ARTICLE

The Cerebellar Deficient Folia (cdf) Gene Acts Intrinsicallyin Purkinje Cell MigrationsChankyu Park,1 Jacqueline H. Finger,1 Jonathan A. Cooper,2 and Susan L. Ackerman1*1The Jackson Laboratory, Bar Harbor, ME2Fred Hutchinson Cancer Research Center, Seattle, WA

Received 7 September 2001; Accepted 20 November 2001

Summary: Cerebellar deficient folia (cdf) is a recentlyidentified mouse mutation causing ataxia and cerebellarabnormalities including lobulation defects and abnormalplacement of a specific subset of Purkinje cells. To un-derstand the etiology of the cerebellar defects in cdfmutant mice, we examined postnatal development of thecdf/cdf cerebellum. Our results demonstrate that Pur-kinje cell ectopia and foliation defects are apparent atbirth, suggesting the cdf mutation disrupts the position-ing of many, but not all, Purkinje cells during develop-ment. In addition to cerebellar abnormalities, we ob-served lamination defects in the hippocampus of cdfmutant mice, although neocortical defects were notseen. Furthermore, ectopic Purkinje cells in cdf/cdf miceexpress an increased level of Dab1 protein, as previouslyobserved in mice with mutations in genes in the reelinsignaling pathway. Lastly, analysis of cdf7ROSA26 chi-meric mice demonstrated that the cdf mutation is intrin-sic to Purkinje cells. We suggest that the cdf gene prod-uct is required in a subset of Purkinje cells, possibly torespond to Reelin signals. genesis 32:32–41, 2002.© 2002 Wiley-Liss, Inc.

Key words: reelin; 5Fki; Dab1; neuronal migration; hip-pocampus; cortex; aggregation chimera

INTRODUCTION

Mouse mutants with neuronal ectopia have providedindispensable experimental models to investigate themolecular mechanisms underlying the neuronal migra-tion process. Studies of the reeler mutant mouse, andother mice with mutations in distinct genes but withsimilar phenotypes, has allowed the identification ofgenes necessary for proper positioning of Purkinje cellsduring cerebellar development (reviewed in Rice andCurran, 1999; Gilmore and Herrup, 2000; Walsh andGoffinet, 2000). Reelin, the product of the reeler gene(reln), is a protein secreted by the Cajal-Retzius cells andthe external granule cells in the developing cerebralcortex and cerebellum, respectively (D’Arcangelo et al.,1995; Ogawa et al., 1995; Miyata et al., 1996). Potentialcomponents of the reelin receptor complex include thevery low-density lipoprotein receptor (VLDLR), the apo-

lipoprotein E receptor 2 (apoER2, also known as LRP8),cadherin-related neuronal receptor (CNR), and �3�1 in-tegrin molecules (D’Arcangelo et al., 1999; Hiesberger etal., 1999; Senzaki et al., 1999; Dulabon et al., 2000).Reelin/receptor binding induces tyrosine phosphoryla-tion of the adapter protein, Dab1, which binds the cy-toplasmic domain of the VLDLR and apoER2 receptorsvia an N-terminal phosphotyrosine binding (PTB) do-main (Trommsdorff et al., 1999). These events in turnactivate a downstream intracellular signaling cascadenecessary for proper neuronal migration (Howell et al.,1999, 2000). Elevation of the level of Dab1 proteinoccurs in reln mutant mice and mice homozygous formutations in both the Vldlr and apoER2 genes, implyingthat the upregulation of Dab1 may be a hallmark ofdisruptions in the reelin signaling pathway (Rice et al.,1998; Trommsdorff et al., 1999). Mice with mutations inreln, Dab1, or apoER2 and Vldlr genes have similarphenotypes that include lamination defects in the neo-cortex, hippocampus, and cerebellum (Caviness and Sid-man, 1973a, 1973b; D’Arcangelo et al., 1995; Howell etal., 1997b; Sheldon et al., 1997; Ware et al., 1997;Trommsdorff et al., 1999). Neuronal migration defectshave also been seen in these brain regions in mice withdisruptions in the genes encoding Cdk5, a neuronal-specific kinase, its activator p35, or combinations ofthese two mutations (Ohshima et al., 1996; Chae et al.,1997). Mice homozygous for a targeted mutation in theCdk5 gene or in the Cdk5r gene, encoding p35, have aninversion of the layers of the cerebral cortex and disrup-tions of olfactory bulb and hippocampal lamination. Inaddition, as in mice with mutations in the reelin path-way, the vast majority of Purkinje cells fail to correctlyposition in Cdk5 mutant mice. Although the connection,if any, between the Cdk5/p35 and reelin signaling path-ways has been elusive, in vitro studies have shown thatthe Cdk5/p35 complex can phosphorylate Dab1

*Correspondence to: Susan L. Ackerman, The Jackson Laboratory, 600Main Street, Bar Harbor, ME 04609.

E-mail: sla@jax.orgGrant sponsors: National Institutes of Health and the NCI.

© 2002 Wiley-Liss, Inc. genesis 32:32–41 (2002)DOI 10.1002/gene.10024

(D’Arcangelo et al., 1999). Moreover, recent studieshave demonstrated synergistic genetic interactions be-tween Cdk5 and Cdk5r, and between Dab1 and Cdk5r,suggesting that Cdk5/p35 and Reelin/Dab1 signalingpathways either interact directly or via the regulation ofdownstream target proteins (Ohshima et al., 2001).

Cerebellar deficient folia (cdf) is a recently identifiedrecessive mutation on mouse chromosome 6 (Cook etal., 1997; Park et al., 2000). Homozygous mutant micehave cerebellar dysgenesis, including hypoplasia and areduced number of folia. Although the cdf mutation doesnot influence the birthdate or numbers of Purkinje cellsin the adult cerebellum, about 40% of Purkinje cells arelocated ectopically in the white matter and inner granulecell layer of cdf mutant mice, suggesting abnormalities inPurkinje cell precursor migration (Beierbach et al.,2001). Unlike other known mutations affecting Purkinjecell placement, cdf causes ectopia of only a specificsubset of Purkinje cells (Beierbach et al., 2001). Expres-sion of the antigen, zebrin II, distinguishes two popula-tions of Purkinje cells in the adult cerebellum: zebrinII-positive and zebrin II negative (Brochu et al., 1990;Eisenman and Hawkes, 1993). In the normal cerebellum,zebrin II-expressing Purkinje cells form parasagittalstripes, alternating with stripes containing zebrin II-neg-ative Purkinje cells. In the cdf mutant cerebellum, theplacement of zebrin II-negative Purkinje cells is specifi-cally affected, suggesting a defect in either the reelinsignaling cascade downstream of reelin or in a neuronalmigration pathway that is independent of reelin (Beier-bach et al., 2001).

