Journal: PLoS ONE

Article Title: Extraembryonic Origin of Circulating Endothelial Cells

doi: 10.1371/journal.pone.0025889

Figure Lengend Snippet: Transverse sections. A) Around the aorta (Ao) a population of QH1 + cells was present: the majority is LEP 100 + macrophages (arrowheads), but some QH1 + /LEP100 − CEC are present (dotted areas, arrow). QH1 + /LEP 100 − CEC are observed in the circulation (red arrowhead). Bar: 40 mm B) Detection of an interstitial QH1 + /LEA − CEC (arrow) and of a QH1 + /LEA + macrophage (arrowhead) in the brain mesenchyme (M), close to the neural epithelium (E). Bar: 20 mm. C) A QH1 + /LEA − CEC (arrow) reaches the endocardium. Note the presence of one quail QH1 + /LEA + (white arrowhead) and two chick QH1 − /LEA + (red arrowheads) macrophages. T: trabecula. Bar: 20 mm. D) Identification of three QH1 + /vWF + CEC in the periaortic mesenchyme (arrows). Note the vWF + endothelium of the aorta (Ao, arrowheads). Bar: 20 mm. E–H) Four examples of vessel-integrated (*) QH1 + /Sambucus nigra + quail CEC (arrows) in the wing (E), the dermis (F), at the vicinity of the dorsal root ganglia (G, DRG) and in the ventral vascular plexus of the neural tube (H). Bar: 20 mm. I) Table 1: Quantification of quail yolk sac derived CEC in the chick embryo. Counting of QH1 + CEC in the embryo shows that the total number of yolk-sac derived CEC (clear gray) increases between 1 and 4 days after grafting. However, as the embryo size expands during the same period, the CEC concentration per mm 3 of embryonic tissue (dark gray) decreases. J) Table 2: Percentage of CEC that reached host endothelium.

Article Snippet: Biotinylated Sambucus nigra lectin (1/400 in PBLEC buffer-PBS pH6.8, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2, 1% triton) recognizes chick and quail EC (Clinisciences, ) but also avian macrophages.

Techniques: Derivative Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Extraembryonic Origin of Circulating Endothelial Cells

doi: 10.1371/journal.pone.0025889

Figure Lengend Snippet: A–D and F) bone marrow cultures. A) Aspect of a quail bone marrow culture after one day; Adipocytes with oil vacuoles (arrow), fibroblasts with filopodia (black arrowhead) and nucleated erythrocytes (red arrowhead) are easily identified. The round cells present in the culture are HC. Bar: 40 mm. B) 8F3/vWF double staining identifies chick CEC (arrow) among 8F3 + /vWF − chick cells (arrowheads). Bar: 20 mm. C) The QH1/vWF double staining shows a chick CEC (arrow) among quail EC (white arrowheads) and HC (green arrowheads). Bar: 20 mm. D) A QH1 − /vWF + /8F3 + CEC is detected in a bone marrow smear. Bar: 10 mm. E) Table 3: Percentage of chick cells invading the bone marrow in the oldest yolk sac chimeras. The number of 8F3/Hoechst cells is compared with the total cell population in cultures. F ) Presence of QH1 + HSC among nucleated erythrocytes in a blood smear. Bar: 20 mm. G) Identification of a group of QH1 − /vWF + /8F3 + CEC in a blood smear. Bar: 20 mm. H) Triple staining on a transverse section through the intestine illustrating the participation of chick QH1 − /Sambucus + /LEP100 − CEC (arrows) to the vascular plexus of smooth muscle layers (M), beneath a villus (V). The quail host vessels are QH1 + /Sambucus + /LEP100 − and appear purple (arrowheads). Bar: 40 mm. I) On a pia mater section, an interstitial QH1 − /Sambucus + /LEP100 − CEC (arrow) is identified among QH1 + HC (arrowheads). Bar: 20 mm. J) An interstitial QH1 − /Sambucus + /LEP100 − CEC (arrow) on a CAM section close to a QH1 + vessel (V) and to QH1 + HC (arrowheads). Note that the endodermal layer (E) of the CAM is underlined by Sambucus and QH1 and appears purple. Bar: 20 mm.

Article Snippet: Biotinylated Sambucus nigra lectin (1/400 in PBLEC buffer-PBS pH6.8, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2, 1% triton) recognizes chick and quail EC (Clinisciences, ) but also avian macrophages.

