The Reelin receptors Apoer2 and Vldlr coordinate the patterning of Purkinje cell topography in the developing mouse cerebellum

The adult cerebellar cortex is comprised of reproducible arrays of transverse zones and parasagittal stripes of Purkinje cells. Adult stripes are created through the perinatal rostrocaudal dispersion of embryonic Purkinje cell clusters, triggered by signaling through the Reelin pathway. Reelin is secreted by neurons in the external granular layer and deep cerebellar nuclei and binds to two high affinity extracellular receptors on Purkinje cells-the Very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer2). In mice null for either Reelin or double null for Vldlr and Apoer2, Purkinje cell clusters fail to disperse. Here we report that animals null for either Vldlr or Apoer2 individually, exhibit specific and parasagittally-restricted Purkinje cell ectopias. For example, in mice lacking Apoer2 function immunostaining reveals ectopic Purkinje cells that are largely restricted to the zebrin II-immunonegative population of the anterior vermis. In contrast, mice null for Vldlr have a much larger population of ectopic Purkinje cells that includes members from both the zebrin II-immunonegative and -immunopositive phenotypes. HSP25 immunoreactivity reveals that in Vldlr null animals a large portion of zebrin II-immunopositive ectopic cells are probably destined to become stripes in the central zone (lobules VI-VII). A small population of ectopic zebrin II-immunonegative Purkinje cells is also observed in animals heterozygous for both receptors (Apoer2(+/-): Vldlr(+/-)), but no ectopia is present in mice heterozygous for either receptor alone. These results indicate that Apoer2 and Vldlr coordinate the dispersal of distinct, but overlapping subsets of Purkinje cells in the developing cerebellum.     

Abnormal HNK-1 expression in the cerebellum of an N-CAM null mouse

Human natural killer antigen-1 (HNK-1) is a carbohydrate epitope associated with sulfoglucuronylglycolipids and glycoproteins. Biochemical analyses have demonstrated associations between the HNK-1 epitope and isoforms of the neural cell adhesion molecule (N-CAM) family. In the cerebellum, HNK-1 is prominently expressed in Purkinje cell dendrites and Golgi cells. Purkinje cell expression of HNK-1 reveals an array of parasagittal stripes and transverse zones. Interestingly, the parasagittal expression pattern of HNK-1 is different from those reported with several other markers such as zebrin II/aldolase C and the small heat shock protein HSP25. N-CAM null knockout mice were used to explore the possible role of the HNK-1/N-CAM interaction during the topographical organization of the cerebellar cortex. N-CAM null mice have no N-CAM immunoreactivity but otherwise the cerebellum appears morphologically normal. Further, in the N-CAM null HNK-1 immunoreactivity is abolished from Purkinje cell dendrites but is retained on Golgi cells and neurons of the cerebellar nuclei. Despite the absence of N-CAM/HNK-1, parasagittal stripes and transverse zones in the cerebellum as revealed by using zebrin II immunocytochemistry appear normal.     

Mouse Model Reveals the Role of RERE in Cerebellar Foliation and the Migration and Maturation of Purkinje Cells

Nuclear receptors and their coregulators play a critical role in brain development by regulating the spatiotemporal expression of their target genes. The arginine-glutamic acid dipeptide repeats gene (Rere) encodes a nuclear receptor coregulator previously known as Atrophin 2. In the developing cerebellum, RERE is expressed in the molecular layer, the Purkinje cell layer and the granule cell layer but not in granule cell precursors. To study RERE''s role in cerebellar development, we used RERE-deficient embryos bearing a null allele (om) and a hypomorphic allele (eyes3) of Rere (Rere ^om/eyes3). In contrast to wild-type embryos, formation of the principal fissures in these RERE-deficient embryos was delayed and the proliferative activity of granule cell precursors (GCPs) was reduced at E18.5. This reduction in proliferation was accompanied by a decrease in the expression of sonic hedgehog (SHH), which is secreted from Purkinje cells and is required for normal GCP proliferation. The maturation and migration of Purkinje cells in Rere ^om/eyes3 embryos was also delayed with decreased numbers of post-migratory Purkinje cells in the cerebellum. During the postnatal period, RERE depletion caused incomplete division of lobules I/II and III due to truncated development of the precentral fissure in the cerebellar vermis, abnormal development of lobule crus I and lobule crus II in the cerebellar hemispheres due to attenuation of the intercrural fissure, and decreased levels of Purkinje cell dendritic branching. We conclude that RERE-deficiency leads to delayed development of the principal fissures and delayed maturation and migration of Purkinje cells during prenatal cerebellar development and abnormal cerebellar foliation and Purkinje cell maturation during postnatal cerebellar development.     

