Otic Epithelium Research Articles

Pioneer statoacoustic neurons guide neuroblast behaviour during otic ganglion assembly

ABSTRACT Cranial ganglia are aggregates of sensory neurons that mediate distinct types of sensation. The statoacoustic ganglion (SAG) develops into several lobes that are spatially arranged to connect appropriately with hair cells of the inner ear. To investigate the cellular behaviours involved in the 3D organization of the SAG, we use high-resolution confocal imaging of single-cell, labelled zebrafish neuroblasts (NBs), photoconversion, photoablation, and genetic perturbations. We show that otic NBs delaminate out of the otic epithelium in an epithelial-mesenchymal transition-like manner, rearranging apical polarity and primary cilia proteins. We also show that, once delaminated, NBs require RhoGTPases in order to perform active migration. Furthermore, tracking of recently delaminated NBs revealed their directed migration and coalescence around a small population of pioneer SAG neurons. These pioneer SAG neurons, not from otic placode origin, populate the coalescence region before otic neurogenesis begins and their ablation disrupts delaminated NB migratory pathways, consequentially affecting SAG shape. Altogether, this work shows for the first time the role of pioneer SAG neurons in orchestrating SAG development.

Enhancer trap lines with GFP driven by smad6b and frizzled1 regulatory sequences for the study of epithelial morphogenesis in the developing zebrafish inner ear.

Live imaging in the zebrafish embryo using tissue-specific expression of fluorescent proteins can yield important insights into the mechanisms that drive sensory organ morphogenesis and cell differentiation. Morphogenesis of the semicircular canal ducts of the vertebrate inner ear requires a complex rearrangement of epithelial cells, including outgrowth, adhesion, fusion and perforation of epithelial projections to generate pillars of tissue that form the hubs of each canal. We report the insertion sites and expression patterns of two enhancer trap lines in the developing zebrafish embryo, each of which highlight different aspects of epithelial cell morphogenesis in the inner ear. A membrane-linked EGFP driven by smad6b regulatory sequences is expressed throughout the otic epithelium, most strongly on the lateral side of the ear and in the sensory cristae. A second enhancer trap line, with cytoplasmic EGFP driven by frizzled1 (fzd1) regulatory sequences, specifically marks cells of the ventral projection and pillar in the developing ear, and marginal cells in the sensory cristae, together with variable expression in the retina and epiphysis, and neurons elsewhere in the developing central nervous system. We have used a combination of methods to identify the insertion sites of these two transgenes, which were generated through random insertion, and show that Targeted Locus Amplification is a rapid and reliable method for the identification of insertion sites of randomly inserted transgenes.

Retinoic acid signaling regulates late stages of semicircular canal morphogenesis and otolith maintenance in the zebrafish inner ear

The vitamin A derivative all-trans retinoic acid (RA) regulates early stages of inner ear development. As the early disruption of the RA pathway results in profound mispatterning of the developing inner ear, this confounds analyses of specific roles in later stages. Therefore, we used the temporal-specific exposure of all-trans RA or diethylaminobenzaldehyde to evaluate RA functions in late otic development. Perturbing late RA signaling causes behavioral defects analogous to those expected in larvae suffering from vestibular dysfunction. These larvae also demonstrate malformations of the semi-circular canals, as visualized through (a) use of the transgenic strain nkhspdmc12a, a fluorescent reporter expressed in otic epithelium; and (b) injection of the fluorescent lipophilic dye DiI. We also noted the altered expression of genes encoding ECM proteins or modifying enzymes. Other malformations of the inner ear observed in our work include the loss or reduced size of the utricular and saccular otoliths, suggesting a role for RA in otolith maintenance. Our work has identified a previously undescribed late phase of RA activity in otic development, demonstrating that vestibular defects observed in human patients in relation to perturbed RA signaling are not solely due to its early disruption in otic development.

Early Deletion of Neurod1 Alters Neuronal Lineage Potential and Diminishes Neurogenesis in the Inner Ear.

