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Rationale : Mutations of SLC26A4 that abrogate pendrin, expressed in endolymphatic sac, cochlea and vestibule, are known to cause autosomal recessive sensorineural hearing loss with enlargement of the membranous labyrinth. This is the first study to demonstrate the feasibility of gene therapy for pendrin-related hearing loss. Methods: We used a recombinant viral vector to transfect Slc26a4 cDNA into embryonic day 12.5 otocysts of pendrin-deficient knock-out ( Slc26a4 ∆/∆ ) and pendrin-deficient knock-in ( Slc26a4 tm1Dontuh/tm1Dontuh ) mice. Results : Local gene-delivery resulted in spatially and temporally limited pendrin expression, prevented enlargement, failed to restore vestibular function, but succeeded in the restoration of hearing. Restored hearing phenotypes included normal hearing as well as sudden, fluctuating, and progressive hearing loss. Conclusion : Our study illustrates the feasibility of gene therapy for pendrin-related hearing loss, suggests differences in the requirement of pendrin between the cochlea and the vestibular labyrinth, and documents that insufficient pendrin expression during late embryonal and early postnatal development of the inner ear can cause sudden, fluctuating and progressive hearing loss without obligatory enlargement of the membranous labyrinth., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Mutations of SLC26A4 are a common cause of hearing loss associated with enlargement of the endolymphatic sac (EES). Slc26a4 expression in the developing mouse endolymphatic sac is required for acquisition of normal inner ear structure and function. Here, we show that the mouse endolymphatic sac absorbs fluid in an SLC26A4-dependent fashion. Fluid absorption was sensitive to ouabain and gadolinium but insensitive to benzamil, bafilomycin and S3226. Single-cell RNA-seq analysis of pre- and postnatal endolymphatic sacs demonstrates two types of differentiated cells. Early ribosome-rich cells (RRCs) have a transcriptomic signature suggesting expression and secretion of extracellular proteins, while mature RRCs express genes implicated in innate immunity. The transcriptomic signature of mitochondria-rich cells (MRCs) indicates that they mediate vectorial ion transport. We propose a molecular mechanism for resorption of NaCl by MRCs during development, and conclude that disruption of this mechanism is the root cause of hearing loss associated with EES.

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB-Efnb2 signaling-deficient mice., (Published by Elsevier Inc.)

Senescence is a form of cellular aging that limits the proliferative capacity of cells. Senescence can be triggered by different stress stimuli, such as DNA damage or oncogene activation. Two recent articles published in Cell have uncovered an unexpected role for cellular senescence during development, as a process that contributes to remodeling and patterning of the embryo. These findings are exciting and have important implications for the understanding of normal developmental and the evolutionary origin of senescence.

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cellular senescence is implicated in several pathological responses in the adult, with important repercussions in tumor suppression, wound healing, and aging. Two studies by Muñoz-Espín et al. and Storer et al. now reveal that senescence contributes to embryonic development, suggesting a primordial role in normal physiology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mutations of SLC26A4 are among the most prevalent causes of hereditary deafness. Deafness in the corresponding mouse model, Slc26a4(-/-), results from an abnormally enlarged cochlear lumen. The goal of this study was to determine whether the cochlear enlargement originates with defective cochlear fluid transport or with a malfunction of fluid transport in the connected compartments, which are the vestibular labyrinth and the endolymphatic sac. Embryonic inner ears from Slc26a4(+/-) and Slc26a4(-/-) mice were examined by confocal microscopy ex vivo or after 2 days of organ culture. Culture allowed observations of intact, ligated or partially resected inner ears. Cochlear lumen formation was found to begin at the base of the cochlea between embryonic day (E) 13.5 and 14.5. Enlargement was immediately evident in Slc26a4(-/-) compared to Slc26a4(+/-) mice. In Slc26a4(+/-) and Slc26a4(-/-) mice, separation of the cochlea from the vestibular labyrinth by ligation at E14.5 resulted in a reduced cochlear lumen. Resection of the endolymphatic sacs at E14.5 led to an enlarged cochlear lumen in Slc26a4(+/-) mice but caused no further enlargement of the already enlarged cochlear lumen in Slc26a4(-/-) mice. Ligation or resection performed later, at E17.5, did not alter the cochlea lumen. In conclusion, the data suggest that cochlear lumen formation is initiated by fluid secretion in the vestibular labyrinth and temporarily controlled by fluid absorption in the endolymphatic sac. Failure of fluid absorption in the endolymphatic sac due to lack of Slc26a4 expression appears to initiate cochlear enlargement in mice, and possibly humans, lacking functional Slc26a4 expression.

