Fig. 3. Localization of an endothelial enhancer in Gata2 intron 4. (A) Schematic representations of Gata2 YAC d16Z (Zhou et al., 1998), the GR22-lacZ plasmid (Zhou et al., 2000) and TKß expression constructs. The lacZ reporter gene (gray box), the Gata2 coding [exon 2 (e2)-e6; black boxes] and two alternative non-coding first exons (1S and 1G; black boxes) are represented. Overlapping fragments of the GR22-lacZ plasmid were excised using different restriction enzymes (BamHI, B; KpnI, K; SalI, Sa; SfiI, S; XbaI, X; XhoI, Xh) and were assayed in founder transgenic analyses. The sizes of the Gata2-enhancer test fragments are indicated. The numbers on the far right refer to the number of embryos with endothelial staining among the total number of transgene-positive embryos. (B,C) Extensive vascular X-gal staining was observed in representative transgenic embryos generated using the GR22-lacZ XhoI-SalI (data not shown) or KpnI-KpnI fragment (B), but not with the KpnI-SfiI fragment (C). (D) A similar lacZ expression pattern was reproduced in transgenic embryos generated using TKBXß (not shown) and TKSXß (D). (E-L) Embryos of E10.5 (E-F;K-L), E12.5 (G), E18.5 (H,I) or postnatal (J) gestation ages from a TKSXß stable transgenic line were stained for ß-galactosidase activity as cryosectioned (E,F,H,I,K,L) or whole-mount (G,J) specimens. X-gal accumulation was detected in the endothelia lining the dorsal aorta (E), umbilical artery (H) and vein (I); in the endocardium (F); and in the vascular network of the yolk sac (G) and postnatal brain (J). Interestingly, clusters of round lacZ-positive cells could be seen budding into the lumen of the dorsal aorta (arrow, E), which is suggestive of early hematopoietic cell formation (see Discussion). (K,L) An anterior transverse embryonic section stained simultaneously for ß-galactosidase activity and for the LEC-specific marker LYVE1. Notice that some cells in and around the anterior cardinal vein (cv) stained for both proteins (arrowheads), indicating that the endothelial enhancer is active in LECs as well as in cardiovascular endothelia.