Seung Hyuk Chung

A key role for the HLH transcription factor EBF2COE2,O/E-3 in Purkinje neuron migration and cerebellar cortical topography

Early B-cell factor 2 (EBF2) is one of four mammalian members of an atypical helix-loop-helix transcription factor family (COE). COE proteins have been implicated in various aspects of nervous and immune system development. We and others have generated and described mice carrying a null mutation of Ebf2, a gene previously characterized in the context of Xenopus laevis primary neurogenesis and neuronal differentiation. In addition to deficits in neuroendocrine and olfactory development, and peripheral nerve maturation, Ebf2 null mice feature an ataxic gait and obvious motor deficits associated with clear-cut abnormalities of cerebellar development. The number of Purkinje cells (PCs) in the Ebf2 null is markedly decreased, resulting in a small cerebellum with notable foliation defects, particularly in the anterior vermis. We show that this stems from the defective migration of a molecularly defined PC subset that subsequently dies by apoptosis. Part of the striped cerebellar topography is disrupted due to cell death and, in addition, many of the surviving PCs, that would normally adopt a zebrin II-negative phenotype, transdifferentiate to Zebrin II-positive, an unprecedented finding suggesting that Ebf2 is required for the establishment of a proper cerebellar cortical map.

Phospholipase cβ4 expression reveals the continuity of cerebellar topography through development

Mediolateral boundaries divide the mouse cerebellar cortex into four transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. Various expression markers reveal this complexity, and detailed maps have been constructed based on the differential expression of zebrin II/aldolase C in a Purkinje cell subset. Recently, phospholipase (PL) Cβ4 expression in adult mice was shown to be restricted to, and coextensive with, the zebrin II‐immunonegative Purkinje cell subset. The Purkinje cell expression of PLCβ4 during embryogenesis and postnatal development begins just before birth in a subset of Purkinje cells that are clustered to form a reproducible array of parasagittal stripes. Double label and serial section immunocytochemistry revealed that the early PLCβ4‐immunoreactive clusters in the neonate are complementary to those previously identified by neurogranin expression. The PLCβ4 expression pattern can be traced continuously from embryo to adult, revealing the continuity of the topographical map from perinatal to adult cerebella. The only exception, as has been seen for other antigenic markers, is that transient PLCβ4 expression (which subsequently disappears) is seen in some Purkinje cell stripes during the second postnatal week. Furthermore, the data confirm that some adult Purkinje cell stripes are composite in origin, being derived from two or more distinct embryonic clusters. Thus, the zone and stripe topography of the cerebellum is conserved from embryo to adult, confirming that the early‐ and late‐antigenic markers share a common cerebellar topography. J. Comp. Neurol. 502:857–871, 2007.

Golgi Cell Dendrites Are Restricted by Purkinje Cell Stripe Boundaries in the Adult Mouse Cerebellar Cortex

Despite the general uniformity in cellular composition of the adult cerebellar cortex, there is a complex underlying pattern of parasagittal stripes of Purkinje cells with characteristic molecular phenotypes and patterns of connectivity. It is not known whether interneuron processes are restricted at stripe boundaries. To begin to address the issue, three strategies were used to explore how cerebellar Golgi cell dendrites are organized with respect to parasagittal stripes: first, double immunofluorescence staining combining anti-neurogranin to identify Golgi cell dendrites with the Purkinje cell compartmentation antigens zebrin II/aldolase C, HNK-1, and phospholipase Cβ4; second, zebrin II immunohistochemistry combined with a rapid Golgi–Cox impregnation procedure to reveal Golgi cell dendritic arbors; third, stripe antigen expression was used on sections of a GlyT2-EGFP transgenic mouse in which reporter expression is prominent in Golgi cell dendrites. In each case, the dendritic projections of Golgi cells were studied in the vicinity of Purkinje cell stripe boundaries. The data presented here show that the dendrites of a cerebellar interneuron, the Golgi cell, respect the fundamental cerebellar stripe cytoarchitecture.

Antigenic compartmentation of the cerebellar cortex in the chicken (Gallus domesticus)

The chick is a well‐understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens—zebrin II/aldolase C and phospholipase Cβ4 (PLCβ4)—has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains (“transverse zones”), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II‐immunopositive/PLCβ4‐immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II–VIa) were distinguished. A central zone (CZ; ∼lobules VIa–VIIIa,b) and a posterior zone (PZ; ∼lobules VIIIa,b–IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II‐immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II‐immunonegative P3‐ stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3−. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds. J. Comp. Neurol. 518:2221–2239, 2010.

