Casein kinase 1α decreases ß-catenin levels at adherens junctions to facilitate wound closure in Drosophila larvae.

Skin wound repair is essential to restore barrier function and prevent infection after tissue damage. Wound-edge epidermal cells migrate as a sheet to close the wound. However, it is still unclear how cell-cell junctions are regulated during wound closure (WC). To study this, we examined adherens junctions during WC in Drosophila larvae. ß-Catenin is reduced at the lateral cell-cell junctions of wound-edge epidermal cells in the early healing stages. Destruction complex components, including Ck1α, GSK3ß and ß-TrCP, suppress ß-catenin levels in the larval epidermis. Tissue-specific RNAi targeting these genes also caused severe WC defects. The Ck1αRNAi -induced WC defect is related to adherens junctions because loss of either ß-catenin or E-cadherin significantly rescued this WC defect. In contrast, TCFRNAi does not rescue the Ck1αRNAi -induced WC defect, suggesting that Wnt signaling is not related to this defect. Direct overexpression of ß-catenin recapitulates most of the features of Ck1α reduction during wounding. Finally, loss of Ck1α also blocked junctional E-cadherin reduction around the wound. Our results suggest that Ck1α and the destruction complex locally regulate cell adhesion to facilitate efficient wound repair.

Drosophila larval epidermal cells only exhibit epidermal aging when they persist to the adult stage.

Holometabolous insects undergo a complete transformation of the body plan from the larval to the adult stage. In Drosophila, this transformation includes replacement of larval epidermal cells (LECs) by adult epidermal cells (AECs). AECs in Drosophila undergo a rapid and stereotyped aging program where they lose both cell membranes and nuclei. Whether LECs are capable of undergoing aging in a manner similar to AECs remains unknown. Here, we address this question in two ways. First, we looked for hallmarks of epidermal aging in larvae that have a greatly extended third instar and/or carry mutations that would cause premature epidermal aging at the adult stage. Such larvae, irrespective of genotype, did not show any of the signs of epidermal aging observed in the adult. Second, we developed a procedure to effect a heterochronic persistence of LECs into the adult epidermal sheet. Lineage tracing verified that presumptive LECs in the adult epidermis are not derived from imaginal epidermal histoblasts. LECs embedded within the adult epidermal sheet undergo clear signs of epidermal aging; they form multinucleate cells with each other and with the surrounding AECs. The incidence of adult cells with mixed AEC nuclei (small) and persistent LEC nuclei (large) increased with age. Our data reveals that epidermal aging in holometabolous Drosophila is a stage-specific phenomenon and that the capacity of LECs to respond to aging signals does exist.

Growth Factor Signaling Regulates Mechanical Nociception in Flies and Vertebrates.

Mechanical sensitization is one of the most difficult clinical pain problems to treat. However, the molecular and genetic bases of mechanical nociception are unclear. Here we develop a Drosophila model of mechanical nociception to investigate the ion channels and signaling pathways that regulate mechanical nociception. We fabricated von Frey filaments that span the subthreshold to high noxious range for Drosophila larvae. Using these, we discovered that pressure (force/area), rather than force per se, is the main determinant of aversive rolling responses to noxious mechanical stimuli. We demonstrated that the RTK PDGF/VEGF receptor (Pvr) and its ligands (Pvfs 2 and 3) are required for mechanical nociception and normal dendritic branching. Pvr is expressed and functions in class IV sensory neurons, whereas Pvf2 and Pvf3 are produced by multiple tissues. Constitutive overexpression of Pvr and its ligands or inducible overexpression of Pvr led to mechanical hypersensitivity that could be partially separated from morphological effects. Genetic analyses revealed that the Piezo and Pain ion channels are required for mechanical hypersensitivity observed upon ectopic activation of Pvr signaling. PDGF, but not VEGF, peptides caused mechanical hypersensitivity in rats. Pharmacological inhibition of VEGF receptor Type 2 (VEGFR-2) signaling attenuated mechanical nociception in rats, suggesting a conserved role for PDGF and VEGFR-2 signaling in regulating mechanical nociception. VEGFR-2 inhibition also attenuated morphine analgesic tolerance in rats. Our results reveal that a conserved RTK signaling pathway regulates baseline mechanical nociception in flies and rats.SIGNIFICANCE STATEMENT Hypersensitivity to touch is poorly understood and extremely difficult to treat. Using a refined Drosophila model of mechanical nociception, we discovered a conserved VEGF-related receptor tyrosine kinase signaling pathway that regulates mechanical nociception in flies. Importantly, pharmacological inhibition of VEGF receptor Type 2 signaling in rats causes analgesia and blocks opioid tolerance. We have thus established a robust, genetically tractable system for the rapid identification and functional analysis of conserved genes underlying mechanical pain sensitivity.

