Variability in the role of the gasbladder in fish audition

The teleost gasbladder is believed to aid in fish audition by transferring pressure components of incoming sound to the inner ears. This idea is primarily based on both anatomical observations of the mechanical connection between the gasbladder and the ear, followed by physiological experiments by various researchers. The gasbladder movement has been modeled mathematically as a pulsating bubble. This study is extending the previous work on fish with a physical coupling of the gasbladder and ear by investigating hearing in two species (the blue gourami Trichogaster trichopterus, and the oyster toadfish Opsanus tau) without a mechanical linkage. An otophysan specialist (the goldfish Carassius auratus) with mechanical coupling, is used as the control. Audiograms were obtained with acoustically evoked potentials (e.g., auditory brainstem response) from intact fish and from the same individuals with their gasbladders deflated. In blue gourami and oyster toadfish, removal of gas did not significantly change thresholds, and evoked potentials had similar waveforms. In goldfish thresholds increased by 33–55 dB (frequency dependent) after deflation, and major changes in evoked potentials were observed. These results suggest that the gasbladder may not serve an auditory enhancement function in teleost fishes that lack mechanical coupling between the gasbladder and the inner ear. Accepted: 28 February 2000     

Hindbrain signals in otic regionalization: walk on the wild side

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this level of complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium and more particularly the adjacent segmented hindbrain, have been implicated in conferring signals required for inner ear development. In this review, we present the current view on the role of hindbrain signals in axial specification of the inner ear. The functional analysis of mutants of hindbrain segmentation genes, as well as the investigation of signaling pathways potentially involved, all point to an essential role of FGF, Wnt and Hh signaling in otic regionalization. However, these data provide conflicting evidence regarding the involvement of hindbrain signals in otic regionalization in fish and in amniotes. We discuss the possible origin of these differences.     

Dual embryonic origin of the mammalian otic vesicle forming the inner ear

The inner ear and cochleovestibular ganglion (CVG) derive from a specialized region of head ectoderm termed the otic placode. During embryogenesis, the otic placode invaginates into the head to form the otic vesicle (OV), the primordium of the inner ear and CVG. Non-autonomous cell signaling from the hindbrain to the OV is required for inner ear morphogenesis and neurogenesis. In this study, we show that neuroepithelial cells (NECs), including neural crest cells (NCCs), can contribute directly to the OV from the neural tube. Using Wnt1-Cre, Pax3(Cre/+) and Hoxb1(Cre/+) mice to label and fate map cranial NEC lineages, we have demonstrated that cells from the neural tube incorporate into the otic epithelium after otic placode induction has occurred. Pax3(Cre/+) labeled a more extensive population of NEC derivatives in the OV than did Wnt1-Cre. NEC derivatives constitute a significant population of the OV and, moreover, are regionalized specifically to proneurosensory domains. Descendents of Pax3(Cre/+) and Wnt1-Cre labeled cells are localized within sensory epithelia of the saccule, utricle and cochlea throughout development and into adulthood, where they differentiate into hair cells and supporting cells. Some NEC derivatives give rise to neuroblasts in the OV and CVG, in addition to their known contribution to glial cells. This study defines a dual cellular origin of the inner ear from sensory placode ectoderm and NECs, and changes the current paradigm of inner ear neurosensory development.     

Auditory saccular sensitivity of the vocal Lusitanian toadfish: low frequency tuning allows acoustic communication throughout the year

A novel form of auditory plasticity for enhanced detection of social signals was described in a teleost fish, Porichthys notatus (Batrachoididae, Porichthyinae). The seasonal onset of male calling coincides with inshore migration from deep waters by both sexes and increased female sensitivity to dominant frequencies of male calls. The closely related Lusitanian toadfish, Halobatrachus didactylus, (Batrachoididae, Halophryninae) also breeds seasonally and relies on acoustic communication to find mates but, instead, both sexes stay in estuaries and show vocal activity throughout the year. We investigated whether the sensitivity of the inner ear saccule of H. didactylus is seasonally plastic and sexually dimorphic. We recorded evoked potentials from populations of saccular hair cells from non-reproductive and reproductive males and females in response to 15–945 Hz tones. Saccular hair cells were most sensitive at 15–205 Hz (thresholds between 111 and 118 dB re. 1 μPa). Both sexes showed identical hearing sensitivity and no differences were found across seasons. The saccule was well suited to detect conspecific vocalizations and low frequencies that overlapped with lateral line sensitivity. We showed that the saccule in H. didactylus has major importance in acoustic communication throughout the year and that significant sensory differences may exist between the two batrachoidid subfamilies.     

