Expression of Clock and Clock-Driven Genes in the Rat Suprachiasmatic Nucleus During Late Fetal and Early Postnatal Development

Home , AVP gene, Hitoshi Okamura

Expression of Clock and Clock-Driven Genes in the Rat Suprachiasmatic Nucleus during Late Fetal and Early Postnatal Development

Zuzana Kovácˇiková,1 Martin Sládek,1 Zdenka Bendová, Helena Illnerová, and Alena Sumová2 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Abstract 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.

Key words circadian clock, suprachiasmatic nucleus, clock genes, arginine-vasopressin, ontogenesis, rat

All mammals exhibit daily rhythms at various The circadian rhythms are controlled by a clock levels, ranging from the molecular to the behavioral. located in the SCN of the hypothalamus (Klein et al., These rhythms persist even in a nonperiodic environ- 1991). The SCN itself exhibits rhythms in the uptake ment with a period close to 24 h and are entrained to the of 2-deoxyglucose, a marker of metabolic activity 24-h day mostly by the LD cycle (Pittendrigh, 1981). (Schwartz, 1991), in electrical activity (Gillette, 1991),

1. These authors contributed equally to the study. 2. To whom all correspondence should be addressed: Alena Sumová, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenˇ ská 1083, 142 20 Prague 4, Czech Republic; e-mail: [email protected].

140 Kovácˇiková et al. / SCN CLOCK RHYTHMICITY DURING ONTOGENY 141 in spontaneous as well as a light-induced expression of the AVP gene transcription (Yambe et al., 2002) of immediate early genes—namely c-fos, a marker of was chosen as a marker of an overt SCN rhythmicity neuronal activity (Kornhauser et al., 1993; Schwartz for its close connection to the core clock mechanism. et al., 1995; Sumová et al., 1998), in the production of CLOCK-BMAL1 heterodimers act through an E-box many peptides—for instance, of arginine-vasopressin enhancer in the vasopressin gene to activate its tran- (AVP) (van den Pol, 1991; Yambe et al., 2002), and other scription (Jin et al., 1999). To detect the AVP transcript rhythms. This SCN rhythmicity is due to SCN mole- levels by in situ hybridization, a probe complemen- cular clockwork (for review, see King and Takahashi, tary to part of the intronic AVP sequence was used 2000; Reppert and Weaver, 2001; Fu and Lee, 2003). instead of the exonic sequence. This approach enabled A set of mammalian clock genes, namely 3 period genes detection of nascent transcripts or hnRNA and thus (Per1, -2, and -3), 2 cryptochrome genes (Cry1 and -2), of a pure transcription of the AVP gene not compro- Clock, Bmal1, casein kinase 1 epsilon (CK1ε), and Rev-erbα, mised by a potential AVP mRNA instability (Yambe are part of the clockwork. With the exception of Clock et al., 2002). and CK1ε, all these genes are expressed in a rhythmic way; the rhythmic expression of Bmal1 is in antiphase to that of Per and Cry genes. Clock genes are thought MATERIALS AND METHODS to be involved in the core clockwork by forming interacting negative and positive transcriptional- Animals translational feedback loops. The mammalian SCN develops gradually (Moore, Female Wistar rats (Bio Test s.r.o., Konarovice, 1991). In the rat, formation of the SCN begins on Czech Republic) were maintained under an LD cycle embryonic day (E) 14 and continues through E17 with 12 h of light and 12 h of darkness per day from the specialized zone of the ventral diencephalic (LD12:12) in a temperature of 23 ± 2 °C and with free germinal epithelium as a component of periventricu- access to food and water for at least 2 months. Light lar cell groups. Synaptogenesis in the SCN progresses was provided by overhead 40-W fluorescent tubes, slowly in the late prenatal and early postnatal periods and illumination was between 50 and 200 lux, and then increases noticeably from postnatal day (P) depending on cage position in the animal room. The 4 to P10. Rhythms in the SCN may appear as early as day the rats were found to be sperm positive was in the late embryonic stage. A day-night variation of designated embryonic day 0 (E0). Birth occurred on metabolic activity monitored by a 2-deoxyglucose average at E21.5. We used day E20 and not E21 for uptake was detected earlier in the fetal rat SCN from prenatal studies so as not to kill pregnant rats during E19 through E21 (Reppert and Schwartz, 1984), of the delivery. At E20, the morning light was not turned AVP mRNA level at E21 (Reppert and Uhl, 1987), and on, and mothers were released into constant dark- in the firing rate of the SCN neurons at E22 (Shibata ness. Starting at expected lights-on, a single mother and Moore, 1987). Although all the above-mentioned was decapitated every 2 h during the circadian cycle, studies indicate the presence of overt rhythms in the and 4 fetuses for expression of 3 clock genes and SCN of rat fetuses, our recent work did not reveal 4 fetuses for expression of another 2 clock genes and detectable rhythms in the expression of clock genes an AVP gene per each time point were sampled. Day and of their products at E19. However, the rhythms in of delivery was designated the postnatal day 0 (P0). Per1, Per2, Cry1, and Bmal1, but not in Clock mRNA, Pups born during the night were sampled during the were expressed at P3 in the SCN (Sládek et al., 2004). next night (P1) or 2 nights later (P2); pups born dur- The aim of the present study was to map the inter- ing the day were sampled during the following day val between E19 and P3, that is, to elucidate when (P1) or 2 days later (P2). On the day of sampling, during the late embryonic and early postnatal stages mothers with their pups were released into constant the expression of various clock genes in the SCN darkness; that is, the morning light was not turned began to be rhythmic. Daily profiles of Per1, Per2, on. At P1, 4 pups for clock gene expression and Cry1, Bmal1, and Clock mRNA were determined in 4 pups for AVP gene expression from 1 litter and at the SCN at E20, P1, and P2. Moreover, to compare P2, only 4 pups for clock gene expression from 1 litter development of core clock gene expression with that per each time point were sampled in darkness every of clock-controlled gene transcription, the SCN daily 2 h throughout the whole circadian cycle. The times profile of AVP heteronuclear (hn) RNA was studied for expected lights-on and lights-off were designated both at E20 and P1. The AVP hnRNA as an indicator CT0 and CT12, respectively. 142 JOURNAL OF BIOLOGICAL RHYTHMS / April 2006

