10,000,000,000 cells 7,000,000,000 people 1984 Conservation of developmental mechanisms.

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Presentation transcript:

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 Alternatively spliced exon 5a Pax6

Paired domain Homeo domain Pax6

Mutant Control Mutant Sisodya et al Nature Genetics 2001

Pax6 gene expression in the developing cortex Rostral Caudal Lateral Medial

Wild-type Mutant

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 Study Delete 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

Pax6 flox allele Ian Simpson EGFP Stop loxP Cre Conditional EGFP reporter

CreER T2 CreER Tamoxifen OFF ON Emx1-CreER T2

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 Pax6 flox/flox Emx1creER controlPax6 cKO Dissect cortex Extract RNA Deep Sequencing

Tamoxifen given at E9.5 E11.5E12.5E13.5

E12.5 GFP / Pax6 immuno control Pax6 cKO

control Pax6 cKO E12.5

Pax6  actin CR CR CR control1control2Pax6cKO1Pax6cKO2 CR E12.5

Tamoxifen given at E9.5 E9.5E11.5E12.5E13.5

E11.5E12.5E13.5 E11.5E12.5E13.5 Tamoxifen given at E9.5 E9.5 E11.5E12.5E13.5 E9.5E11.5E12.5E13.5 * * *

E9.5 E11.5E12.5E13.5 * E9.5 E11.5E12.5E13.5 * E9.5 E11.5E12.5E13.5 Tamoxifen given at E9.5

E9.5E11.5E12.5E13.5 E9.5E11.5E12.5E13.5 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 Collaboration with Genepool

controlPax6 cKO E13.5

Pax6 immuno. E13.5. Sagittal. ControlPax6 cKO Rostral

CR CR controlPax6cKO E13.5 Pax6  actin