Lateral Crista Research Articles

Maturation of type I and type II rat vestibular hair cells in vivo and in vitro.

Vestibular sensory epithelia contain type I and type II sensory hair cells (HCI and HCII). Recent studies have revealed molecular markers for the identification of these cells, but the precise composition of each vestibular epithelium (saccule, utricle, lateral crista, anterior crista, posterior crista) and their postnatal maturation have not been described in detail. Moreover, in vitro methods to study this maturation are not well developed. We obtained total HCI and HCII counts in adult rats and studied the maturation of the epithelia from birth (P0) to postnatal day 28 (P28). Adult vestibular epithelia hair cells were found to comprise ∼65% HCI expressing osteopontin and PMCA2, ∼30% HCII expressing calretinin, and ∼4% HCII expressing SOX2 but neither osteopontin nor calretinin. At birth, immature HCs express both osteopontin and calretinin. P28 epithelia showed an almost adult-like composition but still contained 1.3% of immature HCs. In addition, we obtained free-floating 3D cultures of the epithelia at P1, which formed a fluid-filled cyst, and studied their survival and maturation in vitro up to day 28 (28 DIV). These cultures showed good HC resiliency and maturation. Using an enriched medium for the initial 4 days, a HCI/calretinin+-HCII ratio close to the in vivo ratio was obtained. These cultures are suitable to study HC maturation and mature HCs in pharmacological, toxicological and molecular research.

Tmc Reliance Is Biased by the Hair Cell Subtype and Position Within the Ear.

Hair cells are heterogenous, enabling varied roles in sensory systems. An emerging hypothesis is that the transmembrane channel-like (Tmc) proteins of the hair cell’s mechanotransduction apparatus vary within and between organs to permit encoding of different mechanical stimuli. Five anatomical variables that may coincide with different Tmc use by a hair cell within the ear are the containing organ, cell morphology, cell position within an organ, axis of best sensitivity for the cell, and the hair bundle’s orientation within this axis. Here, we test this hypothesis in the organs of the zebrafish ear using a suite of genetic mutations. Transgenesis and quantitative measurements demonstrate two morphologically distinct hair cell types in the central thickness of a vestibular organ, the lateral crista: short and tall. In contrast to what has been observed, we find that tall hair cells that lack Tmc1 generally have substantial reductions in mechanosensitivity. In short hair cells that lack Tmc2 isoforms, mechanotransduction is largely abated. However, hair cell Tmc dependencies are not absolute, and an exceptional class of short hair cell that depends on Tmc1 is present, termed a short hair cell erratic. To further test anatomical variables that may influence Tmc use, we map Tmc1 function in the saccule of mutant larvae that depend just on this Tmc protein to hear. We demonstrate that hair cells that use Tmc1 are found in the posterior region of the saccule, within a single axis of best sensitivity, and hair bundles with opposite orientations retain function. Overall, we determine that Tmc reliance in the ear is dependent on the organ, subtype of hair cell, position within the ear, and axis of best sensitivity.

Disruption of tmc1/2a/2b Genes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells.

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibular-induced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.SIGNIFICANCE STATEMENT We assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants revealed the presence of two distinct cell types, an upper layer of teardrop-shaped cells that rely on Tmc2a, and a lower layer of gourd-shaped cells that rely on Tmc1/2b.

Gene duplication and functional divergence of the zebrafish otospiralin genes.

