Hair cell development: Commitment through differentiation

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Review

Hair cell development: Commitment through differentiation Matthew W. Kelley Section on Developmental Neuroscience, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 35 Convent Drive, Bethesda, MA 20892, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

The perceptions of sound, balance and acceleration are mediated through the vibration of

Accepted 17 February 2006

stereociliary bundles located on the lumenal surfaces of mechanosensory hair cells located

Available online 13 April 2006

within the inner ear. In mammals, virtually all hair cells are generated during a relatively brief period in embryogenesis with any subsequent hair cell loss leading to a progressive

Keywords:

and permanent loss of sensitivity. In light of the importance of these cells, considerable

Cochlea

effort has been focused on understanding the molecular genetic pathways that regulate

Hearing

their development. The results of these studies have begun to elucidate the signaling

Ear

molecules that regulate several key events in hair cell development. In particular, significant

Stereocilia

progress has been made in the understanding of hair cell commitment, survival and

Thyroid hormone

differentiation. In addition, several aspects of the development of the stereociliary bundle,

bHLH transcription factors

including its elongation and orientation, have recently been examined. This review will summarize results from each of these developmental events and describe the molecular signaling pathways involved. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Mechanosensory hair cells located within the inner ear act as the primary transducers of auditory and vestibular stimuli. In response to selective pressures to perceive small movements or pressure waves, hair cells have developed a unique group of specializations that is most obviously illustrated by the presence of a stereociliary bundle, a group of modified microvilli, located at the lumenal surface. Even the most subtle of movements of the individual stereocilia results in the opening of mechanosensory transduction channels and a subsequent influx of positively charged ions (reviewed in Eatock and Hurley, 2003). The resulting depolarization of the cell results in changes in the rate of tonic release of neurotransmitter from the base of the cell and subsequent changes in the rate of neural activity in the neurons that synapse with those hair cells. Considering the important role of these cells, it is not surprising that

their loss results in significant deficits in hearing and balance. Despite the importance of mechanosensory hair cells, our understanding of the factors that regulate the specification and differentiation of these cells is still fairly limited. However, recent progress has resulted in striking advances in several different aspects of hair cell formation, in particular the commitment of cells to develop as hair cells and subsequent development of the stereociliary bundle. In this review, I will discuss the timing of hair cell commitment and differentiation, using the mouse as a model system, and then present a brief overview of some of the more recent work on several different aspects of hair cell development. Unfortunately, time and space limitations will prevent an exhaustive discussion of all aspects of hair cell development. Readers are directed to recent reviews by Whitlon (2004), Fritzsch and Beisel (2004) and Barald and Kelley (2004), for further information.

E-mail address: [email protected]. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.062

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2. Hair cell development—an extended process The mouse otic placode involutes to form the otocyst around embryonic day 8.5 (E8.5). The first sign of cochlear development, the ventral outgrowth of the cochlear duct, occurs around E11. Hair cell development begins in the vestibular system around E12 and in the cochlea around E13 (reviewed in Barald and Kelley, 2004). Among the earliest definitive markers of developing hair cells are two unconventional Myosins, Myosin VI (Myo6) and Myosin VIIa (Myo7a) (Hasson et al., 1997; Sahly et al., 1997; Montcouquiol and Kelley, 2003). In fact, in the cochlea, the first Myo6-positive cells are present at E13, suggesting that Myo6 is turned on soon after, or concomitant with, hair cell commitment (Montcouquiol and Kelley, 2003). While the first indications of hair cell development are present during the early embryonic period, hair cells do not become functional for another 4 to 5 days, based on studies in the vestibular system (Geleoc and Holt, 2003), and it is postnatal day 14 (P14) before the onset of hearing. Therefore, in some cases, hair cell maturation may take nearly 3 weeks. During this time period, several distinct events must occur. First, the progenitor cells that will develop as hair cells must become committed to a hair cell fate. Following commitment, developing hair cells become dependent on a specific group of transcription factors for their continued survival. At the same time, each developing hair cell must assemble the unique structural and molecular components of the stereociliary bundle and then place that bundle in the appropriate location on the lumenal surface of each cell. Concomitant with, or soon after, the development of the bundle, each developing hair cell must express several unique channel molecules that are required for the cell to maintain homeostasis and to respond to a nearly constant influx of positively charged ions. Further, each hair cell must attract and synapse with ingrowing neurites, both afferent inputs from the developing vestibulo-acoustic nerve and efferent fibers from the hindbrain (reviewed in Simmons, 2002). Finally, subsets of hair cells, such as cochlear outer hair cells, go through an extended period of differentiation that continues through the postnatal period, and includes the development of unique cellular aspects, such as electromotility. Recently, the development of functional aspects of hair cells was examined in two series of studies, one examining vestibular hair cells Geleoc and Holt (2003) and Geleoc et al. (2004) and the second examining development of cochlear hair cells (Marcotti et al., 2003a,b). For the vestibular studies, mouse utricular hair cells were examined between E15 and the early postnatal period. At E15, stereociliary bundles were short with no apparent tip-links, anatomical structures that are believed to be associated with the transduction channels. Moreover, physical deflection of the bundles on 11 E15 and four E16 hair cells failed to elicit any transduction currents, nor did these cells take up FM1-43, a dye that enters hair cells through the transduction channels (Gale et al., 2001; Meyers et al., 2003). In contrast, by E17, stereociliary bundles on developing utricular hair cells had elongated to develop a morphology

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that appeared much more consistent with their mature form. In addition, tip-links were identified in the spaces between individual stereocilia, and hair cells were able to take up FM1-43, suggesting the presence of active transduction apparatus. Further, by E17 or E18, most utricular hair cells had developed a resting membrane potential that was below −50 mV, as well as key electrophysiological properties that are required for hair cell transduction. These results demonstrate a rapid onset of hair cell function between E15 and E17 in utricular hair cells; however, it is important to consider that despite this rapid onset, fully mature hair cell characteristics are not attained until the postnatal period. While a similar study examining both electrophysiological and anatomical aspects of development has not been conducted for hair cells located in other sensory epithelia, the development of inner and outer hair cell membrane properties has been examined (Marcotti et al., 2003a,b). As was observed for vestibular hair cells, cochlear hair cells develop many functional properties long before the onset of hearing. The basis for the early and rapid onset has not been determined, but Marcotti et al. (2003a,b) and Geleoc et al. (2004) suggest that the onset of increased receptor potentials could play a role in the recruitment and stabilization of synaptic contacts with in growing neurites.

