External Granule Layer Research Articles

Regulation of primary cilia disassembly through HUWE1-mediated TTBK2 degradation plays a crucial role in cerebellar development and medulloblastoma growth

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.

Impact of varying maternal dietary folate intake on cerebellar cortex histomorphology and cell density in offspring rats.

The cerebellum has a long, protracted developmental period that spans from the embryonic to postnatal periods; as a result, it is more sensitive to intrauterine and postnatal insults like nutritional deficiencies. Folate is crucial for foetal and early postnatal brain development; however, its effects on cerebellar growth and development are unknown. The aim of this study was to examine the effects of maternal folate intake on the histomorphology and cell density of the developing cerebellum. Twelve adult female rats (rattus norvegicus) were randomly assigned to one of four premixed diet groups: standard (2mg/kg), folate-deficient (0mg/kg), folate-supplemented (8mg/kg) or folate supra-supplemented (40 mg/kg). The rats started their diets 14 days before mating and consumed them throughout pregnancy and lactation. On postnatal days 1, 7, 21 and 35, five pups from each group were sacrificed, and their brains were processed for light microscopic analysis. Histomorphology and cell density of the external granule, molecular, Purkinje and internal granule layers were obtained. The folate-deficient diet group had smaller, dysmorphic cells and significantly lower densities of external granule, molecular, Purkinje and internal granule cells. Although the folate-enriched groups had greater cell densities than the controls, the folate-supplemented group had considerably higher cell densities than the supra-supplemented group. The folate supra-supplemented group had ectopic Purkinje cells in the internal granule cell layer. These findings imply that a folate-deficient diet impairs cellular growth and reduces cell density in the cerebellar cortex. On the other hand, folate supplementation increases cell densities, but there appears to be an optimal dose of supplementation since excessive folate levels may be detrimental.

Tracking of Internal Granular Progenitors Responding to Valproic Acid in the Cerebellar Cortex of Infant Ferrets.

Internal granular progenitors (IGPs) in the developing cerebellar cortex of ferrets differentiate towards neural and glial lineages. The present study tracked IGPs that proliferated in response to valproic acid (VPA) to determine their fate during cerebellar cortical histogenesis. Ferret kits were used to administer VPA (200 μg/g body weight) on postnatal days 6 and 7. EdU and BrdU were injected on postnatal days 5 and 7, respectively, to label the post-proliferative and proliferating cells when exposed to VPA. At postnatal day 20, when the external granule layer was most expanded, EdU- and BrdU-single-labeled cells were significantly denser in the inner granular layer of VPA-exposed ferrets than in controls. No EdU- or BrdU-labeling was found in Purkinje cells and molecular layer interneurons. Significantly higher percentages of NeuN and Pax6 immunostaining in VPA-exposed ferrets revealed VPA-induced differentiation of IGPs towards granular neurons in BrdU-single-labeled cells. In contrast, both EdU- and BrdU-single-labeled cells exhibited significantly greater percentages of PCNA immunostaining, which appeared in immature Bergman glia, in the internal granular layer of VPA-exposed ferrets. These findings suggest that VPA affects the proliferation of IGPs to induce differentiative division towards granular neurons as well as post-proliferative IGPs toward differentiation into Bergmann glia.

Cell division angle predicts the level of tissue mechanics that tune the amount of cerebellar folding.

Modeling has led to proposals that the amount of neural tissue folding is set by the level of differential expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here, we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a distinct critical period during which the folding amount diverges between the two strains. In this period, regional changes in the level of differential expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL varies regionally during the critical period alongside corresponding changes in wavelength. The number of SHH-expressing Purkinje cells predicts the folding amount, but the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential expansion and the thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential expansion along the perimeter and the thickness of the EGL are regionally tuned to set the amount and wavelength of folding.

Syntaxin-1 is necessary for UNC5A-C/Netrin-1-dependent macropinocytosis and chemorepulsion.