Otoconin-90, the principal otoconial matrix protein, provided a tool to investigate the molecular mechanism of otoconial morphogenesis. The endolymphatic sac of the embryonic chick and guinea pig contain otoconia. Here, we show that the embryonic mouse transiently expresses ectopic otoconia in the endolymphatic sac. Massive precipitate of otoconin-90-positive material is detectable in the lumen of the endolymphatic sac between embryonic day 14.5 and 17.5 with frequent accretion into more heavily staining otoconia-like particles. Otoconin-90 was also localized at the surface and the interior of epithelial cells lining the endolymphatic sac as well as incorporated into free floating cells. In contrast, in situ hybridization failed to detect mRNA in the endolymphatic duct and sac, even though the adjacent nonsensory vestibular structures are heavily stained. Because of ample expression of otoconin-90 protein in the absence of the corresponding mRNA, we conclude that the luminal otoconin-90 is imported via longitudinal flow from the vestibular compartments, where both mRNA and protein are strongly expressed. Because of absence of mRNA, the expression of the corresponding protein by the epithelia lining the endolymphatic sac can only be explained by a resorptive process, as previously proposed on the basis of the movement of luminal macromolecules. The data do not support the previous hypothesis that the transient expression of otoconia-like particles of the endolymphatic sac represents a vestigial phenomenon from the amphibian stage, since amphibia express ample mRNA encoding otoconin-22 in the endolymphatic sac system.

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., (Copyright 2003 Wiley-Liss, Inc.)

It has recently been demonstrated that endolymphatic sac (ES) ribosome-rich (dark) cells respond to induced endolymph changes and are thus likely to be involved in endolymph homeostasis. Therefore, we studied the ultrastructural characteristics of rat ES ribosome-rich cells during development in order to determine the cellular distribution of organelles involved in protein metabolism, secretion and absorption, indicative for their contribution to endolymph homeostasis. During embryonal stages ribosome-rich cells contain a limited number and variety of organelles and are predominantly involved in the production of components for cell growth and differentiation. In the young adult stage (P60) three different states of ribosome-rich cells may be distinguished. State A resembles a cell with only limited metabolic activities whereas state B is characterized by numerous different intracellular organelles and is considered to be involved in production and secretion as well as absorption and degradation of complex proteins. A third cellular state, state C, is filled with phagolysosomes and contains very few other organelles. This is considered to be a final (pre)apoptotic state. Autoradiography data suggest that ES ribosome-rich cells are capable of synthesis and secretion of tyrosine-containing proteins and may thus be involved in regulation of the osmolarity of endolymph based on the capacity to bind cations as well as water molecules. In addition, ES ribosome-rich cells appear to synthesize and secrete fucosylated glycoproteins into the endolymph. In conclusion, the present data suggest that ES ribosome-rich cells are actively involved in endolymph homeostasis through secretion and absorption of complex proteins and it is hypothesized that they are able to adapt their function or activities in response to changes in endolymph composition.

Water transport between the perilymph and endolymph is important in regulations of volume and osmotic pressure of the inner ear labyrinth. It is now known that expression of water channels (aquaporins or AQPs) in the cell membrane dramatically increases the ability of water to cross epithelial cells. The aims of the current study were to investigate the cellular localization of AQPs by immunolabeling, and to study the developmental expression and relative abundance of various subtypes of AQPs. We report here that AQP3, AQP7 and AQP9 were expressed in the inner ear. Specific subtypes of AQPs were found in discrete regions expressed by both epithelial cells and fibrocytes in cochlear and vestibular organs. Semi-quantitative measurements showed that AQP4 and AQP1 were the two most abundantly expressed AQP subtypes in the inner ear, and their expressions were dramatically upregulated during development. These data showed a highly localized and largely non-overlapping distribution pattern for different subtypes of AQPs in the inner ear, suggesting the existence of regional subtype-specific water transport pathways, and global regulation of water transport in the inner ear may require concerted actions of multiple types of AQPs.

