Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Glia maintain follower neuron survival during Drosophila CNS development

While survival of CNS neurons appears to depend on multiple neuronal and non-neuronal factors, it remains largely unknown how neuronal survival is controlled during development. Here we show that glia regulate neuronal survival during formation of the Drosophila embryonic CNS. When glial function is impaired either by mutation of the glial cells missing gene, which transforms glia toward a neuronal fate, or by targeted genetic glial ablation, neuronal death is induced non-autonomously. Pioneer neurons, which establish the first longitudinal axon fascicles, are insensitive to glial depletion whereas the later extending follower neurons die. This differential requirement of neurons for glia is instructive in patterning and links control of cell number with axon guidance during CNS development.     

Formation and preservation of cortical layers in slice cultures.

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984-985; Berry and Rogers, 1965, J. Anat. 99:691-709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodeoxyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells continued to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slice, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61-84; Hatten and Mason, 1990, Experientia 46:907-916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cultures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells.     

Neurons reduce glial responses to lipopolysaccharide (LPS) and prevent injury of microglial cells from over-activation by LPS

The microenvironment of the CNS has been considered to tonically inhibit glial activities. It has been shown that glia become activated where neuronal death occurs in the aging brain. We have previously demonstrated that neurons tonically inhibit glial activities including their responses to the bacterial endotoxin lipopolysaccharide (LPS). It is not clear whether activation of glia, especially microglia in the aging brain, is the consequence of disinhibition due to neuronal death. This study was designed to determine if glia regain their responsiveness to LPS once the neurons have died in aged cultures. When cultured alone, glia from postnatal day one rat mesencephalons stimulated with LPS (0.1-1000 ng/mL) produced both nitric oxide (NO) and tumor necrosis factor alpha (TNFalpha), yielding a sigmoid and a bell-shaped curve, respectively. When neuron-containing cultures were prepared from embryonic day 14/15 mesencephalons, the shape of the dose-response curve for NO was monotonic and the bell-shaped curve for TNFalpha production was shifted to the right. After 1 month of culture under conditions where neurons die, the production curves for NO and TNFalpha in LPS-stimulated glia shifted back to the left compared to mixed neuron-glia cultures. Immunostaining of rat microglia for the marker CR3 (the receptor for complement component C3) demonstrated that high concentrations of LPS (1 microg/mL) reduced the number of microglia in mixed-glial cultures. In contrast, reduction of CR3 immunostaining was not observed in LPS-stimulated mixed neuron-glia cultures. Taken together, the results demonstrate that disinhibition of the glial response to LPS occurs after neurons die in aged cultures. Once neurons have died, the responsiveness of glia to LPS is restored. Neurons prevented injury to microglia by reducing their responsiveness to LPS. This study broadens our understanding of the ways in which the CNS microenvironment affects cerebral inflammation.     

The growth of axons in three-dimensional astrocyte cultures

The environment of the adult central nervous system (CNS) does not support axon regeneration. We have been unable to replicate this behaviour using monolayer cultures of glia, so we have developed a technique for three dimensional culture of glial cells. We have examined the growth of axons from embryonic and postnatal retina and dorsal root ganglia (DRG's) through purified three-dimensional astrocyte cultures. Neither postnatal DRG's nor adult retina were able to grow axons through astrocytes from cultures 3 weeks or more old, although some DRG axons grew in astrocyte cultures which were 10 days or less old. However axons from embryonic DRG's and retina grew axons profusely into even elderly astrocyte cultures. All the tissues grew axons into three-dimensional Schwann cell cultures. The behaviour of axons in three-dimensional glial cultures therefore reproduces the behaviour of axons in vivo.     

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.     

Astroglia demonstrate regional differences in their ability to maintain primary dendritic outgrowth from mouse cortical neurons in vitro

To determine whether glia from different regions of the central nervous system (CNS) initiate or maintain primary dendritic growth, embryonic day 18 mouse cortical neurons were co-cultured with rat (postnatal day 4) astroglial cells derived from retina, spinal cord, mesencephalon, striatum, olfactory bulb, retina, and cortex. Axon and dendrite outgrowth from isolated neurons was quantified using morphological and immunohistochemical techniques at 18 h and 1, 3, and 5 days in vitro. Neurons initially extend the same number of neurites, regardless of the source of glial monolayer; however, glial cells differ in their ability to maintain primary dendrites. Homotypic cortical astrocytes maintain the greatest number of primary dendrites. Glia derived from the olfactory bulb and retina maintained intermediate numbers of dendrites, whereas only a small number of primary dendrites were maintained by glia derived from striatum, spinal cord, or mesencephalon. Longer axons were initially observed from neurons grown on glia that did not maintain dendrite number. Axonal length, however, was similar on the various monolayers after 5 days in vitro. Neurons that were grown in media conditioned by either mesencephalic or cortical glia for the first 24 h followed by culture media from glia of the alternate source for 4 days in vitro confirmed that glia maintained, rather than initiated, the outgrowth of the primary dendritic arbor. These results indicate that glial cells derived from various CNS regions differ in their ability to maintain the primary dendritic arbor from mouse cortical neurons in vitro. © 1995 John Wiley & Sons, Inc.     

Formation and preservation of cortical layers in slice cultures

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984–985; Berry and Rogers, 1965, J. Anat. 99:691–709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodexyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells contiuned to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slices, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61–84; Hatten and Mason, 1990, Experientia 46:907–916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cutures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells. © 1992 John Wiley & Sons, Inc.     

