Auditory epithelial migration on the human tympanic membrane: II. The existence of two discrete migratory pathways and their embryologic correlates

The pathways of movement of dye-markings on the tympanic membranes of 30 ears of 15 volunteers were investigated by photography with a Hopkin's rod. Two discrete pathways were identified. Movement on the pars tensa was centrifugal in all directions from the edge of the handle of malleus surface. This supported our previous suggestion that pars tensa epithelial migration is based on growth of the meatal plate. Movement on the more central part of the handle surface itself, however, was always upwards to the pars flaccida epithelium and then, with all dye on the latter, in a posterosuperior direction only. The covering epithelium of the pars flaccida/handle of malleus arises from the fundus of the early embryonic external canal. To explore the possibility that its migration may also commence early, the fundus was examined histologically in 12 embryo ears. It was shaped like an inverted pear to conform to the primordial head of the malleus above and its handle below. There was distinct flattening of the whole fundal epithelium in contrast to the side wall of the external canal, lending support to the concept of a primordial flux of epithelium in the main fundus and its inferior extension. The meatal plate grew from the rim of the fundus producing the pars tensa and deep external canal epithelia. This process, however, was not associated with significant cytologic alterations or increased expression of the human proliferation antigen, Ki-67, at that rim. This suggests that the pars tensa epithelium grows and moves without the active involvement of the epithelium of the pars flaccida/handle of malleus.     

Development of the stratified squamous epithelium of the human tympanic membrane and external canal: the origin of auditory epithelial migration

The development of the stratified squamous epithelium of the tympanic membrane and external canal was studied in 167 embryonic, fetal, and postnatal human ears. It originates as a tube derived from the epithelium of the fundus of the primary external canal (zone 1). The tube is composed of a thin, flat epithelium on the medial side (zone 2), continuous with a thicker one (zone 3) on the lateral side; zone 3 thereafter merges with the external epithelium of the primary external canal (zone 4). Proliferative activity, as indicated by a thickened epithelium, with rete ridges in later fetal life, is present mainly in zones 1 and 3. Cornification at 18 weeks gestation is followed by clearing of keratinous debris to the exterior. Subsequently the canal widens, zone 1 now covering the pars flaccida region, a tongue-shaped area passing inferiorly from it and a part of the postero-superior deep canal adjacent to it; zone 2 covers the pars tensa and zone 3 most of the deep external canal. On the basis of the original embryonic growth, migratory epithelial movement throughout life is postulated to be generated in zone 1 by mitotic interposition and then to pass to zone 2. It then moves en masse through to zone 3, where unilateral progression by mitotic means takes the epithelium up to the cartilaginous canal. Such a pathway is approximately that seen in the marked, living eardrum and canal.     

Auditory development in the mouse: Structural maturation of the middle ear

The development of middle-ear structures in the mouse was examined in nine groups of pups between 1 and 45 days of age. The area of the tympanic membrane (pars tensa and pars flaccida), the length of the lever arms of the malleus and incus, the surface area of the oval window, and the volume of the bulla all showed systematic changes during neonatal life. The area of the oval window reached maturity first and the lever arms achieved 90% of their adult size on day 11. The tympanic membrane achieved the same criterion on day 18. These data help us further to understand the processes that contribute to the functional ontogeny of the middle ear.     

Regeneration of the retina in the adult newt, Triturus cristatus, following surgical division of the eye by a limbal incision.

The regeneration of a retina in adult Triturus cristatus, following surgical division of the eye by a limbal incision, was studied. In agreement with recent reports, it was found that the regenerating retina is dervied from two sources; the retinal pigment epithelium and the pars ciliaris retinae. However, following a limbal incision, most of the retina appears to be derived from the retinal pigment epithelium in the posterior part of the eye. An unexpected finding of this study was that large cystlike spaces form in the fundal regions of the eye, between the regenerating retina and the retinalpigment epithelium. These spaces appear between five and eight days post-operative and persist long enough (25 to 30 days postoperative) to disrupt the fundal portion of the rengenerating retina and to cause it to lag behind the rest of the regenerate, in its development. The relationship of these observations to the genesis of positional markers in the regenerating retina is discussed.     

