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.     

Characters of multituberculates neglected in phylogenetic analyses of early mammals

Multituberculate anatomy is compared with that of other mammals, with an emphasis on the characters that have either been neglected or misinterpreted in previous analyses of early mam mal relationships. These are: brain structure, backward masticatory power stroke (along with aspects of cranial design), and foot structure. New data on ear ossicles and a controversy con cerning multituberculate posture are also discussed. The following characters of multitubercu late skull and lower jaw are interpreted to be related to the backward masticatory power stroke: anterior orbital area roofed dorsally and without a floor (characteristic of advanced multituber culates), parietal postorbital process, lack of the angular process and a more anterior position of the coronoid process and masseteric fossa than in all other mammals. It is argued that the parallel development in the cranial structure of multituberculates and other mammals was lim ited by the backward masticatory power stroke of multituberculates that resulted in different configuration of the masticatory musculature and related osteology. In the postcranial skeleton the parallelism was limited by the structure of the multituberculate foot, in which the calca-neum contacts the fifth metatarsal (MtV) and the middle metatarsal (MtIII) is abducted 30° from the longitudinal axis of the tuber calcanei. Backward masticatory power stroke and related skull design do not show unequivocally whether multituberculates originated from some ‘tri-conodonts’ (a polyphyletic group), or independently from all other mammals from cynodonts. The foot structure refutes the origin of multituberculates from the Morganucodontidae. The brain structure allies the multituberculates with the Triconodontidae, the postcranial skeleton of which remains unknown. New data on ear ossicles suggest close relationships of multituber culates to all modern mammals. Lack of uncontested pre-Kimmeridgian multituberculates dis proves the separate origin of multituberculates from cynodonts.     

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.     

Bapx1 regulates patterning in the middle ear: altered regulatory role in the transition from the proximal jaw during vertebrate evolution

The middle ear apparatus is composed of three endochondrial ossicles (the stapes, incus and malleus) and two membranous bones, the tympanic ring and the gonium, which act as structural components to anchor the ossicles to the skull. Except for the stapes, these skeletal elements are unique to mammals and are derived from the first and second branchial arches. We show that, in combination with goosecoid (Gsc), the Bapx1 gene defines the structural components of the murine middle ear. During embryogenesis, Bapx1 is expressed in a discrete domain within the mandibular component of the first branchial arch and later in the primordia of middle ear-associated bones, the gonium and tympanic ring. Consistent with the expression pattern of Bapx1, mouse embryos deficient for Bapx1 lack a gonium and display hypoplasia of the anterior end of the tympanic ring. At E10.5, expression of Bapx1 partially overlaps that of Gsc and although Gsc is required for development of the entire tympanic ring, the role of Bapx1 is restricted to the specification of the gonium and the anterior tympanic ring. Thus, simple overlapping expression of these two genes appears to account for the patterning of the elements that compose the structural components of the middle ear and suggests that they act in concert. In addition, Bapx1 is expressed both within and surrounding the incus and the malleus. Examination of the malleus shows that the width, but not the length, of this ossicle is decreased in the mutant mice. In non-mammalian jawed vertebrates, the bones homologous to the mammalian middle ear ossicles compose the proximal jaw bones that form the jaw articulation (primary jaw joint). In fish, Bapx1 is responsible for the formation of the joint between the quadrate and articular (homologues of the malleus and incus, respectively) enabling an evolutionary comparison of the role of a regulatory gene in the transition of the proximal jawbones to middle ear ossicles. Contrary to expectations, murine Bapx1 does not affect the articulation of the malleus and incus. We show that this change in role of Bapx1 following the transition to the mammalian ossicle configuration is not due to a change in expression pattern but results from an inability to regulate Gdf5 and Gdf6, two genes predicted to be essential in joint formation.     

On the phylogenetic position of monotremes (Mammalia,Monotremata)

Henosferida from the Middle-Upper Jurassic of Western Gondwana is the most probable sister group for monotremes. They share the derived pretribosphenic structure of lower molars combined with the presumably absent protocone on the upper molars and the plesiomorphic retention of postdentary bones and pseudangular process of the lower jaw. In addition, the two groups share the dental formula with three molars and the position of the Meckel’s groove, which passes ventral to the mandibular foramen. In the course of subsequent evolution, monotremes acquired the mammalian middle ear with three auditory ossicles independently of therian mammals and multituberculates. Jurassic Laurasian Shuotheriidae are probably a sister group of the Gondwanian clade Henosferida + Monotremata. The Jurassic shuotheriid Pseudotribos shows a great plesiomorphic similarity to monotremes in the structure of the pectoral girdle, with a large interclavicle immovably connected to the clavicle. In the lineages leading to therian mammals and multituberculates, the pectoral girdle changed probably independently and in parallel in connection with the establishment of the parasagittal posture of the forelimbs (reduction of the interclavicle, mobile articulation of the interclavicle with clavicle, reduction of the procoracoid, and development of a supraspinous fossa of the scapula) and formation of the mammalian middle ear with three auditory ossicles.     

