Cell Support Research Articles

Cytoskeletal Stability in the Auditory OrganIn Vivo: RhoA Is Dispensable for Wound Healing but Essential for Hair Cell Development

Wound healing in the inner ear sensory epithelia is performed by the apical domains of supporting cells (SCs). Junctional F-actin belts of SCs are thin during development but become exceptionally thick during maturation. The functional significance of the thick belts is not fully understood. We have studied the role of F-actin belts during wound healing in the developing and adult cochlea of mice in vivo. We show that the thick belts serve as intracellular scaffolds that preserve the positions of surviving cells in the cochlear sensory epithelium. Junctions associated with the thick F-actin belts did not readily disassemble during wound healing. To compensate for this, basolateral membranes of SCs participated in the closure of surface breach. Because not only neighboring but also distant SCs contributed to wound healing by basolateral protrusions, this event appears to be triggered by contact-independent diffusible signals. In the search for regulators of wound healing, we inactivated RhoA in SCs, which, however, did not limit wound healing. RhoA inactivation in developing outer hair cells (OHCs) caused myosin II delocalization from the perijunctional domain and apical cell-surface enlargement. These abnormalities led to the extrusion of OHCs from the epithelium. These results demonstrate the importance of stability of the apical domain, both in wound repair by SCs and in development of OHCs, and that only this latter function is regulated by RhoA. Because the correct cytoarchitecture of the cochlear sensory epithelium is required for normal hearing, the stability of cell apices should be maintained in regenerative and protective interventions.

Non-autonomous Cellular Responses to Ototoxic Drug-Induced Stress and Death.

The first major recognition of drug-induced hearing loss can be traced back more than seven decades to the development of streptomycin as an antimicrobial agent. Since then at least 130 therapeutic drugs have been recognized as having ototoxic side-effects. Two important classes of ototoxic drugs are the aminoglycoside antibiotics and the platinum-based antineoplastic agents. These drugs save the lives of millions of people worldwide, but they also cause irreparable hearing loss. In the inner ear, sensory hair cells (HCs) and spiral ganglion neurons (SGNs) are important cellular targets of these drugs, and most mechanistic studies have focused on the cell-autonomous responses of these cell types in response to ototoxic stress. Despite several decades of studies on ototoxicity, important unanswered questions remain, including the cellular and molecular mechanisms that determine whether HCs and SGNs will live or die when confronted with ototoxic challenge. Emerging evidence indicates that other cell types in the inner ear can act as mediators of survival or death of sensory cells and SGNs. For example, glia-like supporting cells (SCs) can promote survival of both HCs and SGNs. Alternatively, SCs can act to promote HC death and inhibit neural fiber expansion. Similarly, tissue resident macrophages activate either pro-survival or pro-death signaling that can influence HC survival after exposure to ototoxic agents. Together these data indicate that autonomous responses that occur within a stressed HC or SGN are not the only (and possibly not the primary) determinants of whether the stressed cell ultimately lives or dies. Instead non-cell-autonomous responses are emerging as significant determinants of HC and SGN survival vs. death in the face of ototoxic stress. The goal of this review is to summarize the current evidence on non-cell-autonomous responses to ototoxic stress and to discuss ways in which this knowledge may advance the development of therapies to reduce hearing loss caused by these drugs.

Apoptosis pattern and alterations of expression of apoptosis-related factors of supporting cells in Kölliker's organ in vivo in early stage after birth in rats.

Kolliker’s organ is a temporary but indispensable structure in the development of the cochlea. Supporting cells (SCs) within it release adenosine 5’-triphosphate (ATP), which may play a crucial role in cochlear development before the onset of hearing. To reveal the apoptosis of Kolliker’s organ in new-born rats, we studied the morphological changes and expression of apoptosis-related factors during early postnatal development. We found SCs in Kolliker’s organ decreased in number and changed in appearance along the cochlea apex-to-base gradient, and the expression of caspase-3, caspase-8, caspase- 9 and bcl-2 in Kolliker’s organ of the cochlea fluctuated along the course of postnatal development, with an expression peak at postnatal day 3. This study demonstrates a time-dependent degeneration of Kolliker’s organ during postnatal cochlea development, which might be triggered by endogenous factors.

