O2A Progenitor Cells Research Articles

CNS-Resident Glial Progenitor/Stem Cells Produce Schwann Cells as well as Oligodendrocytes during Repair of CNS Demyelination

After central nervous system (CNS) demyelination-such as occurs during multiple sclerosis-there is often spontaneous regeneration of myelin sheaths, mainly by oligodendrocytes but also by Schwann cells. The origins of the remyelinating cells have not previously been established. We have used Cre-lox fate mapping in transgenic mice to show that PDGFRA/NG2-expressing glia, a distributed population of stem/progenitor cells in the adult CNS, produce the remyelinating oligodendrocytes and almost all of the Schwann cells in chemically induced demyelinated lesions. In contrast, the great majority of reactive astrocytes in the vicinity of the lesions are derived from preexisting FGFR3-expressing cells, likely to be astrocytes. These data resolve a long-running debate about the origins of the main players in CNS remyelination and reveal a surprising capacity of CNS precursors to generate Schwann cells, which normally develop from the embryonic neural crest and are restricted to the peripheral nervous system.

Expression of oligodendroglial differentiation markers in pilocytic astrocytomas identifies two clinical subsets and shows a significant correlation with proliferation index and progression free survival

The growth pattern of pilocytic astrocytoma (PAs) is unpredictable. Gene expression profiling has recently demonstrated an inverse relationship between myelin basic protein (MBP) expression and progression free survival (PFS) in PAs. We present here the pattern of expression of oligodendroglial differentiation markers (ODMs) in PAs by immunohistochemistry and their correlation with PI and PFS. Sixty-four cases of PA were reviewed and representative sections were stained for Ki-67 and ODMs, including MBP, platelet-derived growth factor receptor-alpha (PDGFR-alpha), Olig-1, and Olig-2. Sections were graded semi-quantitatively for intensity (I: 0-3+) and extent (E: 0-4+) of staining. PI was expressed as a percentage of Ki-67 positive cells. Immunoreactivity of MBP, PDGFR-alpha, Olig-1, and Olig-2 was observed in 84, 56, 97, and 75% of cases, respectively. There was a statistically significant inverse correlation between MBP expression and PI (r (2) = .696, p = .014). A positive correlation was observed between PDGFR-alpha and PI (r (2) = .727, p = .011). Further analysis showed a significant difference in PFS between low expressors [I + E score < or = 3] and high expressors (I + E score > or = 4) for PDGFR-alpha with p < .001. Notably, there was a significant difference in PFS between high expressors of MBP and high expressors of PDGFR-alpha with p < .001. These results suggest that expression of ODMs, especially MBP and PDGFR-alpha, may identify two clinical subsets of PA. In addition, we have shown the expression of 4 different ODMs in PAs, which may support the possibility that PAs arise from oligodendrocyte progenitor/precursor cells probably similar to the O2A progenitor cells in the mouse.

NG2‐positive cells generate A2B5‐positive oligodendrocyte precursor cells

Cellular specification of the oligodendrocyte lineage occurs through a series of stages identified by expression of distinct biochemical characteristics. The best characterized oligodendrocyte progenitor cell (OPC) in vitro is the bipotential O2-A progenitor, identified by labeling with monoclonal antibody A2B5, which proliferates predominantly in response to platelet derived growth factor (PDGF). The cellular ancestors of O2-A progenitor cells are currently unclear. In vivo OPCs can be identified by expression of the cell surface markers NG2 (a sulfated proteoglycan) and platelet derived growth factor receptor alphaR). Substantial evidence supports the generation of oligodendrocytes from NG2(+), PDGFalphaR(+) cells both in vivo and in vitro. The developmental relationship between NG2(+) cells and A2B5(-) positive cells is unknown and it is unclear whether they represent identical, partially overlapping or nonoverlapping populations of cells. Here we show that in cultures of developing brain NG2(+) and A2B5(+) cells arise from overlapping cell populations. NG2(+) cells appear prior to the expression of A2B5(+) cells and generate A2B5(+) cells. We propose that during development NG2(+)/A2B5(-) cells (pre-OPCs) represent the direct ancestor to A2B5(+) O2A progenitor cells (OPCs).

