ogy, Part A 149 (2008) 44–50www.elsevier.com/locate/cbpa
Comparative Biochemistry and Physiol
Ontogeny of melatonin, Per2 and E4bp4 light responsiveness in the chickenembryonic pineal gland
I. Herichová a, J. Monošíková a, M. Zeman a,b,⁎
a Department of Animal Physiology and Ethology, Comenius University Bratislava, Mlynská dolina B2, 842 15 Bratislava, Slovakiab Institute of Animal Biochemistry and Genetics, SASci, 900 28 Ivanka pri Dunaji, Slovakia
Received 17 July 2007; received in revised form 2 October 2007; accepted 2 October 2007Available online 11 October 2007
Biological rhythms in physiological functions are driven bythe circadian system and synchronized to external cycles viaseveral sensory structures. In birds the central part of thecircadian system is composed of the pineal gland, the retina andthe suprachiasmatic nucleus of the hypothalamus (SCN)(Cassone and Menaker, 1984). Unlike in mammals, the avianpineal gland is an autonomous circadian oscillator, is directlyphotosensitive and generates a rhythmic melatonin outputsignal (Takahashi et al., 1989; Zatz et al., 2000). Melatoninplays an important role as a signal mediating information aboutexternal photic conditions to the rest of the body (Gwinner andBrandstätter, 2001).
⁎ Corresponding author. Department of Animal Physiology and Ethology,Comenius University Bratislava, Mlynská dolina B-2, 842 15 Bratislava,Slovakia. Tel.: +421 2 602 96 424; fax: +421 2 654 29 064.
In our previous work we reported that melatonin synthesis bythe pineal gland is rhythmic on embryonic day (ED) 19 and isentrainable by light:dark (LD) or temperature cycles (Zemanet al., 1999, 2004). Expression of the rate limiting enzyme formelatonin synthesis, arylalkylamine N-acetyltransferase, is alsorhythmic in the pineal gland on ED 19 (Herichova et al., 2001).Embryonic development of the avian pineal gland function wasfurther demonstrated by in vitro studies (Lamosova et al., 1995;Csernus et al., 2007).
Molecular circadian oscillations are generated by a complexset of feedback loops formed by clock gene transcripts and theirprotein products (Shearman et al., 2000). In birds, homologousclock genes were cloned (for rev. see Fukada and Okano, 2002)and their functions seem to be similar to those in mammals(Okano et al., 2001).
Chicken Per2 mRNA is expressed rhythmically in the pinealgland on ED 18 when embryos are exposed to a LD cycle(Okabayashi et al., 2003), but levels ofPer2mRNAwere low andarrhythmic when embryos were incubated in constant darkness.These findings raise the question as to whether the pinealcircadian oscillator needs to be stimulated by a synchronizing
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signal during embryonic development in order to becomefunctional.
Previously we showed that a single LD cycle during the firstday post-hatching is sufficient to entrain the avian circadian systemin the chicken, regardless of whether the eggs were previouslyincubated under LD or constant darkness (DD) (Zeman et al.,1999). This finding suggests that a functional embryonicpacemaker can develop in DD. The molecular circadian oscillatorand melatonin synthetic pathways are interconnected in avianpinealocytes (Chong et al., 2000; Okano et al., 2001; Rekasi et al.,2006), therefore the machinery for the molecular pacemakershould develop before or, at a minimum, at the same time asrhythmic melatonin synthesis in the pineal gland.
To elucidate correlations between rhythmic melatoninsynthesis and clock gene expression in the pineal gland wedesigned several experiments focusing on the ontogeny of lightresponsiveness by the pineal gland. We examined the sensitivityof the melatonin signal to light pulses during the subjectivenight and day and compared these responses with the expressionof photoinducible clock genes. To understand the developmen-tal state of the circadian pacemaker, we analyzed the expressionof the photoinducible genes Per2, Bmal1 and E4bp4 in thepineal gland of chick embryo prior to hatching and immediatelypost-hatching under LD and DD conditions.
