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Ras guanyl nucleotide-releasing protein 2 (RASGRP2), one of the Ras guanine exchange factors, is implicated as a critical regulator of inside-out integrin activation in human lymphocytes, neutrophils and platelets. However, the activities of this protein in endothelial cells remain unclear. In the current study, we identify a physiological function in blood vessel formation for XRASGRP2, which is the Xenopus ortholog of mammalian RASGRP2. XRASGRP2 over-expression induced ectopic vascular formation, and XRASGRP2-knockdown embryos showed delayed vascular development. We also investigated the upstream signaling of XRASGRP2 in endothelium formation. XRASGRP2 expression was up-regulated in the presence of VEGF-A and down-regulated following VEGF-A depletion. XRASGRP2 knockdown abolished the ectopic induction of endothelial cells by VEGF-A in the posterior ventral blood island. These results suggest that XRASGRP2 is essential for vascular formation during Xenopus development.
Fig. 1 (Left). XRASGRP expression precedes Ami expression. (A) XRASGRP2 transcripts localized in the anterior cardinal vein (ACV), aortic arch (AA), intersomitic veins (ISV), posterior cardinal veins (PCV), and vascular vitelline network (VVN) at stage 30. (B) XRASGRP2 expression is detected in the PCV and VVN at stage 35. Lines indicate the positions of the sections shown in (G-J). (C) At stage 40, XRASGRP2 expression is restricted to the PCV and VVN. (D) The expression of Ami is weakly detected in the ACV, AA, and VVN at stage 30. (E) Ami expression is evident in the ACV, AA, ISV, PCV, and VVN at stage 35. Lines indicate the positions of the sections shown in (K-N). (F) Ami expression is detected continuously in the PCV, ISV, and VVN until stage 40. (G-J) Histologic section of the embryo shown in (B). (K-N) Histologic section of the embryo shown in (E). Both XRASGRP2 and Ami are expressed in the endothelial cells (PCV and VVN).
Fig. 2 (Right). Ectopic expression of XRASGRP2 affects vascular formation and induces edema. (A) An uninjected control embryo at stage 43. (B) An embryo in which 1 ng of XRASGRP2 mRNA was injected into the dorsal vegetal blastomeres (DV) at the 8-cell stage. The embryo shows edema. (C-F) Whole-mount in situ hybridization for a hematopoietic marker, globin T3, and an endothelial marker, Xmsr, at stage 31. (C) The expression of globin T3 in an uninjected control embryo (ventral view). (D) Expression of globin T3 in an embryo that was co-injected with XRASGRP2 and β- galactosidase (β-gal) into the ventral vegetal blastomeres (VV) at the 8-cell stage. The expression of globin T3 is abolished at the injection site in the VBI. (E) The expression of Xmsr in an uninjected control embryo (ventral view). (F) The expression of Xmsr in an embryo that was co-injected with XRASGRP2 and β-gal into the VV. Ectopic expression of Xmsr is evident at the injection site in the VBI. Arrowheads indicate Xmsr-positive cells.
Fig. 3. XRASGRP2 depletion results in aberrant development of blood vessels. (A) Schematic model for the splice inhibition antisense morpholino oligo- nucleotides (S-MOs). The binding site of MO is represented by a bolded blue line. Arrows indicate the primers used in the RT-PCR to examine the efficacies of the S-MOs. (B) The control MO (c, 40 ng), aS- MO (aS, 40 ng), bS-MO (bS, 40 ng), and S- MO (S, 40 ng, comprising 20 ng aS-MO plus 20 ng bS-MO) were injected into 2- cell-stage embryos, and the embryos were analyzed by RT-PCR at stage 30. The presence of the 312-bp band indicates amplification of the normally spliced mRNA. The intensity of this band is re- duced in both the aS-MO-injected and bS- MO-injected embryos, as compared to the uninjected embryos and control MO- injected embryos, and this band is not detected for the S-MO-injected embryos. This indicates that the S-MO-injected embryos do not produce a functional XRASGRP2 protein. Sample without reverse transcriptase; n uninjected embryos. (C-F) Expression patterns of blood vessel marker genes. The 2-cell-stage embryos were injected with the control MO (40 ng) or S-MO (40 ng) into one blastomere (corresponding to the future right-hand side), and harvested at stage 31. The injected sides are indicated as [Inj(+)] and the uninjected sides are indi- cated as [Inj(-)]. The expression levels of Xflk-1 in the PCV (C, red arrows) and of Xmsr in the ISV (D, black arrows) are diminished in the S-MO-injected side. The expression levels of Xtie2 (E) and Ami (F) in the PCV (red arrows) and VVN (red arrowheads) are diminished in the S-MO-injected side. The expression level of Ami (F) is greatly reduced in the S-MO-in- jected side. No differences are seen in the control MO-injected embryos. (G) The expression of Ami in VVN is gradually mitigated in the S-MO-injected side. (H) Expression of globin T3 in the control MO-injected embryos. S-MO injection does not affect the level of globin T3 expression.
Fig. 4. VEGF-A up-regulates XRASGRP2 expression. Embryos were injected with either 1 ng of VEGF-A mRNA or 20 ng of VEGF-A-MO into the two dorsal-ventral blastomeres (DV) or the two ventral- vegetal blastomeres (VV), together with 200 pg of β-gal mRNA, at the 8-cell-stage. These embryos were prepared for whole-mount in situ hybridization of XRASGRP2 at stage 32. (A-E) Lateral view. (F-I) Ventral view. (A) An uninjected embryo. (B,G) An embryo in which VEGF-A mRNA was injected into the DV. (C). An embryo in which VEGF-A mRNA was injected into the VV. (D,I) An embryo in which VEGF-A-MO was injected into the DV. (E) An embryo in which VEGF- A-MO was injected into the VV. (F) An uninjected embryo. (H) An embryo in which the control MO (20 ng) was injected into the DV. Black arrows indicate inhibition of XRASGRP2 ex- pression in the VVN (D,E) and VBI (I). Black arrowheads indicate ectopic expression of XRASGRP2 in the VVN (B,C) and VBI (G).
Fig. 5. RasGRP2 mediates VEGF-A signaling. Embryos were injected with 200 pg of β-gal mRNA (A,D), 1 ng of VEGF-A mRNA (B,E) or 1 ng of VEGF-A mRNA plus 40 ng of XRASGRP2 S-MO (C,F) into two ventral-vegetal blastomeres at the 8-cell stage. The embryos were cultured until stage 31, for whole-mount in situ hybridization analysis. (A-C) Expression patterns of globin T3. (D-F) Expression patterns of Xmsr. VEGF-A inhibits globin T3 expression in the VBI. Higher-magnification images showing the Xmsr expression patterns in the VBI region are shown (lower panels). (B) VEGF-A-mediated suppression of globin T3 expression is partially rescued by co-injection of the XRASGRP2 S-MO (C). VEGF-A induces ectopic Xmsr expression in the VBI (E). VEGF-A-induced ectopic expression of Xmsr is partially rescued by co-injection of the XRASGRP2 S-MO (F).
Xenopus laevis rasgrp2-b expression in stage 30 embryo
