Figure 1: Expression of Arf6 mRNA in blood vessels and conditional targeting of endothelial Arf6.

(a) In situ hybridization for Arf6 mRNA of neural tube blood vessels. Arrow heads in the left panel indicate vessels sprouting into the neural tube of the wild-type (WT) embryo. (b) In situ hybridization for Arf6 (top and middle panels) and immunostaining for PECAM1 (bottom panels) in dorsal aorta of E13.5 embryos. High-magnification images of the rim of aortas (middle panels) show Arf6 expression in the endothelium of WT embryo. NT, neural tube; SM, smooth muscle layer; E, endothelium; ES, oesophagus; DA, dorsal aorta; L, lung. Scale bar, 100 μm. (c) Schematic representation of the conditional gene targeting strategy for Arf6. Exons for Arf6 are represented by filled black rectangles. The neomycin-resistance selection cassette is represented as ‘neo’. loxP and FRT sites, red and blue triangles, respectively. EV, B; restriction sites for EcoRV and BamHI. Targeting vector-derived restriction sites are shown in bold. The relevant restriction fragments for Southern blotting are shown by horizontal lines with fragment sizes. A probe fragment for Southern blotting is represented by a grey box. Positions of primers used for genomic PCR analysis are shown by arrows. (d) Southern blot analysis of BamHI-digested genomic DNAs from each genotype of mouse tails using probe indicated in c. (e) Genomic PCR analysis of the offspring of a Tie2-Cre;Arf6^flox/+ male and Arf6^flox/flox female pair. DNA fragments amplified from KO, WT and floxed alleles (flox) were detected.