10,000,000,000 cells 7,000,000,000 people
1984
Conservation of developmental mechanisms
Transcription factors
Downstream effectors
5’UTR Paired linker Homeo PST 3’UTR domain domain transactivation domain
Alternativelyspliced exon 5a
Pax6
Paireddomain
Homeodomain
Pax6
Mutant Control
Mutant Mutant
Sisodya et al Nature Genetics 2001
Pax6 gene expression in the developing cortex
Rostral
Caudal
Lateral
Medial
Wild-type
Mutant
CPax6sey/sey.DTy54+ 76.65%N
um
be
r o
f C
ell
s
Fluorescence
BPax6+/+.DTy54+ 69.73%
Non-transgeniccontrol
A
Pax6+/+.DTy54+
purity check
DPax6sey/sey.DTy54+
purity check
E
0%
CPax6sey/sey.DTy54+ 76.65%
CPax6sey/sey.DTy54+ 76.65%N
um
be
r o
f C
ell
s
Fluorescence
BPax6+/+.DTy54+ 69.73%
BPax6+/+.DTy54+ 69.73%
Non-transgeniccontrol
ANon-transgenic
control
A
Pax6+/+.DTy54+
purity check
DPax6+/+.DTy54+
purity check
DPax6sey/sey.DTy54+
purity check
EPax6sey/sey.DTy54+
purity check
E
0%
CPax6sey/sey.DTy54+ 76.65%N
um
be
r o
f C
ell
s
Fluorescence
BPax6+/+.DTy54+ 69.73%
Non-transgeniccontrol
A
Pax6+/+.DTy54+
purity check
DPax6sey/sey.DTy54+
purity check
E
0%
CPax6sey/sey.DTy54+ 76.65%
CPax6sey/sey.DTy54+ 76.65%N
um
be
r o
f C
ell
s
Fluorescence
BPax6+/+.DTy54+ 69.73%
BPax6+/+.DTy54+ 69.73%
Non-transgeniccontrol
ANon-transgenic
control
A
Pax6+/+.DTy54+
purity check
DPax6+/+.DTy54+
purity check
DPax6sey/sey.DTy54+
purity check
EPax6sey/sey.DTy54+
purity check
E
0%
Pax6 expression reporter FACS sorted Pax6-expressing cells
Controls Pax6-/-
control
No
Mutant
+/+ -/-
+/+ -/-
+/+ -/-
Collaboration with Pathway Medicine
Mi et al 2013 Neuron
Martine Manuel Mike Molinek Da Mi John Mason
New StudyDelete Pax6 specifically in the cortex at a chosen time
Look for changes in gene expression that occur rapidly after loss of Pax6
Primary defects
Direct targets
Pax6flox allele Ian Simpson
EGFP
EGFP
StoploxP loxP
Cre
Conditional EGFP reporter
CreERT2
CreER
CreER
TamoxifenOFF
ON
Emx1-CreERT2
Give tamoxifen to pregnant females at E9.5
Collect embryos at E11.5, E12.5 and E13.5
Dissect cortex
Extract RNA
Deep Sequencing
Pax6+/flox Emx1creER Pax6flox/flox Emx1creER
control Pax6 cKO
Dissect cortex
Extract RNA
Deep Sequencing
Tamoxifen given at E9.5
E11.5 E12.5 E13.5
E12.5 GFP / Pax6 immuno
control
control
Pax6 cKO
Pax6 cKO
control Pax6 cKO
E12.5
Pax6
bactin
C R C R C Rcontrol1 control2Pax6cKO1 Pax6cKO2
C RE12.5
Tamoxifen given at E9.5
0
0.2
0.4
0.6
0.8
1
1.2Reduction of Pax6 mRNA levels in Pax6 cKO
embryosra
tio P
ax6
cKO
/ c
ontr
ol
E9.5 E11.5 E12.5 E13.5
E11.5 E12.5 E13.5
E11.5 E12.5 E13.5
Tamoxifen given at E9.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0 Change in Mash1 expression
ratio
Pax
6 cK
O /
con
trol
E9.5 E11.5 E12.5 E13.5
0
0.2
0.4
0.6
0.8
1
1.2Change in Ngn2 expression
ratio
Pax
6 cK
O /
con
trol
E9.5 E11.5 E12.5 E13.5
*
*
*
E9.5 E11.5 E12.5 E13.5
*
E9.5 E11.5 E12.5 E13.5
*
E9.5 E11.5 E12.5 E13.5
Tamoxifen given at E9.5
E9.5 E11.5 E12.5 E13.5
E9.5 E11.5 E12.5 E13.5
Tamoxifen given at E9.5
Also no significant change in Smad4, Cdk4, cyclin D2, MCM3 and MCM6 expression.
R
C
CeM CeL
Rostral
Caudal
Tamoxifen given at E9.5
E12.5
Rostral cortex
Caudal cortex
RNA extraction Pax6 immuno
Collection of samples for RNAseq
controls Pax6 cKO
Control Pax6cKO Control Pax6cKO Control Pax6cKO
Collaboration withGenepool
control Pax6 cKO
E13.5
Pax6 immuno. E13.5. Sagittal.
Control Pax6 cKO
Rostral Rostral
C R C Rcontrol Pax6cKO
E13.5
Pax6
bactin