Otospiralin (OTOSP) is a small protein of unknown function, expressed in fibrocytes of the inner ear and required for normal cochlear auditory function. Despite its conservation from fish to mammals, expression of otospiralin was only investigated in mammals. Here, we report for the first time the expression profile of OTOS orthologous genes in zebrafish (Danio rerio): otospiralin and si:ch73-23l24.1 (designated otospiralin-like). In situ hybridization analyses in zebrafish embryos showed a specific expression of otospiralin-like in notochord (from 14 to 48hpf) and similar expression patterns for otospiralin and otospiralin-like in gut (from 72 to 120hpf), swim bladder (from 96 to 120hpf) and inner ear (at 120hpf). Morpholino knockdown of otospiralin and otospiralin-like showed no strong change of the body structure of the embryos at 5dpf and the inner ear was normally formed. Nevertheless, knockdown embryos showed a reduced number of kinocilia in the lateral crista, indicating that these genes play an important role in kinocilium formation. RT-qPCR revealed that otospiralin is highly expressed in adult zebrafish inner ear comparing to the others analyzed tissues as previously shown for mice. Interestingly, otospiralin-like was not detected in the inner ear which suggests that otospiralin have a more important function in hearing than otospiralin-like. Phylogenetic analysis of otospiralin proteins in vertebrates indicated the presence of two subgroups and supported the functional divergence observed in zebrafish for otospiralin and otospiralin-like genes. This study offers the first insight into the expression of otospiralin and otospiralin-like in zebrafish. Expression data point to an important role for otospiralin in zebrafish hearing and a specific role for otospiralin-like in notochord vacuolization.

Osteochondritis Dissecans of the Humeral Trochlea: Characterization of a Rare Disorder Based on 28 Cases

Background: Osteochondritis dissecans (OCD) of the humeral trochlea is very rare. It may cause pain, mechanical symptoms, and loss of elbow motion, typically in the adolescent athlete. However, little published information is available regarding this condition. Purpose: To describe the clinical presentation, radiographic features, and prognosis of trochlear OCD. Study Design: Case series; Level of evidence, 4. Methods: Over a 10-year period, 28 patients presented to a tertiary pediatric hospital with trochlear OCD. Medical records and imaging were analyzed to characterize presentation, lesions appearances, and outcomes. Results: Mean ± SD age at presentation was 13.4 ± 1.6 years, and 13 of the 28 patients were male. The most common presenting symptom was pain (93%), followed by crepitus (54%). Evidence of trochlear OCD could be seen on initial radiographs in 94% of cases but was commonly missed. Coexisting capitellar OCD lesions were the most common associated abnormalities seen on magnetic resonance imaging (21%). Investigators noted 2 predominant patterns: “typical” trochlear OCD lesions (89%) were located on the lateral crista of the trochlea, 3.1 ± 4.4 mm lateral to the apex of the trochlear groove. This location corresponded to the medial tip of the capitellar epiphyseal ossification center and was not actually on the trochlear ossification center. “Atypical” trochlear OCD lesions (11%) were located more posteromedially. Trochlear OCD lesions in 4 elbows were managed surgically, while the remainder were managed nonoperatively. At mean ± SD follow-up of 13 ± 8 months, 12 patients (43%) were asymptomatic. A further 5 patients had ongoing crepitus but no pain (18%), and 4 patients (14%) underwent surgical treatment for their trochlear OCD (osteochondral fixation, n = 1; drilling/curettage, n = 3); 3 of the 4 patients experienced some improvement in pain. Conclusion: Although rare, trochlear OCD can cause considerable elbow problems. Clinicians should be aware of this differential diagnosis. Plain radiographs should be carefully scrutinized for subtle signs of trochlear OCD, particularly in the repetitive or overhead athlete with elbow pain. Although most patients’ symptoms will improve with activity modification, some may require surgery.

Jag1b is essential for patterning inner ear sensory cristae by regulating anterior morphogenetic tissue separation and preventing posterior cell death.

The sensory patches of the vertebrate inner ear, which contain hair cells and supporting cells, are essential for hearing and balance functions. How the stereotypically organized sensory patches are formed remains to be determined. In this study, we isolated a zebrafish mutant in which the jag1b gene is disrupted by an EGFP insertion. Loss of Jag1b causes cell death in the developing posterior crista and results in downregulation of fgf10a in the posterior prosensory cells. Inhibition of FGFR activity in wild-type embryos also causes loss of the posterior crista, suggesting that Fgf10a mediates Jag1b activity. By contrast, in the anterior prosensory domain, Jag1b regulates separation of a single morphogenetic field into anterior and lateral cristae by flattening cells destined to form a nonsensory epithelium between the two cristae. MAPK activation in the nonsensory epithelium precursors is required for the separation. In the jag1b mutant, MAPK activation and cell flattening are extended to anterior crista primordia, causing loss of anterior crista. More importantly, inhibition of MAPK activity, which blocks the differentiation of nonsensory epithelial cells, generated a fused large crista and extra hair cells. Thus, Jag1b uses two distinct mechanisms to form three sensory cristae in zebrafish.

