Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway

Abstract

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use zebrafish to confirm 5 novel human variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of variants. These findings as well as the prolonged survival of mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia

“…demonstrated that induced larval venous thrombi are fibrin rich [31], suggesting an important role in embryonic hemostasis. In this study our endothelial injury experiments confirm that fibrinogen is indeed required for induced thrombus formation, consistent with our previous studies of F10 deficiency, as well as observed hypofibrinogenemia in At3-deficient fish [29,30]. In all three mutants (fga, f10 and at3), embryonic/larval zebrafish tolerate severe coagulopathies into early adulthood before succumbing, although death occurs at a much later time-point in the fga mutants.…”

“…In mice, fga − / − ; nfe2 − / − results in lethality in the immediate neonatal period, but zebrafish initially survive with a steady decline until ~300 dpf. This trend is consistent with coagulation factor knockouts that are lethal in embryonic mice, but exhibit extended survival in zebrafish . We hypothesize this may be a result of differential species‐specific factors regulating hemostatic balance, which could be leveraged to improve our understanding of coagulopathies.…”

“…In contrast to other coagulation protein deficiencies , loss of fga results in a relatively mild hemorrhagic phenotype in zebrafish. As seen previously , we also did not detect grossly visible bleeding by o‐dianisidine staining in fga − / − larval mutants (data not shown).…”

“…At 2 months of age, 91% of C57BL/6J mice, but only 57% of the mixed 129/CF-1 background, were found to be alive [52]. The fish fga mutant data are in sharp contrast to F10 deficiency, which resulted in death beginning as early as 1 month of age, with complete loss by 4 months [30]. However, this difference is consistent with observations in human patients [1] and mouse knockouts [56][57][58][59] demonstrating that common pathway mutations are more severe than defects in fibrinogen.…”

“…Cross‐regulation of coagulation factors at the mRNA level was identified in the f10 knockout . Loss of F10 resulted in statistically significant increases of fga and at3 mRNA by 75 and 100%, respectively, but no significant effect on f2 .…”