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Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Delphine Moyon^*, Luc Pardanaud^*, Li Yuan, Christiane Bréant and Anne Eichmann

Institut d’Embryologie Cellulaire et Moléculaire CNRS FRE 2160, 49bis, Avenue de la Belle Gabrielle, Nogent-sur-Marne Cedex 94736, France
^* These authors have contributed equally to this work

View larger version (85K): [in a new window]   Fig. 1. Expression of NRP1 becomes restricted to the arterial compartment of the blastodisc. Whole-mount in situ hybridizations with the indicated antisense riboprobes. (A) 4ss, NRP1 is expressed throughout yolk sac blood islands and in the heart anlage (arrow). (B) 14ss, (C) 21ss. NRP1 expression is lost from EC in the anterior lateral mesoderm (asterisk in A-E). Arrowheads in B point to the dorsal aorta. (D) VEGFR2 labels both anterior and posterior lateral mesoderm at the 14ss. (E) 25ss. NRP1 expression is observed in the omphalomesenteric arteries (arrows) and their branches. (F) Schematic representation of the embryonic and extra-embryonic circulation at the 25ss (redrawn from Gilbert, 1994).

View larger version (121K): [in a new window]   Fig. 2. NRP1 and TIE2 label extra-embryonic arterial and venous EC, respectively. (A,B) Whole-mount in situ hybridizations on yolk sacs of E3.5 chick embryos. (A) TIE2 is expressed on veins (arrows), but not on the parallel-running arteries (arrowheads). NRP1 (B) expression is the reciprocal. (C,D) Adjacent sections through the E6 chick allantois. VEGFR2 (C) labels EC of the umbilical artery (A) and vein (V). AS, allantoic stalk. (D) NRP1 (brown staining) is only observed in EC of the umbilical artery, while TIE2 (blue staining) is down-regulated in these EC (arrows) and transcribed in the veins. Mesenchymal cells surrounding arteries are also NRP1 positive (arrowheads). Bar in C, for C and D, 120 µm.

View larger version (117K): [in a new window]   Fig. 3. Intra-embryonic expression of NRP1, TIE2 and VEGFR2. (A,B) Adjacent transverse sections of an E3 quail embryo at the level of the limb bud hybridized with the indicated probes. (A) NRP1 is expressed in motoneurons (arrow) of the neural tube (NT), in future dorsal root and sympathetic ganglia (arrowheads), and in EC of the aorta (Ao). EC of the posterior cardinal vein (cv), omphalomesenteric vein (omv) and umbilical vein (uv) are negative. (B) TIE2 labels all EC. (C,D) Adjacent sections through the hindbrain of an E4 embryo at the level of the otic vesicle (OV). (C) NRP1 is expressed in the internal carotid artery (ica), but not in the anterior cardinal vein (cv). (D) TIE2 shows the complementary expression. (E,F) Adjacent sections through aortic arches (AA) 3, 4 and 5 of an E6 chick embryo hybridized with the indicated probes. (E) VEGFR2 is detectable on the majority of EC in both the aortic arches and the anterior cardinal vein (cv). (F) NRP1 (brown staining) is only detectable in aortic arch EC, few of which (arrows) also express TIE2 (blue staining). (G) Section through the leg bud of an E6 chick embryo. NRP1 (brown staining) is expressed in EC and some mesenchymal cells of the aorta and femoral artery (fa) and is strong in regions of arterial branching (arrows). TIE2 (blue staining) is expressed weakly or not at all in the femoral artery, but is expressed in the cardinal vein (cv) and the peripheral venous plexus of the limb (arrowheads). (H) Higher magnification of the boxed region in G shows the femoral artery EC expressing NRP1, while the underlying venous plexus is TIE2 positive. Capillaries branching from the femoral artery express both markers (arrows). N, notochord. Bars, (A,B,E,F) 130 µm; (C,D) 65 µm; (G) 290 µm; (H) 65 µm.

View larger version (107K): [in a new window]   Fig. 4. Expression of NRP1/TIE2 at later developmental stages. Sections through an E10 quail embryo. (A-C) Sections through connective tissue (A), skeletal muscle (B) and lung (C), double-labeled with NRP1 (brown) and TIE2 (blue) antisense riboprobes. Arteries (arrows), surrounded by a thick wall, are NRP1 positive but show weak TIE2 expression. Veins express TIE2 but not NRP1. Parallel-running streams of capillaries expressing NRP1 or TIE2 can be observed (A,B, arrowheads). (D-F) Triple-labeling of a section through the aorta (Ao) and lymphatic thoracic duct (Th) with the indicated markers. (D) Bright-field view, NRP1 is expressed in EC of the aorta and smaller vessel branching from it (arrow). (E) Dark-field view ; FITC--SMA staining shows a thick coat of positive cells surrounding the branching NRP1-positive vessel. (F) Dark-field view ; Texas Red-labeled QH1-positive EC of the lymphatic thoracic duct are NRP1 negative. (G-I) Section through the myocardium, treated with the same markers. All NRP1-positive arteries (arrows) are surrounded by -SMA+ cells, while NRP1-negative vessels (asterisks) are also -SMA negative. QH1 staining (I) shows the corresponding NRP1-positive arterial, and NRP1-negative venous, EC. Bars, (A) 90 µm; (B and C) 180 µm; (D-F) 95 µm; (G-I) 30 µm.

