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Endoderm is required for vascular endothelial tube formation, but not for angioblast specification

Department of Cell Biology and Anatomy, University of Arizona Health Sciences Center, 1501 N. Campbell Avenue, PO Box 245044, Tucson, AZ 85724, USA

View larger version (74K): [in a new window]   Fig. 1. Endoderm is not necessary for in vivo angioblast specification. (A) Diagram of the dissection used to remove endoderm. The vegetal core (red), comprising future endoderm, was removed from embryos at the onset of gastrulation, and the resulting endoderm-depleted embryos were incubated until stage 34. (B,D,F) Control embryos assayed with X-msr, erg and flk-1 probes, respectively. These show elaborate vascularization, including posterior cardinal veins (pcv; closed arrowheads), intersomitic vessels (is; open arrowheads) and a ventrolateral vascular plexus. (C,E,G) Endoderm-depleted embryos, assayed with X-msr, erg and flk-1 probes, respectively, contain angioblasts (white open arrowheads), but these are not organized into patent blood vessels.

View larger version (29K): [in a new window]   Fig. 2. Endodermless embryos show a marked reduction in expression of endodermal markers but still express endothelial markers. RT-PCR was performed on total RNA from a stage-34 endodermless embryo. Expression levels of the endodermal markers insulin, IFABP and xlhbox8 are either severely reduced or eliminated relative to unmanipulated controls, while the vascular markers flk-1 and erg and the angioblast/hematopoietic cell marker SCL/tal-1, are still present. –RT, RT not performed.

View larger version (39K): [in a new window]   Fig. 3. Embryos depleted of endoderm by treatment with VegT antisense oligonucleotides continue to express vascular markers. RT-PCR analysis of RNA from stage-34 embryos shows a lack of endodermal markers in VegT-treated embryos (labeled VegT–), while vascular markers are still present. Rescue by coinjection of VegT mRNA (labeled Rescue) restores both endodermal and mesodermal markers to control levels, whereas partial rescue with eFGF (labeled VegT-eFGF) restores mesodermal marker expression but has no effect on endodermal markers (Kofron et al., 1999). –RT, RT not performed.

View larger version (38K): [in a new window]   Fig. 4. Animal caps treated with bFGF form mesoderm containing endothelial markers in the absence of detectable endoderm. (A) Animal caps were incubated in bFGF and cultured until the appropriate stage (either 12.5 or 30). Caps were then assayed for early or late markers of endoderm and for endothelial markers using RT-PCR. While the animal caps show expression of both endothelial and mesodermal markers, there is no detectable expression of endodermal markers at either stage. Note that significant expression of endothelial markers is not expected in the stage-12.5 samples. (B) Stage-30 animal caps treated with bFGF express the vascular marker X-msr in discrete patches when assayed by in situ hybridization.

View larger version (63K): [in a new window]   Fig. 5. Endoderm is required for endothelial tube formation. (A) Wild-type stage-37 embryo showing posterior cardinal vein (closed arrowhead), intersomitic vessels (open arrowhead) and a prominent vascular plexus. (B,C) Stage-37 embryos deprived of endoderm at stage 10 contain thick assemblages of angioblasts (closed arrowheads), but do not contain endothelial tubes. (D) Stage-37 embryo deprived of endoderm at stage 10 and rescued by the addition of a vegetal plug of endoderm from a sibling embryo. Note the presence of posterior cardinal veins (closed arrowhead) and intersomitic vessels (open arrowhead). (E) Cross-section through a wild-type stage-37 embryo showing posterior cardinal vein (closed arrowhead) and dorsal aorta (open arrowhead). (F) Cross-section through a stage-37 endodermless embryo showing presence of angioblasts (closed arrowhead) but no assembly into endothelial tubes. All embryos were assayed by in situ hybridization with the vascular marker X-msr. e, endoderm; n, notochord; nt, neural tube.

View larger version (114K): [in a new window]   Fig. 6. Embryos without endoderm lack patent blood vessels. (A-C) 1 µm plastic sections stained with Toluidene Blue. (A) Cross-section through a wild-type stage-37 embryo showing endothelial tubes, including a posterior cardinal vein (closed arrowhead) and dorsal aorta (open arrowhead). (B) Endothelial tubes are not present in stage-37 endodermless embryo but are present in stage-37 embryos that have been rescued by the addition of endoderm (C, closed arrowhead). (D,E) Transmission electron microscopy showing transverse sections through the posterior cardinal veins of a wild-type embryo (D) and an endodermless embryo rescued by the addition of endoderm from a sibling donor embryo (E). Arrows indicate the characteristic thin-walled endothelial cell morphology in each section. Scale bars, 1 µm. Black objects in sections are lipid droplets generated during histological preparation. e, endoderm; n, notochord; nt, neural tube.

View larger version (100K): [in a new window]   Fig. 7. Removal of endoderm in quail embryos does not prevent angioblast formation. (A) Ventral view of quail embryo showing the approximate region from which endoderm was removed. (B,C) Ventral images of fluorescent staining of the endothelial cell marker, QH1 at approximately the 4- and 6-somite stages, respectively. The endoderm-depleted side is on the right. Note the presence of endothelial cells within the manipulated region, but the absence of vascular assembly. The approximate midline of the embryo is indicated by the dashed line. Scale bars, 10 µm.

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