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

Levine, Joel D; Funes, Pablo; Dowse, Harold B; Hall, Jeffrey C

doi:10.1186/1471-2202-3-5

Table 1 Effects of the cry^b mutation on BG-luc and tim-luc reporter activity in isolated body parts under LD 12:12^1,2

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

Genotype³	N_a⁴	Number Rhythmic⁵ (statistical significance)	Period⁶ (Mean ± SEM)	Rhythmicity Index⁷ (Mean ± SEM)	Rhythmicity Statistic⁸ (Mean ± SEM)	Amplitude⁹ (Mean ± SEM)	Phase¹⁰	Activity Counts¹¹ (Mean ± SEM)
Heads
BG-luc; cry ⁺	38	31 (14)	24.7 ± 0.4	0.32 ± 0.03	2.0 ± 0.2	0.10 ± 0.01	3.8,0.9	5759 ± 422
BG-luc; cry ^b	35	4 (1)	22.5 ± 3.5	0.13 ± 0.02	0.8 ± 0.1	0.05 ± 0.01	10.6, 0.7	8382 ± 948
tim-luc; cry ⁺	22	14 (5)	24.7 ± 0.6	0.21 ± 0.03	1.3 ± 0.2	0.10 ± 0.01	3.1,0.8	32024 ± 3766
tim-luc; cry ^b	5	0	-	-	-	-	-	-
Forelegs
BG-luc; cry ⁺	37	33(11)	25.0 ± 0.3	0.45 ± 0.02	2.8 ± 0.2	0.11 ± 0.01	3.7,0.9	1089+93
BG-luc; cry ^b	29	11(4)	25.2 ± 1.2	0.32 ± 0.07	2.0 ± 0.4	0.11 ± 0.01	5.0,0.8	902 ± 181
tim-luc; cry ⁺	24	24(5)	24.6 ± 0.2	0.32 ± 0.03	2.0 ± 0.2	0.12 ± 0.01	1.3,0.9	2605 ± 411
tim-luc; cry ^b	17	4(0)	24.6 ± 3.2	0.02 ± 0.05	0.1 ± 0.3	0.04 ± 0.00	5.7, 0.9	1488 ± 240
Bodies
BG-luc; cry ⁺	32	20(2)	24.2 ± 0.5	0.30 ± 0.03	1.9 ± 0.2	0.10 ± 0.01	5.1,0.9	4549 ± 458
BG-luc; cry ^b	28	5 (0)	20.8 ± 0.4	0.02 ± 0.08	0.1 ± 0.5	0.21 ± 0.09	7.1,0.3	4245 ± 1087
tim-luc; cry ⁺	18	15(4)	24.4 ± 0.2	0.34 ± 0.04	2.1 ± 0.2	0.13 ± 0.01	2.9, 0.9	17678 ± 5053
tim-luc; cry ^b	7	0	-	-	-	-	-	-
Wing
BG-luc; cry ⁺	32	30(14)	25.1 ± 0.1	0.5 ± 0.02	3.1 ± 0.2	0.23 ± 0.01	2.2, 0.9	313+36
BG-luc; cry ^b	25	20 (5)	27.0 ± 0.6	0.2 ± 0.05	1.3 ± 0.3	0.14 ± 0.02	7.5,0.5	333+31
tim-luc; cry ⁺	15	15(7)	25.2 ± 0.2	0.42 ± 0.04	2.6 ± 0.2	0.24 ± 0.02	-0.3, 1.0	497 ± 69
tim-luc; cry ^b	5	5 (0)	26.4 ± 0.4	0.22 ± 0.05	1.4 ± 0.3	0.10 ± 0.01	7.4, 0.9	485+187

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. N_a 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 cry^b 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 cry^b 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 cry^b 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.

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