Uncovering the secreted signals and transcription factors regulating the development of mammalian middle ear ossicles.

The mammalian middle ear comprises a chain of ossicles, the malleus, incus, and stapes that act as an impedance matching device during the transmission of sound from the tympanic membrane to the inner ear. These ossicles are derived from cranial neural crest cells that undergo endochondral ossification and subsequently differentiate into their final functional forms. Defects that occur during middle ear development can result in conductive hearing loss. In this review, we summarize studies describing the crucial roles played by signaling molecules such as sonic hedgehog, bone morphogenetic proteins, fibroblast growth factors, notch ligands, and chemokines during the differentiation of neural crest into the middle ear ossicles. In addition to these cell-extrinsic signals, we also discuss studies on the function of transcription factor genes such as Foxi3, Tbx1, Bapx1, Pou3f4, and Gsc in regulating the development and morphology of the middle ear ossicles.

Histological development and integration of the Zebrafish Weberian apparatus.

BACKGROUND: The Weberian apparatus enhances hearing in otophysan fishes, including Zebrafish (Danio rerio). Several studies have examined aspects of morphological development of the Weberian apparatus and hearing ability in Zebrafish. A comprehensive developmental description including both hard and soft tissues is lacking. This information is critical for both interpretation of genetic developmental analyses and to better understand the role of morphogenesis and integration on changes in hearing ability. RESULTS: Histological development of hard and soft tissues of the Weberian apparatus, including ossicles, ear, swim bladder, and ligaments are described from early larval stages (3.8 mm notochord length) through adult. Results show a strong relationship in developmental timing and maturation across all regions. All required auditory elements are present and morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and changes in shape continue throughout the examined developmental period. CONCLUSIONS: This study provides the first comprehensive histological description of Weberian apparatus development in Zebrafish. Morphological integration was found early, before increases in hearing ability were detected in functional studies (>10 mm total length), suggesting morphological integration precedes functional integration. Further research is needed to examine the nature of the functional delay, and how maturation of the Weberian apparatus influences functionality.

Hox-A2 protein expression in mouse embryo middle ear ossicles.

The origin of the mammalian middle ear ossicles from mandibular and hyoid pharyngeal arches remains controversial and discussed. Two adverse theories are proposed. The first claims that malleus and incus derive from the Meckel's cartilage of the mandibular arch, and stapes from Reichert's cartilage of the hyoid arch. The second postulates that handle of malleus and long process of the incus are derived from the second arch as well as the stapes. Contradictory analyses support alternatively each theory without any experimental evidence. In order to bring new data, we analyzed by immunohistochemistry the expression of Hox-A2 protein in ossicular anlagen in E11 to 13 mouse embryos. HOXA2 gene is known to be expressed in second arch cells and to be absent from mandibular arch derivatives. Surprisingly, Hox-A2 protein was present in all ossicular primordia, as well in Reichert's cartilage. Meckel's cartilage was free of staining. Unlabeled cells were also present in ossicular blastemata. These results suggest that ossicular condensations could arise from mixed cell populations originated in both mandibular and hyoid pharyngeal arches. However, we cannot exclude that diffuse Hox-A2 immunoreactivity could correspond to a secondary expression in craniofacial mesenchyme independently from the branchial origin of cells.

Reassessment of the La Ferrassie 3 Neandertal ossicular chain.

The ossicular chain in La Ferrassie 3 was briefly described in the monograph on the La Ferrassie Neandertal children, but to date has not been the subject of detailed study. We provide new data on these important fossils and re-examine some previous suggestions of derived Neandertal features in the middle ear ossicles based on more limited evidence. The malleus shows a curved lateral margin of the manubrium and a relatively large head. The incus shows a tall articular facet, a depressed area on the medial surface of the body, a straight anterior border of the long process and a more closed angle between the processes. The stapes shows an asymmetrical configuration of the crura, with an anteriorly skewed head, and generally small dimensions, including a smaller and relatively wider stapedial footplate. These same features can also be seen in the few other Neandertal ear ossicles known, suggesting that a consistent anatomical pattern characterizes the Neandertal ossicular chain. While the phylogenetic polarity of many of these features remains to be clarified, the asymmetrical stapes and anteriorly skewed stapedial head appear to be derived Neandertal features. In addition, while the larger malleus head and incus articular facet in La Ferrassie 3 might reflect larger body mass in Neandertals, the larger stapes footplates in Homo sapiens cannot be explained by changes in body mass. Indeed, H. sapiens seems to depart from the general mammalian pattern in combining an increase in stapes footplate size with a decrease in body mass. Although the malleus/incus lever ratio in La Ferrassie 3 is similar to that in H. sapiens, Neandertals appear to be characterized by a slightly different spatial relationship and articulation of the ossicular chain within the tympanic cavity. While only limited inferences can be drawn regarding hearing ability based on the ossicles, the few physiologically relevant dimensions in the La Ferrassie 3 ear bones are similar to H. sapiens.

Development of the skull of the pantropical spotted dolphin (Stenella attenuata).

We describe the bony and cartilaginous structures of five fetal skulls of Stenella attenuata (pantropical spotted dolphin) specimens. The specimens represent early fetal life as suggested by the presence of rostral tactile hairs and the beginnings of skin pigmentation. These specimens exhibit the developmental order of ossification of the intramembranous and endochondral elements of the cranium as well as the functional and morphological development of specific cetacean anatomical adaptations. Detailed observations are presented on telescoping, nasal anatomy, and middle ear anatomy. The development of the middle ear ossicles, ectotympanic bone, and median nasal cartilage is of interest because in the adult these structures are morphologically different from those in land mammals. We follow specific cetacean morphological characteristics through fetal development to provide insight into the form and function of the cetacean body plan. Combining these data with fossil evidence, it is possible to overlie ontogenetic patterns and discern evolutionary patterns of the cetacean skull.

Postnatal development of the middle ear in New Zealand White rabbits: ossicles and tympanic ring.

We studied the postnatal development of the middle ear (ME) in New Zealand White rabbits. Bullae were scanned using a desktop X-ray microtomograph and 3D models of the ME ossicles as well as the tympanic ring (TR) were prepared. In 0, 1, 2 days old rabbits the ossification process was incomplete. We can therefore present quantitative data obtained from older rabbits (ages: 4-180 days) and a qualitative description at the earlier ages. For a number of the measured parameters an exponential curve could be fitted to the data, and the time constant (at which 63% of the final value was obtained) was calculated. The length of the manubrium increased rapidly in a period of about 15 postnatal days, from 1.73 mm to 4.08 mm. The distance between the tip of the malleus and the TR increased rapidly until day 30, from nearly 0 to 1.40 mm. The increase of the surface area within the TR was small as compared to inter-specimen variance, but the ratio [tympanic membrane area]/[TR area] clearly increased (from 1.00 to 1.11), with a time constant of 8.3 days. The area of the stapes footplate (FP) increased rapidly in about 15 days (from 0.72 mm(2) to 1.49 mm(2), time constant 4.8 days). The TR was nearly developed at birth whereas the stapes footplate was quite underdeveloped. The distance between the tip of the malleus and the incudomallear rotation axis increased rapidly until day 20 and varied between 3.47 mm and 5.00 mm. The distance between the tip of the incus and the rotation axis increased until day 133, from 1.39 mm to 1.69 mm. Our study shows that in rabbits the ME is underdeveloped at birth and that the functional geometry develops over the same time course as the ability to hear. The conical shape of the tympanic membrane (TM) is formed by retraction and growth of the manubrium, mainly during the first 40 days after birth.

Developmental and morphological variation in the teleost craniofacial skeleton reveals an unusual mode of ossification.

We investigated the morphology and development of the scleral ossicles within the eyes of three species from three basal teleost orders, namely, the alewife (Alosa pseudoharengus; Clupeiformes), the surface morph of the Mexican tetra (Astyanax mexicanus; Characiformes) and zebrafish (Danio rerio; Cypriniformes). Two morphologies, circular and elongated, and one variation, fused elements, were identified. Zebrafish have small circular ossicles, whereas the alewife and the Mexican tetra have elongated ossicles. Surprisingly in the Mexican tetra these elements fuse at one end forming a continuous element with an antero-ventral opening; this may be typical for the Order Characiformes. Regardless of morphology, the ossicles develop via unilateral perichondral ossification of the scleral cartilage from two centers opposite one another in the eye. This unilateral type of ossification, in which only the perichondrium furthest from the retina contributes to the ossicles, has not previously been reported in any vertebrate. Because either the perichondrium and/or an extension of the perichondrium can transform into the scleral ossicle, we refer to the transitional tissue as periskeletal. Although the functional significance of the different shaped ossicles is unclear, skeletal muscle attaches directly to these bones, implying voluntary control. The morphological and developmental variation of teleost scleral ossicles makes them an ideal system for determining the genetic basis underlying phenotypic variation as well as for studying mechanisms underlying osteogenic and chondrogenic processes in teleosts. These data support our previous finding that scleral ossicles in teleosts may not be homologous to those in other vertebrates, such as reptiles.