Neuronal development in the inner ear is initiated by expression of the proneural basic Helix-Loop-Helix (bHLH) transcription factor Neurogenin1 that specifies neuronal precursors in the otocyst. The initial specification of the neuroblasts within the otic epithelium is followed by the expression of an additional bHLH factor, Neurod1. Although NEUROD1 is essential for inner ear neuronal development, the different aspects of the temporal and spatial requirements of NEUROD1 for the inner ear and, mainly, for auditory neuron development are not fully understood. In this study, using Foxg1Cre for the early elimination of Neurod1 in the mouse otocyst, we showed that Neurod1 deletion results in a massive reduction of differentiating neurons in the otic ganglion at E10.5, and in the diminished vestibular and rudimental spiral ganglia at E13.5. Attenuated neuronal development was associated with reduced and disorganized sensory epithelia, formation of ectopic hair cells, and the shortened cochlea in the inner ear. Central projections of inner ear neurons with conditional Neurod1 deletion are reduced, unsegregated, disorganized, and interconnecting the vestibular and auditory systems. In line with decreased afferent input from auditory neurons, the volume of cochlear nuclei was reduced by 60% in Neurod1 mutant mice. Finally, our data demonstrate that early elimination of Neurod1 affects the neuronal lineage potential and alters the generation of inner ear neurons and cochlear afferents with a profound effect on the first auditory nuclei, the cochlear nuclei.

Extracellular hyaluronate pressure shaped by cellular tethers drives tissue morphogenesis

How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, made by the enzymes uridine 5'-diphosphate dehydrogenase (ugdh) and hyaluronan synthase 3 (has3), drives canal morphogenesis. Charged hyaluronate polymers osmotically swell with water and generate isotropic extracellular pressure to deform the overlying epithelium into buds. The mechanical anisotropy needed to shape buds into tubes is conferred by a polarized distribution of actomyosin and E-cadherin-rich membrane tethers, which we term cytocinches. Most work on tissue morphogenesis ascribes actomyosin contractility as the driving force, while the extracellular matrix shapes tissues through differential stiffness. Our work inverts this expectation. Hyaluronate pressure shaped by anisotropic tissue stiffness may be a widespread mechanism for powering morphological change in organogenesis and tissue engineering.

Otic Neurogenesis in Xenopus laevis: Proliferation, Differentiation, and the Role of Eya1.

Using immunostaining and confocal microscopy, we here provide the first detailed description of otic neurogenesis in Xenopus laevis. We show that the otic vesicle comprises a pseudostratified epithelium with apicobasal polarity (apical enrichment of Par3, aPKC, phosphorylated Myosin light chain, N-cadherin) and interkinetic nuclear migration (apical localization of mitotic, pH3-positive cells). A Sox3-immunopositive neurosensory area in the ventromedial otic vesicle gives rise to neuroblasts, which delaminate through breaches in the basal lamina between stages 26/27 and 39. Delaminated cells congregate to form the vestibulocochlear ganglion, whose peripheral cells continue to proliferate (as judged by EdU incorporation), while central cells differentiate into Islet1/2-immunopositive neurons from stage 29 on and send out neurites at stage 31. The central part of the neurosensory area retains Sox3 but stops proliferating from stage 33, forming the first sensory areas (utricular/saccular maculae). The phosphatase and transcriptional coactivator Eya1 has previously been shown to play a central role for otic neurogenesis but the underlying mechanism is poorly understood. Using an antibody specifically raised against Xenopus Eya1, we characterize the subcellular localization of Eya1 proteins, their levels of expression as well as their distribution in relation to progenitor and neuronal differentiation markers during otic neurogenesis. We show that Eya1 protein localizes to both nuclei and cytoplasm in the otic epithelium, with levels of nuclear Eya1 declining in differentiating (Islet1/2+) vestibulocochlear ganglion neurons and in the developing sensory areas. Morpholino-based knockdown of Eya1 leads to reduction of proliferating, Sox3- and Islet1/2-immunopositive cells, redistribution of cell polarity proteins and loss of N-cadherin suggesting that Eya1 is required for maintenance of epithelial cells with apicobasal polarity, progenitor proliferation and neuronal differentiation during otic neurogenesis.

Opposing effects of Wnt/β-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea

ABSTRACTDuring embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via β-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.

Isolation and Characterization of Mammalian Otic Progenitor Cells that Can Differentiate into Both Sensory Epithelial and Neuronal Cell Lineages.

