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First published online 17 December 2003
doi: 10.1242/dev.00929

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Flow regulates arterial-venous differentiation in the chick embryo yolk sac

¹ Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands
² Inserm U36, Collège de France, 11, Place Marcelin Berthelot, 75005 Paris, France
³ Department of Anatomy, University of Bern, Switzerland
⁴ Department of Physics, Ecole Polytechnique, Palaiseau, France

View larger version (139K): [in a new window]   Fig. 8. Ligation model. Ligation of the right vitelline artery of a 30 ss chick embryo in ovo. (A) Overview of an embryo 24 hours after ligation. Note venularization of the ligated vitelline artery (arrow). The unligated vitelline artery shows enhanced growth across the midline (arrowheads). (B-L) Still images of time-lapse videomicroscopy of another ligated embryo taken at the indicated time points. The future major venous branch can be seen in H (arrow). Note incorporation of an entire branch of the vitelline artery into this vein (arrows, H-L). Asterisks in I-L indicate another arterial segment that becomes venularized. Scale bars: 945 µm in A; 715 µm in B-L.

View larger version (164K): [in a new window]   Fig. 1. Overview of arterial-venous differentiation in the yolk sac. (A) Yolk sac vessels just after the onset of perfusion. The vitelline artery (VA) is just beginning to form. Direction of arterial and venous blood flow is indicated by red and blue arrows respectively. The heart is indicated (*). (B) Schematic representation of vascular system as seen in the boxed area in A. Arteries (red arrows) carry blood away from the heart and veins (blue arrows) carry blood back toward the heart. Note that arteries and veins are in a cis-cis configuration. (C) Embryo 26 hours later than in A. (D) Schematic representation of the vascular system as seen in the boxed area in C. Veins are drawn in white, the direction of venous flow is shown by blue arrows. Arteries are drawn in black, arterial flow is indicated by the red arrows. Note that veins have come to lie parallel to arteries, a venous network is covering the arterial network dorsally. SV, sinus vein. Scale bar: 1100 µm.

View larger version (140K): [in a new window]   Fig. 2. Formation of the vitelline artery. Time-lapse video-microscopy of a 19 ss chick embryo in New culture. Endoderm is facing up. Still images at the indicated times are shown. Note the formation of the vitelline artery (black arrows), which becomes clearly visible at the 21ss (C). Red and blue arrows indicate arterial and venous blood flow, respectively. White arrowheads indicate capillary free-zones. Asterisks show distal progression of vitelline artery formation. (H) The posterior vitelline vein (blue arrow) is formed. Scale bar: 1500 µm.

View larger version (142K): [in a new window]   Fig. 3. Yolk sac vessel configuration and arterial marker expression following perfusion. (A) 12 ss and (B) 19 ss embryos. Whole-mount in situ hybridization with the indicated markers. (A,B) Prior to (A) and just after (B) perfusion, ephrinB2 (A) and NRP1 (B) label the posterior (arterial) pole (arrowheads; A,B). (C-E) 30 ss embryos. (C) Intracardiac FITC-Dextran injection. The left side of the yolk sac is shown. Blood from the vitelline artery (red arrow) flows directly into veins (blue arrow), without passing through a true capillary bed. (D) NRP1 labels the vitelline artery (red arrow), but not the vein (blue arrow). (E) The right side of the yolk sac is shown. Ephrin-B2 expression closely resembles NRP1 expression. The future territory of the posterior vitelline vein has downregulated NRP1 and Ephrin-B2 expression (arrowheads, D and E). (F) 40 ss embryo, the left side of the yolk sac is shown. In vivo protein binding of EphB4-Fc to arteries (A) expressing ephrinB2. Veins (V) are negative and are already interlaced with arteries at this stage. Scale bars: 420 µm in A,B; 400 µm in C-E; 190 µm in F.

View larger version (143K): [in a new window]   Fig. 4. Formation of the posterior vitelline vein. Time-lapse video-microscopy of a 19 ss chick embryo in ovo (dorsal side upwards). Still images taken at the indicated time points. Black arrows show the vitelline artery, black arrowheads the vitelline vein. Red and blue arrows (D-L) indicate direction of arterial and venous flow, respectively. Venous flow is first detected in F; there is rapid formation of the vitelline vein (arrowheads, F-L).

