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Inner ear organoids play a crucial role in hearing research. In comparison to other animal models and 2D cell culture systems, inner ear organoids offer significant advantages for studying the mechanisms of inner ear development and exploring novel approaches to disease treatment. Inner ear organoids derived from human cells are more closely resemble normal human organs in development and function. The 3D culture system of the inner ear organoid enhances cell-cell interactions and mimics the internal environment. In this review, the progress and limitations of organoid culture methods derived from tissue-specific progenitors and pluripotent stem cells (PSCs) are summarized, which may offer new insights into generating organoids that closely resemble the inner ear in terms of morphology and function.

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KEY MESSAGE: 'Sikkim Primitive' maize landrace, unique for prolificacy (7-9 ears per plant) possesses unique genomic architecture in branching and inflorescence-related gene(s), and locus Zm00001eb365210 encoding glycosyltransferases was identified as the putative candidate gene underlying QTL (qProl-SP-8.05) for prolificacy. The genotype possesses immense usage in breeding high-yielding baby-corn genotypes. 'Sikkim Primitive' is a native landrace of North Eastern Himalayas, and is characterized by having 7-9 ears per plant compared to 1-2 ears in normal maize. Though 'Sikkim Primitive' was identified in the 1960s, it has not been characterized at a whole-genome scale. Here, we sequenced the entire genome of an inbred (MGUSP101) derived from 'Sikkim Primitive' along with three non-prolific (HKI1128, UMI1200, and HKI1105) and three prolific (CM150Q, CM151Q and HKI323) inbreds. A total of 942,417 SNPs, 24,160 insertions, and 27,600 deletions were identified in 'Sikkim Primitive'. The gene-specific functional mutations in 'Sikkim Primitive' were classified as 10,847 missense (54.36%), 402 non-sense (2.015%), and 8,705 silent (43.625%) mutations. The number of transitions and transversions specific to 'Sikkim Primitive' were 666,021 and 279,950, respectively. Among all base changes, (G to A) was the most frequent (215,772), while (C to G) was the rarest (22,520). Polygalacturonate 4-α-galacturonosyltransferase enzyme involved in pectin biosynthesis, cell-wall organization, nucleotide sugar, and amino-sugar metabolism was found to have unique alleles in 'Sikkim Primitive'. The analysis further revealed the Zm00001eb365210 gene encoding glycosyltransferases as the putative candidate underlying QTL (qProl-SP-8.05) for prolificacy in 'Sikkim Primitive'. High-impact nucleotide variations were found in ramosa3 (Zm00001eb327910) and zeaxanthin epoxidase1 (Zm00001eb081460) genes having a role in branching and inflorescence development in 'Sikkim Primitive'. The information generated unraveled the genetic architecture and identified key genes/alleles unique to the 'Sikkim Primitive' genome. This is the first report of whole-genome characterization of the 'Sikkim Primitive' landrace unique for its high prolificacy.

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Inner ear morphogenesis requires tightly regulated epigenetic and transcriptional control of gene expression. CHD7, an ATP-dependent chromodomain helicase DNA-binding protein, and SOX2, an SRY-related HMG box pioneer transcription factor, are known to contribute to vestibular and auditory system development, but their genetic interactions in the ear have not been explored. Here, we analyzed inner ear development and the transcriptional regulatory landscapes in mice with variable dosages of Chd7 and/or Sox2. We show that combined haploinsufficiency for Chd7 and Sox2 results in reduced otic cell proliferation, severe malformations of semicircular canals, and shortened cochleae with ectopic hair cells. Examination of mice with conditional, inducible Chd7 loss by Sox2CreER reveals a critical period (~E9.5) of susceptibility in the inner ear to combined Chd7 and Sox2 loss. Data from genome-wide RNA-sequencing and CUT&Tag studies in the otocyst show that CHD7 regulates Sox2 expression and acts early in a gene regulatory network to control expression of key otic patterning genes, including Pax2 and Otx2. CHD7 and SOX2 directly bind independently and cooperatively at transcription start sites and enhancers to regulate otic progenitor cell gene expression. Together, our findings reveal essential roles for Chd7 and Sox2 in early inner ear development and may be applicable for syndromic and other forms of hearing or balance disorders.

