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Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila.

Levine JD, Funes P, Dowse HB, Hall JC - BMC Neurosci (2002)

Bottom Line: Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing.Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and NSF Center for Biological Timing, Brandeis University, Waltham, MA 02454, USA. jlev@brandeis.edu

ABSTRACT

Background: Previously, we reported effects of the cry(b) mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.

Results: Isolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry+ controls. In addition, cry(b) alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions.

Conclusions: These analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression.

Show MeSH
Circular phase analysis of luciferase expression in isolated body parts. The left column shows phase comparisons for BG-luc; cry+ (plotted with asterisk) vs tim-luc; cry+ (plotted with open circles). The right column shows BG-luc; cry+ (plotted as asterisk) vs. BG-luc; cryb (plotted as open circles). All of the data shown here were collected in a light-dark cycle (LD 12:12). The time of lights on is indicated as 0 h on the circle. On these plots time moves forward in a counter-clockwise direction. Phase estimates for each rhythmic specimen are plotted just outside the unit circle and a mean vector summarizes the phase of the group with the mean direction indicating the time and the magnitude of the vector indicating the dispersion (variability) of the individual estimates (see Materials and Methods and [27,32]). The Watson-Williams-Stevens test returns an F-statistic that is used to evaluate whether the vectors are significantly different from one another[32]. Rayleigh's test [27,32] shows each vector is significantly different (p < 0.05) from the  vector (random distribution) with the exception of the BG-luc; cryb vectors shown on the right for heads and bodies in b and c respectively. Beneath each circular plot the difference in hours is given, followed by the mean time for each vector with the number of specimens, n, in brackets, and a p-value for the comparison, a.) comparisons for isolated antennal pairs, b.) isolated heads, c.) isolated bodies. Number of rhythmic samples for each group are given in Table 1 and Table 2. This analysis is continued in Figure 8.
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Figure 7: Circular phase analysis of luciferase expression in isolated body parts. The left column shows phase comparisons for BG-luc; cry+ (plotted with asterisk) vs tim-luc; cry+ (plotted with open circles). The right column shows BG-luc; cry+ (plotted as asterisk) vs. BG-luc; cryb (plotted as open circles). All of the data shown here were collected in a light-dark cycle (LD 12:12). The time of lights on is indicated as 0 h on the circle. On these plots time moves forward in a counter-clockwise direction. Phase estimates for each rhythmic specimen are plotted just outside the unit circle and a mean vector summarizes the phase of the group with the mean direction indicating the time and the magnitude of the vector indicating the dispersion (variability) of the individual estimates (see Materials and Methods and [27,32]). The Watson-Williams-Stevens test returns an F-statistic that is used to evaluate whether the vectors are significantly different from one another[32]. Rayleigh's test [27,32] shows each vector is significantly different (p < 0.05) from the vector (random distribution) with the exception of the BG-luc; cryb vectors shown on the right for heads and bodies in b and c respectively. Beneath each circular plot the difference in hours is given, followed by the mean time for each vector with the number of specimens, n, in brackets, and a p-value for the comparison, a.) comparisons for isolated antennal pairs, b.) isolated heads, c.) isolated bodies. Number of rhythmic samples for each group are given in Table 1 and Table 2. This analysis is continued in Figure 8.

