Axonal Cleaved Caspase-3 Regulates Axon Targeting and Morphogenesis in the Developing Auditory Brainstem.

Caspase-3 is a cysteine protease that is most commonly associated with cell death. Recent studies have shown additional roles in mediating cell differentiation, cell proliferation and development of cell morphology. We investigated the role of caspase-3 in the development of chick auditory brainstem nuclei during embryogenesis. Immunofluorescence from embryonic days E6-13 revealed that the temporal expression of cleaved caspase-3 follows the ascending anatomical pathway. The expression is first seen in the auditory portion of VIIIth nerve including central axonal regions projecting to nucleus magnocellularis (NM), then later in NM axons projecting to nucleus laminaris (NL), and subsequently in NL dendrites. To examine the function of cleaved caspase-3 in chick auditory brainstem development, we blocked caspase-3 cleavage in developing chick embryos with the caspase-3 inhibitor Z-DEVD-FMK from E6 to E9, then examined NM and NL morphology and NM axonal targeting on E10. NL lamination in treated embryos was disorganized and the neuropil around NL contained a significant number of glial cells normally excluded from this region. Additionally, NM axons projected into inappropriate portions of NL in Z-DEVD-FMK treated embyros. We found that the presence of misrouted axons was associated with more severe NL disorganization. The effects of axonal caspase-3 inhibition on both NL morphogenesis and NM axon targeting suggest that these developmental processes are coordinated, likely through communication between axons and their targets.

Dcc Mediates Functional Assembly of Peripheral Auditory Circuits.

Proper structural organization of spiral ganglion (SG) innervation is crucial for normal hearing function. However, molecular mechanisms underlying the developmental formation of this precise organization remain not well understood. Here, we report in the developing mouse cochlea that deleted in colorectal cancer (Dcc) contributes to the proper organization of spiral ganglion neurons (SGNs) within the Rosenthal's canal and of SGN projections toward both the peripheral and central auditory targets. In Dcc mutant embryos, mispositioning of SGNs occurred along the peripheral auditory pathway with misrouted afferent fibers and reduced synaptic contacts with hair cells. The central auditory pathway simultaneously exhibited similar defective phenotypes as in the periphery with abnormal exit of SGNs from the Rosenthal's canal towards central nuclei. Furthermore, the axons of SGNs ascending into the cochlear nucleus had disrupted bifurcation patterns. Thus, Dcc is necessary for establishing the proper spatial organization of SGNs and their fibers in both peripheral and central auditory pathways, through controlling axon targeting and cell migration. Our results suggest that Dcc plays an important role in the developmental formation of peripheral and central auditory circuits, and its mutation may contribute to sensorineural hearing loss.

Role of phosphatase and tensin homolog in the development of the mammalian auditory system.

Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that controls neural stem cell renewal and differentiation and is a potential target for regeneration in the optic nerve. Here we show that it has a critical pattern of expression in the mammalian developing auditory system. PTEN was expressed in the cochlear-vestibular ganglion at embryonic day 10.5 and then progressively in hair cells as they differentiated from the base to the apex of the cochlea. By postnatal day 7, PTEN was downregulated in hair cells and subsequently in the neurons. This very specific, transient expression pattern suggests that PTEN plays a crucial role in the differentiation of the sensory neurons and hair cells and that it is a potential therapeutic target for hearing regeneration.

Origin, evolution and homologies of the Weberian apparatus: a new insight / Origen, evolución y homologías del aparato weberiano: un nuevo acercamiento

The Weberian apparatus is essentially a mechanical device improving audition, consisting of a double chain of ossicles joining the air bladder to the inner ear. Despite being one of the most notable complex systems of teleost fishes and the subject of several comparative, developmental and functional studies, there is still much controversy concerning the origin, evolution and homologies of the structures forming this apparatus. In this paper I provide a new insight on these topics, which takes into account the results of recent works on comparative anatomy, paleontology, and ontogeny as well as of a recent extensive phylogenetic analysis including not only numerous otophysan and non-otophysan extant otocephalans but also ostariophysan fossils such as Chanoides macropoma, Clupavus maroccanus, Santanichthys diasii, Lusitanichthys characiformis, Sorbininardus apuliensis and Tischlingerichthys viohli. According to the evidence now available, the Weberian apparatus of otophysans seems to be the outcome of a functional integration of features acquired in basal otocephalans and in basal ostariophysans, which were very likely not directly related with the functioning of this apparatus, and of features acquired in the nodes leading to the Otophysi and to the clade including the four extant otophysan orders, which could well have been the result of a selection directly related to the functioning of the apparatus.