Brain connectivity requires correct axonal guidance to drive axons to their appropriate targets. This process is orchestrated by guidance cues that exert attraction or repulsion to developing axons. However, the intricacies of the cellular machinery responsible for the correct response of growth cones are just being unveiled. Netrin-1 is a bifunctional molecule involved in axon pathfinding and cell migration that induces repulsion during postnatal cerebellar development. This process is mediated by UNC5 homolog receptors located on external granule layer (EGL) tracts. Biochemical, imaging and cell biology techniques, as well as syntaxin-1A/B (Stx1A/B) knock-out mice were used in primary cultures and brain explants. Here, we demonstrate that this response is characterized by enhanced membrane internalization through macropinocytosis, but not clathrin-mediated endocytosis. We show that UNC5A, UNC5B, and UNC5C receptors form a protein complex with the t-SNARE syntaxin-1. By combining botulinum neurotoxins, an shRNA knock-down strategy and Stx1 knock-out mice, we demonstrate that this SNARE protein is required for Netrin1-induced macropinocytosis and chemorepulsion, suggesting that Stx1 is crucial in regulating Netrin-1-mediated axonal guidance.

Syndecan-3 regulates the time of transition from cell cycle exit to initial differentiation stage in mouse cerebellar granule cell precursors

To analyze the role of syndecan-3 (SDC3), a heparan sulfate proteoglycan, in cerebellum development, we examined the effect of SDC3 on the transition from cell cycle exit to the initial differentiation stage of cerebellar granule cell precursors (CGCPs). First, we examined SDC3 localization in the developing cerebellum. SDC3 was mainly localized to the inner external granule layer where the transition from the cell cycle exit to the initial differentiation of CGCPs occurs. To examine how SDC3 regulates the cell cycle exit of CGCPs, we performed SDC3-knockdown (SDC3-KD) and -overexpression (Myc-SDC3) assays using primary CGCPs. SDC3-KD significantly increased the ratio of p27Kip1+ cells to total cells at day 3 in vitro (DIV3) and 4, but Myc-SDC3 reduced that at DIV3. Regarding the cell cycle exit efficiency using 24 h-labelled bromodeoxyuridine (BrdU) and a marker of cell cycling, Ki67, SDC3-KD significantly increased cell cycle exit efficiency (Ki67-; BrdU+ cells/BrdU+ cells) in primary CGCP at DIV4 and 5, but Myc-SDC3 reduced that at DIV4 and 5. However, SDC3-KD and Myc-SDC3 did not affect the efficiency of the final differentiation from CGCPs to granule cells at DIV3–5. Furthermore, the ratio of CGCPs in the cell cycle exiting stage to total cells, identified by initial differentiation markers TAG1 and Ki67 (TAG1+; Ki67+ cells), was considerably decreased by SDC3-KD at DIV4, but increased by Myc-SDC3 at DIV4 and 5. Altogether, these results indicate that SDC3 regulates the timing of the transition from the cell cycle exit stage to the initial differentiation stage of CGCP.

Parthenolide promotes expansion of Nestin+ progenitor cells via Shh modulation and contributes to post-injury cerebellar replenishment.

Background: Regeneration of injuries occurring in the central nervous system is extremely difficult. Studies have shown that the developing cerebellum can be repopulated by a group of Nestin-expressing progenitors (NEPs) after irradiation injury, suggesting that modulating the mobilization of NEPs is beneficial to promoting nerve regeneration. To date, however, effect of exogenous pharmaceutical agonist on NEPs mobilization remains unknown. Parthenolide (PTL), a sesquiterpene lactone isolated from shoots of feverfew. Although it has been shown to possess several pharmacological activities and is considered to have potential therapeutic effects on the regeneration of peripheral nerve injury, its efficacy in promoting central nervous system (CNS) regeneration is unclear. In this study, we aimed to elucidate the role and possible mechanism of PTL on regeneration in injured CNS after irradiation using a developing cerebellum model. Methods: We investigated the radioprotective effects of PTL on the developing cerebellum by immunoblotting as well as immunofluorescence staining and ROS detection in vivo and in vitro experiments, and then determined the effects of PTL on NEPs in Nestin CFP and Nestin GFP fluorescent mice. Inducible lineage tracing analysis was used in Nestin-CreERT2×ROSA26-LSL YFP mice to label and track the fate of NEPs in the cerebellum after irradiation. Combined with cell biology and molecular biology techniques to determine changes in various cellular components in the cerebellum and possible mechanisms of PTL on NEPs mobilization in the injured developing cerebellum. Results: We found that PTL could attenuate radiation-induced acute injury of granule neuron progenitors (GNPs) in irradiated cerebellar external granule layer (EGL) by alleviating apoptosis through regulation of the cells' redox state. Moreover, PTL increased cerebellar Shh production and secretion by inhibiting the PI3K/AKT pathway, thus promoting expansion of NEPs, which is the compensatory replenishment of granule neurons after radiation damage. Conclusion: Collectively, our results indicate that activation and expansion of NEPs are critical for regeneration of the injured cerebellum, and that PTL is a promising drug candidate to influence this process.

