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Birth Defects Research (Part C) 81:204–214 (2007)

Circadian Clocks During Embryonic and Fetal Development Maria Seron-Ferre,* Gullermo J. Valenzuela, and Claudia Torres-Farfan Circadian rhythmicity is a fundamental characteristic of organisms, which helps ensure that vital functions occur in an appropriate and precise temporal sequence and in accordance with cyclic environmental changes. Living beings are endowed with a system of biological clocks that measure time on a 24-hr basis, termed the circadian timing system. In mammals, the system is organized as a master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus, commanding peripheral clocks located in almost every tissue of the body. At the cell level, interlocking transcription/translation feedback loops of the genes Bmal-1, Clock, Per1–2, and Cry1–2, named clock genes, and their protein products results in circadian oscillation of clock genes and of genes involved in almost every cellular function. During gestation, the conceptus follows a complex and dynamic program by which it is simultaneously fit to develop and live in a circadian environment provided by its mother and to prepare for the very different environment that it will experience after birth. It has been known for a number of years that the mother tells the fetus the time of day and season of the year, and that the fetus uses this information to set the phase of fetal and neonatal circadian rhythms. There is evidence that the maternal rhythm of melatonin is one of the time signals to the fetus. In the last few years, the study of the development of the circadian system has turned to the investigation of the oscillatory expression of clock genes and their possible role in development, and to answering questions on the organization of the fetal circadian system. Emerging evidence shows that clock genes are expressed in the oocyte and during early and late development in embryo/fetal organs in the rat and in a fetal primate. The data available raise the intriguing possibility that the fetal SCN and fetal tissues may be peripheral clocks commanded by separate maternal signals. The rapid methodological and conceptual advances on chronobiology may help to unravel how the developing embryo and fetus faces time in this plastic period of life. Birth Defects Research (Part C) 81:204–214, 2007. VC 2007 Wiley-Liss, Inc.

INTRODUCTION The reliable (and therefore predictable) environmental alternations of day and night and the

seasons originated from the fact that our planet turns about itself in 24 hr and moves around the sun in a year, led to evolution of

biological clocks that measure time on about 24-hr basis (circadian clocks) in practically all living beings. These clocks also time reproduction in seasonal animals, ensuring the best chances for survival to the offspring (Moore-Ede et al., 1982; Edery, 2000; Lincoln et al., 2003). Circadian clocks provide a 24-hr framework for internal temporal organization, separating incompatible physiological functions both at the cellular and also at the organism level. In mammals, the circadian timing system is organized as a hierarchy of oscillators located in most tissues of the body (peripheral oscillators), synchronized by a central rhythm generator located in the suprachiasmatic nucleus (SCN) of the hypothalamus (Fig. 1). The overt circadian rhythms of temperature, cortisol, activity, sleep, and heart rate to which we are familiarized can be viewed as the hands of this clock system. The SCN and peripheral clocks are run by a common transcriptional circuitry of genes (clock genes) that generates rhythmic patterns of gene expression in themselves and on other genes (clock-controlled genes) that are involved in protein expression, metabolism, and/or function depending on the

´n-Ferre ´ is from the Programa de Fisiopatologı´a, Instituto de Ciencias Biome M. Sero ´dicas (ICBM) Facultad de Medicina, Universidad de Chile, Santiago, Chile, and from the Universidad de Tarapaca and Centro de Investigaciones del Hombre en el Desierto (CIHDE), Arica, Chile. G.J. Valenzuela is from the Department of Women’s Health, Arrowhead Regional Medical Center, Colton, CA. C. Torres-Farfan is from the Programa de Fisiopatologı´a, Instituto de Ciencias Biome ´dicas (ICBM) Facultad de Medicina, Universidad de Chile, Santiago, Chile. Grant sponsor: Fondo Nacional de Desarrollo Cientı´fico y Tecnolo ´gico, Fondecyt 106766, Santiago, Chile; Grant sponsor: San Bernardino Medical Foundation, Department of Women’s Health, Arrowhead Regional Medical Center, CA. *Correspondence to: Marı´a Sero ´n-Ferre ´, Ph.D., Salvador 486, Providencia, Santiago, Chile, Programa de Fisiopatologı´a, ICBM, Facultad de Medicina, Universidad de Chile, Casilla 16038, Santiago 9, Santiago, Chile. E-mail: Published online in Wiley InterScience ( DOI: 10.1002/bdrc.20101

