Migration and fate of vestibular melanocytes during the development of the human inner ear.

Melanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph homeostasis. Developmental studies describing the distribution of vestibular melanocytes are scarce, especially in humans. In this study, we investigated the distribution and maturation of the vestibular melanocytes in relation to the developing dark cell epithelium in inner ear specimens from week 5 to week 14 of development and in surgical specimens of the adult ampulla. Vestibular melanocytes were located around the utricle and the ampullae of the semicircular canals before week 7 and were first seen underneath the transitional zones and dark cell areas between week 8 and week 10. At week 10, melanocytes made intimate contact with epithelial cells, interrupting the local basement membrane with their dendritic processes. At week 11, most melanocytes were positioned under the dark cell epithelia. No melanocytes were seen around or in the saccule during all investigated developmental stages. The dark cell areas gradually matured and showed an adult immunohistochemical profile of the characteristic ion transporter protein Na+ /K+ -ATPase α1 by week 14. Furthermore, we investigated the expression of the migration-related proteins ECAD, PCAD, KIT, and KITLG in melanocytes and dark cell epithelium. This is the first study to describe the spatiotemporal distribution of vestibular melanocytes during the human development and thereby contributes to understanding normal vestibular function and pathophysiological mechanisms underlying vestibular disorders.

Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochrome P450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

Retinoic acid degradation shapes zonal development of vestibular organs and sensitivity to transient linear accelerations.

Each vestibular sensory epithelium in the inner ear is divided morphologically and physiologically into two zones, called the striola and extrastriola in otolith organ maculae, and the central and peripheral zones in semicircular canal cristae. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, formation of striolar/central zones is compromised, such that they resemble extrastriolar/peripheral zones in multiple features. Mutants have deficient vestibular evoked potential (VsEP) responses to jerk stimuli, head tremor and deficits in balance beam tests that are consistent with abnormal vestibular input, but normal vestibulo-ocular reflexes and apparently normal motor performance during swimming. Thus, degradation of RA during embryogenesis is required for formation of highly specialized regions of the vestibular sensory epithelia with specific functions in detecting head motions.

Origin of acoustic-vestibular ganglionic neuroblasts in chick embryos and their sensory connections.

The inner ear is a complex three-dimensional sensory structure with auditory and vestibular functions. It originates from the otic placode, which generates the sensory elements of the membranous labyrinth and all the ganglionic neuronal precursors. Neuroblast specification is the first cell differentiation event. In the chick, it takes place over a long embryonic period from the early otic cup stage to at least stage HH25. The differentiating ganglionic neurons attain a precise innervation pattern with sensory patches, a process presumably governed by a network of dendritic guidance cues which vary with the local micro-environment. To study the otic neurogenesis and topographically-ordered innervation pattern in birds, a quail-chick chimaeric graft technique was used in accordance with a previously determined fate-map of the otic placode. Each type of graft containing the presumptive domain of topologically-arranged placodal sensory areas was shown to generate neuroblasts. The differentiated grafted neuroblasts established dendritic contacts with a variety of sensory patches. These results strongly suggest that, rather than reverse-pathfinding, the relevant role in otic dendritic process guidance is played by long-range diffusing molecules.

Genetic interactions support an inhibitory relationship between bone morphogenetic protein 2 and netrin 1 during semicircular canal formation.

The semicircular canals of the mammalian inner ear are derived from epithelial pouches in which epithelial cells in the central region of each pouch undergo resorption, leaving behind the region at the rim to form a tube-shaped canal. Lack of proliferation at the rim and/or over-clearing of epithelial cells in the center of the pouch can obliterate canal formation. Otic-specific knockout of bone morphogenetic protein 2 (Bmp2) results in absence of all three semicircular canals; however, the common crus and ampullae housing the sensory tissue (crista) are intact. The lack of Bmp2 causes Ntn1 (which encodes netrin 1), which is required for canal resorption, to be ectopically expressed at the canal rim. Ectopic Ntn1 results in reduction of Dlx5 and Lmo4, which are required for rim formation. These phenotypes can be partially rescued by removing one allele of Ntn1 in the Bmp2 mutants, indicating that Bmp2 normally negatively regulates Ntn1 for canal formation. Additionally, non-resorption of the canal pouch in Ntn1-/- mutants is partially rescued by removing one allele of Bmp2 Thus, reciprocal inhibition between Bmp2 and netrin 1 is involved in canal formation of the vestibule.

Bone morphogenetic proteins and inner ear development.

