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.

Human auditory ossicles as an alternative optimal source of ancient DNA.

DNA recovery from ancient human remains has revolutionized our ability to reconstruct the genetic landscape of the past. Ancient DNA research has benefited from the identification of skeletal elements, such as the cochlear part of the osseous inner ear, that provides optimal contexts for DNA preservation; however, the rich genetic information obtained from the cochlea must be counterbalanced against the loss of morphological information caused by its sampling. Motivated by similarities in developmental processes and histological properties between the cochlea and auditory ossicles, we evaluate the ossicles as an alternative source of ancient DNA. We show that ossicles perform comparably to the cochlea in terms of DNA recovery, finding no substantial reduction in data quantity and minimal differences in data quality across preservation conditions. Ossicles can be sampled from intact skulls or disarticulated petrous bones without damage to surrounding bone, and we argue that they should be used when available to reduce damage to human remains. Our results identify another optimal skeletal element for ancient DNA analysis and add to a growing toolkit of sampling methods that help to better preserve skeletal remains for future research while maximizing the likelihood that ancient DNA analysis will produce useable results.

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.

Morphogenesis of the middle ear ossicles and spatial relationships with the external and inner ears during the embryonic period.

We describe the three-dimensional morphogenesis of the middle ear ossicles (MEOs) according to Carnegie stage (CS) in human embryos. Seventeen samples including 33 MEOs from CS18 to 23 were selected from the Kyoto Collection. The primordia of the MEOs and related structures were histologically observed and three-dimensionally reconstructed from digital images. The timing of chondrogenesis was variable among structures. The stapes was recognizable as a vague condensation of the mesenchymal cells in all samples from CS18, whereas the malleus and incus were recognizable at CS19. Chondrogenesis of all MEOs was evident in all samples after CS21. The chondrocranium was recognizable in all samples by CS18, and the perichondrium border of the auricular cartilage and otic capsule was distinct in all samples at CS23. At CS19, the MEOs were positioned in the anterior to posterior direction, following the order malleus, incus, stapes, which adjusted gradually during development. The MEOs connected in all samples after CS22. The stapes was located close to the vestibular part of the inner ear, although the basal part was not differentiated into the "footplate" form, even at CS23. The handles of the malleus were close to the tubotympanic recess at CS23, but were distant from the external auditory meatus. Determining the timeline of the formation of MEOs and connection of the external and inner ears can be informative for understanding hearing loss caused by failure of this connection. These data may provide a useful standard for morphogenesis, and will contribute to distinguishing between normal and abnormal MEO development. Anat Rec, 299:1325-1337, 2016. © 2016 Wiley Periodicals, Inc.

Vascular endothelial growth factor signaling affects both angiogenesis and osteogenesis during the development of scleral ossicles.

Intramembranous ossification is a complex multi-step process which relies on extensive interactions among bone cells and surrounding tissues. The embryonic vasculature is essential in regulating endochondral ossification; however, its role during intramembranous ossification remains poorly understood, and in vivo studies are lacking. Previous research from our lab on the development of the intramembranous scleral ossicles has demonstrated an intriguing pattern of vascular development in which the areas of future osteogenesis remain avascular until after bone induction has occurred. Such avascular zones are located directly beneath each of the conjunctival papillae, epithelial structures which provide osteogenic signals to the underlying mesenchyme. Here we provide a high-resolution map of the developing vasculature from the time of ossicle induction to mineralization using a novel technique. We show that vegfa is expressed by the papillae and nearby mesenchymal tissue throughout HH 34-37, when vascular growth is taking place, and is down-regulated thereafter. Localized inhibition of Vegf results in expansion of the avascular zone surrounding the implanted papilla and mispatterning of the scleral ossicles. These results demonstrate that Vegf signaling could provide important insights into the complex relationship between bone and vasculature during intramembranous bone development.

Crural attachment to promontory case report: implications for stapes development.

Understanding of the embryologic origin of the stapes remains controversial. Theories diverge upon whether the entirety of the stapes arises from a single source versus the footplate and suprastructure arising from distinct sources. A 12-year-old boy with left-sided conductive hearing loss had computed tomography of the temporal bone, revealing an inferiorly displaced left stapes, and a nonspecific density in the left Prussak's space. Exploratory tympanotomy revealed the crura of the stapes to be attached to the promontory. The stapes footplate was located in the oval window and was mobile.

