Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

ISL1 is necessary for auditory neuron development and contributes toward tonotopic organization.

A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.

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.

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2, the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

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.

Chromatin remodeler CHD7 is critical for cochlear morphogenesis and neurosensory patterning.

Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using ShhCre/+). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons.

Embryonic staging of bats with special reference to Vespertilio sinensis and its cochlear development.

BACKGROUND: How bats deviate heterochronically from other mammals remains largely unresolved, reflecting the lack of a quantitative staging framework allowing comparison among species. The standard event system (SES) is an embryonic staging system allowing quantitative detection of interspecific developmental variations. Here, the first SES-based staging system for bats, using Asian parti-colored bat (Vespertilio sinensis) is introduced. General aspects of normal embryonic development and the three-dimensional development of the bat cochlea were described for the first time. Recoding the embryonic staging tables of 18 previously reported bat species and Mus musculus into the SES system, quantitative developmental comparisons were performed. RESULTS: It was found that limb bud development of V. sinensis is relatively late among 19 bat species and late limb development is a shared trait of vespertilionid bats. The inner ear cochlear canal forms before the semicircular canal in V. sinensis while the cochlear canal forms after the semicircular canal in non-volant mammals. CONCLUSIONS: The present approach using the SES system provides a powerful framework to detect the peculiarities of bat development. Incorporating the timing of gene expression patterns into the SES framework will further contribute to the understanding of the evolution of specialized features in bats.

Molecular Aspects of the Development and Function of Auditory Neurons.

This review provides an up-to-date source of information on the primary auditory neurons or spiral ganglion neurons in the cochlea. These neurons transmit auditory information in the form of electric signals from sensory hair cells to the first auditory nuclei of the brain stem, the cochlear nuclei. Congenital and acquired neurosensory hearing loss affects millions of people worldwide. An increasing body of evidence suggest that the primary auditory neurons degenerate due to noise exposure and aging more readily than sensory cells, and thus, auditory neurons are a primary target for regenerative therapy. A better understanding of the development and function of these neurons is the ultimate goal for long-term maintenance, regeneration, and stem cell replacement therapy. In this review, we provide an overview of the key molecular factors responsible for the function and neurogenesis of the primary auditory neurons, as well as a brief introduction to stem cell research focused on the replacement and generation of auditory neurons.

Neuronal processes and glial precursors form a scaffold for wiring the developing mouse cochlea.

In the developing nervous system, axons navigate through complex terrains that change depending on when and where outgrowth begins. For instance, in the developing cochlea, spiral ganglion neurons extend their peripheral processes through a growing and heterogeneous environment en route to their final targets, the hair cells. Although the basic principles of axon guidance are well established, it remains unclear how axons adjust strategies over time and space. Here, we show that neurons with different positions in the spiral ganglion employ different guidance mechanisms, with evidence for both glia-guided growth and fasciculation along a neuronal scaffold. Processes from neurons in the rear of the ganglion are more directed and grow faster than those from neurons at the border of the ganglion. Further, processes at the wavefront grow more efficiently when in contact with glial precursors growing ahead of them. These findings suggest a tiered mechanism for reliable axon guidance.

Dual regulation of planar polarization by secreted Wnts and Vangl2 in the developing mouse cochlea.

Planar cell polarity (PCP) proteins localize asymmetrically to instruct cell polarity within the tissue plane, with defects leading to deformities of the limbs, neural tube and inner ear. Wnt proteins are evolutionarily conserved polarity cues, yet Wnt mutants display variable PCP defects; thus, how Wnts regulate PCP remains unresolved. Here, we have used the developing cochlea as a model system to show that secreted Wnts regulate PCP through polarizing a specific subset of PCP proteins. Conditional deletion of Wntless or porcupine, both of which are essential for secretion of Wnts, caused misrotated sensory cells and shortened cochlea - both hallmarks of PCP defects. Wntless-deficient cochleae lacked the polarized PCP components dishevelled 1/2 and frizzled 3/6, while other PCP proteins (Vangl1/2, Celsr1 and dishevelled 3) remained localized. We identified seven Wnt paralogues, including the major PCP regulator Wnt5a, which was, surprisingly, dispensable for planar polarization in the cochlea. Finally, Vangl2 haploinsufficiency markedly accentuated sensory cell polarization defects in Wntless-deficient cochlea. Together, our study indicates that secreted Wnts and Vangl2 coordinate to ensure proper tissue polarization during development.

Long-range cis-regulatory elements controlling GDF6 expression are essential for ear development.

