Rhythms of Drosophila period gene expression in culture (circadian rhythmsyclock geneyring glandyprothoracic glandyluciferase reporting)

The Drosophila clock genes period (per) and timeless (tim) have been studied behaviorally and biochemically, but to date there has been no viable culture system for studying the cell biology of the Drosophila clock. We have cultured pupal ring glands attached to the central nervous system and observed rhythms of period gene expression in the prothoracic gland for 4–7 days. A daily rhythm of Per protein can be entrained by light in culture, even when neural activity is blocked by tetrodotoxin. In cultures maintained for a week in constant darkness, a per-luciferase reporter gene revealed circadian rhythms of bioluminescence. As the first circadian culture system from Drosophila, the prothoracic gland provides unique advantages for investigating the interactions between clock genes and cellular physiology. Recent breakthroughs in defining the molecular components of biological clocks, most notably in Drosophila melanogaster (1–3) and Neurospora crassa (4, 5), originated with genetic screens for mutations that altered overt circadian rhythms (6–8). Two such rhythms in Drosophila are the locomotor activity rhythms of adult f lies (6, 9) and the circadian gating of eclosion at the end of metamorphosis (6, 10). The circadian system that controls the eclosion and locomotor activity rhythms of Drosophila requires the interactions of at least two genes, period (per) (6, 11, 12) and timeless (tim) (8, 13). Daily rhythms of per and tim expression appear to constitute the primary gears in the fly’s molecular circadian clock. Their transcripts accumulate during the day and decrease in abundance in the middle of the night (14, 15). Per and Tim protein levels decline during the day, and increase during the night (16–20). These molecular rhythms are thought to result from a negative feedback mechanism whereby Per and Tim proteins interact, enter the nucleus, and ultimately inhibit the transcription of their own genes (14, 18, 21–24). Light causes rapid destruction of Tim protein and may thereby set the phase of the molecular rhythms (19, 20, 25, 26). While it is clear that these molecular interactions are required for overt behavioral rhythms, the cellular mechanisms by which clock genes mediate their behavioral effects are obscure. We now describe a tissue culture system from Drosophila that provides unique advantages for studying the effects of clock genes on cellular physiology. The pupal prothoracic gland, cultured as a complex with the central nervous system (CNS) for up to 7 days, displays rhythms of per expression that can be entrained by light. Because these rhythms persist in the presence of tetrodotoxin (TTX), they appear to be expressed endogenously by the prothoracic gland cells. Circadian rhythms of per expression were recorded in constant darkness from preparations dissected from a perluciferase reporter strain. The results indicate that the Drosophila prothoracic glands have all the components necessary to turn on per transcription, establish the autoregulatory feedback loop, and be entrained by environmental signals, suggesting that this endocrine tissue can function, in culture, as an autonomous circadian clock. MATERIALS AND METHODS Animal Rearing. Flies were raised in standard medium (27) at 258C in 12-hr lighty12-hr dark cycles (LD). White prepupae were selected at lights-on and aged in humid chambers. Tissue Culturing. The CNS and attached ring gland (RG) were dissected from white prepupae during the first 2 hr after lights-on. They were cultured in Schneider’s medium (GIBCOyBRL) supplemented with 20% fetal bovine serum (GIBCOyBRL), 500 ngyml insulin, 100 unitsyml penicillin, 100 mgyml streptomycin, 20 mgyml gentamycin, 0.25 mgyml fungizone (28), and in some cases, 300 nM TTX (Calbiochem). They were incubated in LD at 258C 6 18C. Medium was replaced every 36 hr. Anti-Per Immunostaining. White-eyed flies (w and w per01) were used for sectioning experiments to eliminate the autofluorescence of eye pigments. Pupae were mounted in Tissue-Tek OCT compound (Miles) and frozen with finely powdered dry ice. Cryosections (8 mm) were fixed with buffered 4% paraformaldehyde and stained with affinity purified rabbit antibody to Per S-peptide (29) and Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch), diluted 1y1000. Incubations were performed for 1 hr at room temperature. Sections were counterstained with 2 mgyml 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes) in PBS and mounted in Fluoromount (Fisher) with 25 mgyml 1,4-diazabicyclo[2.2.2]octane (DABCO; Aldrich). Cultured CNS–RG preparations were fixed with buffered 4% formaldehyde and stained as whole mounts (30). The primary antibody was diluted in 50 mM TriszHCl (pH 7), 0.15 M NaCl, 0.5% Triton X-100, and 2% goat serum, and incubated for 3 hr at room temperature. Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) was diluted 1y500 and incubated for 1 hr at room temperature. Whole mounts were counterstained with DAPI and mounted in Fluoromount with DABCO. Analysis of Immunof luorescence. Stained tissues were viewed on a Zeiss Axioskop equipped with Cy3 and DAPI filter sets (Chroma Technology, Brattleboro, VT). The intensity of Per immunofluorescence in prothoracic gland nuclei of sectioned pupae was measured in digital images acquired under standardized conditions with a Star I cooled chargedcoupled device (CCD) camera (Photometrics, Tucson, AZ). Paired images of Cy3 and DAPI fluorescence were analyzed with IPLAB software (Signal Analytics, Vienna, VA) on a Macintosh Quadra 800. The boundaries of the nuclei of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y944092-5$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: CNS, central nervous system; RG, ring gland; LD, 12-hr lighty12-hr dark cycles; DD, constant darkness; TTX, tetrodotoxin; DAPI, 4,6-diamidino-2-phenylindole. †Present address: Department of Biology, University of New England, 11 Hills Beach Road, Biddeford, ME 04005. §To whom reprint requests should be addressed.