Incudomalleal joint formation: the roles of apoptosis,migration and downregulation

Background  

The middle ear of mammals is composed of three endochondrial ossicles, the stapes, incus and malleus. Joints link the malleus to the incus and the incus to the stapes. In the mouse the first arch derived malleus and incus are formed from a single Sox9 and Type II collagen expressing condensation that later subdivides to give rise to two separate ossicles. In contrast the stapes forms from a separate condensation derived from the second branchial arch. Fusion of the malleus and incus is observed in a number of human syndromes and results in conductive hearing loss. Understanding how this joint forms during normal development is thus an important step in furthering our understanding of such defects.     

Formation of the middle ear: recent progress on the developmental and molecular mechanisms

The middle ear allows animals to hear while moving in an aerial medium. It is composed of a cavity harbouring a chain of three ossicles that transmit vibrations produced by airborne sound in the tympanic membrane into the inner ear, where they are converted into neural impulses. The middle ear develops in the branchial arches, and this requires sequential interactions between the epithelia and the underlying mesenchyme. Gene-inactivation experiments have identified genes required for the formation of different middle ear components. Some encode for signalling molecules, including Endothelin1 and Fgf8, probable mediators of epithelial-mesenchymal interactions. Other genes, including Eya1, Prx1, Hoxa1, Hoxa2, Dlx1, Dlx2, Dlx5, and Gsc, are most likely involved in patterning and morphogenetic processes in the neural crest-derived mesenchyme. Mechanisms controlling formation of a functional tympanic membrane are also discussed. Basically, the tympanic ring, which serves as support for the tympanic membrane, directs invagination of the first pharyngeal cleft ectoderm to form the external acoustic meatus (EAM), which provides the outer layer of the membrane. Gsc and Prx1 are essential for tympanic ring development. While invaginating, the EAM controls skeletogenesis in the underlying mesenchyme to form the manubrium of the malleus, the link between the membrane and the middle ear ossicles.     

Assembling a functional tympanic membrane: signals from the external acoustic meatus coordinate development of the malleal manubrium

In terrestrial mammals, hearing starts with the perception of acoustic pressure by the tympanic membrane. Vibrations in this membrane are then transduced into the inner ear by the ossicle chain of the middle ear, composed of the malleus, incus and stapes. The proper connection of the ossicle chain with the tympanic membrane, provided by the insertion of the manubrium of the malleus into the eardrum, is essential for the functionality of the hearing apparatus. We describe here the mechanisms regulating the development of the manubrium and its integration into the tympanic membrane. We show that the external acoustic meatus (EAM), which eventually forms the outer epithelium of the tympanic membrane, plays an essential role in this developmental process. Histological and expression analyses indicate that the manubrium develops close to the EAM with a similar temporal sequence. In addition, when the middle ear ossicles are allowed to develop in vitro under conditions that do not support further EAM development, the manubrium develops only up to the stage of its induction at the time of explantation. Moreover, genetically or teratogenically derived alterations in the EAM also have an effect on manubrial development. Finally, we show that the EAM is the source of two quite opposite activities, one that induces chondrogenesis and another that represses it. The combination of these two activities results in the proper positioning of the manubrium.     

Ontogenetic and phylogenetic transformations of the ear ossicles in marsupial mammals