The purpose of this study was to determine specific characteristics of endolymphatic sac (ES) cells of the developing rat that are considered to be involved in endolymph homeostasis. Because intermediate filament proteins (IFPs) are regarded as markers of cell differentiation and basal lamina proteins (BLPs) are essential in cell<=>matrix interactions, we determined the presence of IFPs [cytokeratins (CKs) and vimentin] and BLPs [collagen IV, heparan sulphate proteoglycan (HSPG) and laminin] at different developmental stages before and after birth. In addition, we studied the expression of two enzymes of oxidative metabolism: cytochrome oxidase and succinate dehydrogenase. The presence of CKs 8, 18 and 19 in all epithelial cells of the ES during the embryonic stage is characteristic of simple (glandular) epithelial cells. Interestingly, a distinct population of these cells shows additional expression of CK 7, which is a feature of secretory cells. These CK 7-positive cells also contain a high concentration of oxidative enzymes and are rich in mitochondria, indicating that they are light cells. It is suggested that light cells possess specific energy-requiring transport capabilities. Loss of CK 19 expression in the distal part and in a large region of the intermediate part of the ES implies that these cells do not differentiate any further and acquire the capacity to proliferate. Furthermore, prominent co-expression of vimentin with the CKs in the distal part of the ES may confer viscoelastic properties on this epithelium. This may facilitate expansion and thus enable cushioning of pressure fluctuations. Finally, the early prominent occurrence of HSPG in the basal lamina of the ES enables transport of ions. In this light our recent observations of early functioning NaK-ATPases in certain ES cells are interesting.

Objective: The endolymphatic sac (ES) of vertebrates contains varying amounts of a homogeneous substance (HS) that stains deeply with basic aniline dyes. Histochemically, HS is characterized as a carbohydrate-protein complex, being both neutral and acidic in nature. In the present study, deposition of HS in the ES was studied in the guinea pig from the 3rd week of gestation to 104 weeks postnatally in order to find out if HS accumulates with age, at which point during embryonic development this substance appears, if its presence is correlated to the sense of hearing and if the amount of substance in the left versus right ear of one and the same animal is correlated to any degree., Methods: Sixty-nine endolymphatic sacs were evaluated in 38 guinea pigs. The ES specimens were sectioned for light and transmission electron microscopy and the amount of HS filling was categorized in four groups: none, low, medium and a high level of substance., Results: The substance was not discerned until after 7 weeks of gestation, when it filled only a minor part of the distal ES lumen. At 9 weeks gestation the nature of the substance altered, becoming homogeneous, as visualized by osmium-toluidine blue staining and approximately filling the distal half of the luminal space. In the postnatal period, 65% of ES specimens were filled with HS to the intermediate or proximal ES, whereas only 6.5% of the ES specimens were devoid of the substance. The extent of filling of the ES in the prenatal temporal bones was significantly less than postnatally (P < 0.0001, chi2-test). The extent of postnatal filling was not correlated with age. Left and right ears were closely correlated in one and the same animal. Phagocytic cells were often found at the border between clear endolymph and stainable substance., Conclusion: The appearance of HS seemed to coincide temporally with the onset of hearing during the prenatal period indicating that it could play a part in normal inner ear functioning in the guinea pig. The close correlation regarding the level of the HS in the left and right ear, both pre- and postnatally could reflect a general symmetry in endolymph pressure-volume conditions within the inner ear fluid systems, as well as in the environmental hydrostatic pressure in the posterior cranial fossa.

The formation of otoconia in the endolymphatic sac (ES) of the larval newt, Cynops pyrrhogaster, has been studied by light and transmission electron microscopy. Some of the epithelial cells of the ES contain an abundance of swollen vesicles, Golgi complexes, rough endoplasmic reticula and ribosomes at the late larval stages 50 and 51, approximately 26-30 days after eggs are laid. Five days later, at stage 52, crystals are present in the vacuoles between the epithelial cells. Serial sections indicate that these vacuoles actually form small canals which lie in the wall and join the lumen of the ES. Reconstruction of the ES shows that several canals are contained in the ES wall. At stage 56, about 72 days after eggs are laid, a large number of otoconia are present in the ES lumen, while the otoconia disappear from the canals. It appears that the otoconia are first produced in the canals and then released to the lumen. Some epithelial cells of the ES are thought to expel the organic and inorganic material to the canals to form the otoconia in situ. The process of formation of the otoconia in the ES is discussed.

