1. Hypothesis A. ßFTZ-F1 provides the prepupal stage-specific E93 early gene with the competence* to be induced by ecdysone 1) ßFTZ-F1 thus directs the stage-specificity of the E93 response to ecdysone. B. ßFTZ-F1 provides the early genes, the BR-C, E74A and E75A with the competence* to be reinduced by the prepupal ecdysone pulse. *Competence the ability to respond to an inductive signal

2. Evidence in Support of our Hypothesis Staining with anti-ßFTZ-F1 antibodies shows ßFTZ-F1 protein bound to the 2B5, 74EF, 75B and 93F puff loci in prepupal salivary gland polytene chromosomes. [Lavorgna, et al. (1993) PNAS 90: 3004- 3008] Ectopic expression of ßFTZ-F1 provides E93 with the competence to respond to the late larval ecdysone pulse. [Woodard et al. (1994) Cell 79: 607-615] ßFTZ-F1 protein binds E93 genomic sequences. [E. Baehrecke, unpublished]. Induction of BR-C, E74A and E75A transcripts by ecdysone is enhanced significantly by ectopic ßFTZ-F1. [Woodard et al. (1994) Cell 79: 607-615] A Loss-of-function mutation in ßFTZ-F1 results in dramatic reductions in E93, E74A, E75A, and BR-C transcripts at the end of the prepupal stage. [Broadus et al. (1999) Molecular Cell 3: 143-149] A loss-of-function mutation in ßFTZ- F1 results in pupal lethality with defects in larval salivary gland programmed cell death, head eversion, and leg elongation. [Broadus et al. (1999) Molecular Cell 3: 143- 149]

4. The ex17 mutation results in pupal lethality and defects in morphogenesis

5. Mutations in ßFTZ-F1 disrupt leg morphogenesis Control ßFTZ-F1 Mutant

6. Cell Shape Changes During Leg Disc Elongation Courtesy of Condic et al. 1991. Development 111:23-33 ab

9. Comparative Leg Development Control ßFTZ-F1 Mutant

13. Possible Causes of Short Legs 1) Contraction of the muscles is too weak in ßFTZ-F1 mutants. 2) The pupal cuticle is too rigid by the time the muscles contract in ßFTZ-F1 mutants. 3) Connections to the puparium are not sufficiently weakened in ßFTZ-F1 mutants. 4) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants.

15. Leg Extension in ßFTZ-F1 Mutants can be Rescued by a Drop in Pressure Percent of animals with normal leg-length (n = 27)(n = 20) (n = 11) (n = 22)

16. Possible Causes of Short Legs 1) Contraction of the muscles is too weak in ßFTZ-F1 mutants. 2) The pupal cuticle is too rigid by the time the muscles contract in ßFTZ-F1 mutants. 3) Connections to the puparium are not sufficiently weakened in ßFTZ-F1 mutants. --------------------------------------------------------------- 4) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants. RULED OUT

17. Possible Causes of Short Legs 1) Contraction of the muscles is too weak in ßFTZ-F1 mutants. 2) The pupal cuticle is too rigid by the time the muscles contract in ßFTZ-F1 mutants. --------------------------------------------------------------- 3) Connections to the puparium are not sufficiently weakened in ßFTZ-F1 mutants. RULED OUT 4) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants. RULED OUT

18. Conclusions ßFTZ-F1 mutants are unable to generate sufficient internal pressure (at the appropriate time) to extend their legs, evert their heads, and extend their wings. We have been unable to detect ultrastructural abnormalities in the muscles thought to generate this internal pressure. Hypothesis - Perhaps there are defects in the neurons that innervate these muscles.

19. Testing the Hypotheses Hypothesis - There are defects in neurons that innervate the muscles. -Test by examining neurons, perhaps making use of animals expressing neuron-specific GFP. Hypothesis - The pupal cuticle is too rigid by the time the muscles contract in the mutants. -Test by aging the mutant and control animals a bit longer before exposing them to a drop in pressure -Test by measuring the tensile strength of mutant and control pupal cuticle in staged animals.

20. FUTURE DIRECTIONS Legs, etc. - Attempt to rescue ßFTZ-F1-mutant defects by ectopic expression of target genes. Other Projects - Examine the regulation of target genes by ßFTZ-F1 in specific tissues. - Decipher the molecular mechanism by which ßFTZ-F1 provides target genes with the competence to respond to ecdysone.

