Cellular reprogramming with ATOH1, GFI1, and POU4F3 implicate epigenetic changes and cell-cell signaling as obstacles to hair cell regeneration in mature mammals.

Reprogramming of the cochlea with hair-cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, 1 week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming 1 week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.

Tbx2 is a master regulator of inner versus outer hair cell differentiation.

The cochlea uses two types of mechanosensory cell to detect sounds. A single row of inner hair cells (IHCs) synapse onto neurons to transmit sensory information to the brain, and three rows of outer hair cells (OHCs) selectively amplify auditory inputs1. So far, two transcription factors have been implicated in the specific differentiation of OHCs, whereas, to our knowledge, none has been identified in the differentiation of IHCs2-4. One such transcription factor for OHCs, INSM1, acts during a crucial embryonic period to consolidate the OHC fate, preventing OHCs from transdifferentiating into IHCs2. In the absence of INSM1, embryonic OHCs misexpress a core set of IHC-specific genes, which we predict are involved in IHC differentiation. Here we find that one of these genes, Tbx2, is a master regulator of IHC versus OHC differentiation in mice. Ablation of Tbx2 in embryonic IHCs results in their development as OHCs, expressing early OHC markers such as Insm1 and eventually becoming completely mature OHCs in the position of IHCs. Furthermore, Tbx2 is epistatic to Insm1: in the absence of both genes, cochleae generate only OHCs, which suggests that TBX2 is necessary for the abnormal transdifferentiation of INSM1-deficient OHCs into IHCs, as well as for normal IHC differentiation. Ablation of Tbx2 in postnatal, largely differentiated IHCs makes them transdifferentiate directly into OHCs, replacing IHC features with those of mature and not embryonic OHCs. Finally, ectopic expression of Tbx2 in OHCs results in their transdifferentiation into IHCs. Hence, Tbx2 is both necessary and sufficient to make IHCs distinct from OHCs and maintain this difference throughout development.

A convolutional neural-network framework for modelling auditory sensory cells and synapses.

In classical computational neuroscience, analytical model descriptions are derived from neuronal recordings to mimic the underlying biological system. These neuronal models are typically slow to compute and cannot be integrated within large-scale neuronal simulation frameworks. We present a hybrid, machine-learning and computational-neuroscience approach that transforms analytical models of sensory neurons and synapses into deep-neural-network (DNN) neuronal units with the same biophysical properties. Our DNN-model architecture comprises parallel and differentiable equations that can be used for backpropagation in neuro-engineering applications, and offers a simulation run-time improvement factor of 70 and 280 on CPU or GPU systems respectively. We focussed our development on auditory neurons and synapses, and show that our DNN-model architecture can be extended to a variety of existing analytical models. We describe how our approach for auditory models can be applied to other neuron and synapse types to help accelerate the development of large-scale brain networks and DNN-based treatments of the pathological system.

Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues.

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases-the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

Efferent feedback controls bilateral auditory spontaneous activity.

In the developing auditory system, spontaneous activity generated in the cochleae propagates into the central nervous system to promote circuit formation. The effects of peripheral firing patterns on spontaneous activity in the central auditory system are not well understood. Here, we describe wide-spread bilateral coupling of spontaneous activity that coincides with the period of transient efferent modulation of inner hair cells from the brainstem medial olivocochlear system. Knocking out α9/α10 nicotinic acetylcholine receptors, a requisite part of the efferent pathway, profoundly reduces bilateral correlations. Pharmacological and chemogenetic experiments confirm that the efferent system is necessary for normal bilateral coupling. Moreover, auditory sensitivity at hearing onset is reduced in the absence of pre-hearing efferent modulation. Together, these results demonstrate how afferent and efferent pathways collectively shape spontaneous activity patterns and reveal the important role of efferents in coordinating bilateral spontaneous activity and the emergence of functional responses during the prehearing period.

Cell-type identity of the avian cochlea.

In contrast to mammals, birds recover naturally from acquired hearing loss, which makes them an ideal model for inner ear regeneration research. Here, we present a validated single-cell RNA sequencing resource of the avian cochlea. We describe specific markers for three distinct types of sensory hair cells, including a previously unknown subgroup, which we call superior tall hair cells. We identify markers for the supporting cells associated with tall hair cells, which represent the facultative stem cells of the avian inner ear. Likewise, we present markers for supporting cells that are located below the short cochlear hair cells. We further infer spatial expression gradients of hair cell genes along the tonotopic axis of the cochlea. This resource advances neurobiology, comparative biology, and regenerative medicine by providing a basis for comparative studies with non-regenerating mammalian cochleae and for longitudinal studies of the regenerating avian cochlea.

Electron Microscopic Reconstruction of Neural Circuitry in the Cochlea.

Recent studies reveal great diversity in the structure, function, and efferent innervation of afferent synaptic connections between the cochlear inner hair cells (IHCs) and spiral ganglion neurons (SGNs), which likely enables audition to process a wide range of sound pressures. By performing an extensive electron microscopic (EM) reconstruction of the neural circuitry in the mature mouse organ of Corti, we demonstrate that afferent SGN dendrites differ in abundance and composition of efferent innervation in a manner dependent on their afferent synaptic connectivity with IHCs. SGNs that sample glutamate release from several presynaptic ribbons receive more efferent innervation from lateral olivocochlear projections than those driven by a single ribbon. Next to the prevailing unbranched SGN dendrites, we found branched SGN dendrites that can contact several ribbons of 1-2 IHCs. Unexpectedly, medial olivocochlear neurons provide efferent innervation of SGN dendrites, preferring those forming single-ribbon, pillar-side synapses. We propose a fine-tuning of afferent and efferent SGN innervation.

A sensory cell diversifies its output by varying Ca2+ influx-release coupling among active zones.

The cochlea encodes sound pressures varying over six orders of magnitude by collective operation of functionally diverse spiral ganglion neurons (SGNs). The mechanisms enabling this functional diversity remain elusive. Here, we asked whether the sound intensity information, contained in the receptor potential of the presynaptic inner hair cell (IHC), is fractionated via heterogeneous synapses. We studied the transfer function of individual IHC synapses by combining patch-clamp recordings with dual-color Rhod-FF and iGluSnFR imaging of presynaptic Ca2+ signals and glutamate release. Synapses differed in the voltage dependence of release: Those residing at the IHC' pillar side activated at more hyperpolarized potentials and typically showed tight control of release by few Ca2+ channels. We conclude that heterogeneity of voltage dependence and release site coupling of Ca2+ channels among the synapses varies synaptic transfer within individual IHCs and, thereby, likely contributes to the functional diversity of SGNs. The mechanism reported here might serve sensory cells and neurons more generally to diversify signaling even in close-by synapses.

Building inner ears: recent advances and future challenges for in vitro organoid systems.

While inner ear disorders are common, our ability to intervene and recover their sensory function is limited. In vitro models of the inner ear, like the organoid system, could aid in identifying new regenerative drugs and gene therapies. Here, we provide a perspective on the status of in vitro inner ear models and guidance on how to improve their applicability in translational research. We highlight the generation of inner ear cell types from pluripotent stem cells as a particularly promising focus of research. Several exciting recent studies have shown how the developmental signaling cues of embryonic and fetal development can be mimicked to differentiate stem cells into "inner ear organoids" containing otic progenitor cells, hair cells, and neurons. However, current differentiation protocols and our knowledge of embryonic and fetal inner ear development in general, have a bias toward the sensory epithelia of the inner ear. We propose that a more holistic view is needed to better model the inner ear in vitro. Moving forward, attention should be made to the broader diversity of neuroglial and mesenchymal cell types of the inner ear, and how they interact in space or time during development. With improved control of epithelial, neuroglial, and mesenchymal cell fate specification, inner ear organoids would have the ability to truly recapitulate neurosensory function and dysfunction. We conclude by discussing how single-cell atlases of the developing inner ear and technical innovations will be critical tools to advance inner ear organoid platforms for future pre-clinical applications.

The Mechanosensory Transduction Machinery in Inner Ear Hair Cells.

Sound-induced mechanical stimuli are detected by elaborate mechanosensory transduction (MT) machinery in highly specialized hair cells of the inner ear. Genetic studies of inherited deafness in the past decades have uncovered several molecular constituents of the MT complex, and intense debate has surrounded the molecular identity of the pore-forming subunits. How the MT components function in concert in response to physical stimulation is not fully understood. In this review, we summarize and discuss multiple lines of evidence supporting the hypothesis that transmembrane channel-like 1 is a long-sought MT channel subunit. We also review specific roles of other components of the MT complex, including protocadherin 15, cadherin 23, lipoma HMGIC fusion partner-like 5, transmembrane inner ear, calcium and integrin-binding family member 2, and ankyrins. Based on these recent advances, we propose a unifying theory of hair cell MT that may reconcile most of the functional discoveries obtained to date. Finally, we discuss key questions that need to be addressed for a comprehensive understanding of hair cell MT at molecular and atomic levels.

SGN nerve filaments develop synapses with IHCs earlier than with OHCs in C57BL/6 mouse inner ear.

OBJECTIVE: To explore the connections between hair cells and spiral ganglion neurons (SGNs) during the development of the C57BL/6 mouse inner ear. MATERIALS AND METHODS: The specimens of C57BL/6 mouse inner ear, from E15 (embryo day 15) to adult mouse, were collected; immunohistochemistry was employed to explore the frozen sections of specimens. RESULTS: The development of cochlea starts sequentially from the basal turn to the apex turn. Morphological development of SGNs occurs mainly from E16 to P12 (postnatal day 12). Hair cells appear from E18 to P12, and inner hair cells (IHCs) develop earlier than outer hair cells (OHCs). The connections between hair cells and SGNs begin to develop during E18-P1, morphologically resemble mature synapses during P8-P12, and completely mature in adult mice. CONCLUSIONS: The genesis of auditory ribbon synapse occurs from E18 to P1. Synchronized with the development of SGNs and hair cells, the functional filaments remain connected to hair cells, while the spare ones get disconnected from the surface of hair cells. Connections between SGN nerve filaments and IHCs occur earlier than those between SGN nerve filaments and OHCs.

