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Brain Research. 508 (1992) 332 33~ .~'; 1992 Elsevier Science Publishers B.V. All rights reserved 00(16-8993/92/$1)5.(1tl

BRES 25463

Circadian and developmental regulation of Oct-2 gene expression in the suprachiasmatic nuclei Scott A. Rivkees *, David R. Weaver and Steven M. Reppert Laboratory of Det~elopmental Chronobiology, Children's Service, Massachusetts General Hospital and Han~ard Medical School, Boston, MA 02114 (USA) (Accepted 15 September 1992)

Key words: Oct-2 gene expression; Suprachiasmatic nucleus; Circadian rhythm

Oct-2 is a transcriptional activating factor that is expressed in the suprachiasmatic nuclei (SCN), the site of a biological clock. We examined in rats whether Oct-2 gene expression is regulated by the circadian pacemaker or by light using quantitative in situ hybridization. The ontogeny of Oct-2 gene expression in the SCN was also studied. Oct-2 mRNA levels remained constant throughout the circadian cycle. In contrast to c-los mRNA levels which are acutely induced by acute light exposure at night, Oct-2 mRNA levels were not increased by light exposure at night. At gestational day 18, the first age the SCN are anatomically distinct, a prominent Oct-2 hybridization signal was present in the SCN. Our results suggest that Oct-2 is constitutively expressed in the SCN and is present from the time the SCN are discernible as discrete nuclei in fetal brain.

A biological clock located in the hypothalamic suprachiasmatic nuclei (SCN) is responsible for the generation and regulation of mammalian circadian rhythms 14. The SCN manifest endogenous rhythms in metabolic 17'25 and electrical activity 16, and gene expression zT, and drive an array of behavioral and physiological rhythms 14. Retinal pathways relay photic information from the retina to the SCN, leading to the entrainment of circadian rhythms to the 24 h day 3. In rats, the SCN form and begin oscillating as a biological clock prenatally ~5.~7.SCN neurogenesis spans gestation days (GD) 13 to 16 (day 0 = insemination)l'l°: day-night oscillations in SCN metabolic activity are first detectable after the nuclei become apparent on GD 1817. Prior to functional innervation of the SCN by retinohypothalamic pathways, the oscillations of the fetal SCN are entrained by the dam, presumably by the transplacental passage of chemical signals6'~8'29. The molecular events that underlie SCN function and development are largely unknown. With the observations that nocturnal light exposure induces expression of the transcriptional activating factors Fos, Jun-B

and zif-268 in the S C N 2'12'13, there has been increasing interest in the role of transcriptional regulatory factors on SCN function. Recently, another transcriptional activating protein, Oct-2, was shown to be expressed in the SCN of adult rats and in a few other brain regions7. Oct-2 is a member of the POU family of DNA-binding proteins, and regulates the expression of specific genes in peripheral tissues 4'7-9. Oct-2 is also expressed in the neural tube during fetal life7'9, suggesting that it may also have a role during development. To begin to examine the role of Oct-2 in SCN function, we examined whether Oct-2 expression in SCN is regulated by the circadian pacemaker or by light using quantitative in situ hybridization. In addition, we examined the ontogeny of Oct-2 gene expression in the SCN. Sprague-Dawley rats (Zivic Miller, Allison Park, PAl were used in all experiments. Animals were housed in clear plastic cages within well-ventilated light proof compartments in which the lighting cycle could be regulated 19. Food (Purina rodent chow) and water were always available. Animals were kept on an automated

Correspondence: S.M. Reppert, Laboratory of Developmental Chronobiology, Children's Service, Massachusetts General Hospital, Boston, MA 02114, USA. Fax: (1) (617) 726-1694. * Present address: James Whitcomb Riley Hospital for Children, Rm 5984, 702 Barnhill Drive, Indianapolis, IN 46202-5225, USA.

