Retinoic acid degradation shapes zonal development of vestibular organs and sensitivity to transient linear accelerations.

Each vestibular sensory epithelium in the inner ear is divided morphologically and physiologically into two zones, called the striola and extrastriola in otolith organ maculae, and the central and peripheral zones in semicircular canal cristae. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, formation of striolar/central zones is compromised, such that they resemble extrastriolar/peripheral zones in multiple features. Mutants have deficient vestibular evoked potential (VsEP) responses to jerk stimuli, head tremor and deficits in balance beam tests that are consistent with abnormal vestibular input, but normal vestibulo-ocular reflexes and apparently normal motor performance during swimming. Thus, degradation of RA during embryogenesis is required for formation of highly specialized regions of the vestibular sensory epithelia with specific functions in detecting head motions.

Bisphenols disrupt differentiation of the pigmented cells during larval brain formation in the ascidian.

The endocrine disruptor Bisphenol A (BPA), a widely employed molecule in plastics, has been shown to affect several biological processes in vertebrates, mostly via binding to nuclear receptors. Neurodevelopmental effects of BPA have been documented in vertebrates and linked to neurodevelopmental disorders, probably because some nuclear receptors are present in the vertebrate brain. Similarly, endocrine disruptors have been shown to affect neurodevelopment in marine invertebrates such as ascidians, mollusks or echinoderms, but whether invertebrate nuclear receptors are involved in the mode-of-action is largely unknown. In this study, we assessed the effect of BPA on larval brain development of the ascidian Phallusia mammillata. We found that BPA is toxic to P. mammillata embryos in a dose-dependent manner (EC50: 11.8µM; LC50: 21µM). Furthermore, micromolar doses of BPA impaired differentiation of the ascidian pigmented cells, by inhibiting otolith movement within the sensory vesicle. We further show that this phenotype is specific to other two bisphenols (BPE and BPF) over a bisphenyl (2,2 DPP). Because in vertebrates the estrogen-related receptor gamma (ERRγ) can bind bisphenols with high affinity but not bisphenyls, we tested whether the ascidian ERR participates in the neurodevelopmental phenotype induced by BPA. Interestingly, P. mammillata ERR is expressed in the larval brain, adjacent to the differentiating otolith. Furthermore, antagonists of vertebrate ERRs also inhibited the otolith movement but not pigmentation. Together our observations suggest that BPA may affect ascidian otolith differentiation by altering Pm-ERR activity whereas otolith pigmentation defects might be due to the known inhibitory effect of bisphenols on tyrosinase enzymatic activity.

Radial spoke proteins regulate otolith formation during early zebrafish development.

Radial spokes are structurally conserved, macromolecular complexes that are essential for the motility of 9 + 2 motile cilia. In Chlamydomonas species, mutations in radial spoke proteins result in ciliary motility defects. However, little is known about the function of radial spoke proteins during embryonic development. Here, we investigated the role of a novel radial spoke protein, leucine-rich repeat containing protein 23 (Lrrc23), during zebrafish embryonic development. Mutations in lrrc23 resulted in a selective otolith formation defect during early ear development. Similar otolith defects were also present in the radial spoke head 3 homolog ( rsph3) and radial spoke head 4 homolog A ( rsph4a) radial spoke mutants. Notably, the radial spoke protein mutations specifically affected ciliary motility in the otic vesicle (OV), whereas motile cilia in other organs functioned normally. Via high-speed video microscopy, we found that motile cilia formation was stochastic and transient in the OV. Importantly, all the motile cilia in the OV beat circularly, in contrast to the planar beating pattern of typical 9 + 2 motile cilia. We identified the key time frame for motile cilia formation during OV development. Finally, we showed that the functions of radial spoke proteins were conserved between zebrafish and Tetrahymena. Together, our results suggest that radial spoke proteins are essential for ciliary motility in the OV and that radial spoke-regulated OV motile cilia represent a unique type of cilia during early zebrafish embryonic development.-Han, X., Xie, H., Wang, Y., Zhao, C. Radial spoke proteins regulate otolith formation during early zebrafish development.

Otoliths - Accelerometer and seismometer; Implications in Vestibular Evoked Myogenic Potential (VEMP).

