Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss.

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

THOC1 deficiency leads to late-onset nonsyndromic hearing loss through p53-mediated hair cell apoptosis.

Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.

A humanized murine model, demonstrating dominant progressive hearing loss caused by a novel KCNQ4 mutation (p.G228D) from a large Chinese family.

The pathogenic variants in KCNQ4 cause DFNA2 nonsyndromic hearing loss. However, the understanding of genotype-phenotype correlations between KCNQ4 and hearing is limited. Here, we identified a novel KCNQ4 mutation p.G228D from a Chinese family, including heterozygotes characterized by high-frequency hearing loss that is progressive across all frequencies and homozygotes with more severe hearing loss. We constructed a novel murine model with humanized homologous Kcnq4 mutation. The heterozygotes had mid-frequency and high-frequency hearing loss at 4 weeks, and moved toward all frequencies hearing loss at 12 weeks, while the homozygotes had severe-to-profound hearing loss at 8 weeks. The degeneration of outer hair cells (OHCs) was observed from basal to apical turn of cochlea. The reduced K+ currents and depolarized resting potentials were revealed in OHCs. Remarkably, we observed the loss of inner hair cells (IHCs) in the region corresponding to the frequency above 32 kHz at 8-12 weeks. The results suggest the degeneration of OHCs and IHCs may contribute to high-frequency hearing loss in DFNA2 over time. Our findings broaden the variants of KCNQ4 and provide a novel mouse model of progressive hearing loss, which contributes to an understanding of pathogenic mechanism and eventually treatment of DFNA2 progressive hearing loss.

Tight Junctions in the Auditory System: Structure, Distribution and Function.

Tight junctions act as a barrier between epithelial cells to limit the transport of the paracellular substance, which is a required function in various tissues to sequestrate diverse microenvironments and maintain a normal physiological state. Tight junctions are complexes that contain various proteins, like transmembrane proteins, scaffolding proteins, signaling proteins, etc. Defects in those tight junction- related proteins can lead to hearing loss in humans which is also recapitulated in many model organisms. The disruption of the barrier between the endolymph and perilymph caused by tight junction abnormalities will affect the microenvironment of hair cells; and this could be the reason for this type of hearing loss. Besides their functions as a typical barrier and channel, tight junctions are also involved in many signaling networks to regulate gene expression, cell proliferation, and differentiation. This review will summarize the structures, localization, and related signaling pathways of hearingrelated tight junction proteins and their potential contributions to the hearing disorder.

ftr82 is necessary for hair cell morphogenesis and auditory function during zebrafish development.

Damages of sensory hair cells (HCs) are mainly responsible for sensorineural hearing loss, while the pathological mechanism remains not fully understood due to the many potential deafness genes unidentified. ftr82, a member of the largely TRIMs family in fish, has been found specifically expressed in the otic vesicle while its function is still unclear. Here, we investigate the roles of ftr82 in HC development and hearing function utilizing the zebrafish model. The results of in situ hybridization illustrate that ftr82 is always restricted to localize in otic vesicles at different stages. The defects of HCs are observed both in ftr82 morphants and mutants, including significantly decreased crista HCs, shortened cilia as well as remarkably reduced functional HCs in neuromasts, which could be successfully rescued by co-injection of exogenous ftr82 mRNA. The behavior assay of startle response indicates that larvae lacking of ftr82 exhibits lower sensitivity to external sound stimuli. Further research reveals that the loss of HCs is mainly caused by cell apoptosis mediated by caspase-3 activation. Our study demonstrates that ftr82 is a crucial hearing-related gene that regulates the HC morphogenesis and auditory function performing, which provides new insight into the rapid identification of the deafness gene.

The Roles of Solute Carriers in Auditory Function.

Solute carriers (SLCs) are important transmembrane transporters with members organized into 65 families. They play crucial roles in transporting many important molecules, such as ions and some metabolites, across the membrane, maintaining cellular homeostasis. SLCs also play important roles in hearing. It has been found that mutations in some SLC members are associated with hearing loss. In this review, we summarize SLC family genes related with hearing dysfunction to reveal the vital roles of these transporters in auditory function. This summary could help us understand the auditory physiology and the mechanisms of hearing loss and further guide future studies of deafness gene identification.

