The inner ear houses both hearing and balance sensory modalities. The hearing and balance organs consist of similar cell types, including sensory hair cells and associated supporting cells. Previously we showed that Notch1 is required for maintaining supporting cell survival during cochlear maturation. To understand the role of Notch during vestibular maturation, we deleted Notch1 from the vestibular organs of both male and female mice at birth. Histological analyses showed a reduction of supporting cells accompanied by an increase in type II hair cells, indicating a conversion of supporting cells to hair cells. Analysis of mature sensory organs indicate the converted hair cells survive, despite a severe reduction of supporting cells. Vestibular sensory evoked potentials (VsEPs), thought to be generated within the striola regions of the maculae, were absent, indicating that NOTCH1 is critical for striolar function. Specialized type I hair cells in the striola failed to develop the complex calyces typical of these cells. Notch1 mutants did not exhibit vestibular behaviors such as circling and head shaking but showed difficulties with tests of balance and swimming. These results indicate that, unlike the cochlea, supporting cells in balance organs retain the plasticity to convert to hair cells which can survive into adulthood. Despite hair cell survival, vestibular function is compromised likely due to the loss of supporting cells and altered innervation.

The inner ear houses two sensory modalities: the hearing organ, located in the cochlea, and the balance organs, located throughout the vestibular regions of the ear. Both hearing and vestibular sensory regions are composed of similar cell types, including hair cells and associated supporting cells. Recently, we showed that Notch1 is required for maintaining supporting cell survival postnatally during cochlear maturation. However, it is not known whether Notch1 plays a similar role in the balance organs of the inner ear. To characterize the role of Notch during vestibular maturation, we conditionally deleted Notch1 from Sox2-expressing cells of the vestibular organs in the mouse at P0/P1. Histological analyses showed a dramatic loss of supporting cells accompanied by an increase in type II hair cells without cell death, indicating the supporting cells are converting to hair cells in the maturing vestibular regions. Analysis of 6-week old animals indicate that the converted hair cells survive, despite the reduction of supporting cells. Interestingly, measurements of vestibular sensory evoked potentials (VsEPs), known to be generated in the striolar regions of the vestibular afferents in the maculae, failed to show a response, indicating that NOTCH1 expression is critical for striolar function postnatally. Consistent with this, we find that the specialized type I hair cells in the striola fail to develop the complex calyces typical of these cells. These defects are likely due to the reduction in supporting cells, which have previously been shown to express factors critical for the striolar region. Similar to other mutants that lack proper striolar development, Notch1 mutants do not exhibit typical vestibular behaviors such as circling and head shaking, but do show difficulties in some vestibular tests, including the balance beam and forced swim test. These results indicate that, unlike the hearing organ in which the supporting cells undergo cell death, supporting cells in the balance regions retain the ability to convert to hair cells during maturation, which survive into adulthood despite the reduction in supporting cells.

The sensory region of the mammalian hearing organ contains two main cell types—hair cells and supporting cells. During development, Notch signaling plays an important role in whether a cell becomes either a hair cell or supporting cell by mediating lateral inhibition. However, once the cell fate decisions have been determined, little is understood about the role Notch plays in cochlear maturation. Here, we report that deletion ofNotch1from the early postnatal mouse cochlea in both male and female animals resulted in profound deafness at 6 weeks of age. Histologic analyses at 6 weeks revealed significant hair cell and supporting cell loss throughout theNotch1-deficient cochlea. Early analyses revealed a reduction in supporting cells in the outer hair cell region between postnatal day (P) 2 and P6, without a comparable increase in outer hair cell number, suggesting a mechanism other than lateral inhibition. Consistent with this, we found apoptotic cells in the outer supporting cell region of the cochlea at P1 and P2, indicating thatNotch1is required for outer supporting cell survival during early cochlear maturation. Interestingly, inner supporting cell types were not lost afterNotch1deletion. Surprisingly, we do not detect outer hair cell loss inNotch1mutants until after the onset of hearing, around P14, suggesting that hair cell loss is caused by loss of the supporting cells. Together, these results demonstrate thatNotch1is required for supporting cell survival during early maturation and that loss of these cells causes later loss of the hair cells and cochlear dysfunction.SIGNIFICANCE STATEMENTDuring development, Notch signaling has been shown to be critical in regulating the cell fate choices between hair cells and supporting cells. However, little is known about how Notch functions after those cell fate choices are made. Here, we examine the role ofNotch1in the maturing cochlea. We demonstrate that deletion ofNotch1results in profound deafness by 6 weeks of age. Histologic analyses revealed rapid supporting cell death shortly afterNotch1deletion, followed by eventual loss of the hair cells. These results reveal an unexpected role for Notch in supporting cell survival during cochlear maturation.

