An essential signaling cascade for avian auditory hair cell regeneration

Abstract

Hearing loss is a chronic disease affecting millions of people worldwide, yet no restorative treatment options are available. Although non-mammalian species can regenerate their auditory sensory hair cells, mammals cannot. Birds retain facultative stem cells known as supporting cells that engage in proliferative regeneration when surrounding hair cells die. Here, we investigated gene expression changes in chicken supporting cells during auditory hair cell death. This identified a pathway involving the receptor F2RL1, HBEGF, EGFR, and ERK signaling. We propose a cascade starting with the proteolytic activation of F2RL1, followed by matrix-metalloprotease-mediated HBEGF shedding, and culminating in EGFR-mediated ERK signaling. Each component of this cascade is essential for supporting cell S-phase entry in vivo and is integral for hair cell regeneration. Furthermore, STAT3-phosphorylation converges with this signaling toward upregulation of transcription factors ATF3, FOSL2, and CREM. Our findings could provide a basis for designing treatments for hearing and balance disorders.

Keywords: MAPK; cochlea; hearing loss; inner ear; otic; progenitor cell.

Figure 1.. Single-cell RNA sequencing of the regenerating chicken basilar papilla and supporting cell state isolation

(A) scRNA-seq datasets were obtained from basilar papilla sensory epithelia at P7 (baseline control), as well as 12, 16, 20, and 38 h post sisomicin treatment(PST). Apoptotic hair cell demise has been shown to peak between 12 and 20 h, and S-phase entry is detectable as early as 36 h PST. PST, post sisomicin treatment. (B) T-distributed stochastic neighbor embedding (tSNE) plots of all profiled cells representing five time points. CellTrails clustering identifies 20 states. (C) Bar plot showing the absolute size of each state. (D and E) Projection of MYO7A, TECTB, GSN, CSF1R, and HBBA expression onto the tSNE plot. Maximum log₂ expression (Log₂Ex) for each gene is shown on the bottom right of each tSNE plot. Five major epithelial cell groups were identified by specific known markers: hair cells (S1–S7), homogene cells (S8 and S9), supporting cells (S10–S14 + S16–S19), immune cells (S15), and hematopoietic cells (S20). Clusters S10–S14 and S16–S19 were isolated and re-clustered for further analysis.

Figure 2.. Gene expression changes in supporting cells

(A) tSNE plot showing the distribution of 812 identified supporting cells from all five time points (Ctrl, 12, 16, 20, and 38 h PST) and the projection of four distinct supporting cell clusters (S1–S4, color code as indicated). (B) Bar plots illustrating the distribution of cells from each cluster based on the time points. (C and D) Volcano plots showing the differentially expressed genes in S4 (C) and S3 (D) compared with all other clusters. A conservative threshold of 4-fold difference in gene expression was applied. With log₂(x) = 2, solving for x results in x = 4. (E) DotPlot visualization of immune-related genes upregulated in S4, mainly after 20 and 38 h PST. (F) DotPlot visualization of mesenchymal-to-epithelial transition genes, growth factors, G-protein-coupled receptors, and transcription factors differentiallyupregulated in S3. (G and H) HCR in situ detection of mRNA for B3GNTL, TRIM35, and F2RL1 in a representative transversal vibratome section of a control basilar papilla (G) and at 16 h PST (G′ and G″). Dying hair cells at 16 h PST lose B3GNTL expression and upregulate TRIM35 mRNA. F2RL1 mRNA is very low in control supporting cells (G) and strongly expressed at 16 h PST (G′ and G″). HBEGF mRNA is not detectable in control supporting cells (H) and strongly upregulated in supporting cells at 16 h PST (H′ and H″). Each image is representative of 4 biological replicates (specimen), each with at least 3 technical replicates (sections). Scale bars, 20 μm.

Figure 3.. Inhibition of pathway components

(A) Timeline of compound injections, EdU labeling, and assessment of S-phase entry 48 h after sisomicin treatment. SCCI, semicircular canal injection. (B) Box-and-whisker plot showing the fraction of EdU+/SOX2+ medial supporting cells in the control and 48 h post sisomicin treatment, with or without the infusion of antagonist/inhibitor. Each box represents the interquartile range (IQR) between the 25th and 75th percentiles, with the horizontal line inside the box indicating the median. The whiskers extend to 1.5 times the IQR. The dots indicate the individual data points for each condition. The red dots indicate the mean values for each condition. A decrease in supporting cell EdU incorporation was observed when the compounds were infused prior to sisomicin. Error bars represent the standard error of the mean (SEM). Statistical analysis was performed using repeated-measures one-way ANOVA with Bonferroni’s post hoc analysis, with all post hoc comparisons made to the sisomicin condition (n = 10–12 biological replicates and 3 technical replicates per condition). Asterisks indicate statistical significance: ***p < 0.001. PT, post treatment. (C) Transversal chicken basilar papilla section illustrating the morphology in control ears 48 h after sisomicin infusion and robust EdU incorporation. (D–H) Pretreatment of the inner ears with F2RL1 antagonist (D), MMP inhibitor (E), EGFR inhibitor (F), and ERK1/2 inhibitor (G) shows similar morphology as in (C) but low/absent EdU labeling of SOX2-labeled supporting cells. Combination with STAT3 inhibitor (H) did not completely abolish but reduced EdU incorporation compared with controls. Note that EdU incorporation into surrounding cells was not affected by inhibitor treatments. Each image is representative of 4 biological replicates (specimen), each with at least 3 technical replicates (sections). Scale bars, 20 μm.

