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First published online 25 May 2006
doi: 10.1242/dev.02421

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Identification, emergence and mobilization of circulating endothelial cells or progenitors in the embryo

INSERM, Unit 36, F-75005, Paris, France and Collège de France, F-75005, Paris, France.

View larger version (78K): [in a new window]   Fig. 1. Survival of embryonic EPCs in the circulation and emergence of CECs. (A) Schemes illustrating, on the left, the preparation of E2 quail EPCs from somites (S) and splanchnopleural mesoderm (SpM) after enzymatic digestion (E), on the right, the intracardiac injection of cell suspensions in E2 chick hosts. A, aorta. (B-D) Transverse sections of chick embryos that received an intracardiac injection of quail somitic (B,D) or splanchnopleural cells (C). (B,C) Two days after injection, QH1+ECs (arrows) migrate in the mesenchyme close to the mesencephalic epithelium (m). Scale bar: 25 µm. (D) A QH1+EC (brown) is detected in a limb vessel (*) 14 days after the injection. Scale bar: 30 µm. (E) Scheme of ex ovo parabiosis (see text for details). YS, yolk sac. (F) Transverse section in the region of contact between the two embryos: the broken line delimits the quail territory on the left, with QH1+ vessels (*), and the chick territory on the right, with QH1- vessels. Scale bar: 70 µm. The inset is a higher magnification of the outlined region: vascular connections are established as QH1+EC (arrow) and HCs (arrowheads) are present in the chick. Scale bar: 35 µm. (G) Transverse section in a chick embryo showing a QH1+EC (arrow) and a QH1+HC (arrowhead). CV, cardinal vein; N, notochord; *, aorta. Scale bar: 50 µm. (H) Yolk sac section from an E4.5 chick embryo that received an intracardiac injection of E3 quail blood cells: a QH1+EC reaches a vitelline vessel. Scale bar: 30 µm. (I-M) Parabiosis experiments. (I) Aspect of an E15 parabiosis in the egg. The chick (C) and quail (Q) embryos, together with their yolk sac (CYS and QYS, respectively), are wrapped into their respective CAM: the arrow indicates a chick CAM vessel; the arrowhead indicates a quail CAM vessel. Scale bar: 15 mm. (J) When dissected, the two CAMs appear fused (arrow). Scale bar: 20 mm. (K) Blood sample of an E8 chick embryo from a parabiosis: QH1+ cells (arrows) prove the establishment of vascular connections between the embryos. Scale bar: 15 µm. (L,M) Isolated morphologically distinct QH1+ECs present in a thymus (L) and in a lung capillary (asterisk in M) of E14 chick embryos. Scale bar: 25 µm.

View larger version (82K): [in a new window]   Fig. 2. Identification of quail ECs and HCs in chick CAM after parabiosis. (A-C) Sambucus nigra lectin (SAMB)/QH1 double staining. (A) The endothelium (arrow) of a chick vessel (v) stained by SAMB. Scale bar: 25 µm. (B,C) One isolated interstitial quail SAMB+/QH1+EC. Scale bar: 15 µm. (D,E) QH1/VEGFR2 double staining: a QH1+EC expresses VEGFR2 transcripts (arrow); a QH1+HC does not (arrowhead). Scale bar: 25 µm. (F-H) LEP/QH1 double staining: an elongated LEP-/QH1+EC (white arrow) surrounded by chick (LEP+/QH1-, white arrowheads) and quail (LEP+/QH1+, blue arrowheads) macrophages. Scale bar: 25 µm. (I-L) LEA/QH1 double staining: (I-K) two quail LEA-/QH1+ECs (white arrows) present in the vicinity of a chick LEA+/QH1- macrophage (white arrowhead) and a round quail LEA-/QH1+HC (blue arrow). Scale bar: 25 µm. (L) One quail (LEA+/QH1+, blue arrowhead) and one chick (LEA+/QH1-, white arrowhead) macrophage are present. Scale bar: 15 µm. (M-O) GRL2/QH1 double staining: an elongated GRL2-/QH1+EC (white arrow) identified among chick (GRL2+/QH1-, white arrowheads) and quail (GRL2+/QH1+, blue arrowhead) hematopoietic precursors, and a quail GRL2-/QH1+ macrophage (blue arrow). Scale bar: 10 µm. (P-T) QH1+HC distribution. QH1+HCs are found in muscle as extravasated cells (P, arrowheads) or in the bone marrow as osteoclasts (Q, arrowheads). Scale bar: 80 µm. BM, bone marrow; ca, cartilage; mu, muscle. (R-T) QH1/SMA double staining shows that QH1+HCs never reach vessel walls. *, vessel lumen. Scale bar: 70 µm.

