PCP auto count: a novel Fiji/ImageJ plug-in for automated quantification of planar cell polarity and cell counting.

Introdution: During development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and intra-cellular spatial orientation, a feature known as planar cell polarity (PCP). To study the genetic and environmental factors affecting planar cell polarity, investigators must often manually measure cell orientations, which is a time-consuming endeavor. To automate cell counting and planar cell polarity data collection we developed a Fiji/ImageJ plug-in called PCP Auto Count (PCPA). Methods: PCPA analyzes binary images and identifies "chunks" of white pixels that contain "caves" of infiltrated black pixels. For validation, inner ear sensory epithelia including cochleae and utricles from mice were immunostained for ßII-spectrin and imaged with a confocal microscope. Images were preprocessed using existing Fiji functionality to enhance contrast, make binary, and reduce noise. An investigator rated PCPA cochlear hair cell angle measurements for accuracy using a one to five agreement scale. For utricle samples, PCPA derived measurements were directly compared against manually derived angle measurements and the concordance correlation coefficient (CCC) and Bland-Altman limits of agreement were calculated. PCPA was also tested against previously published images examining PCP in various tissues and across various species suggesting fairly broad utility. Results: PCPA was able to recognize and count 99.81% of cochlear hair cells, and was able to obtain ideally accurate planar cell polarity measurements for at least 96% of hair cells. When allowing for a <10° deviation from "perfect" measurements, PCPA's accuracy increased to 98%-100% for all users and across all samples. When PCPA's measurements were compared with manual angle measurements for E17.5 utricles there was negligible bias (<0.5°), and a CCC of 0.999. Qualitative examination of example images of Drosophila ommatidia, mouse ependymal cells, and mouse radial progenitors revealed a high level of accuracy for PCPA across a variety of stains, tissue types, and species. Discussion: Altogether, the data suggest that the PCPA plug-in suite is a robust and accurate tool for the automated collection of cell counts and PCP angle measurements.

Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5-15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

The transcription factor Pou4f3 is essential for the survival of postnatal and adult mouse cochlear hair cells and normal hearing.

Introduction: Hair cells (HCs) of the cochlea are responsible for sound transduction and hearing perception in mammals. Genetic mutations in the transcription factor Pou4f3 cause non-syndromic autosomal dominant hearing loss in humans (DFNA15) which varies in the age of onset depending on the individual mutation. Mouse models with germline deletion or mutations in Pou4f3 have previously demonstrated its critical role in the maturation and survival of cochlear HCs during embryonic development. However, the role of Pou4f3 in auditory function and in the survival or maintenance of cochlear HCs after birth and during adulthood has not been studied. Methods: Therefore, using the inducible CreER-loxP system, we deleted Pou4f3 from mouse cochlear HCs at different postnatal ages, relevant to specific stages of HC maturation and hearing function. Results and discussion: Elevated auditory brainstem response thresholds and significant HC loss were detected in mice with Pou4f3 deletion compared to their control littermates, regardless of the age when Pou4f3 was deleted. However, HC loss occurred more rapidly when Pou4f3 was deleted from immature HCs. Additionally, HC loss caused by Pou4f3 deletion did not affect the number of cochlear supporting cells, but caused a delayed loss of spiral ganglion neurons at 4 months after the deletion. In conclusion, Pou4f3 is necessary for the survival of cochlear HCs and normal hearing at all postnatal ages regardless of their maturation state. Our data also suggest that Pou4f3 indirectly regulates the survival of spiral ganglion neurons.

PCP Auto Count: A Novel Fiji/ImageJ plug-in for automated quantification of planar cell polarity and cell counting.

