A spatial-temporal map of glutamatergic neurogenesis in the murine embryonic cerebellar nuclei uncovers a high degree of cellular heterogeneity.

The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate-mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral-to-medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations.

Cerebellum Lecture: the Cerebellar Nuclei-Core of the Cerebellum.

The cerebellum is a key player in many brain functions and a major topic of neuroscience research. However, the cerebellar nuclei (CN), the main output structures of the cerebellum, are often overlooked. This neglect is because research on the cerebellum typically focuses on the cortex and tends to treat the CN as relatively simple output nuclei conveying an inverted signal from the cerebellar cortex to the rest of the brain. In this review, by adopting a nucleocentric perspective we aim to rectify this impression. First, we describe CN anatomy and modularity and comprehensively integrate CN architecture with its highly organized but complex afferent and efferent connectivity. This is followed by a novel classification of the specific neuronal classes the CN comprise and speculate on the implications of CN structure and physiology for our understanding of adult cerebellar function. Based on this thorough review of the adult literature we provide a comprehensive overview of CN embryonic development and, by comparing cerebellar structures in various chordate clades, propose an interpretation of CN evolution. Despite their critical importance in cerebellar function, from a clinical perspective intriguingly few, if any, neurological disorders appear to primarily affect the CN. To highlight this curious anomaly, and encourage future nucleocentric interpretations, we build on our review to provide a brief overview of the various syndromes in which the CN are currently implicated. Finally, we summarize the specific perspectives that a nucleocentric view of the cerebellum brings, move major outstanding issues in CN biology to the limelight, and provide a roadmap to the key questions that need to be answered in order to create a comprehensive integrated model of CN structure, function, development, and evolution.

ZFP423 regulates early patterning and multiciliogenesis in the hindbrain choroid plexus.

The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423, which encodes a Kruppel-type zinc-finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia/Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate, from which the IV ventricle ChP arises, and, later, in mesenchymal cells, which give rise to the stroma and leptomeninges. Mouse Zfp423 mutants display a marked reduction of the hindbrain ChP (hChP), which: (1) fails to express established markers of its secretory function and genes implicated in its development and maintenance (Lmx1a and Otx2); (2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); and (3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis (Gmnc). Our results propose that Zfp423 is a master gene and one of the earliest known determinants of hChP development.

Ablation of Projection Glutamatergic Neurons in the Lateral Cerebellar Nuclei Alters Motor Coordination in Vglut2-Cre+ Mice.

Cerebellar nuclei (CN) constitute the sole cerebellar output to the rest of the central nervous system and play a central role in cerebellar circuits. Accumulating evidence from both human genetics and animal studies point to a crucial role for CN connectivity in neurological diseases, including several types of ataxia. However, because of the compact and restricted topography and close functional connection between the CN and the cerebellar cortex, identifying cerebellar deficits exclusively linked to CN is challenging. In this study, we have experimentally ablated large projection glutamatergic neurons of the lateral CN and evaluated the impact of this selective manipulation on motor coordination in mice. To this end, through stereotaxic surgery, we injected the lateral CN of Vglut2-Cre+ mice with an adeno-associated virus (AAV) encoding a Cre-dependent diphtheria toxin receptor (DTR), followed by an intraperitoneal injection of diphtheria toxin (DT) to ablate the glutamatergic neurons of the lateral nucleus. Double immunostaining of cerebellar sections with anti-SMI32 and -GFP antibodies revealed GFP expression and provided evidence of SMI32+ neuron degeneration at the site of AAV injection in the lateral nucleus of Vglut2-Cre+ mice. No changes were observed in Vglut2-Cre negative mice. Analysis of motor coordination by rotarod test indicated that the latency to fall was significantly different before and after AAV/DT injection in the Vglut2-Cre+ group. Elapsed time and number of steps in the beam walking test were significantly higher in AAV/DT injected Vglut2-Cre+ AAV/DT mice compared to controls. We demonstrate for the first time that partial degeneration of glutamatergic neurons in the lateral CN is sufficient to induce an ataxic phenotype.

The First 50 Years of Postnatal Neurogenesis in the Cerebellum: a Long Journey Across Phenomena, Mechanisms, and Human Disease.

The discovery by Altman and coworkers of adult-born microneurons in the olfactory bulb and dentate gyrus has triggered a long stream of studies and many attempts to harness adult neurogenesis, promote regeneration after injury, and contrast cognitive decline in the elderly. Likewise, the discovery of postnatal neurogenesis in the cerebellum has provided the framework for many subsequent molecular studies, including investigations of developmental processes and the assessment of GC progenitor (GCP) clonal expansion in the context of human disease. Here, I will briefly discuss some of the discoveries made in the field of cerebellar development over the years building upon the findings of Altman and his colleagues, touching upon signaling pathways that regulate granule cell neurogenesis and their involvement in developmental and neoplastic disorders of the cerebellum.

