Introduction

Like all neurons of the brain and receptor cells of the eye, olfactory system, and taste buds, the ear arises from molecularly transformed embryonic ectoderm. This transformation in the ear is mediated by the expression of transcription factors that expand the proliferation of the precursor population and transforms the cells into neurons or sensory cells (Fritzsch et al. 2006a; Gokoffski et al. 2011). In addition, complex changes of the single layer of cells into a set of tubes and recesses is needed to generate the inner ear labyrinth (Kopecky et al. 2012). The inner ear labyrinth of adult mammals consists of three semicircular canals, the utricle connecting all canals, and the saccular recess (Fig. 1). The base of each canal, the utricle, and the saccule house the five vestibular sensory organs that allow the perception of position and movement in space (Lewis et al. 1985). In addition, only the mammalian ear has the coiled cochlear duct connected via the ductus reuniens with the saccule. The cochlear duct contains the organ of Corti (OC), which translates sound into hearing (Hudspeth 2014) and is connected via the spiral ganglion neurons (SGNs) to the cochlear nuclei of the hindbrain (Nayagam et al. 2011).

Fig. 1
figure 1

Thin-sheet laser imaging microscopy and three-dimensional reconstructions showing a developing left ear viewed laterally (top row, anterior is left) and ventrally (bottom row, anterior is left). Note the dramatic growth of the cochlea to become the largest duct in the mouse ear exceeding the semicircular canals in length and width (E embryonic day, AC anterior canal crista, C cochlear duct, HC horizontal [lateral] canal crista, PC posterior canal crista, S saccule, U utricle). Compiled after Kopecky et al. (2012). Bar 100 μm

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The coordinated transformation of a flat embryonic epithelium into the cellular mosaic of the adult structure requires an orchestrated regulation of a multitude of transcription factors leading to the differential expression of about 17,000 genes that mark the adult inner and outer hair cells (Liu et al. 2014). We are only now beginning to understand the interactions of these factors, mostly through the analysis of expression patterns and phenotypes in mutant mice. The complexity of changes is related to the transformation of a sheath of cells into the vestibular organs and cochlear duct as part of a three-dimensional labyrinth of ducts and recesses (Chang et al. 2004; Fritzsch et al. 2013). The OC is arguably the most sophisticated cellular mosaic of the mammalian body with ten distinct types of cells (Slepecky 1996; Zetes et al. 2012) uniquely distributed in and around the OC to ensure the transformation of sound into electric signals (Hudspeth 2014). In addition, the OC is innervated by a highly patterned set of afferents (Nayagam et al. 2011) and receives a complicated innervation by efferents (Simmons 2002) to apparently modify the sensitivity of the organ to loudness (Sienknecht et al. 2014).

The OC and the unique organization of its innervating sensory neurons into a distinct spiral ganglion (Mao et al. 2014; Nayagam et al. 2011; Sandell et al. 2014) and the organization of efferents into the olivo-cochlear system (Simmons et al. 2011) are features found only in mammals. Interesting transitions of the basilar papilla of tetrapods into an OC exist in egg-laying mammals (Fritzsch et al. 2013). These changes indicate that the expansion of the cochlear duct subsequent to the loss or transformation of the lagena (Luo et al. 2011) was a crucial step in the evolution of the simpler checkerboard cellular mosaic of the basilar papillae of other tetrapods into the complicated organization of the mammalian OC (Fritzsch et al. 2013). Interestingly, the OC of extant Monotremes (Platypus, Spiny Anteater) contains all the cell types of the Eutherian OC, but in a different arrangement. This suggests that the transformation of the tetrapod basilar papilla into a coiled OC had largely occurred in mammalian ancestors. However, these cell types of the OC of Monotremes lie within a short lagena duct that contains at its apex the lagena sensory epithelium, like in reptiles and amphibians. Instead of one row of inner hair cells, two rows of pillar cells, and three rows of Deiter’s cells and outer hair cells throughout the OC, all these elements of the mammalian OC are mostly multiplied in Monotremes, except for the basal part that resembles closely the Eutherian OC (Ladhams and Pickles 1996). In addition to resolving the evolutionary origin of the basilar papilla as a separate sensory epithelium of the lagena recess of tetrapods (Fritzsch et al. 2013), knowledge of the molecular basis of the reorganization of afferents, hair cells, and supporting cells to generate the uniquely mammalian OC out of the tetrapod basilar papilla with its innervating SGNs is the most important next step in understanding the evolution and development of the inner ear part of the mammalian auditory system. As all the central auditory system development (Rubel and Fritzsch 2002) presented in this volume of Cell and Tissue Research hinges directly or indirectly on this evolutionary topic (Fritzsch et al. 2006c; Grothe et al. 2004), this review will provide the molecular development of the OC, cochlear duct and SGN to set the stage for an understanding of the topics explored elsewhere in this special issue.

From placode to SGNs: molecular transformation of ectoderm to neurons that provide the innervation of the OC

The development of vertebrate neuroectoderm out of ectoderm requires bone morphogenetic protein 4 (BMP4) downregulation combined with fibroblast growth factor (Fgf) expression (Delaune et al. 2005; Fritzsch et al. 2006a; Streit et al. 2000). Consolidating the transformation of ectoderm into pro-neurosensory precursors producing neuroectoderm is driven overall by the regulation of similar genes, with Oct6/Pou3f1 being among the earliest transcription factors expressed, followed by Sox2 and other Sox genes to maintain proliferation in a committed population of neuronal precursors (Fritzsch et al. 2006a; Reiprich and Wegner 2014). This continued proliferation of committed pro-neurosensory precursors is achieved in part by preventing premature signals from pro-neural basic helix-loop-helix (bHLH) transcription factors through the expression of Ids and Mycs (Zhu et al. 2014), reinforced by many signals that define the pro-neurosensory region of the otic placode/otocyst, such as Lmx1a (Nichols et al. 2008), Tbx1 (Raft et al. 2004), and various diffusible factors (Chen and Streit 2013; Raft and Groves 2014).

An aspect that is unique to vertebrates is that all major sensory organs, namely eyes, ears, taste buds, and the olfactory system, develop from, or have contributions from the placodes (Northcutt and Gans 1983; O'Neill et al. 2012; Patthey et al. 2014). Placodes might have evolved through the embryonic aggregation of ancestral single-cell neurosensory precursors (Fritzsch and Straka 2014), comparable to the apparent aggregation of all the neurogenic potential of the ectoderm into a single continuous sheet of cells that form the central nervous system (Fritzsch and Glover 2007; Pani et al. 2012). In mammals, the otic placode is initially part of a pan-placodal region that expresses transcription factors necessary to initiate the neurosensory transformation of the embryonic ectoderm (Chen and Streit 2013; Schlosser et al. 2014; Steventon et al. 2014). The size of the otic placode is determined mostly through Wnt signaling (Ohyama et al. 2007), and several other factors cooperate to invaginate the rapidly proliferating otic placode to form the otic cup and later the otocyst, which is completely segregated from the ectoderm (Fritzsch et al. 1998; Romand and Varela-Nieto 2014).