View larger version (122K): [in a new window]   Fig. 3. Behavior of CECs/EPCs. (A-I) QH1+EC distribution. (A) A whole section of a capillary is quail-derived. Scale bar: 50 µm. (B) Chimeric capillary (*) with a QH1+EC (brown) and two chick QH1-ECs (arrowheads). Scale bar: 20 µm. (C) A larger vein (*) with one QH1+EC (arrow). Scale bar: 45 µm. (D) Among lymphatic vessels (*) surrounding a CAM artery (A), one is chimeric (arrow). Scale bar: 90 µm. (E) A QH1+EC (arrow) in the bone marrow. Scale bar: 45 µm. (F-I) Confocal images of whole-mount SAMB/QH1 double staining on chick CAM. (F) A quail SAMB+/QH1+EC (*) integrated in the superficial chick SAMB+/QH1- vascular plexus. Scale bar: 10 µm. (G,H) A SAMB+/QH1+ vascular cord in contact with the chick SAMB+ plexus (arrowhead). Scale bar: 15 µm. (I) A chimeric vascular cord with quail (white arrowheads) and chick (blue arrowheads) regions linked to a chick vessel (V) through a chick vascular bridge (arrow). Scale bar: 40 µm. (J-L) Conserved migratory potential of CECs/EPCs after tissue integration. (J) Chick `parabiotic' liver (grL) grafted onto the CAM of a chick host: one QH1+EC (arrow) is present. (K) A QH1+EC (arrow) is present in the mesenchyme of an E3 chick limb bud grafted next to a chick `parabiotic' liver. Scale bar: 80 µm. (L) Two QH1+ECs (arrows) originating from the grafted `parabiotic' liver have reached a sinusoid of the chick host liver (hL). Scale bar: 15 µm.

View larger version (103K): [in a new window]   Fig. 4. Mobilization of CECs/EPCs in parabiosis (part 1). (A) An E13 chick wing immediately after a wound: the limb is on the CAM (*) and the size of the wound is delimited by two hemorrhagic dots (arrowheads). (B) The wound (arrowhead) 1 day later, after the shortening of feather buds. Scale bar: 5 mm. (C) Transverse section of an E14 wounded wing showing elongated QH1+ECs (arrows) in the interstitium and integrated in a vessel (*) as well as round QH1+ HCs (arrowheads). In a contralateral E14 wing (D), QH1+ECs (arrow) and QH1+HCs (arrowheads) are less numerous. Scale bar: 100 µm. (E,F) Sections of chick CAM from parabiosis 1 day after wounding. (E) After wounding of veins, there are numerous QH1+ECs (arrows) and QH1+HCs (arrowheads). (F) After wounding of arteries, QH1+ECs (arrows) and HCs (arrowheads) are more rare; *, vessel. Scale bar: 40 µm. (G,H) QH1/TUNEL double staining illustrating that QH1+CECs/EPCs (arrow in G) and QH1+HCs (arrowhead in G) are TUNEL-, while apoptotic nuclei are present in the CAM ectoderm (arrowheads in H). Scale bar: 35 µm. (I) Chick wing (W) 6 days after grafting onto the CAM of a `parabiotic' chick embryo. (J) Chick lung (L) 9 days after grafting onto the CAM of a `parabiotic' chick embryo. Scale bar: 3 mm. (K) Transverse section of a grafted limb showing QH1+ECs (arrows) and QH1+HCs (arrowheads) around the cartilage (ca). (L) transverse section of a grafted lung with a QH1+EC (arrow) integrated in a vessel endothelium (*) and QH1+HCs (arrowheads) in the mesenchyme surrounding a parabronchius (p). Scale bar: 100 µm. (M-O) BrdU incorporation in an E15 parabiosis 9 days after grafting of a chick limb bud onto the chick CAM. (M) In a feather bud of the grafted limb, numerous nuclei are BrdU+ (brown dots); however, QH1+ECs (arrows) present in the pulp are BrdU-. Scale bar: 70 µm. (N) In the corresponding `parabiotic' host chick heart, BrdU is incorporated into the myocardium (brown dots), but QH1+ECs (arrows) and QH1+HCs (arrowhead) are BrdU-. Scale bar: 35 µm. (O) A double stained QH1+/BrdU+EC in a myocardiac capillary (c), the brown nucleus (*) is surrounded by the dark blue QH1 staining. Scale bar: 15 µm.

