Rhythm Defects Caused by Newly Engineered Null Mutations in Drosophila's cryptochrome Gene

Abstract

Much of the knowledge about cryptochrome function in Drosophila stems from analyzing the cry b mutant. Several features of this variant's light responsiveness imply either that CRY b retains circadianphotoreceptive capacities or that additional CRY-independent light-input routes subserve these processes. Potentially to resolve these issues, we generated cry knock-out mutants (cry 0 's) by gene replacement. They behaved in an anomalously rhythmic manner in constant light (LL). However, cry 0 flies frequently exhibited two separate circadian components in LL, not observed in most previous cry b analyses. Temperature-dependent circadian phenotypes exhibited by cry 0 flies suggest that CRY is involved in core pacemaking. Further locomotor experiments combined cry 0 with an externally blinding mutation (norpA P24 ), which caused the most severe decrements of circadian photoreception observed so far. cry b cultures were shown previously to exhibit either aperiodic or rhythmic eclosion in separate studies. We found cry 0 to eclose in a solidly periodic manner in light:dark cycles or constant darkness. Furthermore, both cry 0 and cry b eclosed rhythmically in LL. These findings indicate that the novel cry 0 type causes more profound defects than does the cry b mutation, implying that CRY b retains residual activity. Because some norpA P24 cry 0 individuals can resynchronize to novel photic regimes, an as-yet undetermined light-input route exists in Drosophila.

Peripheral circadian clock for the cuticle deposition rhythm in Drosophila melanogaster

“…This result is in contrast to other peripheral tissues, including the antennae, legs, photoreceptor cells, and Malpighian tubules, where CRY plays a role as a core component of the circadian oscillator and the circadian molecular oscillations are absent in cry b or cry 0 flies in DD (9,10,(18)(19)(20)(21). In the photoreceptor cells of the compound eyes, CRY functions as a transcriptional repressor of the CLK-CYCactivated transcription (22).…”

“…However, there is a remarkable difference that, in the peripheral oscillator, CRY functions as a core component besides a photoreceptor. In cry b (a cry loss-of-function mutant) and cry 0 (a KO mutant of cry) flies, molecular oscillations of the clock genes and proteins are absent in peripheral tissues (i.e., the compound eyes, antennae, legs, and Malpighian tubules) (10,(18)(19)(20)(21). In the compound eyes, CRY is suggested to act as a repressor of the CLK/CYC transcriptional activity (22).…”

Synchronization of theDrosophilaCircadian Clock by Temperature Cycles

“…In the case of light, Cry has been implicated as being the crucial factor for synchronization of the peripheral tissues, but it is clear that other photoreceptors also contribute, because isolated tissues synchronize to LD cycles in the absence of functional Cry (Ivanchenko et al 2001;Levine et al 2002a;Dolezelova et al 2007). Given that Cry does have a role in synchronization of the clock, we also tested its potential involvement in temperature synchronization.…”

“…It is not clear whether this "rescue" is only a consequence of altered binding properties between the clock protein Timeless (Tim) and the mutated and perhaps structurally altered Cry B and Per L proteins. The recent observation that Cry loss-of-function mutations are perfectly temperaturecompensated makes it rather unlikely that Cry is also relevant for temperature-compensation in wild-type flies (Dolezelova et al 2007).…”

“…1b) (see, e.g., Wheeler et al 1993;Glaser and Stanewsky 2005), and transcription of many Drosophila genes can be driven by temperature cycles (Boothroyd et al 2007; see below). either mutant or lacking the cry gene but having normal eyes (Stanewsky et al 1998;Dolezelova et al 2007). Similarly, for temperature entrainment, multiple receptor systems could synchronize the clock in parallel.…”