Cerebellar Granule Cell Development Research Articles

Histone bivalency regulates the timing of cerebellar granule cell development.

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

Prenatal and postnatal development of the rat cerebellar granule cells following maternal administration of thyme and tramadol

Prenatal and postnatal development of the rat cerebellar granule cells following maternal administration of thyme and tramadol

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Suppressor of fused controls cerebellum granule cell proliferation by suppressing Fgf8 and spatially regulating Gli proteins.

Cerebellar granule cell (GC) development relies on precise regulation of sonic hedgehog (Shh)-Gli signalling activity, failure of which is associated with motor disorders and medulloblastoma. Mutations in the pathway regulator suppressor of fused (Sufu), which modulates Gli activators and repressors, are linked to cerebellar dysfunction and tumourigenesis. The mechanism by which Sufu calibrates Shh signalling in GCs is unknown. Math1-Cre-mediated deletion of Sufu in mouse GC progenitors (GCPs) demonstrated that Sufu restricts GCP proliferation and promotes cell cycle exit, by promoting expression of Gli3R and suppressing Gli2 levels. Sufu is also required to promote a high threshold of pathway activity in GCPs. Remarkably, central cerebellar lobules are more deleteriously impacted by Sufu deletion, but are less sensitive to downstream genetic manipulations to reduce Gli2 expression or overexpress a Gli3R mimic, compared with anterior lobules. Transcriptome sequencing uncovered new Sufu targets, especially Fgf8, which is upregulated in Sufu-mutant GCPs. We demonstrate that Fgf8 is necessary and sufficient to drive Sufu-mutant GCP proliferation. This study reveals new insights into the spatial and temporal regulation of cerebellar Shh-Gli signalling, while uncovering new targets, such as Fgf8.

Expression of transcription factors and signaling molecules in the cerebellar granule cell development

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) constitute a good model system to investigate proliferation of neural precursors and differentiation of neurons. During development, GCPs proliferate in the outer external granule cell layer (outer EGL) and then exit the cell cycle in the inner EGL to become GCs, which inwardly migrate to the inner granule cell layer (IGL). Misregulation of GCP proliferation or GC differentiation leads to maldevelopment of the cerebellum and the formation of a cerebellar tumor, medulloblastoma. Despite many efforts in this field, the mechanisms underlying GC development remain elusive. In this study, we performed detailed immunostaining in the developing cerebellum, with particular focus on GCPs and GCs, looking at several transcription factors, signaling molecules, cell cycle regulators, some of which are known to regulate neural development. Interestingly, we found distinct distribution patterns of certain proteins within the outer and inner EGL, suggesting the existence of subpopulations of GCPs and GCs in those layers. This study provides a basis for future studies on the cerebellar GC development and medulloblastoma.

Impaired Cerebellar Development in Mice Overexpressing VGF.

VGF nerve growth factor inducible (VGF) is a neuropeptide precursor induced by brain-derived neurotrophic factor and nerve growth factor. VGF is increased in the prefrontal cortex and cerebrospinal fluid in schizophrenia patients. In our previous study, VGF-overexpressing mice exhibited schizophrenia-like behaviors and smaller brain weights. Brain developmental abnormality is one cause of mental illness. Research on brain development is important for discovery of pathogenesis of mental disorders. In the present study, we investigated the role of VGF on cerebellar development. We performed a histological analysis with cerebellar sections of adult and postnatal day 3 mice by Nissl staining. To investigate cerebellar development, we performed immunostaining with antibodies of immature and mature granule cell markers. To understand the mechanism underlying these histological changes, we examined MAPK, Wnt, and sonic hedgehog signaling by Western blot. Finally, we performed rotarod and footprint tests using adult mice to investigate motor function. VGF-overexpressing adult mice exhibited smaller cerebellar sagittal section area. In postnatal day 3 mice, a cerebellar sagittal section area reduction of the whole cerebellum and external granule layer and a decrease in the number of mature granule cells were found in VGF-overexpressing mice. Additionally, the number of proliferative granule cell precursors was lower in VGF-overexpressing mice. Phosphorylation of Trk and Erk1 were increased in the cerebellum of postnatal day 3 VGF-overexpressing mice. Adult VGF-overexpressing mice exhibited motor disability. All together, these findings implicate VGF in the development of cerebellar granule cells via promoting MAPK signaling and motor function in the adult stage.

Recent advances in understanding the mechanisms of cerebellar granule cell development and function and their contribution to behavior.

