Embryonic and Neonatal Mouse Cochleae Are Susceptible to Zika Virus Infection.

Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans.

Opposing effects of Wnt/ß-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea.

During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via ß-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.

Autophagy precedes apoptosis during degeneration of the Kölliker's organ in the development of rat cochlea.

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.

Characterizing a novel vGlut3-P2A-iCreER knockin mouse strain in cochlea.

Precise mouse genetic studies rely on specific tools that can label specific cell types. In mouse cochlea, previous studies suggest that vesicular glutamate transporter 3 (vGlut3), also known as Slc17a8, is specifically expressed in inner hair cells (IHCs) and loss of vGlut3 causes deafness. To take advantage of its unique expression pattern, here we generate a novel vGlut3-P2A-iCreER knockin mouse strain. The P2A-iCreER cassette is precisely inserted before stop codon of vGlut3, by which the endogenous vGlut3 is intact and paired with iCreER as well. Approximately, 10.7%, 85.6% and 41.8% of IHCs are tdtomato + when tamoxifen is given to vGlut3-P2A-iCreER/+; Rosa26-LSL-tdtomato/+ reporter strain at P2/P3, P10/P11 and P30/P31, respectively. Tdtomato + OHCs are never observed. Interestingly, besides IHCs, glia cells, but not spiral ganglion neurons (SGNs), are tdtomato+, which is further evidenced by the presence of Sox10+/tdtomato+ and tdtomato+/Prox1(Gata3 or Tuj1)-negative cells in SGN region. We further independently validate vGlut3 expression in SGN region by vGlut3 in situ hybridization and antibody staining. Moreover, total number of tdtomato + glia cells decreased gradually when tamoxifen is given from P2/P3 to P30/P31. Taken together, vGlut3-P2A-iCreER is an efficient genetic tool to specifically target IHCs for gene manipulation, which is complimentary to Prestin-CreER strain exclusively labelling cochlear outer hair cells (OHCs).

[The function of Kölliker's organ in mammalian cochlear].

Kölliker's organ, which is a transient structure of cochlea during development, in late embryonic and early postnatal period, is one of the signs of cochlear immaturity.Kölliker's organ degradates after the sensory structures become sensitive to external sound. The putative role of Kölliker's organ is important for generating the intrinsic spontaneous activity whichpromotes the development and maturation of a fully functional auditory system.

Role of BDNF and neurotrophic receptors in human inner ear development.

The expression patterns of the neurotrophin, brain-derived neurotrophic factor, BDNF, and the neurotrophic receptors-p75NTR and Trk receptors-in the developing human fetal inner ear between the gestational weeks (GW) 9 to 12 are examined via in situ hybridization and immunohistochemistry. BDNF mRNA expression was highest in the cochlea at GW 9 but declined in the course of development. In contrast to embryonic murine specimens, a decline in BDNF expression from the apical to the basal turn of the cochlea could not be observed. p75NTR immunostaining was most prominent in the nerve fibers that penetrate into the sensory epithelia of the cochlea, the urticule and the saccule as gestational age progresses. TrkB and TrkC expression intensified towards GW 12, at which point the BDNF mRNA localization was at its lowest. TrkA expression was limited to fiber subpopulations of the facial nerve at GW 10. In the adult human inner ear, we observed BDNF mRNA expression in the apical poles of the cochlear hair cells and supporting cells, while in the adult human utricle, the expression was localized in the vestibular hair cells. We demonstrate the highly specific staining patterns of BDNF mRNA and its putative receptors over a developmental period in which multiple hearing disorders are manifested. Our findings suggest that BDNF and neurotrophin receptors are important players during early human inner ear development. In particular, they seem to be important for the survival of the afferent sensory neurons.

Wnt9a Can Influence Cell Fates and Neural Connectivity across the Radial Axis of the Developing Cochlea.

