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Impaired vascular development in the yolk sac and allantois in mice lacking RA-GEF-1
Biochemical and Biophysical Research Communications 387 (2009) 754–759
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Impaired vascular development in the yolk sac and allantois in mice lacking RA-GEF-1 Hoshimi Kanemura a, Takaya Satoh a, Shymaa E. Bilasy a, Shuji Ueda a, Masanori Hirashima b, Tohru Kataoka a,* a b
Division of Molecular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Division of Vascular Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
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Article history: Received 14 July 2009 Available online 25 July 2009 Keywords: Allantois Angiogenesis RA-GEF-1 Rap1 Vascular plexus Yolk sac
a b s t r a c t RA-GEF-1 is a guanine nucleotide exchange factor for the small GTPase Rap1. RA-GEF-1 knockout mice show defects in vascular development starting around 7.5 days post coitum and die by 9.5 days post coitum. Here, we employed in vitro culture systems for allantois explants and endothelial cells to gain insights into the mechanism for RA-GEF-1-mediated regulation of embryonic vascular network formation. The development of the vascular plexus and the accumulation of VE-cadherin at cell–cell junctions were significantly impaired in the RA-GEF-1 knockout allantois and yolk sac. Rap1 activation as visualized by an activation-specific probe was also diminished by RA-GEF-1 knockout. Reduced accumulation of VEcadherin at cell–cell junctions and defects in blood vessel formation in vitro due to the lack of RA-GEF-1 were suppressed by ectopic expression of constitutively activated Rap1. Overall, these results suggest the involvement of Rap1 downstream of RA-GEF-1 in the regulation of vascular network formation in mouse embryos. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Blood vessel formation is a crucial process during embryonic development [1]. Mesoderm-derived angiogenic progenitor cells (angioblasts) migrate to the site of vascularization, differentiate into endothelial cells, and then coalesce to form the primary vascular plexus. This initial process is denoted vasculogenesis. Following this, new capillaries are generated from pre-existing blood vessels through sprouting or intussusceptive angiogenesis. The vascular endothelial growth factor receptor Flk-1 and its ligands are intimately involved in differentiation of haemangioblasts into angioblasts and subsequent vascular plexus formation. Various vascular endothelial growth factor receptors are also responsible for the regulation of angiogenesis. However, signaling mechanisms underlying vascular development during mouse embryogenesis remain largely unknown. The small GTPase Rap1 regulates diverse cellular responses such as proliferation, adhesion, and exocytosis in response to extracellular stimuli [2]. The activity of Rap1 is regulated through the GDP-GTP cycle, which is positively and negatively regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins, respectively. RA-GEF-1 (also called PDZ-GEF1, nRapGEP, and CNRasGEF) [2–4] and its close relative RA-GEF-2 [5] are * Corresponding author. Fax: +81 78 382 5399. E-mail address: [email protected] (T. Kataoka). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.07.108
Rap-specific GEF unique in that they act both upstream and downstream of Ras family GTPases. The Ras/Rap-associating (RA) domains of RA-GEF-1 and RA-GEF-2 interact with Ras family GTPases Rap1 and M-Ras in their GTP-bound active forms. Though these interactions, Rap1 and M-Ras recruit RA-GEFs to the Golgi apparatus and the plasma membrane, respectively, in which RAGEFs activate Rap1 [4,5]. Thus, RA-GEFs serve as a link between Ras family GTPases in intracellular signaling cascades. Although their biochemical properties are similar, expression patterns of RA-GEF-1 and RA-GEF-2 are different from each other. RA-GEF-1 is ubiquitously expressed in mouse tissues whereas the expression level of RA-GEF-2 is particularly high in the spleen and thymus and weak or undetectable in other tissues [5,6]. Therefore, RA-GEF-1 and RA-GEF-2 are likely to be involved in distinct physiological responses. The function of RA-GEF-1 has been investigated through the generation of knockout mice [7]. Mice carrying a null mutation in both RA-GEF-1 alleles in the whole body appeared normal until 7.5 days post coitum (dpc), but died by 9.5 dpc. Severe defects in blood vessel formation in the RA-GEF-1-null yolk sac and embryo proper were observed, which may be a cause for embryonic lethality. However, the mechanism underlying the regulation of embryonic blood vessel formation by RA-GEF-1 remains totally unknown. In contrast to RA-GEF-1 knockout mice, RA-GEF-1 heterozygous mice showed normal phenotypes (Wei et al ., unpublished observations).
H. Kanemura et al. / Biochemical and Biophysical Research Communications 387 (2009) 754–759
Here, we investigate the role of RA-GEF-1 in embryonic vascular network formation by the use of an in vitro culture system of allantoises isolated from wild-type and RA-GEF-1 knockout embryos.
toises of 8.5 dpc embryos were excised, washed with ice-cold HBSS, and then transferred into a 4-well chamber slide (Lab-Tek, NY 14625, USA) containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 IU/ ml penicillin, and 50 lg/ml streptomycin. Explants were cultured for 24 h at 37 °C in an atmosphere of 5% CO2. In some experiments, allantoises at 8.5 dpc were incubated with lentiviral solutions in the presence of polybrene (8 lg/ml), washed with HBSS, and then embedded in collagen I-A (Nitta Gelatin, Tokyo, Japan) in an 8-well chamber slide. Following 24-h culture, pictures were taken using a microscope (BZ-8000, Keyence, Japan). Quantitative reverse transcription-PCR (RT-PCR) analysis. The total cellular RNA was prepared from allantoises or yolk sacs by using the Sepasol-RNA I Super kit (Nacalai tesque, Kyoto, Japan) and reverse-transcribed with the SuperScriptTM III First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR analysis was performed with the Thermal Cycler Dice Real Time system TP800 (Takara Bio, Otsu, Japan). Relative mRNA levels were determined by the comparative Ct method followed by normalization with the b-actin mRNA level in each cDNA sample. Primer sequences are as follows:
Materials and methods Mouse breeding and genotyping. RA-GEF-1 knockout mice were generated and maintained on the C57BL/6 and 129Ola mixed background as described previously [7]. To obtain RA-GEF-1/ embryos, female RA-GEF-1+/ mice were placed with a male RA-GEF1+/ mouse overnight, and the time of vaginal plug appearance was considered as 0.5 dpc. Tail tips, yolk sacs, and embryos were genotyped by polymerase chain reaction (PCR) analysis as described previously [7]. The use and care of animals were reviewed and approved by the Institutional Animal Care and Use Committee of Kobe University. Isolation and culture of allantoises. Allantois culture was performed essentially as described previously [8]. Pregnant females were killed by cervical dislocation at 8.5 dpc, and uterine horns were placed in ice-cold Hank’s balanced salt solution (HBSS). Allan-
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Fig. 1. Vascular network formation and VE-cadherin expression in the cultured allantois explant. (A) Allantoises were isolated from 8.5-dpc wild-type (+/+) and RA-GEF-1 knockout (/) mice and cultured in vitro for indicated times. Endothelial cells were visualized by immunofluorescent staining with an anti-Flk-1 antibody. Scale bars, 100 lm. (B) The length of blood vessels (upper) and the area of the vascular plexus (lower) in a single wild-type (+/+) or RA-GEF-1 knockout (/) allantois explant were measured following 24-h culture in vitro. Results are shown as the means ± SEM from 7 to 10 independent experiments. *1, p = 1.92 105; *2, p = 1.08 106. (C) Allantoises were isolated from 8.5-dpc RA-GEF-1 heterozygous (+/) and RA-GEF-1 knockout (/) mice, cultured in vitro for 24 h, and then stained with anti-VE-cadherin (green) and anti-Flk-1 (red) antibodies. Scale bars, 10 lm. (D) mRNA levels of VE-cadherin and Flk-1 in 24-h cultured wild-type(+/+), RA-GEF-1 heterozygous (+/), and RA-GEF-1 knockout (/) allantoises were determined by quantitative RT-PCR. The mRNA level of VE-cadherin relative to Flk-1 was shown as the means ± SEM from 8 to 9 independent experiments. *1, p = 1.78 102; *2, p = 1.82 102.