Fgf10 expression patterns in the developing chick inner ear

The inner ear is a complex three-dimensional sensorial structure with auditory and vestibular functions. It originates from the otic placode, which invaginates, forming the otic vesicle; the latter gives rise to neurosensory and nonsensory elements of the adult membranous labyrinth. A hypothesis based on descriptive and experimental evidence suggests that the acquisition of discrete sensory patches during evolution of this primordium may be related to subdivision of an early pansensory domain. In order to gain insight into this developmental mechanism, we carried out a detailed analysis of the spatial and temporal expression pattern of the gene Fgf10, by comparing different markers of otic patterning and hair cell differentiation. Fgf10 expression labels a sensory-competent domain included in a Serrate-positive territory from which most of the sensory epithelia arise. Our data show that Fgf10 transcripts are present initially in a narrow ventromedial band of the rudimentary otocyst, extending between its rostral and caudal poles. During development, this Fgf10-expressing area splits repetitively into several separate subareas, creating six of the eight sensory organs present in birds. Only the lateral crista and the macula neglecta were initially Fgf10 negative, although they activated Fgf10 expression after their specification as sensory elements. These results allowed us to determine a timetable of sensory specification in the developing chick inner ear. The comparison of the expression pattern of Fgf10 with those of other markers of sensory differentiation contributes to our understanding of the mechanism by which vertebrate inner ear prosensory domains have arisen during evolution.

Inner ear dysfunction in caspase-3 deficient mice

BackgroundCaspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3-/-) mouse strain.ResultsAverage ABR thresholds of Casp3-/- mice were significantly elevated (P < 0.05) compared to Casp3+/- mice and Casp3+/+ mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P < 0.05) in Casp3-/- mice, whereas Casp3+/- and Casp3+/+ mice showed normal and comparable values to each other. Casp3-/- mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3-/- mice had minimal response to any of the stimuli tested, whereas Casp3+/- mice had an intermediate response compared to Casp3+/+ mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3-/- mice whereas the Casp3+/- and Casp3+/+ mice had normal hair cell numbers.ConclusionsThese results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.

The inner ear phenotype of Volchok (Vlk): An ENU-induced mouse model for CHARGE syndrome

Objective: The Volchok (Vlk) mouse originated as a part of a large-scale ENU program on a C3HeB/FeJ background. The mice present a dominant pattern of inheritance refl ected mainly by circling activity, which led us to search for the causative mutation underlying the vestibular phenotype. Study design: Linkage analysis and subsequent sequencing were used to narrow down the genomic region and uncover the mutation, respectively. Behavioral analysis and examination of the inner ear phenotype was performed using the modifi ed SHIRPA protocol, auditory brainstem response (ABR), immunofl uorescence, light microscopy, scanning electron microscopy (SEM), paint-fi ll and neurofi lament staining techniques. Results: A nonsense mutation was discovered in the Chd7 gene responsible for CHARGE syndrome in humans, which leads to a decrease in Chd7 expression in the nuclei of the vestibular and cochlear cells and structural abnormalities in the inner ear. A mild hearing loss was found in the mutant mice. The malformations detected include a truncated lateral semicircular canal and smaller lateral crista, a decrease in size or absence of the round window in the cochlea and lack of innervations to the posterior crista. Conclusion: Vlk joins a series of previously characterized mice bearing a Chd7 mutation, linking abnormalities found both in ENU-induced mutants and a knock-out mouse model. Combining the information from multiple Chd7 alleles provides a more comprehensive understanding of the mechanisms underlying this human disease.

