IZ - Cardiff Universityorca.cf.ac.uk/90592/7/Figures.pdf · IZ SVZ VZ MZ 0 5 10 15 20 25 Figure 8...
8
A B C D CDH2 Merge Tuj1 CDH2, Tuj1, Pax6 E14.5 CDH2 Pax6 Merge E14.5 CDH4 Merge Tuj1 CDH4, Tuj1, Pax6 CDH4 Pax6 Merge Figure 1 Martinez-Garay et al CDH2 E14.5 CDH4 E15.5 E14.5 E16.5 Cdh2 mRNA Cdh4 mRNA Cdh6 mRNA Cdh11 mRNA Cdh13 mRNA CP CP IZ IZ SVZ SVZ VZ VZ Ctnnb1 mRNA SP SP E13.5 CDH2, CDH4 E13.5 E13.5
Transcript of IZ - Cardiff Universityorca.cf.ac.uk/90592/7/Figures.pdf · IZ SVZ VZ MZ 0 5 10 15 20 25 Figure 8...
A
B C
D
CDH2 MergeTuj1CDH2, Tuj1, Pax6
E14
.5
CDH2 Pax6 Merge
E14
.5
CDH4 MergeTuj1CDH4, Tuj1, Pax6
CDH4 Pax6 Merge
Figure 1 Martinez-Garay et al
CDH2
E14
.5
CDH4
E15
.5
E14
.5E
16.5
Cdh2 mRNA Cdh4 mRNA Cdh6 mRNA Cdh11 mRNA Cdh13 mRNA
CP
CP
IZ
IZ
SVZ
SVZ
VZ
VZ
Ctnnb1 mRNA
SP SP
E13
.5
CDH2, CDH4
E13
.5E
13.5
0
10
20
30
40
50
60
70
Control Cdh2fl/fl Cdh4FF Cdh2/4FF
CA*
*****
Control Cdh2fl/fl Cdh4fl/fl Cdh2/4fl/fl
+
In utero electroporation
Plasmids:Dcx-CRE-i-EGFPDcx-i-GFP
Mice:Cdh2 fl/fl
Cdh4 fl/fl
Cdh2/4 fl/fl
E14.5 E18.5
0
10
20
30
40
50
60
70
% o
f GFP
+ ne
uron
s th
at h
ave
reac
hed
the
top
of th
e C
P
EG
FP T
opro
BCdh2fl/fl Cdh4fl/fl Cdh2/4fl/flControl
MZ
CP
IZ
SVZ
VZ
SP
Figure 2 Martinez-Garay et al
n.s.
WT E14.5 E18.5
SP PD EC1 EC2 EC3 EC4 EC5 p120 βcat/PTP1B
W2AFL Cdh
W2A CdhD134A
D134A Cdh
DCX pro IRES EGFP
BA
EC1EC1
EC2
EC2
W2
D134
D134
Ca2+
Ca2+
CDH2-FL
CDH2-W2A
CDH4-FL
CDH4-W2A
EG
FP T
opro
C
CDH2-D134A CDH4-D134A
EG
FP T
opro
E
DWT E14.5 E18.5
WT E14.5 E18.5
0102030405060708090
EGFP
CDH2-FL
CDH4-FL
CDH2-W2A
CDH4-W2A
CDH2-D134A
CDH4-D134A
% G
FP+
cells
inU
pper
Cor
tical
Pla
te
*** ***
EGFP
CDH2-W2A
CDH4-W2A
CDH2-D13
4A
CDH4-D13
4A
CDH2-FL
CDH4-FL
0
10
20
30
40
50
60
70
80
90
F G
N
GA GLP
EGFP
LCP
IZ
SVZ
VZ
MZ
UCP
EGFP
IZ
SVZ
VZ
MZ
UCP
LCP
Figure 3 Martinez-Garay et al
**
******
0
10
20
30
40
50
60
70
80
90
100
GFP
PTP1BDN
DNCYTO
DNCYTODbcat
DNCYTODPTP1B
WT E14.5 E18.5
SP PD EC1 EC2 EC3 EC4 EC5 p120 βcat/PTP1B
DN-CdhΔβcat
DN-CdhΔPTP1B
DN-PTP1BERPro-richCatalytic domain
C215S
DN-Cdh
DCX pro IRES EGFP
BA
PTP1B
β-catα-cat
+
CDH
EG
FP T
opro
C
DN-CDH DN-CDHDPTP1BDN-PTP1B
WT E14.5 E18.5EGFP
CP
IZ
MZ
SVZ
VZ
DN-CDH + pCIG-aNcatDactin
EG
FP T
opro
DN-CDH + pCIG-aNcatDbcat
EWT E14.5 E18.5
DN-CDH + pCIG-aNcat1-911
DN-CDH + pCIG-aNcat
DN-CDH + pCIGpCIG
CP
IZ
MZ
SVZ
VZ
D F
0
10
20
30
40
50
60
70
pCIG
pCIG
pCIG-‐aNcat
aNcat82-‐906
delta ac8n
aNcat1-‐911
% G
FP+
cells
inC
ortic
al P
late
DN-CDH +pCIG
pCIG-aNcat
pCIG-aNcatDactin
pCIG-aNcatDbcatpCIG
****n.s.
**
*
pCIG-aNcat1-911
***
n.s.
