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Circadian rhythm in clock mutants

ABSTRACT: One hundred ninety wildtype male C57BL/6J mice age 7-10 weeks were purchased from Jackson Laboratory and entrained to a 12:12 light:dark cycle for 2 weeks. Mice were placed in light-tight boxes on a 12:12 LD cycle for 4 weeks, then released into constant darkness. Starting 30 hours after entry into DD (CT18), tissues from 5 (skeletal muscle) or 10 (liver or SCN) wildtype mice were collected every 4 hours for 48 hours, for a total of 12 timepoints. At timepoints 34 through 58 hours in DD, tissues from age-matched male C57BL/6J Clock/Clock homozygous mutant mice that had been treated with the same light entrainment protocol as the wildtype were collected. Tissues were collected from 5 Clock/Clock mutants at each timepoint except for 34 and 46 hours after the onset of DD, when tissues from 10 Clock/Clock mice were collected and run as independent replicates. Mice were sacrificed by cervical dislocation, and the optic nerves were severed in complete darkness; brain dissection was performed using illumination from an infrared viewer (FJW Industries, Palatine, IL). SCNs were dissected out, pooled at a density of 5 per tube in 100 ul RNAlater (Ambion, Austin, TX), frozen on dry ice, and stored at –80 degrees C until use. This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE3751 | GEO | 2007/02/07

SECONDARY ACCESSION(S): PRJNA93919

REPOSITORIES: GEO

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Project description:Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD cycles. We report that a reduced subset of genes were rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially up- or down-regulated specific sets of genes in a fashion that was dependent upon time of day. Our combined approach of utilizing a time of day study and a light/NE pulse microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in pineal physiology. Keywords: circadian; avian; pineal; light; norepinephrine Microarray analysis was performed on pineal samples subjected to one of four experimental groups: cultures placed within a 12-hour light:12-hour dark cycle (LD); cultures placed in continuous darkness (DD); cultures subjected to a 6-hour light pulse beginning at either CT0 or CT12 (i.e., zero or 12 hours after the beginning of subjective day) or that received no light pulse; and cultures subjected to 6 hours of NE treatment beginning at either CT0 or CT12 or that received similarly administered vehicle. For LD and DD groups, time-course sampling was performed such that 6 samples were collected over the course of a day at 4-hour intervals. For the light pulse and NE administration groups, 2 samples were collected over the course of a day, either at CT6 or CT18 (i.e., 6 or 18 hours after the beginning of subjective day). Four biological replicates and two technical replicates (for each biological repeat) were performed for each sample. For the LD and DD groups, time-points ZT18 and CT18 served as the control reference for the time-course analysis. For the light pulse experiment, cultures that did not receive light served as the control. For the NE administration experiment, cultures that received vehicle served as the control. Dye swaps for performed for experimental samples from the light pulse and NE administration experiments.

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Circadian rhythm in clock mutants

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