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Signaling by the hedgehog (Hh) family of secreted growth factors is essential for development of embryonic blood vessels. Embryos lacking Hh function have abundant endothelial cells but fail to assemble vascular cords or lumenized endothelial tubes. However, the role of Hh signaling during later aspects of vascular patterning and morphogenesis is largely unexplored. We have used small molecule inhibitors and agonists to alter activity of the Hh signaling pathway in the chick embryo. When cyclopamine is added after cord formation, aortal cells form tubes, but these are small and disorganized and the density of the adjacent vascular plexus is reduced. Activation of the Hh pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. The number of endothelial cell filopodia is found to correlate with Hh signaling levels. These studies show that Hh signaling levels must be tightly regulated for normal vascular patterning to be achieved.
Figure 1. Characterization of cyclopamine inhibition and SAG activation of hedgehog (Hh) signaling in the chick embryo. In situ hybridization analysis for expression of the hedgehog receptor, Ptch1, as a readout for Hh signaling. A–E: Embryos were treated with increasing doses of cyclopamine (0–200 μM), commencing at the 7-somite stage, until the 10-somite stage (approximately 8 hr). Ptch1 expression was reduced as the concentration of cyclopamine increased. Expression was initially lost from the somites and lateralmesoderm and then more gradually from the notochord and the ventral neural tube. F–J: Embryos treated with increasing concentrations of SAG (0–20 μM), commencing at the 7-somite stage, until the 10-somite stage. Ptch1 expression increased in a concentration-dependent manner. Regions of higher expression in the lateral plate mesoderm are indicated by arrowheads. K–M: Cross-sections through embryos treated as indicated and assayed for Ptch1 by in situ hybridization. K: Control embryo shows wild-type expression pattern of Ptch1 in the notochord, ventral neural tube, ventralsomites, and more weakly in the lateral plate mesoderm. These last two expression domains are directly adjacent to the forming dorsal aortae (DA). L: Embryo treated with 100 μM cyclopamine. Expression of Ptch1 was almost completely absent, except for low levels in the ventral neural tube. M: Embryo treated with 20 μM SAG. Expression of Ptch1 appeared more intense in all regions compared with control. Ptch1 expression was now prominent in more dorsal regions of the somite and in the lateral plate mesoderm, which showed relatively low expression in the control embryo (K). Regions of increased Ptch1 expression in the somite and lateral plate, adjacent to the DA are indicated by the arrowhead. N: Quantitative PCR analysis of Ptch1 transcript levels. Results represent the average relative transcript levels from 3 embryos for each treatment. Both cyclopamine and SAG treatments are statistically different from controls (P < 0.05, Student's t-test, indicated by asterisks).Download figure to PowerPoint
Figure 2. Antibody analysis of vascular formation in quail embryos treated with cyclopamine or SAG. Vascular tissues were detected using QH1 antibody. A–P: The first column shows bright field images of embryos, the second column shows identical embryos stained with QH1, the third column shows magnified images of the vascular plexus region and the fourth column, shows cross-sections through QH1-stained embryos at the level of the fifth somite. A–D: Characterization of embryos at the seven-somite stage, before initiation of treatment. At this stage, a complex network of vascular cords has assembled, but tubulogenesis has only commenced in the most anterior regions of the paired dorsal aortae (DA). Sections at the level of the fifth somite (D) show that angioblasts have assembled at the region of the future DA (da), but tube formation has not started. E–H: Characterization of control embryos at the 10-somite stage at the completion of treatment. The DAs are prominent continuous structures and an elaborate sinusoidal plexus has formed in the adjacent mesoderm. The section image (H) shows the presence of the tubular DA (da). I–L: Characterization of 10-somite stage embryos treated with cyclopamine. Relative to controls, the DA are narrower and much more irregular in appearance. The vascular plexus in less dense and in some places discontinuous. Despite the general disruption of vascular patterning, the section image (L) reveals that tube formation has occurred. M–P: Characterization of 10-somite stage embryos treated with SAG. Relative to controls the DA are wider and the vascular plexus is very dense. The section image at the level of the fifth somite shows the extremely large tubular DA (da). Q: QH1-stained vascular plexus from a control embryo at 10-somites. R: Equivalent region of the vascular plexus from an embryo treated with cyclopamine. In this pronounced example, the plexus has low density and many angioblasts exist in isolated aggregates rather than assembled into cords. S: Confocal image of the isolated angioblast aggregate (indicated by arrow in R), showing the presence of a lumen. QH1 is green, propidium iodide staining for nuclei is red. In panels D, H, L, P, the location of the notochord is indicated by the dotted circle. Scale bar in the first column is 0.5 mm, and in the last column is 100 micrometers. Scale bar = 50 μm in S.Download figure to PowerPoint
