Transcript A. ßFTZ-F1 - Mount Holyoke College

Control of Animal Development
by Steroid Hormones
Craig T. Woodard
Mount Holyoke College
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
20-hydroxyecdysone
Drosophila Life Cycle
Ecdysone Timeline in Drosophila melanogaster
Pupariation
(Entry into Metamorphosis)
Prepupal-Pupal Transition
Destruction of Larval Body Parts
by Programmed Cell Death
Formation of Adult Body Parts by
Morphogenesis
How can a single steroid hormone
elicit different responses at
different times in development?
Ecdysone Timeline in Drosophila melanogaster
Pupariation
(Entry into Metamorphosis)
Prepupal-Pupal Transition
Destruction of Larval Body Parts
by Programmed Cell Death
Formation of Adult Body Parts by
Morphogenesis
How can a single steroid hormone
elicit different responses in
different parts of the body during
development?
Ecdysone Timeline in Drosophila melanogaster
Pupariation
(Entry into Metamorphosis)
Prepupal-Pupal Transition
Destruction of Larval Body Parts
by Programmed Cell Death
Formation of Adult Body Parts by
Morphogenesis
Pupariation
(Entry into
Metamorphosis)
Morphogenesis
of Adult Body Parts
Destruction of
Larval body Parts
by Programmed
Cell Death
Hypothesis
A. ßFTZ-F1 provides the early genes, the BR-C, E74A and
E75A with the competence* to be reinduced by the
prepupal ecdysone pulse.
1) These early genes then direct morphogenesis of adult
body parts.
B. ßFTZ-F1 provides the prepupal stage-specific E93 early
gene with the competence* to be induced by ecdysone.
ßFTZ-F1 thus directs the stage-specificity of the E93
response to ecdysone
1) E93 then directs programmed cell death in larval
body parts.
*Competence the ability to respond to an inductive signal
Pupariation
(Entry into
Metamorphosis)
Morphogenesis
of Adult Body Parts
Destruction of
Larval body Parts
by Programmed
Cell Death
Hypothesis
A. ßFTZ-F1 provides the early genes, the BR-C, E74A and
E75A with the competence* to be reinduced by the
prepupal ecdysone pulse.
1) These early genes then direct morphogenesis of adult
body parts.
B. ßFTZ-F1 provides the prepupal stage-specific E93 early
gene with the competence* to be induced by ecdysone.
ßFTZ-F1 thus directs the stage-specificity of the E93
response to ecdysone
1) E93 then directs programmed cell death in larval
body parts.
*Competence the ability to respond to an inductive signal
Pupariation
(Entry into
Metamorphosis)
Morphogenesis
of Adult Body Parts
Destruction of
Larval body Parts
by Programmed
Cell Death
Evidence in Support of our Hypothesis
•
ßFTZ-F1 protein binds to the
E93, E74A, E75A, and BR-C
genes.
•
Ectopic (over) expression of
ßFTZ-F1 in transgenic larvae
provides E93 with the
competence to respond
(prematurely) to the late larval
ecdysone pulse.
•
Induction of BR-C, E74A and
E75A transcripts by ecdysone is
enhanced significantly by ectopic
expression of ßFTZ-F1.
•
A Loss-of-function mutation in
ßFTZ-F1 results in dramatic
reductions in E93, E74A, E75A,
and BR-C activation at the end of
the prepupal stage.
•
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.
Levels of Early Gene Transcripts are
Reduced in ßFTZ-F1 Mutant
Prepupae
ßFTZ-F1 Mutants Exhibit Pupal
Lethality and Defects in
Morphogenesis
• head eversion
• leg elongation
• wing extension
Mutations in ßFTZ-F1 Disrupt
Leg Morphogenesis
Control
ßFTZ-F1 Mutant
Third Instar Larva
Leg Disc Eversion
Adult
Cell Shape Changes During Leg Disc
Elongation
a
b
Courtesy of Condic et al. 1991. Development 111:23-33
Comparative Leg Development
Control
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
ßFTZ-F1 Mutant
Possible Causes of Short Legs
1) Contraction of the muscles is too weak in
ßFTZ-F1 mutants.
2) There is something wrong with the leg imaginal
discs in ßFTZ-F1 mutants, which prevents
them from extending.
Leg and Wing Length in ßFTZ-F1
Mutants can be Rescued by a Drop
in Pressure
Table 2. Rescue of L eg and Wing Elonga tion in ßFTZ-F1 Mutants by a Drop in Pressure
Control Untreated
41
Mean
Length
8.89
0.44
Std. Error of the
Mean
0.007
Control Treated
28
9.00
0.65
0.123
Mutant Untreated
27
5.31
0.74
0.144
Mutant Treated
32
6.37
1.65
0.292
N
Mean
Length
Std. Deviation
Std. Error of the
Mean
Control Untreated
41
7.74
0.43
0.007
Control Treated
28
7.76
0.42
0.008
Mutant Untreated
27
5.17
0.44
0.008
Mutant Treated
32
5.97
1.26
0.223
LEGS
WINGS
N
Std. Deviation
Untreated vs. Trea ted
Sig. (2-tailed t-test)
0.819
0.002
Significant Difference
Untreated vs. Trea ted
Sig. (2-tailed t-test)
0.441
0.002
“Un treated” anim als were observed at ambient atmosphe ric temperature .
