Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb^-/-, pRb^-/- utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb^-/- cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of ^pRb-/- cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb^-/- cochlea and utricle is centered on E2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb^-/-cochlea or utricle. In pRb^-/- cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involving in proliferation and survival are enriched in pRb^-/-utricle. Clustering analysis showed that the pRb^-/- inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRb^flox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.     

Overactivation of Notch1 signaling induces ectopic hair cells in the mouse inner ear in an age-dependent manner

Background

During mouse inner ear development, Notch1 signaling first specifies sensory progenitors, and subsequently controls progenitors to further differentiate into either hair cells (HCs) or supporting cells (SCs). Overactivation of NICD (Notch1 intracellular domain) at early embryonic stages leads to ectopic HC formation. However, it remains unclear whether such an effect can be elicited at later embryonic or postnatal stages, which has important implications in mouse HC regeneration by reactivation of Notch1 signaling.

Methodology/Principal Findings

We performed comprehensive in vivo inducible overactivation of NICD at various developmental stages. In CAG^CreER+; Rosa26-NICD^loxp/+ mice, tamoxifen treatment at embryonic day 10.5 (E10.5) generated ectopic HCs in the non-sensory regions in both utricle and cochlea, whereas ectopic HCs only appeared in the utricle when tamoxifen was given at E13. When tamoxifen was injected at postnatal day 0 (P0) and P1, no ectopic HCs were observed in either utricle or cochlea. Interestingly, Notch1 signaling induced new HCs in a non-cell-autonomous manner, because the new HCs did not express NICD. Adjacent to the new HCs were cells expressing the SC marker Sox10 (either NICD+ or NICD-negative).

Conclusions/Significance

Our data demonstrate that the developmental stage determines responsiveness of embryonic otic precursors and neonatal non-sensory epithelial cells to NICD overactivation, and that Notch 1 signaling in the wild type, postnatal inner ear is not sufficient for generating new HCs. Thus, our genetic mouse model is suitable to test additional pathways that could synergistically interact with Notch1 pathway to produce HCs at postnatal ages.     

The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear

Precursors of cochlear and vestibular hair cells of the inner ear exit the cell cycle at midgestation. Hair cells are mitotically quiescent during late-embryonic differentiation stages and postnatally. We show here that the retinoblastoma gene Rb and the encoded protein pRb are expressed in differentiating and mature hair cells. In addition to Rb, the cyclin dependent kinase inhibitor (CKI) p21 is expressed in developing hair cells, suggesting that p21 is an upstream effector of pRb activity. p21 apparently cooperates with other CKIs, as p21-null mice exhibited an unaltered inner ear phenotype. By contrast, Rb inactivation led to aberrant hair cell proliferation, as analysed at birth in a loss-of-function/transgenic mouse model. Supernumerary hair cells expressed various cell type-specific differentiation markers, including components of stereocilia. The extent of alterations in stereociliary bundle morphology ranged from near-normal to severe disorganization. Apoptosis contributed to the mutant phenotype, but did not compensate for the production of supernumerary hair cells, resulting in hyperplastic sensory epithelia. The Rb-null-mediated proliferation led to a distinct pathological phenotype, including multinucleated and enlarged hair cells, and infiltration of hair cells into the mesenchyme. Our findings demonstrate that the pRb pathway is required for hair cell quiescence and that manipulation of the cell cycle machinery disrupts the coordinated development within the inner ear sensory epithelia.     

Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity

Background

The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.

Methodology/Principal Findings

We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1^Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1^Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons.

Conclusions/Significance

These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.     

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing ¹. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth ^2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes ^4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development⁶.The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos⁷. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells^8-10. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol¹¹. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Hes1 is a negative regulator of inner ear hair cell differentiation

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, we have focused on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. We report here that targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions which are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells.     

Applying genomics to the avian inner ear: development of subtractive cDNA resources for exploring sensory function and hair cell regeneration

We applied a micro-cDNA-based subtraction method to identify genes expressed in the regenerating sensory epithelia (SE) of the chicken inner ear. Sensory hair cells in the avian utricle SE are in a constant state of turnover, where dying hair cells are replaced by new ones derived from supporting cells. In contrast, hair cells in the cochlea remain quiescent unless damaged. We used this difference to enrich for utricle-specific genes, using reiterative cDNA subtraction and demonstrate enrichment for utricle-specific sequences. A total of 1710 cDNA sequence reads revealed the presence of many cDNAs encoding known structural components of the SE (for example, Harmonin and beta-tectorin), proteins involved in cellular proliferation, such as P311, HIPK2, and SPALT1, among many others of unknown function. These libraries are the first of their kind and should prove useful for the discovery of candidate genes for hearing disorders, regenerative and apoptotic pathways, and novel chicken ESTs.     

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) ^1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions ^{7, 8}. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium ^{9, 10}. Supporting cells are also important mediators of hair cell survival and death ¹¹. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.     

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.     

Transient Receptor Potential Melastatin 1: A Hair Cell Transduction Channel Candidate

Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca²⁺ permeability and ∼100 picosiemens conductance. These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (Trpm1^−/−) or a missense mutation in the pore domain of TRPM1 (Trpm1^{tvrm27/tvrm27}). Hearing thresholds were evaluated in adult (4–5 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in Trpm1^−/− or Trpm1^{tvrm27/tvrm27} mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

Cell cycle regulation in the inner ear sensory epithelia: Role of cyclin D1 and cyclin-dependent kinase inhibitors

Sensory hair cells and supporting cells of the mammalian cochlea and vestibular (balance) organs exit the cell cycle during embryogenesis and do not proliferate thereafter. Here, we have studied the mechanisms underlying the maintenance of the postmitotic state and the proliferative capacity of these cells. We provide the first evidence of the role of cyclin D1 in cell cycle regulation in these cells. Cyclin D1 expression disappeared from embryonic hair cells as differentiation started. The expression was transiently upregulated in cochlear hair cells early postnatally, paralleling the spatiotemporal pattern of unscheduled cell cycle re-entry of cochlear hair cells from the p19^Ink4d/p21^Cip1 compound mutant mice. Cyclin D1 misexpression in vitro in neonatal vestibular HCs from these mutant mice triggered S-phase re-entry. Thus, cyclin D1 suppression is important for hair cell's quiescence, together with the maintained expression of cyclin-dependent kinase inhibitors. In contrast to hair cells, cyclin D1 expression was maintained in supporting cells when differentiation started. The expression continued during the neonatal period when supporting cells have been shown to re-enter the cell cycle upon stimulation with exogenous mitogens. Thereafter, the steep decline in supporting cell's proliferative activity paralleled with cyclin D1 downregulation. Thus, cyclin D1 critically contributes to the proliferative plasticity of supporting cells. These data suggest that targeted cyclin D1 induction in supporting cells might be an avenue for proliferative regeneration in the inner ear.     

Cell Cycle,Differentiation and Regeneration: Where to Begin?

Hair cells, the sensory cells of inner ear, perform essential functions in hearing and balance. However, mammalian hair cells, like most of the CNS neurons, lack the capacity to regenerate. This is in sharp contrast to lower vertebrates in which hair cell regeneration occurs spontaneously through cell division of supporting cells, which leads to hearing restoration. It is believed that the lack of regeneration in mammals is, to a large degree, due to the block of cell cycle re-entry imposed by negative cell growth genes in the inner ear. Recent studies have identified retinoblastoma gene, a well-known tumor suppressor, as the key gene involved in cell cycle exit of inner ear sensory cells. In the inner ear of pRb conditional knockout mice, hair cells undergo continuous cell division, and at the same time differentiate and become functional. Cell division continues in early postnatal cochlea and adult vestibule. Remarkably, the vestibular hair cells without pRb survive, and function at both the cellular and system levels. The time course and effects of pRb inhibition shows that there is a separation between the roles of pRb in cell cycle exit, and subsequent maturation and apoptosis. Those studies reveal distinctly different roles of pRb in the cochlear and vestibular sensory epithelia. The review discusses additional areas to be studied for regeneration of mature hair cells, and highlights the importance of transient and reversible block of pRb function as one of the routes to be explored for regeneration.