supplementary information
www.nature.com/naturecellbiology	 1
DOI: 10.1038/ncb2102
Figure S1 Hypoxia activates Wnt/b-catenin signalling in embryonic cells.
a, ES and P19 EC cells transiently transfected with TOP-Flash reporter
plasmid were cultured under different low O2 levels (0.5% and 3%)
for 16 h (n=6). b, No significant enhancement of FOP-Flash reporter
activity was observed in hypoxic cells (1.5% O2 or other low O2 levels
[data not shown]) over cells cultured under normoxia (n=6). Luciferase
activity from pRL-SV40 reporter co-transfected with TOP-Flash or FOPFlash
reporter plasmids was used for normalization. c, Western blot
analysis of whole cell extracts of ES cells cultured under normoxia or
hypoxia for phosphorylated GSK-3b, and total GSK-3b. Actin served as
the loading control. *= P <0.05, **= P <0.005., Student’s t-test. Error
bars represent S.D.
a
TOP-Flashreporter
activity(fold)
0
0.5
1
1.5
2
2.5
3
3.5
4
*
**
**
ES P19
N
H, 3% O2
H, 0.5% O2
0
0.5
1
1.5
2
2.5
3
3.5
4
FOP-Flashreporter
activity(fold)
b
ES P19
N
H, 1.5% O2
c
N H
actin
pGSK-3β
GSK-3β
ES
FigureS1_Mazumdar (NCB-S19075-T)
Figure S1
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Figure S2 Hypoxia increases Wnt/b-catenin activity in stimulated cells. a,
b, TOP-Flash reporter activity in ES and P19 EC cells treated with either
Wnt-3a CM or LiCl and cultured either under 21% or 1.5% O2 for 16 h
(n=9). Whereas Wnt-3a CM and LiCl stimulates TOP-Flash activity, exposure
to 1.5% O2 further increases TOP-Flash activity in stimulated cells. c, Cellcycle
analyses for ES cells after exposure to normoxia or hypoxia (1.5% O2)
for 24 h or 48 h. Cells were labelled with BrdU, stained with propidium
iodide (PI) and analysed by flow cytometry (n=3 independent experiments).
Increased G1/S ratio of hypoxic ES cells indicates increased accumulation
in G1 stage, and delayed S-phase entry. Note G1/S phase ratio decreases
in ES cells exposed to hypoxia for 48h, as compared to cells cultured under
hypoxia for 24 h. d, Decreased cell death in ES cells cultured under hypoxia
as compared to normoxic control cells (n=3 plates, 9 random fields in each
plate) assessed by a TdT mediated dUTP nick end labelling (TUNEL) assay.
Increased cell survival likely accounts for ES cell expansion under prolonged
hypoxia (Fig. 1g). *= P <0.05., Student’s t-test. Error bars represent S.D.
Reporteractivity(fold)
0
1
2
3
4
5
6
7
0
2
4
6
8
10
12
Reporteractivity(fold)
N + + - + + H
- - + - - +
LiCl - + + - + +
ES P19
N + + - + + H
- - + - - +
Wnt-3a CM - + + - + +
Reporteractivity(fold)
0
2
4
6
8
10
12
* *
ES P19
a b
Figure S2
HN
0
0.5
1
1.5
2
G1/Sratio
24h 48h
ES
*
c
HN
d
24h 48h
ES
%TUNEL+cell/field(Fold)
0
0.4
0.6
0.2
0.8
1
1.2 *
FigureS2_Mazumdar(NCB-S19075-T)
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Figure S3 HIF-1a and ARNT are required for hypoxic activation of Wnt/bcatenin
signalling in embryonic cells. a, qRT-PCR analysis of Wnt-3a target
genes in Arnt +/+, Arnt-/- and Arnt Res ES cells show direct dependence of
Wnt induction on hypoxic ARNT activity (n=3). b, c, Cells were plated at a
density of 104 cells per 60 mm2 and cultured under normoxia or 1.5% O2
for 6 days. Numbers were assessed by cell counts in a hemocytometer at
indicated time points. Hif-1a +/+(b), Arnt+/+ and Arnt Res (c) cells displayed
cell number expansion under hypoxia. To the contrary, hypoxic Hif-1a-/- (b)
and Arnt-/- (c) cells grew at rates comparable to normoxic control cells. *= P
<0.05, **= P <0.005., Student’s t-test. Error bars represent S.D.
