Laura A. Johnston

Drosophila myc regulates cellular growth during development.

Transcription factors of the Myc proto-oncogene family promote cell division, but how they do this is poorly understood. Here we address the functions of Drosophila Myc (dMyc) during development. Using mosaic analysis in the fly wing, we show that loss of dMyc retards cellular growth (accumulation of cell mass) and reduces cell size, whereas dMyc overproduction increases growth rates and cell size. dMyc-induced growth promotes G1/S progression but fails to accelerate cell division because G2/M progression is independently controlled by Cdc25/String. We also show that the secreted signal Wingless patterns growth in the wing primordium by modulating dMyc expression. Our results indicate that dMyc links patterning signals to cell division by regulating primary targets involved in cellular growth and metabolism.

Drosophila myc regulates organ size by inducing cell competition.

Experiments in both vertebrates and invertebrates have illustrated the competitive nature of growth and led to the idea that competition is a mechanism of regulating organ and tissue size. We have assessed competitive interactions between cells in a developing organ and examined their effect on its final size. We show that local expression of the Drosophila growth regulator dMyc, a homolog of the c-myc protooncogene, induces cell competition and leads to the death of nearby wild-type cells in developing wings. We demonstrate that cell competition is executed via induction of the proapoptotic gene hid and that both competition and hid function are required for the wing to reach an appropriate size when dMyc is expressed. Moreover, we provide evidence that reproducible wing size during normal development requires apoptosis. Modulating dmyc levels to create cell competition and hid-dependent cell death may be a mechanism used during normal development to control organ size.

Wingless and Notch regulate cell-cycle arrest in the developing Drosophila wing

In developing organs, the regulation of cell proliferation and patterning of cell fates is coordinated. How this coordination is achieved, however, is unknown. In the developing Drosophila wing, both cell proliferation and patterning require the secreted morphogen Wingless (Wg) at the dorsoventral compartment boundary (reviewed in ref. 1). Late in wing development, Wg also induces a zone of non-proliferating cells at the dorsoventral boundary. This zone gives rise to sensory bristles of the adult wing margin,. Here we investigate how Wg coordinates the cell cycle with patterning by studying the regulation of this growth arrest. We show that Wg, in conjunction with Notch, induces arrest in both the G1 and G2 phases of the cell cycle in separate subdomains of the zone of non-proliferating cells. Wg induces G2 arrest in two subdomains by inducing the proneural genes achaete and scute, which downregulate the mitosis-inducing phosphatase String (Cdc25). Notch activity creates a third domain by preventing arrest at G2 in wg-expressing cells, resulting in their arrest in G1.

Temporal Regulation of Metamorphic Processes in Drosophila by the let-7 and miR-125 Heterochronic MicroRNAs

BACKGROUND The let-7 and lin-4 microRNAs belong to a class of temporally expressed, noncoding regulatory RNAs that function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the rapid evolution of new morphologies. let-7 is highly conserved and in Drosophila is temporally coregulated with the lin-4 homolog, miR-125. Little is known, however, about their requirement outside the nematode or whether they universally control the timing of developmental processes. RESULTS We report the generation of a Drosophila mutant that lacks let-7 and miR-125 activities and that leads to a pleiotropic phenotype arising during metamorphosis. We focus on two defects and demonstrate that loss of let-7 and miR-125 results in temporal delays in two distinct metamorphic processes: the terminal cell-cycle exit in the wing and maturation of neuromuscular junctions (NMJs) at adult abdominal muscles. We identify the abrupt (ab) gene, encoding a nuclear protein, as a bona fide let-7 target and provide evidence that let-7 governs the maturation rate of abdominal NMJs during metamorphosis by regulating ab expression. CONCLUSIONS Drosophila Iet-7 and miR-125 mutants exhibit temporal misregulation of specific metamorphic processes. As in C. elegans, Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit, indicating that its function as a heterochronic microRNA is conserved. The ab gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. Our results suggest that let-7 and miR-125 serve as conserved regulators of events necessary for the transition from juvenile to adult life stages.

Competitive Interactions Between Cells: Death, Growth, and Geography

Winner Takes All? Competition between individual cells plays a role in normal animal development and cell homeostasis. Johnston (p. 1679) reviews two situations of cell competition in Drosophila, one involving epithelial cells in the wing and another involving germline or somatic stem cells. “Loss” in cell competition is evidenced by the “weaker” cells death or displacement. On the other hand, winners may engulf the loser or display enhanced proliferation. Competitive interactions allow cells to sense and eliminate poor-quality cells during development. Competitive interactions between cells are the basis of many homeostatic processes in biology. Some of the best-described cases of competition between cells occur in Drosophila: cell competition, whereby somatic cells within a growing epithelium compete with one another for contribution to the adult, and stem cell competition, in which germline or somatic stem cells vie for residency in the niche. Both types of competition are conserved physiological processes, with much to tell us about how cellular neighborhoods influence cell behavior, and have importance to stem cell biology, regeneration and transplantation, and cancer.

