The archipelago growth suppressor limits apoptosis via the Rb/E2f pathway and is transcriptionally regulated by the Notch receptor Pubblico
Nicholson, Sarah Caroline (2010)
Abstract
The archipelago
growth suppressor limits apoptosis
via the Rb/E2f pathway
and is transcriptionally regulated by the Notch
receptor
By Sarah C. Nicholson
The Drosophila
archipelago ( ago) growth
suppressor encodes the substrate
specificity component of a Skp‐Cullin‐F box (SCF)
ubiquitin ligase that targets the
Cyclin E and dMyc proteins for degradation. Its human ortholog,
Fbw7, is commonly
lost in cancers, suggesting that failure to degrade ago/Fbw7
targets drives excess
tissue growth. While much is known concerning the role of the
ago gene in limiting
growth and proliferation, there remain definite gaps in our
knowledge of ago
functions in the cell. The following questions are addressed here:
(1) what is the
mechanism through which the loss of ago reduces the size of
the adult eye?, and (2)
what signaling pathways are responsible for elevating ago
expression in the
morphogenetic furrow? We find that ago is required to
restrict the apoptotic
activity of the Rb/E2f pathway adjacent to the eye‐specific
morphogenetic furrow
(MF). Thus, loss of ago results in elevated activity of
de2f1 (a transcription factor
necessary for progression into S‐phase), expression of the
pro‐death dE2F1 targets
hid and rpr, and high rates of apoptosis, ultimately
leading to a smaller adult organ.
These phenotypes are modulated by rbf1, de2f1,
hid, and the rbf1/ de2f1 regulators
cyclin
E and dacapo but are independent of
dp53. These data show that ago loss
requires a collaborating block in cell death to efficiently drive
tissue overgrowth. In
light of the apparent requirement for the Ago protein to limit cell
death at the MF,
we next set out to identify developmental signals that regulate
ago expression in
cells within the MF. Expression of ago mRNA and protein is
induced by passage of
the MF, and the Hedgehog ( Hh) and Notch (
N) pathways are required elements of
this inductive mechanism. Cells mutant for N pathway
components, or Hh defective
cells that express reduced levels of the Notch ligand Delta, fail
to upregulate ago
transcription in the region of the MF; reciprocally, ectopic N
activation in eye discs
induces expression of ago mRNA. Furthermore, we find that
the failure to
upregulate ago in N pathway mutant cells correlates
with accumulation of the SCF‐
Ago target Cyclin E in the area of the MF, and this can be rescued
by re‐expression of
ago. This N/Ago/Cyclin E link represents a significant new
cell‐cycle regulatory
mechanism in cells of the developing eye.
The archipelago growth suppressor limits apoptosis via
the Rb/E2f pathway and is transcriptionally regulated by
the Notch receptor
By
Sarah C. Nicholson
B.S. Harding University, 2006
Advisor: Kenneth H. Moberg, Ph.D.
A dissertation submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in
Graduate Division of Biological and Biomedical Science
Genetics and Molecular Biology
2010
Table of Contents
Table of Contents
Chapter 1: Introduction
........................................................................................................
1
I.A. Introduction
.................................................................................................................
2
I.B. Organ size control
......................................................................................................
5
I.C. The fly eye as a model for growth control
........................................................10
I.D. The morphogenetic
furrow...................................................................................14
I.E. Identification of novel growthregulatory genes in the
fly eye.................17
I.F.
archipelago
..................................................................................................................21
I.G. ago
patterns and functions in the eye
...............................................................27
I.H. The Hedgehog
pathway..........................................................................................29
I.I. The Notch
pathway....................................................................................................33
I.J. Objectives
.....................................................................................................................38
I.K. References
..................................................................................................................39
Chapter 2: The archipelago
Tumor Suppressor Gene
Limits Rb/E2FRegulated
Apoptosis in Developing
Drosophila
Tissues...............................................................52
II.A. Abstract
......................................................................................................................54
II.B.
Introduction..............................................................................................................56
II.C.
Results.........................................................................................................................59
II.C1. Loss of ago has different effects on the size of
tissues in the adult head. 59
II.C2. Blocking death increases ago mutant eye size
.................................................... 64
II.C3. ago mutations elevate dE2f activity in the furrow
............................................ 67
II.C4. de2f1 and rbf1 control apoptosis of
ago
cells.......................................................
72
II.C5. Relationship between ago and apoptotic
regulators........................................ 80
II.D. Discussion
.................................................................................................................88
II.E. Experimental
Procedures.....................................................................................92
II.E1. Genetics
................................................................................................................................
92
II.E2. Eye measurements
..........................................................................................................
93
II.E3. Immunohistochemistry & Microscopy
...................................................................
93
II.E4. Real Time
RT‐PCR............................................................................................................
93
II.E5. Cell culture
..........................................................................................................................
94
II.F.
Acknowledgements.................................................................................................95
II.G.
References.................................................................................................................96
Chapter 3: Notch
dependent expression of the
archipelago
ubiquitin ligase
subunit in the Drosophila
eye
........................................................................................
100
III.A.
Abstract..................................................................................................................
102
III.B. Introduction
.........................................................................................................
104
III.C. Results
....................................................................................................................
107
III.C1. ago protein levels and promoter activity are
elevated within the MF ..107
III.C2. Hedgehog and Notch pathway mutations block Ago induction at
the MF
.............................................................................................................................................................112
III.C3. The ago promoter is N‐responsive
.......................................................................120
III.C4. The induction of ago by the N pathway is
required to limited levels of the
SCF‐Ago target Cyclin
E............................................................................................................127
III.D.
Discussion.............................................................................................................
132
III.E. Experimental Procedures ................................................................................ 137
III.E1.
Genetics.............................................................................................................................137
III.E2. Immunohistochemistry and Microscopy
...........................................................138
III.E3. Real‐Time Reverse
Transcriptase‐PCR...............................................................138
III.E4. S2R+ cell manipulations
............................................................................................139
III.E5. Molecular
Cloning.........................................................................................................139
III.F.
Acknowledgements............................................................................................
140
III.G. References
............................................................................................................
141
Chapter 4: Future Experiments and Concluding
Remarks................................... 147
IV.A.
Summary................................................................................................................
148
IV.B. Screen for modifiers of ago
'small
eye'........................................................
151
IV.C. Small molecule testing in Drosophila
...........................................................
154
IV.D. Defining a N/Ago feedback loop
.....................................................................
157
IV.E. Notchdependent ago
expression patterns
in the eye............................ 160
IV.F. Ago/N studies in human
cells..........................................................................
167
IV.G. Concluding
Remarks...........................................................................................
169
IV.H. References
............................................................................................................
170
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