ALBERT

Description: The sequence in which the otoliths and semicircular canals and their associated sensory epithelia appear and develop in the newt are described. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos from stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. A single otolith is first seen in stage 33 embryos (approximately 9 days old); this splits into separate utricular and saccular otoliths at stage 40 (13 days). The lateral semicircular canal is the first to appear, at stage 41 (14 days). The anterior and posterior canals appear approximately one week later and the vestibular apparatus is essentially fully formed at stage 58 (approximately 5 weeks). The data reported here will serve as ground-based controls for fertilized newt eggs flown on the International Microgravity Laboratory-2 Space Shuttle flight, to investigate the influence of microgravity on the development of the gravity-sensing organs.

Description: Otoconia are calcified protein matrices within the gravity-sensing organs of the vertebrate vestibular system. Mammalian otoconia are barrel-shaped with triplanar facets at each end. Reptilian otoconia are commonly prismatic or fusiform in shape. Amphibians have all three otoconial morphologies, barrel-shaped otoconia within the utricle, with prismatic and fusiform otoconia in the saccule. Scanning electron microscopy revealed a sequential appearance of all three otoconial morphologies during larval development of the newt, Cynops pyrrhogaster. The first otoconia appear within a single, developing otolith, and some resemble adult barrel-shaped otoconia. As the larvae hatch, around stages 39-42, the single otolith divides into two anatomically separate regions, the utricle and saccule, and both contain otoconia similar to those seen in the single otolith. Throughout development, these otoconia may have variable morphologies, with serrated surfaces, or circumferential striations with either separated facets or adjacent facets in the triplanar end-regions. Small fusiform otoconia occur later, at stage 51, and only in the saccule. Prismatic otoconia appear later still, at stage 55, and again only in the saccule. Thus, although prismatic otoconia are the most numerous in adult newts, it is the last vestibular otoconial morphology to be expressed.

Description: Pre-mated adult female newts and embryos have been flown on the International Microgravity Laboratory-2 (IML-2) Space Shuttle flight in 1994 (Wiederhold et al., 1992b). With the specimens available from this flight, the calcification of otoliths, ulna, radius and backbone of the flown larvae and adult newts were analyzed. The experiments presented here studied the development of the otoliths on the ground. Otoliths of living newts, from embryo to adult, were observed in situ with the application of a new X-ray and bio-imaging analyzer system. For the establishment of this method, newts at different developmental stages were used. An imaging plate temporarily stores the X-ray energy pattern at the bio-imaging analyzer. A latent image on the imaging plate was transformed into a digital time series signal with an image reader. Acquired digital information was computed with the image processor. The processed information was recorded on film with an image recorder, in order to visualize it on an enlargement computed radiograph. To analyze development of the otoliths, photo-stimulated luminescence level was detected by an image analyzer, using transmitted X-ray photons. A single clump of otoconia could first be seen at stage 33. Stage-36 embryos first have distinguishable otoliths, with the utricle in front and saccule behind. Our results show that this X-ray method detects the otoliths equally as well as sectioning. In the newt, the mandibular/maxillary bone formed before the spine. It is suspected that for the newt embryo, living in water, feeding becomes necessary prior to support of the body.

Description: Statoconia are calcium carbonate inclusions in the lumen of the gravity-sensing organ, the statocyst, of Aplysia californica. The aim of the present study was to examine the role of carbonic anhydrase and urease in statoconia mineralization in vitro. The experiments were performed using a previously described culture system (Pedrozo et al., J. Comp. Physiol. (A) 177:415-425). Inhibition of carbonic anhydrase by acetazolamide decreased statoconia production and volume, while inhibition of urease by acetohydroxamic acid reduced total statoconia number, but had no affect on statoconia volume. Inhibition of carbonic anhydrase initially increased and then decreased the statocyst pH, whereas inhibition of urease decreased statocyst pH at all times examined; simultaneous addition of both inhibitors also decreased pH. These effects were dose and time dependent. The results show that carbonic anhydrase and urease are required for statoconia formation and homeostasis, and for regulation of statocyst pH. This suggests that these two enzymes regulate mineralization at least partially through regulation of statocyst pH.

