Role of p63 and the Notch pathway in cochlea development and sensorineural deafness

Abstract

The ectodermal dysplasias are a group of inherited autosomal dominant syndromes associated with heterozygous mutations in the Tumor Protein p63 (TRP63) gene. Here we show that, in addition to their epidermal pathology, a proportion of these patients have distinct levels of deafness. Accordingly, p63 null mouse embryos show marked cochlea abnormalities, and the transactivating isoform of p63 (TAp63) protein is normally found in the organ of Corti. TAp63 transactivates hairy and enhancer of split 5 (Hes5) and atonal homolog 1 (Atoh1), components of the Notch pathway, known to be involved in cochlear neuroepithelial development. Strikingly, p63 null mice show morphological defects of the organ of Corti, with supernumerary hair cells, as also reported for Hes5 null mice. This phenotype is related to loss of a differentiation property of TAp63 and not to loss of its proapoptotic function, because cochleas in mice lacking the critical Bcl-2 homology domain (BH-3) inducers of p53- and p63-mediated apoptosis—Puma, Noxa, or both—are normal. Collectively, these data demonstrate that TAp63, acting via the Notch pathway, is crucial for the development of the organ of Corti, providing a molecular explanation for the sensorineural deafness in ectodermal dysplasia patients with TRP63 mutations.

Tumor protein p63 (TRP63) is the most ancient member of the p53 family of transcription factors (1) and acts as a key molecule in embryonic development. The structural organization of p63 (2, 3) is similar to p53, containing a transactivation domain (TA), DNA binding domain (DBD), and an oligomerization domain. The expression of p63 is regulated by two distinct promoters giving rise to proteins that either contain (TAp63) or do not contain (ΔNp63) the N-terminal TA domain, which is critical for the transcriptional induction of particular subsets of p63 target genes (2). Both p63 isoforms give rise to differentially spliced mRNAs and proteins, with at least six p63 isoforms (α, β, γ) recognized (1). p63α also contains a sterile α motif (SAM) and a carboxy terminal-inhibitory domain (TID) (4), both absent in p53 (5). The TID binds and inhibits the N-terminal TA domain by masking important N-terminal residues. The importance of this regulatory domain is evident from the identification of mutations in the TAp63α-C terminus in human patients (6).

The ectodermal dysplasia (ED) syndromes are a large and heterogeneous group of inherited human diseases that are characterized by developmental abnormalities of ectodermally derived structures. A large subset of autosomal dominant ED syndromes are caused by heterozygous mutations in the TRP63 gene (7). The prototypic Ectrodactyly, Ectodermal dysplasia, and Cleft lip/palate (EEC) syndrome (Online Mendelian Inheritance in Man: OMIM 604292) has highly variable expression and penetrance. Clinically, EEC patients show ectodermal dysplasia affecting skin, hair, nails and teeth, and facial clefts, as well as frequent lacrimal duct abnormalities, urogenital problems, facial dysmorphism, and hearing loss. Nucleotide sequence analyses have provided evidence for a striking genotype–phenotype correlation with mutations in individual domains of p63 in ED patients (8, 9) The SAM domain seems to be particularly important for skin development, whereas the DBD and TID are crucial for limb development. In general, mutations in EEC patients show substitutions in the DBD that impair p63’s ability to bind to specific target sequences in DNA.

Mice lacking both copies of the Trp63 gene are born lacking limbs, skin, and skin appendages, such as hair shafts, follicles, and sebaceous glands, and consequently die from dehydration shortly after birth (10, 11). ΔNp63 is the predominant isoform in the basal layer of the epidermis (12, 13) and is crucial for the maintenance of epithelial stem cells (10, 11). Accordingly, reconstitution with ΔNp63 at least partially rescues the epidermal defects seen in p63^−/− mice (12). In EEC patients, ΔNp63 mutant proteins accumulate, and these proteins generally show reduced transcriptional activity on skin-specific promoters (compared with TAp63 proteins), although in other ED syndromes ΔNp63 mutant proteins are transcriptionally more active than the WT protein.

