Developmental Neurobiology of the Ear Current Status and Future Directions

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Matthew W. Kelley and Doris K. Wu

The close of the twentieth century marked the dawn of a renaissance in inner ear developmental biology. During the preceding 100 years, the number of scientists willing to accept the challenges of working with one of the smaller structures in the body had remained comparatively small. More recently, however, the emergence of molecular biological techniques, combined with a greater appreciation of the elegance and importance of the inner ear, has led to an increase in the number of scientists who actively study inner ear biology and, more importantly, to a striking increase in the pace of discovery. As a result, now five years into the new century, it seems appropriate to review the remarkable progress that has occurred and to discuss the challenges that still await researchers.

The development of the inner ear has been referred to as one of the most striking examples of cellular morphogenesis in any biological system (Barald and Kelley 2004). From a rather humble beginning as a patch of unremarkable ectodermal cells, the developing ear expands to give rise to a spectrum of specialized cell types and structures that encompass neural, epithelial, secretory, and mechanosensory phenotypes. Along the way, different regions and, ultimately, individual cells, become specified to develop as different parts of the ear through a series of inductive signaling events that require both autocrine and paracrine functions.

The first step in the formation of the ear is the specification of a region of ectoderm as the otic placode (reviewed in Riley and Phillips 2003). Classic embryological studies had examined the role of adjacent tissues in the formation of this placodes. More recently, however, these tissue interactions have been reexamined at a molecular level (Ladher et al. 2000; Mackereth et al. 2004). In Chapter 2 of this volume, Andrew Groves concisely describes the classic experiments of the past century and then provides a critical interpretation of these experiments in light of the emerging molecular data regarding the same developmental process. Following its formation, the otic placode invaginates and ultimately separates from the surface ectoderm to form the spherical otic vesicle (also called the otocyst) (reviewed in Torres and Giraldez 1998). Moreover, as soon as it forms, the otocyst is already regionalized into different developmental compartments or zones (reviewed in Fekete and Wu 2002). As was the case for the placode, classic embryological studies had demonstrated an important role for surrounding tissues, in particular the developing hindbrain and periotic mes-enchyme, in formation and regionalization of the vesicle (reviewed by Fritzsch and Beisel 2001; Fekete and Wu 2002; Liu et al. 2003). In Chapter 3, Mansour and Schoenwolf describe the ongoing assembly of a series of genetic cascades, both in surrounding tissues as well as the otocyst itself, that play a role in these crucial developmental events. This chapter also highlights the power of mouse genetics as a tool for the study of early developmental events in ear formation. By generating animals that carry compound mutations in multiple genes, researchers have been able to demonstrate the existence of redundant genetic signaling that apparently exists to ensure the formation of a relatively normal ear even in the presence of disruptions in individual genes (Maroon et al. 2002; Wright and Mansour 2003). Finally, Chapter 3 also provides an intriguing comparison between the formation of the otocyst and the neural tube. Both structures undergo similar developmental events, including the transition from an initially flat sheet of cells (neural plate and otic placode) to a closed three-dimensional structure with a central lumen. As one might guess, there are commonalties and differences in the molecular pathways used to achieve similar morphogenetic goals.

One of the most striking events that occurs during the initial formation of the otocyst is the specification and delamination of a group of neuroblasts from its ventral region. Almost as soon as these cells leave, they begin to extend neurites back into the otocyst as if they are unwilling to separate fully from their old companions (Carney and Silver 1983; Hemond and Morest 1991a,b). These cells, and their progeny, will go on to give rise to the neurons of the acousti-covestibular (VIIIth cranial) nerve that provide afferent innervation for all aspects of both the auditory and vestibular regions of the ear. Over the past few years, significant progress has been made in establishing the cellular events and molecular cascades that direct these cells from unspecified neuroblasts to mature neurons forming elaborate and precise connections with mechanosensory hair cells in the periphery and auditory and vestibular nuclei in the central nervous system (CNS) (Ma et al. 1998; Liu et al. 2000; Raft et al. 2004). These interactions, as well as stimulating new hypotheses regarding the specification of the initial neuroblast population and its relationship with other cell types in the ear, are described by Pauley, Matei, Beisel, and Fritzsch in Chapter 4.

