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Proc Natl Acad Sci U S A. Mar 14, 2006; 103(11): 4146–4151.
Published online Mar 6, 2006. doi:  10.1073/pnas.0600571103
PMCID: PMC1449661
Developmental Biology

Circadian time-keeping during early stages of development

Abstract

The zebrafish pineal gland is a photoreceptive organ containing an intrinsic central circadian oscillator, which drives daily rhythms of gene expression and the melatonin hormonal signal. Here we investigated the effect of light, given at early developmental stages before pineal gland formation, on the pineal circadian oscillator. Embryos that were exposed to light at 0–6, 10–13, or 10–16 h after fertilization exhibited clock-controlled rhythms of arylalkylamine-N-acetyltransferase (zfaanat2) mRNA in the pineal gland during the third and fourth day of development. This rhythm was absent in embryos that were placed in continuous dark within 2 h after fertilization (before blastula stage). Differences in the phases of these rhythms indicate that they are determined by the time of illumination. Light treatments at these stages also caused a transient increase in period2 mRNA levels, and the development of zfaanat2 mRNA rhythm was abolished by PERIOD2 knock-down. These results indicate that light exposure at early developmental stages, and light-induced expression of period2, are both required for setting the phase of the circadian clock. The 24-h rhythm is then maintained throughout rapid proliferation and, remarkably, differentiation.

Keywords: AANAT, light, period2, pineal, zebrafish

In all organisms, daily rhythms of physiological, biochemical, and behavioral processes are driven by intrinsic circadian oscillators that operate in harmony with the environmental light–dark (LD) cycles. Entrainment and maintenance of these oscillators allow preparation for predicted changes in environmental conditions and, hence, constitute an adaptive advantage. Light is the predominant signal for entrainment of circadian oscillators, enabling their synchronization with the solar 24-h cycle (1).

In mammals, photic signals are perceived by specialized photoreceptive ganglion cells in the retina. These signals entrain and synchronize the master oscillator in the suprachiasmatic nucleus (24). This oscillator is self-sustained; it elicits circadian rhythm outputs with very little damping (5). Peripheral oscillators reside in almost every cell in the body (6); self-sustained independent circadian oscillators have been demonstrated in individual fibroblasts in cultures (79). When disconnected from the master oscillator, peripheral oscillators dampen quickly, probably as a result of desynchronization among individual cells (8).

In nonmammalian vertebrates, the pineal gland is thought to function as a central clock. It is a photoreceptive organ and contains an intrinsic circadian oscillator that drives rhythms of melatonin production (10, 11). Consequently, circadian rhythmicity of melatonin production and photic entrainment of the oscillator can be observed when zebrafish pineal glands are placed in culture (12). Interestingly, peripheral clock-containing tissues in zebrafish were shown to be directly entrained by light without the involvement of the retina or the pineal gland; the mechanism for this photoreception and entrainment is currently unknown (1315). In a zebrafish cell line, a light pulse synchronizes and stabilizes the period of otherwise unsynchronized and stochastic cellular oscillators, thus inducing overt rhythms of gene expression (16). Similarly, in mammalian fibroblast cultures, circadian oscillators in individual cells can be synchronized by a serum shock (8, 9, 17). The existence of physiological and behavioral rhythms in the intact animal may reflect the synchronization of many independent cellular oscillators.

In contrast with terminally differentiated cells of central clock organs that do not divide, such as suprachiasmatic nucleus neurons or pineal photoreceptors, peripheral cells go through cycles of division. The question of how cycles of cell division and circadian oscillators coexist has been the subject of investigation by several research groups. In unicellular organisms, such as cyanobacteria, circadian rhythms of gene expression continue in rapidly dividing (doubling time = 5–10 h) cultures, indicating that the circadian oscillator is not distorted by cell division (1820). Similarly, monitoring rhythmic gene expression in dividing fibroblasts, Nagoshi et al. (9) demonstrated that upon mitosis daughter cells resumed the rhythms of the mother cells. A picture is currently emerging, based on cell culture studies, in which all cells contain autonomous circadian oscillators that are, by and large, not affected by cell division. On the other hand, the timing of cell division seems to be gated by the circadian clock (9, 18, 21, 22).

Here, using an intact animal experimental model, we demonstrate that circadian time is maintained in rapidly dividing and, remarkably, developmentally differentiating cells. Specifically, in zebrafish, light exposure during the first hours of development, at the blastula, gastrula, and early segmentation stages, induces circadian rhythm of gene expression 2–3 days later in the developed pineal gland of posthatched larvae. Moreover, light-induced period2 expression at these early stages appears crucial for this process to take place.

