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First published online 8 February 2006
doi: 10.1242/dev.02269

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Somite-derived cells replace ventral aortic hemangioblasts and provide aortic smooth muscle cells of the trunk

¹ Université Pierre et Marie Curie-Paris6, CNRS UMR7622, Laboratoire de Biologie du Développement, Bat C, 6^ème étage, Case 24, 75252 Paris Cedex 05, France.
² UMR 7128 Laboratoire d'Embryologie Cellulaire et Moléculaire, 49 bis, avenue de la Belle Gabrielle, 94130 Nogent-sur-Marne, France.

View larger version (92K): [in a new window]   Fig. 1. Isochronic and orthotopic graft of segmental plate from quail to chick. (A) Scheme of a graft. (B) AcLDL-DiI labeling of the vascular network; 13 somite-pair embryo; ventral view, truncal level. The whole vascular network is labeled. The arrow indicates the level of the last-formed somite pair. At this level, the paired aortae are positioned lateral to the somites, whereas they are in more central positions at more cephalic levels. Lines indicate the levels of sections in C,D. Scale bar: 800 µm. (C,D) Unilateral last somite + segmental plate removal. 13 somite-pair embryo; cross-sections, QH1 staining. (C) Level of the third last-formed somite. The ectoderm has been removed. (D) Segmental plate level. The right segmental plate has been removed with no damage to the aorta. The aorta is positioned lateral to the segmental plate. Scale bar: 70 µm. (E) Isolated last somite + lateral plate mesoderm, QH1 staining. No QH1+ cell was detected in 18 out of 20 grafts analyzed. Scale bar: 100 µm. Ao, aorta; E, ectoderm; NT, neural tube; S, somite.

View larger version (76K): [in a new window]   Fig. 2. Somite contribution to aortic endothelium remodeling and vascularization of the body wall. (A,B) E2.5 chick host, 1 day post-grafting. QH1 immunohistochemistry DAB stained cross-sections. (A) Caudal region of the grafted embryo. The roof of the right aortic rudiment is colonized with QH1+ cells (arrowheads). QH1+ cells around the neural tube organize into the perineural plexus. No QH1+ signal is visible lateral to the somite. (B) Medial level of the graft. The roof of the aorta (Ao) is entirely of quail origin on the grafted side. QH1+ cells have also colonized the ipsilateral chick host body wall where they give rise to the vascularization of the neural tube, mesonephros (Me), lateral plate (LP) and cardinal vein (CV). Scale bar: 150 µm. (C) E4 chick host, 2 days post-grafting. QH1 immunofluorescence (green) overlaid with Nomarski's interferential contrast. QH1+ cells form a conspicuous plexus around the neural tube. The lateral plate contains numerous QH1+ vessels. The QH1 aortic domain has extended ventrally to the hematopoietic clusters. Scale bar: 150 µm. (Inset) A prominent QH1- hematopoietic cluster of host (chick) origin is underlined by QH1+ ECs (arrow). Some QH1+ cells are integrated in the vascular endothelium (arrowhead). Scale bar: 30 µm. D, dermomyotome; Sc, sclerotome.

View larger version (119K): [in a new window]   Fig. 3. Contribution of the somite to the vascularization of the host and to formation of the smooth muscle tunicae. (A) E6.5 chick host, 5 days after the graft. Cross-section at the trunk level. Triple labeling with QH1 (green)/MEP21 (red)/DAPI (blue). The vascular networks of the neural tube, dorsal root ganglia, dermis, mesonephros, body wall and limb bud derive entirely from the graft. Hematopoiesis has ceased at that stage. The MEP21 immunoreactivity with the coelomic epithelium around the lungs and the heart is a staining artifact. Scale bar: 200 µm. Inset: closer view of the right side of the aorta. The aortic endothelium on the grafted side mostly derives from the graft. Scale bar: 50 µm. (B) E5 chick host, 3.5 days after a bilateral graft of segmental plates. QH1 immunofluorescence overlaid with Nomarski's interferential contrast. Wing bud level. The quail perineural vasculature is conspicuous. ECs of the aorta are entirely quail. Quail ECs are not found in internal organs. Scale bar: 150 µm. (C) Section immediately adjacent to B. Double QCPN (red)/SMA (blue) immunofluorescence. QCPN shows numerous quail cells that surround the notochord and the aorta. Quail cells closest to the aortic lumen express SMA (arrows). Many QCPN+/SMA- cells organize around the SMA+ layer. Scale bar: 70 µm. (D-F) Detail of the ventrolateral side of an E4.5 chick aorta, 3 days after a unilateral graft. QH1 (D) and SMA (E) immunohistochemistry merged to Nomarski's interferential contrast. (F) Overlay of QH1 and SMA signals merged to the Nomarski's picture. The endothelial (green) and smooth muscle (blue) expression domains are clearly separated. The arrowhead indicates the same endothelial cell. Scale bar: 30 µm. H, heart; L, lungs; WB, wing bud.

