Identification of a promoter element within the zebrafish colXalpha1 gene responsive to runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2alphaA2.

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.

Cloning of matrix Gla protein in a marine cartilaginous fish, Prionace glauca: preferential protein accumulation in skeletal and vascular systems.

Matrix Gla protein (MGP) belongs to the family of vitamin K dependent, Gla containing proteins and, in mammals, birds and Xenopus, its mRNA has been previously detected in bone, cartilage and soft tissue extracts, while the accumulation of the protein was found mainly in calcified tissues. More recently, the MGP gene expression was also studied in marine teleost fish where it was found to be associated with chondrocytes, smooth muscle and endothelial cells. To date no information is available on the sites of MGP expression or accumulation in cartilaginous fishes that diverged from osteichthyans, a group that includes mammals, over 400 million years ago. The main objectives of this work were to study the sites of MGP gene expression and protein accumulation by means of in situ hybridization and immunohistochemistry. MGP mRNA and protein were localized as expected not only in cartilage from branchial arches and vertebra but also in the endothelia of the vascular system as well as in the tubular renal endothelium. The accumulation of MGP in non mineralized soft tissues was unexpected and suggests differences in localization or regulation of this protein in shark soft tissues compared to tetrapods and teleosts. Our results also corroborate the hypothesis that in Prionace glauca, as previously shown in mammals, the MGP protein probably also acts as a calcification inhibitor, protecting soft tissues from abnormal and ectopic calcification.

Purification of matrix Gla protein from a marine teleost fish, Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas 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 because both coexist 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 calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, 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. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5'rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted to regions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation.

Cloning and characterization of the cDNA and gene encoding Xenopus laevis osteocalcin.

A full length Xenopus laevis osteocalcin (bone Gla protein, BGP) has been cloned by a combination of reverse transcription and amplification by the polymerase chain reaction, sequenced, and found to encode a polypeptide with 101 amino acid residues, including a 52-residue prepro-region and a 49-residue mature protein. The N-terminal region of the mature Xenopus BGP (xBGP), as deduced from the cDNA, is in full agreement with the sequence of the BGP previously purified from Xenopus long bones. This cDNA was used to clone the xBGP gene and its promoter region. The xBGP gene spans 3727 bp from the site of transcription initiation corresponding to the 5'end of the cDNA to the site of insertion of the poly-A(+) tail, and it contains four exons. This structure is similar to the one obtained for both fish and mammalian BGP genes and indicates that the molecular organization of this gene has been conserved throughout vertebrate evolution. Also similar to other known vertebrate systems, xBGP gene expression is restricted to bone, with no signal for xBGP messenger RNA (mRNA) detected in all other tissues analyzed. The availability of the xBGP promoter will permit to analyze its regulation in a widely used non-mammalian model system for vertebrate development, taking advantage of the availability of sequences for various Xenopus steroid hormone receptors and transcription factors known to affect BGP expression in the mammalian system.