Inactivation of Fgf3 and Fgf4 within the Fgf3/Fgf4/Fgf15 gene cluster reveals their redundant requirement for mouse inner ear induction and embryonic survival.

BACKGROUND: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. RESULTS: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. CONCLUSIONS: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival.

Myc is dispensable for cardiomyocyte development but rescues Mycn-deficient hearts through functional replacement and cell competition.

Myc is considered an essential transcription factor for heart development, but cardiac defects have only been studied in global Myc loss-of-function models. Here, we eliminated Myc by recombining a Myc floxed allele with the Nkx2.5Cre driver. We observed no anatomical, cellular or functional alterations in either fetuses or adult cardiac Myc-deficient mice. We re-examined Myc expression during development and found no expression in developing cardiomyocytes. In contrast, we confirmed that Mycn is essential for cardiomyocyte proliferation and cardiogenesis. Mosaic Myc overexpression in a Mycn-deficient background shows that Myc can replace Mycn function, recovering heart development. We further show that this recovery involves the elimination of Mycn-deficient cells by cell competition. Our results indicate that Myc is dispensable in cardiomyocytes both during cardiogenesis and for adult heart homeostasis, and that Mycn is exclusively responsible for cardiomyocyte proliferation during heart development. Nonetheless, our results show that Myc can functionally replace Mycn We also show that cardiomyocytes compete according to their combined Myc and Mycn levels and that cell competition eliminates flawed cardiomyocytes, suggesting its relevance as a quality control mechanism in cardiac development.

Differential deletion of GDNF in the auditory system leads to altered sound responsiveness.

Glial-derived neurotrophic factor (GDNF) has been proposed as a potent neurotrophic factor with the potential to cure neurodegenerative diseases. In the cochlea, GDNF has been detected in auditory neurons and sensory receptor cells and its expression is upregulated upon trauma. Moreover, the application of GDNF in different animal models of deafness has shown its capacity to prevent hearing loss and promoted its future use in therapeutic trials in humans. In the present study we have examined the endogenous requirement of GDNF during auditory development in mice. Using a lacZ knockin allele we have confirmed the expression of GDNF in the cochlea including its sensory regions during development. Global inactivation of GDNF throughout the hearing system using a Foxg1-Cre line causes perinatal lethality but reveals no apparent defects during formation of the cochlea. Using TrkC-Cre and Atoh1-Cre lines, we were able to generate viable mutants lacking GDNF in auditory neurons or both auditory neurons and sensory hair cells. These mutants show normal frequency-dependent auditory thresholds. However, mechanoelectrical response properties of outer hair cells (OHCs) in TrkC-Cre GDNF mutants are altered at low thresholds. Furthermore, auditory brainstem wave analysis shows an abnormal increase of wave I. On the other hand, Atoh1-Cre GDNF mutants show normal OHC function but their auditory brainstem wave pattern is reduced at the levels of wave I, III and IV. These results show that GDNF expression during the development is required to maintain functional hearing at different levels of the auditory system.

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure, Neural Crest Specification and Anterior-Posterior Axis Extension.

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.

Gαi Proteins are Indispensable for Hearing.

BACKGROUND/AIMS: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary ("hair") bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. METHODS: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. RESULTS: Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing. CONCLUSION: We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes.

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.

Gene expression profiling of the inner ear.

The identification of transcriptional differences has served as an important starting point in understanding the molecular mechanisms behind biological processes and systems. The developmental biology of the inner ear, the biology of hearing and of course the pathology of deafness are all processes that warrant a molecular description if we are to improve human health. To this end, technological innovation has meant that larger scale analysis of gene transcription has been possible for a number of years now, extending our molecular analysis of genes to beyond those that are currently in vogue for a given system. In this review, some of the contributions gene profiling has made to understanding developmental, pathological and physiological processes in the inner ear are highlighted.

Coupling the cell cycle to development and regeneration of the inner ear.

