Pathological mineralization in a zebrafishenpp1mutant exhibits 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.

Retinoic acid-induced premature osteoblast-to-preosteocyte transitioning has multiple effects on calvarial development

“…1C-E), appearing like a subtle craniosynostosis. However, this ectopic mineralization does not seem to occur in intercalvarial regions, but primarily in the adjacent dermis, as described for diseases such as pseudoxanthoma elasticum (PXE) (Apschner et al, 2014;Mackay et al, 2015;Walker et al, 1989). Indeed, both dermal fibroblasts and basal keratinocytes express col1a2 (Fig.…”

Building the backbone: the development and evolution of vertebral patterning

“…Teleost vertebrae can form with normal segmental periodicity when somite patterning is disrupted, as in the tbx6/fused somite mutant (Fleming et al, 2004;Nikaido et al, 2002;van Eeden et al, 1996). In addition, centrum segmentation might be lost in situations in which normal somite patterning is maintained, for example in the over-ossification observed in cyp26b and enpp1 mutants (Apschner et al, 2014;Laue et al, 2008;Spoorendonk et al, 2008). Disruption of the segmentation clock in zebrafish hes6 mutants results in fewer somites and, later in development, fewer vertebrae (Schröter and Oates, 2010), suggesting that vertebral segment number is indeed linked to somite number.…”

Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI

“…To test whether the observed activity of vitamin K is specific to that of abcc6a, we administered vitamin K 1 (80 µM) to dragonfish (enpp1 −/− ) embryos, which feature extensive axial hypermineralisation due to an inability to produce PPi (Apschner et al, 2014;Huitema et al, 2012). In these embryos, vitamin K 1 Fig.…”

“…A possible cause of this discrepancy is the greater propensity for mineralisation to occur in the fibrillar collagen of the notochord sheath. Similarly, the enpp1 −/− zebrafish (dragonfish) features extensive hypermineralisation of the axial skeleton, unlike human patients with GACI (Apschner et al, 2014).…”

“…We have recently described a zebrafish allele, dragonfish (dgf ), with a nonsense mutation in enpp1, resulting in ectopic mineralisation of the skin and axial skeleton in embryos, and the eyes and bulbus arteriosus of the heart in adults similar to GACI symptoms (Apschner et al, 2014 , and the axial hypermineralisation phenotype of dgf mutants was not exacerbated by the grt genotype (data not shown). Furthermore, transgenic overexpression of ENPP1 reduced mineralisation in all embryos, regardless of grt genotype (not shown).…”