Altered Fhod3 expression involved in progressive high-frequency hearing loss via dysregulation of actin polymerization stoichiometry in the cuticular plate.

Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL.

Altered Fhod3 Expression Involved in Progressive High-Frequency Hearing Loss via Dysregulation of Actin Polymerization Stoichiometry in The Cuticular Plate.

Age-related hearing loss (ARHL) is a common sensory impairment with comlex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) and primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer HCs and decrease phalloidin intensities of CP. Ultrastructural analysis revealed shortened stereocilia in the basal turn cochlea. Importantly, the hearing and HC phenotype in TG mice were replicated in KO mice. These findings indicate that Fhod3 plays a critical role in regulating actin dynamics in CP and stereocilia. Further investigation of Fhod3-related hearing impairment mechanisms may facilitate the development of therapeutic strategies for ARHL in humans.

Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development.

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Noncoding Microdeletion in Mouse Hgf Disrupts Neural Crest Migration into the Stria Vascularis, Reduces the Endocochlear Potential, and Suggests the Neuropathology for Human Nonsyndromic Deafness DFNB39.

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival, and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an overexpression of HGF, causes neurosensory deafness. In humans, noncoding variants in HGF are associated with nonsyndromic deafness DFNB39 However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10 bp deletion (del10) in Hgf Male and female mice homozygous for del10 exhibit moderate-to-profound hearing loss at 4 weeks of age as measured by tone burst auditory brainstem responses. The wild type (WT) 80 mV endocochlear potential was significantly reduced in homozygous del10 mice compared with WT littermates. In normal cochlea, endocochlear potentials are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that, in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in Hgf leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and a reduction in the EP, recapitulating human DFNB39 nonsyndromic deafness.SIGNIFICANCE STATEMENT Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously, we reported that human deafness DFNB39 is associated with noncoding variants in the 3'UTR of a short isoform of HGF encoding hepatocyte growth factor. For normal hearing, HGF levels must be fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a Hgf mutant mouse with a small 10 bp deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

Role of Dach1 revealed using a novel inner ear-specific Dach1-knockdown mouse model.

The Dach1 gene is expressed in the inner ear of normal mouse embryos in the area that differentiates into the cochlear stria vascularis (SV). We hypothesised that Dach1 downregulation in the inner ear would lead to SV dysplasia. However, because Dach1 knockout is embryonic lethal in mice, the role of Dach1 in the inner ear is unclear. Here, we established inner ear-specific Dach1-knockdown mice and showed that Dach1 downregulation resulted in hearing loss, reduced endocochlear potential and secondary outer hair cell loss. There were no abnormalities in marginal cells and basal cells in the SV or spiral ligament in inner ear-specific Dach1-knockdown mature mice. However, intermediate cell dysplasia and thinning of the SV were observed. Moreover, dynamic changes in the expression of key genes related to the epithelial-mesenchymal transition were observed in the lateral wall of the cochlear epithelium, which differentiated into the SV in inner ear-specific Dach1-knockdown mice at embryonic stages. In summary, suppression of Dach1 expression in the inner ear caused the epithelial-mesenchymal transition in the lateral wall of cochlear epithelium, resulting in loss of intermediate cells in the SV and SV dysplasia.This article has an associated First Person interview with the first author of the paper.

Role of Neuropilin-1/Semaphorin-3A signaling in the functional and morphological integrity of the cochlea.

Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.

Genome-Wide Association Analysis Identifies Dcc as an Essential Factor in the Innervation of the Peripheral Vestibular System in Inbred Mice.

