The circadian clock is entrained to the diurnal alteration of environmental conditions such as light and temperature, but the molecular mechanism underlying the entrainment is not fully understood. In the present study, we employed a differential display-based screening for a set of genes that are induced by light in the chick pineal gland, a structure of the central clock entrainable to both light and temperature changes. We found that the level of the mRNA encoding chicken heat shock protein 90 α (cHSP90α) was rapidly elevated in the pineal gland within a 5-min exposure of chicks to light. Furthermore, the pineal cHsp90α mRNA was expressed rhythmically under both 12-hr light/12-hr dark (LD) cycles and constant dark (DD) conditions. The total amount of the pineal cHSP90α protein was, however, kept at nearly constant levels under LD cycles, and immunohistochemical analyses of the pineal cHSP90α showed invariable localization at the cytoplasm throughout the day. In vivo measurement of the chick pineal temperature demonstrated its light-dependent and time-of-day-dependent change, and the profile was very similar to that of the pineal cHsp90α mRNA level. These observations suggest that the in vivo temperature change regulates the expression of temperature-responsive genes including cHsp90α in the pineal gland. The temperature change may induce a phase-shift of the pineal clock, thereby facilitating its efficient entrainment to environmental LD cycles.
Daily rhythms in behavior and physiology are observed in a variety of orga nisms from bacteria to humans, and many of them are driven by endogenous oscillators called circadian clocks (Pittendrigh, 1993). The circadian clocks oscillate even in the absence of an external time cue (zeitgeber) with their period lengths slightly different from 24 hr. The circadian clocks are synchronized (entrained) to the environmental 24-hr cycles by several zeitgebers such as light and temperature. As a general feature of the entrainment, light and heat stimuli give a phase-dependent phase-shifting effect on the clock (Zimmerman et al., 1968; Pittendrigh, 1981; Francis and Sargent, 1979; Nakashima, 1987; Takahashi et al., 1989; Edery et al., 1994; Barrett and Takahashi, 1995; Ruby et al., 1999). It is conceivable that the clock systems have evolved so that the clock phase responds similarly to changes in light and temperature, because they are tightly linked to each other in nature. The circadian oscillation is cell-autonomous (Welsh et al., 1995; Nakahara et al., 1997), and the oscillatory mechanism within each cell is based on an autoregulatory feedback loop in which the products of clock genes inhibit their own transcription (Cermakian and Sassone-Corsi, 2000; Allada et al., 2001; Loros and Dunlap, 2001; Ripperger and Schibler, 2001; Williams and Sehgal, 2001; Reppert and Weaver, 2001). In the case of mice, essential components for the circadian oscillation in the central clock reside in individual neurons of the suprachiasmatic nucleus (SCN), where the products of clock genes (mPer1, mPer2, mPer3, mClock, mBmal1, mBmal2, mCry1, mCry2, mCKIε) seem to form interacting negative feedback loops (Shearman et al., 2000). Recent studies on clock systems in non-mammals such as chicken (Fukada and Okano, 2002), frog (Zhuang et al., 2000) and zebrafish (Cermakian et al., 2000) support the idea that the oscillatory mechanisms are conserved among vertebrates.
In contrast to the accumulating knowledge of the oscillatory mechanism, less understood is the input pathway that entrains the circadian clock to environmental cycles of light or temperature. Among several clock-containing tissues of vertebrates, the chick pineal gland has been used as a prominent model for studies of the entrainment mechanisms (Takahashi et al., 1989). This is because the single pineal cell not only retains the clock oscillator but also is equipped with the input pathway for the entrainment to light and temperature cycles (Zatz et al., 1994; Barrett and Takahashi, 1995; Nakahara et al., 1997). In addition, recent molecular analyses of chick pineal clock genes (cPer2, cPer3, cClock, cBmal1, cBmal2, cCry1, cCry2) suggest that the chick pineal and the mouse SCN clock share a similar entrainment mechanism, in which phase-shifts of the clock are closely associated with changes (rise and fall) in the mRNA levels of Per genes (Maywood et al., 1999; Horikawa et al., 2000; Doi et al., 2001; Okano et al., 2001; Yamamoto et al., 2001; Pando and Sassone-Corsi, 2001). Thus, the chick pineal gland represents a good model system to explore the entrainment mechanisms conserved among vertebrate circadian clocks.