Fusion as an Evolutionary Principle of the Vertebrate Labyrinth

This study was undertaken to demonstrate changes in the innervation of vestibular and auditory sense organs with the evolutionary ascent of the vertebrate labyrinth. Dissected labyrinths and their nerve supply prepared by the Sudan black B technique of Rasmussen were examined and photographed with a Canon A100 camera interfaced with a Zeiss operating microscope. In lizards and alligators, the utricular sense organ is represented by 2 small maculae, each with a separate nerve branching off the ampullary nerves to the anterior and lateral canal cristae. These 2 maculae fuse into a bilobed macula with ascent in frogs and pigeons, eventually becoming a single large macula with its nerve supply from the superior vestibular division in guinea pigs, cats, lions, monkeys, and humans. Along with these changes, there is a fusion of the lateral and anterior canal ampullary nerves and of the bifurcating branches of the vertical canal ampullary nerves. The saccular macula is single, but receives a dual innervation from the superior division (anterior ramus) and the inferior division (posterior ramus) of the eighth nerve in alligators, pigeons, guinea pigs, cats, lions, monkeys, and humans. The main innervation is usually from the inferior division; however, saccular innervation is from the inferior division in lizards and from the superior division in frogs. The auditory sense organ is represented by a curved tube with a low-frequency receptor (lagena) at its distal end in lizards, alligators, and pigeons. In mammals, in which there is a coiled cochlea with a variable number of turns, low frequencies are recorded at the apical turn. This configuration may represent fusion of the lagena into the apical end of the auditory sense organ. Fusion of sense organs and of their nerve supply appears to be an evolutionary principle in the vertebrate labyrinth.

Foxg1 is required for proper separation and formation of sensory cristae during inner ear development

The vestibular portion of the inner ear, the three semicircular canals and their sensory cristae, is responsible for detecting angular head movements. It was proposed that sensory cristae induce formation of their non-sensory components, the semicircular canals. Here, we analyzed the inner ears of Foxg1(-/-) mouse mutants, which display vestibular defects that are in conflict with the above model. In Foxg1(-/-) ears, the lateral canal is present without the lateral ampulla, which houses the lateral crista. Our gene expression analyses indicate that at the time when canal specification is thought to occur, the prospective lateral crista is present, which could have induced lateral canal formation prior to its demise. Our genetic fate-mapping analyses indicate an improper separation between anterior and lateral cristae in Foxg1(-/-) mutants. Our data further suggest that a function of Foxg1 in the inner ear is to restrict sensory fate.

Functional and structural changes in the chinchilla cochlea and vestibular system following round window application of carboplatin

OBJECTIVE: In chinchillas, moderate doses of carboplatin administered systemically selectively destroy inner hair cells and type I vestibular hair cells; however, it is unclear whether this unique damage pattern persists if carboplatin is applied directly to the cochlea, how quickly the damage develops and what cell death pathways are involved. STUDY DESIGN: To address these questions, carboplatin (5 mg/ml, 50 µl) was applied to the round window. RESULTS: Carboplatin caused a rapid decline in distortion product otoacoustic emissions, significantly increased compound action potential thresholds and caused massive inner hair cell loss and less severe outer hair cell loss. Hair cell loss was initially more severe in the base than the apex of the cochlea, but by 28 days post-treatment most cochlear hair cells were missing and hair cell density in the utricle, saccule and lateral crista was greatly reduced. At one day post-treatment, many hair cell nuclei were condensed or fragmented indicative of apoptosis, and expressed initiator caspase-8 and executioner caspase-3, but not initiator caspase-9. Carboplatin-treated animals circled towards the treated ear and during the swim test rolled towards the treated ear. CONCLUSION: These results indicate that local application of carboplatin causes loss of hair cells that begins near the base of the cochlea and spreads towards the apex with increasing survival time. Hair cell loss is initiated by caspase-8 followed by executioner caspase-3.

