Hair cell regeneration

The mammalian inner ear largely lacks the capacity to regenerate hair cells, the sensory cells required for hearing and balance. Recent studies in both lower vertebrates and mammals have uncovered genes and pathways important in hair cell development and have suggested ways that the sensory epithelia could be manipulated to achieve hair cell regeneration. These approaches include the use of inner ear stem cells, transdifferentiation of nonsensory cells, and induction of a proliferative response in the cells that can become hair cells.     

Myosin light-chain kinase is necessary for membrane homeostasis in cochlear inner hair cells

The structural homeostasis of the cochlear hair cell membrane is critical for all aspects of sensory transduction, but the regulation of its maintenance is not well understood. In this report, we analyzed the cochlear hair cells of mice with specific deletion of myosin light chain kinase (MLCK) in inner hair cells. MLCK-deficient mice showed impaired hearing, with a 5- to 14-dB rise in the auditory brainstem response (ABR) thresholds to clicks and tones of different frequencies and a significant decrease in the amplitude of the ABR waves. The mutant inner hair cells produced several ball-like structures around the hair bundles in vivo, indicating impaired membrane stability. Inner hair cells isolated from the knockout mice consistently displayed less resistance to hypoosmotic solution and less membrane F-actin. Myosin light-chain phosphorylation was also reduced in the mutated inner hair cells. Our results suggest that MLCK is necessary for maintaining the membrane stability of inner hair cells.     

β4-subunit increases Slo responsiveness to physiological Ca2+ concentrations and together with β1 reduces surface expression of Slo in hair cells

Changing kinetics of large-conductance potassium (BK) channels in hair cells of nonmammalian vertebrates, including the chick, plays a critical role in electrical tuning, a mechanism used by these cells to discriminate between different frequencies of sound. BK currents are less abundant in low-frequency hair cells and show large openings in response to a rise in intracellular Ca(2+) at a hair cell's operating voltage range (spanning -40 to -60 mV). Although the molecular underpinnings of its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Currents from the α (Slo)-subunit alone do not show dramatic increases in response to changes in Ca(2+) concentrations at -50 mV. We have cloned the chick β(4)- and β(1)-subunits and show that these subunits are preferentially expressed in low-frequency hair cells, where they decrease Slo surface expression. The β(4)-subunit in particular is responsible for the BK channel's increased responsiveness to Ca(2+) at a hair cell's operating voltage. In contrast, however, the increases in relaxation times induced by both β-subunits suggest additional mechanisms responsible for BK channel function in hair cells.     

Reactive oxygen species and root hairs in Arabidopsis root response to nitrogen, phosphorus and potassium deficiency

Plant root sensing and adaptation to changes in the nutrient status of soils is vital for long-term productivity and growth. Reactive oxygen species (ROS) have been shown to play a role in root response to potassium deprivation. To determine the role of ROS in plant response to nitrogen and phosphorus deficiency, studies were conducted using wild-type Arabidopsis and several root hair mutants. The expression of several nutrient-responsive genes was determined by Northern blot, and ROS were quantified and localized in roots. The monitored genes varied in intensity and timing of expression depending on which nutrient was deficient. In response to nutrient deprivation, ROS concentrations increased in specific regions of the Arabidopsis root. Changes in ROS localization in Arabidopsis and in a set of root hair mutants suggest that the root hair cells are important for response to nitrogen and potassium. In contrast, the response to phosphorus deprivation occurs in the cortex where an increase in ROS was measured. Based on these results, we put forward the hypothesis that root hair cells in Arabidopsis contain a sensing system for nitrogen and potassium deprivation.     

Hair cell regeneration in the avian auditory epithelium

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in non-mammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.     

Preferential expression of transient potassium current (IA) by 'short' hair cells of the chick's cochlea

We have made a comparative study of the membrane properties of tall and short hair cells isolated from a selected region of the chick's cochlea. Tall hair cells are analogous to inner cochlear hair cells of mammals, and like those, are presynaptic to the majority of afferent neurons in the cochlea. Short hair cells, like mammalian outer hair cells, are the postsynaptic targets of efferent neurons that inhibit the cochlea. Voltage-clamp recordings have revealed that short hair cells have an inactivating potassium (K) current, IA, whereas tall hair cells have little or none. Short hair cells are also sensitive to the cholinergic agonist carbachol, whereas tall hair cells are not. This pattern is in accord with the selective distribution of efferent cholinergic synapses in the cochlea. Although IA is completely inactivated at the resting potential of the short hair cells, cholinergic agonists can hyperpolarize these cells by as much as 30 mV. This hyperpolarization removes inactivation and allows IA to modulate subsequent voltage-dependent processes in short hair cells. It is concluded that IA could increase the high frequency response of the hair cell by decreasing membrane resistance and thus the membrane time constant after inhibition. This will be of particular importance to cochlear function if short hair cells produce voltage-dependent movements, as do mammalian outer hair cells.     

Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule.

Our senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Millions of people suffer from hearing and balance deficits caused by damage to hair cells as a result of exposure to noise, aminoglycoside antibiotics, and antitumor drugs. In some species such damage can be reversed through the production of new cells. This proliferative response is limited in mammals but it has been hypothesized that damaged hair cells might survive and undergo intracellular repair. We examined the fate of bullfrog saccular hair cells after exposure to a low dose of the aminoglycoside antibiotic gentamicin to determine whether hair cells could survive such treatment and subsequently be repaired. In organ cultures of the bullfrog saccule a combination of time-lapse video microscopy, two-photon microscopy, electron microscopy, and immunocytochemistry showed that hair cells can lose their hair bundle and survive as bundleless cells for at least 1 week. Time-lapse and electron microscopy revealed stages in the separation of the bundle from the cell body. Scanning electron microscopy (SEM) of cultures fixed 2, 4, and 7 days after antibiotic treatment showed that numerous new hair bundles were produced between 4 and 7 days of culture. Further examination revealed hair cells with small repaired hair bundles alongside damaged remnants of larger surviving bundles. The results indicate that sensory hair cells can undergo intracellular self-repair in the absence of mitosis, offering new possibilities for functional hair cell recovery and an explanation for non-proliferative recovery.     

Multifrequency forcing of a Hopf oscillator model of the inner ear

In response to a sound stimulus, the inner ear emits sounds called otoacoustic emissions. While the exact mechanism for the production of otoacoustic emissions is not known, active motion of individual hair cells is thought to play a role. Two possible sources for otoacoustic emissions, both localized within individual hair cells, include somatic motility and hair bundle motility. Because physiological models of each of these systems are thought to be poised near a Hopf bifurcation, the dynamics of each can be described by the normal form for a system near a Hopf bifurcation. Here we demonstrate that experimental results from three-frequency suppression experiments can be predicted based on the response of an array of noninteracting Hopf oscillators tuned at different frequencies. This supports the idea that active motion of individual hair cells contributes to active processing of sounds in the ear. Interestingly, the model suggests an explanation for differing results recorded in mammals and nonmammals.     

Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule

Our senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Millions of people suffer from hearing and balance deficits caused by damage to hair cells as a result of exposure to noise, aminoglycoside antibiotics, and antitumor drugs. In some species such damage can be reversed through the production of new cells. This proliferative response is limited in mammals but it has been hypothesized that damaged hair cells might survive and undergo intracellular repair. We examined the fate of bullfrog saccular hair cells after exposure to a low dose of the aminoglycoside antibiotic gentamicin to determine whether hair cells could survive such treatment and subsequently be repaired. In organ cultures of the bullfrog saccule a combination of time‐lapse video microscopy, two‐photon microscopy, electron microscopy, and immunocytochemistry showed that hair cells can lose their hair bundle and survive as bundleless cells for at least 1 week. Time‐lapse and electron microscopy revealed stages in the separation of the bundle from the cell body. Scanning electron microscopy (SEM) of cultures fixed 2, 4, and 7 days after antibiotic treatment showed that numerous new hair bundles were produced between 4 and 7 days of culture. Further examination revealed hair cells with small repaired hair bundles alongside damaged remnants of larger surviving bundles. The results indicate that sensory hair cells can undergo intracellular self‐repair in the absence of mitosis, offering new possibilities for functional hair cell recovery and an explanation for non‐proliferative recovery. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 81–92, 2002; DOI 10.1002/neu.10002     

Sensory interaction in the snailHelix lucorum

The activity of hair cells of statocysts inHelix lucorum was investigated by means of intra- and extracellular recording, applying appropriate stimulation of the organs of balance, optic photoreceptors, and the chemoreceptors of the optic tentacle bulb. Mechanical stimulation of the statocysts evoked a firing reaction in the hair cells as a result of generator potentials occurring at the receptors. The amplitude of generator potentials was proportional to the intensity of the reaction. Stimulating the optic photoreceptors by switching on a light produced a spike response in the hair cells with a short latency of 0.3–2 sec. The latent period of this response was inversely proportional to the intensity of the light. Appropriate stimulation of the chemoreceptors of the optic tentacle bulb caused a faint spike response with a long latent period of 20–40 sec in the hair cells. Illumination and stimulation of the chemoreceptors produced an inhibitory response in the form of bursts of IPSP in 2 out of more than 50 hair cells.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 17–26, January–February, 1986.     

