Aneuploidy-induced cellular behaviors: Insights from Drosophila.

A balanced gene complement is crucial for proper cell function. Aneuploidy, the condition of having an imbalanced chromosome set, alters the stoichiometry of gene copy numbers and protein complexes and has dramatic consequences at the cellular and organismal levels. In humans, aneuploidy is associated with different pathological conditions including cancer, microcephaly, mental retardation, miscarriages, and aging. Over the last century, Drosophila has provided a valuable system for studying the consequences of systemic aneuploidies. More recently, it has contributed to the identification and molecular dissection of aneuploidy-induced cellular behaviors and their impact at the tissue and organismal levels. In this perspective, we review this active field of research, first by comparing knowledge from yeast, mouse, and human cells, then by highlighting the contributions of Drosophila. The aim of these discussions was to further our understanding of the functional interplay between aneuploidy, cell physiology, and tissue homeostasis in human development and disease.

Organogenesis: Cell death matters in size and shape regulation.

Anisotropic growth and large-scale morphogenetic movements contribute to the final size and shape of the adult Drosophila wing. A new study unravels an unexpected contribution of cell death, which follows a spatial and temporal pattern, to the growth of the wing and the acquisition of its elongated shape.

Size precision in insect eyes.

The building of fully functional and well-proportioned individuals relies on the precise regulation of the size of each of their constituting organs. A new study unravels a mechanism that confers precision to size regulation of the adult Drosophila eye through morphogen-mediated modulation of cell survival.

Chromosomal instability-induced cell invasion through caspase-driven DNA damage.

Chromosomal instability (CIN), an increased rate of changes in chromosome structure and number, is observed in most sporadic human carcinomas with high metastatic activity. Here, we use a Drosophila epithelial model to show that DNA damage, as a result of the production of lagging chromosomes during mitosis and aneuploidy-induced replicative stress, contributes to CIN-induced invasiveness. We unravel a sub-lethal role of effector caspases in invasiveness by enhancing CIN-induced DNA damage and identify the JAK/STAT signaling pathway as an activator of apoptotic caspases through transcriptional induction of pro-apoptotic genes. We provide evidence that an autocrine feedforward amplification loop mediated by Upd3-a cytokine with homology to interleukin-6 and a ligand of the JAK/STAT signaling pathway-contributes to amplifying the activation levels of the apoptotic pathway in migrating cells, thus promoting CIN-induced invasiveness. This work sheds new light on the chromosome-signature-independent effects of CIN in metastasis.

A single WNT enhancer drives specification and regeneration of the Drosophila wing.

Wings have provided an evolutionary advantage to insects and have allowed them to diversify. Here, we have identified in Drosophila a highly robust regulatory mechanism that ensures the specification and growth of the wing not only during normal development but also under stress conditions. We present evidence that a single wing-specific enhancer in the wingless gene is used in two consecutive developmental stages to first drive wing specification and then contribute to mediating the remarkable regenerative capacity of the developing wing upon injury. We identify two evolutionary conserved cis-regulatory modules within this enhancer that are utilized in a redundant manner to mediate these two activities through the use of distinct molecular mechanisms. Whereas Hedgehog and EGFR signalling regulate Wingless expression in early primordia, thus inducing wing specification from body wall precursors, JNK activation in injured tissues induce Wingless expression to promote compensatory proliferation. These results point to evolutionarily linked conservation of wing specification and regeneration to ensure robust development of the wing, perhaps the most relevant evolutionary novelty in insects.

Wing regeneration: Single-cell analysis sheds new light.

The developing wing primordium of Drosophila displays a remarkable capacity to regenerate in response to different types of damage. A new study shows that this capacity relies on the activation of a pro-regenerative gene regulatory network in two distinct cell populations within the blastema.

A new and integrated approach to evaluate the environmental and ecotoxicological impact of herbicide mixtures: A case study in maize.

An index-based approach for a comprehensive evaluation of the potential risk for active substances and their mixtures to impact the environment was developed. Some of the indices considered already exist (PRISW-1, Priority Index), while others were created ex novo from indicators available on open-source platforms (PESTi, ECOi, AGROi). These indices maybe used for an evaluation before use of pesticides by farmers and advisers. The present approach was initially validated for herbicides in maize crops, but it can readily be applied to other PPPs and crops. PESTi index underline the physical and chemical characteristics as a whole, not considering the impact of other factors such as application rate or period of application. Hence, this index may underestimate the risk associated to a certain chemical. AGROi has a precautionary approach. The risk associated to a specific mixture derives from a combination of intrinsic characteristics of the chemicals, agronomic impacts, regulation restrictions and potential hazard to water compartment. The ECOi index is focused on the ecotoxicological impact against non-target organisms. The helpfulness of this index stands in its ability to easily discriminate the ecotoxicological impact of chemicals using indicators commonly available in literature and without making complex calculations. PRISW-1 Index discriminate active substances according to their risk against three representative non-target organisms. However, due to the intrinsic characteristics of each pesticide, a high PRISW-1 value could not always mean an easy movement of the chemical via runoff waters. The information deriving from Priority index may certainly help public authorities to select chemicals to be detected in water monitoring campaigns. The application of these indices may represent a valid decision tool for public stakeholders in defining agricultural measures to reduce the externalities of pest control.