Acute Osteochondral Shear Fracture of the Capitellum in a Twelve-Year-Old Patient

The exact prevalence of radiocapitellar joint injuries in adolescents is unknown. The most common elbow disorders in young athletes include overuse injuries, such as osteochondritis dissecans and Panner disease. Fractures of the capitellum and radial neck can also occur in this age group as a result of direct trauma. Marion and Faysse reported only one capitellar fracture in a series of 2,000 elbow fractures in children1. Two fracture patterns of the capitellum have traditionally been described. The first type, a coronal shear fracture often referred to as the Hahn-Steinthal fracture, usually contains a large portion of cancellous bone from the lateral humeral condyle and may include the lateral crista of the trochlea as well2,3. The second type, a pure articular fracture called the Kocher-Lorenz fracture, has little or no subchondral bone attached to the fragment4,5 and is rarely seen in children6. Since the publication of the study by Marion and Faysse1, several cases of capitellar fractures have been described in older adolescents, whose bone more closely resembles that of an adult7-9; however, a pure articular fracture of the capitellum has rarely, if ever, been reported in children who were less than twelve years of age6. There have been two reports describing anterior or articular sleeve fractures of the lateral condyle10,11, and both of these injuries occurred in patients who were less than twelve years of age. Each of these injuries involved a substantial portion of the articular surface of the capitellum, although neither was a pure capitellar injury. The fracture fragment in these patients was composed mainly of the articular surface from the capitellum, in addition to nonarticular epicondylar and metaphyseal portions of the lateral condyle. The …

The interrelationships of Devonian lungfishes (Sarcopterygii: Dipnoi) as inferred from neurocranial evidence and new data from the genus Soederberghia Lehman, 1959

New data are presented on the neurocranial complex (endocranium plus intimately associated bones of the palate) of the Late Devonian (Famennian) ‘rhynchodipterid’ lungfish Soederberghia groenlandica from the Aina Dal Formation (Celsius Bjerg Group) of East Greenland. Only the otic and occipital regions of the braincase are ossified in Soederberghia. The neurocranium of this genus shares a series of derived features with ‘Griphognathus’whitei, including a cranial centrum, fenestrate lateral cristae, and gutters on the ventral surface of the parasphenoid interpreted as accommodating the lateral dorsal aortae. The interrelationships of early lungfishes have been the subject of considerable disagreement. New data from Soederberghia are coupled with a data set focused on the character-rich neurocranial complex in order to examine the systematic utility of this underexploited morphological system. Different methods of phylogenetic inference (maximum parsimony, Bayesian) return broadly consistent results. The Early to Middle Devonian forms Dipnorhynchus, Stomiahykus and Uranolophus are placed among the most basal of lungfishes. ‘Holodontids’ plus ‘rhynchodipterids’ (comprising Griphognathus and Soederberghia) occupy an apical position, and are separated from the earliest lungfishes by a paraphyletic assemblage of taxa generally identified as ‘chirodipterids’ and ‘dipterids.’ This finds broad agreement with the results of previous cladistic studies focused on non-neurocranial data sets, but diverges from functional-adaptive scenarios that posit three lineages of early lungfishes based on aspects of the dentition. As currently defined, both ‘chirodipterids’ and the genus Chirodipterus are heterogeneous assemblages; this analysis fails to find support for the monophyly of either. While this study indicates that Griphognathus is probably paraphyletic, it nevertheless supports a close relationship between the nominal species of this genus and Soederberghia. © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society, 2007, 151, 115–171.

Histomorphologische Untersuchung bei experimentell induzierter Kanalolithiasis

Free otoconia in the posterior semicircular canal (pSCC) are regarded as the main reason for benign paroxysmal positional vertigo. We investigated the distribution of otoconia in the membranous labyrinth of guinea pigs (n = 34) after a defined rotatory/angular kinetic acceleration. The angular kinetic energy of the rotatory experiment produced a dislocation of the otoconia and sometimes of the complete gelatinous otolithic membrane of the utricular and saccular macula. The otoconia could be observed in all three semicircular canals but predominated in the dark cell areas of the utricular side of the lateral SCC. A total obstruction of the semicircular canal could never be seen. Close to the lateral crista, perilymphatic hemorrhage could frequently be observed. The saccular otoconia were located in the amalgamation between endolymphatic membrane and saccule. The results of this histological study can form the basis for different theories about the genesis of canalolithiasis and cupulolithiasis and the vertigo which accompanies them.

