foxg1a is required for hair cell development and regeneration in the zebrafish lateral line.

Mechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Aquatic vertebrates like zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, lateral line hair cells can robustly regenerate following damage. In mammals, the transcription factor Foxg1 functions to promote normal development of the inner ear. Foxg1a is expressed in lateral line sensory organs in zebrafish larvae, but its function during lateral line development and regeneration has not been investigated. Our study demonstrates that mutation of foxg1a results in slower posterior lateral line primordium migration and delayed neuromast formation. In developing and regenerating neuromasts, we find that loss of Foxg1a function results in reduced hair cell numbers, as well as decreased proliferation of neuromast cells. Foxg1a specifically regulates the development and regeneration of Islet1-labeled hair cells. These data suggest that Foxg1 may be a valuable target for investigation of clinical hair cell regeneration.

The people behind the papers - James O'Sullivan and Zoë Mann.

To build a functional inner ear, hair cell morphology must be precisely controlled along the proximo-distal axis. A new paper in Development shows that differential mitochondrial dynamics in proximal versus distal cells impacts on the apical cell surface area - a key aspect of morphology. To find out more about this work, we spoke to first author James O'Sullivan and senior author Zoë Mann, both at King's College London, UK.

Mitochondrial dynamics regulate cell morphology in the developing cochlea.

In multicellular tissues, the size and shape of cells are intricately linked with their physiological functions. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells, which have distinct morphologies and functional properties at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs, are larger than their distal (low-frequency) counterparts, a morphological feature essential for sound perception. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this as a potential mechanism for regulating the shape of developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared with distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC surface area, whereas promotion of fusion increased the distal HC surface area. We identify mitochondrial dynamics as a key regulator of HC morphology in developing inner ear epithelia.

Metabolic Profiling of Cochlear Organoids Identifies α-Ketoglutarate and NAD+ as Limiting Factors for Hair Cell Reprogramming.

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, detailed metabolomic characterizations of the expanding and differentiating organoids are performed. It is found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α-ketoglutarate (α-KG) and nicotinamide adenine dinucleotide (NAD+). Reduced expression of NAD+ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α-KG and NAD+ promotes hair cell reprogramming both in vitro and in vivo. These findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming.

Transdifferentiation is temporally uncoupled from progenitor pool expansion during hair cell regeneration in the zebrafish inner ear.

Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates, including zebrafish, can robustly regenerate hair cells after severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here, we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and we observed gradual regeneration with correct spatial patterning over a 2-week period following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells temporally uncoupled from supporting cell division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear.

MEK/ERK signaling drives the transdifferentiation of supporting cells into functional hair cells by modulating the Notch pathway.

Loss of cochlear hair cells (HCs) leads to permanent hearing loss in mammals, and regenerative medicine is regarded as an ideal strategy for hearing recovery. Limited genetic and pharmaceutical approaches for HC regeneration have been established, and the existing strategies cannot achieve recovery of auditory function. A promising target to promote HC regeneration is MEK/ERK signaling because dynamic shifts in its activity during the critical stages of inner ear development have been observed. Here, we first showed that MEK/ERK signaling is activated specifically in supporting cells (SCs) after aminoglycoside-induced HC injury. We then selected 4 MEK/ERK signaling inhibitors, and PD0325901 (PD03) was found to induce the transdifferentiation of functional supernumerary HCs from SCs in the neonatal mammalian cochlear epithelium. We next found that PD03 facilitated the generation of HCs in inner ear organoids. Through genome-wide high-throughput RNA sequencing and verification, we found that the Notch pathway is the downstream target of MEK/ERK signaling. Importantly, delivery of PD03 into the inner ear induced mild HC regeneration in vivo. Our study thus reveals the importance of MEK/ERK signaling in cell fate determination and suggests that PD03 might serve as a new approach for HC regeneration.

The Suppression of Ubiquitin C-Terminal Hydrolase L1 Promotes the Transdifferentiation of Auditory Supporting Cells into Hair Cells by Regulating the mTOR Pathway.

