Localization of GFP⁻ and GFP⁺endothelial cells in mouse embryos.

E10.5 transgenic embryos were stained with anti-GFP (green) and anti–PECAM-1 (red) antibodies. Nearly all the PECAM-1⁺endothelial cells express GFP, and, in addition, there were PECAM-1⁺ round cells circulating within the vessels, but they were negative for GFP (A-C). (D-L) GFP⁻endothelial cells (arrowheads) were found integrated in the endothelium at the ventral wall of the dorsal aorta, where hematopoietic clusters were formed, whereas the rest of the ECs were GFP⁺. (M-N) expressions of the transcripts for Runx1 were shown by using in situ hybridization (M) or RT-PCR (N). In situ hybridization was performed as described in “Materials and methods,” and red dots indicate the expression of Runx1. The sense probe did not give any signal (data not shown). White-dotted line indicates the outlines of dorsal aorta (M). (N) GFP⁻ VE cadherin⁺CD45⁻Ter119⁻ cells and GFP⁺ VE cadherin⁺ CD45⁻Ter119⁻ cells were sorted from 10.5-dpc transgenic embryos and subjected to RT-PCR using primers specific for Runx1. PCR products were separated on 1% agarose gel and stained with ethidium bromide. The result shown is representative of 3 independent experiments. DA, dorsal aorta.