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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Dev Biol. 2013 Jan 11;376(1):86–98. doi: 10.1016/j.ydbio.2013.01.005

The Atoh1-lineage gives rise to hair cells and supporting cells within the mammalian cochlea

Elizabeth Carroll Driver a, Laura Sillers a, Thomas M Coate a, Matthew F Rose b,1, Matthew W Kelley a
PMCID: PMC3652277  NIHMSID: NIHMS444523  PMID: 23318633

Abstract

The Organ of Corti, located within the mammalian cochlea, contains a precise mosaic of hair cells (HC) and supporting cells (SC), the patterning of which is critical for auditory function. Progenitors of HCs and SCs are found in the same post-mitotic region of the cochlear duct during early stages of cochlear development, and both HCs and SCs are absent in mice lacking the transcription factor Atoh1. Based on existing data, Atoh1 is thought to be the earliest determinant of HC fate, and to have a cell-autonomous role in HC differentiation, but the lineage of Atoh1-positive cells within the cochlear duct remains unclear. To address this issue, we used an inducible Atoh1Cre*PR allele to permanently mark Atoh1-expressing cells at different developmental time points. We found that up to 30% of cells from the Atoh1-lineage develop as SCs, and that the number of Atoh1-positive SCs decreases both spatially and temporally in a pattern consistent with ongoing commitment. Modulation of Notch signaling, necessary for formation of the HC-SC mosaic, changes the percentage of cells from the Atoh1-lineage that develop as either HCs or SCs. The HC-SC ratio is also affected by morphogenesis of the cochlea, as inhibiting the outgrowth of the cochlear duct increases the number of Atoh1-lineage cells that develop as SCs. Our results demonstrate that the Atoh1-lineage is established early in cochlear development, but also show that expression of Atoh1 does not absolutely result in commitment to a HC fate.

Keywords: inner ear, development, cell fate, Organ of Corti

Introduction

Proper auditory function depends on formation of a highly ordered mosaic of hair cells (HC) and supporting cells (SC) within the organ of Corti (OC) of the mammalian cochlea. During development, formation of a post-mitotic region of cells (zone of non-proliferation), containing the progenitors of both HCs and SCs, first distinguishes these cells from other epithelial cells in the cochlear duct (Chen and Segil, 1999). This region, called the prosensory domain, becomes progressively more organized as formation of the rows of inner and outer HCs proceeds, and the HC rows are surrounded by several different types of supporting cells (Driver and Kelley, 2009; Kelley, 2006). Because both HCs and SCs arise from the same physical region of the cochlea, and both prosensory progenitors and immature SCs can transdifferentiate into HCs under some circumstances (Brooker et al., 2006; Kelley et al., 1995; Kiernan et al., 2005; Lanford et al., 2000; White et al., 2006), it has been hypothesized that the prosensory domain represents a common progenitor population, and that subsequent expression of HC and SC-specific genes differentiates prosensory cells into HCs or SCs.

Expression of the basic helix-loop-helix transcription factor Atoh1 is thought to be the earliest determinant of HC fate. Absence of Atoh1 results in a complete loss of both HCs and SCs (Bermingham et al., 1999; Chen et al., 2002), and forced expression of Atoh1 in nonsensory cells within the cochlear duct induces ectopic HCs (Gubbels et al., 2008; Woods et al., 2004; Zheng and Gao, 2000). However, there has been some debate regarding the full extent of changes in the organ of Corti in response to deletion of Atoh1. Some studies have concluded some level of differentiation is retained in Atoh1−/− cochleae. These conclusions are based on either morphological criteria or expression of molecular markers, including Bdnf, Sox2, Prox1 and Jag1, which are expressed specifically in HCs (Bdnf) and SCs (Sox2, Prox1, Jag1) in the late post-natal cochlea (Fritzsch et al., 2005; Dabdoub et al., 2008). However, none of these factors can be strictly considered as a marker of differentiation, as all are initially expressed in undifferentiated cochlear prosensory cells (Kiernan et al., 2005; Morrison et al., 1999; Bermingham-McDonogh et al., 2006; Fariñas et al., 2001). As a result, their continued expression in the epithelium may be an indicator of persistent prosensory cells, rather than differentiated cell types. Similarly, it has been suggested that the extension of spiral ganglion fibers into the cochlea in Atoh1 mutants is an indication of some degree of cellular differentiation (Fritzsch et al., 2005), but since both Bdnf and Nt3 are expressed in the cochlear duct prior to Atoh1 expression (Fariñas et al., 2001), it seems unlikely that further differentiation would be required for fiber extension. Regardless, since Atoh1 is clearly both necessary and sufficient for HC development, it has been the focus of extensive research within the auditory field. But, despite multiple publications, the specific role of Atoh1 remains unclear.

At early stages of cochlear development in the mouse, prior to embryonic day 15 (E15), both Atoh1 transcript and an Atoh1lacZ knock-in reporter indicate broad expression within the cochlea. But beginning around E15, expression is found only in HCs (Woods et al., 2004; Yang et al., 2010). This apparent refinement of Atoh1 expression over developmental time is consistent with the pattern of expression of genes with proneural function, such as Drosophila atonal and achaete-scute (Jarman et al., 1993) and had led to the suggestion that Atoh1 acts a proneural, or, more accurately, pro-hair cell gene within the cochlea (Lanford et al., 1999; Woods et al., 2004) with the effect of its expression being to initiate a HC developmental program. This hypothesis is supported by the increased number of HCs observed in the cochlea in response to deletion of known or likely antagonists of Atoh1, such as the transcription factor Sox2 (Dabdoub et al., 2008) and the Notch signaling pathway (Brooker et al., 2006; Kiernan et al., 2005; Lanford et al., 1999). In addition, several Atoh1-reporter lines have indicated expression in some supporting cell types, an effect that is consistent with an initial broad expression (Matei et al., 2005; Yang et al., 2010). In contrast, localization of ATOH1 by immunohistochemistry or in several other Atoh1-transgenic reporter lines indicated only hair cell-specific expression of Atoh1, leading to the suggestion that ATOH1 acts as a differentiation factor, with expression that is limited to prosensory cells that are already committed to a hair cell phenotype (Chen et al., 2002; Fritzsch et al., 2005; Pan et al., 2011). This conclusion is supported by the relatively normal development of the cochlea prior to the onset of Atoh1 expression, and indications of cellular organization and the presence of supporting cells in vestibular epithelia even in the absence of Atoh1, as previously discussed (Bermingham et al., 1999; Fritzsch et al., 2005).

