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The formation of inner ear sensory epithelia is believed to occur in two steps, initial specification of sensory competent (prosensory) regions followed by determination of specific cell-types, such as hair cells (HCs) and supporting cells. However, studies in which the HC determination factor Atoh1 was ectopically expressed in nonprosensory regions indicated that expression of Atoh1 alone is sufficient to induce HC formation suggesting that prosensory formation may not be a prerequisite for HC development. To test this hypothesis, interactions between Sox2 and Atoh1, which are required for prosensory and HC formation respectively, were examined. Forced expression of Atoh1 in nonprosensory cells resulted in transient expression of Sox2 prior to HC formation, suggesting that expression of Sox2 is required for formation of ectopic HCs. Moreover, Atoh1 overexpression failed to induce HC formation in Sox2 mutants, confirming that Sox2 is required for prosensory competence. To determine whether expression of Sox2 alone is sufficient to induce prosensory identity, Sox2 was transiently activated in a manner that mimicked endogenous expression. Following transient Sox2 activation, nonprosensory cells developed as HCs, a result that was never observed in response to persistent expression of Sox2. These results, suggest a dual role for Sox2 in inner ear formation. Initially, Sox2 is required to specify prosensory competence, but subsequent down-regulation of Sox2 must occur to allow Atoh1 expression, most likely through a direct interaction with the Atoh1 promoter. These results implicate Sox2-mediated changes in prosensory cells as an essential step in their ability to develop as HCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 3-13, 2017.

Mechanosensory hair cells (HCs) residing in the inner ear are critical for hearing and balance. Precise coordination of proliferation, sensory specification, and differentiation during development is essential to ensure the correct patterning of HCs in the cochlear and vestibular epithelium. Recent studies have revealed that FGF20 signaling is vital for proper HC differentiation. However, the mechanisms by which FGF20 signaling promotes HC differentiation remain unknown. Here, we show that mitogen-activated protein 3 kinase 4 (MEKK4) expression is highly regulated during inner ear development and is critical to normal cytoarchitecture and function. Mice homozygous for a kinase-inactive MEKK4 mutation exhibit significant hearing loss. Lack of MEKK4 activityin vivoalso leads to a significant reduction in the number of cochlear and vestibular HCs, suggesting that MEKK4 activity is essential for overall development of HCs within the inner ear. Furthermore, we show that loss of FGF20 signalingin vivoinhibits MEKK4 activity, whereas gain of Fgf20 function stimulates MEKK4 expression, suggesting that Fgf20 modulates MEKK4 activity to regulate cellular differentiation. Finally, we demonstrate, for the first time, that MEKK4 acts as a critical node to integrate FGF20-FGFR1 signaling responses to specifically influence HC development and that FGFR1 signaling through activation of MEKK4 is necessary for outer hair cell differentiation. Collectively, this study provides compelling evidence of an essential role for MEKK4 in inner ear morphogenesis and identifies the requirement of MEKK4 expression in regulating the specific response of FGFR1 during HC development and FGF20/FGFR1 signaling activated MEKK4 for normal sensory cell differentiation.SIGNIFICANCE STATEMENTSensory hair cells (HCs) are the mechanoreceptors within the inner ear responsible for our sense of hearing. HCs are formed before birth, and mammals lack the ability to restore the sensory deficits associated with their loss. In this study, we show, for the first time, that MEKK4 signaling is essential for the development of normal cytoarchitecture and hearing function as MEKK4 signaling-deficient mice exhibit a significant reduction of HCs and a hearing loss. We also identify MEKK4 as a critical hub kinase for FGF20-FGFR1 signaling to induce HC differentiation in the mammalian cochlea. These results reveal a new paradigm in the regulation of HC differentiation and provide significant new insights into the mechanism of Fgf signaling governing HC formation.

MAP3K1 is a serine/threonine kinase that is activated by a diverse set of stimuli and exerts its effect through various downstream effecter molecules, including JNK, ERK1/2 and p38. In humans, mutant alleles of MAP3K1 are associated with 46,XY sex reversal. Until recently, the only phenotype observed in Map3k1tm1Yxia mutant mice was open eyelids at birth. Here, we report that homozygous Map3k1tm1Yxia mice have early-onset profound hearing loss accompanied by the progressive degeneration of cochlear outer hair cells. In the mouse inner ear, MAP3K1 has punctate localization at the apical surface of the supporting cells in close proximity to basal bodies. Although the cytoarchitecture, neuronal wiring and synaptic junctions in the organ of Corti are grossly preserved, Map3k1tm1Yxia mutant mice have supernumerary functional outer hair cells (OHCs) and Deiters' cells. Loss of MAP3K1 function resulted in the downregulation of Fgfr3, Fgf8, Fgf10 and Atf3 expression in the inner ear. Fgfr3, Fgf8 and Fgf10 have a role in induction of the otic placode or in otic epithelium development in mice, and their functional deficits cause defects in cochlear morphogenesis and hearing loss. Our studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear morphogenesis genes. Collectively, our data highlight the crucial role of MAP3K1 in the development and function of the mouse inner ear and hearing., Summary: Map3k1 mutant mice exhibit early-onset profound hearing loss and supernumerary outer hair cells, along with dysregulation of the FGF signaling pathway, accentuating its function in otic epithelium development and morphogenesis.

