PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues

Abstract

Mammalian circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), and current dogma holds that the SCN is required for the expression of circadian rhythms in peripheral tissues. Using a PERIOD2::LUCIFERASE fusion protein as a real-time reporter of circadian dynamics in mice, we report that, contrary to previous work, peripheral tissues are capable of self-sustained circadian oscillations for >20 cycles in isolation. In addition, peripheral organs expressed tissue-specific differences in circadian period and phase. Surprisingly, lesions of the SCN in mPer2(Luciferase) knockin mice did not abolish circadian rhythms in peripheral tissues, but instead caused phase desynchrony among the tissues of individual animals and from animal to animal. These results demonstrate that peripheral tissues express self-sustained, rather than damped, circadian oscillations and suggest the existence of organ-specific synchronizers of circadian rhythms at the cell and tissue level.

Fig. 1.

Generation of mPer2^Luc knockin mice. (A) Diagram of the mPer2 locus, targeting vector, and targeted knockin allele. Exons are indicated by filled blocks with numbers. E, EcoRI; DT, diphtheria toxin A chain; Neo, neomycin resistance gene; triangle, loxP site. (B) Southern blot of DNA from F₂ animals after digestion with EcoRI. The 600-bp 3′ external probe (A) detects a 15.9-kb WT fragment and a 10.2-kb targeted fragment. + indicates WT; L indicates luc knockin allele. (C) PCR genotyping of F₂ animals. Agarose gel electrophoresis reveals the presence of a 230-bp WT (+) allele and a 680-bp knockin allele (L). (D) Northern blot of total RNA extracted from mouse brain probed with a 1.4-kb mPer2 partial cDNA fragment. In contrast to the 7.5-kb WT (+) allele, the larger 11.1-kb band represents the transcript from the targeted (L) allele. (E) Western blot of WT (+/+), mPer2^Luc heterozygote (+/L), and homozygote (L/L) mouse.

Fig. 2.

Representative locomotor activity records from WT and mPer2^Luc/Luc homozygous knockin mice. Animals were maintained on LD12:12 for the first 10 days, indicated by the filled and empty bars above the records, before transfer to DD to measure free-running period. On day 22 in DD conditions, a 6-h light pulse (LP; arrow) was administered at circadian time 16.

Fig. 3.

Real-time analysis of circadian expression of mPER2::LUC. (A) Representative records of bioluminescence showing circadian profiles of mPER2 expression from various tissues from mPer2^Luc knockin animals. Tissues were explanted just before lights off (arrow). Light output (in counts per min) is plotted against previous light onset. (B) Phase map for central and peripheral circadian oscillators of mPer2^Luc knockin mice. The peak of the circadian oscillation was determined during the interval between 12 and 36 h in culture. The average times (± SEM) of peaks were plotted against the time of last lights on (indicated by filled and empty bar). Data for SCN, cornea, liver, pituitary, kidney, retrochiasmatic area (RCA), and lung are shown. (C) Period values of mPER2 rhythms in central and peripheral tissues of mPer2^Luc knockin mice described in B. Mean periods (± SEM) for SCN, cornea, liver, pituitary, kidney, RCA, lung and tail are shown.

Fig. 4.

Long-term persistence circadian rhythms of bioluminescence in peripheral tissues from mPer2^Luc mice. Bioluminescent measurements from cultures of SCN, liver, and lung tissues were recorded for >20 days without media changes or supplements. The first 10 days (Left) and days 11–20 (Right) of data are displayed.

Fig. 5.

Real-time analysis of circadian expression of mPER2::LUC protein in peripheral tissues of SCN-lesioned mPer2^Luc knockin animals. (A) Representative locomotor activity records for two intact control (Upper) and two SCN-lesioned mPer2^Luc knockin (Lower) animals maintained in DD. Complete loss of circadian locomotor activity rhythm is evident in the SCN-lesioned mice. (B) Bioluminescent measurements from static explant cultures of cornea, liver, pituitary, kidney, and lung tissues from SCN-lesioned mPer2^Luc mice maintained in DD. A self-sustained circadian rhythm of bioluminescence in peripheral tissues, equivalent to that observed in nonlesioned mPer2^Luc knockin animals (Fig. 3A), is evident here in SCN-lesioned mPer2^Luc knockin peripheral tissues. Missing data between days 0.5 and 1.2 in cornea and kidney records were the result of a computer malfunction. (C) Circadian oscillation of liver explants from intact control (DD) and SCN-lesioned mice. After 14 days of culture, media were replaced and circadian oscillation was reinitiated in liver explants from both intact and SCN-lesioned mice.

Fig. 6.

Phase and period maps of SCN-lesioned mice. (A) (Top) Phase map of peripheral oscillators in mPer2^Luc LD control mice (n = 8). (Middle) Phase map of peripheral oscillators in mPer2^Luc DD control mice (n = 8). Arrows represent activity onset for each animal. (Bottom) Phase map of peripheral oscillators in mPer2^Luc SCN-lesioned mice (n = 11). The average times (± SEM) of peaks were plotted against the time of last lights on. Each animal is represented by a colored symbol, and tissues from the same animal are connected by lines. Circles represent 12 and 16 days in DD; squares represent 32 days in DD. (B) Circadian period values of mPER2::LUC rhythms in LD control mice (○), DD control mice (•), and SCN-lesioned mice (▴). Mean periods (± SEM) for cornea, liver, pituitary, kidney, and lung are shown. (C) Superimposed plots of bioluminescent data from pituitary and lung for all animals including LD controls, DD controls, and SCN-lesioned mice. The first three cycles in culture are represented; each animal's record is of a different color. Phase desynchronization is evident in individual records of the SCN-lesioned animals for both tissues.