The mammalian inner ear mediates hearing and balance and during development generates both cochleo-vestibular ganglion neurons and sensory epithelial receptor cells, that is, hair cells and support cells. Cell marking experiments have shown that both hair cells and support cells can originate from a common progenitor. Here, we demonstrate the lineage potential of individual otic epithelial cell clones using three cell lines established by a combination of limiting dilution and gene-marking techniques from an embryonic day 12 (E12) rat otocyst. Cell-type specific marker analyses of these clonal lines under proliferation and differentiation culture conditions demonstrate that during differentiation immature cell markers (Nanog and Nestin) were downregulated and hair cell (Myosin VIIa and Math1), support cell (p27Kip1 and cytokeratin) and neuronal cell (NF-H and NeuroD) markers were upregulated. Our results suggest that the otic epithelium of the E12 mammalian inner ear possess multipotent progenitor cells able to generate cell types of both sensory epithelial and neural cell lineages when cultured under a differentiation culture condition. Understanding the molecular mechanisms of proliferation and differentiation of multipotent otic progenitor cells may provide insights that could contribute to the development of a novel cell therapy with a potential to initiate or stimulate the sensorineural repair of damaged inner ear sensory receptors. Anat Rec, 303:451-460, 2020. © 2019 American Association for Anatomy.

Slc26a9P2ACre : a new CRE driver to regulate gene expression in the otic placode lineage and other FGFR2b-dependent epithelia.

Pan-otic CRE drivers enable gene regulation throughout the otic placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We have previously identified Slc26a9 as an otic placode-specific target of the FGFR2b ligands FGF3 and FGF10. We show here that Slc26a9 is otic specific through E10.5, but is not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9P2ACre mice, and observed CRE activity throughout the otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5 to P3. In contrast to analogous mice generated with other pan-otic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9P2ACre provides a useful complement to existing pan-otic CRE drivers, particularly for postnatal analyses.

Expression of MYOSIN VIIA in developing mouse cochleovestibular ganglion neurons

Myosins make up a large super family of motor proteins responsible for actin-based motility in most eukaryotic cells. Myosin VIIA is essential for the development and function of sensory hair cells in the inner ear. The role of Myosin VIIA in the development of cochleovestibular ganglion (CVG) neurons in the mouse is largely unknown. Neurons of the CVG innervate sensory hair cells of the cochlea and vestibular organs to transmit hearing and balance information respectively to the brain. The aim of this study was to characterize the expression of MYOSIN VIIA in the CVG of mouse embryos. Spatiotemporal expression of MYOSIN VIIA was characterized in embryonic (E) mouse inner ear neurons from E9.5 to postnatal (P) day 0. At early stages, when otic neurons begin to delaminate to form the CVG, MYOSIN VIIA was co-expressed with TuJ1, ISLET1 and NEUROD in the otic epithelium and CVG. When CVG neurons were migrating and exiting mitosis, MYSOSIN VIIA was downregulated in a subset of neurons, which were NEUROD-negative and GATA3-positive. After segregation of the CVG, MYOSIN VIIA was observed in a subset of vestibular neurons marked by TUJ1 and absent in cochlear neurons, marked by GATA3. The differential expression of MYOSIN VIIA may indicate a role in inner ear neuron migration and specific labeling of vestibular neurons.

Cilia in the developing zebrafish ear

The inner ear, which mediates the senses of hearing and balance, derives from a simple ectodermal vesicle in the vertebrate embryo. In the zebrafish, the otic placode and vesicle express a whole suite of genes required for ciliogenesis and ciliary motility. Every cell of the otic epithelium is ciliated at early stages; at least three different ciliary subtypes can be distinguished on the basis of length, motility, genetic requirements and function. In the early otic vesicle, most cilia are short and immotile. Long, immotile kinocilia on the first sensory hair cells tether the otoliths, biomineralized aggregates of calcium carbonate and protein. Small numbers of motile cilia at the poles of the otic vesicle contribute to the accuracy of otolith tethering, but neither the presence of cilia nor ciliary motility is absolutely required for this process. Instead, otolith tethering is dependent on the presence of hair cells and the function of the glycoprotein Otogelin. Otic cilia or ciliary proteins also mediate sensitivity to ototoxins and coordinate responses to extracellular signals. Other studies are beginning to unravel the role of ciliary proteins in cellular compartments other than the kinocilium, where they are important for the integrity and survival of the sensory hair cell. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Building a Sound Wall in the Cochlea: Development of the Stria Vascularis

