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4201Development 124, 4201-4212 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV4872

meander tail acts intrinsic to granule cell precursors to disrupt cerebellar

development: analysis of meander tail chimeric mice

Kristin M. Hamre* and Dan Goldowitz

Department of Anatomy and Neurobiology, University of Tennessee, Memphis, 875 Monroe Avenue, Memphis, TN 38163, USA

*Author for correspondence (e-mail: [email protected])

The murine mutation meander tail (gene symbol: mea)causes a near-total depletion of granule cells in the anteriorlobe of the cerebellum, as well as aberrantly locatedPurkinje cells with misoriented dendrites and radial gliawith stunted processes. Whether one, two or all three ofthese cell types is the primary cellular target(s) of themutant gene is unknown. This issue is addressed byexamining cerebella from adult chimeras in which both thegenotype and phenotype of individual cells are marked andexamined. From this analysis, three novel observations aremade. First, genotypically mea/meaPurkinje cells and glialcells exhibit normal morphologies in the cerebella ofchimeric mice indicating that the mea gene acts extrinsi-cally to these two cell populations. Second, few genotypi-cally mea/meagranule cells are present in the anterior lobe

or, unexpectedly, in the posterior lobe. These findingsindicate that the meagene acts intrinsically to the granulecell or its precursors to perturb their development. Third,there are near-normal numbers of cerebellar granule cellsin the chimeric cerebellum. This result suggests thatmea/meacells are out-competed and subsequently replacedby an increased cohort of wild-type granule cells resultingfrom an upregulation of wild-type granule cells in thechimeric environment. We propose that the wild-type alleleof the meagene is critical for the developmental progres-sion of the early granule cell neuroblast.

Key words: Purkinje cell, radial glial cell, cerebellum, chimera,neurological mutant mice, meander tail

SUMMARY

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INTRODUCTION

The meander tailmutant mouse (gene symbol = mea) wasdescribed 20 years ago as an autosomal recessive mutationresulted in a kinky tail and ataxic gait (Hollander and Wagg1977). The ataxic gait was found to be due to the hypoplaof the anterior lobe of the cerebellum with several concomitphenotypic abnormalities (Hollander and Waggie, 197Sidman, 1983; Ross et al., 1990). These abnormalities incluthe misalignment of Purkinje cell somas and dendrites, a deof radially oriented glial processes and foliation abnormalitiwith the absence of several fissures. The most notaphenotype, however, was the almost complete absenceanterior lobe granule cells. Both the restricted nature of phenotypic abnormalities and the fact that these abnormaliare found throughout the anterior lobe make meander tail of the most distinctly compartmentalized of any of the ceebellar mutations.

How the meagene acts to affect development of the ceebellum is currently unknown. It is not known whether the meagene targets granule cell development, or if the loss of grancells is secondary to one of the other phenotypic abnormaliin the mutant. In this paper, we report our analysis intraspecies murine chimeras to address this issue.

Chimeric analyses of mutant gene action are accurate dictors of both where a mutant gene is expressed and ho

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functions, as shown by the recent cloning of the weaver, reelerand staggerergenes (Herrup and Mullen, 1979; D’Arcangeloet al., 1995; Ogawa et al., 1995, Patil et al., 1995; Goldowand Smeyne, 1995; Hamilton et al., 1996; Mullen et al., 1997An important advantage of using experimental murinchimeras to address these issues is that the in situ cellular environmental interactions are preserved in the chimesituation, which spans the developmental history of thorganism. Furthermore, given an appropriate cell markinsystem, all cellular components in the affected structure canassessed to determine if more than one type of cell is primatargeted by a mutant gene.

In the present study, we examined experimental murichimeras to determine whether the meagene acts intrinsicallyor extrinsically to cerebellar granule cells, Purkinje cells anGolgi epithelial cells to create the mutant phenotypes seenthe mea/meacerebellum. In this paper, we document thnormal morphology of genotypically mea/meaPurkinje cellsand Golgi epithelial cells and the paucity of genotypicallmea/meagranule cells in chimeras. We conclude that the meagene acts intrinsically to one cell population, the cerebellgranule cell precursors, but extrinsically to the Purkinje ceand Golgi epithelial cells. Furthermore, the numbers of wildtype granule cells are greatly increased over the expecnumbers in the anterior and posterior lobes of the chimecerebellum indicating an upregulation of non-mutant granu

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K. M. Hamre and D. Goldowitz

cells as well as an out-competition of mutant granule cellsthe posterior lobe. These findings point toward a criticinvolvement of the meagene in the timing of cerebellar granulcell development.

MATERIALS AND METHODS

Mice and production of experimental murine chimerasAll mice used in the present study were bred, born, raised and mtained at the University of Tennessee, Memphis animal care faciMice were given food and water ad libitum and maintained on a 14hour light: dark cycle. The original stock of meander tail mice wobtained from Jackson Laboratory (Bar Harbor, ME) and was on C57BLKS/J (Mus musculus) background. The meander tail(genesymbol = mea) mutation appears to be a true recessive mutation, wa mutant phenotype observed only with two mutant alleles (Hollanand Waggie, 1977; Hamre and Goldowitz, 1996). Homozygous mand female meander tail mice were mated to obtain homozygembryos for use in generating chimeras. The non-mutant compoof chimeras came from one of two sources: either BALB/cJ mice ormice transgenic for a PBR-globin insert (Lo et al., 1987) (GT fglobin transgene). In some cases, GT females were mated wirandom-bred albino male (ICR strain, Harlan Sprague-Dawley, InIndianapolis, IN), to increase the percentage of successful matiThe coat of the chimeras resulting from these latter embryos wagouti and enabled an estimation of percentage chimerism (see T1). In cases where GT × GT matings were used, the coat color wanon-agouti and could not be distinguished from the mea/meacoatcolor in chimeras.