Purkinje cell subtype specification in the cerebellar cortex: early B-cell factor 2 acts to repress the zebrin II-positive Purkinje cell phenotype.

The mammalian cerebellar cortex is highly compartmentalized. First, it is subdivided into four transverse expression domains: the anterior zone (AZ), the central zone (CZ), the posterior zone (PZ), and the nodular zone (NZ). Within each zone, the cortex is further subdivided into a symmetrical array of parasagittal stripes. The most extensively studied compartmentation antigen is zebrin II/aldolase c, which is expressed by a subset of Purkinje cells forming parasagittal stripes. Stripe phenotypes are specified early in cerebellar development, in part through the action of early B-cell factor 2 (Ebf2), a member of the atypical helix-loop-helix transcription factor family Collier/Olf1/EBF. In the murine cerebellum, Ebf2 expression is restricted to the zebrin II-immunonegative (zebrin II-) Purkinje cell population. We have identified multiple cerebellar defects in the Ebf2 null mouse involving a combination of selective Purkinje cell death and ectopic expression of multiple genes normally restricted to the zebrin II- subset. The nature of the cerebellar defect in the Ebf2 null is different in each transverse zone. In contrast to the ectopic expression of genes characteristic of the zebrin II+ Purkinje cell phenotype, phospholipase Cbeta4 expression, restricted to zebrin II- Purkinje cells in control mice, is well maintained, and the normal number of stripes is present. Taken together, these data suggest that Ebf2 regulates the expression of genes associated with the zebrin II+ Purkinje cell phenotype and that the zebrin II- Purkinje cell subtype is specified independently.

hESC-derived Olig2+ progenitors generate a subtype of astroglia with protective effects against ischaemic brain injury

Human pluripotent stem cells (hPSCs) have been differentiated to astroglia, but the utilization of hPSC-derived astroglia as cell therapy for neurological diseases has not been well studied. Astroglia are heterogeneous, and not all astroglia are equivalent in promoting neural repair. A prerequisite for cell therapy is to derive defined cell populations with superior therapeutic effects. Here we use an Olig2-GFP human embryonic stem cell (hESC) reporter to demonstrate that hESC-derived Olig2+ progenitors generate a subtype of previously uncharacterized astroglia (Olig2PC-Astros). These Olig2PC-Astros differ substantially from astroglia differentiated from Olig2-negative hESC-derived neural progenitor cells (NPC-Astros), particularly in their neuroprotective properties. When grafted into brains subjected to global ischaemia, Olig2PC-Astros exhibit superior neuroprotective effects and improved behavioural outcome compared to NPC-Astros. Thus, this new paradigm of human astroglial differentiation is useful for studying the heterogeneity of human astroglia, and the unique Olig2PC-Astros may constitute a new cell therapy for treating cerebral ischaemia and other neurological diseases.

The Subventricular Zone Continues to Generate Corpus Callosum and Rostral Migratory Stream Astroglia in Normal Adult Mice

Astrocytes are the most abundant cells in the CNS, and have many essential functions, including maintenance of blood–brain barrier integrity, and CNS water, ion, and glutamate homeostasis. Mammalian astrogliogenesis has generally been considered to be completed soon after birth, and to be reactivated in later life only under pathological circumstances. Here, by using genetic fate-mapping, we demonstrate that new corpus callosum astrocytes are continuously generated from nestin+ subventricular zone (SVZ) neural progenitor cells (NPCs) in normal adult mice. These nestin fate-mapped corpus callosum astrocytes are uniformly postmitotic, express glutamate receptors, and form aquaporin-4+ perivascular endfeet. The entry of new astrocytes from the SVZ into the corpus callosum appears to be balanced by astroglial apoptosis, because overall numbers of corpus callosum astrocytes remain constant during normal adulthood. Nestin fate-mapped astrocytes also flow anteriorly from the SVZ in association with the rostral migratory stream, but do not penetrate into the deeper layers of the olfactory bulb. Production of new astrocytes from nestin+ NPCs is absent in the normal adult cortex, striatum, and spinal cord. Our study is the first to demonstrate ongoing SVZ astrogliogenesis in the normal adult mammalian forebrain.

