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. 2010 Mar 24;5(3):e9855.
doi: 10.1371/journal.pone.0009855.

Differential expression of the circadian clock in maternal and embryonic tissues of mice

Hamid Dolatshad  1 , Andrew J Cary, Fred C Davis
Affiliations

Differential expression of the circadian clock in maternal and embryonic tissues of mice

Hamid Dolatshad et al. PLoS One. .

Abstract

Background: Molecular feedback loops involving transcription and translation and several key genes are at the core of circadian regulatory cycles affecting cellular pathways and metabolism. These cycles are active in most adult animal cells but little is known about their expression or influence during development.

Methodology/principal findings: To determine if circadian cycles are active during mammalian development we measured the expression of key circadian genes during embryogenesis in mice using quantitative real-time RT-PCR. All of the genes examined were expressed in whole embryos beginning at the earliest age examined, embryonic day 10. In contrast to adult tissues, circadian variation was absent for all genes at all of the embryonic ages examined in either whole embryos or individual tissues. Using a bioluminescent fusion protein that tracks translation of the circadian gene, per2, we also analyzed protein levels. Similar to mRNA, a protein rhythm was observed in adult tissue but not in embryonic tissues collected in-vivo. In contrast, when tissues were placed in culture for the continuous assay of bioluminescence, rhythms were observed in embryonic (E18) tissues. We found that placing embryonic tissues in culture set the timing (phase) of these rhythms, suggesting the importance of a synchronizing signal for the expression of circadian cycles in developing tissues.

Conclusions/significance: These results show that embryonic tissues express key circadian genes and have the capacity to express active circadian regulatory cycles. In vivo, circadian cycles are not expressed in embryonic tissues as they are in adult tissues. Individual cells might express oscillations, but are not synchronized until later in development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Survey of Per2, Bmal1, Cry1 and Clock mRNA expression in whole embryos from embryonic day 10 (E10) to postnatal day 1 (P1).
Pregnant mice were fed adlib and kept on a 12 hr:12 hr Light:Dark cycle. Samples (3–4 embryos per age) were collected at ZT = 5 (lights on = ZT0). The level of each mRNA was measured using quantitative real-time RT-PCR and normalized to gapdh.
Figure 2
Figure 2. Twenty-four-hour expression profiles of Per2 and Bmal1 mRNA in whole embryos and maternal liver during embryogenesis.
Whole embryos were collected every 4 hours for 24 hours on E10-E11 (A), E14-E15 (B) and E18-E19 (C) and mRNA was measured using quantitative real-time RT-PCR. The 0 and 24 hour time points were repeated, independent measures of the same time of day. Maternal livers demonstrated robust variation in both Per2 and Bmal1 at all ages (P<0.0001), consistent with the rhythms expected for these genes. The mRNA of whole embryos failed to show a clear rhythm in either Per2 or Bmal1 at E10-E11 and E18-E19. Low but statistically significant fluctuations were observed for Per2 and Bmal1 at E14-E15 (p<0.01). RNA levels were normalized to the control gene, gapdh. Symbols represent the mean ± standard error of the mean (SEM) of three biological replicates. The maximum maternal liver RNA for each stage of gestation was set to 100 and the rest of maternal and embryonic samples are presented relative to that maximum.
Figure 3
Figure 3. Twenty-four-hour expression profiles of Per2 and Bmal1 mRNA in embryonic (E18-E19) and adult tissues.
mRNA levels of embryonic liver (A), kidney (B) and heart (C) collected every 4 hours, for 24 hours and mRNA was measured using quantitative real-time RT-PCR. Embryonic tissues showed little variation, especially in comparison to the robust changes seen in adult tissues. Adult liver is represented by the same maternal data shown in Figure 2C and by adult kidney and heart from 30-day old mice. RNA levels were normalized to the control gene, gapdh. Symbols represent the mean ± standard error of the mean (SEM) of three biological replicates. For each tissue the maximum adult value was set to 100 and the rest of adult and embryonic samples are presented relative to that maximum.
Figure 4
Figure 4. Circadian rhythms expressed by embryonic heart, kidney and liver in- vitro.
Per2::luc embryonic heart, kidney and liver placed in culture show circadian oscillations in light emission with average periods of 24.36, 25.39 and 22.25. The first day of recording is not shown due to transient activity. Data were detrended as described in methods. Tissues from the same embryo are indicated by the same color line.
Figure 5
Figure 5. Circular phase plots of embryonic liver rhythms in vitro from two experiments showing that in vitro phase is set by the explantation procedure.
Livers were collected from Per2::luc mice and the relative levels of PER2 protein were measured by the recorded light emissions. Phases of in vitro rhythms were determined as described in Methods. The large circles represent the second 24 hours in culture and each small circle represents the phase of peak luminescence of an individual sample. In both experiments the same data are plotted relative to two different references, Maternal Time and Dissection Time. The phases of embryonic liver rhythms were consistently clustered relative to dissection time and not relative to maternal time. A. Livers were collected at two times of day, 12 hours apart, ZT12 (E15.5) and ZT0 (E16). Maternal Time corresponds to clock time since all mothers were entrained to the same light:dark cycle with lights on at 0900 (ZT0) and off at 2100 (ZT12). Red circles represent tissues collected at ZT12 (red star) and black circles represent those collected at ZT0 (black star). For Dissection Time phases are plotted relative to the time of dissection (blue star) regardless of the time of day when dissection was done. B. Two groups of mothers were entrained to different light:dark cycles 12 hours apart (see Fig. S3) and livers were collected at one time of day (1400) and at one embryonic age (E15.25). Because mothers were entrained to different ligh:dark cycles Maternal Time does not correspond to clock time. The red symbols represent embryo samples from mothers with lights off at 1400 and black circles represent those from mothers with lights off at 0200. Because dissections were done at the same time of day for both groups (1400, blue star), Dissection Time corrresponds to clock time. The dissections occurred at different times relative to Maternal Time, ZT0 (black star) or ZT12 (red star). The arrow inside each large circle indicates the average phase of all samples. The arrow's length and the r value indicate the degree of synchrony. P is the probability that the distribution of phases is significantly different from uniform (Rayleigh Test). Each experimental group included samples from at least two pregnant mice.
Figure 6
Figure 6. Acute luciferase activity in embryonic and maternal liver.
Per2::luc mice were used to make protein extracts from maternal and embryonic liver samples every four hours for 24 hours, during E18-19. Luciferase activity was measured as described in Methods. Maternal tissues showed a distinct peak and trough in PER2 levels, at ZT20 and ZT4. In contrast, embryonic liver showed no significant differences during the 24-hour period. Symbols represent the mean ± standard error of the mean (SEM) of three embryos per time point and three technical replicates from a single mother.
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