Not All Inner Ears are the Same: Otolith Matrix Proteins in the Inner Ear of Sub‐Adult Cichlid Fish,Oreochromis Mossambicus,Reveal Insights Into the Biomineralization Process

The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP‐1, otolin‐1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre‐cerebellin‐like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre‐cerebellin‐like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required. Anat Rec, 299:234–245, 2016. © 2015 Wiley Periodicals, Inc.     

Interspecific Variations of Inner Ear Structure in the Deep‐Sea Fish Family Melamphaidae

Inner ear structures are compared among three major genera of the deep‐sea fish family Melamphaidae (bigscales and ridgeheads). Substantial interspecific variation is found in the saccular otoliths, including the presence of a unique otolithic “spur” in the genera Melamphaes and Poromitra. The variation in the saccular otolith is correlated with an increase in the number of hair bundle orientation groups on the sensory epithelia from the genera Scopelogadus to Poromitra to Melamphaes. The diverse structural variations found in the saccule may reflect the evolutionary history of these species. The sensory hair cell bundles in this family have the most variable shapes yet encountered in fish ears. In the saccule, most of the hair bundles are 15–20 μm high, an exceptional height for fish otolithic end organs. These bundles have large numbers of stereovilli, including some that reach the length of the kinocilium. In the utricle, the striolar region separates into two unusually shaped areas that have not been described in any other vertebrates. The brains in all species have a relatively small olfactory bulb and optic tectum, as well as an enlarged posterior cerebellar region that is likely to be involved in inner ear and lateral line (octavolateral) functions. Data from melamphaids support the hypothesis that specialized anatomical structures are found in the ears of some (if not most) deep‐sea fishes, presumably enhancing their hearing sensitivity. Anat Rec, 296:1064–1082, 2013. © 2013 Wiley Periodicals, Inc.     

Spatial Expression of Otolith Matrix Protein‐1 and Otolin‐1 in Normally and Kinetotically Swimming Fish

Kinetosis (motion sickness) has been repeatedly shown to affect some fish of a given clutch following the transition from 1g to microgravity or from hypergravity to 1g. This susceptibility to kinetosis may be correlated with irregular inner ear otolith growth. Otoliths are mainly composed of calcium carbonate and matrix proteins, which play an important role in the process of otolith mineralization. Here, we examine the morphology of otoliths and the expression pattern of the major otolith proteins OMP‐1 and otolin‐1 in a series of hypergravity experiments. In the utricle, OMP‐1 is present in centripetal (medial) and centrifugal (lateral) regions of the meshwork area. In the saccule, OMP‐1 was expressed within a dorsal and a ventral narrow band of the meshwork area opposite to the periphery of the sulcus acusticus. In normal animals, the spatial expression pattern of OMP‐1 reaches more posteriorly in the centrifugal aspect and is considerably broader in the centripetal portion of the utricle compared to kinetotic animals. However, otolin‐1 was not expressed in the utricule. In the saccule, no differences were observed for either gene when comparing normal and kinetotically behaving fish. The difference in the utricular OMP‐1 expression pattern between normally and kinetotically swimming fish indicates a different otolith morphology and thus a different geometry of the otoliths resting on the corresponding sensory maculae. As the utricle is the endorgan responsible for sensing gravity, the aberrant morphology of the utricular otoliths, based on OMP‐1 expression, likely leads to the observed kinetotic behavior. Anat Rec, 298:1765–1773, 2015. © 2015 Wiley Periodicals, Inc.     

Expression patterns of connexin43 protein during facial development in the chick embryo: Associations with outgrowth,attachment, and closure of the midfacial primordia

ABSTRACT Background: In a prior report, evidence was presented for the presence of gap junction proteins [connexin32 and connexin43 (Cx43)] in embryonic facial primordia. The purpose of the present study was, first, to examine in detail the patterns of distribution of Cx43 protein in embryonic chick facial primordia and, second, to consider the possible roles played by this protein during midfacial development. Methods: Chick embryo heads were serially sectioned and processed for immunofluorescent localization of Cx43. The developmental stages examined encompassed the period of formation, enlargement, and union of the facial primordia. Western blot analysis of the facial primordia was also performed. Results: Analysis of serial sections revealed the presence of signal in both epithelium and mesenchyme at sites of attachment in each of the midfacial primordia (i.e., the medial nasal, lateral nasal, and maxillary processes). Furthermore, although signal was concentrated in mesenchyme in the distal tips of the primordia at sites of attachment, immunoreactivity was absent, sparse, or less intense outside the areas of attachment. In some cases (i.e., the maxillary process), immunoreactive signal in mesenchyme did not appear in the distal tip until the primordia approximated each other or contact of the primordia was initiated. Most significantly, signal was also found between the facial primordia in nonprimordial epithelium and mesenchyme at sites where the primordia were joined. Conclusions: These data suggest that the expression of Cx43 protein is spatially and temporally regulated in the facial primordia and that the patterns of expression that were observed are significant to the cascade of events that ultimately lead to the attachment and union of the primordia that form the midface. Anat. Rec. 248:279-290, 1997. © 1997 Wiley-Liss, Inc.     

