CXCL12 is required for stirrup-shaped stapes formation during mammalian middle ear development.

BACKGROUND: The mammalian middle ear comprises a chain of three ossicles-the malleus, incus, and stapes-each of which has a unique morphology for efficiently transmitting sound information. In particular, the stapes, which is attached to the inner ear, is stirrup-shaped with a head and base connected by two crural arches, forming the stapedial foramen, through which the stapedial artery passes. However, how the stapes acquires this critical stirrup shape for association with the stapedial artery during development is not clear. RESULTS: C-X-C motif chemokine ligand 12 (CXCL12) is a chemoattractant essential for cellular movement and angiogenesis. In Cxcl12 -/- embryos, migration of neural crest cells into the prospective middle ear regions and their mesenchymal condensation to form the three ossicles proceed normally in correct alignment with each other and the inner ear. However, in the absence of CXCL12, the stapes loses its stirrup shape and instead exhibits a columnar shape lacking the crural arches and central hole. In addition, although the stapedial artery initially forms during early mesenchymal condensation of the stapes, it degenerates without CXCL12 function. CONCLUSION: CXCL12 plays an essential role in establishing the stirrup-shaped architecture of the stapes, possibly by maintaining the stapedial foramen and stapedial artery throughout development.

The incudopetrosal joint of the human middle ear: a transient morphology in fetuses.

In spite of the amount of research on fetal development of the human middle ear and ear ossicles, there has been no report showing a joint between the short limb of incus and the otic capsule or petrous part of the temporal bone. According to observations of serial histological sections from 65 embryos and fetuses at 7-17 weeks of development, the incudopetrosal joint exhibited a developmental sequence similar to the other joints of ossicles, with an appearance of an interzone followed by a trilaminar configuration at 7-12 weeks, a joint cavitation at 13-15 weeks and development of intraarticular and capsular ligaments at 16-17 weeks. These processes occurred at the same time or slightly later than any other joint. Thus, the joint development might coordinate with vibrating ossicles in utero. The growing short limb of incus appeared to accelerate an expansion of the epitympanic recess of the tympanic cavity. Additional observations of five late-stage fetuses demonstrated the incudopetrosal joint located in the fossa incudis joint changing to syndesmosis. Consequently, a real joint with a cavity existed transiently between the human neurocranium and the first pharyngeal arch derivative (i.e. incus) in contrast to the tympanostapedial joint or syndesmosis between the neurocranium and the second arch derivative. The newly described joint might have an effect on the widely accepted primary jaw concept: the mammalian jaw should thus have been created within the first pharyngeal arch, although the connection with neurocranium by the stapes is of a different origin.

The conserved and divergent roles of Prdm3 and Prdm16 in zebrafish and mouse craniofacial development.

The formation of the craniofacial skeleton is a highly dynamic process that requires proper orchestration of various cellular processes in cranial neural crest cell (cNCC) development, including cell migration, proliferation, differentiation, polarity and cell death. Alterations that occur during cNCC development result in congenital birth defects and craniofacial abnormalities such as cleft lip with or without cleft palate. While the gene regulatory networks facilitating neural crest development have been extensively studied, the epigenetic mechanisms by which these pathways are activated or repressed in a temporal and spatially regulated manner remain largely unknown. Chromatin modifiers can precisely modify gene expression through a variety of mechanisms including histone modifications such as methylation. Here, we investigated the role of two members of the PRDM (Positive regulatory domain) histone methyltransferase family, Prdm3 and Prdm16 in craniofacial development using genetic models in zebrafish and mice. Loss of prdm3 or prdm16 in zebrafish causes craniofacial defects including hypoplasia of the craniofacial cartilage elements, undefined posterior ceratobranchials, and decreased mineralization of the parasphenoid. In mice, while conditional loss of Prdm3 in the early embryo proper causes mid-gestation lethality, loss of Prdm16 caused craniofacial defects including anterior mandibular hypoplasia, clefting in the secondary palate and severe middle ear defects. In zebrafish, prdm3 and prdm16 compensate for each other as well as a third Prdm family member, prdm1a. Combinatorial loss of prdm1a, prdm3, and prdm16 alleles results in severe hypoplasia of the anterior cartilage elements, abnormal formation of the jaw joint, complete loss of the posterior ceratobranchials, and clefting of the ethmoid plate. We further determined that loss of prdm3 and prdm16 reduces methylation of histone 3 lysine 9 (repression) and histone 3 lysine 4 (activation) in zebrafish. In mice, loss of Prdm16 significantly decreased histone 3 lysine 9 methylation in the palatal shelves but surprisingly did not change histone 3 lysine 4 methylation. Taken together, Prdm3 and Prdm16 play an important role in craniofacial development by maintaining temporal and spatial regulation of gene regulatory networks necessary for proper cNCC development and these functions are both conserved and divergent across vertebrates.

