Seasonal cues act through the circadian clock and pigment-dispersing factor to control EYES ABSENT and downstream physiological changes

Abstract

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain to alter seasonal biology are poorly understood. We previously reported that EYES ABSENT (EYA) shows higher levels in cold temperature or short photoperiod and promotes winter physiology in Drosophila. Nevertheless, how EYA senses seasonal cues is unclear. Pigment-dispersing factor (PDF) is a neuropeptide important for regulating circadian output rhythms. Interestingly, PDF has also been shown to regulate seasonality, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of EYA in mediating the function of PDF on seasonal biology. We observed that PDF abundance is lower on cold and short days as compared with warm and long days, contrary to what was previously observed for EYA. We observed that manipulating PDF signaling in eya+ fly brain neurons, where EYA and PDF receptor are co-expressed, modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and EYA-independent mechanisms. At the molecular level, altering PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA promoting its degradation, thus explaining the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations.

Keywords: circadian clock; daily activity rhythm; insect reproductive dormancy; neuropeptide; photoperiod; post-translational modification; seasonal biology; temperature.

Figure 1.. PDF levels are regulated by photoperiod and temperature.

PDF levels in the s-LNv dorsal terminals were analyzed in flies entrained to (A) long-photoperiod (LP, 16:8 LD) at 25°C or short-photoperiod (SP, 8:16 LD) at 10°C, (D) LP or SP at 25°C, and (G) 12:12 LD cycles at 25°C or 10°C. Representative images for these conditions at ZT0 and ZT16 or ZT12 are shown in B, E and H, respectively. Double-plotted graphs of PDF signal for each condition are shown in C, F and I, respectively. White boxes represent lights-on, and black boxes represent lights-off. CircaCompare and RAIN was used to determine rhythmicity and phase for all datasets. MESOR (horizontal lines) and phase (vertical lines) are displayed in the graphs if statistically significant (p < 0.05), except for (F), where the phases detected between CircaCompare and RAIN were different. The phases shown in (F) were calculated using RAIN. Scale bars are 10 μm. Number of brains imaged were LP25: ZT0 n = 18, ZT4 n = 11, ZT8 n = 13, ZT12 n = 18, ZT16 = 9, ZT20 n = 14; SP10: ZT0 n = 9, ZT4 n = 8, ZT8 n = 14, ZT12 n = 14, ZT16 = 18, ZT20 n = 20; SP25 ZT0 n = 6, ZT4 n = 8, ZT8 n = 9, ZT12 n = 6, ZT16 =10, ZT20 n = 3; 12:12 25°C: ZT0 n = 14, ZT4 n = 14, ZT8 n = 15, ZT12 n = 19, ZT16 =10, ZT20 n = 15; 12:12 10°C: ZT0 n = 13, ZT4 n = 14, ZT8 n = 15, ZT12 n = 15, ZT16 =16, ZT20 n = 10.

Figure 2.. Pdf mRNA is sensitive to seasonal cues.

(A) Pdf mRNA (magenta) was detected using fluorescence in situ hybridization (FISH) in whole brains of flies entrained for 7 days to simulated-summer (LP at 25°C) or simulated-winter (SP at 10°C). CD8::GFP was expressed using the Pdf-gal4 driver to detect the PDF-producing LNvs. (B) Representative images of LNvs of flies entrained to LP 25°C (top panel) or SP 10°C (bottom panel). (C) Quantification of the normalized intensity of the Pdf signal at ZT16 is compared between conditions. Unpaired t-test, ** p <0.01. Number of brains imaged were LP25 n = 8, SP10 n = 7 brains. Scale bar in A is 25 μm and 5 μm in B. See also Figure S1 and Table S1.

Figure 3.. Locomotor adaptation to photoperiod and temperature in Pdf-null mutants.

Locomotor activity was monitored in Pdf-null mutants (yw ; Pdf⁰¹) and in flies expressing a wild-type copy of Pdf (genomic rescue, yw ; Pdf{WT} ; Pdf⁰¹) subjected to (A-B) 12:12 LD at 25°C, (C-D) 12:12 LD at 10°C, or (E-F) 8:16 LD at 25°C. (G) Variance in locomotor activity under these environmental conditions as tSNE of the normalized locomotor activity for Rescue (Resc) and the mutant (Pdf⁰¹). Each dot represents an individual fly. (H) Hierarchical clustering of the normalized locomotor activity. Arbitrary cut of the tree at 0.8 (y-axis) generates two distinct groups denotated as black and red labels. White boxes represent lights-on while black boxes represent lights-off in panels A-F. Data were analyzed using a two-way ANOVA with Bonferroni’s multiple comparison test. Number of flies used were yw ; Pdf{WT} ; Pdf⁰¹ 12:12 25°C n = 56, yw ; Pdf{WT} ; Pdf⁰¹ 8:16 25°C n = 30, yw ; Pdf{WT} ; Pdf⁰¹ 12:12 10°C n = 30, yw ; ; Pdf⁰¹ 12:12 25°C n = 55, yw ; ; Pdf⁰¹ 8:16 25°C n = 31, yw ; ; Pdf⁰¹ 12:12 10°C n = 32.

Figure 4.. PDF modulates overall locomotion through eya+ cells.

