Purkinje Cell Migration Research Articles

A neurodevelopmental epigenetic programme mediated by SMARCD3–DAB1–Reelin signalling is hijacked to promote medulloblastoma metastasis

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin–DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

EXTH-79. HIJACKING A NEURODEVELOPMENTAL EPIGENOMIC PROGRAM IN METASTATIC DISSEMINATION OF MEDULLOBLASTOMA

Abstract Normal brain development relies on precise genetic and epigenetic spatiotemporal regulation of gene expression. How dysregulation of neurodevelopment relates to medulloblastoma, the most common pediatric brain tumor, remains elusive. Here, we uncovered a novel neurodevelopmental epigenomic program that regulates Purkinje cell migration in developing cerebellum is hijacked to induce tumor metastatic dissemination in medulloblastoma. Integrating publicly available datasets with our in-house data, unsupervised analyses revealed that BAF60C/SMARCD3, a subunit of SWI/SNF chromatin remodeling complex, promotes tumor cell migration in vitro and metastasis in vivo. Based on analyzing the single-cell RNAseq data of cerebellum developmental trajectory in mice and humans, aligning with the medulloblastoma patients’ datasets, we found that BAF60C/SMARCD3 regulated DAB1-mediated Reelin signaling is involved in Purkinje cell positioning during cerebellum development and medulloblastoma metastasis by orchestrating the cis-regulatory elements (CREs) at the DAB1 gene locus. Moreover, analysis of spatiotemporal gene expression and chromatin architecture in the human and mouse cerebellum demonstrated that transcription activity of the BAF60C/SMARCD3-DAB1 circuit is downregulated in a mature state of cerebellar development, however, is upregulated in metastatic medulloblastoma. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), bi-directionally control BAF60C/SMARCD3 transcriptional regulation by coordinating with the CREs at the BAF60C/SMARCD3 gene locus to form a chromatin hub during developing cerebellar development and medulloblastoma metastatic dissemination. Highly expressed BAF60C/SMARCD3 activates the Reelin/DAB1 signaling pathway downstream Src kinase, which was validated in the pair-wised primary and metastatic tumors from medulloblastoma patients. Preclinical medulloblastoma mouse models revealed that inhibiting Src activity reduces tumor cell migration and metastatic dissemination at a lower and safe dose. Together, these data deepen our understanding of how the developmental program influences disease progression and provide an opportunity for the development of therapeutics for this devastating brain cancer in children.

Endoglycan Regulates Purkinje Cell Migration by Balancing Cell-Cell Adhesion.

The importance of cell adhesion molecules for the development of the nervous system has been recognized many decades ago. Functional in vitro and in vivo studies demonstrated a role of cell adhesion molecules in cell migration, axon growth and guidance, as well as synaptogenesis. Clearly, cell adhesion molecules have to be more than static glue making cells stick together. During axon guidance, cell adhesion molecules have been shown to act as pathway selectors but also as a means to prevent axons going astray by bundling or fasciculating axons. We identified Endoglycan as a negative regulator of cell-cell adhesion during commissural axon guidance across the midline. The presence of Endoglycan allowed commissural growth cones to smoothly navigate the floor-plate area. In the absence of Endoglycan, axons failed to exit the floor plate and turn rostrally. These observations are in line with the idea of Endoglycan acting as a lubricant, as its presence was important, but it did not matter whether Endoglycan was provided by the growth cone or the floor-plate cells. Here, we expand on these observations by demonstrating a role of Endoglycan during cell migration. In the developing cerebellum, Endoglycan was expressed by Purkinje cells during their migration from the ventricular zone to the periphery. In the absence of Endoglycan, Purkinje cells failed to migrate and, as a consequence, cerebellar morphology was strongly affected. Cerebellar folds failed to form and grow, consistent with earlier observations on a role of Purkinje cells as Shh deliverers to trigger granule cell proliferation.

