EYA1—SIX1 complex in neurosensory cell fate induction in the mammalian inner ear

Abstract

The phosphatase-transactivator EYA1 interacts with the homeodomain protein SIX1 to form transcriptional activation complexes, which play essential roles in regulating cell proliferation, survival and induction of sensory and neuronal differentiation programs during inner ear development. Mutations of the Eya1 and Six1 genes cause profound developmental auditory defects in mice and humans. The molecular mechanisms and developmental processes controlled by the EYA1 and SIX1 complex in inner ear development and neurosensory fate induction are the focus of this review.

1. Introduction

Members of the Eyes absent (Eya) and Sine oculis (So/Six) gene families of transcription factors are important regulators for inner ear development and have been implicated in human genetic syndromes with congenital auditory system malformations. The vertebrate genome has at least 4 different Eya genes and 6 So/Six genes with diverse regulatory functions in development and disease (Borsani et al., 1999; Kawakami et al., 2000; Oliver et al., 1995a,b; Xu et al., 1997b). The original Eya (Bonini et al., 1993) and So (Cheyette et al., 1994) genes were reported in Drosophila as key regulators for eye development. Since then, they were identified in vertebrate and invertebrate organisms from worms, frogs, fish, and birds to higher vertebrates (Abdelhak et al., 1997b; Bessarab et al., 2004; Dozier et al., 2001; Hoshiyama et al., 2007; Kawakami et al., 2000; Pineda et al., 2000; Sahly et al., 1999; Seo et al., 1999; Serikaku and O’Tousa, 1994; Xu et al., 1997b; Zimmerman et al., 1997). While So/Six genes encode homeodomain transcription factors with direct DNA-binding capacity, Eya genes encode proteins that affect transcription indirectly by interacting with DNA-binding proteins such as SO/SIX as well as other DNA-binding proteins. The key role of EYA-SO/SIX during development was first revealed by loss- and gain-of-function studies in fruitflies. In Drosophila, eya and so genes function in a molecular network with the fly Pax6 gene eyeless (ey) and dachshund (dach) to regulate eye morphogenesis (Fig. 1) (Treisman, 1999). The components of this molecular network have been highly conserved during evolution with related genes in mammals regulating the development of multiple organ systems, including the inner ear (Xu et al., 2003; Zheng et al., 2003).

Fig. 1.

The EYA-SO/SIX regulatory network. (A) The transcriptional hierarchy among the key regulators in the fly eyes. Feedback loops and protein–protein interactions (double-headed arrows) are indicated. (B) EYA and SIX form transcriptional activation complexes to control cell proliferation and survival as well as to trigger particular differentiation programs. EYA–SIX also interact with other proteins (×), which may vary depending on distinct developmental and cellular contexts to modify the specificity of EYA–SIX function.

The first indication of the importance of Eya and Six genes in auditory system development was the identification of human EYA1 as the gene responsible for Branchio-Oto-Renal (BOR) and Branchio-Oto (BO) syndrome, which are characterized by combinations of craniofacial defects and hearing loss with or without kidney anomalies. Approximately 93% of patients with BOR/BO syndrome show hearing loss, accounting for as many as 2% of profoundly deaf children (Abdelhak et al., 1997a). More recently, mutations in SIX1 and SIX5 have also been identified in BOR/BO patients (Hoskins et al., 2007; Ruf et al., 2004), while mutations in EYA4 cause late-onset autosomal dominant hearing loss at the DFNA10 locus (Pfister et al., 2002; Schonberger et al., 2005; Wayne et al., 2001). Recent studies have provided exciting data suggesting that EYA1 and SIX1 proteins have roles at several stages during neurosensory cell development, including cell fate specification, proliferation, differentiation and maintenance. However, many questions regarding the function and regulation of Eya and So/Six genes in the developing inner ear remain unanswered.

