*5.4. The "PXE" Zebrafish*

Rodent models present remarkable similarity to human diseases and have proven extraordinarily useful. However, like all models, these systems have limitations, i.e., a relatively long life span and the associated cost of development and maintenance. Zebrafish are models that address some of these limitations and provide higher "n" numbers for observation and quantification. Zebrafish carry two orthologs of human *ABCC6* referred to as *abcc6a* and *abcc6b*. These genes encode proteins of 1507 and 1502 amino acids, respectively, which are similar to the human polypeptide at 1503 amino acids. *Abcc6a* and *-b* have ~50% identity and ~70% similarity with the human protein, respectively. The first attempt to generate a zebrafish model used morpholino technology, which is now progressively replaced by genome-targeting methods such as CRISPR/Cas9 because it has often been difficult to discriminate between specific and non-specific effects. Li et al., 2010 reported the expression profiles of both *abcc6a* and *abcc6b* and the lack of phenotype associated with *abcc6b* knockdown [113]. Injection of *abcc6a*-specific morpholinos induced cardiac

and developmental malformations followed by death of the animals. This phenotype could be rescued with injection of mouse *Abcc6* cDNA, suggesting some evolutionary conservation in the physiological function of ABCC6. More recently, van Gils and coworkers described a CRISPR/Cas9 *abcc6a* knockout zebrafish [114]. This zebrafish model showed hypermineralization of the spine starting in the embryonic stage and progressing into adulthood with scoliosis, vertebral and rib mineralization and loss of normal bone architecture. These manifestations are not consistent with the human PXE condition and there was no obvious mineralization in the skin or in the eyes. Thus, the *abcc6a* zebrafish model develops an abnormal calcification phenotype but one that does not recapitulate the human PXE. A new zebrafish model was recently created using the transcription activator-like effector nuclease (TALEN) technique [115]. These animals displayed similar skeletal changes as well as calcification in the ocular Bruch's membrane and a range of other previously unreported manifestations, such as cardiac fibrosis. Remarkably, this phenotype could be attenuated by vitamin K treatment, similar to what had been previously reported in 2015 with another zebrafish model [116].

Overall, zebrafish have shown some utility, notably in the characterization of the 4-phenylbutyrate treatment option for PXE [117], but their evolutionary distance and phenotypic divergence limit their usefulness for understanding the pathophysiology of PXE. However, they could be excellent preliminary testing tools for anti-calcification treatments.