Results

Illumination at Early Embryonic Stages Induces Pineal zfaanat2 Circadian Rhythms at Later Larval Stages.

The development of circadian rhythms of locomotor activity and gene expression in zebrafish depends on exposure to LD cycles (23, 24). The earliest detected rhythms are those of the developing photoreceptive pineal gland. Exposure of zebrafish embryos to light after the pineal gland is formed facilitates the development of clock-controlled rhythms of melatonin and zfaanat2 mRNA in the pineal gland (25, 26). Interestingly however, it has been recently shown that zebrafish embryos are light responsive at an earlier developmental stage, even before the pineal gland is formed (27). To determine whether light reception at this stage has an effect on the development of circadian rhythms, embryos were exposed to different light treatments during the first 16 h of development, before the pineal gland is formed, after which they were placed in constant darkness (DD). Embryos were then collected at 4-h intervals during the third and fourth day of development and pineal zfaanat2 mRNA levels were determined by whole-mount in situ hybridization (ISH).

Zebrafish embryos that were placed in continuous dark within 2 h postfertilization (hpf, before the blastula stage) did not exhibit rhythmic zfaanat2 expression (Fig. 1A). In contrast, embryos that were exposed to light from fertilization to 6 hpf (completion of blastula stage) exhibited 24-h rhythm of zfaanat2 mRNA in the pineal gland during the third and fourth day of development, which was characterized by peak levels at 66 hpf (Fig. 1B). Embryos that were exposed to light at 10–16 hpf (early to mid-segmentation) also exhibited a clock-controlled zfaanat2 mRNA rhythm, characterized by peak levels at 50–54 and 70–78 hpf and nadir at 62 hpf (Fig. 1C). Notably, these two rhythms exhibit an ≈8-h phase difference, indicating that the time of illumination sets the phase of the rhythm. Shorter duration of light exposure (10–13 hpf) induced a lower-amplitude clock-controlled zfaanat2 rhythm as compared to that induced by 6-h light exposure (Fig. 1D). Together, these results indicate that light exposure later than 2 hpf, during the blastula to mid-segmentation stages, facilitates the development of clock-controlled zfaanat2 mRNA rhythms in the pineal gland.

Fig. 1.
Effect of light and zper2 expression at early embryonic stage on development of pineal zfaanat2 mRNA rhythms. Zebrafish embryos, MO-injected and uninjected, were exposed to light for different periods during the first 16 h of development. During the third ...

Light Regulation of zper2 Expression During the First Day of Development.

Light treatment has been shown to increase zper2 mRNA levels in a zebrafish cell line (15) and, recently, in the pineal gland of 24-h embryos (25). To determine the effect of light on the temporal expression pattern of zper2 during the first day of development, embryos were exposed to different photoperiodic regimes and inspected for zper2 mRNA expression at short intervals by whole-mount ISH.

This analysis revealed high levels of zper2 transcript, from one-cell stage to late blastula stages (4 hpf), confirming an earlier report (28). These levels were not affected by light exposure (Fig. 2 and data not shown), in agreement with the lack of transcription before mid-blastula transition (MBT) at 3 hpf (29). These high levels of maternally transferred zper2 mRNA were followed by a rapid decline, the rate of which was affected by light. In embryos that were kept in the dark, zper2 mRNA was undetectable by 5 hpf (Fig. 2 Middle); signal intensity was similar to that obtained with the control sense probe (Fig. 2 Bottom). In embryos that were exposed to light, zper2 mRNA levels were higher than the control group at 5 and 6 hpf; signal intensities of light-treated and control embryos was similar only at 7–8 hpf (Fig. 2 Top). This observation suggests that after transcription activation begins (MBT), light transiently increases the amount of zper2 transcript. It is unknown whether light at this stage delays the degradation of maternal zper2 mRNA or induces de novo zper2 transcription.

Fig. 2.
Effect of light on zper2 expression at the blastula and gastrula stages. Embryos were kept under continuous light or dark and sampled throughout the first 10 h of development; zper2 mRNA was detected by whole-mount ISH. Shown are representative whole-mount ...

After the disappearance of maternal zper2 mRNA, it remained undetectable in embryos that were kept in continuous dark conditions (Fig. 3Lower). In contrast, embryos that were exposed to light for the first time at 10–16 hpf, exhibited a transient increase in zper2 mRNA levels, which declined after lights-off (Fig. 3 Upper). These results indicate that light exposure induces zper2 transcription at this stage.