Techniques: Double Staining, Staining

Journal: PLoS ONE

Article Title: Extraembryonic Origin of Circulating Endothelial Cells

doi: 10.1371/journal.pone.0025889

Figure Lengend Snippet: A) An E5.5 chick embryo (C) developed on a quail yolk sac (QYS). A wound is made in the right wing bud (arrow), which is accessed through a hole in the amnion (*). Bar: 1.2 mm. B, C) Longitudinal sections of wounded wings. Three hours after the operation (B), at the level of the brachial artery (BA), the wound (W) is invaded by an important contingent of chick (blue) and QH1 + quail cells (brown). After 8h (C), the rapid healing of the wound (W) is obvious. Note that numerous QH1 + cells (arrowheads) are present in the wing mesenchyme (B, C). Bar: 85 mm. D) High magnification of the mixed population of brown quail cells (Q) and blue chick cells (C). Bar: 20 mm. E) QH1/LEP 100 double staining permits to discriminate between QH1 + /LEP 100 + macrophages (arrowhead) and QH1 + /LEP 100 − cells (arrow) in the wound. Bar: 40 mm. F–H) Identification of QH1 + cells in the mesenchyme of wounded wings. F) QH1 + cells are not macrophages (arrows). Bar: 20 mm. G) A QH1 + /BEN + HC (arrowhead) is identified close to a non hematopoietic QH1 + /BEN − cell (arrow). H) In the vicinity of a QH1 − /vWF + chick vessel (V), QH1 + /vWF + EC (arrows) are present with QH1 + /vWF − HC (arrowhead). I) In the reverse combination, quail embryo grafted on chick yolk sac, a section of QH1 − /Sambucus + chick vessel (arrow) present among the QH1 + /Sambucus + quail vascular plexus (arrowheads). Bar: 40 mm in F, 30 mm in G–I. J) Table 4: Mobilization of yolk sac derived CEC during wound healing. A wound on wings induces a statistically significant mobilization of CEC (dark gray) by comparison with CEC concentration in contralateral limbs (clear gray). This mobilization is effective as soon as 3h after wounding (right part of the table).

Article Snippet: Biotinylated Sambucus nigra lectin (1/400 in PBLEC buffer-PBS pH6.8, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2, 1% triton) recognizes chick and quail EC (Clinisciences, ) but also avian macrophages.

Techniques: Double Staining, Derivative Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Extraembryonic Origin of Circulating Endothelial Cells

doi: 10.1371/journal.pone.0025889

Figure Lengend Snippet: A) A 9 s.s. half embryo chimera just after the operation: the dotted region corresponds to the grafted quail territory (Q) caudally to the last formed somite. The rest of the chimera is chick (C). Note that the quail and chick neural tubes are aligned (arrow). Bar: 1 mm. B) A half embryo chimera 4 days after the operation: the embryo is well developed with its wing (*) and limb (°) buds; the dotted line shows the limit between chick (C) and quail (Q) territories. The quail allantois (QAL) is present above the head. Bar: 2 mm. C) A cross section at the truncal level showing the chick intrinsic QH1 − /Sambucus + vascularization in the gut (G) and the lungs (Lu). Dorsally, the vascular tree is quail, QH1 + /Sambucus + , in particular around the neural tube (NT), the cardinal veins (*) and the umbilical vein (°). Note that the aortic endothelium (Ao) is chimeric with a part of chick EC located ventrally (arrowhead). Sambucus also labels basement membrane of epithelia in gut, lung and liver (L). N: notochord; M: mesonephros. D) A cross section in the diencephalon. All the vascular network is chick, i. e., QH1 − /Sambucus + in particular around the right eye (E), the diencephalic vesicle (Di) and the right jugular vein (JV). The green dots are QH1 + HC. Ph: Pharynx; T: tongue. Bar: 80 mm in C, D. E) Table 6: Counting of quail CEC identified in chick territories of half embryo chimeras. Less than 3 QH1 + CEC/mm 3 are present. F) Close to the diencephalic epithelium (E), a QH1 + /Sambucus + CEC (arrow) is integrated in a chick QH1 − /Sambucus + vessel (V). G) Identification of a QH1 + /vWF + CEC (arrow) interstitially located in the diencephalic mesenchyme. Bar: 20 mm in F, G. H–K) QH1/Sambucus/BEN triple staining confirms the endothelial nature of a quail purple CEC (arrowhead) in the diencephalic mesenchyme. Bar: 40 mm in H–K.

Article Snippet: Biotinylated Sambucus nigra lectin (1/400 in PBLEC buffer-PBS pH6.8, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2, 1% triton) recognizes chick and quail EC (Clinisciences, ) but also avian macrophages.

Techniques: Staining