Direct and indirect pathways for heterosynaptic interaction underlying developmental synapse elimination in the mouse cerebellum.

Developmental synapse elimination is crucial for shaping mature neural circuits. In the neonatal mouse cerebellum, Purkinje cells (PCs) receive excitatory synaptic inputs from multiple climbing fibers (CFs) and synapses from all but one CF are eliminated by around postnatal day 20. Heterosynaptic interaction between CFs and parallel fibers (PFs), the axons of cerebellar granule cells (GCs) forming excitatory synapses onto PCs and molecular layer interneurons (MLIs), is crucial for CF synapse elimination. However, mechanisms for this heterosynaptic interaction are largely unknown. Here we show that deletion of AMPA-type glutamate receptor functions in GCs impairs CF synapse elimination mediated by metabotropic glutamate receptor 1 (mGlu1) signaling in PCs. Furthermore, CF synapse elimination is impaired by deleting NMDA-type glutamate receptors from MLIs. We propose that PF activity is crucial for CF synapse elimination by directly activating mGlu1 in PCs and indirectly enhancing the inhibition of PCs through activating NMDA receptors in MLIs.

Antibody-assisted selective isolation of Purkinje cell nuclei from mouse cerebellar tissue.

We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability.

In Vivo Monitoring of Fabp7 Expression in Transgenic Zebrafish.

In zebrafish, like in mammals, radial glial cells (RGCs) can act as neural progenitors during development and regeneration in adults. However, the heterogeneity of glia subpopulations entails the need for different specific markers of zebrafish glia. Currently, fluorescent protein expression mediated by a regulatory element from the glial fibrillary acidic protein (gfap) gene is used as a prominent glia reporter. We now expand this tool by demonstrating that a regulatory element from the mouse Fatty acid binding protein 7 (Fabp7) gene drives reliable expression in fabp7-expressing zebrafish glial cells. By using three different Fabp7 regulatory element-mediated fluorescent protein reporter strains, we reveal in double transgenic zebrafish that progenitor cells expressing fluorescent proteins driven by the Fabp7 regulatory element give rise to radial glia, oligodendrocyte progenitors, and some neuronal precursors. Furthermore, Bergmann glia represent the almost only glial population of the zebrafish cerebellum (besides a few oligodendrocytes), and the radial glia also remain in the mature cerebellum. Fabp7 regulatory element-mediated reporter protein expression in Bergmann glia progenitors suggests their origin from the ventral cerebellar proliferation zone, the ventricular zone, but not from the dorsally positioned upper rhombic lip. These new Fabp7 reporters will be valuable for functional studies during development and regeneration.

Phospholipid Scramblase 1 Controls Efficient Neurotransmission and Synaptic Vesicle Retrieval at Cerebellar Synapses.

Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1 -/- synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1 -/- cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

Cerebellar granular neuron progenitors exit their germinative niche via BarH-like1 activity mediated partly by inhibition of T-cell factor.

Cerebellar granule neuron progenitors (GNPs) originate from the upper rhombic lip (URL), a germinative niche in which developmental defects produce human diseases. T-cell factor (TCF) responsiveness and Notch dependence are hallmarks of self-renewal in neural stem cells. TCF activity, together with transcripts encoding proneural gene repressors hairy and enhancer of split (Hes/Hey), are detected in the URL; however, their functions and regulatory modes are undeciphered. Here, we established amphibian as a pertinent model for studying vertebrate URL development. The amphibian long-lived URL is TCF active, whereas the external granular layer (EGL) is non-proliferative and expresses hes4 and hes5 genes. Using functional and transcriptomic approaches, we show that TCF activity is necessary for URL emergence and maintenance. We establish that the transcription factor Barhl1 controls GNP exit from the URL, acting partly through direct TCF inhibition. Identification of Barhl1 target genes suggests that, besides TCF, Barhl1 inhibits transcription of hes5 genes independently of Notch signaling. Observations in amniotes suggest a conserved role for Barhl in maintenance of the URL and/or EGL via co-regulation of TCF, Hes and Hey genes.

Constitutive Neurogenesis and Neuronal Plasticity in the Adult Cerebellum and Brainstem of Rainbow Trout, Oncorhynchus mykiss.