Vertebrate hearing organs manifest cellular asymmetries across the radial axis that underlie afferent versus efferent circuits between the inner ear and the brain. Therefore, understanding the molecular control of patterning across this axis has important functional implications. Radial axis patterning begins before the cells become postmitotic and is likely linked to the onset of asymmetric expression of secreted factors adjacent to the sensory primordium. This study explores one such asymmetrically expressed gene, Wnt9a, which becomes restricted to the neural edge of the avian auditory organ, the basilar papilla, by embryonic day 5 (E5). Radial patterning is disrupted when Wnt9a is overexpressed throughout the prosensory domain beginning on E3. Sexes were pooled for analysis and sex differences were not studied. Analysis of gene expression and afferent innervation on E6 suggests that ectopic Wnt9a expands the neural-side fate, possibly by re-specifying the abneural fate. RNA sequencing reveals quantitative changes, not only in Wnt-pathway genes, but also in genes involved in axon guidance and cytoskeletal remodeling. By E18, these early patterning effects are manifest as profound changes in cell fates [short hair cells (HCs) are missing], ribbon synapse numbers, outward ionic currents, and efferent innervation. These observations suggest that Wnt9a may be one of the molecules responsible for breaking symmetry across the radial axis of the avian auditory organ. Indirectly, Wnt9a can regulate the mature phenotype whereby afferent axons predominantly innervate neural-side tall HCs, resulting in more ribbon synapses per HC compared with abneural-side short HCs with few ribbons and large efferent synapses.SIGNIFICANCE STATEMENT Wnts are a class of secreted factors that are best known for stimulating cell division in development and cancer. However, in certain contexts during development, Wnt-expressing cells can direct neighboring cells to take on specific fates. This study suggests that the Wnt9a ligand may play such a role in the developing hearing organ of the bird cochlea. This was shown through patterning defects that occur in response to the overexpression of Wnt9a. This manipulation increased one type of sensory hair cell (tall HCs) at the expense of another (short HCs) that is usually located furthest from the Wnt9a source. The extraneous tall HCs that replaced short HCs showed some physiological properties and neuronal connections consistent with a fate switch.

Indispensable role of Mdm2/p53 interaction during the embryonic and postnatal inner ear development.

p53 is a key component of a signaling network that protects cells against various stresses. As excess p53 is detrimental to cells, its levels are tightly controlled by several mechanisms. The E3 ubiquitin ligase Mdm2 is a major negative regulator of p53. The significance of balanced p53 levels in normal tissues, at different stages of lifetime, is poorly understood. We have studied in vivo how the disruption of Mdm2/p53 interaction affects the early-embryonic otic progenitor cells and their descendants, the auditory supporting cells and hair cells. We found that p53 accumulation, as a consequence of Mdm2 abrogation, is lethal to both proliferative progenitors and non-proliferating, differentiating cells. The sensitivity of postmitotic supporting cells to excess p53 decreases along maturation, suggesting that maturation-related mechanisms limit p53's transcriptional activity towards pro-apoptotic factors. We have also investigated in vitro whether p53 restricts supporting cell's regenerative capacity. Unlike in several other regenerative cellular models, p53 inactivation did not alter supporting cell's proliferative quiescence nor transdifferentiation capacity. Altogether, the postmitotic status of developing hair cells and supporting cells does not confer protection against the detrimental effects of p53 upregulation. These findings might be linked to auditory disturbances observed in developmental syndromes with inappropriate p53 upregulation.

Dynamic Expression of Sox2, Gata3, and Prox1 during Primary Auditory Neuron Development in the Mammalian Cochlea.

Primary auditory neurons (PANs) connect cochlear sensory hair cells in the mammalian inner ear to cochlear nucleus neurons in the brainstem. PANs develop from neuroblasts delaminated from the proneurosensory domain of the otocyst and keep maturing until the onset of hearing after birth. There are two types of PANs: type I, which innervate the inner hair cells (IHCs), and type II, which innervate the outer hair cells (OHCs). Glial cells surrounding these neurons originate from neural crest cells and migrate to the spiral ganglion. Several transcription factors are known to regulate the development and differentiation of PANs. Here we systematically examined the spatiotemporal expression of five transcription factors: Sox2, Sox10, Gata3, Mafb, and Prox1 from early delamination at embryonic day (E) 10.5 to adult. We found that Sox2 and Sox10 were initially expressed in the proneurosensory cells in the otocyst (E10.5). By E12.75 both Sox2 and Sox10 were downregulated in the developing PANs; however, Sox2 expression transiently increased in the neurons around birth. Furthermore, both Sox2 and Sox10 continued to be expressed in spiral ganglion glial cells. We also show that Gata3 and Prox1 were first expressed in all developing neurons, followed by a decrease in expression of Gata3 and Mafb in type I PANs and Prox1 in type II PANs as they matured. Moreover, we describe two subtypes of type II neurons based on Peripherin expression. These results suggest that Sox2, Gata3 and Prox1 play a role during neurogenesis as well as maturation of the PANs.

Kremen1 regulates mechanosensory hair cell development in the mammalian cochlea and the zebrafish lateral line.