MEDB-88. BAF60C/SMARCD3-mediated novel neurodevelopmental epigenomic program promotes metastatic dissemination in medulloblastoma

Normal brain development relies on precise genetic and epigenetic spatiotemporal regulation of gene expression. How dysregulation of neurodevelopment relates to medulloblastoma, the most common pediatric brain tumor, remains elusive. Here, we uncovered a novel neurodevelopmental epigenomic program that regulates Purkinje cell migration in developing cerebellum is hijacked to induce tumor metastatic dissemination in medulloblastoma. Integrating publicly available datasets with our in-house data, unsupervised analyses revealed that BAF60C/SMARCD3, a subunit of SWI/SNF chromatin remodeling complex, promotes tumor cell migration in vitro and metastasis in vivo. Based on analyzing the single-cell RNAseq data of cerebellum developmental trajectory in mice and humans, aligning with the medulloblastoma patients’ datasets, we found that BAF60C/SMARCD3 regulated DAB1-mediated Reelin signaling is involved in Purkinje cell positioning during cerebellum development and medulloblastoma metastasis by orchestrating the cis-regulatory elements (CREs) at the DAB1 gene locus. Analysis of spatiotemporal gene expression and chromatin architecture in the human and mouse cerebellum demonstrated that transcription activity of the BAF60C/SMARCD3-DAB1 circuit is downregulated in a mature state of cerebellar development, however, is upregulated in metastatic medulloblastoma. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), bi-directionally control BAF60C/SMARCD3 transcriptional regulation by coordinating with the CREs at the BAF60C/SMARCD3 gene locus to form a chromatin hub during developing cerebellar development and medulloblastoma metastatic dissemination. Highly expressed BAF60C/SMARCD3 activates the Reelin/DAB1 signaling pathway downstream Src kinase, which was validated in the pair-wised primary and metastatic tumors from medulloblastoma patients. Preclinical medulloblastoma mouse models revealed that inhibiting Src activity reduces tumor cell migration and metastatic dissemination at a lower and safe dose. Together, these data deepen our understanding of how the developmental program influences disease progression and provide an opportunity for the development of therapeutics for this devastating brain cancer in children.

Loss of CRMP1 and CRMP2 results in migration defects of Purkinje cells in the X lobule of the mouse cerebellum

The three-layered structure of the mammalian cerebellar cortex is generated through the coordinated migration of cerebellar neurons. Purkinje cells migrate and form a three- to four-cell-thick aggregate below the external granule cell layer during the embryonic stage, and align to form a monocellular arrangement in the Purkinje cell layer during the postnatal period. We previously reported the involvement of Cdk5-mediated CRMP2 phosphorylation in Purkinje cell migration and the synergistic roles of two other CRMPs, CRMP1 and CRMP4. In the present study, we investigated the loss of function of CRMP2 along with the synergistic function of CRMP1 in the migration and alignment of Purkinje cells. We found deficits in the migration and alignment of Purkinje cells in lobule X of the cerebella of CRMP1 and CRMP2 double knockout mice. Because lobule X, also called the flocculonodular lobe, is involved in the maintenance of balance equilibrium and muscle tone, we conducted balance beam and grip power tests in these mice and found impaired performance on the balance beam test and lower grip power in CRMP1 and CRMP2 double knockout mice, indicating the importance of these genes in proper cerebellar development.

Alteration of the Dopamine Receptors’ Expression in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant (Naked–Ataxia (NAX)) Mouse

A spontaneous mutation in the lysosomal acid phosphatase (Acp2) enzyme (nax: naked–ataxia) in experimental mice results in delayed hair appearance and severe cytoarchitectural impairments of the cerebellum, such as a Purkinje cell (PC) migration defect. In our previous investigation, our team showed that Acp2 expression plans a significant role in cerebellar development. On the other hand, the dopaminergic system is also a player in central nervous system (CNS) development, including cerebellar structure and function. In the current investigation, we have explored how Acp2 can be involved in the regulation of the dopaminergic pathway in the cerebellum via the regulation of dopamine receptor expression and patterning. We provided evidence about the distribution of different dopamine receptors in the developing cerebellum by comparing the expression of dopamine receptors on postnatal days (P) 5 and 17 between nax mice and wild–type (wt) littermates. To this aim, immunohistochemistry and Western blot analysis were conducted using five antibodies against dopamine receptors (DRD1, –2, –3, –4, and –5) accompanied by RNAseq data. Our results revealed that DRD1, –3, and –4 gene expressions significantly increased in nax cerebella but not in wt, while gene expressions of all 5 receptors were evident in PCs of both wt and nax cerebella. DRD3 was strongly expressed in the PCs’ somata and cerebellar nuclei neurons at P17 in nax mice, which was comparable to the expression levels in the cerebella of wt littermates. In addition, DRD3 was expressed in scattered cells in a granular layer reminiscent of Golgi cells and was observed in the wt cerebella but not in nax mice. DRD4 was expressed in a subset of PCs and appeared to align with the unique parasagittal stripes pattern. This study contributes to our understanding of alterations in the expression pattern of DRDs in the cerebellum of nax mice in comparison to their wt littermates, and it highlights the role of Acp2 in regulating the dopaminergic system.