2. Materials and methods
Hatching eggs of broiler breeders (Gallus gallus domesticus)were incubated in forced draught incubators (BIOS Midi,Sedlčany, Czech Republic) at a temperature of 37.3±0.3 °C andrelative humidity 55–65%. Eggs were turned automaticallyevery two hours. Lighting was provided by an 18 Watt coolwhite fluorescent tube (Osram, Lumilux combi, Germany), withillumination ranging from 40–80 lux at the level of the eggs.The light:dark cycle, light pulses and sampling times are givenseparately for each particular experiment. Two identical forceddraught incubators (BIOS Midi, Sedlčany, Czech Republic)were used in experiments involving different lighting conditionsand light pulses. During the dark-phase, eggs and chicks weretaken from the incubator in complete darkness and decapitationoccurred within 10 s in the neighboring room under very lowintensity red light (15 W, Kodak 1A filter). After decapitationpineal glands were excised, frozen in liquid nitrogen and storedat −80 °C until melatonin measurement or RNA extraction wasperformed. Experimental protocols were approved by theEthical Committee for the Care and Use of Laboratory Animalsat the Comenius University Bratislava, Slovak Republic.
2.1. Experiment 1: Effects of a two hour light pulse on melatoninsynthesis in the pineal glands of 19-day old chick embryos
Incubated eggs (75) were exposed to a 16:8 LD cycle.Sampling was performed in 2–3 h intervals on day 19 ofembryonic development. A single two hour light pulse wasapplied during the dark period at intervals ZT16–ZT18, ZT18–ZT20, ZT20–ZT22 or ZT22–ZT24 (ZT — Zeitgeber time, theonset of the light phase is considered ZT0). Control eggs were
incubated under the original LD regimen (16:8) and maintainedin darkness during the light pulse treatment.
2.2. Experiment 2: Effects of a three hour light pulse on pinealmelatonin synthesis in the pineal glands of 19-day old chickembryos and hatchlings
Hatching eggs (51) were incubated under a 12:12 LD cycle.On ED 19 embryos were exposed to constant darkness and asingle three hour light pulse was applied at intervals ZT18–ZT21, ZT21–ZT24, ZT3–ZT6 or ZT6–ZT9. Control eggs wereincubated under the original LD regimen and during the lightpulse treatment were maintained in the 12:12 photoperiod.
2.3. Experiment 3: Daily rhythms of Per2, Bmal1 and E4bp4expression in the pineal glands of 4-day old chickssynchronized to a LD cycle
Eggs (24) were incubated under a 12:12 LD cycle untilhatching. After hatching, chicks were kept in a brooder roomwith the same LD cycle as in the incubator. The ambienttemperature in the room was kept constant at 37 °C for the first2 days and 34 °C for the next 2 days. Food and water wereavailable ad libitum. Samples were taken over a 24 h cycle in4 h intervals with the first sampling time at ZT14.
2.4. Experiment 4: Daily rhythms of Per2, Bmal1 and E4bp4expression in the pineal glands of 19-day old chick embryossynchronized to a LD cycle or in DD
Eggs (54) were incubated under a 12:12 LD cycle. On ED19, pineal glands were taken at 4 h intervals during thescotophase, with the first sampling at ZT 14. At the end of thescotophase (ZT24) 18 embryos were exposed to the regularlight phase and 18 embryos were incubated in constantdarkness. Pineal glands from both groups were taken at ZT2,ZT6 and ZT10.
2.5. Experiment 5: Effects of 1 h or 3 h light pulses on clockgene expression in the pineal glands of 19-day old chickembryos
Eggs (42) were incubated under a 12:12 LD cycle and on ED19 were exposed to constant darkness. Three hour light pulseswere applied in the middle of the subjective day (ZT3–ZT6) ormiddle of the subjective night (ZT15–ZT18). One hour pulseswere performed at intervals ZT17–ZT18 or ZT5–ZT6. Sampleswere taken at the end of each light pulse. Control eggs wereincubated under the original LD regimen 12:12 in anotherincubator during the light treatment. Control groupswere sampledin the middle of the dark phase or in the middle of the light phase.
