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J Biol Rhythms
2024 Mar 28;:7487304241228617. doi: 10.1177/07487304241228617.
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Functional Analyses of Four Cryptochromes From Aquatic Organisms After Heterologous Expression in Drosophila melanogaster Circadian Clock Cells.
Chen C
,
Tamai TK
,
Xu M
,
Petrone L
,
Oliveri P
,
Whitmore D
,
Stanewsky R
.
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Cryptochromes (Crys) represent a multi-facetted class of proteins closely associated with circadian clocks. They have been shown to function as photoreceptors but also to fulfill light-independent roles as transcriptional repressors within the negative feedback loop of the circadian clock. In addition, there is evidence for Crys being involved in light-dependent magneto-sensing, and regulation of neuronal activity in insects, adding to the functional diversity of this cryptic protein class. In mammals, Crys are essential components of the circadian clock, but their role in other vertebrates is less clear. In invertebrates, Crys can function as circadian photoreceptors, or as components of the circadian clock, while in some species, both light-receptive and clock factor roles coexist. In the current study, we investigate the function of Cry proteins in zebrafish (Danio rerio), a freshwater teleost expressing 6 cry genes. Zebrafish peripheral circadian clocks are intrinsically light-sensitive, suggesting the involvement of Cry in light-resetting. Echinoderms (Strongylocentrotus purpuratus) represent the only class of deuterostomes that possess an orthologue (SpuCry) of the light-sensitive Drosophila melanogaster Cry, which is an important component of the light-resetting pathway, but also works as transcriptional repressor in peripheral clocks of fruit flies. We therefore investigated the potential of different zebrafish cry genes and SpuCry to replace the light-resetting and repressor functions of Drosophila Cry by expressing them in fruit flies lacking endogenous cry function. Using various behavioral and molecular approaches, we show that most Cry proteins analyzed are able to fulfill circadian repressor functions in flies, except for one of the zebrafish Crys, encoded by cry4a. Cry4a also shows a tendency to support light-dependent Cry functions, indicating that it might act in the light-input pathway of zebrafish.
Figure 1. Zebrafish and sea urchin cryptochromes do not abolish constant-light rhythmicity induced by cryb. Male flies were exposed to 2 days of 12 h:12 h LD before being released into LL (~1500 lux 25 °C). Double-plotted actograms show average activity of the genotypes indicated above the plots (progeny of Clk-gal4; cryb flies crossed to UAS-cry; cryb or +; cryb flies). cry + control flies are y w. White areas indicate “lights-on,” and gray areas, “lights-off.” Note that wild-type (y w) and Clk-gal4; UAS-cry; cryb flies become arrhythmic in LL, while cryb flies, as well as those expressing zebrafish or sea urchin cry genes, stay rhythmic. Similar results were obtained with the more restricted Pdf-gal4 driver and at lower light intensities (see Supplementary Table S1).
Figure 2. Zebrafish and sea urchin Cryptochromes do not enhance slow resynchronization of cryb mutants to LD cycles. (a-d) Male flies were exposed to 5 (a) or 4 (b) days of 12 h:12 h LD before delaying the LD cycle by 6 h. After 7 days in this delayed LD cycles, flies were released into DD for 3-4 days. (a, b) Double-plotted actograms on the left show average activity during the entire experiment. Phase plots on the right indicate the daily position of evening activity peak, with error bars indicating SEM. White portions indicate “lights-on,” and gray areas, “lights-off.” “Control” flies are y w combined with the progeny of Clk-gal4; cryb (a) or tim-gal4; cryb (b) flies crossed to y w. “+; cryb
” flies are progeny of Clk-gal4; cryb (a) or tim-gal4; cryb (b) flies crossed to cryb. All other genotypes contain one copy of Clk-gal4; cryb (a) or tim-gal4; cryb (b) as indicated on the top, plus one copy of a UAS-cry transgene (as indicated on the left) in a homozygous cryb mutant background. (c, d) Quantification of the days required for re-entrainment for each of the genotypes shown in (a) and (b). cryb controls were Clk-gal4; cryb (c) or tim-gal4; cryb (d) flies crossed to cryb (+), and flies from a homozygous mutant cryb stock (cryb) (c). Numbers within bars indicate n. Error bars indicate SEM. Significant differences between all genotypes and the controls were determined using the non-parametric Tukey test followed by Dunnett’s test (****p < 0.0001, ns: not significant).
