The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila

Abstract

The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

Figure 1. Data collection of FRET responses and transgenic RNAi screen of ACs potentially coupled to PDF receptor in M cell pacemakers.

(A) Raw FRET imaging data (CY/CC) collected for 10 min (each trace represents an individual cell recorded as an ROI) to show FRET loss in response to a bolus of PDF (marked by arrow) and recoveries to baseline. (B) The scatter plot represents the data shown in 1A and retains its color-coding. For each trace, the maximal deflection from its value at the initial time point is computed as “percent FRET loss” and represented as a single point. Error bars represent SEM. (C) Double-stranded RNAi directed against 11/12 genes known to encode known adenylate cyclases in the Drosophila genome. All genotypes include Pdf-gal4;Epac1camps and one copy of UASRNAi (except for control). Error bars denote SEM. *** p<0.001, ** p<0.01 (compared with control).

Figure 2. A conditional transgenic RNAi test of AC involvement in PDF signaling in M cell pacemakers.

(A) Temperature sensitive gal80 was used to induce knockdown in adult cells only. Flies were raised at 18°C and moved to 29°C for 6 h (inactive), to allow for readable levels of Epac1camps sensor, or >36 h (active). Adult induction of AC3RNAi (gal80ts;AC3RNAi (active)) significantly reduces the PDF response. Adult induction of AC76ERNAi shows no significant difference from control. (B) Genetic confirmation of AC3 involvement was performed using two independently generated RNAi lines against AC3 (GD:AC3 and TRiP:AC3) as well as flies that are deficient for the AC3 gene region (Df(2L)DS6). (C) DH31 responses in M cells from flies with a knockdown of AC3 in combination with Df(2L)DS6. All genotypes include Pdf-gal4;Epac1camps. Error bars denote SEM. *** p<0.001 (compared with control).

Figure 3. Effects of over-expressing AC isoforms on different receptor signaling systems in M pacemakers.

(A) Effects of over-expressing diverse ACs on M cell responses to neuropeptide PDF. (B) Effects of over-expressing AC3 on M cell responses to neuropeptide DH31. (C) Effects of over-expressing AC3 on M cell responses to dopamine. All genotypes include Pdf-gal4;Epac1camps. Error bars denote SEM. *** p<0.001 (compared with control).

Figure 4. Genetic rescues of AC3 knockdown and over-expression effects in M cells.

(A) Rescuing the loss of function state. Flies were raised at 25°C and moved to 18°C as adults for 12–15 h before imaging to reduce levels of AC3 over-expression. The effect of this schedule on the effects of AC3 knockdown (TRiP:AC3RNAi) and AC3 over-expression (UAS-AC3) is shown. The ability of over-expressing AC78C (TRiP:AC3/UASAC78C) and AC3 (TRiP:AC3RNAi/UASAC3) to reverse the knockdown effect of AC3 RNAi are also shown. (B) Rescuing the gain of function state. Two PDF-R over-expression genotypes were tested for their ability to affect AC3 over-expression: a UAS construct (UASPDF-R;UASAC3) and a construct in which PDF-R is driven by its endogenous promotor (UASAC3;PDF-Rmyc). For comparison the effects of co-misexpressing a heterologous neuropeptide receptor is also shown (UASDH31R/UASAC3). All genotypes include Pdf-gal4;Epac1camps. Error bars denote SEM. *** p<0.001, * p<.05 (compared with control).

Figure 5. Both PDF and DH31 responses are affected by altering G_sα60A levels.

(A) PDF responses following knockdown or over-expression of G_sα60A. G_sα60A over-expression effects were also measured in the context of over-expression of PDF-R (UASG_s α 60A;UASPDF-R) or over-expression of DH31R (UASG_s α 60A;UASDH31R). (B) DH31 responses following knockdown or over-expression of G_sα60A. G_sα60A over-expression effects were also measured in the context of over-expression of PDF-R (UASG_s α 60A;UASPDF-R) or over-expression of DH31R (UASG_s α 60A;UASDH31R). All genotypes include Pdf-gal4;Epac1camps. Error bars denote SEM. *** p<0.001, * p<.05 (compared with control).

Figure 6. Effects on PDF responses following RNAi knockdown of scaffolding protein RNAs in M cells.

(A) PDF responses of M pacemakers in flies expressing nervy RNAi. (B) DH31 responses of M pacemakers expressing nervy RNAi. (C) PDF responses of M pacemakers expressing AC3 and nervy RNAi. All transgenic lines are significantly different (<.001) from control and internal comparisons are highlighted by bracketed lines. All genotypes include Pdf-gal4;Epac1camps. Error bars denote SEM. *** p<0.001 (compared with control).

Figure 7. Effects of manipulating G_sα60A and AC3 levels in E cell subgroup.

(A) PDF responses in PDF-R expressing LNd cells (E cells). Flies with the severe PDF-R mutation han⁵³⁰⁴ show no response to PDF. (B) PDF responses in PDF-R expressing LNd cells (E cells). Both knockdown and over-expression of G_sα60A significantly reduce PDF responses in E cells. (C) PDF responses in PDF-R expressing LNd cells (E cells) in genotypes that most severely disrupt M cell PDF responses. Knockdown (Df(2L)/AC3RNAi) and over-expression of AC3 do not affect E cell PDF responses. All genotypes include Mai179-gal4;Epac1camps. Error bars denote SEM. *** p<0.001 (compared with control).

Figure 8. Effects on circadian locomotor activity of altering AC3 in M pacemakers.

(A) Representative locomotor behavior of flies that are heterozygous for the AC3 locus (Df(2L)DS6). (B) Representative locomotor behavior of flies that combine a knockdown of AC3 by RNAi together with a deficiency for the AC3 locus. (C) Representative locomotor behavior of flies over-expressing AC3. (D) Representative locomotor behavior of flies over-expressing PDF-R and over-expressing AC3. Morning anticipation index was calculated as (sum of activity 3 h before lights-on)/(sum of activity 6 h before lights-on). The average morning anticipation index was calculated from three replicates for each genotype. Error bars denote SEM. *** p<0.001 (compared with control). Statistical analysis of morning anticipation is shown in Table 1, and behavioral outcomes for DD are shown in Table 2.

Figure 9. A model for a circadian signalosome comprised of preferential PDF-R:AC3:nervy coupling.

M pacemakers respond to dopamine, DH31, and PDF-R through G_sα-coupled receptors: Activation of each receptor lead to increases in cAMP levels. Both DH31 and PDF receptors signal through G_sα60A, however AC3 alterations affect PDF signaling without affecting DH31 responses. We propose that PDF signals through AC3 to affect circadian function but that dopamine and DH31 couple to other AC isoforms. Likewise, the AKAP nervy preferentially associates with the PDF-R:AC3 signaling complex; other AKAPs support the G_sα-coupled receptors that mediate responsiveness to DA and DH31.