3.

Hair cell commitment

The molecular control of hair cell commitment has been studied most extensively in the mouse cochlea, although valuable insights have also been gained from studies on the avian auditory system during both initial development and regeneration. However, for reasons of clarity, the discussion presented here will be restricted to the mouse cochlea. More thorough reviews of hair cell commitment and regeneration, including avian systems, can be found in Stone and Rubel (2000) and Kelley (2002). Prior to E12, the floor of the cochlear duct is comprised of actively proliferating epithelial cells. The floor of the duct will ultimately give rise to three distinct regions, the organ of Corti and the inner and outer sulci. Although the morphology of cells throughout the floor of the duct appears homogenous at E12, cells that will give rise to the organ of Corti are already positive for the transcription factor Sox2 (Kiernan et al., 2005b) (Fig. 1). Based on presumed differences in developmental potential, the region of the duct that will give rise to the organ of Corti has been termed the prosensory domain. Although specific functional studies have not been conducted, it seems likely that there is a close overlap between the prosensory domain and expression of Sox2 (Kiernan et al., 2005b). Beginning on E12, cells located within the prosensory domain at the apex of the cochlear spiral begin to leave the cell cycle and become permanently postmitotic. As development proceeds through E13 and E14, a wave of terminal mitosis extends from apex to base within the prosensory domain (Ruben, 1967). The factors that specify the wave of terminal mitoses are not fully understood; however, a key regulator of this event is expression of the cell cycle inhibitor, p27kip1 (Chen and Segil,

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Fig. 1 – Hair cell commitment and differentiation. Schematic cross-sections through the base of the cochlear duct. Black circles indicate cell nuclei. Cross-sections in the left hand column indicate patterns of gene expression with approximate embryological times points in mice indicated. Cross-sections in the right hand column illustrate the effects of deletion of the indicated gene. Left hand column: at E12, Sox2 (red) is expressed in a population of cells that correlates with the prosensory domain. Beginning on E13, p27kip1 (green) and Atoh1 (blue) are expressed in a population of cells that appears to largely overlap with the expression of Sox2. As discussed in the text, there is still some debate over the exact pattern of expression for Atoh1. Expression of p27kip1 ultimately becomes restricted to supporting cells while expression of Atoh1 ultimately becomes restricted to hair cells. Following Atoh1, developing hair cells begin to express two Notch ligands, Jag2 and Dll1 (dark green). This leads to activation of Notch1 (Notchicd, purple) in adjacent supporting cells. Concomitant with the onset of expression for Jag2 and Dll1, hair cells also begin to express three transcription factors, Pou4f3, Gfi1 and Barhl1 (orange), that are required for survival and, possibly, differentiation. Right hand column: deletion of Sox2 leads to a disruption in the development of the organ of Corti, expression of p27kip1 and Atoh1, and probably all subsequently expressed hair cell and supporting cell genes. Deletion of p27kip1 leads to additional cellular proliferation and an overproduction of both hair cells and supporting cells. Deletion of Atoh1 leads to a loss of hair cells and a secondary loss of supporting cells as a result of inductive interactions between hair cells and supporting cells. Disruption of Notch pathway signaling leads to an overproduction of hair cells at the expense of supporting cells. Deletion of any member of the Pou4f3/Gfi1/Barhl1 group leads to eventual hair cell death. 1999). Between E12 and E14, expression of p27kip1 extends downwards along the apical-to-basal axis of the cochlea within the prosensory domain. As a result, by E14, the entire

population of Sox2-positive cells is also positive for p27kip1 (Kiernan et al., 2005b). As would be expected, expression of p27kip1 induces an exit from the cell cycle for all cells within

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the Sox2 population (Chen and Segil, 1999). Interestingly, p27kip1 is not expressed in cells located outside of the Sox2 population, and these cells continue to proliferate through the late embryonic to early postnatal period (Ruben, 1967; Kiernan et al., 2005b). As a result of this difference, Chen and Segil (1999) refer to the p27kip1-positive region of the duct as the zone of non-proliferation or ZNP. Based on overlap between expression of p27kip1 and Sox2, it seems likely that the prosensory domain and ZNP probably overlap extensively. Deletion of p27kip1 results in an extended period of proliferation within the ZNP, leading to an overproduction of hair cells and other cell types in the organ of Corti and to profound deficits in hearing (Chen and Segil, 1999) (Fig. 1). In contrast, disruption of Sox2 expression in the inner ear leads to a loss of p27kip1 expression and a complete lack of hair cells (Kiernan et al., 2005b) and probably supporting cells (Fig. 1). Following terminal mitosis, or possibly overlapping with it slightly, cells within the ZNP begin to express the bHLH transcription factor Atoh1 (formerly called Math1) (Bermingham et al., 1999; Lanford et al., 2000; Chen et al., 2002; Woods et al., 2004) (Fig. 1). The full extent of the expression of Atoh1 is a matter of some debate at this point. Conflicting results have suggested that Atoh1 is initially expressed at low levels throughout the ZNP (Lanford et al., 2000; Woods et al., 2004) or alternatively, that Atoh1 is only expressed in the subset of ZNP cells that have already become committed to develop as hair cells (Chen et al., 2002). Finally, it has also been suggested that Atoh1 may be expressed in the cochlea as early as E11, which would suggest that Atoh1 expression could overlap with cellular mitosis (Matei et al., 2005). At this point, each of the Atoh1 results discussed here was obtained using different methods and in different laboratories, so it is difficult to resolve the discrepancies. Hopefully, with further work, a clearer picture of the spatiotemporal pattern of Atoh1 will be obtained. Regardless of its early onset of expression, the role of Atoh1 is fairly well understood. Deletion of Atoh1 results in a complete loss of hair cells and, secondarily, a loss of supporting cells (Bermingham et al., 1999; Woods et al., 2004). However, it is important to note that the effect on supporting cells is indirect and occurs as a result of the loss of hair cells (Woods et al., 2004). Similarly, overexpression of Atoh1, either in the developing organ of Corti, or in the adjacent greater epithelial ridge, leads to a nearly 100% induction of hair cells (Zheng and Gao, 2000; Woods et al., 2004; Jones et al., 2006). Finally, recent results have suggested that overexpression of Atoh1 is sufficient to induce ectopic and, possibly, regenerated hair cells in adult Guinea pigs in vivo (Izumikawa et al., 2005; Kawamoto et al., 2003). These results clearly demonstrate a key role for Atoh1 in the generation of hair cells and in fact, Atoh1 is the earliest gene that has been shown to be sufficient for hair cell formation. It should be noted that expression of Atoh1 is dependent on Sox2, suggesting that Sox2 could act upstream of Atoh1 (Kiernan et al., 2005b), but similar overexpression studies using Sox2 have not been reported. An intriguing issue that should be addressed at this point is the timing of terminal mitosis and the expression of p27kip1, Atoh1 and, the early hair cell marker, Myo6. Terminal mitosis and expression of p27kip1 both occur in a gradient that

