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1 Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France
2 Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA 94720-3200, USA
3 National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
4 Institut Jacques Monod, 2 place Jussieu, 75251 Paris, France
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Genetic analyses have shown that anti-microbial peptide encoding genes are regulated by the Toll and Imd pathways (Tzou et al. 2002; Hoffmann 2003; Hultmark 2003). These two pathways share similarities with the Toll-Like Receptor and Tumor Necrosis Factor Receptor pathways, respectively, which regulate NF-
B in mammals. The Toll pathway is activated mainly by Gram-positive bacteria and fungi, while the Imd pathway responds mainly to Gram-negative bacterial infection. Microarray analyses have shown that the Toll and Imd cascades control the majority of genes regulated by septic injury in addition to anti-microbial peptide encoding genes. The presence of immune-responsive genes independent or only partially dependent on both the Imd and Toll pathways suggested the involvement of other signaling cascades (De Gregorio et al. 2002).
Using a similar approach, Boutros et al. (2002) have shown that in addition to the Toll and Imd pathways, the JAK-STAT and the JNK pathways contribute to the expression of immune response genes. The Drosophila JAK-STAT pathway is involved in multiple developmental events and regulates hemocyte differentiation (Dearolf 1999; Agaisse & Perrimon 2004). This pathway does not regulate anti-microbial peptide genes but affects the expression of a small number of genes induced in the fat body after septic injury (Lagueux et al. 2000). Recently, it has been shown that the JAK-STAT pathway is activated in the fat body by the cytokine Unpaired3 (Upd3), and that it regulates the expression of Turandot (Tot) stress genes in response to septic injury (Agaisse et al. 2003).
In Drosophila, the JNK Mitogen Activated Protein Kinase (MAPK) pathway is induced following immune stimulation (Sluss et al. 1996). Interestingly, both NF-B and JNK branches share the same upstream components, Tak1 and Imd, indicating that the activation of both cascades is tightly linked in Drosophila (Boutros et al. 2002; Silverman et al. 2003; Park et al. 2004). Furthermore, in agreement with a function in wound healing (Rämet et al. 2001; Boutros et al. 2002; Galko & Krasnow 2004), genome profiling indicates that JNK signaling controls the expression of genes involved in cytoskeleton remodeling.
In plants, C. elegans and vertebrates, p38 MAPKs are also involved in the regulation of the immune and stress responses (Asai et al. 2002; Kim et al. 2002). However, little is known about the role of this pathway in the Drosophila immune response. Two p38 MAP Kinases, p38a and p38b, are encoded by the Drosophila genome (Han et al. 1998b). Like mammalian p38, Drosophila p38s are activated in cell culture by stress and inflammatory stimuli, such as UV radiation, high osmolarity, heat-shock, serum starvation and bacterial products (Han et al. 1998a; Zhuang et al. 2005). Flies lacking p38a are viable but are susceptible to some environmental stresses, including heat-shock, oxidative stress and starvation (Craig et al. 2004). However, the precise physiological role of p38 awaits further studies using loss-of-function mutations of the second p38 gene, p38b. Drosophila Mekk1 is a MAPK Kinase Kinase (MAPKKK) similar to the mammalian MEKK4/MTK1. Drosophila mutants lacking Mekk1 show a reduced activation of p38 in vivo and are hypersensitive to some environmental stresses such as elevated temperature and increased osmolarity, suggesting that the Mekk1-p38 pathway is critical for the response to environmental stress in Drosophila (Inoue et al. 2001).
In the present study, we have analyzed the role of Mekk1 in the regulation of genes induced in response to septic injury. Our study demonstrates that the MAPKKK Mekk1 regulates a small subset of target genes induced by septic injury, including Turandot stress genes. Furthermore, Mekk1 mutant flies are susceptible to oxidative stress, suggesting a role of this MAPKKK in the protection against tissue damage and/or protein degradation.