Figure 4.. Inhibition of hair cell regeneration

(A) Timeline of multiple compound injections and assessment of hair cell regeneration 9 days after sisomicin treatment. (B and C) Transversal chicken basilar papilla section illustrating the morphology in control ears (B) and 9 days after sisomicin infusion (C). The insert in (B) shows a high magnification of control hair cells, while the insert in (C) displays the high magnification cytomorphology of newly formed hair cells observed at this specific time point post hair cell death (see also Sato et al.). Each image is representative of 4 biological replicates (specimen), each with at least 3 technical replicates (sections). Scale bars, 20 mm. (D–H) Pretreatment with F2RL1 antagonist (D), EGFR inhibitor (E), MMP inhibitor (F), and ERK1/2 inhibitor (G) shows an absence or only a few newly formed hair cells. The higher magnification insert in (E) shows the immature cytomorphology of these hair cells without hair bundles. Combination with STAT3 inhibitor (H) did not abolish but reduced hair cell regeneration compared with the control (C). Each image is representative of 4 biological replicates (specimen), each with at least 3 technical replicates (sections). Scale bars, 20 μm. (I–L) Box-and-whisker plot showing the number of MYO7A+/hair bundle+ hair cells in the medial (I) and lateral (J) sides, and MYO7A+/hair bundle− (hair bundleless) hair cells in the medial (K) and lateral (L) sides 9 days post treatment, with or without the infusion of antagonist/inhibitor. Each box represents the IQR between the 25th and 75th percentiles, with the horizontal line inside the box indicating the median. The whiskers extend to 1.5 times the IQR. The dots indicate the individual data points for each condition. The red dots indicate the mean values for each condition. Error bars represent the SEM. Statistical analysis was performed using repeated-measures one-way ANOVA with Bonferroni’s post hoc analysis, with all post hoc comparisons made to the sisomicin condition (n = 4 biological replicates and 3 technical replicates per condition). Asterisks indicate statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. PT, post treatment.

Figure 5.. Gene expression and ERK phosphorylation changes show distinct dependencies on different pathway components

(A) Real-time qPCR analysis of gene expression in chicken basilar papillae treated with sisomicin and inhibitors of F2RL1, MMP, EGFR, ERK, and STAT3. The statistics were conducted using repeated-measures one-way ANOVA with Bonferroni’s post hoc analysis, and all post hoc comparisons were done comparing to the sisomicin condition. Asterisks indicate statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; n.s., not significant (n = 4 biological replicates and 3 technical replicates). (B and C) Western blot analysis and quantification of ERK1/2 phosphorylation in individual sensory epithelia of chicken basilar papilla treated with sisomicin and inhibitors of F2RL1, MMP, EGFR, ERK, and STAT3. Inhibition of F2RL1, MMP, and EGFR activity abolished ERK1/2 phosphorylation, while inhibition of STAT3 phosphorylation did not prevent ERK1/2 phosphorylation. Statistical analysis was performed using repeated-measures one-way ANOVA with Bonferroni’s post hoc analysis, with all post hoc comparisons made to the sisomicin condition. Asterisks indicate statistical significance: ***p ≤ 0.001. n = 4 biological replicates with one representative experiment shown in (B).

Figure 6.. Transcription factor dependencies

(A) Real-time qPCR analysis of gene expression inchicken basilar papillae treated with sisomicin and specific pathway component inhibitors. The increased expression of ATF3 (58-fold), FOSL2 (26-fold), and CREM (28-fold) upon treatment with sisomicin was significantly reduced in inner ears treated with inhibitors of F2RL1, MMP, EGFR, ERK1/2, and STAT3. n = 4 biological replicates and 3 technical replicates. (B) The increased expression of KLF6 and TCF24 upon sisomicin treatment was largely unaffected by the different inhibitors. n = 4 biological replicates and 3 technical replicates. The statistics were conducted using repeated-measures one-way ANOVA with Bonferroni’s post hoc analysis, and all post hoc comparisons were done comparing to the sisomicin condition. Asterisks indicate statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 7.. A signaling cascade that leads to proliferative hair cell regeneration

This schematic drawing illustrates the molecular events that initiate avian supporting cell S-phase entry based on our hypotheses, which are supported by the presented experimental evidence. F2RL1 is proteolytically activated in response to hair cell damage. The upregulation of HBEGF mRNA depends on this activation. The activity and the presence of extracellular MMPs are essential for HBEGF shedding, which activates EGFR signaling that extends intracellularly via ERK1/2 and potentially ERK3. We cannot place STAT3 phosphorylation into dependence on ERK signaling, but we show that STAT3 phosphorylation is an essential component contributing to the signaling that culminates in the induction of expression of the transcription factors CREM, ATF3, and FOSL2. We found evidence for an independent pathway that controls the upregulation of KLF6 and TCF24. The illustration was created with BioRender.