]>HEARES2986S0378-5955(98)00018-510.1016/S0378-5955(98)00018-5Elsevier Science B.V.Fig. 1Drawing of a stage 52 larva of the newt Cynops pyrrhogaster, including a three-dimensional reconstruction of the inner ear and endolymphatic system, which are shown at higher magnification below. Abbreviations: UTO, utricular otolith; ES, endolymphatic sac; EO, endolymphatic otolith; ED, endolymphatic duct; SO, saccular otolith.Fig. 2Light micrographs of gross changes in the endolymphatic sac, otic vesicle, midbrain, during development and maturation in the newt Cynops pyrrhogaster. The sections were cut transversely through the middle portion of the endolymphatic sac (1 μm thickness) and stained with 1% toluidine blue. Bar=50 μm. a: Stage 39, an oval shaped endolymphatic sac first appears at the dorsomedial tip of the otic vesicle. The endolymphatic sac is adjacent to the midbrain. b: Stage 41, several tiny vacuoles (arrow in enlarged insert) appear in the sac, indicating that the endolymphatic sac is starting to expand. Scale bar in magnified insert is 10 μm. c: Stage 46, the endolymphatic sac elongates significantly. A slit-like lumen starts forming in the middle portion of the saccule. The endolymphatic duct opens into the otic vesicle. The epithelial lining becomes distinguishable. d: Stage 48, the lumen of the endolymphatic sac expands, and is clearly distinguished from the outer epithelial lining. This section shows that the proximal portion of the endolymphatic sac has extended to the upper end of the medial wall of the otic vesicle. e: Stage 50, the endolymphatic sac lumen enlarges and a number of bright otoconia are present. f: Stage 54, the endolymphatic sac expands and extends markedly forming a large lumen. The lumen of the endolymphatic sac accumulates numerous otoconia nearest the duct entering the otic vesicle (OC and arrow in enlarged insert, scale bar=10 μm). Abbreviations: OT, otic vesicle; MB, midbrain; LM, lumen; EL, epithelial lining; OC, otoconia. Other abbreviations as in Fig. 1.Fig. 3TEM of epithelial cells of the ES at different developmental stages. Bar=1 μm. a: Stage 39, the epithelial cell is of polymorphous type and contains a minimum of cytoplasm. Note the intercellular space is narrow and has a smooth appearance. b: Stage 44, the epithelial cell has a columnar shape. The cytoplasm is not dense with only a few mitochondria seen. The intercellular space contains several vacuoles and primitive microvilli on its surface. The basement membrane blends with the fibrils of the connective tissue (unlabeled arrow). c: Stage 48, the epithelial cell contains dense cytoplasm in which mitochondria are evident. The dilated intercellular space has an irregular surface with finger-like projections where cell junctions form. d: Stage 54, the epithelial cell contains a very dense cytoplasmic matrix and has a fibrous appearance. Abundant cell organelles such as mitochondria, smooth endoplasmic reticulum and Golgi complexes are seen in the cytoplasm. Note many granules and vesicles are also located in the apical and lateral cytoplasm. The intercellular space is loose and has a labyrinthine appearance. Abbreviations: EP, epithelial cell; IS, intercellular space; LV, lipid vesicle; VC, vacuole; M, mitochondria; MV, microvilli; N, nucleus; CJ, cell junction; GR, granules; V, vesicles. Other abbreviations as in Fig. 2.Fig. 4TEM of the lumen of the ES at different stages during development. Bar=5 μm. a: Stage 46, a slit-like lumen goes longitudinally through the middle part of the sac. The intercellular spaces are perpendicular to the surface of the lumen. The vacuoles in the space are fused, which makes the space have a wave-like surface. Note that there is a widened intercellular space between and beneath the epithelial cells. b: Stage 48, the ES lumen expands. This shows an uneven lining formed by microvilli. Floccular material appears in the lumen. c: Stage 50, the lumen dilates markedly. Otoconia are present in the lumen. The spaces where the otoconia were appear as holes, indicating artifacts of processing. The floccular material becomes more dense nearest to the otoconia. The intercellular spaces have a honeycomb