Figure 4. The Medial Angioblasts Give Rise to the Dorsal Aorta, while the Lateral Angioblasts Give Rise to the Posterior Cardinal Vein.

In all experiments, etv2:Kaede embryos were photoconverted at the 14- to 15-somite stages and analyzed at 24–28 hpf. Anterior is to the left; dorsal is up in all panels.

(A and B) Global green to red photoconversion of etv2:Kaede-positive cells demonstrates that all early Kaede⁺ endothelial progenitor cells that were photoconverted contribute to the DA (observed as an overlap of red and green fluorescence), while all late Kaede⁺ cells that originated after photoconversion contribute to the PCV (observed as green-only fluorescence). Note that etv2:Kaede expression pattern is mosaic, and not all endothelial cells express Kaede. In addition, etv2:Kaede is expressed in certain hematopoietic lineages such as neutrophils (red round cells in A and B), which were not included in the analysis.

(C and D) Selected groups of medial etv2:Kaede cells were photoconverted using laser confocal microscopy at the 15-somite stage (arrow, C), and their location was analyzed at 26–28 hpf (arrows, D) using fluorescence microscopy. Note that the labeled medial cells exclusively contribute to the DA, including DA-derived intersegmental vessels (ISV, D). Arrowheads in (C) point to the cells of the lateral line. Red round cells apparent on the right side of (D) correspond to etv2:Kaede expression in hematopoietic lineages. Panels in (C) and (D) represent different embryos.

(E) Global photoconversion of etv2:Kaede in VegfA-RNA-overexpressing embryos. Note that the cells in the DA and PCV positions include both double-positive (red and green) medial-line-derived endothelial cells and green-only lateral-line-derived endothelial cells (arrowheads).

(F) VegfA MO-injected embryos display a single vessel, as observed by global etv2:Kaede photoconversion. Double-positive (red and green) medial-line-derived endothelial cells are largely located in the dorsal most part of the vessel, while green-only lateral-line-derived cells are found largely in the ventral part of the vessel.

(G) Shh RNA-injected etv2:Kaede globally photoconverted embryos display a significant number of green-only lateral-line-derived cells in the DA (arrowhead) and occasional medial-line-derived cells in the PCV (arrow, points to a small double-positive endothelial cell).

(H) Cyclopamine (CyA)-treated etv2:Kaede globally photoconverted embryos have disorganized cells within the vascular cord often with no discernable distinction between the DA and the PCV. Higher frequency of green-only cells can be observed in the dorsal part of the vessel (arrowheads).

(I) Average contribution of medial (red) and lateral (green) globally photoconverted etv2:Kaede cells to the DA and the PCV in wild-type (n = 13 embryos and 335 endothelial cells analyzed), VegfA RNA (n = 17 embryos and 417 cells), Vegf MO (n = 10 embryos and 295 cells), Shh RNA-injected (n = 17 embryos and 247 cells), and CyA-treated (n = 8 embryos and 88 cells) embryos. Cell counts for each embryo are approximate estimates. Embryos treated with 1% DMSO (solvent for CyA) displayed distribution of green and red Kaede+ cells, similar to WT (not shown). In addition to the flat elongated vascular endothelial cells, etv2:Kaede expression was observed in round nonendothelial cells, which most likely correspond to hematopoietic progenitors such as neutrophils (data not shown); these cells were excluded from the analysis. *Statistically significant differences, as calculated using Z-test for two proportions to compare the percentage of green cells contributing to the DA or the percentage of red cells contributing to the PCV between the treated embryos and wild-type controls (p ≤ 0.001).

See also Figure S3.