Smoothened overexpression causes trochlear motoneurons to reroute and innervate ipsilateral eyes.

The trochlear projection is unique among the cranial nerves in that it exits the midbrain dorsally to innervate the contralateral superior oblique muscle in all vertebrates. Trochlear as well as oculomotor motoneurons uniquely depend upon Phox2a and Wnt1, both of which are downstream of Lmx1b, though why trochlear motoneurons display such unusual projections is not fully known. We used Pax2-cre to drive expression of ectopically activated Smoothened (SmoM2) dorsally in the midbrain and anterior hindbrain. We documented the expansion of oculomotor and trochlear motoneurons using Phox2a as a specific marker at E9.5. We show that the initial expansion follows a demise of these neurons by E14.5. Furthermore, SmoM2 expression leads to a ventral exit and ipsilateral projection of trochlear motoneurons. We compare that data with Unc5c mutants that shows a variable ipsilateral number of trochlear fibers that exit dorsal. Our data suggest that Shh signaling is involved in trochlear motoneuron projections and that the deflected trochlear projections after SmoM2 expression is likely due to the dorsal expression of Gli1, which impedes the normal dorsal trajectory of these neurons.

Neurog1 can partially substitute for Atoh1 function in hair cell differentiation and maintenance during organ of Corti development.

Atoh1, a basic helix-loop-helix (bHLH) transcription factor (TF), is essential for the differentiation of hair cells (HCs), mechanotransducers that convert sound into auditory signals in the mammalian organ of Corti (OC). Previous work demonstrated that replacing mouse Atoh1 with the fly ortholog atonal rescues HC differentiation, indicating functional replacement by other bHLH genes. However, replacing Atoh1 with Neurog1 resulted in reduced HC differentiation compared with transient Atoh1 expression in a 'self-terminating' Atoh1 conditional null mouse (Atoh1-Cre; Atoh1(f/f)). We now show that combining Neurog1 in one allele with removal of floxed Atoh1 in a self-terminating conditional mutant (Atoh1-Cre; Atoh1(f/kiNeurog1)) mouse results in significantly more differentiated inner HCs and outer HCs that have a prolonged longevity of 9 months compared with Atoh1 self-terminating littermates. Stereocilia bundles are partially disorganized, disoriented and not HC type specific. Replacement of Atoh1 with Neurog1 maintains limited expression of Pou4f3 and Barhl1 and rescues HCs quantitatively, but not qualitatively. OC patterning and supporting cell differentiation are also partially disrupted. Diffusible factors involved in patterning are reduced (Fgf8) and factors involved in cell-cell interactions are affected (Jag1, Hes5). Despite the presence of many HCs with stereocilia these mice are deaf, possibly owing to HC and OC patterning defects. This study provides a novel approach to disrupt OC development through modulating the HC-specific intracellular TF network. The resulting disorganized OC indicates that normally differentiated HCs act as 'self-organizers' for OC development and that Atoh1 plays a crucial role to initiate HC stereocilia differentiation independently of HC viability.

A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA.

RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels.

The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell.

The inner ear is the hub where hair cells (HCs) transduce sound, gravity, and head acceleration stimuli to the brain. Hearing and balance rely on mechanosensation, the fastest sensory signals transmitted to the brain. The mechanoelectrical transducer (MET) channel is the entryway for the sound-balance-brain interface, but the channel-complex composition is not entirely known. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as MET-complex components. The Pz channels, expressed in HC stereocilia, and cell lines are co-localized and co-assembled with MET complex partners. Mice expressing non-functional Pz1 and Pz2 at the ROSA26 locus have impaired auditory and vestibular traits that can only be explained if the Pzs are integral to the MET complex. We suggest that Pz subunits constitute part of the MET complex and that interactions with other MET complex components yield functional MET units to generate HC MET currents.

Evolution and development of the tetrapod auditory system: an organ of Corti-centric perspective.

