1. March 2, 2011 Introduction to development Gene expression

3. What cellular functions are needed to carry out development?

4. Divide Grow Differentiate Die Move Adhere Secrete Signal Cell biological properties need to be coordinated in space and time

5. Where is the “program” for development encoded?

6. In the genes Other influences on development: Maternal components Environmental signals

7. Why do different cells behave differently?

8. Have different genes? Have different histories? Experience different environments? Chance? Have different gene expression states?

9. Why do different cells behave differently? Have different genes Have different histories Experience different environments Chance Have different gene expression states

10. 05_02_DNA.jpg

11. 05_10_Genes_info.jpg Genes Carry out functions

12. 07_37_Protein.produc.jpg Steps in gene expression

13. 08_03_control.steps.jpg mRNA localization control (tether mRNA to localized proteins) mRNA turnover control (plant microRNAs) Protein turnover control (ubiquitylation and proteasome digestion) Protein localization control Regulation of gene expression

14. 08_03_control.steps.jpg mRNA localization control (tether mRNA to localized proteins) mRNA turnover control (plant microRNAs) Protein turnover control (ubiquitylation and proteasome digestion) Protein localization control Regulation of gene expression

15. coding strand Regulation of transcription: Parts of a gene

16. 08_13_gene.activation.jpg Regulation of transcription

17. Transcription factors Dimer (Two proteins)

18. 05_24_Chromatin pack.jpg Nucleosomes (histones) package DNA Histones affect gene expression

19. 08_14_chromatin.struc.jpg Some transcription factors affect histones

20. 05_30_histone tails.jpg Histone modifications affect gene expression Can affect recruitment of transcription factors to promoter H3K27m 4

21. 08_15_Reg. proteins.jpg Multiple transcription factors regulate most genes

22. 08_18_reporter.gene.jpg Modularity of the Drosophila even-skipped promoter

23. Coordinated regulation of multiple genes Developmental functions

24. Signals can regulate activity of transcription factors

25. 08_23_cell.memory.jpg Maintaining gene expression states (a positive feedback loop)

26. 08_24_chromatin.state.jpg Maintaining gene expression states

27. The histone code also needs to be maintained in daughter cells

28. Maintaining gene expression states – DNA methylation

29. How does one detect which genes a particular cell expresses?

30. 10_14_1_Southrn.blotting.jpg Southern blot

31. 10_14_2_Southrn.blotting.jpg Southern blot – DNA on blot Northern blot – RNA on blot

32. Northern blot hybridization (tests one gene at a time)

33. RT-PCR: 1) Reverse transcription (tests one gene at a time)

34. RT-PCR: 2) Polymerase Chain Reaction (tests one gene at a time)

35. RT-PCR summary (tests one gene at a time) Quantitate on gel or by measuring rate of DNA product accumulation

36. In situ hybridization of developing flowers with ARF6 probe Reveals which cells in a complex tissue express the gene Earlier stages, longitudinal sections Later stages, transverse sections

37. From Wildwater et al., Cell 123: 1337-1349 (2005) Rb-Related expression in Arabidopsis embryos by in situ hybridization Why is this pattern punctate (spotty)?

38. coding strand Making reporter gene fusions Examples: lacZ, GUS, GFP Insert construct into transgenic organism Promoter Reporter gene

39. P ARF6 ::ARF6::GUS fusion expression in ovules Promoter:protein:reporter fusion gene In situ hybridizations

40. 08_18_reporter.gene.jpg Promoter:reporter fusion gene in a transgenic fly embryo

41. Kosman et al., Science 254: 118-122 (1991) anti-Snail anti-Twist Immunolocalization of Snail and Twist proteins in Drosophila embryos Reveals protein location

42. Utility of looking at expression of single genes at a time: Markers of cell type, differentiation Visualization of regulatory events Utility of looking at expression of many genes at once: Global view of tissue identity Comparing different tissues or states Global view of regulatory events