Background: During development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and intra-cellular spatial orientation, a feature known as planar cell polarity (PCP). To study the genetic and environmental factors affecting planar cell polarity investigators must often manually measure cell orientations, which is a time-consuming endeavor. Methodology: To automate cell counting and planar cell polarity data collection we developed a Fiji/ImageJ plug-in called PCP Auto Count (PCPA). PCPA analyzes binary images and identifies "chunks" of white pixels that contain "caves" of infiltrated black pixels. Inner ear sensory epithelia including cochleae (P4) and utricles (E17.5) from mice were immunostained for ßII-spectrin and imaged on a confocal microscope. Images were preprocessed using existing Fiji functionality to enhance contrast, make binary, and reduce noise. An investigator rated PCPA cochlear angle measurements for accuracy using a 1-5 agreement scale. For utricle samples, we directly compared PCPA derived measurements against manually derived angle measurements using concordance correlation coefficients (CCC) and Bland-Altman limits of agreement. Finally, PCPA was tested against a variety of images copied from publications examining PCP in various tissues and across various species. Results: PCPA was able to recognize and count 99.81% of cochlear hair cells (n = 1,1541 hair cells) in a sample set, and was able to obtain ideally accurate planar cell polarity measurements for over 96% of hair cells. When allowing for a <10° deviation from "perfect" measurements, PCPA's accuracy increased to >98%. When manual angle measurements for E17.5 utricles were compared, PCPA's measurements fell within -9 to +10 degrees of manually obtained mean angle measures with a CCC of 0.999. Qualitative examination of example images of Drosophila ommatidia, mouse ependymal cells, and mouse radial progenitors revealed a high level of accuracy for PCPA across a variety of stains, tissue types, and species. Altogether, the data suggest that the PCPA plug-in suite is a robust and accurate tool for the automated collection of cell counts and PCP angle measurements.

Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90'000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

Development of a Triple-Negative Breast Cancer Leptomeningeal Disease Model in Zebrafish.

Leptomeningeal disease occurs when cancer cells migrate into the ventricles of the brain and spinal cord and then colonize the meninges of the central nervous system. The triple-negative subtype of breast cancer often progresses toward leptomeningeal disease and has a poor prognosis because of limited treatment options. This is due, in part, to a lack of animal models with which to study leptomeningeal disease. Here, we developed a translucent zebrafish casper (roy-/-; nacre-/-) xenograft model of leptomeningeal disease in which fluorescent labeled MDA-MB-231 human triple-negative breast cancer cells are microinjected into the ventricles of zebrafish embryos and then tracked and measured using fluorescent microscopy and multimodal plate reader technology. We then used these techniques to measure tumor area, cell proliferation, and cell death in samples treated with the breast cancer drug doxorubicin and a vehicle control. We monitored MDA-MB-231 cell localization and tumor area, and showed that samples treated with doxorubicin exhibited decreased tumor area and proliferation and increased apoptosis compared to control samples.

Mild Therapeutic Hypothermia and Putative Mechanisms of Hair Cell Survival in the Cochlea.

Significance: Sensorineural hearing loss has significant implications for quality of life and risk for comorbidities such as cognitive decline. Noise and ototoxic drugs represent two common risk factors for acquired hearing loss that are potentially preventable. Recent Advances: Numerous otoprotection strategies have been postulated over the past four decades with primary targets of upstream redox pathways. More recently, the application of mild therapeutic hypothermia (TH) has shown promise for otoprotection for multiple forms of acquired hearing loss. Critical Issues: Systemic antioxidant therapy may have limited application for certain ototoxic drugs with a therapeutic effect on redox pathways and diminished efficacy of the primary drug's therapeutic function (e.g., cisplatin for tumors). Future Directions: Mild TH likely targets multiple mechanisms, contributing to otoprotection, including slowed metabolics, reduced oxidative stress, and involvement of cold shock proteins. Further work is needed to identify the mechanisms of mild TH at play for various forms of acquired hearing loss.

Cannabinoid Signaling in Auditory Function and Development.

Plants of the genus Cannabis have been used by humans for millennia for a variety of purposes. Perhaps most notable is the use of certain Cannabis strains for their psychoactive effects. More recently, several biologically active molecules within the plants of these Cannabis strains, called phytocannabinoids or simply cannabinoids, have been identified. Furthermore, within human cells, endogenous cannabinoids, or endocannabinoids, as well as the receptors and secondary messengers that give rise to their neuromodulatory effects, have also been characterized. This endocannabinoid system (ECS) is composed of two primary ligands-anandamide and 2-arachidonyl glycerol; two primary receptors-cannabinoid receptors 1 and 2; and several enzymes involved in biosynthesis and degradation of endocannabinoid ligands including diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL). Here we briefly summarize cannabinoid signaling and review what has been discerned to date with regard to cannabinoid signaling in the auditory system and its roles in normal physiological function as well as pathological conditions. While much has been uncovered regarding cannabinoid signaling in the central nervous system, less attention has been paid to the auditory system specifically. Still, evidence is emerging to suggest that cannabinoid signaling is critical for the development, maturation, function, and survival of cochlear hair cells (HCs) and spiral ganglion neurons (SGNs). Furthermore, cannabinoid signaling can have profound effects on synaptic connectivity in CNS structures related to auditory processing. While clinical cases demonstrate that endogenous and exogenous cannabinoids impact auditory function, this review highlights several areas, such as SGN development, where more research is warranted.