333 light-dark (LD) cycle consisting of 12 h of light per day (lights on from 07.00 to 19.00 h) for at least 1 week prior to study. For adult studies, male rats 60-days old) were placed into constant dim red light (designated as darkness; wavelength > 620 nm; 20 W litho no. 2 fluorescent tubes; Chemical Products, North Warren, PA) at lights-out on the day prior to study. Some animals were exposed to light during nighttime provided by 20 W cool white fluorescent tubes, (ca. 600 lux; Westinghouse, Pittsburgh, PA). Animals were killed at specified times (see below) by decapitation. Specimens were dissected, frozen in cooled 2-methylbutane ( - 20°C), and stored at -80°C. Coronal brain sections (15/xm) were cut in a cryostat, thaw mounted onto slides treated with Vectabond (Vector Labs, Burlingame, CA), and stored at -80°C. For adult studies, consecutive sections spanning the entire hypothalamus were collected. For fetal studies, consecutive sections spanning the entire fetal brain were collected. Oct-2 gene expression was examined using a 1.7 kb antisense cRNA probe complementary to the coding region of the human Oct-2 cDNA, provided by R. Clerc (described in ref. 4). c-fos gene expression was examined using a 1.8 kb antisense cRNA probe complementary to the coding region of the murine c-fos cDNA, provided by M.E. Greenberg. 35S-Labeled antisense and sense cRNA probes were generated by digestion of plasmids with the appropriate restriction endonuclease, followed by in vitro transcription with either SP6, T3 or T7 polymerases (Promega) in the presence of [35S]a-thio-UTP (New England Nuclear). The murine c-fos probe has been previously shown to be effective for in situ hybridization studies in rats 5"29. In situ hybridization was performed, as previously described in detail TM. Sections were sequentially incu-

bated in 4% paraformaldehyde (30 min), 0.2 N HCI (30 min), and acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride (10 min), and dehydrated through ascending alcohol concentrations. Sections were covered with 50/~1 of hybridization buffer 18 containing 1.0 x 107 cpm of probe/ml, glass coverslips were applied, and incubated in a humidified chamber at 55°C overnight. Coverslips were removed the next morning and sections were washed in 2 x SSC (30 min, 20°C), RNAse A (10 /zg/ml for c-fos, 2 txg/ml for Oct-2; Sigma, St. Louis, MO, 60 min, 37°C), 2 x SSC (30 min, 20°C), 0.1 x SSC (60 min, 60°C), and 0.1 x SSC (30 min, 20°C). Sections were then dehydrated in ascending concentrations of alcohol that contained 0.3 M ammonium acetate and air dried. Film autoradiographs were generated by apposing slides to Kodak SB-5 film for 15 days. Emulsion autoradiographs were generated from sections dipped in Kodak NTB-2 emulsion and exposed for 3 weeks. To facilitate identification of anatomical structures, sections were stained with Methylene blue, counterstained with eosin, and examined by light microscopy. The film hybridization signal over the SCN was quantitated by image analysis using a Drexel University Image Processing Center 'Brain Software Package' run on an IBM AT computer connected to a Circon MV 9015-H monochrome microvideo camera, as previously described ~9. Sections with the most intense hybridization signal were identified for each animal; these sections generally corresponded with the level of the midSCN. The optical density (O.D.) of the SCN and adjacent hypothalamus were determined in triplicate for each of 2 sections per animal. Mean relative O.D. values (O.D. of SCN/O.D. of adjacent hypothalamus) were then calculated. We have previously shown that over the range of autoradiographic exposures used in

Fig. 1. Dark-field images of an emulsion autoradiograph generated from a coronal brain section probed for Oct-2 mRNA (left panel). The Nissl stained section used to generate the autoradiograph is shown in the right panel. Areas of specific hybridization appear as white; arrows depict the SCN.