Vestibular otolithic organs are recognized as transducers of head acceleration and they function as such up to their corner frequency or undamped natural frequency. It is well recognized that these organs respond to frequencies above their corner frequency up to the 2-3 kHz range (Curthoys et al., 2016). A mechanics model for the transduction of these organs is developed that predicts the response below the undamped natural frequency as an accelerometer and above that frequency as a seismometer. The model is converted to a transfer function using hair cell bundle deflection. Measured threshold acceleration stimuli are used along with threshold deflections for threshold transfer function values. These are compared to model predicted values, both below and above their undamped natural frequency. Threshold deflection values are adjusted to match the model transfer function. The resulting threshold deflection values were well within in measure threshold bundle deflection ranges. Vestibular Evoked Myogenic Potentials (VEMPs) today routinely uses stimulus frequencies of 500 and 1000 Hz, and otoliths have been established incontrovertibly by clinical and neural evidence as the stimulus source. The mechanism for stimulus at these frequencies above the undamped natural frequency of otoliths is presented where otoliths are utilizing a seismometer mode of response for VEMP transduction.

Using otolith microchemistry and shape to assess the habitat value of oil structures for reef fish.

Over 7500 oil and gas structures (e.g. oil platforms) are installed in offshore waters worldwide and many will require decommissioning within the next two decades. The decision to remove such structures or turn them into reefs (i.e. 'rigs-to-reefs') hinges on the habitat value they provide, yet this can rarely be determined because the residency of mobile species is difficult to establish. Here, we test a novel solution to this problem for reef fishes; the use of otolith (earstone) properties to identify oil structures of residence. We compare the otolith microchemistry and otolith shape of a site-attached coral reef fish (Pseudanthias rubrizonatus) among four oil structures (depth 82-135 m, separated by 9.7-84.2 km) on Australia's North West Shelf to determine if populations developed distinct otolith properties during their residency. Microchemical signatures obtained from the otolith edge using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) differed among oil structures, driven by elements Sr, Ba and Mn, and to a lesser extent Mg and Fe. A combination of microchemical data from the otolith edge and elliptical Fourier (shape) descriptors allowed allocation of individuals to their 'home' structure with moderate accuracy (overall allocation accuracy: 63.3%, range: 45.5-78.1%), despite lower allocation accuracies for each otolith property in isolation (microchemistry: 47.5%, otolith shape: 45%). Site-specific microchemical signatures were also stable enough through time to distinguish populations during 3 separate time periods, suggesting that residence histories could be recreated by targeting previous growth zones in the otolith. Our results indicate that reef fish can develop unique otolith properties during their residency on oil structures which may be useful for assessing the habitat value of individual structures. The approach outlined here may also be useful for determining the residency of reef fish on artificial reefs, which would assist productivity assessments of these habitats.

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.

Striola magica. A functional explanation of otolith geometry.

Otolith end organs of vertebrates sense linear accelerations of the head and gravitation. The hair cells on their epithelia are responsible for transduction. In mammals, the striola, parallel to the line where hair cells reverse their polarization, is a narrow region centered on a curve with curvature and torsion. It has been shown that the striolar region is functionally different from the rest, being involved in a phasic vestibular pathway. We propose a mathematical and computational model that explains the necessity of this amazing geometry for the striola to be able to carry out its function. Our hypothesis, related to the biophysics of the hair cells and to the physiology of their afferent neurons, is that striolar afferents collect information from several type I hair cells to detect the jerk in a large domain of acceleration directions. This predicts a mean number of two calyces for afferent neurons, as measured in rodents. The domain of acceleration directions sensed by our striolar model is compatible with the experimental results obtained on monkeys considering all afferents. Therefore, the main result of our study is that phasic and tonic vestibular afferents cover the same geometrical fields, but at different dynamical and frequency domains.

[Light microscopy of statocyst cell elements from Helix lucorum (space experiment aboard the orbital station "MIR")].

