Radial glia and neural stem cells

During the last decade, the role of radial glia has been radically revisited. Rather than being considered a mere structural component serving to guide newborn neurons towards their final destinations, radial glia is now known to be the main source of neurons in several regions of the central nervous system, notably in the cerebral cortex. Radial glial cells differentiate from neuroepithelial progenitors at the beginning of neurogenesis and share with their ancestors the bipolar shape and the expression of some molecular markers. Radial glia, however, can be distinguished from neuroepithelial progenitors by the expression of astroglial markers. Clonal analyses showed that radial glia is a heterogeneous population, comprising both pluripotent and different lineage-restricted neural progenitors. At late-embryonic and postnatal stages, radial glial cells give rise to the neural stem cells responsible for adult neurogenesis. Embryonic pluripotent radial glia and adult neural stem cells may be clonally linked, thus representing a lineage displaying stem cell features in both the developing and mature central nervous system. This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) NUSUG grant (In vivo screening for genes implicated in glioma formation and development of new animal models of glial tumors) and by Fondazione CARIGE grant (Basi molecolari e cellulari dei gliomi: individuazione di marcatori diagnostici e di nuovi bersagli terapeutici).     

A subset of oligodendrocytes generated from radial glia in the dorsal spinal cord

Many oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ) that also gives rise to motoneurons. Cell fate specification in this region depends on sonic hedgehog (Shh) from the notochord and floor plate. There have been suggestions of an additional source(s) of oligodendrocytes in the dorsal spinal cord. We revisited this idea by Cre-lox fate-mapping in transgenic mice. We found that a subpopulation of oligodendrocytes is generated from the Dbx1-expressing domain of the VZ, spanning the dorsoventral midline. Dbx-derived oligodendrocytes comprise less than 5% of the total; they are formed late during embryogenesis by transformation of radial glia and settle mainly in the lateral white matter. Development of Dbx-derived oligodendrocytes in vitro can occur independently of Shh but requires FGF signalling. Dbx-expressing precursors also generate astrocytes and interneurons, but do not contribute to the ependymal layer of the postnatal spinal cord.     

Immunohistochemical localization of Bis protein in the rat central nervous system

We studied the distribution of Bis (Bcl-2 interacting death suppressor) protein in the adult rat brain and spinal cord using immunohistochemistry. Immunoreactivity was observed in specific neuronal populations in distinct nuclei. The most intensely labeled cells were associated with the motor system, including most cranial nerve motor nuclei, Purkinje cells of the cerebellum, the red nucleus, and the ventral motor neurons of the spinal cord. Bis protein was also expressed in several structures associated with the ventricular system, including the subventricular zone of the lateral ventricle and its rostral extension, in the subcommissural organ, and in tanycytes, radial glial cells in the hypothalamus. Using double-labeling techniques, Bis-immunoreactive cells in the rostral migratory stream, coexpressing Bcl-2, were confirmed as glial fibrillary acidic protein-positive astrocytes comprising the glial tubes. The widespread distribution of Bis suggests that this protein has broader functions in the adult rat central nervous system than previously thought, and that it could be associated with a particular role in the rostral migratory system.J.-H. Lee and M.-Y. Lee contributed equally to this study. This work was supported by the KOSEF through the Cell Death Disease Research Center of MRC at the Catholic University of Korea (R13-2002-005-01001-0) and the Catholic Medical Center Research Foundation grant made in the program year of 2002     

CNS midline cells influence the division and survival of lateral glia in the Drosophila nervous system

Central nervous system (CNS) midline cells are essential for identity determination and differentiation of neurons in the Drosophila nervous system. It is not clear, however, whether CNS midline cells are also involved in the development of lateral glial cells. The roles of CNS midline cells in lateral glia development were elucidated using general markers for lateral glia, such as glial cell missing and reverse polarity, and specific enhancer trap lines labeling the longitudinal, A, B, medial cell body, peripheral, and exit glia. We found that CNS midline cells were necessary for the proper expression of glial cell missing, reverse polarity, and other lateral glia markers only during the later stages of development, suggesting that they are not required for initial identity determination. Instead, CNS midline cells appear to be necessary for proper division and survival of lateral glia. CNS midline cells were also required for proper positioning of three exit glia at the junction of segmental and intersegmental nerves, as well as some peripheral glia along motor and sensory axon pathways. This study demonstrated that CNS midline cells are extrinsically required for the proper division, migration, and survival of various classes of lateral glia from the ventral neuroectoderm.     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.     

Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord

Following tail amputation in urodele amphibians, an ependymal tube, that resembles a developing neural tube, forms from ependymal cells that migrate from the cord stump and elongates by cell proliferation. Expression of the keratin pair 8 and 18 has been observed in the developing urodele nervous system and is maintained in the ependymal cells of the mature cord. We show here that expression of these keratins is not unique to urodeles, but is also observed in the radial glia of the human spinal cord, suggesting that these proteins might play a role both in neural development and regeneration. Analysis of their expression in the regenerating spinal cord following tail amputation shows that their expression, as well as that of glial fibrillary acidic protein (GFAP), is maintained in the ependymal tube during regeneration, though differences in their levels of expression are observed along the anteroposterior axis and appear to be related to the progression of morphogenesis. In addition, we show that following tail amputation the ependymal tube expresses the neural stem cell markers nestin and vimentin, which are undetectable in normal urodele spinal cord. This up-regulation of neural stem cell markers shows that the ependymal cells undergo a phenotypic change. Whereas maintenance of keratin and GFAP expression in the adult ependyma may reflect a higher plasticity of these cells in adult urodeles than in other vertebrates, re-expression of markers of early neural development suggests the occurrence of a dedifferentiation process in the spinal cord in response to injury.Edited by J. Campos-Ortega     

Proliferation and migration of glial precursor cells in the developing rat spinal cord

The mechanisms that control the production and differentiation of glial cells during development are difficult to unravel because of displacement of precursor cells from their sites of origin to their permanent location. The two main neuroglial cells in the rat spinal cord are oligodendrocytes and astrocytes. Considerable evidence supports the view that oligodendrocytes in the spinal cord are derived from a region of the ventral ventricular zone (VZ). Some astrocytes, at least, may arise from radial glia. In this study a 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay was used to identify proliferating cells and examine the location of proliferating glial precursor cells in the embryonic spinal cord at different times post BrdU incorporation. In this way the migration of proliferating cells into spinal cord white matter could be followed. At E14, most of the proliferating cells in the periventricular region were located dorsally and these cells were probably proliferating neuronal precursors. At E16 and E18, the majority of the proliferating cells in the periventricular region were located ventrally. In the white matter the number of proliferating cells increased as the animals increased in age and much of this proliferation occurred locally. BrdU labelling showed that glial precursor cells migrate from their ventral and dorsal VZ birth sites to peripheral regions of the cord. Furthermore although the majority of proliferating cells in the spinal cord at E16 and E18 were located in the ventral periventricular region, some proliferating cells remained in the dorsal VZ region of the cord.     

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of the African lungfish, Protopterus annectens (Dipnoi: Lepidosirenidae)

The distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP), and vimentin, in the brain and spinal cord of the African lungfish, Protopterus annectens, was examined by light microscopy immunoperoxidase cytochemistry. Glial fibrillary acidic protein immunoreactivity is clear and is evident in a radial glial system. It consists of fibers of different lengths and thicknesses that are arranged in a regular radial pattern throughout the central nervous system (CNS). They emerge from generally immunopositive radial ependymoglia (tanycytes), lining the ventricular surface, and are directed from the ventricular wall to the meningeal surface. These fibers give rise to endfeet that are apposed to the subpial surface and to blood vessel walls forming the glia limitans externa and the perivascular glial layer, respectively. GFAP-immunopositive star-shaped astrocytes were not found in P. annectens CNS. In the gray matter of the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependymal layer. Vimentin-immunopositive structures are represented by thin fibers mostly localized in the peripheral zones of the brain and the spinal cord. While a few stained fibers appear in the gray matter, the ependymal layer shows no antivimentin immunostaining. In P. annectens the immunocytochemical response of the astroglial intermediate filaments is typical of a mature astroglia cell lineage, since they primarily express GFAP immunoreactivity. This immunocytochemical study shows that the glial pattern of the African lungfish resembles that found in tetrapods such as urodeles and reptiles. The glial pattern of lungfishes is comparable to that of urodeles and reptiles but is not as complex as that of teleosts, birds, and mammals.     

