Inhibition of a NF-κB/Diap1 Pathway by PGRP-LF Is Required for Proper Apoptosis during Drosophila Development

Abstract

NF-κB pathways are key signaling cascades of the Drosophila innate immune response. One of them, the Immune Deficiency (IMD) pathway, is under a very tight negative control. Although molecular brakes exist at each step of this signaling module from ligand availability to transcriptional regulation, it remains unknown whether repressors act in the same cells or tissues and if not, what is rationale behind this spatial specificity. We show here that the negative regulator of IMD pathway PGRP-LF is epressed in ectodermal derivatives. We provide evidence that, in the absence of any immune elicitor, PGRP-LF loss-of-function mutants, display a constitutive NF-κB/IMD activation specifically in ectodermal tissues leading to genitalia and tergite malformations. In agreement with previous data showing that proper development of these structures requires induction of apoptosis, we show that ectopic activation of NF-κB/IMD signaling leads to apoptosis inhibition in both genitalia and tergite primordia. We demonstrate that NF-κB/IMD signaling antagonizes apoptosis by up-regulating expression of the anti-apoptotic protein Diap1. Altogether these results show that, in the complete absence of infection, the negative regulation of NF-κB/IMD pathway by PGRP-LF is crucial to ensure proper induction of apoptosis and consequently normal fly development. These results highlight that IMD pathway regulation is controlled independently in different tissues, probably reflecting the different roles of this signaling cascade in both developmental and immune processes.

Fig 1. PGRP-LF is mostly expressed in ectodermal derivatives.

(A) PGRP-LF^Gal4, UAS-nlsGFP larvae showing GFP expression in salivary glands, foregut, hindgut and cuticle and to a lesser extend in fat body. UAS-nlsGFP larvae in a wild-type genetic background is shown as a negative control. (B) Relative PGRP-LF mRNA expression in third instar larvae dissected tissues. mRNA level in whole larvae was set to 100 and values obtained with dissected tissues were expressed as a fold of this value. For (B) histograms correspond to the mean value ± SD of three independent experiments. Values indicated by symbols (*) are statistically significant (t-test, p < 0.05). ns: not significantly different.

Fig 2. PGRP-LF inactivation triggers AMP ectopic expression in ectoderm derivatives.

(A) AMPs ectopic expression in PGRP-LF mutant larvae (either PGRP-LF^KO or PGRP-LF^KO/Df(3L)BSC113). AttacinD-Cherry; PGRP-LF^KO third instar larvae. AttacinD-Cherry expression is detected in trachea, hindgut, trachea, cuticle and salivary glands. AttacinD-Cherry larvae in a wild-type genetic background is shown as negative control. (B, C) Relative mRNA levels of AttacinD (B) and Diptericin (C) in third instar larvae tissues. mRNA level in control larvae was set to 1, and values obtained with dissected tissues were expressed as a fold of this value. Ct: cuticle, Hg: hindgut, Fb: fat body, Tr: trachea, Sg: salivary glands. For (A) and (B) histograms correspond to the mean value ± SD of three independent experiments. Values indicated by symbols (*) are statistically significant (t-test, p < 0.05). ns: not significantly different.

Fig 3. AMP expression in infected PGRP-LF mutants.

(A) Overexpression of AttacinD and Diptericin mRNAs in PGRP-LF mutant larvae (either PGRP-LF^KO or PGRP-LF^KO/Df(3L)BSC113) requires a functional PGRP-LC/IMD cascade. Inactivation of IMD, Dredd, Diap2 and Relish, but not dMyd88 or PGRP-LE, completely suppresses both AttacinD and Diptericin ectopic expression in PGRP-LF mutants. Expression of UAS-PGRP-LF under the control of PGRP-LF^Gal4strong suppresses the ectopic expression of AMPs observed in PGRP-LF mutants (B) Ectopic activation of AMP is not detected in mutants for other IMD pathway negative regulators such as Pirk or PGRP-LB. (C) IMD pathway activation, monitored by Diptericin expression, 5h after septic infection with Ecc. Although Diptericin is constitutively expressed at higher levels in uninfected PGRP-LF mutants (PGRP-LF^KO/Df(3L)BSC113) than in wild-type, Diptericin mRNA levels are similar in fat body of wild-type and PGRP-LF mutant flies infected by septic injury. (D) IMD pathway activation, monitored by Diptericin expression, 24h after Ecc oral infection. While PGRP-LF inactivation does not modify IMD pathway inducibility in the midgut of Ecc orally infected flies, it does so in the fat body. For (A), (B), (C) and (D) mRNA level in controls was set to 1, and values obtained with indicated genotypes were expressed as a fold of this value. For (A) (B) (C) and (D) histograms correspond to the mean value ± SD of three independent experiments. Values indicated by symbols (*) are statistically significant (t-test, p < 0.05). ns: not significantly different.

Fig 4. Effects of PGRP-LF inactivation on adult lifespan and ability to survive infection.

