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20
17 N
atu
re A
me
rica
, In
c.,
pa
rt o
f S
pri
ng
er N
atu
re. A
ll ri
gh
ts r
ese
rve
d.
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
Glo
bal h
etG
loba
l CK
O
Perl’s IronH&E
EC het EC CKO
Fibrinogen CD31tMCAO 1 h; reperfusion 1 h
P < 0.05
n = 27
n = 21
Global Gpr124 CKO
Sur
viva
l (%
)
Time (d)
100
80
60
40
20
00 1 2 3 4 5 6 7
HetCKO
HetCKO
P < 0.05
EC Gpr124 CKO
n = 17
n = 19
Time (d)
Sur
viva
l (%
)
100
80
60
40
20
00 1 2 3 4 5 6 7
Infa
rct
size
(%
)
* *
0
20
40
60
80
Gpr124flox/+;ROSA-CreER(global het)
Gpr124flox/–; ROSA-CreER(global CKO)
5 d 5 d3 d1 d
TT
CB
right
field
0
20
40
60
80
100 Hemorrhage
Normaln = 27 n = 21 n = 17 n = 19
Het CKO Het CKO
Inci
den
ce o
f hem
orrh
age
(%
)
Global EC
Gpr124flox/+;Cdh5-CreER(EC het)
Gpr124flox/–;Cdh5-CreER(EC CKO)
TT
CB
right
field
8 8
Hem
orrh
age
are
a (
mm
2)
0
5
10
15
20
****
**
*
n.s.EC Gpr124 CKO
**
**
Re
lativ
e fi
brin
oge
n d
ens
ity
0
2
4
6
8
Global
Het CKO
EC
Het CKO
EC
Het CKO
Global
Het CKO
a b
5 d3 d1 d5 d
c d
Het 1
–2 d
CKO 1–2
d
Het 3
–4 d
CKO 3–4
d
Het 5
–7 d
CKO 5–7
d
e f g
h i j
Gpr124
Gpr124
Gpr124 n n nn n n P P t Gpr124
Gpr124 PGpr124 Gpr124
Gpr124 n Gpr124 n Gpr124 n Gpr124 n P Pt Gpr124
n P t
Q50Q50
Q51Q51
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
Infa
rct s
ize
(%) 60
80
40
20
0
** ****
**
EC het; β-cat
Sulfo-NHS-Biotin CD31
tMCAO 1 h; reperfusion 1 d
Gpr124flox/–;Ctnnb1lox(ex3)/+;
Cdh5-CreER(EC CKO; β-cat)
Gpr124flox/+;Cdh5-CreER
(EC het)
Gpr124flox/+;Ctnnb1lox(ex3)/+;
Cdh5-CreER(EC het; β-cat)
Gpr124flox/–;Cdh5-CreER
(EC CKO)
Log
2 (
fold
cha
ng
e)
Wn
t ta
rget
s
β-ca
t:
WT Gpr1
24
CKO:het
Sfrp1
Nkd1
Dkk3
PrkcbAxin2
Vangl1Apcdd1
Camk2a
Dkk2
Wnt5aNkd2
Ppard
Ratio:
RNA-seq
1.00.6
0.20.0
0.4
Sfrp1
Nkd1
Dkk3
PrkcbAxin2
Vangl1Apcdd1
Camk2a
Dkk2
Wnt5aNkd2
Ppard
Het
Nonstroke
Stroke1 d
Rel
ativ
e e
xpre
ssio
n le
vel
** ****
**
CKO Het CKO
RNA-seq
Re
lativ
e m
RN
A le
vel
*
**
**
FACS-sorted adult brain EC
Global het
Global CKO
0.0
0.5
1.0
1.5
** **
****
Rel
ativ
e s
ign
al d
ens
ity Sulfo-NHS-Biotin
Fibrinogen
3
2
1
0
Rel
ativ
e m
RN
A le
vel **
Primary culture adult brain EC
**
** **
Het + Ad Fc
CKO + Ad Fc
Het + Ad Wnt7b
CKO + Ad Wnt7b
Gpr124 Apcdd10
1
2
3
4
5
Axin2
Rel
ativ
e m
RN
A le
vel
*
* *
* **
*
**
**
**
*
EC het; β-cat
EC het EC CKO
EC CKO; β-cat
–3.00–2.00–1.00
0.00
2.001.00
3.00Gpr124
Axin2
Apcdd1
Cldn5
12
10
8
6
4
2
0
FACS-sorted adult brain EC
PrikcdApcdd1Axin2 Nkd1
a b c d e
5 d 5 d 5 d 5 d
f
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat 10 2 3 4 5
20
0
40
60
80
100
Sur
viva
l (%
)
Time (d)