The protocol for generating experimental murine chimeras has bpreviously detailed (Goldowitz and Mullen, 1982; Goldowitz, 1989aFemale mice were superovulated with injections of gonadotropin frpregnant mare’s serum (dose: 4.0-4.5 IU) followed by the same dof human chorionic gonadotropin (Sigma Chemical Co., St LouMO) approximately 44 hours later. Embryos were collected at theto 8-cell stage and two embryos, one of the mutant and one ofwild-type genotype, were aggregated and cultured overnight. Fuchimeric embryos were implanted into pseudopregnant hosts.

HistologyA total of seven chimeric mice were examined in this study. All miwere allowed to survive a minimum of 3 weeks of age with the oldmouse surviving until over 3 months of age (see Table 1). Mice wexamined for the presence or absence of kinks in the tail and gan estimate of the percent of coat color chimerism.

Mice were then anesthetized with avertin and intracardiaperfused. Chimeras made with a GT embryo were perfused with piologic saline followed by an acetic acid:ethanol (1:3) fixative. Tbrain was postfixed in the same fixative and then dissected out oskull. The tissue was dehydrated through a graded series of ethafollowed by xylenes, infiltrated in several changes of paraffin and thembedded in paraffin. Chimeras made with BALB/c embryos weperfused with physiologic saline followed by paraformaldehydlysine-periodate (PLP) fixative at pH 6.2 (McLean and Nakane, 197The brains were postfixed for 4-6 hours and dissected from the sThe brains were then dehydrated in ethanol and embedded in waxbrains were embedded to be cut in the sagittal plane.

The brains were sectioned using a rotary microtome at 6-8 µm.Every 25th or 50th section was mounted and stained with cresyl vioand coverslips applied to obtain an initial examination of cerebemorphology. Selected sections were mounted for in situ hybridizator immunocytochemistry to demonstrate cell genotype.

DNA in situ hybridization to mark cell genotypeIn GT↔mea/meachimeras, the genotype of individual cells wa

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ascertained by nuclear labeling using in situ hybridization. GT micare transgenic mice in which several hundred copies of a PBR-glogene have been inserted into their genomes (Lo et al., 1987) allowfor easy detection of genotypically GT cells using in situ hybridization. A probe for this insert was biotinylated using a standard nitranslation protocol (Rigby et al., 1977) with biotinylated dUTP (EnzBiochem., New York, NY).

The protocol for in situ hybridization has been previously describe(Goldowitz, 1989a,b). The probe was used at a concentration of ng/µl. Hybridization-positive cells were visualized by processing thsections for alkaline phosphatase histochemistry using nitro-bltetrazolium as the chromogen and BCIP as the substrate. Labeled chad a blue to purple precipitate in their nucleus while unlabeled ceturned light brown in the reaction. The sections were rinsed in distillwater, dehydrated through ethanols and xylenes, and coversapplied with Permount.

Immunocytochemistry to mark cell genotypeIn BALB/c↔mea/meachimeras, cell genotype was marked usingimmunocytochemistry. BALB/c mice do not express a nuclear antigthat is expressed in cell types of other mice of the Mus musculusspecies (Mullen, personal communication), including mea/meamice(see Fig. 3). An antibody to this antigen, B37, was generoussupplied by Dr Richard Mullen.

Standard avidin-biotin immunocytochemical procedures weutilized to label cells (Hamre et al., 1995). Sections were dewaxed aincubated in primary antibody at a dilution of 1:1 in phosphatebuffered saline with 0.3% Triton X-100 (pH=7.2) overnight at roomtemperature. The next day sections were incubated with an anti-mosecondary antibody and avidin-biotin solution from Vector Laboratories, Inc. (Burlingame, CA). The immunolabeling was visualizeusing 0.05% 3,3′ diaminobenzidine hydrochloride plus 0.3%hydrogen peroxide.

Double-labeling of cell genotype and phenotypeDouble-labeling was used to mark both genotype and phenotypethe same cell in GT↔mea/meachimeras. The genotype of each celwas ascertained by DNA in situ hybridization with a protocol identicato that described above except that the cells were visualized wflouroscein-conjugated avidin. In this technique, a ‘sandwich’ wamade whereby repeated applications of biotynylated goat anti-avidD and fluorescein-conjugated avidin were used. Upon completionthe in situ hybridization, sections were rinsed and processed immunocytochemistry to mark cell phenotype.

The phenotype of genotypically mea/meacells was examined byimmunocytochemically labeling sections with either glial fibrillaryacidic protein (GFAP) to examine the radial glia fibers or witcalcium-binding protein (CaBP) to label Purkinje cells. The sectionwere washed with PBS/T and then incubated overnight in primaantibody at room temperature. The anti-GFAP antibody was obtainfrom Immunon (Pittsburgh, PA) and used at a dilution of 8 drops pml in PBS/T. The anti-CaBP antibody was used at a dilution of 1:10(Hamre and Goldowitz, 1996). On the following day, sections weincubated at 37˚C in Texas-red-conjugated goat anti-mouse secondantibody used at a dilution of 1:50. Coverslips were applied wiPBS:glycerol (1:1). To insure that a radial glial process was succefully followed back to its appropriate soma, GFAP-labeled cells wealso stained with the nuclear stain TOTO-3 (Molecular Probes, InEugene, OR) at a concentration of 0.25 M in PBS glycerol. TOTOexcites at a wavelength of approximately 650.

AnalysisTo obtain an estimate of per cent chimerism in the brain of eachthe 7 chimeras, the numbers of labeled and unlabeled cells wcounted from several cell populations. In the GT↔mea/meachimericmice, the following populations were chosen: olfactory bulb granucells, hippocampal granule cells and cerebellar molecular lay

4203The meander tail chimeric cerebellum

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interneurons. In BALB/c↔mea/mea chimeric mice, cerebellarmolecular layer interneurons and Purkinje cells were countedminimum of three sections from each brain was analyzed. distance separating these sections was approximately 500 µm. Asmany of the different neuronal populations as possible were couin a single section. The percentage of genotypically mea/meagranulecells was calculated for both the anterior cerebellum (rostral toprimary fissure) and posterior cerebellum (caudal to the primfissure). An estimate of the percentage of cells that were genocally mea/meawas obtained for each of the sampled neuronal polations. In both molecular layer interneurons and Purkinje cells,attempt was made to separate the two lobes. The paired t-test was usedto determine if the contribution of genotypically mea/meagranulecells was significantly different from the contribution of genotypicamea/mea in other neuronal populations.

