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Differential expression of retinoid receptors in the adult mouse central nervous system
Pergamon
PII:
Neuroscience Vol. 89, No. 4, pp. 1291–1300, 1999 Copyright 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00342-X
DIFFERENTIAL EXPRESSION OF RETINOID RECEPTORS IN THE ADULT MOUSE CENTRAL NERVOUS SYSTEM W. KREZ ~ EL,† P. KASTNER and P. CHAMBON* Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS-INSERM-ULP-Colle`ge de France, BP163, 67404 Illkirch Cedex, C. U. de Strasbourg, Strasbourg, France Abstract––The immunocytochemical distribution of retinoid receptors has been analysed in the mouse adult central nervous system. All retinoic acid receptors (á, â and ã) and retinoid X receptors (á, â and ã) were detected and found to exhibit specific patterns of expression in various areas of the telencephalon, diencephalon and rhombencephalon. The protein localization of several retinoic acid receptors and retinoid X receptors did not correlate with the distribution of the corresponding RNA transcripts, as studied by in situ hybridization and RNase protection assays. This suggests that the expression of retinoid receptors could be post-transcriptionally regulated, which may contribute to their specific localization in the adult nervous system. 1999 IBRO. Published by Elsevier Science Ltd. Key words: retinoid receptors, RAR, RXR, CNS, in situ hybridization, immunohistochemistry.
Biologically active derivatives of vitamin A (essentially retinoic acids) are indispensable for morphogenesis, differentiation and homeostasis of a number of tissues including the nervous system (see Refs 22, 24, 26, 39 and references therein). Complete deficiency in vitamin A during development and after birth is lethal. Deprivation of vitamin A (retinol) in the mouse embryo at 10.5 d.p.c. by inhibition of the synthesis of retinol binding protein (a protein involved in retinol transport) results in cranial neural tube malformations and exencephaly.2 Retinoiddepleted quail40,41 and rat11 embryos also exhibit defects in their CNS. Furthermore, disruption of the retinoic acid response elements of the Hoxa-1 and Hoxb-1 genes causes CNS developmental defects.14,17 In rodents vitamin A deprivation during postnatal life leads to degeneration of the peripheral nervous system (PNS), which affects spinal cord structures, as well as cranial and peripheral nerves.1 However, some of these PNS abnormalities could be due to skeletal malformations.46,58 Retinoic acid (RA) excess also strongly affects neural development in different species, resulting in anencephaly, exencephaly, microphtalmia, and other brain and spinal cord anomalies.31,32,47 *To whom correspondence should be addressed. †Present address: Physiology Unit, Momed, University of Wales, P.O. Box 911, Cardiff CF1 3US, U.K. Abbreviations: D2R, dopamine 2 receptor; ISH, in situ hybridization; MOB, main olfactory bulb; PNS, peripheral nervous system; PPARs, peroxisomal proliferatoractivated receptors; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RBP, retinol binding protein; TRs, thyroid hormone receptors; VAD, vitamin A deficiency; VDR, vitamin D3 receptor.
Nutritional studies have revealed that vitamins in general can modulate several postnatal brain functions, but the contribution of vitamin A in these modulations was not investigated,6,23,34 even though in vitro studies have shown that RA could play a role in the control of expression of several genes involved in neurotransmission processes, including choline acetyltransferase and dopamine receptors.3,15,27,49,55 The retinoid signal is transduced by two families of nuclear receptors: the retinoic acid receptor (RAR) isotypes á, â and ã, which bind and are activated by all-trans and 9-cis retinoic acid, and the retinoid X receptor (RXR) isotypes á, â and ã, whose ligand is 9-cis RA (for review see Refs 7, 8, 19, 21, 33 and 43). In vitro studies have shown that RXRs can act either on their own as homodimers or as heterodimeric partners for a number of nuclear receptors including the RARs, the thyroid hormone receptors (TRs), the vitamin D3 receptor, the peroxisomal proliferatoractivated receptors (PPARs) and several orphan receptors (for review see Refs 8, 20, 43 and 50). Gene knockouts resulting in single or multiple RAR and/or RXR mouse null mutants have revealed that retinoid receptors play a major role in many developmental processes and have established that the retinoid signal is transduced by RAR-RXR heterodimers (see Refs 18, 24 and references therein). Notably, early neural defects, including the lack of hindbrain and cerebellar structures, were characterized in RARá/ RARã double mutants.36 Interestingly, a recent study has shown that mice knocked out for several RARs and RXRs exhibit behavioural abnormalities.29 We have now investigated the expression pattern of RARs and RXRs in adult mouse CNS, as a prerequisite to future studies on the role of retinoids
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and their receptors in postnatal development and function of the brain. EXPERIMENTAL PROCEDURES
Immunocytochemical detection of receptor proteins Brain and cervical spinal cord were prepared from fourmonth-old C57/BL6 male mice (Jackson Laboratories). Ten-micrometre-thick frozen tissue sections were collected on gelatine-coated slides, fixed for 15 min in Zamboni fixative (buffered saturated aqueous solution of picric acid and 4% paraformaldehyde) at room temperature and treated with 1% H2O2 to block endogenous peroxidase. Staining with RXRâ57 and RXRã (TEBU, France) antibodies was as described.57 Polyclonal antibodies specific to all known isoforms of RARá [RPá(F)16], RARâ [RPâ(F)2,52 RARã [RPã(mF)18] and RXRá1 [RPRXá(A)53] were purified by precipitation with ammonium sulphate and filtration on sulfolink gel columns (Pierce, U.S.A.) coated with the corresponding antigenic synthetic peptides. Primary antibodies were revealed using the ABC system (Vector, U.S.A.) according to the manufacturer’s instructions. The specificity of each immunodetection was controlled on brain sections from mutant mice null for the corresponding receptors [RARá,38 RARâ,18 RARã,35 RXRâ25 and RXRã28], or carrying a deletion mutation of the A/B region of RXRá (B. Mascrez and P. Chambon, unpublished observations). In situ hybridization The synthesis of 35S-labelled antisense and sense (as controls) riboprobes from cDNA templates were as described for RARá, â and ã12 and for RXRá, â and ã.13 Ten-micrometre-thick cryosections of four-month-old C57/B6 male mice (Jackson Laboratories) were used for hybridization.10 RNase protection assays Different brain areas of four-month-old C57/BL6 male mice were prepared from isolated brains by cutting the corpus callosum at the hemisphere junction level, and separation of the left and right cortices together with the underlying corpus callosum. The neocortex, the striatum, the hippocampus and all other brain structures were subsequently dissected out. Total RNA was prepared using the isothiocyanate–phenol technique.9 Five micrograms of RNA was used in each hybridization reaction. To prepare a RXRã riboprobe, a cDNA fragment corresponding to nucleotides 304–1497 (numbering according to GenBank
sequence accession no. M84817) was subcloned into the EcoRI site of pBluescript SK() and the probe was synthesized with T7 RNA polymerase using a XbaI-linearized template. The preparation of other riboprobes and hybridization conditions were as described previously.28,35,38,60 Relative transcript levels were determined by densitometric analysis (Bio-Rad GS700 Imaging Densitometer) of RNase protection autoradiograms. After correction for background signal, the intensity of each signal was expressed in arbitrary units relative to the signal given by histone H4 RNA present in the corresponding samples. RESULTS
Distributions of retinoic acid receptor and retinoid X receptor proteins in the adult mouse brain The immunohistochemical distribution of RARá, â and ã, as well as that of RXRá, â and ã, was investigated on adult brain, spinal cord and pituitary sections. With the exception of RARã, for which low levels of protein were detected in most diencephalic and rhombencephalic regions, the distribution of the other receptors was restricted to specific structures of the telencephalon, diencephalon and rhombencephalon. Interestingly, none of the RAR and RXR proteins could be revealed in the mesencephalon, a region in which RARá, RARâ, RXRá and RXRâ transcripts have been detected.13,54 The overlapping patterns of expression of RARs and RXRs are summarized in Table 1, and some typical immunostained sections are displayed in Fig. 1. Telencephalon. Several components of the olfactory system contained cells expressing RARá, RARã and RXRá. These receptors were detected in the main olfactory bulb (MOB) (e.g., RARá in the anterior olfactory nucleus, Fig. 1B), with the exception of RARá in mitral and tufted cells (the principal output cells of the bulb). All three receptors were also found in the targets of MOB efferents, which constitute the primary olfactory cortex (olfactory tubercle, piriform and entorhinal cortex). RARá, RARã and RXRá were also expressed in the targets of primary
Abbreviations used in the figures AcbC AcbSh AO AP Arc BL BLA Ca CA CC CeL CeM ChP CPu DG FR1 Hif Hil
nucleus accumbens core nucleus accumbens shell anterior olfactory nucleus area postrema arcuate hypothalamus basolateral amygdaloid nucleus basolateral amygdaloid nucleus, anterior caudal fields CA1–3 of Ammon’s horn corpus callosum central amygdaloid nucleus central amygdaloid nucleus, medial division choroid plexus caudate putamen dentate gyrus frontal cortex, area 1 hippocampal fissure hilus of the dentate gyrus
IG IGr La Me MHb Par1 Pir Ro Sol TT Tu VC vfu VMH L VIII 1–6 7 12
indusium griseum internal granular layer of the olfactory bulb lateral amygdaloid nucleus medial amygdaloid nucleus medial habenular nucleus parietal cortex piriform cortex rostral solitary tract nucleus tenia tecta olfactory tubercle ventral cochlear nucleus ventral funiculus of spinal cord ventromedial hypothalamic nucleus lamina VIII of the spinal cord cortical layers facial nucleus hypoglossal nucleus
Table 1. Distribution of retinoic acid receptor á, â and ã and retinoid X receptor á, â, and ã receptor proteins in the mouse CNS
Telencephalon Main olfactory bulb Mitral cell layer olfactory bulb Internal plexiform layer olfactory bulb Glomerular layer olfactory bulb Anterior olfactory nucleus Olfactory tubercle Tenia tecta Indusium griseum Cortex Infralimbic Piriform Cingulate, frontal, parietal Entorhinal Perirhinal Hippocampus Field CA1 Field CA2 Field CA3 Dentate gyrus Hilus of the dentate gyrus Striatum Caudate–putamen Accumbens nucleus core Accumbens nucleus shell Amygdala Central amygdaloid nucleus Central amygdaloid nucleus, medial division Amygdaloid basolateral nucleus Amygdaloid lateral nucleus Amygdaloid area, posteromed. Intercalated amygdaloid nucleus, main Diencephalon Thalamus Ventral posterior thalamic nucleus Reticular thalamic nucleus Anterodorsal thalamic nucleus Medial geniculate nucleus Medial habenular nucleus Hypothalamus Arcuate hypothalamus Dorsomedial hypothalamic nucleus Ventromedial hypothalamic nucleus Anterior hypothalamic nucleus Rhombencephalon Cerebellar lobules Pons Facial nucleus Cochlear nucleus Motor trigeminal nucleus Princ. sens. trigeminal nucleus ventrolat. Abducens nucleus Transverse fibre pons Pontine nucleus Medulla oblongata Solitary tract and area postrema Rostroventrolateral reticular nucleus Hypoglossal nucleus Medullar reticular field Spinal cord Laminae II, III Laminae IV–VI Laminae VII–VIII Laminae IX, X Funiculus Pituitary Anterior pituitary Intermediate lobe Posterior pituitary Choroid plexus
RARá
RARâ
RARã
RXRá
RXRâ
RXRã
–* + ++ + –* +++ +++
–* –* –* +* +* – –
++ + +++ ++ +++ – –
+ + ++ + + + +
–* –* –* +* –* –* –*
– – – – – – –
+ – ++(a) – +
–* – – – –
+++ +++ ++(b) + +
+ + +(b) + +
–* –* ++(c) –* –*
– – – – –
+++ +++ ++ – –
– – – – –
+++ +++ ++ +++ +
–* –* –* + +
++ ++ + + –
– – – – –
–* –* –*
+++ +++ +++
++ ++ +
–* –* –*
+ + +
+++ ++ +++
+ + ++ ++ – –
– – – – – –
+ + ++ –* –* –*
+ –* –* + –* ++
–* –* –* –* –* –*
– + ++ – ++ –
++ + ++ ++ ++
– – – – –
+ + + + –
–* –* –* –* +
++ + –* –* +
– – – – –
–* –* –* –*
+++ +++ – –
+ ++ ++ +
–* –* –* –*
–* –* –* –*
+++ +++ +++ ++
+++
–
+
+
–*
–
++ ++ +++ +++ ++ ++ ++
– – – – – – –
+ – + + + + +
++ ++ + + + – –
–* –* –* –* –* + +
– – – – – – –
– – – –
+++ +++ + –
– + – +
++ +* + +
–* –* –* –*
– – – –
– ++ ++ ++ –
– + + + –
+++ ++ ++ ++ ++
–* –* ++ –* –*
–* + + + +
+ ++ ++ ++ ++
++ ++ +++ +
+++ ++ +++ ++
+ + + ++
+ ++ + ++
+ + + ++
+++ – – –
The intensity of the nuclear immunoreaction signal was visually estimated as being either weak (+), medium (++) or strong (+++) by comparison with the strongest signal obtained with the cognate antibody. In all areas scored positively the percentage of immunostained cells was greater than 20%. *Receptor transcripts were readily detected, but the corresponding proteins were either low or undetectable; (a), (b) and (c): receptor proteins were present in layer 5, 2–4 and 6, and in all layers, respectively.
