Role of Wnt and Notch signaling in regulating hair cell regeneration in the cochlea

Abstract

Sensory hair cells in the inner ear are responsible for sound recognition. Damage to hair cells in adult mammals causes permanent hearing impairment because these cells cannot regenerate. By contrast, newborn mammals possess limited regenerative capacity because of the active participation of various signaling pathways, including Wnt and Notch signaling. The Wnt and Notch pathways are highly sophisticated and conserved signaling pathways that control multiple cellular events necessary for the formation of sensory hair cells. Both signaling pathways allow resident supporting cells to regenerate hair cells in the neonatal cochlea. In this regard, Wnt and Notch signaling has gained increased research attention in hair cell regeneration. This review presents the current understanding of the Wnt and Notch signaling pathways in the auditory portion of the inner ear and discusses the possibilities of controlling these pathways with the hair cell fate determiner Atoh1 to regulate hair cell regeneration in the mammalian cochlea.

Keywords

inner ear / cochlea / hair cell / regeneration / Wnt / Notch / signaling pathways

Introduction

Sensorineural hearing loss affects millions of people worldwide. Damage to hair cells (HCs) in the inner ear occurs from exposure to loud noise and environmental and chemical toxins as well as genetic disorders, aging, and some types of medications. Previous studies on hearing loss mainly focused either on preventing HC damage through peripheral protection or on increasing the stimulation of the remaining HCs through prosthetic hearing aids or the use of cochlear implants, which directly send stimuli to the auditory nerves. In recent years, inner ear stem cells, gene therapy, and signaling regulation have been the main approaches used to develop strategies for HC regeneration.

The adult mammalian cochlea contains four rows of HCs, which are interdigitated with multiple layers of supporting cells (SCs) to form the organ of Corti on the basilar membrane (Fig. 1). Despite its sophisticated organization in adults, the organ of Corti completely lacks the ability to regenerate HCs once they are lost [ 1]. Although the cochlea in newborn mammals exhibits potential to regenerate HCs to some extent for a short period during early postnatal life [ 2, 3], hearing loss tends to be permanent and incurable in adult mammals. The inability to regenerate HCs in mammals is likely due to the limited number of Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) or axin2 progenitor cells, which can replace the damaged HCs as well as the complex organization of the organ of Corti.

The sensory epithelium of non-mammalian vertebrates can completely regenerate damaged HCs. In the event of HC damage, adjacent cochlear SCs serve as reservoir for acquiring HC fate [ 4, 5]. In birds, SCs are converted into HCs through mitotic regeneration, where SCs divide first and then differentiate into HCs, or through direct transdifferentiation without any prior cell division [ 6]; the avian cochlea retains the ability to regenerate HCs and completely rebuild the hearing mechanism after damage throughout the adult life of the animal [ 7– 9]. Various molecules and signaling pathways are involved in HC regeneration. Signals regulating the development and formation of HCs during embryonic developmental stages possibly play important roles in HC regeneration; moreover, changes in these signals in the adult mammalian cochlea are the most possible reason for its inability to regenerate HCs after damage. Wnt, Notch, BMP/Smad, FGF, IGF, and Shh signaling cascades guide the specification, patterning, proliferation, and differentiation of sensory epithelia during development [ 10– 14]. These signaling pathways guide the inner ear development by controlling the expression of several transcription factors. In this review, we discuss the common barriers to HC regeneration and the role of the canonical Wnt and Notch signaling pathways. We also highlight recent important findings on reciprocal interaction in regulating HC regeneration.