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Hearing starts, at the cellular level, with mechanoelectrical transduction by sensory hair cells. Sound information is then transmitted via afferent synaptic connections with auditory neurons. Frequency information is encoded by the location of hair cells along the cochlear duct. Loss of hair cells, synapses, or auditory neurons leads to permanent hearing loss in mammals. Birds, in contrast, regenerate auditory hair cells and functionally recover from hearing loss. Here, we characterized regeneration and reinnervation in sisomicin-deafened chickens and found that afferent neurons contact regenerated hair cells at the tips of basal projections. In contrast to development, synaptic specializations are established at these locations distant from the hair cells' bodies. The protrusions then contracted as regenerated hair cells matured and became functional 2 weeks post-deafening. We found that auditory thresholds recovered after 4-5 weeks. We interpret the regeneration-specific synaptic reestablishment as a location-preserving process that might be needed to maintain tonotopic fidelity.

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The ear serves two vital functions of hearing and maintaining balance. It achieves these roles within three major compartments: the outer, the middle, and the inner ear. Embryological development of the ear and its associated structures have been studied in some animal models. Yet, the role of skeletal muscle in ear development and its related structures is largely unknown. Research suggests the outer ear and parts of the inner ear may require skeletal muscle for normal embryogenesis. Here, we describe the role of skeletal muscle in the development of the ear and its associated structures. Moreover, we report the possible consequences of defect in the skeletal muscle of the ear and the clinical correlates of such consequences.

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Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells directed to differentiate into inner ear organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs with human IEOs. We use multiplexed immunostaining and single-cell RNA-sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro-derived otic placode, epithelium, neuroblasts and sensory epithelia. In parallel, we evaluate the expression and localization of crucial markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

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The Notch and Wnt signalling pathways play key roles in the formation of inner ear sensory organs, but little is known about their transcriptional effectors and targets in this context. Here, we perturbed Notch and Wnt activities in the embryonic chicken otic vesicle using pharmacological treatment or in ovo electroporation of plasmid DNA, and used RNA-Seq to analyse the resulting changes in gene expression. Compared to pharmacological treatments, in ovo electroporation changed the expression of fewer genes, a likely consequence of the variability and mosaicism of transfection. The pharmacological inhibition of Notch activity induced a rapid change in the expression of known effectors of this pathway and genes associated with neurogenesis, consistent with a switch towards an otic neurosensory fate. The Wnt datasets contained many genes associated with a neurosensory biological function, confirming the importance of this pathway for neurosensory specification in the otocyst. Finally, the results of a preliminary gain-of-function screening of selected transcription factors and Wnt signalling components suggest that the endogenous programs of otic neurosensory specification are very robust, and in general unaffected by the overexpression of a single factor. Altogether this work provides new insights into the effectors and candidate targets of the Notch and Wnt pathways in the early developing inner ear and could serve as a useful reference for future functional genomics experiments in the embryonic avian inner ear.

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Seed number and harvesting ability in maize (Zea mays L.) are primarily determined by the architecture of female inflorescence, namely the ear. Therefore, ear morphogenesis contributes to grain yield and as such is one of the key target traits during maize breeding. However, the molecular networks of this highly dynamic and complex grain-bearing inflorescence remain largely unclear. As a first step toward characterizing these networks, we performed a high-spatio-temporal-resolution investigation of transcriptomes using 130 ear samples collected from developing ears with length from 0.1 mm to 19.0 cm. Comparisons of these mRNA populations indicated that these spatio-temporal transcriptomes were clearly separated into four distinct stages stages I, II, III, and IV. A total of 23 793 genes including 1513 transcription factors (TFs) were identified in the investigated developing ears. During the stage I of ear morphogenesis, 425 genes were predicted to be involved in a co-expression network established by eight hub TFs. Moreover, 9714 ear-specific genes were identified in the seven kinds of meristems. Additionally, 527 genes including 59 TFs were identified as especially expressed in ear and displayed high temporal specificity. These results provide a high-resolution atlas of gene activity during ear development and help to unravel the regulatory modules associated with the differentiation of the ear in maize.

