Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish

Unconventional myosins have been associated with hearing loss in humans, mice, and zebrafish. Mutations in myosin VI cause both recessive and dominant forms of nonsyndromic deafness in humans and deafness in Snell's waltzer mice associated with abnormal fusion of hair cell stereocilia. Although myosin VI has been implicated in diverse cellular processes such as vesicle trafficking and epithelial morphogenesis, the role of this protein in the sensory hair cells remains unclear. To investigate the function of myosin VI in zebrafish, we cloned and examined the expression pattern of myosin VI, which is duplicated in the zebrafish genome. One duplicate, myo6a, is expressed in a ubiquitous pattern during early development and at later stages, and is highly expressed in the brain, gut, and kidney. myo6b, on the other hand, is predominantly expressed in the sensory epithelium of the ear and lateral line at all developmental stages examined. Both molecules have different splice variants expressed in these tissues. Using a candidate gene approach, we show that myo6b is satellite, a gene responsible for auditory/vestibular defects in zebrafish larvae. Examination of hair cells in satellite mutants revealed that stereociliary bundles are irregular and disorganized. At the ultrastructural level, we observed that the apical surface of satellite mutant hair cells abnormally protrudes above the epithelium and the membrane near the base of the stereocilia is raised. At later stages, stereocilia fused together. We conclude that zebrafish myo6b is required for maintaining the integrity of the apical surface of hair cells, suggesting a conserved role for myosin VI in regulation of actin-based interactions with the plasma membrane.     

Intercellular cross-linkages between the stereociliary bundles of adjacent hair cells in the guinea pig cochlea

Summary Hair cells of the guinea pig organ of Corti have been examined using high resolution scanning electron microscopy. In addition to the extensive array of cross-links between the stereocilia of individual hair cells which have been reported previously, we have seen examples of attachments between the stereocilia of both adjacent inner and adjacent outer hair cells. The implications of these observations are discussed.     

Disruption of the autism-related gene Pak1 causes stereocilia disorganization,hair cell loss,and deafness in mice

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases(PAKs)are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout(Pak1à/à) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell(HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of b II-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1à/àmice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.     

Non-cell-autonomous planar cell polarity propagation in the auditory sensory epithelium of vertebrates

Sensory epithelia of the inner ear require a coordinated alignment of hair cell stereociliary bundles as an essential element of mechanoreceptive function. Hair cell bundle alignment is mediated by core planar cell polarity (PCP) proteins, such as Vangl2, that localize asymmetrically to the circumference of the cell near its apical surface. During early phases of cell orientation in the chicken basilar papilla (BP), Vangl2 is present at supporting cell junctions that lie orthogonal to the polarity axis. Several days later, there is a striking shift in the Vangl2 pattern associated with hair cells that reorient towards the distal (apical) end of the organ. How the localization of PCP proteins transmits planar polarity information across the developing sensory epithelium remains unclear. To address this question, the normal asymmetric localization of Vangl2 was disrupted by overexpressing Vangl2 in clusters of cells. The BP was infected with replication-competent retrovirus encoding Vangl2 prior to hair cell differentiation. Virus-infected cells showed normal development of individual stereociliary bundles, indicating that asymmetry was established at the cellular level. Yet, bundles were misoriented in ears infected with Vangl2 virus but not Wnt5a virus. Notably, Vangl2 misexpression did not randomize bundle orientations but rather generated larger variations around a normal mean angle. Cell clusters with excess Vangl2 could induce non-autonomous polarity disruptions in wild-type neighboring cells. Furthermore, there appears to be a directional bias in the propagation of bundle misorientation that is towards the abneural edge of the epithelium. Finally, regional bundle reorientation was inhibited by Vangl2 overexpression. In conclusion, ectopic Vangl2 protein causes inaccurate local propagation of polarity information, and Vangl2 acts in a non-cell-autonomous fashion in the sensory system of vertebrates.     

Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Stages of ciliogenesis and regulation of ciliary length

Cilia and flagella are highly conserved eukaryotic microtubule-based organelles that protrude from the surface of most mammalian cells. These structures require large protein complexes and motors for distal addition of tubulin and extension of the ciliary membrane. In order for ciliogenesis to occur, coordination of many processes must take place. An intricate concert of cell cycle regulation, vesicular trafficking, and ciliary extension must all play out with accurate timing to produce a cilium. Here, we review the stages of ciliogenesis as well as regulation of the length of the assembled cilium. Regulation of ciliogenesis during cell cycle progression centers on centrioles, from which cilia extend upon maturation into basal bodies. Centriole maturation involves a shift from roles in cell division to cilium nucleation via migration to the cell surface and docking at the plasma membrane. Docking is dependent on a variety of proteinaceous structures, termed distal appendages, acquired by the mother centriole. Ciliary elongation by the process of intraflagellar transport (IFT) ensues. Direct modification of ciliary structures, as well as modulation of signal transduction pathways, play a role in maintenance of the cilium. All of these stages are tightly regulated to produce a cilium of the right size at the right time. Finally, we discuss the implications of abnormal ciliogenesis and ciliary length control in human disease as well as some open questions.     