Phosphorylation of CRMP2 is required for migration and positioning of Purkinje cells: Redundant roles of CRMP1 and CRMP4

Proper migration and positioning of Purkinje cells are important for formation of the developing cerebellum. Although several cyclin-dependent kinase 5 (Cdk5) substrates are known to be critical for ordered neuronal migration, there are no reports of mutant mouse-based, in vivo studies on the function of Cdk5-phosphorylation substrates in migration of Purkinje cells.We focused on the analysis of collapsin response mediator protein 2 (CRMP2), one of the Cdk5 substrates, because a previous study reported migration defects of cortical neurons with shRNA-mediated knockdown of CRMP2. However, CRMP2 KI/KI mice, in which Cdk5-phosphorylation is inhibited, showed little defects in Purkinje cell migration and positioning. We hypothesized compensatory redundant functions of the other CRMPs, and analyzed the migration and positioning of Purkinje cells in the cerebellum in every combination of CRMP1 knockout (KO), CRMP2 KI/KI, and CRMP4 KO mice. Severe disturbance of migration and positioning of Purkinje cells were observed in the triple mutant mice. We also found motor coordination defects in the triple CRMPs mutant mice. These results suggest the importance of both, phosphorylation of CRMP2 by Cdk5 and the redundant functions of CRMP1 and CRMP4 in proper migration and positioning of Purkinje cells in developing cerebellum.

The Ferdinando Rossi Memorial Lecture: Zones and Stripes-Pattern Formation in the Cerebellum.

The cerebellum has a complex architecture-highly reproducible and conserved through evolution. Cerebellar architecture is organized around the Purkinje cell. Purkinje cells in the mouse cerebellum come in many different subtypes, identifiable by expression markers, sensitivity to mutation, etc. These are organized first into five "transverse zones," each of which is further subdivided into dozens of reproducible "stripes." This arrangement serves as the scaffolding to organize afferent topography and restrict the distribution of excitatory and inhibitory interneurons. This brief review will survey some of the mechanisms that lead to the formation of this elaborate pattern during cerebellar development. Pattern formation in the cerebellar cortex is a multistage process that begins early in development with the generation of the various Purkinje cell subtypes, and matures through the dispersal of Purkinje cell clusters into stripes. Two developmental processes will be discussed in particular: the mechanisms that lead to Purkinje cell subtype specification (i.e., how different kinds of Purkinje cells are made) and the role played by Purkinje cell migration in pattern formation (i.e., how these Purkinje cell subtypes end up in a reproducible array of stripes).

Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners.

Cyclin-dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5-null Purkinje cells and Purkinje cells in Wnt1cre -mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7cre -mediated p35; p39 double KO (L7cre -p35f/f ; p39-/- ) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell-autonomous in vivo. We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5-null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non-cell-autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain-derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7cre -p35f/f ; p39-/- mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175-1187, 2017.

GFRα1 Regulates Purkinje Cell Migration by Counteracting NCAM Function

SummaryDuring embryonic development of the cerebellum, Purkinje cells (PCs) migrate away from the ventricular zone to form the PC plate. The mechanisms that regulate PC migration are incompletely understood. Here, we report that the neurotrophic receptor GFRα1 is transiently expressed in developing PCs and loss of GFRα1 delays PC migration. Neither GDNF nor RET, the canonical GFRα1 ligand and co-receptor, respectively, contribute to this process. Instead, we found that the neural cell adhesion molecule NCAM is co-expressed and directly interacts with GFRα1 in embryonic PCs. Genetic reduction of NCAM expression enhances wild-type PC migration and restores migration in Gfra1 mutants, indicating that NCAM restricts PC migration in the embryonic cerebellum. In vitro experiments indicated that GFRα1 can function both in cis and trans to counteract NCAM and promote PC migration. Collectively, our studies show that GFRα1 contributes to PC migration by limiting NCAM function.

Dendritic planarity of Purkinje cells is independent of Reelin signaling.