The cerebellum is the focus of an emergent series of debates because its circuitry is now thought to encode an unexpected level of functional diversity. The flexibility that is built into the cerebellar circuit allows it to participate not only in motor behaviors involving coordination, learning, and balance but also in non-motor behaviors such as cognition, emotion, and spatial navigation. In accordance with the cerebellum’s diverse functional roles, when these circuits are altered because of disease or injury, the behavioral outcomes range from neurological conditions such as ataxia, dystonia, and tremor to neuropsychiatric conditions, including autism spectrum disorders, schizophrenia, and attention-deficit/hyperactivity disorder. Two major questions arise: what types of cells mediate these normal and abnormal processes, and how might they accomplish these seemingly disparate functions? The tiny but numerous cerebellar granule cells may hold answers to these questions. Here, we discuss recent advances in understanding how the granule cell lineage arises in the embryo and how a stem cell niche that replenishes granule cells influences wiring when the postnatal cerebellum is injured. We discuss how precisely coordinated developmental programs, gene expression patterns, and epigenetic mechanisms determine the formation of synapses that integrate multi-modal inputs onto single granule cells. These data lead us to consider how granule cell synaptic heterogeneity promotes sensorimotor and non-sensorimotor signals in behaving animals. We discuss evidence that granule cells use ultrafast neurotransmission that can operate at kilohertz frequencies. Together, these data inspire an emerging view for how granule cells contribute to the shaping of complex animal behaviors.

Meis1 Coordinates Cerebellar Granule Cell Development by Regulating Pax6 Transcription, BMP Signaling and Atoh1 Degradation.

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. Here, we report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in the GC lineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors.SIGNIFICANCE STATEMENT We report that myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse granule cell (GC) development. Here, we show Meis1 is expressed in GC precursors (GCPs) and GCs during development. Our knock-down and conditional knock-out (cKO) experiments and in vitro assays revealed that Meis1 is required for proper cerebellar structure formation and for Pax6 transcription in GCPs and GCs. The Meis1-Pax6 cascade regulates the morphology of GCs. In the cKO cerebella, Smad proteins and bone morphogenic protein (BMP) signaling are severely reduced and Atoh1-expressing GCPs are ectopically detected in the inner external granule layer. These findings suggest that Meis1 regulates degradation of Atoh1 via BMP signaling, contributing to GC differentiation in the inner EGL, and should provide understanding into GC development.

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.

Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos.

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice.

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti-CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive-like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.

Activation of Liver X Receptor Is Protective Against Ethanol-Induced Developmental Impairment of Bergmann Glia and Purkinje Neurons in the Mouse Cerebellum

Cerebellar Purkinje cell and granule cell development are coordinated by Bergmann glia, and are particularly sensitive to ethanol (EtOH) exposure. The liver X receptor (LXR) plays important roles in Bergmann glial development. However, the effect of LXR activation on EtOH-mediated impairment of Bergmann glia and subsequently on Purkinje cell dendritogenesis remains undetermined. Therefore, using immunohistochemistry, quantitative real-time PCR and Western blot, we tested the possible protection of LXR agonist T0901317 (T0) on Bergmann glia and Purkinje cell dendritogenesis in mice exposed to ethanol. Results showed that a brief exposure of EtOH on postnatal day (PD 5) significantly decreased the average body weight of mice at PD 6 without alteration in the brain weight. In EtOH-exposed mice, the number of migrating granule cells in the molecular layer was significantly decreased, and this effect was attenuated by pretreatment of T0. EtOH exposure also resulted in the significant reduction of calbindin-labeled Purkinje cells, their maximum dendrite length, and impairment of Purkinje cell dendritogenesis. Furthermore, EtOH induced the activation of microglia in the Purkinje cell layer and impaired the development of Bergmann glia. However, pretreatment of T0 effectively blocked all of these responses. These responses were found to be mediated by the inhibition of upregulated levels of β-catenin and transcription factor LEF1 in the cerebellum. Overall, the results suggest that activating LXRs on postnatal mice exposed to EtOH is protective to Bergmann glia, and thus may play a critical role in preventing EtOH-induced defects during cerebellar development.

