COUP-TFII is required for morphogenesis of the neural crest-derived tympanic ring.

Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) plays pivotal roles in cell growth, cell differentiation, and cell fate determination. Although genome-wide studies have identified COUP-TFII binding on gene sets mainly involved in neural crest cell (NCC) development and craniofacial morphogenesis, the direct functional connection between COUP-TFII and NCCs in vivo has not been well characterized. In this study, we show that COUP-TFII is expressed in the subpopulation of NCCs and its derivatives, and targeted ablation of COUP-TFII in mouse NCCs results in markedly shortened and bifurcated tympanic rings, which in turn disturb the caudal direction of external acoustic meatus invagination. However, formation of the manubrium of the malleus (MM) in Wnt1-Cre/+;COUP-TFII flox/flox mice is not perturbed, suggesting that the rostral half of the tympanic ring is sufficient to support proper MM development. Interestingly, we found that loss of COUP-TFII up-regulates Sox9 in the tympanic ring primordium and affects the distribution of preosteoblasts before mesenchymal condensation. Together, our results demonstrate that COUP-TFII plays an essential role in regulating the patterning of the NCC-derived tympanic ring.

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.

Middle-ear development. VI: Structural maturation of the rat conducting apparatus.

BACKGROUND: The contribution of middle-ear development to the overall development of hearing has not been explored in great detail. This presentation describes the maturation of conductive elements in the rat middle ear, and provides the basis on which future studies of middle-ear functional development will follow. METHODS: The middle-ear apparatus was examined at nine different ages (between 1 and 80 days postpartum) in Long Evans rats. At each age elements of the conducting apparatus was observed with either light or scanning electron microscopy (SEM), and quantitative measurements were made from video enhanced photomicrographs. Tympanic membrane area and cone depth, the length of the malleus and incus arms, ossicular weight, stapes foot plate and oval window areas, and bulla volume were all measured. Development of the area and lever ratios were derived from these measurements. The data were fitted to exponential equations and the time in days required to reach 90% of the adult level determined. RESULTS: The pars tensa achieved 90% of total area by 17 days. The oval window achieved the 90% criterion by 13 days, while the area ratio was within 10% of its adult size by 8 days. The ossicles took between 26 and 34 days, while bulla volume took 59 days to reach the 90% level. CONCLUSIONS: Middle-ear growth was very orderly and systematic in the data reported. When maturation of the area ratio was considered against development of the endocochlear potential or the round window compound action potential, it was clear that the growth of this important aspect of the middle ear preceded the onset of cochlear function.

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.

A topographic quantitative analysis of the post-natal bone formation in the auditory ossicles of the dog.

The amount and distribution of the post-natal bone deposition in the auditory ossicles and in the left tibia of dogs of varying ages were studied by means of alizarin labelling. The relative amount of fluorescent new-formed bone was expressed as a percentage ratio NB/(NB+PB) of new bone (NB) on the pre-existing bone (PB). The result was that the post-natal bone deposition (1) was larger in the tibia than in the incus, malleus and stapes; (2) significantly decreased with age both in the ossicles and in the tibia; (3) in the stapes it stopped at 3 months, while it was present in the tibia, incus and malleus even at 12 months. In the ossicles the post-natal bone deposition takes place both on the periosteal surfaces of the ossicles and on the internal surfaces of the haversian systems. The first process produces an appositional growth that stops in all three ossicles within the 1st month of post-natal life, the second one produces an internal growth that continues until the age of three months in the stapes, while in the incus and malleus it occurs in small amounts, even in the 12th month of life. In the ossicles all the new-formed bone tissue, periosteal and osteonic, is built up by primary bone (addition bone). In the tibia from 50 days of age the primary bone is gradually replaced by secondary haversian systems as a consequence of remodelling processes.