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Fig. 1. Immunodetection of retinoid receptors in the nervous system. Coronal sections through tenia tecta (A, magnification: 40), main olfactory bulb (B, magnification: 40), caudate–putamen (C, magnification: 40), solitary tract and hypoglossal nucleus (D, G, magnification: 40), left and right hippocampus (E, I, magnification: 40), neocortex (F, magnification: 40), hilus of the dentate gyrus (H, magnification: 40), cervical spinal cord (J, magnification: 100), arcuate hypothalamus (K, magnification: 100) and basolateral amygdaloid nucleus (L, magnification: 40) were immunostained with antibodies specific for RARá (A, B), RARâ (C, D), RARã (E, F), RXRá (G, H), RXRâ (I, J) and RXRã (K, L). The nuclear staining in selected regions (boxes) is shown at 400 magnification in a corner of each panel. In all panels the dorsal region is at the top, except in panel I where it is on the right as indicated by the arrow. For descriptions of panels G and I, see regions depicted on the views D and E, respectively.
Expression of RARs and RXRs in CNS of adult mouse
olfactory cortex projections, e.g., the infralimbic cortex. The strongest expression of RARá in the olfactory system was found in tenia tecta and indusium griseum (e.g., Fig. 1A). The highest levels of retinoid receptors were observed in basal ganglia. Caudate–putamen and the shell and core of the nucleus accumbens were strongly labelled with antibodies specific for RARâ (e.g., Fig. 1C) and RXRã. A large fraction of cells in these regions was also immunopositive for RARã and RXRâ proteins. All RAR and RXR isotypes were detected in the limbic system and limbic-associated cortical structures. In these areas the cells immunostained for RARá, RARã (e.g., Fig. 1E) and RXRâ (e.g., Fig. 1I) were mostly localized in the hippocampus CA fields of Ammon’s horn, whereas the predominant receptor in the dentate gyrus was RARã, together with lower levels of RXRá and RXRâ (see Fig. 1H and Table 1, and data not shown). With the exception of RARâ and RXRâ, all retinoid receptors were also expressed in the hippocampal-associated amygdaloid area (e.g., Fig. 1L for RXRã). All receptors, except for RARâ, were found in the cingulate, frontal, parietal and temporal cortices. However, they were differentially expressed in the various layers, with RARá in layer 5, and RARã (e.g., Fig. 1F) and RXRá in layers 2–4 and 6 of the neocortex. RARã was also expressed in more ventral cortices i.e. perirhinal and entorhinal, whereas RXRâ was detectable throughout all layers of the cortex. Diencephalon. This region was characterized by the mutually exclusive expression patterns of the RARâ/ RXRã and the RARá/RXRâ pairs in hypothalamic and thalamic areas, respectively. Hypothalamic structures showing high immunoreactivity were localized mainly in the periventricular and medial zones of the tuberal region, which are thought to be functionally important in the production of hypophysiotropic hormones (e.g., arcuate nucleus; Fig. 1K) or in mediating somatomotor aspects of complex motivated behaviours in rodents (ventromedial nucleus; see Ref. 48) In the thalamus, signals specific for RARá and RXRâ were detected in ventroposterior and reticular nuclei. However, RXR proteins, in contrast to RXR transcripts (data not shown), could not be detected in other thalamic nuclei expressing RARs, e.g., medial geniculate, possibly due to low protein levels. A weak staining of cells positive for RARã was observed throughout these thalamic areas. Rhombencephalon and spinal cord. Retinoid receptors were detected in all three major subdivisions of the hindbrain. RARá and RARã were found predominantly in cerebellum and pons, whereas medulla oblongata expressed almost exclusively RARâ. RXRá distribution overlapped with that of RARs. In the cerebellum, immunoreactive cells were mostly
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restricted to the cerebellar granular layer. RARâ (e.g., Fig. 1D) and RXRá (e.g., Fig. 1G) were strongly expressed in the solitary tract and area postrema of the medulla oblongata. The expression of these receptors in rostroventrolateral reticular nucleus and solitary tract colocalized with the A1 and A2 noradrenaline cell groups.48 In pons, several motor nuclei (trigeminal motor, abducens, facial and hypoglossal nucleus) and sensory nuclei (cochlear, principal sensory trigeminal nucleus) exhibited relatively high RARá and moderate RARã and RXRá levels of expression. The spinal cord exhibited a quantitatively differential pattern of expression of various receptors. Most of the cells expressed RARã, whose signal was distributed across all the structures of the cervical spinal cord, and particularly strongly in lamina II and III. RXRã was also present in all cell layers, whereas other receptors were more restricted in their distribution, including laminae IV-X for RARá, RARâ and RXRâ (e.g., Fig. 1J) and the motoneurons of laminae VII and VIII for RXRá. In situ distribution of retinoid receptor transcripts in the mouse brain Each of the six riboprobes hybridized specifically to their cognate RNAs, as the in situ hybridization (ISH) signals obtained with antisense probes were always above the background signals obtained with sense controls (data not shown). Moreover, the presence of retinoid receptor transcripts in various brain regions was confirmed by RNase protection analysis (see next section and Fig. 3). The ISH distribution of RARá, RARã, RXRá and RXRâ transcripts was almost ubiquitous, and much wider than anticipated from the immunohistochemical analysis. All of these transcripts, but not the corresponding proteins, were detected, e.g., in the medial amygdaloid nucleus (Fig. 2I, K, L, M). In contrast, RARâ and RXRã RNAs were present only in the brain regions where the corresponding proteins were found (Fig. 2C, J, R and G, N, W, and Table 1). Notably, RARâ and RXRã transcripts were present in the striatum (Fig. 2C, G) and absent from midbrain (data not shown), in which RARâ was found to be expressed before and shortly after birth.54,59 Interestingly, all retinoid receptor transcripts were identified in the striatum (Fig. 2B–G and Fig. 3, lane 3) or hypothalamus (Fig. 2I–N and Fig. 3, lane 7), whereas only RARâ, RARã, RXRâ and RXRã, or RARâ, RARã and RXRã proteins could be immunodetected in these brain regions, respectively (Table 1). In the rhombencephalon, with the exception of RXRâ, the RNA expression patterns correlated with the protein receptor distribution (Fig. 2P–W and Table 1). Note that, as estimated from the ISH data, the levels of RARã and RXRâ transcripts were higher in the cerebellum than in the striatum (compare in Fig. 2D to S and F to U, respectively). In contrast, densitometric analysis of the RNase
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Fig. 2. Expression of retinoid receptor transcripts in the adult brain. Selected planes of coronal sections through (A) caudate–putamen and nucleus accumbens, (H) hippocampus and arcuate hypothalamus, (O) cerebellar lobules and abducens and facial nuclei are presented as bright-field views (top row). The corresponding dark-field in situ hybridization views are shown for RARá (B, I, P), RARâ (C, J, R), RARã (D, K, S), RXRa (E, L, T), RXRâ (F, M, U) and RXRã (G, N, W).
