Entpd5 is essential for skeletal mineralization and regulates phosphate homeostasis in zebrafish

Bone mineralization is an essential step during the embryonic development of vertebrates, and bone serves vital functions in human physiology. To systematically identify unique gene functions essential for osteogenesis, we performed a forward genetic screen in zebrafish and isolated a mutant, no bone (nob), that does not form any mineralized bone. Positional cloning of nob identified the causative gene to encode ectonucleoside triphosphate/diphosphohydrolase 5 (entpd5); analysis of its expression pattern demonstrates that entpd5 is specifically expressed in osteoblasts. An additional mutant, dragonfish (dgf), exhibits ectopic mineralization in the craniofacial and axial skeleton and encodes a loss-of-function allele of ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1). Intriguingly, generation of double-mutant nob/dgf embryos restored skeletal mineralization in nob mutants, indicating that mechanistically, Entpd5 and Enpp1 act as reciprocal regulators of phosphate/pyrophosphate homeostasis in vivo. Consistent with this, entpd5 mutant embryos can be rescued by high levels of inorganic phosphate, and phosphate-regulating factors, such as fgf23 and npt2a, are significantly affected in entpd5 mutant embryos. Our study demonstrates that Entpd5 represents a previously unappreciated essential player in phosphate homeostasis and skeletal mineralization

Characterization of biological mineralization in vitro

Mineralization is an essential requirement for normal skeletal development, which is generally accomplished through the function of two cell types, osteoblasts and chondrocytes. Soft tissues do not mineralize under normal conditions, but under certain pathological conditions some tissues like articular cartilage and cardiovascular tissues are prone to mineralization. The aim of this study was to gain more insight in the processes that take place during the initiation of biological or cell-mediated mineralization and the effectors involved in these processes. Therefore, the mouse embryonal carcinoma-derived ATDC5 cell line was used in our study. It was hypothesized that serum, which is known to contain mineralization inhibitors such as fetuin, inhibits the early stages of mineralization in the cell culture system. Therefore, it was tested whether excluding serum from the medium shortened the cell culture period to induce mineralization, making this model system more suitable for investigation. This resulted in the development of large (sub millimetre size range) mineralizing structures (LMS) in the medium in 2 hours. LMS were shown to contain whole cells, which were embedded in hydroxyapatite and observed to have a stretched morphology. In the presence of serum no LMS were formed, but multiple small mineralized structures were observed inside the cell after 24 hours. Taken together, the results show that excluding serum in the cell culture system enhances rapid crystal growth and suggest that cell-mediated mineralization may start intracellularly. To investigate whether ATDC5 cells themselves or factors released by ATDC5 cells nucleate and/or remodel hydroxy-apatite in the absence of serum, the effect of conditioned medium from ATDC5 cells on the formation of apatite crystal was investigated. It was found that soluble factors released by the ATDC5 cells have the ability to affect the formation of the calcium-phosphate crystal. This suggest that soluble factors released by ATDC5 cells play a role in the growth phase of LMS formation once in the initial phase the crystal nucleus has been formed. Since an imbalance of mineralization may lead to pathological conditions, the ATDC5 cell culture system was also used to test the effect of several agents implicated in bone growth and development as well as pathological mineralization, including the gaseous substance nitric oxide (NO). Therefore, the effect of an NO donor drug, sodium nitroprusside (SNP) on cell mediated mineralization was investigated. It was found that 100 ?M SNP inhibits mineralization. However, the inhibition was not affected by inhibitors of guanylyl cyclase nor mimicked by a cGMP analog. Furthermore, sodium nitroprusside did not inhibit phosphate uptake nor inhibited apoptosis in the ATDC5 cells. Therefore, we subsequently investigated the effect of SNP on mineralization to elucidate the mechanism of action. It was shown that the iron moiety of sodium nitroprusside, rather than nitric oxide, inhibits mineralization

Zebrafish as a unique model system in bone research: the power of genetics and in vivo imaging

For many years bone research has been mainly performed in mice, chicken, cell culture systems or human material from the clinic. In this review, we describe the features of zebrafish (Danio rerio), a relatively new model system in this field. This small teleost offers possibilities which make it a great complement to the mouse: forward genetic screens are possible in fish due to extrauterine development and large brood size, and the recent generation of osteoblast-specific reporter lines allows visualization of osteoblasts in vivo. As key regulators of bone formation are highly conserved between mammals and teleosts, findings in fish likely apply to mammalian osteogenesis and tissue mineralizatio

Trpv5/6 is vital for epithelial calcium uptake and bone formation

Contains fulltext : 92519.pdf (publisher's version ) (Open Access)11 p

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression

Mature osteoblasts dedifferentiate in response to traumatic bone injury in the zebrafish fin and skull

Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration after amputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells