Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea

Alain Dabdoub; Chandrakala Puligilla; Jennifer M. Jones; Bernd Fritzsch; Kathryn S. E. Cheah; Larysa H. Pevny; Matthew W. Kelley

doi:10.1073/pnas.0808175105

New Research In

Edited by Gail R. Martin, University of California, San Francisco, San Francisco, CA, and approved October 2, 2008
↵¹A.D. and C.P. contributed equally to this work. (received for review August 18, 2008)

Abstract

Sox2 is a high-mobility transcription factor that is one of the earliest markers of developing inner ear prosensory domains. In humans, mutations in SOX2 cause sensorineural hearing loss and a loss of function study in mice showed that Sox2 is required for prosensory formation in the cochlea. However, the specific roles of Sox2 have not been determined. Here we illustrate a dynamic role of Sox2 as an early permissive factor in prosensory domain formation followed by a mutually antagonistic relationship with Atoh1, a bHLH protein necessary for hair cell development. We demonstrate that decreased levels of Sox2 result in precocious hair cell differentiation and an over production of inner hair cells and that these effects are likely mediated through an antagonistic interaction between Sox2 and the bHLH molecule Atoh1. Using gain- and loss-of-function experiments we provide evidence for the molecular pathway responsible for the formation of the cochlear prosensory domain. Sox2 expression is promoted by Notch signaling and Prox1, a homeobox transcription factor, is a downstream target of Sox2. These results demonstrate crucial and diverse roles for Sox2 in the development, specification, and maintenance of sensory cells within the cochlea.

The sensory epithelium of the mammalian cochlea (the organ of Corti) develops from a pool of prosensory cells derived from the ventral region of the otocyst. Proper development of the cochlea requires that cochlear progenitor cells transition through states of developmental competence, coordinated cell cycle exit, and cell fate specification and differentiation to generate the distinctly fated cell populations within the highly ordered mosaic of the organ of Corti (1). The signal transduction pathways that coordinate cochlear prosensory specification are only beginning to be identified. Moreover, since these signal transduction pathways are unlikely to be linear cascades, it will also be necessary to determine how different pathways are organized into complex signaling networks that ultimately generate precise and unique responses within individual cochlear prosensory cells.

Sox (SRY related HMG box) proteins are a group of transcription factors that regulate diverse developmental processes; for instance, Sox2 is a universal marker of stem cells and is also expressed in neural progenitor cells at different stages of central nervous system development. Sox2, along with Sox1 and Sox3, comprise the SoxB1 group. Members of this group are thought to maintain neural precursor cells in a progenitor state by inhibiting bHLH-mediated neuronal differentiation (2). Reciprocally, bHLH proteins must suppress expression and activity of SoxB1 proteins to induce cellular differentiation. A recent loss-of-function study demonstrated that Sox2 is required for development of sensory epithelia, including the organ of Corti, within the inner ear (3). However, despite its absolute necessity for the formation of inner ear sensory epithelia, the specific role of Sox2 remains mostly unknown.

In this study, we elucidated the developmental role of Sox2 through gain- and loss-of function studies in addition to identification of both upstream regulators and downstream targets. The results of these experiments enabled us to establish a Sox2 signaling cascade that includes the Notch signaling pathway upstream of Sox2 and the transcription factor Prox1 as a downstream target of Sox2 in the cochlea. Moreover, we present in vitro evidence consistent with a reciprocal antagonistic relationship between Sox2 and Atoh1, a bHLH protein, in which Sox2 expression represses Atoh1-induced hair cell formation while expression of Atoh1 leads to down-regulation of Sox2. These conclusions are supported by the demonstration of ectopic inner hair cells in cochleae from Sox2 hypomorphic mice, suggesting that the level of Sox2 determines, at least in part, the number of cells that develop as inner hair cells. These results suggest that Sox2 expression is required for specification of prosensory cells but that subsequent down-regulation of Sox2 is similarly required for a subset of those cells to differentiate as hair cells.

Results

Sox2 Is Expressed in the Prosensory Region of the Cochlea.

As a first step toward understanding the role of Sox2, we examined its expression in the developing cochlea. Within the cochlear duct at E12.5, Sox2 is expressed in a band of cells located in the medial half of the duct that appear to correspond with the prosensory domain, the population of cells that will give rise to the organ of Corti (Fig. 1A). This was confirmed by comparing the expression of Sox2 with p27^kip1, a known marker of the prosensory domain (4) at E14 (supporting information (SI) Fig. S1). At E16, the band of Sox2 expression within the cochlear duct correlates with the position of the developing organ of Corti (Fig. 1B). In addition, Sox2 is expressed in cells in Kolliker's organ (KO) located adjacent to the medial edge of the developing sensory epithelium. Within the organ of Corti, Sox2 expression begins to be down-regulated in cells that will differentiate as hair cells while it is maintained in cells that will develop as supporting cells—inner phalangeal cells, inner pillar cells, outer pillar cells, Deiters' cells, and Hensens' cells (Fig. 1B). To confirm that down-regulation of Sox2 correlates with hair cell differentiation, expression of Sox2, and the bHLH transcription factor, Atoh1 was compared at E16.5. Sox2 (brown) in Atoh1-positive hair cells (blue) is downregulated (Fig. 1C). At P0, expression of Sox2 is restricted to supporting cells and to a group of cells within KO (Fig. 1D) (5).

Fig. 1.