Drosophila Nociceptive Sensitization Requires BMP Signaling via the Canonical SMAD Pathway.

Nociceptive sensitization is a common feature in chronic pain, but its basic cellular mechanisms are only partially understood. The present study used the Drosophila melanogaster model system and a candidate gene approach to identify novel components required for modulation of an injury-induced nociceptive sensitization pathway presumably downstream of Hedgehog. This study demonstrates that RNAi silencing of a member of the Bone Morphogenetic Protein (BMP) signaling pathway, Decapentaplegic (Dpp), specifically in the Class IV multidendritic nociceptive neuron, significantly attenuated ultraviolet injury-induced sensitization. Furthermore, overexpression of Dpp in Class IV neurons was sufficient to induce thermal hypersensitivity in the absence of injury. The requirement of various BMP receptors and members of the SMAD signal transduction pathway in nociceptive sensitization was also demonstrated. The effects of BMP signaling were shown to be largely specific to the sensitization pathway and not associated with changes in nociception in the absence of injury or with changes in dendritic morphology. Thus, the results demonstrate that Dpp and its pathway play a crucial and novel role in nociceptive sensitization. Because the BMP family is so strongly conserved between vertebrates and invertebrates, it seems likely that the components analyzed in this study represent potential therapeutic targets for the treatment of chronic pain in humans.SIGNIFICANCE STATEMENT This report provides a genetic analysis of primary nociceptive neuron mechanisms that promote sensitization in response to injury. Drosophila melanogaster larvae whose primary nociceptive neurons were reduced in levels of specific components of the BMP signaling pathway, were injured and then tested for nocifensive responses to a normally subnoxious stimulus. Results suggest that nociceptive neurons use the BMP2/4 ligand, along with identified receptors and intracellular transducers to transition to a sensitized state. These findings are consistent with the observation that BMP receptor hyperactivation correlates with bone abnormalities and pain sensitization in fibrodysplasia ossificans progressiva (Kitterman et al., 2012). Because nociceptive sensitization is associated with chronic pain, these findings indicate that human BMP pathway components may represent targets for novel pain-relieving drugs.

Yorkie regulates epidermal wound healing in Drosophila larvae independently of cell proliferation and apoptosis.

Yorkie (Yki), the transcriptional co-activator of the Hippo signaling pathway, has well-characterized roles in balancing apoptosis and cell division during organ growth control. Yki is also required in diverse tissue regenerative contexts. In most cases this requirement reflects its well-characterized roles in balancing apoptosis and cell division. Whether Yki has repair functions outside of the control of cell proliferation, death, and growth is not clear. Here we show that Yki and Scalloped (Sd) are required for epidermal wound closure in the Drosophila larval epidermis. Using a GFP-tagged Yki transgene we show that Yki transiently translocates to some epidermal nuclei upon wounding. Genetic analysis strongly suggests that Yki interacts with the known wound healing pathway, Jun N-terminal kinase (JNK), but not with Platelet Derived Growth Factor/Vascular-Endothelial Growth Factor receptor (Pvr). Yki likely acts downstream of or parallel to JNK signaling and does not appear to regulate either proliferation or apoptosis in the larval epidermis during wound repair. Analysis of actin structures after wounding suggests that Yki and Sd promote wound closure through actin regulation. In sum, we found that Yki regulates an epithelial tissue repair process independently of its previously documented roles in balancing proliferation and apoptosis.

Transcriptional regulation of Profilin during wound closure in Drosophila larvae.

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. Here, we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

Will the wound-healing field earn its wings?

In a recently published issue of Experimental Dermatology, Dr. Nuria Paricio and colleagues review recent advances using the fruit fly, Drosophila melanogaster, as a wound-healing model. They describe many of the advantages of the fly model for gene discovery and functional analysis, highlighting its particular strengths and limitations for studies of wound healing. This commentary assumes that dermatologist-scientists and fly wound-healing researchers share a common field-wide goal of discovering all of the clinically relevant wound-healing genes and understanding in molecular detail how those genes work. We ask: how can we cooperate to achieve this shared goal?

