Hair Cell Mechanotransduction Regulates Spontaneous Activity and Spiral Ganglion Subtype Specification in the Auditory System.

Type I spiral ganglion neurons (SGNs) transmit sound information from cochlear hair cells to the CNS. Using transcriptome analysis of thousands of single neurons, we demonstrate that murine type I SGNs consist of subclasses that are defined by the expression of subsets of transcription factors, cell adhesion molecules, ion channels, and neurotransmitter receptors. Subtype specification is initiated prior to the onset of hearing during the time period when auditory circuits mature. Gene mutations linked to deafness that disrupt hair cell mechanotransduction or glutamatergic signaling perturb the firing behavior of SGNs prior to hearing onset and disrupt SGN subtype specification. We thus conclude that an intact hair cell mechanotransduction machinery is critical during the pre-hearing period to regulate the firing behavior of SGNs and their segregation into subtypes. Because deafness is frequently caused by defects in hair cells, our findings have significant ramifications for the etiology of hearing loss and its treatment.

Development and regeneration of vestibular hair cells in mammals.

Vestibular sensation is essential for gaze stabilization, balance, and perception of gravity. The vestibular receptors in mammals, Type I and Type II hair cells, are located in five small organs in the inner ear. Damage to hair cells and their innervating neurons can cause crippling symptoms such as vertigo, visual field oscillation, and imbalance. In adult rodents, some Type II hair cells are regenerated and become re-innervated after damage, presenting opportunities for restoring vestibular function after hair cell damage. This article reviews features of vestibular sensory cells in mammals, including their basic properties, how they develop, and how they are replaced after damage. We discuss molecules that control vestibular hair cell regeneration and highlight areas in which our understanding of development and regeneration needs to be deepened.

Responses to cell loss become restricted as the supporting cells in mammalian vestibular organs grow thick junctional actin bands that develop high stability.

Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin-GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin-GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia.

Recovery kinetics of dual AAV-mediated human otoferlin expression.

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.

In vivo proliferative regeneration of balance hair cells in newborn mice.

The regeneration of mechanoreceptive hair cells occurs throughout life in non-mammalian vertebrates and allows them to recover from hearing and balance deficits that affect humans and other mammals permanently. The irreversibility of comparable deficits in mammals remains unexplained, but often has been attributed to steep embryonic declines in cellular production. However, recent results suggest that gravity-sensing hair cells in murine utricles may increase in number during neonatal development, raising the possibility that young mice might retain sufficient cellular plasticity for mitotic hair cell regeneration. To test for this we used neomycin to kill hair cells in utricles cultured from mice of different ages and found that proliferation increased tenfold in damaged utricles from the youngest neonates. To kill hair cells in vivo, we generated a novel mouse model that uses an inducible, hair cell-specific CreER allele to drive expression of diphtheria toxin fragment A (DTA). In newborns, induction of DTA expression killed hair cells and resulted in significant, mitotic hair cell replacement in vivo, which occurred days after the normal cessation of developmental mitoses that produce hair cells. DTA expression induced in 5-d-old mice also caused hair cell loss, but no longer evoked mitotic hair cell replacement. These findings show that regeneration limits arise in vivo during the postnatal period when the mammalian balance epithelium's supporting cells differentiate unique cytological characteristics and lose plasticity, and they support the notion that the differentiation of those cells may directly inhibit regeneration or eliminate an essential, but as yet unidentified pool of stem cells.

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.

Characterization of spatial and temporal development of Type I and Type II hair cells in the mouse utricle using new cell-type-specific markers.

The utricle of the inner ear, a vestibular sensory structure that mediates perception of linear acceleration, is comprised of two morphologically and physiologically distinct types of mechanosensory hair cells, referred to as Type Is and Type IIs. While these cell types are easily discriminated in an adult utricle, understanding their development has been hampered by a lack of molecular markers that can be used to identify each cell type prior to maturity. Therefore, we collected single hair cells at three different ages and used single cell RNAseq to characterize the transcriptomes of those cells. Analysis of differential gene expression identified Spp1 as a specific marker for Type I hair cells and Mapt and Anxa4 as specific markers for Type II hair cells. Antibody labeling confirmed the specificity of these markers which were then used to examine the temporal and spatial development of utricular hair cells. While Type I hair cells develop in a gradient that extends across the utricle from posterior-medial to anterior-lateral, Type II hair cells initially develop in the central striolar region and then extend uniformly towards the periphery. Finally, by combining these markers with genetic fate mapping, we demonstrate that over 98% of all Type I hair cells develop prior to birth while over 98% of Type II hair cells develop post-natally. These results are consistent with previous findings suggesting that Type I hair cells develop first and refute the hypothesis that Type II hair cells represent a transitional form between immature and Type I hair cells.

Molecular architecture underlying fluid absorption by the developing inner ear.

