Transcriptomic and epigenomic analyses explore the potential role of H3K4me3 in neomycin-induced cochlear Lgr5+ progenitor cell regeneration of hair cells.

Currently, adult cochlear hair cells (HCs) lack the capacity to regenerate, particularly the hearing damage caused by the HC damage are hard to recover. Remarkably, Lgr5+ inner ear progenitor cells can be activated to proliferate and regenerate hair cells (HCs) in response to injury, but the epigenetic regulatory roles in HC regeneration from Lgr5+ progenitor cells remain unresolved to date. We here investigate the possible roles of H3K4me3 modification in Lgr5+ progenitor cell proliferation and HC regeneration, and identify these differentially expressed genes associated with different binding regions between untreated Lgr5+ progenitor cells (ULPs) and neomycin-treated Lgr5+ progenitor cells (NLPs). Especially, H3K4me3 modification drives 12 genes involved in regulating proliferation and HC regeneration. Interestingly, we find that transcription factors Zeb1, Fev and Prdm5 are enriched in distinct peaks, implying their probable important roles in modulating neomycin-induced Lgr5+ progenitor cell proliferation and HC regeneration. Overall, our study demonstrates the underlying roles of H3k4me3 modification in Lgr5+ progenitor cell proliferation and HCs regeneration, and provides candidate H3K4me3 modification targets and regulators for subsequent studies.

Hippo/YAP signaling pathway protects against neomycin-induced hair cell damage in the mouse cochlea.

The Hippo/Yes-associated protein (YAP) signaling pathway has been shown to be able to maintain organ size and homeostasis by regulating cell proliferation, differentiation, and apoptosis. The abuse of aminoglycosides is one of the main causes of sensorineural hearing loss (SSNHL). However, the role of the Hippo/YAP signaling pathway in cochlear hair cell (HC) damage protection in the auditory field is still unclear. In this study, we used the YAP agonist XMU-MP-1 (XMU) and the inhibitor Verteporfin (VP) to regulate the Hippo/YAP signaling pathway in vitro. We showed that YAP overexpression reduced neomycin-induced HC loss, while downregulated YAP expression increased HC vulnerability after neomycin exposure in vitro. We next found that activation of YAP expression inhibited C-Abl-mediated cell apoptosis, which led to reduced HC loss. Many previous studies have reported that the level of reactive oxygen species (ROS) is significantly increased in cochlear HCs after neomycin exposure. In our study, we also found that YAP overexpression significantly decreased ROS accumulation, while downregulation of YAP expression increased ROS accumulation. In summary, our results demonstrate that the Hippo/YAP signaling pathway plays an important role in reducing HC injury and maintaining auditory function after aminoglycoside exposure. YAP overexpression could protect against neomycin-induced HC loss by inhibiting C-Abl-mediated cell apoptosis and decreasing ROS accumulation, suggesting that YAP could be a novel therapeutic target for aminoglycosides-induced sensorineural hearing loss in the clinic.

The Expression and Roles of the Super Elongation Complex in Mouse Cochlear Lgr5+ Progenitor Cells.

The super elongation complex (SEC) has been reported to play a key role in the proliferation and differentiation of mouse embryonic stem cells. However, the expression pattern and function of the SEC in the inner ear has not been investigated. Here, we studied the inner ear expression pattern of three key SEC components, AFF1, AFF4, and ELL3, and found that these three proteins are all expressed in both cochlear hair cells (HCs)and supporting cells (SCs). We also cultured Lgr5+ inner ear progenitors in vitro for sphere-forming assays and differentiation assays in the presence of the SEC inhibitor flavopiridol. We found that flavopiridol treatment decreased the proliferation ability of Lgr5+ progenitors, while the differentiation ability of Lgr5+ progenitors was not affected. Our results suggest that the SEC might play important roles in regulating inner ear progenitors and thus regulating HC regeneration. Therefore, it will be very meaningful to further investigate the detailed roles of the SEC signaling pathway in the inner ear in vivo in order to develop effective treatments for sensorineural hearing loss.

Characterization of Strip1 Expression in Mouse Cochlear Hair Cells.

