Histochemical and Ultrastructural Study of Developing Gonial Bone With Reference to Initial Ossification of the Malleus and Reduction of Meckel's Cartilage in Mice.

Development of mouse gonial bone and initial ossification process of malleus were investigated. Before the formation of the gonial bone, the osteogenic area expressing alkaline phosphatase and Runx2 mRNA was widely recognized inferior to Meckel's cartilage. The gonial bone was first formed within the perichondrium at E16.0 via intramembranous ossification, surrounded the lower part of Meckel's cartilage, and then continued to extend anteriorly and medially until postnatal day (P) 3.0. At P0, multinucleated chondroclasts started to resorb the mineralized cartilage matrix with ruffled borders at the initial ossification site of the malleus (most posterior part of Meckel's cartilage). Almost all CD31-positive capillaries did not run through the gonial bone but entered the cartilage through the site where the gonial bone was not attached, indicating the forms of the initial ossification site of the malleus are similar to those at the secondary ossification center rather than the primary ossification center in the long bone. Then, the reducing process of the posterior part of Meckel's cartilage with extending gonial bone was investigated. Numerous tartrate-resistant acid phosphatase-positive mononuclear cells invaded the reducing Meckel's cartilage, and the continuity between the malleus and Meckel's cartilage was completely lost by P3.5. Both the cartilage matrix and the perichondrium were degraded, and they seemed to be incorporated into the periosteum of the gonial bone. The tensor tympani and tensor veli palatini muscles were attached to the ligament extending from the gonial bone. These findings indicated that the gonial bone has multiple functions and plays important roles in cranial formation. Anat Rec, 302:1916-1933, 2019. © 2019 American Association for Anatomy.

Region-specific endodermal signals direct neural crest cells to form the three middle ear ossicles.

Defects in the middle ear ossicles - malleus, incus and stapes - can lead to conductive hearing loss. During development, neural crest cells (NCCs) migrate from the dorsal hindbrain to specific locations in pharyngeal arch (PA) 1 and 2, to form the malleus-incus and stapes, respectively. It is unclear how migratory NCCs reach their proper destination in the PA and initiate mesenchymal condensation to form specific ossicles. We show that secreted molecules sonic hedgehog (SHH) and bone morphogenetic protein 4 (BMP4) emanating from the pharyngeal endoderm are important in instructing region-specific NCC condensation to form malleus-incus and stapes, respectively, in mouse. Tissue-specific knockout of Shh in the pharyngeal endoderm or Smo (a transducer of SHH signaling) in NCCs causes the loss of malleus-incus condensation in PA1 but only affects the maintenance of stapes condensation in PA2. By contrast, knockout of Bmp4 in the pharyngeal endoderm or Smad4 (a transducer of TGFß/BMP signaling) in the NCCs disrupts NCC migration into the stapes region in PA2, affecting stapes formation. These results indicate that region-specific endodermal signals direct formation of specific middle ear ossicles.

Early development of the malleus and incus in humans.

It is widely accepted by developmental biologists that the malleus and incus of the mammalian middle ear are first pharyngeal arch derivatives, a contention based originally on classical embryology that has now been backed up by molecular evidence from rodent models. However, it has been claimed in several studies of human ossicular development that the manubrium of the malleus and long process of the incus are actually derived from the second arch. This 'dual-arch' interpretation is commonly presented in otolaryngology textbooks, and it has been used by clinicians to explain the aetiology of certain congenital abnormalities of the human middle ear. In order to re-examine the origins of the human malleus and incus, we made three-dimensional reconstructions of the pharyngeal region of human embryos from 7 to 28 mm crown-rump length, based on serial histological sections from the Boyd Collection. We considered the positions of the developing ossicles relative to the pharyngeal pouches and clefts, and the facial and chorda tympani nerves. Confirming observations from previous studies, the primary union between first pharyngeal pouch and first cleft found in our youngest specimens was later lost, the external meatus developing rostroventral to this position. The mesenchyme of the first and second arches in these early embryos seemed to be continuous, but the boundaries of the developing ossicles proved to be very hard to determine at this stage. When first distinguishable, the indications were that both the manubrium of the malleus and the long process of the incus were emerging within the first pharyngeal arch. We therefore conclude that the histological evidence, on balance, favours the 'classical' notion that the human malleus and incus are first-arch structures. The embryological basis of congenital ossicular abnormalities should be reconsidered in this light.

Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus.

Endochondral ossification is a developmental process by which cartilage is replaced by bone. Terminally differentiated hypertrophic chondrocytes are calcified, vascularized, and removed by chondroclasts before bone matrix is laid down by osteoblasts. In mammals, the malleus is one of three auditory ossicles that transmit vibrations of the tympanic membrane to the inner ear. The malleus is formed from a cartilaginous precursor without growth plate involvement, but little is known about how bones of this type undergo endochondral ossification. Here, we demonstrate that in the processus brevis of the malleus, clusters of osteoblasts surrounding the capillary loop produce bone matrix, causing the volume of the capillary lumen to decrease rapidly in post-weaning mice. Synchrotron X-ray tomographic microscopy revealed a concentric, cylindrical arrangement of osteocyte lacunae along capillaries, indicative of pericapillary bone formation. Moreover, we report that overexpression of Fosl1, which encodes a component of the AP-1 transcription factor complex, in osteoblasts significantly blocked malleal capillary narrowing. These data suggest that osteoblast/endothelial cell interactions control growth plate-free endochondral ossification through 'osteogenic capillaries' in a Fosl1-regulated manner.

Fetal development of the elastic-fiber-mediated enthesis in the human middle ear.

In the human middle ear, the annular ligament of the incudostapedial joint and the insertions of the tensor tympani and stapedius muscles contain abundant elastic fibers; i.e., the elastic-fiber-mediated entheses. Hyaluronan also coexists with the elastic fibers. In the present study using immunohistochemistry, we demonstrated the distribution of elastin not only in the incudostapedial joint but also in the other two joints of the middle ear in adults and fetuses. In adults, the expression of elastin did not extend out of the annular ligament composed of mature elastic fibers but clearly overlapped with it. Electron microscopic observations of the annular ligament demonstrated a few microfibrils along the elastic fibers. Thus, in contrast to the vocal cord, the middle ear entheses seemed not to contain elaunin and oxytalan fibers. In mid-term fetuses (at approximately 15-16 weeks of gestation) before opening of the external acoustic meatus, the incudostapedial joint showed abundant elastic fibers, but the incudomalleolar and stapediovestibular joints did not. At this stage, hyaluronan was not colocalized, but distributed diffusely in loose mesenchymal tissues surrounding the ear ossicles. Therefore, fetal development of elastin and elastic fibers in the middle ear entheses is unlikely to require acoustic oscillation. In late-stage fetuses (25-30 weeks), whose ear ossicles were almost the same size as those in adults, we observed bundling and branching of elastic fibers. However, hyaluronan expression was not as strong as in adults. Colocalization between elastic fibers and hyaluronan appeared to be a result of postnatal maturation of the entheses.

Thyroid hormone receptors control developmental maturation of the middle ear and the size of the ossicular bones.

Thyroid hormone is critical for auditory development and has well-known actions in the inner ear. However, less is known of thyroid hormone functions in the middle ear, which contains the ossicles (malleus, incus, stapes) that relay mechanical sound vibrations from the outer ear to the inner ear. During the later stages of middle ear development, prior to the onset of hearing, middle ear cavitation occurs, involving clearance of mesenchyme from the middle ear cavity while the immature cartilaginous ossicles attain appropriate size and ossify. Using in situ hybridization, we detected expression of Thra and Thrb genes encoding thyroid hormone receptors α1 and ß (TRα1 and TRß, respectively) in the immature ossicles, surrounding mesenchyme and tympanic membrane in the mouse. Thra(+/PV) mice that express a dominant-negative TRα1 protein exhibited deafness with elevated auditory thresholds and a range of middle ear abnormalities including chronic persistence of mesenchyme in the middle ear into adulthood, markedly enlarged ossicles, and delayed ossification of the ossicles. Congenitally hypothyroid Tshr(-/-) mice and TR-deficient Thra1(-/-);Thrb(-/-) mice displayed similar abnormalities. These findings demonstrate that middle ear maturation is TR dependent and suggest that the middle ear is a sensitive target for thyroid hormone in development.

Contribution to morphological knowledge of the development of the human incudo-mallear joint.