Expression of RARs and RXRs in CNS of adult mouse
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tum, cerebellum, pons, hippocampus, hypothalamus, pituitary, as well as from caudal and rostral regions of the spinal cord. The distribution of RARâ and RXRã transcript was more restricted than that of the other receptors, with relatively high levels in the striatum (Fig. 3, lane 3), in good agreement with ISH data (Fig. 2C, G). Transcripts of the other receptors were widely expressed in different brain structures, again in agreement with ISH data. The RAR isoform distribution exhibited some specificity, as RARá2 RNA was found mainly in the hippocampus, where transcript levels were at least six-fold higher (2400 Units) than in other brain regions, as determined from densitometric measurements (Fig. 3, lane 6). Transcripts of RARá1 were detected at low levels (50–300 Units) in all brain regions (Fig. 3). In contrast, high levels of both RARâ2 and RARâ1,3,4 isoform transcripts were found in the striatum (Fig. 3, lane 3). RNase protection assays also revealed the presence of a new tissue-specific RXRá transcript, corresponding to a 300 nucleotide-long protected fragment (see RXRá4 in Fig. 3). This isoform transcript, which is shorter by almost 300 nucleotides in its 5 region than that previously characterized in skeletal muscles and heart (594 nucleotides protected fragment28), may correspond to a novel RXRá isoform (see Discussion) in addition to ubiquitous RXRá1 and testis-specific RXRá2 and 3 isoforms.5,42 Interestingly, RXRá4, as well as RARá1 and RARâ transcripts, were differentially expressed in caudal and rostral spinal cord (Fig. 3, lanes 9 and 10).
DISCUSSION
Fig. 3. Determination of RAR and RXR transcript expression in different brain structures and spinal cord. RNAs were hybridized with receptor specific antisense riboprobes (lanes 1–10). Hybridization with histone H4 antisense riboprobe was used as an internal control. (Ca) and (Ro), caudal and rostral, respectively.
protection assays revealed higher levels of RARã and RXRâ transcripts in the striatum (125, 340 and 2000 Units for RARã1, RARã2, and RXRâ, respectively; Fig. 3, lane 3) than in the cerebellum (80, 110 and 1200 Units for RARã1, RARã2 and RXRâ, respectively; Fig. 3, lane 4). Differences in cell density between striatum and cerebellum may be at the origin of these discrepancies. Analysis of retinoid receptor transcripts in dissected CNS structures To further correlate the expression patterns of retinoid receptor proteins and transcripts, as well as to gain insight into the expression of receptor isoforms, we performed RNase protection analysis of RNA extracted from the olfactory bulb, cortex, stria-
The present analysis reports the protein and transcript expression patterns of the multiple RAR and RXR isotypes in the adult mouse CNS. RARã (and RARá to a lesser degree) and RXRá proteins were the only receptors to be expressed in the different components of the olfactory system, from the mitral and plexiform layers to entorhinal and perirhinal cortices. The functional relevance of this expression is unknown, as no effects of retinoids on functioning of the olfactory system have yet been reported, although retinoid receptors may play a role in the formation of the olfactory pathway which is known to be dependent on RA signalling.30 The gustatory system is also a potential target of retinoid action, as RARâ and RXRá are expressed in the solitary tract and area postrema (Fig. 1D, G, Table 1), which play a role in numerous neurovegetative functions.48 In this respect, the weight deficiency of RARâ null mutant mice18 may possibly result from reduced food consumption. However, a defective RA signalling in the hypothalamo–pituitary axis could also account for this deficiency, as high levels of RARâ and RXRã were found in the hypothalamus and pituitary (Fig. 1K and Table 1; see also Ref. 57).
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Strong expression of RARâ and RXRã in the striatum, a structure implicated, among other processes, in the control of motor functions,48 is the most remarkable feature of RAR and RXR isotype distribution in the brain. This striatal expression persists from early stages of development.13,54 Interestingly, RA appears to be synthesized in mesostriatal and mesolimbic dopaminergic neurons.45 This indicates that the dopaminergic system could be a target for retinoid action in the striatum. In fact, the expression of the dopamine 2 receptor (D2R) can be induced by RA in neuroblastoma or MMQ cell lines15,55 and mice mutated for RARâ and RARã exhibit a decrease in expression of striatal dopamine receptors (D1R and D2R) and locomotor defects.29 Interestingly, the pattern of expression of receptor proteins and transcripts in the adult brain does not correspond to that previously reported for the developing and neonatal CNS.13,54,59 For instance, the relatively strong expression of the RARã protein in the hippocampus and in the striatum is in contrast with its weak expression in discrete regions of the developing brain59 and the apparent absence of its transcripts in fetal and neonatal CNS.54 The absence of RARâ in the adult hippocampal structures also contrasts with the pre- and early postnatal expression of this receptor in these structures.59 Similarly, the lack of RARâ transcripts and protein in midbrains of adult mice is in contrast with the expression of this protein during pre- and neonatal periods.13,54,59 These differences between the distribution pattern of retinoid receptors in early and late postnatal mouse brain most probably reflect a role of retinoids in CNS maturation events. The comparison of the distribution pattern of retinoid receptor transcripts, as judged from ISH and RNase protection studies, with that of the corresponding proteins reveals that the expression of some of these receptors is post-transcriptionally regulated (see ‘‘*’’ in Table 1). For instance, in the olfactory bulbs, there is a discrepancy between a weak and restricted RARâ protein expression and readily detected RARâ transcripts (270 Units, Fig. 3, lane 1), whereas a reverse situation is found in the hypothalamus in which the expression of the RARâ protein is much stronger relative to transcript levels (470 Units for RARâ2; see Fig. 3, lane 7). Evidence indicating that RARâ2 expression could be post-transcriptionally controlled was previously reported.51 Similar mechanisms may operate in the case of RARá, as RARá proteins could not be detected in the striatum, hypothalamus and most of the midbrain structures, even though RARá transcript levels were similar (within a factor of 2) to those found in the neocortex and hindbrain (Fig. 3, compare lanes 2, 3 and 4), in which the protein could be readily revealed (Table 1). Relatively high amounts of RXRá and RXRâ transcripts were also in contrast with low levels of the corresponding
proteins in most brain regions (see for instance the striatum and cerebellum in Table 1 and in Fig. 3). We cannot exclude, however, the presence of the RXRá4 isoform in the regions where its transcript is present (see, e.g., striatum or spinal cord in Fig. 3), as the antibody used to detect the RXRá protein was raised against the N-terminal region of RXRá1, which appears to be absent from RXRá4. Receptor transcript analysis by RNase protection revealed differential, tissue-specific isoform distributions, which were most obvious in the case of RARâ and RARá (Fig. 3). Note, however, that the present study does not provide a quantitative estimation of these differences as the signal intensity was increasing with the size of the protected fragment. It is noteworthy that the RNase protection assay has uncovered a novel RXRá mRNA (see RXRá4 in Fig. 3), in addition to the expected RXRá1 mRNA. Although the exact nature of this novel species is unclear, it may correspond to a new RXRá isoform, as using a RXRá specific probe, at least two new 1.8and 1.5-kb-long RNA species were detected by Northern blot analysis, in addition to the usual 5.6-kb-long transcript (42, and data not shown). In any event, this putative new RXRá isoform appears to be CNS-specific, as it could not be found in other adult tissues (heart, muscle, testis, skin) or in 14.5 day mouse fetus RNA analysed by RNase protection (data not shown). Finally, it is worth stressing that the expression patterns of RARs and RXRs overlap in all CNS structures. This is not surprising, as RAR/RXR heterodimers are most probably the functional units transducing the retinoid signal in vertebrates.8,24,26 However, RXRs may also be implicated in additional signalling pathways, as they can also form functional heterodimers in vitro with other nuclear receptors8,43,44 which are expressed in the CNS (TRá,4 Nur77,56 Nurr1,61 COUP-TFI and ARP-137). In this respect, it is interesting that Nurr1, a putative RXR heterodimerization partner, was shown to be indispensable for the development and maintenance of midbrain dopaminergic neurons.61 The present report provides the basis for future studies aimed at linking CNS functions to the vitamin A nutritional status. CONCLUSIONS
The present study reports a thorough analysis of the expression of retinoid receptors in the adult mouse CNS. Each RAR and RXR receptor exhibits a specific pattern of expression which is different from that reported earlier for pre- and neonatal CNS. These changes may reflect a role of retinoids in the maturation and function of the CNS. Moreover, differences in transcript and protein distribution of several receptors suggest the existence of a post-transcriptional control of RAR and RXR expression.
Expression of RARs and RXRs in CNS of adult mouse Acknowledgements—We thank C. Rochette-Egly for a gift of anti-RARá, â and ã and RXRá antibodies, W. Chin for anti-RXRâ antibody and M. Mark for critical reading of the manuscript. We are also thankful to Betty Feret for excellent technical assistance and to J. M. LaFontaine for help in preparation of the manuscript. This work was supported by funds from the Centre National de la
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Recherche Scientifique (CNRS), the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), the Colle`ge de France, the Hoˆpital Universitaire de Strasbourg, the Association pour la Recherche sur la Cancer (ARC), the Human Frontier Science Program and Bristol-Myers Squibb. W. K. was a recipient of a fellowship from the Ministe`re de la Recherche and the ARC.