Wnt signaling pathway in auditory HC development and regeneration

The Wnt signaling pathway is a highly conserved pathway that regulates pivotal events, such as cell fate determination, proliferation, cell migration, neural patterning, and cell polarity, during development [ 15]. Wnt signaling has been implicated in maintaining stem cell pluripotency. The name “Wnt” is a combination of wingless (the name of the Drosophila segment polarity gene) and integrated (the name of the vertebrate homolog) [ 16]. The binding of secreted Wnt proteins onto the outer surface of the cell triggers the activation of several well-known intracellular signal transduction pathways, including the canonical Wnt pathway, the non-canonical planar cell polarity (PCP) pathway, and the non-canonical Wnt/calcium pathway [ 17]. The activation of the canonical Wnt/b-catenin pathway is associated with coupling of Wnt ligands to the Frizzled receptor and the LRP5/6 co-receptor on the cell surface. This extracellular complex induces the activation of the Disheveled (Dsh) protein inside the cell and causes the breakup of the axin2/glycogen synthase kinase 3b (GSK3b)/adenomatosis polyposis coli (APC) complex, which contains the b-catenin protein. The release of b-catenin from this complex increases the cytoplasmic level of b-catenin and facilitates its subsequent translocation to the cell nucleus [ 18], where this protein combines with the TCF/LEF transcription factor to regulate the expression of downstream Wnt target genes [ 19] (Fig. 2).

The canonical Wnt/b-catenin signaling pathway is indispensable to the control of cell proliferation, specification, and differentiation during development [ 20]. The canonical Wnt/b-catenin pathway and the non-canonical PCP pathway have been studied in the cochlea. During the early stages of cochlear development, the canonical Wnt/b-catenin signaling pathway regulates the fate, proliferation, and differentiation of HCs [ 21, 22]. The inhibition of Wnt/b-catenin signaling by small molecules in explant cultures of the embryonic cochleae reduces the proliferation of prosensory cells [ 21]. Conversely, the activation of Wnt/b-catenin signaling by adding LiCl promotes the formation of the Sox2-expressing prosensory domain and increases the number of HCs [ 21]. These findings have been demonstrated in vivo; in the b-catenin transgenic mice, knockdown of b-catenin expression halts the differentiation of HCs from prosensory cells, whereas overexpression of b-catenin enhances the formation of HCs [ 22]. In addition, the forced expression of b-catenin through retroviral gene transfer to activate the canonical Wnt signaling in chicken otocysts generates ectopic vestibular HCs in the basilar papilla [ 23]. The expression of the leucine rich repeat G-coupled family of receptors (Lgr) and the R-spondin family of ligands has been linked to Wnt signaling [ 24]. During the development of the mammalian cochlea, R-spondin 2 is expressed in the sensory epithelium; the loss of R-spondin 2 disrupts the peripheral pattern and the formation of an extra row of outer HCs [ 25]. The R-spondin ligand binds to the Lgr receptor family, including Lgr4, Lgr5, and Lgr6, to regulate Wnt signaling [ 26, 27]. The spatiotemporal expression of Lgr5 and Lgr6 has been observed during the development of the cochlear duct epithelium; between embryonic day (E)18.5 and postnatal day (P)3, Lgr5 is expressed in the third row of Deiters’ cells, inner pillar cells, inner phalangeal cells, and the lateral greater epithelium region, whereas Lgr6 expression is restricted to the inner pillar cells in the basal turn of the cochlea [ 28, 29]. Treatment of Lgr5⁺ progenitor cells in the neonatal cochlea with Wnt agonists triggers Wnt/b-catenin signaling and induces cell proliferation and differentiation into HCs [ 30].

The non-canonical Wnt/PCP pathway plays an important role during cochlear development. Wnt/PCP signaling mediates the planar polarization of HCs, although its role in HC regeneration remains unclear. The activation of Wnt/PCP signaling does not involve b-catenin or its co-receptor LRP5/6 [ 31]. The Wnt ligand binds directly to Frizzled and their co-receptor to stimulate the intracellular Dsh protein, which forms a complex with the DAAM1 protein. DAAM1 further activates the Rho protein through GTP exchange. The activation of Rho-associated kinase by the Rho protein regulates cytoskeleton formation in the cell [ 32]. The Dsh protein also forms complexes with Rac1 to regulate the binding of profilin to actin, thereby restructuring the cytoskeleton. Rac1 stimulates JNK and leads to actin polymerization [ 33, 34]. In the inner ear, Wnt/PCP signaling regulates the formation and correct orientation of the cochlear stereociliary bundle on the surface of HCs [ 35]. The use of Wnt ligand inhibitors to interrupt Wnt signaling disrupts HC bundles, indicating that secreted Wnt proteins are essential for the proper functioning of the Wnt/PCP signaling pathway in the cochlea [ 36]. Moreover, Wnt5a knockout mice exhibit apparent defects in PCP, with shortened and broader cochleae and an extra row of HCs along the entire cochlear length, particularly in the apical region [ 37, 38]. In addition, the expression of the Wnt receptor Frizzled 3 and 6 is observed in cochlear and vestibular sensory HCs; downregulation of these receptors results in defective PCP in cochlear and vestibular HCs [ 39]. Thus, Wnt/PCP signaling is indispensible to the formation and organization of the cochlear stereociliary bundles on the surface of HCs. The activation of the Wnt/PCP pathway is important for the correct orientation of regenerated HCs; however, the precise mechanism through which the Wnt/PCP pathway participates in HC regeneration must be further investigated.

