Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: reponse to loss of cell partners in the Bronx waltzer mouse

This report is concerned with control of cell shaping, positioning, and cytoskeletal integration in a highly ordered cochlear neuroepithelium. It is largely based on investigations of events that occur during abnormal morphogenesis of the organ of Corti in the Bronx waltzer (bv/bv) mutant mouse. The organ's sensory hair cells and adjacent supporting cells ordinarily construct a spatially elaborate and supracellularly integrated cytoskeletal framework. Large microtubule bundles are connected to cytoskeletal components in neighbouring cells by actin-containing meshworks that link them to substantial arrays of adherens junctions. In bv/bv mice, degeneration and loss of most inner hair cells and outer pillar cells occurs during organ development. These cells flank each side of a row of inner pillar cells that respond by upregulating assembly of their actin-containing meshworks. This only occurs in surface regions where they no longer contact cell types involved in construction of the cytoskeletal framework. The meshworks are larger and exhibit a more extensive sub-surface deployment than is normally the case. Hence, assembly of intercellular cytoskeletal connecting components can proceed without contact with appropriate cell neighbours but termination of assembly is apparently subject to a negative feedback control triggered by successful completion of intercellular connection with the correct cell neighbours. In addition, inner pillar cells compensate for loss of cell neighbours by interdigitating and overlapping each other more extensively than is usually the case to increase opportunities for generating adherens junctions. Certain adherens junctions in the organs of +/+ and bv/bv mice exhibit features that distinguish them from all previously described cell junctions. The dense plaques on their cytoplasmic faces are composed of aligned ridges. We suggest that they are called ribbed adherens junctions. Perturbations of cell shaping and positioning indicate that loss of inner hair cells is the primary consequence of the bv mutation. Most of the other abnormalities can be understood in terms of a secondary sequence of morphogenetic aberrations (precipitated by loss of inner hair cells). These aberrations provide new information about the ways in which supporting cells help to control hair cell positioning.     

Differentiation of the lateral compartment of the cochlea requires a temporally restricted FGF20 signal

A large proportion of age-related hearing loss is caused by loss or damage to outer hair cells in the organ of Corti. The organ of Corti is the mechanosensory transducing apparatus in the inner ear and is composed of inner hair cells, outer hair cells, and highly specialized supporting cells. The mechanisms that regulate differentiation of inner and outer hair cells are not known. Here we report that fibroblast growth factor 20 (FGF20) is required for differentiation of cells in the lateral cochlear compartment (outer hair and supporting cells) within the organ of Corti during a specific developmental time. In the absence of FGF20, mice are deaf and lateral compartment cells remain undifferentiated, postmitotic, and unresponsive to Notch-dependent lateral inhibition. These studies identify developmentally distinct medial (inner hair and supporting cells) and lateral compartments in the developing organ of Corti. The viability and hearing loss in Fgf20 knockout mice suggest that FGF20 may also be a deafness-associated gene in humans.     

Shaping the mammalian auditory sensory organ by the planar cell polarity pathway

The human ear is capable of processing sound with a remarkable resolution over a wide range of intensity and frequency. This ability depends largely on the extraordinary feats of the hearing organ, the organ of Corti and its sensory hair cells. The organ of Corti consists of precisely patterned rows of sensory hair cells and supporting cells along the length of the snail-shaped cochlear duct. On the apical surface of each hair cell, several rows of actin-containing protrusions, known as stereocilia, form a "V"-shaped staircase. The vertices of all the "V"-shaped stereocilia point away from the center of the cochlea. The uniform orientation of stereocilia in the organ of Corti manifests a distinctive form of polarity known as planar cell polarity (PCP). Functionally, the direction of stereociliary bundle deflection controls the mechanical channels located in the stereocilia for auditory transduction. In addition, hair cells are tonotopically organized along the length of the cochlea. Thus, the uniform orientation of stereociliary bundles along the length of the cochlea is critical for effective mechanotransduction and for frequency selection. Here we summarize the morphological and molecular events that bestow the structural characteristics of the mammalian hearing organ, the growth of the snail-shaped cochlear duct and the establishment of PCP in the organ of Corti. The PCP of the sensory organs in the vestibule of the inner ear will also be described briefly.     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

Growth factor regulation of the cell Cycle in developing and mature inner ear sensory epithelia

Growth factors and other extracellular signals regulate cell division in many tissues. Consequently, growth factors may have therapeutic uses to stimulate the production of replacement sensory hair cells in damaged human inner ears, thereby assisting in alleviating hearing loss and vestibular dysfunction. Assessment of the ability of growth factors to stimulate cell proliferation in inner ear sensory epithelia is at an early stage. This paper provides a brief account of what we know regarding growth factor regulation of cell proliferation in developing and mature inner ear sensory epithelia.     