Figure S3
FoldchangeinmRNA
(normalizedto18s)
0
2
4
6
8
10
12
Lef-1 Tcf-1 Dkk-4 Pgk1
N, Arnt+/+
H, Arnt+/+
H, Arnt-/N,
Arnt-/H,
ArntRes
N, ArntRes
**
**
*
a
b
Days in culture
No.ofcellsx104
0 2 4 6
0
25
50
75
100
125
150
175
200
225
N
H
Hif-1α+/+
0 2 4 6
Days in culture
No.ofcellsx104
0
25
50
75
100
125
150
175
200
225
N
H
Hif-1α-/-
No.ofcellsx104
0
25
50
75
100
125
150
175
200
225
N
H
Arnt+/+
Days in culture
0 2 4 6
c
No.ofcellsx104
0
25
50
75
100
125
150
175
200
225
0 2 4 6
Days in culture
N
H
Arnt-/Days
in culture
0 2 4 6
No.ofcellsx104
0
25
50
75
100
125
150
175
200
225
N
H
ArntRes
*
*
* *
****
**
*
*
FigureS3_Mazumdar(NCB-S19075-T)
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Figure S4 ES cell differentiation, HIF-1a/b-catenin interaction and LEF-1
modulation of ES cell growth. a, ES cells treated with (+) or without (-)
N2B27 neuronal growth and differentiation supplements differentiate
into neurons as indicated by the expression of the neuronal marker
doublecortin (DCX) in green. The nuclei are stained with DAPI (blue). b,
Immunoprecipitation with HIF-1a antibody was performed on nuclear
extracts of normoxic or hypoxic (1.5% for 20 h) ES cells. CREB served as
the loading control. c, ES-Lef-1 cells were plated at a density of 104 cells
per 60 mm2, cultured under normoxia and cell numbers assessed over 6
days. Empty virus transduced (VC) cells served as control. Both cell lines
were also treated with DKK-1 (300 ng ml-1). **= P <0.005., Student’s
t-test. Error bars represent S.D.
DCX DAPI Merge
a
Figure S4
5µµm
CREB
HIF-1α
β-catenin
ES
N H N H
1% Input HIF-1αIP:
WB:
b
ES VC+Dkk1
ES VC
ES Lef-1
0 2 4 6
Days in culture
0
50
100
150
200
250
300
No.ofcellsx104
ES Lef-1+Dkk1
c
**
-N2B27+
ES
FigureS4_Mazumdar (NCB-S19075-T)
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supplementary information
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Figure S5 Hypoxic regions in embryonic and adult brain. a, b, Wnt /b-catenin
activity marked by b-galactosidase enzyme staining (blue) in E11.5 BATGAL
reporter mice (a) is closely associated with hypoxic regions marked by
pimonidazole staining (brown) in an adjacent b-galactosidase enzyme stained
(blue) embryonic section (b). (c, d) Magnifications of the boxed region in (a)
and (b). Black line in (d) demarcates highly hypoxic region from the adjacent
light brown area. e-g, b-galactosidase (b-gal) immunostaining in BAT-GAL
reporter line (e) and wildtype control (f) identifies the GCL as active for Wnt/bcatenin
signalling. g, Co-expression of b-galactosidase and Sox2 in neural
stem cells in the SGZ. h-k, India black ink and CD31 mark fewer blood vessels
in the GCL of the DG (j, k) as compared to the Oriens layer of the hippocampus
(h, i). l-n, Pimonidazole immunofluorescence staining (l) and enzymatic
immunodetection (n) indicates the presence of hypoxic pockets in the GCL.
The hippocampus of PBS injected animals served as a negative control (m).
o-q, Expression of CAIX (o), VEGF (p) and stabilization of nuclear HIF-1a (q)
within the GCL of the DG. r-t, Nuclear (arrows) and cytoplasmic distribution
of Cre in the GCL of aCamKII-Cre R1 line (r). Cre negative mice served as
a negative control (s). Colocalization with Sox2+ cells (arrows) indicates the
expression of Cre in neural stem and progenitor cells in the SGZ (t). In e-t,
arrowheads point towards the SGZ, and arrows indicate the relevant cells.
