SUMMARY The spine is a segmented axial structure made of alternating vertebral bodies (centra) and intervertebral discs (IVDs) assembled around the notochord. Here, we show that, prior to centra formation, the outer epithelial cell layer of the zebrafish notochord, the sheath, segments into alternating domains corresponding to the prospective centra and IVD areas. This process occurs sequentially in an anteroposterior direction via the activation of Notch signaling in alternating segments of the sheath, which transition from cartilaginous to mineralizing domains. Subsequently, osteoblasts are recruited to the mineralized domains of the notochord sheath to form mature centra. Tissue-specific manipulation of Notch signaling in sheath cells produces notochord segmentation defects that are mirrored in the spine. Together, our findings demonstrate that notochord sheath segmentation provides a template for vertebral patterning in the zebrafish spine.

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.

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-offunction 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.