Expression of the anaphylatoxin C5a receptor in the oligodendrocyte lineage

Expression of the C5a receptor in the central nervous system has been demonstrated on microglia, astrocytes and neurons. In the present study, we demonstrate C5aR expression in vitro by rat and murine O2-A progenitor cells and oligodendrocytes. We also observed that in vitro differentiation of O2-A progenitors into mature oligodendrocytes is accompanied by down-regulation of C5aR mRNA expression. These results suggest that the C5aR may be a marker for oligodendroglial differentiation and play a role in oligodendrocyte function.

Developmental expression of OSP/claudin-11.

Oligodendrocyte-specific protein (OSP/claudin-11) is a major component of CNS myelin and has been recently added to the claudin family of tight junction proteins. In this study, the developmental expression of OSP/claudin-11 was determined using in situ hybridization, immunohistochemistry (IH), and Western blot analysis. OSP/claudin-11 mRNA was expressed in a bimodal fashion. During prenatal development, OSP/claudin-11 mRNA was abundant in developing meninges, in areas adjacent to cartilage, and in mesoderm. In postnatal animals, OSP/claudin-11 was expressed primarily in developing oligodendrocytes and to a lesser extent, in testes. Double-labeled IH using O2-A progenitor cells revealed that OSP/claudin-11 expression occurs from the early progenitor stage and continues in mature oligodendrocytes. Electron microscopic IH localized OSP/claudin-11 to laminar myelin in the adult CNS. Western blot analysis of OSP/claudin-11 in developing brain revealed the expression of two separate transcripts that were developmentally regulated. These data demonstrate that OSP/claudin-11 expression is highly regulated during development and, therefore, may play an important role in growth and differentiation of oligodendrocytes and other cells outside the CNS.

Developmental expression of OSP/claudin-11

Oligodendrocyte-specific protein (OSP/claudin-11) is a major component of CNS myelin and has been recently added to the claudin family of tight junction proteins. In this study, the developmental expression of OSP/claudin-11 was determined using in situ hybridization, immunohistochemistry (IH), and Western blot analysis. OSP/claudin-11 mRNA was expressed in a bimodal fashion. During prenatal development, OSP/claudin-11 mRNA was abundant in developing meninges, in areas adjacent to cartilage, and in mesoderm. In postnatal animals, OSP/claudin-11 was expressed primarily in developing oligodendrocytes and to a lesser extent, in testes. Double-labeled IH using O2-A progenitor cells revealed that OSP/claudin-11 expression occurs from the early progenitor stage and continues in mature oligodendrocytes. Electron microscopic IH localized OSP/claudin-11 to laminar myelin in the adult CNS. Western blot analysis of OSP/claudin-11 in developing brain revealed the expression of two separate transcripts that were developmentally regulated. These data demonstrate that OSP/claudin-11 expression is highly regulated during development and, therefore, may play an important role in growth and differentiation of oligodendrocytes and other cells outside the CNS. J. Neurosci. Res. 3:284–290, 2000 © 2000 Wiley-Liss, Inc.

GDNF-related factor persephin is widely distributed throughout the nervous system.

Persephin (PSP) is the most recently discovered member of the GDNF family of neurotrophic factors. We have used an RT-PCR approach to start addressing the putative functional significance of PSP by determining sites of its synthesis in the neonatal rat brain. Generally, two transcripts were found. Sequence analysis of the transcripts identifies an 88 bp intronic sequence. Neural tissues analysed included cortex, hippocampus, striatum, diencephalon, mesencephalon, cerebellum, hindbrain and spinal cord as well as superior cervical, dorsal root ganglia, adrenal gland, and PC12 pheochromocytoma cells. As non-neuronal tissues, sciatic nerve, optic nerve, primary astroglial, oligodendroglial, O2A progenitor, and glioma cells (C6, B49) were also included. All tissues/cells except oligodendrocytes and O2A progenitor cells were strongly positive for PSP mRNA. To test the hypothesis of whether PSP might act as a target-derived factor, as suggested for GDNF, the motoneuron-muscle axis has been analysed. PSP is synthesized in skeletal muscle and, to a higher extent, in the spinal cord. Moreover, PSP is synthesized in purified embryonic motoneurons. Together, these data do not support a role for PSP as a typical target-derived neurotrophic factor for motoneurons. We conclude that PSP is synthesized throughout the nervous system and that it is presumably of both astroglial and neuronal origin, in contrast to GDNF and neurturin, which seem to be predominantly of neuronal origin.