To further understand the etiology of cerebellar de-fects in cdf mutant mice, we studied the postnatal de-velopment of the cdf/cdf cerebellum. We also evaluatedcdf mice for abnormalities in the neocortex and hip-pocampus and examined the level of Dab1 in the cdfmutant cerebellum and cortex. Lastly, to determine thesite of action of the cdf mutation, we analyzed chimericmice consisting of cdf mutant cells and wild-type cells.Our results demonstrate that the cdf gene acts intrinsi-cally to Purkinje cells, and mice homozygous for muta-tions in this gene have a subset of phenotypes previouslydescribed in mice with mutations in the reelin/Cdk5pathways.

RESULTS

Defects in Purkinje Cell Placement and FoliationAre Discernable in the P0 cdf Cerebellum

In adult mice homozygous for the cdf mutation, asubset of Purkinje cells, identified by their lack of zebrinII expression, are found in the white matter and internalgranule cell layer of the cerebellum (Cook et al., 1997;Beierbach et al., 2001). In addition, some fissures aremissing in the cdf/cdf adult cerebellum, resulting inabnormal lobulation. To study the process of Purkinjecell layer formation and lobulation in the cdf/cdf cere-bellum, midline sagittal sections of postnatal cerebellafrom cdf/cdf and wild-type littermates were immuno-

stained with antibodies to the Purkinje cell marker, cal-bindin D-28 (Fig. 1).

In wild-type cerebella, four vermal fissures were visi-ble at P0 (Fig. 1). In addition, all Purkinje cells were

FIG. 1. Abnormal postnatal cerebellar development in cdf mutantmice. Midline sagittal sections from wild-type and cdf/cdf cerebellaat P0, P2, P4, P8, and P14 were immunostained with antibodies tocalbindin to visualize Purkinje cells (stained dark brown). Sectionswere counterstained with hematoxylin. Note the Purkinje cell ecto-pia and delayed lobulation in cdf/cdf cerebella compared to wild-type controls. Although the animals used in this study were main-tained on a segregating genetic background of C3H/HeSnJ andCAST/Ei, all the wild-type animals (n � 20) studied showed thelobulation pattern of C3H/HeSnJ mice. Anterior is to the left anddorsal is to the top of each photograph. Fissures are indicated asfollows; ce, precentral; cu, preculminate; pr, primary; i, intercrural;py, prepyramidal; sec, secondary; po, posterolateral. Scale bars:200 �m.

33PURKINJE CELL ECTOPIA IN CDF MUTANT MICE

found in a several-cell-thick Purkinje cell layer below theexternal granule cell layer (EGL), with the white matterfree of Purkinje cells. In contrast, P0 cerebella of cdf/cdfmice lacked fissures, and many Purkinje cells were scat-tered throughout the deep white matter. Two morefissures were formed in the wild-type cerebellum bypostnatal day 2; however the P2 cdf/cdf cerebellumremained unchanged from P0. By P4, Purkinje cells hadaligned into a single cell layer in the wild-type cerebel-lum and the fissures had deepened. In the P4 cdf/cdfcerebellum, the nonectopic Purkinje cells were also in asingle layer, making ectopic cells more apparent. Lobu-lation of the cdf/ cerebellum began between P2 and P4,resulting in a cerebellum with four fissures, similar inappearance to that of wild-type at P0.

Cerebellar lobulation continued to be delayed past thefirst postnatal week in cdf mutant mice. The appearanceof the prepyramidal fissure was finally apparent by P8 inthe cdf/cdf cerebellum. In contrast to the Purkinje celldefects, no apparent defects were observed in the thick-ness of the external granule cell layer (EGL) or theinternal granule cell layer (IGL), nor were there obviousabnormalities in the migration of granule cells from theEGL to IGL in the cdf/cdf cerebellum. In sum, the cdfmutation clearly disrupts the positioning of Purkinjecells during cerebellar development. In addition, cere-bellar foliation is severely delayed with lobulation de-fects in a cdf/cdf cerebellum, largely ascribed to themissing precentral and intercrural fissures and the shal-low existing fissures.

Hippocampal Pyramidal Cell Ectopia in cdfMutant Mice

In addition to cerebellar defects, we observed lamina-tion defects in the hippocampus of cdf mutant mice.Pyramidal cells of CA1, CA2, and CA3 regions of themutant hippocampus were scattered into the externaland deep plexiform layers of the hippocampus, occa-sionally forming an ectopic layer (Fig. 2). The cells re-maining in the pyramidal layer were less densely packedcompared to those in wild-type mice, and occasionallyappeared as isolated cells (Fig. 2D). 5-bromodeoxyuri-dine (BrdU) labeling demonstrated that ectopic pyrami-dal cells are generated at normal times of pyramidal cellneurogenesis (data not shown). The hippocampal de-fects were present in all cdf mutant mice analyzed (n �4). However, the dentate gyrus appeared normal in cdf/cdf mice, demonstrating the specificity of the cdf muta-tion in disruption of the lamination of hippocampalpyramidal cells.

The cdf Mutation Does Not Disrupt RadialMigrations in the Cerebral Cortex

Several mouse mutations that disrupt radial migrationof Purkinje cells also disrupt radial migration of neuronsin the cerebral cortex. To determine if the cdf mutationaffects the migration of both cerebellar and neocorticalneurons, we performed neuronal birthdating analysis.Pregnant dams were injected with BrdU between embry-

onic day (E)12–E17. As in wild-type controls, BrdU-la-beled neurons were observed from injection at E12–E17in the cdf mutant neocortex (Fig. 3, and data not shown).There were no obvious differences between wild-typeand cdf mutant mice in the overall pattern of distributionor in numbers of BrdU–labeled cells in the cerebralcortex, nor were there apparent differences in the thick-ness of the marginal zone (Fig. 3). Thus, in contrast tothe neuronal positioning abnormalities observed in thecerebellum and hippocampus, the radial migration ofneocortical neurons appears normal in cdf/cdf mice.

Dab1 Levels Are Increased in Ectopic PurkinjeCells in cdf Mutant Mice

Dab1 is expressed in the developing neurons of theneocortex, hippocampal pyramidal cells, and cerebellarPurkinje cells. An increased level of Dab1 protein duringembryonic and early postnatal brain development is aphenotypic feature of mutations affecting reelin-directedneuronal migrations (Rice et al., 1998; Trommsdorff etal., 1999). To determine if the level of Dab1 is misregu-lated in the cdf mutant brain, we performed Westernblot analysis and immunohistochemistry on P2 brains(Fig. 4). Densitometry of bands on Western blots dem-onstrated that Dab1 levels in the cdf/cdf cerebellum areincreased approximately twofold relative to the level inwild-type cerebellum, whereas loading controls Cdk5(Fig. 4) and neuron-specific enolase (data not shown) areunchanged (the ratio of Dab1/Cdk5 was 0.45, 0.54, and1.08, in, �/�, cdf/� and cdf/cdf cerebella, respectively).In contrast, no differences in Dab1 levels between cdfmutant and wild-type neocortex were seen by Westernanalysis at P0 or P2 (Fig. 4A and data not shown).