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Results |
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In a previous study, we have identified 400 Drosophila immune regulated genes (DIRGs) through a microarray analysis of the transcriptome after septic injury and natural infection (De Gregorio et al. 2001). To identify whether some of the 400 previously identified DIRGs are controlled by Mekk1, total RNA samples from wild-type (Oregon R) and Mekk1 adult flies, collected after septic injury with a mixture of E. coli and Micrococcus luteus were hybridized to Affymetrix DrosGenome1 GeneChips capable of measuring RNA levels for nearly every gene in the Drosophila genome. Changes in relative transcript levels were measured 1.5, 3, 6, 12, 24 and 48 h after septic injury, ensuring that both early and late gene changes were monitored. Each time series was performed in duplicate for challenged and quadruplet for unchallenged flies. Complete results can be found at: http://www.cgm.cnrs-gif.fr/immunity/enindex.html
Table 1 shows a list of genes that display a change in their expression between wild-type and Mekk1 flies using a two-fold threshold. We found that Mekk1 does not regulate anti-microbial peptides encoding genes, but affects a small group of genes that could not be simply clustered to only one biological function. This list includes the gene coding for the Thiol-Ester Protein II, TepII, which may participate in microbial opsonization (Levashina et al. 2001). Interestingly, the most significantly affected gene was Turandot M (TotM) which was induced seven-fold in wild-type but not in Mekk1 flies (Fig. 1A). TotM belongs to a family of eight Tot genes that encode small secreted proteins of 11–14 kDa sharing weak homology (Ekengren & Hultmark 2001). Previous studies have shown that these genes are induced under stress conditions such as bacterial infection, heat-shock, paraquat feeding and UV exposure, suggesting a role in stress tolerance in Drosophila (Ekengren et al. 2001). Table 1 also shows that Mekk1 represses the expression of a low number of immune genes including Defensin.
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The best characterized member of the Turandot family is TotA which is secreted by the fat body and accumulates in the hemolymph in response to various stress stimuli (Ekengren et al. 2001). TotA was not predicted in the first release of the Drosophila genome and was in consequence not included on the DrosGenome1 GeneChips. To confirm the microarray results, we monitored the mRNA levels of TotA and TotM in Mekk1 flies by Northern blot analysis. Pricking wild-type flies with a mixture of Gram-positive and Gram-negative bacteria strongly activated the expression of TotM and TotA, whereas in Mekk1 mutants, Tot genes expression was dramatically reduced (Fig. 2A,B). However, Diptericin and Drosomycin which encode anti-microbial peptides were induced at a wild-type level in Mekk1 flies. These RNA blots strengthen the microarray analysis and show that induction of both TotA and TotM after septic injury requires Mekk1. To confirm that the lack of induction of TotA was indeed due to the Mekk1 mutation, we performed a rescue experiment with a hsp-Mekk1 construct. Figure 2B clearly shows that over-expression of the Mekk1 cDNA after heat-shock restored a wild-type level of TotA expression in Mekk1 mutants after pricking. In agreement with a previous study (Agaisse et al. 2003), we observed that the basal levels in unchallenged flies and the induction levels after septic injury of TotA expression significantly vary depending on age of the flies, growth conditions and also the genetic background. Therefore, we also exploited the inducible RNA interference technology (RNAi) as an alternative way to analyze the phenotype associated with the knock-down of Mekk1. We generated two independent transgenic fly lines, UAS-Mekk1-IR, containing a GAL4-inducible construct which allows the tissue-specific production of double-stranded RNA (dsRNA) of Mekk1. Figure 2C clearly shows that silencing of Mekk1, using the ubiquitous driver daughterless-GAL4 (da-GAL4) blocked TotA expression after septic injury. In an attempt to determine in which tissue Mekk1 is required for TotA expression, we directed Mekk1 dsRNA synthesis in the fat body using the ppl-GAL4 and in the hemocytes using the hml-GAL4 driver. While we did not observe any effect using hml-GAL4, knock-down of Mekk1 in the fat body using the ppl-GAL4 driver reduced TotA expression, suggesting that Mekk1 is required in the fat body but not in hemocytes (data not shown).