The polarisation of hair cells from the ear of the European bass (Dicentrarchus labrax)

The polarisation of ciliary bundles on the macula of the saccule in the European bass (Dicentrarchus labrax L) has been studied using a scanning electron microscope (SEM). These data show that D. labrax possesses ciliary bundles arranged in four dichotomous quadrants with a standard orientation, comparable to hearing generalists from the order Perciformes. The spacing between ciliary bundles was investigated in three size classes of fish, with the results indicating that the addition of receptor cells in the ear of D. labrax continues for at least the first 2 years of development. The lengths of the kinocilia from ciliary bundles in each quadrant of the macula were also studied, and found to be of uniform length. In addition, we look at the internal structure of the afferent using transmission electron microscopy (TEM), revealing the nucleated cell body and peripheral nerve fibres of the saccule consistent with other TEM examinations of saccular ultrastructure. This information is required to gain an insight into the inner ear of D. labrax, as part of a larger study of the morphology and physiology of the hearing systems of both vertebrate and invertebrate marine animals.     

The ear region of Latimeria chalumnae: functional and evolutionary implications

The anatomy of Latimeria chalumnae has figured prominently in discussions about tetrapod origins. While the gross anatomy of Latimeria is well documented, relatively little is known about its otic anatomy and ontogeny. To examine the inner ear and the otoccipital part of the cranium, a serial-sectioned juvenile coelacanth was studied in detail and a three-dimensional reconstruction was made. The ear of Latimeria shows a derived condition compared to other basal sarcopterygians in having a connection between left and right labyrinths. This canalis communicans is perilymphatic in nature and originates at the transition point of the saccule and the lagena deep in the inner ear, where a peculiar sense end organ can be found. In most gnathostomes the inner ears are clearly separated from each other. A connection occurs in some fishes, e.g. within the Ostariophysi. In the sarcopterygian lineage no connections between the inner ears are known except in the Actinistia. Some fossil actinistians show a posteriorly directed duct lying between the foramen magnum and the notochordal canal, similar to the condition in the ear of Latimeria, so this derived character complex probably developed early in actinistian history. Because some features of the inner ear of Latimeria have been described as having tetrapod affinities, the problem of hearing and the anatomy of the otical complex in the living coelacanth has been closely connected to the question of early tetrapod evolution. It was assumed in the past that the structure found in Latimeria could exemplify a transitional stage in otic evolution between the fishlike sarcopterygians and the first tetrapods in a functional or even phylogenetic way. Here the possibility is considered that the canalis communicans does not possess any auditory function but rather is involved in sensing pressure changes during movements involving the intracranial joint. Earlier hypotheses of a putative tympanic ear are refuted.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

Auditory role of the suprabranchial chamber in gourami fish

Fish hearing specialists (e.g., goldfish, holocentrids, clupeoids, mormyrids) have evolved specialized structures (e.g., Weberian ossicles, swimbladder diverticulae, gas-filled bullae) to enhance their auditory frequency range and threshold sensitivity. The inner ears of anabantoid fish are encased in membranous cranial bones and are protruded into air-filled suprabranchial chambers. This research was intended to test the hypothesis that the gas bubbles inside the suprabranchial chambers may modulate the hearing abilities of anabantoid fish because of their proximity to the membranous bone-encased inner ears. Three species of gourami (blue gourami Trichogaster trichopterus; kissing gourami Helostoma temminckii; dwarf gourami Colisa lalia) were examined. Using the auditory brainstem response recording technique, baseline audiograms tested at 300, 500, 800, 1500, 2500, 4000 Hz were obtained. The air bubbles in the suprabranchial chambers were replaced by water, and the audiograms were remeasured. Thresholds were elevated in all three species. When three blue gouramis were allowed to replenish air into the suprabranchial chambers their hearing abilities returned to baseline levels. These results support the hypothesis that air bubbles in the suprabranchial chambers can affect hearing abilities of gouramis by lowering the thresholds. Accepted: 28 May 1998     

A proposed mechanism for the observed ontogenetic improvement in the hearing ability of hapuka (Polyprion oxygeneios)

Swim bladder extensions and hearing ability were examined in the temperate reef fish Polyprion oxygeneios (hapuka). Using the auditory evoked potential (AEP) technique, hearing thresholds were determined in four age-classes of hapuka, from larvae to juveniles. The youngest age-class had poor hearing abilities, with lowest thresholds of 132 dB re 1 μPa, and a narrow auditory bandwidth (100–800 Hz). Hearing ability improved significantly throughout the remainder of their first year, including decreases in thresholds of up to 27 dB, and an increase in auditory bandwidth (up to 1,000 Hz). Magnetic resonance imaging (MRI) was used to investigate structural mechanisms that may account for this ontogenetic improvement in hearing. These showed rostral extensions of the swim bladder developing early in the juvenile stage, and extending with increasing age closer to the otic capsule. It is suggested that this indirect connection between the swim bladder and the otic capsule could impart pressure sensitivity closer to the inner ear, accounting for the increase in sensitivity seen during development, although further investigation of older fish is required for conclusive evidence. The improvement in hearing ability in hapuka could be potentially related to a unique life history of extended pelagic durations up to 4 years.     

