staggerer phenotype in retinoid-related orphan receptor alpha-deficient mice

Abstract

Retinoid-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme beta-galactosidase. In heterozygous RORalpha+/- mice we found beta-galactosidase activity, indicative of RORalpha protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORalpha gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORalpha is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORalpha. Recently, it was reported that the ataxic mouse mutant staggerer (sg/sg) is caused by a deletion in the RORalpha gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORalpha-/- mice proves identity to sg/sg mice. Although the absence of RORalpha causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORalpha-/- mice display a normal phenotype. However, the pelage hair of both sg/sg and RORalpha-/- is significantly less dense and when shaved shows reluctance to regrow.

Figure 1

Targeting the mouse RORα locus. (A) Schematic representation of the wild-type RORα locus, targeting vector, and recombined locus. In the targeted locus the exon of the second zinc finger of the DNA binding domain (filled box) is disrupted and fused to lacZ encoding β-gal. (B) Diagnostic Southern blot hybridization was performed by using the probe indicated in A. Lanes are homozygous (−/−), heterozygous (+/−), and wild-type (+/+) DNA.

Figure 2

Comparison of physical performance of RORα^−/− and staggerer mice in the motor synchronization learning (rotarod) test. (A) Duration the mice stayed on the rotarod. Wild-type (+/+) (n = 10) and heterozygous RORα knockout mice (+/−) (n = 10) managed to stay on the rod for the entire 180 sec; homozygous RORα knockout mice (−/−) and sg/sg mice never reached this maximal score (n = 12). However, the RORα^−/− mice significantly improved from day 1 to day 10 (Mann–Whitney, P > 0.05), whereas the sg/sg mice did not at all. (B) Strategy used by mice to stay on the rod. Wild-type (n = 10) and RORα^+/− mice (n = 10) used exclusively walking, whereas RORα^−/− mice and sg/sg mice (n = 12) used principally or exclusively hanging, respectively.

Figure 3

Comparison of cellular phenotypes in RORα^−/− (A and D), sg/sg (B), and RORα^+/+ mice (C) with double-immunolabeled preparations (zebrin I/calbindin). In frontal sections of RORα^−/− (A) and sg/sg mice (B) the cerebellar phenotype is identical: the vermis is atrophic but lobulated (large arrows point to the midline), with disorganized PC bodies and thin molecular layer. Note that the immunostained PC projections are densely packed in the medial cerebellar nucleus (MN). All PCs are devoid of zebrin I because the narrow zones marked by arrowheads do not represent zebrin I-positive bands but zones of PC depletion. In contrast, in the RORα^+/+ cerebellum (C) PCs are arranged in a monolayer at the interface of the granular and molecular layers (arrowheads). The molecular layer is thick and contains the parasagittally oriented PC dendrites. Moreover, zebrin I-positive PCs (dark areas caused by quenching of the calbindin immunofluorescence by the immunoprecipitate) are confined to regularly occurring parasagittal bands (the central band is marked by asterisks). (D) High magnification of calbindin-positive PCs in the RORα^−/− vermal cortex. Most of these neurons have apical dendrites that branch into small disoriented dendritic arbors (arrows). (E–G) Electron micrographs illustrating some features of the synaptic arrangement in the RORα^−/− cerebellum. (E) High magnification of the molecular layer neuropil, in a preparation immunostained with anti-GABA antibodies, showing two immunogold-labeled axon terminals (at) apposed to small dendritic profiles. Parallel fibers, isolated or forming small clusters (arrowheads), are present in the layer. (F) Purkinje cell perikaryon (PC). Numerous perikaryal spines (asterisks) are postsynaptic to two axon terminals belonging to climbing fiber varicosities (cf) that have retained their immature pericellular nest location. A large terminal, probably belonging to a basket cell axon (bca), is apposed to the PC between the two climbing fiber varicosities. (G) Granular layer. A mossy fiber rosette (mf) is mostly surrounded by thin astrocytic processes (asterisks), and a few granule cell dendrites (stars). Note the immunogold, GABA-labeled axon terminal (at) almost directly apposed to the mossy fiber, testifying that most granule cell dendrites have disappeared. This terminal belongs to the Golgi cell axon. [Immunofluorescence, bar = 350 μm (A–C) and 70 μm (D); electron microscopy, bar = 0.6 μm (E and G) and 1.3 μm (F).]

Figure 4

Electrophysiological characterization of RORα^−/− PCs. (A1) Two superimposed sweeps of all-or-none CF-mediated EPSCs in normal PCs and one subthreshold response evoked by stimulation of the granular layer at the holding potential of −80 mV. (A2) Same cell recorded in current-clamp mode at −70 mV. (B1) Superimposed sweeps of CF-mediated EPSCs elicited in a multiply innervated RORα^−/− PC held at −80 mV by stimulating the granular layer with progressively increasing strength. (B2) Plot of amplitudes against time of CF-mediated EPSCs recorded at −80 mV in the same cell as in B1. Note the stepwise variation of the responses with progressively increasing stimulation strength. (C1) Same as in B1 using another neuron. (C2) The cell was held in current-clamp mode at −80 mV and the granular layer was stimulated with progressively increasing intensity.

Figure 5

(A–F) Expression of RORα revealed by β-gal activity in RORα^+/− mice. Tissue sections were stained with the β-gal substrate 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) (C) or additionally counterstained with either cresylviolet (A, B, D, and F) or hematoxylin/eosin (E). (A) Retina; gcl, ganglion cells; inl, inner nuclear layer; onl, outer nuclear layer; pel, pigment epithelium layer. (B) Cerebellum; pc, Purkinje cells. (C) Suprachiasmatic nuclei; och, optic chiasm. (D) Testis; ptc, peritubular cells; st, seminiferous tubule. (E) Epididymis; sp, spermatocytes; epi, epithelium. (F) Tail skin; epi, epidermis; seb, sebaceous gland; hf, hair follicle. (G and H) Comparison of a RORα^+/− and a RORα^−/− mouse; note the difference in the appearance of the fur and the partially exposed skin of the RORα^−/− mouse (H).