REFERENCES
1. Aberle S. B. D. (1933) Neurological disturbances in rats reared on diets deficient in vitamin A. J. Nutr. 7, 445–459. 2. Bavik C., Ward S. J. and Chambon P. (1996) Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis. Proc. natn. Acad. Sci. U.S.A. 93, 3110–3114. 3. Berrard S., Faucon Biguet N., Houhou L., Lamouroux A. and Mallet J. (1993) Retinoic acid induces cholinergic differentiation of cultured newborn rat sympathetic neurons. J. Neurosci. Res. 35, 382–389. 4. Bradley D. J., Young W. S. and Weinberger C. (1989) Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary. Proc. natn. Acad. Sci. U.S.A. 86, 7250–7254. 5. Brocard J., Kastner P. and Chambon P. (1996) Two novel RXRá isoforms from mouse testis. Biochem. biophys. Res. Commun. 229, 211–218. 6. Butler P. D., Susser E. S., Brown A. S., Kaufmann C. A. and Gorman J. M. (1994) Prenatal nutritional deprivation as a risk factor in schizophrenia: preclinical evidence. Neuropsychopharmacology 11, 227–235. 7. Chambon P. (1994) The retinoid signaling pathway: molecular and genetic analyses. Semin. Cell Biol. 5, 115–125. 8. Chambon P. (1996) A decade of molecular biology of retinoic acid receptors. Fedn Am. Socs exp. Biol. J. 10, 940–954. 9. Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol– chloroform extraction. Analyt. Biochem. 162, 156–159. 10. De´cimo D., Georges-Labouesse E. and Dolle´ P. (1995) In situ hybridization of nucleic acid probes to cellular RNA. In Genes Probes 2, A Practical Approach (eds Hames B. D. and Higgis S.), pp. 183–210. Oxford University Press, Oxford. 11. Dickman E. D., Thaller C. and Smith S. M. (1997) Temporally-regulated retinoic acid depletion produces specific neural crest, ocular and nervous system defects. Development 124, 3111–3121. 12. Dolle´ P., Ruberte E., Kastner P., Petkovich M., Stoner C. M., Gudas L. J. and Chambon P. (1989) Differential expression of genes encoding alpha, beta and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse. Nature 342, 702–705. 13. Dolle´ P., Fraulob V., Kastner P. and Chambon P. (1994) Developmental expression of murine retinoid X receptor (RXR) genes. Mech. Dev. 45, 91–104. 14. Dupe´ V., Davenne M., Brocard J., Dolle´ P., Mark M., Dierich A., Chambon P. and Rijli F. M. (1997) In vivo functional analysis of the Hoxa-1 3 retinoic acid response element (3 RARE). Development 124, 399–410. 15. Farooqui S. M. (1994) Induction of adenylate cyclase sensitive dopamine D2-receptors in retinoic acid induced differentiated human neuroblastoma SHSY-5Y cells. Life Sci. 55, 1887–1893. 16. Gaub M. P., Rochette-Egly C., Lutz Y., Ali S., Matthes H., Scheuer I. and Chambon P. (1992) Immunodetection of multiple species of retinoic acid receptor alpha: evidence for phosphorylation. Expl Cell Res. 201, 335–346. 17. Gavalas A., Studer M., Lumsden A., Rijli F. M., Krumlauf R. and Chambon P. (1998) Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch. Development 125, 1123–1136. 18. Ghyselinck N. B., Dupe´ V., Dierich A., Messadeq N., Garnier J. M., Rochette-Egly C., Chambon P. and Mark M. (1997) Role of the retinoic acid receptor beta (RARâ) during mouse development. Int. J. dev. Biol. 41, 425–448. 19. Giguere V. (1994) Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr. Rev. 15, 61–79. 20. Glass C. K. (1996) Some new twists in the regulation of gene expression by thyroid hormone and retinoic acid receptors. J. Endocr. 150, 349–357. 21. Gronemeyer H. and Laudet V. (1995) Transcription factors 3: nuclear receptors. Protein Profile 2, 1173–1308. 22. Gudas L. J. (1994) Retinoids and vertebrate development. J. biol. Chem. 269, 15,399–15,402. 23. Heseker H., Kubler W., Pudel V. and Westenhoffer J. (1992) Psychological disorders as early symptoms of a mild-to-moderate vitamin deficiency. Ann. N. Y. Acad. Sci. 669, 352–357. 24. Kastner P., Mark M. and Chambon P. (1995) Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell 83, 859–869. 25. Kastner P., Mark M., Leid M., Gansmuller A., Sugawara W., Grondona J. M., De´cimo D., Krezel W., Dierich A. and Chambon P. (1996) Abnormal spermatogenesis in RXRâ mutant mice. Genes Dev. 10, 80–92. 26. Kastner P., Mark M., Ghynselinck N., Krezel W., Dupe´ V., Grondona J. M. and Chambon P. (1997) Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development. Development 124, 313–326. 27. Kobayashi M., Matsuoka I. and Kurihara K. (1994) Cholinergic differentiation of cultured sympathetic neurons induced by retinoic acid. Induction of choline acetyltransferase-mRNA and suppression of tyrosine hydroxylasemRNA levels. Fedn Eur. biochem. Socs Lett. 337, 259–264. 28. Krezel W., Dupe´ V., Mark M., Dierich A., Kastner P. and Chambon P. (1996) RXRã null mice are apparently normal and compound RXRá+/RXRâ//RXRã/ mutant mice are viable. Proc. natn. Acad. Sci. U.S.A. 93, 9010–9014. 29. Krezel W., Ghyselinck N., Samad T., Dupe´ V., Kastner P., Borrelli E. and Chambon P. (1998) Impaired locomotion and dopamine signaling in retinoid receptor mutant mice. Science 279, 863–867. 30. La Mantia A. S., Colbert M. C. and Linney E. (1993) Retinoic acid induction and regional differentiation prefigure olfactory pathway formation in the mammalian forebrain. Neuron 10, 1035–1048.
1300 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61.
W. Krez˙el et al. Lammer E. J. and Armstrong D. L. (1992) Malformations of hindbrain structures among humans exposed to isotretinoin (13-cis-retinoic acid) during early embryogenesis. In Retinoids in Normal Development and Teratogenesis (ed. Morris-Kay G. M.), pp. 281–295. Oxford University Press, Oxford. Langman J. and Welch G. W. (1967) Excess vitamin A and development of the cerebral cortex. J. comp. Neurol. 131, 15–26. Leid M., Kastner P. and Chambon P. (1992) Multiplicity generates diversity in the retinoic acid signalling pathways. Trends biochem. Sci. 17, 427–433. Levitsky D. A. and Strupp B. J. (1995) Malnutrition and the brain: changing concepts, changing concerns. J. Nutr. 125, 2212S–2220S. Lohnes D., Kastner P., Dierich A., Mark M., Le Meur M. and Chambon P. (1993) Function of retinoic acid receptor gamma in the mouse. Cell 73, 643–658. Lohnes D., Mark M., Mendelsohn C., Dolle´ P., Dierich A., Gorry P., Gansmuller A. and Chambon P. (1994) Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development 120, 2723–2748. Lopes da Silva S., Cox J. J., Jonk L. J., Kruijer W. and Burbach J. P. (1995) Localization of transcripts of the related nuclear orphan receptors COUP-TF I and ARP-1 in the adult mouse brain. Molec. Brain Res. 30, 131–136. Lufkin T., Lohnes D., Mark M., Dierich A., Gorry P., Gaub M. P., Le Meur M. and Chambon P. (1993) High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice. Proc. natn. Acad. Sci. U.S.A. 90, 7225–7229. Maden M. and Holder N. (1992) Retinoic acid and development of the central nervous system. Bioessays 14, 431–438. Maden M., Gale E., Kostetskii I. and Zile M. (1996) Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr. Biol. 6, 417–426. Maden M., Graham A., Gale E., Rollinson C. and Zile M. (1997) Positional apoptosis during vertebrate CNS development in the absence of endogenous retinoids. Development 124, 2799–2805. Mangelsdorf D. J., Borgmeyer U., Heyman R. A., Zhou J. Y., Ong E. S., Oro A. E., Kakizuka A. and Evans R. M. (1992) Characterization of three RXR genes that mediate the action of the 9-cis retinoic acid. Genes Dev. 6, 329–344. Mangelsdorf D. J. and Evans R. M. (1995) The RXR heterodimers and orphan receptors. Cell 83, 841–850. Mangelsdorf D. J., Thummel C., Beato M., Herrlich P., Schu¨tz G., Umesono K., Blumberg B., Kastner P., Mark M., Chambon P. and Evans R. M. (1995) The nuclear receptor superfamily: the second decade. Cell 83, 835–839. McCaffery P. and Drager U. C. (1994) High levels of a retinoic acid-generating dehydrogenase in the mesotelencephalic dopamine system. Proc. natn. Acad. Sci. U.S.A. 91, 7772–7776. Mellanby E. (1943) Nutrition in relation to bone growth and the nervous system. Proc. R. Soc. B 132, 28–54. Morris G. M. (1975) Abnormal cell migration as a possible factor in the genesis of vitamin A-induced craniofacial anomalies. In New Approaches to the Evaluation of Abnormal Embryonic Development (eds Neubert D. and Merker M. J.), pp. 678–687. Georg Thieme, Stuttgart. Paxinos G. (1995) The Rat Nervous System. Academic, San Diego. Pedersen W. A., Berse B., Schuler U., Wainer B. H. and Blusztajn J. K. (1995) All-trans- and 9-cis-retinoic acid enhance the cholinergic properties of a murine septal cell line: evidence that the effects are mediated by activation of retinoic acid receptor-alpha. J. Neurochem. 65, 50–58. Perlmann T. and Evans R. M. (1997) Nuclear receptors in Sicily: all in the famiglia. Cell 90, 391–397. Reynolds K., Zimmer A. M. and Zimmer A. (1996) Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5 -untranslated region. J. Cell Biol. 134, 827–835. Rochette-Egly C., Gaub M. P., Lutz Y., Ali S., Scheuer I. and Chambon P. (1992) Retinoic acid receptor-beta: immunodetection and phosphorylation on tyrosine residues. Molec. Endocr. 6, 2197–2209. Rochette-Egly C., Lutz Y., Pfister V., Heyberger S., Scheuer I., Chambon P. and Gaub M. P. (1994) Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies. Biochem. biophys. Res. Commun. 204, 525–536. Ruberte E., Friederich V., Chambon P. and Morriss-Kay G. (1993) Retinoic acid receptors and cellular retinoid binding proteins. III. Their differential transcript distribution during mouse nervous system development. Development 118, 267–282. Samad T. A., Krezel W., Chambon P. and Borrelli E. (1997) Regulation of dopaminergic pathways by retinoids: activation of D2 receptor by members of the retinoic acid receptor-retinoid X receptor family. Proc. natn. Acad. Sci. U.S.A. 94, 14,349–14,354. Saucedo-Cardenas O. and Conneely O. M. (1996) Comparative distribution of NURR1 and NUR77 nuclear receptors in the mouse central nervous system. J. molec. Neurosci. 7, 51–63. Sugawara A., Yen P. M., Qi Y., Lechan R. M. and Sugawara W. W. (1995) Isoform-specific retinoid-X receptor (RXR) antibodies detect differential expression of RXR proteins in the pituitary gland. Endocrinology 136, 1766–1774. Wolbach S. B. and Bessey O. A. (1941) Vitamin A deficiency and the nervous system. Archs Path. 32, 689–722. Yamagata T., Momoi M. Y., Yanagisawa M., Kumagai H., Yamakado M. and Momoi T. (1994) Changes of the expression and distribution of retinoic acid receptors during neurogenesis in mouse embryos. Devl Brain Res. 77, 163–176. Zelent A., Mendelshon C., Kastner P., Krust A., Garnier J. M., Ruffenbach F., Leroy P. and Chambon P. (1991) Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing. Eur. molec. Biol. Org. J. 10, 71–81. Zetterstro¨m R. H., Solomin L., Jansson L., Hoffer B. J., Olson L. and Perlmann T. (1997) Dopamine neuron agenesis in Nurr1-deficient mice. Science 276, 248–250. (Accepted 9 June 1998)
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Neuroscience Vol. 89, No. 4, pp. 1291–1300, 1999 Copyright 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00342-X
DIFFERENTIAL EXPRESSION OF RETINOID RECEPTORS IN THE ADULT MOUSE CENTRAL NERVOUS SYSTEM W. KREZ ~ EL,† P. KASTNER and P. CHAMBON* Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS-INSERM-ULP-Colle`ge de France, BP163, 67404 Illkirch Cedex, C. U. de Strasbourg, Strasbourg, France Abstract––The immunocytochemical distribution of retinoid receptors has been analysed in the mouse adult central nervous system. All retinoic acid receptors (á, â and ã) and retinoid X receptors (á, â and ã) were detected and found to exhibit specific patterns of expression in various areas of the telencephalon, diencephalon and rhombencephalon. The protein localization of several retinoic acid receptors and retinoid X receptors did not correlate with the distribution of the corresponding RNA transcripts, as studied by in situ hybridization and RNase protection assays. This suggests that the expression of retinoid receptors could be post-transcriptionally regulated, which may contribute to their specific localization in the adult nervous system. 1999 IBRO. Published by Elsevier Science Ltd. Key words: retinoid receptors, RAR, RXR, CNS, in situ hybridization, immunohistochemistry.