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progresses from apex to base between E12 and E14, while expression of Atoh1 begins in the base between E13 and E14 and extends towards the apex. Myo6 protein is first expressed at E13 in the mid-basal turn of the cochlea in a small subset of cells within the ZNP (Montcouquiol and Kelley, 2003). Based on the position and morphology of these cells, they are believed to represent the first committed inner hair cells (Montcouquiol and Kelley, 2003). However, the presence of committed hair cells in the mid-basal region of the cochlea at E13 suggests a very rapid progression between terminal mitosis, acquisition of competence and commitment in these cells. Alternatively, it is possible that the Myo6 positive cells present in the cochlea at E13 represent a population of cells that do not adhere to the same developmental sequence. These could be cells that are not postmitotic, despite expressing Myo6, and in fact, could even be cells that are not committed to develop as hair cells. Unfortunately, at this point, a careful spatial analysis using all the various markers of this important transitional period has not been conducted. As hair cell commitment continues, individual developing hair cells begin to express two members of the Notch signaling pathway, Jagged2 (Jag2) and Delta1 (Dll1) (Lanford et al., 1999; Morrison et al., 1999) (Fig. 1). Both molecules act as ligands that bind to and activate Notch (reviewed in Artavanis-Tsakonas et al., 1999). Similarly, two downstream targets of Notch signaling, HES1 and HES5, are expressed in neighboring cells that ultimately develop as supporting cells (Zheng et al., 2000b; Zine et al., 2001; Lanford et al., 2000). Activation on the Notch signaling pathway regulates the number of cells that develop as hair cells with deletion of Jag2, Dll1, Notch1, HES1, HES5 or combinations thereof, leading to varying levels of overproduction of hair cells (Lanford et al., 1999; Zheng and Gao, 2000; Zine et al., 2001; Kiernan et al., 2005a) (Fig. 1). Moreover, expression of HES5 in supporting cells is disrupted in Jag2 mutants, demonstrating that expression of Jag2 in developing hair cells is required for the activation of the Notch signaling pathway in supporting cells (Lanford et al., 2000). Although a similar effect on HES expression has not been demonstrated for deletion of Dll1, the fact that Dll1 and Jag2 are expressed in similar patterns suggests a redundant mechanism (Morrison et al., 1999; Kiernan et al., 2005a). As discussed, there is some debate regarding the breadth of the initial pattern of Atoh1 expression. Since Atoh1 is a target of Notch regulation in other systems, some insights can be obtained from the analysis of Notch pathways mutants (Gazit et al., 2004; Schonhoff et al., 2004). In fact, despite the overproduction of hair cells, all of which were Atoh1 positive, in Jag2 mutants, no obvious increase in the initial expression of Atoh1 was observed in the cochleae of these animals (Lanford et al., 2000). This result suggests that Atoh1 is a target of Notch regulation in the cochlea as well and that the role of the Notch signaling pathway is to down-regulate Atoh1 in progenitor cells that will not develop as hair cells. In support of this hypothesis, overexpression of HES1 has been shown to be sufficient to inhibit Atoh1-induced hair cell formation in the GER (Zheng and Gao, 2000). Moreover, transient expression of Atoh1 in clusters of GER cells has been shown to induce the formation of small sensory patches that are comprised of both hair cells and supporting cells (Woods et al., 2004). However, if Notch signaling is inhibited in these clusters, then there is a

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significant increase in the number of cells that develop as hair cells, further supporting a role for Notch signaling in downregulation of Atoh1.

4.

Hair cell differentiation and survival

Following commitment and concomitant with the onset of expression of Jag2 and Dll1, developing hair cells begin to express a group of transcription factors that are required for hair cell survival and possibly differentiation (Fig. 1). The first of these factors is Pou4f3, a Pou-domain transcription factor (Xiang et al., 1998). Deletion of Pou4f3 leads to progressive hair cell death that is evident by E18 (Erkman et al., 1996; Xiang et al., 1997). However, expression of early markers of hair cell differentiation, such as Myo6 and Myo7a, is unaffected in Pou4f3 mutants, suggesting that the earliest steps in hair cell differentiation are not dependent on Pou4f3 (Xiang et al., 1998). Soon after the onset of Pou4f3 expression, developing hair cells up-regulate expression of a zinc-finger transcription factor, Gfi1 (Hertzano et al., 2004; Wallis et al., 2003). As was the case for Pou4f3, deletion of Gfi1 leads to a degeneration of hair cells by the early postnatal period (Wallis et al., 2003; Hertzano et al., 2004), but in contrast with Pou4f3 mutants, in which inner and outer hair cells degenerated at roughly the same rate, in Gfi1 mutants outer hair cells degenerate prior to inner hair cells. However, there is a complete degeneration of all hair cells in Gfi1 mutants by P14 (Wallis et al., 2003; Hertzano et al., 2004). As was the case for Pou4f3 mutants, expression of early markers for hair cell development, including Atoh1, Pou4f3, Myosin VI and Myosin VIIa, is unaffected in Gfi1 mutants (Wallis et al., 2003; Hertzano et al., 2004), suggesting that Gfi1 is required for hair cell survival, but not for hair cell commitment. A role for Gfi1 in hair cell differentiation cannot be ruled out. However, the demise of these cells prior to the completion of their differentiation makes this a difficult issue to address. The question of whether Pou4f3 and Gfi1 are members of the same signaling pathway was addressed using microarray profiling of changes in gene expression in cochlea from wild-type and Pouf43 mutant animals (Hertzano et al., 2004). Gfi1 was identified as down-regulated in Pou4f3 mutants and subsequent localization by in situ hybridization confirmed a complete loss of Gfi1 expression in the absence of Pou4f3. These results strongly suggest that Gfi1 is a direct target of Pouf43 in the cochlea. A third transcription factor that is required for hair cell survival is Barhl1, a homeodomain protein that is related to Drosophila barh1 and barh2. Barhl1 is first expressed in developing hair cells in both the cochlea and vestibular epithelia at E14.5 and is maintained through at least P5 (Li et al., 2002). In Barhl1 mutants, there is a progressive loss of hair cells with a fairly unique spatiotemporal pattern. Outer hair cells begin to degenerate in the apex of the cochlea by P6 while outer hair cells in the basal and middle turns appear normal at this time. Inner hair cells are normal in all three turns at P6. By P19, degeneration of outer hair cells has extended to the middle and apical turns, and by P59, there is nearly a complete loss of outer hair cells in the apical and middle turns. However, inner hair cells are present and normal in all three turns at this time point. Interestingly, inner hair cells ultimately degener-

