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SummaryDeveloping neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use 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 find 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 H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2in vitroand in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation 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.

SummaryBrain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, the cerebellum is now recognized to contribute to human cognitive evolution having a broad range of non-motor cognitive functions in addition to motor control. Emerging studies highlight the complexity of human cerebellar histogenesis, compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. Here we report a rapid and simple protocol for the directed derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC), and strategies to decrease culture variability; a common limitation in hPSC studies. Upon transplantation into juvenile mice, early postmitotic hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification (TRAP)-seq, the ATOH1 lineage most closely resembled human cerebellar tissue in the second trimester. Unexpectedly, TRAP-seq identified a heterochronic shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, within granule cell progenitors (GCPs) in the human external granule layer. This molecular divergence may provide the mechanism by which the GCP pool persists into year two post birth in humans, but only lasts for two weeks in mice. Our approach provides a scalablein vitromodel of the human ATOH1 lineage that yields cerebellar granule cells within 48 days as well as a strategy for identifying uniquely human cellular and molecular characteristics.

Significance The conserved proteasome-binding protein PI31 serves as an adapter to couple proteasomes with cellular motors to mediate their transport to distal tips of neurons where protein breakdown occurs. We generated global and conditional PI31 knockout mouse strains and show that this protein is required for protein homeostasis, and that its conditional inactivation in neurons disrupts synaptic structures and long-term survival. This work establishes a critical role for PI31 and local protein degradation in the maintenance of neuronal architecture, circuitry, and function. Because mutations in the PI31 pathway cause neurodegenerative diseases in humans, reduced PI31 activity may contribute to the etiology of these diseases., Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 26S assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.

Prior studies demonstrate that astrotactin (ASTN1) provides a neuronal receptor for glial-guided CNS migration. Here we report that ASTN1 binds N-cadherin (CDH2) and that the ASTN1:CDH2 interaction supports cell–cell adhesion. To test the function of ASTN1:CDH2 binding in glial-guided neuronal migration, we generated a conditional loss of Cdh2 in cerebellar granule cells and in glia. Granule cell migration was slowed in cerebellar slice cultures after a conditional loss of neuronal Cdh2, and more severe migration defects occurred after a conditional loss of glial Cdh2. Expression in granule cells of a mutant form of ASTN1 that does not bind CDH2 also slowed migration. Moreover, in vitro chimeras of granule cells and glia showed impaired neuron–glia attachment in the absence of glial, but not neuronal, Cdh2. Thus, cis and trans bindings of ASTN1 to neuronal and glial CDH2 form an asymmetric neuron–glial bridge complex that promotes glial-guided neuronal migration.

SUMMARY CNS cortical histogenesis depends on polarity signaling pathways that regulate cell adhesion and motility. Here we report that conditional deletion of the Rho GTPase Cdc42 in cerebellar granule cell precursors (GCPs) results in abnormalities in cerebellar foliation revealed by iDISCO clearing methodology, a loss of columnar organization of proliferating GCPs in the external germinal layer (EGL), disordered parallel fiber organization in the molecular layer (ML), and a failure to extend a leading process and form a neuron-glial junction during migration along Bergmann glia (BG). Notably, GCPs lacking Cdc42 had a multi-polar morphology and slowed migration rate. In addition, secondary defects occurred in BG development and organization, especially in the lateral cerebellar hemispheres. By phosphoproteomic analysis, affected Cdc42 targets included regulators of the cytoskeleton, cell adhesion and polarity. Thus, Cdc42 signaling pathways are critical regulators of GCP polarity and the formation of neuron-glial junctions during cerebellar development., Graphical abstract

Accumulating evidence suggests that cerebellar dysfunction early in life is associated with autism spectrum disorder (ASD), but the molecular mechanisms underlying the cerebellar deficits at the cellular level are unclear. Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that often presents with ASD. Here, we developed a cerebellar Purkinje cell (PC) model of TSC with patient-derived human induced pluripotent stem cells (hiPSCs) to characterize the molecular mechanisms underlying cerebellar abnormalities in ASD and TSC. Our results show that hiPSC-derived PCs from patients with pathogenic TSC2 mutations displayed mTORC1 pathway hyperactivation, defects in neuronal differentiation and RNA regulation, hypoexcitability and reduced synaptic activity when compared with those derived from controls. Our gene expression analyses revealed downregulation of several components of fragile X mental retardation protein (FMRP) targets in TSC2-deficient hiPSC-PCs. We detected decreased expression of FMRP, glutamate receptor δ2 (GRID2), and pre- and post-synaptic markers such as synaptophysin and PSD95 in the TSC2-deficient hiPSC-PCs. The mTOR inhibitor rapamycin rescued the deficits in differentiation, synaptic dysfunction, and hypoexcitability of TSC2 mutant hiPSC-PCs in vitro. Our findings suggest that these gene expression changes and cellular abnormalities contribute to aberrant PC function during development in TSC affected individuals.

