Differential gene expression analysis identified determinants of cell fate plasticity during radiation-induced regeneration in Drosophila.

Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. We identified previously a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. We used here a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

Regulators of cell movement during development and regeneration in Drosophila.

Cell migration is a fundamental cell biological process essential both for normal development and for tissue regeneration after damage. Cells can migrate individually or as a collective. To better understand the genetic requirements for collective migration, we expressed RNA interference (RNAi) against 30 genes in the Drosophila embryonic salivary gland cells that are known to migrate collectively. The genes were selected based on their effect on cell and membrane morphology, cytoskeleton and cell adhesion in cell culture-based screens or in Drosophila tissues other than salivary glands. Of these, eight disrupted salivary gland migration, targeting: Rac2, Rab35 and Rab40 GTPases, MAP kinase-activated kinase-2 (MAPk-AK2), RdgA diacylglycerol kinase, Cdk9, the PDSW subunit of NADH dehydrogenase (ND-PDSW) and actin regulator Enabled (Ena). The same RNAi lines were used to determine their effect during regeneration of X-ray-damaged larval wing discs. Cells translocate during this process, but it remained unknown whether they do so by directed cell divisions, by cell migration or both. We found that RNAi targeting Rac2, MAPk-AK2 and RdgA disrupted cell translocation during wing disc regeneration, but RNAi against Ena and ND-PDSW had little effect. We conclude that, in Drosophila, cell movements in development and regeneration have common as well as distinct genetic requirements.

Roles of Membrane and Vesicular Traffic in Regulation of the Hippo Pathway.

The Hippo pathway is a well conserved signaling cascade that modulates cell proliferation and survival in response to external cues such as cell:cell contact, injury, and nutritional status. Models of the Hippo pathway have evolved from a series of genetic interactions defined in the fruit fly Drosophila melanogaster into a complex series of biochemical mechanisms in which transmembrane and cytoskeletal proteins modulate cytoplasmic phosphatase and kinase activities that converge on the serine/threonine kinase Warts (Wts) to regulate nuclear entry of the co-activator protein Yorkie (Yki; vertebrate Yap1). This pathway is well conserved in human cells and broadly implicated in cancer. Progress in understanding biochemical events within the Hippo pathway highlights a need for improved understanding of the cell biological contexts in which these molecular interactions occur. A significant body of data linking Hippo signaling to membranes and proteins involved in intracellular membrane trafficking raise the possibility that some molecular regulatory events occur on the cytoplasmic face of vesicles. In Drosophila, a Yki-vesicle link was solidified by discoveries that cytoplasmic Yki concentrates at late-endosomes and physically interacts with two endosomal adaptor proteins, Myopic (Mop) and Leash. These two proteins are required for Yki to transit the endolysosomal pathway and be turned over in lysosomes. Molecules involved in recruiting and tethering Yki along this endosomal route are not defined but are predicted to play key roles in regulating Yki levels and thus Hippo-responsiveness of cells. As Wts is recruited to the apical membrane by upstream Hippo components, endosomal internalization could also affect complexes involved in Yki phosphorylation events that alter nucleocytoplasmic shuttling. Recent work has revealed an unexpected, non-transcriptional role of membrane-associated Yki in triggering actinomyosin contractility via the myosin-regulatory light chain Spaghetti squash (Sqh). How Yki interacts with the membrane and controls Sqh is unclear, but this mechanism represents a novel regulatory mechanism based on induced localization of Yki to a specific membrane compartment. These and other data will be discussed as we review data linking Yki to membrane and vesicular traffic in development and homeostasis and speculate on missing elements of these membrane-linked Yki regulatory mechanisms.

Ionizing radiation induces stem cell-like properties in a caspase-dependent manner in Drosophila.

Cancer treatments including ionizing radiation (IR) can induce cancer stem cell-like properties in non-stem cancer cells, an outcome that can interfere with therapeutic success. Yet, we understand little about what consequences of IR induces stem cell like properties and why some cancer cells show this response but not others. In previous studies, we identified a pool of epithelial cells in Drosophila larval wing discs that display IR-induced stem cell-like properties. These cells are resistant to killing by IR and, after radiation damage, change fate and translocate to regenerate parts of the disc that suffered more cell death. Here, we report the identification of two new pools of cells with IR-induced regenerative capability. We addressed how IR exposure results in the induction of stem cell-like behavior, and found a requirement for IR-induced caspase activity and for Zfh2, a transcription factor and an effector in the JAK/STAT pathway. Unexpectedly, the requirement for caspase activity was cell-autonomous within cell populations that display regenerative behavior. We propose a model in which the requirement for caspase activity and Zfh2 can be explained by apoptotic and non-apoptotic functions of caspases in the induction of stem cell-like behavior.