appearance. d: Stage 54, the lumen expands its width and length significantly with dense floccular material inside. The relatively thick and long microvilli arise from the luminal surface of the flattened epithelial cells. Abbreviations: FM, floccular material. Other abbreviations as in Figs. 2 and 3.Fig. 5TEM of two morphologies of the ES appearing at stage 54. a: High resolution TEM of a matured microvillus on the luminal surface of an epithelial cell. Note that the lipid bilayer is visible. A small vesicle is also found in the cytoplasm. Bar=0.1 μm. b: TEM of a free floating cell. The cytoplasm of this cell is relatively light with numerous vacuoles and empty mitochondria inside. Long microvilli are present around all the free surfaces of the cell. Bar=1 μm. Abbreviations: LB, lipid bilayer; FF, free floating cell. Other abbreviations as in Figs. 2–4.Fig. 6TEM of the endolymphatic duct during development and maturation. The sections were cut longitudinally through the duct. a: Stage 46, the primitive endolymphatic duct with a narrow lumen within the medial wall of the otic vesicle. Bar=5 μm. b: Stage 50, the lumen of the endolymphatic duct expands at the intermediate portion. Some floccular material is present in the lumen. Note that the luminal surface is relatively smooth with only a few microvilli seen. Bar=2 μm. c: Stage 54, the intermediate portion of the endolymphatic duct. Note that the epithelial cells contain dense cytoplasm. The luminal surface bears numerous microvilli that display an intermeshing appearance. Also note that there are fibrils in the basement membrane. Bar=1 μm. Abbreviations: GC, Golgi complex; F, fibrils. Other abbreviations as in Figs. 2–4.Fig. 7TEM of junctional complex between the epithelial cells of the ES during development. a: Stage 46, tight junctions are formed close to the lumen. The outer layers of the adjacent plasma membrane fuse in the wall of the junction. Bar=0.1 μm. b: Stage 50, a tight junction and an adherent junction are present between the epithelial cells, indicating that junctional complex has developed. Note some granular substance is adhering along the outer wall of the tight junction and cytoplasmic filaments are associated with the adherent junction. Bar=0.2 μm. c: Stage 54, the walls of the tight junction are dense and parallel with a narrow interspace. The cytoplasmic filaments crossing the adherent junction are longer than those at stage 50. Below the junctional complex, both of the cell membranes are closely apposed at some parts. Bar=0.2 μm. Abbreviations: AJ, adherent junction; TJ, tight junction; CF, cytoplasmic filaments. Other abbreviations as in Figs. 2–4.Table 1Abbreviations used in figuresAJadherens junctionFMfloccular materialNnucleusCFcytoplasmic filamentsGCGolgi complexOCotoconiaCJcell junctionGRgranuleOTotic vesicleEDendolymphatic ductISintercellular spaceSOsaccular otolithELepithelial liningLBlipid bilayerTJtight junctionEOendolymphatic otolithLMlumenUTOutricular otolithEPepithelial cellLVlipid vesicleVvesicleESendolymphatic sacMmitochondriaVCvacuoleFfibrilsMBmidbrainFFfree floating cellMVmicrovilliTable 2Quantitative and qualitative characteristics of the developing ESStageDSizeType of cellCytoplasm and cell organellesMicrovilli (μm)Intercellular spaceLumen (μm)Material in lumen391165×25×15polymorphousminimumnarrow, smooth4415columnarnot dense, only a few Mvacuoles4618120×30×25cylindricalincrease in M0.3–0.5fused vacuoles, expanded, wave-like39×3.3×44820150×35×40cylindrical, cuboidalrelatively dense M, scattered R and G0.4–0.6dilated, finger-like protrusions, expandedfloccular material5026160×35×50cuboidaldense, packed M, R, and G vesicles0.5–0.7honeycomb, intercellular digitations120×20×20floccular materialotoconia5448190×40×55cuboidal, flatvery dense with fibrous appearance; abundant M, R, and G vesicles and granules1.0labyrinthine, intercellular digitations, floccular material140×30×25floccular materialotoconiacell debrisfloating cellsD, days after eggs are laid; M, mitochondria; R, ribosome; G, Golgi complex.Table 3Quantitative and qualitative characteristics of the developing EDStageLength (μm)Diameter of lumen (intermediate) (μm)Diameter