Title: Steroid Hormonal Control of Development in Drosophila
1Steroid Hormonal Control of Development in
Drosophila
- Craig T. Woodard
- Mount Holyoke College
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720-hydroxyecdysone
8Drosophila Life Cycle
9How can a single steroid hormone elicit different
responses at different times in development?
10Drosophila Life Cycle
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14- Puffs
- Early
- 2B5
- 74EF
- 75B
- Prepupal early
- 93F
- Mid prepupal
- 75CD
- Genes
- Early
- BR-C
- E74
- E75
- Prepupal early
- E93
- Mid prepupal
- ßFTZ-F1
15Salivary Gland Developmental Northern Analysis
Hours relative to puparium formation
Edysone BR-C E74A E75A E93 ßFTZ-F1
16Hypothesis
- 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
17Hours relative to puparium formation
BR-C E74A E75A E93 ßFTZ-F1
18EXPERIMENTAL 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.
19w wPF-F1
20Hours relative to puparium formation
BR-C E74A E75A E93 ßFTZ-F1
21EXPERIMENTAL 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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25ex17 is a Mutation in ßFTZ-F1
26Expression of wild-type ßFTZ-F1 from a transgene
rescues ex17 mutants
27Levels of early gene transcripts are reduced in
ßFTZ-F1 mutant prepupae
28E93 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
29ßFTZ-F1 mutants fail to histolyze larval salivary
glands
- Normal salivary gland histolysis
30Results of ßFTZ-F1 mutations
- head eversion
- leg elongation
- wing extension
31Mutations in ßFTZ-F1 disrupt leg morphogenesis
32Cell Shape Changes During Leg Disc Elongation
a
b
Courtesy of Condic et al. 1991. Development
11123-33
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35Normal Leg Development
36Comparative Leg Development
Control
ßFTZ-F1 Mutant
37Possible 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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39Leg 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)
40Possible 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
41Possible 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
42Conclusions
- 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.
43Testing 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.
44Ecdysone, ßFTZ-F1, E93 and Programmed Cell
Death(Tissue-Specificity)
45ßFTZ-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
46If 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) ?
47ßFTZ-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
48IN WHICH TISSUES DOES THE EXPRESSION OF
ßFTZ-F1 AFFECT THE ECDYSONE INDUCTION OF BR-C,
E74A, E75A AND E93 TRANSCRIPTION?
49EXPERIMENTAL 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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51RESULTS
- 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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53FUTURE 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.
54Acknowledgements
- 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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56ßFTZ-F1 mutants fail to histolyze larval salivary
glands
57ßFTZ-F1 mutants exhibit pupal lethality and
defects in morphogenesis
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59Ecdysone concentrations
ßFTZ-F1 rp49
Normalized RNA level
Ecdysone concentrations
60Salivary Gland Developmental Northern Analysis
Hours relative to puparium formation
Edysone BR-C E74A E75A E93 ßFTZ-F1