Biologically active derivatives of vitamin A (essentially retinoic acids) are indispensable for morphogenesis, differentiation and homeostasis of a number of tissues including the nervous system (see Refs 22, 24, 26, 39 and references therein). Complete deficiency in vitamin A during development and after birth is lethal. Deprivation of vitamin A (retinol) in the mouse embryo at 10.5 d.p.c. by inhibition of the synthesis of retinol binding protein (a protein involved in retinol transport) results in cranial neural tube malformations and exencephaly.2 Retinoiddepleted quail40,41 and rat11 embryos also exhibit defects in their CNS. Furthermore, disruption of the retinoic acid response elements of the Hoxa-1 and Hoxb-1 genes causes CNS developmental defects.14,17 In rodents vitamin A deprivation during postnatal life leads to degeneration of the peripheral nervous system (PNS), which affects spinal cord structures, as well as cranial and peripheral nerves.1 However, some of these PNS abnormalities could be due to skeletal malformations.46,58 Retinoic acid (RA) excess also strongly affects neural development in different species, resulting in anencephaly, exencephaly, microphtalmia, and other brain and spinal cord anomalies.31,32,47 *To whom correspondence should be addressed. †Present address: Physiology Unit, Momed, University of Wales, P.O. Box 911, Cardiff CF1 3US, U.K. Abbreviations: D2R, dopamine 2 receptor; ISH, in situ hybridization; MOB, main olfactory bulb; PNS, peripheral nervous system; PPARs, peroxisomal proliferatoractivated receptors; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RBP, retinol binding protein; TRs, thyroid hormone receptors; VAD, vitamin A deficiency; VDR, vitamin D3 receptor.
Nutritional studies have revealed that vitamins in general can modulate several postnatal brain functions, but the contribution of vitamin A in these modulations was not investigated,6,23,34 even though in vitro studies have shown that RA could play a role in the control of expression of several genes involved in neurotransmission processes, including choline acetyltransferase and dopamine receptors.3,15,27,49,55 The retinoid signal is transduced by two families of nuclear receptors: the retinoic acid receptor (RAR) isotypes á, â and ã, which bind and are activated by all-trans and 9-cis retinoic acid, and the retinoid X receptor (RXR) isotypes á, â and ã, whose ligand is 9-cis RA (for review see Refs 7, 8, 19, 21, 33 and 43). In vitro studies have shown that RXRs can act either on their own as homodimers or as heterodimeric partners for a number of nuclear receptors including the RARs, the thyroid hormone receptors (TRs), the vitamin D3 receptor, the peroxisomal proliferatoractivated receptors (PPARs) and several orphan receptors (for review see Refs 8, 20, 43 and 50). Gene knockouts resulting in single or multiple RAR and/or RXR mouse null mutants have revealed that retinoid receptors play a major role in many developmental processes and have established that the retinoid signal is transduced by RAR-RXR heterodimers (see Refs 18, 24 and references therein). Notably, early neural defects, including the lack of hindbrain and cerebellar structures, were characterized in RARá/ RARã double mutants.36 Interestingly, a recent study has shown that mice knocked out for several RARs and RXRs exhibit behavioural abnormalities.29 We have now investigated the expression pattern of RARs and RXRs in adult mouse CNS, as a prerequisite to future studies on the role of retinoids
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and their receptors in postnatal development and function of the brain. EXPERIMENTAL PROCEDURES
Immunocytochemical detection of receptor proteins Brain and cervical spinal cord were prepared from fourmonth-old C57/BL6 male mice (Jackson Laboratories). Ten-micrometre-thick frozen tissue sections were collected on gelatine-coated slides, fixed for 15 min in Zamboni fixative (buffered saturated aqueous solution of picric acid and 4% paraformaldehyde) at room temperature and treated with 1% H2O2 to block endogenous peroxidase. Staining with RXRâ57 and RXRã (TEBU, France) antibodies was as described.57 Polyclonal antibodies specific to all known isoforms of RARá [RPá(F)16], RARâ [RPâ(F)2,52 RARã [RPã(mF)18] and RXRá1 [RPRXá(A)53] were purified by precipitation with ammonium sulphate and filtration on sulfolink gel columns (Pierce, U.S.A.) coated with the corresponding antigenic synthetic peptides. Primary antibodies were revealed using the ABC system (Vector, U.S.A.) according to the manufacturer’s instructions. The specificity of each immunodetection was controlled on brain sections from mutant mice null for the corresponding receptors [RARá,38 RARâ,18 RARã,35 RXRâ25 and RXRã28], or carrying a deletion mutation of the A/B region of RXRá (B. Mascrez and P. Chambon, unpublished observations). In situ hybridization The synthesis of 35S-labelled antisense and sense (as controls) riboprobes from cDNA templates were as described for RARá, â and ã12 and for RXRá, â and ã.13 Ten-micrometre-thick cryosections of four-month-old C57/B6 male mice (Jackson Laboratories) were used for hybridization.10 RNase protection assays Different brain areas of four-month-old C57/BL6 male mice were prepared from isolated brains by cutting the corpus callosum at the hemisphere junction level, and separation of the left and right cortices together with the underlying corpus callosum. The neocortex, the striatum, the hippocampus and all other brain structures were subsequently dissected out. Total RNA was prepared using the isothiocyanate–phenol technique.9 Five micrograms of RNA was used in each hybridization reaction. To prepare a RXRã riboprobe, a cDNA fragment corresponding to nucleotides 304–1497 (numbering according to GenBank
sequence accession no. M84817) was subcloned into the EcoRI site of pBluescript SK() and the probe was synthesized with T7 RNA polymerase using a XbaI-linearized template. The preparation of other riboprobes and hybridization conditions were as described previously.28,35,38,60 Relative transcript levels were determined by densitometric analysis (Bio-Rad GS700 Imaging Densitometer) of RNase protection autoradiograms. After correction for background signal, the intensity of each signal was expressed in arbitrary units relative to the signal given by histone H4 RNA present in the corresponding samples. RESULTS
Distributions of retinoic acid receptor and retinoid X receptor proteins in the adult mouse brain The immunohistochemical distribution of RARá, â and ã, as well as that of RXRá, â and ã, was investigated on adult brain, spinal cord and pituitary sections. With the exception of RARã, for which low levels of protein were detected in most diencephalic and rhombencephalic regions, the distribution of the other receptors was restricted to specific structures of the telencephalon, diencephalon and rhombencephalon. Interestingly, none of the RAR and RXR proteins could be revealed in the mesencephalon, a region in which RARá, RARâ, RXRá and RXRâ transcripts have been detected.13,54 The overlapping patterns of expression of RARs and RXRs are summarized in Table 1, and some typical immunostained sections are displayed in Fig. 1. Telencephalon. Several components of the olfactory system contained cells expressing RARá, RARã and RXRá. These receptors were detected in the main olfactory bulb (MOB) (e.g., RARá in the anterior olfactory nucleus, Fig. 1B), with the exception of RARá in mitral and tufted cells (the principal output cells of the bulb). All three receptors were also found in the targets of MOB efferents, which constitute the primary olfactory cortex (olfactory tubercle, piriform and entorhinal cortex). RARá, RARã and RXRá were also expressed in the targets of primary
Abbreviations used in the figures AcbC AcbSh AO AP Arc BL BLA Ca CA CC CeL CeM ChP CPu DG FR1 Hif Hil
nucleus accumbens core nucleus accumbens shell anterior olfactory nucleus area postrema arcuate hypothalamus basolateral amygdaloid nucleus basolateral amygdaloid nucleus, anterior caudal fields CA1–3 of Ammon’s horn corpus callosum central amygdaloid nucleus central amygdaloid nucleus, medial division choroid plexus caudate putamen dentate gyrus frontal cortex, area 1 hippocampal fissure hilus of the dentate gyrus
IG IGr La Me MHb Par1 Pir Ro Sol TT Tu VC vfu VMH L VIII 1–6 7 12
indusium griseum internal granular layer of the olfactory bulb lateral amygdaloid nucleus medial amygdaloid nucleus medial habenular nucleus parietal cortex piriform cortex rostral solitary tract nucleus tenia tecta olfactory tubercle ventral cochlear nucleus ventral funiculus of spinal cord ventromedial hypothalamic nucleus lamina VIII of the spinal cord cortical layers facial nucleus hypoglossal nucleus
Table 1. Distribution of retinoic acid receptor á, â and ã and retinoid X receptor á, â, and ã receptor proteins in the mouse CNS
Telencephalon Main olfactory bulb Mitral cell layer olfactory bulb Internal plexiform layer olfactory bulb Glomerular layer olfactory bulb Anterior olfactory nucleus Olfactory tubercle Tenia tecta Indusium griseum Cortex Infralimbic Piriform Cingulate, frontal, parietal Entorhinal Perirhinal Hippocampus Field CA1 Field CA2 Field CA3 Dentate gyrus Hilus of the dentate gyrus Striatum Caudate–putamen Accumbens nucleus core Accumbens nucleus shell Amygdala Central amygdaloid nucleus Central amygdaloid nucleus, medial division Amygdaloid basolateral nucleus Amygdaloid lateral nucleus Amygdaloid area, posteromed. Intercalated amygdaloid nucleus, main Diencephalon Thalamus Ventral posterior thalamic nucleus Reticular thalamic nucleus Anterodorsal thalamic nucleus Medial geniculate nucleus Medial habenular nucleus Hypothalamus Arcuate hypothalamus Dorsomedial hypothalamic nucleus Ventromedial hypothalamic nucleus Anterior hypothalamic nucleus Rhombencephalon Cerebellar lobules Pons Facial nucleus Cochlear nucleus Motor trigeminal nucleus Princ. sens. trigeminal nucleus ventrolat. Abducens nucleus Transverse fibre pons Pontine nucleus Medulla oblongata Solitary tract and area postrema Rostroventrolateral reticular nucleus Hypoglossal nucleus Medullar reticular field Spinal cord Laminae II, III Laminae IV–VI Laminae VII–VIII Laminae IX, X Funiculus Pituitary Anterior pituitary Intermediate lobe Posterior pituitary Choroid plexus
RARá
RARâ
RARã
RXRá
RXRâ
RXRã
–* + ++ + –* +++ +++
–* –* –* +* +* – –
++ + +++ ++ +++ – –
+ + ++ + + + +
–* –* –* +* –* –* –*
– – – – – – –
+ – ++(a) – +
–* – – – –
+++ +++ ++(b) + +
+ + +(b) + +
–* –* ++(c) –* –*
– – – – –
+++ +++ ++ – –
– – – – –
+++ +++ ++ +++ +
–* –* –* + +
++ ++ + + –
– – – – –
–* –* –*
+++ +++ +++
++ ++ +
–* –* –*
+ + +
+++ ++ +++
+ + ++ ++ – –
– – – – – –
+ + ++ –* –* –*
+ –* –* + –* ++
–* –* –* –* –* –*
– + ++ – ++ –
++ + ++ ++ ++
– – – – –
+ + + + –
–* –* –* –* +
++ + –* –* +
– – – – –
–* –* –* –*
+++ +++ – –
+ ++ ++ +
–* –* –* –*
–* –* –* –*
+++ +++ +++ ++
+++
–
+
+
–*
–
++ ++ +++ +++ ++ ++ ++
– – – – – – –
+ – + + + + +
++ ++ + + + – –
–* –* –* –* –* + +
– – – – – – –
– – – –
+++ +++ + –
– + – +
++ +* + +
–* –* –* –*
– – – –
– ++ ++ ++ –
– + + + –
+++ ++ ++ ++ ++
–* –* ++ –* –*
–* + + + +
+ ++ ++ ++ ++
++ ++ +++ +
+++ ++ +++ ++
+ + + ++
+ ++ + ++
+ + + ++
+++ – – –
The intensity of the nuclear immunoreaction signal was visually estimated as being either weak (+), medium (++) or strong (+++) by comparison with the strongest signal obtained with the cognate antibody. In all areas scored positively the percentage of immunostained cells was greater than 20%. *Receptor transcripts were readily detected, but the corresponding proteins were either low or undetectable; (a), (b) and (c): receptor proteins were present in layer 5, 2–4 and 6, and in all layers, respectively.