Compensatory Proliferation in Drosophila Imaginal Discs Requires Dronc-Dependent p53 Activity

BACKGROUND The p53 transcription factor directs a transcriptional program that determines whether a cell lives or dies after DNA damage. Animal survival after extensive cellular damage often requires that lost tissue be replaced through compensatory growth or regeneration. In Drosophila, damaged imaginal disc cells can induce the proliferation of neighboring viable cells, but how this is controlled is not clear. Here we provide evidence that Drosophila p53 (dp53) has a previously unidentified role in coordinating the compensatory growth response to tissue damage. RESULTS We find that dp53, the sole p53 ortholog in Drosophila, is required for each component of the response to cellular damage, including two separate cell-cycle arrests, changes in patterning gene expression, cell proliferation, and growth. We demonstrate that these processes are regulated by dp53 in a manner that is independent of DNA-damage sensing but that requires the initiator caspase Dronc. Our results indicate that once induced, dp53 amplifies and sustains the response through a positive feedback loop with Dronc and the apoptosis-inducing factors Hid and Reaper. CONCLUSIONS How cell death and cell proliferation are coordinated during development and after stress is a fundamental question that is critical for an understanding of growth regulation. Our data suggest that dp53 may carry out an ancestral function that promotes animal survival through the coordination of responses leading to compensatory growth after tissue damage.

Wingless promotes cell survival but constrains growth during Drosophila wing development

During animal development, organs grow to a fixed size and shape. Organ development typically begins with a rapid growth phase followed by a gradual decline in growth rate as the organ matures, but the regulation of either stage of growth remains unclear. The Wnt/Wingless (Wg) proteins are critical for patterning most animal organs, have diverse effects on development and have been proposed to promote organ growth. Here we report that contrary to this view, Wg activity actually constrains wing growth during Drosophila melanogaster wing development. In addition, we demonstrate that Wg is required for wing cell survival, particularly during the rapid growth phase of wing development. We propose that the cell-survival- and growth-constraining activities of Wg function to sculpt and delimit final wing size as part of its overall patterning programme.

Soluble factors mediate competitive and cooperative interactions between cells expressing different levels of Drosophila Myc

When neighboring cells in the developing Drosophila wing express different levels of the transcription factor, dMyc, competitive interactions can occur. Cells with more dMyc proliferate and ultimately overpopulate the wing, whereas cells with less dMyc die, thereby preventing wing overgrowth. How cells sense dMyc activity differences between themselves and the nature of the process leading to changes in growth and survival during competition remain unknown. We have developed a cell culture-based assay by using Drosophila S2 cells to investigate the mechanism of cell competition. We find that in vitro coculture of S2 cells that express different levels of dMyc leads to cellular interactions that recapitulate many aspects of cell competition in the developing wing. Our data indicate that both cell populations in the cocultures participate in and are required for the competitive process by releasing soluble factors into the medium. We demonstrate that the response of naive cells to medium conditioned with competitive cocultures depends on their potential to express dMyc: Cells that can express high levels of dMyc gain a survival advantage and proliferate faster, whereas cells with lower dMyc levels are instructed to die. We suggest that the ability of cells to perceive and respond to local differences in Myc activity is a cooperative mechanism that could contribute to growth regulation and developmental plasticity in organs and tissues during normal development and regeneration.

An ancient defense system eliminates unfit cells from developing tissues during cell competition

