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Changes in stereociliary morphologies in Shh-bead-implanted BP. (A) E16 BP stained with phalloidin. Rectangles (150 μm × 75 μm) were drawn by the superior edge of the BP at 5%, 25%, 50%, 75%, and 95% positions from the basal end of E9, E11, E13, and E16 control and E16 Shh-beadimplanted ears. Thirty hair cells from each rectangle were randomly selected to measure hair bundle widths. (B-H) Pictures of phalloidin-labeled stereocilia at base (5%), middle (50%), and apex (95%) of E16 control (B-D) and Shh-bead-implanted ears (F-H). (E and I) Paint-filled inner ears of controls (E) and Shh-implanted ears (I) at E9 showing abnormal dorsal vestibular structures (asterisk) and slighlty shorter BP. (J and K) Hair bundle widths of hair cells were plotted as a function of relative distance from the basal end. (J) In controls, the hair bundle width is initially similar along the BP at E9 but gradually increases its width except at the apex. (K) In Shh-bead-implanted ears, hair bundle widths are significantly narrower in the basal regions (5% and 25%) compared with wild-type control or BSA-bead-implanted ears (*P < 0.0001).
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Significance
Sound frequency discrimination is crucial for daily activities throughout the animal kingdom. This process begins at the auditory peripheral organ known as the organ of Corti in mammals and basilar papilla in birds. This frequency tuning is facilitated by specific anatomical and physiological properties, including gradual changes in th...
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... we asked whether this Shh signaling gradient is important for tonotopic organization of the mature BP by perturbing the gradient using beads soaked with Shh protein implanted into chicken otocysts in ovo on 3 consecutive days at embryonic (E) days E2.5, E3.5, and E4.5. The expression domains of Ptch1 and Gli1 were expanded in Shh-implanted ears ( Fig. S1 E and F, arrowheads; n = 4/4 for Ptch1, n = 3/3 for Gli1) compared with controls ( Fig. S1 B and C). The gross morphology of the inner ear was affected as well, exhibiting severely malformed vestibular structures and a slightly shortened cochlea ( Fig. 1 E and I; n = 6/6). The malformed vestibular phenotypes are consistent with ...
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... mature BP by perturbing the gradient using beads soaked with Shh protein implanted into chicken otocysts in ovo on 3 consecutive days at embryonic (E) days E2.5, E3.5, and E4.5. The expression domains of Ptch1 and Gli1 were expanded in Shh-implanted ears ( Fig. S1 E and F, arrowheads; n = 4/4 for Ptch1, n = 3/3 for Gli1) compared with controls ( Fig. S1 B and C). The gross morphology of the inner ear was affected as well, exhibiting severely malformed vestibular structures and a slightly shortened cochlea ( Fig. 1 E and I; n = 6/6). The malformed vestibular phenotypes are consistent with previous mouse models of gain-of-Shh functions ...
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... and E4.5. The expression domains of Ptch1 and Gli1 were expanded in Shh-implanted ears ( Fig. S1 E and F, arrowheads; n = 4/4 for Ptch1, n = 3/3 for Gli1) compared with controls ( Fig. S1 B and C). The gross morphology of the inner ear was affected as well, exhibiting severely malformed vestibular structures and a slightly shortened cochlea ( Fig. 1 E and I; n = 6/6). The malformed vestibular phenotypes are consistent with previous mouse models of gain-of-Shh functions ...
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... analyze the tonotopic properties of hair cells in the developing BP, we sampled hair cells located in the superior side of the BP along the tonotopic axis (Fig. 1A) (20). Stereocilia bundles along the BP are similar in morphology between E9 and E11 ( Fig. 1J and Fig. S2). After E11, the width and length of stereocilia increase at different rates depending on their tonotopic positions such that there is a clear gradation of bundle morphology along the papilla by E16: more numerous short stereocilia ...
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... analyze the tonotopic properties of hair cells in the developing BP, we sampled hair cells located in the superior side of the BP along the tonotopic axis (Fig. 1A) (20). Stereocilia bundles along the BP are similar in morphology between E9 and E11 ( Fig. 1J and Fig. S2). After E11, the width and length of stereocilia increase at different rates depending on their tonotopic positions such that there is a clear gradation of bundle morphology along the papilla by E16: more numerous short stereocilia on basal hair cells compared with the longer and fewer stereocilia on apical hair cells (Figs. 1 B-D and ...