Fig. 3.
Effect of light on zper2 expression during segmentation period. Embryos were kept under constant darkness (Lower) or exposed to light 10–16 hpf (Upper), sampled at 1-h intervals during 10–18 hpf, and zper2 mRNA was detected by whole-mount ...

At the blastula, gastrula, and early segmentation stages, light-induced zper2 transcription seemed ubiquitous. However, as development proceeded, zper2 mRNA showed a restricted expression pattern. At the six-somite stage (12 hpf), before the formation of the neural tube, enhanced expression of zper2 mRNA was detected in the midline of the anterior neural plate, which gives rise to the ventral diencephalons, and the lateral edges of the anterior neural plate (Fig. 4A), which give rise to the pituitary and olfactory placodes (3033). Interestingly, zper2 is expressed at relatively high levels in these structures at later stages (25, 28). After the neural plate condenses medially to form the brain, bilateral zper2 signals at the posterior edge of the anterior neural plate converge dorsally and fuse at the midline where the pineal gland develops (Fig. 4 B and C). Later, expression of zper2 was also detected in the area of the developing eyes where retinal progenitor cells are already located (ref. 31; Fig. 4C).

Fig. 4.
Spatio-temporal expression pattern of zper2 during neurogenesis. Whole-mount ISH analysis of zper2 mRNA in 12- to 22-h embryos after light treatment (dorsal view, anterior to the top) is shown. (A) Enhanced zper2 mRNA expression is detected at the lateral ...

Zebrafish per2 is Required for Light-Induced Rhythms.

Expression of zPER2 in the pineal gland is crucial for the light-induced onset of the pineal clock; knockdown of zPER2 eliminated the light-induced rhythms when light was given during the second day of development, after the pineal gland was formed (25). To determine whether the light-induced zper2 mRNA expression at earlier embryonic stages (Figs. 2 and and3),3), before pineal formation, is important to the development of clock-controlled rhythms in the pineal gland, zPER2 knock-down experiments were performed. Two Morpholino-modified antisense oligonucleotides (MO) were used: One, Per2(AUG)MO, to prevent translation initiation of the zper2 mRNA; and the other, Per2(E4I4)MO, to interfere with zper2 mRNA splicing. Efficiency and specificity of the latter was assessed by PCR analysis using primers directed to exons 3 and 5 of zper2 and corresponding exons of zper1 and zper3. This analysis revealed a 295-bp band in Per2(E4I4)MO-injected embryos and the expected 378-bp zper2 fragment in noninjected embryos; Per2(E4I4)MO had no effect on zper1 and zper3 mRNA splicing (Fig. 1I). Sequence analysis of the 295-bp band indicated that Per2(E4I4)MO injection induced skipping of 84 nucleotides that correspond to the PAS domain of zper2 (28) (Fig. 1J).

MO-injected embryos were exposed to light 0–6 hpf, 10–16 hpf, or 10–13 hpf, photoperiod regimes that facilitate the development of the pineal oscillator (Fig. 1 BD), and then placed in constant darkness. During the third and fourth day of development, embryos were collected at 4-h intervals and subjected to whole-mount ISH for zfaanat2 mRNA. Knock-down of zPER2 using Per2(AUG)MO abolished the clock-controlled zfaanat2 mRNA rhythm that was induced by light exposure 0–6 and 10–16 hpf (Fig. 1 EG), and microinjection of Per2(E4I4)MO eliminated the zfaanat2 rhythm that was induced by light exposure 10–13 hpf (Fig. 1H). An irrelevant MO had no effect on the development of the rhythm (Fig. 1E). The expression of zotx5 mRNA was not affected, indicating that the pineal gland differentiates normally in MO-injected embryos (data not shown).

These results indicate that zPER2, maternal and/or light-induced at early stages of embryonic development, is necessary for the light-induced development of pineal circadian rhythms.

Discussion

The development of clock-controlled rhythms in zebrafish depends on LD cycles. For example, the development of rhythms of locomotor activity requires four LD cycles (23); five to six LD cycles are required for establishing rhythmic zper3 promoter activity in zper3-luc transgenic zebrafish embryos (24); and daily rhythms of cell cycle appear after four LD cycles (34). In contrast, in the current study, a single period of light treatment was sufficient to facilitate the development and to set the phase of the pineal zfaanat2 mRNA rhythm. These differences in the number of required light/dark cycles may be attributed to the difference in monitoring of peripheral clock function (23, 24, 34), as opposed to central clock function in the current study. This finding is supported by previous studies in which the development of pineal rhythms was found to require only one light pulse (25, 26).