The dendritic planarity of Purkinje cells is critical for cerebellar circuit formation. In the absence of Crk and CrkL, the Reelin pathway does not function resulting in partial Purkinje cell migration and defective dendritogenesis. However, the relationships among Purkinje cell migration, dendritic development and Reelin signaling have not been clearly delineated. Here, we use synchrotron X-ray microscopy to obtain 3-D images of Golgi-stained Purkinje cell dendrites. Purkinje cells that failed to migrate completely exhibited conical dendrites with abnormal 3-D arborization and reduced dendritic complexity. Furthermore, their spines were fewer in number with a distorted morphology. In contrast, Purkinje cells that migrated successfully displayed planar dendritic and spine morphologies similar to normal cells, despite reduced dendritic complexity. These results indicate that, during cerebellar formation, Purkinje cells migrate into an environment that supports development of dendritic planarity and spine formation. While Reelin signaling is important for the migration process, it does not make a direct major contribution to dendrite formation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00429-014-0780-2) contains supplementary material, which is available to authorized users.

Impaired structural and functional development of cerebellum following gestational exposure of deltamethrin in rats: role of reelin.

Reelin is an extracellular matrix molecule that is involved in the normal development of the cerebellar lamination, Bergmann glial fibres alignment, Purkinje cell monolayer arrangement and granule cell migration. In this study, we have examined the effects of maternal exposure of deltamethrin (DLT), a type II pyrethroid insecticide, on the structural and functional development of rat cerebellum during postnatal life. DLT (0.75 mg/kg body weight, intraperitoneally dissolved in dimethylsulphoxide) was administered in timed pregnant rats during two different gestational time periods, i.e. gestational days of 7-10 and 11-14, respectively. In DLT exposed rats, a significant overexpression of reelin was observed in the cells of the external granule cell layer (EGL) and internal granule cell layer along with an ectopic expression of reelin in the EGL as well as in the migrating granule cells just below the EGL, revealing an arrest of granule cell migration in this zone. Mis-orientation and hypertrophy of the Bergmann glial fibres further hampered the journey of the granule cells to their final destination. Possibly reelin overexpression also caused misalignment of the Purkinje cells and inhibited the neurite growth leading to a significant decrease in the spine density, main dendritic length and width of the dendritic arbour. Thus, it is proposed that the DLT exerts its neurotoxic effects possibly via the intracellular accumulation and low release of reelin leading to an impaired granule cell and Purkinje cell migration, inhibition of neurite outgrowth and reduced spine density. Such impaired cerebellar development leads to motor coordination deficits.

Purkinje cell loss and motor coordination defects in profilin1 mutant mice

Profilin1 is an actin monomer-binding protein, essential for cytoskeletal dynamics. Based on its broad expression in the brain and the localization at excitatory synapses (hippocampal CA3–CA1 synapse, cerebellar parallel fiber (PF)–Purkinje cell (PC) synapse), an important role for profilin1 in brain development and synapse physiology has been postulated. We recently showed normal physiology of hippocampal CA3–CA1 synapses in the absence of profilin1, but impaired glial cell binding and radial migration of cerebellar granule neurons (CGNs). Consequently, brain-specific inactivation of profilin1 by exploiting conditional mutants and Nestin-mediated cre expression resulted in a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers, and ectopic CGNs. Apart from these findings we noted a loss of PCs and an irregularly shaped PC layer in adult mutants. In this study, we show that PC migration and development are not affected in profilin1 mutants, suggesting cell type-specific functions for profilin1 in PCs and CGNs. PC loss begins during the second postnatal week and progresses until adulthood with no further impairment in aged mutants. In Nestin-cre profilin1 mutants, defects in cerebellar cortex cytoarchitecture are associated with impaired motor coordination. However, in L7-cre mutants, lacking profilin1 specifically in PCs, the cerebellar cortex cytoarchitecture is unchanged. Thereby, our results demonstrate that the loss of PCs is not caused by cell-autonomous defects, but presumably by impaired CGN migration. Finally, we show normal functionality of PF–PC synapses in the absence of profilin1. In summary, we conclude that profilin1 is crucially important for brain development, but dispensable for the physiology of excitatory synapses.