Canal systems in the temporal bone and their right-left differences

The first part of the facial canal is the pars labyrinthica. Its means lateral length is in our material 2.63 mm on the right and 3.03 mm on the left side. The mean width of the pore on the fundus area was 1.3 mm on the right side and 1.07 mm on the left. The geniculate fossa had a mean length of 2.54 mm on the right and of 3.14 mm on the left side. The widths of different areas of the pars labyrinthica and fossa geniculata were also measured. The angle between the first and second parts of the Fallopian canal was estimated with different methods. The mean width of the tympanic part of the facial canal was found to be 1.79 mm on the right side and 1.67 mm on the left, the mean width of the mastoideal part was 1.8 mm on the right side and 1.7 mm on the left. Distances of the mastoideal part of the Fallopian canal to mastoideal cells, ear drum, sigmoid sinus and external surface of the temporal bone were also measured. Measurements of the sigmoid sinus, the bulb of internal jugular vein and the cavum musculi stapedii are included, too. The ranges found in our material are also given.     

Netrin 1 is required for semicircular canal formation in the mouse inner ear

The morphogenetic development of the mammalian inner ear is a complex multistep process, the molecular and cellular details of which are only beginning to be unraveled. We show here that mouse netrin 1, known to be involved in axon guidance and cell migration in the central nervous system, also plays a critical morphogenetic role during semicircular canal formation. netrin 1 is expressed at high levels in the otic epithelium, in cells that will come together to form a fusion plate, a prerequisite for the formation of semicircular canals. In netrin 1 mutant mice, fusion plate formation is severely affected resulting in a reduced anterior semicircular canal and the complete lack of the posterior and lateral canals. Our results suggest that netrin 1 facilitates semicircular canal formation through two different mechanisms: (1) it participates in the detachment of the fusion plate epithelia from the basement membrane, and (2) it stimulates proliferation of the periotic mesenchymal cells which then push the epithelial cell walls together to form the fusion plate.     

Comparative cytokeratin distribution patterns in cholesteatoma epithelium

Cytokeratins (CKs) are known as the intermediate filament proteins of epithelial origin. Their distribution in human epithelia is different according to the type of epithelium, state of growth and differentiation. We used monoclonal mouse antibodies against cytokeratins to study CK expression in the following human tissues: cholesteatoma, middle ear mucosa, glandular epithelium, and meatal ear canal epithelium. Immunohistochemical processing was performed using the labeled steptavidin peroxidase method to demonstrate the presence of CKs in cells of human epidermis. Positive reaction was obtained for CK4, CK34betaE12, CK10, CK14 in skin and cholesteatoma epithelium. However, a more extensive positive reaction with those CKs was observed in cholesteatoma epithelium. Positive immunoreactivity was seen with anti- CK19 in the glandular epithelium. Middle ear mucosa specimens revealed positive immunoreactivity with the antibodies against CK4. The expression of CK4 was definitely positive within the basal layers of the epidermis. The glandular epithelium showed no positive reaction with anti- CK4, anti- CK34betaE12, anti- CK14 and anti-CK10. Immunohistochemistry for CK18 showed no reaction in all examined tissues. Cholesteatoma is known as a proliferative disease in the middle ear which pathogenesis is not completely understood. Keratinocytes express hyperproliferation- associated CKs and after reaching the suprabasal layers they finally undergo apoptosis creating keratinous debris. Cytokeratin expression observed in the epithelium explains proliferative behavior of cholesteatoma which is associated with increased keratinocyte migration. Cytokeratins can be used as potential proliferative markers. It can also allow for searching the usefulness of inhibiting regulators in the treatment of hyperproliferative diseases.     

Postnatal development of the sound-transporting apparatus in the middle ear of rabbits

The middle ear apparatus realized a multiplicatory function during the transfer of the vibrational energy, depending on the area ratio of pars tensa of tympanic membrane and of stapes footplate as well as the lever ratio of long arms of manubrium and of incus. These structures exhibit a size increase during the postnatal development, but the sequel on the multiplicatory function in rabbits is unknown. The middle ear structures of 46 rabbits, 1 to 30 d old, were prepared and measured. The area of pars tensa and the levers of malleus and incus increase with age. After the 10th d of life, no statistical significant growth were measurable. But the calculated multiplicatory factor of single animals indicate the end of the development at the 15th postnatal d. In contrast, the cochlear function attains the adult values not till the 26th d of life. It is 10 d longer than the middle ear growth.     

Middle-ear development: II. Morphometric changes in the conducting apparatus of the chick.

