A bone to pick with zebrafish: chemistry and genetics of osteogenesis

Abstract

The tissues in our bodies are extremely rich in calcium and phosphate – the primary ingredients of bone mineral. The precipitation reaction between these ions to form bone mineral (hydroxyapatite) is so favourable that vertebrate tissues need anti-mineralization factors to prevent ectopic calcification. One of these anti-mineralization factors is pyrophosphate (PPi), a small chemical that mimics the crystal structure of bone, while other factors are vitamin K-dependent proteins (Gla proteins). The ectopic-calcification disorder Pseudoxanthoma Elasticum (PXE) is caused by mutations in the ABCC6 gene, which performs an unknown function in the liver. PXE patients have lower levels of both PPi and vitamin K in their blood compared to healthy subjects but the link between ABCC6 and these factors is not clear.

In this work a zebrafish mutant line with dramatically enhanced mineralization in the spine was found to carry a mutation in the fish version of ABCC6. Treatment with vitamin K successfully prevented this excessive mineralization, confirming the long-speculated link between vitamin K and mineralization (through the Gla proteins). Vitamin K was also effective at treating a similar over-mineralization phenotype seen in zebrafish with a mutation in a separate gene, ENPP1, which has a known function in PPi synthesis. This shows that vitamin K is sufficient to inhibit mineralization in the absence of PPi, a finding which has implications for the still-unknown link between PXE and vitamin K. Finally, the zebrafish gene was found to be active in the bone itself, rather than the liver as it is in humans.

In a separate work, a mutant zebrafish called zwarte piet was identified with almost no hydroxyapatite mineral in the bone. This fish line carries a mutation in a zinc transporter Znt8, and surprisingly, large accumulations of zinc were seen in the bone structure. Zinc is required for bone physiology, but too much zinc could prevent mineralization due to atomic similarities between zinc and calcium. Besides the effect on bone, the loss of this zinc transporter causes blindness, extra pigmentation and swimming difficulties, due to a build-up of zinc in the brain. This build-up leads to accumulation of zinc in specialized zinc-storage compartments called zincosomes, which were here observed for the first time in a living animal.