Novel features of cryptochrome-mediated photoreception in the brain circadian clock of Drosophila

Abstract

In Drosophila, light affects circadian behavioral rhythms via at least two distinct mechanisms. One of them relies on the visual phototransduction cascade. The other involves a presumptive photopigment, cryptochrome (cry), expressed in lateral brain neurons that control behavioral rhythms. We show here that cry is expressed in most, if not all, larval and adult neuronal groups expressing the PERIOD (PER) protein, with the notable exception of larval dorsal neurons (DN2s) in which PER cycles in antiphase to all other known cells. Forcing cry expression in the larval DN2s gave them a normal phase of PER cycling, indicating that their unique antiphase rhythm is related to their lack of cry expression. We were able to directly monitor CRY protein in Drosophila brains in situ. It appeared highly unstable in the light, whereas in the dark, it accumulated in both the nucleus and the cytoplasm, including some neuritic projections. We also show that dorsal PER-expressing brain neurons, the adult DN1s, are the only brain neurons to coexpress the CRY protein and the photoreceptor differentiation factor GLASS. Studies of various visual system mutants and their combination with the cry(b) mutation indicated that the adult DN1s contribute significantly to the light sensitivity of the clock controlling activity rhythms, and that this contribution depends on CRY. Moreover, all CRY-independent light inputs into this central behavioral clock were found to require the visual system. Finally, we show that the photoreceptive DN1 neurons do not behave as autonomous oscillators, because their PER oscillations in constant darkness rapidly damp out in the absence of pigment-dispersing-factor signaling from the ventral lateral neurons.

Figure 6.

Average daily activity profiles of groups of control, GMR-hid, and gl^60J flies before and after a shift in LD schedule using a low (0.08-0.2 lux) intensity of light. Activity profiles are averages for all flies of a given genotype (n = 12-25) over the last 2 d of the corresponding LD schedule. The average morning and evening activity peaks were computed for each day as described in Materials and Methods. GMR-hid flies maintained almost constant activity throughout the light interval, sometimes precluding a clear determination of the activity peaks (especially the morning peak). Entrained gl^60J flies significantly anticipated the light-ON transition in contrast to the GMR-hid or wild-type flies. This anticipation may also account for the apparent lack of acute response of the gl^60J flies to light-ON, again contrasting with the two other genotypes.

Figure 1.

Expression of a cry reporter line (top panel) and the CRY protein (bottom panel) in the larval brain. A-F, PER labeling of third-instar cry-gal4 UAS-gfp larval brains at ZT1.5 (A-C) or ZT10.5 (D-F). A, D, GFP staining. C, F, Anti-PER. B, E, Double labeling with GFP in green and PER in red. PER is expressed in the LNs and DN1s at ZT1.5 (B, C) and in the DN2s at ZT10.5 (E, F). As described previously (Kaneko et al., 1997), there is a fifth larval LN associated to the four PDF-expressing ones, with a similar phase of PER expression in all five cells. No cry-gal4-driven GFP expression was ever detected in either the DN2s or the fifth LN. G-L, CRY labeling of tim-gal4/UAS-gfp (G, H, J-L) or tim-gal4/UAS-cry (I) brains from third-instar larvae reared in the dark. In tim-gal4/UAS-gfp, the two DN1s and four LNs are double labeled (GFP, G, J, and green in K; CRY, H, L, and red in K), but the two DN2s express only GFP. Wild-type CRY levels are very low compared with CRY levels achieved by tim-gal4-driven expression from a UAS-cry construct (compare H and I). The dendritic arborization and dorsal projection of the LNs are more clearly visible in the confocal images in J-L. DP, Dorsal projection of the LNs; DA, dendritic arborization of the LNs. Scale bars, 20 μm.

Figure 2.