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As stated above, Tot genes are induced by various stresses and after septic injury. To determine if Mekk1 also regulates TotA under stress conditions, we compared the level of TotA transcripts after heat-shock, dehydration, mechanical pressure, and osmotic stress in wild-type and in Mekk1 flies. In these experiments, heat-shock, dehydration and mechanical pressure weakly induced TotA expression, whereas osmotic stress had no effect (Fig. 3A). Nevertheless, the weak stimulation of TotA by stress was clearly abolished in Mekk1 flies. Septic injury was by far the strongest and most reliable inducer of TotA (Fig. 3A). Notably, Fig. 3C shows that Gram-negative bacteria induced TotA expression at a higher level than Gram-positive bacteria in agreement with a previous study (Agaisse et al. 2003). However, we observed that an injury without addition of bacteria induced TotA at 40% of the level of septic injury by Gram-negative bacteria. This expression profile significantly differed from that of the anti-bacterial peptide gene Diptericin, which is only weakly induced by clean injury (Lemaitre et al. 1997 and Fig. 3C). This suggests that TotA is induced by a stimulus associated with the injury itself, which is enhanced in presence of Gram-negative bacteria. Consistent with this idea, we observed that TotA was only weakly induced after natural infection of adults by the entomopathogenic fungus B. bassiana (Fig. 3B). Similarly, TotA was not induced after natural infection with the Gram-negative bacteria Erwinia carotovora 15 in larvae (data not shown). Taken together, these findings suggest that Mekk1 is mostly activated by the stress of the injury (wound, oxidative or mechanical stress), and that the higher induction of Tot genes after septic injury is conferred by the concurrent activation of other pathways triggered by the presence of microbial components.
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Recently it has been shown that septic injury activates TotA expression through the Imd and the JAK-STAT pathways (Agaisse et al. 2003). We confirmed that Tot gene induction is clearly blocked in Relish deficient flies that lack a functional Imd pathway but not in Toll deficient flies (Fig. 1B for TotM and Fig. 4A for TotA). However, Mekk1 is unlikely to participate in the Imd pathway, since in contrast to mutations of the MAPKKK Tak1, Mekk1 loss-of-function does not affect Diptericin expression or survival after challenge with Gram-negative bacteria (Figs 2B and 5A).
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The gene encoding the Upd3 cytokine is rapidly induced in hemocytes after septic injury and it is believed that secretion of Upd3 activates the JAK-STAT pathway in the fat body (Agaisse et al. 2003). Figure 4D shows that upd3 expression was not affected in Mekk1 flies after septic injury, indicating that Mekk1 is not involved in the regulation of this cytokine. This result is consistent with the observation that Mekk1 is not required in hemocytes for TotA expression. Altogether, our results indicate that Mekk1 is not a canonical component of the JAK-STAT and Imd pathways. They also underline the complexity of TotA gene regulation that integrate signals from the JAK-STAT and Imd pathways and Mekk1.
Mekk1 protects adult flies from oxidative stress
Mekk1 regulates a small set of genes, such as tepII, TotM and Def, that are supposed to have important functions during immune and stress responses. We further assayed the susceptibility of flies carrying a null allele of Mekk1 to infection by four microorganisms (Fig. 5). We pricked flies with the Gram-negative bacterium Escherichia coli, the Gram-positive bacterium Enterococcus faecalis or the fungus Aspergillus fumigatus and naturally infected flies with the entomopathogenic fungus Beauveria bassiana. As expected, a mutation in Relish induced a high susceptibility to E. coli (Fig. 5A) while a mutation in spaetzle, affecting the Toll pathway, rendered flies susceptible to both Gram-positive and fungal infections (Fig. 5B,C) (Lemaitre et al. 1996; Rutschmann et al. 2002). In sharp contrast, Mekk1 flies showed a survival rate similar to wild-type in all conditions tested (Fig. 5 and data not shown for B. bassiana infection). This result was consistent with our observation that Mekk1 does not affect the expression of Drosophila anti-microbial peptide encoding genes after microbial infection.