The increased level of Dab1 in the Purkinje cells of P2cdf/cdf mice compared to wild-type littermates was con-

FIG. 2. Hippocampal defects in cdf mutant mice. Horizontal sec-tions of the adult hippocampus from wild-type control mice (A, C)and cdf mutant mice (B, D) were stained with luxol fast blue-cresylviolet. Note the disorganization of pyramidal cells in the CA1, CA2,and CA3 regions (arrows). Scale bars: 200 �m.

34 PARK ET AL.

firmed by immunofluorescence (Fig. 4B, C; note thatDab1 is not expressed in other cell types in the cerebel-lum; Howell et al., 1997b; Gallagher et al., 1998; Rice etal., 1998). Interestingly, unlike Dab1 levels in the relnmutants, the level of Purkinje cell-specific Dab1 expres-sion was not uniform in the cdf/cdf cerebellum. Ectopi-cally located Purkinje cells expressed an elevated level ofDab1, whereas Dab1 expression was not upregulated innormally located Purkinje cells. However, no differenceswere observed in the pattern and signal intensity ofDab1 in the neocortex of cdf mutant mice compared towild-type controls (data not shown). The expressionpattern and level of reelin were also compared betweenwild-type and cdf mutant mice by immunohistochemis-try. As expected given the proper positioning of 60% ofthe Purkinje cells in the cdf mutant mouse, no measur-able differences were observed between cdf/cdf andwild-type brains (data not shown).

In addition to cdf mutant animals, we analyzed Dab1expression in 5Fki, a loss-of-function knock-in allele ofthe Dab1 gene (Fig. 4D, E). The 5Fki mutation removesfive putative tyrosine phosphorylation sites in the Dab1protein, and the resulting protein is not tyrosine phos-phorylated (Howell et al., 2000). The phenotype of the

5Fki/5Fki mice is very similar to the Dab1 null or relnmutant mice, with the vast majority of Purkinje cellsremaining ectopic accompanied by an almost completefailure of cerebellar foliation. However, unlike Dab1 lev-els in mice with mutations in reln, apoER2, or Vldlr,Dab1 protein levels in the developing neocortex are onlyslightly elevated (Howell et al., 2000), suggesting thatdownregulation of Dab1 in the neocortex is dependenton reelin but independent of Dab1 tyrosine phosphory-lation. Thus, we anticipated that ectopic neurons in 5Fkimutant brains might have almost normal Dab1 proteinlevels and serve as a control for ectopic neurons in cdfmutants. Indeed, we found that Dab1 levels in the Pur-kinje cells of 5Fki/5Fki mice were only slightly elevatedover wild-type levels. Ectopic Purkinje cells of P2 5Fki/5Fki cerebellum had less Dab1 than corresponding ec-topic cells in cdf/cdf cerebellum (Fig. 4). Furthermore,ectopic Purkinje cells from the cdf/cdf cerebellumtended to be isolated (Fig. 4C), whereas Purkinje cellsfrom the 5Fki/5Fki cerebellum appear more adhesive,resulting in an aggregated cell mass (Fig. 4E). Althoughmost ectopic Purkinje cells are clustered in the deepwhite matter in the P2 5Fki homozygous cerebellum,some are found very close to the external granule celllayer (EGL). Little difference in Dab1 levels was seenbetween Purkinje cells next to the EGL and those thatwere clearly ectopic, suggesting that the level of 5Fmutant Dab1 is independent of the distance of Purkinjecells from reelin-secreting cells of the EGL.

In summary, these results demonstrate that, like mu-tations in members of the reelin signaling pathway thecdf mutation alters levels of Dab1 in ectopically locatedPurkinje cells.

Generation and Analysis of cdf/cdf7ROSA26Chimeras

To understand the primary site of gene action and cellinteractions of the cdf gene, we produced experimentalchimeric mice. Wild-type cells were derived from ho-mozygous ROSA26 embryos and thus were marked by�-galactosidase (�-gal) expression, which we have pre-viously shown to be expressed in all cerebellar cells inthe ROSA26 mouse (Goldowitz et al., 2000). Like othercerebellar mutants, cdf/cdf males exhibit difficulties inbreeding that are likely caused by problems in coordina-tion. Thus, to increase the number of informative (thosecontaining cdf/cdf cells) chimeras, embryos were pro-duced by in vitro fertilization (IVF) using sperm and eggsfrom cdf homozygous mice. IVF was also used to pro-duce ROSA26 homozygous embryos. Embryos were al-lowed to proceed to the 8-cell stage, and pairs compris-ing one embryo from each genotype were fused andimplanted into pseudopregnant mice to produce cdf/cdf7ROSA26 chimeric mice.

Ten animals were produced with highly variegatedcoats. All of these chimeric animals had normal motorbehavior, suggesting that even a contribution of �24%(e.g., chimera 9) of wild-type cells is enough to signifi-cantly improve the locomotion defects associated with

FIG. 3. Neocortical neurons are properly positioned in cdf mutantmice. Comparison of the BrdU labeling pattern in the neocortexbetween wild-type (A, C) and cdf mutant (B, D) mice. Pregnantfemales were injected with BrdU at E12, E13, E14, E15, E16, or E17.Offspring were sacrificed after weaning and brain sections wereimmunostained with BrdU antibodies. At least two control and twomutant animals were analyzed at each time point. No differenceswere seen in the location of BrdU-positive neurons in cdf mutantcortex. Two time points are shown: E14 (A, B) and E16 (C, D). Mostof the BrdU-labeled neurons at both time points were localized inlayers II/ III in both wild-type and cdf mutant mice as indicated bydark brown DAB precipitate. Cortical layers are indicated by Romannumerals. Scale bars: 200 �m.

35PURKINJE CELL ECTOPIA IN CDF MUTANT MICE

the cdf mutation. However, another behavioral pheno-type of cdf mutants, hind-leg clasping (also characteristicof reln and Dab1 mutant mice), was clearly observed infour of the chimeras, suggesting that some movement/coordination abnormalities were still present in a subsetof chimeric animals. These animals had the highest con-tribution of mutant cells, as judged by coat color andhistology (see Table 1).