Alzheimer's Disease (AD) is a neurodegenerative illness without a cure. All current therapies require an accurate diagnosis and staging of AD to ensure appropriate care. Central auditory processing disorders (CAPDs) and hearing loss have been associated with AD, and may precede the onset of Alzheimer's dementia. Therefore, CAPD is a possible biomarker candidate for AD diagnosis. However, little is known about how CAPD and AD pathological changes are correlated. In the present study, we investigated auditory changes in AD using transgenic amyloidosis mouse models. AD mouse models were bred to a mouse strain commonly used for auditory experiments, to compensate for the recessive accelerated hearing loss on the parent background. Auditory brainstem response (ABR) recordings revealed significant hearing loss, a reduced ABR wave I amplitude, and increased central gain in 5xFAD mice. In comparison, these effects were milder or reversed in APP/PS1 mice. Longitudinal analyses revealed that in 5xFAD mice, central gain increase preceded ABR wave I amplitude reduction and hearing loss, suggesting that it may originate from lesions in the central nervous system rather than the peripheral loss. Pharmacologically facilitating cholinergic signaling with donepezil reversed the central gain in 5xFAD mice. After the central gain increased, aging 5xFAD mice developed deficits for hearing sound pips in the presence of noise, consistent with CAPD-like symptoms of AD patients. Histological analysis revealed that amyloid plaques were deposited in the auditory cortex of both mouse strains. However, in 5xFAD but not APP/PS1 mice, plaque was observed in the upper auditory brainstem, specifically the inferior colliculus (IC) and the medial geniculate body (MGB). This plaque distribution parallels histological findings from human subjects with AD and correlates in age with central gain increase. Overall, we conclude that auditory alterations in amyloidosis mouse models correlate with amyloid deposits in the auditory brainstem and may be reversed initially through enhanced cholinergic signaling. The alteration of ABR recording related to the increase in central gain prior to AD-related hearing disorders suggests that it could potentially be used as an early biomarker of AD diagnosis.

The mammalian cochlea is an exceptionally well-organized epithelium composed of hair cells, supporting cells, and innervating neurons. Loss or defects in any of these cell types, particularly the specialized sensory hair cells, leads to deafness. The Notch pathway is known to play a critical role in the decision to become either a hair cell or a supporting cell during embryogenesis; however, little is known about how Notch functions later during cochlear maturation. Uniquely amongst Notch ligands, Jagged1 (JAG1) is localized to supporting cells during cell fate acquisition and continues to be expressed into adulthood. Here, we demonstrate that JAG1 in maturing cochlear supporting cells is essential for normal cochlear function. Specifically, we show that deletion of JAG1 during cochlear maturation disrupts the inner hair cell pathway and leads to a type of deafness clinically similar to auditory neuropathy. Common pathologies associated with disruptions in inner hair cell function, including loss of hair cells, synapses, or auditory neurons, were not observed in JAG1 mutant cochleae. Instead, RNA-seq analysis of JAG1-deficient cochleae identified dysregulation of the Rho GTPase pathway, known to be involved in stereocilia development and maintenance. Interestingly, the overexpression of one of the altered genes, Diaph3 , is responsible for autosomal dominant auditory neuropathy-1 (AUNA1) in humans and mice, and is associated with defects in the inner hair cell stereocilia. Strikingly, ultrastructural analyses of JAG1-deleted cochleae revealed stereocilia defects in inner hair cells, including fused and elongated bundles, that were similar to those stereocilia defects reported in AUNA1 mice. Taken together, these data indicate a novel role for Notch signaling in normal hearing development through maintaining stereocilia integrity of the inner hair cells during cochlear maturation.

