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Steroid Control of Leg Development in Drosophila

Steroid Control of Leg Development in Drosophila. Craig T. Woodard Mount Holyoke College. 20-hydroxyecdysone. Ecdysone. Ecdysone Receptor (EcR). Ultraspiracle (USP). Drosophila Life Cycle. Drosophila Life Cycle. The fruit fly undergoes complete metamorphosis.

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Steroid Control of Leg Development in Drosophila

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  1. Steroid Control of Leg Development in Drosophila Craig T. Woodard Mount Holyoke College

  2. 20-hydroxyecdysone

  3. Ecdysone Ecdysone Receptor (EcR) Ultraspiracle (USP)

  4. Drosophila Life Cycle

  5. Drosophila Life Cycle • The fruit fly undergoes complete metamorphosis. • Development lasts 10-12 days during which the fly embryo develops into larvae, pupa and ecloses into an adult. • Controlled by steroid hormone ecdysone

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

  7. Drosophila Life Cycle

  8. Ecdysone directs metamorphosis Puparium formation Morphogenesis of Adult Body Parts Beginning of imaginal disc morphogenesis Prepupal- pupal transition Gas bubble translation Destruction of Larval body Parts by Programmed Cell Death • High titer of ecdysone at the end of 3rd instar larva initiates entry into metamorphosis • Second high titer at approximately 11 hours APF initiates the Prepupal-Pupal Transition, which includes formation of adult body parts by morphogenesis and destruction of larva body parts through apoptosis

  9. Stages in Drosophila Leg Development • Embryonic Stage • Leg imaginal discs patterned • Puparium Formation (Beginning of Metamorphosis = 0-Hrs. APF) • Ecdysone induces Leg imaginal Discs Eversion and • Elongation • Prepupal-Pupal Transition (~12-Hrs. APF) • Ecdysone induces Pupal Ecdysis, inflating and • Extending Legs

  10. Ecdysone directs leg morphogenesis during metamorphosis Puparium formation (0-Hrs. APF) Leg disc Elongation and Eversion Pupal ecdysis (part of the Prepupal- pupal transition) Leg Extension • High titer of ecdysone at the end of 3rd instar larva initiates leg imaginal disc Elongation and Eversion • Second high titer at approximately 10-12 hours APF initiates pupal ecdysis, which drives leg Extension, and other morphogenetic events of the Prepupal-Pupal Transition

  11. Third Instar Larva Leg Disc Elongation and Eversion Adult

  12. Ecdysone directs leg morphogenesis during metamorphosis Puparium formation (0-Hrs. APF) Leg disc Elongation and Eversion Pupal ecdysis (part of the Prepupal- pupal transition) Leg Extension • High titer of ecdysone at the end of 3rd instar larva initiates leg imaginal disc Elongation and Eversion • Second high titer at approximately 10-12 hours APF initiates pupal ecdysis, which drives leg Extension, and other morphogenetic events of the Prepupal-Pupal Transition

  13. Normal Leg Development

  14. Third Instar Larva Leg Disc Elongation and Eversion Adult

  15. Cell shape changes during leg disc elongation a b Courtesy of Condic et al. 1991. Development 111:23-33

  16. Stubble (Sb) encodes a protease that induces changes in cell shape via activation of the RhoA GTPase, resulting in changes in the actin cytoskeleton

  17. Cell shape changes that drive leg disc elongation fail in Stubble mutants Control 6-Hrs. APF Stubble Mutant 6-Hrs. APF

  18. Cell shape changes that drive leg Disc elongation fail in Stubble mutants Control 6-Hrs. APF Stubble Mutant 6-Hrs. APF

  19. Changes in Actin Cytoskeleton Sb Leg disc Elongation

  20. Imaginal Disc Eversion Pastor-Pareja et al. (2004. Dev. Cell 7: 387-399) propose an updated model for imaginal disc eversion. According to their model, imaginal discs evert by apposing their peripodial side to the larval epidermis, and via invasion of the larval epidermis by cells of the peripodial epithelium and peripodial stalk.

  21. Normal Leg Development

  22. The role of how in leg imaginal disc morphogenesis

  23. The Drosophila how gene has pleiotropic functions during metamorphosis • how (held-out-wings) also named how, struthio, qkr93F • Encodes KH RNA binding protein • Strong similarity to nematode GLD-1 and mouse QK1 • Required for tendon cell differentiation in embryos • how mutants exhibit defects in muscle, muscle attachment, wing development and adult leg development

  24. how mutants show defects in leg development how Mutant Control

  25. how is expressed during metamorphosis

  26. how is expressed in various tissues (including imaginal discs) at the onset of metamorphosis (0-Hrs. APF)

  27. how Mutants undergo normal cell shape changes that drive leg imaginal disc elongation Control 6-Hrs. APF how Mutant 6-Hrs. APF

  28. how Mutants exhibit defects in leg imaginal disc eversion Control 6-Hrs. APF how Mutant 6-Hrs. APF

  29. Control how Mutant

  30. how Mutant how Mutant Control

  31. Sb Leg disc Elongation how Leg disc Eversion

  32. Possible role for how in imaginal disc eversion According to the Pastor-Pareja et al. model, imaginal discs evert by apposing their peripodial side to the larval epidermis, and via invasion of the larval epidermis by cells of the peripodial epithelium and peripodial stalk. During this process, the Jun-N-Kinase (JNK) signaling pathway promotes the apposition of peripodial stalk and larval cells, determines the extent of PEMT and motility of the leading edge/peripodial stalk cells, and helps maintain adhesion between larval and imaginal tissue (Pastor-Pareja et al., 2004). how may play a role in directing interactions between the imaginal disc cells, the cells of the peripodial epithelium and stalk, and larval epithelial cells during disc eversion. Perhaps how regulates expression of genes that play more direct roles in these cell-cell interactions.

  33. The role of ßFTZ-F1 in leg development Control ßFTZ-F1 Mutant

  34. Hypothesis A. ßFTZ-F1, nuclear receptor transcription factor, provides target genes, including the early genes, BR-C, E74A and E75A, with the competence* to be reinduced by the prepupal ecdysone pulse. 1) These early genes then direct morphogenesis of adult body parts. B. ßFTZ-F1provides the prepupal stage-specific E93 early gene with the competence* to be induced by ecdysone. ßFTZ-F1thus directs the stage-specificity of the E93 response to ecdysone 1) E93 then directs programmed cell death in larval body parts. *Competence the ability to respond to an inductive signal

  35. Morphogenesis of Adult Body Parts Pupariation (Entry into Metamorphosis) Destruction of Larval body Parts by Programmed Cell Death

  36. 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. Ectopic expression of ßFTZ-F1 provides E93 with the competence to respond to the late larval ecdysone pulse. ßFTZ-F1 protein binds E93 genomic sequences. Induction of BR-C, E74A and E75A transcripts by ecdysone is enhanced significantly by ectopic ßFTZ-F1. 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. 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 extension. Evidence in Support of our Hypothesis

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