Small GTPase Rap1 Is Essential for Mouse Development and Formation of Functional Vasculature

Background Small GTPase Rap1 has been implicated in a number of basic cellular functions, including cell-cell and cell-matrix adhesion, proliferation and regulation of polarity. Evolutionarily conserved, Rap1 has been studied in model organisms: yeast, Drosophila and mice. Mouse in vivo studies implicate Rap1 in the control of multiple stem cell, leukocyte and vascular cell functions. In vitro, several Rap1 effectors and regulatory mechanisms have been proposed. In particular, Rap1 has been implicated in maintaining epithelial and endothelial cell junction integrity and linked with cerebral cavernous malformations. Rationale How Rap1 signaling network controls mammalian development is not clear. As a first step in addressing this question, we present phenotypes of murine total and vascular-specific Rap1a, Rap1b and double Rap1a and Rap1b (Rap1) knockout (KO) mice. Results and Conclusions The majority of total Rap1 KO mice die before E10.5, consistent with the critical role of Rap1 in epithelial 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 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 endothelium–for 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 vitro studies 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 [4][5][6][7] has raised interest due to its potential significance for human 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 [9][10][11][12], 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 functions in vivo have 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 yeast S. cerevisiae, Rap1 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 involving atypical PKC (aPKC) and Canoe/Afadin [17][18][19]. 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,[25][26][27][28] 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 double Rap1a, Rap1b KO 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 and Afadin.
At E13.5 and E15.5, about 20-50% of Rap1b -/embryos on C57Bl6 background displayed prominent hemorrhage on the side of the head, indicative of cardiovascular defects, while other Rap1b -/had no overt defects ( Fig 1B). Similarly, at E18.5 ( Fig 1C) this bleeding phenotype was only displayed in about 25% of Rap1b -/embryos. With 97% lethality at weaning (Table 1), we conclude that most Rap1b -/mice die perinatally and Rap1b is not absolutely required for embryonic development.

Early embryonic lethality of Rap1a, Rap1b KO mice
In lower organisms Rap1 is critical for morphogenesis [18] and deletion of the only isoform of Rap1 present in these organisms leads to lethality [37]. Since deletion of either Rap1a or Rap1b genes 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. To test if either Rap1 gene is essential for development, we attempted to generate a double Rap1a, Rap1b KO ("Rap1 KO") by intercrossing Rap1a +/and Rap1b +/mice, but no viable Rap1 KO mice were obtained ( Table 2). We therefore examined the effect of Rap1 deletion on embryonic development. We found that at E8.5 all expected embryos were recovered and appeared grossly normal, which indicates that Rap1 is not absolutely required for early development. However at E10.5, a reduced number of Rap1 KO embryos was recovered and only about 20% Rap1 KO embryos appeared viable (Table 3), while the remaining 80% of Rap1 KO embryos appear grossly underdeveloped and abnormal (Fig 2). Thus, we conclude that, unlike in lower organisms [38], Rap1 in mice is not critical for early embryonic events, such as gastrulation; but it is required for further development.

Endothelial-specific double KOs suffer from hemorrhage but branchial arch formation appears normal
To determine the cause of death of EC-Rap1 KO mice, we examined embryos from staged Tie2-Cre +/0 : Rap1a f/+ , Rap1b f/+ intercrosses. At E10.5 we found that a significant fraction of EC-Rap1 KO embryos were defective compared to WT E10.5 embryos (Figs 3 and 4) with a different embryo size suggestive of different times of death. The defective embryos displayed tissue degeneration (Fig 3A-3F) and cranial hemorrhage on their right side (Fig 3D-3F), with vascular rapture in or around branchial arches as likely cause of death. Furthermore, some EC-Rap1 KO embryos displayed dilated microvessels in the vicinity of the neural tube and vascular engorgement of perineural vessels (Fig 4A and 4B). However, the remaining 50% of EC-Rap1 KO embryos appeared viable without obvious vascular or cardiac lesions (Fig 5; normal EC-Rap1 KO). Interestingly, branchial arch formation appeared normal (Fig 5E and 5F) and similar to WT E10.5 mice (Fig 6E and 6F). This is significantly different from KRIT1 or CCM2 KO mice [35,36]. The analysis at E15.5 did not reveal any viable EC-Rap1 KO embryos (Table 4). Considering the vascular phenotype at E10.5 (Fig 3), death likely resulted from vascular defects. Furthermore, viability of Rap1 mutants containing only one of four Rap1 alleles was reduced (Table 4). We observed that about 30% of E15.5 EC-Rap1a +/-Rap1b -/embryos displayed cranial hemorrhage, which contributed to increased lethality (45%) at weaning (Fig 7). No such hemorrhage was observed in EC-Rap1a -/-Rap1b +/embryos. Considering that Rap1b is the major Rap1 isoform in ECs in mice (EC-Rap1a +/-Rap1b -/contains less Rap1 protein than EC-Rap1a -/-R-ap1b +/mutant) [33], and that neither Rap1 isoform is absolutely required in endothelium for survival (Table 4), this suggests that it may be a quantitative effect and that a certain minimal level of Rap1 is critical for vessel development.