Regulated gene expression of hyaluronan synthases during Xenopus laevis development.

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.

Development of form and function in peripheral auditory structures of the zebrafish (Danio rerio).

Investigations of the development of auditory form and function have, with a few exceptions, thus far been largely restricted to birds and mammals, making it difficult to postulate evolutionary hypotheses. Teleost fishes represent useful models for developmental investigations of the auditory system due to their often extensive period of posthatching development and the diversity of auditory specializations in this group. Using the auditory brainstem response and morphological techniques we investigated the development of auditory form and function in zebrafish (Danio rerio) ranging in size from 10 to 45 mm total length. We found no difference in auditory sensitivity, response latency, or response amplitude with development, but we did find an expansion of maximum detectable frequency from 200 Hz at 10 mm to 4000 Hz at 45 mm TL. The expansion of frequency range coincided with the development of Weberian ossicles in zebrafish, suggesting that changes in hearing ability in this species are driven more by development of auxiliary specializations than by the ear itself. We propose a model for the development of zebrafish hearing wherein the Weberian ossicles gradually increase the range of frequencies available to the inner ear, much as middle ear development increases frequency range in mammals.

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.

Rotation of middle ear ossicles during cetacean development.

Cetacean middle ears are unique among mammals in that they have an elongated tympanic membrane, a greatly reduced manubrium mallei, and an incudal crus longum that is shorter than the crus breve. Elongation of the tympanic membrane and reduction of the manubrium is thought to be related to an evolutionary rotation of the incus and malleus out of the plane of the tympanic membrane. We examined if rotation also occurs during ontogeny by comparing the middle ears of two species of dolphins (Delphinus delphis, Stenella attenuata) at different stages of development. We observed that: the incus has the body and crural proportions as in terrestrial mammals early in development; the incudomallear complex rotates approximately 90 degrees following ossification; the tympanic membrane is not elongated until relatively late in development. Therefore, some of the unique characteristics of the cetacean middle ear develop as modifications of an initially terrestrial-like morphology.

Morphometric investigation on osteocytes in human auditory ossicles.

An osteocyte lacunae differential count (1-lacunae with live osteocytes, 2-lacunae with degenerating osteocytes, 3-empty lacunae) was carried out on ear ossicles and clavicles from cadavers as well as on stapes removed by stapedotomy. The distance of the three types of lacunae from the vascular source was also determined by a computer-assisted light microscope. Results showed that the delayed fixation of bone from cadavers does not significantly interfere with osteocyte preservation, at least with the scope of this investigation. The results of osteocyte differential count show that the number of empty lacunae and lacunae with degenerating osteocytes: (a) is significantly higher in ear ossicles than in clavicles, (b) increases with age, (c) is higher in stapes than in incuses and mallei, (d) increases with the distance from the vascular sources in both ear ossicles and clavicles. Additionally it appeared that the process of osteocyte degeneration in ear ossicles is very rapid and widespread, over 40% of the cells being dead within the 2nd year of age. In the light of the recent literature and personal findings, which ascribe to osteocytes the function of mechanical detectors, and considering that bone remodeling occasionally occurs in ear ossicles, it is postulated that osteocyte death in these bones could be a programmed phenomenon (apoptosis?), due to which they lose the ability to react to strains and stresses and achieve the structural stability they need to perform their peculiar stereotyped function.

Coevolution of the mammalian middle ear and neocortex.

Phylogenetic analysis with x-ray computed tomography of fossilized and recent crania implicates differential growth of the neocortex in the evolution and development of the mammalian middle ear. In premammalian tetrapods, the middle ear evolved as a chain of bones attached to the mandible and cranium, but in adult mammals the chain is detached from the mandible and lies behind it. The neocortex evolved concurrently with detachment of the chain. In mammalian development the auditory chain arises connected to the mandible but later detaches, recapitulating the phylogenetic transformation. In modern didelphid development, the auditory chain reaches mature size by the third week after birth and is then separated from the jaw and displaced caudally as the neocortex grows for another 9 weeks.