During evolution, duplication of subnuclei generates broader cerebellar projections

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSC) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate hPSC-PCs that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs, and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hESC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile, mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides the first direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.Significance StatementTo compare global gene expression features of differentiating human pluripotent stem cell-derived Purkinje cells (hPSC-PC) and developing mouse Purkinje cells (PC) we derived hPSC-PCs and compared gene expression datasets from human and mouse PCs. We optimized a differentiation protocol that generated hPSC-PCs most similar in gene expression to mouse P21 PCs. Metagene projection analysis of mouse PC gene expression over postnatal development identified both classical PC marker genes as well as novel mitochondrial and autophagy gene pathways. These key gene expression patterns were conserved in differentiating hPSC-PCs. We further identified differences in timing and expression of key gene sets between mouse and hPSC-PCs and confirmed expression of a novel human PC marker, CD40LG, in human cerebellar tissue.

Postnatal cerebellar development is a precisely regulated process involving well-orchestrated expression of neural genes. Neurological phenotypes associated with CACNA1A gene defects have been increasingly recognized, yet the molecular principles underlying this association remain elusive. By characterizing a dose-dependent CACNA1A gene deficiency mouse model, we discovered that α1ACT, as a transcription factor and secondary protein of CACNA1A mRNA, drives dynamic gene expression networks within cerebellar Purkinje cells and is indispensable for neonatal survival. Perinatal loss of α1ACT leads to motor dysfunction through disruption of neurogenesis and synaptic regulatory networks. However, its elimination in adulthood has minimal effect on the cerebellum. These findings shed light on the critical role of α1ACT in facilitating neuronal development in both mice and humans, and support a rationale for gene therapies for calcium channel-associated cerebellar disorders. Finally, we show that bicistronic expression may be common to the VGCC gene family and help explain complex genetic syndromes.

Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ)-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications., The mechanisms controlling irreversible cell cycle exit in cerebellar granule progenitors (GCPs) have not been fully elucidated. Here, the authors performed RNA-sequencing of GCPs exiting the cell cycle to identify downregulation of Brd4 activity as an early event during cell cycle exit which subsequently regulates Shh activity and is needed for proper cerebellar development

Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

Astrotactins 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking and synaptic plasticity in the brain, as well as in planar polarity pathways in skin. Here, we used glycosylation mapping and protease-protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes (RMs), and found that Astn2 has a cleaved N-terminal signal peptide (SP), an N-terminal domain located in the lumen of the RMs (topologically equivalent to the extracellular surface in cells), two transmembrane helices (TMHs), and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of SP cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second TMH; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

Significance Glial-guided neuronal migration is a key step in the histogenesis of cortical regions in the mammalian brain and requires the expression of adhesion proteins by the migrating neuron and the glial fiber. The neuronal receptor astrotactin (ASTN1) regulates glial-guided migration, but the glial ligand has long been unknown. Here we demonstrate that neuron–glia attachment and neuronal migration depend on glial expression of the neural cadherin (CDH2) and that ASTN1 promotes migration by a direct interaction with neuronal and glial CDH2. Thus, ASTN1 and CDH2 form a cis and trans asymmetric bridge complex in the migration junction that is essential for glial-guided neuronal migration., Prior studies demonstrate that astrotactin (ASTN1) provides a neuronal receptor for glial-guided CNS migration. Here we report that ASTN1 binds N-cadherin (CDH2) and that the ASTN1:CDH2 interaction supports cell–cell adhesion. To test the function of ASTN1:CDH2 binding in glial-guided neuronal migration, we generated a conditional loss of Cdh2 in cerebellar granule cells and in glia. Granule cell migration was slowed in cerebellar slice cultures after a conditional loss of neuronal Cdh2, and more severe migration defects occurred after a conditional loss of glial Cdh2. Expression in granule cells of a mutant form of ASTN1 that does not bind CDH2 also slowed migration. Moreover, in vitro chimeras of granule cells and glia showed impaired neuron–glia attachment in the absence of glial, but not neuronal, Cdh2. Thus, cis and trans bindings of ASTN1 to neuronal and glial CDH2 form an asymmetric neuron–glial bridge complex that promotes glial-guided neuronal migration.