of lumen (low end) (μm)Cell typeMicrovilli (intermediate)Microvilli (lower end)Material in lumen39250046450.50.8duct-likea few501000.81.0lower cuboidalonly a fewa fewfloccular material542001.22.0low cuboidal and flatintermeshinga fewfloccular materialotoconiaDevelopment of the endolymphatic sac and duct in the Japanese red-bellied newt, Cynops pyrrhogasterWenyuanGaoMichael LWiederhold*Jeffery LHarrisonDepartment of Otolaryngology-Head and Neck Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7777, USA*Corresponding author. Tel.: +1 (210) 567 5655; Fax: +1 (210) 567 3617.AbstractThe 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.KeywordsEndolymphatic sac and ductDevelopmentOtoconiaIntercellular space1IntroductionThe endolymphatic sac (ES) and duct (ED), which are part of the membranous labyrinth, exist in most vertebrates. The biological function of this portion of the inner ear has received much attention in mammals. Absorption and endocytosis of endolymph (Fukazawa et al. 1990, 1991, 1995; Hoshikawa et al., 1994), secretion of endolymph and other substances (Friberg et al., 1986; Barbara, 1989; Rask-Andersen et al., 1991), pressure regulation of the endolymphatic compartment (Bagger-Sjöbäck and Rask-Andersen, 1986; Friberg et al., 1986; Barbara et al., 1987; Takumida et al., 1988) and immunoreactive function in the inner ear (Rask-Andersen and Stahle, 1980; Tomiyama and Harris, 1986) have been suggested for the entire endolymphatic system. In amphibians, however, the only function of the ES which is known so far is to store calcium. This stored calcium is used to make otoconia and later to mineralize the skeleton and in egg production (Guardabassi, 1960; Marmo et al., 1986).One way to obtain further knowledge about the ES and ED is to study the embryology of the system. Bast and Anson (1949)found that the human ES forms early in a 7-mm embryo by an anterior-posterior crease in the otic vesicle. Watske and Bast (1950)showed changes in size and position of the ES during its maturation. Apart from the studies by Hultcrantz et al. (1987, 1988)in which the ES and ED were investigated at the light and electron microscopic level in CBA/CBA mouse, few studies have been devoted to the development of this system in mammals. In amphibians, only a few early studies of the embryology of the ED and ES have been reported. Whiteside (1922)demonstrated in Rana temporaria linne, that the ED is a well-differentiated canal, situated at the medial side of the otic vesicle; its upper end expands into a small vesicle to form the ES during the first stage (4 mm long). Dempster (1930)reported that the formation of the ES starts as a dorsal evagination of the closed otic vesicle in 15 mm long cryptobranchidae alleganiensis. Unfortunately, these studies were based on light microscopic observations and did not describe the whole developmental process.The Japanese newt Cynops pyrrhogaster is a favorable species in which to study development, since its vestibular system is similar to those of mammals but develops much more rapidly. The development of the otolith organs and semicircular canals, the gross appearance of the ES and ED and the morphogenic features of otoconia in the utricle and saccule have been investigated in this laboratory (Wiederhold et al. 1992, 1995, 1997; Steyger et al., 1995). However, there is a lack of detailed information concerning the development of the ES and ED. The present study was performed to elucidate the pattern and sequence of events during organogenesis and cytodifferentiation of the ES and ED at the light and electron microscope level. It is hoped that this study will shed light on the function of this system in amphibians.2Materials and methodsNewt embryos of the desired developmental stage were obtained as reported previously (Wiederhold et al., 1995; Steyger et al., 1995). Development stages were determined by observation through a dissecting microscope and comparison with the sequence and stage description reported by Okada and Ichikawa (1947)and Okada (1989).Three embryos or larvae at each stage (from stage 25 to 56) were used as subjects. The specimens were fixed by immersion