Dual-Specificity Phosphatase 14 Regulates Zebrafish Hair Cell Formation Through Activation of p38 Signaling Pathway.

Most cases of acquired hearing loss are due to degeneration and subsequent loss of cochlear hair cells. Whereas mammalian hair cells are not replaced when lost, in zebrafish, they constantly renew and regenerate after injury. However, the molecular mechanism among this difference remains unknown. Dual-specificity phosphatase 14 (DUSP14) is an important negative modulator of mitogen-activated protein kinase (MAPK) signaling pathways. Our study was to investigate the effects of DUSP14 on supporting cell development and hair cell regeneration and explore the potential mechanism. Our results showed that dusp14 gene is highly expressed in zebrafish developing neuromasts and otic vesicles. Behavior analysis showed that dusp14 deficiency resulted in hearing defects in zebrafish larvae, which were reversed by dusp14 mRNA treatment. Moreover, knockdown of dusp14 gene caused a significant decrease in the number of neuromasts and hair cells in both neuromast and otic vesicle, mainly due to the inhibition of the proliferation of supporting cells, which results in a decrease in the number of supporting cells and ultimately in the regeneration of hair cells. We further found significant changes in a series of MAPK pathway genes through transcriptome sequencing analysis of dusp14-deficient zebrafish, especially mapk12b gene in p38 signaling. Additionally, inhibiting p38 signaling effectively rescued all phenotypes caused by dusp14 deficiency, including hair cell and supporting cell reduction. These results suggest that DUSP14 might be a key gene to regulate supporting cell development and hair cell regeneration and is a potential target for the treatment of hearing loss.

Interferon regulatory factor-7 is required for hair cell development during zebrafish embryogenesis.

Interferon regulatory factor-7 (IRF7) is an essential regulator of both innate and adaptive immunity. It is also expressed in the otic vesicle of zebrafish embryos. However, any role for irf7 in hair cell development was uncharacterized. Does it work as a potential deaf gene to regulate hair cell development? We used whole-mount in situ hybridization (WISH) assay and morpholino-mediated gene knockdown method to investigate the role of irf7 in the development of otic vesicle hair cells during zebrafish embryogenesis. We performed RNA sequencing to gain a detailed insight into the molecules/genes which are altered upon downregulation of irf7. Compared to the wild-type siblings, knockdown of irf7 resulted in severe developmental retardation in zebrafish embryos as well as loss of neuromasts and damage to hair cells at an early stage (within 3 days post fertilization). Coinjection of zebrafish irf7 mRNA could partially rescued the defects of the morphants. atp1b2b mRNA injection can also partially rescue the phenotype induced by irf7 gene deficiency. Loss of hair cells in irf7-morphants does not result from cell apoptosis. Gene expression profiles show that, compared to wild-type, knockdown of irf7 can lead to 2053 and 2678 genes being upregulated and downregulated, respectively. Among them, 18 genes were annotated to hair cell (HC) development or posterior lateral line (PLL) development. All results suggest that irf7 plays an essential role in hair cell development in zebrafish, indicating that irf7 may be a member of deafness gene family.

The slc4a2b gene is required for hair cell development in zebrafish.

Hair cells (HCs) function as important sensory receptors that can detect movement in their immediate environment. HCs in the inner ear can sense acoustic signals, while in aquatic vertebrates HCs can also detect movements, vibrations, and pressure gradients in the surrounding water. Many genes are responsible for the development of HCs, and developmental defects in HCs can lead to hearing loss and other sensory dysfunctions. Here, we found that the solute carrier family 4, member 2b (slc4a2b) gene, which is a member of the anion-exchange family, is expressed in the otic vesicles and lateral line neuromasts in developing zebrafish embryos. An in silico analysis showed that the slc4a2b is evolutionarily conserved, and we found that loss of function of slc4a2b resulted in a decreased number of HCs in zebrafish neuromasts due to increased HC apoptosis. Taken together, we conclude that slc4a2b plays a critical role in the development of HCs in zebrafish.