This study aimed to investigate the genetic causes of vestibular dysfunction. We used vestibular sensory-evoked potentials (VsEPs) to characterize the vestibular function of 35 inbred mouse strains selected from the Hybrid Mouse Diversity Panel and demonstrated strain-dependent phenotypic variation in vestibular function. Using these phenotypic data, we performed the first genome-wide association study controlling for population structure that has revealed two highly suggestive loci, one of which lies within a haplotype block containing five genes (Stard6, 4930503L19Rik, Poli, Mbd2, Dcc) on Chr. 18 (peak SNP rs29632020), one gene, deleted in colorectal carcinoma (Dcc) has a well-established role in nervous system development. An in-depth analysis of Dcc-deficient mice demonstrated elevation in mean VsEP threshold for Dcc (+/-) mice (-11.86 dB) compared to wild-type (-9.68 dB) littermates. Synaptic ribbon studies revealed Dcc (-/-) (P0) and Dcc (+/-) (6-week-old) mice showed lower density of the presynaptic marker (CtBP2) as compared to wild-type controls. Vestibular ganglion cell counts of Dcc (-/-) (P0) was lower than controls. Whole-mount preparations showed abnormal innervation of the utricle, saccule, and crista ampullaris at E14.5, E16.5, and E18.5. Postnatal studies were limited by the perinatal lethality in Dcc (-/-) mice. Expression analyses using in situ hybridization and immunohistochemistry showed Dcc expression in the mouse vestibular ganglion (E15.5), and utricle and crista ampullaris (6-week-old), respectively. In summary, we report the first GWAS for vestibular functional variation in inbred mice and provide evidence for the role of Dcc in the normal innervation of the peripheral vestibular system.

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.

Foxi3 Deficiency Compromises Hair Follicle Stem Cell Specification and Activation.

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. Stem Cells 2016;34:1896-1908.

Large-scale phenotyping of noise-induced hearing loss in 100 strains of mice.

A cornerstone technique in the study of hearing is the Auditory Brainstem Response (ABR), an electrophysiologic technique that can be used as a quantitative measure of hearing function. Previous studies have published databases of baseline ABR thresholds for mouse strains, providing a valuable resource for the study of baseline hearing function and genetic mapping of hearing traits in mice. In this study, we further expand upon the existing literature by characterizing the baseline ABR characteristics of 100 inbred mouse strains, 47 of which are newly characterized for hearing function. We identify several distinct patterns of baseline hearing deficits and provide potential avenues for further investigation. Additionally, we characterize the sensitivity of the same 100 strains to noise exposure using permanent thresholds shifts, identifying several distinct patterns of noise-sensitivity. The resulting data provides a new resource for studying hearing loss and noise-sensitivity in mice.

The mouse Foxi3 transcription factor is necessary for the development of posterior placodes.

The inner ear develops from the otic placode, one of the cranial placodes that arise from a region of ectoderm adjacent to the anterior neural plate called the pre-placodal domain. We have identified a Forkhead family transcription factor, Foxi3, that is expressed in the pre-placodal domain and down-regulated when the otic placode is induced. We now show that Foxi3 mutant mice do not form otic placodes as evidenced by expression changes in early molecular markers and the lack of thickened placodal ectoderm, an otic cup or otocyst. Some preplacodal genes downstream of Foxi3-Gata3, Six1 and Eya1-are not expressed in the ectoderm of Foxi3 mutant mice, and the ectoderm exhibits signs of increased apoptosis. We also show that Fgf signals from the hindbrain and cranial mesoderm, which are necessary for otic placode induction, are received by pre-placodal ectoderm in Foxi3 mutants, but do not initiate otic induction. Finally, we show that the epibranchial placodes that develop in close proximity to the otic placode and the mandibular division of the trigeminal ganglion are missing in Foxi3 mutants. Our data suggest that Foxi3 is necessary to prime pre-placodal ectoderm for the correct interpretation of inductive signals for the otic and epibranchial placodes.

Suppression of epithelial differentiation by Foxi3 is essential for molar crown patterning.

