Circadian rhythmicity in mammals is under the control of a molecular pacemaker constituted of clock gene products organized in transcriptional autoregulatory loops. Phase resetting of the clock in response to light involves dynamic changes in the expression of several clock genes. The molecular pathways used by light to influence pacemaker-driven oscillation of clock genes remain poorly understood. We explored the functional integration of both light- and clock-responsive transcriptional regulation at the promoter level of the Period ( Per ) genes. Three Per genes exist in the mouse. Whereas mPer1 and mPer2 are light-inducible in clock neurons of the hypothalamic suprachiasmatic nucleus, mPer3 is not. We have studied the promoter structure of the three mPer genes and compared their regulation. All three mPer promoters contain E-boxes and respond to the CLOCK/brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) heterodimer. On the other hand, only mPer1 and mPer2 promoters contain bona fide cAMP-responsive elements (CREs) that bind CRE-binding protein (CREB) from suprachiasmatic nucleus protein extracts. The mPer1 promoter is responsive to synergistic activation of the cAMP and mitogen-activated protein kinase pathways, a physiological response that requires integrity of the CRE. In contrast, activation of mPer promoters by CLOCK/BMAL1 occurs regardless of an intact CRE. Altogether, these results constitute strong evidence that CREB acts as a pivotal endpoint of signaling pathways for the regulation of mPer genes. Our results reveal that signaling-dependent activation of mPer genes is distinct from the CLOCK/BMAL1-driven transcription required within the clock feedback loop.

Rhythmicity of the rat suprachiasmatic nucleus (SCN), a site of the circadian clock, develops prenatally. A molecular clockwork responsible for the rhythmicity consists of clock genes and their negative and positive transcriptional–translational feedback loops. The aim of the present study was to discover the development of the clockwork during ontogenesis. Daily profiles of Per1 , Per2 , Cry1 , Bmal1 , and Clock mRNA in the SCN of fetuses at the embryonic day (E)19 and of newborn rats at the postnatal day (P)3 and P10 were assessed by the in situ hybridization method. In addition, daily profiles of PER1, PER2, and CRY1 proteins at E19 were assessed by immunohistochemistry. As early as at E19, all the studied clock genes were already expressed in the SCN. However, no SCN rhythm in their expression was detected; Per1 , Cry1 , and Clock mRNA levels were low, whereas Bmal1 mRNA levels were high and Per2 mRNA levels were medium. Moreover, no rhythms of PER1, PER2, and CRY1 were detectable, as no immunoreactive cells were present at E19. At P3, rhythms in Per1 , Per2 , Cry1 , and Bmal1 , but not in Clock mRNA, were expressed in the SCN. The rhythm matured gradually; at P10, the amplitude of Per1 , Per2 , and Bmal1 mRNA rhythms was more pronounced than at P3. Altogether, the data show a gradual development of both the positive and negative elements of the molecular clockwork, from no detectable rhythmicity at E19 to highly developed rhythms at P10.

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbα, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbα and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbα and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbα expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbα, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.

The SCN as a site of the circadian clock itself exhibits rhythmicity. A molecular clockwork responsible for the rhythmicity consists of clock genes and their negative and positive transcriptional-translational feedback loops. The authors’ previous work showed that rhythms in clock gene expression in the rat SCN were not yet detectable at embryonic day (E) 19 but were already present at postnatal day (P) 3. The aim of the present study was to elucidate when during the interval E19-P3 the rhythms start to develop in clock gene expression and in clock-controlled, namely in arginine-vasopressin (AVP), gene expression. Daily profiles of Per1, Per2, Cry1, Bmal1, and Clock mRNA and of AVP heteronuclear (hn) RNA as an indicator of AVP gene transcription were assessed in the SCN of fetuses at E20 and of newborn rats at P1 and P2 by the in situ hybridization method. At E20, formation of a rhythm in Per1 expression was indicated, but no rhythms in expression of other clock genes or of the AVP gene were detected. At P1, rhythms in Per1, Bmal1, and AVP and a forming rhythm in Per2 but no rhythm in Cry1 expression were present in the SCN. The Per1 mRNA rhythm was, however, only slightly pronounced. The Bmal1 mRNA rhythm, although pronounced, exhibited still an atypical shape. Only the AVP hnRNA rhythm resembled that of adult rats. At P2, marked rhythms of Per1, Per2, and Bmal1 and a forming rhythm of Cry1, but not of Clock, expression were present. The data suggest that rhythms in clock gene expression for the most part develop postnatally and that other mechanisms besides the core clockwork might be involved in the generation of the rhythmic AVP gene expression in the rat SCN during early ontogenesis.