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FIGURE 1. Expression of hif-1 during Xenopus development. A, predicted
domains of Xenopus HIF-1 . bHLH, basic helix loop helix binding domain; PAS,
Per-Arnt-Sim domain; TAD-N, N-terminal activation domain; TAD-C, C-terminal
activation domain. B, temporal expression profile of the hif-1 mRNA.
RT-PCR analysis was carried out from stage 1 to 25 using a primer set for
hif-1 . Quality of RNA at each stage was assessed by amplification of the
ubiquitous transcript, ornithine decarboxylase (odc). C, hif-1 expression was
examined by whole mount in situ hybridization of embryos at the stage indicated
in each panel. a, lateral view of stage 23 embryo; b, lateral view of stage
28 embryo; c– e, Stage 33/34 embryos, lateral view (c), ventral view (d), and
higher magnification image (e) of the anterior portion seen in panel d. hif-1
expression in the eyes (yellow arrowhead), brain (yellow arrow), kidneys (red
arrow), somites (yellow asterisk), branchial arches (black arrowhead), myocardium
(red arrowhead), and the ventral blood island (VBI, black asterisk). f, transversal
section of a stage 33/34 embryo at the white line in panel d reveals
hif-1 expressed in myocardium.
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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.
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FIGURE 3. Impaired cardiac development in HIF-1-depleted Xenopus
embryo. A, a representative result of images obtained from embryos uninjected
(control) or injected with hif-1 MO1or hif-1 MO2. Panel a, embryos
without injection possessed a beating heart (white arrowhead) anterior to the
gut. Panels b and c, embryos injected with either hif-1 MO1or hif-1 MO2(40
ng) had either a tissue-free region where the heart should localize or hearts
that did not beat (yellow arrowhead) at stage 42. B, expression of cTnI examined
by in situ hybridization in HIF-1-depleted embryos. The embryos
injected withCMO(40 ng) (a), hif-1 MO1(40 ng) (b), or hif-1 MO2(40 ng) (c)
were analyzed at stage 33/34. Panels show anterior ventral views. Yellow
arrowheads point to the side derived from the blastomere where hif-1 MO
was injected. In hif-1 MO-injected embryos, cTnI was detected in two separate
populations of cardiomyocytes, whereas cTnI was detected at the center
in CMO-injected embryos. Representative eosin-stained cross-sections of
embryos injected with CMO (b), hif-1 MO1 (d), or hif-1 MO2 (f) are shown.
The heart region is indicated by an arrowhead.
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FIGURE 4. Reduction of the expression of early cardiac markers by depletion
of HIF-1 . A, the expression of nkx2.5 (a and b), gata4 (c and d), tbx5 (e
and f), hand1 (g and h), hand2 (i and j), and bmp4 (k and l) was analyzed with
whole mount in situ hybridization analyses at stage 33/34. The embryos uninjected
(a, c, e, g, i, and k) and those injected with hif-1 MO1 (b, d, f, h, j, and
l) are shown. The panel show the anterior ventral view. hif-1 MO1-injected
embryos showed only faint expression of cardiac markers except BMP4. Yellow
arrowheads indicates the side derived from the blastomere where hif-1
MO1 was injected. These results are summarized in Table 3.
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FIGURE 5. HIF-1 regulates transcription of nkx2.5. A, effects of various
transcription factors on nkx2.5 transcriptional activity. Luciferase activity was
measured by the lysates from CV1 cells transfected with the Nkx2.5 promoter-
Luc plasmid and the expression plasmid indicated at the bottom (control
vector (pIRES puro3), mouse Gata4, Smad4, or Hif-1 (P577G)). -Fold differences
in relative luciferase activity were calculated as arbitrary luciferase
activity. Transfection efficiency was normalized by Renilla luciferase activity
derived from null-pRL control plasmid. B, RT-PCR analysis using total RNA
from embryos at stage 1 to 25. BothRNAquality and quantity was assessed by
amplification of ornithine decarboxylase (odc). C, visualization of Nkx2.5 promoter
activity in developing embryos. The Nkx2.5 promoter-Venus (100 pg)
was co-injected with 40 ng of CMO or hif-1 MO1. Green fluorescence
reflected activation of the Nkx2.5 promoter. Fluorescence was observed
around the heart primordial region at stage 20 embryos. Nkx2.5 promoter
activity was suppressed with co-injection with hif-1 MO1, but not with CMO.