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 adult human endolymphatic sac (ES) has been described as a complex network of interconnected tubules. Embryologic examination describes the human ES as a single-lumen, pouch-like structure. Transition from saccular shape to tubules during the entire fetal period has not been previously reported. Tubular ES structure is thought to be unique to humans. Animal investigations describe similar saccular appearance, but without tubules in mature sacs. The authors examined 45 human fetal temporal bones to trace ES development and reviewed six types of animal sacs. Results in humans reveal tubular structure as early as 26 weeks' gestation. Maturation variably occurred in the fetal period and postnatally. For the first time, the tubular system is noted in the animal, the rhesus monkey. These findings suggest that the tubular system may represent more advanced specialized function.

The expression of S-100 protein was analyzed in the human fetal inner ear using immunohistochemical methods. In the 11-week-old human fetus, the cochlea was almost negative for S-100 protein, whereas in the 14- and 15-week-old fetuses, the spiral ligament, Reissner's membrane and spiral limbus were positive for the protein. These results suggest that S-100 protein may be a reliable marker for determining functional maturation of the fetal cochlea and the inner ear. In the 11-, 14- and 15-week fetuses, the epithelial cells of the endolymphatic sac were labelled with S-100 protein. These findings demonstrate that the endolymphatic sac, spiral limbus and spiral ligament in the fetal inner ear have a high activity of S-100 protein, with this presence possibly related to fluid and ion transport of endolymph.

The cytodifferentiation of the human endolymphatic sac was studied in the period between gestational weeks 8 and 20. This period is of particular interest since it covers the major part of the morphological and functional maturation of the human inner ear. The studied time, i.e. 8-20 weeks of gestation, may be divided into three periods. Before week 10, between weeks 11 and 15 and weeks 15-20. While the endolymphatic sac appears as a simple slit-like appendage in the first period, until week 10 with cells of uniform size and shape, a beginning cytodifferentiation occurs in the second period between weeks 10 and 15. Thus the cells attain a more mature shape with different stainability, i.e. light- and dark-staining cells, as well as open lateral intercellular spaces indicating some functional maturity. In the third period, after week 15, the endolymphatic sac more or less seems mature with a rugose appearance in its proximal portion and a more even, slit-like appearance in the distal portion. The cells are differentiated with morphological signs of functional maturation. It may be concluded that the development of the endolymphatic sac roughly follows that of the cochlea which is regarded to be functionally mature in the beginning of the second half of pregnancy.

The expression of laminin was analyzed in the vestibular endorgans of chick embryos using immunohistochemical methods. The basement membrane of the otocyst in the 5-day-old chick embryo was positive for laminin. In the 9-day-old chick embryo, the basement membrane of the macula of the saccule and utricle was positive, but the crista ampullaris showed a very weak positive reaction. The basement membrane of the endolymphatic sac in the 11-day-old chick embryo was strongly positive. These results suggest that laminin may be an essential substance with regard to the pathology of the vestibular endorgans. Moreover, the uneven distribution of laminin suggests that the basement membrane plays a role whose importance is yet to be determined in the physiology of the inner ear.

The distribution of glycoconjugates in the endolymphatic sac (ES) and vestibular end organs from the 12th gestational day (GD) in the developing mouse to the 6th day after birth was analyzed using six biotinylated lectins: wheat germ agglutinin (WGA), Abrus precatorius agglutinin (APA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin 120 (RCA120), Helix pomatia agglutinin (HPA), concanavalin A (conA). In the 13th and 15th GD sections, the luminal contents in the fetal ES were strongly labelled with lectins, while only a small amount of substance in the ES was labelled with lectins after the 17th GD. In the 13th GD sections, before the cupula and otoconia were formed, the fetal ES started to produce glycoconjugates labelled with WGA, APA, RCA120 and ConA. In the 17th GD sections, the immature cupula and otoconia were strongly labelled with WGA, APA and RCA120. Sugar residues stained by lectins detected in the substance of the fetal ES were the same as those found in the immature cupula and otoconia. HPA only stained the ES epithelium around the 13th and 15th GD. The fetal ES may interact with the formation of the cupula and otoconia, especially in the early stage during evolution and that HPA-reactive glycoconjugates may be related to the intracellular elements of the ES epithelium only during a well-defined phase of development.