2.6. Experiment 6: Effects of a 1 h or 3 h light pulse on clockgene expression in the pineal glands of 4-day old chicks
Eggs (32) were incubated in a 12:12 LD cycle until hatching.After hatching, chicks were kept in a brooder room under the
Fig. 1. Effects of 2 h light pulses during the dark phase on pineal melatoninconcentrations in 19-day old chick embryos synchronized to LD 16:8. Samplingtime is expressed as Zeitgeber Time (ZT) and the onset of light phase isconsidered ZT0. The solid line displays the 24 h profile of pineal melatoninlevels, gray bars represent melatonin concentrations after 2 h light pulses endingat the indicated ZT time. The black bar on the bottom of the graph indicates thetime of darkness. Results are given as mean±S.E.M. (n=4–6 per group).
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same 12:12 LD cycle. Ambient temperature was kept at 37 °Cfor the first 2 days and 34 °C for the next 2 days with food andwater available ad libitum. One or three hour light pulses wereapplied at either ZT17 or ZT15, respectively. Samples weretaken at the end of the light pulses. Controls of the same age
Fig. 2. Effects of 3 h light pulses on pineal melatonin levels in 19-day old chick embrtime of sampling is expressed in Zeitgeber Time (ZT), the onset of light phase is conZT24) or subjective day (ZT6 and ZT9). White columns represent concentrations aftethe graph indicate the time of darkness. Results are given as mean±S.E.M. (n=4–6
were kept under the same LD regimen, but during the lighttreatment were exposed to darkness and sampled in the middleof the dark phase.
2.7. Gene expression
Total RNAwas isolated with use of Tri reagent® (MRC, USA)and RNA integrity was examined by agarose gel electrophoresis.All RNA samples were reverse transcribed into cDNA simulta-neously. First-strand cDNA synthesis was carried out using theImProm-II™Reverse Transcription System and random hexamerprimers (0.5 μg per reaction) according to manufacturerinstructions (Promega, USA) using denatured total RNA (1 μg)as a template.
Aliquots of cDNA (0.5–2μLofRT product)were analyzed forgene expression with appropriate primers in 20-μL real time PCRreactions. Primers for the PCR analysis were as follows: for s17(X07257) forward primer 5′-ACACCCGTCTGGGCAACGAC-3′, reverse primer 5′-CCCGCTGGATGCGCTTCATC-3′ (Dragonet al., 2002); for Per2 (AF246956) forward primer 5′-ACC-TAAAGGAAGGCCTGGTG-3′, reverse primer 5′-CGC-TGA-GTAGCTTGCTTGTG-3′; for E4bp4 (AF335427) forwardprimer 5′-TGCCACACAGAAATTGTCATC-3′, reverse primer5′-CAACTCCAGTTTTGCAACCA-3′; for Bmal1 (AF246957)forward primer 5′-ATGGAAGACTGGACTACGCAGAC-3′,reverse primer 5′-ATGCTGGACTGCCATTCTCAATAC-3′.Quantification of cDNA was performed using a QuantiTectSYBR Green PCR Kit (QIAGEN, Germany) and an ABIPRISM®7900HT Sequence Detection System (Applied Biosys-tems, USA). Real time PCR conditions were: 95 °C, 15 min
yos (A) and hatchlings (B). Chick embryos were synchronized to LD 12:12. Thesidered ZT0. Gray columns represent control values during darkness (ZT21 andr a single 3 h light pulse ending at the indicated ZT. Black bars on the bottom ofper group). ⁎Pb0.05 t-test.
Fig. 3. Daily profiles of Per2, Bmal1 and E4bp4 expression in the pineal glandsof 4-day old chicks synchronized to LD 12:12. Samples were taken over the 24 hcycle in 4 h intervals. The time of sampling is expressed in Zeitgeber Time (ZT),the onset of light phase is considered ZT0. Black bar on the bottom of the graphshows the duration of darkness. Results are given as mean±S.E.M. (n=4 pergroup).
