The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line.

To understand the molecular basis of sensory organ development and disease, we have cloned and characterized the zebrafish mutation dog-eared (dog) that is defective in formation of the inner ear and lateral line sensory systems. The dog locus encodes the eyes absent-1 (eya1) gene and single point mutations were found in three independent dog alleles, each prematurely truncating the expressed protein within the Eya domain. Moreover, morpholino-mediated knockdown of eya1 gene function phenocopies the dog-eared mutation. In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts. Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in dog embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Importantly, mutation of the EYA1 or EYA4 gene causes hereditary syndromic deafness in humans. Determination of eya gene function during zebrafish organogenesis will facilitate understanding the molecular etiology of human vestibular and hearing disorders.

Cochlear development: hair cells don their wigs and get wired.

PURPOSE OF REVIEW: Hair cells and spiral ganglion neurons form functional pairings in the cochlea that transduce the mechanical energy of sound into signals that are carried to the brainstem. Mutations of genes affecting the development and maintenance of these two cell populations cause deafness in humans and other animals. This review highlights recent findings regarding the development of hair cell stereocilia and spiral ganglion neurons in the cochlea. RECENT FINDINGS: Genes underlying Usher syndrome 1A have shed light on possible molecular participants in the development and structure of the hair cell stereocilia. Analysis of deaf mouse mutants have uncovered genes involved in stereocilia elongation and the orientation of the stereociliary bundles. Studies on the regulation of spiral ganglion neuronal survival and guidance suggest that the timing of expression of specific growth factors along the cochlear spiral is involved in maintaining the divergence of vestibular and cochlear nerve fibers. SUMMARY: Examining human and mouse genes affecting hearing has not only provided insight into causes of human deafness, but has also opened a window into how stereociliary bundles are constructed and spiral ganglion neurons are preserved and guided during development. Synthesis of information from diverse lines of research pinpoints genes for screening or repair in the genetic medicine of the future and dramatizes the intimate relationship between strict adherence to complex developmental programs and hearing. In addition, future improvements in the efficacy of cochlear implants may depend on the preservation and manipulation of adult spiral ganglion neurons. Developmental mechanisms promise to yield insight into possible interventions to redirect or reconnect spiral ganglion neurons in damaged cochlea.

Differential impact of hypergravity on maturating innervation in vestibular epithelia during rat development.

Over the past decades, the new opportunity of space flights has revealed the importance of gravity as a mechanical constraint for terrestrial organisms as well as its influence on the somatosensory system. The lack of gravitational reference in orbital flight induces changes in equilibrium, with major modifications involving neuromorphological and physiological adaptations. However, few data have illustrated the putative effect of gravity on sensory vestibular epithelial development. We asked if gravity, the primary stimulus of utricles could act as an epigenetic factor. As sensorial deprivation linked to weightlessness is technically difficult, we used a ground-based centrifuge to increase the gravitational vector, in order to hyperstimulate the vestibule. In this study, 3 days after mating, pregnant females were submitted to hypergravity, 2 g (HG). Their embryos were raised, born and postnatally developed under HG. The establishment of connections between primary vestibular afferent neurons and hair cells in the utricle of these young rats was followed from birth to postnatal day 6 (PN6) and compared to embryos developed in normogravity (NG): Immunocytochemistry for neurofilaments and microvesicles revealed the differential effects of gravity on the late neuritogenic and synaptogenic processes in utricles. Taking type I hair cell innervation as a criterion of maturation, we found that primary afferent fibres reached the vestibular epithelium and enveloped hair cells in the same way, both under NG and HG. Thus, this phenomenon of leading growth cones to their epithelial target appears to be dependent on intrinsic genetic properties and not on an external stimulus. In contrast, the maturation of connection processes between type 1 hair cells and the afferent calyx, concerning specifically the microvesicles at their apex, was delayed under HG. Therefore, gravity appears to be an epigenetic factor influencing the late maturation of utricles. These differential effects of altered gravity on the development of the vestibular epithelium are discussed.

Na+ currents in vestibular type I and type II hair cells of the embryo and adult chicken.