View larger version (93K): [in a new window]   Fig. 5. Grafts of early vascular rudiments. (A) Quail donors were at the 14ss. The boxed areas were dissected and grafted. The box labeled omv corresponds to the presumptive territory of the omphalomesenteric vein, while the box labeled oma corresponds to the territory of the omphalomesenteric artery. (B) QH1 (brown) and NRP1 (blue) double-staining of an E4 chick host that had received a graft (G) of territory oma 2 days before. Asterisks mark the connections of the graft to the host. QH1-positive/NRP1-positive EC are observed in the arterial system (arrows), including the aorta (Ao) and brachial artery (ba). QH1-positive/NRP1-negative cells are present in the venous system (arrowheads), including the posterior cardinal vein (cv), the subcardinal vein (scv), the umbilical vein (uv) and the peripheral plexus of the wing (W). M, mesonephros. Bar, 80 µm.

View larger version (177K): [in a new window]   Fig. 6. Grafts of arteries. (A-E) coelomic grafts, (F) dorsal graft. (A,D) Double-staining with QH1 (brown) and NRP1 (blue). (B) Double-staining with QH1 (brown) and ephrinB2 (blue). (C,E) QH1 staining. (F) QCPN/QH1 staining, both the nuclear QCPN and the membranous QH1 labeling are revealed by HRP-staining. (A) Graft (G) of an E5 aorta. QH1-positive/NRP1-positive arterial EC (arrows) have colonized the aorta while QH1-positive/NRP1-negative EC are present in umbilical and omphalomesenteric veins (arrowheads). (B) Section of the same embryo as in A, QH1-positive/ephrinB2-positive EC (arrows) are present in the aorta, while QH1-positive/ephrinB2-negative EC have colonized the subcardinal vein (arrowhead). (C) E15 aorta graft. Only the lateroventral aortic endothelium is colonized. (D) E15 aorta graft. QH1-positive cells in the aorta express NRP1 (arrow). NRP1 expression in the grafted aorta is strongest in the region of EC emigration (Ao, arrow). (E) E11 carotid artery graft. The graft is not visible on this section. QH1-positive EC only colonize the ventrolateral aortic endothelium (arrow). (F) Dorsal graft of an E14 aorta. QH1-positive cells colonize the dorsolateral aortic endothelium (arrow) but appear excluded from the cardinal vein. QCPN-positive/QH1-negative non-endothelial cells (arrowhead) do not migrate. Ao, aorta; cv, cardinal vein; G, graft; M, mesonephros; N, notochord; NT, neural tube; W, wing bud. Bars, (A,D) 140 µm, (B,F) 40 µm, (C,E) 100 µm.

View larger version (98K): [in a new window]   Fig. 7. Grafts of veins. (A,B) Coelomic grafts, (C) dorsal graft. (A) Double-staining with QH1 (brown) and NRP1 (blue) of an E4 chick host that had received a graft of an E5 jugular vein 2 days before. The graft is not visible on this section. Quail EC migrated into the subclavian and brachial artery (ba, arrows), as well as into the umbilical vein (arrowheads). (B) Graft of an E11 jugular vein. QH1-positive EC are found in the umbilical vein (arrowhead). (C) Dorsal graft of an E14 jugular vein. Quail EC have colonized the posterior cardinal vein (arrowhead), which was displaced medially in this graft together with the Wolffian duct (asterisk). G, graft. Bars, (A) 125 µm, (B,C) 90 µm.

View larger version (104K): [in a new window]   Fig. 8 EC plasticity in the absence of a vascular wall. (A,B) QH1 immunoreactivity (A), and phase contrast image (B) of EC isolated from E11 aortae. The majority of the cells are QH1 positive. (C,D) QH1 staining of a chick host that had received a graft of isolated EC, not visible in these sections. (C) Quail EC are found in the aorta (Ao) and the cardinal (cv) and subcardinal veins (scv, arrowheads). (D) Adjacent section, QH1-positive cells are found in the brachial artery (ba, arrows) and in the cardinal and umbilical veins (uv, arrowheads). Bars, (A,B) 85 µm, (C,D) 40 µm.