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Discussion |
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The exact role of Mekk1 in stress signaling remains to be investigated. At this point, we cannot exclude that Mekk1 is part of secondary cascade that branches downstream of the Imd pathway and regulates only a subset of target genes in a situation analogous to the JNK pathway which functions downstream of Tak1 (Boutros et al. 2002; Silverman et al. 2003; Park et al. 2004). Altogether, our work shows that TotA integrates input from multiple signaling pathways. It will be a major challenge to determine how these pathways interact. From this point of view, Tot genes define a new class of immune-inducible genes and provide an easy read-out to monitor JAK-STAT and Mekk1 activities and thus may be used to identify additional signaling components. An important question is whether Mekk1 controls TotA through the activation of p38 MAPKs. It has been shown in vivo and in cell culture that Mekk1 inhibition reduces p38 activation in Drosophila (Inoue et al. 2001; Zhuang et al. 2005). However, p38a and Mekk1 mutants show only partially overlapping phenotypes. For instance, p38a but not Mekk1 flies were vulnerable to hydrogen peroxide (Craig et al. 2004). It is likely that p38b and the existence of other MAPKKK acting upstream of p38 may account for the differences between p38a and Mekk1 mutants. A more thorough depletion of p38 function in vivo by removing both p38a and p38b will allow clarification of the role of this family of MAPK in stress signaling.
Septic injury was the most efficient challenge to stimulate TotA expression. It is therefore tempting to speculate that Mekk1 and its target genes from the Tot family play a role in the response to tissue damage. Mekk1 has already been implicated in survival to high temperature and osmotic stress (Inoue et al. 2001). Here, we show a role for Mekk1 in the resistance to paraquat, an inducer of oxidative stress, in Drosophila. This contrasts with a previous study that indicated that Mekk1 flies show a wild-type resistance to hydrogen peroxide (Craig et al. 2004). This discrepancy could be explained by the fact that in contrast to hydrogen peroxide that can exert its oxidative properties everywhere in the organism, paraquat has to be metabolized inside cells to inhibit mitochondrial complex 1 and subsequently to induce a release of superoxide ions susceptible to damage tissue. Actually, oxidative stress specificities have already been observed in vivo, both at genetic and molecular levels (Monnier et al. 2002; Girardot et al. 2004). Consequently, it is possible that both Mekk1 and JAK-STAT pathways mediate a host response to protect against tissue damage and protein degradation in stressful conditions. The fact that tissue damage can be caused by multiple stimuli, such as infection, injury and environmental stresses may explain the complex regulation of Tot genes by multiple pathways. In agreement with this hypothesis, we found that TotA is only weakly induced by B. bassiana and E. carotovora natural infections, which are known to trigger the Toll and Imd pathways without provoking a major injury (Lemaitre et al. 1997; Basset et al. 2000). However, the situation is probably more complex since we did not observe any induction of TotA in adults upon paraquat feeding (data not shown). This suggests that the susceptibility of Mekk1 mutant flies to paraquat is not directly linked to a lack of TotA expression. Hence, the exact role of Tot genes remains unknown after our study but can be further investigated through the analysis of Mekk1 phenotypes. A significant advantage of the Mekk1 mutation is that flies are perfectly viable under normal conditions, unlike mutations affecting the JAK-STAT pathway.
In conclusion, our study points towards a role for Mekk1 and possibly p38 in the adaptive response to stress associated with injury. The identification of target genes from the Tot family opens the way to genetic screens that should allow the identification of new components of the signaling cascade. Drosophila may provide an excellent model to study the complex interactions between stress and immune pathways.