2. EYA and SO/SIX family proteins

The defining characteristic of the Eya proteins is the presence of a highly conserved 271–273—amino acid C-terminal domain, the Eya domain. The EYA proteins function as both transcription cofactors and phosphatases. The N-terminal domain (NTD) is highly divergent but possesses a transcriptional activation function (Xu et al., 1997a). The conserved Eya domain not only interacts with other proteins but also possesses a catalytic motif belonging to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily of enzymes (Li et al., 2003; Rayapureddi et al., 2003; Tootle et al., 2003). This suggests that EYA is likely to regulate the phosphorylation state of its transcriptional cofactors or itself to directly modify transcriptional output within a cell. However, it is currently not understood whether the two functions depend on each other and how they operate during development in mammals. To date, the DNA damage-related histone H2AX was the only physiological relevant substrate that has been reported for EYA’s tyrosine phosphatase activity (Cook et al., 2009; Krishnan et al., 2009). While the transactivation activity of the EYA–SIX complex represents a nuclear function and EYA’s tyrosine phophsatase activity in DNA damage repair is also a nuclear function, there has been some evidence that the phosphatase activity of the EYAs may participate in a cytoplasmic cellular function. The cytoplasmic EYA4 plays a role in innate immunity via its threonine phosphatase activity (Okabe et al., 2009) and cytoplasmic EYA3 promotes cell motility via its tyrosine phosphatase activity (Pandey et al., 2010). Additional cytoplasmic function has been suggested for EYA proteins, as G protein subunits were isolated as binding partners for EYA2 (Embry et al., 2004; Fan et al., 2000; Ozaki et al., 2002). Future studies involving identification of more binding partners and in vivo substrates of EYA will likely reveal how its functions as both transcriptional coactivator and phosphatase are used throughout development.

The SO/SIX family represents a divergent group of the homeodomain (HD) superfamily of transcription factors and it contains two evolutionarily conserved domains, a 60—amino acid HD and a 110–115—amino acid SO/SIX-specific domain (SD), which interacts with EYA and other proteins (Pignoni et al., 1997). SO/SIX family members have been divided into three subfamilies (SIX1/2, SIX4/5 and SIX3/6) on the basis of Drosophila SO, which belongs to the same subfamily as vertebrate SIX1/2. The SO/SIX HD binds to a TGATAC consensus sequence, differing from the classic TAAT core sequence because it lacks two highly conserved amino acid residues typical of most HDs (Kawakami et al., 1996a,b; Serikaku and O’Tousa, 1994; Spitz et al., 1998). Depending on the context, SO/SIX proteins function as either transcriptional activators (Ohto et al., 1999; Silver et al., 2003) or repressors (Li et al., 2002). When coexpressed with EYA, EYA-SO/SIX interactions lead to formation of a bipartite transcription factor, with the HD of SO/SIX providing the DNA-binding activity and the NTD of EYA providing the transactivation potential, which leads to potent transcriptional activation—even under conditions in which SO/SIX alone acts as a repressor (Ahmed et al., 2012b.; Li et al., 2002; Ohto et al., 1999; Silver et al., 2003).

To date, several binding partners of EYA and SO/SIX proteins have been identified by yeast two-hybrid screens and immunoprecipitation. Recent studies have demonstrated that SOX2 is an essential factor directly interacting with EYA1 or SIX1 both in vitro and in vivo to synergize transcriptional output of EYA1–SIX1 during sensory hair cell induction and neurogenesis in the inner ear (Ahmed et al., 2012a,b; Zou et al., 2008). In addition to SOX2, components of the SWI/SNF chromatin-remodeling complex BRG1 and BAF170 and the helix-loop-helix (bHLH) transcription factors, NEUROG1 and NEUROD1 are found to be binding partners of EYA1 and SIX1 in the inner ear (Ahmed et al., 2012a). Two novel EYA1-binding partners Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) have been recently identified through yeast two-hybrid screens and they may interact with EYA1 during craniofacial development (Landgraf et al., 2010). However, how these protein–protein interactions modify transcriptional activities remains to be elucidated. Since Eya1 and Six1 are coexpressed and interact in neurosensory cells of the inner ear, and mutants of Eya1 and Six1 exhibit a very similar spectrum of deficiencies in mice and humans as well as in lower vertebrates (Abdelhak et al., 1997b; Bricaud and Collazo, 2006; Brugmann et al., 2004; Kozlowski et al., 2005; Laclef et al., 2003; Li et al., 2003,2010; Nica et al., 2006; Ozaki et al., 2004; Ruf et al., 2004; Schlosser, 2005, 2006; Xu et al., 1999; Zheng et al., 2003; Zou et al., 2004), below we will focus on specific functions of EYA1–SIX1 complex during neurosensory cell development.