Zfp423/ZNF423 regulates cell cycle progression, the mode of cell division and the DNA-damage response in Purkinje neuron progenitors.

The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.

Dynamic Expression and New Functions of Early B Cell Factor 2 in Cerebellar Development.

The collier/Olf1/EBF family genes encode helix-loop-helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls, fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.

Layer 4 pyramidal neurons exhibit robust dendritic spine plasticity in vivo after input deprivation.

Pyramidal neurons in layers 2/3 and 5 of primary somatosensory cortex (S1) exhibit somewhat modest synaptic plasticity after whisker input deprivation. Whether neurons involved at earlier steps of sensory processing show more or less plasticity has not yet been examined. Here, we used longitudinal in vivo two-photon microscopy to investigate dendritic spine dynamics in apical tufts of GFP-expressing layer 4 (L4) pyramidal neurons of the vibrissal (barrel) S1 after unilateral whisker trimming. First, we characterize the molecular, anatomical, and electrophysiological properties of identified L4 neurons in Ebf2-Cre transgenic mice. Next, we show that input deprivation results in a substantial (∼50%) increase in the rate of dendritic spine loss, acutely (4-8 d) after whisker trimming. This robust synaptic plasticity in L4 suggests that primary thalamic recipient pyramidal neurons in S1 may be particularly sensitive to changes in sensory experience. Ebf2-Cre mice thus provide a useful tool for future assessment of initial steps of sensory processing in S1.

Neurogenin 2 regulates progenitor cell-cycle progression and Purkinje cell dendritogenesis in cerebellar development.

By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.

Rac1 and rac3 GTPases control synergistically the development of cortical and hippocampal GABAergic interneurons.

The intracellular mechanisms driving postmitotic development of cortical γ-aminobutyric acid (GABA)ergic interneurons are poorly understood. We have addressed the function of Rac GTPases in cortical and hippocampal interneuron development. Developing neurons express both Rac1 and Rac3. Previous work has shown that Rac1 ablation does not affect the development of migrating cortical interneurons. Analysis of mice with double deletion of Rac1 and Rac3 shows that these GTPases are required during postmitotic interneuron development. The number of parvalbumin-positive cells was affected in the hippocampus and cortex of double knockout mice. Rac depletion also influences the maturation of interneurons that reach their destination, with reduction of inhibitory synapses in both hippocampal CA1 and cortical pyramidal cells. The decreased number of cortical migrating interneurons and their altered morphology indicate a role of Rac1 and Rac3 in regulating the motility of cortical interneurons, thus interfering with their final localization. While electrophysiological passive and active properties of pyramidal neurons including membrane capacity, resting potential, and spike amplitude and duration were normal, these cells showed reduced spontaneous inhibitory currents and increased excitability. Our results show that Rac1 and Rac3 contribute synergistically to postmitotic development of specific populations of GABAergic cells, suggesting that these proteins regulate their migration and differentiation.

Early B-cell factors 2 and 3 (EBF2/3) regulate early migration of Cajal-Retzius cells from the cortical hem.

Cajal-Retzius (CR) cells play a crucial role in the formation of the cerebral cortex, yet the molecules that control their development are largely unknown. Here, we show that Ebf transcription factors are expressed in forebrain signalling centres-the septum, cortical hem and the pallial-subpallial boundary-known to generate CR cells. We identified Ebf2, through fate mapping studies, as a novel marker for cortical hem- and septum-derived CR cells. Loss of Ebf2 in vivo causes a transient decrease in CR cell numbers on the cortical surface due to a migratory defect in the cortical hem, and is accompanied by upregulation of Ebf3 in this and other forebrain territories that produce CR cells, without affecting proper cortical lamination. Accordingly, using in vitro preparations, we demonstrated that both Ebf2 and Ebf3, singly or together, control the migration of CR cells arising in the cortical hem. These findings provide evidence that Ebfs directly regulate CR cell development.

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments.

Neural stem (NS) cells are a self-renewing population of symmetrically dividing multipotent radial glia-like stem cells, characterized by homogeneous expansion in monolayer. Here we report that fetal NS cells isolated from different regions of the developing mouse nervous system behave in a similar manner with respect to self-renewal and neuropotency, but exhibit distinct positional identities. For example, NS cells from the neocortex maintain the expression of anterior transcription factors, including Otx2 and Foxg1, while Hoxb4 and Hoxb9 are uniquely found in spinal cord-derived NS cells. This molecular signature was stable for over 20 passages and was strictly linked to the developmental stage of the donor, because only NS cells derived from E14.5 cortex, and not those derived from E12.5 cortex, carried a consistent transcription factor profile. We also showed that traits of this positional code are maintained during neuronal differentiation, leading to the generation of electrophysiologically active neurons, even if they do not acquire a complete neurochemical identity.

ZFP423 coordinates Notch and bone morphogenetic protein signaling, selectively up-regulating Hes5 gene expression.

Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.

Both Schwann cell and axonal defects cause motor peripheral neuropathy in Ebf2-/- mice.

