Disruption of Cryptochrome partially restores circadian rhythmicity to the arrhythmic period mutant of Drosophila

Abstract

The Drosophila melanogaster circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light-dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cry(b) mutation reduces the light sensitivity of the fly's clock, yet locomotor activity rhythms in constant darkness or light-dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when we combined a period-null mutation with cry(b), circadian rhythmicity in locomotor behavior in light-dark cycles, as measured by a number of different criteria, was restored. This effect was significantly reduced in timeless-null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and TIM protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, we have uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period-independent role for timeless in the Drosophila circadian pacemaker. Although we do not yet have a molecular correlate for these apparently iconoclastic observations, we provide a systems explanation for these results based on differential sensitivities of subsets of circadian pacemaker neurons to light.

Fig. 1.

Locomotor activity in LD 12:12 cycles. (A) Canton-S. (B) cry^b.(C) per⁰¹. (D) tim⁰¹. (E) per⁰¹; cry^b. (F) tim⁰¹; cry^b. (G) per⁰¹; tim^UL; cry^b. (H) per⁰¹; tim⁰¹; cry^b. Strains are shown at 18°C (gray) and 29°C (black). The y axis shows activity counts ± SEM. Black and white bars represent the LD regime, with lights on at ZT0 and off at ZT12. A and B are reproduced from ref. . Arrow in the per⁰¹ panel shows the “morning peak” described by Helfrich-Förster (16).

Fig. 2.

Locomotor response to light pulses in LD cycles. A 1-h light pulse (LP, black arrows) was administered on the 5th day of entrainment in wild-type (A), cry^b (B), and per⁰¹; cry^b (C). Black/white bars represent the LD regime. The average locomotor activity for each group of flies from each genotype is shown with a smoothing five-point moving average on the day before (gray) and after (black) the light pulse. Error bars are omitted for clarity. ANOVA on the raw data revealed significant delays for wild-type at ZT15 (F_1,540 = 22.56, P << 0.001) and a nonsignificant advance at ZT21 (F_1,612 = 2.63, P = 0.1). There were no significant phase shifts for cry^b. per⁰¹; cry^b gave significant advances at ZT15 (F_1,540 = 3.91, P = 0.048) and nonsignificant advances at ZT21 (P = 0.45) (see also Fig. 7).

Fig. 3.

Locomotor activity peaks in different T cycles. Average peak time for groups of flies on the 5th day (LD16:16 in B and C from 4th day) is shown for Canton-S (A), cry^b (B), per⁰¹ (C), per⁰¹; cry^b (D), per⁰¹; tim^UL; cry^b (E), tim⁰¹; cry^b (F), and per⁰¹; tim⁰¹; cry^b (G), ±95% confidence limits. “Lights on” occurs at 0 on y axis, and “lights off” is represented by a diagonal black line. The dotted line represents a linear best-fit to the locomotor peaks at each light-dark regime. (H) Slope ± 95% confidence limit of regression lines in A-G. Regression significance: *, P < 0.05; **, P < 0.01.

Fig. 4.

Nuclear localization and TIM levels in wild-type, per⁰¹, per⁰¹; cry^b and cry^b.(A) The localization is shown of TIM (in red) in the l-LN_vs, s-LN_vs, and LN_ds at ZT21. No nuclear staining was detected in neurons of either per⁰¹ or per⁰¹; cry^b mutants. Note that, in cry^b flies, the nuclear accumulation of TIM appears delayed in the l-LN_vs but not in other neurons. In an independent experiment using a different cry^b strain, TIM was predominantly cytoplasmic in the l-LN_vs at ZT21, whereas the other clock neurons showed nuclear staining. These results are representative of 10 brain hemispheres. (Scale bar, 10 μm.) (B) TIM cycles in wild-type and per⁰¹, but does not cycle in cry^b or per⁰¹; cry^b heads in LD cycles. The high molecular weight TIM bands seen at all time points in per⁰¹; cry^b but only present at ZT0 in per⁰¹ heads, are indicative of hyperphosphorylated forms of TIM. The graph shows the normalized mean ± SEM for three independent replicate blots using HSP70 as the loading control (y axis) against ZT. Wild-type (white diamonds, continuous line) per⁰¹ (black dots, hatched and dotted line), per⁰¹; cry^b (gray triangles, hatched lines), and cry^b (white squares, dotted line) blots are shown. Wild-type and cry^b data are from ref. .

Fig. 5.

Photoreceptor connections to l-LN_vs. Projections of confocal z stacks. Axons of a subset of visual photoreceptors end in close proximity to the l-LN_vs. Photoreceptor cells expressing β-galactosidase (green) under control of Rhodopsin3 (Rh3), Rhodopsin5 (Rh5), and Rhodopsin6 (Rh6) promoters extend their axons through the medulla in close proximity of l-LN_vs varicosities (blue arrowhead) labeled with α-PDH antibody (red). The Rh6-driven expression of lacZ also shows projections (arrow) from the Hofbauer-Buchner (HB) eyelets (38). Some neurites from Rh3- and Rh5-expressing cells end in close proximity (yellow arrow heads) to the l-LN_vs. HB projections engage in synapses (magenta arrow heads) with fibers connecting l-LN_vs and s-LN_vs (white square) as described (38). (Scale bar, 20 μm.)