El aparato weberiano es esencialmente un dispositivo mecánico que mejora la audición, consiste en una doble cadena de osículos que unen la cámara de aire al oído interno. A pesar de ser uno de los sistemas complejos más notables de peces teleósteos y objeto de varios estudios comparativos, de desarrollo y funcionales, todavía hay mucha controversia sobre el origen, evolución y homologías de las estructuras que forman este aparato. En este trabajo se proporciona una nueva visión sobre estos temas, que tiene en cuenta los resultados de los últimos trabajos sobre la anatomía comparada, paleontología y la ontogenia, así como de un reciente análisis filogenético amplio que incluyen no sólo numerosos otocéfalos Otofisios y no Otofisios existentes, sino también fósiles Ostariofisios como Chanoides macropoma, Clupavus maroccanus, Santanichthys diasii, Lusitanichthys characiformis, Sorbininardus apuliensis y Tischlingerichthys viohli. Según las pruebas disponibles, el aparato weberiano de Otofisios parece ser el resultado de una integración funcional de las características adquiridas en otocéfalos basales y en ostariofisios basales, los cuales muy probablemente no estén directamente relacionados en el funcionamiento de este aparato, y las características adquiridas en los nodos que condujeron a los Otofisios y al clade incluyendo las cuatro órdenes existentes otofisios, que bien podrían haber sido el resultado de una selección directamente relacionada con el funcionamiento del aparato.

The impact of maternal smoking on fast auditory brainstem responses.

Deficits in auditory processing have been posited as one of the underlying neurodevelopmental consequences of maternal smoking during pregnancy that leads to later language and reading deficits. Fast auditory brainstem responses were used to assess differences in the sensory processing of auditory stimuli among infants with varying degrees of prenatal cigarette exposure. Maternal report of consumption of cigarettes and blood samples were collected in the hospital to assess exposure levels and participants were then seen at 6-months. To participate in the study, all infants had to pass the newborn hearing exam or a clinically administered ABR and have no known health problems. After controlling for participant age, maternal smoking during pregnancy was negatively related to latency of auditory brainstem responses. Of several potential covariates, only perinatal complications and maternal alcohol use were also related to latency of the ABR responses and maternal smoking level accounted for significant unique variance after controlling for these factors. These results suggest that the relationship between maternal smoking may lead to disruption in the sensory encoding of auditory stimuli.

Molecular guidance cues necessary for axon pathfinding from the ventral cochlear nucleus.

During development, multiple guidance cues direct the formation of appropriate synaptic connections. Factors that guide developing axons are known for various pathways throughout the mammalian brain; however, signals necessary to establish auditory connections are largely unknown. In the auditory brainstem the neurons whose axons traverse the midline in the ventral acoustic stria (VAS) are primarily located in the ventral cochlear nucleus (VCN) and project bilaterally to the superior olivary complex (SOC). The circumferential trajectory taken by developing VCN axons is similar to that of growing axons of spinal commissural neurons. Therefore, we reasoned that netrin-DCC and slit-robo signaling systems function in the guidance of VCN axons. VCN neurons express the transcription factor, mafB, as early as embryonic day (E) 13.5, thereby identifying the embryonic VCN for these studies. VCN axons extend toward the midline as early as E13, with many axons crossing by E14.5. During this time, netrin-1 and slit-1 RNAs are expressed at the brainstem midline. Additionally, neurons within the VCN express RNA for DCC, robo-1, and robo-2, and axons in the VAS are immunoreactive for DCC. VCN axons do not reach the midline of the brainstem in mice mutant for either the netrin-1 or DCC gene. VCN axons extend in pups lacking netrin-1, but most DCC-mutant samples lack VCN axonal outgrowth. Stereological cell estimates indicate only a modest reduction of VCN neurons in DCC-mutant mice. Taken together, these data show that a functional netrin-DCC signaling system is required for establishing proper VCN axonal projections in the auditory brainstem.

