Ebf1 is a mouse deafness gene and deletion causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage prior to development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had no detectable auditory brainstem response (ABR) suggesting that this gene is essential for hearing development.

Novel cell types and developmental lineages revealed by single-cell RNA-seq analysis of the mouse crista ampullaris.

This study provides transcriptomic characterization of the cells of the crista ampullaris, sensory structures at the base of the semicircular canals that are critical for vestibular function. We performed single-cell RNA-seq on ampullae microdissected from E16, E18, P3, and P7 mice. Cluster analysis identified the hair cells, support cells and glia of the crista as well as dark cells and other nonsensory epithelial cells of the ampulla, mesenchymal cells, vascular cells, macrophages, and melanocytes. Cluster-specific expression of genes predicted their spatially restricted domains of gene expression in the crista and ampulla. Analysis of cellular proportions across developmental time showed dynamics in cellular composition. The new cell types revealed by single-cell RNA-seq could be important for understanding crista function and the markers identified in this study will enable the examination of their dynamics during development and disease.

Single-Cell Transcriptomic Comparison of Human Fetal Retina, hPSC-Derived Retinal Organoids, and Long-Term Retinal Cultures.

To study the development of the human retina, we use single-cell RNA sequencing (RNA-seq) at key fetal stages and follow the development of the major cell types as well as populations of transitional cells. We also analyze stem cell (hPSC)-derived retinal organoids; although organoids have a very similar cellular composition at equivalent ages as the fetal retina, there are some differences in gene expression of particular cell types. Moreover, the inner retinal lamination is disrupted at more advanced stages of organoids compared with fetal retina. To determine whether the disorganization in the inner retina is due to the culture conditions, we analyze retinal development in fetal retina maintained under similar conditions. These retinospheres develop for at least 6 months, displaying better inner retinal lamination than retinal organoids. Our single-cell RNA sequencing (scRNA-seq) comparisons of fetal retina, retinal organoids, and retinospheres provide a resource for developing better in vitro models for retinal disease.

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.

Effects of 3,3'-Iminodipropionitrile on Hair Cell Numbers in Cristae of CBA/CaJ and C57BL/6J Mice.

This study examines absolute hair cell numbers in the cristae of C57BL/6J mice and CBA/CaJ mice from weaning to adulthood as well as the dose required for 3,3'-iminodiproprionitrile (IDPN)-injury of the cristae in C57BL/6J mice and CBA/CaJ mice, the two mouse strains most commonly used by inner ear researchers. In cristae of CBA/CaJ and C57BL/6J mice, no loss of hair cells was observed up to 24 weeks. In both strains, dose-dependent loss of hair cells was observed 7 days after IDPN treatment of 2-month-old mice (IC50 = 16.1 mmol/kg in C57BL/6J mice vs. 25.21 mmol/kg in CBA/CaJ mice). Four-month-old C57BL/6J mice exposed to IDPN developed dose-dependent vestibular dysfunction as indicated by increased activity and circling behavior in open field tests and by failure to swim 7 days after treatment. IDPN-hair cell injury in C57BL/6J mice and CBA/CaJ mice represents a fast and predictable experimental model for the study of vestibular degeneration and a platform for the testing of vestibular therapies.

Molecular Anatomy of the Developing Human Retina.

Clinical and genetic heterogeneity associated with retinal diseases makes stem-cell-based therapies an attractive strategy for personalized medicine. However, we have limited understanding of the timing of key events in the developing human retina, and in particular the factors critical for generating the unique architecture of the fovea and surrounding macula. Here we define three key epochs in the transcriptome dynamics of human retina from fetal day (D) 52 to 136. Coincident histological analyses confirmed the cellular basis of transcriptional changes and highlighted the dramatic acceleration of development in the fovea compared with peripheral retina. Human and mouse retinal transcriptomes show remarkable similarity in developmental stages, although morphogenesis was greatly expanded in humans. Integration of DNA accessibility data allowed us to reconstruct transcriptional networks controlling photoreceptor differentiation. Our studies provide insights into human retinal development and serve as a resource for molecular staging of human stem-cell-derived retinal organoids.