Tissue growth: Basement membrane degradation triggers cell proliferation.

Building of the Drosophila abdomen relies on the removal of larval cells and expansion, through proliferation, of a population of progenitor epithelial cells. A new study shows that matrix metalloproteinases produced by larval cells drive basement membrane degradation and proliferative growth of the progenitor epithelial population.

Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence.

Aneuploidy, an unbalanced number of chromosomes, is highly deleterious at the cellular level and leads to senescence, a stress-induced response characterized by permanent cell-cycle arrest and a well-defined associated secretory phenotype. Here, we use a Drosophila epithelial model to delineate the pathway that leads to the induction of senescence as a consequence of the acquisition of an aneuploid karyotype. Whereas aneuploidy induces, as a result of gene dosage imbalance, proteotoxic stress and activation of the major protein quality control mechanisms, near-saturation functioning of autophagy leads to compromised mitophagy, accumulation of dysfunctional mitochondria, and the production of radical oxygen species (ROS). We uncovered a role of c-Jun N-terminal kinase (JNK) in driving senescence as a consequence of dysfunctional mitochondria and ROS. We show that activation of the major protein quality control mechanisms and mitophagy dampens the deleterious effects of aneuploidy, and we identify a role of senescence in proteostasis and compensatory proliferation for tissue repair.

The Upd3 cytokine couples inflammation to maturation defects in Drosophila.

Developmental transitions, such as puberty or metamorphosis, are tightly controlled by steroid hormones and can be delayed by the appearance of growth abnormalities, developmental tumors, or inflammatory disorders such as inflammatory bowel disease or cystic fibrosis.1-4 Here, we used a highly inflammatory epithelial model of malignant transformation in Drosophila5,6 to unravel the role of Upd3-a cytokine with homology to interleukin-6-and the JAK/STAT signaling pathway in coupling inflammation to a delay in metamorphosis. We present evidence that Upd3 produced by malignant and nearby cell populations signals to the prothoracic gland-an endocrine tissue primarily dedicated to the production of the steroid hormone ecdysone-to activate JAK/STAT and bantam microRNA (miRNA) and to delay metamorphosis. Upd cytokines produced by the tumor site contribute to increasing the systemic levels of Upd3 by amplifying its expression levels in a cell-autonomous manner and by inducing Upd3 expression in neighboring tissues in a non-autonomous manner, culminating in a major systemic response to prevent larvae from initiating pupa transition. Our results identify a new regulatory network impacting on ecdysone biosynthesis and provide new insights into the potential role of inflammatory cytokines and the JAK/STAT signaling pathway in coupling inflammation to delays in puberty.

Regulation of Anisotropic Tissue Growth by Two Orthogonal Signaling Centers.

The Drosophila wing has served as a paradigm to mechanistically characterize the role of morphogens in patterning and growth. Wingless (Wg) and Decapentaplegic (Dpp) are expressed in two orthogonal signaling centers, and their gradients organize patterning by regulating the expression of well-defined target genes. By contrast, graded activity of these morphogens is not an absolute requirement for wing growth. Despite their permissive role in regulating growth, here we show that Wg and Dpp are utilized in a non-interchangeable manner by the two existing orthogonal signaling centers to promote preferential growth along the two different axes of the developing wing. Our data indicate that these morphogens promote anisotropic growth by making use of distinct and non-interchangeable molecular mechanisms. Whereas Dpp drives growth along the anterior-posterior axis by maintaining Brinker levels below a growth-repressing threshold, Wg exerts its action along the proximal-distal axis through a double repression mechanism involving T cell factor (TCF).

Eiger/TNFα-mediated Dilp8 and ROS production coordinate intra-organ growth in Drosophila.

Coordinated intra- and inter-organ growth during animal development is essential to ensure a correctly proportioned individual. The Drosophila wing has been a valuable model system to reveal the existence of a stress response mechanism involved in the coordination of growth between adjacent cell populations and to identify a role of the fly orthologue of p53 (Dmp53) in this process. Here we identify the molecular mechanisms used by Dmp53 to regulate growth and proliferation in a non-autonomous manner. First, Dmp53-mediated transcriptional induction of Eiger, the fly orthologue of TNFα ligand, leads to the cell-autonomous activation of JNK. Second, two distinct signaling events downstream of the Eiger/JNK axis are induced in order to independently regulate tissue size and cell number in adjacent cell populations. Whereas expression of the hormone dILP8 acts systemically to reduce growth rates and tissue size of adjacent cell populations, the production of Reactive Oxygen Species-downstream of Eiger/JNK and as a consequence of apoptosis induction-acts in a non-cell-autonomous manner to reduce proliferation rates. Our results unravel how local and systemic signals act concertedly within a tissue to coordinate growth and proliferation, thereby generating well-proportioned organs and functionally integrated adults.

