成，第7w嵴上皮分裂分化为4～5层细胞，第8w表层柱状细胞出现纤毛，第13w毛细胞和支持细胞分化明显；第15w胶质膜呈帽状，它由许多平行排列的纵行小管组成；第18w毛细胞可分辨出Ⅰ型和Ⅱ型毛细胞。结论：补充了半规管组织发生的新资料。     

Kanamycin ototoxicity in glutamate transporter knockout mice

Glutamate-aspartate transporter (GLAST), a powerful glutamate uptake system, removes released glutamate from the synaptic cleft and facilitates the re-use of glutamate as a neurotransmitter recycling system. Aminoglycoside-induced hearing loss is mediated via a glutamate excitotoxic process. We investigated the effect of aminoglycoside ototoxicity in GLAST knockout mice using the recorded auditory brainstem response (ABR) and number of hair cells in the cochlea. Kanamycin (100 mg/mL) was injected directly into the posterior semicircular canal of mice. Before the kanamycin treatment, there was no difference in the ABR threshold average between the wild-type and knockout mice. Kanamycin injection aggravated the ABR threshold in the GLAST knockout mice compared with the wild-type mice, and the IHC degeneration was more severe in the GLAST knockout mice. These findings suggest that GLAST plays an important role in preventing the degeneration of inner hair cells in aminoglycoside ototoxicity.     

Ontogenetic Variation in the Bony Labyrinth of Monodelphis domestica (Mammalia: Marsupialia) Following Ossification of the Inner Ear Cavities

Ontogeny, or the development of an individual from conception to death, is a major source of variation in vertebrate morphology. All anatomical systems are affected by ontogeny, and knowledge of the ontogenetic history of these systems is important to understand when formulating biological interpretations of evolutionary history and physiology. The present study is focused on how variation affects the bony labyrinth across a growth series of an extant mammal after ossification of the inner ear chambers. Digital endocasts of the bony labyrinth were constructed using CT data across an ontogenetic sequence of Monodelphis domestica, an important experimental animal. Various aspects of the labyrinth were measured, including angles between the semicircular canals, number of turns of the cochlea, volumes of inner ear constituents, as well as linear dimensions of semicircular canals. There is a strong correlation between skull length and age, but from 27 days after birth onward, there is no correlation with age among most of the inner ear measurements. Exceptions are the height of the arc of the lateral semicircular canal, the angular deviation of the lateral canal from planarity, the length of the slender portion of the posterior semicircular canal, and the length of the canaliculus cochleae. Adult dimensions of several of the inner ear structures, such as the arcs of the semicircular canals, are achieved before the inner ear is functional, and the non‐ontogenetic variation in the bony labyrinth serves as an important source for behavioral, physiological, and possibly phylogenetic information. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Physiologic characteristics of vestibular first-order canal neurons in the cat. I. Response plane determination and resting discharge characteristics.

The response plane and resting rate characteristics of first-order, vestibular, semicircular canal neurons were studied in 67 cats under sodium pentobarbital anesthesia using single-unit recording techniques in the eighth nerve. Five hundred fifty-nine units were classified as to the canal they were associated with by employing an identification technique based on physiologic response patterns to brief, high-level (up to 250 degrees/S2) angular accelerations delivered in various head positions. All horizontal canal neurons had increased firing rates to ampullopetal and all vertical canal units to ampullofugal endolymph flow. The average observed roll and pitch null points for each canal were used to determine the average sensitivity vectors for the right horizontal, anterior, and posterior canals. These sensitivity vectors were at a variance of 4.6-10.2 degrees from those predicted by anatomical measurements (3). The mean resting discharge characteristics of 318 first-order neurons was 36.0 spikes/s (range 0.50-114 spikes/s). No significant difference was noted between horizontal and anterior canal neurons on horizontal and anterior canal neurons on the basis of resting rate. The resting rate of the posterior canal neuron population was significantly lower. The regularity of the resting discharge varied in all three canals and the average coefficient of variation was 0.238 for the population, with a range of 0.298-1.030. The population distribution of all resting-rate statistical parameters appeared to be unimodal, indicating that first-order canal neurons may not be broken into discrete populations on the basis of resting-rate characteristics. Of 47 adequately examined first-order canal neurons, 25 demonstrated a repeatable and predictable alteration in their resting discharge as their position to gravity was reoriented. This alteration was usually nonadapting and varied in magnitude according to the degree of tilt and original starting position. Of 25 canal gravity units, 4 had nearby units from the same canal which were unresponsive to gravity, suggesting the effect was due to a limited distortion of the crista or cupula rather than an overall displacement of the cupula.     