Insm1a Is Required for Zebrafish Posterior Lateral Line Development.

Insulinoma-associated 1 (Insm1), a zinc-finger transcription factor, is widely expressed in the developing nervous system and plays important roles in cell cycle progression and cell fate specification. However, the functions of Insm1 in the embryonic development of the sensory system and its underlying molecular mechanisms remain largely unexplored. Here, through whole-mount in situ hybridization, we found that the zebrafish insm1a gene was expressed in the posterior lateral line (pLL) system, including both the migrating pLL primordium and the deposited neuromast cells. In order to decipher the specific roles of insm1a in zebrafish pLL development, we inhibited insm1a expression by using a morpholino knockdown strategy. The insm1a morphants exhibited primordium migration defects that resulted in reduced numbers of neuromasts. The inactivation of insm1a reduced the numbers of hair cells in neuromasts, and this defect could be a secondary consequence of disrupting rosette formation in the pLL primordium. Additionally, we showed that insm1a knockdown decreased the proliferation of pLL primordium cells, which likely contributed to these pLL defects. Furthermore, we showed that loss of insm1a resulted in elevated Wnt/ß-catenin signaling and downregulation of Fgf target genes in the primordium. Insm1a knockdown also perturbed the expression patterns of chemokine signaling genes. Taken together, this study reveals a pivotal role for Insm1a in regulating pLL development during zebrafish embryogenesis.

Insm1a Regulates Motor Neuron Development in Zebrafish.

Insulinoma-associated1a (insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences that insm1a was a vital regulator of motor neuron development, and provided a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization of insm1a in spinal cord, and primary motor neurons (PMNs) of zebrafish embryos by in situ hybridization, and imaging analysis of transgenic reporter line Tg(insm1a: mCherry)ntu805 . Then we demonstrated that the deficiency of insm1a in zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP), and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout of insm1 impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed that swimming activity was significantly reduced in the insm1a-null zebrafish. Furthermore, we showed that the insm1a loss of function significantly decreased the transcript levels of both olig2 and nkx6.1. Microinjection of olig2 and nkx6.1 mRNA rescued the motor neuron defects in insm1a deficient embryos. Taken together, these data indicated that insm1a regulated the motor neuron development, at least in part, through modulation of the expressions of olig2 and nkx6.1.

c-Myb knockdown increases the neomycin-induced damage to hair-cell-like HEI-OC1 cells in vitro.

c-Myb is a transcription factor that plays a key role in cell proliferation, differentiation, and apoptosis. It has been reported that c-Myb is expressed within the chicken otic placode, but whether c-Myb exists in the mammalian cochlea, and how it exerts its effects, has not been explored yet. Here, we investigated the expression of c-Myb in the postnatal mouse cochlea and HEI-OC1 cells and found that c-Myb was expressed in the hair cells (HCs) of mouse cochlea as well as in cultured HEI-OC1 cells. Next, we demonstrated that c-Myb expression was decreased in response to neomycin treatment in both cochlear HCs and HEI-OC1 cells, suggesting an otoprotective role for c-Myb. We then knocked down c-Myb expression with shRNA transfection in HEI-OC1 cells and found that c-Myb knockdown decreased cell viability, increased expression of pro-apoptotic factors, and enhanced cell apoptosis after neomycin insult. Mechanistic studies revealed that c-Myb knockdown increased cellular levels of reactive oxygen species and decreased Bcl-2 expression, both of which are likely to be responsible for the increased sensitivity of c-Myb knockdown cells to neomycin. This study provides evidence that c-Myb might serve as a new target for the prevention of aminoglycoside-induced HC loss.

Effects of the prolonged vertical tube on the separation performance of a cyclone.