A central to peripheral progression of cell cycle exit and hair cell differentiation in the developing mouse cristae.

The inner ear contains six distinct sensory organs that each maintains some ability to regenerate hair cells into adulthood. In the postnatal cochlea, there appears to be a relationship between the developmental maturity of a region and its ability to regenerate as postnatal regeneration largely occurs in the apical turn, which is the last region to differentiate and mature during development. In the mature cristae there are also regional differences in regenerative ability, which led us to hypothesize that there may be a general relationship between the relative maturity of a region and the regenerative competence of that region in all of the inner ear sensory organs. By analyzing adult mouse cristae labeled embryonically with BrdU, we found that hair cell birth starts in the central region and progresses to the periphery with age. Since the peripheral region of the adult cristae also maintains active Notch signaling and some regenerative competence, these results are consistent with the hypothesis that the last regions to develop retain some of their regenerative ability into adulthood. Further, by analyzing embryonic day 14.5 inner ears we provide evidence for a wave of hair cell birth along the longitudinal axis of the cristae from the central regions to the outer edges. Together with the data from the adult inner ears labeled with BrdU as embryos, these results suggest that hair cell differentiation closely follows cell cycle exit in the cristae, unlike in the cochlea where they are uncoupled.

A transient wave of BMP signaling in the retina is necessary for Müller glial differentiation.

The primary glial cells in the retina, the Müller glia, differentiate from retinal progenitors in the first postnatal week. CNTF/LIF/STAT3 signaling has been shown to promote their differentiation; however, another key glial differentiation signal, BMP, has not been examined during this period of Müller glial differentiation. In the course of our analysis of the BMP signaling pathway, we observed a transient wave of Smad1/5/8 signaling in the inner nuclear layer at the end of the first postnatal week, from postnatal day (P) 5 to P9, after the end of neurogenesis. To determine the function of this transient wave, we blocked BMP signaling during this period in vitro or in vivo, using either a BMP receptor antagonist or noggin (Nog). Either treatment leads to a reduction in expression of the Müller glia-specific genes Rlbp1 and Glul, and the failure of many of the Müller glia to repress the bipolar/photoreceptor gene Otx2. These changes in normal Müller glial differentiation result in permanent disruption of the retina, including defects in the outer limiting membrane, rosette formation and a reduction in functional acuity. Our results thus show that Müller glia require a transient BMP signal at the end of neurogenesis to fully repress the neural gene expression program and to promote glial gene expression.

Hair cell generation by notch inhibition in the adult mammalian cristae.

Balance disorders caused by hair cell loss in the sensory organs of the vestibular system pose a significant health problem worldwide, particularly in the elderly. Currently, this hair cell loss is permanent as there is no effective treatment. This is in stark contrast to nonmammalian vertebrates who robustly regenerate hair cells after damage. This disparity in regenerative potential highlights the need for further manipulation in order to stimulate more robust hair cell regeneration in mammals. In the utricle, Notch signaling is required for maintaining the striolar support cell phenotype into the second postnatal week. Notch signaling has further been implicated in hair cell regeneration after damage in the mature utricle. Here, we investigate the role of Notch signaling in the mature mammalian cristae in order to characterize the Notch-mediated regenerative potential of these sensory organs. For these studies, we used the γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), in conjunction with a method we developed to culture cristae in vitro. In postnatal and adult cristae, we found that 5 days of DAPT treatment resulted in a downregulation of the Notch effectors Hes1 and Hes5 and also an increase in the total number of Gfi1(+) hair cells. Hes5, as reported by Hes5-GFP, was downregulated specifically in peripheral support cells. Using lineage tracing with proteolipid protein (PLP)/CreER;mTmG mice, we found that these hair cells arose through transdifferentiation of support cells in cristae explanted from mice up to 10 weeks of age. These transdifferentiated cells arose without proliferation and were capable of taking on a hair cell morphology, migrating to the correct cell layer, and assembling what appears to be a stereocilia bundle with a long kinocilium. Overall, these data show that Notch signaling is active in the mature cristae and suggest that it may be important in maintaining the support cell fate in a subset of peripheral support cells.

Notch signaling in mammalian hair cell regeneration.

In the inner ear, Notch signaling has been shown to have two key developmental roles. The first occurs early in otic development and defines the prosensory domains that will develop into the six sensory organs of the inner ear. The second role occurs later in development and establishes the mosaic-like pattern of the mechanosensory hair cells and their surrounding support cells through the more well-characterized process of lateral inhibition. These dual developmental roles have inspired several different strategies to regenerate hair cells in the mature inner ear organs. These strategies include (1) modulation of Notch signaling in inner ear stem cells in order to increase hair cell yield, (2) activation of Notch signaling in order to promote the formation of ectopic sensory regions in normally non-sensory regions within the inner ear, and (3) inhibition of Notch signaling to disrupt lateral inhibition and allow support cells to transdifferentiate into hair cells. In this review, we summarize some of the promising studies that have used these various strategies for hair cell regeneration through modulation of Notch signaling and some of the challenges that remain in developing therapies based on hair cell regeneration.

Notch prosensory effects in the Mammalian cochlea are partially mediated by Fgf20.

Hearing loss is becoming an increasingly prevalent problem affecting more than 250 million people worldwide. During development, fibroblast growth factors (FGFs) are required for inner ear development as well as hair cell formation in the mammalian cochlea and thus make attractive therapeutic candidates for the regeneration of sensory cells. Previous findings showed that Fgfr1 conditional knock out mice exhibited hair cell and support cell formation defects. Immunoblocking with Fgf20 antibody in vitro produced a similar phenotype. While hair cell differentiation in mice starts at embryonic day (E)14.5, beginning with the inner hair cells, Fgf20 expression precedes hair cell differentiation at E13.5 in the cochlea. This suggests a potential role for Fgf20 in priming the sensory epithelium for hair cell formation. Treatment of explants with a gamma-secretase inhibitor, DAPT, decreased Fgf20 mRNA, suggesting that Notch is upstream of Fgf20. Notch signaling also plays an early role in prosensory formation during cochlear development. In this report we show that during development, Notch-mediated regulation of prosensory formation in the cochlea occurs via Fgf20. Addition of exogenous FGF20 compensated for the block in Notch signaling and rescued Sox2, a prosensory marker, and Gfi1, an early hair cell marker in explant cultures. We hypothesized that Fgf20 plays a role in specification, amplification, or maintenance of Sox2 expression in prosensory progenitors of the developing mammalian cochlea.

Regulated reprogramming in the regeneration of sensory receptor cells.

Vision, olfaction, hearing, and balance are mediated by receptors that reside in specialized sensory epithelial organs. Age-related degeneration of the photoreceptors in the retina and the hair cells in the cochlea, caused by macular degeneration and sensorineural hearing loss, respectively, affect a growing number of individuals. Although sensory receptor cells in the mammalian retina and inner ear show only limited or no regeneration, in many nonmammalian vertebrates, these sensory epithelia show remarkable regenerative potential. We summarize the current state of knowledge of regeneration in the specialized sense organs in both nonmammalian vertebrates and mammals and discuss possible areas where new advances in regenerative medicine might provide approaches to successfully stimulate sensory receptor cell regeneration. The field of regenerative medicine is still in its infancy, but new approaches using stem cells and reprogramming suggest ways in which the potential for regeneration may be restored in individuals suffering from sensory loss.

Dynamic expression of Lgr5, a Wnt target gene, in the developing and mature mouse cochlea.