This study is based on the examination of histological sections of specimens of different ages and of adult ossicles from macerated skulls representing a wide range of taxa and aims at addressing several issues concerning the evolution of the ear ossicles in marsupials. Three-dimensional reconstructions of the ear ossicles based on histological series were done for one or more stages of Monodelphis domestica, Caluromys philander, Sminthopsis virginiae, Trichosurus vulpecula, and Macropus rufogriseus. Several common trends were found. Portions of the ossicles that are phylogenetically older develop earlier than portions representing more recent evolutionary inventions (manubrium of the malleus, crus longum of the incus). The onset of endochondral ossification in the taxa in which this was examined followed the sequence; first malleus, then incus, and finally stapes. In M. domestica and C. philander at birth the yet precartilaginous ossicles form a supportive strut between the lower jaw and the braincase. The cartilage of Paauw develops relatively late in comparison with the ear ossicles and in close association to the tendon of the stapedial muscle. A feeble artery traverses the stapedial foramen of the stapes in the youngest stages of M. domestica, C. philander, and Sminthopsis virginiae examined. Presence of a large stapedial foramen is reconstructed in the groundplan of the Didelphidae and of Marsupialia. The stapedial foramen is absent in all adult caenolestids, dasyurids, Myrmecobius, Notoryctes, peramelids, vombatids, and phascolarctids. Pouch young of Perameles sp. and Dasyurus viverrinus show a bicrurate stapes with a sizeable stapedial foramen. Some didelphids examined to date show a double insertion of the Tensor tympani muscle. Some differences exist between M. domestica and C. philander in adult ossicle form, including the relative length of the incudal crus breve and of the stapes. Several differences exist between the malleus of didelphids and that of some phalangeriforms, the latter showing a short neck, absence of the lamina, and a ventrally directed manubrium. Hearing starts in M. domestica at an age in which the external auditory meatus has not yet fully developed, the ossicles are not fully ossified, and the middle ear space is partially filled with loose mesenchyme. The ontogenetic changes in hearing abilities in M. domestica between postnatal days 30 and 40 may be at least partially related to changes in middle ear structures.     

Scaling of the marsupial middle ear and its functional significance

The marsupial middle ear performs an anatomical impedance matching for acoustic energy travelling in air to reach the cochlea. The size of the middle ear sets constraints for the frequencies transmitted. For generalized placental mammals, it has been shown that the limit for high-frequency hearing can be predicted on the basis of middle ear ossicle mass, provided that the ears fulfil requirements of isometry. We studied the interspecific size variation of the middle ear in 23 marsupial species, with the following measurable parameters: skull mass, condylobasal length, ossicular masses for malleus, incus and stapes, tympanic membrane area, oval window area, and lever arm lengths for malleus and incus. Our results show that the middle ear size grows with negative allometry in relation to body size and that the internal proportions of the marsupial middle ear are largely isometric. This resembles the situation in placental mammals and allows us to use their isometric middle ear model to predict the high-frequency hearing limit for marsupials. We found that the isometry model predicts the high-frequency hearing limit for different marsupials well, indicating that marsupials can be used as auditory models for general therian mammalian hearing. At very high frequencies, other factors, such as the inner ear, seem to constrain mammalian hearing.     

Auditory ossicles from southwest Asian Mousterian sites

The present study describes and analyzes new Neandertal and early modern human auditory ossicles from the sites of Qafzeh and Amud in southwest Asia. Some methodological issues in the measurement of these bones are considered, and a set of standardized measurement protocols is proposed. Evidence of erosive pathological processes, most likely attributed to otitis media, is present on the ossicles of Qafzeh 12 and Amud 7 but none can be detected in the other Qafzeh specimens. Qafzeh 12 and 15 extend the known range of variation in the fossil H. sapiens sample in some metric variables, but morphologically, the new specimens do not differ in any meaningful way from living humans. In most metric dimensions, the Amud 7 incus falls within our modern human range of variation, but the more closed angle between the short and long processes stands out. Morphologically, all the Neandertal incudi described to date show a very straight long process. Several tentative hypotheses can be suggested regarding the evolution of the ear ossicles in the genus Homo. First, the degree of metric and morphological variation seems greater among the fossil H. sapiens sample than in Neandertals. Second, there is a real difference in the size of the malleus between Neandertals and fossil H. sapiens, with Neandertals showing larger values in most dimensions. Third, the wider malleus head implies a larger articular facet in the Neandertals, and this also appears to be reflected in the larger (taller) incus articular facet. Fourth, there is limited evidence for a potential temporal trend toward reduction of the long process within the Neandertal lineage. Fifth, a combination of features in the malleus, incus, and stapes may indicate a slightly different relative positioning of either the tip of the incus long process or stapes footplate within the tympanic cavity in the Neandertal lineage.     