TAp63 also exerts critical functions in the development and function of the heart (14) and oocytes (15, 16). The latter suggests a role for this gene in female infertility (17). Only limited information is available for the role of p63 in development and maintenance of other tissues/organs. Although the relationship between ΔNp63 mutations and ED is consistent with the epidermal defects seen in the p63^−/− mice, the role of mutant p63 isoforms in the conductive and sensorineural deafness seen in some patients (8, 18) is unclear. Because a deaf EEC patient presented with abnormalities in the morphology of the cochlea (18), we have investigated the role of p63 in the development of the inner ear and, in particular, the organ of Corti. Our studies, using gene expression analysis and morphological examination of gene-targeted mice, demonstrate that TAp63, through activation of the Notch signaling pathway, is important for normal development of the cochlea.

Results

Deafness in ED Patients and Cochlea Morphology in p63-Null Mice.

To evaluate the incidence of deafness among ED patients with p63 mutations, we analyzed a cohort of 112 patients: 17% of these were affected by deafness, albeit to different extents. Table 1 summarizes the relative incidence of deafness for each ED subgroup. Additional analysis on two new patients revealed by audiometric tests a mild hearing loss, both conductive and sensorineural (Fig. S1 A and B). Because the p63-deficient mice phenocopy to a substantial extent the symptoms observed in ED patients, we investigated whether these mice show cochlear abnormalities, consistent with the human deafness phenotype. To this end, we compared p63^−/− and WT embryos from embryonic day (E)14.5 and E18.5 (adult p63^−/− mice could not be investigated, because the pups die a few hours after birth). H&E staining of sections from embryos showed no structural abnormalities in the cochlea of p63^−/− E14.5 embryos (Fig. S2 A and B). However, morphological defects were clearly detected in the cochlear duct (scala media), scala tympani, and scala vestibuli (Fig. 1 A and B) in E18.5 p63^−/− embryos.

Table 1.

Incidence of deafness in EEC-like patients

p63-associated syndrome	Patients affected, % (n)	Trp63 mutations
EEC syndrome	7 (11/86)	DBD: R204, S272, R279, R304, D312, and ins 3′ end.
RHS syndrome	20 (2/10)	TID: S541 and frameshift.
AEC syndrome	38 (6/16)	SAM: L514, C522, G530, I537, and frameshift
Total EEC-Like	17 (19/112)

AEC, Ankyloblepharon Ectodermal dysplasia Cleft lip/palate; RHS, Rapp-Hodgkin Syndrome.

Fig. 1.

Morphological and functional defects in cochlea of p63-deficient mice. (A and B) H&E-stained sections of murine WT and p63^−/− E18.5 embryos. The p63^−/− embryo shows an abnormal structure of the scalae tympani and media compared with the corresponding WT samples. (Scale bars, 350 μm.) (C) TAp63α and ΔNp63α expression in cochlea and epidermis of WT embryos. Semiquantitative PCR analysis shows much higher expression of TAp63α in cochlear tissue compared with the expression of ΔNp63α. In contrast, expression of only ΔNp63α was seen in the skin; expression was normalized to the expression of actin. (D–F) Immunofluorescence staining of cochlear sections of mouse embryos (E18.5): WT (D), p63^−/− (E), and TAp63^−/− (F). MyoVIIa (green), Cx26 (red), and DAPI nuclei (blue) staining. (Scale bars, 20 μm.) E and F show supernumerary inner and outer hair cells in both p63^−/− and TAp63^−/− embryos. The staining for Cx26 protein suggests that the p63^−/− embryos lack intercellular junctions compared with the WT embryos in which connections were correctly formed; the same effect was observed in TAp63^−/− (F). (G) Auditory evoked potential analysis shows a variable delay of the fifth wave cerebral cortex potentials in response to 8-KHz stimulation. (H) Comparative study of the width (w) and length (L) of the mouse Cochlea by CT images (Fig. S4). (I) Histological analysis of at least three sections (base, middle, apex) from each cochlea TAp63^−/− mice revealed the presence of supernumerary IHC/OHC cells. The supernumerary cells were present at least in 50% of section (n= 6 mice analyzed for each group), ratio shows defective sections over total sections analyzed in TAp63^−/− mice.

