Cytokine Signaling Mediates UV-Induced Nociceptive Sensitization in Drosophila Larvae  Daniel T. Babcock, Christian Landry, Michael J. Galko  Current Biology  Volume 19, Issue 10, Pages 799-806 (May 2009) DOI: 10.1016/j.cub.2009.03.062 Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 1 Larval Responses to Thermal Stimuli and UV Radiation (A) Diagram of a thermal probe illustrating the thermal control unit, feedback design (arrows), and brass tip. Box shows area of interest in (B). (B) Schematic diagram illustrating the major components of the heat probe. (C) Aversive response of third-instar w1118 larvae to stimulation at various temperatures. Behaviors were classified as “no response” (white, > 20 s), “slow withdrawal” (gray, between 5 and 20 s), or “fast withdrawal” (black, < 5 s). n = 50 for each temperature tested. (D) Average withdrawal latency at different temperatures. 44°C = 9.0 s ± 1.5 s and 48°C = 1.5 s ± 0.8 s. n = 50 for each temperature. Error bars indicate standard error of the mean. The median withdrawal time was significantly different between groups (p < 0.001, by log-rank test). (E) Survival rate of third-instar larvae to adulthood after treatment with increasing doses of ultraviolet radiation. n = 75 for each group. Current Biology 2009 19, 799-806DOI: (10.1016/j.cub.2009.03.062) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 2 Epidermal Damage Induced by UV Radiation (A–P) Whole-mount staining showing the cellular effects of mock treatment (A–D) or UV treatment on larval tissues at 4 hr (E–H), 24 hr (I–L), or 48 hr (M–P) after exposure. (A, E, I, and M) Epidermal cell membranes of w1118;ppk1.9-Gal4, UAS-eGFP larvae labeled with anti-Fasciclin III. (B, F, J, and N) Class IV dendritic-arborization sensory neurons labeled in the same ppk1.9-Gal4, UAS-eGFP larvae as in (A), (E), (I), and (M). (C, G, K, and O) X-Gal staining of msn-lacZ larvae. (D, H, L, and P) Apoptotic cells of w1118 larvae labeled with an activated caspase-3 antibody (purple) and anti-Fasciclin III (green). Scale bar in (P), 100 μm for all panels. Current Biology 2009 19, 799-806DOI: (10.1016/j.cub.2009.03.062) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 3 Thermal Allodynia and Hyperalgesia after Tissue Damage (A and B) Comparison of response thresholds between UV-treated and mock-treated w1118 larvae. (A) Response to the highest normally innocuous temperature (38°C; see Figure 1B) at specified times after UV-induced tissue damage. White = no response, gray = response between 5 and 20 s, and black = response in less than 5 s. Nociceptive threshold distributions are significantly different (p < 0.001, by Fisher's exact test) from those of mock-treated larvae (asterisks) at 8, 16, and 24 hr after UV exposure. Mock-treated larvae were assessed 24 hr after mock irradiation so that their response could be compared with the greatest response seen in UV-treated larvae. (B) Responses to decreasing temperatures below the normal nociceptive threshold 24 hr after tissue damage. Gray = mock treated, and hatched = UV treated. (C and D) Comparison of withdrawal latencies between UV-treated larvae and mock-treated w1118 larvae. (C) Response to a normally noxious temperature (45°C) at specified times after UV-induced tissue damage. White = no response, gray = response between 5 and 20 s, black = response in less than 5 s. Nociceptive threshold classification is significantly different (asterisks) from that of mock-treated larvae at 8 (p < 0.001) and 16 (p = 0.039) hours after UV exposure. Mock-treated larvae were assessed 8 hr after mock irradiation so that their response could be compared with the greatest response seen in UV-treated larvae. (D) Average withdrawal latency to 45°C stimulation at various times after tissue damage. Error bars indicate standard error of the mean. Mean withdrawal was significantly different (p < 0.001, by Student's t test) from that of mock-treated larvae (asterisks) at 8 hr after exposure. n = 50 for all conditions. Current