Prior studies demonstrate that Astrotactin (ASTN1) provides a neuronal receptor for glial-guided CNS migration. Here we report that ASTN1 binds N-cadherin (CDH2) and that the ASTN1:CDH2 interaction supports cell-cell adhesion. To test the function of ASTN1:CDH2 binding in glial-guided neuronal migration, we generated a conditional loss ofCdh2in cerebellar granule cells and in glia. Granule cell migration was slowed in cerebellar slice cultures after a conditional loss of neuronalCdh2, and more severe migration defects occurred after a conditional loss of glialCdh2. Expression of a mutant form of ASTN1 that does not bind CDH2, in granule cells, also slowed migration. Moreover,in vitrochimeras of granule cells and glia showed impaired neuron-glia attachment in the absence of glial, but not neuronal,Cdh2. Thus,cisandtransbindings of ASTN1 to neuronal and glial CDH2 form an asymmetric neuron-glial bridge complex that promotes glial-guided neuronal migration.

Significance Neurogenetic studies demonstrate that copy number variations (CNVs) in the ASTN2 gene occur in patients with neurodevelopmental disorders (NDDs), including autism spectrum. Here, we show that ASTN2 associates with recycling and degradative vesicles in cerebellar neurons, and binds to and promotes the endocytic trafficking and degradation of synaptic proteins. Overexpression of ASTN2 in neurons increases synaptic activity and reduces the levels of ASTN2 binding partners, an effect dependent on its FNIII domain, which is recurrently perturbed by CNVs in patients with NDDs. These findings suggest that ASTN2 is a key regulator of dynamic trafficking of synaptic proteins and lend support to the idea that aberrant regulation of protein homeostasis in neurons is a contributing cause of complex NDDs., Surface protein dynamics dictate synaptic connectivity and function in neuronal circuits. ASTN2, a gene disrupted by copy number variations (CNVs) in neurodevelopmental disorders, including autism spectrum, was previously shown to regulate the surface expression of ASTN1 in glial-guided neuronal migration. Here, we demonstrate that ASTN2 binds to and regulates the surface expression of multiple synaptic proteins in postmigratory neurons by endocytosis, resulting in modulation of synaptic activity. In cerebellar Purkinje cells (PCs), by immunogold electron microscopy, ASTN2 localizes primarily to endocytic and autophagocytic vesicles in the cell soma and in subsets of dendritic spines. Overexpression of ASTN2 in PCs, but not of ASTN2 lacking the FNIII domain, recurrently disrupted by CNVs in patients, including in a family presented here, increases inhibitory and excitatory postsynaptic activity and reduces levels of ASTN2 binding partners. Our data suggest a fundamental role for ASTN2 in dynamic regulation of surface proteins by endocytic trafficking and protein degradation.

Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are up-regulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of up-regulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNA interference in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped ∼2-fold less in the Pcsk9(-/-)-lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.

The architectonics of the mammalian brain arise from a remarkable range of directed cell migrations, which orchestrate the emergence of cortical neuronal layers and pattern brain circuitry. At different stages of cortical histogenesis, specific modes of cell motility are essential to the stepwise formation of cortical architecture. These movements range from interkinetic nuclear movements in the ventricular zone, to migrations of early-born, postmitotic polymorphic cells into the preplate, to the radial migration of precursors of cortical output neurons across the thickening cortical wall, and the vast, tangential migrations of interneurons from the basal forebrain into the emerging cortical layers. In all cases, actomyosin motors act in concert with cell adhesion receptor systems to provide the force and traction needed for forward movement. As key regulators of actin and microtubule cytoskeletons, cell polarity, and adhesion, the Rho GTPases play critical roles in CNS neuronal migration. This review will focus on the different types of migration in the developing neocortex and cerebellar cortex, and the role of the Rho GTPases, their regulators and effectors in these CNS migrations, with particular emphasis on their involvement in radial migration. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 528–553, 2011

Medulloblastoma (MB) is the most common malignant pediatric brain tumor and is thought to arise from genetic anomalies in developmental pathways required for the normal maturation of the cerebellar cortex, notably developmental pathways for granule cell progenitor (GCP) neurogenesis. Over the past decade, a wide range of studies have identified genes and their regulators within signaling pathways, as well as non-coding RNAs, that have critical roles in both normal cerebellar development and pathogenesis. These include the Notch, Wnt/β-catenin, Bone Morphogenic Proteins (Bmp) and Sonic Hedgehog (Shh) pathways. In this review, we highlight the function of these pathways in the growth of the cerebellum and the formation of MB. A better understanding of the developmental origins of these tumors will have significant implications for enhancing the treatment of this important childhood cancer.