Development of the cochlea during embryogenesis occurs from prosensory cells at E14.5, which leads to the formation of both supporting cells as well as inner (IHC) and outer hair cells (OHC). At the molecular level, Sex determining region Y-box 2 (Sox2) is expressed in supporting cells, whereas Myo-VIIa is found in IHC and OHC. In p63^−/− E14.5 embryos, we identified Sox2 (Fig. S2 C and F), Myosin-VIIa (Fig. S2 I and K), and Connexin 26 (Cx26), a cell-specific marker of the stria vascularis (Fig. S2 D and G) in germinative prosensory cells and in the organ of Kolliker. Whereas Sox2 and Myo-VIIa appeared to be normally expressed at this stage, Cx26 showed a slightly altered distribution compared with E14.5 WT embryos (Fig. S2 D and G).

At E18.5, Sox2 expression was detected in supporting cells, at the base of the organ of Corti (Fig. S3 A and D) in both WT and p63^−/− mice. Cx26 was expressed in WT mice, as previously reported (19), in supporting and stria vascularis cells, with modest overexpression seen in the p63^−/− embryos (Fig. S3 B and E), where it was abnormally localized below the stria. The distribution of supporting cells was similar in WT and p63^−/− mice. Staining of hair cells with Myo-VIIa in E18.5 embryos showed an abnormal number of hair cells in p63^−/− mice (Fig. 1 D and E): four OHC and two IHC were identified in p63^−/− mice instead of the regular three OHC and one IHC seen in the WT. Moreover, both IHC and OHC in p63^−/− embryos lacked the characteristic cilia, which are clearly visible in the sections from WT embryos. The abnormal number of hair cells in p63^−/− mice suggests that p63 transcriptional target genes are essential for the normal development of the cochlea.

Selective Involvement of TAp63 Isoform in Cochlea Development.

Whereas ΔΝp63 is normally expressed in epithelia, TAp63 was reported to be restricted to oocytes and cardiomyocytes (14–16). To investigate the importance of each of these p63 isoforms in the cochlea, we performed RT-PCR on surgical extract from whole cochlea from E18.5 WT mice. Interestingly, this analysis revealed a strong expression of TAp63 in the cochlea (Fig. 1C), as well as in oocytes (15), whereas, as reported, ΔΝp63 was the predominant isoform expressed in the skin.

According to this result, we analyzed the cochlear morphology of selective TAp63^−/− mice. As shown in Fig. 1F, the TAp63^−/− mouse shows the same abnormalities of the full p63^−/−. Also in this case, in fact, supernumerary OHC and IHC are evident; the TAp63^−/− mouse cochlea show a higher number of HC (Fig. S2O).

TAp63^−/− Mice Have Sensorineural Deafness.

Auditory evoked potential analysis performed on a cohort of WT and TAp63^−/− mice showed an altered response to 8-KHz stimulation, with a variable delay of cerebral cortex potentials, especially on the fifth wave (Fig. 1G). Furthermore, computerised axial tomography (CT-scan) analysis of mice cochleae demonstrated that this organ is lightly smaller in size in knockout compared with the WT mice (Fig. 1H; sample images in Fig. S4). The analysis of IHC/OHC number, in cochleae from TAp63^−/− and WT mice, using at least three cochlea sections located near the apex, middle, and base of the organ, did not reveal defects in WT mice, whereas all of the TAp63 mice show 50% to approximately 70% sections with supernumerary IHC/OHC cells (Fig. 1I). This suggests that TAp63 ablation (or total p63 ablation) could have different penetrance, supporting the different degrees of hearing loss.

p63 Regulation of the Cochlea Does Not Involve a Cell Death Mechanism.

