Du Pasquier D et al. (2006), Survivin increased vascular development during ...

XB-ART-252

Differentiation 2006 Jun 01;745:244-53. doi: 10.1111/j.1432-0436.2006.00073.x.

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Survivin increased vascular development during Xenopus ontogenesis.

Du Pasquier D , Phung AC , Ymlahi-Ouazzani Q , Sinzelle L , Ballagny C , Bronchain O , Du Pasquier L , Mazabraud A .

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Survivin is a member of the inhibitor of apoptosis proteins (IAP) family. These proteins contain one to three zinc-binding motifs termed bacculoviral IAP-binding repeats (BIRs). Survivin contains a single BIR motif. Contrary to other members that directly interact with caspases and inhibit apoptosis, Survivin is believed to have both antiapoptotic and proliferative functions. In mammals, Survivin is not detected in most adult tissues except in endothelial cells of newly formed capillaries and large blood vessels. Importantly, Survivin is highly expressed in all common human cancers. To gain a better view of Survivin expression and function during development, we used the amphibian Xenopus developmental model. We show that the genomes of X. laevis, X. tropicalis, Zebrafish, fugu pufferfish, and rainbow trout encode two different Survivin genes (Su1 and Su2), contrary to mammalian genomes, which encode a single one. In X. laevis, these two genes have a differential spatiotemporal transcription pattern. Transgenic expression of Su1 leads to an enlargement of tadpole's blood vessels with an increase in the number of endothelial cells. This effect requires a functional BIR domain and the p34/cdc2 phosphorylation site. It does not seem to rely on the antiapoptotic activity of Su1 as it is not observed in tadpoles overexpressing other antiapoptotic factors such as XIAP or BclXL. We conclude that Su1 ubiquitous gain of function leads directly or indirectly to an increase in blood vessels size via the proliferation of endothelial cells.

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Species referenced: Xenopus
Genes referenced: aagab bcl2l1 birc5 birc5l cdk1 uqcc6 xiap

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	Fig. 1 Multiple sequence alignment of Xenopus and human Survivin. The bacculoviral IAPbinding repeats domain is highly conserved between these proteins (arrow) as well as the p34/cdc2 phosphorylation site (arrowhead).
	Fig. 2 Evolutionary relationships of vertebrates Survivin 1, Survivin 2, and XIAP families of bacculoviral IAP binding repeats (BIR)-containing proteins. In this rooted neighbor-joining tree, only the BIR domain was used for comparison. XIAP BIR3 is used as an outgroup. The different families are shaded. Hs, Homo sapiens; Gg, Gallus gallus; Cf, Canis familiaris; Rn, Rattus norvegicus; Ss, Sus scrofa; Xl, Xenopus laevis; Xt, Xenopus tropicalis; Dr, Danios rerio; Ci, Ciona intestinalis; Tr, Takifugu rubripes; Om, Oncorhynchus mykiss; Bt, Bow taurus; Mm, Mus musculus.
	Fig. 3 Expression levels of Survivin 1 and Survivin 2 (Su1 and Su2) transcripts in Xenopus. RT-PCR analysis were performed to detect Su1 and Su2 mRNA. (A) Stages 1â€“35 embryos (Niewkoop and Faber, 1994). (B) Premetamorphic (stage 56) and metamorphic tissues (stages 61â€“63). (C) Premetamorphic (stage 56), metamorphic tissues (stage 63), and adult tissues. (D) Several adult tissues. (E) Premetamorphic (stage 56) and metamorphic tissues (stages 62). L8 ribosomal protein (rpl8) or Ornithine Decarboxylase (odc) transcripts were used to normalize amplification products. Ad, adult; Te, testis; Br, brain; Lu, lung; Bl, blood; In, intestine; Ov, ovary; Sk, skin; St, stomach; Li, liver; Mu, muscle; D. Mu, dorsal muscle; C. Mu, caudal muscle; D. Ma, dorsal marrow; C. Ma, caudal marrow.
	Fig. 4 Dissymmetric vascular system of half-sided CMVâ€“GFPSu1 transgenic tadpoles. (A) Ventral view of a half-sided CMVâ€“GFP transgenic tadpole under UV light showing GFP expression pattern restricted to the left side of the animal. Arrowhead shows the limit of the GFP signal. (B) Ventral view of the same animal under bright light showing symmetric aortic crosses (arrowheads). (C) Half-sided CMVâ€“Su1GFP heart (H) under UV light, GFP expression is restricted to the left side of the animal (arrowhead). (D) Dissymmetric aortic crosses (arrowheads) of the same animal observed under bright light. (E) Dorsal view of a half-sided CMVâ€“Su1GFP tadpole (black boxes are enlarged in (G) and (H)). (F) Ventral view of the kidney (K) of the same transgenic showing the dissymmetric kidney arteries (arrowheads). Dorsal views of the eye vasculatures on the transgenic side (G) or wild-type side (H) showing the increased vessel size in the transgenic side. Scale bars : 0.4 cm in (A, B, E); 0.14 cm in (C, D, F); 0.1 cm in (G, H).
	Fig. 5 Survivin 1 (Su1) transgenic expression increases aortic cross diameter. (A) Ventral view of a full CMVâ€“Su1GFP transgenic tadpole. Black arrow: distance between the eyes, Black arrowheads: positions where the aortic crosses diameters were measured, scale bar50.2 cm. The white arrowheads indicate the head arteries (shown in Fig. 6). (B) Relative aortic crosses diameter of wild-type (WT) or transgenic tadpoles. All transgenics were ubiquitous (CMV promoter) and expressed: GFP, Su1â€“GFP, Su1â€“T34Aâ€“GFP (unphosphorylable form), Su1â€“T34Eâ€“GFP (phosphomimetic mutation), Su1â€“C84Aâ€“GFP (bacculoviral IAP binding repeats loss of function mutation), XIAPâ€“GFP, XR11â€“GFP. Aortic crosses diameters were measured and normalized relative to the distance between the tadpoleâ€™s eyes (n430 for each group). po0,01 or po0,001.
	Fig. 6 Survivin 1 transgenic expression increases the number of endothelial cells in blood vessel walls. (A, B) Pictures of a crosssection of a transgenic tadpole showing the position of the head arteries (arrows in (B), black box in (A) enlarged in (B) (scale bar50.15 cm in (A), 0.02 cm in (B))). (C) Picture of head artery at a higher magnification (scale bar50.003 cm). The arrow indicates an endothelial cell nucleus. (D) Chart showing the mean numbers of wall cell nuclei in 100 mm tissue width, counted on 30 serial crosssections of five transgenics or wild-type animals.
	Fig. 7 Detection of Survivin 1 mRNA by in situ hybridization. In situ hybridization was performed on a tail cross-section with a fulllength cDNA as an antisense probe ((B) enlarged in (D)). Hybridization signal is detected in the main tail artery. No signal is detected in this artery with a Sense probe hybridization, performed as a control ((A) enlarged in (C)).