To determine the percentage chimerism within thecerebellum, cerebellar sections were processed for de-tection of �-gal, which identifies genetically wild-typecells. To clearly distinguish Purkinje cells, sections wereconcurrently immunostained for calbindin D-28. �-galreactivity was identified in various cell types distin-guished by immunohistological markers, morphologiesof cells and/or their position in the cerebellum. These

FIG. 4. Increased expression of the Dab1 protein in ectopic Purkinje cells of the cdf mutant cerebellum. (A) Western blot analysis of proteinextracts from P2 neocortexes and cerebella using antibodies to Dab1 (B3) reveals �2� increased Dab1 levels in cdf/cdf cerebellacompared to the cdf/� or �/� cerebella. Differences in the neocortical levels of Dab1 protein among different genotypes were notobserved. Similar results were observed in three independent experiments. Cdk5 antibodies were used as a loading control. P2 cerebellafrom wild-type (B) and cdf mutant littermate (C); and wild-type (D) and 5Fki mutant littermate (E) were concurrently immunostained withanti-Dab1 antibody and signals were detected with an FITC-conjugated secondary antibody. Camera exposure times are equivalent foreach panel. An elevated level of fluorescence signal was seen in ectopic Purkinje cells (arrowheads) in the white matter of the cdf/cdfcerebellum (n � 3). Normally placed Purkinje cells in the cdf/cdf cerebellum (arrows) had Dab1 protein levels similar to Purkinje cells in thewild-type control cerebellum (arrows; B). Lobules in the 5Fki/5Fki cerebellum fail to form and the majority of Purkinje cells are ectopicallylocated. The Dab1 signal intensity differences between ectopic cdf/cdf Purkinje cells (arrowheads; C) and those properly positioned in thecdf mutant or wild-type cerebellum (B, C; arrows) are more pronounced than the differences in levels between Purkinje cells in the 5Fkimutant (E; arrowheads) and its wild-type control (D; arrows). Also note that the difference between Dab1 expression levels in Purkinje cellsin the deep white matter versus those directly underneath EGL (open arrowhead) in 5Fki/5Fki cerebella (E) is less than that seen betweenectopic and nonectopic Purkinje cells in the cdf/cdf cerebellum (C). Scale bars � 200 �m.

36 PARK ET AL.

cells included Purkinje cells, granule cells and deepcerebellar neurons, as well as other nonneuronal cells.The ratio of wild-type to mutant granule and Purkinjecells was similar in each chimera, and cells of bothgenotypes were relatively evenly distributed throughoutthe rostral-to-caudal extent of the chimeric cerebellum(Table 2).

The cdf Mutation Is Intrinsic to Purkinje Cells

To assess the influence of wild-type cells on the num-ber of ectopic Purkinje cells, the relationship betweenthe percentages of ROSA26 cells and ectopic Purkinjecells was addressed. In cdf mutant mice, �40% of Pur-kinje cells are ectopically located (Beierbach et al.,2001). In the chimeras, the percentage of ectopic Pur-kinje cells ranged from 2% to 23%, and the number ofboth cdf mutant Purkinje and cdf mutant granule cellswas correlated with the number of ectopic Purkinje cellsin a linear fashion (r � 0.93 for Purkinje cells and r �0.98 for granule cells), with only chimera 9 deviatingslightly. However, when the numbers of ectopic cellswere compared to the total number of mutant Purkinjecells, the percentage of mutant cells that were ectopicwas approximately 40% in the majority of chimeras (Ta-ble 2). Of particular interest are the chimeras with low

contributions (ranging from 4.8 to 8%) of mutant neu-rons (X3, 5, 6, 7, 8). The percentage of mutant cells thatwere ectopically positioned in the cerebellum of theseanimals was 47, 42, 47, 42, and 80%, respectively, sug-gesting that even a large contribution of wild-type neu-rons failed to influence the position of the subset ofmutant cells that will become ectopic.

To directly evaluate the target cells and cell interac-tions affected by the cdf mutation, the �-gal expressionpattern was analyzed in all cdf/cdf7ROSA26 chimeras.As expected since only a subset of Purkinje cells areectopic in the mutant cerebellum, a mixture of both�-gal positive and negative Purkinje cells were found inthe Purkinje cell layer of chimeric cerebella (Fig. 5). Incontrast, wild-type Purkinje cells were not found in thedeep white matter, the white matter within the folia, orin the internal granule layer. This result demonstratesthat, although only a subpopulation of cells is affected,the effect of the cdf mutation on the position of Purkinjecells is strictly cell autonomous.

DISCUSSION

Histopathological characterization of mutant mice withneuronal migration abnormalities and the identificationof the underlying molecular defect in these strains havecontributed significantly to our understanding of themolecular mechanisms in brain development (reviewedin Hatten, 1999; Rice and Curran, 1999). Similarly, theanalysis of experimental mutant chimeric animals con-taining both mutant and wild-type cells has been aneffective method to understand whether phenotypes ofmutant cells are determined in a cell-autonomous man-

Table 1Behavioral Phenotypes of cdf/cdf 7 ROSA26 Chimeras

Chimera no.Coat variegation

(% cdf/cdf) Ataxia Hind leg clasping

1 60–70 No Yes2 20–30 No No3 40–50 No No4 80–90 No Yes5 40–50 No No6 40–50 No No7 30–40 No No8 40–50 No No9 80–90 No Yes

10 60–70 No Yes

Table 2Phenotype and Percentage Chimerism in cdf/cdf

7 ROSA26 Chimeras*

Chimerano.

Granule cells(% cdf/cdf) Purkinje cells

Anterior Posterior % cdf/cdf % Ectopic

1 48.0 4.9 58.6 4.9 39.7 13.0 17.3 8.3 (41)a

2 16.7 4.7 20.3 5.4 16.8 10.5 7.7 6.2 (47)3 10.1 5.8 12.4 8.6 8.0 2.4 3.8 2.5 (47)4 67.7 6.8 68.3 3.6 71.0 9.0 23.4 8.6 (33)5 11.6 8.9 20.6 8.7 4.8 3.4 2.0 2.5 (42)6 16.1 8.2 20.4 5.1 7.1 5.0 3.2 2.8 (47)7 9.0 6.6 9.3 2.3 6.2 4.2 2.6 3.1 (42)8 15.0 7.5 21.7 5.1 4.9 6.0 3.9 2.6 (80)9 81.0 9.0 88.1 8.5 75.6 9.2 17.1 7.1 (22)

10 41.6 14.7 42.0 15.3 38.0 8.2 15.8 7.9 (42)

* standard deviation.aThe percentage of cdf/cdf cells that are ectopic is shown within

parentheses.

FIG. 5. Purkinje cell misplacement in cdf/cdf mice is cell autono-mous. High magnification of calbindin-immunostained ectopic Pur-kinje cells in the deep white matter from chimera 10. Note the lackof �-gal reactivity in ectopic Purkinje cells (arrowheads) versus thepresence of both �-gal-positive and -negative Purkinje cells in thePurkinje cell layer (arrows). The same result was observed in allchimeras regardless of mutant contribution. Scale bar � 200 �m.