The Wnt signaling pathway is a recurring theme in tissue development and homeostasis. Its specific roles during inner ear development are just emerging, but few studies have characterized Wnt target genes. Lgr5, a member of the G protein-coupled receptor family, is a Wnt target in the gastrointestinal and integumentary systems. Although its function is unknown, its deficiency leads to perinatal lethality due to gastrointestinal distension. In this study, we used a knock-in reporter mouse to examine the spatiotemporal expression of Lgr5 in the cochlear duct during embryonic and postnatal periods. In the embryonic day 15.5 (E15.5) cochlear duct, Lgr5-EGFP is expressed in the floor epithelium and overlapped with the prosensory markers Sox2, Jagged1, and p27(Kip1). Nascent hair cells and supporting cells in the apical turn of the E18.5 cochlear duct express Lgr5-EGFP, which becomes downregulated in hair cells and subsets of supporting cells in more mature stages. In situ hybridization experiments validated the reporter expression, which gradually decreases until the second postnatal week. Only the third row of Deiters' cells expresses Lgr5-EGFP in the mature organ of Corti. Normal cochlear development was observed in Lgr5(EGFP/EGFP) and Lgr5(EGFP/+) mice, which exhibited normal auditory thresholds. The expression pattern of Lgr5 contrasts with another Wnt target gene, Axin2, a feedback inhibitor of the Wnt pathway. Robust Axin2 expression was found in cells surrounding the embryonic cochlear duct and becomes restricted to tympanic border cells below the basilar membrane in the postnatal cochlea. Both Lgr5 and Axin2 act as Wnt targets in the cochlea because purified Wnt3a promoted and Wnt antagonist suppressed their expression. Their differential expression among cell populations highlights the dynamic but complex distribution of Wnt-activated cells in and around the embryonic and postnatal cochlea.

Notch signaling specifies prosensory domains via lateral induction in the developing mammalian inner ear.

During inner ear morphogenesis, the process of prosensory specification defines the specific regions of the otic epithelium that will give rise to the six separate inner ear organs essential for hearing and balance. The mechanism of prosensory specification is not fully understood, but there is evidence that the Notch intercellular signaling pathway plays a critical role. The Notch ligand Jagged1 (Jag1) is expressed in the prosensory domains, and mutation of Jag1 impairs sensory formation. Furthermore, pharmacological inhibition of Notch in vitro during prosensory specification disrupts the prosensory process. Additionally, activation of Notch by cDNA electroporation in chick otocysts results in formation of ectopic sensory patches. Here we test whether Notch activity is sufficient for prosensory specification in the mouse, using a Cre-/loxP approach to conditionally activate the Notch pathway in nonsensory regions of the inner ear epithelia during different stages of otic vesicle morphogenesis. We find that broad ectopic activation of Notch at very early developmental stages causes induction of prosensory markers throughout the entire otic epithelium. At later stages of development, activation of Notch in nonsensory regions leads to induction of sensory patches that later differentiate to form complete ectopic sensory structures. Activation of Notch in isolated nonsensory cells results in lateral induction of Jag1 expression in neighboring cells and spreading of prosensory specification to the adjacent cells through an intercellular mechanism. These results support a model where activation of Notch and propagation through lateral induction promote prosensory character in specific regions of the developing otocyst.

A method for stabilizing RNA for transfection that allows control of expression duration.

RNA transfection methods have not proven to be as popular as DNA methods due to the highly transient nature of the RNA inside the cell. However, there are many advantages in using RNA for gene over-expression, such as the rapidity of expression, the ability to express in all cell types without the need for cell-type-specific promoters, and the ability to analyze the effects of gene over-expression in a transient manner. Therefore, we have developed a method (StabiLizingUtr: SLU) to stabilize the RNA for varying durations, using specific sequences from the 3'UTR of the Venezuelan equine encephalitis virus (VEEV). We have designed a plasmid for cloning genes upstream from repeated stabilizing sequences to generate mRNA with one or more VEEV-stabilizing sequence motifs. We demonstrate this method in several cell and tissue types, including the mammalian cochlea, a tissue that has been difficult to transfect with other methods.

Expression patterns of FGF receptors in the developing mammalian cochlea.