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b-actin sense, 50 -ATGAAGATCAAGATCATTGCTCCTC-30 ; b-actin antisense, 50 -ACATCTGCTGGAAGGTGGACAG-30 ; Flk-1 sense, 50 -GATGC AGGAAACTACACGGTCA0 -30 ; Flk-1 antisense, 50 -TCCATAGGCTGTTG GCTTCCACA-30 ; VE-Cadherin sense, 50 -CACTGCTTTGGGAGCCTT C-30 ; VE-Cadherin antisense, 50 -GGGGCAGCGATTCATTTTTCT-30 . Immunohistochemistry. Whole embryos were dissected at 8.5 dpc and fixed in 4% paraformaldehyde (PFA) overnight. Embryos were washed three times with phosphate-buffered saline containing 0.2% (v/v) Triton X-100 (PBST) for 30 min at 4 °C, blocked in PBST containing 1% bovine serum albumin (BSA) for 1 h at room temperature, and incubated with primary antibodies in PBST containing 1% BSA at 4 °C overnight. Embryos were washed three times with PBST for 30 min at 4 °C and twice for 30 min at room temperature, followed by incubation with secondary antibodies in PBST containing 1% BSA at 4 °C overnight. Subsequently, embryos were washed three times with PBST for 30 min at 4 °C and twice for 30 min at room temperature. Yolk sacs and allantoises were peeled off and flat-mounted on slide glasses. Cultured allantoises were fixed in 2% PFA for 20 min and permeabilized in phos-
phate-buffered saline (PBS()) containing 0.02% (v/v) Triton X-100 for 40 min. Fixed allantoises were blocked by 3% BSA in PBS() and immunolabeled with primary and secondary antibodies as described above. Antibodies against Flk-1 (clone AVAS 12a1, rat, BD Bioscience-Pharmingen, San Diego, CA; 1:50), PECAM-1 (clone MEC 13.3, rat, BD Bioscience-Pharmingen, San Diego, CA; 1:50), PECAM-1 (clone 2H8, Armenian hamster, Endogen; 1:1000), VEcadherin (C-19, goat, Santa Cruz Biotechnology; 1:50), VE-cadherin (clone 11D4.1, rat, BD Bioscience-Pharmingen, San Diego, CA; 1:1000), Rap1 (121, rabbit, Santa Cruz Biotechnology; 1:200), and the V5 epitope tag (Nacalai tesque, Kyoto, Japan; 1:1000) were commercially available. Secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Cy3, or Cy5 (Molecular Probes and Jackson ImmunoResearch) were used for the detection. Samples were analyzed by confocal laser scanning microscopy (LSM510 META; Carl Zeiss). In situ detection of Rap1 activation. Cultured allantoises were fixed in formaldehyde buffer (50 mM Hepes/NaOH, pH7.4, 150 mM NaCl, 20 mM MgCl2, 0.05% (v/v) Tween 20, 0.1% (v/v) Tri-
Fig. 2. Vascular network formation and VE-cadherin expression in the yolk sac. (A) Endothelial cells in 8.5-dpc and 9.0-dpc wild-type (+/+) and RA-GEF-1 knockout (/) yolk sacs were visualized by immunofluorescent staining with an anti-PECAM-1 antibody. Scale bars, 500 lm. (B) Wild-type (+/+) and RA-GEF-1 knockout (/) yolk sacs at 8.5 dpc were stained with anti-VE-cadherin (green) and anti-PECAM-1 (red) antibodies. Scale bars, 10 lm. (C) mRNA levels of VE-cadherin and PECAM-1 in wild-type(+/+) and RA-GEF-1 knockout (/) yolk sacs were determined by quantitative RT-PCR. Mean values of the mRNA level of VE-cadherin relative to PECAM-1 from two independent experiments are shown.