Description: Otoconia are calcified protein matrices within the gravity-sensing organs of the vertebrate vestibular system. These protein matrices are thought to originate from the supporting or hair cells in the macula during development. Previous studies of mammalian calcitic, barrel-shaped otoconia revealed an organized protein matrix consisting of a thin peripheral layer, a well-defined organic core and a flocculent matrix inbetween. No studies have reported the microscopic organization of the aragonitic otoconial matrix, despite its protein characterization. Pote et al. (1993b) used densitometric methods and inferred that prismatic (aragonitic) otoconia have a peripheral protein distribution, compared to that described for the barrel-shaped, calcitic otoconia of birds, mammals, and the amphibian utricle. By using tannic acid as a negative stain, we observed three kinds of organic matrices in preparations of fixed, decalcified saccular otoconia from the adult newt: (1) fusiform shapes with a homogenous electron-dense matrix; (2) singular and multiple strands of matrix; and (3) more significantly, prismatic shapes outlined by a peripheral organic matrix. These prismatic shapes remain following removal of the gelatinous matrix, revealing an internal array of organic matter. We conclude that prismatic otoconia have a largely peripheral otoconial matrix, as inferred by densitometry.

Description: The gravity-sensing organ of Aplysia californica consists of bilaterally paired statocysts containing statoconia, which are granules composed of calcium carbonate crystals in an organic matrix. In early embryonic development, Aplysia contain a single granule called a statolith, and as the animal matures, statoconia production takes place. The objective of this study was to determine the effect of hypergravity on statoconia production and homeostasis and explore a possible physiologic mechanism for regulating this process. Embryonic Aplysia were exposed to normogravity or 3 x g or 5.7 x g and each day samples were analyzed for changes in statocyst, statolith, and body dimensions until they hatched. In addition, early metamorphosed Aplysia (developmental stages 7-10) were exposed to hypergravity (2 x g) for 3 weeks, and statoconia number and statocyst and statoconia volumes were determined. We also determined the effects of hypergravity on statoconia production and homeostasis in statocysts isolated from developmental stage 10 Aplysia. Since prior studies demonstrated that urease was important in the regulation of statocyst pH and statoconia formation, we also evaluated the effect of hypergravity on urease activity. The results show that hypergravity decreased statolith and body diameter in embryonic Aplysia in a magnitude-dependent fashion. In early metamorphosed Aplysia, hypergravity decreased statoconia number and volume. Similarly, there was an inhibition of statoconia production and a decrease in statoconia volume in isolated statocysts exposed to hypergravity in culture. Urease activity in statocysts decreased after exposure to hypergravity and was correlated with the decrease in statoconia production observed. In short, there was a decrease in statoconia production with exposure to hypergravity both in vivo and in vitro and a decrease in urease activity. It is concluded that exposure to hypergravity downregulates urease activity, resulting in a significant decrease in the formation of statoconia.

Description: 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.

Description: To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

Description: 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.

Description: The gravity sensors of all molluscs phylogenetically below the cephalopods are spherical organs called statocysts. The wall of the sphere contains mechanosensory cells whose sensory cilia project into the lumen of the cyst. The lumen is filled with fluid and dense "stones", the statoconia or statoliths, which sink under the influence of gravity to load, and stimulate, those receptor cells which are at the bottom. The statoconia of Aplysia californica are shown to be calcified about a lamellar arrangement of membranes. Similar lamellar membrane arrangements are seen within the receptor cells, and their possible role in the formation of the statoconia is discussed. SEM of unfixed statoconia reveals plate-like crystallization on their surface. Elemental analysis shows a relatively high Sr content, which is of interest, since others have recently reported that Sr is required in the culture medium of several laboratory reared molluscs in order for the statoconia to develop.