Finally, Atoh1 expression has also been reported in spiral ganglion neurons, suggesting possible early expression in a hypothesized neurosensory progenitor (Matei et al., 2005). However, some of these data were derived using a transgenic mouse line that has been reported to include ectopic expression (Lumpkin et al., 2003) and several studies from other laboratories have failed to replicate this finding (Basch et al., 2011; Lu et al., 2011; Yamamoto et al., 2011; Yang et al., 2010). To more directly examine the expression pattern of Atoh1 in the developing cochlea, we used an inducible Atoh1Cre*PR knock-in allele (Rose et al., 2009) to mark the cochlear lineage of Atoh1-mRNA expressing cells at different developmental time points. Our results indicate that early in cochlear development, up to 30% of cells that initiate Atoh1 promoter activity develop as SCs rather than HCs, consistent with a proneural role for Atoh1. In addition, labeling of Atoh1-expressing cells at later developmental time points revealed a rapid down regulation of Atoh1 promoter activity and subsequent protein expression that contrasts with previous results. Positive or negative modulation of the Notch signaling pathway increases or reduces the percentage of SCs in the Atoh1-lineage, respectively, demonstrating for the first time that Notch signaling can influence cells within the cochlea that are already positive for Atoh1. Finally, inhibition of cochlear duct extension also alters the ratio of Atoh1-expressing HCs to SCs, demonstrating that spatial rearrangements involved with growth of the cochlear duct serve an important role in cellular patterning within the sensory epithelium.

Materials and methods

Mice

Male mice carrying both the Atoh1Cre*PR and a reporter allele, R26RlacZ, or R26RtdTomato (Madisen et al., 2010; Rose et al., 2009; Soriano, 1999) were mated with CD1 females (Charles River). In vivo RU486 injections into pregnant females were performed as previously described (Rose et al., 2009). All mice were maintained according to animal care and use protocols of the either the Center of Comparative Medicine of Baylor College of Medicine or the National Institutes of Health.

Cochlear explant cultures

Embryos were collected from timed pregnant females and cochlear explant cultures established between embryonic day 13 (E13) and post-natal day 0 (P0) as previously described (Driver and Kelley, 2010; Montcouquiol and Kelley, 2003; Yamamoto et al., 2009). RU486 (Mifepristone, Sigma) was dissolved in DMSO and added to the culture media at the indicated concentration for 1, 4, or 24 hours; the total DMSO concentration was less than or equal to 0.2%. The RU486-containing media was then removed and replaced with control media after rinsing, and the explants cultured for another 4 to 6 DIV. DAPT and blebbistatin were both from EMD. Recombinant mouse DLL1-Fc chimeric protein was obtained from R&D Systems, and added at 1.25, 2.5, 5, or 10 μg/ml. Paired controls were treated with the equivalent concentration of control IgG2A Fc protein. For blebbistatin-treated explants, cochleae were bisected at approximately 50% of the cochlear length, and only the basal portion of the explant was cultured.

Gene expression analysis and immunohistochemistry

Whole mount cochleae and cochlear explants were fixed in 4% paraformaldehyde. Staining for β-galactosidase activity was performed as previously described (Rose et al., 2009). Antibodies used were as follows: anti-dsRed (Clontech), anti-MyosinVI (Proteus Biosciences), anti-MyosinVIIa (Developmental Studies Hybridoma bank), anti-Sox2, Santa Cruz, anti-p75NTR and anti-Prox1 (Covance), and anti-S100A1 (Thermo Scientific). All secondary antibodies and labeled phalloidin were from Invitrogen. Atoh1 mRNA expression was determined by in situ hybridization as previously described (Driver et al., 2008).

For determination of relative expression levels of Atoh1, wild-type cochleae were dissected at E13, E15, P0, and P5. Cochleae were treated with thermolysin to separate the tissue layers, and the epithelia were manually dissociated. For P0 samples, only the most apical and basal quadrants of the epithelia were collected. At least three independent samples from each stage, composed of tissue pooled from 3 or 4 cochleae, were compared in the qPCR analysis. E15 was used as the reference sample. The relative level of MyoVI was also determined as a positive control for the presence of HCs. Hes1 and Hes5 relative expressions were determined from wild-type E13 CD1 cochlear explants treated with 5 or 10 μM blebbistatin or 0.1% DMSO (control), beginning after 1 DIV, for a total of 6 DIV. Total RNA was extracted using the Arcturus Picopure kit (Applied Biosystems). Relative gene expression levels were determined using TaqMan gene expression assays (Applied Biosystems) and the ΔΔCT method, with Gapdh as an internal control. Statistically significant different levels of expression were determined using one-way ANOVA followed by Tukey’s test.

Anti-ATOH1 polyclonal antibody and immunohistochemistry

A chicken anti-peptide antibody (Aves Labs) was generated against a synthetic peptide corresponding to amino acids 330–351 of mouse ATOH1 (CZRSHRSDGEFSPHSHYSDSDEAS). The antibody was affinity purified, then diluted to 1.5 mg/ml. During the optimization of staining procedures with this antibody, we found that it provides optimal detection of ATOH1 in briefly fixed tissue. Wild type E14-P0 mouse cochleae for sections were fixed for 30 minutes in 4% paraformaldehyde prior to embedding. For blocking, samples were treated with a combination of 10% normal goat serum and 10% Henblocker reagent (Aves Labs). Anti-ATOH1 was added at 150–300 ng/ml (1:10,000–1:20,000) overnight at 4°C. Sections were rinsed extensively prior to incubation with anti-chicken secondary antibodies.

Analysis of cell fate

The lengths of cochlear explants were measured along the IHC row, and confocal Z-stack images taken at 5, 25, 50, 75, and 95% of the total length. For the bisected explants used in the blebbistatin treatment experiments, cells were counted at the 5, 50, and 95% positions (equivalent to 5, 25, and 50% positions of intact explants). All tdTomato-positive cells of the sensory epithelium within the image field (approximately 200 μm wide) at each distance were counted, and classified as IHCs, OHCs, ISCs, or OSCs. The total number of HCs (MyoVI or MyoVIIa-positive cells) was also determined. Data are the average percentage of cell type found, plus or minus standard error of the mean (SEM). Results are from at least three separate experiments, with at least two explants from each experiment. Statistical significance of changes in cell fate was determined by two-tailed paired student’s t-test. Statistical significance of differences among SCs and HCs at different basal-to-apical positions was determined using one-way ANOVA followed by Tukey’s test.