The ontogeny of various middle-ear structures was examined in 11 groups of chicks between 10 days embryonic and adult. Measurements of the tympanic membrane surface area and height, columella length, and that of the columella footplate, annular ligament, and oval window area were obtained using video micrographs and computer digitization techniques. The oval window matures first at 53 days post-hatching, whereas the columella achieves adult size at 74 days. The tympanic membrane surface area is the last middle-ear variable studied to reach adult size (79 days post-hatch). The columella increases its length from 0.63 mm (10 days embryonic) to 2.73 mm in the adult. The tympanic membrane area expands by 280% whereas the columellar footplate area increases by 11x. As a result, the pressure amplification of the middle ear due to the tympanic membrane/columellar footplate area ratio improves by over 400%. These data further contribute to our understanding of the functional development of the middle ear.     

Anatomy and physics of the exceptional sensitivity of dolphin hearing (Odontoceti: Cetacea)

During the past 50 years, the high acoustic sensitivity and the echolocation behavior of dolphins and other small odontocetes have been studied thoroughly. However, understanding has been scarce as to how the dolphin cochlea is stimulated by high frequency echoes, and likewise regarding the ear mechanics affecting dolphin audiograms. The characteristic impedance of mammalian soft tissues is similar to that of water, and thus no radical refractions of sound, nor reflections of sound, can be expected at the water/soft tissue interfaces. Consequently, a sound-collecting terrestrial pinna and an outer ear canal serve little purpose in underwater hearing. Additionally, compared to terrestrial mammals whose middle ear performs an impedance match from air to the cochlea, the impedance match performed by the odontocete middle ear needs to be reversed to perform an opposite match from water to the cochlea. In this paper, we discuss anatomical adaptations of dolphins: a lower jaw collecting sound, thus replacing the terrestrial outer ear pinna, and a thin and large tympanic bone plate replacing the tympanic membrane of terrestrial mammals. The paper describes the lower jaw anatomy and hypothetical middle ear mechanisms explaining both the high sensitivity and the converted acoustic impedance match.     

A Late Role for bmp2b in the Morphogenesis of Semicircular Canal Ducts in the Zebrafish Inner Ear

Background

The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.

Methodology/Principal Findings

We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b^−/−) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.

Conclusions/Significance

Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.     

Biomechanics of the tympanic membrane

The tympanic membrane is a key component of the human auditory apparatus which is a complex biomechanical system, devoted to sound reception and perception. Over the past 30 years, various bioengineering approaches have been applied to the ear modeling and particularly to the middle part. The tympanic membrane, included in the middle ear, transfers sound waves into mechanical vibration from the ear canal into the middle ear. Changes in structure and mechanical properties of the tympanic membrane due to middle ear diseases or damages can deteriorate sound transmission. An accurate model of the tympanic membrane, which simulates the acoustic-mechanical transmission, could improve clinical surgical intervention. In this paper a detailed survey of the biomechanics and the modeling of the tympanic membrane focusing on the finite element method is conduced. Eight selected models are evaluated and compared deducing the main features and most design parameters from published models, mainly focusing on geometric, constraint and material aspects. Non-specified parameters are replaced with the most commonly employed values. Our simulation results (in terms of modal frequencies and umbo displacement), compared with published numerical and experimental results, show a good agreement even if some scattering appears to indicate the need of further investigation and experimental validation.     

Congenital anomalies of the ear resulting from cyclophosphamide treatment in the rat

A single dose of cyclophosphamide (20 mg/kg) administered on day 12 of gestation to CF rats resulted in microtia in 97.5% of fetuses at term. The ears of affected fetuses were low set and dorsally placed. Histological examination revealed persistence of meatal plug, branching of the primordium of external acoustic meatus, periotic hemorrhages, narrowing of tympanic cavity, presence of only one to two primordia of middle ear ossicles, hypoplasia of stapedial artery, a maximum of two turns of cochlea and under differentiation of the organ of Corti and the semicircular ducts. Hemorrhages in and around the region of the ear were extensive. Bilaterally symmetrical distribution of microtia and consistency of associated anomalies suggest that this is a good animal model for further investigation into the pathogenetic mechanisms of these ear anomalies.     

