PDFR and CRY signaling converge in a subset of clock neurons to modulate the amplitude and phase of circadian behavior in Drosophila

Abstract

Background: To synchronize their molecular rhythms, circadian pacemaker neurons must input both external and internal timing cues and, therefore, signal integration between sensory information and internal clock status is fundamental to normal circadian physiology.

Methodology/principal findings: We demonstrate the specific convergence of clock-derived neuropeptide signaling with that of a deep brain photoreceptor. We report that the neuropeptide PDF receptor and the circadian photoreceptor CRYPTOCROME (CRY) are precisely co-expressed in a subset of pacemakers, and that these pathways together provide a requisite drive for circadian control of daily locomotor rhythms. These convergent signaling pathways influence the phase of rhythm generation, but also its amplitude. In the absence of both pathways, PER rhythms were greatly reduced in only those specific pacemakers that receive convergent inputs and PER levels remained high in the nucleus throughout the day. This suggested a large-scale dis-regulation of the pacemaking machinery. Behavioral rhythms were likewise disrupted: in light:dark conditions they were aberrant, and under constant dark conditions, they were lost.

Conclusions/significance: We speculate that the convergence of environmental and clock-derived signals may produce a coincident detection of light, synergistic responses to it, and thus more accurate and more efficient re-setting properties.

Figure 1. PDFR-MYC and CRYPTOCHROME are precisely co-expressed in the same subsets of clock neurons.

PDFR-MYC fly brains were triple-stained with anti-MYC (green), anti-PER (magenta), and anti-PDF (blue) antibodies (A, C, E), or double-stained with anti-MYC (green) and anti-CRY (blue) antibodies (B, D, F). (A) Three LNds of six show strong PDFR-MYC staining (white arrowheads), whereas the others show no PDFR-MYC (magenta arrowheads). (B) Three LNds express both PDFR-MYC and CRY (arrowheads). (C) The 5^th s-LNv showed strong staining of PDFR-MYC (arrow). (D) Nine LNv stained with anti-CRY antibody, three of these were also stained with anti-MYC. By reference to results shown in panel C, we assigned the strongest MYC expressing neuron to the 5^th s-LNv (arrow). The two remaining MYC(+) neurons are marked with white arrowheads: By size, we speculate these are l-LNv. Two CRY(+) l-LNvs (by size) were not detected with anti-MYC antibody (magenta arrowheads). (E) Six of the 17 DN1s show PDFR-MYC staining at strong levels (white arrowheads), whereas the remaining ones show little or no MYC staining. (F) Six of 15 DN1ps express both PDFR-MYC and CRY (arrowheads). Asterisks (in A, B, D, F) - non-specific staining by either anti-MYC or anti-CRY rabbit antibodies. Scale bars, 10 µm. (G) A summary diagram of the precise PDFR and CRY co-expression in discreet subsets of the three major pacemaker cell groups.

Figure 2. Lack of PDF and CRY signaling causes arrhythmic behavior under DD.

Group averaged actograms of each genotype under constant darkness (DD) following LD cycles. (A) w¹¹¹⁸ flies; (B) pdfr⁵³⁰⁴ single mutant flies; (C) pdfr⁵³⁰⁴; ; cry^b/01 double mutant flies; (D) cry-G4⁽¹⁹⁾; cry^b ss Pdf⁰¹double mutant flies. The double mutant flies fail to maintain free running rhythms. For the experiment shown here, the numbers of animals averaged are 16 (A), 15 (B), 29 (C), and 24 (D).

Figure 3. Daily locomotor activities under LD cycles reveal genetic interactions between PDF and CRY signaling pathways.

Averaged activity of various genotype flies for a six-day-period under 8∶16 LD (A–D), 12∶12 LD (E–H), and 16∶8 LD (I–L) entrainment conditions. (A, E, I) w¹¹¹⁸ control flies; (B, F, J) pdfr⁵³⁰⁴ single mutant flies; (C, G, K) pdfr⁵³⁰⁴; ; cry^b/01 double mutant flies; (D, H, L) cry-G4⁽¹⁹⁾; cry^b ss Pdf⁰¹ double mutant flies. Both double mutant flies display lack of anticipatory peaks under LD cycles. Note that, in pdfr single mutants, the longer the day length becomes the more pronounced the advanced evening phenotype. For the experiment shown, the numbers of animals averaged are 32 (A), 31 (B), 32 (C), 32 (D), 30 (E), 14 (F), 15 (G), 32 (H), 32 (I), 31 (J), 31 (K), and 32 (L).

