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Hypothesis

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.

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Hypothesis

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  1. Hypothesis A. ßFTZ-F1provides the prepupal stage-specific E93 early gene with the competence* to be induced by ecdysone 1) ßFTZ-F1thus 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

  2. 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 [E. Baehrecke, unpublished].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] Evidence in Support of our Hypothesis

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

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

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

  6. Comparative Leg Development Control ßFTZ-F1 Mutant

  7. 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.

  8. 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)

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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 Acknowledgements

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

  16. 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

  17. 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

  18. hsßFTZ-F1 Control hsßFTZ-F1 Control hsßFTZ-F1 Control hsßFTZ-F1 Control hsßFTZ-F1 Control SG gut fat CNS SG

  19. 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 Acknowledgements

  20. Normal Leg Development

  21. 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 Acknowledgements

  22. 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

  23. Third Instar Larva Leg Disc Eversion Adult

  24. 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

  25. 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

  26. 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

  27. Gut tissue control tissue mutant tissue

  28. hsßFTZ-F1 Control hsßFTZ-F1 Control hsßFTZ-F1 Control hsßFTZ-F1 Control hsßFTZ-F1 Control SG gut fat CNS SG

  29. 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

  30. Third Instar Larva Leg Disc Eversion Adult

  31. 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

  32. 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

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