Copyright ©The Histochemical Society, Inc.

Immunohistochemical Characterization of the Orphan Nuclear Receptor ROR{alpha} in the Mouse Nervous System

Hidetoshi Ino

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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ROR{alpha} is an orphan nuclear receptor. A deletion mutation in the ROR{alpha} gene leads to severe cerebellar defects, known as the staggerer mutant mouse. Although previous in situ hybridization (ISH) studies have shown that ROR{alpha} is highly expressed in the cerebellum, especially in Purkinje cells, and in the thalamus, sufficient immunohistochemical (IHC) study has not yet been presented. I demonstrate here the IHC analysis of ROR{alpha} using a specific anti-ROR{alpha} antibody, in adult and developing mouse nervous system. ROR{alpha} immunoreactivity was observed in the Purkinje cell and molecular layers of the cerebellum. The co-localization of ROR{alpha} with calbindin D28K (CaBP) and parvalbumin indicates that ROR{alpha}-positive cells were Purkinje cells, stellate cells, and basket cells. In addition to the cerebellum, strong to medium ROR{alpha} immunoreactivity was found in the thalamus, cerebral cortex (mainly in the layer IV), dorsal cochlear nucleus (DCN), suprachiasmatic nucleus (SCN), superior colliculus, spinal trigeminal nucleus, and retina. The immunostaining was restricted in nuclei of neurons. Developmentally, ROR{alpha} immunoreactivity was observed in the cerebellum and thalamus from embryonal day 16 (E16). The distribution of ROR{alpha} immunoreactivity and ROR{alpha} mRNA hybridization signal was almost coincident. However, the intensity of hybridization signal was not always parallel to that of immunoreactivity.

(J Histochem Cytochem 52:311–323, 2004)

Key Words: ROR{alpha} • retinoic acid receptor • retinoid • cerebellum • thalamus • dorsal cochlear nucleus • immunohistochemistry


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NUCLEAR RECEPTORS are ligand-activated transcription factors, which include receptors for steroids, thyroid hormone, retinoids, and vitamin D3. Some nuclear receptors have no known ligands and therefore are called orphan nuclear receptors. ROR{alpha} (retinoic acid receptor-related orphan receptor-alpha) is an orphan member of nuclear receptors showing high homology in the DNA-binding domain with retinoic acid receptors (Becker–André et al. 1993Go). It activates transcription as a monomer and a homodimer (Carlberg et al. 1994Go). ROR{alpha} forms the ROR/RZR subfamily with the closely related orphan nuclear receptors RORß (Carlberg et al. 1994Go) and ROR{gamma} (Hirose et al. 1994Go).

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. 1962Go). 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 1979Go,1981Go; Herrup 1983Go). Staggerer mice carry a deletion mutation in the ROR{alpha} gene that prevents translation of the ligand-binding homology domain (Hamilton et al. 1996Go; Matysiak–Scholze and Nehls 1997Go), and ROR{alpha}-knockout mice show similar symptoms to staggerer mice (Dussault et al. 1998Go; Steinmayr et al. 1998Go).

Distribution of ROR{alpha} mRNA expression is revealed by Northern blotting and ISH analyses in the mouse brain (Matsui et al. 1995Go). ROR{alpha} is expressed in specific areas of the brain, including the cerebellum, thalamus, and olfactory bulb. By E14–E15, ROR{alpha} is already highly expressed in Purkinje cells (Hamilton et al. 1996Go; Nakagawa et al. 1997Go). In contrast, RORß is highly expressed in the retina, SCN, and pineal body (Schaeren–Wiemers et al. 1997Go). ROR{gamma} is highly expressed in the skeletal muscle and thymus but not in the nervous system (Hirose et al. 1994Go). ROR{alpha} has multiple isoforms produced by alternative RNA processing at the amino-terminal region (Becker–André et al. 1993Go; Giguère et al. 1994Go; Matysiak–Scholze and Nehls 1997Go).

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{alpha} in the adult and developing mouse brain, examining the validity of IHC data by comparison with ISH data.