334 this experiment, film relative O.D. values are linearly related to emulsion grain density 29. Oct-2 gene expression was first examined in the SCN of adult animals. Analysis of film autoradiographs showed that a specific Oct-2 hybridization signal is present in the SCN, but not in the adjacent hypothalamus or other hypothalamic nuclei. Emulsion autoradiographs confirmed that Oct-2 mRNA was expressed throughout the entire extent of the anatomical boundaries of the SCN with relatively higher levels in the ventral than the dorsal region (Fig. 1). A small number of cells expressing Oct-2 mRNA also appeared to be scattered throughout the hypothalamus on emulsion autoradiographs. The specificity of the hybridization signal was validated in several ways. First, a hybridization signal was apparent over the SCN using antisense probes, but not with sense probes. Second, treatment of brain sections with RNAse prior to hybridization resulted in loss of the hybridization signal. Third, hybridization using anti-sense probes was observed over the white pulp of the spleen, as would be expected, since Oct-2 is expressed in B-lymphocytes (data not shown; refs. 4, 9, 26). A hybridization signal was also seen over the medial mammilary bodies of adult rat brain (data not shown), as previously reported by He et al. 7. Finally, antisense cRNA probes generated from the 1.7 kb mouse Oct-2 cDNA (provided by L. Corcoran) produced hybridization patterns in rat brain similar to those observed with probes generated from the human Oct-2 cDNA used for these studies (data not shown). We next examined whether Oct-2 gene expression shows circadian variation or is regulated by light. Oct-2 mRNA levels were assessed at different phases of the circadian cycle and after light exposure at night, c-fos mRNA levels were assessed on adjacent tissue sections from the same animals; c-los mRNA expression is acutely induced by light exposure at night ~2'13. The temporal expression of the Oct-2 gene was assessed in constant darkness. Animals were killed at 11.00, 18.00, 23.00 and 05.00 h (n = 4 per interval in each of two separate experiments). With each experiment, all sections were processed in a single in situ hybridization run and exposed to film for the same duration. In each animal at each time point, a clear Oct-2 hybridization signal was apparent over the SCN (Fig. 1); a relatively less intense c-fos hybridization signal was apparent over the SCN (data not shown). Image analysis showed that neither Oct-2 nor c-los mRNA levels varied significantly with the time of day in constant darkness (Fig. 2; P > 0.05, ANOVA). Along with the circadian studies, we examined whether Oct-2 gene expression is influenced by light.

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Clock Time (hours) Fig. 2. c-los (top) and Oct-2 (bottom) hybridization signals from animals studied in constant darkness (solid symbols) and after light exposure at night (open symbols). Mean _+S.E.M. of SCN relative optical densities (O.D. of S C N / O . D . of adjacent hypothalamus) for 3-4 animals is depicted at each time point; the study shown is representative of two such studies. Adjacent sections from the same animal were probed for c-fos and Oct-2 mRNA using antisense cRNA probes. A: animals previously maintained in a light-dark cycle were studied in constant darkness. (Stippled area of bar represents daytime; solid area represents nighttime). B: open area of bar represents the period of light exposure at night.

Rats in constant darkness were exposed to light beginning at 23.00 h, the time at which photic induction of c-los expression is maximal t2"13. Animals were killed at 30, 60, 180 or 360 rain after light exposure (n = 3 animals per time point). In agreement with published reports ts, a robust c-los hybridization signal was apparent 30 rain after the onset of light exposure; c-los levels steadily declined thereafter (Fig. 2). In contrast, no significant changes in Oct-2 m R N A levels were observed at the intervals examined. A tendency for Oct-2 m R N A levels to rise with prolonged light exposure was noted, but this trend was not statistically significant (Fig. 2; P > 0.05, ANOVA). Next, the expression of Oct-2 in the developing SCN was examined. Oct-2 m R N A levels were assessed in the fetal hypothalamus at gestational days (GD) 12, 14, 16, 17, 18, 20, and on postnatal day 1 (day 0 = day of birth). At least two fetuses from pregnant dams

335 killed between 12.00 and 15.00 h in LD were examined at each gestational age. In contrast to the adult hypothalamus, Oct-2 m R N A was more widely expressed in the hypothalamus of the fetus (Fig. 3). On G D 12, 14, 16 and 17, a hybridization signal was present at the base of the third ventricle; this region is the site of origin of SCN neurons prior to nuclear settling 15. With increasing age the hybridization signal became more restricted. At G D 18, the first age the SCN were anatomically distinct, a prominent hybridization signal was present within the SCN: hybridization was also apparent over the supraoptic nuclei. At postnatal day 1, Oct-2 m R N A expression in the hypothalamus was observed predominantly in the SCN region. Our results show that Oct-2 gene expression does not vary over the course of the day, nor is it acutely induced by light exposure at night. These data suggest that this octamer transcription factor is constitutively expressed in the SCN and not acutely regulated by light. Our developmental studies show that the Oct-2 gene is expressed in the SCN from the time of its appearance as a discrete nucleus in fetal rat brain.