Results

In vivo induction of Atoh1Cre*PR indicates that supporting cells arise from the Atoh1 lineage

To determine whether the Atoh1 promoter is ever active in cells that do not differentiate into hair cells, we used an inducible Atoh1Cre*PR knock-in allele (Rose et al., 2009) crossed to either the R26RlacZ or R26REYFP Cre reporter alleles to permanently mark cells of the Atoh1 lineage. We induced activity of Cre*PR by administering RU486 to pregnant females at a variety of different time-points when differentiation of cells within the cochlea is known to occur. In contrast to the Atoh1-LacZ knock-in reporter allele ((Bermingham et al., 1999), Fig. 1A), in which virtually all hair cells are positive for β-galactosidase at P0, only a few cells in the Atoh1Cre*PR; R26RlacZ cochlea showed evidence of β-gal activity when examined at P0 (Fig. 1B). This was the case whether Cre*PR activity was induced by repeated low-dose RU486 injections over several days (e.g. from E9.5 to E17.5) or higher-dose injections on a single day (due to the stochastic and dose-dependent nature of Cre*PR induction). Most of the β-gal-positive cells were HCs, identifiable by their shape and position within the OC (Fig. 1B, C). Similar results were obtained in Atoh1Cre*PR; R26RtdTomato cochleae (Fig. 1D). However, some labeled cells appear to be SCs based on their morphology and position in the OC matrix. For instance, in Fig. 1E, one tdTomato-positive SC (arrowhead), presumably a Dieters’ cell based on its position between the second and third rows of OHCs, is present. Other examples of tdTomato-labeled SCs are shown in Fig. 1F and 1G. The low number of labeled cells in all cochleae examined is likely due to inefficient activation of the Cre*PR protein in the inner ear. The difference between the rates of R26R recombination in the cochlea compared to the brain (Rose et al., 2009) may be caused by the relatively limited vascular perfusion in the cochlea at these stages, which could restrict the amount of RU486 that reaches the cells in the developing OC.

Figure 1. In vivo induction of Atoh1Cre*PR labels both hair cells and supporting cells.

Figure 1

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A. Surface view of the base of the cochlear epithelium from an Atoh1lacZ knock-in reporter at P0. Atoh1 is only found in HCs. Scale bar (same for (B, D), 20 μm). B–C. β-galactosidase activity in in vivo induced Atoh1Cre*PR; R26RlacZ cochleae. RU486 was injected daily into pregnant females from 9.5–17.5 dpc, then cochleae were dissected and stained at P0. B. Low magnification view, similar to (A). Few cells in the OC are labeled. C. High magnification of a β-galactosidase-positive OHC (arrow). Scale bar (same for (E–G), 10 μm). D. Low magnification view of an Atoh1Cre*PR; R26RtdTomato cochlea. Cre*PR activity was induced with a single injection of RU486 at 14.5 dpc. Both IHCs (arrows) and OHCs (open arrows) are tdTomato-positive. E. High magnification view of tdTomato-positive cells from the Atoh1-lineage. IHCs and OHCs are labeled (arrows), as well as one SC, identifiable as a Dieters’ cell (arrowhead) based on its morphology and position between OHCs. F, G. Z-stack confocal projections. F. One IHC and one OHC, identified by position and phalloidin-labeled stereocilia bundles (green), are tdTomato-positive (arrows). A Dieters’ cell is also labeled by tdTomato (arrowhead), and has a strongly Sox2-positive nucleus (blue). G. tdTomato positive OHC (arrow), and inner phalangeal cell (arrowhead). MyosinVI (green) labels all HCs.

Supporting cells are found in the Atoh1-lineage in cochlear explants

Because so few cells were labeled by in vivo induction of Cre*PR activity, which precluded meaningful statistical analysis of the number of labeled SCs, we used a cochlear explant protocol (Montcouquiol and Kelley, 2003) and the sensitive Cre-activated R26RtdTomato reporter allele to increase the number of labeled cells (Madisen et al., 2010). Atoh1Cre*PR/+; R26RtdTomato/+ cochlear explants were established, usually at E13, and Cre*PR activity was induced with RU486 at specific time-points and for different durations of the culture period. We found that cochlear hair cells were tdTomato-labeled in a dose-dependent manner. In cochlear explants treated with high concentrations of RU486 (1 μM or 100 nM) for 24 hours beginning at E13, nearly all hair cells (identified by co-expression of MyoVI) were tdTomato-positive (Fig. 2A–G). However, a significant number of tdTomato-positive cells within these explants developed as SCs rather than HCs. In explants treated with 1 μM or 100 nM RU486 the number of tdTomato-positive cells SCs comprised just over 30% of the total tdTomato-positive cells (Fig. 2D–F, H). With lower doses of RU486, both the total number of labeled HCs and the percentage of tdTomato-labeled cells that developed as SCs decreased (Fig. 2G, H); no tdTomato-expressing cells were ever observed in the absence of RU486.

Figure 2. Atoh1-expressing cells develop as both hair cells and supporting cells.

Figure 2

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Atoh1Cre*PR; R26RtdTomato cochlear explants established at E13, induced for the first 24 hours in culture and maintained for 6 DIV. A–C. Low magnification views. A. Most HCs along the length of the explants are tdTomato-positive (magenta). B. MyosinVI (MyoVI) labeling of HCs in the same cochlear explant (green). C. Merge of (A) and (B) shows overlap (white) of tdTomato and MyoVI labeling. Scale bar, (A–C), 200 μm. D–F. Higher magnification views labeled as in (A–C). Nearly all HCs are tdTomato-positive, although MyoVI labeling indicates a few HCs that are not (arrows). In addition, some tdTomato-positive cells have a SC morphology and are negative for MyoVI (open arrowheads). Scale bar, (D–F), 25 μm. G. The average percentage of total HCs (MyoVI-positive) also expressing tdTomato in cochlear explants treated with the indicated concentrations of RU486. H. The percentage of total tdTomato-positive cells that develop as SCs following treatment with the indicated concentrations of RU486. I. Z-stack confocal projection of cells from a similar explant as in (A–F). Sox2 is strongly expressed in the nuclei of supporting cells. Open arrowheads indicate tdTomato-labeled supporting cells with bright Sox2-positive nuclei and lumenal projections. Scale bar, 10 μm.

