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

Integrin Adhesions Suppress Syncytium Formation in the Drosophila Larval Epidermis

Funding Information: We thank members of M.J.G.’s lab for comments; Jodie Polan for confocal assistance; Guy Tanentzapf, Andreas Wodarz, and Talila Volk, for fly stocks/antibodies; the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and the Kyoto stock center for fly strains; and the Developmental Studies Hybridoma Bank for antibodies. This work was supported by a March of Dimes Basil O’Connor Award (5-FY06-588) and NIH R01 GM083031 to M.J.G., NIH R01 GM084103 to J.L.K., and European Research Council Starting Grant (2007-StG-208631) to A.J. Publisher Copyright: © 2015 Elsevier Ltd. All rights reserved.Integrins are critical for barrier epithelial architecture. Integrin loss in vertebrate skin leads to blistering and wound healing defects. However, how integrins and associated proteins maintain the regular morphology of epithelia is not well understood. We found that targeted knockdown of the integrin focal adhesion (FA) complex components β-integrin, PINCH, and integrin-linked kinase (ILK) caused formation of multinucleate epidermal cells within the Drosophila larval epidermis. This phenotype was specific to the integrin FA complex and not due to secondary effects on polarity or junctional structures. The multinucleate cells resembled the syncytia caused by physical wounding. Live imaging of wound-induced syncytium formation in the pupal epidermis suggested direct membrane breakdown leading to cell-cell fusion and consequent mixing of cytoplasmic contents. Activation of Jun N-terminal kinase (JNK) signaling, which occurs upon wounding, also correlated with syncytium formation induced by PINCH knockdown. Further, ectopic JNK activation directly caused epidermal syncytium formation. No mode of syncytium formation, including that induced by wounding, genetic loss of FA proteins, or local JNK hyperactivation, involved misregulation of mitosis or apoptosis. Finally, the mechanism of epidermal syncytium formation following JNK hyperactivation and wounding appeared to be direct disassembly of FA complexes. In conclusion, the loss-of-function phenotype of integrin FA components in the larval epidermis resembles a wound. Integrin FA loss in mouse and human skin also causes a wound-like appearance. Our results reveal a novel and unexpected role for proper integrin-based adhesion in suppressing larval epidermal cell-cell fusion - a role that may be conserved in other epithelia.publishersversionpublishe

Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila

Pain signaling in vertebrates is modulated by neuropeptides like Substance P (SP). To determine whether such modulation is conserved and potentially uncover novel interactions between nociceptive signaling pathways we examined SP/Tachykinin signaling in a Drosophila model of tissue damage-induced nociceptive hypersensitivity. Tissue-specific knockdowns and genetic mutant analyses revealed that both Tachykinin and Tachykinin-like receptor (DTKR99D) are required for damage-induced thermal nociceptive sensitization. Electrophysiological recording showed that DTKR99D is required in nociceptive sensory neurons for temperature-dependent increases in firing frequency upon tissue damage. DTKR overexpression caused both behavioral and electrophysiological thermal nociceptive hypersensitivity. Hedgehog, another key regulator of nociceptive sensitization, was produced by nociceptive sensory neurons following tissue damage. Surprisingly, genetic epistasis analysis revealed that DTKR function was upstream of Hedgehog-dependent sensitization in nociceptive sensory neurons. Our results highlight a conserved role for Tachykinin signaling in regulating nociception and the power of Drosophila for genetic dissection of nociception

Involvement of Skeletal Muscle Gene Regulatory Network in Susceptibility to Wound Infection Following Trauma

Despite recent advances in our understanding the pathophysiology of trauma, the basis of the predisposition of trauma patients to infection remains unclear. A Drosophila melanogaster/Pseudomonas aeruginosa injury and infection model was used to identify host genetic components that contribute to the hyper-susceptibility to infection that follows severe trauma. We show that P. aeruginosa compromises skeletal muscle gene (SMG) expression at the injury site to promote infection. We demonstrate that activation of SMG structural components is under the control of cJun-N-terminal Kinase (JNK) Kinase, Hemipterous (Hep), and activation of this pathway promotes local resistance to P. aeruginosa in flies and mice. Our study links SMG expression and function to increased susceptibility to infection, and suggests that P. aeruginosa affects SMG homeostasis locally by restricting SMG expression in injured skeletal muscle tissue. Local potentiation of these host responses, and/or inhibition of their suppression by virulent P. aeruginosa cells, could lead to novel therapies that prevent or treat deleterious and potentially fatal infections in severely injured individuals

