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Steroid Hormonal Regulation of Development in Drosophila. Craig T. Woodard Mount Holyoke College. 20-hydroxyecdysone. Drosophila Life Cycle. How can a single steroid hormone elicit different responses at different times in development?. Drosophila Life Cycle. Puffs Early 2B5 74EF 75B - PowerPoint PPT Presentation
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Steroid Hormonal Regulation of Development in Drosophila
Craig T. Woodard
Mount Holyoke College
20-hydroxyecdysone
Drosophila Life Cycle
How can a single steroid hormone How can a single steroid hormone elicit different responses at elicit different responses at
different times in development?different times in development?
Drosophila Life Cycle
Puffs
Early
2B5
74EF
75B
Prepupal early
93F
Mid prepupal
75CD
Genes
Early
BR-C
E74
E75
Prepupal early
E93
Mid prepupal
ßFTZ-F1
Edysone
BR-C
E74A
E75A
E93
ßFTZ-F1
Hours relative to puparium formation
Salivary Gland Developmental Northern Analysis
HypothesisA. 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
Hours relative to puparium formation
BR-C
E74A
E75A
E93
ßFTZ-F1
EXPERIMENTAL DESIGN
• Transformant Flies called P[F-F1] were used that express a high level of ßFTZ-F1 protein upon heat shock.
• Control w1118 and transformant w;P[F-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.
w
w;P[F-F1]
Hours relative to puparium formation
BR-C
E74A
E75A
E93
ßFTZ-F1
EXPERIMENTAL DESIGN
• Transformant Flies called P[F-F1] were used that express a high level of ßFTZ-F1 protein upon heat shock.
• Control w1118 and transformant w;P[F-F1] mid-third instar larvae were heat shocked for 30 min. and the salivary glands were immediately dissected in oxygenated Robb’s 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.
ex17 is a Mutation in ßFTZ-F1
Expression of wild-type ßFTZ-F1 from a transgene
rescues ex17 mutants
Levels of early gene transcripts are reduced in ßFTZ-F1 mutant
prepupae
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
ßFTZ-F1 mutants fail to histolyze larval salivary glands
• Normal salivary gland histolysis
Results of ßFTZ-F1 mutations
• head eversion
• leg elongation
• wing extension
Mutations in ßFTZ-F1 disrupt leg morphogenesis
Cell Shape Changes During Leg Disc Elongation
Courtesy of Condic et al. 1991. Development 111:23-33
a b
Normal Leg Development
Comparative Leg Development
Control
ßFTZ-F1 Mutant
Possible Causes of Short Legs1) 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.
0102030405060708090
100
controluntreated
mutantuntreated
controltreated
mutanttreated
Leg Extension in ßFTZ-F1 Mutants can be Rescued by a Drop in Pressure
Percent of animals
with normal leg-length
(n = 27) (n = 20) (n = 11) (n = 22)
Possible Causes of Short Legs1) 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
Possible Causes of Short Legs1) 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
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.
Testing the HypothesesHypothesis - 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.
Ecdysone, ßFTZ-F1, E93 and
Programmed Cell Death
(Tissue-Specificity)
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
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) ?
ßFTZ-F1 is not required for E93 transcription in larval gut tissue
mutant tissuecontrol tissue
E93
rp49
0 2 4 6 8 10 12 14
E93
rp49
0 2 4 6 8 10 12 14
IN WHICH TISSUES DOES THE EXPRESSION OF ßFTZ-F1 AFFECT
THE ECDYSONE INDUCTION OF BR-C, E74A, E75A AND E93
TRANSCRIPTION?
EXPERIMENTAL DESIGN
• Transformant Flies called P[F-F1] were used that express a high level of ßFTZ-F1 protein upon heat shock.
• Control w1118 and transformant w;P[F-F1] mid-third instar larvae were heat shocked for 30 min. and the various tissues were immediately dissected in oxygenated Robb’s 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.
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
CONDITIONCNS GUT IMAGINALDISCS
FAT SALIVARYGLANDS
w1118
[-Ecd] -- -- -- -- --w;P[F-F1][-Ecd] + -- -- + --W1118
[+Ecd] -- -- -- -- --w;P[F-F1][+Ecd] + + -- + +
FUTURE DIRECTIONSLegs, 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.
Acknowledgements
• Mount Holyoke College• Tina M. Fortier**• Samara Brown**• Zareen Gauhar • Dana Cruz• Michael Chapman• Jennifer R. McCabe• Priya Vasa• Lynn L’Archeveque• 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
ßFTZ-F1 mutants fail to histolyze larval salivary glands
ßFTZ-F1 mutants exhibit pupal lethality and defects in
morphogenesis
Ecdysone concentrations
ßFTZ-F1
rp49
Ecdysone concentrations
Normalized RNA level
Edysone
BR-C
E74A
E75A
E93
ßFTZ-F1
Hours relative to puparium formation
Salivary Gland Developmental Northern Analysis