The five vestibular organs of the inner ear derive from patches of prosensory cells that express the transcription factor SOX2 and the Notch ligand JAG1. Previous work suggests that JAG1-mediated Notch signaling is both necessary and sufficient for prosensory formation and that the separation of developing prosensory patches is regulated by LMX1a, which antagonizes Notch signaling. We used an inner ear-specific deletion of the Rbpjκ gene in which Notch signaling is progressively lost from the inner ear to show that Notch signaling, is continuously required for the maintenance of prosensory fate. Loss of Notch signaling in prosensory patches causes them to shrink and ultimately disappear. We show this loss of prosensory fate is not due to cell death, but rather to the conversion of prosensory tissue into non-sensory tissue that expresses LMX1a. Notch signaling is therefore likely to stabilize, rather than induce prosensory fate.

The transcription factor SOX2 is required for the formation of hair cells and supporting cells in the inner ear and is a widely used sensory marker. Paradoxically, we demonstrate via fate-mapping that initially SOX2 primarily marks nonsensory progenitors in the cochlea, and is not specific to all sensory regions until late otic vesicle stages. SOX2 fate-mapping reveals an apical-to-basal gradient of SOX2 expression in the sensory region of the cochlea, reflecting the pattern of cell cycle exit. To understand SOX2 function, we undertook a timed-deletion approach, revealing that early loss of SOX2 severely impaired morphological development of the ear; whereas later deletions resulted in sensory disruptions. During otocyst stages SOX2 shifted dramatically from a lateral to medial domain over 24-48hrs, reflecting the nonsensory-to-sensory switch observed by fate-mapping. Early loss or gain of SOX2 function leads to changes in otic epithelial volume and progenitor proliferation, impacting growth and morphological development of the ear. Our study demonstrates a novel role for SOX2 in early otic morphological development, and provides insights into the temporal and spatial patterns of sensory specification in the inner ear.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5, a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin (hspn)] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13hspn/hspn mice have malformations similar to those of Dlx5-/- embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13hspn/hspn embryos, indicating that the Slc25a13hspn deletion affects otic-specific enhancers of Dlx5 In addition, transheterozygous Slc25a13+/hspn Dlx5+/- mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13hspn/hspn and Dlx5-/-embryos, verifying a cis-acting effect of the Slc25a13hspn deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13hspn deleted region failed to phenocopy the defects of Slc25a13hspn/hspn mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13hspn/hspn mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear.

Heterozygous Bmp4 mutations in humans and mice cause severe ocular anterior segment dysgenesis (ASD). Abnormalities include pupil displacement, corneal opacity, iridocorneal adhesions, and variable intraocular pressure, as well as some retinal and vascular defects. It is presently not known what source of BMP4 is responsible for these defects, as BMP4 is expressed in several developing ocular and surrounding tissues. In particular, BMP4 is expressed in the ciliary margins of the optic cup which give rise to anterior segment structures such as the ciliary body and iris, making it a good candidate for the required source of BMP4 for anterior segment development. Here, we test whether ciliary margin-derived BMP4 is required for ocular development using two different conditional knockout approaches. In addition, we compared the conditional deletion phenotypes with Bmp4 heterozygous null mice. Morphological, molecular, and functional assays were performed on adult mutant mice, including histology, immunohistochemistry, in vivo imaging, and intraocular pressure measurements. Surprisingly, in contrast to Bmp4 heterozygous mutants, our analyses revealed that the anterior and posterior segments of Bmp4 conditional knockouts developed normally. These results indicate that ciliary margin-derived BMP4 does not have a major role in ocular development, although subtle alterations could not be ruled out. Furthermore, we demonstrated that the anterior and posterior phenotypes observed in Bmp4 heterozygous animals showed a strong propensity to co-occur, suggesting a common, non-cell autonomous source for these defects.