C 2007 Wiley-Liss, Inc. V


Figure 1. Schematic representation of the organization of the mammalian circadian system and the entrainment pathways to the SCN of the hypothalamus and peripheral oscillators (adrenal gland, heart, and kidney). The SCN is entrained by the light:dark cycle, whereas peripheral clocks phase are entrained by the SCN through humoral and neuronal signals. Feeding and metabolic signals might contribute to the entrainment of peripheral clocks.

cell or organ (Reppert and Weaver, 2002; Okamura et al., 2002; Richter et al., 2004; Levi and Schibler, 2007). Circadian clocks are entrained (reset) by Zeitgebers (time givers), environmental cues that act by shifting the pattern of clock gene expression and therefore the phase of the overt circadian rhythms. The SCN is connected with the retina by a monosynaptic pathway, the retinohypothalamic tract that allows entrainment by the light/ dark cycle. Peripheral clocks are entrained directly by the SCN through some neurohumoral signals and by other Zeitgebers, such as body temperature and feeding time (Levi and Schibler, 2007).

In mammals, the conceptus follows a complex and dynamic program by which it is simultaneously fit to develop and live in a circadian environment provided by its mother and to prepare for the very different environment that it will experience after birth. The conceptus knows time of day and also knows time of year, programming physiology for the environment encountered after birth, as shown in seasonal animals like sheep (Ebling et al., 1989), hamsters and voles (Gorman et al., 2001), and deer (Adam et al., 1994). During gestation, the close relationship between mothers and their progeny ensures an adequate development and a suc-

cessful transition to the postnatal life. The mother supplies oxygen, nutrients, and hormones that influence the development of the embryo/fetus and also provide environmental cues since body temperature and many metabolic pathways and hormones in the mother present circadian rhythms entrained to the external environment. Additional time cues may be provided by uterine activity rhythms. Circadian rhythms of fetal heart rate, respiratory movements, fetal movements, and hormones are detected in the human and in other species. Since it is known that the fetal SCN oscillates in utero (reviewed in Seron-Ferre et al., 1993; Davis and Reppert, 2001; Weinert, 2005), several questions arise from the new findings in circadian physiology. Does the conceptus have an operating circadian system similar to that of the adult? What are the signals entraining fetal circadian rhythms? What is the importance of the circadian system during development and for postnatal life? The following paragraphs review the molecular mechanisms driving circadian clocks and the questions that have been addressed to understand the ontogeny and organization of the circadian system and its possible function during development and in the preparation for postnatal events.

CIRCADIAN CLOCKS: MOLECULAR MECHANISMS At the cell level, circadian rhythms are driven by the interlocking selfregulatory interaction of a set of genes (Bmal-1, brain and muscle ARNT-like protein 1, also known as Mop3), Per1–2, Cry1–2, and Clock (circadian locomotor output cycle kaput), called ‘‘clock genes,’’ and their protein products, schematically represented in Figure 2. The positive arm of the circuit is the heterodimer of the proteins CLOCK:BMAL-1. This complex binds E-box elements (CACGTG/T) at the promoter region of Per1–2 and Cry1–2, inducing their transcription (Lee et al., 2001). The negative regulators are the trans-

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lated CRY and PER proteins, that complex with casein kinase e and translocate into the nucleus, interacting with the CLOCK:BMAL1 complex and thus blocking their own transcription. Phosphorylation of the PER and CRY proteins by casein kinase e controls their proteasomal degradation, delaying the formation of CRY:PER complexes and determining the length of the cycle (Lowrey et al., 2000; Vanselow et al., 2006). The CLOCK:BMAL1 heterodimer also induces the transcription of Reverba and Rora genes, which interact with Rev-erb/Ror elements (RREs) in the promoter of Bmal1, repressing and driving its transcription, respectively (Preitner et al., 2002). Additional regulators are a further negative loop generated by the transcription of the dec1 (sharp2/stra13) and dec2 (sharp1) genes, which are also driven by CLOCK:BMAL1 via Eboxes in their promoters (Grechez-Cassiau et al., 2004). DEC1 and DEC2 proteins may block circadian gene expression, in part by the formation of a nonfunctional heterodimer with BMAL1 (and hence inhibiting the expression of all genes dependent on an E-box), as well as play a role in light induction of genes in the SCN (Honma et al., 2002). The circadian oscillation of clock gene expression controls the expression of genes involved in multiple cellular functions in the 24-hr period (clock-controlled genes, CCGs) by at least two mechanisms, direct interaction with E-boxes in the promoters of these genes, and through the regulation of other CCGs that are in turn transcription factors, like DBP (reviewed in Reppert and Weaver, 2002; Okamura et al., 2002; Richter et al., 2004; Levi and Schibler, 2007). An important breakthrough was the finding of circadian expression of clock genes in practically every tissue studied (liver, pancreas, kidney, and heart) and also in cell lines (Oishi et al., 1998a, 1998b). These tissues are referred to as peripheral oscillators or peripheral clocks. Importantly, recent experiments show that under culture