H. Kanemura et al. / Biochemical and Biophysical Research Communications 387 (2009) 754–759
ton X-100, and 3.7% formaldehyde) on ice for 1 min and washed three times with wash buffer A (50 mM Hepes/NaOH, pH7.4, 150 mM NaCl, 20 mM MgCl2, and 0.05% (v/v) Tween 20). Explants were then incubated with GST-RalGDS-V5 3 (10 lg/ml) in probe buffer (50 mM Hepes/NaOH, pH7.4, 150 mM NaCl, 20 mM MgCl2, 0.05% (v/v) Tween 20, and 0.02% (v/v) Triton X-100) on ice for 10 min. After washing three times with wash buffer A, cells were fixed again in formaldehyde buffer on ice for 8 min. Subsequently, explants were washed three times with PBS(–) supplemented with 0.05% (v/v) Tween 20. GST-RalGDS-V5 3 was detected with an anti-V5 antibody by immunofluorescent analysis. Preparation of recombinant lentiviruses. 293T cells were maintained in DMEM supplemented with 10% FBS, 50 IU/ml penicillin, and 50 lg/ml streptomycin. cDNAs for N-terminally FLAG- and DsRed-tagged human Rap1A(G12V) and Rap1B(G12V) were subcloned into the BamHI site of CSII-CMV-MCS (a generous gift from Dr. Hiroyuki Miyoshi, RIKEN Bioresource Center, Tsukuba, Japan), generating CSII-CMV-FLAG-DsRed-Rap1A(G12V) and CSII-CMVFLAG-DsRed-Rap1B(G12V), respectively. Lentiviruses were prepared as described in the provider’s protocol. Briefly, 5 106 293T cells were seeded onto a 10-cm culture plate. At 75% confluency, cells were transfected with 17 lg of CSII-CMV-FLAG-DsRedRap1A(G12V) or CSII-CMV-FLAG-DsRed-Rap1B(G12V), 10 lg of the packaging plasmid (pCAG-HIVgp), and 10 lg of the VSV-Gand Rev-expressing plasmid (pCMV-VSV-G-RSV-Rev) using the calcium phosphate precipitation method. After 12–16 h, the culture medium was replaced by 5 ml of DMEM containing 10 lM forskolin. Forty-eight hours later, supernatants were filtrated through 0.45-lm filters (Millex-HV filter; Millipore) and centrifuged twice at 50,000g for 2 h at 20 °C. The virus pellet was resuspended in HBSS and stored at 80 °C.Primary culture of allantois cells. OP9 mouse stroma cells were cultured in a-Modified Eagle’s Minimum Essential Medium (a-MEM) supplemented with 20% FBS, 50 IU/ml penicillin, and 50 lg/ml streptomycin. Allantoises at 8.5 dpc were digested in a solution containing 0.5% trypsin and 0.53 mM EDTA.
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One-thousand allantois cells were incubated with lentiviral solutions in the presence of polybrene (8 lg/ml), washed with HBSS, and then seeded onto mitomycin C-treated OP9 cells in a-MEM in an 8-well chamber slide. Cells were cultured for 7 days with medium change every 3 days, washed with PBS(), and fixed in 4% PFA for 20 min at room temperature. Immunostaining was performed as described above. Morphometric and statistical analysis. The length of blood vessels and the area of the vascular plexus in allantois cultures were measured using a microscope (BZ-8000, Keyence, Japan) after immunostaining of Flk-1. Results Previously, we demonstrated that RA-GEF-1 knockout mice failed to develop vascular systems and died by 9.5 dpc [7]. To gain insights into the mechanism underlying defective vascular network formation in RA-GEF-1 knockout mice, we cultured allantois explants in vitro [9,10]. The allantois was isolated from 8.5-dpc wild-type and RA-GEF-1 knockout mice and cultured for 24 h (Fig. 1A). At 8.5 dpc, the wild-type allantois has a network of primitive blood vessels as shown by immunostaining of Flk-1. The formation of the blood vessel network in the 8.5-dpc RA-GEF-1 knockout allantois was similar to the wild-type. Following 24-h culture, the Flk-1-positive capillary-like vessels extended their network in wild-type allantois explants. In marked contrast, Flk-1-positive cells failed to develop the blood vessel network, and isolated clusters of these cells were observed in RA-GEF-1 knockout explants. The length of blood vessels and the area of the vascular plexus in wild-type and RA-GEF-1 knockout allantois explants were measured following 24-h culture, demonstrating that suppression of vascular network development was statistically significant by both criteria (Fig. 1B). VE-cadherin is localized at adherence junctions in vascular endothelial cells and required for vascular morphogenesis [11].
Fig. 3. Rap1 activation in the cultured allantois explant. Allantoises were isolated from 8.5-dpc RA-GEF-1 heterozygous (+/) and RA-GEF-1 knockout (/) mice, cultured in vitro for 0 h (A) or 24 h (B), and then stained with an activation-specific probe for Rap1GTP (green), anti-Rap1 (red) and anti-Flk-1 (blue) antibodies. Scale bars, 50 lm.
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Localization of VE-cadherin at cell–cell junctions was evident in endothelial cells of RA-GEF-1+/ allantois explants after 24-h culture in vitro, whereas VE-cadherin was not detected at cell–cell junctions in RA-GEF-1 knockout endothelial cells (Fig. 1C). The relative expression level of VE-cadherin was significantly decreased in RA-GEF-1 knockout cells as assessed by quantitative RT-PCR (Fig. 1D). Therefore, a lowered expression or aberrant subcellular localization of VE-cadherin may cause defective vascular development in the RA-GEF-1 knockout allantois. The vascular network at 9.0 dpc in the yolk sac was next examined because the allantois degenerates after 8.5 dpc. In the 8.5-dpc wild-type yolk sac, PECAM-1-positive endothelial cells formed the primitive vascular plexus (Fig. 2A). Endothelial cells in the RA-GEF1 knockout yolk sac also formed the primitive vascular plexus, but the vasculogenesis processes seemed to be slightly impaired (Fig. 2A). At 9.0 dpc, larege vitelline vessels and a meshwork of small vessels were formed in the wild-type, but not RA-GEF-1 knockout, yolk sac (Fig. 2A). VE-cadherin was localized at cell–cell junctions of PECAM-1-positive monolayered endothelial cells in the 8.5-dpc wild-type yolk sac (Fig. 2B). In contrast, endothelial cells in the RA-GEF-1 knockout yolk sac were smaller, more spherical in shape, and less organized compared to wild-type cells (Fig. 2B). Moreover, localization of VE-cadherin at cell–cell junctions was lost, and the relative VE-cadherin mRNA level was reduced in the RA-GEF-1 knockout yolk sac (Fig. 2B and C). Therefore, a loss of VE-cadherin at cell–cell junctions may be responsible for impaired vascular development also in the RAGEF-1 knockout yolk sac. The involvement of Rap1 downstream of RA-GEF-1 was next examined in cultured allantois explants. The activation of endogenous Rap1 was visualized by immunofluorescent staining by the use of activation-specific probe as previously reported for Cdc42 and Rac1 [12,13] (Fig. 3). Rap1 was indeed activated in Flk-1-positive endothelial cells in RA-GEF-1+/ allantois explants whereas no Rap1 activation was detected in RA-GEF-1 knockout cells. Rap1 activation in RA-GEF-1+/ endothelial cells was sustained during 24-h culture in vitro, however Rap1 remained inactivated in RAGEF-1 knockout cells. Furthermore, the accumulation of VE-cadherin at cell–cell junctions was recovered by ectopic expression of constitutively activated Rap1 in PECAM-1-positive RA-GEF-1 knockout endothelial cell culture (Fig. 4A). Extension of blood vessel-like processes consisting of PECAM-1-positive endothelial cells were observed following 24-h culture of wild-type allantois explants in collagen gel, which was enhanced by ectopic expression of constitutively activated Rap1 (Fig. 4B). In contrast, RA-GEF-1 knockout allantoises extended only a few processes. Ectopic expression of constitutively activated Rap1 again rescued this phenotype (Fig. 4B). Collectively, it is likely that Rap1 is indeed involved in the RA-GEF-1-mediated signaling pathway and regulates embryonic blood vessel formation.
