Abstract
Background: The tissue factor pathway regulates vascular structure in the adult through regulation of thrombin generation and signaling. Deletion of tissue factor pathway inhibitor (TFPI) in the mouse results in early intrauterine lethality, precluding study of vascular development. In addition, the carboxyl terminus of TFPI may have a TF-independent effect on vascular structure. We sought to determine the role of TFPI and its carboxyl terminus in vascular development of zebrafish. Methods and results: TFPI mRNA localization studies demonstrated expression in the developing kidney, gut, and vasculature of the zebrafish. We further assessed the role of TFPI in zebrafish angiogenesis, by injecting translational and splice-inhibiting morpholino (MO) oligonucleotides directed against zebrafish TFPI mRNA into embryos from the double transgenic zebrafish Tg(Fli1GFP); Tg(GATA1dsRed) expressing GFP in vascular endothelium and dsRed in erythroid cells. Knockdown of TFPI protein expression was subsequently confirmed by immunoblotting. In a dose-dependent manner, multiple non-overlapping MOs targeting the translation initiation site of TFPI independently and synergistically led to the formation of abnormally targeted intersegmental arteries which crossed somite boundaries by 48 hours post fertilization (hpf). Additionally, a plethoric caudal vein plexus with increased honeycombing was present by 24 hpf. This latter aspect of the phenotype was most striking after a MO targeted against a splice site involving the Kunitz 3 and carboxyl terminus domains of zebrafish TFPI was injected alone. Conclusions: TFPI is expressed during zebrafish development. Morpholino knockdown of TFPI expression alters both arterial and venous patterning in the developing zebrafish, indicating a fundamental role for TFPI in targeting of newly-formed blood vessels. Moreover, different domains of the TFPI protein may preferentially act on distinct vascular networks.
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