Macroglial cell development in embryonic rat brain: studies using monoclonal antibodies, fluorescence activated cell sorting, and cell culture

Astrocytes, ependymal cells, and oligodendrocytes have been shown to develop on the same schedule in dissociated cell cultures of early embryonic rat brain as in vivo. Subsequent studies showed that there are two major types of astrocyte (type-1 and type-2), which, in cultures of perinatal optic nerve, develop as two distinct lineages. In such cultures, type-2 astrocytes and oligodendrocytes develop from the same, bipotential, (O-2A) progenitor cells, which differentiate into type-2 astrocytes in 10% fetal calf serum (FCS) and into oligodendrocytes in less than or equal to 0.5% FCS. In light of these findings, we now have extended our studies on macroglial cell development in rat brain and show the following: (i) The first astrocytes to develop have a type-1 phenotype, while astrocytes with a type-2 phenotype do not develop until almost 2 weeks later, just as in the optic nerve. (ii) Most importantly, type-2 astrocytes, like the other macroglial cells, develop on the same schedule in cultures of early embryonic (less than or equal to E15) brain as they do in vivo. (iii) By contrast, both oligodendrocytes and type-2 astrocytes develop prematurely in cultures of E17 brain, and FCS influences this development in the same way it does in perinatal optic nerve cultures. (iv) Type-2 astrocyte precursors are labeled by the A2B5 monoclonal antibody, as shown previously for oligodendrocyte precursors in brain and for O-2A progenitor cells in optic nerve. Taken together with our previous findings, these results suggest that oligodendrocytes and type-2 astrocytes in brain develop from bipotential O-2A progenitor cells, whose choice of developmental pathway and timing of differentiation depend on mechanisms that operate independently of brain morphogenesis.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Irradiation in vitro discriminates between different O-2A progenitor cell subpopulations in the perinatal central nervous system of rats

The effects of X irradiation on oligodendrocyte-type-2-astrocyte (O-2A) progenitor cells derived from different regions of the perinatal central nervous system (CNS) of rats were investigated in vitro. The O-2A progenitor cells can differentiate into either oligodendrocytes or type-2 astrocytes. The depletion of these cells could lead to demyelination, seen as a delayed reaction after irradiation of the CNS in vivo. To quantify cell survival, O-2A progenitor cells were grown on monolayers of type-1 astrocytes. Monolayers of type-1 astrocytes stimulate O-2A progenitor cells to divide. O-2A progenitor cells were irradiated in vitro and clonogenic cell survival was measured. The O-2A progenitor cells derived from perinatal optic nerve were quite radiosensitive in contrast to O-2A progenitor cells derived from perinatal spinal cord and perinatal corpus callosum. Furthermore, O-2A progenitor cells derived from the optic nerve formed smaller colonies, with most colonies showing early differentiation into oligodendrocytes. In contrast, more than half of the colonies derived from corpus callosum did not show any differentiation after 2 weeks in vitro and kept growing. These differences support the view that perinatal O-2A progenitor cells derived from the optic nerve are committed progenitor cells while the O-2A progenitor cells derived from the perinatal corpus callosum and the perinatal spinal cord have more stem cell properties.     

Extracellular matrix-associated molecules collaborate with ciliary neurotrophic factor to induce type-2 astrocyte development

O-2A progenitor cells give rise to both oligodendrocytes and type-2 astrocytes in vitro. Whereas oligodendrocyte differentiation occurs constitutively, type-2 astrocyte differentiation requires extracellular signals, one of which is thought to be ciliary neurotrophic factor (CNTF). CNTF, however, is insufficient by itself to induce the development of stable type-2 astrocytes. In this report we show the following: (a) that molecules associated with the extracellular matrix (ECM) cooperate with CNTF to induce stable type-2 astrocyte differentiation in serum-free cultures. The combination of CNTF and the ECM-associated molecules thus mimics the effect of FCS, which has been shown previously to induce stable type-2 astrocyte differentiation in vitro. (b) Both the ECM-associated molecules and CNTF act directly on O-2A progenitor cells and can induce them to differentiate prematurely into type-2 astrocytes. (c) ECM-associated molecules also inhibit oligodendrocyte differentiation, even in the absence of CNTF, but this inhibition is not sufficient on its own to induce type-2 astrocyte differentiation. (d) Whereas the effect of ECM on oligodendrocyte differentiation is mimicked by basic fibroblast growth factor (bFGF), the effect of ECM on type-2 astrocyte differentiation is not. (e) The ECM-associated molecules that are responsible for inhibiting oligodendrocyte differentiation and for cooperating with CNTF to induce type-2 astrocyte differentiation are made by non-glial cells in vitro. (f) Molecules that have these activities and bind to ECM are present in the optic nerve at the time type-2 astrocytes are thought to be developing.     