The number of cerebellar granule cells was analyzed in expmental chimeras and wild-type control (BALB/c and GT) cerebeto determine if the numbers of granule cells in chimeric cerebwere equivalent to those seen in the wild-type species. Bothanterior lobe and posterior lobes were examined. The numbegranule cells was estimated in a single mid-sagittal section of cerebellum as well as in three sections in more lateral regions overmis in the chimeric cerebella. Sections from the non-chimecontrol cerebella were matched for similar mediolateral locatioThe estimate of granule cell number was obtained by determingranule cell density and multiplying this number by the area of granule cell layer. Granule cell density was the mean from 10 coin each lobe. Each count was made using a grid measuring 720µm2

that was randomly placed throughout the lobe. The area of granule cell layer in each lobe was determined using a camera-ludrawing tube and the MacMeasure (NIH Image) computer progrThe number of granule cells in chimeric cerebella was comparecontrol mice of the appropriate non-mutant genotype, which westimated in the same manner. A t-test was conducted to determinif the number of granule cells was reduced in chimeric mice relato controls.

The qualitative analysis of the Purkinje cells and radial glial ceinvolved recording and comparing the phenotypes of genotypicmutant and wild-type cells. The genotypic label was always 1-2 dof hybridization signal over the nucleolus of Purkinje cells nucleus of radial glial cells. Because of the large size of Purkcell nuclei, the label was typically present in only one section tcontained the nucleolus, while the cell nucleus was visible for tor three sections. Thus a given Purkinje cell could be misclassas unlabeled (mutant) when in fact it was labeled (non-mutaTherefore, each Purkinje cell was examined in three serial sectto reconstruct the whole cell. The examination of more than section insured the correct classification of genotypically mut(unlabeled) cells. The phenotype was examined using immunocchemistry for CaBP or GFAP. The phenotype of Purkinje cells w

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Table 1. Genotype, age at killing and the percentage of thechimeric mouse that is genotypically meander tail based

on the coat color of the seven chimeras used in the presenstudy

Number Genotype Age at sacrifice Percent meander t

1 GT↔mea/mea 21.5 days 95% mea2 GT↔mea/mea 21.5 days 50% mea3 GT↔mea/mea 21.5 days N.A.*4 GT↔mea/mea 21.5 days 10% mea5 BALB/c↔mea/mea 108 days 25 % mea6 BALB/c↔mea/mea 28.5 days 35% mea7 BALB/c↔mea/mea 50 days 30% mea

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assessed using CaBP immunocytochemistry to mark Purkinje soma and their dendritic expansions. The phenotype of radial gcells were clearly seen with GFAP immunolabeling. TOTOlabeled all cell nuclei, which insured that radial glial processes cobe traced back to their soma. Both the small size of radial glial ceand the fact that their processes were typically found in only osection, obviated the need for examination of more than one secfor the analysis of any given radial glial cell. Analysis of the doublabeled material was conducted using a confocal laser scannmicroscope in the Department of Anatomy and Neurobiology at University of Tennessee. Initial scans of sections were madedetermine the presence or absence of cells with abnormal phtypes, with more intensive analysis conducted on a smaller numof cells.

RESULTS

The cerebella of meander tail chimeric mice appearphenotypically normalChimeric mice were generated between genetically homogous meander tailembryos and either globin transgenic (GTembryos (Lo et al., 1987) or BALB/c embryos. IGT↔mea/meachimeras, the non-mutant cells were labelewith a probe to the globin transgenic insert using in situ hybrization. In BALB/c↔mea/meachimeras, the mutant cells werelabeled immunocytochemically by virtue of the fact thBALB/c cells do not posses a neuronal nuclear antigen thapresent in most other mice including the meander tail mo(Mullen, personal communication). In this manner, a compmentary picture was obtained: in GT↔mea/meachimeras, thegenotypically wild-type cells were labeled while, inBALB/c↔mea/meachimeras, the genotypically mutant cellwere labeled.

A total of 7 chimeric mice were examined, 4 GT↔mea/meachimeras and 3 BALB/c↔mea/meachimeras. The chimericmice ranged in age from 21 days to over 3 months at the tof killing. All chimeric mice exhibited a normal tail phenotypwith no evidence of any kinks or bends (compare Fig. 1A wB). Behaviorally, the chimeric mice also appeared normal: ataxia or other sign of a movement disorder was observed.initial estimation of chimerism was made based on coat coAs shown in Table 1, the percentage of genotypically meantail coat ranged from 10% to over 50%.

The gross morphology of the cerebellum looked normalchimeric mice (compare Fig. 1C with 1D), with one exceptioIn one chimeric cerebellum, an extra fissure was seen inright hemisphere dividing the crus into two smaller lobes. Tabnormal phenotype is most likely due to normal variatioparticularly since this phenotype is the opposite to that fouin homozygous meander tailmice, in which one or morefissures are absent (Ross et al., 1990). In all other casesfoliation appeared normal with no observable alterations in number of fissures. Furthermore, there were no apparent continuities between the anterior and posterior aspects ofcerebellum as is strikingly apparent in the meander tail cebellum.