Semiquantitative Vestibulotoxicity to Subablative and Ablative Treated Gerbil Cristae Using Gentamicin or Streptomycin

Semiquantitative histological evaluation was performed on inner ears from Mongolian gerbils to study vestibular damages using ototoxic drugs. Comparisons were made between animals receiving five daily injections of gentamicin/gelfoam slurry, streptomycin/gelfoam slurry, saline/gelfoam slurry and noninjected controls. For gentamicin treated ears, when percent decrease of nuclei in sensory cell layer was compared, the damage to the sensory cells was significantly more severe in the posterior crista than in the superior and lateral crista from two of three gerbils. For streptomycin treated ears, when percent decrease of nuclei in sensory cell layer was compared, the damage to the posterior crista was similar to superior and lateral crista. These results indicate that our models are useful for detecting different vestibulotoxicity among three cristae.

Differential expression of Na,K-ATPase alpha and beta subunit genes in the developing zebrafish inner ear.

We have used whole-mount in situ hybridization to analyze Na,K-ATPase alpha and beta subunit gene expression in the developing zebrafish ear. Four alpha1-like (alpha1a.1, alpha1a.2, alpha1a.4, and alpha1a.5) and two beta (beta1a and beta2b) subunit genes are expressed in ear beginning at mid-somitogenesis. Each gene exhibits a distinct spatial and temporal expression pattern. The alpha1a.1 gene was ubiquitously expressed in the otic epithelium from mid-somitogenesis to 24 hr postfertilization (hpf). Expression of this gene was gradually reduced and by 48 hpf, alpha1a.1 transcripts were no longer detectable in the ear. The alpha1a.2 and alpha1a.5 genes were expressed in regions that correspond to the anterior macula, lateral crista, and semicircular canal projections up to 48 hpf. At later stages, expression of these genes was limited to cells in the dorsolateral septum and semicircular canal projections. alpha1a.4 and beta1a transcripts were ubiquitously expressed during ear development and were present in most otic tissues at 5 days postfertilization (dpf). Expression of the beta2b gene, on the other hand, was restricted to subsets of cells that form sensory epithelia. These results strongly suggest different functional roles for individual Na,K-ATPase genes in zebrafish ear development. Na,K-ATPase genes are likely to represent useful markers for the analysis of zebrafish otogenesis.

Diversification of brain and sense organ morphology in Antarctic dragonfishes (Perciformes: Notothenioidei: Bathydraconidae).

In the subzero shelf waters of Antarctica, fishes of the perciform suborder Notothenioidei dominate the fish fauna and constitute an adaptive radiation and a species flock. The 16 species of dragonfishes of the family Bathydraconidae live from surface waters to nearly 3,000 m and have the greatest overall depth range among notothenioid families. We examined the anatomy and histology of the brain, retina, and cephalic lateral line system of nine bathydraconid species representing 8 of the 11 known genera. We evaluate these data against a cladogram identifying three clades in the family. We provide a detailed drawing of the brain and cranial nerves of Gymnodraco acuticeps and Akarotaxis nudiceps. Bathydraconid brain morphology falls into two categories. Brains of most species are similar to those of generalized perciforms and some basal notothenioids (Class I). However, brains of deep-living bathydraconids (members of the tribe Bathydraconini minus Prionodraco) have a reduced telencephalon and tectum that renders the neural axis visible - the stalked brain morphology (Class II). All bathydraconids have duplex (rod and cone) retinae but there is considerable interspecific variation in the ratio of cones:rods and in the number of cells in the internal nuclear layer. Retinal histology reflects habitat depth but is not tightly coupled to phylogeny. Although the deep-living species of Bathydraconini have rod-dominated retinae, the retinae of some sister species are photopic. An expanded cephalic lateral line system is also characteristic of all members of the Bathydraconini as exemplified by Akarotaxis. This morphology includes large lateral line pores, wide membranous canals, hypertrophied canal neuromasts, and large anterodorsal lateral line nerves, eminentia granulares, and crista cerebellares. The saccular otoliths are also enlarged in members of this tribe. Neural diversification among bathydraconids on the Antarctic shelf has not involved the evolution of sensory specialists. Brain and sense organ morphologies do not approach the specialized condition seen in primary deep-sea fishes or even that of some secondary deep-sea fishes including sympatric non-notothenioids such as liparids (snailfishes) and muraenolepidids (eel cods). The brains and sense organs of bathydraconids, including the deep-living species, reflect their heritage as perciform shorefishes.