Emx2 and early hair cell development in the mouse inner ear

Emx2 is a homeodomain protein that plays a critical role in inner ear development. Homozygous null mice die at birth with a range of defects in the CNS, renal system and skeleton. The cochlea is shorter than normal with about 60% fewer auditory hair cells. It appears to lack outer hair cells and some supporting cells are either absent or fail to differentiate. Many of the hair cells differentiate in pairs and although their hair bundles develop normally their planar cell polarity is compromised. Measurements of cell polarity suggest that classic planar cell polarity molecules are not directly influenced by Emx2 and that polarity is compromised by developmental defects in the sensory precursor population or by defects in epithelial cues for cell alignment. Planar cell polarity is normal in the vestibular epithelia although polarity reversal across the striola is absent in both the utricular and saccular maculae. In contrast, cochlear hair cell polarity is disorganized. The expression domain for Bmp4 is expanded and Fgfr1 and Prox1 are expressed in fewer cells in the cochlear sensory epithelium of Emx2 null mice. We conclude that Emx2 regulates early developmental events that balance cell proliferation and differentiation in the sensory precursor population.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Development and evolution of inner ear sensory epithelia and their innervation

The development and evolution of the inner ear sensory patches and their innervation is reviewed. Recent molecular developmental data suggest that development of these sensory patches is a developmental recapitulation of the evolutionary history. These data suggest that the ear generates multiple, functionally diverse sensory epithelia by dividing a single sensory primordium. Those epithelia will establish distinct identities through the overlapping expression of genes of which only a few are currently known. One of these distinctions is the unique pattern of hair cell polarity. A hypothesis is presented on how the hair cell polarity may relate to the progressive segregation of the six sensory epithelia. Besides being markers for sensory epithelia development, neurotrophins are also expressed in delaminating cells that migrate toward the developing vestibular and cochlear ganglia. These delaminating cells originate from multiple sites at or near the developing sensory epithelia and some also express neuronal markers such as NeuroD. The differential origin of precursors raises the possibility that some sensory neurons acquire positional information before they delaminate the ear. Such an identity of these delaminating sensory neurons may be used both to navigate their dendrites to the area they delaminated from, as well as to help them navigate to their central target. The navigational properties of sensory neurons as well as the acquisition of discrete sensory patch phenotypes implies a much more sophisticated subdivision of the developing otocyst than the few available gene expression studies suggest.     

FGF信号通路在内耳发育调控和毛细胞再生中的作用

内耳是感受听觉和平衡觉的复杂器官。在内耳发育过程中,成纤维生长因子(fibroblast growth factor, FGF)信号通路参与了听基板的诱导、螺旋神经节(statoacoustic ganglion, SAG)的发育以及Corti器感觉上皮的分化。FGF信号开启了内耳早期发育的基因调控网络,诱导前基板区域以及听基板的形成。正常表达的FGF信号分子可促进听囊腹侧成神经细胞的特化,但成熟SAG神经元释放的过量FGF5可抑制此过程,形成负反馈环路使SAG在稳定状态下发育。FGF20在Notch信号通路的调控下参与了前感觉上皮区域向毛细胞和支持细胞的分化过程,而内毛细胞分泌的FGF8可调控局部支持细胞分化为柱细胞。人类FGF信号通路异常可导致多种耳聋相关遗传病。此外,FGF信号通路在低等脊椎动物毛细胞自发再生以及干细胞向内耳毛细胞诱导过程中都起到了关键作用。本文综述了FGF信号通路在内耳发育调控以及毛细胞再生中的作用及其相关研究进展,以期为毛细胞再生中FGF信号通路调控机制的阐明奠定理论基础。     

Mechanical forces shaping the development of the inner ear

The inner ear is one of the most complex structures in the mammalian body. Embedded within it are the hearing and balance sensory organs that contain arrays of hair cells that serve as sensors of sound and acceleration. Within the sensory organs, these hair cells are prototypically arranged in regular mosaic patterns. The development of such complex, yet precise, patterns require the coordination of differentiation, growth, and morphogenesis, both at the tissue and cellular scales. In recent years, there is accumulating evidence that mechanical forces at the tissue, the cellular, and the subcellular scales coordinate the development and organization of this remarkable organ. Here, we review recent works that reveal how such mechanical forces shape the inner ear, control its size, and establish regular cellular patterns. The insights learned from studying how mechanical forces drive the inner ear development are relevant for many other developmental systems in which precise cellular patterns are essential for their function.     