Epithelial morphogenesis generates the shape of the tooth crown. This is driven by patterned differentiation of cells into enamel knots, root-forming cervical loops and enamel-forming ameloblasts. Enamel knots are signaling centers that define the positions of cusp tips in a tooth by instructing the adjacent epithelium to fold and proliferate. Here, we show that the forkhead-box transcription factor Foxi3 inhibits formation of enamel knots and cervical loops and thus the differentiation of dental epithelium in mice. Conditional deletion of Foxi3 (Foxi3 cKO) led to fusion of molars with abnormally patterned shallow cusps. Foxi3 was expressed in the epithelium, and its expression was reduced in the enamel knots and cervical loops and in ameloblasts. Bmp4, a known inducer of enamel knots and dental epithelial differentiation, downregulated Foxi3 in wild-type teeth. Using genome-wide gene expression profiling, we showed that in Foxi3 cKO there was an early upregulation of differentiation markers, such as p21, Fgf15 and Sfrp5. Different signaling pathway components that are normally restricted to the enamel knots were expanded in the epithelium, and Sostdc1, a marker of the intercuspal epithelium, was missing. These findings indicated that the activator-inhibitor balance regulating cusp patterning was disrupted in Foxi3 cKO. In addition, early molar bud morphogenesis and, in particular, formation of the suprabasal epithelial cell layer were impaired. We identified keratin 10 as a marker of suprabasal epithelial cells in teeth. Our results suggest that Foxi3 maintains dental epithelial cells in an undifferentiated state and thereby regulates multiple stages of tooth morphogenesis.

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.

Foxi transcription factors promote pharyngeal arch development by regulating formation of FGF signaling centers.

The bones of the vertebrate face develop from transient embryonic branchial arches that are populated by cranial neural crest cells. We have characterized a mouse mutant for the Forkhead family transcription factor Foxi3, which is expressed in branchial ectoderm and endoderm. Foxi3 mutant mice are not viable and display severe branchial arch-derived facial skeleton defects, including absence of all but the most distal tip of the mandible and complete absence of the inner, middle and external ear structures. Although cranial neural crest cells of Foxi3 mutants are able to migrate, populate the branchial arches, and display some elements of correct proximo-distal patterning, they succumb to apoptosis from embryonic day 9.75 onwards. We show this cell death correlates with a delay in expression of Fgf8 in branchial arch ectoderm and a failure of neural crest cells in the arches to express FGF-responsive genes. Zebrafish foxi1 is also expressed in branchial arch ectoderm and endoderm, and morpholino knock-down of foxi1 also causes apoptosis of neural crest in the branchial arches. We show that heat shock induction of fgf3 in zebrafish arch tissue can rescue cell death in foxi1 morphants. Our results suggest that Foxi3 may play a role in the establishment of signaling centers in the branchial arches that are required for neural crest survival, patterning and the subsequent development of branchial arch derivatives.

Canonical Notch signaling is not necessary for prosensory induction in the mouse cochlea: insights from a conditional mutant of RBPjkappa.

The mammalian organ of Corti consists of a highly organized array of hair cells and supporting cells that originate from a common population of prosensory progenitors. Proper differentiation of this complex cellular mosaic requires lateral inhibition mediated by Notch signaling. Several studies have implicated Notch signaling in the earlier induction of the prosensory domain that lies along the length of the cochlear duct, and which forms before the onset of hair cell and supporting cell differentiation. To investigate the role of Notch signaling in prosensory domain formation, we conditionally inactivated the transcriptional mediator of canonical Notch signaling, RBPjκ, throughout the inner ear. Although RBPjκ mutants have severe vestibular defects and a shortened cochlear duct, markers of the prosensory domain appear at the normal time and location in the cochlea of RBPjκ mutants. Despite the lack of RBPjκ, hair cell and supporting cell markers also appear at appropriate times in the cochlea, suggesting that RBPjκ is dispensable for differentiation of the cochlear sensory epithelium. However, we also observed that differentiating hair cells and supporting cells rapidly die in RBPjκ mutants, suggesting a requirement of RBPjκ for cell survival in this tissue. Finally, in contrast to the chick basilar papilla, ectopic activation of Notch signaling did not induce ectopic sensory patches in nonsensory regions of the cochlea. Our results indicate that canonical Notch signaling is not necessary for prosensory specification in the mouse cochlea, suggesting that other signaling pathways may specify this highly derived sensory organ.

SOX9 controls epithelial branching by activating RET effector genes during kidney development.

