Table 1.

Animal Models of EphrinB2-EphB4-RASA1 Signaling Axis

EFNB2 Animal Models
Animal Model	Genotype	Phenotype	Description	Ref
Mouse	ephrinB2tlacZ	Defects in angiogenesis by both arteries and veins in the capillary networks for the head and yolk sac as well as in myocardial trabeculation	Vasculogenesis occurred normally between E8.5 and E9.0; apparent pronounced disorganization at E10; defect in yolk sac angiogenesis by E9; absence of internal carotid arterial branches; defective angiogenesis of venous capillaries in the head; capillary ingrowth into the neural tube failed to occur	[2]
Mouse	ephrinB2ΔC/ΔC(ephrinB2-ΔC-HA fusion protein)	Able to restore guidance of migrating cranial neural crest cells, but have defects in vasculogenesis and angiogenesis and midgestation lethality	Lethal at midgestation (E10.5-E11) with growth retardation and inflation of the pericard; normal formation of branchial arches; defects in vascular morphogenesis	[17]
Mouse	ephrinB2lacZ/loxp;Tie2-Cre+(endothelium and endocardium specific deletion of ephrinB2)	Angiogenic remodeling defect identical to ephrinB2 global knockout mice(Efnb2tml/tml)	Growth retardation; swollen but still beating heart with little blood flow; disorganized and less developed vasculature compared to WT; arrest in development of arteries and veins at the primary plexus stage in yolk sac; failure in anterior cardinal vein (ACV) assembly and remodeling; initial head plexus forms but remains in disorganized primitive stage	[20]
Mouse	CAGp-ephrin-B2 Tg (gloal overexpression of ephrinB2)	Abnormal segmental arrangement of intersomitic vessels; die of acute aortic dissecting aneurysms at neonatal stages	Thin vascular wall in the ascending aorta and aortic arch; ECs have a flat morphology with a budlike structure; SMCs and elastic bands are missing, while microvessels are seen in these spaces; abnormal vasculature in ascending and arch portion of the aorta	[25]
Mouse	Tie-2p-ephrin-B2 Tg (vascular ECs specific overexpression of ephrinB2)	Cranial hemorrhages with incomplete penetrance	Intracerebral bleeding	[25]
Mouse	ephrinB2ΔC-βgal/ΔC-βgal (ephrinB2 C-terminal truncation and βgal fusion protein)	Severe hypospadias and incomplete midline fusion of the primitive cloaca with incomplete penetrance (heterozygotes) Failure in cloacal septation with complete penetrance (homozygotes)	Severe anorectal malformations with an absence of the terminal-most hindgut and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder.	[21]
		Grossly normal with no early vascular defects, but die within the first day of birth due to cardiac defects	Normal chamber formation and vascular connection; thickened cardiac valves; disrupted pathfinding of anterior commissure axons; defective maturation of cardiac valve leaflets.	[18]
Mouse	ephrinB2T/T (ephrinB2 with C-terminal truncation)	Embryonic lethal with a failure in cardiovascular development	Visibly abnormal, appearing growth-retarded and necrotic, had enlarged pericardia, displayed abnormal heart and vascular structures	[18]
Mouse	Efnb2ΔPC/vSMC (mural cell specific deletion of ephrinB2)	Perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries	Develop to term but die shortly after birth due to respiratory failure; edema and extensive hemorrhaging in the skin; pale intestines and blood filling mesenteric lymphatics; hemorrhaging in the lung; comma- and S-shaped bodies developed normally, but the more mature glomerular tufts were poorly organized	[24]
Mouse	Efnb2GFP/GFP; Tg (ECs specific expression of ephrinB2 in the background of Efnb2 null)	Similar growth retardation, and heart morphogenesis defects as observed in Efnb2 null mice, but the early lethality associated with Efnb2 null mutation is partially rescued	Lethality earlier than E9.5 likely due to mixed genetic background of mice used in this study; partial rescue of the lethal phenotype of the Ephb2 null mice by E10.5; bleeding in the pericardial cavity; intersomitic vessel network appeared fused dorsally and poorly branched	[23]
Mouse	Efnb2CR/GFP; Tie2-CreTg/+ (vascular ECs specific expression of ephrinB2 in the background of Efnb2 null)	Perinatal lethality; normal angiogenesis; exhibit tracheoesophageal and urorectal fistulae as well as an incorrectly positioned thymus, while other bilateral structures including lungs, vena cava, and clavicles appear to be the same as controls.	Overtly normal at E9.5 without signs of angiogenic defects; normal remodeling of the primitive vascular plexus into hierarchically branched vessels at E9.5; survived to birth but died perinatally; no defects in branchial arches morphogenesis or cranial or trunk neural crest development	[22]

