Evolutionary relationships and Spatiotemporal expression of Zebrafish TF-f3b:(A) Phylogenetic tree of tissue factor (F3) from zebrafish (Danio), mouse (Mus), rabbit (Oryc), and human (Homo). (B) Protein and DNA percent identity of F3 from zebrafish (Danio), mouse (Mus), rabbit (Oryc), and human (Homo). (C) mRNA expression from whole embryos demonstrates that zebrafish f3a is expressed in early embryonic stages. Beta-actin was used to normalize the expression of f3a.

f3a knockdown by Morpholino approach: (A) schematic diagram of f3a antisense morpholino oligonucleotide (MO) targeting the exon-intron junction (exon 3); design and synthesis by GeneTools (Oregon, United States). (B) The molecular targeting and efficiency of f3a MO-injected with 1 and 2 ng of f3a was assessed by PCR and quantified using ImageJ.

MO-mediated knockdown of f3a expression results in morphological abnormalities: (A) Zebrafish embryos at 1-or 2-cell stage were injected with control and f3a MO and examined morphologically at 52 hpf. MO phenotypes: control MO injected (control MO), and mild, moderate, and severe phenotypes. (B) The morphological abnormalities were quantified. Numbers in the bars represent the ratios used to calculate the percentages. Data were pooled from three independent experiments.

f3a knockdown shows bleeding phenotype: (A) Lateral and dorsal views of head, and lateral view of tail in live embryos. The arrows depict brain and tail bleeding at 52 hpf and (B) quantification of the same. Numbers in the bars represent the ratios used to calculate the percentages. (C) Bright-field images of zebrafish embryos stained with O-dianisidine (OD) and imaged at 48 hpf. Black arrows indicate sites where abnormal accumulation of hemoglobinized blood was detected in the head region (control MO and f3a MO). (D) Percentage of cerebral hemorrhage in embryos at 52 hpf. Numbers in the bars represent the ratios used to calculate the percentages. Data were pooled from three independent experiments.

f3a knockdown shows delayed angiogenesis: To study vascular development, endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX) embryo were used to knockdown f3a. (A) Images represent MO-mediated knockdown of f3a expression resulting in morphological abnormalities. MO phenotypes: control MO injected (control MO), and mild, moderate, and severe phenotypes. Differences in caudal vein plexus (CVP) in the trunk regions are indicated by white arrows. (B) Schematic diagram depicts to identify Caudal vein (CV), caudal vein plexus (CVP) and Central aorta (CA). (C)Tg(fli1:Myr-mCherry) line was used to quantify CVP loop number and Sprouting number from the CVP at 48 hpf. Embryos were randomly picked from ∼60 MO-injected (6 from control and 9 from f3a MO) embryos from control and f3a.