Deposition and role of thrombospondin in the histogenesis of the cerebellar cortex

The patterns of deposition of thrombospondin (TSP), a trimeric extracellular matrix glycoprotein, were determined during the initial establishment of the external granule cell layer and the subsequent inward migration of granule cells forming the molecular and (internal) granule cell layers. The early homogeneous deposition of TSP became restricted to the rhombic lip in the region of granule cell exit from the neuroepithelium, and was present between migrating granule cells. During the later inward migration of granule cells, little TSP was associated with dividing granule cells; it was enriched in premigratory granule cells. With the cessation of migration, TSP was lost except in association with fasciculating axons in the molecular layer where staining persisted briefly. At the EM level, TSP was associated with the leading process of granule cells as they associated with Bergmann glial cells and migrated through the molecular layer. TSP was present within granule cell axons; Purkinje cells and their dendrites, as well as Bergmann glial fibers and endfeet were negative for TSP. When anti-TSP antibodies were added to explant cultures of cerebellar cortex during active granule cell migration, a dose-dependent inhibition of migration was observed. In control cultures, granule cells migrated into the (internal) granule cell layer, while granule cells exposed to anti-TSP antibodies were arrested within the external granule cell layer. These results suggest that TSP plays an important role in the histogenesis of the cerebellar cortex by influencing granule cell migration.     

Sequential expression and differential function of multiple adhesion molecules during the formation of cerebellar cortical layers

We have correlated the times of appearance of the neural cell adhesion molecule (N-CAM), the neuron-glia cell adhesion molecule (Ng-CAM), and the extracellular matrix protein, cytotactin, during the development of the chicken cerebellar cortex, and have shown that these molecules make different functional contributions to granule cell migration. Immunofluorescent staining showed distinct spatiotemporal expression sequences for each adhesion molecule. N-CAM was present at all times in all layers. However, the large cytoplasmic domain polypeptide of N-CAM was always absent from the external granular layer and was enriched in the molecular layer as development proceeded. Ng-CAM began to be expressed in the premigratory granule cells just before migration and later disappeared from cell bodies but remained on parallel fibers. Cytotactin, which is synthesized by glia and not by neurons, appeared first in a speckled pattern within the external granular layer and later appeared in a continuous pattern along the Bergmann glia; it was also enriched in the molecular layer. After we established their order of appearance, we tested the separate functions of these adhesion molecules in granule cell migration by adding specific antibodies against each molecule to cerebellar explant cultures that had been labeled with tritiated thymidine and then measuring the differential distribution of labeled cells in the forming layers. Anti-N-CAM showed marginal effects. In contrast, anti-Ng-CAM arrested most cells in the external granular layer, while anti-cytotactin arrested most cells in the molecular layer. Time course analyses combined with sequential addition of different antibodies in different orders showed that anti-Ng-CAM had a major effect in the early period (first 36 h in culture) and a lesser effect in the second part of the culture period, while anti-cytotactin had essentially no effect at the earlier time but had major effects at a later period (18-72 h in culture). The two major stages of cerebellar granule cell migration thus appear to be differentially affected by distinct adhesion molecules of different cellular origins, binding mechanisms, and overall distributions. The results indicated that local cell surface modulation of adhesion molecules of different specificities at defined stages and sites is essential to the formation of cerebellar cortical layers.     

Monoclonal antibody (M2) to glial and neuronal cell surfaces

A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface Of all GFA protein-positive astrocytes and on more immature oligodendrocytes, that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxinpositive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO₂. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures I. A PKH26 Labeling Study in Microexplant and Organotypic Cultures

Granule cells were dissociated from early postnatal mouse cerebella and labeled with a fluorescent dye probe PKH26. Small number of the labeled cells were mixed with cerebellar cortical microexplant cultures or transplanted into cerebellar cortical organotypic explants, and their time-dependent morphological changes during cultures were examined with fluorescence microscopy. Granule cell neurons first extended asymmetrical short bipolar processes in both cultures, and migrated actively in microexplant cultures. After elongation of symmetrically bipolar long and thin neurites, they sprouted short thick processes from cell bodies and migrated perpendicular to neurite bundles that were devoid of glia in microexplant cultures, or migrated vertically inward into the internal granular layer in the organotypic explant. During such migrations, they extended short thick processes in front and thin processes behind the cell body. The latter processes were connected to thin long neurites with T- or Y-shaped junctions in both cultures. Finally, they extended many short thick processes from cell bodies in both cultures. Such behaviors of granule cell neurons in microexplant cultures were, thus, similar to those in organotypic explant cultures despite of the absence of Bergmann glial cells. These migration patterns may be closely related to migration of granule cells in histogenesis of the cerebellar cortex.     