Molecular mechanisms governing the development of the mammalian cochlea, the hearing organ, remain largely unknown. Through genome sequencing in 3 subjects from 2 families with nonsyndromic cochlear aplasia, we identified homozygous 221-kb and 338-kb deletions in a noncoding region on chromosome 8 with an approximately 200-kb overlapping section. Genomic location of the overlapping deleted region started from approximately 350 kb downstream of GDF6, which codes for growth and differentiation factor 6. Otic lineage cells differentiated from induced pluripotent stem cells derived from an affected individual showed reduced expression of GDF6 compared with control cells. Knockout of Gdf6 in a mouse model resulted in cochlear aplasia, closely resembling the human phenotype. We conclude that GDF6 plays a necessary role in early cochlear development controlled by cis-regulatory elements located within an approximately 500-kb region of the genome in humans and that its disruption leads to deafness due to cochlear aplasia.

Characterization of the development of the mouse cochlear epithelium at the single cell level.

Mammalian hearing requires the development of the organ of Corti, a sensory epithelium comprising unique cell types. The limited number of each of these cell types, combined with their close proximity, has prevented characterization of individual cell types and/or their developmental progression. To examine cochlear development more closely, we transcriptionally profile approximately 30,000 isolated mouse cochlear cells collected at four developmental time points. Here we report on the analysis of those cells including the identification of both known and unknown cell types. Trajectory analysis for OHCs indicates four phases of gene expression while fate mapping of progenitor cells suggests that OHCs and their surrounding supporting cells arise from a distinct (lateral) progenitor pool. Tgfßr1 is identified as being expressed in lateral progenitor cells and a Tgfßr1 antagonist inhibits OHC development. These results provide insights regarding cochlear development and demonstrate the potential value and application of this data set.

The acquisition of positional information across the radial axis of the cochlea.

The mammalian cochlea detects sound and transmits this information to the brain. A cross section through the cochlea reveals functionally distinct epithelial domains arrayed around the circumference of a fluid-filled duct. Six major domains include two on the roof of the duct (Reissner's membrane medially and the stria vascularis laterally) and four across the floor of the duct, including the medial and lateral halves of the sensory domain, the organ of Corti. These radial domains are distinguishable in the embryonic cochlea by differential expression of transcription factors, and we focus here on a subset of the factors that can influence cochlear fates. We then move upstream of these genes to identify which of five signaling pathways (Notch, Fgf, Wnt, Bmp, and Shh) controls their spatial patterns of expression. We link the signaling pathways to their downstream genes, separating them by their radial position, to create putative gene regulatory networks (GRNs) from two time points, before and during the time when six radial compartments arise. These GRNs offer a framework for understanding the acquisition of positional information across the radial axis of the cochlea, and to guide therapeutic approaches to repair or regenerate distinct cochlear components that may contribute to hearing loss.

Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea.

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.

Unidirectional and stage-dependent roles of Notch1 in Wnt-responsive Lgr5+ cells during mouse inner ear development.

Wnt and Notch signaling play crucial roles in the determination of the prosensory domain and in the differentiation of hair cells (HCs) and supporting cells during mouse inner ear development; however, the relationship between the two signaling pathways in the mouse cochlea remains largely unknown. Here, we investigated the interactions between Notch and Wnt signaling on the basis of the bidirectional regulation of Notch1 specifically in Wnt-responsive Lgr5+ progenitors during different cochlear development stages. We found that the downregulation of Notch1 in Lgr5+ cells from embryonic day (E) 14.5 to E18.5 can drive the quiescent Lgr5+ cells to re-enter the cell cycle and differentiate into extra HCs, whereas the upregulation of Notch1 expression did not affect the proliferation or differentiation of otic progenitor cells. No effect was observed on the upregulation or downregulation of Notch1 in Lgr5+ cells from E10.5 to E14.5. We concluded that the roles of Notch1 in Wnt-responsive Lgr5+ cells are unidirectional and stage dependent and Notch1 serves as a negative regulator for Lgr5+ progenitor activation during cochlear differentiation. Our findings improved the understanding of the interactions between Notch and Wnt signaling in cochlear development.

Genomic architecture of Shh-dependent cochlear morphogenesis.