The future cochlear duct in mice begins its growth at the ventral tip of the elongated otocyst (Fig. 1) around embryonic day (E) 11.5 and finishes its elongation around birth (Kopecky et al. 2012). Similar growth of the cochlear duct occurs in humans, albeit with a different timetable (Kopecky and Fritzsch 2013). As the cochlear duct elongates, SGNs delaminate apparently from three principal areas of the cochlear duct (Fig. 2): the ductus reuniens that later separates the cochlear duct from the saccular recess, the middle turn, and the apex (Yang et al. 2011). These delaminating neurons are characterized by their expression of neurotrophins (Durruthy-Durruthy et al. 2014; Farinas et al. 2001). The neurons continue to proliferate and eventually shut down neurotrophin expression, after they become post-mitotic, and express neurotrophin receptors instead (Fritzsch et al. 2002). Neurotrophins released from the sensory epithelia and neurotrophin receptors expressed in developing SGNs are essential for neuronal survival (Fritzsch et al. 2004). This expression of neurotrophins in the prosensory region and in early delaminating neuroblasts suggests a possible lineage relationship of hair cells and sensory neurons (Farinas et al. 2001), now expanded to neuronal and hair cell differentiation transcription factors and their interactions (Fritzsch et al. 2010b; Ma et al. 2000; Pan et al. 2012b). The migrating SGNs have to digest their way through the basal lamina surrounding the developing cochlear duct possibly with enzymes similar to those used in the neural crest (Mao et al. 2014) and respond to stop signals provided by Schwann cells to reside in Rosenthal’s canal inside the developing ear (Yang et al. 2011). In the absence of Schwann cells, SGNs migrate in part outside the ear, like vestibular ganglion neurons (Mao et al. 2014). “Birthdating” with 3H-thymidine (Ruben 1967) and BrdU (Matei et al. 2005) has established that SGNs become post-mitotic in a base-to-apex progression between E10.5 to E12.5. Several recent reviews have expertly summarized much of what is known about the gene regulation of the neuronal development of the ear (Appler and Goodrich 2011; Coate and Kelley 2013; Raft and Groves 2014; Yang et al. 2011), and the reader is referred to these more detailed accounts. Here, we concentrate only on the molecular regulation of SGN formation and development as relevant for the viability and patterned projection of these neurons to connect precisely the OC with the cochlear nuclei, the molecularly least understood aspect of SGN development.

Fig. 2
figure 2

The developing ear has neurosensory precursors that share expression of brain-derived neurotrophic factor (Bdnf, a-c), irrespective of whether they develop into hair cells or neurons (AC anterior canal crista, HC horizontal [lateral] canal crista, DR ductus reuniens, Vgl vestibular ganglion). In control animals, all sensory epithelia are positive for Bdnf (a), but only the canal cristae and the apex of the cochlea retain Bdnf expression in Atoh1 null mice (b), indicating that the fate of sensory cells varies in different epithelia. In contrast to Atoh1 null mice, Neurog1 null mice never develop ganglion neurons and show an enlargement of the utricle (U) but a severe reduction of the saccule (S, c). Whereas only canal cristae and apical precursors remain Bdnf-positive in Atoh1 null mice (b), mice homozygotic for Atoh1LacZ show profound expression in sensory precursors nearly equivalent to that of control mice at this early stage (d). Delaminating neurons from the ear are only positive for Bdnf (stars in a, b), but some neurons are also positive for enhanced green fluorescent protein (eGFP) expression driven by an Atoh1 enhancer element (Vgl in e) that also labels all vestibular hair cells (e). At later stages, this Atoh1-eGFP expression expands not only to all hair cells of the OC  (IHC inner hair cell, OHC outer hair cell), but also to almost all inner pillar cells (IPC, f). Compiled after Fritzsch et al. (2005b) and Matei et al. (2005). Bar 100 μm (a–e), 25 μm (f)

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Among genes that have been shown to be essential for the formation of SGNs and their patterned projection are mostly transcription factors, but also several diffusible factors such as Shh (Bok et al. 2007), Wnts (Ohyama et al. 2007), and Fgfs (Wright and Mansour 2003). Among the transcription factors, several are more global and locally expressed that specify first the pre-placodal region and subsequently the otic placode and the prosensory region of the otocyst. One of the most important preplacodal transcription factors is Eya1/Six1, which is now known to regulate the expression of downstream pro-neurogenic factors such as Neurog1 (Ahmed et al. 2012). Several factors are selectively expressed in the otic placode such as Foxi factors that are essential for Fgf signaling (Edlund et al. 2014). Fgfs and their receptors are, in turn, essential for placode differentiation (Pirvola et al. 2000). Analogous to the role of Pax6 for eye development (Gehring 2011; Lamb 2013) are Pax2/8 genes for ear development (Bouchard et al. 2010). Mutational analysis has shown that the loss of Pax2 results in the loss of the cochlea (Burton et al. 2004) or its transformation into a featureless sack without SGNs (Bouchard et al. 2010). In contrast, the earlier-expressed Pax8 has no direct effect on ear development, apparently because of compensation by the later expressed Pax2. Eliminating both Pax2 and Pax8 results in the severe truncation of ear development at the otocyst stage without any formation of neurosensory cells, indicating a molecularly unclear connection of Pax2/8 genes to the formation of ear ganglion cells, possibly via an interaction with Pou domain factors as in general neurogenesis (Zhu et al. 2014). Incidentally, Pax2 plays a similar role in retinal ganglion cell development (Cross et al. 2011; Torres et al. 1996), which requires the bHLH transcription factor Atoh7, an ortholog of the hair cell differentiation transcription factor Atoh1, for normal differentiation (Mao et al. 2013; Prasov and Glaser 2012). These similarities might be related to the conservation of these molecular cascades in the sense of deep homology (Shubin et al. 2009) for eye and ear development and evolution (Fritzsch and Piatigorsky 2005).

Among other genes essential for SGN development are Gata3 (Karis et al. 2001), Neurog1 (Ma et al. 2000), Neurod1 (Kim et al. 2001), Tbx1 (Raft et al. 2004), and Pou4f1 (Huang et al. 2001). Gata3 is uniquely expressed in the ear, and mutations truncate the cochlear duct and result, if eliminated early enough, in the complete absence of all neurosensory development (Duncan and Fritzsch 2013). In contrast, the later conditional deletion of Gata3 in the differentiated SGNs causes severe disruption of their projection (Appler et al. 2013; Duncan and Fritzsch 2013). Another gene uniquely expressed in the ear is Foxg1, and its loss truncates severely the formation of both the sensory epithelia and the sensory neurons, consistent with its alleged function in enhancing the proliferation of precursors (Pauley et al. 2006). Other early expressed genes of the ganglion neurons are known, but their function has not been analyzed in detail, and they might mostly function to modify the pro-neural signal such as Delta/Notch and the downstream bHLH transcription factors (Raft and Groves 2014; Yang et al. 2011).