The importance of the current study lies in the observation that photic signals during early embryonic stages, blastula to early segmentation (2–16 hpf), affect the circadian oscillator that drives rhythmic expression of zfaanat2 mRNA in the pineal gland 2–3 days later. It was recently shown that light induces translation of 4–6 photolyase and zper2 in zebrafish embryos as early as the blastula and gastrula stages (27). 4–6 photolyase induction was proposed to play a role in resistance to light-induced DNA damage (27). The current study provides another biological significance to this photoreception, relating to the development of a functional circadian clock. In addition, it suggests that the clock phase is not set by a developmental process and is not an inherited feature as previously suggested (35), but that it needs to be entrained by an external light cue.

As mentioned above, cell culture studies indicate that circadian oscillators are generally not affected by cell division. This is reflected here, in the intact developing embryo, by the maintenance of a 24-h rhythm throughout the many cell cycles that occur during the first day after fertilization. However, a more important implication of the current data is that the phase of the circadian oscillator is maintained throughout differentiation.

Zebrafish are unique in that light-entrainable circadian oscillators are found in almost every cell; light pulses synchronize the rhythms of clock genes expression in cell and organ cultures (13, 14, 36). The mechanism underlying the observed effect of light on the embryo’s oscillator is currently unknown. However, the fact that light is effective only after MBT suggests that transcription of new genes is required for the effect of light on the clock. A recent study of individual zebrafish cells in culture, using zper1 promoter activity as a marker, revealed that a pulse of light synchronizes the oscillator and stabilizes its period to 24 h (16). Thus, it is hypothesized here that embryonic cells contain stochastic, free-running, oscillators that, via a transcriptional-dependent mechanism, are synchronized and stabilized by light and are then maintained throughout proliferation and differentiation.

The results of the current study suggest that zper2 transcription mediates this synchronizing effect of light. Levels of zper2 mRNA were up-regulated by light at the blastula, gastrula, and early segmentation stages, confirming earlier observations (27). Moreover, the effect of these early light treatments on the development of clock-controlled rhythms in the pineal gland was abolished by blocking translation initiation (Fig. 1 F and G) and by deleting part of the PAS domain (Fig. 1H). Interestingly, deletion in the PAS B and PAC subdomains abolished light-entrainment in per2 mutant mice (37, 38). On the other hand, maternally transferred zPER2 mRNA alone is not sufficient for the development of circadian rhythms, as indicated by the arrhythmic zfaanat2 expression in embryos that were transferred to constant darkness before the MBT stage (Fig. 1A). Thus, zPER2 is induced by light in embryonic cells that are not classical photoreceptors (15, 27) and, by a still unknown mechanism, is important for the entrainment and synchronization of circadian oscillators.

Although the effect of light on peripheral oscillators was not studied here, the ubiquitous expression of zper2 after light treatment suggests that this early light exposure may affect peripheral clocks as well as the pineal clock (39). It is unclear why peripheral and central clocks develop so early. However, given accumulating reports on gating of cell division by the circadian clock and the proposed evolutionary link between these two systems (40), it is conceivable that this early development is important for cell cycle-dependent timed events that occur during development. In the zebrafish larvae, daily variations in cell division (34) and cell death (41) were reported. Furthermore, the entrance to S phase occurs in daily, light-regulated rhythms (34). The same phenomenon was seen in a zebrafish cell line, suggesting a cell-autonomous circadian clock-regulation of the cell-cycle (34). Accurate timing of cell-cycle-dependent decisions such as proliferation, differentiation, and death during embryogenesis has a profound impact on the final phenotype of the organism (42). The possible involvement of the circadian oscillator in timing these events emphasizes the importance of a functional circadian oscillator throughout embryonic development.

In summary, the results of the current study suggest that the zebrafish embryo possesses cellular circadian oscillators from very early stages and that light exposure is required for synchronization and stabilization of these oscillators. Zebrafish per2, which exhibits ubiquitous expression at these stages, is required for this light-induced process. The oscillators maintain a synchronized 24-h rhythm throughout the rapid proliferation and differentiation, which is reflected here by the free-running clock-controlled rhythms of zfaanat2 mRNA in the pineal gland 2–3 days later. Thus, a vertebrate circadian oscillator is maintained during differentiation.

Materials and Methods

Fish and Embryos.