Exogenous Material in the Inner Ear of the Adult Port Jackson Shark,Heterodontus portusjacksoni (Elasmbranchii)

Previous studies have suggested that the inner ear of some benthic species of elasmobranchs contain only exogenous material within their otoconial organs, a unique feature within vertebrates. However, these examinations have not accounted for the possibility of otoconial degeneration or used modern experimental methods to identify the materials present. Both of these issues are addressed in this study using inner ear samples from the adult Port Jackson shark, Heterodontus portusjacksoni. A comparison of the otoconial mass in fixed specimens over short and medium time scales reveals that over those timescales the degeneration of calcium carbonate‐based otoconia does not occur and confirms that calcium carbonate‐based otoconia are not found within the otoconial organs of H. portusjacksoni. Additionally, microanalysis of the chemical composition and ultrastructure of the otoconial mass using energy dispersive X‐ray microanalysis and scanning electron microscopy confirms that the entire otoconial mass is comprised of exogenous silicon dioxide particles, bound within a carbon matrix. This exogenous material is suggested to play an equivalent role to the otoconia found in other species of elasmobranchs, and allows both hearing and vestibular control to occur in benthic sharks that spend their lives foraging within a sandy substrate. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Growth and turnover of rat otoconia as revealed by labeling with tetracycline

Background: The formation process of otoconia remains controversial, and the turnover rate of mammalian otoconia has not been determined. Methods: Tetracycline was administered as a tracer for calcium, and the growth and turnover of rat otoconia were examined by fluoresence microscopy. Results: Exposure of rats to tetracycline from 15.5 gestational day to 3-day postpartum resulted in incorporation of the drug into central portions of all otoconia, in both maculae sacculi and utriculi. When postnatal rats were injected subcutaneously with tetracycline, uptake of the drug into otoconia depended on the age of rats at injection. Apparent fluorescence was emitted from the periphery of all otoconia when tetracycline was injected into 7-day-old or younger rats. However, very little or no fluorescence was observed when this reagent was administered 10 days after birth. No fluorescence was detected when rats of 12 days of age or older were given this antibiotic. Otoconia that had been labeled with tetracycline during gestation were monitored during subsequent development, and it was found that all otoconia retained labelling in their central portions for at least 12 months. No otoconia that were not labeled with tetracycline were found. Conclusions: These findings indicate that: (1) all otoconia grow synchronously during late gestation and the neonatal period (up to about 10 days after birth) by accretion, (2) no new otoconia are formed subsequently, and (3) essentially no turnover of otoconia occurs and probably no turnover of calcium takes place under normal conditions once otoconia have formed. © 1995 Wiley-Liss, Inc.     

Embryogenesis and early skeletogenesis in the antarctic bullhead notothen,Notothenia coriiceps

Background: Environmental temperature influences rates of embryonic development, but a detailed staging series for vertebrate embryos developing in the subzero cold of Antarctic waters is not yet available from fertilization to hatching. Given projected warming of the Southern Ocean, it is imperative to establish a baseline to evaluate potential effects of changing climate on fish developmental dynamics. Results: We studied the Bullhead notothen (Notothenia coriiceps), a notothenioid fish inhabiting waters between ‐1.9 and +2 °C. In vitro fertilization produced embryos that progressed through cleavage, epiboly, gastrulation, segmentation, organogenesis, and hatching. We compared morphogenesis spatially and temporally to Zebrafish and medaka. Experimental animals hatched after about 6 months to early larval stages. To help understand skeletogenesis, we analyzed late embryos for expression of sox9 and runx2, which regulate chondrogenesis, osteogenesis, and eye development. Results revealed that, despite their prolonged developmental time course, N. coriiceps embryos developed similarly to those of other teleosts with large yolk cells. Conclusions: Our studies set the stage for future molecular analyses of development in these extremophile fish. Results provide a foundation for understanding the impact of ocean warming on embryonic development and larval recruitment of notothenioid fish, which are key factors in the marine trophic system. Developmental Dynamics 245:1066–1080, 2016. © 2016 Wiley Periodicals, Inc.     

