8 research outputs found
Quantification of PER intensity in the nucleus and cytoplasm of PDF(+) s-LNvs in LD cycles.
<p>(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) (nβ=β5βΌ6).</p
Lack of PDF and CRY signaling causes weak, short behavioral rhythms under LL.
<p>Group-averaged actograms of each genotype. (A) <i>w<sup>1118</sup>; ; cry<sup>b/01</sup></i> single mutant flies; (B) <i>pdfr<sup>5304</sup></i> single mutants; (C) <i>pdfr<sup>5304</sup>; ; cry<sup>b/01</sup></i> double mutant flies; (D) <i>cry-G4<sup>(19)</sup></i>; <i>cry<sup>b</sup> ss Pdf<sup>01</sup></i> double mutant flies.</p
PER molecular rhythms in double mutants under different light intensity LL conditions.
<p>(AβD) low intensity; (EβH) high intensity. (A, E) PDF(+) s-LNv; (B, F) 5<sup>th</sup> 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. (BβD, FβH) For the other cells examined, a statistically-significant amplitude rhythm was shown under low light conditions for the 5<sup>th</sup> 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<sup>th</sup> s-LNv occurred at CT77: nuclear amplitude rhythm β 2.2-fold; cytoplasmic amplitude rhythm β 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 β 3.3-fold; cytoplasmic amplitude rhythm β 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<sup>th</sup> s-LNv occurred at CT77 and cytoplasmic peak occurred at CT71: nuclear amplitude rhythm β 1.8-fold; cytoplasmic amplitude rhythm β 1.4-fold. ANOVA test indicated the group difference was not significant (Pβ=β0.0527). (G) In the ITP(+) LNd, the nuclear peak occurred at CT83 and the cytoplasmic peak at CT77: nuclear amplitude rhythm β 2.3-fold; cytoplasmic amplitude rhythm β 1.4-fold. ANOVA test revealed that the difference in this group is not significant (Pβ=β0.08). (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018974#pone-0018974-t002" target="_blank">Table 2</a>.</p
Lack of PDF and CRY signaling causes arrhythmic behavior under DD.
<p>Group averaged actograms of each genotype under constant darkness (DD) following LD cycles. (A) <i>w<sup>1118</sup></i> flies; (B) <i>pdfr<sup>5304</sup></i> single mutant flies; (C) <i>pdfr<sup>5304</sup>; ; cry<sup>b/01</sup></i> double mutant flies; (D) <i>cry-G4<sup>(19)</sup></i>; <i>cry<sup>b</sup> ss Pdf<sup>01</sup></i>double 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).</p
PDFR-MYC and CRYPTOCHROME are precisely co-expressed in the same subsets of clock neurons.
<p>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<sup>th</sup> 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<sup>th</sup> 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 Β΅m. (G) A summary diagram of the precise PDFR and CRY co-expression in discreet subsets of the three major pacemaker cell groups.</p
Summary of behaviors under constant conditions.
<p>Summary of behaviors under constant conditions.</p
Daily locomotor activities under LD cycles reveal genetic interactions between PDF and CRY signaling pathways.
<p>Averaged activity of various genotype flies for a six-day-period under 8βΆ16 LD (AβD), 12βΆ12 LD (EβH), and 16βΆ8 LD (IβL) entrainment conditions. (A, E, I) <i>w<sup>1118</sup></i> control flies; (B, F, J) <i>pdfr<sup>5304</sup></i> single mutant flies; (C, G, K) <i>pdfr<sup>5304</sup>; ; cry<sup>b/01</sup></i> double mutant flies; (D, H, L) <i>cry-G4<sup>(19)</sup></i>; <i>cry<sup>b</sup> ss Pdf<sup>01</sup></i> double mutant flies. Both double mutant flies display lack of anticipatory peaks under LD cycles. Note that, in <i>pdfr</i> 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).</p
LD Molecular rhythms in the 5<sup>th</sup> s-LNv and LNd are deranged in the double mutants.
<p>At various time-points, PER levels were monitored in the nucleus (filled histograms) and cytoplasm (open histograms) of the 5<sup>th</sup> s-LNv (A, B) and the ITP(+) LNd (C, D). (A) In the 5<sup>th</sup> s-LNv of <i>w<sup>1118</sup>; ; cry<sup>b/01</sup></i>, PER levels in the nucleus and cytoplasm are robustly cycling: nuclear amplitude rhythm β 19.4-fold; cytoplasmic amplitude rhythm β 9.0-fold. ANOVA test revealed that the differences in nuclear staining levels are significant (P<0.0001). (B) In the 5<sup>th</sup> s-LNv of <i>pdfr<sup>5304</sup>; ; cry<sup>b/01</sup></i>, PER staining is always found in the nucleus with very low amplitude rhythms and no phase difference between nucleus and cytoplasm: nuclear amplitude rhythm β2.4-fold; cytoplasm amplitude rhythm β 3.0-fold. ANOVA test revealed that the difference in this group is significant (Pβ=β0.03). (C) In the ITP(+) LNd of <i>w<sup>1118</sup>; ; cry<sup>b/01</sup></i>, PER levels in the nucleus and cytoplasm are robustly cycling, nuclear amplitude rhythm β 19.0-fold; cytoplasmic amplitude rhythm β 11.4-fold. ANOVA test revealed that the difference in this group is significant (P<0.0001). (D) In the ITP(+) LNd of <i>pdfr<sup>5304</sup>; ; cry<sup>b/01</sup></i>, PER staining is always found in the nucleus with very low amplitude rhythms and no phase difference between nucleus and cytoplasm: nuclear amplitude rhythm β 3.8-fold; cytoplasmic amplitude rhythm β 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018974#pone-0018974-t002" target="_blank">Table 2</a>.</p