Striatin-interacting protein 1 (Strip1) is a core component of the striatin interacting phosphatase and kinase (STRIPAK) complex, which is involved in embryogenesis and development, circadian rhythms, type 2 diabetes, and cancer progression. However, the expression and role of Strip1 in the mammalian cochlea remains unclear. Here we studied the expression and function of Strip1 in the mouse cochlea by using Strip1 knockout mice. We first found that the mRNA and protein expression of Strip1 increases as mice age starting from postnatal day (P) 3 and reaches its highest expression level at P30 and that the expression of Strip1 can be detected by immunofluorescent staining starting from P14 only in cochlear HCs, and not in supporting cells (SCs). Next, we crossed Strip1 heterozygous knockout (Strip +/-) mice to obtain Strip1 homozygous knockout (Strip1-/-) mice for studying the role of Strip1 in cochlear HCs. However, no Strip1-/- mice were obtained and the ratio of Strip +/- to Strip1+/+ mice per litter was about 2:1, which suggested that homozygous Strip1 knockout is embryonic lethal. We measured hearing function and counted the HC number in P30 and P60 Strip +/- mice and found that they had normal hearing ability and HC numbers compared to Strip1+/+ mice. Our study suggested that Strip1 probably play important roles in HC development and maturation, which needs further study in the future.

Knockdown of Foxg1 in Sox9+ supporting cells increases the trans-differentiation of supporting cells into hair cells in the neonatal mouse utricle.

Foxg1 plays important roles in regeneration of hair cell (HC) in the cochlea of neonatal mouse. Here, we used Sox9-CreER to knock down Foxg1 in supporting cells (SCs) in the utricle in order to investigate the role of Foxg1 in HC regeneration in the utricle. We found Sox9 an ideal marker of utricle SCs and bred Sox9CreER/+Foxg1loxp/loxp mice to conditionally knock down Foxg1 in utricular SCs. Conditional knockdown (cKD) of Foxg1 in SCs at postnatal day one (P01) led to increased number of HCs at P08. These regenerated HCs had normal characteristics, and could survive to at least P30. Lineage tracing showed that a significant portion of newly regenerated HCs originated from SCs in Foxg1 cKD mice compared to the mice subjected to the same treatment, which suggested SCs trans-differentiate into HCs in the Foxg1 cKD mouse utricle. After neomycin treatment in vitro, more HCs were observed in Foxg1 cKD mice utricle compared to the control group. Together, these results suggest that Foxg1 cKD in utricular SCs may promote HC regeneration by inducing trans-differentiation of SCs. This research therefore provides theoretical basis for the effects of Foxg1 in trans-differentiation of SCs and regeneration of HCs in the mouse utricle.

Hair cell regeneration from inner ear progenitors in the mammalian cochlea.

Cochlear hair cells (HCs) are the mechanoreceptors of the auditory system, and because these cells cannot be spontaneously regenerated in adult mammals, hearing loss due to HC damage is permanent. However, cochleae of neonatal mice harbor some progenitor cells that retain limited ability to give rise to new HCs in vivo. Here we review the regulatory factors, signaling pathways, and epigenetic factors that have been reported to play roles in HC regeneration in the neonatal mammalian cochlea.

Frizzled-9+ Supporting Cells Are Progenitors for the Generation of Hair Cells in the Postnatal Mouse Cochlea.

Lgr5+ cochlear supporting cells (SCs) have been reported to be hair cell (HC) progenitor cells that have the ability to regenerate HCs in the neonatal mouse cochlea, and these cells are regulated by Wnt signaling. Frizzled-9 (Fzd9), one of the Wnt receptors, has been reported to be used to mark neuronal stem cells in the brain together with other markers and mesenchymal stem cells from human placenta and bone marrow. Here we used Fzd9-CreER mice to lineage label and trace Fzd9+ cells in the postnatal cochlea in order to investigate the progenitor characteristic of Fzd9+ cells. Lineage labeling showed that inner phalangeal cells (IPhCs), inner border cells (IBCs), and third-row Deiters' cells (DCs) were Fzd9+ cells, but not inner pillar cells (IPCs) or greater epithelial ridge (GER) cells at postnatal day (P)3, which suggests that Fzd9+ cells are a much smaller cell population than Lgr5+ progenitors. The expression of Fzd9 progressively decreased and was too low to allow lineage tracing after P14. Lineage tracing for 6 days in vivo showed that Fzd9+ cells could also generate similar numbers of new HCs compared to Lgr5+ progenitors. A sphere-forming assay showed that Fzd9+ cells could form spheres after sorting by flow cytometry, and when we compared the isolated Fzd9+ cells and Lgr5+ progenitors there were no significant differences in sphere number or sphere diameter. In a differentiation assay, the same number of Fzd9+ cells could produce similar amounts of Myo7a+ cells compared to Lgr5+ progenitors after 10 days of differentiation. All these data suggest that the Fzd9+ cells have a similar capacity for proliferation, differentiation, and HC generation as Lgr5+ progenitors and that Fzd9 can be used as a more restricted marker of HC progenitors.