Cell cycle exit and acquirement of a postmitotic state is essential for the proper development of organs. In the present review, we examine the role of the cell cycle control in the sensory epithelia of the mammalian inner ear. We describe the roles of the core cell cycle regulators in the proliferation of prosensory cells and in the initiation and maintenance of terminal mitosis of the sensory epithelia. We also discuss how other intracellular signalling may influence the cell cycle. Finally, we address the question of whether manipulations of the cell cycle may have the potential to create replacement cells for the damaged inner sensory epithelia.

Correction: Transcription factor induced conversion of human fibroblasts towards the hair cell lineage.

[This corrects the article DOI: 10.1371/journal.pone.0200210.].

Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss.

The precision of sound information transmitted to the brain depends on the transfer characteristics of the inner hair cell (IHC) ribbon synapse and its multiple contacting auditory fibers. We found that brain derived neurotrophic factor (BDNF) differentially influences IHC characteristics in the intact and injured cochlea. Using conditional knock-out mice (BDNF(Pax2) KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNF(Pax2) KO IHCs showed a normal maturation. Likewise, in BDNF(Pax2) KO membrane capacitance (ΔC(m)) as a function of inward calcium current (I(Ca)) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔC(m), but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNF(Pax2) KO IHCs. Maximal ΔC(m) correlated with a loss of IHC ribbons in these cochlear turns and a reduced activity of the auditory nerve (auditory brainstem response wave I). Remarkably, a noise-induced loss of IHC ribbons, followed by reduced activity of the auditory nerve and reduced centrally generated wave II and III observed in control mice, was prevented in equally noise-exposed BDNF(Pax2) KO mice. Data suggest that BDNF expressed in the cochlea is essential for maintenance of adult IHC transmitter release sites and that BDNF upholds opposing afferents in high-frequency turns and scales them down following noise exposure.

N-myc controls proliferation, morphogenesis, and patterning of the inner ear.

Myc family members play crucial roles in regulating cell proliferation, size, and differentiation during organogenesis. Both N-myc and c-myc are expressed throughout inner ear development. To address their function in the mouse inner ear, we generated mice with conditional deletions in either N-myc or c-myc. Loss of c-myc in the inner ear causes no apparent defects, whereas inactivation of N-myc results in reduced growth caused by a lack of proliferation. Reciprocally, the misexpression of N-myc in the inner ear increases proliferation. Morphogenesis of the inner ear in N-myc mouse mutants is severely disturbed, including loss of the lateral canal, fusion of the cochlea with the sacculus and utriculus, and stunted outgrowth of the cochlea. Mutant cochleas are characterized by an increased number of cells exiting the cell cycle that express the cyclin-dependent kinase inhibitor p27(Kip1) and lack cyclin D1, both of which control the postmitotic state of hair cells. Analysis of different molecular markers in N-myc mutant ears reveals the development of a rudimentary organ of Corti containing hair cells and the underlying supporting cells. Differentiated cells, however, fail to form the highly ordered structure characteristic for the organ of Corti but appear as rows or clusters with an excess number of hair cells. The Kölliker's organ, a transient structure neighboring the organ of Corti and a potential source of ectopic hair cells, is absent in the mutant ears. Collectively, our data suggest that N-myc regulates growth, morphogenesis, and pattern formation during the development of the inner ear.

Cell and tissue therapy in regenerative medicine.

Cell therapy is one of the most promising future techniques in the medical arsenal for the repair of damaged or destroyed tissue. The diseases which cell therapy can target are very varied: Hormonal dysfunction, such as diabetes and growth hormone deficiency; neurodegenerative diseases, such as Parkinson's, Alzheimer's and Huntington's; and cardiovascular lesions, such as myocardial infarction, peripheral vascular ischaemia; as well as lesions in the cornea, skeletal muscle, skin, joints and bones etc. The objective of cell therapy is to restore the lost function rather than produce a new organ, which could cause duplicity and undesirable effects. Several resources of cells can be used to restore the damaged tissue, such as resident stem cells, multipotent adult progenitor cells or embryonic stem cells. Some cell therapies have been established and approved for clinical use, such as artificial skin derived from keratinocytes, derived from chondrocyte, cells of the corneal limbus or pancreatic islet transplantation. These therapies have had good results, although the scarcity of the starting material may represent a serious limitation. Other therapies under research, using pluripotent stem cells, have been modest so it is useful to review the protocols and try to improve the outcomes. In this chapter we will review the new advances made in this way.

