Fig. 1. Endothelial GATA-2 expression in Gata2-GFP knock-in embryos. (A-R) GFP expression in Gata2-GFP embryos at E9.5 (A), E10.5 (B-D,G-L), E11.5 (M-O), E18.5 (E,F) and postnatal day 1 (P1; P-R) was monitored by direct fluorescence (A), indirect immunofluorescence (B-D,F-R) or light (E) microscopy. (A) Robust GFP fluorescence is visualized in the heart and the dorsal aorta, a vessel formed by vasculogenesis (arrowhead), of a whole-mount embryo orientated with its head (not shown) facing to the left, towards the tail bud. (B-D,F) Transverse embryonic cryosections were stained for GFP using Alexa Fluor 488-conjugated secondary antibody. GFP immunoreactivity was detected in the intersomitic vessels (B, arrowheads) in the tail region of an embryo, in the endothelia lining the aortic sac (C), in the thin-walled umbilical vein and thick-walled umbilical artery (E,F), and in the endocardium of the heart ventricle (C), as well as in the vessels that begin to invade the neural tube, a typical example of sprouting angiogenesis (D). (E) A phase-contrast image of F. (G-L) The intersomitic vessels and the aorta in the tail region of an embryo co-stained for GFP (G,J) or PECAM (H,K) antigens using CY3- or Alexa Fluor 488-conjugated secondary antibodies, respectively. Coincidence of anti-PECAM and anti-GFP staining demonstrates that Gata2 is expressed in endothelial cells (I,L). Boxed areas in G-I are magnified in J-L. (M,N) Transverse embryonic cryosections were stained for GFP (N) or VEGFR3 (M) using CY2- or CY3-conjugated secondary antibodies, respectively. Clustered cells in the vicinity of the anterior cardinal vein expressed both VEGFR3 and GFP (arrowheads). Notice that, although GFP immunofluorescence was detected strongly in endothelia of the dorsal aorta and cardinal vein, both of these blood vessels stained only weakly, in comparison to LECs, with anti-VEGFR3 antibody. (O) An adjacent section was co-stained with anti-PROX1 and anti-VEGFR3 antibodies using CY2- or CY3-conjugated secondary antibodies, respectively. Notice that VEGFR3-positive cells displayed anti-PROX1 nuclear staining (arrowhead), thus confirming their LEC identity. (P-R) P1 postnatal intestines and mesentery were sectioned and stained for VEGFR3 (P) and GFP (Q) expression as described above. Coincidence of staining in lymphatic vessels (arrowheads) is distinct from blood vessels that stained only for GFP (arrows). The nuclei in panels L,O and R were co-labeled with DAPI. h, heart; tb, tail bud; as, aortic sac; ven, ventricle; mv, mesencephalic vesicle; uv, umbilical vein; ua, umbilical artery; cv, cardinal vein; da, dorsal aorta.