Middle ear congenital cholesteatoma: systematic review, meta-analysis and insights on its pathogenesis.

PURPOSE: Congenital cholesteatoma (CC) presents as a white pearl-like lesion behind a normal tympanic membrane (TM), without a history of otorrhea, infection, perforation or previous otologic surgery. Several recent studies provided new data improving this pathology characterization. The aim of this paper is to expand the knowledge about CC and to provide new insights on its pathogenesis. METHODS: The study consisted of two main research parts: (1) systematic review and meta-analysis; (2) medical literature review englobing anatomy, histology, embryology and congenital pathology of the ear. RESULTS: The search strategy identified a total of 636 papers. Seventy retrospective studies were included. A total of 1497 cases were studied and the mean age was 6.58 years, with a male-female ratio of 3:1, 34% were asymptomatic, 26% had hearing loss and 2% had facial dysfunction/paralysis. The overall estimate for antero-superior quadrant involvement was 0.70 [95% confident interval (CI) 0.64-0.76], in the postero-superior quadrant was 0.60 (95% CI 0.52-0.69), in the antero-inferior quadrant was 0.32 (95% CI 0.23-0.41), in the postero-inferior quadrant was 0.38 (95% CI 0.29-0.47), in the attic was 0.53 (95% CI 0.43-0.63) and in the mastoid was 0.33 (95% CI 0.26-0.41). More advanced Potsic stages were present in older patients. The most likely inclusion place seems to be between the pars flaccida and the upper quadrants of the pars tensa. CONCLUSIONS: During the last decades, a substantial improvement in CC diagnosis and management had been achieved. The presented mechanism seems to explain most of middle ear CC.

Developmental aspects of the tympanic membrane: Shedding light on function and disease.

The ear drum, or tympanic membrane (TM), is a key component in the intricate relay that transmits air-borne sound to our fluid-filled inner ear. Despite early belief that the mammalian ear drum evolved as a transformation of a reptilian drum, newer fossil data suggests a parallel and independent evolution of this structure in mammals. The term "drum" belies what is in fact a complex three-dimensional structure formed from multiple embryonic cell lineages. Intriguingly, disease affects the ear drum differently in its different parts, with the superior and posterior parts being much more frequently affected. This suggests a key role for the developmental details of TM formation in its final form and function, both in homeostasis and regeneration. Here we review recent studies in rodent models and humans that are beginning to address large knowledge gaps in TM cell dynamics from a developmental biologist's point of view. We outline the biological and clinical uncertainties that remain, with a view to guiding the indispensable contribution that developmental biology will be able to make to better understanding the TM.

Single origin of the epithelium of the human middle ear.

OBJECTIVE: The epithelium lining the human middle ear and adjacent temporal bone cavity shows a varying morphological appearance throughout these cavities. Its embryologic origin has long been debated and recently got attention in a newly proposed theory of a dual embryologic origin. The epithelial morphology and its differentiating capabilities are of significance in future mucosa-targeted therapeutic agents and could affect surgical approaches of the temporal bone. This study aims to analyze reported murine histological findings that led to the theory of a dual epithelial embryological origin and immunohistochemically investigate whether such an epithelial embryological origin in the human fetal middle ear could be true. METHODS: By combining a sagittal sectioning technique and immuno-histochemical staining, a comprehensive immuno-histological overview of the fetal human middle ear during a critical stage of tympanic cavitation was provided. A critical analysis of previously reported findings leading to the theory of a dual epithelial embryological origin and a comparison of these findings to the findings in the human fetal middle ear was performed. RESULTS: The reported findings and critical analysis provide multiple arguments for an entirely endodermal embryonic origin of the epithelium lining the tympanic cavity. CONCLUSION: Different morphological epithelial appearances throughout the tympanic and temporal bone cavities could be explained by different stages of epithelial differentiation rather than different embryologic origin and endodermal rupture does not seem to be a necessity for these cavities to form.

Chronic otitis media is initiated by a bulla cavitation defect in the FBXO11 mouse model.

Auditory bulla cavitation defects are a cause of otitis media, but the normal cellular pattern of bulla mesenchyme regression and its failure are not well understood. In mice, neural-crest-derived mesenchyme occupies the bulla from embryonic day 17.5 (E17.5) to postnatal day 11 (P11) and then regresses to form the adult air-filled bulla cavity. We report that bulla mesenchyme is bordered by a single layer of non-ciliated epithelium characterized by interdigitating cells with desmosome cell junctions and a basal lamina, and by Bpifa1 gene expression and laminin staining of the basal lamina. At P11-P12, the mesenchyme shrinks: mesenchyme-associated epithelium shortens, and mesenchymal cells and extracellular matrix collagen fibrils condense, culminating in the formation of cochlea promontory mucosa bordered by compact non-ciliated epithelial cells. FBXO11 is a candidate disease gene in human chronic otitis media with effusion and we report that a bulla cavitation defect initiates the pathogenesis of otitis media in the established mouse model Jeff (Fbxo11Jf/+ ). Persistent mesenchyme in Fbxo11Jf/+ bullae has limited mesenchymal cell condensation, fibrosis and hyperplasia of the mesenchyme-associated epithelium. Subsequent modification forms fibrous adhesions that link the mucosa and the tympanic membrane, and this is accompanied by dystrophic mineralization and accumulation of serous effusion in the bulla cavity. Mouse models of bulla cavitation defects are important because their study in humans is limited to post-mortem samples. This work indicates new diagnostic criteria for this otitis media aetiology in humans, and the prospects of studying the molecular mechanisms of murine bulla cavitation in organ culture.

Genetic variation in thyroid folliculogenesis influences susceptibility to hypothyroidism-induced hearing impairment.

Maternal and fetal sources of thyroid hormone are important for the development of many organ systems. Thyroid hormone deficiency causes variable intellectual disability and hearing impairment in mouse and man, but the basis for this variation is not clear. To explore this variation, we studied two thyroid hormone-deficient mouse mutants with mutations in pituitary-specific transcription factors, POU1F1 and PROP1, that render them unable to produce thyroid stimulating hormone. DW/J-Pou1f1dw/dw mice have profound deafness and both neurosensory and conductive hearing impairment, while DF/B-Prop1df/df mice have modest elevations in hearing thresholds consistent with developmental delay, eventually achieving normal hearing ability. The thyroid glands of Pou1f1 mutants are more severely affected than those of Prop1df/df mice, and they produce less thyroglobulin during the neonatal period critical for establishing hearing. We previously crossed DW/J-Pou1f1dw/+ and Cast/Ei mice and mapped a major locus on Chromosome 2 that protects against hypothyroidism-induced hearing impairment in Pou1f1dw/dw mice: modifier of dw hearing (Mdwh). Here we refine the location of Mdwh by genotyping 196 animals with 876 informative SNPs, and we conduct novel mapping with a DW/J-Pou1f1dw/+ and 129/P2 cross that reveals 129/P2 mice also have a protective Mdwh locus. Using DNA sequencing of DW/J and DF/B strains, we determined that the genes important for thyroid gland function within Mdwh vary in amino acid sequence between strains that are susceptible or resistant to hypothyroidism-induced hearing impairment. These results suggest that the variable effects of congenital hypothyroidism on the development of hearing ability are attributable to genetic variation in postnatal thyroid gland folliculogenesis and function.

A requirement for Fgfr2 in middle ear development.

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air-filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain-of-function disorders are clinically characterized, those in the FGFR2 loss-of-function disorder lacrimo-auriculo-dento-digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra-articular ligaments of the incudomalleal joint is derived from Scx-expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome.

Neural crest contributions to the ear: Implications for congenital hearing disorders.

Congenital hearing disorders affect millions of children worldwide and can significantly impact acquisition of speech and language. Efforts to identify the developmental genetic etiologies of conductive and sensorineural hearing losses have revealed critical roles for cranial neural crest cells (NCCs) in ear development. Cranial NCCs contribute to all portions of the ear, and defects in neural crest development can lead to neurocristopathies associated with profound hearing loss. The molecular mechanisms governing the development of neural crest derivatives within the ear are partially understood, but many questions remain. In this review, we describe recent advancements in determining neural crest contributions to the ear, how they inform our understanding of neurocristopathies, and highlight new avenues for further research using bioinformatic approaches.

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.

Morphogenesis of the Middle Ear during Fetal Development as Observed Via Magnetic Resonance Imaging.