Modulation of Fgf3 dosage in mouse and men mirrors evolution of mammalian dentition.

A central challenge in evolutionary biology is understanding how genetic mutations underlie morphological changes. Because highly calcified enamel enables preservation of detailed dental features, studying tooth morphology enables this question to be addressed in both extinct and extant species. Previous studies have found that mutant mice can have severe abnormalities in tooth morphology, and several authors have explored the evolutionary implications of tooth number modifications in mutants. However, although they can potentially shed much light on evolutionary mechanisms, anomalies in tooth shape remain poorly studied. Here, we report that alterations in dosage of the Fgf3 gene cause morphological changes in both genetically engineered mutant mice and in human patients. By comparing the dental morphologies in mice and humans carrying Fgf3 mutations with primitive rodent and primate fossils, we determined that decreases in dosage of Fgf3 lead to phenotypes that resemble the progressive reappearance of ancestral morphologies. We propose that modifications in the FGF signaling pathway have played an important role in evolution of mammalian dentition by giving rise to new cusps and interconnecting cusps by new crests. We anticipate that our multidisciplinary study will advance the detailed correlation of subtle dental modifications with genetic mutations in a variety of mammalian lineages.

Meis2 Is Required for Inner Ear Formation and Proper Morphogenesis of the Cochlea.

Meis genes have been shown to control essential processes during development of the central and peripheral nervous system. Here we have explored the roles of the Meis2 gene during vertebrate inner ear induction and the formation of the cochlea. Meis2 is expressed in several tissues required for inner ear induction and in non-sensory tissue of the cochlear duct. Global inactivation of Meis2 in the mouse leads to a severely reduced size of the otic vesicle. Tissue-specific knock outs of Meis2 reveal that its expression in the hindbrain is essential for otic vesicle formation. Inactivation of Meis2 in the inner ear itself leads to an aberrant coiling of the cochlear duct. By analyzing transcriptomes obtained from Meis2 mutants and ChIPseq analysis of an otic cell line, we define candidate target genes for Meis2 which may be directly or indirectly involved in cochlear morphogenesis. Taken together, these data show that Meis2 is essential for inner ear formation and provide an entry point to unveil the network underlying proper coiling of the cochlear duct.

Deletion of BDNF in Pax2 Lineage-Derived Interneuron Precursors in the Hindbrain Hampers the Proportion of Excitation/Inhibition, Learning, and Behavior.

Numerous studies indicate that deficits in the proper integration or migration of specific GABAergic precursor cells from the subpallium to the cortex can lead to severe cognitive dysfunctions and neurodevelopmental pathogenesis linked to intellectual disabilities. A different set of GABAergic precursors cells that express Pax2 migrate to hindbrain regions, targeting, for example auditory or somatosensory brainstem regions. We demonstrate that the absence of BDNF in Pax2-lineage descendants of Bdnf Pax2 KOs causes severe cognitive disabilities. In Bdnf Pax2 KOs, a normal number of parvalbumin-positive interneurons (PV-INs) was found in the auditory cortex (AC) and hippocampal regions, which went hand in hand with reduced PV-labeling in neuropil domains and elevated activity-regulated cytoskeleton-associated protein (Arc/Arg3.1; here: Arc) levels in pyramidal neurons in these same regions. This immaturity in the inhibitory/excitatory balance of the AC and hippocampus was accompanied by elevated LTP, reduced (sound-induced) LTP/LTD adjustment, impaired learning, elevated anxiety, and deficits in social behavior, overall representing an autistic-like phenotype. Reduced tonic inhibitory strength and elevated spontaneous firing rates in dorsal cochlear nucleus (DCN) brainstem neurons in otherwise nearly normal hearing Bdnf Pax2 KOs suggests that diminished fine-grained auditory-specific brainstem activity has hampered activity-driven integration of inhibitory networks of the AC in functional (hippocampal) circuits. This leads to an inability to scale hippocampal post-synapses during LTP/LTD plasticity. BDNF in Pax2-lineage descendants in lower brain regions should thus be considered as a novel candidate for contributing to the development of brain disorders, including autism.