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... E11, the width and length of stereocilia increase at different rates depending on their tonotopic positions such that there is a clear gradation of bundle morphology along the papilla by E16: more numerous short stereocilia on basal hair cells compared with the longer and fewer stereocilia on apical hair cells (Figs. 1 B-D and J and 2 D-F). In Shh-beadimplanted ears, this gradation is disrupted in the base and middle regions of the papilla with narrower stereocilia bundle widths and somewhat taller stereocilia in the middle region ( Fig. 1 F-H). ...
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... stereocilia phenotype was first quantified by comparing the width of the entire stereocilia bundle between control and Shh-bead-implanted ears. The bundle width of basal hair cells in Shh-treated ears is significantly narrower than that of control hair cells and is comparable to that of stereocilia located in the middle region of controls (Fig. 1K), suggesting that ectopic Shh activation in the otocyst resulted in basal hair cells adapting more apical characteristics. Furthermore, we counted the total number of stereocilia per hair cell using scanning electron micrographs of hair cells from three different regions of the papilla (base, mid, and apex). At E16, hair cells in ...
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... there is precedence for a model in which sequential signaling from the midline and spiral ganglion sources of Shh is required for tonotopic patterning of the cochlea. The requirement for consecutive days of Shh delivery in ovo to elicit a modulation of the normal tonotopic patterning supports this hypothesis ( Figs. 1 and 2). Alternatively, it is equally likely that other signaling molecules, yet to be identified, are involved in tonotopic organization. ...
Citations
... High-frequency HCs at the proximal end of the BP are significantly larger that the low-frequency distal HCs at the opposite end. Specification of a HC's 'tonotopic identity' relies on the coordinated signalling between graded glucose metabolism and morphogen signalling prior to their terminal differentiation at around embryonic day (E) 6-8 Thiede et al., 2014;Son et al., 2015;O'Sullivan et al., 2023). Once instructed of their tonotopic identity, little is known about the effector mechanisms that generate the size differences between proximal and distal HCs in the mature epithelium. ...
In multicellular tissues, cell size and shape are intricately linked with physiological function. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells (SCs), which have distinct morphologies at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs are larger than their distal (low-frequency) counterparts, a morphological feature essential for frequency tuning. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this a potential mechanism for cell size regulation in developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared to distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC size, whilst promotion of fusion increased the distal HC size. We identify mitochondrial dynamics as a key regulator of HC size and morphology in developing inner ear epithelia.
Summary Statement
Mitochondrial remodelling drives developmental changes in cell size in the auditory sensory epithelium. Our data reveal a fundamental mechanism regulating cell size and frequency-place coding in the developing cochlea.
... 64 During embryonic development, regional identity is mirrored in tonotopic gene expression pattern of genes such as Fst, Hmga2, and A2m. 63,65,66 However, during organoid development, none of the candidate genes were detected in HCLCs nor in SCLCs ( Figures S3A-S3C). Together, these findings suggest that cues mediating apex-to-base regional identity are not mediated by the self-guiding protocol. ...
Inner ear organoids recapitulate development and are intended to generate cell types of the otic lineage for applications such as basic science research and cell replacement strategies. Here, we use single-cell sequencing to study the cellular heterogeneity of late-stage mouse inner ear organoid sensory epithelia, which we validated by comparison with datasets of the mouse cochlea and vestibular epithelia. We resolved supporting cell sub-types, cochlear-like hair cells, and vestibular type I and type II–like hair cells. While cochlear-like hair cells aligned best with an outer hair cell trajectory, vestibular-like hair cells followed developmental trajectories similar to in vivo programs branching into type II and then type I extrastriolar hair cells. These results highlight the transcriptional accuracy of the organoid developmental program but will also inform future strategies to improve synaptic connectivity and regional specification.
... www.nature.com/scientificreports/ growth factor (TGF)-β ligands, is present in the lateral portion of the PSD at E13.5 and E14.5 34,35 (quantified in Fig. S5). The Fst gene forms three isoforms with varying affinities to different TGF-β ligands, but all strongly bind Activin A with a strong preference for BMPs 36 . ...
