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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.
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AAß and TKANß) used
to functionally localize the Gata2 endothelium-specific enhancer
element. Sub-fragments of Gata2 intron 4 were individually cis-linked
to a TK promoter-lacZ reporter gene. The positions of relevant
restriction enzyme sites (AlwNI, A; ApaI, Ap; NcoI,
N; RsrII, R, SfiI, S; XbaI, X) and the restriction
fragment lengths (in bp) are indicated. The numbers on the right indicate the
number of embryos with cardiovascular ß-gal staining/total number of
transgene-positive embryos. The arrows represent the positions of the primer
pairs used to amplify the 167 bp VE enhancer (see
Fig. 5). (B-E) E10.5
embryos bearing TKSRß (B) or TKAAß (C) transgenes showed widespread
endothelial ß-gal staining, whereas the TKSX
