Figure legends supplemental figures
Supplemental figure 1.
(a) Staining pattern of cre-protein is similar to GFAP-staining of astrocytes but does
not resemble NeuN-staining of neurons in mouse hippocampus. (b) Cre protein does
not
colocalize
with
NeuN-positve
neurons
(scalebar
100µm)
(c)
Upper:
GFAPcre+/VHL+f/+f are smaller than widtype littermates. Lower: A dome-shaped head
in GFAPcre+/VHL+f/+f mice (arrowhead) indicates the presence of hydrocephalus in
those animals. (d) Analysis of Evans blue (EB) leakage into the CNS indicates a
significantly impaired blood-brain barrier function in GFAPcre+/VHL+f/+f (1.73 ± 0.2fold increase (n=7) of OD620/g brain) and GFAPcre+/VHL+f/+f/HIF-1α+f/+f (1.46 ± 0.2fold increase (n=4)) compared to GFAPcre-negative mice. GFAPcre+/VHL+f/+f/HIF2α+f/+f mice have no significantly altered blood-brain barrier permeability (1.22 ± 0.1fold change (n=5; Mann-Whitney-test)) (e) Upper graph: hearts of GFAPcre+/VHL+f/+f
(167.1 ± 10.8 mg (n=7)) and of GFAPcre+/VHL+f/+f/HIF-1α+f/+f (158.0 ± 7.9 (n=5)) are
significantly larger than hearts of control (GFAPcre-; 130.0 ± 2.5 mg (n=9)) and of
GFAPcre+/VHL+f/+f/HIF-2α+f/+f mice (135.0 ± 8.9 mg (n=4;
Mann-Whitney-test)).
Lower graph: spleens are significantly enlarged in GFAPcre+/VHL+f/+f (244.2 ± 31.9
mg (n=6)) and in GFAPcre+/VHL+f/+f/HIF-1α+f/+f animals (173.0 ± 14.5 mg (n=5))
compared to control mice (GFAPcre-: 77.7 ± 8.4 mg (n=9)). Spleens are not
significantly different in GFAPcre+/VHL+f/+f/HIF-2α+f/+f animals (88 ± 0.01 mg (n=4);
data represents means ± S.E.M.). (f) Hydrocephaly is detectable in three week old
GFAPcre+/VHL+f/+f
and
GFAPcre+/VHL+f/+f/HIF-1α+f/+f
animals.
In
GFAPcre+/VHL+f/+f/HIF-2α+f/+f mice ventricles are not enlarged (representative Nissl
staining, 50x magnification).
Supplemental figure 2.
Expression of VEGF mRNA (a) and GLUT-1 mRNA (b) in livers is not significantly
different
between
control
(GFAPcre-;
n=11),
GFAPcre+/VHL+f/+f
(n=6),
GFAPcre+/VHL+f/+f/HIF-1α+f/+f (n=6) and GFAPcre+/VHL+f/+f/HIF-2α+f/+f (n=5) animals
(data represents means ± S.E.M.). (c) VEGF mRNA is significantly upregulated in
brains of GFAPcre+/VHL+f/+f (n=10) and GFAPcre+/VHL+f/+f/HIF-1α+f/+f mice (n=6)
compared to control animals (GFAPcre-; n=8). Double knockout of VHL and HIF-1α
does not change VEGF expression significantly. In contrast, double knockout of VHL
and HIF-2α (n=10) significantly reduces VEGF upregulation, and double knockout of
VHL and VEGF (n=5) reduces it further to wildtype levels (data represent means ±
S.E.M.). (d) Immunofluorescence staining in the hippocampus shows that VEGF
upregulation in GFAPcre+/ GFAPcre+/VHL+f/+f mice increases vessel numbers as
visualized by the endothelial marker CD34 (red). Double knockout of VHL and HIF-2α
reduces vessel numbers whereas double knockout of VHL and HIF-1α has
essentially no effect. In GFAPcre+/VHL+f/+f/VEGF+f/+f mice vessel number is further
reduced (representative picture of 3 animals each genotype; astrocytes are stained
with anti-GFAP (green); nuclei are stained with DAPI; scalebars 100µm). (e) 2Photon
microscopy
shows
the
increased
cerebral
vessel
number
in
GFAPcre+/VHL+f/+f and GFAPcre+/VHL+f/+f/HIF-1α+f/+f mutant animals. No difference
is
observed
between
wildtype
and
GFAPcre+/VHL+f/+f/HIF-2α+f/+f
animals
(representative images of cranial windows; larger image 400x, scalebar 50µm; inset
200x, scalebar 200µm). (f) In vivo measurements of brain capillary diameter (right)
and the blood velocity in the brain capillaries (left) by 2-photon microscopy
demonstrate that both GFAPcre+/VHL+f/+f and GFAPcre+/VHL+f/+f/HIF-1α+f/+f animals
have significantly thicker capillaries and that bloodflow is significantly slower
compared to wildtype (GFAPcre-) and GFAPcre+/VHL+f/+f/HIF-2α+f/+f mice (n=4 each
genotype; Kruskal-Wallis-test).