The tetrapod auditory system transmits sound through the outer and middle ear to the organ of Corti or other sound pressure receivers of the inner ear where specialized hair cells translate vibrations of the basilar membrane into electrical potential changes that are conducted by the spiral ganglion neurons to the auditory nuclei. In other systems, notably the vertebrate limb, a detailed connection between the evolutionary variations in adaptive morphology and the underlying alterations in the genetic basis of development has been partially elucidated. In this review, we attempt to correlate evolutionary and partially characterized molecular data into a cohesive perspective of the evolution of the mammalian organ of Corti out of the tetrapod basilar papilla. We propose a stepwise, molecularly partially characterized transformation of the ancestral, vestibular developmental program of the vertebrate ear. This review provides a framework to decipher both discrete steps in development and the evolution of unique functional adaptations of the auditory system. The combined analysis of evolution and development establishes a powerful cross-correlation where conclusions derived from either approach become more meaningful in a larger context which is not possible through exclusively evolution or development centered perspectives. Selection may explain the survival of the fittest auditory system, but only developmental genetics can explain the arrival of the fittest auditory system. [Modified after (Wagner 2011)].

Hair cell morphological patterns and polarity organization in the sea lamprey vestibular cristae.

The inner ear of the sea lamprey was examined by scanning electron microscopy, antibody labeling with tubulin, Myo7a, Spectrin, and Phalloidin stain to elucidate the canal cristae organization and the morphology and polarity of the hair cells. We characterized the hair cell stereocilia bundles and their morphological polarity with respect to the kinocilia. We identified three types of hair cells. In Type 1 hair cells, the kinocilia were slightly longer than the tallest stereocilia. This type was located along the medial bank of the crista and their polarity, based on kinocilia location, was uniformly pointed ampullipetally. Type 2 hair cells that had kinocilia that were much longer than the stereocilia, were most abundant in the central region of the crista. This type of hair cell displayed variable polarity. Type 3 hair cells had extremely long kinocilia (~40-50 µm long) and with extremely short stereocilia. They were mostly located in the lateral zone crista and displayed ampullipetal polarity. Myo7a and tubulin antibodies revealed that hair cells and vestibular afferents are distributed across the canal cristae in the lamprey, covering the area of cruciate eminence; a feature that is absent in more derived vertebrates. Spectrin shows hair cells of varying polarities in the central zone. In this zone, some cells followed the main polarity vector (lateral) like those in medial and lateral zones, whereas other cells displayed polarities that carried up to 40° from the main polarity vector.

Genetic and pharmacologic alterations of claudin9 levels suffice to induce functional and mature inner hair cells.

Hearing loss is the most common form of sensory deficit. It occurs predominantly due to hair cell (HC) loss. Mammalian HCs are terminally differentiated by birth, making HC loss incurable. Here, we show the pharmacogenetic downregulation of Cldn9, a tight junction protein, generates robust supernumerary inner HCs (IHCs) in mice. The putative ectopic IHCs have functional and synaptic features akin to typical IHCs and were surprisingly and remarkably preserved for at least fifteen months >50% of the mouse's life cycle. In vivo, Cldn9 knockdown using shRNA on postnatal days (P) P1-7 yielded analogous functional putative ectopic IHCs that were equally durably conserved. The findings suggest that Cldn9 levels coordinate embryonic and postnatal HC differentiation, making it a viable target for altering IHC development pre- and post-terminal differentiation.

The Piezo channel is central to the mechano-sensitive channel complex in the mammalian inner ear.

The inner ear is the hub where hair cells transduce sound, gravity, and head acceleration stimuli carried by neural codes to the brain. Of all the senses, hearing and balance, which rely on mechanosensation, are the fastest sensory signals transmitted to the central nervous system. The mechanoelectrical transducer (MET) channel in hair cells is the entryway for the sound-balance-brain interface, but the channel's composition has eluded biologists due to its complexity. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as central components of the MET complex. The Pz channel subunits are expressed in hair-cell stereocilia, are co-localized and co-assembled, and are essential components of the MET complex in vitro and in situ, including integration with the transmembrane channel (Tmc1/2) protein. Mice expressing non-functional Pz1 and Pz2, but not functional Pz1 at the ROSA26 locus under the control of hair-cell promoters, have impaired auditory and vestibular traits that can only be explained if Pz channel multimers are integral to the MET complex. We affirm that Pz protein subunits constitute MET channels and that functional interactions with components of the MET complex yield current properties resembling hair-cell MET currents. Our results demonstrate Pz is a MET channel component central to interacting with MET complex proteins. Results account for the MET channel pore and complex.

The role of sensory organs and the forebrain for the development of the craniofacial shape as revealed by Foxg1-cre-mediated microRNA loss.