Rac-deficient cerebellar granule neurons die before they migrate to the internal granule layer

Granule neurons are the most common cell type in the cerebellum. They are generated in the external granule layer and migrate inwardly, forming the internal granule layer. Small Rho GTPases play various roles during development of the nervous system and may be involved in generation, differentiation and migration of granule neurons. We deleted Rac1, a member of small Rho GTPases, by GFAP-Cre driver in cerebellar granule neurons and Bergmann glial cells. Rac1flox/flox; Cre mice showed impaired migration and slight reduction in the number of granule neurons in the internal granule layer. Deletion of both Rac1 and Rac3 resulted in almost complete absence of granule neurons. Rac-deficient granule neurons differentiated into p27 and NeuN-expressing post mitotic neurons, but died before migration to the internal granule layer. Loss of Rac3 has little effect on granule neuron development. Rac1flox/flox; Rac3+/−; Cre mice showed intermediate phenotype between Rac1flox/flox; Cre and Rac1flox/flox; Rac3−/−; Cre mice in both survival and migration of granule neurons. Rac3 itself seems to be unimportant in the development of the cerebellum, but has some roles in Rac1-deleted granule neurons. Conversely, overall morphology of Rac1+/flox; Rac3−/−; Cre cerebella was normal. One allele of Rac1 is therefore thought to be sufficient to promote development of cerebellar granule neurons.

P75NTR prevents the onset of cerebellar granule cell migration via RhoA activation.

Neuronal migration is one of the fundamental processes during brain development. Several neurodevelopmental disorders can be traced back to dysregulated migration. Although substantial efforts have been placed in identifying molecular signals that stimulate migration, little is known about potential mechanisms that restrict migration. These restrictive mechanisms are essential for proper development since it helps coordinate the timing for each neuronal population to arrive and establish proper connections. Moreover, preventing migration away from a proliferative niche is necessary in maintaining a pool of proliferating cells until the proper number of neuronal progenitors is attained. Here, using mice and rats, we identify an anti-migratory role for the p75 neurotrophin receptor (p75NTR) in cerebellar development. Our results show that granule cell precursors (GCPs) robustly express p75NTR in the external granule layer (EGL) when they are proliferating during postnatal development, however, they do not express p75NTR when they migrate either from the rhombic lip during embryonic development or from the EGL during postnatal development. We show that p75NTR prevented GCP migration by maintaining elevated levels of active RhoA. The expression of p75NTR was sufficient to prevent the migration of the granule cells even in the presence of BDNF (brain-derived neurotrophic factor), a well-established chemotactic signal for this cell population. Our findings suggest that the expression of p75NTR might be a critical signal that stops and maintains the GCPs in the proliferative niche of the EGL, by promoting the clonal expansion of cerebellar granule neurons.

Endothelial Rbpj Is Required for Cerebellar Morphogenesis and Motor Control in the Early Postnatal Mouse Brain.

Intercellular influences are necessary for coordinated development and function of vascular and neural components in the brain. In the early postnatal period after birth, the mammalian cerebellum undergoes extensive morphogenesis - developing its characteristic lobules, organizing its diverse cell types into defined cellular layers, and establishing neural circuits that support cerebellar function, such as coordinated movement. In parallel, the cerebellar vasculature undergoes extensive postnatal growth and maturation, keeping pace with the expanding neural compartment. Endothelial deletion of Rbpj leads to neurovascular abnormalities in mice, including arteriovenous (AV) shunts that supplant capillaries and instead direct high-pressure/high-flow arterial blood directly to veins. Gross and histopathological cerebellar abnormalities, associated with these Rbpj-mediated brain AV malformations (AVMs), led to our hypothesis that early postnatal morphogenesis and lamination of cerebellum was perturbed in mice harboring endothelial Rbpj deficiency from birth. Here, we show that endothelial Rbpj-mutant mice developed enlarged vascular malformations on the cerebellar surface, by 2-week post-Rbpj deletion. In addition, outgrowth of cerebellar lobules was impaired through decreased cell proliferation, but not increased apoptosis, in the external granule layer. Molecular layer thickness was reduced, and the Purkinje layer was affected, by decreased Purkinje cell number, primary dendrite length, and dendritic arbor density. Endothelial deletion of Rbpj also led to impaired motor behaviors, consistent with abnormal cerebellar morphogenesis and lamination. Thus, our data suggest that Rbpj is required, in early postnatal vascular endothelium, to ensure proper cerebellar outgrowth, morphogenesis, and function in mice.

