Limits...
The role of survivin in angiogenesis during zebrafish embryonic development.

Ma ACh, Lin R, Chan PK, Leung JC, Chan LY, Meng A, Verfaillie CM, Liang R, Leung AY - BMC Dev. Biol. (2007)

Bottom Line: Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene.Efficacy of SurUTR and SurATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids.The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Hong Kong, Hong Kong. h0025231@hkusua.hku.hk <h0025231@hkusua.hku.hk>

ABSTRACT

Background: Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (SurUTR) and sequences flanking the initiation codon (SurATG) of zebrafish survivin-1 gene were injected into embryos at 1-4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:EGFP)y1 embryos.

Results: In embryos co-injected with SurUTR and SurATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of SurUTR and SurATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression.

Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.

Show MeSH

Related in: MedlinePlus

Regulation of survivin expression by vascular endothelial growth factor (VEGF) at 96 hpf. (a): Sub-intestinal vessels in uninjected Tg(fli1:EGFP)y1 embryos. (b): Injection of human VEGF (2 ng) gave rise to ectopic angiogenesis (arrows). There was no observable ectopic angiogenesis in the ISV (c): Axial and inter-segmental vessels in untreated Tg(fli1:EGFP)y1 embryos. (d): Tg(fli1:EGFP)y1 embryos treated with VEGF tyrosine kinase receptor inhibitor (VEGFTKRI, 25 μmol/L) showing defective sprouting of inter-segmental vessels. (e): Injection of survivin mRNA did not reverse the defects seen in VEGFTKRI treated embryos. (f): Histogram showing the average survivin mRNA expression (expressed in fold-change) in untreated and VEGFTKRI treated embryos as well as in embryos injected with human VEGF. Results expressed in mean ± S.E.M. (n = 3 experiments using at least 20 embryos per experiments). When the three groups of data were compared using Kruskal-Wallis Test, p-value = 0.016. When the data of uninjected vs VEGFTKRI treated embryos were compared using Mann-Whitney U Test, p-value = 0.037.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1884147&req=5

Figure 5: Regulation of survivin expression by vascular endothelial growth factor (VEGF) at 96 hpf. (a): Sub-intestinal vessels in uninjected Tg(fli1:EGFP)y1 embryos. (b): Injection of human VEGF (2 ng) gave rise to ectopic angiogenesis (arrows). There was no observable ectopic angiogenesis in the ISV (c): Axial and inter-segmental vessels in untreated Tg(fli1:EGFP)y1 embryos. (d): Tg(fli1:EGFP)y1 embryos treated with VEGF tyrosine kinase receptor inhibitor (VEGFTKRI, 25 μmol/L) showing defective sprouting of inter-segmental vessels. (e): Injection of survivin mRNA did not reverse the defects seen in VEGFTKRI treated embryos. (f): Histogram showing the average survivin mRNA expression (expressed in fold-change) in untreated and VEGFTKRI treated embryos as well as in embryos injected with human VEGF. Results expressed in mean ± S.E.M. (n = 3 experiments using at least 20 embryos per experiments). When the three groups of data were compared using Kruskal-Wallis Test, p-value = 0.016. When the data of uninjected vs VEGFTKRI treated embryos were compared using Mann-Whitney U Test, p-value = 0.037.

Mentions: 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).


The role of survivin in angiogenesis during zebrafish embryonic development.

Ma ACh, Lin R, Chan PK, Leung JC, Chan LY, Meng A, Verfaillie CM, Liang R, Leung AY - BMC Dev. Biol. (2007)

Regulation of survivin expression by vascular endothelial growth factor (VEGF) at 96 hpf. (a): Sub-intestinal vessels in uninjected Tg(fli1:EGFP)y1 embryos. (b): Injection of human VEGF (2 ng) gave rise to ectopic angiogenesis (arrows). There was no observable ectopic angiogenesis in the ISV (c): Axial and inter-segmental vessels in untreated Tg(fli1:EGFP)y1 embryos. (d): Tg(fli1:EGFP)y1 embryos treated with VEGF tyrosine kinase receptor inhibitor (VEGFTKRI, 25 μmol/L) showing defective sprouting of inter-segmental vessels. (e): Injection of survivin mRNA did not reverse the defects seen in VEGFTKRI treated embryos. (f): Histogram showing the average survivin mRNA expression (expressed in fold-change) in untreated and VEGFTKRI treated embryos as well as in embryos injected with human VEGF. Results expressed in mean ± S.E.M. (n = 3 experiments using at least 20 embryos per experiments). When the three groups of data were compared using Kruskal-Wallis Test, p-value = 0.016. When the data of uninjected vs VEGFTKRI treated embryos were compared using Mann-Whitney U Test, p-value = 0.037.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1884147&req=5

Figure 5: Regulation of survivin expression by vascular endothelial growth factor (VEGF) at 96 hpf. (a): Sub-intestinal vessels in uninjected Tg(fli1:EGFP)y1 embryos. (b): Injection of human VEGF (2 ng) gave rise to ectopic angiogenesis (arrows). There was no observable ectopic angiogenesis in the ISV (c): Axial and inter-segmental vessels in untreated Tg(fli1:EGFP)y1 embryos. (d): Tg(fli1:EGFP)y1 embryos treated with VEGF tyrosine kinase receptor inhibitor (VEGFTKRI, 25 μmol/L) showing defective sprouting of inter-segmental vessels. (e): Injection of survivin mRNA did not reverse the defects seen in VEGFTKRI treated embryos. (f): Histogram showing the average survivin mRNA expression (expressed in fold-change) in untreated and VEGFTKRI treated embryos as well as in embryos injected with human VEGF. Results expressed in mean ± S.E.M. (n = 3 experiments using at least 20 embryos per experiments). When the three groups of data were compared using Kruskal-Wallis Test, p-value = 0.016. When the data of uninjected vs VEGFTKRI treated embryos were compared using Mann-Whitney U Test, p-value = 0.037.
Mentions: 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).

Bottom Line: Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene.Efficacy of SurUTR and SurATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids.The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Hong Kong, Hong Kong. h0025231@hkusua.hku.hk <h0025231@hkusua.hku.hk>

ABSTRACT

Background: Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (SurUTR) and sequences flanking the initiation codon (SurATG) of zebrafish survivin-1 gene were injected into embryos at 1-4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:EGFP)y1 embryos.

Results: In embryos co-injected with SurUTR and SurATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of SurUTR and SurATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression.

Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.

Show MeSH
Related in: MedlinePlus