Figure 3. Zebrafish and sea urchin cryptochromes do not restore robust light-dependent Tim degradation in cryb mutants. Male flies were kept in 12 h:12 h LD cycles, before being exposed to a 2-h light pulse (LP, ~1500 lux) starting at ZT19. Brains of light-pulsed and non-pulsed control flies were dissected at ZT21 and incubated with Tim antibodies. Tim levels in all clock neuronal groups (apart from the LPN) were determined and compared between light-pulsed and dark controls in the genotypes indicated. Note that in cryb mutants expressing Drosophila cry in all clock neurons (Clk-gal4/UAS-cry; cryb), Tim levels are drastically reduced after the LP compared to dark controls. In contrast, Tim levels are always high in cryb mutants expressing no or any of the zebrafish or sea urchin cry genes. Only zebrafish cry4-expressing flies show a consistent (yet not significant) reduction of Tim in all clock neuronal groups after the LP. At least 12 brain hemispheres were analyzed for each condition and genotype. To test statistical significance of intensity differences between the two time points, a two-way ANOVA with Sidak’s post-comparison was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s., no significance. Error bars indicate SEM.
Figure 4. Zebrafish and sea urchin Cryptochromes do not restore light-dependent period-luciferase oscillations in cryb mutants. Male BG-luc flies were placed individually in the wells of microtiter plates filled with food and luciferin. Bioluminescence was measured hourly using a TopCount reader (PerkinElmer) during 3 days of LD (a, b), followed by 3 days of DD (a) as described in the Materials and Methods section. Except for BG-luc control flies (yellow), all other genotypes were homozygous mutant for cryb, which diminishes the robust bioluminescence oscillations emitted by BG-luc flies during LD (compare yellow and gray tracks in the upper panels of (a) and (b) (Stanewsky et al., 1998). To test the ability to restore BG-luc oscillations, Drosophila cry and the 4 heterologous cry genes were expressed in the BG-luc; cryb mutant background using tim-gal4. (a) Raw bioluminescence data showing that except for Drosophila cry none of the tested cry genes is able to restore robust BG-luc oscillations. (b) Data of the LD part only were de-trended and cosine-fitted (see Material and Methods) to reveal more subtle differences. Dark and white bars above the plots indicate dark and light periods, respectively. Numbers in parentheses indicate n.
Figure 5. Zebrafish Cry1a, Cry3, and SpuCry function as transcriptional repressors of period-luciferase expression in Drosophila. The ability to repress per transcription was tested by overexpressing the various cry genes in flies carrying the transcriptional per-luc reporter plo, which contains only 5’-flanking regulatory DNA sequences of the per gene (Brandes et al., 1996). Bioluminescence emanating from male plo flies was measured during 2 days of LD followed by 5 days of DD as described in the legend of Figure 4. Control flies (yellow “tim >” in [a] and “+” in [b]) carry 1 copy of tim-gal4 on chromosome 2 and 1 copy of plo on chromosome 3. Test flies in addition carry 1 copy of the respective UAS-cry construct on chromosome 2. As a positive control for repression, we also crossed UAS-per (on chromosome 3) to tim-gal4; plo flies (gray in each panel). (a) Raw averaged bioluminescence recordings from flies with the genotypes indicated to the right. Upper panel: Controls (yellow and gray) and tim-gal4; plo flies expressing Drosophila cry (blue), and zebrafish cry1a (orange), a strong repressor. Middle panel: tim-gal4; plo flies expressing SpuCry (green) and zebrafish cry3 (red) showing medium repression. Lower panel: tim-gal4; plo flies expressing zebrafish cry4, encoding a weak repressor. White and black bars above each panel indicate times of light and darkness. (b) Quantification of the average expression level for each genotype for the data is shown in (a). Genotypes and color codes as in (a). Numbers in bars indicate n, and error bars SEM. Data represent results from 3 independent experiments. Significant differences between all genotypes and the tim-gal4; plo controls (“+”) were determined using the non-parametric Tukey test followed by Dunnett’s test (**p < 0.005, ****p < 0.0001, ns: not significant).
