Human Cryptochrome Light Responsivity Table 1. Heterologous CRY expression does not restore a wild-type arrhythmic phenotype to cryb mutant flies. cry transgene present, Light ( ): no driven Total Rhythmic conditions Genotype expression flies (n) flies (%) Period (h) Power Activity yw;;cryb ss LL 15 80 24,8 6 0,2 74 6 7 20 6 3 w 10F;;cryb ss (HsDmcry) 14 93 25,5 6 0,2 115 6 9 22 6 2 yw/w;tim-gal4/+ 16 6 27,0 22 14 yw/w;tim-gal4+/;2M cryb ss/cryb ss HsDmcry 14 71 25,0 6 0,5 88 6 17 26 6 4 w 10F/yw;tim-gal4/+;cryb ss HsDmcry 19 100 25,2 6 0,2 117 6 6 27 6 2 w/yw;tim-gal4/26.9A;cryb ss Hscry1 12 100 25,9 6 0,2 95 6 14 32 6 2 yw;;cryb ss 12 100 24,9 6 0,2 101 6 10 19 6 4 W 11 9 25,0 65 47 w;;31.2A cryb ss (Hscry1) 6 100 24,8 6 0,5 86 6 9 18 6 4 w;tim-gal4/+;31.2A cryb ss/cryb ss Hscry1 16 81 25,0 6 0,2 108 6 11 40 6 6 w;26.9A;cryb ss (Hscry1) 10 60 24,7 6 0,3 65 6 11 30 6 6 w;tim-gal4/26.9A;cryb ss Hscry1 16 81 25,3 6 0,2 104 6 9 22 6 2 w 26.13C;;cryb ss (Hscry1) 10 100 24,8 6 0,2 92 6 11 15 6 1 w 26.13C/+;tim-gal4/+;cryb ss Hscry1 16 100 25,3 6 0,2 113 6 9 18 6 2 w;26.16;cryb ss (Hscry1) 11 100 25,1 6 0,1 94 6 10 12 6 2 w; tim-gal4/26.16;cryb ss Hscry1 15 100 25,6 6 0,1 120 6 7 17 6 1 w;41.4;cryb ss (Hscry1) 9 67 25,0 6 0,2 99 6 9 18 6 2 w; tim-gal4/41.4;cryb ss Hscry1 16 100 25,7 6 0,1 125 6 6 15 6 1 DD w;tim-gal4/+ 17 94 24,3 6 0,2 106 6 10 33 6 2 w;tim-gal4/+;2M HsDmcry 16 94 24,8 6 0,1 92 6 10 24 6 2 yw/w;tim-gal4/+;2M cryb ss/cryb ss HsDmcry 18 94 24,4 6 0,1 103 6 6 26 6 2 w 10F;tim-gal4/+ HsDmcry 10 90 24,7 6 0,1 105 6 8 29 6 3 yw/w;tim-gal4/+;31.2A/+ Hscry1 19 100 24,4 6 0,1 109 6 7 35 6 2 yw/w;tim-gal4/26.9A Hscry1 28 89 24,8 6 0,1 93 6 7 30 6 2 Actogramms of rhythmicity conferred by Hscry constructs in LL and DD. Three day old adult flies expressing HsCRY1, HsDM in both wild type and cryb mutant genetic backgrounds were entrained for 72 hours to 12 hr day/night cycles and analyzed for free running period in LL and DD for 10 days. Rhythmicity and period length are shown. The mean values of circadian period (h), associated powers (see Methods) and activities (number of events per 0.5 h) are given 6 s.e.m. doi:10.1371/journal.pone.0031867.t001 Therefore, DmCRY is not the primary photoreceptor implicated Gal46Hscry1 and tim-Gal46Hscry1 F1 lines. This indicates a light- in this response to white light. independent role of HsCRY1 in the acceleration of larval We next measured the effect of HsCRY1 and HsDM on eclosion development in addition to a light-dependent one. Interestingly, the time in heterozygous expressing F1 flies (Fig. 1b). In LL, different HsCRY1 expressing lines grown in red light showed no developmental time was significantly shorter for both HsCRY1 (by significant difference in eclosion times (peak after 11 days for all lines). By contrast, responsiveness in blue light is accelerated in HsCRY1 3 days) and HsDM (by 1.5 days) expressing flies as compared to wild expressing lines (peak one day in advance of wt and cry mutants). type flies (Fig. 2a). Time to eclosion was also accelerated in flies that carried the constructs in the cryb genetic background, suggesting this Surprisingly, the response in HsCRY1 expressing lines was even more pronounced at shorter wavelengths of light (near - UV) than at effect is not due to interaction between heterologous and endogenous similar intensities of blue light, with peak of eclosion occuring two cryptochromes. Interestingly, HsCRY1 did also accelerate develop- days in advance of wt and 3 days in advance of cryb and cry0 mutants ment in DD (Fig. 2b), although to a reduced extent as compared to (Fig. 2c). LL. Wavelength dependence of the eclosion phenotypes mediated by Taken together, these results indicate that the effect of HsCRY1 HsCRY1 and DmCRY, respectively, was investigated in near UV in this developmental response occurs to a large extent (peak at 380 nm), blue (peak at 450 nm) and red (peak at 660 nm) independently of light (as seen in flies kept in complete darkness). light (Fig. 2c). Here, HsCRY1 was either expressed under control of However, the enhanced response of HsCRY1 expressing flies as the timeless (tim) promoter or under the control of the daughterless (da) compared to wt or cry mutant flies in white, blue and UV/A, but promoter (using the uas-Gal4 expression system). The da promoter is a not in red light, is consistent with an additional light activation of strong promoter expressed in all cell types [34]. ensuring HsCRY1 HsCRY1. expression throughout the fly and not just in cell types expressing the clock gene tim. As a further negative control, tim-gal46uas-Hscry1 flies were tested in a cry knockout mutant background (cry0; [32]). As in Transcriptome analysis of Hscry1 expressing transgenic flies previous experiments (Fig. 2a,b), there was acceleration of time to eclosion in HsCRY1 expressing lines (peak activity is one day in Mammalian Type II cryptochrome has been shown to function advance) even in complete darkness, both in the case of da- as a transcriptional repressor in mammalian cell cultures [21] and PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e31867