Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin

GABAergic Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture. Although the mechanisms of PC differentiation have been well studied, the precise origin and initial specification mechanism of PCs remain to be clarified. Here, we identified a cerebellar and spinal cord GABAergic progenitor-selective cell surface marker, Neph3, which is a direct downstream target gene of Ptf1a, an essential regulator of GABAergic neuron development. Using FACS, Neph3⁺ GABAergic progenitors were sorted from the embryonic cerebellum, and the cell fate of this population was mapped by culturing in vitro. We found that most of the Neph3⁺ populations sorted from the mouse E12.5 cerebellum were fated to differentiate into PCs while the remaining small fraction of Neph3⁺ cells were progenitors for Pax2⁺ interneurons, which are likely to be deep cerebellar nuclei GABAergic neurons. These results were confirmed by short-term in vivo lineage-tracing experiments using transgenic mice expressing Neph3 promoter-driven GFP. In addition, we identified E-cadherin as a marker selectively expressed by a dorsally localized subset of cerebellar Neph3⁺ cells. Sorting experiments revealed that the Neph3⁺ E-cadherin^high population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2⁺ interneurons were enriched in the Neph3⁺ E-cadherin^low population. Taken together, our results identify two spatially demarcated subregions that generate distinct cerebellar GABAergic subtypes and reveal the origin of PCs in the ventricular zone of the cerebellar primordium.     

Localization of the type 1 corticotropin releasing factor receptor (CRF-R1) in the embryonic mouse cerebellum

Corticotropin releasing factor (CRF) is present in the adult, as well as in the embryonic and postnatal rodent cerebellum. Further, the distribution of the type 1 CRF receptor has been described in adult and postnatal animals. The focus of the present study is to determine the distribution and cellular relationships of the type 1 CRF receptor (CRF-R1) during embryonic development of the cerebellum. Between embryonic day (E)11 and E12, CRF-R1 immunoreactive puncta are uniformly distributed in the ventricular zone, the site of origin of Purkinje cells, nuclear neurons, and GABAergic interneurons, as well as the germinal trigone, the birthplace of the precursors of granule cells. Between E13 and 18, the distribution of immunolabeled puncta decreases in both the ventricular zone and the germinal trigone and increases in the intermediate zone, as well as in the dorsal aspect of the cerebellar plate. Between E14 and 18, antibodies that label specific populations of cerebellar neurons were combined with the antibody for the receptor to determine the cellular elements that expressed CRF-R1. At E14, CRF-R1 immunoreactivity is co-localized in neurons immunolabeled with PAX-2, an antibody that is specific for GABAergic interneurons. These neurons continue to express CRF-R1 as they migrate dorsally toward the cerebellar surface. Between E16 and 18, Purkinje cells, immunolabeled with calbindin, near the dorsal surface of the cerebellum express CRF-R1 in their cell bodies and apical processes. CRF has been shown to have a depolarizing effect on adult and postnatal Purkinje cells. Further, CRF has been shown to contribute to excitability of hippocampal neurons during embryonic development by binding to CRF-R1; depolarization induced excitability appears to be critical for cell survival. The location of the type one CRF receptor and the presence of its primary ligand, CRF, in the germinal zones of the cerebellum and in migrating neurons suggest that this receptor/ligand interaction could be important in the regulation of neuronal survival through cellular mechanisms that lead to depolarization of embryonic cerebellar neurons.     

Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

Anatomy of zebrafish cerebellum and screen for mutations affecting its development

The cerebellum is important for the integration of sensory perception and motor control, but its structure has mostly been studied in mammals. Here, we describe the cell types and neural tracts of the adult zebrafish cerebellum using molecular markers and transgenic lines. Cerebellar neurons are categorized to two major groups: GABAergic and glutamatergic neurons. The Purkinje cells, which are GABAergic neurons, express parvalbumin7, carbonic anhydrase 8, and aldolase C like (zebrin II). The glutamatergic neurons are vglut1⁺ granule cells and vglut2^high cells, which receive Purkinje cell inputs; some vglut2^high cells are eurydendroid cells, which are equivalent to the mammalian deep cerebellar nuclei. We found olig2⁺ neurons in the adult cerebellum and ascertained that at least some of them are eurydendroid cells. We identified markers for climbing and mossy afferent fibers, efferent fibers, and parallel fibers from granule cells. Furthermore, we found that the cerebellum-like structures in the optic tectum and antero-dorsal hindbrain show similar Parvalbumin7 and Vglut1 expression profiles as the cerebellum. The differentiation of GABAergic and glutamatergic neurons begins 3 days post-fertilization (dpf), and layers are first detectable 5 dpf. Using anti-Parvalbumin7 and Vglut1 antibodies to label Purkinje cells and granule cell axons, respectively, we screened for mutations affecting cerebellar neuronal development and the formation of neural tracts. Our data provide a platform for future studies of zebrafish cerebellar development.     

