The endoplasmic reticulum unfolded protein response varies depending on the affected region of the tissue but independently from the source of stress

Abstract

Accumulation of unfolded proteins and calcium dyshomeostasis induces endoplasmic reticulum (ER) stress, which can be resolved by the unfolded protein response (UPR). We have previously reported that activation of the PERK/ATF4 branch of the UPR, by overexpressing Presenilin in part of the vestigial domain of Drosophila wing imaginal discs, induces both a caspase-dependent apoptosis and a Slpr/JNK/Dilp8-dependent developmental delay that allows compensation of cell death in the tissue. Recently, dDad1 depletion in Drosophila in engrailed-expressing cells of wing imaginal discs was also reported to activate the PERK/ATF4 branch but induced Mekk1/JNK-dependent apoptosis. Here, we assessed whether the stressed cell location in the wing imaginal disc could explain these differences in response to chronic ER stress or whether the stress source could be responsible for the signaling discrepancy. To address this question, we overexpressed a Rhodopsin-1 mutant prone to aggregate either in vestigial- or engrailed-expressing cells. We observed similar responses to the Presenilin overexpression in the vestigial domain and to the dDad1 depletion in the engrailed domain. Therefore, the consequences of a PERK/ATF4 branch activation depend on the position of the cell in the Drosophila wing imaginal disc, suggesting interactions of PERK signaling with developmental pathways involved in the determination or maintenance of wing domains.

Keywords: Apoptosis; Homeostasis; PERK; UPR; Wing imaginal disc.

Fig. 1

Rh-1^G69D expression at the wing disc dorso-ventral boundary induces both cell death and JNK pathway activation in a PERK/ATF4-dependent manner. a Projections from confocal stacks of male third-instar larval wing imaginal discs immunostained with an anti-activated Dcp-1 antibody (top) and expressing the TRE reporter of JNK signaling (bottom). From left to right, genotypes are vg-Gal4, TRE-red-2L/+ (Ctrl), vg-Gal4, TRE-red-2L/+; UAS-Rh1^G69D/+ (Rh1^G69D), vg-Gal4, TRE-red-2L/+; UAS-Rh1^G69D/UAS-Atf4-RNAi (Rh1^G69D, Atf4^RI) and vg-Gal4, TRE-red-2L/+; UAS-Rh1^G69D/UAS-Perk-RNAi (Rh1^G69D, Perk^RI). b Quantification of apoptosis and JNK reporter stainings on wing discs of the previous genotypes. Error bars indicate S.E.M. Statistical difference with the control is indicated by * if p < 5% or *** if p < 10⁻³ (Mann-Whitney U test)

Fig. 2

Rh-1^G69D expression-induced apoptosis in the wing disc dorso-ventral boundary is JNK independent. a Projections from confocal stacks of male third-instar larval wing imaginal discs immunostained with an anti-activated Dcp-1 antibody. From left to right, genotypes of control discs on the top row are vg-GAL4/+, vg-GAL4/UAS-bsk-RNAi, and vg-GAL4/UAS-hep-RNAi. From left to right, wing imaginal discs expressing ectopically Rh1^G69D (bottom row) display the following genotypes: vg-GAL4/+; UAS-Rh1^G69D/+, vg-GAL4/UAS-bsk-RNAi; UAS-Rh1^G69D/+ and vg-GAL4/UAS-hep-RNAi; UAS-Rh1^G69D/+. b Quantification of apoptosis stainings on wing discs for the previously described genotypes allowing Rh-1^G69D ectopic expression. Error bars indicate S.E.M. The Mann-Whitney U test revealed no statistic difference

Fig. 3

Dilp8 expression in wing imaginal disc cells expressing Rh-1^G69D depends on the Rac1/Slpr/JNK pathway. a Fluorescence intensity of GFP using a fire lookup table (ImageJ) reflecting the expression of a dilp8::EGFP reporter transgene in projections from confocal stacks of male third-instar larval wing imaginal discs. (Top row) From left to right, control genotypes are vg-GAL4/+; dilp8^MI00727/+, vg-GAL4/UAS-Mekk1-RNAi; dilp8^MI00727/+, vg-GAL4/UAS-slpr-RNAi; dilp8^MI00727/+ and vg-GAL4/UAS-bsk-RNAi; dilp8^MI00727/+. (Bottom row) From left to right, Rh-1^G69D expressing wing disc genotypes are vg-GAL4/+; dilp8^MI00727/UAS-Rh1^G69D, vg-GAL4/UAS-Mekk1-RNAi; dilp8^MI00727/UAS-Rh1^G69D, vg-GAL4/UAS-slpr-RNAi; dilp8^MI00727/UAS-Rh1^G69D and vg-GAL4/UAS-bsk-RNAi; dilp8^MI00727/UAS-Rh1^G69D. b Quantification of Dilp8::EGFP was performed for the previously described genotypes allowing Rh-1^G69D ectopic expression. Error bars represent the S.E.M. The asterisks indicate significant differences between the indicated genotype and its control (p < 10⁻³, Mann-Whitney U test). cdilp8 RNA levels measured by quantitative reverse PCR. Data represent mean ± S.E.M. of three independent experiments. RNA was extracted from control (vg-GAL4/+, open bar), from ER stressed (vg-GAL4/+; UAS-Rh1^G69D/+, black bar), or from Rac1-depleted ER-stressed (vg-GAL4/UAS-rac1-RNAi; UAS-Rh1^G69D/+, gray bar) wing imaginal discs. ** indicates a significant difference with the control (p < 1%, Student’s t test)

Fig. 4

Rh-1^G69D expression in the wing posterior compartment activates both JNK signaling and apoptosis in a PERK/ATF4-dependent manner. a Projections from confocal stacks of male third-instar larval wing imaginal discs expressing the TRE reporter of JNK signaling (top) and immunostained with an anti-activated Dcp-1 antibody (bottom). From left to right, genotypes are en-GAL4, TRE-red-2L/+; UAS-Rh1^G69D/+ (Rh1^G69D), en-GAL4, TRE-red-2L/+; UAS-Rh1^G69D/UAS-Atf4-RNAi (Rh1^G69D, Atf4^RI) and en-GAL4, TRE-red-2L/+; UAS-Rh1^G69D/UAS-Perk-RNAi (Rh1^G69D, Perk^RI). b Quantification of apoptosis (top) and JNK reporter (bottom) stainings on wing discs of the previous genotypes. Error bars indicate S.E.M. Statistical difference with the control is indicated by * if p < 5% or *** if p < 10⁻³ (Mann-Whitney U test)

Fig. 5

Rh-1^G69D expression in the wing posterior compartment induces a Mekk1/JNK-dependent apoptosis. a Projections from confocal stacks of male third-instar larval wing imaginal discs immunostained with an anti-activated Dcp-1 antibody. From left to right, genotypes of wing imaginal discs expressing ectopically Rh1^G69D are en-GAL4/+; UAS-Rh1^G69D/+, en-GAL4/UAS-bsk-RNAi; UAS-Rh1^G69D/+ and en-GAL4/UAS-Mekk1-RNAi; UAS-Rh1^G69D/+. b Quantification of apoptosis stainings on wing discs of the previous genotypes. Error bars indicate S.E.M. Statistical difference with the control is indicated by *** (p < 10⁻³, Mann-Whitney U test)

Fig. 6

Proposed model of ER stress pathways in Drosophila. Mutated aggregation-prone Rh1 induces different pathways depending on the tissue location. As in the compartment posterior to the morphogenetic furrow of the eye imaginal disc (a, green domain), ectopic Rh1^G69D triggers a JNK-dependent cell death in the posterior compartment (blue domain) but not on the dorso-ventral frontier (orange line) of the wing imaginal disc (b). Both mutant Rh1^G69D and depletion of dDad1, which is essential to N-glycosylation, induce the same unfolded protein response to ER stress in the posterior compartment of wing discs. On the dorso-ventral frontier, mutant Rh1^G69D activates a different unfolded protein response to ER stress, which is identical to the signaling induced by overexpressing Psn. The exact pattern of expression of vg-GAL4 (left) and en-GAL4 (right), which drive the different transgenes used in this study, is revealed with a UAS-GFP reporter transgene (c). Our data together with the literature strongly suggest that the ER stress source is not crucial to specify the cell response, while cell location is