Figure 1.
PER and TIM labeling in wild-type (left panels) and pdf01 (right panels) larval brains in LD. Larvae were entrained in LD conditions for 5 d (25°C) before dissection of 15–20 brain hemispheres for each time point. The GFP reporter expressed under tim-gal4 driver control was used to label each neuronal group. A, PER immunoreactivity in wild-type and pdf01 mutant brains at ZT0 (light on) and ZT12 (light off). Representative regions of the LNs and DNs are shown as projections of confocal stacks. In the wild type at ZT0, five LNs and two DN1s are strongly labeled, whereas the DN2s are only weakly labeled. At ZT12, no more PER labeling is visible in LNs and DN1s, whereas DN2s show stronger labeling. PER labeling in the LNs and the DN1s is unchanged in pdf01 mutants compared with wild type. In the DN2s of the mutant, however, no clear difference in labeling was observed between ZT0 and ZT12. Note also the heterogeneity in PER localization at both time points in the mutant. B, Time course of PER oscillations in different groups of larval clock neurons. Brains were dissected every 4 h during an LD cycle. The white and black boxes on x-axes indicate light and dark periods, respectively. PER labeling was quantified in the four PDF-positive LNs, the fifth LN, and the DN1s and DN2s, as indicated. Error bars represent the SEM for each neuronal group. Very similar results were obtained in two other experiments for each genotype. Although ANOVA found significant effects of time on PER levels in the DN2s of the pdf0 mutant, their oscillations were clearly blunted. For instance, in the experiment shown here, post hoc Dunnett's test concluded that the ZT4 time point differed only from the ZT8 time point (p < 0.05) in the mutant, while differing from ZT0 (p < 0.05), ZT12, ZT16, and ZT20 (p < 0.001) in the wild type. C, Time course of TIM oscillations in the larval DN2s. This was obtained as in B in an experiment in which TIM and PER were both labeled. Again, ANOVA found significant effects of time on TIM levels in the DN2s of the pdf0 mutant, but post hoc Dunnett's test concluded that the ZT0 time point was not significantly different from any other time point (p > 0.05) in the mutant, while differing from all other time points (p < 0.001) except ZT20 in the wild type.