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.     

Development of midline glial populations at the corticoseptal boundary

Three midline glial populations are found at the corticoseptal boundary: the glial wedge (GW), glia within the indusium griseum (IGG), and the midline zipper glia (MG). Two of these glial populations are involved in axonal guidance at the cortical midline, specifically development of the corpus callosum. Here we investigate the phenotypic and molecular characteristics of each population and determine whether they are generated at the same developmental stage. We find that the GW is derived from the radial glial scaffold of the cortex. GW cells initially have long radial processes that extend from the ventricular surface to the pial surface, but by E15 loose their pial attachment and extend only part of the way to the pial surface. Later in development the radial morphology of cells within the GW is replaced by multipolar astrocytes, providing supportive evidence that radial glia can transform into astrocytes. IGG and MG do not have a radial morphology and do not label with the radial glial markers, Nestin and RC2. We conclude that the GW and IGG have different morphological and molecular characteristics and are born at different stages of development. IGG and MG have many phenotypic and molecular characteristics in common, indicating that they may represent a common population of glia that becomes spatially distinct by the formation of the corpus callosum.     

Distribution of glial-associated proteins in the developing chick auditory brainstem

In the avian brainstem, nucleus magnocellularis (NM) projects bilaterally to nucleus laminaris (NL) in a pathway that facilitates sound localization. The distribution of glia during the development of this pathway has not previously been characterized. Radial glia, astrocytes, and oligodendrocytes facilitate many processes including axon pathfinding, synaptic development, and maturation. Here we determined the spatiotemporal expression patterns of glial cell types in embryonic development of the chick auditory brainstem using glial-specific antibodies and histological markers. We found that vimentin-positive processes are intercalated throughout the NL cell layer. Astrocytes are found in two domains: one in the ventral neuropil region and the other dorsolateral to NM. GFAP-positive processes are primarily distributed along the ventral margin of NL. Astrocytic processes penetrate the NL cell layer following the onset of synaptogenesis, but before pruning and maturation. The dynamic, nonoverlapping expression patterns of GFAP and vimentin suggest that distinct glial populations are found in dorsal versus ventral regions of NL. Myelination occurs after axons have reached their targets. FluoroMyelin and myelin basic protein (MBP) gradually increase along the mediolateral axis of NL starting at E10. Multiple GFAP-positive processes are directly apposed to NM-NL axons and MBP, which suggests a role in early myelinogenesis. Our results show considerable changes in glial development after initial NM-NL connections are made, suggesting that glia may facilitate maturation of the auditory circuit.     

Intermediate filament proteins in the developing chick spinal cord

The distribution of different intermediate filament (IF) proteins in the embryonic chick spinal cord was examined at several stages of development using immunohistochemical techniques, analytic gel electrophoresis, and electron microscopy. We have found that: (1) the fibroblast-type IF protein (vimentin) is present in virtually all of the replicating neuroepithelial cells of the early neural tube, as well as in radial glia, astrocytes, and Schwann cells in later stages of development; (2) the fibroblast-type IF protein is not detectable in definitive neurons; (3) the neurofilament proteins are first detectable in postmitotic neuroblasts at about the time of initial axon formation and they are restricted to neurons; (4) the astrocyte-type IF protein (glial fibrillary acidic protein) is in definitive astrocytes, but not in radial glia; (5) the prekeratin proteins are restricted to cells of the leptomeninges; and (6) the muscle-type IF protein (desmin) is restricted to vascular tissue in and around the developing spinal cord. These findings suggest that the fibroblast-type IF protein is the only IF protein in the early neuroepithelial cells and that the progeny of these cells will follow one of three different patterns of IF protein expression: (1) continued expression of only the fibroblast-type IF protein (radial glia); (2) expression of both the fibroblast-type IF protein and the astrocyte-type IF protein (astrocytes); or (3) expression of only the neurofilament proteins (neurons).     

