Roles of Wnt8a during formation and patterning of the mouse inner ear.

Fgf and Wnt signalling have been shown to be required for formation of the otic placode in vertebrates. Whereas several Fgfs including Fgf3, Fgf8 and Fgf10 have been shown to participate during early placode induction, Wnt signalling is required for specification and maintenance of the otic placode, and dorsal patterning of the otic vesicle. However, the requirement for specific members of the Wnt gene family for otic placode and vesicle formation and their potential interaction with Fgf signalling has been poorly defined. Due to its spatiotemporal expression during placode formation in the hindbrain Wnt8a has been postulated as a potential candidate for its specification. Here we have examined the role of Wnt8a during formation of the otic placode and vesicle in mouse embryos. Wnt8a expression depends on the presence of Fgf3 indicating a serial regulation between Fgf and Wnt signalling during otic placode induction and specification. Wnt8a by itself however is neither essential for placode specification nor redundantly required together with Fgfs for otic placode and vesicle formation. Interestingly however, Wnt8a and Fgf3 are redundantly required for expression of Fgf15 in the hindbrain indicating additional reciprocal interactions between Fgf and Wnt signalling. Further reduction of Wnt signalling by the inactivation of Wnt1 in a Wnt8a mutant background revealed a redundant requirement for both genes during morphogenesis of the dorsal portion of the otic vesicle.

Role of Gbx2 and Otx2 in the formation of cochlear ganglion and endolymphatic duct.

The boundary of gene expression of transcription factors often plays a role in making a signaling center in development. In the otic vesicle, Gbx2 is expressed in the dorso-medial region including the endolymphatic duct, and Otx2 in the ventral region. Fgf10 is expressed between their expression boundaries, and the cochleovestibular ganglion develops close to the medial side of the Fgf10 expressing domain. Similar expression patterns are observed in the central nervous system, where Otx2 and Gbx2 expression abut at the mid-hindbrain boundary, and the repressive interaction between Otx2 and Gbx2 defines the mid-hindbrain boundary. These analogous expression patterns raise a question about the role of the interaction between Gbx2 and Otx2 in the otic vesicle. To address this, we misexpressed Gbx2 and Otx2 to the otic epithelium. Ectopic Gbx2 expression could repress Otx2 expression and vice versa. In addition, Fgf10 expression was repressed and cochlear ganglion formation was interfered with. Moreover, endolymphatic duct was severely hypomorphic in the Otx2 misexpressing embryos. These results suggest that the interaction between Gbx2 and Otx2 in developing inner ear defines Fgf10 expression domain to induce the cochlear ganglion. It is also suggested that Gbx2 expression is important for the formation of the endolymphatic duct.

Gbx2 is required for the morphogenesis of the mouse inner ear: a downstream candidate of hindbrain signaling.

Gbx2 is a homeobox-containing transcription factor that is related to unplugged in Drosophila. In mice, Gbx2 and Otx2 negatively regulate each other to establish the mid-hindbrain boundary in the neural tube. Here, we show that Gbx2 is required for the development of the mouse inner ear. Absence of the endolymphatic duct and swelling of the membranous labyrinth are common features in Gbx2-/- inner ears. More severe mutant phenotypes include absence of the anterior and posterior semicircular canals, and a malformed saccule and cochlear duct. However, formation of the lateral semicircular canal and its ampulla is usually unaffected. These inner ear phenotypes are remarkably similar to those reported in kreisler mice, which have inner ear defects attributed to defects in the hindbrain. Based on gene expression analyses, we propose that activation of Gbx2 expression within the inner ear is an important pathway whereby signals from the hindbrain regulate inner ear development. In addition, our results suggest that Gbx2 normally promotes dorsal fates such as the endolymphatic duct and semicircular canals by positively regulating genes such as Wnt2b and Dlx5. However, Gbx2 promotes ventral fates such as the saccule and cochlear duct, possibly by restricting Otx2 expression.

The role of Pax2 in mouse inner ear development.

The paired box transcription factor, Pax2, is important for cochlear development in the mouse inner ear. Two mutant alleles of Pax2, a knockout and a frameshift mutation (Pax21Neu), show either agenesis or severe malformation of the cochlea, respectively. In humans, mutations in the PAX2 gene cause renal coloboma syndrome that is characterized by kidney abnormalities, optic nerve colobomas and mild sensorineural deafness. To better understand the role of Pax2 in inner ear development, we examined the inner ear phenotype in the Pax2 knockout mice using paint-fill and gene expression analyses. We show that Pax2-/- ears often lack a distinct saccule, and the endolymphatic duct and common crus are invariably fused. However, a rudimentary cochlea is always present in all Pax2 knockout inner ears. Cochlear outgrowth in the mutants is arrested at an early stage due to apoptosis of cells that normally express Pax2 in the cochlear anlage. Lack of Pax2 affects tissue specification within the cochlear duct, particularly regions between the sensory tissue and the stria vascularis. Because the cochlear phenotypes observed in Pax2 mutants are more severe than those observed in mice lacking Otx1 and Otx2, we postulate that Pax2 plays a key role in regulating the differential growth within the cochlear duct and thus, its proper outgrowth and coiling.