*
*
EC het
EC CKO
EC het; β-cat
EC CKO; β-cat
*
n = 9
n = 10
n = 16
n = 12
g h
EC het EC CKO
EC CKO; β-catEC h
et
EC CKO
EC het
; β-c
at
EC CKO
; β-c
at
i j
Gpr124 Axin2 Gpr124
P t Gpr124Cldn5, Gpr124
n P P tGpr124
n n P Pt
Ctnnb1lox(ex3)/+;Cdh5-CreER Ctnnb1+/+ Cdh5-CreER n Gpr124 n
Gpr124
n P tGpr124
n n n n P tGpr124 P
nP t
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
. Col
IV C
d31
tMCAO 1 h; reperfusion 5 d
Desmin CD31
Het
CK
O
tMCAO 1 hreperfusion 1 d
Pdgfb in situ
EC
het
EC
CK
O
tMCAO 1 hreperfusion 1 d
CD31
tMCAO 1 h; reperfusion 5 d
tMCAO 1 h; reperfusion 5 d
Rel
ativ
e C
ldn5
mR
NA
*****
*
FACS-sorted brain ECNo stroke
0
1
2
3
Pdgfr-β Desmin
Rel
ativ
e pe
ricyt
eco
vera
ge ****
* **
0.0
0.5
1.0
1.5
2.0
Rel
ativ
e P
dg
fbm
RN
A *
FACS-sortedCd31+ brain EC1 d poststroke
0.5
0.0
1.0
1.5
Rel
ativ
e E
CM
depo
sitio
n ****
**Laminin Col IV
tMCAO 1 h; reperfusion 5 d
0.00.51.01.52.02.5
Mic
rove
ssel
dens
ity (
% a
rea)
tMCAO 1 h reperfusion 5 d
0
1
2
3
4
EC het EC CKO
EC het EC CKO
EC het EC CKO
EC CKO; β-catEC het; β-cat
Pdg
fr-β
CD
31
tMCAO 1 h; reperfusion 5 d FACS-sortedCd31+ brain EC
Rel
ativ
e P
dg
fbm
RN
A
0.50.0
1.01.52.02.5 *
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
EC het
EC CKO
EC het
EC β-c
at
a b c d e f g
EC het; β-cat EC CKO; β-cat
Lam
inin
CD
31
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
EC het; β-cat EC CKO; β-cat
h i j k
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
Cldn5Gpr124
n n n nP P t
Gpr124
Gpr124n n n
n P P t Pdgfb in situGpr124
Pdgfb Gpr124n n P
t Pdgfb Gpr124n P t
n P tGpr124
n
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
Global het (flox/+)Global CKO (flox/–)
P < 0.02
Time (d)
Sur
viva
l (%
)
0
20
40
60
80
100
0 2 12 16 20 24 28
Normal mouse brain Mouse GL261 glioblastoma
H&
EB
righ
tfie
ld
H&
EB
righ
tfie
ld 150
100
50
0
*
Not compensatedfor edema
80
60
40
20
0
Compensatedfor edema
0.8
0.6
0.4
0.2
0.0
Global EC
Het Het CKOCKO
Global EC
Het Het CKOCKO
Global EC
Het Het CKOCKO
*
** **
Normal EC Tumor EC
0.0
0.5
1.0
1.5
Gpr124 CD31 DAPI Gpr124 CD31 DAPI
Global Gpr124 CKO
Gpr124flox/–;ROSA-CreER
Gpr124flox/+;ROSA-CreER
EC Gpr124 CKO
Gpr124flox/–;Cdh5-CreER
Gpr124flox/+;Cdh5-CreER
Rel
ativ
e G
pr1
24 m
RN
A
EC CKOEC het
Intr
a-t
umor
aled
em
a/to
tal a
rea
Tum
or
volu
me
(m
m3)
Tu
mo
r vo
lum
e (m
m3)
n.s. n.s.******
a b c d
e f g h i
Gpr124 nP Gpr124
Gpr124Gpr124- n n
P P t Gpr124
Gpr124n Gpr124 n Gpr124 n Gpr124 n P t
Gpr124 n Gpr124 n Gpr124 n Gpr124n P P t
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
*
**** **
**
mIg
G C
D3
1 D
AP
IB
right
field
H&
E
n.s. n.s.
n.s.
