1. Shiva Swamynathan sks47@Pitt.edu Eye Development and Gene Expression

2. Why study eye development & gene expression? Aniridia PAX6 Anophthalmia SOX2 Iridocorneal Fusion Microphthalmia PAX6, OTX2, CHX10 & RAX Keratolenticular Fusion Opaque Cornea Axenfeld-Rieger Syndrome PITX2 Peters Anomaly FOXC1, PAX6, PITX2, or CYP1B1 Anophthalmia RAX

3. Cell lineage during vertebrate eye development

4. Drosophila eye transcriptome transcription factor genes are highly expressed Structural and pigment biosynthesis genes genes involved in intracellular signaling, neurotransmission, and the visual process

5. Whitney Heavner and Larysa Pevny. 2012. Cold Spring Harb Perspect Biol 2012;4:a008391 Transcription factor network that establishes the eye field

6. Gene expression in the developing optic vesicle

7. Transcription factors regulating corneal development Swamynathan, S.K. 2013. J. Ophthalmol. 103947. PMID: 23533700

8. Mature cornea is formed by 8-10 weeks after birth E12.5E14.5E16.5 E17.5E18.5Adult

9. Number of differentially regulated genes in the cornea between different time points Stephens et al 2013. J. Bio. Chem. 288:34304-24

10. Cornea derives cells from the surface ectoderm and neural crest mesenchyme Swamynathan, S.K. 2013. J. Ophthalmol. 103947. PMID: 23533700

11. KLF4 IRF1 ELF3 Ets homologous factor HEB KLF5 JunB Rev-erbA-a IRF6 Oxysterols receptor LXR-b Pax6 Transcription Factors in the Mouse Cornea Highly expressed in the cornea Highly expressed in the cornea Norman et al. 2004. IOVS. 45: 429-40 PMID: 14744882

12. Swamynathan et al., 2007. Mol. Cell. Biol. 27:182-194 Kenchegowda et al 2011. Dev. Biol. 356:5-18 Wild Type Klf4CN Functions of Klf4 and Klf5 in the ocular surface Wild Type Klf5CN

13. Gene, transcript and protein structure of Pax6

14. Pax6 regulatory networks during early eye development Neuroectoderm lineage Surface ectoderm lineage

15. Pax6 is essential for normal corneal development Ramaesh et al., 2005. Exp.Eye Res. 81: 224-35

16. Mort et al. 2011. PMID: 22220198 Corneal functions of Pax6 are dosage-dependent

17. Role of Pitx2 in corneal development Gage et al. 2014. Dev Dyn. PMID: 25044936 Paired-like homeodomain transcription factor PITX2 in E14.5 P42 PITX2 Mutants Overabundance of mesenchyme Malformed stroma and endothelium Absence of anterior chamber Corneo-lenticular fusion

18. Pitx2 helps establish corneal avascularity Gage et al. 2014. Dev Dyn. PMID: 25044936

19. Corneal epithelium and the lens are both derived from the head surface ectoderm Surface Ectoderm forms the Lens & the Corneal Epithelium

20. Lens and Cornea Share Transcriptional Networks

21. Genetic pathways inducing lens differentiation

22. Pax6 and Sox2 cooperatively regulate chicken δ1-crystallin gene expression Kamachi et al 2001. Genes Dev. 15: 1272-86

23. Sox2 regulates cell-specific target genes through distinct partner factors Kamachi et al 2001. Genes Dev. 15: 1272-86

24. Organization of retinal circuits

25. Whitney Heavner and Larysa Pevny. 2012. Cold Spring Harb Perspect Biol 2012;4:a008391 Transcription factors regulating temporal progression of mouse retinogenesis

26. Stages of photoreceptor development

27. OTX2 plays an essential role in vertebrate retinogenesis Otx2-/- WT PN 0 PN 9 Otx2-/- WT Otx2 expression in developing retina E12.5 E17.5 PN6

28. Temporal functions of Otx2 in the mouse retina OTX2 mutations are associated with blinding ocular defects and brain abnormalities

29. OTX2 gene network in the developing retina

30. Rx expression, phenotypes and regulation overexpression Xenopus Mouse Xenopus Human Xenopus

31. Essential roles of LHX2 in eye development Lhx2 is a LIM domain (a double-zinc finger motif found in a variety of proteins)- containing homeobox gene expressed in B-cells, forebrain and neural retina LHX2 coregulates the expression of Pax6, Six3 and Rax in the optic vesicle LHX2 is essential for eye development

32. Human NRL gene and protein Nrl is required for rod photoreceptor development

33. Optic vesicle and RPE specification

34. MITF: A big gene associated with small eyes

35. Summary Large-scale expression studies suggest that about a thousand genes are expressed in a preferential manner in the eye, compared with the rest of the body A series of homeobox transcription factors including Otx2, Sox2, Lhx2, Rax and Pax6 play a major role in different stages of eye development Eyefield is specified in early embryos by overlapping expression of Otx2 and Sox2 in the neural ectoderm, which in turn activate expression of Lhx2, Rax and Pax6, resulting in the formation of the optic vesicle Inductive interaction between neural ectoderm and surface ectoderm results in formation of the lens placode, defined by the expression of Pax6 and Sox2 Lens and cornea share many transcriptional networks, consistent with their origin from the surface ectoderm Pitx2 and Foxc1 play major roles in anterior segment formation Sox2 regulates cell-specific target genes by cooperating with distinct factors such as Pax6, Oct3/4 and Brn2 Pax6 plays a major role in early retinogenesis, and formation of lens and cornea. Cone cells are the default photoreceptor cell type. NRL expression is essential for rod cell formation Mitf plays a critical role in RPE formation

37. Expression of homeobox genes in the mature vertebrate retina