3. Eya1 and Six1 family genes in early neurosensory lineage development

The inner ear is a beautiful yet complex sensory organ that originates from the otic vesicle (also called otocyst). Its development begins ~E8.0 in mice as a placodal thickening (the otic placode) in the surface ectoderm next to the hindbrain neuronal tube that is induced by the adjacent dorsal hindbrain and the underlying mesoderm. The otic placode invaginates to form the otic cup and vesicle, which subsequently undergoes elaborate processes that include patterning, cellular growth, apoptosis, migration and differentiation to eventually give rise to different regions of the inner ear. In the mouse, the basic architecture of the inner ear is fully established by E14.5 (Morsli et al., 1998).

The otocyst is specified and diversified by combinatorial gene expression and gives rise to three main cell types: neuronal, sensory (future hair cells and supporting cells) and nonsensory. The neuronal lineage is the earliest cell fate specified within the otic epithelium (~E9.0–E9.5) (Fekete and Wu, 2002). Soon after the otocyst forms, the neuroblasts delaminate from the anteroventral region and migrate into the adjacent mesoderm where they coalesce to form the auditory and vestibular ganglion (gVIII), which provides the sensory innervation for the inner ear (Barald and Kelley, 2004). During the delamination of otic neuroblasts, gene expression studies suggest that the otocyst simultaneously becomes regionally patterned along several different axes, also referred to as compartmentalization (Fekete and Wu, 2002). From ~E11.0, the ventral region of the otocyst elongates and coils to form the cochlear duct.

The essential roles of Eya1 and Six1 in neurosensory cell lineage development have been demonstrated by loss-of-function studies in mice. Eya1 and Six1 are coexpressed in otic placode from as early as E8.5–8.75 and in neuroblast precursors before their delamination. In the otocyst, Eya1 and Six1 are coexpressed in the ventral region where vestibular and auditory sensory epithelia form and in delaminating neuroblasts as well as the vestibuloacoustic ganglion (Fig. 2) (Xu et al., 1997a,b; Zheng et al., 2003). At later stages, these two genes are expressed in all the six sensory epithelia of the inner ear. Inactivation of either gene leads to malformation of the outer, middle and inner ears (Xu et al., 1999; Zheng et al., 2003). The inner ear development in Eya1^−/− or Six1^−/− embryos arrested at the otocyst stage and all components of the inner ear failed to form due to increased cell death and reduced cell proliferation in the otic epithelium (Xu et al., 1999; Zheng et al., 2003; Zou et al., 2006). Both genes are not only required for normal growth and maintenance but also for regional specification of the otocyst, as all sensory or neuronal markers failed to be expressed in their mutant otocysts (Xu et al., 1999; Zheng et al., 2003; Zou et al., 2006), thus causing ventral shift of expression of dorsal markers.

Fig. 2.

Schematic drawing of Eya1 and Six1 expression domains in relation to Sox2 expression in the otocyst and developing cochlea, cristae and maculae and their possible roles in sensory cell development. In the otocyst, Eya1 (red), Six1 (red) and Sox2 (green) are co-expressed in the ventral otocyst where the prosensory epithelia are formed. These genes may act together to specify a common prosensory area in the otocyst at this early stage by turning on other sensory genes including Jag1, Lfng, Bmp4 and Fgfs, which are not expressed in Eya1- or Six1-null embryos. In the developing cochlea, at E12.5, the floor of cochlear duct will give rise to the organ of Corti (oc), the greater epithelial ridge (GER) and lesser epithelial ridge (LER). Eya1 and Sox2 are coexpressed in the prosenory progenitors of the OC, and this colocalization suggests that both genes may act together to regulate the cell cycle of the prosensory progenitors. Six1 expression disappears in the postmitotic progenitors but reappears in differentiating hair cells in a pattern similar to that of Atoh1. Once the sensory progenitor cells become postmitotic, they express p27Kip1, and at around E13.5–14.5, Atoh1 is activated in the postmitotic progenitors that become committed to hair cell fate and is coexpressed with Eya1 and Six1 in the differentiating hair cells. However, Sox2 expression becomes restricted to supporting cells and border cells in the GER. Based on published data, Eya1, Six1 and Sox2 act together to regulate hair cell commitment by activating Atoh1; however, Eya1 and Six1 but not Sox2 act to maintain or upregulate Atoh1 expression during hair cell differentiation. Similar expression pattern of these genes is found in the sensory cell development of the cristae and maculae.