Discussion Two closely related Rap1 proteins, Rap1A and Rap1B, exist in mammals, sharing more than 90% amino acid identity. Recently, Rap1A and Rap1B have been implicated in the regulation of angiogenesis through the analysis of knockout mice. Rap1A knockout mice showed an impaired angiogenic response to fibroblast growth factor 2 in Matrigel plug assay [14] and reduced neovascularization capacity in the hind limb ischemia model [15]. Furthermore, suppression of Rap1A expression indeed inhibited migration and adhesion of endothelial cells [14,15]. Similar effects of Rap1B abrogation were observed in vivo and in vitro [15,16]. Here we show that Rap1 under the control of RA-GEF-1 regulates blood vessel for-
Fig. 4. Rescue of VE-cadherin expression and blood vessel formation by ectopic expression of a constitutively activated mutant of Rap1. (A) Allantoises were isolated from 8.5-dpc wild-type (+/+) and RA-GEF-1 knockout (/) mice, and the trypsin-dispersed cell suspension was prepared. Allantois cells were infected with lentiviruses expressing DsRed or DsRed-Rap1A(G12 V), cultured with mouse stroma OP9 cells for 7 days, and then stained with anti-VE-cadherin (green) and antiPECAM-1 (blue) antibodies. Scale bars, 10 lm. (B) Allantoises were isolated from 8.5-dpc wild-type (+/+) and RA-GEF-1 knockout (/) mice, infected with lentiviruses expressing DsRed, DsRed-Rap1A(G12 V), or DsRed-Rap1B(G12 V), embedded in collagen gel, and cultured for 24 h. The expression of DsRed, DsRedRap1A(G12 V), or DsRed-Rap1B(G12 V) was shown in insets. Lower panels show the expression of DsRed and immunofluorescent staining of a wild-type (+/+) blood vessel-like process with an anti-PECAM-1 (green) antibody. Scale bars; 100 lm (for upper and middle panels), 500 lm (for insets of upper and middle panels), 10 lm (for lower panels).
mation at an embryonic stage. Considering that activated forms of both Rap1A and Rap1B could rescue defective vascular formation in RA-GEF-1 knockout allantois (Fig. 4B), two Rap1 subtypes may have redundant functions. A pivotal role of Rap1 in the control of cell–cell adhesion, particularly the formation of E- or VE-cadherin-containing adherens junctions, has been described [17–20]. During the initial step of junction formation, GEFs for Rap1, such as C3G and DOCK4, seem to be responsible for Rap1 activation and subsequent cadherin recruitment to the plasma membrane [21]. RA-GEF-2 (PDZ-GEF2) also acts as an activator of Rap1A and Rap1B, which in turn regulate adherens junction maturation and E-cadherin expression, respectively [22]. The involvement of the cAMP-responsive Rap
H. Kanemura et al. / Biochemical and Biophysical Research Communications 387 (2009) 754–759
GEF Epac was also examined by the use of an Epac-specific cAMP analogue because integrity of VE-cadherin-mediated endothelial cell junctions is regulated by intracellular cAMP. In fact, Epac activated Rap1 in endothelial cells in response to cAMP, leading to the potentiation of cell adhesion and a decrease in the permeability of the cell monolayer [23–25]. Here we show that the RA-GEF-1 knockout allantois showed vascular morphologies similar to the VE-cadherin knockout allantois in culture [10] (Fig. 1A). In addition, VE-cadherin localized in cell–cell junctions was diminished in endothelial cells in the RA-GEF-1 knockout allantois and yolk sac (Figs. 1C and 2B). Thus, RA-GEF-1 may be a Rap1 GEF responsible for the regulation of VE-cadherin expression and localization in embryonic blood vessel formation. Reduction in the VE-cadherin mRNA level (Figs. 1D and 2C), at least in part, may account for the loss of VE-cadherin in cell–cell junctions. In addition, the expression level of VE-cadherin on the cell surface may be regulated through its endocytosis because endocytosis of E-cadherin is suppressed by Rap1 [26]. Given a crucial role of VE-cadherin in the prevention of disassembly of nascent embryonic blood vessels following vascular epithelialization [10], RA-GEF-1 may regulate VE-cadherin-mediated maintenance of nascent blood vessels. In marked contrast to RA-GEF-1, RA-GEF-2 knockout mice showed no gross abnormalities in growth characteristics and were also fertile [6]. However, the high level expression of RA-GEF-2 in the thymus and spleen suggested a crucial role of RA-GEF-2 in the immune system. In fact, tumor necrosis factor a-induced activation of lymphocyte function-associated antigen 1 was almost completely abolished in RA-GEF-2 knockout B lymphocytes. Therefore, RA-GEF-1 and RA-GEF-2 may have distinct roles in mouse development and physiology although they exhibit similarities in domain structure and biochemical properties. Further analysis will be required to clarify the RA-GEF-1-specific mechanism underlying the regulation of vascular formation in embryos. References [1] W. Risau, Mechanisms of angiogenesis, Nature 386 (1997) 671–674. [2] J.L. Bos, J. de Rooij, K.A. Reedquist, Rap1 signalling: adhering to new models, Nat. Rev. Mol. Cell Biol. 2 (2001) 369–377. [3] Y. Liao, K. Kariya, C.D. Hu, M. Shibatohge, M. Goshima, T. Okada, Y. Watari, X. Gao, T.G. Jin, Y. Yamawaki-Kataoka, T. Kataoka, RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans, J. Biol. Chem. 274 (1999) 37815– 37820. [4] Y. Liao, T. Satoh, X. Gao, T.G. Jin, C.D. Hu, T. Kataoka, RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1 GTP and enhances Rap1-dependent B-Raf activation, J. Biol. Chem. 276 (2001) 28478–28483. [5] X. Gao, T. Satoh, Y. Liao, C. Song, C.D. Hu, K. Kariya, T. Kataoka, Identification and characterization of RA-GEF-2, a Rap guanine nucleotide exchange factor that serves as a downstream target of M-Ras, J. Biol. Chem. 276 (2001) 42219–42225. [6] Y. Yoshikawa, T. Satoh, T. Tamura, P. Wei, S.E. Bilasy, H. Edamatsu, A. Aiba, K. Katagiri, T. Kinashi, K. Nakao, T. Kataoka, The M-Ras-RA-GEF-2-Rap1 pathway
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Impaired vascular development in the yolk sac and allantois in mice lacking RA-GEF-1 Hoshimi Kanemura a, Takaya Satoh a, Shymaa E. Bilasy a, Shuji Ueda a, Masanori Hirashima b, Tohru Kataoka a,* a b
Division of Molecular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Division of Vascular Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
a r t i c l e
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Article history: Received 14 July 2009 Available online 25 July 2009 Keywords: Allantois Angiogenesis RA-GEF-1 Rap1 Vascular plexus Yolk sac
