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

ADP-ribosylation factor GTPases are activated by guanine nucleotide exchange factors including Gbf1 (Golgi brefeldin A-resistant factor 1) 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 that arises from -ethyl--nitrosourea (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 locus, resulting in a leucine → arginine substitution (L1246R) in the HDS2 domain. The mutant phenotype is mimicked by 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 mutants can be prevented partially and temporally by treating with the endoplasmic reticulum stress/apoptosis inhibitor tauroursodeoxycholic acid or by knocking down the proapoptotic gene Therefore, endothelial endoplasmic reticulum stress and subsequent apoptosis induced by deficiency may account for the vascular collapse and hemorrhage.