Positional information encoded in signaling molecules is essential for early patterning in the prosensory domain of the developing cochlea. The sensory epithelium, the organ of Corti, contains an exquisite repeating pattern of hair cells and supporting cells. This requires precision in the morphogen signals that set the initial radial compartment boundaries, but this has not been investigated. To measure gradient formation and morphogenetic precision in developing cochlea, we developed a quantitative image analysis procedure measuring SOX2 and pSMAD1/5/9 profiles in mouse embryos at embryonic day (E)12.5, E13.5, and E14.5. Intriguingly, we found that the pSMAD1/5/9 profile forms a linear gradient up to the medial ~ 75% of the PSD from the pSMAD1/5/9 peak in the lateral edge during E12.5 and E13.5. This is a surprising activity readout for a diffusive BMP4 ligand secreted from a tightly constrained lateral region since morphogens typically form exponential or power-law gradient shapes. This is meaningful for gradient interpretation because while linear profiles offer the theoretically highest information content and distributed precision for patterning, a linear morphogen gradient has not yet been observed. Furthermore, this is unique to the cochlear epithelium as the pSMAD1/5/9 gradient is exponential in the surrounding mesenchyme. In addition to the information-optimized linear profile, we found that while pSMAD1/5/9 is stable during this timeframe, an accompanying gradient of SOX2 shifts dynamically. Last, through joint decoding maps of pSMAD1/5/9 and SOX2, we see that there is a high-fidelity mapping between signaling activity and position in the regions that will become Kölliker’s organ and the organ of Corti. Mapping is ambiguous in the prosensory domain precursory to the outer sulcus. Altogether, this research provides new insights into the precision of early morphogenetic patterning cues in the radial cochlea prosensory domain.
... Although the importance of Shh signaling in cochlear development has been studied in mammals and non-mammals, little is known about the role of Shh in hair cell regeneration after trauma in non-mammalian vertebrates. As in the mammalian cochlea, it potentially plays a role in development and tonotopic organization [85,139]. ...
Sensorineural hearing loss is caused by damage to sensory hair cells and/or spiral ganglion neurons. In non-mammalian species, hair cell regeneration after damage is observed, even in adulthood. Although the neonatal mammalian cochlea carries regenerative potential, the adult cochlea cannot regenerate lost hair cells. The survival of supporting cells with regenerative potential after cochlear trauma in adults is promising for promoting hair cell regeneration through therapeutic approaches. Targeting these cells by manipulating key signaling pathways that control mammalian cochlear development and non-mammalian hair cell regeneration could lead to regeneration of hair cells in the mammalian cochlea. This review discusses the pathways involved in the development of the cochlea and the impact that trauma has on the regenerative capacity of the endogenous progenitor cells. Furthermore, it discusses the effects of manipulating key signaling pathways targeting supporting cells with progenitor potential to promote hair cell regeneration and translates these findings to the human situation. To improve hearing recovery after hearing loss in adults, we propose a combined approach targeting (1) the endogenous progenitor cells by manipulating signaling pathways (Wnt, Notch, Shh, FGF and BMP/TGFβ signaling pathways), (2) by manipulating epigenetic control, and (3) by applying neurotrophic treatments to promote reinnervation.
... LRP2 also modulates sonic hedgehog signaling in mouse and is required for normal development of the inner ear and eye. 13 Additionally, in SV marginal cells, LRP2 is the receptor for aminoglycoside, which is cytotoxic to inner hair cells. 14 Our RNAseq data suggest a defect in the SV leading to hearing loss in patients with LRP2 mutations. ...
Hereditary deafness and retinal dystrophy are each genetically heterogenous and clinically variable. Three small unrelated families segregating the combination of deafness and retinal dystrophy were studied by exome sequencing (ES). The proband of Family 1 was found to be compound heterozygous for NM_004525.3: LRP2: c.5005A > G, p.(Asn1669Asp) and c.149C > G, p.(Thr50Ser). In Family 2, two sisters were found to be compound heterozygous for LRP2 variants, p.(Tyr3933Cys) and an experimentally confirmed c.7715 + 3A > T consensus splice‐altering variant. In Family 3, the proband is compound heterozygous for a consensus donor splice site variant LRP2: c.8452_8452 + 1del and p.(Cys3150Tyr). In mouse cochlea, Lrp2 is expressed abundantly in the stria vascularis marginal cells demonstrated by smFISH, single‐cell and single‐nucleus RNAseq, suggesting that a deficiency of LRP2 may compromise the endocochlear potential, which is required for hearing. LRP2 variants have been associated with Donnai–Barrow syndrome and other multisystem pleiotropic phenotypes different from the phenotypes of the four cases reported herein. Our data expand the phenotypic spectrum associated with pathogenic variants in LRP2 warranting their consideration in individuals with a combination of hereditary hearing loss and retinal dystrophy.
... Each hair cell has a bundle of stereocilia protruding from its surface that is responsible for detecting sound vibrations. These stereocilia are shorter in the hair cells at the cochlear base and longer toward the apex (3)(4)(5). In terms of number, hair cells at the base have more stereocilia per bundle than those at the apex (4,6). ...