Regulation of oligodendrocyte differentiation: protein kinase C activation prevents differentiation of O2A progenitor cells toward oligodendrocytes.

Oligodendrocytes differentiate on a specific schedule in vivo in order to myelinate axons at the precise time and at the appropriate position. The current study was undertaken to obtain further insight as to how this timed appearance is regulated intracellularly. We observed that exposure of O2A progenitor cells in culture to phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C, PKC) inhibited their differentiation to oligodendrocytes by suppressing the expression of specific myelin markers at the O4-stage. To positively identify a role of PKC per se in differentiation, the use of a minimal medium with low serum content turned out to be essential. This was demonstrated by showing that the inhibitory effect of PMA on oligodendrocyte differentiation could be completely abolished by a combined action of insulin, triiodothyronine (T3), hydrocortisone and other components of a chemically defined medium (CDM). Furthermore, the PMA-mediated inhibition of oligodendrocyte differentiation could be partially restored by activation of the cAMP signal transduction pathway. The results indicate that PKC plays a crucial role in the differentiation of O2A progenitor cells toward oligodendrocytes: PKC activation prevents differentiation of O2A progenitor cells, whereas differentiation toward oligodendrocytes is dependent on other signaling compounds which may counteract the PKC signal transduction route.

Cellular distribution of branched-chain amino acid aminotransferase isoenzymes among rat brain glial cells in culture.

The first step in the catabolism of branched-chain amino acids (BCAA), reversible transamination, is catalyzed by one of the two isoforms of branched-chain amino acid aminotransferase (BCAT). The mitochondrial isoenzyme (BCATm) is widely distributed among tissues, whereas the cytosolic isoenzyme (BCATc) is restricted to only a few organs. Remarkably, BCATc is the prominent isoenzyme found in brain. The physiological significance of the subcellular compartmentation of BCAT is still not understood. To contribute to the elucidation of the cellular distribution of the two isoenzymes in brain, we used cultured rat glial cells in an immunocytochemical study to determine the pattern of BCAT isoenzyme expression by glial cells. Antiserum against BCATm generated a punctate staining pattern of astroglial cells, confirming the mitochondrial location of this isoenzyme. In contrast, the cytosol of galactocerebroside-expressing oligodendroglial cells and O2A progenitor cells displayed intense staining only for BCATc. In addition, subpopulations of astroglial cells exhibited BCATc immunoreactivity. The presence of BCATm in astrocytes is consistent with the known ability of these cells to oxidize BCAA. Furthermore, our results on BCATc provide support for the hypothesis that BCATs are also involved in nitrogen transfer from astrocytes to neurons.

Axonal neuregulin signals cells of the oligodendrocyte lineage through activation of HER4 and Schwann cells through HER2 and HER3.

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor-inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite-type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.

Interaction between NG2 proteoglycan and PDGF α‐receptor on O2A progenitor cells is required for optimal response to PDGF