The structure and composition of all layers within thchimeric cerebellum also appeared phenotypically normalboth the anterior and posterior lobes as shown in Fig. 1Purkinje cells were arranged in a monolayer and an eadefinable granule cell layer and molecular layer were presAs expected, the disappearance of the EGL was complete

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enotype of a homozygous meander tailmouse and a chimeric mouse.ozygous meander tailmouse showing that the tail has kinks and bends.a/meachimeric mouse (chimera no. 6). The BALB/c component is

l component in misty/grey. Significant intermixing can be seen between coat color. The tail demonstrates no evidence of any kinks or bends. (C)ction through the cerebellum from an mea/meamouse. Arrow marks the lobe of the cerebellum (rostral to the primary fissure) appears markedly of a definable granule cell layer in the anterior lobe. (D) Example of arebellum from the BALB/c↔mea/meachimeric mouse shown in B. Thesure. The anterior lobe appears normal in all respects with a clearly

layer and a Purkinje cell monolayer. Scale bar (C,D) = 500 µm.

the time the mice were killed. Thus, no obvious abnormalitor developmental delays were observed in the cerebellachimeric mice.

There are two typical explanations to account for the normappearing cerebellum in +/+↔mea/meachimeric mice. First,despite evidence of chimerism from coat color, the cerebellathese mice were not chimeric. Second, the meagene works viaa cell extrinsic mechanism that is cured by the presence of cells in the chimeric environment. These possibilities aaddressed below.

Genotypically meander tail cells participate in thecolonization of the brains of chimeric miceTo determine if genotypically mea/meacells populated eitherthe brain or the cerebellum in any of these chimeric animathe percentage of cells that were genotypically mea/meawasexamined in several neuronal populations. The genotype ofcells was ascertained using in situ hybridization histochemisor immunocytochemistry depending on the strain of mouused for the wild-type contribution. The analyzed populatiowere chosen either because of their similarity in generattime to cerebellar granule cells (hippocampal granule cells aolfactory bulb granule cells) or in their origin to cerebellagranule cells (Purkinje cells and molecular layer interneuron

Evidence of chimerism was found in hippocampal granucells, olfactory bulb granule cells, cerebellar Purkinje cells amolecular layer interneurons (Figs 2-4). All GT↔mea/meaand BALB/c↔mea/meachimeras had genotypicallymea/mea cells present outsideand within the cerebellum. Thus,a lack of brain chimerism was notresponsible for the normalappearance of the chimericcerebella.

Chimeric mice lackgenotypically meander tailgranule cells in the anteriorlobe of the cerebellumThe normal morphology of thechimeric cerebellum, in particularthe apparently normal number ofgranule cells, may be due to anextrinsic effect of the mea genethat was corrected in the chimericenvironment. If this was the case,then genotypically mea/meagranule cells should be foundwithin the anterior lobe of thechimeric cerebellum. The per-centage of genotypicallymea/mea granule cells wasestimated in the anterior lobe ofthe cerebellum (defined as rostralto the primary fissure) to assessthis possibility.

We found, contrary to expecta-tions, that few genotypicallymea/meacells were found in theanterior lobe of the cerebellum

Fig. 1. Comparison of the ph(A) Example of an adult hom(B) Example of a BALB/c↔mealbino while the meander taithe two genotypes based onExample of a mid-sagittal seprimary fissure. The anteriorabnormal. Note the absencemid-sagittal section of the cearrow denotes the primary fisdefined internal granule cell

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(Figs 3-5). This was consistent in both GT↔ mea/meachimeras where few unlabeled (genotypically mea/mea)granule cells were present and in BALB/c↔mea/meachimeraswhere very few if any labeled (genotypically mea/mea) granulecells were observed. These results were not due to the faito appropriately mark mea/meagranule cells. Using the globintransgenic marker, we found that less than 1% of granule cwas scored as unlabeled in a GT mouse while no cells wscored as labeled in the mea/mea cerebellum (see Fig. 4C,D).Using the B37 antibody marker, we found that all cells lackimmunolabeling in the BALB/c cerebellum while virtuallyevery cerebellar cell with a nucleus in a section from mea/meamice was immunolabeled (see Fig. 5C-G). Furthermore, near-complete labeling of granule cells in control material athe complementary results obtained with the GT and Bmarkers diminish the likelihood that there is an alteration the expression of the B37 antigen due to the chimeric enronment (e.g., downregulation of the B37 antigen in +/+ ceor an upregulation in −/− cells).

The most dramatic examples of the exclusion of mea/meacells from the granule cell population were found iBALB/c↔mea/meachimeras where virtually the only labeledcells present in the IGL were the Golgi cells while labelegranule cells were almost completely absent. The lack of getypically mea/meagranule cells was consistent throughout a5 lobules of the anterior lobe. Additionally, there was no sinificant variation in the number of labeled granule cells


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of chimeric mice show evidence of chimerism as seen in americ mouse (chimera no. 1). All cell nuclei turn light brown in the ine labeled cells have one or more blue/purple dots over the nucleus.yrus of the hippocampal formation has labeled cells (arrowheads)nlabeled cells (arrows). In this chimeric animal, over 50% of the dentateunlabeled, i.e., of meander tail origin. (B) The olfactory bulb of thisws intermixing of labeled (arrowheads) and unlabeled (arrows) granule 35 µm in A and 20 µm in B.

sections measured at various mediolateral regions of the ebellar vermis. A statistical analysis of the percentage granule cells in the anterior lobe of chimeras used the averpercentage chimerism for the brain (see Fig. 3) comparedthe percentage chimerism for the anterior lobe granule ceThe average percentage chimerism consisted of the mchimerism of all non-granule cell populations that weanalyzed. No significant differences existed between thpopulations. Furthermore, the percentages of chimerismvarious regions of the brain and the cerebellar granule cellspositively correlated in wild-type, intraspecific chimeras (danot shown). The percentage of genotypically mea/meagranulecells was significantly lower in the anterior lobe (P<0.001,d.f.=6). This finding is not consistent with the hypothesis ththe meagene acts extrinsically to cerebellargranule cells but supports the opposite con-clusion, i.e., that the meagene acts intrinsi-cally to the granule cells or their precursorsto result in the virtual elimination of anteriorlobe granule cells.

