Analysis of the cell-cell interactions that control type-2 astrocyte development in vitro

Oligodendrocytes and type-2 astrocytes develop sequentially from O-2A progenitor cells in the rat CNS. We have reproduced this sequential development in a simplified, serum-free in vitro system: in cultures of newborn optic nerve cells treated with platelet-derived growth factor to maintain O-2A progenitor cell proliferation, progenitor cells differentiate into oligodendrocytes during the first week in vitro and into type-2 astrocytes during the second week. Thus all of the signals needed for type-2 astrocyte development are made by serum-free optic nerve cultures, indicating that neurons are not required. By manipulating the cellular composition of the cultures, we provide evidence that type-2 astrocyte development does not depend on oligodendrocytes, but instead requires non-O-2A lineage cells, which are also responsible for timing this development.     

In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the expression of viral RNA and proteins, implying that replication was inhibited at some step between penetration and initiation of genomic functions, perhaps at the stage of uncoating. We therefore examined the possibility that protein kinases and phosphatases, which influence cellular regulation during cAMP-induced differentiation, may be responsible for the phenomenon of coronavirus suppression in oligodendrocytes. Evidence was obtained indicating that normal processing of the phosphorylated nucleocapsid protein is inhibited in differentiated oligodendrocytes, consistent with the notion that JHMV replication might be arrested during uncoating.     

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.     

Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-1 astrocytes

O-2A progenitor cells are bipotential glial precursors that give rise to both oligodendrocytes and type-2 astrocytes on a precise schedule in the rat CNS. Studies in culture suggest that oligodendrocyte differentiation occurs constitutively, while type-2 astrocyte differentiation requires an exogenous inducer such as fetal calf serum. Here we describe a rat brain cell culture system in which type-2 astrocytes develop on schedule in the absence of exogenous inducers. Coincident with type-2-astrocyte development, the cultures produce an approximately 20 kd type-2-astrocyte-inducing factor(s). Purified cultures of type-1 astrocytes can produce a similar factor(s). Under conditions where they produce type-2-astrocyte-inducing factor(s), both brain and type-1 astrocyte cultures produce a factor(s) with ciliary neurotrophic (CNTF)-like activity. Purified CNTF, like the inducers from brain and type-1 astrocyte cultures, prematurely induces type-2 astrocyte differentiation in brain cultures. These findings suggest that type-2 astrocyte development is initiated by a CNTF-like protein produced by type-1 astrocytes.     

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.     

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.     

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.     

Heterotypic and homotypic cellular interactions influencing the growth and differentiation of bipotential oligodendrocyte-type-2 astrocyte progenitors in culture

Cell populations highly enriched in oligodendrocyte-type-2 astrocyte (O-2A) progenitors (so defined by their ability to bind the monoclonal antibodies LB1 and O4, and by the lack of expression of the differentiated glial markers galactocerebroside and glial fibrillary acidic protein (GFAP) were obtained from rat mixed cortical glial cultures. The O-2A progenitors were grown at low density (2 X 10(4) cells/cm2) in BME + 10% fetal calf serum (FCS) on a poly-L-lysine (PLL) substrate (controls) or on a substrate of purified type-1 astrocytes (AS) killed by air drying (K-AS), in order to analyze the effects of the interaction between the two cell types on the growth and differentiation of the immature O-2A cells, independently of the mitogenic soluble factors (e.g., platelet-derived growth factor; see Raff, 1989, Science 243, 1450-1455) secreted by type-1 AS. While on PLL most of the progenitors differentiated into GFAP+ type-2 AS within 1 week, on K-AS they largely differentiated into GalC+ oligodendrocytes (OL). On the latter substrate, however, the precursors achieved a higher density, due to higher proliferative activity. The additional observation, that when immature O-2A cells were seeded at high density (greater than 5 X 10(4) cells/cm2) on PLL their differentiation into OL was much more pronounced than in cultures of lower density, indicates that there is a close correlation between the density of immature O-2A cells and lineage decision, and that the increased OL differentiation of the immature O-2A cells on K-AS is at least partly related to the higher density achieved by the cells on this substrate. The enhanced proliferation of immature O-2A cells on K-AS did not appear to be related to platelet-derived growth factor or fibroblast growth factor remaining attached to the substrate, nor to known components of the extracellular matrix (ECM), such as heparan sulfate, chondroitin sulfate, laminin, or fibronectin, but was probably due to other components of a polypeptide nature present in the ECM produced by type-1 AS. A cell-free ECM was in fact almost as mitogenic as the K-AS substrate, and the mitogenic activities of both K-AS and AS-ECM were similarly inhibited by a set of enzymatic (pronase, trypsin) and physicochemical (heat, pH) treatments.     

Establishment of mouse oligodendrocyte/type-2 astrocyte lineage cell line by transfection with origin-defective simian virus 40 DNA.