Charcot-Marie-Tooth neuropathies are frequent hereditary disorders of the nervous system and most cases remain without a molecular definition. Mutations in transcription factors have been previously associated to various types of this disease. Mice carrying a null mutation in Ebf2 transcription factor present peripheral nerve abnormalities. To get insight into Ebf2 function in peripheral nervous system, here we characterize the peripheral neuropathy affecting these mice. We first show that Ebf2 is largely expressed in peripheral nerve throughout postnatal development, its expression being not only restricted to non-myelin forming Schwann cells, but also involving myelin forming Schwann cells and the perineurium. As a consequence, the onset of myelination is delayed and Schwann cell differentiation markers are downregulated in Ebf2-/- mice. Later in development, myelin pathology appears less severe and characterized by isolated clusters of hypomyelinated fibers. However, we find defects in the nerve architecture, such as abnormalities of the nodal region and shorter internodal length. Furthermore, we demonstrate a significant decrease in axonal calibre, with a lack of large calibre axons, and a severe impairment of motor nerve conduction velocity and amplitude, whereas the sensory nerve parameters are less affected. Interestingly, a clinical case with peripheral motor neuropathy and clinical features similar to Ebf2-/- mice phenotype was associated with a deletion encompassing EBF2 human genomic locus. These findings demonstrate that Ebf2 is a new molecule implicated in peripheral nerve development and a potential candidate gene for peripheral nerve disorders.

Expression of mutant beta2 nicotinic receptors during development is crucial for epileptogenesis.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal form of epilepsy characterized by seizures occurring during non-REM sleep. We have developed and characterized the first mouse model for ADNFLE type III carrying the V287L mutation of the beta2 subunit of neuronal nicotinic receptor. Mice expressing mutant receptors show a spontaneous epileptic phenotype by electroencephalography with very frequent interictal spikes and seizures. Expression of the mutant beta2 subunit is driven by a neuronal-specific tetracycline-controlled promoter, which allows planned silencing of transgene expression in a reversible fashion and tracking the involvement of mutant receptor in crucial phases of epileptogenesis. We found that restricted silencing during development is sufficient to prevent the occurrence of epileptic seizures in adulthood. Our data indicate that mutant nicotinic receptors are responsible for abnormal formation of neuronal circuits and/or long-lasting alteration of network assembly in the developing brain, thus leading to epilepsy.

Upregulation of Neural Cell Adhesion Molecule 1 and Excessive Migration of Purkinje Cells in Cerebellar Cortex.

Purkinje cells (PCs) are large GABAergic projection neurons of the cerebellar cortex, endowed with elaborate dendrites that receive a multitude of excitatory inputs. Being the only efferent neuron of the cerebellar cortex, PCs project to cerebellar nuclei and control behaviors ranging from movement to cognition and social interaction. Neural cell adhesion molecule 1 (NCAM1) is widely expressed in the embryonic and postnatal development of the brain and plays essential roles in neuronal migration, axon pathfinding and synapse assembly. However, despite its high expression levels in cerebellum, little is known to date regarding the role(s) of NCAM1 in PCs development. Among other aspects, elucidating how the expression of NCAM1 in PCs could impact their postnatal migration would be a significant achievement. We analyzed the Acp2 mutant mouse (nax: naked and ataxia), which displays excessive PC migration into the molecular layer, and investigated how the excessive migration of PCs along Bergmann glia could correlate to NCAM1 expression pattern in early postnatal days. Our Western blot and RT-qPCR analysis of the whole cerebellum show that the protein and mRNA of NCAM1 in wild type are not different during PC dispersal from the cluster stage to monolayer formation. However, RT-qPCR analysis from FACS-based isolated PCs shows that Ncam1 is significantly upregulated when PCs fail to align and instead overmigrate into the molecular layer. Our results suggest two alternative interpretations: (1) NCAM1 promotes excessive PC migration along Bergmann glia, or (2) NCAM1 upregulation is an attempt to prevent PCs from invading the molecular layer. If the latter scenario proves true, NCAM1 may play a key role in PC monolayer formation.

EBF2 regulates osteoblast-dependent differentiation of osteoclasts.

Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts.

Origins, Development, and Compartmentation of the Granule Cells of the Cerebellum.

Granule cells (GCs) are the most numerous cell type in the cerebellum and indeed, in the brain: at least 99% of all cerebellar neurons are granule cells. In this review article, we first consider the formation of the upper rhombic lip, from which all granule cell precursors arise, and the way by which the upper rhombic lip generates the external granular layer, a secondary germinal epithelium that serves to amplify the upper rhombic lip precursors. Next, we review the mechanisms by which postmitotic granule cells are generated in the external granular layer and migrate radially to settle in the granular layer. In addition, we review the evidence that far from being a homogeneous population, granule cells come in multiple phenotypes with distinct topographical distributions and consider ways in which the heterogeneity of granule cells might arise during development.