Localization of the Rev-ErbA orphan receptors in the brain

Brain Research 743 Ž1996. 315–319

Short communication

Localization of the Rev-ErbA orphan receptors in the brain Tommi Kainu

a, )

˚ Gustafsson , Eva Enmark b , Jan-Ake

b,c

, Markku Pelto-Huikko

a,d

a

c

Tampere UniÕersity Medical School, P.O. Box 607, Tampere, Finland b Center for Biotechnology, Karolinska Institute, Huddinge, Sweden Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden d Department of Neurology, UniÕersity of Kuopio, Kuopio, Finland Accepted 9 April 1996

Abstract In the present study, we report the localization of the Rev-ErbAa and b nuclear orphan receptors, two closely related members of the nuclear hormone receptor superfamily, in the brain. Both Rev-ErbA variant mRNAs were highly expressed in the olfactory bulb, the hippocampus, and in the granular cells of the cerebellum, areas enriched also in other nuclear orphan receptors. Furthermore, the a-isoform was found in high amounts in the frontal cortex, the superficial gray layer of the superior colliculus, and the stria terminalis. Lower expression was observed in the nucleus accumbens, the caudate-putamen, and in some thalamic and brainstem nuclei. The b-variant, in contrast, was only moderately expressed in the cortex, mainly in the striate and retrosplenial cortices. In addition, moderate levels of Rev-ErbAb mRNA were seen in various thalamic, pontine and brainstem nuclei. We conclude that the two Rev-ErbA isoforms share a partly similar pattern of expression in the brain, especially in areas that also contain other nuclear orphan receptors and that otherwise the localization of the two receptor subtypes is differential. Keywords: Orphan receptor; Transcriptional regulation; Thyroid hormone

The number of nuclear orphan receptors, proteins resembling ‘classic’ nuclear hormone receptors, i.e., the steroid and thyroid hormone, retinoic acid and vitamin D receptors, but devoid of a known function andror ligand, has grown extensively during the past few years Žreviewed in w16x.. Many of these newly characterized proteins have been proposed to function as modulators of the ‘classic’ nuclear hormone receptors Žfor review see w15x.. One such nuclear orphan receptor is Rev-ErbAa , which was first identified on the basis of its unique genomic localization; on the opposite strand of the thyroid hormone receptor ŽTR. a gene w12x. Further investigations have revealed that this unusual genomic arrangement serves a functional purpose in the sense that Rev-ErbAa modifies the alternate splicing of the TRa transcript in favor of the a 1 subtype w13x. Of the two TR variants only a 1 is a transcriptional activator w8,10,17x, while a 2 , although unable to bind T3 , can inhibit the transactivation of T3-responsive genes by the other TRs w9,12x. By increasing the cellular

) Corresponding author. Fax: q358 Ž31. 215-7332; E-mail: [email protected]

TRa 1rTRa 2 ratio in T3 target cells Rev-ErbAa serves, thus, as a positive regulator of T3 function. Recently, we and others reported the finding of a nuclear orphan receptor that shows a striking homology to Rev-ErbAa , hence termed Rev-ErbAb Žalso BD73 and RVR; w5–7,18x.. In their reports, Forman et al. and Retnakaran et al. also show that both Rev-ErbA isoforms are able to modify the transcriptional activity of the RORrRZR nuclear orphan receptors w7,18x. Furthermore, a recent study by Bonnelye et al. suggests a role for Rev-ErbAb in controlling neural development w2x. With reference to the well-known effects of thyroid hormone on the development and function of the central nervous system, and these previous reports on Rev-ErbAb w2,7,18x, we thought it interesting to examine the localization of both Rev-Erb variants in the brain as this might provide information relevant to their possible physiological functions in the nervous system. Nine adult male Sprague–Dawley rats were decapitated, after which their brains were excised and frozen on a block of dry ice. Serial 14 mm thick coronal, frontal, and sagittal sections representing different brain areas were cut with a Microm HM-500 cryostat and the sections were thawed on

0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 0 5 0 7 - 0

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Probe On ŽFischer Scientific, PA, USA. glasses. Four oligonucleotide probes directed against nucleotides 1021– 1065 and 1791–1835, and nucleotides 544–591 and 937– 984 of the rat Rev-ErbAa and b-genes, respectively w6,12x were used in this study. The sequences exhibited less than 60% homology with any other known gene, when compared against the known sequences in the GenBank database. Also, several control probes with the same length, similar GC-content and specific activity were used to ascertain the specificity of the hybridizations. The probes were labelled with a-w 33 PxdATP using terminal deoxynucleotidyltransferase as previously described w4x. The sec-

tions were briefly air dried and hybridized at 428C for 18 h with 8–10 = 10 6 cpmrml of the probe in a mixture containing 4 = SSC Ž1 = SSC s 0.15 M NaCl, 0.015 M sodium citrate., 50% formamide, 1 = Denhardt’s solution Ž0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin and 0.02% Ficoll., 1% sarkosyl, 0.02 M phosphate buffer ŽpH 7.0., 10% dextran sulphate, 500 mgrml heat denatured salmon sperm DNA and 200 mM dithiothreitol. After hybridization, the sections were rinsed 4 times at 558C in 1 = SSC for 15 min each and subsequently left to cool for 1 h at room temperature. The sections were dipped in distilled water, dehydrated with 60 and 90% ethanol and

