Zebrafish as outgroup model to study evolution of scavenger receptor class B type I functions

Background and aims: Scavenger receptor class B1 (SCARB1) -also known as the high-density lipoprotein (HDL) receptor -is a multi-ligand scavenger receptor that is primarily expressed in liver and steroidogenic organs. This receptor is known for its function in reverse cholesterol transport (RCT) in mammals and hence disruption leads to a massive increase in HDL cholesterol in these species. The extracellular domain of SCARB1 -which is important for cholesterol handling -is highly conserved across multiple vertebrates, except in zebrafish.Methods: To examine the functional conservation of SCARB1 among vertebrates, two stable scarb1 knockout zebrafish lines, scarb1 715delA (scarb1-1 nt) and scarb1 715_716insGG (scarb1 +2 nt), were created using CRISPR-Cas9 technology.Results: We demonstrate that, in zebrafish, SCARB1 deficiency leads to disruption of carotenoid-based pigmen-tation, reduced fertility, and a decreased larvae survival rate, whereas steroidogenesis was unaltered. The observed reduced fertility is driven by defects in female fertility (-50 %, p < 0.001). Importantly, these alter-ations were independent of changes in free (wild-type 2.4 +/- 0.2 mu g/mu l versus scarb1-/-2.0 +/- 0.1 mu g/mu l) as well as total (wild-type 4.2 +/- 0.4 mu g/mu l versus scarb1-/-4.0 +/- 0.3 mu g/mu l) plasma cholesterol levels. Uptake of HDL in the liver of scarb1- /-zebrafish larvae was reduced (-86.7 %, p < 0.001), but this coincided with reduced perfusion of the liver. No effect was observed on lipoprotein uptake in the caudal vein. SCARB1 deficient ca-naries, which also lack carotenoids in their plumage, similarly as scarb1-/-zebrafish, failed to show an increase in plasma free-and total cholesterol levels.Conclusion: Our findings suggest that the specific function of SCARB1 in maintaining plasma cholesterol could be an evolutionary novelty that became prominent in mammals, while other known functions were already present earlier during vertebrate evolution.