Whole-mount in-situ hybridization was performed to examine survivin mRNA expression in zebrafish embryos at 26 hpf. Survivin was detected diffusely throughout the developing brain and neural tube. It was also expressed at the vicinity of the axial vasculature from which the inter-segmental vessels arise (Figure 1a–b). This was further confirmed in histological sectioning in which the areas corresponding to the developing axial vasculature and neural tube showed increased expression relative to the adjacent tissues (Figure 1b, insert). Furthermore, double in-situ hybridization showed that survivin was expressed in the developing axial vasculature dorsal to the intermediate cell mass (ICM), where gene encoding for embryonic hemoglobin α was expressed. The pattern was remarkably similar to that of flk1, a VEGF receptor tyrosine kinase (Figure 1c–d).

The role of survivin during embryonic development was investigated by knocking-down its function using MOs. The phenotypic penetrance of survivin MOs was dose- and time-dependent. At 22 hpf, when injected with either 3 ng Sur_UTR or 3 ng Sur_ATG-MOs (referred as Sur_UTR^mo and Sur_ATG^mo embryos), most embryos had a relatively normal morphology (Figure 2a,c). However, at 48 hpf, 74.8 ± 7.3% and 72.0 ± 4.0% of embryos manifested "characteristic phenotypes" with reduced eye and head sizes and a mildly curved tail (Figure 2b,d). There was no overt tissue necrosis in these embryos. At 6 ng of either MOs, increasing numbers of embryos became severely deformed and died shortly after 48 hpf (Figure 2d, insert). Co-injecting Sur_ATG + Sur_UTR-MOs (3 ng each) resulted in specific phenotypes in 79.4 ± 7.2% embryos without increase in toxicity or mortality as compared with 3 ng of either MO alone (Figure 2e). The combination regimen remained significantly less toxic than that of Sur_UTR-MO at 6 ng. In all subsequent experiments, Sur_ATG and Sur_UTR-MOs were co-injected at 3 ng each (referred as Sur_UTR+ATG^mo embryos). Only embryos with characteristic phenotypes were investigated while those which were severely deformed were excluded from analysis.

We have previously shown that survivin is significantly up-regulated in a zebrafish chordin morphant in which the ICM was expanded 7. Therefore, we first examined the effects of survivin knock-down on vascular formation in Tg(fli1:EGFP)^y1 embryos. In uninjected embryos, the axial circulation (AC), inter-segmental vessels (ISV), dorsal longitudinal anastomotic vessels (DLAV), vertebral and sub-intestinal vessels (SIV) were readily observable (Figure 3a,c). In Sur_UTR+ATG^mo embryos, the dorsal aorta and posterior cardinal vein were patent, indicative of intact vasculogenesis (see additional file 1: Wild-type embryos and file 2: Survivin morphants). However, the development of vertebral and ISV was perturbed with defective or total absence of sprouting as well as failure to form the DLAV and SIV (Figure 3b,d). These defects were seen in all 54 Sur_UTR+ATG^mo embryos observed (n = 3 separate experiments) with the characteristic phenotypes. The results were confirmed using microangiography in which defective ISV sprouting and failure to form the DLAV, as well as defective inner optic circle (IOC) and optic veins (OV) of the developing eyes were seen in the Sur_UTR+ATG^mo embryos (Figure 3e–h). Similar patterns of angiogenesis defects were observed when either Sur_UTR or Sur_ATG morpholinos were injected (data not shown).

As a member of the IAP family, survivin has been shown to inhibit apoptosis by regulating caspase activity 12. Therefore; we investigated if there was increased apoptosis in the Sur_UTR+ATG^mo embryos as measured by TUNEL assay. At both 24 and 48 hpf, increased TUNEL staining was detected in the developing neural tube and the brain (not shown), with significant, albeit weaker, staining at the vicinity of the axial vasculature (Figure 3i,j). The increased apoptosis was further confirmed by specific caspase-3 activity which was significantly increased in 48 hpf Sur_UTR+ATG^mo embryos (299.1 ± 8.3 arbitrary units) compared with control embryos injected with a random sequence MO at 6 ng (103.0 ± 2.3 arbitrary units, n = 3 experiments using 240 embryos, p < 0.05).