CONCLUSION: At the time of birth, the incudo-mallear joint is completely developed. OBJECTIVE. To study the development of the incudo-mallear joint in human embryos and fetuses. MATERIALS AND METHODS. In all, 46 temporal bones with ages between 9 mm and newborns were studied. The preparations were cut in a series and dyed using Martins' trichrome technique. RESULTS. The incudo-mallear joint acquires the characteristics of a saddle joint at 10 weeks of development. The cartilage that covers the articular surfaces is formed by different strata that develop successively: the superficial stratum at 14 weeks, the transitional between 15 and 19 weeks, and the radial from 20 weeks. The subchondral bone develops between weeks 25 and 28 by the mechanisms of apposition and extension of the periosteal and endosteal bones, but it is not until week 30 that it completely covers the articular surfaces, consisting of bone fascicles whereby the lines of force will be transmitted. The articular capsule is formed as from the inter-zone. The surface zone develops the capsular ligament, and the internal surface develops the synovial membrane. Even though it is not consistent, the primordium of the meniscus is seen at 18 weeks.

Tympanic ossicles and pharyngeal arches.

We have performed a study on 11 human embryos regarding the development of the tympanic ossicles and their relationship with the first pharyngeal arch. After performing measurements to date the embryos and foetuses chronologically, we performed a meticulous dissection of the temporal bones. Subsequently, they were fixed in 10% formol, decalcified with 2% nitric acid, embedded in Paraplast, sectioned in 7-mm sequences and stained with Martin's trichrome technique. In the 21- and 24-mm cranium-raquis (CR) length human embryos, we have observed the head of the malleus and the body of the incus close to Meckel's cartilage, in addition to the handle of the malleus, the long limb of the incus and the stapes. Between them there was a mesenchymal band inside the primordium of the tympanic cavity. In the 27-mm CR embryo, the various components of the malleus and incus were fusing, and in the 30-mm CR embryo the union was complete. From our observations, we can conclude that the malleus and the incus are derived from the first and second pharyngeal arches.

Development of the dynamic structure (force lines) of the middle ear ossicles in human foetuses.

OBJECTIVES: To study the ontogenic development of the organisation of the human middle ear ossicles structure. MATERIAL AND METHODS: 46 human temporal bones of ages varying from 32 days post-conception to newborns. RESULTS: The development of the structural organisation of the malleus begins at 16 weeks via two cortical fascicles situated in the neck; at 21 weeks they extend towards the head, at 23 weeks to the lateral process and at 24 weeks to the handle. In the handle, the force lines are transmitted via three cardinal fascicles, two of them via the cortical fascicle and one via the centre, which starts after 29 weeks' development and is consolidated after 31 weeks. In the incus the force lines start at 16 weeks via two cortical fascicles situated in the long process, which progressively extend in a rostro-caudal direction between 17 and 20 weeks. At 21 weeks they occupy the whole extension of the long process and at 22 weeks the fusion of both cortical fascicles begins. From 30 weeks onwards it is strengthened by the crossing of bone trabeculae from one cortical to another. Two fascicles come out of the incus body, surrounding the medullary cavity and going in the direction of the short process. In the beginning, the stapes have two cortical fascicles in their crura. The remodelling process makes the internal cortical fascicle disappear and after 31 weeks all the force lines run through the external cortical fascicle. The tympanic membrane of the stapes footplate undergoes a remodelling process and after 28 weeks bony trabeculae are deposited. In newborns (40 weeks), the ossicles' structure is cavitary and has not been completed. The fan-shaped trabecular fascicle, which starts in the articular facets of the malleus and the incus, still has to develop.

Joint formation in the middle ear: lessons from the mouse and guinea pig.

The malleus, incus and stapes form an ossicle chain in the mammalian middle ear. These ossicles are articulated by joints that link the chain together. In humans and mice, fusion of the ossicles leads to hearing loss. However, in the adult guinea pig the malleus and incus are normally found as a single complex. In this report, we investigate how the malleus and incus form during mouse and guinea pig development. The murine malleus and incus develop from a single condensation that splits to form the two ossicles. Even before a morphological split, we show that the ossicles have distinct genetic identities and joint markers are expressed. In the guinea pig embryo, joint formation is initiated but no cavitation is observed, resulting in a single complex divided by a thin suture. The malleal-incudo complex in the guinea pig is, therefore, not caused by a defect in joint initiation.

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.

The discomallear ligament and the anterior ligament of malleus: an anatomic study in human adults and fetuses.