a b s t r a c t RA-GEF-1 is a guanine nucleotide exchange factor for the small GTPase Rap1. RA-GEF-1 knockout mice show defects in vascular development starting around 7.5 days post coitum and die by 9.5 days post coitum. Here, we employed in vitro culture systems for allantois explants and endothelial cells to gain insights into the mechanism for RA-GEF-1-mediated regulation of embryonic vascular network formation. The development of the vascular plexus and the accumulation of VE-cadherin at cell–cell junctions were significantly impaired in the RA-GEF-1 knockout allantois and yolk sac. Rap1 activation as visualized by an activation-specific probe was also diminished by RA-GEF-1 knockout. Reduced accumulation of VEcadherin at cell–cell junctions and defects in blood vessel formation in vitro due to the lack of RA-GEF-1 were suppressed by ectopic expression of constitutively activated Rap1. Overall, these results suggest the involvement of Rap1 downstream of RA-GEF-1 in the regulation of vascular network formation in mouse embryos. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Blood vessel formation is a crucial process during embryonic development [1]. Mesoderm-derived angiogenic progenitor cells (angioblasts) migrate to the site of vascularization, differentiate into endothelial cells, and then coalesce to form the primary vascular plexus. This initial process is denoted vasculogenesis. Following this, new capillaries are generated from pre-existing blood vessels through sprouting or intussusceptive angiogenesis. The vascular endothelial growth factor receptor Flk-1 and its ligands are intimately involved in differentiation of haemangioblasts into angioblasts and subsequent vascular plexus formation. Various vascular endothelial growth factor receptors are also responsible for the regulation of angiogenesis. However, signaling mechanisms underlying vascular development during mouse embryogenesis remain largely unknown. The small GTPase Rap1 regulates diverse cellular responses such as proliferation, adhesion, and exocytosis in response to extracellular stimuli [2]. The activity of Rap1 is regulated through the GDP-GTP cycle, which is positively and negatively regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins, respectively. RA-GEF-1 (also called PDZ-GEF1, nRapGEP, and CNRasGEF) [2–4] and its close relative RA-GEF-2 [5] are * Corresponding author. Fax: +81 78 382 5399. E-mail address: [email protected] (T. Kataoka). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.07.108
Rap-specific GEF unique in that they act both upstream and downstream of Ras family GTPases. The Ras/Rap-associating (RA) domains of RA-GEF-1 and RA-GEF-2 interact with Ras family GTPases Rap1 and M-Ras in their GTP-bound active forms. Though these interactions, Rap1 and M-Ras recruit RA-GEFs to the Golgi apparatus and the plasma membrane, respectively, in which RAGEFs activate Rap1 [4,5]. Thus, RA-GEFs serve as a link between Ras family GTPases in intracellular signaling cascades. Although their biochemical properties are similar, expression patterns of RA-GEF-1 and RA-GEF-2 are different from each other. RA-GEF-1 is ubiquitously expressed in mouse tissues whereas the expression level of RA-GEF-2 is particularly high in the spleen and thymus and weak or undetectable in other tissues [5,6]. Therefore, RA-GEF-1 and RA-GEF-2 are likely to be involved in distinct physiological responses. The function of RA-GEF-1 has been investigated through the generation of knockout mice [7]. Mice carrying a null mutation in both RA-GEF-1 alleles in the whole body appeared normal until 7.5 days post coitum (dpc), but died by 9.5 dpc. Severe defects in blood vessel formation in the RA-GEF-1-null yolk sac and embryo proper were observed, which may be a cause for embryonic lethality. However, the mechanism underlying the regulation of embryonic blood vessel formation by RA-GEF-1 remains totally unknown. In contrast to RA-GEF-1 knockout mice, RA-GEF-1 heterozygous mice showed normal phenotypes (Wei et al ., unpublished observations).
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Here, we investigate the role of RA-GEF-1 in embryonic vascular network formation by the use of an in vitro culture system of allantoises isolated from wild-type and RA-GEF-1 knockout embryos.
toises of 8.5 dpc embryos were excised, washed with ice-cold HBSS, and then transferred into a 4-well chamber slide (Lab-Tek, NY 14625, USA) containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 IU/ ml penicillin, and 50 lg/ml streptomycin. Explants were cultured for 24 h at 37 °C in an atmosphere of 5% CO2. In some experiments, allantoises at 8.5 dpc were incubated with lentiviral solutions in the presence of polybrene (8 lg/ml), washed with HBSS, and then embedded in collagen I-A (Nitta Gelatin, Tokyo, Japan) in an 8-well chamber slide. Following 24-h culture, pictures were taken using a microscope (BZ-8000, Keyence, Japan). Quantitative reverse transcription-PCR (RT-PCR) analysis. The total cellular RNA was prepared from allantoises or yolk sacs by using the Sepasol-RNA I Super kit (Nacalai tesque, Kyoto, Japan) and reverse-transcribed with the SuperScriptTM III First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR analysis was performed with the Thermal Cycler Dice Real Time system TP800 (Takara Bio, Otsu, Japan). Relative mRNA levels were determined by the comparative Ct method followed by normalization with the b-actin mRNA level in each cDNA sample. Primer sequences are as follows:
Materials and methods Mouse breeding and genotyping. RA-GEF-1 knockout mice were generated and maintained on the C57BL/6 and 129Ola mixed background as described previously [7]. To obtain RA-GEF-1/ embryos, female RA-GEF-1+/ mice were placed with a male RA-GEF1+/ mouse overnight, and the time of vaginal plug appearance was considered as 0.5 dpc. Tail tips, yolk sacs, and embryos were genotyped by polymerase chain reaction (PCR) analysis as described previously [7]. The use and care of animals were reviewed and approved by the Institutional Animal Care and Use Committee of Kobe University. Isolation and culture of allantoises. Allantois culture was performed essentially as described previously [8]. Pregnant females were killed by cervical dislocation at 8.5 dpc, and uterine horns were placed in ice-cold Hank’s balanced salt solution (HBSS). Allan-
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Fig. 1. Vascular network formation and VE-cadherin expression in the cultured allantois explant. (A) Allantoises were isolated from 8.5-dpc wild-type (+/+) and RA-GEF-1 knockout (/) mice and cultured in vitro for indicated times. Endothelial cells were visualized by immunofluorescent staining with an anti-Flk-1 antibody. Scale bars, 100 lm. (B) The length of blood vessels (upper) and the area of the vascular plexus (lower) in a single wild-type (+/+) or RA-GEF-1 knockout (/) allantois explant were measured following 24-h culture in vitro. Results are shown as the means ± SEM from 7 to 10 independent experiments. *1, p = 1.92 105; *2, p = 1.08 106. (C) Allantoises were isolated from 8.5-dpc RA-GEF-1 heterozygous (+/) and RA-GEF-1 knockout (/) mice, cultured in vitro for 24 h, and then stained with anti-VE-cadherin (green) and anti-Flk-1 (red) antibodies. Scale bars, 10 lm. (D) mRNA levels of VE-cadherin and Flk-1 in 24-h cultured wild-type(+/+), RA-GEF-1 heterozygous (+/), and RA-GEF-1 knockout (/) allantoises were determined by quantitative RT-PCR. The mRNA level of VE-cadherin relative to Flk-1 was shown as the means ± SEM from 8 to 9 independent experiments. *1, p = 1.78 102; *2, p = 1.82 102.
