Patterning and morphogenesis of the vertebrate inner ear

The positional cues for formation of individual inner ear components are dependent on pre-established axial information conferred by inductive signals from tissues surrounding the developing inner ear. This review summarizes some of the known molecular pathways involved in establishing the three axes of the inner ear, anterior-posterior (AP), dorsal-ventral (DV) and medial-lateral (ML). Signals required to establish the AP axis of the inner ear are not known, but they do not appear to be derived from the hindbrain. In contrast, the hindbrain is essential for establishing the DV axis of the inner ear by providing inductive signals such as Wnts and Sonic hedgehog. Signaling from the hindbrain is also required for the formation of the ML axis, whereas formation of the lateral wall of the otocyst may be a result of first establishing both the AP and DV axes. In addition, this review addresses how genes induced within the otic epithelium as a result of axial specification continue to mediate inner ear morphogenesis.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

Building inner ears: recent advances and future challenges for in vitro organoid systems

While inner ear disorders are common, our ability to intervene and recover their sensory function is limited. In vitro models of the inner ear, like the organoid system, could aid in identifying new regenerative drugs and gene therapies. Here, we provide a perspective on the status of in vitro inner ear models and guidance on how to improve their applicability in translational research. We highlight the generation of inner ear cell types from pluripotent stem cells as a particularly promising focus of research. Several exciting recent studies have shown how the developmental signaling cues of embryonic and fetal development can be mimicked to differentiate stem cells into “inner ear organoids” containing otic progenitor cells, hair cells, and neurons. However, current differentiation protocols and our knowledge of embryonic and fetal inner ear development in general, have a bias toward the sensory epithelia of the inner ear. We propose that a more holistic view is needed to better model the inner ear in vitro. Moving forward, attention should be made to the broader diversity of neuroglial and mesenchymal cell types of the inner ear, and how they interact in space or time during development. With improved control of epithelial, neuroglial, and mesenchymal cell fate specification, inner ear organoids would have the ability to truly recapitulate neurosensory function and dysfunction. We conclude by discussing how single-cell atlases of the developing inner ear and technical innovations will be critical tools to advance inner ear organoid platforms for future pre-clinical applications.Subject terms: Cell biology, Somatic system, Stem-cell research     

Origins of inner ear sensory organs revealed by fate map and time-lapse analyses

The inner ear develops from a simple ectodermal thickening called the otic placode into a labyrinth of chambers which house sensory organs that sense sound and are used to maintain balance. Although the morphology and function of the sensory organs are well characterized, their origins and lineage relationships are virtually unknown. In this study, we generated a fate map of Xenopus laevis inner ear at otic placode and otocyst stages to determine the developmental origins of the sensory organs. Our lineage analysis shows that all regions of the otic placode and otocyst can give rise to the sensory organs of the inner ear, though there were differences between labeled quadrants in the range of derivatives formed. A given region often gives rise to cells in multiple sensory organs, including cells that apparently dispersed from anterior to posterior poles and vice versa. These results suggest that a single sensory organ arises from cells in different parts of the placode or otocyst and that cell mixing plays a large role in ear development. Time-lapse videomicroscopy provides further evidence that cells from opposite regions of the inner ear mix during the development of the inner ear, and this mixing begins at placode stages. Lastly, bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta (TGF-beta) family, is expressed in all sensory organs of the frog inner ear, as it is in the developing chicken ear. Inner ear fate maps provide a context for interpreting gene expression patterns and embryological manipulations.     

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.     

Programmed cell death in the development of the vertebrate inner ear

Programmed cell death is known to be an essential process for accurate ontogeny during the normal development of the inner ear. The inner ear is a complex sensory organ responsible for equilibrium and sound detection in vertebrates. In all vertebrates, the inner ear develops from a single ectodermic patch on the surface of the embryo's head, which undergoes a series of morphological changes to give rise to the complex structure of the adult inner ear. Enlargement and morphogenesis of the inner ear primordium is likely to depend on cellular division, growth, migration, differentiation and apoptosis. Here we describe the regions of programmed cell death that contribute to the final morphological aspect of the adult inner ear. The few studies that focus on the molecules that control this process during inner ear development indicate that the molecules and intracellular signaling pathways activated during the apoptotic response in the inner ear are similar to the previously described for the nervous system. In this review, we will describe some of the growth factors and key pathways that regulate pro- and anti-apoptotic signals and how they cross talk to determine the apoptotic or survival fate of cells in the development of the inner ear.     

Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear

An early and crucial event in vertebrate inner ear development is the acquisition of axial identities that in turn dictate the positions of all subsequent inner ear components. Here, we focus on the role of the hindbrain in establishment of inner ear axes and show that axial specification occurs well after otic placode formation in chicken. Anteroposterior (AP) rotation of the hindbrain prior to specification of this axis does not affect the normal AP orientation and morphogenesis of the inner ear. By contrast, reversing the dorsoventral (DV) axis of the hindbrain results in changing the DV axial identity of the inner ear. Expression patterns of several ventrally expressed otic genes such as NeuroD, Lunatic fringe (Lfng) and Six1 are shifted dorsally, whereas the expression pattern of a normally dorsal-specific gene, Gbx2, is abolished. Removing the source of Sonic Hedgehog (SHH) by ablating the floor plate and/or notochord, or inhibiting SHH function using an antibody that blocks SHH bioactivity results in loss of ventral inner ear structures. Our results indicate that SHH, together with other signals from the hindbrain, are important for patterning the ventral axis of the inner ear. Taken together, our studies suggest that tissue(s) other than the hindbrain confer AP axial information whereas signals from the hindbrain are necessary and sufficient for the DV axial patterning of the inner ear.     

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.     

Molecular mechanisms of inner ear development

The inner ear is a structurally complex vertebrate organ built to encode sound, motion, and orientation in space. Given its complexity, it is not surprising that inner ear dysfunction is a relatively common consequence of human genetic mutation. Studies in model organisms suggest that many genes currently known to be associated with human hearing impairment are active during embryogenesis. Hence, the study of inner ear development provides a rich context for understanding the functions of genes implicated in hearing loss. This chapter focuses on molecular mechanisms of inner ear development derived from studies of model organisms.     

Celastrol enhances Atoh1 expression in inner ear stem cells and promotes their differentiation into functional auditory neuronal-like cells

We aimed to investigate the beneficial effect of Celastrol on inner ear stem cells and potential therapeutic value for hearing loss. The inner ear stem cells were isolated and characterized from utricular sensory epithelium of adult mice. The stemness was evaluated by sphere formation assay. The relative expressions of Atoh1, MAP-2 and Myosin VI were measured by RT-PCR and immunoblotting. The up-regulation of MAP-2 was also analysed with immunofluorescence. The in vitro neuronal excitability was interrogated by calcium oscillation. The electrophysiological property was determined by inward current recorded on patch clamp. Our results demonstrated that Celastrol treatment significantly improved the viability and proliferation of mouse inner ear stem cells, and facilitated sphere formation. Moreover, Celastrol stimulated differentiation of mouse inner ear stem cells to neuronal-like cells and enhanced neural excitability. Celastrol also enhanced neuronal-like cell identity in the inner ear stem cell derived neurons, as well as their electrophysiological function. Most notably, these effects were apparently associated with the upregulation of Atoh1 in response to Celastrol treatment. Celastrol showed beneficial effect on inner ear stem cells and held therapeutic promise against hearing loss.     

Otitis Media Impacts Hundreds of Mouse Middle and Inner Ear Genes

Objective

Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition.

Methods

To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes.

Results

Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear.

Conclusion

Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media.     

An X-radiographic and SEM study of the osseous inner ear of multituberculates and monotremes (Mammalia): implications for mammalian phylogeny and evolution of hearing

Multituberculate petrosals with well-preserved, three-dimensional internal anatomy from the Late Cretaceous/early Paleocene Bug Creek Anthills, Montana, U.S.A., are described from X-radiographic and SEM images, as well as from conventional visual observations, and are compared with the anatomy of the osseous inner ear in monotremes and in primitive non-therian and therian mammals. Results of this study indicate that: (1) the cochlea of at least some multituberculates retained a lagena, previously known only in monotremes among mammals; (2) an enlarged vestibule evolved in several lineages of multituberculates independently, and hence is not a synapomorphy of the order; (3) the cochlear canal lacks osseous laminae in support of the short, wide basilar membrane, which was probably inefficient in responding to high-frequency airborne vibrations; and (4) consequently, bone-conducted hearing in some multituberculate species may have been important in interpretation of their surroundings. Comparisons with the inner ear of monotremes and primitive therians indicate that curvature of the cochlea and cribriform plates for passage of vestibulocochlear nerve branches through the petrosal are unlikely homologues between monotremes and therians. From non-therian to therian mammals, there is a distinct morphological gap in the inner ear transition, characterized by acquisition of a number of neomorphs in the therian inner ear; an intermediate stage has yet to be discovered.     