Figure 2. Schematic representation of the core components of a circadian clock. The positive arm of the clock, the heterodimer CLOCK:BMAL1, initiates transcription of the genes Per 1–3 and Cry 1–2. Upon translation, the complex of PER: CRY is phosphorylated by casein kinase e (CK e) and transported to the nucleus inhibiting transcription. A second loop is formed by the transcription factors REVERBa and ROR. [Redrawn from Merrow et al. (2006).]

conditions, the clock gene oscillatory process lasts for weeks in these tissues, underlying its endogenous nature (Yoo et al., 2004). Microarray analysis has shown circadian oscillation in an important part of the transcriptome involving almost every cellular biochemical pathway (Panda et al., 2002). The link between biochemical processes and clock genes is not completely understood; however, transcriptional coactivators like PGC-1a have been proposed to be involved (Liu et al., 2007). A consistent observation is that in vivo, clock genes in the peripheral oscillators have a phase delay of 4- to 8-hr relative to their oscillation in the SCN (Balsalobre et al., 2000; Damiola et al., 2000; Stokkan et al., 2001). In rats and mice, destruction of the SCN eliminates circadian rhythms of clock gene expression in peripheral organs (Sakamoto et al., 1998). However, the experiments cannot distinguish whether SCN lesions abolish circadian rhythms within an individual or results in asynchronous rhythms between individuals. SCN transplant can restore the rhythm of locomotor activity but not endocrine rhythms in hamster (MeyerBernstein et al., 1999), suggesting that blood-borne and/or other nonneural (e.g., behavioral) rhy-

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thms may be responsible for the communication of the SCN with some but not all peripheral oscillators. This is further supported in parabiosis experiments linking SCN-lesioned mice to intact mice (Guo et al., 2005). In these experiments, parabiosis induced recovery of rhythms of mPer1, mPer2, and mBmal1 gene expression in liver and kidney, but not in spleen, heart, or skeletal muscle, of the SCN-lesioned mice. Feeding may provide a time cue for some peripheral clocks, as the phase displayed by clock genes in the liver, pancreas, kidney, and heart is modified in rodents subjected to feeding restricted to a few hours every day (Balsalobre et al., 2000; Stokkan et al., 2001; Le Minh et al., 2001; Balsalobre, 2002). These animals increase both locomotor activity and core body temperature in anticipation of the timed daily meal. Therefore, in addition to endocrine and metabolic signals, biophysical signals like locomotor activity and body temperature may provide Zeitgebers for peripheral clocks (reviewed by Stratmann and Schibler, 2006). In summary, as depicted in Figure 1, the circadian timing system in mammals is organized as a hierarchy of peripheral oscillators synchronized by the SCN of the hypothalamus (Guo et al., 2006). The end result is the separation of vital functions in an appropriate and precise temporal sequence within the 24-hr period, hence ensuring predictive adaptation to cyclic environmental changes (Pittendrigh, 1981; Moore-Ede et al., 1982).