Fetuses and pups were killed by rapid decapitation. et al., 2004). The sections were hybridized for 20 h at The brains were removed, immediately frozen on dry 60 °C (Per1, Per2, Clock, and AVP), 58 °C (Bmal1), and ice, and stored at –80 °C. Pup brains were sectioned 55 °C (Cry1). Following a posthybridization wash, into 5 and/or 1 series (at P1) and 5 series (at P2) of the sections were dehydrated in ethanol and air dried. 12-µm-thick slices for each brain in alternating order Finally, the slides were exposed to film BIOMAX MR throughout the whole rostrocaudal extent of the SCN (Kodak) for 8 to 10 days and developed using the and processed for in situ hybridization to determine developer Fomatol LQN and fixer FOMAFIX (FOMA, levels of Per1, Per2, Cry1, Bmal1, and Clock mRNAs Hradec Králové, Czech Republic). As a control, in situ and AVP hnRNA. The fetal brains were cut into hybridization was performed in parallel with sense 3 series of 12-mm-thick slices for each brain and probes (apart from Per2 and AVP) on sections con- processed for in situ hybridization as well to deter- taining the SCN. For each age, the whole daily profile mine levels of Per1, Per2, Cry1, Bmal1, and Clock of a clock gene expression was measured using the mRNAs and AVP hnRNA. For all ages, each gene same labeled probe. All sections hybridized with the expression at each time point was determined in 4 of probe were processed simultaneously under identical the brains. conditions. For the determination of AVP gene expres- All experiments were conducted under license no. sion at E20 and P1, the same labeled probe was used A5228-01 with the U.S. National Institutes of Health for both ages. Therefore, only AVP hnRNA profiles at and in accordance with the Animal Protection Law of E20 and P1 were compared by 2-way analysis of vari- the Czech Republic (license no. 1020/491/A/00). ance (ANOVA); in all other profiles for each age, only 1-way ANOVA was used to compare absolute values In Situ Hybridization Histochemistry within the profile. Autoradiographs of sections were analyzed using The cDNA fragments of rat rPer1 (980 bp; corre- an image analysis system (ImagePro, Olympus, New sponds to nucleotides 581-1561 of the sequence in Hyde Park, NY) to detect the relative optical density GenBank accession no. AB002108), rat rPer2 (1512 bp; (OD) of the specific hybridization signal. In each corresponds to nucleotides 369-1881 of the sequence animal, the mRNA or hnRNA level was quantified in GenBank accession no. NM 031678), rat rBmal1 bilaterally, always at the midcaudal SCN section con- (841 bp; identical to nucleotides 257-1098 of the taining the strongest hybridization signal. Each mea- sequence in GenBank accession no. AB012600), rat surement was corrected for a nonspecific background rClock (1158 bp; identical to nucleotides 167-1325 of by subtracting the OD values from the same adjacent the sequence in GenBank accession no. AB019258), area in the hypothalamus. This area was expected mouse mCry1 (719 bp; corresponds to nucleotides to be free of the specific signal and thus served as an 1074-1793 of the sequence in GenBank accession no. internal standard. The background signal of the area NM 007771), and rat rAVP (506 bp; identical to was consistently low and did not exhibit marked nucleotides 796-1302 of the intronic sequence in changes with age or the time of day. Finally, slides GenBank accession no. X01637) were used as tem- were counterstained with cresyl violet to check the plates for in vitro transcription of complementary presence and the midcaudal position of the SCN in RNA probes. The rPer1, rPer2, and mCry1 fragment- each section. In no case did in situ hybridization containing vectors were generously donated by yield any specific signal using a sense probe. Professor H. Okamura (Kobe University School of Data were expressed as a mean of OD from Medicine, Japan), and rBmal1, rClock, and rAVP 4 animals ± SEM; the OD for each animal was calcu- were cloned in our laboratory. Briefly, cDNA frag- lated as a mean of the left and right SCN values. ments were yielded from the rat hypothalamic total RNA. After reverse transcription, cDNA was amplified Statistical Analysis by standard polymerase chain reaction and ligated into vector pGem-T-Easy and pBluescript, respectively. Data on clock gene mRNA profiles were analyzed For AVP probe, primers designed to amplify intronic by 1-way ANOVA for the time difference. Data on region were used. The cloned inserts were sequenced AVP hnRNA were analyzed by 2-way ANOVA for to verify the amplified products. age and time differences and by 1-way ANOVA for The probes were labeled by using α-[35S]thio-UTP, only time differences. Subsequently, the Student- and the in situ hybridization was performed as Newman-Keuls multiple range test was used, with described previously (Shearman et al., 2000; Sládek p < 0.05 being required for significance. A rhythm Kovácˇiková et al. / SCN CLOCK RHYTHMICITY DURING ONTOGENY 143