Cranial development is critically influenced by the relative growth of distinct elements. Previous studies have shown that the transcription factor Foxg1 is essential the for development of the telencephalon, olfactory epithelium, parts of the eye and the ear. Here we investigate the effects of a Foxg1-cre-mediated conditional deletion of Dicer1 and microRNA (miRNA) depletion on mouse embryos. We report the rapid and complete loss of the telencephalon and cerebellum as well as the severe reduction in the ears and loss of the anterior half of the eyes. These losses result in unexpectedly limited malformations of anterodorsal aspects of the skull. We investigated the progressive disappearance of these initially developing structures and found a specific miRNA of nervous tissue, miR-124, to disappear before reduction in growth of the specific neurosensory areas. Correlated with the absence of miR-124, these areas showed numerous apoptotic cells that stained positive for anticleaved caspase 3 and the phosphatidylserine stain PSVue® before the near or complete loss of those brain and sensory areas (forebrain, cerebellum, anterior retina, and ear). We conclude that Foxg1-cre-mediated conditional deletion of Dicer1 leads to the absence of functional miRNA followed by complete or nearly complete loss of neurons. Embryonic neurosensory development therefore depends critically on miRNA. Our data further suggest that loss of a given neuronal compartment can be triggered using early deletion of Dicer1 and thus provides a novel means to genetically remove specific neurosensory areas to investigate loss of their function on morphology (this study) or signal processing within the brain.

Sustained Loss of Bdnf Affects Peripheral but Not Central Vestibular Targets.

The vestibular system is vital for proper balance perception, and its dysfunction contributes significantly to fall-related injuries, especially in the elderly. Vestibular ganglion neurons innervate vestibular hair cells at the periphery and vestibular nuclei and the uvula and nodule of the cerebellum centrally. During aging, these vestibular ganglion neurons degenerate, impairing vestibular function. A complete understanding of the molecular mechanisms involved in neurosensory cell survival in the vestibular system is unknown. Brain-derived neurotrophic factor (BDNF) is specifically required for the survival of vestibular ganglion neurons, as its loss leads to early neuronal death. Bdnf null mice die within 3 weeks of birth, preventing the study of the long-term effects on target cells. We use Pax2-cre to conditionally knock out Bdnf, allowing mice survival to approximately 6 months of age. We show that a long-term loss of Bdnf leads to a significant reduction in the number of vestibular ganglion neurons and a reduction in the number of vestibular hair cells. There was no significant decrease in the central targets lateral vestibular nucleus (LVN) or the cerebellum at 6 months. This suggests that the connectivity between central target cells and other neurons suffices to prevent their loss despite vestibular hair cell and ganglion neuron loss. Whether the central neurons would undergo eventual degeneration in the absence of Bdnf remains to be determined.

Developmental Changes in Peripherin-eGFP Expression in Spiral Ganglion Neurons.

The two types of spiral ganglion neurons (SGNs), types I and II, innervate inner hair cells and outer hair cells, respectively, within the mammalian cochlea and send another process back to cochlear nuclei in the hindbrain. Studying these two neuronal types has been made easier with the identification of unique molecular markers. One of these markers, peripherin, was shown using antibodies to be present in all SGNs initially but becomes specific to type II SGNs during maturation. We used mice with fluorescently labeled peripherin (Prph-eGFP) to examine peripherin expression in SGNs during development and in aged mice. Using these mice, we confirm the initial expression of Prph-eGFP in both types I and II neurons and eventual restriction to only type II perikarya shortly after birth. However, while Prph-eGFP is uniquely expressed within type II cell bodies by P8, both types I and II peripheral and central processes continue to express Prph-eGFP for some time before becoming downregulated. Only at P30 was there selective type II Prph-eGFP expression in central but not peripheral processes. By 9 months, only the type II cell bodies and more distal central processes retain Prph-eGFP expression. Our results show that Prph-eGFP is a reliable marker for type II SGN cell bodies beyond P8; however, it is not generally a suitable marker for type II processes, except for central processes beyond P30. How the changes in Prph-eGFP expression relate to subsequent protein expression remains to be explored.

Effects of Neurod1 Expression on Mouse and Human Schwannoma Cells.