in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h. After a triple-rinse in 0.1 M cacodylate buffer, the specimens were post-fixed in 1% osmium tetroxide (pH 7.4) for 1 h, and again rinsed in buffer. Specimens were dehydrated with increasing concentrations of ethanol (35% to 100%, 15 min each). A transition fluid of propylene oxide was used (45 min) before infiltration and embedding in Epon. The polymerization process required three days in an oven (37, 45, 60°C for 24 h each).Serial transverse sections (1 μm thickness) were cut through the ES and ED. The sections were stained with 1% toluidine blue, mounted on slides and cover slipped. The sections were examined under an Olympus BH-2 light microscope. Ultrathin sections (90 nm) were cut transversely at the middle portion of the ES and ED. The sections were stained with uranyl acetate and Reynold's lead citrate (Reynolds, 1963), and examined with a Philips 301 electron microscope.The care and use of the animals reported in this study were approved by the University of Texas Health Science Center at San Antonio's Institutional Animal Care and Use Committee.3Results3.1Development of the endolymphatic sac (ES)3.1.1Gross structureThe endolymphatic system is much larger, relatively, in adult and developing amphibians, than in mammals. Fig. 1 (in Table 1 are the abbreviations used in the figures) shows a drawing of a stage 52 Cynops larva and a three-dimensional reconstruction of the inner ear and ES and ED. Stage 52 is approximately 28 days after oviposition and 16 days after the larvae usually hatch from the egg. At this stage, the two ESs are separate but represent large projections from the dorsomedial aspect of the saccule. As early as stage 31, the precursor of the ES can be recognized as a slight extension of the otic vesicle (see Wiederhold et al., 1995) and both the ES and ED expand greatly from stages 50 to 56. In an adult newt, the ES from the two sides join to form a continuum across the top of the brainstem (see Koike et al., 1995).Before stage 38, serial sections did not reveal an ES. At stage 39, the ES first appears as an oval capsule at the dorsomedial tip of the otic vesicle (Fig. 2a). The ES is about 65 μm long, 25 μm wide and 15 μm thick. Serial sections did not show a lumen inside the ES. At stage 41, several tiny vacuoles (arrow in insert of Fig. 2b) appear in the sac, indicating the ES is ready to expand. At stage 46, the ES elongates markedly, to approximately 120 μm long and 30 μm wide. The capsule boundary appears in 25 serial sections. Its proximal portion extends to the upper part of the medial wall of the otic vesicle. A narrow slit-like lumen can barely be seen in the middle of the ES (LM in Fig. 2c). At stage 48, the ES increases in size with a long and short diameter of 150 μm and 35 μm, respectively. The whole ES continued to display its outline in about 40 serial sections with the expanded lumen clearly distinguished from the wall of the ES (LM in Fig. 2d). At stage 50 the ES is 160 μm long, 35 μm wide and 50 μm thick. The lumen becomes much wider than that at stage 48. In the lumen, a number of crystals (otoconia) can now be seen (OC in Fig. 2e). The birefringence of these otoconia display the same colors in polarized light microscopy as do the saccular and utricular otoconia, indicating that they are, indeed, crystalline. At stage 54, the ES forms a large bellow-like pouch, which is 190 μm long, 40 μm wide and 55 μm thick. The sac extends from the middle portion of the medial wall of the otic vesicle to the dural membrane. The large lumen cavity contains numerous otoconia piled together (arrow in magnified insert in Fig. 2f).3.1.2Epithelial cell and intercellular spaceAt stage 39, the epithelial cells are of a polymorphous type and contain a minimum of cytoplasm with very few organelles (Fig. 3a). At stage 44, the epithelial cells have a columnar shape. The cell cytoplasm has increased in size but is not dense, with only a few mitochondria inside (Fig. 3b). At stage 46, the epithelium of the ES contains a single layer of cylindrical cells. The content of mitochondria appears increased. A few microvilli appear on the