Steroid Hormonal Regulation of Development in Drosophila

1. Steroid Hormonal Regulation of Development in Drosophila Craig T. Woodard Mount Holyoke College

7. 20-hydroxyecdysone

8. Drosophila Life Cycle

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

10. Drosophila Life Cycle

14. Puffs Early 2B5 74EF 75B Prepupal early 93F Mid prepupal 75CD Genes Early BR-C E74 E75 Prepupal early E93 Mid prepupal ßFTZ-F1

15. Salivary Gland Developmental Northern Analysis Hours relative to puparium formation Edysone BR-C E74A E75A E93 ßFTZ-F1

16. Hypothesis A. ßFTZ-F1provides 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-F1provides 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

17. Hours relative to puparium formation BR-C E74A E75A E93 ßFTZ-F1

18. EXPERIMENTAL DESIGN • Transformant Flies called P[F-F1] were used that express a • high level of ßFTZ-F1 protein upon heat shock. • Control w1118 and transformant w;P[F-F1] late-third instar • larvae were heat shocked for 30 min. and then allowed to • recover at 25˚ C for 2 hrs. • Salivary glands were dissected. • Total RNA was extracted from the salivary glands • 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.

19. w w;P[F-F1]

20. Hours relative to puparium formation BR-C E74A E75A E93 ßFTZ-F1

21. EXPERIMENTAL DESIGN • Transformant Flies called P[F-F1] were used that express a • high level of ßFTZ-F1 protein upon heat shock. • Control w1118 and transformant w;P[F-F1] mid-third instar • larvae were heat shocked for 30 min. and the salivary glands • were immediately dissected in oxygenated Robb’s saline. • The salivary glands were then cultured in the presence of • oxygen at 25˚ C for 2 hr with or without ecdysone. • Total RNA was extracted from the salivary glands 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.

25. ex17 is a Mutation in ßFTZ-F1

26. Expression of wild-type ßFTZ-F1 from a transgene rescues ex17 mutants

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

28. E93 transcription is greatly reduced in ßFTZ-F1 mutant salivary glands control tissue mutant tissue E93 rp49 E93 rp49 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

29. Normal salivary gland histolysis ßFTZ-F1 mutants fail to histolyze larval salivary glands

30. head eversion leg elongation wing extension Results of ßFTZ-F1 mutations

31. Mutations in ßFTZ-F1 disrupt leg morphogenesis

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

35. Normal Leg Development

36. Comparative Leg Development Control ßFTZ-F1 Mutant

37. 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.

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

40. 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. RULEDOUT

41. 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. RULEDOUT

42. 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.

43. 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.

44. Ecdysone, ßFTZ-F1, E93 and Programmed Cell Death(Tissue-Specificity)

45. ßFTZ-F1is required for E93 transcription in larval salivary glands control tissue mutant tissue E93 rp49 E93 rp49 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

46. If E93 is required for a complete programmed cell death response, how does destruction of the larval gut start at the beginning of metamorphosis (before ßFTZ-F1 is expressed) ?

47. ßFTZ-F1 is not required for E93 transcription in larval gut tissue control tissue mutant tissue E93 rp49 E93 rp49 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

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

49. EXPERIMENTAL DESIGN • Transformant Flies called P[F-F1] were used that express a • high level of ßFTZ-F1 protein upon heat shock. • Control w1118 and transformant w;P[F-F1] mid-third instar • larvae were heat shocked for 30 min. and the various tissues • were immediately dissected in oxygenated Robb’s saline. • The tissues 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.