Three dimensional reconstruction of the mouse cerebellum in Hedgehog-driven medulloblastoma models to identify Norrin-dependent effects on preneoplasia

Spontaneous mouse models of medulloblastoma (MB) offer a tractable system to study malignant progression in the brain. Mouse Sonic Hedgehog (Shh)-MB tumours first appear at postnatal stages as preneoplastic changes on the surface of the cerebellum, the external granule layer (EGL). Here we compared traditional histology and 3DISCO tissue clearing in combination with light sheet fluorescence microscopy (LSFM) to identify and quantify preneoplastic changes induced by disrupting stromal Norrin/Frizzled 4 (Fzd4) signalling, a potent tumour inhibitory signal in two mouse models of spontaneous Shh-MB. We show that 3DISCO-LSFM is as accurate as traditional histology for detecting Norrin/Fzd4-associated changes in PNL formation in Ptch+/− mice and EGL hyperplasia in Neurod2-SmoA1+/− mice. Moreover, we show that the anti-tumour effect of Norrin/Fzd4 signalling is restricted to the posterior region of the cerebellum and is characterized by defective neural progenitor migration away from the EGL. In conclusion, 3DISCO-LSFM is a valid way to monitor tumour initiation events in mouse MB models and reveals an unanticipated regional restriction of stromal signalling in constraining tumour initiation.

Pleiotrophin, nitric oxide and glutamate AMPA receptors in chick cerebellum morphogenesis.

Avian cerebellum, a highly conserved, laminated and foliated structure, provides an excellent model for developmental studies. During the intermediate embryonic stages, granule cell progenitor proliferation and the inwards migration of post-mitotic granule cells have been implicated in the morphogenesis of cerebellar cortex cytoarchitecture and foliation. The present study questioned the spatio-temporal expression pattern of pleiotrophin, an extracellular matrix growth factor, during the morphogenesis of embryonic cerebellum, and the roles of ionotropic AMPA glutamate receptors and the diffusible neuromodulator nitric oxide (NO) in the proliferation pattern of EGL granule cell progenitors. To this end, the density of proliferating cells in the developing embryonic external granule layer (EGL) was determined following acute treatment with AMPA receptor antagonist CNQX or NO synthase inhibitor L-NAME, at embryonic stages HH38-41 (E12-E15 days), by means of BrdU immunohistochemistry and double immunofluorescence. Importantly, at earlier stages, pleiotrophin-like immunoreactivity showed high expression levels in the EGL that gradually decreased, persisting within the growing folia apices, later in development. Interestingly, blockage of AMPA receptors had no effect; while NOS inhibition resulted in transient age- and region-specific increases of EGL granule progenitor cell proliferation at earlier stages, but decreased the post-mitotic granule cells at folia apices, at a later stage HH41 (E15 day). Overall, NO had a transient anti-proliferative effect in EGL similar to mammalian cerebellum, acting as a modulator of the EGL function at different stages, suggesting its possible implication in complex processes guiding cerebellar cytoarchitecture and folia formation.

Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells.

Brain development is regulated by conserved transcriptional programs across species, but little is known about the divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with nonhuman primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSCs). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice.

Cell Type-specific Knockout with Gli1-mediated Cre Recombination in the Developing Cerebellum.

The inducible Cre-loxP system provides a useful tool for inducing the selective deletion of genes that are essential for proper development and enables the study of gene functions in properly developed animals. Here, we show that inducible Cre-loxP driven by the Gli1-promoter can induce cell-type-specific deletion of target genes in cerebellar cortical neurons. We used reporter mice containing the YFP (yellow fluorescence protein) gene at the Gt(ROSA)26Sor locus with a loxP-flanked transcriptional stop sequence, in which successful Cre-mediated excision of the stop sequence is indicated by YFP expression in Cre-expressing cells. Administration of tamoxifen during early postnatal days (P4~7) induces Cre-dependent excision of stop sequences and allows YFP expression in proliferating neuronal progenitor cells in the external granule layer and Bergmann glia in the Purkinje cell layer. A substantial number of YFP-positive progenitor cells in the external granule layer migrated to the internal granule cell layer and became granule cell neurons. By comparison, injection of tamoxifen during late postnatal days (P19~22) induces YFP expression only in Bergmann glia, and most granule cell neurons were devoid of YFP expression. The results indicate that the Gli1 promoter is temporarily active in progenitor cells in the external granule layer during the early postnatal period but constitutively active in Bergmann glia. We propose that the Gli1-mediated CreER system can be applied for the conditional deletion of genes of interest from cerebellar granule cell neurons and/or Bergmann glia.