was considered when the 1-way ANOVA revealed a the maximum nor the minimum levels thus clustered significant effect of time and at the same time the into separate intervals. The same holds true for the maximum and minimum values clustered into 2 sep- Cry1 (Fig. 2G) and Bmal1 (Fig. 2J) mRNA profiles. arate, roughly out-of-phase time intervals. The inter- The Cry1 mRNA level at CT10 was significantly vals included at least 2 successive maximum and higher than those at CT14, 20, and 24. The Bmal1 minimum values, respectively. mRNA level at CT22 was significantly elevated when compared with those at CT2, 6, and 20 and the level at CT10 was significantly higher than those at CT6 and 20. Hence, at E20 only, forming of an initial RESULTS rhythm in Per1 mRNA expression was indicated. In 1-day-old rats, the 1-way ANOVA also revealed Daily Profiles of Clock Gene mRNAs a significant effect of time for Per1, Per2, and Bmal1 (p < 0.01) (Fig. 2B, E, K) and Cry1 (p < 0.05) (Fig. 2H). Figure 1 shows representative in situ hybridiza- Per1 mRNA levels at CT7, 8, and 9 were significantly tion studies of Per1, Per2, Cry1, and Bmal1 mRNA in elevated as compared with those at CT16, 22, and 23 the SCN of 20-day-old fetuses and 1- and 2-day-old (Fig. 2B). Hence, elevated and also low values clus- rats as well as Clock mRNA in the SCN of 20-day-old tered into 2 separate, roughly out-of-phase time fetuses and 2-day-old rats at CT8 (i.e., during the intervals. Per2 mRNA levels at CT7, 8, 9, and 13 were subjective day) and CT16 (i.e., during the subjective significantly higher than that at CT22 (Fig. 2E). The night). From these and other similar autoradio- maximum level at CT9 was significantly higher than graphs, relative OD levels, that is, relative mRNA levels at CT15, 19, and 22 but not those at CT16, 17, amounts of the above mentioned clock genes, were or 21. Although elevated values fell into 1 interval, estimated. this was not the case for low levels. For the Cry1 In 20-day-old fetuses, the 1-way ANOVA revealed mRNA profile, the post hoc analysis did not reveal a significant effect of time for Per1, Per2, and Bmal1 significant differences among various time points (p < 0.01) (Fig. 2A, 2D, 2J) and Cry1 (p < 0.05) (Fig. 2G) (Fig. 2H). Bmal1 mRNA levels at CT16, 17, 19, 21, 22, but not for Clock (Fig. 2M). Per1 mRNA at CT2 was and 23 were significantly elevated when compared significantly higher than all other values except those with those at CT7, 8, 11, and 13, but they did not dif- at CT0 and CT4 (Fig. 2A). Elevated levels might thus fer from that at CT9 (Fig. 2K). The elevated and also begin to cluster in the CT0-4 interval. Per2 mRNA low values thus clustered into 2 separate time inter- levels at CT4, 8, and 12 were significantly elevated as vals, with the exception of the CT9 value. It appears compared with the value at CT10 (Fig. 2D). Neither that at P1, a slight rhythm in Per1 expression, a pronounced rhythm in Bmal1 expression, and a forming rhythm in Per2 expression were already present. In 2-day-old rats, the 1-way ANOVA revealed a significant effect of time for Per1, Per2, Cry1, and Bmal1 mRNA profiles (p < 0.01) (Fig. 2C, 2F, 2I, 2L) but not for the Clock mRNA profile (Fig. 2N). Per1 mRNA levels at CT2, 4, 6, and 8 were significantly higher than those at CT16, 18, 20, and 22 (Fig. 2C). Hence, the elevated values as well as low ones fell into 2 separate time intervals of almost polar oppo- sites. Per2 mRNA levels at CT6, 8, 10, and 12 were significantly elevated as compared with those at CT18, 20, 22, and 24 (Fig. 2F). The maximum and minimum values clustered into 2 separate, roughly out-of-phase time intervals, as was the case with Per1 mRNA. Cry1 mRNA levels at CT10 and CT14 were Figure 1. Representative autoradiographs of coronal brain sec- significantly higher than the minimum value at CT22 tions at the level of the SCN; Per1, Per2, Cry1, and Bmal1 mRNAs (Fig. 2I). Elevated values might thus start to cluster in 20-day-old fetuses (E20) and in 1-day-old (P1) and 2-day-old (P2) rats, and Clock mRNA at E20 and P2 were examined at CT8 into 1 time interval. Bmal1 mRNA levels at CT16 and or CT16 by in situ hybridization. CT18 were significantly elevated when compared 144 JOURNAL OF BIOLOGICAL RHYTHMS / April 2006