Although Neurog1 is clearly an essential gene for the induction of neuronal development in the ear, alternative interpretations for the origin of neurons from the ear and neural crest have recently been proposed (Freyer et al. 2011). To put this recent claim about the dual origin of the inner ear neurons into perspective, one needs first to realize that neurons have been traced with regard to their delamination from the ear by using neurotrophins (Fig. 2), an expression not found in neural crest derived neurons. Moreover, inner ear neurons require factors for their development or express genes that are only present in the developing ear and not in the adjacent neural crest (Pax2, Pax8, Gata3, Foxg1, Bdnf; Bouchard et al. 2010; Pauley et al. 2006; Rubel and Fritzsch 2002). However, some genes, such as Neurog1, are common to neuralcrest-derived and ear-derived neurons (Ma et al. 1998). Consistent with the ubiquitous expression of some markers of ear-derived neurons, enhancers isolated from transcription factors expressed in the neural crest have been claimed to show that a variable number of neurons and even hair cells derive from the neural crest (Freyer et al. 2011). Unfortunately, alternative interpretations of these results, such as an altered expression of the artificial construct, have not been ruled out. More recent analysis of neural crest contributions to the neurosensory cells of the developing ear by using more specific techniques have not verified such contributions (Sandell et al. 2014). Most recently, the genetic ablation of the complete neural crest has failed to show noticeable deficits in the neurosensory development of the ear (Mao et al. 2014), as would be expected  based on the claims of substantial contributions of the neural crest to neurosensory ear development (Freyer et al. 2011). At the moment, the numerous studies that directly show the delamination of neurons from the ear by using ear specific markers (Bdnf, Ntf3; Fig. 2) or demonstrate the loss of neurons by using ear-specific transcription factors (Gata3) in combination with ear-specific Cre lines such as Foxg1 (Duncan and Fritzsch 2013), neither of which is expressed in the neural crest (Karis et al. 2001; Pauley et al. 2006), appear not to be compatible with a neuralcrest-derived contribution to the neurosensory cells of the ear. In addition, the neuralcrest-derived hypothesis cannot explain why nearly all neurons of the vestibular system depend on TrkB (Fritzsch et al. 1995), much like other placode-derived neurons (Fritzsch et al. 1997b), but unlike neural-crest-derived neurons (von Bartheld and Fritzsch 2006). Indeed, all inner ear sensory neurons are uniquely dependent on two neurotrophin receptors, namely TrkB and TrkC, and their two ligands, namely Bdnf and Ntf3 (Fritzsch et al. 2006b; Rubel and Fritzsch 2002), and this unique dependency is not shared with any neuralcrest-derived sensory neurons (von Bartheld and Fritzsch 2006).

The transformation of ectoderm into sensory neurons depends on the expression of a number of genes, but some form an interesting cascade of gene expression. Sox2 is needed for Neurog1 expression (Puligilla et al. 2010), which, in turn, regulates the expression of Neurod1, a gene necessary for the differentiation of most, but not all sensory neurons (Kim et al. 2001; Krüger et al. 2006), as initially claimed (Liu et al. 2000). Among the genes regulated by Neurod1 to ensure proper differentiation are Pou4f1 (Brn3a), a gene needed for proper fiber growth (Huang et al. 2001), and the neurotrophin receptors TrkB/TrkC, which are needed for the survival of sensory neurons (Farinas et al. 2001). Many other genes expressed in differentiating neurons have been identified, but their role in the differentiation and development of the targeted projections of SGNs has not yet been fully clarified through genetic analysis in mutant mice (Appler and Goodrich 2011; Yang et al. 2011).

One feature unique to SGNs is their specific topographically precise order of projection to the cochlear nuclei and the orderly projection to the two types of hair cells of the OC to form the neuroanatomical basis of the tonotopic map of the auditory system (Nayagam et al. 2011). The vast majority of SGNs differentiate as Type I SGNs that terminate exclusively at the single row of inner hair cells in an overlapping fashion of 10-30 fibers converging on a single hair cell. A minority of 5-8 % of SGNs develop into Type II SGNs that project seemingly exclusively to the three rows of outer hair cells, each fiber innervating many outer hair cells over a distance of several hundred micrometers. Despite this diffuse peripheral projection (Fritzsch 2003), the central projection in the cochlear nuclei appears to be in parallel to and is as well organized for Type II as that for Type I SGNs (Nayagam et al. 2011). Numerous ideas of the way that this peripheral segregation of Type I fibers to inner hair cells and Type II fibers to outer hair cells comes about have been proposed (Bulankina and Moser 2012; Echteler et al. 2005). However, detailed analysis of this process has revealed that the segregation of Type I and Type II neurons might begin much earlier (Bruce et al. 1997; Koundakjian et al. 2007) than previously suggested. Indeed, certain mutations indicate deviation from the normal development of Type II afferents prior to reaching the outer hair cells (Fritzsch et al. 2010a), indicating that Type II SGNs might be genetically determined to project differently rather than being randomly sorted upon reaching the OC with their processes, as some previous ideas have implied.

An interesting set of data seemed  to provide a solution for this problem by indicating that neurotrophins have differential effects on Type II versus Type I fibers (Ernfors et al. 1995). However, more detailed investigations have concluded that this differential effect is only a quantitative correlation and does not fit to the distribution pattern of either neurotrophins or their receptors (Farinas et al. 2001; Yang et al. 2011). Moreover, genetic manipulations that lead to the almost complete ablation of inner hair cells indicates that the majority of spiral ganglion afferents have the capacity to grow to outer hair cells, if forced (Pan et al. 2012a ;  Jahan et al. 2013), but normally receive a stop signal around the inner hair cells, a signal that is possibly related either to the unique co-expression of Ntf3 and Bdnf in the inner hair cells and that develops shortly before birth (Farinas et al. 2001) or to the expression of EphA4 (Defourny et al. 2013). Markers for Type II fibers such as Peripherin are able to distinguish Type II fibers only after they have projected differentially to the outer hair cells and thus cannot be causally linked to the segregation process (Nayagam et al. 2011). Despite plausible ideas conerning this subject, the molecular basis of the segregation of these types of afferents remains still unclear. Indeed, more recent data indicate that a combination of early neuronally expressed genes such as Prox1 (Fritzsch et al. 2010a) and interactions of growing afferents with neuralcrest-derived Schwann cells (Mao et al. 2014) ultimately regulate the growth of the two types of spiral ganglion fibers to the OC and within the OC (Fig. 3). Given the role of Schwann cells in guiding afferents to the OC, we can reasonably assume that afferent interactions with the Schwanncell-like supporting cells play a major role in afferent fiber segregation, as is so obvious in mouse mutants with deficits in the development of supporting cells (Puligilla et al. 2007). Until molecules have been identified beyond those of the single candidate gene (Defourny et al. 2013), more data on the effects of upstream regulators of such genes will be found to affect proper afferent growth such as Gata3, Pax2, Foxg1, and others.