According to some reports, movement of the malleus, resulting from anterior hypertension on the discomallear ligament (DML), could produce aural symptoms related with damage to middle ear structures. The aim of this study was to examine the topographic relationship of the DML and the anterior ligament of malleus (ALM). Four fetuses and 16 adult hemi-sectioned heads were used to determine the anatomic-clinical relevance of DML and ALM in temporomandibular disorder. In fetal specimens, the DML was distinctly interposed between the malleus and the disc of the temporomandibular joint (TMJ), and the ALM had a structure apparently composed of the superior and inferior lamellae, running anteriorly in continuation with the sphenomandibular ligament (SML) through the future petrotympanic fissure (PTF). In all adult specimens, the DML was inserted into the malleus, and it expanded broadly toward the disc and capsular region of the TMJ in a triangular shape and inserted into the disc and capsule of the TMJ. The two-lamellae structure of the ALM was not distinguishable in adult specimens. The overstretched ALM resulted in movement of the malleus in five cases, but similar tension applied to the DML did not cause any movement of the malleus. This result provides an indication of the clinical significance of the ALM, a ligamentous structure continuous with the SML. It is apparent that the ALM has the potential to cause aural symptoms as a result of damage to the middle ear structure.

Malleal processus brevis is dispensable for normal hearing in mice.

The mammalian middle ear cavity contains a chain of three ossicles (the malleus, incus, and stapes), which develop from the mesenchyme of the first two branchial arches. Mice deficient in the Msx1 homeobox gene exhibit craniofacial abnormalities, including the absence of the malleal processus brevis that is normally attached to the upper part of the tympanic membrane. Here, we show that the expression of Msx1 and Msx2 overlaps in the malleal primordium during early embryonic development. A functional redundancy of Msx1 and Msx2 in the development of the middle ear is suggested by the stronger hypomorphism in the malleus of Msx1(-/-)/Msx2(-/-) embryos, including the absence of the malleal manubrium and the malleal processus brevis. The expression of Bmp4, a known downstream target of Msx1 in several developing craniofacial organs, was down-regulated in the malleal primordium, particularly in the region of the developing malleal manubrium, of Msx1 and Msx1(-/-)/Msx2(-/-) embryos. Msx genes, thus, appear to act in a cell autonomous manner, possibly by regulating Bmp4 expression, in the formation of the malleus. Transgenic rescue of the cleft palate of Msx1(-/-) mice overcame the neonatal lethality and allowed Msx1(-/-) mice to grow into adulthood but retain the phenotype of the absence of the malleal processus brevis. The availability of this animal model for the first time allowed us to measure auditory evoked potentials to assess the functional significance of the malleal processus brevis. The results demonstrated unimpaired auditory function in Msx1(-/-) mice. In addition, mutant mice appeared normal in balance behavior and in the vestibular evoked potential screening test. These results indicate that the malleal processus brevis is not necessary for sound transmission and seems dispensable for normal hearing and balance in mammals.

Fetal development of the human tympanic ossicular chain articulations.

OBJECTIVES: To obtain further knowledge on the morphogenesis of the articulations in the tympanic ossicular chain in humans. MATERIAL AND METHODS: In 25 temporal bones of human fetuses the structural development of incudomallear, incudostapedial and stapediovestibular articulations was studied. The chronological ages were between the 7th week (21 mm) and the 29th week (270 mm). RESULTS AND DISCUSSION: Incudomallear articulation showed diarthrosis and sellar joint characteristics. It showed a homogenous interzone in the 7th week of development, a three-layered interzone in the 8th week, the first cavitation signs in the 9th week and the presence of an articular cavity in the 10th week. The presence of a hyaline cartilage covering articular surfaces was observed starting in the 20th week of development. Incudostapedial articulation showed typical characteristics of a diarthrosis and spheroidal joint with a homogenous interzone at the 7th week, showing similar characteristics for 12 weeks, and completed its cavitation at the 16th week. We observed hyaline cartilage on articular surfaces from 29 weeks. Stapediovestibular articulation showed typical characteristics of syndesmosis. The annular ligament primordium derived from cartilage differentiation, both from stapedial footplate and from the surrounding otic capsule, into mesenchyme and its subsequent transformation into fibrous tissue, reaching definitive characteristics from the 12th week.