Introduction The function of a tissue and, therefore, the health of an organism can be compromised by the presence of mutant or unfit cells. Cell competition is a mechanism that has evolved to prevent such cells from contributing to tissues. Two widely studied models of cell competition are Myc-induced supercompetition, whereby cells with increased levels of Myc outcompete and actively kill neighboring wild-type cells, and competition in which wild-type cells eliminate cells carrying a Minute mutation, a class of mutants in Drosophila affecting ribosomal protein genes. Minute cells are viable, but when surrounded by wild-type cells they are eliminated by apoptosis. Relative cell vigor or fitness is believed to be a critical feature assessed in cell competition, but the mechanisms that underlie the recognition and elimination of the less-fit “loser” cell remain mysterious. TRR/NFκB modules mediate the elimination of loser cells in two distinct contexts of cell competition. (Top) The relatively less-fit cells in mosaic tissues (green) are eliminated via cell competition. (Bottom) The competitive context influences the TRR/NFκB signaling module that is triggered in the loser cells. This module then selectively activates a proapoptotic inducer that kills the loser cells. Rationale The recognition and elimination of unfit or mutant cells in cell competition is reminiscent of the detection of pathogens by the innate immune system. In Drosophila, the Toll and immune deficiency (IMD) signaling pathways govern the innate immune response to a broad range of pathogens and activate the NFκB transcription factor homologs Relish (Rel), Dorsal (dl), or Dorsal-related immunity factor (Dif). The conceptual similarities between innate immunity and cell competition led us to investigate whether the Toll and IMD pathways were required for cell competition in Drosophila wing discs. Results Analysis of both Myc-induced and Minute-induced cell competition revealed requirements for two related but distinct cohorts of components from the IMD and Toll pathways. Both signaling cohorts required the extracellular ligand Spätzle and noncanonical Toll-related receptors (TRRs) and led to elimination of the less-fit loser cells by inducing NFκB-dependent activation of proapoptotic genes. However, our analysis uncovered interesting differences between the signaling module deployed in each competitive context. In Myc-induced competition, elimination of wild-type loser cells required four of the nine TRRs encoded in the Drosophila genome (Toll-2, Toll-3, Toll-8, and Toll-9) in nonredundant roles. By contrast, elimination of RpL14–/+ cells in Minute-induced competition required only Toll-3 and Toll-9. Furthermore, the NFκB factor activated downstream of the TRRs was also context-dependent. Signal transduction within wild-type loser cells led to selective activation of Relish, whereas the death of RpL14–/+ loser cells in Minute-induced competition required Dorsal and Dif. These results suggest that signaling from the different TRR subsets influenced which NFκB factor was activated. Finally, although in each competitive context apoptosis of the relatively less-fit cells was induced, the specific death-inducing gene expressed was determined by specifically activated NFκB factor. Conclusion In two genetically distinct contexts of cell competition, the ancient innate immune defense response system is activated and drives the elimination of the cells perceived as relatively less fit. In each competition paradigm, different signaling modules are employed, suggesting that the genetic identity of the competing cell populations influences the pathway that is activated. Our results thus provide evidence for evolutionary adaptation of TRR-NFκB signaling modules in an organismal surveillance system that measures internal tissue fitness rather than external pathogenic stimuli. Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. Here we report that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. We find that Toll-related receptors (TRRs) and the cytokine Spätzle (Spz) lead to NFκB-dependent apoptosis. Diverse “loser” cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. Our findings demonstrate a functional repurposing of components of TRRs and NFκB signaling modules in the surveillance of cell fitness during development. Components of the innate immune system are repurposed to eliminate weak cells during development. [Also see Perspective by Morata and Ballesteros-Arias] Cell competition and immunity Even in what appears to be homogeneous tissue, cell variability exists. The presence of mutant cells can compromise the functional integrity of a tissue and ultimately the organisms health. Cell competition is an internal cell surveillance mechanism that monitors cell fitness to eliminate compromised cells and prevent them from contributing to the tissue. However, how cells recognize fitness differences has remained elusive. Meyer et al. report that this recognition uses signaling pathways from the innate immune response system. Cell-cell differences in fitness activate distinct NF-κB/Rel factors in the weaker population that lead to activation of distinct pro-apoptotic genes, leading to cell death in the losing cells. Science, this issue 10.1126/science.1258236

Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis

Cell competition is a conserved mechanism that regulates organ size and shares properties with the early stages of cancer. In Drosophila, wing cells with increased Myc or with optimum ribosome function become supercompetitors that kill their wild-type neighbors (called losers) up to several cell diameters away. Here, we report that modulating STAT activity levels regulates competitor status. Cells lacking STAT become losers that are killed by neighboring wild-type cells. By contrast, cells with hyper-activated STAT become supercompetitors that kill losers located at a distance in a manner that is dependent on hid but independent of Myc, Yorkie, Wingless signaling, and of ribosome biogenesis. These results indicate that STAT, Wingless and Myc are major parallel regulators of cell competition, which may converge on signals that non-autonomously kill losers. As hyper-activated STATs are causal to tumorigenesis and stem cell niche occupancy, our results have therapeutic implications for cancer and regenerative medicine.