The trabecular meshwork (TM), a tissue residing in the iridocorneal angle of the eye, is the primary site of aqueous humor outflow and often develops abnormally in children with anterior segment dysgenesis (ASD). However, the cellular mechanisms underlying both normal and pathophysiological TM formation are poorly understood. Here, we improve the characterization of TM development via morphological and molecular analyses. We first assessed the TM of wild-type C57BL/6J mice at multiple time points throughout development (E15.5–P21). The morphology of TM cells, rate of cell division, presence of apoptotic cell death, and age of onset of an established TM marker (αSMA) were each assessed in the developing iridocorneal angle. We discovered that TM cells are identifiable histologically at P1, which coincided with both the onset of αSMA expression and a significant decrease in TM precursor cell proliferation. Significant apoptotic cell death was not detected during TM development. These findings were then used to assess two mouse models of ASD. Jag1 and Bmp4 heterozygous null mice display ASD phenotypes in the adult, including TM hypoplasia and corneal adherence to the iris. We further discovered that both mutants exhibited similar patterns of developmental TM dysgenesis at P1, P5, and P10. Our data indicate that P1 is an important time point in TM development and that TM dysgenesis in Jag1 and Bmp4 heterozygous null mice likely results from impaired TM cell migration and/or differentiation.

Neurons of the cochleovestibular ganglion (CVG) transmit hearing and balance information to the brain. During development, a select population of early otic progenitors express NEUROG1, delaminate from the otocyst, and coalesce to form the neurons that innervate all inner ear sensory regions. At present, the selection process that determines which otic progenitors activate NEUROG1 and adopt a neuroblast fate is incompletely understood. The transcription factor SOX2 has been implicated in otic neurogenesis, but its requirement in the specification of the CVG neurons has not been established. Here we tested SOX2's requirement during inner ear neuronal specification using a conditional deletion paradigm in the mouse. SOX2 deficiency at otocyst stages caused a near-absence of NEUROG1-expressing neuroblasts, increased cell death in the neurosensory epithelium, and significantly reduced the CVG volume. Interestingly, a milder decrease in neurogenesis was observed in heterozygotes, indicating SOX2 levels are important. Moreover, fate-mapping experiments revealed that the timing of SOX2 expression did not parallel the established vestibular-then-auditory sequence. These results demonstrate that SOX2 is required for the initial events in otic neuronal specification including expression of NEUROG1, although fate-mapping results suggest SOX2 may be required as a competence factor rather than a direct initiator of the neural fate.

Expression levels of Notch signaling molecules are increased in synovium from patients with rheumatoid arthritis (RA). However, it is not known which cell type(s) in RA synovium have Notch activation or if they play a pathogenetic role in RA. Here, we used Hes1-GFP/TNF-transgenic (TNF-Tg) mice to investigate the role of cells with active Notch signaling (GFP+) in RA. The number of GFP+ cells was significantly increased in synovium in Hes1-GFP/TNF-Tg mice and about 60% of them were F4/80+ macrophages expressing the inflammatory macrophage (M1) marker. TNF-Tg mice transplanted with Hes1-GFP/TNF-Tg bone marrow (BM) had significantly more GFP+ cells in their synovium than in BM. Intraarticular injection of Hes1-GFP/TNF-Tg or Hes1-GFP+ BM macrophages into WT and TNF-Tg mice showed the highest synovial GFP+ cells in the TNF-Tg mice that received Hes1-GFP/TNF-Tg cells. Thapsigargin (THAP), a Notch inhibitor, decreased TNF-induced M1 and increased M2 numbers and reduced joint lesion, synovial M1s, and GFP+ cells in Hes1-GFP/TNF-Tg mice. THAP did not affect M1s from mice carrying a constitutively active Notch1. Thus, the main cells with activated Notch signaling in the inflamed synovium of TNF-Tg mice are M1s derived from BM and targeting them may represent a new therapeutic approach for patients with inflammatory arthritis. © 2017 American Society for Bone and Mineral Research.

