title: Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE).
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 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.
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3548CT Utrecht, The Netherlands.
authors.forename: Alexander
authors.initials: A
authors.lastname: Apschner
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3548CT Utrecht, The Netherlands.
authors.forename: Leonie F A
authors.initials: LF
authors.lastname: Huitema
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3548CT Utrecht, The Netherlands.
authors.forename: Bas
authors.initials: B
authors.lastname: Ponsioen
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3548CT Utrecht, The Netherlands.
authors.forename: Josi
authors.initials: J
authors.lastname: Peterson-Maduro
authors.affiliation: Hubrecht Institute - KNAW & UMC Utrecht, 3548CT Utrecht, The Netherlands. WUR, Experimental Zoology, 3700AH Wageningen, The Netherlands. Institute of Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, University of Münster, 48149 Münster, Germany. s.schulte@hubrecht.eu.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulte-Merker
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002114:Calcinosis / Q000150:complications* / Q000188:drug therapy / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002828:Choristoma / Q000201:enzymology / Q000473:pathology; D012968:Etidronic Acid / Q000494:pharmacology / Q000627:therapeutic use; D000089703:Fibroblast Growth Factor-23; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009672:Notochord / Q000187:drug effects / Q000473:pathology; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D010710:Phosphates / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics*; D011561:Pseudoxanthoma Elasticum / Q000150:complications* / Q000188:drug therapy / Q000201:enzymology*; D011755:Pyrophosphatases / Q000235:genetics*; D061205:Vascular Calcification / Q000150:complications* / Q000188:drug therapy / Q000201:enzymology; D015027:Zebrafish / Q000235:genetics*
chemical_list: D015415:Biomarkers; C000717427:FGF23 protein, human; D010710:Phosphates; D000089703:Fibroblast Growth Factor-23; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases; D012968:Etidronic Acid; D002118:Calcium
keywords: Ectopic mineralization; GACI; Generalized arterial calcification of infancy; PXE; Pseudoxanthoma elasticum; Pyrophosphate; Zebrafish