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

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

Primary Cilia in the Murine Cerebellum and in Mutant Models of Medulloblastoma

Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.     

Bergmann Glia Function in Granule Cell Migration During Cerebellum Development

Granule cell migration influences the laminar structure of the cerebellum and thereby affects cerebellum function. Bergmann glia are derived from radial glial cells and aid in granule cell radial migration by providing a scaffold for migration and by mediating interactions between Bergmann glia and granule cells. In this review, we summarize Bergmann glia characteristics and the mechanisms underlying the effect of Bergmann glia on the radial migration of granule neurons in the cerebellum. Furthermore, we will focus our discussion on the important factors involved in glia-mediated radial migration so that we may elucidate the possible mechanistic pathways used by Bergmann glia to influence granule cell migration during cerebellum development.     

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

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

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.     

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.     

The enzymehistochemical maturation of the cerebellar cortex of rats after early postnatal X-irradiation

Summary The enzymehistochemical maturation of the cerebellar cortex was studied at 8, 16 and 30 days in rats whose cerebellar area was irradiated with 1,000 r at 2 days of age, and was compared with that in normal animals.After irradiation, the granule cells and basket and stellate neurones are absent due to lethal damage to their precursors. Only large neurones, glial cells and bloodvessels are present in a disorderly arrangement; the laminar pattern of the cortex and specialized structures such as the glomeruli do not develop.Enzymehistochemically the absence of specialized structures such as the molecular layer and the glomeruli is reflected in a uniformly strong diffuse activity of most of the dehydrogenases studied. AMPase-activity shows a strong positive correlation with the presence of granule cells. High subcortical activity of AChE in the posterior half of the vermis is presumed to be located within mossy fibres which lack their normal goal: the granule cells. Capillaries in the irradiated cortex show a close spatial relation to Purkinje cells. The development of the cytoplasmic organization of the Purkinje cells as demonstrated by TPPase and AcPase activities proceeds in a normal way. Some enzymes can be used as markers of certain cell-types in the irradiated cortex: ICDH and G6PDH of Bergmann glial cells, AcPase of Golgi cells.It is suggested that enzymehistochemical studies of experimentally disturbed development may lead to better understanding of normal maturation.     

The localization of mercury and metallothionein in the cerebellum of rats experimentally exposed to methylmercury

Summary Rats were dosed with methylmercuric chloride, either by gastric gavage (5 × 10 mg kg^-1 body weight over a 15-day period), or in their drinking water (20 mg methylmercuric chloride l^–1 for 14 or 42 days). Localization of mercury within the cerebellum was performed with a silver physical development technique, and metallothionein with dinitrophenyl hapten-sandwich immunohistochemistry. Mercury was detected in structurally undamaged Purkinje neurones and adjacent Bergmann glial cells; no mercury was detected in granule cells even though these small cells nearest the Purkinje layer had a high incidence of pyknotic nuclei. In general, metallothionein was detected mainly in Bergmann glial cells, Purkinje cells, astrocytes and glial cells of white matter; no metallothionein was detected in granule cells. We hypothesized that the resistance of Purkinje cells to methylmercuric chloride reflects their ability to transform organic mercurials to inorganic mercury that, in turn, induces the synthesis of radical-scavenging metallothionein molecules.     

Suppression of death receptor signaling in cerebellar Purkinje neurons protects neighboring granule neurons from apoptosis via an insulin-like growth factor I-dependent mechanism

Neuronal apoptosis contributes to the progression of neurodegenerative disease. Primary cerebellar granule neurons are an established in vitro model for investigating neuronal death. After removal of serum and depolarizing potassium, granule neurons undergo apoptosis via a mechanism that requires intrinsic (mitochondrial) death signals; however, the role of extrinsic (death receptor-mediated) signals is presently unclear. Here, we investigate involvement of death receptor signaling in granule neuron apoptosis by expressing adenoviral, AU1-tagged, dominant-negative Fas-associated death domain (Ad-AU1-deltaFADD). Ad-AU1-deltaFADD decreased apoptosis of granule neurons from 65 +/- 5 to 27 +/- 2% (n = 7, p < 0.01). Unexpectedly, immunocytochemical staining for AU1 revealed that <5% of granule neurons expressed deltaFADD. In contrast, deltaFADD was expressed in >95% of calbindin-positive Purkinje neurons ( approximately 2% of the cerebellar culture). Granule neurons in proximity to deltaFADD-expressing Purkinje cells demonstrated markedly increased survival. Both granule and Purkinje neurons expressed insulin-like growth factor-I (IGF-I) receptors, and deltaFADD-mediated survival of granule neurons was inhibited by an IGF-I receptor blocking antibody. These results demonstrate that the selective suppression of death receptor signaling in Purkinje neurons is sufficient to rescue neighboring granule neurons that depend on Purkinje cell-derived IGF-I. Thus, the extrinsic death pathway has a profound but indirect effect on the survival of cerebellar granule neurons.     

Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.     

The small GTPases RhoA and Rac1 regulate cerebellar development by controlling cell morphogenesis,migration and foliation

The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology.     