Responses of hair cells to statocyst rotation

A new technique is described for stimulating hair cells of the Hermissenda statocyst. The preparation and recording apparatus can be rotated at up to 78 rpm while recording intracellular potentials. Hair cells in front of the centrifugal force vector depolarize in response to rotation. Hair cells in back of the centrifugal force vector hypoerpolarize in response to rotation. Mechanisms by which the hair cell generator potential might arise are examined.     

The responses of primary and secondary sensory hair cells in the squid statocyst to mechanical stimulation

Summary Intracellular recordings were obtained from primary and secondary sensory hair cells in the anterior transverse crista segment of the squid (Alloteuthis subulata) statocyst during imposed displacements of the overlying cupula. The secondary sensory hair cells were depolarized by ventral movements of the cupula and hyperpolarized by dorsal cupula movements. The displacement/response curve was asymmetric around the zero position and sigmoidal in shape, similar to that already described for vertebrate hair cells. The cells are estimated to have a sensitivity of at least 0.5 mV per degree angle of cilia displacement. The responses showed pronounced adaptation and could be blocked by bath applied alcohols, such as heptanol or octanol, or by high concentrations of aminoglycosides.The primary sensory hair cells were depolarized by dorsal movements of the cupula, usually responding with a burst of action potentials. The displacement/response curve was also sigmoidal in shape and the firing pattern showed strong adaptation to maintained displacements of the cupula.The cupula itself appeared to be irregular in shape, extending much further into the statocyst cavity in its central part than at its edges. This is likely to result in differences in the responses of the underlying hair cells along the length of the crista ridge.     

Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen

The lowermost portion of the resting (telogen) follicle consists of the bulge and secondary hair germ. We previously showed that the progeny of stem cells in the bulge form the lower follicle and hair, but the relationship of the bulge cells with the secondary hair germ cells, which are also involved in the generation of the new hair at the onset of the hair growth cycle (anagen), remains unclear. Here we address whether secondary hair germ cells are derived directly from epithelial stem cells in the adjacent bulge or whether they arise from cells within the lower follicle that survive the degenerative phase of the hair cycle (catagen). We use 5-bromo-2'-deoxyuridine to label bulge cells at anagen onset, and demonstrate that the lowermost portion of the bulge collapses around the hair and forms the secondary hair germ during late catagen. During the first six days of anagen onset bulge cells proliferate and self-renew. Bulge cell proliferation at this time also generates cells that form the future secondary germ. As bulge cells form the secondary germ cells at the end of catagen, they lose expression of a biochemical marker, S100A6. Remarkably, however, following injury of bulge cells by hair depilation, progenitor cells in the secondary hair germ repopulate the bulge and re-express bulge cell markers. These findings support the notion that keratinocytes can "dedifferentiate" to a stem cell state in response to wounding, perhaps related to signals from the stem cell niche. Finally, we also present evidence that quiescent bulge cells undergo apoptosis during follicle remodeling in catagen, indicating that a subpopulation of bulge cells is not permanent.     

Involvement of follicular stem cells in forming not only the follicle but also the epidermis

The location of follicular and epidermal stem cells in mammalian skin is a crucial issue in cutaneous biology. We demonstrate that hair follicular stem cells, located in the bulge region, can give rise to several cell types of the hair follicle as well as upper follicular cells. Moreover, we devised a double-label technique to show that upper follicular keratinocytes emigrate into the epidermis in normal newborn mouse skin, and in adult mouse skin in response to a penetrating wound. These findings indicate that the hair follicle represents a major repository of keratinocyte stem cells in mouse skin, and that follicular bulge stem cells are potentially bipotent as they can give rise to not only the hair follicle, but also the epidermis.     

Profiling drug-induced cell death pathways in the zebrafish lateral line

Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while d-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades.     

Notch Signaling Limits Supporting Cell Plasticity in the Hair Cell-Damaged Early Postnatal Murine Cochlea

In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.