In addition to Wnt, several other genes regulate HC planar polarization and orientation in the developing mammalian cochlea. The loss of Vangl2 and Scrb1 affects the position and anchoring of the kinocilium and significantly disrupts the orientation of HC stereociliary bundles in the mouse cochlea [ 40]. Testin and PTK7 interact with Vangl2 to regulate HC orientation in the inner ear. The absence of Testin results in aberrant orientation of vestibular HCs and defective cellular patterning in the cochlea [ 41, 42]. Similarly, Ankrd6, which is a functional mammalian homolog of the Drosophila PCP gene diego, interacts with Vangl2 to regulate HC polarity in the cochlea. The loss of Ankrd6 causes PCP defects in inner ear HCs [ 43]. Kif3a, a subunit of microtubule motor protein, controls auditory HC planar polarization and mutation in Kif3a, leading to lack of kinocilium, a shortened cochlear duct, and defective HC bundle morphology [ 44]. Moreover, Rho GTPase cdc42, a candidate regulator of PCP, controls the apical polarization of auditory HCs and is indispensible for stereociliogenesis in the immature cochlea [ 45]. Wnt regulates some of these factors; for example, Wnt5a promotes the proper subcellular distribution of Ltap/Vangl2 and interacts genetically with Ltap/Vangl2 for uniform orientation of the stereocilia [ 38]. The Wnt co-receptor receptor-like tyrosine kinase (Ryk) regulates HC polarity by modulating the degradation of the core planar cell polarity component Vangl2. The Ryk receptor transduces the Wnt5a signal by forming a complex with Vangl2 [ 46]. Thus, the canonical Wnt/b-catenin and non-canonical Wnt/PCP signaling pathways regulate HC formation during inner ear development, and their potential roles in HC regeneration must be further explored.

Implication of canonical Wnt signaling in auditory HC regeneration

Active canonical Wnt signaling facilitates HC regeneration, which is robust in non-mammalian vertebrates. In the lateral line of zebrafish, cell clusters comprising the sensory organ, called neuromast, instantly begin to proliferate and regenerate after ototoxic insult and subsequent treatment with the Wnt activator 1-azakenpaullone [ 47]. The activation enhances the production of sensory HCs and concomitantly increases the neuromast size; by contrast, treatment of zebrafish with the Wnt inhibitor dkk1b suppresses the proliferation and regeneration of sensory HCs [ 48]. The chicken basilar papilla shows a similar pattern of HC regeneration by Wnt activation after HC damage [ 49]. The gene expression profiles of regenerating SCs from the zebrafish lateral line demonstrate that Wnt/b-catenin signaling is not activated immediately after damage but is instead upregulated in a later regenerative response [ 50].

