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- Osteocalcin and Matrix Gla Protein in developing teleost teeth. Identification of sites of mRNA and protein accumulation at single cell resolutionPublication . Ortiz-Delgado, J. B.; Simes, D; Gavaia, Paulo J.; Sarasquete, C.; Cancela, LeonorIn this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence ofBGP andMGPmRNAin teethwas revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules,whilemRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP andMGPgene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.
- Purification, biochemical characterization and localization at single cell resolution of Matrix Gla Protein (MGP) and Bone Gla Protein (BGP) in the teleost fish Argyrosomus regiusPublication . Simes, D; Cancela, LeonorMatrix Gla protein (MGP) and Bone Gla Protein (BGP, osteocalcin) belong to the family of vitamin K dependent (VKD), Gla containing proteins. Matrix Gla protein (MGP) is a 10-15 kDa secreted protein with 4-5 residues of the Ca2+ binding y-carboxyglutamic acid residue (depending on the species). MGP was previously found to accumulate within the organic matrix of mammalian bone, from which it was originally purified. In mammals, birds and Xenopus, its mRNA was previously detected in extracts of bone, cartilage and soft tissues (mainly heart and kidney) while the protein was found to accumulate mainly in bone. However, at that time it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells since both co-exist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calciflcation. Interestingly, MGP was also found to accumulate in vértebra of shark, a cartilaginous fish. But to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. BGP is a small secreted protein with 46-50 residues including three potential Ca24 binding y-carboxyglutamic acid residues. This protein is the most abundant non-collagenous protein found in mammalian bone from which it was first isolated. BGP has been known for a number of years to be present in teleost fishes. nevertheless there is little information about the regulation of expression and tissue localization of this protein in lower vertebrate organisms and in particular in fish. This report describes, for the first time the identification of MGP in a teleost fish, with the protein purification and molecular cloning of MGP and BGP from the same teleost fish, Argyrosomus regius. The obtention of valuable biochemical tools such as specific antibodies and mRNA/DNA probes also permitted the study of tissue distribution/accumulation for MGP/BGP by Northern analysis, in situ hybridization and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in chondrocytes from branchial arches, with no expression detected in the different bone-like mineralized tissues analyzed while BGP mRNA was mainly located in bony tissues as expected. Accordingly,the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in chondrocyte-containing tissues. However and in contrast with results obtained for Xenopus and higher vertebrates, the protein does not significantly accumulate in vertebra of non-osteocytic teleost fish (such as A. regius) but only in its calcified cartilage. As previously seen in mammals and Xenopus, MGP was also found in soft tissues predominantly in heart and kidney. Our results indicate, in addition, that the presence of MGP mRNA in heart tissue is due, at least in físh, to the expression of the MGP gene in two specific cell types only, smooth muscle and endothelial cells, while no expression was found in the striated muscle fibers of the ventricle. In light of these results and in agreement with recent information on expression of the MGP gene in these same cell types in mammalian aorta, it is likely that the presence of MGP mRNA previously detected in Xenopus, birds and mammalian heart tissue may be due to expression of the gene only in smooth muscle and endothelial cells. Our results provide clear evidence that fish represem a valuable model system to study MGP/BGP gene expression and regulation, to bring further insight into its mode of action at the molecular levei throughout evolution.
- Osteocalcin and Matrix Gla Protein in zebrafish (Danio rerio) and Senegal sole (Solea senegalensis): comparative gene and protein expression during larval development through adulthoodPublication . Gavaia, Paulo J.; Simes, D; Ortiz-Delgado, J. B.; S B Viegas, Carla; Pinto, Jorge; Kelsh, R. N.; Sarasquete, C.; Cancela, LeonorBone Gla protein (Bgp or osteocalcin) and matrix Gla protein (Mgp) are important in calcium metabolism and skeletal development, but their precise roles at the molecular level remain poorly understood. Here, we compare the tissue distribution and accumulation of Bgp and Mgp during larval development and in adult tissues of zebrafish (Danio rerio) and throughout metamorphosis in Senegal sole (Solea senegalensis), two fish species with contrasting environmental calcium levels and degrees of skeletal reorganization at metamorphosis. Mineral deposition was investigated in parallel using a modified Alizarin red/Alcian blue protocol allowing sensitive simultaneous detection of bone and cartilage. In zebrafish, bgp and mgp mRNAs were localized in all mineralized tissues during and after calcification including bone and calcified cartilage of branchial arches. Through immunohistochemistry we demonstrated that these proteins accumulate mainly in the matrix of skeletal structures already calcified or under calcification, confirming in situ hybridization results. Interestingly, some accumulation of Bgp was also observed in kidney, possibly due to the presence of a related protein, nephrocalcin. Chromosomal localization of bgp and mgp using a zebrafish radiation hybrid panel indicated that both genes are located on the same chromosome, in contrast to mammals where they map to different chromosomes, albeit in regions showing synteny with the zebrafish location. Results in Senegal sole further indicate that, during metamorphosis, there is an increase in expression of both bgp and mgp, paralleling calcification of axial skeleton structures. In contrast with results obtained for previously studied marine fishes, in zebrafish and Senegal sole Mgp accumulates in both calcified tissues and non-mineralized vessel walls of the vascular system. These results suggest different patterns of Mgp accumulation between fish and mammals.