cKO embryos display a variety of defects in the yolk sac mesoderm.

<p>A–B, D–E) Whole mount immunofluorescence of 9.5 <i>dpc</i> WT (A–B) or mutant (D–E) yolk sacs (YS) using the endothelial marker PECAM (green) and the vascular smooth muscle marker (αSMA) demonstrates that the large disorganized vessels in the cKO (D) are not surrounded by αSMA (E). C, F) Section immunofluorescence of WT (C) and cKO (F) 9.5 <i>dpc</i> yolk sacs demonstrates loss of αSMA in the cKO. G–L) Section immunofluorescence of VEGFA (green) demonstrates relatively uniform VEGF levels in the 9.0, 9.25 and 9.5 <i>dpc</i> WT yolk sac (G–I) while VEGF distribution in the visceral endoderm of the mutant is progressively diminished at each stage (J–L). M) A Western blot of whole yolk sacs at the indicated stages. The ratio of VEGFA to GAPDH signal intensities for the cKO relative to each stage-matched WT control is displayed under each band. N) Cleaved Caspase-3 staining was used to assess the percentage of cell death in the yolk sacs layers of WT and cKO sections at 8.5 and 9.0 <i>dpc</i>. A significant increase in apoptosis was observed in the cKO mesoderm (ME) at 9.0 <i>dpc</i>. O) Phosphohistone-H3 (PH-3) staining was similarly used to assess proliferation and a significant decrease in proliferation was found in the cKO yolk sac mesoderm at 9.0 <i>dpc</i>. *** = p<0.001, ** = p<0.01; error bars = standard error; dotted line is drawn between the visceral endoderm (VE) and mesoderm derivatives (ME) on yolk sac sections.