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Fig. 3. Inhibition of KLF2 function results in reduced Flk1 expression
in the Xenopus embryo. (A-C) Whole-mount in situ
hybridization analysis of Flk1, Erg and Klf2 expression in
Xenopus embryos (stage 34, lateral view). For each gene, expression
is observed in the endothelial cells of the major developing vessels,
including the posterior cardinal vein (PCV), intersomitic vessels (IS), aortic
arches (AA) and in the forming plexus on the flank of the embryo (PL).
(D,E) Klf2 MO effectively blocks translation of a control
Klf2 transcript. (D) Bright-field and fluorescent images of embryos
injected with a control transcript in which the 5' UTR of Klf2
was fused to the coding sequences of GFP (Klf2-GFP). (E) Bright-field
and fluorescent images of embryos injected with Klf2-GFP transcript
plus Klf2 MO (25 ng). Note that GFP reporter fluorescence is greatly
inhibited by Klf2 MO treatment. (F) Embryo injected with 50 ng
of a control MO and assayed for expression of Flk1 transcripts. The
inset is a higher magnification view, centered on the developing posterior
cardinal vein. (G,H) Two different embryos injected with 50 ng of
Klf2 MO and assayed for expression of Flk1. Klf2 MO-injected
embryos show a dramatic reduction of Flk1 expression. (I)
Embryo injected with 500 pg of GFP mRNA and assayed for Flk1
transcripts. (J,K) Two different embryos injected with 250 pg of mRNA
encoding a dominant-repressor form of KLF2 (DR-KLF2) and assayed for
Flk1 transcripts. Embryos expressing the DR-KLF2 construction show a
dramatic reduction of Flk1 transcripts. (L) qRT-PCR analysis
reveals significant reduction in Flk1 transcript levels in
Klf2 MO-treated embryos. Results shown are the average of three
separate embryos for each MO treatment. (M-M'') Klf2 MO
treatment eliminates vascular tubes. Histological section through a stage 42
embryo injected with Klf2 MO. Somites (s) and notochord (nc) are
indicated. Scale bar: 100 µm. Injected side is to the right (arrowhead).
M' and M'' show enlargements of the region of the posterior
cardinal vein on the untreated and treated sides, respectively. Although the
pronephric duct (pnd) is visible on both sides, no tube corresponding to the
posterior cardinal vein (pcv) is visible on the treated side.
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-crystallin reporter construction that serves
as a marker for transgenesis. (B) Fluorescent image of the trunk region
of a typical transgenic embryo (stage 45) showing GFP expression in minor
blood vessels. The gut (g) is indicated for orientation. (C)
Fluorescent image of entire transgenic embryo (stage 47) showing GFP
expression throughout the vasculature. (D) Bright-field view of embryo
transgenic for the ETS mutant construction. (E) Fluorescent image of
embryo shown in D. Mutation of the conserved ETS site dramatically reduces
detectable GFP fluorescence in the vasculature. (F,G) Fluorescent
images of embryos transgenic for the KLF mutant construction, showing major
reduction in GFP reporter expression. To allow direct comparison, images in
B,F,G were collected at the same magnification and exposure. (H)
Mutation of the KLF binding site reduces Flk1 reporter expression in
an endothelial cell line. The luciferase coding region was substituted for GFP
in the Flk1 reporter construction and transfected into a mouse
endothelial cell line (bEnd.3). Relative luciferase activity of wild-type and
mutant constructions is indicated. Assays were carried out in quadruplicate.
(I) Three copies of the ETS/KLF binding sequence, or equivalent ETS and
KLF mutated sequences, were inserted upstream of a minimal promoter in a
luciferase reporter construction and the resulting plasmids transfected into
bEnd.3 cells. Mutations of either the ETS or KLF binding sites result in a
reduction in relative luciferase activity. Assays were carried out in
quadruplicate.
