XB-IMG-138000
Xenbase Image ID: 138000
|
FIGURE 2. The gain- or loss-of-function of HIF-1 . A, effects of injection of
hif-1 mRNAs indicated at the top on vascular development. Panels a–d,
Xenopus embryos un-injected; panels e–h, embryos injected with hif-1
(P558G) mRNA (750 pg); and panels i and j, embryos injected with hif-1 antisense
morpholino 1 (hif-1 MO1) (40 ng).MOormRNAwere injected into one
of the two vegetal-dorsal blastomeres at the 8-cell stage. The embryos were
assayed at tail-bud stage 33/34 with wholemountdouble in situ hybridization
for tie-2 and -globin, endothelial and hematopoietic markers, respectively.
Panels a, e, and i, lateral views; and panels c, g, and k, ventral views. Panels b, f,
and j, enlarged vascular vitelline network (VVN); and panels b, h, and l,
enlarged VBI formation. Positive signal was detected with BM purple (blue)
and DAB (brown) for tie-2 and -globin, respectively. VVN meshwork was
increased in hif-1 (P558G) mRNA-injected embryos compared with the
control. In contrast, knockdown with MO reduced the VVN and VBI regions.
B, schematic presentation of 5 UTR-HIF(N)-Venus and the region against
which MO are designed. Transcript of 5 UTR-HIF(N)-Venus corresponds to a
single transcript of the 5 -UTR (102 bp) and mRNA of hif-1 N-terminal (100
amino acids) andmRNAcoding Venus. C, effect of hif-1 MO1and hif-1 MO2
on in vitro translated expression of HIF-1 fused to Venus. pCS2 -5 UTRHIF(
N)-Venus (0.5 g) was transcribed and translated in vitro in the absence of
MOs (lane 1) or in the presence of CMO (lane 2) and hif-1 MO1 and hif-1
MO2 at the concentrations indicated on at the top (lanes 3–6). HIF-1 -Venus
was analyzed by immunoblotting (IB) with anti-GFP antibody. Both hif-1
MO1 and hif-1 MO2 inhibit translation of hif-1 in vitro. D, in vivo translation
of HIF(N)-Venus (200 pg) is specifically inhibited by hif-1 MOs (40 ng).
Embryos (8-cell stage) were injected with pCS2 -5 UTR-HIF(N)-Venus and
hif-1 MOs. pCS2 -5 UTR-HIF(N)-Venus plus CMO (left panels), hif-1 MO1
(middle panels), and hif-1 MO2 (right panels) are shown. Embryos were
observed by fluorescence microscopy. BF, bright field; GFP, green fluorescence
from Venus. E, the lysates from embryos injected with pCS2 -5 UTRHIF(
N)-Venus and the MOs as indicated at the top were subjected to immunoblot
analysis using anti-GFP antibody to examine the effect of MOs on the
transcription of HIF-1 tagged with Venus. -Tubulin was used as loading
control. F, the endogenous HIF-1 protein level in Xenopus was reduced in
hif-1 MO-injected embryos. Panel a, the lysates obtained from Xenopus
embryos were subjected to immunoprecipitation (IP) with the antibody indicated
at the top and followed by immunoblotting with anti-HIF-1 antibody.
Panel b, Xenopus embryos injected with hif-1 MOs (MO1 or MO2) as
described under “Experimental Procedures” was analyzed for expression of
HIF-1 byimmunoprecipitationandIBusinganti-HIF-1 antibody. Panel c,quantitative
analysis of the results of panel b obtained from three independent experiments.
Relative expression of HIF-1 in embryos treated with hif-1 MOto that
of those treated with CMO was calculated and expressed as mean S.D. Image published in: Nagao K et al. (2008) Copyright © 2008. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
Image source: Published Larger Image Printer Friendly View |