Immunohistochemical Characterization of the Orphan Nuclear Receptor ROR in the Mouse Nervous System
Department of Neurobiology (C1), Graduate School of Medicine, Chiba University, Chiba, Japan
Correspondence to: Hidetoshi Ino, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: ino{at}med.m.chiba-u.ac.jp
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Summary |
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(J Histochem Cytochem 52:311323, 2004)
Key Words: ROR retinoic acid receptor retinoid cerebellum thalamus dorsal cochlear nucleus immunohistochemistry
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Introduction |
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Homozygous staggerer mutant mice show severe cerebellar defects, including degeneration of granule cells and ectopic Purkinje cells reduced in number and size with poor dendritic arbors (Sidman et al. 1962). Evidence suggests that the effect of the staggerer gene mutation appears intrinsically in Purkinje cells and that the degeneration of granule cells is a secondary phenomenon (Herrup and Mullen 1979
,1981
; Herrup 1983
). Staggerer mice carry a deletion mutation in the ROR
gene that prevents translation of the ligand-binding homology domain (Hamilton et al. 1996
; MatysiakScholze and Nehls 1997
), and ROR
-knockout mice show similar symptoms to staggerer mice (Dussault et al. 1998
; Steinmayr et al. 1998
).
Distribution of ROR mRNA expression is revealed by Northern blotting and ISH analyses in the mouse brain (Matsui et al. 1995
). ROR
is expressed in specific areas of the brain, including the cerebellum, thalamus, and olfactory bulb. By E14E15, ROR
is already highly expressed in Purkinje cells (Hamilton et al. 1996
; Nakagawa et al. 1997
). In contrast, RORß is highly expressed in the retina, SCN, and pineal body (SchaerenWiemers et al. 1997
). ROR
is highly expressed in the skeletal muscle and thymus but not in the nervous system (Hirose et al. 1994
). ROR
has multiple isoforms produced by alternative RNA processing at the amino-terminal region (BeckerAndré et al. 1993
; Giguère et al. 1994
; MatysiakScholze and Nehls 1997
).
Although these data analyzing mRNA expression levels are impressive, they provide no information on the protein itself. It has remained unknown whether mRNA expression levels correlate with protein levels temporally and spatially. Subcellular localization of the protein is also unknown. I demonstrate here the IHC analysis of ROR in the adult and developing mouse brain, examining the validity of IHC data by comparison with ISH data.
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Materials and Methods |
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Preparation of Brain Extracts
Brain tissues collected from adult ddY albino mice were homogenized with a Teflonglass homogenizer in one volume of ice-cold 2%SDS, 2% Triton X-100, 1 mM EDTA in 50 mM Tris-HCl, pH 7.6, or 1 mM EDTA in 5 M guanidine HCl.
Western Blotting Analysis
The brain extracts (50 µg) and aliquots of the GST fusion proteins were denatured, applied to 10% SDS-PAGE, and blotted onto polyvinylidene difluoride filters (Immobilon P; Millipore, Bedford, MA). The filters were incubated with the goat polyclonal anti-ROR1 antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000) or mouse monoclonal anti-GST antibody (B14; Santa Cruz; 1:1000), followed by incubation with horseradish peroxidase-conjugated anti-goat IgG(H+L) or anti-mouse IgG(H+L) antibody (Vector Laboratories, Burlingame, CA; 1:2000). Immunoreactivity was visualized with the ECL Plus Western blotting detection reagents (Amersham).
Preparation of Tissue Sections
Adult, neonatal, and fetal ddY albino mice were used. Tissues were prepared as described previously (Ino 2003). Briefly, adult and neonatal mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.5, via the heart under pentobarbital anesthesia. Brains sliced at approximately 5-mm thickness were further fixed in the same solution at 4C for 2 days. Fetal mice (E16) were rinsed with saline and cut into two or three pieces and fixed in the same solution at 4C for 2 days. The tissues were then transferred to distilled water and incubated at 4C overnight. The tissues were boiled in distilled water for 1.53 min for antigen retrieval and then immersed in 30% sucrose in PBS at 4C overnight. The antigen retrieval procedure was critical for successful immunostaining for ROR
(Ino 2003
). The tissues were frozen in crushed dry ice. Cryostat sections (10-µm) were prepared, placed on 0.02% poly-L-lysine-coated glass slides, and air-dried. Frozen sections, used for ISH and IHC, were prepared by the identical procedure. All animals were treated and cared for in accordance with the guidelines established by the Animal Care and Use Committee of Chiba University.