In contrast to its non-mammalian counterpart, the adult mammalian cochlea has no potential for spontaneous regeneration. Nevertheless, various studies on the role of canonical Wnt signaling in the mammalian cochlea suggested its possible involvement in this pathway in HC regeneration. Lgr5 is a Wnt downstream target gene that labels adult stem cells across a diverse range of tissue [ 51, 52]. Chai et al. [ 30] explored the existence of Lgr5⁺ progenitor cells in the embryonic and neonatal mice cochleae. Lgr5⁺ progenitor cells in the inner ear are quiescent in nature but assume a proliferative state when isolated and cultured in vitro; these cells form colonies that differentiate into HCs [ 30, 53]. Lgr5⁺ cells can act as HC progenitors both in vivo and in vitro because of their ability to self-renew, proliferate, and regenerate HCs [ 2, 30, 53, 54]. Wnt agonists promote the proliferative capacity of Lgr5⁺ progenitor cells to form HCs in vitro, whereas Wnt antagonists reduce the ability of these cells to proliferate and regenerate HCs [ 30, 53]. Similarly, other in vivo approaches have used transgenic mice; the conditional expression of b-catenin enhances the proliferation of Lgr5⁺ progenitor cells and the formation of cell foci [ 30, 55]. Selective ablation of HCs in transgenic neonatal mice also triggers SCs to adopt a HC fate and spontaneously regenerate HCs in the cochlea; however, these regenerated HCs eventually die because of lack of survival factors [ 2]. A follow-up study demonstrated that after deliberate HC damage, newly regenerated HCs emerge from Lgr5⁺ inner pillar and third-row Deiters cells [ 3]. Compared with neonatal cochlea, the undamaged adult cochleae does not proliferate and regenerate in response to the conditional expression of b-catenin [ 55]. Moreover, no HC regeneration was observed when the cochlear HCs were selectively ablated at one week of age [ 2]. Another study determined the expression of the Wnt downstream target gene Axin2 in tympanic border cells of the mammalian cochlea. Axin2 expression indicates active canonical Wnt signaling in various tissue [ 56, 57]. In the neonatal cochlea, Axin2-expressing tympanic border cells, which lie beneath the sensory epithelium, possess proliferative and regenerative potential in vitro. In the presence of a Wnt activator, these cells rapidly enter into a proliferative state and differentiate to form HCs and SCs [ 58]. Thus, active Wnt signaling influences the proliferation and regeneration of Lgr5⁺ progenitor cells in the neonatal cochleae.

Atoh1, a well-known determinant of HC fate [ 59, 60], is possibly associated with canonical Wnt signaling in HC regeneration. A 3′ enhancer region of Atoh1 serves as a targeted binding site for b-catenin to regulate Atoh1 expression [ 61]. Likewise, overexpressing b-catenin through virus-mediated gene transfer in Lgr5⁺ neurospheres upregulates Atoh1 expression and enhances HC formation [ 53]. Hence, Wnt signaling regulates Atoh1 expression by acting upstream and activating the proliferation and differentiation of HCs. Kuo et al. suggested another integrative strategy for HC regeneration; the combined expression of b-catenin and Atoh1 in Lgr5⁺ progenitor cells could increase the proliferation and HC regeneration capacities of the postnatal cochleae by 10-fold. In contrast to previous reports, these newly regenerated HCs could survive until adulthood [ 62]. Bmi, which is a member of the polycomb protein family, regulates the proliferation of SCs and Lgr5⁺ progenitors in neonatal mice; the targeted deletion of Bmi downregulates Wnt signaling in the cochlea [ 63]. Wnt/b-catenin signaling plays a protective role against neomycin-induced HC loss [ 64]. In summary, Wnt/b-catenin signaling promotes HC regeneration in mammals; however, this activity has only been established in the neonatal cochlea and must be further investigated in damaged mature cochleae.

Wnt activation promotes HC proliferation and differentiation into prosensory cells through upregulation of the cell cycle regulator cyclin D1 [ 21]; conversely, cyclin-dependent kinase inhibitors (CDKIs) can negatively control cell cycle progression and contribute to the loss of regenerative potential in SCs by preventing the cells from reentering the cell cycle. During embryonic development, the Notch–Hes1 pathway promotes cochlear prosensory cell formation by CDKI 1B (p27kip1) transcriptional downregulation [ 65], suggesting that Notch and p27kip1 exhibit related functions during prosensory determination. p27kip1 causes cell arrest in the G₁ phase of the cell cycle [ 66]. This protein is first expressed in the primordial organ of Corti during embryonic development (E12 and E14) and then blocks cell division in the progenitors of SCs and HCs [ 67]. The targeted disruption of the p27kip1 gene allows SCs to proliferate and regenerate supernumerary HCs in the postnatal and adult cochlea [ 67, 68]. Flow-sorted isolation of p27/GFP⁺ cells from the embryonic cochlea of p27kip1 transgenic reporter mice shows that these cells express other sensory lineage markers, such as Sox2, and differentiate into HCs in vitro [ 69]; hence, these cells can also potentially act as HC progenitors.