View larger version (81K): [in a new window]   Fig. 4. Clonal analysis of the vascular endothelial lineage and culture of purified endothelial cells. (A) SNV-based, non-replicative retroviral vectors. 5' and 3' long terminal repeats (LTR) are shown as open boxes. An arrow indicates the direction of transcription from the promoter in the left-hand LTR. Bold lines between LTRs denote SNV sequences. Encapsidation sequences required for the packaging of virions are indicated as . Provirus sizes and putative translations of the reporter genes are indicated below the viral structure. (B) Six-day-old embryo inoculated at E4 with a mix of lacZ- and PLAP-carrying vectors. Double histochemistry to reveal the reporter genes and double immunofluorescence with anti-MEP21 (orange) and anti-SMA (blue) mAbs. Cross-section through the aorta. lacZ (black arrowhead) or PLAP (white arrowhead) infections are restricted to the endothelium. (Inset) Two infected cells expressing PLAP framed in B. Scale bars: 50 µm and 20 µm in inset. (C) FACS analysis after AcLDL-DiI and CD45 immunostaining. The frame points to the AcLDL-DiI+/CD45- cell population, i.e. the endothelial cells, selected for culture. (D) Cultures of purified endothelial cells without growth factors (left column), supplemented with VEGF (middle column) or TGFß (right column) at two different time points. Upper line, second day of culture; lower line, fifth day of culture. Triple staining with MEP21 (red)/SMA (green)/DAPI (blue). Two days of culture: purified endothelial cells uniformly express MEP21, although cell sizes in the different culture conditions may vary. Five days of culture: MEP21 expression has decreased and SMA becomes expressed. Disappearance of MEP21 is total in the control medium without growth factors. The VEGF supplementation produces numerous cells co-expressing MEP21 and SMA. With TGFß supplementation, most of the cells have lost MEP21 expression. Some cells, however, retain a MEP21 signal and thus express both markers. Scale bar: 25 µm.

View larger version (127K): [in a new window]   Fig. 5. Formation of the aorta and molecular characterization of somite angioblasts. (A-E) Cross-sections from caudal (A) to cephalic (E) levels in an 18-somite quail embryo. QH1 staining merged to Nomarski's interferential contrast. (A) Primitive streak level. QH1+ angioblasts visible at a lateral position (arrowhead) aggregate against the endoderm and organize into groups of cells. (B) Segmental plate level. QH1+ groups organize into vessels that progressively fuse. These structures remain positioned lateral to the segmental plate. (C) Nascent somite level. The paired aortae have formed. A few QH1+ angioblasts are now detected in a dorsal position. (D) Epithelialized somite level. The paired aortae are now underneath the somites. Dorsal QH1+ angioblasts become more numerous. (E) Dermomyotome and sclerotome have separated. Conspicuous QH1+ cells are around the Wolffian duct (arrow) and in close association with the aortic roof (arrowhead). Scale bar: 100 µm. (F-I). EC-specific markers in chick (F-H) and quail (I) somites. (F-H) GATA2, VEGFR2 and SCL/TAL1 in situ hybridization. All markers delineate a quadrant of cells in the dorsolateral aspect of the epithelial somite (arrow). GATA2 is also present in the epidermis (Sheng and Stern, 1999; Minko et al., 2003). (I) QH1 immunohistochemistry. Scale bar: 50 µm. D, dermomyotome; E, ectoderm; En, endoderm; M, mesoderm; Sc, sclerotome; So, somatopleural mesoderm; Sp, splanchnopleural mesoderm; SP, segmental plate; WD, Wolffian duct.

View larger version (45K): [in a new window]   Fig. 6. Developmental history of the aorta in relationship with endothelial remodeling and smooth muscle formation. (A) Before fusion of the aortae, aortic ECs (red) derive from the splanchnopleura. The dorsolateral quadrant of the somite displays a population of ECs (yellow). (B) Immediately before fusion, the initial roof of splanchnopleural origin (red) has been replaced by ECs from the somite (yellow). (C) After fusion, the roof is of somite origin, the sides and floor remain of splanchnopleural origin. (D) During early hematopoiesis, the floor begins to loose endothelial markers and acquires of hematopoietic traits (Jaffredo et al., 1998). (E) At the hematopoietic clusters stage, clusters are budding into the lumen, whereas some HCs ingress into the mesentery. At the same time, somitic ECs replace the initial floor, whereas numerous somitic ECs are found in the floor either underneath the clusters. At this time, cells of somite origin reach the aorta (gray). When these cells reach the aorta, they begin to express SMA (green). (F) Completion of hematopoiesis. The aortic floor has disappeared and is replaced by somitic ECs. Aortic ECs are entirely of somite origin. Somite cells in abluminal position now express SMA (green).