Expression of Prox1 defines regions of the avian otocyst that give rise to sensory or neural cells.

The simple primordium of the inner ear (otocyst) differentiates into many cell types, including sensory neurons and hair cells. We examined expression of the divergent homeobox transcription factor, cProx1, during otocyst development in chickens. Nuclear cProx1 protein is not evident in the otic placode but emerges in the otic cup by stage 12. At stage 16, cProx1-positive nuclei are scattered continuously throughout the neuroepithelium, from anteroventral to posteromedial. These labeled cells are neural precursors; they express betaIII-tubulin and migrate to the cochleovestibular ganglion between stages 13 and 21. By stage 18, two areas develop a dense pattern of cProx1 expression in which every nucleus is labeled. These areas emerge at the anterior and posterior extremes of the band of scattered cProx1 expression and express the sensory markers cSerrate1 and Cath1 by stage 23. Four discrete patches of dense cProx1 expression appear by stage 23 that correspond to the future superior crista, lateral crista, saccular macula, and posterior crista, as confirmed by immunolabeling for hair cell antigen (HCA) by stage 29. The remaining sensory epithelia display a dense pattern of cProx1 expression and label for HCA by stage 29. In the basilar papilla, nuclear cProx1 expression is down-regulated in most hair cells by stage 37 and in many supporting cells by stage 40. Our findings show that regions of the otocyst that give rise to neurons or hair cells are distinguished by their relative density of cProx1-positive nuclei, and suggest a role for cProx1 in the genesis of these cell types.

The cell adhesion molecule BEN defines a prosensory patch in the developing avian otocyst.

The distribution of the cell adhesion molecule BEN in the developing chick inner ear is described. BEN is first detected in the otic placode at stage 11. As the placode begins to invaginate, BEN becomes concentrated in a ventromedial region extending from the anterior to the posterior end of the otic pit. BEN expression levels increase in this region as the pit closes to form the otocyst, and distinct boundaries become defined along the dorsal and ventral edges of the ventromedial band of BEN expression. BEN expression also becomes concentrated dorsally within the otic epithelium as the pit closes and is observed in the condensing otic ganglion. By stage 22, the ventromedial band of BEN expression splits into two distinct regions, a small caudal patch within which the posterior crista will develop, and a larger anterior patch. By stage 26, this larger anterior patch of cells expressing BEN becomes subdivided into five separate areas corresponding to the regions within which the anterior crista, the lateral crista, the utricle, the saccule, and both the basilar papilla and lagenar macula form. Hair cells only develop within these regions defined by BEN distribution. The data suggest that the ventromedial patch of BEN expression observed from stage 11 onwards defines a single sensory competent zone from which all sensory organs of the inner ear develop. BEN immunoreactivity in the inner ear declines after stage 38. In response to noise exposure, upregulation of BEN expression is mainly detected in regions of the posthatch papilla where the damage is severe and regenerating hair cells are not observed. The regenerating hair and supporting cells do not express BEN, highlighting a molecular difference between the processes of development and regeneration.