Genetic duplication of tissue factor reveals subfunctionalization in venous and arterial hemostasis

Tissue factor (TF) is an evolutionarily conserved protein necessary for initiation of hemostasis. Zebrafish have two copies of the tissue factor gene (f3a and f3b) as the result of an ancestral teleost fish duplication event (so called ohnologs). In vivo physiologic studies of TF function have been difficult given early lethality of TF knockout in the mouse. We used genome editing to produce knockouts of both f3a and f3b in zebrafish. Since ohnologs arose through sub- or neofunctionalization, they can unmask unknown functions of non-teleost genes and could reveal whether mammalian TF has developmental functions distinct from coagulation. Here we show that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF results in lethal hemorrhage by 2-4 months despite normal embryonic and vascular development. Larval vascular endothelial injury reveals predominant roles for TFa in venous circulation and TFb in arterial circulation. Finally, we demonstrate that loss of TF predisposes to a stress-induced cardiac tamponade independent of its role in fibrin formation. Overall, our data suggest partial subfunctionalization of TFa and TFb. This multigenic zebrafish model has the potential to facilitate study of the role of TF in different vascular beds. Author summary Tissue factor (TF) is a critical component in the initiation of blood coagulation. It is exposed upon blood vessel injury, which leads to blood clot formation and cessation of hemorrhage. TF is functionally conserved and likely required for survival across all vertebrate species. Due to an ancestral genomic duplication event in the teleost class of fish, zebrafish have two copies of tissue factor (referred to as ohnologs) on separate chromosomes. We exploited this aspect of the zebrafish model to study real time developmental phenotypes previously suggested in mouse studies, with the surprising finding of that the f3 ohnologs have evolved separate functions. We saw no evidence for TF in blood vessel formation, however, we did see an obvious role in protecting the integrity of the vascular space around the heart. We found that clotting was completely blocked in the total absence of TF, but loss of each individual ohnolog led to differential deficiencies in the arterial and venous circulations. This type of evolution is known as subfunctionalization and presents an opportunity to further understand differences between blood clot formation based on vascular location.

Automated summary

By using genome editing to generate knockouts of both f3a and f3b in zebrafish, it was found that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF leads to lethal hemorrhage, and TFa and TFb play predominant roles in venous and arterial circulation, respectively. Loss of TF predisposes to stress-induced cardiac tamponade.