Identification and Characterization of Novel Regulators of Salvador-Warts-Hippo Signaling in Drosophila Público

Robinson, Brian (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/cj82k786j?locale=pt-BR
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Abstract

Patterned growth is fundamental to proper metazoan development, and deregulated growth is implicated in human diseases including cancer. We use the fruit fly Drosophila melanogastor as a model system to identify growth regulatory mechanisms in developing epithelia. In an effort to understand how defects in apicobasal polarity of epithelial cells influences cellular proliferation, we performed a genetic screen against an overgrowth phenotype elicited by the transmembrane protein Crumbs (Crb), a protein known for its role in apicobasal polarity formation. Interestingly, we find that Crb-driven wing overgrowth is sensitive to the genetic dose of the Salvador-Warts-Hippo (SWH)-pathway, a conserved growth regulatory network that function across metazoans to control organ size. We show that in Drosophila epithelia, Crb controls the levels and localization of the cytoskeleton-associated FERM-domain protein Expanded (Ex), a protein that regulates SWH-activity in cells. Moreover, we show that Crb integrates these effects through a domain that is distinct from those required apicobasal polarity regulation, allowing for Crb to integrate junctional polarity signals with a growth signals. In an effort to define whether SWH-activation also exists in mutants that are defective in Crb turnover and hyper-accumulate Crb, we analyzed SWH-activity in mutants on the endolysosomal pathway. We find that, like those tissues in which Crb is overexpressed, endolysosomal mutants display ectopic activation of Yki in cells and altered levels of Ex. In the final part of my thesis work I have focused on analyzing the role of taiman (tai) in regulating SWH-activity. taiman is the Drosophila ortholog of human AIB1 (Amplified In Breast cancer-1), which potentiates nuclear steroid signaling and is amplified in several human cancers. We identified tai as a genetic modifier of Crb-driven overgrowth and find that like Crb, Tai can activate SWH-signaling in cells. Moreover, we show that Tai and Yorkie (Yki), the transcriptional effector of the SWH-pathway, shows a mutual reliance on each other in their effects on cells: Tai cannot fully activate SWH-signaling without Yki, while reciprocally, Yki cannot drive overgrowth in the absence of Tai. In summary, these studies highlight three novel inputs into SWH-signaling in Drosophila and further delineate factors and mechanisms that control SWH signaling in cells.

Table of Contents

Table of Contents
Chapter 1: An Introduction .................................................................................................. 1

Growth Control as a Biological Question....................................................................................................2
Drosophila as a model organism to understand organ size--â€control ...............................................4
Features of Drosophila ..................................................................................................................................4
Genetic Manipulation in Drosophila ........................................................................................................5
GAL4--â€UAS system.......................................................................................................................................5
FLP--â€FRT system ..........................................................................................................................................6
Genetic Analysis in Drosophila...................................................................................................................7
Epithelia in Drosophila ............................................................................................................................... 11
Polarity Control in Drosophila Imaginal Disc Epithelia................................................................ 13
The 'apical membrane determinant' crumbs............................................................................... 15
Growth Control in Drosophila....................................................................................................................... 21
Cell Division .................................................................................................................................................... 22
Cell Growth...................................................................................................................................................... 24
Cell Death (Apoptosis) ............................................................................................................................... 25
Pathways implicated in growth control in Drosophila ...................................................................... 29
The MAP Kinase Pathways ....................................................................................................................... 29
The Salvador--â€Warts--â€Hippo (SWH) Pathway .................................................................................... 32
The JAK--â€STAT Pathway.............................................................................................................................. 34
The Notch Pathway...................................................................................................................................... 35
The Ecdysone Pathway .............................................................................................................................. 37
Neoplastic growth in Drosophila............................................................................................................ 38
Scope and Significance..................................................................................................................................... 46
Chapter 2: Dominant--Modifiers of Crumbs--driven Overgrowth ...........................47
Introduction ......................................................................................................................................................... 48
Results .................................................................................................................................................................... 50
Overexpression of crbi drives overgrowth of the Drosophila wing......................................... 50
A candidate--â€based approach reveals dominant modifiers of crbi--â€driven overgrowth .. 53
A discovery--â€based screen reveals genetic loci that reproducibly modify crbi--â€driven
overgrowth...................................................................................................................................................... 57
Discussion ............................................................................................................................................................. 61
Experimental Procedures............................................................................................................................... 65
Chapter 3: The apical membrane determinant Crumbs acts via the FERM--
domain protein Expanded to control SWH signaling in Drosophila .....................67
Introduction ......................................................................................................................................................... 68
Results .................................................................................................................................................................... 70
The intracellular domain of crb requires yki to drive tissue growth ..................................... 70
crbi expression elevates Yki activity in developing epithelia .................................................... 73
crbi downregulates Ex protein levels in wing disc cells............................................................... 77
The juxtamembrane domain of Crb regulates Ex in vivo............................................................. 86
Effects of crb loss on Ex protein and Yki activity............................................................................ 93
Modification by Crb--â€interacting factors ............................................................................................. 98
Effect of crb loss on Yki activity .......................................................................................................... 101
Discussion .......................................................................................................................................................... 104
Experimental Procedures............................................................................................................................ 108
Chapter 4: Blockade of the Endolysosomal Pathway Affects SWH--signaling via
the c--Jun N--Terminal MAP Kinase Pathway in Drosophila ................................... 110
Introduction ...................................................................................................................................................... 111
Results ................................................................................................................................................................. 114
ept mutant cells display altered yki--â€activity .................................................................................. 114
Loss of both early and late endocytic nTSG elevates yki--â€activity in vivo .......................... 117
Endocytic neoplastic tumor suppressor genes require yki to overgrow .......................... 121
Endocytic neoplastic tumor suppressor genes elevate JNK activity in vivo..................... 123
Discussion .......................................................................................................................................................... 128
Experimental Procedures............................................................................................................................ 131

Chapter 5: The Nuclear Receptor Co--Activator Taiman promotes a Yorkie--
dependent growth program in Drosophila ................................................................ 132
Introduction ...................................................................................................................................................... 133
Results ................................................................................................................................................................. 135
taiman regulates imaginal disc growth............................................................................................ 135
Excess tai elevates yki--â€activity............................................................................................................. 142
Loss of tai reduces yki--â€activity ............................................................................................................ 145
yki is required for tai to elevate ex--lacZ and drive imaginal discs growth ....................... 147
tai growth phenotypes occur independently of the β--â€HLH and ecdysone signaling .... 149
tai functions genetically downstream of core pathway components to control organ
size ................................................................................................................................................................... 154
Discussion .......................................................................................................................................................... 158
Experimental Procedures............................................................................................................................ 161
Chapter 6: Future Directions .......................................................................................... 163
Future Directions............................................................................................................................................ 164
The Crbi Dominant--â€Modifier Screen.................................................................................................. 166
The Crb--â€Ex Regulatory Network......................................................................................................... 169
Endocytic Regulation of SWH--â€signaling .......................................................................................... 172
Taiman ........................................................................................................................................................... 175
Concluding Remarks...................................................................................................................................... 178

References ............................................................................................................................ 181

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