The Origin of the Mammalian Middle Ear

A functional explanation is presented for the shift of the reptilianarticular and quadrate into the mammalian middle ear to becomethe malleus and incus. Modification of the masticatory apparatusof therapsids results in reduction of stresses on the jaw jointand consequently in reduction of posterior elements of the jaw.In the late therapsid, Bienotherium, the quadrate and post-dentaryjaw bones resemble the mammalian malleus and incus which togetherform a lever. The therapsid articular possesses a downturnedretroarticular process (for insertion of M. depressor mandibulae)homologous with the manubrium (force lever arm) of the malleus.About the time of origin of the mammalian (dentarysquamosal)jaw joint and following the origin of the mammalian depressor,the reptilian depressor is lost. This allows the enlarging reptiliantympanum to become attached to the retroarticular process. Thenew lever system thus formed by articular and quadrate increasesthe sensitivity of the ear and the reptilian one-bone systemis replaced. In early mammals the reflected lamina of the angularmigrates posteriorly with the angle of the dentary so that itcontacts and assumes support of the tympanum. Non-homology ofthe monotreme and therian depressors indicates a multiple originof the mammalian middle ear.     

Study of age-related changes in Middle ear transfer function

Abstract

Osteoporosis (OP) is common with advancing age. Several studies have shown a strong correlation between OP and otosclerosis. However, no studies have investigated OP of the malleus, incus or stapes in the human middle ear, its effect on middle ear transfer function. Here, we investigate whether these three ossicles develop OP, and how this affects middle ear transfer function. The effect of OP on middle ear transfer function was investigated in simulations based on a finite element (FE) method. First, the FE model used in our previous study was refined, and optimized by introducing viscoelastic properties to selected soft tissues of the middle ear. Then, the FE model was used to simulate OP of the three ossicles and assess its influence on middle ear transfer function. Other possible age-related changes, such as stiffness of the joints or ligaments in the middle ear, were also investigated. The results indicated that OP of the ossicles could increase the high frequency displacement of both the umbo and stapes footplate (FP). However, the stiffness of the middle ear soft tissue can lead to the decrease of middle ear gain at lower frequencies. Furthermore, loosening of these joints or ligaments could increase displacement of the umbo and stapes FP. In conclusion, although age-related hearing loss is most commonly conceived of as sensorineural hearing loss (SNHL), we found that age-related changes may also include OP and changes in joint stiffness, but these will have little effect on middle ear transfer function in elderly people.     

Details of human middle ear morphology based on micro‐CT imaging of phosphotungstic acid stained samples

A multitude of morphological aspects of the human middle ear (ME) were studied qualitatively and/or quantitatively through the postprocessing and interpretation of micro‐CT (micro X‐ray computed tomography) data of six human temporal bones. The samples were scanned after phosphotungstic acid staining to enhance soft‐tissue contrast. The influence of this staining on ME ossicle configuration was shown to be insignificant. Through postprocessing, the image data were converted into surface models, after which the approaches diverged depending on the topics of interest. The studied topics were: the ME ligaments; morphometric and mechanical parameters of the ossicles relating to inertia and the ossicular lever arm ratio; the morphology of the distal incus; the contact surface areas of the tympanic membrane (TM) and of the stapes footplate; and the thickness of the TM, round window of the cochlea, ossicle joint spaces, and stapedial annular ligament. Some of the resulting insights are relevant in ongoing discussions concerning ME morphology and mechanical functions, while other results provide quantitative data to add to existing data. All findings are discussed in the light of other published data and many are relevant for the construction of mechanical finite element simulations of the ME. J. Morphol. 276:1025–1046, 2015. © 2015 Wiley Periodicals, Inc.     

Middle-ear dynamics before and after ossicular replacement

The mechanism of hearing involves conduction of mechanical vibrations along the ossicular chain to the inner ear. An acoustic wave is collected and transformed as it passes down the ear canal and impacts on the tympanic membrane (ear drum). The drum is connected to the inner-ear by three ossicle bones (malleus, incus, and stapes) in a complex arrangement, which serves to further transform the mechanical vibration before it reaches the cochlea of the inner ear. What is the mechanical function of the ossicular chain, and what are the biomechanical consequences of surgical reconstruction with prostheses? To answer these questions, a three-dimensional finite element model of the outer ear canal and middle ear was generated. The dynamical behaviour was predicted for the normal ear, and an ear reconstructed with partial and total ossicular replacement prostheses. For the normal ear, stapes amplitudes of 1x10(-8) m at low frequencies decrease to 4x10(-10)m at approximately 3kHz with several resonance peeks in between, most significantly at approximately 1kHz. Thereafter a further resonance is predicted at 4kHz associated with the ear canal. The behaviour is changed fundamentally by adding a prosthesis; the partial replacement increases the vibratory coupling of the drum and the stapes compared to the normal ear whereas the total replacement does the opposite, and is predicted to have the disadvantage of bringing several new resonances of the ossicular chain into the hearing range. It is hypothesised that the function of the malleus-incus-stapes arrangement is to link the drum to the oval window with the flexibility required for impedance matching but the rigidity to prevent unconstrainable resonances from occurring in the hearing range. If this is true, then the structural stiffness of ossicular chain is the critical design variable for middle-ear replacement prostheses.     