Cadherin 23 is a component of the transient lateral links in the developing hair bundles of cochlear sensory cells

Cadherin 23 is required for normal development of the sensory hair bundle, and recent evidence suggests it is a component of the tip links, filamentous structures thought to gate the hair cells' mechano-electrical transducer channels. Antibodies against unique peptide epitopes were used to study the properties of cadherin 23 and its spatio-temporal expression patterns in developing cochlear hair cells. In the rat, intra- and extracellular domain epitopes are readily detected in the developing hair bundle between E18 and P5, and become progressively restricted to the distal tip of the hair bundle. From P13 onwards, these epitopes are no longer detected in hair bundles, but immunoreactivity is observed in the apical, vesicle-rich, pericuticular region of the hair cell. In the P2-P3 mouse cochlea, immunogold labeling reveals cadherin 23 is associated with kinocilial links and transient lateral links located between and within stereociliary rows. At this stage, the cadherin 23 ectodomain epitope remains on the hair bundle following BAPTA or La(3+) treatment, but is lost following exposure to the protease subtilisin. In contrast, mechano-electrical transduction is abolished by BAPTA but unaffected by subtilisin. These results suggest cadherin 23 is associated with transient lateral links that have properties distinct from those of the tip-link.     

Cochlear stem/progenitor cells from a postnatal cochlea respond to Jagged1 and demonstrate that notch signaling promotes sphere formation and sensory potential

Hair cells and supporting cells of the mammalian cochlea terminally differentiate during development. Recent in vitro evidence suggests the presence of hair cell progenitors in the postnatal cochlea. Phenotypic properties of these cells and factors that promote their ability to generate spheres in aggregate cultures have not been reported. We define an in vitro system that allows stem/progenitor cells harvested from the early postnatal cochlea to develop into spheres. These spheres contain Abcg2, Jagged1 and Notch1 positive progenitor cells that can divide and generate new hair cell-like cells, i.e. immunopositive for specific hair cell markers, including Myosin VI, Myosin VIIa, Math1 and ability to uptake FM1-43.We demonstrate that reducing Notch signaling with a gamma secretase inhibitor decreases the number of spheres generated following treatment of the stem/progenitor cell cultures. Additionally, activation of Notch by an exogenous soluble form of a Notch ligand, i.e. Jagged1 protein, promotes sphere formation and the sensory potential of cochlear stem/progenitor cells. Our findings suggest that Notch1/Jagged1 signaling plays a role in maintaining a population of Abcg2 sensory stem/progenitor cells in the postnatal cochlea.     

Interconnections between the stereovilli of the fish inner ear

Summary We investigated at the electron-microscopic level the hair bundles of the macula organs in the inner ear of two species of teleost fish (Rutilus rutilus, Scardinius erythrophthalmus). All hairs (stereovilli) are interconnected by extracellular material that, similar to the cell coat, is well contrasted by application of tannic acid/osmium.The kinocilium is connected to the longest stereovilli via filaments. Filaments also connect the stereovilli at their bases. More distally all stereovilli are linked by thin filaments or very short solid connectors.The interconnections of the stereovilli in fish show greater variations, and are different in structural detail and distribution, compared to the interconnections described for amphibians and mammals. The interconnections are discussed in the context of their structural and functional significance and variations in terms of organ- and species specificity. Certain forms of interconnections are tentatively interpreted to be characteristic for developing hair bundles.     

Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear

The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.     

Saccular potentials of the vocal plainfin midshipman fish, <Emphasis Type="Italic">Porichthys notatus</Emphasis>

The plainfin midshipman fish, Porichthys notatus, is a vocal species of teleost fish that generates acoustic signals for intraspecific communication during social and reproductive behaviors. All adult morphs (females and males) produce single short duration grunts important for agonistic encounters, but only nesting males produce trains of grunts and growls in agonistic contexts and long duration multiharmonic advertisement calls to attract gravid females for spawning. The midshipman fish uses the saccule as the main acoustic endorgan for hearing to detect and locate vocalizing conspecifics. Here, I examined the response properties of evoked potentials from the midshipman saccule to determine the frequency response and auditory threshold sensitivity of saccular hair cells to behaviorally-relevant single tone stimuli. Saccular potentials were recorded from the rostral, medial and caudal regions of the saccule while sound was presented by an underwater speaker. Saccular potentials of the midshipman, like other teleosts, were evoked greatest at a frequency that was twice the stimulus frequency. Results indicate that midshipman saccular hair cells of non-reproductive adults had a peak frequency sensitivity that ranged from 75 (lowest frequency tested) to 145 Hz and were best suited to detect the low frequency components (≤105 Hz) of midshipman vocalizations.     

