TF protein and DNA sequences from human (Homo sapiens), rabbit (Oryctolagus cuniculus), mouse (Mus musculus), and zebrafish (Danio rerio) were retrieved from the National Center for Biotechnology Information (NCBI), European Molecular Biology Laboratory (EMBL), and Ensembl genome databases. Sequence alignment and construction of phylogenetic tree based on neighbor-joining method was performed using ClustalW2 (Hung and Weng, 2016).

The following zebrafish lines were used in this study: Tübingen (ZIRC [Zebrafish International Resource Center], Eugene, Oregon), Tg (fli1a:nEGFP)y7 (Tg(gata1:dsRed)sd2 (Eisa-Beygi et al., 2018), and Tg(kdrl:mCherry-CAAX)y171(Borovina et al., 2010). Adult fish were maintained under a constant temperature of 28.0 °C and were subjected to 14-h light: 10-h ark photoperiod. All animal studies performed in these facilities were under the Medical College of Wisconsin–approved protocol for Animal Use Application. Freshly fertilized embryos were procured through natural breeding of adult zebrafish and were kept at 28.0°C in 1X E3 embryo medium (E3 medium) containing 5 mmol/L NaCl, 0.17 mmol/L KCl, 0.33 mmol/L CaCl₂, 0.33 mmol/L MgSO₄, and 0.05% methylene blue. In some instances, embryos were treated with 0.003% of 1-phenyl-2-thiourea (Sigma-Aldrich), starting at 24 h postfertilization (hpf), to minimize pigmentation.

Wild-type strain zebrafish were maintained at 23.5°C on a 14 h light/10 h dark cycle. At the time of mating, breeding males and females were separated overnight before letting them spawn naturally in the morning to allow for synchronization of developmental stages. Fertilized eggs were grown at 28.0°C and staged using previously defined criteria (Kimmel et al., 1995; Machikhin et al., 2020). Samples from 9 different developmental stages, 2 hpf to 72 hpf, were collected by snap freezing the embryos in dry ice.

RNA extraction was performed as described by De Jong et al. (2010). In brief, tubes with embryos from different timepoints were kept in liquid nitrogen and ground individually with a liquid nitrogen pre-chilled metal micro-pestle (Carl Roth). The pestle was lifted slightly and 200 μl Qiazol (Qiagen, United States) was added. The pestle was placed back into the tube with Qiazol and the homogenate was allowed to thaw. Before removal, the pestle was washed with an additional 100 μl Qiazol to rinse off any material that might have stuck to the pestle. The homogenate was vortexed vigorously for 30 s, left at room temperature for at least 5 min, and then spun down quickly for 30 s 60 μl chloroform was added to the homogenate, vortexed for 15 s and kept at room temperature for 3 min. The partly separated mixture was transferred to a pre-prepared phase-lock gel heavy containing tube and centrifuged for 15 min at 12,000 × g. The aqueous phase was transferred to a new 1.5 ml tube. RNA was purified by column precipitation according to the RNeasy MinElute Cleanup kit (Qiagen, United States). At the end of the procedure, RNA was eluted in 12 μl nuclease-free water.

Double-stranded cDNA was synthesized using the QuantiTect Reverse transcription kit (Qiagen, United States). For RT-PCR analysis of f3a, the forward and reverse primers used were 5′- ACG​TGG​AGT​CCA​AAA​CCA​AC -3′ and 5′- CAG​CGC​TGT​AAT​AGG​CCT​TC -3′ and for Beta-actin (control), 5′- CGA​GCT​GTC​TTC​CCA​TCC​A -3′ and 5′- TCA​CCA​ACG​TAG​CTG​TCT​TTC​TG -3′. Transcript levels were quantified by SYBR™ Green PCR Master Mix (IDT, United States). For amplification by PCR, the initial denaturing step at 94°C for 5 min was followed by 40 amplification cycles of 30 s at 94°C; 30 s at 60°C; 60 s at 72°C, and a final extension period of 10 min at 72°C. f3a expression at different embryonic stages was calculated using beta-actin as a normalizing control and the ΔΔCt method.

An antisense MO targeting the exon-intron junction (exon 3) of f3a (MO:f3a) was designed to knockdown the expression of f3a. As a negative control, we used a standard control MO (control-MO) specific to a human β-globin intron mutation. All MO solutions were synthesized by GeneTools (Oregon, United States). The MO sequences are as follows:

f3a-MO: 5′- AAA​CAA​CTA​AAC​ACT​GAC​TGT​CAT​G-3′

Control-MO: 5′-CCT​CTT​ACC​TCA​GTT​ACA​ATT​TAT​A-3′

All MO solutions were briefly heated at 65°C and resuspended in 1X Danieau buffer (58 mmol/L NaCl, 0.7 mmol/L KCl, 0.4 mmol/L MgSO4, 0.6 mmol/L Ca(NO₃)₂, 5.0 mmol/L HEPES, pH 7.6), and 0.1% (w/v) phenol red dye (Sigma-Aldrich, United States), to a final concentration of 8 ng/nl. Embryos at 1 to 2-cell stage were positioned in individual grooves made on a 1.0% agarose gel and were initially injected at concentrations ranging from 0.5 to 2 ng/nl.