Postnatal development of the sound-transporting apparatus in the middle ear of rabbits

The middle ear apparatus realized a multiplicatory function during the transfer of the vibrational energy, depending on the area ratio of pars tensa of tympanic membrane and of stapes footplate as well as the lever ratio of long arms of manubrium and of incus. These structures exhibit a size increase during the postnatal development, but the sequel on the multiplicatory function in rabbits is unknown. The middle ear structures of 46 rabbits, 1 to 30 d old, were prepared and measured. The area of pars tensa and the levers of malleus and incus increase with age. After the 10th d of life, no statistical significant growth were measurable. But the calculated multiplicatory factor of single animals indicate the end of the development at the 15th postnatal d. In contrast, the cochlear function attains the adult values not till the 26th d of life. It is 10 d longer than the middle ear growth.     

Hind limb movements of the American alligator (Alligator mississippiensis) and postural grades

Middle ear ontogeny in Monodelphis domestica is investigated to understand better both the immediate consequences for suckling in a neonate marsupial and the epigenetic factors that constrain morphogenesis. Neonates of Monodelphis possess neither mammalian (dentarysquamosal) nor reptilian (quadrate-articular) jaw articulations, nor does the contact between the incus and crista parotica offer a joint surface ( contra Maier, 1987). Elasticity in Meckel's cartilage allows minimal deflection of the lower jaw. Observation of the developmental rate of the individual elements reveals that mandibular arch derivatives (malleus, incus and tensor tympani) are on a slightly faster ontogenetic schedule than hyoid arch derivatives (stapes and m. stapedius).     

The influence of muscles activation on the dynamical behaviour of the tympano-ossicular system of the middle ear

The human ear is a complex biomechanical system and is divided into three parts: outer, middle and inner ear. The middle ear is formed by ossicles (malleus, incus and stapes), ligaments, muscles and tendons, which transfers sound vibrations from the eardrum to the inner ear, linking with mastoid and Eustachian tube. In this work, a finite element modelling of the tympano-ossicular system of the middle ear was developed. A dynamic study based on a structural response to harmonic vibrations, for a sound pressure level (SPL) of 110, 120 and 130 dB SPL applied in the eardrum, is presented. The connection between the ossicles is made using a contact formulation. The model includes the different ligaments considering its hyperelastic behaviour. The activation of the muscles is based on the constitutive model proposed by previous work. The harmonic responses of displacement and pressure obtained on the stapes footplate, for a frequency range between 100 Hz and 10 kHz, are obtained simulating the muscle activation. The results are compared considering the passive and active states. The results are discussed and they are in accordance with audiological data published with reference to the effects of the middle ear muscles contraction.     

Technical note: a landmark-based approach to the study of the ear ossicles using ultra-high-resolution X-ray computed tomography data

Previous study of the ear ossicles in Primates has demonstrated that they vary on both functional and phylogenetic bases. Such studies have generally employed two-dimensional linear measurements rather than three-dimensional data. The availability of Ultra- high-resolution X-ray computed tomography (UhrCT) has made it possible to accurately image the ossicles so that broadly accepted methodologies for acquiring and studying morphometric data can be applied. Using UhrCT data also allows for the ossicular chain to be studied in anatomical position, so that it is possible to consider the spatial and size relationships of all three bones. One issue impeding the morphometric study of the ear ossicles is a lack of broadly recognized landmarks. Distinguishing landmarks on the ossicles is difficult in part because there are only two areas of articulation in the ossicular chain, one of which (the malleus/incus articulation) has a complex three-dimensional form. A measurement error study is presented demonstrating that a suite of 16 landmarks can be precisely located on reconstructions of the ossicles from UhrCT data. Estimates of measurement error showed that most landmarks were highly replicable, with an average CV for associated interlandmark distances of less than 3%. The positions of these landmarks are chosen to reflect not only the overall shape of the bones in the chain and their relative positions, but also functional parameters. This study should provide a basis for further examination of the smallest bones in the body in three dimensions.     