OBJECTIVES: The objective was to explore the effect of the proneuronal transcription factor neurogenic differentiation 1 (Neurod1, ND1) on Schwann cells (SC) and schwannoma cell proliferation. METHODS: Using a variety of transgenic mouse lines, we investigated how expression of Neurod1 effects medulloblastoma (MB) growth, schwannoma tumor progression, vestibular function, and SC cell proliferation. Primary human vestibular schwannoma (VS) cell cultures were transduced with adenoviral vectors expressing Neurod1. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) uptake. STUDY DESIGN: Basic science investigation. RESULTS: Expression of Neurod1 reduced the growth of slow-growing but not fast-growing MB models. Gene transfer of Neurod1 in human schwannoma cultures significantly reduced cell proliferation in dose-dependent way. Deletion of the neurofibromatosis type 2 (Nf2) tumor-suppressor gene via Cre expression in SCs led to increased intraganglionic SC proliferation and mildly reduced vestibular sensory-evoked potentials (VsEP) responses compared to age-matched wild-type littermates. The effect of Neurod1-induced expression on intraganglionic SC proliferation in animals lacking Nf2 was mild and highly variable. Sciatic nerve axotomy significantly increased SC proliferation in wild-type and Nf2-null animals, and expression of Neurod1 reduced the proliferative capacity of both wild-type and Nf2-null SCs following nerve injury. CONCLUSION: Expression of Neurod1 reduces slow-growing MB progression and reduces human SC proliferation in primary VS cultures. In a genetic mouse model of schwannomas, we find some effects of Neurod1 expression; however, the high variability indicates that more tightly regulated Neurod1 expression levels that mimic our in vitro data are needed to fully validate Neurod1 effects on schwannoma progression. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E259-E270, 2021.

Neurod1 regulates survival and formation of connections in mouse ear and brain.

The developing sensory neurons of the mammalian ear require two sequentially activated bHLH genes, Neurog1 and Neurod1, for their development. Neurons never develop in Neurog1 null mice, and most neurons die in Neurod1 null mutants, a gene upregulated by Neurog1. The surviving neurons of Neurod1 null mice are incompletely characterized in postnatal mice because of the early lethality of mutants and the possible compromising effect of the absence of insulin on peripheral neuropathies. Using Tg(Pax2-cre), we have generated a conditional deletion of floxed Neurod1 for the ear; this mouse is viable and allows us to investigate ear innervation defects of Neurod1 absence only in the ear. We have compared the defects in embryos and show an ear phenotype in conditional Neurod1 null mice comparable with the systemic Neurod1 null mouse. By studying postnatal animals, we show that Neurod1 not only is necessary for the survival of most spiral and many vestibular neurons, but is also essential for a segregated central projection of vestibular and cochlear afferents. In the absence of Neurod1 in the ear, vestibular and cochlear afferents enter the cochlear nucleus as a single mixed nerve. Neurites coming from vestibular and cochlear sensory epithelia project centrally to both cochlear and vestibular nuclei, in addition to their designated target projections. The peripheral innervation of the remaining sensory neurons is disorganized and shows collaterals of single neurons projecting to multiple endorgans, displaying no tonotopic organization of the organ of Corti or the cochlear nucleus. Pending elucidation of the molecular details for these Neurod1 functions, these data demonstrate that Neurod1 is not only a major factor for the survival of neurons but is crucial for the development of normal ear connections, both in the ear and in the central nervous system.

Neuronal Migration Generates New Populations of Neurons That Develop Unique Connections, Physiological Properties and Pathologies.

Central nervous system neurons become postmitotic when radial glia cells divide to form neuroblasts. Neuroblasts may migrate away from the ventricle radially along glia fibers, in various directions or even across the midline. We present four cases of unusual migration that are variably connected to either pathology or formation of new populations of neurons with new connectivities. One of the best-known cases of radial migration involves granule cells that migrate from the external granule cell layer along radial Bergman glia fibers to become mature internal granule cells. In various medulloblastoma cases this migration does not occur and transforms the external granule cell layer into a rapidly growing tumor. Among the ocular motor neurons is one unique population that undergoes a contralateral migration and uniquely innervates the superior rectus and levator palpebrae muscles. In humans, a mutation of a single gene ubiquitously expressed in all cells, induces innervation defects only in this unique motor neuron population, leading to inability to elevate eyes or upper eyelids. One of the best-known cases for longitudinal migration is the facial branchial motor (FBM) neurons and the overlapping inner ear efferent population. We describe here molecular cues that are needed for the caudal migration of FBM to segregate these motor neurons from the differently migrating inner ear efferent population. Finally, we describe unusual migration of inner ear spiral ganglion neurons that result in aberrant connections with disruption of frequency presentation. Combined, these data identify unique migratory properties of various neuronal populations that allow them to adopt new connections but also sets them up for unique pathologies.

Topologically correct central projections of tetrapod inner ear afferents require Fzd3.

Inner ear sensory afferent connections establish sensory maps between the inner ear hair cells and the vestibular and auditory nuclei to allow vestibular and sound information processing. While molecular guidance of sensory afferents to the periphery has been well studied, molecular guidance of central projections from the ear is only beginning to emerge. Disorganized central projections of spiral ganglion neurons in a Wnt/PCP pathway mutant, Prickle1, suggest the Wnt/PCP pathway plays a role in guiding cochlear afferents to the cochlear nuclei in the hindbrain, consistent with known expression of the Wnt receptor, Frizzled3 (Fzd3) in inner ear neurons. We therefore investigated the role of Wnt signaling in central pathfinding in Fzd3 mutant mice and Fzd3 morpholino treated frogs and found aberrant central projections of vestibular afferents in both cases. Ear transplantations from knockdown to control Xenopus showed that it is the Fzd3 expressed within the ear that mediates this guidance. Also, cochlear afferents of Fzd3 mutant mice lack the orderly topological organization observed in controls. Quantification of Fzd3 expression in spiral ganglion neurons show a gradient of expression with Fzd3 being higher in the apex than in the base. Together, these results suggest that a gradient of Fzd3 in inner ear afferents directs projections to the correct dorsoventral column within the hindbrain.

Spiral Ganglion Neuron Projection Development to the Hindbrain in Mice Lacking Peripheral and/or Central Target Differentiation.

We investigate the importance of the degree of peripheral or central target differentiation for mouse auditory afferent navigation to the organ of Corti and auditory nuclei in three different mouse models: first, a mouse in which the differentiation of hair cells, but not central auditory nuclei neurons is compromised (Atoh1-cre; Atoh1f/f ); second, a mouse in which hair cell defects are combined with a delayed defect in central auditory nuclei neurons (Pax2-cre; Atoh1f/f ), and third, a mouse in which both hair cells and central auditory nuclei are absent (Atoh1-/-). Our results show that neither differentiated peripheral nor the central target cells of inner ear afferents are needed (hair cells, cochlear nucleus neurons) for segregation of vestibular and cochlear afferents within the hindbrain and some degree of base to apex segregation of cochlear afferents. These data suggest that inner ear spiral ganglion neuron processes may predominantly rely on temporally and spatially distinct molecular cues in the region of the targets rather than interaction with differentiated target cells for a crude topological organization. These developmental data imply that auditory neuron navigation properties may have evolved before auditory nuclei.

Prickle1 regulates neurite outgrowth of apical spiral ganglion neurons but not hair cell polarity in the murine cochlea.

In the mammalian organ of Corti (OC), the stereocilia on the apical surface of hair cells (HCs) are uniformly organized in a neural to abneural axis (or medial-laterally). This organization is regulated by planar cell polarity (PCP) signaling. Mutations of PCP genes, such as Vangl2, Dvl1/2, Celsr1, and Fzd3/6, affect the formation of HC orientation to varying degrees. Prickle1 is a PCP signaling gene that belongs to the prickle / espinas / testin family. Prickle1 protein is shown to be asymmetrically localized in the HCs of the OC, and this asymmetric localization is associated with loss of PCP in Smurf mutants, implying that Prickle1 is involved in HC PCP development in the OC. A follow-up study found no PCP polarity defects after loss of Prickle1 (Prickle1-/-) in the cochlea. We show here strong Prickle1 mRNA expression in the spiral ganglion by in situ hybridization and ß-Gal staining, and weak expression in the OC by ß-Gal staining. Consistent with this limited expression in the OC, cochlear HC PCP is unaffected in either Prickle1C251X/C251X mice or Prickle1f/f; Pax2-cre conditional null mice. Meanwhile, type II afferents of apical spiral ganglion neurons (SGN) innervating outer hair cells (OHC) have unusual neurite growth. In addition, afferents from the apex show unusual collaterals in the cochlear nuclei that overlap with basal turn afferents. Our findings argue against the role of Prickle1 in regulating hair cell polarity in the cochlea. Instead, Prickle1 regulates the polarity-related growth of distal and central processes of apical SGNs.