Patterned Neuroprotection in the Inpp4awbl Mutant Mouse Cerebellum Correlates with the Expression of Eaat4

The weeble mutant mouse has a frame shift mutation in inositol polyphosphate 4-phosphatase type I (Inpp4a). The phenotype is characterized by an early onset cerebellar ataxia and neurodegeneration, especially apparent in the Purkinje cells. Purkinje cell loss is a common pathological finding in many human and mouse ataxic disorders. Here we show that in the Inpp4a^wbl mutant, Purkinje cells are lost in a specific temporal and spatial pattern. Loss occurs early in postnatal development; however, prior to the appearance of climbing fibers in the developing molecular layer, the mutant has a normal complement of Purkinje cells and they are properly positioned. Degeneration and reactive gliosis are present at postnatal day 5 and progress rapidly in a defined pattern of patches; however, Inpp4a is expressed uniformly across Purkinje cells. In late stage mutants, patches of surviving Purkinje cells appear remarkably normal with the exception that the climbing fibers have been excessively eliminated. Surviving Purkinje cells express Eaat4, a glutamate transporter that is differentially expressed in subsets of Purkinje cells during development and into adult stages. Prior to Purkinje cell loss, reactive gliosis and dendritic atrophy can be seen in Eaat4 negative stripes. Our data suggest that Purkinje cell loss in the Inpp4a^wbl mutant is due to glutamate excitotoxicity initiated by the climbing fiber, and that Eaat4 may exert a protective effect.     

FGF/FGFR2 Signaling Regulates the Generation and Correct Positioning of Bergmann Glia Cells in the Developing Mouse Cerebellum

The normal cellular organization and layering of the vertebrate cerebellum is established during embryonic and early postnatal development by the interplay of a complex array of genetic and signaling pathways. Disruption of these processes and of the proper layering of the cerebellum usually leads to ataxic behaviors. Here, we analyzed the relative contribution of Fibroblast growth factor receptor 2 (FGFR2)-mediated signaling to cerebellar development in conditional Fgfr2 single mutant mice. We show that during embryonic mouse development, Fgfr2 expression is higher in the anterior cerebellar primordium and excluded from the proliferative ventricular neuroepithelium. Consistent with this finding, conditional Fgfr2 single mutant mice display the most prominent defects in the anterior lobules of the adult cerebellum. In this context, FGFR2-mediated signaling is required for the proper generation of Bergmann glia cells and the correct positioning of these cells within the Purkinje cell layer, and for cell survival in the developing cerebellar primordium. Using cerebellar microexplant cultures treated with an FGFR agonist (FGF9) or antagonist (SU5402), we also show that FGF9/FGFR-mediated signaling inhibits the outward migration of radial glia and Bergmann glia precursors and cells, and might thus act as a positioning cue for these cells. Altogether, our findings reveal the specific functions of the FGFR2-mediated signaling pathway in the generation and positioning of Bergmann glia cells during cerebellar development in the mouse.     

Zebrin II / Aldolase C Expression in the Cerebellum of the Western Diamondback Rattlesnake (Crotalus atrox)

Aldolase C, also known as Zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+), alternating with stripes of Purkinje cells with little or no expression (ZII-). The patterns of ZII+ and ZII- stripes in the cerebellum of birds and mammals are strikingly similar, suggesting that it may have first evolved in the stem reptiles. In this study, we examined the expression of ZII in the cerebellum of the western diamondback rattlesnake (Crotalus atrox). In contrast to birds and mammals, the cerebellum of the rattlesnake is much smaller and simpler, consisting of a small, unfoliated dome of cells. A pattern of alternating ZII+ and ZII- sagittal stripes cells was not observed: rather all Purkinje cells were ZII+. This suggests that ZII stripes have either been lost in snakes or that they evolved convergently in birds and mammals.     

Increased Noradrenergic Metabolism in the Cerebellum of the Mouse Mutant Dystonia Musculorum

Abstract: The neurological mouse mutant dystonia musculorum exhibits bizarre appendicular and truncal dystonia without known cerebellar histopathology. We evaluated striatal dopamine and cerebellar norepinephrine metabolism in this mutant and compared the results with those obtained in wild-type BALB/c and B6C3 controls. Tyrosine hydroxylase activity and dopamine metabolite levels (homovanillic acid and 3,4-dihydroxyphenylacetic acid) in the striatum of the mutant were similar to controls. Tyrosine hydroxylase activity and the steady-state level of 3-methoxy-4-hydroxyphenethyleneglycol, a metabolite of norepinephrine, in the cerebellum were 38% and 42-66%, respectively, greater in the mutant. However, the level of norepinephrine was similar (∼350 ng/g). Further, a Purkinje cell-specific marker, cGMP-dependent protein kinase, was unchanged in the mutant and no Purkinje cell pathology was observed with light microscopy. The lack of Purkinje cell derangement and similar levels of cerebellar norepinephrine and cGMP-dependent protein kinase activity suggest that increased norepinephrine metabolism in the cerebellum of this mutant is not a morphological response to gross target cell loss during morphogenesis. The observed changes may be a reaction to abnormal impulse traffic or altered input/output pathways to the mutant cerebellum during its development.     

Granule cell raphes in the developing mouse cerebellum

The cerebellar cortex of many vertebrates shows a striking parasagittal compartmentation that is thought to play a role in the establishment and maintenance of functional cerebellar connectivity. Here, we demonstrate the existence of multiple parasagittal raphes of cells in the molecular layer of the developing cerebellar cortex of postnatal mouse. The histological appearance and immunostaining profile of the raphe cells suggest that they are migrating granule cells. We therefore conclude that the granule cell raphes previously described in birds also exist in a mammalian species. The raphes in mouse are visible on nuclear stains from around birth to postnatal day 6 and are frequently found at the boundaries of Purkinje cell segments that differentially express cadherins ("early-onset" parasagittal banding pattern). A similar relation between the raphe pattern and various markers for the early-onset banding pattern has been found in the chicken cerebellum. One of the cadherins mapped in the present study (OL-protocadherin) continues to be expressed in specific Purkinje cell segments until at least postnatal day 14. At this stage of development, the borders of the OL-protocadherin-positive Purkinje cell segments coincide with the borders of Purkinje cell segments that express zebrin II, a marker for the "late-onset" parasagittal banding pattern which persists in the adult cerebellum. These findings demonstrate that the early-onset banding pattern, as reflected in the complementary arrangement of raphes/Purkinje cell segments, and the late-onset pattern of zebrin II expression share at least some positional cues during development.     

Biphasic dispersion of clones containing Purkinje cells and glia in the developing chick cerebellum.

The cerebellum is a highly conserved structure which exhibits patterns of gene expression and axonal connections that are organized into parasagittal domains. These aspects of the mature cerebellum are presaged during embryonic development by the expression patterns of vertebrate homologs of Drosophila segmentation genes. We wished to determine whether the parasagittal domains of gene expression are compartments of lineage restriction. To this end, a clonal analysis of the chick cerebellum was conducted with a complex retroviral library. From embryonic day (E) 8 to E12, clones derived from the more medial portion of the cerebellar ventricular zone (VZ) were observed to spread preferentially in the mediolateral direction, crossing the boundaries of the parasagittal domains of gene expression. In late embryonic and posthatch periods, VZ clones were found to comprise Purkinje cells, glial cells, or both types of cells. At these later times, clonally related glial cells formed tight parasagittal clusters, while clonally related Purkinje cells were scattered extensively in the anteroposterior direction. We propose that a subset of the cerebellar VZ clones, those with medial origins, undergoes a biphasic dispersion: an early phase of mediolateral dispersion and a late phase of anteroposterior dispersion. This novel pattern of clonal dispersion suggests that the cerebellar VZ is not partitioned into parasagittal domains of lineage restriction. It leaves open the possibility that the later dispersion along the anteroposterior axis results from the parasagittal patterns of gene expression in the developing cerebellar cortex.     