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.     

干细胞治疗感音神经性耳聋

治疗内耳疾病的主要困难之一是找到耳蜗毛细胞或者螺旋神经元丢失所导致的听力损失的治疗方法。本文讨论使用干细胞替代感觉细胞丢失为目的的几个治疗策略。作者最近在成年内耳中发现了可以分化为毛细胞的干细胞,发现了胚胎干细胞可在体外转化为毛细胞并表达毛细胞标记物。在动物模型中,成年内耳干细胞、神经干细胞和胚胎干细胞来源的前体细胞可分化成为毛细胞和神经细胞。本文将讨论使用干细胞再生损伤毛细胞的不同方法,介绍几种可行的动物模型,并讨论发展基于干细胞的细胞替代疗法治疗内耳损伤中存在的困难。     

Biophysical basis for inner ear decompression sickness.

Isolated inner ear decompression sickness (DCS) is recognized in deep diving involving breathing of helium-oxygen mixtures, particularly when breathing gas is switched to a nitrogen-rich mixture during decompression. The biophysical basis for this selective vulnerability of the inner ear to DCS has not been established. A compartmental model of inert gas kinetics in the human inner ear was constructed from anatomical and physiological parameters described in the literature and used to simulate inert gas tensions in the inner ear during deep dives and breathing-gas substitutions that have been reported to cause inner ear DCS. The model predicts considerable supersaturation, and therefore possible bubble formation, during the initial phase of a conventional decompression. Counterdiffusion of helium and nitrogen from the perilymph may produce supersaturation in the membranous labyrinth and endolymph after switching to a nitrogen-rich breathing mixture even without decompression. Conventional decompression algorithms may result in inadequate decompression for the inner ear for deep dives. Breathing-gas switches should be scheduled deep or shallow to avoid the period of maximum supersaturation resulting from decompression.     

Expression and functions of FGF ligands during early otic development

Classical studies have postulated the action of an endomesodermal signal initiating inner ear induction, subsequently followed by a neural tube-derived signal to complete the process of otic placode formation in the surface ectoderm. Members of the Fibroblast growth factor (FGF) gene family have been implicated in these processes. In this review, expression analysis and recent experimental evidence for candidate inner ear FGF ligands during inner ear induction is discussed. Careful examination of the spatiotemporal expression patterns of different FGFs during inner ear induction reveals that the sequential appearance of FGF members in the endoderm and/or mesoderm is followed by expression in the posterior hindbrain in all vertebrate species analysed to date. Experimental manipulations have demonstrated the sufficiency and/or necessity of some FGFs during different steps of inner ear induction in vitro and in vivo. Combining the advantages of the molecular tools and approaches available in different experimental systems such as zebrafish, chicken or mouse will eventually lead to a complete understanding of how FGFs control inner ear induction in vertebrates.     

Progressive restriction of otic fate: the role of FGF and Wnt in resolving inner ear potential

The development of the vertebrate inner ear is an emergent process. Its progression from a relatively simple disk of thickened epithelium within head ectoderm into a complex organ capable of sensing sound and balance is controlled by sequential molecular and cellular interactions. Fibroblast growth factor (FGF) and Wnt signals emanating from mesoderm and neural ectoderm have been shown to direct inner ear fate. However, the role of these multiple signals during inner ear induction is unclear. We demonstrate that the action of the FGFs and Wnts is sequential, and that their roles support a model of hierarchical fate decisions that progressively restrict the developmental potential of the ectoderm until otic commitment. We show that signalling by Fgf3 and Fgf19 is required to initiate a proliferative progenitor region that is a precursor to both the inner ear and the neurogenic epibranchial placodes. Significantly, we find that only after FGF action is attenuated can the subsequent action of Wnt signalling allow otic differentiation to proceed. In addition, gain and loss of function of Wnt-signalling components show a role for this signalling in repressing epibranchial fate. This interplay of signalling factors ensures the correct and ordered differentiation of both inner ear and epibranchial systems.