Deletion of Jdp2 enhances Slc7a11 expression in Atoh-1 positive cerebellum granule cell progenitors in vivo

BackgroundThe cerebellum is the sensitive region of the brain to developmental abnormalities related to the effects of oxidative stresses. Abnormal cerebellar lobe formation, found in Jun dimerization protein 2 (Jdp2)-knockout (KO) mice, is related to increased antioxidant formation and a reduction in apoptotic cell death in granule cell progenitors (GCPs). Here, we aim that Jdp2 plays a critical role of cerebellar development which is affected by the ROS regulation and redox control.ObjectiveJdp2-promoter-Cre transgenic mouse displayed a positive signal in the cerebellum, especially within granule cells. Jdp2-KO mice exhibited impaired development of the cerebellum compared with wild-type (WT) mice. The antioxidation controlled gene, such as cystine-glutamate transporter Slc7a11, might be critical to regulate the redox homeostasis and the development of the cerebellum.MethodsWe generated the Jdp2-promoter-Cre mice and Jdp2-KO mice to examine the levels of Slc7a11, ROS levels and the expressions of antioxidation related genes were examined in the mouse cerebellum using the immunohistochemistry.ResultsThe cerebellum of Jdp2-KO mice displayed expression of the cystine-glutamate transporter Slc7a11, within the internal granule layer at postnatal day 6; in contrast, the WT cerebellum mainly displayed Sla7a11 expression in the external granule layer. Moreover, development of the cerebellar lobes in Jdp2-KO mice was altered compared with WT mice. Expression of Slc7a11, Nrf2, and p21Cip1 was higher in the cerebellum of Jdp2-KO mice than in WT mice.ConclusionJdp2 is a critical regulator of Slc7a11 transporter during the antioxidation response, which might control the growth, apoptosis, and differentiation of GCPs in the cerebellar lobes. These observations are consistent with our previous study in vitro.

Plexin-B2 controls the timing of differentiation and the motility of cerebellar granule neurons.

Plexin-B2 deletion leads to aberrant lamination of cerebellar granule neurons (CGNs) and Purkinje cells. Although in the cerebellum Plexin-B2 is only expressed by proliferating CGN precursors in the outer external granule layer (oEGL), its function in CGN development is still elusive. Here, we used 3D imaging, in vivo electroporation and live-imaging techniques to study CGN development in novel cerebellum-specific Plxnb2 conditional knockout mice. We show that proliferating CGNs in Plxnb2 mutants not only escape the oEGL and mix with newborn postmitotic CGNs. Furthermore, motility of mitotic precursors and early postmitotic CGNs is altered. Together, this leads to the formation of ectopic patches of CGNs at the cerebellar surface and an intermingling of normally time-stamped parallel fibers in the molecular layer (ML), and aberrant arborization of Purkinje cell dendrites. There results suggest that Plexin-B2 restricts CGN motility and might have a function in cytokinesis.

TMOD-06. LOSS OF DICER COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE METASTATIC MEDULLOBLASTOMA

To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a previously characterized glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and tumors were histologically indistinguishable from human medulloblastoma (MB). The minimum genetic mutations required for MB formation were Dicer and Trp53. Nf1 was dispensable, while additional loss of Pten produced more invasive tumors and leptomeningeal metastases. The time window for initiation of tumorigenesis was until the 2nd postnatal week, coinciding with the disappearance of the external granule layer (EGL), where cerebellar granule neuron precursors (CGNPs) undergo proliferation. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the EGL, suggesting that the tumors may arise from CGNPs. However, targeting a subset of CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Analysis of tumor transcriptome and MB subtype-specific genes and markers show that Dicer tumors most resemble extremely high risk p53-mutated SHH MB. Small RNA and mRNA sequencing analyses showed downregulation of microRNAs and dysregulation of its targets such as N-Myc. These studies demonstrate a role for microRNAs in MB development and show a fully penetrant genetic mouse model of highly metastatic MB.