TAp63 can trigger apoptosis, and this is dependent on the direct transcriptional induction of the proapoptotic BH3-only proteins Puma and Noxa (20). To test the possibility that the additional IHC and OHC present in p63^−/− mice could result from the loss of p63-mediated apoptosis (21, 22), we examined cochleae from single Puma^−/−, Noxa^−/−, or double Puma^−/−Noxa^−/− knockout mice. Figure 2 A–C shows that, in all these mice, the numbers of hair cells are normal. Therefore, the increased IHC and OHC cells in the p63^−/− mice are unlikely to result from a failure of p63-mediated apoptosis, but alternative mechanisms must be involved.

Fig. 2.

Mice lacking Puma, Noxa, or both BH3-only proteins have normal hair cells in the cochlea; TAp63 induces Notch-related genes. Immunofluorescence staining of cochlear sections from E18.5 noxa^−/− (A), puma^−/− (B), and puma^−/−;noxa^−/− (C) mouse embryos stained with MyoVIIa (green), Cx26 (red), and DAPI nuclei (blue). (Scale bars, 20 μm.) (D) Prox1, Hairy and enhancer of split 5 (Hes5), and Atonal homolog 1 (Atoh1) mRNA levels are regulated by TAp63α. Transcriptional activity (RT-qPCR) of TAp63α in doxycycline-inducible (24 h) TAp63α cell lines. Tet-on cells lacking p63-inducible constructs were used as a control, and β-actin was used as an internal standard for 2^ΔΔCt calculations. (E) Western blot of Tet-on induced SaOs2 cells, expressing HA-tagged TAp63α. p21 expression was analyzed as a positive control, and expression of tubulin was used as a loading control.

TAp63 Regulates the Notch Pathway via Hairy and enhancer of split 5 (Hes5) and Atonal homolog 1 (Atoh1) in Cochlea Development.

To identify the molecular mechanisms leading to the morphological defects in the cochlea of p63^−/− embryos, we performed a gene expression microarray analysis using SaOs-2 cells transfected with TAp63α or ΔNp63α. In particular, we searched for genes known to be involved in cochlear neuroepithelial development. A comparative gene function map provided evidence for the involvement of the Notch pathway, which is known to be critical for cochlea development (23–25) (Fig. S5 A and B). Indeed, Hes1, Hes7, Hes5, Atoh1, prospero homeobox 1 (Prox1), Jagged 1 (Jag1), Jag2, Connexin 26 (Cx26), Notch1 and Notch3 have all been validated as differentially expressed genes by RT–quantitative PCR (RT-qPCR) in this Tet-On inducible system as well as in cells transiently transfected with expression constructs for TAp63α (Fig. 2 D and E). Remarkably, Prox1 and Atoh1 were up-regulated ∼fivefold, and Hes5 by nearly 35-fold in response to TAp63α overexpression (Fig. 2 D).

This suggests that TAp63 selectively transactivated genes may be important in cochlear neuroepithelial development, and we focused specifically on Hes5, Atoh1 (Math1), and Prox1. We first identified in silico p53-like responsive elements in their promoters (Mathinspector software; Genomatix). The analysis of the Hes5 −2,000 bp (from transcription start site, TSS) promoter region showed the presence of a putative p63 responsive element (RE) located at −1,098/1,066 bp upstream of the TSS (Fig. 3A). This entire region was therefore cloned upstream of luciferase ORF and the resulting construct transfected into SaOs-2 cells. Cotransfection of WT TAp63 significantly enhanced luciferase reporter activity, whereas DBD TAp63α mutants (R280C, R279H, S272N, R304W) were inactive, SAM domain mutants (G530V, I537T, Q536L) showed a significant loss of function, and a TAp63α N-terminal domain mutant (Q634X) showed a gain of function activity (Fig. 3B) (26, 27). Confirming the in silico detection of a p63 binding site within the Hes5 promoter, ChIP assays on Saos-2 Tet-On expressing TAp63α-HA showed binding of TAp63α to this site (Fig. 3D, Lower); the MDM2 promoter was used as a positive control (Fig. 3D, Upper; Fig. 3C for protein control).