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b-actin sense, 50 -ATGAAGATCAAGATCATTGCTCCTC-30 ; b-actin antisense, 50 -ACATCTGCTGGAAGGTGGACAG-30 ; Flk-1 sense, 50 -GATGC AGGAAACTACACGGTCA0 -30 ; Flk-1 antisense, 50 -TCCATAGGCTGTTG GCTTCCACA-30 ; VE-Cadherin sense, 50 -CACTGCTTTGGGAGCCTT C-30 ; VE-Cadherin antisense, 50 -GGGGCAGCGATTCATTTTTCT-30 . Immunohistochemistry. Whole embryos were dissected at 8.5 dpc and fixed in 4% paraformaldehyde (PFA) overnight. Embryos were washed three times with phosphate-buffered saline containing 0.2% (v/v) Triton X-100 (PBST) for 30 min at 4 °C, blocked in PBST containing 1% bovine serum albumin (BSA) for 1 h at room temperature, and incubated with primary antibodies in PBST containing 1% BSA at 4 °C overnight. Embryos were washed three times with PBST for 30 min at 4 °C and twice for 30 min at room temperature, followed by incubation with secondary antibodies in PBST containing 1% BSA at 4 °C overnight. Subsequently, embryos were washed three times with PBST for 30 min at 4 °C and twice for 30 min at room temperature. Yolk sacs and allantoises were peeled off and flat-mounted on slide glasses. Cultured allantoises were fixed in 2% PFA for 20 min and permeabilized in phos-
phate-buffered saline (PBS()) containing 0.02% (v/v) Triton X-100 for 40 min. Fixed allantoises were blocked by 3% BSA in PBS() and immunolabeled with primary and secondary antibodies as described above. Antibodies against Flk-1 (clone AVAS 12a1, rat, BD Bioscience-Pharmingen, San Diego, CA; 1:50), PECAM-1 (clone MEC 13.3, rat, BD Bioscience-Pharmingen, San Diego, CA; 1:50), PECAM-1 (clone 2H8, Armenian hamster, Endogen; 1:1000), VEcadherin (C-19, goat, Santa Cruz Biotechnology; 1:50), VE-cadherin (clone 11D4.1, rat, BD Bioscience-Pharmingen, San Diego, CA; 1:1000), Rap1 (121, rabbit, Santa Cruz Biotechnology; 1:200), and the V5 epitope tag (Nacalai tesque, Kyoto, Japan; 1:1000) were commercially available. Secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Cy3, or Cy5 (Molecular Probes and Jackson ImmunoResearch) were used for the detection. Samples were analyzed by confocal laser scanning microscopy (LSM510 META; Carl Zeiss). In situ detection of Rap1 activation. Cultured allantoises were fixed in formaldehyde buffer (50 mM Hepes/NaOH, pH7.4, 150 mM NaCl, 20 mM MgCl2, 0.05% (v/v) Tween 20, 0.1% (v/v) Tri-
Fig. 2. Vascular network formation and VE-cadherin expression in the yolk sac. (A) Endothelial cells in 8.5-dpc and 9.0-dpc wild-type (+/+) and RA-GEF-1 knockout (/) yolk sacs were visualized by immunofluorescent staining with an anti-PECAM-1 antibody. Scale bars, 500 lm. (B) Wild-type (+/+) and RA-GEF-1 knockout (/) yolk sacs at 8.5 dpc were stained with anti-VE-cadherin (green) and anti-PECAM-1 (red) antibodies. Scale bars, 10 lm. (C) mRNA levels of VE-cadherin and PECAM-1 in wild-type(+/+) and RA-GEF-1 knockout (/) yolk sacs were determined by quantitative RT-PCR. Mean values of the mRNA level of VE-cadherin relative to PECAM-1 from two independent experiments are shown.