Figure 4. Behavioral responses to changes in light schedules.

Flies entrained to a 12∶12 LD cycle were given an eight-hour phase delay. (A) w¹¹¹⁸; (B) pdfr⁵³⁰⁴; ; cry^b double mutants; (C) cry^b single mutants; (D) pdfr⁵³⁰⁴; ; cry^b flies carrying a ∼70 kB pdfr-myc transgene. Green arrowheads mark anticipatory behavior of the original light schedule after the delay. Red arrowheads mark the onset of activity by pdfr⁵³⁰⁴; ; cry^b flies following lights-on in the original and delayed schedules. Note that cry mutant flies require more cycles to re-entrain to such phase changes , and that pdfr⁵³⁰⁴; ; pdfr-myc, cry^b (D) displayed a cry^b-like phenotype, indicating rescue of the double mutant behavioral defect. For the experiment shown, the numbers of animals averaged are 16 (A), 16 (B), 16 (C), and 32 (D).

Figure 5. Behavioral defects of pdfr⁵³⁰⁴; ; cry^b double mutants are reversed by restoring normal pdfr expression.

Flies with either the 70 kB pdfr-myc transgene or GAL4-driven pdfr expression in the clock network in the pdfr⁵³⁰⁴; ; cry^b double mutant were tested for LD behavior. (A) w¹¹¹⁸; (B) pdfr⁵³⁰⁴; ; cry^b double mutant flies; (C) cry^b mutant flies; (D) pdfr⁵³⁰⁴; ; pdfr-myc, cry^b; (E) pdfr⁵³⁰⁴; uas-pdfr/+ ; cry^b (control flies for GAL4-mediated rescue); (F) pdfr⁵³⁰⁴; uas-pdfr/Pdf-GAL4 ; cry^b (pdfr expression restored specifically in PDF cells in the double mutants); (G) pdfr⁵³⁰⁴; uas-pdfr/cry(39)-GAL4 ; cry^b (pdfr expression restored specifically in clock cells in the double mutants); (H) pdfr⁵³⁰⁴; uas-pdfr/tim(uas)-GAL4 ; cry^b (pdfr expression restored in clock neurons and many other brain cells in the double mutants). For the experiment shown, the numbers of animals averaged are 32 (A), 31 (B), 29 (C), 28 (D), 21 (E), 32 (F), 20 (G), and 31 (H).

Figure 6. LD Molecular rhythms in the 5^th s-LNv and LNd are deranged in the double mutants.

At various time-points, PER levels were monitored in the nucleus (filled histograms) and cytoplasm (open histograms) of the 5^th s-LNv (A, B) and the ITP(+) LNd (C, D). (A) In the 5^th s-LNv of w¹¹¹⁸; ; cry^b/01, PER levels in the nucleus and cytoplasm are robustly cycling: nuclear amplitude rhythm – 19.4-fold; cytoplasmic amplitude rhythm – 9.0-fold. ANOVA test revealed that the differences in nuclear staining levels are significant (P<0.0001). (B) In the 5^th s-LNv of pdfr⁵³⁰⁴; ; cry^b/01, PER staining is always found in the nucleus with very low amplitude rhythms and no phase difference between nucleus and cytoplasm: nuclear amplitude rhythm –2.4-fold; cytoplasm amplitude rhythm – 3.0-fold. ANOVA test revealed that the difference in this group is significant (P = 0.03). (C) In the ITP(+) LNd of w¹¹¹⁸; ; cry^b/01, PER levels in the nucleus and cytoplasm are robustly cycling, nuclear amplitude rhythm – 19.0-fold; cytoplasmic amplitude rhythm – 11.4-fold. ANOVA test revealed that the difference in this group is significant (P<0.0001). (D) In the ITP(+) LNd of pdfr⁵³⁰⁴; ; cry^b/01, PER staining is always found in the nucleus with very low amplitude rhythms and no phase difference between nucleus and cytoplasm: nuclear amplitude rhythm – 3.8-fold; cytoplasmic amplitude rhythm – 2.8-fold. ANOVA test revealed that the difference in this group is significant (P<0.0001). Results from post-hoc statistical tests are presented in Table 2.