Here we present spatio-temporal localization of Kremen1, a transmembrane receptor, in the mammalian cochlea, and investigate its role in the formation of sensory organs in mammal and fish model organisms. We show that Kremen1 is expressed in prosensory cells during cochlear development and in supporting cells of the adult mouse cochlea. Based on this expression pattern, we investigated whether Kremen1 functions to modulate cell fate decisions in the prosensory domain of the developing cochlea. We used gain and loss-of-function experiments to show that Kremen1 is sufficient to bias cells towards supporting cell fate, and is implicated in suppression of hair cell formation. In addition to our findings in the mouse cochlea, we examined the effects of over expression and loss of Kremen1 in the zebrafish lateral line. In agreement with our mouse data, we show that over expression of Kremen1 has a negative effect on the number of mechanosensory cells that form in the zebrafish neuromasts, and that fish lacking Kremen1 protein develop more hair cells per neuromast compared to wild type fish. Collectively, these data support an inhibitory role for Kremen1 in hair cell fate specification.

Hepatocyte Growth Factor-c-MET Signaling Mediates the Development of Nonsensory Structures of the Mammalian Cochlea and Hearing.

UNLABELLED: The stria vascularis is a nonsensory structure that is essential for auditory hair cell function by maintaining potassium concentration of the scala media. During mouse embryonic development, a subpopulation of neural crest cell-derived melanocytes migrates and incorporates into a subregion of the cochlear epithelium, forming the intermediate cell layer of the stria vascularis. The relation of this developmental process to stria vascularis function is currently unknown. In characterizing the molecular differentiation of developing peripheral auditory structures, we discovered that hepatocyte growth factor (Hgf) is expressed in the future stria vascularis of the cochlear epithelium. Its receptor tyrosine kinase, c-Met, is expressed in the cochlear epithelium and melanocyte-derived intermediate cells in the stria vascularis. Genetic dissection of HGF signaling via c-MET reveals that the incorporation of the melanocytes into the future stria vascularis of the cochlear duct requires c-MET signaling. In addition, inactivation of either the ligand or receptor developmentally resulted in a profound hearing loss at young adult stages. These results suggest a novel connection between HGF signaling and deafness via melanocyte deficiencies. SIGNIFICANCE STATEMENT: We found the roles of hepatocyte growth factor (HGF) signaling in stria vascularis development for the first time and that lack of HGF signaling in the inner ear leads to profound hearing loss in the mouse. Our findings reveal a novel mechanism that may underlie human deafness DFNB39 and DFNB97. Our findings reveal an additional example of context-dependent c-MET signaling diversity, required here for proper cellular invasion developmentally that is essential for specific aspects of auditory-related organogenesis.

The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals.

Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs.

BMP regulates regional gene expression in the dorsal otocyst through canonical and non-canonical intracellular pathways.

The inner ear consists of two otocyst-derived, structurally and functionally distinct components: the dorsal vestibular and ventral auditory compartments. BMP signaling is required to form the vestibular compartment, but how it complements other required signaling molecules and acts intracellularly is unknown. Using spatially and temporally controlled delivery of signaling pathway regulators to developing chick otocysts, we show that BMP signaling regulates the expression of Dlx5 and Hmx3, both of which encode transcription factors essential for vestibular formation. However, although BMP regulates Dlx5 through the canonical SMAD pathway, surprisingly, it regulates Hmx3 through a non-canonical pathway involving both an increase in cAMP-dependent protein kinase A activity and the GLI3R to GLI3A ratio. Thus, both canonical and non-canonical BMP signaling establish the precise spatiotemporal expression of Dlx5 and Hmx3 during dorsal vestibular development. The identification of the non-canonical pathway suggests an intersection point between BMP and SHH signaling, which is required for ventral auditory development.

Dcc Mediates Functional Assembly of Peripheral Auditory Circuits.

Proper structural organization of spiral ganglion (SG) innervation is crucial for normal hearing function. However, molecular mechanisms underlying the developmental formation of this precise organization remain not well understood. Here, we report in the developing mouse cochlea that deleted in colorectal cancer (Dcc) contributes to the proper organization of spiral ganglion neurons (SGNs) within the Rosenthal's canal and of SGN projections toward both the peripheral and central auditory targets. In Dcc mutant embryos, mispositioning of SGNs occurred along the peripheral auditory pathway with misrouted afferent fibers and reduced synaptic contacts with hair cells. The central auditory pathway simultaneously exhibited similar defective phenotypes as in the periphery with abnormal exit of SGNs from the Rosenthal's canal towards central nuclei. Furthermore, the axons of SGNs ascending into the cochlear nucleus had disrupted bifurcation patterns. Thus, Dcc is necessary for establishing the proper spatial organization of SGNs and their fibers in both peripheral and central auditory pathways, through controlling axon targeting and cell migration. Our results suggest that Dcc plays an important role in the developmental formation of peripheral and central auditory circuits, and its mutation may contribute to sensorineural hearing loss.

