A novel intronic TCOF1 pathogenic variant in a Chinese family with Treacher Collins syndrome.

BACKGROUND: Treacher Collins syndrome (TCS; OMIM 154500) is a craniofacial developmental disorder. METHODS: To investigate the genetic features of a four-generation Chinese family with TCS, clinical examinations, hearing tests, computed tomography, whole-exome sequencing (WES), Sanger sequencing, reverse transcription (RT)-PCR, and the Minigene assay were performed. RESULTS: The probands, an 11-year-old male and his cousin exhibited typical clinical manifestations of TCS including conductive hearing loss, downward slanting palpebral fissures, and mandibular hypoplasia. Computed tomography revealed bilateral fusion of the anterior and posterior stapedial crura and malformation of the long crura of the incus. WES of both patients revealed a novel heterozygous intronic variant, i.e., c.4342 + 5_4342 + 8delGTGA (NM_001371623.1) in TCOF1. Minigene expression analysis revealed that the c.4342 + 5_4342 + 8delGTGA variant in TCOF1 caused a partial deletion of exon 24 (c.4115_4342del: p.Gly1373_Arg1448del), which was predicted to yield a truncated protein. The deletion was further confirmed via RT-PCR and sequencing of DNA from proband blood cells. A heterozygous variant in the POLR1C gene (NM_203290; exon6; c.525delG) was found almost co-segregated with the TCOF1 pathogenic variant. CONCLUSIONS: In conclusion, we identified a heterozygous TCOF1 splicing variant c.4342 + 5_4342 + 8delGTGA (splicing) in a Chinese TSC family with ossicular chain malformations and facial anomalies. Our findings broadened the spectrum of TCS variants and will facilitate diagnostics and prognostic predictions.

AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial.

BACKGROUND: Autosomal recessive deafness 9, caused by mutations of the OTOF gene, is characterised by congenital or prelingual, severe-to-complete, bilateral hearing loss. However, no pharmacological treatment is currently available for congenital deafness. In this Article, we report the safety and efficacy of gene therapy with an adeno-associated virus (AAV) serotype 1 carrying a human OTOF transgene (AAV1-hOTOF) as a treatment for children with autosomal recessive deafness 9. METHODS: This single-arm, single-centre trial enrolled children (aged 1-18 years) with severe-to-complete hearing loss and confirmed mutations in both alleles of OTOF, and without bilateral cochlear implants. A single injection of AAV1-hOTOF was administered into the cochlea through the round window. The primary endpoint was dose-limiting toxicity at 6 weeks after injection. Auditory function and speech were assessed by appropriate auditory perception evaluation tools. All analyses were done according to the intention-to-treat principle. This trial is registered with Chinese Clinical Trial Registry, ChiCTR2200063181, and is ongoing. FINDINGS: Between Oct 19, 2022, and June 9, 2023, we screened 425 participants for eligibility and enrolled six children for AAV1-hOTOF gene therapy (one received a dose of 9 × 1011 vector genomes [vg] and five received 1·5 × 1012 vg). All participants completed follow-up visits up to week 26. No dose-limiting toxicity or serious adverse events occurred. In total, 48 adverse events were observed; 46 (96%) were grade 1-2 and two (4%) were grade 3 (decreased neutrophil count in one participant). Five children had hearing recovery, shown by a 40-57 dB reduction in the average auditory brainstem response (ABR) thresholds at 0·5-4·0 kHz. In the participant who received the 9 × 1011 vg dose, the average ABR threshold was improved from greater than 95 dB at baseline to 68 dB at 4 weeks, 53 dB at 13 weeks, and 45 dB at 26 weeks. In those who received 1·5 × 1012 AAV1-hOTOF, the average ABR thresholds changed from greater than 95 dB at baseline to 48 dB, 38 dB, 40 dB, and 55 dB in four children with hearing recovery at 26 weeks. Speech perception was improved in participants who had hearing recovery. INTERPRETATION: AAV1-hOTOF gene therapy is safe and efficacious as a novel treatment for children with autosomal recessive deafness 9. FUNDING: National Natural Science Foundation of China, National Key R&D Program of China, Science and Technology Commission of Shanghai Municipality, and Shanghai Refreshgene Therapeutics.

Prognostic implications of alpha-fetoprotein and C-reactive protein elevation in hepatocellular carcinoma following resection (PACE): a large cohort study of 2770 patients.

