Hypothesis
description
Transcript of Hypothesis
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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
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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: 3004-3008]
• Ectopic expression of ßFTZ-F1 provides E93 with the competence to respond to the late larval ecdysone pulse. [Woodard et al. (1994) Cell 79:
607-615]
• 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 [E. Baehrecke, unpublished].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: 607-615]
• 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: 143-149]
• 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: 143-149]
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The ex17 mutation results in pupal lethality and defects in
morphogenesis
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Mutations in ßFTZ-F1 disrupt leg morphogenesis
Control ßFTZ-F1 Mutant
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Cell Shape Changes During Leg Disc Elongation
Courtesy of Condic et al. 1991. Development 111:23-33
a b
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Comparative Leg Development
Control
ßFTZ-F1 Mutant
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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.
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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)
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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
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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
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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 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.
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FUTURE DIRECTIONSLegs, etc.- Attempt to rescue ßFTZ-F1-mutant defects by
ectopic expression of target genes.
Other Projects- Examine 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**
• 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
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Levels of early gene transcripts are reduced in ßFTZ-F1 mutant
prepupae
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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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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
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SG gut fat CNS SG
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
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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
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Normal Leg Development
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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
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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
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Third Instar Larva
Leg Disc Eversion
Adult
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Larval and Pupal Stages of Drosophila Development
A B C D E F
A. First instar larvaB. Second instar larvaC. Third instar larvaE. PrepupaF. Early pupa
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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
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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
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Gut tissue
mutant tissuecontrol tissue
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SG gut fat CNS SG
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
hsßF
TZ
-F
1C
ontrol
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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
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Third Instar Larva
Leg Disc Eversion
Adult
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Larval and Pupal Stages of Drosophila Development
A B C D E F
A. First instar larvaB. Second instar larvaC. Third instar larvaE. PrepupaF. Early pupa
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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
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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
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Gut tissue
mutant tissuecontrol tissue
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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
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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
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Fig C. ECR Expression in Tissues
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THE CHEMICAL STRUCTURE OF ECDYSONE
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Ecdysone Timeline in Drosophila melanogaster
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IN WHICH OTHER TISSUES DOES THE EXPRESSION OF ßFTZ-F1
AFFECT THE ECDYSONE INDUCTION OF BR-C, E74A, E75A AND E93
TRANSCRIPTION?
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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.
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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.
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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?