In order to find out the cellular pathways for the entrainment of the clock, it is a fruitful approach to investigate a set of genes that are induced by light or temperature change. In the present study, we aimed at extensive screening for light-induced genes in the chick pineal gland. To this end, the pineal glands were isolated from chicks that were exposed to a light stimulus at various circadian time (CT) points, and the pineal RNA preparations were subjected to differential display analyses. We found the light-dependent increase in the mRNA level of the pineal cHsp90α gene, which is known to be induced by a heat stimulus (Wolfe and Zatz, 1994). In vivo measurement of the chick pineal temperature revealed a light-dependent increase in the temperature, and its profile was similar to that of the cHsp90α mRNA level. Implications of the in vivo temperature change for the photic-entrainment of the pineal clock are discussed.
MATERIALS AND METHODS
Animals were treated in accordance with the guidelines of the University of Tokyo. Newly hatched chicks were purchased from a local supplier, and maintained under various light/dark conditions. The light was provided by fluorescent lamps to give a constant light intensity of ∼300 lux at the level of chicks. The ambient temperature was kept at 28°C±0.5°C irrespective of light/dark conditions. Pineal glands were isolated from decapitated chicks, frozen in liquid nitrogen, and kept at −80°C until subsequent analyses. All of the procedures during the dark-period were performed under dim red light (>640nm).
Differential display-based cloning
Differential display analysis was performed as previously described (Doi et al., 2001). Briefly, total RNA was extracted from each pool of the isolated pineal glands by using guanidine thiocyanate (Takanaka et al., 1998), and it was treated with DNase I (Takara Shuzo, Kyoto, Japan) to digest contaminating genomic DNA. One micro gram of the total RNA was reverse transcribed by using ThermoScript (Invitrogen, CA, USA) with an anchored oligo (dT) primer (H-T11A, H-T11C or H-T11G; RNAimage kit, GenHunter, TN, USA) at 55°C for 60min. One-hundredth of this reaction mixture was subjected to PCR in a reaction mixture (10 μl) composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM each of the dNTPs, 1unit AmpliTaq DNA polymerase (Applied Biosystems, Tokyo, Japan), 1 μM anchored oligo (dT) primer (H-T11A, H-T11C or H-T11G), 1 μM H-AP primer (one of eighty kinds of H-AP primers; RNAimage kit, GenHunter). The amplification reaction was comprised of an initial denaturation step of 94°C for 5 min, and 30 cycles of 94°C for 30 sec, 36°C for 2 min and 72°C for 30 sec, with a final extension step of 72°C for 7 min. The PCR products were subjected to 7.5% native polyacrylamide gel electrophoresis (PAGE), stained with SYBR Green I (Molecular Probes, OR, USA) and the band intensities were quantified by FLA-2000 bioimage analyzer (Fuji Film, Tokyo, Japan). The PCR products selected were cut out from the gel and eluted by boiling in 50 μl of distilled water at 95°C for 5 min. An aliquot (2 μl) of the eluted DNA solution was subjected to the secondary PCR under the same conditions as those in the initial analysis. The reamplified DNA fragment was sub-cloned into pCR2.1 TOPO vector (Invitrogen) and sequenced.
Quantitative reverse transcription (RT)-PCR analysis
Quantitative RT-PCR analyses were performed as previously described (Hirota et al., 2001). Primer pairs used were as follows; for cHsp90α (GenBank accession number; X07265), 5′-GATGA TGAGC AGTAT GCTTG G-3′ (forward) and 5′-TGGTC TTGTT GAGCT CTTCC-3′ (reverse); for chicken TATA-box binding protein (cTbp, GenBank accession number; D83135), 5′-GTCGA ATATA ATCCC AAGCG-3′ (forward) and 5′-TCTGC TCGAA CTTTA GCACC-3′ (reverse). The optimal cycle numbers for quantitative analyses were 17 and 21 for cHsp90α and cTbp, respectively.