D, activation of mouse Nkx2.5 promoter (9.0 kbp) in developing Xenopus
embryos. Xenopus embryos were injected with the pGL4.10–9kb Nkx2.5 promoter
(Nkx2.5 promoter-Luc) or promoter-less luciferase reporter gene
(pGL4.10). Dual luciferase assay were performed in the embryos at the neural
stage, stage 19–20 when Nkx2.5 mRNA expression peaked. E, the effect of
injection of either CMO or hif-1 MO on Nkx2.5 promoter activity. The Nkx2.5
promoter-Luc (100 pg) and pRL-null (2 pg) were co-injected with either 40 ng
of CMO or hif-1 MO1. Embryos were harvested at the neural stage, stage
19–20. For each group, 15 pools containing 5 embryos each were used. Luciferase
activities are shown, with error bars representing the mean S.E. (n
15 each group). **, p 0.01 versus CMO). F, RT-PCR analysis for examining
expression of nkx2.5 mRNA using total RNA from embryos at stage 20. Both
RNA quality and quantity was assessed by amplification of ornithine decarboxylase
(odc).
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HIFFIGURE
6. NKX2.5 functions downstream of HIF-1. A, cardia bifida in HIF-
1-depleted embryos was rescued by expression of nkx2.5. Embryos were
injected with hif-1 MO1 (40 ng) and nkx2.5 mRNA (0, 125, and 250 pg). The
expression pattern of cTnI was analyzed by in situ hybridization at stage 33/34
to detect the precise migration of cardiomyocytes. Cardia bifida caused by
depletion of HIF-1 was restored by co-injection of nkx2.5 mRNA. B, depletion
of NKX2.5 results in cardia bifida in Xenopus embryos. CMO-injected embryos
(a), nkx2.5 MO-injected embryos (b), and hif-1 MO1-injected embryos (c)
were examined for cTnI by in situ hybridization at stage 33/34. NKX2.5-depleted
embryos showed a similar staining pattern of cTnI to HIF-1-depleted
embryos. Yellow arrowheads indicate the side derived from the blastomere
where hif-1 MO1 was injected. C, the incidence of cardia bifida in nkx2.5
MO-injected embryos, hif-1 MO-injected embryos, or CMO-injected
embryos.
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Figure 1S. Classification of the embryos showing cardia bifida at stage 33/34. HIF-1α-depleted embryos at showing cardia bifida stage 33/34 in Figure 3B were separated into three groups according to the degree of reduced cardiac troponin I (TnIc) expression in the side derived from the blastomere where HIF-1α MO1 was injected. TnIc mRNAs were visualized using BM purple (blue). TypeI, II, and III reflect faint reduction, mild reduction, and severe reduction, respectively. Reduced expression of TnIc was found in the side (yellow arrowhead) derived from the blastomere where HIF-1α MO1 was injected.
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Figure 2S. Defect of myocardial cell differentiation and heart development in embryos injected with HIF-1α MO. Whole mount in situ hybridization analysis was carried out at stage 42 to detect cardiac troponin I (TnIc) mRNA expression in embryos injected with 40 ng of CMO (a) or HIF-1α MO1 (b,c) at 8-cell stage. TnIc mRNAs were visualized (blue) on transverse sections of the embryos injected with CMO (d) or HIF-1α MO1 (e,f). TnIc was asymmetrically detected in the embryos injected with HIF-1α MO1 compared to than those injected with CMO. This asymmetrical expression of TnIc reflects the interference of heart development (smaller heart in IF-1α MO1-injected embryos than CMO-injected embryos). nc, notochord; en, endoderm; h, heart
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Figure 3S. Reduced expression of GATA4 in HIF-1α–depleted embryos. A, RT-PCR analysis using total RNA from embryos at stage 1 to stage 25. Both RNA quality and quantity was assessed by amplification of ornithine decarboxylase (ODC). PCR was carried out using the following primers; 5’-AGTGCTACTGCTGCTACCTC-3’ and 5’-ACTGTAGGAGACCTCTCTGC-3’ (55°C annealing, 30 cycles). B, The effect of injection of either CMO or HIF-1α MO1 on GATA4 promoter activity. Similarly to Nkx2.5, Xenopus embryos were injected with the pGL4.10-1.5kb GATA4 promoter (GATA4 promoter-Luc) and promoter-less luciferase reporter gene (pGL4.10) together with either 40 ng CMO or HIF-1α MO1. **P<0.01 vs. CMO. The mouse GATA4 5’-flanking fragment was isolated from a mouse genome using PCR with the following primer pair: 5’-TCCGGCTTGAAGCTTGCTCCCGGC-3’ and 5’-GGAACCTGCAGGCCCTGATTCTGAC-3’. The 1.5kbp fragment was inserted into pGL4.10 vector with EcoRV and HindIII site.
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hif1a (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
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hif1a (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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hif1a (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33 and 34, lateral view, anterior left, dorsal up.
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