Development of the chick's auditory ossicle, the columella.

The avian middle ear contains a single auditory ossicle that is composed of a bony columella and a cartilaginous extracolumella. The columellar footplate (clipeolus) inserts into the oval window at its proximal end. The extracolumella attaches to the tympanic membrane at its distal end. The present study was undertaken to determine the patterns of growth, chondrification, and ossification of the chick's auditory ossicle before and after hatching.

Middle ear development. III: Morphometric changes in the conducting apparatus of the Mongolian gerbil.

Middle-ear structural ontogeny was examined in 12 age groups of Mongolian gerbils between 2 and 42 days after birth. Measurements of tympanic membrane surface area; depth of the tympanic membrane cone; the lengths of the malleus and incus long processes; and stapes footplate, annular space, and oval window areas were obtained using video micrographs and computer digitization techniques. The incus long process matured first at 3.5 days after birth, while the pars flaccida surface area was the last middle-ear variable studied to reach adult size (26 days after birth). The incus long process increased its length by 30% from 0.5 mm to 0.65 mm. The malleus long process, however, demonstrated much more relative growth (47%). Pars tensa area expanded from 6.35 mm2 at two days after birth to its adult size of 16.9 mm2 and the stapes footplate expanded by 50%. The developmental changes observed in middle-ear anatomy are then discussed with regard to their contribution to the functional maturation of both the middle ear and more central auditory function.

[Morphological studies of postnatal changes of the sound transport apparatus in the middle ear of humans and rabbits]. / Morphologische Untersuchungen über postnatale Veränderungen des Schallantransportapparates im Mittelohr bei Mensch und Kaninchen.

The dimensions of the tympanic membrane and middle ear ossicles, respectively, were measured, in order to investigate the postnatal development of sound transport function. In human we observed only a small increase of the tympanic membrane area between the 7th postconceptional month and the 32nd year of life. This growth has no functional effect on the sound transport. In contrast, in rabbits all measured parameters with the exception of the stapes plate area show an increase until the 12th day of life. Thus, the amplification factor doubles in this species during the first 2 weeks of life. The temporal parallelism of the middle ear development with the ontogenesis of the acoustic function seems to exist in humans, but does not in rabbits.

Quantitative evaluation of postnatal bone growth in the auditory ossicles of the dog.

The postnatal bone growth (appositional and internal) in the auditory ossicles and in the tibia of three mongrel dogs was studied by means of tetracycline and alizarin labeling. The appositional growth is characterized by an increase in the external dimensions of the auditory ossicles within the first month of postnatal life. No trace of bone deposition was found after this period of time. The patterns of internal growth are: 1) the rate of primary osteon formation shows the same value in the incus and malleus; 2) these values are always less than those in the primary osteons of the diaphysis of the tibia of the same dog; 3) the time required for the formation of these osteons in the auditory ossicles, like in the other parts of the skeleton, increases significantly with age.

A topographic quantitative analysis of the post-natal bone formation in the auditory ossicles of the dog.

The amount and distribution of the post-natal bone deposition in the auditory ossicles and in the left tibia of dogs of varying ages were studied by means of alizarin labelling. The relative amount of fluorescent new-formed bone was expressed as a percentage ratio NB/(NB+PB) of new bone (NB) on the pre-existing bone (PB). The result was that the post-natal bone deposition (1) was larger in the tibia than in the incus, malleus and stapes; (2) significantly decreased with age both in the ossicles and in the tibia; (3) in the stapes it stopped at 3 months, while it was present in the tibia, incus and malleus even at 12 months. In the ossicles the post-natal bone deposition takes place both on the periosteal surfaces of the ossicles and on the internal surfaces of the haversian systems. The first process produces an appositional growth that stops in all three ossicles within the 1st month of post-natal life, the second one produces an internal growth that continues until the age of three months in the stapes, while in the incus and malleus it occurs in small amounts, even in the 12th month of life. In the ossicles all the new-formed bone tissue, periosteal and osteonic, is built up by primary bone (addition bone). In the tibia from 50 days of age the primary bone is gradually replaced by secondary haversian systems as a consequence of remodelling processes.