BACKGROUND: Routine clinical staging for hepatocellular carcinoma (HCC) incorporates liver function, general health, and tumor morphology. Further refinement of prognostic assessments and treatment decisions may benefit from the inclusion of tumor biological marker alpha-fetoprotein (AFP) and systemic inflammation indicator C-reactive protein (CRP). METHODS: Data from a multicenter cohort of 2770 HCC patients undergoing hepatectomy were analyzed. We developed the PACE risk score (Prognostic implications of AFP and CRP Elevation) after initially assessing preoperative AFP and CRP's prognostic value. Subgroup analyzes were performed in BCLC cohorts A and B using multivariable Cox analysis to evaluate the prognostic stratification ability of the PACE risk score and its complementary utility for BCLC staging. RESULTS: Preoperative AFP ≥ 400ng/mL and CRP ≥ 10 mg/L emerged as independent predictors of poorer prognosis in HCC patients who underwent hepatectomy, leading to the creation of the PACE risk score. PACE risk score stratified patients into low, intermediate, and high-risk groups with cumulative 5-year overall (OS) and recurrence-free survival (RFS) rates of 59.6%/44.9%, 43.9%/38.4%, and 20.6%/18.0% respectively (all P < 0.001). Increased PACE risk scores correlated significantly with early recurrence and extrahepatic metastases frequency (all P < 0.001). The multivariable analysis identified intermediate and high-risk PACE scores as independently correlating with poor postoperative OS and RFS. Furthermore, the PACE risk score proficiently stratified the prognosis of BCLC stages A and B patients, with multivariable analyses demonstrating it as an independent prognostic determinant for both stages. CONCLUSION: The PACE risk score serves as an effective tool for postoperative risk stratification, potentially supplementing the BCLC staging system.

Full-color persistent room temperature phosphorescent elastomers with robust optical properties.

Persistent room temperature phosphorescent materials with unique mechanical properties and robust optical properties have great potential in flexible electronics and photonics. However, developing such materials remains a formidable challenge. Here, we present highly stretchable, lightweight, and multicolored persistent luminescence elastomers, produced by incorporating ionic room temperature phosphorescent polymers and polyvinyl alcohol into a polydimethylsiloxane matrix. These prepared elastomers exhibit high optical transparency in daylight and emit bright persistent luminescence after the removal of 365 nm excitation. The homogeneous distribution of polymers within the matrix has been confirmed by confocal fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. Mechanical property investigations revealed that the prepared persistent luminescence elastomers possess satisfactory stretchability. Impressively, these elastomers maintain robust optical properties even under extensive and repeated mechanical deformations, a characteristic previously unprecedented. These fantastic features make these persistent luminescence elastomers ideal candidates for potential applications in wearable devices, flexible displays, and anti-counterfeiting.

Glutamate-aspartate transporter dysfunction enhances aminoglycoside-induced cochlear hair cell death via NMDA receptor activation.

Glutamate is a crucial neurotransmitter for hearing transduction in the cochlea, but excess glutamate is detrimental to the survival of cochlear sensory cells. Glutamate-aspartate transporter (GLAST) is the major transporter for glutamate removal; however, its role in aminoglycoside-induced hair cell loss is not well studied. In the present study, we first investigated the localization and expression of GLAST over the course of development of the mouse cochlea, and we found that inhibition of GLAST increased hair cell death. However, when the glutamate receptor NMDAR was inhibited by D-AP5, hair cell death was no longer increased by the GLAST inhibitor. Our results indicate that GLAST inhibition aggravates damage to cochlear hair cells, which may occur via NMDAR, and this suggests new clinical strategies for ameliorating the ototoxicity associated with the dysfunction of glutamate metabolism.

MPZL2 variant analysis with whole exome sequencing in a cohort of Chinese hearing loss patients.

