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

11. 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

12. 20-hydroxyecdysone

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

15. Drosophila Life Cycle

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

18. Drosophila Life Cycle

20. Ecdysone directs metamorphosis Puparium formation Prepupal- pupal transition High titer of ecdysone at the end of 3 rd 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 Morphogenesis of Adult Body Parts Destruction of Larval body Parts by Programmed Cell Death Gas bubble translation Beginning of imaginal disc morphogenesis

21. Third Instar Larva Leg Disc Elongation and Eversion Adult

22. Stages in Drosophila Leg Development Embryonic Stage Leg imaginal discs patterned Puparium Formation (Beginning of Metamorphosis = 0-Hrs. APF) Ecdysone induces Leg imaginal Disc Eversion and Elongation Prepupal-Pupal Transition (~12-Hrs. APF) Ecdysone induces Pupal Ecdysis, inflating and Extending Legs Pupal Stage Ecdysone directs refinement of legs: Joints, bristles and claws develop

23. Ecdysone directs leg morphogenesis during metamorphosis Puparium formation (0-Hrs. APF) Pupal ecdysis (part of the Prepupal- pupal transition) High titer of ecdysone at the end of 3 rd 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 Leg Extension Leg disc Elongation and Eversion

24. Third Instar Larva Leg Disc Elongation and Eversion Adult

25. Ecdysone directs leg morphogenesis during metamorphosis Puparium formation (0-Hrs. APF) Pupal ecdysis (part of the Prepupal- pupal transition) High titer of ecdysone at the end of 3 rd 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 Leg Extension Leg disc Elongation and Eversion

26. Normal Leg Development

27. Third Instar Larva Leg Disc Elongation and Eversion Adult

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

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

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

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

32. Sb Leg disc Elongation Changes in Actin Cytoskeleton

33. 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. Imaginal Disc Eversion

34. Normal Leg Development

35. The role of how in leg imaginal disc morphogenesis

36. The Drosophila how gene has pleiotropic functions during metamorphosis how (held-out-wings) also named who, 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.

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

38. how is expressed during metamorphosis

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

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

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

44. Control how Mutant

45. Control how Mutant

46. how is required for leg imaginal disc Eversion Sb Leg disc Elongation how Leg disc Eversion

47. 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). OUR HYPOTHESIS 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.

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

49. βFTZ-F1 ecdysoneActivates genes Developmental processes βFTZ-F1 βFTZ-F1 encodes an orphan nuclear receptor It encodes a protein that functions as a competence factor and mediates the late prepupal developmental response to ecdysone.

50. 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-F1 provides the prepupal stage-specific E93 early gene with the competence* to be induced by ecdysone. ßFTZ-F1 thus 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

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

52. Evidence in Support of our Hypothesis Staining with anti-ßFTZ-F1 antibodies shows ßFTZ-F1 protein bound to the BR-C, E74A, E75A and E93 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.

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

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

55. ßFTZ-F1 mutants fail to complete metamorphosis head eversion leg development wing development

56. Mutations in ßFTZ-F1 result in defective leg development Control ßFTZ-F1 Mutant

57. ßFTZ-F1 Mutants undergo normal cell shape changes that drive leg imaginal disc elongation

59. Leg extension fails at the prepupal-pupal transition in ßFTZ-F1 mutants Control ßFTZ-F1 Mutant

60. Possible Causes of Short Legs 1) Contraction of the muscles is too weak in ßFTZ-F1 mutants. 2) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants, which prevents them from extending.

62. Leg and wing length in ßFTZ-F1 mutants can be rescued by reduced external pressure Significant Difference

63. Leg and wing length in ßFTZ-F1 mutants can be rescued by reduced external pressure

64. Possible Causes of Short Legs 1) Contraction of the muscles is too weak in ßFTZ-F1 mutants. --------------------------------------------------------------- 2) There is something wrong with the leg imaginal discs in ßFTZ-F1 mutants. RULED OUT

65. Possible Causes of Short Legs 1) Contraction of the muscles is too weak in ßFTZ-F1 mutants. This is supported by our careful observations of control and ßFTZ-F1 mutant animals going through pupal ecdysis. The ßFTZ-F1 mutants exhibit severe defectsin the muscle contractions that occur during pupal ecdysis.

66. 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. ßFTZ-F1 is required for the muscle movements of pupal ecdysis, which generates internal pressure (at the appropriate time), which drives extention of legs and wings, and eversion of the head. Hypothesis - Perhaps there are defects in the muscles that generate pressure.