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6alpha localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6alpha inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to "pull" the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6alpha.

During mammalian corticogenesis a series of transient cell layers establish laminar architectonics. The preplate, which forms from the earliest-generated neurons, separates into the marginal zone and subplate layer. To provide a systematic screen for genes involved in subplate development and function, we screened lines of transgenic mice, generated using bacterial artificial chromosome methodology (GENSAT Project), to identify transgenic lines of mice that express the enhanced green fluorescent protein (EGFP) reporter in preplate neurons destined for the subplate. Gene expression profiling of RNA purified from EGFP-positive neurons identified over 200 genes with enriched expression in future subplate neurons. Major classes of subplate-enriched genes included genes involved in transcriptional processes, cortical development, cell and axon motility, protein trafficking and steroid hormone signaling. Additionally, we identified 10 genes related to degenerative diseases of the cerebral and cerebellar cortex. Cre recombinase--based fate mapping of cells expressing Phosphodiesterase 1c (Pde1c) revealed beta-galactosidase positive cells in the ventricular zone, as well as the subplate, suggesting that subplate neurons and cortical projection neurons may be derived from common progenitors. These experiments therefore reveal genetic markers, which identify subplate neurons from the earliest stages of their development, and genes with enriched expression in subplate neurons during early stages of corticogenesis.

Transgenic mouse lines are characterized with Cre recombinase driven by promoters of CNS-specific genes using bacterial artificial chromosome (BAC) constructs. BAC-Cre constructs for 10 genes (Chat,Th,Slc6a4,Slc6a2,Etv1,Ntsr1,Drd2,Drd1,Pcp2, andCmtm5) produced 14 lines with Cre expression in specific neuronal and glial populations in the brain. These Cre driver lines add functional utility to the >500 BAC-EGFP (enhanced green fluorescent protein) transgenic mouse lines that are part of the Gene Expression Nervous System Atlas Project.

The neuronal circuits of the cerebellar cortex are essential for motor and sensory learning, associative memory formation, and the vestibular ocular reflex. In children and young adults, tumors of the granule cell, the medulloblastomas, represent 40% of brain tumors. We report the differentiation of E14 ES cells into mature granule neurons by sequential treatment with secreted factors (WNT1, FGF8, and RA) that initiate patterning in the cerebellar region of the neural tube, bone morphogenic proteins (BMP6/7 and GDF7) that induce early granule cell progenitor markers (MATH1, MEIS1, ZIC1), mitogens (SHH, JAG1) that control proliferation and induce additional granule cell markers (Cyclin D2, PAX2/6), and culture in glial-conditioned medium to induce markers of mature granule neurons (GABAα 6 r), including ZIC2, a unique marker for granule neurons. Differentiated ES cells formed classic “T-shaped” granule cell axons in vitro , and implantation of differentiated Pde1c-Egfp- BAC transgenic ES cells into the external granule cell layer of neonatal mice resulted in the extension of parallel fibers, migration across the molecular layer, incorporation into the internal granule cell layer, and extension of short dendrites, typical of young granule cells forming synaptic connections with afferent mossy fibers. These results underscore the utility of treating ES cells with local, inductive signals that regulate CNS neuronal development in vivo as a strategy for cell replacement therapy of defined neuronal populations.