Statocyst epithelial lining of terrestrial pulmonary snail Helix lucorum is a spatially arranged structure consisting of 13 cell ensembles. Each ensemble has a sensory cell surrounded by companion cells. The sensory cell on the anterior statocyst pole is star-shaped due to multiple protoplasmatic protrusions on its body. The remaining 12 polygon-shaped cells form 3 tires along the statocyst internal perimeter: anterior, middle or equatorial and posterior. There are 4 cells in each tire. Topography of every sensory cell on the statocyst internal surface was described as well as cell nuclei size and form, nucleoli number and patterns of cytoplasm vacuolization. Space free of sensory cells is occupied by supporting or intercalary cells. Exposure to space microgravity over 40, 43, 102 and 135 days aboard the orbital station MIR affected morphology of the sensory cells. Specifically, this appeared as reductions in cell height and, consequently, extension of the statocyst cavity internal diameter and volume in the space-flown snails.

Otolith end organ projections to auditory neurons in the descending octaval nucleus of the goldfish, Carassius auratus: a confocal analysis.

The distribution of axons from the saccule, lagena, and utricle to descending octaval nucleus neurons that project to the auditory midbrain in the goldfish is reported. We have divided these auditory projection neurons, located in the dorsal portion of the descending octaval nucleus (dDO), into two groups, medial and lateral, each of which contains several neuronal populations based on morphology and location. At most levels of the dDO, there are three medial and three lateral populations; the rostral dDO contains an additional lateral population. The saccule provides input to each of the seven medial and lateral populations but appears to be the exclusive/nearly exclusive source of primary input to the most dorsal cell group of the medial population. Along with the saccule, the lagena and utricle each supply the remaining six medial and lateral populations. Neurons in each of these populations receive input from more than one end organ. One medial and one lateral population include neurons that receive remarkably large contacts from utricular afferents. Overall, the results reveal a more substantial input from the lagena and utricle to the main first-order auditory nucleus in the goldfish than was previously recognized, suggest this nucleus is composed of functionally distinct populations, and relate to functional and evolutionary issues about hearing in early vertebrates.

Dynamics of freely oscillating and coupled hair cell bundles under mechanical deflection.

In vitro, attachment to the overlying membrane was found to affect the resting position of the hair cell bundles of the bullfrog sacculus. To assess the effects of such a deflection on mechanically decoupled hair bundles, comparable offsets were imposed on decoupled spontaneously oscillating bundles. Strong modulation was observed in their dynamic state under deflection, with qualitative changes in the oscillation profile, amplitude, and characteristic frequency of oscillation seen in response to stimulus. Large offsets were found to arrest spontaneous oscillation, with subsequent recovery upon reversal of the stimulus. The dynamic state of the hair bundle displayed hysteresis and a dependence on the direction of the imposed offset. The coupled system of hair bundles, with the overlying membrane left on top of the preparation, also exhibited a dependence on offset position, with an increase in the linear response function observed under deflections in the inhibitory direction.

The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle.

Otoliths are biomineralised structures required for the sensation of gravity, linear acceleration and sound in the zebrafish ear. Otolith precursor particles, initially distributed throughout the otic vesicle lumen, become tethered to the tips of hair cell kinocilia (tether cilia) at the otic vesicle poles, forming two otoliths. We have used high-speed video microscopy to investigate the role of cilia and ciliary motility in otolith formation. In wild-type ears, groups of motile cilia are present at the otic vesicle poles, surrounding the immotile tether cilia. A few motile cilia are also found on the medial wall, but most cilia (92-98%) in the otic vesicle are immotile. In mutants with defective cilia (iguana) or ciliary motility (lrrc50), otoliths are frequently ectopic, untethered or fused. Nevertheless, neither cilia nor ciliary motility are absolutely required for otolith tethering: a mutant that lacks cilia completely (MZovl) is still capable of tethering otoliths at the otic vesicle poles. In embryos with attenuated Notch signalling [mindbomb mutant or Su(H) morphant], supernumerary hair cells develop and otolith precursor particles bind to the tips of all kinocilia, or bind directly to the hair cells' apical surface if cilia are absent [MZovl injected with a Su(H)1+2 morpholino]. However, if the first hair cells are missing (atoh1b morphant), otolith formation is severely disrupted and delayed. Our data support a model in which hair cells produce an otolith precursor-binding factor, normally localised to tether cell kinocilia. We also show that embryonic movement plays a minor role in the formation of normal otoliths.

