XB-ART-39761Dev Biol. May 15, 2009; 329 (2): 269-79.
Rasip1 is required for endothelial cell motility, angiogenesis and vessel formation.
Ras proteins are small GTPases that regulate cellular growth and differentiation. Components of the Ras signaling pathway have been shown to be important during embryonic vasculogenesis and angiogenesis. Here, we report that Rasip1, which encodes a novel Ras-interacting protein, is strongly expressed in vascular endothelial cells throughout development, in both mouse and frog. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial specific. siRNA-mediated reduction of Rasip1 severely impairs angiogenesis and motility in endothelial cell cultures, and morpholino knockdown experiments in frog embryos demonstrate that Rasip1 is required for embryonic vessel formation in vivo. Together, these data identify Rasip1 as a novel endothelial factor that plays an essential role in vascular development.
PubMed ID: 19272373
PMC ID: PMC2683470
Article link: Dev Biol.
Genes referenced: aplnr cad cald1 cdh5 erg kdr pecam1 rasip1
Morpholinos referenced: rasip1 MO1
Article Images: [+] show captions
|Fig. 5. Rasip1 knockdown in frog embryos results in failure of blood vessel formation. Expression of Xenopus tropicalis Rasip1 transcripts in frog embryos by in situ hybridization marks the developing embryonic blood vessels (in all panels anterior is to the left). (A–D) Vascular markers reveal the embryonic vasculature at stages indicated, including angioblasts (white arrowheads in A, B) and developing blood vessels, such as the posterior cardinal vein (black arrow, B–D). (A) vegfr2; (B) erg, vascular ETS factor; (C) ve-cad, vascular endothelial cadherin; D) msr, vascular G-protein coupled receptor. (E–H) Rasip1 initially marks scattered angioblasts (E, st.25), but progressively marks aggregating flank vessels (F, st.28). (G) As vessels form, Rasip1 marks all embryonic frog vessels examined, including the flank plexus (red arrow), endocardium (red arrowhead), cardinal veins (black arrow) and ISVs (black arrowheads). (H) Expression of Rasip1 declines slightly in vessels as they mature (including data not shown). (I) Schematic of microinjection of Rasip1 morpholino injections into blastomeres of early cleavage stage embryo. (J) Semi-quantitative RT-PCR shows the knockdown of Rasip1 transcript expression in radially injected embryos (24 cycles). (K–N) msr in situ hybridization of injected embryo, outlining vasculature on either uninjected (K, M) or injected (L, N) sides. (K, M) Uninjected side of embryo displays major blood vessels, including the prominent cardinal vein (black arrow) and ISVs. (M) Higher magnification of embryo in panel K, showing ISVs sprouting from the cardinal vein on the uninjected side (black arrowheads). (L, N) MO-injected side of embryos shows a severe reduction in vascular structures, including absence of cardinal vein (yellow arrow) and reduced plexus vessels (thick yellow arrow). (N) Higher magnification of embryo in panel L, showing complete absence of both ISVs (white arrowhead) and cardinal vein (yellow arrow). Note that faint outline of ISVs in panel N results from ISVs from uninjected side appearing through the dorsal fin tissue (white arrowhead). (O) Transverse sections through injected embryos show that cardinal veins and ISVs are lost on the injected side (arrowheads) but not the uninjected side (arrows). (P) Quantification of observations in K–O. Total number of ISVs and flank plexus branch points were counted from the injected versus uninjected sides of MO-injected embryos. Y axis for ISVs is to left; Y axis for flank branch points is to right. The scale bars represent 250 μm (A–H), 100 μm (K, L), and 50 μm (M–O), respectively.|
|Fig. 5. Rasip1 expression at stg 32|
|Fig. S8. Schematic of morpholino injection into X. tropicalis and summary of resulting vascular disruptions. A) Rasip1-MO and Alexa488 lineage tracer are co-injected at 8 ng/cell into two blastomeres of 4–8 cell stage embryos. Embryos are allowed to develop to desired stage: st.20–21 to assess angioblast specification, st.25–27 to assess vasculogenesis in progress, or st.33–35 to assess vessels following formation. MO injection is followed by Alexa488 fluorescence (B, C). Once fixed at desired stage, embryos are analyzed by whole mount in situ hybridization (WISH) for the vascular markers msr (D, E), flk1 (F, G), erg (H, I) or VE-cadherin (J, K).|