Casein Kinase 1δ (CK1δ) is a serione/threonine kinase required for cell cycle progression, circadian rhythm, vesicle trafficking, and neurite outgrowth [1]. CK1δ is also a therapeutic target in various cancers, Alzheimer's disease, alcoholism, and sleep disorders [1]. To examine the role of CK1δ in brain development, we used cerebellar granule cell progenitors (GCPs) as a model system. GCPs are the most abundant neurons in the mammalian brain and are one of two principal neurons in the cerebellar circuitry [2]. CK1δ is expressed in GCPs during peak times of proliferation (postnatal day 6-postnatal day 8). To probe a role for CK1δ in proliferation of GCPs during this time, we assayed proliferation in GCPs lacking CK1δ, after knockdown of CK1δ by RNAi methodology or in purified GCPs treated with highly specific CK1δ inhibitor [3]. In all three cases,3H-thymidine incorporation assays showed reduced levels of proliferation. Given CK1δ's role in GCP neurogenesis, we anticipated that CK1δ levels would decrease as GCPs exit the cell cycle. Indeed, we found that CK1δ protein but not mRNA levels dropped during cell cycle exit, which suggested that CK1δ is targeted for degradation during this time. Importantly, biochemical assays demonstrate that CK1δ is targeted for degradation via the Anaphase Promoting Complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase, which has well-established roles in mitotic exit and G1 progression [2]. APC/C is also active in differentiating and differentiated cells [4]. APC/C associates with one of two activators termed Cdc20 or Cdh1, which recruit substrates to bring them into close proximity of the E2 enzyme bound to APC/C [4]. We report that Cdh1 binds to CK1δ to initiate APC/C dependent ubiqutination. In vitro ubiquitination assays containing purified APC/C and CK1δ demonstrate that APC/C mediates CK1δ polyubiquitination in vitro. APC/C mediate ubiquitination of CK1δ was dependent on two N-terminal destruction boxes in CK1δ as mutation of these sites abrogated ubiquitination in vitro [2]. To demonstrate a requirement for CK1δ in vivo we deleted CK1δ in GCPs in the cerebellum [2]. Deletion of the APC/C activator Cdh1 in GCPs increased CK1δ levels, suggesting that CK1δ is turned over in GCPs [2]. Collectively, these studies suggest that APC/C targets CK1δ for destruction in vitro and in vivo and that APC/CCdh1 is an important regulator of GCP proliferation by controlling CK1δ. Our studies therefore suggest that APC/C-mediated degradation of CK1δ functions in multiple steps in CNS neuronal differentiation. CK1δ has been linked to neurite outgrowth [5] and thus it will be important to determine whether APC/C mediated degradation of CK1δ occurs in axons or dendrites. Prior studies demonstrated that APC/C inhibition in postmitotic neurons [4] increases neurite outgrowth while CK1δ inhibition reduces neurite outgrowth in cell lines [5]. Thus, CK1δ could be one of the substrates, which APC/C targets during neurite outgrowth, and whose levels rise during APC/C inhibition or depletion. It will be important to determine whether CK1δ protein levels are modulated by APC/C active in postmitotic neurons. Interestingly, there are two forms of the APC/C that are active in postmitotic neurons, APC/CCdh1 and APC/CCdc20 [4]. APC/CCdh1 represses axonal growth [4] while APC/CCdc20 activity controls dendritic morphogenesis [4]. APC/CCdc20 is localized to centrosomes in postmitotic neurons. Given the finding that CK1δ is localized to centrosomes [5] it will be interesting to determine whether APC/CCdc20 is able to induce CK1δ destruction at centrosomes. An alternative could be that centrosome bound CK1δ is protected from APC/C mediated degradation as other APC/C substrates cannot be ubiquitinated and degraded when bound to microtubules [6]. As centrosomal proteins often have roles in migration it will be important to determine whether APC/C mediated control of CK1δ is linked to migration of neuronal precursors. Consistent with a role for CK1δ in neuronal migration we found that CK1δ inhibition reduced GCP migration ex vivo (unpublished observations). Figure 1 Model of CK1δ and APC/C during proliferation and differentiation of GCPs In addition, since CK1δ has a role in ciliogenesis1 and APC/CCdc20 has been reported to be required for primary cilia formation [7], the APC/C may interact with CK1δ in primary cilia. It will be interesting to determine whether APC/CCdc20 induces CK1δ degradation within cilia. Interestingly, since the primary cilium is required for Hedgehog (Hh) pathway signaling as we found that CK1δ inhibition or disruption reduced Hh signaling in GCPs [2], it will be essential to determine whether the APC/C-CK1δ interaction is important for Hh signaling in GCPs. Future studies will determine the importance of the APC/C-CK1δ interaction in various signaling pathways including Hh and WNT, where CK1δ has been implicated [1]. Furthermore, it will be critical to determine whether the APC/C-CK1δ interaction is dysregulated in various neurological diseases.

The manuscript by Tsai et al. (935–945) is a tour de force analysis of a controversial issue in developmental neurobiology, namely the molecular basis of the devastating human brain malformation, type I lissencephaly (Lis1) (Jellinger, K., and A. Rett. 1976. Neuropadiatrie. 7:66–91). For several decades, defects in neuronal migration have been assumed to underlie all defects in cortical histogenesis. In the paper by Tsai et al., the authors use a variety of elegant approaches, including the first real-time imaging of cortical neurons with reduced levels of LIS1, to demonstrate that LIS1 and dynactin act as regulators of dynein during cortical histogenesis. A loss of LIS1 results in both a failure to exit the cortical germinal zone and abnormal neuronal process formation. Thus, the primary action of the mutation is to disrupt the production of neurons in the developing brain as well as their migration.

