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First published online 4 August 2004
doi: 10.1242/dev.01304

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Hedgehog signaling is essential for endothelial tube formation during vasculogenesis

Steven A. Vokes^1,*, Tatiana A. Yatskievych¹, Ronald L. Heimark², Jill McMahon³, Andrew P. McMahon³, Parker B. Antin¹ and Paul A. Krieg^1,

¹ Department of Cell Biology and Anatomy, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, AZ 85724-5044, USA
² Department of Surgery, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, AZ 85724-5044, USA
³ Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA

View larger version (94K): [in a new window]   Fig. 1. (A) Endoderm was removed from one half of stage 5 quail embryos with the unmanipulated half serving as an internal control. (B) The embryos were then assayed at the seven- to eight-somite stage, after tube formation had occurred, using whole-mount staining with QH1 antibody and subsequent sectioning and analysis with deconvolution microscopy. (C) Endoderm was removed from one half of the embryo (typically at stage 5, but at three somites in this example) with the other half serving as a control. (B) As shown by QH1 staining of vascular cells in the eight-somite embryo, removal of endoderm results in failure of vascular assembly and tube formation.

View larger version (98K): [in a new window]   Fig. 2. Expression of Hedgehog signaling components correlates with vascular assembly. (A) RT-PCR analysis of five-somite chick tissues, showing presence of Shh in the endoderm and absence in non-axial mesoderm. VEGF is present in both the mesoderm and endoderm. (B,C) Shh in situ hybridization on a two-somite chick embryo showing low levels of endodermal expression (indicated by arrowheads) and an eight-somite chick embryo showing greatly increased endodermal staining (arrowheads). (D) Transverse section through an eight-somite chick embryo showing Shh expression in the endoderm (arrowheads). (E,F) In situ hybridization analysis of a sixsomite quail embryo with Ptch1 (bright field) and QH-1 (fluorescence) showing that Ptch1 is present in angioblasts that have not yet formed tubes (arrowheads). (G-J) Transverse sections through seven-somite chick embryos analyzed by in situ hybridization. The expression of Shh in the endoderm (G, arrowhead), but not in the dorsal aorta (da). (H-J) The Hedgehog receptors Ptch1 and Ptch2, and the transducer Smo are present in endothelial cells of seven-somite embryos.

View larger version (94K): [in a new window]   Fig. 3. Hedgehog signaling is essential for vascular assembly. Quail embryos treated with 100 µM cyclopamine from the two-somite until the eight-somite stage (B) show severe deficiencies in vascular assembly when assayed with the endothelial cell specific antibody, QH-1. Dorsal aortae formation (arrowheads) is not present and there is almost complete lack of vascular assembly when compared with embryos treated with control media (A).

View larger version (105K): [in a new window]   Fig. 4. Mouse embryos lacking SMO have severe defects in vascular tube formation. Vascular cells are visualized using an antibody that recognizes VEGFR2. Smo mutant embryos fail to form anterior dorsal aortae, although abundant VEGFR2-positive angioblasts are still present. (A,D) Wild-type embryo at four-somites contains patent dorsal aortae (arrowheads). (B,C,E) Mutant embryos (four to eight somites) show no apparent anterior dorsal aortae, either in wholemount embryos (arrowheads in B,C) or section (E, arrowheads show numerous angioblasts, but no dorsal aortae). (F) Posterior section through embryo depicted in E shows that dorsal aortae in mutant embryos (indicated by black arrowheads) appear histologically normal at the caudal end of the embryo. (G) Transverse section through additional wild-type and (H) mutant embryos showing complete lack of a patent dorsal aorta (arrowheads) in the mutant embryo. Transverse sections through branchial arch of wild-type (I) and mutant (J) sibling embryos at 11 somites showing severe vascular defects. The mutant embryo contains only a few, very poorly formed, endothelial tubes (white arrowheads), while an equivalent section through a wild-type embryo shows well-formed blood vessels (white arrowheads), including the primary head vein (hv), dorsal aorta (da) and first branchial arch artery (ba). Both sections are approximately 90 µm anterior to the start of the heart. (K,L) In situ hybridization for Shh expression on wild-type (K) or mutant (L) embryos at approximately eight somites showing equivalent expression of Shh in endoderm.

View larger version (73K): [in a new window]   Fig. 5. Sonic hedgehog (SHH) signaling is sufficient to rescue vascular assembly in the absence of endoderm. The panels on the left show a whole-mount view of QH-1 stained quail embryos, while those on the right show deconvolution microscope sections through the same or similar embryos. (A,B) The control side of embryos contains a robust vascular plexus with patent tubes (arrowhead in B). (C-E) By contrast, the side lacking endoderm contains unassembled clusters of angioblasts that do not contain a lumen (arrowheads in D,E; asterisks indicate control beads). (F,G) Beads containing SHH rescue vascular assembly and tube formation (arrowhead in G). (H,I) VEGF causes massive endothelial cell proliferation, but is not able to rescue vascular assembly or tubulogenesis. QH-1-positive cells form apparent sheets of tissue (arrowhead in I). (J,K) When both SHH and VEGF beads are added (not in field of vision), a robust tubular vascular plexus is formed. The arrowhead in K indicates a longitudinal section through a connecting blood vessel. Scale bar: 15 µm.

View larger version (48K): [in a new window]   Fig. 6. Vascular assembly in cell culture in response to exogenous SHH. (A) RT-PCR analysis detects Ptch1, Smo and Vegfr2 transcripts in bEnd3 cells (lane 1) and Eoma cells (lane 2). (B) Confluent bEnd3 cells were switched to serum-free DMEM or (C) DMEM plus 2 µg/ml of SHH. SHH induces assembly of vascular endothelial cells into tube-like structures. (D) RT-PCR analysis of Vegf transcripts. The cDNA was synthesized from untreated and treated bEND3 cultures 48 hours after the addition of SHH or VEGF. cDNA synthesized from adult mouse heart total RNA was used as a positive control. (E) bEnd3 cells were assayed for proliferation by BrdU labeling, following addition of SHH at concentrations indicated. Data were averaged from three separate experiments. Addition of SHH does not increase endothelial cell proliferation.

View larger version (17K): [in a new window]   Fig. 7. Model for vascular assembly. (A) In the unmanipulated embryo, both the mesodermal and endodermal layers contribute VEGF, although the mesodermal contribution is greater (as indicated by the larger red arrows). The presence of VEGF leads to the proliferation of angioblasts (black dots) in the mesodermal layer. In combination with SHH signaling from the endoderm (blue arrows), angioblasts assemble into a vascular network with patent tubes. (B) When endoderm is experimentally removed from embryos, angioblasts are specified, but are unable to organize into vascular tubes. (C) The addition of SHH beads (blue circles) to endodermless embryos is sufficient to rescue vascular tubulogenesis. When VEGF beads (red circles) are added in combination with SHH beads, a more complete vascular plexus is formed, suggesting that VEGF from the endoderm (small red arrows) is also important for vascular development.