37PURKINJE CELL ECTOPIA IN CDF MUTANT MICE

ner or by cellular environments (reviewed by Mullen etal., 1997).

Unlike other known cerebellar mutants that disruptPurkinje cell positioning, the cdf mutation uniquely af-fects the position of a specific subset of Purkinje cells,primarily those not expressing zebrin II, in the adultcerebellum. In our study of postnatal cerebellar devel-opment of cdf mutant mice, Purkinje cell ectopia wasalready visible at P0. Preliminary neural counting datafrom a small number of midline postnatal sagittal sec-tions showed that the percentage of the ectopic orcdf-dependent Purkinje cells was approximately 40%(data not shown), which is similar to that reported in theadult cdf mutant cerebellum (Beierbach et al., 2001).The proportion of ectopic Purkinje cells was consistentamong sections from different developmental stagesfrom P4, when ectopic Purkinje cells are clearly identi-fiable, through P14 (data not shown), suggesting that asin the reln and Dab1 mutants, the survival of ectopicPurkinje cells is not directly affected by the cdf mutation(Goldowitz et al., 1997). These results, along with ourprevious work demonstrating that ectopic Purkinje cellsare generated at normal times of Purkinje cell neurogen-esis (Beierbach et al., 2001), suggest that the cdf muta-tion disrupts embryonic Purkinje cell positioning with-out affecting survival or specification. Analysis ofpostnatal cerebellar development of cdf mutant micealso demonstrated a dramatic delay in cerebellar foliationwith the absence of the precentral and the intercruralfissures. In addition, our analysis of cdf/cdf7ROSA26chimeras demonstrated that the degree of proper cere-bellar lobulation in these animals was correlated withthe percentage of wild-type cerebellar Purkinje and gran-ule cells (data not shown). The delayed cerebellar lobu-lation and hypoplasia in cdf/cdf animals may arise fromthe reduced number of normally positioned Purkinjecells during postnatal cerebellar development, which inturn could result in poor proliferation or apoptosis ofgranule cells in the EGL, leading to the lobulation abnor-malities. For example, reln, Dab1, Vldlr and apoER2 andCdk5 mutant mice display almost complete failure ofboth Purkinje cell layer formation and cerebellar lobula-tion. However, these mutations also result in a dramaticdecrease in granule cell numbers, a likely secondaryeffect of extensive Purkinje cell ectopia. ExperimentalPurkinje cell ablation also results in severe foliation de-fects combined with a reduction in granule cell prolifer-ation (Smeyne et al., 1995). Although no obvious differ-ences in EGL thickness between cdf/cdf and wild-typecerebella during postnatal development were observed,the hypoplasia of the cdf mutant cerebellum suggeststhat the number of granule cells in the cdf mutant cere-bellum is likely reduced, probably as a secondary effectof Purkinje cell ectopia. The delayed and reduced lobu-lation of the cdf mutant cerebellum is consistent with aprimary defect in the Purkinje cells.

The absence of ectopic wild-type (ROSA26) Purkinjecells in cdf/cdf7ROSA26 chimeras also indicates thatthe cdf mutation is intrinsic to the Purkinje cell. The cdf

gene product is thus likely to be expressed in, andfunction in, Purkinje cells. This contrasts with the cellnonautonomous mechanism of some cerebellar mutants.For example, experimental chimera analysis of reln miceshowed that cell migrations are not cell intrinsic and arethus caused by deficiency in the extracellular environ-ment (Yoshiki and Kusakabe, 1998). The identificationof the reeler gene product as a secreted molecule ex-plains the cell nonautonomous phenotype of reln mu-tant cells (D’Arcangelo et al., 1995). Similarly, mutationof Unc5h3 results in ectopic Purkinje and granule cellsin the midbrain and brainstem (Ackerman et al., 1997).Analysis of Unc5h3/7ROSA26 chimeras demon-strated that Purkinje cell positioning abnormalities aresecondary to granule cell migration defects (Goldowitzet al., 2000). On the other hand, a study of Cdk5/

aggregation chimeras revealed that like cdf mutants, themigration defects caused by the absence of the Cdk5protein are cell autonomous (Ohshima et al., 1999).

In addition to Purkinje cell ectopia, two other aspectsof the cdf mutant phenotype are similar to those of reelinpathway mutants. First, pyramidal cells are displaced inthe cdf mutant hippocampus, especially in the CA1 re-gion. Similarly, these cells are disorganized in the reelermutant mice although this disorganization is more se-vere with the formation of a bilaminate hippocampalstructure (Caviness and Sidman, 1973a). A hypomorphicallele of Dab1 also affects the CA1 region without ap-parent disruption of other parts of the hippocampus (T.Herrick and J.A.C., unpublished results), suggesting thatthe CA1 region might be most sensitive to reductions inReelin-Dab1 signaling. Further, reln/ mice, like cdfmutants, also display a decrease in packing density ofhippocampal neurons. Second, Dab1 expression is mis-regulated in ectopic Purkinje cells in the cdf mutantcerebellum. Dab1 is increased approximately twofold inthe cdf/cdf cerebellum, whereas Dab1 was upregulated5- to 13-fold in whole-brain extracts from both reln andreelin receptor Vldlr/apoER2 double mutants (Rice etal., 1998; Trommsdorff et al., 1999). This smaller in-crease may reflect the fact that only 40% of Purkinje cellsare ectopic in the cdf mutant cerebellum, in contrast togreater than 95% Purkinje cell ectopia in reelin pathwaymutants. Furthermore, the correctly positioned cdf mu-tant Purkinje cells adjacent to the reelin-secreting EGLhave normal Dab1 levels. Recently, Howell and col-leagues (2000) showed that although the phenotype ofanimals expressing the nontyrosine phosphorylated ver-sion of Dab1 was identical to the Dab1-null mutant,Dab1 levels in embryonic cerebral cortex were onlyslightly higher than in wild type. This result suggests thatcorrect Purkinje cell placement requires Dab1 tyrosinephosphorylation, whereas Dab1 levels are downregu-lated by reelin independently (or partly independently)of Dab1 tyrosine phosphorylation. Thus, the reelin re-ceptors generate two signals, one dependent on Dab1tyrosine phosphorylation to regulate neuronal positionand one independent of Dab1 tyrosine phosphorylationthat contributes to Dab1 protein downregulation. In this

38 PARK ET AL.

study we show that Purkinje cells of the 5Fki mutantexpress Dab1 at slightly elevated levels, relative to wild-type, regardless of their position relative to the EGL. Thissuggests that exposure to reelin and subsequent down-regulation of Dab1 protein levels can occur even inPurkinje cells that are distant from reelin made in theEGL. The fact that Dab1 levels are greater in ectopicPurkinje cells in the cdf mutant cerebellum is consistentwith this subset of cells (zebrin II-negative) lacking theability to sense the reelin signal; the zebrin II-positivePurkinje cells can sense the reelin signal and respond bypositioning correctly and downregulating Dab1 expres-sion. Although these results are suggestive of a role ofthe cdf gene in the reelin pathway, it is also possible thatDab1 upregulation may be an indirect consequence ofPurkinje cell ectopia. In the absence of mice with Pur-kinje cell migration defects that clearly do not disturbreelin signaling, identification of the developmentalpathways in which cdf resides awaits molecular analysisof the cdf gene product.

