The Taiman transcriptional coactivator engages Toll signals to promote apoptosis and inter-tissue invasion in Drosophila 公开

Byun, Phil K. (Fall 2018)

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Tissue morphogenesis and remodeling is a tightly choreographed phenomenon fundamental to the development of multicellular organisms. Among the numerous developmental cues that can guide morphogenesis, steroid hormones stand out as distinct from local morphogen gradients for their ability to cause organism-wide transcriptional changes in response to systemic hormonal pulses. In Drosophila, the steroid hormone ecdysone (Ec) controls a number of tissue morphogenic events such as fusion of the thoracic discs into an intact dorsal thorax, activation and movement of hemocytes and immune cells, and overall cell growth. Ec exerts these effects by binding to its cognate receptor, the Ec receptor (EcR). Activation of EcR homologs in humans, such as the estrogen and androgen receptors, is often associated with invasive cancers. We have discovered that ectopic expression of an EcR co-activator called taiman (tai) can transform a normal wing epithelial to invade and penetrate the neighboring thorax. This unique and novel phenotype can be modified using alleles of known genetic interactors of tai such as EcR, yorkie (yki; the nuclear effector of the Hippo pathway), pvf2 and pvf3 (PDGF/VEGF related proteins 2 and 3). To ascertain a more complete landscape of the transcriptional changes induced by tai expression in invasive wing cells, we performed two different screens: (1) a genetic suppressor screen using genetic deficiencies (deletions) that tile across the entire Drosophila 2nd chromosome, (2) and an RNA-sequencing (RNA-seq) analysis of transcripts altered in Tai-expressing pupal wing cells relative to control wings cells. These parallel screens revealed that the Toll and immune deficient (IMD) pathways, which control expression of innate immunity and apoptotic genes, are active in Tai-expressing cells. Each of these pathways is activated by binding of ligand to cell-surface receptors: the Toll family of receptors is bound by secreted, processed Spätzle (Spz) ligands, and transmembrane peptidoglycan recognition proteins (PGRPs) serve as IMD receptors. We find that Tai expression in wing cells elicits a systemic Toll/IMD response in the absence of a pathogen, a phenomenon referred to as “sterile inflammation” that is often associated with locally invasive Drosophila tumors. Based upon published work linking Toll and IMD pathways to competitive killing of neighboring cells by faster growing “super-competitors” that overexpress dMyc, we posited that Tai-expressing wing cells express immune ligands, specifically the Spz proteins, and “kill” their way through the thoracic epidermis and into underlying tissue by activating Toll in these cells. Consistent with this hypothesis, a Toll/IMD reporter is activated in thoracic cells adjacent to invasive Tai-expressing wing cells and this correlates spatially with elevated apoptosis. Moreover, loss of function alleles of factors that act downstream of the Toll receptor dominantly suppress Tai-driven wing invasion. Intriguingly, a strong loss-of-function allele of the IMD pathway inhibitor caspar (casp) dominantly suppressed Tai invasion, implying that elevated IMD activity can prevent invasion. Upon further investigation, I found that Tai-expression hyper-sensitizes wing cells to casp dosage, and that casp heterozygosity causes Tai-expressing cells to undergo apoptosis, which in turn prevents them from invading thoracic tissue. These data led to my model that Tai-expressing cells elevate expression of Spz proteins, which kill neighboring cells, and Casp, which protects Tai cells from Spz-mediated death. When Casp is reduced, Tai-expressing cells succumb to Toll-driven cell death. In summary, these studies show a novel inter-tissue invasion model driven by an EcR co-activator Taiman that non-autonomously induces Toll-mediated killing of neighboring cells but the differing threshold for IMD activation protects Tai-expressing cells from apoptotic fate. Similar mechanism of local invasion is seen in human cancers where pro-inflammatory signals have been linked to invasive behavior of cancer cells, including breast and prostate. In the future, this novel aspect of Tai function may provide insight into immune-based interactions that contribute to the competitive advantage of human tumors overexpressing Tai homologs.

Table of Contents

Table of Contents

Chapter 1: An Introduction                                                                                   1

Cellular movement and tissue invasion as a Biological Question                1

Features of Drosophila melanogaster                                                           5

Border cell migration and cancer metastasis                                                6

Taiman and Ecdysone Receptor                                                                    9

Tissue Fusion                                                                                                 12

The Hippo pathway                                                                                       13

Apoptosis and caspases in Drosophila                                                         16

Innate immunity in Drosophila                                                                     18

Sterile Inflammation                                                                                     22

Chapter 2:    The Taiman transcriptional coactivator engages Toll

signals to promote apoptosis and inter-tissue invasion in Drosophila                 24       

Introduction                                                                                                   25

Results                                                                                                           28

Discussion                                                                                                     61

Material and Methods                                                                                   67

Chapter 3: A deficiency screen for uncovering dominant modifiers of

Tai-driven wing invasion                                                                                 71      

Introduction                                                                                                   72

Results                                                                                                           74

Discussion                                                                                                     82

Material and Methods                                                                                   83

Chapter 4: Requirement of steroid hormone production                                   84

in the wing epithelium for proper growth                                                        

Introduction                                                                                                   85

Results                                                                                                           89

Discussion                                                                                                     98

Material and Methods                                                                                   100           

Chapter 5: Future directions & concluding remarks                                         101

           Additional deficiency screens                                                                       102

           Reactive oxygen species                                                                               104

           Autonomous vs. Non-autonomous                                                                106

           Additional binding partners of Tai in the nucleus                                         107

           Role of shd and local steroid level in wound regeneration                           109

           Concluding remarks                                                                                      112

Reference                                                                                                                  113

Appendix                                                                                                                  126

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