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.

Development of the Human Incus With Special Reference to the Detachment From the Chondrocranium to be Transferred into the Middle Ear.

The mammalian middle ear represents one of the most fundamental features defining this class of vertebrates. However, the origin and the developmental process of the incus in the human remains controversial. The present study seeks to demonstrate all the steps of development and integration of the incus within the middle ear. We examined histological sections of 55 human embryos and fetuses at 6 to 13 weeks of development. At 6 weeks of development (16 Carnegie Stage), the incus anlage was found at the cranial end of the first pharyngeal arch. At this stage, each of the three anlagen of the ossicles in the middle ear were independent in different locations. At Carnegie Stage 17 a homogeneous interzone clearly defined the incus and malleus anlagen. The cranial end of the incus was located very close to the otic capsule. At 7 and 8 weeks was characterized by the short limb of the incus connecting with the otic capsule. At 9 weeks was characterized by an initial disconnection of the incus from the otic capsule. At 13 weeks, a cavity appeared between the otic capsule and incus. Our results provide significant evidence that the human incus developed from the first pharyngeal arch but independently from Meckel's cartilage. Also, during development, the incus was connected with the otic capsule, and then it was detached definitively. The development of the incus in humans provides evidence that this ossicle is homologous to the quadrate. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

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.

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.

[Ontogenic development of the incudostapedial joint]. / Desarrollo ontogénico de la articulación incudoestapedial.

OBJECTIVE: To study the development of the incudostapedial joint in human embryos and foetuses. MATERIAL AND METHOD: 46 temporal bones with specimens between 9 mm and newborns were studied. The preparations were sliced serially and dyed using the Martins trichrome technique. RESULTS: The incudostapedial joint takes on the characteristics of a spheroidal joint at 16 weeks of development. The cartilage covering the articular surfaces is formed by different strata that develop in succession: the superficial stratum at 19 weeks, the transitional between 20 and 23 weeks, and the radial from 24 weeks on. The subchondral bone develops after 29 weeks by the mechanisms of apposition and extension of the periosteal and endosteal bones, but it is not until week 34 that it completely covers the articular surfaces, following constitution of the bone fascicles transmitting the lines of force. The articular capsule is formed from the inter-zone, the surface zone develops the capsular ligament, and the internal surface develops the synovial membrane. CONCLUSIONS: At the time of birth, the incudostapedial joint is completely developed.

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.

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.

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.

Desarrollo del oído medio humano entre la 5a y la 17a semana de gestación / Human middle ear development between the 5th and the 17th week of pregnancy

Se realizó un estudio de la embriogénesis del oído medio humano entre la 5a y la 17a semana de gestación, utilizando 50 individuos (9 embriones y 41 fetos), los cuales fueron fijados, realizándose técnicas de tinción corrientes (hematoxilina-eosina), histoquímicas, tricrómicas e inmunohistoquímicas (anticuerpos monoclonales antilaminina, anticolágeno IV, antineurofilamentos). Seis fetos fueron procesados para microscopía electrónica de barrido, intentando obtener la mayor información posible del material humano. El análisis de los resultados reveló que el desarrollo del oído medio humano es mas tardío que el de los animales, distinguimos un crecimiento interesante de la tuba auditiva y como esta recubre a los huesecillos formando sus mesos, describimos la fijación de la platina del estribo a la pared medial de la caja timpánica en un período específico del desarrollo y la posterior formación, por muerte celular programada de la ventana oval y un nuevo pericondrio, hecho llamativo en la embriogénesis. En los embriones y fetos estudiados se describe la formación de nudos epidérmicos en la membrana timpánica (en su capa epitelial), dentro del desarrollo normal de esta estructura. Este hallazgo, no ha sido comunicado en la literatura. Con el conocimiento actual y el avance en la tecnología, se puede utilizar técnicas mas avanzadas de tinciones histológicas, inmunohistoquímicas y el uso de microscopio de barrido, para esclarecer las interrogantes del desarrollo del oído medio humano, agregando nueva información a lo tradicionalmente conocido

[Some structural characteristics of articulation of the incus and stapes in man]. / Nekotorye osobennosti stroeniia nakovalnestremennogo sochleneniia cheloveka.

Structural characteristics of human incudostapedial articulation (ISA) have been studied on a series of histological sections from temporal bone decalcified pyramids in three planes and by means of macro-micropreparation of tympanal structural elements from 96 human fetuses at various terms of gestation. It was established that the surface of the lenticular process has a spherical form. Its length surpasses its width. The articular surface on the head of the stapes presents a dome-shaped lacuna. The capsule is usually thicker in its posterior part than in the anterior one. It can endure deviation of the long lenticular process up to 3 mm without rupture. When the deviation overruns 3.5 mm, the articular bursa and round ligament of the base of the stapes break. This property of the capsule should be kept in mind when operating on the stapes.

[The morphometry of the ear ossicles in humans during development]. / Zur Morphometrie der Gehörknöchelchen beim Menschen im Rahmen der Entwicklung.

The dimensions and the mass of the auditive ossicles was determined bilaterally in 100 human fetuses, of each sex aged from 21 to 40 weeks and 20 individual adults aged 18 to 40 years. It was found that the development of auditive ossicles in human is not completed during fetal life. The analyzed parameters of malleus (a, b, c, d1, d2, e) in the over fetal life period increased adequately by 14.02%, 11.22%, 16.70%, 12.80%, 12.01%, 21.98%, incus by (a, b, c1, c2, d, e) - 7.61%, 11.48%, 11.40%, 23.59%, 12.14%, 14.94%, stapes by (a, b, c, d, e, f) - 6.28%, 7.66%, 8.40%, 4.54%, 4.54%, 4.16%. In the over fetal life period increase of weight of malleus by 22.05%, incus by 26.49%, stapes by 11.57% was also observed. Described parameters of ossicles system are very important with respect to classification to the operations improving hearing in the system convecting of vibration of the tympanic membrane in children.