0
10
20
30
40
50
60
70
Figure 4 Martinez-Garay et al
% G
FP+
cells
inC
ortic
al P
late
EGFPDN-CDH
DN-CDHDPTP1BDN-PTP1B
*
0
10
20
30
40
50
60
70
80
90100
DN-CDHDbcat
DN-CDHDbcat
*****
***n.s.
0
2
4
6
8
10
12
1 2
BA
+
In utero electroporation
pCIGDCX-DN-Cdh-iEGFPDCX-Cdh2-W2A-iEGFPDCX-Cdh4-W2A-iEGFPDCX-Cdh2-D134A-iEGFPDCX-Cdh4-D134A-iEGFP
E14.5 E18.5+ GalNacT2-dsRed
Plasmids:
E14.5
+3d
In utero electroporation
DCX-i-EGFPDCX-DN-Cdh
live imaging
Plasmids:
+ dsRed-Centrin
C
EG
FP G
olgi
WT E14.5 E18.5
EGFP
*
DN-CDH
**
CDH2-W2A
**
CDH4-W2A
*
*
CDH2-D134A
*
*
CDH4-D134A
*
*
WT E14.5, slice culture at E17.5
EG
FPds
Red
-Cen
trin
DN
-CD
Hds
Red
-Cen
trin
* ***
*
* * * * *
t=0
t=0
20m 40m 60m 80m
20m 40m 60m 80m
D
Figure 5 Martinez-Garay et al
Pro
cess
leng
ht in
mm
0
20
40
60
80
140
120
100
EGFP
CDH2-W2A
CDH4-W2A
CDH2-D134A
CDH4-D134A
DN-CDH
E*
F
mig
ratio
n sp
eed
in m
m /
hour
EGFP
*
DN-CDH0
8
10
12
6
4
2
0
20
40
60
80
100
120
140
1 2 3 4 5 6
+
E14.5
+2d
In utero electroporation
Actin-GFP+ mCherry
live imaging
Plasmids:
Actin-EGFP+ Dcx-Cdh2-D134A+ mCherry
or
B
A
C
t = 0 t = 01 hr 2 hrs 3 hrs 1 hr 2 hrs 3 hrs
control CDH2-D134A
EG
FP-a
ctin
m
Che
rry
WT E14.5 E16.5 WT E14.5 E16.5
Figure 6 Martinez-Garay et alE
GFP
-act
in
mC
herr
y
control - 1 hour control - 2 hours CDH2-D134A - 1 hour CDH2-D134A - 2 hours
+
E14.5
+2-3d
In utero electroporation
Plasmids:
+ Calyculin A
Image35 min
Image50 min
Dcx-i-GFPDcx-DN-Cdh
B
A
EG
FPD
N-C
DH
* * * * * *
* * * * * *
t=0
t=0
10m
5m
20m
10m
30m
15m
40m
20m
50m
25m
WT E14.5, slice culture at E17.5
Figure 7 Martinez-Garay et al
C
% G
FP+
cells
EGFP
*
DN-CDH0
10
20
30
40
50
60
70
80
90
100
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110%
Control DN-Cdh
noresponse
retract
translocate
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
20
40
60
80
100
120
140
160
180
A
B
EG
FP H
A B
FP
single section CDH2-EGFP HA-LIS1 CDH2-EGFP
+ HA-LIS1CDH2FL-EGFP + HA-LIS1 + BFP
A’
A’’
A’ A’ A’ A’
A’’ A’’ A’’ A’’
D
EG
FP % G
FP+
cells
in C
P
WT E14.5 E18.5
DN-CDH +
GFP0!
5!
10!
15!
20!
25! *
DN-CDH +
LIS1
EWT E14.5 E18.5
DN-CDH + EGFP DN-CDH + LIS1
EG
FP
DN-CDH + EGFP DN-CDH + LIS1
CP
IZ
SVZ
VZ
MZ
0
5
10
15
20
25
Figure 8 Martinez-Garay et al
Lis1
inte
nsity
in le
adin
g pr
oces
s/Li
s in
tens
ity in
som
a
GFP
DN-CDH
0
0.2
0.4
0.6
0.8
1
HA-LIS1 + DN-CDHHA-LIS1 + EGFP
B’B’
B’
B’
B’’
B’’
B’’
B’’
EG
FP H
A
CWT E14.5 E17.5
F
Pro
cess
leng
ht in
mm
*
0
20
40
60
80
180
G
WT E14.5 E17.5
DN-CDH +
GFP
DN-CDH +
LIS1
160
140
120
100
1.2
1.4*