A permanent glial cell line has been established from the neonatal mouse primary mixed glial cell cultures by transfection with replication origin-defective simian virus 40 DNA. This cell line, designated OS3, has morphological similarity to type-2 astrocyte and expresses an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), when cultured in the presence of 10% calf serum (CS). OS3 cells do not express the O4 antigen, galactocerebroside (GalC) and A2B5 under this culture condition. When cultured in a medium containing 2% CS or a chemically defined medium, these cells undergo morphological transformation. Some of these cells express O4 antigen and/or GalC, and the percentage of GFAP positive cells decreases under these conditions. Thus depending on the culture conditions, the OS3 cells display either type-2 astrocyte properties or immature oligodendrocyte characteristics. Furthermore, the OS3 cells show similar responses to the various growth factors as do oligodendrocyte/type-2 astrocyte (O-2A) progenitors. Therefore, the OS3 cell line is an unique mouse bipotential permanent O-2A lineage cell line which may be useful to analyze the developmental properties of these glial cells.     

In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with [3H]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.     

Substance P Receptors on O-2A Progenitor Cells and Type-2 Astrocytes In Vitro

Abstract: Bradykinin- and substance P (SP)-stimulated second messenger studies in isolated subsets of neuroglia showed bradykinin-stimulated synthesis of phospho- inositides (PI) in type-1 astrocytes and oligodendrocytes. SP-stimulated PI accumulation was restricted to oligoden- drocyte/type-2 astrocyte progenitor cells and type-2 astrocytes. These data were confirmed by analysis of calcium transients in single cells. In a regional study, SP-stimulated PI accumulation in primary astrocyte cultures was restricted to white matter. We conclude that regional heterogeneity in the expression of peptide receptors in cultures of primary astrocytes arises from a restricted distribution on subsets of macroglia. SP receptors restricted on cells of the oligodendrocyte/type-2 astrocyte type-2 lineage in vitro, coupled with in vivo observations by others, suggests that SP receptor expression is conserved on subsets of macroglia in vitro and possibly reactive astrocytes in vivo.     

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.     

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.     

The complex relationship between cell division and the control of differentiation in oligodendrocyte-type-2 astrocyte progenitor cells isolated from perinatal and adult rat optic nerves

By studying the response of a well-defined progenitor cell to two well-defined mitogens, we have been able to provide a dramatic example of the complex relationship which can exist between the control of cell division and the control of differentiation.In previous studies we have described the development of the oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell, a glial progenitor cell isolated from the rat optic nerve. Although originally described as a bipotential cell, we have recently identified a new differentiation pathway in this lineage. We have found that O-2A^perinatal progenitors, with properties appropriate for early development, give rise to O-2A^adult progenitors, which have stem cell-like properties more appropriate to the physiological needs of adult animals. Our studies thus indicate that the population of O-2A^perinatal progenitors is tripotential, and also suggests a possible developmental origin for self-renewing stem cells. Moreover, the properties of O-2A^adult progenitor cells may provide a cellular biological basis for understanding the failure of remyelination in multiple sclerosis.The division of both O-2A^perinatal and O-2A^adult progenitors is stimulated by type-1 astrocytes (which are themselves derived from a separate glial lineage) but this cell-cell interaction promotes different programs of differentiation in the two progenitor populations. The effects of type-1 astrocytes on perinatal and adult progenitors appears to be mediated by platelet-derived growth factor (PDGF), and this mitogen will also induce different programs of differentiation in the two progenitor populations. Moreover, the patterns of differentiation promoted by PDGF are different from those promoted by fibroblast growth factor (FGF), demonstrating that the modulation of division can be distinguished from the modulation of differentiation.     

Heparin is a unique marker of progenitors in the glial cell lineage.

The oligodendrocyte-type-2 astrocyte progenitor cells (precursors of oligodendrocytes and type-2 astrocytes) are an excellent system in which to study differentiation as they can be manipulated in vitro. Maintenance of oligodendrocyte-type-2 astrocyte progenitor cells requires basic fibroblast growth factor, a growth factor whose action normally depends on a heparan sulfate coreceptor. Biochemical analysis revealed a most surprising result: that the oligodendrocyte-type-2 astrocyte progenitors did not synthesize heparan sulfate, the near ubiquitous N-sulfated cell surface polysaccharide, but the chemically related heparin in a form that was almost completely N- and O-sulfated. The heparin was detected in the pericellular fraction of the cells and the culture medium. In contrast the differentiated glial subpopulations (oligodendrocytes and type-2 astrocytes) synthesized typical heparan sulfate but with distinctive fine structural features for each cell type. Thus heparin is a unique differentiation marker in the glial lineage. Previously heparin has been found only in a subset of mature mast cells called the connective tissue mast cells. Its presence within the developing nervous system on a precise population of progenitors may confer specific and essential recognition properties on those cells in relation to binding soluble growth and/or differentiation factors and the extracellular matrix.     

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.     

Cyclic AMP regulates the rate of differentiation of oligodendrocytes without changing the lineage commitment of their progenitors

Oligodendrocytes differentiate in primary cultures of rat brain cells on a specific schedule similar to that observed in vivo. We show that the pace of this developmental schedule is accelerated by the addition of the cyclic AMP analogs dibutyryl cAMP (dbcAMP) or 8-bromo cAMP. Dibutyryl cAMP also inhibits DNA synthesis in A2B5-positive oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, consistent with the relationship between cessation of proliferation and onset of differentiation observed in vivo and in vitro. Treatment of cultures with dbcAMP has no effect on the proportion of O-2A progenitors that become oligodendrocytes rather than type 2 astrocytes and thus does not affect progenitor lineage decisions. Thus, cyclic AMP analogs accelerate the differentiation of cells apparently already determined to become oligodendrocytes.     

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.     

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.