[The first appearance of Meckel's cartilage in the fetus]. / Le cartilage de Meckel au début de la période foetale.

Meckel's cartilage plays an important role in the topographical organisation and in the differentiation of the facial structure during the embryonal and even much later during the foetal period. Our observations on serial sections carried out in two human foetuses aged 12 and 16 weeks indicate that the two dorsal (tympanic) and ventral (mandibular) branches of Meckel's cartilage are perfectly defined at 16 weeks. In the dorsal branch, the primordia of the incus and of head of the malleus are still composed on non-ossified cartilage. In the ventral branch, it is also possible to describe at 16 weeks three posterior, medial and anterior parts which are composed of cartilage. The initiating role played by the ventral part of Meckel's cartilage on the ossification of the mandible leads during the embryonal period to the formation of the mandibular primary growth center, which is therefore clearly defined in our first stage at 12 weeks. The partial fibrous evolution and the regression of the major part of the ventral branch of Meckel's cartilage only start after 16 weeks of intrauterine life.

Computerized 3-dimensional study of the embryologic development of the human masticatory muscles and temporomandibular joint.

PURPOSE: In this study, the development of human embryonic temporomandibular joint (TMJ) and masticatory muscles were investigated by using computed 3-dimensional reconstructions. MATERIALS AND METHODS: Sixteen human embryos and fetuses, ranging from 6.5 to 107 mm crown-rump length, were examined. RESULTS: At 10 weeks, a band of mesenchyme extending from the attachment of the lateral pterygoid muscle to the condylar process was observed to pass through the medial side of the condylar process to attach to the malleus. The temporal, masseter, and pterygoid muscles develop from the so called "temporal muscle" primordium, and the temporal muscle was in continuity with the masseter muscle until 14 weeks of fetal life. CONCLUSIONS: The study shows that the muscles of mastication arise from a single primordium. It also confirms the presence of a ligamentous attachment between the lateral pterygoid muscle and the malleus.

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.

Maintenance of regional histodifferentiation patterns and a spatially restricted expression of type X collagen in rat Meckel's cartilage explants in vitro.

The major, central portion of Meckel's cartilage undergoes fibrous transformation and contributes to the sphenomandibular ligament, whereas its distal end undergoes endochondral ossification ultimately giving rise to inner-ear ossicles. This regional histodifferentiation of Meckel's cartilage is known to be associated with the spatially restricted expression of type X collagen. The objective of this study was to determine if this unique histodifferentiation is regulated by local environmental factors or by a preprogrammed genetic mechanism. Meckel's cartilage, and condylar cartilage used for comparison, were isolated from 17-day-old rat embryos and from newborn rats, respectively. The cartilage explants were maintained in vitro for 50 days with or without supplementation with 10% fetal bovine serum. When the explants were cultured under serum-free conditions, well-regulated cartilage development was observed. Expression of type X collagen, a differentiation marker for hypertrophic cartilage, was restricted to the distal end of Meckel's cartilage, whereas type II and IX collagens were found uniformly along the entire explant. Matrix calcification was examined histochemically using alizarin red S staining and found to be restricted to the distal end of Meckel's cartilage. Both Meckel's and condylar cartilage cultured with 10% fetal bovine serum developed unregulated dysmorphogenesis. These data suggest that, although Meckel's cartilage has an intrinsic potential to differentiate to its terminal stage, external regulatory factors can significantly influence its normal development at the molecular level.

[Oto-mandibular ligaments: disco-mallear and malleo-mandibular ligaments]. / Les ligaments oto-mandibulaires: ligaments disco-malléaire et malléo-mandibulaire.

Phylogenesis, embryology and anatomy are emphasizing that two ligaments are found between the temporo-mandibular joint and the middle ear. These two oto-mandibular ligaments are named the disco-mallear ligament and the malleo-mandibular ligament. Originating from the first arch, these ligaments are not involved in the otologic manifestations of the temporo-mandibular joint syndrome. The disco-mallear ligament is a brake applied to the anterior excursion of the disc. When this disco-mallear ligament is stretched the disc can be displaced anteriorly breeding disc displacement, hypermobility and temporo-mandibular dislocation. The malleo-mandibular ligament is a remainder of the Meckel's cartilage: its role is not clear. When excessive forces are applying on the mandible, ossicles dislocation can be seen the forces being transmitted through it.