Embryonic hematopoiesis modulates the inflammatory response and larval hematopoiesis in Drosophila
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
- Centre National de la Recherche Scientifique, France
- Institut National de la Santé et de la Recherche Médicale, France
- Université de Strasbourg, France
Figures
Figure 1 with 4 supplements
Gcm hinders Jak/Stat-mediated melanotic tumor formation.
(a) Third instar larvae of the indicated genotypes (Note that all the detailed genotypes are in the Supplementary Methods, Fly strains and genetics section). White arrowheads indicate the melanotic …
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Figure 1—source data 1
p-values for all figures.
- https://doi.org/10.7554/eLife.34890.007
Figure 1—figure supplement 1
Gcm is not expressed in the second hematopoietic wave.
Control lineage tracing in the lymph gland of Wandering L3 using the lymph gland specific Dot> gtrace line (Kimbrell et al., 2002) (a,a’). Lineage tracing in Wandering L3 using gcmgtrace (b,b’) and h…
Figure 1—figure supplement 2
Crystal cell phenotype in gcm26 embryos.
(a–b’) Immunolabelling of gcm26/CyOactinGFP (a,a’) and gcm26 homozygous embryos (b,b’) (DAPI in blue, GFP in green, PPO1 crystal cell marker in red). (a,b) show merge of the three channels and (a’,b’…
Figure 1—figure supplement 3
Crystal cell phenotype in gcm KD larvae.
(a) gcm RNAi efficiency in S2 cells detected by GFP intensity in gcm GOF + gcm KD transfected S2 cells as compared to controls (n = 9). GFP signal measured upon transfection with pPac-gal4, pUAS-gcm …
Figure 1—figure supplement 4
Gcm inhibits Jak/Stat-mediated melanotic tumor formation.
(a–c) Immunolabelling of hemocytes from Wandering L3 of the mentioned genotypes (DAPI in blue, Phalloidin in green, Srp in red, lamellocyte marker L4 in white), (n > 3). Strong Phalloidin labelling …
Figure 2
Gcm induces the expression of Jak/Stat inhibitors and hinders Jak/Stat-mediated melanotic tumor formation.
(a–c) Immunolabelling of S2 cells transfected with pPac-gal4 and pUAS-RFP (Control, (a), or pPac-gal4, pUAS-RFP and pUAS- hopTum-l (hopTum-l GOF, (b), or pPac-gal4, pUAS-RFP, pUAS- hopTum-l and pPac-…
Figure 3
Embryonic hemocytes signal to the lymph gland.
(a) Embryonic hemocytes (red) in early and late embryos as well as in a third instar larva. The lymph gland (blue) histolyzes at the larva to pupa transition. (b) Number and percentage of lymph …
Figure 4 with 1 supplement
Embryonic hemocytes signal through Upd2 and Upd3.
(a) Lymph gland phenotypes. (b,c) Lymph gland immunolabelling as in Figure 3c–f. (d) Tumor penetrance. (e–f) Relative expression levels of upd2 and upd3 in hemocytes from wandering L3 larvae of the …
Figure 4—figure supplement 1
Interaction between Jak/Stat pathway, Gcm and Upd2/Upd3 cytokines.
(a) Tumor penetrance in double heterozygous female larvae hopTum-l/upd2Δ and hopTum-l/upd3Δ. (b,b’) upd2 and upd3 expression levels in hemocytes sorted from stage 16 control embryos (srp(hemo)Gal4/UA…
Figure 5 with 1 supplement
Inflammatory response upon systemic and conditional Jak/Stat activation.
(a) Tumor penetrance, phenotype expressivity assessed as number of tumors/larva and tumor size of control animals (gcm> ), systemic hopTum-l, conditional hopTum-l (gcm> UAS-hopTum-l) and conditional …
Figure 5—figure supplement 1
Phenotypes induced by conditional activation of the Jak/Stat pathway in the embryonic hemocytes.