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BACKGROUND: In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. LMO4, an LIM domain-only transcriptional regulator, is drawing an increasing amount of interest for its multiple roles regarding human embryonic development and the modulation of ototoxic side effects of cisplatin including cochlear apoptosis and hearing loss. The aim of the present study is to further explore the role of lmo4a in zebrafish inner ear development and thus explore its functional role. METHODS: The Spatial Transcript Omics DataBase was referred to in order to evaluate the expression of lmo4a during the first 24 h of zebrafish development. In situ hybridization was applied to validate and extend the expression profile of lmo4a to 3 days post-fertilization. The morpholino (MO) knockdown and CRISPR/Cas9 knockout (KO) of lmo4a was applied. Morphological analyses of otic vesical, hair cells, statoacoustic ganglion and semicircular canals were conducted. The swimming pattern of lmo4a KO and MO zebrafish was tracked. In situ hybridization was further applied to verify the expression of genes of the related pathways. Rescue of the phenotype was attempted by blockage of the bmp pathway via heat shock and injection of Dorsomorphin. RESULTS: lmo4a is constitutively expressed in the otic placode and otic vesicle during the early stages of zebrafish development. Knockdown and knockout of lmo4a both induced smaller otocysts, less hair cells, immature statoacoustic ganglion and malformed semicircular canals. Abnormal swimming patterns could be observed in both lmo4a MO and KO zebrafish. eya1 in preplacodal ectoderm patterning was downregulated. bmp2 and bmp4 expressions were found to be upregulated and extended in lmo4a morphants, and blockage of the Bmp pathway partially rescued the vestibular defects. CONCLUSIONS: We concluded that lmo4a holds a regulative effect on the Bmp pathway and is required for the normal development of zebrafish inner ear. Our study pointed out the conservatism of LMO4 in inner ear development between mammals and zebrafish as well as shed more light on the molecular mechanisms behind it. Further research is needed to distinguish the relationships between lmo4 and the Bmp pathway, which may lead to diagnostic and therapeutic approaches towards human inner ear malformation.

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Dentin matrix protein 1 (Dmp1) is a highly phosphorylated, extracellular matrix protein that is extensively expressed in bone and teeth but also found in soft tissues, including brain and muscle. However, the functions of Dmp1 in the mice cochlea are unknown. Our study showed that Dmp1 was expressed in auditory hair cells (HCs), with the role of Dmp1 in those cells identified using Dmp1 cKD mice. Immunostaining and scanning electron microscopy of the cochlea at P1 revealed that Dmp1 deficiency in mice resulted in an abnormal stereociliary bundle morphology and the mispositioning of the kinocilium. The following experiments further demonstrated that the cell-intrinsic polarity of HCs was affected without apparent effect on the tissue planer polarity, based on the observation that the asymmetric distribution of Vangl2 was unchanged whereas the Gαi3 expression domain was enlarged and Par6b expression was slightly altered. Then, the possible molecular mechanisms of Dmp1 involvement in inner ear development were explored via RNA-seq analysis. The study suggested that the Fgf23-Klotho endocrine axis may play a novel role in the inner ear and Dmp1 may regulate the kinocilium-stereocilia interaction via Fgf23-Klotho signaling. Together, our results proved the critical role of Dmp1 in the precise regulation of hair bundle morphogenesis in the early development of HCs.

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The EYA1 gene is essential for normal inner ear development and affects the development and function of the inner ear in a dose-dependent manner. However, the mechanisms regulating EYA1 gene expression are not well understood. Recently, miRNAs have become recognized as important regulators of gene expression. In this study, we identified miR-124-3p through a microRNA (miRNA) target prediction website and found that miR-124-3p and its target site in the EYA1 3' untranslated region (3'UTR) are conserved in most vertebrates. Both in vivo and in vitro, the interaction of miR-124-3p with the EYA1 3'UTR exerts a negative regulatory effect. Microinjection of agomiR-124-3p into zebrafish embryos resulted in a phenotype of reduced auricular area, suggesting inner ear dysplasia. In addition, injection of agomiR-124-3p or antagomiR-124-3p caused abnormal hearing function in zebrafish. In conclusion, our results suggest that miR-124-3p can affect inner ear development and hearing function in zebrafish by regulating EYA1.