(A) Survival analysis of control, PGRP-LF^KO/Df(3L)BSC113 and Dredd^D55; PGRP-LF^KO/Df(3L)BSC113 mutants raised in axenic conditions. Ectopic activation of IMD pathway in PGRP-LF mutants reduces lifespan in axenic conditions. (B) Survival analysis of control, PGRP-LF^KO/Df(3L)BSC113, PGRP-LB^KO and PGRP-LC^E12 mutants after septic injury with Ecc. PGRP-LF mutants are as susceptible as controls but less susceptible than PGRP-LC^E12 mutants. (C) Survival analysis of controls and mutants after oral infection with Ecc. PGRP-LF mutants (by either PGRP-LF^KO/Df(3L)BSC113 or PGRP-LF^Gal4strong; UAS-PGRP-LF^RNAi/UAS-Dicer2) and PGRP-LB^KO are highly susceptible to Ecc oral infection. IMD pathway inactivation, via Dredd^D55 mutation, partially suppresses Ecc induced lethality of PGRP-LF mutants. Survival curves are representative of at least five independent trials. Error bars indicate SD.

Fig 5. Adult PGRP-LF mutants display abdominal cuticle and male genitalia defects.

(A) Dorsal (d) and ventral (v) views of male or female abdomens showing incomplete epidermis differentiation (white arrow) and abnormal male genitalia orientation (white bar). (B) Elimination of larval epidermal (LECs) cells is not taking place in PGRP-LF mutants. LECs are labeled with the Ddc^Gal4 driver combined with UAS-GFP. LECs are prominently distributed along the surface of the dorsal abdomen of control pupae at 24 hours APF, but by 48 hours APF most of LECs have been eliminated. By 72 hours APF, only very few intact LECs remains. At 48h and 72h APF, PGRP-LF^KO and Drice^Δ1 mutant pupae present many persistent LECs that accumulate along the midline of the abdomen (white arrow), and at segmental borders (arrowhead). (C) Defective LEC cell death in PGRP-LF mutant cuticle. Pictures show the lateral abdomen of 24h hours APF pupae stained with the anti-cleaved Dcp-1 antibody which labels caspase-activating LECs (arrows). The dashed lines indicate the boundary between the histoblasts and the LECs. (D) Quantification of dying LECs for 6 pupae 24h hours APF is shown in the histogram. For (D) histograms correspond to the mean value ± SD of six samples. Values indicated by symbols (*) are statistically significant (t-test, p < 0.05).

Fig 6. Genitalia rotation is impaired in PGRP-LF mutants.

(A) Time-lapse series of genitalia rotation in His2Av-mRFP/+ (control), His2Av-mRFP/+; PGRP-LF^KO/Df(3L)BSC113 and His2Av-mRFP/+; Drice^Δ1/Drice^Δ1 pupae. Ventral is towards the top in all panels. A and S indicate respectively anus and sexe primordia locations. (B) The genitalia angle (θ) in control (blue), PGRP-LF^KO/Df(3L)BSC113 (black) and Drice^Δ1mutant pupae (gray) was measured every 30 minutes, and the mean angle is shown. Error bars indicate SD (control, n = 5; PGRP-LF^KO, n = 6, Drice^Δ1 n = 5). Velocity (V = dθ /dt) was quantified by measuring θ as a function of time t in control (blue), PGRP-LF^KO/Df(3L)BSC113 (black) and Drice^Δ1 mutant (gray).

Fig 7. Activation of Diap1 expression in PGRP-LF mutants and IMD gain-of-function cells.

(A) Dorsal views of third instar larvae and pupal cases showing ectopic expression of Diap1-GFP4.3 in PGRP-LF mutant LECs, trachea and hindguts. See also S7 Fig. (B) Expression of Diap1 mRNA is higher in hindgut, trachea and cuticle from PGRP-LF mutant larvae than from controls. (C) Diap1-GFP4.3 expression is induced in cells overexpressing IMD. Dorsal epidermis (top images) and fat body (bottom images) of en^Gal4, UAS-RFP/+ (control) and en^Gal4, UAS-RFP/+; UAS-IMD/+ (UAS-IMD) larvae are shown. In LECs and fat body cells, IMD expression induce Diap1-GFP4.3 cell autonomously (arrows). (D) Expression of Diap1 mRNA is induced following overexpression of IMD and PGRP-LCa in adult fat body. Abdomen from 6d old females of the following genotypes were dissected and analyzed by q-RT-PCR: Cg^Gal4/nlsUAS-GFP (control), Cg^Gal4/+; UAS-IMD/+ (UAS-IMD) and Cg^Gal4/+; UAS-PGRP-LCa/+ (UAS-PGRP-LCa). For (B) and (D) mRNA level in control was set to 1, and values obtained with tissues or larvae of indicated genotypes were expressed as a fold of this value. For (B) and (D) histograms correspond to the mean value ± SD of three independent experiments. Values indicated by symbols (*) are statistically significant (t-test, p < 0.05). ns: not significantly different.