Cerebellar Hypoplasia in Mice Lacking Selenoprotein Biosynthesis in Neurons

Selenium exerts many, if not most, of its physiological functions as a selenocysteine moiety in proteins. Selenoproteins are involved in many biochemical processes including regulation of cellular redox state, calcium homeostasis, protein biosynthesis, and degradation. A neurodevelopmental syndrome called progressive cerebello-cortical atrophy (PCCA) is caused by mutations in the selenocysteine synthase gene, SEPSECS, demonstrating that selenoproteins are essential for human brain development. While we have shown that selenoproteins are required for correct hippocampal and cortical interneuron development, little is known about the functions of selenoproteins in the cerebellum. Therefore, we have abrogated neuronal selenoprotein biosynthesis by conditional deletion of the gene encoding selenocysteyl tRNA^[Ser]Sec (gene symbol Trsp). Enzymatic activity of cellular glutathione peroxidase and cytosolic thioredoxin reductase is reduced in cerebellar extracts from Trsp-mutant mice. These mice grow slowly and fail to gain postural control or to coordinate their movements. Histological analysis reveals marked cerebellar hypoplasia, associated with Purkinje cell death and decreased granule cell proliferation. Purkinje cell death occurs along parasagittal stripes as observed in other models of Purkinje cell loss. Neuron-specific inactivation of glutathione peroxidase 4 (Gpx4) used the same Cre driver phenocopies tRNA^[Ser]Sec mutants in several aspects: cerebellar hypoplasia, stripe-like Purkinje cell loss, and reduced granule cell proliferation. Parvalbumin-expressing GABAergic interneurons (stellate and/or basket cells) are virtually absent in tRNA^[Ser]Sec-mutant mice, while some remained in Gpx4-mutant mice. Our data show that selenoproteins are specifically required in postmitotic neurons of the developing cerebellum, thus providing a rational explanation for cerebellar hypoplasia as occurring in PCCA patients.     

The development of molecular compartmentation in the cerebellar cortex

The cerebellum is subdivided into hundreds of discrete modules defined by their connectivity and molecular signatures. Cerebellar compartmentation arises very early in development through the formation of multiple populations of chemically distinct Purkinje cells that migrate in a coordinated fashion to form parasagittal bands of cells. Different Purkinje cell bands are then innervated by discrete subpopulations of cerebellar afferents. Because of its stereotyped and strikingly beautiful organization the cerebellum is an excellent model in which to explore genetic/epigenetic aspects of pattern formation in the central nervous system.     

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.     

Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice

Lesions in the cerebellar vermis abolish acquisition of fear-conditioned bradycardia in animals and human patients. The δ2 glutamate receptor (GluD2) is predominantly expressed in cerebellar Purkinje cells. The mouse mutant ho15J carries a spontaneous mutation in GluD2 and these mice show a primary deficiency in parallel fiber-Purkinje cell synapses, multiple innervations of Purkinje cells by climbing fibers, and impairment of long-term depression. In the present study, we used ho15J mice to investigate the role of the cerebellum in fear-conditioned bradycardia. We recorded changes in heart rate of ho15J mice induced by repeated pairing of an acoustic (conditioned) stimulus (CS) with an aversive (unconditioned) stimulus (US). The mice acquired conditioned bradycardia on Day 1 of the CS-US phase, similarly to wild-type mice. However, the magnitude of the conditioned bradycardia was not stable in the mutant mice, but rather was exaggerated on Days 2–5 of the CS-US phase. We examined the effects of reversibly inactivating the cerebellum by injection of an antagonist against the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR). The antagonist abolished expression of conditioned responses in both wild-type and ho15J mice. We conclude that the GluD2 mutation in the ho15J mice affects stable retention of the acquired conditioned bradycardia.     

Increased Density of Dystrophin Protein in the Lateral Versus the Vermal Mouse Cerebellum

Dystrophin, present in muscle, also resides in the brain, including cerebellar Purkinje neurons. The cerebellum, although historically associated with motor abilities, is also implicated in cognition. An absence of brain dystrophin in Duchenne muscular dystrophy (DMD) and in the mdx mouse model results in cognitive impairments. Localization studies of cerebellar dystrophin, however, have focused on the vermal cerebellum, associated with motor function, and have not investigated dystrophin distribution in the lateral cerebellum, considered to mediate cognitive function. The present study examined dystrophin localization in vermal and lateral cerebellar regions and across subcellular areas of Purkinje neurons in the mouse using immunohistochemistry. In both vermal and lateral cerebellum, dystrophin was restricted to puncta on somatic and dendritic membranes of Purkinje neurons. The density of dystrophin puncta was greater in the lateral than the vermal region. Neither the size of puncta nor the area of Purkinje neuron somata differed between regions. Results support the view that cognitive deficits in the DMD and the mdx model may be mediated by the loss of dystrophin, particularly in the lateral cerebellum. Findings have important implications for future studies examining the neurophysiological sequelae of neuronal dystrophin deficiency and the role of the lateral cerebellum in cognition.     