Previous studies on the NG2 chondroitin sulfate proteoglycan have shown that NG2 is expressed on A2B5-positive O2A progenitor cells, which are known to respond to platelet-derived growth factor (PDGF). In the accompanying paper (Nishiyama et al.; J Neurosci Res 43:299–314, 1996) we show that on O2A progenitors in the embryonic and newborn rat brain, NG2 and PDGF α-receptor display an extensive co-localization which becomes less pronounced as the brain matures past the first postnatal week. The present communication describes the relationship between NG2 and PDGF α-receptor in vitro. NG2 and PDGF α-receptor are highly co-localized on A2B5-positive O2A cells isolated from neonatal rat cerebrum. Mimicking the situation in vivo, the level of expression of the two molecules and the extent of co-localization decline as these cells differentiate into O4-positive pre-oligodendrocytes. However, maintenance of the cells in a progenitor state by treatment with bFGF results in increased levels of both NG2 and PDGF α-receptor on the cell surface, suggesting that expression of the two molecules may be coordinately regulated. Furthermore, NG2 can be co-immunoprecipitated from radiolabeled O2A extracts with a rabbit antibody to PDGF α-receptor, indicating the presence of a molecular complex that includes NG2 and the receptor. Finally, antibody-patching and subsequent down-regulation of NG2 results in reduced expression of PDGF α-receptor and diminishes the proliferative response of the cells to PDGF. These findings suggest that correct co-expression of the NG2 proteoglycan and PDGF α-receptor on the surface of O2A progenitor cells is important for the cells' ability to respond effectively to the mitogenic stimulus of PDGF. © 1996 Wiley-Liss, Inc.

Interaction between NG2 proteoglycan and PDGF ?-receptor on O2A progenitor cells is required for optimal response to PDGF

Previous studies on the NG2 chondroitin sulfate proteoglycan have shown that NG2 is expressed on A2B5-positive O2A progenitor cells, which are known to respond to platelet-derived growth factor (PDGF). In the accompanying paper (Nishiyama et al.; J Neurosci Res 43:299-314, 1996) we show that on O2A progenitors in the embryonic and newborn rat brain, NG2 and PDGF alpha-receptor display an extensive co-localization which becomes less pronounced as the brain matures past the first postnatal week. The present communication describes the relationship between NG2 and PDGF alpha-receptor in vitro. NG2 and PDGF alpha-receptor are highly co-localized on A2B5-positive O2A cells isolated from neonatal rat cerebrum. Mimicking the situation in vivo, the level of expression of the two molecules and the extent of co-localization decline as these cells differentiate into O4-positive pre-oligodendrocytes. However, maintenance of the cells in a progenitor state by treatment with bFGF results in increased levels of both NG2 and PDGF alpha-receptor on the cell surface, suggesting that expression of the two molecules may be coordinately regulated. Furthermore, NG2 can be co-immunoprecipitated from radiolabeled O2A extracts with a rabbit antibody to PDGF alpha-receptor, indicating the presence of a molecular complex that includes NG2 and the receptor. Finally, antibody-patching and subsequent down-regulation of NG2 results in reduced expression of PDGF alpha-receptor and diminishes the proliferative response of the cells to PDGF. These findings suggest that correct co-expression of the NG2 proteoglycan and PDGF alpha-receptor on the surface of O2A progenitor cells is important for the cells' ability to respond effectively to the mitogenic stimulus of PDGF.

Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.

A detailed comparison in the developing rat central nervous system between the distribution of the NG2 proteoglycan and the alpha-receptor for platelet-derived growth factor (PDGF) shows that these two molecules are co-expressed by glial progenitor cells of the O2A lineage and can serve as reliable markers for identification of O2A cells in vivo. Our mapping experiments indicate that NG2-positive, PDGF alpha-receptor positive O2A cells are abundant throughout the developing central nervous system in both white and gray matter. The earliest cells immunoreactive for either of the two markers are found adjacent to the central canal of the embryonic day 15 (E15) spinal cord. These cells express only PDGF alpha-receptor and not NG2. By E17, process-bearing cells expressing both NG2 and PDGF alpha-receptor in a highly co-localized fashion are found throughout the central nervous system. The first postnatal week marks the peak in the number of NG2 and PDGF alpha-receptor immunoreactive cells, as well as the peak in the level of expression and the extent of co-localization of the two molecules. After the first week, the level of expression of both NG2 and PDGF alpha-receptor declines, although both molecules continue to be expressed in the adult brain. On O2A cells in the mature brain, NG2 and PDGF alpha-receptor are not as well co-localized at the subcellular level as they are on O2A cells in the younger brain. The functional consequences of co-localization and subsequent dissociation of NG2 and PDGF alpha-receptor on maturing O2A progenitors are investigated in the accompanying paper (Nishiyama et al.: J Neurosci Res 43:315-330, 1996).

