10,000,000,000 cells 7,000,000,000 people

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

umbe

r of C

ells

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

umbe

r of C

ells

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

umbe

r of C

ells

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

umbe

r of C

ells

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

/ co

ntro

l

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 /

cont

rol

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 /

cont

rol

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