... These stereocilia are shorter in the hair cells at the cochlear base and longer toward the apex (3)(4)(5). In terms of number, hair cells at the base have more stereocilia per bundle than those at the apex (4,6). In mammals, the angle of the V-shaped stereocilia on outer hair cells is wider at the base, growing gradually narrower toward the apex (7,8). ...
... Although various morphological and molecular features associated with frequency discrimination have been identified, the underlying mechanisms by which tonotopy is established during development remain poorly understood. Recently, sonic hedgehog (SHH) signaling from the ventral midline (i.e., the notochord and floor plate) was proposed to initiate the establishment of tonotopy in both birds and mammals (4). An increasing gradient of SHH from base to apex confers regional identity to the primordial cochlea (4,5). ...
The cochlea’s ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region’s frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.
... In one patient, who initially showed a response upon DPOAE (YUHL457: IV-2) ( Figure 13D-E), the signal-to-noise ratio was increased bilaterally ( Figure 13F). Tinnitus was identified in two patients; the symptom improved according to the Tinnitus Handicap Inventory (THI) questionnaire ( Figure 13G) [27][28][29][30][31][32][33][34][35][36]. No critical side effects or other safety issues were observed in these patients after 3 months of treatment. ...
... Immunoblotting and immunofluorescence were performed as described previously [33,34] were from commercial sources. Immunoblotting was performed using primary antibodies at a 1:1,000 dilution, followed by corresponding anti-isotype secondary antibodies (Cell Signaling Technology, 7074P2 and 7076P2) at a 1:2,000 dilution. ...
Intracellular accumulation of mutant proteins causes proteinopathies, which lack targeted therapies. Autosomal dominant hearing loss (DFNA67) is caused by frameshift mutations in OSBPL2. Here, we show that DFNA67 is a toxic proteinopathy. Mutant OSBPL2 accumulated intracellularly and bound to macroautophagy/autophagy proteins. Consequently, its accumulation led to defective endolysosomal homeostasis and impaired autophagy. Transgenic mice expressing mutant OSBPL2 exhibited hearing loss, but osbpl2 knockout mice or transgenic mice expressing wild-type OSBPL2 did not. Rapamycin decreased the accumulation of mutant OSBPL2 and partially rescued hearing loss in mice. Rapamycin also partially improved hearing loss and tinnitus in individuals with DFNA67. Our findings indicate that dysfunctional autophagy is caused by mutant proteins in DFNA67; hence, we recommend rapamycin for DFNA67 treatment.
Abbreviations: ABR: auditory brainstem response; ACTB: actin beta; CTSD: cathepsin D; dB: decibel; DFNA67: deafness non-syndromic autosomal dominant 67; DPOAE: distortion product otoacoustic emission; fs: frameshift; GFP: green fluorescent protein; HsQ53R-TG: human p.Q53Rfs*100-transgenic: HEK 293: human embryonic kidney 293; HFD: high-fat diet; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NSHL: non-syndromic hearing loss; OHC: outer hair cells; OSBPL2: oxysterol binding protein-like 2; SEM: scanning electron microscopy; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: transgenic; WES: whole-exome sequencing; YUHL: Yonsei University Hearing Loss; WT: wild-type.
... Cluster E13.5-3-1 cells are differentiating SGNs expressing Shh and Epha5 (Figure 6C). Shh is transiently expressed in SGNs and is crucial for cochlear development (Bok et al., 2013;Liu et al., 2010;Son et al., 2015). Finally, cluster E13.5-3-3 and E13.5-3-4 cells together are defined as differentiating VGNs expressing Tlx3 ( Figure 6C). ...
Inner ear vestibular and spiral ganglion neurons (VGNs and SGNs) are known to play pivotal roles in balance control and sound detection. However, the molecular mechanisms underlying otic neurogenesis at early embryonic ages have remained unclear. Here, we use single-cell RNA sequencing to reveal the transcriptomes of mouse otic tissues at three embryonic ages, embryonic day 9.5 (E9.5), E11.5, and E13.5, covering proliferating and undifferentiated otic neuroblasts and differentiating VGNs and SGNs. We validate the high quality of our studies by using multiple assays, including genetic fate mapping analysis, and we uncover several genes upregulated in neuroblasts or differentiating VGNs and SGNs, such as Shox2, Myt1, Casz1, and Sall3. Notably, our findings suggest a general cascaded differentiation trajectory during early otic neurogenesis. The comprehensive understanding of early otic neurogenesis provided by our study holds critical implications for both basic and translational research.