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ton X-100, and 3.7% formaldehyde) on ice for 1 min and washed three times with wash buffer A (50 mM Hepes/NaOH, pH7.4, 150 mM NaCl, 20 mM MgCl2, and 0.05% (v/v) Tween 20). Explants were then incubated with GST-RalGDS-V5 3 (10 lg/ml) in probe buffer (50 mM Hepes/NaOH, pH7.4, 150 mM NaCl, 20 mM MgCl2, 0.05% (v/v) Tween 20, and 0.02% (v/v) Triton X-100) on ice for 10 min. After washing three times with wash buffer A, cells were fixed again in formaldehyde buffer on ice for 8 min. Subsequently, explants were washed three times with PBS(–) supplemented with 0.05% (v/v) Tween 20. GST-RalGDS-V5 3 was detected with an anti-V5 antibody by immunofluorescent analysis. Preparation of recombinant lentiviruses. 293T cells were maintained in DMEM supplemented with 10% FBS, 50 IU/ml penicillin, and 50 lg/ml streptomycin. cDNAs for N-terminally FLAG- and DsRed-tagged human Rap1A(G12V) and Rap1B(G12V) were subcloned into the BamHI site of CSII-CMV-MCS (a generous gift from Dr. Hiroyuki Miyoshi, RIKEN Bioresource Center, Tsukuba, Japan), generating CSII-CMV-FLAG-DsRed-Rap1A(G12V) and CSII-CMVFLAG-DsRed-Rap1B(G12V), respectively. Lentiviruses were prepared as described in the provider’s protocol. Briefly, 5 106 293T cells were seeded onto a 10-cm culture plate. At 75% confluency, cells were transfected with 17 lg of CSII-CMV-FLAG-DsRedRap1A(G12V) or CSII-CMV-FLAG-DsRed-Rap1B(G12V), 10 lg of the packaging plasmid (pCAG-HIVgp), and 10 lg of the VSV-Gand Rev-expressing plasmid (pCMV-VSV-G-RSV-Rev) using the calcium phosphate precipitation method. After 12–16 h, the culture medium was replaced by 5 ml of DMEM containing 10 lM forskolin. Forty-eight hours later, supernatants were filtrated through 0.45-lm filters (Millex-HV filter; Millipore) and centrifuged twice at 50,000g for 2 h at 20 °C. The virus pellet was resuspended in HBSS and stored at 80 °C.Primary culture of allantois cells. OP9 mouse stroma cells were cultured in a-Modified Eagle’s Minimum Essential Medium (a-MEM) supplemented with 20% FBS, 50 IU/ml penicillin, and 50 lg/ml streptomycin. Allantoises at 8.5 dpc were digested in a solution containing 0.5% trypsin and 0.53 mM EDTA.
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One-thousand allantois cells were incubated with lentiviral solutions in the presence of polybrene (8 lg/ml), washed with HBSS, and then seeded onto mitomycin C-treated OP9 cells in a-MEM in an 8-well chamber slide. Cells were cultured for 7 days with medium change every 3 days, washed with PBS(), and fixed in 4% PFA for 20 min at room temperature. Immunostaining was performed as described above. Morphometric and statistical analysis. The length of blood vessels and the area of the vascular plexus in allantois cultures were measured using a microscope (BZ-8000, Keyence, Japan) after immunostaining of Flk-1. Results Previously, we demonstrated that RA-GEF-1 knockout mice failed to develop vascular systems and died by 9.5 dpc [7]. To gain insights into the mechanism underlying defective vascular network formation in RA-GEF-1 knockout mice, we cultured allantois explants in vitro [9,10]. The allantois was isolated from 8.5-dpc wild-type and RA-GEF-1 knockout mice and cultured for 24 h (Fig. 1A). At 8.5 dpc, the wild-type allantois has a network of primitive blood vessels as shown by immunostaining of Flk-1. The formation of the blood vessel network in the 8.5-dpc RA-GEF-1 knockout allantois was similar to the wild-type. Following 24-h culture, the Flk-1-positive capillary-like vessels extended their network in wild-type allantois explants. In marked contrast, Flk-1-positive cells failed to develop the blood vessel network, and isolated clusters of these cells were observed in RA-GEF-1 knockout explants. The length of blood vessels and the area of the vascular plexus in wild-type and RA-GEF-1 knockout allantois explants were measured following 24-h culture, demonstrating that suppression of vascular network development was statistically significant by both criteria (Fig. 1B). VE-cadherin is localized at adherence junctions in vascular endothelial cells and required for vascular morphogenesis [11].
Fig. 3. Rap1 activation in the cultured allantois explant. Allantoises were isolated from 8.5-dpc RA-GEF-1 heterozygous (+/) and RA-GEF-1 knockout (/) mice, cultured in vitro for 0 h (A) or 24 h (B), and then stained with an activation-specific probe for Rap1GTP (green), anti-Rap1 (red) and anti-Flk-1 (blue) antibodies. Scale bars, 50 lm.
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Localization of VE-cadherin at cell–cell junctions was evident in endothelial cells of RA-GEF-1+/ allantois explants after 24-h culture in vitro, whereas VE-cadherin was not detected at cell–cell junctions in RA-GEF-1 knockout endothelial cells (Fig. 1C). The relative expression level of VE-cadherin was significantly decreased in RA-GEF-1 knockout cells as assessed by quantitative RT-PCR (Fig. 1D). Therefore, a lowered expression or aberrant subcellular localization of VE-cadherin may cause defective vascular development in the RA-GEF-1 knockout allantois. The vascular network at 9.0 dpc in the yolk sac was next examined because the allantois degenerates after 8.5 dpc. In the 8.5-dpc wild-type yolk sac, PECAM-1-positive endothelial cells formed the primitive vascular plexus (Fig. 2A). Endothelial cells in the RA-GEF1 knockout yolk sac also formed the primitive vascular plexus, but the vasculogenesis processes seemed to be slightly impaired (Fig. 2A). At 9.0 dpc, larege vitelline vessels and a meshwork of small vessels were formed in the wild-type, but not RA-GEF-1 knockout, yolk sac (Fig. 2A). VE-cadherin was localized at cell–cell junctions of PECAM-1-positive monolayered endothelial cells in the 8.5-dpc wild-type yolk sac (Fig. 2B). In contrast, endothelial cells in the RA-GEF-1 knockout yolk sac were smaller, more spherical in shape, and less organized compared to wild-type cells (Fig. 2B). Moreover, localization of VE-cadherin at cell–cell junctions was lost, and the relative VE-cadherin mRNA level was reduced in the RA-GEF-1 knockout yolk sac (Fig. 2B and C). Therefore, a loss of VE-cadherin at cell–cell junctions may be responsible for impaired vascular development also in the RAGEF-1 knockout yolk sac. The involvement of Rap1 downstream of RA-GEF-1 was next examined in cultured allantois explants. The activation of endogenous Rap1 was visualized by immunofluorescent staining by the use of activation-specific probe as previously reported for Cdc42 and Rac1 [12,13] (Fig. 3). Rap1 was indeed activated in Flk-1-positive endothelial cells in RA-GEF-1+/ allantois explants whereas no Rap1 activation was detected in RA-GEF-1 knockout cells. Rap1 activation in RA-GEF-1+/ endothelial cells was sustained during 24-h culture in vitro, however Rap1 remained inactivated in RAGEF-1 knockout cells. Furthermore, the accumulation of VE-cadherin at cell–cell junctions was recovered by ectopic expression of constitutively activated Rap1 in PECAM-1-positive RA-GEF-1 knockout endothelial cell culture (Fig. 4A). Extension of blood vessel-like processes consisting of PECAM-1-positive endothelial cells were observed following 24-h culture of wild-type allantois explants in collagen gel, which was enhanced by ectopic expression of constitutively activated Rap1 (Fig. 4B). In contrast, RA-GEF-1 knockout allantoises extended only a few processes. Ectopic expression of constitutively activated Rap1 again rescued this phenotype (Fig. 4B). Collectively, it is likely that Rap1 is indeed involved in the RA-GEF-1-mediated signaling pathway and regulates embryonic blood vessel formation.