Mutations of SLC26A4 are a common cause of hearing loss associated with enlargement of the endolymphatic sac (EES). Slc26a4 expression in the developing mouse endolymphatic sac is required for acquisition of normal inner ear structure and function. Here, we show that the mouse endolymphatic sac absorbs fluid in an SLC26A4-dependent fashion. Fluid absorption was sensitive to ouabain and gadolinium but insensitive to benzamil, bafilomycin and S3226. Single-cell RNA-seq analysis of pre- and postnatal endolymphatic sacs demonstrates two types of differentiated cells. Early ribosome-rich cells (RRCs) have a transcriptomic signature suggesting expression and secretion of extracellular proteins, while mature RRCs express genes implicated in innate immunity. The transcriptomic signature of mitochondria-rich cells (MRCs) indicates that they mediate vectorial ion transport. We propose a molecular mechanism for resorption of NaCl by MRCs during development, and conclude that disruption of this mechanism is the root cause of hearing loss associated with EES.

Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear.

In the inner ear, cochlear and vestibular sensory epithelia utilize grossly similar cell types to transduce different stimuli: sound and acceleration. Each individual sensory epithelium is composed of highly heterogeneous populations of cells based on physiological and anatomical criteria. However, limited numbers of each cell type have impeded transcriptional characterization. Here we generated transcriptomes for 301 single cells from the utricular and cochlear sensory epithelia of newborn mice to circumvent this challenge. Cluster analysis indicates distinct profiles for each of the major sensory epithelial cell types, as well as less-distinct sub-populations. Asynchrony within utricles allows reconstruction of the temporal progression of cell-type-specific differentiation and suggests possible plasticity among cells at the sensory-nonsensory boundary. Comparisons of cell types from utricles and cochleae demonstrate divergence between auditory and vestibular cells, despite a common origin. These results provide significant insights into the developmental processes that form unique inner ear cell types.

A historical to present-day account of efforts to answer the question: "what puts the brakes on mammalian hair cell regeneration?".

Hearing and balance deficits often affect humans and other mammals permanently, because their ears stop producing hair cells within a few days after birth. But production occurs throughout life in the ears of sharks, bony fish, amphibians, reptiles, and birds allowing them to replace lost hair cells and quickly recover after temporarily experiencing the kinds of sensory deficits that are irreversible for mammals. Since the mid 1970s, researchers have been asking what puts the brakes on hair cell regeneration in mammals. Here we evaluate the headway that has been made and assess current evidence for alternative mechanistic hypotheses that have been proposed to account for the limits to hair cell regeneration in mammals.

Specializations of intercellular junctions are associated with the presence and absence of hair cell regeneration in ears from six vertebrate classes.

Sensory hair cell losses lead to hearing and balance deficits that are permanent for mammals, but temporary for nonmammals because supporting cells in their ears give rise to replacement hair cells. In mice and humans, vestibular supporting cells grow exceptionally large circumferential F-actin belts and their junctions express E-cadherin in patterns that strongly correlate with postnatal declines in regeneration capacity. In contrast, chicken supporting cells retain thin F-actin belts throughout life and express little E-cadherin. To determine whether the junctions in chicken ears might be representative of other ears that also regenerate hair cells, we investigated inner ears from dogfish sharks, zebrafish, bullfrogs, Xenopus, turtles, and the lizard, Anolis. As in chickens, the supporting cells in adult zebrafish, Xenopus, and turtle ears retained thin circumferential F-actin belts and expressed little E-cadherin. Supporting cells in adult sharks and bullfrogs also retained thin belts, but were not tested for E-cadherin. Supporting cells in adult Anolis exhibited wide, but porous webs of F-actin and strong E-cadherin expression. Anolis supporting cells also showed some cell cycle reentry when cultured. The results reveal that the association between thin F-actin belts and low E-cadherin is shared by supporting cells in anamniotes, turtles, and birds, which all can regenerate hair cells. Divergent junctional specializations in supporting cells appear to have arisen independently in Anolis and mammals. The presence of webs of F-actin at the junctions in Anolis appears compatible with supporting cell proliferation, but the solid reinforcement of the F-actin belts in mammals is associated with its absence.

MYC gene delivery to adult mouse utricles stimulates proliferation of postmitotic supporting cells in vitro.

The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and the degradation-resistant T58A mutant of c-Myc (c-MycT58A) triggered significant levels of supporting cell S-phase entry as assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the proliferative response. The number of BrdU-labeled cells plateaued between 5-7 days after infection, and then decreased ~60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and differentiation may restore regenerative potential to supporting cells within the adult mammalian inner ear.

Over half the hair cells in the mouse utricle first appear after birth, with significant numbers originating from early postnatal mitotic production in peripheral and striolar growth zones.