67. Abdominal muscles Play an important role in metamorphosis Larval origin but a subset persists after puparium formation to drive morphogenetic events of pupal ecdysis Majority of larval muscles are destroyed during prepupal or early pupal stages and replaced by adult structures from muscle precursor cells

68. Musculature Five dorsal muscles Four lateral transverse muscles Segment border muscles Ventral muscles

69. Abdominal Muscle Activity Contractions that occur during pupal ecdysis result in: -separation of the pupal cuticle from the puparium (pupal case) -shortening of the prepupal body -translocation of the mid-abdominal gas bubble -build up of hydrostatic pressure to aid head eversion, leg and wing extension

70. We have been unable to defects in muscle structure in ßFTZ- F1 mutants (but we are still looking!) ßFTZ-F1 Mutant 12-Hrs. APF

71. 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. ßFTZ-F1 is required for the muscle movements of pupal ecdysis, which generates internal pressure (at the appropriate time), which drives extention of legs and wings, and eversion of the head. We have been unable to detect ultrastructural abnormalities in the muscles thought to generate this internal pressure. New Hypothesis - Perhaps there are defects in the neurons that innervate these muscles.

72. 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.

73. Results Control 0-Hrs. APF ßFTZ-F1 Mutant 0-Hrs. APF ln:longitudinal nerve; sn:segmental nerve; pg:peripheral glia

74. w; P [w+, Nrv2]; FTZ-F1 17 /DfCat 14hr w; P [w+, Nrv2]; + 14hr ln:longitudinal nerve; sn:segmental nerve; pg:peripheral glia Control 14- Hrs. APF ßFTZ-F1 Mutant 14-Hrs. APF

75. Wild-Type Control

76. ßFTZ-F1 Mutant

78. ETH triggers Pupal Ecdysis Recent findings by colleagues have elucidated the role of Ecdysis Triggering Hormone (ETH) in triggering pupal ecdysis. ETH is a peptide hormone, released into the circulation by specific cells called the INKA cells of the epitracheal gland in response to ecdysone. ETH acts on the Central Nervous System, inducing the pupal ecdysis behavioral sequence.

79. Zitnan, D. et al. J Exp Biol 2003;206:1275-1289 PETH-immunoreactive Inka cells (stained orange/red) in different holometabolous inse cts

80. Pupal Ecdysis behavioral sequence and Leg Extension Pupariatium Formation (Entry into Metamorphosis) Target Genes in INKA cells Sb how Leg disc Elongation Leg disc Eversion ETH CNS

81. FUTURE DIRECTIONS how - We are examining expression patterns of genes in the JNK signaling pathway in how mutants vs. controls (Blanca Carbajal). -We are examining how mutant leg imaginal discs using TEM (Antonina Kruppa) ßFTZ-F1 -We are examining the transcription of genes encoding neuropeptides in ßFTZ-F1 mutants vs. controls (Melanie Ayerh and Kori Matsuura). - We are continuing to examine motor neurons and muscles in ßFTZ-F1 mutants (Hyowon Choi). - We are attempting to decipher the molecular mechanism by which ßFTZ-F1 provides target genes with the competence to respond to ecdysone (Antonina Kruppa).

82. FUTURE DIRECTIONS (cont.) Other Genes - We are examining Tis-11 mutants, which have abnormal legs (Hyowon Choi). - Erika Power is examining genetic interactions between genes involved in leg development.

83. Acknowledgments Mount Holyoke College Tina Fortier Jennifer McCabe Priya Vasa Nicole Whyte Rizwana Islam Jodi McKenzie Melanie Ayerh Monique Killins MANY other MHC independent research students! University of Maryland Biotechnology Institute Eric Baehrecke Runa Chatterjea Susan Klinedinst Special Thanks for Technical Assistance Maria Bledzka Lezek Bledzki Samara Brown George Cobb Austin Federa Rachel Fink Janice Gifford Peter Gruber Diane Kelly Stan Rachootin Marian Rice Marinko Sremac Funding National Science Foundation Howard Hughes Medical Institute Mount Holyoke College Biology Department and Biochemistry Program

85. ßFTZ-F1 mutants fail to histolyze larval salivary glands

86. ßFTZ-F1 mutants exhibit pupal lethality and defects in morphogenesis

88. Ecdysone concentrations ßFTZ-F1 rp49 Ecdysone concentrations Normalized RNA level

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

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

92. Morphogenesis of Adult Body Parts Destruction of Larval body Parts by Programmed Cell Death Pupariation (Entry into Metamorphosis) Target Genes? Cell Death Genes how Sb

93. Conclusions who mutants show multiple lethal phases and pleiotropic effects during metamorphosis. who is expressed during metamorphosis. who mutant leg discs undergo proper cell shape changes during morphogenesis but do not extend fully at the prepupal- pupal transition. This defect is associated with inappropriate orientation of leg imaginal discs. The pleiotropic function of who suggests that KH proteins play essential roles in development of numerous cell types.

94. Acknowledgments Mount Holyoke College Tina Fortier Craig Woodard University of Maryland Biotechnology Institute Runa Chatterjee Susan Klinedinst Eric Baehrecke