“Treated” anim als were subjected to reduced pressure, as described in Materials and Method s.
For an exp lana tion o f how leg and wing leng th were recorded, see Materials and Methods .
Significant Difference
Leg and Wing Length in ßFTZ-F1
Mutants can be Rescued by a Drop in
Pressure
Control Untreated vs. Treated
75
Untreated
Treated
50
25
75
25
5
10
10
.5
9
9.
5
8
8.
5
7
7.
5
6
75
Untreated
Treated
50
25
9.
9
8
5
8.
7
5
7.
5
6
6.
5
5
5
10
10
.5
Denticle Belt Reached by Legs
5.
9
5
10
10
.5
9.
8
5
8.
7
5
7.
5
6.
5.
6
0
5
0
Percent of Animals
Untreated
Treated
25
Mutant Untreated vs. Treated
100
75
50
6.
5.
Denticle Belt Reached by Wings
Mutant Untreated vs. Treated
100
5
5
5
10
10
.5
9
9.
5
8
8.
5
7
7.
5
6
6.
5
Denticle Belt Reached by Legs
5
Untreated
Treated
50
0
5.
5
0
Percent of Animals
Control Untreated vs. Treated
100
Percent of Animals
Percent of Animals
100
Denticle Belt Reached by Wings
Possible Causes of Short Legs
1) Contraction of the muscles is too weak in
ßFTZ-F1 mutants.
--------------------------------------------------------------2) There is something wrong with the leg imaginal
discs in ßFTZ-F1 mutants, which prevents them
from extending.
RULED OUT
Possible Causes of Short Legs
1) Contraction of the muscles is too weak in
ßFTZ-F1 mutants.
This is supported by our careful observations of
control and ßFTZ-F1 mutant animals going
through the Prepupal-Pupal Transition.
The ßFTZ-F1 mutants exhibit severe defects
in muscle contractions.
Conclusions: Morphogenesis
ßFTZ-F1 directs the muscle movements that
generate internal pressure (at the appropriate
time), which drives extention of legs and wings,
and eversion of the heads.
We are attempting to determine which ßFTZ-F1
target genes are involved in these processes.
Pupariation
(Entry into
Metamorphosis)
Morphogenesis
of Adult Body Parts
Target Genes?
Destruction of
Larval body Parts
by Programmed
Cell Death
ßFTZ-F1 Mutants Fail to Activate
E93 in the Larval Salivary glands
control
ßFTZ-F1 mutant
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 Destroy
Larval Salivary Glands
• Normal salivary gland histolysis
ßFTZ-F1
Mutant
ßFTZ-F1 Mutants are Defective
in DNA Fragmentation
Mutant
Ectopic Expression of
ßFTZ-F1 Induces Premature Activation of E93
w
w;P[F-F1]
Ectopic Expression of
ßFTZ-F1 Induces Premature Cell
Death
Ectopic Expression of ßFTZ-F1
Activates Cell Death Genes
(an Apaf-1 homolog)
(a Caspase)
LOADING CONTROL
Induction of Cell Death by
ßFTZ-F1 Requires E93
Activation of Cell Death Genes by
ßFTZ-F1 Requires E93
Conclusions: Programmed Cell
Death
ßFTZ-F1 enables ecdysone to activate E93 in cells
(such as those in the larval salivary gland) that
are to be destroyed by programmed cell death.
E93 then activates other genes that direct
programmed cell death.
Thus, the right cells are destroyed at the right time.
Pupariation
(Entry into
Metamorphosis)
Morphogenesis
of Adult Body Parts
Target Genes?