Supplemental figure 3.
(a) Survival of GFAPcre+/VHL+f/+f/VEGF+f/+f mice (n=19) is significantly reduced
compared to wildtype mice (GFAPcre-; n=62), but extended compared to
GFAPcre+/VHL+f/+f animals (data represents median survival (weeks)). (b) The
hydrocephaly
of
the
GFAPcre+/VHL+f/+f
mice
is
completely
rescued
in
GFAPcre+/VHL+f/+f/VEGF+f/+f double knockout animals (representative T1 weighed
fMRI picture).
Supplementary Methods:
Primer Sequences for RNA analyses:
EPO: (F): 5′-TGCGACAGTCGAGTTCTGGA-3′
(R): 5′-TCTGCACAACCCATCGTGAC-3′
Probe: 5′- (6~FAM)AGGTACATCTTAGAGGCCAAGGAGGCAGAAA-(BHQ)-3′
VEGF (F): 5′-ATCCGCATGATCTGCATGG-3′
(R): 5′-AGTCCCATGAAGTGATCAAGTTCA-3′
Probe: 5′-(6~FAM)TGCCCACGTCAGAGAGCAACATCAC-(BHQ)-3′
Primer Sequences for DNA recombination analyses:
Location of the primers see (42)
VHL:
VHL F1: 5′-CTGGTACCCACGAAACTGTC-3′
VHL F2: 5′-CTAGGCACCGAGCTTAGAGGTTTGCG-3′
VHL R: 5′-CTGACTTCCACTGATGCTTGTCACAG-3′
Product size: VHL 2-lox allele: 460bp, 1-lox allele: 260bp; wt allele 290bp
HIF-1α:
HIF-1α F1: 5′-TTGGGGATGAAAACATCTGC-3′
HIF-1α F2: 5′-GCAGTTAAGAGCACTAGTTG-3′
HIF-1α R: 5′-GGAGCTATCTCTCTAGACC-3′
Product size: HIF-1α 1-lox allele: 270bp; 2-lox allele 260bp; wt allele 240bp
HIF-2α:
HIF-2α F1: 5′-CAGGCAGTATGCCTGGCTAAT TCCAGTT-3′
HIF-2α F2: 5′-CTTCTTCCATCATCTGGGATCTGGGACT-3′
HIF-2α R: 5′-GCTAACACTGTACTGTCTGAAAGAGTAGC-3′
Product size: HIF-2α 1-lox allele: 360bp; 2-lox allele 460bp; wt allele 430bp
Supplemental Figure 1
GFAP
b
NeuN
GFAPcre-
c
NeuN / Cre
GFAPcre-
OD620nm/g brain (fold increase)
GFAPcre+/VHL
n.s.
Heart weight (g)
n.s.
