1. Lecture 7 Advanced epistasis Ectopic expression in Drosophila Morphogens: secreted ligands

2. Epistasis headlesstwinhead

3. Epistasis headless hltwinhead th hl lf th lf phenotype no head; two tails no tail; two heads

4. Epistasis headless hltwinhead th hl lf th lf phenotype no head; two tails no tail; two heads Head Tail

5. Advanced Epistasis Why do I always assume that the most epistatic step is at the end of the pathway?

6. Advanced Epistasis Why do I always assume that the most epistatic step is at the end of the pathway? Can we always assume this?

7. Example biochemical pathway ABCDE WXY Z E is an essential metabolite

8. Phenotype of mutants in W, X, Y, Z all can not grow without E in the media.

9. Phenotype of mutants in W, X, Y, Z all can not grow without E in the media.

10. Phenotype of double mutants in W, X, Y, Z.

11. Order of epistasis Z epi to Y epi to X epi to W

12. Example biochemical pathway ABCDE WXY Z What intermediates accumulates in the mutant?

13. Accumulated intermediates

14. Double mutants

15. Order of epistasis W epi to X epi to Y epi to Z

16. Order of epistasis W epi to X epi to Y epi to Z Z epi to Y epi to X epi to W

17. Order of epistasis W epi to X epi to Y epi to Z Z epi to Y epi to X epi to W When you assay along the pathway When you assay at the end of the pathway

18. Ectopic expression in Drosophila Heat-shock promoter fusion genes Binary or two component system Flip-mediated ectopic expression Combination system I Combination system II

19. Heat-shock promoter fusion gene YFG hsp Qualities A) high level of expression B) expressed in all cells C) expression is transient

20. Binary or two component system Fly stock A: UAS lineFly stock B: Driver line YFG GAL4 binding sites UASGAL4 GAL4 Promoter or Enhancer YFG GAL4 Progeny

21. Binary or two component system Fly stock A: UAS lineFly stock B: Driver line YFG GAL4 binding sites UASGAL4 GAL4 Promoter or Enhancer YFG GAL4 Progeny GAL4

22. How do you make different GAL4 drivers? A. Construct them Known promoter GAL4

23. How do you make different GAL4 drivers? B. Screen for them by enhancer trapping. lacZ p P-element transposase promoter: very very weak expression-basically no expression by itself. Enhancer detector

24. Enhancer structure of a portion of the genome Gut Brain Gonad

25. Insertion of a enhancer detector lacZ Gonad Gut Brain

26. Insertion of a enhancer detector Gonad Gut Brain lacZ

27. How do you make different GAL4 drivers? B. Screen for them by enhancer trapping. GAL4 p P-element transposase promoter: very very weak expression-basically no expression by itself. Enhancer detector

28. Qualities of Binary system Control temporal and spatial pattern of YFG Expression is not transient Nonexpression of YFG from UASGAL4 allows transformants to be established

29. YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression FLP hsp Heat shock and induce FLP expression

30. YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression

31. YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression

32. YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression Results in a mosaic pattern of expression of YFG: recombination does not occur in all cells

33. Combination system I YFG FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 Promoter or enhancer

34. Combination system I YFG FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 Promoter or enhancer Induce FLP expression

35. Combination system I YFG FRT UASGAL4 GAL4 Promoter or enhancer

36. What happens? Cell expressing GAL4Cells that have undergone a site specific recombination event

37. What happens?

38. Combination system II GAL4 FRT y+y+, CD2 Tubulin promoter YFG UASGAL4 FLP hsp

39. Combination system II GAL4 FRT y+y+, CD2 Tubulin promoter YFG UASGAL4 FLP hsp Induce FLP expression

40. Combination system II GAL4 FRT Tubulin promoter YFG UASGAL4

41. Combination system II GAL4 FRT Tubulin promoter YFG UASGAL4 Results in clones of cells that express YFG

42. Morphogen Alan Turing-a father of computational sciences In 1952 publishes a paper entitled “A. M. Turing on the chemical basis of morphogenesis.” Not recommended reading

43. [morphogen] Simplified Turing morphogen model

44. [morphogen] Simplified Turing morphogen model Fate A Fate B

45. Morphogen Long range action Forms a concentration gradient of activity Affect polarity Concentration dependent response Different fates at different concentrations

46. Suspected secreted morphogens  Tumor growth factor TGF/decapentaplegic dpp WNT/wingless wg Sonic hedgehog etc./hedgehog hh

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

48. One eyed pinhead Schier et al. 1997 Development

49. Get similar phenotypes in mice by altering BMP signalling Gilbert Developmental Biology 7th ed

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

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

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

53. DPP a morphogen? Long range action YES Forms a concentration gradient of activity YES Affect polarity YES Concentration dependent response YES Different fates at different concentrations YES

54. Wing imaginal discs

55. A P

56. DPP is expressed in gradient Wing imaginal disc dpp mRNA DPP protein

57. Spalt is a DPP responsive gene Increase [DPP] and spalt response widens

58. Spalt and octomotor blind are DPP responsive SPT OMB

59. Spalt and octomotor blind are DPP responsive SPT OMB SPT

60. Long range action DPP expressed in this cell

61. Long range action DPP expressed in this cell Directly Affects expression in this cell

62. Signal relay system DPP expressed in this cell indirectly

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

64. Combination system I tkv Q253D FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 ubiquitous expression

65. UAS-tkv gf CD2 expressionOMB expression

66. UAS-tkv gf CD2 expressionOMB expression

67. FLP mediated mitotic recombination with a tkv lf allele

68. TKV is required right out to the edge of OMB expression domain

69. Signal relay system DPP expressed in this cell indirectly

70. no tail expression squint sqt Zebrafish example

71. no tail expression squint sqt Zebrafish example one eyed pinhead mutant

72. no tail expression squint sqt Zebrafish example one eyed pinhead mutant Wild-type cell

73. no tail expression squint sqt Zebrafish example one eyed pinhead mutant Wild-type cell