While all forms of glaucoma are characterized by a specific pattern of retinal ganglion cell death, they are clinically divided into several distinct subclasses, including normal tension glaucoma, primary open angle glaucoma, congenital glaucoma, and secondary glaucoma. For each type of glaucoma there are likely numerous molecular pathways that control susceptibility to the disease. Given this complexity, a single animal model will never precisely model all aspects of all the different types of human glaucoma. Therefore, multiple animal models have been utilized to study glaucoma but more are needed. Because of the powerful genetic tools available to use in the laboratory mouse, it has proven to be a highly useful mammalian system for studying the pathophysiology of human disease. The similarity between human and mouse eyes coupled with the ability to use a combination of advanced cell biological and genetic tools in mice have led to a large increase in the number of studies using mice to model specific glaucoma phenotypes. Over the last decade, numerous new mouse models and genetic tools have emerged, providing important insight into the cell biology and genetics of glaucoma. In this review, we describe available mouse genetic models that can be used to study glaucoma-relevant disease/pathobiology. Furthermore, we discuss how these models have been used to gain insights into ocular hypertension (a major risk factor for glaucoma) and glaucomatous retinal ganglion cell death. Finally, the potential for developing new mouse models and using advanced genetic tools and resources for studying glaucoma are discussed.

Purpose To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear. Methods An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). The differentiation of NICD-expressing hair cells was assessed at postnatal day (P) 6, 11 and 20, using histological and molecular markers for hair cells, as well as supporting cells/progenitor cells. We also examined whether the effects of Notch were mediated by SOX2, a gene expressed in supporting cells and a likely downstream target of Notch, by crossing an inducible form of SOX2 to the Gfi1-Cre. Results Activation of Notch1 in developing auditory hair cells causes profound deafness. The NICD-expressing hair cells switch off a number of hair cell markers and lose their characteristic morphology. Instead, NICD-expressing hair cells adopt a morphology resembling supporting cells and upregulate a number of supporting cell markers. These effects do not appear to be mediated by SOX2, because although expression of SOX2 caused some hearing impairment, the SOX2-expressing hair cells did not downregulate hair cell markers nor exhibit a supporting cell-like phenotype. Conclusions Our data show that Notch signaling inhibits hair cell differentiation and promotes a supporting cell-like phenotype, and that these effects are unlikely to be mediated by SOX2.

In mammals, formation of the auditory sensory organ (the organ of Corti) is restricted to a specialized area of the cochlea. However, the molecular mechanisms limiting sensory formation to this discrete region in the ventral cochlear duct are not well understood, nor is it known whether other regions of the cochlea have the competence to form the organ of Corti. Here we identify LMO4, a LIM-domain-only nuclear protein, as a negative regulator of sensory organ formation in the cochlea. Inactivation of Lmo4 in mice leads to an ectopic organ of Corti (eOC) located in the lateral cochlea. The eOC retains the features of the native organ, including inner and outer hair cells, supporting cells, and other nonsensory specialized cell types. However, the eOC shows an orientation opposite to the native organ, such that the eOC appears as a mirror-image duplication to the native organ of Corti. These data demonstrate a novel sensory competent region in the lateral cochlear duct that is regulated by LMO4 and may be amenable to therapeutic manipulation.

Sox2 regulates the self-renewal of multiple types of stem cells. Recent studies suggest it also plays oncogenic roles in the formation of squamous carcinoma in several organs, including the esophagus where Sox2 is predominantly expressed in the basal progenitor cells of the stratified epithelium. Here, we use mouse genetic models to reveal a mechanism by which Sox2 cooperates with microenvironmental signals to malignantly transform epithelial progenitor cells. Conditional overexpression of Sox2 in basal cells expands the progenitor population in both the esophagus and forestomach. Significantly, carcinoma only develops in the forestomach, where pathological progression correlates with inflammation and nuclear localization of Stat3 in progenitor cells. Importantly, co-overexpression of Sox2 and activated Stat3 (Stat3C) also transforms esophageal basal cells but not the differentiated suprabasal cells. These findings indicate that basal stem/progenitor cells are the cells of origin of squamous carcinoma and that cooperation between Sox2 and microenvironment-activated Stat3 is required for Sox2-driven tumorigenesis.

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to cross-talk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.