Sox2 is expressed in the prosensory domain and inhibits hair cell formation. (A-D) Immunohistochemistry using anti-Sox2 (red in A,B, and D and brown in C) and actin (green in A, B, and D) on mid-modiolar cross-sections of WT cochleae shows Sox2 expression at E12.5, E16, and P0. Sox2 is broadly expressed in the prosensory cells at E12.5 (A); however, by E16 Sox2 levels are downregulated in cells that will subsequently acquire a hair cell fate (B, arrows). (C) Co-localization of Sox2 (brown) and Atoh1 (β-gal staining in blue) in the organ of Corti of Atoh1^LacZ^/+ mice at E16.5 demonstrates downregulation of Sox2 in Atoh1-positive hair cells. (D) By P0 the expression of Sox2 is restricted to supporting cells (Inset: organ of Corti) with weak expression in a subset of cells within KO. Sox2 overexpression inhibits hair cell formation. (E) Low-magnification confocal image of a cochlear explant culture transfected with Sox2.nucEGFP. Explants are comprised of three regions, KO, the SE, and the LER, see Experimental Procedures for details. Transfected cells are typically present in all three regions (arrows in KO and LER, and arrowhead in SE). (F) High-magnification confocal image of a cochlear prosensory cell transfected with Sox2.nucEGFP (arrowhead). The transfected cell is negative for the hair cell marker Myo6 (red); (Inset) Z-stack confocal cross-section of the cell illustrated in F. Although the cell is located in the hair cell layer, it is negative for Myo6. KO, Kolliker's organ; LER, lesser epithelial ridge; SE, sensory epithelium; OC, organ of Corti; IHC, inner hair cell; O1-O3, outer hair cells; IPh, inner phalangeal cell; IP, inner pillar cell; OP, outer pillar cell; D1-D3, Deiters' cells; HeC, Hensens' cells. (Scale bars, A, B, D, 20 μm; C, 10 μm; E, 50 μm; F, 10 μm.)

Forced Expression of Sox2 Inhibits Hair Cell Formation.

Previous results had demonstrated that Sox2 expression is necessary for development of all of the cell types within the organ of Corti, including mechanosensory hair cells and non-sensory supporting cells (3). To determine whether the observed down-regulation of Sox2 is necessary for hair cell formation, prosensory cells were transfected with Sox2.nucEGFP at E13 resulting in maintenance of expression of Sox2. The expression vector also contained an internal ribosome entry site (IRES) sequence which resulted in the generation of independent transcripts for nuclear-localized enhanced green fluorescent protein (nuclear EGFP) under the control of the same promoter. Cochlear explants were maintained for 6 days in vitro and cells that maintained expression of Sox2 in the sensory epithelium were analyzed using expression of Myosin6 (Myo6) as a marker for hair cell development. While Myo6 is expressed in a number of different tissues throughout the body, within the inner ear, it is expressed in all hair cells and in no other cell types (6). Quantification of transfected cells indicated that 99.4% of cells transfected with Sox2 in the sensory epithelium were inhibited from developing as hair cells (Fig. 1 E and F and Table S1) suggesting that maintenance of high levels of Sox2 expression inhibits hair cell formation. In contrast, 50.1% of prosensory cells transfected with a control vector expressing only nuclear EGFP developed as hair cells (Table S1). Overexpression of Sox2 had no effect on support cell fate (data not shown) demonstrating a specific inhibition of hair cell fate by Sox2.

An Antagonistic Relationship Between Sox2 and Atoh1.

Following a period of co-expression with Atoh1, Sox2 becomes down-regulated in progenitor cells that will develop as hair cells. The timing of Sox2 down-regulation correlates with increased expression of Atoh1 in the same cells. This pattern of expression, along with the demonstrated existence of an antagonistic relationship between SoxB1 and bHLH transcription factors in the CNS (2), prompted us to investigate whether a similar relationship exists between Sox2 and Atoh1. As a first step, P19 embryonic carcinoma cells, which express Sox2 endogenously (Fig. 2 A and B), were transfected with Atoh1.EGFP containing an IRES sequence (Fig. 2C) and assayed for expression of Sox2 after 24 h. Transfected cells were consistently Sox2-negative suggesting that Atoh1 antagonizes Sox2 expression (97% of Atoh1-transfected cells were negative for Sox2 expression; n = 4, t = 63) (Fig. 2 A-D).

Fig. 2.

An antagonistic relationship between Sox2 and Atoh1. (A-D) P19 cells, labeled with DAPI (blue in A), endogenously express Sox2 (red in B). However, the cell transfected with Atoh1.EGFP (asterisk in A-D) is negative for Sox2 expression (B-D). Sox2 antagonizes Atoh1 in vitro. (E-G) Cells within KO or the LER transfected with Atoh1.EGFP alone are positive for Myo6 (red) indicating development as hair cells (E-F) while Sox2-transfected cells never develop into hair cells (G). (H-J) Cells within the LER that are co-transfected with Sox2.nucEGFP and Atoh1.EGFP. One cell is positive for Myo6 (red), while the other is not, illustrating the antagonistic interaction between Sox2 and Atoh1. Early hair cell differentiation and formation of extra hair cells in Sox2 hypomorphic cochlea. (K-O) Cross-sections of the basal turn of the cochlea from Sox2^+/+ and Sox2^EGFP/LP mice at E15.5. (K) Anti-Myo6 labeling shows the presence of a single inner hair cell in the Sox2^+/+ cochlea (arrowhead). (L) In contrast, the Sox2^EGFP/LP cochlea contains a single inner hair cell (arrowhead) and two outer hair cells (arrows). (M and N) Analysis of hair cells (labeled with anti-Myo6) in cochleae from E18 littermates. Progressive decrease in the level of Sox2 activity result in an increase in the number of extra inner hair cells (stars in N). (O) Quantification of extra inner hair cells in Sox2^+/+ (n = 5), Sox2^EGFP^/+ (n = 5), and Sox2^EGFP/LP (n = 3 from 2 mice) littermate cochleae indicates a significant increase in extra inner hair cells in Sox2^EGFP/LP cochlea (P < 0.001). Error bars are S.E.M. IHC, inner hair cell; O1-O3, outer hair cells. (Scale bars, A-J, 10 μm; K-N, 20 μm.)