Compartmentation of the cerebellar nuclei of the mouse

The cerebellar nuclei integrate inhibitory input from Purkinje cells with excitatory input from mossy and climbing fiber collaterals and are the sole cerebellar output. Numerous studies have shown that the cerebellar cortex is highly compartmentalized into hundreds of genetically determined, reproducible topographic units--transverse zones and parasagittal stripes--that can be identified through the expression patterns of numerous molecules. The Purkinje cell stripes project to the cerebellar nuclei. However, there is no known commensurate topographic complexity in the cerebellar nuclei. Rather, conventional anatomical descriptions identify four major subdivisions--the medial, anterior and posterior interposed, and lateral nuclei--together with a few intranuclear subdivisions. To begin to address the apparent complexity gap, we have used a panel of antigens and transgenes to reveal a reproducible molecular heterogeneity in the mouse cerebellar nuclei. Based on the differential expression patterns, singly and in combination, a new cerebellar nuclear topographic map has been constructed. This reveals the subdivision of the cerebellar nuclei into at least 12 reproducible expression domains. We hypothesize that such heterogeneity is the counterpart of the zones and stripes of the cerebellar cortex.

A novel transverse expression domain in the mouse cerebellum revealed by a neurofilament-associated antigen.

The mammalian cerebellum is composed of a highly reproducible array of transverse zones, each of which is subdivided into parasagittal stripes. By using a combination of Purkinje cell antigenic markers and afferent tracing, four transverse zones have been identified: the anterior zone (AZ: approximately lobules I-V), the central zone (CZ: approximately lobules VI-VII), the posterior zone (PZ: approximately lobules VIII-dorsal IX) and the nodular zone (NZ: approximately ventral lobule IX+lobule X). Neurofilament-associated antigen (NAA) is an epitope recognized by a monoclonal antibody, which is expressed strongly in association with neurofilaments. During perinatal cerebellar development, anti-NAA immunocytochemistry reveals novel features of cerebellar organization. In particular, the CZ is reproducibly subdivided into anterior and posterior components. Between embryonic day 17 and postnatal day 7 NAA immunoreactivity is expressed selectively by a parallel fiber bundle that is restricted to lobule VII, thereby distinguishing the CZ anterior (lobules VIa, b) from the CZ posterior (lobule VII). The novel restriction boundary at lobule VII/VIII, which is also reflected in the morphology of the external granular layer and aligns with a gap in the developing Purkinje cell layer, precedes the morphological appearance of the posterior superior fissure between lobules VIb and VII. In addition, afferent axons to the CZ terminate in an array of parasagittal stripes that is probably a specific climbing fiber projection. Thus, the transverse zone architecture of the mouse cerebellum is more complex than had previously been appreciated.

A novel somatostatin-immunoreactive mossy fiber pathway associated with HSP25-immunoreactive purkinje cell stripes in the mouse cerebellum.

Somatostatin 28 immunoreactivity (Sst28‐ir) identifies a specific subset of mossy fiber terminals in the adult mouse cerebellum. By using double‐labeling immunohistochemistry, we determined that Sst28‐ir is associated with presynaptic mossy fiber terminal rosettes, and not Purkinje cells, Golgi cells, or unipolar brush cells. Sst28‐ir mossy fibers are restricted to the central zone (lobules VI/VII) and nodular zone (lobules IX, X) of the vermis, and the paraflocculus and flocculus. Within each transverse zone the mossy fiber terminal fields form a reproducible array of parasagittal stripes. The boundaries of Sst28‐ir stripes align with a specific array of Purkinje cell stripes revealed by using immunocytochemistry for the small heat shock protein HSP25. In the cerebellum of the homozygous weaver mouse, in which a subpopulation of HSP25‐ir Purkinje cells are located ectopically, the corresponding Sst28‐ir mossy fiber projection is also ectopic, suggesting a role for a specific Purkinje cell subset in afferent pattern formation. Likewise, in the scrambler mutant mouse, Sst28‐ir mossy fibers show a very close association with HSP25‐ir Purkinje cell clusters. HSP25 itself does not appear to be critical for normal patterning, however: in the KJR mouse, which does not express cerebellar HSP25, Sst28 expression appears to be normal. Likewise, the Purkinje cell patterning antigens zebrin II and HSP25 are expressed normally in both Sst‐ and Sst‐receptor knockout mice, suggesting that somatostatinergic transmission is not necessary for Purkinje cell stripe formation. J. Comp. Neurol. 517:524–538, 2009.