Fig. 1. Both Rev-ErbAa ŽA. and b ŽB. are localized to the olfactory bulb. However, the a form has a higher expression. Of the different layers of the olfactory bulb, mRNA for both isoforms can be observed in the internal granular layer Žarrow. and the olfactory nerve layer Žarrowhead.. Rev-ErbAa mRNA can be observed in the stria medullaris thalamus Žst. and the anterior thalamic Žat. nuclei ŽC.. Both receptor forms, a ŽD. and b ŽE., have a high expression in the dentate gyrus Ždg. of the hippocampus, and a moderate expression in the hippocampal areas CA1–CA4 Ž1–4.. In the thalamus both receptor variants are seen in the dorsal lateral ŽDlg. and medial geniculate ŽMg. nuclei. Re-ErbAb is additionally seen in the reticular thalamic nucleus Žrt. and in the anterior pretectal area Žap..

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air dried. Thereafter the sections were covered with Amersham b-max autoradiography film ŽAmersham, Buckinghamshire, UK.. The autoradiography films were developed using LX24 developer and AL4 fixative ŽKodak, Rochester, NY, USA.. Alternatively, the sections were dipped in NTB2 emulsion ŽKodak. diluted 1:1 with distilled water and exposed at y208C. The sections were developed with D19 developer ŽKodak., fixed with G333 fixative ŽAgfa Gevaert, Leverkusen, Germany. and coverslipped with a mixture of glycerol and phosphate buffered saline Ž3:1.. The distribution of the Rev-ErbA nuclear orphan receptors was largely similar, but also differences in their expression existed. In the olfactory system, both isoforms were moderately expressed in the internal granular layer of the olfactory bulb ŽFig. 1a,b. and in the primary olfactory cortex. Low mRNA levels of both receptors could be observed in the olfactory nerve layer of the olfactory bulb ŽFig. 1a,b. and in the anterior olfactory nucleus. The various subnuclei of the anterior olfactory nucleus did not differ in their content of the Rev-ErbA mRNAs. In addition, a moderate expression of both receptors could be seen in the taenia tecta and in the olfactory tubercle. Only Rev-ErbAa mRNA could be observed in the caudate-putamen and the stria terminalis. Furthermore, in the nucleus accumbens a moderate expression of RevErbAa could be seen Ždata not shown.. In the thalamus, Rev-ErbAa mRNA was observed in the anterior and ventroposterior thalamic nuclei and in the stria medullaris thalamus ŽFig. 1c.. Both Rev-ErbA variants had a moderate expression in the medial and dorsal lateral geniculate nuclei ŽFig. 1d,e.. Rev-ErbAb was additionally expressed in the anterior and olivary pretectal nuclei ŽFig. 1e.. In the hypothalamus, Rev-ErbAa mRNA was expressed in the paraventicular and suprachiasmatic nuclei. All the hippocampal areas exhibited high levels of both Rev-ErbA mRNAs ŽTable 1.. The strongest expression of both subtypes was observed in the granular cells of dentate gyrus ŽFig. 1d,e.. Although both receptor subtypes were expressed in the cerebral cortex their relative mRNA levels differed in various cortical areas. However, in all cortical areas the expression of Rev-ErbAa was higher than that of the b-subtype. Rev-ErbAa had a high expression in the frontal and fronto-parietal cortices, while the expression of the b-variant was highest in the striatal and retrosplenial cortices. Both receptor isoforms had a high expression in the post-cingulate cortex. Of the cortical layers, the pyramidal cell layer exhibited the strongest expression of both receptors ŽFig. 2a–d.. The superficial gray area of the superior colliculus contained high amounts of both RevErbA mRNAs ŽFig. 2e,f., while only Rev-ErbAb was expressed in the nucleus ruber and in the pars compacta of the substantia nigra. Of all the brain areas, by far the strongest expression of both Rev-ErbA isoforms was in the cerebellar cortex, where the expression of both receptor variants was confined to the granular cell layer ŽFig. 2c,d.. In the brain-

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Table 1 The Rev-ErbA orphans in the different brain areas. The relative levels of expression are indicated by the number of plusses

Olfactory system: Internal granular layer of olfactory bulb Primary olfactory cortex Olfactory nerve layer of olfactory bulb Anterior olfactory nucleus Taenia tecta Olfactory tubercle Forebrain: Caudate-putamen Stria terminalis Nucleus accumbens Thalamus: Geniculate nuclei Anterior thalamic nucleus Ventroposterior thalamic nucleus Nucleus habenularis Pretectal nuclei Hypothalamus: Paraventricular nucleus Suprachiasmatic nucleus Hippocampus: Dentate gyrus CA1 area CA2–CA4 Cerebral cortex: Frontal cortex Fronto-parietal cortex Striatal cortex Retrosplenial cortex Post-cingulate cortex Tegmentum: Superficial gray layer of the superior colliculus Nucleus ruber Substantia nigra, pars compacta Cerebellum: Granular cell layer Brainstem: Ventral cochlear nucleus Spinal nuclei of the trigeminal nerve Pontine nucleus Inferior olive