epithelial cell luminal surface. At stage 48, the epithelial cells are of both cuboidal and cylindrical types. The epithelial cells contain relatively dense cytoplasm in which many mitochondria, as well as scattered ribosomes and Golgi complexes can be found (Fig. 3c). At stage 50, the epithelial cells are cuboidal, with a height of about 8 μm and a width of 7 μm. The dense cytoplasm contains an abundance of vesicles surrounded by Golgi complexes and ribosomes. Mitochondria are packed together in the cytoplasm of some epithelial cells (see Fig. 4c). At stage 54, cells are cuboidal at the proximal portion but more flattened at the intermediate and distal portion. The cytoplasm is of high density with a fibrous appearance. Abundant cytoplasmic organelles such as mitochondria, ribosomes, Golgi complexes, endoplasmic reticulum are distributed in the cytoplasm. Numerous granules are found in the apical and lateral cytoplasm (GR in Fig. 3d) at this stage. These granules, 0.2–0.4 μm in diameter, are usually round or oval and surrounded by a limiting membrane.The epithelial cells are separated by intercellular spaces (ISs). At stage 39, these spaces are narrow and have a smooth surface, with nothing inside (Fig. 3a). At stage 44, the ISs are radially distributed from the center of the ES with some vacuoles filled in the space (see Fig. 3b). Primitive microvilli are occasionally found on the lateral surface of the intercellular spaces (MV in Fig. 3b). At stage 46, the vacuoles in the IS are fused, which makes the space expand and have a wave-like surface (see Fig. 4a). At stage 48, the ISs dilate irregularly, in which finger-like protrusions arise from adjacent surfaces (CJ in Fig. 3c). The ISs expand markedly and have a honeycomb-like appearance at stage 50 (see Fig. 4c). Numerous protrusions arise from the surface of the space to form intercellular digitations. At stage 54, floccular material is found in the widened irregular IS (not illustrated). The IS is loose and contains many digitating projections, which display a labyrinthine appearance (Fig. 3d).3.1.3LumenIn Fig. 4a, the slit-like lumen of the ES lies longitudinally in the middle of the ES at stage 46. The lumen is approximately 39 μm long and 3.3 μm wide and displays its outline in only four serial sections. The luminal surfaces of the epithelial cells bear a few short microvilli, which are about 0.3–0.5 μm long. At stage 48, the ES lumen becomes wider and longer than before. The epithelial surface is uneven with several short protrusions and microvilli in the lumen (MV in Fig. 4b). The microvilli are longer than before (0.4–0.6 μm). Floccular material (endolymph) appears in the lumen (FM in Fig. 4b). This material is not dense and is evenly distributed within the lumen. Crystals were not found in the lumen in serial sections at this stage. At stage 50, the ES lumen dilates markedly and is about 120 μm long, 20 μm wide and 20 μm thick. The luminal surface is irregular with several protrusions into the lumen (not illustrated). The microvilli on the luminal surface are relatively long (0.5–0.7 μm) and curved. Otoconia are first found in the lumen at stage 50. The spaces where the crystals were appear bright under TEM, indicating that these spaces are ghosts of dislodged or dissolved otoconia. The endolymph becomes dense at places adjacent to the otoconia. At stage 54, the ES lumen forms a large cavity (Fig. 4d) that is about 140 μm long, 30 μm wide and 25 μm thick. In the lumen, numerous otoconia pile together. The size of individual otoconia is similar to that at stage 50. The floccular material is dense and contains occasional cell debris. Thick and long microvilli (1 μm long and 0.1–0.2 μm wide) arise from the luminal surface of the epithelial cell. At high magnification, these microvilli have a triple layered unit membrane with the middle layer more dense, indicating a lipid bilayer (LB in Fig. 5a). The floccular material close to the microvilli and the otoconia sometimes becomes very condensed as seen in Fig. 5a. Free floating cells first appear in the ES lumen at this stage. The cytoplasm of this cell is relatively light with numerous vacuoles inside. Long