ate in Barhl1 mutants as well, but this does not occur until approximately P300. The timing of degeneration in these three mutants suggests that Pou4f3 and Gfi1 may regulate hair cell differentiation and/ or survival while Barhl1 is most likely involved exclusively in hair cell survival, since most Barhl1 mutant mice actually have relatively good levels of hearing at both 1 and 3 months of age (Li et al., 2002). For Pou4f3 and Gfi1, it is not clear at this point whether these genes function in both survival and differentiation pathways, or if the death of these cells is an indirect affect of defects in differentiation. Some insight into the role of Pou4f3 can be gained through an examination of the phenotype in an Israeli family with late onset non-syndromic hearing loss as a result of a mutation in POU4F3, the human homolog of mouse Pou4f3 (Vahava et al., 1998). In these individuals, there is an 8 base pair deletion in the homeodomain that leads to a dominant mutation. The late onset of hearing loss in these individuals suggests that POU4F3 may play a greater role in hair cell survival; however, further studies, and in particular the generation of a mouse model for the human mutation, are clearly required.

5.

Development of the stereociliary bundle

One of the most striking and important components of each mechanosensory hair cell is the stereociliary bundle located at the lumenal surface. The bundle itself is comprised of between 50 and 200 individual stereocilia, each of which is a modified microvillus containing a dense core of filamentous actin (reviewed in Frolenkov et al., 2004). The arrangement of the stereocilia, and therefore the shape of the bundle, varies between different sensory epithelia and between species. The most common bundle shape is round, but more elaborate shapes, such as “)” and “W” also occur. However, regardless of the shape of the bundle, individual stereocilia within each bundle are always arranged in a staircase pattern such that the tallest stereocilia are located at one side of the bundle with progressively shorter stereocilia located in adjacent rows. As will be discussed in the section on planar cell polarity, the shape and orientation of these bundles play a key role in hair cell sensitivity. In addition to a variable number of stereocilia, every bundle also contains a single true cilium referred to as the kinocilium. The kinocilium is always located directly adjacent to the tallest row of stereocilia; however, functional studies have demonstrated that it does not play a role in mechanotransduction (Hudspeth and Jacobs, 1979). In fact, stereociliary bundles in the mammalian cochlea lose their kinocilia during the early postnatal period, demonstrating that these structures are not required for normal bundle function. However, as will be discussed in the next section, it is believed that the kinocilium may play a role in determining the polarization of the each bundle. The assembly and patterning of the stereociliary bundle are clearly key steps in the development of a hair cell. In addition, the unique shape of this structure has long fascinated structural biologist. Recently, it was discovered that an active actin treadmill maintains the height of each individual stereocilium (Schneider et al., 2002). Individual actin monomers are shuttled to the distal tip of each

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stereocilium and incorporated into the dense actin core (Fig. 2). Over time, these monomers move downward towards the lumenal surface of the cell as more monomers are incorporated at the tip. Interestingly, the rate of incorporation correlates with the height of the individual stereocilia, such that taller stereocilia incorporate and treadmill actin at a faster rate than shorter stereocilia (Rzadzinska et al., 2004). However, regardless of the height of the stereocilia, the constant treadmilling results in a complete turnover of actin monomers within each stereocilia every 48 to 96 h. Considering that stereocilia must be maintained throughout the life of the organism, these results suggest that there is a constant demand on each hair cell to efficiently traffic actin monomers to the distal tip of the stereocilia and to incorporate those monomers into the actin core. In fact, inhibition of actin polymerization with Cytochalasin D results in a rapid shortening of stereocilia, demonstrating the importance of actin trafficking and incorporation in the maintenance of stereocilia height (Rzadzinska et al., 2004). Further results, from a somewhat unexpected source, have led to significant progress in the identification and

Fig. 2 – Growth and maintenance of stereocilia. A cross-section through the apical tip of a single stereocilium. Actin monomers (> in green), along with Espin molecules (green or black ovals), are translocated to the distal tip of the stereocilia where they are incorporated into highly cross-linked actin filaments. Myosin XVa (red) and Whirlin (blue) are both localized to the distal tip of the stereocilia and play key roles in actin incorporation. Over time, an actin treadmill carries incorporated actin monomers and Espin molecules towards the proximal end of the stereocilia.