Fig. 3
figure 3

Normal pattern of organ of Corti (OC) innervation around birth (a, d) and aberrations induced with three mutations (b, c, e). The normal pattern appears as a highly regular set of tunnel crossing fibers (TC) that project to three bundles forming between adjacent Deiter’s cells (a, d). Note that the fibers, as they grow to outer hair cells (OHC), always turn toward the base (a, d), and that the majority of radial fibers (RF) end at inner hair cells (IHC). Targeted deletion of Prox1 in spiral ganglion neurons (SGNs) by using Nestin-Cre (Nes-Cre)  results in aggregation of all tunnel crossing fibers between the outer pillar cells (OP) and the third row of Deiter’s cells (D3). Note that most inner pillar cells (IP), outer pillar cells (OP), and Deiter’s cells (D) remain positive for Prox1, indicating a selective effect of Prox1 expression in SGNs to drive fiber sorting. Altering the properties of supporting cells in Fgfr3 null mice results in the disorganization of afferents to hair cells (c). Most disruptive for the pattern of innervation is the lack of Schwann cells (e). Radial fibers extend from the more centrally migrated SGNs either to bypass entirely the hair cells (HC) of the OC to end up at the lateral wall (LW) or to form a disorganized innervation of the OC (NF+Tub neurofilament plus tubulin, e). Data are from Mao et al. (2014) and Yang et al. (2011). Bars 20 μm (a–c), 100 μm (d, e)

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SGNs carry local hair cell activity to the cochlear nuclei to translate the frequency of sound dispersed along the OC into the tonotopic map used by the central auditory system to derive cues relevant for directional hearing and other aspects of sound processing. Numerous ideas regarding the way that such a map can develop based on local electric signals have been proposed (Bulankina and Moser 2012; Tritsch et al. 2007). However, the central projections of SGNs to the cochlear nuclei develop as early as E12.5 in mice (Fig. 4; Fritzsch 2003; Karis et al. 2001) and have basal and apical segregation even at E14.5 (Jahan et al. 2010a). Based on these data (Fig. 4), a topological representation of the OC onto the cochlear nuclei develops in early embryos many days prior to the onset of such electrical phenomena (Fritzsch 2003; Tritsch et al. 2007). Even the disruption of hair cell differentiation in certain mutants has little effect on the overall initial topology of the peripheral target onto the cochlear nuclei (Xiang et al. 2003), suggesting that developmental mechanisms that do not require electrical activity might generate at least a coarse tonotopic map that might be refined by activity later in life (Leake et al. 2002). Mutations in transcription factors such as Neurod1 (Jahan et al. 2010a) or Gata3 (Appler et al. 2013; Duncan and Fritzsch 2013) cause the disruption of central and peripheral projections prior to the onset of electric activity and with or without defects in the OC. Notably, Type II afferents have a highly different peripheral distribution extending to many outer hair cells instead of to a single inner hair cell (Fritzsch 2003). Despite this different peripheral organization, the central projection seems to reflect a similar topological restriction that is parallel to, and nearly identical to, the adjacent Type I afferent fibers (Nayagam et al. 2011), indicating that the central projections are sorted independently from peripheral fiber distribution. Such topological segregation of afferents might reflect the birth dates of SGNs, as has been suggested for afferent segregations in other developing systems (Fritzsch et al. 2005a). Another factor seems to be the competition for connections that can be altered in neurotrophin mutants and that result in an expansion of central projections in areas devoid of afferents from other areas of the OC (Fritzsch et al. 1997a). Coordinated analyses of the afferent birth dates with the development of central and peripheral connections are needed to substantiate these suggestions.

Fig. 4
figure 4

Spiral ganglion axons reach the cochlear nuclei around E12.5 (a), showing that the initial projection are clearly separated from vestibular axons of the posterior vertical canal crista (PC). Vestibular afferents extend early past the boundaries of the cochlear nuclei into the brainstem and the cerebellum, whereas cochlear afferents reach the anteroventral (AVCN) and dorsal cochlear (DCN) nuclei (Cne cochlear nerve, Co cochlear application, eff olivocochlear efferent bundle, PC posterior canal crista application). A segregated projection of base and apex is established already in E14.5 embryos (b) but appears to become refined in early neonates (c). Modified after Fritzsch et al. (2005a) and Jahan et al. (2010a). Bars 100 μm

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In summary, SGNs are specified in early ear development as a subset of cells of the antero-ventral neurosensory part of the developing ear. Proliferating precursors delaminate mostly from three areas of the growing cochlear duct to assemble in the spiral ganglion that parallels and partially drives the spiraling of the cochlear duct. In embryos, SGNs develop a topological projection to the cochlear nuclei (Fig. 4) that predates the formation of, and is independent of, hair cell differentiation. SGNs interact with Schwann cells to stop their migration and to develop ordered projections to the OC. Within the OC, afferents apparently interact with supporting cells to sort out the Type II projection to the outer hair cells (Fig. 3). The detailed projections both to the cochlear nuclei (Fig. 4) and to the OC (Fig. 3) require a currently only partially understood set of factors for normal development, but neither the full complement of factors needed, nor the detailed interactions to generate the tonotopic projection to the cochlear nucleus and the sorting of Type I versus Type II afferents are clear at the moment.