3.0
p=0.029
p=0.0011
2.5
2.0
1.5
1.0
0.5
0
-
+
GFAPcre-
+
+
p=0.0079
0.18
0.16
0.14
0.12
n.s.
GFAPcre-
GFAPcre+/VHL+f/+f
GFAPcre+/
VHL+f/+f/HIF-2α+f/+f
GFAPcre+/
VHL+f/+f/HIF-1α+f/+f
Spleen weight (g)
p=0.0010
0.4
p=0.0004
0.3
0.2
0.1
0.0
GFAPcre-
GFAPcre+/
GFAPcre+/
VHL+f/+f/
VHL+f/+f
HIF-1α+f/+f
GFAPcre+/
VHL+f/+f/
HIF-2α+f/+f
+
GFAPcre+/ GFAPcre+/ GFAPcre+/
VHL+f/+f
VHL+f/+f/
VHL+f/+f/
HIF-1α+f/+f HIF-2α+f/+f
e
p=0.0010
0.20
Evans blue permeability into CNS
EB
+f/+f
Cortex
NeuN / Cre
d
GFAPcre+/VHL+f/+f
d
Hippocampus
GFAP-cre+
Cre
GFAP-cre+
a
Supplemental Figure 2
b
Liver
1.50
1.25
n.s.
n.s.
n.s.
1.00
0.75
0.50
0.25
0.00
GFAP-cre+/
GFAP-creVHL+f/+f
rel. VEGF expression
c
GFAP-cre+/ GFAP-cre+/
VHL+f/+f/
VHL+f/+f/
HIF-1α+f/+f
HIF-2α+f/+f
Brain
17.5
15.0
Liver
rel. Glut-1 expression
rel. VEGF expression
a
2.00
1.75
1.50
n.s.
n.s.
n.s.
1.25
1.00
0.75
0.50
0.25
0.00
GFAP-cre-
GFAP-cre+/
VHL+f/+f
GFAP-cre+/ GFAP-cre+/
VHL+f/+f/
VHL+f/+f/
HIF-1α+f/+f
HIF-2α+f/+f
d
n.s.
p<0.0001
p<0.0001
n.s.
12.5
10.0
GFAP-cre-
GFAP-cre+/VHL+f/+f/HIF-2α+f/+f
GFAP-cre+/VHL+f/+f/VEGF+f/+f
7.5
GFAP/
CD34/
DAPI
5.0
2.5
0.0
GFAPcre- GFAPcre+/ GFAPcre+/ GFAPcre+/ GFAPcre+/
VHL+f/+f/
VHL+f/+f/
VHL+f/+f/
VHL+f/+f
+f/+f
+f/+f
HIF-1α
HIF-2α
VEGF+f/+f
GFAP-cre+/VHL+f/+f
GFAP-cre+/VHL+f/+f/HIF-1α+f/+f
Supplemental Figure 2
GFAPcre+/VHL
e GFAPcre-
+f/+f
GFAPcre+/VHL+f/+f/HIF-2α +f/+f
f
GFAPcre+/VHL+f/+f/HIF-1α +f/+f
5
p<0.0001
n.s.
4
3
2
1
0
GFAPcre- GFAPcre+/ GFAPcre+/ GFAPcre+/
VHL+f/+f
VHL+f/+f/
VHL+f/+f/
HIF-1α+f/+f HIF-2α+f/+f
Velocity (mm/sec)
Diameter (µm)
n.s.
p<0.0001
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
n.s.
p<0.0001
p<0.0001
n.s.
GFAPcre- GFAPcre+/ GFAPcre+/ GFAPcre+/
VHL+f/+f
VHL+f/+f/
VHL+f/+f/
HIF-1α+f/+f HIF-2α+f/+f
Supplemental figure 3
a
b
GFAPcreGFAPcre+/VHL+f/+f/VEGF+f/+f
GFAPcre+/VHL+f/+f
Percent Survival
100
p<0.0001
80
60
40
20
0
GFAPcre+/VHL+f/+f/VEGF+f/+f
0
5
10
15
Age (weeks)
20