Rev-ErbAa

Rev-ErbAb

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stem, Rev-ErbAa mRNA could be observed in low amounts in the ventral cochlear nucleus and in the spinal nuclei of the trigeminal nerve, whereas Rev-ErbAb was expressed in the pontine nucleus and in the inferior olive. In our earlier report describing the cloning of RevErbAb w6x, we already observed that the localization of the b-variant differed from that reported for the a-type in periphereal tissues. Thus, the results of this study that show regional differences in the expression of the two isoforms in the brain are not unexpected. In addition, although the amino acid sequences of the two Rev-ErbA variants are highly homologous, their hinge and ligand-binding domains show less similarity than is the case for other receptor subtypes w5–7,18x. Therefore, our results

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combined with these earlier observations suggest that the two Rev-ErbA variants may have specific roles of their own. Both TRa transcripts share a similar localization in the brain w3x and the localization of Rev-ErbAa is similar to this. Thus, the hypothesis suggested by Lazar et al. that Rev-ErbAa controls the alternative splicing of the TRa transcript w15x gains further support from results of this study. With regard to the fact that only the a 1 variant is a thyroid hormone receptor w8,10,17x, while the a 2 isoform is a negative regulator of trans-activation of T3-responsive genes by other TRs in vitro w9,11x, by increasing the cellular TRa 1rTRa 2 ratio, Rev-ErbAa could serve as a positive regulator of the effects of T3 on neurons.

The brain areas where the two subtypes are co-expressed, i.e., the hippocampus, the cerebellum and the olfactory bulb are enriched also in other orphan receptor types Žreviewed in w14x.. The presence of several other members of the nuclear receptor superfamily in the same brain areas as the Rev-ErbAs is interesting in view of the reports of Forman et al. and Retnakaran et al. demonstrating that the Rev-ErbA receptors are constitutive negative regulators of transcription by repressing the transcriptional activity of other orphans, namely RORa w7,18x. RORs, recently characterized members of the same superfamily of receptors as the Rev-ErbAs, are like the majority of orphans, potent activators of gene transcription w1x. However, when RORa is co-expressed with Rev-ErbAs its transcrip-

Fig. 2. In coronal brain sections, it can be observed that the expression of the a form ŽA. is much higher than that of the b variant ŽB. in the cortex. The arrow points to the pyramidal cell layer, where both isoforms have their highest expression in the cerebral cortex. In sagittal sections of the brain, Rev-ErbAa ŽC. and b ŽD. can be observed to have their highest expression in the granular cell layer of the cerebellar cortex Žarrowhead.. The differences in the expression of the isoforms in the cerebral cortex are also demonstrated here; in addition to being more highly expressed in the cortex the a form also has a more extensive distribution: while the b form is seen only in the striatal Žst. and retrosplenial Žrs. cortices, mRNA for the a form is seen also in the frontal cortex Žf.. In the superior colliculus, both mRNAs, a ŽE. and b ŽF., are expressed solely in the superficial gray area ŽSuG., with the a form having a higher level of expression also in this brain area.

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tional activity is suppressed w7,18x. In addition, of the two Rev-ErbA variants the a-type seems to be more potent in suppressing RORa mediated transcription w7x. In conclusion, in this study we have demonstrated the localization of the Rev-ErbA orphan receptors in the brain. The discrete and differential localization of the two receptor variants suggests important roles for both receptor isoforms in the brain.

w7x

w8x

w9x

Acknowledgements w10x

This study was supported by a grant from the Swedish Medical Research Council ŽGrant 13X-2819.. The skillful technical assistance of Miss Anne Maatta ¨¨ ¨ is gratefully appreciated by the authors. References w1x Becker-Andre, ´ M., Andre, ´ E. and DeLamarter, J.F., Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences, Biochem. Biophys. Res. Commun., 194 Ž1993. 1371–1379. w2x Bonnelye, E., Vanacker, J.-M., Desbiens, X., Begue, A., Stehelin, D. and Laudet, V., Rev-Erbb, a new member of the nuclear receptor superfamily, is expressed in the nervous system during chicken development, Cell Growth Differ., 5 Ž1994. 1357–1365. w3x Bradley, D.J., Young III, W.S. and Weinberger, C., Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary, Proc. Natl. Acad. Sci. USA, 86 Ž1989. 7250– 7254. ˚ Friberg, K, Bean, A.J. and Hokfelt, w4x Dagerlind, A., T., Sensitive ¨ messenger RNA detection using unfixed tissue-combined radioactive and non-radioactive in situ hybridization histochemistry. Histochemistry, 98 Ž1992. 39–49. w5x Dumas, B., Harding, H.P., Choi, H.-S., Lehmann, K.A., Chung, M., Lazar, M.A. and Moore, D.D., A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb, Mol. Endocrinol., 8 Ž1994. 996–1005. ˚ w6x Enmark, E., Kainu, T., Pelto-Huikko, M. and Gustafsson, J.-A., Identification of a novel member of the nuclear receptor superfamily

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