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Fig. 1. Immunodetection of retinoid receptors in the nervous system. Coronal sections through tenia tecta (A, magnification: 40), main olfactory bulb (B, magnification: 40), caudate–putamen (C, magnification: 40), solitary tract and hypoglossal nucleus (D, G, magnification: 40), left and right hippocampus (E, I, magnification: 40), neocortex (F, magnification: 40), hilus of the dentate gyrus (H, magnification: 40), cervical spinal cord (J, magnification: 100), arcuate hypothalamus (K, magnification: 100) and basolateral amygdaloid nucleus (L, magnification: 40) were immunostained with antibodies specific for RARá (A, B), RARâ (C, D), RARã (E, F), RXRá (G, H), RXRâ (I, J) and RXRã (K, L). The nuclear staining in selected regions (boxes) is shown at 400 magnification in a corner of each panel. In all panels the dorsal region is at the top, except in panel I where it is on the right as indicated by the arrow. For descriptions of panels G and I, see regions depicted on the views D and E, respectively.
Expression of RARs and RXRs in CNS of adult mouse
olfactory cortex projections, e.g., the infralimbic cortex. The strongest expression of RARá in the olfactory system was found in tenia tecta and indusium griseum (e.g., Fig. 1A). The highest levels of retinoid receptors were observed in basal ganglia. Caudate–putamen and the shell and core of the nucleus accumbens were strongly labelled with antibodies specific for RARâ (e.g., Fig. 1C) and RXRã. A large fraction of cells in these regions was also immunopositive for RARã and RXRâ proteins. All RAR and RXR isotypes were detected in the limbic system and limbic-associated cortical structures. In these areas the cells immunostained for RARá, RARã (e.g., Fig. 1E) and RXRâ (e.g., Fig. 1I) were mostly localized in the hippocampus CA fields of Ammon’s horn, whereas the predominant receptor in the dentate gyrus was RARã, together with lower levels of RXRá and RXRâ (see Fig. 1H and Table 1, and data not shown). With the exception of RARâ and RXRâ, all retinoid receptors were also expressed in the hippocampal-associated amygdaloid area (e.g., Fig. 1L for RXRã). All receptors, except for RARâ, were found in the cingulate, frontal, parietal and temporal cortices. However, they were differentially expressed in the various layers, with RARá in layer 5, and RARã (e.g., Fig. 1F) and RXRá in layers 2–4 and 6 of the neocortex. RARã was also expressed in more ventral cortices i.e. perirhinal and entorhinal, whereas RXRâ was detectable throughout all layers of the cortex. Diencephalon. This region was characterized by the mutually exclusive expression patterns of the RARâ/ RXRã and the RARá/RXRâ pairs in hypothalamic and thalamic areas, respectively. Hypothalamic structures showing high immunoreactivity were localized mainly in the periventricular and medial zones of the tuberal region, which are thought to be functionally important in the production of hypophysiotropic hormones (e.g., arcuate nucleus; Fig. 1K) or in mediating somatomotor aspects of complex motivated behaviours in rodents (ventromedial nucleus; see Ref. 48) In the thalamus, signals specific for RARá and RXRâ were detected in ventroposterior and reticular nuclei. However, RXR proteins, in contrast to RXR transcripts (data not shown), could not be detected in other thalamic nuclei expressing RARs, e.g., medial geniculate, possibly due to low protein levels. A weak staining of cells positive for RARã was observed throughout these thalamic areas. Rhombencephalon and spinal cord. Retinoid receptors were detected in all three major subdivisions of the hindbrain. RARá and RARã were found predominantly in cerebellum and pons, whereas medulla oblongata expressed almost exclusively RARâ. RXRá distribution overlapped with that of RARs. In the cerebellum, immunoreactive cells were mostly
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restricted to the cerebellar granular layer. RARâ (e.g., Fig. 1D) and RXRá (e.g., Fig. 1G) were strongly expressed in the solitary tract and area postrema of the medulla oblongata. The expression of these receptors in rostroventrolateral reticular nucleus and solitary tract colocalized with the A1 and A2 noradrenaline cell groups.48 In pons, several motor nuclei (trigeminal motor, abducens, facial and hypoglossal nucleus) and sensory nuclei (cochlear, principal sensory trigeminal nucleus) exhibited relatively high RARá and moderate RARã and RXRá levels of expression. The spinal cord exhibited a quantitatively differential pattern of expression of various receptors. Most of the cells expressed RARã, whose signal was distributed across all the structures of the cervical spinal cord, and particularly strongly in lamina II and III. RXRã was also present in all cell layers, whereas other receptors were more restricted in their distribution, including laminae IV-X for RARá, RARâ and RXRâ (e.g., Fig. 1J) and the motoneurons of laminae VII and VIII for RXRá. In situ distribution of retinoid receptor transcripts in the mouse brain Each of the six riboprobes hybridized specifically to their cognate RNAs, as the in situ hybridization (ISH) signals obtained with antisense probes were always above the background signals obtained with sense controls (data not shown). Moreover, the presence of retinoid receptor transcripts in various brain regions was confirmed by RNase protection analysis (see next section and Fig. 3). The ISH distribution of RARá, RARã, RXRá and RXRâ transcripts was almost ubiquitous, and much wider than anticipated from the immunohistochemical analysis. All of these transcripts, but not the corresponding proteins, were detected, e.g., in the medial amygdaloid nucleus (Fig. 2I, K, L, M). In contrast, RARâ and RXRã RNAs were present only in the brain regions where the corresponding proteins were found (Fig. 2C, J, R and G, N, W, and Table 1). Notably, RARâ and RXRã transcripts were present in the striatum (Fig. 2C, G) and absent from midbrain (data not shown), in which RARâ was found to be expressed before and shortly after birth.54,59 Interestingly, all retinoid receptor transcripts were identified in the striatum (Fig. 2B–G and Fig. 3, lane 3) or hypothalamus (Fig. 2I–N and Fig. 3, lane 7), whereas only RARâ, RARã, RXRâ and RXRã, or RARâ, RARã and RXRã proteins could be immunodetected in these brain regions, respectively (Table 1). In the rhombencephalon, with the exception of RXRâ, the RNA expression patterns correlated with the protein receptor distribution (Fig. 2P–W and Table 1). Note that, as estimated from the ISH data, the levels of RARã and RXRâ transcripts were higher in the cerebellum than in the striatum (compare in Fig. 2D to S and F to U, respectively). In contrast, densitometric analysis of the RNase
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Fig. 2. Expression of retinoid receptor transcripts in the adult brain. Selected planes of coronal sections through (A) caudate–putamen and nucleus accumbens, (H) hippocampus and arcuate hypothalamus, (O) cerebellar lobules and abducens and facial nuclei are presented as bright-field views (top row). The corresponding dark-field in situ hybridization views are shown for RARá (B, I, P), RARâ (C, J, R), RARã (D, K, S), RXRa (E, L, T), RXRâ (F, M, U) and RXRã (G, N, W).
