Function of bidirectional sensitivity in the otolith organs established by transcription factor Emx2.

Otolith organs of the inner ear are innervated by two parallel afferent projections to the brainstem and cerebellum. These innervations were proposed to segregate across the line of polarity reversal (LPR) within each otolith organ, which divides the organ into two regions of hair cells (HC) with opposite stereociliary orientation. The relationship and functional significance of these anatomical features are not known. Here, we show regional expression of Emx2 in otolith organs, which establishes LPR, mediates the neuronal segregation across LPR and constitutes the bidirectional sensitivity function. Conditional knockout (cKO) of Emx2 in HCs lacks LPR. Tmie cKO, in which mechanotransduction was abolished selectively in HCs within the Emx2 expression domain also lacks bidirectional sensitivity. Analyses of both mutants indicate that LPR is specifically required for mice to swim comfortably and to traverse a balance beam efficiently, but LPR is not required for mice to stay on a rotating rod.

Emx2 regulates hair cell rearrangement but not positional identity within neuromasts.

Each hair cell (HC) precursor of zebrafish neuromasts divides to form two daughter HCs of opposite hair bundle orientations. Previously, we showed that transcription factor Emx2, expressed in only one of the daughter HCs, generates this bidirectional HC pattern (Jiang et al., 2017). Here, we asked whether Emx2 mediates this effect by changing location of hair bundle establishment or positions of HCs since daughter HCs are known to switch positions with each other. We showed this HC rearrangement, redefined as two processes named Rock and Roll, is required for positional acquisition of HCs. Apical protrusion formation of nascent HCs and planar polarity signaling are both important for the Rock and Roll. Emx2 facilitates Rock and Roll by delaying apical protrusion of its nascent HCs but it does not determine HCs' ultimate positions, indicating that Emx2 mediates bidirectional HC pattern by changing the location where hair bundle is established in HCs.

Live imaging of hair bundle polarity acquisition demonstrates a critical timeline for transcription factor Emx2.

Directional sensitivity of hair cells (HCs) is conferred by the aymmetric apical hair bundle, comprised of a kinocilium and stereocilia staircase. The mother centriole (MC) forms the base of the kinocilium and the stereocilia develop adjacent to it. Previously, we showed that transcription factor Emx2 reverses hair bundle orientation and its expression in the mouse vestibular utricle is restricted, resulting in two regions of opposite bundle orientation (Jiang et al., 2017). Here, we investigated establishment of opposite bundle orientation in embryonic utricles by live-imaging GFP-labeled centrioles in HCs. The daughter centriole invariably migrated ahead of the MC from the center to their respective peripheral locations in HCs. Comparing HCs between utricular regions, centriole trajectories were similar but they migrated toward opposite directions, suggesting that Emx2 pre-patterned HCs prior to centriole migration. Ectopic Emx2, however, reversed centriole trajectory within hours during a critical time-window when centriole trajectory was responsive to Emx2.

Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

Retinoic acid degradation shapes zonal development of vestibular organs and sensitivity to transient linear accelerations.

Each vestibular sensory epithelium in the inner ear is divided morphologically and physiologically into two zones, called the striola and extrastriola in otolith organ maculae, and the central and peripheral zones in semicircular canal cristae. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, formation of striolar/central zones is compromised, such that they resemble extrastriolar/peripheral zones in multiple features. Mutants have deficient vestibular evoked potential (VsEP) responses to jerk stimuli, head tremor and deficits in balance beam tests that are consistent with abnormal vestibular input, but normal vestibulo-ocular reflexes and apparently normal motor performance during swimming. Thus, degradation of RA during embryogenesis is required for formation of highly specialized regions of the vestibular sensory epithelia with specific functions in detecting head motions.

Genetic interactions support an inhibitory relationship between bone morphogenetic protein 2 and netrin 1 during semicircular canal formation.

The semicircular canals of the mammalian inner ear are derived from epithelial pouches in which epithelial cells in the central region of each pouch undergo resorption, leaving behind the region at the rim to form a tube-shaped canal. Lack of proliferation at the rim and/or over-clearing of epithelial cells in the center of the pouch can obliterate canal formation. Otic-specific knockout of bone morphogenetic protein 2 (Bmp2) results in absence of all three semicircular canals; however, the common crus and ampullae housing the sensory tissue (crista) are intact. The lack of Bmp2 causes Ntn1 (which encodes netrin 1), which is required for canal resorption, to be ectopically expressed at the canal rim. Ectopic Ntn1 results in reduction of Dlx5 and Lmo4, which are required for rim formation. These phenotypes can be partially rescued by removing one allele of Ntn1 in the Bmp2 mutants, indicating that Bmp2 normally negatively regulates Ntn1 for canal formation. Additionally, non-resorption of the canal pouch in Ntn1-/- mutants is partially rescued by removing one allele of Bmp2 Thus, reciprocal inhibition between Bmp2 and netrin 1 is involved in canal formation of the vestibule.