Prenatal auditory enrichment with species-specific calls and sitar music modulates expression of Bcl-2 and Bax to alter programmed cell death in developing chick auditory nuclei.

Postnatal auditory stimulation influences early perceptual learning. Previously we reported morphological effects of prenatal auditory stimulation by species-specific and sitar musical sounds on the chick brainstem auditory nuclei-nucleus magnocellularis and nucleus laminaris. At hatching, these two nuclei of auditory enriched embryos showed higher neuronal numbers, amongst other morphological changes. There were also increases in synaptophysin and syntaxin1 expressions in the sound enriched groups and modulation of the developmental expression of transcription factors c-Fos and c-Jun. We hypothesized that prenatal auditory enrichment may have reduced embryonic apoptosis in these nuclei with possible alteration of molecular mechanisms enhancing the postsynaptic neuron's ability to survive. In the present study, therefore, we examined apoptotic cell death by TUNEL technique and Bcl-2 expression using immunohistochemistry and immunoblotting. In the controls, a peak percentage in the TUNEL-positive cells was noted in the auditory nuclei at embryonic day 12, which was reduced at embryonic day 16. Bcl-2 immunoreactivity decreased from embryonic day 8 to embryonic day 12 overlapping the period of embryonic cell death in these nuclei. The stimulated groups, however, showed fewer apoptotic neurons and higher Bcl-2 level than that in the controls. On the other hand, Bax immunohistochemistry showed correlated reverse changes compared to Bcl-2 expression. Thus prenatal extra-acoustic stimulation appears to alter Bcl-2 and Bax expression to support cell survival and differentiation, thereby augmenting the development of auditory nuclei.

Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner ear.

Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors--and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation.

Ventral otic cell lines as developmental models of auditory epithelial and neural precursors.

Conditionally immortal cell lines were established from the ventral otocyst of the Immortomouse at embryonic day 10.5 and selected to represent precursors of auditory sensory neural and epithelial cells. Selection was based upon dissection, tissue-specific markers, and expression of the transcription factor GATA3. Two cell lines expressed GATA3 but possessed intrinsically different genetic programs under differentiating conditions. US/VOT-E36 represented epithelial progenitors with potential to differentiate into sensory and nonsensory epithelial cells. US/VOT-N33 represented migrating neuroblasts. Under differentiating conditions in vitro the cell lines expressed very different gene expression profiles. Expression of several cell- and tissue-specific markers, including the transcription factors Pax2, GATA3, and NeuroD, differed between the cell lines in a pattern consistent with that observed between their counterparts in vivo. We suggest that these and other conditionally immortal cell lines can be used to study transient events in development against different backgrounds of cell competence.

Ontogenetic expression of trk neurotrophin receptors in the chick auditory system.

Neurotrophins and their cognate receptors are critical to normal nervous system development. Trk receptors are high-affinity receptors for nerve-growth factor (trkA), brain-derived neurotrophic factor and neurotrophin-4/5 (trkB), and neurotrophin-3 (trkC). We examine the expression of these three neurotrophin tyrosine kinase receptors in the chick auditory system throughout most of development. Trks were localized in the auditory brainstem, the cochlear ganglion, and the basilar papilla of chicks from embryonic (E) day 5 to E21, by using antibodies and standard immunocytochemical methods. TrkB mRNA was localized in brainstem nuclei by in situ hybridization. TrkB and trkC are highly expressed in the embryonic auditory brainstem, and their patterns of expression are both spatially and temporally dynamic. During early brainstem development, trkB and trkC are localized in the neuronal cell bodies and in the surrounding neuropil of nucleus magnocellularis (NM) and nucleus laminaris (NL). During later development, trkC is expressed in the cell bodies of NM and NL, whereas trkB is expressed in the nerve calyces surrounding NM neurons and in the ventral, but not the dorsal, dendrites of NL. In the periphery, trkB and trkC are located in the cochlear ganglion neurons and in peripheral fibers innervating the basilar papilla and synapsing at the base of hair cells. The protracted expression of trks seen in our materials is consistent with the hypothesis that the neurotrophins/tyrosine kinase receptors play one or several roles in the development of auditory circuitry. In particular, the polarized expression of trkB in NL is coincident with refinement of NM terminal arborizations on NL.