Each pool of the isolated pineal glands (n=5) was homogenized with 400 μl of ice-cold 20 mM Tris-HCl buffer (pH 7.4) containing 2.5 mM EDTA, 100 mM NaCl, 1 μg/ml leupeptin, 1 μg/ml aprotinin and 0.1 mM phenylmethylsulfonyl fluoride, and the homogenate was centrifuged at 700 g for 10 min. These manipulations for the pineal glands isolated during the dark period were performed under dim red light (>640 nm). The supernatant contained almost all of the detectable cHSP90α protein expressed in the pineal gland (data not shown). An aliquot of the supernatant was subjected to sodium dodecyl sulfate (SDS)-PAGE (7.5% acrylamide) after boiling for 8 min in SDS-PAGE sample buffer (10 mM Tris-HCl, 6% [v/v] glycerol, 2% [w/v] SDS, 50 mM DTT, 2 mM EDTA, 0.002% [w/v] Coomassie Brilliant Blue, pH 6.8). Proteins in the gel were electro-transferred to a polyvinylidene difluoride membrane (Immobilon transfer membrane; Millipore Corp., MA, USA), which was then incubated for 1hr at 37°C in blocking solution I (10 mM sodium phosphate, 150 mM NaCl, 4% bovine serum albumin, pH 7.4), followed by incubation at 4°C overnight with HSP90α-specific antibody (final concentration, 400 ng/ml; StressGen Biotechnologies, Victoria, Canada) diluted in a reaction buffer (10 mM sodium phosphate, 150 mM NaCl, 0.1% bovine serum albumin, pH 7.4). After washing 3 times for 5 min each with the reaction buffer, the blot was incubated for 1 hr at 37°C with alkaline phosphatase-conjugated anti-rabbit IgG antibody (New England Biolabs, MA, USA) diluted in the reaction buffer (1:2,500 dilution). The immunoreactivities were visualized by using CDP-star detection system (New England Biolabs).
Preparation of tissue sections and immunofluorescence analysis
The isolated pineal glands were fixed with 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 4hr at 4°C. Tissues isolated during the dark period were fixed in complete darkness. The fixed tissues were incubated for cryoprotection in dehydrating solution I (10 mM sodium phosphate, 10% sucrose [w/v], 140mM NaCl, pH 7.2) at 4°C for 4 hr, and subsequently incubated in dehydrating solution II (10 mM sodium phosphate, 30% sucrose [w/v], 140 mM NaCl, pH 7.2) at 4°C overnight. Then, the tissues were embedded in a 2:1 (v/v) mixture of dehydrating solution II and OCT mounting medium (Sakura, Tokyo, Japan), frozen by using liquid nitrogen, and stored at −80°C until use. Finally, 10-μm-thick sections were cut out from the frozen tissues, mounted on gelatin-coated glass slides, and air-dried. The sections on glass slides were pretreated with blocking solution II (10 mM sodium phosphate, 1% bovine serum albumin, 3% horse normal serum, 3% goat normal serum, 0.05% Tween20, 140 mM NaCl, 1 mM MgCl2, pH 7.4) for 30 min at room temperature, and then incubated with a mixture of rabbit anti-HSP90α antibody (final concentration, 1 μg/ml; Stress-Gen Biotechnologies) and mouse anti-pinopsin antibody P9 (1:400 dilution; Okano et al., 1997) diluted in blocking solution II at 4°C overnight. After rinsing with phosphate-buffered saline buffer (10 mM sodium phosphate, 140 mM NaCl, 1 mM MgCl2, pH 7.4), the sections were incubated with a mixture of fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (final concentration, 1.2 μg/ml; Vector Laboratories, CA, USA) and Texas Red-conjugated anti-mouse IgG antibody (final concentration, 6 μg/ml; Vector Laboratories) diluted in blocking solution II at 4°C overnight. The sections were coverslipped with Vectashield mounting medium (Vector Laboratories) containing 4′, 6-diamidino-2-phenylindole (DAPI) for nuclear staining.