Cool OtOprotective Ear Lumen (COOL) Therapy for Cisplatin-induced Hearing Loss.

HYPOTHESIS: Localized cooling of the external ear has a protective effect on the susceptibility to cisplatin-induced hearing loss. BACKGROUND: We previously demonstrated significant protection from cisplatin-induced hearing loss using cool water ear canal irrigation. However, the study was limited to a single bolus injection of cisplatin and an acute time period. Here, we examined the application of localized cooling of the ear canal with repeated doses of cisplatin, over an expanded period of time, and using two methods of cooling. METHODS: Twenty-four guinea pigs (12 male and 12 female) underwent auditory physiological testing (auditory brainstem response and distortion product otoacoustic emissions at 8-32 kHz) and pre/postadministration of cisplatin. Cisplatin (4 mg/kg i.p.) was administered in 3 weekly single injections for a total of 12 mg/kg. While anesthetized, the left ears of the guinea pigs were exposed to either cool water (22°C; ICS Water Caloric Irrigator), a cool ear bar (15°C, cooled by a Peltier device; TNM, Scion NeuroStim), or left uncooled as a sham control. The animals were tested 3 days post each dosage and 1 month post the final dose. At the end of the experiment the animals were euthanized for histological evaluation. RESULTS: We found that hearing loss was significantly reduced, and hair cell survival greatly improved, in animals that received cooling treatments compared to cisplatin-only control animals. No significant difference was observed between the two methods of cooling. CONCLUSION: Localized cooling of the ear canal during administration of cisplatin mitigated loss of auditory function and loss of hair cells.

Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues.

Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.

Comparison of ethylenediaminetetraacetic acid and rapid decalcificier solution for studying human temporal bones by immunofluorescence.

OBJECTIVES: The pervasiveness of hearing loss and the development of new potential therapeutic approaches have led to increased animal studies of the inner ear. However, translational relevance of such studies depends upon verification of protein localization data in human samples. Cadavers used for anatomical education provide a potential research resource, but are limiting due to difficulties in accessing sensory tissues from the dense temporal bones. This study seeks to reduce the often months-long process of decalcification and improve immunofluorescent staining of human cadaveric temporal bones for research use. METHODS: Temporal bones were decalcified in either (a) hydrochloric acid-containing RDO solution for 2 days followed by 0.5 M ethylenediaminetetraacetic acid (EDTA) for 3 to 5 additional days, or (b) 0.5 M EDTA alone for 2 to 4 weeks. Image-iT FX signal enhancer (ISE) was used to improve immunofluorescent signal-to-noise ratios. RESULTS: The data indicate that both methods speed decalcification and allow for immunolabeling of the extranuclear proteins neurofilament (heavy chain), myosin VIIa, oncomodulin and prestin. However, RDO decalcification was more likely to alter structural morphology of sensory tissues and hindered effective labeling of the nuclear proteins SRY-box transcription factor 2 and GATA binding protein 3. CONCLUSIONS: Although both approaches allow for rapid decalcification, EDTA appears superior to RDO for preserving cytoarchitecture and immunogenicity. LEVEL OF EVIDENCE: NA.

Approaches for the study of epigenetic modifications in the inner ear and related tissues.

DNA methylation and histone modifications such as methylation, acetylation, and phosphorylation, are two types of epigenetic modifications that alter gene expression. These additions to DNA regulatory elements or to the tails of histones can be inherited or can also occur de novo. Since epigenetic modifications can have significant effects on various processes at both the cellular and organismal level, there has been a rapid increase in research on this topic throughout all fields of biology in recent years. However, epigenetic research is relativity new for the inner ear field, likely due to the limited number of cells present and their quiescent nature. Here, we provide an overview of methods used to detect DNA methylation and histone modifications with a focus on those that have been validated for use with limited cell numbers and a discussion of the strengths and limitations for each. We also provide examples for how these methods have been used to investigate the epigenetic landscape in the inner ear and related tissues.