Meander tail chimeras exhibit fewgenotypically mea/mea granule cellsin the posterior lobe of thecerebellumThe effects of the meander tailmutation onposterior lobe granule cells in the chimeramay be a more complex issue. In mea/meamice, there is a 20-30% reduction in thenumber of granule cells in the posterior lobeof the cerebellum (Hamre and Goldowitz,1996). One expectation would be that geno-typically mea/meagranule cells are presentin percentages similar, although slightlyreduced, to those found in other neuronalpopulations. Additionally, because of thecompartmentalized nature of the mea/meaphenotype, we expected that the percentageof genotypically mea/meagranule cells inthe posterior lobe should be significantlygreater than the percentage of cells found inthe anterior lobe.

Surprisingly, a survey of chimericcerebella showed that few genotypicallymea/meacells were found in the posteriorlobe (Fig. 5) except for regions thatcontained small ‘nests’ of genotypicallymea/meagranule cells in the internal granulecell layer (IGL). The nests were restricted inlocation to the distal region of lobule IX infive out of the seven chimeric brainsanalyzed. In two chimeras, additional nestswere found in the distal regions of lobulesVII and VIII. In regions of the posterior lobethat did not harbor nests of granule cells, nomore than 10% of the granule cells weregenotypically mea/mea. In regions contain-ing granule cell nests, the percentage ofgenotypically mea/mea granule cellsincreased to 30-40%, which was similar tothe percentage chimerism in other neuronal

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populations. However, this contribution was relatively minimaas the total number of genetically mea/meagranule cells in theposterior lobe never approached the percentage of mea/meacells seen in other neuronal populations. As found for thanterior lobe, a paired t-test showed that genotypicallmea/meagranule cells in the posterior lobe were significantlyreduced compared to the chimerism in other neuronal poputions (P<0.001, d.f.=6). Thus, genotypically mea/meacellswere significantly under-represented in both the anterior anthe posterior lobes of the chimeric cerebellum.

The deficit in mea/meagranule cell colonization of theposterior lobe, however, was consistently less severe than deficit of granule cell colonization in the anterior lobe (Figs 35). There was a significantly lower percentage of mea/mea

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on of the percentage chimerism in several neuronal populationsrain from each chimeric mouse. The percentage of each population thatea/meais shown in both GT↔mea/meachimeras (top graph) and ineachimeras (bottom graph). The asterisks (*) represent populations

ntage of the cells that were genotypically mea/meais below 1%. Thenotypically mea/meagranule cells in either the anterior or posterior lobe

ss than that in any other population examined (P<0.001). Also note thatgenotypically mea/meagranule cells in the anterior lobe compared to the0.05).

granule cells in the anterior lobe than in the posterior lo(P<0.05, d.f.=6). These results are unlikely to be due totendency for wild-type cells to preferentially colonize thanterior lobe because we see the same results in two diffechimeric combinations and similar findings have been reporusing the injection of immortalized cells to the mea/meacer-ebellum (Rosario et al., 1997).

Meander tail chimeras have a normal number ofgranule cellsOne expectation from the foregoing results is that there wobe a deficit in the total number of granule cells in the meantail chimeric cerebellum due to the absence of genotypicamea/meagranule cells. This has been the outcome in chimeanalyses of weaver mice, a mutation that also intrinsicatargets granule cells (Goldowitz and Mullen, 1982; Goldowit1989a). This, however, was not the case in mea/meachimericmice. The number of granule cells in a midsagittal section westimated in chimeric mice, and in controlmice of the two non-mutant genotypes. Asshown in Fig. 6, neither the GT↔ mea/meacerebellum, nor the BALB/c↔mea/meacer-ebellum, demonstrated any substantialdecrease in granule cell number relative totheir respective controls. No significant loss ofgranule cells was observed in either theanterior or posterior lobes. Additionally, theseresults are found across several sections (seeFig. 6, bottom panel) in the cerebellar vermisdemonstrating that this finding is consistentthroughout the vermis. These results are inter-preted to mean that the production of geneti-cally wild-type granule cells is upregulated togenerate a near-normal number of cerebellargranule cells. An additional conclusion is thatthe wild-type neuroblasts destined for theposterior lobe are able to out-compete thegenotypically mea/meaneuroblasts and thus,virtually exclude the genotypically mea/meacells from the posterior lobe granule cell pop-ulation.

Genotypically mutant Purkinje cellsexhibit normal morphology in meandertail chimeric cerebellaIn addition to the dramatic effects on cerebel-lar granule cells, the mea gene also affectsPurkinje cells (Ross et al., 1990; Napieralskiand Eisenman, 1996). Wild-type Purkinje cellsomas are aligned in a monolayer at theinterface between the granule cell andmolecular layers, with dendrites projectingthrough the molecular layer towards the pialsurface. In contrast, in the anterior lobe ofhomozygous meander tailcerebella, Purkinjecell somas are not aligned in a monolayer butare scattered throughout all regions of theanterior lobe, with some somas located nearthe pial surface in the presumptive molecularlayer and others deep near the white matter.The dendrites of these cells can be stunted, and

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Analysis of chimeric cerebella allowed for the assessmentwhether the Purkinje cell phenotypes found in homozygomeander tail mice were intrinsic or extrinsic to the cerebellPurkinje cells. The Purkinje cells in the anterior lobe ochimeric cerebella were aligned in a phenotypically normmonolayer (Figs 1D, 7B). The action of the meagene on thedendritic phenotype of mea/meaPurkinje cells was examinedby marking cell genotype using in situ hybridization and susequently highlighting dendritic phenotype using immunoctochemistry with an antibody to calcium-binding protein(Hamre and Goldowitz, 1996). In this analysis, 45 Purkincells from the anterior lobe of the cerebellum were recostructed from serial sections (see Materials and Methods). this total, 23 of these cells were identified as genotypica