Reciprocal Negative Regulation Between Lmx1a and Lmo4 Is Required for Inner Ear Formation.

LIM-domain containing transcription factors (LIM-TFs) are conserved factors important for embryogenesis. The specificity of these factors in transcriptional regulation is conferred by the complexes that they form with other proteins such as LIM-domain-binding (Ldb) proteins and LIM-domain only (LMO) proteins. Unlike LIM-TFs, these proteins do not bind DNA directly. LMO proteins are negative regulators of LIM-TFs and function by competing with LIM-TFs for binding to Ldb's. Although the LIM-TF Lmx1a is expressed in the developing mouse hindbrain, which provides many of the extrinsic signals for inner ear formation, conditional knock-out embryos of both sexes show that the inner ear source of Lmx1a is the major contributor of ear patterning. In addition, we have found that the reciprocal interaction between Lmx1a and Lmo4 (a LMO protein within the inner ear) mediates the formation of both vestibular and auditory structures. Lmo4 negatively regulates Lmx1a to form the three sensory cristae, the anterior semicircular canal, and the shape of the utricle in the vestibule. Furthermore, this negative regulation blocks ectopic sensory formation in the cochlea. In contrast, Lmx1a negatively regulates Lmo4 in mediating epithelial resorption of the canal pouch, which gives rise to the anterior and posterior semicircular canals. We also found that Lmx1a is independently required for the formation of the endolymphatic duct and hair cells in the basal cochlear region.SIGNIFICANCE STATEMENT The mammalian inner ear is a structurally complex organ responsible for detecting sound and maintaining balance. Failure to form the intricate 3D structure of this organ properly during development most likely will result in sensory deficits on some level. Here, we provide genetic evidence that a transcription factor, Lmx1a, interacts with its negative regulator, Lmo4, to pattern various vestibular and auditory components of the mammalian inner ear. Identifying these key molecules that mediate formation of this important sensory organ will be helpful for designing strategies and therapeutics to alleviate hearing loss and balance disorders.

Directional selectivity of afferent neurons in zebrafish neuromasts is regulated by Emx2 in presynaptic hair cells.

The orientation of hair bundles on top of sensory hair cells (HCs) in neuromasts of the lateral line system allows fish to detect direction of water flow. Each neuromast shows hair bundles arranged in two opposing directions and each afferent neuron innervates only HCs of the same orientation. Previously, we showed that this opposition is established by expression of Emx2 in half of the HCs, where it mediates hair bundle reversal (Jiang et al., 2017). Here, we show that Emx2 also regulates neuronal selection: afferent neurons innervate either Emx2-positive or negative HCs. In emx2 knockout and gain-of-function neuromasts, all HCs are unidirectional and the innervation patterns and physiological responses of the afferent neurons are dependent on the presence or absence of Emx2. Our results indicate that Emx2 mediates the directional selectivity of neuromasts by two distinct processes: regulating hair bundle orientation in HCs and selecting afferent neuronal targets.

Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells.

The asymmetric location of stereociliary bundle (hair bundle) on the apical surface of mechanosensory hair cells (HCs) dictates the direction in which a given HC can respond to cues such as sound, head movements, and water pressure. Notably, vestibular sensory organs of the inner ear, the maculae, exhibit a line of polarity reversal (LPR) across which, hair bundles are polarized in a mirror-image pattern. Similarly, HCs in neuromasts of the zebrafish lateral line system are generated as pairs, and two sibling HCs develop opposite hair bundle orientations. Within these sensory organs, expression of the transcription factor Emx2 is restricted to only one side of the LPR in the maculae or one of the two sibling HCs in neuromasts. Emx2 mediates hair bundle polarity reversal in these restricted subsets of HCs and generates the mirror-image pattern of the sensory organs. Downstream effectors of Emx2 control bundle polarity cell-autonomously via heterotrimeric G proteins.

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.

Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Conserved role of Sonic Hedgehog in tonotopic organization of the avian basilar papilla and mammalian cochlea.

Sound frequency discrimination begins at the organ of Corti in mammals and the basilar papilla in birds. Both of these hearing organs are tonotopically organized such that sensory hair cells at the basal (proximal) end respond to high frequency sound, whereas their counterparts at the apex (distal) respond to low frequencies. Sonic hedgehog (Shh) secreted by the developing notochord and floor plate is required for cochlear formation in both species. In mice, the apical region of the developing cochlea, closer to the ventral midline source of Shh, requires higher levels of Shh signaling than the basal cochlea farther away from the midline. Here, gain-of-function experiments using Shh-soaked beads in ovo or a mouse model expressing constitutively activated Smoothened (transducer of Shh signaling) show up-regulation of apical genes in the basal cochlea, even though these regionally expressed genes are not necessarily conserved between the two species. In chicken, these altered gene expression patterns precede morphological and physiological changes in sensory hair cells that are typically associated with tonotopy such as the total number of stereocilia per hair cell and gene expression of an inward rectifier potassium channel, IRK1, which is a bona fide feature of apical hair cells in the basilar papilla. Furthermore, our results suggest that this conserved role of Shh in establishing cochlear tonotopy is initiated early in development by Shh emanating from the notochord and floor plate.

Pou3f4-mediated regulation of ephrin-b2 controls temporal bone development in the mouse.

The temporal bone encases conductive and sensorineural elements of the ear. Mutations of POU3F4 are associated with unique temporal bone abnormalities and X-linked mixed deafness (DFNX2/DFN3). However, the target genes and developmental processes controlled by POU3F4 transcription factor activity have remained largely uncharacterized. Ephrin-B2 (Efnb2) is a signaling molecule with well-documented effects on cell adhesion, proliferation, and migration. Our analyses of targeted mouse mutants revealed that Efnb2 loss-of-function phenocopies temporal bone abnormalities of Pou3f4 hemizygous null neonates: qualitatively identical malformations of the stapes, styloid process, internal auditory canal, and cochlear capsule were present in both mutants. Using failed/insufficient separation of the stapes and styloid process as a quantitative trait, we found that single gene Efnb2 loss-of-function and compound Pou3f4/Efnb2 loss-of-function caused a more severe phenotype than single gene Pou3f4 loss-of-function. Pou3f4 and Efnb2 gene expression domains overlapped at the site of impending stapes-styloid process separation and at subcapsular mesenchyme surrounding the cochlea; at both these sites, Efnb2 expression was attenuated in Pou3f4 hemizygous null mutants relative to control. Results of immunoprecipitation experiments using chromatin isolated from nascent middle ear mesenchyme supported the hypothesis of a physical association between Pou3f4 and specific non-coding sequence of Efnb2. We propose that Efnb2 is a target of Pou3f4 transcription factor activity and an effector of mesenchymal patterning during temporal bone development.

Ephrin-B2 governs morphogenesis of endolymphatic sac and duct epithelia in the mouse inner ear.

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB-Efnb2 signaling-deficient mice.

Auditory ganglion source of Sonic hedgehog regulates timing of cell cycle exit and differentiation of mammalian cochlear hair cells.

Neural precursor cells of the central nervous system undergo successive temporal waves of terminal division, each of which is soon followed by the onset of cell differentiation. The organ of Corti in the mammalian cochlea develops differently, such that precursors at the apex are the first to exit from the cell cycle but the last to begin differentiating as mechanosensory hair cells. Using a tissue-specific knockout approach in mice, we show that this unique temporal pattern of sensory cell development requires that the adjacent auditory (spiral) ganglion serve as a source of the signaling molecule Sonic hedgehog (Shh). In the absence of this signaling, the cochlear duct is shortened, sensory hair cell precursors exit from the cell cycle prematurely, and hair cell differentiation closely follows cell cycle exit in a similar apical-to-basal direction. The dynamic relationship between the restriction of Shh expression in the developing spiral ganglion and its proximity to regions of the growing cochlear duct dictates the timing of terminal mitosis of hair cell precursors and their subsequent differentiation.

Progression of neurogenesis in the inner ear requires inhibition of Sox2 transcription by neurogenin1 and neurod1.