To further demonstrate the efficacy of Sur_UTR and Sur_ATG MO binding to survivin mRNA, embryos were co-injected with a 5'UTR survivin:GFP plasmid (50 pg) and Sur_UTR+ATG-MOs (3 ng each). Injecting the plasmid alone lead to GFP expression in 79.7 ± 9.4% embryos (Figure 4a,c). Co-injection of the plasmid with Sur_UTR+ATG-MOs completely abolished protein translation and hence GFP expression in all embryos tested (Figure 4b,d). A splice site MO (Sur_SS-MO (12 ng)) not only induced similar morphological changes as in Sur_UTR+ATG^mo embryos (smaller head and eye size and mildly curved tail) but also induced defective angiogenesis as shown in Tg(fli1:EGFP)^y1 embryos (61.7%, n = 3 experiments using 159 embryos) (Figure 4e–f). Angiogenesis defects were seen in ISV as well as OV/IOC of the developing eyes (not shown). A relatively high dose of MO (12 ng) was used as lower doses produced less phenotypic penetrance and at 12 ng, there was no excessive mortality. In the Sur_SS^MO embryos, RT-PCR confirmed defective splicing of part of the intron, as shown by a larger PCR transcript which was verified by bi-directional DNA sequencing (Figure 4g,h). Whether defective splicing could be induced by lower doses of this MO has not been examined. Finally, defective sprouting or failure to form the DLAV occurred in all Sur_UTR+ATG^mo embryos and co-injecting survivin mRNA (30 pg) with Sur_UTR+ATG-MOs rescued the vascular defect in 47 out of 58 embryos in three separate experiments (81%) (Figure 4i–l).

VEGF plays an important role in angiogenesis during zebrafish embryonic development 8. In-vitro studies have shown that survivin mediates the proliferative and anti-apoptotic effects of VEGF in endothelial cells 9. Therefore; we investigated if survivin expression during embryogenesis is regulated by VEGF. Exogenous human VEGF protein (2 ng) was injected into zebrafish embryos at one-cell stage 10. Angiogenesis was examined in the sub-intestinal vessels at 96 hpf, where the vasculature was well-developed and any ectopic structures could be readily detectable. In 78 out of 110 embryos (70%) (from three separate experiments), VEGF induces ectopic angiogenesis which was associated with a significant up-regulation of survivin mRNA expression (Figure 5a–b,f). We also incubated embryos with a VEGF receptor inhibitor (VEGFTKR) at one-cell stage. VEGFTKR (25 μmol/L) induced defective angiogenesis in all treated embryos at 48 hpf (Figure 5c–d) and could not be rescued by survivin mRNA injection (30 pg) (Figure 5e). Survivin mRNA expression was significantly down-regulated in these embryos (Figure 5f).

Wild-type zebrafish (Danio rerio) were obtained from local aquarium and were maintained and raised under standard conditions at 28°C. Transgenic Tg(fli1:EGFP)^y1 embryos were used to track endothelial cell populations. Anti-sense morpholinos (MO) (Gene-Tools, OR, USA) were designed to target the 5'untranslated region (UTR) or sequences flanking and including the initiation codon (ATG) of the zebrafish survivin gene. A splice-site (SS) MO was designed to target the exon2-intron junction of the survivin gene (Sur_SS-MO). A random sequence MO was used as a control as described previously (Table 1). Procedures for micro-injection, whole mount in-situ hybridization, microangiography, TUNEL and caspase-3 activity assays have been described previously 72526.