Developmental origin and fate of middle ear structures.

Results from developmental and phylogenetic studies have converged to facilitate insight into two important steps in vertebrate evolution: (1) the ontogenetic origin of articulating elements of the buccal skeleton, i.e., jaws, and (2) the later origins of middle ear impedance-matching systems that convey air-borne sound to the inner ear fluids. Middle ear ossicles and other skeletal elements of the viscerocranium (i.e., gill suspensory arches and jaw bones) share a common origin both phylogenetically and ontogenetically. The intention of this brief overview of middle-ear development is to emphasize the intimate connection between evolution and embryogenesis. Examples of developmental situations are discussed in which cells of different provenance, such as neural crest, mesoderm or endoderm, gather together and reciprocal interactions finally determine cell fate. Effects of targeted mutagenesis on middle ear development are described to illustrate how the alteration of molecularly-controlled morphogenetic programs led to phylogenetic modifications of skeletal development. Ontogenetic plasticity has enabled the diversification of jaw elements as well as middle ear structures during evolution. This article is part of a special issue entitled "MEMRO 2012".

Ossicular fusion and cholesteatoma in auriculo-condylar syndrome: in vivo evidence of arrest of embryogenesis.

Auriculo-condylar syndrome (ACS) is a rare condition affecting first branchial arch structures. The types of hearing loss and temporal bone findings in ACS have not been reported. We describe a 14-year-old male with constricted pinnae, mandibular dysostosis, glossoptosis, a high-arched palate, hearing loss, and cholesteatoma. Computed tomography imaging demonstrated malleoincudal joint ankylosis. The fused malleoincudal complex was removed during mastoidectomy for cholesteatoma. Electron microscopy and histopathology of the joint suggested the fusion was congenital. This is the first report of ossicular fusion and cholesteatoma in ACS and the most detailed in vivo evidence of disruption of embryogenesis during malleoincudal joint formation.

The origin of the stapes and relationship to the otic capsule and oval window.

BACKGROUND: The stapes, an ossicle found within the middle ear, is involved in transmitting sound waves to the inner ear by means of the oval window. There are several developmental problems associated with this ossicle and the oval window, which cause hearing loss. The developmental origin of these tissues has not been fully elucidated. RESULTS: Using transgenic reporter mice, we have shown that the stapes is of dual origin with the stapedial footplate being composed of cells of both neural crest and mesodermal origin. Wnt1cre/Dicer mice fail to develop neural crest-derived cartilages, therefore, have no middle ear ossicles. We have shown in these mice the mesodermal stapedial footplate fails to form and the oval window is induced but underdeveloped. CONCLUSIONS: If the neural crest part of the stapes fails to form the mesodermal part does not develop, indicating that the two parts are interdependent. The stapes develops tightly associated with the otic capsule, however, it is not essential for the positioning of the oval window, suggesting that other tissues, perhaps within the inner ear are needed for oval window placement.

Induction of the chick columella and its integration with the inner ear.

BACKGROUND: The auditory complex of the chick, like that of humans, is made of intimate and highly ordered connections between the inner ear, the middle ear, and the outer ear. Unlike mammals, the middle ear of chick has only one ossicle, known as the columella. The independent lineages of the two suggest that some mechanism must exist that ensures the connectivity between the inner ear and the columella; however, the basis of integration is not known. RESULTS: Using quail-chick chimeras, we demonstrate that columella development depends on signaling interactions. Specifically, both pharyngeal endoderm and cranial paraxial mesoderm can alter the morphology of the columella. Only a discrete region of pharyngeal endoderm exerts this patterning activity, and this region is specified by the overlying paraxial mesoderm. CONCLUSIONS: Paraxial mesoderm is also used in the induction of the inner ear, thus we propose that this overlapping source of signalling cues in both middle and inner ear development may underlie the integration of these structures.

Three-dimensional observation of the mouse embryo by micro-computed tomography: Meckel's cartilage, otocyst, and/or muscle of tongue.