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understanding of the molecular players that regulate actin trafficking and stereocilia height. Geneticists working to identify the genes that lead to non-syndromic and syndromic forms of hereditary deafness have identified a number of genes that play key roles in stereocilia formation or maintenance (Table 1). A complete review of the roles of all of these genes is beyond the scope of this review, but a brief discussion of several recent exciting studies will highlight the kind of advances that have been made. For a more complete review of non-syndromic deafness genes that affect stereociliary bundle development, please see Frolenkov et al. (2004). Among the genes listed in Table 1 are Myosin XVa, Whirlin and Espin. Mutations in any one of these genes lead to deafness in both humans and mice (see Table 1 for references). Moreover, there is a very similar phenotype, the presence of short stereocilia and the absence of a staircase, in all three mouse mutants. The similarity of these phenotypes suggested that the three molecules might be acting in the same molecular pathway and the specific effect on stereocilia length suggested that the filamentous actin that forms the core of each stereocilium could be involved. In support of this hypothesis, previous results had demonstrated that Espin acts as an actin bundling protein (Bartles et al., 1996). Considering the importance of the addition of actin monomers to the maintenance of stereocilia height, these observations suggested a possible role for MyosinXVa, Whirlin and Espin at the tips of the stereocilia. In fact, immunolocalization or transfection of postnatal hair cells with MyosinXVa or Whirlin fusion proteins demonstrated specific localization of both molecules just at the tips of stereocilia (Belyantseva et al., 2005). In contrast, Espin is initially localized to the tips of the stereocilia but then becomes incorporated along the length in a pattern similar to incorporation of actin monomers (Rzadzinska et al., 2004). MyosinXVa is an atypical myosin motor while Whirlin is a scaffolding protein with three PDZ domains that presumably mediate protein–protein interactions (reviewed in Frolenkov et al., 2004; Lin et al., 2005). Localization of Whirlin to the tips of stereocilia is dependent on MyosinXVa, while MyosinXVa localization is unaffected in Whirlin mutants, suggesting that a MyosinXVa motor regulates the localization of Whirlin to the stereocilia tips (Belyantseva et al., 2005). Finally, transfection of MyosinXVa into hair cells in MyosinXVa mutants or of Whirlin into hair cells in Whirlin mutants results in a restoration of the stereocilia height and the graded staircase pattern, demonstrating the specific roles of each protein (Belyantseva et al., 2005). Overall, these results suggest that MyosinXVa acts as the motor that shuttles actin monomers and Espin to the tips of the stereocilia where they are incorporated into the actin core. Whirlin most likely acts as a bridging protein, utilizing its PDZ domains to bind to both MyosinXVa and the Espin/actin complex. However, it should be noted that while an interaction between MyosinXVa and Whirlin has been demonstrated (Belyantseva et al., 2005), similar interactions between Whirlin and Espin have not, suggesting that other molecules could also be involved in the formation and targeting of this complex. None of these molecules is uniquely expressed in the ear, yet mutations in each lead to non-syndromic deafness. These observations suggest that hair cells are

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Table 1 – Summary of genes identified through mouse and/or human mutations that lead to defects in stereociliary bundle development Gene

Biological role

γ-actin Diaphanous1 Espin Cadherin23

Cytoskeletal formation Cytoskeletal formation Cytoskeletal formation Cellular adhesion

Protocadherin Cellular adhesion 15 MyosinVI Motor protein MyosinVIIa

Motor protein

MysoinXVa Harmonin Sans Whirlin Tmie Pi Tde Tlc

Motor protein Scaffold Scaffold Scaffold Transmembrane Unknown Unknown Unknown

Human locus

Mouse model

References

DFNA20, A26

N/A

Morell et al., 2000; Zhu et al., 2003

DFNA1

N/A

Lynch et al., 1997; Higashida et al., 2004

DFNB36

Jerker

Zheng et al., 2000a; Naz et al., 2004

DFNB12, USH1F

Waltzer

DFNB23, USH1F

Ame's waltzer

DFNA22, B37

Snell's waltzer

DFNB2, A11, USH1B DFNB3 DFNB18, USH1C USH1G DFNB31 DFNB6 DFNB25 ?

Shaker-1, Headbanger Shaker-2 Deaf circler Jackson circler Whirler Spinner Pirouette Tasmanian Devil Tailchaser

Bolz et al., 2001; Bork et al., 2001; Di Palma et al., 2001 Ahmed et al., 2003b; Alagramam et al., 2001; Hampton et al., 2003 Ahmed et al., 2003a; Avraham et al., 1995; Melchionda et al., 2001; Mohiddin et al., 2004 Weil et al., 1995; Gibson et al., 1995; Self et al., 1998; Rhodes et al., 2004 Probst et al., 1998; Wang et al., 1998 Verpy et al., 2000; Bitner-Glindzicz et al., 2000 Weil et al., 2003 Mburu et al., 2003 Mitchem et al., 2002; Naz et al., 2002 Deol, 1956; Odeh et al., 2004 Erven et al., 2002 Kiernan et al., 1999

For each gene, the biological role, if known, is listed along with the human locus, name of any available mouse models and important references are listed. This table was modified form the table presented in Frolenkov et al. (2004).

uniquely sensitive to disruptions in this signaling complex. The reasons for this sensitivity are unknown but could be related to the constant actin turn over in the stereocilia or possibly, to a lack of functional redundancy in hair cells, although the reasons for such a difference between hair cells and other cells are not clear.

6.

Polarization of the stereociliary bundle

As discussed, each stereociliary bundle is arranged in a staircase pattern with the kinocilium asymmetrically located adjacent to the tallest row of stereocilia. Pioneering studies by Hudspeth and colleagues (Hudspeth and Corey, 1977; Hudspeth and Jacobs, 1979) demonstrated that the bundle is directionally sensitive such that deflection of the bundle towards the tallest row of stereocilia results in a depolarization of the cell while deflections towards the shortest row lead to hyperpolarization. Surprisingly, deflections in a plane perpendicular to axis of the staircase do not change the resting state of the cell. These results demonstrate that each bundle is directionally sensitive. The basis for this sensitivity appears to be the presence of tip links that stretch between the top of one stereocilium to the shaft of the next highest stereocilium (Assad et al., 1991). Tip links are oriented parallel with the staircase, providing the structural basis for directional sensitivity of the bundle (Pickles et al., 1984). In most hair cell epithelia, stereociliary bundles are uniformly oriented such that all the cells within a specific region will be similarly stimulated by a single stimulus. For instance, lateral line neuromasts in most fishes are comprised of two populations of hair cells with opposite polarities that are grouped into two opposing regions within the neuromast

(reviewed in Lewis and Davies, 2002). As a result, a single stimulus will lead to depolarization of one population of hair cells and hyperpolarization of the other population. One of the most striking examples of uniform hair cell orientation is the arrangement of stereociliary bundles in the mammalian cochlea. All of the hair cells are oriented such that the plane of sensitivity is perpendicular with the cochlear spiral. This orientation correlates with the deflection of the basilar membrane that is generated in response to the progression of a traveling wave within the cochlear duct. As a result, misorientated stereociliary bundles lead to a decrease in sensitivity and hearing perception (Fujita, 1990; Yoshida and Liberman, 1999). These results emphasize the importance of the generation of appropriate stereociliary bundle orientation for normal auditory function. The development of stereociliary bundle orientation has been described in several different systems, including the mammalian cochlea and utricle and the avian basilar papilla (Cotanche and Corwin, 1991; Denman-Johnson and Forge, 1999; Dabdoub et al., 2003; Montcouquiol et al., 2003) (Fig. 3). In addition, stereociliary bundle orientation has also been examined during hair cell regeneration in the chick basilar papilla (Cotanche and Corwin, 1991). The results from all of these studies indicate a conserved two step process for bundle orientation. Prior to the formation of the bundle, each developing hair cell contains a single true (microtubulebased) cilium located in the center of its lumenal surface. This cilium will develop as the kinocilium. The first step in the process is a centrifugal migration of the developing kinocilium towards the outer edge of the lumenal surface. The direction of this movement is non-random and shows a significant bias towards the final orientation of the bundle. However, the direction of this movement can deviate from the bundle's final