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Experimental procedures |
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Oregon R, Canton S, w Canton S and w1118 flies were used as wild-type controls. Exact genotypes of the flies analyzed in this study are: spaetzlerm7 (spz); RelishE20 (Rel) w; Mekk1Ur36 (Mekk1); spzrm7, RelE20 (Rel, spz) Toll1RXA/TollR632 (Tl); hopmsv1, hopTum (Inoue et al. 2001; Lemaitre et al. 1996). spzrm7, RelE20 and Mekk1Ur36 are strong or null alleles of spz, Rel and Mekk1 (Hedengren et al. 1999). The hop alleles used, msv1 and Tum, have been previously described (Agaisse et al. 2003). Rescue experiments were performed with flies carrying both the Mekk1 mutation and an hsp-Mekk1 P transgene on the same chromosome (Inoue et al. 2001). RNAi transgenic fly lines of Mekk1 were obtained using the inducible RNAi method. A 500 bp-long cDNA fragment (nucleotides 1–500 of the coding sequence) was amplified by PCR, and inserted as an inverted repeat (IR) in a modified pUAST transformation vector, pUAST-R57 as described in Leulier et al. (2002). Transformation of Drosophila embryos was carried out in w1118 fly stocks. The UAS-Mekk1-IR1 and UAS-Mekk1-IR2 are independent insertions, both located on the second chromosome. The da-GAL4 driver expresses GAL4 strongly and ubiquitously (Leulier et al. 2002). Hemolectin (hml)-GAL4 is an hemocyte specific GAL4 driver (Goto et al. 2003) and pumpless (ppl)-GAL4 expresses GAL4 in the fat body (Colombani et al. 2003).
Infection and stress experiments
For septic injury and natural infection experiments, we used Drosophila adults, aged 2–4 days at 25 °C and reared under the same conditions as many environmental parameters cause considerable variability in TotA expression. Septic injury was performed by pricking the thorax of the flies with a needle previously dipped into a concentrated mixed culture of Escherichia coli and Micrococcus luteus. Natural infection by the entomopathogenic fungus Beauveria bassiana was initiated by shaking anesthetized flies in a Petri dish containing a sporulating culture of the fungus (Lemaitre et al. 1997). Natural infections by Erwinia carotovora 15 were performed by incubating Drosophila larvae in a mixture of crushed banana and bacteria (Basset et al. 2000). For survival experiments, flies were incubated at 25 °C after infection. For Northern and microarray analysis, flies were incubated at 25 °C and collected at specific times after infection.
Other stress inductions were performed as follows: for dehydration, flies were placed in an empty vial for 120 min; mechanical pressure was applied by squeezing flies for 1 h with a sponge plug without rupture of the cuticle; flies were heat-shocked during 3 h at 37 °C; osmotic stress was applied by placing flies on a vial containing 5 M NaCl food.
Northern blot analysis
Total RNA extraction, Northern blotting experiments and Northern quantifications were performed as described in Lemaitre et al. (1997).
Quantitative RT-PCR
For TotA, upd3 and rp49 mRNA quantification from whole animals, RNA was extracted using RNA TrizolTM. cDNAs were synthesized from 1 µg of total RNA using SuperScript II (Invitrogen) and PCR was performed using dsDNA dye SYBR Green I (Roche Diagnostics). Primer pairs for TotA (forward 5'-GCA CCC AGG AAC TAC TTG ACA TCT-3', and reverse 5'-GAC CTC CCT GAA TCG GAA CTC-3'), for upd3 (forward 5'-GGC CCG TTT GGT TCT GTA GA-3', and reverse 5'-GTA GAT TCT GCA GGA TCC TT-3') and control rp49 (forward 5'-GAC GCT TCA AGG GAC AGT ATC TG-3', and reverse 5'-AAA CGC GGT TCT GCA TGA G-3') were used to detect target gene transcripts. SYBR Green analysis was performed on a Lightcycler (Roche). All samples were analyzed in duplicate and the amount of mRNA detected was normalized to control rp49 mRNA values. We used normalized data to quantify the relative levels of TotA mRNA according to cycling threshold analysis (
Ct).