Table 1Acrophases of best fitted cosine curves and cosinor analysis
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followed by 50 cycles of 94 °C, 15 s; 49 °C, 30 s and 72 °C, 30 s.Expression of Per2, E4bp4 and Bmal1 were normalized relativeto ribosomal protein S17 mRNA expression.
2.8. Melatonin radioimmunoassay
Pineal melatonin was measured following methanol extrac-tion. Melatonin radioimmunoassay (Fraser et al., 1983) wasvalidated for chick embryo pineals in our laboratory (Zeman etal., 1999). We used [3H]-labeled melatonin with a specificactivity 3.056 TBq/mmol (NEN Du Pont, Germany) and sheepmelatonin antiserum (G/S/704 8483 Stockgrand Ltd., Univer-sity of Surrey, U.K.). Intra-and interassay coefficients ofvariation were 8.1 and 10.7%, respectively and the detectionlimit of the assay was 1.4 pg/tube.
2.9. Statistics
Daily profiles were fitted to a cosinor curve with a 24 hperiod. When experimental data significantly matched thecosinor curve, its parameters were calculated with 95%confidence limits: mesor (the time series mean), amplitude(one-half the peak-trough difference expressed herein relative tothe mesor) and acrophase (peak time referenced to the time oflights on in the animal facility). Since we determined geneexpression in relative units, only acrophase is determined for therhythmic patterns of Per2, Bmal1 and E4bp4 expression.Goodness of fit (R-value) of the approximated curves wereestimated by ANOVA (Nelson et al., 1979; Klemfuss andClopton, 1993). Data within one time point were compared byan unpaired Student's t-test.
3. Results
We tested the effects of 2 h light pulses on melatonin levelsin the pineal glands of 19-day old embryos incubated under 16:8LD conditions. Pineal melatonin levels in the embryonic chickshowed a distinct daily rhythm (cosinor, Pb0.01). Exposure tolight did not decrease melatonin content in the pineal gland atthis stage of development (Fig. 1). The only time when atendency for decreased pineal melatonin concentrations wasobserved was at the end of the dark phase.
To further address the question of whether the pineal gland ismore sensitive to light at the end of the dark phase, we designedan experiment in which three hour light pulses were appliedduring the transition from the darkness to light. We observedthat light pulses were effective at reducing pineal melatonin in19-day old embryos when applied at ZT 21–24, and during thesubjective light phase when embryos are held in constantdarkness, but not at ZT 18–21 (Fig. 2A). The results differconsiderably from those found in hatchlings (Fig. 2B). In thelater case, light pulses were effective at reducing pinealmelatonin levels throughout the dark phase and during thesubjective light phase. Nighttime melatonin levels in the pinealmelatonin rhythm were two times higher in hatchlings than in19-day old chick embryos.
To correlate the effects of light pulses on melatonin synthesiswith clock gene expression, we measured the daily profiles ofPer2, Bmal1 and E4bp4 expression in the pineal glands of 4-day old chicks and 19-day old chick embryos. Expression ofPer2 mRNA was highest during the early daytime, whileexpression of E4bp4 and Bmal1 mRNA was in antiphase withPer2 in 4-day old chicks (Fig. 3). The daily rhythm of E4bp4expression was advanced by approximately 2.75 h in relation toBmal1 expression. Detailed data on the respective rhythmparameters are given in Table 1.
Daily profiles of Per2, Bmal1 and E4bp4 expression inembryonic pineal glands were measured over a 24 h cycleduring ED 19 under both LD and DD conditions (Fig. 4, Table 1).Expression of Per2 was rhythmic under both LD and DDconditions, with the amplitude being substantially lower underDD than LD. Bmal1 did not express a rhythmic pattern ofexpression on ED 19, and the profile of its expression did notdiffer between LD and DD. Expression of E4bp4 was rhythmicunder LD conditions, but cosinor analysis did not reveal arhythmic pattern in constant darkness. We observed a slightdecrease in E4bp4 expression during subjective day in
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comparison with subjective night, but the trend was notsignificant.