BACKGROUND: Hearing loss is a genetically heterogeneous disease with more than 100 genes identified. Pathogenic variants in the MPZL2 gene cause autosomal recessive non-syndromic hearing loss. MPZL2 patients showed mild to moderate progressive hearing loss with onset age around 10 years old. To date, four pathogenic variants have been identified. AIMS: To explore the clinical characteristics and variants of MPZL2-related hearing loss, and summarize the prevalence rate in overall hearing loss patients. MATERIAL AND METHODS: To determine the prevalence of MPZL2-related hearing loss in the Chinese population, we analyzed MPZL2 variants of whole exome sequencing data derived from a cohort of 385 hearing loss patients. RESULTS: Overall, homozygous MPZL2 variants were identified in 5 sporadic cases (diagnostic rate = 1.30%). A novel missense variant c.52C > T;p.Leu18Phe was identified in one other patient with compound heterozygous mutations in MPZL2, but the pathogenicity was uncertain according to the American College of Medical Genetics guidelines (2015). A patient homozygous for the c.220C > T,p.Gln74Ter variant showed congenital profound hearing loss at all frequencies, a phenotype different from previous reports. CONCLUSIONS: Our results enriched the mutation and phenotype spectrum of MPZL2-related hearing loss. Comparisons between allele frequencies of MPZL2:c.220C > T;p.Gln74Ter and other common deafness variants suggested that MPZL2:c.220C > T;p.Gln74Ter should be included in the group of common deafness variants for prescreening.

P2X7 receptor is required for the ototoxicity caused by aminoglycoside in developing cochlear hair cells.

Aminoglycoside antibiotics (AGAs) are widely used in life-threatening infections, but they accumulate in cochlear hair cells (HCs) and result in hearing loss. Increases in adenosine triphosphate (ATP) concentrations and P2X7 receptor expression were observed after neomycin treatment. Here, we demonstrated that P2X7 receptor, which is a non-selective cation channel that is activated by high ATP concentrations, may participate in the process through which AGAs enter hair cells. Using transgenic knockout mice, we found that P2X7 receptor deficiency protects HCs against neomycin-induced injury in vitro and in vivo. Subsequently, we used fluorescent gentamicin-Fluor 594 to study the uptake of AGAs and found fluorescence labeling in wild-type mice but not in P2rx7-/- mice in vitro. In addition, knocking-out P2rx7 did not significantly alter the HC count and auditory signal transduction, but it did inhibit mitochondria-dependent oxidative stress and apoptosis in the cochlea after neomycin exposure. We thus conclude that the P2X7 receptor may be linked to the entry of AGAs into HCs and is likely to be a therapeutic target for auditory HC protection.

Quantifying the spatial nonstationary response of influencing factors on ecosystem health based on the geographical weighted regression (GWR) model: an example in Inner Mongolia, China, from 1995 to 2020.

The identification of ecosystem health and its influencing factors is crucial to the sustainable management of ecosystems and ecosystem restoration. Although numerous studies on ecosystem health have been carried out from different perspectives, few studies have systematically investigated the spatiotemporal heterogeneity between ecosystem health and its influencing factors. Considering this gap, the spatial relationships between ecosystem health and its factors concerning climate, socioeconomic, and natural resource endowment at the county level were estimated based on a geographically weighted regression (GWR) model. The spatiotemporal distribution pattern and driving mechanism of ecosystem health were systematically analysed. The results showed the following: (1) the ecosystem health level in Inner Mongolia spatially increases from northwest to southeast, displaying notable global spatial autocorrelation and local spatial aggregation. (2) The factors influencing ecosystem health exhibit significant spatial heterogeneity. Annual average precipitation (AMP) and biodiversity (BI) are positively correlated with ecosystem health, and annual average temperature (AMT) and land use intensity (LUI) are estimated to be negatively correlated with ecosystem health. (3) Annual average precipitation (AMP) significantly improves ecosystem health, whereas annual average temperature (AMT) significantly worsens eco-health in the eastern and northern regions. LUI negatively impacts ecosystem health in western counties (such as Alxa, Ordos, and Baynnur). This study contributes to extending our understanding of ecosystem health depending on spatial scale and can inform decision-makers about how to control various influencing factors to improve the local ecology under local conditions. Finally, this study also proposes some relevant policy suggestions and provides effective ecosystem preservation and management support in Inner Mongolia.

Analysis of the impact of traditional ethnic villages in Hani area on sustainable development.