STAT, Wingless, and Nurf-38 determine the accuracy of regeneration after radiation damage in Drosophila.

We report here a study of regeneration in Drosophila larval wing imaginal discs after damage by ionizing radiation. We detected faithful regeneration that restored a wing disc and abnormal regeneration that produced an extra wing disc. We describe a sequence of changes in cell number, location and fate that occur to produce an ectopic disc. We identified a group of cells that not only participate in ectopic disc formation but also recruit others to do so. STAT92E (Drosophila STAT3/5) and Nurf-38, which encodes a member of the Nucleosome Remodeling Factor complex, oppose each other in these cells to modulate the frequency of ectopic disc growth. The picture that emerges is one in which activities like STAT increase after radiation damage and fulfill essential roles in rebuilding the tissue. But such activities must be kept in check so that one and only one wing disc is regenerated.

Drosophila Wnt and STAT Define Apoptosis-Resistant Epithelial Cells for Tissue Regeneration after Irradiation.

Drosophila melanogaster larvae irradiated with doses of ionizing radiation (IR) that kill about half of the cells in larval imaginal discs still develop into viable adults. How surviving cells compensate for IR-induced cell death to produce organs of normal size and appearance remains an active area of investigation. We have identified a subpopulation of cells within the continuous epithelium of Drosophila larval wing discs that shows intrinsic resistance to IR- and drug-induced apoptosis. These cells reside in domains of high Wingless (Wg, Drosophila Wnt-1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) activity and would normally form the hinge in the adult fly. Resistance to IR-induced apoptosis requires STAT and Wg and is mediated by transcriptional repression of the pro-apoptotic gene reaper. Lineage tracing experiments show that, following irradiation, apoptosis-resistant cells lose their identity and translocate to areas of the wing disc that suffered abundant cell death. Our findings provide a new paradigm for regeneration in which it is unnecessary to invoke special damage-resistant cell types such as stem cells. Instead, differences in gene expression within a population of genetically identical epithelial cells can create a subpopulation with greater resistance, which, following damage, survive, alter their fate, and help regenerate the tissue.

Drosophila C-terminal Src kinase regulates growth via the Hippo signaling pathway.

The Hippo signaling pathway is involved in regulating tissue size by inhibiting cell proliferation and promoting apoptosis. Aberrant Hippo pathway function is often detected in human cancers and correlates with poor prognosis. The Drosophila C-terminal Src kinase (d-Csk) is a genetic modifier of warts (wts), a tumor-suppressor gene in the Hippo pathway, and interacts with the Src oncogene. Reduction in d-Csk expression and the consequent activation of Src are frequently seen in several cancers including hepatocellular and colorectal tumors. Previous studies show that d-Csk regulates cell proliferation and tissue size during development. Given the similarity in the loss-of-function phenotypes of d-Csk and wts, we have investigated the interactions of d-Csk with the Hippo pathway. Here we present multiple lines of evidence suggesting that d-Csk regulates growth via the Hippo signaling pathway. We show that loss of dCsk caused increased Yki activity, and our genetic epistasis places dCsk downstream of Dachs. Furthermore, dCsk requires Yki for its growth regulatory functions, suggesting that dCsk is another upstream member of the network of genes that interact to regulate Wts and its effector Yki in the Hippo signaling pathway.

Scribble acts in the Drosophila fat-hippo pathway to regulate warts activity.

Epithelial cells are the major cell-type for all organs in multicellular organisms. In order to achieve correct organ size, epithelial tissues need mechanisms that limit their proliferation, and protect tissues from damage caused by defective epithelial cells. Recently, the Hippo signaling pathway has emerged as a major mechanism that orchestrates epithelial development. Hippo signaling is required for cells to stop proliferation as in the absence of Hippo signaling tissues continue to proliferate and produce overgrown organs or tumors. Studies in Drosophila have led the way in providing a framework for how Hippo alters the pattern of gene transcription in target cells, leading to changes in cell proliferation, survival, and other behaviors. Scribble (Scrib) belongs to a class of neoplastic tumor suppressor genes that are required to establish apical-basal cell polarity. The disruption of apical-basal polarity leads to uncontrolled cell proliferation of epithelial cells. The interaction of apical basal polarity genes with the Hippo pathway has been an area of intense investigation. Loss of scrib has been known to affect Hippo pathway targets, however, its functions in the Hippo pathway still remain largely unknown. We investigated the interactions of Scrib with the Hippo pathway. We present data suggesting that Drosophila scrib acts downstream of the Fat (Ft) receptor, and requires Hippo signaling for its growth regulatory functions. We show that Ft requires Scrib to interact with Expanded (Ex) and Dachs (D), and for regulating Warts (Wts) levels and stability, thus placing Scrib in the Hippo pathway network.