In postnatal mice, SCs are postmitotic in vivo, whereas p27/GFP⁺ cells cultured in vitro show that p27kip1 downregulation promotes the differentiation of SCs into HCs [ 70]. The use of short hairpin RNA-expressing vectors to knockdown p27kip1 expression induces cell cycle reentry in postmitotic SCs to regenerate HCs [ 71]. Another study demonstrated p27kip1 expression in the mature cochleae and the forced expression of Skp2, which stimulates the transition of G₁ to G₀ cell cycle via p27kip1 and cyclin E ubiquitination [ 72]; p27kip1 along with Atoh1 results in mitotic division and transdifferentiation of SCs into HCs in the mature auditory epithelium [ 73]. In addition, the sustained expression of p27kip1 maintains the mature organ of Corti in a quiescent state. Tamoxifen-induced deletion of p27kip1 in mice shows that the absence of p27kip1 is sufficient to induce proliferation of SCs in the neonatal and adult cochleae [ 74]. In previous reports, manipulation of p27kip1 expression in SCs can promote proliferation and transdifferentiation into HCs. By contrast, Walters et al. demonstrated that HC-specific deletion of p27kip1 in the neonatal mouse cochlea promotes autonomous cell proliferation and generation of new inner and outer HCs from the residing HCs; these newly regenerated HCs survive to adult age and express mature HC markers [ 75]. This finding suggests that p27kip1 expression in HCs is sufficient to maintain a quiescent state, and the targeted deletion of p27kip1 triggers existing HCs to divide and regenerate new HCs in the mature mouse cochlea. The newly regenerated HCs express the characteristic synaptic and stereociliary markers and can survive into adulthood. p27kip1 knockout mice also exhibit normal hearing, as measured by evoked auditory brainstem response; therefore, these newly generated HCs possibly contribute to the normal functioning of the cochlea [ 75].

The targeted disruption of CDKI p19 (Ink4d) in postnatal mice also results in aberrant re-entry of HCs into the cell cycle, although these regenerated HCs cells do not survive into adult age [ 65]. The deletion of Ink4d along with CDKI p21 (Cip1) triggers the re-entry of auditory HCs in the cell cycle during the early postnatal period, leading to the formation of supernumerary HCs. However, the aberrant cell cycle re-entry activates the DNA damage response pathway, which is followed by p53-mediated cell apoptosis [ 76].

Retinoblastoma protein (Rb1) is another candidate cell cycle repressor that regulates cell cycle exit in HCs. Rb1 suppresses Skp2 activity and stabilizes p27kip1 (a downstream target of Rb1), which inhibits the kinase activity associated with cyclin E and A and arrests cell cycle progression [ 77]. The disruption of Rb1 during embryonic development (E17 to E18) and early postnatal age promotes continuous cell division in the mammalian cochlea, thereby regenerating new HCs from the division of preexisting HCs [ 78]. In adult mice, the targeted deletion of Rbl2, another member of the retinoblastoma protein family (pRb), forms extra rows of HCs and SCs in the apex, although these newly regenerated SCs do not transdifferentiate to form HCs in the adult cochleae [ 79]. These studies highlight important factors that prevent cell cycle progression and inhibit the proliferation and transdifferentiation of cochlear SCs into HCs. Conversely, modulating the expression of these factors may enhance regenerative potential in the mature mammalian cochlea.