Catecholaminergic Innervation of Central and Peripheral Auditory Circuitry Varies with Reproductive State in Female Midshipman Fish,Porichthys notatus

In seasonal breeding vertebrates, hormone regulation of catecholamines, which include dopamine and noradrenaline, may function, in part, to modulate behavioral responses to conspecific vocalizations. However, natural seasonal changes in catecholamine innervation of auditory nuclei is largely unexplored, especially in the peripheral auditory system, where encoding of social acoustic stimuli is initiated. The plainfin midshipman fish, Porichthys notatus, has proven to be an excellent model to explore mechanisms underlying seasonal peripheral auditory plasticity related to reproductive social behavior. Recently, we demonstrated robust catecholaminergic (CA) innervation throughout the auditory system in midshipman. Most notably, dopaminergic neurons in the diencephalon have widespread projections to auditory circuitry including direct innervation of the saccule, the main endorgan of hearing, and the cholinergic octavolateralis efferent nucleus (OE) which also projects to the inner ear. Here, we tested the hypothesis that gravid, reproductive summer females show differential CA innervation of the auditory system compared to non-reproductive winter females. We utilized quantitative immunofluorescence to measure tyrosine hydroxylase immunoreactive (TH-ir) fiber density throughout central auditory nuclei and the sensory epithelium of the saccule. Reproductive females exhibited greater density of TH-ir innervation in two forebrain areas including the auditory thalamus and greater density of TH-ir on somata and dendrites of the OE. In contrast, non-reproductive females had greater numbers of TH-ir terminals in the saccule and greater TH-ir fiber density in a region of the auditory hindbrain as well as greater numbers of TH-ir neurons in the preoptic area. These data provide evidence that catecholamines may function, in part, to seasonally modulate the sensitivity of the inner ear and, in turn, the appropriate behavioral response to reproductive acoustic signals.     

Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.     

A fate map of chick otic cup closure reveals lineage boundaries in the dorsal otocyst

The vertebrate inner ear is structurally complex, consisting of fluid-filled tubules and sensory organs that subserve the functions of hearing and balance. The epithelial parts of the inner ear are derived from the otic placode, which deepens to form a cup before closing to form the otic vesicle. We fate-mapped the rim of the otic cup to monitor the cellular movements associated with otocyst formation and to aid in interpreting the changing gene expression patterns of the early otic field. Twelve sites around the rim, defined as positions of a clock face, were targeted by iontophoretic injection of fluorescent, lipophilic dye. Labeled cells were imaged 24 and 48 h after injection. The data show that the entire dorsal rim of the otic cup becomes the endolymphatic duct (ED), while the posteroventral rim becomes the lateral otocyst wall. Two intersecting boundaries of lineage restriction were identified near the dorsal pole: one bisecting the ED into anterior and posterior halves and the other defining its lateral edge. We hypothesize that signaling across compartment boundaries may play a critical role in duct specification. This model is discussed in the context of mouse mutants that are defective in both hindbrain development and ED outgrowth.     

Tbx1 and Brn4 regulate retinoic acid metabolic genes during cochlear morphogenesis

Background  

In vertebrates, the inner ear is comprised of the cochlea and vestibular system, which develop from the otic vesicle. This process is regulated via inductive interactions from surrounding tissues. Tbx1, the gene responsible for velo-cardio-facial syndrome/DiGeorge syndrome in humans, is required for ear development in mice. Tbx1 is expressed in the otic epithelium and adjacent periotic mesenchyme (POM), and both of these domains are required for inner ear formation. To study the function of Tbx1 in the POM, we have conditionally inactivated Tbx1 in the mesoderm while keeping expression in the otic vesicle intact.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

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.     

Tissue-specific requirements for FGF8 during early inner ear development

Several members of the FGF gene family have been shown to intervene from various tissue sources to direct otic placode induction and otic vesicle formation. In this study we define the roles of FGF8, found in different expression domains during this process, in mice and chickens. By conditional inactivation of Fgf8 in distinct tissue compartments we demonstrate that Fgf8 is required in the mesoderm and endoderm during early inner ear development. In the chicken embryo, overexpression of Fgf8 from various tissue sources during otic specification leads to a loss of otic tissue. In contrast ectopic overexpression of Fgf10, a major player during murine otic induction, does not influence otic vesicle formation in chicken embryos but results in the formation of ectopic structures with a non-otic character. This study underlines the crucial role of a defined Fgf8 expression pattern controlling inner ear formation in vertebrates.