PER cycling in larval brain neurons overexpressing cry. A-F, cry-gal4/UAS-cry (A, B), pdf-gal4/UAS-cry (C, D), and tim-gal4/UAS-cry (E, F) third instar larval brains labeled at ZT0-1 (A, C, E) or ZT9-10 (B, D, F) with anti-PER antibodies (red). The cry-gal4/UAS-cry and pdf-gal4/UAS-cry brains were double labeled with anti-PDF antibodies (A-D, green). The DN2s always appear very close to the horizontal end-segment of the PDF-labeled dorsal projection from the LNs. The GFP reporter in panels E and F (green) allows even surer identification of the DN2s, but that was possible only with the tim-gal4 driver. Note that because of competition, the presence of the UAS-gfp transgene in these larvae might lower expression from UAS-cry, and thus strengthens the comparison with the other two genotypes. Weakly labeled DN2s could sometimes be seen at ZT0 in cry-gal4/UAS-cry and pdf-gal4/UAS-cry brains (as illustrated in C). Similar results were obtained in at least two independent experiments for each genotype, both with and without anti-PDF colabeling (or GFP coexpression). PER immunoreactivity was quantified in the LNs, DN1s, and DN2s (G) for pdf-gal4/UAS-cry and tim-gal4/UAS-cry larval brains dissected at either ZT0 or ZT9. The front and back histograms correspond to pdf-gal4/UAS-cry and tim-gal4/UAS-cry values, respectively. Error bars indicate SEMs. At least 19 hemispheres were scored per sample, except for tim-gal4/UAS-cry at ZT9 (n = 12). Scale bar, 40 μm.

Figure 3.

Expression of a cry reporter line and the CRY protein in the adult brain. A-F, Anti-PER labeling at ZT1-2 in a cry-gal4 UAS-gfp brain. A-C, Confocal projection of the region of l-LN_vs and LN_ds. A, B, Green, GFP; B, Red, C, PER. All LN_vs and LN_ds were always GFP positive. The arrow indicates a GFP-positive cell without PER expression, which was reproducibly observed within the LN_d cluster. D-F, Confocal projection of the dorsal brain. D, E, Green, GFP; E, Red, F, PER. Most of the DN1s (11.4 ± 0.5 of 15.2 ± 0.6; n = 5 brain hemispheres) expressed GFP. In the DN3 group, comprising a total of 30-40 cells (Kaneko et al., 1997; Kaneko and Hall, 2000), the number of clearly GFP-positive cells was only 8.5 ± 0.4 (n = 11). Two or three of these stood out with much stronger GFP expression and a much larger cell body. The DN2s lie very close to the dorsal projection (DP) of the s-LN_vs. The many additional PER-expressing cells are probably glial (Kaneko, 1998), except possibly for the three cells indicated by an arrow. As judged from their number and position, they could correspond to the novel cluster of three TIM-positive cells described by Kaneko and Hall (2000). One (thicker arrow) is GFP positive and displays an axon-like projection (data not shown). G-L, Anti-CRY labeling. G-I, Confocal projection of the region of the lateral neurons in a cry-gal4 UAS-gfp brain. G, H, Green, GFP; H, Red, I, CRY. After rearing in LD, adult flies were kept for 3 d in DD before dissection. All four s-LN_vs were always labeled with the anti-CRY antibodies, whereas only variable proportions of the l-LN_vs and LN_ds were labeled, usually more weakly than the s-LN_vs. J-L, Confocal projection of the dorsal brain of an adult tim-gal4/UAS-gfp brain. J, K, Green, GFP; K, Red, L, CRY. Flies were reared in constant darkness throughout. An average of 6.1 ± 0.3 DN1s were CRY positive (n = 8 brain hemispheres). Two of them were usually much more strongly labeled than the others, as shown here. Double labeling of wild-type brains with anti-CRY and anti-GLASS antibodies (data not shown) allowed us to identify them as larva-originating DN1s (Fig. 4). The dorsal projections of the s-LN_vs were labeled in seven of eight brain hemispheres. Scale bars, 20 μm.

Figure 4.