Selective Killing of RAS-Malignant Tissues by Exploiting Oncogene-Induced DNA Damage.

Several oncogenes induce untimely entry into S phase and alter replication timing and progression, thereby generating replicative stress, a well-known source of genomic instability and a hallmark of cancer. Using an epithelial model in Drosophila, we show that the RAS oncogene, which triggers G1/S transition, induces DNA damage and, at the same time, silences the DNA damage response pathway. RAS compromises ATR-mediated phosphorylation of the histone variant H2Av and ATR-mediated cell-cycle arrest in G2 and blocks, through ERK, Dp53-dependent induction of cell death. We found that ERK is also activated in normal tissues by an exogenous source of damage and that this activation is necessary to dampen the pro-apoptotic role of Dp53. We exploit the pro-survival role of ERK activation upon endogenous and exogenous sources of DNA damage to present evidence that its genetic or chemical inhibition can be used as a therapeutic opportunity to selectively eliminate RAS-malignant tissues.

Ask1 and Akt act synergistically to promote ROS-dependent regeneration in Drosophila.

How cells communicate to initiate a regenerative response after damage has captivated scientists during the last few decades. It is known that one of the main signals emanating from injured cells is the Reactive Oxygen Species (ROS), which propagate to the surrounding tissue to trigger the replacement of the missing cells. However, the link between ROS production and the activation of regenerative signaling pathways is not yet fully understood. We describe here the non-autonomous ROS sensing mechanism by which living cells launch their regenerative program. To this aim, we used Drosophila imaginal discs as a model system due to its well-characterized regenerative ability after injury or cell death. We genetically-induced cell death and found that the Apoptosis signal-regulating kinase 1 (Ask1) is essential for regenerative growth. Ask1 senses ROS both in dying and living cells, but its activation is selectively attenuated in living cells by Akt1, the core kinase component of the insulin/insulin-like growth factor pathway. Akt1 phosphorylates Ask1 in a secondary site outside the kinase domain, which attenuates its activity. This modulation of Ask1 activity results in moderate levels of JNK signaling in the living tissue, as well as in activation of p38 signaling, both pathways required to turn on the regenerative response. Our findings demonstrate a non-autonomous activation of a ROS sensing mechanism by Ask1 and Akt1 to replace the missing tissue after damage. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration.

Chromosomal Instability Induces Cellular Invasion in Epithelial Tissues.

Most sporadic carcinomas with high metastatic activity show an increased rate of changes in chromosome structure and number, known as chromosomal instability (CIN). However, the role of CIN in driving invasiveness remains unclear. Using an epithelial model in Drosophila, we present evidence that CIN promotes a rapid and general invasive behavior. Cells with an abnormal number of chromosomes delaminate from the epithelium, extend actin-based cellular protrusions, form membrane blebs, and invade neighboring tissues. This behavior is governed by the activation of non-muscle Myosin II by Rho kinase and by the expression of the secreted EGF/Spitz ligand. We unravel fundamental roles of the mitogen-activated protein kinase pathways mediated by the Fos proto-oncogene and the Capicua tumor suppressor gene in the invasive behavior of CIN-induced aneuploid cells. Our results support the proposal that the simple production of unbalanced karyotypes contributes to CIN-induced metastatic progression.

Epithelial tumors: Growing from within.

The growth of epithelial tumors is often governed by cell interactions with the surrounding stroma. Drosophila has been instrumental in identifying the relevant molecular elements mediating these interactions. Of note is the role of the TNF ligand Eiger, released from recruited blood cells, in activating the JNK tumor-promoting pathway in epithelial tumors. JNK drives the transcriptional induction of mitogenic molecules, matrix metalloproteases and systemic signals that lead to tumor growth, tissue invasiveness and malignancy. Here we review our findings on a tumor-intrinsic, Eiger- and stroma-independent mechanism that contributes to the unlimited growth potential of tumors caused either by chromosomal instability or impaired cell polarity. This newly identified mechanism, which was revealed in an experimental condition in which contacts between tumor cells and wild-type epithelial cells were minimized, relies on interactions between functionally distinct tumor cell populations that activate JNK in a cell-autonomous manner. We discuss the impact of cell interaction-based feedback amplification loops on the unlimited growth potential of epithelial tumors. These findings are expected to contribute to the identification of the relevant cell populations and molecular mechanisms to be targeted in drug therapy.