An integrated gene regulatory network controls stem cell proliferation in teeth.

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.

Age-Dependent Auditory Processing Deficits after Cochlear Synaptopathy Depend on Auditory Nerve Latency and the Ability of the Brain to Recruit LTP/BDNF.

Age-related decoupling of auditory nerve fibers from hair cells (cochlear synaptopathy) has been linked to temporal processing deficits and impaired speech recognition performance. The link between both is elusive. We have previously demonstrated that cochlear synaptopathy, if centrally compensated through enhanced input/output function (neural gain), can prevent age-dependent temporal discrimination loss. It was also found that central neural gain after acoustic trauma was linked to hippocampal long-term potentiation (LTP) and upregulation of brain-derived neurotrophic factor (BDNF). Using middle-aged and old BDNF-live-exon-visualization (BLEV) reporter mice we analyzed the specific recruitment of LTP and the activity-dependent usage of Bdnf exon-IV and -VI promoters relative to cochlear synaptopathy and central (temporal) processing. For both groups, specimens with higher or lower ability to centrally compensate diminished auditory nerve activity were found. Strikingly, low compensating mouse groups differed from high compensators by prolonged auditory nerve latency. Moreover, low compensators exhibited attenuated responses to amplitude-modulated tones, and a reduction of hippocampal LTP and Bdnf transcript levels in comparison to high compensators. These results suggest that latency of auditory nerve processing, recruitment of hippocampal LTP, and Bdnf transcription, are key factors for age-dependent auditory processing deficits, rather than cochlear synaptopathy or aging per se.

Analysis of mouse kreisler mutants reveals new roles of hindbrain-derived signals in the establishment of the otic neurogenic domain.

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB--a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain--in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis.

Lower ototoxicity and absence of hidden hearing loss point to gentamicin C1a and apramycin as promising antibiotics for clinical use.

Spread of antimicrobial resistance and shortage of novel antibiotics have led to an urgent need for new antibacterials. Although aminoglycoside antibiotics (AGs) are very potent anti-infectives, their use is largely restricted due to serious side-effects, mainly nephrotoxicity and ototoxicity. We evaluated the ototoxicity of various AGs selected from a larger set of AGs on the basis of their strong antibacterial activities against multidrug-resistant clinical isolates of the ESKAPE panel: gentamicin, gentamicin C1a, apramycin, paromomycin and neomycin. Following local round window application, dose-dependent effects of AGs on outer hair cell survival and compound action potentials showed gentamicin C1a and apramycin as the least toxic. Strikingly, although no changes were observed in compound action potential thresholds and outer hair cell survival following treatment with low concentrations of neomycin, gentamicin and paromomycin, the number of inner hair cell synaptic ribbons and the compound action potential amplitudes were reduced. This indication of hidden hearing loss was not observed with gentamicin C1a or apramycin at such concentrations. These findings identify the inner hair cells as the most vulnerable element to AG treatment, indicating that gentamicin C1a and apramycin are promising bases for the development of clinically useful antibiotics.

Efficient transfer of embryonic stem cells into the cochlea via a non-invasive vestibular route.

CONCLUSION: Cell transplantation into the utriculus provides an efficient and non-invasive route to introduce embryonic stem (ES) cells into the vestibular and cochlear portions of the inner ear. OBJECTIVE: The transfer of stem cells into the inner ear for therapeutic purposes is an important approach to cure damage to the cochlea and vestibulum. A key issue is to provide an entry point for cell transplants into the inner ear that does not affect its physiologic functions. The aim of this study was to examine the feasibility of transferring ES cells into the inner ear via the utriculus. MATERIALS AND METHODS: ES cells were injected via utriculostomy into the mouse inner ear. The distribution of the injected cells was determined using a beta-galactosidase marker gene expressed by the ES cells. RESULTS: Injected ES cells were found within the perilymph of the scala tympani and vestibuli. Moreover, ES cells were detected close to the cochlear sensory epithelium and spiral limbus.