Impairment of Cargo Transportation Caused by gbf1 Mutation Disrupts Vascular Integrity and Causes Hemorrhage in Zebrafish Embryos.

Abstract ADP-ribosylation factor (ARF) GTPases are activated by guanine nucleotide exchange factors including Golgi Brefeldin A-resistant factor 1 (Gbf1) and play important roles in regulating organelle structure and cargo-selective vesicle trafficking. However, the developmental role of Gbf1 in vertebrates remains elusive. In this study, we report the zebrafish mutant line tsu3994 that arises from ENU-mediated mutagenesis and is characterized by prominent intracerebral and trunk hemorrhage. The mutant embryos develop hemorrhage accompanied by fewer pigments and shorter caudal fin at day 2 of development. The hemorrhage phenotype is caused by vascular breakage in a cell-autonomous fashion. Positional cloning identifies a T>G nucleotide substitution in the 23rd exon of the gbf1 locus, resulting in a leucine>arginine substitution (L1246R) in the HDS2 domain. The mutant phenotype is mimicked by gbf1 knockouts and morphants, suggesting a nature of loss-of-function. Experimental results in mammalian cells show that the mutant form Gbf1(L1246R) is unable to be recruited to the Golgi apparatus and fails to activate Arf1 for recruiting COPI complex. The hemorrhage in tsu3994 mutants can be prevented partially and temporally by treating with the endoplasmic reticulum (ER) stress/apoptosis inhibitor tauroursodeoxycholic acid (TUDCA) or by knocking down the proapoptotic gene baxb. Therefore, endothelial ER stress and subsequent apoptosis induced by gbf1 deficiency may account for the vascular collapse and hemorrhage.