In addition to the inner ear, EYA1 or SIX1 is required for normal cell proliferation and survival for several other organs that were affected in their mutants. Recent studies have suggested that SIX1 may promote proliferation by interaction with cell cycle control genes such as CyclinA1, CyclinD1, and c-Myc (Coletta et al., 2004; Ford et al., 1998; Li et al., 2003; Yu et al., 2006) and that EYA1 may modulate apoptosis and survival decisions by dephosphorylating histone H2AX (Cook et al., 2009). Future studies are necessary to characterize the precise roles of Eya1 and Six1 in regulating proliferation and cell survival.

Recently, studies have identified Sox2 as a key gene for sensory and neuronal cell specification in the inner ear (Kiernan et al., 2005; Puligilla et al., 2010). Eya1 and Six1 are coexpressed with Sox2 in the otic placode and the ventral otocyst (Fig. 2) (Zheng et al., 2003; Zou et al., 2008). All three genes have early roles in the inner ear and are required for inner ear morphogenesis (Puligilla et al., 2010; Zou et al., 2004). New insights into the important roles of EYA1–SIX1 complex in combination with SOX2 in triggering neuronal and sensory developmental programs have recently been obtained (Ahmed et al., 2012a,b). Below, EYA1–SIX1 complex and its interaction with SOX2 in inducing sensory and neuronal cell fate as well as their differentiation will be discussed.

4. EYA1–SIX1 complex in inducing hair cell development

Besides the early roles of EYA1 and SIX1 in inner ear development, the EYA1–SIX1 transcriptional activation complex has recently emerged as a key regulator of hair cell differentiation. The ventral portion of the otocyst elongates and begins to coil ~E12 to reach a full 1.5 turns of cochlear duct by E17.5. Previous studies have revealed that the prosensory progenitors that give rise to the entire organ of Corti are specified between E12–E14 in a region termed the prosensory domain that is distinctively marked by the expression of cyclin-dependent kinase inhibitor p27^Kip1 (Chen and Segil, 1999; Fekete, 2000; Ruben, 1967) (Fig. 2). The progenitor cells exit the cell cycle from apex toward base of the cochlear duct between E12 to E14 (Chen et al., 2002; Lee et al., 2006). Functional studies have shown that Sox2 is required for specifying the precursors, as inner ear structures derived from the prosensory progenitors are not formed in Sox2-deficient mice (Kiernan et al., 2005). Nonsensory cells develop adjacent to the sensory epithelium and reside within the greater and lesser epithelial ridges (GER/LER) flanking the organ of Corti. Eya1 and Six1 are coexpressed with Sox2 in the entire cochlear duct by E12.5 and are required for cochlear duct formation, as inner ear development arrests at otocyst stage in their null mutants. All three genes also show differential expression in the nonsensory epithelium with Eya1 and Sox2 in the GER and Six1 in both the GER and LER, suggesting that they may play essential roles in regulating differentiation of the organ of Corti.

In the nascent organ of Corti, hair cell differentiation initiates from base toward apex, as expression of the hair cell differentiation factor gene Atoh1 begins in the base of the cochlea between E13.5 and E14.5, and reaches the apex at ~E17.5 (Chen et al., 2002). Recent findings have provided evidence that such developmental patterns of Atoh1 expression are likely to be achieved by combinatorial regulation of Eya1, Six1 and Sox2 (Ahmed et al., 2012b), as discussed below.

Between E12.5 and 13.5 when the progenitors within the prosensory domain begin to exit the cell cycle, a high level of Eya1 and Sox2 expression becomes restricted to the postmitotic progenitors, whereas Six1 expression disappears in the postmitotic progenitors within the prosensory domain (Fig. 2). From E13.5, Six1 shows a gradient of expression that is similar to the pattern of Atoh1 but appears to occur before the onset of Atoh1 expression (Ahmed et al., 2012b). This pattern of Six1 expression in the organ of Corti suggests that it may serve as a critical positive regulator for stimulating Atoh1 activation near the basal cochlear duct. Supporting evidence for this hypothesis comes from both gain- and loss-of-function studies in mice.

In a gain-of-function experiment in cochlear explants, Ahmed et al. (2012b) demonstrated that nonsensory epithelial cells within the GER electroporated with Eya1/Six1 expression plasmids can be induced to become sensory hair cells. Approximately 89% of the electroporated cells differentiated into Myo7a⁺ hair cells. One would expect all the ectopic hair cells to express Atoh1, however this was not the case. The vast majority (~ 61%) were in fact Atoh1⁻, suggesting that there exists an Atoh1-independent pathway for hair cell development. Unlike Atoh1, all the ectopic hair cells were POU4F3⁺. Before this study, ATOH1 was the only known and accepted factor responsible for hair cell fate determination (Bermingham et al., 1999). This study reveals that there is an Atoh1 - dependent and -independent pathway for hair cell development, and POU4F3 appears to be the common downstream factor that is activated in both pathways. The authors further demonstrated that the ability of EYA1/SIX1 to induce endogenous Atoh1 expression to induce a hair cell fate depends on endogenous SOX2 activity.

