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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.

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Bivariate phase analysis of isolated body parts. These data are the same as shown in Figure 4. 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). The axes for each plot describe an x-y plane with the origin occurring at the center of the unit circle plotted within the plane for reference. The point (0,1) defines the beginning of the subjective day, or time 0. Time moves in a counter clock-wise direction on this circle. Each point denotes the head of a vector that summarizes the phase of an individual specimen. The tail of this vector would extend from the origin to the plotted point, with the direction indicating the mean peak time across cycles and the magnitude (distance from the origin) describing the variability of the peaks for each specimen. However, these tails are not plotted to simplify the appearance of the figure. A mean vector is calculated and fully plotted to show the mean phase time for each group of points as well as the mean variability (indicated by its length). Below each plot: the length of each mean vector is given by r; the mean time of each vector is given by phi and the p-value used to assess whether the phase differs between groups is obtained by Hotelling's two-sample test (for more detail see [32]). This analysis is continued in Figure 10.
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Figure 9: Bivariate phase analysis of isolated body parts. These data are the same as shown in Figure 4. 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). The axes for each plot describe an x-y plane with the origin occurring at the center of the unit circle plotted within the plane for reference. The point (0,1) defines the beginning of the subjective day, or time 0. Time moves in a counter clock-wise direction on this circle. Each point denotes the head of a vector that summarizes the phase of an individual specimen. The tail of this vector would extend from the origin to the plotted point, with the direction indicating the mean peak time across cycles and the magnitude (distance from the origin) describing the variability of the peaks for each specimen. However, these tails are not plotted to simplify the appearance of the figure. A mean vector is calculated and fully plotted to show the mean phase time for each group of points as well as the mean variability (indicated by its length). Below each plot: the length of each mean vector is given by r; the mean time of each vector is given by phi and the p-value used to assess whether the phase differs between groups is obtained by Hotelling's two-sample test (for more detail see [32]). This analysis is continued in Figure 10.

Mentions: The analysis of phase presented in Figure 7 and Figure 8 relies on an average estimate of phase for each specimen and it neglects any variability in the occurrence of peaktime associated with the daily rhythm in bioluminescence. As noted above, the mean vectors depicted within each panel of Figures 7,8 represent a vector average of the individual phase estimates with the direction of the vector indicating the mean time and the length of the vector indicating the variability (dispersion) of the estimates. An alternative analysis, presented in Figures 9,10, preserves the intraspecimen variability. This analysis, called a bivariate analysis [32], represents an individual specimen as a vector in the x-y plane. The position of each point (plotted as an asterisk or an open circle) is determined by the variability of the occurrence of the peak for that individual record. Thus, the points that fall on the diameter of the circle are precisely consistent from day to day, while those falling closer to the origin indicate greater variability. The position of the point within the circle describes the mean peak time for each specimen. In this way, each point indicates the head of a vector that summarizes the phase for a particular specimen. The tail of the vector would connect the point to the origin but we do not plot the vectors this way because the figure would be difficult to interpret. A statistical comparison between the two groups (in this case per-luc v tim-luc or per-luc; cry+ v per-luc; cryb) is obtained by first calculating a mean vector that connects the origin to the center of the cloud of points defined by the respective group and then testing whether or not these representative vectors are different from one another.


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)

Bivariate phase analysis of isolated body parts. These data are the same as shown in Figure 4. 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). The axes for each plot describe an x-y plane with the origin occurring at the center of the unit circle plotted within the plane for reference. The point (0,1) defines the beginning of the subjective day, or time 0. Time moves in a counter clock-wise direction on this circle. Each point denotes the head of a vector that summarizes the phase of an individual specimen. The tail of this vector would extend from the origin to the plotted point, with the direction indicating the mean peak time across cycles and the magnitude (distance from the origin) describing the variability of the peaks for each specimen. However, these tails are not plotted to simplify the appearance of the figure. A mean vector is calculated and fully plotted to show the mean phase time for each group of points as well as the mean variability (indicated by its length). Below each plot: the length of each mean vector is given by r; the mean time of each vector is given by phi and the p-value used to assess whether the phase differs between groups is obtained by Hotelling's two-sample test (for more detail see [32]). This analysis is continued in Figure 10.
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Related In: Results  -  Collection

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Figure 9: Bivariate phase analysis of isolated body parts. These data are the same as shown in Figure 4. 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). The axes for each plot describe an x-y plane with the origin occurring at the center of the unit circle plotted within the plane for reference. The point (0,1) defines the beginning of the subjective day, or time 0. Time moves in a counter clock-wise direction on this circle. Each point denotes the head of a vector that summarizes the phase of an individual specimen. The tail of this vector would extend from the origin to the plotted point, with the direction indicating the mean peak time across cycles and the magnitude (distance from the origin) describing the variability of the peaks for each specimen. However, these tails are not plotted to simplify the appearance of the figure. A mean vector is calculated and fully plotted to show the mean phase time for each group of points as well as the mean variability (indicated by its length). Below each plot: the length of each mean vector is given by r; the mean time of each vector is given by phi and the p-value used to assess whether the phase differs between groups is obtained by Hotelling's two-sample test (for more detail see [32]). This analysis is continued in Figure 10.
Mentions: The analysis of phase presented in Figure 7 and Figure 8 relies on an average estimate of phase for each specimen and it neglects any variability in the occurrence of peaktime associated with the daily rhythm in bioluminescence. As noted above, the mean vectors depicted within each panel of Figures 7,8 represent a vector average of the individual phase estimates with the direction of the vector indicating the mean time and the length of the vector indicating the variability (dispersion) of the estimates. An alternative analysis, presented in Figures 9,10, preserves the intraspecimen variability. This analysis, called a bivariate analysis [32], represents an individual specimen as a vector in the x-y plane. The position of each point (plotted as an asterisk or an open circle) is determined by the variability of the occurrence of the peak for that individual record. Thus, the points that fall on the diameter of the circle are precisely consistent from day to day, while those falling closer to the origin indicate greater variability. The position of the point within the circle describes the mean peak time for each specimen. In this way, each point indicates the head of a vector that summarizes the phase for a particular specimen. The tail of the vector would connect the point to the origin but we do not plot the vectors this way because the figure would be difficult to interpret. A statistical comparison between the two groups (in this case per-luc v tim-luc or per-luc; cry+ v per-luc; cryb) is obtained by first calculating a mean vector that connects the origin to the center of the cloud of points defined by the respective group and then testing whether or not these representative vectors are different from one another.

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
Related in: MedlinePlus