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orientation by as much as 45°. By the time the developing kinocilium reaches the lateral edge of the lumenal surface, adjacent developing stereocilia are present. Next, deviations from the final orientation are corrected through a gradual rotation of the bundle that is referred to as reorientation. As a result of these two processes, all stereociliary bundles within a specific population of hair cells develop with a uniform orientation. The generation of uniform orientation of cells or of a specific aspect of each cell, such as the stereociliary bundle, is referred to as planar cell polarity, or PCP. While the orientation of stereociliary bundles is one of the best examples of PCP in a vertebrate system, multiple examples of PCP exist in invertebrates systems, such as the uniform orientation of wing hairs in Drosophila. As a result, many of the molecules that regulate PCP were initially identified in fruit flies (reviewed in Fanto and McNeill, 2004; Klein and Mlodzik, 2005). Not surprisingly, orthologs for many of these genes were identified in vertebrates; however, a specific role for these genes in PCP had not been examined. Beginning in 2003, work from several laboratories demonstrated a role for two PCP orthologs, Vangl2 and Celsr1, in the orientation of stereociliary bundles. The role of Vangl2, a vertebrate ortholog of the Drosophila PCP gene van gogh/strabismus, was determined through an analysis of cochleae from Looptail mice (Montcouquiol et al., 2003). The Looptail phenotype, which includes an open neural tube, is caused by a point mutation within the Vangl2 gene (Kibar et al.,

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2001; Murdoch et al., 2001a). The Vangl2 protein is novel and contains four transmembrane domains and a PDZ-binding domain at its carboxy terminus. In Looptail mutants, a serine to asparagine transition at amino acid position 464 (referred to as Vangl2Lp) results in an apparent loss of function with no detectable Vangl2 protein in Vangl2Lp mutants (Montcouquiol et al., unpublished observations). Analysis of stereociliary bundles in cochleae from E18.5 Vangl2Lp homozygotes indicated normal overall development of the bundles, including the formation of a staircase pattern and normal bundle morphology. However, many of the bundles showed severe deviations in orientation (Montcouquiol et al., 2003). There was a direct correlation between the severity of the mis-orientation and the position of the hair cells within the organ of Corti. Hair cells located in the first outer hair cell row were statistically unchanged from control, while hair cells located in the second and third row of outer hair cells showed progressively greater deviations in orientation. Interestingly, bundle orientations on inner hair cells were as severely affected as third row outer hair cells. Celsr1 is one of three vertebrate orthologs of the Drosophila PCP gene flamingo, and codes for an atypical cadherin. Two mouse strains carrying mutations in Celsr1, Spincycle and Crash, were generated as a result of an ENU mutagenesis screen (Curtin et al., 2003). Heterozygous mice exhibited behaviors consistent with vestibular defects, while animals homozygous for either mutation had an open neural tube phenotype that was similar to Looptail. Analysis of the inner ears from animals that were heterozygous or homozygous for either the Spincycle or Crash mutation indicated the presence of misoriented stereociliary bundles, with a greater number of misoriented cells in homozygotes, indicating a gene dosage effect. A

Fig. 3 – Development and regulation of stereociliary bundle orientation. (A) Prior to hair cell development, each cell has a centrally located true cilium (red) that will eventually develop as the kinocilium. Based on similar studies in flies, Celsr1 (purple) and Vangl2 (green) are thought to be evenly distributed around the lumenal surface of the cell. Distal and proximal sides of the cell are illustrated to the right. (B) Prior to centrifugal migration of the developing kinocilium, Celsr1 is thought to become restricted to proximal and distal sides of the cell, while Vangl2 is thought to become restricted to the proximal side. These changes in protein distribution generate an instructional vector that directs the kinocilium to move towards the distal edge of the cell. While the direction of this migration is non-random, it can deviate by as much as 45° (area indicated in blue) from the final orientation of the stereociliary bundle. (C) During and following the period of centrifugal migration, the stereociliary bundle (black circles) begins to develop adjacent to the kinocilium. Once centrifugal migration is completed, any mistakes in orientation are corrected through a gradual rotation of the bundle that is referred to as reorientation. Wnt7a exists in a gradient (yellow) that decreases towards the distal side of the rows of outer hair cells, and disruption of the Wnt7a signal has been shown to inhibit the reorientation process. (D) Gradual reorientation of the bundle results in a mature bundle with correct orientation.

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comparison of the extent of misorientation between Vangl2 and Celsr1 mutants indicates relatively comparable levels of disruption (Montcouquiol et al., in press). Interestingly, the overall pattern of the disruption, with first row outer hair cells unaffected and third row outer hair cells and inner hair cells most affected, is also similar between the two mutants. These results strongly suggest that the two molecules act within the same signaling pathway, although genetic epistasis experiments will be required to confirm this suggestion. The nature of the mutations in Spincycle and Crash is intriguing (Curtin et al., 2003). In both, a single point mutation results in a single amino acid change, asparagine to lycine at codon 1110 in Spincylce and aspartate to glycine at codon 1040 in Crash. The Crash mutation is located within one of the extracellular cadherin repeats while the Spincycle mutation is located in a linker region that may be required for calcium binding. Although the nature of these mutations has not been determined, it seems likely that these mutations would probably lead to hypomorphic versions of Celsr1 rather than to a complete loss of function. However, the homozygous phenotype for these mutants is comparable with Vangl2Lp, which, as discussed, appears to be a complete loss of function, albeit as a result of a single amino acid change as well. Moreover, Spincycle and Crash heterozygotes have a vestibular phenotype that has not been reported in Vangl2Lp heterozygous animals. These results suggest that the PCP process may be more sensitive to perturbations in Celsr1 than in Vangl2, an observation that is consistent with similar results in Drosophila. Vangl2 and Celsr1 represent orthologs for Drosophila PCP genes, demonstrating a conservation in the molecular basis of this pathway. However, additional results have identified two new components of the PCP pathway in vertebrates. Scribble1 is the first identified mammalian ortholog of the Drosophila gene scribble, and is mutated in Circletail mice, so named for the curly tails present in heterozygotes (Murdoch et al., 2003). Scrb1Crc homozygotes have an open neural tube and mild PCP defects that are restricted to the third row of outer hair cells (Murdoch et al., 2001b; Montcouquiol et al., 2003). However, mice that are doubly heterozygous for Vangl2Lp and Scrb1Crc have PCP defects that are comparable to the defects observed in animals that are homozygous for mutations in either Vangl2 or Celsr1 (Montcouquiol et al., 2003). These results demonstrate a strong genetic interaction between Vangl2 and Scrb1 and suggest a potential physical interaction between the two proteins. Surprisingly, flies with mutations in scribble display no PCP defects and instead have defects in apical-basal polarization (Bilder and Perrimon, 2000). It cannot be ruled out that Circletail mice have apical–basal defects in addition to PCP defects; however, the existing data certainly demonstrate that the PCP defects, in particular the open neural tube, are more profound. These results suggest that the Scrb1 protein has been incorporated into the PCP pathway in vertebrates. It seems possible that the apical–basal functions of Scribble may be carried out by an, as yet unidentified, additional Scribble ortholog. More recently, PTK7, a novel receptor tyrosine kinase, has also been shown to regulate PCP (Lu et al., 2004). Mice with complete loss of PTK7 expression have an open neural and a mild cochlear PCP defect that is restricted to third row outer hair cells. The overall extent of the defect in PTK7 mutants is