To directly address the requirement of Eya1/Six1 in hair cell development, the authors used an inducible system to delete these genes in vivo by crossing transgenic mice expressing tamoxifen regulated Cre recombinase under control of the 1.4 kb Atoh1 enhancer (Atoh1CreERT2) with either Eya1^flox or Six1^flox mice. No hair cells were induced when either gene function was abolished and hair cells that had already formed were eventually lost when cultured in vitro.

The molecular link between Eya1, Six1, Sox2 and Atoh1 has been revealed (Fig. 3). SOX2 and SIX1 are DNA-binding proteins and binding sites for SOX2 and SIX1 were found in the 1.4 kb Atoh1 enhancer, which is located ~3.4 kb 3′ of the Atoh1 coding sequence and contains two conserved elements (element A and B) and can direct expression of reporter transgenes to the inner ear hair cells (Chow et al., 2006; Helms et al., 2000). Two specific regions being the essential elements for mediating SOX2- or SIX1- dependent transcriptional activation were found to be located in the conserved element A and B respectively. The authors found that EYA1, SIX1 and SOX2 cooperatively interact via direct protein–protein interaction and that the ability of each transcription factor to induce the endogenous Atoh1 expression is partially dependent upon the other. Endogenous SOX2 activity in the GER cells is not only necessary for endogenous Atoh1 activation but also for activating the 1.4 kb Atoh1-GFP reporter. The authors proposed that SIX1 may stimulate the initial expression of Atoh1 in cooperation with SOX2 and EYA1 to initiate hair cell differentiation within the organ of Corti. In the absence of SIX1, EYA1/SOX2 alone may not lead to the formation of an active complex capable of transcriptional activation, which explains why in the primordial organ of Corti, Atoh1 activation is not initiated in all prosensory precursors that express both EYA1 and SOX2. This study also revealed that SOX2 represses POU4F3⁺ cells to differentiation into MYO7A⁺ hair cell (Fig. 3).

Fig. 3.

EYA1–SIX1 complex in inducing hair cell fate. (A) Molecular relationships among the key transcription factors for hair cell differentiation. A direct interaction between EYA1/SIX1/SOX2 proteins coordinately regulates Atoh1 expression, and that POU4F3 is a common downstream factor of the Atoh1-dependent and -independent pathways. Dashed lines indicate that SOX2 may repress Pou4f3 or downstream factors of Pou4f3 to inhibit hair cell differentiation. (B) Possible mechanisms for Atoh1 activation by Sox2/Eya1/Six1. EYA1/SIX1 in collaboration with SOX2 activity in prosensory progenitors can induce Atoh1 activation via direct binding to the Sox and Six binding sites within enhancer A and B, respectively. These three factors may directly interact (model a), or EYA1 may bridge SIX1 and SOX2 (model b). These three factors may also form an active complex to regulate Atoh1 activation via enhancer A (model c), whereas EYA1/SIX1 efficiently upregulate Atoh1 via enhancer B. Question mark indicates that the involvement of the factor is unclear.

5. Molecular mechanism by which EYA1–SIX1 complex drives neuronal differentiation

New findings have revealed that EYA1–SIX1 appears to operate through different mechanisms to induce neuronal versus sensory fates (Ahmed et al., 2012a), with the former induced several days earlier during development than the latter. In the auditory system, neuronal precursors become distributed in a spiral pattern (referred to as the spiral ganglion) that mirrors the distribution of their peripheral targets, the mechanosensory hair cells located along the cochlea. Spiral ganglion neurons extend dendritic processes that innervate both inner and outer hair cells with 95% of all afferent synapses occurring on inner hair cells. Loss of spiral ganglion neurons is believed to contribute to decreased hearing acuity and presence of intact spiral ganglion neurons is required for cochlear implant function. Despite their crucial role in auditory function, the molecular pathways that mediate spiral ganglion neuron formation are not fully understood.