Differences between otolith- and semicircular canal-activated neural circuitry in the vestibular system.

In the last two decades, we have focused on establishing a reliable technique for focal stimulation of vestibular receptors to evaluate neural connectivity. Here, we summarize the vestibular-related neuronal circuits for the vestibulo-ocular reflex, vestibulocollic reflex, and vestibulospinal reflex arcs. The focal stimulating technique also uncovered some hidden neural mechanisms. In the otolith system, we identified two hidden neural mechanisms that enhance otolith receptor sensitivity. The first is commissural inhibition, which boosts sensitivity by incorporating inputs from bilateral otolith receptors, the existence of which was in contradiction to the classical understanding of the otolith system but was observed in the utricular system. The second mechanism, cross-striolar inhibition, intensifies the sensitivity of inputs from both sides of receptive cells across the striola in a single otolith sensor. This was an entirely novel finding and is typically observed in the saccular system. We discuss the possible functional meaning of commissural and cross-striolar inhibition. Finally, our focal stimulating technique was applied to elucidate the different constructions of axonal projections from each vestibular receptor to the spinal cord. We also discuss the possible function of the unique neural connectivity observed in each vestibular receptor system.

Origin of inner ear hair cells: morphological and functional differentiation from ciliary cells into hair cells in zebrafish inner ear.

Auditory and vestibular functions in vertebrates depend on the transduction of sound vibration or head acceleration into electrical responses in inner ear hair cells. Mechanoelectrical transduction occurs at the tip of stereocilia, which are polarized to form an orientational arrangement that determines directional sensitivity. It remains to be clarified when and how premature hair cells acquire their specialized structure and function in living animals. The developmental origin of inner ear hair cells has been studied in vivo in zebrafish embryos. Tether cells, a small number of ciliated cells associated with an "ear stone" (or otolith) in the embryonic zebrafish inner ear, are believed to be precocious hair cells. However, whether or not tether cells acquire hair bundles and mechanosensitivity remains unknown. In the present study, we investigated the morphological and functional development of tether cells. Immunohistochemical examination revealed that stereocilia appeared on the tether cell apex in a polarized arrangement at 22 h postfertilization (hpf). Labeling with FM1-43, a marker of functional mechanotransduction channels, and the in vivo electrophysiological recording of mechanotransducer responses in the developing inner ear demonstrated that tether cells acquired direction-selective mechanosensitivity at 23 hpf. These results revealed that tether cells begin to function as hair cells within an hour after the appearance of a polarized array of stereociliary bundles. Thus, the ciliary cells morphologically and functionally differentiate into the first sensory hair cells in the inner ear of the zebrafish.

Identification of starmaker-like in medaka as a putative target gene of Pax2 in the otic vesicle.

Otoliths in bony fishes are involved in the function of the ear in the senses of balance and hearing. In a large-scale random in situ hybridization screen of genes expressed in the medaka developing ear, we identified starmaker-like (stm-l) gene, a novel homologue of zebrafish starmaker and human dentine sialo-phosphoprotein (dspp) gene. Despite the absence of sequence similarity between these genes, here we describe their similar genomic structure and expression patterns hinting for a conserved function. In medaka fry, stm-l is expressed in various organs such as otoliths, teeth, gills, and kidney. Additionally, our results provide evidence that stm-l is a putative downstream target gene of Pax2 transcription factor and Pax2 itself has a promoting function in otolith formation.

Altered chondrocyte differentiation and extracellular matrix homeostasis in a zebrafish model for mucolipidosis II.

