Browsing by Person "Hilbig, Reinhard"

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Zur Genese der Innenohr-Otolithen bei Knochenfischen (Oreochromis mossambicus) unter besonderer Berücksichtigung von Matrixproteinen
(2013) Weigele, Jochen; Hilbig, Reinhard
Vertebrate calcified structures are involved in the construction of several tissues e.g. bones, teeth, scales and inner ear otoliths. The underlying biomineralization process largely differs from parameters of non-biological mineralization issues as they occur e.g. in geology. These differences are mainly based on proteins, which control the incorporation and aggregation of inorganic substances. In contrast to bones, which are composed cellular and consist of calcium phosphate, the completely acellular inner ear otoliths and otoconia are built out of calcium carbonate. The fish otoliths serve as counter-mass in the sensory organs of hearing and balance. These ear stones are an appropriated system to investigate the process of biomineralization, because their mineralization takes part in the closed environment of the inner ear and they are easy to remove without any other tissue residues. The inner ear is highly conserved among the vertebrates, from fish up to humans. The vertebrate inner ear is a complex labyrinth containing multiple sensory organs, the semicircular canals with the rotation-sensing cristae and the otolithic organs. The otolithic organs (utricule, saccule and lagena) of teleosts are saccular structures. The fish utricule serve primary vestibular function in contrast to the saccule and lagena, which serve auditory functions. Little is known about the formation of otoliths and otoconia but several proteins involved in their biomineralization are identified. These proteins serve only 0.1-10% in the weight of otoliths, but they accomplish several important function during the biomineralization process. These proteins build a matrix, which directly controls the biomineralization e.g. regulation of the crystals strength and the selection of the crystal polymorph. Several proteins of fish otoliths, and in their orthologous structures in mammals, the otoconia, are identified like otoconin, otolith matrix protein-1 (OMP-1), otolin-1, starmaker, SPARC, myosin light chain 9, neuroserpin and osteopontin, but their function during otolith or otoconia mineralization is only partly known. The aim of this study is the analysis of the regulation, synthesis and deposition of otolith matrix proteins in the cichlid fish Oreochromis mossambicus, with special attention on gravitational effects. Moreover, the function of the otolith matrix proteins, if they occur in other mineralization processes, like in bones and teeth, is also of interest. The present dissertation adduced mainly the following results: 1. Occurrence of otolith matrix proteins in the inner ear of sub-adult cichlid fish (Oreochromis mossambicus) was detected with an enhanced protein purification technique In a set of experiments, the composition of this proteinous matrix was analyzed by mass spectrometry in a shotgun approach. Therefore an enhanced protein purification technique was developed that can exclude any contamination of proteins from body fluids. In the use of that method eight proteins were identified, the common otolith matrix proteins OMP-1, otolin-1, neuroserpin, SPARC and otoconin, and three previously unidentified alpha tectorin, otogelin and transferrin. Moreover, it was able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin like protein) known from other fish species. In further analysis it was shown, that the absence of the otolith matrix protein starmaker corresponds to calcitic otoliths and that even pre-cerebellin like protein did not occur during the whole otolith development in the inner ear. Moreover, the source of another matrix protein, neuroserpin, which was identified in the hypothalamus was also focused on. In consequence, generally some known otolith proteins seem to be not required in all fish species and further functional studies of the new identified proteins during otolith development are required. 2. Formation of the inner ear during embryonic and larval development of a cichlid fish (Oreochromis mossambicus) characterized otolith matrix protein expression and morphological markers The formation of otoconia and otoliths is discussed to be a stepwise process and it is in fish generally divided in an aggregation of the otolith primordia from precursor particles and in a growth process continuing throughout life. In the present dissertation, the complex transition between these two steps was investigated. Therefore the developmental profiles of the otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC otoconin, alpha tectorin, otogelin and transferrin) was analyzed during the complete embryonic and larval development of the cichlid fish Oreochromis mossambicus, parallel to the morphology of inner ear and especially otoliths. In a set of experiments, it was able to prove that the formation of otoliths is a highly regulated temporal and spatial process, which takes part during the complete fish embryonal and larval development. Based on the data eight phases of otolith differentiation were defined, from the primordia to definitively structured otolith. 