Afferent innervation patterns of the pigeon horizontal crista ampullaris

The vestibular semicircular canals are responsible for detection of rotational head motion although the precise mechanisms underlying the transduction and encoding of movement information are still under study. In the present investigation, we utilized neural tracers and immunohistochemistry to quantitatively examine the topology and afferent innervation patterns of the horizontal semicircular canal crista (HCC) in pigeons (Columba livia). Two hundred and eighty-six afferents from five horizontal canal organs were identified of which 92 units were sufficiently labeled and isolated to perform anatomical reconstructions. In addition, a three-dimensional contour map of the crista was constructed. Bouton afferents were located only in the peripheral regions of the receptor epithelium. Bouton afferents had the most complex innervation patterns with significantly longer and more numerous branches as well as a higher branch order than any other fiber type. Bouton fibers also contained significantly more bouton terminals than did dimorph afferents. Calyx afferents were located only in the apex and central planar regions. Calyx fibers had the largest axonal diameters yet the smallest fiber lengths and innervation areas, the fewest number of branches, the lowest branch order, and the fewest total number of terminals of all fiber types. Dimorph afferents were located throughout the central crista with afferent terminations that were larger and more complex than calyx fibers but less so than bouton fibers. Overall, the pigeon HCC morphology and innervation shares many common features with those of other animal classes.     

A cell therapy approach to substitute neural elements in the inner ear

Three different donor tissues were tested for their capacity to survive, integrate and differentiate in the adult inner ear. Surviving embryonic dorsal root ganglion cells were found within the spiral ganglion neuron region and along the auditory nerve fibers. In the presence of exogenous nerve growth factor (NGF), the dorsal root ganglion cells formed extensive growth of neurites that seemed to contact the host neurons. Adult neural stem cells survived relative poorly in the inner ear whereas embryonic stem cells showed a somewhat greater capacity for survival and integration. Overall, the survival rate of implanted tissue was quite low in the cochlea. It is concluded that an inner ear cell therapy approach based on the implantation of exogenous cells will require that important survival factors are identified and supplied. In addition, it is possible that the physical properties of the cochlea, e.g., fluid-filled compartments and very limited space for cell proliferation, are unfavorable, at least in the normal cochlea.     

Inner ear localization of mRNA and protein products of COCH, mutated in the sensorineural deafness and vestibular disorder, DFNA9.

Missense mutations in the COCH gene, which is expressed preferentially at high levels in the inner ear, cause the autosomal dominant sensorineural deafness and vestibular disorder, DFNA9 (OMIM 601369). By in situ hybridization of mouse and human inner ear sections, we find high-level expression of COCH mRNA in the fibrocytes of the spiral limbus and of the spiral ligament in the cochlea, and in the fibrocytes of the connective tissue stroma underlying the sensory epithelium of the crista ampullaris of the semicircular canals. A polyclonal antibody against the human COCH protein product, cochlin, was raised against the N-terminal 135 amino acid residues of cochlin, corresponding to the Limulus factor C-homology (cochFCH) domain; this domain harbors all five known point mutations in DFNA9. On western blots of human fetal cochlear extracts, anti-cochlin reacts with a cochlin band of the predicted full-length size as well as a smaller isoform. Immunohistochemistry performed with anti-cochlin shows staining predominantly in the regions of the fibrocytes of the spiral limbus and of the spiral ligament in mouse and in human fetal and adult tissue sections. These sites correspond to those areas that express COCH mRNA as determined by in situ hybridization, and to the regions of the inner ear which show histological abnormalities in DFNA9. The fibrocytes expressing mRNA and protein products of COCH are the very cell types which are either absent or markedly reduced and replaced by eosinophilic acellular material in temporal bone sections of individuals affected with DFNA9.