To examine whether Sox2 antagonizes the effects of Atoh1, cells within KO or the lesser epithelial ridge (LER, located lateral to the sensory epithelium) were transfected with either Atoh1 alone or a combination of Atoh1 and Sox2. Transfection of Atoh1.EGFP resulted in expression of the hair cell marker Myo6 in 97.2% of transfected cells (Fig. 2 E and F and Table S1) (7, 8) while cells transfected with Sox2.nucEFGP did not express hair cell markers (Fig. 2G and Table S1). To confirm that transfection with Atoh1.EGFP results in the induction of a hair cell fate, transfected cells were also assayed for the expression of a second hair cell marker, Myosin7a (6) and for the presence of a stereociliary bundle. Nearly all Atoh1.EGFP-transfected cells expressed Myosin7a and had a stereociliary bundle (Fig. S2). In contrast, only 50.1% of cells co-transfected with Atoh1.EGFP and Sox2.nucEGFP developed as hair cells as compared with 97.2% when Atoh1 was singly transfected (Fig. 2 H-J and Table S1). These results suggest that even though Sox2 is necessary for the expression of all or most aspects of the prosensory domain, Sox2 also acts as an antagonist by inhibiting the ability of Atoh1 to induce hair cell formation. If this is the case, then reducing, but not eliminating, Sox2 activity might lead to premature formation and over-production of hair cells due to a decrease in the antagonistic effect on Atoh1 function. To test this hypothesis, we examined cochleae from hypomorphic Sox2^EGFP/LP mice which express Sox2 at approximately 30% of normal levels (9). At E15.5, wild-type cochleae contain only a single row of Myo6 positive inner hair cells (Fig. 2K). This single row of inner hair cells extends along the basal half of the cochlear duct. This pattern is consistent with the known basal-to-apical gradient of hair cell differentiation. In contrast, in cochleae from E15.5 Sox2^EGFP/LP littermates, a single row of inner hair cells and two rows of outer hair cells were clearly evident in the basal half of the cochlear duct (Fig. 2L). Moreover, by E18 a significant increase in the number of inner hair cells was observed along the entire length of the basal-to-apical axis of the cochlea in Sox2^EGFP/LP mice (P < 0.001) (Fig. 2 M-O). The increase in inner hair cell number was not at the expense of support cells as marked my Prox1 or Sox2 (data not shown). No significant change was observed in the length of the sensory epithelium or in the number of outer hair cells. Moreover, the epithelium was continuous along its entire length. A comparison between cochleae from animals with different levels of decreased Sox2 activity demonstrated a dose-dependent effect on the number of additional inner hair cells (Fig. 2 O).

Sox2 Induces Prox1 Expression in the Cochlea.

The initial onset and pattern of expression for Sox2 coincide with, but precedes and is slightly larger than, that of Prox1, a homeodomain transcription factor that is expressed in the developing sensory epithelium and also becomes restricted to supporting cell nuclei by P0 (10, 11) (Fig. 3 A and A′). Since expression of Prox1 overlaps with Sox2 in the lateral region of the cochlear duct (Fig. 3B), we speculated that Sox2 might be required for Prox1 expression. To examine this hypothesis, expression of Prox1 was determined in cochleae from Sox2^Lcc/Lcc (light coat and circling) mice (3). Consistent with this hypothesis, Prox1 expression was absent in cochleae from Sox2^Lcc/Lcc mice at both E15.5, a time point just after the normal onset of Prox1 expression (Fig. 3 C and D), and P0 (Fig. S3). To determine whether Sox2 induces Prox1 expression, Sox2 was ectopically expressed in cells within cochlear explants. Forced expression of Sox2.nucEGFP induced expression of Prox1 in 100% of transfected cells (n = 4, t = 103) located throughout the cochlea including KO (Fig. 3 E-G) and the LER (Fig. 3 H-J). In addition, cells transfected with Sox2.nucEGFP in the developing sensory epithelium show an apparent increase in Prox1 expression (Fig. S4). In contrast, cells transfected with nuc.EGFP alone did not express Prox1 (data not shown). To ascertain if down-regulation of Prox1 is required for hair cell formation, cochlear prosensory cells were transfected with Prox1.nucEGFP; 95.5% of cells that maintained expression of Prox1 were inhibited from developing as hair cells as assayed by Myo6 (Fig. 3 K and L and Table S1), suggesting that the inhibitory effects of Sox2 could be mediated, at least in part, by Prox1. To test this hypothesis directly, non-prosensory cells located in KO or the LER were co-transfected with Atoh1.EGFP and Prox1.nucEGFP as described previously. Only 4.5% of co-transfected cells developed as hair cells (Fig. 3M and Table S1), strongly supporting the hypothesis that Prox1 plays a key role in the antagonistic effects of Sox2.