In the cochlea, spiral ganglion neurons play a critical role in hearing as they form the relay between mechanosensory hair cells in the inner ear and cochlear nuclei in the brainstem. The proneural basic helix-loop-helix transcription factors Neurogenin1 (Neurog1) and NeuroD1 have been shown to be essential for the development of otocyst-derived inner ear sensory neurons. Here, we show neural competence of nonsensory epithelial cells in the cochlea, as ectopic expression of either Neurog1 or NeuroD1 results in the formation of neuronal cells. Since the high-mobility-group type transcription factor Sox2, which is also known to play a role in neurogenesis, is expressed in otocyst-derived neural precursor cells and later in the spiral ganglion neurons along with Neurog1 and NeuroD1, we used both gain- and loss-of-function experiments to examine the role of Sox2 in spiral ganglion neuron formation. We demonstrate that overexpression of Sox2 results in the production of neurons, suggesting that Sox2 is sufficient for the induction of neuronal fate in nonsensory epithelial cells. Furthermore, spiral ganglion neurons are absent in cochleae fromSox2Lcc/Lccmice, indicating that Sox2 is also required for neuronal formation in the cochlea. Our results indicate that Sox2, along with Neurog1 and NeuroD1, are sufficient to induce a neuronal fate in nonsensory regions of the cochlea. Finally, we demonstrate that nonsensory cells within the cochlea retain neural competence through at least the early postnatal period.

A significant proportion of hearing loss and deafness is caused by defects in the structure or function of cells within the organ of Corti. Identification of the molecular factors that regulate the development of this structure should provide valuable insights regarding inner ear formation and the signaling pathways that underlie congenital auditory deficits. In addition, targeted modulation of these same factors could be developed as therapies for hair cell regeneration.Results from experiments using transgenic and mutant mice, as well as in-vitro techniques, have identified genes and signaling pathways that are required to either specify unique auditory cell types, such as hair cells or supporting cells, or to generate the highly ordered cellular pattern that is characteristic for the organ of Corti. In particular, the hedgehog and fibroblast growth factor signaling pathways modulate the formation of the progenitor cells that will give rise to the organ of Corti. SRY-box containing gene 2, a transcription factor that is required for the formation of the cochlear progenitor cell population, has paradoxically been shown to also act as an inhibitor of hair cell development. Finally, the motor protein myosin II regulates extension of the organ of Corti and the alignment of hair cells and supporting cells into ordered rows.A better understanding of the signaling pathways that direct different aspects of cochlear development, such as specific of cell fates or cellular patterning, offers the potential to identify new pathways or molecules that could be targeted for therapeutic interventions.

In mammals, auditory perception is initially mediated through sensory cells located in a rigorously patterned mosaic of unique cell types located within the coiled cochlea. Identification of the factors that direct multipotent progenitor cells to develop as each of these specialized cell types has the potential to enhance our understanding of the development of the auditory system and to identify potential targets for regenerative therapies. Recent results have identified specific signaling molecules and pathways, including Notch, Hedgehog, Sox2 and Fgfs, that guide progenitor cells to develop first as a sensory precursor, referred to as a prosensory cell, and subsequently as one of the specialized cell types within the sensory mosaic.

Deletion of fibroblast growth factor receptor 3 (Fgfr3) leads to hearing impairment in mice due to defects in the development of the organ of Corti, the sensory epithelium of the Cochlea. To examine the role of FGFR3 in auditory development, cochleae from Fgfr3−/− mice were examined using anatomical and physiological methods. Deletion of Fgfr3 leads to the absence of inner pillar cells and an increase in other cell types, suggesting that FGFR3 regulates cell fate. Defects in outer hair cell differentiation were also observed and probably represent the primary basis for hearing loss. Furthermore, innervation defects were detected consistent with changes in the fiber guidance properties of pillar cells. To elucidate the mechanisms underlying the effects of FGFR3, we examined the expression of Bmp4, a known target. Bmp4 was increased in Fgfr3−/− cochleae, and exogenous application of bone morphogenetic protein 4 (BMP4) onto cochlear explants induced a significant increase in the outer hair cells, suggesting the Fgf and Bmp signaling act in concert to pattern the cochlea. Developmental Dynamics 236:1905–1917, 2007. Published 2007 Wiley-Liss, Inc.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development(1-8).