In birds, type I and type II hair cells differentiate before birth. Here we describe that chick hair cells, from the semicircular canals, begin expressing a voltage-dependent Na current (INa) from embryonic day 14 (E14) and continue to express the current up to hatching (E21). During this period, INa was present in most (31/43) type I hair cells irrespective of their position in the crista, in most type II hair cells located far from the planum semilunatum (48/63), but only occasionally in type II hair cells close to the planum semilunatum (2/35). INa activated close to -60 mV, showed fast time- and voltage-dependent activation and inactivation, and was completely, and reversibly, blocked by submicromolar concentrations of tetrodotoxin (Kd = 17 nM). One peculiar property of INa concerns its steady-state inactivation, which is complete at -60 mV (half-inactivating voltage = -96 mV). INa was found in type I and type II hair cells from the adult chicken as well, where it had similar, although possibly not identical, properties and regional distribution. Current-clamp experiments showed that INa could contribute to the voltage response provided that the cell membrane was depolarized from holding potentials more negative than -80 mV. When recruited, INa produced a significant acceleration of the cell membrane depolarization, which occasionally elicited a large rapid depolarization followed by a rapid repolarization (action-potential-like response). Possible physiological roles for INa in the embryo and adult chicken are discussed.

Apoptosis during chick inner ear development: some observations by TEM and TUNEL techniques.

In order to clarify the occurrence, distribution and possible role of apoptosis during inner ear development, the ultrastructural aspects (by TEM) (at 9-19 incubation day and 1 day after hatching) and the distribution of the apoptotic phenomenon (by the TdT-mediated dUTP nick end-labeling technique), were studied in the crista ampullaris of chick embryo at 5-19 days of incubation to hatching and of postnatal 1-day old chick. We found, in the sensorial epithelium, dark supporting cells in chick embryos and mainly dark hair cells in postnatal chicks, both with ultrastructural features consistent with those of apoptosis. The presence of apoptotic phenomena was confirmed by the TUNEL technique. According to our findings, it is hypothesized that apoptosis in the inner ear may be involved: 1) at first, in macroscopic remodelling of the membranous labyrinth in early developmental stages, 2) later, in the correct differentiation of the hair and of the supporting cells, leading to characteristic cellular pattern formation and 3) finally, in physiological cell turnover of the postnatal chicken sensorial epithelium of the crista.

Hair cells without supporting cells: further studies in the ear of the zebrafish mind bomb mutant.

Each sensory hair cell in the ear is normally surrounded by supporting cells, which separate it from the next hair cell. In the mind bomb mutant, as a result of a failure of lateral inhibition, cells that would normally become supporting cells differentiate as hair cells instead, creating sensory patches that consist of hair cells only. This provides a unique opportunity to pinpoint the functions for which supporting cells are required in normal hair cell development. We find that hair cells in the mutant develop an essentially normal cytoskeleton, with a correctly structured hair bundle and well-defined planar polarity, and form apical junctional complexes with one another in standard epithelial fashion. They fail, however, to form a basal lamina or to adhere properly to the adjacent non-sensory epithelial cells, which overgrow them. The hair cells are eventually expelled from the ear epithelium into the underlying mesenchyme, losing their hair bundles in the process. It is not clear whether they undergo apoptosis: many cells staining strongly with the TUNEL procedure are seen but do not appear apoptotic by other criteria. Supporting cells, therefore, are needed to hold hair cells in the otic epithelium and, perhaps, to keep them alive, but are not needed for the construction of normal hair bundles or to give the hair bundles a predictable polarity. Moreover, supporting cells are not absolutely required as a source of materials for otoliths, which, though small and deformed, still develop in their absence.

The combined effects of trkB and trkC mutations on the innervation of the inner ear.

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer Dil. In the vestibular system, trkC+/- heterozygosity aggravates the trkB-/- mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthal's canal of the cochlea. trkC-/- mutation alone or in combination with trkB+/- heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB-/- mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB-/- mutant mouse combined with trkC+/- heterozygosity. In addition, the trkB-/- mutation combined with trkC+/- heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC-/- mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type: trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.

Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development.

The Brn-3 subfamily of POU-domain transcription factor genes consists of three highly homologous members-Brn-3a, Brn-3b, and Brn-3c-that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes plays distinctive roles in the somatosensory, visual, and auditory/vestibular systems.

Effects of microgravity on vestibular ontogeny: direct physiological and anatomical measurements following space flight (STS-29).

Does space flight change gravity receptor development? The present study measured vestibular form and function in birds flown as embryos for 5 days in earth orbit (STS-29). No major changes in vestibular gross morphology were found. Vestibular response mean amplitudes and latencies were unaffected by space flight. However, the results of measuring vestibular thresholds were mixed and abnormal responses in 3 of the 8 flight animals raise important questions.