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Preparation of Fusion Proteins
Mouse ROR{alpha} (nt 1142–1587), rat RORß (nt 1381–1765), and mouse ROR{gamma} (nt 1213–1618) cDNA fragments containing the carboxy-terminal regions were prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) according to the sequence data (GenBank accession numbers S82720, L14610, and U43508, respectively) and subcloned into pGEX-6P (Amersham Biosciences; Piscataway, NJ). ROR{alpha} (aa 347–467), RORß (aa 337–459), and ROR{gamma} (aa 393–516) glutathione S-transferase (GST) fusion proteins were prepared and affinity-purified with glutathione–Sepharose (Amersham) according to the manufacturer's protocol.

Preparation of Brain Extracts
Brain tissues collected from adult ddY albino mice were homogenized with a Teflon–glass 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-ROR{alpha}1 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 2003Go). 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 1.5–3 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{alpha} (Ino 2003Go). 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{alpha} (nt 617–1587), rat RORß (nt 737–1765), and mouse ROR{gamma} (nt 647–1618) 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. 1994Go). 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 2003Go). Briefly, sections were immersed in 0.3% Triton X-100 in PBS at RT for 2–3 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{alpha}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.


    Results
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 Results
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 Literature Cited
 
Western Blotting Analysis with the Anti-ROR{alpha} Antibody
First I examined the specificity of the anti-ROR{alpha} antibody (C-16) used in this study because the carboxy-terminal region of ROR{alpha}, which is recognized by this antibody, is partly identical to those of RORß and ROR{gamma}. Figure 1A shows that this antibody was specific for ROR{alpha} and crossreacted neither for RORß nor ROR{gamma}.



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Figure 1

Specificity of the anti-ROR{alpha} antibody (A). ROR{alpha}, RORß, and ROR{gamma} GST-fusion proteins were incubated with the anti-ROR{alpha}1 (C-16) or anti-GST (B-14) antibody. The GST-fusion proteins were affinity-purified with glutathione-Sepharose. B-14 reactivity indicates that each fusion protein was successfully produced. The anti-ROR{alpha} antibody reacted to ROR{alpha} but not to RORß and ROR{gamma}. Western blotting analysis of mouse brain extracts with the anti-ROR{alpha} antibody (B). Proteins were extracted with 2% SDS, 2% Triton X-100, 1 mM EDTA in 50 mM Tris-HCl, pH 7.6 (Lane 1) or 1 mM EDTA in 5 M guanidine-HCl (Lane 2). A lower band (38–40 kD) seen only in Lane 1 may represent degradation products of ROR{alpha}. The molecular weight of the upper bands (arrow) corresponds to 53–59 kD. Theoretical molecular weights of mouse ROR{alpha} are 58.8 kD (ROR{alpha}1) and 53.4 kD (ROR{alpha}4).

 
In brain proteins extracted with 2% SDS, 2% Triton X-100, 1 mM EDTA in 50 mM Tris HCl, pH 7.6, two bands (53–59 kD and 38–40 kD) were recognized by the anti-ROR{alpha} antibody (Figure 1B, Lane 1). In contrast, in brain proteins extracted with 1 mM EDTA in 5 M guanidine-HCl, only the upper band was recognized (Figure 1B, Lane 2). The lower band in Lane 1 may correspond to degradation products. Because the upper band is broad, it may be composed of multiple protein species. The upper band shows a good correlation with the theoretical molecular weights of mouse ROR{alpha}1 (58.8 kD) and ROR{alpha}4 (53.4 kD).

Localization of ROR{alpha} in the Adult Mouse Nervous System
I demonstrate the localization of ROR{alpha} mRNA and protein by ISH and IHC with the specific antisense riboprobe and antibody. ROR{alpha} mRNA has been already reported to be abundantly expressed in the cerebellum, especially in Purkinje cells, and thalamus (Matsui et al. 1995Go).

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{alpha} 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{alpha} immunoreactivity between them was less apparent. Strong hybridization signal was also observed in the thalamus, and simultaneously strong nuclear ROR{alpha} immunoreactivity was located in this region, except for the reticular thalamic nucleus (Figures 2C and 2D). In the cerebral cortex, ROR{alpha} 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{alpha}-positive cells in the piriform cortex mainly existed in layer II (data not shown).



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Figure 2

ROR{alpha} in adult mouse cerebellum, thalamus, and cerebral cortex. Parasagittal sections of the cerebellum (A,B), and coronal sections of the thalamus showing the reticular, ventral posterolateral, and ventral posteromedial thalamic nuclei (C,D) and the cerebral cortex (E,F). ROR{alpha} was detected by immunohistochemistry (IHC; A,C,E) and by in situ hybridization (ISH; B,D,F). In the cerebellum, ROR{alpha}-positive cells were observed in the Purkinje cell and molecular layers but not in the granule cell layer (A). Strong hybridization signal was observed in Purkinje cells and, with lower intensity, in cells of the molecular layer (B). Both ROR{alpha} protein and mRNA were detected in the ventral posterolateral and ventral posteromedial thalamic nuclei but not in the reticular thalamic nucleus (C,D). In the cerebral cortex, intense ROR{alpha} immunoreactivity was observed in layer VI (E). Inset in F shows a higher-magnification view of the area indicated by the box. M, molecular layer; P, Purkinje cell layer; G, granule cell layer; Rt, reticular thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Bar = 200 µm.

 

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Table 1

Distribution of ROR{alpha} and RORß in the mouse nervous system and pituitary glanda

 
In addition to the cerebellum, thalamus and cerebral cortex, conspicuous ROR{alpha} immunoreactivity was observed in the DCN (Figure 3A) . Although ROR{alpha} mRNA expression was also observed in this region, the level was lower than in Purkinje cells and in the thalamus and was approximately equivalent to that in the molecular layer of the cerebellum (Figure 3B). Cells showing strong ROR{alpha} immunoreactivity were located in the superficial region of the DCN.



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Figure 3

ROR{alpha} in the adult mouse DCN, SCN, superior colliculus, and retina. Coronal sections of the DCN (A,B) and SCN (C,D), superior colliculus and pineal body (E,F), and longitudinal sections of the retina (G,H). ROR{alpha} was detected by immunohistochemistry (IHC; A,C,E,G) and by in situ hybridization (ISH; B,D,F,H). In the DCN, ROR{alpha} immunoreactivity was observed in the superficial layer (A). In the retina, ROR{alpha} immunoreactivity was detected in ganglion cells and cells of the inner nuclear layer (G). Although ROR{alpha} mRNA expression was also observed in these portions, the intensity was lower than in Purkinje cells and in the thalamus. Insets in B,F,H show higher-magnification views of the areas indicated by the boxes. ox, optic chiasma; SC, superior colliculus; PI, pineal body; G, retinal ganglion cells, IN, inner nuclear layer; ON, outer nuclear layer. Bars: A–F = 200 µm; G,H = 100 µm.

 
In addition to the above regions, moderate ROR{alpha} immunoreactivity was observed in the suprachiasmatic nucleus (SCN), superior colliculus, spinal trigeminal nucleus, and retina (Figures 3C, 3E, and 3G; Table 1). In the retina, ganglion cells and cells of the inner nuclear layer showed positive immunoreactivity. In either case, moderate to low levels of ROR{alpha} mRNA expression were observed (Figures 3D, 3F, 3H; Table 1). In any portion, ROR{alpha} immunoreactivity was observed in nuclei.

The data are summarized in Table 1. By comparison, the distribution of RORß mRNA expression is also shown. ROR{gamma} 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{alpha}-positive Cells
I performed the classification of cells showing ROR{alpha} 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 1981Go; Celio 1990Go). Therefore, CaBP and parvalbumin were used as cell markers for the classification of cell types in the cerebellum. Purkinje cells were clearly ROR{alpha}-positive (Figures 4A–4C , arrows). In the molecular layer, all ROR{alpha}-positive cells were parvalbumin-positive (Figures 4D–4F, arrowheads). Therefore, these ROR{alpha}-positive cells were stellate cells or basket cells. In the granule cell layer, no ROR{alpha}-immunoreactive cells were found, which indicates that granule cells as well as Golgi cells were ROR{alpha}-negative.



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Figure 4

Co-localization of ROR{alpha} with CaBP and parvalbumin in adult mouse cerebellum and DCN. Parasagittal sections of the cerebellum (A–F) and coronal sections of the DCN (G–L). Localization of ROR{alpha} (A,D,G,J), CaBP (B,H), and parvalbumin (E,K) immunoreactivity. (A,B), (D,E), (G,H),and (J,K) are identical sections. The two images are merged in C,F,I,L. CaBP-positive Purkinje cells were ROR{alpha}-positive (C, arrows). Parvalbumin-positive Purkinje cells (F, arrows), stellate cells, and basket cells (F, arrowheads) were ROR{alpha}-positive. CaBP-positive ectopic Purkinje cells were ROR{alpha}-positive (I, arrows). Parvalbumin-positive cells, which may be cartwheel cells and stellate cells, were ROR{alpha}-positive (L). (A,D,G,J) Labeled with Alexa Fluor488 (green); and (B,E,H,K) labeled with Texas Red (red). Bar = 50 µm.

 
In the DCN, cells sporadically found in the superficial region showed intense CaBP immunoreactivity; these are ectopic Purkinje cells (Purkinje-like cells, Figure 4H) (Celio 1990Go; Hurd and Feldman 1994Go). These cells were ROR{alpha}-positive (Figures 4G and 4I, arrows). Except for ectopic Purkinje cells, no CaBP immunoreactivity was found. In contrast, many cells showed moderate parvalbumin immunoreactivity, and these parvalbumin-positive cells were ROR{alpha}-positive (Figures 4J–4L).

The classification of ROR{alpha}-positive cells in the thalamus using NeuN as a specific neuronal marker was performed. Figures 5A–5C clearly show that ROR{alpha} immunoreactivity was found only in NeuN-positive neurons, but not all NeuN-positive neurons were ROR{alpha}-positive. NeuN-positive and parvalbumin-positive neurons in the reticular thalamic nucleus were ROR{alpha}-negative (Figures 5D–5F), which indicates that parvalbumin is independent of ROR{alpha} expression.



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Figure 5

Co-localization of ROR{alpha} with NeuN in the adult mouse thalamus. Coronal sections of the thalamus around the reticular thalamic nucleus (Rt)–ventral posterolateral thalamic nucleus (VPL) boundary. Localization of ROR{alpha} (A,D), NeuN (B),and parvalbumin (F) immunoreactivity. (A,B) and (D,E) are identical sections, respectively. The two images are merged in C,F. NeuN-positive neurons in the VPL were ROR{alpha}-positive, but neurons in the Rt were ROR{alpha}-negative (A,B). Parvalbumin-positive neurons in the Rt were ROR{alpha}-negative (D,E). (A,D) Labeled with Alexa Fluor488, (green); (B,E) Labeled with Texas Red (red). Bar = 100 µm.

 
Localization of ROR{alpha} in the Developing Mouse Nervous System
I examined the localization of ROR{alpha} mRNA and immunoreactivity in the developing mouse nervous system using mice from E16 to postnatal day 21 (P21). As early as E16, both strong hybridization signal and immunoreactivity were found in the cerebellum and thalamus (Figures 6A–6D) . At E16 and P0, ROR{alpha}-positive immature Purkinje cells with small- to medium-sized nuclei were not yet arranged in a single layer (Figures 6A and 6E). At P7, ROR{alpha} immunoreactivity was observed in Purkinje cells having large nuclei and forming the monolayer architecture (Figure 6F). In addition to Purkinje cells, ROR{alpha}-positive cells with small nuclei were observed in the molecular layer in the vicinity of the Purkinje cell layer (Figure 6F). In rats, the generation of basket cells peaks around P7 and the generation of stellate cells is delayed several days (Altman 1972Go). Given that the timetable of the cerebellar neurogenesis in rats is similar to that in mice, the majority of these ROR{alpha}-positive cells in the molecular layer are basket cells. At E16, P0, or P7, no ROR{alpha} immunoreactivity was observed in the external germinal layer. At P14 and P21, ROR{alpha}-positive cells in the molecular layer increased in number and spread over the entire area of the molecular layer (Figures 6G and 6H). The distribution of ROR{alpha}-positive cells was almost identical to that of adult mice.