Cell Death Genes
Destruction of
Larval body Parts
by Programmed
Cell Death
Acknowledgments
•
Mount Holyoke College
•
•
•
•
Leg Morphogenesis
Tina M. Fortier**
Priya Vasa
Paejonette Jacobs
•
•
•
•
•
•
•
•
•
E93 and Programmed Cell
Death
Tina M. Fortier**
Samara Brown**
Zareen Gauhar
Paejonette Jacobs
Gitanjali Chimalakonda
Kimberly Bucknor
Michael Chapman
Yashaswi Shrestha
•
•
•
•
•
Mutagenesis of ßFTZ-F1
Jennifer R. McCabe
Lynn L’Archeveque
Margaret Lobo
Emily McNutt
•
•
•
ßFTZ-F1 Gene Structure
Dana Cruz
Tetyanya Obukhanych
•Mechanism of ßFTZ-F1 Action
•Diyya Mathur
•
University of Utah
•
•
•
Carl Thummel
Julie Broadus
Bart Endrizzi
•Genome-Wide Functions of ßFTZ-F1
•Katie McMenimen
•
•Vidya Anegundi
•
•Rhiana Menen
•
•Other
•Cindy Chang
•Jacque Miller
University of Maryland
Eric Baehrecke
Cheng Yu Lee
•
•
•
•
Special Thanks for Technical Assistance
Rachel Fink
Diane Kelly
Tamara Hjermstad
•
This research was funded by the
National Science Foundation
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
Induction of cell death by
ßFTZ-F1 requires E93
Levels of early gene transcripts are
reduced in ßFTZ-F1 mutant
prepupae
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
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
hsßFTZ-F1
Control
hsßFTZ-F1
Control
hsßFTZ-F1
Control
hsßFTZ-F1
Control
hsßFTZ-F1
Control
SG
SG gut fat CNS
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
Normal Leg Development
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
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
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
Third Instar Larva
Leg Disc Eversion
Adult
Larval and Pupal Stages of
Drosophila Development
A B
C
D
E
A. First instar larva
B. Second instar larva
C. Third instar larva
E. Prepupa
F. Early pupa
F
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
Gut tissue
control tissue
E93
mutant tissue
E93
rp49
rp49
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14
Gut tissue
control tissue
mutant tissue
hsßFTZ-F1
Control
hsßFTZ-F1
Control
hsßFTZ-F1
Control
hsßFTZ-F1
Control
hsßFTZ-F1
Control
SG
SG gut fat CNS
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
Third Instar Larva
Leg Disc Eversion
Adult
Larval and Pupal Stages of
Drosophila Development
A B
C
D
E
A. First instar larva
B. Second instar larva
C. Third instar larva
E. Prepupa
F. Early pupa
F
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
Gut tissue
control tissue
E93
mutant tissue
E93
rp49
rp49
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14
Gut tissue
control tissue
mutant tissue
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
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
Fig C. ECR Expression in Tissues
THE CHEMICAL STRUCTURE OF ECDYSONE
Ecdysone Timeline in Drosophila melanogaster
IN WHICH OTHER TISSUES DOES
THE EXPRESSION OF ßFTZ-F1
AFFECT THE ECDYSONE INDUCTION
OF BR-C, E74A, E75A AND E93
TRANSCRIPTION?
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.
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.
How can a single steroid hormone
elicit different responses at
different times in development?
Induction of cell death by
ßFTZ-F1 requires E93
Control Untreated vs. Treated
75
Untreated
Treated
50
25
75
25
9
9.
5
10
10
.5
8
8.
5
75
Untreated
Treated
50
25
9.
9
8
5
8.
7
5
7.
5
6
6.
5
5
5
10
10
.5
Denticle Belt Reached by Legs
5.
9
5
10
10
.5
9.
8
5
8.
7
5
7.
5
6.
5.
6
0
5
0
Percent of Animals
Untreated
Treated
25
Mutant Untreated vs. Treated
100
75
50
7
7.
5
Denticle Belt Reached by Wings
Mutant Untreated vs. Treated
100
6
6.
5
5
5.
5
5
10
10
.5
9
9.
5
8
8.
5
7
7.
5
6
6.
5
Denticle Belt Reached by Legs
5
Untreated
Treated
50
0
5.
5
0
Percent of Animals
Control Untreated vs. Treated
100
Percent of Animals
Percent of Animals
100
Denticle Belt Reached by Wings
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)
Leg and Wing Length in ßFTZ-F1
Mutants can be Rescued by a Drop
in Pressure
Table 2. Rescue of L eg and Wing Elonga tion in ßFTZ-F1 Mutants by a Drop in Pressure
Control Untreated
41
Mean
Length
8.89
0.44
Std. Error of the
Mean
0.007
Control Treated
28
9.00
0.65
0.123
Mutant Untreated
27
5.31
0.74
0.144
Mutant Treated
32
6.37
1.65
0.292
N
Mean
Length
Std. Deviation
Std. Error of the
Mean
Control Untreated
41
7.74
0.43
0.007
Control Treated
28
7.76
0.42
0.008
Mutant Untreated
27
5.17
0.44
0.008
Mutant Treated
32
5.97
1.26
0.223
LEGS
WINGS
N
Std. Deviation
Untreated vs. Trea ted
Sig. (2-tailed t-test)
0.819
0.002
Untreated vs. Trea ted
Sig. (2-tailed t-test)
0.441
0.002
“Un treated” anim als were observed at ambient atmosphe ric temperature .
“Treated” anim als were subjected to reduced pressure, as described in Materials and Method s.
For an exp lana tion o f how leg and wing leng th were recorded, see Materials and Methods .
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: 143149]
ßFTZ-F1 mutants exhibit pupal
lethality and defects in
morphogenesis
Induction of Cell Death by
ßFTZ-F1 Requires E93