From placode to OC: locating and differentiating the OC in the correct position in the cochlear duct to translate sound into hearing

Like the SGNs, the OC appears to be derived entirely from the cochlear duct and thus exemplifies an in situ transformation of pro-neurosensory epithelial cells into the most complex of all cellular mosaics in the mammalian body. In order to understand the necessary steps needed to achieve this transformation and to appreciate the outcome of genetic manipulations in various mouse mutants, the cellular organization of the OC needs to be fully comprehended (Slepecky 1996). In contrast to some theoretical attempts to explain the cellular mosaic of the OC as a simple checkerboard mosaic of alternating hair cells and supporting cells (Sprinzak et al. 2011), a pattern that applies to vestibular organs or to the basilar papilla of vertebrates, the OC is only partially organized like a checkerboard (Fig. 5). More to the point, only the outer compartment of  outer pillar cells, Deiter’s cells of rows 1 and 2, and outer hair cells form a checkerboard pattern, except for the most extreme aspects of the base and the apex of the OC (Fig. 5). In contrast, the inner compartment, which consists of inner pillar cells, inner hair cells, inner phalangeal cells, and border cells, shows a markedly different cellular distribution pattern. In the human cochlea, a continuous row of ∼6000 inner pillar cells abuts an equally continuous row of ∼4000 outer pillar cells (Fig. 5). Obviously, simple lateral inhibition via the Delta/Notch system can drive the checkerboard mosaic of the outer compartment (Sprinzak et al. 2011). However, the continuity of cells as a single row of adjacent, broadly contacting, inner pillar cells (Zetes et al. 2012) that is not numerically matched to any other cell types of the OC is not easily explained by simple lateral inhibition models. Likewise, adult inner hair cells form a single row of broadly contacting cells (Slepecky 1996), a feature that is again difficult to reconcile with the lateral inhibition model. Obviously, the broad cellular contacts of inner hair cells and inner pillar cells must develop despite the Delta/Notch activity that should counteract the formation of adjacent and broad contacts. Clearly, Delta/Notch-based models require significant modifications (Moody 2007) to serve as an explanation for the boundary between the inner and outer compartments and the continuous approximation of continuous rows of inner hair cells and inner pillar cells.

Fig. 5
figure 5

The OC is often depicted in most theoretical work as a simple checkerboard of alternating hair cells and supporting cells (a), so that each hair cell is surrounded by four supporting cells, and each supporting cell is surrounded by four hair cells. This checkerboard pattern exists, however, only in the “outer compartment”of the OC with alternating rows of outer hair cells (OHC) and Deiter’s cells/outer pillar cells (D1-3, OPC). This outer compartment is separated by a single row of adjacent inner pillar cells (IPC) from the inner compartment, consisting of two alternating cell types, the inner hair cell (IHC) and the inner phalangeal cells (IPhC). Note that inner phalangeal cells seem to be organized in pairs flanking either medially or laterally the IHCs according to more recent data from transmission electron microscopy. Note also the numerical match of the outer compartment elements in humans, whereas neither IHC nor IPC fit to the numbers of the outer compartments (b). In particular, the continuity of IPCs in a single row and the finding that two supporting cells (IPC, OPC) are adjacent to and touching each other is difficult to reconcile with simple interpretations of the Delta/Notch interaction of lateral inhibition. Modified after Slepecky (1996), Spoendlin and Schrott (1988), and Zetes et al. (2012)

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Comparative data show that the basilar papilla of tetrapods, which is the precursor of the mammalian OC, has the checkerboard organization of vertebrate vestibular organs, and both can regenerate hair cells upon loss (Golub et al. 2012). It remains unclear how  the exclusive organization of the single row of broadly contacting inner hair cells and equally broadly contacting inner pillar cells, uniquely characterized by p75 (Ngfr) expression (von Bartheld et al. 1991), evolved. Likewise, how this altered organization of the OC might relate to its inability to regenerate are unclear. Although acknowledging that this major aspect of OC cellular development and evolution remains unresolved at the moment, we will summarize here our molecular understanding beyond recent reviews summarizing certain aspects covered here only in part (Fritzsch et al. 2011; Groves et al. 2013; Raft and Groves 2014). Delta/Notch signaling (Imayoshi and Kageyama 2014; Zine et al. 2014) is undoubtedly an important player for certain aspects of the patterning of the OC. However, we will concentrate on the pro-neural genes, which are ultimately responsible for much of the cellular neurosensory development (Imayoshi and Kageyama 2014). As will be apparent in this overview, pro-neural genes are also responsible for the continued patterning of the OC and the proper differentiation of hair cells. Indeed, the initial upregulation of an important gene for hair cell differentiation, Atoh1, can happen in the complete absence of the canonical Delta/Notch signaling but requires lateral inhibition for sustained expression (Basch et al. 2011). Unfortunately, the factors defining the position of the OC and the distribution of the inner and outer hair cells remain unclear. In contrast to the loss of function effects, the ubiquitous expression of Atoh1 leads to nearly ubiquitous differentiation of hair cells that nevertheless form some semblance of an OC in the correct position (Kelly et al. 2012).

As outlined in multiple reviews, the proper positioning of the OC and its cellular elements is crucial for the function of the OC. To emphasize this point about the importance of cell type and positioning, even slight changes in the numbers and distribution of outer pillar cells through the manipulation of Fgfr3 signaling (Puligilla et al. 2007), the reduction or loss of the inner hair cells in Srrm4 mutants (Nakano et al. 2012), or the loss of the outermost row of outer hair cells in Fgf20 mutants (Huh et al. 2012) can render the OC dysfunctional. Attempts to regenerate the OC require an understanding of the signals that position the OC properly and differentiate the correct hair cell and supporting cell type in the correct position in the correct numbers and correct proximity, aspects of OC development we are just beginning to understand (Jahan et al. 2013; Kopecky et al. 2013). Given the unique distribution and number of inner pillar cells, we can fairly say that an understanding of OC development requires minimally a causal explanation as to the way that the position and distribution of the single row of inner pillar cells and inner hair cells (Fig. 5) is achieved, in addition to the checkerboard patterning of the outer hair cell compartment that appears to fit so well with the lateral inhibition model (Sprinzak et al. 2011).

In addition to the position of cells and their distribution pattern in a radial organization, there exists also a  systematic longitudinal variation of cells within the OC (Slepecky 1996). Whereas these variations are functionally meaningful, as they may help to tune the OC to different frequencies (Slepecky 1996), the way in which this comes about molecularly is only understood to a limited extent and is mostly correlative with a causality still to be explored. One such correlation is cell cycle exit with the associated onset of differentiation (Jahan et al. 2013).

The OC shows a unique pattern of cell cycle exit followed by a diverse pattern of gene expression that regulates the differentiation process. Whereas the proliferation of hair cells progresses from the apex toward the base (Ruben 1967), the expression of Atoh1 and the differentiation of hair cells starts near the base and progresses bi-directionally toward the base and the apex (Chen et al. 2002; Matei et al. 2005). Importantly, changes in the expression of Atoh1 result invariably in the disorganization of hair cells and hair cell types in areas with such alterations of expression. For example, in Lmx1a mutants, the expression of Atoh1 is delayed in the basal turn, and the entire basal turn of the cochlea develops into a hybrid set of disorganized vestibular- and cochlear-like hair cells that do not show the patterning of the OC into recognizable inner and outer hair cells (Nichols et al. 2008). In contrast, the apex shows a more normal timing of Atoh1 expression and develops into a near normal OC. Similar but less profound disorganization of the OC occurs when Atoh1 is prematurely expressed in mutants null for Neurog1 (Matei et al. 2005) or Neurod1 (Jahan et al. 2010b). Manipulation of the cell cycle by mutating the bHLH transcription factor N-Myc results in premature cell exit but delayed expression of Atoh1 and the disorganization of apical hair cells, so that no OC organization is apparent in the apex  (Kopecky et al. 2013). Combined, these data indicate that the timing of cell cycle exit relative to the expression of genes that affect the timing and level of expression of Atoh1 is crucial for normal OC development (Jahan et al. 2013). This basic idea has recently been used to explain the limited changes in the OC observed after combined Hey1 and Hey 2 knockout (Benito-Gonzalez and Doetzlhofer 2014).