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It has been established for almost 30 years that the retinoic acid receptor (RAR) signalling pathway plays essential roles in the morphogenesis of a large variety of organs and systems. Here, we used a temporally controlled genetic ablation procedure to precisely determine the time windows requiring RAR functions. Our results indicate that from E8.5 to E9.5, RAR functions are critical for the axial rotation of the embryo, the appearance of the sinus venosus, the modelling of blood vessels, and the formation of forelimb buds, lung buds, dorsal pancreatic bud, lens, and otocyst. They also reveal that E9.5 to E10.5 spans a critical developmental period during which the RARs are required for trachea formation, lung branching morphogenesis, patterning of great arteries derived from aortic arches, closure of the optic fissure, and growth of inner ear structures and of facial processes. Comparing the phenotypes of mutants lacking the 3 RARs with that of mutants deprived of all-trans retinoic acid (ATRA) synthesising enzymes establishes that cardiac looping is the earliest known morphogenetic event requiring a functional ATRA-activated RAR signalling pathway.

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High incidence of chronic otitis media is associated with human craniofacial syndromes, suggesting that defects in the formation of the middle ear and associated structures can have a knock-on effect on the susceptibility to middle ear inflammation. Patients with branchio-oto-renal (BOR) syndrome have several defects in the ear leading to both sensorineural and conductive hearing loss, including otitis media. 40% of BOR syndrome cases are due to Eya1 haploinsufficiency, with mouse models affecting Eya1, mimicking many of the defects found in patients. Here, we characterize the onset, consequences, and underlying causes of chronic otitis media in Eya1 heterozygous mice. Cavitation defects were evident in these mice from postnatal day (P)11 onwards, with mesenchyme around the promontory and attic regions of the middle ear space. This mesenchyme was still prominent in adult Eya1 heterozygous mice, while the wild-type littermates had fully aerated ears from P14 onwards. MicroCT analysis highlighted a significantly smaller bulla, confirming the link between bulla size defects and the ability of the mesenchyme to retract successfully. Otitis media was observed from P14, often presenting unilaterally, resulting in hyperplasia of the middle ear mucosa, expansion of secretory cells, defects in the motile cilia, and changes in basal epithelial cell markers. A high incidence of otitis media was identified in older mice but only associated with ears with retained mesenchyme. To understand the impact of the environment, the mouse line was rederived onto a super-clean environment. Cavitation defects were still evident at early stages, but these generally resolved over time, and importantly, no signs of otitis media were observed at 6 weeks. In conclusion, we show that a small bulla size is closely linked to defects in cavitation and the presence of retained mesenchyme. A delay in retraction of the mesenchyme predates the onset of otitis media, making the ears susceptible to its development. Early exposure to OM appears to exacerbate the cavitation defect, with mesenchyme evident in the middle ear throughout the animal's life. This highlights that permanent damage to the middle ear can arise as a consequence of the early onset of OM.

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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.

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BACKGROUND: The vitamin A derivative all-trans retinoic acid (RA) regulates early stages of inner ear development. As the early disruption of the RA pathway results in profound mispatterning of the developing inner ear, this confounds analyses of specific roles in later stages. Therefore, we used the temporal-specific exposure of all-trans RA or diethylaminobenzaldehyde to evaluate RA functions in late otic development. RESULTS: Perturbing late RA signaling causes behavioral defects analogous to those expected in larvae suffering from vestibular dysfunction. These larvae also demonstrate malformations of the semi-circular canals, as visualized through (a) use of the transgenic strain nkhspdmc12a, a fluorescent reporter expressed in otic epithelium; and (b) injection of the fluorescent lipophilic dye DiI. We also noted the altered expression of genes encoding ECM proteins or modifying enzymes. Other malformations of the inner ear observed in our work include the loss or reduced size of the utricular and saccular otoliths, suggesting a role for RA in otolith maintenance. CONCLUSION: Our work has identified a previously undescribed late phase of RA activity in otic development, demonstrating that vestibular defects observed in human patients in relation to perturbed RA signaling are not solely due to its early disruption in otic development.