Notch signaling pathway in auditory HC development and regeneration

The Notch signaling pathway is an evolutionarily conserved pathway that influences cell patterning, proliferation, differentiation, and apoptosis in various tissue [ 80]. Mammals possess four different types of Notch receptors (Notch 1–4) and five Notch ligands (Jagged 1, Jagged 2, and Dll 1, 3, and 4) [ 81– 83]. The activity of Notch signaling depends solely on the interaction of Notch receptors with their ligands, all of which are highly conserved transmembrane proteins [ 84, 85]. Notch receptors contain two binding domains, namely, intracellular cytoplasmic domain and another domain exposed outside the cell surface for the binding of the Notch ligand. The Notch pathway becomes activated when the Notch transmembrane receptor combines with the transmembrane ligand protein expressed on the extracellular surface of neighboring cells. This interaction stimulates the proteolytic cleavage of Notch receptor, liberating the Notch intracellular domain (NICD), which then translocates into the nucleus to associate with the DNA binding protein CSL (also called RBP-J in mammals) and form an active transcriptional complex. The subsequent association of the Mastermind-like (MAML) family of transcriptional co-activators with this complex induces the transcription of Notch-targeted genes, such as Myc, P21, and HES family members (Fig. 3). HES acts as the primary Notch receptor and affects cell fate decision. Expressing HES downregulates the expression of tissue-specific transcriptional activators and ultimately influences cell proliferation, differentiation, and apoptosis [ 86, 87].

In the inner ear, Notch signaling plays crucial roles during the development and patterning of sensory organs. The Notch signaling pathway operates via two modes, lateral induction and lateral inhibition. In lateral induction, the signaling between the Notch ligand and Notch receptor on adjacent prosensory cells follows a positive-feedback loop; this loop activates Notch ligand expression in neighboring cells to promote prosensory cell formation during early cell development [ 88, 89]. In lateral inhibition, Notch signaling between the Notch ligand and Notch receptor is negatively regulated [ 90]. Cells that express numerous Notch ligands on their surfaces are committed to a specific cell fate, such that providing signals to neighboring cells to reduce the expression of Notch ligands prevents them from adopting the same cell fate. Notch lateral inhibition is used during later stages of development when the HCs and SCs differentiate [ 91].

During early otic development Notch signaling specifies the prosensory domain via lateral induction. The expression of Jagged 1 during prosensory specification is indispensable; the targeted deletion of Jagged 1 downregulates the expression of prosensory markers, such as Sox2 and P27kip1, resulting in the formation of a malformed cochlear duct with reduced numbers of HCs [ 92, 93]. Moreover, in vitro treatment with the g-secretase inhibitor DAPT prevents cochlear explants from forming a prosensory region [ 94]. The conditional activation of Notch signaling during early development induces the expression of prosensory markers, and its activation at later stages stimulates the formation of ectopic sensory patches containing both HCs and SCs [ 95– 97]. In chick otocysts, virus-mediated transient overexpression of NICD stimulates ectopic sensory patches in the cochlear duct [ 89]. Another study on developing chick cochlea reported that DAPT-mediated Notch inhibition disrupts the maintenance of prosensory patch development but does not influence the initial specification of the prosensory domain [ 98]. During the later stages of cochlear development, Notch signaling organizes the formation of mosaic-like patterns of sensory HCs and adjacent SCs through lateral inhibition [ 92, 99]. Conditional deletion of the Notch ligands Jagged 2 and Dll1 results in the production of supernumerary HCs, suggesting that both ligands work synergistically to control HC differentiation in the cochlea [ 100, 101]. A study suggested that Jagged 1 stimulates lower levels of Notch activity compared with Dll1, resulting in the differential expression of the Notch target genes Hey1 and Hes5 in chick inner ear [ 102]. Thus, Notch signaling is shown to be indispensable and sufficient for prosensory specification and HC patterning during development.

Implication of Notch signaling in auditory HC regeneration

The Notch signaling pathway has been examined because of its participation in the proliferation and differentiation of HCs in the prosensory domain during development. The mechanism through which this pathway regulates developmental processes is relevant to HC regeneration because it may be used to induce regenerative potential in the mammalian cochlea. In the zebrafish lateral line, the expression level of Notch-targeted genes decreases immediately after neomycin treatment; thus, the regenerating neuromasts have low levels of Notch activity during cell division [ 50] and more active Notch signaling with further regeneration. The application of g-secretase inhibitor causes excessive regeneration of HCs in the zebrafish lateral line [ 103]. Cell proliferation is suppressed by the Notch signaling pathway by regulating the expression of cell cycle inhibitors. In the mammalian cochlea, Notch signaling acts upstream of the cell cycle inhibitor p27kip1. During the regeneration of neuromasts, the downregulation of Notch activity is associated with reduced expression of cell cycle inhibitors [ 50, 65, 92, 93]. In the case of HC damage in mature avian basilar papilla, the transcription of Notch pathway genes is upregulated in the actively dividing region at days 1 and 4 of gentamicin treatment; the addition of the Notch inhibitor DAPT increases the regeneration of HCs, at the expense of SCs, through mitotic division and direct transdifferentiation. By contrast, the constitutive overexpression of the Notch pathway in SCs causes the cells to maintain a quiescent state, thereby inhibiting HC regeneration [ 104]. Hence, after the initiation of HC regeneration in birds, Notch signaling further controls the behavior of SCs.