The central nervous system of Pacific salmon retains signs of embryonic structure throughout life and a large number of neuroepithelial neural stem cells (NSCs) in the proliferative areas of the brain, in particular. However, the adult nervous system and neurogenesis studies on rainbow trout, Oncorhynchus mykiss, are limited. Here, we studied the localization of glutamine synthetase (GS), vimentin (Vim), and nestin (Nes), as well as the neurons formed in the postembryonic period, labeled with doublecortin (DC), under conditions of homeostatic growth in adult cerebellum and brainstem of Oncorhynchus mykiss using immunohistochemical methods and Western Immunoblotting. We observed that the distribution of vimentin (Vim), nestin (Nes), and glutamine synthetase (GS), which are found in the aNSPCs of both embryonic types (neuroepithelial cells) and in the adult type (radial glia) in the cerebellum and the brainstem of trout, has certain features. Populations of the adult neural stem/progenitor cells (aNSPCs) expressing GS, Vim, and Nes have different morphologies, localizations, and patterns of cluster formation in the trout cerebellum and brainstem, which indicates the morphological and, obviously, functional heterogeneity of these cells. Immunolabeling of PCNA revealed areas in the cerebellum and brainstem of rainbow trout containing proliferating cells which coincide with areas expressing Vim, Nes, and GS. Double immunolabeling revealed the PCNA/GS PCNA/Vim coexpression patterns in the neuroepithelial-type cells in the PVZ of the brainstem. PCNA/GS coexpression in the RG was detected in the submarginal zone of the brainstem. The results of immunohistochemical study of the DC distribution in the cerebellum and brainstem of trout have showed a high level of expression of this marker in various cell populations. This may indicate: (i) high production of the adult-born neurons in the cerebellum and brainstem of adult trout, (ii) high plasticity of neurons in the cerebellum and brainstem of trout. We assume that the source of new cells in the trout brain, along with PVZ and SMZ, containing proliferating cells, may be local neurogenic niches containing the PCNA-positive and silent (PCNA-negative), but expressing NSC markers, cells. The identification of cells expressing DC, Vim, and Nes in the IX-X cranial nerve nuclei of trout was carried out.

Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

Generation and enrichment of cerebellar GABAergic interneurons from human induced pluripotent stem cells and intracellular calcium measurements.

GABAergic interneurons are inhibitory neurons of the CNS, playing a fundamental role in neural circuitry and activity. Here, we provide a robust protocol for the successful enrichment of human cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs; generating embryoid bodies; and differentiating and enriching for cerebellar GABAergic neurons (cGNs), with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators, followed by intracellular calcium imaging and analyses. For complete details on the use and execution of this protocol, please refer to Pilotto et al.1.

Intrinsic and synaptic determinants of receptive field plasticity in Purkinje cells of the mouse cerebellum.

Non-synaptic (intrinsic) plasticity of membrane excitability contributes to aspects of memory formation, but it remains unclear whether it merely facilitates synaptic long-term potentiation or plays a permissive role in determining the impact of synaptic weight increase. We use tactile stimulation and electrical activation of parallel fibers to probe intrinsic and synaptic contributions to receptive field plasticity in awake mice during two-photon calcium imaging of cerebellar Purkinje cells. Repetitive activation of both stimuli induced response potentiation that is impaired in mice with selective deficits in either synaptic or intrinsic plasticity. Spatial analysis of calcium signals demonstrated that intrinsic, but not synaptic plasticity, enhances the spread of dendritic parallel fiber response potentiation. Simultaneous dendrite and axon initial segment recordings confirm these dendritic events affect axonal output. Our findings support the hypothesis that intrinsic plasticity provides an amplification mechanism that exerts a permissive control over the impact of long-term potentiation on neuronal responsiveness.

Cerebellar Purkinje cells in male macaques combine sensory and motor information to predict the sensory consequences of active self-motion.

Accurate perception and behavior rely on distinguishing sensory signals arising from unexpected events from those originating from our own voluntary actions. In the vestibular system, sensory input that is the consequence of active self-motion is canceled early at the first central stage of processing to ensure postural and perceptual stability. However, the source of the required cancellation signal was unknown. Here, we show that the cerebellum combines sensory and motor-related information to predict the sensory consequences of active self-motion. Recordings during attempted but unrealized head movements in two male rhesus monkeys, revealed that the motor-related signals encoded by anterior vermis Purkinje cells explain their altered sensitivity to active versus passive self-motion. Further, a model combining responses from ~40 Purkinje cells accounted for the cancellation observed in early vestibular pathways. These findings establish how cerebellar Purkinje cells predict sensory outcomes of self-movements, resolving a long-standing issue of sensory signal suppression during self-motion.

Specialized connectivity of molecular layer interneuron subtypes leads to disinhibition and synchronous inhibition of cerebellar Purkinje cells.

Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex and are vital to cerebellar processing. MLIs are thought to primarily inhibit Purkinje cells (PCs) and suppress the plasticity of synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Here, we find that two recently recognized MLI subtypes, MLI1 and MLI2, have a highly specialized connectivity that allows them to serve distinct functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond timescale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent behavior and learning. The synchronous firing of electrically coupled MLI1s and disinhibition provided by MLI2s require a major re-evaluation of cerebellar processing.

Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10 µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

Climbing fibers provide essential instructive signals for associative learning.

Supervised learning depends on instructive signals that shape the output of neural circuits to support learned changes in behavior. Climbing fiber (CF) inputs to the cerebellar cortex represent one of the strongest candidates in the vertebrate brain for conveying neural instructive signals. However, recent studies have shown that Purkinje cell stimulation can also drive cerebellar learning and the relative importance of these two neuron types in providing instructive signals for cerebellum-dependent behaviors remains unresolved. In the present study we used cell-type-specific perturbations of various cerebellar circuit elements to systematically evaluate their contributions to delay eyeblink conditioning in mice. Our findings reveal that, although optogenetic stimulation of either CFs or Purkinje cells can drive learning under some conditions, even subtle reductions in CF signaling completely block learning to natural stimuli. We conclude that CFs and corresponding Purkinje cell complex spike events provide essential instructive signals for associative cerebellar learning.

Choline supplementation mitigates effects of bilirubin in cerebellar granule neurons in vitro.

BACKGROUND: Premature infants may suffer from high levels of bilirubin that could lead to neurotoxicity. Bilirubin has been shown to decrease L1-mediated ERK1/2 signaling, L1 phosphorylation, and L1 tyrosine 1176 dephosphorylation. Furthermore, bilirubin redistributes L1 into lipid rafts (LR) and decreases L1-mediated neurite outgrowth. We demonstrate that choline supplementation improves L1 function and signaling in the presence of bilirubin. METHODS: Cerebellar granule neurons (CGN) were cultured with and without supplemental choline, and the effects on L1 signaling and function were measured in the presence of bilirubin. L1 activation of ERK1/2, L1 phosphorylation and dephosphorylation were measured. L1 distribution in LR was quantified and neurite outgrowth of CGN was determined. RESULTS: Forty µM choline significantly reduced the effect of bilirubin on L1 activation of ERK1/2 by 220% (p = 0.04), and increased L1 triggered changes in tyrosine phosphorylation /dephosphorylation of L1 by 34% (p = 0.026) and 35% (p = 0.02) respectively. Choline ameliorated the redistribution of L1 in lipid rafts by 38% (p = 0.02) and increased L1-mediated mean neurite length by 11% (p = 0.04). CONCLUSION: Choline pretreatment of CGN significantly reduced the disruption of L1 function by bilirubin. The supplementation of pregnant women and preterm infants with choline may increase infant resilience to the effects of bilirubin. IMPACT: This article establishes choline as an intervention for the neurotoxic effects of bilirubin on lipid rafts. This article provides clear evidence toward establishing one intervention for bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into the mechanism of the ameliorative effect of choline on bilirubin neurotoxicity.

Cellular development and evolution of the mammalian cerebellum.

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

A transcriptomic taxonomy of mouse brain-wide spinal projecting neurons.

The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.

Lifelong restructuring of 3D genome architecture in cerebellar granule cells.

The cerebellum contains most of the neurons in the human brain and exhibits distinctive modes of development and aging. In this work, by developing our single-cell three-dimensional (3D) genome assay-diploid chromosome conformation capture, or Dip-C-into population-scale (Pop-C) and virus-enriched (vDip-C) modes, we resolved the first 3D genome structures of single cerebellar cells, created life-spanning 3D genome atlases for both humans and mice, and jointly measured transcriptome and chromatin accessibility during development. We found that although the transcriptome and chromatin accessibility of cerebellar granule neurons mature in early postnatal life, 3D genome architecture gradually remodels throughout life, establishing ultra-long-range intrachromosomal contacts and specific interchromosomal contacts that are rarely seen in neurons. These results reveal unexpected evolutionarily conserved molecular processes that underlie distinctive features of neural development and aging across the mammalian life span.