ANTISENSE

GD 16

Octamer binding proteins are members of the POU family of transcriptional activating proteins 8. In mammals, more than 10 specific octamer binding proteins and several of the genes that encode them have been identified, each with specific regional and developmental patterns of expression 4'7'9'22-24. To date, Oct-2 remains the only POU family member identified in the SCN. Two differentially spliced products (Oct-2a and Oct-2b) of a single Oct-2 gene have been reported 9. The Oct-2 probe used in our studies detects both Oct-2 gene splice products, thus either Oct-2a or Oct-2b may be expressed in the SCN. Octamer binding proteins contain a 70 amino acid POU-specific and a 60 amino acid homeobox domain, and bind to a specific octamer nucleotide motif (ATTT G C A T ) present in the promoter and enhancer regions of certain genes 4'8'2~. Oct-2 is a bona fide transcriptional activating factor and has been shown to regulate the expression of immunoglobulin genes 4,2L26. The observation that Oct-2 gene expression in the SCN remains relatively constant suggests that the octamer transcription factor may regulate housekeeping genes in the SCN that are not under circadian or photic

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GD 20

Fig. 3. Developmental appearance of Oct-2 mRNA at the base of the third ventricle at gestational days (GD) 16, 18 and postnatal day 1. Negative images of autoradiographs produced from coronal brain sections are shown. Images generated from adjacent sections using asS-labeled antisense (left column) and sense (middle column) probes are shown. Sections adjacent to those used to generate antisense images were stained and are shown on the right.

336

regulation. This is in contrast to cqbs expression which is acutely regulated by light, and may play a role in entrainment mechanisms 2'12'13'2°. The potential target gene(s) of Oct-2 proteins in the SCN await identification. During development, several genes encoding octamer binding proteins are expressed at early fetal stages and may play critical roles in structural development 7'11'22-24. Oct-2 mRNA has been detected in the diencephalon of rats at GD 13 7, and the Oct-2 protein has been shown to be expressed in murine brain as early as GD 8 9. During the period of SCN neurogenesis, we have found that Oct-2 mRNA is present at the base of the third ventricle, which is the site of origin of SCN neurons ~5. It is thus possible that this DNA binding protein is expressed in SCN progenitor cells and may influence their development. Thus, while Oct2 gene expression appears not to be acutely regulated by light or the circadian clock in the SCN, this gene may nevertheless be important for SCN formation and function. This work was supported by NIH Grants HD14227 (to S.M.R.), K08 HD00924 (to S.A.R) and a Genentech/Lawson Wilkins Pediatric Endocrine Society Award (to S.A.R.). The authors wish to thank Mr. Jim Deeds for technical assistance and Dr. Lynn M. Corcoran for useful discussions. l Altman, J. and Bayer, S.A., Development of the diencephalon in the rat. I. An autoradiographic study of the time of origin and settling patterns of neuron of the hypothalamus, J. Comp. Neurol., 182 (1978) 945-971. 2 Aronin, N., Sagar, S., Sharp, F. and Schwartz, W.J., Light regulates expression of a Fos-related protein in rat suprachiasmatic nuclei, Proc. Natl. Acad. Sci. USA, 87 (1990) 5859-5962. 3 Card, J.P. and Moore, R.Y., The organization of visual circuits influencing the circadian activity of the suprachiasmatic nucleus. In D.C. Klein, R.Y. Moore and S.M. Reppert (Eds.), Suprachiasmatie Nucleus. The Mind's Clock, Oxford University Press, New York, pp. 51-76. 4 Clerc, R.G., Corcoran, L.M., LeBowitz, J.H., Baltimore, D. and Sharp, P.A., The B-cell-specific Oct-2 protein contains POU boxand homeo box-type domains, Genes Dev., 2 (1988) 1570-1581. 5 Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474-476. 6 Davis, F.C. and Mannion, J., Entrainment of hamster pup circadian rhythms by prenatal melatonin injections to the mother, Am. J. Physiol., 255 (1988) R439-R448. 7 He, X., Treacy, M.N., Simmons, D.M., Ingraham, H.A., Swanson, L,W. and Rosenfeld, M.G., Expression of a large family of POU-domain regulatory genes in mammalian brain development, Nature, 340 (1989) 35-42. 8 Herr, W., Sturm, R.A., Clerc, R.G., Corcoran, L.M., Baltimore, D., Sharp, P.A., Ingraham, H.A., Rosenfeld, M.G., Finney, M., Ruvkin, G., et al., A large conserved region in the mammalian Pit-l, Oct-2, and Caenorhabditis elegans unc-86 gene products, Genes De~,., 2 (1988) 1513-1516.