Over the past two decades, molecular genetic studies have enabled a common conceptual framework for the development and basic function of the nervous system. These studies, and the pioneering efforts of mouse geneticists and neuroscientists to identify and clone genes for spontaneous mouse mutants, have provided a paradigm for understanding complex processes of the vertebrate brain. Gene cloning for human brain malformations and degenerative disorders identified other important central nervous system (CNS) genes. However, because many debilitating human disorders are genetically complex, phenotypic screens are difficult to design. This difficulty has led to large-scale, genomic approaches to discover genes that are uniquely expressed in brain circuits and regions that control complex behaviors. In this review, we summarize current phenotype- and genotype-driven approaches to discover novel CNS-expressed genes, as well as current approaches to carry out large-scale, gene-expression screens in the CNS.

Neuronal migrations along glial fibers provide a primary pathway for the formation of cortical laminae. To examine the mechanisms underlying glial-guided migration, we analyzed the dynamics of cytoskeletal and signaling components in living neurons. Migration involves the coordinated two-stroke movement of a perinuclear tubulin 'cage' and the centrosome, with the centrosome moving forward before nuclear translocation. Overexpression of mPar6alpha disrupts the perinuclear tubulin cage, retargets PKCzeta and gamma-tubulin away from the centrosome, and inhibits centrosomal motion and neuronal migration. Thus, we propose that during neuronal migration the centrosome acts to coordinate cytoskeletal dynamics in response to mPar6alpha-mediated signaling.

The establishment of neural circuits in the spinal cord depends on the differentiation of functionally distinct types of neurons in the embryonic neural tube. A number of genes have recently been shown to control the generation of dorsal interneurons through inductive signals provided by the roof plate. The roof plate is a transient signaling center on the dorsal midline of the neural tube that coordinates dorsal CNS development through the action of local peptide signals, primarily the bone morphogenic proteins (BMPs) and the Wingless-related genes (Wnts). The role of the roof plate has become evident through studies of mutations of genes in these gene families, and through several spontaneously occurring mouse mutants, including dreher(J) (dr(J)), all of which cause dorsal neural tube defects. We previously demonstrated that the roof plate is missing in the dreher mouse. Positional cloning of the dreher locus demonstrated that an inactivating point mutation in the LIM homeodomain (HD) transcription factor encoded by the Lmx1a gene, is responsible for the dreher(J) phenotype [Nature, 403 (2000) 764]. Here we report that Lmx1a is first expressed at E8.5 in a small number of cells in the lateral neural plate. As the neural tube closes, Lmx1a expression is restricted to the roof plate. In dr(J)/dr(J), although non-functional Lmx1a is correctly expressed at E8.5-E9.5, its expression is lost in the spinal cord roof plate by E10.5. Coincident with the loss of Lmx1a expression, Bmp expression fails, and the generation and differentiation of the dorsal-most spinal cord neurons, the dl1 interneurons, is abnormal. In dr(J)/dr(J) embryos, defects are evident in the number of dl1 progenitors, as well as in their migration to form the lateral and medial nuclei, and axon patterning, through mechanisms that apparently involve defects in early steps of neuronal polarity. Consistent with the general hypothesis that a failure of roof plate formation and function results in deficits in dorsal patterning of the neural tube, the dreher affects the generation and differentiation of the dl1 interneuron population.

Previous studies showed that the serine/threonine kinase Unc51.1 is one of the earliest genes in neuronal differentiation and is required for granule cell axon formation. To examine the mechanism of Unc51.1 regulation of axon extension, we have identified two direct binding partners. The first, SynGAP, a negative regulator of Ras, is expressed within axons and growth cones of developing granule cells. Overexpression of SynGAP blocks neurite outgrowth by a mechanism that involves Ras-like GTPase cascade. The second binding partner is a PDZ domain-containing scaffolding protein, Syntenin, that binds Rab5 GTPase, the activity of which is attenuated by SynGAP. Thus, our results demonstrate that the Unc51.1-containing protein complex governs axon formation via Ras-like GTPase signaling and through regulation of the Rab5-mediated endocytic pathways within developing axons.

Disrupted-in-Schizophrenia-1 ( DISC-1 ) is a gene whose mutant truncation is associated with major psychiatric illness with a predominance of schizophrenic symptomatology. We have cloned and characterized rodent DISC-1. DISC-1 expression displays pronounced developmental regulation with the highest levels in late embryonic life when the cerebral cortex develops. In yeast two-hybrid analyses, DISC-1 interacts with a variety of cytoskeletal proteins. One of these, NudE-like (NUDEL), is associated with cortical development and is linked to LIS-1, the disease gene for a form of lissencephaly, a disorder of cortical development. The disease mutant form of DISC-1 fails to bind NUDEL. Expression of mutant, but not wild-type, DISC-1 in PC12 cells reduces neurite extension. As schizophrenia is thought to reflect defects in cortical development that are determined by cytoskeletal protein activities, the cellular disturbances we observe with mutant DISC-1 may be relevant to psychopathologic mechanisms.