(a) Tumor penetrance in conditional hopTum-l animals (UAS-hopTum-l) using srp(hemo)> , sn> , gcm> and gcm(hemo)> drivers as compared to hopTum-l/+ (n > 50). (b) Tumor expressivity assessed as tumor …
Figure 6
Jak/Stat activation in the embryonic hemocytes activates the Jak/Stat pathway in Wandering L3 muscles.
(a–c) Muscle immunolabelling in Wandering larvae of the indicated genotypes. The Phalloidin labelling (in green) highlights the striated muscle fibers rich in actin filament, Stat92E is in red and …
Figure 7
The inflammatory response upon wasp infestation is exacerbated in gcm KD animals.
(a) Histogram representing the percentage of total, partial and no wasp egg encapsulation (n > 30). (b) Lethality of the parasitic wasp after infestation of Drosophila larvae (n = 4) (> 200 animals).
Figure 8 with 1 supplement
Gcm impacts the development and the inflammatory response of hemocytes.
Relative expression levels of plasmatocyte and lamellocyte markers, Jak/Stat inhibitor and cytokines in hemocytes from stage 16 mutant embryos (srp(hemo)> RFP,gcm [Cattenoz et al., 2016]) or from …
Figure 8—figure supplement 1
Gcm is not induced in circulating hemocytes nor in lymph glands of 3rd instar larvae upon wasp infestation.
Immunolabelling of hemocytes from third instar larvae gcm> GFP without (a) or after wasp infestation (b). (DAPI in blue, GFP in green and Phalloidin in gray). (c) Immunolabelling of the lymph gland …
Figure 9
Impact of the wasp infestation on the hemocytes.
(a) Schematic of the correspondence between the timeline after egg laying (AEL) and the larval developmental stage. The wasp infestation was carried out at the L2 stage. (b–c) RFP signals from HmlΔRF…
Figure 10
Wasp infestation recruits first the embryonic hemocytes and then the LG hemocytes.
Timeline summarizing the sequence of events occurring in the immune system of the larva in normal conditions (top panel) and upon wasp infestation (lower panel). In normal conditions, only embryonic …
Figure 11
Schematic model on the molecular cascade leading to tumor formation upon Jak/Stat activation in the embryonic hemocytes.
(a) gcm> hopTum-l activates the Jak/Stat pathway exclusively in the hemocytes at embryonic stage. This leads to the production of the Upd2/Upd3 pro-inflammatory cytokines in those cells and to the …
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Tables
Key resources table
| Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|
| Mouse anti-L4 | Pr. I. Ando | Mouse anti-L4 | (1/30) |
| Rabbit anti-PH3 | Upstate biotechnology #06–570 | Rabbit anti-PH3 | (1/1000) |
| Mouse anti-Hemese | Pr. I. Ando | Mouse anti-Hemese | (1/30) |
| Chicken anti-GFP | abcam #13970 | Chicken anti-GFP | (1/500) |
| Rabbit anti-Serpent | Trebuchet, unpublished | Rabbit anti-Serpent | (1/1000) |
| Rabbit anti-Stat92E | Dr. D. Montell | Rabbit anti-Stat92E | (1/100) |
| Rabbit anti-PPO1 | Dr. WJ. Lee | Rabbit anti-PPO1 | (1/100) |
| Rabbit anti-RFP | abcam #62341 | Rabbit anti-RFP | (1/500) |
| S2 cells | ATCC Ref: CRL-1963 | S2 ATCC | |
| Effectene Transfection Reagent | Qiagen | Effectene Transfection Reagent | |
| WT | Bloomington #5905 | w1118 | |
| hopTum-l | Bloomington #8492 | hopTum-l/FM7c | Point mutation that constitutively activates the Jak/Stat pathway |