Effects of light pulses during the dark phase and subjectivelight phase were studied. Both one and three hour light pulses,applied in the middle of the subjective day increased Per2expression in the pineal glands of 19-day old embryos (Fig. 5).No changes were observed in E4bp4 expression after 1 h or 3 hlight pulses in 19-day old embryos. Light pulses applied in themiddle of the dark phase did not influence the expression ofclock genes (Per2, E4bp4) in the pineal gland of 19-day oldchick embryos (Fig. 6). In 4-day old chicks, light pulses causeda significant increase in both Per2 and E4bp4 expression(Fig. 6, Table 2).
4. Discussion
Our previous work demonstrated that the circadian oscillatorin the chick pineal is functional at the end of embryonicdevelopment and is able to entrain to light:dark cycles andtemperature cycles (Zeman et al., 1999, 2004). The rhythm ofmelatonin synthesis in the embryonic pineal gland persists inconstant darkness under in vivo conditions (Zeman et al., 1999)and in vitro conditions (Lamosova et al., 1995).
In the present study, we show the persistence of rhythmicPer2 expression in constant darkness in 19-day old chickembryos previously synchronized to LD cycle (Fig. 4), a stagewhere expression of Per2 is not rhythmic in the heart of chickembryo (Herichova et al., 2006), implying that the circadianoscillator in the pineal gland develops earlier than it does inperipheral tissues. Expression of E4bp4 and Bmal1 mRNA inthe pineal gland was not rhythmic in constant darkness. Thepossible reason why the pineal oscillator is still able to drivemelatonin rhythm can rise from a redundancy within thecircadian system and the fact that the functional analog ofBmal1 can play a role as a positive regulating factor in themolecular circadian feedback loop. Post-hatching, Per2, E4bp4and Bmal1 show pronounced daily rhythms with theirrespective expected acrophases (Chong et al., 2000, 2003;Doi et al., 2001; Okano et al., 2001).
Our results demonstrate that light pulses have greater effectson melatonin production and clock gene expression during thesubjective day than during the subjective night in chickembryos. A two hour light pulse was unable to suppressmelatonin synthesis in the pineal gland of 19-day old embryos(Fig. 1) and a three hour light pulse was effective only at the endof the dark phase (Fig. 2). Post-hatching, melatonin synthesis in
Fig. 4. Daily rhythms of Per2 (A), Bmal1 (B) and E4bp4 (C) expression in thepineal gland of 19-day old chick embryos synchronized to a 12:12 LD cycle(solid line) and after release into constant darkness (dashed line). Eggs wereincubated under LD 12:12 and during ED 19 half of the eggs were placed indarkness. Samples were taken over the 24 h cycle in 4 h intervals, time isexpressed as Zeitgeber Time (ZT) and the onset of light phase is considered ZT0.The black bars on the bottom of the graph represent dark phase, and the whitebar light phase. Results are given as mean±S.E.M. (n=6 per group). ⁎⁎Pb0.01t-test.
Fig. 5. Effects of one (dark gray columns) and three (pale gray columns) hourlight pulses during the subjective day on clock gene expression in the pinealgland of 19-day old chick embryos previously synchronized to LD 12:12. At theend of darkness, ED 19 chick embryos (n=6) were exposed to either theregularly scheduled light phase (L) or incubated in the dark (n=18). Time isexpressed in Zeitgeber Time (ZT) and the onset of light phase is considered ZT0.Embryos incubated in the dark were subsequently exposed to either a three hour(3 h) light pulse (from ZT3 to ZT6, n=6), a one hour (1 h) pulse (from ZT5 toZT6, n=6), or no light pulse (n=6, SL). Black columns represent values duringthe subjective day, white columns during the objective light phase. Results aregiven as mean±S.E.M. Asterisks indicate significant increases in mRNA levelsafter a light pulse. (⁎Pb0.05, t-test).