Bone morphogenetic proteins (BMPs) are the largest subfamily of the transforming growth factor-ß superfamily, and they play important roles in the development of numerous organs, including the inner ear. The inner ear is a relatively small organ but has a highly complex structure and is involved in both hearing and balance. Here, we discuss BMPs and BMP signaling pathways and then focus on the role of BMP signal pathway regulation in the development of the inner ear and the implications this has for the treatment of human hearing loss and balance dysfunction.

Conserved and divergent development of brainstem vestibular and auditory nuclei.

Vestibular function was established early in vertebrates and has remained, for the most part, unchanged. In contrast, each group of tetrapods underwent independent evolutionary processes to solve the problem of hearing on land, resulting in a remarkable mixture of conserved, divergent and convergent features that define extant auditory systems. The vestibuloacoustic nuclei of the hindbrain develop from a highly conserved ground plan and provide an ideal framework on which to address the participation of developmental processes to the evolution of neuronal circuits. We employed an electroporation strategy to unravel the contribution of two dorsoventral and four axial lineages to the development of the chick hindbrain vestibular and auditory nuclei. We compare the chick developmental map with recently established genetic fate-maps of the developing mouse hindbrain. Overall, we find considerable conservation of developmental origin for the vestibular nuclei. In contrast, a comparative analysis of the developmental origin of hindbrain auditory structures echoes the complex evolutionary history of the auditory system. In particular, we find that the developmental origin of the chick auditory interaural time difference circuit supports its emergence from an ancient vestibular network, unrelated to the analogous mammalian counterpart.

Formation of the Periotic Space During the Early Fetal Period in Humans.

The inner ear is a very complicated structure, composed of a bony labyrinth (otic capsule; OC), membranous labyrinth, with a space between them, named the periotic labyrinth or periotic space. We investigated how periotic tissue fluid spaces covered the membranous labyrinth three-dimensionally, leading to formation of the periotic labyrinth encapsulated in the OC during human fetal development. Digital data sets from magnetic resonance images and phase-contrast X-ray tomography images of 24 inner ear organs from 24 human fetuses from the Kyoto Collection (fetuses in trimesters 1 and 2; crown-rump length: 14.4-197 mm) were analyzed. The membranous labyrinth was morphologically differentiated in samples at the end of the embryonic period (Carnegie stage 23), and had grown linearly to more than eight times in size during the observation period. The periotic space was first detected at the 35-mm samples, around the vestibule and basal turn of the cochlea, which elongated rapidly to the tip of the cochlea and semicircular ducts, successively, and almost covered the membranous labyrinth at the 115-mm CRL stage or later. In those samples, several ossification centers were detected around the space. This article thus demonstrated that formation of the membranous labyrinth, periotic space (labyrinth), and ossification of the OC occurs successively, according to an intricate timetable. Anat Rec, 301:563-570, 2018. © 2018 Wiley Periodicals, Inc.

CT Scan Imaging of the Human Fetal Labyrinth: Case Series Data Throughout Gestation.

OBJECTIVES: The inner ear in humans reaches its final configuration and its adult size during fetal life. According to the literature, this occurs between 18 and 25 weeks of amenorrhea (WA). Our goal is to clarify the course of inner ear size development. METHODS: Using computed tomography (CT) scanner, we studied 13 measurements in the inner ear of a collection of 153 fetuses from 21 to 40 WA. RESULTS: We found no side-related differences or sexual dimorphism in the measurements. Cochlear and vestibular bone measurements did not show growth from 21 to 40 WA, with the exception of the lateral semicircular canal (LSCC) bony island, which grows until 25 WA. Internal auditory canal (IAC) and cochlear aqueduct (CA) growth are correlated with gestational age. As our cochlear measurements are similar to those of infants and adults, in accordance with the literature we conclude that the cochlea has reached its adult size before 21 WA. The continuous growth of the IAC and CA is linked to petrous ossification that continues during fetal gestation and after birth. CONCLUSION: We confirm that the cochlea reaches its adult size during the second trimester of fetal life.

Anatomical and physiological development of the human inner ear.