THE CIRCADIAN SYSTEM AND CIRCADIAN CLOCKS THROUGHOUT DEVELOPMENT Earlier studies on development of the circadian system were centered on development of the master clock (SCN) and on investigating the hands of the clock; i.e., the presence of overt circadian rhythms either in the late gestation fetus or in the newborns. Cir-


cadian rhythms of several physiological functions are present in fetuses of precocial species like human, rhesus monkey, and sheep: 1) in the human, fetal heart rate, fetal breathing, fetal movements, and plasma cortisol; 2) in rhesus fetuses circulating plasma dehydroepiandrosterone sulfate (DHAS); and 3) in fetal sheep fetal breathing, plasma prolactin. and arginine vasopressin (AVP) in cerebrospinal fluid (reviewed in Seron-Ferre et al., 1993, 2001). In these species, fetal SCN neurogenesis and SCN innervation by the retinohypothalamic tract (RHT) is completed by midgestation (reviewed in SeronFerre et al., 2001) and the fetal SCN shows circadian rhythms of metabolic activity (using the 14 C-labelled 2-deoxy-D-glucose uptake) (Reppert and Schwartz, 1984) and of c-FOS (Constandil et al., 1995; Breen et al., 1996). In contrast, in altricial species (pups are very immature at birth), like rats and hamsters, overt circadian rhythms of temperature, behavioral rhythms like locomotor activity, drinking, and the plasma corticosterone rhythm develop postnatally. In these species, SCN neurogenesis is completed close to birth and innervation by RTH is completed postnatally (reviewed by Davis and Reppert, 2001); notwithstanding, the fetal SCN presents day/night rhythms in metabolic activity (Reppert and Schwartz, 1983; Davis and Gorski, 1985), an abundance of messenger RNA (mRNA) for vasopressin (Reppert and Uhl, 1987), and of spontaneous neural activity (recorded in slice preparations; Shibata and Moore, 1987). Altogether, these evidences demonstrate oscillatory function of the fetal SCN in altricial and precocial species and the presence of overt circadian rhythms during fetal life in the latter. A characteristic common to precocial and altricial species is that fetal SCN rhythms appear synchronized to clock time in individuals from the same litter and between individuals from different mothers. Rhythms are synchronized between twins in

human and sheep pregnancy (Parraguez et al., 1996; Maeda et al., 2006). In addition, postnatal rhythms are synchronized between newborns from the same litter (mice, rats, and hamsters). A series of experiments in rats and hamsters demonstrate that fetal SCN rhythms persist but within-litter synchrony is lost after maternal SCN ablation, indicating that the SCN rhythm is generated endogenously in the fetus but is entrained by circadian signals controlled by the maternal SCN. Similarly, lesions of the maternal SCN result in desynchronization of postnatal behavioral rhythms within a litter. To summarize, these series of experiments demonstrate that the fetal SCN has endogenous oscillatory properties, and is entrained by a signal controlled by the maternal SCN. A second finding is that the phase of postnatal behavioral rhythms in rats and hamsters is set by signals experienced during gestation (reviewed by Davis and Reppert, 2001). The underlying concept was of a fetal circadian system organized with the fetal SCN as master clock entrained by a nonphotic signal derived from the mother. The new tools provided by the understanding of the molecular events underlying circadian rhythms and the recent awareness that circadian clocks are present in most peripheral tissues may challenge this view. Oscillation of circadian clocks accompanies the growing oocyte, and thereafter. The clock genes, Per-2 and Bmal-1, and their proteins oscillate in follicles and corpus luteum in the rat ovary and may play a role in steroidogenesis (Karman and Tischkau, 2006; Fahrenkrug et al., 2006). A full complement of clock genes oscillate in the rat oviduct (Johnson et al., 2002; Kennaway et al., 2003), which carries the ovulated oocyte through fertilization and early embryonic development, and in the uterus, where it will implant (Johnson et al., 2002; Horard et al., 2004). The functional significance of these genes in early development and uterine preparation for