Table 2Effectiveness of light pulses applied during the subjective day (SL) andsubjective dark (SD) on Per2 and E4bp4 mRNA expression and melatoninsynthesis in the avian pineal gland
Per2 E4bp4 melatonin
SL SD SL SD SL SD
Embryos x – – – x –Hatchling x a x x b x x x
– non-effective, x effective.a Yoshimura et al. (2000).b Doi et al. (2001).
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the pineal gland is sensitive to light pulses and pineal andplasma concentrations decrease immediately after light expo-sure (Binkley, 1988).
Fig. 6. Effects of one (dark gray columns) and three (pale gray columns) hour light puof 19-day old chick embryos (A, B) and 4-day old chicks (C, D). Chicks and embZeitgeber Time (ZT) and the onset of light phase is considered ZT0. Three hour lighZT17 to ZT18. Black columns correspond to the dark phase. Results are given as mealevels after light pulses. (⁎Pb0.05, t-test).
Photosensitivity of Per2 expression in the pineal of 19-dayold chick embryos was phase dependent in a similar way likemelatonin synthesis. One and three hour light pulses increasedPer2 expression during the subjective day, but were ineffectiveduring the subjective night (Figs. 5 and 6). Expression of apreviously reported light inducible gene, E4bp4, was found tobe unresponsive to light on ED19. This finding demonstratesthat photoinduction of Per2 and E4bp4 does not share the samemechanism of activation. After hatching, both genes areresponsive to light pulses throughout the 24 h cycle (Doi et al.,2001; Yoshimura et al., 2000).
Melatonin synthesis as well as Per2 expression is lightsensitive during the phase of day when light is expected.Photosensitivity during the subjective light phase and theinverse effects of light on melatonin and Per2 expression can
lses applied during the dark phase on clock gene expression in the pineal glandsryos were previously synchronized to a 12:12 LD cycle. Time is expressed int pulses were applied from ZT15 to ZT18 and one hour pulses were given fromn±S.E.M. (n=4–6 per group). Asterisks indicate a significant increase in mRNA
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arise from the functional relationship between melatoninsynthesis and Per2 expression, however the exact mechanismis presently unclear. Melatonin synthesis in the pineal gland isregulated by an autonomous pacemaker and by sympatheticinnervation from the SCN (Cassone and Menaker, 1984). Inbirds, melatonin synthesis is inhibited during the day bynorepinephrine and stimulated during the dark phase via cAMP(Zatz et al., 2000). Light exhibits direct and synchronizingeffects on melatonin synthesis, and these are based upon theeffects on mRNA transcription, posttranscriptional modificationand proteosomal proteolysis of arylalkylamine N-acetyltrans-ferase (Zatz and Mullen, 1988; Bernard et al., 1997; Iuvoneet al., 2002). All of the aforementioned regulatory stepscontribute to of the ultimate response of the embryonic pinealgland to light.
Per2 is the only gene studied until now that shows rhythmicexpression under LD in an avian embryo (Okabayashi et al.,2003). Our study demonstrates that rhythmic expression of Per2persists in DD by ED 19 and the amplitude of Per2 mRNAexpression is lower in DD in comparison with LD. Damping wasmore pronounced in chick embryos compared with two weeksold chick (Doi et al., 2001). These findings suggest that rhythmicPer2 expression and possibly a rhythmic circadian oscillatoritself is more dependent on the presence of synchronizing lightcues at this stage of development than postnatally.
Our data suggests that components of the avian pinealpacemaker became functional in a stepwise fashion. Earlydevelopment of the avian circadian systemmay play an importantdevelopmental role, particularly during the first days afterhatching by providing internal synchronizing cues for thedeveloping embryo.
Acknowledgements
This study was supported by a grants APVV-20-022704,VEGA 1/4328/07 and VEGA 1/4343/07. We thank Dr. Paul A.Bartell for critical reading.
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