Fig. 3.

p63 drives Hes5 and Atoh1 promoters. (A) The Hes5 gene structure shows the presence of a putative p53/p63 RE localized at −988, −966 from the TSS, in the promoter sequence. All promoters analyses were performed by MathInspector professional release 8.0.5, March 2011; Matrix Family Library Version 8.3, October 2010. (B) SaOs2 cells were transiently cotransfected with expression constructs for WT TAp63α-HA or TAp63α-HA mutant (G530V, I537T, Q536L, R280C, R304W, S272N, R279H, Q566fsX94, and Q634X) plus the hHes5-luc reporter vector. There was an increase in luciferase activity in cells transfected with TAp63α-HA WT, more pronounced with the TAp63α-Q634X-HA mutant, but not in cells transduced with the vectors encoding mutants for the DBD (mean ± SD, n = 3). (C) Western blot analysis was performed to verify TAp63a protein expression. (D) ChIP analysis shows the binding of TAp63α to the putative p53/p63-RE. ChIP on the MDM2 promoter was used as positive control. (E) The Atoh1 gene structure shows the presence of two p53 RE: RE-I5′ localized at −1,682, −1,660 from the TSS; and RE-II3′ in the Enhancer-A. (F) ChIP analysis of the Atoh1 p53-Res. TAp63α binds only to the p53-RE Atoh1 enhancer sequence; the MDM2 promoter was used as a positive control. (G) Western blot showing TAp63α protein expression. (H) Luciferase activity was increased by TAp63α-HA and TAp63α Q634X plasmids. SaOs2 cells were transiently cotransfected with an hAtoh1-luc expression vector, TAp63α-HA, and TAp63α-HA mutants (G530V, I537T, R280C, R304W, S272N, R279H, Q566fsX94, and Q634X) (mean ± SD, n = 3).

Atoh1 regulatory sequences (28) have characteristic canonical promoter sequences that are located at the 5′ end of the gene and two downstream sequences, called Enhancer-A and Enhancer-B, which are located ∼3 kb from the stop codon. These two sequences are separated by ∼400 bp and span ∼500 bp each. Their strong conservation between human and mouse highlights their importance in gene expression regulation. Indeed, these enhancer sequences are responsible for timing and tissue specificity of gene expression during embryogenesis (28). The in silico analysis identified p63RE within the promoter and Enhancer-A (Fig. 3E); the p63RE in the Enhancer-A bound TAp63 by ChIP (Fig. 3F; Fig. 3G for protein control) and responded to TAp63 in a Luciferase assay, though not to p63 mutants (Fig. 3H). These results support the hypothesis that this region is important for the expression of this p63 target gene in vivo (28). In contrast, analysis of the Prox1 putative promoter did not reveal the presence of any responsive element.

To test the possibility that TAp63α drives Hes5 transcription, we treated H1299 cells with LBH589, a histone deacetylase inhibitor, to induce TAp63 (29) (Fig. 4A). LBH589 induced TAp63 expression by ∼15-fold, compared with ΔNp63, and resulted in the induction of both Hes5 (∼15-fold) and p21 (∼40-fold), the latter known to be a p63 target gene. To confirm the relationship between TAp63 and Hes5, Atoh1, and Prox1, we extracted RNA from cochleae of p63^−/− E18.5 embryos and found that the levels of Hes5 mRNA were considerably (∼10-fold) lower than in WT mice (Fig. 4B); a reduction is also shown for Atoh1 and Prox1, thereby providing a direct link between p63 and these genes in vivo. Furthermore, the protein analysis, using cochlear extracts, revealed Hes5 and Atoh1 depletion in p63^−/− E18.5 samples (Fig. 4C), as well as Atoh1 induction in TAp63 transfected cells (Fig. 4D). Studies in transfected TAp63 cells showed a significant Hes5 induction (Fig. S6D), in which a mechanism of nuclear relocalization is also evident (Fig. S6 A–C).