Evolution of the mammalian middle ear.

The structure and evolution of the mandible, suspensorium, and stapes of mammal-like reptiles and early mammals are examined in an attempt to determine how, why, and when in phylogeny the precursors of the mammalian tympanic bone, malleus, and incus (postdentary jaw elements and quadrate) came to function in the reception of air-borne sound. The following conclusions are reached: It is possible that at no stage in mammalian phylogeny was there a middle ear similar to that of "typical" living reptiles, with a postquadrate tympanic membrane contracted by an extrastapes. The aquamosal sulcus of cynodonts and other therapsids, usually thought to have housed a long external acoustic meatus, possibly held a depressor mandibulae muscle. In therapsids an air-filled chamber (recessus mandibularis of Westoll) extended deep to the reflected lamina and into the depression (external fossa) on the outer aspect of the angular element. A similar chamber was present in sphenacodontids but pterygoideus musculature occupied the small external fossa. The thin tissues superficial to the recessus mandibularis served as eardrum. Primitively, vibrations reached the stapes mainly via the anterior hyoid cornu, but in dicynodonts, therocephalians, and cynodants vibrations passed mainly or exclusively from mandible to quadrate to stapes and the reflected lamina was a component of the eardrum. In the therapsid phase of mammalian phylogeny, auditory adaptation was an important aspect of jaw evolution. Auditory efficiency, and sensitivity to higher sound frequencies were enhanced by diminution and loosening of the postdentary elements and quadrate, along with transference of musculature from postdentary elements to the dentary. These changes were made possible by associated modifications, including posterior expansion of the dentary. Establishment of a dentary-squamosal articulation permitted continuation of these trends, leading to the definitive mammalian condition, with no major change in auditory mechanism except that in most mammals (not monotremes) the angular, as tympanic, eventually bcame a non-vibrating structure.     

The ear in subterranean insectivora and rodentia in comparison with ground-dwelling representatives. I. Sound conducting system of the middle ear.

Compared to acoustically unspecialized mammals (soricids and murids), the middle ear of subterranean insectivores and rodents (twelve species of six families examined) was clearly distinguished and characterized by many common features: rather round and relatively larger eardrum without a pars flaccida; reduced gonial; loose or no connection between the malleus and the tympanic bone; reduced and straightened transversal part of the malleus; enlarged incus; increased and rather flat incudo-mallear joint; rather parallel position of the mallear manubrium and incudal crus longum in some species (and their fusion in bathyergids); reduced or even missing middle ear muscles. Convergent occurrence of these structural features in taxa of different origin and their generally derived character suggest that they cannot be categorized as degenerative. The form of the stapes can be considered as a non-adaptive trait; it was taxon specific yet remarkably polymorphous in some species and exhibited no convergent features among subterranean mammals. Structural retrogression resulting in a columella-like stapes was observed in some species lacking the stapedial artery. The stapedial base was relatively larger than in unspecialized mammals. The subterranean mammals did not exhibit conspicuously enlarged eardrums as would be required for sensitive tuning to low frequencies. It is, however, argued that while selective pressures in the subterranean ecotope promoted hearing of low frequencies, hearing sensitivity did not have to be enhanced.     

Ear ossicle morphology of the Jurassic euharamiyidan Arboroharamiya and evolution of mammalian middle ear