We identified SCs based on multiple criteria: cell morphology (nuclei located in the basal layer and a thin projection to the lumenal surface of the epithelium), negative staining for MyosinVI or MyosinVIIa, strong expression of Sox2 in the nucleus (Fig. 2D–F, I, open arrowheads), and absence of a stereocilia bundle (Fig. 3A–C). To further confirm that these tdTomato-positive cells developed as SCs, we evaluated the expression of other SC markers. tdTomato-labeled cells with SC morphology found between the IHC and OHC rows were also positive for p75NTR, a marker of pillar cells (PCs, Mueller et al., 2002, Fig. 3D–F). Labeled SCs located in the OHC region also expressed Prox1 and S100A1, both normally expressed by Dieters’ cells (Bermingham-McDonogh et al., 2006), whereas those SCs adjacent to the IHC row were negative for both Prox1 and S100A1 (Fig. 3G–L).

Figure 3. tdTomato-labeled cells express markers of supporting cells.

Figure 3

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Atoh1Cre*PR; R26RtdTomato cochlear explants established at E13, treated with 10 nM RU486 for the first 24 hours in culture, and maintained for 6 DIV. tdTomato-positive cells are shown in red, and HCs are marked with MyoVI or MyoVIIa (blue). Open arrowheads indicate tdTomato-labeled cells with SC morphology. A. Shallow confocal Z-stack projection of the lumenal surface of a cochlear explant. Three tdTomato-positive, MyoVI-negative cells are shown. B. F-actin-rich stereocilia bundles present on HCs are stained with phalloidin (green). C. Merge of (A) and (B). The tdTomato-labeled cells lack stereocilia bundles (open arrowheads). D. A single MyoVIIa-negative cell is present between the IHC and OHC rows (open arrowhead). E. Pillar cells are labeled with anti-p75NTR (green). F. Merge of (D) and (E). p75NTR labeling coincides with tdTomato staining. D′–F′. Z-stack confocal reconstructions from the images shown in (D–F). The XY location of the Z-stack is indicated by the brackets in (D–F). The p75NTR and tdTomato-positive, MyoVIIa-negative cell has characteristic PC location and morphology. G. Several tdTomato-positive, MyoVIIa-negative cells in the OHC or PC region (open arrowheads), or IHC region (arrow) are shown. H. Anti-Prox1 staining in the nuclei of SCs in the OHC and PC regions (green). I. Merge of (G) and (H). tdTomato-labeled cells found near OHCs and PCs are Prox1-positive (open arrowheads), but a labeled SC adjacent to the IHCs is Prox1-negative (arrow). J. Z-stack confocal reconstruction showing two tdTomato-positive SCs. K. S100A1 marks the IHCs, PCs, and Dieters’ cells (green). L. Merge of (J) and (K). The SC next to the IHCs is S100A1- negative (arrow), but the tdTomato-positive outer PC is S100A1-positive (open arrowhead).

To determine whether Atoh1 expression in SCs decreases over developmental time, RU486 was applied at later stages. In contrast to the 30.3 ± 2.1% of labeled cells that developed as SCs in explants established at E13, when RU486 was applied for 24 hours starting at E15, only 6.9 ± 0.9% of tdTomato-positive cells developed as SCs (Fig. 4A-C). For E17, the number of SCs dropped to 1.4 ± 0.7%. This result is consistent with a rapid down-regulation of Atoh1 in cells that will develop as SCs.

Figure 4. Cell fate specification occurs in a basal to apical gradient.

Figure 4

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A. Low magnification view of an Atoh1Cre*PR; R26RtdTomato cochlear explant established at E15 and maintained for 4 DIV. RU486 was added for the first 24 hours of culture. The positions along the cochlear length at which cells were counted are indicated. Scale bar, 200 μm. B. Image taken at 5% of the cochlear length, measured from the base. With few exceptions (arrows), MyoVIIa-positive HCs are also tdTomato-positive. Few tdTomato-labeled SCs are seen, and those present are mostly found near the rows of OHCs (OSCs, open arrowheads). Only one ISC is observed in this field (closed arrowhead). Scale bar (same for C), 25 μm. C. Image from the 95% position. Many more labeled ISCs (closed arrowheads) are visible, along with several tdTomato-positive OSCs (open arrowheads). Arrows indicate unlabeled HCs. D. The percentage of total tdTomato-positive cells that develop as SCs at each position along the cochlear length, from base to apex, at E15. More labeled SCs are found at apical positions. E. Relative percentages of total tdTomato-labeled SCs that develop as either ISCs or OSCs, based on position along the duct. The percentage of ISCs increases toward the apex.

Cellular commitment occurs in basal-to-apical and medial-to-lateral gradients

Several aspects of cellular differentiation within the OC have been described as occurring in gradients that extend from base to apex. To establish whether determination of cell fate follows a similar pattern, we next examined whether the fates of Atoh1-expressing cells vary along the long axis of the cochlea over developmental time. When RU486 induction was started at E13, the percentage of labeled SCs was fairly constant regardless of basal-to-apical location, which is consistent with a rapid onset of Atoh1 mRNA expression from base to apex. However, a developmental gradient was apparent when induction was started at E15 (Fig. 4A–D). In comparison to the most basal region, the percentage of tdTomato-positive cells that developed as SCs was 6 times greater at the most apical position (Fig. 4D). In E17 induced explants, tdTomato-positive SCs were restricted entirely to the apical-most (95%) portion of the cochlea, and even in that region the total number of labeled SCs had decreased to 5.8 ± 2.1% (not shown).

In addition to the developmental gradient from the base to apex of the OC, at any position along that axis, cell fate determination is also thought to occur in a gradient that extends along the medial-to-lateral (IHC to OHC) axis as well. To examine this, tdTomato-labeled SCs along the length of the cochlea were classified as either inner supporting cells (ISCs), which included phalangeal cells, border cells, and inner pillar cells, or OSCs, which included outer pillar cells, Dieters’ cells, and Hensen’s cells, at different positions along the basal-to-apical axis in explants treated with RU486 starting on E15. Consistent with the hypothesis that medially positioned cells of the organ of Corti are specified prior to laterally positioned cells, a much higher percentage of Atoh1-labeled SCs were observed in the OSC region in the base of the cochlea while in the apex the ratio of labeled ISC:OSC approached 1:1 (Figure 4E).