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tum, cerebellum, pons, hippocampus, hypothalamus, pituitary, as well as from caudal and rostral regions of the spinal cord. The distribution of RARâ and RXRã transcript was more restricted than that of the other receptors, with relatively high levels in the striatum (Fig. 3, lane 3), in good agreement with ISH data (Fig. 2C, G). Transcripts of the other receptors were widely expressed in different brain structures, again in agreement with ISH data. The RAR isoform distribution exhibited some specificity, as RARá2 RNA was found mainly in the hippocampus, where transcript levels were at least six-fold higher (2400 Units) than in other brain regions, as determined from densitometric measurements (Fig. 3, lane 6). Transcripts of RARá1 were detected at low levels (50–300 Units) in all brain regions (Fig. 3). In contrast, high levels of both RARâ2 and RARâ1,3,4 isoform transcripts were found in the striatum (Fig. 3, lane 3). RNase protection assays also revealed the presence of a new tissue-specific RXRá transcript, corresponding to a 300 nucleotide-long protected fragment (see RXRá4 in Fig. 3). This isoform transcript, which is shorter by almost 300 nucleotides in its 5 region than that previously characterized in skeletal muscles and heart (594 nucleotides protected fragment28), may correspond to a novel RXRá isoform (see Discussion) in addition to ubiquitous RXRá1 and testis-specific RXRá2 and 3 isoforms.5,42 Interestingly, RXRá4, as well as RARá1 and RARâ transcripts, were differentially expressed in caudal and rostral spinal cord (Fig. 3, lanes 9 and 10).
DISCUSSION
Fig. 3. Determination of RAR and RXR transcript expression in different brain structures and spinal cord. RNAs were hybridized with receptor specific antisense riboprobes (lanes 1–10). Hybridization with histone H4 antisense riboprobe was used as an internal control. (Ca) and (Ro), caudal and rostral, respectively.
protection assays revealed higher levels of RARã and RXRâ transcripts in the striatum (125, 340 and 2000 Units for RARã1, RARã2, and RXRâ, respectively; Fig. 3, lane 3) than in the cerebellum (80, 110 and 1200 Units for RARã1, RARã2 and RXRâ, respectively; Fig. 3, lane 4). Differences in cell density between striatum and cerebellum may be at the origin of these discrepancies. Analysis of retinoid receptor transcripts in dissected CNS structures To further correlate the expression patterns of retinoid receptor proteins and transcripts, as well as to gain insight into the expression of receptor isoforms, we performed RNase protection analysis of RNA extracted from the olfactory bulb, cortex, stria-
The present analysis reports the protein and transcript expression patterns of the multiple RAR and RXR isotypes in the adult mouse CNS. RARã (and RARá to a lesser degree) and RXRá proteins were the only receptors to be expressed in the different components of the olfactory system, from the mitral and plexiform layers to entorhinal and perirhinal cortices. The functional relevance of this expression is unknown, as no effects of retinoids on functioning of the olfactory system have yet been reported, although retinoid receptors may play a role in the formation of the olfactory pathway which is known to be dependent on RA signalling.30 The gustatory system is also a potential target of retinoid action, as RARâ and RXRá are expressed in the solitary tract and area postrema (Fig. 1D, G, Table 1), which play a role in numerous neurovegetative functions.48 In this respect, the weight deficiency of RARâ null mutant mice18 may possibly result from reduced food consumption. However, a defective RA signalling in the hypothalamo–pituitary axis could also account for this deficiency, as high levels of RARâ and RXRã were found in the hypothalamus and pituitary (Fig. 1K and Table 1; see also Ref. 57).
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Strong expression of RARâ and RXRã in the striatum, a structure implicated, among other processes, in the control of motor functions,48 is the most remarkable feature of RAR and RXR isotype distribution in the brain. This striatal expression persists from early stages of development.13,54 Interestingly, RA appears to be synthesized in mesostriatal and mesolimbic dopaminergic neurons.45 This indicates that the dopaminergic system could be a target for retinoid action in the striatum. In fact, the expression of the dopamine 2 receptor (D2R) can be induced by RA in neuroblastoma or MMQ cell lines15,55 and mice mutated for RARâ and RARã exhibit a decrease in expression of striatal dopamine receptors (D1R and D2R) and locomotor defects.29 Interestingly, the pattern of expression of receptor proteins and transcripts in the adult brain does not correspond to that previously reported for the developing and neonatal CNS.13,54,59 For instance, the relatively strong expression of the RARã protein in the hippocampus and in the striatum is in contrast with its weak expression in discrete regions of the developing brain59 and the apparent absence of its transcripts in fetal and neonatal CNS.54 The absence of RARâ in the adult hippocampal structures also contrasts with the pre- and early postnatal expression of this receptor in these structures.59 Similarly, the lack of RARâ transcripts and protein in midbrains of adult mice is in contrast with the expression of this protein during pre- and neonatal periods.13,54,59 These differences between the distribution pattern of retinoid receptors in early and late postnatal mouse brain most probably reflect a role of retinoids in CNS maturation events. The comparison of the distribution pattern of retinoid receptor transcripts, as judged from ISH and RNase protection studies, with that of the corresponding proteins reveals that the expression of some of these receptors is post-transcriptionally regulated (see ‘‘*’’ in Table 1). For instance, in the olfactory bulbs, there is a discrepancy between a weak and restricted RARâ protein expression and readily detected RARâ transcripts (270 Units, Fig. 3, lane 1), whereas a reverse situation is found in the hypothalamus in which the expression of the RARâ protein is much stronger relative to transcript levels (470 Units for RARâ2; see Fig. 3, lane 7). Evidence indicating that RARâ2 expression could be post-transcriptionally controlled was previously reported.51 Similar mechanisms may operate in the case of RARá, as RARá proteins could not be detected in the striatum, hypothalamus and most of the midbrain structures, even though RARá transcript levels were similar (within a factor of 2) to those found in the neocortex and hindbrain (Fig. 3, compare lanes 2, 3 and 4), in which the protein could be readily revealed (Table 1). Relatively high amounts of RXRá and RXRâ transcripts were also in contrast with low levels of the corresponding
proteins in most brain regions (see for instance the striatum and cerebellum in Table 1 and in Fig. 3). We cannot exclude, however, the presence of the RXRá4 isoform in the regions where its transcript is present (see, e.g., striatum or spinal cord in Fig. 3), as the antibody used to detect the RXRá protein was raised against the N-terminal region of RXRá1, which appears to be absent from RXRá4. Receptor transcript analysis by RNase protection revealed differential, tissue-specific isoform distributions, which were most obvious in the case of RARâ and RARá (Fig. 3). Note, however, that the present study does not provide a quantitative estimation of these differences as the signal intensity was increasing with the size of the protected fragment. It is noteworthy that the RNase protection assay has uncovered a novel RXRá mRNA (see RXRá4 in Fig. 3), in addition to the expected RXRá1 mRNA. Although the exact nature of this novel species is unclear, it may correspond to a new RXRá isoform, as using a RXRá specific probe, at least two new 1.8and 1.5-kb-long RNA species were detected by Northern blot analysis, in addition to the usual 5.6-kb-long transcript (42, and data not shown). In any event, this putative new RXRá isoform appears to be CNS-specific, as it could not be found in other adult tissues (heart, muscle, testis, skin) or in 14.5 day mouse fetus RNA analysed by RNase protection (data not shown). Finally, it is worth stressing that the expression patterns of RARs and RXRs overlap in all CNS structures. This is not surprising, as RAR/RXR heterodimers are most probably the functional units transducing the retinoid signal in vertebrates.8,24,26 However, RXRs may also be implicated in additional signalling pathways, as they can also form functional heterodimers in vitro with other nuclear receptors8,43,44 which are expressed in the CNS (TRá,4 Nur77,56 Nurr1,61 COUP-TFI and ARP-137). In this respect, it is interesting that Nurr1, a putative RXR heterodimerization partner, was shown to be indispensable for the development and maintenance of midbrain dopaminergic neurons.61 The present report provides the basis for future studies aimed at linking CNS functions to the vitamin A nutritional status. CONCLUSIONS
The present study reports a thorough analysis of the expression of retinoid receptors in the adult mouse CNS. Each RAR and RXR receptor exhibits a specific pattern of expression which is different from that reported earlier for pre- and neonatal CNS. These changes may reflect a role of retinoids in the maturation and function of the CNS. Moreover, differences in transcript and protein distribution of several receptors suggest the existence of a post-transcriptional control of RAR and RXR expression.
Expression of RARs and RXRs in CNS of adult mouse Acknowledgements—We thank C. Rochette-Egly for a gift of anti-RARá, â and ã and RXRá antibodies, W. Chin for anti-RXRâ antibody and M. Mark for critical reading of the manuscript. We are also thankful to Betty Feret for excellent technical assistance and to J. M. LaFontaine for help in preparation of the manuscript. This work was supported by funds from the Centre National de la
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Recherche Scientifique (CNRS), the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), the Colle`ge de France, the Hoˆpital Universitaire de Strasbourg, the Association pour la Recherche sur la Cancer (ARC), the Human Frontier Science Program and Bristol-Myers Squibb. W. K. was a recipient of a fellowship from the Ministe`re de la Recherche and the ARC.