Mucolipidosis II (ML-II) is a pediatric disorder caused by defects in the biosynthesis of mannose 6-phosphate, the carbohydrate recognition signal responsible for targeting certain acid hydrolases to lysosomes. The mechanisms underlying the developmental defects of ML-II are largely unknown due in part to the lack of suitable animal models. To overcome these limitations, we developed a model for ML-II in zebrafish by inhibiting the expression of N-acetylglucosamine-1-phosphotransferase, the enzyme that initiates mannose 6-phosphate biosynthesis. Morphant embryos manifest craniofacial defects, impaired motility, and abnormal otolith and pectoral fin development. Decreased mannose phosphorylation of several lysosomal glycosidases was observed in morphant lysates, consistent with the reduction in phosphotransferase activity. Investigation of the craniofacial defects in the morphants uncovered striking changes in the timing and localization of both type II collagen and Sox9 expression, suggestive of an accelerated chondrocyte differentiation program. Accumulation of type II collagen was also noted within misshapen cartilage elements at later stages of development. Furthermore, we observed abnormal matrix formation and calcium deposition in morphant otoliths. Collectively, these data provide new insight into the developmental pathology of ML-II and suggest that altered production and/or homeostasis of extracellular matrix proteins are integral to the disease process. These findings highlight the potential of the zebrafish system in studying lysosomal disease pathogenesis.

Adaptation of orientation of central otolith-only neurons.

Otolith-only neurons were recorded extracellularly in the vestibular nuclei before and after cynomolgus monkeys were held on-side for up to 3 hr. The aim was to determine whether the polarization vectors of these neurons reorient toward the spatial vertical as do canal-otolith convergent neurons. Otolith input was characterized by tilting the animal 30 degrees from the upright position while positioning the head in different directions in yaw. This determined the response vector orientation (RVO), that is, the projection of the otolith polarization vector onto the head horizontal plane. Changes in the RVO of otolith-only neurons ranged from 2 degrees -16 degrees , which was on average considerably less than the changes previously noted in canal-otolith convergent vestibulo-only (VO) and vestibular plus saccade (VPS) neurons, which ranged up to 109 degrees. Some of the otolith-only neurons had marked sensitivity changes. These findings suggest that otolith-only neurons tend to maintain a head-fixed orientation during prolonged head tilts relative to gravity. In contrast, canal-convergent VO and VPS neurons optimize their response vector orientation to gravity when the head is oriented for prolonged periods.

Plasma membrane calcium ATPase required for semicircular canal formation and otolith growth in the zebrafish inner ear.

Fish otoliths consist of >90% calcium carbonate, the accretion of which depends on acellular endolymph. This study confirms the presence of plasma membrane calcium ATPase 1a isoform (Atp2b1a) in the auditory and vestibular system of a teleost fish. As shown by in situ hybridization, zebrafish atp2b1a is expressed mainly in larval otic placode and lateral-line neuromast as well as in the hair cells within the adult zebrafish inner ear chamber. Zebrafish atp2b1a knockdown by antisense morpholinos reduced the number of hair cells and produced malformation of semicircular canals and smaller otoliths. These defects coincide with unbalanced body orientation. The formation of smaller otoliths in atp2b1a morphants may stem from an impairment of calcium supply in the endolymph. However, otolith formation persists in most morphants, suggesting that other zebrafish Atp2b isoforms or paracellular pathways may also transport calcium into the endolymph. These results suggest that Atp2b1a plays an important role for normal development of the auditory and vestibular system as well as calcium transport in the inner ear of zebrafish.

The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear.

In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized 'ear stones' (or otoliths). Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

Effect of initial size on daily growth and survival in freshwater Chondrostoma nasus larvae: a field survey.

Effects of initial size on the growth and survival of a freshwater fish, Chondrostoma nasus, were tested in a field survey, where individually tagged larvae were introduced into a potential nursery habitat. Characteristics of individual daily growth trajectories were utilized as a basis to explain growth, as well as survival patterns, in relation to ontogeny. Initial size only affected growth rates during the larval phase. Survival patterns could not be explained solely based on size-selective mortality processes because prey-predator interactions played a major role as well. This is confirmed by the Lande-Arnold selection model because directional, as well as stabilizing gradients, showed significant values. Thus, for the 0+ year freshwater fish, inherited size-specific effects were a significant advantage for growth performance and survival in early ontogeny. As fish grew older, however, other effects such as compensatory growth and prey-predator interactions apparently gained in importance.

State of the art: 3T imaging of the membranous labyrinth.

This article reviews the anatomy of the membranous labyrinth and demonstrates the ability of high-resolution MR imaging at 3T to visualize the neurosensory epithelium by using the latest fast spin-echo techniques.