CNS myelinogenesis in vitro: time course and pattern of rat oligodendrocyte development.

Oligodendrocyte precursor cells that develop into myelin-forming cells of the central nervous system (CNS) were cultured from newborn rat brain to study how they proliferate and differentiate in normal conditioning medium, and their cell development was characterized by scanning electron microscopy (SEM) observation and immunocytochemical studies. We have identified A2B5-negative pre-O2A progenitor cells (so-called "type-1" oligodendrocytes) in the secondary cultures on the astrocyte feeder layer. These cells are very small (diameter: 3.5 microns), round, and glossy, and develop into the process-bearing O2A progenitor cells (called "type-2" oligodendrocytes), which also express myelin basic protein (MBP) both in the cell body and in their cell processes. Finally, they develop into mature oligodendrocytes (called "type-3" oligodendrocytes). After MBP expression is elicited in these cells and MBP accumulates in the cell process in the area in contact with the axon, these cells are capable of forming the myelin sheath. Therefore, we examined the mechanism of myelin-sheath formation of "type-3" oligodendrocytes using video time-lapse movies, and demonstrated that these cells initially sent out processes to search for axons several times before the onset of myelination. Then thick filopodia extended towards the axon, and at the same time, the axonal part of neuron moved forward. Finally the ruffling lamellipodial parts wrapped up the axon similarly to a transverse wave with the secured thick filopodial process on the axon acting as scaffolding. These results suggest that our experimental systems are useful in studying normal oligodendrocyte development and their cellular biochemistry, as well as investigating the mechanism of myelin formation by oligodendrocytes.

A role for the acetylcholine receptor-inducing protein ARIA in oligodendrocyte development.

ARIA acetylcholine receptor-inducing activity protein, is a member of a family of ligands that includes the Neu differentiation factor, heregulin, and glial growth factor. These ligands all act through one or more receptor tyrosine kinases of approximately 185 kDa. In some conditions these ligands promote proliferation, whereas in others they induce differentiation. ARIA was originally isolated from chick brain on the basis of its ability to induce synthesis of nicotinic acetylcholine receptors in skeletal muscle. In this paper we show that ARIA is expressed in the subventricular zone of the rat brain and that it enhances the development of oligodendrocytes from bipotential (O2A) glial progenitor cells. We have also found that ARIA induces tyrosine phosphorylation of a 185-kDa protein in O2A progenitor cells. ARIA does not increase bromodeoxyuridine incorporation by oligodendrocytes but is mitogenic when added to Schwann cells in vitro. Thus, ARIA accelerates the formation of oligodendrocytes in vitro and is expressed where it could exercise the same influence in vivo.

Regulation of the POU domain gene SCIP during cerebral cortical development

The mammalian cerebral cortex is patterned into layers of neurons that share characteristic morphologies, physiological properties, and axonal connections. Neurons in the various layers are thought to acquire their lamina-specific identities shortly before the time of their final mitosis in the cortical ventricular zone. In order to investigate the molecular basis of laminar patterning in the CNS, we have performed in situ hybridization studies of the POU homeodomain gene SCIP (also known as Tst-1 or Oct-6), which is expressed in proliferating Schwann cells in the PNS and O2A progenitor cells in the developing CNS. In the CNS of adult rats, SCIP is expressed at high levels in the cerebral cortex, specifically in layer 5 pyramidal neurons that form subcortical axonal connections. SCIP is both temporally and spatially regulated during cortical development. Its initial expression in the intermediate zone and cortical plate is correlated with the early migration and differentiation of layer 5 neurons. SCIP hybridization was not, however, observed within the ventricular zone during the period of neurogenesis. SCIP is also expressed at high levels in the neurons of cortical layer 2/3, during their migration and differentiation within the cortical plate. This expression in the upper layers is apparently downregulated during postnatal periods, with the adult pattern apparent by postnatal day 30 (P30). POU domain genes are thought to play a role in cell lineage and cell fate decisions in several systems; thus, SCIP may serve a function in generating discrete laminar phenotypes in the developing cerebral cortex. In addition, since SCIP is a putative repressor of myelin gene expression, our results suggest that SCIP plays a role in regulating transcription in differentiated CNS neurons as well as in proliferating glial precursors.