Figure 2. Daily profiles of Per1 (A,B,C), Per2 (D,E,F), Cry1 (G,H,I), Bmal1 (J,K,L), and Clock (M,N) mRNA levels in the SCN of 20-day- old fetuses (E20; A,D,G,J,M) and 1-day-old (P1; B,E,H,K) and 2-day-old (P2; C,F,I,L,N) rats maintained in LD12:12 and released into darkness at the time of the expected DL transition (CT0). The brain sections were assayed for mRNA by in situ hybridization. Data are expressed as mean ± SEM from 4 animals. Per1 mRNA: on E20, the level at CT2 was significantly higher than all other levels (p < 0.05) except those at CT0 and CT4; on P1, levels at CT7, 8, and 9 were higher than those at CT16, 22, and 23 (p < 0.05); on P2, levels at CT2, 4, 6, and 8 were higher than those at CT16, 18, 20, and 22 (p < 0.05). Per2 mRNA: on E20, levels at CT4, 8, and 12 were significantly higher than that at CT10 (p < 0.05); on P1, levels at CT7, 8, 9, and 13 were higher than that at CT22 (p < 0.05); and the maximum level at CT9 was higher than levels at CT15, 19, and 22 (p < 0.01). Cry1 mRNA: on E20, the level at CT10 was significantly higher than those at CT14, 20, and 24 (p < 0.01); on P2, levels at CT10 and CT14 were higher than that at CT22 (p < 0.05). Bmal1 mRNA: on E20, the level at CT22 was higher than those at CT6 and CT20 (p < 0.01); on P1, levels at CT16, 17, 19, 21, and 23 were higher than those at CT7, 8, 11, and 13 (p < 0.01); on P2, levels at CT16 and CT18 were higher than those at CT8 and CT10 (p < 0.05). Kovácˇiková et al. / SCN CLOCK RHYTHMICITY DURING ONTOGENY 145