3. Spatial expression of otolith matrix protein-1 and otolin-1 in normally and kinetotically swimming fish Repeatedly it has been shown that some fish of a given batch reveal motion sickness (a kinetosis) following the transition from 1g to microgravity or from hypergravity (centrifuge) to 1g. It was suggested that the susceptibility to kinetosis is correlated with an irregular inner ear otolith growth (e.g. leading to asymmetric otoliths or to an abnormally otolith morphology). Otoliths are mainly composed of calcium carbonate and matrix proteins, which play an important role in the process of otolith mineralization. Concerning the morphology of otoliths ? thus possibly leading to the susceptibility to kinetosis - the expression of the major otolith proteins OMP-1 and otolin-1 was of special interest. In a series of hypergravity experiments, the spatial distribution of OMP-1 and otolin-1 in the utricule and saccule of kinetotically and normally behaving fish was analyzed by means of in-situ hybridization on transverse cryostat sections. In the utricule, OMP-1 was mainly observed in centripetal (medial) and centrifugal (lateral) macula, the regions of the meshwork areas. In the saccule, OMP-1 was expressed within a dorsal and a ventral narrow band of the meshwork area opposite to the periphery of the otoliths sulcus acusticus. Quantifying the OMP-1 positive areas, it was investigated that the expression in the utricule reached farther posterior in the centrifugal aspect and was considerably broader in the centripetal portion of the utricle in normally swimming fish as compared to kinetotic animals. Differences in the saccule were not observed for both genes. The difference in the utricular OMP-1 expression pattern between normally and kinetotically swimming fish indicates a different otolith morphology and thus a different geometry of the otoliths resting on the corresponding sensory maculae. As the utricle is the endorgan responsible for sensing gravity, an aberrant morphology of the utricular otoliths, based on OMP-1, thus might lead to kinetotic behavior. 4. Developmental expression of SPARC and the osteopontin-like protein in the cichlid fish Oreochromis mossambicus. Bones are mainly composed of calciumhydroxyapatite and a proteinous matrix. In this dissertation the non-collagenous bone matrix proteins - the fish osteopontin orthologous protein (osteopontin-like protein; OP-L) and SPARC - were of special interest. The current knowledge about the expression of OP-L is fragmentary. Therefore, first the expression pattern of OP-L in the cichlid fish Oreochromis mossambicus was analyzed in detail during its larval development. OP-L expression was only identified in those tissues that underwent ossification in the developing bones and teeth, in contrast to the mammalian osteopontin, which was found, beside the calcified tissues, also in several soft tissues. Therefore, it was focused on the developmental appearance of SPARC and OP-L in the whole cranial and post cranial development. In a set of experiments, it was able to show, that there is a fundamental difference in the formation of the cartilages of splanchnocranium and all other cartilages, concerning the SPARC expression. SPARC functions in the cascade, regulating cartilage differentiation of the splanchnocranium, the eldest part of the fish skeleton, whereas it was not detected in that of the chondrocranium and axial skeleton. These findings are probably based on the different evolutionary background of the splanchnocranium. Moreover, it was able to show that during osteogenesis initial expression of the bone matrix proteins OP-L and SPARC coincidentally appear with the initial calcium carbonate deposition. This result indicates a co-occurrence of the initial calcium deposition and the appropriation of bone matrix proteins during osteogenesis. 5. The anosteocytic bone syncytium in fish: A new intracellular ossification detected by molecular biological, histological and enzyme-histochemical methods The bones of most fish are referred as ?acellular? or anosteocytic bone and no osteocytes occur within this osseus. It was assumed, that during the development the osteoblasts, which secrete collagen and ground substance, recede from the mineralizing front (preosseous matrix) and never become trapped as osteocytes in mammals do. Therefore it was assumed, that this is the only difference to mammalians. Typical osteoblasts with an alkaline phosphatase activity in fish only occur at the outer surface of the outgrowing bone parts. Therefore, the cells that might be responsible for the further growth of the anosteocytic bones, up to now have not been investigated. In this dissertation it was shown, that the osseus of the anosteocytic bone itself comprised mRNA and shows a cytoplasmic character. Furthermore, the results suggest that the formation of the osseus may possibly take part intracellular in a syncytium. Based on the findings concerning the occurrence RNA and carbonic anhydrase activity, which accompany with a change in the bone mineral system and additionally with a fractional occurrence of a plasma membrane between the spindle shaped osteocytes and the osseus, a new model for the intracellular bone formation in teleost fish was developed.