comparable with the defect in Circletail mutants. These results suggest that PTK7 represents a second novel mediator of PCP in vertebrates; however, it has been noted that possible PCP phenotypes in flies with mutations in the Drosophila homolog of PTK7, off-track, have not been thoroughly examined. Another difference between vertebrate and invertebrate PCP is the role of Wnt/wg proteins. The Wnt/wg family is comprised of 19 Wnt genes in vertebrates and 7 wg genes in Drosophila (reviewed in Widelitz, 2005). All are secreted glycoproteins that have been shown to mediate multiple biological events during development. Wnt/wg molecules act as ligands for the Frizzled/frizzled class of transmembrane receptors. Frizzleds have been shown to play a key role in PCP in Drosophila (reviewed in Klein and Mlodzik, 2005) and one of the mammalian orthologs, Frizzled6, has been shown to mediate orientation of hair follicles on the skin (Guo et al., 2004). However, the role of Wnt/wg in PCP appears to differ between flies and vertebrates. In flies, wg is not required for the establishment of many polarized structures including the fly wing. In contrast, two vertebrate developmental events that have been shown to act through the PCP pathway, convergence and extension of the neural plate and neural tube closure, are dependent on Wnt signaling (reviewed in Montcouquiol et al., in press). These results suggest a role for Wnt in vertebrate PCP that may have been lost in Drosophila. The role of the PCP pathway in neural tube closure also explains the strong correlation between neural tube and PCP defects in the different mutant mice discussed in this section. The potential role of Wnt signaling in PCP in vertebrates is also supported by a recent study which identified a role for Wnts, and most probably Wnt7a, in the regulation of the reorientation phase of outer hair cell stereociliary bundles in the cochlea (Dabdoub et al., 2003). First, in situ hybridization demonstrated an asymmetric expression of Wnt7a on the modiolar edge of the outer hair cell domain Moreover, addition of Wnt7a-conditioned media or factors that inhibit Wnt7a signaling, including secreted frizzled related protein 1 (sFRP1) or wnt inhibitory factor (WIF), leads to an inhibition in the reorientation of outer hair cells. Two of the more intriguing aspects of the development of PCP within the cochlea are the questions of whether an organizing center exists and if so, the nature of the signal that arises from that center. The theory that uniformly oriented populations of cells will be generated through the existence of an asymmetrically located organizing center that acts to initiate polarization has existed at least since the work of Wolpert (1969) and probably even long before that. The regular structure of the organ of Corti would appear to make it well suited for regulation through an organizing center, and the demonstration that the severity of PCP defects directly correlates with the position of each hair cell is very consistent with this hypothesis. In addition, since PCP is not disrupted in the first row outer hair cells in any of the known mutants, the data would suggest that these cells might act as the organizing center. Alternatively, the demonstration that Wnt7a, a mediator of stereociliary bundle reorientation, becomes restricted to pillar cells (Dabdoub et al., 2003), suggests that these cells could act as the organizing center. In either case, the progressive loss of PCP with distance from the pillar cell/first row outer hair cell region suggests that Vangl2, Scrb1, Celsr1

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and PTK7 all play a role in the conveyance of the polarizing signal from one cell to the next and are not required for the actual formation of the putative organizing center. The nature of the signal that arises from the organizing center is harder to infer. Historically, polarizing signals were hypothesized to be secreted factors that would diffuse from a point or line source, generating a concentration gradient across the cells in question. Those cells would be able to detect the change in concentration across the diameter of each cell and respond accordingly. However, recent work in the Drosophila wing has suggested that a secreted, diffusible factor may not be required, at least in systems where all of the cells are polarized (Amonlirdviman et al., 2005). In contrast, in a structure such as the Drosophila retina, in which uniformly polarized ommatidia are separated by unpolarized epithelial cells, it is less clear how a non-diffusible signal could act to uniformly orient a large population of cells (reviewed in Fanto and McNeill, 2004). The existing data for the organ of Corti do not provide any meaningful insights regarding the nature of the signal in this system. All of the known PCP mutants are transmembrane or intracellular proteins. Wnt7a, a secreted molecule, has been shown to modulate some aspects of stereociliary bundle PCP, and is asymmetrically located at one edge of the population of outer hair cells; however, a role for a gradient of Wnt7a has not been demonstrated yet.