Total RNA extraction (De Jong et al., 2010) and cDNA synthesis were performed from control- and f3a- MO injected embryos as described before (Peterson and Freeman, 2009). For PCR-based gene expression analysis of f3a, the forward and reverse primers used were 5′- ACG​TGG​AGT​CCA​AAA​CCA​AC -3′ and 5′- TCC​GTC​ACA​TGC​AGC​TTT​GT -3′; for f3b, 5′- CTT​GGG​GAC​CCA​AAC​CTG​TC -3′ and 5′- TCC​AGT​CGG​TTA​AAC​TCC​GC -3′, and for GAPDH (control), 5′- GTG​GAG​TCT​ACT​GGT​GTC​TTC -3′ and 5′- GTG​CAG​GAG​GCA​TTG​CTT​ACA -3′. Each amplification reaction was separated on a 1.5% agarose gel stained with ethidium bromide-stain and the bands were visualized using UV Transilluminator (Chemidoc System, United States). Gels were quantified using ImageJ software (Schneider et al., 2012).

As described by Paffett-Lugassy and Zon (Paffett-Lugassy and Zon, 2005), O-dianisidine staining was used to detect the presence of hemoglobin within intact 52 hpf zebrafish embryos injected with Control or f3a MOs. Dechorionated or hatched embryos were stained in the dark for 30 min at room temperature within a solution containing O-dianisidine (0.6 mg/ml), 0.01 M sodium acetate (pH 4.5), 0.65% H₂O₂, and 40% (vol/vol) ethanol. After staining, embryos were washed with RO water and then fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature. Pigments were removed from fixed embryos by incubating in a solution of 0.8% KOH, 0.9% H₂O₂, and 0.1% Tween-20 for 30 min at room temperature. Embryos were then washed with phosphate-buffered saline (PBS) containing 0.1% Tween-20, and then fixed again in 4% PFA for 3 h before storage in PBS at 4°C. All embryos were positioned in dorsal recumbency and imaged using a Keyence BZ-X700 fluorescent microscope (Japan).

Endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX) were used to study vascular development. In brief, Tg (fli1a:nEGFP)y7 (Tg(gata1:dsRed)sd2 (Eisa-Beygi et al., 2018), and Tg(kdrl:mCherry-CAAX)y171(Borovina et al., 2010) lines were maintained at 23.5°C on a 14 h light/10 h dark cycle. At the time of mating, breeding males and females were separated overnight before letting them spawn naturally in the morning. Embryos at 1 to 2-cell stage were positioned in individual grooves made on a 1.0% agarose gel and were initially injected at concentrations ranging from 1 to 2 ng/nl.

For bright-field/fluorescent microscopy, wild-type or transgenic embryos were first embedded in 1.0% low melting agarose in E3 medium and 30 μg/ml tricaine mesylate. Embryos were mounted on 35 mm glass-bottom Petri dishes and imaged using Keyence BZ-X700 fluorescent microscope (Japan). A Texas Red filter cube (OP-87765, Keyence) was used to detect mCherry and dsRed (red fluorescent protein)-labeled cells, a GFP filter cube (OP-87763, Keyence) was used to detect GFP/EGFP-labeled tissues, and a 4′,6-diamidino-2-phenylindole (DAPI) filter cube (OP-87762, Keyence) was used to image DAPI-stained samples. Z-series bright-field and fluorescent images were acquired, and composite images were generated using the BZ-X Image Analyzer software. Brightness and contrast were adjusted using Fiji Software.

Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, United States) by Student’s t test (2 groups; nonparametric). Bleeding and morphological phenotypes were expressed as percentage differences from respective controls.