The distribution of glycoconjugates in the 11 to 16 weeks old human fetal endolymphatic sac (ES) was analyzed using six biotinylated lectins; Wheat germ agglutinin (WGA), Abrus precatorius agglutinin (APA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin 120 (RCA120), Helix pomatia agglutinin (HPA), Concanavalin A (ConA). In the 11 week old human fetus, fluorescent reactions with WGA, APA, RCA120 and ConA were detected in the ES. There was almost no reaction with HPA and UEA-I. In the 14 week old human fetus, however fluorescent reactions with HPA and UEA-I appeared. This result suggests that the presence of glycoconjugates changes during the maturation process of the ES. Glycoconjugates detected with HPA were related to the epithelial cell elements of the ES epithelium. The reaction with UEA-I suggests that the stainable substance present in the ES lumen may be secreted locally by the ES itself.

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.

The development of perisaccular blood vessels is described during embryology and the postnatal period of the mouse. Primitive sinusoidal vessels already appear at the early otocyst stage as the future endolymphatic sac is formed. Before birth the vasculature attains a more mature appearance with tubular, somewhat fully developed blood vessels. At this stage a primitive basement membrane is also formed. Soon after birth the blood vessels appear mature with developed fenestrations and micropores, giving them an appearance comparable to blood vessels in other fluid transporting organs.

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 paper describes an investigation of the influence of gravity on the early differentiation of gravity receptors in Xenopus embryos and larvae. There is evidence that the expression of crystals in the saccus endolymphaticus was statistically greater when the embryos developed in near weightlessness (hypogravity) than on earth. The function of these crystals is unknown but they may contribute to the functioning of the vestibular apparatus.

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.

During certain stages of mammalian inner ear development, small crystallized bodies which resemble otoconia may be found in the endolymphatic sac. In order to examine whether the endolymphatic sac plays any part in the process of degradation and dissolution of otoconia, we made an electron-microscopic examination on the endolymphatic sac of fetuses and adult guinea pigs injected with streptomycin sulfate (SM). In 30-day-old fetal guinea pigs we found miniature otoconium-like bodies (OLBs) in the endolymphatic sac and a giant OLB in the endolymphatic duct. In adult animals we found no otoconia in the endolymphatic sac following SM intoxication. However, the results suggested that both the dark cells of the utricle, as well as the non-sensory epithelium of the saccule, may be engaged in the absorption and dissolution of otoconia.

In the present investigation we studied the morphology o the endolymphatic sac in guinea pig fetuses (age 20-, 30-, 45-, 60-days-old and newborns). Twenty-day and 30-days-old guinea pig fetuses often displayed small prismatic or hexagonally shaped granules, presumably representing miniature otoconia. The granules appeared freely in the lumen of the endolymphatic sac as well as incorporated in the cytoplasm of the freely floating cells or macrophages. The origin of these "sac otoconia' as well as the possible role of the endolymphatic sac in statoconis turnover and metabolism is discussed.

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.

The venous vascular anatomy of the endolymphatic sac in human embryos was examined. The endolymphatic sac was found to be covered by sinusoid-like blood vessels arising from the sigmoid sinus. A rich and extensive capillary network was present on the epithelial surface of the endolymphatic sac. Connections between this capillary bed and the vein in the paravestibular canaliculus were seen. The blood of the endolymphatic sac can therefore drain either into the vein of the vestibular aqueduct in the paravestibular canaliculus or directly into the sigmoid sinus. The vessels lying on the endolymphatic sac are thin-walled and irregular. The endothelial cells lies in direct contact with the epithelial cells of the endolymphatic sac. The reduction of the dense capillary bed in the young embryo to only a few vessels in the order embryo is described.

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.