implantation is unknown (reviewed by Boden and Kennaway, 2006). On the other hand, the unfertilized mouse oocyte, as shown in Figure 3, expresses the mRNAs of the six canonical clock genes (Per 1 and 2, Cry 1 and 2, Clock, and Bmal1). After fertilization, expression of these mRNAs decreases between two-cell and 16-cell stages to be reinitiated at the blastocyst stage (Ko et al., 2000; Johnson et al., 2002). What follows after implantation is just beginning to be explored. Using imaging techniques, Saxena et al. (2007) studied the expression of the clock gene Per-1 throughout gestation in the uterus of pregnant rats (Fig. 3). Wild-type female rats were mated with a heterozygous transgenic male carrying the Per-1 promoter coupled to firefly luciferase; therefore, some of the fetuses expressed the Per-1::luc construct. Although the study was aimed at establishing the feasibility of the technique, some amazing observations were produced. Using 120-sec exposure, Per1::luc expression was detected at dusk at 10 days of gestation and a dawn/dusk difference was observed at 12 days. A sharp rise in fluorescence was detected at this age, obligating reduced exposure time to 1 sec for measurements at subsequent ages. Overall, the authors found an exponential increase in Per-1::luc expression from day 10 to day 21, the end of gestation, and in postnatal day 1. It is not known which intrauterine tissues (placenta, embryo, or fetal organs) contributed to the intrauterine fluorescence observed. However, after birth, there was strong internal organ fluorescence attributed mainly to the liver, a very large organ in the newborn (and also in the fetus), besides weak skin fluorescence (Saxena et al., 2007). It is of interest to note that fluorescence was higher at dusk than at dawn at late gestation, whereas the pattern is reversed in the newborn. This promising technique in addition to more conventional techniques offers the possibility to identify which fetal organ(s) is the

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Figure 3. A: Expression of clock genes at different embryonic stages. [Data from Johnson et al. (2002) and Ko et al. (2000)]. B: Bioluminescence from heterozygous Per1::luc embryos imaged in utero throughout gestation and from two heterozygous newborns and two wild-type newborns. [Modified from Saxena et al. (2007).]

source of fluorescence at the different gestational ages and whether this fluorescence is accompanied by oscillatory expression of other clock genes. The ontogeny of oscillatory mRNA expression of clock genes in the fetal SCN and in a few fetal peripheral tissues has been recently studied in rat, mice, hamster, rabbit, and the capuchin monkey. Studies in the rat and hamster demonstrate that the core elements of the circadian system, the clock genes Per1, Per2, Cry1, Bmal1, and Clock, are expressed in the rat and hamster fetal SCN (Sladek et al., 2004; Li and Davis, 2005). However, there

was no clear circadian rhythm of any of the clock gene mRNAs or PER1, PER2, and CRY1 proteins in the fetal SCN during fetal life, whereas a clear oscillation of mRNA and protein was detected at three to 10 days of postnatal age in the rat and of Bmal1, Cry1, and Per1 also in the hamster SCN. At this postnatal age, the pattern of expression of clock genes was akin to that in the adult SCN. Similar observations were obtained for Per1 and Per 2 in the rat and mice (Shimomura et al., 2001; Ohta et al., 2002).The lack of oscillatory expression in the fetal rodent SCN is surprising, given the presence of cyclic metabolic activity already

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discussed. The source of this discrepancy remains to be established. The SCN of another altricial species, the rabbit, show oscillatory expression of clock genes at day 7 postnatal, the only time studied (Caldelas et al., 2007). This is of interest since rabbit pups show a rhythm of body temperature by day 2 of postnatal age, and show anticipatory activity to the daily mother visit for feeding soon after birth (reviewed in Gonzalez-Mariscal, 2007). In contrast to the lack of oscillation of clock genes in rats and hamster fetal SCN, as shown in Figure 4A, Bmal1 and Per-2 expression in the capuchin fetal SCN is oscillatory at


Figure 4. A: Oscillatory expression of Bmal-1 and Per-2 measured by RT-PCR in the fetal capuchin monkey SCN and adrenal gland at the indicated hours. Fetal adrenal gland (right upper panels): mean 6 SE of Bmal-1 and Per-2 expression measured by RT-PCR at 0800 hr (n 5 5), 1400 hr (n 5 5), and 2000 hr (n 5 3). *Different from 0800 hr and 1400 hr, p \ 0.05, ANOVA and Tukey’s test; yDifferent from 1400 hr and 2000 hr, p \ 0.001, ANOVA, and Tukey’s test. In each graph the value at 0800 hr is repeated in the next 24 hr. The dark bars indicate lights-off hours. B: Clock gene expression (Bmal-1, Per-2, Cry-2, and Clock) in capuchin monkey pituitary, thyroid, brown fat, and pineal. Fetuses were euthanized at 0800, 1400, and 2000 hr. [Redrawn from TorresFarfan et al. (2006a).]