Eya1 and Six1 stay coexpressed in differentiating cochlear neurons in the spiral ganglia. As discussed above, both genes are required for the neuroblast specification as Neurog1 and Neurod1 are not expressed in Eya1;Six1 double mutant otocyst, which still express dorsal marker such as Dlx5 (Ahmed et al., 2012a).

Recent studies have shown that the cochlear nonsensory GER cells are not only competent to develop as sensory hair cell fate but also as neurons since ectopic expression of Neurog1 or NeuroD1 within these cells is sufficient to induce a neuronal phenotype (Puligilla et al., 2010). Overexpression of Sox2 in the GER cells can also induce a neuronal phenotype (Puligilla et al., 2010). However, neither factor alone nor combination of all three factors together is capable of inducing Neurofilament⁺ cells, indicating that additional factors are necessary for promoting neuronal differentiation. In a recent gain-of-function study, Ahmed et al. (2012a) demonstrated that overexpression of both Eya1 and Six1 is sufficient to convert cochlear GER cells into neurons that not only express Neurog1, Neurod1 and TuJ1 but also mature neuronal markers such as Neurofilament. The authors further demonstrated that overexpression of Eya1/Six1 can also covert non-neuronal epithelial cells within the otocyst, head ectoderm and 3T3 fibroblast cells into neurons (Ahmed et al., 2012a).

When Eya1 and Six1 are ectopically expressed in the GER cells, only ~11% of transfected cells were induced to neurons (Ahmed et al., 2012a), while the other ~89% of transfected cells were induced to differentiate into hair cells (Ahmed et al., 2012a). Interestingly, EYA1 and SIX1 were found to interact directly with the SWI/SNF chromatin-remodeling subunits BRG1 and BAF170 to drive neurogenesis cooperatively in 3T3 cells and cochlear non-sensory epithelial cells. SOX2 also cooperates with these factors to mediate neuronal differentiation, as when all five factors are overexpressed together, ~100% of transfected GER cells or 3T3 cells became neurons. Furthermore, the ATPase activity of BRG1 is required for not only EYA1- and SIX1-induced ectopic neurogenesis but also normal neurogenesis in the otocyst (Ahmed et al., 2012a). Thus, EYA1 and SIX1 appear to be key transcription factors in initiating the neuronal developmental program, mostly likely by recruiting and interacting with the SWI/SNF chromatin-remodeling complex to specifically mediate Neurog1 and Neurod1 transcription (Fig. 4). EYA1 and SIX1 activities are also required for neuronal differentiation based on their synergistic interactions with NEUROG1 or NEUROD1 during neuronal maturation (Fig. 4) (Ahmed et al., 2012a). How the SWI/SNF chromatin-remodeling complex is recruited to chromatin, and the direct targets of EYA1-SIX1 in neuronal differentiation are interesting areas for future studies.

Fig. 4.

Possible mechanism for Neurog1 and Neurod1 activation by Eya1/Six1. EYA1/SIX1 in collaboration with the SWI/SNF complex in progenitor cells induce Neurog1—Neurod1 activation. Eya1/Six1 also interact with Neurog1 to regulate Neurod1 activation. In addition, Eya1/Six1 interact with Neurod1 to regulate neuronal differentiation. Sox2 works cooperatively with Eya1/Six1 or the SWI/SNF complex to promote neuronal differentiation. These factors are physically associated.

6. Conclusion and perspectives

The studies summarized here have begun to elucidate the important functions that Eya1 and Six1 play during multiple stages of inner ear development. These multiple functions such as maintaining a proliferative state of cells, triggering neuronal and sensory hair cell differentiation programs, and mediating cell survival, suggest a diversity of activity at the cellular level. This diversity is reflected at the molecular level by complex modes of interaction with chromatin, multiple binding partners and sophisticated transcriptional activation and repression activities as well as dephosphorylation and phosphorylation activities. However, their precise mode of action and direct downstream targets in mediating proliferation and survival of neurosensory progenitors and inducing neuronal and sensory differentiation programs in the inner ear have left much to be revealed. Future studies of cell type- and stage-specific knockouts, genetic lineage tracing, proteomics and ChIP-seq analysis are essential for fully understanding how the EYA–SIX complexes work in different biological processes and disease states. New findings of a robust neuronal or hair cell induction via interaction with different factors may not only have new therapeutic implications but also reveal a way to reprogram new hair cells or auditory neurons for restoring sensory or neuronal function in the inner ear.