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Fig. 4.Genotypically mea/meagranule cells are virtually absentfrom the anterior lobe of the cerebellum. (A) A confocal image froa GT↔mea/meachimeric mouse (no. 1). All cell nuclei arehighlighted with the TOTO-3 nuclear marker and appear blue. Algenotypically GT cells have a green dot over the nucleus (seeMethods and Results). (B) Photomicrograph of the sameGT↔mea/meachimeric mouse as shown in A labeled with in situhybridization and viewed with standard light microscopy. In both and B, the molecular layer has obvious chimerism as shown by tintermixing of the labeled (arrowheads) with the unlabeled (arrowcells. In the granule cell layer, by contrast, virtually every cell isgenotypically wild-type (GT) (arrowheads) as evidenced by theabsence of unlabeled cells. (C) Example of the in situ labeling incontrol GT cerebellum. Note that all cells are labeled. (D) Exampof the in situ labeling with the GT probe in an mea/meamousedemonstrating that no labeled cells are found. ML, molecular layPC, Purkinje cell layer; GC, granule cell layer and * marks the piasurface. Scale bar: 35 µm in A, 30 µm in B, and 50 µm in C and D.

mea/mea. In all 23 cases, the Purkinje cells were part of Purkinje cell monolayer and their dendrites were orientedthe appropriate direction, towards the pial surface, demostrating that genotypically mea/meaPurkinje cells appearedphenotypically normal in all respects in the chimeric enviroment (Fig. 7). Thus, the abnormal placement of Purkinje csomas and alterations in dendritic orientation observedhomozygous meander tail mice are corrected in the chimeenvironment demonstrating that the meagene acts extrinsic toPurkinje cells.

Genotypically mutant Golgi epithelial cells exhibitphenotypically normal radial glial fibers in meandertail chimeric cerebellaIn the cerebellum of a non-mutant mouse, the somas of Goepithelial cells are located near the Purkinje cell somas wtheir radial glial fibers running radially, towards the piasurface. In the anterior lobe of homozygous meander tailmice,there is an absence of radially oriented glial processes (Rosal., 1990). Instead, these fibers appear shortened and thickeand sometimes take a convoluted path towards the pial surfIn addition, the cell somas are more scattered and do not fa discrete layer (Fig. 8A).

The morphology of the radially oriented glial fibers iGT↔mea/meachimeras was comparable to that found in nomutant mice (Fig. 8B). The cell bodies were located nePurkinje cell somas with long, thin, radial processes extendtowards the pial surface. In double label studies, the genotof the cells was marked using in situ hybridization and tphenotype was marked using immunocytochemistry with antibody against GFAP. All genotypically mea/meaglia cellswere seen to exhibit a phenotypically normal morphologThus, the mutant phenotype of the radial glial cells is correcin the chimeric environment demonstrating that the meageneacts extrinsically to Golgi epithelial cells to alter theplacement and process morphology.

DISCUSSION

In this study, we show that the mea gene acts intrinsically tothe granule cells or their precursors to cause the near tdepletion of anterior lobe granule cells in the mea/meacer-ebellum. This finding is robust and reproducible. The samresults are obtained from two different chimeric combinatioand seen in chimeras where either the genotypically mutanwild-type granule cells are labeled. Additional support for thgranule cell intrinsic action of the meagene is derived fromwork by Snyder and colleagues using the injection of immotalized cells into the neonatal mea/mea cerebellum (Rosario etal., 1997). They find that these immortalized cells havegranule cell phenotype and can integrate into the anterior posterior lobes of the mea/mea cerebellum. Thus, the mea/meaphenotype is not due to cell extrinsic, environmental defebut to intrinsic properties of mutant granule cell precursors

Furthermore, the effect of the meagene on cerebellar cellsis specific to granule cells. Genotypically mutant Purkinje cein the anterior lobe appear identical to wild-type Purkinje cein the chimeric cerebellum indicating that the abnormalitiobserved in homozygous meander tailmice are secondary tothe loss of granule cells. Finally, genotypically mea/mearadial

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glial also exhibit a non-mutant phenotype in the chimeric enronment with processes extending radially toward the psurface. Therefore, the changes in these cells found in homogous meander tailmice would also be secondary to other alteations in cerebellar morphology and not due to the meageneacting intrinsically to the Golgi epithelial cells.

Other research supports the important role of granule cor their precursors in the EGL on the subsequent developmof Purkinje and Golgi epithelial cells. There is a relatively largbody of literature demonstrating that the depletion of granucell precursors results in Purkinje cell ectopia and dendrdysmorphology (e.g. Altman and Anderson, 1972; Shimadaal., 1975; Woodward et al., 1975; Yamano et al., 1978; Altma1982). Several lines of experimental evidence also suggest a normal cohort of EGL or granule cells is critical for phentypically normal glial cell morphology. For example, in thweavermutation, there is severe depletion of cerebellar grancells and abnormal radial glia (Rakic and Sidman, 1973). Itknown that the cerebellar granule cell is the target of tweaver mutant gene (Goldowitz, 1989a). Examination oweaver glial cells in a chimeric setting has shown that gentypically mutant Golgi epithelial cells exhibited normal morphology (Goldowitz, 1989a; Hatten et al., 1986). Further suportive evidence comes from studies that disrupted the EGLinjections of the mitotic poison cytosine arabinoside and fouthat radial glia were stunted and mispositioned in a mansimilar to that seen in the mea/meacerebellum (Shimada et al.,1975; Yamano et al., 1980, 1983; Ono et al., 1990). The resof the present study on the meagene add further support to thegrowing body of data that indicates an important granule ceglial cell interaction in the establishment of normal glia cemorphology.