Figure 3. Quantitation of the structure of the dorsal aortae (DA) and the vascular plexus in quail embryos with inhibited and activated hedgehog (Hh) signaling. A: Relative width of the DA in control, cyclopamine and SAG-treated embryos. Measurements were made from photographs of QH1 stained whole-mount embryos. Measurements were made for each DA at five positions along the AP axis, including at least three embryos for each treatment. B: Measurements of DA area were made by analysis of digital photographs of QH1 stained sections using Image J. Measurements were made at five positions along the AP axis, including at least 3 embryos for each treatment. C: Measurements of DA perimeter (the length of the inside of the DA vessel wall) were made by Image J analysis of the same digital photographs used in (B). Measurements were made at five positions along the AP axis, including at least three embryos for each treatment. D: Area of the vascular plexus was determined by Image J analysis of digital photographs. At least five embryos were assayed for each treatment. In all cases, the measurements for cyclopamine and SAG treated embryos are significantly different from control values (P < 0.05, Student's t-test, indicated by asterisks).Download figure to PowerPoint
Figure 4. The number of cells comprising vascular structures is little changed after inhibition or activation of hedgehog (Hh) signaling. After experimental treatment, quail embryos were stained with QH1 to detect angioblasts and DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride) to mark nuclei. A: Endothelial cell (EC) number was determined by counting number of nuclei corresponding to QH1 stained cells. Five sections were counted for each embryo, with at least three embryos for each treatment. The number of ECs in SAG treated embryos is significantly different from controls and cyclopamine-treated embryos (asterisk). B–D: Determination of number of ECs comprising the dorsal aortae (DA). Confocal images of sections at approximately the level of the fifth somite. From left to right the panels show DAPI stain, QH1 stain and the merged image. Representative images are shown for a single DA in control (B), cyclopamine-treated (C) and SAG-treated embryos (D). Scale bar = 100 μm. E: Number of cells contributing to the wall of the DA. DAPI-stained nuclei contained in the wall of the vessels (QH1 stained) were counted. Cells in both DA were counted in at least five sections per embryo, for at least three embryos for each treatment. Despite differences in the size of the vessels, the number of ECs comprising the walls was unchanged by the different treatments.Download figure to PowerPoint
Figure 5. Relative number of EC filopodia correlates with hedgehog (Hh) signaling levels. After experimental treatment, quail embryos were stained with QH1 to mark endothelial cells (ECs) and images of the vascular plexus were obtained using confocal microscopy. A: Image of region of vascular plexus from posterior of embryo, adjacent to dorsal aorta (just out of frame at top), of control embryo. B: Inverted image (ECs are black) of region outlined in box in A. Filopodia extending from ECs are indicated by open arrowheads. C: Image of vascular plexus of cyclopamine treated embryo. Plexus region is equivalent to that in control. D: Inverted image of region outlined in box in C showing filopodia marked with arrowheads. E: Image of vascular plexus of SAG-treated embryo. Plexus region is equivalent to that in control. F: Inverted image of region outlined in box in E showing filopodia marked with arrowheads. G: Quantitation of relative number of filopodia following different treatments. Total number of filopodia were counted and then normalized against length of perimeter (edge). Length of perimeter was calculated using Image J. Plexus on both sides of midline was counted for at least three embryos for each treatment. Results show that the relative number of filopodia correlates with the level of Hh signaling and that both cyclopamine and SAG treatments are significantly different from controls (P < 0.05, Student's t-test, indicated by asterisks).Download figure to PowerPoint
Figure 6. Vegfa but not Notch1 expression levels change when hedgehog (Hh) signaling is altered. In situ hybridization analysis of chick embryos was used to examine expression of Vegfa (A–C) and Notch1 (D–F) after treatment with Hh inhibitor (cyclopamine, 100 μM) or activator (SAG, 20 μM). A: Embryo treated with cyclopamine showing reduced expression of Vegfa compared with controls. B: Control embryo showing normal Vegfa expression pattern. C: Embryo treated with SAG showing increased expression of Vegfa compared with controls. D–F: Embryos treated with either cyclopamine (D) or SAG (F), showed Notch1 expression patterns indistinguishable from control embryos (E). Arrows indicate Notch1 expression in the endothelium.Download figure to PowerPoint
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