(A-B) Average locomotor activity of flies in constant darkness at 25°C where the PDF receptor was knocked down in eya+ cells (eya > Pdfr-RNAi red line and box) compared to controls (eya / + and Pdfr-RNAi / +, black and gray lines, respectively). (C-D) Average locomotor activity of flies where a membrane tethered version of PDF was expressed in eya+ cells (eya > t-PDF red line and box) compared to the expression of a scrambled version of the peptide (eya > t-SCR black line and box). (E-G) Confocal images of fly brain expressing endogenous GFP tagged PER (green) and stained against EYA (magenta). Red arrows points DN1, DN2 and LNd clock populations. Scale bar is 25 μm. Data in B were analyzed with Kruskal-Wallis test followed by Dunnett’s multiple comparison test, Mann-Whitney in D. Number of flies used were eya / + n = 29, eya > Pdfr-RNAi n = 32, Pdfr-RNAi / + n = 28, eya > t-SCR n = 31, eya > t-PDF n = 31, eya / + n = 30. See also Figures S2 and S3.

Figure 5.. PDF and EYA have opposite effects in the IPCs to modulate ovary size under summer-like conditions (16:8 LD at 25°C)

(A) Expression of CD8::GFP was driven in the Pdfr+ cells using a Pdfr-gal4 driver. Nc82 antibody was used as counterstaining. Expression of CD8::GFP is observed in several segments of the brain, including the pars intercerebralis, containing the IPCs (inset). Scale bar is 50μm. (B) SMART-seq dataset from dilp2+ isolated IPCs from Fly Cell Atlas as UMPA, colored by Pdfr (green) and eya (red) expression. Coexpression is shown as yellow. Color bar denotes the relationship between color intensity and relative expression levels. (C) EYA staining (upper right panel) in brains of flies expressing CD8::GFP in the Pdfr+ cells (Pdfr > CD8::GFP, upper left panel). Merged image is observed in the bottom panel. Scale is 5 μm. (D) Comparison of ovary size in mm² while knocking down eya (dilp2 > eya-RNAi) or knocking down Pdfr (eya > Pdfr-RNAi) compared to genetic controls (black and grey boxes). Data was analyzed using a one-way ANOVA followed by Holm-šidák’s multiple comparison test. Number of flies used were dilp2 / + n = 40, dilp2 > eya-RNAi n = 28, eya-RNAi / + n = 34, dilp2 > Pdfr-RNAi n = 45, UAS-Pdfr-RNAi / + n = 32.

Figure 6.. PDF modulates EYA protein but not eya mRNA expression.

(A) eya mRNA daily oscillations in Pdf-null mutants (yw ; ; Pdf⁰¹ flies, red dots, RAIN p = 0.833) and in flies expressing a Pdf genomic rescue (yw ; Pdf{WT} ; Pdf⁰¹, white dots, RAIN p = 0.0078) in 12:12 LD cycles at 25°C. (B) Representative western blot and (C) quantification of EYA levels in whole-head lysates from Pdf null-mutant (red dots, RAIN p = 0.677) and in the genomic rescue (white dots, RAIN p = 0.039) at ZT0, 4, 8, 12, 16 and 20 in 12:12 LD 25°C cycles. (D) Representative western blot and (E) quantification of EYA levels from whole head lysates of yw (genetic control, white boxes), Pdf-null mutants (red boxes) and the genomic rescue (grey boxes) at ZT4 and ZT16 in 12:12 LD 25°C condition. (F) Representative western blot and (G) quantification of EYA levels in head lysates of flies where the PDF receptor was knocked down in eya+ cells (eya > Pdfr-RNAi, red boxes) and controls (eya / + and Pdfr-RNAi / +, white and grey boxes, respectively) at ZT4 and ZT16. (H) Representative western blot and (I) quantification of EYA levels in flies expressing a membrane-tethered version of PDF in eya+ cells (eya > t-PDF, red boxes) compared to a scrambled peptide as control (eya > t-SCR, white boxes) at ZT4 and ZT16. Data were analyzed using a two-way ANOVA followed by Tukey’s post hoc test in B and C, and šídák’s multiple comparison test in E, G and I. HSP70 was used for normalization in all cases. n > 3 biological replicates, each consisting of around 50 flies for each genotype.

Figure 7.. PKA phosphorylates EYA and mediates its degradation

(A) PDF receptor triggers the activation of adenylate cyclase (AC) which produces cAMP. The binding of cAMP to the regulatory subunits of the protein kinase A (PKA-R) releases the catalytic subunits of the kinase (PKA-C) permitting their action. EYA has potential sites for PKA phosphorylation. (B) Drosophila S2 cells co-expressing EYA-FLAG and the active C1 domain of PKA tagged with V5 (PKA-C1-V5 + condition) or EYA alone. Samples were then subjected to phosphatase treatmet (+λPP) and compared to samples that were mock treated (−λPP). Immunoprecipitated EYA was detected using α-FLAG in the input. α-V5 and α-HSP70 were used to detect PKA-C1 and HSP70 (n>3, representative image is shown). (C) Representative western blot of a CHX chase assay in S2 cells co-expressing EYA-FLAG alone or with PKA-C1-V5. (D) Quantification showing the rates of EYA degradation in absence (black line) or presence (red line) of PKA-C1. Data analyzed with two-way ANOVA with Holm-šídák’s multiple comparison test. Each dot represents an independent experiment (n = 3, * p<0.05).