EPHB4 Animal Models
Animal Model	Genotype	Phenotype	Description	Ref
Mouse	EphB4taulacZ/taulacZ	Cardiovascular defects and embryonic lethality with very high penetrance	Growth retardation, arrested heart development, and lack of blood flow by E9.5-E10; degeneration and necrosis throughout the embryo by E10.5; incomplete heart looping at E8.75-E9.0; arrest of the remodeling of the primitive dilated vessels in head; disrupted development of the ACV and both arterial and venous intersomitic vessels; heart failed to increase in size, with incomplete cardiac looping and endocardium expansion; angiogenic remodeling arrest in the yolk sac with the phenotype being more severe on the venous than the arterial sides	[6]
Mouse	Ephb4f/f back crossed with smMHC-Cre-IRES-eGFP tg (SMC specific EphB4 knockout)	Hypotension in male but not female knockout mice	significantly reduced systolic pressure and mean arterial pressure with normal heart rate compared to WT in male but not female knockout mice	[37]

RASA1 Animal Models
Animal Model	Genotype	Phenotype	Description	Ref
Mouse	Rasa1−/− (RASA1 null)	Embryonic lethal by day E10.5 with blood vessel abnormalities	16-somite stage: thinning of dorsal aorta, aberrant ventral branches resemble inter-segmental arteries; E9.5: yolk sacs visibly abnormal (rough wrinkled appearance), embryonic vasculature exhibits local ruptures	[57]
Mouse	Rasa1R780Q/R780Q (knock-in expressing a mutant RASA1 that lacks an arginine finger)	Mid-gestation embryonic lethal with blood vessel abnormalities	E9.5: embryos small and less developed, distended pericardial sac, wrinkled yolk sac, no organized blood vessel networks, irregularly shaped dorsal aorta, severely disrupted and irregularly shaped inter-segmental arteries	[60]
Mouse	RASA1fl/fl/Uber2cre (conditional rasa1 floxed allele and an inducible Cre-ERT2 transgene under the control of a ubiquitin promoter)	Lymphatic vessel abnormalities, death from chylothorax	8 weeks post-tamoxifen administration to adult mice: hyperplasia of initial lymphatics, dilation of initial and collecting lymphatics, contractility of conducting or collecting lymphatics, chylothorax, in some cases chylous ascites	[63]
		Complete loss of RasGAP expression	Complete loss of RasGAP expression, exon 17 spliced exclusively to exon 19 (not to other downstream exons)	[61]
		Death from chylothorax, lymphatic vessel abnormalities, increased density of lymphatic vessels within and inside the chest wall	Death by chylothorax by 8 months post-tamoxifen administration, chyle found in peritoneal cavity, dye injected into the footpads/tail leaked into peritoneal cavity instead of draining into thoracic duct, dye did not follow the typical route of lymph drainage to inguinal LN (drained into an expansive lymph vessel network instead of taking a direct route to the LN), increased density of lymphatic vessels within and inside chest wall facing the pleural cavity, dilated/increased number of lymphatic vessels in skin and lungs, no pulsatile activity in hyperplastic draining lymphatics, majority of their lymph vessels not lack smooth muscle	[62]
		Impaired development of LV valves, chylothorax	TM injection at 9 weeks: LVs ineffective as pumps, downstream valves re-opened inappropriately during the diastolic phase of vessel contraction and were unable to close properly in response to adverse intra-luminal pressure leading to back-leak, LV leaflets contained reduced amounts of ECM proteins; TM between E12.5-E14.5: complete absence of the LV system at E19.5 as a result of LEC apoptosis, TM at E15.5: mesenteric LVs that lack valves and appear dilated with increased # of cells	[64]
Mouse	RASA1fl/fl/Prox1er2cre (conditional floxed rasal allele and lymph vessel specific inducible CreERT2 transgene)	Lymphatic vessel abnormalities, chylothorax	TM injection at 2 months: dilated increased number of lymphatic vessels in skin, no evidence of early lethality, chylothorax, or chylous ascites; TM at E15: chylothorax, death, lymph vessel hyperplasia due to increased LEC proliferation, phenotype rescued by administration of anti-VEGF-3 antibody	[62]
		Lymphatic valve dysnfunction and LV leaflet changes	The majority of valves dysfunctional including both mesenteric and popliteal LV valves, LV leaflets contained reduced amounts of ECM proteins	[64]
Mouse	RASA1fl/R780Q/Uber2cre(conditional floxed rasa1 knock in allele with an inducible CreERT2 transgene)	Chylothorax, LV hyperplasia	TM injection at E15.5: death by to chylothorax, mesenteric LVs lack valves, sometimes appear dilated with increased numbers of cells	[62]
Zebrafish	Knock down of RASA1 homologs using antisense MOs	Cell death in the head region, improper formation of the caudal vascular plexus	Widespread cell death in head region and improper formation of caudal vascular plexus resulting in enlarged caudal vascular deformity (caused arterial blood flow to abruptly return to the posterior cardinal vein), phenotype rescued by administration of PI3K-TORC1 chemical inhibitors (but not MEK1/2 inhibitor)	[8]