2. 10,000,000,000 cells 7,000,000,000 people

3. 1984 Conservation of developmental mechanisms

4. Transcription factors Downstream effectors

5. 5’UTR Paired linker Homeo PST 3’UTR domain domain transactivation domain Alternatively spliced exon 5a Pax6

6. Paired domain Homeo domain Pax6

7. Mutant Control Mutant Sisodya et al Nature Genetics 2001

9. Pax6 gene expression in the developing cortex Rostral Caudal Lateral Medial

10. Wild-type Mutant

11. Pax6 expression reporter FACS sorted Pax6-expressing cells

12. Controls Pax6 -/- control No Mutant +/+ -/- +/+ -/- +/+ -/- Collaboration with Pathway Medicine

13. Mi et al 2013 Neuron

14. 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

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

16. CreER T2 CreER Tamoxifen OFF ON Emx1-CreER T2

17. 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

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

19. E12.5 GFP / Pax6 immuno control Pax6 cKO

20. control Pax6 cKO E12.5

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

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

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

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

26. 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.

27. R C CeM CeL Rostral Caudal Tamoxifen given at E9.5 E12.5

28. Rostral cortex Caudal cortex RNA extraction Pax6 immuno Collection of samples for RNAseq

29. controls Pax6 cKO Control Pax6cKO Collaboration with Genepool

32. controlPax6 cKO E13.5

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

34. CR CR controlPax6cKO E13.5 Pax6  actin