Applying very high resolution microfocus X-ray CT and 3-D reconstruction to the human auditory apparatus.

Conventional high-resolution X-ray computed tomography (XCT) is an important medical technique because it provides sectional images (tomograms) of internal structures without destroying the specimen. However, it is difficult to observe and to analyze fine structures less than a few cubic millimeters in size because of its low spatial resolution of 0.4 mm. Overcoming this problem would not only enable visualization of human anatomical structures in living subjects by means of computer images but would make it possible to obtain the equivalent of microscopic images by XCT without making microscopic sections of biopsy material, which would allow the examination of the entire body and detection of focal lesions at an early stage. Bonse et al. and Kinney et al. studied absorption contrast microtomography by using synchrotron radiation and achieved 8-microns spatial resolution in human cancellous bone. Recently, Momose et al. reported examining the soft tissue of cancerous rabbit liver by a modification of the phase-contrast technique using synchrotron radiation with a spatial resolution of 30 microns (ref. 4). However, the equipment for synchrotron radiation requires a great deal of space and is very expensive. Aoki et al., on a different tack, reported microtomography of frog embryos by using a conventional laboratory microfocus X-ray source with a spot size of about 2 microns (ref. 5). As no human tomographic studies by superresolution microfocus XCT (MFXCT) using a normal open-type X-ray source have been reported, we tried using MFXCT with a maximum experimental spatial resolution of 2.5 microns, especially designed for industrial use, on the auditory ossicles of a human fetus, the smallest and lightest bones in the skeletal system. No XCT studies of fetal auditory ossicles have been reported to date. The fine tomograms with three-dimensional reconstructions obtained showed the existence of an apparently previously undescribed joint between the tympanic ring and the anterior process of the malleus. We hope the early development of this MFXCT for clinical use will make a great contribution to medicine.

Immunoelectron microscopic analysis of a novel carbohydrate differentiation antigen (CDA-3C2) in the developing rat olfactory and otic systems.

A carbohydrate differentiation antigen (CDA-3C2) exhibits a highly specific and restricted pattern of expression during rat embryogenesis. In the periphery of the embryo, this antigen is associated transiently with the lateral ectoderm but is retained only in the olfactory and otic epithelium throughout morphogenesis. At the light microscopic level, CDA-3C2 immunoreactivity appears mostly along cell periphery and in the extracellular matrix. The aim of the present study was to determine the specific cellular and subcellular distribution of CDA-3C2 in vivo in order to identify potential sites of cellular and tissue function of the antigen during embryogenesis. There was a strikingly similar subcellular distribution of CDA-3C2 in the developing otic and olfactory systems, found mostly along cell membranes, microvillar projections and acellular secretions of the epithelium. Mature sensory components of the epithelia were not immunoreactive, whereas supportive cells and their secreted structures were densely stained. The highly coincident nature of CDA-3C2 in both sensory epithelia suggests that this carbohydrate epitope, and possibly its carrier macromolecule, participate in a morphogenetic function common to these two sensory epithelia.

Immunocytochemical analysis of a novel carbohydrate differentiation antigen (CDA-3C2) associated with olfactory and otic systems during embryogenesis in the rat.