The development of normally positioned and ectopicPurkinje cells in the cdf cerebellum and the upregulationof Dab1 only in ectopic cells provide evidence that atleast two independently controlled developmental pop-ulations of Purkinje cells exist. Biochemical heterogene-ity of Purkinje cells has been well demonstrated by thedifferential expression of various markers in predomi-nantly parasagittal stripes (reviewed in Sotelo and Was-sef, 1991; Herrup and Kuemerle, 1997). This heteroge-neity suggests potential differential functions among thecells expressing different markers. Indeed, the Purkinjecell degeneration mutants leaner and nervous cause theloss of Purkinje cells in alternating parasagittal compart-ments of the cerebellar cortex (Edwards et al., 1994;Heckroth and Abbott, 1994). The fact that predomi-nantly zebrin II-negative Purkinje cells are affected bythe cdf mutation suggests that Purkinje cells have dis-tinct migratory mechanisms that may contribute to theformation of parasagittal stripes and perhaps to func-tional compartmentalization within the cerebellum(Beierbach et al., 2001). Similarly, the existence of asubset of ectopic pyramidal neurons predominantly inthe CA1 region of the hippocampus, rather than thesevere disruption of the hippocampus and dentate gyruscaused by reelin pathway mutations, may indicate diver-sity in the migration mechanisms of hippocampal neu-rons. Our BrdU-birthdating study revealed that the neo-cortex of cdf mutant mice is not inverted as seen in relnand Cdk5 mutants, although we cannot completely ex-clude the possible existence of subtle defects. Also, theabsence of ectopic neurons in layer I (the marginal zone)suggests that the preplate has split properly. These re-sults suggest that the cdf gene may function cell auton-omously within some cerebellar Purkinje cells and hip-pocampal pyramidal cells but is not required in themigration of cortical neurons. It is also possible that theloss of the cdf gene product is compensated in thecdf/cdf neocortex (and in the majority of Purkinje cells)by another, functionally redundant, protein.

MATERIALS AND METHODS

MiceThe cdf mutant mouse has been described previously

(Cook et al., 1997; Park et al., 2000). cdf/cdf homozy-gous animals were produced from heterozygous inter-crosses maintained on a segregating C3H/HeSnJ andCAST/Ei background. Genotyping was performed as de-scribed (Park et al., 2000). The day of birth was consid-ered P0. C57BL/6J-Gtrosa26 (ROSA26) mice comprisedthe wild-type component of the chimera and was thestrain used to mark cell genotype in chimeras (Friedrichand Soriano, 1991). ROSA26 mice were obtained fromthe colony maintained at The Jackson Laboratory (BarHarbor, ME). Dab1tm2JAC (5Fki) mice were genotyped asdescribed (Howell et al., 2000). All animal procedureswere approved by the Animal Care and Use Committeeat The Jackson Laboratory.

Histology and Morphological Analysis

Adult cdf/cdf mice and phenotypically normal mice(�/� or �/cdf) were deeply anesthetized and transcar-dially perfused with Bouin’s fixative. Brains were dis-sected and embedded in paraffin wax in sagittal, coronal,or horizontal orientations. Brains were serially sectionedand 7 �m sections were mounted on egg-white/glycerin(1:1) coated slides. The sections were deparaffinized inxylene and rehydrated through graded alcohol. Everyfifth section was stained with luxol fast blue-cresyl violetor hematoxylin and eosin.

Immunohistochemistry

Brains from P0-P14 mice were fixed in methanol/acetic acid (3:1) overnight prior to paraffin embedding.Sections (7 �m) were mounted on poly L-lysine coatedslides (Sigma). The sections were deparaffinized in xy-lene and rehydrated through graded alcohol. Sectionswere rinsed in PBS and then incubated overnight at 4°Cwith rabbit anti-mouse calbindin D-28 antibodies(1:1500, Swant). Sections were subsequently processedusing standard procedures (Ackerman et al., 1997) priorto colorimetric detection with DAB (Sigma) and coun-terstaining with hematoxylin. For Dab1 and reelin immu-nofluorescence, the brains were fixed in 4% paraformal-dehyde overnight, embedded in OCT (Tissue-TeK), andfrozen in liquid nitrogen. Cryostat sections (10 �m) wereincubated with anti-mDab1 B3 (1:200; Howell et al.,1997a) or anti-reelin (1:10; Ogawa et al., 1995) antibod-ies. After washing, the sections were incubated with fluo-rescein (FITC) or Cy3-conjugated anti-rabbit (mDab1) oranti-mouse (reelin) secondary antibodies (Jackson Immu-noResearch).

Neuronal Birthdating Analysis

Pregnant dams were injected intraperitoneally with5-bromodeoxyuridine (BrdU; 50 �g/g body weight;Sigma) diluted in 7 mM NaOH. At 21–28 days of age, thebrains from offspring were fixed in methanol/acetic acid

39PURKINJE CELL ECTOPIA IN CDF MUTANT MICE

(3:1) overnight. Paraffin-embedded brains were sagittallysectioned and mounted on poly L-lysine (Sigma) coatedslides. Sections were deparaffinized and rehydrated asabove. After rinsing in PBS, DNA was denatured in 4 NHCl for 20 min. Sections were rinsed in PBS and incu-bated overnight with an anti-BrdU monoclonal antibody(1:50; Dako). Sections were processed as described (Pr-zyborski et al., 1998) and antibody-binding sites wererevealed using colorimetric detection with DAB. Sec-tions were counterstained with hematoxylin.

Western Blot Analysis

The neocortex and cerebellum were dissected fromP2 mice and quick frozen in liquid nitrogen in lysisbuffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl(pH 7.4), 1 mM EDTA, 14 mM 2-mercaptoethanol, 2 mMPMSF, 20 �M leupeptin, and 50 mM NaF). After homog-enization, the homogenate was cleared by centrifugationat maximum speed for 30 min in a 4°C microcentrifuge.The protein extracts were pooled by genotype and ana-lyzed by SDS-PAGE and electroblotted onto nitrocellu-lose membranes. The blots were incubated with anti-Dab1 (1:1000) antibody. Antibodies to Cdk4 (1:500;Santa Cruz Biotechnology) and neuron-specific enolase(1:1,000; Scytek) were used as a loading controls. Theblots were developed using the ECL kit (Amersham).Quantification of the protein level was performed bydensitometry (Computing Densitometer, Molecular Dy-namics).