As discussed, treatment with RU486 for 24 hours at E13 resulted in uniform labeling of hair cells, therefore 1 and 4 hour treatments of 1 nM RU486 at E13 were used in an effort to determine whether the onset of Atoh1 expression occurs in a gradient or not. Following either a 1 or 4 hour pulse of RU486, a slight, but not statistically significant, basal-to-apical gradient in the percentage of tdTomato-positive HCs was observed (Fig 5A–C). However, the highest percentage of tdTomato-positive cells was observed at the 25% from the base position rather than at the 5% position (Fig. 5A, C), indicating either a higher number of Atoh1-expressing cells, or cells expressing a higher level of Atoh1, or both at that location. This result is consistent with previous studies that have shown the earliest markers of cell differentiation not at the extreme base but slightly more apically (Rubel, 1978). In contrast, we did not detect any differences in the proportion of IHCs versus OHCs that were labeled at any position along the cochlear duct, and so did not observe a gradient in HC labeling along the IHC to OHC axis. It may be that the temporal resolution of Atoh1Cre*PR induction by RU486 is insufficient to detect this gradient, or the gradient may not exist. Interestingly, some tdTomato-positive OHCs were present at the most apical positions of the cochlear duct even following a 1 hour pulse on E13 (Fig. 5B). This suggests that progenitors at the apex of the cochlear that will develop as OHCs are already positive for Atoh1 at that time, and will be maintained in an undifferentiated state for several days.

Figure 5. HCs along the length of the cochlea are Atoh1-positive at E13.

Figure 5

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A–B. Images from an Atoh1Cre*PR; R26RtdTomato cochlear explant established at E13, treated with 1 nM RU486 for 1 hour, and then maintained for an additional 6 DIV. A. Image from the 25% position. The highest percentage of tdTomato-labeled cells is found in this mid-basal region. B. Image from the 95% position. Some OHCs are found in the Atoh1-lineage, even after only a 1 hour RU486 treatment at E13 (arrows). C. The average percentage of MyoVIIa-positive HCs also labeled with tdTomato in explants treated with 1 nM RU486 for either 1 or 4 hours at E13. Differences among the percentage of labeled cells at each position were not statistically significant from one another.

Atoh1-promoter activity is rapidly down-regulated in developing hair cells

Previous assessments of the duration of Atoh1-expression in developing hair cells have obtained differing results, with some studies indicating down-regulation in the early postnatal period (Lanford et al., 2000) while others reported Atoh1 expression into adulthood (Matei et al., 2005). To examine when Atoh1 expression becomes down-regulated in HCs in this mouse model, the percentage of labeled HCs was examined following induction of Cre*PR at different developmental ages. As discussed, induction of Cre*PR at a concentration of 100 nM RU486 at E13 resulted in an average of 98% of HCs being labeled (Fig. 2G). The same induction regime at E15 marked only 89% of the HCs, suggesting that some cells were already negative for Atoh1, but that most HCs were still expressing Atoh1. However, by E17 only about half of the HCs were labeled, and this number was further reduced to a quarter of HCs at P0 (Fig. 6A). Parsing of these data based on spatial distribution along the basal-to-apical axis revealed a pronounced gradient in the down-regulation of Atoh1-promoter activity (Fig. 6B). At E17, the gradient of tdTomato-positive HCs increased from a minimum of 20% of total hair cells in the base to 80% of total HCs in the apex (Fig. 6B). By P0, 80% of HCs in the apex were still labeled while virtually no labeled HCs were observed in the base (Fig. 6D–G). In contrast with the onset of Atoh1 promoter activity, which occurs nearly simultaneously along the length of the cochlea (Fig. 5), the down-regulation of Atoh1 expression occurs in a pronounced basal-to-apical gradient.

Figure 6. Atoh1 expression in hair cells decreases over developmental time.

Figure 6

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A. The average percentage of MyoVIIa-positive HCs that are also positive for tdTomato in cochlear explants treated with 100 nM RU486 for 24 hours at E13, E15, E17, or P0. B. Same data as in (A), but shown as measured at each position along the cochlear length, from base to apex. More tdTomato-positive cells are found at the apex. C–D. Low magnification images from an Atoh1Cre*PR; R26RtdTomato explants treated with RU486 at E17 (C) and P0 (D), stained for MyoVIIa (green) and tdTomato (magenta). E–G. High magnification views of the explant in (D). E. At 5% of the cochlear length from the base, no tdTomato-positive cells are observed. F. A few tdTomato-labeled cells are present at the 50% position. G. Most but not all (arrows) MyoVIIa-positive HCs are also tdTomato-labeled at the 95% position. A single SC is tdTomato-positive (open arrowhead).

ATOH1 protein is absent from HCs by P5

The results presented above suggested a rapid decrease in Atoh1-promoter activity that contrasts with previous reports. To determine whether this decrease in promoter activity leads to a decrease in ATOH1 protein, we generated an antibody against ATOH1 that specifically labels HC nuclei. ATOH1-positive cell nuclei were first detectable in cochlear sections at E14 (Fig. 7A), in positions that approximate the locations of developing IHCs, and are consistent with previous results from reporter lines (Chen et al., 2002; Lumpkin et al., 2003). By E16, strong expression of ATOH1 protein was observed in all HCs in both the apex and the base of the cochlea (Fig. 7B, C). In contrast, in P0 cochlear sections, ATOH1 expression in HCs at the base of the cochlea was nearly undetectable, though the staining was still strong in HCs at the apex (Fig. 7D-E). These data are consistent with the observed decrease in Atoh1 promoter activity in Atoh1Cre*PR cochleae and suggest that ATOH1 protein is rapidly down-regulated in a basal-to-apical gradient beginning by P0 (Fig. 6). However, mRNA for Atoh1 is still detectable in both the apex and the base of the cochlea at P0, although the level of expression does appear to be somewhat reduced in the base (Fig. 7G, H). By P5, all HCs were negative for ATOH1 protein (Fig. 7I). We obtained similar results from quantitative RT-PCR for Atoh1 performed on isolated cochlear epithelia at the same ages (Fig. 7M), which showed that the level of Atoh1 in the P0 base is significantly lower compared to E15, and is almost undetectable at P5 (p<0.01). Specificity of the ATOH1 antibody was confirmed using cochlea from Atoh1lacZ/lacZ embryos (Fig. 7L), which lack the entire Atoh1 coding domain (Ben-Arie et al., 1997).

Figure 7. ATOH1 protein expression.

Figure 7

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A. Section from the base of an E14 cochlea. Three ATOH1-positive nuclei are present (red, arrows). B–F, I. Sections from cochleae at the indicated ages, stained with antibodies against ATOH1 (red) and MyoVI (green). F-actin stained by phalloidin is shown in blue. B, C. E16 cochlea. Strong staining for the ATOH1 antibody is present in all HCs (also positive for MyoVI), at both the apex and the base. D–F. P0 sections. D. Low magnification view of two turns of the cochlea. MyoVI staining marks HCs in the apex and base (arrows). Non-specific background staining from the ATOH1 antibody is present on the surface of the stria vascularis (open arrowheads). E. Apical section from (D). Strong ATOH1 signal is present in the OHCs (bracket), but slightly weaker staining is found in the IHC (arrow). F. ATOH1 staining is almost absent in a section from the base at P0 in both the IHC and OHCs. G, H. In situ hybridizations for Atoh1 transcript. Atoh1 mRNA is still present at the apex (G) and base (H) of a P0 cochlea. I. ATOH1 protein is not detectable at the apex of the cochlea by P5. J. ATOH1 protein (red) in the mid-base of an E16 Atoh1+/+ cochlea. K. Same section as in (J), also showing MyoVI staining (blue), weak Sox2 staining in the HC nuclei, and strong Sox2 staining in the SC nuclei (green). L. In a section from an E16 Atoh1−/−cochlea, no reactivity for either ATOH1 (red) or MyoVI (blue) is visible. Sox2-positive cells remain (green). M. Relative expression levels of Atoh1 and MyoVI in the cochlear epithelium at E13, E15, P0, and P5 as determined by qPCR. Values are presented as relative to E15. Error bars represent SEM. *p<0.01.

Notch signaling regulates Atoh1-cell fate

Notch signaling plays a key role in determining the number of hair cells that form within the organ of Corti. However, it has not been resolved whether Notch signaling specifically regulates the expression of Atoh1 (Chen et al., 2002). To determine whether activation of Notch signaling induces Atoh1-expressing cells to develop as SCs, the percentage of tdTomato-positive cells that develop as SCs was assayed following inhibition of Notch signaling using a β-secretase inhibitor N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT). E13 explants were treated with 10 nM RU486 for 24 hours to label all cells expressing Atoh1 during that time. 50 μM DAPT was then added beginning at 3 DIV, equivalent to E16. The addition of DAPT was delayed in order to allow any residual RU486 to be completely cleared, so that cells newly expressing Atoh1 would not be tdTomato-positive. In cochlear explants treated with DAPT, the percentage of labeled SCs decreased to 5%, as compared to about 20% in paired controls (p<0.0001), consistent with a conversion of most SCs from the Atoh1-positive lineage to a HC fate (Fig. 8A, B, E). In addition, an increase in the number of tdTomato-negative HCs was observed in DAPT-treated explants, suggesting that unlabeled, previously Atoh1-negative, SC progenitors were also converting to a HC fate in response to inhibition of Notch signaling (Fig. 8D, p<0.001). The total number of tdTomato-positive cells was not significantly different between control and DAPT-treated explants.

Figure 8. Modulation of Notch signaling alters the fates of Atoh1-positive cells.

Figure 8

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A–D. Atoh1Cre*PR; R26RtdTomato cochlear explants established at E13 and maintained for 6 DIV. RU486 was added for the first 24 hours of culture. All images are from the 25% of cochlear length position. Explants are stained for tdTomato (magenta) and anti-MyoVIIa (green). Open arrowheads indicate tdTomato-positive SCs, and arrows show non-tdTomato labeled HCs. A. Control explant. B. Explant treated with 50 μM DAPT from 3 to 6 DIV. Some tdTomato, MyoVI-positive HCs are adjacent to other HCs with no apparent SCs in between; more tdTomato-negative, MyoVI-positive HCs are also observed (arrows). Few tdTomato-labeled SCs are present (open arrowheads). C. Explant treated with 5 μg/ml control IgG2A Fc. D. Explant treated with 5 μg/ml DLL1-Fc protein, beginning after 1 DIV. E. Percentages of all tdTomato-labeled cells exhibiting a SC morphology, and percentages of all MyoVIIa-positive cells also labeled with tdTomato. Data are from paired explants from multiple experiments, and expressed as means ± SEM. DLL1-Fc data represent the average from all explants treated with 2.5 to 10 μg/ml DLL1-Fc. *p <0.05, ** p <0.005, *** p <0.00001. F. Average change in the % of tdTomato-labeled cells with a SC morphology in DLL1-treated explants vs. control Fc-treated. The increase in labeled SCs is significant for all concentrations of DLL1 except 1.25 μg/ml (p<0.05), but no dose response is evident.

Conversely, in explants with increased levels of Notch signaling as a result of treatment with a soluble form of the Notch ligand Delta-like1 (DLL1), the percentage of tdTomato-positive cells that developed as SCs increased. Recombinant DLL1-Fc chimeric protein was added at varying concentrations beginning at E14, after explants had been treated for 24 hours with 10 nM RU486. A small but statistically significant increase in the percentage of labeled SCs was observed in explants treated with 2.5, 5, or 10 μg/ml of DLL1-Fc, as compared to paired controls treated with an equivalent concentration of control Fc protein, with an average increase from 19.7% to 23.3% (Fig. 7C-F, p<0.005). We also observed a small decrease in the percentage of tdTomato-labeled HCs (Fig. 8E, p<0.005). This result demonstrates that treating cochlear explants with exogenous DLL1-Fc causes some Atoh1-expressing cells that had been fated to become HCs to be converted to SCs, although fewer cells were converted from HC to SC than were converted from SC to HC by the addition of DAPT, perhaps because it is more difficult to convert a committed HC to a SC, as compared to converting a less-committed SC to a HC.