In mammals, Notch signaling prevents cell cycle re-entry. Notch inhibition promotes the proliferation of resident SCs in postmitotic cochlear explants [ 105]; hence, Notch signaling positively regulates the expression of cell cycle inhibitors in postmitotic cochleae. Aminoglycoside-mediated cochlear damage increases the expression levels of Jagged1, Hes1, and Hes5, thereby activating Notch signaling [ 106]. In the postnatal cochleae, the blockade of Notch signaling by g-secretase inhibitor results in the transdifferentiation of adjacent SCs into HCs, which possess the same characteristics as immature HCs [ 107]. In the noise-induced HC damage model, the deliberate pharmacological inhibition of Notch signaling with g-secretase inhibitor upregulates the expression of Atoh1 transcription factor, which subsequently triggers HC regeneration in the damaged cochleae [ 108]. Some elements of the Notch signaling pathway are also expressed in the adult damaged mammalian cochlea, and Notch activity immediately increases after ototoxic damage [ 106, 109, 110]. An in vivo functional study reported that the inhibition of Notch activity in adult noise-damaged cochleae stimulates HC regeneration and improves hearing ability [ 111]. However, a recent study on 6-day-old mouse cochleae showed completely opposite results with the in vitro exposure of explant cultures to the Notch inhibitor [ 112]. The discrepancy in the results could be due to the fact that in vitro experimental conditions do not completely mimic in vivo conditions. Maass et al. used the in vitro approach with noise damage method to induce hearing loss in adult mice [ 112]. Other groups followed the in vivo approach and used ototoxic drugs to induce hearing loss in adult mice [ 106, 109, 110]. Thus, different conditions may cause a lack of Notch signaling in the adult cochlea. Apart from the damage model, few studies also highlighted that the conditional deletion of the Rbpsuh gene or the addition of the Notch inhibitor DAPT causes SCs to take on a HC fate in undamaged cochleae [ 113– 115]. These findings in mammalian and non-mammalian species ultimately imply that Notch signaling inhibits the differentiation of SCs into HCs and presents a potential for developing strategies to regulate the mechanism of Notch lateral inhibition in SCs and promote HC regeneration in the mammalian cochlea.

Wnt and Notch signaling — an integrative approach in auditory HC regeneration

The Wnt and Notch signaling pathways are involved in animal development, from embryonic to adult stage; early pivotal events, such as cell proliferation, differentiation, and specification, have been well studied. Zak et al. comprehensively reviewed the role of the Wnt and Notch signaling pathways in the developing cochlea [ 14]. The mechanisms through which Wnt and Notch regulate developmental events have attracted recent attention in the context of HC regeneration. The canonical Wnt pathway is activated during the early development of the cochlear prosensory region and controls both proliferation and HC differentiation within the cochlear duct [ 21]. Moreover, Notch activity is crucial in early developmental events, and the Notch ligand Jagged1 stimulates lateral induction to control sensory cell fate in the prosensory region [ 85, 92, 95, 98, 116].

Much research has demonstrated that both the Wnt and Notch signaling pathways work in parallel for cell fate determination in multiple tissue [ 3, 115, 117, 118]. At the molecular level, the interaction between the Wnt and Notch signaling pathways was first identified by Katoh et al.; in their study, the 5′ promoter region of the mammalian Notch ligand Jagged1 ortholog contains specific TCF/LEF sites, where b-catenin binds and positively regulates Jagged1 expression [ 119]. This study showed that Jagged1 is an evolutionarily conserved target of the canonical Wnt signaling pathway. In the mouse otic placode, a reciprocal interaction was observed between the Wnt and Notch signaling pathways. Wnt signaling positively regulates Notch target genes, namely, Jagged1, Notch1, and Hes1, by acting upstream, and inhibition of Wnt signaling results in a smaller otic placode [ 117]. A recent study in the zebrafish lateral line reported that Notch signaling hinders the proliferation and differentiation of SCs into HCs in localized regions by inhibiting Wnt activity [ 47]. These observations suggest an interplay between the Wnt and Notch pathways during development; however, further functional studies must be performed to elucidate their interaction.