In vivo measurement of the local temperature adjacent to the pineal gland
The chick body temperature at a local region adjacent to the pineal gland was measured by using a small sensor tip of thermistor (globular shaped, ø 1.0 mm; 503ET-1, Ishizuka Electronics, Japan), which was connected to a digital monitor (DM1001C, Ishizuka Electronics, Japan) with a pair of electric wires (ø 0.8 mm, 1.0 m long) in a single silicon tube (1 mm in inside diameter, 2 mm in outside diameter; AS ONE, Tokyo, Japan). Fourteen-days-old chicks were subjected to the in vivo temperature measurement. The sensor tip was surgically placed in a brain position close to the pineal gland (1.0 mm anterior and 1.0 mm lateral to the protuberantia occipitalis interna, 1.0 mm ventral to the cortical surface), and the connecting tube was attached to the skull with the aid of dental cement (GC Corp., Tokyo, Japan) so that the sensor was fixed at the position. Activities of the chick were restricted by placing it in a small columnar arena (80 mm in diameter, 80 mm high) with a cover of a circular cloth loosely bound to the neck of the bird like a collar. Food and water were freely available, and the ambient temperature was monitored and kept at 28°C±0.5°C.
Identification of chick pineal Hsp90α as a light responsive gene
To explore the molecular mechanism underlying the phase-dependent phase-shifting effect of light on the circa-dian clock, we employed a differential display-based screening of the chick pineal genes that are induced by light in a phase-dependent manner. Newly hatched chicks were raised in LD cycles for 7days and transferred to DD on day 8. Then animals were exposed to a 90-min light stimulus from various CT points (CT0, CT14 or CT20) on day 8, and the pineal glands were collected from animals (30 chicks each) at two distinct time periods after the light onset (20–50 min and 60–90 min after the light onset). The control animals were maintained in the dark, and their pineal glands were collected at CT1.5, CT15.5 and CT21.5. This sampling schedule was chosen to identify various types of light-responsive genes such as those showing a CT-dependent light-induction and those induced rapidly or slowly after the light onset. We screened 5,699 bands of the PCR products and found 99 bands as light-induced products. As shown in Table 1, these products were classified into four types on the basis of the time point(s) when they were light-induced: (i) induced only at CT14 (early night), (ii) induced only at CT20 (the late night), (iii) induced both at CT14 and CT20 (night), but not at CT0 (morning), and (iv) induced at every time point tested. All the light-induced products showed higher band intensities at 60min than those at 20min after the light onset. We cloned three products, two from type (iv) (Fig. 1) and one from type (i) (Doi et al., 2001) because these three showed remarkable light induction. Sequencing analyses revealed that the latter one was derived from a message of cE4bp4 gene (Doi et al., 2001). The sequences of the former two products (indicated by arrows in Fig. 1A and 1B) matched completely the cHsp90α cDNA sequence (GenBank accession no. X07265) within the 339 bases (+2513 to +2851; +1 indicates transcription initiation site) and within the 646 bases (+1650 to +2295), respectively. These results strongly suggest that the mRNA levels of the pineal cHsp90α increase profoundly in response to the light stimulus given at CT0, 14 and 20.
Classification of PCR products in differential display analyses
Light-dependent and time-of-day-dependent expression of cHsp90α mRNA
A time course of the light-dependent cHsp90α induction was examined by quantitative RT-PCR analyses, in which the mRNA levels of the chick pineal cHsp90α were monitored during and after 60-min light exposure from CT20 (Fig. 2A). The cHsp90α mRNA level began to increase within 5min after the light onset, and at 90 min it reached a peak level ∼4 fold higher than the basal level. Then, the cHsp90α mRNA level decreased gradually and reached to a level close to that of the control (dark-kept) animals within 3 hr after the light offset. To examine whether the pineal cHsp90α expression shows a CT-dependent light response, chicks were exposed to a 60-min light stimulus at various CT points of a day (CT2, CT6, CT10, CT14, CT18 and CT22), and the level of the pineal cHsp90α mRNA was evaluated at 60 min after the light onset (Fig. 2B). The light stimuli given at CT10, CT14, CT18 and CT22 produced significant increases in the level of the pineal cHsp90α mRNA, but no significant effect was observed by light given at early morning to midday (CT2 and CT6) under the experimental conditions. In addition to the acute response to light, a daily change in the pineal cHsp90α mRNA level was examined. In a LD cycle (Fig. 3; on day 8), the pineal cHsp90α mRNA exhibited a profound daily fluctuation with a peak in the early morning, and a similar fluctuation with lower amplitude was observed in animals maintained in DD (Fig. 3; on day 9). These results indicate that the expression of cHsp90α is regulated not only by the external light but also by the circa-dian clock.
