As deletion of eitherRap1aorRap1bgenes is not essential for development, as some KOs survive to adulthood, we hypothesized that there is a redundancy in Rap1a and Rap1b function in vertebrates. morphogenesis. At that time point, about 50% of Tie2-double Rap1 KOs appear grossly normal and develop normal vasculature, while the remaining 50% suffer tissue degeneration and show vascular abnormalities, including hemorrhages and engorgement of perineural vessels, albeit Mouse monoclonal to TDT with normal branchial arches. However , no Tie2-double Rap1 KO embryos are present at E15. 5, with hemorrhages a likely cause of death. Therefore , at least one Rap1 allele is required for development prior to the formation of the vascular system; and in endotheliumfor the life-supporting function of the vasculature. == Introduction == The evolutionarily conserved and ubiquitously expressed small GTPase Rap1 is at the crossroads of signaling pathways that govern key cellular processes. Downstream from multiple receptors, Rap1 activity is spatiotemporally regulated by a network of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) acting in a tissue-specific manner [1]. In a GTP-bound form, activated Rap1 interacts with a number of effectors to control cell-substrate adhesion, cell-cell adhesion and junction formation [2], and cell polarity [3]. In vitrostudies have implicated KRIT1/CCM1, a protein mutated in cerebral cavernous malformations (CCM), RASIP1 and an actin-cytoskeleton linker Canoe/Afadin as Rap1 effectors controlling cell-cell junction formation and maintenance. In particular, Rap1 interaction with KRIT1 [47] has raised interest due to its potential significance for human MC-Val-Cit-PAB-vinblastine disease [8]. KRIT1, one of three proteins whose autosomal mutations have been linked with CCM; a neurovascular malformation syndrome that leads to seizures and lethal stroke [912], is a multi-domain protein that links cortical actin cytoskeleton with integral membrane proteins, and interacts with CCM2 [13]. In vitro, in endothelial cells (ECs), Rap1 facilitates localization of KRIT1 to cell-cell junctions and interaction with junctional proteins [5, 14, 15]. However , whether Rap1-KRIT1 interaction plays a physiological role in the development of CCM is unknown. Rap1 functionsin vivohave been studied in several model organisms. In lower organisms, a single Rap1 ortholog plays a central role in the development of cell polarity: in budding yeastS. cerevisiae, Rap1 MC-Val-Cit-PAB-vinblastine ortholog Bud1/Rsr controls positioning of the bud [16] and in Drosophila, Rap1 controls apico-basal polarity during mesoderm formation and dorsal closure via its interactions with a protein network MC-Val-Cit-PAB-vinblastine involving atypical PKC (aPKC) and Canoe/Afadin [1719]. In higher organisms, two highly homologous isoforms of Rap1 exist: Rap1a and Rap1b. The two Rap1 isoforms are encoded by separate genes [20, 21] and murine genetic deletion models of both have been described [8]. Deletion of either isoform leads to partial embryonic lethality and bleeding [22, 23]. While deletion of either Rap1 isoform does not limit the lifespan of surviving adult mice, several defects in neurological [24] and immune responses [23, 2528] and hematopoiesis [29] have been described. Some of the most significant defects observed in Rap1 knockout (KO) mice involve their cardiovascular functions: platelet function [22], angiogenesis [30, 31], smooth muscle contractility and vessel tone [32]. The similarity of some of the phenotypes of the two Rap1 isoforms KOs suggested functional redundancy. To determine if the two isoforms have similar functions, we attempted to generate doubleRap1a, Rap1bKO mice. In this paper, for the first time to our knowledge, we report on the phenotype of these mice. Because of the bleeding phenotype in total Rap1-deficient embryos we have been particularly interested in the role of Rap1 in the vasculature. We have made endothelial lineage restricted Rap1 KO mice and demonstrated a critical role of Rap1 in endothelial cells in angiogenesis [32, 33] and, more recently, regulation of endothelial function and blood pressure [34]. Interestingly, molecular mechanisms underlying these defects in adult mice implicate Rap1 in regulation of the signaling aspect of adhesion receptors [32, 33], rather than in their role in promoting cell adhesion. However , the role of Rap1 in vasculature during morphogenesis and development is not known. To address this knowledge gap, here we describe the phenotype of Rap1 endothelial-specific KO mice and analyze it in the context of Rap1 effectors, KRIT1 MC-Val-Cit-PAB-vinblastine and Afadin. == Methods == == Animal generation and husbandry == All mouse procedures were performed according to approved Medical College of Wisconsin or Indiana University School of Medicine Institutional Animal Use and Care Committee protocols. Generation ofRap1b-/-mice and endothelial-specific Rap1 KO mice (EC-Rap1 KO; Tie2-Cre+/0Rap1af/fRap1bf/f) (with a.
Acyltransferases