Formation of the middle ear: recent progress on the developmental and molecular mechanisms

The middle ear allows animals to hear while moving in an aerial medium. It is composed of a cavity harbouring a chain of three ossicles that transmit vibrations produced by airborne sound in the tympanic membrane into the inner ear, where they are converted into neural impulses. The middle ear develops in the branchial arches, and this requires sequential interactions between the epithelia and the underlying mesenchyme. Gene-inactivation experiments have identified genes required for the formation of different middle ear components. Some encode for signalling molecules, including Endothelin1 and Fgf8, probable mediators of epithelial-mesenchymal interactions. Other genes, including Eya1, Prx1, Hoxa1, Hoxa2, Dlx1, Dlx2, Dlx5, and Gsc, are most likely involved in patterning and morphogenetic processes in the neural crest-derived mesenchyme. Mechanisms controlling formation of a functional tympanic membrane are also discussed. Basically, the tympanic ring, which serves as support for the tympanic membrane, directs invagination of the first pharyngeal cleft ectoderm to form the external acoustic meatus (EAM), which provides the outer layer of the membrane. Gsc and Prx1 are essential for tympanic ring development. While invaginating, the EAM controls skeletogenesis in the underlying mesenchyme to form the manubrium of the malleus, the link between the membrane and the middle ear ossicles.     

Intermediate epithelium lining the mouse auditory tube

In the newborn mouse, the auditory tube is lined throughout the pharyngeal orifice to the tympanic orifice with ciliated columnar epithelium. In the adult mouse, the tube is divided into membranous and cartilaginous parts. The membranous part is covered by the ciliated columnar epithelium, while the cartilaginous part by varying types of epithelium ranging from ciliated columnar to stratified squamous type. It is suggested that the varying types of epithelium correspond to the 'intermediate epithelium', and that ciliated columnar epithelium transforms in part to stratified squamous epithelium by passing through the intermediate epithelium.     

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.     

Mucin histochemistry of the anal canal epithelium. Studies of normal anal mucosa and mucosa adjacent to carcinoma

Summary The epithelial lining of the anal canal is of colo-rectal type in the upper part and squamous in the lower part, while the middle zone is called the anal transitional zone (ATZ). This zone is characterized by an epithelium which bears a resemblance to that of the anal glands and shows little mucus secretion. The histochemical properties of the mucins in the epithelium of 39 anal canals, resected for ano-rectal adenocarcinoma, basaloid carcinoma, squamous carcinoma and malignant melanoma were investigated. The study reveals that (1) the mucin composition of the ATZ epithelium corresponds to that of the anal glands, being characterized by a mixture of sulpho- and sialomucins with scarcity or absence ofO-acylated sialic acids; and (2) cases with carcinomas located near the dentate line show changes in the mucin composition of the adjoining anal canal epithelium, regardless of tumour type. In colo-rectal type mucosa, these mucins consist of increasing amounts of sialomucins with a predominance ofN-acyl derivatives, and in the anal could be detected in the ATZ epithelium. It is concluded that rectal and anal glands in the anal canal are exposed to stimuli which alter the normal process of glycoprotein synthesis and secretion. The changes seem to be secondary to tumour growth and independent of the histological type of tumour.     

Hyaluronan as a propellant for epithelial movement: the development of semicircular canals in the inner ear of Xenopus.

The membranous labyrinth of the inner ear, with its three semicircular canals, originates from a simple spheroidal otic vesicle. The process is easily observed in Xenopus. The vesicle develops three dorsal outpocketings; from the two opposite faces of each outpocketing pillars of tissue are protruded into the lumen; and these paired 'axial protrusions' eventually meet and fuse, to form a column of tissue spanning the lumen of the outpocketing like the hub of a wheel, with a tube of epithelium forming the semicircular canal around the periphery. Each axial protrusion consists of epithelium encasing a core of largely cell-free extracellular matrix that stains strongly with alcian blue. In sections, at least 60% of the stainable material is removed by treatment with Streptomyces hyaluronidase. When Streptomyces hyaluronidase is microinjected into the core of a protrusion in vivo, the protrusion collapses and the corresponding semicircular canal fails to form. Hyaluronan (hyaluronic acid) in the core of the protrusion therefore seems to be essential in driving the extension of the protrusion. Autoradiography with tritiated glucosamine indicates that the hyaluronan-rich matrix is synthesised by the epithelium covering the tip of the protrusion; the basal lamina here appears to be discontinuous. These findings indicate that the epithelium of the axial protrusion propels itself into the lumen of the otocyst by localised synthesis of hyaluronan. Hyaluronan may be used in a similar way in the development of other organs, such as the heart and the secondary palate.     

Circling mouse, a spontaneous mutant in the inner ear.

A spontaneous mutant was established in the ICR mouse strain. The affected mice became hyperactive at about 7 days of age, and then showed circling behavior. The body weight decreased significantly 2 weeks after birth, and developmental defects were revealed in the middle ear, cochlea, cochlear nerve, and semicircular canal areas. The mutation was inherited by an autosomal single recessive gene and is referred to as cir.