Carbohydrate differentiation antigens are known to display specific patterns of expression during mammalian development and are thought to participate in significant morphogenetic events. In the present study, two monoclonal antibodies that react with a novel carbohydrate differentiation antigen (CDA-3C2) were used to analyze, by light microscopy, the spatiotemporal distribution of this unique high molecular weight antigen during embryogenesis in the rat. Correlative analysis of the development of peripheral neural structures, in which CDA-3C2 was expressed, was carried out with an anti-neurofilament antibody. Enzymatic digestion, combined with Western blots, reveal that the CDA-3C2 epitope is a carbohydrate which is carried on a high molecular weight glycoprotein with a mass of greater than 1 million Daltons. Characteristic of carbohydrate antigens, immunoreactivity was found in several distinct cellular patterns: only along the apical border of cells, along lateral and basal membranes of cells, and extracellular-like staining in the mesenchyme. During neurulation, CDA-3C2 showed differential staining in the ectoderm, distinguishing lateral from neural regions. Following closure of the neural tube, there was a striking specificity of expression of CDA-3C2 in the periphery, found almost exclusively in olfactory and otic epithelial structures. While CDA-3C2 is found in placode-derived tissues that subserve sensory transduction, it appears to be primarily associated with the supportive cells (and their secretions) in both otic and olfactory regions and less so with the sensory cells. The data suggest that a unique carbohydrate antigen on a large macromolecule may play a role in neurulation and/or morphogenesis of the placode-derived otic and olfactory structures.

Developmental regulation of a neurite-promoting factor influencing statoacoustic neurons.

The present study investigated a target-derived, neurite-promoting factor (NPF) released by the developing chick otocyst and its effects on statoacoustic ganglia (SAG). SAG explants cultured in the absence of otocysts produced little neurite outgrowth at all stages of development examined (E4-E13). However, extensive neurite outgrowth was seen when E4-E6 SAG were cultured in the presence of otocysts of the same age. The amount of neurite outgrowth observed in cocultures steadily decreased at later developmental stages. E7-E9 cocultures produced less outgrowth and E10-E13 cocultures produced the least outgrowth compared to E4-E6 cocultures. Additionally, otocysts from older stages were unable to promote outgrowth of E4 SAG. Thus, the level of the factor released by the otocysts declined during development. In contrast, neurite outgrowth was promoted when E10-E15 SAG were cocultured in the presence of younger stage otocysts. Our data indicate that the release of NPF from chick otocysts decreased from E6 to E13, although the ability of SAG neurons to respond to the NPF was maintained throughout development.

Synapsin I and Synaptophysin expression during ontogenesis of the mouse peripheral vestibular system.

Synapsin I and Synaptophysin are selectively localized in axonal endings of CNS neurons where they are associated with small synaptic vesicle membranes. The development of expression of these 2 proteins was studied by immunocytochemistry during ontogenesis of the peripheral vestibular system in the mouse. Both proteins are localized in vestibular ganglion neurons and in their peripheral sensory extensions as early as gestational day 14. While the entire periphery of these fibers is labeled during embryogenesis, both proteins are subject to relocation during the postnatal maturation of these fibers. In the mature vestibular receptors they disappear from the fibers themselves but are found concentrated in their intraepithelial endings and in the neuronal cell body. These observations show that the distribution pattern of Synapsin I and Synaptophysin in peripheral extensions of vestibular afferent neurons during development is identical to that described in axonal processes of CNS neurons. This suggests that the peripheral processes of the vestibular afferent neurons present structural and biochemical characteristics of axons. These characteristics are consistent with a bimodal sensory and secretory function of mature endings.

Cell polarity changes and migration during early development of the avian peripheral auditory system.

The intracellular location of organelles was studied in avian embryos immediately before and during the initial detachment of cells from the dorsoanterolateral wall of the otocyst, using light and electron microscopy. The Golgi apparatus was silver-impregnated, and its location within the otic epithelium was determined quantitatively. The present study demonstrates that a sub-population of cells of the dorsoanterolateral otic epithelium changes the intracellular location of its organelles, particularly the Golgi apparatus and the centrosome, from an apical to a basal position. Concomitantly, cells lose specializations characteristically present at apical (tight junctions, microvilli) and basal (basal lamina) surfaces. At basolateral cell surfaces, filopodia form ahead of the Golgi apparatus and centrosome and penetrate the previously continuous underlying basal lamina. Thereafter, cells detach from the otocyst and migrate medially toward the hind-brain. Thus, concomitant with changes in surface polarity, the cells that comprise the dorsoanterolateral wall of the otocyst undergo profound changes in the intracellular location of their organelles, especially the Golgi apparatus and the centrosome, so that by the time cells detach from the otic epithelium a reversal in their "normal" internal polarity has occurred. We suggest that the change in cell polarity may be related to the mechanisms that allow cells to leave the otocyst.