Characterization of Lgr5+ Progenitor Cell Transcriptomes after Neomycin Injury in the Neonatal Mouse Cochlea

Lgr5+ supporting cells (SCs) are enriched hair cell (HC) progenitors in the cochlea. Both in vitro and in vivo studies have shown that HC injury can spontaneously activate Lgr5+ progenitors to regenerate HCs in the neonatal mouse cochlea. Promoting HC regeneration requires the understanding of the mechanism of HC regeneration, and this requires knowledge of the key genes involved in HC injury-induced self-repair responses that promote the proliferation and differentiation of Lgr5+ progenitors. Here, as expected, we found that neomycin-treated Lgr5+ progenitors (NLPs) had significantly greater HC regeneration ability, and greater but not significant proliferation ability compared to untreated Lgr5+ progenitors (ULPs) in response to neomycin exposure. Next, we used RNA-seq analysis to determine the differences in the gene-expression profiles between the transcriptomes of NLPs and ULPs from the neonatal mouse cochlea. We first analyzed the genes that were enriched and differentially expressed in NLPs and ULPs and then analyzed the cell cycle genes, the transcription factors, and the signaling pathway genes that might regulate the proliferation and differentiation of Lgr5+ progenitors. We found 9 cell cycle genes, 88 transcription factors, 8 microRNAs, and 16 cell-signaling pathway genes that were significantly upregulated or downregulated after neomycin injury in NLPs. Lastly, we constructed a protein-protein interaction network to show the interaction and connections of genes that are differentially expressed in NLPs and ULPs. This study has identified the genes that might regulate the proliferation and HC regeneration of Lgr5+ progenitors after neomycin injury, and investigations into the roles and mechanisms of these genes in the cochlea should be performed in the future to identify potential therapeutic targets for HC regeneration.

Characterization of the Transcriptomes of Lgr5+ Hair Cell Progenitors and Lgr5- Supporting Cells in the Mouse Cochlea.

Cochlear supporting cells (SCs) have been shown to be a promising resource for hair cell (HC) regeneration in the neonatal mouse cochlea. Previous studies have reported that Lgr5+ SCs can regenerate HCs both in vitro and in vivo and thus are considered to be inner ear progenitor cells. Lgr5+ progenitors are able to regenerate more HCs than Lgr5- SCs, and it is important to understand the mechanism behind the proliferation and HC regeneration of these progenitors. Here, we isolated Lgr5+ progenitors and Lgr5- SCs from Lgr5-EGFP-CreERT2/Sox2-CreERT2/Rosa26-tdTomato mice via flow cytometry. As expected, we found that Lgr5+ progenitors had significantly higher proliferation and HC regeneration ability than Lgr5- SCs. Next, we performed RNA-Seq to determine the gene expression profiles of Lgr5+ progenitors and Lgr5- SCs. We analyzed the genes that were enriched and differentially expressed in Lgr5+ progenitors and Lgr5- SCs, and we found 8 cell cycle genes, 9 transcription factors, and 24 cell signaling pathway genes that were uniquely expressed in one population but not the other. Last, we made a protein–protein interaction network to further analyze the role of these differentially expressed genes. In conclusion, we present a set of genes that might regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these might serve as potential new therapeutic targets for HC regeneration.