View larger version (120K): [in a new window]   Fig. 6. Mercox casts and QH1 whole-mount staining of the developing vitelline artery. (A,B) Scanning electron micrographs of mercox-filled vitelline artery (VA). (C,D) QH1 staining. QH1 endothelial surface staining shows the same morphology as the mercox cast of the vessel lumen. (A) Asterisks show capillary free-zones. Arrowheads indicate disconnected arterial capillaries that are perfused from distal parts and that correspond to the blood filled `spots' in Fig. 5. (B,D) Higher magnification of the boxed areas in A, C, respectively. Blind ending sacs (B,D, arrows) are present on both sides of the vitelline artery and there are corresponding protrusions on opposite capillary segments (B, double arrows). Note the presence of a capillary sprout (Sp) dorsal to the vitelline artery (VA). Scale bars: 160 µm in A; 70 µm in B; 125 µm in C; 20 µm in D.

View larger version (154K): [in a new window]   Fig. 5. Reconnection of disconnected arterial capillaries to the venous system. Still images of time-lapse video microscopy of a 30 ss chick embryo in ovo taken at the indicated time points. Red and blue arrows indicate direction of arterial and venous flow, respectively. (A-I) There are blood filled `spots' at both side of the vitelline artery (VA). Venous flow at this stage is only detected in two small veins (blue arrows). Sprouts originating from these `spots' cross over the VA (D-H, black arrows). As soon as the sprouts connect to the vein, the blood filled `spots' are flushed out (compare H with I, black arrows). The same process can be observed more distally (compare J-L, black arrows). Scale bar: 455 µm.

View larger version (43K): [in a new window]   Fig. 7. No-flow and incision models. (A) Bright field photomicrograph of a no-flow yolk sac of an E5 quail embryo. Note the absence of arteries and veins. (B,C) Whole-mount in situ hybridization of an E5 quail embryo no-flow yolk sac with an ephrinB2 anti-sense riboprobe. Part of the capillary plexus expresses ephrinB2 (B), other parts are ephrinB2 negative (C). (D) Incision model. 35 ss embryo 24 hours after incision. The incision site separating the embryo from the left side of the yolk sac is indicated (stippled area). A single vitelline vein (VV) has developed on the left side. The left vitelline artery (VA) is absent. The right VA shows increased growth. Asterisk indicates the heart. Scale bars: 300 µm in A; 50 µm in B,C; 600 µm in D.

View larger version (72K): [in a new window]   Fig. 9. Whole-mount in situ hybridization of ligated embryos with arterial markers. Ligations were performed for the indicated time periods, and embryos were hybridized with the indicated antisense riboprobes. Arrows (A-H) show ligation site. (A-F) Arterial markers. Note arterial expression on the left side of all embryos and progressive downregulation of NRP1 (A-D) and ephrinB2 (E), as well as the absence of binding of EphB4-Fc (F) on the ligated side. (G-J) Venous markers. (G,H) No upregulation of venous marker expression is detectable after 1 hour of ligation. (I,J) 12 hours after ligation. Tie2 and NRP2 mRNA are not expressed in the anterior (I) or posterior (J) left vitelline arteries (A) that cross over the midline, although they are both expressed in venularized arteries (arrowheads) on the ligated side. Scale bars: 530 µm in A,C,D; 510 µm in B; 725 µm in E; 230 µm in F; 700 µm in G,J; 880 µm in H; 435 µm in I.

View larger version (93K): [in a new window]   Fig. 10. Removal of the ligation tool restores arterial marker expression. (A) Vessel integrity 4 hours after ligation demonstrated by Fitcdextran injection. Note retrograde perfusion of the ligated vitelline artery. Boxed region is shown in Movie 6 at http://dev.biologists.org/supplemental. (B,C,F) The ligation tool was removed after the indicated time points, and embryos were fixed 6 hours later. Whole-mount in situ hybridization with NRP1 antisense riboprobe shows re-expression after 30 minutes (A) and 4 hours (B), but not after 6 hours (F). (D,E) Photomicrographs of a live embryo treated as the embryo in B. Note decreased size of the vascular plexus on the ligated side. (E) Higher magnification of the boxed region in D. Arterial blood flow is shown in red, venous blood flow in blue. Arrows indicate ligation site. Scale bars: 760 µm in A; 725 µm in B,C,F; 1400 µm in D; 780 µm in E.

View larger version (182K): [in a new window]   Fig. 11. Application of ephrinB2-Fc and EphB4-Fc on the allantois. (A-I) Ephrin-B2 application. (J-L) EphB4 application. Still images of time-lapse video-microscopy taken at the indicated time points. Arteries (A) and veins (V) in the application area are indicated. Note increased venous branching (C,D, arrows), and rapid formation of arterial-venous shunts after ephrinB2-Fc application (arrowheads in F-I). Note enlargement of the artery in EphB4-Fc treated embryos (J-L) as well as formation of arterial-venous shunts (arrowhead, L). Scale bar: 830 µm.