Pokes, sunburn, and hot sauce: Drosophila as an emerging model for the biology of nociception.

The word "nociception" is derived from the Latin "nocere," which means "to harm." Nociception refers to the sensory perception of noxious stimuli that have the potential to cause tissue damage. Since the perception of such potentially harmful stimuli often results in behavioral escape responses, nociception provides a protective mechanism that allows an organism to avoid incipient (or further) damage to the tissue. It appears to be universal in metazoans as a variety of escape responses can be observed in both mammalian and non-mammalian vertebrates, as well as diverse invertebrates such as leeches, nematodes, and fruit flies (Sneddon [2004] Brain Research Review 46:123-130; Tobin and Bargmann [2004] Journal of Neurobiology 61:161-174; Smith and Lewin [2009] Journal of Comparative Physiology 195:1089-1106). Several types of stimuli can trigger nociceptive sensory transduction, including noxious heat, noxious chemicals, and harsh mechanical stimulation. Such high-threshold stimuli induce the firing of action potentials in peripheral nociceptors, the sensory neurons specialized for their detection (Basbaum et al. [2009] Cell 139:267-284). In vertebrates, these action potentials can either be relayed directly to a spinal motor neuron to provoke escape behavior (the so-called monosynaptic reflex) or can travel via spinal cord interneurons to higher-order processing centers in the brain. This review will cover the establishment of Drosophila as a system to study various aspects of nociceptive sensory perception. We will cover development of the neurons responsible for detecting noxious stimuli in larvae, the assays used to assess the function(s) of these neurons, and the genes that have been found to be required for both thermal and mechanical nociception. Along the way, we will highlight some of the genetic tools that make the fly such a powerful system for studies of nociception. Finally, we will cover recent studies that introduce new assays employing adult Drosophila to study both chemical and thermal nociception and provide an overview of important unanswered questions in the field.

The insulin receptor regulates the persistence of mechanical nociceptive sensitization in flies and mice.

Early phase diabetes is often accompanied by pain sensitization. In Drosophila, the insulin receptor (InR) regulates the persistence of injury-induced thermal nociceptive sensitization. Whether Drosophila InR also regulates the persistence of mechanical nociceptive sensitization remains unclear. Mice with a sensory neuron deletion of the insulin receptor (Insr) show normal nociceptive baselines; however, it is uncertain whether deletion of Insr in nociceptive sensory neurons leads to persistent nociceptive hypersensitivity. In this study, we used fly and mouse nociceptive sensitization models to address these questions. In flies, InR mutants and larvae with sensory neuron-specific expression of RNAi transgenes targeting InR exhibited persistent mechanical hypersensitivity. Mice with a specific deletion of the Insr gene in Nav1.8+ nociceptive sensory neurons showed nociceptive thermal and mechanical baselines similar to controls. In an inflammatory paradigm, however, these mutant mice showed persistent mechanical (but not thermal) hypersensitivity, particularly in female mice. Mice with the Nav1.8+ sensory neuron-specific deletion of Insr did not show metabolic abnormalities typical of a defect in systemic insulin signaling. Our results show that some aspects of the regulation of nociceptive hypersensitivity by the insulin receptor are shared between flies and mice and that this regulation is likely independent of metabolic effects.

Pvr and distinct downstream signaling factors are required for hemocyte spreading and epidermal wound closure at Drosophila larval wound sites.

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster larvae, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. Here, we discover a role for the platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, we performed a genetic screen in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure (WC), another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. Our data reveal that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal WC or hemocyte spreading.

Circulating blood cells function as a surveillance system for damaged tissue in Drosophila larvae.

Insects have an open circulatory system in which the heart pumps blood (hemolymph) into the body cavity, where it directly bathes the internal organs and epidermis. The blood contains free and tissue-bound immune cells that function in the inflammatory response. Here, we use live imaging of transgenic Drosophila larvae with fluorescently labeled blood cells (hemocytes) to investigate the circulatory dynamics of larval blood cells and their response to tissue injury. We find that, under normal conditions, the free cells rapidly circulate, whereas the tissue-bound cells are sessile. After epidermal wounding, tissue-bound cells around the wound site remain sessile and unresponsive, whereas circulating cells are rapidly recruited to the site of damage by adhesive capture. After capture, these cells distribute across the wound, appear phagocytically active, and are subsequently released back into circulation by the healing epidermis. The results demonstrate that circulating cells function as a surveillance system that monitors larval tissues for damage, and that adhesive capture, an important mechanism of recruitment of circulating cells to inflammatory sites in vertebrates, is shared by insects and vertebrates despite the vastly different architectures of their circulatory systems.

Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration.

Aa robust inflammatory response to tissue damage and infection is conserved across almost all animal phyla. Neutrophils and macrophages, or their equivalents, are drawn to the wound site where they engulf cell and matrix debris and release signals that direct components of the repair process. This orchestrated cell migration is clinically important, and yet, to date, leukocyte chemotaxis has largely been studied in vitro. Here, we describe a genetically tractable in vivo wound model of inflammation in the Drosophila melanogaster embryo that is amenable to cinemicroscopy. For the first time, we are able to examine the roles of Rho-family small GTPases during inflammation in vivo and show that Rac-mediated lamellae are essential for hemocyte motility and Rho signaling is necessary for cells to retract from sites of matrix- and cell-cell contacts. Cdc42 is necessary for maintaining cellular polarity and yet, despite in vitro evidence, is dispensable for sensing and crawling toward wound cues.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An assay for chemical nociception in Drosophila larvae.

Chemically induced nociception has not yet been studied intensively in genetically tractable models. Hence, our goal was to establish a Drosophila assay that can be used to study the cellular and molecular/genetic bases of chemically induced nociception. Drosophila larvae exposed to increasing concentrations of hydrochloric acid (HCl) produced an increasingly intense aversive rolling response. HCl (0.5%) was subthreshold and provoked no response. All classes of peripheral multidendritic (md) sensory neurons (classes I-IV) are required for full responsiveness to acid, with class IV making the largest contribution. At the cellular level, classes IV, III and I showed increases in calcium following acid exposure. In the central nervous system, Basin-4 second-order neurons are the key regulators of chemically induced nociception, with a slight contribution from other types. Finally, chemical nociception can be sensitized by tissue damage. Subthreshold HCl provoked chemical allodynia in larvae 4 h after physical puncture wounding. Pinch wounding and UV irradiation, which do not compromise the cuticle, did not cause chemical allodynia. In sum, we developed a novel assay to study chemically induced nociception in Drosophila larvae. This assay, combined with the high genetic resolving power of Drosophila, should improve our basic understanding of fundamental mechanisms of chemical nociception. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Crawling wounded: molecular genetic insights into wound healing from Drosophila larvae.

For animals, injury is inevitable. Because of this, organisms possess efficient wound healing mechanisms that can repair damaged tissues. However, the molecular and genetic mechanisms by which epidermal repair is accomplished remain poorly defined. Drosophila has become a valuable model to study epidermal wound healing because of the comprehensive genetic toolkit available in this organism and the similarities of wound healing processes between Drosophila and vertebrates. Other reviews in this Special Issue cover wound healing assays and pathways in Drosophila embryos, pupae and adults, as well as regenerative processes that occur in tissues such as imaginal discs and the gut. In this review, we will focus on the molecular/genetic control of wound-induced cellular processes such as inflammation, cell migration and epithelial cell-cell fusion in Drosophila larvae. We will give a brief overview of the three wounding assays, pinch, puncture, and laser ablation, and the cellular responses that ensue following wounding. We will highlight the actin regulators, signaling pathways and transcriptional mediators found so far to be involved in larval epidermal wound closure and what is known about how they act. We will also discuss wound-induced epidermal cell-cell fusion and possible directions for future research in this exciting system.

Injury-induced cold sensitization in Drosophila larvae involves behavioral shifts that require the TRP channel Brv1.

Nociceptive sensitization involves an increase in responsiveness of pain sensing neurons to sensory stimuli, typically through the lowering of their nociceptive threshold. Nociceptive sensitization is common following tissue damage, inflammation, and disease and serves to protect the affected area while it heals. Organisms can become sensitized to a range of noxious and innocuous stimuli, including thermal stimuli. The basic mechanisms underlying sensitization to warm or painfully hot stimuli have begun to be elucidated, however, sensitization to cold is not well understood. Here, we develop a Drosophila assay to study cold sensitization after UV-induced epidermal damage in larvae. Larvae respond to acute cold stimuli with a set of unique behaviors that include a contraction of the head and tail (CT) or a raising of the head and tail into a U-Shape (US). Under baseline, non-injured conditions larvae primarily produce a CT response to an acute cold (10°C) stimulus, however, we show that cold-evoked responses shift following tissue damage: CT responses decrease, US responses increase and some larvae exhibit a lateral body roll (BR) that is typically only observed in response to high temperature and noxious mechanical stimuli. At the cellular level, class III neurons are required for the decrease in CT, chordotonal neurons are required for the increase in US, and chordotonal and class IV neurons are required for the appearance of BR responses after UV. At the molecular level, we found that the transient receptor potential (TRP) channel brivido-1 (brv1) is required for these behavioral shifts. Our Drosophila model will allow us to precisely identify the genes and circuits involved in cold nociceptive sensitization.