Netrin 1 mediates protective effects exerted by insulin-like growth factor 1 on cochlear hair cells

Sensorineural hearing loss (SNHL) is mainly caused by the damage of cochlear hair cells (HCs). As HCs and supporting cells (SCs) do not proliferate in postnatal mammals, the loss of HCs and SCs is irreversible, emphasizing the importance of preserving their numbers to prevent SNHL. It is known that insulin-like growth factor 1 (IGF1) is instrumental in the treatment of SNHL. Our previous study indicates that IGF1 protects HCs against aminoglycoside by activating IGF1 receptor and its two major downstream pathways, PI3K/AKT and MEK/ERK, in SCs, which results in the upregulation of the expression of the Netrin1-encoding gene (Ntn1). However, the mechanisms underlying IGF1-induced protection of HCs via SC activation as well as the role of NTN1 in this process have not been elucidated. Here, we demonstrated that NTN1, similar to IGF1, promoted HC survival. NTN1 blocking antibody attenuated IGF1-induced HC protection from aminoglycoside, indicating that NTN1 is the effector molecule of IGF1 signaling during HC protection. In situ hybridization demonstrated that IGF1 potently induced Ntn1 expression in SCs. NTN1 receptors were abundantly expressed in the cochlea; among them, UNC5B mediated IGF1 protective effects on HCs, as NTN1 binding to UNC5B inhibited HC apoptosis. These results provide new insights into the mechanisms underlying IGF1 protection of cochlear HCs, suggesting a possibility of using NTN1 as a new treatment for SNHL.

Allicin protects auditory hair cells and spiral ganglion neurons from cisplatin - Induced apoptosis

Cisplatin is a broad-spectrum anticancer drug that is commonly used in the clinic. Ototoxicity is one of the major side effects of this drug, which caused irreversible sensorineural hearing loss. Allicin, the main biologically active compound derived from garlic, has been shown to exert various anti-apoptotic and anti-oxidative activities in vitro and in vivo studies. We took advantage of C57 mice intraperitoneally injected with cisplatin alone or with cisplatin and allicin combined, to investigate whether allicin plays a protective role in vivo against cisplatin ototoxicity. The result showed that C57 mice in cisplatin group exhibited increased shift in auditory brainstem response, whereas the auditory fuction of mice in allicin + cisplatin group was protected in most frequencies, which was accordance with observed damages of outer hair cells (OHCs) and spiral ganglion neurons (SGNs) in the cochlea. Allicin markedly protected SGN mitochondria from damage and releasing cytochrome c, and significantly reduced pro-apoptosis factor expressions activated by cisplatin, including Bax, cleaved-caspase-9, cleaved-caspase-3and p53. Furthermore, allicin reduced the level of Malondialdehyde (MDA), but increased the level of superoxide dismutase (SOD). All data suggested that allicin could prevent hearing loss induced by cisplatin effectively, of which allicin protected SGNs from apoptosis via mitochondrial pathway while protected OHCs and supporting cells (SCs) from apoptosis through p53 pathway.

Ultrastructural study of the neural microcircuits in the sensory epithelium of the paratympanic organ of the chicken.

The paratympanic organ (PTO) is a sensory organ located in the medial wall of the tympanic cavity of birds. The organ looks like a small tapering vesicle, and is equipped with a sensory epithelium formed by supporting cells (SCs) and Type II hair cells (Type II-HCs). The function of the PTO has not yet been precisely defined. The prevailing current hypothesis is that the PTO assesses the air pressure exerted on the external surface of the tympanic membrane. The PTO could may thus function as a barometer and, in flying birds, also as an altimeter. The afferent synapses of the PTO of chicken were described in detail in a previous paper. Reciprocal synapses between efferent nerve endings (ENEs) and the HCs were also observed, suggesting the existence of local microcircuits. The aim of this work was to provide a more detailed ultrastructural description of these microcircuits in the PTO of chicken. We observed for the first time: (1) reciprocal synapses between the HCs and the afferent nerve endings (ANEs); (2) presence of two distinct types of ENEs; (3) reciprocal synapses between the HCs and both types of ENEs. Overall, these results indicate that a complex processing of the incoming sensory signals may occur in the PTO. This thus suggests that the PTO may perform more complex functions than those supposed until now. We hypothesize that the PTO could have a role in the low-frequency sound perception.