If this aspect of regulated and differential delay between hair cell cycle exit and expression of Atoh1 in the differentiation of hair cells is so critical for the normal development of the OC, one would expect that several transcription factors need to cooperate to stabilize this pattern against all changes to prevent misguided differentiation leading to a non-functional OC. Despite significant efforts to detect the regulatory network for cell cycle exit, we have at  hand only factors that maintain the non-proliferative state of hair cells once they exit the cell cycle (Chen et al. 2002), factors that are needed for overall OC development such as Sox2 (Kiernan et al. 2005), various Delta/Notch ligands (Doetzlhofer et al. 2009; Kiernan et al. 2006), Pax2 (Bouchard et al. 2010), micro RNA (Kersigo et al. 2011; Soukup et al. 2009), and Gata3 (Duncan and Fritzsch 2013; Karis et al. 2001). Levels of Gata3 and Sox2 define hair cell differentiation in their interaction with Atoh1 (Dabdoub et al. 2008; Duncan and Fritzsch 2013), and micro RNA is crucial for normal hair cell differentiation (Groves et al. 2013; Kersigo et al. 2011). In contrast to this data, no information is available on the molecular definition of cell cycle exit beyond the effects of other bHLH genes such as Neurog1 (Matei et al. 2005) and N-Myc (Kopecky et al. 2013). Clearly, neither gene defines hair cells, but each plays an as yet to be specified role at some point in the precursors of hair cells. In mutants for either gene, ectopic hair cells form in the greater epithelial ridge of the middle turn and the ductus reuniens, both areas with extensive delamination of SGNs (Fig. 2).

The hypothesis of the neurosensory evolution and the development of the vertebrate hair cells and neurons as being a split of an ancestral neurosensory cell through gene duplication and diversification (Fritzsch et al. 2006a; Fritzsch and Straka 2014; Pan et al. 2012b) has been confirmed for the vestibular part of the ear through lineage tracing (Raft and Groves 2014; Raft et al. 2007). However, more clear-cut data are needed for the more derived development of the OC, despite obvious indirect evidence to its validity (Matei et al. 2005). This inability to verify the clonal relationship of SGNs and OC cells adds to the overall unusual development of the OC and might tie into its unusual pattern of cells and its inability to regenerate, whereas mammalian vestibular organs have some capacity to regenerate (Golub et al. 2012). In this context, we need to stress that simple manipulations, such as the removal of Neurod1, can change surviving sensory neurons into hair cells, indicating the lasting plasticity of neurons to differentiate into hair cells, a potential that is normally suppressed by Neurod1 (Jahan et al. 2010b). These data support the notion that cellular decision making is not a simple process of expressing a single gene that drives differentiation of a specific cell type, but rather is specified by an intricate cascade of interacting transcription factors with a coordinated change in expression over time to guide the development of various cell types (Fritzsch et al. 2006a; Imayoshi and Kageyama 2014; Reiprich and Wegner 2014).

The simple finding that the expression of the bHLH gene Atoh1 can induce differentiation in almost any cell of the developing ear (Kelly et al. 2012), including developing neurons, if not suppressed by other factors (Jahan et al. 2010b), has led to the uncomplicated perspective that the proper expression of Atoh1 is all that is needed to generate and, by logical extension, to regenerate hair cells of the OC (Woods et al. 2004). Whereas this certainly applies to sensory epithelia with a simple checkerboard mosaic of hair cells and supporting cells, such a simple mosaic is limited to the outer compartment in the OC (Fig. 5). Consistent with the unusual organization of the inner compartment is an unusual distribution of several factors that play a role in neurosensory development: Bmp4 and various Fgfs. Whereas Bmp4 and Fgf10 are, for example, known to overlap in canal cristae (Chang et al. 2004; Pauley et al. 2003), they are differentially distributed in the OC (Fig. 6). Mutations of Bmp4 (Ohyama et al. 2010) and Fgf20 (Huh et al. 2012) result in the differential loss of hair cells and supporting cells, indicating that the patterned expression of these two diffusible factor classes has different positional information for the various cell types of the OC, presumably in interaction with local cell-cell interactions (Doetzlhofer et al. 2009). Consistent with this idea is the finding that alteration in the patterning of the OC always results in a changed expression pattern of Fgf8 (Jahan et al. 2010b; Nichols et al. 2008), which in turn regulates the differentiation of supporting cells into a different phenotype (Jahan et al. 2013). An essential feature for the normal patterning of the OC seems to be the positional distribution of these diffusible factors with regard to the medial and lateral edge of the OC (Groves and Fekete 2012). Intriguingly, the expression of some Fgfs is, in turn, maintained by the normally differentiating OC (Pan et al. 2011, 2012a). The developing OC is possibly part of a feedback loop whereby diffusible factors outside the OC define, in their interactive gradients (Srinivasan et al. 2014), both the position and type of cells. Apparently, the differentiating OC releases an as yet to be identified signal to maintain such expression for the continued reinforcement of differentiation, fine-tuned by the proper cellular interactions via the Delta/Notch lateral inhibition system.

Fig. 6
figure 6

Distribution of OC cells and origin and hypothetical gradients of several diffusible factors such as fibroblast growth factors (Fgf) and bone morphogenetic protein 4 (BMP4) in control (a, b) and Neurod1 null ears (c, d). Note that the conversion of outer hair cells into Fgf8-positive hair cells in Neurod1 null mice results in the transformation of the surrounding Deiter’s cells into pillar-cell-like cells (white arrows in d), indicating that a second center of Fgf8 diffusion can disrupt the cellular mosaic of the outer compartment of the OC (c, d). In essence, diffusible factors cooperate with lateral inhibition and selected expression of other genes to coordinate the normal development of the OC (Tub tubulin). Misexpression of only one diffusible factor (Fgf8 in some OHCs) can override all other interactions leading to a disrupted development of the OC (Jahan et al. 2010b). a, c, d Modified after Groves and Fekete (2012) and Jahan et al. (2013). b Unpublished data. Bar 100 μm (b, d)