O2A progenitor cells transplanted into the neonatal rat brain develop into oligodendrocytes but not astrocytes.

The differentiation of the bipotential O2A progenitor cell into an oligodendrocyte or a type 2 astrocyte has been well documented in cell cultures of various regions of the central nervous system. The appropriate tools to prove its existence in vivo have been lacking. We report on an in vitro-in vivo approach that combines stable labeling of an enriched population of cultured O2A progenitors by the fluorescent dye fast blue, followed by their transplantation into neonatal rat brains, which allowed us to study the influence of the brain microenvironment on their lineage decision. The grafted cells survived well and 21 days after grafting nearly all were positive for the oligodendroglial marker galactocerebroside. Surprisingly, the fast blue-positive grafted cells did not stain for the astroglial marker glial fibrillary acidic protein. These results indicate that the O2A progenitor's plasticity is restricted by the in vivo environment, resulting in the developmental exclusion of the type 2 astrocyte initially described in vitro.

Injury‐induced Regulation of Ciliary Neurotrophic Factor mRNA in the Adult Rat Brain

Ciliary neurotrophic factor (CNTF) is a pleiotropic molecule that acts as a neurotrophic factor for a wide range of embryonic neurons as well as a differentiation factor for sympathetic neuroblasts and O2A progenitor cells in culture. CNTF messenger RNA (mRNA) is present at very low levels in the normal adult rat central nervous system (CNS), but is dramatically up-regulated after an aspiration lesion of dorsal hippocampus and overlying cortex, in the area coincident with glial scar. The increased level of CNTF mRNA in lesioned hippocampus is maximal by 3 days and is sustained for up to 20 days, the longest time point examined. In contrast, mRNA levels for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were slightly decreased during the same period. In situ hybridization experiments revealed that cells expressing CNTF mRNA were concentrated at the margin of the wound, and also present within the gelfoam which filled the lesion cavity. This distribution of CNTF-expressing cells corresponded very closely to that of cells expressing high levels of glial fibrillary acidic protein mRNA at the wound site. Paralleling the observed increase in CNTF mRNA, increased levels of CNTF-like neurotrophic activity were apparent in soluble extracts of the lesioned tissues. This neurotrophic activity for ciliary ganglion neurons was completely blocked by the addition of neutralizing antiserum against CNTF. Basic fibroblast growth factor, which has been shown by others to increase after a similar lesion paradigm (Frautschy et al., Brain Res., 553, 291-299, 1991), does not contribute appreciably to this trophic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Astrocyte precursors in neonatal rat spinal cord cultures

Cultures of newborn rat spinal cord contain multiple types of astrocytes. By using a combination of cultures enriched for glial precursors and clonal analysis, we have identified a particular astrocyte precursor that gives rise to morphologically distinct classes of astrocytes. This astrocyte precursor labels with the monoclonal antibody A2B5, is highly migratory, proliferates in response to serum and platelet-derived growth factor, and differentiates into process-bearing astrocytes, many of which subsequently assume a "pancake"-shaped morphology. A2B5+ astrocyte precursors share antigenic and migratory characteristics with previously described O2A progenitor cells but differ in their response to regulatory factors, including serum and coculture with type 1 astrocytes. More importantly, these astrocyte precursors do not give rise to oligodendrocytes. In their proliferative response to serum and their capacity to differentiate into astrocytes, these glial precursors resemble type 1 astrocyte precursors from optic nerve. However, unlike type 1 astrocyte precursors, these cells are A2B5+, highly migratory, and do not give rise to fibroblast-like astrocytes. Neonatal rat spinal cord cultures contain approximately twice the number of the A2B5+ astrocyte precursors than O2A progenitor cells. By contrast, the majority of A2B5+ cells in postnatal day 7 optic nerve cultures are O2A progenitors. The presence of large numbers of A2B5+ astrocyte precursors in rat spinal cord cultures may reflect the more complex cytoarchitecture of the spinal cord compared to the optic nerve.

The primary structure of NG2, a novel membrane-spanning proteoglycan.

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.