Interaction of astrochondrin with extracellular matrix components and its involvement in astrocyte process formation and cerebellar granule cell migration

We have recently characterized a chondroitin sulfate proteoglycan from the murine central nervous system which is expressed by astrocytes in vitro and carries the L2/HNK-1 and L5 carbohydrate structures. In the present study, we provide evidence that its three core proteins of different size are similar in their proteolytic peptide maps and thus designate this group of structurally related molecules astrochondrin. During development, astrochondrin and the L5 carbohydrate were hardly detectable in the brain of 14-d-old mouse embryos by Western blot analysis. Expression of astrochondrin and the L5 epitope was highest at postnatal day 8, the peak of cerebellar granule cell migration and Bergmann glial process formation, and decreased to weakly detectable levels in the adult. Immunocytochemical localization of astrochondrin in the cerebellar cortex of 6-d-old mice showed association of immunoreactivity with the cell surface of astrocytes, including Bergmann glial processes and astrocytes in the internal granular layer or prospective white matter. Endfeet of astrocytes contacting the basal lamina of endothelial and meningeal cells and contact sites between Bergmann glial processes and granule cells also showed detectable levels of astrochondrin. Furthermore, granule cell axons in the molecular layer were astrochondrin immunoreactive. In the adult, astrochondrin immunoreactivity was weakly present in the internal granular layer and white matter. Both Fab fragments of polyclonal antibodies to astrochondrin and monovalent fragments of the L5 monoclonal antibody reduced the formation of processes of mature GFAP- positive astrocytes on laminin and collagen type IV, but not on fibronectin as substrata. Interestingly, the initial attachment of astrocytic cell bodies was not disturbed by these antibodies. Antibodies to astrochondrin also reduced the migration of granule cells in the early postnatal mouse cerebellar cortex. In a solid phase radioligand binding assay, astrochondrin was shown to bind to the extracellular matrix components laminin and collagen type IV, being enhanced in the presence of Ca2+, but not to fibronectin, J1/tenascin or other neural recognition molecules. Furthermore, astrochondrin interacted with collagen types III and V, less strongly with collagen types I, II, and IX, but not with collagen type VI. The interaction of astrochondrin with collagen types III and V was saturable and susceptible to increasing ionic strength, and could be competed by chondroitin sulfate, heparin, and dextran sulfate, but not by hyaluronic acid, glucose-6-phosphate, or neuraminic acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Postnatal expression of Doublecortin (Dcx) in the developing cerebellar cortex of mouse

We have investigated the expression of Doublecortin (Dcx) protein in the developing cerebellum of mouse from postnatal 2nd day to postnatal 22nd day and in young adults by immunohistochemistry. Strong expression of Dcx was present in the inner zone of the external granule cell layer, and remained strong while postmitotic granule cell precursors were present in this transitory layer. Descending granule cell precursors exhibited Dcx immunostaining not only while migrating but for a short time also after their settlement. Dcx-immunostained cells appeared in deep cerebellocortical territories and in the cerebellar white matter during the first postnatal week. These bipolar cells were arranged in the sagittal plane and built up transitory migratory streams during the second postnatal week and their number gradually decreased during the third postnatal week. Upward migration of bipolar cells was observed while leaving the migratory streams, penetrating the internal granule cell layer and the molecular layer. These cells were considered as precursors of late migrating molecular layer interneurons. However, a proportion of Dcx-immunostained cells underwent a bipolar-to-multipolar dendritic remodellation and - on the basis of strong morphological similarities - was taken for "multipotent progenitor cells", described recently in the neocortex of adult rat.     