DAN directs endolymphatic sac and duct outgrowth in the avian inner ear.

Bone morphogenetic proteins (BMPs) are expressed in the developing vertebrate inner ear and participate in inner ear axial patterning and the development of its sensory epithelium. BMP antagonists, such as noggin, chordin, gremlin, cerberus, and DAN (differential screening-selected gene aberrative in neuroblastoma) inhibit BMP activity and establish morphogenetic gradients during the patterning of many developing tissues and organs. In this study, the role of the BMP antagonist DAN in inner ear development was investigated. DAN-expressing cell pellets were implanted into the otocyst and the periotic mesenchyme to determine the effects of exogenous DAN on otic development. Similar to the effects on the inner ear seen after exposure of otocysts to the BMP4 antagonist noggin, semicircular canals were truncated or eliminated based upon the site of pellet implantation. Unique to the DAN implantations, however, were effects on the developing endolymphatic duct and sac. In DAN-treated inner ears, endolymphatic ducts and sacs were merged with the crus or grew into the superior semicircular canal. Both the canal and endolymphatic duct and sac effects were rescued by joint implantation of BMP4-expressing cells. Electroporation of DAN antisense morpholinos into the epithelium of stage 15-17 otocysts, blocking DAN protein synthesis, resulted in enlarged endolymphatic ducts and sacs as well as smaller semicircular canals in some cases. Taken together, these data suggest a role for DAN both in helping to regulate BMP activity spatially and temporally and in patterning and partitioning of the medial otic tissue between the endolymphatic duct/sac and medially derived inner ear structures.

Development of the endolymphatic sac and duct in the Japanese red-bellied newt, Cynops pyrrhogaster.

The development and maturation of the endolymphatic sac (ES) and duct (ED) were studied in the newt Cynops pyrrhogaster. The ES first appears as an oval capsule at the dorsal-medial tip of the otic vesicle at stage 39, about 11 days after oviposition. The ES consists of polymorphous epithelial cells with a minimum of cytoplasm. The intercellular space (IS) between the epithelial cells is narrow and has a smooth surface. At stage 44, the size of the ES increases as many vacuoles in the IS become filled. At stage 46, 18 days after oviposition, the ES elongates markedly and a slit-like lumen is found in the ES. The epithelium contains a few cell organelles which are scattered in the cytoplasm. The vacuoles in the IS are fused, which expands the IS. Two days later (stage 48), floccular material (endolymph) is present in the expanded lumen. The IS dilates and has a wide and irregular appearance. At stage 50, approximately 26 days after oviposition, the ES extends and expands significantly and crystals (otoconia) can now be seen in the widened lumen of the ES. The cytoplasm of the cuboidal epithelial cells contains an abundance of vesicles surrounded by ribosomes and Golgi complexes. Intercellular digitations are formed in the expanded IS. At stage 54, the ES forms a large bellow-like pouch. Numerous otoconia accumulate in the lumen. Free floating cells and cell debris can be seen in the lumen at this stage. The epithelial cells contain numerous cytoplasmic organelles which are evenly distributed in the cytoplasm. Granules are found in the apical and lateral cytoplasm. The IS is loose and displays a labyrinthine appearance. The primitive ED first appears as a connection between the ES and the saccule but no lumen is present inside at stage 39. At stage 46, a narrow lumen is formed in the ED, which corresponds to the formation of the ES lumen. At stage 50, as the ED extends, floccular material is seen in the lumen. At stage 54, the ED bears numerous microvilli on its luminal surface. Otoconia and endolymph are present in the ED. Tight junctions between the epithelial cells are formed at stage 46. A fully developed intercellular junctional complex is produced at stage 54. Based on the development of the ES and ED, the maturation of function of the ES and ED are discussed.

The utriculo-endolymphatic valve in pediatric temporal bones.

