The Hippo pathway transcription factor Yorkie: novel regulators and invasive potential in Drosophila. Open Access
Daniel Barron (Spring 2018)
Published
Abstract
The manner in which organisms regulate the growth of their organs is a fundamental question in biology that has broad implications for our understanding of development and cancer. The fruit fly Drosophila melanogaster is a relevant and powerful model system with which to study the signal transduction pathways governing growth. The Hippo signaling pathway has been demonstrated to play a central role in the regulation of tissue and organ size during development. The molecular nature of this pathway was first uncovered in Drosophila melanogaster through genetic screens to identify regulators of cell growth and cell division. The pathway is strongly conserved in humans, rendering Drosophila a suitable and efficient model system to better understand the molecular nature of this pathway. Despite numerous studies that have described the relationships between different Hippo pathways components, a complete picture of its regulation is lacking. The LC8 family of small ~8kD proteins are highly conserved and interact with multiple protein partners in eukaryotic cells. LC8-binding modulates target protein activity, often through induced dimerization via LC8:LC8 homodimers. Although many LC8-interactors have roles in signaling cascades, LC8’s role in developing epithelia is poorly understood. Using the Drosophila wing as a developmental model, I find that the LC8 family member Cut up (Ctp) is primarily required to promote epithelial growth, which correlates with effects on the pro-growth factor dMyc and two genes, diap1 and bantam, that are classic targets of the Hippo pathway coactivator Yorkie. Genetic tests confirm that Ctp supports Yorkie-driven tissue overgrowth and indicate that Ctp acts through Yorkie to control bantam (ban) and diap1 transcription. Quite unexpectedly however, Ctp loss has inverse effects on ban and diap1: it elevates ban expression but reduces diap1 expression. Although LC8 complexes with Yap1, a Yorkie homolog, in human cells, an orthologous interaction was not detected in Drosophila cells. Collectively these findings reveal that that Drosophila Ctp is a required regulator of Yorkie-target genes in vivo and suggest that Ctp may interact with a Hippo pathway protein(s) to exert inverse transcriptional effects on Yorkie-target genes. In addition to their role in promoting hyperplastic growth, Yorkie orthologs in humans have been associated with cancer invasion and metastasis. In the final part of this dissertation, I present preliminary data indicating that Yorkie-overexpressing clones in the Drosophila wing disc extend actin-rich protrusions into neighboring tissue that are consistent with invadopodia. I also show that Yorkie overexpressing tissue have elevated levels of ECM degrading matrix metalloproteases. Together, these data suggest a new in vivo model with which to study invadopodia and indicate that Yorkie may be capable of driving a pro-invasive gene expression program.
Table of Contents
Chapter 1: Introduction………………………………………………………………... 1
Growth control as a biological question………………………………………………...….2
Drosophila melanogaster as a model organism to study the regulation of organ size.........4
Features of Drosophila………………………………………………...…..………4
Imaginal discs………………………………………………...……………..……..5
Genetic manipulation toolkit…………………………………………………...….6
GAL4-UAS………………………………………………...…………………...…6
FLP-FRT………………………………………………...………………………...7
Genetic Analysis in Drosophila………………………………………………...…9
Growth control in Drosophila………………………………………………...…...……..13
Cell growth………………………………………………...………………..……14
Cell division………………………………………………...……………………15
Cell death………………………………………………………………….......…16
The Hippo Pathway: growth control and human cancer……………………………….…17
The molecular features of the Hippo signaling pathway…………………………17
Cellular inputs that alter Hippo signaling………………………………………...21
Deregulation of Hippo pathway alters several cancer related processes………….22
Increased cellular proliferation…………………………………………...24
Inhibition of apoptosis…………………………………………………....24
Deregulation of cellular differentiation………………………………..…25
Hippo pathway signaling suppresses cancerous phenotypes in cell culture………25
Hippo pathway components contribute to tumorigenesis in mouse models………26
Molecular alterations of Hippo pathway components in human cancer…..……...27
YAP/TAZ activation is a prognostic indicator in cancer patients………..………32
Hippo pathway and non-cancer disease processes………………………………..32
Potential therapeutics targeting the Hippo pathway………………………………33
Conclusions: YAP/TAZ in Human Cancer………………………………………34
Remaining Questions and Emerging Hypotheses……………...…………………35
Chapter 2: Inverse regulation of two classic Hippo pathway target genes
in Drosophila by the dimerization hub protein Ctp……………………………………38
Introduction…………………….……………………………………...…………………39
Results……………………………………………………………………………………44
Ctp is required for imaginal-disc derived adult tissues to grow to normal size……44
Ctp loss elevates apoptosis and division of wing disc cells and reduces
their size……………………………………………………………………….....48
Ctp is dispensable for multiple signaling pathways but genetically
interacts with Yorkie…………………………..…………………………………63
Ctp loss reduces thread/diap1 transcription……………………………...………71
Ctp loss elevates transcription of the bantam microRNA locus…………..………75
Epistatic relationship between yki and ctp in control of th/diap1
and ban reporters………………………………………………………...……….78
v
Discussion…………………………………………………………..……………………85
Materials and Methods……………………………………………………………...……88
Chapter 3: Yki-overexpressing clones extend actin-rich protrusions and secrete matrix metalloproteases……………………………………………………………...…90
Introduction………………………………………………………………………91
Results……………………………………………………………………………93
Discussion………………………………………………………………………104
Materials and Methods……………………………………………….…………107
Chapter 4: Concluding remarks………..………………………………………….…108
References…………………………………………………………………………...…115
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