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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 tim-luc-reported activity in rhythmic isolated body parts continued. Details for this figure are the same as in Figure 5. e.) averaged data for cry+ or cry5 rhythmic isolated forelegs as tabulated in Table 1.
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Figure 6: cryb vs. cry+: average tim-luc-reported activity in rhythmic isolated body parts continued. Details for this figure are the same as in Figure 5. e.) averaged data for cry+ or cry5 rhythmic isolated forelegs as tabulated in Table 1.

Mentions: In Figures 3,4,5,6, plots of average counts for each tissue are presented, based on the rhythmic subset of specimens tabulated in Tables 1 and 2 for LD 12:12. These average signals indicate once again that cryb impinges on clock-gene cyclings. For example, with the exception of the BG-luc-reported wing data shown in Figure 4d, simple inspection of the raw data column suggests that cryb reduces or eliminates such cyclings under LD 12:12 (compare the raw data for cry+ v cryb for example, in Figure 3a, 3b, or 3c). The appearance of rhythmicity in the cryb specimens is mostly revealed after detrending and normalization (see Figures 3,4,5,6 the second column labeled normalized). However, in the case of heads and bodies – for which there were very few rhythmic cryb samples (Table 1) – no rhythmicity is evident in the averaged plots. One explanation for this could be that the separate rhythms are not in phase with one another or are noisy; thus the average may not appear to be rhythmic. Nevertheless, applying autocorrelation to the averaged time-courses revealed daily cyclings to have occurred in all cases except for the average taken from the rhythmic subset of BG-luc; cryb bodies. These results show that rhythmicity is evident in the mean signal as well as when it was initially tabulated on a specimen by specimen basis (see Materials and Methods). Nevertheless, there might not have been agreement between these two views of rhythmicity for any of the tissues (or between different approaches to the analysis of phase as described below). Although it is unlikely that arrhythmic specimens would give rise to a mean rhythm, it is conceivable that rhythmic specimens might give rise to an arrhythmic mean.


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 tim-luc-reported activity in rhythmic isolated body parts continued. Details for this figure are the same as in Figure 5. e.) averaged data for cry+ or cry5 rhythmic isolated forelegs as tabulated in Table 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC103668&req=5

Figure 6: cryb vs. cry+: average tim-luc-reported activity in rhythmic isolated body parts continued. Details for this figure are the same as in Figure 5. e.) averaged data for cry+ or cry5 rhythmic isolated forelegs as tabulated in Table 1.
Mentions: In Figures 3,4,5,6, plots of average counts for each tissue are presented, based on the rhythmic subset of specimens tabulated in Tables 1 and 2 for LD 12:12. These average signals indicate once again that cryb impinges on clock-gene cyclings. For example, with the exception of the BG-luc-reported wing data shown in Figure 4d, simple inspection of the raw data column suggests that cryb reduces or eliminates such cyclings under LD 12:12 (compare the raw data for cry+ v cryb for example, in Figure 3a, 3b, or 3c). The appearance of rhythmicity in the cryb specimens is mostly revealed after detrending and normalization (see Figures 3,4,5,6 the second column labeled normalized). However, in the case of heads and bodies – for which there were very few rhythmic cryb samples (Table 1) – no rhythmicity is evident in the averaged plots. One explanation for this could be that the separate rhythms are not in phase with one another or are noisy; thus the average may not appear to be rhythmic. Nevertheless, applying autocorrelation to the averaged time-courses revealed daily cyclings to have occurred in all cases except for the average taken from the rhythmic subset of BG-luc; cryb bodies. These results show that rhythmicity is evident in the mean signal as well as when it was initially tabulated on a specimen by specimen basis (see Materials and Methods). Nevertheless, there might not have been agreement between these two views of rhythmicity for any of the tissues (or between different approaches to the analysis of phase as described below). Although it is unlikely that arrhythmic specimens would give rise to a mean rhythm, it is conceivable that rhythmic specimens might give rise to an arrhythmic mean.

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