To understand the evolutionary relationships across different species, we compared the mouse, human and rabbit TF gene and protein sequences to the TF paralogs (f3a and f3b). Multiple sequence alignment (ClustalW) revealed that zebrafish f3a protein was more closely related to that of mouse and human compared with f3b (Figure 1A) and was more identical to human F3 compared to f3b (Figure 1B). RT-PCR analysis of expression during embryonic developmental stages showed that f3a was detected as early as 2 h (64-cell stage) and in larvae at up to 36 hpf (Figure 1C). Previous report indicate detecting f3a transcripts at 64-cell stage, which suggest maternal contribution to larvae at this stage (Kimmel et al., 1995). Between 2.75 hpf to 6 hpf, both maternal and zygotic expression appears to be present because f3a was strongly transcribed compared to earlier (2 hpf) and later stages (10, 18, and 24 hpf) (Figure 1C). These observations indicate f3a transcription crossover from maternal to zygotic stages (between 2 hpf to 6 hpf). From 28 hpf to 36 hpf the expression of f3a was relatively stable, suggesting that f3a expression contributes to the development of yolk sac, circulation, pigmentation, heartbeat, circulation in the aortic arch, and fins (Kimmel et al., 1995). At 48 hpf and 72 hpf expression was low compared to 36 hpf, suggesting f3a might not play a significant role in hatching and protruding mouth stages.

Previously, Zhou and colleagues reported that f3b knockdown in zebrafish exhibits defective vasculogenesis (Zhou et al., 2011). Because f3a′s role during embryonic development was not known, we therefore examined its role by mRNA knockdown using MO antisense oligonucleotides (Gene Tools, United States) (Figure 2A). Embryos were collected freshly and 1 and 2 ng of f3a MO, targeted exon-intron junction (e3-i3), was injected at 1- to 2-cell stage. Twenty-four hours later, RNA was isolated from the injected embryos and expression of f3a and f3b analyzed by PCR. Control group embryos were injected with a random sequence of standard MO. Our data revealed lower PCR transcript levels of f3a, but not f3b, at both 1 and 2 ng levels in injected embryos, suggesting specificity of the targeted knockdown towards f3a (Figure 2B).

The f3a morphants, injected with 2 ng of MO, were examined at 52 hpf for any defect in morphological appearance. Control MO-injected embryos appeared to be normal, with properly developed head and eyes, and elongated body axis, and a long tail (Figure 3A). In contrast, embryos injected with 2 ng of f3a MO exhibited a shortened body axis and a short, crooked tail. Based on the degree of severity, we segregated them as mild, moderate, and severe and quantified the severity. On an average, 11.45% (15/131) showed mild phenotype with shortened body axis. Moderate defect in morphological appearance was observed in 17.55% (23/131), with a partially curved tail. Severe morphological defect was observed in 27.5% (36/131) of embryos, with curved body axis and defectively developed tail (Figure 3B).

As TF is a triggering factor for activation and coagulation cascade, in a separate experiment, we checked whether the knockdown of f3a showed bleeding phenotype. We observed embryos at 52 hpf, which showed bleeding mostly in the head and tail regions (Figure 4A). Control MO-injected embryos appeared normal with no visible bleeding. Conversely, f3a MO-injected embryos showed 5.44% (8/147) and 9.52% (14/147) bleeding in tail and head regions, respectively (Figure 4B). With the next set of f3a and standard MO injection, at 52 hpf, we stained the embryos with O-dianisidine to detect hemoglobin-containing cells (Figure 4C); around 7.5% had bleeding in the head. No bleeding was observed in control MO-injected embryos at 52 hpf (Figures 4C,D). Common cardinal vein (CCV) region also stained with O-dianisidine due to the presence of a pool of blood cells and heart. This region appears stained irrespective of phenotype, and therefore, we did not consider the CCV region for bleeding score. These findings confirm that, similar to f3b (Zhou et al., 2011), f3a plays a role in vascular stability in our zebrafish model.

Next, we investigated the effect of f3a knockdown on vasculogenesis and angiogenesis using endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX) and imaged vascular development. Interestingly, we did not observe any defect in vessel formation at 48 hpf between control and f3a-MO-injected groups (Figure 5A), suggesting that f3a might not be involved in initial development of central aorta (CA) and no major deficit in vasculogenesis. During zebrafish embryonic angiogenesis, caudal vein plexus (CVP) and inter-segmental vessel (ISV) formation are the two obvious vessel patterns observed. In f3a knockdown embryos, the development of CVP was greatly abrogated (Figure 5C). Quantification of loop formation at mean CVP showed a 3-fold decrease in f3a morphants versus controls at 48 hpf (Figure 5C). The CVP forms during a very active period of angiogenic sprouting from beginning at 25 hpf when venous endothelial cells of the posterior cardinal vein sprout and migrate ventrally, then fuse with neighboring tip cells (Figure 5B). Thus, we looked at the difference in the CVP angiogenic sprouting at 48 hpf. Like number of CVP, the mean spouting from CVP was 4-fold lower in f3a injected MO than the control MO-injected group (Figure 5C). Occasionally, we also observed a defect in central arteries (CtAs) and primordial midbrain channel (PMBC) in f3a morphant CVP defective embryos. However, we did not see any impairment in the intersegmental vessel (ISV) development. These findings support the notion that f3a is required for angiogenesis.