1. Control of Animal Development by Steroid Hormones
Craig T. Woodard
Mount Holyoke College

7. 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

8. 20-hydroxyecdysone

9. Drosophila Life Cycle

10. Ecdysone Timeline in Drosophila melanogaster
Pupariation
(Entry into Metamorphosis)
Prepupal-Pupal Transition
Destruction of Larval Body Parts by Programmed Cell Death
Formation of Adult Body Parts by Morphogenesis

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

12. Ecdysone Timeline in Drosophila melanogaster
Pupariation
(Entry into Metamorphosis)
Prepupal-Pupal Transition
Destruction of Larval Body Parts by Programmed Cell Death
Formation of Adult Body Parts by Morphogenesis

13. How can a single steroid hormone elicit different responses in different parts of the body during development?

14. Ecdysone Timeline in Drosophila melanogaster
Pupariation
(Entry into Metamorphosis)
Prepupal-Pupal Transition
Destruction of Larval Body Parts by Programmed Cell Death
Formation of Adult Body Parts by Morphogenesis

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

16. Hypothesis
A. ßFTZ-F1 provides the early genes, the 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

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

18. Hypothesis
A. ßFTZ-F1 provides the early genes, the 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

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

20. Evidence in Support of our Hypothesis
ßFTZ-F1 protein binds to the E93, E74A, E75A, and BR-C genes.
Ectopic (over) expression of ßFTZ-F1 in transgenic larvae provides E93 with the competence to respond (prematurely) to the late larval ecdysone pulse.
Induction of BR-C, E74A and E75A transcripts by ecdysone is enhanced significantly by ectopic expression of ßFTZ-F1.
A Loss-of-function mutation in ßFTZ-F1 results in dramatic reductions in E93, E74A, E75A, and BR-C activation 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 elongation.

21. Levels of Early Gene Transcripts are Reduced in ßFTZ-F1 Mutant Prepupae

22. ßFTZ-F1 Mutants Exhibit Pupal Lethality and Defects in Morphogenesis
head eversion
leg elongation
wing extension

23. Mutations in ßFTZ-F1 Disrupt Leg Morphogenesis
Control ßFTZ-F1 Mutant

24. Third Instar Larva
Leg Disc Eversion
Adult

25. Cell Shape Changes During Leg Disc Elongationb
Courtesy of Condic et al Development 111:23-33

28. Comparative Leg Development
Control
ßFTZ-F1 Mutant

30. 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.

32. Leg and Wing Length in ßFTZ-F1 Mutants can be Rescued by a Drop in Pressure
Significant Difference
Significant Difference

33. Leg and Wing Length in ßFTZ-F1 Mutants can be Rescued by a Drop in Pressure

34. 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.
RULED OUT

35. 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 the Prepupal-Pupal Transition.
The ßFTZ-F1 mutants exhibit severe defects in muscle contractions.

36. Conclusions: Morphogenesis
ßFTZ-F1 directs the muscle movements that generate internal pressure (at the appropriate time), which drives extention of legs and wings, and eversion of the heads.
We are attempting to determine which ßFTZ-F1 target genes are involved in these processes.

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

39. ßFTZ-F1 Mutants Fail to Activate E93 in the Larval Salivary glands
control
ßFTZ-F1 mutant
E93
rp49
E93
rp49

40. ßFTZ-F1 Mutants Fail to Destroy Larval Salivary Glands
Normal salivary gland histolysis

41. ßFTZ-F1 Mutants are Defective in DNA Fragmentation

43. Ectopic Expression of ßFTZ-F1 Induces Premature Activation of E93w
w;P[F-F1]

44. Ectopic Expression of ßFTZ-F1 Induces Premature Cell Death

45. Ectopic Expression of ßFTZ-F1 Activates Cell Death Genes
(an Apaf-1 homolog)
(a Caspase)
LOADING CONTROL

46. Induction of Cell Death by ßFTZ-F1 Requires E93

47. Activation of Cell Death Genes by ßFTZ-F1 Requires E93

48. Conclusions: Programmed Cell Death
ßFTZ-F1 enables ecdysone to activate E93 in cells (such as those in the larval salivary gland) that are to be destroyed by programmed cell death.
E93 then activates other genes that direct programmed cell death.
Thus, the right cells are destroyed at the right time.

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

50. Acknowledgments Mount Holyoke College University of Utah
Leg Morphogenesis
Tina M. Fortier**
Priya Vasa
Paejonette Jacobs
E93 and Programmed Cell Death
Samara Brown**
Zareen Gauhar
Michael Chapman
Biology 340 Classes
Mutagenesis of ßFTZ-F1
Jennifer R. McCabe
Lynn L’Archeveque
Margaret Lobo
Emily McNutt
ßFTZ-F1 Gene Structure
Dana Cruz
Tetyanya Obukhanych
Petra Scamborova
University of Utah
Carl Thummel
Julie Broadus
Bart Endrizzi
University of Maryland
Eric Baehrecke
Cheng Yu Lee
Special Thanks for Technical Assistance
George Cobb
Rachel Fink
Janice Gifford
Tamara Hjermstad
Diane Kelly
This research was funded by the National Science Foundation
Mechanism of ßFTZ-F1 Action
Diyya Mathur
Genome-Wide Functions of ßFTZ-F1
Katie McMenimen
Vidya Anegundi
Rhiana Menen
Other
Cindy Chang
Jacque Miller

51. 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

53. Induction of cell death by ßFTZ-F1 requires E93

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

55. Salivary glands control tissue mutant tissue E93 rp49 E93 rp49

56. Gut tissue control tissue mutant tissue E93 E93 rp49 rp49

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

58. 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

59. Normal Leg Development

62. 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

63. 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

64. Third Instar Larva
Leg Disc Eversion
Adult

65. 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

66. Gut tissue control tissue mutant tissue E93 rp49 E93 rp49

67. Gut tissue control tissue mutant tissue E93 rp49 E93 rp49

68. Gut tissue
control tissue
mutant tissue

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

75. Gut tissue control tissue mutant tissue E93 E93 rp49 rp49

79. Third Instar Larva
Leg Disc Eversion
Adult

80. 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

81. Gut tissue control tissue mutant tissue E93 rp49 E93 rp49

82. Gut tissue control tissue mutant tissue E93 rp49 E93 rp49

83. Gut tissue
control tissue
mutant tissue

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

87. 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

88. Fig C. ECR Expression in Tissues

89. THE CHEMICAL STRUCTURE OF ECDYSONE

90. Ecdysone Timeline in Drosophila melanogaster

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

92. 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.

93. EXPERIMENTAL DESIGN
Transformant Flies called P[F-F1] were used that express a
high level of ßFTZ-F1 mRNA upon heat shock.
Control w1118 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.

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

96. Induction of cell death by ßFTZ-F1 requires E93

99. 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)

100. Leg and Wing Length in ßFTZ-F1 Mutants can be Rescued by a Drop in Pressure

102. 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: ]
Ectopic expression of ßFTZ-F1 provides E93 with the competence to respond to the late larval ecdysone pulse. [Woodard et al. (1994) Cell 79: ]
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: ]
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: ]
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: ]

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

104. Induction of Cell Death by ßFTZ-F1 Requires E93