Notch Signaling between Cerebellar Granule Cell Progenitors.

Cerebellar granule cells (GCs) are cells which comprise over 50% of the neurons in the entire nervous system. GCs enable the cerebellum to properly regulate motor coordination, learning, and consolidation, in addition to cognition, emotion and language. During GC development, maternal GC progenitors (GCPs) divide to produce not only postmitotic GCs but also sister GCPs. However, the molecular machinery for regulating the proportional production of distinct sister cell types from seemingly uniform GCPs is not yet fully understood. Here we report that Notch signaling creates a distinction between GCPs and leads to their proportional differentiation in mice. Among Notch-related molecules, Notch1, Notch2, Jag1, and Hes1 are prominently expressed in GCPs. In vivo monitoring of Hes1-promoter activities showed the presence of two types of GCPs, Notch-signaling ON and OFF, in the external granule layer (EGL). Single-cell RNA sequencing (scRNA-seq) and in silico analyses indicate that ON-GCPs have more proliferative and immature properties, while OFF-GCPs have opposite characteristics. Overexpression as well as knock-down (KD) experiments using in vivo electroporation showed that NOTCH2 and HES1 are involved cell-autonomously to suppress GCP differentiation by inhibiting NEUROD1 expression. In contrast, JAG1-expressing cells non-autonomously upregulated Notch signaling activities via NOTCH2-HES1 in surrounding GCPs, eventually suppressing their differentiation. These findings suggest that Notch signaling results in the proportional generation of two types of cells, immature and differentiating GCPs, which contributes to the well-organized differentiation of GCs.

Argentic staining reveals changes in cerebellar tissue organisation by prenatal glucocorticoid administration in rats.

It was almost 150 years ago that Golgi revolutionised histology with silver-based stains. Major advances in knowledge of the nervous system became possible because of silver impregnations. Silver staining combined with classical histological staining, cytochemistry methods, and electron microscopy is useful for studying mechanisms and components at subcellular, cellular, and tissue levels. Despite the advantages of silver staining, its use has decreased over time. The aim of this work was to use argentic staining to study the cerebellar effects of controversial prenatal glucocorticoid (GC) therapy. At postnatal day 12 (P12), the cerebellum of corticosterone (CC)-treated rats impregnated with AgNOR staining exhibited diminished thickness of the external granule layer (EGL) and irregular Purkinje cell arrangement. There was a greater number of nucleoli and nucleolar organiser regions (NORs) in 24% of Purkinje cells. Cerebellar granule neuron progenitor (CGNP) cells of the EGL showed a decrease in cellular density (confirmed by proliferating cell nuclear antigen [PCNA] immunolocalization) and NORs. At postnatal day 6 (P6), the Golgi-Kopsch technique allowed us to observe disturbances in the distribution pattern of CGNP cells (during proliferation, migration, and differentiation) and premature growth of the Bergmann glia. Our findings reveal disturbances in the cerebellar development program with early cellular and tissue changes.

The role of SCFSkp2 and SCFβ-TrCP1/2 in the cerebellar granule cell precursors.

A proper balance between proliferation and differentiation of cerebellar granule cell precursors (GCPs) is required for appropriate cerebellar morphogenesis. The Skp1-Cullin1-F-box (SCF) complex, an E3 ubiquitin ligase complex, is involved in polyubiquitination and subsequent degradation of various cell cycle regulators and transcription factors. However, it remains unknown how the SCF complex affects proliferation and differentiation of GCPs. In this study, we found that the scaffold protein Cullin1, and F-box proteins Skp2, β-TrCP1 and β-TrCP2 are expressed in the external granule layer (EGL). Knockdown of these molecules in the EGL showed that Cullin1, Skp2 and β-TrCP2 enhanced differentiation of GCPs. We also observed accumulation of cyclin-dependent kinase inhibitor p27 in GCPs when treated with a Cullin1 inhibitor or proteasome inhibitor. Furthermore, knockdown of p27 rescued enhancement of differentiation by Cullin1 knockdown. These results suggest that the SCF complex is involved in the maintenance of the proliferative state of GCPs through p27 degradation. In addition, inhibition of Cullin1 activity also prevented cell proliferation and enhanced accumulation of p27 in Daoy cells, a cell line derived from the sonic hedgehog subtype of medulloblastoma. This suggested that excess degradation of p27 through the SCF complex causes overproliferation of medulloblastoma cells.