Mentions: 1. The experimental design is described in Krishnan et al [23] and in Materials and Methods. LD 12:12 refers to a light-dark cycle with 12 hours of light and 12 hours of darkness. 2. Individual body parts were isolated by dissection and placed immediately in cell culture medium containing luciferin substrate for analysis under LD 12:12 (see Materials and Methods) 3. Fly strains are described in Stanewsky et al [20,21]. 4. Na is number of specimens analyzed. This analysis was applied to samples previously reported elsewhere [23]. The number of cry+ specimens was increased over the number previously reported as follows 10 additional heads, 9 additional forelegs and 15 additional bodies; the number of cryb specimens is unchanged. 5. Each sample is evaluated separately and considered rhythmic based on the correlogram and the requirement that rhythmicity falls between 18–40 hours according to spectral analysis (see Materials and Methods and also see [27,30]). We asked whether cryb affects rhythmicity in these body parts. Chi-squared tests showed significant effects of the mutation on rhythmicity with BG-luc or tim-luc for all body parts (p < .02) except the wing (p > .05 for both reporters). As discussed in the text, the finding of rhythmicity does not necessarily indicate statistical significance for the rhythm. The numbers in parentheses indicate how many of the specimens we called rhythmic also displayed statistically significant rhythmicity (the height of the peaks in the correlogram were above the 95% confidence line). The remainder of the rhythmic specimens were determined to be rhythmic because of the sinusoidal shape of the correlogram. See the text for further discussion about our criteria for rhythmicity (also see [23]). 6. The estimate of circadian period is assessed by mesa [33] for each individual. Mean and standard error of the mean (SEM) are tabulated from the individual estimates. 7. The rhythmicity index (RI) is a measure of the strength of the rhythm obtained from the autocorrelation function as described in Levine et al [27], see also [31]. Like the estimate of period, the RI is given as a mean with SEM based on the values obtained for each individual rhythmic sample. The cryb mutation significantly reduces the RI value for each reporter in every body part (t-test, p < .001). Note that these tests could not be performed for tim-luc specimens from isolated heads or bodies because there were no rhythmic samples to evaluate. 8. The Rhythmicity Statistic (RS) is calculated as a ratio of the RI to the absolute value of the 95% confidence line for the correlogram obtained for each individual with means and SEM tabulated as above for RI (see Figure 2 and Figure 3, for examples). The RS provides a quick indicator of whether the rhythm is statistically significant (RS ≥ 1) or not (see Materials and Methods). 9. Amplitude is a measure of the distance from the peak (or trough) to the mean in the detrended and normalized rhythmic data (see Materials and Methods for more details) 10. The two numbers given here represent the mean phase, or the direction in which the phase vector points and the correlation coefficient describing the distribution of phases among the specimens, or the length of the vector . Phase is determined for the group of rhythmic individuals using circular statistics [22,30]. See Figure 7 for example. 11. Mean expression level is given as mean ± SEM for counts per second of bioluminescence/hour.


Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila.

Levine JD, Funes P, Dowse HB, Hall JC - BMC Neurosci (2002)

Circular phase analysis of luciferase expression in isolated body parts. The left column shows phase comparisons for BG-luc; cry+ (plotted with asterisk) vs tim-luc; cry+ (plotted with open circles). The right column shows BG-luc; cry+ (plotted as asterisk) vs. BG-luc; cryb (plotted as open circles). All of the data shown here were collected in a light-dark cycle (LD 12:12). The time of lights on is indicated as 0 h on the circle. On these plots time moves forward in a counter-clockwise direction. Phase estimates for each rhythmic specimen are plotted just outside the unit circle and a mean vector summarizes the phase of the group with the mean direction indicating the time and the magnitude of the vector indicating the dispersion (variability) of the individual estimates (see Materials and Methods and [27,32]). The Watson-Williams-Stevens test returns an F-statistic that is used to evaluate whether the vectors are significantly different from one another[32]. Rayleigh's test [27,32] shows each vector is significantly different (p < 0.05) from the  vector (random distribution) with the exception of the BG-luc; cryb vectors shown on the right for heads and bodies in b and c respectively. Beneath each circular plot the difference in hours is given, followed by the mean time for each vector with the number of specimens, n, in brackets, and a p-value for the comparison, a.) comparisons for isolated antennal pairs, b.) isolated heads, c.) isolated bodies. Number of rhythmic samples for each group are given in Table 1 and Table 2. This analysis is continued in Figure 8.
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Related In: Results  -  Collection