Many non-mammalian vertebrates produce hair cells throughout life and recover from hearing and balance deficits through regeneration. In contrast, embryonic production of hair cells declines sharply in mammals where deficits from hair cell losses are typically permanent. Hair cell density estimates recently suggested that the vestibular organs of mice continue to add hair cells after birth, so we undertook comprehensive counting in murine utricles at different ages. The counts show that 51% of the hair cells in adults arise during the 2 weeks after birth. Immature hair cells are most common near the neonatal macula's peripheral edge and striola, where anti-Ki-67 labels cycling nuclei in zones that appear to contain niches for supporting-cell-like stem cells. In vivo lineage tracing in a novel reporter mouse where tamoxifen-inducible supporting cell-specific Cre expression switched tdTomato fluorescence to eGFP fluorescence showed that proteolipid-protein-1-expressing supporting cells are an important source of the new hair cells. To assess the contributions of postnatal cell divisions, we gave mice an injection of BrdU or EdU on the day of birth. The labels were restricted to supporting cells 1 day later, but by 12 days, 31% of the labeled nuclei were in myosin-VIIA-positive hair cells. Thus, hair cell populations in neonatal mouse utricles grow appreciably through two processes: the progressive differentiation of cells generated before birth and the differentiation of new cells arising from divisions of progenitors that progress through S phase soon after birth. Subsequent declines in these processes coincide with maturational changes that appear unique to mammalian supporting cells.

Variations in shape-sensitive restriction points mirror differences in the regeneration capacities of avian and mammalian ears.

When inner ear hair cells die, humans and other mammals experience permanent hearing and balance deficits, but non-mammalian vertebrates quickly recover these senses after epithelial supporting cells give rise to replacement hair cells. A postnatal decline in cellular plasticity appears to limit regeneration in mammalian balance organs, where declining proliferation responses are correlated with decreased spreading of supporting cells on artificial and native substrates. By culturing balance epithelia on substrates that differed in flexibility, we assessed spreading effects independent of age, showing a strong correlation between shape change and supporting cell proliferation. Then we made excision wounds in utricles cultured from young and old chickens and mice and compared quantified levels of spreading and proliferation. In utricles from young mice, and both young and old chickens, wounds re-epithelialized in <24 hours, while those in utricles from mature mice took three times longer. More cells changed shape in the fastest healing wounds, which accounted for some differences in the levels of proliferation, but inter-species and age-related differences in shape-sensitive restriction points, i.e., the cellular thresholds for shape changes that promote S-phase, were evident and may be particularly influential in the responses to hair cell losses in vivo.

Lime pretreatment of switchgrass at mild temperatures for ethanol production.

To improve the enzymatic digestibility of switchgrass at mild temperatures, lime pretreatment of switchgrass was explored at 50 and 21 degrees Celsius, and compared with that at 121 degrees Celsius. The effects of residence time, lime loading, and biomass washing on the sugar production efficiency were investigated. Pretreatments were evaluated based on the yields of biomass-derived sugars in the subsequent enzymatic hydrolysis. Under the best pretreatment conditions (50 degrees Celsius, 24 h, 0.10 g Ca(OH)(2)/g raw biomass, and wash intensity of 100 ml water/g raw biomass), the yields of glucose, xylose, and total reducing sugars reached 239.6, 127.2, and 433.4 mg/g raw biomass, which were respectively 3.15, 5.78, and 3.61 times those of untreated biomass. The study on calcium-lignin bonding showed that calcium ions crosslinked lignin molecules under alkaline conditions, which substantially decreased lignin solubilization during pretreatment, but the resulting high lignin contents of the pretreated biomass did not compromise the improvement of enzymatic digestibility.

Bioenergy from Coastal bermudagrass receiving subsurface drip irrigation with advance-treated swine wastewater.

Coastal bermudagrass (Cynodon dactylon L.) may be a potentially important source of bio-based energy in the southern US due to its vast acreage. It is often produced as part of a waste management plan with varying nutrient composition and energy characteristics on fields irrigated with livestock wastewater. The objective of this study was to determine the effect of subsurface drip irrigation with treated swine wastewater on both the quantity and quality of bermudagrass bioenergy. The treated wastewater was recycled from an advanced treatment system and used for irrigation of bermudagrass in two crop seasons. The experiment had nine water and drip line spacing treatments arrayed in a randomized complete block-design with four replicates. The bermudagrass was analyzed for calorific and mineral contents. Bermudagrass energy yields for 2004 and 2005 ranged from 127.4 to 251.4MJ ha(-1). Compared to irrigation with commercial nitrogen fertilizer, the least biomass energy density was associated with bermudagrass receiving treated swine wastewater. Yet, in 2004 the wastewater irrigated bermudagrass had greater hay yields leading to greater energy yield per ha. This decrease in energy density of wastewater irrigated bermudagrass was associated with increased concentrations of K, Ca, and Na. After thermal conversion, these compounds are known to remain in the ash portion thereby decreasing the energy density. Nonetheless, the loss of energy density using treated effluent via SDI may be offset by the positive influence of these three elements for their catalytic properties in downstream thermal conversion processes such as promoting a lesser char yield and greater combustible gas formation.