Chimera Production

Eight-cell-stage embryos were obtained by in vitrofertilization as described (Sztein et al., 1997). Chimericmice were generated by aggregation of one 8-cell-stagemutant and one 8-cell-stage wild-type embyro as previ-ously described (Mullen and Whitten, 1971; Goldowitzand Mullen, 1982). Fused embryos at the blastocyst stagewere implanted into pseudopregnant mice.

Genotype Identification of Neurons in ChimericMice

All chimeras were analyzed at 2–3 months of age.Chimeras were deeply anesthetized and transcardiallyperfused with 4% paraformaldehyde (PFA). The brainswere postfixed in 4% PFA overnight, infiltrated with 30%sucrose in PBS, embedded in OCT (Tissue-TeK), andfrozen in the cryostat. Cryostat sections (10–15 �m)were mounted on poly-L-lysine (Sigma) coated slides.Sections were dried at room temperature, rinsed withPBS, 0.2% Triton X-100, and incubated with rabbit anti-mouse calbindin D-28 antibodies (1:1500, Swant) for 8 hat room temperature. Sections were rinsed in PBS andthen lightly fixed in 1% PFA for 2 min. Sections werewashed with HEPES buffer (pH 6.9) and incubated with0.1% X-gal staining solution (pH 6.9) overnight at 30°Cfor �-galactosidase (�-gal) reactivity as previously de-scribed (Oberdick, 1994). Sections were washed, andcolorimetric detection of calbindin immunoreactivity

was performed as described with DAB (Goldowitz et al.,2000) prior to counterstaining with nuclear fast red.

Estimation of Chimerism in the Cerebellum

Percentage chimerism was defined as the percentageof cells within an individual population that are geno-typically cdf/cdf. To estimate the percentage chimerismin the Purkinje cell population, all calbindin-immunopo-sitive Purkinje cells were counted from 10 equallyspaced sagittal sections. Purkinje cells were determinedas genotypically �/� if they possessed several bluepuncta of �-gal reaction product and as genetically cdf/cdf if they lacked �-gal reactivity. To estimate the per-centage chimerism within the granule cell population,�-gal-positive and negative cells were counted using a40� objective within a 20,000 um3 grid (50 � 50 � 8�m) randomly superimposed throughout the granulecell layer from five equally spaced sagittal sections.

ACKNOWLEDGMENTS

We thank Chantal Longo-Guess and Priscilla Kronstadtfor technical assistance, The Jackson Laboratory biolog-ical imaging service for assistance in imaging, JenniferSmith for help with figure preparation, and Drs. ThomasGridley, Timothy O’Brien, and Juergen Naggert for com-ments on the manuscript. This work was supported byNIH grants to S.L.A and J.A.C., a NIH postdoctoral fel-lowhip to J.A.E, and a core grant from the NCI to TheJackson Laboratory.

LITERATURE CITED

Ackerman SL, Kozak LP, Przyborski SA, Rund LA, Boyer BB, KnowlesBB. 1997. The mouse rostral cerebellar malformation gene en-codes an UNC-5-like protein. Nature 386:838–842.

Beierbach E, Park C, Ackerman SL, Goldowitz D, Hawkes R. 2001.Abnormal dispersion of a Purkinje cell subset in the mouse mutantcerebellar deficient folia (cdf). J Comp Neurol 436:42–51.

Brochu G, Maler L, Hawkes R. 1990. Zebrin II: a polypeptide antigenexpressed selectively by Purkinje cells reveals compartments inrat and fish cerebellum. J Comp Neurol 291:538–552.

Caviness VS Jr, Sidman RL. 1973a. Retrohippocampal, hippocampaland related structures of the forebrain in the reeler mutant mouse.J Comp Neurol 147:235–254.

Caviness VS Jr, Sidman RL. 1973b. Time of origin or corresponding cellclasses in the cerebral cortex of normal and reeler mutant mice: anautoradiographic analysis. J Comp Neurol 148:141–151.

Chae T, Kwon YT, Bronson R, Dikkes P, Li E, Tsai LH. 1997. Micelacking p35, a neuronal specific activator of Cdk5, display corticallamination defects, seizures, and adult lethality. Neuron 18:29–42.

Cook SA, Bronson RT, Donahue LR, Ben-Arie N, Davisson MT. 1997.Cerebellar deficient folia (cdf): a new mutation on mouse chro-mosome 6. Mamm Genome 8:108–112.

D’Arcangelo G, Homayouni R, Keshvara L, Rice DS, Sheldon M, CurranT. 1999. Reelin is a ligand for lipoprotein receptors. Neuron24:471–479.

D’Arcangelo G, Miao GG, Chen SC, Soares HD, Morgan JI, Curran T.1995. A protein related to extracellular matrix proteins deleted inthe mouse mutant reeler. Nature 374:719–723.

Dulabon L, Olson EC, Taglienti MG, Eisenhuth S, McGrath B, Walsh CA,Kreidberg JA, Anton ES. 2000. Reelin binds �3�1 integrin andinhibits neuronal migration. Neuron 27:33–44.

40 PARK ET AL.

Edwards MA, Crandall JE, Leclerc N, Yamamoto M. 1994. Effects ofnervous mutation on Purkinje cell compartments defined by Ze-brin II and 9-O-acetylated gangliosides expression. Neurosci Res19:167–174.

Eisenman LM, Hawkes R. 1993. Antigenic compartmentation in themouse cerebellar cortex: zebrin and HNK-1 reveal a complex,overlapping molecular topography. J Comp Neurol 335:586–605.

Friedrich G, Soriano P. 1991. Promoter traps in embryonic stem cells:a genetic screen to identify and mutate developmental genes inmice. Genes Dev 5:1513–1523.

Gallagher E, Howell BW, Soriano P, Cooper JA, Hawkes R. 1998.Cerebellar abnormalities in the disabled (mDab1-1) mouse.J Comp Neurol 402:238–251.

Gilmore EC, Herrup K. 2000. Cortical development: receiving reelin.Curr Biol 10:R162–R166.

Goldowitz D, Cushing RC, Laywell E, D’Arcangelo G, Sheldon M, SweetHO, Davisson M, Steindler D, Curran T. 1997. Cerebellar disorga-nization characteristic of reeler in scrambler mutant mice despitepresence of reelin. J Neurosci 17:8767–8777.

Goldowitz D, Hamre KM, Przyborski SA, Ackerman SL. 2000. Granulecells and cerebellar boundaries: analysis of Unc5h3 mutant chi-meras. J Neurosci 20:4129–4137.