Uneven distribution of NG2 cells in the rat cerebellar vermis and changes in aging

We describe by NG2 (neuron-glia chondroitin sulphate proteoglycan 2) immunocytochemistry an uneven distribution of NG2 glial cells in the rat cerebellum, being them more represented in the central lobules of the cerebellar vermis, belonging to the cerebrocerebellum. The cerebellar distribution of NG2 cells changes in aging rats, in which the area where the cells appear to be densely scattered throughout all cerebellar layers involves also more rostral and caudal lobules. In addition, in aging rats, in the most rostral and caudal lobules belonging to the spinocerebellum, punctate reaction product is present at the apical pole of Purkinje cells, i.e. in the area where the majority of synapses between olivary climbing fibers and Purkinje cells occur. Data suggest that the different distribution of NG2 cells is correlated to differences in physiology among cerebellar areas and reflects changes during aging.Key words: cerebellum, aging, NG2 glia.     

Cerebellar development: afferent organization and Purkinje cell heterogeneity.

Olivo- and spinocerebellar maps in the adult cerebellum of small rodents are discontinuous, with sharp boundaries. Cortical Purkinje cells constitute a heterogeneous population, organized into parasagittal, mutually exclusive compartments. The boundaries of the intrinsic cortical compartments and those of the projectional maps are congruent. During development; (i) The incoming olivary fibres, once they penetrate in the cerebellar parenchyma, are attracted toward their ultimate terminal fields, without passing through a stage of random dispersion. (ii) Migrating Purkinje cells and inferior olivary neurons begin, asynchronously, to express cellular markers in an independent manner, giving rise to a transient compartmentation of the cerebellar cortex and the inferior olivary complex respectively. In both instances, the biochemical heterogeneity disappears during the first postnatal week, simultaneously with the acquisition of adult-like cerebellar maps. (iii) The formation of the maps is an early event, prior to the establishment of the synaptology of the cerebellar cortical circuitry. Moreover, the organization of the spinocerebellar projection in adult mutant mice does not depend on the presence of granule cells (staggerer) but on the presence of normal Purkinje cells (weaver), indicating that synaptogenesis with their target neurons is not involved in the process of map formation. The matching of region specific chemical labels between incoming afferent fibres and heterogeneous sets of Purkinje cells is the most appealing mechanism for the formation of cerebellar maps.     

The organization of the corticonuclear and olivocerebellar climbing fiber projections to the rat cerebellar vermis: The congruence of projection zones and the zebrin pattern

The zonal organization of the corticonuclear and the olivocerebellar climbing fiber projections to the vermis of the cerebellum of the rat was compared to the pattern of zebrin-positive and zebrin-negative bands in material double-stained for zebrin II and for different anterograde tracers injected in subnuclei of the inferior olive, or retrograde tracers injected in the cerebellar and vestibular target nuclei of the Purkinje cells of the vermis. Projection zones A₁, A_X, X, B, C_X in the vermis and A₂ (accessory A zone) and C₂ in the hemisphere were defined by their efferent corticonuclear and their afferent climbing fiber connections, and were found to share the same topographical framework with the zebrin pattern.     

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca²⁺-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca²⁺-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.     

The cerebellar deficient folia (cdf) gene acts intrinsically in Purkinje cell migrations

Cerebellar deficient folia (cdf) is a recently identified mouse mutation causing ataxia and cerebellar abnormalities including lobulation defects and abnormal placement of a specific subset of Purkinje cells. To understand the etiology of the cerebellar defects in cdf mutant mice, we examined postnatal development of the cdf/cdf cerebellum. Our results demonstrate that Purkinje cell ectopia and foliation defects are apparent at birth, suggesting the cdf mutation disrupts the positioning of many, but not all, Purkinje cells during development. In addition to cerebellar abnormalities, we observed lamination defects in the hippocampus of cdf mutant mice, although neocortical defects were not seen. Furthermore, ectopic Purkinje cells in cdf/cdf mice express an increased level of Dab1 protein, as previously observed in mice with mutations in genes in the reelin signaling pathway. Lastly, analysis of cdf <-->ROSA26 chimeric mice demonstrated that the cdf mutation is intrinsic to Purkinje cells. We suggest that the cdf gene product is required in a subset of Purkinje cells, possibly to respond to Reelin signals.