The stria vascularis is a specialized epithelial structure of the mammalian cochlea that produces endolymph, the potassium‐rich fluid responsible for the positive endocochlear potential of the cochlear lumen. This positive extracellular potential is the major driving force for proper signal transduction by sensory cells in the ear, and thus normal hearing. The stria vascularis is the “battery” of the auditory organ: it is localized in the lateral wall of the cochlea, and consists of a multilayered highly vascularized epithelium. It is composed of three main cell types with distinct embryonic origins: the marginal cells derived from the otic epithelium , the intermediate cells which are specialized melanocyte‐like cells derived from migratory neural crest cells, and the basal cells that are derived from otic mesenchyme. Mesoderm‐derived blood vessels are interspersed between these cells throughout the stria. Defects in the development of the stria vascularis are part of a number of inheritable deafness syndromes in humans, including Hirschsprung disease and Waardenburg, Jervell and Lange‐Nielsen, Bartter and SeSAME syndromes. Strial degeneration is estimated to cause more than 30% of age related hearing loss or presbycusis, which affects between 25–40% of the population over 65 years and over 60% of the population over 80 years old. The causes of strial atrophy remain poorly understood. Over the past decade, a great effort has been made to understand the development of the sensory cells in the mammalian cochlea with the aim of applying their developmental principles to regenerative therapies. By contrast, very little is known about the developmental potential of the stria vascularis, or even the mechanisms of strial degeneration due to aging. Our focus is to uncover the normal development of the stria, the cellular interactions that lead to its formation, and the potential for regeneration in cases of congenital strial deafness.Support or Funding InformationHearing Health FoundationThe Hartwell FoundationNIDCD R01DC015785This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CTCF deficiency causes expansion of the sensory domain in the mouse cochlea

The cochlea in the mammalian inner ear is a sensitive and sharply organized sound-detecting structure. The proper specification of neurosensory-competent domain in the otic epithelium is required for the formation of mature neuronal and sensory domains. Genetic studies have provided many insights into inner ear development, but there have been few epigenetic studies of inner ear development. CTCF is an epigenetic factor that plays a pivotal role in the organization of global chromatin conformation. To determine the role of CTCF in the otic sensory formation, we made a conditional knockout of Ctcf in the developing otic epithelium by crossing Ctcffl/fl mice with Pax2-Cre mice. Ctcf deficiency resulted in extra rows of auditory hair cells in the shortened cochlea on mouse embryonic day 14.5 (E14.5) and E17.5. The massive and ectopic expression of sensory specifiers such as Jag1 and Sox2 indicated that the sensory domain was expanded in the Ctcf-deficient cochlea. Other regulators of the sensory domain such as Bmp4, Gata3, and Fgf10 were not affected. These results suggest that CTCF plays a role in the regulation of the sensory domain in mammalian cochlear development.

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.

Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing.

The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1Cre mice with Exoc5f/f mice for hair cell-specific deletion (Gfi1Cre/+;Exoc5f/f) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5f/f). Gfi1Cre/+;Exoc5f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.

Claudin7b is required for the formation and function of inner ear in zebrafish.

Zebrafish has become an excellent model for studying the development and function of inner ear. We report here a zebrafish line in which claudin 7b (cldn7b) locus is interrupted by a Tol2 transposon at its first intron. The homozygous mutants have enlarged otocysts, smaller or no otoliths, slowly formed semicircular canals, and insensitiveness to sound stimulation. These abnormal phenotypes and hearing loss of inner ear could be mostly rescued by injection of cldn7b-mRNA into one-cell stage homozygous mutant embryos. Mechanistically, cldn7b-deficiency interrupted the formation of apical junction complexes (AJCs) in otic epithelial cells of inner ear and the ion-homeostasis of endolymph, which then led to the loss of proper contact between otoliths and normally developed hair cells in utricle and saccule or aberrant mechanosensory transduction. Thus, Cldn7b is essential for the formation and proper function of inner ear through its unique role in keeping an initial integrity of otic epithelia during zebrafish embryogenesis.

Pioneer neurog1 expressing cells ingress into the otic epithelium and instruct neuronal specification.