Purkinje Cell Heterotopy With Cerebellar Hypoplasia in Two Free-Living American Kestrels (Falco sparverius)

Two wild fledgling kestrels exhibited lack of motor coordination, postural reaction deficits, and abnormal propioception. At necropsy, the cerebellum and brainstem were markedly underdeveloped. Microscopically, there was Purkinje cells heterotopy, abnormal circuitry, and hypoplasia with defective foliation. Heterotopic neurons were identified as immature Purkinje cells by their size, location, immunoreactivity for calbindin D-28 K, and ultrastructural features. The authors suggest that this cerebellar abnormality was likely due to a disruption of molecular mechanisms that dictate Purkinje cell migration, placement, and maturation in early embryonic development. The etiology of this condition remains undetermined. Congenital central nervous system disorders have rarely been reported in birds.

Crk and Crk-Like Play Essential Overlapping Roles Downstream of Disabled-1 in the Reelin Pathway

Reelin controls neuronal positioning in the developing brain by binding to the two lipoprotein receptors, very-low-density lipoprotein receptor and apolipoprotein E receptor 2, to stimulate phosphorylation of Disabled-1 (Dab1) by the Fyn and Src tyrosine kinases. Crk and Crk-like (CrkL) have been proposed to interact with tyrosine phosphorylated Dab1 to mediate downstream events in the Reelin pathway. However, these adaptor proteins are widely expressed, and they fulfill essential functions during embryonic development. To address their specific roles in Reelin-mediated neuronal migration, we generated mutant mice, by Cre-loxP recombination, lacking Crk and CrkL in most neurons. These animals displayed the major anatomic features of reeler including, cerebellar hypofoliation, failure of Purkinje cell migration, absence of preplate splitting, impaired dendritic development, and disruption of layer formation in the hippocampus and cerebral cortex. However, proximal signaling involving tyrosine phosphorylation and turnover of Dab1 occurred normally in the mutant mouse brain and in primary cortical neurons treated with Reelin. In contrast, two downstream signaling events, Reelin-induced phosphorylation of C3G and Akt, were not observed in the absence of Crk and CrkL in mouse embryonic cortical neurons. These findings place C3G and Akt phosphorylation downstream of Crk and CrkL, which play essential overlapping functions in the Reelin signaling pathway.

The anatomy of the cerebellar nuclei in the normal and scrambler mouse as revealed by the expression of the microtubule-associated protein kinesin light chain 3

Conventional kinesin is a motor protein complex including two heavy chains and two light chains (KLC). Junco et al. (Junco, A., Bhullar, B., Tarnasky, H.A. and van der Hoorn, F.A., 2001. Kinesin light-chain KLC3 expression in testis is restricted to spermatids. Biol. Reprod. 64, 1320–1330). recently reported the isolation of a novel KLC gene, klc3. In the present report, immunohistochemistry has been used to characterize the expression of KLC3 in the cerebella of normal and scrambler (scm) mutant mice. In cryostat sections through the cerebellum of the normal adult mouse immunoperoxidase stained for KLC3, reaction product is deposited in the nuclei and somata of deep cerebellar nuclear neurons. No other structures are stained in the cerebellum. Strong and specific KLC3 expression is observed in the adult cerebellum in all three major cerebellar nuclei—medial, interposed, and lateral. Double immunofluorescence studies reveal that KLC3 immunoreactivity is colocalized with both endosomes and GW bodies. KLC3 immunohistochemistry has been exploited to study the organization of the cerebellar nuclei in scrambler mice, in which disruption of the mdab1 gene results in severe foliation defects due to Purkinje cell ectopia, with most Purkinje cells clumped in centrally located clusters. Despite the severe failure of Purkinje cell migration, the cerebellar nuclei appear normal in scrambler mutant mice, suggesting that their topography is dependent neither on normal Purkinje cell positioning nor the Reelin signaling pathway.

Dissection of the Cellular and Molecular Events that Position Cerebellar Purkinje Cells: A Study of themath1Null-Mutant Mouse