Probable diphyletic formation of the mammalian middle ear: Monotremata and Theria

The first steps in the formation of the middle ear of the mammalian type, with the tympanum and three auditory ossicles, have only been passed by higher cynodonts. They have an incipient malleus, which developed from the anterior process of the articulare rather than the retroarticular process, which is rudimentary in cynodonts. The tympanic bone is formed of the anterior projections of the angulare. In some gorgonopians, the retroarticular process is elongated and curved anteriorly, resembling the malleus of mammals; however, this is only convergent similarity.     

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.     

Morphology of the middle ear of golden moles (Chrysochloridae)

The middle ear structures of nine species of golden moles (family Chrysochloridae) were examined under the light microscope. Auditory structures of several of these species are described here for the first time in detail, the emphasis being on the ossicular apparatus. Confirming previous observations, some golden moles (e.g. Amblysomus species) have ossicles of a morphology typical of mammals, whereas others ( Chrysospalax , Chrysochloris , Cryptochloris and Eremitalpa species) have enormously hypertrophied mallei. Golden moles differ in the nature and extent of the interbullar connection, the shape of the tympanic membrane and that of the manubrium. The stapes has an unusual orientation, projecting dorsomedially from the incus. It has been proposed that hypertrophied ossicles in golden moles are adapted towards the detection of seismic vibrations. The functional morphology of the middle ear apparatus is reconsidered in this light, and it is proposed that adaptations towards low-frequency airborne hearing might have predisposed golden moles towards the evolution of seismic sensitivity through inertial bone conduction. The morphology of the middle ear apparatus sheds little light on the disputed ordinal position of the Chrysochloridae.     

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.     

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).     

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.     

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.     

The Inner Ear of Two Late Cretaceous Multituberculate Mammals, and Its Implications for Multituberculate Hearing

The inner ear of the Late Cretaceous multituberculates Nemegtbaatar gobiensis and Chulsan-baatar vulgaris is described from serial sections and enlarged models. The size and proportions of the inner ear as a whole are as expected for extant small mammals. The lengths of the cochlea (Nemegtbaatar gobiensis, 3.0 mm, Chulsanbaatar vulgaris, 2.0 mm) are comparable to those of other multituberculates, when ratios of length of the cochlea to skull length are calculated. The vestibule is not as expanded in the two taxa as in Lambdopsalis, ?Meniscoessus, and ?Catopsalis; the estimated volume for Nemegtbaatar gobiensis is 9 mm³. A slightly laterally curved, anteriomedially directed cochlea, relatively robust ear ossicles, and the estimations of the area of the tympanic membrane and stapedial footplate in Chulsanbaatar suggest high-frequency hearing but a relatively low sensitivity to low-decibel sounds. The semicircular canals of Nemegtbaatar and Chulsanbaatar are fully developed; the size of the anterior, posterior, and lateral canals and their angles and proportions are comparable to those of extant mammals of similar size. The anterior semicircular canal of Nemegtbaatar forms a smooth half-circle and thus is more derived than the angular canal of Ornithorhynchus. The notable differences between the ratio of the width of the lateral semicircular canal to skull length and the size of the vestibule in Nemegtbaatar and the Paleocene multituberculate Lambdopsalis bulla are probably related to different modes of life.     

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.     

Evolutionary analysis of the middle and inner ear of Late Jurassic multituberculates

We describe three previously unreported specimens of petrosal bones of paulchoffatiid multituberculate mammals, collected from strata of Late Jurassic age in the Guimarota lignite mine of Leiria, west-central Portugal. The new fossils allow correction, supplementation, and confirmation of anatomical details, thus refining knowledge of general adaptation in the ear region among Jurassic multituberculates. Virtually all observed characters in the paulchoffatiid otic region are primitive relative to homologous features seen among Late Cretaceous and younger representatives of the Multituberculata; we recognize few unique otic specializations in paulchoffatiids that would preclude ancestry to later multituberculates. The plesiomorphic nature of paulchoffatiid ear regions provides no evidence in support of the hypothesis of a special, sister-group relationship between multituberculates and Late Cretaceous/Cenozoic marsupials plus placentals. Used in isolation, objective evidence derived from paulchoffatiid ear regions is consistent with interpretation of multituberculate divergence from other mammals predating the stem to living monotremes and postdating the stem to extinct morganucodontids. More broadly based comparative studies among Mesozoic mammals, however, suggest that independent acquisition of similarly advanced mammalian features was a pervasive theme among evolutionary histories of early mammals, probably including multituberculates. Although the phylogenetic position of multituberculates relative to other mammalian groups has yet to be unequivocally resolved, we suggest that a very early divergence of the group remains a distinct possibility.     

The relationships of mammals

A cladistic analysis generates alternative hypotheses regarding both the origin and the interrelationships of mammals to those most widely accepted at the present time. It is proposed that the tritylodontids are more closely related to mammals than is Probainognathus ; that the non-therian mammals do not constitute a monophyletic group; and that the monotremes are related to the modern therians, the ear ossicles among other characters having evolved only once. The multituberculates may be related to the monotremes.
It is argued that the current views are variously based on an overemphasis of superficial dental similarities, misinterpretation of the structure of the mammalianbraincaseand too readyacceptance of parallel evolution amongstthe groups concerned. The hypotheses proposed here are apparently much more parsimonious.     

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.