Drosophila Insulin receptor regulates the persistence of injury-induced nociceptive sensitization.

Diabetes-associated nociceptive hypersensitivity affects diabetic patients with hard-to-treat chronic pain. Because multiple tissues are affected by systemic alterations in insulin signaling, the functional locus of insulin signaling in diabetes-associated hypersensitivity remains obscure. Here, we used Drosophila nociception/nociceptive sensitization assays to investigate the role of Insulin receptor (Insulin-like receptor, InR) in nociceptive hypersensitivity. InR mutant larvae exhibited mostly normal baseline thermal nociception (absence of injury) and normal acute thermal hypersensitivity following UV-induced injury. However, their acute thermal hypersensitivity persists and fails to return to baseline, unlike in controls. Remarkably, injury-induced persistent hypersensitivity is also observed in larvae that exhibit either type 1 or type 2 diabetes. Cell type-specific genetic analysis indicates that InR function is required in multidendritic sensory neurons including nociceptive class IV neurons. In these same nociceptive sensory neurons, only modest changes in dendritic morphology were observed in the InRRNAi -expressing and diabetic larvae. At the cellular level, InR-deficient nociceptive sensory neurons show elevated calcium responses after injury. Sensory neuron-specific expression of InR rescues the persistent thermal hypersensitivity of InR mutants and constitutive activation of InR in sensory neurons ameliorates the hypersensitivity observed with a type 2-like diabetic state. Our results suggest that a sensory neuron-specific function of InR regulates the persistence of injury-associated hypersensitivity. It is likely that this new system will be an informative genetically tractable model of diabetes-associated hypersensitivity.

Cellular and genetic analysis of wound healing in Drosophila larvae.

To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution.

Novel Assay for Cold Nociception in Drosophila Larvae.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (≤10 ºC), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated. Here we describe and characterize the first noxious cold (≤10 ºC) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Drosophila caspase activity is required independently of apoptosis to produce active TNF/Eiger during nociceptive sensitization.

Tumor necrosis factor (TNF) signaling is required for inflammatory nociceptive (pain) sensitization in Drosophila and vertebrates. Nociceptive sensitization in Drosophila larvae following UV-induced tissue damage is accompanied by epidermal apoptosis and requires epidermal-derived TNF/Eiger and the initiator caspase, Dronc. Major gaps remain regarding TNF function in sensitization, including the relationship between apoptosis/tissue damage and TNF production, the downstream signaling in this context, and the target genes that modulate nociceptive behaviors. Here, apoptotic cell death and thermal nociceptive sensitization are genetically and procedurally separable in a Drosophila model of UV-induced nociceptive sensitization. Activation of epidermal Dronc induces TNF-dependent but effector caspase-independent nociceptive sensitization in the absence of UV. In addition, knockdown of Dronc attenuated nociceptive sensitization induced by full-length TNF/Eiger but not by a constitutively soluble form. UV irradiation induced TNF production in both in vitro and in vivo, but TNF secretion into hemolymph was not sufficient to induce thermal nociceptive sensitization. Downstream mediators of TNF-induced sensitization included two TNF receptor-associated factors, a p38 kinase, and the transcription factor nuclear factor kappa B. Finally, sensory neuron-specific microarray analysis revealed downstream TNF target genes induced during thermal nociceptive sensitization. One of these, enhancer of zeste (E(z)), functions downstream of TNF during thermal nociceptive sensitization. Our findings suggest that an initiator caspase is involved in TNF processing/secretion during nociceptive sensitization, and that TNF activation leads to a specific downstream signaling cascade and gene transcription required for sensitization. These findings have implications for both the evolution of inflammatory caspase function following tissue damage signals and the action of TNF during sensitization in vertebrates.