Essential role for Gα ₁₃ in endothelial cells during embryonic development

Abstract

Toward identifying the roles of protease-activated receptor-1 (PAR1) and other G protein-coupled receptors important for vascular development, we investigated the role of G␣ 13 in endothelial cells in the mouse embryo. LacZ inserted into G␣13 exon 1 was highly expressed in endothelial cells at midgestation. Endothelialspecific G␣13 knockout embryos died at embryonic days 9.5-11.5 and resembled the PAR1 knockout. Restoration of G␣ 13 expression in endothelial cells by use of a Tie2 promoter-driven G␣13 transgene rescued development of endothelial-specific G␣ 13 knockout embryos as well the embryonic day 9.5 vascular phenotype in G␣ 13 conventional knockouts; transgene-positive G␣13 ؊/؊ embryos developed for several days beyond their transgene-negative G␣ 13 ؊/؊ littermates and then manifested a previously uncharacterized phenotype that included intracranial bleeding and exencephaly. Taken together, our results suggest a critical role for G␣ 13 in endothelial cells during vascular development, place G␣13 as a candidate mediator of PAR1 signaling in this process, and reveal roles for G␣ 13 in other cell types in the mammalian embryo.F ormation of blood vessels during mammalian embryonic development is a complex and highly regulated process. Angioblasts proliferate, migrate, and differentiate to form primitive vascular structures composed of endothelial cells. These structures remodel by sprouting, branching, growing, and regressing, and mature by the recruitment and differentiation of pericytes and smooth muscle cells (1-3). Studies to define the molecular signals that orchestrate vascular development have focused mainly on receptor tyrosine kinases and integrins and their ligands (1,(4)(5)(6)(7)(8)(9). Less is known regarding the roles of G protein-coupled receptors (GPCRs) in this process (10-16).Protease-activated receptor-1 (PAR1), a GPCR for thrombin, plays an important role in the formation and͞or maintenance of blood vessels in mouse embryos, a role likely attributable to PAR1 function in endothelial cells (11,14). PAR1 triggers a host of cellular responses through heterotrimeric G proteins of the Gq͞11, Gi͞o, and G12͞13 families; which of these pathways is important for proper vascular development is unknown. Conventional knockout of the gene-encoding G␣ 13 caused death of mouse embryos at midgestation with a phenotype grossly similar to, but more penetrant than, the PAR1 phenotype (17). We report a key role for G␣ 13 in endothelial cells at midgestation, suggesting that loss of G␣ 13 signaling in endothelial cells may account for the embryonic phenotype associated with PAR1 deficiency. Our results also unmask roles for G␣ 13 in other cell types. Materials and Methods Mutant Mouse Strains. Generation of the G␣13-lacZ knockin allele.Gna13 (herein designated G␣ 13 ) gene fragments from a bacterial artificial chromosome from a 129͞SvJ mouse genomic library, along with the lacZ gene, were used to construct a G␣ 13 -lacZ knockin allele as described in Supporting Methods, which is published as supporting informat...

A Role for the G12 Family of Heterotrimeric G Proteins in Prostate Cancer Invasion

“…In addition to the study noted above demonstrating a role for G 12 proteins in breast cancer metastasis (30), reports have appeared suggesting a role for these proteins in neutrophil (25) and fibroblast migration (50). Further, studies on the role of G 12 signaling in development have suggested that genetic ablation of the G 12 proteins results in impaired cell migration (21)(22)(23)(24). Interestingly, some of these studies that were conducted in Drosophila and zebrafish, revealed that signaling through the G 12 family is required for the cell shape changes and movements required for normal gastrulation.…”

“…G 12 Signaling Promotes Prostate Cancer Cell Invasion-Since previous studies have suggested a role for the G 12 proteins in cell migration (21)(22)(23)(24)(25) and cancer cell invasion (30), we next examined the role of G 12 signaling in prostate cancer cell invasion. We found that expression of the activated forms of G␣ 12 or G␣ 13 in the PC3 (Fig.…”

“…These findings led to the hypothesis that GPCRs may signal through the G 12 proteins to promote tumorigenesis and tumor cell growth (20). Interestingly, however, studies that examined the role of the G 12 proteins in development found that G 12 proteins were not required for cell growth but were critical for cell movement in the developing embryo (21)(22)(23)(24)(25). Since similar cellular movements underlie cancer cell invasion (26 -29), these findings suggest that G 12 signaling may also play a role in cancer metastasis.…”

Protease-activated Receptor-1 (PAR1) Acts via a Novel Gα13-Dishevelled Axis to Stabilize β-Catenin Levels

“…In contrast, G␣ 12 Ϫ/Ϫ mice appear viable and grow normally. In addition, during vascular development, a selective and critical role for G␣ 13 in endothelial cells has been demonstrated (55). It therefore appears that G␣ 13 plays a significant role in PAR1-induced developmental processes.…”

Identification of Mouse 8-Cell Embryo Stage-Specific Genes by Digital Differential Display

“…The Nsd1 (M.m.168965) belongs to an emerging family of proteins, Nsd1is a developmental regulatory protein that is essential for early post-implantation development [24]. Gna13 (M.m.193925) plays an important role in embryonic vascular development as a candidate mediator of protease-activated receptor-1 (PAR1) signaling [25]. Architectural chromatin protein HMGA2 (M.m.157190) is highly expressed in male meiotic cells.…”