Over the past decade, genetic analyses have yielded a more molecular view of neuronal migration and its role in central nervous system development. We now realize that many of the molecular mechanisms that guide migrations in invertebrates are recapitulated in the vertebrate nervous system. These mechanisms guide dorsoventral and anterior-posterior migrations and merge with radial migratory pathways that are prominent in the development of the mammalian cortex. This review discusses the choreography of these different migratory mechanisms within the context of genetic approaches that have defined their molecular mechanisms.

The cortical regions of the brain are laminated as a result of directed migration of precursor cells along glia during development. Previously, we have used an assay system to identify astrotactin as a neuronal ligand for migration on glial fibers. To examine the function of astrotactin in vivo, we generated a null mutation by targeted gene disruption. The cerebella of astrotactin null mice are approximately 10% smaller than wild type. In vitro and in vivo cerebellar granule cell assays show a decrease in neuron-glial binding, a reduction in migration rates and abnormal development of Purkinje cells. Consequences of this are poorer balance and coordination. Thus, astrotactin functions in migration along glial processes in vivo, a process required for generating laminar structures and for the development of synaptic partner systems.

In the developing cerebellar cortex, granule neuron precursors (GNPs) proliferate and commence differentiation in a superficial zone, the external granule layer (EGL). The molecular basis of the transition from proliferating precursors to immature differentiating neurons remains unknown. Notch signaling is an evolutionarily conserved pathway regulating the differentiation of precursor cells of many lineages. Notch2 is specifically expressed in proliferating GNPs in the EGL. Treatment of GNPs with soluble Notch ligand Jagged1, or overexpression of activated Notch2 or its downstream target HES1, maintains precursor proliferation. The addition of GNP mitogens Jagged1 or Sonic Hedgehog (Shh) upregulates the expression of HES1, suggesting a role for HES1 in maintaining precursor proliferation.

In the vertebrate central nervous system (CNS), a cascade of signals that originates in the ectoderm adjacent to the neural tube is propagated by the roof plate to dorsalize the neural tube. Here we report that the phenotype of the spontaneous neurological mutant mouse dreher (dr) results from a failure of the roof plate to develop. Dorsalization of the neural tube is consequently affected: dorsal interneurons in the spinal cord and granule neurons in the cerebellar cortex are lost, and the dorsal vertebral neural arches fail to form. Positional cloning of dreher indicates that the LIM homeodomain protein, Lmx1a, is affected in three different alleles of dreher. Lmx1a is expressed in the roof plate along the neuraxis during development of the CNS. Thus, Lmx1a is required for development of the roof plate and, in turn, for specification of dorsal cell fates in the CNS and developing vertebrae.

SUMMARY We have used a combination of quail-chick fate-mapping techniques and dye labelling to investigate the development of the avian cerebellum. Using Hoxa2 as a guide for the microsurgical construction of quail-chick chimaeras, we show that the caudal boundary of the presumptive cerebellum at E6 maps to the caudal boundary of rhombomere 1. By fate mapping the dorsoventral axis of rhombomere 1, we demonstrate that granule cell precursors are generated at the rhombic lip together with neurons of the lateral pontine nucleus. DiI-labelling of cerebellum explants reveals that external germinal layer precursors have a characteristic unipolar morphology and undergo an orientated, active migration away from the rhombic lip, which is apparently independent of either glial or axon guidance or ‘chain’ formation.

Cerebellar granule neurons, the most abundant class of CNS neurons, have a critical role in cerebellar function. Granule neurons are generated at the dorsal border of the mesencephalon and metencephalon, the rhombic lip. In the mouse embryo, rhombic lip cells express a number of granule neuron markers, notably the bHLH transcription factor Math1. Dorsal midline cells adjacent to the rhombic lip express Bmp6, Bmp7 and Gdf7, three genes encoding peptide growth factors of the bone morphogenetic protein (BMP) family. These BMPs induced the expression of granule neuron markers in cultured neural tissue. Moreover, BMP-treated neural cells formed mature granule neurons after transplantation into the early postnatal cerebellum, suggesting that BMPs initiate the program of granule cell specification.

The conservation of transcriptional regulatory mechanisms across species, combined with the restricted expression of these molecules in time and space within the embryo, has offered new insights into CNS cell specification. Studies examining transcriptional control in the generation of specific cell classes within the cerebellar cortex have been particularly elucidative.