Hair cell regeneration in the avian auditory epithelium

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in non-mammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.     

Notch signaling during cell fate determination in the inner ear

In the inner ear, Notch signaling has been proposed to specify the sensory regions, as well as regulate the differentiation of hair cells and supporting cell within those regions. In addition, Notch plays an important role in otic neurogenesis, by determining which cells differentiate as neurons, sensory cells and non-sensory cells. Here, I review the evidence for the complex and myriad roles Notch participates in during inner ear development. A particular challenge for those studying ear development and Notch is to decipher how activation of a single pathway can lead to different outcomes within the ear, which may include changes in the intrinsic properties of the cell, Notch modulation, and potential non-canonical pathways.     

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius'' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.     

p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti

Strict control of cellular proliferation is required to shape the complex structures of the developing embryo. The organ of Corti, the auditory neuroepithelium of the inner ear in mammals, consists of two types of terminally differentiated mechanosensory hair cells and at least four types of supporting cells arrayed precisely along the length of the spiral cochlea. In mice, the progenitors of greater than 80% of both hair cells and supporting cells undergo their terminal division between embryonic day 13 (E13) and E14. As in humans, these cells persist in a non-proliferative state throughout the adult life of the animal. Here we report that the correct timing of cell cycle withdrawal in the developing organ of Corti requires p27(Kip1), a cyclin-dependent kinase inhibitor that functions as an inhibitor of cell cycle progression. p27(Kip1) expression is induced in the primordial organ of Corti between E12 and E14, correlating with the cessation of cell division of the progenitors of the hair cells and supporting cells. In wild-type animals, p27(Kip1) expression is downregulated during subsequent hair cell differentiation, but it persists at high levels in differentiated supporting cells of the mature organ of Corti. In mice with a targeted deletion of the p27(Kip1) gene, proliferation of the sensory cell progenitors continues after E14, leading to the appearance of supernumerary hair cells and supporting cells. In the absence of p27(Kip1), mitotically active cells are still observed in the organ of Corti of postnatal day 6 animals, suggesting that the persistence of p27(Kip1) expression in mature supporting cells may contribute to the maintenance of quiescence in this tissue and, possibly, to its inability to regenerate. Homozygous mutant mice are severely hearing impaired. Thus, p27(Kip1) provides a link between developmental control of cell proliferation and the morphological development of the inner ear.     

The modulation of cell adhesion molecule expression and intercellular junction formation in the developing avian inner ear

The cells that constitute the membranous labyrinth in the vertebrate inner ear are all derived from a single embryonic source, namely, the otocyst. The mature inner ear epithelia contain different regions with highly differentiated cells, displaying a highly specialized cytoarchitecture. The present study was designed to determine the presence of adherens-type intercellular junctions in this tissue and study the expression of cell adhesion molecules (CAMs) associated with these junctions, namely, A-CAM and L-CAM, in the developing avian inner ear epithelia. The results presented here show that throughout the early otocyst, A-CAM is coexpressed with L-CAM. The formation of asymmetries between sensory and nonsensory areas in the epithelium is accompanied by the modulation of CAMs expression and the assembly of intercellular junctional complexes. A-CAM and L-CAM display reciprocal expression patterns, the former being expressed mostly in the mosaic sensory epithelium, while L-CAM becomes conspicuous in the nonsensory areas but its expression in the sensory region is markedly reduced. Adherens-type junctions and numerous desmosomes are found in the junctional complexes of early otocyst cells. The former persist to maturity of the various inner ear epithelia, whereas desmosomes disappear from junctional complexes of hair cells but remain in the intercellular junctional complexes of all other cell types in the membranous labyrinth. Thus, adherens type intercellular junctions comprise the only defined cytoskeleton-bound junction in mature hair cells. A-CAM-positive cells are also found in the region of the acoustic ganglion in early developmental stages but not in the mature neural elements.