The mammalian cochlea develops from a ventral outgrowth of the otic vesicle in response to Shh signaling. Mouse embryos lacking Shh or its essential signal transduction components display cochlear agenesis; however, a detailed understanding of the transcriptional network mediating this process is unclear. Here, we describe an integrated genomic approach to identify Shh-dependent genes and associated regulatory sequences that promote cochlear duct morphogenesis. A comparative transcriptome analysis of otic vesicles from mouse mutants exhibiting loss (Smoecko ) and gain (Shh-P1) of Shh signaling reveal a set of Shh-responsive genes partitioned into four expression categories in the ventral half of the otic vesicle. This target gene classification scheme provides novel insight into several unanticipated roles for Shh, including priming the cochlear epithelium for subsequent sensory development. We also mapped regions of open chromatin in the inner ear by ATAC-seq that, in combination with Gli2 ChIP-seq, identified inner ear enhancers in the vicinity of Shh-responsive genes. These datasets are useful entry points for deciphering Shh-dependent regulatory mechanisms involved in cochlear duct morphogenesis and establishment of its constituent cell types.

SOX2 is required for inner ear growth and cochlear nonsensory formation before sensory development.

The transcription factor sex determining region Y-box 2 (SOX2) is required for the formation of hair cells and supporting cells in the inner ear and is a widely used sensory marker. Paradoxically, we demonstrate via fate mapping that, initially, SOX2 primarily marks nonsensory progenitors in the mouse cochlea, and is not specific to all sensory regions until late otic vesicle stages. SOX2 fate mapping reveals an apical-to-basal gradient of SOX2 expression in the sensory region of the cochlea, reflecting the pattern of cell cycle exit. To understand SOX2 function, we undertook a timed-deletion approach, revealing that early loss of SOX2 severely impaired morphological development of the ear, whereas later deletions resulted in sensory disruptions. During otocyst stages, SOX2 shifted dramatically from a lateral to medial domain over 24-48 h, reflecting the nonsensory-to-sensory switch observed by fate mapping. Early loss or gain of SOX2 function led to changes in otic epithelial volume and progenitor proliferation, impacting growth and morphological development of the ear. Our study demonstrates a novel role for SOX2 in early otic morphological development, and provides insights into the temporal and spatial patterns of sensory specification in the inner ear.

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.

Open chromatin dynamics in prosensory cells of the embryonic mouse cochlea.

Hearing loss is often due to the absence or the degeneration of hair cells in the cochlea. Understanding the mechanisms regulating the generation of hair cells may therefore lead to better treatments for hearing disorders. To elucidate the transcriptional control mechanisms specifying the progenitor cells (i.e. prosensory cells) that generate the hair cells and support cells critical for hearing function, we compared chromatin accessibility using ATAC-seq in sorted prosensory cells (Sox2-EGFP+) and surrounding cells (Sox2-EGFP-) from E12, E14.5 and E16 cochlear ducts. In Sox2-EGFP+, we find greater accessibility in and near genes restricted in expression to the prosensory region of the cochlear duct including Sox2, Isl1, Eya1 and Pou4f3. Furthermore, we find significant enrichment for the consensus binding sites of Sox2, Six1 and Gata3-transcription factors required for prosensory development-in the open chromatin regions. Over 2,200 regions displayed differential accessibility with developmental time in Sox2-EGFP+ cells, with most changes in the E12-14.5 window. Open chromatin regions detected in Sox2-EGFP+ cells map to over 48,000 orthologous regions in the human genome that include regions in genes linked to deafness. Our results reveal a dynamic landscape of open chromatin in prosensory cells with potential implications for cochlear development and disease.

CTCF deficiency causes expansion of the sensory domain in the mouse cochlea.

The cochlea in the mammalian inner ear is a sensitive and sharply organized sound-detecting structure. The proper specification of neurosensory-competent domain in the otic epithelium is required for the formation of mature neuronal and sensory domains. Genetic studies have provided many insights into inner ear development, but there have been few epigenetic studies of inner ear development. CTCF is an epigenetic factor that plays a pivotal role in the organization of global chromatin conformation. To determine the role of CTCF in the otic sensory formation, we made a conditional knockout of Ctcf in the developing otic epithelium by crossing Ctcffl/fl mice with Pax2-Cre mice. Ctcf deficiency resulted in extra rows of auditory hair cells in the shortened cochlea on mouse embryonic day 14.5 (E14.5) and E17.5. The massive and ectopic expression of sensory specifiers such as Jag1 and Sox2 indicated that the sensory domain was expanded in the Ctcf-deficient cochlea. Other regulators of the sensory domain such as Bmp4, Gata3, and Fgf10 were not affected. These results suggest that CTCF plays a role in the regulation of the sensory domain in mammalian cochlear development.