Preparation of Probes
Mouse ROR (nt 6171587), rat RORß (nt 7371765), and mouse ROR
(nt 6471618) cDNAs were prepared by RT-PCR and subcloned into pGEM-T (Promega; Madison, WI) or pBluescript II (Stratagene; La Jolla, CA). Digoxigenin (DIG)-labeled antisense and sense riboprobes were prepared with DIG RNA labeling mix (Roche Diagnostics; Mannheim, Germany) and SP6, T3 or T7 RNA polymerase using linearized plasmids as templates. Riboprobes were hydrolyzed with alkaline (in 40 mM sodium bicarbonate, 60 mM sodium carbonate, and 5 mM dithiothreitol at 60C for 20 min) to an average size of 300 nucleotides.
In Situ Hybridization
ISH was performed as described previously with some alterations (Ino et al. 1994). Sections were immersed in 0.3% Triton X-100 in PBS at room temperature (RT) for 2 hr. The adult tissue sections were incubated with 1 µg/ml proteinase K in PBS at 37C for 10 min. After washing with PBS, the sections were immersed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.5, at RT for 10 min. After washing with PBS, the sections were immersed in 0.2 N HCl at RT for 10 min, immersed in 0.1 M triethanolamine HCl, pH 8.0, at RT for 5 min, and immersed in freshly prepared 0.25% acetic anhydrate in 0.1 M triethanolamine-HCl, pH 8.0, at RT for 10 min. After washing with PBS, the sections were incubated in 50% formamide in 2 x SSC at RT for 2 hr. The sections were hybridized with approximately 0.5 µg/ml DIG-labeled riboprobes in the hybridization solution (50% formamide, 0.2 mg/ml E. coli tRNA, 1 x Denhardt's solution, 10% dextran sulfate, 0.6 M NaCl, 0.25% SDS, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.6) covered with Parafilm at 50C for 40 hr in a humid chamber saturated with 50% formamide. The sections were incubated in 50% formamide in 2 x SSC at 50C for 1 hr and immersed in 0.5 M NaCl, 1 mM EDTA in 10 mM Tris-HCl, pH 8.0 (TNE), at RT for 30 min. Unhybridized riboprobes were digested with 10 µg/ml ribonuclease A in TNE at 37C for 10 min. The sections were washed with TNE at RT for 30 min, with 2 x SSC at 50C for 20 min, and twice with 0.2 x SSC at 50C for 20 min. After immersing in 0.15 M NaCl and Tris-HCl, pH 7.5 (buffer 1), the sections were blocked with 1.5% blocking reagent (Roche) in buffer 1. Immunoreaction was performed with anti-DIG-AP Fab fragments (Roche; 1:2000) in the blocking solution at RT overnight. After washing with buffer 1 and 0.1 M NaCl, 50 mM MgCl2 and 0.1 M Tris-HCl, pH 9.5 (buffer 2), the sections were stained with 0.45 mg/ml nitroblue tetrazolium and 0.175 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate in buffer 2 at 37C for 6 hr.
Immunohistochemistry
IHC was performed as described previously (Ino 2003). Briefly, sections were immersed in 0.3% Triton X-100 in PBS at RT for 23 hr and blocked in 5% skim milk in PBS at RT for several hours. Immunoreaction was performed with primary antibodies in the blocking solution at RT overnight. Primary antibodies for IHC were goat polyclonal anti-ROR
1 (C-16; 1:1000) and mouse monoclonals anti-CaBP (CB-955; Sigma, St. Louis, MO; 1:1000), anti-parvalbumin (PARV-19; Sigma; 1:1000), and anti-neuronal nuclei (NeuN; Chemicon, Temecula, CA; 1:1000). After washing with PBS, the sections were incubated with biotin-conjugated anti-goat IgG(H+L) antibody (Vector; 1:200), followed by reaction with the Vectastain ABC kit (Vector). The reaction was developed with 3,3'-diaminobenzidine, nickel sulfate, and hydrogen peroxide. For double staining after incubation with primary antibodies, the sections were incubated with biotin-conjugated anti-goat IgG(H+L) antibody (Vector; 1:200), followed by incubation with Alexa Fluor488-conjugated streptavidin (Molecular Probes, Eugene, OR; 1:400) and Texas Red-conjugated anti-mouse IgG(H+L) antibody (Vector; 1:200). The sections were observed by fluorescence microscopy.
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Results |
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Localization of ROR in the Adult Mouse Nervous System
I demonstrate the localization of ROR mRNA and protein by ISH and IHC with the specific antisense riboprobe and antibody. ROR
mRNA has been already reported to be abundantly expressed in the cerebellum, especially in Purkinje cells, and thalamus (Matsui et al. 1995
).