Neuroanatomical Tracing Techniques in the Ear: History, State of the Art, and Future Developments.

The inner ear has long been at the cutting edge of tract tracing techniques that have shaped and reshaped our understanding of the ear's innervation patterns. This review provides a historical framework to understand the importance of these techniques for ear innervation and for development of tracing techniques in general; it is hoped that lessons learned will help to quickly adopt transformative novel techniques and their information and correct past beliefs based on technical limitations. The technical part of the review presents details of our protocol as developed over the last 30 years. We also include arguments as to why these recommendations work best to generate the desired outcome of distinct fiber and cell labeling, and generate reliable data for any investigation. We specifically focus on two tracing techniques, in part developed and/or championed for ear innervation analysis: the low molecular multicolor dextran amine tract tracing technique and the multicolor tract tracing technique with lipophilic dyes.

Inner ear hair cells deteriorate in mice engineered to have no or diminished innervation.

The innervation of the inner ear critically depends on the two neurotrophins Ntf3 and Bdnf. In contrast to this molecularly well-established dependency, evidence regarding the need of innervation for long-term maintenance of inner ear hair cells is inconclusive, due to experimental variability. Mutant mice that lack both neurotrophins could shed light on the long-term consequences of innervation loss on hair cells without introducing experimental variability, but do not survive after birth. Mutant mice with conditional deletion of both neurotrophins lose almost all innervation by postnatal day 10 and show an initially normal development of hair cells by this stage. No innervation remains after 3 weeks and complete loss of all innervation results in near complete loss of outer and many inner hair cells of the organ of Corti within 4 months. Mutants that retain one allele of either neurotrophin have only partial loss of innervation of the organ of Corti and show a longer viability of cochlear hair cells with more profound loss of inner hair cells. By 10 months, hair cells disappear with a base to apex progression, proportional to the residual density of innervation and similar to carboplatin ototoxicity. Similar to reports of hair cell loss after aminoglycoside treatment, blobbing of stereocilia of apparently dying hair cells protrude into the cochlear duct. Denervation of vestibular sensory epithelia for several months also resulted in variable results, ranging from unusual hair cells resembling the aberrations found in the organ of Corti, to near normal hair cells in the canal cristae. Fusion and/or resorption of stereocilia and loss of hair cells follows a pattern reminiscent of Myo6 and Cdc42 null mice. Our data support a role of innervation for long-term maintenance but with a remarkable local variation that needs to be taken into account when attempting regeneration of the organ of Corti.

Beyond generalized hair cells: molecular cues for hair cell types.

Basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial for inner ear neurosensory development. The proneural TF Atoh1 regulates the differentiation of hair cells (HCs) whereas Neurog1 and Neurod1 regulate specification and differentiation of neurons, respectively, but also affect HC development. Expression of Delta and Jagged ligands in nascent HCs and Notch receptors in supporting cells induce supporting cell differentiation through the regulation of neurogenic bHLH TFs (such as Hes1, Hes5) and suppression of limited Atoh1 expression. In sensorineural hearing loss, HCs are lost followed by supporting cells and progressive degeneration of neurons, at least in rodents. Regaining complete hearing may require reconstituting the organ of Corti from scratch, including the two types of HCs, inner and outer hair cells with the precise sorting of two types of afferent (type I and II) and efferent (lateral and medial olivo-cochlear) innervation. We review effects of bHLH TF dosage and their cross-regulation to differentiate HC types in the organ of Corti. We categorize findings of specific gene expressions in HCs: 1. as markers without meaning for the regeneration task, 2. as stabilizers who are needed to maintain or complete differentiation, and 3. as decision-making genes, expressed and acting early enough to be useful in this process. Only one TF has been characterized that fits the last aspect: Atoh1. We propose that temporal and intensity variations of Atoh1 are naturally modulated to differentiate specific types of HCs. Importantly, the molecular means to modify the Atoh1 expression are at least partially understood and can be readily implemented in the attempts to regenerate specific types of HCs.