Inhibition of cochlear outgrowth increases the number of Atoh1-expressing SCs

Because growth, morphogenesis, and patterning of the cochlea all occur simultaneously, we hypothesized that disrupting the outgrowth of the cochlear epithelium might also influence cell fate decisions, possibly as a result of changes in Notch signaling caused by altered cell-cell interactions. Extension of the cochlear duct had been previously shown to depend on the activity of non-muscle Myosin II, which can be blocked specifically by the pharmacological inhibitor blebbistatin (Yamamoto et al., 2009). Therefore, E13 cochlear explant extension assays were established as described previously (Wang et al., 2005; Yamamoto et al., 2009). Explants were treated for 24 hours with RU486 and then cultured with or without 10 μM blebbistatin for the remaining 5 DIV. As expected, explants treated with blebbistatin were much shorter than paired controls (Fig. 9A, B), and the pattern of the sensory epithelium was disrupted, lacking the normally ordered rows of cells (Fig. 9C, D). In addition, the percentage of tdTomato-positive SCs increased from 8% in controls to 12% in paired blebbistatin explants (Fig. 9E, p<0.005). The lower percentage of labeled SCs in both controls and blebbistatin-treated explants (compared to other explants treated with 10 nM RU486) is likely due to the removal of the apical portion of the cochlea (which contains more tdTomato-positive SCs) as a precondition for the extension assay. This result demonstrates a role for cochlear outgrowth in the determination of cell fates within the OC.

Figure 9. Inhibition of cochlear outgrowth leads to change in fates of Atoh1-positive cells.

Figure 9

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A, B. Basal halves of E13 explants treated with 10 nM RU486 for 24 hrs. and maintained for 6 DIV; stained for tdTomato (magenta) and MyoVIIa (green). The control (A) shows normal extension and patterning while the paired explant treated with 10 μM blebbistatin beginning after 1 DIV is markedly shortened in length and disruption in cellular patterning is apparent. Dashed lines indicate position of high magnification images in (C, D). C, D. Open arrowheads indicate tdTomato- positive SCs and arrows show tdTomato-negative HCs as in previous figures. C. Control explant. HCs and SCs are present in ordered rows, and the separation of IHCs and OHCs is obvious. D. Blebbistatin-treated explant. The rows of HCs are disorganized, with both regions of closely packed HCs and gaps where no HCs are present. There is no discernible separation between IHCs and OHCs. Several tdTomato-positive SCs are visible (open arrowheads). E. Percentages of all tdTomato-labeled cells exhibiting a SC morphology, and percentages of all MyoVIIa-positive cells also labeled with tdTomato. Data are from paired explants from multiple experiments, and expressed as means ± SEM. *p <0.05, ** p<0.01 F. Relative expression levels of Hes1 and Hes5 in blebbistatin-treated cochlear explants. Explants were established and treated as in (A–D), and relative gene expression levels determined by qPCR after 6 DIV. Hes5 expression is significantly increased relative to control at 10 μM blebbistatin (p <0.05). Error bars represent SEM.

One possible explanation for the increase in Atoh1-positive SCs could be aberrant Notch-mediated lateral inhibition; since the cochlear epithelium fails to extend normally, the cells of the epithelium remain at a higher density with a greater number of cell-cell contacts. As a result, Atoh1-positive cells that would normally be fated to become HCs may remain in contact with other Atoh1-positive cells that have already begun to express the Notch ligands DLL1 and JAG2. Prolonged contact could lead to increased levels of Notch signaling and therefore to a conversion to a SC fate. To examine this hypothesis, we performed quantitative rt-PCR (qPCR) for the Notch target genes Hes1 and Hes5 (Zheng et al., 2000; Zine et al., 2001). While Hes1 expression was unchanged in explants treated either with 3 or 10 μM blebbistatin, Hes5 expression was increased significantly at 10 μM blebbistatin (p < 0.05) compared to controls (Fig. 9F). This result is consistent with the hypothesis that inhibition of cochlear outgrowth leads to an aberrant increase in Notch signaling, with resulting changes to the fates of cells within the OC (Murata et al., 2009). However, we cannot rule out the possibility that changes in expression of Hes5 arise as a result of prolonged contacts between HCs and SCs which occurs in response to inhibition of extension.

Discussion

While Atoh1 is known to be necessary for hair cell formation, questions regarding the specific role of this protein have persisted since the initial description of the inner ear phenotype in Atoh1 mutant mice. The results presented here demonstrate that up to 30% of cells within the OC that are initially Atoh1-positive become SCs, not HCs, confirming that ATOH1 acts at the level of HC specification, and that Atoh1 expression does not irreversibly commit prosensory cells to a HC fate. Rather, the onset of Atoh1 expression marks the beginning of a dynamic temporal process in which initially low levels of Atoh1 are gradually increased, or inhibited, through multiple signaling pathways. ATOH1 is known to bind to one of its own enhancers leading to the formation of a strong positive feedback loop (Helms et al., 2000), while recent studies have identified several signaling pathways, including Notch signaling, Inhibitors of differentiation (Ids), Sox2, Neurogenin1, and NeuroD (Dabdoub et al., 2008; Doetzlhofer et al., 2009; Jahan et al., 2010; Jones et al., 2006; Kiernan et al., 2005; Raft et al., 2007), which play a role in suppressing the activity of ATOH1. Cells able to maintain expression of Atoh1, a process that probably requires the continued presence of ATOH1 itself, will develop as HCs, while cells that are unable to maintain or increase their initial Atoh1 expression fail to complete the HC program. Although canonical Wnt signaling has been implicated, the factors that initiate Atoh1 expression are not known, nor is the process by which individual cells are selected to maintain/increase Atoh1 expression.

One signaling pathway that clearly plays a role in Atoh1 regulation is the Notch signaling pathway. Blocking Notch with DAPT resulted in a significant reduction in the percentage of Atoh1-positive SCs cells, as nearly all Atoh1-positive cells within the sensory epithelium developed as HCs. These results suggest that many Atoh1-expressing cells that would have become SCs were converted to HCs. Atoh1-positive SCs that remained in DAPT treated explants were largely localized to the pillar cell (PC) region, located between the IHCs and OHCs, consistent with recent results (Doetzlhofer et al., 2009) that showed that PC fate is dependent on FGF signaling, rather than Notch signaling. An increased number of tdTomato-negative HCs were observed in DAPT-treated explants, but it did not appear that any cells were recruited from outside the sensory epithelium to become HCs. The additional tdTomato-negative HCs were found intermingled with the tdTomato-positive HCs, rather than located near the medial or lateral edges of the OC. Therefore, these additional HCs were presumably derived from unlabeled, Atoh1-negative, SCs that later adopted a HC fate in response to inhibition of Notch signaling.