Experimental Sey mouse chimeras reveal the developmental deficiencies of Pax6-null granule cells in the postnatal cerebellum

Pax6 has been implicated in cerebellar granule cell development, however the neonatal lethality of the Sey/Sey mutant has precluded a more detailed study of this late developing neuronal type. In this study we use experimental mouse chimeras made from wildtype and Pax6-null embryos to circumvent early lethality and assess the developmental potential of mutant cells in the construction of the cerebellum. We have identified the granule cell as a direct target of mutant gene action, with glia and Purkinje cells being affected in what is largely a non-cell autonomous manner.Most dramatically, in postnatal day 21 (P21) chimeras, mutant cells are largely absent in the anterior and posterior cerebellum while present in central lobules, but amidst disorganized cerebellar architecture. Analysis of P0/1 and P10 chimeras demonstrates a profound temporally based defect where mutant cells colonize the anterior and posterior EGL but fail to migrate to the IGL. Mutant granule cells in the central lobules can reach the IGL in an abnormal manner, with large streams of cells forming raphes through the molecular layer.These studies provide new insights into the role of Pax6 in postnatal cerebellar development that pinpoint the granule cell as an intrinsic target of the mutant gene and key events in the life of the developing granule cell that depend upon normal Pax6 expression.

Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development

Semaphorins and Plexins are cognate ligand-receptor families that regulate important steps during nervous system development. The Plexin-B2 receptor is critically involved in neural tube closure and cerebellar granule cell development, however, its specific ligands have only been suggested by in vitro studies. Here, we show by in vivo and in vitro analyses that the two Semaphorin-4 family members Sema4C and Sema4G are likely to be in vivo ligands of Plexin-B2. The Sema4C and Sema4G genes are expressed in the developing cerebellar cortex, and Sema4C and Sema4G proteins specifically bind to Plexin-B2 expressing cerebellar granule cells. To further elucidate their in vivo function, we have generated and analyzed Sema4C and Sema4G knockout mouse mutants. Like Plexin-B2−/− mutants, Sema4C−/− mutants reveal exencephaly and subsequent neonatal lethality with partial penetrance. Sema4C−/− mutants that bypass exencephaly are viable and fertile, but display distinctive defects of the cerebellar granule cell layer, including gaps in rostral lobules, fusions of caudal lobules, and ectopic granule cells in the molecular layer. In addition to neuronal defects, we observed in Sema4C−/− mutants also ventral skin pigmentation defects that are similar to those found in Plexin-B2−/− mutants. The Sema4G gene deletion causes no overt phenotype by itself, but combined deletion of Sema4C and Sema4G revealed an enhanced cerebellar phenotype. However, Sema4C/Sema4G double mutants showed overall less severe cerebellar phenotypes than Plexin-B2−/− mutants, indicating that further ligands of Plexin-B2 exist. In explant cultures of the developing cerebellar cortex, Sema4C promoted migration of cerebellar granule cell precursors in a Plexin-B2-dependent manner, supporting the model that a reduced migration rate of granule cell precursors is the basis for the cerebellar defects of Sema4C−/− and Sema4C/Sema4G mutants.

Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival

Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.

BDNF-Mediated Cerebellar Granule Cell Development Is Impaired in Mice Null for CaMKK2 or CaMKIV

The Ca(2+)/calmodulin-activated kinases CaMKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca(2+). To address the biological functions of Ca(2+) signaling via these kinases during brain development, we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here, we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of cAMP response element-binding protein (CREB) correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons. We show in vivo and in vitro that the absence of either CaMKK2 or CaMKIV disrupts the ability of developing cerebellar granule cells in the external granule cell layer to cease proliferation and begin migration to the internal granule cell layer. Furthermore, loss of CaMKK2 or CaMKIV results in decreased CREB phosphorylation (pCREB), Bdnf exon I and IV-containing mRNAs, and brain-derived neurotrophic factor (BDNF) protein in cerebellar granule cell neurons. Reexpression of CaMKK2 or CaMKIV in granule cells that lack CaMKK2 or CaMKIV, respectively, restores pCREB and BDNF to wild-type levels and addition of BDNF rescues granule cell migration in vitro. These results reveal a previously undefined role for a CaMKK2/CaMKIV cascade involved in cerebellar granule cell development and show specifically that Ca(2+)-dependent regulation of BDNF through CaMKK2/CaMKIV is required for this process.

A Molecular Program for Contralateral Trajectory: Rig-1 Control by LIM Homeodomain Transcription Factors

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.