An inducer protein may control the timing of fate switching in a bipotential glial progenitor cell in rat optic nerve

In rat optic nerve, oligodendrocytes and type-2 astrocytes develop from a common (O-2A) progenitor cell. The first oligodendrocytes differentiate at birth, while the first type-2 astrocytes differentiate in the second postnatal week. We previously showed that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell. Here we provide evidence that the timing of type-2 astrocyte differentiation, by contrast, may depend on an inducing protein that appears late in the developing nerve. We show that extracts of 3- to 4-week-old, but not 1-week-old, rat optic nerve contain a protein (apparent Mr approximately 25,000) that induces O-2A progenitor cells in culture to express glial fibrillary acidic protein (GFAP), an astrocyte-specific marker in the rat central nervous system.     

Development and regeneration in the central nervous system

As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.     

Reconstitution of a developmental clock in vitro: a critical role for astrocytes in the timing of oligodendrocyte differentiation

The rat optic nerve contains three types of macroglial cells: type 1 astrocytes first appear at embryonic day 16 (E16), oligodendrocytes at birth (E21), and type 2 astrocytes between postnatal days 7 and 10. The oligodendrocytes and type 2 astrocytes develop from a common, bipotential O-2A progenitor cell. We show here that although O-2A progenitor cells in E17 optic nerve prematurely stop dividing and differentiate into oligodendrocytes within 2 days in culture, when cultured on a monolayer of type 1 astrocytes, they continue to proliferate; moreover, the first cells differentiate into oligodendrocytes after 4 days in vitro, which is equivalent to the time that oligodendrocytes first appear in vivo. Our findings suggest that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell that counts cell divisions that are driven by a growth factor (or factors) produced by type 1 astrocytes.     

Coexistence of perinatal and adult forms of a glial progenitor cell during development of the rat optic nerve

We have studied the developmental appearance of the O-2A(adult) progenitor cell, a specific type of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell that we have identified previously in cultures prepared from the optic nerves of adult rats. O-2A(adult) progenitors differ from their counterparts in perinatal animals (O-2A perinatal progenitor cells) in antigenic phenotype, morphology, cell cycle time, rate of migration, time course of differentiation into oligodendrocytes or type-2 astrocytes and sensitivity to the lytic effects of complement in vitro. In the present study, we have found that O-2A(adult) progenitor-like cells first appear in the developing optic nerve approximately 7 days after birth and that by 1 month after birth these cells appear to be the dominant progenitor population in the nerve. However, the perinatal-to-adult transition in progenitor populations is a gradual one and O-2A(adult) and O-2A perinatal progenitors coexist in the optic nerve for 3 weeks or more. In addition, cells derived from optic nerves of P21 rats express characteristic features of O-2adult and O-2A perinatal progenitors for extended periods of growth in the same tissue culture dish. Our results thus indicate that the properties that distinguish these two types of O-2A progenitors from each other are expressed in apparently identical environments. Thus, these cells must either respond to different signals present in the environment, or must respond with markedly different behaviours to the binding of identical signalling molecules.     

A role for platelet-derived growth factor in normal gliogenesis in the central nervous system

The bipotential progenitor cells (O-2A progenitors) that produce oligodendrocytes and type-2 astrocytes in the developing rat optic nerve are induced to proliferate in culture by type-1 astrocytes. Here, we show that the astrocyte-derived mitogen is platelet-derived growth factor (PDGF). PDGF is a potent mitogen for O-2A progenitor cells in vitro. Mitogenic activity in astrocyte-conditioned medium comigrates with PDGF on a size-exclusion column, competes with PDGF for receptors, and is neutralized by antibodies to PDGF. PDGF dimers can be immunoprecipitated from astrocyte-conditioned medium, and mRNA encoding PDGF is present in rat brain throughout gliogenesis. We propose that astrocyte-derived PDGF is crucial for the control of myelination in the developing central nervous system.     

Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O- 2Aperinatal progenitor cells

We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.     

PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.     

Expression of fibronectin and laminin by different types of mouse glial cells cultured in a serum-free medium

The expression of fibronectin and laminin by cultured glial cells was studied. The glial culture from neonatal mouse cerebra maintained in a chemically defined, serum-free medium consisted of type-1 astrocytes, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, oligodendrocytes and type-2 astrocytes. Double-labelling immunofluorescent experiments performed using the mixed glial culture indicated that fibronectin and laminin are expressed in different patterns among the glial subtypes. The staining intensities with anti-fibronectin or anti-laminin antibodies decreased in the order: type-1 astrocytes, O-2A progenitor cells and type-2 astrocytes. Both molecules were deposited in a fibrillar matrix underneath type-1 astrocytes, whereas only intracytoplasmic localization of these molecules was observed with O-2A progenitor cells and type-2 astrocytes. Western blot analysis showed that glial fibronectin has a slightly higher molecular weight than mouse plasma fibronectin (230 kDa) and that glial laminin is a variant with a 220 kDa B chain present and the 400 kDa A chain missing. Using enzyme-linked immunosorbent assays (ELISA), these molecules were detected in the glial extracellular matrix at the concentration of 4 ng/10⁶ cells. A large amount of fibronectin (82 ng/10⁶ cells) was secreted into the culture medium, while secretion of laminin was not detected.     

In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells

We have been studying the differing characteristics of oligodendrocyte- type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O- 2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.     

Identification of an adult-specific glial progenitor cell

We have found that glial progenitor cells isolated from the optic nerves of adult rats are fundamentally different from their counterparts in perinatal animals. In our studies on bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, we have seen that O-2Aadult progenitor cells can be distinguished from O-2Aperinatal progenitors by their morphology and antigenic phenotype, their much longer cell cycle time (65 h versus 18 h), slower rate of migration rate (4 microns h-1 versus 21 microns h-1), and their time course of differentiation into oligodendrocytes or type-2 astrocytes in vitro (less than or equal to 3 days versus greater than 5 days). At least some of the differences between O-2Aadult and O-2Aperinatal progenitor cells appear to be clearly related to the differing cellular requirements of the adult and perinatal central nervous system (CNS). The properties of the O-2Aadult progenitor cells may make these cells ideally suited for the needs of the adult CNS, where rapid exponential increases in the number of oligodendrocytes and O-2A progenitor cells would be inappropriate. However, the properties of the O-2Aadult progenitor cells are such that they may not be able to replace oligodendrocytes in sufficient numbers to repair extensive or recurrent damage in the adult brain, such as in patients suffering from the human demyelinating disease multiple sclerosis. Moreover, available information about other tissues suggests that the transition from perinatal to adult progenitor cell types may represent a developmental mechanism of general importance.     

A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: in vivo evidence for two distinct astrocyte lineages

We have shown previously that three antibodies--anti-galactocerebroside (GC), anti-glial fibrillary acidic protein (GFAP), and the A2B5 monoclonal antibody--can be used to help distinguish three classes of glial cells in the rat optic nerve: oligodendrocytes are GC+, GFAP-, almost all type-1 astrocytes are A2B5-, GFAP+, and almost all type-2 astrocytes are A2B5+, GFAP+. In the present study we have used these antibodies to examine the timing and sequence of the development of the three types of glial cells in vivo. We show that type-1 astrocytes first appear at embryonic Day 16 (E16), oligodendrocytes at birth (E21), and type-2 astrocytes between postnatal Days 7 and 10 (P7-10). Moreover, we demonstrate quantitatively that astrocytes in the optic nerve develop in two waves, with more than 95% of type-1 astrocytes developing before P15 and more than 95% of type-2 astrocytes developing after P15. Finally, we provide indirect evidence that type-2 astrocytes do not develop from type-1 astrocytes in vivo, supporting previous direct evidence that the two types of astrocytes develop from two serologically distinct precursor cells in vitro.     

Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions

The clonal development of oligodendrocytes was studied by culturing individual oligodendrocyte--type-2 astrocyte (O-2A) progenitor cells on monolayers of type-1 astrocytes, which stimulate O-2A progenitor cells to divide. Oligodendrocytes developed by a proliferative lineage in which clonal progeny differentiated together after a number of cell divisions. Most O-2A progenitor cells had similar cell cycle times (1-2 days), but their proliferative capacity varied greatly: some divided only once while others divided up to eight times before differentiating. sister cells behaved similarly when recultured separately on astrocyte monolayers. These findings are consistent with the cell-division-counting hypothesis previously proposed to explain the timing of oligodendrocyte differentiation. They also unambiguously establish the phenotype of O-2A progenitor cells in vitro and demonstrate that these cells respond directly to growth factors produced by type-1 astrocyte monolayers.     

Infection by coronavirus JHM of rat neurons and oligodendrocyte-type-2 astrocyte lineage cells during distinct developmental stages.

Primary telencephalic cultures derived from neonatal Wistar Furth rats were able to support the growth of coronavirus JHM if a viable neuronal population was maintained. This occurred under serum-free defined, but not serum-supplemented, growth conditions. The importance of neurons in establishing infections in mixed cultures was confirmed by immunocytochemical and electron microscopic studies. Glia, although more abundant than neurons in these cultures, were less frequently infected during the initial 48 h postinoculation. The two glial lineages present in mixed telencephalic cultures were separated into type-1 astrocytes and oligodendrocyte-type-2 astrocyte (O-2A) lineage cells and individually assessed for their ability to support virus growth. Infection could not be established in type-1 astrocytes regardless of the culture conditions employed, consistent with our previous study (S. Beushausen and S. Dales, Virology 141:89-101, 1985). In contrast, infections could be initiated in selected O-2A lineage cells grown in serum-free medium. Virus multiplication was however significantly reduced by preconditioning the medium with mixed telencephalic or enriched type-1 astrocyte cultures, suggesting that intercellular interactions mediated by soluble factor(s) can influence the infectious process in O-2A lineage cells. This presumption was supported by eliciting similar effects with basic fibroblast growth factor and platelet-derived growth factor, two central nervous system cytokines known to control O-2A differentiation. The presence of these cytokines, which synergistically block O-2A cells from differentiating into oligodendrocytes was correlated with specific and reversible resistance to JHM virus (JHMV) infection. These data, combined with our finding that accelerated terminal differentiation of the oligodendrocyte phenotype confers resistance to JHMV (Beushausen and Dales, Virology, 1985), suggest that the permissiveness of O-2A cells for JHMV is restricted to a discrete developmental stage.     

Specific Expression of N-Acetylaspartate in Neurons, Oligodendrocyte-Type-2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro

To test the specificity of N-acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both 1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type-1 astrocytes, meningeal cells, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type-1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O-2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for 1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or their precursors.     

PDGF and intracellular signaling in the timing of oligodendrocyte differentiation

In the rat optic nerve, bipotential O-2A progenitor cells give rise to oligodendrocytes and type 2 astrocytes on a precise schedule. Previous studies suggest that PDGF plays an important part in timing oligodendrocyte development by stimulating O-2A progenitor cells to proliferate until they become mitotically unresponsive to PDGF, stop dividing, and differentiate automatically into oligodendrocytes. Since the loss of mitotic responsiveness to PDGF has been shown not to be due to a loss of PDGF receptors, we have now examined the possibility that the unresponsiveness results from an uncoupling of these receptors from early intracellular signaling pathways. We show that (a) although PDGF does not stimulate newly formed oligodendrocytes to synthesize DNA, it induces an increase in cytosolic Ca2+ in these cells; (b) a combination of a Ca2+ ionophore plus a phorbol ester mimics the effect of PDGF, both in stimulating O-2A progenitor cell division and in reconstituting the normal timing of oligodendrocyte differentiation in culture; and (c) the same combination of drugs does not stimulate newly formed oligodendrocytes to proliferate, even in the presence of PDGF or dibutyryl cAMP. The most parsimonious explanation for these results is that O-2A progenitor cells become mitotically unresponsive to PDGF because the intracellular signaling pathways from the PDGF receptor to the nucleus are blocked downstream from the receptor and some of the early events that are triggered by receptor activation.