We describe the development of the human inner ear with the invagination of the otic vesicle at 4 weeks gestation (WG), the growth of the semicircular canals from 5 WG, and the elongation and coiling of the cochlea at 10 WG. As the membranous labyrinth takes shape, there is a concomitant development of the sensory neuroepithelia and their associated structures within. This review details the growth and differentiation of the vestibular and auditory neuroepithelia, including synaptogenesis, the expression of stereocilia and kinocilia, and innervation of hair cells by afferent and efferent nerve fibres. Along with development of essential sensory structures we outline the formation of crucial accessory structures of the vestibular system - the cupula and otolithic membrane and otoconia as well as the three cochlea compartments and the tectorial membrane. Recent molecular studies have elaborated on classical anatomical studies to characterize the development of prosensory and sensory regions of the fetal human cochlea using the transcription factors, PAX2, MAF-B, SOX2, and SOX9. Further advances are being made with recent physiological studies that are beginning to describe when hair cells become functionally active during human gestation. This article is part of a Special Issue entitled .

Insm1 promotes neurogenic proliferation in delaminated otic progenitors.

INSM1 is a zinc-finger protein expressed throughout the developing nervous system in late neuronal progenitors and nascent neurons. In the embryonic cortex and olfactory epithelium, Insm1 may promote the transition of progenitors from apical, proliferative, and uncommitted to basal, terminally-dividing and neuron producing. In the otocyst, delaminating and delaminated progenitors express Insm1, whereas apically-dividing progenitors do not. This expression pattern is analogous to that in embryonic olfactory epithelium and cortex (basal/subventricular progenitors). Lineage analysis confirms that auditory and vestibular neurons originate from Insm1-expressing cells. In the absence of Insm1, otic ganglia are smaller, with 40% fewer neurons. Accounting for the decrease in neurons, delaminated progenitors undergo fewer mitoses, but there is no change in apoptosis. We conclude that in the embryonic inner ear, Insm1 promotes proliferation of delaminated neuronal progenitors and hence the production of neurons, a similar function to that in other embryonic neural epithelia. Unexpectedly, we also found that differentiating, but not mature, outer hair cells express Insm1, whereas inner hair cells do not. Insm1 is the earliest known gene expressed in outer versus inner hair cells, demonstrating that nascent outer hair cells initiate a unique differentiation program in the embryo, much earlier than previously believed.

Morphogenesis of the inner ear at different stages of normal human development.

This study examined the external morphology and morphometry of the human embryonic inner ear membranous labyrinth and documented its three-dimensional position in the developing embryo using phase-contrast X-ray computed tomography and magnetic resonance imaging. A total of 27 samples between Carnegie stage (CS) 17 and the postembryonic phase during trimester 1 (approximately 6-10 weeks after fertilization) were included. The otic vesicle elongated along the dorso-ventral axis and differentiated into the end lymphatic appendage and cochlear duct (CD) at CS 17. The spiral course of the CD began at CS18, with anterior and posterior semicircular ducts (SDs) forming prominent circles with a common crus. The spiral course of the CD comprised more than two turns at the postembryonic phase, at which time the height of the CD was evident. A linear increase was observed in the length of anterior, posterior, and lateral SDs, in that order, and the length of the CD increased exponentially over the course of development. Bending in the medial direction was observed between the cochlear and vestibular parts from the latero-caudal view, with the angle decreasing during development. The position of the inner ear was stable throughout the period of observation on the lateral to ventral side of the rhombencephalon, caudal to the pontine flexure, and adjacent to the auditory ganglia. The plane of the lateral semicircular canal was approximately 8.0°-14.6° with respect to the cranial caudal (z-)axis, indicating that the orientation of the inner ear changes during growth to adulthood.

Fgf10 is required for specification of non-sensory regions of the cochlear epithelium.

The vertebrate inner ear is a morphologically complex sensory organ comprised of two compartments, the dorsal vestibular apparatus and the ventral cochlear duct, required for motion and sound detection, respectively. Fgf10, in addition to Fgf3, is necessary for the earliest stage of otic placode induction, but continued expression of Fgf10 in the developing otic epithelium, including the prosensory domain and later in Kolliker׳s organ, suggests additional roles for this gene during morphogenesis of the labyrinth. While loss of Fgf10 was implicated previously in semicircular canal agenesis, we show that Fgf10(-/+) embryos also exhibit a reduction or absence of the posterior semicircular canal, revealing a dosage-sensitive requirement for FGF10 in vestibular development. In addition, we show that Fgf10(-/-) embryos have previously unappreciated defects of cochlear morphogenesis, including a somewhat shortened duct, and, surprisingly, a substantially narrower duct. The mutant cochlear epithelium lacks Reissner׳s membrane and a large portion of the outer sulcus-two non-contiguous, non-sensory domains. Marker gene analyses revealed effects on Reissner׳s membrane as early as E12.5-E13.5 and on the outer sulcus by E15.5, stages when Fgf10 is expressed in close proximity to Fgfr2b, but these effects were not accompanied by changes in epithelial cell proliferation or death. These data indicate a dual role for Fgf10 in cochlear development: to regulate outgrowth of the duct and subsequently as a bidirectional signal that sequentially specifies Reissner׳s membrane and outer sulcus non-sensory domains. These findings may help to explain the hearing loss sometimes observed in LADD syndrome subjects with FGF10 mutations.