Neural patterning involves regionalised cell specification. Recent studies indicate that cell dynamics play instrumental roles in neural pattern refinement and progression, but the impact of cell behaviour and morphogenesis on neural specification is not understood. Here we combine 4D analysis of cell behaviours with dynamic quantification of proneural expression to uncover the construction of the zebrafish otic neurogenic domain. We identify pioneer cells expressing neurog1 outside the otic epithelium that migrate and ingress into the epithelialising placode to become the first otic neuronal progenitors. Subsequently, neighbouring cells express neurog1 inside the placode, and apical symmetric divisions amplify the specified pool. Interestingly, pioneer cells delaminate shortly after ingression. Ablation experiments reveal that pioneer cells promote neurog1 expression in other otic cells. Finally, ingression relies on the epithelialisation timing controlled by FGF activity. We propose a novel view for otic neurogenesis integrating cell dynamics whereby ingression of pioneer cells instructs neuronal specification.

Distribution of neurosensory progenitor pools during inner ear morphogenesis unveiled by cell lineage reconstruction.

Reconstructing the lineage of cells is central to understanding how the wide diversity of cell types develops. Here, we provide the neurosensory lineage reconstruction of a complex sensory organ, the inner ear, by imaging zebrafish embryos in vivo over an extended timespan, combining cell tracing and cell fate marker expression over time. We deliver the first dynamic map of early neuronal and sensory progenitor pools in the whole otic vesicle. It highlights the remodeling of the neuronal progenitor domain upon neuroblast delamination, and reveals that the order and place of neuroblasts' delamination from the otic epithelium prefigure their position within the SAG. Sensory and non-sensory domains harbor different proliferative activity contributing distinctly to the overall growth of the structure. Therefore, the otic vesicle case exemplifies a generic morphogenetic process where spatial and temporal cues regulate cell fate and functional organization of the rudiment of the definitive organ.

Expression patterns of Irx genes in the developing chick inner ear.

The vertebrate inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. The molecular patterning of the developing otic epithelium creates various positional identities, consequently leading to the stereotyped specification of each neurosensory and non-sensory element of the membranous labyrinth. The Iroquois (Iro/Irx) genes, clustered in two groups (A: Irx1, Irx2, and Irx4; and B: Irx3, Irx5, and Irx6), encode for transcriptional factors involved directly in numerous patterning processes of embryonic tissues in many phyla. This work presents a detailed study of the expression patterns of these six Irx genes during chick inner ear development, paying particular attention to the axial specification of the otic anlagen. The Irx genes seem to play different roles at different embryonic periods. At the otic vesicle stage (HH18), all the genes of each cluster are expressed identically. Both clusters A and B seem involved in the specification of the lateral and posterior portions of the otic anlagen. Cluster B seems to regulate a larger area than cluster A, including the presumptive territory of the endolymphatic apparatus. Both clusters seem also to be involved in neurogenic events. At stages HH24/25-HH27, combinations of IrxA and IrxB genes participate in the specification of most sensory patches and some non-sensory components of the otic epithelium. At stage HH34, the six Irx genes show divergent patterns of expression, leading to the final specification of the membranous labyrinth, as well as to cell differentiation.

Temporal coupling between specifications of neuronal and macular fates of the inner ear

The inner ear is a complex organ comprised of various specialized sensory organs for detecting sound and head movements. The timing of specification for these sensory organs, however, is not clear. Previous fate mapping results of the inner ear indicate that vestibular and auditory ganglia and two of the vestibular sensory organs, the utricular macula (UM) and saccular macula (SM), are lineage related. Based on the medial-lateral relationship where respective auditory and vestibular neuroblasts exit from the otic epithelium and the subsequent formation of the medial SM and lateral UM in these regions, we hypothesized that specification of the two lateral structures, the vestibular ganglion and the UM are coupled and likewise for the two medial structures, the auditory ganglion and the SM. We tested this hypothesis by surgically inverting the primary axes of the otic cup in ovo and investigating the fate of the vestibular neurogenic region, which had been spotted with a lipophilic dye. Our results showed that the laterally-positioned, dye-associated, vestibular ganglion and UM were largely normal in transplanted ears, whereas both auditory ganglion and SM showed abnormalities suggesting the lateral but not the medial-derived structures were mostly specified at the time of transplantation. Both of these results are consistent with a temporal coupling between neuronal and macular fate specifications.