The effects of glutamate receptor antagonists on cerebellar granule cell survival and development

N-Methyl- d-aspartate (NMDA) receptor stimulation promotes neuronal survival and differentiation under both in vitro and in vivo conditions. We studied the effects of various NMDA receptor antagonists acting at different NMDA receptor binding sites and non-NMDA receptor antagonists on the development and survival of cerebellar granule cell (CGC) culture. Only three of the drugs tested induced neurotoxicity—MK-801 (non-competitive NMDA channel blocking antagonist), ifenprodil (an antagonist of the NR2B site and polyamine site of the NMDA receptor) and L-701.324 (full antagonist at glycine site), while CGP-37849 (a competitive NMDA antagonist), (+)-HA-966 (a partial agonist of the glycine site of the NMDA receptor), and NBQX (a competitively acting AMPA receptor antagonist) were not toxic at any concentration (1–100 μM) used. Among these drugs, only MK-801 was toxic for the immature CGC on second day in vitro (2DIV), and toxicity was diminished parallel to the neuronal maturation. In more mature neurons (7DIV), MK-801 demonstrated some neuroprotection, which diminished spontaneously occurring neuronal death in culture. Neither NMDA nor glutamate were able to prevent the neurotoxic effect of MK-801 at 2DIV. MK-801, ifenprodil and L-701.324 induced DNA fragmentation on 2DIV in CGC culture measured by the TUNEL method. The BOC-D-FMK, the universal caspase inhibitor, completely reversed MK-801-induced DNA fragmentation, suggesting an apoptotic pathway of MK-801-induced cell death. Neurite outgrowth as a characteristic feature of the development of CGC was diminished after treatment with MK-801, ifenprodil and L-701.324. In conclusion, the results of the present study demonstrate that only nonselective channel blocker MK-801 decreases cell viability, induces apoptosis and inhibits neurite outgrowth of CGC in a development-dependent manner.

Characterization of a cerebellar granule progenitor cell line, E tC.1, and its responsiveness to 17-β-estradiol

Mouse cerebellar development occurs at late embryonic stages and through the first few weeks of postnatal life. Hormones such as 17-β-estradiol (E2) have been implicated in cerebellar development, through the expression of E2 receptors (ER). However, the role of E2 in the development and function of cerebellar neurons has yet to be fully elucidated. To gain insight into E2's actions on the developing cerebellum, we characterized a cloned neuronal cell line, E tC.1, derived from late embryonic cerebellum for its neural properties and responsiveness to E2. Our results revealed that E tC.1 cells express markers characteristic of neural progenitor cells such as Nestin, Musashi, and Doublecortin (DCX), and of the granule cell lineage such as Math1 and Zipro1. The ER alpha and beta (ERα and ERβ) were also identified in this cell line. Functionality of ERs was verified using an Estrogen Response Element (ERE)-Luciferase reporter plasmid. E2 modulated ERα, FMRP, and IL-6, which were expressed in these cells. However, E2 did not induce changes in neural proteins nor induce maturation of E tC.1 cells. CREB and ERK 1/2 protein kinases were not modulated by E2 either. Interestingly, E tC.1 expressed active p450 Aromatase (P450arom), which was confirmed by the aromatization of androstenedione (AD) to E2 and other estrogen metabolites. Collectively, our results show that the E tC.1 cell line may serve as a model to study early development of cerebellar progenitor granule cells, and their responsiveness to E2.

Nuclear Factor I Coordinates Multiple Phases of Cerebellar Granule Cell Development via Regulation of Cell Adhesion Molecules

A central question is how various stages of neuronal development are integrated as a differentiation program. Here we show that the nuclear factor I (NFI) family of transcriptional regulators is expressed and functions throughout the postmitotic development of cerebellar granule neurons (CGNs). Expression of an NFI dominant repressor in CGN cultures blocked axon outgrowth and dendrite formation and decreased CGN migration. Inhibition of NFI transactivation also disrupted extension and fasciculation of parallel fibers as well as CGN migration to the internal granule cell layer in cerebellar slices. In postnatal day 17 Nfia-deficient mice, parallel fibers were greatly diminished and disoriented, CGN dendrite formation was dramatically impaired, and migration from the external germinal layer (EGL) was retarded. Axonal marker expression also was disrupted within the EGL of embryonic day 18 Nfib-null mice. NFI regulation of axon extension was observed under conditions of homotypic cell contact, implicating cell surface proteins as downstream mediators of its actions in CGNs. Consistent with this, the cell adhesion molecules ephrin B1 and N-cadherin were identified as NFI gene targets in CGNs using inhibitor and Nfi mutant analysis as well as chromatin immunoprecipitation. Functional inhibition of ephrin B1 or N-cadherin interfered with CGN axon extension and guidance, migration, and dendritogenesis in cell culture as well as in situ. These studies define NFI as a key regulator of postmitotic CGN development, in particular of axon formation, dendritogenesis, and migratory behavior. Furthermore, they reveal how a single transcription factor family can control and integrate multiple aspects of neuronal differentiation through the regulation of cell adhesion molecules.