Zur Lokalisation und Funktion des Calciumtransporters im Innenohr von Knochenfischen (Oreochromis mossambicus)
(2014) Knie, Miriam; Hilbig, Reinhard
In fish the perception of gravity takes place in the otolithic organs of the inner ear. These structures are filled with endolymph fluid and posses a sensory macula epithelium on which the otolith, the so called inner ear stone, is attached. The otoliths mainly consist of calcium carbonate which is embedded in a protein matrix. The otolith growth is subjected to a diurnal rhythm. Up to now, it is not clear how the calcium ions, which are needed for the otolith minerlisation, are transported through the sensory epithelium into the endolymph. The aim of this study was to analyze the localization and function of the plasmamembrane-calcium ATPase (PMCA) as a candidate for the calcium transporting protein in the inner ear sensory epithelia of Oreochromis mossambicus with regard to the otolith biomineralization. This study was carried out using larval cichlids in different developmental stages, because in the course of the larval development, the otoliths are characterised by a rapid increase in size and dynamic rearrangements. In addition, further mineralised tissues like the teeth and the skull bones have been analysed. Moreover, the organs generally participating in the calcium metabolism like gills, intestine and kidney have been examined in the same developmental profiles. All analysed epithelia and tissues are charaterised by a co-expression of the PMCA-isoforms 1 to 4 throughout the larval development. Based on the results of this study it can be assumed that each of the four PMCA-isoforms fullfills a specialised function in the examined epithelia and tissues. In the inner ear sensory epitehlia PMCA 1 is involved in the transport of calcium ions into the endolymph, whereas isoform 2 rather performs the function of a "housekeeping gene" and is important for the maintenance of the basal calcium concentration. In the bones, the calcium export function is mainly performed by the isoforms 1 and 4, the basal calcium levels are kept stable by isoform 3. Thus the regulation of the calcium ions may be a complex and delicately regulated process. The results of the expression analysis - a specific reaction with the sense-, as well as the antisense-probe - led to the assumption that, in addition to the sense-RNA, also naturally occuring antisense-RNA is transcribed for the four plasmamembrane-calcium ATPase isoforms in Oreochromis mossambicus. These hypothesis was tested with the Northern Blot method, thereby further clues for the existence of natural antisense-RNA have been obtained. Naturally occuring antisense-transcripts can either directly serve as a template for transcription or influence the expression of the corresponding sense-RNA. To check if this is also true for Oreochromis mossambicus quantitative in-situ hybridizations in the context of the diurnal otolith growth rhythm have been performed. By the help of this method it was possible to observe changes in the expression levels of both transcripts, as well as changes in the ratio between sense- and antisense-RNA. Herefrom it was deducible, that the regulation of PMCA in the inner ear sensory epithelia may occur via the transcription rate, as well as by the expression of the antisense-transcripts themselves, in this case probably the ratio between the sense- and antisense-RNA matters. Which of the regulatory mechanisms known for naturally occuring antisense-RNA is acting in detail requires further research.