A systems-level interrogation identifies regulators of Drosophila blood cell number and survival.

Richelle Sopko,Katja Brückner,You Bin Lin,Brandy Alexander,Norbert Perrimon,Kalpana Makhijani,Utpal Banerjee

doi:10.1371/journal.pgen.1005056

Abstract

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

Highlights

The regulation of cell number varies greatly and typically depends on developmental and environmental stimuli that determine the intracellular balance of pro- and anti-death, and proliferative signals [1,2,3]
We use a simple Drosophila model of cell number control, which centers on PDGF/VEGF receptor signaling
Performing a genome-wide RNAi screen under PDGF/VEGF Receptor (Pvr)-sensitized conditions, we identify regulators of cell number that have not been found in conventional screens

Summary

Introduction

The regulation of cell number varies greatly and typically depends on developmental and environmental stimuli that determine the intracellular balance of pro- and anti-death, and proliferative signals [1,2,3]. Understanding the complex signaling networks that regulate cell survival is an important yet incompletely accomplished goal [4,5], which can be facilitated by studying a simple model organism. Blood cells in the fruitfly Drosophila melanogaster have been instrumental in the discovery of fundamental concepts in immunity, hematopoiesis and wound healing [6,7,8,9,10,11], but they are a convenient model to study mechanisms that regulate cell number. The Drosophila PDGF/VEGF Receptor (Pvr), a member of the Receptor Tyrosine Kinase (RTK) family, controls anti-apoptotic survival signaling in Drosophila blood cells (hemocytes) in vivo and in the embryonic cell line Kc in culture [12]. Drosophila Pvr parallels roles of the vertebrate family of PDGF/VEGF Receptors in development and disease [12,21,23,24,25,26]

Methods

Results

Discussion

Conclusion