Tissue communication in a systemic immune response of Drosophila

Several signaling pathways, including the JAK/STAT and Toll pathways, are known to activate blood cells (hemocytes) in Drosophila melanogaster larvae. They are believed to regulate the immune response against infections by parasitoid wasps, such as Leptopilina boulardi, but how these pathways control the hemocytes is not well understood. Here, we discuss the recent discovery that both muscles and fat body take an active part in this response. Parasitoid wasp infection induces Upd2 and Upd3 secretion from hemocytes, leading to JAK/STAT activation mainly in hemocytes and in skeletal muscles. JAK/STAT activation in muscles, but not in hemocytes, is required for an efficient encapsulation of wasp eggs. This suggests that Upd2 and Upd3 are important cytokines, coordinating different tissues for the cellular immune response in Drosophila. In the fat body, Toll signaling initiates a systemic response in which hemocytes are mobilized and activated hemocytes (lamellocytes) are generated. However, the contribution of Toll signaling to the defense against wasps is limited, probably because the wasps inject inhibitors that prevent the activation of the Toll pathway. In conclusion, parasite infection induces a systemic response in Drosophila larvae involving major organ systems and probably the physiology of the entire organism.     

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival.     

Tousled-like kinase regulates cytokine-mediated communication between cooperating cell types during collective border cell migration

Collective cell migration is emerging as a major contributor to normal development and disease. Collective movement of border cells in the Drosophila ovary requires cooperation between two distinct cell types: four to six migratory cells surrounding two immotile cells called polar cells. Polar cells secrete a cytokine, Unpaired (Upd), which activates JAK/STAT signaling in neighboring cells, stimulating their motility. Without Upd, migration fails, causing sterility. Ectopic Upd expression is sufficient to stimulate motility in otherwise immobile cells. Thus regulation of Upd is key. Here we report a limited RNAi screen for nuclear proteins required for border cell migration, which revealed that the gene encoding Tousled-like kinase (Tlk) is required in polar cells for Upd expression without affecting polar cell fate. In the absence of Tlk, fewer border cells are recruited and motility is impaired, similar to inhibition of JAK/STAT signaling. We further show that Tlk in polar cells is required for JAK/STAT activation in border cells. Genetic interactions further confirmed Tlk as a new regulator of Upd/JAK/STAT signaling. These findings shed light on the molecular mechanisms regulating the cooperation of motile and nonmotile cells during collective invasion, a phenomenon that may also drive metastatic cancer.     

Pleiotropy of the Drosophila JAK pathway cytokine Unpaired 3 in development and aging

The Janus kinase (JAK) pathway is an essential, highly re-utilized developmental signaling cascade found in most metazoans. In vertebrates, the JAK intracellular cascade mediates signaling by dozens of cytokines and growth factors. In Drosophila, the Unpaired (Upd) family, encoded by three tandemly duplicated genes, is the only class of ligands associated with JAK stimulation. Unpaired has a central role in activation of JAK for most pathway functions, while Unpaired 2 regulates body size through insulin signaling. We show here that the third member of the family, unpaired 3 (upd3), overlaps upd in expression in some tissues and is essential for a subset of JAK-mediated developmental functions. First, consistent with the known requirements of JAK signaling in gametogenesis, we find that mutants of upd3 show an age-dependent impairment of fertility in both sexes. In oogenesis, graded JAK activity stimulated by Upd specifies the fates of the somatic follicle cells. As upd3 mutant females age, defects arise that can be attributed to perturbations of the terminal follicle cells, which require the highest levels of JAK activation. Therefore, in oogenesis, the activities of Upd and Upd3 both appear to quantitatively contribute to specification of those follicle cell fates. Furthermore, the sensitization of upd3 mutants to age-related decline in fertility can be used to investigate reproductive senescence. Second, loss of Upd3 during imaginal development results in defects of adult structures, including reduced eye size and abnormal wing and haltere posture. The outstretched wing and small eye phenotypes resemble classical alleles referred to as outstretched (os) mutations that have been previously ascribed to upd. However, we show that os alleles affect expression of both upd and upd3 and map to untranscribed regions, suggesting that they disrupt regulatory elements shared by both genes. Thus the upd region serves as a genetically tractable model for coordinate regulation of tandemly duplicated gene families that are commonly found in higher eukaryotes.     

Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. Here, we describe our analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. We show that upd2 is expressed in a pattern essentially identical to that of upd and demonstrate that the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, we developed a reporter based on a natural JAK/STAT pathway responsive enhancer and show that ectopic upd2 expression can effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to our understanding of cytokine signalling in Drosophila.     

Differential activities of the Drosophila JAK/STAT pathway ligands Upd, Upd2 and Upd3

JAK/STAT signalling in vertebrates is activated by multiple cytokines and growth factors. By contrast, the Drosophila genome encodes for only three related JAK/STAT ligands, Upd, Upd2 and Upd3. Identifying the differences between these three ligands will ultimately lead to a greater understanding of this disease-related signalling pathway and its roles in development. Here, we describe the analysis of the least well characterised of the Upd-like ligands, Upd3. We show that in tissue culture-based assays Upd3-GFP is secreted from cells and appears to interact with the extracellular matrix (ECM) in a similar manner to Upd, while still non-autonomously activating JAK/STAT signalling. Quantification of each of the Upd-like ligands in conditioned media has allowed us to determine the activity of equal amounts of each ligand on JAK/STAT ex vivo and reveals that Upd is the most potent ligand in this system. Finally, investigations into the effects of ectopic expression of Upd3 in vivo have confirmed its ability to activate pathway signalling at long-distance.     

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.     

Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury

To characterize the features of JAK/STAT signaling in Drosophila immune response, we have identified totA as a gene that is regulated by the JAK/STAT pathway in response to septic injury. We show that septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, we demonstrate that totA activation also requires the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-KB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals.     

The UPD3 cytokine couples environmental challenge and intestinal stem cell division through modulation of JAK/STAT signaling in the stem cell microenvironment

In Drosophila, the replacement of spent enterocytes (ECs) relies on division of intestinal stem cells (ISCs) and differentiation of their progeny, the enteroblasts (EBs). Recent studies have revealed a role for JAK/STAT signaling in the modulation of the rate of ISC division in response to environmental challenge. Here, we demonstrate the critical role of the UPD3 cytokine in the JAK/STAT-dependent response to enteric infection. We show that upd3 expression is activated in ECs and in EBs that massively differentiate in response to challenge. We show that the UPD3 cytokine, which is secreted basally and accumulates at the basement membrane, is required for stimulation of JAK/STAT signaling in EBs and visceral muscles (VMs). We further show that stimulation of ISC division requires active JAK/STAT signaling in EBs and VMs, but apparently not in ISCs. Our results suggest that EBs and VMs modulate the rate of the EGFR-dependent ISC division through upd3-dependent production of the EGF ligands Spitz and Vein, respectively. This study therefore supports the notion that the production of the UPD3 cytokine in stem cell progeny (ECs and EBs) stimulates intestinal stem cell division through modulation of JAK/STAT signaling in the stem cell microenvironment (EBs and VMs).     

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.     

A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation

Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. We have generated a transgenic animal with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand upd. The system has enabled us to identify the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture.     

ROS-Induced JNK and p38 Signaling Is Required for Unpaired Cytokine Activation during Drosophila Regeneration

Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. We genetically induced cell death in wing imaginal discs, monitored the production of ROS and analyzed the signals required for repair. We found that cell death generates a burst of ROS that propagate to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. Our findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.