Gata2 is required for the development of inner ear semicircular ducts and the surrounding perilymphatic space

Gata2 has essential roles in the development of many organs. During mouse inner ear morphogenesis, it is expressed in otic vesicle and the surrounding periotic mesenchyme from early on, but no defects in the ear development of Gata2 null mice have been observed before lethality at embryonic day (E) 10.5. Here, we used conditional gene targeting to reveal the role of Gata2 at later stages of inner ear development. We show that Gata2 is critically required from E14.5–E15.5 onward for vestibular morphogenesis. Without Gata2 the semicircular ducts fail to grow to their normal size and the surrounding mesenchymal cells are not removed properly to generate the perilymphatic space. Gata2 is the first factor known to control the clearing of the vestibular perilymphatic mesenchyme, but interestingly, it is not required for the formation of the cochlear perilymphatic areas, suggesting distinct molecular control for these processes. Developmental Dynamics 239:2452–2469, 2010. © 2010 Wiley‐Liss, Inc.     

Chondrocyte‐specific Smad4 gene conditional knockout results in hearing loss and inner ear malformation in mice

Smad4 is the central intracellular mediator of transforming growth factor‐β (TGF‐β) signaling, which plays crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. Conventional Smad4 gene knockout results in embryonic lethality, precluding its use in studies of the role of Smad4 in inner ear development. We used chondrocyte‐specific Smad4 knockout mice (Smad4^Co/Co) to investigate the function of Smad4 in inner ear development. Smad4^Co/Co mice were characterized by a smaller cochlear volume, bone malformation, and abnormalities of the osseous spiral lamina and basilar membrane. The development of the hair cells was also abnormal, as evidenced by the disorganized stereocilia and reduced density of the neuronal processes beneath the hair cells. Auditory function tests revealed the homozygous Smad4^Co/Co mice suffered from severe sensorineural hearing loss. Our results suggest that Smad4 is required for inner ear development and normal auditory function in mammals. Developmental Dynamics, 2009. © 2009 Wiley‐Liss, Inc.     

The role of Zic genes in inner ear development in the mouse: Exploring mutant mouse phenotypes

Background: Murine Zic genes (Zic1–5) are expressed in the dorsal hindbrain and in periotic mesenchyme (POM) adjacent to the developing inner ear. Zic genes are involved in developmental signaling pathways in many organ systems, including the ear, although their exact roles haven't been fully elucidated. This report examines the role of Zic1, Zic2, and Zic4 during inner ear development in mouse mutants in which these Zic genes are affected. Results: Zic1/Zic4 double mutants don't exhibit any apparent defects in inner ear morphology. By contrast, inner ears from Zic2^kd/kd and Zic2^Ku/Ku mutants have severe but variable morphological defects in endolymphatic duct/sac and semicircular canal formation and in cochlear extension in the inner ear. Analysis of otocyst patterning in the Zic2^Ku/Ku mutants by in situ hybridization showed changes in the expression patterns of Gbx2 and Pax2. Conclusions: The experiments provide the first genetic evidence that the Zic genes are required for morphogenesis of the inner ear. Zic2 loss‐of‐function doesn't prevent initial otocyst patterning but leads to molecular abnormalities concomitant with morphogenesis of the endolymphatic duct. Functional hearing deficits often accompany inner ear dysmorphologies, making Zic2 a novel candidate gene for ongoing efforts to identify the genetic basis of human hearing loss. Developmental Dynamics 243:1487–1498, 2014. © 2014 Wiley Periodicals, Inc.     