microvilli (2 μm) are present around all the free surface of the cell (Fig. 5b).3.2Development of the endolymphatic duct (ED)Before stage 38, no ED was found in serial sections. At stage 39, a primitive ED is present as a connection, about 25 μm long, between the ES capsule and the medial side of the otic vesicle (ES in Fig. 2a). Serial sections did not show a lumen inside this connection. At stage 41, the ED extends and expands, but still no lumen was found. A true short and thin tube (ED lumen), which joins the ES lumen and passes into the otic vesicle lumen at the upper end of the saccule (SC), is present at stage 46 (LM in Fig. 2c,Fig. 6a). The ED is about 55 μm long and has a lumen diameter of 0.5 μm. The ED is lined with a single layer of duct-like epithelial cells which rest on a smooth basement membrane. A large nucleus with its long axis parallel to the duct can be seen in the cytoplasm. Intercellular spaces are present between the epithelial cells. At the low end of the ED, near the saccule, the duct dilates to 0.8 μm in diameter. Microvilli are seen only on the luminal surface of this dilated end of the ED (ED in Fig. 6a).At stage 50, the ED extends its length to about 100 μm in length with a lumen diameter of 0.8 μm. At the low end of the ED, the diameter of the lumen extends to 1.0 μm. The lower cubical epithelial cells of the ED contain more cytoplasm than before. The nucleus usually lies at the base. Some floccular material (FM in Fig. 6b) can now be seen in the lumen. The luminal surface of the ED is relatively smooth and bears only a few microvilli (Fig. 6b).At stage 54, the ED is about 200 μm long with a lumen diameter of 1.2 μm. At the low end of the ED, close to the saccule, the diameter of the lumen extends to 2 μm. In the low portion of the ED, the epithelial cells bear a few short microvilli which rise perpendicularly from the apical surface. In the intermediate and transitional portion, the epithelial cells bear numerous relatively long and thick microvilli. These microvilli protrude into the lumen at different directions and touch or overlap each other, which makes the lumen have an intermeshing appearance (MV in Fig. 6c). The epithelial cell becomes low cubical. The cytoplasm is rather dense and has many mitochondria, Golgi complexes, vesicles and vacuoles. The nucleus is irregular in shape with condensed nucleoplasm inside. Floccular material and otoconia are occasionally found in the lumen of the ED. A well-defined and dilated IS can be seen between the epithelial cells.3.3Development of junctional complexes between the epithelial cellsBefore stage 45, there is no sign of junctions between the adjacent epithelial cells in the ES and ED. The epithelial cells are connected to each other with a smooth or rugose IS. At stage 46 (Fig. 7a), tight junctions first appear in the lateral space between the epithelial cells close to the luminal surface of the ES. The outer layers of the adjacent plasma membrane fuse in the wall of the junction. The junction is rather short (0.2 μm long). Very fine filamentous material fills the interspace of the junction. Below the junction, the IS expands and has an irregular surface.At stage 50 (Fig. 7b), the length of the tight junction increases (0.4 μm). In the cytoplasm of the adjacent cells close to the junction, some granular substance adheres to the outer wall of the junction. Below the tight junction, an adherent junction 0.15 μm long is formed, indicating that a junctional complex has developed. The adherent junction consists of two dark cytoplasmic plaques very close to the innermost layer of the plasma membrane (AJ in Fig. 7b). Several parallel filaments traverse across the junction and extend to the cytoplasm of both cells (CF in Fig. 7b). Below the junctional complex, cellular digitations can be seen in the lateral intercellular space.At stage 54 (Fig. 7c), the cell walls of the tight junction are parallel with a narrow intercellular space of about 200 Å. In the adhering junction, there are two oval plaques of dense material in the cytoplasm close to the inner layer of the plasma membrane. The crossing parallel filaments become longer and have