Factor X Mutant Zebrafish Tolerate a Severe Hemostatic Defect in Early Development Yet Develop Lethal Hemorrhage in Adulthood

Factor X (F10) deficiency is a rare inherited bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted knockout of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering further studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in development. The zebrafish model is characterized by external development, optical transparency, ability to generate thousands of offspring at low cost, and a highly characterized vasculature. We have used these advantages for more in depth study of the role of the coagulation cascade in developmental regulation of hemostasis and vasculogenesis.

We generated a 17 base pair deletion in the zebrafish f10 locus by genome editing with TALENs. Although indistinguishable morphologically from f10^+/+ and f10^+/- siblings at early stages, f10^-/- mutants demonstrated progressive lethality between 1 and 5 months of age. Extensive hemorrhage was identified in multiple tissues starting at 3-4 weeks of age, particularly the brain. Notably, intracranial hemorrhage is a common feature in multiple zebrafish mutants with various vascular defects, including anomalies of endothelial differentiation and specification, apoptosis, and vessel integrity/permeability. Gross inspection of f10 mutant embryos and larvae in the first week of life revealed no apparent defects in circulation or vascular development. Expression of arterial and venous endothelial markers were examined by in situ hybridization at 24 and 72 hours post fertilization, the time period during which axial and intersegmental vessels form, along with early establishment of the vascular network. Markers included ephb2a, cdh5, flk1, flt4, and ephb4, and expression patterns were indistinguishable between mutants and wild type siblings. Acridine orange staining at 5 days post fertilization (dpf) did not detect any dysregulation of apoptosis. Hemoglobin staining found no specific hemorrhage in vehicle or warfarin treated mutants. However, 3 dpf f10^-/- mutants did not develop occlusive thrombi in response to laser-mediated venous endothelial injury, indicating that F10 is required for hemostasis. We used quantitative PCR to measure transcription of f10 and downstream coagulation factors in 3 dpf larvae. f10 mRNA was undetectable in homozygous mutants, presumably due to nonsense-mediated decay as a consequence of the TALEN-induced frameshift mutation. fga (fibrinogen alpha) and at3 (antithrombin III) mRNAs were increased by 1.8 and 2.3-fold, respectively (p<0.05), when compared to wild type siblings. Prothrombin (f2) mRNA transcription was slightly decreased, although the effect was not statistically significant.

In summary, we have produced a zebrafish model of human F10 deficiency that exhibits a spontaneous adult lethal bleeding phenotype, although early embryonic/larval survival is unaffected despite an underlying severe hemostatic defect. Further study of this mutant may identify species specific factors enabling this early survival. qPCR analysis in mutant larvae suggests that the level of F10 mRNA or protein indirectly regulates other coagulation factors during development. We have also used this model to evaluate the longstanding hypothesis that coagulation cascade mediated thrombin signaling is required for embryonic vasculogenesis. Thus far we have not found evidence for defects in vessel development or integrity, apoptosis, or endothelial differentiation/specification. Taken together, these data suggest that the effects of F10 loss are restricted to hemostasis, and thus therapies should continue to be focused on this in deficient patients. Further study as to why mutant embryos and larvae are able to tolerate what would likely cause lethal hemorrhage in mammals, as well as small molecule screens using this model, could potentially lead to innovative therapeutic modalities for patients with bleeding disorders.