OTX2 regulates the expression of TAp63 leading to macular and cochlear neuroepithelium development.

OTX proteins, homologs of the Drosophila orthodenticle (Otd), are important for the morphogenesis of the neuroectoderm, and for the central nervous system formation. OTX1 and OTX2 are important for the cochlea and macula development, indeed when OTX1 is knocked down, these organs undergo developmental failure. Moreover OTX2 transfection revert this effect in OTX1(-/-) mice. The TA isoform of TP63, involved in Notch regulation pathway, has a critical function in the cochlear neuroepithelium differentiation. TAp63 positively regulates Hes5 and Atoh1 transcription. This pathway has been also demonstrated in p63(-/-) mice, and in patients p63 mutated, affected by Ectodermal Dysplasia (ED, OMIM 129810). These patients are affected by mild sensorineural deafness, most likely related to the mutation in p63 gene impairing the Notch pathway. We demonstrated the role of OTX2 on TAp63 regulation necessary for the correct formation of macular neuroepithelium and we confirmed the impairment of vestibular function caused by p63 mutations. Although the abnormalities found in our patient were still at a subclinical extent, aging could exacerbate this impairment and cause a decrease in quality of life.

Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea.

Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea.

Development of the stria vascularis and potassium regulation in the human fetal cochlea: Insights into hereditary sensorineural hearing loss.

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9-W18) and show the cochlear expression dynamics of key potassium-regulating proteins. At W12, MITF+/SOX10+/KIT+ neural-crest-derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+-ATPase-positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy.

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.

Distribution and development of peripheral glial cells in the human fetal cochlea.

The adult human cochlea contains various types of peripheral glial cells that envelop or myelinate the three different domains of the spiral ganglion neurons: the central processes in the cochlear nerve, the cell bodies in the spiral ganglia, and the peripheral processes in the osseous spiral lamina. Little is known about the distribution, lineage separation and maturation of these peripheral glial cells in the human fetal cochlea. In the current study, we observed peripheral glial cells expressing SOX10, SOX9 and S100B as early as 9 weeks of gestation (W9) in all three neuronal domains. We propose that these cells are the common precursor to both mature Schwann cells and satellite glial cells. Additionally, the peripheral glial cells located along the peripheral processes expressed NGFR, indicating a phenotype distinct from the peripheral glial cells located along the central processes. From W12, the spiral ganglion was gradually populated by satellite glial cells in a spatiotemporal gradient. In the cochlear nerve, radial sorting was accomplished by W22 and myelination started prior to myelination of the peripheral processes. The developmental dynamics of the peripheral glial cells in the human fetal cochlea is in support of a neural crest origin. Our study provides the first overview of the distribution and maturation of peripheral glial cells in the human fetal cochlea from W9 to W22.

PTEN regulation of the proliferation and differentiation of auditory progenitors through the PTEN/PI3K/Akt-signaling pathway in mice.

The organ of Corti, which is the sensory organ of hearing, consists of a single row of inner hair cells and three rows of outer hair cells in mice. The auditory hair cells develop from auditory progenitors. Hair cell development is related to several genes, including PTEN. Homozygous null mutant (PTEN(-/-)) mice die at around embryonic day 9, when hair cells are extremely immature. Moreover, in heterozygous PTEN knockout mice, it was found that PTEN regulates the proliferation of auditory progenitors. However, little is known about the molecular mechanism underlying this regulation. In the present study, we generated PTEN conditional knockout in the inner ear of mice and studied the aforementioned molecular mechanisms. Our results showed that PTEN knockout resulted in supernumerary hair cells, increased p-Akt level, and decreased p27(kip1) level. Furthermore, the presence of supernumerary hair cells could be explained by the delayed withdrawal of auditory progenitors from the cell cycle. The increased p-Akt level correlates with p27(kip1) downregulation in the cochlea in the Pax2-PTEN mice. The reduced p27(kip1) could not maintain the auditory progenitors in the nonproliferative state and some progenitors continued to divide. Consequently, additional progenitors differentiated into supernumerary hair cells. We suggest that PTEN regulates p27(kip1) through p-Akt, thereby regulating the proliferation and differentiation of auditory progenitors.