Fig. 4.

p63 drives Hes5 expression in the cochlea. (A) Treatment of H1299 cells with LBH589 for 24 h induced the expression of TAp63, resulting in the transcriptional induction of Hes5, as determined by RT-qPCR analysis (mean ± SD, n = 3). (B) Transcriptional analysis on cochlea tissue biopsies from WT and p63^−/− mouse embryos (E18.5). Expression levels of Hes5 proved to be dependent on TAp63, and Hes5 mRNA levels were ∼10 times lower in cochlea of p63^−/− embryos compared with those from WT embryos; additionally, Prox1 and Atoh1 expression were lower in p63^−/− embryos. (C) Protein expression of Hes5 and Atoh1 in WT and TAp63^−/− embryos. C shows a lower expression of Hes5 in TAp63^−/− sample; Cx26 protein was used as control. (D) Up-regulation of Atoh1 in TAp63 transfected H1299 cells. Parp protein was analyzed as loading control for nuclear protein extract.

Interestingly, the supernumerary hair cell phenotype observed in our p63^−/− mice (Fig. 1) is also seen in ASPP2^−/− mice (Fig. S6E) a regulator of p63 function (30), and is highly reminiscent of that found in Hes5^−/− mice (31). This is consistent with the notion that the lack of TAp63-mediated regulation of the Hes5 promoter with consequent reduction of Notch expression is responsible for this developmental defect.

Discussion

Notch signaling is critical for the development of many tissues and organs. This pathway is activated by ligand–receptor interactions, resulting in transcriptional activation of target genes, including Hes1 and Hes5 (23, 24, 32). Specifically, the Notch pathway is involved in neuroepithelial differentiation of the inner ear, in the organ of Corti (25, 33, 34). Here, the auditory sensory epithelium is the specialized region of the cochlea that transduces sound. The sensory neuroepithelia are characterized by a mosaic of supporting cells and hair cells, both IHC and OHC, which are arranged into highly ordered rows. Several components of the Notch signaling pathway are known to be expressed in the developing organ of Corti. Importantly, interference with their expression or mutation of the corresponding genes leads to an abnormal increase in hair cells (25, 35), highlighting a role for Notch activation in the regulation of progenitors that develop into hair cells. Previous studies have established the importance of the basic helix–loop–helix (bHLH) Notch target genes Hes1, Hes5, and Math1 in the developing ear. Math1-deficient mice die shortly after birth (36) with complete disruption of hair cell development (37). Hes1 and Hes5 were shown to negatively regulate the differentiation of inner ear hair cells and, accordingly, Hes5-deficient mice have supernumerary OHC (31).

We have previously demonstrated that p63 can regulate components of the Notch pathway by direct transcriptional induction of Jag1 and Jag2 during epidermal differentiation (21), further implicating a link between p63 and Notch in epidermal development (38). Here we identify Hes5 and Atoh1 as two Notch-related transcriptional targets of TAp63 that are involved in the differentiation of the organ of Corti. Hes5 is involved in the formation of the hair cells in the organ of Corti (31), and here we demonstrate that it is primarily regulated by TAp63. TAp63 also regulates another bHLH transcription factor, Atoh1, which also plays an important role in the differentiation of hair cells. Atoh1 is expressed in a transient population of actively proliferating progenitor cells (39), and Atoh1-deficient mice are characterized by the absence of auditory sensory hair cells (37). We show that TAp63 regulates Atoh1 via binding to the enhancer A, a region known to be critical for time- and tissue-specific expression of this gene. The morphological comparison of the inner ear of p63^−/− and WT mouse embryos also confirmed the involvement of p63 in the late stages of the formation of the organ of Corti. Mice lacking Puma and/or Noxa, the BH3-only proteins that are transcriptionally induced by TAp63 and essential for its ability to trigger apoptosis, had normal cochlea and organs of Corti. This demonstrated that loss of a nonapoptotic mechanism activated by TAp63 must be the cause of the defects in ear development in the p63^−/− embryos. Indeed, H&E staining of E18.5 embryos showed disorganization of the cochlea, especially the scala medium and timpani, and a minor thickening of the stria vascularis, with supernumerary IHC and OHC, as well as an abnormal distribution of Cx26 (Fig. 1 D–F). This inhibition of hair progenitor cell by Notch signaling is therefore essential for correct morphological and functional development of the inner ear. Accordingly, we propose a model whereby TAp63 regulates the formation of the organ of Corti by transcriptional induction of Hes5/Atoh1 (Fig. S7A), thereby regulating this process of lateral inhibition. Indeed, both Hes5^−/− and p63^−/− mice develop supernumerary hair cells (Fig. S7B) (31) as a result of abnormal differentiation of supporting cells into hair cells, because the block by Hes5 has been removed by the ablation of p63.