90% gestation (Torres-Farfan et al., 2006a). The peaks of Bmal1 and Per-2 expression were in antiphase, Bmal-1 peaking at the beginning of the night and Per-2 peaking about 10 hr later, at the end of the night. This 10-hr interval between the increases of Bmal-1 and Per-2 in the fetal capuchin SCN is longer than that reported in the SCN of adult animals (Oishi et al., 1998a; Zylka et al., 1998; Lincoln et al., 2002;

Johnston et al., 2005; Watanabe et al., 2006), and it is reminiscent of the antiphase pattern described in most peripheral clocks mentioned in the preceding section. A second fetal tissue studied was the fetal adrenal. As in the SCN, Bmal-1 and Per-2 oscillation was detected. A surprising finding was that the phase of oscillation was similar to that detected in the fetal SCN, in contrast with the phase delay between SCN and pe-

ripheral clocks in the adult (Oishi et al., 1998a; Zylka et al., 1998; Lincoln et al., 2002; Johnston et al., 2005; Watanabe et al., 2006). In both fetal SCN and adrenal glands, the clock gene oscillation was accompanied by a circadian output. We found a rhythm of DHAS, a steroid secreted by the fetal adrenal and rhythms of expression of the MT1 melatonin receptor in the fetal SCN and adrenal gland. In addition, another

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Figure 5. Distribution of the mean hour of drinking in individual pups between three and six weeks of age. Pups were born to dams sham operated (control), pinealectomized during pregnancy, and pinealectomized dams receiving daily melatonin replacement. Each symbol represents a pup. The length of the arrow indicates the degree of synchrony of each group of pups. An absent arrow indicates no synchrony. [Redrawn from Bellavia et al. (2006).]

marker of fetal adrenal function, expression of the steroidogenic enzyme 3b hydroxysteroid dehydrogenase showed clock time– related changes (Torres-Farfan et al., 2006a). Of note, the promoter of the MT1 receptor has Eboxes and retinoid orphan receptor elements (ROREs), suggesting that it could be a clock-controlled gene (Ueda et al., 2005; Johnston et al., 2007). Thus, in the capuchin, the fetal SCN and the fetal adrenal show circadian oscillation of two clock genes and evidence of oscillatory function downstream of these genes. Recently, several authors reported the oscillatory expression of clock genes in the adrenal gland of adult rodents (Ishida et al., 2005; Watanabe et al., 2006; Torres-Farfan et al., 2006b) and primate (Lemos et al., 2006), providing strong evidence for the presence of a peripheral clock in this tissue. This possibility is highly relevant, in light of evidence supporting a role for plasma glucocorticoids in the entrainment of other oscillators (Balsalobre et al., 2000), as well as in control of metabolic rhythms in other organs such as liver (Oishi et al., 2005) and adipocytes (AlonsoVale et al., 2005). Expression of clock genes in the adrenal gland may participate in the generation of the corticosterone rhythm, as mutant mice defective in adrenal

clock genes (double Per2/Cry1 knockout), show loss of hypothalamus-pituitary-adrenal axis rhythmicity. In these mice, transplant of adrenals from wild-type to mutant mice rescues the corticosterone rhythm in light:dark conditions (Oster et al., 2006). Whether the fetal adrenal gland is a powerful circadian clock remains to be investigated. Clock genes were also expressed in the other capuchin fetal tissues investigated (pituitary, thyroid, and brown adipose tissue) as shown in Figure 4B, although oscillatory expression was not measured. These are tissues that exert several functions in fetal life, either producing hormones (pituitary, thyroid) (Grumbach and Kaplan, 1998; Polk and Fisher, 1998) or accruing to serve as thermogenic substrate in the newborn (adipose tissue) (Cannon and Nedergaard, 2004), whereas the fetal pineal starts secreting melatonin after birth (Kennaway et al., 1992). The fetal pineal did not express Per-2 but expressed Bmal-1 and Clock and had a significantly higher expression of Cry-2 than the other tissues tested. Whether the absence of Per-2 expression coupled to high Cry-2 expression relates to the lack of pineal melatonin synthesis during fetal life needs to be explored. The only other fetal tis-