The utriculo-endolymphatic valve (UEV) is located in the posterior wall of the utricle at its junction with the utricular duct and was first described in a human fetus by Bast in 1928. Although different theories about its normal position and function have been postulated, the function of the UEV remains unclear. In the present investigation we studied 118 temporal bones from 70 children to determine whether there were differences in the position of the valve and by inference, its function between children and adults. Premortem ages ranged from newborn to 10 years (mean age, 11.6 months). All temporal bones were fixed in 10% formalin, decalcified and processed by the celloidin technique. Specimens were sectioned in a horizontal plane at a thickness of 20 microns. Every tenth section was stained with hematoxylin-eosin and studied by light microscopy. The position of the UEV was then classified as closed or open. Valves damaged by preparation or having an uncertain position were classified as artifact. The chi-square test was used to determine a correlation between the position of the valve and the status of the rest of the endolymphatic system and whether or not endolymphatic hydrops was present in the cochlear and vestibular systems. The UEV was closed in 39 temporal bones (33.1%) and open in 13 (11.0%). Artifacts were found in 66 bones (55.9%). In the group of patients with a collapsed ductus reuniens the UEV was closed in 38% of the specimens, suggesting that the UEV prevented loss of endolymph from the pars superior, but these findings were not statistically significant.

Descriptive and experimental analysis of the epithelial remodellings that control semicircular canal formation in the developing mouse inner ear.

The inner ear of the mouse develops from a roughly spherical epithelial vesicle, the otocyst, which undergoes a series of complex shape changes to produce the functionally important parts of an adult inner ear; in particular, a coiled cochlea--which houses the auditory apparatus, a saccule and utricle containing sensors of gravity and linear acceleration, and three precisely shaped and oriented semicircular canals, with which angular acceleration is detected. This paper follows the development of the shape of the mouse inner ear from simple otocyst until a stage when the vesicle has become a rather squat miniature model of its adult self. We have been able to visualize clearly these complex shape changes by injecting an opaque marker into the lumina of a series of fixed ears. We have further concentrated on the mechanism of formation of the semicircular canals using light-, electron-microscopic, and dye-marking techniques. Classic embryological texts describe the canals developing from outpocketings of the epithelial ear rudiment, whose opposite walls meet, fuse, and "disappear" in the central canal plate region to leave a tube of epithelium encircling their margin. To trace the fate of these "disappearing" epithelial cells we have dye-marked all of the otic epithelial cells at a stage prior to canal formation in mouse embryos which we then grow in roller culture until their canals have formed. From these marking experiments we show that the disappearing cells of the canal plate neither die nor become transformed into mesenchymal cells, but rather, most are retracted back into the canal tube epithelium at either side of the site of fusion. We speculate that this mechanism of epithelial resorption may be a common way of "losing" epithelial cells during embryonic morphogenetic remodellings.

Unilateral duplication of the vestibular aqueduct.

A case of unilateral duplication of the vestibular aqueduct is presented. This very rare abnormality can be explained either by an excessive induction by the rhombencephalon or by an abnormal complete division of the endolymphatic duct into utricular and saccular parts.

Embryology of the human endolymphatic duct and sac.

Endolymphatic sac specimens from human embryos, ranging in age from 8 to 20 weeks, were examined in order to get a good understanding of the embryological development. The gross development can be separated into three phases, with the first phase terminating around week 10, the second phase between weeks 11 and 15-16 and the third phase lasting to week 20. While presenting as an immature oval appendage emanating from the vestibule in the first phase, the sac then develops into its more mature shape with crypts and folds within the epithelial lining. The third period is characterized by splitting up of the lumen into separate tubules. The epithelial cells differentiate particularly during the second and third phases with light- and dark-staining cells as well as granulated cells. It is evident that the development of the endolymphatic sac is not finished by week 20, even though it has attained many of its adult characteristics.

[Experimental study on early development of rat ear in vitro using whole embryo culture].

Rat embryos were explanted on late 11 day of gestation and cultured for 24 hours in rotating bottles with the yolk sac opened. Rat serum was used as culture medium and culture bottles were filled with 5% CO2 + 95% O2 gas mixture as gas phase. At the time of explantation and the end of the culture period, differentiation and growth of the embryos were monitored by counting somites and measuring crown rump length. About part of the embryos, protein determinations were made to measure growth. These data were compared with values found for 12 and 13 day embryos. The results for cultured embryos showed slight retardation in their differentiation and slight depression in the growth. At the same time to study early inner ear development in vitro, 9 of the other cultured embryos were serially sectioned and observed by light microscopy. In the cultured embryos endolymphatic duct elongated and acoustico-facial ganglion enlarged remarkably. Otocysts became flattened and elongated ventrally. Vestibular and cochlear portion were identifiable in the otocysts. Nerves arose from the ganglion reached to brain centrally and wall of pharynx peripherally. There was no significant difference in inner ear development between 13 day and the cultured embryos. This culture system should prove useful for studies on early inner ear development of mammalian embryos.

