The Notch Ligands Dll4 and Jagged1 Have Opposing Effects on Angiogenesis

The Notch Ligands Dll4 and Jagged1 Have Opposing Effects on Angiogenesis
Rui Benedito, Cristina Roca, Inga Sorenson, Susanne Adams, Achim Gossler, Marcus Fruttiger, and Ralf H. Adams
Cell 137, 1124-1135, June 12, 2009 ©2009 Elsevier Inc.

The authors’ principle aim is to characterize how the transmembrane protein Jagged1, which is expressed on the surface of epithelial cells, affects angiogenesis through the Notch signaling pathway. They conclude that Jagged1 is a competitive inhibitor of Dll4 and is an important upregulator of angiogenesis during fetal development, growth and tissue regeneration.

The Notch signaling pathway is a highly conserved cellular system that controls differentiation and proliferation, especially in relation to stem cells and fetal development. Angiogenesis during development, growth and tissue regeneration is controlled partly by circulatory growth factors (i.e. vascular endothelial growth factor) but also locally by transmembrane proteins on the surfaces of endothelial cells themselves, such as Delta-like ligand 4 (Dll4) and the protein of interest in this paper, Jagged1. These transmembrane proteins act through the Notch pathway to either upregulate or downregulate primary features of angiogenesis, including sprouting and branching. Dll4 has previously been shown to be an important downregulator of angiogenesis through the Notch pathway and is upregulated by VEGF as a negative control mechanism. Jagged1, in contrast, had not been extensively studied and its effect on angiogenesis was less clearly understood.

The researchers visualize, characterize and quantify angiogenesis through microscopic staining techniques. They principally study retinal vascularization in genetically-modified mice strains in order to compare angiogenesis between wildtype and Jagged1 knock-out/knock-in transgenic mice. Below is a summary of a few of the most relevant figures from the paper.
Figure 1: Comparison of retinal vascularization in wildtype vs. Jagged1 knock-out mice using fluorescent staining specific for endothelial cells. The mice are genetically engineered to only activate the Jagged1 gene (if present) after exposure to tamoxifen. Tamoxifen is administered after birth and the mice retinas are extracted and stained with EC-specific antibodies 1-3 days after the tamoxifen injection. The wildtype mice exhibit +44% vasculature length, +65% branch points and +32% surface area covered by vasculature.
Figure 4: Comparison of retinal vascularization in wildtype vs. Jagged1-overexpressing mice using fluorescent staining. Three stains are used: α-Jagged1 to specifically target Jagged1 proteins, isolectin B4 for total endothelial cell staining and Topro3 for nuclear staining. The images in the top rank show that Jagged1 is much more highly expressed in the mutant mice strain and suggests that Jagged1 overexpression leads to more dense and branched retinal vasculature. The images in the bottom rank show that Jagged1 proteins are much more highly concentrated in endothelial stalk cells than tip cells.
Figure 5: An analysis of the effect of Jagged1 on the genetic targets of the Notch signaling parthway, Hey1 and Hes1 genes. In (B) Hey1 expression in Jagged1-overexpression mutants is identified by green fluorescence and is notably much more highly concentrated in endothelial stalk cells than tip cells, in concurrence with Figure 4. (D) identifies Hes1 expression. Hes1 expression in endothelial cells (blue) is marked by a yellow box and by a red box in non-ECs. In the Jagged1-overexpression mutants the relative number of yellow boxes increased while the overall vasculature noticeably decreases, indicating Hes1 activation by Jagged1.

The authors conclude that angiogenesis requires, among other factors, a tightly-controlled coordination between Dll4 and Jagged1 expression on endothelial cell surfaces. The paper seems successful in identifying the importance of Jagged1 as a stimulatory protein for angiogenesis and is also successful in characterizing which cells in growing blood vessels express the most/least Jagged1 on their surfaces. My only criticism is that the paper relies heavily on quantitative imaging techniques which, as we have learned, can be tricky and somewhat “hand-wavy”. I understand that angiogenesis research is well-suited to imaging analysis and I’m sure the researchers took every precaution they could, however I think that presentation of Southern and Northern blot results for the downstream Notch genes would augment the paper’s argument. Otherwise, bravo!