Abstract
The circadian clock plays a vital role in monarch butterfly (Danaus plexippus) migration by providing the timing component of time-compensated sun compass orientation, a process that is important for successful navigation. We therefore evaluated the monarch clockwork by focusing on the functions of a Drosophila-like cryptochrome (cry), designated cry1, and a vertebrate-like cry, designated cry2, that are both expressed in the butterfly and by placing these genes in the context of other relevant clock genes in vivo. We found that similar temporal patterns of clock gene expression and protein levels occur in the heads, as occur in DpN1 cells, of a monarch cell line that contains a light-driven clock. CRY1 mediates TIMELESS degradation by light in DpN1 cells, and a light-induced TIMELESS decrease occurs in putative clock cells in the pars lateralis (PL) in the brain. Moreover, monarch cry1 transgenes partially rescue both biochemical and behavioral light-input defects in cryb mutant Drosophila. CRY2 is the major transcriptional repressor of CLOCK:CYCLE-mediated transcription in DpN1 cells, and endogenous CRY2 potently inhibits transcription without involvement of PERIOD. CRY2 is co-localized with clock proteins in the PL, and there it translocates to the nucleus at the appropriate time for transcriptional repression. We also discovered CRY2-positive neural projections that oscillate in the central complex. The results define a novel, CRY-centric clock mechanism in the monarch in which CRY1 likely functions as a blue-light photoreceptor for entrainment, whereas CRY2 functions within the clockwork as the transcriptional repressor of a negative transcriptional feedback loop. Our data further suggest that CRY2 may have a dual role in the monarch butterfly's brain—as a core clock element and as an output that regulates circadian activity in the central complex, the likely site of the sun compass.
Highlights
In insects, circadian clocks regulate the timing of numerous biological events [1]
When the temporal profile of CRY2 staining in the pars intercerebralis (PI), pars lateralis (PL), and dorsal and ventral optic lobe (OL) was analyzed over the circadian cycle, we found a significant circadian oscillation of cytoplasmic CRY2 staining in PL (p, 0.05), PI (p, 0.01), and OL (p, 0.01), which was most pronounced in OL (Figure 6F), with peak staining at circadian time (CT) 15
Collectively, our results provide several lines of evidence suggesting that monarch CRY1 functions in vivo as a circadian photoreceptor, whereas CRY2 functions as a transcriptional repressor for the butterfly clockwork
Summary
Circadian clocks regulate the timing of numerous biological events [1]. Some examples of critical circadian rhythm outputs in holometabolous insects include the time of day of adult eclosion, the seasonal timing of reproductive diapause, and time-compensated sun compass navigation. The resultant PER and TIM proteins form heterodimers that translocate back into the nucleus to repress their own transcription via inhibitory effects on CLK and CYC. We have been developing this species as a model to examine the role of the circadian clock in time-compensated sun compass orientation and in the seasonal induction of the migratory generation [12]. Using clock protein expression patterns, we previously identified the location of circadian clock cells in the dorsolateral protocerebrum (pars lateralis [PL]) of the butterfly [13], which expresses PER, TIM, and a Drosophila-like CRY (designated CRY1; see below). A CRY1 pathway may connect the circadian clock to neurosecretory cells in the pars intercerebralis (PI) for the initiation of the migratory state [12,13].
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