As its morphogenesis continues, a subset of epithelial cells within the otocyst become specified to develop as the sensory patches that will actually perceive sound and movement (reviewed in Whitfield et al. 1997). Subsequently, individual cells within these patches become specialized to develop as either mechanosensory hair cells or as surrounding nonsensory cells, which are collectively referred to as supporting cells. Intriguingly, but perhaps not surprisingly considering the limited number of developmental signaling pathways, the same molecular pathway appears to regulate both types of decisions (Adam et al. 1998; Haddon et al. 1998; Lanford et al. 1999). The Notch pathway is a nearly ubiquitously expressed signaling cascade that is used in multiple developing and mature systems to sort homogeneous progenitor cells into different cell fates (reviewed in Schweisguth 2004). In Chapter 5, Lanford and Kelley examine the role of Notch in the ear in light of expression and functional data in the ear and recent progress in our understanding of the different cofactors and signaling events that mediate this intriguing signaling pathway.

The final two chapters in this volume examine an exciting emerging field in inner ear development, the development of the stereociliary bundle located on all mechanosensory hair cells. In Chapter 6, Bryant, Forge, and Richardson describe the morphological process of hair-cell differentiation, including the development of the stereociliary bundle, while in Chapter 7, Hertzano and Avra-ham review insights into the development of the inner ear that have been obtained through the identification of genetic mutations that underly human non-syndromic and syndromic deafness. Surprisingly, a number of these mutations have direct effects on the development of stereociliary bundles. Although the stereociliary bundle is comprised of modified microvilli that are not dissimilar from the microvilli located on many developing and mature epithelial cells, the striking arrangement of these cells into a staircase pattern with a specific plane of polarization, and the exquisite sensitivity of this structure, suggests that it may be unique. In fact, as Hertzano and Avraham discuss, the recent explosion in the identification and understanding of the molecular factors that regulate the formation of these bundles has its roots in the field of human genetics, and more specifically in the study of mutations that lead to auditory and/or vestibular defects (A. Wang et al. 1998; Zheng et al. 2000; Naz et al. 2004). Many of these genes play a crucial role in the formation of the stereociliary bundle, and the ongoing studies of their molecular function has led to valuable insights into the cell biology of bundle development (Belyantseva et al. 2003; Sekerkova et al. 2004; Rzadzinska et al. 2004). These studies also demonstrate the power of genetics in developmental biology and highlight the opportunity to learn about unique cell types or structures through the identification of nonsyndromic genetic mutations in both humans and mice.

Although the chapters in this book strikingly describe the progress that has occurred in recent years, it is important to consider that many questions remain unanswered and that there is much work to be done. Perhaps the most glaring deficits exist in our understanding, or lack of understanding, of the factors that generate heterogeneity throughout the ear. For instance, while considerable efforts have been devoted to the examination of the factors that specify the sensory patches, we know relatively little about the level and degree of heterogeneity in the nonsensory regions of the ear. As an example, the endolymphatic duct and the semicircular canals are both located in the dorsal region of the inner ear. Fate mapping studies in chicken show that the endolymphatic duct is derived from the dorsal region of the otic cup, whereas cells in the three semicircular canals are derived mostly from the posterolateral region of the otic cup (Bri-gande et al. 2000). These two regions of the otic cup are molecularly distinct from each other, suggesting that their fates are restricted early in development

(W. Wang et al. 1998, 2001; Acampora et al. 1999; Depew et al. 1999), but the factors that specify either structure are unclear. Similarly, the specification of the two nonsensory structures in the mammalian cochlea, the stria vascularis and Reissner's membrane, are largely unknown.

Similar heterogeneities exist among various sensory patches. Ampullae differ from saccule or utricle and both clearly differ from auditory epithelia. Recent results have suggested that the Wnt signaling pathway, and more specifically b-catenin, may play a role in the determination of vestibular versus auditory sensory patches (Stevens et al. 2003), but this discovery serves as only a potential tip of the iceberg. Heterogeneities are even found within individual sensory patches. Vestibular epithelia contain type I and type II hair cells, while auditory epithelia such as the avian basilar papilla and the mammalian cochlea contain at least two types of hair cells (tall and short in birds, inner and outer in mammals). Similarly, at least four different types of supporting cells can be identified in the mammalian cochlea, and it seems likely that similar supporting cell heterogeneities exist in other sensory patches.

A second area of uncertainty is the developmental relationship between me-chanosensory hair cells and the neurons that innervate them. Existing molecular data suggest that the progenitors for both populations of cells arise from the same anterior-ventral region of the otocyst (reviewed in Fritzsch and Beisel 2001; Fekete and Wu 2002; Fritzsch et al. 2002), but it is unclear whether any clonal relationship, such as has been observed for sensory cells and innervating neurons in invertebrates (Hartenstein and Posakony 1990; Ghysen and Dambly-Chaudiere 1993; Parks and Muskavitch 1993; Jan and Jan 1995; Zeng et al. 1998), exists between the two cell types. Lineage data in chicken generated using replication-incompetent retroviruses indicate that common precursors can give rise to both neurons and hair cells (Satoh and Fekete 2004), but the number of reported clones is small, and it is unclear whether the neurons and hair cells that derive from a common precursor actually communicate with one another, as would be expected by analogy with invertebrates.