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Figure 6

ROR{alpha} in developing mouse brain. Parasagittal sections of cerebellum (A,B,E–H) and coronal sections of thalamus (C,D) from E16 (A–D), P0 (E), P7 (F), P14 (G), and P21 (H) mice. ROR{alpha} was detected by immunohistochemistry (A,C,E–H) and by in situ hybridization (ISH; B,D). Bar = 200 µm.

 
Using parvalbumin as a cell marker, a detailed examination of the development of ROR{alpha}-positive cells in the cerebellum was performed (Figure 7) . At E16 and P0, no parvalbumin was detected in the cerebellum (data not shown). At P7, parvalbumin was detected only in Purkinje cells, and therefore ROR{alpha}-positive cells in the molecular layer were parvalbumin-negative (Figures 7A–7C, arrowheads). At P14, several but not all ROR{alpha}-positive cells in the molecular layer exhibited parvalbumin immunoreactivity (Figures 7D–7F, arrows), and at P21 the number of ROR{alpha}- and parvalbumin-double positive cells in the molecular layer further increased (Figures 7G–7I, arrows). As shown in Figures 4D–4F, almost all ROR{alpha} and parvalbumin immunoreactivity in the molecular layer was overlapped in the adult. The extent of the distribution of ROR{alpha}-positive cells in the molecular layer appeared to be restricted within the arborization of Purkinje dendrites, and this extent spread in accordance with the maturation of Purkinje cells during development.



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Figure 7

Co-localization of ROR{alpha} with parvalbumin in developing mouse cerebellum. Parasaggital sections of the cerebellum from P7 (A–C), P14 (D–F) and P21 (G–I) mice. Localization of ROR{alpha} (A,D,G) and parvalbumin (B,E,H). (A,B), (D,E), and (G,H) are identical sections. The two images are merged in C,F,I. At P7, Purkinje cells were ROR{alpha}- and parvalbumin-double positive (C), but ROR{alpha}-positive cells in the molecular layer were parvalbumin-negative (C, arrowheads). At P14 and P21, stellate/basket cells in the deep layer of the molecular layer were ROR{alpha}- and parvalbumin-double positive (F,I, arrows), whereas stellate cells in the superficial layer of the molecular layer were ROR{alpha}-positive but still parvalbumin-negative (F,I, arrowheads). (A,D,G) Labeled with Alexa Fluor488 (green); (B,E,H) labeled with Texas Red (red). Bar = 50 µm.

 

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 Discussion
 Literature Cited
 
Distribution of ROR{alpha} Immunoreactivity
I demonstrated here the successful IHC staining of ROR{alpha}, whose distribution was closely coincident to that of ROR{alpha} mRNA expression. For the IHC detection of ROR{alpha}, antigen retrieval was critical. I used the antigen retrieval method in which tissues were boiled en bloc in distilled water before preparation of frozen sections (Ino 2003Go). ROR{alpha} immunoreactivity was located in cell nuclei and was mainly distributed in the cerebellum (especially in Purkinje cells, stellate cells, and basket cells), thalamus, and DCN. Although the distributions of ROR{alpha} mRNA and protein closely coincided with each other, their levels were not parallel. Both strong hybridization signal for ROR{alpha} mRNA and strong immunoreactivity of ROR{alpha} protein were seen in Purkinje cells, whereas strong immunoreactivity of the protein but weak hybridization signal of the mRNA were observed in stellate cells and basket cells of the cerebellum and in cells of the DCN. Although the hybridization signal of the mRNA in the DCN was weaker than that in the thalamus, the immunoreactivity of the protein was conversely stronger. It is unknown why there was an imbalance between mRNA and protein levels. Translation levels may be different from cell to cell.