mNegative control-PBS
50µµm
l Pimonidazole-FITC
50µµm
Pimonidazole-DABn
CAIXo
50µµm
q HIF-1αp VEGF
50µµm
a b
d
50µµm
2.5µµm
50µµm
2.5µµm
Figure S5
India Ink
India Ink
ββ-gal Negative control
e f g
50µµm 50µµm
DG
h
OrienslayerDentategyrus
j k
i
r s t
Negative control
50µµm
DAPI, CD31
50µµm
50µµm
Cre, DAPI
50µµm
DAPI, CD31
50µµm
BAT-GAL ββ-gal
c
FigureS5_Mazumdar (NCB-S19075-T)
50µµm
50µµm50µµm
50µµm
50µµm
Cre, Sox2
ββ-gal, Sox2
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Figure S6 In vivo deletion of Hif-1a impairs adult hippocampal neurogenesis
and Wnt/b-catenin signalling. a, qRT-PCR confirmation of Hif-1a deletion in
the adult hippocampal extracts of gHif-1aD/D mice (n=3-4 in each group). b,
PCR genotyping indicating Cre mediated deletion of Hif-1a marked by the
absence of 2loxP band and the presence of Cre and 1loxP bands (gHif-1aD/D
mice). The recombination efficiency of Cre is variable. Weak 2loxP band
was detected in some Hif-1aD/D mice (data not shown). c, b-galactosidase
enzyme (X-gal) staining of the dentate gyrus of Hif-1af/f, BAT-GALTg (c
upper panel) and gHif-1aD/D, BAT-GALTg (c lower panel). d, 4 fold reduction
in Wnt activity as assessed by X-gal staining in the dentate gyrus of adult
gHif-1aD/D compared to gHif-1af/f control animals (n=3, 5 sections per
animal). e, DCX (green) and BrdU (red) double positive cells (white arrows)
reveal post-mitotic neurons in the SGZ and GCL of gHif-1af/f (upper) and
gHif-1aD/D mice (lower). f, g, In vivo deletion of neuronal Hif-1a impairs
adult hippocampal neuronal morphology (n=3). *= P <0.05, **= P <0.005.,
Student’s t-test. Error bars represent S.E.M.. h, Stereotactic injection of
D-GSK3-b-catenin lentivirus (HIV- DGSK3-b-catenin-IRES-Zsgreen) into the
adult DG (12-16 weeks) is detected by green fluorescence, which is absent
in PBS injected adult DG. i, Quantification of BrdU+ (i left panel) and DCX+
(i right panel) cells in nHif-1aD/D DG transduced with high titer D-GSK3-bcatenin
lentivirus. Following 2 weeks of recovery, mice were injected with
BrdU (100 mg Kg-1) i.p daily for 4 days. Control virus treated nHif-1aD/D and
nHif-1af/f mice served as controls (n=3-5 per group). Statistical significance
(i) was computed using one-way ANOVA. nHif-1aD/D animals transduced
with high titer D-GSK3-b-catenin lentivirus displayed remarkable increase in
BrdU+ and DCX+ cell counts as compared to control virus treated nHif-1aD/D
animals, and is approaching significance (# indicates P=0.06). Error bars
represent S.E.M.
b
Cre
Hif-1α 2loxP
Hif-1α 1loxP
gHif-1αf/f gHif-1α∆/∆
Hif-1α
a
**
FoldchangeinmRNA
(normalizedto18s)
0
0.2
0.4
0.6
0.8
1
1.2 gHif-1αf/f
gHif-1α∆/∆
DCX, BrdU
gHif-1αf/fgHif-1α∆/∆ 20µµm
e
DCX, DAPI
nHif-1ααf/f nHif-1αα∆∆//∆∆
20µµm
f
**
nHif-1αf/f
nHif-1α∆/∆
%ofDCX+cells
withneurite/field
0
25
50
75
100
125
g
Figure S6
**
gHif-1αf/f
gHif-1α∆/∆
%ofDGXgal+cells/field
0
25
50
75
100
125
0
4
8
12
16
20
No.ofBrDU+cells/field
*
nHif-1αf/f + CV nHif-1α∆/∆ + CV
0
10
20
30
40
50
No.ofDCX+cells/field
nHif-1α∆/∆ +∆GSK-3-β-catenin
*60
ih
Dentate gyrus
nHif-1αf/f
HIV-∆GSK β-catenin-IRES-Zsgreen
PBS injected
70 µµm
c d
gHif-1αf/fgHif-1α∆/∆
X-gal staining
#
#
Mazumdar_Fig. S6 (NCB-S19075-T)
100µµm
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Figure S7 Uncropped blots
Figure 1b Figure 1f Figure 2e
Figure 2f
Figure 3d Figure S1c
Figure S4b
Figure S6b
© 2010 Macmillan Publishers Limited. All rights reserved.