A New Zebrafish Model for Pseudoxanthoma Elasticum

Abstract

Calcification of various tissues is a significant health issue associated with aging, cancer and autoimmune diseases. There are both environmental and genetic factors behind this phenomenon and understanding them is essential for the development of efficient therapeutic approaches. Pseudoxanthoma elasticum (PXE) is a rare genetic disease, a prototype for calcification disorders, resulting from the dysfunction of ABCC6, a transport protein found in the membranes of cells. It is identified by excess calcification in a variety of tissues (e.g., eyes, skin, arteries) and currently it has no cure, known treatments target the symptoms only. Preclinical studies of PXE have been successful in mice, proving the usefulness of animal models for the study of the disease. Here, we present a new zebrafish (Danio rerio) model for PXE. By resolving some ambiguous assemblies in the zebrafish genome, we show that there are two functional and one non-functional paralogs for ABCC6 in zebrafish (abcc6a, abcc6b.1, and abcc6b.2, respectively). We created single and double mutants for the functional paralogs and characterized their calcification defects with a combination of techniques. Zebrafish deficient in abcc6a show defects in their vertebral calcification and also display ectopic calcification foci in their soft tissues. Our results also suggest that the impairment of abcc6b.1 does not affect this biological process.

Significance of Premature Vertebral Mineralization in Zebrafish Models in Mechanistic and Pharmaceutical Research on Hereditary Multisystem Diseases

“…Abcc6a−/− larvae showed advanced vertebral calcification in both larvae and adult fish, contrary to what was observed for abcc6b mutants, and these findings were similar to those reported by MacKay et al and Van Gils et al (Figure 2) [55,57]. No gene expression analyses were performed [59].…”

“…For some of these disorders, such as PXE or GACI, the vertebral phenotype is the main-if not the only-feature of the zebrafish mutants and knockouts [53,55,[57][58][59][60]. PXE is considered a hallmark disease with respect to studying ectopic soft tissue mineralization, c.q., hydroxyapatite precipitation, occurring in several genetic and acquired diseases as well as during aging [50,60,106].…”

“…In early embryonic stages, abcc6a was expressed ubiquitously; at 48 h post fertilization (hpf) and 4 dpf, expression was noted in the heart, operculum, and cleithrum as well as in the ear and notochord [58]. [57][58][59][60], except for the model developed by Li et al [53]. In the adult models, excessive (inter)vertebral calcification was observed.…”

“…Finally, Czimer et al used CRISPR/Cas9 editing to create frameshift alleles, p.Arg60Serfs*183 and p.Cys205Leufs*4, in the abcc6a and abcc6b genes, respectively, both leading to a premature termination codon [59]. Abcc6a−/− larvae showed advanced vertebral calcification in both larvae and adult fish, contrary to what was observed for abcc6b mutants, and these findings were similar to those reported by MacKay et al and Van Gils et al (Figure 2) [55,57].…”

Zebrafish Models to Study Ectopic Calcification and Calcium-Associated Pathologies

“…They also displayed higher osteoclast activity at sites of ectopic calcification, and bisphosphonates could efficiently rescue associated phenotypes [ 30 ]. Zebrafish abcc6a mutants showed defects in vertebral calcification and displayed ectopic calcification in soft tissues [ 32 ]. Since Abcc6a is present in the eyes and heart of the zebrafish, extensive calcification of the ocular sclera and Bruch’s membrane and a fibrotic heart were also observed in the abcc6a mutants [ 33 ].…”