I further demonstrated the existence of ROR{alpha} protein in the retina (especially in ganglion cells and in the inner molecular layer) and SCN. An experiment using ROR{alpha}-knockout mice, in which truncated ROR{alpha} fused with ß-galactosidase lacking normal ROR{alpha} function is generated instead of ROR{alpha}, 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. 1998Go). 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{alpha} 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. 1997Go). Because ROR{alpha} mRNA expression in the molecular layer is low compared to Purkinje cells, ß-galactosidase activity may have not been detected in the ROR{alpha}-knockout mice. I believe that ROR{alpha} protein exists in stellate cells and basket cells of normal mice. In addition, Steinmayr et al. (1998)Go demonstrated ß-galactosidase activity in the testis, epididymis, and skin (epidermis, hair follicle, and sebaceous gland). I observed ROR{alpha} 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{alpha} isoforms in humans (Becker–André et al. 1993Go; Giguère et al. 1994Go) and at least two in mice (Matysiak–Scholze and Nehls 1997Go). 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{alpha} Immunoreactivity
In the cerebellum, the ROR{alpha} 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{alpha}-negative. In the DCN, ROR{alpha} exists in ectopic Purkinje cells. From the size of ROR{alpha}-positive nuclei and their distribution, most ROR{alpha}-positive cells in the DCN may be cartwheel cells (Wouterlood and Mugnaini 1984Go), and stellate cells may be also ROR{alpha}-positive (Wouterlood et al. 1984Go). 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 1987Go).

Studies on Staggerer Mutant Mice and Possible Physiological Roles of ROR{alpha}
Analyses of staggerer mutant mice have provided suggestions about the physiological role of ROR{alpha}. Studies using chimera mice suggest that the defects intrinsically exist in Purkinje cells but not in granule cells (Herrup and Mullen 1979Go,1981Go; Herrup 1983Go; Soha and Herrup 1995Go), which is supported by the distribution of ROR{alpha} 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. 2000Go). Purkinje cells of staggerer mice show characteristic features of embryonal Purkinje cells, such as embryonal cell-surface carbohydrate patterns (Hatten and Messer 1978Go; Trenkner 1979Go; Edelman and Chuong 1982Go), multiple innervation by climbing fibers (Crepel et al. 1980Go; Mariani and Changeux 1980Go), and NMDA responses (Dupont et al. 1984Go). It is likely that ROR{alpha} is essential for the maturation of Purkinje cells during development. Although the role of ROR{alpha} after maturation remains unknown, ROR{alpha} 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. 1991Go; Doulazmi et al., 1999Go; Hadj–Sahraoui et al. 2001Go).

In addition to Purkinje cells, defects in staggerer mice have been reported in the DCN (Berrebi et al. 1990Go), inferior olive (Shojaeian et al. 1985Go), and olfactory bulb (Monnier et al. 1999Go). 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{alpha} in these portions, because both ROR{alpha} 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 1986Go). In contrast to the central nervous system, I found neither ROR{alpha} 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 1992Go), accounts for the immunodysfunction of staggerer mice. However, this does not exclude another possibility, that ROR{alpha} is involved in normal development of the immune system. It would be interesting to examine whether there are any abnormalities in the ROR{alpha}-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{alpha} mRNA and protein are observed. This fact has been attracting attention because a high level of ROR{alpha} 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{alpha} and conceals the staggerer phenotype in these portions. This is plausible; however, evidence has not been presented. In addition, I showed ROR{alpha} 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 1974Go; Landis and Sidman 1978Go), the above explanation is not sufficient. Possibly the absence of ROR{alpha} appears as phenotype only in restricted cell types. At least, ROR{alpha} and RORß are not always necessary for survival or function of neurons, because many neurons, such as hippocampal neurons, lack both ROR{alpha} and RORß (ROR{gamma} is not expressed in the nervous system).


    Footnotes
 
Received for publication March 19, 2003; accepted October 23, 2003


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 Literature Cited
 

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