Discussion Two closely related Rap1 proteins, Rap1A and Rap1B, exist in mammals, sharing more than 90% amino acid identity. Recently, Rap1A and Rap1B have been implicated in the regulation of angiogenesis through the analysis of knockout mice. Rap1A knockout mice showed an impaired angiogenic response to fibroblast growth factor 2 in Matrigel plug assay [14] and reduced neovascularization capacity in the hind limb ischemia model [15]. Furthermore, suppression of Rap1A expression indeed inhibited migration and adhesion of endothelial cells [14,15]. Similar effects of Rap1B abrogation were observed in vivo and in vitro [15,16]. Here we show that Rap1 under the control of RA-GEF-1 regulates blood vessel for-
Fig. 4. Rescue of VE-cadherin expression and blood vessel formation by ectopic expression of a constitutively activated mutant of Rap1. (A) Allantoises were isolated from 8.5-dpc wild-type (+/+) and RA-GEF-1 knockout (/) mice, and the trypsin-dispersed cell suspension was prepared. Allantois cells were infected with lentiviruses expressing DsRed or DsRed-Rap1A(G12 V), cultured with mouse stroma OP9 cells for 7 days, and then stained with anti-VE-cadherin (green) and antiPECAM-1 (blue) antibodies. Scale bars, 10 lm. (B) Allantoises were isolated from 8.5-dpc wild-type (+/+) and RA-GEF-1 knockout (/) mice, infected with lentiviruses expressing DsRed, DsRed-Rap1A(G12 V), or DsRed-Rap1B(G12 V), embedded in collagen gel, and cultured for 24 h. The expression of DsRed, DsRedRap1A(G12 V), or DsRed-Rap1B(G12 V) was shown in insets. Lower panels show the expression of DsRed and immunofluorescent staining of a wild-type (+/+) blood vessel-like process with an anti-PECAM-1 (green) antibody. Scale bars; 100 lm (for upper and middle panels), 500 lm (for insets of upper and middle panels), 10 lm (for lower panels).
mation at an embryonic stage. Considering that activated forms of both Rap1A and Rap1B could rescue defective vascular formation in RA-GEF-1 knockout allantois (Fig. 4B), two Rap1 subtypes may have redundant functions. A pivotal role of Rap1 in the control of cell–cell adhesion, particularly the formation of E- or VE-cadherin-containing adherens junctions, has been described [17–20]. During the initial step of junction formation, GEFs for Rap1, such as C3G and DOCK4, seem to be responsible for Rap1 activation and subsequent cadherin recruitment to the plasma membrane [21]. RA-GEF-2 (PDZ-GEF2) also acts as an activator of Rap1A and Rap1B, which in turn regulate adherens junction maturation and E-cadherin expression, respectively [22]. The involvement of the cAMP-responsive Rap
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GEF Epac was also examined by the use of an Epac-specific cAMP analogue because integrity of VE-cadherin-mediated endothelial cell junctions is regulated by intracellular cAMP. In fact, Epac activated Rap1 in endothelial cells in response to cAMP, leading to the potentiation of cell adhesion and a decrease in the permeability of the cell monolayer [23–25]. Here we show that the RA-GEF-1 knockout allantois showed vascular morphologies similar to the VE-cadherin knockout allantois in culture [10] (Fig. 1A). In addition, VE-cadherin localized in cell–cell junctions was diminished in endothelial cells in the RA-GEF-1 knockout allantois and yolk sac (Figs. 1C and 2B). Thus, RA-GEF-1 may be a Rap1 GEF responsible for the regulation of VE-cadherin expression and localization in embryonic blood vessel formation. Reduction in the VE-cadherin mRNA level (Figs. 1D and 2C), at least in part, may account for the loss of VE-cadherin in cell–cell junctions. In addition, the expression level of VE-cadherin on the cell surface may be regulated through its endocytosis because endocytosis of E-cadherin is suppressed by Rap1 [26]. Given a crucial role of VE-cadherin in the prevention of disassembly of nascent embryonic blood vessels following vascular epithelialization [10], RA-GEF-1 may regulate VE-cadherin-mediated maintenance of nascent blood vessels. In marked contrast to RA-GEF-1, RA-GEF-2 knockout mice showed no gross abnormalities in growth characteristics and were also fertile [6]. However, the high level expression of RA-GEF-2 in the thymus and spleen suggested a crucial role of RA-GEF-2 in the immune system. In fact, tumor necrosis factor a-induced activation of lymphocyte function-associated antigen 1 was almost completely abolished in RA-GEF-2 knockout B lymphocytes. Therefore, RA-GEF-1 and RA-GEF-2 may have distinct roles in mouse development and physiology although they exhibit similarities in domain structure and biochemical properties. Further analysis will be required to clarify the RA-GEF-1-specific mechanism underlying the regulation of vascular formation in embryos. References [1] W. Risau, Mechanisms of angiogenesis, Nature 386 (1997) 671–674. [2] J.L. Bos, J. de Rooij, K.A. Reedquist, Rap1 signalling: adhering to new models, Nat. Rev. Mol. Cell Biol. 2 (2001) 369–377. [3] Y. Liao, K. Kariya, C.D. Hu, M. Shibatohge, M. Goshima, T. Okada, Y. Watari, X. Gao, T.G. Jin, Y. Yamawaki-Kataoka, T. Kataoka, RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans, J. Biol. Chem. 274 (1999) 37815– 37820. [4] Y. Liao, T. Satoh, X. Gao, T.G. Jin, C.D. Hu, T. Kataoka, RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1 GTP and enhances Rap1-dependent B-Raf activation, J. Biol. Chem. 276 (2001) 28478–28483. [5] X. Gao, T. Satoh, Y. Liao, C. Song, C.D. Hu, K. Kariya, T. Kataoka, Identification and characterization of RA-GEF-2, a Rap guanine nucleotide exchange factor that serves as a downstream target of M-Ras, J. Biol. Chem. 276 (2001) 42219–42225. [6] Y. Yoshikawa, T. Satoh, T. Tamura, P. Wei, S.E. Bilasy, H. Edamatsu, A. Aiba, K. Katagiri, T. Kinashi, K. Nakao, T. Kataoka, The M-Ras-RA-GEF-2-Rap1 pathway
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