Transcript presentationPC - Mount Holyoke College

Steroid Hormonal Control 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
Genes
Early
2B5
74EF
75B
Prepupal early
93F
Mid prepupal
75CD
Early
BR-C
E74
E75
Prepupal early
E93
Mid prepupal
ßFTZ-F1
Salivary Gland Developmental Northern Analysis
Hours relative to puparium formation
Edysone
BR-C
E74A
E75A
E93
ßFTZ-F1
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
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
E93
rp49
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
a
b
Courtesy of Condic et al. 1991. Development 111:23-33
Normal Leg Development
Comparative Leg Development
Control
ßFTZ-F1 Mutant
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.
Leg Extension in ßFTZ-F1 Mutants can
be Rescued by a Drop in Pressure
100
90
80
70
Percent
60
of animals
with normal 50
leg-length
40
30
20
10
0
control
untreated
mutant
untreated
control
treated
mutant
treated
(n = 27)
(n = 20)
(n = 11)
(n = 22)
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
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
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 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.
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
E93
rp49
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
control tissue
mutant tissue
E93
E93
rp49
rp49
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14
IN WHICH TISSUES DOES THE
EXPRESSION OF ßFTZ-F1 AFFECT
THE ECDYSONE INDUCTION OF BRC, 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
CONDITION
w1118
[-Ecd]
w;P[F-F1]
[-Ecd]
W1118
[+Ecd]
w;P[F-F1]
[+Ecd]
CNS
GUT
-+
-+
---+
IMAGINAL
DISCS
-----
FAT
-+
-+
SALIVARY
GLANDS
---+
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.
Acknowledgements
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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!
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University of Utah
Carl Thummel
Eric Baehrecke
Julie Broadus
Bart Endrizzi
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Special Thanks for Technical
Assistance
Rachel Fink
Diane Kelly
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ßFTZ-F1 mutants fail to histolyze
larval salivary glands
ßFTZ-F1 mutants exhibit pupal
lethality and defects in
morphogenesis
Ecdysone concentrations
ßFTZ-F1
rp49
Normalized
RNA level
Ecdysone concentrations
Salivary Gland Developmental Northern Analysis
Hours relative to puparium formation
Edysone
BR-C
E74A
E75A
E93
ßFTZ-F1