1. Lecture 8 Lab results/lab report The morphogen problem Nuclear gradients and linear pathways  TGF and Brinker Three ‘habits’ of signaling pathways

2. Results are posted on the web Go to results page

3. Click and find your files

4. Instruction to authors

6. Turnitin.com To be marked the lab report must be submitted to turnitin.com minus the references. Class ID: see web site Enrollment password: see web site Due April 5, 2012

7. The problem with Morphogens 100% 50% Percent bound/ response Concentration

8. The problem with Morphogens 100% 50% Percent bound/ response Concentration

9. The problem with Morphogens 100% 50% Percent bound/ response Concentration

10. The problem with Morphogens 100% 50% Percent bound/ response Concentration Cooperative

11. The problem with Morphogens 100% 50% Percent bound/ response Concentration Cooperative

12. The primacy of secreted morphogens Teleman and Cohen Cell 103, 971

13.  TGF/DPP pathway  TGF/DPP ligand One eyed pinhead oep Co-receptor in zebrafish Type1. thickvein Type2, punt Smads

14. The primacy of secreted morphogens Teleman and Cohen Cell 103, 971

15. Problems with primacy of secreted morphogens Changes to gene expression is ultimately a nuclear event. Most morphogen signaling pathways are linear. Gradient on gradient on gradient. Bicoid rules.

16. Toll Dorsal Pathway Gilbert 7th Ed.

17. Gradients Spatzle is activated in a graded manner. Toll is active in a graded manner. Pelle kinase is active in a graded manner. Cactus is degraded in a graded manner. Dorsal enters the nucleus in a graded manner.

18. Dorsal nuclear gradient Stathopoulos and Levine Dev. Biol. 246, 57

19. Interpretation in the nucleus of the gradient Stathopoulos and Levine Dev. Biol. 246, 57

20. Interpretation in the nucleus of the gradient Convert low affinity site to high affinity Stathopoulos and Levine Dev. Biol. 246, 57

21. Interpretation in the nucleus of the gradient Stathopoulos and Levine Dev. Biol. 246, 57

22. Dorsal patterns the dorsal ventral axis Stathopoulos and Levine Current opinion in Genetics and Development 14, 477

23. Different Dorsal dependent regulatory elements Stathopoulos and Levine Current opinion in Genetics and Development 14, 477

24. Different Dorsal dependent regulatory elements Stathopoulos and Levine Current opinion in Genetics and Development 14, 477

25. Problem with Dorsal as model morphogen The Toll Dorsal pathway activated at syncytial blastoderm stage. Look at DPP again.

26.  TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads

27.  TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads P

28.  TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads P P

29.  TGF/DPP pathway  TGF/DPP ligand Type1. thickvein Type2, punt Smads P P Nucleus Mad Medea

30. What do Mad and Medea do in the nucleus? Activate expression of an inhibitory Smad called Dad Repress Brinker expression

31. Brinker? Identified as a DPP regulated gene required for the repression of DPP regulated genes.

32. Brinker expression repressed by DPP pathway FRT UbiGFP mad Campbell and Tomlinson Cell 96, 553

33. FRT UbiGFP brk XH Brinker represses Octomotor blind and Spalt Campbell and Tomlinson Cell 96, 553

34. Brinker epistasis Jazwinska et al., Cell 96, 563

35. Brinker is a nuclear protein that is repressed by DPP such that it is expressed in a DPP anti- gradient. Sal and omb are repressed by different concentrations of Brinker. Campbell and Tomlinson Cell 96, 553

36. Brinker DNA binding domain bound to DNA

37. Questions about Brinker How do Mad and Medea both activate and repress transcription? How does the DPP pathway regulate Brinker expression to create the anti- gradient?

38. Schnurri is required for expression of DPP responsive genes Schnurri phenotype is suppressed by brinker mutant wtshnbrkshn, brk Marty et al., Nature cell biol. 2, 745

39. Expression in shn brk mutants shn Sal expression shn Brinker expression shn brk Sal expression GFP Marty et al., Nature cell biol. 2, 745

40. DPP pathway has two branches Marty et al., Nature cell biol. 2, 745

41. Looking for the brinker regulatory element Muller et al., Cell 113, 221

42. 24 bases required for repression Pyrowolakis et al., Dev. Cell 7, 229

43. Mad Medea and Schnurri bind to the silencer Pyrowolakis et al., Dev. Cell 7, 229

44. Brinker is not the only gene repressed Pyrowolakis et al., Dev. Cell 7, 229

45. Brinker is a nuclear protein that is repressed by DPP such that it is expressed in a DPP anti- gradient. Sal and omb are repressed by different concentrations of Brinker. Bicoid rules Campbell and Tomlinson Cell 96, 553

46. The three ‘habits’ Barolo and Posakony 2002 Looking for common themes in the organization of signaling pathways.

47. Trying to explain the precision of expression Example wingless expression

48. The three ‘habits’ Activator insufficiency Cooperative activation Default repression

49. SPRE-binding transcription factor SPRE: Signaling pathway response elements

50. SPRE-binding transcription factor SPRE: Signaling pathway response elements HH: Ci/Gli WNT: Lef/Tcf Notch: Su(H)/CBF1

51. Other factors Barolo and Posakony Genes and Dev. 16, 1167

52. 3 habits model Barolo and Posakony Genes and Dev. 16, 1167

53. Basis for the proposal of the model Barolo and Posakony Genes and Dev. 16, 1167

54. Activator insufficiency SPRE-binding transcription factor can not activate transcription alone. Tissue culture vs in vivo

55. Cooperative activation SPRE-binding transcription factors require other transcription factors for the activation of transcription. The interaction is cooperative

56. Default repression In the absence of ligand SPRE-binding factors repress transcription.

57. 3 habits model Barolo and Posakony Genes and Dev. 16, 1167

58. Switching from default repression to activation Barolo and Posakony Genes and Dev. 16, 1167

59. 3 habits model Barolo and Posakony Genes and Dev. 16, 1167

60. Consequences of the 3 ‘habits’ Barolo and Posakony Genes and Dev. 16, 1167

61. Trying to explain the precision of expression Wingless is regulated by the HH pathway

62. DPP pathway and the three habits Pyrowolakis et al., Dev. Cell 7, 229