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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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cryb vs. cry+:average BG-luc reported activity in rhythmic isolated body parts continued. All of the details and definitions of the panels in this figure are the same as in Figure 3. d) Averaged data for cry+ or cryb wings. These are assessed for rhythmicity on a specimen by specimen basis as tabulated in Table 1. e)same as d) for isolated forelegs.
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Figure 4: cryb vs. cry+:average BG-luc reported activity in rhythmic isolated body parts continued. All of the details and definitions of the panels in this figure are the same as in Figure 3. d) Averaged data for cry+ or cryb wings. These are assessed for rhythmicity on a specimen by specimen basis as tabulated in Table 1. e)same as d) for isolated forelegs.

Mentions: We evaluated these various isolated tissues under a series of successive 12-hr-light12-hr-dark cycles (LD 12:12). These environmental conditions are the least likely to reveal deficits in the pattern of clock gene expression because the light-dark cycle provides a dominant stimulus to the clock (clock time is normally synchronized to this environmental cue) and is thought to increase the amplitude of clock gene cycling [20,28,29]. Rhythmicity was assessed for each specimen by autocorrelation analysis [30,27]. This analysis provides a quantitative estimate of rhythmicity with statistical confidence; whereas the confidence interval is based solely on the number of data points used in the analysis without relying on the variability in the data set or any other feature of the measured values, the criterion for statistical confidence is the same for all individual or group analyses within an experiment (see [27] for more detail). In addition, the autocorrelation read-outs may be used in a less stringent (and more subjective) manner to evaluate rhythmicity, based on the shape of a given plot – even when statistical significance is not achieved (see [27] for discussion of this feature of applying autocorrelation). This qualitative criterion is conservative from at least one perspective: If we imagine that cryb's effect might be an elimination of per-luc and tim-luc cyclings (given the mutant's isolation phenotype [21]), elementary scrutiny of correlogram plots increases the chance that data from a given specimen could be judged rhythmic, thereby decreasing the likelihood that we would go overboard in evaluating mutationally induced damage to the clock system. Figure 2 provides an example of why we use a system of qualitative criteria to assess rhythmicity; while the correlogram of a pair of forelegs shown on the top row indicates a statistically significant rhythm, the foreleg pair shown on the bottom row is tabulated as rhythmic even though the correlogram does not achieve statistical significance. In an effort to include any specimen that could be judiciously apprehended as rhythmic, we applied this conservative criterion to obtain the data shown in Tables 1 and 2. We also show our analysis in graphic form for data averaged from each tissue of a given type to illustrate effects of cryb in cases for which only the rhythmic specimens were included (Figures 3 and 4; also see Materials and Methods).


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)

cryb vs. cry+:average BG-luc reported activity in rhythmic isolated body parts continued. All of the details and definitions of the panels in this figure are the same as in Figure 3. d) Averaged data for cry+ or cryb wings. These are assessed for rhythmicity on a specimen by specimen basis as tabulated in Table 1. e)same as d) for isolated forelegs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC103668&req=5

Figure 4: cryb vs. cry+:average BG-luc reported activity in rhythmic isolated body parts continued. All of the details and definitions of the panels in this figure are the same as in Figure 3. d) Averaged data for cry+ or cryb wings. These are assessed for rhythmicity on a specimen by specimen basis as tabulated in Table 1. e)same as d) for isolated forelegs.
Mentions: We evaluated these various isolated tissues under a series of successive 12-hr-light12-hr-dark cycles (LD 12:12). These environmental conditions are the least likely to reveal deficits in the pattern of clock gene expression because the light-dark cycle provides a dominant stimulus to the clock (clock time is normally synchronized to this environmental cue) and is thought to increase the amplitude of clock gene cycling [20,28,29]. Rhythmicity was assessed for each specimen by autocorrelation analysis [30,27]. This analysis provides a quantitative estimate of rhythmicity with statistical confidence; whereas the confidence interval is based solely on the number of data points used in the analysis without relying on the variability in the data set or any other feature of the measured values, the criterion for statistical confidence is the same for all individual or group analyses within an experiment (see [27] for more detail). In addition, the autocorrelation read-outs may be used in a less stringent (and more subjective) manner to evaluate rhythmicity, based on the shape of a given plot – even when statistical significance is not achieved (see [27] for discussion of this feature of applying autocorrelation). This qualitative criterion is conservative from at least one perspective: If we imagine that cryb's effect might be an elimination of per-luc and tim-luc cyclings (given the mutant's isolation phenotype [21]), elementary scrutiny of correlogram plots increases the chance that data from a given specimen could be judged rhythmic, thereby decreasing the likelihood that we would go overboard in evaluating mutationally induced damage to the clock system. Figure 2 provides an example of why we use a system of qualitative criteria to assess rhythmicity; while the correlogram of a pair of forelegs shown on the top row indicates a statistically significant rhythm, the foreleg pair shown on the bottom row is tabulated as rhythmic even though the correlogram does not achieve statistical significance. In an effort to include any specimen that could be judiciously apprehended as rhythmic, we applied this conservative criterion to obtain the data shown in Tables 1 and 2. We also show our analysis in graphic form for data averaged from each tissue of a given type to illustrate effects of cryb in cases for which only the rhythmic specimens were included (Figures 3 and 4; also see Materials and Methods).

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