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sue studied to date is the rat fetal liver. Ontogeny of oscillation of the clock genes, Per1, Per2, RevErba, Cry1, Bmal1, and Clock, in the rat fetal liver, was studied by Sladek et al. (2007) finding prenatal (20 days of gestational age) oscillation of Rev-Erba but not of the other genes. A more adult pattern of oscillation was attained between 10 to 20 days of age. Of note, the authors show oscillation of Per-1 in the liver at postnatal day 2, coinciding with the findings in neonatal rats of Figure 3 (from Saxena et al., 2007). The emerging picture from the data accrued in the last few years is that clock genes are expressed very early in development and most likely in many embryonic/fetal tissues. Whether they represent functional circadian clocks in the sense that we understand in the adult or serve other functions is presently unknown. Parsimony is used widely in biology, and it is conceivable that during development some of the clock genes may serve functions not related to circadian timekeeping (Miller et al., 2007).

MATERNAL SIGNALS ENTRAINING FETAL AND NEWBORN CIRCADIAN RHYTHMS As described before, the fetal rhythms in precocial species or neonatal rhythms in altricial species and the rhythms of expression Bmal-1 and Per-2 in the fetal capuchin SCN and fetal adrenal gland were entrained to clock time. A recent experiment, using clock gene mutant mice, showed loss of synchrony in the postnatal rhythm of activity in heterozygous pups of arrhythmic double knockout mPer1Brdn/Per2Brdnmice and mPer2Brdn/mCry1 mice (Jud and Albrecht, 2006), confirming again that the rhythm of activity is not induced by the mother but it requires maternal signals for synchronization. As mentioned before, fetuses are subject to a variety of nonphotic stimuli originating from the mother, including circadian


fluctuations in hormones, nutrients, and uterine motility. Maternal signals are most likely redundant, and may have different targets in the embryo/fetus during development. There is evidence for two maternal signals acting during gestation on entrainment of newborn rhythms: maternal feeding time and maternal melatonin. In SCN-lesioned pregnant rats, restricting feeding to a 4-hr period restores synchronization of the pups’ drinking rhythms (Weaver and Reppert, 1989). As already discussed, the time of food availability changes the phase of the rhythms of corticosterone and locomotor activity (Krieger et al., 1977), induces an anticipatory increase in body temperature (Recabarren et al., 2005), and in addition changes the phase of clock gene expression in liver without changing the phase in the SCN (Balsalobre, 2002). The maternal metabolic signal has not been identified. However, the more recent findings that body temperature and corticosterone shift the phase of clock genes in fibroblasts (Balsalobre et al., 2000; Le Minh et al., 2001; Balsalobre, 2002) and in the liver (Balsalobre et al., 2000) open interesting possibilities to identify the Zeitgeber mediating the effects of maternal restricted feeding on the newborns. The second maternal signal studied, melatonin, is one of the few maternal hormones crossing the placenta without being altered. Melatonin is produced by the mother in a circadian fashion. It has been named the hormone of the night, as it is secreted with almost a square wave pattern, concentration very low at daytime, increasing sharply at the beginning of the night (when lights are off), and decreasing abruptly at dawn (when lights are on). The duration of the night increase in melatonin, signals the length of the photoperiod and thus season of the year. Pineal melatonin synthesis starts postnatally in the rat (Deguchi, 1975), sheep (Nowak et al., 1990), and human (Kennaway et al., 1992); however, maternal melatonin generates a

Figure 6. Schematic representation of the proposed entrainment pathway of capuchin monkey fetal SCN and fetal peripheral oscillators. The fetal SCN is entrained by the rhythm of maternal melatonin, whereas the fetal adrenal and possibly other fetal peripheral clocks are phase-entrained by the maternal SCN through humoral or metabolic signals that cross the placenta. [Based on data of Torres-Farfan et al. (2006a).]

