A Combinatorial Role of Angiopoietin-1 and Orphan Receptor TIE1 Pathways in Establishing Vascular Polarity during Angiogenesis  Siobhan Loughna, Thomas N Sato  Molecular Cell  Volume 7, Issue 1, Pages 233-239 (January 2001) DOI: 10.1016/S1097-2765(01)00171-X

Figure 1 Polarized Branching and Left–Right Asymmetry in Vascular Network Formation (A) Three-dimensional schematic diagram of a vessel and its branches. Vessels can branch out from any point in any direction in a polarized manner. (B) Schematic diagram of the principal vessels of the embryonic venous system. The vasculature shows left–right symmetry early on in development but with increasing right-sided dominance and vessels either undergoing regression or enlargement; the venous system shows increasing left–right asymmetry as development proceeds. The umbilicovitelline system forms the vitelline veins, umbilical veins, ductus venosus, and hepatoportal system. The cardinal veins form the rest of the venous system (modified from Bannister et al. 1995). Polarized vessel branching establishes this asymmetric vascular network. ACV, anterior cardinal vein; AV, azygos vein; CCV, common cardinal vein; CIV, common iliac veins; DV, ductus venosus; HV, hemizygous vein; HpV, hepatic vein; IJV, internal jugular vein; IPC, interpostcardinal; ISC, intersubcardinal; IVC, inferior vena cava; LGV, left gonadal vein; LRV, left renal vein; LSV, left suprarenal vein; PCV, posterior cardinal vein; PV, portal vein; S, subclavian; SCV, subcardinal vein; SupCV, supracardinal vein; SIV, superior intercostal vein, SV; sinus venosus; SVC, superior vena cave; UV, umbilical vein; and VV, vitelline vein. Molecular Cell 2001 7, 233-239DOI: (10.1016/S1097-2765(01)00171-X)

Figure 2 Polarized Expression of Ang-1 in the Atrium and Sinus Venosus at E9.5 (A) Schematic diagram of the heart and connecting major vessels at E9.5. The inflow portion of the heart is the caudally located sinus venosus. The left and right horns of the sinus venosus connect to the developing systemic venous circulation via the left and right common cardinal veins, respectively. The anterior cardinal veins, which drain cranial regions, and the posterior cardinal veins, which drain caudal regions, both connect to the common cardinal veins. Further connections to the sinus venosus are the vitelline veins from the yolk sac and umbilical veins from the placenta (modified from Edwards 1998). (B and C) In situ hybridization whole-mount (B) and section (C) of an E8.5 embryo. Ang-1 is expressed symmetrically to the sinus venosus of the heart (yellow arrowheads). (D–G) In situ hybridization on tissue sections of an E9.5 embryo. Ang-1 is expressed in the atrium (D) and sinus venosus (E), except to their ventral left walls (yellow arrows). Both TIE1 (F) and TIE2 (G) are expressed uniformly in the endothelial layer of sinus venosus. LA, left atrium; RA, right atrium; LHS SVH, left-hand side sinus venosus horn; and RHS SVH, right-hand side sinus venosus horn. The scale bar is 100 μm. Molecular Cell 2001 7, 233-239DOI: (10.1016/S1097-2765(01)00171-X)

Figure 3 Requirement of Ang-1 and TIE1 Specifically for Normal Right-Side Venous Formation at E9.5 (A) Ang-1 is an activating ligand for the TIE2 receptor, whereas Ang-2 is an antagonist for this pathway. Tie1 is an orphan receptor. (B) Whole embryos immunostained with a panendothelial marker, CD31. A distinctive normal RHS anterior cardinal vein can be seen in all genotypes (outlined with yellow dotted line) except Ang-1−/− TIE1−/−, where no continuous vessel lumen could be detected. Instead, a network of small disorganized vessels is detected (red arrows). On the LHS, all genotypes have a normal anterior cardinal vein (yellow dotted line). This was confirmed on sectioning with analysis using differential interference contrast (DIC) optics. A normal anterior cardinal vein (ACV) and posterior cardinal vein (PCV) can be seen for all genotypes on the left side and right side (yellow arrows) except for Ang-1−/− TIE1−/−, where both of these vessels are abnormal specifically on the RHS (red arrows). Three to ten embryos of each genotype were analyzed, each of 20–25 somites. The scale bar is 100 μm. (C) High magnification of CD31 immunostained Ang-1−/− TIE1−/− sections using DIC optics. Red arrows point to abnormal anterior and posterior cardinal veins on the RHS. Note the cluster of CD31-positive cells and the very small discontinuous lumen. On the LHS, both vessels exhibit normal large and distinctive lumen (yellow arrows). The scale bar is 25 μm. (D) Schematic diagram showing the failure to form the RHS anterior cardinal and posterior cardinal veins in the Ang-1−/− TIE1−/− knockout. Scattered endothelial cells (black dots) were seen, and the only small discontinuous vessel lumens (red) were observed. All other genotypes had normal anterior and posterior cardinal veins on both the RHS and LHS. ACV, anterior cardinal vein; CCV, common cardinal vein; and PCV, posterior cardinal vein. In addition to the presented genotypes, Ang-1−/− TIE2−/−, Ang-2−/− TIE2−/−, and Ang-2−/− TIE1−/− embryos and all the double-heterozygous embryos were also studied, and they exhibit no difference in the RHS and LHS vascular system at E9.5 (data not shown). Molecular Cell 2001 7, 233-239DOI: (10.1016/S1097-2765(01)00171-X)

Figure 4 Ang-1/Tie1 Pathway Is Involved in an Early Phase of the Right-Hand Side Anterior Cardinal Vein Formation Whole embryos immunostained with a panendothelial marker, CD31 (A, B, C, G, and H). Sections of the wild-type embryos shown in (A)–(C) analyzed using DIC optics (D–F). Although difficult to detect in the whole wild-type E8.5 embryo (A), the primitive anterior cardinal vein can be seen as a very small indistinct lumen on the right side on sections (yellow arrowhead), whereas a lumen could not yet be detected on the left side (D). By E9.0 in a wild-type embryo, the anterior cardinal vein can be seen in the whole embryo (yellow dotted line) (B) and on sections (yellow arrowheads) (E). In a E9.5 wild-type embryo, the anterior cardinal vein is a distinct large vessel that is clearly detected in both the whole embryo (C) and on sections (yellow arrowheads) (F). An abnormal anterior cardinal vein is seen on the right side of an E9.0 Ang-1−/−TIE1−/− embryo (G). The vessel appears tortuous and thinner than a wild-type control (H). Anterior cardinal vein on the left-hand side of an E9.0 Ang-1−/− TIE1−/− embryo was normal (data not shown). Three embryos of each genotype were analyzed. The scale bar is 100 μm. DA, dorsal aorta; LHS SVH, left-hand side sinus venosus horn; and RHS SVH, right-hand side sinus venosus horn. Molecular Cell 2001 7, 233-239DOI: (10.1016/S1097-2765(01)00171-X)