Steroid Hormonal Control of Development in Drosophila

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Steroid Hormonal Control of Development in
Drosophila

• Craig T. Woodard
• Mount Holyoke College

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20-hydroxyecdysone
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Drosophila Life Cycle
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How can a single steroid hormone elicit different
responses at different times in development?
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Drosophila Life Cycle
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• Puffs
• Early
• 2B5
• 74EF
• 75B
• Prepupal early
• 93F
• Mid prepupal
• 75CD

• Genes
• Early
• BR-C
• E74
• E75
• Prepupal early
• E93
• Mid prepupal
• ßFTZ-F1

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

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Hours relative to puparium formation
BR-C E74A E75A E93 ßFTZ-F1
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EXPERIMENTAL DESIGN

• Transformant Flies called PF-F1 were used that
  express a
• high level of ßFTZ-F1 protein upon heat shock.
• Control w1118 and transformant wPF-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.

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w wPF-F1
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Hours relative to puparium formation
BR-C E74A E75A E93 ßFTZ-F1
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EXPERIMENTAL DESIGN

• Transformant Flies called PF-F1 were used that
  express a
• high level of ßFTZ-F1 protein upon heat shock.
• Control w1118 and transformant wPF-F1
  mid-third instar
• larvae were heat shocked for 30 min. and the
  salivary glands
• were immediately dissected in oxygenated
  Robbs 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.

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ex17 is a Mutation in ßFTZ-F1
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Expression of wild-type ßFTZ-F1 from a transgene
rescues ex17 mutants
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Levels of early gene transcripts are reduced in
ßFTZ-F1 mutant prepupae
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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
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ßFTZ-F1 mutants fail to histolyze larval salivary
glands

• Normal salivary gland histolysis

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Results of ßFTZ-F1 mutations

• head eversion
• leg elongation
• wing extension

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Mutations in ßFTZ-F1 disrupt leg morphogenesis
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Cell Shape Changes During Leg Disc Elongation
a
b
Courtesy of Condic et al. 1991. Development
11123-33
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Normal Leg Development
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Comparative Leg Development
Control
ßFTZ-F1 Mutant
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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.

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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)
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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.
• RULED OUT

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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.
• RULED OUT

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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.

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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.

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Ecdysone, ßFTZ-F1, E93 and Programmed Cell
Death(Tissue-Specificity)
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ßFTZ-F1 is 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
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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) ?
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ßFTZ-F1 is not required for E93 transcription in
larval gut tissue
mutant tissue
control 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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IN WHICH TISSUES DOES THE EXPRESSION OF
ßFTZ-F1 AFFECT THE ECDYSONE INDUCTION OF BR-C,
E74A, E75A AND E93 TRANSCRIPTION?
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EXPERIMENTAL DESIGN

• Transformant Flies called PF-F1 were used that
  express a
• high level of ßFTZ-F1 protein upon heat shock.
• Control w1118 and transformant wPF-F1
  mid-third instar
• larvae were heat shocked for 30 min. and the
  various tissues
• were immediately dissected in oxygenated
  Robbs 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.

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RESULTS

• Northern hybridization results show that the
  induction of E93 by
• ßFTZ-F1 expression differs from tissue to tissue
  in mid-third instar larvae.

Induction of E93 by ßFTZ-F1in late-third
instar larvae
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FUTURE DIRECTIONS

• Legs, etc.
• - Attempt to rescue ßFTZ-F1-mutant defects by
  ectopic expression of target genes.
• Other Projects
• - Continue examining 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.

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Acknowledgements

• Mount Holyoke College
• Tina M. Fortier
• Samara Brown
• Zareen Gauhar
• Dana Cruz
• Michael Chapman
• Jennifer R. McCabe
• Priya Vasa
• Lynn LArcheveque
• Margaret Lobo
• Emily McNutt
• Tetyanya Obukhanych
• Petra Scamborova
• Diyya Mathur
• Biology 340 Class!

• University of Utah
• Carl Thummel
• Eric Baehrecke
• Julie Broadus
• Bart Endrizzi
• Special Thanks for Technical Assistance
• Rachel Fink
• Diane Kelly

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ßFTZ-F1 mutants fail to histolyze larval salivary
glands
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ßFTZ-F1 mutants exhibit pupal lethality and
defects in morphogenesis
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Ecdysone concentrations
ßFTZ-F1 rp49
Normalized RNA level
Ecdysone concentrations
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Salivary Gland Developmental Northern Analysis
Hours relative to puparium formation
Edysone BR-C E74A E75A E93 ßFTZ-F1