Figure 3. AVP heteronuclear (hn) RNA levels in 20-day-old fetuses (E20) and in 1-day-old rats (P1) maintained in LD12:12 and sampled in darkness either at CT2 (i.e., during the subjective day) or at CT16 (i.e., during the subjective night). Representative coronal brain sections at the level of the SCN examined by in situ hybridization are depicted. Note that in contrast to the SCN, the signal in supraoptic nuclei does not vary with time.

with those at CT8 and CT10 (Fig. 2L). Elevated levels and the low ones thus clustered into 2 separate time intervals. The Clock mRNA profile did not show any rhythm whatsoever. It appears that at P2, rhythms in Figure 4. Daily profiles of AVP heteronuclear (hn) RNA in the Per1, Per2, and Bmal1 expression were fully present, SCN of 20-day-old fetuses (A) and in 1-day-old (P1) rats (B). Rats were maintained in LD12:12 and released into darkness at CT0. while the rhythm in Cry1 expression might only start The brain sections were assayed for hnRNA by in situ hybridiza- to form. tion. Data are expressed as mean ± SEM from 4 animals. For P1, AVP hnRNA levels at CT2 and CT22 were significantly higher than those at CT10, 12, 14, 16, 18, and 20 (p < 0.05). Daily Profiles of AVP hnRNA