Three-dimensional observation during embryogenesis is possible with micro-computed tomography, but there are no observations of organ size. In this paper, three examples of three-dimensional observation of organs by micro-CT are tried. At 13.0 days post-coitum, mouse embryos were fixed in 4% paraformaldehyde for 24 h and stained enbloc by osmium tetroxide overnight. The embryos were then embedded in paraffin using standard methods for 24 h. Specimens were analyzed by micro-computed tomography and image processing was performed. The entire Meckel's cartilage and its relation in the mandible, as well as the complex structure of the otocyst, are easily visualized. Although it is difficult to extract detailed structures of the tongue muscles, it is possible to identify the inner and external tongue muscles. Relation among the organs and other are easily visualized. Three-dimensional observation by micro-computed tomography is an important technology for visualization of embryogenesis and could be used in organ culture.

Evolutionary development of the middle ear in Mesozoic therian mammals.

The definitive mammalian middle ear (DMME) is defined by the loss of embryonic Meckel's cartilage and disconnection of the middle ear from the mandible in adults. It is a major feature distinguishing living mammals from nonmammalian vertebrates. We report a Cretaceous trechnotherian mammal with an ossified Meckel's cartilage in the adult, showing that homoplastic evolution of the DMME occurred in derived therian mammals, besides the known cases of eutriconodonts. The mandible with ossified Meckel's cartilage appears to be paedomorphic. Reabsorption of embryonic Meckel's cartilage to disconnect the ear ossicles from the mandible is patterned by a network of genes and signaling pathways. This fossil suggests that developmental heterochrony and gene patterning are major mechanisms in homplastic evolution of the DMME.

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.

[Connections between bone marrow and mesenchyme of the middle ear]. / Conexiones entre la médula ósea de los osículos y el mesénquima del oído medio.

OBJECTIVE: To investigate the presence of connections between the bone marrow of the ossicles and the mesenchyme that fills the future tympanic cavity. MATERIAL AND METHODS: Ninety temporal bones from embryos and foetuses were examined, selecting 15 aged between 20th to 30th weeks of development, to show connections between ossicle marrow and mesenchyme. RESULTS: The connections are transitory and appear in the malleus and the incus between 20th to 24th weeks of development, while in the stapes appear later, being between 24th to 28th weeks. CONCLUSIONS: These connections may have an important role in the phagocytosis of the mesenchymal remains and join in the detritus elimination mechanisms produced during the regression.

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.

Development of the stapes and associated structures in human embryos.

The objective of this study was to clarify the development of the stapes in humans and its relationship with the cartilage of the second branchial arch. The study was carried out in 25 human embryos between 6 and 28 mm crown-rump length. The stapes develops at the cranial end of the second branchial arch through an independent anlage of the cartilage of this arch. Between the stapedial anlage and the cranial end of the Reichert's cartilage there is a formation called the interhyale, the internal segment of which gives rise to the tendon of the stapedial muscle. The stapedial anlage is a unique formation with two distinct parts: the superior part that will comprise the base and the inferior part that will be crossed by the stapedial artery during embryonic development and will constitute the limbs and the head of the stapes. According to the results, the otic capsule is not involved in formation of the base of the stapes.

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.

[Ontogenic peculiarities of the human tympanic ossicular chain]. / Peculiaridades en la organización de la cadena osicular timpánica humana a lo largo de su ontogenia.

We have studied the development of the tympanic ossicles in 40 embryo-foetal human series aged between 32 days (6 mm) and newborn. Once performed the measurements to date chronologically embryos and foetuses, we did a meticulous dissection of temporal bones. After fix in 10% formol, decalcified with 2% nitric acid, embedded in Paraplast, sectioned in a sequence of 7 mm, and stained with Martin's trichrome. The tympanic ossicles are developed in the mesenchyme of the two first pharyngeal archs. The head of the malleus, the body and the short limb of the incus arise from the first arch while the handle of the malleus, the long limb of the incus and the mass of the stapes arise from the second arch. The vestibular side of the stapedial footplate develops in the otic capsule. The tympanic ossicles develop from endochondral ossification, while anterior process of the malleus has the membranous ossification. In their ontogenia 6 stages are observed. First stage, the formation of their sketch by mesenchimal condensation, in the second stage, "pre-cartilaginous", the cells of the primordia are differentiated into condroblasts, in the third stage "cartilaginous" the ossicles show a cartilaginous structure, in the forth stage the primary ossification centers are developed, in the fifth stage the ossicles arise in the periostic annulus and inside the endochondral bone, and in the last stage the osseous tissue grows until it acquires a compact osseous structure.