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Intermediate cells of the stria vascularis are neural crest derived melanocytes. They are essential for the establishment of the endocochlear potential in the inner ear, which allows mechanosensory hair cells to transduce sound into nerve impulses. Despite their importance for normal hearing, how these cells develop and migrate to their position in the lateral wall of the cochlea has not been studied. We find that as early as E10.5 some Schwann cell precursors in the VIIIth ganglion begin to express melanocyte specific markers while neural crest derived melanoblasts migrate into the otic vesicle. Intermediate cells of both melanoblast and Schwann cell precursor origin ingress into the lateral wall of the cochlea starting at around E15.5 following a basal to apical gradient during embryonic development, and continue to proliferate postnatally.

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BACKGROUND: Stimulating maize ear development is an effective way of improving yield. However, limited information is available regarding the regulation of sink strength change from weak to strong at the same position of maize plants. Here, a novel method for stimulating development combined with physiological assays and proteomics was applied to explore the regulation of ear strengthened development. RESULTS: By blocking pollination of the upper ear of maize hybrid Suyu 41, the adjacent lower ear was dramatically stimulated at 4 days after pollination (DAP). Tandem mass tag (TMT)-based proteomics identified 173 differentially expressed proteins (fold change >1.2 or <0.83, P < 0.05) from 7793 total proteins. Gene ontology annotations indicated that several pathways showed noticeable changes, with a preferential distribution to cell wall remodeling, hormone signals and lipid metabolism in the stimulated kernels. Cell wall remodeling was highly mediated by chitinase, exhydrolase II and xyloglucan enotransglucosylase/hydrolase, and accompanied by increased sucrose and glucose content. A series of lipoxygenase proteins were significantly upregulated, causing a significant alteration in lipid metabolism. Hormone signals were influenced by the expression of the proteins involved in indole-3-acetic acid (IAA) transport, zeatin (ZT) biosynthesis and abscisic acid (ABA) signal response, and increased IAA, ZT and ABA content. CONCLUSION: The critical time for understanding the mechanism by which ear growth is stimulated is 4 DAP. Comparative proteomics and physiological analysis revealed that lipid metabolism enhancement, cell wall remodeling and changes in hormone signaling (IAA, ZT and ABA) were all important in stimulating early ear development. Proper regulation of these pathways may improve ear development, resulting in increased maize yield. © 2022 Society of Chemical Industry.

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We investigated DAB1-protein deficiency in the inner-ear development of yotari in comparison to humans and wild-type (wt) mice by immunofluorescence for the expression of connexins (Cxs) and the pannexin Panx1. The spatial and temporal dynamics of Cx26, Cx32, Cx37, Cx40, Cx43, Cx45, and Panx1 were determined in the sixth and eighth weeks of human development and at the corresponding mouse embryonic E13.5 and E15.5, in order to examine gap junction intercellular communication (GJIC) and hemichannel formation. The quantification of the area percentage covered by positive signal was performed for the epithelium and mesenchyme of the cochlear and semicircular ducts and is expressed as the mean ± SD. The data were analysed by one-way ANOVA. Almost all of the examined Cxs were significantly decreased in the cochlear and semicircular ducts of yotari compared to wt and humans, except for Cx32, which was significantly higher in yotari. Cx40 dominated in human inner-ear development, while yotari and wt had decreased expression. The Panx1 expression in yotari was significantly lower than that in the wt and human inner ear, except at E13.5 in the mesenchyme of the wt and epithelium and mesenchyme of humans. Our results emphasize the relevance of GJIC during the development of vestibular and cochlear functions, where they can serve as potential therapeutic targets in inner-ear impairments.

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Abstract Mucopolysaccharidoses (MPS) are lysosomal diseases caused by deficiencies in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Sensorineural hearing impairment is a common feature in MPS patients, but there is no consensus on its etiology. For this reason, we aimed to identify genes and pathways related to hearing loss and to correlate them with gene expression data in MPS. We used HPO and Disgenet to identify candidate genes. We constructed the network with string and Cytoscape, and hub genes were identified in Cytohubba. Expression data were obtained from the MPSBase website. We found the NDUFA gene family as the major hub genes and 114 enriched pathways related to hearing loss. These genes and biological pathways may serve as potential candidates for clinical studies to better understand hearing impairment mechanisms in lysosomal storage diseases like mucopolysaccharidosis.