Fig. 1.

Endothelial-specific knockout of Smad4 does not affect arterial identity but results in the loss of Ephb4 expression. a, b Representative E10.5 whole-mount images (a) and transverse sections (b) from wild-type Smad4^+/+ (n = 17), heterozygous Smad4^EC/+; (n = 12) and homozygous Smad4^EC/EC (n = 10) embryos all expressing the arterial Dll4in3:LacZ transgene (five litters in total). Robust transgene expression, specific to arterial endothelial cells, was seen in all embryos regardless of Smad4 genotype. Grey scale bars are 500 μm, black scale bars are 100 μm. c, d Representative E10.5 whole-mount images (c) and transverse sections (d) from wild-type Smad4^+/+ (n = 9), heterozygous Smad4^EC/+; (n = 9) and homozygous Smad4^EC/EC (n = 8) embryos also transgenic for the venous marker Ephb4^LacZ (four litters total). Robust X-gal activity is detected in the veins of Smad4^+/+ embryos but is reduced in Smad4^EC/+ embryos and absent in Smad4^EC/EC. Red box denotes zoomed region, grey numbers on bottom right denote the number of embryos similar to picture shown. Grey scale bars are 500 μm, black scale bars are 100 μm. Outliers are shown in Supplementary Figure 1e. e, f Expression of the venous endothelial cell markers EPHB4 (e) and COUP-TFII (f) in transverse sections from E10.5 Smad4^+/+ and Smad4^EC/EC embryos. In addition to venous endothelial cells, COUP-TFII is expressed by arterial smooth muscle cells and other mesenchymal cells (as reported by You et al.⁷). White scale bars are 100 μm. ^EC indicates Tie2:Cre-mediated deletion, ^+/+ indicates Cre−, ^EC/+ indicates Cre+,Smad4^fl/+ and ^EC/EC indicates Cre+;Smad4^fl/fl. da = dorsal aorta, ica = internal carotid artery, isa = intersomitic arteries, isv = intersomitic vessel, baa = branchial arch arteries, nt = neural tube, cv = cardinal vein, cev = branches of cerebral venous plexus. See also Supplementary Figure 1