a microtubule-like appearance (CF in Fig. 7c). Below the junctional complex, both of the cell membranes are closely apposed at some point (TJ in Fig. 7c) indicating that multiple cell junctional complexes are formed.4DiscussionThe membranous labyrinth of the inner ear derives from ectoderm. In C. pyrrhogaster, a small protuberance of the otic vesicle appears as a precursor of the ES, on the dorsal-medial aspect of the saccule at stage 31, about 7 days after the eggs are laid (Wiederhold et al., 1995). In the present study, a true egg-like capsule, which is composed of epithelial cells of the ES and a connection between the capsule and the saccule, which forms the primitive ED, are seen at stage 39, approximately 11 days after oviposition. It is believed that the protuberance of the otic vesicle extends to produce the ED and its upper end expands to form the ES during development.Before the 1980's, investigations on the development of the inner ear only briefly mentioned or failed to describe the process of organogenesis of the ES and ED in mammals (Ruben, 1967; Sher, 1971). In amphibians, earlier descriptions of the development of C. pyrrhogaster showing histological sections through the brain of embryos up to stage 42 did not mention the ES and ED (e.g. Okada and Ichikawa, 1947). In this study, the whole sequence of maturation of the ES and ED has been demonstrated, both concerning organogenesis and epithelial cell cytogenesis (Table 2Table 3).From Tables 2 and 3, it is known that a slit-like lumen of the ES is formed at stage 46. At the same time, a true lumen of the ED, which joins the ES lumen at its upper end and the lumen of the otic vesicle at the low end, first appears. Several days later (stages 48 and 50), floccular material (endolymph) is found in the expanded ES lumen and the ED. It is believed that the entire endolymphatic system is in communication with the other parts of the inner ear at this period. The floccular material flows into the ES from the saccule, or out to the otic vesicle lumen from the ES, by passing through the ED at stage 46. Apparently, in Cynops, the ES and ED play their biological function only from this stage.The cytodifferentiation of the epithelial cells in Cynops occurs from stage 39 to stage 54. During the process, the height of the cell reduces but the cytoplasm becomes larger with increasing number of cytoplasmic organelles. This indicates that the metabolic activity within the epithelial cells of the ES increases during development. At stage 54, the cytoplasm of the epithelial cells is of high density with a fibrous appearance. Abundant cytoplasmic organelles such as mitochondria, ribosomes, Golgi complexes and endoplasmic reticulum are distributed in the cytoplasm. The fine structure of the epithelial cells at stage 54 is similar to that of the epithelial cells at stages 56 and 58 (a nearly adult newt). Thus, it is concluded that the epithelial cells in the newt C. pyrrhogaster have reached maturation at stage 54.In the present study, the cytoplasm of the epithelial cells of the ES contains numerous vesicles at stage 50 and many granules at stage 54. These vesicles and granules are surrounded by a limiting membrane and located close to the apical cell membrane. Salamat et al. (1980)have also observed vesicles in the cells of the labyrinthine wall of the rat. They believed that the content within the vesicles would be expelled into the labyrinthine lumen. In amphibians, Kawamata et al. (1987)found that most epithelial cells of the ES in the tree frog, Hyla arborea japonica, contain granules. These granules, varying in size, are surrounded by a limiting membrane and apposed to the apical membrane of the cell. Kawamata suggested that these granules are released into the lumen by the epithelial cells. In a histological and ultrastructural study, Dahlmann and Düring (1995) found many ribosomes in some epithelial cells of the ES of the rat. They suggested that supranuclear accumulation of organelles, especially ribosomes, Golgi complexes and vesicles in the cytoplasm are interpreted as signs of secretory activity. In this study, numerous ribosomes and Golgi complexes are