Many studies have shown the importance of the fibroblast growth factor (FGF) family of factors in the development of the mammalian cochlea. There are four fibroblast growth factor receptors (FGFR1-4) and all four are expressed in the cochlea during development. While there are examples in the literature of expression patterns of some of the receptors at specific stages of cochlear development there has been no systematic study. We have assembled a full analysis of the patterns of receptor expression during cochlear development for all four Fgfrs using in situ hybridization. We have analyzed the expression patterns from embryonic day 13.5 through postnatal ages. We find that Fgfr1, 2, and 3 are expressed in the epithelium of the cochlear duct and Fgfr4 is limited in its expression to the mesenchyme surrounding the duct. We compare the receptor expression pattern to markers of the sensory domain (p27kip1) and the early hair cells (math1).

Delta/notch-like EGF-related receptor (DNER) is expressed in hair cells and neurons in the developing and adult mouse inner ear.

The Notch signaling pathway is known to play important roles in inner ear development. Previous studies have shown that the Notch1 receptor and ligands in the Delta and Jagged families are important for cellular differentiation and patterning of the organ of Corti. Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a novel Notch ligand expressed in developing and adult CNS neurons known to promote maturation of glia through activation of Notch. Here we use in situ hybridization and an antibody against DNER to carry out expression studies of the mouse cochlea and vestibule. We find that DNER is expressed in spiral ganglion neuron cell bodies and peripheral processes during embryonic development of the cochlea and expression in these cells is maintained in adults. DNER becomes strongly expressed in auditory hair cells during postnatal maturation in the mouse cochlea and immunoreactivity for this protein is strong in hair cells and afferent and efferent peripheral nerve endings in the adult organ of Corti. In the vestibular system, we find that DNER is expressed in hair cells and vestibular ganglion neurons during development and in adults. To investigate whether DNER plays a functional role in the inner ear, perhaps similar to its described role in glial maturation, we examined cochleae of DNER-/- mice using immunohistochemical markers of mature glia and supporting cells as well as neurons and hair cells. We found no defects in expression of markers of supporting cells and glia or myelin, and no abnormalities in hair cells or neurons, suggesting that DNER plays a redundant role with other Notch ligands in cochlear development.

CLRN1 is nonessential in the mouse retina but is required for cochlear hair cell development.

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5-6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT-PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT-PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.

Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation.

Usher syndrome 3A (USH3A) is an autosomal recessive disorder characterized by progressive loss of hearing and vision due to mutation in the clarin-1 (CLRN1) gene. Lack of an animal model has hindered our ability to understand the function of CLRN1 and the pathophysiology associated with USH3A. Here we report for the first time a mouse model for ear disease in USH3A. Detailed evaluation of inner ear phenotype in the Clrn1 knockout mouse (Clrn1(-/-)) coupled with expression pattern of Clrn1 in the inner ear are presented here. Clrn1 was expressed as early as embryonic day 16.5 in the auditory and vestibular hair cells and associated ganglionic neurons, with its expression being higher in outer hair cells (OHCs) than inner hair cells. Clrn1(-/-) mice showed early onset hearing loss that rapidly progressed to severe levels. Two to three weeks after birth (P14-P21), Clrn1(-/-) mice showed elevated auditory-evoked brainstem response (ABR) thresholds and prolonged peak and interpeak latencies. By P21, approximately 70% of Clrn1(-/-) mice had no detectable ABR and by P30 these mice were deaf. Distortion product otoacoustic emissions were not recordable from Clrn1(-/-) mice. Vestibular function in Clrn1(-/-) mice mirrored the cochlear phenotype, although it deteriorated more gradually than cochlear function. Disorganization of OHC stereocilia was seen as early as P2 and by P21 OHC loss was observed. In sum, hair cell dysfunction and prolonged peak latencies in vestibular and cochlear evoked potentials in Clrn1(-/-) mice strongly indicate that Clrn1 is necessary for hair cell function and associated neural activation.