Figure 3 shows representative in situ hybridiza- hnRNA levels might thus fall in the CT0-8 and again tion studies of AVP hnRNA in the SCN of 20-day-old in the CT22-24 interval, whereas low levels might fetuses and 1-day-old rats at CT2 during the subjec- fall in the CT10-20 interval. Apparently, in 1-day-old tive day and at CT16 during the subjective night. rats, a circadian rhythm in AVP hnRNA expression From these and other similar autoradiographs, rela- was present. tive OD levels, that is, relative AVP hnRNA amounts, were estimated. The daily profile of AVP hnRNA of 20-day-old DISCUSSION fetuses (Fig. 4A) was compared with that of 1-day- old rats (Fig. 4B). The 2-way ANOVA revealed a sig- In agreement with our previous study (Sládek nificant effect of age (F = 172.1, p < 0.01), effect of et al., 2004), the rhythmic expression of canonical time (F = 6.4, p < 0.01), as well as a significant inter- core clock genes in the rat SCN could be clearly action effect (F = 4.1, p < 0.01). The AVP hnRNA pro- detected only after birth. In 20-day-old fetuses, Per1 file of 20-day-old fetuses thus differed significantly expression was still as low as that in 19-day-old from that of 1-day-old rats. In 20-day-old fetuses, the fetuses, but a rhythm of Per1 mRNA might start to 1-way ANOVA did not reveal a significant effect of form. In 1-day-old rats, the Per1 mRNA rhythm was time, and hence no circadian rhythm in AVP hnRNA clearly discernible, with higher levels clustered in the expression was detected. In 1-day-old rats, the effect CT7-9 interval. However, only in 2-day-old rats was of time was by now highly significant (p < 0.01). The a more robust rhythm in Per1 expression, similar in AVP hnRNA levels at CT2 and again at CT22 were waveform and phase to those in 3- and 10-day-old significantly higher than those at CT10, 12, 14, 16, 18, rats (Sládek et al., 2004) already present. At P2, analo- and 20, while levels at CT0, 4, 6, 8, and 24 did not gously as at P3 and P10, maximum values fell in the differ significantly from that at CT22. Elevated AVP daytime CT2-8 interval. Similarly, in the adult mouse 146 JOURNAL OF BIOLOGICAL RHYTHMS / April 2006