Axon formation in developing cerebellar granule neurons in situ is spatially and temporally segregated from subsequent neuronal migration and dendrite formation. To examine the role of local environmental cues on early steps in granule cell differentiation, the sequence of morphologic development and polarized distribution of membrane proteins was determined in granule cells isolated from contact with other cerebellar cell types. Granule cells cultured at low density developed their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, first extending a unipolar process, then long, thin bipolar axons, and finally becoming multipolar, forming short dendrites around the cell body. Axonal- and dendritic-specific cytoskeletal markers were segregated to the morphologically distinct domains. The cell surface distribution of a specific class of endogenous glycoproteins, those linked to the membrane by a glycosylphosphatidyl inositol (GPI) anchor, was also examined. The GPI-anchored protein, TAG-1, which is segregated to the parallel fiber axons in situ, was found exclusively on granule cell axons in vitro; however, two other endogenous GPI-anchored proteins were found on both the axonal and somatodendritic domains. These results demonstrate that granule cells develop polarity in a cell type-specific manner in the absence of the spatial cues of the developing cerebellar cortex. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 223–236, 1997.

Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI:http://dx.doi.org/10.7554/eLife.00400.001.

The specification of diverse classes of neurons is critical to the development of the cerebellar cortex. Here, we describe the purification of early embryonic precursors of cerebellar granule neurons from the rhombic lip, the dorsal aspect of the midbrain/hindbrain region. Isolation of rhombic lip cells reveals a homogenous population of precursor cells that express general neuronal markers and the granule cell marker RU49, but fail to extend neurites or express differentiation markers. Differentiation is induced by coculture with external germinal layer (EGL) cells, or their membranes, suggesting that a local inducing factor acts after formation of the EGL. Thus, proliferating precursors within the rhombic lip are specified to be granule cells very early, with the availability of an inducing factor increasing over the course of development.

In the neurological mutant mouse weaver , granule cell precursors proliferate normally in the external germinal layer of the cerebellar cortex, but fail to differentiate. Granule neurons purified from weaver cerebella have greatly reduced G protein–activated inwardly rectifying K + currents; instead, they display a constitutive Na + conductance. Expression of the weaver GIRK2 channel in oocytes confirms that the mutation leads to constitutive activation, loss of monovalent cation selectivity, and increased sensitivity to three channel blockers. Pharmacological blockade of the Na + influx in weaver granule cells restores their ability to differentiate normally. Thus, Na + flux through the weaver GIRK2 channel underlies the failure of granule cell development in situ.

To characterize CNS neuronal precursor migration along astroglial fibers, we examined the motility of the migratory leading process and cytoskeletal-based mechanisms of locomotion of early postnatal mouse cerebellar granule neurons in vitro. To visualize the surface motility of the leading process, granule neurons were labeled with the fluorescent lipophilic dye, PKH-26, and imaged by time lapse fluorescence microscopy. The motile behavior and cytoskeletal organization of the migrating neuron had several distinctive features. As the migrating neuron moved along the glial fiber, the leading process rapidly extended, projecting up to 40 microns, and retracted, withdrawing towards the cell soma. Broad lamellipodia were common along the entire length of the leading process, giving it a ruffled appearance. Within the cell soma, a cage-like distribution of microtubules encircled the nucleus and actin filaments formed a subcortical rim underneath the plasma membrane. Disruption of actin filaments with cytochalasin B inhibited migration, suggesting involvement of actin subunit assembly in neuronal migration. Both microtubules and actin filaments were heavily concentrated in the leading process; the leading process did not show the development of a distinct actin-rich domain at its tip.

In vitro methods for studying interactions between axons and their target cells are presented. The methods maximize the number of cultures that can be produced by limiting the volume and area of the cultures. Small cultures promote cell-cell Interactions and permit rapid conditioning of medium. In addition, valuable reagents added to these microcultures are conserved. The methods include: (a) the manufacture of 40-μl well-volume, coverslip-bottomed culture dishes with plating area of less than 24 mm 2 the dishes allow the small working distances of high-resolution light microscopy; (b) a micromethod to test for the Involvement of secreted factors in cell-cell interactions; cells on different surfaces are cocultured in shared medium; (c) a method to plate explant sources of neurites at a controlled distance from target cells to facilitate neurite identification and to control the timing of growth cone-target cell contacts; and (d) nonisotopic in situ hybridization for chamber-slide cultures combined with immunolabeling of cells in the hybridized culture. These methods can be used in culture assays to identify cell types or molecules involved in a variety of neuronal or, more generally, cell-cell interactions.