Time course of organ of Corti degeneration after noise exposure

From our permanent collection of plastic-embedded flat preparations of chinchilla cochleae, 22 controls and 199 ears from noise-exposed animals were used to determine when, postexposure, hair cell (HC) and supporting cell (SC) degeneration were completed. The exposed ears were divided into four groups based on exposure parameters: 0.5- or 4-kHz octave band of noise at moderate (M) or high (H) intensities. Postexposure survival ranged from <1 h to 2.5 y. Ears fixed ≤ 0–12 h postexposure were called 'acute'. For 'chronic' ears, postexposure survival was ≥7 d for groups 0.5M and 4M, ≥ 1 mo for the 4H group and ≥7 mo for the 0.5H group. The time course of inner-ear degeneration after noise exposure was determined from data in the 0.5H and 4H groups because these groups contained ears with intermediate survival times. Outer hair cells (OHCs) began dying during the exposure. OHC loss slowed down beyond 1 mo but was still present. Conversely, much inner hair cell loss was delayed until 1–3 wk postexposure. Outer pillar and inner pillar losses were present at a low level in acute ears but increased exponentially thereafter. These results are the first to demonstrate quantitatively that hair cells (HCs) and supporting cells (SCs) may continue to degenerate for months postexposure.With short postexposure survivals, the remaining SCs often had pathological changes, including: buckled pillar bodies, shifted Deiters' cell (DC) nuclei, detachment of DCs from the basilar membrane and/or splitting of the reticular lamina. These pathological changes appeared to allow endolymph and perilymph to intermix in the fluid spaces of the organ of Corti, damaging additional HCs, SCs and nerve fibers. This mechanism may account for some postexposure degeneration. In ears exposed to moderate noise, some of these SC changes appeared to be reversible. In ears exposed to high-level noise, these changes appeared to indicate impending degeneration.

Quantitative Analysis of Supporting Cell Subtype Labeling Among CreER Lines in the Neonatal Mouse Cochlea.

Four CreER lines that are commonly used in the auditory field to label cochlear supporting cells (SCs) are expressed in multiple SC subtypes, with some lines also showing reporter expression in hair cells (HCs). We hypothesized that altering the tamoxifen dose would modify CreER expression and target subsets of SCs. We also used two different reporter lines, ROSA26 tdTomato and CAG-eGFP, to achieve the same goal. Our results confirm previous reports that Sox2 CreERT2 and Fgfr3-iCreER T2 are not only expressed in neonatal SCs but also in HCs. Decreasing the tamoxifen dose did not reduce HC expression for Sox2 CreERT2 , but changing to the CAG-eGFP reporter decreased reporter-positive HCs sevenfold. However, there was also a significant decrease in the number of reporter-positive SCs. In contrast, there was a large reduction in reporter-positive HCs in Fgfr3-iCreER T2 mice with the lowest tamoxifen dose tested yet only limited reduction in SC labeling. The targeting of reporter expression to inner phalangeal and border cells was increased when Plp-CreER T2 was paired with the CAG-eGFP reporter; however, the total number of labeled cells decreased. Changes to the tamoxifen dose or reporter line with Prox1 CreERT2 caused minimal changes. Our data demonstrate that modifications to the tamoxifen dose or the use of different reporter lines may be successful in narrowing the numbers and/or types of cells labeled, but each CreER line responded differently. When the ROSA26 tdTomato reporter was combined with any of the four CreER lines, there was no difference in the number of tdTomato-positive cells after one or two injections of tamoxifen given at birth. Thus, tamoxifen-mediated toxicity could be reduced by only giving one injection. While the CAG-eGFP reporter consistently labeled fewer cells, both reporter lines are valuable depending on the goal of the study.