Migration Behavior of Rodent Granule Neurons in the Presence of Antibody to the 4C5 Antigen

Abstract: We have reported the production of monoclonal antibody 4C5, which recognizes a cell surface antigen, the 4C5 antigen, involved in granule cell migration processes. In the present study, we investigated in a more precise manner the role of the 4C5 antigen in the different types of granule cell migrations that take place during cerebellar development. When cerebellar explant cultures derived from 10-day-old rats were performed for 2 days in the presence of monoclonal antibody 4C5, vertical granule cell migration, occurring in the presence of glia, was not significantly inhibited. In contrast, when monoclonal antibody 4C5 was included in the medium of microexplant cultures derived from 4-day-old mice and maintained for 4 days in vitro, granule cell migrations that occurred both parallel and perpendicular to the neurite bundles that were free of glia were inhibited. Moreover, a stronger inhibitory effect of the antibody was observed on migration perpendicular to the neurite bundles compared with the parallel type of migration. Our results indicate that the 4C5 antigen differentially affects the different developmental stages and types of granule cell migration during rodent cerebellar development.     

Granule cell migration in developing rat cerebellum. Influence of neonatal hypo- and hyperthyroidism.

The rate of cerebellar granule cell migration is altered by neonatal hypo- and hyperthyroidism in a manner similar to previously reported effects on the growth of granule cell axons, the parallel fibers, suggesting that the two processes may be intimately linked. Altered rates of granule cell acquisition in these experimental animals reflect changes in germinal cell proliferation in the external granular layer (EGL), movement of postmitotic cells within the EGL, as well as the rate and time course of granule cell migration. Results of this study support the hypothesis that granule cells migrate to the internal granular layer by translocation of the cell body through the descending portion of the growing parallel fiber, rather than by amoeboid-like migration of the perikaryon trailing the elongating parallel fiber behind.     

Effects of long-term hypothyroidism in the morphology and synaptic organization of cerebellar ectopic granule cells

Abundant ectopic granule cells scattered in the cerebellar molecular layer have been observed in 30-day-old hypothyroid rats. Their morphological features indicate that they must be regarded as mature heterotopic cells arrested during their migration towards the granular layer. As their impoverished dendritic trees are identical to those seen in controls, it is unlikely that the lack of thyroid hormones played a major role in the deficient dendritic outgrowth. The study of 180-day-old hypothyroid rats revealed that although ectopic granule cells remained quite numerous, their number per unit surface was lesser than in the 30-day-old hypothyroid group. This finding may be related to the capacity displayed by heterotopic neurons to establish synaptic contacts with the components of the molecular layer. This was inferred by the presence of a peculiar synaptic cell investment formed by axosomatic and somatodendritic contacts in 180-day-old hypothyroid rats which shows that the surviving ectopic granule cells manage to adapt to an adverse milieu.     

Progressive restriction of cell fates in relation to neuroepithelial cell mingling in the mouse cerebellum

Neurogenesis in the cerebellum proceeds through a temporal series of cell production from two separate epithelia, the ventricular zone (VZ) and the external granule cell layer (EGL). Using the laacZ cell lineage tracer in transgenic mice, we describe cellular clones whose dates of birth span the entire period of cerebellar development and deduce a sequence of cell dispersion leading to the final allocation of cells in the cerebellum. Clones probably labeled early during neural tube formation show that individual progenitors can give rise to all cerebellar cell types. The distribution of clonally related granule cells in these clones indicates a mediolateral organization of EGL progenitors already established before the allocation of the EGL progenitors to the cerebellum. Clones restricted to the cerebellar VZ show that the VZ derives progenitors for deep nuclei and multipotent cortical progenitors, which lose their systematic lineage relationship when longitudinal cell intermingling in the cerebellar VZ becomes more limited. The small clones also show that cell dispersion is radial in the internal granule layer and tangential in the molecular layer. Together, the data demonstrate the broad maintenance of the relative order of cells from neural tube stages to the adult cerebellum.     

Expressions of amyloid precursor protein, synaptophysin and presenilin-1 in the different areas of the developing cerebellum of rat