Subcellular and developmental expression of alternatively spliced forms of fibroblast growth factor 14

Fibroblast growth factors (FGFs) 11–14 comprise a subfamily of FGFs with poorly defined biological function. Here we characterize two isoforms of FGF14 (FGF14-1a and FGF14-1b) that result from the alternative usage of two different first exons. We demonstrate that these isoforms have differential subcellular localization and that they are differentially expressed in various adult tissues. Using in situ hybridization we show that Fgf14 is widely expressed in brain, spinal cord, major arteries and thymus between 12.5 and 14.5 days of mouse embryonic development. We also show that during cerebellar development, Fgf14 is first observed at postnatal day 1 in post mitotic granule cells, and later in development, in migrating and post migratory granule cells. The developmental expression pattern of Fgf14 in the cerebellum is complementary to that of Math1, a marker for proliferating granule cells in the external germinal layer.     

Astroglial cells provide a template for the positioning of developing cerebellar neurons in vitro

Indirect immunocytochemical staining with antisera raised against purified glial filament protein and a neurofilament polypeptide was used to study cell interactions between astrocytes and neurons dissociated from embryonic and early postnatal cerebellum. Staining with antibodies raised against purified glial filament protein revealed that greater than 99% of all processes present in cerebellar cultures during the 1st wk in vitro were glial in origin. After 1 wk in culture, unstained processes that were presumably neuronal were observed. Stained astroglial processes formed a dense network that served as a template for cerebellar neurons, identified by indirect immunocytochemical localization of tetanus toxin. More than 90% of neurons from postnatal days 1 or 7 were positioned within one cell diameter of a glial process. In contrast, less than 40% of the neurons dissociated from early embryonic cerebellum were located adjacent to a glial process. Staining with antibodies raised against purified glial filament protein also revealed differences in astroglial morphology that were under developmental regulation. Astroglial cells from embryonic cerebellum were fewer in number and had thick, unbranched processes. Those from postnatal day 1 were more slender, branched, and stellate. Those from postnatal day 7 were highly branched and stellate. Some veil-like astroglial processes were also observed in cells from postnatal animals. These morphological changes were also observed when cells from embryonic day 13 were maintained for a week in vitro. No specific staining of embryonic or postnatal cerebellum cells was observed with antibodies raised against purified neurofilament polypeptides.     

Cerebellum- and forebrain-derived stem cells possess intrinsic regional character

The existence of stem cells in the adult nervous system is well recognized; however, the potential of these cells is still widely debated. We demonstrate that neural stem cells exist within the embryonic and adult cerebellum. Comparing the potential of neural stem cells derived from the forebrain and cerebellum, we find that progeny derived from each of these brain regions retain regional character in vitro as well as after homotopic transplantation. However, when ectopically transplanted, neurosphere-derived cells from either region are largely unable to generate neurons. With regard specifically to embryonic and adult cerebellar stem cells, we observe that they are able to give rise to neurons that resemble different select classes of cerebellar subclasses when grafted into the perinatal host cerebellum. Most notably, upon transplantation to the perinatal cerebellum, cerebellar stem cells from all ages are able to acquire the position and mature electrophysiological properties of cerebellar granule cells.     

Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family

By DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase Calpha (PKCalpha) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCalpha activation in PCs.     

Induction of excitatory and inhibitory presynaptic differentiation by GluD1

The δ subfamily of ionotropic glutamate receptor subunits consists of GluD1 and GluD2. GluD2, which is selectively expressed in cerebellar Purkinje neurons, has been shown to contribute to the formation of synapses between granule neurons and Purkinje neurons through interaction with Cbln1 (cerebellin precursor protein1) and presynaptic Neurexin. On the other hand, the synaptogenic activity of GluD1, which is expressed not in the cerebellum but in the hippocampus, remains to be characterized. Here, we report that GluD1 expressed in non-neuronal HEK cells, induced presynaptic differentiation of granule neurons through its N-terminal domain in co-cultures with cerebellar neurons, similarly to GluD2. We also show that GluD1 rescued the defect of synapse formation in GluD2-knockout Purkinje neurons, indicating the functional similarity of GluD1 and GluD2. In contrast, GluD1 expression alone did not induce presynaptic differentiation in co-cultures of HEK cells with hippocampal neurons. However, when Cbln1 was exogenously added to the culture medium, GluD1 induced presynaptic differentiation of not only glutamatergic presynaptic terminals but also GABAergic ones. Cbln1 is not expressed in hippocampal neurons but is expressed in entorhinal cortical neurons projecting to the hippocampus. In co-cultures of HEK cells expressing GluD1 and entorhinal cortical neurons, both glutamatergic and GABAergic presynaptic terminals were formed on the HEK cells without exogenous application of Cbln1. These results suggest that GluD1 might contribute to the formation of specific synapses in the hippocampus such as those formed by the projecting neurons of the entorhinal cortex.     