In the cerebellum, strong hybridization signal was observed in Purkinje cells. Hybridization signal was also observed in the molecular layer, although the intensity was weaker than in Purkinje cells, but not in the granule cell layer (Figure 2B) . IHC showed strong ROR immunoreactivity in Purkinje cells and in cells of the molecular layer (Figure 2A). In either case, immunostaining was located in nuclei. Although the mRNA level in Purkinje cells was much greater than that in cells of the molecular layer, a difference in ROR
immunoreactivity between them was less apparent. Strong hybridization signal was also observed in the thalamus, and simultaneously strong nuclear ROR
immunoreactivity was located in this region, except for the reticular thalamic nucleus (Figures 2C and 2D). In the cerebral cortex, ROR
mRNA and protein were found mainly in layer IV (Figures 2E and F). They were also observed in the rostral part of the piriform cortex but not in the caudal part, and in the entrhinal cortex (Table 1). ROR
-positive cells in the piriform cortex mainly existed in layer II (data not shown).
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The data are summarized in Table 1. By comparison, the distribution of RORß mRNA expression is also shown. ROR mRNA expression was not observed in the mouse nervous system and pituitary gland (data not shown). No hybridization signal was observed with the sense riboprobes (data not shown).
Classification of ROR-positive Cells
I performed the classification of cells showing ROR immunoreactivity in the cerebellum, DCN, and thalamus by double fluorescence immunostaining. Among cerebellar neurons, Purkinje cells are CaBP- and parvalbumin-double positive, and stellate cells and basket cells are parvalbumin-positive but CaBP-negative (Celio and Heinzmann 1981
; Celio 1990
). Therefore, CaBP and parvalbumin were used as cell markers for the classification of cell types in the cerebellum. Purkinje cells were clearly ROR
-positive (Figures 4A4C
, arrows). In the molecular layer, all ROR
-positive cells were parvalbumin-positive (Figures 4D4F, arrowheads). Therefore, these ROR
-positive cells were stellate cells or basket cells. In the granule cell layer, no ROR
-immunoreactive cells were found, which indicates that granule cells as well as Golgi cells were ROR
-negative.
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The classification of ROR-positive cells in the thalamus using NeuN as a specific neuronal marker was performed. Figures 5A5C
clearly show that ROR
immunoreactivity was found only in NeuN-positive neurons, but not all NeuN-positive neurons were ROR
-positive. NeuN-positive and parvalbumin-positive neurons in the reticular thalamic nucleus were ROR
-negative (Figures 5D5F), which indicates that parvalbumin is independent of ROR
expression.
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Discussion |
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I further demonstrated the existence of ROR protein in the retina (especially in ganglion cells and in the inner molecular layer) and SCN. An experiment using ROR
-knockout mice, in which truncated ROR
fused with ß-galactosidase lacking normal ROR
function is generated instead of ROR
, showed that ß-galactosidase activity is detected not only in the Purkinje cells and thalamus but also in the retinal ganglion cell and SCN (Steinmayr et al. 1998
). The present data are consistent with their study in these points. In that study, however, ß-galactosidase activity in the molecular layer of the cerebellum was not shown. ROR
mRNA expression is observed in the molecular layer of normal developing mice from P14, when stellate cells and basket cells have already been generated (Nakagawa et al. 1997
). Because ROR
mRNA expression in the molecular layer is low compared to Purkinje cells, ß-galactosidase activity may have not been detected in the ROR
-knockout mice. I believe that ROR
protein exists in stellate cells and basket cells of normal mice. In addition, Steinmayr et al. (1998)
demonstrated ß-galactosidase activity in the testis, epididymis, and skin (epidermis, hair follicle, and sebaceous gland). I observed ROR
immunoreactivity in the epidermis, hair follicle, and sebaceous gland, but not in the testis and epididymis (data not shown). Although the reason for the discrepancy is unknown, further pursuit of this problem was not performed in the present study.
There are at least four ROR isoforms in humans (BeckerAndré et al. 1993
; Giguère et al. 1994
) and at least two in mice (MatysiakScholze and Nehls 1997
). These isoforms have distinct amino-termini, produced by differential promoter usage and alternative slicing. The present ISH data did not distinguish these isoforms because I used the common region for the probe, and the IHC data also did not distinguish the isoforms because the antibody recognizes the common carboxy-terminus.