Interplay of proliferation and proapoptotic and antiapoptotic factors is revealed in the early human inner ear development.

HYPOTHESIS: Spatiotemporal interplay of factors controlling proliferation, differentiation and apoptosis within the developing human inner ear is essential for labyrinth morphogenesis and development of vestibular and cochlear functions. BACKGROUND: Studies on the early human inner ear development are scarce and insufficient. METHODS: The immunolocalization of Ki-67, Bcl-2, caspase-3, and IGF-1 was analyzed in 6 human inner ears, 5 to 10 gestational weeks old. Statistical data were analyzed using the Kruskal-Wallis test. RESULTS: During the analyzed period, the otocyst has transformed into cochlear duct and saccule ventrally and semicircular canals and utricle dorsally. Initial differentiation of sensorineural fields characterized organ of Corti, maculae, and cristae ampullares. Intense (50%) and evenly distributed proliferation Ki-67 in the otocyst decreased to 24% to 30% and became spatially restricted within the membranous labyrinth epithelium. Simultaneously, expression of antiapoptotic Bcl-2 protein increased in sensorineural fields of organ of Corti, macula, and crista ampullaris. Throughout the investigated period, apoptotic caspase-3 positive cells were mainly distributed at the luminal and basal surfaces of labyrinth epithelium. An inhibitor of apoptosis IGF-1 co-expressed with Bcl-2 and increased in the sensorineural fields with advancing development. CONCLUSION: The described expression pattern indicates roles for cell proliferation in the growth of the inner ear and Bcl-2 in differentiation of sensorineural fields and protection from apoptosis. Both IGF-1-and caspase-3-mediated apoptosis seem to contribute to proper morphogenesis, differentiation, and innervations of sensorineural fields within the cochlea, semicircular canals, saccule, and utricle. Alterations in spatiotemporal interplay of investigated factors might lead to disturbances of vestibular and cochlear function.

Responses to cell loss become restricted as the supporting cells in mammalian vestibular organs grow thick junctional actin bands that develop high stability.

Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin-GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin-GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia.

Vestibular development in marsupials and monotremes.

The young of marsupials and monotremes are all born in an immature state, followed by prolonged nurturing by maternal lactation in either a pouch or nest. Nevertheless, the level of locomotor ability required for newborn marsupials and monotremes to reach the safety of the pouch or nest varies considerably: some are transferred to the pouch or nest in an egg (monotremes); others are transferred passively by gravity (e.g. dasyurid marsupials); some have only a horizontal wriggle to make (e.g. peramelid and didelphid marsupials); and others must climb vertically for a long distance to reach the maternal pouch (e.g. diprotodontid marsupials). In the present study, archived sections of the inner ear and hindbrain held in the Bolk, Hill and Hubrecht collections at the Museum für Naturkunde, Berlin, were used to test the relationship between structural maturity of the vestibular apparatus and the locomotor challenges that face the young of these different mammalian groups. A system for staging different levels of structural maturity of the vestibular apparatus was applied to the embryos, pouch young and hatchlings, and correlated with somatic size as indicated by greatest body length. Dasyurids are born at the most immature state, with the vestibular apparatus at little more than the otocyst stage. Peramelids are born with the vestibular apparatus at a more mature state (fully developed semicircular ducts and a ductus reuniens forming between the cochlear duct and saccule, but no semicircular canals). Diprotodontids and monotremes are born with the vestibular apparatus at the most mature state for the non-eutherians (semicircular canals formed, maculae present, but vestibular nuclei in the brainstem not yet differentiated). Monotremes and marsupials reach the later stages of vestibular apparatus development at mean body lengths that lie within the range of those found for laboratory rodents (mouse and rat) reaching the same vestibular stage.

Tracing Sox10-expressing cells elucidates the dynamic development of the mouse inner ear.