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Figure 7: Circular phase analysis of luciferase expression in isolated body parts. The left column shows phase comparisons for BG-luc; cry+ (plotted with asterisk) vs tim-luc; cry+ (plotted with open circles). The right column shows BG-luc; cry+ (plotted as asterisk) vs. BG-luc; cryb (plotted as open circles). All of the data shown here were collected in a light-dark cycle (LD 12:12). The time of lights on is indicated as 0 h on the circle. On these plots time moves forward in a counter-clockwise direction. Phase estimates for each rhythmic specimen are plotted just outside the unit circle and a mean vector summarizes the phase of the group with the mean direction indicating the time and the magnitude of the vector indicating the dispersion (variability) of the individual estimates (see Materials and Methods and [27,32]). The Watson-Williams-Stevens test returns an F-statistic that is used to evaluate whether the vectors are significantly different from one another[32]. Rayleigh's test [27,32] shows each vector is significantly different (p < 0.05) from the vector (random distribution) with the exception of the BG-luc; cryb vectors shown on the right for heads and bodies in b and c respectively. Beneath each circular plot the difference in hours is given, followed by the mean time for each vector with the number of specimens, n, in brackets, and a p-value for the comparison, a.) comparisons for isolated antennal pairs, b.) isolated heads, c.) isolated bodies. Number of rhythmic samples for each group are given in Table 1 and Table 2. This analysis is continued in Figure 8.
Mentions: 1. The experimental design is described in Krishnan et al [23] and in Materials and Methods. LD 12:12 refers to a light-dark cycle with 12 hours of light and 12 hours of darkness. 2. Individual body parts were isolated by dissection and placed immediately in cell culture medium containing luciferin substrate for analysis under LD 12:12 (see Materials and Methods) 3. Fly strains are described in Stanewsky et al [20,21]. 4. Na is number of specimens analyzed. This analysis was applied to samples previously reported elsewhere [23]. The number of cry+ specimens was increased over the number previously reported as follows 10 additional heads, 9 additional forelegs and 15 additional bodies; the number of cryb specimens is unchanged. 5. Each sample is evaluated separately and considered rhythmic based on the correlogram and the requirement that rhythmicity falls between 18–40 hours according to spectral analysis (see Materials and Methods and also see [27,30]). We asked whether cryb affects rhythmicity in these body parts. Chi-squared tests showed significant effects of the mutation on rhythmicity with BG-luc or tim-luc for all body parts (p < .02) except the wing (p > .05 for both reporters). As discussed in the text, the finding of rhythmicity does not necessarily indicate statistical significance for the rhythm. The numbers in parentheses indicate how many of the specimens we called rhythmic also displayed statistically significant rhythmicity (the height of the peaks in the correlogram were above the 95% confidence line). The remainder of the rhythmic specimens were determined to be rhythmic because of the sinusoidal shape of the correlogram. See the text for further discussion about our criteria for rhythmicity (also see [23]). 6. The estimate of circadian period is assessed by mesa [33] for each individual. Mean and standard error of the mean (SEM) are tabulated from the individual estimates. 7. The rhythmicity index (RI) is a measure of the strength of the rhythm obtained from the autocorrelation function as described in Levine et al [27], see also [31]. Like the estimate of period, the RI is given as a mean with SEM based on the values obtained for each individual rhythmic sample. The cryb mutation significantly reduces the RI value for each reporter in every body part (t-test, p < .001). Note that these tests could not be performed for tim-luc specimens from isolated heads or bodies because there were no rhythmic samples to evaluate. 8. The Rhythmicity Statistic (RS) is calculated as a ratio of the RI to the absolute value of the 95% confidence line for the correlogram obtained for each individual with means and SEM tabulated as above for RI (see Figure 2 and Figure 3, for examples). The RS provides a quick indicator of whether the rhythm is statistically significant (RS ≥ 1) or not (see Materials and Methods). 9. Amplitude is a measure of the distance from the peak (or trough) to the mean in the detrended and normalized rhythmic data (see Materials and Methods for more details) 10. The two numbers given here represent the mean phase, or the direction in which the phase vector points and the correlation coefficient describing the distribution of phases among the specimens, or the length of the vector . Phase is determined for the group of rhythmic individuals using circular statistics [22,30]. See Figure 7 for example. 11. Mean expression level is given as mean ± SEM for counts per second of bioluminescence/hour.

Bottom Line: Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing.Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and NSF Center for Biological Timing, Brandeis University, Waltham, MA 02454, USA. jlev@brandeis.edu

ABSTRACT

Background: Previously, we reported effects of the cry(b) mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.

Results: Isolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cry(b) mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry+ controls. In addition, cry(b) alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions.

Conclusions: These analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression.

Show MeSH