**
* ***
Not compensated for edema
n.s.
Mic
rove
ssel
den
sity
(% a
rea
)
**
4
2
1
0
3
n.s.
0.6
0.4
0.2
0.0Intr
atu
mor
al e
dem
a(f
ract
ion
of t
ota
l tu
mo
r a
rea)
120
160
0
80
40
Tu
mo
r vo
lum
e (m
m3)
0
20
40
80
60
Tum
or v
olu
me
(m
m3)
Compensated for edema Compensated for edema
Rel
ativ
e m
RN
A le
vel
Apcdd1Axin2
*****
**
***
0.0
0.5
1.0
1.5
Mou
se Ig
G d
ens
ity (
a.u
.)
**
** ****
50
100
150
250
200
EC CKO-tumor EC
EC het-tumor ECEC het-normal EC
EC CKO-normal EC
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
EC het
EC CKO
Het; β
-cat
CKO; β-c
at
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
EC het
EC CKO
EC het
; β-c
at
EC CKO; β
-cat
Gpr124flox/–;Ctnnb1lox(ex3)/+;
Cdh5-CreER(EC CKO; β-cat)
Gpr124flox/+;Cdh5-CreER
(EC het)
Gpr124flox/–;Cdh5-CreER
(EC CKO)
Gpr124flox/+;Ctnnb1lox(ex3)/+;
Cdh5-CreER(EC het; β-cat)
a b
d e f g
c
Gpr124Axin2 Apcdd1
Gpr124 n n
P P tGpr124 n
n n nn
P tn n n
n P P t Gpr124n n n
nn n n n P
t
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
Gpr124
n P tGpr124
n P P t PdgfbGpr124 n n
P t
n Pt Gpr124
Gpr124n
P t
Pdgfb
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
b
Pdg
fr-β
CD
31
** ****
20
40
60
80
100
0
Col
IV C
D3
1La
min
in C
D3
1C
ldn
5 C
D3
1
Cldn5 Pgdfb
Gpr124
CKO
Fzd
LRP5/6Wnt7
ECM
ECM disruptionlaminin, collagen IV
?
CNS pathologic injury
Reck
Canonical Wnt–β-catenin signaling
CNSendothelium
?
Activatedβ-catenin
Rescues?
Rel
ativ
e C
ldn5
sig
nal d
en
sity
** ****
Desmin
***
**
**
0.0
0.5
1.0
1.5
2.0
Re
lativ
e E
CM
dep
osi
tion
Glu
t1 C
D3
1 D
AP
I
Pdgfr-β
* **
Col IVLaminin
1.5
1.0
0.0
0.5
Rel
ativ
e pe
ricy
te c
over
age
0.0
0.5
1.5
2.0
1.0
EC CKO; β-cat
EC CKO; β-cat
EC CKO; β
-cat
EC het; β-cat
EC het; β-cat
EC het
; β-c
at
EC CKO
EC CKO
EC CKO
EC het
EC CKO; β-catEC het; β-catEC CKOEC het
EC het
EC CKO; β-catEC het; β-cat
EC CKOEC het
EC het
EC het
; β-c
at
EC CKO
EC het
EC CKO
; β-c
at
EC het
; β-c
at
EC CKO
EC het
EC CKO
; β-c
at
EC het
; β-c
at
EC CKO
EC het
Glu
t1+
vess
els
(%)
Glut1(tumor)
Pericyte
loss
Angiogenesistumor only
BBB breakdown, hemorrhagestroke, tumor
Tight-junctioncompromise
a
c d
gf
h j
i
EC het
Tumor EC
*
0.0
0.5
1.0
1.5
2.0
Re
lativ
e P
dg
fb m
RN
A
EC CKO
EC CKO
; β-c
at
e
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
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7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
Gpr124
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
ure
. All
rig
hts
re
serv
ed
.
Pdgfb in situ
201
7 N
atu
re A
mer
ica,
Inc.
, p
art
of
Sp
rin
ger
Nat
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