Aminergic Signaling Controls Ovarian Dormancy in Drosophila

Abstract

In response to adverse environmental conditions many organisms from nematodes to mammals deploy a dormancy strategy, causing states of developmental or reproductive arrest that enhance somatic maintenance and survival ability at the expense of growth or reproduction. Dormancy regulation has been studied in C. elegans and in several insects, but how neurosensory mechanisms act to relay environmental cues to the endocrine system in order to induce dormancy remains unclear. Here we examine this fundamental question by genetically manipulating aminergic neurotransmitter signaling in Drosophila melanogaster. We find that both serotonin and dopamine enhance adult ovarian dormancy, while the downregulation of their respective signaling pathways in endocrine cells or tissues (insulin producing cells, fat body, corpus allatum) reduces dormancy. In contrast, octopamine signaling antagonizes dormancy. Our findings enhance our understanding of the ability of organisms to cope with unfavorable environments and illuminate some of the relevant signaling pathways.

Figure 1

Serotonergic signaling promotes dormancy regulating IIS, whereas octopamine inhibits dormancy. (A) Knocking down the serotonin receptor 5-HT1A in the IPCs strongly reduces dormancy levels (cf. Supplementary Figure S1A), suggesting that serotonin promotes dormancy. (B) Constitutive activation of serotoninergic neurons for 11 days at 12 °C and 16 h:8 h L:D increases ovarian dormancy, confirming that serotonin is a positive regulator of dormancy. Figure 1A and B show percentage dormancy (mean ± binomial SE); assays were performed with 5–7 replicates per genotype, each replicate consisting of ~60 females. ***p < 0.001. (C–F) Expression levels (mean ± standard error [SE]) of dilp2, dilp3, dilp5 in dilp2 > 5-HT1A-RNAi and 4E-BP in dilp2(p) > 5-HT1A-RNAi females at 12 °C as compared to controls. *p < 0.05; **p < 0.01. (G) Downregulation of octopaminergic signaling via OAMB RNAi in the IPCs does not affect dormancy (also see Supplementary Figure S1B). (H) In contrast, however, constitutive activation of octopaminergic neurons decreases dormancy levels. (I) Knockdown of GBR in the IPCs with dilp2-GAL4 has no effect on dormancy (but see Supplementary Figure S1C for a positive yet inconsistent result with dilp2(p)-GAL4). (J) In support of the notion that GABA does not affect dormancy, constitutive activation of GABAergic neurons is ineffective in regulating dormancy. (K) Overexpression of Upd2 in fat body with cg-GAL4, a manipulation that blocks GABA-mediated inhibition of dILP release from the IPCs, does not affect dormancy (also see Supplementary Figure S1D). Shown are the percentage of females in dormancy (mean ± binomial SE); assays were performed with 5–10 replicates per genotype, each replicate consisting of ~50 females. ***p < 0.001.

Figure 2

Dopamine is a positive regulator of dormancy. (A) Dormancy-inducing conditions (exposure of females to 12 °C and either to 8 h:16 h or 12 h:12 h L:D during 11 days) significantly increases dopamine levels. Shown are mean dopamine levels ± SE (3 replicates per condition, with 500 females each). p-values from t-test: **p < 0.01; ***p < 0.001. (B) Mutations that impact dopamine synthesis and/or signaling (ple⁴, Ddc^hyp, DopR1^hyp) inhibit flies from entering dormancy, whereas e¹ mutant females, which exhibit doubled dopamine levels, show enhanced dormancy. Displayed is the percentage of dormancy (mean ± binomial SE); assays were performed with 4–7 replicates per genotype, each replicate consisting of ~50 females. ***p < 0.001. (C) Ovarian development under dormancy conditions in Ddc^hyp and e¹ mutants as compared to controls. Photographs show representative examples of ovarian development and levels of vitellogenesis after 11 days at 12 °C (scale bars = 0.2 mm). (D) Constitutive activation of dopaminergic neurons increases dormancy. Shown is the percentage of dormancy (mean ± binomial SE); assays were performed with 5–6 replicates per genotype, each replicate consisting of ~60 females. **p < 0.01; ***p < 0.001.

Figure 3

Dopamine promotes dormancy via DopR1 and PKA in IPCs, CA and fat body. (A) Knockdown of DopR1 in the IPCs with dilp2-GAL4 reduces dormancy (also see Supplementary Figure S2A). (B,C) Downregulation of DopR1 but not of D2R in both CA (B) and fat body (C) decreases dormancy (also cf. Supplementary Figure S2B,C). (D–F) Downregulation of PKA signaling in the IPCs, CA and fat body substantially reduces dormancy levels (also see Supplementary Figure S2D–F). Figure 3A–F show the percentage of females in dormancy (mean ± binomial SE); assays were performed with 5–6 replicates per genotype, each replicate consisting of ~60 females. ***p < 0.001. (G) Ovarian development of females expressing mutated forms of PKA-R in the CA. Pictures show representative levels of vitellogenesis after 11 days at 12 °C (scale bars = 0.2 mm).

Figure 4

Impaired PKA signaling in the CA alters expression of transcripts involved in IIS and JH signaling. mRNA abundance of transcripts involved IIS and JH signaling upon downregulation of PKA signaling in the CA via expression of mutated forms of PKA-R (PKA-R33 or PKA-R35) which constitutively inhibit PKA. (A and D) mRNA levels of 4E-BP, a transcriptional readout of IIS pathway activity; increased IIS is expected to decrease 4E-BP levels. Jon25Bii and obp99b are transcriptional readouts of JH signaling; (B and E) show the mRNA levels of Jon25Bii, which is normally induced by JH; and (C and F) show the mRNA levels of obp99b, which is typically downregulated by JH. mRNA levels (mean ± SE) were measured in females kept at 12 °C for 11 days (4 replicates per genotype, each consisting of 10 females). p-values from ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Model of the aminergic signaling control of Drosophila dormancy. (A) Under normal, non-dormancy conditions, dILPs and JH promote reproduction and ovarian growth at the expense of reduced somatic maintenance; under these conditions, serotonin and dopamine signaling in IPCs, CA and fat body is reduced, thus inhibiting the dormancy response. (B) Under dormancy-inducing conditions, in contrast, serotonin and dopamine inhibit the production and/or release of dILPs in the IPCs, thereby causing the downregulation of systemic IIS (and JH signaling) and thus promoting entry into the dormancy state. Similarly, in the CA, dopamine/DopR1 activate PKA signaling which reduces JH synthesis and/or release, thus favoring dormancy induction. Likewise, increased activity of dopaminergic signaling in the fat body promotes reproductive dormancy, perhaps by inhibiting processes required for ovarian maturation (e.g., vitellogenesis). In contrast to serotonin and dopamine, octopamine (not shown in the model) likely represents a dormancy antagonist (see Results). While several of the regulatory connections in this model are hypothetical and remain to be worked out in more detail, our study provides clear evidence that serotonin and dopamine signaling act in key endocrine tissues to promote dormancy entry in Drosophila.