In the initial characterization of the meander tailmutation,the phenotype was described as restricted to the anterior of the cerebellum (Ross et al., 1990). Recent evidence

Fig. 5. Genotypically mea/meagranule cells are virtually absentfrom the anterior lobe and under-represented in the posterior lobethe cerebellum as shown in a BALB/c↔mea/meachimericcerebellum (chimera no. 6). Genotypically mutant (mea/mea) cellsare labeled with a ring outlining the nucleus. (A) In the anterior lobof the cerebellum, no labeled granule cells are found. Otherpopulations of cerebellar neurons, including molecular layerinterneurons and Purkinje cells, are labeled (arrows). (B) In theposterior lobe of the cerebellum, genotypically mea/meagranulecells are present but are mainly found at the distal tips of lobules Vand IX. One such region is shown here, taken from the distal regioof lobule VIII. Examples of labeled cells (arrows) are seen in themolecular layer (ML), Purkinje cell layer and granule cell layer(GC). (C) Example of the immunolabeling seen with the B37antibody in the cerebellum from an mea/meamouse. Note thecomplete labeling of the granule cell layer. (D) Example of theimmunolabeling seen with the B37 antibody in the cerebellum froma BALB/c mouse. Note the absence of immunolabeling. The boxeC and D highlight the region shown in E and G, respectively. (E) Bimmunolabeling pattern seen in an mea/meamouse. Note theimmunolabeled granule cells (GC), Purkinje cells (arrow) andmolecular layer interneurons. (F) Cresyl violet staining illustrates tcell density of mea/meagranule cells in the region of lobule VIIIfrom a neighboring section to that shown in C and E. (G) Absenceany immunolabeling in a cerebellum from a BALB/c mouse. Anunlabeled Purkinje cell is denoted by the arrow. Scale bar: 50 µm inA and B, 800 µm in C and D and 30 µm in E-G.

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shown that this description is an over-simplification. The modramatic abnormalities are found in the anterior lobe, bchanges in the distribution of Purkinje cells (Napieralski anEisenman, 1996) and a 20-30% reduction in granule cnumber (Hamre and Goldowitz, 1996) are also found in tposterior lobe. Similarly, differences in the distribution ogenetically mea/meagranule cells are seen between anteriand posterior lobes of the chimeric mouse cerebellum. Whboth posterior and anterior lobes show significantly fewgenotypically mea/meagranule cells compared to any otheneuronal population, there is a significantly greater percenta

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ric cerebella exhibit near-normal numbers of cerebellar granule cells. shows the mean number of granule cells in the anterior and posteriord from a midsagittal section of the cerebella of control GT and BALB/c

mice, and experimental GT↔mea/mea(labeled GT X) anda/mea(labeled BALB/c X) chimeras. No significant differences in thenule cells were found between control and experimental groups. In the

, the number of cells in the anterior and posterior lobes of each chimericompared to the control values from either GT (nos 1-4) or BALB/c (nosm. The granule cell population in parasagittal sections from the same

region of the cerebellar vermis was estimated in chimeric and control/c mice. For each analyzed section in each chimera, the number ofwas divided by the mean control number of granule cells. The meansntrol group was used as the 100% value. The bars represent the mean ofns with the associated standard error of the mean for each chimera. Allbella have granule cell numbers that are at least 87% of normal withric cerebella greater than their respective controls. GT↔mea/mea

numbers 1-4 and BALB/c↔mea/meachimeras are numbers 5-7. In bothnterior lobe is defined as lobules I-V, rostral to the primary fissure whilelobe is defined as lobules VI-X, caudal to the primary fissure.

of mea/meagranule cells surviving in the posterior lobe thaanterior lobe. Thus, a meander tail phenotype is not restricto the anterior lobe, but is also present in the posterior loThis finding further supports the hypothesis that the effectsthe meagene are not strictly compartmentalized, but are fouin an anterior-to-posterior gradient throughoutthe cerebellum.

Two unexpected findings of this study are theunder-representation of genotypically mea/meagranule cells in the posterior lobe, and theapparent upregulation in the number of non-mutant granule cells in the chimeric cerebellum.In two chimeric mice that had over 50% contri-butions from genotypically mea/meaneurons incerebellar molecular layer neurons andelsewhere, we expected that the posterior lobewould consist of approximately 50% mea/meagranule cells (chimera numbers 1 and 2 in Fig.3). However, what we found was that in theposterior lobe as a whole, only 10% of thegranule cells were genotypically mea/mea. Thus,genetically mea/meagranule cell precursors thatwould normally colonize the posterior lobe of themea/meacerebellum are developmentally disad-vantaged and fail to make a significant contribu-tion to the granule cell population in the chimera.The flip side of this picture is that there are near-normal total numbers of granule cells in both theanterior and posterior lobes of the chimeric cer-ebellum, with approximately 90% of thesegranule cells being genetically wild type (Fig. 6).Thus, the number of wild-type granule cells isgreater than expected leading to a second con-clusion: there are factors in the cerebellar envi-ronment that regulate granule cell productionand, in the face of developmentally compromisedmea/meagranule cell precursors, there is anupregulated production of developmentallycompetent wild-type granule cells.

These conclusions raise two obviousquestions. First, what are the factors that regulategranule cell number? Second, what is the natureof the mea/meagranule cell defect that impedesits development in the face of +/+ granule cells?

Purkinje cells have been shown to regulategranule cell number by influencing both granulecell proliferation in the EGL and granule cellsurvival in the IGL. This latter mechanism is dueto a target-based interaction such that the size ofthe Purkinje cell population determines the sizeof the surviving granule cell population (Wettsand Herrup, 1983; Chen and Hillman, 1989;Vogel et al., 1989). This is an unlikely mechanismin the chimeric cerebellum since there is adepletion in the number of granule cells in themea/mea cerebellum (Hamre and Goldowitz,1996) while Purkinje cells are present in normalnumbers (Hamre and Goldowitz, 1996; Napieral-ski and Eisenman, 1996). Furthermore, the devel-opmental profile seen in the mea/mea EGL wouldargue against this possibility (see below).