Activation of Wnt/β-Catenin Signalling Affects Differentiation of Cells Arising from the Cerebellar Ventricular Zone

Development of the cerebellum proceeds under the precise spatio-temporal control of several key developmental signalling pathways, including the Wnt/β-catenin pathway. We recently reported the activity of Wnt/β-catenin signalling in the perinatal cerebellar ventricular zone (VZ), a germinal centre in the developing cerebellum that gives rise to GABAergic and glial cells. In order to investigate the normal function of Wnt/β-catenin signalling in the VZ and the cell lineages it gives rise to, we used a combination of ex vivo cerebellar slice culture and in vivo genetic manipulation to dysregulate its activity during late embryonic development. Activation of the pathway at the cerebellar ventricular zone led to a reduction in the number of cells expressing the glial lineage markers Sox9 and GFAP and the interneuron marker Pax2, but had no consistent effect on either proliferation or apoptosis. Our findings suggest that activation of the Wnt/β-catenin pathway in the cerebellar ventricular zone causes a shift in the cell types produced, most likely due to disruption of normal differentiation. Thus, we propose that regulation of Wnt/β-catenin signalling levels are required for normal development of cells arising from the cerebellar ventricular zone during late embryogenesis.     

ABCA2 is a marker of neural progenitors and neuronal subsets in the adult rodent brain

The notion that the ATP-binding cassette transporter-A2 (ABCA2) may be involved in brain sterol homeostasis and is associated with early onset Alzheimer's disease led us to explore its neural expression. Our data support and extend the previous reports on ABCA2 expression by oligodendrocytes. They evidence that ABCA2 (i) is located in intracellular vesicles, identified in transfected cells as lysosome-related organelles only partially overlapping with classical endolysosomes; (ii) is a marker of neural progenitors as it is expressed in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampal formation, sites of continual neurogenesis in the adult brain, and in nestin(+) cells differentiated in vitro from embryonic stem cells; (iii) persists, in the adult rodent brain, in a subset of GABAergic and glutamatergic neurons. Considering that the latter are targets of Alzheimer's lesions, these data provide a new rationale to explore the neuropathological consequences of ABCA2 functional dysregulations.     

Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum

Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input - that from the inferior olive via climbing fibres and that from the granule neurons via parallel fibres. The precursors of granule neurons proliferate at the surface of the developing cerebellumin the external granule layer (EGL), which persists until postnatal day 14 in the mouse [1]. PCs are thought to provide trophic support for granule neurons [2][3] and to stimulate the proliferation of cells in the EGL [4], but the signalling molecules that mediate these cell-cell interactions have not been identified. I show here that PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and that dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes of which expression is upregulated in response to Hedgehog signalling (see [5] and references therein). Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies [6] disrupted cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulated BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL.     

Age-related changes of structures in cerebellar cortex of cat

We studied the structures of the cerebellar cortex of young adult and old cats for age-related changes, which were statistically analysed. Nissl staining was used to visualize the cortical neurons. The immunohistochemical method was used to display glial fibrillary acidic protein (GFAP)-immunoreactive (IR) astrocytes and neurofilament-immunoreactive (NF-IR) neurons. Under the microscope, the thickness of the cerebellar cortex was measured; and the density of neurons in all the layers as well as that of GFAP-IR cells in the granular layer was analysed. Compared with young adult cats, the thickness of the molecular layer and total cerebellar cortex was significantly decreased in old cats, and that of the granular layer increased. The density of neurons in each layer was significantly lower in old cats than in young adult ones. Astrocytes in old cats were significantly denser than in young adult ones, and accompanied by evident hypertrophy of the cell bodies and enhanced immunoreaction of GFAP substance. Purkinje cells (PCs) in old cats showed much fewer NF-IR dendrites than those in young adults. The above findings indicate a loss of neurons and decrease in the number of dendrites of the PCs in the aged cerebellar cortex, which might underlie the functional decline of afferent efficacy and information integration in the senescent cerebellum. An age-dependent enhancement of activity of the astrocytes may exert a protective effect on neurons in the aged cerebellum