This study reveals the expressions of Alzheimer's disease-related amyloid precursor protein, presenilin-1, and a presynaptic marker protein, synaptophysin, in the archi-, paleo- and neocerebellum during the postnatal development of the rat. The Western blot results demonstrate a gradual increase in the soluble amyloid precursor protein level in the archicerebellum during the first 3 weeks, while in the neo- and paleocerebellum the levels reach a plateau as early as the 1st week. Immunohistochemically, the protein is present in the deep part of the external granule cell layer and the internal granule cell layer in the newborn animal, while in 3-week-old animals the staining appears mainly in the perikarya and dendrites of the Purkinje cells. The level of synaptophysin increases progressively from postnatal day 7 up to 3 weeks in the archi- and paleocerebellum, and up to 6 weeks in the neocerebellum. Immunohistochemically, the amyloid precursor protein staining appears first in the inner part of the molecular layer and in the internal granule cell layer. In a 3-week-old animal, synaptophysin staining is present in all areas of the cerebellar molecular layer and in the internal granule cell layer. The presenilin-1 immunohistochemical reaction appeared equally in the archi-, paleo- and neocerebellum. Much of the staining is present in the glial cells and Purkinje cells. Less immunoreactivity is observed in the Golgi cells and granule cells. It is concluded that the postnatal expressions of soluble and membrane-bound amyloid precursor protein, synaptophysin and presenilin-1 are regulated differently during the ontogenetical development of the archi-, paleo- and neocerebellum of rat. Further, the amyloid precursor protein and presenilin-1 may be present in cells which do not degenerate in Alzheimer's disease.     

Transient accumulations of glycosaminoglycans in rat and chicken cerebellar cortex during development

Modifications of glycosaminoglycans at neuropile of rat and chicken cerebellum during development were histochemically studied. The application of Alcian Blue staining techniques and enzymatic degradations permitted to reveal in both species that in earlier stages of cerebellar development hyaluronic acid is present throughout neuropile of entire cerebellum but it accumulated preferentially at the medullary region and around precursory Purkinje cells where it showed a mucoid-like appearance. This substance was related with cell migration and aligning processes. At the middle of cerebellar development, around 2nd postnatal week in rat and 12-16 embryonary days in chick, a new polyanionic transient accumulation, presumably chondroitinsulphate, became present at the medullary region following the longitudinal axis of folium and limiting the forming granular layer, being this substance mainly related with polarity processes by controlling or guiding the growing cones of afferent fibers, which enter massively to cerebellar cortex. It disappeared as myelination progressed. Also from the middle stage of development onward, beside glycosaminoglycans, other polyanionic substances were present at the molecular and granular layer neuropile and at the cytoplasm of some nerve cells. These macromolecules were rather related with nerve cell differentiation and maturation.     

Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4-/- cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4-/- cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4-/- cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4-/- cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.     

Trio Is a Key Guanine Nucleotide Exchange Factor Coordinating Regulation of the Migration and Morphogenesis of Granule Cells in the Developing Cerebellum

Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.     

人体小脑神经元发育的定量观察

目的:研究人体小脑神经元的发育过程。方法:应用体视学方法,对18例不同时期人体小脑组织Golgi染色后进行观察,观测小脑皮质分层出现的时间,观测并计算神经元的数密度、体密度和表面积密度。结果:6月龄时,小脑皮质出现较明显的分子层、蒲肯野细胞层和颗粒层;星形细胞、篮状细胞、蒲肯野细胞、颗粒细胞和高尔基细胞的的数密度随月龄/年龄的增长而减少,体密度和表面积密度随月龄/年龄的增长而增加,但这些减小和增大是不等速的,6-8月龄变化最明显。结论:人体小脑神经元的发育呈现快慢交替、不均速发展,6～8月是小脑神经元发育的重要时期。     

Alterations in Actin-Binding β-Thymosin Expression Accompany Neuronal Differentiation and Migration in Rat Cerebellum

The β₄-and β₁₀-thymosins, recently identified as actin monomer-sequestering proteins, are developmentally regulated in brain. Using specific mRNA and protein probes, we have used in situ hybridization and immunohis-tochemical techniques to investigate the distribution of the β-thymosin mRNAs and their proteins in developing rat cerebellum. Early in postnatal development, both β-thymosin mRNAs were expressed at highest levels in the postmitotic, premigratory granule cells of the external granular layer; expression diminished as granule cells migrated to and differentiated within the developing internal granular layer. In addition, both β-thymosin proteins were present in bundles of cerebellar afferent fibers in the white matter at this time. Throughout the maturation period, both proteins were present in elongating parallel fibers in the upper portion of the molecular layer. Later in cerebellar development, thymosin β₄, but not thymosin β₁₀, was expressed in Golgi epithelial cells and Bergmann processes. Thymosin β₄ was expressed in a small population of cells with microglial morphology scattered throughout the gray and white matter. Thymosin β₁₀ was detected in an even smaller population of glia. Expression of thymosin β₄ and thymosin β₁₀ in premigratory granule cells and in growing neuronal processes is consistent with the possibility that both β-thymosins are involved in the dynamics of actin polymerization during migration and process extension of neurons.