Wdpcp, a protein required for both planar cell polarity and ciliogenesis, regulates cell polarity and alignment via direct modulation of the actin cytoskeleton., Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration., Author Summary Cilia are microscopic cell surface hair-like protrusions that can act as antennae to mediate cell signaling. Mutations disrupting ciliogenesis can cause many developmental anomalies associated with syndromes known as “ciliopathies.” Some developmental defects, such as limb polydactyly, arise from disruption of cilia-transduced sonic hedgehog signaling, while other defects, such as aberrant patterning of hair cells in the inner ear, arise from disrupted Wnt signaling resulting in modulation of planar cell polarity (PCP)—a process whereby cells are polarized and aligned. While ciliopathy phenotypes would suggest that cilia are involved in modulating PCP, the mechanistic link between cilia and PCP has been elusive. Our study using a mouse model carrying a mutation in Wdpcp, a gene required for both ciliogenesis and PCP, suggest that Wdpcp modulation of PCP involves interactions with the actin cytoskeleton separate from its function in ciliogenesis. We observe Wdpcp localization in cilia, where it is required for recruitment of proteins essential for ciliogenesis. Wdpcp interacts with Sept2, and is also found in actin filaments, where it regulates actin dynamics essential for PCP. Together, these findings show that PCP regulation by Wdpcp is distinct from its function in ciliogenesis and involves direct modulation of the actin cytoskeleton.

Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters’ cells in the Fgfr3 P244R/ + mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.

The mammalian inner ear is a highly complex structure containing six distinct sensory organs. Each sensory organ comprises mechanosensory hair cells and nonsensory supporting cells arranged in a cellular mosaic. Hair cells and supporting cells are both derived from progenitor cells located in developing sensory patches. Hair cell differentiation is initiated through the expression of the transcription factor Atoh1. Initially, most progenitor cells within each sensory patch express Atoh1; however, subsequent cell–cell interactions lead to the downregulation of Atoh1 in cells that will develop into supporting cells. In hair cells, the expression of Atoh1 leads to the initiation of a signaling cascade that promotes hair cell development and survival.

Sox2 is a high-mobility transcription factor that is one of the earliest markers of developing inner ear prosensory domains. In humans, mutations in SOX2 cause sensorineural hearing loss and a loss of function study in mice showed that Sox2 is required for prosensory formation in the cochlea. However, the specific roles of Sox2 have not been determined. Here we illustrate a dynamic role of Sox2 as an early permissive factor in prosensory domain formation followed by a mutually antagonistic relationship with Atoh1, a bHLH protein necessary for hair cell development. We demonstrate that decreased levels of Sox2 result in precocious hair cell differentiation and an over production of inner hair cells and that these effects are likely mediated through an antagonistic interaction between Sox2 and the bHLH molecule Atoh1. Using gain- and loss-of-function experiments we provide evidence for the molecular pathway responsible for the formation of the cochlear prosensory domain. Sox2 expression is promoted by Notch signaling and Prox1, a homeobox transcription factor, is a downstream target of Sox2. These results demonstrate crucial and diverse roles for Sox2 in the development, specification, and maintenance of sensory cells within the cochlea.

The DNA damage/replication checkpoints act by sensing the presence of damaged DNA or stalled replication forks and initiate signaling pathways that arrest cell cycle progression. Here we report the cloning and characterization of Xenopus orthologues of the RFCand PCNA-related checkpoint proteins. XRad17 shares regions of homology with the five subunits of Replication factor C. XRad9, XRad1, and XHus1 (components of the 9-1-1 complex) all show homology to the DNA polymerase processivity factor PCNA. We demonstrate that these proteins associate with chromatin and are phosphorylated when replication is inhibited by aphidicolin. Phosphorylation of X9-1-1 is caffeine sensitive, but the chromatin association of XRad17 and the X9-1-1 complex after replication block is unaffected by caffeine. This suggests that the X9-1-1 complex can associate with chromatin independently of XAtm/XAtr activity. We further demonstrate that XRad17 is essential for the chromatin binding and checkpoint-dependent phosphorylation of X9-1-1 and for the activation of XChk1 when the replication checkpoint is induced by aphidicolin. XRad17 is not, however, required for the activation of XCds1 in response to dsDNA ends.

The canonical Wnt/β-catenin signaling pathway is known to play crucial roles in organogenesis by regulating both proliferation and differentiation. In the inner ear, this pathway has been shown to regulate the size of the otic placode from which the cochlea will arise; however, direct activity of canonical Wnt signaling as well as its function during cochlear mechanosensory hair cell development had yet to be identified. Using TCF/Lef:H2B-GFP reporter mice and transfection of an independent TCF/Lef reporter construct, we describe the pattern of canonical Wnt activity in the developing mouse cochlea. We show that prior to terminal mitosis, canonical Wnt activity is high in early prosensory cells from which hair cells and support cells will differentiate, and activity becomes reduced as development progresses. Using an in vitro model we demonstrate that Wnt/β-catenin signaling regulates both proliferation and hair cell differentiation within the developing cochlear duct. Inhibition of Wnt/β-catenin signaling blocks proliferation during early mitotic phases of development and inhibits hair cell formation in the differentiating organ of Corti. Conversely, activation increases the number of hair cells that differentiate and induces proliferation in prosensory cells, causing an expansion of the Sox2-positive prosensory domain. We further demonstrate that the induced proliferation of Sox2-positive cells may be mediated by the cell cycle regulator cyclin D1. Lastly, we provide evidence that the mitotic Sox2-positive cells are competent to differentiate into hair cells. Combined, our data suggest that Wnt/β-catenin signaling has a dual function in cochlear development, regulating both proliferation and hair cell differentiation.