... Modifying hedgehog signaling interferes with axial patterning of the zebrafish otic vesicle [38] Ectopic Shh signaling induces apical hair cell identities in the basal and middle regions of the avian basilar papilla [39] Constitutive activation of Shh signaling hinders HC differentiation in developing murine cochleae [20] Inhibition of hedgehog signaling in cochlear explants results in an expanded sensory domain and formation of ectopic hair cells [19] Modifying Shh signaling does not seem to be an effective strategy to promote regeneration Inhibition of FGF signaling in E5-E9 chicks results in overproduction of HCs through non-proliferative mechanisms. FGF inhibition increases the number of Sox2+ HCs in the embryonic basilar papilla, suggesting that the formation of extra hair cells is due to transdifferentiation [41] Fgfr3 is restricted to supporting cells in the mature basilar papilla [42] Fgfr3 expression is downregulated in the mature basilar papilla following damage to hair cells [42] Fgfr1 hypomorphs lack 3rd-row OHCs [43] Fgfr1 plays a role in prosensory specification [44], In the embryo, Fgfr3 is expressed in the area of the cochlear duct that gives rise to pillar cells, OHCs, and Deiter's cells, but Fgfr3 is confined to pillar cells by birth [45], Activation of Fgfr3 with Fgf17 inhibits OHC differentiation without affecting IHCs [46], Pan Fgf inhibition decreases expression of Atoh1 in murine cochlear explants [47], Fgfr3-/-mice lack a row of pillar cells, but have an ectopic additional row of Deiters cells and an additional row of OHCs which appear to have normal bundle morphology [48] FGF signaling seems to be important signaling to modulate to promote HC regeneration, however, the results seem to be receptor- ...
Noise-induced, drug-related, and age-related disabling hearing loss is a major public health problem and affect approximately 466 million people worldwide. In non-mammalian vertebrates, the death of sensory hair cells (HCs) induces the proliferation and transdifferentiation of adjacent supporting cells into new HCs; however, this capacity is lost in juvenile and adult mammalian cochleae leading to permanent hearing loss. At present, cochlear implants and hearing devices are the only available treatments and can help patients to a certain extent; however, no biological approach or FDA-approved drug is effective to treat disabling hearing loss and restore hearing. Recently, regeneration of mammalian cochlear HCs by modulating molecular pathways or transcription factors has offered some promising results, although the immaturity of the regenerated HCs remains the biggest concern. Furthermore, most of the research done is in neonates and not in adults. This review focuses on critically summarizing the studies done in adult mammalian cochleae and discusses various strategies to elucidate novel transcription factors for better therapeutics.
... Interestingly, some of the tonotopic features are conserved between the chicken and mouse. For example, the length of the hair bundle, which are composed of tens of stereocilium arranged in a stair-case pattern, is shorter at the base and gradually longer toward the apex, whereas the width of hair bundle is wider at the base and gradually narrower toward the apex (Son et al., 2015;Moon et al., 2020). ...
... BMP7 and DNER were shown to be expressed gradually higher towards the apex and regulate hair bundle morphology along the tonotopic axis in chicken basilar papilla (Kowalik and Hudspeth, 2011;Mann et al., 2014). KCNJ2, which encodes an inward rectifier potassium ion channel, is known to be a bona fide marker of apical hair cells in chicken cochlea (Son et al., 2015). TECTB, which is categorized as a supporting cell marker in chicken cochlea (Janesick et al., 2021), was shown to be expressed in a base-to-apex increasing gradient in mouse cochlea (Son et al., 2012;Yoshimura et al., 2014). ...
Alternative splicing (AS) refers to the production of multiple mRNA isoforms from a single gene due to alternative selection of exons or splice sites during pre-mRNA splicing. It is a primary mechanism of gene regulation in higher eukaryotes and significantly expands the functional complexity of eukaryotic organisms, contributing to animal development and disease. Recent studies have shown that AS also influences functional diversity by affecting the transcriptomic and proteomic profiles in a position-dependent manner in a single organ. The peripheral hearing organ, the cochlea, is organized to detect sounds at different frequencies depending on its location along the longitudinal axis. This unique functional configuration, the tonotopy, is known to be facilitated by differential gene expression along the cochlear duct. We profiled transcriptome-wide gene expression and AS changes that occur within the different positions of chick cochlea. These analyses revealed distinct gene expression profiles and AS, including a splicing program that is unique to tonotopy. Changes in the expression of splicing factors PTBP3 , ESRP1 , and ESRP2 were demonstrated to contribute to position-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS at different positions by different RNA-binding proteins. These data, along with gene ontology (GO) analysis, represent a comprehensive analysis of the dynamic regulation of AS at different positions in chick cochlea.