Wnt activation followed by Notch inhibition promotes mitotic hair cell regeneration in the postnatal mouse cochlea

Hair cell (HC) loss is the main cause of permanent hearing loss in mammals. Previous studies have reported that in neonatal mice cochleae, Wnt activation promotes supporting cell (SC) proliferation and Notch inhibition promotes the trans-differentiation of SCs into HCs. However, Wnt activation alone fails to regenerate significant amounts of new HCs, Notch inhibition alone regenerates the HCs at the cost of exhausting the SC population, which leads to the death of the newly regenerated HCs. Mitotic HC regeneration might preserve the SC number while regenerating the HCs, which could be a better approach for long-term HC regeneration. We present a two-step gene manipulation, Wnt activation followed by Notch inhibition, to accomplish mitotic regeneration of HCs while partially preserving the SC number. We show that Wnt activation followed by Notch inhibition strongly promotes the mitotic regeneration of new HCs in both normal and neomycin-damaged cochleae while partially preserving the SC number. Lineage tracing shows that the majority of the mitotically regenerated HCs are derived specifically from the Lgr5+ progenitors with or without HC damage. Our findings suggest that the co-regulation of Wnt and Notch signaling might provide a better approach to mitotically regenerate HCs from Lgr5+ progenitor cells.

Extensive Supporting Cell Proliferation and Mitotic Hair Cell Generation by In Vivo Genetic Reprogramming in the Neonatal Mouse Cochlea.

The generation of hair cells (HCs) from the differentiation of proliferating supporting cells (SCs) appears to be an ideal approach for replacing lost HCs in the cochlea and is promising for restoring hearing after damage to the organ of Corti. We show here that extensive proliferation of SCs followed by mitotic HC generation is achieved through a genetic reprogramming process involving the activation of β-catenin to upregulate Wnt signaling, the deletion of Notch1 to downregulate Notch signaling, and the overexpression of Atoh1 in Sox2(+) SCs in neonatal mouse cochleae. We used RNA sequencing to compare the transcripts of the cochleae from control mice and from mice with β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion in Sox2(+) SCs. We identified the genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. Moreover, the proliferation of SCs induced by Notch1 deletion disappears after deleting β-catenin in Notch1 knock-out Sox2(+) cells, which further demonstrates that Notch signaling is an upstream and negative regulator of Wnt signaling. We show here that the extensive proliferation of supporting cells (SCs) and the subsequent mitotic hair cell (HC) generation is achieved through a genetic reprogramming process involving activation of β-catenin to upregulate Wnt signaling, deletion of Notch1 to downregulate Notch signaling, and overexpression of Atoh1 in Sox2(+) SCs in neonatal mice cochleae. By comparing the transcripts of the cochleae among controls, β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion group, we identified multiple genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. This provides a better understanding of the mechanisms behind mitotic HC generation and might provide new approaches to stimulating mitotic HC regeneration.

Co-regulation of the Notch and Wnt signaling pathways promotes supporting cell proliferation and hair cell regeneration in mouse utricles.

This work sought to determine the crosstalk between the Notch and Wnt signaling pathways in regulating supporting cell (SC) proliferation and hair cell (HC) regeneration in mouse utricles. We cultured postnatal day (P)3 and P60 mouse utricles, damaged the HCs with gentamicin, and treated the utricles with the γ-secretase inhibitor DAPT to inhibit the Notch pathway and with the Wnt agonist QS11 to active the Wnt pathway. We also used Sox2-CreER, Notch1-flox (exon 1), and Catnb-flox (exon 3) transgenic mice to knock out the Notch pathway and activate the Wnt pathway in Sox2+ SCs. Notch inhibition alone increased SC proliferation and HC number in both undamaged and damaged utricles. Wnt activation alone promoted SC proliferation, but the HC number was not significantly increased. Here we demonstrated the cumulative effects of Notch inhibition and Wnt activation in regulating SC proliferation and HC regeneration. Simultaneously inhibiting Notch and overexpressing Wnt led to significantly greater SC proliferation and greater numbers of HCs than manipulating either pathway alone. Similar results were observed in the transgenic mice. This study suggests that the combination of Notch inhibition and Wnt activation can significantly promote SC proliferation and increase the number of regenerated HCs in mouse utricle.