GFI1 functions to repress neuronal gene expression in the developing inner ear hair cells.

Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.

Recent advancements in understanding the role of epigenetics in the auditory system.

Sensorineural deafness in mammals is most commonly caused by damage to inner ear sensory epithelia, or hair cells, and can be attributed to genetic and environmental causes. After undergoing trauma, many non-mammalian organisms, including reptiles, birds, and zebrafish, are capable of regenerating damaged hair cells. Mammals, however, are not capable of regenerating damaged inner ear sensory epithelia, so that hair cell damage is permanent and can lead to hearing loss. The field of epigenetics, which is the study of various phenotypic changes caused by modification of genetic expression rather than alteration of DNA sequence, has seen numerous developments in uncovering biological mechanisms of gene expression and creating various medical treatments. However, there is a lack of information on the precise contribution of epigenetic modifications in the auditory system, specifically regarding their correlation with development of inner ear (cochlea) and consequent hearing impairment. Current studies have suggested that epigenetic modifications influence differentiation, development, and protection of auditory hair cells in cochlea, and can lead to hair cell degeneration. The objective of this article is to review the existing literature and discuss the advancements made in understanding epigenetic modifications of inner ear sensory epithelial cells. The analysis of the emerging epigenetic mechanisms related to inner ear sensory epithelial cells development, differentiation, protection, and regeneration will pave the way to develop novel therapeutic strategies for hearing loss.

Macromolecular and electrical coupling between inner hair cells in the rodent cochlea.

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in 'mini-syncytia'. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.

Characterization of the development of the mouse cochlear epithelium at the single cell level.

Mammalian hearing requires the development of the organ of Corti, a sensory epithelium comprising unique cell types. The limited number of each of these cell types, combined with their close proximity, has prevented characterization of individual cell types and/or their developmental progression. To examine cochlear development more closely, we transcriptionally profile approximately 30,000 isolated mouse cochlear cells collected at four developmental time points. Here we report on the analysis of those cells including the identification of both known and unknown cell types. Trajectory analysis for OHCs indicates four phases of gene expression while fate mapping of progenitor cells suggests that OHCs and their surrounding supporting cells arise from a distinct (lateral) progenitor pool. Tgfßr1 is identified as being expressed in lateral progenitor cells and a Tgfßr1 antagonist inhibits OHC development. These results provide insights regarding cochlear development and demonstrate the potential value and application of this data set.

Cochlear supporting cells function as macrophage-like cells and protect audiosensory receptor hair cells from pathogens.

To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.

Direct reprogramming adult fibroblast into cells with partial inner ear hair cell characteristics through cell activation and signal directed approach.

Loss of inner ear hair cell (HC) is an irreversible process in mammals and is the most common cause of human hearing and balance disorders especially in the elderly. Cell therapy based on highly scalable generation of HC linage and inner ear transplantation is one of the most promising therapeutic approaches for HC impairment. For fibroblast is quite abundant and readily available in human body, it is an ideal endogenous cell source to generate HC lineage for transplantation purpose. In the present study, by using a cell activation and signaling directed method, we demonstrate that adult fibroblast can be direct reprogrammed into a kind of cell which expresses lots of HC markers. At the same time, an intermediate progenitor stage exists during such a lineage conversion and activation of FGF pathway is critical for its formation. Although these reprogrammed cells still lack some of the key features of HC such as mechanosensitive ion channel hence have not acquired the functional properties of HC, the findings reported here raise the possibility of reprogramming endogenous fibroblasts into functional HC for regenerative purpose.

Notch-mediated lateral induction is necessary to maintain vestibular prosensory identity during inner ear development.

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 acquisition of positional information across the radial axis of the cochlea.

The mammalian cochlea detects sound and transmits this information to the brain. A cross section through the cochlea reveals functionally distinct epithelial domains arrayed around the circumference of a fluid-filled duct. Six major domains include two on the roof of the duct (Reissner's membrane medially and the stria vascularis laterally) and four across the floor of the duct, including the medial and lateral halves of the sensory domain, the organ of Corti. These radial domains are distinguishable in the embryonic cochlea by differential expression of transcription factors, and we focus here on a subset of the factors that can influence cochlear fates. We then move upstream of these genes to identify which of five signaling pathways (Notch, Fgf, Wnt, Bmp, and Shh) controls their spatial patterns of expression. We link the signaling pathways to their downstream genes, separating them by their radial position, to create putative gene regulatory networks (GRNs) from two time points, before and during the time when six radial compartments arise. These GRNs offer a framework for understanding the acquisition of positional information across the radial axis of the cochlea, and to guide therapeutic approaches to repair or regenerate distinct cochlear components that may contribute to hearing loss.