Recently, our research group has utilized serial histological sections to investigate the morphogenesis of the middle ear, which corresponds to the period of middle ear ossicle (MEO) cartilage formation. However, research regarding middle ear development during the post-embryonic period has been limited. In the present study, we investigated morphogenesis of the middle ear in human fetuses with a crown-rump length (CRL) between 37 and 197 mm using high-resolution magnetic resonance imaging (MRI). Our findings indicated that the morphology of the MEOs is similar during fetal development and adulthood; further, growth of the MEOs nearly ceases once a CRL of 150 mm is attained. In each MEO, ossification spreads from a single center. The malleus and Meckel's cartilage could be discriminated in samples exhibiting a CRL of 145 mm based on differences in MRI signal intensity. In samples with a CRL of 86 mm, the tympanic cavity (TC) appeared as a thin yet distinct structure attached to the external auditory meatus at the convex surface. Only the handle of the malleus was covered by the TC, while the incus and stapes contacted the cavity at the region of articulation between the two ossicles only, even after a CRL of 145 mm had been attained. Thus, although the TC increased in both diameter and thickness, coverage did not extend across all three MEOs during the observation period. These data are expected to provide a useful standard for morphogenesis and may aid researchers in distinguishing between normal and abnormal development. Anat Rec, 301:757-764, 2018. © 2017 Wiley Periodicals Inc.

Differing contributions of the first and second pharyngeal arches to tympanic membrane formation in the mouse and chick.

We have proposed that independent origins of the tympanic membrane (TM), consisting of the external auditory meatus (EAM) and first pharyngeal pouch, are linked with distinctive middle ear structures in terms of dorsal-ventral patterning of the pharyngeal arches during amniote evolution. However, previous studies have suggested that the first pharyngeal arch (PA1) is crucial for TM formation in both mouse and chick. In this study, we compare TM formation along the anterior-posterior axis in these animals using Hoxa2 expression as a marker of the second pharyngeal arch (PA2). In chick, the EAM begins to invaginate at the surface ectoderm of PA2, not at the first pharyngeal cleft, and the entire TM forms in PA2. Chick-quail chimera that have lost PA2 and duplicated PA1 suggest that TM formation is achieved by developmental interaction between a portion of the EAM and the columella auris in PA2, and that PA1 also contributes to formation of the remaining part of the EAM. By contrast, in mouse, TM formation is highly associated with an interdependent relationship between the EAM and tympanic ring in PA1.

Fetal facial nerve course in the ear region revisited.

PURPOSE: The aim of this study was to re-examine the structures that determine course of the facial nerve (FN) in the fetal ear region. MATERIALS AND METHODS: We used sagittal or horizontal sections of 28 human fetuses at 7-8, 12-16, and 25-37 weeks. RESULTS: The FN and the chorda tympani nerve ran almost parallel until 7 weeks. The greater petrosal nerve (GPN) ran vertical to the distal FN course due to the trigeminal nerve ganglion being medial to the geniculate ganglion at 7 weeks. Afterwards, due to the radical growth of the former ganglion, the GPN became an anterior continuation of the FN. The lesser petrosal nerve ran straight, parallel to the FN at 7 weeks, but later, it started to wind along the otic capsule, possibly due to the upward invasion of the tympanic cavity epithelium. Notably, the chorda tympanic nerve origin from the FN, and the crossing between the vagus nerve branch and the FN, was located outside of the temporal bone even at 37 weeks. The second knee of the FN was not evident, in contrast to the acute anterior turn below the chorda tympanic nerve origin. In all examined fetuses, the apex of the cochlea did not face the middle cranial fossa, but the tympanic cavity. CONCLUSION: Topographical relation among the FN and related nerves in the ear region seemed not to be established in the fetal age but after birth depending on growth of the cranial fossa.

Major evolutionary transitions and innovations: the tympanic middle ear.

One of the most amazing transitions and innovations during the evolution of mammals was the formation of a novel jaw joint and the incorporation of the original jaw joint into the middle ear to create the unique mammalian three bone/ossicle ear. In this review, we look at the key steps that led to this change and other unusual features of the middle ear and how developmental biology has been providing an understanding of the mechanisms involved. This starts with an overview of the tympanic (air-filled) middle ear, and how the ear drum (tympanic membrane) and the cavity itself form during development in amniotes. This is followed by an investigation of how the ear is connected to the pharynx and the relationship of the ear to the bony bulla in which it sits. Finally, the novel mammalian jaw joint and versatile dentary bone will be discussed with respect to evolution of the mammalian middle ear.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Ontogenetic explanation for tegmen tympani dehiscence and superior semicircular canal dehiscence association. / Explicación ontogénica para la asociación entre dehiscencia del tegmen tympani y dehiscencia del canal semicircular superior.

