Coagulation Factor IIIa (f3a) Knockdown in Zebrafish Leads to Defective Angiogenesis and Mild Bleeding Phenotype

Aim: Tissue factor (TF), an initiator of the extrinsic coagulation pathway, is crucial for embryogenesis, as mice lacking TF are embryonically lethal (E10.5). This lethality may be attributed to defects in vascular development and circulatory failure, suggesting additional roles for TF in embryonic development beyond coagulation. In this study, we characterized the role of one of the TF paralogs (f3a) using a zebrafish model. Methods: To understand the TF evolution across different species, we performed molecular phylogenetic and sequence homology analysis. The expression of f3a during embryonic developmental stages was determined by RT-PCR. Endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX) was used to image the vascular development. The role of f3a during embryonic development was investigated by mRNA knockdown using Morpholinos (MO), an antisense-based oligonucleotide strategy. The f3a morphants were examined at 52 hpf for defects in morphological appearance, bleeding, and vascular patterning. Results: Spatiotemporal expression of f3a by qPCR revealed expression in all developmental stages, suggesting that f3a transcripts are both maternally and zygotically expressed. High expression of f3a from 28 hpf to 36 hpf confirmed the role of in the development of the yolk sac, circulation, and fins. f3a MO-injected embryos showed morphological abnormalities, including shorter body lengths and crooked tails. O-dianisidine staining showed f3a MO-injected embryos exhibited bleeding in the trunk (5.44%) and head (9.52%) regions. Using endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX), imaging of caudal vein plexus, which forms immediately following the onset of circulation and sprouting, showed a 3-fold decrease in f3a morphants versus controls at 48 hpf, suggesting a potential role for f3a in flow-induced angiogenesis.