Care for adult zebrafish and production of embryos was as described (25, 43). Embryos were collected immediately after spawning, within 2 hpf, and placed in a temperature- and light-controlled incubator (28.5°C, 12 Wm2). In studies designed to determine the effect of light at early embryonic stages on gene expression, embryos were exposed to different photoperiodic regimes as indicated in Results. Developmental stages were determined according to Kimmel et al. (44). No difference in the rate of development was observed between embryos raised under continuous dark or different light regimes. Handling of embryos was avoided throughout the experiments to eliminate possible time-giving events (23).

Morpholino Design and Injection.

Zebrafish per2 knock-down experiments were performed by using morpholino-modified anti-sense oligonucleotides (MO; Gene Tools, Philomath, OR). One MO was designed toward the AUG flanking sequences to block initiation of zper2 mRNA translation [Per2(AUG)MO, 5′-GGTCTTCAGACATCGGACTTGGGTT-3′]. This MO has <50% identity with the ATG flanking regions of zper1 and zper3. Another MO was designed toward the exon 4–intron 4 boundary to interfere with splicing [Per2(E4I4)MO, 5′-TGCAGATGTACTTACAGTGTTTTTG-3′]. This MO has 80% and 68% identity with the corresponding region of zper1 and zper3, respectively. An additional MO against AUG flanking sequences of EGFP (EGFP MO, 5′-ACAGCTCCTCGCCCTTGCTCACCAT-3′) was used for control injections. MOs were injected (2 nl, 0.5 mM) into one-cell-stage embryos at the yolk and cytoplasm interface. In each experiment, 100–400 embryos were microinjected with MO as described (25).

RNA Isolation and RT-PCR Analysis.

Efficacy of MOs directed against the splice site was evaluated by using RT-PCR. Uninjected and Per2(E4I4)MO-injected embryos were sampled 3 h after lights on, and total RNA was extracted (EZ-RNA Total RNA Isolation kit, Biological Industries, Beit-Haemek, Israel) according to the manufacturer’s protocol. After DNase treatment, total RNA was used as template to prepare cDNA using M-MLV reverse transcriptase and Random Hexamer primers (Promega). DNA fragments were then PCR-amplified by using primers directed to exons 3 and 5 of zper2 and corresponding exons of zper1 and zper3 (Per2-E3F 5′-AAGCCAAGACGCAGAAAGAG-3′ and Per2-E5R 5′-CCACAAACTTGGCATTCTTG-3′; Per1-E3F 5′-CGCCAGAGAGGAAGATGAAG-3′ and Per1-E5R 5′-ACACAACCTTCCCAGACAGG-3′; Per3-E4F 5′-GGTGCAGGAGATGAAGAAGC-3′ and Per3-E6R 5′-TGACGTCCTGATGGTACAGC-3′). PCR products were cut out from a gel, subcloned into pGEM-T easy (Promega) and sequenced (ABI PRISM 3100 Genetic Analyzer, AME Bioscience).

Whole Mount in Situ Hybridization.

Clones of zper2 (25) and zfaanat2 (43) were used as templates to prepare digoxygenin (DIG)-labeled antisense riboprobe (DIG RNA labeling kit, Roche). Whole-mount ISH was performed as described (25) except for the proteinase K (10 μg/ml) treatment, which was shortened to 3–5 min in embryos younger than 16 hpf.

Observation, Quantification, and Statistical Procedure.

After whole-mount ISH, embryos/larvae were placed in 70% glycerol for observation and photography using a SZX12 dissecting stereoscope with a DP70 digital camera (Olympus). Identical microscopic and camera settings were used for recording signal intensity. The zfaanat2 signal, expressed as optical density, was calculated by using imagej software (National Institutes of Health, Bethesda) as described (25). Differences in signal intensities between treatments and time points throughout the 24-h cycle were evaluated by using ANOVA. Specific comparisons were performed by using multicomparison Tukey’s test. Results are expressed as mean total optical density ± standard error.

Acknowledgments

We thank Profs. Nava Zisapel (Tel Aviv University), Jack Falcon (Centre National de la Recherche Scientifique, Paris), and David C. Klein (National Institutes of Health, Bethesda) for their helpful discussions. This work was supported by United States–Israel Binational Science Foundation (Jerusalem) Grant 2001132 and the Adams Super Center for Brain Studies, Tel Aviv University, Tel Aviv, Israel.

Abbreviations

ISH
in situ hybridization
hpf
hours postfertilization
MBT
mid-blastula transition
LD
light–dark.

Footnotes

Conflict of interest statement: No conflicts declared

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