Dynamic regulation of the microtubule and actin cytoskeleton in zebrafish epiboly

Gastrulation is a key developmental stage with striking changes in morphology. Coordinated cell movements occur to bring cells to their correct positions in a timely manner. Cell movements and morphological changes are accomplished by precisely controlling dynamic changes in cytoskeletal proteins, microtubules, and actin filaments. Among those cellular movements, epiboly produces the first distinct morphological changes in teleosts. In this review, I describe epiboly and its mechanics, and the dynamic changes in microtubule networks and actin structures, mainly in zebrafish embryos. The factors regulating those cytoskeletal changes will also be discussed.     

Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea

Summary The distribution and polarity of actin in sensory hair cells of the chinchilla cochlea has been determined by decoration of actin filaments with myosin sub fragment S₁. Decorated actin filaments of the same polarity were present within the stereocilia above the cuticular plate. However the filaments in the rootlets and the thin filaments projecting laterally from the rootlets into the cuticular plate did not decorate with S₁. Decorated actin filaments were present within the cuticular plate, and near the plasma-membrane filaments of opposite polarity were observed. In the cross-striated region at the base of the cuticular plate of inner hair cells, decorated filaments were present in the dense bands of the cross-striations but the thin filaments perpendicular to the dense bands were not decorated. These results are discussed with respect to the two mechanisms that have been suggested for actin-myosin mediated movement of the stereocilia of inner-ear sensory cells.     

Applying genomics to the avian inner ear: development of subtractive cDNA resources for exploring sensory function and hair cell regeneration

We applied a micro-cDNA-based subtraction method to identify genes expressed in the regenerating sensory epithelia (SE) of the chicken inner ear. Sensory hair cells in the avian utricle SE are in a constant state of turnover, where dying hair cells are replaced by new ones derived from supporting cells. In contrast, hair cells in the cochlea remain quiescent unless damaged. We used this difference to enrich for utricle-specific genes, using reiterative cDNA subtraction and demonstrate enrichment for utricle-specific sequences. A total of 1710 cDNA sequence reads revealed the presence of many cDNAs encoding known structural components of the SE (for example, Harmonin and beta-tectorin), proteins involved in cellular proliferation, such as P311, HIPK2, and SPALT1, among many others of unknown function. These libraries are the first of their kind and should prove useful for the discovery of candidate genes for hearing disorders, regenerative and apoptotic pathways, and novel chicken ESTs.     

Regression analysis of four physical characteristics—stature,length of thigh,length of leg and length of foot of the male population in Sichuan Province

Using four physical characteristics of 394 adult male individuals in the Sichuan Province of China — stature, length of thigh, length of leg and length of foot, a series of linear regression equations and ternary regression equations have been established. Meanwhile three stepwise regression equations also have been established including a quarternary regression equation. Generally speaking, the established multiple equations in this study should be applied as much as possible, where there are two or three independent variables, because they predict more effectively in the individual identification of forensic practice.     

Structures transmitting stimulatory force to the sensory hairs of vestibular ampullae of fishes and frog

Summary Serial sections of the vestibular ampullae of two species of fish and one species of frog were investigated by electron microscopy. The kinocilium is the only connection between the sensory cells and the auxiliary structure (cupula). The cupula possesses canals that traverse its entire height. Each canal contains a single kinocilium in its proximal part; distally, it is filled with material that stains with colloidal silver. The matrix of the cupula consists of filaments running perpendicular to the canals. These filaments do not stain with colloidal silver. The kinocilium is connected to the wall of the canal via structures that differ in the studied species of fish and frog. The filamentous links between the kinocilium and the longest stereovilli of the sensory hair bundle are similar in all the investigated species. The stereovilli are interconnected by basal and shaft links, and by horizontal and oblique tip connectors, similar to those described by other authors for macula organs and the organ of Corti, although differences in structural details, especially of the horizontal tip and the shaft connectors, are present. Some of these are species specific and some are related to the position of the sensory cell in the epithelium and/or specific to the organ (ampulla or macula organ). Some attachment sites of the links are associated with osmiophilic submembranous material. These differences in the structure, distribution and attachment sites of the links are possibly of functional importance.     

The responses of primary and secondary sensory hair cells in the squid statocyst to mechanical stimulation

Summary Intracellular recordings were obtained from primary and secondary sensory hair cells in the anterior transverse crista segment of the squid (Alloteuthis subulata) statocyst during imposed displacements of the overlying cupula. The secondary sensory hair cells were depolarized by ventral movements of the cupula and hyperpolarized by dorsal cupula movements. The displacement/response curve was asymmetric around the zero position and sigmoidal in shape, similar to that already described for vertebrate hair cells. The cells are estimated to have a sensitivity of at least 0.5 mV per degree angle of cilia displacement. The responses showed pronounced adaptation and could be blocked by bath applied alcohols, such as heptanol or octanol, or by high concentrations of aminoglycosides.The primary sensory hair cells were depolarized by dorsal movements of the cupula, usually responding with a burst of action potentials. The displacement/response curve was also sigmoidal in shape and the firing pattern showed strong adaptation to maintained displacements of the cupula.The cupula itself appeared to be irregular in shape, extending much further into the statocyst cavity in its central part than at its edges. This is likely to result in differences in the responses of the underlying hair cells along the length of the crista ridge.