Recent advances in hair cell regeneration research.

PURPOSE OF REVIEW: This review discusses recent progress in research that seeks to understand the regeneration of hair cells and highlights findings that may hold importance for the eventual development of regenerative therapies for hearing and balance impairments. RECENT FINDINGS: Signaling via the Notch receptor and the basic helix-loop-helix transcription factors has important roles in the development and regeneration of hair cells. The cytoskeletal properties and cell-matrix interactions of supporting cells in mice of different ages may hold part of the explanation for the age-related differences in their proliferative responses to damage and the differences between mammals and nonmammals in hair cell regeneration. Progress also has been made in deriving stem cells from inner ear tissues and other sources and in the evaluation of their potential uses as sources of new hair cells and as tools for biomedical research. SUMMARY: Much has been accomplished since the discovery of postembryonic hair cell production and hair cell regeneration in nonmammals decades ago. No therapies for hair cell regeneration are under clinical trials, but research is yielding potentially important discoveries that are likely to lead to the development of therapeutic methods for inducing hair cell regeneration in the mammalian inner ear.

Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds.

Debilitating hearing and balance deficits often arise through damage to the inner ear's hair cells. For humans and other mammals, such deficits are permanent, but nonmammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F-actin that bracket the apical junctions between supporting cells in the gravity-sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, whereas their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F-actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F-actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement and that the postnatal reinforcement of the F-actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration.

Performance of lactating dairy cows fed varying levels of total mixed ration and pasture.

Two, 8-week experiments, each using 30 lactating Holstein cows, were conducted to examine performance of animals offered combinations of total mixed ration (TMR) and high-quality pasture. Experiment 1 was initiated in mid October 2004 and Experiment 2 was initiated in late March 2005. Cows were assigned to either a 100% TMR diet (100:00, no access to pasture) or one of the following three formulated partial mixed rations (PMR) targeted at (1) 85% TMR and 15% pasture, (2) 70% TMR and 30% pasture and (3) 55% TMR and 45% pasture. Based on actual TMR and pasture intake, the dietary TMR and pasture proportions of the three PMR in Experiment 1 were 79% TMR and 21% pasture (79:21), 68% TMR and 32% pasture (68:32), and 59% TMR and 41% pasture (59:41), respectively. Corresponding proportions in Experiment 2 were 89% TMR and 11% pasture (89:11), 79% TMR and 21% pasture (79:21) and 65% TMR and 35% pasture (65:35), respectively. Reducing the proportion of TMR in the diets increased pasture consumption of cows on all PMR, but reduced total dry matter intake compared with cows on 100:00. An increase in forage from pasture increased the concentration of conjugated linoleic acids and decreased the concentration of saturated fatty acids in milk. Although milk and milk protein yields from cows grazing spring pastures (Experiment 2) increased with increasing intakes of TMR, a partial mixed ration that was composed of 41% pasture grazed in the fall (Experiment 1) resulted in a similar overall lactation performance with increased feed efficiency compared to an all-TMR ration.

Variation in Ruminant Preference for Alfalfa Hays Cut at Sunup and Sundown.

Diurnal variation in the concentration of total nonstructural carbohydrates (TNC) occurs in plants as a result of photosynthesis. Ruminants have been shown to prefer tall fescue (Festuca arundinacea Schreber) hays cut in the afternoon but the effect of morning vs. evening cutting had not been tested in legumes. To test for diurnal variation in preference for alfalfa (Medicago sativa L.), we harvested six times in the midbud stage. Harvests were paired so that each time a cutting of alfalfa was made at sundown (PM) another was made the next morning at sunup (AM). We harvested in this manner three times resulting in six hays. The hays were field dried, baled, and chopped prior to their use 3 to 6 mo after harvest. Three experiments were conducted [Exp. 1, sheep (Ovis aries); Exp. 2, goats (Capra hircus hircus); and Exp. 3, cattle (Bos taurus)] utilizing six animals in each case. During an adaptation phase, hays were offered alone as meals. In the experimental phase, every possible pair of hays (15 pairs) was presented for a meal. Data were analyzed by multidimensional scaling as well as by traditional analyses. Multidimensional scaling indicated that the animals were basing selection on at least two criteria. Variables associated with preference through multiple regression varied across experiments but significant coefficients were found between preference and nitrate, protein, carbohydrate fractions, lignin, and cellulose. Coefficients varied depending on which other variables were in the model; however, carbohydrates were associated with positive coefficients. Shifting hay mowing from early in the day to late in the day was effective in increasing forage preference as expressed by short-term dry matter intake.