In Vivo Interplay between p27Kip1, GATA3, ATOH1, and POU4F3 Converts Non-sensory Cells to Hair Cells in Adult Mice.

Hearing loss is widespread and persistent because mature mammalian auditory hair cells (HCs) are nonregenerative. In mice, the ability to regenerate HCs from surrounding supporting cells (SCs) declines abruptly after postnatal maturation. We find that combining p27Kip1 deletion with ectopic ATOH1 expression surmounts this age-related decline, leading to conversion of SCs to HCs in mature mouse cochleae and after noise damage. p27Kip1 deletion, independent of canonical effects on Rb-family proteins, upregulated GATA3, a co-factor for ATOH1 that is lost from SCs with age. Co-activation of GATA3 or POU4F3 and ATOH1 promoted conversion of SCs to HCs in adult mice. Activation of POU4F3 alone also converted mature SCs to HCs in vivo. These data illuminate a genetic pathway that initiates auditory HC regeneration and suggest p27Kip1, GATA3, and POU4F3 as additional therapeutic targets for ATOH1-mediated HC regeneration.

Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes.

The mammalian cochlea is a highly specialized organ within the inner ear. Sensory hair cells (HC) in the cochlea detect and transduce sound waves into electrical impulses that are sent to the brain. Studies of the molecular pathways regulating HC formation are hindered by the very sparse nature of HCs, where only ~3300 are found within an entire mouse cochlea. Current cell lines mimic certain aspects of HCs but lack terminal HC marker expression. Here we successfully "pseudo-immortalized" cochlear progenitor cells using the "conditional reprogramming" technique. These cells, termed "Conditionally Reprogrammed Otic Stem Cells" (CR-OSC), are able to bypass the senescence inherent to cochlear progenitor cells without genetic alterations, allowing for the generation of over 15 million cells from a single cochlea. These cells can be differentiated and up-regulate both early and terminal differentiation genes associated with HCs, including the terminal HC differentiation marker prestin. CR-OSCs also respond to known HC cues, including upregulation of HC genes in response to Atoh1 overexpression, and upregulation of prestin expression after thyroid hormone application. Overall, we describe the creation of a HC line capable of regulated expression of HC genes that can easily be recreated in any laboratory from any mouse of interest.

Sox2-CreER mice are useful for fate mapping of mature, but not neonatal, cochlear supporting cells in hair cell regeneration studies.

Studies of hair cell regeneration in the postnatal cochlea rely on fate mapping of supporting cells. Here we characterized a Sox2-CreER knock-in mouse line with two independent reporter mouse strains at neonatal and mature ages. Regardless of induction age, reporter expression was robust, with CreER activity being readily detectable in >85% of supporting cells within the organ of Corti. When induced at postnatal day (P) 28, Sox2-CreER activity was exclusive to supporting cells demonstrating its utility for fate mapping studies beyond this age. However, when induced at P1, Sox2-CreER activity was also detected in >50% of cochlear hair cells, suggesting that Sox2-CreER may not be useful to fate map a supporting cell origin of regenerated hair cells if induced at neonatal ages. Given that this model is currently in use by several investigators for fate mapping purposes, and may be adopted by others in the future, our finding that current protocols are effective for restricting CreER activity to supporting cells at mature but not neonatal ages is both significant and timely.

A Sox10(rtTA/+) Mouse Line Allows for Inducible Gene Expression in the Auditory and Balance Organs of the Inner Ear.