Discussion
Rap1 is a positive regulator of adhesion and a critical regulator of polarity-dependent morphogenesis in lower organisms [16,17,19]. In this paper we demonstrate that Rap1 is essential for   early morphogenesis in mice. Deletion of Rap1a and Rap1b separately has only a small effect on initial development and is not required for embryonic morphogenesis (Table 3). Global deletion of both Rap1 genes leads to major malformation and death before mid-gestation (E10.5). This phenotype is consistent with Rap1 playing a key role in adhesion and polarity in vivo also in higher organisms. Furthermore, we demonstrate that Rap1 in endothelium is critically required for vessel formation, as endothelial-specific deletion of both Rap1 isoforms leads to engorgement of perineural vessels, hemorrhage and embryonic lethality between E10.5 and E13.5. Therefore Rap1 is critical for both: tissue and vessel morphogenesis.
Earlier studies in lower organisms demonstrated the importance of the single Rap1 ortholog in tissue morphogenesis through its critical role in the regulation of polarity. Epithelial polarity, required for collective cell migration and tissue morphogenesis, is maintained by cell-cell junctions: tight junctions and cadherin-based adherens junctions, and, physically connected to them, polarized cytoskeleton networks [39]. Rap1 functional interaction with actin cytoskeleton linker Canoe/Afadin is essential to Drosophila morphogenesis [40]. Mechanistically, Rap1 localizes Afadin to cadherin in adherens junctions [19,41]. Global deletion of Afadin leads to developmental defects during gastrulation and embryonic lethality after E9.5, with loss of structures derived from ectoderm and mesoderm and improper adherens junctions' organization [42]. The severity of Afadin -/phenotype exceeds that of Rap1 KO, suggesting the existence of other regulatory mechanisms in the absence of Rap1.
Vessel stability is critical for embryo growth and development. Stabilization of vessels to allow circulation and withstand shear stress forces coincides with dynamic regulation of growth and remodeling. Thus, vascular permeability is tightly regulated and changes in permeability lead to pathologies, including hemorrhage and edema [43]. Our studies show that Rap1 is a critical regulator of vascular stability. Endothelial KO of both Rap1 isoforms leads to localized hemorrhage and tissue degeneration in 50% of double EC-Rap1 KO at mid-gestation. Several Rap1 effectors including KRIT1, RASIP1 and Afadin have been implicated in regulation of vascular permeability by controlling cell-cell junctions. Endothelial deletion of Afadin leads to a defect in postnatal angiogenesis in vivo and reduced VEGFR2 signaling [44], similarly to what we reported in EC-Rap1 KO mice [33]. Reduced viability of EC-Afadin KO mice at weaning has been attributed to angiogenesis defect during development [44], however no specific developmental vascular phenotypes have been described. RASIP1, another proposed junctional effector of the EPAC-Rap1 signaling axis, has been implicated in vascular lumen formation [45][46][47] and more recently, in stabilization of cell-cell junctions required for formation of normal vasculature [48]. RASIP1-KO vessels initially form but are unable to sustain circulation beyond E10.5 in mice, leading to focal hemorrhage and lethality [48]. Similarity of RASIP1 and Afadin KO phenotypes to that of EC-Rap1 KO mice suggests that functional interaction of these molecules is important for murine vascular development. However, approximately half of EC-Rap1 KO embryos are able to form functional vessels and appear grossly normal at midgestation ( Fig 5). This, again, suggests a regulatory rather than critical role of endothelial Rap1 in early vasculogenesis. KRIT1 has been implicated as a cell-cell junction target of Rap1 required for vascular stability [6,49]. Global deletion of KRIT1and CCM2 leads to early and severe vascular pathologies that result in embryo lethality mid-gestation. Vascular defects in KRIT1-null embryos include dilatation of brain vessels and a branchial arch formation defect [35,50] and endothelial-specific deletion of KRIT1 leads to lethality before E12.5 due to failed vascular development [51]. Global or endothelial-specific CCM2 KO mice die of cardiovascular defects, such as: insufficient vascular lumen formation, defective arteriogenesis and heart malformation [36,52]. All three CCM genes are essential for embryonic angiogenesis [14,53,54]. Interestingly, here we find that EC-Rap1 KO phenotype is distinct from that of KRIT1, as branchial arches form normally in these embryos (Fig 4). Therefore, Rap1 and CCM proteins may not act in the same signaling pathways during development. However, these interactions may still be pertinent in the regulation of cerebral vascular integrity; consequently, their disruption might result in postnatal development of brain hemorrhagic lesions [8,55].
In addition to directly controlling the function of the above putative effectors, Rap1 may regulate vascular stability via additional mechanisms. We have recently shown that Rap1 is a critical regulator of mechanosensing complexes and shear stress-regulated vessel homeostasis in adult mice [34]. Interestingly, vascular defects observed in endothelial KRIT1-deficiency have also been associated with a defect in endothelial flow response [51]. It is possible that observed EC-Rap1 KO bleeding and embryo lethality might arise from physiologic/hemodynamic abnormality and defective mechanosensing functions, a hypothesis to address in future studies. In addition to the mechanosensing function in endothelium, Rap1b controls vascular tone and blood pressure by limiting smooth muscle contractility [32]. Interestingly, the phenotype of total Rap1b -/mice: hypertension and cardiac hypertrophy, phenocopies that of EC-Ra-p1a +/-Rap1b -/mice. This suggests that similar hemodynamic defects may contribute to the observed perinatal lethality of pups of the two genotypes.
This study has provided an insight into discrete and redundant functions of two Rap1 isoforms. During initial development, the functions of Rap1a and Rap1b are at least somewhat redundant, as development of the majority of embryos is supported upon single isoform deletion (Table 3) [22,23]. However, later in development Rap1b -/-, but not Rap1a -/-, embryos develop hemorrhages (Fig 1) and Rap1a -/embryos develop edema [23](and data not shown). This suggests that the Rap1b isoform may be a more critical regulator of vascular stability during development. While this bleeding phenotype is not observed in EC-Rap1b KO, additional deletion of one Rap1a allele results in a bleeding phenotype similar to that of global Rap1b KO   (Fig 7). This suggests that Rap1 in other, non-endothelial cells is required for vessel homeostasis. Such a conclusion is supported by our findings of the importance of Rap1b in smooth muscle cells in maintenance of vascular tone [32]. Thus, additional studies of Rap1 in other