The middle ear bones of Mesozoic mammals are rarely preserved as fossils and the morphology of these ossicles in the earliest mammals remains poorly known. Here, we report the stapes and incus of the euharamiyidan Arboroharamiya from the lower Upper Jurassic (～160 Ma) of northern China, which represent the earliest known mammalian middle ear ossicles. Both bones are miniscule in relation to those in non‐mammalian cynodonts. The skull length/stapedial footplate diameter ratio is estimated as 51.74 and the stapes length as the percentage of the skull length is 4%; both numbers fall into the stapes size ranges of mammals. The stapes is “rod‐like” and has a large stapedial foramen. It is unique among mammaliaforms in having a distinct posterior process that is interpreted as for insertion of the stapedius muscle and homologized to the ossified proximal (stapedial) end of the interhyal, on which the stapedius muscle attached. The incus differs from the quadrate of non‐mammalian cynodonts such as morganucodontids in having small size and a slim short process. Along with lack of the postdentary trough and Meckelian groove on the medial surface of the dentary, the ossicles suggest development of the definitive mammalian middle ear (DMME) in Arboroharamiya. Among various higher‐level phylogenetic hypotheses of mammals, the one we preferred places “haramiyidans” within Mammalia. Given this phylogeny, development of the DMME took place once in the allotherian clade containing euharamiyidans and multituberculates, probably independent to those of monotremes and therians. Thus, the DMME has evolved at least three times independently in mammals. Alternative hypothesis that placed “haramiyidans” outside of Mammalia would require independent acquisition of the DMME in multituberculates and euharamiyidans as well as parallel evolution of numerous derived similarities in the dentition, occlusion pattern, mandibles, cranium, and postcranium between the two groups and between “haramiyidans” and other mammals. J. Morphol. 279:441–457, 2018. © 2016 Wiley Periodicals, Inc.     

Auditory systems of Heteromyidae: functional morphology and evolution of the middle ear.

Middle ears (515) from 26 species of the rodent family Heteromyidae - genera Dipodomys, Microdipodops, Perognathus, and Liomys - were studied both grossly and histologically, for qualitative and quantitative comparisons. Middle ear modifications characteristic of each genus are qualitatively described. Quantitative comparisons are made among the 26 species in the study. Some correlations between middle ear size and other measurements are discussed. The middle ear is an acoustical transformer that for best efficiency must match the impedance of the cochlea to the impedance of the air in the external auditory meatus. It accomplishes this by a pressure increase and a velocity decrease through the combined effects of the lever and areal ratios; however, because the important consideration is a matching of two impedances rather than an absolute pressure increase, the pressure transformer ratio is a less informative measure of the middle ear's efficiency than is the impedance transform ratio. The impedance transformer mechanism is explained (from a morphological point of view), and equations are presented. Dipodomys, Microdipodops, and Perognathus have a theoretical transmission (at the resonant frequency) of 94-100% of the incident acoustical energy; Liomys, 78-80%. The areal ratio of stapes footplate to 2/3 tympanic membrane is remarkably constant among the species, varying only from 0.04 to 0.07: in Dipodomys and Microdipodops this small ratio is due to the very large tympanic membrane; in Perognathus and Liomys it is due to the extremely small stapes footplate. The lever ratio of incus to malleus varies from 0.28 to 0.33 in Dipodpmys and Microdipodops, from 0.37 to 0.46 in Perognathus, and from 0.55 to 0.60 in Liomys. In addition, the middle ear volumes and the morphology of tympanic membrane, ossicles, ligaments, and muscles, all combine to minimize both mass and stiffness. All these data suggest middle ear mechanisms which are very efficient over a broad frequency range. The middle ear modifications found in heteromyids are adaptive in predator avoidance, especially in areas of little natural cover; nevertheless, contrary to expectations, there is no firm relationship between habitat and the extent of these modifications in the 26 species. However, environment did apparently plan an important role in the evolution of the family, and this is discussed.     

Middle ear defects associated with the double knock out mutation of murine goosecoid and Msx1 genes.

A number of developmental regulatory genes, including homeobox genes, are dynamically expressed in the mammalian cephalic ectomesenchyme during craniofacial morphogenesis. Owing to the vast amount of gene knock out experiments, functions of such genes are now being revealed in the mammalian skeletal patterning process. The murine goosecoid (Gsc) and Msx1 genes are expressed during craniofacial development and each mutant mouse displays intriguing facial abnormalities including those of middle ear ossicles, suggesting that both genes play roles in spatial programming of craniofacial regions. In order to examine whether these genes could function in concert to direct particular craniofacial morphogenesis, double knock out mice were analyzed. The phenotype of the double mutant mice was restricted to the first arch derivatives and was apparently additive of the single gene mutant mice, implying region specific genetic interactions of these homeobox genes expressed in overlapping regions of middle ear forming ectomesenchyme. Our results also suggested that the patterning of distal portions of the malleus depends on the tympanic membrane, for which normal expressions of both the genes are prerequisite.