Sox2 is required for proper neuronal formation in the CNS, but the molecular mechanisms involved are not well characterized. Here, we addressed the role of Sox2 in neurogenesis of the developing chicken inner ear. Overexpressing Sox2 from a constitutive (ß-actin) promoter induces the expression of the proneural gene, Neurogenin1 (Ngn1); however, the expression of a downstream target of Ngn1, Neurod1, is unchanged. As a result, there is a reduction of neural precursors to delaminate and populate the developing cochleo-vestibular ganglion. In contrast, overexpression of either Ngn1 or Neurod1 is sufficient to promote the neural fate in this system. These results suggest that high levels of Sox2 inhibit progression of neurogenesis in the developing inner ear. Furthermore, we provide evidence that Ngn1 and Neurod1 inhibit Sox2 transcription through a phylogenetically conserved Sox2 enhancer to mediate neurogenesis. We propose that Sox2 confers neural competency by promoting Ngn1 expression, and that negative feedback inhibition of Sox2 by Ngn1 is an essential step in the progression from neural precursor to nascent neuron.

Elongation factor 1 alpha1 and genes associated with Usher syndromes are downstream targets of GBX2.

Gbx2 encodes a DNA-binding transcription factor that plays pivotal roles during embryogenesis. Gain-and loss-of-function studies in several vertebrate species have demonstrated a requirement for Gbx2 in development of the anterior hindbrain, spinal cord, inner ear, heart, and neural crest cells. However, the target genes through which GBX2 exerts its effects remain obscure. Using chromatin immunoprecipitation coupled with direct sequencing (ChIP-Seq) analysis in a human prostate cancer cell line, we identified cis-regulatory elements bound by GBX2 to provide insight into its direct downstream targets. The analysis revealed more than 286 highly significant candidate target genes, falling into various functional groups, of which 51% are expressed in the nervous system. Several of the top candidate genes include EEF1A1, ROBO1, PLXNA4, SLIT3, NRP1, and NOTCH2, as well as genes associated with the Usher syndrome, PCDH15 and USH2A, and are plausible candidates contributing to the developmental defects in Gbx2(-/-) mice. We show through gel shift analyses that sequences within the promoter or introns of EEF1A1, ROBO1, PCDH15, USH2A and NOTCH2, are directly bound by GBX2. Consistent with these in vitro results, analyses of Gbx2(-/-) embryos indicate that Gbx2 function is required for migration of Robo1-expressing neural crest cells out of the hindbrain. Furthermore, we show that GBX2 activates transcriptional activity through the promoter of EEF1A1, suggesting that GBX2 could also regulate gene expression indirectly via EEF1A. Taken together, our studies show that GBX2 plays a dynamic role in development and diseases.

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.

Redundant functions of Rac GTPases in inner ear morphogenesis.

Development of the mammalian inner ear requires coordination of cell proliferation, cell fate determination and morphogenetic movements. While significant progress has been made in identifying developmental signals required for inner ear formation, less is known about how distinct signals are coordinated by their downstream mediators. Members of the Rac family of small GTPases are known regulators of cytoskeletal remodeling and numerous other cellular processes. However, the function of Rac GTPases in otic development is largely unexplored. Here, we show that Rac1 and Rac3 redundantly regulate many aspects of inner ear morphogenesis. While no morphological defects were observed in Rac3(-/-) mice, Rac1(CKO); Rac3(-/-) double mutants displayed enhanced vestibular and cochlear malformations compared to Rac1(CKO) single mutants. Moreover, in Rac1(CKO); Rac3(-/-) mutants, we observed compromised E-cadherin-mediated cell adhesion, reduced cell proliferation and increased cell death in the early developing otocyst, leading to a decreased size and malformation of the membranous labyrinth. Finally, cochlear extension was severely disrupted in Rac1(CKO); Rac3(-/-) mutants, accompanied by a loss of epithelial cohesion and formation of ectopic sensory patches underneath the cochlear duct. The compartmentalized expression of otic patterning genes within the Rac1(CKO); Rac3(-/-) mutant otocyst was largely normal, however, indicating that Rac proteins regulate inner ear morphogenesis without affecting cell fate specification. Taken together, our results reveal an essential role for Rac GTPases in coordinating cell adhesion, cell proliferation, cell death and cell movements during otic development.

Mechanotransduction in mouse inner ear hair cells requires transmembrane channel-like genes.

Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1(Δ) mice) were deaf and those with a deletion of Tmc2 (Tmc2(Δ) mice) were phenotypically normal, Tmc1(Δ)Tmc2(Δ) mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1(Δ)Tmc2(Δ) mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.