Fig. 3.

Sox2 regulates Prox1 expression. (A and A′) Labeling of Prox1 (red) and actin (green) on a mid-modiolar cross-section from a WT cochlea at P0 shows expression of Prox1 in a subset of support cell nuclei; IP, OP, and D1-D3. (B) Double-immunolabeling using anti-Prox1 (red) and anti-Sox2 (green) indicates overlap of Prox1 and Sox2 expression in the lateral region including PC and DC nuclei. (C and D) Prox1 is absent in cochleae from Sox2^Lcc/Lcc mice. (C) Anti-Prox1 (red) staining labels nuclei of prosensory cells (circle) at E15.5. (D) In contrast with WT, Prox1 staining is absent in Sox2^Lcc/Lcc mutant cochlea at E15.5. DAPI (blue) staining in C and D shows cell nuclei. Sox2 induces ectopic expression of Prox1. (E-J) Sox2.nucEGFP transfected in cells within KO (E-G), or the LER (H-J) are positive for Prox1 (red). (K and L) Forced expression of Prox1 inhibits hair cell formation. Cochlear prosensory cells were transfected with Prox1.nucEGFP. (K) Low-magnification view of confocal image of explant culture illustrating Prox1.nucEGFP transfection (green) in KO and SE (arrowheads). (L) High-magnification view of the SE demonstrating that Prox1.nucEGFP expressing cells (arrowheads) are negative for the hair cell marker Myo6 (red). (M) Co-transfection with Prox1.nucEGFP and Atoh1.EGFP results in inhibition of Atoh1. Despite expression of Atoh1, the cell is negative for Myo6 (red). IHC, inner hair cell; IP, inner pillar cell; OP, outer pillar cell; D1-D3, Deiters' cell; O1-O3, outer hair cells; IPh, inner phalangeal cell; HeC, Hensens' cell; KO, Kolliker's organ; LER, lesser epithelial ridge; SE, sensory epithelium. (Scale bars, A, 20 μm; A′-B, 10 μm; C-K, 20 μm, L, 10 μm, M, 20 μm.)

Notch Signaling Regulates Expression of Sox2.

Jagged1-mediated activation of the Notch pathway has been shown to be required for specification of most inner ear prosensory domains (12). To determine whether expression of Sox2 is dependent on Notch signaling, activation of the Notch pathway was inhibited in cochlear explant cultures beginning at E13 by exposure to the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), which blocks Notch signaling. DAPT treatment in cochlear explants leads to a reduction in the expression of both Sox2 (Fig. 4 A and B) and consequently Prox1 (Fig. 4 C and D), suggesting that Notch signaling is required to maintain Sox2 and Prox1 expression within prosensory cells. However DAPT treatment also results in a significant increase in the number of hair cells as previously reported (13) (Fig. 4 E and F) suggesting that the loss of Sox2 and Prox1 could be a result of changes in cell fate rather than a direct effect of the Notch pathway on Sox2/Prox1 expression in prosensory cells. Therefore, to determine whether activation of Notch is sufficient to induce expression of Sox2 and/or Prox1, Notch1 intracellular domain (NICD) was ectopically expressed in cells within KO or the LER. An average of 96.4% of NICD.EGFP-transfected cells were positive for Sox2 expression (Fig. 4G; n = 3, t = 51). None of the cells transfected with the control vector expressing EGFP alone were positive for Sox2 (n = 3, t = 47; data not shown). However, no NICD.EGFP-transfected cells were observed to express Prox1 (data not shown). These results suggest that activation of the Notch signaling pathway acts to induce ectopic expression of Sox2, but that additional factors are required for the subsequent activation of Prox1 in the same cells. This result is also consistent with the observation that Prox1 is only expressed in a subset of prosensory cells. Furthermore, NICD overexpression in the sensory epithelium was inhibitory to hair cell formation (n = 5, t = 35; data not shown) similar to results obtained for Sox2 overexpression. To confirm that Notch signaling acts upstream of Sox2, we examined Notch1 expression in Sox2^Lcc/Lcc mice at E15.5. As expected, expression of Notch1 is present in these mutants (Fig. 4H). Taken together, our data suggest that while the Notch signaling pathway is independent and upstream of Sox2, it plays a role in the regulation of Sox2 expression. However, our results do not exclude an additional role for Notch signaling downstream of Sox2.

Fig. 4.

Notch signaling is required for Sox2 expression. (A-F) Inhibition of Notch signaling using the γ-secretase inhibitor, DAPT on cochlear explant cultures beginning at E13 results in the reduction of Sox2 (A and B) and Prox1 (C and D) expression. (E and F) In contrast, Myo6 labeling indicates an increase in the number of inner and outer hair cells in DAPT-treated explant cultures. (G) Forced expression of NICD.EGFP in cells within the LER induces Sox2 (yellowish red nuclei) expression. (H) Notch1 expression (brown) is present in E15.5 Sox2^Lcc/Lcc mutant cochleae. (Scale bars, 20 μm.)

Specification of the Prosensory Domain Is Independent of Atoh1.