Analysis of mRNA expression using oligonucleotide arrays
Total RNA was extracted from 25 flies for each time point using Trizol reagent (GibcoBRL). Gene expression analysis was performed using the Affymetrix Drosophila GeneChipTM, using the laboratory methods in the Affymetrix GeneChip expression manual. Briefly, double stranded cDNA was synthesized using 2 g of RNA. Biotin-labeled cRNA was synthesized using BioArray high yield RNA transcript labeling kit (Enzo) and 15 g of fragmented RNA were hybridized to each array. The arrays were washed using the EukGW2 protocol on the GeneChip Fluidics Station 400 series and scanned using the GeneArray scanner. Gene expression analysis was performed using multiple arrays, and multiple independent mRNA samples for each time point.
Data Analysis: Genes are represented on the DrosGenome1 chip by one or more transcripts, which in turn are represented by a probe set. Each probe set consisted of 14 pairs of perfect match (PM) and mismatch (MM) oligos. Data were collected at the transcript level, but for ease in the text, the data is referred to by gene. Intensity data for each feature on the array was calculated from the images generated by the GeneChip scanner using the GeneChip Microarray Suite. This intensity data was loaded into a MySQL database, where information on each of the features was also stored. The difference between the perfect match and mismatch oligos (probe pair) was calculated and the mean PM—MM intensity for each array was set to a constant value by linearly scaling array values. The mean intensity of individual probe pairs was calculated across all 34 arrays, and the log2 ratio of each value to this mean was stored. Next, all log ratios for each probe pair set (transcript) were averaged creating one measurement for each transcript on each array. The final dataset was generated by averaging data for each transcript on replicate arrays and subtracting the value of the uninfected sample from each measurement. We restricted our analysis to the 400 DIRGs already identified by De Gregorio et al. (2001). A threshold of 2 in two different time points was used to select the genes affected by the Mekk1 mutation.
Oxidative stress resistance tests
We used 50 mL vials containing 1 mL of a solid medium composed of 1.3% low melting agarose, 1% sucrose and 10 mM paraquat. These compounds were incorporated at 45 °C to avoid loss of oxidative activity. Three-to five-day-old males were placed by groups of 30 in these vials and maintained at 26 °C. Dead flies were counted twice a day until the end of the experiment. For each experimental condition, at least three vials of 30 males were used for each genotype. In addition, to minimize genetic background effects, all the lines used in these experiments (with the exception of the Mekk1Ur36 mutation, which is not associated to a visible marker) were previously out-crossed for at least four generations against a w Canton S reference line. For heat-shock rescue experiments, flies were heat-shocked 30 min at 37 °C before transfer to vials containing paraquat medium. To ensure a sustained expression of the transgene, subsequent 20 min 37 °C heat-shocks were performed on these flies every 24 h.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work. 
Communicated by: Konrad Basler * Correspondence: E-mail: lemaitre{at}cgm.cnrs-gif.fr
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References |
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Received: 18 October 2005
Accepted: 27 December 2005
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), Mekk1 (
), Rel (
) and spz (x) flies after different types of infection are presented. One hundred 2–4 days old adults were pricked with a needle dipped into a suspension of one of the following microbes: (A) E. coli, (B) E. faecalis(C) A. fumigatus. The infected flies were incubated at 25 °C and transferred to fresh tubes 72 h after treatment.
) or Mekk1, hsp-Mekk1/Mekk1 flies (
) compared to w control flies (
). When 37 °C heat-shocks were performed every 24 h, we observed a survival rescue for Mekk1, hsp-Mekk1/Mekk1 flies (•), compared to w control flies (