Genetic mouse models provide invaluable tools for discerning gene function in vivo. Tetracycline-inducible systems (Tet-On/Off) provide temporal and cell-type specific control of gene expression, offering an alternative or even complementary approach to existing Cre/LoxP systems. Here we characterized a Sox10(rtTA/+) knock-in mouse line which demonstrates inducible reverse tetracycline trans-activator (rtTA) activity and Tet-On transgene expression in the inner ear following induction with the tetracycline derivative doxycycline (Dox). These Sox10(rtTA/+) mice do not exhibit any readily observable developmental or hearing phenotypes, and actively drive Tet-On transgene expression in Sox10 expressing cells in the inner ear. Sox10(rtTA/+) activity was revealed by multiple Tet-On reporters to be nearly ubiquitous throughout the membranous labyrinth of the developing inner ear, and notably absent from hair cells, tympanic border cells, and ganglion neurons following postnatal Dox inductions. Interestingly, Dox-induced Sox10(rtTA/+) activity declined with induction age, where Tet-On reporters became uninducible in adult cochlear epithelium. Co-administration of the loop diuretic furosemide was able to rescue Dox-induced reporter expression, though this method also caused significant cochlear hair cell loss. Surprisingly, Sox10(rtTA/+) driven reporter expression in the cochlea persists for at least 54 days after cessation of neonatal induction, presumably due to the persistence of Dox within inner ear tissues. These findings highlight the utility of the Sox10(rtTA/+) mouse line as a powerful tool for functional genetic studies of the auditory and balance organs in vivo, but also reveal some important considerations that must be adequately controlled for in future studies that rely upon Tet-On/Off systems.

Auditory hair cell-specific deletion of p27Kip1 in postnatal mice promotes cell-autonomous generation of new hair cells and normal hearing.

Hearing in mammals relies upon the transduction of sound by hair cells (HCs) in the organ of Corti within the cochlea of the inner ear. Sensorineural hearing loss is a widespread and permanent disability due largely to a lack of HC regeneration in mammals. Recent studies suggest that targeting the retinoblastoma (Rb)/E2F pathway can elicit proliferation of auditory HCs. However, previous attempts to induce HC proliferation in this manner have resulted in abnormal cochlear morphology, HC death, and hearing loss. Here we show that cochlear HCs readily proliferate and survive following neonatal, HC-specific, conditional knock-out of p27(Kip1) (p27CKO), a tumor suppressor upstream of Rb. Indeed, HC-specific p27CKO results in proliferation of these cells without the upregulation of the supporting cell or progenitor cell proteins, Prox1 or Sox2, suggesting that they remain HCs. Furthermore, p27CKO leads to a significant addition of postnatally derived HCs that express characteristic synaptic and stereociliary markers and survive to adulthood, although a portion of the newly derived inner HCs exhibit cytocauds and lack VGlut3 expression. Despite this, p27CKO mice exhibit normal hearing as measured by evoked auditory brainstem responses, which suggests that the newly generated HCs may contribute to, or at least do not greatly detract from, function. These results show that p27(Kip1) actively maintains HC quiescence in postnatal mice, and suggest that inhibition of p27(Kip1) in residual HCs represents a potential strategy for cell-autonomous auditory HC regeneration.

In vivo visualization of Notch1 proteolysis reveals the heterogeneity of Notch1 signaling activity in the mouse cochlea.

Mechanosensory hair cells (HCs) and surrounding supporting cells (SCs) in the mouse cochlea are important for hearing and are derived from the same prosensory progenitors. Notch1 signaling plays dual but contrasting and age-dependent roles in mouse cochlear development: early lateral induction and subsequent lateral inhibition. However, it has been difficult to directly visualize mouse cochlear cells experiencing various levels of Notch1 activity at single cell resolution. Here, we characterized two knock-in mouse lines, Notch1(Cre (Low)/+) and Notch1(Cre (High)/+) , with different Cre recombinase activities, that can detect Notch1 receptor proteolysis or Notch1 activity at high and low thresholds, respectively. Using both lines together with a highly sensitive Cre reporter line, we showed that Notch1 activity is nearly undetectable during lateral induction but increases to medium and high levels during lateral inhibition. Furthermore, we found that within the neonatal organ of Corti, the vast majority of cells that experience Notch1 activity were SCs not HCs, suggesting that HCs kept undetectable Notch1 activity during the entire lineage development. Furthermore, among SC subtypes, ∼85-99% of Deiters' and outer pillar cells but only ∼19-38% of inner pillar cells experience medium and high levels of Notch1 activity. Our results demonstrate that Notch1 activity is highly heterogeneous: 1) between lateral induction and inhibition; 2) between HC and SC lineages; 3) among different SC subtypes; 4) among different cells within each SC subtype. Such heterogeneity should elucidate how the development of the cochclear sensory epithelium is precisely controlled and how HC regeneration can be best achieved in postnatal cochleae.