7. Thyroid hormone and differentiation of the organ of corti Although cellular patterning is largely complete within the organ of Corti by postnatal day 0 in mice, the onset of hearing does not occur until P14. During this time period, there is progressive differentiation of different cell types within the organ of Corti and cochlear duct, including hair cells and supporting cells. The factors that regulate this differentiation are still largely unknown; however, a number of studies have demonstrated an important role for thyroid hormone in regulating this process. Hypothyroidism and iodine deficiencies in pregnant women have long been known to cause auditory deficits in children (reviewed in Sher et al., 1998). In the 1970s and 1980s, Deol (1973) and Uziel et al. (1981, 1980) addressed these issues by creating hypothyroidism in pregnant rats using the thyroid hormone inhibitors propylthiouracil and methimazole. Analysis of the pups generated from these rats indicated immature cochleae with progressive loss of hair cells and defects in the formation of the tectorial membrane. However, because hypothyroidism had been generated systemically in these animals, it was not clear whether the inner ear defects were a direct result of the disruption in the thyroid hormone signaling pathway or a secondary effect of systemic hypothyroidism. The actions of thyroid hormone are mediated through binding and activation of thyroid hormone receptors (TRs), members of the steroid/thyroid family of transcription factors (reviewed in Forrest and Vennstrom, 2000). The TRs are encoded by two genes, TRα and TRβ, each of which can generate multiple splice variants. The direct effects of thyroid hormone signaling in inner ear development were recently examined through analysis of the cochleae from mice with

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targeted deletions of TRα, TRβ or both (Rusch et al., 1998; Rusch et al., 2001). TRα mutants are apparently completely normal in all respects. In contrast, TRβ mutants are deaf although morphological development of the cochlea appears normal. Deafness appears to be a result of a delay in the maturation of an important potassium conductance in inner hair cells, although further analysis may ultimately reveal other defects. In order to eliminate possible functional redundancy between TRα and TRβ in the cochlea, animals lacking both TR genes were generated. These animals are viable but have a significant hearing loss that slightly exceeds the hearing loss observed in TRβ single mutants (Rusch et al., 2001). In addition, morphological analysis of inner ears from TRα/β double mutants revealed morphological defects that were comparable to those observed in hypothyroid animals. These included delayed maturation of hair cells and supporting cells, as well as, significant overgrowth of the tectorial membrane and disruption of the striated sheet matrix, a component of the tectorial membrane. In addition, endocochlear potential was also significantly reduced. Cochlear maturation was not completely inhibited in TRα/β double mutants, as analysis of adult cochleae demonstrated opening of the tunnel of Corti and thinning of the inner sulcus, two indications of maturation. However, the delay appears to permanently affect the morphology of the tectorial membrane, which may be a primary cause of the observed auditory defects. TRs are activated by ligand binding. Although the primary product of the thyroid gland is thyroid hormone, also referred to as T4 because of the presence of 4 iodine molecules, the biologically active form of the molecule is actually T3. The conversion of T4 to T3 is mediated by a family of deiodinases that includes three genes, Dio1, Dio2 and Dio3 (reviewed in Hernandez and St Germain, 2003). Dio2 is particularly intriguing because it is expressed in the cochlea in a pattern that is complimentary to TRβ (Campos-Barros et al., 2000). Moreover, Dio2 is expressed in, or near, vascularized tissues in the cochlear duct, including the stria vascularis and the hebenula perforata. As a result, Dio2 may act to convert blood-born T4 into T3 that would then be available within the cochlea. Consistent with this hypothesis, cochleae from mice with a targeted deletion in Dio2 are morphologically similar to cochleae from TRα/β double mutants, and these animals also have a similar level of hearing impairment (Ng et al., 2004). Virtually all of the reported effects of disruption of thyroid signaling within the cochlea are apparently related to a delay in overall maturation. Since TRs are transcription factors, a next logical step would be to attempt to identify specific genes that are regulated through TR binding. In fact, such a study was recently carried out by Thomas Weber in the laboratory of Marlies Knipper (Weber et al., 2002). Previous work from the same laboratory had identified a delay in the development of distortion product otoacoustic emissions (DPOAEs) in hypothyroid animals (Knipper et al., 2000). Since DPOAEs are largely a result of outer hair cell motility, this result suggested a possible role for TRs in the regulation of molecules related to motility. Working from this premise, the authors examined the expression of Prestin, an outer hair cell motor protein, in hypothyroid animals. Prestin is normally turned on in rat outer hair cells around P0 and is strongly expressed by P8. However, the onset of Prestin

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expression was significantly delayed in hypothyroid rats, although Prestin levels eventually reached levels that were comparable with control animals. Since TRs act as transcription factors, the authors examined the promoter region of the Prestin gene to determine if any TR binding sites were present. And in fact, two potential Thyroid Response Elements (TRE) were identified in the Prestin promoter. The authors then confirmed that TRα bound to the Prestin TRE through mobility shift assays. These results suggest that the expression of Prestin, as well as other molecules associated with ongoing differentiation within the organ of Corti, may be directly modulated through the binding of activated TRs. As discussed, Prestin levels eventually reach normal or near normal levels in hypothyroid animals, suggesting that TRs act as modulators of Prestin expression, rather than as direct activators. However, the existence of defective DPOAEs in adult animals that were hypothyroid during the early postnatal period suggested that a critical period exists for the onset of Prestin, and probably other genes as well, and that a change in the timing of expression of different genes results in a permanent deficit.

8.

Summary

Hair cells within the organ of Corti go through an extended period of differentiation that begins soon after terminal mitosis and determination of cell fate and extends, in the mouse at least, for another 2.5 weeks. The onset of hair cell development begins with the determination of a subset of cells within the cochlear duct as hair cells. The factors that regulate hair cell commitment are still debatable, but the transcription factor Atoh1 is the only single molecule that is known to be sufficient to generate hair cells. Following commitment, one of the first events that occurs is the onset of expression of a number of genes that become necessary for hair cell survival. These include the transcription factors Pou4f3, Gfi1 and Barhl1. Each of these genes is expressed exclusively in hair cells within the cochlea, suggesting that they play a specific role in hair cell survival. The reasons for this dependence are unclear but could reflect a unique aspect of hair cell physiology, such as their need to maintain a high metabolic rate. One of the most fascinating aspects of hair cell morphology is the stereociliary bundle that serves as a primary mechanotransducer. Research from a number of different fields including human genetics and invertebrate development has resulted in exciting findings regarding the molecular factors that mediate different aspect of the development of the stereociliary bundle. In particular, several murine homologs of human non-syndromic deafness genes, including Espin, Whirlin and MyosinXVa, act together to maintain stereocilia height while planar cell polarity genes, such as Vangl2, Celsr1, Scrb1 and PTK7, coordinate the orientation of the bundle. Finally, the timing of particular development events during the extended differentiation of both inner and outer hair cells is regulated through the action of thyroid hormone and thyroid hormone receptors. Although many of the events involved with hair cell differentiation will apparently occur even in the absence of thyroid hormone, the timing of these events is crucial for normal function, resulting in profound deafness in animals in which the thyroid

signaling pathway is disrupted. The results summarized here indicate both how far we have come in our understanding of how the cochlea develops, and how far we have to go before we can completely understand the complex signaling pathways that must be coordinated to generate a functional auditory system.

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