Full size image

The way that these known and unknown factors combine to regulate the spatiotemporal expression progression of Atoh1, which, in turn, seems to define major aspects of OC development, is not yet established in its detailed causality. Indeed, some aspects of Atoh1 expression defy simple explanations of OC patterning, e.g., the expression of Atoh1 in a large subset of inner pillar cells, a unique single row of adjacent supporting cells (Figs. 2 and 5). The initial observation of this expression of Atoh1 in these cells (Matei et al. 2005) could have been the result of a problem with the incomplete regulation of the enhancer fragment used in the reporter line, despite the fact that it requires prior expression of Atoh1 protein to drive this enhancer (Chen et al. 2002). However, subsequent data obtained by using different molecular strategies have meanwhile confirmed the original finding (Driver et al. 2013; Yang et al. 2010), raising the intriguing possibility that Atoh1 expression, if counteracted by numerous co-expressed supporting-cell-differentiating bHLH genes (Benito-Gonzalez and Doetzlhofer 2014; Doetzlhofer et al. 2009), is mostly compatible with inner pillar cell differentiation. In other words, even in the ear, Atoh1 expression will only drive hair cell differentiation if the cellular context of the gene expression is correct. This, in turn, stresses the intricate interaction of the levels of expression of various bHLH genes as the driver for differential hair-cell-type development of the OC (Jahan et al. 2013). These data also stress the uniqueness of inner pillar cells, as outlined in the introduction to this section. Inner pillar cells might be the single row of Atoh1-lacZ-positive cells remaining in Atoh1 null mice (Fritzsch et al. 2005b), as suggested also in conditional deletions of Atoh1 prior to hair cell differentiation (Pan et al. 2011). Indeed, inner pillar cells might play a unique role in the extension of the OC (Yamamoto et al. 2009).

In summary, the mammalian OC is a molecularly highly derived end organ in which the ubiquitous Delta/Notch signaling has been modified through the action of unknown factors, including the re-patterning of diffusible factors such as Fgfs and Bmps. This re-patterning generates continuous rows of adjacent hair cells and supporting cells with broad contact to each other in the inner compartment and a near ideal checkerboard pattern of alternating outer hair cells and Deiter’s cells in the outer compartment (Fig. 5). Available evidence suggests that the overall patterning of the OC is tied into the unconventional cell cycle exit and Atoh1 upregulation mode of the OC relative to spiral ganglion cells (Matei et al. 2005), but details of causality are unclear. A notable cell, unique to the OC, is the inner pillar cell, which also has unique molecular signatures, such as p75 and Atoh1 expression, that are not shared with other supporting cells. Moreover, its continuous contact with surrounding supporting cells (basal and apical inner pillar cells in the longitudinal extent, inner phalangeal cells and outer pillar cells in the radial plane) makes this cell highly unusual. Notably, the inner pillar cell seems to be the only cell remaining long-term in the otherwise completely degenerated OC of Pou4f3 (Brn3c) null mutant mice (Pauley et al. 2008; Xiang et al. 2003) or mice with conditional deletion of Atoh1 (Pan et al. 2012a, 2011). Given this highly derived development of the inner compartment of the OC, we urgently need to understand the way that it becomes organized to allow the regeneration of a functional OC for which the position of the inner pillar cell over the bony lip of Rosenthal’s canal, with the single row of inner hair cell being immediately medial to it, appears to be essential for OC function. In essence, we need to be able to explain causally the way that a cell that expresses Atoh1 (Fig. 2) can be flanked by other supporting cells and does not differentiate as a hair cell but remains an inner pillar cell. Solving this problem requires the generation of mutant mice in which the fate of the inner pillar cells is altered. This might elucidate the molecular conditions that normally specify this crucial cell for OC function and development, possibly including the extension of the basilar papilla of tetrapods to become the OC of eutherian mammals.

From OC to “placode-like” epithelium and back: neurosensory loss in transcription factor mutants, the aging population, and attempts to regenerate an OC

Hair cells are progressively lost with age, typically starting from the base and progressing toward the apex. Various chemicals and loud sound can accelerate this process, and several models using chemicals and sound to ablate hair cells have been designed to study what happens in the OC upon sudden or progressive loss of hair cells. We concentrate here on hair cell loss following the mutation of genes that are demonstrably necessary for the continued viability of hair cells. Chief amongst these genes are four transcription factors, Atoh1, Pou4f3, Gfi1, and Barhl1. Mice with mutations in these genes lose all their hair cells, but in a different pattern of temporal and spatial progression. Similar to the differences between these genes and their potential regulation, directly or indirectly, by Atoh1 (Ikeda et al. 2014), manipulations of Atoh1 by different approaches lead to somewhat different results. Loss of Atoh1 through replacement with a LacZ reporter or conditional deletion prior to differentiation leads to the absence of all hair cell differentiation, but many LacZ-positive cells (Bermingham et al. 1999) are retained along the entire length of the OC (Fritzsch et al. 2005b), instead of  the complete loss of all cells, as previously suggested (Chen et al. 2002). Whereas many cells die via apoptosis in Atoh1 null mutants, the overall cochlear length extension, attributed by some to the convergent extension and intercalation of OC cells (Kelly and Chen 2009), is normal. Likewise, the expression of factors expressed in and adjacent to the OC, such as neurotrophins (Fritzsch et al. 2005b), Fgf10, and Bmp4 (Pan et al. 2011), is initially normal in Atoh1 null mutants. However, the loss of hair cells causes the loss of Fgf10 expression in the greater epithelial ridge and the absence of Fgf8 expression.  It also results in an  expansion of Bmp4-positive Claudius cells to replace the OC by a flat epithelium composed of cells that normally never differentiate as part of the OC (Pan et al. 2012a). Most intriguing is the persistence of a single row of cells along the entire length of the OC; this possibly represents the inner pillar cells (Fritzsch et al. 2005b; Pan et al. 2011), which are now well characterized as being mostly Atoh1-positive (Driver et al. 2013; Matei et al. 2005) and important for extension of the OC (Yamamoto et al. 2009). Interestingly, in Foxg1 null mutant, the OC is truncated and composed of multiple rows of hair cells, whereas the pillar cells are barely recognizable (Pauley et al. 2006). The lack of pillar cell differentiation to drive the extension of the OC, and not the inability of the multiple rows of hair cells to conduct convergent extension movements, is perhaps the reason for this short and wide OC.

Intriguingly, a few pillar cells surrounded by Myo7a-positive cells differentiate, even in the complete absence of Atoh1 in areas that show, in earlier stages, the persistent expression of Sox2 (Pan et al. 2011). These data show that Atoh1 is a critical differentiation factor, but that some differentiation toward an OC can occur transiently in its absence. This point is particularly obvious in mutants in which Atoh1 is replaced by Neurog1, a related bHLH factor expressed in neurosensory precursors but not in differentiated hair cells (Jahan et al. 2012). These mice show a pattern of Neurog1 expression consistent with the pattern of LacZ markers replacing Atoh1 (Fritzsch et al. 2005b) but with a major difference: a patchy development of cells show a large tuft of microvilli similar to  undifferentiated hair cells generated in micro-RNA-deficient mice (Soukup et al. 2009). Moreover, the loss of Neurog1-expressing cells is patchy instead of showing a continuous row as in Atoh1 null mice (Fritzsch et al. 2005b), indicating that some residual signaling of Neurog1 partially differentiates cells of an OC through lateral inhibition via the Delta/Notch signaling pathway (Jahan et al. 2012).

