Pathological mineralization in a zebrafish enpp1 mutant exhibits features of Generalized Arterial Calcification of Infancy (GACI) and Pseudoxanthoma Elasticum (PXE)
Apschner, A., Huitema, L.F., Ponsioen, B., Peterson-Maduro, J., Schulte-Merker, S.
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.