To date, a developmental role for TAp63 has only been clearly documented in oocytes, where it promotes apoptosis upon DNA damage (15, 20), or in the heart (14). Here we show that p63 is also important for the maturation of the cochlea; this activity of p63 requires transcriptional regulation of cell differentiation and not the induction of its predominant apoptosis inducing target genes. Auditory analysis in two new patients demonstrated a sensorineural component in hearing loss previously not reported. Further studies performed on TAp63^−/− mice, demonstrated a variable but consistent sensorineural hearing loss, and histological examination confirmed the presence of supernumerary IHC/OHC, in all TAp63^−/− mice cochleae. These data imply that TAp63 mutations lead to modification of sensorineural epithelium by affecting the integrity of the Hes5/Atoh1 pathway and contribute with different degrees to the hearing loss seen in a subset of patients with ED.

Materials and Methods

Mice.

p63 knockout mice (11) and mice lacking Puma, Noxa (40), or both Puma and Noxa (41) and TAp63 (42), have been described. Mice lacking p63 were generated electroporating JI Es cells, which were microinjected into blastocysts from C57BL/6 mice. Heterozygous mice have been crossed with C57BL/6 for more than 10 generations. Mice lacking Puma or Noxa were generated on a C57BL/6 background, using C57BL/6-derived ES cells. TAp63 knockout mice were in a C57BL/6 background. Animal experiment have been performed in the Department of Experimental Medicine and Surgery, and approved by the Department Board, according Italian law 116/92, September 29, 2011.

Cell Culture, Transfection, and Plasmid.

SaOs-2 cells with doxycycline-inducible expression of HA-TAp63a and HA-DNp63a were generated and treated as previously described (43). The luciferase reporter plasmid (hHes5-luc) was modified from the pGL3-Basic vector (Promega) by inserting the putative hHes5 promoter sequence containing the p53RE between Sac1 and Xho1 sites. Similarly, the amplified hAtoh1 enhancer sequence was inserted in pGL3 using NheI sites.

Western Blot, Immunostaining, and Confocal Microscopy.

See refs. 12 and 44 for embryo preparation and confocal analysis. Subsequently, sections were incubated for 2 h with the following primary antibodies: anti-p63 (H129 sc-8344 and H137 sc-8243; Santa Cruz), anti-MyoVIIa (25-6790, Proteus), anti-Sox2 (AB5603; Millipore), and anti-Cx26 (13-8100; Invitrogen). See ref. 26 for Western blot preparation. All membranes were incubated for 2 h with the following antibodies: anti-Hes5 (ab25374; Abcam), anti-Math1 or Atoh1 (ab27667; Abcam;), anti-Cx26 (13-8100; Invitrogen), anti-p63 (Y4A3 p3362; Sigma-Aldrich), and anti-Parp (sa250; Biomol).

Real-Time qPCR, Semiquantitative RT-PCR, and ChIP.

See ref. 44 for PCR analysis and Table S1 for specific PCR primer sequences. SaOs TAp63α-inducible cells (5 × 10⁶) were cross-linked for 10 min in a solution containing 1% formaldehyde, and ChIP assays were performed using a MAGnify ChIP system (Invitrogen), according to the manufacturer’s instructions. Luciferase reporter assays and immunoblot analysis were performed as described previously (44).

Auditory Evoked Potential.

Mice were anesthetized by i.m. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed in a dark, electrically shielded room. Stainless steel electrodes were inserted s.c. into the vertex (positive pole), one retroauricular region (negative pole), and the opposite retroauricular region (ground) of each mouse. Acoustic stimuli, consisting of tone bursts at frequencies of 8 kHz, were delivered to each mouse. Amplaid MK12 (Amplifon) instruments was used to perform the analysis and elaborate data.