SCN, elevated mRNA levels clustered in the CT3-12 increasing the rhythm maximum, decreasing the interval (Reppert and Weaver, 2001). No rhythmic minimum, or both. Also, in the fetal SCN of Syrian Per2 expression could be detected in 20-day-old hamsters, molecular oscillations equivalent to those fetuses. In 1-day-old rats, formation of a rhythm was observed in adults were not detected (Li and Davis, already indicated, with elevated Per2 mRNA levels 2005). However, Ohta and colleagues (2002, 2003) falling in the CT7-13 interval. In 2-day-old rats, a reported on rhythms in Per1 and Per2 expression in more robust Per2 mRNA rhythm was present, similar the fetal rat SCN. The above-mentioned authors sam- to those in 3- and 10-day-old rats (Sládek et al., 2004). pled 20-day-old fetuses at 4-h intervals and found At P2 and P3, high levels fell in the CT6-12 interval, peak levels of Per1 mRNA at ZT8 and Per2 mRNA and at P10, they fell in the CT6-14 interval. at ZT12 and ZT16. In our more densely sampled Analogously, in adult mice, high levels were clus- 20-day-old fetuses, only a forming rhythm in Per1 tered in the CT5-12 interval (Reppert and Weaver, expression with an indicated peak at CT2 (but no 2001). No rhythms in Cry1 expression could be detected rhythm in Per2 expression) was present. Since Ohta and in 20-day-old fetuses or 1-day-old rats. In 2-day- colleagues (2002, 2003) presented only relative values old rats, formation of a rhythm was indicated, with pertaining to the maximum expression, a detailed elevated levels clustered in the CT10-14 interval. In comparison of their data with the present study is not 3- and 10-day-old rats (Sládek et al., 2004) and in possible. The discrepancy between both data might adult mice (Reppert and Weaver, 2001), high Cry1 possibly be explained by how pregnant rats were mRNA levels fell in CT8-12, CT8-16, and CT8-14 maintained. In our study, rats were released into con- intervals, respectively. Similarly as at E19 (Sládek stant darkness before sampling, whereas in the Ohta et al., 2004), expression of Bmal1 was high at E20, but and colleagues’ studies, rats were killed during no rhythm could be detected. In 1- and 2-day-old an LD cycle. Although the fetal clock is believed to rats, however, a clear Bmal1 mRNA rhythm was pre- be entrained by a maternal cue and probably does sent, with maximum values falling at night to not use an LD cycle for synchronization (Davis and CT16-23 and CT16-18 intervals, respectively. In 3- and Mannion, 1988; Reppert and Weaver, 1991; Weaver 10-day-old rats (Sládek et al., 2004) and in adult mice and Reppert, 1995), the direct effect of light on the (Reppert and Weaver, 2001), maximum values were developing fetal circadian clock cannot be excluded. clustered in CT14-20, CT14-22, and CT12-21 intervals, Biologically relevant light wavelengths may reach the respectively (i.e., always during the night hours). fetus in the uterus and influence its development Expression of Clock did not reveal any rhythm in (Jacques et al., 1987). 20-day-old fetuses or 2-day-old rats, as was the case No rhythm of the clock-controlled expression of in 3- and 10-day-old and adult rats. At all ages, Clock the AVP gene could be detected in 20-day-old fetuses. appeared to be expressed constitutively rather than However, a significant rhythm of AVP hnRNA with in a cyclical manner. Nevertheless, when adult pronounced amplitude appeared in the SCN of rats are maintained under a short photoperiod, Clock 1-day-old rats. Elevated levels fell in the CT0-8 interval expression may also become rhythmic (Sumová and again in the CT22-24 interval and low levels et al., 2003). in the CT10-20 interval. In adult rats, AVP hnRNA The data indicate that since the 1st appearance of levels start to rise after CT21, are elevated until CT9, rhythms in clock gene expression in the rat SCN, the and become undetectable at CT13 and CT17; the AVP phase of the rhythms is roughly in agreement with mRNA rhythm is phase delayed by about 4 h, as that of 10-day-old rats (Sládek et al., 2004) and of compared with the AVP hnRNA rhythm (Yambe adult mice (Reppert and Weaver, 2001). The phase et al., 2002). It seems that since the 1st appearance might rather be set by the mother than by an LD of clock-controlled AVP gene expression, the phase cycle. Although a newborn rat can perceive light and of the rhythm has been roughly in phase with that respond to a photic stimulus by induction of the c-fos in adult rats, similarly as has been the case with gene in the SCN, it does not yet exhibit any rhythm rhythmic clock gene expression. Our data indicate in the SCN photosensitivity (Bendová et al., 2004). that the rhythm of AVP gene expression in the rat The data also confirm our previous finding that SCN develops only after E20. Reppert and Uhl (1987) rhythms in canonical clock gene expression in the rat found a significant difference between a daytime SCN emerge mostly post- and not prenatally (Sládek AVP mRNA level at CT5 and a nighttime level at et al., 2004). Maturation of the rhythms may proceed CT17 already in the SCN of 21-day-old fetuses. In our by increasing the rhythms’ amplitude, either by study, we used in situ hybridization with an intronic Kovácˇiková et al. / SCN CLOCK RHYTHMICITY DURING ONTOGENY 147

