The blue-light photoreceptor CRYPTOCHROME is expressed in a subset of circadian oscillator neurons in the Drosophila CNS

Abstract

In the fruit fly Drosophila melanogaster, CRYPTOCHROME (CRY) functions as a photoreceptor to entrain circadian oscillators to light-dark cycles and as a transcription factor to maintain circadian oscillator function in certain peripheral tissues. Given the importance of CRY to circadian clock function, we expected this protein to be expressed in all oscillator cells, yet CRY cellular distribution and subcellular localization has not been firmly established. Here we investigate CRY spatial expression in the brain using a newly developed CRY antibody and a novel set of cry deletion mutants. We find that CRY is expressed in s-LNvs, l-LNvs, and a subset of LNds and DN1s, but not DN2s and DN3s. CRY is present in both the nucleus and the cytoplasm of these neurons, and its subcellular localization does not change over the circadian cycle. Although CRY is absent in DN2s and DN3s, cry promoter activity and/or cry mRNA accumulation can be detected in these neurons, suggesting that CRY levels are regulated posttranscriptionally. Oscillators in DN2s and DN3s entrain to environmental light-dark cycles, which implies that they are entrained indirectly by retinal photoreceptors, extraretinal photoreceptors, or other CRY-expressing cells.

Figure 1

GP23 antiserum detects CRY in fly head extracts. A. Western containing head extracts from hs-cry flies that were heat induced (+) or uninduced (−) at 37°C for 30 min. A ∼60 kDa band corresponding to CRY is detected. B. Western containing head extracts from wild-type flies that received (+) or did not receive (−) a 6hr light pulse at CT12 on the third day of DD before being collected at CT18. C. Western blot of white, cry^b and heat induced hs-cry flies collected at the times shown during a 12hr light: 12hr dark cycle and probed with GP23. The CRY and non-specific (ns) bands are shown. D. Western blot of white, cry^b and heat induced hs-cry flies collected at the times shown during the first day of DD and probed with GP23. The CRY band is shown. In panels B-D, proteins were extracted using the lab's standard procedure (see Material and Methods). All experiments were replicated at least three times with similar results.

Figure 2

Expression and structure of cry deletion mutants. A. qPCR of cry mRNA from the heads of w (WT), cry^b and cry excision flies (A1, A6, B1, B2, D2, D3, E2, I1) collected at ZT5 (white bars) or ZT17 (black bars) under LD conditions. Relative cry mRNA levels were quantified as described in Materials and Methods. Error bars are ±SD. B. Western blots of head extracts from w (WT), cry^b and excision flies (A1, A6, B1, B2, D2, D3, E2, I1) collected at CT19 during the first day of DD were probed with GP23. In panels A and B, B2 is a precise excision control. C. Structural characterization of cry deletion mutants. Diagram of the cry gene (top) showing the first two exons (white boxes), intron 1 (dashed line), upstream sequence (black line), the P{XP}cry^d10630 insert (triangle), the transcription start site (+1) and the translation start site (ATG). The extent of deleted sequences the A2, B1 and I1 strains are shown as gray lines.

Figure 3

CRY expression in adult brains. Brains were dissected from w flies collected at CT19 on the third day of DD, immunostained with CRY (GP23) and PER antisera, and imaged via confocal microscopy. A minimum of six brains were analyzed with similar results. A. Images of a 2μm z-stack projection through the left hemisphere are shown, where dorsal is at the top. Colocalization of CRY (green) and PER (red) immunofluorescence is seen in the merged image as yellow. Arrows indicate CRY and/or PER immunostaining in oscillator neurons. s-LN_v, small ventrolateral neurons; l-LN_vs, large ventrolateral neurons; LN_ds, dorsolateral neurons; DN1, dorsal neuron1s; DN2, dorsal neuron 2s; DN3, dorsal neuron 3s. B. A compressed stack of six 1 μm optical sections through the lateral protocerebrum is shown. Colocalization of CRY (green) and PER (red) immunofluorescence is seen in the merged image as yellow. Arrows indicate CRY and/or PER immunostaining in oscillator neurons. Oscillator neurons are labeled as in panel A. C. A compressed stack of six 1μm optical sections through the dorsal protocerebrum is shown. Colocalization of CRY (green) and PER (red) immunofluorescence is seen in the merged image as yellow. Arrows indicate CRY and/or PER immunostaining in oscillator neurons. Oscillator neurons are labeled as in panel A. D. Images of a 2μm z-stack projection through the right hemisphere are shown, where dorsal is at the top. CRY and PER immunostaining and oscillator cells are as described in panels A-C, CRY-IR is also detected in the Pars Intercerebralus (PI) and the medial protocerebrum (MP).

Figure 4

CRY expression in cry^b and cry deletion strains. Brains were dissected from cry deletion strains cry^ΔI2 and cry^ΔA2, cry^b mutant flies, and cry B2 (precise excision) flies collected at CT19 on the third day of DD, immunostained with CRY (GP23) and PER or PDF antisera, and imaged via confocal microscopy. A minimum of four cry^ΔI2, four cry^ΔA2, and six cry^b brains were analyzed with similar results. A. A 2μm z-stack projection through the left hemisphere of a cry^ΔI2 fly is shown. Colocalization of PER (green) and CRY (red) immunofluorescence is seen in the merged image as yellow. Arrows indicate PER and/or CRY immunostaining in oscillator neurons, which are labeled as in panel A of Fig. 3. B. A compressed stack of 6−8 1μm optical sections through the Pars Intercerebralus is shown. A merged image of CRY (green) and PER (red) immunofluorescence is shown. C. A compressed stack of 3−6 1μm optical sections through the lateral protocerebrum of cry B2 and cry^ΔA2 flies is shown. Colocalization of PDF (green) and CRY (red) immunofluorescence in s-LN_vs is seen in the merged image as yellow. D. A compressed stack of 3−4 2μm optical sections from the right brain hemispheres of cry^b flies is shown. Colocalization of CRY (green) and PER (red) immunofluorescence in the merged image is shown as yellow. Arrows indicate PER and/or CRY immunostaining in oscillator neurons, which are labeled as in panel A of Fig. 3.

Figure 5

CRY subcellular localization over the circadian cycle. A. Brains were dissected from w flies collected every 4 hours starting at CT1 on the third day of DD, immunostained with CRY (GP23) and PER or PDF antisera, and imaged via confocal microscopy. A minimum of five brains were analyzed for each region with similar results. A. 1μm optical sections of DN₁s in the dorsal protocerebrum and LN_ds, l-LN_vs and s-LN_vs in the lateral protocerebrum. Colocalization of CRY (green) and PER (red) immunofluorescence is seen in the merged images as yellow. B. A compressed stack of 2−31μm optical sections showing s-LN_vs in the lateral protocerebrum. Colocalization of PDF (magenta) and CRY (green) immunofluorescence is seen in the merged image as pink.