Study of Cerebellar Tissue Following Induction of Aging in Mice By Di-Galactose / Estudio del Tejido Cerebeloso tras la Inducción del Envejecimiento en Ratones por D-Galactosa

SUMMARY: The cerebellum is a crucial area of the hindbrain that plays an essential role in balancing, excitement control, and subtle and accurate functions. Studies have shown that long-term use of D-galactose in mice, as with the symptoms of aging, causes morphological and functional disorders in the brain. This study was performed to evaluate the changes in the cerebellum cortex tissue and the measurement of reactive oxygen species (ROS) in the cerebellum following the induction of aging in mice by D-galactose. Accordingly, subjects were randomly assigned into two groups: Normal saline group and Aging group (D-galactose). To create an aging model, D- galactose, and saline solution (sodium chloride 0.9 %) were used. After completing the preparation and passage of the tissue, the cerebellum specimens were cut in 5 microns thickness and then stained with hematoxylin-eosin stain and finally examined under a Nikon microscope. Quantitative variables were analyzed by SPSS software using T-test. In the observations of cerebellum tissue samples, in the aged induced group by D-galactose, the most changes were observed in the Neuron purkinjense (Purkinje cells) layer. In the observations of the cerebellum tissue samples of aging group induced by D-galactose, the most changes were observed in the Neuron purkinjense, and the arrangement and placement of these cells were disorientated. The nucleus positioning was not central, and the Neuron purkinjense induced by aging were seen in different morphological forms. Necrosis, Chromatolysis, and Pyknosis were found. Based on the results, D-galactose (induction of aging) causes pathological changes in the cerebellar cortex, especially in the Neuron purkinjense layer.

El cerebelo es un área crucial del rombencéfalo que desempeña un papel esencial en el equilibrio, el control de la excitación y las funciones sutiles y precisas. Los estudios han demostrado que el uso a largo plazo de D-galactosa en ratones, al igual que con los síntomas del envejecimiento, provoca trastornos morfológicos y funcionales en el cerebro. Este estudio se realizó para evaluar los cambios en el tejido de la corteza del cerebelo y la medición de especies reactivas de oxígeno (ROS) en el cerebelo luego de la inducción del envejecimiento en ratones por D-galactosa. En consecuencia, los sujetos fueron asignados aleatoriamente a dos grupos: grupo de solución salina normal y grupo de envejecimiento (D-galactosa). Para crear un modelo de envejecimiento, se utilizaron D-galactosa y solución salina (cloruro de sodio al 0,9 %). Después de completar la preparación y el paso del tejido, las muestras de cerebelo se cortaron en un grosor de 5 µm y luego se tiñeron con tinción de hematoxilina-eosina y finalmente se examinaron bajo un microscopio Nikon. Las variables cuantitativas se analizaron mediante el software SPSS utilizando la prueba T. En las observaciones de muestras de tejido de cerebelo, en el grupo envejecido inducido por D-galactosa, la mayoría de los cambios se observaron en la capa de neuronas purkinjenses (células de Purkinje). En las observaciones de las muestras de tejido del cerebelo del grupo de envejecimiento inducidas por D-galactosa, la mayoría de los cambios se observaron en las neuronas purkinjenses, y la disposición y ubicación de estas células estaban desorientadas. El posicionamiento del núcleo no era central y las neuronas purkinjenses inducidas por el envejecimiento se observaban en diferentes formas morfológicas. Se encontró necrosis, cromatólisis y picnosis. Según los resultados, la D-galactosa (inducción del envejecimiento) provoca cambios patológicos en la corteza cerebelosa, especialmente en la capa de neuronas purkinjenses.

Regulation of early cerebellar development.

The study of cerebellar development has been at the forefront of neuroscience since the pioneering work of Wilhelm His Sr., Santiago Ramón y Cajal and many others since the 19th century. They laid the foundation to identify the circuitry of the cerebellum, already revealing its stereotypic three-layered cortex and discerning several of its neuronal components. Their work was fundamental in the acceptance of the neuron doctrine, which acknowledges the key role of individual neurons in forming the basic units of the nervous system. Increasing evidence shows that the cerebellum performs a variety of homeostatic and higher order neuronal functions beyond the mere control of motor behaviour. Over the last three decades, many studies have revealed the molecular machinery that regulates distinct aspects of cerebellar development, from the establishment of a cerebellar anlage in the posterior brain to the identification of cerebellar neuron diversity at the single cell level. In this review, we focus on summarizing our current knowledge on early cerebellar development with a particular emphasis on the molecular determinants that secure neuron specification and contribute to the diversity of cerebellar neurons.

Human fetal cerebellar cell atlas informs medulloblastoma origin and oncogenesis.

Medulloblastoma (MB) is the most common malignant childhood brain tumour1,2, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella3-5 have not fully defined the compositional heterogeneity of MBs. Here we undertook single-cell profiling of freshly isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group 3 and metastatic tumours. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group 3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group 3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group 3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs show potential cell populations predisposed to transformation and regulatory circuitries underlying tumour cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.