9 Hatzopoulos, A.K., Stoykova, A.S., Erselius. J.R., Goulding, M.. Neuman, T. and Gruss, P., Structure and expression of the mouse Oct2a and Oct2b, two differentially spliced products of the same gene, Development, 109 (1990) 349-362. 10 lfft, J.D., An autoradiographic study of the time of final division of neurons in the rat hypothalamic nuclei, J. Comp. Neurol., 144 (1972) 193-204. 11 Kessel, M. and Gruss, P., Murine developmental control genes, Science, 249 (1990) 374-379. 12 Kornhauser, J., Nelson, D., Mayo, K. and Takahashi, J., Photic and circadian regulation of c-los gene expression in the hamster suprachiasmatic nucleus, Neuron, 5 (1990) 127-134. 13 Kornhauser, J., Nelson, D., Mayo, K. and Takahashi, J., Regulation of jun-B messenger RNA and AP-1 binding activity by light and a circadian clock, Science. 255 (1992) 1581-1584. 14 Moore-Erie, M.C., Su[zman, F.M. and Fuller, C.A., The Clocks That Time Us, Harvard University Press, Cambridge, MA, 1982. 15 Moore, R.Y., Shibata, S. and Bernstein, M.E., Developmental anatomy of the circadian system. In S.M. Reppert (Ed.), Det,elopment of Circadian Rhythmicity and Photoperiodism in Mammals, Perinatology Press, Ithaca, NY, 1989, pp. 1-24. 16 Prosser, R.A. and Gillette, M.U., The mammalian circadian clock in the suprachiasmatic nuclei is reset in vitro by cAMP, J. Neurosci., 9 (1989) 1073-1081. 17 Reppert, S.M. and Schwartz, W.J., Maternal coordination of the fetal biological clock in utero, Science, 220 (1983) 969-971. 18 Reppert, S.M., Weaver, D.R., Stehle, J.H. and Rivkees, S.A., Molecular cloning and characterization of a rat At-adenosine receptor that is widely expressed in brain and spinal cord, Mol. Endocrinol., 5 (1991) 1037-1048. 19 Rivkees, S.A., Fox, C.A., Jacobson, C.D. and Reppert, S.M., Anatomic and functional development of the suprachiasmatic nuclei in the gray short-tailed opossum, Z Neurosci., 8 (1988) 4269-4276. 20 Rusak, B., Robertson, H., Wisden, W. and Hunt, S., Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science, 248 (1990) 1237-1240. 21 Ruvkun, G. and Finney, M., Regulation of transcription and cell identity by POU domain proteins, Cell, 64 (1991) 475-478. 22 Scholer, H.R., Bailing, R., Hatzopoulos, A.K., Suzuki, N. and Gruss, P., A family of octamer-specific proteins present during mouse emhryogenesis: evidence for germline-specific expression of an Oct factor, EMBO J., 8 (1989) 2543-2550. 23 Scholer, H.R., Bailing, R., Hatzopoulos, A.K., Suzuki, N. and Gruss, P., Octamer binding proteins confer transcriptional activity in early mouse embryogenesis, EMBO J., 8 (1989) 2551-2557. 24 Scholer, H.R., Ruppert, S., Suzuki, N., Chowdhury, K. and Gruss, P.. New type of POU domain in germ line-specific protein Oct-4, Nature, 344 (1990) 435-439. 25 Schwartz, W.J. and Gainer, H., Suprachiasmatic nucleus: use of 14C-labeled deoxyglucose uptake as a functional marker, Science, 197 (1977) 1089-1091. 26 Staudt, L.M., Singh, H., Sen, R., Wirth, T., Sharp, P.A. and Baltimore, D., A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes, Nature, 323(1986) 640643. 27 Uhl, G.R. and Reppert, S.M., Suprachiasmatic nucleus vasopressin messenger RNA: circadian variation in normal and Brattleboro rats, Science, 232 (1986) 390-393. 28 Weaver, D.R. and Reppert, S.M., Periodic feeding of SCN-lesioned pregnant rats entrains the fetal biological clock, Det,. Brain Res., 46 (1989) 291-296. 29 Weaver, D.R., Rivkees, S.A. and Reppert, S.M., DI-Dopamine receptors activate c-fos expression in the fetal suprachiasmatic nuclei, Proc. Natl. Acad. Sci. USA, 89 (1992) 9201-9204.

Circadian and developmental regulation of Oct-2 gene expression in the suprachiasmatic nuclei.

Oct-2 is a transcriptional activating factor that is expressed in the suprachiasmatic nuclei (SCN), the site of a biological clock. We examined in rat...
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