probe and thus detected hnRNA, that is, nascent article, and Professor Hitoshi Okamura (Kobe AVP transcript and hence AVP gene transcription University School of Medicine, Japan) for his generous (Sherman et al., 1986; Yambe et al., 2002). The diurnal gift of the plasmid templates used for the synthesis changes of AVP mRNA levels in the SCN may be of rPer1, rPer2, and mCry1 riboprobes. Our work is mostly regulated by transcriptional activities (Carter supported by the Grant Agency of the Czech Republic, and Murphy, 1992) but also by mRNA stability asso- Grant Nos. 309021241 and 309050350, Research Project ciated with changes in the length of the poly-A-tail Nos. LC554 and AV0Z 50110509, and by the EU (Robinson et al., 1988; Carter and Murphy, 1989). 6th Framework Project EUCLOCK No. 018741. Therefore, it cannot be excluded that mRNA degra- dation might contribute to the finding of diurnal AVP REFERENCES mRNA changes in 21-day-old fetuses. It is also possible that a circadian variation in AVP Bendová Z, Sumová A, and Illnerová H (2004) expression might start as early as during the very late Development of circadian rhythmicity and photoperi- prenatal period. From a comparison of the profiles of odic response in subdivisions of the rat suprachiasmatic clock gene expression with the profile of AVP gene nucleus. Brain Res 148:105-112. expression at P1, it seems that the rhythms of AVP Carter DA and Murphy D (1989) Independent regulation of neuropeptide mRNA level and poly(A) tail length. J Biol hnRNA and Bmal1 mRNA were the most pronounced Chem 264:6601-6603. in 1-day-old rats. It is tempting to speculate that Carter DA and Murphy D (1992) Nuclear mechanisms the rhythm in Bmal1 expression, which peaked during mediate rhythmic changes in vasopressin mRNA expres- the subjective night, might be the driving force for sion in the rat suprachiasmatic nucleus. Mol Brain Res the rhythm in AVP gene expression. However, it also 12:315-321. Davis FC and Mannion J (1988) Entrainment of hamster cannot be excluded that some maternal cues might pup circadian rhythms by prenatal melatonin injections trigger the rhythm of AVP expression in the very late to the mother. Am J Physiol 255:R439-R448. prenatal stage. The rhythms of AVP gene expression Fu L and Lee CC (2003) The circadian clock: pacemaker and and AVP production (Jácˇ et al., 2000; Sumová et al., tumour suppressor. Nat Rev Cancer 3:350-361. 2000) in the adult rat SCN are present, similar to the Gillette MU (1991) SCN electrophysiology in vitro: rhyth- rhythm of endogenous c-Fos production (Sumová mic activity and endogenous clock properties. In Suprachiasmatic Nucleus: The Mind’s Clock, Klein DC, et al., 1998), in the dorsomedial (dm) but not in the Moore RY, and Reppert SM, eds, pp 125-143, New York, ventrolateral (vl) subdivision of the SCN. And during Oxford University Press. postnatal development, it is just the endogenous Jácˇ M, Kiss A, Sumová A, Illnerová H, and Jezˇová D (2000) rhythm in c-Fos production, a marker of the dm-SCN Daily profiles of arginine vasopressin mRNA in the rhythmicity, which develops earlier postnatally than suprachiasmatic, supraoptic and paraventricular nuclei of the rat hypothalamus under various photoperiods. the vl-SCN rhythm in c-Fos photoinduction (Bendová Brain Res 887:472-476. et al., 2004). Jacques SL, Weaver DR, and Reppert SM (1987) Penetration In conclusion, our data indicate that the detectable of light into the uterus of pregnant animals. Photochem rhythmic expression of clock genes in the rat SCN Photobiol 45:637-641. starts mostly after birth. We cannot, however, Jin X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ, and exclude the possibility of oscillations in clock gene Reppert SM (1999) A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian expression in a relatively small number of cells in the clock. Cell 96:57-68. fetal SCN. The appearance of a pronounced rhythm King DP and Takahashi JS (2000) Molecular genetics of in the AVP hnRNA already in the SCN of 1-day-old circadian rhythms in mammals. Annu Rev Neurosci rats suggests that other mechanisms besides rhyth- 23:713-742. mic clock gene expression might contribute to gener- Klein DC, Moore RY, and Reppert SM, eds (1991) Supra- chiasmatic Nucleus: The Mind’s Clock, New York, Oxford ation of the rhythm in AVP gene transcription during University Press. the early developmental stage. Kornhauser JM, Mayo KE, and Takahashi JS (1993) Immediate-early gene expression in a mammalian circadian pacemaker: the suprachiasmatic nucleus. In ACKNOWLEDGMENTS Molecular Genetics of Biochemical Rhythms, Young MW, ed, pp 271-307, New York, Dekker. Li X and Davis FC (2005) Developmental expression of clock The authors thank Lucie Heppnerová and Eva genes in the Syrian hamster. Dev Brain Res 158:31-40. Suchanová for their excellent technical assistance, Moore RY (1991) Development of the suprachiasmatic Mr. John Novotney for his careful reading of the nucleus. In Suprachiasmatic Nucleus: the Mind’s Clock, 148 JOURNAL OF BIOLOGICAL RHYTHMS / April 2006

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