In mammals, hearing loss is irreversible due to the lack of the regenerative capacity of the auditory epithelium. However, stem/progenitor cells in mammalian cochleae may be a therapeutic target for hearing regeneration. The ubiquitin proteasome system plays an important role in cochlear development and maintenance. In this study, we investigated the role of ubiquitin C-terminal hydrolase L1 (UCHL1) in the process of the transdifferentiation of auditory supporting cells (SCs) into hair cells (HCs). The expression of UCHL1 gradually decreased as HCs developed and was restricted to inner pillar cells and third-row Deiters' cells between P2 and P7, suggesting that UCHL1-expressing cells are similar to the cells with Lgr5-positive progenitors. UCHL1 expression was decreased even under conditions in which supernumerary HCs were generated with a γ-secretase inhibitor and Wnt agonist. Moreover, the inhibition of UCHL1 by LDN-57444 led to an increase in HC numbers. Mechanistically, LDN-57444 increased mTOR complex 1 activity and allowed SCs to transdifferentiate into HCs. The suppression of UCHL1 induces the transdifferentiation of auditory SCs and progenitors into HCs by regulating the mTOR pathway.

Emx2 lineage tracing reveals antecedent patterns of planar polarity in the mouse inner ear.

The planar polarized organization of hair cells in the vestibular maculae is unique because these sensory organs contain two groups of cells with oppositely oriented stereociliary bundles that meet at a line of polarity reversal (LPR). EMX2 is a transcription factor expressed by one hair cell group that reverses the orientation of their bundles, thereby forming the LPR. We generated Emx2-CreERt2 transgenic mice for genetic lineage tracing and demonstrate Emx2 expression before hair cell specification when the nascent utricle and saccule constitute a continuous prosensory domain. Precursors labeled by Emx2-CreERt2 at this stage give rise to hair cells located along one side of the LPR in the mature utricle or saccule, indicating that this boundary is first established in the prosensory domain. Consistent with this, Emx2-CreERt2 lineage tracing in Dreher mutants, where the utricle and saccule fail to segregate, labels a continuous field of cells along one side of a fused utriculo-saccular-cochlear organ. These observations reveal that LPR positioning is pre-determined in the developing prosensory domain, and that EMX2 expression defines lineages of hair cells with oppositely oriented stereociliary bundles.

Pcolce2 overexpression promotes supporting cell reprogramming in the neonatal mouse cochlea.

Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.

AAV-mediated Gpm6b expression supports hair cell reprogramming.

Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.

Localization of cadherins in the postnatal cochlear epithelium and their relation to space formation.

The sensory epithelium of the cochlea, the organ of Corti, has complex cytoarchitecture consisting of mechanosensory hair cells intercalated by epithelial support cells. The support cells provide important trophic and structural support to the hair cells. Thus, the support cells must be stiff yet compliant enough to withstand and modulate vibrations to the hair cells. Once the sensory cells are properly patterned, the support cells undergo significant remodeling from a simple epithelium into a structurally rigid epithelium with fluid-filled spaces in the murine cochlea. Cell adhesion molecules such as cadherins are necessary for sorting and connecting cells in an intact epithelium. To create the fluid-filled spaces, cell adhesion properties of adjoining cell membranes between cells must change to allow the formation of spaces within an epithelium. However, the dynamic localization of cadherins has not been properly analyzed as these spaces are formed. There are three cadherins that are reported to be expressed during the first postnatal week of development when the tunnel of Corti forms in the cochlea. In this study, we characterize the dynamic localization of cadherins that are associated with cytoskeletal remodeling at the contacting membranes of the inner and outer pillar cells flanking the tunnel of Corti.

Dync1li1 is required for the survival of mammalian cochlear hair cells by regulating the transportation of autophagosomes.

Dync1li1, a subunit of cytoplasmic dynein 1, is reported to play important roles in intracellular retrograde transport in many tissues. However, the roles of Dync1li1 in the mammalian cochlea remain uninvestigated. Here we first studied the expression pattern of Dync1li1 in the mouse cochlea and found that Dync1li1 is highly expressed in hair cells (HCs) in both neonatal and adult mice cochlea. Next, we used Dync1li1 knockout (KO) mice to investigate its effects on hearing and found that deletion of Dync1li1 leads to early onset of progressive HC loss via apoptosis and to subsequent hearing loss. Further studies revealed that loss of Dync1li1 destabilizes dynein and alters the normal function of dynein. In addition, Dync1li1 KO results in a thinner Golgi apparatus and the accumulation of LC3+ autophagic vacuoles, which triggers HC apoptosis. We also knocked down Dync1li1 in the OC1 cells and found that the number of autophagosomes were significantly increased while the number of autolysosomes were decreased, which suggested that Dync1li1 knockdown leads to impaired transportation of autophagosomes to lysosomes and therefore the accumulation of autophagosomes results in HC apoptosis. Our findings demonstrate that Dync1li1 plays important roles in HC survival through the regulation of autophagosome transportation.