This question also highlights a greater need for studies of lineage, fate mapping, and cell movement, especially in mammals, in which the relative inaccessibility of the inner ear has limited our ability to generate meaningful data about these important questions. The inner ear undergoes dynamic morphogenesis during development. Gene expression data alone are insufficient for the full comprehension of the developmental processes involved in the formation of this intricate organ. Two recent fate mapping studies in Xenopus laevis (Kil and Collazo 2001) and chicken (Brigande et al. 2000) have indicated that dynamic cell movements occur during inner ear development, suggesting that there is much to be learned from these approaches. Encouragingly, the first cell lineage studies in a mammalian (mouse) ear have recently been reported using ultrasound backscatter microscopy techniques (Brigande and Fekete, personal communication).

As the chapters in this book emphasize, the pace of discovery at the molecular level has increased dramatically, in particular in terms of our understanding of the earliest events in ear development. Ironically, however, the crucial roles for many of these genes in early ear development have also proven to be a major impediment to our understanding of the molecular factors that regulate later developmental events. All biologically developing systems utilize a combination of a relatively limited number of molecular signaling pathways, but unique con-textually based responses to those pathways generate diverse heterogeneities at all levels from the determination of the three basic germ layers through organogenesis. Therefore, disruption of a single molecular signaling pathway may have multiple profound effects of different developmental events even within a single organ, but the first effect of this disruption may negate the analyses of later effects of this same pathway.

A good example of this is the role of Fgfrl. Complete deletion of Fgfr1 leads to early embryonic lethality prior to inner ear formation (Deng et al. 1994; Yamaguchi et al. 1994), but a conditional deletion of Fgfr1 that is limited to the inner ear and small number of other structures reveals a specific role for this gene in the development of the cochlea (Pirvola et al. 2002). The repeated use of conserved signaling pathways highlights the need for the generation of tissue-specific mutants and the examination of specific pathways at different developmental time points.

The importance of the development of these tools is emphasized by multiple examples of studies that attempted to generate mouse models for human diseases by simply disrupting genes that were known to cause human syndromic or non-syndromic deafness, but instead resulted in novel and unexpected consequences. For instance, mutations in EYA1, PAX2, and PENDRIN have been associated with branchio-oto-renal, renal coloboma, and Pendred syndromes, respectively, all of which can cause syndromic forms of human deafness (Abdelhak et al. 1997; Everett et al. 1997; Li et al. 1998; Sanyanusin et al. 1995). The knockout mouse models for each of these genes, however, show more severe inner ear defects than observed in human patients (Torres et al. 1996; Xu et al. 1999; Everett et al. 2001; Burton et al. 2004). Perhaps the most striking example of this phenomenon is the observation that mutations in the gap junction protein GJB2 lead to nonsyndromic deafness in humans while deletion of the mouse homolog, Connexin 26, results in lethality prior to implantation as a result of placental defects (Gabriel et al. 1998). All of these phenotypic differences could be attributable to species differences or to the fact that some of the mutations in the human genes result in hypomorphic versions of the genes rather than the functional nulls generated in the mouse models. These results demonstrate the crucial need for the ability to regulate gene deletion both spatially and temporally using mice that express Cre-recombinase under the control of ear specific promoters.

Finally, as more and more candidate molecules that are important for normal inner ear functions are identified, it will be crucial to gain an in-depth understanding of the cellular events that are mediated by these molecules. A number of studies, particularly in the area of hair cell biology and stereociliary bundle formation, have certainly advanced in this direction (Belyantseva et al. 2003;

Rzadzinska et al. 2004; Sekerkova et al. 2004). Therefore, despite the technical challenges and the requirement for the development of novel and unique methodologies, the recent advances in our current understanding of molecular basis for mechanotransduction clearly make these efforts worthwhile.

The potential benefits of an increased understanding of the cell biology of the inner ear are perhaps no more obvious than when one considers the potential application of this knowledge to the generation of therapies for both congenital and acquired deafness. Genetic analyses in both humans and mice have demonstrated that both the hair cells and the supporting cells are crucial for normal auditory function. Yet, our understanding of how these cells develop and function is still extremely limited. Considering the potential impact of regenerative therapies for auditory or vestibular dysfunction, a more comprehensive understanding of both hair cells and supporting cells is crucial.

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