View larger version (27K): [in a new window]   Fig. 5. Quantification of the QH1+ cell mobilization. (A) Kinetics of the wound healing process: a specific, rapid and significant increase in the number of QH1+ECs in wounded wings in comparison with control limbs or with contralateral wings is observed. The specificity of this mobilization is attested by the similar number of QH1+ECs found in livers and hearts isolated from control or wounded chick embryos. (B) During the wound healing process, the QH1+HC number did not vary significantly between control, contralateral and wounded wings. (C) Wounds on CAMs lead to a significant increase in the QH1+EC number when veins are injured compared with control CAM or wounded arteries. (D) In grafting experiments on CAM, the QH1+EC number is significantly greater in the grafted limb buds than in the viscera. (E-G) VEGF-treated CAM assays: while the number of vessel branches is significantly increased in VEGF-treated CAMs (E), by comparison with PBS-treated CAMs (E), the overall QH1+EC number (F) and the number of QH1+ECs per branch (G) do not vary significantly between treated and control CAMs.

View larger version (82K): [in a new window]   Fig. 6. Mobilization of CECs/EPCs in parabiosis (part 2). (A) An endothelin 1-producing CHO nodule (No), 5 days after cellular implantation onto a `parabiotic' chick CAM. Capillaries are organized in a spoke-wheel formation (*) converging to the nodule. Scale bar: 1 mm. (B) The nodule (No) is wrapped in the CAM, and QH1+ECs (arrows) participate in its vascularization. Scale bar: 160 µm. (C) Higher magnification of QH1+ECs invading the nodule (arrows): one cell is in a capillary endothelium (*), two are located interstitially. Scale bar: 20 µm. (D-G) `Parabiotic' chick CAM 4 days after VEGF (D) or PBS (E) treatment: increased vessel branching (*) is apparent in D compared with E. Scale bar: 1.5 mm. (F,G) The vessel density (*) is higher in VEGF-treated (F) than in PBS-treated (G) CAM. Although the inflammatory response leads to an invasion of a great number of QH1+HCs (brown dots) in both treatments, the number of QH1+ECs (arrows) is similar. Scale bar: 90 µm. (H,I) Mobilization in absence of bone marrow: wounds on `parabiotic' chick wings (H) and grafts of chick limb buds onto `parabiotic' chick CAM (I) lead to an invasion of QH1+ECs (arrows) and QH1+HCs (arrowheads). ca, cartilage. Scale bars: 90 µm in H; 70 µm in I.

View larger version (11K): [in a new window]   Fig. 7. Mobilization in the absence of bone marrow. The wound healing process results in the mobilization of significantly more QH1+ECs in injured wings compared with control or contralateral limbs.