Submicroscopic study of the vestibular dark cell area in human fetuses.

The purpose of this study was to determine the characteristic ultra-microstructure of the vestibular dark cell area related to inner ear metabolism at mid-term human embryonic development. This is when the general inner ear structures apparently attain their final development. Two types of epithelial cells, dark cells and light cells, are discernible in the vestibular dark cell areas. The morphology of the dark cells is described and their role in the metabolism of endolymph and otoconia is indicated. Little is known about the nature and presence of the light cells in mammals. The present study has revealed the ultra-microstructure of the light cells and indicates their secretory function in otoconia metabolism. The dark and light cells seem to be closely related to each other in the metabolic function of the dark cell area.

The pre- and postnatal maturation of the epithelium in the endolymphatic sac. An electron microscopic survey.

The cellular development of the endolymphatic sac was studied in the CBA/CBA mouse, starting from day 10 of gestation following the different stages of maturation up to an adult age of one month. The first immature cylindrical cells lining the future sac in several cell layers are seen at day 12 of gestation. At day 18 of gestation, a true sac appears and a floccular precipitate is frequently found in its lumen together with signs of increased activity in the still immature epithelial cells. Approximately one day before birth the first signs of the future light and dark cells can be distinguished. At day 4 post partum the cells are more differentiated with some showing signs of secretory activity indicating that these cells start to function at this stage. Eight days after birth differentiation into distinguishable almost mature light and dark cells is seen. Two days later these epithelial cells have obtained a fully mature appearance. At 14 days after birth widened lateral intercellular spaces separating the epithelial cells can be visualized and a few free floating cells are found in the sac lumen. The sac epithelium is thus considered to have completed its maturation process at this stage.

The development of the endolymphatic duct and sac. A light microscopical study.

The development and maturation of the endolymphatic sac were studied in the CBA/CBA mouse. The otocyst is developed at gestational day 10 and the primitive endolymphatic sac is present as a large slit-like appendage at day 12 of gestation. At day 18 the endolymphatic sac is clearly detached from the rest of the otocyst, forming a true sac. The epithelial lining consists of only one layer of immature cells containing large vesicles. The endolymphatic sac is surrounded by a rich network of vessels. One day before birth, the epithelial lining is uneven and the first signs of differentiation into light and dark cells is visible. This situation is more pronounced 2 days post partum when the sac also seems to be filled with a stainable material. At day 6 post partum the otic capsule fuses around the sac, forming the vestibular aqueduct. At 14 days post partum the sac is mature, with clearly developed light and dark cells and widened lateral intercellular spaces, constituting the rugose epithelium. The lumen is filled with a stainable precipitate and a few free-floating cells.

[The early development of the labyrinth (author's transl)]. / Das frühembryonale Wachstum des Labyrinths.

The developmental kinetics of the inner ear of 2- to 30-mm embryos are examined. The development of the inner ear can be shown as being constructively correlated with the development of the brain and thus being an ontogenic requisite for the structural development of the embryo. The development kinetic findings, as shown here, demonstrate that the functions of the inner ear have already started during its development and differentiation.

Development of endolymph during maturation of the mammalian inner ear. A preliminary report.

The development of the elemental composition in the endolymphatic space was investigated during embryologic and early post natal maturation of the CBA/CBA mouse. At birth the elemental distribution was similar in the endo- and perilymphatic spaces. Mature composition of endolymph was reached 6--8 days post partum. The maturation of endolymph corresponded well in time with the morphological maturation of the stria vascularis.

[Comparative studies of the endolymphatic sac and duct in embryonic skull (author's transl)]. / Vergleichende Untersuchungen über den Ductus und Saccus endolymphaticus am embryonalen Schädel.

The endolymphatic sac and duct were studied in 25 embryonic ears of humans between the 10th and 25th week of development. In the early stage the anlage develops as a protrusion medio-caudally from the utricle. During further development vacuoles appear within the connective tissue surrounding the duct and sac which eventually unite with the endolymphatic system enlarging the endolymphatic space as an answer of the connective tissue to the expansion and proliferation of the endothelium. In older specimens (fronto-occipital diameter: 40 mm) we find a tongue-like projection of the wall of the duct into the utricle known as the endolymphatic valve. This structure is covered with a thickened folded epithelium. It is stabilized by fibrous, cartilaginous, and bony tissue. Under normal conditions it therefore seems improbable that it can serve as a valve as supposed by other authors. The proliferation of endothelium and a concentration of blood vessels in this might indicate a higher metabolic activity with a biochemical filtering mechanism.