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Previously, we described the DNA microarray screening of vascular endothelial cells that were formed by treatment of aggregates prepared from Xenopus animal cap cells with activin and angiopoietin-2. One of the genes identified in this screening showed homology to human RASGRP2 which plays a role in the regulation of GTP-GDP exchange of the Ras and Rap proteins, and was named XRASGRP2. In the present study, we analyzed the expression pattern of xrasgrp2 during Xenopus embryogenesis. The xrasgrp2 mRNA was expressed after stage 24, as assessed by stage PCR analysis. Whole-mount in situ hybridization showed that xrasgrp2 mRNA was located in the vascular region of the embryo. Loss-of-function analysis revealed that the formation of blood and endothelial cells in the explants transplanted into Xenopus embryos was inhibited by antisense morpholino oligonucleotides that block xrasgrp2 translation. These results suggest that XRASGRP2 plays a role in angiogenesis in Xenopus embryos.
Deduced amino acid sequence of XRASGRP2. Regions 1, 2, 3, and 4 indicate the N-terminal Ras-GEF domain, Ras interaction domain, Ca2+-binding site, and diacylglycerol-binding site, respectively.
Phylogenetic relationships among the RASGRP proteins. The evolutionary tree is calculated by the neighbor-joining method . The evolutionary distance is shown as the total of the branch lengths (horizontal lines). D, dog; H, human; P, chimpanzee; M, mouse; R, rat; C, cattle; G, chick; Z, zebrafish; X, Xenopus.
Expression patterns of the xrasgrp2 mRNA. (A) Temporal expression pattern of xrasgrp2 mRNA assessed by RT-PCR. ODC expression serves as a quantitative control. (B) Spatial expression patterns of xrasgrp2 mRNA (upper panel) and X-msr mRNA (lower panel). Whole-mount in situ hybridization was performed using stage 35 embryos. ISV, intersomitic vein; PCV, posterior cardinal vein; AA, aortic arch; VVN, vascular vitelline network. (C) Sections of the transplants were hybridized with DIG-labeled xrasgrp2 probes, which were visualized with NBT/BCIP using the Ventana Discovery system. The right panel shows a higher magnification of the transplanted region. V and D indicate ventral axis and dorsal axis, respectively. Arrowheads indicate blood vessels.
Loss-of-function analysis of XRASGRP2. The aggregates derived from eggs injected with the antisense morpholino oligonucleotides (MO or 5miss MO) were transplanted into the lateral regions of the host embryos. MO inhibits xrasgrp2 translation but 5miss MO does not. Arrows indicate the locations of the explants.