Identification and Characterization of Zebrafish Tissue Factor and Its Role in Embryonic Blood Vessel Development.

Tissue factor (TF) is a trans-membrane glycoprotein that is the cellular receptor for coagulation factor VII/VIIa. Activation of factor VII by TF is well established as the primary regulator of blood coagulation. Although TF has been implicated in such diverse processes as angiogenesis and tumor metastasis, these other functions have been difficult to study, as the knockout of murine TF is lethal at day 8.4 of embryonic life. Because of their hardiness, their rapid maturation sequence and their optical clarity, the zebrafish is an increasingly popular model for studying embryonic development. In order to study the role of TF in embryonic development we have cloned zebrafish TF cDNA. The 1.36kb orthologous cDNA predicts an ORF encoding a 293 amino acid (aa) protein with a 26 amino acid (aa) signal peptide. The predicted mature protein contains a 230 aa extra-cellular domain, a 20 aa trans-membrane segment, and a 17 aa cytoplasmic tail. The predicted amino acid sequence of zebrafish TF has 31% and 32% overall identity with human and mouse TF respectively. The zebrafish TF gene, like the human and mouse genes, has 6 exons spanning 14kb of genomic DNA. Whole-mount in situ hybridization with a TF anti-sense Riboprobe revealed that TF is detected as early as 18 hours post-fertilization (hpf) in developing head structures and brain. At 20 hpf, TF is expressed in the vascular system and highly expressed in the hindbrain. By 48 hpf, TF is seen in the brain, heart, liver, lens and gut. To investigate the expression of zebrafish TF in vivo, we generated transgenic zebrafish lines that express enhanced green fluorescent protein (EGFP) under the control of the TF promoter. A ∼ 2.0 kb promoter fragment upstream of the first exon of the TF gene was sufficient to drive expression of EGFP in a manner that recapitulated the expression of native TF. We inhibited TF expression by injecting anti-sense oligonucleotide morpholinos into the Tg(fli1:EGFP) line, in which there is robust expression of EGFP in vascular endothelial cells and their angioblast precursors. At 24 hpf the development of trunk capillary beds was delayed and, at 48 hpf, most of the morphants developed pericardial edema and impaired formation of inter-somitic vessels. These studies demonstrate that TF is expressed in multiple organs during zebrafish embryogenesis and that TF expression is necessary for the proper migration of endothelial cells into developing blood vessels during the process of embryonic vasculogenesis.