Alterations in the Development of Rat Cerebellum and Impaired Behavior of Juvenile Rats after Neonatal 6-OHDA Treatment

The effects of neonatal systemic administration of the neurotoxin 6-hydroxydopamine (6-OHDA) on cerebellum development and behavior were studied in juvenile rats. The methods employed were immunohistochemistry, in situ hybridization, ligand binding, and behavioral testing. The results revealed, for the first time, that 6-OHDA treatment alters Bergmann glial cells and reduced the expression GABA_A receptor subtypes α1 and α6 especially in granule cells. The Bergmann glial cells were abnormally located and structurally different (e.g., no intimate associations with Purkinje cells). Significant microglial activation was also observed. The animals showed impairment in behavior, especially in their orientation to a novel environment. Recent data on neuron–glia interactions support the conclusion that the observed structural changes in Bergmann glia and granular neurons disrupted the normal functioning of the Purkinje cells which then in turn resulted in the impaired sensory-motor coordination at least in juvenile rats. This paper is a summary of previously published work and some recent data in this field obtained at our laboratory. Special issue dedicated to Dr. Simo S. Oja     

Interactions between cerebellar Purkinje cells and their associated astrocytes

Some neurons, including cerebellar Purkinje cells, are completely ensheathed by astrocytes. When granule cell neurons and functional glia were eliminated from newborn mouse cerebellar cultures by initial exposure to a DNA synthesis inhibitor, Purkinje cells lacked glial sheaths and there was a tremendous sprouting of Purkinje cell recurrent axon collaterals, terminals of which hyperinnervated Purkinje cell somata, including persistent somatic spines, and formed heterotypical synapses with Purkinje cell dendritic spines, sites usually occupied by parallel fiber (granule cell axon) terminals. Purkinje cells in such preparations failed to develop complex spikes when recorded from intracellularly, and their membrane input resistances were low, making them less sensitive to inhibitory input. If granule cells and oligodendrocytes were eliminated, but astrocytes were not compromised, sprouting of recurrent axon collaterals occurred and their terminals projected to Purkinje cell dendritic spines, but the Purkinje cells had astrocytic sheaths, their somata were not hyperinnervated, the somatic spines had disappeared, complex spike discharges predominated, and membrane input resistance was like that of Purkinje cells in untreated control cultures. When cerebellar cultures without granule cells and glia were transplanted with granule cells and/or glia from another source, a series of changes occurred that included stripping of excess Purkinje cell axosomatic synapses by astrocytic processes, reduction of heterotypical axospinous synapses in the presence of astrocytes, disappearance of Purkinje cell somatic spines with astrocytic ensheathment, and proliferation of Purkinje cell dendritic spines after the introduction of astrocytes. Dendritic spine proliferation was followed by formation of homotypical axospinous synapses when granule cells were present or persistence as unattached spines in the absence of granule cells. The results of these studies indicate that astrocytes regulate the numbers of Purkinje cell axosomatic and axospinous synapses, induce Purkinje cell dendritic spine proliferation, and promote the structural and functional maturation of Purkinje cells.     

Sonic hedgehog regulates the growth and patterning of the cerebellum.

The molecular bases of brain development and CNS malignancies remain poorly understood. Here we show that Sonic hedgehog (Shh) signaling controls the development of the cerebellum at multiple levels. SHH is produced by Purkinje neurons, it is required for the proliferation of granule neuron precursors and it induces the differentiation of Bergmann glia. Blocking SHH function in vivo results in deficient granule neuron and Bergmann glia differentiation as well as in abnormal Purkinje neuron development. Thus, our findings provide a molecular model for the growth and patterning of the cerebellum by SHH through the coordination of the development of cortical cerebellar cell types. In addition, they provide a cellular context for medulloblastomas, childhood cancers of the cerebellum.     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.     

Is laminin-1 a guidance cue for cerebellar granule cell migration?

Laminin-1 is a glycoprotein found in the basement membrane of many tissues. In the cerebellum of rodents, it has also been localized along Bergmann glial fibers, where it is thought to be involved in promoting granule cell migration by enhancing adhesion and neurite outgrowth along these fibers. Recent reports, however, indicate that laminin-1 is not present on Bergmann fibers, but instead is associated with blood vessels and meninges. Furthermore, attempts to block granule cell migration using antibodies against laminin-1 have yielded conflicting results. In this report, we provide further evidence that laminin-1 is associated exclusively with blood vessels and meninges in the cerebellum of postnatal rats. In addition, we show that adhesion and neurite outgrowth of granule cells was impeded on laminin-coated surfaces. In fact, cerebellar cells dramatically and consistently avoided laminin-1 regions of patterned surfaces. Cells did adhere to laminin regions if it was coadsorbed with polylysine or tested in serum-containing medium. Avoidance of laminin-1 regions in culture was not, however, blocked by pretreatment with laminin-1 antibodies. By comparison, mouse neuroblastoma cells adhered preferentially to laminin-1 regions in serum-free medium, a response which was blocked by laminin-1 antibodies. These results indicate that laminin-1 is not involved in granule cell migration along Bergmann glial fibers. Instead, they suggest that laminin-1 may function as a repulsive guidance cue preventing granule cells from following inappropriate pathways during development. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 72–84, 1997     

The monolayer formation of Bergmann glial cells is regulated by Notch/RBP-J signaling

The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia.