10,000,000,000 cells 7,000,000,000 people 1984 Conservation of developmental mechanisms.
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Transcript of 10,000,000,000 cells 7,000,000,000 people 1984 Conservation of developmental mechanisms.
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10,000,000,000 cells 7,000,000,000 people
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1984
Conservation of developmental mechanisms
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Transcription factors
Downstream effectors
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5’UTR Paired linker Homeo PST 3’UTR domain domain transactivation domain
Alternativelyspliced exon 5a
Pax6
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Paireddomain
Homeodomain
Pax6
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Mutant Control
Mutant Mutant
Sisodya et al Nature Genetics 2001
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Pax6 gene expression in the developing cortex
Rostral
Caudal
Lateral
Medial
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Wild-type
Mutant
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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
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Controls Pax6-/-
control
No
Mutant
+/+ -/-
+/+ -/-
+/+ -/-
Collaboration with Pathway Medicine
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Mi et al 2013 Neuron
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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
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Pax6flox allele Ian Simpson
EGFP
EGFP
StoploxP loxP
Cre
Conditional EGFP reporter
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CreERT2
CreER
CreER
TamoxifenOFF
ON
Emx1-CreERT2
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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
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Tamoxifen given at E9.5
E11.5 E12.5 E13.5
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E12.5 GFP / Pax6 immuno
control
control
Pax6 cKO
Pax6 cKO
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control Pax6 cKO
E12.5
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Pax6
bactin
C R C R C Rcontrol1 control2Pax6cKO1 Pax6cKO2
C RE12.5
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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
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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
*
*
*
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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
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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.
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R
C
CeM CeL
Rostral
Caudal
Tamoxifen given at E9.5
E12.5
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Rostral cortex
Caudal cortex
RNA extraction Pax6 immuno
Collection of samples for RNAseq
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controls Pax6 cKO
Control Pax6cKO Control Pax6cKO Control Pax6cKO
Collaboration withGenepool
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control Pax6 cKO
E13.5
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Pax6 immuno. E13.5. Sagittal.
Control Pax6 cKO
Rostral Rostral
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C R C Rcontrol Pax6cKO
E13.5
Pax6
bactin