21. Acknowledgements Mount Holyoke College Tina M. Fortier** Priya Vasa Samara N. Brown** **put this presentation together Thanks to these folks from the University of Utah for help in making the movies. Carl S. Thummel Pamela Reid

23. Levels of early gene transcripts are reduced in ßFTZ-F1 mutant prepupae

24. Salivary glands control tissuemutant tissue E93 rp49 E93 rp49 0 2 4 6 8 10 12 14

25. Gut tissue mutant tissuecontrol tissue E93 rp49 0 2 4 6 8 10 12 14 E93 rp49 0 2 4 6 8 10 12 14

26. SG gut fat CNS SG hs ßFTZ-F1 Control hs ßFTZ-F1 Control hs ßFTZ-F1 Control hs ßFTZ-F1 Control hs ßFTZ-F1 Control

27. Acknowledgements Mount Holyoke College Tina M. Fortier** Samara N. Brown** Michael Chapman Priya Vasa Dana Cruz Zareen Gauhar Thanks to these folks from the University of Utah for help in making the movies. Carl S. Thummel Pamela Reid

28. Normal Leg Development

31. Acknowledgements Mount Holyoke College Tina M. Fortier** Samara N. Brown** Michael Chapman Jennifer R. McCabe Priya Vasa Dana Cruz Zareen Gauhar Lynn L’Archeveque Margaret Lobo Emily McNutt Tetyanya Obukhanych Petra Scamborova University of Utah Carl S. Thummel Eric H. Baehrecke Julie Broadus Bart Endrizzi

32. Hypothesis A. ßFTZ-F1 provides the prepupal stage-specific E93 early gene with the competence* to be induced by ecdysone 1) ßFTZ-F1 thus directs the stage-specificity of the E93 response to ecdysone. B. ßFTZ-F1 provides the early genes, the BR-C, E74A and E75A with the competence* to be reinduced by the prepupal ecdysone pulse. *Competence the ability to respond to an inductive signal

33. Third Instar Larva Leg Disc Eversion Adult

34. Larval and Pupal Stages of Drosophila Development A B C D E F A. First instar larva B. Second instar larva C. Third instar larva E. Prepupa F. Early pupa

35. Gut tissue mutant tissuecontrol tissue E93 rp49 0 2 4 6 8 10 12 14 E93 rp49 0 2 4 6 8 10 12 14

36. Gut tissue mutant tissuecontrol tissue E93 rp49 0 2 4 6 8 10 12 14 E93 rp49 0 2 4 6 8 10 12 14

37. Gut tissue mutant tissuecontrol tissue

40. SG gut fat CNS SG hs ßFTZ-F1 Control hs ßFTZ-F1 Control hs ßFTZ-F1 Control hs ßFTZ-F1 Control hs ßFTZ-F1 Control

44. Gut tissue mutant tissuecontrol tissue E93 rp49 0 2 4 6 8 10 12 14 E93 rp49 0 2 4 6 8 10 12 14

48. Third Instar Larva Leg Disc Eversion Adult

49. Larval and Pupal Stages of Drosophila Development A B C D E F A. First instar larva B. Second instar larva C. Third instar larva E. Prepupa F. Early pupa

50. Gut tissue mutant tissuecontrol tissue E93 rp49 0 2 4 6 8 10 12 14 E93 rp49 0 2 4 6 8 10 12 14

51. Gut tissue mutant tissuecontrol tissue E93 rp49 0 2 4 6 8 10 12 14 E93 rp49 0 2 4 6 8 10 12 14

52. Gut tissue mutant tissuecontrol tissue

55. Leg Extension in ßFTZ-F1 Mutants can be Rescued by a Drop in Pressure Percent of animals with normal leg-length

56. BACKGROUND The life cycle of Drosophila melanogaster has a duration of ten to twelve days, during which the embryo develops into a larvae to a stationary pupa and finally ecloses into the adult fly. This transition from larvae to adult is known as metamorphosis and is controlled by the steroid hormone, ecdysone. The Life Cycle of Drosophila melanogaster

57. Fig C. ECR Expression in Tissues

58. THE CHEMICAL STRUCTURE OF ECDYSONE

59. Ecdysone Timeline in Drosophila melanogaster

60. IN WHICH OTHER TISSUES DOES THE EXPRESSION OF ßFTZ-F1 AFFECT THE ECDYSONE INDUCTION OF BR-C, E74A, E75A AND E93 TRANSCRIPTION?

61. What is the molecular mechanism by which ßFTZ-F1 exerts its function to regulate early gene expression? Does ßFTZ-F1 induce expression of the ecdysone-receptor complex to facilitate the induction of the early genes? To test this hypothesis, in vitro experiments and Northern blot hybridization analysis was used to see if there is any ECR induction in the mid-third instar larval tissues.

62. EXPERIMENTAL DESIGN Transformant Flies called P[F-F1] were used that express a high level of ßFTZ-F1 mRNA upon heat shock. Control w 1118 and transformant w;P[F-F1] mid-third instar larvae were heat shocked for 30 min and the tissues were immediately dissected in oxygenated Robb’s saline. The organs were then cultured in the presence of oxygen at 25 C for 2 hr with or without ecdysone. Total RNA was extracted from the tissues and analyzed for E93 mRNA by Northern blot hybridization. The Northern blot was also probed with rp49 (gene encoding ribosomal protein) as a control for loading and transfer.

63. How can a single steroid hormone elicit different responses at different times in development?