PER and GLASS expression in wild-type, gl^60J, and GMR-hid adult brains. Labeling was done on brains collected at CT0. A-C, Anti-PER. D-M, Anti-PER (green) and anti-GLASS (red) colabeling. The two DN2s usually formed a close pair, more ventral and in a different plane of focus than the DN1s, as described previously (Kaneko et al., 1997) and as shown here (A-F). However, cell identification was not always certain (hence the “?” in panels B and E). The number of GLASS-positive cells in the wild-type dorsal clusters was 22.2 ± 0.4 (n = 9). An additional five to six cells appeared weakly but very reproducibly GLASS positive in that region (D, F, arrows). The DN1^*s, which do not express GLASS, presumably correspond to the two larval DN1s. They were often in a more dorsal position than the other DN1s. The dorsal neurons are the only cells that coexpress PER and GLASS in adult brains. G-I, Half-brains. K-M, Higher magnification of LN_d regions. The largest GLASS-expressing cluster (variably positioned, depending on the orientations of the optic lobe and central brain on the slide) and the DNs are in a different frontal plane than the smallest GLASS-expressing cluster and the LNs. The thinly boxed areas in G and I were thus taken from another differently focused picture. In gl^60J brains, the anti-GLASS antibody labeled a completely different set of scattered and mostly ventral cells (E, H, L). There was reproducibly a single colabeled cell (E). From its very dorsal localization and its persistence in the mutant, we tentatively identify it as one of the two DN1^*s (which do not express GLASS in the wild type; see D). No GLASS-expressing cell seemed to be missing in GMR-hid brains (F, I, M). Quantification of PER-expressing dorsal neurons in control, gl^60J, and GMR-hid adult brains is shown in N. Significant differences relative to control are indicated (^*p < 0.05; ^**p < 0.001; Student's t test; one-tailed, after testing for differences in variance). Because it was not always possible to distinguish between the three subgroups of dorsal neurons as easily as on the examples shown in A-F, all of these PER-expressing cells were systematically counted together. gl^60J mutant brains display less (PER only) neurons than control or GMR-hid flies, because one of the presumptive DN1^*s is colabeled in the mutant but not in the other two strains. Scale bars: A-F, K-M, 10 μm; G-I, 50 μm.

Figure 5.

Actograms of individual control and visual system-defective flies submitted to a shift in LD schedule at either high- or low-light intensity. Light intensity was 80-200 lux for the top panel and 0.08-0.2 lux for the bottom panel. For the first half of the experiment, lights were on from 8:00 P.M. to 8:00 A.M., almost completely antiphase to the normal lab schedule that the flies had previously been exposed to. The LD schedule was then shifted by 8 hr on the seventh day by delaying the light-OFF transition, as indicated by the broken lines. At low light intensity, the glass^60J fly clearly took longer than the w, GMR-hid, and norpA^p24 to entrain to both the first LD schedule and the subsequent 8 hr shift. These actograms are representative of two to three independent experiments, each with at least 12 flies for each experimental condition.

Figure 7.

Average activity profiles of gl^60J cry^b and GMR-hid; cry^b flies entrained with intense (∼500 lux) white light. Note that GMR-hid; cry^b behaved differently from gl^60J cry^b flies, in that their activity was neither suppressed during the day nor transiently induced by the light-OFF transition. These profiles were obtained by averaging the activity of 16 flies of each genotype during the fourth full day of entrainment.

Figure 8.

PER cycling in the DN1s of wild-type and pdf⁰¹ adult brains. A-D, Dorsal region from wild-type (A, C) and pdf⁰¹ (B, D) adult brains stained with anti-PER antibodies at either CT36 (A, B) or CT48 (C, D). Similar results were obtained in two independent experiments for each genotype, one of which extended to CT74 (i.e., >3 full days of DD after the last dark cycle of the entraining LD schedule). E, Quantization of PER levels in the DN1s from the experiment illustrated in A-D. PER levels are mean values over an average of 16 brain hemispheres per sample. Error bars indicate SEMs.