The inner ear is constituted by complicated cochlear and vestibular compartments, which are derived from the otic vesicle, an embryonic structure of ectodermal origin. Although the inner ear development has been analyzed using various techniques, the developmental events have not been fully elucidated because of the intricate structure. We previously developed a Sox10-IRES-Venus mouse designed to express green fluorescent protein under the control of the Sox10 promoter. In the present study, we showed that the Sox10-IRES-Venus mouse enabled the non-destructive visualization and understanding of the morphogenesis during the development of the inner ear. The expression of the transcription factor Sox10 was first observed in the invaginating otic placodal epithelium, and continued to be expressed in the mature inner ear epithelium except for the hair cells and mesenchymal cells. We found that Sox10 was expressed in immature hair cells in the developing inner ear, suggesting that hair cells were generated from the Sox10-expressing prosensory cells. Furthermore, we demonstrated that scattered Sox10-expressing cells existed around the developing inner ear, some of which differentiated into pigmented melanocytes in the stria vascularis, suggesting that they were neural crest cells. Further analyzing the Sox10-IRES-Venus mice would provide important information to better understand the development of the inner ear.

Molecular architecture of the chick vestibular hair bundle.

Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

Mutanlallemand (mtl) and Belly Spot and Deafness (bsd) are two new mutations of Lmx1a causing severe cochlear and vestibular defects.

Mutanlallemand (mtl) and Belly Spot and Deafness (bsd) are two new spontaneous alleles of the Lmx1a gene in mice. Homozygous mutants show head tossing and circling behaviour, indicative of vestibular defects, and they have short tails and white belly patches of variable size. The analysis of auditory brainstem responses (ABR) showed that mtl and bsd homozygotes are deaf, whereas heterozygous and wildtype littermates have normal hearing. Paint-filled inner ears at E16.5 revealed that mtl and bsd homozygotes lack endolymphatic ducts and semicircular canals and have short cochlear ducts. These new alleles show similarities with dreher (Lmx1a) mutants. Complementation tests between mtl and dreher and between mtl and bsd suggest that mtl and bsd are new mutant alleles of the Lmx1a gene. To determine the Lmx1a mutation in mtl and bsd mutant mice we performed PCR followed by sequencing of genomic DNA and cDNA. The mtl mutation is a single point mutation in the 3' splice site of exon 4 leading to an exon extension and the activation of a cryptic splice site 44 base pairs downstream, whereas the bsd mutation is a genomic deletion that includes exon 3. Both mutations lead to a truncated LMX1A protein affecting the homeodomain (mtl) or LIM2-domain (bsd), which is critical for LMX1A protein function. Moreover, the levels of Lmx1a transcript in mtl and bsd mutants are significantly down-regulated. Hmx2/3 and Pax2 expression are also down-regulated in mtl and bsd mutants, suggesting a role of Lmx1a upstream of these transcription factors in early inner ear morphogenesis. We have found that these mutants develop sensory patches although they are misshapen. The characterization of these two new Lmx1a alleles highlights the critical role of this gene in the development of the cochlea and vestibular system.

Analysis of miR-376 RNA cluster members in the mouse inner ear.

Mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1) are associated with a spectrum of non-syndromic to syndromic hearing loss. PRPS1 transcript levels have been shown to be regulated by the microRNA-376 genes. The long primary RNA transcript of the miR-376 RNA cluster members undergo extensive and simultaneous A → I editing at one or both of two specific sites (+4 and +44) in particular human and mouse tissues. The PRPS1 gene, which contains target sites for the edited version of miR-376a-5p within its 3'UTR, has been shown to be repressed in a tissue-specific manner. To investigate whether the transcription of Prps1 is regulated by miR-376 cluster members in the mouse inner ear, we first quantified the expression of the mature miR-376 RNAs by quantitative real-time-PCR. The spatio-temporal patterns of miR-376 expression were assessed by in situ hybridization. Finally, we examined whether A →I editing of pri-miR-376 RNAs occurs in mouse inner ear by direct sequencing. Our data showed that the miR-376a-3p, b-3p, c-3p are present in mouse embryonic inner ears and intensive expression of miR-376a-3p/b-3p was detected in the sensory epithelia and ganglia of both auditory and vestibular portions of the inner ear. In adult inner ear, the expression of miR-376a-3p/b-3p is restricted within ganglion neurons of auditory and vestibular systems as well as the cells in the stria vascularis. Only unedited pri-miR-376 RNAs were detected in the cochlea suggesting that the activity of PRPS1 in the inner ear may not be regulated through the editing of miR-376 cluster.