Rapid economic development and accelerated urbanization have seriously affected the development of traditional ethnic villages in China. We used the minimum cumulative resistance (MCR) model based on land use, landscape pattern, and ecosystem service value (ESV) to evaluate the spatio-temporal dynamics of sustainable development in Hani traditional ethnic villages from 1995 to 2020. By analyzing changes in sustainability indicators in the Hani area and different buffer zones, this paper aims to assess the impact of ethnic villages in the Hani Area on sustainable development and provide recommendations for the sustainable development of traditional ethnic Hani villages. The results indicated that: (1) The area of construction land and landscape fragmentation in the Hani area significantly increased and the value of ecosystem services and levels of sustainable development decreased each year during the study period; (2) The area of cropland in the 2 km buffer zone of the traditional ethnic villages increased, the degree of landscape fragmentation, the value of ecosystem services, and the level of sustainable development were lower than in the 4 km buffer zone during the study period. This is due to population increases in traditional ethnic Hani villages, as well as the intensive reclamation of cropland, increased construction land, and landscape fragmentation. We suggest that the Hani should implement scientific land planning and management policies to protect the local ecosystem and realize the sustainable development of traditional ethnic Hani villages.

Novel WFS1 mutations in patients with low-to-middle frequency hearing loss.

BACKGROUND: Hearing loss (HL) is the most common sensorineural disorder in human. It is estimated that genetic factors contribute to over 50% of prelingual hearing loss. Most of dominant HHL patients manifest postlingual progressive hearing loss that mainly affect high frequencies. However, mutations in a few dominant HL genes, such as WFS1, TECTA and DIAPH1, cause distinct audiogram that primarily affects the low and middle frequencies. METHODS: We recruited twelve independent HL families with worse low or middle frequency audiograms. Each proband of these families was excluded for pathogenic mutations in GJB2, SLC26A4, and MT-RNR1 genes. Mutation screening was performed by whole exome sequencing. Next, candidate variants were validated in each family by sanger sequencing. RESULTS: Six heterozygous WFS1 variants were identified in six families, including three novel mutations (c.2519T > G, p.F840C; c.2048T > G, p.M683R and c.2419A > C, p.S807R) and three previously reported variants (c.2005T > C, p.Y669H; c.2590G > A, p.E864K and c.G2389A, p.D797 N). All the novel mutations were absent in 100 ethnically matched controls and were predicted to be deleterious by multiple algorithms. CONCLUSIONS: We identified three novel and three previously reported WFS1 mutations in six unrelated Chinese families. Our findings enriched the genotype-phenotype spectrum of WFS1 related NSHL. Additional genotype-phenotype correlation study will clarify the detailed phenotypic range caused by WFS1 mutations.

Quantifying the impact of the Grain-for-Green Program on ecosystem service scarcity value in Qinghai, China.

Studying the impact of large-scale ecological projects, such as the Grain-for-Green Program (GGP), on ecosystem services (ES) is currently a frontier and hot topic of ecological research. The GGP can directly change land use and land cover, thus affecting ES. By comparing the changes of ecosystem service value (ESV) and ecosystem service scarcity value (ESSV) in Qinghai before and after the implementation of the GGP, this paper clarified the impact of the GGP on Qinghai from the angles of ecology and economics. This paper quantified and evaluated the land use dynamics, ESV, and ESSV in Qinghai from 1995 to 2020. The results showed that in the past 25 years, the total annual Normalized Difference Vegetation Index (NDVI) of Qinghai showed a trend of sustained growth. From 1995 to 2020, the ESV increased by 6.80%. After considering supply and demand, the ESSV showed a continuous upward trend, increasing by 719.38%. After implementation of the GGP, the increase of NDVI inhibited the increase of the ESSV. These findings from evaluation of the effect of the GGP implementation provide a theoretical basis for future policy implementation and, in particular, a reference for the evaluation of the ESV and the ESSV in Qinghai.

Generation of innervated cochlear organoid recapitulates early development of auditory unit.

Functional cochlear hair cells (HCs) innervated by spiral ganglion neurons (SGNs) are essential for hearing, whereas robust models that recapitulate the peripheral auditory circuity are still lacking. Here, we developed cochlear organoids with functional peripheral auditory circuity in a staging three-dimensional (3D) co-culture system by initially reprogramming cochlear progenitor cells (CPCs) with increased proliferative potency that could be long-term expanded, then stepwise inducing the differentiation of cochlear HCs, as well as the outgrowth of neurites from SGNs. The function of HCs and synapses within organoids was confirmed by a series of morphological and electrophysiological evaluations. Single-cell mRNA sequencing revealed the differentiation trajectories of CPCs toward the major cochlear cell types and the dynamic gene expression during organoid HC development, which resembled the pattern of native HCs. We established the cochlear organoids with functional synapses for the first time, which provides a platform for deciphering the mechanisms of sensorineural hearing loss.

Loss of Mst1/2 activity promotes non-mitotic hair cell generation in the neonatal organ of Corti.