richly distributed around the vesicles and granules in the apical cytoplasm of the epithelial cells at stage 50. These findings strongly suggest that, in the newt, the epithelial cells of the ES are secretory after the animals reach stage 50.The intercellular spaces (ISs) between epithelial cells of the ES have received much attention (Friberg et al., 1985; Takumida et al., 1988). This study demonstrates the developmental process of this structure. At stage 39, the primitive IS is narrow and has a smooth surface. Vacuoles are found in the IS at stage 41. At stage 46, about 18 days after the eggs are laid, the IS starts to expand. The intercellular spaces dilate evidently and have an irregular appearance at stage 48. After stage 50, intercellular digitations arise from the surface of the IS. The IS is honeycomb-like between the epithelial cells. At stage 54, the IS contains many intercellular digitations and displays a loose labyrinth appearance.The function of the intercellular spaces of the ES is still not known. Many morphological and experimental studies indicate that the lining of the ES and ED may represent a fluid transporting epithelium with an ability to transfer water and solutes to and from the ES and ED (Lundquist, 1965; Adlington, 1967; Rask-Andersen et al., 1981; Bagger-Sjöbäck and Rask-Andersen, 1986). Friberg et al. (1985)and Takumida et al. (1988)suggested that the intercellular spaces of the ES may form a pathway for transepithelial water flow from the ES to blood vessels. According to their assumption, the epithelial cells may actively transfer salt from the ES lumen to the IS, and then water is drawn through local osmotic force from the lumen to the intercellular space. Based on the `longitudinal theory' (Guild, 1927), endolymph flows from the vestibule out to the ED and ES. Friberg et al. (1985)confirmed that cessation of longitudinal flow to the ES by surgery would make the intercellular spaces collapse. They considered that this cessation of flow also arrests transepithelial movement of fluid. In the present study, the intercellular spaces are not dilated before stage 45, when the lumen of the ED and ES have not yet been formed. After the lumen of the ED and ES have developed at stage 46, the intercellular spaces start to expand. The IS dilates evidently at stage 48 when abundant floccular material appears in the lumen. This finding suggests that fluid transport across the sac epithelium may be initiated at the same time the IS starts to dilate.Tight junctions first appear in the epithelium at stage 46, about the same time the lumen is formed. At stage 50, adherent junctions are present below the tight junctions, indicating that a junctional complex has developed. At stage 54, fully developed multi-junctional complexes are present between the epithelial cells. Tight junctions act as a permeability barrier to fluids and ions (Claude and Goodenough, 1973; Bagger-Sjöbäck and Anniko, 1984). Anniko and Bagger-Sjöbäck (1982) reported that the tight junctions of the mouse have developed before formation of the high potassium concentration in the endolymph. In the present study, tight junctions are formed at stage 46, at which time a slit lumen of the ES and very narrow ED have just developed. Apparently, formation of the tight junctions occurs before endolymph is available in the ES lumen. These tight junctions probably play an important role in maintaining ion gradients between the endolymphatic lumen and lateral and extralabyrinthine space, and the elevated Ca2+ concentration in endolymph which is necessary for formation of otoconia, a major function of the ES in amphibians.An interesting finding in this study is that so many microvilli touch and overlap each other, which gives an intermeshing appearance to the lumen when the ED reaches maturity. The functional significance of this organization of the microvilli is not known, but would suggest an active secretory function.AcknowledgementsThese studies were supported by the NASA Space Biology Program (Grants NAG 2-952 and NAG 10-0180) and the National Science Foundation (Grant BIN-95-29136). 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