The results described above demonstrate the molecular signaling cascade that specifies prosensory cells within the organ of Corti. Since it has been reported that hair cells and supporting cells are absent in Atoh1^−/− cochlea (7, 14), we wanted to examine if the formation of the prosensory domain is affected in these mutants. The prosensory marker Sox2 is expressed at E16.5 in the Atoh1^−/− cochlea (Fig. 5 A and B) (3). Since we demonstrated that Notch signaling is upstream of Sox2, we examined the expression of the Notch ligand, Jagged1 in the Atoh1^−/− mice. Jagged1 is expressed in the area that is topographically equivalent to the organ of Corti (Fig. 5 C and D), suggesting that at least the initial phase of Notch signaling is unaffected in these mutants. Finally, since we provide evidence that Sox2 is upstream of Prox1 we examined the expression of Prox1. Prox1 is expressed in the Atoh1^−/− cochlea at E16.5 similar to its expression in Atoh1^+/− cochlea (Fig. 5 E and F). In fact, the expression pattern of Prox1 at E16.5 in Atoh1^−/− cochlea is similar to its expression in undifferentiated sensory epithelia at E14.5 of WT cochleae (10). These data suggest that the prosensory domain in the cochlea is specified independent of Atoh1 and the Notch-Sox2-Prox1 signaling cascade is intact in the Atoh1 mutants.

Fig. 5.

The prosensory domain is present in Atoh1 mutants. Sox2, Jagged1, and Prox1 were used as markers for the prosensory domain. Immunolabeling was done on apical cochlear sections at E16.5. (A and B) Sox2 immunolabeling (brown) in Atoh1^LacZ^/+ and Atoh1^LacZ/LacZ (mutant) cochleae shows no change in Sox2 expression. The X-gal staining (blue) illustrates reporter activity for Atoh1. (C-F) Expression of Jagged1 (red in C and D) and Prox1 (red in E and F) are also present in the Atoh1 mutants. OC, organ of Corti; SG, spiral ganglion. (Scale bars, 20 μm.)

Discussion

Sox2 is one of the earliest transcription factors expressed in the prosensory domain of the otocyst. Mutations in SOX2 lead to sensorineural deafness in humans (15) and to deafness and absence of sensory structures in the inner ears of mice (3). We show here that the role of Sox2 is complex and includes both inductive and antagonistic effects. Our results suggest that the initial expression of Sox2 plays a direct role in establishing the prosensory domain within the cochlea and that Sox2 is permissive for the development of hair cells. The inability of Sox2, or other members of its signaling cascade (Notch signaling, and Prox1), to induce Atoh1 expression suggests that either Sox2 functions in a separate pathway from Atoh1 and unknown factors regulate the initiation of Atoh1 expression or additional factors are required along with Sox2 to induce Atoh1 expression. However, following the onset of Atoh1 expression, our data demonstrate that Sox2 levels become downregulated in differentiating hair cells and that an antagonistic interaction between Sox2 and Atoh1 plays a role in this down-regulation. Similar effects have been observed in the CNS where SoxB1 genes generally become down-regulated upon differentiation (2) and constitutive expression of SoxB1 genes inhibits differentiation (2, 16). In addition, our data indicate that Sox2 inhibits inner hair cell formation in a dose dependent manner, suggesting that the relative ratio of Sox2 to Atoh1 is important for the regulation of inner, and possibly outer, hair cell fate.

Consistent with this hypothesis is the demonstration of premature hair cell differentiation and the formation of extra inner hair cells in cochleae from Sox2 hypomorphic mice, suggesting that under these circumstances, while sufficient Sox2 is expressed for formation of the prosensory domain, the amount of Sox2 is insufficient for its subsequent role as an Atoh1 antagonist. In addition, while forced expression of Atoh1 alone results in a strong induction of expression of the hair cell markers Myosin6 and Myosin7a and the development of a stereociliary bundle in non-sensory cells, co-expression of Sox2 along with Atoh1 significantly reduces the number of transfected cells that express hair cell markers. These data suggest that the capacity of endogenous Atoh1 to direct the commitment of hair cells from prosensory cells seems to be based, at least in part, on its ability to repress Sox2.

Furthermore, we demonstrate that Prox1 is a downstream target of Sox2 and that forced expression of Prox1 resulted in the inhibition of hair cell development. Similarly, co-transfection of Prox1 and Atoh1 resulted in inhibition of the ability of Atoh1 to induce expression of the hair cell marker Myo6 in non-sensory cells suggesting that the inhibitory action of Sox2 is mediated through its ability to induce Prox1 expression. However, endogenous Prox1 expression in the cochlea is restricted to the lateral domain of the sensory epithelium—the outer hair cell region (10, 11)—while data from the Sox2^EGFP/LP hypomorphic mice indicated that the inner hair cell (Prox1-negative) region is most affected. These results suggest that while Sox2 acts to inhibit Atoh1 through Prox1 in the outer hair cell region, it also acts, either directly or through an unidentified intermediary targets, to inhibit Atoh1 in the inner hair cell region.

While the factors that determine the specific number of cells that will develop as hair cells are not known, the relatively invariant number of hair cells within the mammalian cochlea strongly suggests a highly specific regulatory mechanism. One possible factor could be the expression of Id (Inhibitors of differentiation) genes which have been shown to antagonize bHLH function and are specifically down-regulated as cells begin to develop as hair cells (8). These data suggest that down-regulation of Id expression in a subset of progenitor cells could allow Atoh1 activity to increase sufficiently to effectively antagonize and down-regulate Sox2, leading to induction of hair cell formation.