Overexpression of XIAP inhibits cisplatin-induced hair cell loss.

Cisplatin is a platinum-containing drug with ototoxicity commonly used clinically and has significant efficacy against a variety of solid tumors. One of the most important mechanisms of ototoxicity is that cisplatin induces apoptosis of hair cells. According to relevant literature, X-linked inhibitor of apoptosis protein (XIAP, anti-apoptotic protein) could inhibit the apoptotic pathway. We hypothesized that this protein might protect cochlear hair cells from cisplatin-induced injury. To figure it out, we treated cochlea of normal mice with various concentrations of cisplatin to observe the response and morphology of hair cells and determine a reasonable concentration. Next, Western Blot and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) experiments were conducted to make an investigation about the expression of XIAP protein and mRNA. In addition, we constructed and identified XIAP overexpressing mice. Finally, we treated cochlear tissues of normal and overexpressing mice with cisplatin to investigate the cyto-protection of XIAP on hair cells, respectively. It was found that 50 µmol/L cisplatin resulted in significant loss and disorganization of hair cells, while simultaneously downregulating the protein and mRNA of XIAP. In XIAP overexpressing mice, the loss and disorganization of hair cells were significantly lessened. These results showed that XIAP can lessen cisplatin-induced hair cell loss and play a role in otoprotection.

Puerarin attenuates cisplatin-induced apoptosis of hair cells through the mitochondrial apoptotic pathway.

Puerarin, one of the main components of Pueraria lobata, has been reported to possess a wide range of pharmacological activities, including anti-inflammatory, antioxidative and anti-apoptotic effects. However, the role of puerarin in ototoxic drug-induced hair cell injury has not been well characterized. This study explored whether puerarin protects against cisplatin-induced hair cell damage and its potential mechanisms. The viability of puerarin-treated HEI-OC1 cells was assessed by CCK8 assay. Reactive oxygen species (ROS) was estimated with flow cytometric analysis using Cellrox Green fluorescent probe. Apoptosis-related protein levels were detected by western blot analysis. Immunostaining of the organ of Corti was performed to determine mice cochlear hair cell survival. Our results showed that puerarin improved cell viability and suppressed apoptosis in the cisplatin-damaged HEI-OC1 cells and cochlear hair cells. Mechanistic studies revealed that puerarin attenuated mitochondrial apoptosis pathway by regulating apoptotic related proteins, such as Bax and cleaved caspase-3, and attenuated ROS accumulation after cisplatin damage. Moreover, puerarin was involved in regulating the Akt pathway in HEI-OC1 cells in response to cisplatin. Our results demonstrated that puerarin administration decreased the sensitivity to apoptosis dependent on the mitochondrial apoptotic pathway by reducing ROS generation, which could be used as a new protective agent against cisplatin-induced ototoxicity.

Interferon regulatory factor-7 is required for hair cell development during zebrafish embryogenesis.

Interferon regulatory factor-7 (IRF7) is an essential regulator of both innate and adaptive immunity. It is also expressed in the otic vesicle of zebrafish embryos. However, any role for irf7 in hair cell development was uncharacterized. Does it work as a potential deaf gene to regulate hair cell development? We used whole-mount in situ hybridization (WISH) assay and morpholino-mediated gene knockdown method to investigate the role of irf7 in the development of otic vesicle hair cells during zebrafish embryogenesis. We performed RNA sequencing to gain a detailed insight into the molecules/genes which are altered upon downregulation of irf7. Compared to the wild-type siblings, knockdown of irf7 resulted in severe developmental retardation in zebrafish embryos as well as loss of neuromasts and damage to hair cells at an early stage (within 3 days post fertilization). Coinjection of zebrafish irf7 mRNA could partially rescued the defects of the morphants. atp1b2b mRNA injection can also partially rescue the phenotype induced by irf7 gene deficiency. Loss of hair cells in irf7-morphants does not result from cell apoptosis. Gene expression profiles show that, compared to wild-type, knockdown of irf7 can lead to 2053 and 2678 genes being upregulated and downregulated, respectively. Among them, 18 genes were annotated to hair cell (HC) development or posterior lateral line (PLL) development. All results suggest that irf7 plays an essential role in hair cell development in zebrafish, indicating that irf7 may be a member of deafness gene family.