Inhibition of H3K9me2 Reduces Hair Cell Regeneration after Hair Cell Loss in the Zebrafish Lateral Line by Down-Regulating the Wnt and Fgf Signaling Pathways.

The activation of neuromast (NM) supporting cell (SC) proliferation leads to hair cell (HC) regeneration in the zebrafish lateral line. Epigenetic mechanisms have been reported that regulate HC regeneration in the zebrafish lateral line, but the role of H3K9me2 in HC regeneration after HC loss remains poorly understood. In this study, we focused on the role of H3K9me2 in HC regeneration following neomycin-induced HC loss. To investigate the effects of H3K9me2 in HC regeneration, we took advantage of the G9a/GLP-specific inhibitor BIX01294 that significantly reduces the dimethylation of H3K9. We found that BIX01294 significantly reduced HC regeneration after neomycin-induced HC loss in the zebrafish lateral line. BIX01294 also significantly reduced the proliferation of NM cells and led to fewer SCs in the lateral line. In situ hybridization showed that BIX01294 significantly down-regulated the Wnt and Fgf signaling pathways, which resulted in reduced SC proliferation and HC regeneration in the NMs of the lateral line. Altogether, our results suggest that down-regulation of H3K9me2 significantly decreases HC regeneration after neomycin-induced HC loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus H3K9me2 plays a critical role in HC regeneration.

Gene Transfer into the Chicken Auditory Organ by &lt;em&gt;In Ovo&lt;/em&gt; Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hrafter electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Characterization of Lgr5+ progenitor cell transcriptomes in the apical and basal turns of the mouse cochlea.

Lgr5+ supporting cells (SCs) are enriched hair cell (HC) progenitors in the cochlea, and several studies have shown a difference in the proliferation and HC regeneration ability of SCs between the apical and basal turns. However, the detailed differences between the transcriptomes of the apical and basal Lgr5+ SCs have not yet been investigated. We found that when isolated by FACS, Lgr5+ cells from the apex generated significantly more HCs and had significantly higher proliferation and mitotic HC regeneration ability compared to those from the base. Next, we used microarray analysis to determine the transcriptome expression profiles of Lgr5+ progenitors from the apex and the base. We first analyzed the genes that were enriched and differentially expressed in Lgr5+ progenitors from the apex and the base. Then we analyzed the cell cycle genes and the transcription factors that might regulate the proliferation and differentiation of Lgr5+ progenitors. Lastly, to further analyze the role of differentially expressed genes and to gain an overall view of the gene network in cochlear HC regeneration, we created a protein-protein interaction network. Our datasets suggest the possible genes that might regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these genes might provide new therapeutic targets for HC regeneration in the future.

Synergistic action of nectins and cadherins generates the mosaic cellular pattern of the olfactory epithelium.

In the olfactory epithelium (OE), olfactory cells (OCs) and supporting cells (SCs), which express different cadherins, are arranged in a characteristic mosaic pattern in which OCs are enclosed by SCs. However, the mechanism underlying this cellular patterning is unclear. Here, we show that the cellular pattern of the OE is established by cellular rearrangements during development. In the OE, OCs express nectin-2 and N-cadherin, and SCs express nectin-2, nectin-3, E-cadherin, and N-cadherin. Heterophilic trans-interaction between nectin-2 on OCs and nectin-3 on SCs preferentially recruits cadherin via α-catenin to heterotypic junctions, and the differential distributions of cadherins between junctions promote cellular intercalations, resulting in the formation of the mosaic pattern. These observations are confirmed by model cell systems, and various cellular patterns are generated by the combinatorial expression of nectins and cadherins. Collectively, the synergistic action of nectins and cadherins generates mosaic pattern, which cannot be achieved by a single mechanism.

