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. 2020 Jan 20;9:e51595. doi: 10.7554/eLife.51595

Figure 4. Plasmatocytes promote muscle Dome activity.

(A) Muscle (MHCYFP) and plasmatocytes (srpHemo-3xmCherry) in 7-day-old flies. Plasmatocytes are found in close proximity to adult muscles. One representative fly of 5 is shown. Scale bar = 500 µm. (B) Legs and plasmatocytes in 7-day-old 10xSTAT92E-GFP;srpHemo-3xmCherry flies. Muscle with high JAK-STAT activity (green) is surrounded by plasmatocytes (magenta). One representative fly of 5 is shown. Scale bar = 100 µm. (C) STAT activity and plasmatocytes in legs from control (10xSTAT92E-GFP;crq-Gal4 >CD8-mCherry/+) and upd3-overexpressing (10xSTAT92E-GFP;crq-4>CD8mCherry/UAS-upd3) flies. One representative fly of 10–14 is shown. Scale bar = 100 µm. Graph shows mean fluorescence intensity (MFI). Unpaired T-Test: ***p<0.0001. (D) STAT activity and plasmatocytes in legs from control (10xSTAT92E-GFP;crq-Gal80ts > CD8-mCherry/+) and plasmatocyte-depleted (10xSTAT92E-GFP;crq-Gal80ts > CD8 mCherry/rpr) flies. One representative fly of six is shown. Scale bar = 250 µm. (E) Western blot analysis of STAT-driven GFP in legs from 7-day-old control (10xSTAT92E-GFP;crq-Gal80ts > CD8-mCherry/+) and plasmatocyte-depleted (10xSTAT92E-GFP;crq-Gal80ts > CD8 mCherry/rpr flies). One representative experiment of three is shown. Graph shows STAT-GFP/α-tubulin for control (crq-Gal80ts/+) and plasmatocyte-depleted (crq-Gal80ts > rpr) leg samples. Unpaired T-Test: *p=0.0121. (F) Lifespan of crq-Gal80ts/+, crq-Gal80ts > rpr, upd2Δ upd3Δ;crq-Gal80ts/+, and upd2Δ upd3Δ;crq-Gal80ts > rpr flies at 29°C. Log-Rank test (crq-Gal80ts/+ vs. crq-Gal80ts > rpr): χ2 = 101.7, ***p<0.0001; Wilcoxon test (crq-Gal80ts/+ vs. crq-Gal80ts > rpr): χ2 = 107.8, ***p<0.0001; Log-Rank test (crq-Gal80ts/+ vs. upd2 Δ upd3Δ;crq-Gal80ts > rpr): χ2 = 60.03, ***p<0.0001; Wilcoxon test (crq-Gal80ts/+ vs. upd2 Δ upd3Δ;crq-Gal80ts > rpr): χ2 = 80.97, ***p<0.0001. (G) Actin (Phalloidin) and neutral lipid (LipidTox) in thorax samples from 14-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2 Δ upd3Δ;crq-Gal80ts/+, and upd2 Δ upd3Δ;crq-Gal80ts > rpr flies. One representative fly per genotype shown of 6 analysed per group. Scale bar = 50 µm. (H) Lifespan of crq-Gal4/+ and crq-Gal4 >upd1 IR flies at 29°C. Log-Rank test: χ2 = 31.36, ***p<0.0001; Wilcoxon test: χ2 = 22.17, ***p=0.0001. (I) Expression by qRT-PCR of upd1, upd2 and upd3 in thorax samples of crq-Gal4/+ and crq-Gal4 >upd1 IR flies, data from four independent samples of each genotype.. Unpaired T-Test (upd1): ns p=0.848, unpaired T-Test (upd2): *p=0.0449 and unpaired T-Test (upd3): **p=0.0038. (J) Lifespan of crq-Gal4/+, upd2 Δ upd3Δ;UAS-upd1-IR/+, and upd2Δ upd3Δ;crq-Gal4 >upd1 IR flies at 29°C. Log-Rank test (crq-Gal4/+ vs. upd2 Δ upd3Δ;crq-Gal4 >upd1 IR): χ2 = 41.12, ***p<0.0001; Wilcoxon test (crq-Gal4/+ vs. upd2Δ upd3Δ;crq-Gal4 >upd1 IR): χ2 = 54.47, ***p<0.0001 Log-Rank test (crq-Gal4/+ vs. upd2 Δ upd3Δ;UAS-upd1-IR/+): χ2 = 14.46, ***p<0.0001; Wilcoxon test (crq-Gal4/+ vs. upd2Δ upd3Δ;UAS-upd1-IR/+): χ2 = 19.99, ***p<0.0001. P values in C, E, H from unpaired T-test.

Figure 4.

Figure 4—figure supplement 1. Further characterisation of plasmatocyte-depleted flies.

Figure 4—figure supplement 1.

(A) Plasmatocyte depletion in crq-Gal80ts > rpr flies (right) and controls (left) at 24 hr, 48 hr and 72 hr at 29°C. One out of four flies is shown. Scale bar = 250 µm. (B) Quantification of mCherry-positive plasmatocytes in crq-Gal80ts > rpr flies (right) and controls (left) by flow cytometry after 72 hr at 29°C. n = 3 per group was analysed. Unpaired T-Test: *p=0.0337. (C) Negative geotaxis assay of 14-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2∆upd3∆;UAS-rpr/+ and upd2∆upd3∆;crq-Gal80ts > rpr flies. Points represent mean climbing height of individual vials analysed (~20 flies/vial). Unpaired T-Test: *p=0.0194.
Figure 4—figure supplement 2. Further characterisation of requirements for specific Upds.

Figure 4—figure supplement 2.

(A) Glucose + trehalose and glycogen in 7-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2Δ upd3Δ;crq-Gal80ts/+, and upd2Δ upd3Δ; crq-Gal80ts > rpr flies. (B) TLC of triglyceride in 7-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2Δ upd3Δ;crq-Gal80ts/+, and upd2Δ upd3Δ;crq-Gal80ts > rpr flies, n = 2–3 samples per genotype. (C) Western blot analysis of STAT-driven GFP in legs from 7-day-old w1118, upd3Δ, upd2Δ, and upd2Δ upd3Δ flies. One representative experiment of two is shown. (D) STAT activity and plasmatocytes in legs from 7-day-old control (crq-Gal4/+), upd1 knockdown (crq-Gal4 >upd1 IR), and upd3 knockdown (crq-Gal4 >upd3 IR) flies. One representative fly is shown of 5–7 imaged for each genotype. Scale bar = 100 µm. Mean fluorescence intensity (MFI) is shown for all flies imaged. (E) Western blot analysis of STAT-driven GFP in legs from 7-day-old control (crq-Gal4/+), upd1 knockdown (crq-Gal4 >upd1 IR), and upd3 knockdown (crq-Gal4 >upd3 IR) flies. One of two independent experiments is shown. (F) Western blot analysis of STAT-driven GFP in thorax from 7-day-old crq-Gal4/+, crq-Gal4 >upd1, upd2Δupd3Δ;crq-Gal4/+ and upd2Δupd3Δ;crq-Gal4 >upd1 IR flies. P values in A-F from unpaired T-test.