OBJECTIVES: To analyze the ontogeny of the superior semicircular canal and tegmen tympani and determine if there are common embryological factors explaining both associated dehiscence. METHODS: We analyzed 77 human embryological series aged between 6 weeks and newborn. Preparations were serially cut and stained with Masson's trichrome technique. RESULTS: The tegmental prolongation of tegmen tympani and superior semicircular canal originate from the same structure, the otic capsule, and have the same type of endochondral ossification; while the extension of the squamous prolongation of tegmen tympani runs from the temporal squama and ossification is directly of intramembranous type. The nuclei of ossification of the superior and external semicircular canals and accessory of tegmen collaborate in the ossification of the tegmental extension and by growth extend to the tegmental prolongation. This fact plus the fact that both structures share a common layer of external periosteum could explain the coexistence of lack of bone coverage in tegmen and superior semicircular canal. CONCLUSION: The development of the semicircular canal and tegmen tympani could explain the causes of the association of both dehiscences.

Evolution of the mammalian middle ear: a historical review.

Here we present a brief, historical review of research into the mammalian middle ear structures. Most of their essential homologies were established by embryologists, notably including Reichert, during the 19th century. The evolutionary dimension was confirmed by finds of fossil synapsids, mainly from the Karroo of South Africa. In 1913, Ernst Gaupp was the first to present a synthesis of the available embryological and paleontological data, but a number of morphological details remained to be solved, such as the origin of the tympanic membrane. Gaupp favoured an independent origin of the eardrum in anurans, sauropsids, and mammals; we support most of his ideas. The present review emphasizes the problem of how the mammalian middle ear structures that developed at the angle of the lower jaw were transferred to the basicranium; the ontogenesis of extant marsupials provides important information on this question.

The development of the mammalian outer and middle ear.

The mammalian ear is a complex structure divided into three main parts: the outer; middle; and inner ear. These parts are formed from all three germ layers and neural crest cells, which have to integrate successfully in order to form a fully functioning organ of hearing. Any defect in development of the outer and middle ear leads to conductive hearing loss, while defects in the inner ear can lead to sensorineural hearing loss. This review focuses on the development of the parts of the ear involved with sound transduction into the inner ear, and the parts largely ignored in the world of hearing research: the outer and middle ear. The published data on the embryonic origin, signalling, genetic control, development and timing of the mammalian middle and outer ear are reviewed here along with new data showing the Eustachian tube cartilage is of dual embryonic origin. The embryonic origin of some of these structures has only recently been uncovered (Science, 339, 2013, 1453; Development, 140, 2013, 4386), while the molecular mechanisms controlling the growth, structure and integration of many outer and middle ear components are hardly known. The genetic analysis of outer and middle ear development is rather limited, with a small number of genes often affecting either more than one part of the ear or having only very small effects on development. This review therefore highlights the necessity for further research into the development of outer and middle ear structures, which will be important for the understanding and treatment of conductive hearing loss.

Development and Integration of the Ear.

The perception of our environment via sensory organs plays a crucial role in survival and evolution. Hearing, one of our most developed senses, depends on the proper function of the auditory system and plays a key role in social communication, integration, and learning ability. The ear is a composite structure, comprised of the external, middle, and inner ear. During development, the ear is formed from the integration of a number of tissues of different embryonic origin, which initiate in distinct areas of the embryo at different time points. Functional connections between the components of the hearing apparatus have to be established and maintained during development and adulthood to allow proper sound submission from the outer to the middle and inner ear. This highly organized and intimate connectivity depends on intricate spatiotemporal signaling between the various tissues that give rise to the structures of the ear. Any alterations in this chain of events can lead to the loss of integration, which can subsequently lead to conductive hearing loss, in case of outer and middle ear defects or sensorineural hearing loss, if inner ear structures are defective. This chapter aims to review the current knowledge concerning the development of the three ear compartments as well as mechanisms and signaling pathways that have been implicated in the coordination and integration process of the ear.

Endoscopic anatomy of the pediatric middle ear.

Traditionally, otologists have aimed to produce a clean, dry, safe ear with the best possible hearing result. More recently, "less invasively" has been added to this list of goals. The development of small-diameter, high-quality rigid endoscopes and high-definition video systems has made totally endoscopic, transcanal surgery a reality in adult otology and a possibility in pediatric otology. This article reviews the anatomy of the pediatric middle ear and its surrounding airspaces and structures based on the work of dozens of researchers over the past 50 years. It will focus on the developmental changes in ear anatomy from birth through the first decade, when structure and function change most rapidly. Understanding the limits and possibilities afforded by new endoscopic technologies, the pediatric otologist can strive for results matching or exceeding those achieved by more invasive surgical approaches.