Mammalian sensory hair cells (HCs) have limited capacity for regeneration, which leads to permanent hearing loss after HC death. Here, we used in vitro RNA-sequencing to show that the Hippo signaling pathway is involved in HC damage and self-repair processes. Turning off Hippo signaling through Mst1/2 inhibition or Yap overexpression induces YAP nuclear accumulation, especially in supporting cells, which induces supernumerary HC production and HC regeneration after injury. Mechanistically, these effects of Hippo signaling work synergistically with the Notch pathway. Importantly, the supernumerary HCs not only express HC markers, but also have cilia structures that are able to form neural connections to auditory regions in vivo. Taken together, regulating Hippo suggests new strategies for promoting cochlear supporting cell proliferation, HC regeneration, and reconnection with neurons in mammals.

The heterogeneity of mammalian utricular cells over the course of development.

BACKGROUND: The inner ear organ is a delicate tissue consisting of hair cells (HCs) and supporting cells (SCs).The mammalian inner ear HCs are terminally differentiated cells that cannot spontaneously regenerate in adults. Epithelial non-hair cells (ENHCs) in the utricle include HC progenitors and SCs, and the progenitors share similar characteristics with SCs in the neonatal inner ear. METHODS: We applied single-cell sequencing to whole mouse utricles from the neonatal period to adulthood, including samples from postnatal day (P)2, P7 and P30 mice. Furthermore, using transgenic mice and immunostaining, we traced the source of new HC generation. RESULTS: We identified several sensory epithelial cell clusters and further found that new HCs arose mainly through differentiation from Sox9+ progenitor cells and that only a few cells were produced by mitotic proliferation in both neonatal and adult mouse utricles. In addition, we identified the proliferative cells using the marker UbcH10 and demonstrated that in adulthood the mitotically generated HCs were primarily found in the extrastriola. Moreover, we observed that not only Type II, but also Type I HCs could be regenerated by either mitotic cell proliferation or progenitor cell differentiation. CONCLUSIONS: Overall, our findings expand our understanding of ENHC cell fate and the characteristics of the vestibular organs in mammals over the course of development.

Novel homozygous variant in the PDZD7 gene in a family with nonsyndromic sensorineural hearing loss.

Hearing loss is the most common sensory neural disorder in humans, and according to a WHO estimation, 5.5% (466 million) of people worldwide have disabling hearing loss. In this study, a Chinese family with prelingual sensorineural hearing loss was investigated. The affected individuals showed moderately severe hearing loss at all frequencies. Using target genome enrichment and high-throughput sequencing, the homozygous variant c.2372del; p.(Ser791fs) was identified in PDZD7. This variant lies in exon 15 of PDZD7 and results in a frame shift followed by an early stop codon. It is classified as pathogenic according to the ACMG/AMP guidelines and ClinGen specifications. Our study expands the pathogenic variant spectrum of PDZD7 and strengthens the clinical importance of this gene in patients with moderately severe hearing loss.

Identification of a novel CNV at the EYA4 gene in a Chinese family with autosomal dominant nonsyndromic hearing loss.

BACKGROUND: Hereditary hearing loss is a heterogeneous class of disorders that exhibits various patterns of inheritance and involves many genes. Variants in the EYA4 gene in DFNA10 are known to lead to postlingual, progressive, autosomal dominant nonsyndromic hereditary hearing loss. PATIENTS AND METHODS: We collected a four-generation Chinese family with autosomal-dominant nonsyndromic hearing loss (ADNSHL). We applied targeted next-generation sequencing (TNGS) in three patients of this pedigree and whole-genome sequencing (WGS) in the proband. The intrafamilial cosegregation of the variant and the deafness phenotype were confirmed by PCR, gap-PCR and Sanger sequencing. RESULTS: A novel CNV deletion at 6q23 in exons 8-11 of the EYA4 gene with a 10 bp insertion was identified by TNGS and WGS and segregated with the ADNSHL phenotypes. CONCLUSIONS: Our results expanded the variant spectrum and genotype‒phenotype correlation of the EYA4 gene and autosomal dominant nonsyndromic hereditary hearing loss in Chinese Han individuals. WGS is an accurate and effective method for verifying the genomic features of CNVs.

A humanized murine model, demonstrating dominant progressive hearing loss caused by a novel KCNQ4 mutation (p.G228D) from a large Chinese family.