The working definition of the cochlear prosensory domain is a population of cells with the unique ability to develop as either hair cells or supporting cells. However, the factors that specify this domain have not been determined. Here we show that the prosensory domain, marked to differing degrees by Jagged1, Sox2, and Prox1, forms normally in the absence of Atoh1. These results are consistent with the suggestion that Atoh1 acts as a pro-hair cell rather than a prosensory gene (7). In contrast, both Jagged1 and Sox2 are expressed in patterns that are consistent with prosensory specification and disruption of either results in the absence of most or all prosensory cells (3, 12). Moreover, Sox2 expression is downregulated in the cochleae of Jagged1 conditional mutants (12), while Jagged1 expression persists in Sox2^Lcc/Lcc mice (A. Pelling and K.S.E.C., unpublished data), suggesting that Jagged1 acts upstream of Sox2. This conclusion is supported by our results demonstrating that Notch signaling is upstream of Sox2. These results, along with the results of forced expression of Notch-ICD in chick inner ear (17), suggest that activation of one of the Notch receptors acts to induce prosensory identity. Here we demonstrate that this probably occurs through induction of Sox2. This is in contrast to the eye and neocortex, where it has been shown that Sox2 appears to act upstream of Notch signaling (9, 18). However, the demonstration that inhibition of Notch signaling within an existing prosensory domain leads to down-regulation of Sox2 expression suggests that the role of Notch in Sox2 expression extends beyond the period of Sox2 induction. Moreover, we hypothesize that the extra hair cells observed in Notch1 conditional mutant cochleae (19) might occur, at least in part, because the loss of Notch1 leads to a decrease in the level of Sox2 within the existing prosensory domain. Since Sox2 acts to antagonize Atoh1, the loss of Sox2 could result in increased activity of Atoh1 and, as a result, to an increase in the number of cells that develop as hair cells, similar to the phenotype in Sox2 hypomorphic mice.

Our gain-of-function experiments using KO and the LER as model cell populations to examine the signaling pathways that specify prosensory cell fate within the otocyst suggest a signaling cascade in which Sox2 is expressed in response to Jagged1-mediated activation of Notch, and that Sox2 in turn induces expression of Prox1. However, it is important to note that while forced expression of either NICD or Sox2 leads to induction of Sox2 or Prox1 respectively, data on the endogenous expression patterns of activated Notch, Sox2, and Prox1 (1) which are not exactly overlapping, suggest that additional unidentified modifiers act to modulate the expression of each target.

In summary, the results presented here demonstrate that Sox2 is centrally situated in inner ear development functioning both as a node, receiving signals from several inputs, including Notch signaling, and as a junction, directing signals to different effectors that apparently play unique roles in sensory development. Additional work to identify other molecular components of the Sox2 pathway should identify crucial co-factors (20, 21) that determine the different molecular effects of Sox2 in different cells and at different developmental time points during inner ear development. In addition to the upstream Notch signaling that we describe, it would be interesting to investigate whether Wnt and/or FGF signaling, both known to play early roles in inner ear development (22–24), also converge to activate Sox2 expression as they do in developing neural plate (25).

Experimental Procedures

Organotypic Explant Cochlear Cultures.

Cochlear cultures from E13 embryos were established as described previously (26) (see SI Methods for more details). To inhibit notch signaling, the γ-secretase inhibitor N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) was added to explants at a concentration of 50 μM and replenished every day for six days. At the conclusion of each experiment, explants were fixed in 4% paraformaldehyde for 30 min and analyzed by immunohistochemistry.

Cell Cultures.

Transfections in P19 cells were carried out using Lipofectamine2000 reagent (Invitrogen) according to manufacturer's instructions. Cells were fixed in 4% PFA for 20 min and analyzed by immunohistochemistry using indicated antibodies. Cell nuclei were counterstained with DAPI nuclear stain (Molecular Probes, 1:5000).

Immunohistochemistry.

Immunocytochemistry was performed on cochleae and cochlear explant cultures as described previously (7, 8) (see SI Methods for more details).

Expression Constructs.

See SI Methods for details.

Electroporation.

Individual cells in cochlear explants were transfected using square-wave electroporation as described previously (8) (See SI Methods for more details).

Quantification of Changes in Cell Fate.

See SI Methods for details.

Generation of Sox2^Lcc/Lcc, Atoh1^lacZ/lacZ, and Sox2^LP/EGFP Mice.

Sox2^Lcc/Lcc (3), Atoh1^lacZ/lacZ (14), and Sox2^LP/EGFP (9) mutants were generated by crossing Sox2^+/Lcc, Atoh1^+/LacZ, and Sox2^+/EGFP x Sox2^+/LP mice respectively.

Acknowledgments

We thank Drs. A. Kiernan, A. Pelling, and T. Friedman for reading the manuscript and C. Woods, CW. Kramer, T. Dennison, and Dr. EC. Driver for technical assistance. K.S.E.C. was supported by the Research Grants Council of Hong Kong HKU7385/02M, B.F. by National Institutes of Health Grant R01 DC005590, L.H.P. by National Institutes of Health Grant R01 EYO1861. This work was supported by the National Institute on Deafness and Other Communication Disorders intramural program.