Wild-type (WT) embryos at 24 hpf were fixed with 4% paraformaldehyde (PFA) and dehydrated. After stepwise re-hydration, the embryos were incubated in pre-hybridization buffer (50% formamide, 5 × SSC, 50 μg/ml heparin, 0.1% Tween20, 5 mg/ml rRNA in phosphate-buffered saline, PBS) at 65°C followed by overnight incubation with digoxigenin (DIG)- (either flk-1 or survivin-1) and fluorescein-labeled riboprobe (α-embryonic globin) at 65°C. The embryos were washed and incubated with alkaline phosphatase (AP) conjugated anti-DIG antibody (Roche Molecular Biochemicals, Mannheim, Germany) for overnight at 4°C. Blue color was developed using NBT/BCIP (Roche Molecular Biochemicals, Mannheim, Germany) as substrate and the reaction was stopped with 0.5 mM EDTA in PBT. AP was destroyed by washing the stained embryos with 0.1 M glycine-HCl, pH 2.2 in PBT for 10 min twice. Background staining was removed by washing the embryos in absolute ethanol with continuous monitoring. After re-hydration to PBT, embryos were incubated with AP conjugated anti-fluorescein antibody (Roche Molecular Biochemicals, Mannheim, Germany) for overnight at 4°C and red color was developed using INT/BCIP (Roche Molecular Biochemicals, Mannheim, Germany) as substrate.

The full length sequence of zebrafish survivin including the 3' UTR was amplified by PCR (Table 1) from cDNA of 24 hpf embryos and subcloned into pGem-T vector (pGEM-T Vector Systems, Promega, Madison, WI, USA). A 623 bp anti-sense survivin mRNA riboprobe was synthesized from linearized vector containing the insert. A digoxigenin labeled mRNA probe was synthesized by SP6 RNA polymerase according the manufacturer's protocols (Roche Applied Science, Indianapolis, IN, USA). The size and integrity of the synthesized riboprobe was confirmed by RNA formaldehyde gel electrophoresis. Histological assessment of stained embryos was performed on 5–7-μm paraffin sections.

The 5'UTR of survivin, including the target sequences of Sur_UTR-MO and Sur_ATG-MO, were amplified from 24 hpf wild-type embryo cDNA (Table 1). PCR products were gel purified and cloned in frame and immediately upstream of the GFP coding sequence into vector pcDNA3.1/CT-GFP-TOPO (Invitrogen, Carlsbad, CA, USA) and transformed into chemically competent E. coli TOP10 (Invitrogen, Carlsbad, CA, USA). Plasmids containing the 5'UTR-survivin:GFP fusion sequence were isolated and the sequence of the DNA inserts verified using the GFP reverse primer (5'-GGG TAA GCT TTC CGT ATG TAG C-3').

The complete coding sequence of survivin was TA-cloned into pGEM-T vector (pGEM-T Vector Systems, Promega, Madison, WI, USA) and the orientation of the insert confirmed by PCR (Table 1). mRNA transcripts were synthesized from the T7 promoter of the Sal I digested pGEMT-Sur sequence using the mMessage mMachine Kit (Ambion, Austin, TX, USA).

Embryos were treated with an inhibitor of vascular endothelial growth factor receptor tyrosine kinase (VEGFRTK inhibitors, Calbiochem, EMD Bioscience, CA, USA). The embryos were incubated in inhibitor solution at 25 μmol/L (stock solution in DMSO at 10 mmol/L) from one-cell stage onwards. They were dechorionated at 24 hpf with continuous exposure to inhibitors until 48 hpf. Control experiments were set up from the same clutches of embryos and were exposed to equal volume of DMSO for comparison.

Human VEGF protein (BD Bioscience, Bedford, MA, USA) was prepared in 1 mg/mL in water. Embryos at 1–4 cell stage were injected with VEGF (2 ng) into the yolk sac and its effect on angiogenesis was examined at 96 hpf.

cDNA from 48 and 96 hpf embryos were reverse transcribed from RNA and Q-PCR for survivin was performed using the SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA). Expression level was presented as fold-change calculated using the comparative C_T method as described 27 with β-actin as the internal reference. Primer sequences for Q-PCR were shown in Table 1.

Results were expressed as mean ± SEM unless otherwise stated. Comparisons between groups of data were evaluated by Mann-Whitney U and Kruskal-Wallis Test where appropriate. P-value of less than 0.05 was considered statistically significant.