Two forms of cerebellar glial cells interact differently with neurons in vitro

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.     

Activation of glial FGFRs is essential in glial migration, proliferation, and survival and in glia-neuron signaling during olfactory system development

Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.     

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

Studies on effects of co-culturing on the development of embryonic and postnatal cerebellar cells

Experiments were conducted to compare the in vitro development of different cerebellar cells in primary surface cultures to that observed in their co-cultures. Single cultures of embryonic (E.19) and postnatal (P 7) cerebellar cells as well as their co-cultures were established. Changes in cell-size distribution and in high-affinity GABA-uptake were studied during the first three weeks in the different cultures. Morphological analyses showed that in single cultures of embryonic (E 19) cells a number of large and middle-size neurons survived on the top of confluent monolayer of large flattened astroglial cells. In cultures of postnatal (P 7) cerebellar cells small tetanus toxin positive non-GABAergic neurons proved to be the most abundant cell-constituents while no confluent feeder-layer of non-neuronal cells was formed. In co-cultures both embryonic and postnatal morphological features were observed but we could not observe any improvement in either survival or maturation of the neuronal types studied. A transient increase in glial GABA-uptake, however, was observed in both postnatal and co-cultures soon after plating of postnatal cerebellar cells.     

Developmental regulation of glial cell phagocytic function during Drosophila embryogenesis

The proper removal of superfluous neurons through apoptosis and subsequent phagocytosis is essential for normal development of the central nervous system (CNS). During Drosophila embryogenesis, a large number of apoptotic neurons are efficiently engulfed and degraded by phagocytic glia. Here we demonstrate that glial proficiency to phagocytose relies on expression of phagocytic receptors for apoptotic cells, SIMU and DRPR. Moreover, we reveal that the phagocytic ability of embryonic glia is established as part of a developmental program responsible for glial cell fate determination and is not triggered by apoptosis per se. Explicitly, we provide evidence for a critical role of the major regulators of glial identity, gcm and repo, in controlling glial phagocytic function through regulation of SIMU and DRPR specific expression. Taken together, our study uncovers molecular mechanisms essential for establishment of embryonic glia as primary phagocytes during CNS development.     

Differential Distribution of Purine Metabolizing Enzymes Between Glia and Neurons

Abstract: Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5'-AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthlne oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine-guanine phosphoribosyltransferase and production of adenosine by S-adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S-adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 ± 61 pmol/min/10⁷ cells), nucleoside phosphorylase (187 ± B pmol/min/10⁷ cells), and adenosine deaminase (233 ± 32 pmol/min/10⁷ cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.     

Spatiotemporal heterogeneity of CNS radial glial cells and their transition to restricted precursors

Radial glia are among the first cells that develop in the embryonic central nervous system. They are progenitors of glia and neurons but their relationship with restricted precursors that are also derived from neuroepithelia is unclear. To clarify this issue, we analyzed expression of cell type specific markers (BLBP for radial glia, 5A5/E-NCAM for neuronal precursors and A2B5 for glial precursors) on cortical radial glia in vivo and their progeny in vitro. Clones of cortical cells initially expressing only BLBP gave rise to cells that were A2B5+ and eventually lost BLBP expression in vitro. BLBP is expressed in the rat neuroepithelium as early as E12.5 when there is little or no staining for A2B5 and 5A5. In E13.5-15.5 forebrain, A2B5 is spatially restricted co-localizing with a subset of the BLBP+ radial glia. Analysis of cells isolated acutely from embryonic cortices confirmed that BLBP expression could appear without, or together with, A2B5 or 5A5. The numbers of BLBP+/5A5+ cells decreased during neurogenesis while the numbers of BLBP+/A2B5+ cells remained high through the beginning of gliogenesis. The combined results demonstrate that spatially restricted subpopulations of radial glia along the dorsal-ventral axis acquire different markers for neuronal or glial precursors during CNS development.     

Misexpression of basic helix-loop-helix genes in the murine cerebral cortex affects cell fate choices and neuronal survival

To investigate the role(s) of basic helix-loop-helix genes (bHLH) genes in the developing murine cerebral cortex, Mash1, Math2, Math3, Neurogenin1 (Ngn1), Ngn2, NeuroD, NeuroD2 and Id1 were transduced in vivo into the embryonic and postnatal cerebral cortex using retrovirus vectors. The morphology and location of infected cells were analyzed at postnatal stages. The data indicate that a subset of bHLH genes are capable of regulating the choice of neuronal versus glial fate and that, when misexpressed, they can be deleterious to the survival of differentiating neurons, but not glia.     

Calcium and glial cell death

Calcium (Ca2+) homeostasis is crucial for development and survival of virtually all types of cells including glia of the central nervous system (CNS). Astrocytes, oligodendrocytes and microglia, the major glial cell types in the CNS, are endowed with a rather sophisticated array of Ca2+-permeable receptors and channels, as well as store-operated channels and pumps, all of which determine Ca2+ homeostasis. In addition, glial cells detect functional activity in neighbouring neurons and respond to it by means of Ca2+ signals that can modulate synaptic interactions. Like in neurons, Ca2+ overload resulting from dysregulation of channels and pumps can be deleterious to glia. In this review, we summarize recent advances in the understanding Ca2+ homeostasis in glial cells, the consequences of its alteration in cell demise as well as in neurological and psychiatric disorders that experience glial cell loss.