Defective vascular morphogenesis and mid-gestation embryonic death in mice lacking RA-GEF-1

doi:10.1016/j.bbrc.2007.08.149

Biochemical and Biophysical Research Communications

Volume 363, Issue 1, 9 November 2007, Pages 106-112

https://doi.org/10.1016/j.bbrc.2007.08.149 Get rights and content

Abstract

A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1^−/− mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1^−/− yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1^−/− embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.

Section snippets

Materials and methods

Construction of the targeting vector. An RA-GEF-1 genomic DNA fragment was cloned from a 129/Sv mouse genomic bacterial artificial chromosome library (Invitrogen, Carlsbad, CA). Exon 15 of the RA-GEF-1 gene was flanked with a loxP site at its 5′ end and loxP-neomycin-resistant cassette (TK-neo)-loxP at its 3′ end (Fig. 1A). The resulting vector contains 5′ and 3′ arms of 7.1- and 4.1-kb RA-GEF-1 genomic sequences, respectively, for homologous recombination.

Gene targeting and generation of

Generation of the RA-GEF-1 null allele

The RA-GEF-1^flox allele was created in mouse 129/Ola-derived ES cells by homologous recombination (Fig. 1A). One out of 95 G418-resistant ES cell clones was verified to carry the RA-GEF-1^flox allele (Fig. 1B). This ES clone was used to derive RA-GEF-1^flox/+ mice, whose genotypes were verified by Southern blot hybridization and PCR (Fig. 1C). Subsequently, the RA-GEF-1⁻ allele was generated through Cre-mediated deletion of exon15-loxP-TK-neo (Fig. 1C). Embryo genotypes were determined by PCR

Discussion

During embryogenesis, the vascular system is formed by two main processes, vasculogenesis and angiogenesis. In vasculogenesis, the endothelial cells differentiate from endothelial progenitor cells and coalesce into a primary vascular plexus. During angiogenesis, the primitive vasculature is remodeled to form the more complex vasculature [17], [20]. We demonstrate that blood island formation and hematopoiesis are not affected until E8.5, but blood islands fail to fuse with each other to form a

Acknowledgments

We are grateful to Dr. Hitoshi Niwa for providing ES cells, Dr. Jun-ichi Miyazaki for providing CAG-cre transgenic mice, Dr. Kenji Araishi for valuable experimental assistance, and Dr. Shuji Ueda for valuable advice. This work was supported by Grants-in-Aid for Scientific Research in Priority Areas 17014061 and 18016018 and for Scientific Research 17390078, 17370050, and 18790223, and by a 21st Century COE Program from the Ministry of Education, Science, Sports, and Culture of Japan.

References (28)

Y. Liao et al.
RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans
J. Biol. Chem.
(1999)
Y. Liao et al.
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.
(2001)
X. Gao et al.
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.
(2001)
K. Sakai et al.
A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission
Biochem. Biophys. Res. Commun.
(1997)
E.L. George et al.
Fibronectins are essential for heart and blood vessel morphogenesis but are dispensable for initial specification of precursor cells
Blood
(1997)
G.L. Radice et al.
Developmental deffects in mouse embryos lacking N-cadherin
Dev. Biol.
(1997)
J.L. Bos et al.
Rap1 signalling: adhering to new models
Nat. Rev. Mol. Cell Biol.
(2001)
T. Kinashi
Intracellular signalling controlling integrin activation in lymphocytes
Nat. Rev. Immunol.
(2005)
M. Duchniewicz et al.
Rap1A-deficient T and B cells show impaired integrin-mediated cell adhesion
Mol. Cell. Biol.
(2006)
M. Chrzanowska-Wodnicka et al.
Rap1b is required for normal platelet function and hemostasis in mice
J. Clin. Invest.
(2005)

Y. Ohba et al.

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis

EMBO J.

(2001)

J.R. Crittenden et al.

CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation

Nat. Med.

(2004)

Y. Yoshikawa et al.

The M-Ras-RA-GEF-2-Rap1 pathway mediates tumor necrosis factor alpha dependent regulation of integrin activation in splenocytes

Mol. Biol. Cell

(2007)

M. Tadano et al.

Congenital semilunar valvulogenesis defect in mice deficient in phospholipase C epsilon

Mol. Cell. Biol.

(2005)

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Defective vascular morphogenesis and mid-gestation embryonic death in mice lacking RA-GEF-1

Abstract

Section snippets

Materials and methods

Generation of the RA-GEF-1 null allele

Discussion

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Blood

Dev. Biol.

Rap1 signalling: adhering to new models

Nat. Rev. Mol. Cell Biol.

Intracellular signalling controlling integrin activation in lymphocytes

Nat. Rev. Immunol.

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Mol. Cell. Biol.

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J. Clin. Invest.

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EMBO J.

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Nat. Med.

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Mol. Biol. Cell

Congenital semilunar valvulogenesis defect in mice deficient in phospholipase C epsilon

Mol. Cell. Biol.