This partial differentiation of an OC is even more obvious in mice in which delayed deletion of Atoh1 occurs through a Cre driven by its own enhancer (Matei et al. 2005), generating “self-terminating” Atoh1 expression (Pan et al. 2012a). Interestingly enough, in these mice, the inner hair cells and the third row of outer hair cells are almost completely lost, suggesting the differential need of specific levels of Atoh1 expression for inner hair cells. Altered Atoh1  levels might , in turn, induce the proper differentiation of surrounding inner phalangeal cells to release the Fgf20 needed for the development of the third row of outer hair cells (Huh et al. 2012). Whereas all these data on the conditional and full-deletion mutants of Atoh1 are inherently consistent, some more recent data are not. Conditional deletion of Atoh1 by using inducible Cre results in the rapid and complete loss of hair cells, both partially differentiated hair cells and undifferentiated hair cells (Cai et al. 2013; Chonko et al. 2013), suggesting a critical phase of continued dependency on Atoh1 once differentiation has been initiated. Atoh1-positive differentiating hair cells seem to be necessary for the continuous differentiation of supporting cells, which rapidly die in these mice. Why this induced Cre also affects the apical hair cells that have not yet expressed significant amounts of Atoh1 (Chen et al. 2002; Matei et al. 2005) remains unclear. The induced expression of Cre might have adverse effects on undifferentiated hair cells as previously reported for neurons (Forni et al. 2006); additional work is needed to rule this out.

In summary, these data suggest a difference in the overall response of OC development when Atoh1 is completely absent in mice null for Atoh1 (Bermingham et al. 1999; Fritzsch et al. 2005b) or with a deletion of Atoh1 prior to its expression (Pan et al. 2011), as compared with the delayed loss of Atoh1 by using various non-inducible (Pan et al. 2012a) and inducible Cre lines (Cai et al. 2013; Chonko et al. 2013). Much of the interpretation presented here of various Atoh1 mutants is in agreement with data collected in mice with a delayed deletion of genes necessary for hair cell maintenance. This is discussed below.

Pou4f3 (aka Brn3c, Brn3-1) is a Pou domain transcription factor that is essential for hair cell maintenance and, indirectly, for sensory neuron maintenance (Xiang et al. 2003). Without this transcription factor, hair cells form and start to differentiate but eventually all degenerate in late embryos and early neonates (Hertzano et al. 2004; Xiang et al. 2003). Despite the complete and rapid loss of all hair cells, which results in a nearly completely flat epithelium, some pillar cells, possibly inner pillar cells judging from the distance to the habenula perforata, survive for as long as 6 months. Most interesting is the expression change of Atoh1 in these mutants. First, Atoh1 remains expressed in cells that are possibly undifferentiated hair cells, for several weeks (Pauley et al. 2008). In addition, cells surrounding the tubulin-positive pillar cells express Atoh1-LacZ even in 6-month-old animals. What remains unclear is whether the loss of hair cells results in an altered expression of Atoh1-LacZ in these cells, as suggested for tamoxifen-induced conditional Atoh1 deletion (Cai et al. 2013), or whether some hair cells survive as undifferentiated cells in the absence of Pou4f3. Pou4f3 is regulated by multiple transcription factors, including Atoh1 (Ikeda et al. 2014). The ability of Pou4f3 to induce hair cell differentiation is directly proportional to the binding of multiple transcription factors to various enhancer elements (Ikeda et al. 2014), providing a glimpse of the way that the altered expression levels of Atoh1 (Jahan et al. 2013) might affect the differentiation of hair cells. Clearly, the expression of Atoh1 without Pou4f3 cannot initiate the late differentiation of hair cells in 6-month-old animals, raising issues concerning the use of Atoh1 expression to differentiate hair cells in humans carrying mutant Pou4f3 genes.

Overall similar effects of a delayed loss of hair cells have been reported in mice null for the zinc finger transcription factor Gfi1 (Hertzano et al. 2004; Wallis et al. 2003). As in the loss of Pou4f3, which seems to regulate the expression of Gfi1, a progressive base to apex loss of hair cells occurs in late embryos and neonates. In contrast to all these factors, the homeodomain-containing transcription factor Barhl1 shows a different progression, both in time and in place. Hair cell degeneration starts after the onset of hearing and progresses from the apex toward the base in Barhl1 mutant mice (Li et al. 2002). Moreover, like Atoh1 (Jahan et al. 2012; Pan et al. 2012a), Barhl1 seems to positively regulate its own expression (Chellappa et al. 2008).

These data indicate that hair cell loss can be caused by several factors, progressing either from base to apex or vice versa. More detailed analysis of the way that the loss of hair cells affects the overall retention of supporting cells is needed if we are to better understand the clinical significance of the loss of hair cells in human patients with mutations in these factors. However, remarkably, the simple delayed loss of Atoh1 or loss of several other transcription factors can cause the rapid demise of hair cells leading to a nearly flat epithelium, as is also observed in many human cases of long-term hair cell loss. Equally remarkable is that in most of these mice with genetically induced hair cell loss, substantial and long-lasting innervation of parts of the OC (Fritzsch et al. 2005b; Pan et al. 2011; Xiang et al. 2003) occurs that resembles more that of human hearing loss conditions compared with hair cell loss induced by other means (Alam et al. 2007).

Why do these data matter for current attempts to return hearing to the deaf who have lost their hair cells? The ways in which the limited expression or hypomorphic function of the above and several other genes can be combined in a single person are manifold, and the resultant progressive hair cell loss differs in a spatio-temporal progression. In part, such loss of hair cells will depend on the unique genetic predisposition of an individual, as recently demonstrated (Ishimura et al. 2014), but will also in part depend on the unique combined exposure to loud sound and ototoxic chemicals throughout life. We are not yet in a position to go beyond personalized medicine and to move toward an individualized age-related hearing loss assessment by combining both genetic predisposition and life accumulations of insults into a predictability matrix in order to estimate overall risk. Such information is needed to provide the correct therapy for a given person at a given age. Depending on the residual presence of hair cells or remaining supporting cells, including in particular inner pillar cells, various strategies could be designed to restore iteratively the lost hair cells and to reconstitute a semblance of an OC that is functionally meaningful (Zine et al. 2014). Attempts aimed at understanding the molecular composition of the various types of hair cells through hair-cell-specific gene expression analysis (Liu et al. 2014) will help to elucidate the way that the diverse hair cell types develop (Jahan et al. 2013).