Granule cell precursors in the external germinal layer (EGL) of the cerebellum have been proposed to be a major player in the migration and positioning of Purkinje cells through the expression of the Netrin-like receptor Unc5h3 and the extracellular matrix molecule Reelin. To explore the role of the EGL on these processes, we made use of the math1 null-mutant mouse in which the EGL does not form. In the absence of the EGL, we find three populations of ectopic Purkinje cells. First, we find 1% of all Purkinje cells in a supracerebellar position at the dorsal midline. Second, we find 7% of all Purkinje cells in the inferior colliculus, similar to what is seen in the Unc5h3 mutant. Our finding that Unc5h3 expression is not disrupted in these cells supports the proposed role of EGL granule cell precursors in establishing the anterior cerebellar boundary through the expression of Unc5h3. Third, we find 20% of all Purkinje cells positioned deep to the cerebellar cortex as seen in the reeler mutant. However, unlike the reeler mutant, where 5% of the Purkinje cells migrate successfully, we find that in the math1 null that 72% of the Purkinje cells migrate successfully. This finding demonstrates that Purkinje cell migration is not solely dependent on Reelin signaling from the EGL and is likely caused by Reelin signals emanating from the nuclear transitory zone or the ventricular zone, or both.

The Community Effect and Purkinje Cell Migration in the Cerebellar Cortex: Analysis of Scrambler Chimeric Mice

The Disabled-1 protein in mouse is known to be an intercellular signaling component of the Reelin molecular pathway that subserves neuronal migration in several structures in the brain and spinal cord. The scrambler mutant mouse, which is phenotypically identical to the reeler mouse, is due to a mutation in the disabled-1 gene (Howell et al., 1997; Sheldon et al., 1997). The Purkinje cells of the cerebellum express Disabled-1 and experience a massive failure of migration in the scrambler mutant mouse (Howell et al., 1997; Sheldon et al., 1997; Gallagher et al., 1998; Rice et al., 1998). We sought to define the developmental basis of this mutation by studying the Purkinje cell population in experimental mouse aggregation chimeras using a cell marker that permitted the identification of neurons derived from the mutant lineage. We found that a genetically normal component to the environment cannot assist scrambler mutant Purkinje cells in the migratory process. However, the presence of a mutant component to the environment can cause the ectopia of wild-type Purkinje cells. There appears to be a linear relationship between the percentage of the cerebellum that is genetically mutant and the number of wild-type Purkinje cells that express a mutant phenotype. These studies point to the interplay between cell-intrinsic and cell-extrinsic properties in the migration of neurons to form laminated structures during CNS development.

Evaluation of superior vermal Purkinje cell placement in mental illness

Background: A number of neuroimaging and neuropathological studies have reported abnormalities in the cerebellar vermis in schizophrenia and bipolar disorder. In an effort to further understand vermal abnormalities in mental illness, we have analyzed ectopic placement of Purkinje-like cells. Methods: The superior cerebellar vermis was evaluated in 39 cases of severe mental illness [schizophrenia ( n = 12), bipolar disease ( n = 12), and depression ( n = 15)]. We also examined 9 subjects with polysubstance abuse and 15 normal controls. All normally placed Purkinje cells and displaced Purkinje-like cells (i.e., in the internal granule layer and intrafoliar white matter) were counted in the same foliar field. The ratio of displaced Purkinje-like cells to total Purkinje cells and Purkinje cell density were calculated. Results: No significant difference in the ratio of displaced to normally placed Purkinje cells or in Purkinje cell density between groups of subjects was found. Conclusions: Our study does not support a hypothesis of abnormalities of Purkinje cell migration or other events related to their displacement as a basis for the vermal abnormalities reported previously in schizophrenia and bipolar disorder.

Purkinje cell compartments in the reeler mutant mouse as revealed by Zebrin II and 90-acetylated glycolipid antigen expression.

The cerebellum is organized into a series of parasagittally aligned bands that may be revealed histologically in the adult mouse by largely complementary immunostaining of Purkinje cells sets with the monoclonal antibodies Zebrin II (ZII; antigen:aldolase C) and P-path (PP; antigen:90-acetyl glycolipids). We compared the normal staining pattern using these markers and an antibody to calbindin with that found in the reeler mutants (rl/rl), in which most Purkinje cell migration is halted beneath the cerebellar white matter. The results revealed that Purkinje cells in reeler mutants, despite their ectopic location in large subcortical masses, show a clear tendency to distribute into alternating zones that either stain for Zebrin II or for P-path, with variable transition zones of mixed labeling. However, the estimated number of zones was fewer than in the normal adult cortex: roughly 7-9 zones are revealed per side in the mutant compared with 14 major divisions in wild type mice. These results raise the possibility that neurons destined to express these markers are segregated during their migration and that the final phase of migration into the cortex might involve further splitting or interdigitation between cell sets expressing the two antigens.