Analyses of the chick pineal HSP90α protein
A possible change in cHSP90α protein level was examined by evaluating the total amount of HSP90α protein expressed in the chick pineal gland. In immunoblot analysis (Fig. 4), the expression of cHSP90α protein was detected in the pineal gland of chicks that were maintained in LD cycles at a constant room temperature, and the levels showed no significant change throughout the day in contrast to the profound fluctuation of its mRNA level.
Although the total amount of the cHSP90α protein accumulated in the whole pineal tissue was apparently constant (Fig. 4), the level of the cHSP90α protein may diurnally fluctuate within a subset of the pineal cells. To test this, localization of cHSP90α protein in the chick pineal gland was investigated by immunohistochemical analyses (Figs. 5 and 6). Examination of a possible colocalization of cHSP90α with pinopsin, a pineal specific photoreceptive molecule (Okano et al., 1994) revealed cHSP90α immunoreactivity in almost all the pineal cells including the follicular pinealocytes (indicated by solid arrowheads in Fig. 5) whose rudimentary outer segments showed pinopsin immunoreactivity (indicated by open arrowheads in Fig. 5). This suggests cHSP90α protein expression in the photosensitive clock cells in the pineal gland (Takahashi et al., 1989; Okano et al., 1994; Bernard et al., 1997). Pineal cHSP90α proteins were localized mainly in the cytosol and diffusely in the nucleus in the follicular and parafollicular pinealocytes (indicated by arrows in Fig. 5). The immunohistochemical analysis of the temporal change in the localization demonstrated that cHSP90α immunoreactivities were kept at a nearly constant level in immunopositive pineal cells of the pineal glands isolated at ZT1, ZT7, ZT13 and ZT19 (Fig. 6). At every ZT point tested, the subcellular localization of cHSP90a protein was mainly cytoplasmic as observed at ZT6 (Fig. 5A). These observations indicate that not only the total amount of the pineal cHSP90α protein but also its sub-cellular localization in each pineal cell is kept almost constant all day long in LD cycles.
Light-dependent and time-of-day-dependent changes in the in vivo temperature of the pineal gland
Considering that the expression of heat shock proteins is generally responsive to a temperature change, we assumed that the changes in the mRNA level of the pineal cHsp90α (Figs. 2 and 3) might be ascribed to the changes in the in vivo temperature of the pineal gland (designated TP). To test this, TP was measured continuously with the aid of a thermometer fixed at a brain position close to the pineal gland of the chick (see Materials and Methods). When chicks were maintained in LD cycles, TP exhibited an overt diurnal fluctuation. As shown in Fig. 7A, TP rapidly decreased and reached trough levels of 39.2–39.4°C within 1 hr after the light offset (on day 14 and 15). Then TP gradually increased during the late dark period. After the light onset (on day 15), TP was rapidly elevated and kept at 40.5-40.9°C during the light period. TP also exhibited a diurnal fluctuation in constant dark condition (on day 16) albeit with a lower peak/trough ratio (Fig. 7A). In response to light exposure during the nighttime (on day 16), TP rapidly increased by 1.1°C from 39.9°C to 41.0°C (Fig. 7A). Notably, TP began to increase within 2min after the light onset, and at 20 min it reached a plateau of 40.9–41.0°C (Fig. 7B). The light-dependent and time-of-day-dependent changes of the pineal temperature (Figs. 7A and 7B) indicate a possible association of the expression profile of the pineal cHsp90α mRNA with the temperature change of the pineal gland.
The differential display-based screening of light-induced genes in the chick pineal gland resulted in identification of the light-induced products, which were classified into four types on the basis of the CT-dependent pattern of the light-induction (Table 1). It is most probable that the pineal gland has a gene expression system discriminating the circadian time when the chick is exposed to a light stimulus (Doi et al., 2001). Here we showed that the two products with remarkable light-responsiveness at every CT point tested were derived from a message of cHsp90α gene (Fig. 1), and demonstrated that the level of the pineal cHsp90α mRNA changed in light-dependent and time-of-day-dependent manners (Figs. 2 and 3). Monitoring the in vivo temperature of the pineal gland demonstrated a close link between the profiles of the pineal temperature and the cHsp90α mRNA level (Figs. 2, 3 and 7). Although it is unclear whether the in vivo temperature change of 1–2°C (Fig. 7) is responsible for the cHsp90α expression, the data presented here strongly suggest that the gene expression mechanism sensitive to a temperature change operates in the light-dependent and time-of-day-dependent manners in vivo.
Contrary to the mRNA level exhibiting pronounced diurnal changes (Fig. 3), the total amount of the pineal cHSP90α protein was kept at nearly constant level under the LD cycle condition (Figs. 4 and 6). Such a constant level expression of HSP90α protein has been seen in several rabbit tissues such as cerebellum, retina, thymus and testis (Quraishi and Brown, 1995), where HSP90α is an abundant protein constituting ∼2% of the total cellular protein under unstressed conditions (Lai et al., 1984). Very interestingly, the total amount of HSP90α protein expressed in these tissues showed no significant change even after a heat stimulus that elevated the rate of its protein synthesis (Clark and Brown, 1982). On the other hand, a heat-stimulus-dependent increase in the total amount of the HSP90α protein was observed in tissues such as muscle, heart and kidney, showing a low-level expression of HSP90α protein under unstressed conditions (Quraishi and Brown, 1995). These observations support an idea that the high level expression of HSP90α protein in certain tissues would mask the upregulation of its protein synthesis therein. This idea may account for the constant level of cHSP90α protein in the chick pineal gland (Figs. 4 and 6), although alternative explanations are also possible. For example, the rates of synthesis and degradation of cHSP90α protein may be balanced, or alternatively, the amount of the pineal cHSP90α protein is regulated at the translational level, not at the transcriptional level.
The constant expression level of cHSP90α protein implies invariable cHSP90α activities in the pineal gland, excluding a possible function of cHSP90α as a zeitnehmer (Roenneberg et al., 1999) or a state variable in the pineal circadian clock. However, considering the fact that some of clock proteins are expressed at constant levels in the clock-containing tissues (Ripperger et al., 2000; Kloss et al., 2001; Chilov et al., 2001), it is still possible for cHSP90α protein to participate in the chick pineal clock system. Indeed, human HSP90α is known to interact with BMAL1 and NPAS2 (Hogenesch et al., 1997), both of which play pivotal roles in the vertebrate circadian clock system (Bunger et al., 2000; Reick et al., 2001). A potential involvement of the cHSP90α protein in the circadian oscillation and/or in the light-entrainment is also suggested by colocalization of a photoreceptive molecule pinopsin and cHSP90α protein in the follicular pinealocyte (Fig. 5) that contains both the circadian oscillator and the photic-input pathway (Okano et al., 1997; Bernard et al., 1997; Nakahara et al., 1997).
In previous studies, the effect of temperature on the pineal clock was examined in in vitro experiments with cultured chick pineal cells, and it was clearly demonstrated that a transient increase of temperature from 36.7°C to 40.0°C (Zatz et al., 1994) or from 37.0°C to 39.5°C (Barrett and Takahashi, 1995) induces a phase-shift of the clock in a CT-dependent manner. Such a temperature-dependent phase-shift is observed in fungi (Francis and Sargent, 1979; Nakashima, 1987), insects (Zimmerman et al., 1968; Edery et al., 1994), reptiles (Underwood, 1985), and mammals (Ruby et al., 1999). In the present study, we demonstrated that the in vivo temperature of the chick pineal gland fluctuated between 39.2°C and 40.9°C under LD cycles (Fig. 7A), and that it rapidly increased from 39.9°C to 41.0°C upon receiving a light stimulus (Fig. 7B). Although it is difficult to examine whether the in vivo temperature increase in the dark can also induce a phase-shift of the pineal clock, the in vivo temperature increase would likely contribute to the phase-shift of the pineal clock. Then it is conceivable that the cooperative effects of light and temperature signals would allow an efficient entrainment of the pineal clock to environmental LD cycles. Further analyses of the heat-responsive pathways in the clock structures might be impor tant to know the molecular mechanisms underlying the phase-shift of the clock and the temperature compensation, both of which are functionally prerequisite for the circadian clock.
We thank Dr. K. Kokame at National Cardiovascular Center Research Institute (Osaka, Suita, Japan) for technical advice in the differential display analysis. This work was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.