rhythm of melatonin in the fetal circulation (Yellon and Longo, 1988). Melatonin receptors are present in the human, capuchin monkey, and rodent fetal SCN in addition to other sites in the human fetal brain (Weaver and Reppert, 1996; Thomas et al., 2002), fetal kidney (Drew et al., 1998), and as mentioned, in the capuchin fetal adrenal gland and fetal SCN (Torres-Farfan et al., 2004, 2006a). Experimental evidence supports maternal melatonin being an entraining signal for postnatal behavioral rhythms in hamsters and rats and for the rhythm of clock gene expression in the capuchin SCN. Timed injection

of melatonin into SCN-lesioned maternal hamsters restores synchrony in the rhythm of activity of the newborns (Davis and Mannion, 1988). Similarly, maternal melatonin replacement during late gestation restores synchronization of the rhythm of drinking water in 25-day-old pups of pinealectomized mothers (Bellavia et al., 2006) (Fig. 4). The site of action of melatonin is presumed to be the developing fetal SCN. Direct evidence for maternal melatonin as a strong Zeitgeber for the fetal capuchin SCN was obtained by us in the capuchin monkey (TorresFarfan et al., 2006a). Suppression of maternal melatonin secretion by exposing pregnant females to constant light during the last third of gestation, shifted Bmal-1 and MT1 melatonin receptor expression in the fetal capuchin monkey SCN and this shift was reverted by daily melatonin replacement. Altogether, these data support a role for maternal melatonin as a Zeitgeber for the fetal primate SCN, and rat and hamster SCN. However, as usually happens in research, experiments answer one question and raise a few more. In the experiments in the capuchin just described, neither adrenal clock gene expression nor markers of circadian fetal adrenal function were modified by suppressing maternal melatonin. This observation is in conflict with a vision of the fetal SCN being a master clock entrained by the mother that commands a putative fetal adrenal clock and probably other fetal peripheral clocks not yet identified. It rather suggests that, at least in the capuchin monkey, the fetal SCN is not a master clock for the fetal adrenal. More research is needed to identify whether circadian clocks are present in other fetal organs and whether they are commanded by the fetal SCN. An intriguing possibility, that requires further investigation, is that during fetal life the fetal SCN and distinct fetal organs may be peripheral clocks to the mother, and entrained by diverse maternal signals, one of them being melatonin for the fetal SCN (Fig. 6).

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IMPORTANCE OF THE CIRCADIAN SYSTEM DURING DEVELOPMENT AND FOR PREPARATION FOR POSTNATAL LIFE An important fact is that 20% of the working population is subjected to a shift-work schedule, with no exceptions being made for working pregnant women. The relationship between exposure to changed photoperiod and pregnancy and fetal outcome is not clear. However, an increased risk of preterm delivery and low weight at birth in women under system shift-work schedule has been demonstrated (Zhu et al., 2004; Croteau et al., 2006). The increased incidence of metabolic syndrome in shift workers (Karlsson et al., 2001) may alter the maternal environment. Additionally, the growing evidence of the long-term impact of pregnancy environment programming susceptibility to diseases that appear in adult life, such as diabetes, hypertension, and metabolic syndrome (Barker et al., 2006; Hammer and Stewart, 2006; Fowden et al., 2006; Nathanielsz, 2006), further stimulates the need to understand the development of the circadian system. Although the very fact that a number of clock gene knockout animals are born, progress to adulthood, and reproduce suggests that development was not interrupted, the study of these clock gene mutant models indicates that the integrity of the circadian system is paramount for normal metabolic function(s) during adult life. For instance, lack of Clock expression is transduced to adipocyte hypertrophy and lipid engorgement of hepatocytes, with prominent glycogen accumulation, while plasma exhibits hypercholesterolemia, hypertriglyceridemia, hyperglycemia, hypoinsulinemia, and lower corticosterone concentration than wild-type mice (Turek et al., 2005; Shimba et al., 2005). It is interesting to point out recent findings in the human of association of clock gene defects to advanced sleep syndrome (Jones

et al., 1999; Toh et al., 2001; Xu et al., 2007) and breast cancer (Chen et al., 2005). Altogether, the evidence available supports that the normal circadian system is involved in several physiological processes and also in metabolic balance; whether these mechanisms are operating in fetal life remains to be investigated. Moreover, understanding the circadian factors helping life in utero has practical importance for neonatal care of preterm infants abruptly deprived of circadian signals. In this regard, the positive impact of exposing the preterm newborn to a light:dark cycle is being recognized, while the possible therapeutic use in preterm infants of melatonin is being proposed (Rivkees, 2003; Jan et al., 2007). The circadian timing system is emerging as an important factor during development. Rapid methodological advances combining in vivo and in vitro experiments, together with the incorporation of new findings on the mechanisms regulating circadian clocks in the adult, may help to unravel how the developing embryo/fetus faces time in this plastic period of life.

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