In contrast, we were able to convert a smaller percentage of Atoh1-positive HCs to SCs by adding an exogenous Notch-ligand, DLL1-Fc. The difference in efficacy of the two manipulations of Notch activity is probably mostly due to inefficient activation of Notch signaling by the DLL1-Fc protein, but may also reflect the inherent tendency of prosensory cells to adopt a HC fate. In order to be driven towards a SC fate, a cell must be actively inhibited from becoming a HC. Further, once a prosensory cell begins to express Atoh1/ATOH1 robustly, it is likely difficult to divert that cell from HC differentiation. Finally, these results demonstrate that modulation of Atoh1 activity is an important consequence of Notch signaling within the inner ear. While this had been assumed for some time, a direct demonstration had not been presented.

Concomitant with Notch-mediated sorting of Atoh1-positive cells into the proper mosaic of HCs and SCs is active outgrowth and extension of the cochlear duct. Our results using the Myosin II inhibitor blebbistatin indicate that coordination between these two processes is important for patterning of the OC. When normal extension of the cochlear epithelium is blocked, the proportion of Atoh1-expressing cells that develops as SCs increases. Our hypothesis that this increase in SCs is due to an increase in Notch signaling is supported by the increased expression of the Notch target Hes5 in blebbistatin-treated explants. This suggests that the separation of developing HCs that arises during cochlear extension acts to limit the effects of Notch signaling, most likely as a result of changes in the spatial relationships between developing prosensory cells. Previous studies have shown that the average number of cell contacts per HC drops from six to four as the cochlea extends (McKenzie et al., 2004), a process that is presumably disrupted when extension is inhibited. Finally, the observation that in blebbistatin-treated explants Hes5, which is expressed in Dieters’ cells, increased, while Hes1, which is primarily found in Hensen’s cells (Murata et al., 2009), was unchanged, suggests that the increase in SCs is probably a result of HCs converting to Dieters’ cells; however, the patterning defects that arise from blebbistatin treatment make it difficult to determine specific SC phenotypes. Regardless, these results demonstrate a direct link between cochlear outgrowth and cell fate, implicating physical rearrangements of developing prosensory cells as an important factor in limiting the number of HCs within the OC.

The observation that Notch signaling was disrupted when cochlear outgrowth was inhibited may provide a mechanistic explanation for some of the phenotypes observed in mouse mutants with shortened cochleae. Deletion of either Neurog1 or FoxG1 results in cochleae that are approximately one-half of normal length (Ma et al., 2000; Pauley et al., 2006) and while the overall number of hair cells are reduced, patterning defects in these mutants are comparable to phenotypes from mice with mutations in Notch1, Dll1, Jag2, HES1 or HES5 (Brooker et al., 2006; Kiernan et al., 2005; Lanford et al., 1999; Zheng et al., 2000; Zine et al., 2001). While patterning defects in shortened cochleae almost certainly arise simply because cells with the prosensory domain cannot physically extend, these results suggest that disruptions in Notch signaling may also contribute to an overabundance of hair cells towards the apical portion of the cochlea in these mutants.

By varying the timing of Cre*PR induction, we were able to visualize a largely basal-to-apical gradient (actually initiating in the mid-basal region) in the restriction of Atoh1-expression to HCs, confirming that determination of cell fate proceeds in a gradient that precedes the gradient of cellular differentiation. However, while modulation of Atoh1 expression, and subsequent effects on cell fate appear to occur as a prolonged process that begins on E13 and is not fully completed until E17 or later, the onset of Atoh1-expression apparently occurs much more rapidly. Cre*PR induction for as little as one hour on E13 was sufficient to label cells along the entire length of the cochlea, including cells that would ultimately develop as OHCs located in the extreme apex of the OC. Similarly, while an expected medial-to-lateral gradient in SC specification was observed, no such gradient was observed in terms of HC specification. These results are consistent with previous work that reported rapid onset of Atoh1 expression along the length of the cochlea at E13.5 based on expression of lacβ under the control of the endogenous Atoh1 promoter (Woods et al., 2004; Yamamoto et al., 2009). In contrast, the gradient in Atoh1 expression observed by in situ hybridization (Lanford et al., 1999; Woods et al., 2004) or in several transgenic Atoh1-reporter lines (Chen et al., 2002; Matei et al., 2005) more likely reflects the subsequent establishment of strong expression of Atoh1 in cells that have become committed to develop as hair cells. The difference in timing between these two events (the initial onset of the HC commitment program via a uniform low level of Atoh1 expression along the length of the cochlear duct versus the cell fate determination, commitment, and differentiation that will not be completed in some of these cells until E17 or later) suggests that ongoing inhibition of ATOH1 is seemingly necessary to prevent cellular specification and commitment in more apical regions of the duct. These results are consistent with recent work demonstrating the importance of various pathways and molecules, including Inhibitors of differentiation (Ids), Sox2, Neurogenin1, and NeuroD (Dabdoub et al., 2008; Jahan et al., 2010; Jones et al., 2006; Raft et al., 2007), in suppressing the activity of ATOH1. In particular, Sox2 and several Id molecules are down-regulated in basal-to-apical gradients that precede HC commitment.

Finally, the demonstration that ATOH1 protein is absent from the basal HCs as early as P0, while residual Atoh1 mRNA is still clearly detectable, suggests that additional levels of regulation of ATOH1 protein may be present. In other contexts, ATOH1 can be the target of proteasome-mediated degradation in response to either Wnt or Bmp signaling (Tsuchiya et al., 2007; Zhao et al., 2008). Since both of these factors are present within the developing cochlear duct, active degradation of ATOH1 protein may play a role in a rapid termination of ATOH1 activity even while Atoh1 mRNA is still present within developing HCs. While further studies are clearly required, these results suggest intriguing new possibilities regarding the spatial and temporal resolution of ATOH1 activity within the cochlea.

  • Atoh1 is expressed in both hair and supporting cell precursors of the cochlea

  • Activation of Notch signaling inhibits Atoh1 expression and inhibits hair cell fate

  • Inhibition of cochlear outgrowth results in changes in cell fate

  • ATOH1 protein is down-regulated prior to Atoh1 mRNA in developing hair cells

Acknowledgments

We thank H. Zoghbi for sharing reagents prior to publication, T. Okano for help with statistical analysis, and S. Raft, S. Maricich, and M. Kelly for critical comments on the manuscript. This work was supported by The Howard Hughes Medical Institute (M.F.R.), and the Intramural Research Program at the National Institute on Deafness and Other Communication Disorders.

Footnotes

The authors declare no conflict of interest.

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Contributor Information

Thomas M. Coate, Email: coatet@nidcd.nih.gov.

Matthew W. Kelley, Email: kelleymt@nidcd.nih.gov.

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