REFERENCES
1. Aberle S. B. D. (1933) Neurological disturbances in rats reared on diets deficient in vitamin A. J. Nutr. 7, 445–459. 2. Bavik C., Ward S. J. and Chambon P. (1996) Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis. Proc. natn. Acad. Sci. U.S.A. 93, 3110–3114. 3. Berrard S., Faucon Biguet N., Houhou L., Lamouroux A. and Mallet J. (1993) Retinoic acid induces cholinergic differentiation of cultured newborn rat sympathetic neurons. J. Neurosci. Res. 35, 382–389. 4. Bradley D. J., Young W. S. and Weinberger C. (1989) Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary. Proc. natn. Acad. Sci. U.S.A. 86, 7250–7254. 5. Brocard J., Kastner P. and Chambon P. (1996) Two novel RXRá isoforms from mouse testis. Biochem. biophys. Res. Commun. 229, 211–218. 6. Butler P. D., Susser E. S., Brown A. S., Kaufmann C. A. and Gorman J. M. (1994) Prenatal nutritional deprivation as a risk factor in schizophrenia: preclinical evidence. Neuropsychopharmacology 11, 227–235. 7. Chambon P. (1994) The retinoid signaling pathway: molecular and genetic analyses. Semin. Cell Biol. 5, 115–125. 8. Chambon P. (1996) A decade of molecular biology of retinoic acid receptors. Fedn Am. Socs exp. Biol. J. 10, 940–954. 9. Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol– chloroform extraction. Analyt. Biochem. 162, 156–159. 10. De´cimo D., Georges-Labouesse E. and Dolle´ P. (1995) In situ hybridization of nucleic acid probes to cellular RNA. In Genes Probes 2, A Practical Approach (eds Hames B. D. and Higgis S.), pp. 183–210. Oxford University Press, Oxford. 11. Dickman E. D., Thaller C. and Smith S. M. (1997) Temporally-regulated retinoic acid depletion produces specific neural crest, ocular and nervous system defects. Development 124, 3111–3121. 12. Dolle´ P., Ruberte E., Kastner P., Petkovich M., Stoner C. M., Gudas L. J. and Chambon P. (1989) Differential expression of genes encoding alpha, beta and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse. Nature 342, 702–705. 13. Dolle´ P., Fraulob V., Kastner P. and Chambon P. (1994) Developmental expression of murine retinoid X receptor (RXR) genes. Mech. Dev. 45, 91–104. 14. Dupe´ V., Davenne M., Brocard J., Dolle´ P., Mark M., Dierich A., Chambon P. and Rijli F. M. (1997) In vivo functional analysis of the Hoxa-1 3 retinoic acid response element (3 RARE). Development 124, 399–410. 15. Farooqui S. M. (1994) Induction of adenylate cyclase sensitive dopamine D2-receptors in retinoic acid induced differentiated human neuroblastoma SHSY-5Y cells. Life Sci. 55, 1887–1893. 16. Gaub M. P., Rochette-Egly C., Lutz Y., Ali S., Matthes H., Scheuer I. and Chambon P. (1992) Immunodetection of multiple species of retinoic acid receptor alpha: evidence for phosphorylation. Expl Cell Res. 201, 335–346. 17. Gavalas A., Studer M., Lumsden A., Rijli F. M., Krumlauf R. and Chambon P. (1998) Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch. Development 125, 1123–1136. 18. Ghyselinck N. B., Dupe´ V., Dierich A., Messadeq N., Garnier J. M., Rochette-Egly C., Chambon P. and Mark M. (1997) Role of the retinoic acid receptor beta (RARâ) during mouse development. Int. J. dev. Biol. 41, 425–448. 19. Giguere V. (1994) Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr. Rev. 15, 61–79. 20. Glass C. K. (1996) Some new twists in the regulation of gene expression by thyroid hormone and retinoic acid receptors. J. Endocr. 150, 349–357. 21. Gronemeyer H. and Laudet V. (1995) Transcription factors 3: nuclear receptors. Protein Profile 2, 1173–1308. 22. Gudas L. J. (1994) Retinoids and vertebrate development. J. biol. Chem. 269, 15,399–15,402. 23. Heseker H., Kubler W., Pudel V. and Westenhoffer J. (1992) Psychological disorders as early symptoms of a mild-to-moderate vitamin deficiency. Ann. N. Y. Acad. Sci. 669, 352–357. 24. Kastner P., Mark M. and Chambon P. (1995) Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell 83, 859–869. 25. Kastner P., Mark M., Leid M., Gansmuller A., Sugawara W., Grondona J. M., De´cimo D., Krezel W., Dierich A. and Chambon P. (1996) Abnormal spermatogenesis in RXRâ mutant mice. Genes Dev. 10, 80–92. 26. Kastner P., Mark M., Ghynselinck N., Krezel W., Dupe´ V., Grondona J. M. and Chambon P. (1997) Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development. Development 124, 313–326. 27. Kobayashi M., Matsuoka I. and Kurihara K. (1994) Cholinergic differentiation of cultured sympathetic neurons induced by retinoic acid. Induction of choline acetyltransferase-mRNA and suppression of tyrosine hydroxylasemRNA levels. Fedn Eur. biochem. Socs Lett. 337, 259–264. 28. Krezel W., Dupe´ V., Mark M., Dierich A., Kastner P. and Chambon P. (1996) RXRã null mice are apparently normal and compound RXRá+/RXRâ//RXRã/ mutant mice are viable. Proc. natn. Acad. Sci. U.S.A. 93, 9010–9014. 29. Krezel W., Ghyselinck N., Samad T., Dupe´ V., Kastner P., Borrelli E. and Chambon P. (1998) Impaired locomotion and dopamine signaling in retinoid receptor mutant mice. Science 279, 863–867. 30. La Mantia A. S., Colbert M. C. and Linney E. (1993) Retinoic acid induction and regional differentiation prefigure olfactory pathway formation in the mammalian forebrain. Neuron 10, 1035–1048.
1300 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61.
W. Krez˙el et al. Lammer E. J. and Armstrong D. L. (1992) Malformations of hindbrain structures among humans exposed to isotretinoin (13-cis-retinoic acid) during early embryogenesis. In Retinoids in Normal Development and Teratogenesis (ed. Morris-Kay G. M.), pp. 281–295. Oxford University Press, Oxford. Langman J. and Welch G. W. (1967) Excess vitamin A and development of the cerebral cortex. J. comp. Neurol. 131, 15–26. Leid M., Kastner P. and Chambon P. (1992) Multiplicity generates diversity in the retinoic acid signalling pathways. Trends biochem. Sci. 17, 427–433. Levitsky D. A. and Strupp B. J. (1995) Malnutrition and the brain: changing concepts, changing concerns. J. Nutr. 125, 2212S–2220S. Lohnes D., Kastner P., Dierich A., Mark M., Le Meur M. and Chambon P. (1993) Function of retinoic acid receptor gamma in the mouse. Cell 73, 643–658. Lohnes D., Mark M., Mendelsohn C., Dolle´ P., Dierich A., Gorry P., Gansmuller A. and Chambon P. (1994) Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development 120, 2723–2748. Lopes da Silva S., Cox J. J., Jonk L. J., Kruijer W. and Burbach J. P. (1995) Localization of transcripts of the related nuclear orphan receptors COUP-TF I and ARP-1 in the adult mouse brain. Molec. Brain Res. 30, 131–136. Lufkin T., Lohnes D., Mark M., Dierich A., Gorry P., Gaub M. P., Le Meur M. and Chambon P. (1993) High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice. Proc. natn. Acad. Sci. U.S.A. 90, 7225–7229. Maden M. and Holder N. (1992) Retinoic acid and development of the central nervous system. Bioessays 14, 431–438. Maden M., Gale E., Kostetskii I. and Zile M. (1996) Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr. Biol. 6, 417–426. Maden M., Graham A., Gale E., Rollinson C. and Zile M. (1997) Positional apoptosis during vertebrate CNS development in the absence of endogenous retinoids. Development 124, 2799–2805. Mangelsdorf D. J., Borgmeyer U., Heyman R. A., Zhou J. Y., Ong E. S., Oro A. E., Kakizuka A. and Evans R. M. (1992) Characterization of three RXR genes that mediate the action of the 9-cis retinoic acid. Genes Dev. 6, 329–344. Mangelsdorf D. J. and Evans R. M. (1995) The RXR heterodimers and orphan receptors. Cell 83, 841–850. Mangelsdorf D. J., Thummel C., Beato M., Herrlich P., Schu¨tz G., Umesono K., Blumberg B., Kastner P., Mark M., Chambon P. and Evans R. M. (1995) The nuclear receptor superfamily: the second decade. Cell 83, 835–839. McCaffery P. and Drager U. C. (1994) High levels of a retinoic acid-generating dehydrogenase in the mesotelencephalic dopamine system. Proc. natn. Acad. Sci. U.S.A. 91, 7772–7776. Mellanby E. (1943) Nutrition in relation to bone growth and the nervous system. Proc. R. Soc. B 132, 28–54. Morris G. M. (1975) Abnormal cell migration as a possible factor in the genesis of vitamin A-induced craniofacial anomalies. In New Approaches to the Evaluation of Abnormal Embryonic Development (eds Neubert D. and Merker M. J.), pp. 678–687. Georg Thieme, Stuttgart. Paxinos G. (1995) The Rat Nervous System. Academic, San Diego. Pedersen W. A., Berse B., Schuler U., Wainer B. H. and Blusztajn J. K. (1995) All-trans- and 9-cis-retinoic acid enhance the cholinergic properties of a murine septal cell line: evidence that the effects are mediated by activation of retinoic acid receptor-alpha. J. Neurochem. 65, 50–58. Perlmann T. and Evans R. M. (1997) Nuclear receptors in Sicily: all in the famiglia. Cell 90, 391–397. Reynolds K., Zimmer A. M. and Zimmer A. (1996) Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5 -untranslated region. J. Cell Biol. 134, 827–835. Rochette-Egly C., Gaub M. P., Lutz Y., Ali S., Scheuer I. and Chambon P. (1992) Retinoic acid receptor-beta: immunodetection and phosphorylation on tyrosine residues. Molec. Endocr. 6, 2197–2209. Rochette-Egly C., Lutz Y., Pfister V., Heyberger S., Scheuer I., Chambon P. and Gaub M. P. (1994) Detection of retinoid X receptors using specific monoclonal and polyclonal antibodies. Biochem. biophys. Res. Commun. 204, 525–536. Ruberte E., Friederich V., Chambon P. and Morriss-Kay G. (1993) Retinoic acid receptors and cellular retinoid binding proteins. III. Their differential transcript distribution during mouse nervous system development. Development 118, 267–282. Samad T. A., Krezel W., Chambon P. and Borrelli E. (1997) Regulation of dopaminergic pathways by retinoids: activation of D2 receptor by members of the retinoic acid receptor-retinoid X receptor family. Proc. natn. Acad. Sci. U.S.A. 94, 14,349–14,354. Saucedo-Cardenas O. and Conneely O. M. (1996) Comparative distribution of NURR1 and NUR77 nuclear receptors in the mouse central nervous system. J. molec. Neurosci. 7, 51–63. Sugawara A., Yen P. M., Qi Y., Lechan R. M. and Sugawara W. W. (1995) Isoform-specific retinoid-X receptor (RXR) antibodies detect differential expression of RXR proteins in the pituitary gland. Endocrinology 136, 1766–1774. Wolbach S. B. and Bessey O. A. (1941) Vitamin A deficiency and the nervous system. Archs Path. 32, 689–722. Yamagata T., Momoi M. Y., Yanagisawa M., Kumagai H., Yamakado M. and Momoi T. (1994) Changes of the expression and distribution of retinoic acid receptors during neurogenesis in mouse embryos. Devl Brain Res. 77, 163–176. Zelent A., Mendelshon C., Kastner P., Krust A., Garnier J. M., Ruffenbach F., Leroy P. and Chambon P. (1991) Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing. Eur. molec. Biol. Org. J. 10, 71–81. Zetterstro¨m R. H., Solomin L., Jansson L., Hoffer B. J., Olson L. and Perlmann T. (1997) Dopamine neuron agenesis in Nurr1-deficient mice. Science 276, 248–250. (Accepted 9 June 1998)