Induction and Function of the JNK Pathway around Puncture Wounds

<div><p>(A–C) Larvae carrying the JNK pathway reporter <i>puc-lacZ,</i> which expresses a nuclear β-galactosidase, were mock-wounded (A) or puncture wounded (B and C), and then cultured for the indicated times before staining with X-gal to visualize reporter activity (blue). There is little reporter activity in unwounded epidermis (A), but 4 h after wounding the reporter is expressed in a gradient emanating from the wound, with highest expression in the row of epidermal nuclei at the wound margin and decreasing levels in surrounding nuclei out to five cell diameters away. At 24 h (C), reporter expression has declined.</p> <p>(D–F) Larvae carrying the JNK pathway reporter <i>msn-lacZ,</i> treated as above. Wounding-induced reporter expression is seen out to seven cell diameters.</p> <p>(G–I) Larvae carrying <i>msn-lacZ</i> and <i>A58-Gal4</i> and <i>UAS-bsk^DN</i> transgenes (to inactivate the JNK pathway in larval epidermis), treated as above. Reporter induction is inhibited, but the scab forms normally.</p> <p>(J and K) Larvae carrying <i>msn-lacZ</i> and either <i>UAS-bsk^DN</i> alone as control (J) or <i>A58-Gal4</i> and <i>UAS-bsk^DN</i> transgenes (K), wounded as above and analyzed 24 h later by immunostaining for Fasciclin III and β-galactosidase. Reporter induction is inhibited in (K), but epidermal cells have oriented toward the wound, and although nuclear β-galactosidase staining is faint, careful inspection shows that the cells closest to the wound have fused to form a syncytium. Syncytium formation was confirmed using the <i>A58-Gal4>UAS-GFP.nls</i> marker.</p> <p>(L and M) Larvae carrying <i>msn-lacZ</i> and either <i>UAS-bsk^DN</i> alone as control (L) or <i>A58-Gal4</i> and <i>UAS-bsk^DN</i> transgenes (M), wounded and analyzed 24 h later by TEM. Note that the epidermis in M has failed to spread across the wound gap and is still discontinuous (asterisks). No cuticle has been synthesized in the wound gap, but the cuticle flanking the wound appears thickened.</p> <p>Bar in (I), 100 μm (for [A–I]). Bar in (K), 50 μm (for [J and K]). Bar in (M), 5 μm (for [L and M]).</p></div

Ultrastructural Analysis of Puncture Wound Healing

<div><p>(A) Schematic of unwounded epidermis showing the cell monolayer, its apical cuticle lining, and basal lamina. White ovals indicate nuclei.</p> <p>(B) Schematic of recently wounded epidermis showing a plug of cell debris in the wound gap. Cells and ruptured cuticle at the wound margin are shown.</p> <p>(C–S) TEM sections of unwounded (C–F) and wounded (G–S) larvae at the times indicated after wounding. Transverse sections through each wound site are shown (C, G, J, N, and Q) along with close-ups of the boxed regions at right. c, cuticle; d, debris; e, epidermis; ec, epicuticle; m, muscle; n, epidermal nucleus; p, plug; pc, procuticle; s, scab; t, trachea</p> <p>(C) Pre-wounding. The epidermis and cuticle are intact.</p> <p>(D) Apical surface of epidermal cell showing villi (arrowhead) that secrete cuticle.</p> <p>(E) Basal surface. Arrowhead, basal lamina.</p> <p>(F) Epidermal cuticle. The epicuticle (top three layers) overlies the striated procuticle layer.</p> <p>(G) 1 h postwounding. The epidermis and cuticle are discontinuous but the gap is filled with a plug (outlined by dashed line) of cell debris. The epidermis has partially separated from the cuticle beyond the wound margin (asterisks).</p> <p>(H) The plug contains highly vesiculated cell debris.</p> <p>(I) The epidermis separating (asterisk) from overlying cuticle appears vesiculated and is presumably necrotic.</p> <p>(J) 2 h postwounding. The outer portion of the plug has melanized to form an electron-dense scab (outlined by white dashed line). The epidermis and cuticle are still discontinuous.</p> <p>(K) Debris, including a necrotic trachea, in the plug. The plug is not bounded by a membrane or basal lamina.</p> <p>(L) Portion of scab showing melanized debris and trachea.</p> <p>(M) Close-up of a lamellipodium (bracket) extending into a plug at the outer edge of another 2-h wound. Note basal lamina (arrowhead) along the lamellipodium.</p> <p>(N) 8 h postwounding. The epidermis has migrated across the gap to reestablish continuity, and has secreted new cuticle beneath the scab.</p> <p>(O) A region of epidermal cell cytoplasm near wound plug debris contains vesiculated material (outlined by dashed line) that is probably phagocytosed debris.</p> <p>(P) The newly established epidermis under the wound has a continuous basal lamina (arrowhead) and apical villi (arrow) secreting cuticle.</p> <p>(Q) 24 h postwounding. The new cuticle underlying the scab is thicker and the scab is more electron dense. Four nuclei in close apposition are in a syncytium because there are no membranes separating them.</p> <p>(R) Portion of scab and old cuticle. Note that cuticle in contact with the scab is melanized.</p> <p>(S) Cytoplasmic extension (arrowhead) engulfing debris at the basal surface of the epidermis of another 24-h wound.</p> <p>Bar, 10 μm (C, G, J, N, and Q), 0.33 μm (D and E), 0.83 μm (F), 1 μm (H, M, and P), 2 μm (I and O), 1.67 μm (K and L), 4 μm (R), 1.25 μm (S).</p></div

Scab Formation and Resolution during Puncture Wound Healing

<div><p>(A) Puncture wound assay. L3 larvae are punctured at the dorsal midline with a 100-μm diameter pin; they are then cultured and the healing wounds analyzed as shown.</p> <p>(B–E) Photomicrographs of heat-killed L3 larvae before wounding (B) and at the indicated times after wounding (C–E). Note larval growth during wound healing. Anterior is up.</p> <p>(F) L2 larva wounded as above and analyzed in L3, 60 h after wounding. Wounding in L2 allows visualization of late stages of wound healing without the complication of pupariation, which begins about 48 h after wounding in the standard L3 assay.</p> <p>(G) A mock-wounded L2 larva visualized 60 h after wounding. Note that it and the wounded larva (F) grew to a similar extent.</p> <p>(H–M) Close-up images of (B–G) showing unwounded cuticle (H and M) or wound sites (I–L, boxed regions in C–F) to show detail of scab. Micrographs are of living larvae taken shortly before the corresponding images of the whole heat-killed larvae above. Bar, 500 μm (for B–G), 50 μm (for H–M).</p> <p>(N) Timing of wound responses. Solid lines, time response was most often observed; dashed extensions to left, time response was occasionally observed; dashed extensions to right, time response was diminishing; BL, basal lamina.</p></div