Neuronal or glial progeny: regional differences in radial glia fate

The precursor function of the ubiquitous glial cell type in the developing central nervous system (CNS), the radial glia, is largely unknown. Using Cre/loxP in vivo fate mapping studies, we found that radial glia generate virtually all cortical projection neurons but not the interneurons originating in the ventral telencephalon. In contrast to the cerebral cortex, few neurons in the basal ganglia originate from radial glia, and in vitro lineage analysis revealed intrinsic differences in the potential of radial glia from the dorsal and ventral telencephalon. This shows that the progeny of radial glia not only differs profoundly between brain regions but also includes the majority of neurons in some parts of the CNS.     

Regulated vnd expression is required for both neural and glial specification in Drosophila.

The Drosophila embryonic CNS arises from the neuroectoderm, which is divided along the dorsal-ventral axis into two halves by specialized mesectodermal cells at the ventral midline. The neuroectoderm is in turn divided into three longitudinal stripes--ventral, intermediate, and lateral. The ventral nervous system defective, or vnd, homeobox gene is expressed from cellularization throughout early neural development in ventral neuroectodermal cells, neuroblasts, and ganglion mother cells, and later in an unrelated pattern in neurons. Here, in the context of the dorsal-ventral location of precursor cells, we reassess the vnd loss- and gain-of-function CNS phenotypes using cell specific markers. We find that over expression of vnd causes significantly more profound effects on CNS cell specification than vnd loss. The CNS defects seen in vnd mutants are partly caused by loss of progeny of ventral neuroblasts-the commissures are fused and the longitudinal connectives are aberrantly positioned close to the ventral midline. The commissural vnd phenotype is associated with defects in cells that arise from the mesectoderm, where the VUM neurons have pathfinding defects, the MP1 neurons are mis-specified, and the midline glia are reduced in number. vnd over expression results in the mis-specification of progeny arising from all regions of the neuroectoderm, including the ventral neuroblasts that normally express the gene. The CNS of embryos that over express vnd is highly disrupted, with weak longitudinal connectives that are placed too far from the ventral midline and severely reduced commissural formation. The commissural defects seen in vnd gain-of-function mutants correlate with midline glial defects, whereas the mislocalization of interneurons coincides with longitudinal glial mis-specification. Thus, Drosophila neural and glial specification requires that vnd expression by tightly regulated.     

Changes in progenitor populations and ongoing neurogenesis in the regenerating chick spinal cord

The chick spinal cord can regenerate following injury until advanced developmental stages. It is conceivable that changes in stem/progenitor cell plasticity contribute to the loss of this capacity, which occurs around E13. We investigated the contribution of proliferation, phenotypic changes in radial glia progenitors, and neurogenesis to spinal cord regeneration. There was no early up-regulation of markers of gliogenic radial glia after injury either at E11 or E15. In contrast, increased proliferation in the grey matter and up-regulation of transitin expression following injury at E11, but not E15, suggested high levels of plasticity within the E11 spinal cord progenitor population that are lost by later stages. Changes in neural progenitors with development were also supported by a higher neurosphere forming ability at E11 than at E15. Co-labelling with doublecortin and neuron-specific markers and BrdU in spinal cord sections and dissociated cells showed that neurogenesis is an ongoing process in E11 chick spinal cords. This neurogenesis appeared to be complete by E15. Our findings demonstrate that the regeneration-competent chick spinal cord is less mature and more plastic than previously believed, which may contribute to its favourable response to injury, and suggest a role for neurogenesis in maintaining regenerative capacity.     

Neuroanatomical and functional analysis of neural tube formation in notochordless Xenopus embryos; laterality of the ventral spinal cord is lost.

Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15% of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.