Li et al. provides the first piece of evidence for the direct interaction between the Wnt and Notch signaling pathways in the postnatal cochleae. The localized or conditional inhibition of Notch signaling in the postnatal cochlea accelerates the proliferation and differentiation of the pool of resident SCs into HCs. A large number of newly regenerated HCs is derived from Lgr5⁺ SCs, which are known to be responsive to Wnt signaling [ 115]. In addition, the simultaneous inhibition of Wnt and Notch signaling decreases the number of HCs generated through proliferation. This study proposed a model for HC regeneration after Notch inhibition in the neonatal cochlea (Fig. 4), which either follows the mitotic generation of HCs through the activated Wnt signaling or the direct transdifferentiation of SCs into HCs without the involvement of Wnt [ 115]. However, another study indicated that Notch inhibition fails to stimulate the conversion of SCs into HCs in the explant culture of b-catenin knockout mice [ 3]; hence, Wnt signaling is required for direct transdifferentiation of SCs into HCs, and Notch signaling alone is insufficient to convert resident SCs into HCs. These observations imply that Notch inhibition promotes the accumulation of b-catenin in SCs to positively regulate the expression of the Atoh1 transcription factor and form functional HCs. Atoh1 is a well-known master switch that determines HC fate [ 59]; in the absence of b-catenin, the expression of Atoh1 is not sufficient to induce the differentiation of SCs into HCs.

Atoh1 is a Wnt target gene, and its role in determining HC fate has been well studied [ 59, 60, 120]. In neural progenitor cells, b-catenin upregulates the expression of Atoh1 by interacting with the 3′ enhancer region of Atoh1 [ 61]. In the developing cochlea, Wnt activation promotes the expression of Atoh1 and generates supernumerary HCs [ 22], suggesting that b-catenin is an upstream regulator of Atoh1 expression and promotes HC formation. Thus, developmental studies imply that Wnt signaling promotes the formation of functional HCs by positively regulating Atoh1 expression, whereas Notch signaling promotes the formation of SCs by inhibiting Atoh1 expression. However, the mechanism through which Atoh1 interacts with the Wnt and the Notch signaling pathways during the postnatal period remains unclear. A recent study showed that the co-activation of b-catenin and Atoh1 generates tenfold more HCs in the neonatal cochlea compared with previous reports [ 62], suggesting that the constitutive expression of b-catenin and Atoh1 induces the proliferation and differentiation of localized SCs into HCs in postnatal cochleae.

Collectively, these findings suggest that, together with Atoh1, the manipulation of the Wnt and Notch signaling pathways have the potential to induce HC regeneration in postnatal ages. Thus, a strategy is required to first activate Wnt signaling in mitotically quiescent SCs to stimulate their proliferation, then to regulate Atoh1 expression and inhibit Notch signaling to promote the differentiation of SCs into HCs. However, at this point, much research is still needed to gain further understanding of the relationship between the Wnt and Notch signaling pathways, for the inducement of regenerative potential in mature mammalian cochleae.

Conclusions

In recent years, research on HC regeneration has been focused on developing approaches to treat sensorineural hearing loss. The momentum built up by current studies on the role of signaling pathways in regenerating sensory epithelium will likely drive the development of therapeutic interventions for hearing loss in the near future. The involvement of Wnt and Notch signaling during proliferation, differentiation, and regeneration of sensory HCs has been the focus of much research. Thus far, most studies focused on neonates, whose neighboring SCs show a limited capacity to acquire HC fate after HC damage. Understanding the modulatory roles of Wnt and Notch, together with other signaling pathways, is critical to induce regenerative potential in the mature mammalian cochleae.

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