Feedback amplification loop drives malignant growth in epithelial tissues.

Interactions between cells bearing oncogenic mutations and the surrounding microenvironment, and cooperation between clonally distinct cell populations, can contribute to the growth and malignancy of epithelial tumors. The genetic techniques available in Drosophila have contributed to identify important roles of the TNF-α ligand Eiger and mitogenic molecules in mediating these interactions during the early steps of tumor formation. Here we unravel the existence of a tumor-intrinsic-and microenvironment-independent-self-reinforcement mechanism that drives tumor initiation and growth in an Eiger-independent manner. This mechanism relies on cell interactions between two functionally distinct cell populations, and we present evidence that these cell populations are not necessarily genetically different. Tumor-specific and cell-autonomous activation of the tumorigenic JNK stress-activated pathway drives the expression of secreted signaling molecules and growth factors to delaminating cells, which nonautonomously promote proliferative growth of the partially transformed epithelial tissue. We present evidence that cross-feeding interactions between delaminating and nondelaminating cells increase each other's sizes and that these interactions can explain the unlimited growth potential of these tumors. Our results will open avenues toward our molecular understanding of those social cell interactions with a relevant function in tumor initiation in humans.

Boundary Dpp promotes growth of medial and lateral regions of the Drosophila wing.

The gradient of Decapentaplegic (Dpp) in the Drosophila wing has served as a paradigm to characterize the role of morphogens in regulating patterning. However, the role of this gradient in regulating tissue size is a topic of intense debate as proliferative growth is homogenous. Here, we combined the Gal4/UAS system and a temperature-sensitive Gal80 molecule to induce RNAi-mediated depletion of dpp and characterise the spatial and temporal requirement of Dpp in promoting growth. We show that Dpp emanating from the AP compartment boundary is required throughout development to promote growth by regulating cell proliferation and tissue size. Dpp regulates growth and proliferation rates equally in central and lateral regions of the developing wing appendage and reduced levels of Dpp affects similarly the width and length of the resulting wing. We also present evidence supporting the proposal that graded activity of Dpp is not an absolute requirement for wing growth.

Phototransformation of the Herbicide Propanil in Paddy Field Water.

When irradiated in paddy-field water, propanil (PRP) undergoes photodegradation by direct photolysis, by reactions with •OH and CO3•-, and possibly also with the triplet states of chromophoric dissolved organic matter. Irradiation also inhibits the nonphotochemical (probably biological) degradation of PRP. The dark- and light-induced pathways can be easily distinguished because 3,4-dichloroaniline (34DCA, a transformation intermediate of considerable environmental concern) is produced with almost 100% yield in the dark but not at all through photochemical pathways. This issue allows an easy assessment of the dark process(es) under irradiation. In the natural environment, we expect PRP photodegradation to be important only in the presence of elevated nitrate and/or nitrite levels, e.g., [NO3-] approaching 1 mmol L-1 (corresponding to approximately 60 mg L-1). Under these circumstances, •OH and CO3•- would play a major role in PRP phototransformation. Because flooded paddy fields are efficient denitrification bioreactors that can achieve decontamination of nitrate-rich water used for irrigation, irrigation with such water would both enhance PRP photodegradation and divert PRP dissipation processes away from the production of 34DCA, at least in the daylight hours.

A Drosophila model of myeloproliferative neoplasm reveals a feed-forward loop in the JAK pathway mediated by p38 MAPK signalling.

Myeloproliferative neoplasms (MPNs) of the Philadelphia-negative class comprise polycythaemia vera, essential thrombocythaemia and primary myelofibrosis (PMF). They are associated with aberrant numbers of myeloid lineage cells in the blood, and in the case of overt PMF, with development of myelofibrosis in the bone marrow and failure to produce normal blood cells. These diseases are usually caused by gain-of-function mutations in the kinase JAK2. Here, we use Drosophila to investigate the consequences of activation of the JAK2 orthologue in haematopoiesis. We have identified maturing haemocytes in the lymph gland, the major haematopoietic organ in the fly, as the cell population susceptible to induce hypertrophy upon targeted overexpression of JAK. We show that JAK activates a feed-forward loop, including the cytokine-like ligand Upd3 and its receptor, Domeless, which are required to induce lymph gland hypertrophy. Moreover, we present evidence that p38 MAPK signalling plays a key role in this process by inducing expression of the ligand Upd3. Interestingly, we also show that forced activation of the p38 MAPK pathway in maturing haemocytes suffices to generate hypertrophic organs and the appearance of melanotic tumours. Our results illustrate a novel pro-tumourigenic crosstalk between the p38 MAPK pathway and JAK signalling in a Drosophila model of MPNs. Based on the shared molecular mechanisms underlying MPNs in flies and humans, the interplay between Drosophila JAK and p38 signalling pathways unravelled in this work might have translational relevance for human MPNs.