The pathogenic variants in KCNQ4 cause DFNA2 nonsyndromic hearing loss. However, the understanding of genotype-phenotype correlations between KCNQ4 and hearing is limited. Here, we identified a novel KCNQ4 mutation p.G228D from a Chinese family, including heterozygotes characterized by high-frequency hearing loss that is progressive across all frequencies and homozygotes with more severe hearing loss. We constructed a novel murine model with humanized homologous Kcnq4 mutation. The heterozygotes had mid-frequency and high-frequency hearing loss at 4 weeks, and moved toward all frequencies hearing loss at 12 weeks, while the homozygotes had severe-to-profound hearing loss at 8 weeks. The degeneration of outer hair cells (OHCs) was observed from basal to apical turn of cochlea. The reduced K+ currents and depolarized resting potentials were revealed in OHCs. Remarkably, we observed the loss of inner hair cells (IHCs) in the region corresponding to the frequency above 32 kHz at 8-12 weeks. The results suggest the degeneration of OHCs and IHCs may contribute to high-frequency hearing loss in DFNA2 over time. Our findings broaden the variants of KCNQ4 and provide a novel mouse model of progressive hearing loss, which contributes to an understanding of pathogenic mechanism and eventually treatment of DFNA2 progressive hearing loss.

Integrating supply and demand factors for estimating ecosystem services scarcity value and its response to urbanization in typical mountainous and hilly regions of south China.

In the process of rapid urbanization, the decline in ecosystems' physical supply value is a direct result of the increasing demands of human development. The ecosystem services value combined with supply and demand factors can be used to obtain the ecosystem services scarcity value (ESSV). These ecosystems with larger ESSVs warrant increased protection, or at the very least a development plan to ensure a balance between their continued existence and human usage. However, urbanization and other developmental effects have often caused the ESSV of those regions to be disregarded, which produces an imbalance in the ecosystem. This paper evaluated the spatial differentiation of ESSV based on heterogeneous units in the Yuebei region, China. Then, a spatial response analysis of the ESSV to urbanization according to the spatial correlation model and Getis-Ord Gi* was conducted. The results showed that the ecosystem service physical supply value declined due to urban sprawl in 1990-2015, but the ESSV increased sharply during the same time with its acceleration being particularly strong after 2000. Areas with higher ESSV were concentrated in the northern region due to a higher prevalence of woodland. Urbanization accelerated significantly after 2000, and it is found that similar tendencies of ESSV and urbanization occurred over the same period. Surprisingly, the ESSV was negatively correlated with urbanization in local regions, and their interaction continued to increase over time. The spatial hotspot map of the ESSV and urbanization also formed an opposite pattern. In addition, the grid number of High-Low and Low-High types is more than half of the total number of aggregated grids. According to the results, understanding the variation in the ESSV clearly due to urbanization and identifying their interaction in local regions can provide a more specific direction for future resource adjustment and ecosystem service improvements.

The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea.

Sensory hair cells (HCs) are highly susceptible to damage by noise, ototoxic drugs, and aging. Although HCs cannot be spontaneously regenerated in adult mammals, previous studies have shown that signaling pathways are involved in HC regeneration in the damaged mouse cochlea. Here, we used a Notch antagonist (DAPT), a Wnt agonist (QS11), and recombinant Sonic hedgehog (SHH) protein to investigate their concerted actions underlying HC regeneration in the mouse cochlea after neomycin-induced damage both in vivo and in vitro. With DAPT, the numbers of HCs increased, and supporting cell (SC) proliferation was seen in both the intact and damaged cochlear sensory epithelia, while these numbers were unchanged in the presence of QS11. When simultaneously treated with DAPT and QS11, the number of HCs increased dramatically, and much greater SC proliferation was seen in the cochlear epithelium. In transgenic mice with both Notch1 conditional knockout and ß-catenin over-expression, cochlear SC proliferation and HC regeneration were more obvious than in either Notch1 knockout or ß-catenin over-expressing mice separately. When cochleae were treated with DAPT, QS11, and SHH together, SC proliferation was even greater, and this proliferation was seen in both the HC region and the greater epithelial ridge. High-throughput RNA sequencing was used to identify the differentially expressed genes between all groups, and the results showed that the SHH and Wnt signaling pathways are involved in SC proliferation. Our study suggests that co-regulation of the Notch, Wnt, and SHH signaling pathways promotes extensive cell proliferation and regeneration in the mouse cochlea.