This article aims at the gas flow into the dustbin of conventional cyclones, the prolonged cyclone (attaching a vertical tube at the bottom of the dust outlet) is proposed by some researchers, which can make flow with dust enter into the tube and separate further. The Reynolds stress transport model (RSTM) has been employed to predict the gas flow fields of the conventional and prolonged cyclones. The tangential velocity, axial velocity profiles and turbulent kinetic energy profiles are presented, and the downward flow rates into the dustbin of the three cyclones are compared. The separation performances of these three cyclones are tested. The result indicates that the tangential velocity, axial velocity and turbulent kinetic energy in the dustbin reduce greatly when the prolonged vertical tube attaching into the dust outlet, which can avoid the re-entrainment of already separated dust effectively. Furthermore, the prolonged vertical tube increases the separation space of dusts. The downward flow rate into the dustbin of the prolonged cyclone decreases compared with the conventional cyclone. The experimental results show that the prolonged vertical tube can improve the separation efficiency by a slightly increased pressure drop. However, for an even longer tube, the separation efficiency is slightly reduced. Thus, there is an optimal tube length for a given cyclone.

Reduced TRMU expression increases the sensitivity of hair-cell-like HEI-OC-1 cells to neomycin damage in vitro.

Aminoglycosides are ototoxic to the cochlear hair cells, and mitochondrial dysfunction is one of the major mechanisms behind ototoxic drug-induced hair cell death. TRMU (tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase) is a mitochondrial protein that participates in mitochondrial tRNA modifications, but the role of TRMU in aminoglycoside-induced ototoxicity remains to be elucidated. In this study, we took advantage of the HEI-OC-1 cell line to investigate the role of TRMU in aminoglycoside-induced cell death. We found that TRMU is expressed in both hair cells and HEI-OC-1 cells, and its expression is significantly decreased after 24 h neomycin treatment. We then downregulated TRMU expression with siRNA and found that cell death and apoptosis were significantly increased after neomycin injury. Furthermore, when we down-regulated TRMU expression, we observed significantly increased mitochondrial dysfunction and increased levels of reactive oxygen species (ROS) after neomycin injury, suggesting that TRMU regulates mitochondrial function and ROS levels. Lastly, the antioxidant N-acetylcysteine rescued the mitochondrial dysfunction and cell apoptosis that was induced by TRMU downregulation, suggesting that ROS accumulation contributed to the increased aminoglycosides sensitivity of HEI-OC-1 cells after TRMU downregulation. This study provides evidence that TRMU might be a new therapeutic target for the prevention of aminoglycoside-induced hair cell death.

Accelerating bioelectric functional development of neural stem cells by graphene coupling: Implications for neural interfacing with conductive materials.

In order to govern cell-specific behaviors in tissue engineering for neural repair and regeneration, a better understanding of material-cell interactions, especially the bioelectric functions, is extremely important. Graphene has been reported to be a potential candidate for use as a scaffold and neural interfacing material. However, the bioelectric evolvement of cell membranes on these conductive graphene substrates remains largely uninvestigated. In this study, we used a neural stem cell (NSC) model to explore the possible changes in membrane bioelectric properties - including resting membrane potentials and action potentials - and cell behaviors on graphene films under both proliferation and differentiation conditions. We used a combination of single-cell electrophysiological recordings and traditional cell biology techniques. Graphene did not affect the basic membrane electrical parameters (capacitance and input resistance), but resting membrane potentials of cells on graphene substrates were more strongly negative under both proliferation and differentiation conditions. Also, NSCs and their progeny on graphene substrates exhibited increased firing of action potentials during development compared to controls. However, graphene only slightly affected the electric characterizations of mature NSC progeny. The modulation of passive and active bioelectric properties on the graphene substrate was accompanied by enhanced NSC differentiation. Furthermore, spine density, synapse proteins expressions and synaptic activity were all increased in graphene group. Modeling of the electric field on conductive graphene substrates suggests that the electric field produced by the electronegative cell membrane is much higher on graphene substrates than that on control, and this might explain the observed changes of bioelectric development by graphene coupling. Our results indicate that graphene is able to accelerate NSC maturation during development, especially with regard to bioelectric evolvement. Our findings provide a fundamental understanding of the role of conductive materials in tuning the membrane bioelectric properties in a graphene model and pave the way for future studies on the development of methods and materials for manipulating membrane properties in a controllable way for NSC-based therapies.