Zinc is an essential element for the maintenance of redox homeostasis and cell cycle in murine auditory hair cells.

A nutrition deficiency is one of the various causes of hearing loss. Zinc is an essential element for cell proliferation, antioxidant reactions, and the maintenance of hearing ability. Our previous studies have reported that the auditory brainstem response (ABR) threshold is increased in mice fed with zinc-deficient diets. However, the molecular mechanism of zinc involved in auditory system remains to be elucidated. In the present study, we examined the detrimental effects of zinc deficiency on cell cycle progression in murine auditory cells (HEI-OC1). The treatment of HEI-OC1 cells with 0.5 µM TPEN (N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine) for 24 h inhibited cell proliferation, accumulation of reactive oxygen species (ROS), and induction of apoptosis. The cell proliferation block was caused by a G1/S phase arrest. Supplementation of the cell growth medium with 5 µM ZnCl2 after exposure to TPEN attenuated ROS accumulation and the arrest caused by the zinc deficiency. The ABR threshold was elevated in mice fed with a zinc-deficient diet. Additionally, we observed an increased expression of p21 and decreased expression of cyclin E and pRb in the spiral ganglion (SG), the organ of Corti (OC), Limbus (L), and stria vascularis (SV) in the zinc-deficient mouse cochlea. These results indicated that zinc is an essential nutrient for proliferation via the cell cycle and that a dysregulation of the cell cycle may cause hearing loss.

Cy3-ATP labeling of unfixed, permeabilized mouse hair cells.

ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced-but did not disappear altogether-in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

Isolation of sensory hair cell specific exosomes in human perilymph.

Evaluation of hearing loss patients using clinical audiometry has been unable to give a definitive cellular or molecular diagnosis, hampering the development of treatments of sensorineural hearing loss. However, biopsy of inner ear tissue without losing residual hearing function for pathologic diagnosis is extremely challenging. In a clinical setting, perilymph can be accessed, potentially allowing the development of fluid based diagnostic tests. Recent approaches to improving inner ear diagnostics have been focusing on the evaluation of the proteomic or miRNA profiles of perilymph. Inspired by recent characterization and classification of many neurodegenerative diseases using exosomes which not only are produced in locally in diseased tissue but are transported beyond the blood brain barrier, we demonstrate the isolation of human inner ear specific exosomes using a novel ultrasensitive immunomagnetic nano pom-poms capture-release approach. Using perilymph samples harvested from surgical procedures, we were able to isolate exosomes from sensorineural hearing loss patients in only 2-5 µL of perilymph. By isolating sensory hair cell derived exosomes through their expression level of myosin VIIa, we for the first-time sample material from hair cells in the living human inner ear. This work sets up the first demonstration of immunomagnetic capture-release nano pom-pom isolated exosomes for liquid biopsy diagnosis of sensorineural hearing loss. With the ability to isolate exosomes derived from different cell types for molecular characterization, this method also can be developed for analyzing exosomal biomarkers from more accessible patient tissue fluids such as plasma.

High-throughput screening on cochlear organoids identifies VEGFR-MEK-TGFB1 signaling promoting hair cell reprogramming.

Hair cell degeneration is a major cause of sensorineural hearing loss. Hair cells in mammalian cochlea do not spontaneously regenerate, posing a great challenge for restoration of hearing. Here, we establish a robust, high-throughput cochlear organoid platform that facilitates 3D expansion of cochlear progenitor cells and differentiation of hair cells in a temporally regulated manner. High-throughput screening of the FDA-approved drug library identified regorafenib, a VEGFR inhibitor, as a potent small molecule for hair cell differentiation. Regorafenib also promotes reprogramming and maturation of hair cells in both normal and neomycin-damaged cochlear explants. Mechanistically, inhibition of VEGFR suppresses TGFB1 expression via the MEK pathway and TGFB1 downregulation directly mediates the effect of regorafenib on hair cell reprogramming. Our study not only demonstrates the power of a cochlear organoid platform in high-throughput analyses of hair cell physiology but also highlights VEGFR-MEK-TGFB1 signaling crosstalk as a potential target for hair cell regeneration and hearing restoration.