Goldowitz D, Mullen RJ. 1982. Nuclear morphology of ichthyosismutant mice as a cell marker in chimeric brain. Dev Biol 89:261–267.

Hatten ME. 1999. Central nervous system neuronal migration. AnnuRev Neurosci 22:511–539.

Heckroth JA, Abbott LC. 1994. Purkinje cell loss from alternatingsagittal zones in the cerebellum of leaner mutant mice. Brain Res658:93–104.

Herrup K, Kuemerle B. 1997. The compartmentalization of the cere-bellum. Annu Rev Neurosci 20:61–90.

Hiesberger T, Trommsdorff M, Howell BW, Goffinet A, Mumby MC,Cooper JA, Herz J. 1999. Direct binding of Reelin to VLDL recep-tor and ApoE receptor 2 induces tyrosine phosphorylation ofdisabled-1 and modulates tau phosphorylation. Neuron 24:481–489.

Howell BW, Gertler FB, Cooper JA. 1997a. Mouse disabled (mDab1): aSrc binding protein implicated in neuronal development. EMBO J16:121–132.

Howell BW, Hawkes R, Soriano P, Cooper JA. 1997b. Neuronal positionin the developing brain is regulated by mouse disabled-1. Nature389:733–737.

Howell BW, Herrick TM, Cooper JA. 1999. Reelin-induced tryosinephosphorylation of disabled 1 during neuronal positioning. GenesDev 13:643–648.

Howell BW, Herrick TM, Hildebrand JD, Zhang Y, Cooper JA. 2000.Dab1 tyrosine phosphorylation sites relay positional signals duringmouse brain development. Curr Biol 10:877–885.

Miyata T, Nakajima K, Aruga J, Takahashi S, Ikenaka K, Mikoshiba K,Ogawa M. 1996. Distribution of a reeler gene-related antigen inthe developing cerebellum: an immunohistochemical study withan allogeneic antibody CR- 50 on normal and reeler mice. J CompNeurol 372:215–228.

Mullen RJ, Hamre KM, Goldowitz D. 1997. Cerebellar mutant mice andchimeras revisited. Perspect Dev Neurobiol 5:43–55.

Mullen RJ, Whitten WK. 1971. Relationship of genotype and degree ofchimerism in coat color to sex ratios and gametogenesis in chi-meric mice. J Exp Zool 178:165–176.

Oberdick J. 1994. Evidence for a genetically encoded map of functionaldevelopment in the cerebellum. Histochemistry 102:1–14.

Ogawa M, Miyata T, Nakajima K, Yagyu K, Seike M, Ikenaka K,Yamamoto H, Mikoshiba K. 1995. The reeler gene-associated an-tigen on Cajal-Retzius neurons is a crucial molecule for laminarorganization of cortical neurons. Neuron 14:899–912.

Ohshima T, Gilmore EC, Longenecker G, Jacobowitz DM, Brady RO,Herrup K, Kulkarni AB. 1999. Migration defects of cdk5/ neu-rons in the developing cerebellum is cell autonomous. J Neurosci19:6017–6026.

Ohshima T, Ogawa M, Veeranna, Hirasawa M, Longenecker G, IshiguroK, Pant HC, Brady RO, Kulkarni AB, Mikoshiba K. 2001. Synergis-tic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developingmouse brain. Proc Natl Acad Sci U S A 98:2764–2769.

Ohshima T, Ward JM, Huh CG, Longenecker G, Veeranna, Pant HC,Brady RO, Martin LJ, Kulkarni AB. 1996. Targeted disruption of thecyclin-dependent kinase 5 gene results in abnormal corticogen-esis, neuronal pathology and perinatal death. Proc Natl Acad Sci US A 93:11173–11178.

Park C, Longo CM, Ackerman SL. 2000. Genetic and physical mappingof the cerebellar deficient folia (cdf) locus on mouse chromosome6. Genomics 69:135–138.

Przyborski SA, Knowles BB, Ackerman SL. 1998. Embryonic phenotypeof Unc5h3 mutant mice suggests chemorepulsion during the for-mation of the rostral cerebellar boundary. Development 125:41–50.

Rice DS, Curran T. 1999. Mutant mice with scrambled brains: under-standing the signaling pathways that control cell positioning in theCNS. Genes Dev 13:2758–2773.

Rice DS, Sheldon M, D’Arcangelo G, Nakajima K, Goldowitz D, CurranT. 1998. Disabled-1 acts downstream of Reelin in a signalingpathway that controls laminar organization in the mammalianbrain. Development 125:3719–3729.

Senzaki K, Ogawa M, Yagi T. 1999. Proteins of the CNR family aremultiple receptors for Reelin. Cell 99:635–647.

Sheldon M, Rice DS, D’Arcangelo G, Yoneshima H, Nakajima K, Miko-shiba K, Howell BW, Cooper JA, Goldowitz D, Curran T. 1997.Scrambler and yotari disrupt the disabled gene and produce areeler-like phenotype in mice. Nature 389:730–733.

Smeyne RJ, Chu T, Lewin A, Bian F, S. Crisman S, Kunsch C, Lira SA,Oberdick J. 1995. Local control of granule cell generation bycerebellar Purkinje cells. Mol Cell Neurosci 6:230–251.

Sotelo C, Wassef M. 1991. Cerebellar development: afferent organiza-tion and Purkinje cell heterogeneity. Philos Trans R Soc Lond BBiol Sci 331:307–313.

Sztein JM, Farley JS, Young AF, Mobraaten LE. 1997. Motility of cryo-preserved mouse spermatozoa affected by temperature of collec-tion and rate of thawing. Cryobiology 35:46–52.

Trommsdorff M, Gotthardt M, Hiesberger T, Shelton J, Stockinger W,Nimpf J, Hammer RE, Richardson JA, Herz J. 1999. Reeler/Dis-abled-like disruption of neuronal migration in knockout micelacking the VLDL receptor and ApoE receptor 2. Cell 97:689–701.

Walsh CA, Goffinet AM. 2000. Potential mechanisms of mutations thataffect neuronal migration in man and mouse. Curr Opin GenetDev 10:270–274.

Ware ML, Fox JW, Gonzalez JL, Davis NM, Lambert de Rouvroit C,Russo CJ, Chua SC Jr, Goffinet AM, Walsh CA. 1997. Aberrantsplicing of a mouse disabled homolog, mdab1, in the scramblermouse. Neuron 19:239–249.

Yoshiki A, Kusakabe M. 1998. Cerebellar histogenesis as seen in iden-tified cells of normal-reeler mouse chimeras. Int J Dev Biol 42:695-700.

41PURKINJE CELL ECTOPIA IN CDF MUTANT MICE