Pathological mineralization in a zebrafish enpp1 mutant exhibits features of Generalized Arterial Calcification of Infancy (GACI) and Pseudoxanthoma Elasticum (PXE)

Abstract

Abstract 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 has to be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements or 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. We here provide a detailed analysis of a zebrafish mutant - dragonfish (dgf), for ectonucleoside pyrophophatase/phosphodiesterase 1 (enpp1), a protein critical 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. Similarly to human patients, we here show that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues, most notably: the skin, cartilage elements, the heart, intracranial space, and the notochord sheet. Using transgenic reporter lines we demonstrate that those ectopic mineralizations 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 calcifications at time points when osteoclasts are not present yet in wildtype siblings. Treatment with the bisphosphonate etidronate is suitable to rescue aspects of the dgf phenotype and we detected deregulated expression of genes 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 remote from its site of expression.