Footnotes

³To whom correspondence may be addressed. E-mail: adabdoub{at}ucsd.edu or puligillac{at}nidcd.nih.gov
Author contributions: A.D., C.P., and M.W.K. designed research; A.D., C.P., and J.M.J. performed research; A.D., C.P., B.F., K.S.E.C., and L.H.P. contributed new reagents/analytic tools; A.D. and C.P. analyzed data; and A.D., C.P., and M.W.K. wrote the paper.
↵²Present address: Department of Surgery, Division of Otolaryngology, School of Medicine, University of California San Diego, 9500 Gilman Drive, 0666, La Jolla, CA 92093.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0808175105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA

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1. Woods C,
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(2004) Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat Neurosci 7:1310–1318.
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1. Jones JM,
2. Montcouquiol M,
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1. Taranova OV,
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1. Bermingham-McDonogh O,
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(2006) Expression of Prox1 during mouse cochlear development. J Comp Neurol 496:172–186.
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1. Puligilla C,
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1. Kiernan AE,
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3. Gridley T
(2006) The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PloS Genet 2:27–38.
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1. Takebayashi S,
2. et al.
(2007) Multiple roles of Notch signaling in cochlear development. Dev Biol 307:165–178.
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↵
1. Bermingham NA,
2. et al.
(1999) Math1: An essential gene for the generation of inner ear hair cells. Science 284:1837–1841.
OpenUrl Abstract/FREE Full Text
↵
1. Hagstrom SA,
2. et al.
(2005) SOX2 mutation causes anophthalmia, hearing loss, and brain anomalies. Am J Med Genet A 138:95–98.
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1. Graham V,
2. Khudyakov J,
3. Ellis P,
4. Pevny L
(2003) SOX2 functions to maintain neural progenitor identity. Neuron 39:749–765.
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1. Daudet N,
2. Lewis J
(2005) Two contrasting roles for Notch activity in chick inner ear development: Specification of prosensory patches and lateral inhibition of hair-cell differentiation. Development 132:541–551.
OpenUrl Abstract/FREE Full Text
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1. Bani-Yaghoub M,
2. et al.
(2006) Role of Sox2 in the development of the mouse neocortex. Dev Biol 295:52–66.
OpenUrl CrossRef PubMed
↵
1. Kiernan AE,
2. Cordes R,
3. Kopan R,
4. Gossler A,
5. Gridley T
(2005) The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear. Development 132:4353–4362.
OpenUrl Abstract/FREE Full Text
↵
1. Ambrosetti DC,
2. Scholer HR,
3. Dailey L,
4. Basilico C
(2000) Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer. J Biol Chem 275:23387–23397.
OpenUrl Abstract/FREE Full Text
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1. Dailey L,
2. Basilico C
(2001) Coevolution of HMG domains and homeodomains and the generation of transcriptional regulation by Sox/POU complexes. J Cell Physiol 186:315–328.
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1. Riccomagno MM,
2. Takada S,
3. Epstein DJ
(2005) Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh. Genes Dev 19:1612–1623.
OpenUrl Abstract/FREE Full Text
↵
1. Ohyama T,
2. Mohamed OA,
3. Taketo MM,
4. Dufort D,
5. Groves AK
(2006) Wnt signals mediate a fate decision between otic placode and epidermis. Development 133:865–875.
OpenUrl Abstract/FREE Full Text
↵
1. Schimmang T
(2007) Expression and functions of FGF ligands during early otic development. Int J Dev Biol 51:473–481.
OpenUrl CrossRef PubMed
↵
1. Takemoto T,
2. Uchikawa M,
3. Kamachi Y,
4. Kondoh H
(2006) Convergence of Wnt and FGF-signals in the genesis of posterior neural plate through activation of the Sox2 enhancer N-1. Development 133:297–306.
OpenUrl Abstract/FREE Full Text
↵
1. Dabdoub A,
2. et al.
(2003) Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea. Development 130:2375–2384.
OpenUrl Abstract/FREE Full Text

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Table of Contents

Submit

[1] ↵
Kelley MW
(2006) Regulation of cell fate in the sensory epithelia of the inner ear. Nat Rev Neurosci 7:837–849.
OpenUrl CrossRef PubMed

[2] Kelley MW

[3] ↵
Bylund M,
Andersson E,
Novitch BG,
Muhr J
(2003) Vertebrate neurogenesis is counteracted by Sox1–3 activity. Nat Neurosci 6:1162–1168.
OpenUrl CrossRef PubMed

[4] Bylund M,

[5] Andersson E,

[6] Novitch BG,

[7] Muhr J

[8] ↵
Kiernan AE,
et al.
(2005) Sox2 is required for sensory organ development in the mammalian inner ear. Nature 434:1031–1035.
OpenUrl CrossRef PubMed

[9] Kiernan AE,

[10] et al.

[11] ↵
Chen P,
Segil N
(1999) p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development 126:1581–1590.
OpenUrl Abstract

[12] Chen P,

[13] Segil N

[14] ↵
Hume CR,
Bratt DL,
Oesterle EC
(2007) Expression of LHX3 and SOX2 during mouse inner ear development. Gene Expr Patterns 7:798–807.
OpenUrl CrossRef PubMed

[15] Hume CR,

[16] Bratt DL,

[17] Oesterle EC

[18] ↵
Avraham KB,
et al.
(1995) The mouse Snell's waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner hair cells. Nat Genet 11:369–375.
OpenUrl CrossRef PubMed

[19] Avraham KB,

[20] et al.

[21] ↵
Woods C,
Montcouquiol M,
Kelley MW
(2004) Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat Neurosci 7:1310–1318.
OpenUrl CrossRef PubMed

[22] Woods C,

[23] Montcouquiol M,

[24] Kelley MW

[25] ↵
Jones JM,
Montcouquiol M,
Dabdoub A,
Woods C,
Kelley MW
(2006) Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J Neurosci 26:550–558.
OpenUrl Abstract/FREE Full Text

[26] Jones JM,

[27] Montcouquiol M,

[28] Dabdoub A,

[29] Woods C,

[30] Kelley MW

[31] ↵
Taranova OV,
et al.
(2006) SOX2 is a dose-dependent regulator of retinal neural progenitor competence. Genes Dev 20:1187–1202.
OpenUrl Abstract/FREE Full Text

[32] Taranova OV,

[33] et al.

[34] ↵
Bermingham-McDonogh O,
et al.
(2006) Expression of Prox1 during mouse cochlear development. J Comp Neurol 496:172–186.
OpenUrl

[35] Bermingham-McDonogh O,

[36] et al.

[37] ↵
Puligilla C,
et al.
(2007) Disruption of fibroblast growth factor receptor 3 signaling results in defects in cellular differentiation, neuronal patterning, and hearing impairment. Dev Dyn 236:1905–1917.
OpenUrl CrossRef PubMed

[38] Puligilla C,

[39] et al.

[40] ↵
Kiernan AE,
Xu J,
Gridley T
(2006) The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PloS Genet 2:27–38.
OpenUrl

[41] Kiernan AE,

[42] Xu J,

[43] Gridley T

[44] ↵
Takebayashi S,
et al.
(2007) Multiple roles of Notch signaling in cochlear development. Dev Biol 307:165–178.
OpenUrl CrossRef PubMed

[45] Takebayashi S,

[46] et al.

[47] ↵
Bermingham NA,
et al.
(1999) Math1: An essential gene for the generation of inner ear hair cells. Science 284:1837–1841.
OpenUrl Abstract/FREE Full Text

[48] Bermingham NA,

[49] et al.

[50] ↵
Hagstrom SA,
et al.
(2005) SOX2 mutation causes anophthalmia, hearing loss, and brain anomalies. Am J Med Genet A 138:95–98.
OpenUrl

[51] Hagstrom SA,

[52] et al.

[53] ↵
Graham V,
Khudyakov J,
Ellis P,
Pevny L
(2003) SOX2 functions to maintain neural progenitor identity. Neuron 39:749–765.
OpenUrl CrossRef PubMed

[54] Graham V,

[55] Khudyakov J,

[56] Ellis P,

[57] Pevny L

[58] ↵
Daudet N,
Lewis J
(2005) Two contrasting roles for Notch activity in chick inner ear development: Specification of prosensory patches and lateral inhibition of hair-cell differentiation. Development 132:541–551.
OpenUrl Abstract/FREE Full Text

[59] Daudet N,

[60] Lewis J

[61] ↵
Bani-Yaghoub M,
et al.
(2006) Role of Sox2 in the development of the mouse neocortex. Dev Biol 295:52–66.
OpenUrl CrossRef PubMed

[62] Bani-Yaghoub M,

[63] et al.

[64] ↵
Kiernan AE,
Cordes R,
Kopan R,
Gossler A,
Gridley T
(2005) The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear. Development 132:4353–4362.
OpenUrl Abstract/FREE Full Text

[65] Kiernan AE,

[66] Cordes R,

[67] Kopan R,

[68] Gossler A,

[69] Gridley T

[70] ↵
Ambrosetti DC,
Scholer HR,
Dailey L,
Basilico C
(2000) Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer. J Biol Chem 275:23387–23397.
OpenUrl Abstract/FREE Full Text

[71] Ambrosetti DC,

[72] Scholer HR,

[73] Dailey L,

[74] Basilico C

[75] ↵
Dailey L,
Basilico C
(2001) Coevolution of HMG domains and homeodomains and the generation of transcriptional regulation by Sox/POU complexes. J Cell Physiol 186:315–328.
OpenUrl CrossRef PubMed

[76] Dailey L,

[77] Basilico C

[78] ↵
Riccomagno MM,
Takada S,
Epstein DJ
(2005) Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh. Genes Dev 19:1612–1623.
OpenUrl Abstract/FREE Full Text

[79] Riccomagno MM,

[80] Takada S,

[81] Epstein DJ

[82] ↵
Ohyama T,
Mohamed OA,
Taketo MM,
Dufort D,
Groves AK
(2006) Wnt signals mediate a fate decision between otic placode and epidermis. Development 133:865–875.
OpenUrl Abstract/FREE Full Text

[83] Ohyama T,

[84] Mohamed OA,

[85] Taketo MM,

[86] Dufort D,

[87] Groves AK

[88] ↵
Schimmang T
(2007) Expression and functions of FGF ligands during early otic development. Int J Dev Biol 51:473–481.
OpenUrl CrossRef PubMed

[89] Schimmang T

[90] ↵
Takemoto T,
Uchikawa M,
Kamachi Y,
Kondoh H
(2006) Convergence of Wnt and FGF-signals in the genesis of posterior neural plate through activation of the Sox2 enhancer N-1. Development 133:297–306.
OpenUrl Abstract/FREE Full Text

[91] Takemoto T,

[92] Uchikawa M,

[93] Kamachi Y,

[94] Kondoh H

[95] ↵
Dabdoub A,
et al.
(2003) Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea. Development 130:2375–2384.
OpenUrl Abstract/FREE Full Text

[96] Dabdoub A,

[97] et al.

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