A novel combination of drug therapy to protect residual hearing post cochlear implant surgery

Conclusions A cocktail combining NAC, Mannitol, and Dexamethasone may be used to prevent loss of residual hearing post-implantation. There is a window of opportunity to treat the cochlea before the onset of cell death in HCs. Objective Inner ear trauma caused by cochlear implant electrode insertion trauma (EIT) initiates multiple molecular mechanisms in hair cells (HCs) or support cells (SCs), resulting in initiation of programmed cell death within the damaged tissues of the cochlea, which leads to loss of residual hearing. In earlier studies L-N-acetylcysteine (L-NAC), Mannitol, and dexamethasone have been shown independently to protect the HCs loss against different types of inner ear trauma. These three molecules have different otoprotective effects. The goal of this preliminary study is to test the efficacy of a combination of these molecules to enhance the otoprotection of HCs against EIT. Methods OC explants were dissected from P-3 rats and placed in serum-free media. Explants were divided into control and experimental groups. Control group: (1) untreated controls; (2) EIT. Experimental group: (1) EIT + L-NAC (5, 2, or 1 mM); (2) EIT + Mannitol (100, 50, or 10 mM); (3) EIT + Dex (20, 10, or 5 μg/mL); (4) EIT + L-NAC + Mannitol + Dex. After EIT was caused in an in-vitro model of CI, explants were cultured in media containing L-NAC alone, Mannitol alone, or Dex alone at decreasing concentrations. Concentrations of L-NAC, Mannitol, and Dex that showed 50% protection of hair cell loss individually were used as a combination in experimental group 4. Results There was an increase of total hair cell (THC) loss in the EIT OC explants when compared with control group HC counts or the tri-therapy cochlea. This study defined the dosage of L-NAC, Mannitol, and Dex for the survival of 50% protection of hair cells in vitro. Their combination provided close to 96% protection, demonstrating an additive effect.

Bmi1 Regulates the Proliferation of Cochlear Supporting Cells Via the Canonical Wnt Signaling Pathway.

Cochlear supporting cells (SCs), which include the cochlear progenitor cells, have been shown to be a promising resource for hair cell (HC) regeneration, but the mechanisms underlying the initiation and regulation of postnatal cochlear SC proliferation are not yet fully understood. Bmi1 is a member of the Polycomb protein family and has been reported to regulate the proliferation of stem cells and progenitor cells in multiple organs. In this study, we investigated the role of Bmi1 in regulating SC and progenitor cell proliferation in neonatal mice cochleae. We first showed that knockout of Bmi1 significantly inhibited the proliferation of SCs and Lgr5-positive progenitor cells after neomycin injury in neonatal mice in vitro, and we then showed that Bmi1 deficiency significantly reduced the sphere-forming ability of the organ of Corti and Lgr5-positive progenitor cells in neonatal mice. These results suggested that Bmi1 is required for the initiation of SC and progenitor cell proliferation in neonatal mice. Next, we found that DKK1 expression was significantly upregulated, while beta-catenin and Lgr5 expression were significantly downregulated in neonatal Bmi1-/- mice compared to wild-type controls. The observation that Bmi1 knockout downregulates Wnt signaling provides compelling evidence that Bmi1 is required for the Wnt signaling pathway. Furthermore, the exogenous Wnt agonist BIO overcame the downregulation of SC proliferation in Bmi1-/- mice, suggesting that Bmi1 knockout might inhibit the proliferation of SCs via downregulation of the canonical Wnt signaling pathway. Our findings demonstrate that Bmi1 plays an important role in regulating the proliferation of cochlear SCs and Lgr5-positive progenitor cells in neonatal mice through the Wnt signaling pathway, and this suggests that Bmi1 might be a new therapeutic target for HC regeneration.