The effects of a parasitic copepod on the recent larval growth of a fish inhabiting rocky coasts

Parasites can infect larval, juvenile or adult marine fishes; however, the effects of parasites on the growth and condition of fish larvae have seldom been investigated. This study analysed the effects of a parasitic copepod on the larval growth of the Chilean triplefin Helcogrammoides chilensis (Tripterygiidae) based on the microstructure of the sagittal otoliths. Fish larvae were collected during the austral spring of 2010 off central Chile. Their body length ranged from 5.1 to 16.6?mm (2 to 57?days old). They were parasitised by a penellid larval copepod that was always externally attached to the ventral side of the fish's gut. The prevalence of the copepod ranged from 2.7?% to 20.8?%, with one to four parasites per fish larva. Relationships between otolith size (radius, perimeter) and larval size were equal for parasitised and unparasitised fish larvae (P?>?0.05). Larval growth was also similar for unparasitised (0.21?mm/day) and parasitised fish larvae (0.19?mm/day) (P?>?0.05). However, a comparison of same-aged larvae showed that the larvae with copepods were smaller in both length and estimated body volume than the larvae without copepods. The Recent Otolith Growth Index, indicated that larval H. chilensis with copepods showed a reduction in recent growth and condition compared with those without evidence of copepods (P?相似文献   

Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase

The vestibular system of the inner ear is responsible for the perception of motion and gravity. Key elements of this organ are otoconia, tiny biomineral particles in the utricle and the saccule. In response to gravity or linear acceleration, otoconia deflect the stereocilia of the hair cells, thus transducing kinetic movements into sensorineural action potentials. Here, we present an allelic series of mutations at the otoconia-deficient head tilt (het) locus, affecting the gene for NADPH oxidase 3 (Nox3). This series of mutations identifies for the first time a protein with a clear enzymatic function as indispensable for otoconia morphogenesis.     

Role of osteopontin in the rodent inner ear as revealed by in situ hybridization

Osteopontin (OPN) is considered to be a non-collagenous bone matrix protein that is involved in the ossification process. However, OPN has recently been observed in ectopic sites such as the kidney and nervous tissue. In the present study, the expression of OPN mRNA was examined in the rat and mouse inner ear by nonradioisotopic in situ hybridization. Signals of OPN mRNA were observed in the marginal cells of the stria vascularis, spiral ganglion cells, vestibular sensory hair cells, and vestibular dark cells. OPN protein was detected only in the otoconia by immunohistochemistry. The presence of OPN mRNA in the cochlea and vestibular dark cells may indicate that OPN is involved in the regulation of ions in the inner ear fluid. Findings in the saccule and utricle suggest that OPN is one of the protein components of the rodent otoconia and that the vestibular sensory hair cells are involved in the production of otoconia.     

The development of the mammalian outer and middle?ear

The mammalian ear is a complex structure divided into three main parts: the outer; middle; and inner ear. These parts are formed from all three germ layers and neural crest cells, which have to integrate successfully in order to form a fully functioning organ of hearing. Any defect in development of the outer and middle ear leads to conductive hearing loss, while defects in the inner ear can lead to sensorineural hearing loss. This review focuses on the development of the parts of the ear involved with sound transduction into the inner ear, and the parts largely ignored in the world of hearing research: the outer and middle ear. The published data on the embryonic origin, signalling, genetic control, development and timing of the mammalian middle and outer ear are reviewed here along with new data showing the Eustachian tube cartilage is of dual embryonic origin. The embryonic origin of some of these structures has only recently been uncovered (Science, 339, 2013, 1453; Development, 140, 2013, 4386), while the molecular mechanisms controlling the growth, structure and integration of many outer and middle ear components are hardly known. The genetic analysis of outer and middle ear development is rather limited, with a small number of genes often affecting either more than one part of the ear or having only very small effects on development. This review therefore highlights the necessity for further research into the development of outer and middle ear structures, which will be important for the understanding and treatment of conductive hearing loss.     

Immunolocalization of basal lamina components during development of chick otic and optic primordia

Immunolocalization of laminin, fibronectin, and type IV collagen was examined during early morphogenetic shape changes of the avian inner ear and eye. The ear was studied from formation of the otic placode to invagination of the otic pit and the eye from the optic vesicle stage to formation of an optic cup. Distribution and intensity of immunoreactivity were compared in the two organ primordia and in adjacent epithelial layers. Laminin formed a continuous layer at the basal surface of the otic ectoderm and adjacent neural tube at all stages. The basal surfaces of the optic and lens epithelia also were continuously covered with laminin throughout development. The otic placode became attached to the neural ectoderm through a single layer of fibronectin and collagen IV between the layers of laminin. The ring-like attachment between the edges of the optic cup and lens primordium had the same structure. In addition, the central regions of the optic and lens primordia were attached by fibrils containing type IV collagen, whereas finer strands containing fibronectin and laminin also connected the otic epithelium and neural tube. The results are discussed in terms of models of invagination for the two primordia. © 1993 Wiley-Liss, Inc.