Inhibition of ARC decreases the survival of HEI-OC-1 cells after neomycin damage in vitro.

Hearing loss is a common sensory disorder mainly caused by the loss of hair cells (HCs). Noise, aging, and ototoxic drugs can all induce apoptosis in HCs. Apoptosis repressor with caspase recruitment domain(ARC) is a key factor in apoptosis that inhibits both intrinsic and extrinsic apoptosis pathways; however, there have been no reports on the role of ARC in HC loss in the inner ear. In this study, we used House Ear Institute Organ of Corti 1 (HEI-OC-1) cells, which is a cochlear hair-cell-like cell line, to investigate the role of ARC in aminoglycoside-induced HC loss. ARC was expressed in the cochlear HCs as well as in the HEI-OC-1 cells, but not in the supporting cells, and the expression level of ARC in HCs was decreased after neomycin injury in both cochlear HCs and HEI-OC-1 cells, suggesting that reduced levels of ARC might correlate with neomycin-induced HC loss. We inhibited ARC expression using siRNA and found that this significantly increased the sensitivity of HEI-OC-1 cells to neomycin toxicity. Finally, we found that ARC inhibition increased the expression of pro-apoptotic factors, decreased the mitochondrial membrane potential, and increased the level of reactive oxygen species (ROS) after neomycin injury, suggesting that ARC inhibits cell death and apoptosis in HEI-OC-1 cells by controlling mitochondrial function and ROS accumulation. Thus the endogenous anti-apoptotic factor ARC might be a new therapeutic target for the prevention of aminoglycoside-induced HC loss.

A novel analgesic approach to optogenetically and specifically inhibit pain transmission using TRPV1 promoter.

Chronic pain is a pathological condition that results in significant loss of life quality, but so far no specific treatment for chronic pain has been developed. Currently available analgesia drugs are either not specific enough or have severe side effects. Therefore a non-invasive approach with high specificity to inhibit nociception becomes essential. In this study, a recombinant virus (AAV5-TRPV1-ArchT-eGFP) was constructed and injected into the mouse dorsal root ganglion (DRG). The Transient Receptor Potential Vanilloid type 1 (TRPV1) channel promoter was used to selectively express inhibitory light-sensitive pump ArchT (the archaerhodopsin from Halorubrum strain TP009) in nociceptive DRG neurons. The successful transfer of ArchT gene was confirmed by a robust expression of green florescent protein in the DRG neurons. In vivo behavioral tests demonstrated that both the mechanical paw withdrawal threshold and the radiant heat evoked paw withdrawal latency were significantly increased upon illumination by a 532 nm green laser light to the paw of a viral-vector injected mice, while the same laser light did not induce any observable change in naïve mice. In conclusion, we have established a novel analgesic approach that can noninvasively and selectively inhibit pain transmission using an acute and controllable optogenetics method. This study may shed light on the application of a novel optogenetic strategy for the treatment of pain.

Study of the filtration performance of a plain wave fabric filter using response surface methodology.

The gas-solid two-phase flows in the plain wave fabric filter were simulated by computational fluid dynamics (CFD) technology, and the warps and wefts of the fabric filter were made of filaments with different dimensions. The numerical solutions were carried out using commercial computational fluid dynamics (CFD) code Fluent 6.1. The filtration performances of the plain wave fabric filter with different geometry parameters and operating condition, including the horizontal distance, the vertical distance and the face velocity were calculated. The effects of geometry parameters and operating condition on filtration efficiency and pressure drop were studied using response surface methodology (RSM) by means of the statistical software (Minitab V14), and two second-order polynomial models were obtained with regard to the effect of the three factors as stated above. Moreover, the models were modified by dismissing the insignificant terms. The results show that the horizontal distance, vertical distance and the face velocity all play an important role in influencing the filtration efficiency and pressure drop of the plane wave fabric filters. The horizontal distance of 3.8 times the fiber diameter, the vertical distance of 4.0 times the fiber diameter and Reynolds number of 0.98 are found to be the optimal conditions to achieve the highest filtration efficiency at the same face velocity, while maintaining an acceptable pressure drop.