| HmlΔRFP | Makhijani et al., 2011 | HmlΔRFP | |
| UAS-hopTum-l | Harrison et al., 1995 | UAS-hopTum-l/CyO, twilacZ | Reporter line for hop Tum-l over-expression |
| Gcm> GFP | Soustelle and Giangrande, 2007 | gcmGal4,UAS-mCD8 GFP/CyO,Tb | Driver specific to embryonic hemocytes and glia, gcm hypomorphic mutation |
| Gcm KD | Bloomington #31519 | UAS-gcmRNAi | dsRNA reporter line for gcm down-regulation |
| Gcm GOF | Bernardoni et al., 1997 | UAS-gcmF18A | Reporter line for gcm over-expression |
| Gcm26 | Vincent et al., 1996 | gcm26/CyOactinGFP | Null gcm mutation |
| Upd2Δ | Bloomington #55727 | upd2Δ | 4.7 kb deletion |
| Upd3Δ | Bloomington #55728 | upd3Δ | Imprecise excision |
| Upd2 KD | Bloomington #33988 | UAS-upd2RNAi | dsRNA reporter line for upd2 down-regulation |
| Upd3 KD | Bloomington #32859 | UAS-upd3RNAi | dsRNA reporter line for upd3 down-regulation |
| Upd2 GOF | Jiang et al., 2009 | UAS-upd2/CyO | Reporter line for upd2 over-expression |
| Upd3 GOF | Jiang et al., 2009 | UAS-upd3/CyO | Reporter line for upd3 over-expression |
| Ptp61F KD | Bloomington #32426 | UAS-Ptp61FRNAi | dsRNA reporter line for Ptp61F down-regulation |
| Socs36E KD | Bloomington #35036 | UAS-Socs36ERNAi | dsRNA reporter line for Socs36E down-regulation |
| Socs44A KD | Bloomington #42830 | UAS-Socs44ARNAi | dsRNA reporter line for Socs44A down-regulation |
| Ptp61Fa GOF | Müller et al., 2005 | UAS-Ptp61Fa/CyO | Reporter line to over-express the cytoplasmic splicing isoform |
| Ptp61Fc GOF | Müller et al., 2005 | UAS-Ptp61Fc/TM3 | Reporter line to over-express the nuclear splicing isoform |
| Gcm(hemo)> | Cattenoz et al., 2016 | gcmGal4,UAS-mCD8GFP, repoGal80/CyO | gcm driver not expressed in glia, hypomorphic mutation |
| sn> | Zanet et al., 2012 | snGal4 | Singed driver, specific to embryonic hemocytes |
| srp(hemo)> | Brückner et al., 2004 | srp(hemo)Gal4 | Serpent driver specific to embryonic hemocytes |
| dot> | Bloomington #67608 | DotGal4 | Dorothy driver specifically expressed in embryonic and larval lymph gland |
| lz> GFP | Bloomington #6314 | lzGal4,UAS-mCD8GFP | Lozenge driver expressed in crystal cells |
| 10xStat92E-GFP | Bloomington #26198 | 10xStat92E-GFP | Reporter line for STAT activity, 10 Stat92E binding sites driving GFP expression |
| Gtrace | Bloomington #28282 | UAS-FLP;;Ubi-p63E (FRT.STOP)Stinger | This line allows the analysis of lineage-traced expression of Gal4 drivers |
| Ubi> | Bloomington #32551 | UbiGal4 | Expresses GAL4 in all cells |
| RFP | Bloomington #30556 | UAS-RFP | Inserted on the second chromosome |
| Gtrace-LacZ | Bloomington #6355 | P{Act5C> polyA> lacZ.nls1}3 | |
| Fiji | Schindelin et al., 2012 | Fiji | |
| pPac-gcm | Miller et al., 1998 | pPac-gcm | |
| repoGFP | Laneve et al., 2013 | 4.3 kb repo-GFP | |
| pUAS-hopTum-l | Harrison et al., 1995 | pUAS-hopTum-l | |
| pUAS-gcmRNAi | Vienna Drosophila Resource Center (VDRC) #dna1452 | pUAS-gcmRNAi |
Additional files
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Supplementary file 1
details the materials and methods used in the manuscript
- https://doi.org/10.7554/eLife.34890.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.34890.022
