Interconnections between the stereovilli of the fish inner ear

Summary The maculae utriculi and sacculi of the inner ear from the European roach (Rutilus rutilus) were investigated by transmission electron microscopy. The stereovilli of peripherally and centrally located sensory cells differ in several features that suggest a developmental gradient. The stereovilli of the peripheral sensory cells, shown to be differentiating cells by other research groups, are short and less steeply graded in height than in central hair cells. All stereovilli in both kinds of hair bundles are interconnected. In the central bundles of stereovilli basal, tip, and vertical connectors are separated by unconnected regions. In contrast, filaments and sometimes other additional structures connect the stereovilli of peripheral bundles over their entire length, but vertical connectors are usually absent. Osmiophilic material occurring inside peripheral stereovilli is interpreted to be monomeric actin. Central and peripheral hair bundles also differ in their reaction to ruthenium red and cationized ferritin. Only the stereovilli of the central cells can be fused by these polycations. Ruthenium red also discriminates between supporting and sensory cells indicating differences in amount or distribution of extracellular material. Hair bundles, intermediate in properties and position between central and peripheral sensory cells, were also found, so that it became possible to propose a scheme of developmental steps leading from microvilli or microvillus-like stereovilli to the fully differentiated hair bundle.     

Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.     

The mechanism of sensory transduction in the sensilla of the trochanteral hair plate of the cockroach,Periplaneta americana

Summary The trochanteral hair plate of the cockroach leg contains approximately 60 hair sensilla that are deflected by a joint membrane during flexion of the leg. Previous work has shown that the organ is a mechanoreceptor which limits leg flexion during walking by reflex connections to flexor and extensor motoneurons. Functional analysis of the largest sensilla has shown that their behaviour may be well approximated by a velocity detector followed by a unidirectional rectifier.We report here the results of an examination of the largest sensilla by scanning and transmission electron microscopy in an attempt to correlate the structure with the known functional elements. Each hair is innervated by a single sensory dendrite which is surrounded by an electron dense dendritic sheath. The dendrite terminates below the hair shaft in a tubular body containing a parallel array of microtubules embedded in an electron dense matrix, while the dendritic sheath extends beyond the tubular body to form the walls of the ecdysial canal. At the proximal end of the tubular body the dendritic sheath and sensory dendrite are anchored to the cuticular socket by a fibrous dome which seems to form a fulcrum around which the tubular body can be deflected by movements of the hair. We suggest that the basis for the detection of velocity may be mechanical differentiation by a fluid space between the dendritic sheath and the tubular body. The structure is also discussed with relation to the mechanism of sensory transduction and the possible causes of the unidirectional sensitivity.Supported by the Canadian Medical Research Council. The authors gratefully acknowledge the expert technical assistance of Sita Prasad     

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.     

The effect of monoamine depleting drugs upon the synaptic bars in the inner ear of the bullfrog (Rana catesbeiana)

Summary Reserpine and guanethidine produce a highly significant reduction in electron density of the synaptic bars in the sensory cells of the bullfrog labyrinth. When amphetamine is administered simultaneously with guanethidine, the density of the synaptic bars is similar to those of untreated frogs. p-Chloramphetamine has no significant effect upon the electron density of synaptic bars. These observations are discussed in the light of what is known of the biological effects of these drugs, and are taken to indicate that the synaptic bars could be intracellular storage sites for a monoamine that mediates the synaptic contacts between the sensory cells and afferent nerve fibres. It is suggested that the monoamine involved is a catecholamine.Both of us thank Mrs. J. Birch and Miss J. Sutcliffe for their technical assistence. One of us (M. P. O.) was supported by a U.S. National Research Council Senior Research Associateship (1967–1968) during the earlier phase of this work, and we are both indebted to the Italian Consiglio Nazionale delle Ricerche for a grant (No. 69.01697.119.3) which financed the latter stages of this study.     

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.     

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.     

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.     

Neural crest and placode interaction during the development of the cranial sensory system

In the vertebrate head, the peripheral components of the sensory nervous system are derived from two embryonic cell populations, the neural crest and cranial sensory placodes. Both arise in close proximity to each other at the border of the neural plate: neural crest precursors abut the future central nervous system, while placodes originate in a common preplacodal region slightly more lateral. During head morphogenesis, complex events organise these precursors into functional sensory structures, raising the question of how their development is coordinated. Here we review the evidence that neural crest and placode cells remain in close proximity throughout their development and interact repeatedly in a reciprocal manner. We also review recent controversies about the relative contribution of the neural crest and placodes to the otic and olfactory systems. We propose that a sequence of mutual interactions between the neural crest and placodes drives the coordinated morphogenesis that generates functional sensory systems within the head.     

BMP-signaling regulates the generation of hair-cells

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, yet there is no understanding of the role of BMP4 in this process. The present work was aimed at exploring the effects of BMP-signaling on the development of hair-cells. For this purpose, we studied gene expression, cell proliferation and cell death in isolated chick otic vesicles that were grown in vitro in the presence of recombinant BMP4 or the BMP-inhibitor Noggin. Cath1 was used as a marker for hair-cell specification. BMP4 reduced the number of Cath1-cells and, conversely, Noggin increased the size of the sensory patches and the number of Cath1-positive cells. The effect of BMP4 was irreversible and occurred before hair-cell specification. Lfng and Fgf10 were expressed in the prosensory domain before Cath1, and their expression was expanded by Noggin. At these stages, modifications of BMP activity did not respecify non-sensory epithelium of the otic vesicle. The expression of Bmp4 at sensory patches was suppressed by BMP4 and induced by Noggin suggesting an autoregulatory loop. Analysis of BrdU incorporation during 6 and 18 h indicated that the effects of BMP4 were due to its ability to reduce the number of actively proliferating progenitors and inhibit cell fate specification. BMP4 induced cell death within the prosensory domain of the otic vesicle, along with the expression of Msx1, but not Msx2. On the contrary, BMP-inhibition with Noggin favored hair-cell specification without changes in the overall cell proliferation. We propose that about the stage of terminal division, the balance between BMP and BMP-inhibitory signals regulates survival and specification of hair-cell precursors, the final number of sensory hair-cells being limited by excess levels of BMPs. The final size of sensory patches would hence depend on the balance between BMP4 and opposing signals.     

Modes of neuronal arbor enlargement in the ear of a postembryonic fish,Astronotus ocellatus

New hair cells are added during postembryonic life in several species of fishes and birds. The production of new hair cells appears to require enlargement of eighth nerve arbors during growth since, at least in fish, eighth nerve neurons are added more slowly than hair cells or not at all. This situation provides an intriguing opportunity to study the mechanisms of growth of the neuronal arbors. In this paper, we report the results of studies on the postembryonic growth of eighth nerve dendritic arbors in the saccular epithelium of the cichlid fish Astronotus ocellatus. Arbor sizes and shapes were compared in small and large fish using the axonal tracer cobaltouslysine. Our data suggest that postembryonic eighth nerve arbors enlarge in 2 ways. First, arbors add new terminal endings to their distal ends. Second, whole new branches appear to be added at locations up to hundreds of micrometers proximal to the terminal endings. These 2 modes of growth suggest that more than one mechanism may be operative in controlling arbor enlargement.     

Observations on the primary sensory ending of tenuissimus muscle spindles in the cat

Summary The arrangement of preterminal and terminal axon branches in the primary sensory endings of cat tenuissimus muscle spindles was studied using whole-mount and serial-section techniques. Although in every case one firstorder preterminal branch was supplied exclusively to the bag₁ type of intrafusal muscle fibre, the preterminal branching patterns differed considerably in detail.Terminals varied widely in size and location. Their precise form varied according to their position on the intrafusal muscle fibres rather than their relationship to preterminal branches. Terminals derived from separate preterminal branches remained separate and did not fuse with themselves or each other. Individually bag₁ fibres had most terminals, chain fibres least. The surface of the muscle fibres were differentially indented by the terminals, least in bag₁ fibres and most in chain fibres.The results are discussed in relation to mechanosensory transduction and to the factors involved in determining the form of the primary ending.     

MicroRNAs and regeneration: Let-7 members as potential regulators of dedifferentiation in lens and inner ear hair cell regeneration of the adult newt

MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.