Biology 2009 19, 799-806DOI: (10.1016/j.cub.2009.03.062) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 4 Dronc Activity in Epidermal Cells Is Required for Thermal Allodynia (A–I) Whole-mount staining of w1118;A58-Gal4/+ (A–C), w1118;UAS-droncIR/+ (D–F), and w1118;UAS-droncIR/+;A58-Gal4/+ larvae (G–I). (A, D, and G) Epidermal cell membranes (green) labeled with anti-Fasciclin III. (B, E, and H) Apoptotic cells (red) labeled with an activated caspase-3 antibody. (C,F, and I) Merged panels. The scale bar in (I) represents 100 μm for (A)–(I). (J) Response to the highest normally innocuous temperature (38°C) 24 hr after UV exposure. Larvae bearing both the A58-Gal4 and UAS-DroncIR inserts are significantly less sensitive (p < 0.001, by Fisher's exact test) than the parental control strains when tested for thermal allodynia. (K) Response to a normally noxious temperature (45°C, see Figure 1C) 8 hr after UV exposure. Larvae bearing both the A58-Gal4 and UAS-DroncIR inserts displayed no statistically significant differences from the parental control lines or the w1118 control line (Figure 3D) when tested for thermal hyperalgesia (p = 0.659, by one-way ANOVA). (L and M) Response to the highest normally innocuous temperature (38°C) 24 hr after UV exposure (L) or to a normally noxious temperature (45°C) 8 hr after UV exposure (M). Larvae lacking hemocytes (Pxn-Gal4 > UAS-hid) displayed no differences from the parental control lines when tested for thermal allodynia or hyperalgesia. n = 30 for each group in (J) through (M). Error bars indicate standard error of the mean. Current Biology 2009 19, 799-806DOI: (10.1016/j.cub.2009.03.062) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 5 Thermal Allodynia Is Induced by TNF Signaling (A) Response to the highest normally innocuous temperature (38°C) of eiger mutants or larvae lacking either Eiger or Wengen in epidermal cells (A58-Gal4) or in nociceptive sensory neurons (ppk1.9-Gal4) 24 hr after UV treatment. (B) Response of control larvae to a normally innocuous temperature (38°C) and reponse of larvae with ectopic expression of Eiger in class IV sensory neurons in the absence of UV treatment. White = no response within 20 s, gray = response between 5 and 20 s, and black = response in less than 5 s. n = 30 for each condition. An asterisk indicates p < 0.001 in comparison to the wild-type by Fisher's exact test. (C–E) Whole-mount staining of egr1/egr3 larvae. (C) Epidermal cell membranes (green) labeled with anti-Fasciclin III. (D) Apoptotic cells (red) labeled with an activated caspase-3 antibody. (E) Merged panel. (F) Response to innocuous (38°C) and noxious (48°C) temperatures in the absence of tissue damage. Neither eiger mutants nor control larvae responded within 20 s to normally subthreshold temperatures (white bars). However, all larvae within all groups rapidly withdrew from noxious stimuli (black bars), suggesting that normal nociception is not impaired in eiger mutants. Epidermal knockdown of Eiger and nociceptive sensory neuron knockdown of Wengen also did not affect normal nociception. Error bars indicate standard error of the mean. n = 25 for each condition. The scale bar in (E) represents 100 μm for (C–E). Current Biology 2009 19, 799-806DOI: (10.1016/j.cub.2009.03.062) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 6 Model Top-down illustration depicting the relationship between UV-induced epidermal damage and changes in underlying sensory neurons. Ultraviolet light is absorbed by epidermal cells (1), which eventually undergo apoptosis and activate Dronc (2). As a result, Eiger is produced (3) and released from epidermal cells (4) and ultimately activates Wengen on the nociceptive sensory neuron membrane (5). This activation leads to intracellular signaling that sensitizes the nociceptor (6) to normally subthreshold stimuli. VNC = ventral nerve cord. Current Biology 2009 19, 799-806DOI: (10.1016/j.cub.2009.03.062) Copyright © 2009 Elsevier Ltd Terms and Conditions