Congenital abnormalities of the kidney and urinary tract are some of the most common defects detected in the unborn child. Kidney growth is controlled by the GDNF/RET signalling pathway, but the molecular events required for the activation of RET downstream targets are still poorly understood. Here we show that SOX9, a gene involved in campomelic dysplasia (CD) in humans, together with its close homologue SOX8, plays an essential role in RET signalling. Expression of SOX9 can be found from the earliest stages of renal development within the ureteric tip, the ureter mesenchyme and in a segment-specific manner during nephrogenesis. Using a tissue-specific knockout approach, we show that, in the ureteric tip, SOX8 and SOX9 are required for ureter branching, and double-knockout mutants exhibit severe kidney defects ranging from hypoplastic kidneys to renal agenesis. Further genetic analysis shows that SOX8/9 are required downstream of GDNF signalling for the activation of RET effector genes such as Sprouty1 and Etv5. At later stages of development, SOX9 is required to maintain ureteric tip identity and SOX9 ablation induces ectopic nephron formation. Taken together, our study shows that SOX9 acts at multiple steps during kidney organogenesis and identifies SOX8 and SOX9 as key factors within the RET signalling pathway. Our results also explain the aetiology of kidney hypoplasia found in a proportion of CD patients.

BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea.

The mammalian inner ear detects sound with the organ of Corti, an intricately patterned region of the cochlea in which one row of inner hair cells and three rows of outer hair cells are surrounded by specialized supporting cells. The organ of Corti derives from a prosensory domain that runs the length of the cochlear duct and is bounded by two nonsensory domains, Kölliker's organ on the neural side and the outer sulcus on the abneural side. Although much progress has been made in identifying the signals regulating organ of Corti induction and differentiation, less is known about the mechanisms that establish sensory and nonsensory territories in the cochlear duct. Here, we show that a gradient of bone morphogenetic protein (BMP) signaling is established in the abneural-neural axis of the cochlea. Analysis of compound mutants of Alk3/6 type I BMP receptors shows that BMP signaling is necessary for specification of the prosensory domain destined to form the organ of Corti. Reduction of BMP signaling in Alk3/6 compound mutants eliminates both the future outer sulcus and the prosensory domain, with all cells expressing markers of Kölliker's organ. BMP4 upregulates markers of the future outer sulcus and downregulates marker genes of Kölliker's organ in cochlear organ cultures in a dose-dependent manner. Our results suggest BMP signaling is required for patterning sensory and nonsensory tissue in the mammalian cochlea.

Unraveling inner ear induction by gene manipulation using Pax2-Cre BAC transgenic mice.

One of the biggest drawbacks of conventional mouse knockout techniques in the study of the inner ear is that loss of a gene of interest may cause embryonic lethality before the inner ear develops. Thus, there is a need for an inner ear-specific gene manipulation system for loss- and gain-of-function analysis in the mouse inner ear. We generated a Pax2-Cre BAC transgenic line in which Cre recombinase expression recapitulates Pax2 expression in the presumptive otic ectoderm. Here, we present a brief summary of a recent model of inner ear induction suggested by the results of inner ear-specific gene modification using Pax2-Cre mice.

Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti.

The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here, we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that-unlike other Hes/Hey factors-Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.

Notch signaling augments the canonical Wnt pathway to specify the size of the otic placode.

The inner ear derives from a patch of ectoderm defined by expression of the transcription factor Pax2. We recently showed that this Pax2(+) ectoderm gives rise not only to the otic placode but also to the surrounding cranial epidermis, and that Wnt signaling mediates this placode-epidermis fate decision. We now present evidence for reciprocal interactions between the Wnt and Notch signaling pathways during inner ear induction. Activation of Notch1 in Pax2(+) ectoderm expands the placodal epithelium at the expense of cranial epidermis, whereas loss of Notch1 leads to a reduction in the size of the otic placode. We show that Wnt signaling positively regulates Notch pathway genes such as Jag1, Notch1 and Hes1, and we have used transgenic Wnt reporter mice to show that Notch signaling can modulate the canonical Wnt pathway. Gain- and loss-of-function mutations in the Notch and Wnt pathways reveal that some aspects of otic placode development - such as Pax8 expression and the morphological thickening of the placode - can be regulated independently by either Notch or Wnt signals. Our results suggest that Wnt signaling specifies the size of the otic placode in two ways, by directly upregulating a subset of otic genes, and by positively regulating components of the Notch signaling pathway, which then act to augment Wnt signaling.