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A second, more likely means of Purkinje cell regulation ogranule cell number in the mea/meachimeric cerebellum maybe due to a two-way interaction between granule cell precsors and Purkinje cells. Purkinje cells have a powerfinfluence on the mitotic activity of granule cell precursor

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Fig. 7.Genotypically meander tail Purkinje cellsexhibit normal morphology in chimeric cerebella.(A) In mea/meamice, Purkinje cells (dark-brownstaining somas) in the anterior lobe exhibit severalabnormal features. The cell somas are not arrangedin a monolayer, but are jumbled throughout theputative molecular and granule cell layers. Also,dendrites are often oriented inappropriately withsome facing away from the pial surface and crossingthe white matter towards the granule cells in theposterior lobe (arrowhead). (B) Confocal images ofthe Purkinje cells from the anterior lobe of aGT↔mea/meachimeric mouse. Genotypically GT(+/+) cells have a green/yellow dot over the nucleuswhile mea/meacells appear unlabeled. Purkinjecells are immunolabeled with an antibody tocalcium-binding protein and are red. Note that allPurkinje cells, both +/+ (arrowhead) and mea/mea(arrow), have a normal monolayer arrangement withtheir dendrites oriented normally towards the pialsurface. Scale bar: 45 µm in A and 10 µm in B.

(Smeyne et al., 1995) such that when Purkinje cells depleted, or developmentally compromised, the mitotic activin the EGL is severely diminished (Messer and Hatch, 19Sonmez and Herrup, 1984; Smeyne et al., 1995). This influeof Purkinje cells on granule cells may be reciprocated winformation provided by granule cell neuroblasts to Purkincells that, in some manner, ‘communicates’ either the numof mitotically active precursors or the number of granule cebeing generated. A comparison of the phenotype of weavermeander tail chimeras lends additional support for the two-wcommunication between Purkinje cells and mitotically actigranule cells. In homozygous weaver mice, granule cellscomplete their cell cycle in the proliferative zone of the EGlikely generating normal numbers of granule cells, and diethe premigratory, postmitotic zone of the EGL (Smeyne aGoldowitz, 1989). In chimeras made between wild-type ahomozygous weaverembryos, there is a deletion of virtuallyall wv granule cells with no evidence for an upregulation +/+ cells (Goldowitz, 1989a). In contrast, in wildtype↔mea/meachimeras, near-normal numbers of granucells are generated, even in the absence of any contribufrom the cells of the meagenotype. We hypothesize that thiis accomplished in the following manner. Given the substatial contribution of genotypically mea/meacells to the rest of

Fig. 8. Genotypically meander tailradial glial fibers have normalmorphology in the chimeric cerebella. (A) radial glial fibers in theanterior lobe of anmea/meamouse exhibit shortened processes withfibers that often take a tortuous path towards the pial surface(arrowheads). Additionally, the placement of cell bodies is notrestricted to the Purkinje cell layer, but is scattered throughout theputative molecular and granule cell layers (arrows). Large clear spare Purkinje cell somas. (B) Confocal image of radial glial fibers inthe anterior lobe of a GT↔mea/meachimeric mouse. Radial glialfibers are labeled with GFAP and appear blue in this image.Genotypically GT cells are labeled with a dot over the nucleus thagreen in this image. Both genotypically +/+ cells (white arrowheadand genotypically mea/meacells (white arrow) appear normal in thechimeric environment with processes extending radially towards thpial surface. Scale bar: 55 µm in A, and 20 µm in B.

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the brain (including other neuronal populations in the cereblum), we must conclude that at some point in the developmof the cerebellum there is a depletion of mea/meacells enteringthe granule cell lineage. This would create a deficit in tnumber of EGL cells and result in a diminished signal frogranule cell precursors to the Purkinje cells. The Purkinje ce

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This hypothesis leaves open the key questions of when how granule cell neuroblasts are affected by the action of meagene and, conversely, the role of the normal allele of mea gene in directing granule cell development. The eadevelopment of the EGL is compromised in homozygomeander tailmice. Napieralski and Eisenman (1993) founthat phenotypic differences in mea/meaEGL developmentwere demonstrable as early as 3 days prior to birth (by Eand we have found that the mea/meaEGL has significantlydecreased cell numbers as early as embryonic day 17 (Haand Goldowitz, 1996). Furthermore, we have found differencin the morphology of the EGL are present at the time of initial formation (E12-13) (unpublished observations). Thelatter findings suggest that the meander tailmutation disruptsthe progress of granule cell precursors at the time of thegress from the germinal trigone. It would make sense thais at this time in development when the preferential selectof +/+ neuroblasts to enter the granule cell lineage occursthe chimeric environment. This ‘developmental competitiowould yield a population of granule cell neuroblasts that apredominantly of the +/+ genotype. This competition wouoccur very early in development and create a depleted pooEGL neuroblasts that are then upregulated during the perioEGL proliferation and granule cell neurogenesis resultingthe near-normal number of granule cells in the chimeric cebellum.

At present, the developmental event disrupted by the meagene is open to speculation. One possibility is that the normallele of the meagene is critical for the specification of earlymembers of the granule cell lineage. It is believed froanalysis of interspecies chimeras that anterior lobe grancells are developmentally advanced compared to posterior lgranule cells (Goldowitz, 1989b), and may well need specinstructions that later generated precursor cells do not requThe meagene may code for such a critical molecule. Othpossibilities include a role for the meagene in the migratorypath taken by granule cell precursors destined to colonizeanterior lobe or in the process of naturally occurring cell dein the EGL. These potential mechanisms of meagene actionrequire further experimental analysis.

In summary, this study demonstrates that, while the meandertail gene has pleiotropic effects on the cerebellum, only effects on the granule cells are due to the direct, intrinsic acof the meagene. Alterations in cerebellar foliation, Purkinjcell placement and dendritic morphology, and radial glial mophology are all secondary to the loss of granule cell precursFurther, due to the developmentally compromised nature ofgenotypically mea/meagranule cell precursors, the geneticallwild-type cells are upregulated and overproduced resultinga phenotypically normal cerebellum. These results lead to hypothesis that the mea gene acts to disrupt the very earldevelopment of granule cell neuroblasts through an as unknown mechanism.

The authors wish to thank Dr Richard Mullen for the gift of thB37 antibody and Richard Cushing for technical assistance. We wish to acknowledge the expert staff at the confocal facility at tUniversity of Tennessee, Memphis (supported by PHS grS10RR08385 to Andrea Elberger). Grant support for this project w

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(Accepted 29 July 1997)

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