Cells Showing ROR Immunoreactivity
In the cerebellum, the ROR immunoreactivity was observed in Purkinje cells, stellate cells, and basket cells, which was clearly demonstrated by the coexistence with CaBP and parvalbumin. Granule cells and Golgi cells were ROR
-negative. In the DCN, ROR
exists in ectopic Purkinje cells. From the size of ROR
-positive nuclei and their distribution, most ROR
-positive cells in the DCN may be cartwheel cells (Wouterlood and Mugnaini 1984
), and stellate cells may be also ROR
-positive (Wouterlood et al. 1984
). However, further reliable classification of the cell type in the DCN was difficult because no useful cell marker to distinguish them was available and the cytoarchitecture is not so clear as in the cerebellum. Cartwheel cells share some features common to Purkinje cells, such as cerebellin immunoreactivity (Mugnaini and Morgan 1987
).
Studies on Staggerer Mutant Mice and Possible Physiological Roles of ROR
Analyses of staggerer mutant mice have provided suggestions about the physiological role of ROR. Studies using chimera mice suggest that the defects intrinsically exist in Purkinje cells but not in granule cells (Herrup and Mullen 1979
,1981
; Herrup 1983
; Soha and Herrup 1995
), which is supported by the distribution of ROR
mRNA and protein.
Purkinje cells of staggerer mice are reduced in size and number, ectopic in location, and rudimentary in dendritic arborization. However, the neurogenesis of Purkinje cells is not affected in staggerer mice and the reduced number is due to cell death after differentiation (Vogel et al. 2000). Purkinje cells of staggerer mice show characteristic features of embryonal Purkinje cells, such as embryonal cell-surface carbohydrate patterns (Hatten and Messer 1978
; Trenkner 1979
; Edelman and Chuong 1982
), multiple innervation by climbing fibers (Crepel et al. 1980
; Mariani and Changeux 1980
), and NMDA responses (Dupont et al. 1984
). It is likely that ROR
is essential for the maturation of Purkinje cells during development. Although the role of ROR
after maturation remains unknown, ROR
may be necessary for survival of mature Purkinje cells, because heterozygous staggerer mice, despite a lack of overt clinical phenotype, show progressive Purkinje cell degeneration with age (Zanjani et al. 1991
; Doulazmi et al., 1999
; HadjSahraoui et al. 2001
).
In addition to Purkinje cells, defects in staggerer mice have been reported in the DCN (Berrebi et al. 1990), inferior olive (Shojaeian et al. 1985
), and olfactory bulb (Monnier et al. 1999
). Cartwheel cells are eliminated in the staggerer DCN. Reduction in size of glomerular and external and internal plexiform layers and reduction in number of mitral cells are observed in the staggerer olfactory bulb. Decreased cell numbers are found in the staggerer inferior olive. These abnormalities in the central nervous system are likely to be directly influenced by the absence of ROR
in these portions, because both ROR
mRNA and protein are detected there. Alternatively, the cell loss may be due to an indirect effect, as in the case of granule cells of the cerebellum.
In addition to the central nervous system, abnormalities have been also observed in the staggerer immune system, including the thymus and spleen (Trenkner and Hoffmann 1986). In contrast to the central nervous system, I found neither ROR
mRNA nor protein in the thymus and spleen of adult and young mice (data not shown). There is a possibility that a distinct genetic locus, a small thymus located between the staggerer and shorter-ear loci on chromosome 9 (Heinlein and Wolle 1992
), accounts for the immunodysfunction of staggerer mice. However, this does not exclude another possibility, that ROR
is involved in normal development of the immune system. It would be interesting to examine whether there are any abnormalities in the ROR
-knockout mouse immune system.
Although staggerer mutant mice display severe defects in Purkinje cells, no apparent change was observed in the thalamus, SCN, and retina, where high to moderate levels of ROR mRNA and protein are observed. This fact has been attracting attention because a high level of ROR
mRNA expression in the thalamus has been previously known. One possible explanation is that RORß, highly expressed in the thalamus, SCN, and retina, compensates the role of ROR
and conceals the staggerer phenotype in these portions. This is plausible; however, evidence has not been presented. In addition, I showed ROR
immunoreactivity in the stellate cells and basket cells of the cerebellum with no detectable RORß mRNA expression. Because stellate cells and basket cells of staggerer mice appear to be unaffected (Sotelo and Changeux 1974
; Landis and Sidman 1978
), the above explanation is not sufficient. Possibly the absence of ROR
appears as phenotype only in restricted cell types. At least, ROR
and RORß are not always necessary for survival or function of neurons, because many neurons, such as hippocampal neurons, lack both ROR
and RORß (ROR
is not expressed in the nervous system).
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Footnotes |
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