Figure 7. Quantification of PER intensity in the nucleus and cytoplasm of PDF(+) s-LNvs in LD cycles.

(A) PER staining in single focal plane images of the PDF(+) s-LNvs at various time points. s-LNvs were chosen by size and PDH immunoreactivity. All four genotypes of flies show normal cycling of PER in the s-LNvs. (B and C) Quantifications of the mean pixel intensities of PER in the nucleus (filled histograms) and cytoplasm (open histograms) at ZT14 (B) and ZT23 (C) (n = 5∼6).

Figure 8. Lack of PDF and CRY signaling causes weak, short behavioral rhythms under LL.

Group-averaged actograms of each genotype. (A) w¹¹¹⁸; ; cry^b/01 single mutant flies; (B) pdfr⁵³⁰⁴ single mutants; (C) pdfr⁵³⁰⁴; ; cry^b/01 double mutant flies; (D) cry-G4⁽¹⁹⁾; cry^b ss Pdf⁰¹ double mutant flies.

Figure 9. Light intensity affects the phase of LL rhythmic behavior of the double mutants.

(A–I) Group-averaged actograms of each genotype. Low light intensity – top row. Middle light intensity – middle row. High light intensity – bottom row. (A–C) w¹¹¹⁸; ; cry^b/01 flies; (D–F) pdfr⁵³⁰⁴; ; cry^b/01 flies; (G–I) cry^b flies. (J) Averaged phase markers under different light intensity LL conditions; arrow widths display standard errors of the populations. Statistical analyses were performed between behavioral peak phases under different light intensity conditions of the same genotype flies. ANOVA test for both double mutant flies showed that the difference was significant (P<0.0001). Tukey-Kramer multiple comparisons post-hoc test results revealed P<0.0001 for [L vs M] and for [L vs H] for both double mutant genotypes. For the experiment shown, the numbers of animals averaged are 27 (A), 30 (B), 32 (C), 30 (D), 32 (E), 30 (F), 30 (G), 32 (H), and 32 (I).

Figure 10. PER molecular rhythms in double mutants under different light intensity LL conditions.

(A–D) low intensity; (E–H) high intensity. (A, E) PDF(+) s-LNv; (B, F) 5^th s-LNv; (C, G) ITP(+) LNd; (D, H) DN1. Filled histograms - nuclear values and open histograms - cytoplasmic values (B, C, F, G). (A, E) PDF(+) s-LNv (magenta) showed robust PER (green) staining rhythms under both light intensity conditions. (B–D, F–H) For the other cells examined, a statistically-significant amplitude rhythm was shown under low light conditions for the 5^th s-LNv and for the ITP(+) LNd. None of the cells showed a significant amplitude rhythm under high light intensity. (B) At low intensity, both nuclear and cytoplasmic peaks in the 5^th s-LNv occurred at CT77: nuclear amplitude rhythm – 2.2-fold; cytoplasmic amplitude rhythm – 2.5-fold. ANOVA test revealed that the difference in this group is significant (P<0.0003). (C) In the ITP(+) LNd, both nuclear and cytoplasmic peaks occurred at CT77: nuclear amplitude rhythm – 3.3-fold; cytoplasmic amplitude rhythm – 2.0-fold. ANOVA test revealed the group difference is significant (P<0.0001). (D) PER(+) DN1 neurons were counted at four time points under low light intensity conditions. The rhythm in PER(+) DN1 didn't show 24 hour rhythms. ANOVA test P value was 0.0038. (F) At high intensity, nuclear peak in the 5^th s-LNv occurred at CT77 and cytoplasmic peak occurred at CT71: nuclear amplitude rhythm – 1.8-fold; cytoplasmic amplitude rhythm – 1.4-fold. ANOVA test indicated the group difference was not significant (P = 0.0527). (G) In the ITP(+) LNd, the nuclear peak occurred at CT83 and the cytoplasmic peak at CT77: nuclear amplitude rhythm – 2.3-fold; cytoplasmic amplitude rhythm – 1.4-fold. ANOVA test revealed that the difference in this group is not significant (P = 0.08). (H) PER(+) DN1 neurons were counted at four time points under high intensity conditions. The rhythm in PER(+) DN1 showed a peak at CT65, but it was not statistically significant. Results from post-hoc statistical tests are presented in Table 2.